In this article we will discuss about:- 1. Meaning of Lactic Acid Bacteria 2. Activities of Lactic Acid Bacteria in Foods.
Meaning of Lactic Acid Bacteria:
The term lactic acid bacteria (LAB) has no strict taxonomic significance, although the LAB have been shown by serological techniques and 16S ribosomal RNA cataloguing to be phylogenetically related. They share a number of common features: they are Gram-positive, non-spore forming rods or cocci; most are aero tolerant anaerobes which lack cytochromes and porphyrins and are therefore catalase- and oxidase-negative.
Some do take up oxygen through the mediation of flavoprotein oxidases and this is used to produce hydrogen peroxide and/or to re-oxidize NADH produced during the dehydrogenation of sugars. Cellular energy is derived from the fermentation of carbohydrate to produce principally lactic acid.
To do this, they use one of two different pathways and this provides a useful diagnostic feature in their classification (Figure 9.1). Homofermenters produce lactate as virtually a single product from the fermentation of glucose.
They follow the Emden-Meyerhof-Parnas (EMP) glycolytic pathway whereby the six-carbon molecule glucose is phosphorylated and isomerized before cleavage by the enzyme aldolase into glyceraldehyde-3-phosphate.
This is then converted to pyruvate during which ATP is produced by substrate-level phosphorylation at two sites to give an overall yield of two molecules of ATP for every molecule of glucose fermented. In order to regenerate the NAD+ consumed in the oxidation of glyceraldehyde-3-phosphate, pyruvate is reduced to lactate using NADH.
Heterofermenters produce roughly equimolar amounts of lactate, ethanol/ acetate, and carbon dioxide from glucose. They lack aldolase and transform the hexose, glucose, into a pentose, ribose, by a sequence involving oxidation and decarboxylation. The pentose is cleaved into glyceraldehyde phosphate and acetyl phosphate by the enzyme phosphoketolase.
The triose phosphate is converted into lactate by the same sequence of reactions as occurs in glycolysis to give two molecules of ATP. The fate of the acetyl phosphate depends on the electron acceptors available. In the absence of alternatives, acetyl phosphate fulfills this role and is reduced to ethanol while regenerating two molecules of NAD+ from NADH.
In the presence of oxygen, NAD+ can be regenerated by NADH oxidases and peroxidases, leaving acetyl phosphate available for conversion to acetate. This provides another site for substrate level phosphorylation and increases the overall ATP yield of hetero-fermentation from one to two molecules ATP per molecule of glucose dissimilated.
When this is possible, the increased yield of ATP is reflected in a faster growth rate and a higher molar growth yield. The same effect can be achieved with other electron acceptors, for example fructose which is reduced to mannitol. Heterofermenters and homo-fermenters can be readily distinguished in the laboratory by the ability of hetero-fermenters to produce carbon dioxide in glucose-containing media.
The principal genera of the lactic acid bacteria are described in Table 9.4. Lactobacillus is recognized as being phylogenetically very heterogeneous and this is evidenced by the broad range of %GC values exhibited within the genus.
Some non-acidoduric, heterofermentative lactobacilli have been recently reclassified in the new genus Carnobacterium and there is likely to be significant further refinement of the genus in the future.
Currently the lactobacilli are subdivided into three groups: obligate homofermenters, facultative heterofermenters and obligate heterofermenters. The obligate homofermenters correspond roughly to the Thermo bacterium group of the Orla-Jensen classification scheme and include species such as Lb. acidophilus, Lb. delbruckii and Lb. helveticus.
They ferment hexoses almost exclusively to lactate but are unable to ferment pentoses. The facultative heterofermenters ferment hexoses via the EMP pathway to lactate but have an inducible phosphoketolase which allows them to ferment pentoses to lactate and acetate.
They include some species important in food fermentation such as Lb. plantarum, Lb. casei, and Lb. sake. Obligate heterofermenters which include Lb. brevis, Lb. fermentum and Lb. kefir use the phosphoketolase pathway for hexose fermentation.
Leuconostoc is treated as a separate genus on morphological grounds as its members are typically irregular cocci. This is not entirely satisfactory since the vexed question, ‘When does a short rod become a coccus?’ often arises; for example, Lactobacillus confuses was originally classed as a Leuconostoc.
It is possible to distinguish leuconostocs from most heterofermentative lactobacilli by two phenetic characteristics: their production of only d-lactate and inability to produce ammonia from arginine.
The genus Pediococcus also includes species of importance in food fermentations such as P. pentosaceus and, until fairly recently, P. halophilus now in a genus of its own as Tetragenococcus halophilus. Nucleic acid studies of the streptococci have shown that they comprise three distinct groups worthy of genus status.
The enterococci now form the genus Enterococcus although the faecal strains of S. bovis and S. equinus which also react with the group D antisera used in Lancefield’s classical serological classification scheme are not included.
What were known as Lancefield’s group N streptococci, the lactic or dairy streptococci, are now members of the genus Lactococcus and a number of these which were considered distinct Streptococcus species are now classified as subspecies of Lactococcus lactis. The yoghurt starter Streptococcus salivarius subsp. thermophilus does not possess the Group N antigen and remains in the genus Streptococcus.
Some authors also include Bifidobacterium among the lactic acid bacteria although this has less justification as they are quite distinct both phylogenetically and biochemically. For example hexose fermentation by bifidobacteria follows neither the EMP glycolytic pathway nor the phosphoketolase pathway but produces a mixture of acetic and lactic acids.
Activities of Lactic Acid Bacteria in Foods:
1. Antimicrobial Activity of Lactic Acid Bacteria:
Lactic acid bacteria are often inhibitory to other micro-organisms and this is the basis of their ability to improve the keeping quality and safety of many food products. The principal factors which contribute to this inhibition are presented in Table 9.5. By far the most important are the production of lactic and acetic acids and the consequent decrease in pH.
Bacteriocins are bactericidal peptides or proteins which are usually active against species closely related to the producing organism. Production of bacteriocins by lactic acid bacteria has been extensively studied in recent years and a number have been described.
Interest in them stems from the fact that they are produced by food- grade organisms and could therefore be regarded as ‘natural’ and hence more acceptable as food preservatives. A number of promising candidates have been found but many others have a spectrum of activity which is too limited to be of any practical utility.
The only bacteriocin to find application in the food industry to-date is nisin, produced by certain strains of Lactococcus lactis. In the UK and some other countries it has been used as a food preservative since the early 1950s, although USFDA approval was granted more recently, in 1988.
It has a relatively broad spectrum of activity against Gram-positive bacteria and has also been shown to be active against some Gram-negatives when their outer membrane has been damaged by thermal shock or treatment with a chelating agent such as EDTA.
Bacterial spores are particularly sensitive and its principal application has been to inhibit their outgrowth in products such as processed cheeses and canned foods. In vegetative bacteria it has been shown to act by creating pores in the plasma membrane through which there is a leakage of cytoplasmic components and a breakdown of the trans-membrane potential.
Nisin is a polypeptide containing 34 amino acids and is remarkably heat stable at acid pH. It belongs to a group of antibiotics known as lantibiotics, most of which are produced by non-lactic acid bacteria and are characterized by the possession of unusual amino acids such as lanthionine (3,3′-thiodialanine) and β-methyl lanthionine (Figure 9.2).
These are produced by a series of post translational modifications to a pre-pro-peptide which is then cleaved to remove a leader peptide. Production of many bacteriocins appears to be a plasmid-encoded function but the gene coding for nisin has been cloned and sequenced from both chromosomal and plasmid DNA.
Introduction of the ability to produce nisin into a chosen starter organism may prove useful in some fermented foods where competition from other Gram-positives needs to be controlled, although this is not desirable in cheese- making where nisin production could inhibit the lactobacilli that contribute to cheese maturation.
Hydrogen peroxide is well known for its antimicrobial properties. Since lactic acid bacteria possess a number of flavoprotein oxidases but lack the degradative enzyme catalase, they produce hydrogen peroxide in the presence of oxygen. This will confer some competitive advantage as they have been shown to be less sensitive to its effects than some other bacteria.
Accumulation of hydrogen peroxide has been demonstrated in some fermented foods but its effects are, in general, likely to be slight. Lactic acid fermentations are essentially anaerobic processes so hydrogen peroxide formation will be limited by the amount of oxygen dissolved in the substrate at the start of fermentation.
It may be, however, that at this critical initial stage of a fermentation hydrogen peroxide production provides an important additional selective advantage. In milk, hydrogen peroxide is also known to potentiate the lacto-peroxidase antimicrobial system.
Heterofermentative LAB produce ethanol, another well-established antimicrobial. It may make some contribution to the inhibition of competitors, although its concentration in lactic fermented products is generally low.
There are a number of other factors which may, like ethanol, give LAB a selective advantage in some situations. In most cases however their contribution is likely to be negligible, particularly when compared to the ability of LAB to produce lactic acid in quantities up to around 100 milli-molar and a pH in the range 3.5 to 4.5.
2. Health-promoting Effects of Lactic Acid Bacteria:
Fermented foods have long had a reputation for being positively beneficial to human health in a way that ordinary foods are not. Ilya Metchnikoff, the Russian founder of the theory of phagocytic immunity, was an early advocate of this idea based on his theories on disharmonies in nature.
He held that the human colon was one such disharmony since intestinal putrefaction by colonic bacteria produced toxins which shorten life. One solution to this which he advocated in his book ‘The Prolongation of Life’, published in 1908, was the consumption of substantial amounts of acidic foods particularly yoghurt.
He thought that the antimicrobial activity of the lactic acid bacteria in these products would inhibit intestinal bacteria in the same way they inhibit putrefaction in foods and attributed the apparent longevity of Bulgarian peasants to their consumption of yoghurt.
Since then a number of claims have been made for lactic acid bacteria, particularly in association with fermented milks (Table 9.6). So much so, that live cultures of lactic acid bacteria (and some others such as Bifidobacterium spp.) consumed in foods are frequently termed ‘probiotics’ (Greek: for life).
Much of the evidence available on these putative benefits is however inadequate or contradictory at present, and many remain rather ill defined.
Several studies have shown improved nutritional value in grains as a result of lactic fermentation, principally through increasing the content of essential amino acids. Such improvements however may be of only marginal importance to populations with a varied and well balanced diet.
It has also been reported that fermentation of plant products reduces levels of anti-nutritional factors which they may contain such as cyanogenic glycosides and phytic acid, although this effect is often the result of other aspects of the process such as soaking or crushing rather than microbial action. Some have claimed that fermentation of milks increases the bioavailability of minerals, although this is disputed.
One area where there is good evidence for a beneficial effect is in the ability of fermented milks to alleviate the condition known as lactose intolerance. All human infants possess the enzyme lactase (β-galactosidase) which hydrolyses the milk sugar lactose into glucose and galactose which are then absorbed in the small intestine.
In the absence of this enzyme when milk is consumed, the lactose is not digested but passes to the colon where it is attacked by the large resident population of lactose-fermenting organisms producing abdominal discomfort, flatulence and diarrhoea. Only people of north European origin and some isolated African and Indian communities maintain high levels of gut β-galactosidase throughout life.
In most of the world’s population it is lost during childhood and this precludes the consumption of milk and its associated nutritional benefits. If however lactase- deficient individuals take milk in a fermented form such as yoghurt, these adverse effects are less severe or absent.
This is not simply a result of reduced levels of lactose in the product since many yoghurts are fortified with milk solids so that they have lactose contents equivalent to fresh milk. It appears to be due to the presence of β-galactosidase in viable starter organisms, as pasteurized yoghurts show no beneficial effect.
In the gut, the ingested cells become more permeable in the presence of bile and this allows them to assist the body in the hydrolysis of lactose. The protective role of the gut’s microflora has been discussed already and there is evidence that ingested lactic acid bacteria can contribute to this.
Yoghurt has been shown to have a strong inhibitory effect on the growth of coliform bacteria in the stomach and duodenum of piglets and studies of human infants with diarrhoea have shown that the duration of illness was shorter in those groups given yoghurt than in control groups.
However, the usual starter organisms in yoghurt, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus are not bile tolerant and do not colonize the gut. They will persist in the alimentary tract and be shed in the stools only as long as they are being ingested, so that any improving effect is likely to be transient.
Recently attention has focused on lactic acid bacteria such as Lactobacillus acidophilus and bifidobacteria such as Bifidobacterium longum which can colonize the gut and these organisms have been included in yoghurts and other fermented milks.
Pathogen inhibition in vivo by LAB unable to colonize the gut must be by mechanisms broadly similar to those which apply in vitro. With those organisms able to colonize the gut, the masking of potential attachment sites in the gut may also be involved.
Lactic acid bacteria have been reported to stimulate the immune system and various studies have described their ability to activate macrophages and lymphocytes, improve levels of immunoglobulin A (IgA) and the production of gamma interferon. These effects may contribute to a host’s resistance to pathogens and to the anti-tumour activity noted for LAB, mainly Lactobacillus acidophilus, in some animal models.
An additional or alternative possible mechanism proposed for the anti-tumour effect is the observed reduction in activity of enzymes such as β- glucuronidase, azoreductase and nitro-reductase in faecal material when LAB are ingested.
These enzymes, produced by components of the intestinal flora, can convert pro-carcinogens to carcinogens in the gut and their decreased activity is probably due to inhibition of the producing organisms by LAB.
High levels of serum cholesterol are established as a predisposing factor for coronary heart disease. It has been suggested that consumption of fermented milks has a hypocholestaemic action and some have suggested a variety of mechanisms by which this can occur. The evidence is however weak at present and this area requires further study.
3. The Malo-lactic Fermentation:
LAB can de-carboxylate L -malic acid to produce L-lactate in a reaction known as the malo-lactic fermentation (Figure 9.3). This process is particularly associated with wines, where malic acid can form up to half the total acid, and its effect is to reduce substantially a wine’s acidity.
It is particularly encouraged in wines from cool regions which tend to have a naturally high acidity and, although less desirable in wines from warmer regions, it is often promoted to provide bacteriological stability to the bottled product. It may also modify and improve the body and flavour of a wine.
A natural malo-lactic fermentation can be encouraged by refraining from sulfiting the new wine and leaving it on the yeast lees (sediment) for longer than usual. Commercial starter cultures are also available usually consisting of strains of Leuconostoc oenos.
Until recently it was unclear how LAB derive any benefit from performing this reaction. Substrate-level phosphorylation does not occur (it is not therefore, strictly speaking, correct to call it a fermentation) and the free energy of the decarboxylation reaction is low.
It now seems that the reaction conserves energy through a proton motive force generated across the cell membrane by the transport of malate, lactate and protons.