166 Modern Food Microbiology the products studied by these workers were as follows: 3.65–4.40 for yogurts, 4.1–4.9 for buttermilk, 4.18–4.70 for sour creams, and 4.80–5.10 for cottage cheese samples In another study, commercially produced yogurts in Ontario were found to contain the desired 1:1 ratio of coccus to rod in only 15% of 152 products examined.5 Staphylococci were found in 27.6% and coliforms in around 14% of these yogurts Twenty-six percent of the samples had yeast counts more than 1,000/g and almost 12% had psychrotroph counts more than 1,000/g In his study of commercial unflavored yogurt in Great Britain, Davis11 found counts of the two starters to range from a low of around 82 million to a high of over billion/g, and the final pH to range from 3.75 to 4.20 The antimicrobial activities of lactic acid bacteria are discussed further in Chapters and 13 Kefir is prepared by the use of kefir grains, which contain one or more bacterial species of the genera Acetobacter, Lactobacillus, Lactococcus, Leuconostoc, and one or more yeast species of the genera Candida, Kluyveromyces, and Saccharomyces These symbionts are held together by coagulated protein.18 The important Lactobacillus spp in kefir are: L kefiri, L parakefiri, L kefiranofaciens subsp kefiranofaciens, and L kefiranofaciens subsp kefirgranum.75 The last two are responsible for the production of kefiran (a water-soluble polysaccharide), which accounts for about 24% of kefir grains.75 Kumiss is similar to kefir except that mare’s milk is used, the culture organisms not form grains, and the alcohol may reach 2% Acidophilus milk is produced by the inoculation of an intestinal implantable strain of L acidophilus into sterile skim milk The inoculum of 1–2% is added, followed by holding the product at 37◦ C until a smooth curd develops A popular variant of this product that is produced commercially in the United States consists of adding a concentrated implantable strain culture of L acidophilus to a pasteurized and cold vat of whole milk (or skim or 2% milk), and it is bottled immediately It has the pH of normal milk and is more palatable than the more acidic product The numbers of L acidophilus should be in the 107 –108 /ml range.32 Bulgarian buttermilk is produced in a similar manner by the use of L bulgaricus as the inoculum or starter, but unlike L acidophilus, L bulgaricus is not implantable in the human intestines A summary of fermented milk is presented in Table 7–4 Butter contains around 15% water, 81% fat, and generally less than 0.5% carbohydrate and protein Although it is not a highly perishable product, it does undergo spoilage by bacteria and molds The main source of microorganisms for butter is cream, whether sweet or sour, pasteurized or nonpasteurized The biota of whole milk may be expected to be found in cream because as the fat droplets rise to the surface of milk, they carry up microorganisms The processing of both raw and pasteurized creams to yield butter brings about a reduction in the numbers of all microorganisms, with values for finished cream ranging from several hundred to over 100,000/g having been reported for finished salted butter Salted butter may contain up to 2% salt, and this means that water droplets throughout may contain an effective level of about 10%, thus making this product even more inhibitory to bacterial spoilage.32 Bacteria cause two principal types of spoilage in butter The first is a condition known as “surface taint” or putridity This condition is caused by Pseudomonas putrefaciens as a result of its growth on the surface of finished butter It develops at temperatures within the range 4–7◦ C and may become apparent within 7–10 days The odor of this condition is apparently due to certain organic acids, especially isovaleric acid Surface taint along with an apple odor is caused also by Chryseobacterium joostei.34 The second most common bacterial spoilage condition of butter is rancidity This condition is caused by the hydrolysis of butterfat with the liberation of free fatty acids Lipase from sources other than microorganisms can cause the effect The causative organism is Pseudomonas fragi, although P fluorescens is sometimes found Bacteria may cause three other less common spoilage conditions in butter Malty flavor is reported to be due to the growth of Lactococcus lactis var maltigenes Skunklike Milk, Fermentation, and Fermented and Nonfermented Dairy Products 167 Table 7–4 Some Fermented Milk Products Foods and Products Raw Ingredients Acidophilus milk Bulgarian buttermilk Milk Cheeses (ripened) Kefir Milk curd Milk Kumiss Raw mare’s milk Taette Milk Tarhana∗ Yogurt† Wheat meal and yogurt Milk, milk solids Bioghurt Milk, milk solids ∗ Similar to Kishk in Syria and Kushuk in Iran † Also yoghurt (matzoon in Armenia; leben in Egypt; Fermenting Organisms Where Produced Lactobacillus acidophilus L delbrueckii subsp bulgaricus Lactic starters Lactococcus lactis, L delbrueckii subsp bulgaricus, “Torula” spp Lactobacillus leichmannii, L delbrueckii subsp bulgaricus, “Torula” spp S lactis var taette Many countries Balkans, other areas Lactics L delbrueckii subsp bulgaricus, S salivarius subsp thermophilus L acidophilus, Lactococcus lactis Worldwide Southwestern Asia Russia Scandinavian peninsula Turkey Worldwide Worldwide naja in Bulgaria; gioddu in Italy; dadhi in India) odor is reported to be caused by Pseudomonas mephitica; black discolorations of butter have been reported to be caused by P nigrifaciens Butter undergoes fungal spoilage rather commonly by species of Cladosporium, Alternaria, Aspergillus, Mucor, Rhizopus, Penicillium, and Geotrichum, especially G candidum (Oospora lactis) These organisms can be seen growing on the surface of butter, where they produce colorations referable to their particular spore colors Black yeasts of the genus Torula also have been reported to cause discolorations on butter The microscopic examination of moldy butter reveals the presence of mold mycelia some distances from the visible growth The generally high lipid content and low water content make butter more susceptible to spoilage by molds than by bacteria Cottage cheese undergoes spoilage by bacteria, yeasts, and molds The most common spoilage pattern displayed by bacteria is a condition known as slimy curd Alcaligenes spp have been reported to be among the most frequent causative organisms, although Pseudomonas, Proteus, Enterobacter, and Acinetobacter spp have been implicated Penicillium, Mucor, Alternaria, and Geotrichum all grow well on cottage cheese, to which they impart stale, musty, moldy, and yeasty flavors The shelf life of commercially produced cottage cheese in Alberta, Canada was found to be limited by yeasts and molds.59 Although 48% of fresh samples contained coliforms, these organisms did not increase upon storage in cottage cheese at 40◦ F for 16 days For more on fermented dairy products, see references 52, 54 168 Modern Food Microbiology Cheeses Most but not all cheeses result from a lactic fermentation of milk In general, the process of manufacture consists of two important steps: Milk is prepared and inoculated with an appropriate lactic starter The starter produces lactic acid, which, with added rennin, gives rise to curd formation The starter for cheese production may differ depending on the amount of heat applied to the curds S salivarius subsp thermophilus is employed for acid production in cooked curds (up to 60◦ C) because it is more heat tolerant than either of the other more commonly used lactic starters; or a combination of S salivarius subsp thermophilus and L lactis subsp lactis is employed for curds that receive an intermediate cook The curd is shrunk and pressed, followed by salting, and, in the case of ripened cheeses, allowed to ripen under conditions appropriate to the cheese in question Although most ripened cheeses are the product of metabolic activities of the lactic acid bacteria, several well-known cheeses owe their particular character to other related organisms In the case of Swiss cheese, a mixed culture of L delbrueckii subsp bulgaricus and S salivarius subsp thermophilus is usually employed along with a culture of Propionibacterium shermanii or P freundenreichii added to function during the ripening process in flavor development and eye formation (See Figure 7–1(C) and (D)) for a summary of propionibacteria pathways and Figure 7–4 for pathway in detail.) These organisms have been reviewed extensively by Hettinga and Reinbold.33 For blue cheeses such as Roquefort, the curd is inoculated with spores of Penicillium roqueforti, which effect ripening and impart the blue-veined appearance characteristic of this type of cheese In a similar fashion, either the milk or the surface of Camembert cheese is inoculated with spores of Penicillium camemberti Two coryneform bacteria of the genus Brachybacterium have been recovered from the surfaces of French Gruy`ere and Beaufort cheeses65 but the role these organisms play in the ripening process is unclear In a study of L monocytogenes in European red smear cheese (soft, semisoft, and hard), 5.8% of 329 test samples contained Listeria spp with 6.4% being L monocytogenes and 10.6% L innocua.60 Eight samples contained >100 L monocytogenes/cm2 ; and two samples contained 104 cfu/cm2 There are over 400 varieties of cheeses representing fewer than 20 distinct types, and these are grouped or classified according to texture or moisture content, whether ripened or unripened, and if ripened, whether by bacteria or molds The three textural classes of cheeses are hard, semihard, and soft Examples of hard cheeses are all cheddar, Provolone, Romano, Parmesan, Gruy`ere, Emmental, and Edam All hard cheeses are ripened by bacteria over periods ranging from to 16 months Semihard cheeses include Muenster, Roquefort, Limburger, and Gouda and are ripened by bacteria over periods of 1–8 months Blue and Roquefort are two examples of semihard cheeses that are mold ripened for 2–12 months Limburger is an example of a soft bacteria-ripened cheese, and Brie and Camembert are examples of soft mold-ripened cheeses Among unripened cheeses are cottage, cream, Mozzarella, and Neufchatel The low moisture content of hard and semihard ripened cheeses makes them insusceptible to spoilage by most organisms, although molds can and grow on these products as would be expected Some ripened cheeses have sufficiently low oxidation–reduction potentials to support the growth of anaerobes It is not surprising to find that anaerobic bacteria sometimes cause the spoilage of these products when aw (water activity) permits growth to occur Clostridium spp., especially C pasteurianum, C butyricum, C sporogenes, and C tyrobutyricum, have been reported to cause late gassiness of cheeses One of these (C tyrobutyricum) is well established as the cause of a butyric acid Milk, Fermentation, and Fermented and Nonfermented Dairy Products 169 Figure 7–5 Inhibition of Clostridium tyrobutyricum in processed cheese spread (cheese blend B) by 0.5% and 1.0% of HBS polyphosphate Reprinted with permission from J Food Protect., (c) held by the Int Assoc Food Protect., Des Moines, IA, USA Source: Loessner et al.41 fermentation or the late-blowing defect in cheeses such as Gouda and Emmentaler.39 With 0.5% of a long-chain polyphosphate mixture, growth of C tyrobutyricum was inhibited for at least days and could not be detected after 16–50 days (see Figure 7–5) Growth was completely inhibited by 1.0% polyphosphate,41 and it was due to the sequestration of Ca2+ /Mg2+ by poly-P, which led to filamentous cells and lysis An aerobic sporeformer, Paenibacillus polymyxa, has been reported to cause gassiness This condition is the result of CO2 being produced from lactic acid For the years 1973–1992, there were 32 cheese-associated disease outbreaks in the United States with 1,700 cases and 58 deaths with 52 of the latter caused by L monocytogenes in the 1985 California outbreak.4 The most common vehicle was soft cheeses, and improper pasteurization was common DISEASES CAUSED BY LACTIC ACID BACTERIA Although the beneficial aspects of the lactic acid bacteria to human and animal health are unquestioned, some of these bacteria are associated with human illness This subject has been reviewed by Aguirre and Collins,2 who noted that around 68 reports of involvement of lactobacilli in human clinical illness were made over about a 50-year period Several species of leuconostocs were implicated in about 27 reports in years, the pediococci in 18 reports over years, and the enterococci in numerous reports The enterococci are the third leading cause of nosocomial (hospital acquired) infections, with E faecalis and E faecium being the two most common species It appears that lactic acid bacteria are opportunists that are not capable of initiating infection in normal healthy individuals To determine whether vancomycin-resistant enterococci (VRE) existed in ground beef and pork in Germany, 555 samples were examined for VRE, and overall their incidence in ground beef was too low to be a significant source of nosocomial infections.38 170 Modern Food Microbiology REFERENCES Adler, B.B., and L.R Beuchat 2002 Death of Salmonella, Escherichia coli 0157:H7, and Listeria monocytogenes in garlic butter as affected by storage temperature J Food Protect 65:1976–1980 Aguirre, M., and M.D Collins 1993 Lactic acid bacteria and human clinical infection J Appl Bacteriol 75:95–107 Allen, S.H.G., R.W Killermeyer, R.L Stjernholm, and H.G Wood 1964 Purification and properties of enzymes involved in the propionic acid fermentation J Bacteriol 87:171–187 Altekruse, S.F., B.B Timbo, J.C Mobray, N.H Bean, and M.E Potter 1998 Cheese-associated outbreaks of human illness in the United States, 1973 to 1992: Sanitary manufacturing practices protect consumers J Food Protect 61:1405– 1407 Arnott, D.R., C.L Duitschaever, and D.H Bullock 1974 Microbiological evaluation of yogurt produced commercially in Ontario J Milk Food Technol 37:11–13 Beumer, R.R., J.J.M Cruysen, and I.R.K Birtantie 1988 The occurrence of Campylobacter jejuni in raw cows’ milk J Appl Bacteriol 65:93–96 Bodnaruk, P.W., R.G Williams, and D.A Golden 1998 Survival ofYersinia enterocolitica during fermentation and storage of yogurt J Food Sci 63:535–537 Brown, W.V., and E.B Collins 1977 End products and fermentation balances for lactic streptococci grown aerobically on low concentrations of glucose Appl Environ Microbiol 33:38–42 Carvajal, M., A Bolanos, F Rojo, and I M´endez 2003 Aflatoxin M1 in pasteurized and ultrapasteurized milk with different fat content in Mexico J Food Protect 66:1885–1892 10 Centers for Disease Control and Prevention 2002 Outbreak of Campylobacter jejuni infections associated with drinking unpasteurized milk procured through a cow-leasing program—Wisconsin, 2001 Morb Mort Wkly Rept 51:548–549 11 Davis, J.G 1975 The microbiology of yoghurt In Lactic Acid Bacteria in Beverages and Food, ed J.G Carr, C.V Cutting, and G.C Whiting, 245–263 New York: Academic Press 12 Doelle, H.A 1975 Bacterial Metabolism New York: Academic Press 13 Douglas, S.A., M.J Gray, A.D Crandall, and K.J Boor 2000 Characterization of chocolate milk spoilage patterns J Food Protect 63:516–521 14 Doyle, M.P., and D.J Roman 1982 Prevalence and survival of Campylobacter jejuni in unpasteurized milk Appl Environ Microbiol 44:1154–1158 15 Food and Drug Administration, United States 1995 Grade A pasteurized milk ordinance Washington, D.C.: U.S Department of Health and Human Services, Public Health Service 16 Foster, E.M., F.E Nelson, M.L Speck, R.N Doetsch, and J.C Olson 1957 Dairy Microbiology Englewood Cliffs, N.J.: Prentice-Hall 17 Frank, J.F 2001 Milk and dairy products, In Food Microbiology: Fundamentals and Frontiers, 2nd ed., ed M.P Doyle, L.R Beuchat, and T.J Montville, 111–126 Washington, DC: ASM Press 18 Garrote, G.L., A.G Abraham, and G.L de Antoni 2000 Inhibitory power of kefir: The role of organic acids J Food Protect 63:364–369 19 Gilliland, S.E., and M.L Speck 1974 Frozen concentrated cultures of lactic starter bacteria: A review J Milk Food Technol 37:107–111 20 Gitter, M., R Bradley, and P.H Blampied 1980 Listeria monocytogenes infection in bovine mastitis Vet Rec 107:390–393 21 Goodenough, E.R., and D.H Kleyn 1976 Qualitative and quantitative changes in carbohydrates during the manufacture of yoghurt J Dairy Sci 59:45–47 22 Grant, I.R., H.J Ball, and M.T Rowe 2002a Incidence of Mycobacterium paratuberculosis in bulk raw and commercially pasteurized cows’ milk from approved dairy processing establishments in the United Kingdom Appl Environ Microbiol 68:2428–2435 23 Grant, I.R., E.I Hitchings, A McCartney, F Ferguson, and M.T Rowe 2002b Effect of commercial-scale high-temperature, short-time pasteurization on the viability of Mycobacterium paratuberculosis in naturally infected cows’ milk Appl Environ Microbiol 68:602–607 24 Grant, I.R., H.J Ball, S.D Neill, and M.T Rowe 1996 Inactivation of Mycobacterium paratuberculosis in cows’ milk at pasteurization temperatures Appl Environ Microbiol 62:631–636 ... and Fermented and Nonfermented Dairy Products 167 Table 7–4 Some Fermented Milk Products Foods and Products Raw Ingredients Acidophilus milk Bulgarian buttermilk Milk Cheeses (ripened) Kefir Milk... lactis subsp lactis is employed for curds that receive an intermediate cook The curd is shrunk and pressed, followed by salting, and, in the case of ripened cheeses, allowed to ripen under conditions... J.C Olson 1957 Dairy Microbiology Englewood Cliffs, N.J.: Prentice-Hall 17 Frank, J.F 2001 Milk and dairy products, In Food Microbiology: Fundamentals and Frontiers, 2nd ed. , ed M.P Doyle, L.R Beuchat,