Center for Dairy Research ( 0 ~ College of AgIculhsal and ute SCiences ? lkliV9lSily of WISCOOSir>Madlson : I . ~ AnnuaJReport . . Babcock Hall Addition and Remodel New Facilities & Expanded Capabilities 1990-1.991 Center for ~:~Z}~:~~:,~~~OR. Annual Report July 1, 1990 ? June 30, 1991 September 1, 1991 Center for Dairy Research 1605 Linden Drive Madison, WI 53706 Direct your inquiries about CDR to : Center for Dairy Research University of Wisconsin-Madison 1605Linden Dr., Rm.. 241 Madison, Wisconsin 53706 Phone: 6081262-2217 The material in this annual report has been prepared for organizations funding CDR and for fellow dairy researchers. Research reports contained in this document describe projects in progress and interpretations of data gathered to date. This is not a peer-reviewed publication. Materials within this report should not be reprinted, refer enced, or publicized without written consent of the author. For information on how to contact the author call Sarah Quinones at 6081262?2217. ii Contents Letter from the Director vii Chapter 1. Overview of CDR 1 Chapter 2. Research Progress Reports 9 Dairy Poods Safety and Quality Progress Reports Overview.??..?._?.?...?._ _ t 1 Behavior of Listeria monocytogenes in ultrafiltered milk with and without added lactic add bacteria; fE. Marth 12 Behavior of foodbome pathogens in the presence of lactic add bacteria; fR. Marth 14 The growth and survival of Borrelia burgdorjeri. in milk; GM. Farrell 16 Prevention of survival and growth of ps.thogens in milk and cheese by enhancement of the activity of lactoferrin and lysozyme; E.A. Johnson .?.?...?.?.?.??.?..??????????.??.?????.?..?....??????????.?......., , 11 Effects of heat treatment and cheesemaking variables on pathogen survival and growth; f.A.Johnson 24 Generation and roles of proline in providing flavor and pathogen protection in cheese; E.A.Johnson 34 Identification of environmental sources of Listeria monocytogenes in dairy product manufacturing plants and development of HACCP programs designed to prevent Listeria contamination of dairy products; E.A.Johnson 38 Control of Listeria monocytogenes surface colonization of cheese; E.A.]ohnson 40 Growth inhibition of milkbome pathogens by fatty adds; E.A.Johnson 41 Cheese Technology Progress Reports Overview 43 Economic benefits of increasing the quality of milk used for cheese in Wisconsin; E.Jesse 45 Development of an economic engineering microcomputer model for analysis of cheese plant operation; B.W. Gould 47 iii I Center for Dairy Research I ( Physical and thermal properties of different cheeses; S. GunRSekaran .48 Development of a systematic approach for producing cheese as a food ingredient; N.F. Olson 51 Effect of fat, moisture, and salt on the freezing qualities of Cheddar-type cheeses; W.L. Wendorff 52 Construction of a gene bank of Lactobacillus helveticus CNRZ 32: cloning and characterization of the aminopeptidase and threonine aldolase genes; J.L. Steele 53 . Effectof starter culture produced glutathione on cheese flavor development; J.L Steele 54 Enhancing flavor characteristics and maturation rate of cheese by selected enzymatic and microbial treabnents; N.F. Olson 57 The peptide hydrolase system of Bifidobacterium species; M. EI-SOda 59 Autolytic characteristics of cheese ripening bacteria; M.E.Johnson 63 Mechanisms of injury to Streptococcus 1at:tis during spray drying; M.R'- Etzel 64 Effect of post-processing on cell viability, cell permeability, and enzyme activity of Lactobacillus helveticus cheese starter culture adju.l\ct; MaR. Etzel 66 Inactivation kinetics of Lactobacillus helveticus and its intracellular enzymes; C. Buijsse ...f ?????????????????????????????????????????????????????????????????????????????????????????????1' ????????????? 69 Improving the flavor ofenzyme-modified cheeses by control of lipase action in supercritical COz:R.W.Hartel 71 Development of process technology and flavor enhancement of reduced-fat cheese; M.E.Johnson 73 Development of basic technology for improving the flavor and consumer acceptability of reduced-sodium Cheddar cheese; R.C.. Uttdsay , 77 Heat-resistance of pediococci and lactobacilli isolated from raw milk and Cheddar cheese; M.E. Johnson 79 Control of color formation in smoked cheese; W.L. Wendorff 81 Milk Component Utilization Progress Reports I. Milkfat Utilization Overview 83 Milk: a point of entry into the human diet for mevalonate suppressive plant secondary metabolites; C.E. Elson 85 Modification of milkfat composition by production of null mutants for acetyl-eoA carboxylase in transgenic animals; R.D. Bremel 89 iv Annual Report 1990-1991 II Expression of bovine alpha 5(1) casein in milk and. tissues of transgenic mice; R.D. Brernel 91 Development of improved. processes for enhanced. melt properties and flavor stability of cold-spreadable butter and other dairy-based. food products;.R.C. lindsay 93 Incorporation of butterfat fractions into chocolate and confectionery; R.W. Hartel ..?.................................................................................................... .. ............. .. .............. 95 Relationship between butterfat short-chain fatty acids and lipoprotein metabolism in the rat; D.M. Neg 97 Use of immobilized. enzymes in the treatment of milkfat; c.G. HiU, Jr 100 Modification of butterfat by lipolytic reactions in novel reaction system; J.P. Chen 104 Interesterification of butterfat with gel-entrapped. cells; J.P. Chen 105 Enzymic modification of butterfat in supercritica1 CO 2 ; R.W. Hartel 106 Demand analysis of dairy products using cross-sectional data; B. W. Gould 108 Development and maintenance of dairy product related database; B. W. Gould 110 Development of a Wisconsin input-output model for dairy product industry analysis; B.W. Gould 111 A new technology for milkfat products; R.W. Hartel 112 II. Non-fat Solids (Whey> Utilization Overview , 113 Effect of protein and non-protein components on thermal gelation Conversion of whey components to commercially valuable products; Removal of lipids from cheese whey; J.P. Olen 114 of whey protein concentrates; S. Damodaran 115 D.C. Cameron 116 Construction of a D(->-Iactic dehydrogenase negative strain of Lactobacillus heIveticus; J.L. Steele 119 New Product and Process Development Progress Reports Overview 121 Use of novel immobilized. beta-galactosidase reactor to hydrolyze the lactose constituent of skim milk; c.G. Hill, Ir 122 Freeze concentration of fluid dairy products; R.W. Hartel 124 New dairy foods with added calcium and modified. salt; J.L. Greger 130 CLA: a newly recognized anticarcinogen isolated from dairy products; M.W. Pariza 132 v I Center for DRiry ReseRrch I ( Chapter 3. Worldwide Information and Technology Exchange Program (WITEP) ..; 133 Overview 135 e Scientist Exchange Programs 136 Scimtism 'Visitin.g CDR 136 111 CDRresearcher visits to other dairy research programslconferenceslcourses 140 Seminars and Conferences 144 PublicationslInforD1ation Dissemination 147 Chapter 4. 1990 CDR Research PublicationslPresentations 149 Appendix A: Index of Principal Investigators and Academic DepartJn.ents 159 vi Letter from the Director Dear Colleagues: The research and information/technology transfer activities of the Center for Dairy Research (CDR), which were initiated in 1986,were designed as multi-disdplinary programs to address specific needs in four focus areas: milk component utilization, cheese technology, food safety, and process and product development. It is impossible to trace or verify the use of all the CDR research outputs, but some examples of CDR research that have been adopted or evaluated by the dairy industry are: 1. techniques developed in cooperation with Land a Lakes to minimize calcium lactate crystallization on cheese, 2. development of Wisconsin Style Havarti? cheese, which is being manufactured and marketed commercially, 3. development of whey fermentation techniques to produce two unique polysaccharides (gums) that have potential food and non-food uses, - 4. development of lactic add bacterial strains that produce only L-Iactic add, a key ingredient in the emerging biodegradable plastics industry, 5. building a basis for industrial quality-control practices by determining the effects of environmental factors on survival of Listeria monocytogenes in milk clotting enzyme preparations, 6. developing technologies being utilized by cheese manufacturers and culture supply houses for manufacturing lowfat cheeses and determining the factors that effect flavor quality of lowfat and low-sodium cheeses, 7. computer programs that have been or are being used by the cheese industry to evaluate economics of milk standardization and cheese manufacturing. The research program summarized in this report describes the continuation of our efforts in our four focus areas. Accomplishments in each of the areas are summarized preceding the project reports. The activities of CDR's Worldwide Information and Technology Exchange Program (WITEP) illustrates our commitment to communicating the information/technology generated by the research program to the dairy industry. The research and the information/technology transfer functions outlined in this report result from finandal and technical support from the Wisconsin Milk Marketing Board and the National Dairy Promotion and Research Board, input from industry advisors, careful plan ning by CDR committees and researchers, and, most importantly, the creativity and dedica tion of the researchers and the WITEP and administrative staff. We invite your comments and suggestions to further improve our programs. Sincerely, '::Z~ ~r; Norman F. Olson Professor of Food Sdence Director, Center for Dairy Research vii ) I Annual Report 1.990-1991. II Overview of CDR CDR was established in 1986 to: ? Provide the technical expertise to enhance the economic well-being of the dairy industry. ? Re-establish a focus on dairy research at University of Wisconsin?Madison. ? Foster multidisciplinary research and information/technology transfer related to dairy manufacturing. ? Facilitate the integration of research between milk production, processing and marketing. CDR Structure CDR is composed of three functional areas - research programs, the Administrative Program and the Worldwide Information and Technology Exchange Program - plus a committee structure for administrative and planning purposes. il Program Advisory Committees ........................ Technical Advisoryg Committee U I I I I CentralOffice II Support UW Food Science Extension direct line of supervision/participation close interaction Organizational Components of CDR 3 I Center for Dairy Research r----,.-----------? The Research Program CDR-sponsored research covers dozens of dairy-related topics and spans disciplines from genetic engineering to economics. In spite of this diversity, CDR research is focused on four areas of emphasis. These areas are milk components and their uses, cheese technology, dairy product and process development, and dairy foods safety. Milk Components and Their Usea- Milkfat and Nonfat Solids LMilkfat Research on milkfat sponsored by CDR brings an integrated approach to the entire spectrum of milldat needs. Demand analysis research in CDR and the Department of Agricultural Economics predicts future milkfat use and serves as a guide for other milkfat projects. These projects include work on milk composition modification, milkfat fractionation, and nutritional evaluation of milkfat. U. Nonfat Solids Cooperation and collaborative research between researchers at CDR and the Departments of Food Science, Oaemical Engineering and Agricultural Economics is addressing utilization of whey proteins and lactose (ultrafiltration permeate). Patent applications dealing with production of polysaccharide from whey and whey permeate have been filed. Industrial evaluation of the polysaccharides is underway. Cheese Technology Cheese-related research includes researchers from CDR and Departments of Food Science, Agricultural Economics and Food Microbiology and Toxicology. Development and improvement of specialty cheeses (including lowfat and low-sodium varieties) involves selection and characterization of bacteria, making the necessary genetic modifications to obtain desired characteristics, manufacture of cheese to evaluate cheesema1dng cultures and cheese quality, and economic analyses to assess feasibility of industry adoption. Dairy Process and Product Development Process and product development has focused on nutritionally-enhanced dairy foods. Studies on calcium-enriched foods and bioavailability of calcium by the Departments of Nutritional Sciences and Food Science indicated the feasibility of increasing available calcium levels in cottage cheese and in suppressing bitterness imparted by added calcium salts. An economic-engineering microcomputer model has been developed by CDR and Agricultural Economics researchers to be used for evaluating processes from CDR research and for use by industrial firms. Dairy Foods Safety Dairy food safety research is conducted in cooperation with the Departments of Food Microbiology and Toxicology (Food Research Institute) and Food Science. In the future virtually all of this research will be carried out in the Food Research Institute. Past work has focused on factors affecting survival and growth of pathogens and on basic research, such as differentiating virulent and avirulent strains of L. monocytogenes. 4 ) I Annual Report 1990-1991 II The Worldwide Information and Technology Exchange Program (WITEP) Information and technology transfer is an important component of CDR. WIlEP provides this capability through publications, workshops, seminars, conferences, and scientist exchanges. The Administrative Program The Administrative Program provides the personnel, payroll, fiscal, and word processing support necessary for all other CDR activities. Committees Administrative Committee: The Administrative Committee is responsible for policy formulation and appointment of the CDR Director. Its members (FY1990-1991) are: Norman Olson, Director, CDR Dean Smith, Associate Dean of Graduate School Leo Walsh, Dean, College of Agricultural and Life Sciences David Ward, Board Member, WMMB Janet Williams, Director of Research, NDPRB Neal Jorgensen (ex-officio), Associate Dean, College of Agricultural and Life Sciences Leslie Lamb (ex-offido), Vice-President of Business Development, WMMB The Technical Advisory Committee (TAC) is charged with planning the research program of CDR, evaluating and approving research projects for scientific merit and assisting the CDR Director in evaluating annual and final reports of projects. Membership is: Robert Bremel, Department of Dairy Science Janet Greger, DepartrJ:lentof Nutritional Sciences Richard Hartel, Department of Food Science Charles Hill, [r., Department of Chemical Engineering Edward Jesse, Department of Agricultural Economics Eric Johnson, Department of Food Microbiology and Toxicology (Food Research Institute) Robert Lindsay, Department of Food Science John Nelson, Department of Food Microbiology and Toxicology (Food Research Institute) Norman Olson, CDR Director, Professor of Food Science Thomas Szalkucki, Administrative Officer, CDR Mark Johnson (ex-officio),CDR Neal Jorgensen (ex-offido), Associate Dean, College of Agricultural and Life Sciences Janet Williams (ex-offido), Director of Research, NDPRB Leslie Lamb (ex-offici5?, Vice-President of Business Development, WMMB 5 I Center for Dlli", Research I ( CDR Fadlities CDR, along with the UW Department of Food Science and the UW Dairy Plant, is housed in Babcock Hall on the UW-Madison campus. The first phase of a planned two-phase construction and remodeling projectat Babcock was recently completed, giving CDR the space to expand its programs and research capabilities. A 1S,ooo..square-foot addition to Babcock Hall and extensive remodeling of the existing building was completed at the end of June, 1991. The construction has quadrupled CDR's available laboratory space. Prior to the work CDR had just two laboratories; it now hasa total of nine, including five new laboratories for chemistry, engineering, and microbiological research. The additional space will allow CDR to add new equipment, such as an ion-exchange amino acid analyzer, while a new media-transfer room equipped with biological hoods will make it possible to conduct sensitive in-house microbiological research. Phase II improvements, which will involve further Babcock remodeling, are included in a University bUdget request and are expected to begin within a few years. CDR Staff Director - Norman F. Olson, Ph.D. Administrative Officer - Tom Szalkucki Research Staff David Bogenrief, assistant researcher Cheese as a food ingredient, functional and analytical testing of cheese Mahmoud Buazzi, laboratory technician Carol Chen, associate researcher Specialty cheeses, cheese ripening, pilot plant supervision Jyh-Ping Chen, assistant scientist Whey protein recovery David Everett, graduate student Jeremy Foltz, graduate student Ronald Gajewski, laboratory technician Brian Gould, associate scientist Process economics John Jaeggi, assistant researcher Specialty cheeses, cheese manufacture Janice Johnson, graduate student Mark Johnson, senior scientist Specialty cheeses, cheese ripening, cheese microbiology Eric Kettner, graduate student Dale McGill, research specialist Norman Olson, CDR director Cheese ripening, cheese functional properties 6 Annual Report 1990-1991 Hong Pai, graduate student Lisa Pannell, graduate student Michael Thomsen, graduate student William Tricomi, assistant researcher Cheese ripening, cheese analysis, laboratory supervision Baokang Yang, graduate student Visiting Researchers Carla Buijsse, visiting research intern, Wageningen Agricultural University, The Netherlands Jose Coca, visiting senior scientist, University of Oveido, Spain Moustafa El?Shenawy, visiting assistant scientist, National Research Center Academy of Scientific Research and Technology, Egypt Morsi El-Soda, visiting senior scientist, Alexandria University, Egypt Geraldine Farrell, visiting assistant scientist, St. Angela's College of Education, Ireland Patrick Fox, guest lecturer/mentor, University College, Cork, Ireland James Harper, visiting senior scientist/mentor, The Ohio State University Kamal Kamaly, visiting research associate, Monoufia University, Egypt Bertrand Lanher, honorary fellow, National Institute of Agricultural Research( INRA), France Peter Linklater, guest lecturer/mentor, University of New South Wales, Australia Worldwide Information and Technology Exchange Program Sarah Quinones, program coordinator Charles Behnke, information management consultant Beth Carney, program assistant David Gaeuman, editor Administrative Program Melinda Adams, program coordinator MaryAnn Crosby, fiscal clerk Cindy Genschaw, fiscal clerk Cannen Huston, fiscal clerk Lisa Lucas, program assistant Shirley Miller, word processing operator Al Muelling, program assistant Paula Rozman, program assistant Brenda Williams, fiscal clerk 7 8 )l----------------------! 'fM""UH tu,"a dOJ ~ua:J I 6 S+Joda~ ~OarOJd 40JDaSa~ ~a:> 01 )}----------------------1 ) I Annual Report 1990-1991 II Dairy Foods Safety and Quality Progress Reports Maintaining the high quality ofdairy products through prevention of foodbome infection is a primary goal of the dairy product safety area. Marth's group continues to report on theability ofspecific pathogens to survive and grow when inoculated into various dairy products. Results of these projects, which address the behavior ofknown dairy foods pathogens as well as organisms with the potential to contami nate dairy products, document Borrelia burgdor{eri in milk, Yersinia enterocolitica and Escherichia coli 0157:H7 in the presenceof lactic acid bacteria, and Listeria monocytoge.nes in UF milk with and without lactic add bacteria. Johnson's group reports on six projects focusing onmethods of prevention and inhibition of pathogen survival and growth. Three of the reports are final reports, and summarize the results of their investigations of theeffects of heat, lysozyme, and lactoferrin on pathogen survival and growth, as well as the role of proline in providing pathogen protection in cheese. Their results, which demonstrate the inability of proline overproduction to provide osmotic tolerance and the significance of acid adaptation for pathogen survival, are quite interesting. Although it is incomplete, their work on minimal growth requirements and cell surface analysis of L. monocytogenes helps explain this organism's tenacity and persistence. Further information regarding L. monocytoge.nes growth requirements is found in their newly initiated project investigating control of L. monocytogenes on surface-ripened cheeses. Initial data reportedon another project demonstrates the ability of some fatty acids and monoglycerides from milkfat to inhibit L. m0"!Ocytogenes. 11 I Center for Dairy Research I ( FINAL REPORT Behavior of Listeria monocytogenes in Ultrafiltered Milk With and Without Added Lactic Add Bacteria Personnel: Elmer H. Marth, professor, Dept. of Food Science (PI); Fathy E. El-Gazzar, visiting scientist, Dept. of Food Science Funding and Funding Codes: National Dairy Promotion and Research Board, MAR89-2 Dates: Jan. 1,1990 to Dec.31, 1990 Objectives: 1. Detennine the growth/survival behaviorof L. monocytogenes in ultrafiltered milk (concentrated 2Xto 5X> during storage at refrigeration tem perature (e.g.,4-C) and at temperatures usedin cheesemaking (e.g.,32" - 40-c). 2. Detennine the growth/survival behaviorof L monocytogenes in ultrafiltered milk (concentrated 2X to 5x) inoculated with lactic addbacteria and held at temperatures used in cheesemaking (e.g., 32"-40-c). 3. Determine the growth/survival of L. monocytogenes in permeate resulting from ultrafiltration of milk (concentration 2X to 5X). This objective was originally to determine the heat-resistance of L. monocytogenes in ultrafiltered milk of various concentrations. It was modified because little or no information is available on the behavior of L monocytogenes in permeate, while considerable information is already available on the heat-resistance of L. monocytogenes. Summary: Ultrafiltration (UP) can be used to concentrate milk which can then be converted to such varieties of cheese as mozzarella, Cheddar, cottage, Havarti, brick and Colby. The principal advantages of using UP milk are a 16% - 20% increase in cheese yield; reduction in costs of energy, equipment, and labor; improved consis tency of cheese flavor; and possible production of new by-products. Ultrafiltration increases the content of protein in UP milk over that in unfiltered milk and hence its buffering capacity is increased. As a result, the amount oflactic add that starter culture bacteria must produce to cause a unit change in pH is increased considerably over that needed when unfiltered milk is used. Presently, infor mation on behavior of L. monocytogenes in UP milk and resultant penneate with or without added lactic acid bacteria is limited. Hence, this work was done to provide the dairy industry with infonnation to help control this pathogen in dairy products. TyndaIlized. (steamed for 0.5 h, held overnight at 25-C, then steamed again) skim milk and retentate (concentrated 5X or 2X by volume) and permeate from ultrafiltered skim milk were inoculated with ca. 10'-1OS cfu/ml L. monocytogenes strains California or V7 and incubated with or without ca. 1CY-10'cfu mesophilic lactic add bacteria / mI. Samples were incubated at 32" or 40-c. There was a significant effect of using lactic add bacteria on inhibition of L. monocytogenes in all the three products tested. The inhibition was greater in permeate, compared to unfiltered skim milk and retentate. Inactivation was not complete, how ever, and both strains of L. monocytogenes sur vived up to 36 h of lactic acid fermentation. The buffering capacity of retentate apparently played a role in determining the extent of inactivation of the pathogen, with greater buffering capacity resulting in less inactivation. L. monocytogenes alone grew well in permeate and retentate. Both strains of the pathogen behaved similarly in the three products with or without added lactic acid bacteria. L. monocytogenes not only survived the lactic fermentation of skim milk, and retentate and penneate from UP milk, but also survived for several weeks in refrigerated fennented products 12 ) -------1 Annual Report 1990-1991 II stored at 4-C. Thelength of survival time depended on the product and strain of the pathogen tested. Both strains grew faster and achieved higher (ca. 1-2orders of magnitude) populations at 4-C in pasteurizedretentate of either concentration than in skim milk. The pathogen grew in pasteurized penneate at 4-C and attained maximum populations ofca. 10' to 10'/mI. L. monocytogenes populations in samples incubated at 30?to 4O-Cwere similar for skim milk, retentate, and permeate (ca. 10' to lOS/mIl. Significance to the Dairy IndustJ.y: Delivery of safe food to the consumer must be the first priority of the dairy industry. To accomplish that, it is necessary to know how pathogens behave during manufacture and storage ofdairy foods. With the knowledge provided by our results, appropriate action can be taken by industry and other organizations to minimize the risk from L. monocytogenes to UF milk. Results of this project should help the dairy industry use listeria-free ultrafiltered milk for cheesemaking. Publications: El-Gazzar, F.E.,H.F. Bohner, and E.H. Marth. 1991. Growth of Listeria monocytogenes at 4?, 3r and 4O-Cin skim milk and in retentate and permeate from ultrafiltered skim milk. [:Food Prot. 54:338-342,348. El-Gazzar, F.E., H.F. Bohner, and E.H. Marth. 1991. Antagonism between Listeria monocytogenes and mesophilic lactic acid bacteria during fermentation of skim milk and retenlate and permeate from ultrafiltered skim milk. [: Dairy Sci. (submitted). El-Gazzar, F.E.and E.H. Marth. 1991. Ultrafiltra tion and reverse osmosis in dairy technology: A review.'. Food Prot. (accepted). Presentations: El-Gazzar, F.E. and E.H. Marth. 1991. Behavior of listeria monocytogenes in ultro.fi1tered milkwith and without added lactic acid bacteria. Poster Session, CDR Cheese Research and Technology Conference, March 6-7,1991, Madison, WI. 13 I Center for Dairy Research I ( FINAL REPQRT Behavior of Foodbome Pathogens in the Presence of Lactic Acid Bacteria Personnel: E.H. Marth, emeritus professor, Dept. of Food Science;Seham Farrag and Jane Wenzel, graduate students, Dept. of Food Science Fundin& and Fundin& Codes: Wisconsin Milk Marketing Board, 89-28 Dates: Dec. 1, 1989-March 1,1992 OQjgctives: 1. Determine the behavior of Yminia enterocolitial and Escherichia coli 0157:H7 in the presence of starter cultures during their prepara tion with external or internal control of the pH of the culture medium. Starter cultures to be studied include Streptococcus cremoris, Streptococ cus 1actis, Leuconostoc cremoris, Streptococcus thermophilus or lActobacillus bulgaricus. They will be evaluated singly and as mixtures used in commercial practice. Substrates to be used include whey-based, skim..milk-based, or nonfat dry-milk-based starter culture media for external pH control and several typical commercially prepared starter culture media using an internal pH control system. The pH values to be studied are 5.4-5.6, 5.8-6.0, and 6.0-6.2. 2. Determine the behavior of Y. enterocolitial and E. coli 0157:H7 in raw and pasteurized milk when lactic acid bacteria are added to such milks. Lactic acid bacteria to be studied include the commercial raw milk inoculant, L. cremoris, Lactobacillus addophilus or Bifidobacterium lrifidum. The ratio of number of cells of pathogen to number of cells of lactic acid bacterium needed for control of the pathogen will be determined. 3. Determine the effectiveness of the activated lactoperoxidase/thiocyanate system in raw milk for control of Y.enterocolitica and E. coli 0157:H7. Lactic acid bacteria may playa role in activation of the system. Sullll1Ul1'J'i Behavior of Yersinia enterocolitial in a medium with internal pH control (lPCM-l) and contain ing Streptococcus cremoris (Lactococcus L:u:tis subsp. cremoris) was determined. Samples of IPCM-l were inoculated to contain, per milliliter, ca. 10'CPU E. coli 0157:H7 or Y. enterocolitica and S. cremoris (ca. 2.9 x 1()'5), and incubated at 25? and 3O'Crespectively. Yminia enterocolitica . controls contained ca. 10'CPU/mI after 18 h regardless of incubation temperature. IPCM-l is ready for use when a pH of 5.5 is reached after 16-18h of incubation. Escherichia coli was affected less by presence of S.cremoris than was Y. enterocolitial. After 18 h of incubation at 25? and 3O'C,populations of E. coli had increased to 1()1 and 10'CPU / ml, respectively, in samples of IPCM-lcontaining the pathogen and the lactic culture. Although Y. enterocolitica was more sensitive than E. coli to the effects of S. cremoris, lOS-lO' of the pathogen were present per milliliter of treatment samples after 18 h of incubation at either temperature. Although some inhibition was caused by presence of S. cremoris, substantial numbers of E.coli 0157:H7 and Y. enterocolitica were present in IPCM-l when it is said to be ready for use. E. coli 0157:H7 strain 933 isolated from an outbreak of human illness was used to study how this pathogen is affected by the lactoperoxidase system of milk. Lactoperoxidase (LP) was added to SSM medium (a liquid culture . medium) at the rate of 5 mg/mI. To activate the LP system, thiocyanate concentration in raw milk was determined spectrophotometrically as ferric thiocyanate, with theconcentration of thiocyanate ion being 1.09 ppm. This was adjusted upward to 14.5 ppm by addition of sodium thiocyanate. Also ~02 (14.5 ppm) was added to milk samples. Both substrates (raw milk and SSM medium) were inoculated to contain lot CPU of E. coli 0157:H7/mI (levell) or lQll CPU /mI (level 2). Control samples were 14 II ) I Annual Reporll990-1991 prepared at 0, 2,4, 6, 12, and 24 h when incuba tion was at 3O"C and at 0, 2, 4, 6, 12, 24,48, 72,96, and 120 h when incubation was at 4"C. Reductions in numbers of the pathogen ?ow inoculum), when compared to populations in controls at the same time, after 2 h were 4.5 and 4.4 orders of magnitude for SSM and raw milk, respectively. Maximum reduction of population at 30"C was observed 12 h after the system was activated. When the large inoculum was used, a reduction in population of 1.5 and 1.8 orders of magnitude for SSM and raw milk, respectively, was observed after 6 and 12 h. After 12 h, surviving cells resumed growth and the popula tions in treated and untreated samples were similar. At 4"C, for both raw milk and SSM, reduction in the population of the pathogen started after 4 h and continued until theend of the incubation period (120 h), when the pathogen was com pletely inactivated in both substrates. Si&nificance to the Dairy Industry: Escheridria coli 0157:H7 is associated with various animals, including dairy cows. Thus, the organ ism hasappeared in raw milk and can enter the factory environment In humans, this pathogen causes bloody diarrhea, hemolytic uremic syndrome and other disorders. Yersinia enterocolitica also is associated with animals, primarily but not exclusively with swine. In humans, the bacterium causes gastroenteritis characterized by pain in the lower right quad rant of the abdomen. This is often thought to be appendidtis and has led to needless appendecto mies. Outbreaks of this form ofillness have been related to contaminated chocolate milk and pasteurized milk. Y.enterocolitica also grows quite well at refrigeration temperatures. Much of Wisconsin's milk is converted to cheese. To accomplish this, starter cultures (lactic add bacteria) are prepared and added to milk. Such starter cultures often are prepared using a medium in which the pH is controlled with buffers. This is beneficial for the lactic acid bacteria, but the protective effect (against gTc)wth of unwanted bacteria) of the acid has been minimized. Thus, it becomes important to know how E. coli 0157:H7 and Y.enterocolitica would 1.Ci do should they be chance contaminants in the starter culture. With such information, needed precautions can be taken to prevent problems with these pathogens in our Wisconsin cheese. Publications: Wenzel, J.M. and E.H. Marth (1991). Behavior of Escherichia coli 0157:H7 or Yemnia enterocolitica in the presence of SC in a medium with internal pH control. Lebensm. Wiss. Technol.24:164-168 I Center for Dairy Research I ( FINAL REPORT The Growth and Survival of Borrelia burgdorferi in Milk Personnel: Geraldine M. Farrell, visiting assistant scientist, CDR; Elmer H. Marth, professor, Dept. of Food Science Funding: WITEP ~ Sept 1, 1989-Sept. 1, 1990 Objectives: The principal objective of this research program was to determine the growth!survival of Borrelia burgdorferi in milk. Willy Burgdorfer, in 1982, established that this organism is the etiologic agent of Lyme disease. Many of the components of the Lyme complex of disorders have been recognized for decades in Europe, but their OCCUITence in North America appears to be more recent (1). In man, Lyme disease may involve the skin, the joints, the cardiac muscle and!or the nervous system. The symptoms may be brief and inconsequential or chronic and severely disabling (1). Lymedisease is now the most commonly reported vector-borne disease in the U.S.A. (2), and is primarily transmitted by ticks of the Ixodes species (3). Research over the last five years has shown the presence of B. burgdorferi or antibodies to this bacterium in a number ofanimals that are in dose association with humans (4). Included in this group of animals are cows, where the organism has been isolated from urine, co lostrum, and many of the internal organs. In laboratory experiments, B.burgdorferi infection has been acquired by routes that did not involve the tick vector. These findings raise the question as to whether milk will support the growth! survival of the Lyme pathogen. Summary: Sterile whole, low fat, protein-fortified skim, and skim milk samples were inoculated with one of three strains of B. burgdorferi and stored at 34"C, for 16 days. The numbers of B.burgdorferi were determined using the most probable number (MPN) technique and the Barbour-Stoermer Kelly medium. Experiments were also carried out at a storage temperature of 5"C, in skim milk, over a period of 45 days. Thelog of the micro bial count vs. time was used to ascertain the Do value for each strain in each milk at both storage temperatures. Results indicate that B.burgdorferi did not grow under the test conditions. At 34"C, the organism did not remain viable after 12 days, however at5"Cviable counts were recorded for up to 46 days of storage. Si&nificance to the Dairy Industry; The safety of dairy foods is a major concern for consumers. Because Borrelia burgdor[eri, the cause of Lyme disease in humans, can infect dairy cows, the likelihood exists that the bacte rium can appear in raw milk. Evidence in the literature suggests that infection via the oral route is possible. When this research was begun, nothing was known about the behavior of the pathogen in milk. Our results indicate that it does not grow in milk (the good news), but it can survive in milk at 34"Cfor at least 12 days and in milk at 5"Cfor at least 46 days (the bad news). Publications: Farrell, G.M, A.E. Yousef, and E.H. Marth. 1990. Survival of Borrelia burgdorferi in whole, lowfat and skim milk when stored at 34"Cand in skim milk at 5"C. J. Food Prot. (submitted) Farrell, G.M. and E.H. Marth. 1990. Borrelia burgdorferi - A pathogen of increasing importance. . Whole pasteurized milk was obtained from the Department of Food Science. Bacteria and growth conditions: Bacterial strains tested were mainly isolates from outbreaks of food poisoning or spoilage and were obtained mostly from faculty-of the Food Research Institute. Bacteria were grown as previously described (Hughey and Johnson, 1987). Activity of lysozyme against pathogens and spoilage bacteria: The antibacterial activity of lysozyme was evaluated by lysis of cells in phosphate buffer (PB)or milk, and by growth inhibition in culture media, milk, and cheese. Lysozyme hydrochloride was dissolved in 67 mM sodium phosphate buffer (PB), pH 6.4, and filter sterilized. It was used at 100 mg/I of milk. Milk alterations: To decrease the concentrations of metals in whole milk, the pH of the milk was lowered to pH 6.2 with concentrated citric acid, and it was dialyzed overnight at 4?C in two changes of 10 mM EGTA (Sigma Chemical Co., St. Louis, MO) using dialysis tubing with a MW cutoff 6,OOO-S,OOO (Spectrum Medical Industries, Inc., Los Angeles, CA). The milk was dialyzed the next day against two changes of 20 mM sodium phosphate buffer, pH 6.4. Certain milk batches were further treated with Chelex 100 exchange resin (BioRad Laboratories, Richmond, CA). The Chelex was prepared by repeated 17 -- -- -- I Center fot Dairy Research I ( Figure 1. Inhibition of0ostridium botulinum (a) and reduction ofgrowth rates ofYersinia enterocolitica (b) by lysozyme (L), lactoferrin (LF), and sodium bicarbonate (N). a. 0.9 .-. E I: = 0.7 '0 '0 8 I: III O.S j ~ 0.3 -= i .. 0.1 e Mll?AJ.a..Beluga +EDTA,5mM +Lactofenin. 1 mg/ml +Lysozyme. 100ppm -e-- - -------.-- -o.r 0 2 3 4 s Days b. Inhibition at Yersinia enterocolitJca WA by lysozyme and lactaterrin 0.6 i _ o.s - e 0.4 c 0 co e w 0.3 (J Z OIl( CD a: 0 0.2 en CD OIl( 0.1 0.0 ., o , j 20 j' i 6040 TIME (HOURS) j 80 I 100 18 6 I control --.-.,.. iysazyme (Ll Iacloferrin (l.F) L~LF -- L+N -:-a-- LF+N --- L+LF+N ) I Annual &port 1990-1991 II Figure 2. Reduction ofgrowth mtesof various L. monocytogenes stminsin brain heart infusion bylysozyme (100ppm) (L), 1actoferrin (05 mgjml) (LF),andsodium bicarbonate (1 mgjml) (N). L. moooeytogeoes V7 1.0 I I control - lysozyme IL) L+NaHC03 - e ---- - ? l.actof&rrin L+ LF = ----0-- = 0.6 ..?._._. L+N u~ ~ ? LF+L+N "" '-' "" Q,l J b? -. . " ~ = .. I:l; 0.4 .c " ...--.... ?????-.j e {Il l :a:-;-:.,::.~ .???. _1I' .?.?.-. .c " -------..~.......... -< r l ."..,.?--------... ?:~..... :;:::-.* 0.0 0 20 40 60 80 Time (bours) 0.8 i L. moneeytogenes Califoroia ? Control i - Lysozyme IL) ........_. L+NaHC03 - Lactoferrin (IF) I::: L+LF LF+N --...... L+LF+N .... ~. o 20 40 Time (hours) 60 I 80 I - 0.61 Ii:\ e = = "" "" '-' Q,l OA ~ = I:l; ,Q .. e {Il 0.2 .J I .;r;'-" ? ,Q -e 0.0 , 'i i' 19 I~~~~~I ( Figure 3. Lysozyme inhibition ofL. monocytogenes Scott A in wlwle milkand milktreated to remove metals (see textfor experimental details). In certain incubations, L. monocytogenes was sublethaUy heated in milk(60?Cfor 15s) prior toaddition ofinhibitors. Control (+0 treatment) Milk - metalsl + heat + LZ t + LZ Milk - Milk + Milk - meJls + heat (-LZ) 100 Time (h) washings in 05 M sodium acetate until a pH of 6.3 was reached. The chelex (50 g) was then washed five times with ultrapure water (500-800 ml water per wash). The resin was used at SOg/ 100 ml milk. The milk was allowed to mix with the chelex in a beaker on a stirplate in the coldroom overnight, and stored refrigerated until use. The pH of the demineralized milk was 6.0-6.2. When Ca was added back to demineralized milk, the milk was dialyzed against 20 mM sodium succinate buffer, pH 6.4, to avoid precipitation of calcium phosphate complexes. Metals (Ca0 2 , Mg0 2 , and Zn0 2 ) were dissolved in distilled water and filter sterilized. They were used at concentrations similar to levels found in whole milk. Metal concentrations were determined by inductively coupled plasma spectroscopy OCP spectroscopy) on 5 g samples. Preparation of Camembert-type and Cheddar type cheeses: Camembert-type and Cheddar type cheeses were prepared as described in another paper (Kihm and Johnson, CDR annual report> except that lysozyme (100 ppm final concentration) was added as follows. For Cheddar, 0.06 g of lysozyme hydrochloride 20 (Societa Produtti Antibiotici, Milan, Italy) was dissolved in 5 ml of PB and added to 1 liter of milk after the addition of L.monocytogenes and starter culture. For Camembert, 2 g of lysozyme was dissolved in 20 ml of PB and added to the milk vat containing 16-18 I after the addition of L.monocytogenes, starter culture, and rennet. Results Growth inhibition by lysozyme in media: Lysozyme showed antibacterial activity against some pathogens important in milk and cheese safety including Clostridium botulinum and Yersinia enterocolitica (Fig. 1). Lysozyme together with lactoferrin inhibitied the germination of C. botulinum spores (Fig. 1). Other pathogens and spoilage organisms were also inhibited by lysozyme including Campylobacter and Pseudo monas sp, (data not shown). As demonstrated in an earlier study (Hughey et al., 1989), lysozyme also had activity against several strains of Listeria monocytogenes in culture media (Fig. 2). How ever, lysozyme was inactive against the patho gens in milk. In the following experiments, we focused on ways to sensitize L. monocytogenes as this pathogen is most important in dairy food safety. ) 1Annual Report 1990-1991 II Table 1. Metal analyses of whole and demineralized milk samples. Concentration (ppm) lA.. Metal analysis of demlnerallzed mUk. Metal Whole mJlk Dialyzed mJlk Dialyzed + Chelex p 979 563 474 K 1839 <7 <7 Ca 1170 325 100 Mg 118 21 <1 S 285 195 157 zn 3.5 1.5 <0.1 Fe <0.13 <0.13 <0.12 Cu <0.29 <0.28 <0.28 Na 448 773 899 lB. Metal analysis of refortlfled m.lIk. Whole mJlk Dialyzed + Dialyzed + Dialyzed + Dialyzed + Chelex Chelex + Ca Chelex+ Mg Chelex + zn P 923 516 529 470 506 K 1559 <2.5 <2.5 <2.5 <2.5 Ca 1056 9.3 667 4.2 7.8 Mg 104 <0.4 <0.4 82 <0.4 S 287 148 156 140 146 Zn 3.4 <0.04 <0.04 <0.04 3.0 Fe 0.21 0.06 0.08 <0.05 0.08 Cu <0.1 <0.1 <0.1 <0.1 <0.1 Na 371 739 734 665 722 Values for metals in IB were generally lower in dtalyzed preparations owing to longer dialysis and and a higher cone.of Chela (75 g per 100 ml of milk compared to 50 g per 100 ml milk in IN. Effect of milk alterations on inactivation of L. monocytogenes by lysozyme: In repeated determinations, L. monocytogenes was found to be completely resistant to inactivation by lysozyme in whole or skim milk for at least 6 days at 4?C. However, L. monocytogenes was sensitive to lysis in phosphate buffer (pH 6.4) at 4?C (Fig. 3). We carried out a series of experiments to understand the basis of resistance of the pathogen to ly sozyme in milk. Heat treatment of the cells at 60?C for 15 s in PB followed by addition to whole milk at 4?C did not lead to inactivation of L. monocytogenes. Lysozyme did not promote inactivation of these heat treated cells in the milk (Fig. 3). Removal of metals from the milk slightly improved inactivation of L. monocytogenes by lysozyme (Fig. 3). When the cells were first heat-treated in PB at 60?C for 15 s and then added to cold demineral ized milk the activity of lysozyme was dramati cally increased (Fig. 3). This combination of heat-treatment and demineralization resulted in a five-log decrease in viable cells of L. monocytogenes by lysozyme after 90 h (Fig. 3). Analysis of demineralized milk for metal content by ICP spectroscopy indicated that the contents of Ca, Mg, and Zn were significantly reduced compared to whole milk (Table tal. Resistance 21 I~~~-~I ( Figure 4. Protection against inactivation ofL monocytogenes in demineralized milkbyreplacement of various metlJ1s {Ca, 666ppm; Mg, 83ppm; Zn, 3.0 ppmJ. Metals were replaced at about the concentrations that are found in milk(determined by inductively coupled plasma aCP)emission spectroscopy). Protection against lysis by added metals in deionized milk 8 10 r; -------==.:=---~--- , 107~ 0 - i :I~' ----01"10- ---'""'C- ..--- ...-n metals + Ca -~ Col - 10 6 {I,/ metals + Mg for 15-20 containing the pathogens entered into the cheese sec.l on survival of pathogens in milk and in vats. The 1983 outbtreak was described by the cheeses. Centers for Disease Control as "homemade". In summary, our review of cheese-borne outbreaks 2. Quantitatively determine heat resistances of in the U.S. did not identify any that were caused pathogens of concern in cheeses, including by inadequate thermal processing. strains of Listeria monocytoge:nes, Salmonella, and enteropathogenic E.scherichia coli. b. Fate of pathogens in Cheddar and Camembert cheeses prepared from heat-treated Summary: milk. a. Review of safety ofcheeses prepared from Experimental Methods heat-treated milk. Bacterial strains: Salmonella heidelberg, Listeria To define pathogens of concern in cheeses monocytogenes Scott A, and Escherichia coli commercially prepared from heat-treated milk, 0157:H7 were used in this study. S. heidelberg E. A. Johnson and J. H. Nelson conducted an and L monocytogenes were grown in trypticase extensive review of the safety record of cheeses. soy broth (Difco Laboratories, Detroit, MI) and E. Total US. production of cheeses increased from coli was grown in Luria broth (10 g tryptone, 5 g 1 billion pounds in 1948 to more than 5 billion yeast extract, and 5 g NaO perliter distilled pounds in 1988 (Fig. 1). Of total cheese produc water) for 16-20 h at 30?C. The bacteria were tion, an estimated 13% or 704 million pounds harvested by centrifugation and resuspended in was made from heat-treated milk including the 67 mM sodium phosphate buffer, pH 6.4 (PB) varieties sharp Cheddar, Swiss, Parmesan, and prior to their addition to the cheesemilk. Romano. Pathogen heat treatments: Pathogens were Our review of the cheese safety from 1948 cultured, harvested and suspended in buffer. through 1988 revealed very few confirmed They were added (10'/ml) to whole, non incidences of food poisonings caused by con homogenized pasteurized milk preheated to sumption of cheeses. During this 4Q-year period 60?C, 62.5?C, or 650C and held for 15 s, Controls in which more than 100 billion pounds of cheese were also added to milk which was first heated were produced, seven outbreaks of food poison to 6QOC and then cooled prior to addition of ings were attributed to cheese (Table 1). Three pathogens. This milk was used for cheese preparation. 24 )l----------------i Annual Report 1990-1991 I Figure 1. United States production ofnatural cheese, 1948-1988. United States Production of Cheese 0000' i 5000 -! 4000 c - o ~ 8 3000 j - 2000 i 1000 o J ' ut"'9'A" ':..=: , ? ? I 1940 1950 1960 1970 1980 1990 ? ? ? Total ? American Italian Cheddar Year Table1. Foodpoisoning oUlb"aksassociated withcows' milkcheese in the United States. 1948-1987 Year Cheese variety 1958 Colby 1958 Cheddar 1965 Cheddar. Kuminost. Monterey 1976 Cheddar 1983 Homemade "Mexican style" 1985 "Mex1can style" Pathogen ,a. aureus a ayreus ,a. aureus Salmonella Streptococcus zooeptdemtcus L. monocyto@nes Cases 200 >339 16 >100 25 I Center for Dairy Research I Cheese Preparation Cheddar-type: For preparation of Cheddar-type cheese, 1% lactic starter was added to the milk which was then incubated at 31.5?C (88.7lF')for 45 min. Single strength calf rennet (200 f.Ll suspended in 5 ml water, Marschall Products) was then added, and the milk was allowed to coagulate for 30 min. The coagulum was cut into cubes -0.6 em (0.25 in.) square and the whey expelled with gentle stirring for 15 min. Curd was then cooked by raising the temperature from 31.5? to 38.9OC (88.70-102.2?F') over 30 min and holding at 38.9?C (l02.2?F') for 45 min. Wheywas removed and the cheese curd com pressed in cheesecloth. The pH of the whey at draining ranged from 6.0 to 6.2. One gram samples of curd were taken, ground in 9 ml phosphate buffer in a Whirl-pak bag, and plated for pathogen enumeration. The curd was pressed in a beaker, salted to a final concentra tion of -1.8% (w/w), and stored at SOC (41?F') overnight. The next day the Cheddar-type cheese was divided into four equal samples (-25 g) and packaged tightly in Whirl-Pak bags. These were incubated at 5?C (41?F) for up to eight weeks and sampled biweekly for pathogen enumeration. Camembert: Camembert-type cheese was prepared consistent with recommendations of Dr. M. E. Johnson of the Center for Dairy Re search. Because of equipment limitations it was not possible to heat-treat the cells directly in the vats. About 16-18 liters of whole unhomogenized pasteurized milk were poured into each of two 20 liter vats. The vats were placed in a warm water bath and allowed to equilibrate at 33?C (91.4?F) for 30 to 60 min. Two percent lactic starter and 4.5 ml of undiluted rennet suspension were added to each vat. The milk was mixed well and allowed to set for 50 min at 33 QC (91.4?F). The curd was cut into -1.90 em cubes (-0.75 in.) and allowed to stand for 15 20 min with gentle mixing every 5 min. Curds and whey were then poured into hoops on draining racks covered with cheese cloth. After five hours the filled cheese hoops were turned over, covered with cheesecloth, and left over night at ambient temperature. The next day the cheese wheels were removed from the hoops, rolled in cheese salt to completely cover all surfaces and were held for 24 h at 5?C (41?F) on cheesecloth-eovered draining racks. The wheels 26 ( were then rolled in a suspension of Penicillium CQmembertii to cover all surfaces. Wheels were then placed on a cheesec1oth-covered draining rack set atop a tub half-filled with water en closed in a large plastic bag (method described by Dr. M. E. Johnson, pers. comm.), and incu bated at 13?C (55.4?F') until mold growth covered the wheels (10-14 days). The wheels were then wrapped in aluminum foil and stored at 5?C (41?F). Enumeration of Pathogens To enumerate pathogens, milk and cheese samples were suspended and diluted in PB. Salmonella was plated on trypticase soy agar with a 10-15 ml Hektoen enteric agar (Difco) overlay. The rich medium underlay was used to help recover the stressed cells. E. coliwas plated on trypticase soyagar + MgSO..(t.5 gIl) which was overlayed with MacConkey agar (OOco). When no colonies appeared on the initial plates, enrichments were carried out for Salmonella and E. coliaccording to FDA methodologies. L. monocytogenes was initially enumerated on tryptose phosphate agar + 1% sodium pyruvate (TPBA +P). However, since starter also grew well on TPBA + P, GBNA agar was used in certain experiments. Enrichments were carried out according to FDA Usteria methodology. Twenty-five g of cheese was incubated at 300C for 48h in 225 ml of listeria enrichment broth (LEB) + 0.2 ml of 1.2% Acriflavin. One ml of the primary enrichment was added to 9 ml of LEB + 2.5% of Acriflavin and incubated at 30?C for 24 48h. The secondaryenrichment was streaked onto LPM agar plates, which were incubated at 370C for 48 h. No colonies indicated negative primary and secondary enrichments. Individual colonies were streaked on tJyptose agar and colonies subjected to confirmatory tests includ ing catalase, tumbling motility, umbrella-form growth, bile esculin reaction, litmus milk reac tion, and methyl red test. Serological confirma tions were also carried out in two independent experiments. Heat injury of the pathogens was determined by plating on media designed to detect heat-injured cells. S. heidelbeTg and L. monocytogenes were grown overnight at 30?C in trypticase soy broth, harvested by centrifugation, and resuspended in PB. E. coliwas grown overnight in Luria broth at 30?C and harvested in the same manner. The ) I Annual Report 1990-1991 II Table 2. Effect of htQ.t-tTe4tment at 65?C (149?FJ/15 s on surofoal of pathogens in lllboratory-prepared Cheddar-type cheese. Survival of Pathogens (cfu/g) 1. monocytDgenes S. heidelberg E.colt Week Heated Control Heated Control Heated Control o (ld cheese) <100 6.4 x 1()4 <100 7.5 x1()4 <100 4.0 xlOS 2 NO'" 8.4 x 1()4 NO'" 2.0 x 1()2 NO" 4.7 xl()4 ? 4 NO'" 4.8 xlO? NO'" <100 NO'" 1.7 x lOS 6 NO" NAb NO'" <100 NO'" <100 8 NO'" NAb xo- <100 NO'" NAb -ND. Not detected: enrichment was negatiVe by FDA fLlsterla) or BAM procedures ?.. heidelber~ and It~. see Matel1a1sand Methods for details. "NA. Counts not available. However. other experiments In this study showed that I.e. monocyto'lSmes and ~. ~ can survive In Cheddar-type cheese (see figures). cell suspensions (40 to 125 JJl) were added to 4.0 ml of milk preheated at 6(JO, 62.5?, or 65?C (initial concentration of cells was lOS to 10' cells per ml), Aliquots were taken after ISs of heat-treatment, and immediatelydiluted in cold PB. L monocytogenes was plated on tryptose--phosphate agar + 1% sodium pyruvate or tryptose phos phate agar + 5% sodium chloride. S. heidelberg and E. coli were plated on the same media except 1% sodium chloride was used. The plates were incubated at 370Cfor three days and colonies were counted. Results and Discussion: Pathogen survival in Cheddar-type cheese was markedly affected by milk heat-treatment. S. heidelberg and L monocytogenes heat-treated in milk at 60?C (1400F) or 62.5?C (1445?F) died off in the cheese compared to the unheated controls (Fig. 2-4), and no cells were recovered at 6SOC (14~F) =0 lIeated + LA E 10 3 ...i 10:1 lot nealed + LZ + LA 10' 0 10 20 30 40 Time (h) in phosphate buffer (data not shown), but sensitization was not observed in milk. These results suggest that the cell surface and cell . membrane are affected by heat. Our data also suggest that lactic acid and lysozyme are the primary components in milk and cheese that inactivate the sensitized cells. More work is needed to understand themechanisms by which pathogens are sensitized to inhibitors by heat. SilWificance to the Dairy Industry: Heat-treatment of milk can exert a Significant role in producing microbiologically safe cheese. Research has indicated that milk heat-treatment at 65.0-to 65.6?C (149--150?F) for 16-18 swill prevent the growth in cheese of pathogenic organisms which are major threats to cheese safety. A review of the literature carried out during this study indicated that cheese is a remarkably safe 32 food and identified only six illness outbreaks transmitted in US. produced cheese during 1948-1989. During this period, the US. pro duced more than 100 billion pounds of natural cheese. An examination of the epidemiological literature indicated that three organisms, Salmo nella, Listeria monocytogenes, and Escherkhia coli 0157:H7, were of potentially high risk to the cheese industry. L monocytogenes is widespread in the dairyplantsand has been found in several dairy products. The FDA and USDA have enforced a zero tolerance level for L. monocytogenes. Salmonella and E. coli have caused important foodbome disease outbreaks in cheeses. Effective methods must be developed to keep these pathogens out of dairy products. Our research has provided important advances in this direction. The results of this study indicate that heat-treatment of pathogens in cheesemilk significantly affect their subsequent survival. )>--------------------11 Annual Report 1990-1991 II Publications: Johnson, E. A., J. H. Nelson, and M. Johnson (1990). Microbiological safety of cheese made from heat-treated milk. I. Introduction and historical review. [. Food Prot. 53:441-452. Johnson, E. A, J. H. Nelson, and M. Johnson (1990). Microbiological safety of cheese made from heat-treated milk. D. Microbiology. J. Food Prot. 53:519-540 Johnson, E. A, J. H. Nelson, and M. Johnson (1990). Microbiological safety of cheese made from heat-treated milk. m. Effects on process ing. J. Food Prot. 53:610-623 Kihm, D., and E. A Johnson. Survival of Listeria monocytogmes, Salmonell4 heidelberg, and Escherichia coli 0157:H7 in Cheese Prepared from Heat-treated Milk. Submitted for publication. Presentations: Johnson, E.A. ''Effectiveness of antibacterial substances against pathogens:' Food Research Institute Annual Meeting, May 1990, University of Wisconsin-Madison, Madison, WI Kihm, D.J. and E.A. Johnson. "Influence of heat treatments on survival of pathogens in cheese." Poster at the Food Research Institute Annual Meeting, May 1990, University of Wisconsin Madison, Madison, WI Johnson, E.A "Survival of L. monocytogenes in cheese-like environments." Food Research Institute Annual Meeting, May 1991, University of Wisconsin-Madison, Madison, WI Kihm, D., and E. A. Johnson. Use of heat treatment and lysozyme to control Usteria monocytogenes in milk and Camembert cheese. In preparation. 33 I Center for Dairy Research I~-----------------------{C FINAL REPORT Generation and Roles of Proline in Providing Flavor and Pathogen Protection in Cheese Personnel: Eric A. Johnson, associate professor, Food Microbiology and Toxicology; Greg Leyer, research assistant Fundin~ and Fundin& Codes: National Dairy Promotion and Research Board, 88-6 July 1989-June 1991. Objective: 1. Determine the role of proline overproduction and increased osmotolerance of S. typhimllrillm on the pathogen's persistence in cheese. 2. Determine the role of acidic adaptation in providing pathogen protection in cheese. Summary: a. Role of proline in protecting against osmotic stress. It is well known that accumulation of proline in Salmonella can alleviate growth inhibition imposed by osmotic stress. Bacteria canaccumu late proline to high intracellular concentrations by increased net synthesis or by enhanced uptake from the medium. To understand the importance of proline in protecting against osmotic stress, we obtained a proline overproducing mutant of Salmonella typhimllrium (TL 978)from Dr. Laszlo Csonka, Purdue University. To determine if the proline overproducing mutant has a competitive edge over wild type S. typhimurillm TLl, we prepared a synthetic medium (Medium 63) with various salt and proline concentrations. Cells were incubated at 370C and growth was monitored by following absorbence at 660 nm. We observed that in most cases, strain TL 978 initiated growth more rapidly than the wild-type, but within a day or two, the wild type reached a similar optical density (data not shown). To better understand the role of proline overpro duction against osmotic stress in a food system, Cheddar-type cheese was prepared with Salmo nella -inoculated milk. Salmonella was added at an initial concentration of 100CFU/ml. After cheese preparation, the cheese was salted to a final concentration of 13% NaO. The cheese had a moisture content of 49%. The cheese was then incubated at an abusive temperature of 3QOC, and samples were taken over a five-week period. We found that at the 13% NaO and high moisture level, the wild type and the proline overproducer grew equally well (Table 1). We repeated this experiment with cheese that was brined to increase the salt content to 4.6%. Under these conditions, the cells died off over time at a fairly equal rate (Table 2). These results suggest that in cheese, overcoming salt concen tration by proline overproduction is not the only factor necessary to allow pathogen growth, and this area needs further study. In particular, we are finding evidence that adaptation to acid is important for pathogen survival in cheese. b. Role of acid adaptation in protecting against acid stress. It appears that overcoming osmotic stress may not be the most important factor in determining Salmonella persistence in cheese. The ability of a pathogen to overcome acid stress in fermented milk products undoubtedly aids in its ability to survive and grow. In this portion of the study we have examined the ability of S. typhimurium to become adapted to higher acidity. We have also looked at the role of acidic adaptation in increasing the organism's tolerance to lactic acid, and also affect its survivability in dairy fermenta tions. In the course of these studies, we have also identified the role of acidic adaptation in enhanc ing thermal tolerance, and have confirmed the stimulatory effect of pyruvate in recovering acid injured cells. S. typhimurium can be adapted to acidic condi tions by a brief exposure to a higher acid environ ment. The optimum pH for this adaptation occurs at a pH of 5.8. We were interested in 34 Anmtal Report 1990-1 Table 1. Growth of wUd type (TLl) andmutant (TL978) Salmonella in cheese (1.3% NaCI) Table2. Death ofSalmonella in brined cheese (4.6% NaCI) DiLl 0 mmm 1Ll 1L978 cFV/gm 5.4 x lOS 9.2 x ID4 DiLl 0 ?.tmiD. 1Ll 'IL978 CFV/@l 5.7 x 1()5 2.2 x 1()5 3 1Ll 'IL978 5.9x loe 1.3 x loe 4 1Ll 'IL978 7.2 x 1()3 7.3 x 1()3 7 1Ll 'IL978 4.1 x 10 1 6.2 x 10 1 9 1Ll 'IL978 3.1 x 1()3 1.3 x 1()3 21 1Ll 'IL978 7.0x 10 7 8.9x 10 7 16 1Ll 'IL978 2.65x lOll 1.78 x lOll 35 1Ll 'IL978 2.5 x 10" 8.3 x 10 1 24 1Ll 'IL978 9.2 x 10 1 6.4 x lOll 38 1LI 'IL978 <10 <10 determining the effect that acidic adaptation has on S. typhimurium's survival in a lactic acid acidified buffer system (Figure 1). The acid adapted cells survive approximately 10' fold better than their unadapted counterparts. Since a large difference in survival was observed in the "in vitro" system, we were interested to see what comparative survival rates would be in a fermentation study. To do this, sterilized skim milk was inoculated(l% vIv) with a 12 hour culture of Lactococcus lacHs and Streptococcus thermophUus (1:1). Once the pH of the skim milk had dropped to about 5.0 due to the fermenta tion by the starter eul~,either adapted or unadapted cells were added and their surviv ability monitored as the pH kept decreasing. The adapted cells survived about 1,000fold better in this test system (Figure 2). The next step is to determine the comparative viabilities of these cells in a cheese system. The results indicate that the adapted cells survived better in this system as well (experiment is still in progress). The ability of an inducing signal, such as higher acidity, to affect other stress responses was also examined. To test for interaction of stress responses, we adapted the cells to acid, and evaluated thermal inactivation in milk at 55?C and 57.s?C (Figure 3). Unexpectedly, the acid adapted cells also demonstrated more thermal tolerance. This may be an important mechanism affecting pathogen persistence in cheese made from heat-treated milk. In the course of recovering acid-injured cells from the test buffer system, we found that the .presence of 0.1% pymvate dramatically im proved survival in minimal and complex media. Figure 4 shows the effect of a 0.1% pymvate supplementation to LB medium on the recovery of add-adapted and acid-unadapted cells when exposed the lactic acid buffer system. After only 20 minutes of exposure, the media with the pymvate supplementation enhanced recovery about 10,000fold. Significance to the Dairy Industry: Salmonella in mozzarella cheese recently caused a foodbome illness outbreak that had serious economic consequences for the cheese industry. Dr. Paul Blake (director, Infectious Diseases, Centers for Disease Control, Atlanta, GA) has indicated in personal communication (EAJ) that the CDC is concerned about future contamina tion of dairy products with salmonellae. The completion of this project provided us with some interesting information regarding eradication of Salmonella from dairy products. We found that add tolerance was a critical factor for survival, whereas salt tolerance was relatively unimpor tant. Adaptation to add also affected other survival properties including heat resistance. Knowledge of acid production in cheese could provide a predictive method for the safety of the cheese, and could eventually lead to improved safety. 35 -- Center for Dairy Research Figu~ 1. Suroival ofacid-adapted andnon-adapte.d cultures ofSalmonella typhimurium in 1.20 % lactic acid, pH3.84. t0 3 10 2 Add-adapted 10 1 Non-adapted 10? -; .e i: tO'l i 10'2 tt bO 10,3 .9 10-4 to,S 10,6 0 20 40 60 80 Time(minutes) Figure 2. Survival ofadd-adapted andnon-adapted cultures ofSalmonella typhirnurium during anactive fermentation ofmille. 10 3 Add-adapted 10 2 Non-adapted \ I 10I iii 10? 'E ~ ::2 10. 1 trJ ~ co 10'2 0 o-l 10. 3 10. 4 10. 5 0 2 .3 4 5 6 Time(Hours) 36 ))----------------.--.,1 Annual Reporl1990-1991 II Figure 3. Survival ofacid-adapted andnon-adapted cultures ofSalmonella typhimurium during therinal itUlCtifXltion. 1000 ...-------------------, --0- Add-adapted -55"C --+- Non-adapted - 55"'C --6 Acid-adapted- 575"C --.- NOIHdapted: 575"'C .01 .001 .ron -+-.-.-.,.--~r__ ......_-......,...--......_--I o 2 468 10 12 Time(Minutes) Figure 4. Effect of0.1 % pyruvate supplementation in LB medium onrecovery ofacid-adapted and non adapted Salmonella typhimuriumin 1.20% lactic acid,pH3.84. 1000~---------------_ lOOK----t......llll::;::::::::=~ 10 --a- Add-adapted-U + 0.1%pyruvate --0- Add-adapted- LB --.-No~pted-LB+ 0.1%pyruvate --e- Non-adapted- LB .001 .0001 30 10 20 Time (Minutes) 37 I Center for Dairy Research If-------------------------? Identification of Environmental Sources of Listeria monocytogenes in Dairy . Product Manufacturing Plants and Development of HACCP Programs Designed to Prevent Listeria Contamination of Dairy Products Personnel; Eric A. Johnson, assistant professor, Food Microbiology and Toxicology; Ranga Premaratne, postdoctoral research associate; Alvaro Quinones, research specialist Fundini and Fundini Codes; Wisconsin Milk Marketing Board, IV-65 Jan. 1,1990 - Nov. 30,1991. Objectives: (a). Detennine the location and prevalence of Usteria in dairy plants. (b). Characterize various L. monocytogenes dairy isolates for properties that would influence their persistence in dairy plants, including heat resistance, growth, and survival at refrigerated temperatures, and resistance to sanitizers, adds, alcohols, and. other inhibitory agents. (c). Characterize the surface structures from Usteria dairy isolates. (d). Determine mechanisms by which L. monocytogenes colonizes equipment and other surfaces in dairy plants. (e). Communicate useful information to the dairy industry. Summary: Twenty-nine isolates were obtained from the dairy environment and from foodbome out breaks involving dairy products. These were evaluated for resistance to sanitizers and other inhibitory agents, sensitivity to heat and cold, and nutrient requirements. The isolates were also examined for cell surface components that contributed to resistance and adherence charac teristics. Of various sanitizers tested, quaternary ammonium sanitizers were most effective against the environmental and pathogenic L. monocytogenes isolates. Resistance of L. mOnocytogenes strains to lactic, acetic, and hydro chloric adds (0.2 Mandl M) was tested inbrain heart infusion and tryptic soy media. Acetic add was the most effective in inhibiting L. monocytogenes. Some strains developed resis tance to the adds and sanitizers, indicating an adaptive response. Nutritional requirements of the environmental and pathogenic isolates were examined. For this purpose, a chemically defined minimal medium was developed. L. monocytogenes requires leucine, isoleucine, arginine, methi onine, valine, cysteine, riboflavin, biotin, thia mine, and thioctic add as growth factors. Growth is also strongly stimulated by iron. Glucose and glutamine are required as primary sources of carbon and nitrogen. All strains grew wen after two transfers in minimal defined medium at IS- and 370C. Strains V1, Ohio, California, ScottA and. certain dairy isolates adapted much more rapidly to the minimal growth requirements than most of the environ mental isolates. Cells survived much more poorly in minimal medium supplied with a carbon source compared to media not containing an energy source ora medium supplemented with chitin or cell walls of Lactococcus lacHs. These results suggest that survival is enhanced in cells starved for an energy source. The role of cell surface proteins in adherence and survival has been examined by electrophoretic analysis of surface polypeptides. We have found that L. monocytogenes synthesizes specific extracellular proteins in response to iron and energy starva tion. By labeling exposed proteins with N hydroxysucdnimide, experiments also indicate that L. monocytogenes contains high concentra tions of a protein that is exposed to the surface and may be involved in adhesion and resistance to antimicrobial agents. Si&nificanceto the Dairy Industry: This research project has identified locations of Listeria monocytogenes in dairy plants. We have also determined important factors involved in survival of L. monocytogenes in dairy plants and in eradication of L. monocytogenes by sanitizers. 38 )1-----------------1 Annual Report 1.990-1.991. II Publications: 1. Nelson, J.H. (1990). Where are Listeria likely to be found in dairy plants? Dairy, Food and Erro. Sanitation 10:344-345 2. Premaratne, R, W.-L. Un, and E.A. Johnson (1991). Improved chemically defined medium for Listeritl monocytogenes. submitted to Applied andEnf'. Microbiology Presentations: 1. Quinones, A., R. Premaratne, and E.A. Johnson. "'Survival and inactivation of Listeritl monocytogenes in dairy products.R Poster at CDR Cheese Researchand Technology Conference, March 6-7, 1991, Madison, WI ? 39 I Center for Dairy Research If-------------------------(( Control of Listeria monocytogenes Surface Colonization of Cheese Personnel: Eric A. Johnson, assistant professor, Food Microbiology and Toxicology; Ann Larson, research specialist; Myeng-Ae Yu, research associate; Martin Krause, student assistant Funding and Funding Codes: National Dairy Promotion and ResearchBoard, EJN91 Jan. 1,1991-~.31, 1992 Objectives: (a). To identify nutrients/components in cheese which limit growth of L. monocytogenes and to determine ifnutrient-limited conditions influ ence the physiology and survival of L. monocytogenes in cheese. (b). To isolate yeasts and ripening bacteria associated with the surfaces of ripened cheeses and detennine competitive activity against Listeria. To develop a competitive exclusion microflora, particularly yeasts, which inhibit L monocytogenes on cheese. The system will be designed primarily to outcompete L. mOnDcytogenes for limiting nutrients for growth and survival. (c). To develop an antimicrobial solution against L monDcytogenes containing GRAS or permitted preservatives that could be applied to the surface of the cheese. Sununary: Listeria monocytogenes continues to persist as a health concern in dairy products, particularly cheeses. L. monocytogenes has been demonstrated to be widespread in dairy plants, and coloniza tion of cheeses primarily occurs after heat treatments, probably during cheesemaking, ripening, or packaging. In this study, we have made progress in identifying the growth limiting nutrients in cheese. Preliminary evidence using cheese extracts as sources of energy or amino acids in minimal medium indicates that energy 40 availability and the limited ability of L monocytogenes to tolerate add probably limit growth of the pathogen in Camembert cheese. Previous studies in our laboratory showed that in minimal medium, only a few sugars could replace glucose including fructose, cellobiose, and trehalose, but these would not be expected to occur in growth-promoting levels in cheese. Interestingly, the amino sugars glucosamine, N acetylglucosamine, and N-acetylmuramic add supported growth in the absence of glucose. We have found evidence that chitin and cell walls of starter bacteria (Lactococcus lactis supported survival of L. monocytogenes, which suggests that the pathogen may obtain carbon and energy sources during colonization of some foods such as cheeses by assimilating bacteria or molds that are present. We have also begun to isolate brevibacteria and yeasts from cheeses and are testing their ability to compete with L. monocytogenes on the surface of cheeses. Significance to the Dairy Industr.,y: Listeria persists as a pathogen of concern in dairy products and has caused economic hardship to the dairy industry mostly because of its class I recalls. This project will identify nutrients important in the colonization of surface-ripened cheeses and will develop a preservation system to prevent Listeria contamination. Presentations: 1. Johnson, E.A. "Survival of L. mOnDcytogenes in cheese-like environments." Food Research Institute Annual Meeting, May 1991, University ofWisconsin-Madison, Madison, WI )l------------------j AnnuIII Report 1990-1991 II Growth Inhibition of Mllkbome Pathogens by Fatty Adds Personnel: Eric A. Johnson, assistant professor, Food Microbiology and Toxicology; Lib-ling Wang, research associate; Katrina Olszewski, student research assistant Fundina and Fundin~Codes: National Dairy Promotion and Research Board, EJ90-1 July 1, 1990-June 30, 1992 Ol:zjectives: (a). To determine the in vitro susceptibility of foodborne pathogens to fatty adds-commonly present in bovine milk triglycerides. (b). To develop methods to specifically release inhibitory fatty acids with lipolytic enzymes. (c). To determine if the inhibitory systems influence pathogen growth and survival in Cheddar cheese and surface-ripened cheeses. Summary: We have examined the in vitro susceptibility of milkbome pathogens to fatty acids and monoglycerides commonly present in milkfat. Certain gram-negative pathogens including Salmonella typhimurium and Escherichia coli 0157:H7 were resistant to several fatty adds. Interestingly, we found that certain fatty acids were strongly inhibitory against Listeria monocytogenes. In our preliminary screening of theeffect of various fatty adds and monoglycerides on the growth of Listeria monocytogenes in Brain Heart Infusion broth (BHI) at pH 6.0, it was found that monolaurin, C12:0, C18:2, CLA (conjugated linoleic acids) and C18:3 at the concentration of 10 ppm, 20 ppm, 200 ppm and 200 ppm, respectively, completely inhibited L. monocytogenes for 6 days at 37"C . In whole milk and skim milk, CLA had a bacterio static effect on L. monocytogenes. The length of the lag period was proportional to the concentra tion of CLA, and at concentrations of 50 ppm, 100 ppm, 200 ppm and 300 ppm, the lag periods 41 were 5 hr, 9 hr, 20 hr and 38 hr, respectively, at 300C. CLA (100 ppm) extended the lag phase for 7 days at refrigeration temperature. In compari son, monolaurin was bacteriocidal at low temperature (4 "C)in skim milk, but L. monocytogenes grew slowly in whole milk with monolaurin. Preliminary experiments have indicated that inhibitory levels of fatty adds can be produced through lipolysis of fats in cheese. Our results suggest that fatty acids could be used as inhibitory agents against L. monocytogenes in dairy foods. Si&"ificance to the Dairy Industry: Usteria persists as a pathogen of concern in dairy products. Findings of this study suggest that certain fatty acids are important in control ling survival of L.monocytogenes in cheese. Presentations: 1. Wang, L.-L and E.A. Johnson. "Inhibition of Listeria monocytogenes by fatty acids." lAMFES Annual Meeting, July 21, 1991, Louisville, KY I Center/or Dairy Research 1-----------------------? 42 Annual Report 1990-1991 Cheese Technology Progress Reports The integrated cheese research program provides the industry with basic information on economics, technology and flavor control that is essential to an industry dealing with a more highly segmented market in terms of cheese types and consumers. The final report by Jesse et al, correlates the significance of milk quality parameters to the economics of cheese production. Their research emphasizes the importance of multiple component pricing for proper transmission of market place signals. Gould reports the development of a "user-friendly" economic engineering model designed for use by cheese plant managers to maximize the profitabilityof large or small dairy plants. The program is presently being evaluated in commercial cheese plants. With the evolution of worldwide dairy/cheese industries, plants will have to weigh economic options (i.e, cheese types> with greater speed and accuracy. This user friendly program will greatly assist the decision making process. Factors affecting physical characteristics of cheeses and their measurement are being investi gated in three projects. Gunasekaran reports on progress towards development of an instrumentative measurement system for determining the physical properties of cheese. In a related project, Olson describes the effect of variations in specific compositional parameters .on the properties of cheese. Wendorff's project will be using similar test methodology to establish the effects of fat, moisture and salt on the qualities of Cheddar and lowfat and low sodium Cheddar-type cheeses subjected to fro.zen storage. These projects are designed to aid the cheese industry in controlling the properties of cheese used as a food ingredient. Twelve individual but interrelated projects in the cheese technology area deal with flavor control, mechanisms of flavor development (desirable and undesirable), and the measure ment/detection of flavor compounds in cheese. These projects build on the findings of CDR sponsored research plus findings from other laboratories concerning the beneficial effects of selected bacteria and enzymes on control and enhancement of cheese flavor quality and intensity. The basic research by Steele on cloning and characterization of bacterial enzymes is the next logical step in verifying the importance of selected enzymes in cheese ripening. This will allow the cheese research team to systematically focus on the enzymes most essential for cheese ripening and flavor control. The same systematic approach is being used to evaluate the impact of glutathione, which has been suggested as an important contributor to flavor of certain cheeses. Characterization of selected promising bacterial strains for enzymatic activities, and relating those activities to cheese flavor quality and intensity is being continued through projects of El-Soda, Olson, and Johnson. Selection of the best bacterial strains must be followed by processes to convert the cultures into commercially usable form. Three reports by Et.zel's group describe post-processing treatments that regulate cell viability and enzymatic activities and yield cultures with tailor-made flavor producing properties. The research goals also include the optimization of processes to produce spray-dried cultures which can be shipped and used conveniently in cheese plants. Flavor improvement of certain cheese varieties requires controlled liberation of fatty acids. Hartel's report on lipolysis in supercritical CO 2 provides options to enhance lipolysis and confers specificity to regulate ratios of fatty acids that are released. These capabilities can give the cheese industry alternatives for tailor making cheese products as food ingredients. 43 I Center for Dairy Research I-------------------------l( Three reports by Johnson and Undsay summarize progress on controlling and enhancing flavor and body characteristics of lowfat and low-sodium cheeses. Infonnation generated from these projects is being used routinely by the cheese industry. The growth of nonstarter bacteria in these cheese types must be carefully controlled, as indicated in Lindsay's report. The report by Johnson on heat-resistance of bacteria will provide methods to minimize this contamination and its adverse effects on cheese flavor. In the specialty cheese area, Wendorff reports the initial findings on consumers' surface color preference for commercial smoked Cheddar and Swiss cheese. Benz(a)pyrene analysis of these and experimental cheeses will be used to develop a recommended procedure for the production of smoked specialty cheeses. 44 )r----------------1 Annual Report 1990-1991 II FINAL BEPORT Economic Benefits of Increasing the Quality of Milk Used for Cheese in Wisconsin Personnel: Ed Jesse (ProjectCoordinator), professor, Dept. of Ag. Economics; Matt Holt (Principal Investigator; statistical analysis), associate professor, Dept of Ag. Economics; p.e Vasavada, professor, UW-River Falls Dept. of Food Science;Hugo DeGroote, research assistant, Dept. of Ag. Economics; Drs. Arden Hardie, LynnJohnson, and Mr. Loren Cropp, Wisconsin Dairy Herd Improvement Cooperative Fundins and Fundins Codes: Wisconsin Milk Marketing Board, 88-50 July 1, 1988-Dec. 31, 1990(Extended) Objectives: 1. Estimate the physical relationship between casein recovery (i.e., casein retained in cheese as a proportion of total protein) and milk quality as measured by somatic cell count (SCQ. 2. Estimate how casein recovery is influenced by individual cow and herd factors such as breed, stage of lactation, total solids content, and selected herd management practices and charac teristics. 3. Determine the effects of milk holding time and the commingling of milk of differing quality on casein recovery. 4. Estimate variation in milk value as related to the measurable physical characteristics gener ated from objectives (1)-(3). Specifically, deter mine the monetary value of: (a) milk quality as measured by SCC; (b) selected cow and herd characteristics associated with high levels of casein recovery; and (c) changes in plant storage practices to increase casein recovery. 5. Estimate potential revenue gains to Wisconsin dairy fanners from increasing the casein recov ery as indicated by (4). Summary: The overall objective of this study was to mea sure the economic value of improving the quality of milk used for cheese in Wisconsin. Information relating to milk composition, cow characteristics, and dairy herd management practices was collected from commercial dairy herds in Wisconsin. A relatively new test for .casein was employed that simulated commercial cheesemaking in deriving the casein content of milk. This information was used in regression analyses to evaluate factors affecting casein-to-protein ratio and somatic cell count. Using the estimated regression parameters and assumptions pertain ing to cheese yields and prices, the economic influence of selected factors affecting casein recovery was simulated. Major conclusions include: ? Tests involving the effect of milk holding time showed that milk quality was largely unaffected by length of storage up to six days. However, large increases in bacteria count suggest cheese quality could suffer. ? Seasonality was a critical factor in casein-to protein ratios (C/P). Based on variability in CIP, cheese yields would be expected to vary season ally by one-half pound per hundredweight. ? Other factors exhibiting a major influence on CIP included somatic cell count, cowage, and breed of cow. The effect of these factors was small relative to the seasonal effect. ? SCC exhibited a pronounced seasonal pattern, showing significantly higher values during the summer months. 45 I Center for Dairy Research II------------------------{( ? sec was significantlyand positively influ enced by cowage and stage of lactation. The effect on sec was significant and negative for milk weight. Milking practices that are known to reduce the incidence of mastitis significantly reduced sec. Measured in terms of its direct effect on recover able casein and cheese yield, the economic benefit of low sec is small in comparison to quality premiums offered by cheese plants. But quality premiums tied to sec tend to simulta neously reward other factors associated with higher cheese yields. And milk with low sec has other economically beneficial effects on cheesemakers. Perhaps the most important implication of this research is that multiple component pricing is critical to proper transmission of marketplace signals. Milk is not homogeneous with respect to its value in cheese. More important, neither is protein. Consequently, consistent with milk testing technology, multiple component pricing should price casein; not measures of milk components and quality that are imperfectly correlated with casein. Significance to the Dairy Industry: This research does not purport to tell cheese plants how they should price their milk. But it does suggest that quality premiums based.on sec are rewarding farmers for something other than associated cheese value. Currently, most quality premium payment plans value sec, either directly or indirectly, higher than can be justified according to what we found to be the economic value of sec in cheesemaking. We found that, based.strictly on its value in making cheese, milk with a sec of 100,000cells/mI. is worth about 6 cents per hundredweight more than milk with a secof 750,000. A typical quality premium program in Wisconsin would value milk with secat 100,000 from 25 to 75 cents per hundredweight more than milk with secat 750,000. Our results measure the effect of sec on cheese yield na with several factors that are positively correlated with sec; in particular, stage of lactation and age of cow. When these factors are not separately accounted for, then the ~ effect of sec is larger. In pricing herd milk, the value of low sec is twice that associated with individual cow milk, but still considerably less than implied by quality premiums. Also, our results do not account for some negative effects of sec that do not show up in a reduced casein/protein ratio. With a very high sec, cheese quality can be negatively affected, possibly resulting in down-grading. Some studies have shown that secaffects butterfat recovery as well as casein recovery, but results are mixed. High sec reduces curd rigidity, reqUiring larger amounts of rennet and resulting in higher manufacturing costs. All of these factors mean that low-seC milk may reduce cheesemaking costs or increase cheese value apart from the direct effect of secon C/P ratios. Fmally, we must emphasize that our results do not in any way suggest that dairy fanners can or should ignore high secin their herds. Somatic cell count is a measure of herd health. Healthy cows produce more milk, and more milk means more money. But our results do suggest that the benefits of lower somatic cell count accrue more to the dairy farmer in the fonn of larger milk volume than to the cheesemaker in the form of higher cheese yields. Pricing signals to dairy fanners should be related to marketplace values. Perhaps the most impor tant implication of this research is that multiple component pricing is critical to proper transmis sion of marketplace signals. And, to the extent possible given milk testing technology, multiple component pricing should price casein; not measures ofmilk components and quality that are imperfectly correlated with casein. Presentations: Jesse, Ed, and Matt Holt, "Cheese Yield Econom ics: Further Evidence," Cheese Research and Technology Conference, Center for Dairy Research, University of Wisconsin-Madison, Madison, WI, March 6-7,1991. 46 II ))..--------------------1 Annual Reporl1990-1991 Development of an Economic Engineering Microcomputer Model for Analysis of Cheese Plant Operation Personnel: Brian W. Gould, associate scientist, CDR and Dept. of Ag. Economics; Michael Thomsen, graduate student, Dept. of Ag. Economics Fundini and Fundini Codes: Wisconsin Milk Marketing Board: Basic Research, 133-R124 Continuing Objectives: 1. Develop the necessary economic and financial principles for use in the computer model; 2. Extend the model to include cash flow concepts and provide for a multi-period analysis of new products or processes; and 3. Apply the computer model to several dairy plants. Summary: A research objective of the Center is to evaluate the economic feasibility of alternative processes and products. In fulfilling this general objective, we have initiated research into the development of computer models that allows for an economic analysis of new process and products. As a result of this activity a computer program, CHEESE-ECO, written in PASCAL code allows the user to input plant specific data such as yields, hours of labor, capacity utilization of plant, milk price, cheese price, etc. From this data, single period net return values are calcu lated. Data input and modification is achieved within a spreadsheet type of environment This program has been developed so as to enable the user to recall previously entered data and to update or modify any input requirement, process change, price change etc., and to analyze the economic impacts. We are currently devel oping a users manual for the software and are undertaking efforts to pretest the program. In developing the above model we decided not to extend the analysis to include general ac counting and cash flow principles (e.g., objective 2). Instead we allocated considerable time to making the current version of the model as user friendly as possible. We have also incorporated within the model the milk standardization routines developed by Kerrigan and Johnson which allow one to determine "net" milk price and which cheese yields are determined by the milk characteristics. Over the next year we intend to use 4-5active cheese plants to apply this model to actual plant settings. Significance to the Dairy Industry: Industry has expressed considerable interest in the use of the computer model. With the use of this model a plant manager will be able to quickly answer questions as to the impacts of changes in plant configuration or product line on net returns. Given the nature of the model it can be applied to both large and small dairy plant environments. Presentations: B.W. Gould and M. Thomsen, "Development of a Computer Program for Economic Analysis of Cheese Plant Operation", Cheese Research and Technology Conference, Center for Dairy Research, March 6-7, 1991, Madison Wisconsin 47 I Center for Dairy Research -------------~c Physical and Thermal Properties of Different Cheeses Personnel: S. Gunasekaran, assistant professor, Depts. of Ag. Engineering and Food Science; Norman Olson, director, COR; J.P. Chen, assistant scientist, COR; M.M. Ak,graduate student, Dept. of Ag. Engineering Funding and Fundinl; Codes: National Dairy Promotion and Research Board, 89-1 May I, 1990-April30, 1993 Objgctives: 1. Develop instrumentation and measurement systems for physical and thermal property evaluations of cheese. 2. Experimentally determine physical (textural and/or rheological) and thermal properties of cheese. 3. Investigate the effect of various cheese compositional factors and test conditions on physical and thermal properties. Summary: The research has focused on fulfilling the first objective. Much of the effort was on updating the Instron Model 1130 machine with computer data acquisition capability. Evaluation of the Instron was performed in the uniaxial compres sion mode to determine the most appropriate testing conditions. The effects of several experi mental parameters, such as sample size (more specifically the aspect ratio, defined as the diameter to length ratio of the sample), deforma tion rate (the crosshead speed), and friction at the sample-machine interface, have been investi gated with a particular emphasis on determining the interaction or interdependencies among these factors. Twenty-day-old Cheddar cheese was used in the experiments. Test samples were cut from the block ofcheese using a specially made cork borer type cutter. The samples were 0.75 inches in diameter and the length was either 0.75 inches or 1.15 inches, giving two aspect ratios of 1.0 or 0.65, respectively. The cut samples were indi vidually wrapped in plastic to prevent surface drying until testing. The tests were conducted after allowing suffi cient time for the samples to equilibrate to room temperature (23?0. Six crosshead speeds (deformation rates) in the range of 0.09 to 5.0 in.! min. were used. Two sample-machine interfacial conditions, lubricated and nonlubricated, were also employed. Lubricated tests were conducted by applying generous amount of paraffin oil at the sample-machine interfaces. These 2 x 6 x 2 factorial (aspect ratio x crosshead speed x interfacial friction) experiments were replicated four times. Because of the large deformations employed in the experiments, true (or Hencky) strain and true stress rather than engineering strain and stress were used for analysis. The effects of crosshead speed on the response of Cheddar cheese to uniaxial compression are presented in Figures 1 and 2. It is clear that the crosshead speed exerted a profound influence on the deformation behavior of Cheddar cheese. The higher the crosshead speed, the larger the stress required to achieve a certain strain in both lubricated and nonlubricated samples at both aspect ratios. Cheese behaved more like a solid when com pressed at a higher rate. At slower speeds, particularly at 0.09 in/min., stress growth leveled off after a strain of 03. At higher speeds it continued to increase. This might indicate that at slower speeds stress relaxation was taking place as the deformation continued to progress. Therefore, the shape of the stress-strain curve depends strongly on the balance between stress relaxation and stress growth due to compression. In all cases it appears that Cheddar cheese started yielding at strains approximately be tween 0.8 and 1.0. The yielding was determined from the presence of a zero-slope region on the plots. The yield stress was larger for nonlubricated samples than for lubricated ones 48 ? ?? ?? ? ?? )>----------------------1 Annual Reporl1990-1991 II Figure 1. Stress-strain relationship ofCheddar cheese ofdifferent aspect ratios when thesample-machine interface was lubricated. O-Q-.......,.--r---.--r---r--.....--.....,.----r-~~ 0.0 0.2 0.4 0.6 0.8 1.0 1.2 True strain (in.lin.) Figure 2. Stress-strain relationship ofCheddar cheese ofdifferent asped ratios when thesample-machine interface wasnonlubricated. 20 Crosshead speed (in./min)/ 'Nonlubncatad Aspect ratio liJ 0.27/1.0 15 ??? D ?? 1.011.0 ? ? 5.0/1.0 ? ? 'u;- ..e, ....... 0.27/0.65 ? ----------------------1 Annual Report 1990-1991 11 Autolytic Characteristics of Cheese Ripening Bacteria Personnel: Mark Johnson, senior scientist, CDR; K.M. Kamaly, visiting assistant scientist, CDR Fundins and Fundins Codes: National Daily Promotion and Research Board, MAR89-3 April 1, 1991-March31, 1992 Qkiect:ives: To accelerate the ripening ofCheddar cheese through enhanced autolytic activity of starter and non-starterbacteria that provide enzymes important to cheese ripening. SuJnInaJy: At the time of this writing, this project was just getting underway. It is therefore too early to report any progress. Significance to the Dairy Industry: Knowledge of cell autolytic characteristics, which regulate release of enzymes from specific organisms important to cheese ripening, should speed the ripening process and enable the cheesemaker to better control the quality of ripened cheese. Better quality will enhance sales. 63 I Cmter for DtUty Rnellt'ch Ii....-----------------------'\( Mechanisms of Injury to Streptococcus lactis During Spray Drying Personnel: M.R. Etzel, assistant professor, Depts. of Food Science and Chern. Engineering; W.-Y. Fu, graduate student Fundinz and Fundinz Codes: National Dairy Promotion and Research Board, ETZ91 Jan. 1991-June 30, 1993 0Qjectiyes: 1. Establish methods to grow Streptococcus lactis CI0 and prepare the cells for drying. Spray dry the culture suspension. 2. Collect samples of the partially-dried culture at different positions in the spray dryer to discover the regions that are critical to culture injury and inactivation. 3. Use the data to determine the mechanisms of injury to the culture. Summary: At the time of this writing, the graduate student has been on the project less than one week. This report will briefly summarize the motivation, objectives, experimental approach and industry impact of this project. The demand for lowfat cheese is increasing as consumers become more health conscious. Lowfat cheeses frequently have a bitter taste and a weak or nonexistent aroma. The challenge for the dairy industry lies in developing lowfat cheeses with an attractive aroma and flavor. Active spray-dried starter cultures can be added during the cheesemaking process as an adjunct to normal cultures. These cultures contain active enzymes which leak out during cheese ripening, increasing the rate of formation and the intensity of desirable flavor. However, the dried cultures must not disturb the normal fennentation process. This is avoided in spray dried cultures, which may exhibit a 4-5 hour lag time before producing add. After this time, fermentation has ended because the normal starter cultures have consumed all the fermentable sugar. In this research, we seek to produce dry and stable starter culture adjuncts which can be used on a large scale to enhance flavor development and reduce the bitterness in lowfat and normal fat cheeses. To work effectively, the starter culture adjunct must be reversibly injured but not killed. Understanding the mechanisms that produce active dried cultures with long lag times is the critical step in developing this new technology. Streptococcus lactis C10 was chosen for this research because much is known about the glycolysis pathway, and some promising results from spray drying experiments have been published. Very little has been published on the mechanism of reversible injury to cells during spray drying. In the most relevant study, the lag-time before the return of glycolysis in spray dried Streptococcus lactis C10 with 63% viability was about five hours longer than that of the fresh culture (Foster EM., J. Dairy Sci. 196245 1290). However, the data were difficult to reproduce and the mechanism of injury was not investigated. In this research, we will collect samples of the partially-dried culture solution at different points during the spray drying process. These samples will be assayed for cell viability and the rate of lactic acid production, as well as for permeability to and intracellular concentrations of 2-phosphoglycerate, 3-phosphoglycerate, phosphoenolpyruvate, and lactate dehydrogenase, which are all components of the glycolysis pathway. These experiments should provide a chronology for the order of occurrence of injuries, such as a longer lag time, an increase in cell-membrane permeability, and a loss in viability. They will also help to determine the 64 effects of atomization, free..water removal, and bound-water removal. Knowing the mechanism of injury, we will suggest growth and processing conditions to enhance or decrease the extent of . injury. Any theories relating reversible injury to the lag time before lactic acid production must involve the glycolysis pathway. An injury which increases the cell-membrane permeability to phosphoenolpyruvate would probably cause a long lag time because phosphoenolpyruvate is needed for lactose to enter the cell. However, more phosphoenolpyruvate cannot be synthesized without lactose entry into the cell. Replenishing this constituent could be a very slow process, and may account for the long lag times in spray dried cultures. Significance to the Dairy Industry: This research will develop new technology to stimulate the flavor development and reduce the bitterness of cheese. This is espedaUy important for lowfat cheeses which frequently have a bitter taste and a weak or nonexistent aroma. Our technology involves the addition of active spray dried starter culture adjunct during cheesemaking. During cheese ripening, the Annual Report 1990-1991 active enzymes in the adjunct are thought to leak out of the cell due to cell waillysis and accelerate the development of desirable cheese flavor. In this way, we can improve the flavor development in lowfat cheese without over produdng acid or competing with the normal starter culture. Lastly, this new technology may be generally useful in understanding the mechanisms of injury to normal starter cultures during spray drying. The latter can lead to improved processes to produce lactic starter cultures with active acid-producing capabilities in a less expensive (spray-dried), stable fonn. 65 I Center for Did", Research II-----------------------~C Effect of Post-processing on Cell Viability, Cell Permeability, and Enzyme Activity of Ladobacillus helveticus Cheese Starter Culture Adjunct Personnel: M.R. Etzel, assistant professor, Depts. of Food.Science and Chem. Engineering; J.A.C. Johnson, graduate student Fundin; and Fundin; Codes: National Dairy Promotion and Research Board; ETZ89--6 Oct. 1989-Sept. 30, 1992 Objectives: The purpose of this research is to develop a new, large-scaJe technology for preparing microbial cells to stimulate desirable flavor development in cheese. This is especially important for lowfat cheese which tends to have a bitter taste and a weak aroma. Our procedure is based on the addition of dried, non-viable L. helveticus cells as a starter culture adjunct during the cheesemaking process. Because the adjuncts are notviable, they will not produce lactic add or compete with the normal cultures for substrates and nutrients. However, the adjuncts still contain active intracellular enzymes which can leak out of the now permeable cell wall, thus enhandng the development of desirable cheese flavor. Addition of heat-treated and freeze-treated L.helveticus during cheese making has already been shown to accelerate and improve flavor development dUring cheese ripening. We hope to produce dry and stable L. helveticus cells which can be used on a large scale to enhance flavor development in cheese. Summary: In order to produce and preserve inactive starter culture adjuncts, the conventional methods of freezing and freeze-drying were first considered. However, these techniques are both time consuming and costly. Spray drying, on the other hand, is a much more economical process, espedally on a large-scale basis. It is a continuous operation and the particles are dried within a few seconds which minimizes thermal degradation. Because the product is dry and occupies a small volume, transportation and storage costs are reduced as well. For these reasons, spray drying became the method of choice. The first experiments performed examined the effect of various spray drying conditions on cell viability. L. helveticus cultures were cultivated and harvested, and then dispersed in condensed skim milk having solids concentrations ranging from 15% to 34%. For each concentration, powder samples were collected at various spray dryer outlet air temperatures. A plot of the residual cell viability as a function of these parameters is shown in Figure 1. It was found that as both the outlet air temperature and the solids content increased, cell viability decreased. To produce non-viable adjuncts for lowfat cheese manufacture, one would want to utilize starter culture adjuncts spray dried in a high solids feed solution at a high outlet air temperature. Conversely, samples collected at low outlet temperatures had residual viabilities as high as 50%. Therefore, not only is spray drying a good. technique for produdng cultures with low viability, but it may also be used to produce high viability cultures which may then be useful as normal cultures. The intracellular enzyme activity of dried cultures was also determined as a function of the outlet air temperature. Aminopeptidase, dipeptidase, and proteolytic activities were measured using methods established by the Center for Dairy Research ("Proteolysis, Dipeptidase, and Aminopeptidase Enzyme Activity Assays," C. Chen and A. Macedo, 1989). The activities of several aminopeptidases and dipeptidases were measured, however, because the results were similar, only the lysine aminopeptidase and glycine-tyrosine dipeptidase activities were plotted in Figure 2. As shown, viability greatly decreases with increasing outlet air temperature whereas 66 Annual Report 1990-1991 Figure 1. L. helveticus cells were dispersed in different concentrations of condensed skimmilk. The solutions were spray dried and samples were collected at several outlet air temperatures. Residual activity was determined byNINo x 100, where No is theinitial activity andN istheactivity after spray drying. Population counts were obtained using theStandard Plate Count Method. 100 10 l ~ ~ == is ~ i ! 1 0.1 0.01 -. 15.0% Solids -.. 20.1% Solids .... ;... 25.1%SoIds -... 29.7%SoIds .....??34%SoIids ?, , \. , \ . ?? . \ ...... , T,in =22O"C . ~, , , 0.001 60 70 80 90 100 110 120 130 Spray Dryer Outlet Air Temperature (0C) Figure 2. Effect ofspray dryer outlet airtemperature on the cell viability and intracellular enzyme activity of L. helveticus. ...... - -.-Lysine - .....Glycine?tyrosine ??????? Proteolysis ____ Viability - ...... 70 80 90 100 110 Spray Dryer Outlet AirTemperature (0C) 67 Centerlot' DRiry ReseRrch intracellular enzyme activity decreases only slightly. As discussed in the project objectives, this is exactly what is desired for the production of lowfat cheese. SiGNficanceto the Dairy Industry: Spray drying is a promising new technology for manufacturing dairy starter culture adjuncts. Using this technology, starter culture adjuncts can be produced with viabilities as low as 0.03% and high intracellular enzyme activity. This may enable the development of lowfat cheese with a flavor and texture as attractive as in normal fat varieties. The adjuncts do not produce lactic add or compete with the normal starter cultures, yet they contain active intracellular enzymes which can leak into the . cheese during ripening to enhance desirable flavor development. In addition to low viabilities, cultures with viabilities as high as 50% can be produced via spray drying technology. This may be useful for the large-scale production and distribution of normal, viable starter cultures. Publications; Johnson, J.A.c. and M.R. Etzel. Inactivation of lactobadlli during spray drying. To appear in Al.Ch.E. Symposium Series, 1991 Presentations: J.A.C. Johnson and M.R.. Etzel. "Inactivation of Lactobacilli During Spray Drying," A.I.Ch.E. Annual Meeting, Nov. 16, 1990,Chicago, IL, paper306b J.A.C. Johnson and M.R.. Etzel. "Inactivation of Microorganisms During Spray Drying," CDR Cheese Research and Technology Conference, March 6, 1991,Madison, WI, poster 313 J.A.c. Johnson and M.R.. Etzel. "Inactivation of Microorganisms During Spray Drying," Al.Ch.E. Conference of Food Engineering, March 11, 1991,Chicago, IL, poster lIB.10 J.A.C. Johnson and M.R.. Etzel. "Inactivation of Dairy Starter Cultures During Spray Drying," I.F.T.Annual Meeting, June 2, 1991, Dallas, 'IX, paper 35 J.A.C. Johnson and M.R. Etzel. "Spray-drying and Freeze-drying for the Large-scale Production of Microbial Products," A.I.Ch.E. Annual Meeting, Nov. 17-22,1991,Los Angeles, CA 68 )}---------------~I AmnIa'Report 1990-1991 I FINAL REPORT Inactivation Kinetics of L4ctobacillus helveticus and its Intracellular Enzymes (This study relates to one aspect of NDPRB project E1Z89~, Effect of Post-processing on Cell Viability, Cell PermPl1.bility, and Enzyme ActivityoflActobacillus helveticus Cheese Starter Culture Adjunct.) Personnel: Carla Buijsse,research intern, CDR; M..R. Etzel, assistant professor, Dept. of Food Science Fundingi Wl1EP Dates: April 1, 1991-Aug. I, 1991 Objectiyesi 1. To measure the thermal inactivation kinetics of intracellular proteases, dfpeptidasee, and aminopeptidases as a function of moisture content of lActobacillus helveticus cells during spray drying. 2. To integrate this data into an existing computer model designed to predict the potential for differential inactivation of these enzymes during spray drying. Summar,y: The demand for lowfat cheese is increasing as consumers become more health conscious and decrease their fat consumption. However, so far, lowfat cheeses do not have as attractive an aroma and flavor as the normal fat varieties. These characteristics may be improved by adding inactive starter culture adjuncts during the cheesemaking process. The inactive cultures contain active enzymes which are believed to leak out during cheese ripening, increasing the rate of formation and intensity of desirable flavor compounds. lActobacillus helveticus, a common dairy starter culture, has proven to be a suitable microorganism for improving flavor through this process. Still, to generate end products with a consistent quality, it is necessary to find suitable preservation methods to obtain ....ready to use" bacteria. Drying is one of the methods used for this purpose. To produce dry L. helveticus with the desired properties, the effects of drying conditions on cell viability, intracellular enzyme activity, and permeability should be known. Controlling the relative production of flavor compounds is key to the use of this new technology. The drying process can affect the viability of the cells and the relative activity of the various flavor producing enzymes because the cells experience heating and dehydration during drying. Therefore, these effects (thermal treatment and dehydration) have to be investigated before the drying process can be controlled. In this study, the thermal inactivation kinetics of L helveticus cells and their intracellular proteases, aminopeptidases, and p. galactosidases were measured as a function of moisture content. Proteases are enzymes that form bitter-flavored peptides in cheese, whereas aminopeptidases degrade peptides into amino acids which give desirable flavors. P galactosidase allows the L. helveticus cells to produce lactic acid. To determine inactivation kinetics of L. helveticus and its intracellular enzymes, experiments were 69 I Center tor Dairy Research If-----------------------\.( Table 1. Critical tempemtuTes at which cell viability OT specifU: enzyme activity begins to decmzse. 84 % moisture 40 % moisture viability protease aminopeptidase ~galactosidase carried out on samples (either cell paste or cell paste mix) with different combinations of moisture contents, heating temperatures, and heating times. Cell paste is defined as the washed cells harvested by batch centrifugation of the growth media. The cell paste mixture is cell paste with maltodextrin and sufficient water to achieve the desired moisture. The moisture contents of the samples were either 84% or 40%. Heating temperatures were from SOOC to 9QOC, with heating times from 1 to 16 minutes. Viability was defined as the number of colony forming units (cfu) per gram of sample, and specific activity as units per mg protein. For the aminopeptidases and P-galactosidase, a decrease in the moisture content of the samples increased thermoresistance, which resulted in less enzyme inactivation. No significant difference was found in the viability decrease between samples of different moisture contents after heating. Heating had no effect on the proteases, as inactivation was not found in any of the samples. The amount of data generated by this work is insufficient to quantitatively describe the kinetics. Itis, however, possible to find some qualitative relations. For the two moisture contents, tolerable and intolerable combinations of heating temperature and heating time were found. The "critical temperatures" for the microorganisms and the enzymes, where viability of the cells and specific activity start to decrease, are shown in Table 1. From this data, it is evident that viability can be greatly decreased during heating without significant destruction of aminopeptidase activity. Therefore. it should be possible to cause cell inactivation with minimal inactivation of desirable enzymes. $i&Jlificance to the Dairy Industry: The results of this study provide guidelines for more definitive treatments to produce inactivated bacterial cells that will contain sufficient enzymes to accelerate ripening. Additional data should allow integration of the inactivation kinetics into a computer model that can predict conditions needed for cell inactivation with minimal inactivation of desirable enzymes. This will allow production of dried, "tailor-made" cheese ripening bacteria that are convenient for commercial use. 70 )"""---------------------11 AnnulIl Report 1990-1991 I Improving the Flavor of Enzyme-modified Cheeses by Control of Lipase Action in Supercritical CO 2 Personnel: Richard W. Hartel, associate professor, Dept. of Food Science; Janice M. Johnson, graduate student Fundins and Fundini Codes: Cheese Research Institute July 1, 1989 to June 30, 1992 Objectives: 1. To assess the feasibility of using supercritical (SC) CO 2 as a means to control lipolytic action on butterfat. 2. To detennine the specificity of lipase action on butterfat in SC CO 2 as influenced by the type of lipase used, the level of water as an entrainer, and the reactor operating temperature and pressure. 3. To develop a fundamental understanding of how lipase action on butterfat in SC CO 2 may be controlled to improve the flavor balance and fatty acid profile of enzyme modified butterfat. Sunurw:y: Uberation of fatty acids from triacylgiycerides -------------------...1 Annual Reporl1990-1991 II Development of Basic Technology for Improving the Flavor and Consumer Ac ceptability of Reduced-sodium Cheddar Cheese Personnel: R.C. lindsay, professor, Dept. of Food Science; E.A. Johnson, associate professor, Food Research Institute; G. Leyer; Food Research Institute, S. McDonald, and B. Guthrie, graduate students, Dept. of Food Science, University of Wisconsin-Madison; S.L. Taylor, professor of Food Science, R W. Hutkins, assistant professor of Food SCience, J.E. Stratton, graduate student, University of Nebraska lincoln Funding and Fundinl: Code: Wisconsin Milk Marketing Board, 88-41 July 1988-June 1991 Objectives: 1. To investigate the flavor chemistry and microbiology of theproduction of unclean, Strecker-type flavor compounds in low-salt Cheddar cheese made with conventional and UF- and RQ- retentate supplemented milk. 2. To develop methods to analytically measure the ability of starter and cheese ripening lactic acid bacteria to produce unclean, Strecker-type flavor compounds, and to screen various lactic cultures and wild isolates for this metabolic activity. 3. To investigate the potential for growth of food pathogens that are capable of causing histamine or botulinal toxigenesis in low-salt Cheddar cheese, and to characterize the behavior of these organisms in low-salt Cheddar cheese. Summary; Each collaborator in this project is responsible for conducting research in a specific area of the project. E.A. Johnson (UW Food Research Institute) is responsible for research on the botulinal safety issues of low-salt cheese. S.L. Taylor and R W. Hutkins (University of Ne braska-lincoln) are responsible for research on the histamine safety issues of low-salt cheese. Re. Lindsay is responsible for the flavor chemis try and microbiology of low-salt Cheddar cheeses where techniques for overcoming the production of unclean flavors are sought. ~, This past year, research has focused on the refinement of analytical methods to measure flavor compounds associated with the unclean flavor systems of reduced-sodium Cheddar cheese. These studies have been augmented by investigations into the development of refined procedures for screening wild isolates and commercially-used starter culture organisms using a semi-defined nutrient medium contain ing specific precursors for indiVidual unclean flavor compounds. It has been found that the aromatic amino acids serve as the precursors for intermediates that are in fact the direct precur sors for the offending flavor compounds. With anaerobic incubation, it is possible to determine the relative rate of production of, unclean flavor compounds which characterizes a particular culture. Non-starter lactics resembling fecal-source lactobacilli appear to be particularly active p-cresol- and indole-producing organisms. Development of a method to measure alpha dicarbonyl compounds has provided an analyti cal method to address desirable flavor com pound formation in low-salt cheeses also. When used in concert with screening against unclean 77 I Center for Dairy Research 1'--------------------------'\( flavor-producing organisms, lactic microorgan isms can be selected to comprise adjunct starter systems for cheesemaking that overcome prob lems with lack of flavor and unclean flavors. Inoculations of low-salt Cheddar cheeses with Clostridium botulinum mixed spore suspensions followed by incubation at 27"C for up to three weeks showed that sodium chloride reduction did not increase the hazard over control cheese. Only one sample containing 2% NaO tested positive, and repeated trials did not induce toxin formation in samples. It was found that arginine supplementation stimulated germination and outgrowth of C. botulinum in low-salt cheese and full-salt cheese. Lactobacillus buchneri inoculated into reduced-sodium cheese increased in counts by two logs during ripening at 7"C for 180 days, but histamine levels remained very low (less than 5 mg/l00g) indicating little concern for normally handled low-salt Cheddar cheese. Siif1ificance to the Dairy Industry: Information on a relatively unknown group of detrimental flavor compounds found in low-salt cheeses will be gained, and identification of the proper lactic acid bacteria for producing clean cheese flavors in low-salt products will be determined. This work will help processors create quality, reduced-sodium cheeses that are free of the flavor defects sometimes found in low-salt cheeses today. 78 )....-----------------:1 Annual Report 1990-1991 II Heat-resistance of Pediococci and Lactobacilli Isolated from Raw Milk and Cheddar Cheese Personnel: Mark Johnson, senior scientist, CDR; Moustafa EI-Shenawy, visiting assistant scientist, CDR Fundi", and Funding Codes: National Dairy Promotion and Research Board, MJ90-1 Nov. 1,1~t.31,1991 Objectives: 1. To determine the heat-resistance of particular (defect causing) pediococci and lactobacilli of dairy origin. 2. To determine the suitability of currently used methods and media for the isolation of pediococd and lactobacilli after thermal stress. Summary: More than 300 lactobacilli and five pediococci isolates have been obtained from raw milk and commercially produced Cheddar cheese. The cheese samples were selected on the basis of an apparent defect, i.e, unclean or off-flavors, cracks, splits, sweet holes, or calcium lactate crystals. The bacteria were originally isolated on Rogosa. SL agar, a selective medium for lactoba dIU and pediococci, and individually purified on MRS agar. All isolates are being carried on MRS agar slants for storage. Each isolate was initially characterized according to a negative catalase test and cell morphology. Lactobacilli are rod-shaped and usually in chains when grown on synthetic media, pediococci are coed in tetrads. All lactobacilli were tested for homofermentative or heterofermentative ability based on the production ofCO 2 using APT broth to which 1.0% glucose and .5% sodium acetate had been added. Heterofennentative lactobacilli produced gas that collected within a Durham tube inverted in a large culture tube. With several of the heterofermentative isolates, gas production was minimal or took several days or was not evident until the culture tube was shaken. These cultures would have been misclassified using published standard proce dures. A more rapid and definite test has been developed and is currently being evaluated with our isolates and cultures obtained from the American Type Culture Collection. The test is based on the ability to fennent citrate in a specific medium within 48 hours. Most of the work hasthus far concentrated on the heterofermentative lactobacilli. Twenty-four heterofermentative lactobacilli have been iso lated and all have been characterized according to the API SOL Lactobacillus identification system. These 24 isolates have been divided into five distinct groups according to metabolic activities against several different substrates. The ability of heterofermentative lactobacilli to racemize lactic acid has also been tested. Only one group was found to be racemase positive. One group was able to remove L(+) lactic acid from the test system without racemization. It is possible that the lactic acid is being converted to acetic acid and CO 2 , This assumption is cur rently under investigation. Eleven heterofermentative and two homofermentative lactobacillus isolates have been tested for heat sensitivity. None of the cultures tested have survived minimum pasteur ization (161"F/15 sec). Research on heat sensi tivity will continue on all lactobacilli to define both D and Z values. Heat injury and repair will also be determined. Research will continue to also identify all isolates for bacteriocin production. Homofennentative lactobacilli will also be characterized with the API SOL Lactobacillus identification system. SilP'ificance to the Dairy Industry: The results of this study will provide informa tion on the effectiveness of using high-tempera ture pasteurization to drastically reduce particu lar non-starter lactic acid bacteria levels and thus decrease the incidence of calcium lactate crystal formation on cheese. Controlling crystal forma 79 ,---------------------~C I CenterforDtdry Research tion is of great importance to the shredded cheese industry since vacuum packaging can not be used and cheesemakers are reluctant to alter their cheese production methods. Similar information will help to detennine the effectiveness of reducing bacterial numbers in producing more shelf-stable high moisture, reduced-fat and reduced-sodium cheese. Information will be generated to provide for the best isolation and quantitative techniques for enumeration of pediococd/lactobacilli from pasteurized milk and cheese. Such information is necessary for effective quality control in cheese plants. This study may also generate a collection of bacteriocin producing bacteria. These bacteria will be used in other studies to determine their usefulness in controlling the growth of undesir able bacteria in cheese or other dairy products. 80 )r----------------------i[ Annual Report 1.990-2992 II Control of Color Formation in Smoked Cheese Personnel: William L. Wendorff, assistant professor, Dept. of Food Science (PI); William Riha, research assistant, Dept.of Food Science Fundini and Fundin& Code: National Dairy Promotion and Research Board, WEN90-2R Sept. 1, 1990to June 30,1992 Objectives: 1. Determine the effect of temperature on smoke color reaction of cheese products. 2. Determine what effect the curing of cheese (protein breakdown) has on the formation of smoke color in cheese products. 3. Determine if smoked cheese can be produced with liquid smoke to match traditionally cold smoked cheese, but without the polycyclic aromatic hydrocarbons present in traditionally smoked products. Summary: Surface color is one of the major attributes affecting the consumer acceptance of smoked cheeses. To better understand consumer percep tions and preference for smoked cheese color, numerous samples of commercial smoked Cheddar and Swiss cheeses were evaluated by consumer panelists. The surface color of these smoked cheeses were also analyzed with a Hunter colorimeter. Hunter L values correlated well with the panel perception of surface color for Cheddar (R2 = 0.949)but not as well for Swiss (R2 = 0.794). The a and b values alone were not effective indicators of consumer perception of surface color. Smoked cheeses with L values between 32 to 48 were preferred by theconsumer panelists. Hunter color dimen sions can be used effectively to predict the acceptability of surface color in smoked cheeses. The commercial samples of smoked cheeses are also being analyzed for benz(a)pyrene to deter mine if any of the natural vaporous smoked cheeses contain detectable quantities of polycy clic aromatic hydrocarbons. Preliminary results indicate that cold-smoked cheeses do not contain any detectable levels of benz(a)pyrene. In the second phase of the study, the effects of temperature of reaction, level of smoke carbon yls and degree of curing in the cheese are being evaluated as they relate to formation of smoke color. Results of these experiments will be used to formulate recommended smoke processing procedures for selected varieties of smoked Specialty cheeses. Siawificanceto the Dairy Indusb:y: Several varieties of cheese can be smoked or smoke-flavered to provide an array of specialty cheeses for the retail market. With better knowl edge of consumer perceptions and preferences concerning smoked cheese color and an under standing of the methods of controlling the formation of smoke color in the manufacturing process, the dairy industry should be able to expand the specialty cheese market by produc ing better quality smoked cheeses. With the proper use of natural smoke flavorings, any cheese plant could produce quality smoked cheeses without the problems involved in maintaining a smokehouse and smoke generator or any major capital expenditures. Presentations: "Smoked cheese color as related to consumer acceptance and objective measurements," W.E. Riha and W.L. Wendorff, 86th Annual ADSA Meeting, Logan, UT, 1991. 81 I Center for Dairy Research 1-------------------------\( 82 )>---------------------1 Annual Report 1990-1991 II Milk Component Utilization Progress Reports Part 1. Milkfat The breadth of CDR's integrated milkfat utilization research is reflected in the following progress reports, which include projects on milk composition control, fractionation and improvement of milkfat properties, nutritional aspects of milkfat, and demand analysis of milk products. The final report from Elson's project describes the isolation of cholesterol suppressive compounds from alfalfa, and confirms the hypocholesterolemic effect of ~-ionone. These compounds are closely related to carotenoids which-are readily passed into milk, but analytical confirmation of their presence in milk and milk products has not been completed. Bremel reports progress on both of his projects to alter the composition of milk in transgenic mice. One project is directed at lowering milkfat production by incorporation of a complementary gene sequence (anti-sense gene) for a key enzyme in milkfat synthesis, In his other project, Breme1 has successfully expressed bovine a S(1)casein in the milk of transgenic mice. lindsay and Hartel continued their characterization of milkfat fractions obtained by controlled crystallization. A cold-spreadable butter was produced from selected fractions, demonstrating the utility of the fractionation and characterization information. Commercial use of the information on various milkfat fractions will be greatly enhanced by a database which will be prepared and disseminated in 1991-1992. In an associated project, Hartel reports continued progress in incorporating butterfat fractions into chocolate (partial 20-30% cocoa butter substitution) and confectionery products (50%coconut oil replacement>. Information obtained thus far on crystallization behavior of cocoa butter with milkfat fractions should be useful to confectionery manufacturers in selecting and using milkfat fractions. Also working with milkfat fractions, Ney reports the results of rat feeding studies which indicate that reduction of 16-and 18-earbon saturated fatty acid content of milkfat will reduce its hypercholesterolemic effect. The potential for modifying milkfat at the processing plant to enhance selected attributes has been evaluated in several projects. Hill and Amundson demonstrate the technical feasibility of using immobilized lipase for either hydrolysis or interesterification of milkfat. Preliminary economic assessment of their hollow fiber and axial annular reactors indicate they are a commercially viable alternative to conventional production of lipolyzed butteroils. The reaction rate for immobilized lipase in an axial-annular reactor was six-fold greater than the traditional soluble lipase in a batch reactor. A more efficient immobilized lipase stirred-tank process to incorporate an unsaturated fatty acid (linoleic) into milkfat (interesterification) illustrates commercial applicability of these systems. Chen reports on the hydrolytic reaction kinetics and selectivity of lipases entrapped in reversed micelles and polymer gels. These systems provide enhanced control of the composition of the lipolyzed milkfat. In another project, Chen reports methods to increase intracellular lipase in Rhizopus delemar through controlled growth and processing conditions. After treatment, the immobilized R. delemar cells are used to lipolyze milkfat in a continuous stirred tank reactor, producing a much higher ratio (3~ fold) of short-chain to long-ehain fatty acids than the traditional soluble lipases. This suggests that the experimental treatments can yield greater potency of milkfat flavor for food ingredient use. Hartel and Parkin update their progress in evaluating the enzymatic modification of butterfat in supercritical CO 2 , Both hydrolysis and acyl-exchange are being investigated. Data obtained in initial supercritical CO 2 acyl-exchange studies indicate control of reaction specificity compared to atmospheric conditions. 83 !I------------------------l( I Center for Dairy Research Three projects by Gould's group relating to dairy product demand are included in this section since they deal, in part, with estimating future demand for milkfat These projects, however, also have broader implications. One project comprises the successful development of a database system using the QUICKSILVERdatabase program, and allows market analysis for a variety of dairy products using aggregate time-series data. Household level demand analysis for butter and four other food fats and oils was completed and published, as was the analysis of the two-stage purchase decision process for at-home consumption of cheese. The demand analysis also examined the role of household demographics on the purchase of cheese, and showed that changes in cheese price consistently affect consumption by current consumers more than potential new purchasers. These types of analyses will be used to guide our research on modifying milk composition by genetics, feeding, and processing technologies. The premise that an integrated milkfat research program is essential appears to be warranted from the research results to date. Process modification of milkfat is promising, but nutritional and demand analysis plus some of the processing results suggest that milk and milkfat composition will have to be regulated at the cow. 84 )>--------------------1 Annual Report 1990-1991 II FINAL REPORT Milk: A Point of Entry into the Human Diet for Mevalonate-suppressive Plant Secondary Metabolites Personnel: Charles E. Elson, professor, Dept. of Nutritional Sciences; Naji Abuinneileh, visiting scientist, Jordan University of Technology and Science; Ge Gugeyu, graduate student, Dept. of Nutritional Sciences Funding and Funding Codes: Wisconsin Milk Marketing Board, IV-61 Jan. 1,1989-~.31, 1990 objectives: Original objectives 1. Isolate and identify constituents of alfalfa which suppress the synthesis of mevalonic acid (MVA). 2. Confirm that the MVA-suppressive constitu ents are passed into milk. 3. To determine which milk products contain plant-derived MVA-suppressive constituents. 4. To assess the impact of diverse dairy products (identified in objective 3) on mevalonate metabo lism in experimental animals. Modification of original objectives The active MVA-suppressive constituents of alfalfa referred to in original objectives 1 and 2 are heat labile. Thermal decomposition during gas-liquid chromatographic analysis of milk extracts precludes identification. NMR analysis of the compounds, isolated by HPlC, will confirm their identities. Summary: Epidemiological surveys supported by animal studies suggest that milk consumption is associ ated with decreased cardiovascular and cancer risk. The predominant forage in dairy rations, alfalfa, is reported to lower the serum cholesterol levels of experimental animals. Fiber and phytosterol constituents of alfalfa are presumed to be responsible, as both decrease the absorp tion of dietary cholesterol. Our studies argue against this presumption because the adaptive response to decreased cholesterol absorption, an increase in cholesterol synthesis, fails to take place when the forage is added to an animal's diet. Numerous studies demonstrate that animals fed diets containing alfalfa have lower serum cholesterol levels. For example, the blood cholesterol level of chicks fed a cholesterol-free diet supplemented with 5% alfalfa meal was 13% lower than that of chicks fed the alfalfa-free control diet. The finding that led to our study was that hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity, assayed under conditions that maximize activity, was reduced by more than 50%. HMGR catalyzes the synthesis of mevalonic acid, the rate-limiting substrate in the synthesis of cholesterol. Alfalfa is a rich source of secondary plant metabolites closely related to metabolites we have shown to have anticarcinogenic and cholesterol-suppressive actions. We have isolated and tentatively identified four second ary products present in alfalfa, each of which acts to suppress mevalonate biosynthesis. These products are closely related to carotenoids, which are readily passed into milk. We believe the cholesterol-suppressive action of milk can be traced to these secondary products. Further, there is an emerging, if somewhat reluctant, 85 I Centerfor Dairy Research I ( Table 1. Impact ofalfalfa metabolites on blood cholesterol, apoproteins, triglycerides andglucose concentrations. Units are mgldl. I2ia Cholesterol APoJnoteins Trildvcerides GlucQse Total HDL A-I .. B Control 189?,9 10S?,5 151?4 34.5:t3.0 80?,10 235:t8 PE solubles 148?5 97?5 147?7 29.0?2.9 76?6 236?8 PE-MAinsolubles 141;t6 96?8 149%.3 26.7?3.0 75?6 237;t6 Band 1 121;t6 89?7 151%.3 23.4?2.2 66;t6 211?7 PE-MAsolubles 131?,5 9S?7 148?3 24.9?2.7 7S?7 237?7 MA solubles 176?7 l00?5 149?4 32.1?1.2 79?5 201?8 Table 2. Impact ofalfalfa metabolites onhepatic 3-hydro:ry-3-methylglutaryl coenzyme A reductase activity. HMGR; aetjvity pmolmevalonate/mg microsomal protein/min Control PE solubles PE-MAinsoluble Band 1 PE-MAsolubles MA solubles 1232?179 10SO?123 1086?.277 870?,109 858? 93 1053? 81 recognition that non-nutrient constituents of food play dietary roles as anticarcinogens. The recent recognition of the essential role mevalonate plays in neoplastic tissue growth lends credence to our belief that plant-derived secondary products present in milk play roles in health maintenance. For the research project reported here, we fractionated the constituents of alfalfa according to their solubilities in organic solvents. The fractionation consisted of sequential extraction with petroleum ether (PE solubles), methyl alcohol (MA solubles) and water. A feeding trial gave evidence that the major cholesterol-sup pressive constituent(s} were fractionated into the PE solubles. We then subfractionated the PE soluble by washing its more polar constituents into methyl alcohol (pE-MA insolubles and PE MA solubles). A feeding trial showed that the PE-MA insolubles fraction contained the major inhibitory activity. The constituents of the PE MA insolubles were separated by chromatogra phy on silica gel G plates with a mobile phase of petroleum ether:diethyl ether:acetic acid (95/5/ OS). A microbial assay showed that mevalonate suppressive activity was confined to band 1 containing the more polar of these apolar constituents of alfalfa. The results of a combined feeding trial are shown on Tables 1 and 2. Cholesterol-free diets supplemented with 200 ppm of the dried fractions were fed to layer pullets for four weeks; blood was collected after which the birds were fasted and refed to maxi mally induce HMGR activity. 86 )l-----------------I Annual Report 1990-1991 II Band 1 constituents were fractionated by high performance liquid chromatography on a C-18 column with acetonitrile as the mobile phase. The mevalonate-suppressive activities of the individual fractions were tested in the microbial assay; constituents of the four fractions with this activity were tentatively identified by mass spectroscopy (Table 3). Metabolite #1, tentatively 2,6,6-trimethyl-l cyclohexen-l-yl-3-butenone, and commercial b ionone (FW 192.3) areco-eluted from the C-18 column. We examined the hypocholesterolemic impact of p-ionone in two trials. In the first, the impact of 250 ppm p-ionone was compared with that of the 250 ppm geraniol, another isoprenoid present in alfalfa. The diets were fed to four week-old pullets for four weeks (Table 4). The finding that p-ionone effectively lowered blood cholesterol was confirmed in a second study. In this trial, two-week-old cockerels were fed diets containing 100 ppm geraniol or p-ionone for four weeks. In parallel groups, 20 ppm pentobarbital was fed to induce a hepatic microsomal P450 monooxygenase that degrades the isoprenoids. The cockerels were fasted for two days and refed for three days prior to sacrifice to maximize HMGR activity. During the refeeding phase, mevinolin (500 ppm) was added to the diets of subsets of the birds. Mevinolin, a competitive inhibitor of HMGR, induces the synthesis of the enzyme. The results of this trial are shown on TableS. Data on Tables 4 and 5 confirms that p-ionone suppresses HMGR activity leading to a reduc tion in the blood cholesterol levels of male and female chicks which were fed a cholesterol-free diet. Pentobarbital reversed the effects of {3 ionone and geraniol. Pentobarbital induces the enzymic activities that degrade the isoprenoids. This finding therefore provides a confirmation of the cholesterol-suppressive action of the isoprenoids. Consistent with the findings of others, rnevinolin induced HMGR activity in washed microsomes. The other finding of substance is that {3-iononeattenuated the mevinolin-mediated induction. Continuing studies address three issues: 1. We are in the process of confirming the identities of the four metabolites using NMR and mass spectroscopy. 2. The molecular structures of p-ionone and the related metabolites are similar to those of metabolites which block cholesterol synthesis at a site between famesyl PP and lanosterol in the sterol pathway. We are testing the hypothesis that the suppression of HMGR activity shown in our studies represents a secondary response. 3. We will develop HPLC techniques for the assay of milk products for the presence of the aforementioned metabolites. Table 3. Tentative identification ofmevalonate-suppressirJe constituents ofPE-MA solubles iso1Jlted from alfalfa? Constituent mJe+ carbon hydrogen oxygen #1 192+ 13 20 1 #2 232+ 16 24 1 #3 236+ 16 28 1 #4 246+ 17 26 1 #I. 2.6.6-trimethyl-I-cyclohexen-l-yl-3-butenone #2. 2.6.6-trimethyl-l-cyclohexen-l-yl-3.5-dipentene-3.5-dimethyl-2-000 #3. 2.6.6-trimethyl-I-cyclohexen-l-yl-pentane-3.5-dimethyl-2-one #4. 2.4.6.6-tetramethyl-I-cyclohexen-I-yl-3.5-dipentene-3.5-dimethyl-2-one 87 I Center for Dairy Research I---------------------~C Table 4. Impact ofdiets containing 250 ppm fJ-ionone or geraniol onlipid metabolism ofeight-week old pullets. ~ Cholesterol fmaldO Total HDL p HMGR moJlmg microsomal protein/min Control 120?7 70?3 468;t41 Ji-Ionone 80?2 56?,12 1l0?19 Geraniol 70;t4 54?3 252?50 Table 5. Impact of 100 ppm fJ-ionone and geraniol oncholesterol metabolism and interactions with pentobarbital and mevinolin. Treatment Cbolesterol fmaldD HMGR Total HDL pmoJlmg protein/min Control 133?4 72?9 1064? 41 Ji-Ionone 1I3?3 80?9 562?150 Geraniol 125.? 3 75?6 838? 90 Pentobarbital 133?7 73?9 1029?120 Ji-Ionone + Pentobarbital 130?8 77?5 912?230 Geraniol + Pentobarbital 135.? 3 72?3 1016?,250 Control + Mevinolin 138?3 68?3 1519?130 p-Ionone+ Mevinolin 122?10 77?6 1I00?, 60 Significance to the Dairy Industry: "Single fat" studies demonstrate convincingly the cholesterol-elevating and to a lesser degree, cancer-promoting actions of butterfat. It is a rare individual who consumes a diet in which butterfat, the sole dietary fat, provides 40%of energy, e.g. the basis of the above studies. Dairy fats contribute 12%of the 166 g of fat and 14%of the 481 mg of cholesterol available per capita (1984). Animal studies employing diets reflect ing this level of contribution of dairy fat to total fat and cholesterol intake paint a very different picture of the role of dairy products in health maintenance. Nevertheless, public concern forces the industry to focus (negatively) on reducing the fat content of dairy products. A 88 positive focus on the role of dairy products in the diet, akin to that on calcium, may evolve from our studies. Publications: Abuirmeileh, N. and CE. Elson (1990). The potential of the mevalonate-reversible isopre noid-mediated suppression of Halobacterium halobium growth by pentobarbital. FASEB J. 4:A927 Yu, G., W.A. Altmann, and CE. Elson (1991). Mevalonate-suppressive plant secondary me tabolites isolated from alfalfa. FASEBJ. 5:A948 )1----------------------1 AnlUlal Report 1990-1991 II Modification of Milkfat Composition by Production of Null Mutants for Acetyl CoA Carboxylase in Transgenic Animals Personnel: Robert Bremel, professor, Dept. of Dairy Science; Glenn Staffeld, senior research specialist; Greg Bleck, graduate student; Jan Heideman, research associate; Ed Metcalf, laboratory assistant, UW-Madison; Ki-Han Kim, professor, Dept. of Biochemistry, Purdue University Fundin.: and FundinK Codes: National Dairy Promotion and Research Board, 89-1 July 1, 1989-June 30, 1992 Objectives: 1. To produce anti-sense genes for acetyl-CoA carboxylase, which is the controlling enzyme for fat synthesis in the mammary gland, in gene constructs driven by mammary-specific pro moter and enhancer sequences (Purdue). 2. To produce transgenic mice expressing anti sense acetyl-eoA carboxylase gene in their mammary glands, and to analyze the composi tion of the milk produced by these animals (Wisconsin). Summa.ry: During the past year, progress has been made in several areas. The researchers at Purdue Univer sity have continued to make new ribozyme constructs with modifications based on in vitro results and publications from other laboratories. Also, they have continued to test the new constructs using in vitro methods. These new constructs include addition of an 1,IQO-bp fragment which contains definite termination and poly-adenylation signals, as well as exon 4 of a-lactalbumin. We hope this fragment will increase stability of the RNA produced. Three constructs containing various ribozymes with this fragment and the a-lactalbumin promoter were synthesized.. R51 is the active form of the double ribozyme directed to bases 146 and 159 described previously; R52 is the inactive (in verted control) form of the double ribozyme, and R53 is the single ribozyme directed to base 159 (active form). During the coming year they plan to examine the effectof the exon 4 fragment on RNA stability, as well as to investigate theeffect of length of complementary sequences on the efficiency of ribozyme activity. The transgenic facility in Madison has continued to work on generating transgenic mice. At present we have one male founder mouse carrying the P16 ribozyme construct behind the bovine a-lactalbumin promoter. This founder has been bred, and offspring are being screened for presence of the transgene. We currently have four positive offspring, one of which is a female, which will be further bred to obtain mice homozygous for the transgene. Also, any female offspring which are bred will have their milk tested for enzyme and fat levels. More injections are being made using the new ribozyme con structs. Also, we have recently received clear ance to obtain a bovine mammary cell line from Penn State University. This cell line will also be used in testing various ribozyme constructs in experiments using transient transfection. We expect to begin these experiments in the next month or so. We have been trying to use the MacT cell tine obtained from Md;i1l University. However this line has not proven to be appropri ate for these studies due to physiological irregu larities and poor transfection rates. 89 I Center for Dairy Research f......----------------? Silr;IUficance to the Dairy Industry: Advances in genetic technology have made it possible to make substantial changes in the composition of milk through genetic engineer ing, It has recently been shown to be possible to produce so called "null mutants," animals in which a genehas been selectively inactivated by inserting a gene containing the sequence comple mentary to the sequence of the mRNA coding for the gene to be nullified. The objective of this project is to produce anti-sense genes for a key enzyme in the synthesis of milkfat. With these genetic constructions it should be possible to dramatically alter the fat composition of milk. Presentations: Bremel, Robert D. "Expression of a-lactalbumin and ~lactogIobulin as indexes if milk-producing ability of heifers." Molecular Biology and Milk Production Meeting, sponsored by Dairy Re search and Development Corporation. June 1991, Melbourne, Australia. Bremel, Robert D. "Characteristics of the bovine a-lactalbumin prototypic mammary expression promoter." Molecular Biology and Milk Produc tion Meeting, sponsored by Dairy Research and Development Corporation. June 1991, Melbourne, Australia. 90 II )>----------------------1 Annual Reporl1990-1991 Expression of Bovine Alpha S{t) Casein in Milk and Tissues of Transgenic Mice Personnel: Robert Bremel, professor, Dept. of Dairy Science; Heng-Cherl Yom, graduate student, Dept. of Dairy Science; Gregory Bleck,graduate student, Dept of Dairy Science; Neal Fust, professor, Dept. of Meat and Animal Science Fundins and FunginS Codes: Wisconsin Milk Marketing Board, IV-64 July 1, 1988-Sept. 30, 1991 Objectives: ? To insert the alpha 5(1) casein gene from cows into mice and evaluate production, expression, and transmission of the transgene to offspring. ? To produce a general-purpose test system with which we can determine the utility of various promoter and/or other genetic constructs prior to their use with milk proteins. ? To study the expression of beta- and kappa caseins under the control of a known mammary specific promoter. Summary: a 5(1) casein transgenic production, expression We have produced, by microinjection, three lines of transgenic mice (two males and one female) carrying bovine a 5(1) casein cDNA under the control of mouse mammary tumor virus (MM1V) promoter/enhancer (5' flanking region), and SV40 early splicing region and polyadenylation site (3' flanking region). Transgenic mice were screened by Southern blot and/or polymerase chain reaction. All three lines transmitted the bovine transgene into their offspring approximately at the expected 50% Mendelian rate. Analysis of Tissues for Expression of Transgene Mammary biopsies from both hetero- and ho mozygous mice for bovine a-easein were per formed at day eight of pregnancy, lactation day 10, and day 11 after dexamethasone injection. The tissues were homogenized and RNAs were isolated. Northern blot analysis showed that the transgene was expressing in all three lines. All the mice from line 27 expressed the highest level among the lines and and approximately 60%for the line 42. The expression level varied among the lines, however, it was similar within the same line. The expression was lactation-specific. Lactating mice were sacrificed at day 10 of lactation and various tissues were removed. Northern blot analysis showed the transgene was specific in mammary glands expressing at high level, although trace level of expression was observed in salivary gland, kidney, lung. Ho mozygous mice expressed the transgene approxi mately 70%higher than heterozygous mice and also responded to dexamethasone injection thus further increasing the message by approximately 70%. Expression of bovine a 5(1) casein in Milk of Transgenic Mice Lactating offspring, from two male founder, expressed a high level of bovine a 5(1) casein Oine 27: 0.2 mg/ml, line 42: 0.13 mg/ml) in their milk as determined by Western blot analysis using chicken anti-casein antibodies. These antibodies did not cross-react with any mouse milk proteins. The expression of the transgene in lactating mice was further induced by dexamethasone injection and analyzed by West ern blot. The results showed that 0.5 mg/ ml (line 27) and 0.3 mg/ml (line 42) of bovine a 5(1) casein were expressed into their milk, indicating a 2.0 to 25 fold increase compared to that without induction. Milk from homozygous mice con tained higher levels of the bovine casein at 0.4 mg/ ml (line 27) and 0.25mg/ml Qine42) than that of heterozygous mice in the same line. Dexamethasone injection further increased the bovine casein level in their milk, reaching 0.8 mg/ ml (line 27) and 0.5 mg/ ml (line 42). This is the highest level of cDNA expression in the literature. Development of a Mammary Specific Promoter Isolation and Coning of Bovine a-lactalbumin: A bovine genomic DNA library was screened using a cDNA of bovine a-lactalbumin. Positive DNA clones were isolated and characterized. The 91 I Center for Dairy Research I---------------------~C Une Sex Transmiuion(lJ') Fo Fl >F2 27 40 42 Male Female Male 43 67 8 sr 54 48 SubTotal: Number of Offspring (173 total) Hetero Homo 51 12 63 14 24 9 138 3S Homozygous mice were produced by mating either mother x son or daughter x father. Total number of transgenic mice is 173 . 1...--- "best" clone identified contained the whole a lactalbumin coding sequence along with addi tional control regions of the gene. Construction of Bovine a-lactalbumin Vectors: The 5' flanking region "promoter region" of a lactalbumin hasbeen placed into two different vectors. This 2.1 kb fragment was placed into both the vector Pic 20R and Bluescript I-----------------1 Annual Report 1990?1991 II Development of Improved Processes for Enhanced Melt Properties and Flavor Stability of Cold-spreadable Butter and Other Dairy-based Food Products Personnel: R.e. Undsay, professor, Dept. of Food Science; R.W. Hartel, associate professor, Dept. of Food Science; K. Kaylegian and D. Grall, graduate students, Dept. of Food Science Fundinl and Fundin& Code: Wisconsin Milk Marketing Board, 88-56 July 1988-June 1991 objectives: 1. To devise processes using controlled crystalli zation and/or supercritical carbon dioxide extraction to obtain a range of fractions from milkfat ranging from very low-melting to very high-melting. 2. To investigate and characterize the funda mental molecular structure of milkfat fractions that are suitable for incorporation into cold spreadable butter and other food products. 3. To investigate means for efficiently stabilizing flavors of milkfat fractions to be used in cold spreadable butter and other food products. 4. To devise processes to yield improved cold spreadable butter that exhibits suitable cold spreadability and flavor stability. Summary: By letter from J.A. O'Donnell on July 6,1988, the scope of the project was broadened to a variety of milkfat fractions obtained from the available milkfat fractionation techniques, and the scope also extended to a study of the physical, chemi cal, and sensory properties of these fractions. This fundamental information was to be col lected to allow application of milkfat fractions to a wide variety of potential users. R. lindsay is responsible for the systematic characterization of milkfat fractions, and the development of cold spreadable butter applications for the fractions as an example of the utility of the fundamental data collected. R. Hartel is responsible for the supercritical carbon dioxide extraction and separation technology for milkfat, and for developing basic data on the crystallization of milkfat from the various systems. This past year, work continued on the character ization of milkfat fractions obtained from molten milkfat or acetone solution of milkfat using either single temperature conditions or sequen tial temperature adjustments in a downward fashion. In addition to the original lot of milkfat analyzed, a second lot of milkfat was similarly fractionated and the fractions characterized by gas chromatographic analysis of triglycerides and fatty acids as well as by differential scanning calorimetry and solid fat content profiles using NMR techniques. Rather similar data were obtained for the two series of fractions. Research was also focused on extraction kinetics for supercritical carbon dioxide processing of milkfat. A packed-bed extractor was employed to obtain up to 14 fractions for each extraction. Higher concentrations of short-chain fatty acids were found in the fractions removed earliest, while higher-melting fractions comprised the predominance of the materials remaining in the extractor at the conclusion of the extraction. Kinetics of melt crystallization were studied using a lab-scale batch crystallizer at various agitation rates, temperatures, and cooling rates. The number of crystals increased rapidly during the early stages of crystallization, indicating a high rate of nucleation. During the early stages, crystals also grew quite rapidly. After the initial rapid crystallization, nucleation and crystal growth decreased substantially. High agitation rates result in smaller average crystal size than low agitation rates. 93 I Center for Dmrtl Research 1---------------------""1C Milldat fractions were selected from milkfat crystallization and acetone solution crystalliza tion for incorporation into butters with a range of spreadability properties. Two butters pre pared with high-melting fractions from the melt, low-melting fractions from the melt, and a portion of a very high-melting fraction from acetone fractionation yielded properties of spreadability similar to tub margarine when tested at refrigerator and room temperatures. Si&nificance to the Dairy Industty: These fundamental data of milkfat fractions will allow selection of fractions that are functional in food formulations. Further, the profiling data on these fractions will allow matching and selection of fractions that simulate the valuable melting and textural characteristics of high-value fats. A milkfat fraction database will be developed and will describe the practical functional proper ties of the various fractions for potential food industry uses. The use of milldat could be expanded through increased incorporation into modem foods, such as microwave foods. The project will also provide technology and infor mation on improved methods for the fraction ation of milkfat so that the dairy industry can provide specific fractions to other segments of the food industry. 94 )--------------------1 Atmulll Reporl1990-1991 II Incorporation of Butterfat Fractions into Chocolate and Confedionery Personnel: Richard W. Hartel, associate professor, Dept. of Food Science; Christine Bunting and Serpil Metin, graduate students, Dept. of Food Science; Poaline Tjuradi and Lori Slowinsld, undergraduate researchers Fundin& and Fundin& Codes: National Dairy Promotion and Research Board, Competitive Grant July 1, 1989-June 30, 1992 Ol2jectives: 1. To investigate the fat crystallization behavior of mixed fat systems of interest to the chocolate industry to determine the feasibility of using butterfat fractions as cocoa butter replacers or extenders. 2. To enhance the utilization of butterfat by optimizing the use of butterfat fractions in chocolate products. 3. To investigate the use of butterfat fractions in confections to potentially replace the vegetable fats in current use. Sununary; Objective 1: Research in this area has focused on the development of techniques for comparing mixtures of butterfat fractions and cocoa butter. In particular, the use of NMR spectroscopy to evaluate solid fat content of various mixtures has been applied. In addition, the DSChas been used to quantify the changes in melting profiles of the various cocoa butter/ milkfat fraction products. This information is useful in under standing the physical chemistry of these mix tures. From these results, a clearer picture of the effects of butterfat components on cocoa butter crystallization will arise. This year we have emphasized the effects of tempering procedures on the mixed crystallization results. Our past experiences in this area have shown that incon sistent results may arise from using improper tempering procedures for comparisons, yet no standard techniques apply for our fat mixtures. Our results this year show the importance of tempering profile on development of the final crystalline product. Differences in solid fat content (NMR) and melting profile (DSC) have been observed for different time-temperature treatments and for different blends of cocoa butter and milkfat fractions. Objective 2: The main objective of this project is to enhance the utilization of butterfat by study ing the useof appropriate butterfat fractions in milk chocolate. Various chocolates are made with incorporation of butterfat fractions and evaluated for physical and organoleptic charac teristics. The research thus far has focused primarily on sample preparation and developing methods to analyze various chocolate character istics that could be affected by altering the fat composition of the product. These characteris tics have included tempering profile, viscosity, and hardness or "snap". In this past year, we have also developed procedures for looking at changes in melting properties of various ch0co lates made with fractions using DSCanalysis. A temper meter type device is being constructed where we can follow the rate of heat evolution in a tempered chocolate as the massis cooled similar to commercial temper meters. Our results show that the tempering conditions required for each fraction added to our base chocolate varied depending on how much was added and what type of fraction was used. In addition, the high melting fraction (HMF) (solid at >3O?C) from melt crystallization, when added at a 20% cocoa butter replacement level, does not temper. Rather, the butterfat crystallizes sepa rately from the cocoa butter giving an unaccept able chocolate. This difference is also seen in DSCmelting profiles. The medium (MMF> (20 to JOOC) and low (LMF> ? 200C) fractions from melt crystallization have been incorporated into our base chocolate up to 20% replacement levels. 95 I---------------------~C I Center fOT Dairy Research Good quality chocolates were made, however significant differences in tempering profiles were required. Several more chocolates will be made with increasing levels of fractions. Objective 3: Incorporation of these butterfat fractions into confectionery products is also an important goal of this research. In the past year, an undergraduate student was involved in setting up the equipment necessary to manufac ture caramels with the addition of these butterfat fractions. Significant differences in caramels were measured for different cook temperatures and different additions of milkfat fractions. These differences included hardness of the final caramel, cold flow of the caramel, butter inten sity and flavor. All caramels prepared with half of the coconut oil in the control formulation replaced by milkfat fraction were described as having increased overaU quality compared to the control when cooked at 112.70C. The perceived butter intensity of the MMF and LMF caramels was increased while the HMF caramel at 112.70C had a lower butter intensity than the control. Work is continuing in this area. Silmificance to the Dairy Industry: Our work on potential applications of butterfat fractions in confectionery and chocolate contin ues. Various butterfat fractions made by melt crystallization under different conditions are being investigated in chocolate formulations and caramels. Several milk chocolates have been made with the incorporation of up to 30% additional butterfat fraction. That is, the fraction replaces some of the cocoa butter that would normally be present. These chocolates had different properties than the control milk choco late made with all cocoa butter. Their melting properties change depending on the association of cocoa butter and milkfat triglycerides. The extent of these differences is dependent on the particular butterfat fraction used. A combina tion of basic and applied research is being used to investigate these applications of milkfat fractions to chocolates. The applied aspectsof incorporating fractions into a chocolate base allow information on practical applications to be developed. The basic aspects of this research, including studying the mutual crystallization properties of cocoa butter and milkfat fractions using NMR and DSC techniques, allow a better understanding of why various milkfat fractions behave as they do when added to chocolates. 96 III Report 1990-1991 I Relationship between Butterfat Short-chain Fatty Acids and Lipoprotein Metabolism in the Rat Personnel: Denise Ney, associate professor, Dept. of Nutritional Sciences; Hui Chuan Lai, graduate student, Dept. of Nutritional Sciences; Mike Grahn, research specialist, Dept. of Nutritional Sciences Funding and Funding Codes: Wisconsin Milk Marketing Board, 90-17 , July1, 1990-Dec. 31, 1991 Objectives: Original Objectives: 1. To determine if the short-chain fatty acid component of butterfat is the fraction associated with its neutral or positive effect on plasma cholesterol levels in rats and hamsters fed diets with and without added cholesterol and chelle add. 2. To determine if lower fasting plasma and VLOL triglyceride levels in rats fed butterfat versus beef tallow are assodated with differ ences in post-heparin plasma lipoprotein lipase activity. Modifications of Objectives: 1. To determine the effects of ingesting a diet containing low-melting-point or high-melting point milkfat fractions on plasma and hepatic cholesterol and triglyceride levels in fasted rats fed diets with and without added cholesterol and cholic add. Hamsters will not be used in this study. 2. To determine the effects of ingesting a diet containing low-melting-point or high-melting point milkfat fractions on postprandial plasma triglyceride catabolismin the rat. This objective is similar to original objective 2, except that studies will be conducted using a postprandial instead of a fasted animal model. Summary: A preliminary experiment was conducted to compare the fatty acid profile of milkfat frac tions (intact, low-melting-point and high melting-point) obtained by two different frac tionation techniques. We compared the solvent (acetone) and two-step temperature crystalliza tion techniques used respectively by Dr. Undsay and Dr. Hartel of the Department of Food Science at UW-Madison. Our analysis of the fatty acid composition of the milkfat fractions indicated that the solvent crystallization tech nique resulted in the largest differences in the saturated fatty acid profiles of the high- and low melting-point fractions (Table 1). Therefore, we proceeded to use the solvent crystallization technique to produce 35 kg each of low-melting point and high-melting-point milkfat fractions for subsequent experiments. Thiswork was done by my research specialist, Mike Grahn, in Dr. Undsay's laboratory over a period of four months. Two experiments were conducted to assess the effects associated with ingestion of milkfat fractions on plasma and hepatic cholesterol and triglyceride levels. Rats were fed diets contain ing 16% fat (wt!wt) from 16% com oil or from 2% com oil plus 14% intact milkfat, 14% low melting-point milkfat, 14% high-melting-point milkfat, or 14% palm oil for three weeks. In the first experiment, a total of 10 dietary treatments were arranged in a 5 x 2 design with respect to type of dietary fat and the presence or absence of 1% cholesterol and 0.2% cholic acid in the diets. In the second experiment, an additional dietary group was included in which the low- and high melting-point milkfat fractions were recom bined. The purpose of this additional dietary group was to assess if the response to ingestion of the milkfat fractions was related to the frac tionation technique. The results of these two experiments have addressed objective 1. We conclude that a. The response to ingestion of intact milkfat and milkfat fractions is due to differences in the fatty 97 I Center for Dairy Research I ( Table 1. Fatty acid composition ofmilkfat fractions (% butyl esters). Ltndsay: Solvent Hartel (acetone) crystallization Temperature (2-step) crystalJzation Low High Low Middle High Fatty .Acid Intact Melting Melting Melting Melting Melting 4:0 4.3 5.2 3.5 4.1 2.5 2.4 6:0 2.5 2.9 2.1 2.3 1.6 1.4 8:0 1.5 1.8 1.2 1.6 1.1 1.1 10:0 3.2 3.8 2.7 3.0 2.5 2.3 12:0 3.6 4.3 3.4 3.3 3.4 2.9 14:0 11.3 11.4 11.6 10.5 12.3 11.0 14:1 0.9 1.2 0.7 1.0 0.7 0.6 16:0 29.7 22..a .afiJl(61%)* 28.8 35.2 35.9 16:1 1.5 1.9 1.2 1.6 1.0 1.1 18:0 13.0 aa lU(89%)* 10.9 16.0 18.1 18:1 22.6 aQJ.. lB...1. (40%)" 25.4 17.0 16.3 18:2 2.5 3.3 1.7 3.4 3.7 3.1 18:3 0.5 0.7 Others 2.9 2.3 1.3 4.3 3.1 3.8 C<10 11.5 .iaz 9.J2..(31%)** 11.0 7.7 1.2 % Sat FA 69.1 60.5 71.0 64.3 74.5 75.1 ? The percent tnerease in 16:0 and 18:0 fatty acid composition of the high-melting-point fraction compared to the low-melting-point fraction. ?? The percent decrease in 18:1 and C<10 fatty acid composition of the h1gh-meltlng-point fraction compared to the low-meltmg-point fraction. add profile and/or triglyceride structure of the Experiments are currently in progress to deter milkfat samples. The differing responses cannot mine the effects of milkfat versus other dietary be attributed to the processing techniques used saturated fats on changes in plasma lipid concen to fractionate the milkfat, trations after ingestion of a meal (i.e, postpran dial changes) in rats. Previous experiments used b. There is a trend that animals fed diets con animals which were fasted prior to sampling of taining high-melting-point milkfat fractions tissues. Current evidence suggests that alter enriched in 16- and 18-earbon saturated fatty ations in postprandial lipid concentrations and adds had higher plasma cholesterol levels than impaired lipid clearance may contribute to the animals fed low-melting-point milkfat fractions accumulation of arterial lipids seen in which were enriched in oleic add (18:1) and atherogenesis. The study of postprandial short-chain fatty acids. The trend of lower changes in lipid concentrations using the rat is a cholesterol levels with ingestion of the low better model for the study of human lipid melting-point milkfat fractions appears to be metabolism than is the use of a fasted rat model. related to the removal of the saturated fatty acids Thus, this revised experimental design will provide new information regarding the relation rather than the presence and modest enrichment of short-ehain fatty acids in the low-melting ship between ingestion of dietary milkfat, point milkfat fractions. This research supports plasma lipid concentrations, and atherosclerosis. the notion that reduction of the 16- and 18 This research will also provide preliminary data carbon saturated fatty acid content of milkfat to design a human study to assess the effects will reduce the hypercholesterolemic effect associated with ingestion of different milkfat associated with ingestion of milkfat, fraction on blood lipid levels. 98 ))-----------------------11 Annual Report 1990-1991 II Si&nificanceto the Dairy Industry: The dairy industry is faced with a surplus of milldat which is projected to increase in future years. One approach to this problem is to increase the marketability of milkfat by improv ing its Ilnutritional image" and by enhancing the performance of milkfat or milkfat fractions as food ingredients. CDR is conducting research on the technology needed for fractionation of milkfat. Work is also being done to characterize the chemical, physical, and functional properties of different milkfat fractions. Our research extends and compliments work conducted at CDR by providing new informa tion regarding the nutritional effects of defined milldat fractions on lipid metabolism. This information is needed because it will help identify which components of milldat have a negative, neutral, or positive effect on lipid metabolism relative to other dietary fats. Our work is coordinated with CDR research on milkfat processing technology. Thus, if new milkfat products or food ingredients were developed, information aboutthe nutritional properties of the milkfat would be available. The American public currently demonstrates great interest in issues related to diet, nutrition, and health. In order to market dairy products to consumers, the dairy industry needs valid information about the effects of milkfat con sumption on plasma lipids. Results from this project should provide important nutrition information for the marketing of dairy products. Publications: 1. Ney, D.M. (1991) Potential for enhancing the nutritional properties of milkfat, J. Dairy Sci. (in press) 2. Ney, D.M., a-c. Lai, J.B. Lasekan, and M. Lefevre. (1991) Interrelationship of plasma triglycerides and high density lipoprotein size and composition in rats fed different dietary saturated fats, ] Nutr. (in press) 3. Lai, H.-e., J.B.Lasekan, H. Yang, M.K. Oayton, and D.M. Ney (1991), In vivo determi nation of triglyceride secretion using radioactive glycerol in rats fed different dietary saturated fats, Upids (in press) 99 I Center for Dairy Research I-------------------------t( Use of Immobilized Enzymes in the Treatment of Milldat Personnel: Charles G. Hill, Ir., professor, Depts. of Chern. Engineering and Food Science; Oyde H. Amundson, professor, Depts, of Food Science and Agricultural Engineering; Hugo S.Garcia, graduate student, Dept. of Food Science; F. Xavier Malcata, graduate student, Dept. of Chern. Engineering; Hector R. Reyes, graduate student, Dept. of Chern. Engineering Fundini and Fundina Codes; National Dairy Promotion and Research Board, 89-3; CONACYT (Mexico); INVorAN (Portugal); AT&T; Institute of Food Technologists July 1, 1989-June 30,1992 Objectives: The objective of this study is to perform a preliminary assessment of the technical feasibil ity of utilizing immobilized enzyme technology to bring about hydrolysis and interesterification reactiens to change the composition of milkfat. Specific tasks to be accomplished include: ? Develop appropriate experimental procedures for the immobilization of selected lipases; ? Accumulate kinetic data for model systems; ? Characterize in quantitative fashion relevant adsorption and rate phenomena: in particular, develop rate expressions for both the overall hydrolysis reaction and the release of each type of fatty add as functions of temperature and pH; ? Assess the economic feasibility of using immobilized enzyme technology on a commercial scale to effect hydrolysis and interesterification reactions. Sul1U1lA[yj Our experimental effort to characterize the enzymatic modification ofmilkfat has involved several components. FX, Malcata has investi gated both flat sheet and hollow fiber membrane reactors in which the membrane containing an immobilized enzyme is used to bring about the hydrolysis reaction. HS. Garda has focused on the use of an axial-annular flow reactor to study the kinetics of the hydrolysis of butteroil. H. R. Reyes has been studying interesterification reactions in a model system. Results obtained in each of these components of our program during 199Q..91 are described below. Hollow Fiber Membrane Reactor Studies The major thrust of this research component has involved technical and economic assessments of a continuous process for the production of lipolyzed butterfat using an immobilized lipase from Aspergillus niger. This enzyme is immobi lized by adsorption on microporous polypro pylene hollow fibers. Thephysical configuration of the reactor resembles a miniature shell-and tube heat exchanger. Butterfat flows through the shellside of the reactor while an aqueous buffer solution flows concurrently through the interior of the fibers. The enzyme is entrapped within the walls of the fiber. Characterization of the overall rate of hydrolysis was accomplished using ethanolic titration of the fatty adds released. Measurements of the rates of release of the individual fatty add residues (i.e., C4:0, C6:0, C8:O, CI0:0, C12:0, C14:O, C16:O, CI8:0, CI8:1, and CI8:2) are based on HPLC analyses. Several rate expressions based on proposed mechanisms were assessed for their ability to fit the data. The best fit rate expression for the lipase-cata lyzed hydrolysis ofbutterfat corresponds to (0 a MichaiHis-Menten mechanism with competitive inhibition by a product of the reaction, (ii) a 100 )1---------------------11 Annual Repent 1990-1991 II Gaussian distribution (approximately centered at CI0:0) for v"'"" as a function of the length of the hydrocarbon backbone (with an additive correc tion to account for the number of double bonds), (iii) constant values for the Michaelis and inhibition constants and (iv) the existence of two interconvertible active forms of lipase, one of which is subject to irreversible deactivation. Axial-Annular Flow Membrane Reactor Studies The major thrust of this research component has involved characterization of the performance of this reactor in quantitative terms. Characterization of the adsorption of a lipase from Candida cylindTtlUll on a microporous polypropylene membrane indicates that physical adsorption of this enzyme can be described by the classical Langmuir model. For temperatures in the range 25--45?C, the amount of protein adsorbed on the membrane reaches a maximum of 15 mgtcm1. When butteroil is used as a substrate, 45?C is the minimum feasible operating temperature because of problems arising from partial crystal lization of high-melting fractions of the milkfat, even at 400C. At 45?C the half-life of the enzyme is estimated to be ca. 4.5 days. Enzymatic activity was observed when water was suspended and!or dissolved in plain butteroil. This result suggests that for hydropho bic binding (immobilization by adsorption) of the enzyme to the polymeric support, the active site remains exposed. Hence, the usual two phase emulsion system is not required to obtain hydrolytic activity. At very low water concentrations (ca. 1%), water itself becomes the limiting reagent, achieving nearly complete conversion at a reactor space time of two hours. As the water content of the substrate emulsion was increased (but below 10% water), greater production of free fatty acids was observed. Above this water concentration, reaction rates decreased dramatically, probably due to a change in the hydrophobicity of the local environment of the enzyme. Overall, very high reaction rates were obtained with the axial-annular reactor. Space times of only four hours were required to reach the same levels of conversion that would require ca. one day in a batch reactor employing a soluble enzyme dispersed in a conventional emulsion. Studies of Interesterification Reactions This component of the research focused on interesterification reactions catalyzed by an immobilized lipase supported on solid particles suspended in a liquid mixture of octanoic acid and an oil. In these studies olive oil was used initially as a model substrate. Subsequent work employed butteroil as the substrate. For the same feedstock and operating conditions, lipase immobilized on polypropylene powder gave higher rates of reaction than either free lipase or lipase immobilized on Celite. At low water concentrations, the rate of interesterification increased with increasing concentration of water. A larger initial concen tration of water accelerates the rate of hydrolysis at the expense of a greater accumulation of by products (lower glycerides). A larger ratio of octanoic add to olive oil increases the rate of interesterification and the extent of incorporation of octanoic add into the glycerides of olive oil. Observed interesterification reactions of milkfat and octanoic.acid were slower than the corre sponding reactions with olive oil. The interesterification reaction of milkfat and lino lenic acid proceeded at a faster rate than that of milkfat and octanoic acid (presumably because of the selectivity of the lipase toward long-chain fatty acids). Significance to the Dairy Industry: To date our research efforts have demonstrated the technical feasibility of utilizing immobilized lipase technology to produce products with modified milkfat compositions. The differences in composition resulting from hydrolysis and interesterification reactions have important implications with respect to nutrition, flavor generation, and potential anticancer activity. The use of this technology offers the intriguing possibility of being able to tailor-make products for selected segments of the population, in particular those individuals who are especially health conscious from a dietary standpoint or are high risk candidates for cardiovascular or other health problems. 101 I Center for Dili." Research 1~-----------------------lC Utilization of an immobilized lipase reactor in the flavor industry would lead to continuous production of lipolyzed butteroil, a primary ingredient used for preparation of dairy flavors. A process utilizing the immobilized lipase should offer significant practical advantages over the traditional process employing a soluble lipase. These advantages include facilitation of a shift to continuous as opposed to batch produc tion and provision of better control, both for the process and the quality of the product. This technology can be used in the controlled devel opment of flavor in processing feedstocks for the production of certain typesof specialty cheeses, e.g., blue and Italian. Since the products result ing from the use of this technology contain little free water, savings associated with concentration or dehydration of the hydrolyzate can be Signifi cant. Moreover, the enzyme does not need to be thermally inactivated once the feedstock has . beenconverted to the desired extent. The hollow fiber and axial annular reactors offer intriguing possibilities for commercial develop ment. Preliminary economic assessments indi cate that they will provide commerdally viable alternatives to conventional technology for the production of lipolyzed butteroils. Publications: Malcata, F. X, H. R. Reyes, H. S. Garcia, e.G. Hill, and e.H. Amundson (1990). Immobilized lipase reactors for modification of fats and oils a review. J. Am. Oil Chern. Soc. 67(12), 890-910 Garcia, H 5., H. R. Reyes, F. X. Malcata, e.G. Hill, and e. H. Amundson ?1990). Determina tion of the major free fatty acids in milkfat using a three-component mobile phase for HPLC analysis. Milchwiss. 45(12), 757-759 Garcia, H.5., e.G. Hill, and C.H. Amundson. 1991. Partial characterization of the action of an A. niger lipase on butteroil emulsions. Accepted for publication in J. Food Sci. Malcata, F. X., e. G. Hill, and e. H. Amundson. Use of a lipase immobilized in a membrane reactor to hydrolyze the glycerides of butteroil. Accepted for publication in Biotechnol. Bioeng. Malcata, F. X., H. S. Garcia, e. G. Hill, and e. H. Amundson. Hydrolysis of butteroil by immobi lized lipase using a hollow-fiber reactor. Part I: Adsorption studies. Submitted to Biotechnol. Bioeng. Malcata, F.X.,e.G. Hill, and C H. Amundson. Hydrolysis of butteroil by immobilized lipase using a hollow-fiber reactor. Part II: Uniresponse kinetic studies. Submitted to Biotechnol. Bioeng. Malcata, F.X.,e.G. Hill, and C H. Amundson. Hydrolysis of butteroil by immobilized lipase using a hollow-fiber reactor. Part ill: Multiresponse kinetic studies. Submitted to Biotechnol. Bioeng. Malcata, F. X, e.G. Hill, and C H. Amundson. Hydrolysis of butteroil by immobilized lipase using a hollow-fiber reactor. Part IV: Effects of temperature. Submitted to Biotechnol. Bioeng. Malcata, F. X., H.R. Reyes, H.5. Garcia, e.G. Hill, and e.H. Amundson. The kinetics and mecha nisms of reactions catalyzed by immobilized lipases. Submitted to Enz: Microb. Technol. Garcia, H. S., F. X. Malcata, C G. Hill, and C H. Amundson. Use of Candida rugosa lipase immo bilized in a spiral wound membrane reactor for the hydrolysis of milkfat. Submitted to Enz: Microb. Technol. Presentations: Garcia, H.5., CH Amundson, and CG. Hill, Jr. "Partial characterization of the action of an A. niger lipase on butteroil emulsions," paper presented at the 1990 meeting of the Institute of Food Technologists. Reyes, HR., CG. Hill, [r., and CH Amundson. "Kinetics of the interesterification reactions of olive oil in the presence of lipase from Pseudo monas cepacia immobilized on a hydrophobic support," paper presented at the 1990 AIChE meeting. Malcata, rx, CG. Hill, Jr., and CH Amundson. "Hydrolysis of butterfat using a membrane reactor," paper presented at the 1990 Annual Meeting of the Institute of Food Technologists. Malcata, F.X., CG. Hill, Jr., and CH. Amundson. "Enzymatic hydrolysis of butteroil using a hollow fiber reactor," paper presented at the 1990 AIChE meeting. 102 Annual Report 1990-1991 Malcata, F'x', e.G. Hill, [r., and e.R Amundson. "Kinetic studies of the hydrolysis of butterfat catalyzed by an immobilized lipase," paper presented at the 1991annual meeting of the American Oil Chemists' Society. Garcia, HS., C.G. Hill,[r., and eH. Amundson. "Hydrolysis of butteroil with C. cylindracetllipase immobilized on microporous polypropylene," paper presented at the 1991 Annual Meeting of the Institute of Food Technologists. Malcata, F.X.,H.S. Garcia, e.G. Hill, [r., and C.R Amundson. "Adsorption of A. niger lipase on a microporous polypropylene membrane for hydrolysis of butteroil," paper presented at the 1991 Annual Meeting of the Institute of Food Technologists. 103 ,L....- -(( I Center for Dairy Research Modification of Butterfat by Lipolytic Reactions in Novel Reaction System Personnel: J.P. Chen, assistant scientist, CDR; H. Pai, graduate student, Dept. of Food Science; B.K.Yang, graduate student, Dept. of Food Science Fundina; and Fundina; Codes: Wisconsin Milk Marketing Board: Basic Research, 133-P644 July 1, 1990-June 30, 1991 Objectives: To study the reaction kinetics and selectivity of lipolytic reactions on milkfat with microbial lipases entrapped in lecithin reversed micelles and synthetic gel matrices Summary: We have carried out enzymatic hydrolysis of milkfat with Candidtz cylindracea lipase ina homogeneous microemulsion containing water, oil, and a surfactant (soybean lecithin). The oil phase was butteroil or butteroil in isooctane, Theoptimum reaction temperature was at 55"C and the optimum molar ratio of water to surfac tant (R value) was 11 for both systems. Upase showed enhanced thermal stability in reversed micelles at R=6.7 compared to in buffer. Re versed micelles with entrapped lipase can be retained with ultrafiltration membranes where short-chain fatty adds were concentrated as well. The profile of the freefatty adds liberated in the reaction was influenced by the reaction tempera ture, enzyme concentration, and the water content. Thebest reaction conditions for gener ating short-ehain fatty adds were at R=18.7,400 mg/ml enzyme concentration, 45"C, and longer reaction time. The molar percentage of short chain fatty adds (butyric to capric adds) was increased from 6% for freeenzyme system to 17% for lipase in reversed micelles. In a different system, Candidtz cylindracea or Rhizopus arrhizus lipase was entrapped in polymer gels prepared from photo-cross-linkable resin prepolymers. The immobilized biocatalysts were used in milkfat hydrolysis and were effective in enhandng the flavor of the lipolyzed product. The molar percentage of short-chain fatty acids liberated in the reaction increased significantly after enzyme immobiliza tion, with the high molecular weight hydropho bic resin (ENTP-4000) yielding the best perfor mance (molar percentage increased 7.6 fold compared to free enzyme system). This may be due to different diffusion rates of milkfat triglyc erides into the gel. Changes in reaction condi tions such as low reaction temperature, low bulk substrate concentration, high enzyme concentra tion in the gel, and long reaction time could promote flavor development in this system. We are currently studying glycerolysis in these systems, with the goal of produdng mono- or diglycerides from milkfat, which are valuable surfactants useful in various food applications. Significance to the Dairy Industry: Current methods of controlling the free fatty acid profile of lipolyzed milkfat rely solely on the specificity of the enzyme used, which is limited by the commercial availability of an effective lipase at a reasonable cost. The immobilized lipase biocatalysis developed in this project can help to solve this problem by offering a conve nient means for controlling flavor development of lipolyzed milkfat. The immobilized enzyme can also be used in a continuous bioreactor to cut down the processing cost. Publications: Chen, J.P. and H. Pai (1991). Hydrolysis of milkfat with Upase in Reversed Micelles. J. Food Sci. 56, 234-237 Presentations: Pai, H. and J.P. Chen. "Enzymatic hydrolysis of milkfat in reversed micelle systems." Presented at 1990 annual meeting, American Institute of Chemical Engineers, Chicago, IL, Nov. 1990. Pai, H. and J.P. Chen. "Controlled lipolysis of milkfat in lecithin microemulsions." Presented at 52nd annual meeting, Institute of Food Tech nologists, DalJas, TX, June, 1991. Yang, B.K. and J.P. Chen. "Production of Dairy Flavor by controlled hydrolysis of milkfat with gel-entrapped microbial lipase." Presented at Biocatalysis for the 90s conference, Lake Buena Vista, FL, June, 1991. 104 ))...----------------1 AnnUli' Report 1990-1991 II Interesterification of Butterfat with Gel-entrapped Cells Personnel: J.P. Chen, assistant scientist, CDR; B.K.Yang, graduate student, Dept. of Food Science; Dale Mc.<;ill, research specialist, CDR Funding and Funding Codes: National Dairy Promotion and Research Board, CHN9Q..1 July 1, 1990-June 30,1993 Objectives: To study the production of intracellular lipase from fungus cells and the use of immobilized cells with bound lipase activity in lipolytic reactions of millcfal To determine the influence of gel hydrophobicity on the reaction rates and specificity of lipolytic . reactions with butterfat triglycerides. Summary: We first investigated the production of intracel lular lipase from Rhi:zopus delemar. The best medium composition for production of intracel lular lipase by Rhi:zopus delenul1' contained oleic acid and no glucose. The technique of cell immobilization using polyurethane foam bio mass support particles was studied as a means of further enhancing intracellular lipase activity. Fungal mycelia were found to strongly adhere to the polyurethane foam matrix during growth to form immobilized cell biocatalysts, which, after cell membrane permeabilization and drying, exhibited a lipase activity 33 times greater than that of cells grown in a freely suspended state. Upase within the immobilized cells showed properties similar to purified extracellular Rhizopus delem.ar lipase but was more thermo stable than the free enzyme. The biocatalyst was used for triglyceride hydrolysis in a continuous stirred-tank reactor. In this format it was found that the flavor profile of lipolyzed milkfat could be easily controlled. To study the effects of gel properties on lipase activity, we entrapped microbiallipases in homologous gels prepared from photo-cross linkable resin prepolymers which have hydro phobic (ENTP) or hydrophilic (ENT) characteris tics with varying chain length. The influence of supporting materials on substrate specificity was studied by measuring the hydrolysis activity with tributyrin or olive oil as the substrate. Hydrophobic gels with the longest chain length generally gave the highest activity yield. The ratio of activity on tributyrin to olive oil changed dramatically from 0.3 for free enzymes to 14.8 for C. cylindraaa lipase in ENTP-200 gel and to 16.3 for R. arrhizus lipase in ENTP-4000 gel. This substrate specificity was pH and temperature dependent and the ratio of reaction rate canbe increased to 35 at 20?C or pH 8.5. This high selectivity exceeds the substrate selectivity of almost all commercially available lipases. Significance to the Dairy Industry: To cut down the cost associated with immobi lized lipase preparations, using whole cells with their intracellular lipase immobilized in situ is a reasonable way in minimizing the cost associ ated with enzyme purification. We have discov ered a way to enhance the intracellular lipase production by R. arrhizus, which will make the immobilized cells approach more attractive. Furthermore, entrapping fungus cells in a synthetic gel can protect the cells from damage by the mechanical forces when theyare used in a bioreactor. The gel also provides a way to control the reaction rates and specificity in milkfat interesterifications based on the ability to control gelhydrophobicity. This work has the potential to provide an economical system for carrying out controlled interesterifications of milkfat and to produce useful products for food and non-food applications. Publications: Chen, J.P. and S.D. Mc.<;ill. Enzymatic hydroly sis of triglycerides by Rhizopus delemar immobi lized on Biomass Support Particles. Food Biotech nology, in press. Presentations: Yang, B.K.and J.P. Chen. "Influence of the gel matrix on the hydrolysis of triglycerides with immobilized lipase." Presented at 52nd annual meeting, Institute of Food Technologists, Dallas, TX,June, 1991. 105 I Centn for Did,., Research Ii--------------------------\( Enzymic Modification of Butterfat in Supercritical CO 2 Personnel: Richard W. Hartel, associate professor, Dept. of Food Science (co-PI); Dr. Kirk L. Parkin, associate professor, Dept of Food Science (co-PI); Janice M. Johnson, graduate student; Michelle Mani, graduate student, Dept. of Food Science Fundins and Fundinz Codes: National Dairy Promotion and Research Board, 88-1;Wisconsin Milk Marketing Board, 88-40 July 1,1988 to June 30, 1991 (no-cost extension requested to October 31, 1991) . Objectives: 1. Assess the feasibility of using supercritical (SO fluids as a means to control lipase action on butterfat. 2. Determine the specificity of lipase action on butterfat in SC CO 2 as influenced by the reactor operating temperature and pressure, as well as the level of an aqueous entrainer. 3. Develop a fundamental understanding of how lipase action on butterfat in SC cqmay be controlled by determining the reaction kinetics and specificities under various processing conditions. Summary: The objectives of this project are to combine fractionation of butterfat using supercritical (SC) CO 2 with enzymic modification (hydrolysis or acyl-exchange) of these fractions to produce specific butterfat fractions for use as food ingredients. Currently, research is focused on enzymatic hydrolysis of model triacylglyceride (TAG) systems underSC cqconditions, acyl-exchange reactions in anhydrous media at ambient conditions and under supercritical CO 2 conditions. Enzymatic hydrolysis of model TAG systems is being studied underSCcqconditions to determine rate and extent of enzymic hydrolysis. Experiments performed in a batch reaction vessel at 40<><: and 4,500 psi with CO 2 saturated with water at these conditions showed that both tricaprin and tripalmitin were hydrolyzed by lipase (crude porcine pancreatiC> under these conditions. After three hours at these conditions, 11.9%of the palmitic add in the original tripalmitin was hydrolyzed. Our work is continuing in this area with plans to study different TAG's, vary water content in the SC cqreactor and to vary the reactor pressure and temperature to determine rates of hydrolysis. We have continued our studies on the fatty acyl substrate preference for pancreatic lipase cata lyzed acyl-exchange reactions in anhydrous butteroil. Previous data indicated that free fatty add levels above 250 mM were inhibitory to this reaction. Fatty acyl groups were used in the form of TAG, free fatty adds (FFA) and fatty add methyl esters (FAME). No substrate inhibition was observed for fatty acyl groups esterified as methyl and glycerol esters at levels up to 1 M. At 50 mM acyl group, there was no inhibition using free fatty adds as substrates and the enzyme activities on the various fonns of substrates were similar at this level. At 500 mM, the relative activities were>15:9:1 for TAG:FAME:FFA as substrates, respectively. This information on substrate preference for acyl-exchange reaction with butteroU in anhy drous media will be adapted. to studies on this reaction in SC CO 2 , Initial studies have been done in SC CO 2 using butteroil and tricapryIin as acyl donor substrate using Upozyme TN as the catalyst. TricapryIin and butteroil were added at a ratio of acyl groups of about 1:4. Conditions of reaction were six hours at SOOC and 5,000 psi, with no mechani cal mixing of reactor contents. The control of water content in the reaction appears to be important in regulating enzyme action under these conditions. Almost no activity is observed ifno water is added to the reactor, whereas substantial activity is obtained when water is 106 )1-------------------11 Annual Report 1.990-1.991. II introduced into the reaction vessel. Under these conditions, the amount of tricaprylin reacting with butteroil was 35-50%. A change in reaction specificity was observed in SC CO 2 compared to the reaction in anhydrous butteroil at SOoC and atmospheric pressure. The relative specificity for the release of fatty acids from butteroil is en hanced in the SC CO 2 reactor by factors of 1.28 for caprylic, 1.19for capric, 1.39 for lauric, 2.33 for myristic, 1.54for stearic, and 1.43 for oleic acids, compared to the atmospheric reactor. The relative rates for reactivity toward butyric, caproic, and palmitic acids were 0.44,0.76, and 0.76 respectively in the SC C02' compared to the atmospheric reactor. Based on this data, it appears that SC CO 2 offers an added dimension of control over reaction specificity that can be obtained under normal (atmospheric) anhydrous reaction conditions. The nature of this selectivity is under continuing evaluation. Understanding the mechanism of selectivity would allow for a systematic design of reaction conditions to yield butteroil derivatives with unique properties. SiBJUficance to the Dairy Industry: The modification of butterfat through a combina tion of extraction in SC CO 2 and enzymic reac tion has the potential for producing tailor-made fat fractions for use as food ingredients. If successful, this technology will allow modifica tion of butterfat to aid in reduction of excess butterfat supplies by providing fractions that are designed for specific food applications. Our studies involve determining the feasibility of performing these enzymic reactions under SC cq conditions, determining the rates and extents of these reactions and comparing the specificity of reactions under these conditions with those obtained under atmospheric condi tions. 107 I Center for Dairy Resea.rch ]'-----------------------(( Demand Analysis of Dairy Products Using Cross-sectional Data Personnel: Brian W. Gould, associate scientist, CDR; Eric Kettner, graduate student, Dept. of Ag. Economics Fundin; and Fundin; Codes: Wisconsin Milk Marketing Board: Basic Research, 133-P583 ContinUing Objectives: 1. Obtain and make accessible the 1965,1977, and 1987 USDA Food Consumption Surveys; 2. Fonnulate a dynamic model of dairy product demand using the above surveys and incorporat ing various socio-economic factors in a demand systems framework; and 3. Quantify the impacts of variables that are most important in explaining individual intakes of selected nutrients, and determine the chang ing role of dairy products in the intake of these nutrients. The emphasis is on a disaggregated list ofdairy products as well as other food aggregates so as to cover the total food budget. Sununary: To analyze the household level demand for dairy products we continued with our use of time series data to refine the methodologies that will be used in the cross-sectional analysis. The demand for butter was analyzed using aggregate quarterly disappearance data for the years 1962 1987. Five food fats and oils commodities were delineated in the demand system: butter, vegetable shortenings, cooking and salad oils, margarine, and lard. The results of this research have been published in the American Journal of Agricultural Economics. The demographic variables included in the analysis were percent of the population under the age of 5 years, percent of the population between the ages of 5 and 13, percent of the population over the age of 65, the median number of years of schooling completed, and the percent of the population that is nonwhite. We analyzed the impacts of changes in the demographic profile of the U.S. population on the intake and type of dietary fat obtained directly from food fats and oils. Due to delays in the release of the 1987 Household Food Consumption Survey, we used the 1987 Bureau of Labor Statistics, Consumer Expenditure Survey in an analysis of the at-home consump tion of cheese. We estimated an econometric model that examines the two stage purchase decision making process, Le., decision to pur chase and the level of purchase, within an econometric model that allows for the separation of these two stages. More than 5,000 households across the US. are used in this analysis in which such factors as ethnicity, income, and thecompo sition of the household in tenns of the age and sex of the household members are used as explanatory variables that separately affect the decision of a household to purchase cheese and the level of expenditure given the decision to purchase. The unique aspects of this research is that with the econometric model as specified, we can answer questions as to the differing signifi cance of the explanatory variables in the two stages of the purchase process. The research paper generated out of this effort has been accepted for publication in the Amerialn Journal ofAgricultural Economics. A third research effort concerned with the analysis of dairy product demand was under taken with the cooperation of Professor Robin Douthitt, associate professor in the Department of Consumer Sciences, University of Wisconsin Madison. In this research we investigate the role of the household's age/sex composition on the demand for specific types of cheese: all cheese, natural Cheddar, American processed, Italian, eyed, and cream cheese. The data used in this analysis consisted of household level dairy product diary data obtained from the Market Research Corporation of America. More than 4,700 households were 108 II ))-----------------------1 Annual Report 1990-1991 used in this analysis. Actual cheese quantities purchased as well as calculated prices, demo graphic characteristics, and household age/sex composition variables were used as explanatory variables. Similar to previous research we assume a two stage cheese purchase process which allows for differentiation of the whether-to-purchase and the amount-to-purchase decisions. Household ethnicity significantly impacts purchase prob ability while its impact on the level of consump tion was negligible. Income and household composition effects also varied by consumption stage and types of cheese analyzed. The price elastidties estimated in this analysis show that changes in cheese price consistently affect the consumption of current consumers more than potential new purchasers. These results have important implications for cheese manufacturers . attempting to expand the consumption of their products. A research paper generated out of this research has beensubmitted for publication in the American Journal ofAgricultural Economics. Si&Jlificance to the Dairy Industry: Public concern with health implications of dietary fat intake has become a major factor affecting the consumption of dairy products. The consumption of lowfat milk, for example, has increased to the point where after 1987 the per capita consumption of reduced fat milks has exceeded the consumption of whole milk. Butter consumption hasalso declined relative to the consumption of vegetable basedoils. In contrast to these trends, the consumption of cheese, both at-home and away-from-home, has beenincreas ing. This project will attempt to answer the question as to where future demand for dairy products will be and what are the factors affect ing such demand. Publications: B.W.Gould, T.L. Cox, and F. Perali (1991). Demand for Food Fats and Oils: The Role of Demographic Variables and Government Donations. American Journal ofAgricultural Economics, Vol. 73(1):212-221 B.W.Gould (1992). At-Home Consumption of Cheese: An Analysis Using a Purchase Infre quency Model, forthcoming in American Journal ofAgricultural Economics, Vol. 74(1) B.W.Gould and R.A. Douthitt (1991). The Impact of Household Composition on the Demand for Cheese, submitted for publication in American Journal ofAgricultural Economics Presentations: B.W.Gould, "An Overview of the Availability of Household Level Data for Dairy Product De mand Analysis," paper presented at the 1991 annual meeting of the American Cultured Dairy Products Institute, Charleston, S.c., March. 109 I Center for Dairy Research 1-1-------------------------( Development and Maintenance of Dairy Product Related Database Personnel: Brian W. Gould, associate scientist, CDR and Dept. of Ag. Economics; Jeremy Foltz, graduate student, Dept. of Ag. Economics Fundins and FUndini Codes Wisconsin Milk Marketing Board: Basic Research, 133-R126 Continuing Objectives: 1. Develop a "stand alone" database system for the collection of dairy product time series data. 2. Initiate the collection and cataloging of appropriate time series data. 3. Develop a user's manual to accompany the software. Summcuy: This project involved the development of a database system that allows for easyinput and retrieval of historical dairy product data useful for program and policy analysis. Using the database system, WISCMIS, as a starting point, the database software developed in this project greatly improves upon the previous system in terms of its ability to add, delete, and manipulate aggregate daily product data. The program has been written using the QUICKSILVER database program and has been compiled so it can run without the need to purchase this software. It is designed to allow the user to enter, edit, and manipulate dairy product data files with differ ent structures. Extensive search capabilities have been incorporated into the model. SiiJUficance to the Dairy Industry: The software developed as a result of this project will allow for an analysis of the market for a variety of dairy products via the use of aggregate tiIne-series data. Given the nature of this program, the user will be able to run this com puter model "in the background" and with a few keystrokesbe able to seeand!or print file(s} coveringa variety of dairy product related topics. 110 )r----------------------I Annual Report 1990-1991 II Development of a Wisconsin Input-output Model for Dairy Product Industry Analysis Personnel: Brian W. Gould, associate scientist, CDR and Dept. of Ag. Economics Fundin~ and Fundin~Codes: Wisconsin Milk Marketing Board: Basic Research, 133-R125 Continuing Objectives: 1. Develop an input-output model of the state of . Wisconsin with a detailed dairy processing sector; 2. Develop the necessary software for use of the input-output model on a microcomputer; and 3. Estimate impact multipliers of changes in the final demand for dairy products. Summary: This project required the use of an input-output (1-0) model of the state of Wisconsin in which county level data is available. As is the case for all input-output models, the Wisconsin 1-0 model is a demand driven system of equations where a change in final demand - exports (domestic or foreign), household demand, new business investment, and government current and capital expenses- provide an economic stimulus in the economy which eventually results in a new equilibrium. The new equilib rium provides information as to the level of economic output as well as payments to primary inputs or value-added sectors. This project uses the Wisconsin 1-0 model to estimate various types of multipliers resulting from the activity of the dairy processing sector. These multipliers include income, employment, value-added, output, and import multipliers and provide an indication as to the impacts on income, employment, etc. to a change in final demand. Because of the late release of new software and data, the most recent Wisconsin input-output model could not be purchased until late May, 1991. As such, work on this project has been just started. We antidpate little delay in obtaining the objectives indicated above, and a final dairy industry 1-0 model should be available by the end of 1991. Sianificance to the Dairy Industry: This research will provide information as to the macro-economic impacts of changes in the dairy processing industry on the state of Wisconsin. This project will result in a tool that will be able to answer such questions as the employment impacts from increased exports of Wisconsin diary products and the additional product value resulting from this increased export demand. Such information is essential for the Wisconsin dairy products industry for planning, and to demonstrate the industry's importance to the state's economy when requesting public policy changes that will benefit the state's dairy prod uct industry. 111 I Center/orDairy Research I------------------------I( 112 Annual Report 1.990-1.991. Milk Component Utilization Progress Reports Part II. Nonfat Solids 0Nhey) Profitable use of cheese whey and its constituents continues to be the focus for non-fat milk component utilization research. Limited but specific whey protein projects have been selected to complement projects at other research institutions. Projects on utilization of non protein components, primarily lactose, evolved from previous economic analyses of potential products or from direct industry input. Cameron updates his work highlighting the development of two microbial polysaccharide gums produced by fermentation of whey permeate with selected organisms. Interest by commercial firms prompted pilot-plant scale production of the two polysaccharides. Evaluation of the samples hasbeen or is presently being performed by industrial firms. In another whey fermentation project, Steele's group is proceeding with its genetic screening of their lActobacillus helveticus CNRZ 32 gene bank in order to construct an organism able to produce L-lactic add from whole sweet whey. This research was initiated because commercial firms were seeking bacterial strains which would produce only L-Iacticacid. Lowfat whey protein isolate production is reported by Chen, who is developing a liquid liquid extraction process for the removal of fat from whole sweet whey. Damodaran's project to investigate the effects of protein and non-protein components on the thermal gelation of whey protein concentrates just got underway. Although none of these projects will use all the excess whey and whey permeate being produced, they have the potential to incrementally utilize some of the excess through production of value-added products. 113 I- I Center for Dairy Research Removal of Lipids from Cheese Whey Personnel: J.P. Chen, assistant scientist, CDR; Dale McGill, research specialist, CDR FundinS and FundinS Codes; Wisconsin Milk Marketing Board: Basic Research, 133-R127 July I, 199().June 30, 1991 Objectives: To develop an efficient and economical process for the removal of fat from cheese whey using liquid-liquid extraction. To determine phase compositions, the partitions of individual whey proteins in aqueous two phase systems, and the influence of system parameters on the partitions. Summary: We continued the previous study which used aqueous two-phase systems containinga poly mer and a salt. Lipid removal was very efficient in this system according to total lipid analysis, where values close to zero were obtained for the clarified whey protein phase. Sodium citrate has been tried as an alternative salt for forming the phase since this salt is biodegradable, creating less of a waste disposal problem than the potas sium phosphate used previously. Lipids could still be efficiently removed from whey in this system, but the recovery of whey proteins needs to be optimized to increase the yield of this process. The location of lipids can be confined to a region close to the interface in the bottom phase when a low molecular weight PEG is chosen. This will have the advantage of increas ing the recovery of whey proteins and minimiz ing the loss of chemicals. ----i( We also measured the partition coefficients of individual whey proteins (a-lactalbumin and ~ lactoglobulin, bovine serum albumin, immuno globulin G, lactoferrin) in the two-phase sys tems. In general, a-lactalbumin and ~lacto? globulin showed different preference for the phases, depending on the phase compositions and processing conditions (polymer molecular weight, polymer concentration, salt concentra tion, pH, the addition of NaG). Under the best conditions, 100% pure p-Iactoglobulin could be obtained in the bottom phase while 80% pure a lactalbumin could be obtained in the top phase. The aqueous two-phase system may be used to produce whey protein concentrates enriched in one of the major whey proteins simultaneously during lipid removal, or may be used separately for whey protein fractionation. Significance to the Dairy IndustJ:y: The major limitations for increased utilization of whey proteins are the cost of production of low fat, low-lactose whey protein isolates, off-flavor, and poor functional properties of whey protein products that are available in the market. One of the rudimentary causes of these problems can be traced:back to the residual fat in cheese whey. In view of the fact that current methods of remov ing fat from cheese whey have either been associated with high protein loss or involve high additional processing cost, the new process developed in this study may provide a more efficient means to solve the lipids removal problem. 114 II )r---------------------I Annual Report 1990-1991 Effect of Protein and Non-protein Components on Thermal Gelation of Whey Protein Concentrates Personnel: Srinivasan Damodaran, associate professor, Dept. of Food Science; Balaji Krishnaswamy, graduate student, Dept. of Food Science Fundini: and Fundin~ Codes: National Dairy Promotion and Research Board, SRN90-1 July 1, 1990-June 30, 1993 Objectives: 1. To elucidate the effects of various non-protein components (minerals, lactose, etc.) of WPC on the gelling properties of WPC. 2. To elucidate the influence of the relative amounts of ~lactoglobulin and a-lactalbumin on the gelling properties of WPC. SuIlUT\Cll"$ A graduate student was appointed on Sept. 1, 1990 as research assistant for this project. Cur rently, we are working on optimizing experi mental conditions for studying whey protein gelation. Once this is done, we will be systemati cally studying the various factors that affect gelation of WPC. Siamificance to the Dairy Industry: Gelation is a functional property of a food protein that has major applications in formulated food products. This research will provide better understanding of the gelation characteristics of whey proteins in the presence of various whey components. The research is expected to gener ate a database on the gelling properties of WPCS, which will be highly useful to food industries. Using this database, specific WPC formulations canbe manufactured for specific food applica tions. This will enhance the utilization of whey protein in formulated foods. 115 I CenUr for Dairy Raearch 1~-----------------------tC Conversion of Whey Components to Commercially Valuable Products Personnel: Douglas C. Cameron, assistant professor, Dept. ofChem. Engineer ing; EN. Lightfoot, professor, Dept. of Chern. Engineering; Timothy A. Cooper and Dina Dogger, graduate students, Dept. ofChern, Engineer ing; Stacey Tiedemann, undergrad uate hourly; Marianella Salapata, hourly graduate student. fundinG and FundinG Codes: Wisconsin Milk Marketing Board, 88-53 July 1988-April30, 1992 (Extended) Objectives: 1. Develop a database of products that can be derived from lactose and whey minerals. 2. Identify products for which whey compo nents are the preferred substrates. 3. Perform an economicevaluation to identify the most promising products for further development. 4. Develop technologically feasible laboratory scale processes for the production of selected products. 5. Make recommendations for further develop ment and commercialization of the laboratory processes. 6. Addition: Conduct pilot-scale fermentations using the two most promising polysaccharide producing organisms in order to 1) produce sufficient quantities of both polysaccharides for actual industrial testing 2) examine the technical feasibility of commercializing the production of polysac charides from whey permeate. Summary: The success of our research in the area of micro bial gum production from whey permeate has led to the need for an economical evaluation of our two most promisinggums. These two gums, zooglan and an anionic galactornannan which we call Lactan?, show excellent potential in becoming marketable products to the dairy industry as well as other industries. Therefore, we performed a preliminary economic analysis of the production of these gums from whey permeate. A more thorough analysis will follow. This analysis took into consideration such variables as capital investment, operating costs, fermentation costs, and product recoveryand purification. A pretax return on investment of 50% was used to determine the minimum selling price. The analysis revealed that the cost of fermentation is the single largest contributor to overall costs. The analysis also showed that even modest increases in gum productivity greatly improve the feasibility of the gum production process. For the Lactan? gum, a minimum selling price of $B.30/kg could gener ate a pretax return on investment of 50%. This price is at the low end of the microbial polysac charide scale, indicating a realistic chance that the actual selling price of the polysaccharide would enable the process to have a sufficient rate of return to interest investors. The minimum selling price for zooglan was$15.36/kg. Research is progressing on our metal-binding experiments involving zooglan. Previous researchers have shown that zooglan, due to its anionic nature, reversibly binds toxic metals. However, it has been difficult to implement a practical metal binding procedure with zooglan owing to the difficulty in recovering the gum after metal has been released. We have begun work on a novel technique that allows the gum to be easily recovered and reused. Initial results are promising. This technique will broaden the number of end-uses for zooglan and increase its 116 )1----------------------1 Amaual Report 1990-1991 I potential for commercial production. A patent describing improved recovery procedures for zooglan as well as broader uses for the gum was submitted to the patent office in September. . During October through December we carried out eight pilot-scale polysaccharide fermenta tions in the UW-Biochemistry Pilot Plant. The purpose of these fermentations was to obtain large quantities of zooglan and LactanTM for application testing in our laboratory and for distribution to industrial polysaccharide users for testing. A secondary purpose was to gain experience in the large-scale production and purification of microbial polysaccharides. Such experience will help us to identify potential difficulties in transferring this technology to industry. Seven of the eight pilot-scale fermentations used whey permeate which we prepared from fresh commercial whey using an APV ultrafiltration system provided by the Center for Dairy Re search (synthetic whey was used for the first fermentation). For Lactan?, two 80liter fermen tations and two 300 liter fermentations were performed. Over 6 kg of Lactan? was recovered and purified. For the zooglan, three 300 liter and one 80liter fermentation were performed. Approximately 5 kg of zooglan was obtained. Zooglan produced from permeate is able to retain greater thickening efficiency when ex posed to salts and acids than zooglan produced from a defined medium. This surprising yet positive result further enhances the appeal of whey permeate as a fermentation substrate. Si&J1ificance to the Dairy Industry: The purpose of this project is to develop eco nomical new products from whey permeate. We have developed two new microbial gumsthat are produced with good yields and productivities from whey permeate. Microbial gums have numerous industrial and food applications, including uses in dairy products such as cottage cheese and cream cheese. There fore, widespread implementation of gum production from whey permeate could serve two very important functions to the dairy industry of Wisconsin: 1) It could utilize a large percentage of the whey that is presently disposed of, thus reducing the burden and cost of whey disposal, and possibly add value to the whey stream in general. 2} It could benefit the dairy industry directly by locally producing gums that are needed in other dairy products. Our preliminary economic analysis reveals that gum production from whey is likely to be economically feasible. Pyblications: 1. JR. Flatt, 1990. Microbial Production of Novel Polysaccharides from Lactose in Whey Permeate. Ph.D. Thesis, Department of Chemi cal Engineering, University of Wisconsin, Madison, WI. Patent Applications Submitted: Hardin, R.S.,J.H. Flatt and D.C. Cameron, 1990. Polysaccharide producing organism and novel galactomannan polysaccharide. U.S. patent submitted May 1990. J.H. Flatt and T.A. Cooper, 1990. Zooglan Polysaccharide. U.S. patent submitted Septem ber 1990. Presentations: D.C Cameron 1990. "Polysaccharide Production from Whey." Presentation at the WMMB Nonfat Solids Research Advisory Committee meeting, November 6, 1990, Madison, WI. 2. T.A. Cooper, J.H. Flatt, EN. Ughtfoot and D.C Cameron. "Characterization and produc tion of a superior succinylated zooglan polysac charide from whey by Zoogloea ramigera 115." Poster presented at the UW Bioprocess and Metabolic Engineering Consortium Annual Meeting, September 13, 1990, Madison, WI. JR. Flatt, T.A. Cooper, EN. Ughtfoot and D.C. Cameron. 1990. "Production of a High Quality Polysaccharide from Lactose in Whey Dairy Waste by Zoogloea ramigera: Fermentation Kinetics and Control." Paper 48A, AIChE Annual Meeting, November 11-16, 1990,Chi cago,IL. 117 I Centerto,. DlJiry ReselJ,.ch II---------------------~C J.H. flatt, RH. Hardin, J.M. Gonzales, T.A. Cooper, D.E. Dogger, E.N. Lightfoot and D.C. Cameron. 1990. '1'roduction of a Novel Po lysaccharide by a Newly Isolated Lactose Utilizing Microorganism." Paper 301E, AIChE Annual Meeting, November 11-16, 1990,Chi cago,IL. flatt, J. H., R. S. Hardin, J. M. Gonzales, and D. C. Cameron, (1990) "Polysaccharide Production from Lactose bya Newly Isolated SoilBacte rium". NA1O-Advanced Study Workshop on New Biosynthetic Biodegradable Polymers of Industrial Interest from Microorganisms, Sitges, Spain, May 26-31. J.H. Flatt, "Microbial Production of a Novel Polysaccharide from Lactose in Whey Perme ate." Presentation at the UW Bioprocess and Metabolic Engineering Consortium Annual Meeting, September 14, 1990, Madison, WI. T.A. Cooper, D. Dogger, EN. Lightfoot, and D.C. Cameron, "Pilot Plant Production ofTwo Microbial Polysaccharides (Laetan'DI and Zooglan) from Whey Permeate." Presentation at the CDR conference, 1991, Madison. 118 ))--------------------11 AnntIal Report 1990-1991 II Construction of a D(-)-lactic Dehydrogenase Negative Strain of Lactobacillus helveticus Personnel: James L Steele, assistant professor, Dept. of Food Science; Tarun Bhowrnik, research associate, Dept. of Food Science; Ann Caven, graduate student, Dept. of Food Science Funding and Funding Codes: Wisconsin Milk Marketing Board, 90-19 July 1990-June 30,1993 Objectives: 1. To characterize lactic acid productionby LActo/:1Qdllus he1veticus and its lactic dehydrOgenases (LDH). 2. To clone and characterize the D-LDH gene of L helveticus. 3. To construct a derivative of the cloned characterized D-LDH genewhich contains an internal deletion. 4. To use the deleted D-LDH geneto construct a D-LDH negative derivative of L helveticus; this strain would produce only L-lactic add. Summa:lJ': Lactic acid production by L. he1veticus CNRZ 32 during growth in reconstituted whole whey has been examined. Production of both 0(+ and L(+)-lactic acid was found to begin approxi mately 9 hours after inoculation. F'mal concen trations of 0(+and L(+Hactic acid were 15 and 5.0 grams/liter, respectively. Studies to optimize the production of lactic add from whey have been initiated. Factors to be examined are pH control, whey protein concentration, and atmo sphere. L he1veticus CNRZ 32 was determined to contain independent D-LDH and L-LDH enzymes. Both LDH's were determined to be nicotinamide adenine dinucleotide-dependent and fructose diphosphate-independent. A previously constructed gene bank, a collection of DNA fragments likely to contain every gene from a given organism, of L. helveticus CNRZ 32 was screened for the D-LDH gene. Three methods were attempted before we were suc cessful in identifying a D-LDH positive clone. The clone was confinned by comparing the protein produced by the clone to the L helveticus protein. The clone (pSUWl00) contains 6.0 kilobases (kb) of lactobacilli DNA. A restriction endonuclease map of the cloned DNA has been constructed (Figure 1). The location of the D LDH gene on the cloned segment of DNA has been narrowed down to 1.5 kb by employing TnS mutagenesis 1 mm) due to this decreasing driving force and the relatively long times required (up to 4-5 hr), A more efficient process is described below. Table 1 shows a summary of some experimental conditions and results for ice crystal growth under controlled heat balance conditions for skim milk in semi-batch crystallization. Here, controlled heat balance conditions were main tained by decreasing refrigerant temperature, increasing agitation rate and removing crystal slurry as growth proceeded. Ice crystal products with average crystal sizes ranging from 0.5 to 25 mm were grown in a period of several hours depending on the initial growth conditions. Final product concentrations ranging from 13% to 29% total solids were obtained under these conditions. Typical curves for average size vs. time are shown in Figure 1. The rate of growth was controlled primarily by control of refriger ant temperature with higher growth rates at higher subcoolings. Conditions of growth were found where secondary nucleation was not a . problem. That is, any secondary nuclei that formed at these conditions were not stable in the extremely high growth environment and there fore dissolved rather than grew. This resulted in a uniform distribution of spherical to disk shaped ice crystals that were relatively easy to separate from concentrate (however, no filtration studies were done to prove this). The curves shown in Figure 1 demonstrate the efficiency of growing ice crystals under these controlled heat balance conditions. For compari son, typical batch crystallization experiments result in size vs. time curves that start out with high slopes but then the increases in average size with time start to decrease as the equilibrium conditions are approached. Eventually, the ice crystals will stop growing as a thermal equilib rium is attained. By maintaining constant heat balance conditions, the growth rate of ice crystals can be maintained for long periods of time resulting in large crystal sizes in relatively short periods of time. Although these results are quite promising, several difficulties must be overcome prior to the development of this technology. These include 1.) control of the process and experimental conditions, 2.) elimination of product foaming and 3.) development of a continuous process based on these principles. These problems, l25 I Center for Dairy Resellrch I ( Table 1. Experimental conditions and results for ice crystal growth under heat equilibrium conditions in skim milkin batch crystallization. Run Seeds (Tb? TJ Holding C r L Nucleation r (ml) (OC) TIme (h) (%) (mm) RM6 20 0.7 7.0 13.3 0.52 No RM2 20 1.4 7.0 16.9 0.59 Few RMll 10' 1.5 6.5 17.9 0.64 Yes RM12 5 1.5 7.0 21.2 2.57 No RM13 10 1.5 6.5 17.3 1.32 No RM3 20 2.4 6.0 19.4 0.42 Yes RM4 20 2.4 6.0 22.6 0.71 Few RMI0 10 2.5 6.5 21.6 0.84 Few RM14 1 2.5 5.5 21.9 1.34 No RM5 20 3.4 6.5 26.6 0.57 Yes RM9 10 3.4 2.5 20.3 0.50 Few RM7 20 1-4.2 4.0 27.4 1.35 No RM8 7 4.3 4.5 29.1 0.53 Yes ? Seeds formed by spontaneous nucleatton m 10% lactose and added as slurry. .. rr b ? TJ:: bulk solution temperature minus refr1gerant temperature ? C f = final concentration: r,.:: final average crystal siZe Figure 1. Growth of icecrystals in skim milkat various refrigerant sUbcoolings under heat balance conditions. Refrigerant subcooling is the bulksolution temperature minustherefrigerant temperature. EffECTS OF REFRIGERANT SUBCOOLING 1.2 1.0 e e .. 0.8 ... N ... 0.6 ... m c &:I: 0.4 .... =c 0.2 0.0 4.2?e 1.4?C ? 0.7?t 0 100 200 300 400 500 TIME, min 126 )....-----------------------i[ Annual Reporl1990-1991 II particularly the development of a continuous process, are the subject of future work in freeze concentration processes. Summary: The current process for growing large ice crystals based on maintaining a high level of heat balance in a suspension crystallizer shows great promise for future developments in freeze concentration technology. Further work is required for development of this unique technol ogy and for outlining the ice crystal growth kinetics for a variety of fluid dairy products under a variety of conditions in an attempt to optimize the freeze concentration process. A significant amount of effort will be required to develop this technique into a commercial technology. Nevertheless, the potential gains from an economic freeze concentration process warrant continued efforts in this area. Objective 2 Nucleation of ice crystals is critical to the devel opment of freeze concentration technologies. Providing the seed stock from which to grow large ice crystals suitable for separation is crucial to the efficient operation of the process. Under standing the mechanisms of nucleation that may occur during the suspension growth of ice crystals to large size is also important. Defining the conditions under which nucleation becomes important during the growth process will allow operation of the suspension type crystallizer at optimal growth conditions. For example, lower temperatures will cause the ice crystals to grow more rapidly. However, nucleation may occur under these conditions, resulting in an unaccept able product crystal size distribution unsuitable for efficient separation. Outlining the conditions under which nucleation is suppressed will allow the most rapid growth conditions that yield a suitable size distribution for separation. In order to study these phenomena, several experimental techniques were utilized to observe the basic mechanisms of nucleation as well as the practical sense of the conditions under which nucleation will occur in a suspension crystallizer. Experimental: Several experiments were per formed to evaluate both primary (heteroge neous) and secondary nucleation processes during ice crystallization. Heterogeneous nucleation occurs when subcoolings are high, and is caused by the presence of foreign material (dust, ete.) present in the solution during crystal lization. Secondary nucleation occurs due to the presence of existing ice crystals, and is usually caused by crystal collisions that produce micro scopic ice fragments which can grow into stable nuclei. Secondary nucleation occurs at much lower subcoolings than heterogeneous nucle ation, so methods to measure them are different. Heterogeneous nucleation was studied using a batch crystallization process where a lactose solution was cooled to a low temperature until nucleation occurred spontaneously. The point at which nucleation occurred was noted (nucle ation temperature), and the change in size and crystal shape of these nuclei were followed using photomicroscopy. Several experimental condi tions were studied mcluding cooling rate and refrigerant temperature, agitation rate, and lactose concentration (10% or 20%). These experiments were conducted to determine the optimal conditions for providing nuclei for subsequent growth experiments. Secondary nucleation was studied using two different techniques. The first employed a photomicroscopic cell that allowed controlled contacts between a parent ice crystal and a glass cover slip in a controlled environment. The temperature at which these contacts produced nuclei was taken as a measure of the critical subcooling required for secondary nucleation. A batch suspension crystallizer was also used to study secondary nucleation where a single parent seed crystal was placed in an agitated solution held at a spedfied subcooling. The critical subcooling required for initiation of secondary nucleation due to contacts between the seed crystal and the impeller was determined by sequentially lowering the bulk temperature. Results: When a seed crystal was slid across a glass cover Slip in sugar solutions at the appro priate subcoolings, a crop of secondary nuclei could be observed along the path of contact. These nuclei were generated by the sliding collision of the seed crystal and the surface and were likely due to the disruption of a semi ordered layer at the surface of the seed crystal. This adsorption layer during crystal growth is thought to be where the water molecules are rearranging from the liquid water phase to become incorporated into the crystal lattice structure. Any contact at this point causes small clusters of water molecules to pass into the bulk solution where, under the appropriate condi 127 I Center /01' Daity Research I---------------------~C Figure 2. Effects ofwhey protein in aqueous dairyproducts onthe critical subcooling temperature for contact nucleation. 1.6 -U 0 - 0 lZ .... ..:I 0 0 U III I:) Cl) ..:I c1 .... to .... C' U ? Whey Extract ? Whey Powcler 1.4 ? Nonfat Dried Milk 0 Skim Milk ? A ? 1.2 1.0 0.8 0.6 0.4 +-...........--......-r-...........-.,.-_.............,r-"I'.................,....., 0.0 0.5 1.0 1.5 2.0 WIlEY PROTEIH (') tions, they will survive and grow to appear as secondary nuclei. The critical subcooling for nucleation was found to be between 0.016?and 0.033OC (bulk temperature minus freezing point) for both lactose and sucrose solutions with very little dependence on lactose concentration. Slightly higher critical subcoolings for secondary nucleation were found when using the batch suspension technique due to differences in collision intensity and probability. For lactose, the critical subcoolings increased from 0.14?to O.23?C as lactose concentration increased from 5% to 20%. Several dairy products were evalu ated with this technique, including solutions of whey extract, whey powder, and nonfat dry milk at various concentrations, as well as a skim milk sample. Increasing critical subcooling with concentration was found for each of these samples for concentrations between 6.8%and 17.8%(w/w). Regression analysis on sample composition detennined that the major influence on critical subcooling was due to the whey protein content in the samples. To test this hypothesis, a sample solution was made up with 6.8% whey extract to which 2.5g of whey protein concentrate (34.5%protein) was added (called test sample A). The results of this study are shown in Figure 2 where the critical subcooling for secondary nucleation is plotted against the whey protein content. Obviously, the whey protein content plays an important role in deter mining nucleating capability of crystals growing in fluid dairy products. This result is important in determining the conditions under which a suspension crystallizer can be operated without secondary nucleation occurring. Heterogeneous nucleation of ice from lactose solutions was studied for the purpose of deter mining conditions under which seed nuclei could be produced for use in subsequent growth experiments. These experiments involved cooling agitated lactose solutions at varying rates, noting when nucleation occurred (nucleation tempera ture) and the size and shape of ice nuclei. It was found that nucleation was not reproducible, as expected, due to the sporadic nature of the crystal formation. This has been observed previously in almost every study on nucleation. Thus, for identical conditions, nucleation occurred at different times and temperatures. One way to characterize this nucleation process involves plotting the mean crystal size of the nuclei against the temperature at which nucle ation occurred. These plots gave reasonably straight lines indicating that the size of nuclei 128 )}.-----------------[ An,..,," Report 1990-1991 II produced depended on when they were initially formed and how many were produced. Condi tions that gave lower nucleation temperatures resulted in smaller crystals since many more crystals were formed at the higher driving force. Significant differences in crystal size (and thus, nucleation rate) were found with varying agitation rate and lactose concentration. Higher stirring rates promoted nucleation at higher temperatures resulting in larger ice crystals while higher lactose concentration inhibited nucleation resulting in lower nucleation tem peratures and smaller ice nuclei. All nuclei generated from these studies had reasonably well defined disk-shaped crystals after 10 minutes of growth that were between 50 and 100 J,Un in size, depending on conditions. These nuclei were quantified by size analysis for use as seeds in growth experiments. Summary: These results demonstrate the nature of the secondary nucleation event of ice crystals in fluid dairy products. It has been observed that the magnitude of the critical subcooling temperature above which secondary nucleation is negligible depends on the type of contacting system used to observe the nucleation event. This indicates that, at this point, each freeze concentration system will require an individual study to determine the critical subcooling point for any given dairy product. Further work will be required to fully understand the mechanism of secondary nucleation so that this event can be predicted from first principles. It has also been observed that the magnitude of the critical subcooling depends on the type of fluid dairy product and its concentration. That is, this boundary will be continually changing dUring the freeze concentration process as the product becomes more and more concentrated. The results indicate that the whey protein content plays the dominant role in determining the magnitude of the critical subcooling with an increase in whey protein content resulting in a direct increase in the critical subcooling tempera ture for secondary nucleation. Further work will be necessary to detail the exact mechanism of this interaction. Significance to the Dairy Industry: Freeze concentration is a relatively new tech nique for concentrating fluid foods. Due to its low temperature operation, it has several advan tages, particularly for heat sensitive foods, over the traditional techniques of evaporation and reverse osmosis. At the present time, however, the economics of freeze concentration do not compare favorably with evaporation or reverse osmosis. Further research on the fundamentals of ice nucleation and growth in fluid dairy products is necessary to allow the full advan tages of freeze concentration to be determined. The results of this study show that control of the heat balance in an ice crystallizer can result in rapid growth of ice crystals to product size for efficient separation. These results may allow the development ofmore economicaIly attractive means of concentrating fluid dairy products using freeze concentration. Publications: Shi, Y.,Liang, B.and R.W. Hartel. 1990.Crystalli zation of Ice From Aqueous Solutions in Suspension Crystallizers, ACS Symposium Series 438,316. Hartel, R.W. and MS. Chung. Contact Nucle ation of Ice in Fluid Dairy Products, submitted to ]. Food Engineering (1991). Chung, Myong-Soo. 1990.Formation ofContact Secondary Nuclei From Crystals in Sugar Solutions and Fluid Dairy Products, MS thesis, UW-Madison. Presentations: Shi, Y., Liang, B.and R.W. Hartel. 1989. /lIce Crystal Morphology During Growth From Aqueous Solutions in a Suspension Reactor," American Institute of Chemical Engineers National Meeting, San Francisco, CA, Nov. 1989. Shi, Y..;B.LiangandR.W.HarteI.1989. IICrystal lization of Ice From Aqueous Solutions in Suspension CrystaDizers," PACIFICHEM meeting (joint ACS), Honolulu, HA, Dec. 1989. Shi, Y, Liang, B. and R.W. HarteI.1990. "Nucle ation and Growth of Ice Crystals in a Batch Suspension Crystallizer," submitted for presen tation at Industrial Crystallization symposium to be held in Sept. 1991,Garmisch, West Germany. Espinel, L. and R.W. Hartel, "Freeze Concentra tion of Fluid Dairy Products," accepted for presentation at Annual AIChE Meeting, Los Angeles, CA, Nov. 1991. 129 I Center/or Dairy Research 1-------------------------\( New Dairy Foods with Added Calcium and Modified Salt Personnel: J.L. Greger, professor, Dept. of Nutritional Sciences; Emily Tseng, graduate research assistant; Lisa Cole, assistant research specialist Fundin.: and Fundin.: Codes: National Dairy Promotion and Research Board, GRLE90 July 9O-Dec. 31, 1992 (Extended) Objectives: Low-sodium cheeses are being developed and advertised as a way to reduce the risk of hyper tension. Recent research shows that it may not be the sodium alone but also the chloride in salt that promotes hypertension. Objective one of this project is to assess whether substitution of KCI for NaG or substitution of NaHC0 3 for NaO in cheese reduces the tendency for cheese with excess NaG to promote hypertension in an established, sensitive animal model. We hypoth esize that: 1) animals fed diets in which regular cottage cheese is a major component will de velop significantly higher blood pressures than animals fed cottage cheese with low levels of sodium and chloride, but the "unsalted" product will taste flat to consumers; 2) animals fed a cottage cheese "salted" with NaHC0 3 or KO will experience less elevation in blood pressure than animals fed cottage cheese "salted" with NaO; 3) taste panels will prefer cottage cheese "salted" with NaCHO! rather than KO. A second objective is to determine whether changing additives used. in the production of cheese could improve bioavailability of calcium in the cheese. We hypothesize that calcium in traditional cottage cheese will be less bioavailable than calcium in the products developed in this work, i.e., cheese in which the level of chloride has beenreduced. Sununaty: Two hundred pounds of curd for cottage cheese was prepared in the Center for Dairy Research and split into quarters. Then the salt added to the cream was varied so that four types of cottage cheese were produced: 1. Regular cottage cheese "salted" with sodium chloride (1.47moles Na and G perg dry cottage cheese). 2. Cottage cheese "salted" with sodium acetate (an equimolar amount of sodium as the regular cottage cheese). 3. Cottage cheese "salted" with potassium chloride (an equimolar amount of chloride as the regular cottage cheese). 4. Cottage cheese to which no salt was added to the cream <0.02 moles Na and 0 per g dry cottage cheese). The cottage cheeses were dried and incorporated into four diets in which the cottage cheese provided all the protein (20%by weight) in the diets. An additional four standardized, semi purified diets were prepared using lactalbumin as a protein source. The sodium, chloride, calcium, phosphorus, fat and protein levels of the lactalbumin diets were adjusted to be similar to those of the cottage cheese-based diets. The eight diets are being fed to Sprague Dawley rats for two months. Blood pressures of rats are being monitored throughout the study. Plasma 130 Annual Report 1.990-1.991. Figure 1. Theeight diets fed to Sprague Dawley rats in this study. Cottage cheese (CC)-based diets No salt added KCL added NaHCO s added RegularCC With NaCI Lactalbumin (L)-based diets No salt added KCL added NaHCO s added NaCI added renin, urinary excretion ofelectrolytes and calcium, and absorption and tissue retention of calcium will also be assessed. Si&Dificance to the Dair.y Industry: In"Promoting Health/Preventing Disease: Year 2000 Objectives for theNation," the US. Department of Health and Human Services lists several major goals relevant to this project: ? To promote increased intake of calcium. ? To promote reduced intake of salt. ? To double the sale of low-salt, processed foods. These federal initiatives suggest that the demand for nutritionally designed dairy products will be increasing. Before the dairy industry embarks on production of more low-sodium dairy products, we need to consider and extend recent research on the relationship of sodium, chloride, potassium and calcium to hypertension and osteoporosis. Cottage cheese is a logical product to study and develop as a nutritionally designed food because it is already appealing to health conscious consumers. Moreover, FDAplans to require data from nutritional studies to support health claims for food products in the future. Publications: (Work based on previous CDR project that was the progenitor of this project) Puspitasari N.L., K. Lee, and J.L. Greger (1991). Calcium fortification of cottage cheese. J. Dairy Sci. 74:In press. Kaup S.M., J.L Greger, and K. Lee (1991). Nutritional evaluation with an animal model of cottage cheese fortified with calcium and guar gum. J. Food Sci. In press. 131 I Center for Dairy Research 1-------------------\( CLA:A Newly Recognized Anticarcinogen Isolated from Dairy Products Personnel: Michael W. Panza, director, Food Research Institute, and professor and chair, Dept. of Food Toxicology and Microbiology; Hania Benjamin, associate scientist; Karen Albright, research specialist, Jayne Storkson, research specialist; Wei Liu, research specialist, Food Research Institute Fundin~and Fundin~Codes: 'Wisconsin Milk Marketing Board, 89-27 Dec. 1, 1990-March1, 1996 Objectives: To determine: 1. the function of CLA in protecting cell mem branes from oxidation; 2. the effects in regulating/modulating various key cell membrane enzymesand enzyme sys tems; 3. the mechanism whereby CLA inhibits mold growth; 4. the optimal way to synthesize CLA for commercial applications. The overall goals of this project are to determine the role of CLA in health maintenance and its optimal use in food preservation. Su11'U1lD[)"i Work toward each of the specific objectives has commenced. It is too early to report specific findings, but progress is being made. Significance to the Dairy Industry: Dairy products are a principal dietary source of CLA. Hence, work aimed at possible health benefits of CLA is expected to enhance the already fine image of dairy products as impor tant for sound health. The fact that the CLA is a component of dairy fat is particularly intriguing in this regard. Additionally, CLA is potentially of great importance as a natural antioxidant and mold inhibitor for use in food systems. 132 )-------------------------i Chapter3 Worldwide Information and Technology Exchange Program (WITEP) 133 I Centerfor Dairy Research ~-----------------I,.C 134 )r----------------------I Annual Report 1990-1991 II Overview of WITEP The purpose of the Worldwide Information and Technology Program is to help make CDR's research program even more effective by facilitating interactions with other research programs and transferring information and applied technology to the dairy industry. In the past three years, the WITEP has continued evolving to address the changing communication and technology transfer needs at CDR. The following reports summarize the activities WITEP employs to achieve these goals. These include: 1. Scientist Exchange Programs 2. Seminars and Conferences 3. Publications/Information Dissemination - annual reports, a newsletter for the dairy processing industry, training videotapes, information databases, directories, and other resources. Plans and activities for the coming year: Along with the three activity areas listed above, there are some additions to highlight: Additional technology transfer activities: The coming year will include more technology transfer activities than past years. Two new staff members are joining WITEP. One, a specialist in cheese technology transfer, will interact directly with the Wisconsin cheese industry on questions they have regarding their operations. This individual will also work closely with UW researchers to help transfer new technology from the lab to the cheese plant. The second new staff member will be responsible for putting the results of UW milkfat research in a format that is accessible to the dairy foods industry. Workshops and trainini seminars: WITEP will host two training programs this year. A whey functionality workshop and a lowfat cheese seminar are scheduled for the autumn of 1991. Both will focus on providing information and technology that will help food processors manufacture consistently high-quality dairy products. 135 I Center for Dairy Research ------------------i( Scientist Exchange Program. A goal of WITEP is to promote research collaboration between UW researchers and scientists at other dairy research programs. This results in an exchange of knowledge and skills between researchers and, most importantly, a long-term working relationship. WITEP fadlitates these partnerships by setting up contacts, coordinating visits, and funding exchanges. In many cases collaborative research projects are the result. The Scientist Exchange Program consists of a variety of exchange activities, including hosting visiting scientists from both academia and industry, sponsoring CDR staff on visits to other other research institutions or training programs, and hosting mentors experienced senior researchers who can offer suggestions and research guidance to the CDR staff. Mentors also sometimes teach a dairy foods course at the UW during their visits. From year to year the number of visiting scientists to CDR fluctuates depending on the availability of host scientists, lab space, and availability of funding. It also depends on how much time a visitor needs to arrange a leave from his/her institution. Scientists visiting CDR: Carla Buijsse, Researcher, Dept. of Food Science, laboratory of dairying, Wageningen Agricultural University, Wageningen, The Netherlands. Period of stay: April 1, 1991 - August 1, 1991. Host: Mark Etzel, Asst. Professor, Dept. of Food Science. Visit rq>Ort See research report Differential inactivation of intracellular enzymes during spray drying of starter cultures, Chapter 2. Morsi El-Soda, Professor, Dept. of Agriculture Industries, Alexandria University, Alexandria, Egypt. Period of stay: August 1, 1990- October 31,1990. Host: N. F. Olson, Professor, Dept. of Food Science. Visit report See research report Acceleration of cheese ripening using liposome-entrapped enzymes, Chapter 2. Note from the WITEP Coordinator: Each year Dr. El-Soda works with the cheese research group at Laval University, Laval, Canada before coming to CDR. Dr. El-Soda's participation in our research program is a collaborative bridge between CDR the Laval. During his visit to CDR this past year, Dr. El-Soda was accompanied by a Laval PhD candidate Mouhsine EI Abboudi, who conducted cheese ripening research during his stay at CDR. Mr. El 136 Anmlal Report 1990-1991 Abboudi's visit was funded by Laval. Currently (August, 1991)the Laval cheese research group and CDR researchers are preparing a proposal to conduct joint cheese research. Geraldine Farrell, Post-doctoral Researcher, Sligo, Ireland. Period of stay: September 1, 1989 - August 30, 1990. Host: Elmer R Marth, professor, Dept. of Food Science. Visit report See research report The growth and suruival ofBorrelia burgdorferi in milk, Chapter 2. Peter Linklater, retired. Former manager of dairy manufacturing research at the Australian Dairy Research and Development Corporation, New South Wales, Australia. Period of stay: March 1 - March 22, 1991. Visit report Activities enga&ed in while at theCDR: As Mentor to CDR researchers, Dr. Unklater discussed the following areas with CDR staff: whey utilization, drying of starter cultures,.cheese as a food ingredient, database development/management, whey proteins, economics of cheese manufacture, cheese starters. What were the project's objectives and how were they met? 1. Dr. LinkIater alsoserved as a mentor to graduate students, as well as to the dairy research faculty. He pointed out strengths in their work which they had perhaps not recognized, and suggested some potentially useful contacts. 2. He examined the relationship between the CDR and the dairy manufacturing industry, and made a number of constructive suggestions to the Director. These included a proposal to improve communication among the scientists and the Industry. 3. He suggested that consideration be given to providing the Industry with a wider range of technical information. This could be done be accessing the FIRA database at Leatherhead in the U.K., or some similar U.s. facility. There is also a store of under utilized information among researchers. This could be tapped without imposing directly on their time. 4. Presented a paper on "Computer aided cheese manufacture" at the 1991 CDR Cheese Research and Technology Conference. How did these efforts contribute to CDR's research pfQ&Tam? 1. Conveyed to the dairy faculty some facets of work done in other countries and on other problems. 2. Provided objective views on ways to take research closer to the Industry. 137 Center for Dairy Research 3. Suggested some possible expansion of infonnation processing in WITEP. Publications produced as a result of research: "Computer Aided Cheese Manufacture." Proceedings of the CDR Cheese Research and Technology Conference 1991. Patrick. Fox,Professor and Chair of Dept. of Food and Dairy Chemistry, University College, Cork, Ireland. Period of stay: August 23 - September 3, 1990. Visit report: As Mentor to CDR staff Dr. Fox, met with CDR researchers to discuss research projects. He presented a week-long dairy chemistry course, Food Science #375/875, to 35 students who came from both the UW and industry, and presented a dairy chemistry lecture at Schreiber Foods, Inc. Green Bay, WI. Evaluations of the course were excellent. Students said they appreciated the opportunity to attend a course by one of the world's leading scientists in the area of dairy chemistry. The course was videotaped and is being offered to food science departments and industry through a brochure and advertisements in trade journals and publications. Note from the WITEP Coordinator: Dr. Fox arranged to have one of his doctoral students spend one year (starting August 1991) at CDR to do cheese research with Dr. Norm Olson's research group. w. James Harper, Emeritus professor, Dept. of Food Science, the Ohio State University, Columbus, Ohio. Consultant to various dairy foods organizations. Period of stay: March I, 1991-May 31, 1991. Project Objectives: As CDR Program Improvement/ mentor, the objective of Dr. Harper's visit was to assist in the overall development of CDR. Specific Aims Includedj 1. To transfer basic knowledge of milk components and their interaction to scientists who have not had previous opportunities for in-depth study of dairy products. 2. To assist in the development of procedures to evaluate the research projects and programs of CDR. 3. To evaluate projects and programs of CDR, with special attention to transferring basic science to commercial applications. 4. To provide suggestions for further research that might open new directions for milk utilization. 138 )l--------------------1[ Annual Report 1990-1991 II 5. To develop position papers on the basic research that will benefit dairy technology 10-20years in the future. 6. Assist with the development of a national dairy researcher's database and a collaborative network associated with the WITEP program. Major Accomplishments: 1. Aiding in the professional development of research personnel engaged in a variety of research programs, including: - whey utilization - bioscience -cheese - engineering Interactions with staff included: J.P. Chen, Mark Johnson, Carol Chen, Dave Bogenrief, Doug Cameron, Mark Etzel, Rich Hartel, Jim Steele, Bob Bremel, BillWendorff, Bob lindsay, Norm Olson, Sarah Quinones, graduate students, and technicians. 2. Assisting in information and data management through helping with the development of - text management data bases - inter-relational data bases 3. Assisting in the development of networks among researchers, including the development of a national Directory of Dairy Researchers grouped by their specific focus and research interests. 4. Serving as a facilitator to assist in program development and helping to improve the liaison between the CDR and one of the major funding agencies - WMMB. 5. Direct input into selected research programs and program development, including: - ion exchange membrane application to whey proteins - genetic modification of milk composition and role of unique cows - development of data base for milkfat fractions and transfer of technology for milkfat fraction applications - development of a structured program for the transfer of the exopolysaccharide technology developed by Doug Cameron - enzyme catalyzed modification of milkfat; method development - role of glutathione in cheese ripening 6. Serving as a gate-keeper with respect to research directions, and developing procedures for determining long-term research needs and directions. 139 I Center for Dlliry ReselU'Ch 1------------------------1( How did these efforts contribute to the CDR's Research Program? This visit will have both tangible and intangible effects on the CDR research program through the professional development by CDR staff achieved as a result of the interaction. Two tangible effects will be: 1. Improvement in the knowledge of the CDR researchers relating to dairy product and process fundamentals, as well as an improved awareness of the key industrial concepts important to the eventual commercial adaptation of their research. 2. Improved skills in information organization through the databases developed during this visit Note from the WITEP Coordinator: Dr. Harper developed the program for our whey protein workshop, which will be held October 21-23,1991. He also assisted with identifying mentor and scientist exchange candidates. CDR researcher visits to other dairy research programs/conferences/courses: Program participant: Jim Steele, Assistant Professor, Dept of Food Science. Conference attended: Third Symposium on Lactic Add Bacteria September 17-21, 1990 Wageningen, The Netherlands R=arch project's) which the conference prouam pertains to: Construction of a Gene Bankof Lactobacillus helveticus CNRZ 32: Ooning and characterization of the aminopeptidase and threonine aldolase genes. Purpose for attending the conference: 1) To discuss recent advances in our knowledge of the peptidase enzyme system of . lactic add bacteria with expertsin the field. 2) To meet and discuss possible collaborative research with Dr. Finn Vogensen (also attending the conference) from the Royal Veterinary and Agricultural University, Copenhagen, Denmark. How does this information benefit CDR's research and information transfer programs. and the dairy foods industry? By discussing recent ad vances in our knowledge of the peptidase enzyme system of lactic acid bacteria with experts in the field, updated information on the basic biology and methods of study of these enzymes were obtained. Examples include the nucleotide sequence of the X-proyl dipeptidyl aminopeptidase from Lactococcus lactis, 140 )>----------------------1 Annual Report 1990-1991 II substrates for positive selection of peptidase deficient mutants, and cloning strategies for peptidase genes. This information will benefit CDR and the dairy foods industry by insuring that the scientists directing CDR sponsored research are up-to-date and making meaningful contributions to our understanding of how lactic acid bacteria influence the quality of dairy products. Discussions of possible collaborative research with Dr. Finn Vogensen from Denmark will be continued when he arrives for a visit to Madison on the 17th and 18th of October. A component of this collaboration may be Dr. Vogensen taking a research sabbatical in Madison. Strengthening the ties between CDR and other dairy research centers worldwide should increase the quality of the dairy related information generated by the CDR research effort and hence the information available for transfer to our dairy foods industry. !'Yote from the WITEPCoordinator: At this time Dr. Vogensen is considering spending six months to a year on a research sabbatical at CDR (working with Dr.Steele) starting June, 1992. Program participant: Brian W. Gould, Associate Scientist, CDR Conference attended: Annual conference of the American Cultured Dairy Products Institute, March 3-5, 1991,Charleston, South Carolina Research project(s) which the conference program pertains to: Dairy Product Demand Analysis Using Cross-Sectional Data Purpose for attendinl; the conference: To present summary of data sources for conducting analysis of dairy product demand. How does this information benefit CDR's research and information transfer programs. and the daily foods industry? Provide information so that individual firms may use publicly available data to analyze the demand for the products they are producing and to identify those individuals or households that consume their products. Program participant: Robert D. Bremel, Professor of Dairy Science Dairy center visited: Dairy Research and Development Center Ruakura Research Center Hamilton, New Zealand June 21-30,1990 141 I Center for Dldry Research li--------------------------\( Scientists with whom you discussed research: Richard Wilkins, Steve Davis, Colin Prosser, Phil L'Huillier Specific purpose for visiting this research center: This research center is actively pursuing the modification of milk through the use of transgenic technology and through the use of ribozymes. Description of &meral research areas that were discussed and the likelihood of collaborative research resulting: An ongoing collaboration has already been established. This provided the opportunity to see their laboratory and to learn more about their test systems. It was agreed. that the two research groups would continue to share genetic constructs when that is useful. The groups also will be working to establish a system to measure the alpha-lactalbumin and beta-lactoglobulin content in blood of heifers. Note from the WlIEP Coordinator: Dr. Bremel has agreed. to present a seminar on campus that conveys his observations and conclusions gained from these visits. Program participant: Carol Chen, CDR Associate Researcher Course attended: Using and Programming in Paradox. Minneapolis, MN May 21-23, 1991 Research project(s) to which the training will be applied: All CDR cheese research projects ? Lowfat cheese development ? Cheese as a Food Ingredient ? Industry Cheesemaking Specific training objectives: Learn how to set-up tables, queries and write applications in Paradox, a relational database. How does this information benefit CDR's research and information transfer programs. and the daily foods industry? The goal of this project is to set-up a functioning database which will store all cheesemaking information: manufacturing schedules, sensory analysis, chemical analysis and physical analysis. This information will be able to be recalled, and sorted with ease. Ideally all individuals involved with "cheese technology" will be 142 )>-----------------1 Annual Report 1990-1991 II networked together. This database will save researchers time when putting data together for summarization and will ensure that data is not lost. Program participant: Dale McGill, CDR Research Specialist Course attended: Biological Research Course Waters Division of Millipore Milford, MA 01757 July 23-25,1990 Research projects to which the trainin& will be applied: Whey utili:zDtion and butterfat modification, Dr. J.P. Chen. Specific trainin& objectives: The three-day lecture course was intended" to cover the various chromatographic methods which pertain to High Pressure Liquid Chromatography. The course is offered free to anyone who purchases an HPLC from Waters Division of Millipore. Objective was to learn as much as possible about use of the HPLC system. How does this information benefit CDR's research and information transfer programs. and the daily foods industIy? The removal of lipids from cheese whey will improve the functionalities of whey protein concentrates and facilitate the production of WPC using ultrafiltration. The flavor of the WPC will also be improved. Chromatography is a versatile tool in the laboratory and it is quite complex. HPLC requires training and an understanding of the aspects described in this course. Program participant: Usa Pannell, Research Assistant with Dr. Norm Olson Food Science Department Dairy research program visited: Centre de recherche STELA University Laval Quebec, CANADA July 22-July 28,1990 Purpose of visitinl' To learn how to use a computer program which will locate possible origins of peptides in the caseins and whey proteins, given amino acid composition of the peptide. This program will be useful in CDR cheese ripening research. 143 ---l( I Centertot' DlJiry Resetlt'ChlI Training/topics discussed: In addition to a thorough discussion of the computer program, hands-on use assured competency for future use. Discussion of the protein research program at Laval provided insights for CDR's research program. No collaborative research appeared to be warranted in the area at the present time. The researchers at Laval were instructed on the fabrication of milkfat microcapsules previously used in CDR research. Laval is planning to continue this research but using a different technique, micrefluidization, to produce the microcapsules. Briefly describe the valuable aspect of this scientist exchange: The computer program for predicting the sequence of peptides will save significant time in CDR research on cheese proteolysis and ripening. It will aid in determining the source of ripening, the peptides generated during cheese ripening, the release and disappearance of amino adds and, hence, the source of flavor compounds. This information can be used to systematically control proteolysis and flavor development by selecting enzymes that will release the desired amino adds. Seminars and Conferences An objective of WITEP is transferring dairy research information and technology to the dairy industry. Among the mechanisms used to accomplish this, WITEP coordinates an annual research conference, industry-oriented seminars, and research oriented seminars. CDR Annual Research Conference: This past year,CDR hosted its annual Cheese Research andTechnology Conference, March 6-7, 1991 at the Holiday Inn-West Towne, Madison, WI. In addition to the speakers, there was an extensive research project poster session, a training aids exhibit, a poster exhibit of the research programs at the six national dairy foods research centers, and a cheese buffet comprised of award winning cheeses primarily from Wisconsin. The conference was attended by 290 members of the dairy foods industry and academia. Evaluations of the conference showed that the conference was a tremendous success and that the speakers and various exhibits were successful in transferring information on cheese research/technology and highlighting CDR's research and outreach programs. The conference proceedings proved to be useful and informative both during the conference and as a resource for subsequent use. 144 Annual Reporl1990-1991 The pt'QlP'am of presentatiQn follows: Session L Milk and Cheese Quality - Impact on Cheese Marketing Tests you should be usingin yourmilkmonitoring program, Robert L. Bradley Taking Control of Communications with YourProducer, Darrel E. Johnson, D.V.M. Canadian Microbiological Standards in Cheese - HIlOe They Been a Success? Karl F. Weiss Specifications from theBuyer's Perspective, Jeff Giffin. New Labeling Requirements for Cheese Products, Floyd D. Gaibler Session U. Cheese Technology and Research A'OOid Cheese Defects byGetting Back to theBasks of Cheesemaki.ng, Norman F. Olson Computer-aided Cheese Manufacture, Peter M. Linklater Making Quality lDwfat Cheese, Mark E. Johnson Cheese tasting/evaluation of current luwfat cheese problems, Ronald E. Simard Banquet and keynote address: TheChanging International Marketplace andIts Effect on the U.S. Dairy Industry, David P. Hammer Session UL Considerations for Profitability in the Cheese Industry MilkQualityandCheese Yields: Further Evidence, Ed Jesse HDouble Loop Standardimtion/ A Software/Hardware Combination Designed to Monitor and Control theCheesemaking Process, David McKenna Development of a Computer Program for Economic Analysis of Cheese Plant Operations, Brian W. Gould Factors Influencing Costs and Profitability ofCheese and Whey Product Manufacture, David M. Barbano Session IV. On the Research Horizon Anticarcinogenic Fatty Acids in Cheese, Michael W. Panza Potential forModifying Milk Composition through Milk Feeding Practices, Ric R. Grummer What to Expect from Future Cheese Cultures, James L. Steele Controlling Body andTexture ofMozzarella Cheese: Microbiological and Chemical Methods, Craig Oberg 145 I Centn'for Did", Research Ii------------------------l( Seminars: WITEP targets research topics with an applied nature for seminar presentations. A new practice in the past year is inviting industry personnel to meet one-on-one with the seminar speaker. This hasbeen an effective means of making the researcher more accessible to industry and facilitating a more effective information/technology transfer. Applied Dairy Chemistry (August 27-31, 1990): Pat Fox, Chairman, Dept of Food and Dairy Chemistry, University College, Cork Ireland. This was also offered as a course to UW dairy foods students and to industry. This was a major success with 38 students attending. The course was videotaped and is being sold as a training video. Thus far, 16 copies have been sold. Controlling thePhysical Properties ofMozmrella Cheese (January 10, 1991): Paul Kindstedt, Asst. Professor, Dept. of Animal Science, University of Vermont, Burlington, VT. Attended by 31 from UW and industry; 2S were from industry. This was videotaped and hasbeen a very popular selling video. Thus far 26 copies of the video have been sold. Cheese Technology Preview (Apri124, 1991): Means of Determining pH During Cheesema.king, Fritz Buss, Nelson-Jameson, Inc, Marshfield, WI. Biofilms: Formation and Destruction in the Dairy Plant Environment. Dr. Ed Zottola, Minnesota Dairy Foods Research Center, St.Paul, MN. Effect of Curd Formation on Cheese Yield. Robert Selman, Pfizer, Inc. Dairy Products Division, Milwaukee, WI. There were 35 people from industry and the UW attending. To reach a larger audience, a training videotape on pH measurement was produced in conjunction with the seminar. Thus far 28 copies of the video have been sold to organizations that can now provide training on pH use to their entire technical staffs. CDR research-oriented. seminars: Membrane Separation Research at theDept. ofChemical Engineering, University ofOviedo, Oviedo, Spain (July 24, 1990): Dr. Jose Coca, Professor, Chemical Engineering, University of Oviedo, CDR visiting scientist in FY90. Survi'Otll of Borrelia lmrgdorfrri in whole, lcwfat, andskimmilkat34?C andin skimmilkat S?C (August 22,1990): Geraldine Farrell, National University of Ireland, lecturer in food science and technology, CDR visiting scientist. Hydrolysis of lactose in skimmilkbybetR-galactosidase immobilized in a spiral flow reactor (August 24, 1990): Andrew P. Bakken, Research Assistant, Dr. Charles Hill, Dept. of Chemical Engineering, UW-Madison. 146 )f----------------I Annual Report 1990-1991 II Publications/Information Dissemination Annual Report The CDR annual report is published to provide our funding agencies and the dairy industry with a comprehensive and condse yearly summaryof our research and communications activities. This document has been evaluated with a survey of the readers. Surveyresults have been highly positive about the annual report and describe it as a useful resource, In addition, other UW researchers and administrators are kept abreast of CDR with the annual report. This helps promote the research team approach among researchers and alsogives CDR visibility to UW and state leaders/ administrators. UW Dairy Pipeline newsletter The UWDIliry Pipeline is CD~s primary outreach tool to Wisconsin's cheese manufacturing indUStry. Published quarterly, this training newsletter provides applied infonnation to dairy manufacturers. The Pipeline has several regular contributors who are leaders in academic dairy research/extension. It alsocarries articles from industry and regulatory agency representatives. The question/answer column, 1'he Curd Oinie,., features UW cheese scientists addressing specific cheese related problems. In addition the newsletter includes listings of CDR's research projects and a CDR calendar of events. The distribution of the newsletter has grown to more than 700. CDR Videotape Catalog This past year WITEP compiled a list ofour videotapes and developed a catalog. The fonnat includes a brief description of the video and its cost. The catalog also includes a section of training videos offered by the other national dairy foodsresearch centers. This has been a very useful device for responding to video requests and informing industry of the selection of training videos available. UW dairy researchers directory This publication is a listing of all the UW dairy foods researchers; it lists the research areas ofeach scientist and includes their addresses and phone numbers. National dairy researchers directory In the past year WITEP has begun development of an national dairy foods researchers directory. This is currently housed in a Paradox software database but will ultimately be placed in an appropriate fonnat for distribution. 147 I Center for Dairy Research ---------------......;( UW dairy foods research projects list The purpose of the research projects list is to provide the dairy industry, funding agencies, and UW researchersI administrators with an up-to-date list of what dairy foods research projects are underway at the UW. This list includes projects receiving funding from the graduate school and other state and federal agendes as well as industry funding. The list helps keep researchers abreast of what the various research groups on campus are doing. Conference proceedings Our annual conference is always a key means of communicating to the industry. A very popular feature has been our conference proceedings publication which is prepared in advance of the conference so that those attending have it in hand for reviewing and taking notes on the various presentations. This publication is requested subsequent to the conference as well. Databases WTIEP has developed two databases in the past year. The Cheese database consists research notes and references accumulated over the years by Dr. Norman F. Olson, Director of CDR and The Cheese Research Institute. This database will be available to industry when its approximately 25,000 individual records have been entered. It will also be used by CDR cheese researchers and UW extension personnel to assist with research and answering cheese industry questions. 'The second database, CDRPUBS, contains all the publications and presentations generated by CDR funded or administered research. In the next phase of development for these databases, personnel who will use these databases will be thoroughly trained in their use. 148 )--------------------------1 Chopter4 1990 CDR Research Publications/Presentations 149 I Center for Dairy Research I-----------------------I.( 150 I )f----------------------II .Annual Report 1990-1991 il 1990 CDR Research PublicationsIPresentations The following are all 1990 publications and presentations resulting from projects administered through CDR. Abstracts or reprints are available through CDR; call Sarah Quinones at 608/262-2217. I. Dairy Product Safety and Quality 1. EI-Shenawy, M., and E. Marth. (1990). Behavior of Listeria monocytogenes in the presence of gluconic acid and during preparation of cottage cheese curd using gluconic acid. I. Dairy Sci. 73:1429-1438. 2. EI-Shenawy, M., H. Garcia, and E. Marth. (1990). Inhibition and inactivation of Listeria monocytogenes by the lactoperoxidase system in raw milk, buffer or a semi synthetic medium. Milclrwissenschaft.45 (10): 638-641. 3. Farrag, S., F. El-Gazzar, and E. Marth. (1990). Fate of Listeria monocytogenes in sweetened condensed and evaporated milk during storage at 7 and 21"C. ,. Food Protection. 53(9): 747-750. 4. Johnson, E.A. (1990). "Effectiveness of antibacterial substances against pathogens." University of Wisconsin Food Research Institute Annual Meeting. Madison, WI. 5. Johnson, E.A. (1990). ''Lysozyme to control growth and survival of Listeria monocytogenes:' International Life Sciences Institute-Nutrition Foundation Colloquium. Washington, D.C. 6. Johnson, E.A., J. Nelson, and M. Johnson. (1990). Microbiological safety of cheese made from heat-treated milk, Part I. Executive summary, introduction, and history. l. ofFood Protection. 53(5): 441-452. 7. Johnson, E.A., J. Nelson, and M. Johnson. (1990). Microbiological safety of cheese made from heat-treated milk, Part Il. Microbiology. [. Food Protection. 53 (5): 519-540. 8. Johnson, E.A., J. Nelson, and M. Johnson. (1990). Microbiological safety of cheese made from heat-treated milk, Part m. Technology, discussion, recommendations, blbliography.]. ofFood Protection. 53 (5): 610-623. 9. Kihm, D., and E.A. Johnson. (1990). "Influence of heat-treatments on survival of pathogens in cheese:' University of Wisconsin Food Research Institute Annual Meeting. Poster presentation. Madison, WI. 10. Lammerding, A., K. Glass, A. Gendron-Fitzpatrick, and M. Doyle. (1990). Virulence of Listeria monocytogenes in pregnant animal models.]. Food Protection. 53: 902. 11. Lee, S., A. Yousef, and E. Marth. (1990). Thermal inactivation of Borrelia burgdorferi, the cause of Lyme Disease.]. ofFood Protection. 53 (4): 296-299. 151 I Center tor Dairy Research I~-----------------------\( 12. Leyer, G., and E. Johnson. (1990).Regulation of neurotoxin in Clostridium botulinum type E by nitrogen. Arch. Microbiol. 154:443-447. 13. Nelson, J. (1990).Where are Usteria likely to be found in dairy plants? Dairy, Food and Environmental Sanitation. 10(6):344-345. 14. Pearson, L, and E. Marth. (1990). Listeria monocytogenes - threat to a safe food supply: A review.T. Dairy Sci. 73: 912-928. 15. Pearson, L, and E. Marth. (1990). Behavior of Listeria monocytogenes in the presence of methylxanthines-caffeine and theobromine. ]. Food Protection. 53 (1): 47-50. 16. Pearson, L., and E. Marth. (1990). Behavior of Listeria monocytogenes in the presence of cocoa, carrageenan, and sugar in a milk medium incubated with and without agitation. ]. Food Protet:tion. 53 (1): 30-37. 17. Pearson, L, and E. Marth. (1990). Inhibition of Listeria monocytogenes by cocoa in a broth medium and neutralization of this effect by casein. J. Food. Protection. 53 (1): 38 46. 18. Wenzel, J., and E. Marth. (1990). Behavior of Listeria monocytogenes in the presence of Streptococcus lattis in a medium with internal pH control. ]. Food Protet:tion. 53: 918 923. 19. Wenzel, J., and E. Marth. (1990). Behavior of Listeria monocytogenes at 4 and 7"<: in raw milk inoculated with a commercial culture of lactic acid bacteria. Mikhwissenschaft. 45 (12): 772-774. 20. Wenzel, J., and E. Marth. (1990).Changes in populations of Listeria monocytogenes in a medium with internal pH control containing Streptococcus cremoris. [. Dairy Sci.-73 (12):3357-3365. 21. Yousef, A, and E. Marth. (1990). Fate of Listeria monocytogenes during the manufacture and ripening of parmesan cheese.]. Dairy Sci. 73 (12): 3351-3356. II. Cheese Technology 22. Bhowmik, T.,and E. Marth. (1990). Esterases of Micrococcus speciesddentification and Partial Characterization. J. Dairy Science. 73 (1): 33-40. 23. Bhowmik, T.,and E. Marth. (1990). Peptide-hydrolysing enzymes of Pediococcus species. Microbios. 62: 197-211. 24. Bhowmik, T., and E. Marth. (1990). Role of Micrococcus and Pediococcus species in cheese ripening: A Review. ]. Dairy Science. 73 (4): 859-866. 25. Bhowmik, T.,and E. Marth. (1990). I>-galactosidase of Pediococcus species: Induction, purification, and partial characterization. Appl. Microbiol. Biotechnol. 33: 317 323. 152 )>---------------------------1 Annual Report 1.990-1.991. II 26. Bhowmik, T., B. Riesterer, M. van Boeke),and E. Marth. (1990). Characteristics of low-fat Cheddar cheese made with added Micrococcus or Pediococcus species. Milchwissenschaft.45 (4):230-235. 27. Bhowmik, T., J. Royneberg, J. Steele, and M. Johnson. (1990>. Construction and characterization of a lactose-negative, proteinase-negative derivative of Uzctococcus lactis ssp. cremoris E8 and its use as a starter adjunct in low-fat cheese manufacture (Paper 045). Annual Meeting of American Dairy Science Association.f.Dairy Science 73: (SuppI. 1) 84. 28. Oemons, J., and M.R. Etzel. (199(}). "Inactivation of Lactobadlli during spray drying." AIChEAnnual Meeting. Chicago, IL. 29. Etzel, M.R. (1990). "Freeze-drying and spray drying of microorganism." Biotechnology and Industry Symposium. Pewaukee, WI. 30. Etzel, M.R. (1990). "Freeze-drying and Spray-drying of microorganisms." Industrial and Environmental Biotechnology Seminar Series. Madison, WI. 31. Etzel, M.R. (1990). "Inactivation of microorganisms during drying:' Invited speaker at the Bioprocess and Metabolic Engineering Consortium Annual Meeting. Madison, WI. 32. Etzel, M.R., and J. Oemons. (1990). "Drying of dairy starter cultures." Invited speaker at Chr. Hansen's Laboratory, Inc, Milwaukee, WI. 33. Johnson, J.A.C., and M.R. Etzel. (1990). "Inactivation of lactobacilli during spray drying (Paper 306b)." AIChEAnnual Meeting. Chicago, IL. 34. Johnson, J.M. (1990). The effect of sodium, calcium, and magnesium chloride salts on the chymosin-induced coagulation of skim milk. M.Sc. Thesis. UW-Madison Food Science Department. 35. Johnson, M.E., B. Riesterer, C. Chen, W. Tricomi, and N. Olson. (199()). Effect of packaging and storage conditions on calcium lactate crystallization on the surface of Cheddar cheese.f. Dairy Science. 73: 3033-3041. 36. Johnson, M.E., B. Riesterer, and N. Olson. (1990). Influence of nonstarter bacteria on calcium lactate crystallization on the surface of Cheddar cheese.f. Dairy Science. 73 (S): 1145-1149. 37. Khalid, N., M. El-Soda, and E. Marth. (1990). Esterases of Lactobacillus helveticus and Uzctobacillus delbrueckii ssp. bulgaricus.f. Dairy Sdence. 73: 2711-2719. 38. I their enzymes and role in ripening and spoilage of cheese: A review.f. Dairy Science. 73: 2669-2684. 39. Khalid, N., and E. Marth. (1990). Partial purification and characterization of an aminopeptidase from Uzctobadllus helveticus CNRZ 32. System. Appl. Microbial. 13: 311 319. 40. Khalid, N., and E. Marth. (1990). Proteolytic activity by strains of I..tu:tobadllus plantarum and Lactobacillus casei.]. Dairy Science: 73 (12): 3068-3076. 153 I Center for Dairy Research ~----------------(C 41. Khalid, N., and E. Marth. (199()). Purification and partial characterization of prolyl-dipeptidyl aminopeptidase from LActobacillus helveticus CNRZ 32. Applied and Environmentlll Microbiology. 56 (2):381-388. 42. Kim, 5., M. Kim, and N. Olson. (1990). Interactive effect of hydrogen sulfide (H2S) production from cysteine and methanethiol production from methionine in milk-fat coated microcapsules containing Breuibacterium linens. J. Dairy Research. 57: 579-585. 43. Kim,S., N. Olson, and T. Richardson. (1990).Polymerization and gelation of thiolated Ji-lactoglobulin at ambient temperature induced by oxidation by potassium iodate. MilchwissensduJft. 45 (10):627-631. 44. Kim,S., N. Olson, and T. Richardson. (1990).Thiolation of Ji-Iactoglobulin with N acetylhomocysteine thiolactone (N-AHTL) and 5-acetlylmercaptosuccinic anhydride. MilchwissensduJft. 45 (9):580-583. 45. Olson, N. (1990).The impact of lactic acid bacteria on cheese flavor. FEMS .Microbiology Reuiews. 87: 131-148. 46. Olson, N., and M. Johnson. (1990). Light cheese products: Characteristics and economics. Food Technology. (10): 93-96. 47. Paluch, L. (1990). Cheddar cheese made from milk pasteurized at 73.3"C,75.6"C, and 77.8"C. M.Sc. Thesis. UW-Madison Food Science Department. 48. Spangler, P., L Jensen, C. Amundson, N. Olson, and CHill. (1990).Gouda cheese made from ultrafiltered milk: Effects of concentration factor rennet concentration, and coagulation temperature. J. Dairy Science. 73: 1420-1428. . 49. Steele, J.L. (1990). ''Peptidases of lActobacillus helveticus:' A Brown Bag seminar at the Department of Bacteriology, University of Wisconsin-Madison, October 25. SO. Wendorff, W. (1990). Companion manual for Wisconsin cheesemaker's video short course. UW-Madison Food Science Department. 51. Wendorff, W., and B.Joppa. (1990). A compilation of lectures and references for the Wisconsin Cheesemaker's short course. UW-Madison Food Science Department Cooperative Extension and Center for Dairy Research. 52. Wendorff, W. (1990). Hydrogen peroxide in milk - cheesemaker beware. UW Dairy Pipeline. 2 (1): 1-3. 53. Wendorff, W. (1990). "Hydrogen peroxide adulteration of milk:' Wisconsin Laboratory Association, Annual Convention. Milwaukee, WI. 54. Wendorff, W. (1990).Mastitis - hidden cost for cheesemakers. UW Dairy Pipeline. 2 (2): 1-3. 154 )f---------------------I Annual Report 1990-1991 II III. Milk Component Utilization 55. Abuirmeileh, N., and C. Elson. (1990). The potential of the mevalonate-reversible isoprenoid-mediated suppression of H. Halobium growth by pentobarbital. FASEBJ. 4: A927. 56. Bleck, G., and R. Bremel. (1990). Isolation and cloning of a gene encoding bovine a-lactalbumin (Paper No 1'242).J. Dairy Sci. 73 (Suppl. 1): 200. 57. Bremel, R. (1990). "Genetic engineering of milk. It Proceedings of FIrSt Bulgarian American Symposium. Borovetz, Bulgaria. 58. Cameron, D.C. (1990). "Microbial production of polysaccharides from lactose." Midwest Biotechnology Symposium. St. Paul, MN. 59. Cameron, D.C. (1990). ''Polysaccharide production from whey." Presentation at the WMMB nonfat solids research advisory committee meeting. Madison, WI. 60. Chen, J. (1990). Novel affinity-based processes for protein purification. A review. J. ofFermentation andBiotechnology. 70 (3): 199-209. 61. Cooper, T.A, J.H. Flatt, EN. Lightfoot, and D.C. Cameron. (1990). "Characterization and production of a superior succinylated zoogIan polysaccharide from whey by Zoogloea ramigenz 115." Poster presented at the UW Bioprocess and Metabolic Engineering Consortium Annual Meeting. Madison, WI. 62. Elliot, J. (1990). Modification of butteroil by lipase-catalyzed acyl-exchange reaction in anhydrous media. MSc. Thesis. UW-Madison Food Science Department. 63. Elliot, J., M. Mani, S. Kuo, and K. Parkin. (1990). "Lipase-mediated transesterifications of butteroil in nonaqueous media," 51st 1FTAnnual Meeting. Anaheim, CA. 64. Flatt, JR. (1990). Microbial production of novel polysaccharides from lactose in whey permeate. Ph.D. Thesis. UW-Madison Chemical Engineering Department. 65. Flatt, J.H. (1990). "Microbial production of a novel polysaccharide from lactose in whey permeate." Presentation at the UW Bioprocess and Metabolic Engineering Consortium Annual Meeting. Madison, WI. 66. Flatt, J.H., and T.A. Cooper. (1990). Zooglan Polysaccharide. U.S. patent submitted September 1990. 67. Flatt, J., T. Cooper, E. Lightfoot, and D. Cameron. (1990). "Production of a high quality polysaccharide from lactose in whey dairy waste by Zoogloea ramigera: Fermentation kinetics and control:' A1ChE Annual Meeting. Conference Extended Abstracts (paper 48A). Chicago, IL. 68. Flatt, J., R. Hardin, J. Gonzalez, and D. Cameron. (1990). "Polysaccharide production from lactose by a newly isolated soil bacterium," NATO Advanced Research Workshop on New Biosynthetic Biodegradable Polymers of Industrial Interest from Microorganism. Sitges, Spain. 1"55 I Center for Dairy Research ----------------{( 69. Flatt, J., R. Hardin, J. Gonzalez, T. Cooper, D. Dogger, E. Lightfoot, and D. Cameron. (1990). "Production of a novel polysaccharide by a newly isolated lactose utilizing microorganism." AIOtE Annual Meeting. Conference Extended Abstracts (paper 301E). Chicago, IL. 70. Garda, H., C. Amundson, and C. Hill. (1990). "Partial characterization of the action of an A. niger lipase on butteroil emulsions." Annual Meeting of the Institute of Food Technologists. Anaheim, CA. 71. Garda, H., H. Reyes, F. Malcata, C. Hill, and C. Amundson. (1990). Determination of the major free fatty acids in milkfat using a three-component mobile phase for HPLC analysis. Milchwissen?haft. 45 (12): 755-822. 72.Gould, B.,T. Cox, and F. Perali. (1990).The demand for fluid milk products in the U.S. - A demand systems approach. Western Journal of Agricultural Economics 15 (1): 1 12. 73. Hardin, RS., J.H. Flatt, and D.C. Cameron. (1990). Polysaccharide producing organism and novel galactomannan polysaccharide. US. patent submitted May 1990. 74. tai, H.-e., OM. Ney, J.B. Lasekan, and M.I<.Cayton. (1990). In vivo determination of triglyceride secretion in rats fed different dietary saturated fats using [2-S"H]-glycerol. FASEBJ. 4: 2297. 75. Lai, H.-c. (1990). Plasma lipoprotein composition and hepatic triglyceride secretion in response to ingestion of dietary saturated fats in rats. M.Sc. Thesis. UW Madison Nutritional Sciences Department. 76. Malcata, F., C. Hill, and C. Amundson. (1990). "Enzymatic hydrolysis of butteroil using a hollow fiber reactor." AIChE Meeting. Chicago, IL. 77. Malcata, F., C. Hill, and C. Amundson. (1990). "Hydrolysis of butterfat using a membrane reactor." Annual Meeting of tile Institute of Food Technologists. Anaheim, CA. 78. Malcata, F., H. Reyes, H. Garda, C. Hill, and C. Amundson. (1990). Immobilized lipase reactors for modification of fats and oils - A review. ]. Am. Oil Chern. Soc. 67 (12): 89Q..910. 79.Mao, F., and R. Bremel. (1990). Development of sensitive a-lactalbumin, ~ lactoglobulin and a s1-easein EUSAs for mammary explant culture study (Paper No P241). J. Dairy Science. 73 (Suppl. 1): 200. 80. Mao, F:, and R. Bremel. (1990). Genetic variation of prolactin dependent milk protein regulation: a needle biopsy model (Paper No P240). J. Dairy Science. 73 (Suppl, 1): 200. . 81. Ney, D.M. (1990). "Effects of milkfat on human lipid metabolism:' Wisconsin Dairy Technology Society. Milwaukee, WI. 82. Ney, D.M. (1990). "Effects of milkfat on human lipid metabolism." Dean Foods Annual Research Meeting. Eagle Ridge, IL. 156 Anmud Report 83. Ney, D.M. (1990). "Milkfat - nutritional issues of the 1990's." Wisconsin Dairy Products Association Annual Meeting. Madison, WI. 84. Ney, D.M. (1990). Potential for enhancing the nutritional properties of milkfat. Symposium - The role of nutritional &: health benefits to marketing dairy products. 1990 ADSA Annual Meeting. [: Dairy Science. 73:(5uppl.1) 98. 85. Ney, D.M. (1990). ''Relative effects of milkfat on lipoprotein composition and in vivo hepatic triglyceride secretion in rats." 81st Amer. Oil Chern. Soc. Annual Meeting. Baltimore, MD. 86. Ney, D.M., H.'<:. Lai, M. Lefevre, and J.B. Lasekan. (1990). Relative effects of milkfat on lipoprotein composition and in vivo hepatic triglyceride secretion in rats. INFORM. 1 (4):333-334. 87. Pai, H., and J. Chen. (1990). "Enzymatic hydrolysis of milkfat in reversed micelle systems." Annual Meeting of the American Institute of Chemical Engineers. Chicago, IL. 88. Reyes, H., C. Hill, and C. Amundson. (1990). "Kinetics of the interesterification reactions of olive oil in the presence of lipase from Pseudomonas cepacia immobilized on a hydrophobic support:' AIChE Meeting. Chicago, IL' 89. Wang, C; and J. Chen. (1990). "Isolation of lactoferrin and immunoglobulin G from cheese whey by affinity microfiltration (Paper No 82)." IFf Annual Meeting. Conference Abstracts, p. 119. Anaheim, CA. 90. White, T. (1990). Solubilities of selected fatty acids in supercritical carbon dioxide. M.Sc. Thesis. UW-Madison Food Science Department. New Product and Process Development 91. Bakken, A. (1990). Novel immobilized J3-galactosidase reactors for the hydrolysis of lactose in skim milk. Ph.D. Thesis. UW-Madison Chemical Engineering Department. 92. Bakken, A., C. Hill, and C. Amundson. (1990). "A novel immobilized J3 galactosidase reactor to hydrolyze the lactose constituent of skim milk: m. 13 Galactosidase from Bacillus circulans" AIChE National Meeting. Chicago, IL. 93. Bakken, A., C. Hill, and C. Amundson. (1990). Use of novel immobilized 13 galactosidase reactor to hydrolyze the lactose constituent of skim milk. Biotechnol. Bioeng. 36:293-309. 94. Behling, A., and J. Greger. (1990). Importance of lactose in yogurt for mineral utilization. l- Agricultural andFood Chemistry. 38 (1): 200-204. 95. Brummel,S., and K. Lee. (1990). Soluble hydrocolloids enable fat reduction in process cheese spread. I. Food Sci. 55 (5):1290-1293. 157 I Center for Dairy Research 1-----------------------;( 96. Chung, M.S. (1990). Formation of contact secondary nuclei from crystals in sugar solutions and fluid dairy products. M.5c. Thesis. UW-Madison Food Science Department. 97. Greger, J., S. Kaup, C. Powers, and K. Lee. (1990). Bioavailability of calcium from calcium-fortified cottage cheese. FASEB J. 4 (3): 1474. 98. Lee, K.,J. Greger, and N. Puspitasari. (1990). "Minimizing bitter flavor defects in cottage cheese fortified with calcium." 23rd International Dairy Congress. Montreal, Canada. 99. Puspitasari, N.L. (1990). Suppression of bitter flavors in calcium fortified cottage cheese with hydrocolloids. M.5c. Thesis. UW-Madison Food Science Department. 100.Reykdal, O. (1990). Soluble, dialyzable and ionic calcium in milk, spinach and calcium fortified cottage cheese with correlation to bioassay. M.Sc. Thesis. UW- Madison Food Science Department. . 101.Shi, Y., B. Uang, and R. Hartel. (1990). Crystallization kinetics of alpha-lactose monohydrate in a continuous cooling crystallizer. J. Food Science. 55 (3):817-820. 102 Shi, Y.,B.Liang, and R. Hartel. (1990). Crystallization of ice from aqueous solutions in suspension crystallizers. In: Crystalliz.t1.tion lIS a separation process (Editors: Myerson, AS. and K. Toyokura). ACS Symposium Series 438. Chp 23: 316-328. 103. Shi, Y.,B. Uang, and R. Hartel. (1990). "Nucleation and growth of ice crystals in a 'batch suspension crystallizer." Industrial Crystallization Symposium. Garmisch, West Germany. Other Topics 104.Gould, B., and W. Saupe. (1990). Changes in the distribution ofincome and wealth of farm households: Evidence from Wisconsin panel data. North Central Journal of Agricultural Economics. 12 (1): 31-46. 105.Olson, N. (1990). Food research: past, present, and future. J. Dairy Science. 73 (5): 1193-1200. 106. Wendorff, W. (1990). "Education and training for people working in the industry - short courses, extension:' International Dairy Congress. Montreal, Canada. 107. Wendorff, W., Price, W.V. (1990). The dairy school - A century of service to the industry (1890-1990). Produced by the Department of Food Science, College of Agricultural and Ufe Sciences, Cooperative Extension, and University of Wisconsin Extension. 158 )1----------------------1 Annuid Reporl1990-1991 II Appendix A: Index of Principallnvestigafors and Academic Departments I. Principal Investigators Bremel, Robert Professor Department of Dairy Science 263-5652 Cameron, Douglas Assistant Professor Department of Chemical Engineering 262-8931 Chen,J.P Assistant Scientist COR 262-2253 Damodaran, Srinivasan Associate Professor Department of Food Science 263-2012 El-Soda, Morsi Senior Visiting Scientist CDR 262-5970 Elson, Charles Professor Department of Nutritional Sciences 262-1332 Etzel, Mark Assistant Professor Departments of Food Science and Chemical Engineering 263-2083 First, Neal Professor Department of Meat and Animal Science 2634307 Gould, Brian Associate Scientist Greger, Janet Professor Department of Nutritional Sciences 262-9972 Gunasekaran, Sundaram Assistant Professor Deparbnents of Agricultural Engineering and Food Science 262-1019 Hanel, Richard Associate Professor Department of Food Science 263-1965 Hill, Charles Jr. Professor and Chair Department of Chemical Engineering 262-1092 Jesse, Edward Professor and Chair Department of Agricultural Economics 263-4472 Johnson, Eric Associate Professor Department of Food Microbiology and Toxicol ogy 263-7944 Johnson, Mark Senior Scientist CDR 262..Q275 Lightfoot, Edwin Professor Department of Chemical Engineering 262-6934 COR and Deparbnent of Agricultural Economics 263-3212 continued next page... 159 I Center for DlI.iry Research 1-----------------------\( Lindsay, Robert Professor Department of Food Science 263-2568 Marth, Elmer Emeritus Professor Departments of Food Science and Food Microbiology and Toxicology 263-7280 Ney, Denise Associate Professor Department of Nutritional Sciences 262-4386 Olson, Norman Director, CDR Professor Department of Food Science 262-5970 panza, Michael Director, Food Research Institute Professor Department of Food Microbiology and Toxicol ogy 263-77TJ Parkin, Kirk Associate Professor Department of Food Science 263-2011 Steele, James Assistant Professor Department of Food Science 262-5960 Wendorff, William Assistant Professor Department of Food Science Extension dairy manufacturing specialist 263-2015 II. Academic Departments Agricultural Economics, Department of 263-4472 Agricultural Engineering, Department of 262-3310 Chemical Engineering, Department of 262-1092 Dairy Science, Department of 263-3308 Food Microbiology and Toxicology, Department of; (Food Research Institute) 263-77TJ Food Science, Department of 262-3046 Meat and Animal Science, Department of 263-4300 Nutritional Sciences, Department of 262-2727 160