S. thermophilus and L. delbrueckii subsp. bul-garicus both make lactic acid during the yogurt fermentation.They are homofermentative, meaning lactic acid is the primary end-product from sugar metabolism, and both ferment lactose in a similar manner. Moreover, the specific means by which lactose metabolism occurs in these bacteria not only dictate product formation, but also have an important impact on the health-promoting activity these bacteria provide (discussed later).
The first step involves transport of lactose across the cell membrane. As reviewed in Chapter 2, there are two general routes by which this step can occur in lactic acid bacteria. Mesophilic lactococci (i.e., Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris) use the phosphoenolpyru-vate (PEP)-dependent phosphotransferase system (PTS), in which lactose is phosphory-lated during its transport across the cytoplasmic membrane. The high energy phosphate group of PEP, following its transfer via a protein cascade, serves as the phosphoryl group donor for the phosphorylation reaction. It also provides the driving force for lactose transport.The product that accumulates in the cytoplasm, therefore, is lactose-phosphate, with the phosphate attached to carbon 6 on the galactose moiety. In contrast, S. thermophilus and L. delbrueckii subsp. bulgaricus, the thermophilic culture bacteria, use a secondary transport system (called LacS) for lactose uptake.Transport of lactose occurs via a symport mechanism, with the proton gradient serving as the driving force (although this is not the entire story, as discussed below). Lactose is not modified during transport and instead accumulates inside the cell as free lactose.
For both the PTS and LacS systems, the next step is hydrolysis, but since the substrates are different, the hydrolyzing enzymes must be different, too. In lactococci, hydrolysis of lactose-phosphate occurs by phospho-p-galactosidase, forming glucose and galactose-6-phosphate. Glucose is subsequently phosphorylated (using ATP as the phosphoryl group donor) by glucokinase (or hexokinase) to form glucose-6-phosphate. The latter then feeds directly into the Embden Meyerhof glycolytic pathway, ultimately leading to lactic acid.The other product of the phospho-p-galactosidase reaction, galactose-6-phosphate, is metabolized simultaneously by a parallel pathway called the tagatose pathway, which also results in formation of lactic acid.
In S. thermophilus and L. delbrueckii subsp. bulgaricus, the intracellular lactose that accumulates is hydrolyzed by a p-galactosidase, releasing glucose and galactose. The glucose is metabolized to lactic acid, the same as in the lactococci. However, most strains of S. ther-mophilus and L. delbrueckii subsp. bulgaricus lack the ability to metabolize galactose and instead secrete galactose back into the milk.This is despite the observation that a pathway for galactose metabolism indeed exists in these bacteria.
The reason why this pathway does not function, especially in S. thermophilus, and why this apparent metabolic defect occurs, has been the subject of considerable study. It now appears that the genes coding for the enzymes necessary to metabolize galactose, the so-called Leloir pathway, are not transcribed or expressed. Rather, these genes are strongly repressed, and the cell responds by excreting the unfermented galactose that it has accumu-lated.The extracellular galactose is also not metabolized even later, although genes coding for a galactose transport system apparently exist.
Although it would appear that S. ther-mophilus is being wasteful by not making efficient use of both monosaccharide constituents of lactose, this is not the case. When S. ther-mophilus grows in milk, where the lactose concentration is 5% (more than 140 mM), or in yogurt mix, which has an even higher lactose concentration due to added milk solids, sugar limitation is not an issue. Rather, growth cessation in yogurt occurs due to low pH or low temperature, not sugar availability. Interestingly, when S. thermophilus excretes galactose it does so via the LacS permease, the same system that is responsible for lactose uptake.Thus, LacS acts as an exchange system, trading an out-bound galactose for an in-coming lactose (Chapter 2). This precursor-product exchange reaction spares the cell of energy that it would normally spend to transport lactose. In fact, S. thermo-philus may be so well-adapted to growth in milk that the galactose efflux reaction is kinetically favored, even in variant or mutant strains where the galactose genes are de-repressed and galactose pathway enzymes are expressed. In yogurt, galactose accumulation, even at concentrations as high as 0.7% to 1.0%, is ordinarily of no major consequence (the exception may be for individuals prone to cataracts, since dietary galactose may exacerbate that condition). However in cheese manufacture, galactose can be the cause of serious technological problems, and galac-tose-fermenting strains of S. thermophilus may be of considerable value (Chapter 5).
In some yogurt cultures, Lactobacillus hel-veticus is used instead of L. delbrueckii subsp. bulgaricus. This organism deals with lactose and galactose in a somewhat similar manner. Transport of lactose, as noted above, occurs via a LacS that has nearly the same activity as in S. thermophilus. Galactose efflux occurs after hydrolysis by p-galactosidase, but not nearly to the same extent. Instead, some of the galactose is metabolized via the Leloir pathway at the same time as glucose (Chapter 2). In addition, extracellular galactose can be transported and metabolized. However, little of the secreted galactose is fermented because the fermentation period is so short. Even most of the lactose is left unfermented. Since most yogurt is made with high solids milk, the lactose concentration in yogurt is often higher than that of milk, with levels of 6% to 7%.
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