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Lactic Acid Bacteria

Lactobacillus

Lactobacillus acidophilus  The bacterium is a member of the normal flora of humans,  found in the oral cavity, the small intestine, and the vaginal epithelium, where it is thought to play a beneficial role. The organism is generally the first bacterium listed as present in probiotic concoctions.

from: textbook of Bacteriology. "The Good, the Bad, and the Deadly". (SCIENCE Magazine - Vol 304: p. 1421). 

Lactic Acid Bacteria (LAB) are Gram-positive, non-sporeforming cocci, coccobacilli or rods with a DNA base composition of less than 53mol% G+C. They generally are non respiratory and lack catalase. They ferment glucose primarily to lactic acid, or to lactic acid, CO2 and ethanol. All LAB grow anaerobically, but unlike most anaerobes, they grow in the presence of O2 as "aerotolerant anaerobes". Although they lack catalase, they possess superoxide dismutase and have alternative means to detoxify peroxide radicals, generally through peroxidase enzymes.

Although many genera of bacteria produce lactic acid as a primary or secondary end-product of fermentation, the term Lactic Acid Bacteria is conventionally reserved for genera in the order Lactobacillales, which includes Lactobacillus, Leuconostoc, Pediococcus, Lactococcus and Streptococcus, in addition to Carnobacterium, Enterococcus, Oenococcus, Tetragenococcus, Vagococcus, and Weisella.

Because they obtain energy only from the metabolism of sugars, lactic acid bacteria are restricted to environments in which sugars are present. They have limited biosynthetic ability, having evolved in environments that are rich in amino acids, vitamins, purines and pyrimidines, so they must be cultivated in complex media that fulfill all their nutritional requirements. Most are free-living or live in beneficial or harmless associations with animals, although some are opportunistic pathogens. They are found in milk and milk products and in decaying plant materials. They are normal flora of humans in the oral cavity, the intestinal tract and the vagina, where they play a beneficial role. 

A few LAB are pathogenic for animals, most notably some members of the genusStreptococcus. In humans, Streptococcus pyogenes is a major cause of disease (strep throat, pneumonia, and other pyogenic infections, scarlet fever and other toxemias), Streptococcus pneumoniae causes lobar pneumonia, otitis media and meningitis; some viridans and nonhemolytic oral streptococci play a role in dental caries and may be an insidious cause of endocarditis. The pathogenic streptococci are dealt with elsewhere in the text. This chapter deals primarily with LAB in association with food and dairy microbiology, to a lesser extent with LAB as beneficial components of the human normal flora and probiotics.

Lactic acid bacteria are among the most important groups of microorganisms used in food fermentations. They contribute to the taste and texture of fermented products and inhibit food spoilage bacteria by producing growth-inhibiting substances and large amounts of lactic acid. As agents of fermentation LAB are involved in making yogurt, cheese, cultured butter, sour cream, sausage, cucumber pickles, olives and sauerkraut, but some species may spoil beer, wine and processed meats.


Differential characteristics of lactic acid bacteria based on morphology and physiology
Genus

Lacto-
bacillus

Entero-
coccus

Lacto-
coccus

Leuconostoc

Pedio-
coccus

Strepto-
coccus

Characteristic






Morphology

rods

cocci

cocci

cocci

cocci in
tetrads

cocci

CO2 from glucose*

±

+

Growth






at 10°C

±

+

+

+

±

at 45°C

±

+

±

±

in 6.5% NaCl

±

+

±

±

at pH 4.4

±

+

±

±

+

at pH 9.6

+

Lactic acid 
configuration

D, L, DL

L

L

D

L, DL

L

+ positive; - negative; ± varies between species 
*
test for homo- or heterofermentation of glucose: - homofermentation 
+ heterofermentation

Metabolism

The essential feature of LAB metabolism is efficient carbohydrate fermentation coupled to substrate-level phosphorylation. Adenosine triphosphate (ATP) generated is subsequently used for biosynthesis. LAB as a group exhibit an enormous capacity to degrade different carbohydrates and related compounds. Generally, the predominant end product is lactic acid (>50% of sugar carbon). However, LAB adapt to various conditions and change their metabolism accordingly. This may lead to significantly different end-product patterns.

Based on sugar fermentation patterns, two broad metabolic categories of LAB exist: homofermentative and heterofermentative. The first category, homofermentative LAB, includes some lactobacilli and most species of enterococci, lactococci, pediococci, streptococci, tetragenococci, and vagococci, that ferment hexoses by the Embden-Meyerhof (E-M) pathway. The second category, heterofermentative LAB, includes leuconostocs, some lactobacilli, oenococci, and weissella species. The apparent difference on the enzyme level between these two categories is the presence or absence of the key cleavage enzymes of the E-M pathway (fructose 1,6-diphosphate) and the PK pathway (phosphoketolase).  


Homolactic Fermentation

Under conditions of excess glucose and limited oxygen, homolactic LAB catabolize one mole of glucose in the Embden-Meyerhof pathway to yield two moles of pyruvate. Intracellular redox balance is maintained through the oxidation of NADH, concomitant with pyruvate reduction to lactic acid. This process yields two moles of ATP per glucose consumed. Representative homolactic LAB genera includeLactobacillus, Lactococcus, Enterococcus, Streptococcus and Pediococcus species.

The transport and phosphorylation of sugars occur by (1) transport of free glucose and phosphorylation by an ATP-dependent hexose kinase (other sugars, such as mannose and fructose, enter the major pathways at the level of glucose-6-phosphate or fructose-6-phosphate after isomerization or phosphorylation or both); or (2) the phosphoenolypyruvate (PEP) sugar phosphotransferase system (PTS), in which PEP is the phosphoryl donor for the uptake of sugar. Some species of LAB use the PTS for transport of galactose only; others use the PTS for all sugars.



 


The pathway of homolactic acid fermentation in Lactic Acid Bacteria


Heterolactic Fermentation

Heterofermentative LAB utilize the phosphoketolase pathway (pentose phosphate pathway) to dissimilate sugars. One mole of glucose-6-phosphate is initially dehydrogenated to 6-phosphogluconate and subsequently decarboxylated to yield one mole of CO2. The resulting pentose-5-phosphate is cleaved into one mole glyceraldehyde phosphate (GAP) and one mole acetyl phosphate. GAP is further metabolized to lactate as in homofermentation, with the acetyl phosphate reduced to ethanol via acetyl-CoA and acetaldehyde intermediates. Theoretically, end- products (CO2, lactate and ethanol) are produced in equimolar quantities from the catabolism of one mole of glucose. Obligate heterofermentative LAB include Leuconostoc, Oenococcus, Weissella, and certain lactobacilli. 


The pathway of heterolactic acid fermentation in Lactic Acid Bacteria


Lactic acid bacteria have a very limited capacity to synthesize amino acids using inorganic nitrogen sources. They are therefore dependent on preformed amino acids being present in the growth medium as a source of nitrogen. The requirement for amino acids differs among species and strains within species. Some strains are prototrophic for most amino acids, whereas others may require 13–15 amino acids. Since the quantities of free amino acids present in their environment are not sufficient to support the growth of bacteria to a high cell density, they require a proteolytic system capable of hydrolyzing peptides and proteins in order to obtain essential amino acids. All dairy lactococci used for acidification of milk (e.g., in cheese manufacture) have proteolytic activity. The lactococcal proteolytic system consists of enzymes outside the cytoplasmic membrane, transport systems, and intracellular peptidases. The proteolytic activity of LAB contributes additionally to the development of the flavor, aroma and texture of fermented products. For many varieties of cheeses, such as Swiss and Cheddar, desirable "flavor tones" are derived by proteolysis.

Fermentation of Foods by Lactic Acid Bacteria

Many human foods are plants or animal products which have been fermented by lactic acid bacteria, since these bacteria possess properties that can benefit food production or conversion. The acidic and organoleptic properties of fermented foods result from the metabolic activities of these microorganisms. Foods such as ripened cheeses, fermented sausages, sauerkraut and pickles have not only a greatly extended shelf life compared to the raw materials from which they are derived, but also aroma and flavor characteristics contributed directly or indirectly by the fermenting organisms.

Fermented dairy products have been made for thousands of years, but only within the last century have the microbiological bases of these fermentations been elucidated. Lactic acid bacteria are the principal organisms involved in fermenting dairy products. Prior to the availability of starter cultures, milk fermentations relied on the LAB naturally present in raw milk. The first commercial starter cultures were unknown mixes of microbes from raw milk that were prepared in Denmark around end of the 19th century. In the 1930s and 40s, the idea of pure single-strain starter cultures evolved. 

Fermented dairy products are enjoying increased popularity as convenient, nutritious, stable, natural, and healthy foods. Lactic acid bacteria are the principal organisms involved in the manufacture of cheese, yogurt, buttermilk, cottage cheese, sour cream and cultured butter. In some fermented dairy products, additional bacteria, referred to as secondary microflora, are added to produce carbon dioxide, which influences the flavor and alters the texture of the final product.

Sausage is one of the oldest processed meat products. The writings of ancient Egyptians described the preservation of meat by salting and sun drying. The ancient Babylonians, Greeks, and Romans used sausage as a food source during times of war. Microorganisms were recognized as being important to the production of sausages about 1921. In the 1940s and 1950s, pure microbial starter cultures consisting of lactic acid bacteria became available but their use was not widespread until the early 1980s.

The fermentation of vegetables, a practice that originated in the Orient, has been used as a means of preserving food for more than 2,000 years. In the third century B.C., during the construction of the Great Wall of China, the Chinese produced fermented vegetables (cabbages, radishes, turnips, cucumbers, etc.) on a large scale. The most common fermented vegetables available in the United States are pickles, sauerkraut, and olives. Carrots, cauliflower, celery, okra, onions, and sweet and hot peppers also are sold as fermented vegetable products.

Generally LAB that are important in the fermentation of food products (dairy, meat, vegetables, fruits, and beverages), include only certain species of the genera Lactobacillus, Lactococcus, Streptococcus, Leuconostoc and Pediococcus.  Some of these species are also members of normal flora of the mouth, intestine, and vagina of mammals.


Involvement of lactic acid bacteria in the manufacture of fermented dairy products

Product

Principal acid producers

Secondary microflora

Cheese

Colby, cheddar, cottage, cream

Lactococcus lactissubsp. cremoris

None

Lactococcus lactissubsp. lactis

 

Blue

Lactococcus lactissubsp. cremoris

Cit+ Lactococcus lactissubsp. lactis Penicillium roqueforti

Lactococcus lactissubsp. lactis

 

Mozzarella, provolone, Romano, parmesan

Streptococcus thermophilus

None

Lactobacillus delbrueckii  subsp. bulgaricus 

 

Lactobacillus helveticus

 

Swiss

Streptococcus thermophilus

Propionibacterium freudenreichii subsp. shermanii

Lactobacillus delbrueckii subsp. bulgaricus 

 

Lactobacillus helveticus

 

Fermented milk

Yogurt

Streptococcus thermophilus

None

Lactobacillus delbrueckii subsp. bulgaricus 

 

Buttermilk

Lactococcus lactissubsp. cremoris

Leuconostoc sp.  Cit+ Lactococcus lactis subsp. lactis 

Lactococcus lactissubsp. lactis

 

Sour cream

Lactococcus lactissubsp. cremoris

None

Lactococcus lactissubsp. lactis




Lactobacilli

Lactobacillus 
is very heterogeneous genus, encompassing species with a large variety of phenotypic, biochemical, and physiological properties.
Most species of lactobacilli are homofermentative, but some are heterofermentative. The genus has been divided into three major subgroups and over 70 species are recognized. Group I lactobacilli are obligately homofermentative and produce lactic acid as a major end product (>85%) from glucose. They are represented by L. delbrueckii and L. acidophilus. They grow at 45oC but not at 15oC. Group II, also homofermentative, grow at 15oC and show variable growth at 45oC. Represented by L. casei and L. plantarum, they can produce more oxidized fermentations (e.g. acetate) if O2 is present. Group III lactobacilli are heterofermentative. They produce lactic acid from glucose, along with CO2 and ethanol. Aldolase is absent and phosphoketolase is present. Representative species include L. fermentum, L. brevis and L. keferi.

Lactobacilli are often found in dairy products, and some species are used in the preparation of fermented milk products. For example L. delbrueckii subspbulgaricus are used in the preparation of yogurt; L. acidophilus is used in the preparation of acidophilus milk; L. helveticus, as well as L. delbrueckii subspbulgaricus, are used to make Swiss, Mozzarella, provolone, Romano, and parmesan cheeses. Other species are used in the production of sauerkraut, silage and pickles. The lactobacilli are usually more resistant to acidic conditions than are other LAB, being able to grow at pH values as low as 4. This enables them to continue to grow during natural lactic fermentations when the pH has dropped too low for other LAB to grow, so they are often responsible for the final stages of many lactic acid fermentations.

Many sausage fermentations include a Lactobacillus species. L. plantarum is used in starter cultures for the manufacture of summer sausage, pepperoni and salami.

The natural fermentation of cabbage to make sauerkraut involves L. brevis and L. plantarum in the final succession of microbe. The starter cultures for manufacture of cucumber pickles includes L. brevis and L. plantarumL. plantarum is the most essential species in pickle production, as it is for sauerkraut. Like cucumbers, olives are fermented under conditions similar to those of other vegetable products. The microbial population responsible for the fermentation of olives differs from that of sauerkraut and pickles mainly because the higher salt concentration of the brine prevents many salt-sensitive strains from growing and provides an advantage to salt-tolerant strains. LAB become prominent during the intermediate stage of fermentation. L. mesenteroides and P. cerevisiae are the first lactics to become predominant, followed by lactobacilli, with L. plantarum and L. brevis being the most important.


Streptococci and Lactococci

Note on Streptococcal Classification. Since 1985, members of the diverse genus Streptococcus have been reclassified into Lactococcus, Vagococcus, Enterococcus, and Streptococcus, based on biochemical characteristics, as well as ssRNA analysis. Historically, streptococci were segregated into serological groups based on the presence of specific carbohydrate antigens. Antigenic groups, or Lancefield groups (named for Rebecca Lancefield, a pioneer in Streptococcus taxonomy), are designated by letters A through O. Lancefield groups have proven to correlate well with the current taxonomic definitions. The beta-hemolytic streptococci found in humans contain the group A antigen, while "fecal streptococci" (enterococci) contain the group D antigen. Group B streptococci, usually found in animals, are a cause of mastitis in cows, and have been implicated in human infections.  "Lactic streptococci" (streptococci and lactococci) contain the group N antigen and are nonpathogenic. However, Lactococcus has been defined as a genus separate from Streptococcus. As lactic acid bacteria, Lactococcus lactis and Streptococcus thermophilus are the cornerstones of cheese manufacture.

Streptococcus thermophilus

Streptococcus thermophilus is an alpha-hemolytic species of the viridans group. The bacterium is found in milk and milk products. It is not a probiotic (it does not survive the stomach) and generally is used in the production of yogurt and the manufacture of several types of cheese, especially Italian and Swiss cheeses. The organism is a moderate thermophile with an optimal growth rate at 45 °C. Although S. thermophilus is closely related to other pathogenic streptococci (such as S. pneumoniae and S. pyogenes), S. thermophilus is classified as a non-pathogenic, alpha-hemolytic species that is part of the viridan's group. It is closely related to S. salivarius in the oral cavity.

Lactococcus lactis

Lactococcus
 is a genus of of LAB with five major species formerly classified as Group N streptococci. The type species for the genus is L. lactis, which has two subspecies, lactis and cremoris. Lactococci differ from other lactic acid bacteria by their pHsalt and temperature tolerances for growth. 

Lactococcus lactis is critical for manufacturing cheeses such as Cheddar, cottage, cream, Camembert, Roquefort and Brie, as well as other dairy products like cultured butter, buttermilk, sour cream and kefir. The bacterium can be used in single strain starter cultures, or in mixed strain cultures with other lactic acid bacteria such as Lactobacillus and Streptococcus.

Bacteriocins and LAB

Class I bacteriocins or lantibiotics are small peptides containing the unusual dehydroamino acids and thioether amino acids lanthionin and 3-methyllanthionine, which are synthesized by Gram-positive bacteria during posttranslational modifications. These peptides are thought to attach to the membrane of target cells and, by an as yet unknown conformational rearrangement, lead to increased permeability and disruption of the membrane potential. There are two types of lantibiotics, types A and B. The lantibiotics produced by LAB all belong to type A, which are elongated screw-shaped peptides, whereas type B lantibiotics are mainly globular. Nisin produced by Lactococcus lactis ssp. lactis has been studied extensively. It has a broad spectrum of activity against Gram-positive bacteria. The primary target is believed to be the cell membrane. Unlike some other antimicrobial peptides, nisin does not need a receptor for its interaction with the cell membrane; however, the presence of a membrane potential is required.


Starter cultures


Starter cultures consisting of lactic acid bacteria are added at the beginning of the cheesemaking process. Lactic acid bacteria are essential for manufacture of cheese, yogurt, sour cream, cultured butter and most fermented milk products.

Starter cultures play an essential part in the manufacture of fermented dairy products. They produce the lactic acid that coagulates milk and they contribute to texture, moisture content, freedom from pathogenic microorganism, and taste of the product. The rate of acid production is critical in the manufacture of certain products, e.g. Cheddar cheese. Depending on the product, especially in mechanized cheese production units, starters may also be required to produce acid at a consistently fast rate through the manufacturing period each day and every day. The negative redox potential created by starter growth in cheese also aids in preservation and the development of flavor in Cheddar and similar cheeses. Additionally antibiotic substances, now referred to as bacteriocins, produced by starters, e.g. nisin, may also have a role in preservation. 

Ecology of starter bacteria 
Most starters in use to today have their origins as lactic acid bacteria naturally present as part of the contaminating microflora of milk. These bacteria probably came from vegetation in the case of lactococci or the intestinal tract in the case of bifidobacteria, enterococci and Lactobacillus acidophilus

Modern starter cultures developed from the practice of retaining small quantities of whey or cream from the successful manufacture of a fermented product on a previous day and using this as the inoculum or starter for the following day’s production. In the foods industry his practice has been referred to as "back-slopping".

Classification of starter  cultures 
While the microbes used in the manufacture of fermented dairy products are generally lactic acid bacteria, Propionibacterium shermanii and Bifidobacteriumspp. which are not lactic acid bacteria, are also used. In addition, other bacteria including Brevibacterium linens, responsible for the flavor of Limburger cheese; and molds (Penicillium species) are used in the manufacture of Camembert, Roquefort and blue cheeses.


Probiotics and Lactic Acid Bacteria

Probiotics are products designed to deliver potentially beneficial bacterial cells to the microbiotic ecosystem of humans and other animals. Strains of lactic acid bacteria are the most common microbes employed as probiotics, especially Lactobacillus and Bifidobacterium species, but lactococci, some enterococci and some streptococci are also included as probiotics.

Lactic acid-producing Bacteria Used as Probiotics

Lactobacillus 
Lactobacillus species are facultative anaerobes. They grow in the presence of O2, however, and may convert it to H2O or H2O2
Lactobacilli normally predominate in the small intestine, and they are known for their beneficial effects which may antagonize potential pathogens. Of the more than 100 Lactobacillus species, the following are commonly used probiotics:
L. acidophilus 

L. fermentum
L. paracasei 
L. brevis
L. gasseri
L. plantarum 
L. bulgaricus
L. helveticus
L. reuteri
L. casei
L. jensenii
L. rhamnosus
L. crispatus
L. johnsonii
L. salivarius

Bifidobacterium 
Bifidobacterium is not included in the traditional Lactic Acid Bacteria due to its genetic unrelatedness, but the bacterium has a habitat that overlaps with LAB, and it has a metabolism that produces lactic acid as a primary end-product
 of fermentation.  Bifidobacteria are strictly anaerobic and normally vie for predominance in the large intestine. Among 30 species, those recognized as probiotics include:
B. adolescentis
B. breve
B. longum
B. animalis
B. infantis
B. thermophilum
B. bifidum
B. lactis
 


Bifidobacterium longum. Bifidobacteria are an obligately anaerobic bacteria, not classified with the lactic acid bacteria, but which occupy similar habitats and produce lactic acid as a sole end-product. They are a prominant Gram-positve bacterium in the large intestine (colon). Bifidobacterium infantum is the predominant bacterium in the intestine of breast-fed infants because mother's milk contains a specific growth factor that enriches for the growth of the bacterium.


Streptococcus 
Streptococcus species are not typically associated with health benefits and some are highly pathogenic. However, one facultative anaerobic species, Streptococcus thermophilus, is known to promote health. It is one of the two primary species found in yogurt cultures, the other being L. bulgaricus

Enterococcus 
Found in a number of probiotic products, the facultative anaerobe Enterococcus faecium is invariably a component of the normal intestinal microbiota and is considered a beneficial microbe. However, E. faecium has evolved from a relatively nonpathogenic commensal bacterium to the third most common cause of hospital-acquired infections and now accounts for over 10% of enterococcal clinical isolates. Furthermore, it has developed extensive resistance to antibiotics, which it is capable of transferring to other bacteria.
 


The human body, primarily the gastrointestinal tract, is home to a large number of different species of bacteria, and it is likely we could not survive without their presence. Two of the most common bacteria that comprise the intestinal microbiota ("normal flora") are Lactobacillus acidophilus and Bifidobacterium bifidum. Hence, they are a main component of probiotics.

The indigenous bacteria of humans serve a wide range of functions, which include manufacture of some B vitamins and vitamin K, synthesis of some digestive enzymes (e.g. lactase), competition with pathogens for colonization sites, production of antibacterial and antifungal substances that protect against harmful organisms, production of chemicals that have been shown to be anti-carcinogenic, and stimulation of the development and activity of the immune system. 

The natural balance of the body’s bacteria can be upset by several factors such as certain medicines, antibiotics and steroids, increased acidity in the digestive system caused by stress, lack of sleep and poor diet, constipation or diarrhea, yeast overgrowth, fatigue, IBS and other intestinal conditions.

It has been been suggested, in a few cases proven, that one way to combat these conditions is by supplementation of the diet with probiotic bacteria in natural foods or artificial supplements.

Probiotics have been recommended or suggested for patients receiving radiation treatment, individuals who have recurrent thrush, vaginal yeast infections, or urinary tract infections, persons suffering from irritable bowel syndrome (IBS) or other bowel problems, for travelers abroad to protect against food poisoning and during any period where antibiotics may be taken.


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