Although this chapter is devoted to milk and other dairy products, it begins with a discussion of fermentation because of the importance of this process to dairy products.
FERMENTATION
Background
Numerous food products owe their production and characteristics to the fermentative activities of microorganisms. Many foods such as ripened cheeses, pickles, sauerkraut, and fermented sausages are preserved products in that their shelf life is extended considerably over that of the raw materials from which they are made. In addition to being made more shelf stable, all fermented foods have aroma and flavor characteristics that result directly or indirectly from the fermenting organisms. In some instances, the vitamin content of the fermented food is increased along with an increased digestibility of the raw materials. The fermentation process reduces the toxicity of some foods (for example, gari and peujeum), whereas others may become extremely toxic during fermentation (as in the case of bongkrek). From all indications, no other single group or category of foods or food products is as important as these are and have been relative to nutritional well-being throughout the world.
The microbial ecology of food and related fermentations has been studied for many years in the case of ripened cheeses, sauerkraut, wines, and so on, and the activities of the fermenting organisms are dependent on the intrinsic and extrinsic parameters of growth discussed in Chapter 3. For example, when the natural raw materials are acidic and contain free sugars, yeasts grow readily, and the alcohol they produce restricts the activities of most other naturally contaminating organisms. If, on the other hand, the acidity of a plant product permits good bacterial growth and at the same time the product is high in simple sugars, lactic acid bacteria may be expected to grow, and the addition of low levels of NaCl will ensure their growth preferential to yeasts (as in sauerkraut fermentation).
Products that contain polysaccharides but no significant levels of simple sugars are normally stable to the activities of yeasts and lactic acid bacteria due to the lack of amylase in most of these organisms.
To effect fermentation, an exogenous source of saccharifying enzymes must be supplied. The use of barley malt in the brewing and distilling industries is an example of this. The fermentation of sugars
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to ethanol that results from malting is then carried out by yeasts. The use of koji in the fermentation of soybean products is another example of the way in which alcoholic and lactic acid fermentations may be carried out on products that have low levels of sugars but high levels of starches and proteins.
Whereas the saccharifying enzymes of barley malt arise from germinating barley, the enzymes of koji are produced by Aspergillus oryzae growing on soaked or steamed rice or other cereals (the commercial product takadiastase is prepared by growing A. oryzae on wheat bran). The koji hydrolysates may be fermented by lactic acid bacteria and yeasts, as is the case for soy sauce, or the koji enzymes may act directly on soybeans in the production of products such as Japanese miso.
Defined and Characterized
The word fermentation has had many shades of meaning in the past. According to one dictionary definition, it is “a process of chemical change with effervescence . . . a state of agitation or unrest . . . any of various transformations of organic substances.” The word came into use before Pasteur’s studies on wines. Prescott and Dunn56 and Doelle12have discussed the history of the concept of fermentation, and the former authors note that in the broad sense in which the term is commonly used, it is “a process in which chemical changes are brought about in an organic substrate through the action of enzymes elaborated by microorganisms.” It is in this broad context that the term is used in this chapter. In the brewing industry, a top fermentation refers to the use of a yeast strain that carries out its activity at the upper parts of a large vat, such as in the production of ale; a bottom fermentation requires the use of a yeast strain that will act in lower parts of the vat, such as in the production of lager beer.
Biochemically, fermentation is the metabolic process in which carbohydrates and related compounds are partially oxidized with the release of energy in the absence of any external electron acceptors.
The final electron acceptors are organic compounds produced directly from the breakdown of the carbohydrates. Consequently, incomplete oxidation of the parent compound occurs, and only a small amount of energy is released during the process. The products of fermentation consist of some organic compounds that are more reduced than others.
The Lactic Acid Bacteria
This group is composed of 13 genera of Gram-positive bacteria at this time:
Carnobacterium Oenococcus Enterococcus Pediococcus Lactococcus Paralactobacillus Lactobacillus Streptococcus Lactosphaera Tetragenococcus Leuconostoc Vagococcus
Weissella
With the enterococci and lactococci having been removed from the genus Streptococcus, the member of this genus of most importance in foods is S. salivarius subsp. thermophilus. S. diacetilactis has been reclassified as a citrate-utilizing strain of Lactococcus lactis subsp. lactis.
Related to the lactic acid bacteria but not considered to fit the group are genera such as Aerococcus, Microbacterium, and Propionibacterium, among others. The last genus has been reduced by the transfer
Milk, Fermentation, and Fermented and Nonfermented Dairy Products 151
of some of its species to the new genus Propioniferax, which produces propionic acid as its principal carboxylic acid from glucose.80
The history of our knowledge of the lactic streptococci and their ecology has been reviewed by Sandine et al.63 These authors believe that plant matter is the natural habitat of this group, but they note the lack of proof of a plant origin for Lactococcus cremoris. It has been suggested that plant streptococci may be the ancestral pool from which other species and strains developed.47
Although the lactic acid group is loosely defined with no precise boundaries, all members share the property of producing lactic acid from hexoses. As fermenting organisms, they lack functional heme-linked electron transport systems or cytochromes, and they obtain their energy by substrate-level phosphorylation while oxidizing carbohydrates; they do not have a functional Krebs cycle.
Kluyver divided the lactic acid bacteria into two groups based on end products of glucose metabolism. Those that produce lactic acid as the major or sole product of glucose fermentation are designated homofermentative (Figure 7–1(A)). The homolactics are able to extract about twice as much energy from a given quantity of glucose as are the heterolactics. The homofermentative pat-tern is observed when glucose is metabolized but not necessarily when pentoses are metabolized, for some homolactics produce acetic and lactic acids when utilizing pentoses. Also the homofermentative
Figure 7–1 Generalized pathways for the production of some fermentation products from glucose by various organisms. (A) Homofermentative lactics; (B) heterofermentative lactics; (C) and (D) Propionibacterium (see Figure 7–3); (E) Saccharomyces spp.; (F) Acetobacter spp.; and (G) Acetobacter “overoxidizers.”
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Table 7–1 Some of the Homo- and Heterofermentative Lactic Acid Bacteria
Homofermentative Heterofermentative
Lactobacillus Lactobacillus
L. acetotolerans L. brevis
L. acidipiscis L. buchneri
L. acidophilus L. cellobiosus
L. alimentarius L. coprophilus
L. casei L. fermentum
L. hilgardii
L. coryniformis L. sanfranciscensis
L. curvatus L. trichoides
subsp. curvatus L. pontis
subsp. melibiosus L. fructivorans
L. delbrueckii L. kimchii
subsp. bulgaricus L. paralimentarius subsp. delbrueckii L. panis
subsp.lactis L. sakei
L. fuchuensis subsp.sakei
L. helveticus subsp.carnosus
L. jugurti Leuconostoc
L. jensenii L. argentinum
L. kefiranofaciens L. citreus
subsp. kefiranofaciens L. fallax
subsp. kefirgranum L. carnosum
L. leichmannii L. gelidum
L. mindensis L. inhae
L. plantarum L. kimchii
L. salivarius L. lactis
Lactococcus L. mesenteroides
L. lactis subsp. cremoris
subsp. lactis subsp. dextranicum
subsp. cremoris subsp. mesenteroides
subsp. diacetylactis Carnobacterium
subsp. hordniae C. divergens
L. garvieae C. gallinarum
L. plantarum C. mobile
L. raffinolactis C. piscicola
Paralactobacillus C. viridans
P. selangorensis Oenococcus
Pediococcus O. oeni
P. acidilactici Weissella
P. claussenii W. cibaria
P. pentosaceus W. confusa
P. damnosus W. hellenica
P. dextrinicus W. halotolerans
P. inopinatus W. kandleri
P. parvulus W. kimchii
(continued)
Milk, Fermentation, and Fermented and Nonfermented Dairy Products 153
Table 7–1 (continued)
Streptococcus W. minor
S. bovis W. thialandensis
S. salivarius W. paramesenteroides
subsp. salivarius W. viridescens subsp. thermophilus W. koreensis Tetragenococcus
T. halophilus T. muriaticus Vagococcus
V. fluvialis V. salmoninarum
character of homolactics may be shifted for some strains by altering growth conditions such as glucose concentration, pH, and nutrient limitation.8,42
Those lactics that produce equal molar amounts of lactate, carbon dioxide, and ethanol from hexoses are designated heterofermentative (Figure 7–1(B)). All members of the genera Pediococcus, Strep-tococcus, LacStrep-tococcus, and Vagococcus are homofermenters, along with some of the lactobacilli.
Heterofermenters consist of Leuconostoc, Oenococcus, Weissella, Carnobacterium, Lactosphaera, and some lactobacilli (Table 7–1). The heterolactics are more important than the homolactics in pro-ducing flavor and aroma components such as acetylaldehyde and diacetyl (Figure 7–2).
The genus Lactobacillus was subdivided historically into three subgenera: Betabacterium, Strepto-bacterium, and Thermobacterium. All of the heterolactic lactobacilli in Table 7–1 are betabacteria. The streptobacteria (for example, L. casei and. plantarum) produce up to 1.5% lactic acid with an optimal growth temperature of 30◦C, whereas the thermobacteria (such as L. acidophilus and L. delbrueckii subsp. bulgaricus) can produce up to 3% lactic acid and have an optimal temperature of 40◦C.43
More recently, the genus Lactobacillus has been arranged into three groups based primarily on fermentative features.70Group 1 includes obligate homofermentative species (L. acidophilus, L. del-brueckii subsp. bulgaricus, etc.). These are the thermobacteria, and they do not ferment pentoses.
Group 2 consists of facultative heterofermentative species (L. casei, L. plantarum, L. sakei; etc.).
Members of this group ferment pentoses. Group 3 consists of the obligate heterofermentative species, and it includes L. fermentum, L. brevis, L. reuteri, L. sanfranciscensis, and others. They produce CO2 from glucose. The lactobacilli can produce a pH of 4.0 in foods that contain a fermentable carbohydrate, and they can grow up to a pH of about 7.1.70
In terms of their growth requirements, the lactic acid bacteria require preformed amino acids, B vitamins, and purine and pyrimidine bases—hence their use in microbiological assays for these compounds. Although they are mesophilic, some can grow below 5◦C and some as high as 45◦C. With respect to growth pH, some can grow as low as 3.2, some as high as 9.6, and most grow in the pH range 4.0–4.5. The lactic acid bacteria are only weakly proteolytic and lipolytic.69
The cell mucopeptides of lactics and other bacteria have been reviewed by Schleifer and Kandler.64 Although there appear to be wide variations within most of the lactic acid genera, the homofermentative lactobacilli of the subgenus Thermobacterium appear to be the most homogeneous in this regard in having l-lysine in the peptidoglycan peptide chain and d-aspartic acid as the interbridge peptide. The lactococci have similar wall mucopeptides.
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Figure 7–2 The general pathway by which acetoin and diacetyl are produced from citrate by group N lactococci and Leuconostoc spp. Pyruvate may be produced from lactate, and acetyl coenzyme A (CoA) from acetate.
Molecular genetics have been employed by McKay and co-workers to stabilize lactose fermentation by L. lactis. The genes responsible for lactose fermentation by some lactic cocci are plasmidborne, and loss of the plasmid results in the loss of lactose fermentation. In an effort to make lactose fermentation more stable, lac+genes from L. lactis were cloned into a cloning vector, which was incorporated into a Streptococcus sanguis strain.28Thus, the lac genes from L. lactis were transformed into S. sanguis via a vector plasmid, or transformation could be effected by use of appropriate fragments of DNA through which the genes were integrated into the chromosome of the host cells.29In the latter state, lactose fermentation would be a more stable property than when the lac genes are plasmidborne.
Metabolic Pathways and Molar Growth Yields
The end-product differences between homo- and heterofermenters when glucose is attacked are a re-sult of basic genetic and physiological differences (Figure 7–1). The homolactics possess the enzymes aldolase and hexose isomerase but lack phosphoketolase (Figure 7–1(A)). They use the Embden–
Meyerhof–Parnas (EMP) pathway toward their production of two lactates/glucose molecule. The
Milk, Fermentation, and Fermented and Nonfermented Dairy Products 155
heterolactics, on the other hand, have phosphoketolase but do not possess aldolase and hexose iso-merase, and instead of the EMP pathway for glucose degradation, these organisms use the hexose monophosphate or pentose pathway (Figure 7–1(B)).
The measurement of molar growth yields provides information on fermenting organisms relative to their fermentation substrates and pathways. By this concept, the microgram dry weight of cells produced per micromole of substrate fermented is determined as the molar yield constant, indicated by Y . It is tacitly assumed that essentially none of the substrate carbon is used for cell biosynthesis, that oxygen does not serve as an electron or hydrogen acceptor, and that all of the energy derived from the metabolism of the substrate is coupled to cell biosynthesis.25 When the substrate is glucose, for example, the molar yield constant for glucose, YG, is determined by
YG= g dry weight of cells moles glucose fermented
If the adenosine triphosphate (ATP) yield or moles of ATP produced per mole of substrate used is known for a given substrate, the amount of dry weight of cells produced per mole of ATP formed can be determined by
YAT P= g dry weight of cells/moles ATP formed moles substrate fermented
A large number of fermenting organisms has been examined during growth and found to have YATP= 10.5 or close thereto. This value is assumed to be a constant, so that an organism that ferments glucose by the EMP pathway to produce 2 ATP/mole of glucose fermented should have YG= 21 (i.e., it should produce 21 g of cells dry weight/mole of glucose). This has been verified for E. faecalis, Saccharomyces cerevisiae, Saccharomyces rosei, and L. plantarum on glucose (all YG= 21, YATP= 10.5, within experimental error). A study by Brown and Collins8indicates that YGand YATPvalues for Lactococcus lactis subsp. lactis biovar diacetylactis and Lactococcus lactis subsp. cremoris differ when cells are grown aerobically on a partially defined medium with low and higher levels of glucose, and further when grown on a complex medium. On a partially defined medium with low glucose levels (1–7µmol/ml), values for L. lactis subsp. lactis biovar diacetylactis were YG= 35.3 and YATP= 15.6, whereas for L. lactis subsp. cremoris, YG= 31.4 and YATP= 13.9. On the same medium with higher glucose levels (1–15µmol/ml), YGfor L. lactis subsp. lactis biovar diacetylactis was 21, YATPvalues for these two organisms on the complex medium with glucose 2µmol/ml were 21.5 and 18.9 for L.
lactis subsp. lactis biovar diacetylactis and L. lactis subsp. cremoris, respectively. Anaerobic molar growth yields for enterococcal species on low levels of glucose have been studied by Johnson and Collins.36Zymomonas mobilis utilizes the Entner–Doudoroff pathway to produce only 1 ATP/mole of glucose fermented (YG= 8.3, YATP= 8.3). If and when the produced lactate is metabolized further, the molar growth yield would be higher. Bifidobacterium bifidum produces 2.5–3 ATP/mole of glucose fermented resulting in YG= and YATP= 13.71
ACETIC ACID BACTERIA
These Gram-negative bacteria belong to the family Acetobacteriaceae, and to the alpha-subclass of Proteobacteria. The recognized genera are: Acetobacter, Asaia, Acidomonas, Gluconobacter, Glu-conacetobacter, and Kozakia.79With the exception of Asaia, they produce large quantities of acetic acid from ethanol, and can grow in the presence of 0.35% acetic acid. The metabolic pathway employed
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by the acetic acid producing strains is shown in Figure 7–1(F) and (G). Asaia, on the other hand, pro-duces little or no acetic acid from ethanol, and its species do not grow in the presence of 0.35% acetic acid.79The three recognized species oxidize acetate and lactate to CO2and water.
DAIRY PRODUCTS
Milk
Milk is used throughout the world as a human food in at least one form, and from at least one of a number of different mammals. Bovine milk is typical of other milk types and it is the basis of the discussion that follows. Many of the aspects of milk microbiology not covered below have been presented or reviewed by Frank17and Murphy and Boor.50
Composition
From the general chemical composition of cow’s milk in Table 7–2, some differences between this product and red meats in Table 4–9 are readily evident. The protein content of milk is considerably lower (3.5 vs. 18.0%) while the carbohydrate content is considerably higher (14.9 vs. ca. 1.0%). The higher structural protein content of red meats enables these products to exist as solids. Although the average water content near the surface of fresh meats of ca. 75.5% is lower than the average of 87%
for milk, the aw of both products is near 1.0. The milk of goats and sheep is similar in composition to that of cows.
Milk protein consists mainly of casein, and it exists in several classes:α, β, etc. If milk pH falls below 4.6, the casein precipitates. Although casein represents 80–85% of total milk protein, when precipitation occurs, the liquid portion is referred to as whey. The remaining proteins are found in whey and they include serum albumin, immunoglobulins,α-lactalbumin, etc. Milk carbohydrate is principally lactose and its content is fairly consistent among breeds of milk cows at around 5.0%.
Although lactose is the main sugar, smaller quantities of glucose and citric acid exist. The fat content varies between ca. 3.5 and 5.0% depending upon cattle breed, and it consists mainly of triglycerides composed of C14, C16, C18, and C18:1fatty acids. Smaller quantities of diglycerides and phospholipids occur. Milk lipids exist largely in the form of fat globules that are surrounded by a phospholipid layer.
The ash content of around 0.7% consists of a relatively high level of Ca2+and a lower level of Fe2+. Overall, the nonfat solids in cow’s milk average ca. 9.0% while total solids range between 12.5 and 14.5%, and average ca. 12.9% depending upon breed.
Table 7–2 Average Chemical Composition (%) of Whole Bovine Milk (Summarized from the Literature)
Water 87.0
Protein 3.5
Fat 3.9
Carbohydrate 4.9
Ash 0.7
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The pH of fresh whole milk is around 6.6 but it may reach ca. 6.8 from a cow that has mastitis.
Mastitis is an infection of the udder that is most often caused by Streptococcus agalactiae and S. uberis but sometimes by Staphylococcus aureus or Streptococcus dysgalactiae. Fresh milk from a mastitic cow typically contains leucocytes (white blood cells)>106/ml in contrast to nonmastitic milk that contains leucocytes around 70,000/ml.
Milk contains a very adequate supply of B vitamins with pantothenic acid and riboflavin being the two most abundant. Vitamins A and D are added for human consumption, and their presence has no known effect on the activity of microorganisms.
Overall, the chemical composition of whole cow’s milk makes it an ideal growth medium for heterotrophic microorganisms, including the nutritionally fastidious Gram-positive lactic acid bacteria.
How the milk microbiota utilize these constituents and bring about its spoilage is covered below under spoilage.
Processing
Milk is processed in a number of ways to produce a variety of products such as cream, cheese, and butter. Whole fresh milk is processed to produce a number of fluid products. Skim milk (0.5% fat) or reduced fat milk (up to 2.0% fat) is produced by high-speed centrifugation following heating to ca. 100◦F to remove butter fat as cream, or by use of skim milk to which the desired fat content is added. The latter is pasteurized either at 150–155◦F (65.5–68.3◦C) for 30 minutes or at 166–175◦F (74.4–79.4◦C) for 15 sec prior to cooling to around 4◦C.78
Evaporated milk is produced by the removal of about 60% water from whole milk which results in the lactose content being about 11.5%. Sweetened condensed milk is produced by the addition of sucrose or glucose before evaporation. This leads to a product with a sugar content of about 54%
or>64% in solution.
In the United States, grade A raw milk that is to be pasteurized should not have an APC that exceeds 300,000 cfu/ml for commingled or blended milk, or should not exceed 100,000/ml for milk from an individual producer. After pasteurization, the APC should not exceed 20,000 cfu/ml, and the coliform count should not exceed 10/ml.15 Raw milk should not be held longer than 5 days at 40◦F (4.4◦C) prior to pasteurization.
Chocolate milk is processed at a slightly higher temperature than unflavored milk (75◦C for 15 sec rather than 72◦C). A study of chocolate milk from four plants revealed that the APC was higher at 14 days post-processing than unflavored milk even though the initial numbers for both types were essentially the same.13 On day 14, 76.1% of unflavored and 91.6% of chocolate milk had APCs
>20,000 cfu/ml with 26.1% of the former and 53.7% of the latter products having APCs >106cfu/ml.
These investigators suggested that the chocolate flavor powder contributed to increased growth.13The higher numbers were not due to higher numbers in the chocolate powder per se.
Pasteurization
The objective of milk pasteurization is the destruction of all disease-causing microorganisms. En-dospores of pathogens such as Clostridium botulinum and spoilage organisms such as Clostridium tyrobutyricum, C. sporogenes, or Bacillus cereus are not destroyed. Although pathogens can be de-stroyed by nonthermal means, milk pasteurization is achieved solely by heating.
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The low temperature-long time (LTLT) method consists of heating the coolest part to 145◦F (63◦C) for 30 minutes. This is referred to as the batch method. The other more widely used method is the high temperature-short time (HTST) method, and it consists of heating to 161◦F (72◦C) for 15 sec. This is the flash method, and it is inherently less destructive than the batch method. The basis for the heating time and temperature is the thermal death time (TDT) of the most heat-resistant non-sporeforming milk-borne pathogens. Prior to 1950, the LTLT method involved heating at 143◦F for 30 minutes, which was the TDT of Mycobacterium tuberculosis. However, after the discovery of the Q fever agent (Coxiella burnetti) and the determination of its presence in bovine, goat, and sheep milk, the LTLT method was changed so that it involved heating at 145◦F for 30 minutes to correspond to the TDT of this pathogen. In properly pasteurized milk, the naturally occurring enzyme alkaline phosphatase is destroyed.76
UHT (ultra-high temperature) is another thermal treatment that destroys non-sporeforming pathogens in milk, but in addition some sporeformers are severally reduced in numbers. The UHT treatment is achieved by heating at temperatures of 275–284◦F (135–140◦C) for a few sec (the mini-mum treatment is 130◦C for 1 sec). UHT-treated milk is commercially sterile with a shelf life of 40–45 days at 40◦F when aseptically packaged in sterile containers.7 UHT-treated whole milk is said to be more flavorful, due apparently to formation of some Maillard products.
Although pasteurized milk is free of non-sporeforming pathogens, it is not sterile. The efficacy of either LTLT or HTST to destroy the mycobacterial subspecies that is associated with Crohn’s disease in humans has been called in to question, and this is discussed further below under milk-borne diseases.
Most if not all Gram-negative bacteria (especially psychrotrophs) are destroyed along with many Gram positives. Thermoduric Gram positives belonging to the genera Enterococcus, Streptococcus (especially Streptococcus salivarius subsp. thermophilus), Microbacterium, Lactobacillus, Mycobac-terium, CorynebacMycobac-terium, and most if not all sporeformers survive. Among the survivors are a number of psychrotrophic species of the genus Bacillus.45
General Microbiota of Milk
Theoretically, milk that is secreted to the udder of a healthy cow should be free of microorganisms.
However, freshly drawn milk is generally not free of microorganisms. Numbers of several hundred to several thousand cfu/ml are often found in freshly drawn milk, and they represent the movement up the teat canal of some and the presence of others at the lower ends of teats. Although the APC of milk from healthy cows is generally<103cfu/ml, numbers of 104/ml are not uncommon.50
Milk-Borne Pathogens
Since it is such an excellent nutrient source and because milk-producing animals may harbor organisms that cause human diseases, it is not surprising that raw milk can be a source of diseases.
Some of the most obvious are the animal diseases below to which humans are susceptible and which may occur in milk of cows:
Brucellosis Anthrax
Tuberculosis Listeriosis Salmonellosis Q fever
Campylobacteriosis Crohn’s disease (?) Enterohemorrhagic colitis Staph./Strep. Mastitis