Lactic Acid Starter Culture


Table of Contents

  • Lactic acid bacteria (LAB) serve multiple purposes in the culinary, agricultural, and therapeutic industries.
  • Using LAB in food fermentation is one of the oldest known methods of food preservation. Around 8,000 to 10,000 years ago, fermented milk products, such as yoghurt and cheese, were introduced into the human diet.
  • Prior to the 20th century, food fermentation was unregulated; however, the discovery and classification of LAB have altered perceptions around food fermentation.
  • Properties like as nutritional, environmental, and adhesional modifications have enabled LAB to adapt to and thrive in a variety of habitats, including food matrices such dairy products, meats, vegetables, and sourdough bread.
  • LAB are also prevalent occupants of human mucosal surfaces, including the mouth cavity, vagina, and gastrointestinal system.
  • Numerous useful molecules, including as organic acids, polyols, exopolysaccharides, and antibacterial compounds, are produced by metabolic processes.
  • LAB are categorised as Gram-positive bacteria with low Guanine + Cytosine (G + C) concentration, acid tolerance, non-motility, non-spore formation, and rod- or cocci-shaped cells.
  • The primary purpose of LAB is to make lactic acid, which acidifies the meal. Therefore, the primary use of LAB is as starter cultures in the food industry, which produces a vast array of fermented dairy, meat, fish, fruit, vegetable, and cereal products.
  • LAB also contribute to the flavour, texture, and nutritional value of fermented foods via the production of aroma components and their use as adjunct cultures; the production or degradation of exopolysaccharides, lipids, and proteins; the production of nutritional components such as vitamins; and the promotion of therapeutic effects and their use as probiotics.
  • In addition, they suppress spoilage and harmful microorganisms and are therefore utilised as bioprotective cultures.

Lactic acid bacteria

  • Early bacteriologists employed the term “Lactic Acid Bacteria” to refer to the microorganisms that promptly ruined ordinary lactic acid fermented meals.
  • In developed nations, the vast majority of lactic acid fermentation has been accumulated in dairy and vegetable products, whereas in developing nations, and especially in Africa, lactic acid fermentation dominates all indigenous processing of cereals such as maize, sorghum millet, and root crops such as cassava.
  • Generally, lactic acid bacteria (LAB) are utilised in the production of fermented dairy products. However, Propionibacterium shermanii and Bifidobacterium spp., which are not lactic acid bacteria but do create lactic acid, are also utilised.
  • In addition, Brevibacterium linens, which is responsible for the flavour of Limburger cheese, and moulds (Penicillium species) are employed to produce Camembert, Roquefort, and Stilton cheeses.
  • Throughout the years, lactic acid fermentation methods have persisted in Africa due to the following advantages of this technology:
    • It is a domestic gadget that enhances food safety.
    • In Africa, it serves as a low-cost technique of food preservation.
    • It enhances the nutritional value and digestibility of dietary raw materials in Africa. In addition, lactic acid fermentations have endured due to traditional beliefs, the product’s flavour, appearance, and shelf life.
  • Currently, LAB is being utilised to produce food diversity by modifying the flavour, texture, and appearance of raw materials.
  • The sour aromatic flavours given by lactic acid fermentation are desirable characteristics in fermented items and lend the products a natural appearance. It has also been shown that lactic acid bacteria colonise the human intestinal mucosa, resulting in a positive impact.
  • Currently, there are sixteen genera of (LAB). Recent research has addressed the importance of many species of the genera Enterococcus, Lactobacillus, Lactococcus, Oenococcus, Oenococcus, Ped iococcus, Streptococcus, and Tetragenococcus in food fermentation.
  • The composition of these microorganisms varies from one product to the next. Consequently, numerous forms of fermented milk have evolved. Variables include milk heat treatment, fermentation temperature, inoculum concentration, and milk concentration.
  • According to these conditions, various forms of lactic acid bacteria become predominate, e.g., those that produce various flavour components. As beginning cultures, many different genera of LAB are used. The categories include:

Genera Leuconostocs

  • The original taxonomy of bacteria placed the genus Leuconostoc close to Streptococcus, as it was mostly based on morphology, albeit in a separate genus as the “heterofermentative cocci,” also known as the betacocci by Orla- Jensen.
  • They create D (-) lactate from glucose, in contrast to L (+) lactate produced by lactococci and DL-lactate produced by heterofermentative lactobacilli, with whom they share many properties.
  • Leuconostoc is the most common LAB on plants, with Leuconostoc mesenteroides subsp. mesenteroides being the most prevalent isolate. All Leuconostocs species can convert glucose into lactic acid, carbon dioxide, and aromatic chemicals (ethanol and acetic acid).
  • Typically, these microorganisms are combined with lactic acid bacteria (LAB) in multiple or mixed strain cheese starting cultures to develop flavour components. Leuconostoc cremoris, Leuconostoc citrovorum, Leuconostoc dextranicum.
  • Leuconostocs species contribute significantly to the flavour of fermented dairy products. The importance of Leuconostoc mesenteroides subsp. cremoris and Lecon. lactis in starter cultures is widely acknowledged.
  • Leuconostocs, unlike lactococci, are hetrofermentative and produce carbon dioxide from glucose and typically fructose.
  • While the creation of carbon dioxide is unwanted in Cheddar cheese, it is good in some varieties, such as Emmental.
  • On microscopic examination, leuconostocs often appear as Gram-positive cocci with a similar size and shape to lactococci (occurring in pairs and typically in short chains).
  • Since leuconostocs develop on Rogosa agar, there is a tendency to think that these cultures are contaminated, for example with lactobacilli, if tiny rods are present.
  • Leuconostocs, in contrast to lactococci, do not create ammonia from arginine and instead produce the D isomer of lactic acid. Leuconostocs, with a few exceptions, develop poorly in milk and are incapable of decreasing litmus prior to coagulation in litmus milk media.
  • Time-consuming and laborious, isolating and identifying leuconostocs in starters requires the use of Rogosa agar to acquire initial isolates, according to the author. Fermentation of carbohydrates and identification of the lactic acid isomer are useful components of an identification protocol.

Genera Streptococcus

  • Due to their link with a significant range of human and animal diseases, streptococci were among the first bacteria to be identified by microbiologists.
  • The genus Streptococcus was initially described based on morphological, serological, physiological, and biochemical properties, and it included a wide range of organisms, such as the extremely dangerous bacteria S. preumoniae, S. pyogenes, and S. agalactiae; the intestinal group D streptococci. S. faecalis and S. faecium, as well as the economically significant group N starting bacteria S. cremoris and S. lactis.
  • Streptococcus members are Gram-positive organisms that typically form pairs or chains.
  • In 1937, Sherman split the genus based on physiological and growth features, in particular temperature constraints on growth. Sherman identifies four categories: (1) pyogenic, (2) viridans, (3) enterococcus, and (4) lactic.
  • Relationships between species have been demonstrated to overlap, rendering this classification rather obsolete.
  • The streptococcus is categorised as a thermophile that grows at temperatures of 45°C or higher and is commonly utilised in the manufacturing of yoghurt and some cheeses, such as Mozzarella.
  • The only species of Streptococcus detected in starter cultures is Streptococcus thermophilus. It has recently been utilised extensively in the manufacturing of Cheddar cheese.
  • Along with lactococci, it is a component of certain DVI/DVS cultures that generates acid fast upon scalding. Its integration into Cheddar-cultures also reduces the manufacturing costs of DVI/DVS cultures and helps to manage retail prices.
  • Temperatures around 15 degrees Fahrenheit inhibit plant growth. Cocci are cells of Streptococcus thermophilus and rods are cells of Lactobacillus delbrueckii subsp. bulgaricus in the making of yoghurt.
  • Strains of Streptococcus thermophilus, like lactococci and many leuconostocs, are catalase-negative, coccus-shaped, and occur in pairs and chains. In general, most strains generate lengthy chains.
  • From glucose, only L-lactic acid and no carbon dioxide are generated. Some strains produce urease and are capable of generating CO2 from urea.
  • Due to the fact that Streptococcus thermophilus and Streptococcus thermophilus-like organisms can thrive in the regeneration section of pasteurisers, cheese may occasionally have elevated quantities.
  • Strains that produce urease have the ability to cause cheese to separate. In addition, the inability of numerous strains to digest galactose can result in cheese containing significant concentrations of a fermentable starch that could be exploited by NSLAB for gas production.
  • The probable relationship with Streptococcus thermophilus should be evaluated when analysing instances of open texture or obvious gas production in cheese.
  • NSLAB and Str. thermophilus-like organisms that had grown to high cell densities in the regeneration section of pasteurizers are most likely to blame for the intermittent problems of excessive early acidification experienced by mozzarella manufacturers utilising extended production runs with pasteurised milk. The capacity of various strains to utilise galactose varies.
  • Utilizing non-galactose fermenting strains will result in products with elevated quantities of this lowering sugar. Due to the fact that galactose and other reducing sugars react with amino acids in the Maillard reaction, it is customary to choose only galactose-utilizing strains to limit the likelihood of unfavourable colour changes in heated products.
  • DNA has revealed the presence of Streptococcus salivarius, which is often found in saliva. DNA hybridization suggests that it is similar to Streptococcus thermophilus. In this manner, Streptococcus thermophilus was classified as a subspecies of Streptococcus salivarius for several years.
  • However, it is now considered that Streptococcus thermophiles, despite their similarities, are sufficiently different to warrant species identification. Streptococcus thermophilus is sensitive to low salt concentrations, specifically sodium chloride values of approximately 2%.
  • This sensitivity is essential for the use of DVI/DVS cultures in Cheddar and other similar cheeses. Once the salt concentration in moisture hits 2 to 3 percent, lactose consumption and acid generation cease.
  • M17 medium, which is widely used in lactococci investigations as a component of concentrates, is not optimal for the development of specific strains unless its glycerophosphate concentration is reduced.
  • Large parts of the isolates from pasteurizers are much less sensitive to salt and generally grow well on M17 agar.-like strains are significantly less sensitive to salt and typically grow well on M17 agar.

Genera Lactobacillus

  • The Lactobacillus genus contains a significant number of rod-shaped, Gram-positive, catalase-negative bacteria. There are species that are homofermentative and species that are hetrofermentative.
  • Some species primarily create L-lactate from glucose, while others produce D-lactate. Due to the fact that racemase is an isomerase enzyme and some strains demonstrate substantial racemase activity, D/L lactic acid is also generated. Strains may also display coccoid morphology, which can lead to confusion with leuconostocs and possibly lactococci.
  • Yogurt and mozzarella cheese are made with lactobacilli as starter cultures. In addition, they are used as starter subordinates to accelerate the ripening of Cheddar and similar cheeses, to reduce the rate of bitterness intensity, and as probiotics in yoghurt-like products.
  • Along with Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus is commonly employed as a starter in yoghurt manufacturing. This subspecies is homofermentative, produces around 2% w/v lactic acid in milk, has an optimal temperature of 42°C, and flourishes at 45°C and higher temperatures.
  • It cannot develop in low salt concentrations and is susceptible to bile salts. Lactobacillus acidophilus, which is normally found in the digestive tract, is not often used as a starter; instead, it is widely employed as a probiotic.
  • This bacteria is homofermentative, producing large amounts of D-lactic acid in milk, has an optimal temperature of 37 degrees Celsius, and is reasonably tolerant to oxygen compared to Bifidobacterium species that are usually used in conjunction with this organism.
  • At temperatures below 20 °C, growth is minimal, and the vast majority of strains do not develop at 15 °C. Because Lactobacillus acidophilus produces D-lactate, its use in newborn feeding has raised some concerns.
  • Lactobacillus casei is a common resident of the small intestine and is bile-resistant. It is employed as a probiotic despite its presence in some starter cultures and as one of several non-starter lactic acid bacteria (NSLAB) often found in Cheddar cheese.
  • Rogosa agar is commonly used as a lactobacilli isolation medium. Lactobacillus helveticus is commonly combined with other thermophilic lactic acid bacteria in the manufacturing of fermented milk products such as Emmental cheese, Mozzarella, and yoghurt.
  • In addition to Lb. delbrueckii subsp. bulgaricus, Lactobacillus helveticus uses galactose, which can be advantageous if products without reducing sugars are required.
  • Since numerous strains have been shown to have proline-iminopeptidase-like activity, Lactobacillus helveticus has been utilised to manufacture modified “Cheddar-type cheese” with some of the “sweetness” characteristics of Swiss cheeses such as Emmental.
  • Recently, designated strains have been used as starting subordinates to reduce bitterness in a variety of cheeses, to improve or perhaps accelerate maturation.
  • Due to peptidase action on starter-derived hydrophobic peptides, bitterness is diminished. The species is homofermentative and produces high D/L lactic acid concentrations in milk.
  • Many strains develop around 45°C, despite the fact that lower temperatures 42-43°C generally yield greater recoveries when employing selective media such as Rogosa or modified MRS agars for counting. display no or minimal growth at 15 °C (some unusual strains may require many weeks to grow at 15 °C or below).

Genera Lactococcus

  • These bacteria were categorised as members of the genus Streptococcus and given the name lactic streptococci.
  • By their unique reactivity with Group N antiserum and their tolerance to cold, salt, and dyes, they were distinguished from other streptococci, some of which are pathogens.
  • It is currently understood that serotyping lactic LAB is of little use in species separation; strains of the same species may react differently with different sera, and some strains may lack group antigen.
  • The ovoid shape of lactococci can be difficult to understand since the cells are sometimes elongated along the plane of chain formation, causing some lactococci to be misclassified as lactobacilli.
  • The lactococci’s most likely habitat is dairy products. They are non-motile, coccus-shaped, homofermentative bacteria that thrive at 10°C but not 45°C and convert glucose to L-lactic acid. Due to their efficient uptake and fermentation of lactose, several strains become well-adapted to developing in milk.
  • Strains able to consume citrate and produce diacetyl were classified as S. diacetylactis, and then as Lc. lactis subsp. lactis. Lactococci are widely used and have the longest tradition in industrial starting culture technique.
  • Strains of Lc. lactis produce nisin, a bacteriocin with a reasonably broad spectrum of activity against gram-positive bacteria, including Clostridium botulinum and its spores. Using the system described for lactic streptococci, it is possible to differentiate lactococci by species or biovariant.
  • Lactococci will not cultivate on Rogosa agar. There are available differential, but not selective, media that can be utilised for quality control and strain separation. The medium created by Reddy et al. in 1972, Reddys’ Differential Agar, is still useful.
  • This medium includes the unique components lactose, calcium citrate, L-arginine, and pH indicator bromcresol purple. This indicator is yellow under acidic conditions and blue/purple under alkaline conditions.
  • Lactococcus lactis subsp. cremoris forms yellow colonies due to acid generation from lactose. While creating acid, Lactococcus lactis subsp. lactis also creates ammonia from arginine.
  • The ammonia neutralises the acid and ultimately causes an alkaline reaction, resulting in colonies with a blue/purple hue. Also producing a blue/purple colony is Lactococcus lactis subsp. lactis biovar. diacetylactis.
  • Due to the fact that some strains of Lactococcus lactis subsp. lactis show only minimal arginase activity, it may be possible to detect these strains by the use of better streaking techniques on this medium.

Metabolic activity of lactic acid bacteria

  • Lactic acid bacteria (LAB) are typically mesophilic, but can grow at temperatures as low as 5 oC and as high as 45 oC. Also, whereas the majority of bacteria thrive between pH 4.0 and 4.5, some are also active at pH 9.6 and pH 3.2.
  • Strains are often weakly proteolytic and lipolytic, and their growth requires amino acids, purine and pyrimidine bases, and B vitamins.
  • Since they lack functional electron transport chains and a functional Krebs cycle, all LAB get their energy through substrate-level phosphorylation.
  • The lactic acid generated may be L (+), D(-) or a combination of both. Note that D(-) lactic acid is contraindicated for newborns and young children (WHO, 1974).
  • The routes for hexoses separate homofermentative and heterofermentative lactic acid bacteria.
  • Homofermenters, including Pediococcus, Streptococcus, Lactococcus, and some Lactobacillus, create lactic acid as the sole byproduct of glucose fermentation.
  • This may change, however, under different development conditions especially when the substrate is pentose.
  • Homofermenters utilise the Glycolysis pathway to produce two moles of lactate per mole of glucose and nearly twice the amount of energy per mole of glucose as heterofermenters.
  • Through the hexose monophosate or pentose route, heterofermenters such as Weisella and Leuconostoc and certain Lactobacillus create equal amounts of lactate, CO2, and ethanol from glucose.

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