Presence and Activity of Psychrotrophic Microorganisms in Milk and Dairy Products: A Review 1

Transcription

1 172 Journal of Food Protection, Vol. 45, No.2. Pages !,February 1982) Copyright, International Association of Milk, Food, and Environmental Sanitarians Presence and Activity of Psychrotrophic Microorganisms in Milk and Dairy Products: A Review 1 M.A. COUSIN Animal Sciences Department and Food Sciences Institute, Purdue University. West Lafayette, Indiana (Received for publication March 18, 1981) ABSTRACT The presence and metabolic activity of psychrotrophic microorganisms in milk and dairy products are reviewed. Problems involved in adequately defining the microorganisms and temperatures of growth are discussed. The sources and incidences of psychrotrophs in milk and dairy products and methods to control these microorganisms are presented. Methods ranging from simple plate counting techniques to detection of metabolites produced by the psychrotrophs are reviewed. Alterations of protein, lipid and carbohydrate fractions of milk and their effects on the keeping quality of milk and dairy products are discussed. Finally, additional research areas are suggested. Extended refrigerated storage of milk on the farm, in transport, at the dairy plant, and in the supermarket have required an increased shelf-life for fluid milk. Milk is collected on alternate days from dairy farms in most areas of the United States. However, the decrease in number of dairy farms and dairy processing plants causes some raw milk to be transported for long distances before it reaches a processing plant. Once the milk reaches the processing plant, it may be stored for 3 to 4 days before it is processed. This is particularly true if the milk arrives at a processing plant on Friday, since some plants operate only on a 5-day schedule per week. After processing, the pasteurized milk is coded for a 10 to 14 last-day-of-sale beginning with the date of processing. Generally, the milk is expected to remain acceptable for about 5 days past the last-day-of-sale. Hence, the total age of the milk before consumption could be 20 to 21 days. Storage of milk for long periods at refrigeration temperatures has resulted in new quality problems for the dairy industry. These problems are related to growth and metabolic activities of microorganisms at low temperatures. These microorganisms, which are termed psychrotrophs, are ubiquitous in nature and common Journal paper No of the Purdue University Agricultural Experiment Station. contaminants of milk ( ,328,330,333). As these psychrotrophs increase in number throughout refrigerated storage, enzymes are synthesized during microbial growth in the milk. These enzymes, many of which are heat-stable, biochemically alter the milk eventually causing spoilage. Prevention of psychrotrophs from entering milk is technically possible, but it is difficult to achieve in practice because human errors result in milk contamination. This review of psychrotrophs in milk and dairy products will summarize and organize the voluminous information generated on these microorganisms into 10 categories: (a) definition, (b) temperature, (c) types of microorganisms, (4) sources, (e) incidence in milk, (ft methods to enumerate, (g) biochemical changes in milk, (h) milk and dairy product keeping quality, (l) control and inhibition and (J) future research. DEFINITION The definition as well as the terminology for microorganisms that are able to grow at temperatures close to 0 C has confused microbiologists since the beginning of this century. In 1887, Forster first observed bacterial growth at 0 C, but it was not until 1902 that Schmidt-Nielsen termed these microorganisms psychrophiles (121,122,310,318,332,378). This term is derived from the Greek words psychros, which means cold, and philos, which means loving. Therefore, the term psychrophile implied that the microorganism grows best at low temperatures and is cold-loving. Cryophile and rhigophile are other names, derived from Greek words, that have been used for microorganisms that grow at low temperatures (122). However, several investigators have objected to use of these terms since they imply a preference for growing at low temperatures when these microorganisms actually grow better at 20 C or higher ( ,310,324,332). Unlike the terms mesophile and thermophile, which define microbial growth at the optimum temperatures, psychrophiles have been defined in many different ways JOURNAL OF FOOD PROTECTION. VOL. 45. FEBRUARY 1982

2 PSYCHROTROPHS IN DAIRY PRODUCTS 173 based on: (a) optimum growth temperature, (b) growth at low temperatures, (c) methods of enumeration and (d) other criteria unrelated to temperature (;378). The optimum temperature of growth can refer to that temperature at which reproduction is most rapid, maximum populations are reached or a combination of these two occur. Morita (214) has defined psychrophilic bacteria as microorganisms having an optimum growth temperature of 15 C, a maximum grmvth temperature of 20 C and a minimum growth temperature of 0 C or below. This is also the classical definition used in most microbiology textbooks. However, most microbiologists think of psychrophiles as microorganisms that can grow at low temperatures. Stokes (;310) stated that the best way to identify psychrophiles was by observing visible growth at 0 C within 1 week, although the temperature where growth was most rapid was closer to 30 C or lower. A similar definition was proposed by Elliott and Michener (76). Historically, the temperature of isolation or enumeration of microorganisms that grow at low temperatures has served as a definition (378). The temperature and time of incubation and medium used would result in growth of different microorganisms and tabulation of different microbial counts. Psychrophiles have been defined by classifying them into groups based on criteria other than temperature, such as: psychrophiles are generally gram-negative rods, psychrophiles are usually asporogenous, psychrophiles are non-acidforming, and so on ( ,378). Since the term "psychrophiles" incorrectly labels microorganisms capable of growth at low temperatures, other names have been proposed (;310,318,332). Psychrocartericus or cold-conquering proposed by Kruse in 1910 and again by Rubentschik in 1925, psychrotolerant or cold-tolerant by Horowitz-Wlassowa and Grinberg in 1933, eurythermic or capable of growing over a wide range by ZoBell in 1934, and psychrotrophic or cold-thriving by Eddy in 1966, are a few of the names that have been proposed (74,144,310,330). Mossell suggested that the word psychrotrophic should be used for microorganisms able to grow on solid media at 5 C or below regardless of their optimum growth temperature (74.244). Morita (214) suggested that those mesophilic microorganisms that could grow at 0 C are more correctly termed psychrotolerant or psychrotrophic than psychrophilic. Most of the literature published before 1960 probably refers to psychrotrophs rather than psychrophiles. The term psychrophile should refer only to those microorganisms whose optimum temperature of growth or minimum temperature of generation is achieved by low temperatures (74,144). In the dairy industry, and to some extent other food industries, psychrotrophs are defined as those microorganisms able to grow at 7 C or less regardless of optimum growth temperatures (159,319,332). The terms psychrophile, psychrotroph and cryophile are still used interchangeably by microbiologists; however, in this paper psychrotroph will be used to identify microorganisms able to grow at 7 C or less, regardless of optimum temperature and psychrophile will be used for microorganisms that conform to Morita's definition (214). TEMPERATURE Growth temperatures Though psychrotrophs can grow at temperatures close too C, their optimum temperature is much higher. Elliott and Michener (76) reported that optima for growth of most microorganisms are 20 to 30 C with some having optima of 30 to 45 C and very few of 15 Cor below. The minimum growth temperature for psychrotrophic bacteria has been reported as -10 C (122,318). Kraft and Rey (159) listed the minimum growth temperatures for microorganisms in foods as -10 C for most bacteria, -18 C for most molds and -12 C for most yeasts. The maximum growth temperature for psychrotrophs usually is stated as 30 C, but some have maxima of 37 to 45 C (116,122,310). Normally, growth at a low temperature is characterized by a long lag phase and a slow logarithmic phase (76,95,122). The lag phase is shortest at the optimum temperature and becomes increasingly longer as temperature is lowered. The optimum growth temperature can be obtained by determining the temperature at which the generation time is shortest. Some of the generation times reported for psychrotrophs and psychrophiles are presented in Table 1. After examining these data, it is evident that only Vibrio marinus is a psychrophile and all other organisms are psychrotrophs since their generation times decrease significantly as temperature increases. The rate of growth in liquid media at subzero temperatures is listed in Table 2. Inniss (123) also reported that growth was observed on solid media for two gram-positive cocci and one gram-negative rod at -2 C, for four yeasts at -4.5 C and for five Bacillus species at -7 to -10 C. The psychrophiles that have been studied at subzero temperatures may grow at even lower temperatures; however, toxicity of the antifreeze used in the media has prevented further investigation. Y ano et al. (381) studied generation times for psychrotrophic bacteria in refrigerated raw milk and found a range of 6.6 to 12.7 hat 5 C and 12.2 to 26.1 at 0 C. Therefore, the keeping quality of the milk was about 2 to 5 days at 5 C and approximately 4 to 13 days at 0 C. Some microorganisms with longer generation times at a given temperature did not attain the same population levels as those for microorganisms with longer generation times (94). Response to low temperatures Temperature is one of the most important variables affecting microbial growth within natural or controlled laboratory environments. However, much of the literature on the physiology of growth at low temperatures was generated from experiments that used higher temperatures than the environments from which the microorganisms were isolated. Therefore, present researchers who JOURNAL OF FOOD PROTECTION. VOL. 45, FEBRUARY 1982

3 174 COUSIN TABLE 1. Generation times (h) at temperature (C) of psychrophiles and psychrotrophs. Microorganism Reference number Bacillus coagulans Bacillus spp. (GpA) Bacillus spp. (GpB) 295 Clostridium hastiforme 21 Enterobacter aerogenes (#48) Pseudomonas sp. (#92) Pseudomonas sp. (#69) Pseudomonas fragi 236 Pseudomonas ftuorescens 236 Pseudomonas ftuorescens Pseudomonas sp Pseudomonas sp. (#92) a a3c TABLE 2. Generation times (days) at subzero temperatures (C) of psychrophiles. Organism Reference number Generation time Temperature (C) (days) Bacillus spp. Bacillus spp. Bacillus spp to -7 Bacillus cryophilusll study growth of microorganisms at low temperatures interpret earlier studies with scientific caution and present studies with the strict definition of psychrophiles and psychrotrophs. In an effort to explain growth at low temperatures, many investigators have used the Arrhenius equation to describe how chemical reactions are affected by temperature (12,115,123,124,214). By using this procedure, Ingraham (120) postulated that a psychrophile could be distinguished from a mesophile since temperatare affected the resultant growth curve. When the bacterial growth rate is substituted for the reaction rate in the Arrhenius equation, the J.t-values generated will be lower for psychrophilic than for mesophilic microorganisms. Subsequent research has challenged this theory since no significant differences were noted between a psychrophile, a psychrotroph and a mesophile of the same genus (115,214). Though ~A-values do not adequately describe the growth characteristics of psychrophiles, psychrotrophs and mesophiles, the Arrhenius curve of log-specific growth rate versus 1/"K appears significant (115). The Arrhenius curve for psychrophiles is linear to 0 C and below, but that for psychrotrophs deviates from linearity at 4-5 C and for mesophiles it deviates from linearity at even higher temperatures (115,124). Protein synthesis can be affected by temperature. The ability to carry out protein synthesis at low temperatures has been linked to the ribosomal content of the cell (123,124,214). Inniss and Ingraham (124) reported a positive correlation between ribosomal denaturation temperature and the maximum temperature of growth for some psychrophiles. However, all ribosomes of psychrophiles did not become inactivated at elevated temperatures (123). Ribosomes from a psychrotrophic Pseudomonas species remained active at 2 to 9 C, whereas those from a mesophilic Escherichia coli did not (214). Therefore, some researchers have postulated that microorganisms synthesize increased amounts of enzymes to adjust to growth at low temperatures in response to reduced enzyme activity (76,115). Some psychrotrophs exhibit a preferential release of proteolytic and lipolytic enzymes at low temperatures (76,378). Likewise, some enzymes from psychrophilic and psychrotrophic microorganisms are temperature-sensitive and may be responsible for establishing maximum growth temperatures (123,124,213). The ability of microorganisms to grow at low temperatures has been linked to cell permeability and substrate uptake ( ,214). Substrate uptake was reduced for psychrotrophic Vibrio species at low temperature; however, some studies indicated that substrate uptake by psychrophiles was independent of temperature (115). Other investigators have shown that sugar uptake by psychrophiles occurs maximally at 0 C and decreases with increasing temperature (115). Solute transport may be affected by the degree of unsaturation of fatty acid side chains in membrane lipids (115). Psychrophiles and psychrotrophs generally have higher levels of unsaturated fatty acids than do mesophiles (115,123, ). The minimum growth temperature probably is not determined by the fatty acid composition since experiments with Escherichia coli have shown that this organism can grow at 12 and 37 C with the same lipid composition ( ). JOURNAL OF FOOD PROTECTION. VOL. 45. FEBRUARY 1982

4 PSYCHROTROPHS IN DAIRY PRODUCTS 175 Gill (91) has shown no significant differences in amounts of extractable lipids or phospholipids when psychrotrophic Pseudomonas jluorescens was grown at temperature extremes from 3 to 30 C. Further research with P. fluorescens suggested that the increase in saturation of lipids at higher temperatures may result from the microorganism's inability to control the mechanism in response to environmental changes (92). No physiological basis exists for a change to occur in the amount or type of lipids with a change in temperature. Normally, the activities regulating the enzymes vary so that the fatty acid composition remains constant regardless of temperature fluctuation. The significance or reality of a change in unsaturation of cell lipids due to decreased temperatures has not been resolved. Moderate temperature changes can alter physiological or permeability functions of microorganisms that grow at low temperatures. The permeability barrier of the cell can be damaged sufficiently to allow leakage of intracellular components or total cell lysis (123,124,214). In psychrophiles, leakage of protein, DNA, RNA and amino acids was noted at 20 C (123,214). Lysis of marine Vibrio species has been revealed by phase optics when temperatures exceed 20 C and for some species leakage and lysis occur simultaneously after 95% of the cells are dead (214). No information is available on whether psychrotrophs react in a similar manner at elevated temperatures. Early literature on growth of bacteria at low tern perature reported that high cell yields were obtained and this increase in cell yield was attributed to the increased solubility and availability of oxygen at low temperatures (76,122,297). Later research on marine psychrophiles has shown that the minimum uptake of oxygen and the maximum cell yield occurred at the optimum growth temperature for the microorganisms (115,214). The data indicated that the psychrophiles increased the amounts of respiratory enzymes produced, resulting in growth maintenance, but decreased cell yield at suboptimum temperatures. Herbert and Bhakoo (115) reported that cell yield varied depending on the microorganisms studied. Pseudomonas species produced 0.25 mg of cells (dry weight) per mg of carbon at 16 C with a growth rate of 0.20; however, Vibrio AF-1, which had a growth rate of 0.05 at 15 C, produced 0.60 mg of cells (dry weight) per mg of carbon. Therefore, the rapidly growing psychrophiles may be trading cell yield for the rapid rate of growth. More research is needed before definite correlations between cell yield, oxygen utilization and growth rate can be established. Frank et al. (84) grew two pseudomonads at 2 and 30 C and found no difference in cell size, protein content, DNA or RNA, but the growth rate was 10-fold less at 2 than at 30 C. These authors concluded that growth at low temperatures may be possible because the structure and enzymes are similar to those at higher temperatures. Temperature adaptability Early reports of adaptation of microorganisms to low-temperature growth are rare (76,122,324). Generally, the ability of microorganisms to grow at 0 C is a specific property of some microorganisms and cannot be acquired by culturing microorganisms at lower temperatures. Azuma et al. (9) reported that attempts to adapt bacteria to low-temperature growth by serial transfers have been unsuccessful. Temperature adaptability of psychrotrophic Pseudomonas species were studied using thermal gradient incubator and changes observed suggested that adaptation depended on the microorganism's ability to metabolize substrates normally (.383). Conclusions from this research indicated that microorganisms that are transferred to lower growth temperatures may need an induction period in which biochemical changes necessary for substrate metabolism, enzyme synthesis or cell permeability take place. In an effort to understand how temperature affects the growth of microorganisms, mutant strains have been developed. Generally two types of mutants are used: (a) a mutant which has a different temperature growth range from that of the parent strain because both the minimum and maximum growth temperatures have been increased or decreased and (b) a mutant in which either the maximum or minimum growth temperature differs from that ofthe parent strain (123). There is genetic evidence that a mesophile can be converted to a psychrotroph by transduction or exposure to ultraviolet light and that a psychrotroph can be converted to a mesophile by exposure to ultraviolet light (214). However, Morita (214) concluded that a true psychrophile cannot be made by these methods since psychrophiles contain more than one thermolabile enzyme and several membrane differences when compared to mesophiles. Olsen and Metcalf (238) observed a change in the minimum and maximum growth temperature of Pseudomonas aeruginosa after a process involving transduction via a Pseudomonas bacteriophage P x 4. The transfer to a lower minimum growth temperature of 0 C as opposed to 11 C also caused a decrease in the maximum growth temperature from 44 to 32 C. This new mutant had a doubling time of 4 to 6 h at 3.5 C. Azuma et al. (9) also produced a mutant P. aeruginosa that grew at 6 C within 2 days and at 0 C within 8 days. The parent strain, which was not subjected to ultraviolet radiation, showed no growth at 10 C after 10 days incubation. Two types of temperature-sensitive mutants have been used to study the biochemical basis for growth at low temperatures and for minimum growth temperatures (123,124). Cold-sensitive mutants, which have an elevated minimum growth temperature, differed from the parent strain in only the lower temperature range of growth (124). From experiments using these mutants, the sensitivity to enzyme feedback was greater at lower than higher temperatures and the ability to grow at low temperatures depended on the properties of various proteins (123,124). Heat-sensitive mutants, which have a lowered maximum temperature, have been studied extensively (123). Their mechanism of adjustment to JOURNAL OF FOOD PROTECTION, VOL. 45. FEBRUARY 1982

5 176 COUSIN temperature involves decreased protein thermostability or lack of enzyme synthesis at the old temperature maximum. The isolation of only one temperature- sensitive mutant from psychrophilic parent strains has been reported (123). Undoubtably, mutants will continually be used to understand growth and adaptability to low temperatures. TYPES OF PSYCHROTROPHIC MICROORGANISMS General classifications Psychrotrophs, which are ubiquitous in nature, include bacteria, yeasts, and molds (311). These microorganisms may include long or short rods, cocci, or vibrios; gram-positive or gram-negative bacteria; sporeformers or nonsporeformers; and aerobic, facultative anaerobic or anaerobic microorganisms. Since many of the early investigators incorrectly labeled psychrotrophs as psychrophiles or failed to identify the isolates, it is difficult to tabulate all genera that have been isolated from milk and dairy products. Some researchers described the morphological characteristics and gram reaction; whereas, others used additional tests to identify the genera. Characteristics that have been used to identify psychrotrophic isolates are: Gram reaction, cell shape, motility, oxidative-fermentation (0/F) of carbohydrates by the Hugh-Liefson methods, production of ammonia from arginine, oxidase reaction according to Kovac's test, lipolysis, proteolysis and action on litmus milk (242). Otte et a!. (244) used a microtiter primary test system to identify bacteria to genus level using Gram stain, morphology, acid fastness, spore formation, motility, catalase and oxidase tests and the 0/F test. These miniaturized tests were used to classify to the genus level 98.So/oof the cultures isolated from milk and dairy products. Gram-negative rods The early literature reported that microorganisms that spoil milk at temperatures slightly above freezing are predominantly gram-negative, non-sporeforming, catalase-positive rods (121,310,316). Bacteria in the genus Pseudomonas normally are most frequently encountered in raw milk (71.121,132,148,180,207,289,305,310,314, 316,320,323,328,329,330,333,378). Other gram-negative bacteria that have been isolated from milk and dairy products are listed in Table 3. These gram-negative rods also increase in numbers during cold-storage, leading to deterioration and/or spoilage of the milk. Most of these gram-negative rods, especially P. fluorescens, are associated with proteolysis and/or lipolysis in milk and dairy products (71,100,148,164,168,207,209,235,242,305, 317,320,328,333,352,378). Gram-negative facultative anaerobic rods of the family Enterobacteriaceae collectively are referred to as coli-aerogenes or coliform bacteria (323,329). Species of Enterobacter and Klebsiella are the Enterobacteriaceae most frequently isolated from refrigerated raw milk TABLE 3. Gram-negative bacteria isolated from milk and dairy products. Genus Reference numbers Product(s) Achromobacte,Jl Acinetobacte,b Aeromonas Alcaligenes Chromobacterium Citrobacter Cytophaga Enterobacte, Escherichia Flavobacterium Klebsiella Pseudomonas Serratia Vibrio 58, 100, 121, 122, 126, 207, 289, 305, 310, 316, 320, 328, 330, 352, 378 Raw and pasteurized milks and cream, butter 209, 223, 314, 333 Raw and pasteurized milk and cream 6, 58, 207, 209, 328, 333 Raw and pasteurized milk and butter 100, , 126, 223, 289, 305, 310, 314, 316, 320, 328, 330, 352,Raw and pasteurized milk and 378 cream and butter 126, 207,223, 316 Raw milk 207, 317, 329, 349, 378 Raw and pasteurized milk 209 Rawmilk 6, , 207, 209, 328, 329, 330, 333, 378 Raw and pasteurized milk and butter 126, 207, 316, 349, 378 Raw milk 6, 100, 121, , 223, , 310, , , 352, 378 Raw and pasteurized milk and 20~316,31~320,32~349 6, 71, , , 180, 207, , 235, 289, 305, 310, ,32~323, 328,329,33~ , 209, 316, butter Raw milk and cream, butter, cottage cheese Raw and pasteurized milk and cream and butter Raw milk Butter agenus is no longer recognized in Bergey's Manual ofdeterminative Bacteriology, 8th edition, and most have been reclassified as Alcaligenes or Pseudomonas species. bsome authors rename Alcaligenes and Achromobacter species as Acinetobacter species. However, Bergey's Manual of Determinative Bacteriology, 8th edition, lists Alcaligenes as a genus of uncertain affiliation. centerobacter species were formerly called Aerobacter species. JOURNAL OF FOOD PROTECTION. VOL. 45, FEBRUARY 1982

6 PSYCHROTROPHS IN DAIRY PRODUCTS 177 (126,207,209,316,320,328,329,330,333,338). After Thomas (323) tabulated some research data on the incidence of coli-aerogenes bacteria in refrigerated raw milk, he noted that 5 to 33 o/o of the psychrotrophic microflora consisted of these organisms. Although these bacteria can ferment lactose to acid and gas at 37 C, research has indicated that psychrotrophic coliforms can contribute to proteolytic spoilage of milk (207,209,323,329). Presence of coliforms has been used to assess the sanitary production of milk and dairy products since these organisms are an indication of fecal contamination of milk and cream (320,329). Gram-positive bacteria Gram-positive bacteria have been isolated from raw milk; however, they are usually present in smaller numbers than the gram-negative bacteria. Table 4 lists the major gram-positive bacteria that have been isolated from milk and dairy products. Species of Micrococcus, Bacillus and Arthrobacter are the most frequently isolated gram-positive microorganisms (6,99, , 131,148,180,207,235,293,314,333,378). Witter (378) stated that early investigators were reluctant to include gram-positive microorganisms as psychrotrophs. Unfortunately, some of this thinking is still present today. However, presence of gram-positive bacteria has become more important in pasteurized milk and dairy products because some can form spores; they and others can survive the heat treatments given the products. Therefore, they have been linked to the possible spoilage ofthese products (58,98,200). Chung and Cannon (46) reported that 83.3% of raw milk samples analyzed contained from 2 to 900 spores/ml; however, these spores exhibited an 8- to 14-day lag phase at 7 C with generation times of 22 to 26 h. Similar lag and generation times were reported by other investigators: Bacillus coagulans grew in pasteurized milk in 13 to 17 days at 2 C with a generation time of 24 to 30 h (98) and Bacillus species have a long lag time with generation times of 15 to 20 h in pasteurized milk at 7 C (200). Thomas (323) reported that the generation time of Bacillus species was about 45 h at 5 C, compared to 7 h for some gram-negative rods. Hence, spoilage by TABLE 4. Gram-positive bacteria isolated from milk and dairy products. Genus Reference numbers Sources Arthrobacter 6,131,328,330,333,378 Raw milk, butter Bacillus 58, 99,131,207,293,305,314,316,333,349,378 Raw and pasteurized milk and cream Clostridium 21,61 Raw milk Corynebacterium 310,314,349 Raw and pasteurized milk and cream Lactobacillus 121, 122, 126, 378 Raw milk Microbacterium 58,209,349 Pasteurized milk Micrococcus ~5~ 121, 14~23~31~31~333,34~378 Raw and pasteurized milk Sarcina 378 Pasteurized milk Staphylococcus 148 Raw milk Streptococcus 58,121, ,20~349,378 Raw and milk TABLE S. Isolation ofbacillus species from milk and dairy products. ;;;:- R: ~ ~ "0 ] 13 "" ~ N R: s: ;::: s: c c ~ ~ ::1 ::l ~ "' ::l :::! ~ "'... ~ ~ "' ~ ~ Organism "" "' "' <11 Cl:: "" "" ~ Cl:: ~ ~ Cl:: Cl:: "" B. cereus B. circulans B. coagulans B.finnis + + B. lentus + + B. lichenifonnis B. macerans + + B. megaterium B. pantothenticus + B.polymixa + B.pumilus B. subtilis a Reference. ~ JOURNAL OF FOOD PROTECTION. VOL. 45, FEBRUARY 1982

7 178 COUSIN these sporeformers is slower and requires prolonged storage at refrigeration temperatures and selective conditions. Of all the species isolated from milk and dairy products, Bacillus cereus was of major concern since it caused a 'bitty cream' defect, food poisoning and reactions with the methylene blue test (49,57,62,200,248). Use of higher pasteurization temperatures and longer cold storage of pasteurized milk have led to concern about heat-resistant or thermoduric bacteria. Thermoduric psychrotrophs are usually gram-positive rods and cocci which belong to the genera Arthrobacter, Bacillus, Clostridium, Corynebacterium, Lactobacillus, Microbacterium, Micrococcus or Streptococcus (305,353,365). The significance of these microorganisms in pasteurized milk and dairy products depends on numerous variables: type present, cell numbers, total flora, product shelf life, storage temperature, etc. (365). Presently, the importance of all these factors has not been resolved. Fungi Psychrotrophic yeasts and molds, which can present problems, have been found in some dairy products. Psychrotrophic Candida, Saccharomyces, Rhodotorula, Torulopsis and Trichosporon species have been isolated from butter and, to a lesser extent, milk (122,254,316, 320,328). Psychrotrophic Aspergillus, Cladosporium, Geotrichum and Penicillium species have been associated with defects in butter and cream (320,328). Stokes (310) reported that species of Altemaria, Mucor and Rhizopus can be psychrotrophic. Pathogenic microorganisms Growth of psychrotrophs in foods usually does not result in illness since organisms able to cause foodborne illness rarely grow below 10 C, and seldom below 3.3 C. Stewart (308) reported that few B. cereus strains can grow below 10 C and most grow readily between 15 and 35 C although germination can occur as low as -8 C. Coghill and Juffs (49) reported that some strains of B. cereus germinated and increased by one to three log cycles in milk at 7 C within 10 days. Since no B. cereus isolates from milk were reported to grow below 10 C in a 1960 study, Mikolajcik ()00) postulated that continued use of refrigerated milk storage has allowed these sporeformers to adapt to outgrowth at low temperatures. Cox (57) reported that B. cereus species can grow between 4.5 and 7.5 C, and therefore, are important because some strains have been implicated in foodborne disease. Since Yersinia enterocolitica, serotype 0:8, was documented as causing foodborne illness from consumption of contaminated chocolate milk, research has focused on this organism, which can grow at temperatures as low as 0 C (176,288). Raw milk was implicated in two outbreaks caused by Y. enterocolitica in Canada U 76). Schiemann ()88) studied the incidence of Y. enterocolitica in cheese and found both Cheddar and Italian cheeses to be negative for this organism although 18.2 o/o of vats of cheese milk contained Y. enterocolitica. The organisms did not survive if proper acid developed in the cheese. Hanna et al. (107) reported that five strains of Y. enterocolitica did not survive heating.at 60 C for several minutes. Therefore, adequate pasteurization should destroy Y. enterocolitica and problems that result would be due to post-pasteurization contamination. The significance of this organism to milk has not been adequately assessed. Other pathogens have been isolated from milk and dairy products; however, these microorganisms had grown at higher temperatures and the pathogen or its toxin was simply present in the products during refrigerated storage. Mikolajcik ()03) reviewed information on pathogens that have been implicated in foodborne disease of fermented dairy products. Staphylococcus aureus and enteropathogenic E. coli multiplied at some point in the cheese-making process and were not of concern during refrigerated storage because these temperatures were below their normal growth ranges. Sharpe and Bramley 092) briefly reviewed reports of pathogens and viruses in raw milk and concluded that pasteurization destroys all pathogens, except Clostridium per.fringens, which is present in very small numbers and does not multiply because milk is aerobic in the natural state. In a survey of retail fluid milk products, Jones and Langlois (132) isolated only one pathogen, C. peifringens, which averaged less than one microorganism per milliliter of milk. The only known pathogens that are important because they can grow at low temperatures are B. cereus and Y. enterocolitica. More research is needed on both of these bacteria before their true significance in raw milk, pasteurized milk and dairy products can be determined. SOURCESOFPSYCHROTROPHS Forster in 1892 demonstrated that psychrotrophic bacteria are widely distributed in nature in both winter and summer. He found psychrotrophs in fresh and salt water, in and on fish, in meat, milk (1000/ml), garden soil (140,000/ml}, street dirt, canal water (1000/ml) and meadow water (122,332). Later investigators isolated psychrotrophs from soil, vegetables, meat, fish, milk, flour and air (122). Hence, many investigators have confirmed that psychrotrophs are ubiquitous in nature and that soil, water, plants and animals form the natural habitats of these organisms (122,310,311,316,318,330, ). Soil and vegetation Most psychrotrophic microorganisms in milk and dairy products usually come from soil, water and vegetation (330). Soil, grass and hay contain psychrotrophs that, at times, exceed 1 x 10 7 /g (332). Psychrotrophic colony counts on soil have ranged from 3 to 200 million/g (318,330). Thomas 018) reported that coryneforms and Arthrobacter spp. constituted 60o/o, while JOURNAL OF FOOD PROTEC110N. VOL. 45, FEBRUARY 1982

8 PSYCHROTROPHS IN DAIRY PRODUCTS 179 Pseudomonas, Alcaligenes, Achromobacter and Flavobacterium made up the remaining 40o/o, of soil psychrotrophs. Grass, hay, barley and oats contain from 5 x 10 5 to 2 x 10 8 psychrotrophs/g ( ). Water Water is also a source of psychrotrophic contamination in milk and dairy products. Treated farm water supplies can have psychrotrophic counts of 1 x 10 2 /ml for chlorinated water (332). Pseudomonas, Achromobacter, Alcaligenes and Flavobacterium dominated the psychrotrophic flora in water with Chromobacterium, Bacillus and coliforms in lesser numbers (316,318,330). Counts of 1 x 10 7 /ml or more of Flavobacterium have been found in chilled water around milk cans, especially water which is changed or chlorinated infrequently (316, ). Many of the strongly lipolytic and caseolytic psychrotrophs found in milk have their origin from the water supply (316,318). Morse et al. (217) reported that the psychrotrophic count of water ranged from less than 10 to more than 100,000 microorganisms/ml, with a median of 10 to 560/ml. Air Very few ofthe psychrotrophs found in milk have been isolated from air in clean milking parlors or dairies (316,318,330). Dust collected from milking equipment surfaces and from cereal grains contained from 1 to 300 million psychrotrophs/ g (330). At milking time, in a fairly clean barn, 1 x 101 to 1 x 10 4 psychrotrophs per square foot of exposed surface were collected during 10 min (316). The main sources of airborne microorganisms in dairy plants are worker activity, ventilating fans, drains and dust (327). Fecal contamination Fecal contamination, because of poor milking conditions, may contribute psychrotrophs to milk since both fresh and dry manure contain millions of such bacteria per gram (318,330,332). Swabs have revealed large numbers of psychrotrophic bacteria on teats and udders of cows after disinfection. Morse et a!. (216) reported that milk from cows, the udders of which had been washed with the highest concentration of sanitizer, had the lowest total counts. Bacterial count and udder cleanliness were correlated, but this did not account for all the contamination in milk. Dairy equipment Poorly cleaned and sanitized dairy farm and processing plant equipment probably constitute major sources of contamination of raw milk with psychrotrophs (316,318,324,330,332, ,378). Rarely did psychrotrophic counts exceed 1 X 10 4 /ft 2 for swabs of steam-sterilized milking equipment, but 29o/o of the time chemically disinfected equipment gave counts which exceeded this value (318,332). Methods of cleaning and sanitizing milking equipment resulted in a different microflora on the surface (347). When steam or boiling- water immersion were used, 60% of the isolates were Micrococcus and Corynebacterium spp. while only 14o/owere aerobic sporeforming rods and less than 10%, gram-negative rods. With quaternary ammonium sanitizer, gram-negative rods predominated. Thomas and Thomas ( ) studied the bacterial flora of farm bulk milk tanks and found a high incidence of gram-negative rods, especially in poorly cleaned tanks. Small numbers of Streptococcus, Micrococcus and Corynebacterium species were found. Aerobic sporeforming rods constituted about 10o/o of the total population in some tanks. These authors concluded that milk collected from farms using good cleaning methods usually had lower numbers of gram-negative rods than milk from farms with poorly cleaned equipment. Rinses of equipment also have given high psychrotrophic counts (316,318,330,332, ). Gram-negative rods and Streptococcus spp. were present in large numbers in high-count rinses of dairy equipment (347). The same type of microflora was observed for pipeline milking plants. Most Micrococcus and Corynebacterium species failed to grow within a 72-h period. Thomas and Thomas ( ) noted that gram-negative rods were dominant in rinses from equipment in milk plants; however, some Micrococcus and Streptococcus species were observed in poorly cleaned plants. The psychrotrophic counts were higher for milk tanks than for pipelines. The coliform content of rinses from dairy equipment has been attributed to improper cleaning of equipment rather than to infected udders, manure, dust or untreated water (348). E. coli, Klebsiella aerogenes, Klebsiella cloacae and Citrobacter freundii were the major coliforms isolated from dairy farm equipment rinses. Some of the strains were psychrotrophic; some E. coli were heatresistant and survived pasteurization (348). Investigators have shown that the psychrotrophic population of farm milk tanks generally is less than that of the milk plants, but the proportion of psychrotrophs in the total population is often higher for bulk tanks (344). Valves in pipes and tanks can contribute to the psychrotrophic population (316,318,332,346). Rubber parts of milking machines contained from 10 to 117 times more bacteria than did the metal parts of milking machines (346). In fact, Thomas and Thomas (346) concluded that milking equipment caused the most microbial contamination of any source for farm bulk tank milk. Thomas and Thomas (349) reported that thermoduric counts of over 10,000/ft 2 were indicators of milk residue build-up on improperly cleaned equipment. Since aseptically drawn milk does not contain thermoduric bacteria, their presence in milk is a result of contamination from soil, dust, animal feeds, manure and surface water. Additional references can be obtained from a two part bibliography on bacterial aspects of bulk-collected milk (350,351). Dairy product contamination Post-pasteurization contamination of milk and dairy products generally results from improperly cleaned and sanitized equipment as well as from airborne contamina- JOURNAL OF FOOD PROTECTION, VOL. 45, FEBRUARY 1982

9 180 COUSIN tion (324). Improperly sanitized bottles and containers have been implicated as additional sources of recontamination of pasteurized milk. Airborne contamination can add psychrotrophs to the milk and at 7.2 C, one psychrotroph in a half-gallon carton of milk could increase to 1 x 10 6 psychrotrophs/ml in 8 days or less. In overcrowded dairy cases where the temperature may be closer to 10 to 12 C, this can happen in less time (324). Thomas and Druce (327) reported that unsanitary equipment, water and air are the major sources of psychrotrophic contamination in butter. Aerial contamination is important in butter processing because butter is often exposed to air for extended periods during packing and printing. Since water is used often in butter manufacture, recontamination with psychrotrophs, especially lipolytic organisms, can cause spoilage. INCIDENCE OF PSYCHROTROPHS IN MILK Occurrence of psychrotrophic microorganisms in raw milk varies depending on the type and number of microorganisms present, conditions under which the milk was produced and the temperature and length of storage time before it is processed. Milk produced under sanitary conditions usually contains less than loo/o of the total microbial flora as psychrotrophs, but milk produced under unsanitary conditions can contain more than 7So/o psychrotrophs (332). Reports from Scotland and England have shown that psychrotrophic colony counts of< 1 x 10 3 /ml were obtained when milk came from farms where steam-sanitation of milking equipment and careful cleaning and disinfection of bulk tanks were practiced (332). A study of bulk-collected milk in a section of Minnesota showed that 63o/o of the samples contained less than 3000 psychrotrophs/ml (332). Age of milk and psychrotrophic colony counts Alternate Day (AD) collection of milk resulted in higher psychrotrophic colony counts than in milk that was collected daily, but this was deemed insignificant if refrigeration was good (318, ). Counts of SO psychrotrophs/ml of milk have been reported immediately after milking (180). Psychrotrophic counts ranging from 1700 to 49,000/ml were observed for day-old milk. Two-day-old milk had from 4300 to 71,000 psychrotrophs/ml. Psychrotrophic counts of 400,000; 2.1 million; and 11 million/ml of raw milk stored at 5 C for 1, 2 and 3 days, respectively. have been recorded (180). Psychrotrophic populations and conditions of milk production Milk produced under sanitary conditions usually does not have a rapid increase in psychrotrophs when held at 4 C or less. Milk that is produced under unsanitary conditions, however, has a rapid increase in psychrotrophic microorganisms. The increase is not the result of the initial number of psychrotrophs but rather the presence of actively multiplying psychrotrophs (330). Thomas et al. (330) reported that 3 h after milking, psychrotrophic counts ranged from 0 to 13,000/ml and after 72 h of holding at 3 to 5 C, the numbers ranged from 10 to 29,000,000/ml. Low-count milks had microflora composed of Micrococcus and Corynebacterium species. High-count milks had proteolytic, lipolytic and/or fluorescent pseudomonads present in large numbers (347). Streptococcus species made up 10 to 20o/o of the isolates regardless of total bacterial count. Also, aerobic sporeformers sometimes accounted for 10 to 12o/o of the microflora. Thomas and Thomas (343) reported that thermoduric bacteria, which were dominated by aerobic sporeforming rods, comprised 25o/o of the microbial population for pipeline milk plants and 1 o/o for farm bulk tanks. Therefore, the incidence of certain types of bacteria in raw milk depended upon sanitation on the farm and at the processing plant. Durr (73) observed that when milks obtained directly after milking contained counts of less than 25,000 to more than 100,000 psychrotrophic microorganisms/ml and were stored up to 4 days at 2 to 8 C, the number of psychrotrophic bacteria increased. Milk containing 100,000 to 500,000 psychrotrophs/ml could not be stored for more than 2 or 3 days at 4 C without the count increasing to more than 1 to 2 million/mi. Milk delivered twice daily to the processing plant had psychrotrophic counts of 400 to 17,000/ml which increased to 4500 to several million/ml after refrigeration for 14 to 48 h {)07). Muir et al. {)21) observed that safe refrigerated storage was about 72 h at less than 8 C. After 96 h at8 C, 93.8o/oofmilk had more thansx 10 6 CFU/ml compared to 12.2o/ofor milk held at 4 C. Ogawa {)35) studied the psychrotrophic counts at various times after milking. Psychrotrophic counts of raw milk obtained by hand-milking were 10 to 100; for machine milking, 1 x 103; at collection depots, 1 x 10 4 to 1 x 10 5 ; and at the milk plants, tx 10' to 1 x 10 8 /ml for milk obtained either by hand- or machine-milking, but organoleptic changes were seldom detectable at this stage. Kikuchi and Matsui (149) reported that the direct microscopic count for bulk-collected raw milk from bucket milk and pipeline systems were 6.3 x 10 5 microorganisms/ml and 6.9 x 10 5 microorganisms/ml, respectively. More samples of pipeline milk contained psychrotrophs than did bucket milk. Thomas and Thomas (332) reported that the bacterial content of milk upon arrival at the dairy plant was higher than that of milk sampled at the farm; the increase was greater than 1 x 10 4 CFU/ml. Seasonal variation Seasonal variation in the numbers and types of microorganisms in milk has been reported. Psychrotrophic contamination was reported to be higher in milk in the summer than in the winter because poorly cleaned milk tanks can contaminate milk with microorganisms whose optimum growth temperature is achieved by summer temperatures (149,332). Kikuchi and Matsui (149) observed that Micrococcus species accounted for JOURNAL OF FOOD PROTECTION. VOL. 45, FEBRUARY 1982

10 PSYCHROTROPHS IN DAIRY PRODUCTS % ofthe population in the summer milks, compared to 18.9% in the winter milks. Also, the coryneform bacteria were in milk in greater numbers in the summer than in other seasons. Audrey and Frazier (6) found that a sevenfold increase in the total number of bacteria occurred when the cows went from barn-feeding to pasture-feeding in the spring. The flora of milk from barn-fed cows consisted mainly of species of Arthrobacter, Pseudomonas and Micrococcus; however, Flavobacterium species becam,e dominant when the cows were put out for pasture-feeding. Psychrotrophic microorganisms after pasteurization Early researchers concluded that psychrotrophic bacteria do not survive laboratory or commercial pasteurization (180,317,325,378). Thomas and Druce (325) reported that none of the psychrotrophic cultures isolated from milk and milk products refrigerated at 7 C or below survived pasteurization treatments, but some cultures isolated from milk and dairy products held at 8 to 10 C were heat-resistant. Research within the last decade bas identified two groups of microorganisms which can survive pasteurization temperatures, namely sporeforming and non-sporeforming gram-positive bacteria. Washam et al. (365) isolated heat-resistant microorganisms from pasteurized milk and classified those microorganisms that survived four exposures to 71.7 C for 16 sec and then grew at 7.2 Cas thermoduric psychrotrophs. The sporeformers were identified as species of Bacillus and nonsporeformers, as species of Corynebacterium and Arthrobacter. Thermoduric psychrotrophs have been reported by several researchers in recent years (J1,205,305,322,344,347,349,353). Thermoduric psychrotrophs identified as Bacillus subtilis, Lactobacillus casei, Micrococcus jlavus and Streptococcusfaecalis were isolated from laboratory-pasteurized milk (353). These organisms had generation times of 7 to 27 h at 1. 7 to 7.2 C, and data showed they could reach sufficient population levels to cause defects. Micrococcus and Bacillus species were dominant in raw milk supplies (322,344). These thermodurics were present in milk as a result of use of poorly cleaned equipment (31,344,347, 349). Mikolajcik and Simon (205) examined 109 raw milk samples and noted that only 13% of the milks heated to 80 C for 12 min contained psychrotrophic spore counts of 10 or more per milliliter. After storage at 7 C for 7 days, 58% of the samples had psychrotrophic spore counts of 10 or more per milliliter and an average of 340 psychrotrophic spores/mi. In another study, no psychrotrophs were recovered from any milk immediately after pasteurization; however, after storage at 7 to 7.2 C for 7 to 10 days psychrotrophic counts were between < 1 and 100,000/ml (253,367). With the increased use of high temperature - short time pasteurization and the extended refrigerated storage of milk, the sporeforming psychrotrophs may become more important to the dairy industry. Information on the incidence of psychrotrophic sporeformers is limited, but new information is surfacing. Some investigators have postulated that psychrotrophic sporeformers may be variants of mesophilic sporeformers that have adapted themselves to growth at low temperatures (31,200,293,294,323). H.T.S.T. pasteurization probably promotes spore germination and under acceptable conditions, outgrowth of spores and vegetative cell multiplication proceed (49). A double heating of milk at 80 C for 10 min separated by an anaerobic incubation of 1 to 24 h did not reduce the spore loads of B. cereus and B. subtilis because the germination rates varied for these species (38). Psychrotrophic spores are less heat-resistant than mesophilic spores (61,196,294). A correlation exists between the temperature at which the spore is produced and the degree of tbermolability (61). Though sporeformers survive pasteurization, a long lag period and slow growth at 7 Cis evident (200,201). Some isolates have generation times of 15 to 20 hat 7.2 C; other species can double in about 7 h at this temperature (200,353). Many researchers consider that spoilage by Bacillus species is not a practical problem until after 2 weeks of refrigerated storage (31,323). Overcast and Atmaram (248) observed that previous growth of P. fluorescens and Pseudomonas fragi in skimmilk stimulated germination and outgrowth of B. cereus spores after activation at 80 C for 15 sec. Mikolajcik and Simon (205) found it unlikely that gram-negative counts of less than 25,000/ml would produce stimulatory substances for outgrowth of spores. Earlier studies on the correlation between psychrotrophic growth in raw milk and subsequent growth in pasteurized milk have shown no relationships between pre- and post-pasteurization microbial growth at 7.2 C (247,367). In fact, excessive growth of P. fragi in milk had an inhibitory effect on subsequent psychrotrophic growth (247). Maxcy (191) reported that freshly pasteurized milk did not contain gram-negative bacteria and their presence in the milk was a result of post-pasteurization contamination. Later research has indicated that some gramnegative bacteria may survive pasteurization temperatures (305, ). Stadhouders (305) included Alcaligenes tolerans among the thermoresistant bacteria in milk. This gram-negative bacterium could cause deterioration of uncontaminated pasteurized milk stored at 6 to 20 C. Some strains of coliform organisms, especially E. coli I are heat-resistant (348). Also, research with Pseudomonas indicated that some could survive heat treatments used in the pasteurization of milk when initial psychrotrophic populations were large enough (368). Since most growth appeared after 7 days, the psychrotrophs may have been injured and needed time to recover before multiplication began or to make adjustments to the new heat-treated environment. Higher temperatures for shorter times were less effective in destroying the Pseudomonas species than were lower temperatures for longer times because conditions created JOURNAL OF FOOD PROTECTION. VOL 45, FEBRUARY 1982

11 182 COUSIN in the milk at high temperatures may favor recovery of injured cells. When psychrotrophic cultures are partially inactivated by pasteurization, especially in processing milk for cheese, the survivors are most fastidious in their requirement for nutrients, are more sensitive to ph and have an increased lag phase at low temperatures (112). Since large numbers of psychrotrophic bacteria are found in pasteurized milk after 10 to 16 days of refrigerated storage, the possibility exists that bacteria apparently inactivated by pasteurization may recover their ability to grow upon extended cold storage (60). Dab bah et al. (60) found that Pseudomonas species were able to recover and grow normally in milk after a heat-treatment of 55 C for 30 min. However, recovery could not be demonstrated after heating at 60 C for 30 min. Some bacteria that appeared to be killed by heat could recover in a favorable medium after incubation for long periods. Resuscitation of bacteria that initially appear to have been killed by heat may be important when milk contains heat-resistant microorganisms and the milk or milk products undergo extended refrigerated storage before consumption. Post-pasteurization contamination contributes most microorganisms which eventually grow and spoil pasteurized milk during refrigerated storage. In Britain, psychrotrophic counts at 5 to 7 C usually do not exceed 100/ml in freshly pasteurized bottled milk (325). Oliveria and Parmelee (237) found at least 20 psychrotrophs/ml in freshly pasteurized commercial and laboratory milk samples in the United States. If efficient high temperature -short time pasteurization was used, psychrotrophic counts of the bottled milk were usually < 10/ml. Many samples of pasteurized milk with low initial psychrotrophic counts contained actively multiplying strains. One study, using 64 samples of pasteurized milk from nine dairies, showed that after 72 h at 7 C, 67% of the milks contained actively multiplying psychrotrophs (326). Jones and Langlois (132) observed that highest psychrotrophic counts were in June and October and the lowest in December and March for retail milk. METHODSTOENUMERATEPSYCHROTROPHS Numerous incubation times and temperatures for psychrotrophic counts appear in the literature. Since no precise definition for psychrotrophic bacteria exists, enumeration procedures for these microorganisms have been difficult to establish. Presently, Standard Methods for the Examination of Dairy Products recommends incubating plates at 7 C for 10 days (185). Since the recommended incubation time and temperature have changed with most editions of Standard Methods, differences of opinion still exist among workers in the field (18). Bauman and Reinbold (19) examined differences in results caused by incubation at 5 to 7 C for 7 to 10 days. Incubation at 7 C for 7 days or at 5 to 7 C for 10 days gave colonies that were large enough to count. As the incubation temperature approaches the optimum temperature for growth, plate counts are expected to increase and the same is true for increased incubation time. Incubation at 7 C for 10 days gave the highest count, but this may not be the best enumeration procedure. Reproducibility of psychrotrophic counts from different laboratories would be greater if one time-temperature combination were adopted by all researchers. One disadvantage of the "standard" method is that it requires a long time and the product may already be consumed by the time the count is realized. As a result, other methods have been evaluated for obtaining the psychrotrophic count. Selective media using inhibitors or dyes Since most psychrotrophs of importance to the dairy industry are gram-negative bacteria, efforts to inhibit gram-positive bacteria have focused on the use of antibiotics, dyes and other chemicals. These procedures normally used incubation temperatures between 20 and 30 C for 2 to 3 days (319). The major disadvantage is that gram-negative mesophiles cannot be differentiated from gram-negative psychrotrophs (262,319,332). Lightbody (I ) experimented with media containing penicillin for detection of spoilage microorganisms in milk and cream. Most gram-negative rods were resistant to 2.5 I. U. of penicillin; however, so were some Micrococcus and Arthrobacter species (177). Since most gram-negative rods are penicillin-resistant and do not survive pasteurization, use of agar with 2.5 to 10 I.U. penicillin and subsequent incubation at 30 to 32 C for 3 days was deemed an acceptable way to measure post-pasteurization contamination of milk, cream and butter (178). Some thermoduric microorganisms were resistant to penicillin, namely Micrococcus, Microbacterium, Bacillus and Alcaligenes species. Use of the penicillin-resistant count at 5 C correlated well with the psychrotrophic count (179). Other methods using 1 to 2.5 I.U. penicillin were acceptable as rapid methods to determine psychrotrophic populations in milk (180,319, 340). Waes (362) found that Plate Count Agar containing 1 I.U. penicillin, which was incubated at 25 C for 3 days, was the best rapid method for enumeration of psychrotrophs in pasteurized milk. Addition of penicillin to ammonium lactate agar with subsequent incubation at 28 C failed to detect sufficient numbers of psychrotrophs in pasteurized milk (319). Freeman et al. (86) tested 32 dyes and 26 chemicals for their ability to inhibit gram-positive bacteria and only five chemicals were considered to be effective: sodium desoxycholate, alkyldimethyl benzyl ammonium chloride, methyl dodecyl trimethyl ammonium chloride, alphabromo-lauric acid and alpha-bromo-myristic acid. All plates in these tests were incubated at 23 C for 72 h. Since 0.5% sodium desoxycholate (SDC) inhibited gram-positive bacteria, a method for enumeration of gram-negative contaminants was developed in which 1 JOURNAL OF FOOD PROTECTION. VOL. 45, FEBRUARY 1982

12 PSYCHROTROPHS IN DAIRY PRODUCTS 183 ml of milk was added to 9 ml of 0.5% SDC, the mixture was allowed to react for a few minutes, then was plated with Plate Count Agar and plates were incubated at 32 C for 48 h (30). Blankenagel and Humbert (29) developed a rapid method for determining post-pasteurization contamination of milk. In this procedure, a disc was saturated with milk and placed on the surface of a plate containing 2 ppm of crystal violet, 50 ppm of 2, 3, 5 triphenyl tetrazolium chloride and 1% gelatin. After 24 h at 22.2 C, discs that were partially or entirely red were positive for growth of gram-negative bacteria. Proteolysis could also be detected by the clear zones around the disc. Brant et al. (35) tried several media containing inhibitors such as triphenyl tetrazolium chloride, crystal violet, nacconol wetting agent, cetrimide, diamide and other chemicals. However, some of the media were inhibitory to E. coli and Pseudomonas vulgaricus. They questioned the value of using one selective medium for the nonhomogenous psychrotrophic flora. Since gram-negative microorganisms are more resistant to dyes than gram-positive microorganisms, various methods of enumerating psychrotrophs have been based on media that incorporate these substances (88,180,181, ,300,340). Fung and Miller (88) found that bromothymol blue, o-cresolphthalein, janus green, methylene blue and safranin 0 allowed gramnegative but not gram-positive microorganisms to grow. In this study, crystal violet inhibited Pseudomonas and Alcaligenes species, E. coli and gram-positive bacteria. Of 17 inhibitory chemicals and dyes, only crystal violet and neotetrazolium chloride at 2 mg/1 proved reliable for enumerating psychrotrophic bacteria (.100). LUck and Hopkins (181) reported no significant difference from the standard method when using Standard Methods Agar plus 5% skimmilk plus 1% Andrade's indicator (acid fuchsin in 1 N NaOH). In Europe, violet-red bile agar has been used for estimating the psychrotrophic count of milk and cottage cheese (340). Hankin and Dillman (104) suggested a rapid test for "potential psychrotrophs" that screened for oxidasepositive pseudomonads. Plates including serial dilutions of pasteurized dairy products were incubated at 32 C for 48 h, then flooded with 2 ml of an oxidase reagent. After standing for 10 to 15 min, only colonies that turned blue were counted. Species of Pseudomonas, Alcaligenes and Aeromonas were positive, but coliforms, Streptococcus and Staphylococcus species plus most Bacillus were negative and some Micrococcus species were weakly positive (.119,340). Incubation at 32 C may be too high for most psychrotrophs; therefore, 25 C has been recommended (104,319). Suiface plating Since the incubation times for psychrotrophic pour plates has traditionally been 7 to 10 days, several investigators have attempted to reduce the incubation time by using surface plating. A surface plating method with incubation at 6 C for 5 days was proposed by Punch and Olson (18,262). Counts by this procedure correlated with those from pour plates incubated at 6 C for 8 days. Surface plating with incubation at 10 C for 10 days has been used to enumerate psychrotrophs (319). Gramnegative rods in pasteurized milk were surface-inoculated onto a selective medium containing alkyl aryl sulfonate and incubated at 32 C for 48 h (319). Colonies on such plates were larger, more uniform and easier to count than those on poured plates. The relative error and variability of the surface plate method were slightly less than those for the poured plate method. Spiral surface plate methods have been produced which provide an alternative automated test to replace the pour plate methods (96,185,337). Since agar must be held at 45 C to remain liquefied, investigators have searched for substitutes that will solidify at lower temperatures to eliminate the need for surface plating. Parker et al. (251) used carrageenan as a gelling agent since it solidifies at 30 C. Media using this gelling agent were not rigid and allowed microorganisms such as Pseudomonas species to spread. Incubation at elevated temperatures Recommendations have been made for incubation of psychrotrophic plates at temperatures higher than 7 C to reduce the time necessary to obtain results. W aes (.162) suggested holding the plates at 17 C for 16 h followed by incubation at 7 C for 3 days. This method was suitable for detecting psychrotrophs, but it was laborious. LUck (180) reported that studies made by his coworkers with plate incubation at 27 or 32 C for 2 days gave a good indication of the psychrotrophic count. Other workers suggested indirectly counting psychrotrophs by determining the difference in counts between plates incubated at 27 and 37 C, but this method can lead to negative counts. Juffs (133) noted that a 4-day psychrotrophic count (24 h at 15 C followed by 72 h at 5-7 C) gave results comparable to the standard method of incubating plates at 7 C for 10 days. After comparing eight different tests using time-temperature variations, LUck and Hopkins (181) recommended the following methods: 3 days at 15 C, 2 days at 15 C plus 1 day at 7 C, and 1 day at 17 C plus 3 days at 7 C. Since most psychrotrophs.grow well at 21 C and most mesophiles grow slowly, Oliveria and Parmelee (237) developed a plating method to enumerate psychrotrophs that used a 25-h incubation at 21 C. Counts obtained by this method correlated well with the standard psychrotrophic count and over 95% of the microorganisms enumerated by this method were confirmed as psychrotrophs. Griffiths et al. (96) applied this method to enumeration of psychrotrophs in pasteurized double cream. When the data were analyzed for the two psychrotrophic counts (25 hat 21 C and 14 days at 6 C), a correlation coefficient of was found. Bauman and Reinbold (19) also found that an incubation temperature of20 C gave the highest psychrotrophic counts. Preliminary incubation The use of preliminary incubation of milk at temperatures above 10 C for h has been JOURNAL OF FOOD PROTECTION. VOL. 45, FEBRUARY 1982

13 184 COUSIN recommended as an acceptable modification to various methods for microbiological evaluation (69,332). The temperatures for preincubation have ranged from 12.5 to 22 C, but the times have been from 16 to 18 h. Preincubation has been used before reduction tests, Standard Plate Counts at 32 C and psychrotrophic plate counts at various temperatures. Preliminary incubation assists in detection of post-pasteurization contamination, determination of keeping quality and assessment of plant sanitation. However, the types of microorganisms and their rates of multiplication affect the results obtained. Johns (129) recommended preincubation of 16 h at 17 C before doing microbiological testing of raw milk. Preincubation of samples at 13 C for 18 h was useful to indicate processing plant sanitation since gram-negative rods outgrow other microorganisms under these conditions (130). Morse et al. (215) also used an 18-h preincubation at 13 C to determine bacterial quality of raw milk. Though this preliminary incubation increased the Standard Plate Count at 32 C, the increase was not enough to cause concern. Randolph et al. (268) reported considerable increases in counts after using preliminary incubation for Standard Plate, psychrotrophic, crystal violet tetrazolium and penicillin-tetrazolium counts. A test to detect post-pasteurization contamination of HTST-pasteurized milk included a preincubation step of 24 h at 25 C before the milk was inoculated onto Plate Count Agar containing benzalkon A-SO% (162). The test results correlated well with reduced shelf-life of pasteurized milk stored at 7 C. Llick (180) noted that preliminary incubation was of little value for milk with high initial counts. A specialized preincubation method is the Moseley Keeping Quality Test, which is used to determine shelf-life rather than to detect specific concentrations of microorganisms (28,185,218). A sample of milk is plated, then the milk is held at 7.2 C for 7 days before being plated and plates are incubated at 32 C (218). The milk is also tasted after 7 days at 7.2 C. The major disadvantage of this method is the time involved, approximately 10 days. Flemingham and Juffs (81) found that the Moseley test more accurately reflected the presence of psychrotrophs than did the methylene blue reduction test. The Moseley test correlated well with the organoleptic acceptability of the milk after 10 days storage at 7.2 C. A total count of 1 x 10 7 organisms/ml after 7 days at 7.2 C was used as the point at which the milk failed the Moseley test. Reduction tests Tests designed to measure the metabolic activity of microorganisms, such as dye reduction methods, have been used to measure quality of milk (180). The results of the metabolic tests cannot be interpreted in terms of bacterial numbers. These tests only serve as an index of microbial loads and are dependent on metabolic rates of the microorganisms. The methylene blue test has been both hailed and scorned for its correlation to the plate count (180). This test may not be applicable to the psychrotrophic count since many psychrotrophs are not able to reduce methylene blue. Caruso and Feder (40) found that a triphenyltetrazolium chloride medium was more sensitive than the methylene blue test in detecting psychrotrophs in raw milk. The resazurin test has been used as a way to predict keeping quality of milk; it suffers from some of the same shortcomings as the methylene blue test (1 80). Catchick and Gibson (42) developed a 16-h test for detecting post-pasteurization contamination based on the resazurin dye. Nine milliliters of milk were added to 1 ml of sterilized 5% sodium desoxycholate and incubated at 32 C for 16 h. After adding 1 ml of resazurin, the tubes were incubated at 37 C for 6 h before they were compared to a color standard. A modification of this method involved incubation at 32 instead of 37 C (252). This modified method failed to detect 17.6% of milk samples which were moderately contaminated after pasteurization and detected 8.3% of milk samples which had only slight post-pasteurization contamination. Blankenagel (28) further modified the test to include incubation at 25 C for 18 h. Druce and Thomas (69) reported that gram-negative psychrotrophs represented by species of Pseudomonas, Alcaligenes, Flavobacterium and Enterobacter gave poor resazurin reduction results. Detection of metabolites produced by psychrotrophs Microorganisms growing in milk produce biochemical changes in the carbohydrates, proteins and fat by their metabolic activity. Therefore, some research efforts have been directed at detection of these metabolites or components resulting from metabolic activity because analytical techniques are available with which many samples can be prepared, analyzed and evaluated with good sensitivity and reproducibility (114). Bassette et al. (11) characterized bacteria in milk by measuring volatile growth products with a gas-liquid chromatograph (GLC). These authors concluded that some volatile growth products were unique as fingerprints, whereas others were not. Pierami and Stevenson (257) also used GLC to detect metabolites produced in milk by Alcaligenes viscolactis, P. fragi, Pseudomonas perolens and Bacillus pumilus. Of the chemicals detected by GLC, acetaldehyde was the best indicator of large populations of psychrotrophs in milk; however, no differences were observed from the controls until counts of 1 x 10 5 to 1 x 10 6 psychrotrophs/ml were attained. Lee et al. (175) found that high-resolution gas chromatography combined with mass spectrometric analysis gave profiles that were unique to Pseudomonas and Moraxella species. Complex mixtures of organic compounds produced by the microorganisms during spoilage could be assigned to specific microorganisms. When Heeschen (113) and Heeschen et al. (114) detected metabolites of glycolysis, proteolysis and lipolysis in an effort to enumerate bacteria in milk, the presence of lactic acid, ammonia, free fatty acids and pyruvate correlated well with the biochemical status of microorganisms in milk. They concluded that since pyruvate is a key metabolite produced during degrada- JOURNAL OF FOOD PROTECTION. VOL. 45, FEBRUARY 1982

14 PSYCHROTROPHS IN DAIRY PRODUCTS 185 tion of carbohydrates, proteins and fat and is present at a concentration of about 1.5 ppm in normal milk, the change in amount of pyruvate can be used to detect the presence of microorganisms in milk. Since pyruvate concentrations in milk are independent of lactation, air and pasteurization, Tolle et al. (;154) reported that this method could be used to determine the quality of milk before and after incubation. Levels of 1 x 10s to 1 x 10& psychrotrophs/ml could be detected when the pyruvate concentration was greater than 1.5 ppm. A pyruvate value of 1.3 ± 0.5 ppm may not provide evidence for poor sanitation on the farm, but it may be of value in assessing the handling and storage of milk (;176). Marshall and Harmon (186) suggested that psychrotrophs are variable in their ability to produce pyruvate. Also, use of a pyruvate difference test (pyruvate concentration after incubation minus pyruvate concentration before incubation) was required since some microorganisms can metabolize pyruvate. Psychrotrophic plate counts correlated significantly with pyruvate content only when raw milk from normal quarters was analyzed (186). This study left many unanswered questions that must be resolved before application to raw milk can be proposed. Zandstra and devries (;185) questioned use of the pyruvate test for refrigerated milk since it does not give an estimation of the number of contaminants in milk due to delayed metabolic activity at refrigeration temperatures. Pyruvate content of milk refrigerated for 1 to 3 days did not indicate the possible cell count. Therefore, it may be necessary to use several different assessment methods since a single method does not give enough information on the keeping quality of milk. V asavada and White (;158) screened psychrotrophs for the ability to reduce diacetyl Of 268 isolates studied, only 25.7% reduced diacetyl and 13% produced acetylmethylcarbinol. Hence, this method would be of little value in detecting the presence of psychrotrophs in milk and dairy products. Several plating methods have been developed to estimate the number of proteolytic or lipolytic microorganisms in milk and dairy products. Lawrence et al. (172) developed a tributyrin emulsion assay for detecting microbial lipases that was 90 times more sensitive than the milkfat assay. In addition to tributyrin, several indicator dyes, such as Nile blue sulfate, Victoria blue, Analine blue and neutral red have been used in media containing some type of lipid (340). However, some of these dyes are inhibitory to microorganisms. Proteolytic microorganisms have been detected by use of sodium caseinate in Standard Methods Agar (188). This medium was reported to be more sensitive than Standard Methods Agar plus sterile skimmilk for detecting proteolytic microorganisms since early stages of paracasein precipitation can be detected. This method is free of the false-positive zones of skimmilk agar caused by acid development. Standard Methods Agar plus 10o/o sterile skimmilk is normally the method used to detect proteolysis (;140). Juffs and Madsen (143) noted that a percentage of proteolytic organisms could not be enumerated on penicillin-resistant or bromcresol purple agars. Rapid screening methods To eliminate the long incubation periods for obtaining psychrotrophic counts, work has centered on rapid methods for obtaining counts. Microcolonies formed on the surface of dried Yeastrel-milk agar plates have been counted in 72 h, using a simple microscope with a magnification of ><40 (;119). Electronic microcolony counting was suggested, using nutrient gelatin incubated at 6 C for 5 days; gelatin was overlayed with 2 ml of HCl/HCHO, liquefied at 30 to 35 C; formalin was then added and the medium was liquefied and counted in a coulter counter (180,332). Another method involved a membrane ftlter technique to enumerate gram-negative bacteria. Filters were placed on yeast extract glucose medium and were incubated initially at 21 C for 12 h followed by incubation for 72 hat 10 C. Colonies were stained with methylene blue and observed microscopically (18). Cady et al. (;19) noted that early data on use of automated impedance measurements (a procedure in which microorganisms growing in liquid media produce chemical changes which alter electrical resistance of the solution) provided a possible 9- to 14-h impedance-based keeping quality test. The comparison between impedance measurements and plate counts was 50% from milks with counts over 1 x 10" organisms/mi. 80% for milks with counts of 1 x 10 3 to 1 x 10" organisms/ml and 32% for milks with counts lower than 1 x 10 3 organisms/mi. Juffs and Babel (140) reported that the microscopic colony count differed from the standard psychrotrophic count and that care must be exercised in interpreting microscopic colony counts. Analysis of variance showed that the microscopic colony counts differed from the psychrotrophic count at P >0.01 since incubation for less than 12 h at 21 C underestimated the psychrotrophic count and incubation beyond 20 h at 21 C overestimated the count. A microtiter count method was modified for use in enumerating mesophiles, psychrotrophs and coliforms in raw and pasteurized milk (41). This method required less time, media and space than did standard methods. Another screening method used catalase activity as an indicator of psychrotrophs since most were catalasepositive (189). Two milliliters of a 0.03%-hydrogen peroxide solution were added to milk which was then incubated for 2 hat 25 C, then 2 ml of 70% TCA were added and finally the percentage of hydrogen peroxide that was degraded was determined. Comparison of various methods for detection or enumeration of psychrotrophs Many tests have been developed over the years to determine the microbial quality and shelf-life of milk and dairy products. Since all these tests have specific JOURNAL OF FOOD PROTECTION, VOL. 45, FEBRUARY 1982

15 186 COUSIN advantages and disadvantages, dairy processors have doubts about the best method to use for their operations. Recent reviews have been published on the various methods that can be used (28,109, ) and on comparisons of some of the methods (48,81,129,240,241, 268,372). Blankenagel (28) proposed the following criteria for the ideal test to detect keeping quality of pasteurized milk. (a) The ideal test would be accurate and give the exact number of microorganisms in the product, including a differentiation between thermodories and contaminants. (b) The ideal test would provide results rapidly, preferably within a day. (c) The ideal test would be simple to do and economically feasible. After reviewing various methods to measure milk quality (agar plate count, coliform and enterococcus counts, reduction methods, thermoduric and psychrotrophic counts and preliminary incubation tests), Hartely et al. (1 09) concluded that no single test can determine everything that is necessary to assess the microbial quality of milk and dairy products. Thomas and Thomas ( ) reviewed the microbial testing of bulk-collected milk. Since the total count at 32 C for 48 h usually does not include those microorganisms responsible for milk spoilage, plates should be incubated at a low temperature, such as 27 C for 4 days (335). Various simplified methods for counting colonies include: roll-tube, plate loop, agar strip, drop plate, electronic microcolony, Frost little plate and direct microscopic count ( ). Specific types of microorganisms in milk have been enumerated: psychrotrophic, thermoduric, coliform, proteolytic, lipolytic, gram-negative bacteria, pseudomonads and oxidasepositive bacteria (339,340). The last two articles in the series included discussions of tests to determine conditions of production and an overview of standards (341,342). After examining several procedures for evaluating raw milk, Johns (129) concluded that a temperature of 32 C was too high for some psychrotrophs to grow and 7 C for 10 days was too long to wait for a count. The coliform test failed to measure keeping quality of the milk. The cytochrome oxidase and catalase tests correlated well with gram-negative rods, but exclusion of the rest of the flora has been questioned. A method using preincubation for 16 h at 17 C was suggested. Randolph et al. (268) evaluated three procedures for enumerating psychrotrophs (psychrotroph count, crystal violet tetrazolium agar and penicillin tetrazolium agar) and found highly significant correlations between them after preliminary incubation. However, procedures using selective agents would have limited value for milk with low counts. Orr et al. (240,241) compared a number of tests used to assess milk production and raw milk quality. These authors found that the psychrotrophic count gave a reliable indication of sanitation during milk production. Other methods that gave a good correlation with the standard psychrotrophic count were one with plate count agar plus 2.5 I.U. penicillin/ml and agars to detect proteolytic and lipolytic microorganisms. In the final analysis, Orr et al. (241) concluded that the mesophilic plate count at 30 C for 72 h and the thermoduric count gave reliable indications of production methods and pasteurized milk quality. Comparisons of the Moseley test with other microbiological tests produced significant relationships between the Moseley test and these four tests: the modified 4-day Moseley test at 7 C, the inhibitory test of Freeman et al. (86), the organoleptic test and the psychrotrophic count at 7 C for 10 days. The Moseley test did not correlate well with the test of Oliveria and Parmelee (237), Blankenagel's inhibitory test (28) or coliform counts. White et al. (372) noted that results of the microscopic colony count incubated at 7 C for 48 hand at 21 C for 13.5 h correlated better with each other than with results of the psychrotrophic count of 10 days at 7 C. After preliminary incubation at 13 C for 18 h, results of the microscopic colony count at 21 C correlated only slightly better with those of the psychrotrophic count. These authors found that the degree of proteolysis, as measured by the Hull test, was directly related to bitter flavors in milk. INHIBITION AND CONTROL OF PSYCHROTROPHS The dairy industry has been faced with the control of psychrotrophs for many years. Though prevention of psychrotrophs from entering milk is the best method and one that is technically feasible, it is difficult to achieve. Human errors that result in improperly cleaned, sanitized and handled equipment allow psychrotrophs to enter raw and pasteurized milks. Hence, control must focus on destruction of psychrotrophs or prevention of their growth in milk. Some general reviews on control of psychrotrophs already appear in the literature (202,377). Cleaning and sanitation of equipment Since proper cleaning and sanitizing of dairy equipment are important for production of milk with acceptable microbial quality, control of psychrotrophs should begin at the farm level. Thomas (322) noted that the number of gram-negative bacteria was lowest on equipment that was steam-sterilized or cleaned with hot detergent-hypochlorite solutions. Detergent-quaternary ammonium compound solutions were not as effective in controlling gram-negative bacteria as the detergenthypochlorite solutions. Since energy conservation is becoming important, Bigalke (22) studied use of low-temperature cleaning of dairy equipment. The conclusions from this study were: (a) equipment can be effectively cleaned if the final wash temperature is not lower than 40.6 C, (b) energy saving is significant and (c) temperatures of cleaning of 40.6 C or higher had little effect on microbiological quality of milk. Inactivation of spores with chlorine solutions or steam takes considerably longer than for vegetative cells (147,308,364). Keogh and Hedrick (147) reported JOURNAL OF FOOD PROTECTION, VOL 45, FEBRUARY 1982

16 PSYCHROTROPHS IN DAIRY PRODUCTS 187 that B. subtilis spores were destroyed in 30 min at 93 C with 300 ppm of sodium dichloroisocyanurate, but this compound was corrosive to stainless steel. Other acidic and basic sanitizers were ineffective at the same temperature-time combination even at higher concentrations. Other researchers have observed that vegetative cells of B. cereus were destroyed by 10 ppm of chlorine in 30 sec at ph 7.0 and 25 C (364). Spores of B. cereus had D-values ranging from 0.5 to 0.1 min at ph 7.0 and 25 C for solutions containing 100 to 200 ppm of chlorine. D-values were slightly greater for B. pumilus and Bacillus laterosporus spores. Greater sporicidal action was achieved when the temperature was increased to 75 C and the ph was lowered to 5.2. Temperature Dairy processors in European countries use a process called thermisation, a special temperature treatment for milk refrigerated before processing, to prevent psychrotrophs from growing in milk (27,56,384). This process normally involves heating the milk to 63 to 66 C for 15 sec immediately after arrival at the processing plant. In fact, Zall (384) has suggested that milk be heated on the farm before cooling and storage to prevent psychrotrophic growth. Ordinary pasteurization time-temperature relationships destroy most psychrotrophs (180,317, 325,378), except those that have been reported as being heat resistant (31,323,365). Low temperatures reduce the rate of microbial growth (32,356). Tompkin (356) has compiled various generation times for psychrotrophic growth between 0 and 32 C, and concluded that these organisms take longer to grow when the temperature is reduced. Since temperature control is critical for adequate shelf-life of dairy products, Bodyfelt and Davidson (32) examined temperatures in dairy cases of Oregon food stores and found that 35%exceeded 7.2 C and 75% exceeded 4.4 C. These authors concluded that improved temperature control was necessary to prevent deterioration of milk. Control of sporeformers Sporeformers in pasteurized milk have become more important because some grow at refrigeration temperatures. Mikolajcik and Koka (204) demonstrated that heat-shocking Bacillus spores increased the rate of germination and outgrowth in milk. Spores of B. cereus were activated by pasteurization of milk and outgrowth was observed within a short time (377). However, spores of B. subtilis and Bacillus licheniformis demonstrated no outgrowth in pasteurized milk within 3 h. Davies and Wilkinson (62) isolated a germinant for B. cereus, which was produced as a result of pasteurization. Franklin (85) observed that the temperature coefficient for change in a reaction rate was greater for bacterial spore destruction than for chemical change; therefore, heating to a higher temperature for a shorter time should result in bacterial spore destruction. This principle is used for ultra-high temperature processing of milk. Spores produced by psychrotrophs have lower D-values than do spores produced by mesophiles (61,196); however, they can still survive milk pasteurization (294). Investigators have suggested variations of the tyndallization process as a means of destroying sporeformers in milk (38,294). Shehata and Collins (294) suggested a process in which milk was heated at 87.8 C for 20 sec, stored at 32 C for 4 h, and reheated at 76.7 C for 20 sec. Since the number of spores was reduced by two or more log cycles, the shelf-life of milk could be increased by 2 to 3 days. Other investigators observed no effect on B. cereus and B. subtilis by a double heat treatment of 10 min at 80 C separated by an anaerobic incubation of 1 to 24 h at 30 C (38). The storage life of milk was not changed by these treatments. Another way to reduce the number of spores in milk is by physically removing them from the milk. Langeveld (161) reduced the number of spores of two strains of Clostridium tyrobutyricum in milk by using a bactofuge. This method removed 98.5% of these anaerobic spores from milk. Antibacterial systems in milk The lactoperoxidase system is a naturally occurring inhibitory system in raw milk that can reduce the bacterial content of milk (24,25,26,275,276). The lactoperoxidase system, which consists of lactoperoxidase, thiocyanate and hydrogen peroxide, inhibited lactic streptococci and gram-negative rods, especially pseudomonads (26,275). Lactoperoxidase is present in bovine milk, but thiocyanate content depends on the feed consumed by the cows (24). Hydrogen peroxide, which is the limiting factor in this system, can be produced by lactic acid bacteria or added directly or generated by glucose oxidase enzymes. The antibacterial effect depends on the amount of thiocyanate present and the temperature. When the thiocyanate in milk has been depleted, the bacteria can start to multiply, usually within 4 h at 30 C and 72 h at 5 C (24,26). The antibacterial component, thought to be an intermediate of thiocyanate oxidation, is readily destroyed at 60 C for 15 min and is not present in pasteurized milk (24,25,26,275). Bjorck et al. (25) experi~ented with this system as a temporary preservative for raw milk in developing countries. Results of these studies demonstrated that the length of bacteriostasis is temperature-dependent: 7 to 8 hat 30 C, 11 to 12 hat 25 C, 15 to 16 hat 20 C and 24 to 26 h at 15 C. The quality of treated milk, measured by the resazurin test, showed a significant improvement over that of the untreated control. Reiter and Marshall (275) found that milk treated by the lactoperoxidase system produced cheeses which had normal flavor whereas the cheeses made from control milks became rancid within 4 months of storage. Since the lactoperoxidase system inhibited gram-negative psychrotrophs in milk, Reiter et al. (276) studied the inhibitory effect of this system within the abomasum of the calf. A hydrogen peroxide-producing strain of Lactobacillus lactis and E. coli were inoculated into milk and fed to a calf. Analyses of samples withdrawn from the abomasum at particular JOURNAL OF FOOD PROTECTION, VOL. 45, FEBRUARY 1982

17 188 COUSIN intervals indicated that sufficient hydrogen peroxide was produced by L. lactis to activate the lactoperoxidase system and subsequently damage the inner membrane of E. coli, resulting in a leakage of amino acids and potassium followed by inhibition of glucose and amino acid uptake (275,276). Several articles have been published on inhibition of microorganisms by lactic acid bacteria (11, 70,118,139, 260,261,374). Babel (11) concluded that Leuconostoc cremoris can be used to inhibit gram-negative psychrotrophs in milk to be used for manufacturing cheese and butter since this lactic acid bacterium produced a metabolite which inhibited some Pseudomonas species and coliforms. Dubois et al. (70) found that both Leconostoc and Streptococcus species were inhibitory to microorganisms isolated from ground meat. Since these and other reports indicated that lactic acid bacteria, especially citrate-fermenting bacteria, exhibited antimicrobial effects on psychrotrophs, White and Shilotri (374) inoculated raw milk in farm bulk tanks with citrate-fermenting bacteria (Streptococcus lactis subsp. diacetylactis and L. cremoris). The results of this research were disappointing because this treatment did not reduce the number of psychrotrophs, but the shelf-life of products made from this milk was increased by1 day. It has been postulated that lactic acid bacteria produce antimicrobial compounds (118,261) or hydrogen peroxide U39,260), which inhibit other microorganisms. Hosono et al. (118) isolated an inhibitory substance produced by Lactobacillus acidophilus. The antibiotic substance was a peptide, containing 11 different amino acids, with a molecular weight close to 3,500. A heat-stable amine with a molecular weight of approximately 700 daltons was isolated from a medium containing Streptococcus thermo phi/us (261). This amine inhibited P. fluorescens, P. aeruginosa and B. subtilis. Other researchers have shown that hydrogen peroxide produced by lactic acid bacteria can inhibit psychrotrophic bacteria (139,260). Price and Lee (260) found that a Lactobacillus species inhibited Pseudomonas species within 48 h at 30 C. Since the ph of the medium was not lowered below ph 6.3, these authors speculated that hydrogen peroxide produced by the Lactobacillus species inhibited the Pseudomonas species. The inhibition of psychrotrophs by lactic acid bacteria was decreased after catalase was added, suggesting that inhibition was due to hydrogen peroxide (139). However, acid development by the lactic acid bacteria would limit the use of the milk to cultured products. Miscellaneous control methods Various additives have been examined for their effectiveness in inhibiting psychrotrophs (65,220,284). Moustafa and Collins (220) researched selected food additives for their bactericidal effect on P. fragi and found that only potassium sorbate was effective at a suitable concentration (0.3%) at ph 5.5. Similar results were reported for P. fluorescens; however, the concentration of sorbate and ph of the medium determined ultimate growth (284). Gram-negative and some grampositive bacteria were inhibited by SO IJg of nalidixic acid/ml in milk to be used for growth of lactic acid bacteria (65). This concentration of nalidixic acid was not inhibitory to the lactic cultures. Cunningham (59) observed that psychrotrophic counts on raw meat were reduced by minimal exposure to microwave energy (915 MHZ). P. aeruginosa was the most radiation-resistant psychrotroph; it needed an exposure of25 sec (temperature of75 C) before the log of bacterial cells reached essentially zero. Applicability of this method of control to dairy products may be limited. BIOCHEMICAL CHANGES CAUSED BY PSYCBROTROPBS Psychrotrophic microorganisms are important in milk because they cause spoilage by biochemically altering the constituents of milk. Microorganisms that are growing at low temperatures carry out many biochemical activities involved in cell synthesis and maintenance. Incubation at low temperatures for weeks or months may be necessary for external detection of such biochemical reactions as carbohydrate fermentation and gelatin liquefaction (122). Many psychrotrophic organisms produce diffusible or nondiffusible pigments during growth at low temperatures. Fermentation of glucose and other sugars with the liberation of acid or acid and gas has been observed for some psychrotrophs grown at 0 C. Psychrotrophs can decompose urea, hydrolyze starch, reduce nitrate to nitrite and hydrolyze proteins and lipids at 0 C or lower. The temperature of incubation determines how soon the biochemical reaction will be evident (122). In general, psychrotrophs exhibit all the biochemical activities evident at higher temperatures, but these reactions occur at reduced rates at temperatures as low as 0 C. Growth of psychrotrophic bacteria is primarily responsible for limiting the keeping quality of milk and dairy products held at temperatures below 7 C. Slight biochemical changes occur in the early growth phase of psychrotrophs, resulting in a lack of freshness or a stale taste. Upon subsequent cold storage a variety of defects become apparent. Development of these off-flavors and -odors is usually a result of proteolysis and/ or lipolysis by psychrotrophs. These two degradative reactions of psychrotrophs in milk are of great concern to dairy manufacturers and processors. The literature abounds with reports of psychrotrophs isolated from milk and dairy products that have shown lipolysis and/or proteolysis (148,180,198,235,242,318, ,332,333, 352,361). Lipolysis Lipolytic microorganisms or their enzymes are important in the dairy industry because they can produce rancid flavors and odors in milk and dairy products that make these foods unacceptable to consumers (64,66,170, JOURNAL OF FOOD PROTECTION. VOL. 45, FEBRUARY 1982

18 PSYCHROTROPHS IN DAIRY PRODUCTS ). Lipase production by psychrotrophs varies with the species, as does the optimum temperature, optimum ph and enzyme specificity (333). Overcast and Skean (249) studied 25 pure cultures of lipolytic microorganisms at 4 C and found that 17 produced rancid flavor and/or bitterness in milk; six of them in 4 days, eight in 8 days, and the remainder in 12 days. The bitterness probably was a result of both proteolysis and lipolysis. P. fragi readily hydrolyzed milkfat at 7 C within 6 to 9 days (270). The liberated short-chain fatty acids probably served as the substrate for subsequent esterification. These short-chain fatty acids may be responsible for development of fruity flavors in milk and cottage cheese (271). These researchers examined the gas liquid chromatographic profile of milk after growth of P. jragi. Reddy et al. (272) isolated six esterases from P. jragi and all had different specificities for the acyl-side chain of the substrate. Lipase activity was reported for most of the psychrotrophs isolated from milk, cottage cheese and cream (90). This activity correlated well with an increase in acid degree value and off-flavors. In contrast, isolates of thermoduric bacteria did not increase either the acid degree value or the off-flavors in milk. Muir et al. (221,222) observed that small differences in temperature between 4 and 8 C have a significant effect on growth and lipolysis of psychrotrophic species. After 48 h at 6 C, most milk had counts of more than 1.7 x 10 7 CFU/ml and these counts correlated well with the concentration of free fatty acids (FF A). Fluorescent Pseudomonas species and Flavobacterium and Alcaligenes species were the most active lipolytic bacteria (223). Amounts of FF A increased in all milks that were inoculated with psychrotrophic bacteria and incubated at 2 to 10 C for 24 to 48 h (128). Therefore, FF A production is not temperature-dependent. Stewart et al. (309) isolated species of Pseudomonas, Acinetobacter and Moraxella that produced lipases capable of hydrolyzing tributryin and milkfat in media at both 6 and 25 C. These authors suggested that the fatty acids released by microbial lipases would be further degraded to carbonyls and other volatile compounds, resulting in development of offflavors in milk and dairy products. Fruity flavors were produced by psychrotrophic Pseudomonas species, especially by ethylbutyrate and ethylhexanoate esters (117). Sultzer (313) found that psychrotrophic species of Pseudomonas and Alcaligenes could oxidize saturated fatty acids. Some investigators studied specific lipolytic organisms and their enzymes for conditions of growth as well as enzyme specificity. Different strains of P. fragi had unequal abilities to produce lipase under the same growth conditions. Maximum lipase production occurred at 15 C, or below, after 3 or more days of incubation (227). McCaskey (192) found that the lipase from Achromobacter lipolyticum (genus is no longer recognized) liberated more caprylic, capric, lauric and myristic acids from milkfat than other fatty acids from butyric to palmitic. Lipase specificity was also shown by Mencher (195) with P. fragi, which hydrolyzed only glycerol esters of fatty acids in the presence of a water-fat interface. This enzyme also exhibited a specificity for the 1, 3-position of triglycerides. No significant difference was observed in the esterase patterns of exo- and endoenzymes obtained from the same culture; therefore, Yano and Morichi (380) suggested using the esterase patterns for classification of psychrotrophs. Breuil and Kushner (36,37) studied the lipase and esterase activity of two Acinetobacter species isolated from water, one psychrotroph and one mesophile. They observed that 90 to 95% of the lipase activity was extracellar and 95% of esterase activity was intracellular. Production of lipase was affected by the nutrient composition of the medium, but that of esterase was not (36). In addition to nutrients, temperature and ph influenced production of lipase; 20 C and ph 6.6 stimulated production of this enzyme. The crude enzyme was more temperature-stable than the purified enzyme (37). San Clemente and Vadehra (285) developed an assay for microbial lipase that centered around a constant ph measurement. This method was praised as being accurate, reproducible, rapid and sensitive for studying enzyme activity. A lipase isolated from P. fluorescens exhibited high activity against tributyrin between 25 and SO C, but activity decreased rapidly at temperatures above and below this range (160). At 30 C, the optimum temperature, ph 8.0 was optimum for enzyme activity. This enzyme had broad specificity, but the activity decreased as chain length of the fatty acid increased. Gas chromatographic analysis of milk inoculated with P. fluorescens has shown that the largest amounts of short chain fatty acids were released at 5 to toe. Heat inactivation of lipases Inactivation of lipase by heat has been important to dairy processors because enzymes that survive pasteurization can be detrimental to keeping quality of products. Driessen and Stadhouders (67a) found that some lipases were inactivated at 52.5 to 57.5 C, but others were heat-resistant with ad-value of 16 min at 130 C. Lipases of Pseudomonas, Achromobacter and Serratia species were heat-resistant, but those of Alcaligenes and Flavobacterium species were not (307,325). Lipases from several psychrotrophs were evaluated for their ability to survive ordinary pasteurization of 72 C for 15 to 20 sec (66). Results indicated that 0.3 to 170 min were required before 90% inactivation of the lipases was achieved. Ultra-high temperature (UHT) treatment of about 150 C was necessary to destroy lipolytic and proteolytic enzymes of psychrotrophic bacteria (151). Kishonti (150) observed that 40 o/o of the psychrotrophic bacteria, mainly species of Pseudomonas, Alcaligenes and Aerobacter, produced enzymes that retained 75% of their activity after heating for 2 min at 90 C. Eighty percent of the total lipase activity as found in cream (151). When this cream was heated at 90 C for 2 min and churned into butter, the butter became rancid in 2 days at 5 C. JOURNAL OF FOOD PROTECTION, VOL. 45, FEBRUARY 1982

19 190 COUSIN Many of the published reports on the heat stability of lipases have used Pseudomonas species as test organisms. Studies by Nashif and Nelson (226) on the lipase of P. fragi revealed that appreciable lipase activity remained after heating at 61.6 or 71.6 C for 30 min, but complete inactivation was accomplished at 99 C for 30 min. Law et al. (169) found that 75o/o of the lipase from psychrotrophs in raw milk survived pasteurization; lipases from P. fluorescens and P. fragi survived 10 min at 100 C. Complete destruction of the lipases was obtained by autoclaving milk at 121 C for 15 min. Lipase activity was detected in milk containing 6 x 10 7 CFU/ml of pseudomonds, but this activity was reduced 90% by heating the milk at 70 C for 20 sec (,115). Knaut (155) observed that lipases from P. fluorescens species were stable above 100 C. A heat-treatment of 98 C for 14 to 25 min was necessary to inactivate the lipases of some Pseudomonas species, including P. fluorescens and P. fragi (234). However, after ultrafiltration ofthe milk, the lipases were rapidly inactivated at 55 and 70 C, leading O'Donnel (234) to conclude that thermostability is probably due to a weak association between the lipase and an unidentified stabilizing factor. Pinheiro et al. (258) found that heat-inactivation of enzymes was different for members of the same genus since strains of P. fragi and Pseudomonas mucidolens produced lipases that were more heat stable than those of P. fluorescens. Twenty minutes at 99 C were needed for total inactivation of a lipase of P. fluorescens; some activity was observed after a heat treatment of 95 C for 10 min (67). Some research on heat inactivation of lipases has suggested that two phases are involved in the loss of enzymatic activity (5,68). Andersson et al. (5) studied inactivation of a lipase from a psychrotrophic strain of P. fluorescens and concluded that it followed a first order reaction in nutrient broth, but was biphasic in milk. They hypothesized that the lipase may be complexing with an unidentified component of the milk, which helps stabilize the enzyme and prevents destruction by heat. Earlier work by Driessen and Stadhouders (68) also showed that the exocellular lipases of a P. fluorescens strain lost activity in two stages. They postulated that either two enzymes were present or that one enzyme had different active sites. A Q 10 of for the first or rapid stage of lipase inactivation suggested that protein was denatured, but a Q 10 of 1.93 for the second or slow stage of inactivation was indicative of a chemical reaction. Phospholipase production by psychrotrophs Only glycolytic, lipolytic or proteolytic activities were assumed to be involved in degradation of milk by psychrotrophic microorganisms; however, recent research has suggested that phospholipases may be important in milk spoilage. Fox et al. (82) isolated 58 microorganisms, which produced phospholipase C, from fresh and spoiled homogenized milk. Most isolates were species of Pseudomonas, particularly P. fluorescens. The remainder of the isolated cultures included species of Alcaligenes, Acinetobacter, Flavobacterium, Enterobacter, Citrobacter. Bacillus and two unidentified yeasts. These authors postulated that phospholipase was degrading the milkfat globule membrane and thus increasing the susceptibility of milkfat to the action of lipases. Owens (250) also isolated species of Pseudomonas, Bacillus, Alcaligenes, coliforms and corynebacteria that produced phospholipase as demonstrated by the lecithinase reaction on agar containing egg yolk. B. cereus and B. cereus var. mycoides were shown to be responsible for the production of the 'bitty cream' defect in pasteurized milk. One explanation of this reaction suggested that these species of Bacillus produced enzymes that hydrolyzed lecithins present in the milkfat globule membrane, resulting in aggregation of the fat. Proteolysis Changes in milk proteins as a result of psychrotrophic growth or enzymatic action are important in the keeping quality of milk and its products at refrigeration temperatures. Release of various nitrogen components or degradation of the individual protein fractions have been observed when studying proteolysis caused by psychrotrophic enzymes. Data indicate that most raw milk supplies probably contain heat-stable proteases or bacteria able to produce them (1,303). These proteases can attack casein and whey proteins, leading to bitter flavor development and coagulation of milk. The success of UHT pasteurization could be hindered by presence of these heat-stable enzymes in milk. Some investigators reported that bacterial proteases were not detected when bulk milk was stored at 4 C in tanks which were emptied 3 to 4 times a week (286). Pseudomonas spp. produced protease upon further storage at 4 C, but greater amounts were elaborated at 10 to 20 C. Van der Zant and Moore (,157) found no relation between psychrotrophic populations and proteolytic activity. Juffs et al. (142) also reported that proteolytic activity was not proportional to microbial growth since a peak of proteolysis in the early log phase was observed and was more pronounced at 3 than at 28 C. Proteolytic enzymes were absent if the medium lacked a source of organic nitrogen. Large psychrotrophic populations are not required for production of significant amounts of heat-resistant proteases (J). A recent review of proteolytic enzymes produced by psychrotrophs in milk and the effects of their enzymes on dairy products was published by Law (164). Production and characterization ofproteases Several investigators have isolated proteolytic enzymes from psychrotrophic species and tried to characterize them. Peterson and Gunderson (256) purified a proteolytic enzyme from P. fluorescens, which had been isolated from a frozen chicken pie. This culture liquefied gelatin rapidly and attacked casein in addition to being lipolytic and amylolytic. The enzyme was produced in largest quantity at 0 C and production decreased as the temperature increased to 30 C. The proteolytic activity of JOURNAL OF FOOD PROTECTION. VOL 45, FEBRUARY 1982

20 PSYCHROTROPHS IN DAIRY PRODUCTS 191 the isolated enzyme increased as the temperature increased. Several strains of Pseudomonas were found to be highly proteolytic by Huskey (119). The most proteolytic culture produced the largest amount of proteolysis in raw, heated and renneted raw skimmilks. Raw skimmilk usually had more proteolytic activity than heated milk, but after trichloracetic acid (fca) precipitation the heated samples had more proteolysis than the unheated, suggesting that intermediate amino acid forms in the raw skimmilk could have been precipitated by TCA. McCaskey (192) reported that proteolytic activity of P. fluorescens was retarded in milk stored at 4.4 C for 7 days with or without added glucose, but growth of the organism was not retarded to the same degree as activity of the enzyme. Enzymes in the cell-free broth were not inactivated by holding at 62.8 C for 82 min. The percentage of protein in the whey from the milk also increased during 7 days at 4.4 C. Mayerhofer et al. (190) characterized a heat-stable, extracellular protease from P. fluorescens P26 which caused off-flavors in milk as well as release oftyrosine. Proteinase from P. aeruginosa was active in the ph range of 5.5 to 9.0 with maximum activity at ph 7.3 (141). This enzyme lost 6% of its activity when heated for 30 min at 63 C and 36 o/o at 72 C for 15 sec and was completely inactivated by boiling for 2 min. At 2 C this proteolytic enzyme was stable for 1 month and casein was readily hydrolyzed at this temperature. Characteristics of some proteases that have been isolated from psychrotrophs are listed in Table 6. Gebre-Egziabher et al. (89) found that each of the proteases isolated from several Pseudomonas strains had different degrees of heatresistance. Heating at 121 C for 2 min destroyed less than 40% of the activity of most of these proteases. Though they most actively hydrolyzed casein at 40 C, about 60 to 80o/'oofthe maximum activity occurred at 25 C and there was some activity at 4 to 7 C. Similar results have been observed by other researchers (1,254,279). Adams et al. W reported that a protease produced by Pseudomonas MC60 was 4000 times more heat-resistant than spores of Bacillus stearothermophilus. Less than 10% of the protease from this culture was destroyed in 4 sec at 149 C. This protease was most active at 45 C and retained 25% of that activity at 25 C. UHT-milk containing this protease developed bitter flavor within 3 days with 18 units of protease/ml and within 14 to 32 days with 0.89 unit of protease/mi. Patel et al. (254) also found that a protease from a Rhodotorula species had considerable activity at 5 to 7 C although the optimum activity was observed at 20 C. Several studies were done to explain the relationship of nutrients and environmental factors to production of proteases by psychrotrophic microorganisms (),138,145, 146,193,208). Production of proteases was reduced by addition of sugars although growth was increased (138,145,193). Amino acids enhanced production of proteases (193). As a result of these studies, the extracellular proteases were believed to supply carbon for growth rather than amino acids for protein synthesis. Keen and Williams (1 45) observed that both growth and protease activities were increased when the culture was aerated. Other research reports indicated that aeration may or may not result in increased protease production ()15). The optimum ph and temperature for protease production depends on the species and strain (fable 7). Various metal ions (aluminum, cadmium, copper, mercury, lead, silver, tin, zinc) and reagents reacting with TABLE 6. Characteristics ofproteases produced by psychrotrophs. Optimum Molecular Temperature-time Microorganism ph weight of inactivation Pseudomonas sp , C-10 min Pseudomonas sp C-8 min Pseudomonas MC a 149C-1.5 min Rhodotorula sp. 7 looc-20 min Pseudomonas sp. B C- >2 min Pseudomonas B C- >2 min a Figure is not given in the paper. TABLE 7. Optimum temperature and ph for protease production by psychrotrophs. Microorganism Optimum temperature P. fluorescens 28 P Cytophaga 20 C P. fluorescens P. aeruginosa P. fluorescens AR Pseudomonas P31 45 Pseudomonas P12 37 ao/oactivity ofthe maximum observed at optimum temperature. hmedium was at a constant ph of7.0±0.2. CMedium was skimmilk. Optimum ph o/o Activity at4-7ca < <5... b 33 b c 0.2 c 5 Reference Reference JOURNAL OF FOOD PROTECTION, VOL.4S,FEBRUARY 1982

21 -~~ ~ ~----~ COUSIN sulfhydryl groups (mercaptoethanol, dithiothreitol, iodoacetamide, iodoacetic acid) caused complete or partial inhibition of the protease from P. jluorescens AR-11 "). However, ethylenediaminetetraacetic acid (EDTA) slightly increased protease activity. Richardson and Te Whaiti (279) observed that EDT A inactivated proteases of the four Pseudomonas strains, which they studied. The most common proteolytic activity in milk was reported as clotting (138,146). luffs (138) also observed peptonization and occasionally alkaline reactions. Though they isolated Pseudomonas lachrymans from a cucumber, Keen et al. (146) reported that the protease was active against casein and caused clotting of milk. Two general review papers (93,267) have presented information on production and molecular biology of extracellular enzymes. Some of this information is applicable to the protease produced by psychrotrophic microorganisms. Hydrolysis of milk proteins Many investigators around the world have studied milk protein degradation by psychrotrophic bacteria or by their proteolytic enzymes. Analysis of milk which had supported growth of three psychrotrophic Pseudomonas spp. gave modified electrophoretic patterns for casein (245,298). P. fluorescens, P. fragi, Flavobacterium sp. and Achromobacter sp. altered the caseins in skimmilk at 0 and 5 C with the largest change caused by P. fragi (225). Electrophoretic analysis of hydrolyzed casein revealed that {3- and a-caseins were attacked preferentially by proteases from psychrotrophs (50,63,153,156,208,212, 266,379). Kiuru et al. U53) found that changes in casein composition were observed when flavor defects first appeared in milk. Part of the as-casein was removed and probably was associated with the {3-casein since the amount of this fraction increased. Workers in Japan "79) noted that milks that were inoculated with Pseudomonas and Flavobacterium species and incubated for 7 days at 5 C showed decreases in {3- and a-casein while the amounts of unabsorbed and early-eluted TABLE 8. Protein degradation by psychrotrophic microorganisms. Protease/ Microorganism Reference a- (3- fractions increased. After 14 days at 5 C, bands of a- and {3-casein, as well as early moving components during the second phase of proteolysis, were evident in gels by appearance of bands that migrated in front of that of a-casein (156,266). Cousin and Marth (50) observed similar results with Pseudomonas, Flavobacterium and Micrococcus species. Hydrolysis of casein began by the appearance of fast-moving electrophoretic bands. The a and {3-caseins were degraded by these psychrotrophs, but the selectivity and degree of hydrolysis differed (Table 8). DeBeukelar et al. (63) also reported that DEAE-cellulose chromatograms showed some early eluting fractions that were attributed to proteolysis by psychrotrophic bacteria. {3-casein was degraded to a greater extent than a-casein by psychrotrophic bacteria (156,265,266). Overcast (246) observed some distinct changes in the electrophoretic pattern of casein when milk was inoculated with P. fragi, P. fluorescens and Pseudomonas putrefaciens and complete disappearance of {3-casein was observed after 42 days at 3 to 5 C. Work with B. cereus showed that {3-casein was degraded more readily at 30 C than the other casein fractions, but degradation of whole casein and as-casein was also very rapid (194). Kiuru et al. (152) found /3 and x:-caseins were proteolyzed by the native flora of milk. An Aerobacter sp. hydrolyzed x:-casein and clotted the milk. These changes in the casein fractions were noted after the psychrotrophic count reached 6 x 10 7 CFU/ml or more. Isolated as- and {3-caseins were quite susceptible to proteolysis, but were less so in the whole casein mixture. {3-casein was more susceptible to proteolysis as the temperature was lowered and as-casein became more susceptible when the micellar structure was disrupted by removal of the calcium phosphate colloid (83). Other researchers reported that individual caseins were more susceptible to proteolysis than was native casein (259). Some of the work on casein hydrolysis used enzymes or cell-free extracts instead of the psychrotrophic cells. When whole casein, and. a- and {3-caseins were incubated Whey )( y (j.a a-5 Pseudomonas sp C +d Pseudomonas sp P. fluorescens Pseudomonas sp Pseudomonas sp P.fluorescens 28 P Flavobacterium sp Flavobacterium sp Micrococcus sp Cytophaga 20 C Pseudomonas sp Aeromonas crefers to most active degradation. drefers to degradation. erefers to slight degradation. a a-lactalbumin. b [J-lactaglobulin (+)e JOURNAL OF FOOD PROTEC110N. VOL. 45, FEBRUARY 1982

22 PSYCHROTROPHS IN DAIRY PRODUCTS 193 with cell-free extracts from micrococci which were isolated from Cheddar cheese, as-casein was degraded, but (3-casein showed only slight hydrolysis after 2 days at 30 C. {3-casein had disappeared completely from whole casein after 2 days at 30 C, but part of the a-casein remained ()30). Ledford and Chen (174), working with proteases from milk, rennet and Streptococcus faeca/is var. /iquefaceins, found that as- and {3-caseins were degraded with {3-casein, being degraded first at 32 C for 20 h. Law et al. (166) studied gelatin of UHT milk by proteases from P.jluorescens and noted that after 3 days and a count of 5 x 10 7 CPU /ml, 20% of the {3-casein had been degraded and x:-casein was lost, but as-casein remained. Eight proteases produced nearly identical patterns of milk protein degradation, starting with a decrease in x:- and y-caseins followed by subsequent decreases in {3- and asl caseins ()79). Degradation of whey proteins by psychrotrophs had been observed less frequently. Whey proteins were not degraded by the psychrotrophs used in the research (50,63,166,208,279,379). However, other workers (J45, 246,298) reported that two fractions of whey protein were more vulnerable to psychrotrophic growth and these were probably proteose peptone fractions. In addition, some new electrophoretically-different fractions appeared after 14, 28 and 42 days at 3 to 5 C, depending on the Pseudomonas species studied. Adams et al. ())observed that psychrotrophic Pseudomonas species caused decreases in both a-lactalbumin and (3-lactoglobulin, ranging from 0 to 39% degradation after 13 days at 5 C, depending on the species and strain. Degree of proteolysis Some researchers studied the degree of proteolysis caused by psychrotrophs or their proteases by measuring different nitrogen components released into the growth medium. Nakanishi and Tanabe ()25) studied changes in milk protein caused by psychrotrophs during coldstorage and observed that casein nitrogen in milk decreased, whereas nonprotein nitrogen (NPN) increased during storage. Other investigators reported an increase in both noncasein nitrogen (NCN) and NPN in cold-stored milk treated with heat-resistant proteases isolated from psychrotrophic bacteria (1). Knaut (156) observed that the values for formol nitrogen and NPN were greater in milks inoculated with Pseudomonas spp. than in control milks. Amino acid concentrations were different for control milks and those milks inoculated with Pseudomonas spp. Pseudomonas organisms which were inoculated into milk grew rapidly at 6 to 9 C and increased production of pyruvate and ammonia fre quently were observed (113). Aerobic s poreformers had a much slower growth rate than nonsporeforming psychrotrophs, but still produced considerable proteolysis. Patel et al. ()54) assumed that casein was degraded to only proteose-peptones since the NPN level remained essentially constant and the decrease in casein nitrogen corresponded to the increase in proteose-peptone nitrogen. Richardson and Newstead ()78) reported that NPN increased with increasing protease concentration. Bitterness and significant proteolysis, measured by electrophoresis, were evident in these milks. Though no relationship was found between psychrotrophic populations and proteolysis, a relationship existed between populations and production of soluble nitrogen, tyrosine and tryptophan (357). An increase in proteolysis, measured by the Hull test, occurred in high temperature-short time pasteurized milk which had protease added before or after pasteurization or had P. fluorescens P26 added before pasteurization (373). Studies have shown that the rate, type and breakdown products of proteolysis differ for various psychrotrophic bacteria (80,225,246). Proteolysis of milk by B. subtilis differed from that by P. fluorescens (80). P. fluorescens gave high ninhydrin values at comparable NPN levels and the difference became larger as the NPN values increased. Proteolytic activity was greater for Pseudomo nas nigrijicans and P. putrefaciens than for Pseudomonas cohaerens at both 10 and 25 C ()25). Miller and Kandler (J06) studied the amino acid and nitrogen content of raw milks that contained pure cultures of Pseudomonas, Alcaligenes, Micrococcus and Escherichia and found that a population of 100 million psychrotrophs/ml gave evidence of quantitative changes in low molecular weight nitrogen compounds. Different amino acid patterns after psychrotrophic growth were speciesdependent. Ami1lo acids released by proteases have been identified in a few studies. Knaut (154) used an amino acid analyser to identify the free amino acids in milk after growth of three Pseudomonas species. Lysine, leucine, ornithine, phenylalanine, valine, methionine, glutamine, serine and isoleucine were detected. Other amino acids were detected in trace amounts or not at all. Amino acids liberated from (3-casein after inoculation and incubation with a protease of Micrococcus freudenreichii were mainly lysine, glutamine, proline, valine, leucine, threonine, serine, alanine and methionine (J13). Reimerdes et al. ()73) studied the specificity for L-phenylalanine- 4-nitroanilide hydrolysis. This specificity could be used to design an assay for proteases produced by P. fluorescens. Thermostabi/ity and inactivation of psychrotrophic proteases Researchers within recent years have attempted to determine why proteases are thermostable and develop methods to inactivate these enzymes. Barach et al. (15) observed that calcium and zinc increase thermal stability of Pseudomonas proteases. They suggested that the ability to survive extreme temperatures probably resulted from the structural flexibility of the enzyme and the interaction of the cations in allowing renaturation to occur during heating (13). Barach et al. (14) investigated the possibility of using low temperatures to inactive proteases since these enzymes were stable to high temperatures. Heating purified protease from Pseudomonas species for 10 min JOURNAL OF FOOD PROTECTION. VOL. 45, FEBRUARY 1982

23 194 COUSIN at 55 C destroyed more than 90% of the proteolytic activity, but the same treatment of a crude protease destroyed only 70% of the activity. The inactivation was independent of protease concentration (14,369). West et al. (369) reported that the best method of inactivating heat-resistant proteases was to heat for 1 hat 55 C since this resulted in inactivation of 87 to 90o/'o of the protease in milk. No enzyme reactivation was observed after this treatment. In studies of this low temperature inactivation of proteases, Barach et al. (16) observed that a temperature of 55 to 60 C caused the protease to lose activity. Inactivation appeared to be a two-stage process beginning with a conformational change of the enzyme structure followed by aggregation with casein micelles to form a complex. The conformational change did little to decrease protease activity, but complex formation resulted in enzyme inactivation. At high enzyme concentrations, autolysis also caused protease inactivation. Marshall and Marstiller (187) used this concept to test the degree of inactivation of seven proteases from P. jluorescens. Six of 10 filtrates were less than 50% inactivated by 60 min at 40 C, but one P. jluorescens protease was completely inactivated. These authors reported little lability at 50, 60, or 70 C. Detection of proteolysis Proteases in milk and dairy products have made determination of proteolysis an important area of research. Presently, a rapid and reliable method is not available for use in determining proteolysis of milk. Juffs (134) studied the tyrosine value as a method for detecting proteolysis in milk, but found no relationship between psychrotrophs or proteolytic psychrotrophs and the tyrosine value. Further research by Juffs (135) showed that detection of proteolysis in cold-stored milk by measuring the tyrosine value after preincubation at 30 C was not reliable since non-psychrotrophs could cause an increase in the population and tyrosine value. Use of the tyrosine value for determining proteolysis and milk quality has limited application since counts greater than 1 x 10 6 CFU/ml were necessary before changes in this value were detected (137). Lawrence and Sanderson (173) developed an agar diffusion slide assay that used a thin layer of caseinate agar for quantitating proteolytic enzymes. Concentrations as low as 0.1 j.jg/ml could quantitatively be estimated by this method. Though this method was developed for detecting microbial rennet substitutes, it may have some use for detecting proteases from psychrotrophs since it was highly reproducible and suitable for assaying a large number of samples. Several methods have been proposed as replacements for the Kjeldahl nitrogen determination. Koops et al. (1 58) developed a method for determining nitrogen in milk that used a colorimetric determination of ammonia after sample digestion. These authors reported that there was good correlation between Kjeldahl and colorimetric nitrogen determinations. An advantage of this method is that 16 samples can be analyzed per hour. KEEPING QUALITY OF MILK AND DAIRY PRODUCTS Since multiplication rate and activity of bacteria are reduced at low temperatures, keeping quality of milk is improved by refrigerated storage. However, psychrotrophs multiply and become active at refrigeration temperatures, causing spoilage of milk and dairy products. Many researchers have concluded that psychrotrophic contamination and keeping quality are closely related. Overcast ~45) observed three stages in the development of off-flavors: (a) milk started to lack freshness, (b) milk became stale and (c) rancid, fruity and bitter flavors occurred. Off-flavors have been detected organoleptically in milk in less than 5 days at 1 to 4.4 C. The most common defects observed were fruity and rancid flavors ~96,318,330,355). Putrid, potato, cheesy, bitter, unclean, soapy and fishy flavors have been associated with proteolysis and/or lipolysis by psychrotrophs (126,180,264,28L296,318,330,355,378). A beef broth-type flavor was produced by a proteolysate of P. jluorescens as a result of lactose-protein breakdown and browning of milk (197). Bitter flavors usually accompany protein degradation ~96). Unclean and putrid flavors and odors may be attributed to protein breakdown into bitter peptides and further decomposition of amino acids to putrid endproducts. Soapy and rancid flavors usually are a result of lipid breakdown to even-numbered fatty acids, such as: butyric, caproic, caprylic, capric and lauric. Patel and Blankenagel ~53) attributed flavor defects in milk to: (a) endproducts of microbial metabolism, (b) constituents of lysed bacterial cells, (c) heat stable enzymes and (d) growth of thermoduric microorganisms. In addition to flavor changes in milk, ropiness was sometimes noted before off-flavors developed ~64,317,318,378). Milk Raw and pasteurized milks usually spoil when held at refrigeration temperatures because of the action of psychrotrophic contaminants (75). The populations needed to cause detectable changes in milk varies among genera, among species and within a genus, but levels at which flavor changes occurred were similar at 6 and 20 C ~64,332). Flavor changes were observed when populations were less than 100 million/ml ~64). Generally, populations ranged from 5 x 10 6 to 20 x 10 6 /ml at the time a detectable change was observed. Another report stated that off-flavors in milk were only detectable after the maximum stationary growth phase was completed and at this time the populations usually exceeded 2 x 10 8 /ml (333). Ogawa ~35) observed that organoleptic changes seldom were detectable in milk after 7 days of storage at 5 to 7 C when the population approximated 1 x 10 7 to 1 x 10 8 /ml. Milk spoilage by psychrotrophs was reported in a range of populations of 1 x 10 2 to 1 x 10 9 /ml (314). Population (per ml) at the time when definite off-flavors were observed have been reported for some psychrotrophs: Pseudomonas, 5.2 to 200 million; JOURNAL OF FOOD PROTECTION. VOL. 45, FEBRUARY 1982

24 PSYCHROTROPHS IN DAIRY PRODUCTS 195 Alcaligenes, 2.5 to 14 million; Flavobacterium, 8.3 to 120 million; coliforms, 2.7 to 150 million and yeasts, 2.5 to 14 million a63). Tolle et al. (355) observed that P..fragi and P..fluorescens at populations of about 5 x 10 6 organisms/ml caused unclean flavors in milk held at 6 C. They also reported that other psychrotrophs produced similar off-flavors at 1 x 106 to 108 organisms/mi. Most psychrotrophs will produce a detectable flavor change when the population exceeds 1 million/ml according to Richter (281). However, some investigators (105,253,299) have found that some milk with high counts was still acceptable, indicating that the number of bacteria was not as important as the types of bacteria which were able to degrade milk components. Patel and Blankenagel (253) observed that milk still was acceptable 14 days after pasteurization with a count of 5 x 10 7 organisms/mi. Low temperature has been an important control for prolonging the shelf-life of pasteurized milk. Smith et al. a99) observed that milks with high psychrotrophic counts usually had shorter shelf-life than milk with low psychrotrophic counts. However, microbial spoilage was dependent on the number of microorganisms contaminating the milk, length of lag phase of growth, rate of growth at storage temperature and type of microorganism present. Finley et al. (79) noted that as the storage temperature increased, the keeping quality decreased. Eighty-one percent of milk held at 0 C remained acceptable for over 3 weeks, but only 15% of that held at 7.2 C was acceptable for more than 1 week. Similar results were reported by Hankin et al. (105). Milk stored at 1.7 C, was good for 17.5 days and was spoiled primarily by proteolytic microorganisms, but that stored at 5.6 C was spoiled by proteolytic and acid-producing microorganisms within 12.1 days. Only 4% of the milk was acceptable after 1 week at 10 C since acid-producers and coliforms caused flavor defects. The flavor score of the milk decreased as the age of the milk increased (105a). Factors, in addition to microbiological deterioration, which affect the shelf-life of milk are enzymatic and physical deterioration (125). White et al. (,171) and White and Bulthaus (.170) inoculated raw milk with 1 x 105 proteolytic psychrotrophs/ml and incubated the milks at 5 to 25 C for 24 h before pasteurization and storage at 4 C. The flavor scores decreased and the Hull value, which measured proteolysis, increased over the 22-day storage period at 4 C. After 14 days, this milk was unacceptable on the basis of flavor scores and high proteolysis values. Proteolytic activity and acceptability do not always correlate well with microbial populations (75). Thermoduric psychrotrophs which survive pasteurization and grow in milk can affect the keeping quality of milk. Bodyfelt (31) has speculated that 20 to 25% of shelf-life problems with pasteurized milk may be due to heat-resistant psychrotrophs. Langeveld et al. (163) reported that some milk was spoiled by aerobic sporeforming strains of Bacillus circulans which fermented lactose. Psychrotrophic counts of 1 x 105 to 1 x 10 6 heat-resistant organisms/ml have been reported after 14 days of storage at 7 C aos). Bitter, fruity, rancid, sour, yeasty, putrid and unclean off-flavors have been attributed to growth of thermoduric psychrotrophs in milk (21,31,200,293,365). Organoleptic defects were noted when thermoduric psychrotrophs reached populations of 3 to 4 million CFU /ml when milk was held at 7.2 C for 6 days ()53). B. cereus strains have been implicated in bitty cream and sweet curdling defects of pasteurized milk (45,57,200,248,305). The curd usually develops only at the bottom of the container of milk and may result from casein degradation (45). Overcast and Atmaram a48) reported that previous growth of Pseudomonas species and higher temperatures of pasteurization may stimulate germination and outgrowth of B. cereus. Various chemical tests have been used for determining the keeping quality of refrigerated raw milk. Among them are titratable acidity, ph, protein stability, tyrosine and tryptophan content, coagulation during boiling and methylene blue and resazurin reduction, but all are not truly indicative of keeping quality (330). A test that takes into account the number and type of psychrotrophic contaminants, rate of increase of psychrotrophs during storage and psychrotrophic activity in producing spoilage is necessary (326). Thomas and Thomas (.133), after summarizing various surveys, recommended the following standards for keeping quality of raw milk: (a) an initial colony count of < 10 4 /ml determined by incubation at 7 C for 10 days would be satisfactory, (b) initial counts ranging from 1 x 10 4 to 10 5 /ml shows unsatisfactory production and/or refrigeration of milk. Maxcy (191) observed that about 90% of the thermoduric bacteria contributing to the total plate count (at 32 C) of pasteurized milk are of minor significance in spoiling milk. The gram-negative contaminants are of greater importance; therefore, the total plate count after pasteurization will not determine the keeping quality of milk. Watrous et al. (,166) made similar observations with pasteurized milk and concluded that a test with incubation at 4.4 or 7.2 C for 5 to 10 days was a good indicator of keeping quality. Since neither initial flavor scores nor microbial counts were useful indicators of keeping quality of pasteurized milk, Hankin et al. (1 OS) suggested that more discriminating tests must be developed for routine work in dairy plants. Ultra-high temperature (UHn processed milk and cream UHT processing of milk and cream is used to extend the shelf-life of these products under refrigeration. Problems with bitter flavor development and coagulation have been reported since enzymes produced by psychrotrophs can survive UHT-processing (2,20,166, 278,282,301,303). Bengtsson et al. (20) reported that a protease from a Pseudomonas species degraded casein and split glucomacropeptide from the x-casein moiety. A protease from P. fluorescens caused rapid breakdown of x-casein to para-x-casein and eventually (J- and as-casein were degraded (1 66). Formation of para-x-casein resulted JOURNAL OF FOOD PROTECTION, VOL.45, FEBRUARY 1982

25 1% COUSIN in a rennet-like clot since it was unable to stabilize the casein (),301). Adams et al. ())noted that milk which had extensive ~e-casein proteolysis coagulated during UHT treatment. Richter et al. ()82) used the Hull test to measure proteolytic activity in UHT-treated cream. Very little proteolytic activity was detected after 12 h at 37 C, but activity was observed after 1-2 days. The Hull test was not sufficiently sensitive to detect low levels of proteolysis. Richardson and Newstead ()78) found that milks were unacceptable after 3 months due to development of bitter flavors. Increase in the nonprotein nitrogen to total nitrogen (NPN:TN) ratio correlated with an increase in bitter flavor. Other authors have cited similar bitter flavor development accompanying milk protein degradation()). Cream Lipolytic spoilage of cream was noted as early as 1932 when raw cream held at 6 C yielded rancid butter (317). Investigators found that a bitter flavor had developed because of marked bacterial growth in raw cream held at 1.7 C for 10 days. An increase in the number of lipolytic pseudomonads and achromobacteria (no longer a recognized genus) was noted while the number of micrococci had decreased (317). N ashif and Nelson ()28) noted that over SO% of the lipase from P. fragi was not inactivated by pasteurizing cream at 71.5 C for 30 min. Of the total lipase activity, 80% was concentrated in the cream (J 51). Thomas (320), in a review on market cream, stated that deterioration of cream was characterized by lipolysis and development of a bitter flavor. Psychrotrophic yeasts and molds also lipolize cream. Heatresistant lipases can cause the deterioration of market cream. Cream was considered to be deteriorated by Tekinson et al. (314) when colony counts were 1 x 10 6 CFU/ml or greater. The microbial flora at 5 C was composed of species of Pseudomonas, Alcaligenes, Acinetobacter and Aeromonas with Pseudomonas species dominating the population. P. fluorescens and Pseudomonas viscosus have caused lipolytic taints and bitter flavors in cream (125). More recently B. cereus has become important in the possible spoilage of cream (57). Butter Butter usually is not a suitable medium for growth of microorganisms because of the limited amount of water and the presence of salt (327). Pseudomonas species can cause defects in butter, especially P..fragi which produces fruity odors and rancidity, P. putrefaciens which produces proteolytic changes and putrid defects, and P. nigri,ficiens which produces a black surface discoloration of butter (328). Lipases, which survive pasteurization, can cause deterioration of butter. Nashif and Nelson ()28) found that butter containing residual lipase underwent considerable fat hydrolysis even when stored at -10 C. Butter can develop such defects as: putrid, cheesy, musty and fishy tastes and skunk-like and apple odors due to psychrotrophic bacteria (328). White and Marshall (373) found that the protease from P. jluorescens P26 had no appreciable effect on butter. Deeth and Fitzgerald (64) reported that lipolysis in cream resulted in frothing during churning of butter. Rancid cream required 10 to SO% longer to churn than fresh cream. Problems in separation can result when rancid cream is used. Cheese In recent years, the quality of refrigerated raw milk for cheesemaking has been of concern, especially in regard to bacterial numbers and types, coagulation by rennet, starter activity and the quality of the finished cheese. Psychrotrophic counts for raw milk for cheese production have ranged from 1 x 10 4 to 1 x 1Q&/ml (44,321,331). The time required for coagulation of milk by rennet increased when milk is refrigerated for long periods (8,43,210,269,283,360). Anita (8) found that after 1, 2 and 3 days at 2 to 3 C, the renneting time for milk increased by 4, 7 and 11 o/o, respectively. Similar increases in rennetting time were reported by Rapp and Calbert ()69). The reduced tendency for milk to coagulate upon addition of rennet was overcome by adding calcium salts or by acidification of milk ()83,375). Recent research by Cousin and Marth (55) contradicted these earlier reports since coagulation time of milk by rennet was reduced when previous psychrotrophic growth had occurred in the milk. This may result from casein hydrolysis since additional research by these authors demonstrated that (3- and a-caseins were being degraded by the psychrotrophs possibly making ~e-casein more accessible to rennet coagulation (SO). Law et al. (165) noted differences in renneting times of milk with and without psychrotrophic growth, but they did not attribute these differences to psychrotrophs. Some workers have reported that lactic acid bacteria do not grow well in milk which has been stored cold; whereas, other researchers claim that it is a better medium for growth. Mocquot and Ducluzeau ()10) believed that cold-stored milk was a poor growth medium for lactic bacteria. The same belief was held by other researchers (10,291,321). Sellars ()91) noted that fractional components of protein degradation can sometimes inhibit lactic cultures. Evidence for stimulation of lactic cultures in cold-stored milk that has psychrotrophic growth can also be found in the literature (47,102,157,302). Nath and Ledford ()29,230) and Nath and Wagner ()31) found that capsular material from micrococci stimulated acid production in milk by Lactobacillus and Streptococcus species and suggested that it was due to proteolysis and lipolysis by micrococci. Cells of Micrococcus F4 removed hydrogen peroxide, but the capsular material was less effective than whole cells in destroying hydrogen peroxide. Cousin and Marth ($3,54) observed that previous psychrotrophic growth in milk resulted in increased acid production by S. lactis, 10 URNAL OF FOOD PROTECTION. VOL. 45. FEBRUARY 1982

26 PSYCHROTROPHS IN DAIRY PRODUCTS 197 Streptococcus cremoris, S. thermophilus and Lactobacillus bulgaricus. This increase was attributed to proteolysis by the psychrotrophs, which could have supplied the lactic acid bacteria with usable nitrogen fractions. Several reports on production of cheese from cold-stored milk indicated that there were no problems in manufacturing cheese of good quality (7,8,34,44,101, 210,321,360). Camembert-type cheese made from milk with psychrotrophic counts ranging from 1 x 101 to 1 x 10 6 CFU/ml did not suffer from loss of quality (101). However, other researchers observed that cheeses made from milk of inferior quality did suffer from flavor and textural changes due to psychrotrophic growth, which produced heat-resistant proteases and lipases that were active in the cheese (.14,51, 72,97,219,236,290,306, 331). Cousin and Marth (51) observed that Cheddar cheese made from milks with previous growth of psychrotrophic Pseudomonas and Flavobacterium species took less time to manufacture, had firmer curds and became unacceptable after 6 months of aging at 10 C in contrast to control cheeses. The differences that were observed were attributed to proteolysis. Cousins et al. (56) noted that proteolysis of cheesemilk can affect cheese manufacture. However, Law et al. (165) concluded that numbers of psychrotrophs present in raw milk are not apt to reduce the quality of Cheedar cheese by production of proteases. Instead, these cheeses became rancid because ofthe lipases present. FF A, produced by lipolysis of milk fat, are important in Cheddar cheese flavor (225,255). However, excessive lipolysis in cheese, resulting from heat-resistant lipases of psychrotrophic origin, can cause off-flavors (56,64,67, 67a,72,87,103,168,169,258,304,307). Cheese which was made from milk that was stored at 3.5 C for 48 h before pasteurization and manufacture into cheese, showed marked organoleptic defects, including a high content of free nonvolatile fatty acids (219). Soft cheese made from milk that was stored at 5 C was more compact, more bland and became rancid faster than control cheeses. This cheese had greater concentrations of long-chain saturated fatty acids, unsaturated methyl ketones and volatile fatty acids than control cheese (72). Bills and Day (23) also reported that rancid cheese had greater concentrations of FFA than normal cheese; 10 times more butyric through linolenic were reported for rancid compared to control cheeses. Major volatile compounds of Cheddar cheese increased faster and to a much greater degree in raw milk cheese than in pasteurized milk cheese (287). Psychrotrophic microorganisms or their enzymes in milk used for cheese manufacture can result in cheese with various defects. Ohren and Tuckey (236) observed that the best cheeses were made from milks having psychrotrophic counts between 1 x 10 3 and 2 x 10 4 CFU/ml. Large numbers of bacteria in milk caused off-flavors, but no bacteria resulted in flavorless cheese (277). Intense Cheddar cheese flavor developed faster in cheese that contained bacteria other than lactobacilli (167,274). However, Hattowska (110) reported that psychrotrophic growth in milk before pasteurization and cheesemaking apparently produced enzymes that inhibited acid and flavor development in cheese. Bitter flavors in cheese have been attributed to proteases in milk or cheese (165,373). Rancid, soapy, yeasty, butyric and fruity flavors have been reported as the result of lipolytic activity in cheese (51, 72,168, 169,258,274). Flavor changes in cheese have been found as early as after 2-3 months of ripening and as late as after 9 months (51,103,168,169,274). Proteolytic psychrotrophs in milk have led researchers to study the yield of cheese after the growth of psychrotrophic bacteria in milk. Feuillat et al. (78) reported that the partial solubilization of proteose peptones and amino acids caused a 5% decrease in yield of nitrogen. Reduced cheese yields were reported by several other investigators from milks with counts as low as 2 x 10 5 CFU/ml (4,232,239,280,384). Usually, proteolytic and lipolytic strains of Pseudomonas and Bacillus were used. Allauddin et al. (4) correlated the decrease in yield with an increase in NPN of milk. Yates and Elliot (.182) measured the protein lost in whey and then concluded that it was a result of growth of psychrotrophic bacteria that resulted in a decreased cheese yield. Law et al. (165) noted that 1 x 10 7 CFU/ml of Pseudomonas and Acinetobacter species caused a slight degree of {t- and x-casein breakdown, but concluded that this breakdown was not sufficient to cause losses in cheese yield. Magdoub et al. (182) studied the possibility of using cell-free filtrates of proteolytic species of Bacillus to accelerate cheese ripening. These authors concluded that stimulation of lactic acid bacteria resulted from proteolysis of the milk by the filtrates thus reducing the ripening period for Ras cheese by 50%. The quality of the cheeses was rated as equal to or better than control cheeses. Juffs (136) experimented with proteases from Pseudomonas species as coagulants of milk for Cheddar cheese manufacture. The body of these cheeses was soft because whey expulsion was reduced and proteolytic activity decreased as acidity increased. These experimental cheeses were evaluated as having bitter and unclean flavors. Cottage cheese Most of the information relating to psychrotrophic growth and cottage cheese quality concerns product spoilage. Contaminated water can be a source of cottage cheese spoilage organisms since the curd is washed and held at low temperatures (233,317). Psychrotrophs are responsible for a gelatinous or slimy curd defect of cottage cheese which can be accompanied by a putrid or fruity odor or flavor. Surface spoilage of cottage cheese was reported to develop slowly at 3.5 C (.117). Mann (183) found that yeasts and molds limited the shelf-life at 4.4 C to 14 to 16 days. Other workers have studied the chemical constituents responsible for fruity flavor caused JOURNAL OF FOOD PROTECTION, VOL. 45, FEBRUARY 1982

27 198 COUSIN by P. fragi in cottage cheese. Marth (184) discussed spoilage of cottage cheese by psychrotrophic bacteria, molds and yeasts. Pseudomonas, Achromobacter, Flavo bacterium, Alcaligenes, Escherichia and Enterobacter were claimed to be the genera most likely to cause defects in cottage cheese, with Pseudomonas being most often encountered. Bonner and Harmon (33) noted that similar organisms caused cottage cheese spoilage, but ph of 5.4 could control growth. Principal defects in cottage cheese were slime formation, surface discoloration, off-odors and off-flavors (184). Since pasteurization kills psychrotrophic bacteria that spoil cottage cheese, their presence in the cheese represents post-pasteurization contamination by equipment, water, air or personnel (77). Hankin et al. (106) found that 26.9o/oof cottage cheese samples were unacceptable before the code date expired because psychrotrophic bacteria, usually proteolytic and lipolytic gram-negative rods, grew and reduced quality. White and Marshall (373) observed that either P. fluorescens P26 or its protease in milk before pasteurization caused proteolysis and off-flavors in cottage cheese made from the milk. The off-flavors for the cottage cheese, made from protease-treated milk, were different than those reported for samples from milk treated with P. fluorescens P26. Increases in soluble nitrogen and bitter flavor were associated with increases in psychrotrophic bacteria in cottage cheese (312). Cousin and Marth (52) observed increases in NPN and noncasein nitrogen in milks inoculated with psychrotrophs before cottage cheese production. These cottage cheeses had reduced manufacturing times and increased curd firmness similar to Cheddar cheese made by the same researchers (51). Milk with whey separation before cutting resulted in abnormal curd containing porous and spongy structures (211). The proteases from psychrotrophs could have altered casein so a firm curd was not formed. The yield was decreased slightly if milks that had previous psychrotrophic growth were used in the manufacture of cottage cheese (211,384). Buttermilk and yogurt Psychrotrophic organisms, whether added to milk in low numbers together with the starter culture, added as a suspension of dead cells or allowed to act on milk and then removed, all had a deleterious effect on flavor development in buttermilk by mixed starter cultures (108). Vasayada and White (359) found a decrease in the percentage of psychrotrophs in commercial buttermilk, which probably resulted from the acidic nature of the product. Wang and Frank (363) analyzed fresh buttermilk for psychrotrophic counts and found a range of< 10 to 3 x 10 2 CFU/ml. After refrigeration for 17 days the counts were still relatively low, < 10 to > 4 x 10 CFU /mi. Isolated psychrotrophs were identified as Pseudomonas, Enterobacter, Acinetobacter, Escherichia and Actinobacillus. Otte et al. (243) observed similiar results for buttermilks since psychrotrophic counts were usually about 1 x 10 4 CFU/ml. Haukka and Harper (111) noted that acetaldehyde and diacetyl contents of buttermilks were lower for milks inoculated with P. fragi than for control milks. Wang and Frank (363) noted that psychrotrophs could reduce diacetyl but no consistent pattern of reduction was noted. Yogurt had unacceptable flavor scores when made from milk with previous psychrotrophic growth (52). Firmer curds and batch failures also were observed when yogurt was made from milk that had been stored to allow psychrotrophic growth. Zall (384) experimented with heating milk on the farm to 74 C for 15 sec. Yogurt and buttermilks made from these milks did not suffer from quality defects. FUTURE RESEARCH After a review of this magnitude, some people may feel that too much research has been done on psychrotrophs, but others may envision the research that is still needed. Future areas in which research should be focused include: (a) response and adaption of microorganisms to low temperature growth, especially research aimed at psychrotrophs instead of psychrophiles; (b) significance in milk and dairy products of pathogens that grow at low temperatures; (c) rapid, reliable and economical tests to detect psychrotrophs in milk and dairy products; (d) acceptable methods to control and/or inhibit psychrotrophs and their enzymes in milk on the farm and at the dairy plant and (e) methods that correlate biochemical activity of psychrotrophs with detection of possible keeping quality problems of milk and dairy products. REFERENCES 1. Adams, D. M., J. T. Barach, and M. L. Speck Heat resistant proteases produced in milk by psychrotrophic bacteria of dairy origin. I. Dairy Sci. 58: Adams. D. M., I. T. Barach, and M. L. Speck Effect of psychrotrophic bacteria from raw milk on milk proteins and stability of milk proteins to ultra high temperature treatment. J. Dairy Sci. 59: Alichanidis, E., and A. T. Andrews Some properties of the extracellular protease produced by the psychrotrophic bacterium Pseudomonasftuorescens strain AR-11. Biochim. Biophys. Acta 485: Allauddin, M., B. E. Langlois, J. O'Leary, and C. Hicks Effects of raw milk quality on yield of Cheddar cheese. Proc. 71st Annual Meeting, American Dairy Science Association., p Andersson, R. E., C. B. Hedlund. and U. Jonsson Thermal inactivation of a heat-resistant lipase produced by the psychrotrophic bacterium Pseudomonas ftuorescens. I. Dairy Sci. 62: Andrey, J., Jr., and W. C. Frazier Psychrophiles in milk held two days in farm bulk cooling tanks. J. Dairy Sci. 42: Annibaldi, S., R. Guidetti, R. Mora, and M. Pecorari Effect of subclinical mastitis on the cheesemaking properties of milk. 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28 PSYCHROTROPHS IN DAIRY PRODUCTS Azuma, Y., S. B. Newton, and L. D. Witter Production of psychrophilic mutants from mesophilic bacteria by ultraviolet irradiation. J. Dairy Sci. 45: Babel, F. J Slow acid production by lactic acid cultures: A review. J. Dairy Sci. 38: Babel, F. I Antibiosis by lactic culture bacteria. I. Dairy Sci. 60: Baig, I. A., and J. W. Hopton Psychrophilic properties and the temperature characteristics of growth of bacteria. J, Bacteriol. 100: Barach, I. T., and D. M. Adams Thermostability at ultrahigh temperatures of thermolysin and a protease from a psychrotrophic Pseudomonas. Biochim. Biophys. Acta 485: Barach, J. T., D. M. Adams, and M. L. Speck Low temperature inactivation in milk of heat resistant proteases from psychrotrophic bacteria. I. Dairy Sci. 59: Barach, J. T., D. M. Adams, and M. L. Speck Stabilization of a psychrotrophic Pseudomonas protease by calcium against thermal inactivation in milk at ultrahigh temperature. Appl. Environ. 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Dolezalek Some relationships between milk and the quality of Emmental cheese. Prumipl Potrovin 25: (Dairy Sci. Abst. 36:3828). 44. Chapman, H. R., M. E. Sharpe, and B. A. Law Some effects of low temperature storage of milk on cheese production and Cheddar cheese flavor. Dairy Indus. Int. 41: Choudhery, A. K., and E. M. Mikolajcik Bacilli in milk. 2. Sweet curd formation. J. Dairy Sci. 54: Chung, B. H., and R. Y. Cannon Psychrotrophic spore forming bacteria in raw milk supplies. I. Dairy Sci. 54:448 (Abst.). 47. Claydon, T.J., and H. C. Fryer.l%0. Effect ofraw milk storage and bacterial development on subsequent lactic culture activity. Appl. Microbic!. 8: Coghill, D. and H. S. Juffs Comparison of the Moseley keeping quality test for pasteurized milk and cream products with other tests of shorter duration. Aust. J. Dairy Techno!. 34: Coghill, D., and H. S. Juffs Incidence of psychrotrophic sporeforming bacteria in pasteurized milk and cream products and effect of temperature on their growth. Aust. J. Dairy Techno!. 3: Cousin, M.A., and E. H. Marth Changes in milk protein caused by psychrotrophic bacteria. Milchwissenschaft 32: Cousin, M. A., and E. H. Marth Cheddar cheese made from milk that was precultured with psychrotrophic bacteria. J. Dairy Sci. 60: Cousin, M. A., and E. H. Marth Cottage cheese and yogurt manufactured from milks precultured with psychrotrophic bacteria. Cultured Dairy Products 1.12(1):15-18, Cousin, M.A., and E. H. Marth Lactic acid production by Streptococcus lactis and Streptococcus cremoris 1n milk precultured with psychrotrophic bacteria. J. Food?rot. 40: Cousin, M.A., and E. H. Marth Lactic acid production by Streptococcus thennophilus and Lactobacillus buigaricus m milk precu!tured with psychrotrophic bacteria. J. Food Prot. 40: Cousin, M.A., and E. H. Marth Psychrotrophic bacteria JOURNAL OF FOOD PROTECTION, VOL. 45, FEBRUARY 1982

29 200 COUSIN cause changes in stability of milk to coagulation by rennet and heat. J. Dairy Sci. 60: Cousins, C. M., M. E. Sharpe, and B. A. Law The bacteriological quality of milk for Cheddar cheesemaking. Dairy Indus. Int. 42(7):12-13, 15, Cox, W. A Problems associated with bacterial spores in heat-treated milk and dairy products. J. Soc. Dairy Techno!. 28: Credit, C., R. Hedeman, P. Heywood, and D. Westhoff Identification of bacteria isolated from pasteurized milk following refrigerated storage. J. Milk Food Techno!. 35: Cunningham, F. E Influence of microwave radiation on psychrotrophic bacteria. J. Food Prot. 43: Dabbah, R., W. A. Moats, and J. F. Mattick Factors affecting resistance to heat and recovery of heat-injured bacteria. J. Dairy Sci. 52: Davies, F. L Heat resistance of Bacillus species. J. Soc. Dairy Techno!. 28: Davies, F. L., and G. Wilkinson A germinant for Bacillus cereus derived from pasteurized milk. Pages In A. N. Barker, J. Wolf, D. J. Ellar, G. J. 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A., D. B. Emmons, and A. R. Yates The influence of the bacterial quality of milk on the properties of dairy products. A review. J. Inst. Can. Sci. Techno!. 7(1): Elliot, R. P., and H. D. Michener Factors affecting the growth of psychrophilic microorganisms in foods. A review. USDA Technical Bulletin No Emmons, D. B Recent research in the manufacture of cottage cheese. Part H. Dairy Sci. Abst. 25: Feuillat, M., S. LeGuennec and A. Olsson Contribution ll: 'etude de Ia proteolyse des laits refrigeres et incidences sur le rendement d 'une fabrication de fromages a p~te molle. Le Lait 56(558): Finley, R. D., H. B. Warren, and R. E. Hargrove Sorage stability of commercial milk. J. Milk Food Techno!. 31: Fish, N. L., P. J. Pinkston, and T. J. Claydon Comparison of milk proteolysis by Bacillus subtilis protease and by Pseudomonasfluorescens. J. Dairy Sci. 52: Flemingham, D., and H. S. Juffs Comparison and evaluation of keeping quality tests for pasteurized milk and cream products. Aust. J. 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Muir Rapid plate counting techniques for enumeration of psychrotrophic bacteria in pasteurized double cream. 1. Soc. Dairy Techno!. 33(1): Grooserhode, J Chemical, physical and technological changes in raw milk caused by deep cooling. Deutsche Milchwirtschaft 26: (Dairy Sci. Abst. 37:4614). 98. Grosskopf, J. C., and W. J. Harper Role of psychrophilic sporeformers in long life milk. J. Dairy Sci. 52:897 (Abst.). 99. Grosskopf, J. C., and W. J. Harper Role of psychrophilic sporeformers in long life milk. Milchwissenschaft 29: Gyllenberg. H. G., and E. Eklund A taxonomic survey of the psychrophilic bacteria in milk and milk products. Milchwissenschaft 21: Raisch, K., M. Hermann, and F. Kiermeier Suitability of deep-cooled milk for cheesemaking. Milchwissenschaft 26: Hall. N. J Influence of Bacterium coli on starter production. XIV Int. Dairy Congress. II: Hamilton, D. N., and W. W. 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30 PSYCHROTROPHS IN DAIRY PRODUCTS Hankin, L., and W. F. Dillman A rapid test to find "potentially" psychrophilic organisms in pasteurized dairy products. J. Milk Food Technol. 31: Hankin, L., W. F. Dillman, and G. R. Stephens Keeping quality of pasteurized milk for retail sale related to code date, storage temperature, and microbial counts. J. Food Prot. 40: losa. Hankin, L., W. F. Dillman, and G. R. Stephens Relation of code dates to quality of milk sold in retail markets. J. Food Prot. 40: Hankin, L., G. R. Stephens, and W. F. Dillman Quality control significance of special media for enumeration of microbial groups in cottage-type cheese. J. Milk Food Technol. 38: Hanna, M. 0., J. C. Stewart, Z. L. Carpenter, arid C. Vanderzant Heat resistance of Yersinia enterocolitica in skim milk. J. Food Sci. 42: Harper, W. J Cultured products flavor with emphasis on biological factors. Cultured Dairy Prod. J. 3: Hartley, J. C., E. R. Vedamuthu, and G. W. Reinbold Bacteriological methods for evaluation of raw milk quality. A review. II. Bacterial tests used to measure milk quality. J. Milk Food Technol. 32: Hattowska, H Influence of ordinary contamination of cheese milk prior to pasteurization on the growth of lactic bacteria after pasteurization. XVIII Int. Dairy Congress. 1E:S21 (Abst.) Haukka, J. J., and W. J. Harper The effect of the psychrotrophic organisms and the age of milk on the production of acetaldehyde and diacetyl in cultured buttermilk. Meijeritieteellinen Aikakanskirja (1978) 36:1-10. (Dairy Sci. Abst. 41:7853) Heather, C. D., and C. Vanderzant Effects of temperature and time of incubating and ph of plating medium on enumerating heat-treated psychrophilic bacteria. J. Dairy Sci. 40: Heeschen, W Biochemische Veriinderungen von Lebensmitteln durch psychrotrophe Mikroorganismen. Arch. Leben smittelhyg. 23: Heeschen, W., A. Tolle, H. Wernery, and J. Reichmuth Determination of metabolites of bacterial activity in milk. Advances in Automated Analysis, Proceedings of the 1972 Technicon International Congress 7: Herbert, R. A., and M. Bhakoo Microbial growth at low temperatures. Pages In A. D. Russell and R. Fuller (eds.) Cold tolerant microbes in spoilage and the environment. Academic Press, New York Higoshi, H., S. Hamada, and M. Doi Studies on the growth temperature of psychrotrophic bacteria: growth at various incubation temperatures of psychrotrophic Pseudo monas originated from raw milk and raw meat. Jap. J. Veterinary Sci. 37: (Dairy Sci. Abst. 38:406) Hosono, A., and J. A. Elliott Properties of crude ethyl ester-forming enzyme preparations from some lactic acid and psychrotrophic bacteria. J. Dairy Sci. 57: Hosono, A., K. Yastuki. and F. Tokita Isolation and characterization of an inhibitory substance against Escherichia acidophilus. Milchwissenschaft 32: Huskey, G. E Characterization of gelatinous caseinateproducing psychrophilic bacteria. Diss. Abst. Sect. B. 27: Ingraham, J. L Newer concepts of psychrophilic bacteria. Pages In Proceedings low temperature microbiology symposium Campbell Soup Company, Camden, New Jersey Ingraham, J. L Temperature relationships. Pages In I. C. Gunsalus and R. Y Stanier (ed.) The bacteria. Vol. IV: The physiology of growth. Academic Press, New York Ingraham, J. L., and J. L. Stokes Psychrophilic bacteria. Bacteriol. Rev. 23: Inniss, W. E Interaction of temperature and psychrophilic microorganisms. Ann. Rev. Microbiol. 29: Inniss, W. E., and J. L. Ingraham Microbial life at low temperatures: Mechanisms and molecular aspects. Pages In D. J. Kushner (ed.). Microbial life in extreme environments. Academic Press, New York Jackson, A. C Short shelf-life milk products. J. Soc. Dairy Techno). 31: Jayashankar, S. R., A. T. Dundani, and K. K. lya Studies on psychrophilic bacteria in milk. Int. Dairy Congress 17, B:S Jayne-Williams, D. J., and J. G. Franklin Bacillus spores in milk-part 1. Dairy Sci. Abst. 22: Jensen, H. 0., and M. S. Hansen Investigations on producer milk with low microbial counts stored at various temperatures with particular regard to lipolytic activity. Maelkeritidende 87: (Dairy Sci. Abst. 36:3160) Johns, C. K Bacteriological testing of milk for regulatory purposes usefulness of current procedures and recommendations for change. II. Bacteriological testing of raw milk for regulatory purposes. J. Milk Food Techno I. 34: Johns, C. K Use of counts after preliminary incubation to improve raw milk quality for a Denver plant. J. Milk Food Technol. 38: Johns, C. K., and G. B. Landerkin Effects of preliminary incubation on the bacterial flora of bulk tank milks produced in three different Canadian milk sheds. J. Dairy Sci. 52: Jones, F. T., and B. E. Langlois Microflora of retail fluid milk products. J. Food Prot. 40: Juffs, H. S A four-day count for psychrotrophs. Aust. J. Dairy Technol. 25: Juffs, H. S Proteolysis detection in milk. I. Interpretation of tyrosine value data for raw milk supplies in relation to natural variation, bacterial counts, and other factors. J. Dairy Res. 40: Juffs, H. S Proteolysis detection in milk. II. The effect of preincubation of raw and laboratory pasteurized bulk milk samples on tyrosine value and its relationship with bacterial populations. J. Dairy Res. 40: Juffs, H. S Influence of proteinases produced by Pseudomonas aeruginosa and Pseudomonas.fluorescens on manufacture and quality of Cheddar cheese. Aust. J. Dairy Technol. 29: Juffs, H. S Proteolysis detection in milk. III. Relationships between bacterial populations, tyrosine value and organoleptic quality during extended cold storage of milk and cream. J. Dairy Res. 42: Juffs, H. S Effects of temperature and nutrients on proteinase production by Pseudomonas.fluorescens and Ps. aeruginosa in broth and milk. J. Appl Bacteriol. 40: Juffs, H. S., and F. J. Babel Inhibition of psychrotrophic bacteria by lactic cultures in milk stored at low temperatures. J. Dairy Sci. 58: Juffs, H. S., and F. J. Babel Rapid enumeration of psychrotrophic bacteria in raw milk by the microscopic colony count. J. Milk Food Technol. 38: Juffs, H. S., and H. W. Doelle Some properties of the extracellular proteolytic enzymes of the milk-spoilage organism Pseudomonas aeruginosa ATCC J. Dairy Res. 35: Juffs, H. S., A. C. Hayward. and H. W. Doelle Growth and proteinase production in Pseudomonas spp. cultivated under various conditions of temperature and nutrition. J. Dairy Res. 35: Juffs, H. S., and 0. Madsen Incidence of proteolytic and some other bacterial populations in raw milk produced in South Eastern Queensland. Aust. J. Dairy Technol. 29: Kandler, Zur Definition der "psychrophilen" Bakterien. Milchwissenschaft 21: Keen, N. T., and P. H. Williams Effect of nutritional JOURNAL OF FOOD PROTECTION, VOL. 45, FEBRUARY 1982

31 202 COUSIN factors on extracellular protease production by Pseudomonas lachrymans. Can. J. Micro bioi. 13: Keen, N. T., P. H. Williams, and J. C. Walker Characterization of a protease produced by Pseudomonas lachrymans. Phytopathology 57: Keogh, M. K., and T. I. Hedrick Spore destruction with hot chemical solutions. J. Milk Food Technol. 34: Kikuchi, M., and Y. Matsui Changes in the number of bacteria and bacterial flora in bulk-collected milk. Jap. J. Zootechnical Sci. 45: (Food Sci. Techno). Abst. 7:6P1329) Kikuchi, M., and Y. Matsui Bacteriological quality of bulk-collected raw milk. 1. Seasonal variation of bacterial counts and 2. Seasonal variations of bacterial flora. Jap. J. Dairy Food Sci. A119-A Kishonti, E Influence of heat resistant lipases and proteases in psychrotrophic bacteria on product quality. Int. Dairy Fed. Annual Bull. 36: Kishonti, E., and G. Sjostrom Influence of heat resistant lipases and proteases in psychrotrophic bacteria on product quality. XVIII Int. Dairy Congress IE: 501 (Abst.) Kiuru, K., E. Eklund, H. Gyllenberg, and M. Antila Die proteolytische Aktivitat der psychrotrophen Mikroorganismen in der Hofbehaltermilch. Milchwissenschaft 26: Kiuru, K. E. Eklund, H. Gyllenberg, and M. Antila Psychrotrophic microorganisms in farm tank milk and their proteolytic action. XVIII Int. Dairy Congress 1E: 108 (Abst.) Knaut, T Die proteolytischen Eigenschaften der Pseudomonas Arten beim Wachstum in der Milch. Milchwissenschaft 22: Knaut, T Heat resistance of Pseudomonas lipases in milk. XX Int. Dairy Congress E: (Abst.) Knaut, T., and G. Bruderer Elektrophoretische Veriinderungen das Kaseins beim W achstum von Pseudomonas Arten in der Milch. Milchwissenschaft 20: Koburger, J, A., and T. J. Claydon Identification of substances in milk cultures of Pseudomonas fluorescens which stimulate lactic starter cultures. J. Dairy Sci. 44: Koops, J., H. Klomp, and R. H. C. Elgersma Rapid determination of nitrogen in milk and dairy products by colorimetric estimation of ammonia following an accelerated digestion procedure. Neth. Milk Dairy J. 29: Kraft, A. A., and C. R. Rey Psychrotrophic bacteria in foods: An update. Food Techno!. 33: Landaas, A., and P. Solberg Production and characterization of lipase from a fluorescent pseudomonad. XX Int. Dairy Congress E: (Abst.) Langeveld, L. P.M Effect of the bactofugation of milk on the butyric acid fermentation in Gouda cheese. Neth. Milk Dairy J. 26: Langeveld, L. P. M., F. Cuperus, P. Van Breemen, and J. Dijkers A rapid method for the detection of post-pasteurization contamination in HTST pasteurized milk. Neth. Milk Dairy J. 30: Langeveld, L. P. M., F. Cuperus, and J. Stadhouders Bacteriological aspects of the keeping quality at 5 C of reinfected and noninfected pasteurized milk. Neth. Milk Dairy J. 27: Law, B. A Reviews of the progress of dairy science: Enzymes of psychrotrophic bacteria and their effects on milk and milk products. J. Dairy Res. 46: Law, B. A., A. T. Andrews, A. J. Cliffe, M. E. Sharpe, and H. R. Chapman Effect of proteolytic raw milk psychrotrophs on Cheddar cheese making with stored milk. J. Dairy Res. 46: Law, B. A., A. T. Andrews, and M. E. Sharpe Gelation of ultra-high temperature-sterilized milk by proteases from a strain of Pseudomonas fluorescens.isolated from raw milk. J. Dairy Res. 44: Law, B. A., M. Castanon, and M. E. Sharpe The effect of non-starter bacteria on the chemical composition and flavor of Cheddar cheese.j. Dairy Res. 43: Law, B. A., C. M. Cousins, M. E. Sharpe, and F. L. Davies Psychrotrophs and their effects on milk and dairy products. Pages In A. D. Russell and R. Fuller (ed.) Cold tolerant microbes in spoilage and the environment. Academic Press, New York Law, B. A., M. E. Sharpe, and H. R. Chapman The effect of lipolytic gram-negative psychrotrophs in stored milk on the development of rancidity in Cheddar cheese. J. Dairy Res. 43: Lawrence, R. C Microbiallipases and related esterases. Part I. Detection, distribution and production of microbial lipases. Dairy Sci. Abst. 29(1): La\\Tence, R. C Microbiallipases and related esterases. Part II. Estimation of lipase activity. Characterization of Iipases. Recent work concerning their effect on dairy products. Dairy Sci. Abst. 29(2): Lawrence, R. C., T. F. Fryer, and B. Reiter Rapid method for the quantitative estimation of microbial lipases. Nature 213: Lawrence, R. C., and W. B. Sanderson A micro-method for the quantitative estimation of rennets and other proteolytic enzymes.j. Dairy Res. 36: Ledford, R. A., and J. H. Chen Degradation of caseins by proteases from cow's milk, rennet and Streptococcus faecalis var.liquefaciens. I. Dairy Sci. 50:947 (Abst.) Lee, M. L., D. L. Smith, and L. R. Freeman High-resolution gas chromatographic profiles of volatile organic compounds produced by microorganisms at refrigeration temperatures. Appl. Environ. Microbiol. 37: Lee, W. H An Assessment of Yersinia enterocolitica and its presence in foods. J. Food Prot. 40: Lightbody, L. G Media containing penicillin for detecting spoilage organisms in milk and cream. Aust. J. Dairy Techno!. 19: Lightbody, L. G Media containing penicillin for detecting post-pasteurization contamination. Aust. J. Dairy Techno!. 20: Lightbody, L. G The effect of low-temperature storage of pasteurized milk on bacterial counts using penicillin agar. Aust. J. Dairy Technol. 21: Liick, H Bacteriological quality tests for bulk-cooled milk. A review. Dairy Sci. Abst. 34: Liick, H., and F. Hopkins Comparison of different plate count tests for determining the number of psychrotrophs. South African I. Dairy Techno!. 7: Magdoub, M. N. I., A. E. Sherata, E. 0. Fayed, and A. A. Hofi Effect of filtrates from milk cultures of proteolytic sporeformers on bacteriological quality of Ras cheese during ripening. Dairy Indus. Int. 44:5 & Mann. E. J Cottage cheese. Dairy Ind. 38: Marth, E. H Spoilage of cottage cheese by psychrophilic microorganisms. Cultured Dairy Prod. J. 5: Marth, E. H. (ed.) Standard methods for the examination of dairy products, 14th ed. American Public Health Association, Washington, D.C Marshall, R. T., and C. C. Harmon The automated pyruvate method as a quality test for grade A milk. J. Food Prot. 41: Marshall, R. T., and J. K. Marstiller Low temperature lability vs. high temperature stability of proteases from Pseudomonas fluorescens MS. J. Dairy Sci. 63 (Suppl. 1): 57 (Abst.) Martley, F. G., S. R. Jayeshankar, and R. C. Lawrence An improved agar medium for the detection of proteolytic organisms in total bacterial counts. J. Appl. Bacteriol. 33: Matsumoto, M., K. Zinbo, and M. Haruta Catalase test for detection of psychrotrophic organisms in UHT - treated milk. JOURNAL OF FOOD PROTECTION. VOL.45, FEBRUARY 1982

32 PSYCHROTROPHS IN DAIRY PRODUCTS 203 Annual Report of Tokyo Metropolitan Research Laboratory of Public Health 23: (Dairy Sci. Abst. 38(10):6580) Mayerhofer,H.J.,R. T.Marshall, C. H. White, and M. Lu Characterization of heat-stable protease of Pseudomonas fluorescens P26. Appl. Microbiol. 25: Maxcy, R. B Nature and growth response of the microflora of pasteurized milk. J. Milk Food Technol. 30: McCaskey, T. A Microbial and enzymatic activity in raw milk held at low temperatures. Diss. Abst. 4046B McDonald, I. J. and A. K. Chambers Regulation of proteinase formation in a species of Micrococcus. Can. J. Microbiol. 12: Melachouris, N. P., and S. L. Tuckey Properties of a milkclotting microbial enzyme. J. Dairy Sci. 51: Mencher, J. R Purification and characterization of the lipase of Pseudomonas fragi. Diss. Abst. 4047B Michels, M. J. M., and F. M. W. Visser Occurrence and thermo-resistance of spores of psychrophilic and psychrotrophic aerobic sporeformers in soil and foods. J. Appl. Bacteriol. 41: Mickelsen, R., N. L. Fish, and T. J. Claydon Some chemical and flavor characteristics of a milk proteolysate of Pseudomonas fluorescens. J. Dairy Sci. SO: Mikawa, K., and T. Hoshino Effect of temperature on proteolytic and lipolytic activities of psychrotrophic bacteria in milk. Jap. J. Dairy Sci. 22:A176-A186. (Dairy Sci. Abst. 36:3162) Mikolajcik, E. M Thermodestruction of Bacillus spores in milk. J. Milk Food Technol. 33: Mikolajcik, E. M Psychrotrophic sporeformers: A possible keeping-quality problem in market milk. Amer. Dairy Rev. 40(4):34A,34D Mikolajcik, E. M Psychrotrophic bacteria and dairy product quality. 1. Major organisms involved and defects produced. Cultured Dairy Prod. J.14(4): Mikolajcik, E. M Psychrotrophic bacteria -and dairy product quality. 2. Detection of problem organisms and their control. Cultured Dairy Prod. J. 15(1): Mikolajcik, E. M Psychrotrophic bacteria and dairy product quality. 3. Organisms of public health importance in fermented dairy foods. Cultured Dairy Prod. J.15(2): Mikolajcik, E. M., and M. Koka Bacilli in milk I. Spore germination and growth. J. Dairy Sci. 51: Mikolajcik, E. M., and N. T. Simon Heat resistant psychrotrophic bacteria in raw milk and their growth at 7 C. J. Food Prot. 41: Miller, 1., and 0. Kandler Der Einfluss der Rohmilchflora auf dem Gehalt der Milch an freien Aminosiiuren und anderen Ioslichen Stickstoffverbindungen. Milchwissenschaft 22: Milliere, J. B., M.S. Ben Rachid and A. Chadli Bacteries psychrotrophes du lait cru. Archiv. de I'Institut Pasteur de Tunis 50: Milliere, J. B. et 1. Veillet-Poncet Caracterisation des systmnes enzymatiques protedytiques de deux souches psychrotrophes isolees de laits crus refrigeres. Le Lait 59( ): Milliere, J. B., and L. Veillet-Poncet Determination de Ia flore bacterienne caseolytique psychrotrophe des laits crus refrigeres. Le Lait 59( ): Mocquot, G., and R. Ducluzeau The influence of cold storage of milk on its microflora and its suitability for cheesemaking. Pages In J. Hawthorn and E. J. Rolfe (ed.) Low temperature biology of foodstuffs. Pergamon Press, Oxford Mohamed, F. 0., and R. Bassette Quality and yield of cottage cheese influenced by psychrotrophic microorganisms in milk. J. Dairy Sci. 62: Moreno, V., and F. V. Kosikowski Degradation of (J-casein by micrococcal cell-free preparations. J. Dairy Sci. 56: Moreno, V., and F. V. Kosikowski Peptides, amino acids, and amines liberated from (J-casein by micrococcal cell-free preparations. J. Dairy Sci. 56: Morita, R. Y Psychrophilic bacteria. Bacteriol. Rev. 39: Morse, P.M., H. Jackson, C. H. McNaughton, A. G. Leggatt, G. B. Landerkin, and C. K. Johns Investigation of factors contributing to the bacterial count of bulk tank milk. I. Influence of two-day storage and effect on results of preliminary incubation. J. Dairy Sci. 51: Morse, P.M., H. Jackson, C. H. McNaughton, A. G. Leggatt, G. B. Landerkin, and C. K. Johns Investigation of factors contributing to the bacterial count of bulk tank milk. II. Bacteria in milk from individual cows. J. Dairy Sci. 51: Morse, P.M., H. Jackson, C. H. McNaughton, A. G. Leggatt, G. B. Landerkin, and C. K. Johns Investigation of factors contributing to the bacterial count of bulk tank milk. Ill. Increase in count from cow to bulk tank, and effects of refrigerated storage and preliminary incubation. J. Dairy Sci. 51: Moseley, W Improving and maintaining shelf-life of dairy products. Dairy Ice Cream Field 158(2):44, 46, Mourques, R., J. P. Accolas, and J. Auclair Use of refrigerated milk from bulk tanks for cheese manufacture. II. Manufacture of Gruyere cheese. Revue Lait Fr. 243:289-93, (Dairy Sci. Abst. 31:3039) Moustafa, H. H., and E. B. Collins Effects of selected food additives on growth of Pseudomonas fragi. J. Dairy Sci. 52: Muir, D. D., M. E. Kelley, and J.D. Phillips The effect of storage temperature on bacterial growth and lipolysis in raw milk. J. Soc. Dairy Technol. 31: Muir, D. D., M. E. Kelly, J.D. Phillips, and A. G. Wilson The quality of blended raw milk in creameries in South-West Scotland. J. Soc. Dairy Technol. 31: Muir, D. D., J.D. Phillips, D. G. Dalgleish The lipolytic and proteolytic activity of bacteria isolated from blended raw milk. J. Soc. Dairy Technol. 32: Nakae. T Characterization and classification of psychrotrophic bacteria in milk by means of temperaturegradient incubation. Milchwissenschaft 25: Nakanishi, T., and T. Tanabe Studies on psychrotrophic bacteria in cows' milk. II. Changes of protein in cows' milk by psychrotrophic bacteria during low temperature storage. Jap. J. Dairy Sci. 19:A75-A87. (Dairy Sci. Abst. 33:2004) Nashif, S. A., and F.' E. Nelson The lipase of Pseudomonas fragi. I. Characterization of the enzyme. J. Dairy Sci. 36: Nashif, S. A., and F. E. Nelson The lipase of Pseudomonas fragi. II. Factors affecting lipase production. J. Dairy Sci. 36: Nashif, S. A., and F. E. Nelson The lipase of Pseudomonas fragi. Ill. Enzyme action in cream and butter. J. Dairy Sci. 36: Nath, K. R., and R. A. Ledford Stimulation ofthe rate of acid production by lactobacilli in media containing a capsular substance from micrococci. J. Dairy Sci. 54: Nath, K. R., and R. A. Ledford Caseinolytic activity of micrococci isolated from Cheddar cheese. J. Dairy Sci. 55: Nath, K. R., and B. J. Wagner Stimulation of lactic acid bacteria by a Micrococcus isolate: Evidence for multiple effects. Appl. Microbiol. 26: Nelson, P. J., and R. T. Marshall Microbial proteolysis sometimes decreases yield of cheese curd. J. Dairy Sci. 60 (Suppl. 1):35-36 (Abst.). JOURNAL OF FOOD PROTECTION, VOL. 45, FEBRUARY 1982

33 204 COUSIN 233. Nielsen, V. H Prolonging cottage cheese life. Am. Dairy Rev. 31(6) 54, 56, O'Donnell, E. T Heat resistance of lipase enzymes produced by psychrotrophic bacteria XX. Int. Dairy Congress E: 307 (Abst.) Ogawa, M Contamination of raw milk with psychrophilic bacteria and its effect on milk quality. Ja:p. J. Dairy Sci. 16:A168-A176. (Dairy Sci. Abst. 30: 1697) Ohren, J. A., and S. L. Tuckey Relation of flavor development in Cheddar cheese to chemical changes in the fat of cheese. J. Dairy Sci. 52: Oliveria, J. S., and C. E. Parmelee Rapid enumeration of psychrotrophic bacteria in raw and pasteurized milk. J. Milk Food Technol. 39: Olsen, R. H., and E. S. Metcalf Conversion of mesophilic to psychrophilic bacteria. Science 162: Onuorah, E. C., C. L. Hicks, and J. O'Leary Effect of milk storage on Cheddar cheese yield. J. Dairy Sci. 63 (Suppl. '1): (Abst.) Orr, M. J., G. McCrae, and S. Baines The value of milk tests in assessing production methods. Milk Ind. 76(6): Orr, M. J., G. McCrae, and S. Baines A comparison of tests used in assessing milk quality. Milk Ind. 77: Orr, M. J., R. M. McLarty, M. E. McCance, and S. Baines Alternate day collection of bulk milk. Dairy Ind. 29: Otte, 1., G. Suhren, W. Heeschen, and A. Tolle Zur Microflora von Buttermilch, sauer Sahne und Speisequark. Milchwissenschaft 34: Otte, 1., A. Tolle, and G. Hahn Zur Analyse der Mikroflora von Milch and Milchprodukten. 2. Miniaturisierte Primartests zur Bestimmung der Gattung. Milchwissenschaft 34: Overcast, W. W Extending shelf-life by controlling psychrophiles. Amer. Dairy Rev. 29(1): Overcast, W. W Psychrophilic microorganisms and keeping quality of milk and its products. J. Dairy Sci. 51: Overcast, W. W., and G. A. Adams Growth of certain psychrophilic bacteria in pasteurized milk as influenced by previous excessive psychrophilic growth in the raw milk. J. Milk Food Technol. 29: Overcast, W. W., and K. Atmaram The role of Bacillus cereus in sweet curdling of fluid milk. J. Milk Food Technol. 37: Overcast, W. W., and J. D. Skean Growth of certain lipolytic microorganisms at 4 C and their influence on free fat acidity and flavor of pasteurized milk. J. Dairy Sci. 42: Owens, J. J Lecithinase positive bacteria in milk. Proc. Biochem.13(1):13-14, Parker, E. T., J. B. Bernsteinas, and J. H. Green Increased recovery of psychrophilic bacteria by the use of a new medium with lower solidifying temperature. Appl. Microbiol. 16: Parmelee, C. E Early detection of psychrotrophs in pasteurized milk. Dairy Ice Cream Field 157(8): Patel, G. B., and G. Blankenagel Bacterial counts of raw milk and flavor of the milk after pasteurization and storage. J. Milk Food Technol. 35: Patel. G. B., W. M. Ingledew, and G. Blankenagel A heat stable microbial proteolytic enzyme produced in milk. Can Inst. Food Sci. Technoi.J 5: Patton. S Volatile acids and the aroma of Cheddar cheese. J. Dairy Sci. 46: Peterson, A. C., and M. F. Gunderson Some characteristics of proteolytic enzymes from Pseudomonas jluorescens. Appl. Microbiol. 8: Pierani, R. M., and K. E. Stevenson Detection of metabolites produced by psychrotrophic bacteria growing in milk. J. Dairy Sci. 59: Pinheiro, A. J. R., B. J. Liska, and C. E. Parmelee Heat stability of lipases of selected psychrophilic bacteria in milk and Purdue Swiss-type cheese. J. Dairy Sci. 48: Poznanski, S., J. Lenoir, and G. Mocquot La proteolyse de Ia caseine par les enzymes intracellulaires de certaines bacteries. Le Lait 45: Price, R. J., and J. S. Lee Inhibition of Pseudomonas species by hydrogen peroxide producing lactobacilli. J. Milk Food Technol. 33: Pulusani, S. R., D. R. Rao, and G. R. Sunki Antimicrobial activity of lactic cultures: partial purification and characterization of antimicrobial compound(s) produced by Streptococcus thermophilus. J. Food Sci. 44: Punch, J. D., and J. C. Olson, Jr Comparison between standard methods procedure and a surface plate method for estimating psychrophilic bacteria in milk. J. Milk Food Technol. 27: Punch, J. D., J. C. Olson, Jr., and E. L. Thomas Preliminary observations on population levels of pure cultures of psychrophilic bacteria necessary to induce flavor or physical change in pasteurized milk. J. Dairy Sci. 44: Punch, J. D., J. C. Olson, Jr., and E. L. Thomas Psychrophilic bacteria. III. Population levels associated with flavor or physical change in milk. J. Dairy Sci. 48: Purschell, M., and C. Pollack Proteolytischer Abbau der Milcheiweissstoffe durch Bakterien. 2 Mitt. Die Wirkung von psychrophilen und milchsaurebildenben Bakterien auf die Eiweissstoffe in der Milch. Die Nahrung 16: Purschell, M., and M. Pospisil Proteolytischer Abbau der Milcheiweissstoffe durch Bakterien. 1 Mitt. Die Wirkung von aeroben Sporenbildnern auf die Eiweissstoffe in der Milch. Die Nahrung 12: Ramaley, R. F Molecular biology of extracellular enzymes. Adv. Appl. Microbiol. 25: Randolph, H. E., B. K. Chakraborty, 0. Hampton, and D. L. Bogart Microbial counts of individual producer and commingled grade A raw milk. J. Milk Food Techno). 36: Rapp, H., and H. E. Calbert Influence of the bulk handling of raw milk on its rennet coagulation time. J. Dairy Sci. 37:637 (Abst.) Reddy, M. C., D. D. Bills, and R. C. Lindsay Ester production by Pseudomonas fragi. II. Factors influencing ester levels in milk cultures. Appl. Microbiol. 17: Reddy, M. C., D. D. Bills, R. C. Lindsay, L. M. Libby, A. Miller Ill, and M. E. Morgan Ester production by Pseudomonas fragi. I. Identification and quantification of some esters produced in milk cultures. J. Dairy Sci. 51: Reddy, M. C., R. C. Lindsay, and M. W. Montgomery Ester production by Pseudomonas fragi. IV. Demonstration of esterase activity. Appl. Microbiol. 20: Reimerdes, E. H., F. Petersen, and G. Kielwein Milchproteinasen. 9. Proteinasepektren von Caseinmicellen, Milchserum, Rinderblutserum, und Pseudomonas jluorescens. Milchwissenschaft 34: Reiter, B., T. F. Fryer, A. Pickering, H. R. Chapman, R. C. Lawrence, and M. E. Sharp The effect of the microbial flora on the flavour and free fatty acid composition of Cheddar cheese. J. Dairy Res. 34: Reiter, B., and V. M. Marshall Bactericidal activity of the lactoperoxidase system against psychrotrophic Pseudomonas spp. in raw milk. Pages In A. D. Russell and R. Fuller (ed.). Cold tolerant microbes in spoilage and the environment. Academic Press, New York Reiter, B.. V. M. Marshall, and S. M. Philips The antibiotic activity of the lactoperoxidase-thiocyanete-hydrogen peroxide system in the calf abomasum. Res. Vet. Sci. 20: Reiter, B., and M. E. Sharpe Relationship of the microflora to the flavor of Cheddar cheese. J. Appl. Bacteriol. 34: URNAL OF FOOD PROTECTION. VOL. 45, FEBRUARY 1982

34 PSYCHROTROPHS IN DAIRY PRODUCTS Richardson, B. C., and D. F. Newstead Effect of heat-stable protease on the storage life of UHT milk. New Zealand J. Dairy Sci. Techno!. 14: Richardson, B. C., and I. E. TeWhaiti Partial characterization of heat-stable extracellular proteases for some psychrotrophic bacteria from raw milk. New Zealand J. Dairy Sci. Technol.13: Richter, R The effect of psychrotrophic bacteria on cheese manufacture. Amer. Dairy Rev. 41(8):48, Richter. R Psychrotrophic bacteria and shelf-life. Amer. Dairy Rev. 41(7): Richter, R. L., R. H. Schmidt, K. L. Smith, L. E. Mull, and S. L. Henry Proteolytic activity in ultra-pasteurized, aseptically packaged whipping cream. J. Food Prot. 42: Ritter, W Problems of the production of cheese from refrigerated milk with particular reference to Emmental cheese. Industrie Aliment, Pinerolo 9: (Dairy Sci. Abst. 32:3247) Robach, M. C Effect of potassium sorbate on the growth of Pseudomonas fluorescens. J. Food Sci. 43: San Clemente, C. L., and D. V. Vadehra. 1%7. Instrumental assay of microbial lipase at constant ph. Appl. Microbiol. 15: Sandvik, 0.. and K. Fossum Accumulation of bacterial proteinases during storage of milk due to selection of psychrophilic bacteria. Meieriposten 52: (Dairy Sci. Abst. 25:3519) Scarpellino, R., and F. V. Kosikowski Evolution of volatile compounds in ripening raw and pasteurized milk Cheddar cheese observed by gas chromatography. J. Dairy Sci. 45: Schiemann, D. A Association of Yersinia enterocolitica with the manufacture of cheese and occurrence in pasteurized milk. Appl. Environ. Microbiol. 36: Schultze, W. D., and J. C. Olson, Jr Studies on psychrophilic bacteria. I. Distribution in stored commercial dairy products. J. Dairy Sci. 43: Scott, R The cheese process. Microbiology and enzymology. Proc. Biochem. 7: Sellars, R. L Maximizing cultured product yields: The success factor. Dairy Ice Cream Field 160(2):68F-68H, 69, Sharpe, M. E., and A. J. Bramley Incidence of human pathogenic bacteria and viruses in raw milk. Dairy Ind. Int. 42(9): Shehata, T. E., and E. B. Collins Isolation and identification of psychrophilic species of Bacillus from milk. Appl. Microbiol. 21: Shehata. T. E., and E. B. Collins Sporulation and heat resistance of psychrophilic strains of Bacillus. J. Dairy Sci. 55: Shehata, T. E., A. Duran, and E. B. Collins Influence of temperature on the growth of psychrophilic strains of Bacillus. J. Dairy Sci. 54: Shipe, W. F., R. Bassette, D. D. Deane, W. L. Dunkley, E. G. Hammond, W. J. Harper. D. H. Kleyn, M. E. Morgan, J. H. Nelson. and R. A. Scanlan Off flavors of milk: Nomenclature. standards, and bibliography. J. Dairy Sci. 61: Sinclair, N. A., and J. L. Stokes.l%3. Role of oxygen in the high cell yields of psychrophiles and mesophiles at low temperatures. J. Bacteriol. 85: Skean, J.D., and W. W. Overcast Changes in the paper electrophoretic protein patterns of refrigerated skim milk accompanying growth of three Pseudomonas species. Appl. Micro bioi. 8: Smith. K. L., L. E. Mull, C. B. Lane, and A. J. Baggott, Jr Keeping quality of milk exposed to high temperature as experienced during transport in automobiles. J. Milk Food Technol. 35: Smith, T. L., and L. D. Witter Evaluation of inhibitors for rapid enumeration of psychrotrophic bacteria. J. Food Prot. 42: Snoeren, T. H. M., C. A. van der Spek, R. Dekker, and P. Both Proteolysis during the storage of UHT-sterilized whole milk. I. Experiments with milk heated by the direct system for 4 sec at 142 C. Neth. Milk Dairy J. 33: Speck, M. L Starter culture growth and action in milk. J. Dairy Sci. 42: Speck, M. L., and D. M. Adams Heat resistant proteolytic enzymes from bacterial sources. J. Dairy Sci. 59: Stadhouders, J Technological aspects of the quality of raw milk. Neth. Milk Dairy J. 26: Stadhouders, J Microbes in milk and dairy products. An ecological approach. Neth. Milk Dairy J. 29: Stadhouders, J., E. DeVries, and H. Mulder The thermal resistance of Iipases in connection with cheese-making. XV Int. Dairy Congress 3: Stadhouders, J., and H. Mulder. 1%0. Fat hydrolysis and cheese flavor. IV. Fat hydrolysis in cheese from pasteurized milk. Neth. Milk Dairy 1.14: Stewart, D. B Factors influencing the incidence of B. cereus spores in milk. J. Soc. Dairy Techno!. 28(2): Stewart, D. B., J. G. Murray, and S. D. Neill Lipolytic activity of organisms isolated from refrigerated bulk milk. Int. Dairy Fed. Annu. Bull. 86: :HO. Stokes, J. L General biology and nomenclature of psychrophilic microorganisms. Pages InN. E. Gibbons, (ed.). Recent progress in microbiology. University of Toronto Press, Canada Stokes, J. L., and M. L. Redmond Quantitative ecology of psychrophilic microorganisms. Appl. Microbiol. 14: Stone, W. K., and D. M. Naff Increases in soluble nitrogen and bitter flavor development in cottage cheese. J. Dairy Sci. 50: Sultzer, B. M Oxidative activity of psychrophilic and mesophilic bacteria on saturated fatty acids. J. Bacteriol. 82: Tekinson, 0. C., and J. Rothwell A study of the effect of storage at 5 C on the microbial flora of heat-treated market cream. J. Soc. Dairy Technol. 27(2): TeWhaiti, I. E., and T. F. Fryer Production and heat stability in milk of proteinases and Iipases of psychrotrophic Pseudomonas. XX. Int. Dairy Congress E: (Abst.) Thomas. S. B Psychrophilic microorganisms in milk and dairy products. Part L Dairy Sci. Abst. 20: Thomas, S. B Psychrophilic microorganisms in milk and dairy products. Part II. Dairy Sci. Abst. 20: Thomas. S. B Sources, incidence and significance of psychrotrophic bacteria in milk. Milchwissenschaft 21: Thomas. S. B Methods of assessing the psychrotrophic bacterial content of milk. 1: Appl. Bacteriol. 32:269-2% Thomas, S. B Psychrotrophic microorganisms in market cream. A review. Part I. Dairy Ind. 35: Thomas, S. B The suitability ofrefrigerated bulk collected milk for cheesemaking. Dairy Ind. 36: Thomas, S. B The microflora of bulk collected milk. Part 1. Dairy Ind. 39: Thomas, S. B The microflora of bulk collected milk. Part 2. Dairy Ind. 40: Thomas, S. B., and R. G. Druce Psychrotrophic bacteria in refrigerated milk. Part I. Dairy Ind. 34: Thomas, S. B., and R. G. Druce. 1%9. Psychrotrophic bacteria in refrigerated milk. Part II. Dairy Ind. 34: Thomas, S. B., and R. G. Druce Psychrotrophic bacteria in refrigerated milk. Part III. Dairy Ind. 34: Thomas, S. B., and R. G. Druce Psychrotrophic microorganisms in butter. A review. Part I. Dairy Ind. 36: Thomas, S. B., and R. G. Druce Psychrotrophic microorganisms in butter. A review. Part II. Dairy Ind. 36: JOURNAL OF FOOD PROTEC170N. VOL. 45, FEBRUARY 1982

35 206 COUSIN 329. Thomas, S. B., and R. G. Druce The incidence and significance of coli-aerogenes bacteria in refrigerated bulk collected milk. Dairy Ind. 37: Thomas, S. B., R. G. Druce, and A. Davies The significance of psychrotrophic bacteria in raw milk. Dairy Ind. 31: Thomas, S. B., R. G. Druce, and M. Jones Influence of production conditions on the bacteriological quality of refrigerated farm bulk tank milk. A review. J. Appl. Bacteriol. 34: Thomas, S. B., and B. F. Thomas Psychrotrophic bacteria in refrigerated bulk-collected raw milk. Part I. Dairy Ind. 38: Thomas, S. B., and B. F. Thomas Psychrotrophic bacteria in refrigerated bulk-collected raw milk. Part II. Dairy Ind. 38:61-64,66,68, Thomas, S. B.. and B. F. Thomas The bacteriological grading of bulk collected milk. Part 2: General principles. Dairy Ind. 40: Thomas, S. B., and B. F. Thomas The bacteriological grading of bulk collected milk. Part 3: The total colony count. Dairy Ind. 40: Thomas, S. B.. and B. F. Thomas The bacteriological grading of bulk collected milk. Part 4: Simplified methods of determining colony counts. Dairy Ind. 40: Thomas, S. B., and B. F. Thomas The bacteriological grading of bulk collected milk. Part 5: Further simplified methods for determining colony counts. Dairy Ind. 40: , Thomas, S. B., and B. F. Thomas The bacteriological grading of bulk collected milk. Part 6: The direct microscopic count. Dairy Ind. 40: Thomas, S. B., and B. F. Thomas The bacteriological grading of bulk collected milk. Part 7: Thermoduric, psychrotrophic and coli-aerogenes colony counts. Dairy Ind. 40: Thomas, S. B., and B. F. Thomas The bacteriological grading of bulk collected milk. Part 8: Differential and selective agar media. Dairy Ind. 40: Thomas, S. B., and B. F. Thomas The bacteriological grading of bulk collected milk. Part 9: Bacterial tests as indices of conditions of production. Dairy Ind. 40: Thomas, S. B., and B. F. Thomas The bacteriological grading of bulk collected milk. Part 10: Standards. Dairy Ind. 40: Thomas, S. B., and B. F. Thomas The bacterial content of farm bulk milk tanks. Dairy Ind. Int. 41(5): Thomas, S. B., and B. F. Thomas The bacterial content of farm bulk milk tanks. Dairy Ind. Int. 41: Thomas, S. B., and B. F. Thomas The bacterial content of milking machines and pipeline milking plants. A review. Dairy Ind. Int. 42(4): Thomas, S. B.. and B. F. Thomas The bacterial content of milking machines and pipeline milking plants. Part II of a review. Dairy Ind. Int. 42(5):16,18-19, Thomas, S. B., and B. F. Thomas The bacterial content of milking machines and pipeline milking plants. Part Ill of a review: Composition of microflora. Dairy Ind. Int. 42(7):19, 21, Thomas, S. B., and B. F. Thomas The bacterial content of milking machines and pipeline plants. Part IV. Coli-aerogenes bacteria. Dairy Ind. Int. 42(11):25, 28-30, Thomas, S. B., and B. F. Thomas The bacterial content of milking machines and pipeline milking plants. Part V. Thermoduric organisms. Dairy Ind. Int. 43(5): 17-21, Thomas, S. B., and B. F. Thomas A selective bibliographical guide to the literature on bacteriological aspects of bulk milk collection. Part I. Dairy Ind. Int. 44(10:37, Thomas, S. B.. and B. F. Thomas A selective bibliographical guide to the literature on bacteriological aspects of bulk milk collection. Part 2. Dairy Ind. Int. 44(11:21, 23, 27, Thomas, S. B., B. F. Thomas, and D. Ellison Milk bacteria which grow at refrigerator temperatures. Dairy Ind. 14: , Tinuoye, 0. L., and L. G. Harmon Growth of thermoduric psychrotrophic bacteria in refrigerated milk. Amer. Dairy Rev. 37(9):26, 28, Tolle, A., W. Heeschen, H. Wernery, J. Reichmuth and G. Suhren Die Pyruvat bestimmung-ein neuer Weg rtir Messung der bakteriologischen Wertigkeit von Milch. Milchwissenschaft 27: Tolle, A., G. Suhren, I Otte, and W. Heeschen Zur Bakteriologie und Sensorik der pasteurisierten Trinkmilch. Milchwissenschaft 34: Tompkin, R. B Refrigeration temperature as an environmental factor influencing the microbial quality of food. A review. Food Techno). 27(12):54-56, Vander Zant, W. C., and A. V. Moore The influence of certain factors on the bacterial counts and proteolytic activities of several psychrophilic organisms. J. Dairy Sci. 38: Vasavada, P. C., and C. H. White A screening method for bacterial reduction of diacetyl. J. Dairy Sci. 60: Vasavada, P. C., and C. H. White Quality of commercial buttermilk. J. Dairy Sci. 62: Vitagliano, M., V. M. Radogna, and A. M. Leone Cheesemaking from refrigerated milk. Latte 45: (Dairy Sci. Abst. 34:4450) Von Bockelmann, I Lipolytic and psychrotrophic bacteria in cold-stored milk. XVIII Inter. Dairy Congress 1E:106 (Abst.) Waes, G Some observations on the enumeration of psychrotrophic bacteria in milk. Dairy Ind. 21: Wang, J. J., and J. F. Frank Characterization of psychrotrophic bacteria isolated from commercial buttermilk. J. Dairy Sci. 63 (Suppl. 1):39 (Abst.) Wang, M. Y., E. B. Collins, and J. C. Lob ben Destruction of psychrophilic strains of bacillus by chlorine. J. Dairy Sci. 56: Washam, C.J., H. C. Olson, and E. R. Vedamuthu Heatresistant psychrotrophic bacteria isolated from pasteurized milk. J. Food Prot. 40: Watrous, G. H., Jr., S. E. Barnard, and W. W. Coleman II Survey of the actual and potential bacterial keeping quality of pasteurized milk from SO Pennsylvania Dairy Plants. J. Milk Food Techno!. 34: Watrous. G. H., Jr., S. E. Barnard, and W. W. Coleman II.197l. Bacterial concentrations in raw milk, immediately after laboratory pasteurization and following 10 days storage at 7.2 C. J. Milk Food Techno!. 34: Weckbach, L. S., and B. E. Langlois Effect of heat treatments on survival and growth of a psychrotroph and on nitrogen fractions in milk. J. Food Prot. 40: West, F. B., D. M. Adams, and M. L. Speck Inactivation of heat resistant proteases in normal ultrahigh temperature sterilized skim milk by a low temperature treatment. J. Dairy Sci. 61: White, C. H., and M. Bulthaus Changes in milk caused by addition of protease-producing psychrotrophs to raw milk. J. Dairy Sci. 63 (Suppl. 1):56-57 (Abst.) White, C. H., M. Bulthaus, and R. T. Marshall Defects caused by adding protease-producing psychrotrophs to raw milk. J. Dairy Sci. 62 (Suppl.1):46 (Abst.) White, C. H., W. T. Gillis, D. L. Sirnmler, M. K. Gala!, J. R. Walsh, and J. T. Adams Evaluation of raw milk quality tests. J. Food Prot. 41: White, C. H., and R. T. Marshall Reduction of shelf-life of dairy products by a heat-stable protease from PseudomonilS J1uorescens P26. J. Dairy Sci. 56: JOURNAL OF FOOD PROTECTION, VOL 45, FEBRUARY 1982