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 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 pattern 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.” 152 Modern Food Microbiology Table 7–1 Some of the Homo- and Heterofermentative Lactic Acid Bacteria Homofermentative Lactobacillus L acetotolerans L acidipiscis L acidophilus L alimentarius L casei L coryniformis L curvatus subsp curvatus subsp melibiosus L delbrueckii subsp bulgaricus subsp delbrueckii subsp.lactis L fuchuensis L helveticus L jugurti L jensenii L kefiranofaciens subsp kefiranofaciens subsp kefirgranum L leichmannii L mindensis L plantarum L salivarius Lactococcus L lactis subsp lactis subsp cremoris subsp diacetylactis subsp hordniae L garvieae L plantarum L raffinolactis Paralactobacillus P selangorensis Pediococcus P acidilactici P claussenii P pentosaceus P damnosus P dextrinicus P inopinatus P parvulus Heterofermentative Lactobacillus L brevis L buchneri L cellobiosus L coprophilus L fermentum L hilgardii L sanfranciscensis L trichoides L pontis L fructivorans L kimchii L paralimentarius L panis L sakei subsp.sakei subsp.carnosus Leuconostoc L argentinum L citreus L fallax L carnosum L gelidum L inhae L kimchii L lactis L mesenteroides subsp cremoris subsp dextranicum subsp mesenteroides Carnobacterium C divergens C gallinarum C mobile C piscicola C viridans Oenococcus O oeni Weissella W cibaria W confusa W hellenica W halotolerans W kandleri W kimchii (continued) Milk, Fermentation, and Fermented and Nonfermented Dairy Products 153 Table 7–1 (continued) Streptococcus S bovis S salivarius subsp salivarius subsp thermophilus Tetragenococcus T halophilus T muriaticus Vagococcus V fluvialis V salmoninarum W minor W thialandensis W paramesenteroides W viridescens W koreensis 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, Streptococcus, Lactococcus, 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 producing flavor and aroma components such as acetylaldehyde and diacetyl (Figure 7–2) The genus Lactobacillus was subdivided historically into three subgenera: Betabacterium, Streptobacterium, 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.70 Group includes obligate homofermentative species (L acidophilus, L delbrueckii subsp bulgaricus, etc.) These are the thermobacteria, and they not ferment pentoses Group consists of facultative heterofermentative species (L casei, L plantarum, L sakei; etc.) Members of this group ferment pentoses Group 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 154 Modern Food Microbiology 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.28 Thus, 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.29 In 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 result 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 not possess aldolase and hexose isomerase, 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 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 Collins8 indicates that YG and YATP values 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 (1–7 µmol/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 (1–15 µmol/ml), YG for L lactis subsp lactis biovar diacetylactis was 21, YATP values for these two organisms on the complex medium with glucose µmol/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.36 Zymomonas mobilis utilizes the Entner–Doudoroff pathway to produce only 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, Gluconacetobacter, and Kozakia.79 With 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 ... 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... mucopeptides 154 Modern Food Microbiology 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... 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