Lactic Acid Bacteria; Definition, Classification and Characteristics

Over the past 150 years, nearly 50,000 scientific publications on lactic acid bacteria have appeared in SCI-indexed journals, including 3,453 articles published in 2023 alone (Web of Science, 2024). Today, thousands of scientists continue to study the subject intensively, and important discoveries are still being made.

Given that the global starter culture market, in which lactic acid bacteria play a central role, is worth billions, the importance of this group of microorganisms becomes clear.

Unfortunately, a definitive definition and family-level classification of lactic acid bacteria are difficult to establish. Recent taxonomic developments and accumulated genetic data suggest that they represent ancient Bacillus-like soil organisms that adapted to new, nutrient-rich ecological environments.

Although they lost many metabolic activities during adaptation, their fermentation systems, acid production and acid tolerance have enabled lactic acid bacteria to compete effectively with other microorganisms.

According to the biological classification;

LABs are members of;

• Bacteria kingdom,
• Firmicutes phylum,
• Bacilli class,
• Lactobacillales order.

The image below shows the traditional family and genus classification of these bacteria, together with some of their distinguishing characteristics.

Changes in the Classification of Lactic Acid Bacteria Since 2020

The image above reflects a classification of lactic acid bacteria that was used for many years. As genome sequencing advanced, it became clear that the species grouped under Lactobacillus were more diverse than their shared name suggested. Zheng and colleagues (2020) reorganized the former broad genus into 25 genera: the redefined Lactobacillus, the previously described Paralactobacillus, and 23 new genera proposed in their study.

These changes also affect the species names encountered later in this article. The species formerly known as Lactobacillus casei and L. rhamnosus are now called Lacticaseibacillus casei and Lacticaseibacillus rhamnosus, respectively. Likewise, L. plantarum became Lactiplantibacillus plantarum, L. brevis became Levilactobacillus brevis, and L. reuteri became Limosilactobacillus reuteri.

Lactobacillus kefir, found in kefir, was renamed Lentilactobacillus kefiri in the 2020 taxonomic revision. The two species used as examples in the discussion of genome size, L. sanfranciscensis and L. parakefiri, are now named Fructilactobacillus sanfranciscensis and Lentilactobacillus parakefiri. Other species, including Lactobacillus acidophilus, L. delbrueckii, L. helveticus, L. johnsonii, and Lactobacillus kefiranofaciens, remain in Lactobacillus. In other words, the genus name did not change for every species once placed there.

The revision went beyond genus names. Leuconostocaceae, shown as a separate family in the image, was incorporated into an expanded Lactobacillaceae in the 2020 classification. The image’s Lactobacillus row also represents the genus in its former broad sense. Its characteristics should therefore not be read as traits shared by every species now assigned to different genera.

A further change to the genus Lactococcus followed in 2021. Li and colleagues elevated Lactococcus lactis subsp. cremoris to species rank as Lactococcus cremoris. This is separate from Zheng and colleagues’ 2020 revision and does not change the names Lactococcus lactis subsp. lactis or Leuconostoc mesenteroides subsp. cremoris. These revisions explain why older scientific publications still use the previous names.

Former name	Current name
Lactobacillus casei	Lacticaseibacillus casei
Lactobacillus plantarum	Lactiplantibacillus plantarum
Lactobacillus rhamnosus	Lacticaseibacillus rhamnosus
Lactobacillus brevis	Levilactobacillus brevis
Lactobacillus reuteri	Limosilactobacillus reuteri
Lactobacillus kefir	Lentilactobacillus kefiri
Lactobacillus sanfranciscensis	Fructilactobacillus sanfranciscensis
Lactobacillus parakefiri	Lentilactobacillus parakefiri
Lactococcus lactis subsp. cremoris	Lactococcus cremoris

Sources: Zheng et al. (2020), https://doi.org/10.1099/ijsem.0.004107; Li et al. (2021), https://doi.org/10.1099/ijsem.0.004727.

Some Characteristics of Lactic Acid Bacteria

Lactic acid bacteria from the genera Carnobacterium, Enterococcus, Lactobacillus (dairy, meat, vegetable and cereal products), Lactococcus (dairy products), Leuconostoc (vegetables and dairy products), Oenococcus (wine), Pediococcus (vegetables and meat products), Streptococcus and Weissella (dairy products) are commonly used as starter cultures in the food industry.

Lactic acid bacteria are generally non-spore-forming, heterotrophic, anaerobic, microaerophilic, Gr (+), immobile, rod or spherical-shaped bacteria with relatively high acid resistance. The G+C ratio in their DNA is below 55%. LAB genomes are characterized by small sizes ranging from 1.23 Mb (Lactobacillus sanfranciscensis) to 4.91 Mb (Lactobacillus parakefiri).

By fermenting carbohydrates in a homofermentative or heterofermentative way, they can produce acetic acid, formic acid, ethanol and CO₂, although the main product is lactic acid. According to optimum growth temperatures, LAB can be divided into mesophilic and thermophilic.

Mesophilic LAB can grow optimally at 20-30^oC, while thermophilic LAB can grow optimally between 30-45^oC. This is probably why products from Western and Northern European countries have predominantly mesophilic LABs, while products from subtropical countries have thermophilic LAB.

Milk and plants (vegetables, fruits and grains) form the natural habitats of lactic acid bacteria. However, they can also be found naturally on warm-blooded animals’ mucosal surfaces, such as the oral cavity, intestine and colon.

A LAB strain can be isolated from many different environments, including humans, plants, animals and dairy products. This reflects the wide distribution and strong adaptability of LAB. Despite their GRAS (Generally Recognized As Safe) status, increasing antibiotic resistance and the potential transfer of this resistance to pathogenic bacteria pose a risk that should not be overlooked.

Since lactic acid bacteria lack cytochrome and electron transport proteins, they cannot obtain energy through aerobic respiration. Therefore, substrate-level phosphorylation reactions during glycolysis are the primary source of ATP.

Although there are significant differences in how some genera and species use and metabolize certain carbohydrates, lactic acid bacteria generally lack metabolic diversity. Lactic acid bacteria can ferment carbohydrates in two ways.

Homofermentative LABs metabolize the carbon source via the Embden-Meyerhof-Parnas (EMP) pathway, producing 2 moles of ATP and 2 moles of pyruvate per mole of hexose. Pyruvate is then reduced to lactate by lactate dehydrogenase, converting over 90% of the initial hexose to lactic acid.

Heterofermentative LAB metabolize carbohydrates through the pentose phosphate, Leloir and phosphoketolase pathways, producing 1 mole of ATP, at least 50% lactic acid, and 30–50% ethyl alcohol and/or acetic acid, together with CO₂.

LABs are well adapted to using lactose as a source of energy and carbon and using casein as a nitrogen source. Lactic acid bacteria, which cannot use inorganic nitrogen, can metabolize proteins and peptides to meet their amino acid requirements.

Lactic acid bacteria have highly efficient systems that break casein subfractions into smaller fragments, supplying the amino acids needed for growth. In addition to supporting growth, their proteolytic activity has a strong influence on ripening and flavor, particularly in cheese production.

In general, lactic acid bacteria have very low lipolytic activity, mainly acting on mono- and diglycerides. In some cheeses and in butter, a limited degree of lipolysis may be desirable to increase the concentration of volatile fatty acids and develop the characteristic taste and aroma.

Although citrate metabolism leading to diacetyl (butter aroma) and acetoin/2,3-butanediol is common in many LAB, the genera Lactococcus and Leuconostoc are particularly important technologically.

Citrate metabolism is the most important factor in forming aroma and flavor in dairy products. Citrate metabolism requires large amounts of pyruvate relative to the NAD+ regeneration requirement.

The needed pyruvate is provided by the breakdown of citrate naturally found in milk (~1.5 mg/mL). The important point here is that the pH of the environment must be lower than 6.0 for citrate to be taken into the cell.

LAB contribute to aroma formation in fermented products through three main metabolic pathways: glycolysis, lipolysis and proteolysis. Lactate is the main product of lactose conversion, while some of the intermediate pyruvate is converted through alternative routes into compounds such as diacetyl, acetoin, acetaldehyde or acetic acid.

LAB contributes relatively little to lipolysis, but proteolysis is the key biochemical pathway for flavor formation. The degradation of caseins by rennet enzymes and cell envelope proteinases and peptidases produces small peptides and free amino acids.

For specific flavor development, these amino acids can also be converted to various alcohols, aldehydes, acids, esters and sulfur compounds.

Lactic acid bacteria species used as starter culture in dairy products are given in the table below;

Species	Dairy Product	Role	Reference
Lactococcus lactis ssp. lactis	butter, cheese, and buttermilk	acidity, bacteriocin production	Akgül (2020), Broome et al. (2003), Wouters et al. (2002)
Lactococcus lactis ssp. lactis biovar diacetylactis	butter, gouda, edam, sour cream, buttermilk	acidity and aroma	Akgül (2020), Wood (1997), Leroy and De Vusyt (2004)
Lactococcus cremoris	butter, some cheese types, buttermilk	acidity	Akgül (2020), Weerkamp et al. (1996)
Streptococcus thermophilus	yogurt, some hard and semi-hard cheese types,	acidity	Broome et al. (2003), Beresford et al. (2001)
Lactobacillus acidophilus	yogurt, kefir, butter, and some cheese types	probiotic character	Erkaya et al. (2015), Briggiler-Marcó et al. (2007)
Lactobacillus delbrueckii ssp. bulgaricus	yogurt, grana, emmental, mozzarella, taiogga, kefir	acidity	Slattery et al. (2010), Accolas and Auclair (1983), Dellaglio et al. (1994), Kılıç (2014)
Lactobacillus delbrueckii ssp. lactis	some cheese types	acidity	Broome et al. (2003), Giraffa et al. (2010)
Lactobacillus helveticus	cheddar, kefir	aroma	Slattery et al. (2010)
Lacticaseibacillus casei	some cheese types, kefir	probiotic character	Minelli et al (2004), Kongo (2013)
Lactiplantibacillus plantarum	yogurt, some cheese types	probiotic character	Leroy and De Vusyt (2004)
Lacticaseibacillus rhamnosus	butter, some cheese types	probiotic character, exopolysaccharide production	Akgül (2020), Coppola et al. (2005)
Levilactobacillus brevis	feta, kefir	aroma	Terzić-Vidojević et al. (2015), Leroy and De Vuyst (2004)
Limosilactobacillus reuteri	cheddar	exopolysaccharide production	Lynch et al. (2014)
Lactobacillus johnsonii	yogurt	probiotic character	Leroy and De Vusyt (2004)
Lentilactobacillus kefiri	kefir	probiotic character	Leroy and De Vusyt (2004)
Lactobacillus kefiranofaciens	kefir	probiotic character	Leroy and De Vusyt (2004)
Leuconostoc mesenteroides ssp. cremoris	butter, sour cream, some cheese types, edam, gouda, kefir	aroma	Akgül (2020), Weerkamp et al. (1996) Slattery et al. (2010)
Enterococcus faecium	some cheese types, Izmir tulum cheese	aroma, probiotic character	Terzić-Vidojević et al. (2015), Yerlikaya and Akbulut (2019)
Enterococcus faecalis	Iran UF cheese, cebreiro	aroma, probiotic character	Pirouzian et al. (2012), Centeno et al. (1999)
Enterococcus durans	Izmir tulum cheese	probiotic character	Yerlikaya and Akbulut (2019)
Weissella cibaria	cheddar	exopolysaccharide production	Lynch et al. (2014)

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