Different lactic acid bacteria used as starter cultures in foods and their properties

1. Lactic acid
bacteria used as
starter cultures in
foods and their
properties
M. ANZA Adamou

2. Introduction
– Lactic acid bacteria (LAB) are commonly detected in various habitats
such as foodstuffs, gut and mucous membranes of humans and animals,
and in many environmental niches. In the fermentations of yogurt,
cheese, salami, sourdough bread, and wines, LAB are the key organisms
providing both desired sensory changes and increased shelf life and
product safety (Björkroth & Koort, 2016). The ability of lactic bacteria to
be involved in different fermentation processes results from the
heterogeneity of the groups. In the following we will discuss about the
group of lactic acid bacteria used in food technology as well as their
properties.

3. Characteristics of lactic acid bacteria
(LAB)
– Typical LAB are Gram-positive, non-spore-forming rods or cocci producing lactic
acid as the major end product of fermentation of glucose. There have been
frequent changes in the taxonomic classification of LAB, but most classification
schemes agree that genera in the order Lactobacillales, which includes
Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and Streptococcus, in
addition to Carnobacterium, Enterococcus, Oenococcus, Tetragenococcus,
Vagococcus, and Weissella, are the genera of LAB (O'Bryan, Crandall, Ricke, &
Ndahetuye, 2015).

4. Genera of Lactic Acid Bacteria Important to
Food Fermentations adapted from (Erten, 2016)
Old Classification New Classification
Lactobacillus Lactobacillus
Carnobacterium
Leuconostoc Leuconostoc
Oenococcus
Streptococcus Streptococcus thermophilus
Lactococcus
Enterococcus
Vagococcus
Pediococcus Pediococcus
Aerococcus
Tetragenococcus

5. Characteristics of lactic acid bacteria
(LAB)
– Based on sugar fermentation patterns, two broad metabolic categories of LAB
exist: homofermentative and heterofermentative. The main products of
fermentation include organic acids, alcohol, and carbon dioxide, although LAB
may also produce aromatic molecules, vitamins, or bioactive peptides (O'Bryan
et al., 2015).
– Those that produce lactic acid as the major or sole product of glucose
fermentation are designated homofermentative. Those that produce equal
amounts of lactic acid, ethanol and CO2 are termed heterofermentative. The
homolactics are able to extract about twice as much energy from a given
quantity of glucose as the heterolactics (Erten, 2016).

6. Characteristics of lactic acid bacteria
(LAB)
– All members of Pediococcus, Lactococcus, Streptococcus, Vagococcus, along
with some lactobacilli are homofermenters. Carnobacterium, Oenococcus,
Enterococcus, Weissella and Leuconostoc and some Lactobacilli are
heterofermenters. The heterolactics are more important than the homolactics
in producing flavour and aroma components such as acetylaldehyde and
diacetyl (Erten, 2016).

7. Characteristics of lactic acid bacteria
(LAB)
– LAB have a very limited capacity to synthesize amino acids using inorganic
nitrogen sources and are therefore dependent on preformed amino acids being
present in the growth medium; this requirement for amino acids differs among
species and strains within species, with some strains requiring as many as 15
preformed amino acids. Because free amino acids are not typically present in
sufficient concentrations in the environment, the LAB require a proteolytic
system capable of hydrolyzing peptides and proteins to obtain their essential
amino acids. The proteolytic activity of LAB contributes additionally to the
development of the flavor, aroma, and texture of fermented products; for
instance, many varieties of cheeses, such as Swiss and Cheddar, rely on
proteolysis for their desirable flavor notes (O'Bryan et al., 2015).

8. Metabolism of lactic acid bacteria
adapted from (O'Bryan et al., 2015)

9. Lactococcus
– The genus Lactococcus comprises seven species, Lactococcus lactis (including
the subspecies cremoris, lactis, and hordniae), L. garvieae, L. piscium, L.
plantarum, and L. raffinolactis, L. chungangensis, and the quite recently
characterized L. fujiensis.
– Species identification is based on physiological, chemotaxonomic, and
molecular biological criteria. Morphologically, lactococci are all non-motile,
gram-positive cocci of 0.5–1.5 µm in size, forming short chains. They are
mesophilic, facultative anaerobes, with an optimum growth of Temperature
near 30°C, ferment hexoses homofermentatively producing l (+) lactic acid, and
have complex growth requirements (Erten, 2016; LAHTINEN, OUWEHAND,
SALMINEN, & WRIGHT, 2011).

10. Lactococcus
– Some physiological characteristics differentiating the species are listed in Table.
In practice, it is often difficult to distinguish between L. lactis and L. garvieae.
Pyrrolidonylarylamidase activity has been considered specific for the latter
species, but L. lactis strains having this activity are also frequently met. Thus
molecular biological identification is usually necessary to obtain a reliable
identification (LAHTINEN et al., 2011).
– One species in particular L. lactis, is among the most important of all lactic acid
bacteria (and perhaps one of the most important organisms involved in food
fermentations, period). L. lactis is used as a starter culture for most of the hard
cheeses and many of the cultured dairy products produced around the world
(Erten, 2016).

11. Physiological Characteristics
Differentiating Lactococcus Species
adapted from (LAHTINEN et al., 2011)

12. Lactococcus
– Thus far Lactococcus lactis ssp. lactis and cremoris are the only lactococci used
as dairy starters. The potential of a few strains of L. lactis ssp. hordniae, L.
raffinolactis, L. garvieae, L. piscium, and L. plantarum in dairy applications has
been assessed. The strains were characterized for phage resistance, lactose
fermentation, and growth in milk supplemented with glucose and casein
hydrolysate.
– The most promising were the L. raffinolactis strains; however, even they lacked
proteinase activity, and the activity was not expressed even after the
introduction of the proteinase-associated plasmid from L. lactis ssp. cremoris. L.
garvieae has been proposed for starter culture preparations, due to its
prevalence in artisanal Italian cheeses and its potential contribution to the
typical sensory characteristics of traditional cheeses (LAHTINEN et al., 2011).

13. Lactobacillus
– Lactobacillus is the largest genus within the group of lactic acid bacteria. To date
(July 2010), it contains 168 species, some of which are used in the manufacture of
fermented dairy, sourdough, meat, and vegetable foods, or used as probiotics.
Lactobacilli are Gram-positive, catalase-negative, non-spore forming, rod-shaped
bacteria that produce lactic acid as the major end product of fermentation (Calasso
& Gobbetti, 2011; De Angelis & Gobbetti, 2016)
– Ecologically, lactobacilli occupy a wide range of habitats. They are frequently found
in dairy and meat environments, in juice and fermented beverages, and in grains
and cereal products. Their presence in the animal and human gastrointestinal tract
(as well as in the stomach, mouth) has led to the suggestion that these bacteria
have broad “probiotic activity”. In foods, they are involved not only in much
important fermentation, but are also frequently implicated in spoilage of fermented
and non-fermented foods (Erten, 2016).

14. Lactobacillus
– Although most species are mesophilic, the genus also contains species that are
psychrotrophic, thermoduric, or thermophilic. Temperature optima varies
widely, from 30°C to 45°C but they can grow over a range of 5– 53 C. They also
are aciduric with an optimum growth pH of 5.5–5.8; in general, they can grow at
a pH <5 (C. A. Batt, 2014; Erten, 2016). Acid-tolerance is a common trait of
lactobacilli (many strains actually prefer an acidic environment), and some also
are ethanol-tolerant or bile-tolerant. Some species show high tolerance to salt,
osmotic pressure, and low water activity. Most species are aero tolerant,
whereas others require more strict anaerobic conditions (Erten, 2016).

15. Group classification of lactobacilli adapted
from (C. A. Batt, 2014)

16. Lactobacillus
– Given the diversity of metabolic properties exhibited by members of the
Lactobacillus genus, they are found in a number of fermented food products. In
these products, the lactobacilli contribute to their preservation, nutrition
availability, and flavor. Lactobacilli are added as deliberate starters or take part
in the fermentation as a result of their being natural contaminants of the
starting substrates. A number of dairy products are produced using
Lactobacillus either alone or in combination with other lactic acid bacteria.
Acidophilus milk is an example of a fermented dairy product and L. acidophilus
is the organism used to produce it. Lactobacillus bulgaricus in combination with
Streptococcus thermophilus is used to produce yogurt, and a balance between
these two starters can affect product quality. Vegetables are fermented with
lactobacilli to produce products, including pickles, olives, and sauerkraut.

17. Lactobacillus
– Lactobacillus spp. play an essential role in breadmaking and a number of unique
strains have been identified in products, most notably sourdough bread. Typical
species of lactobacilli identified in sourdough bread include L. acidophilus,
Lactobacillus farciminis, L. delbrueckii subsp. delbrueckii, L. casei, L. plantarum,
Lactobacillus rhamnosus, L. brevis, Lactobacillus sanfrancisco, and L. fermentum.
The exact composition of most sourdough breads is not known and attempts to
blend starters to mimic a particular product are sometimes less than satisfactory.
Traditional sourdough fermentations are carried out by ‘backslopping,’ a process
in which a small fraction of a prior batch is used to start the next batch. The
indigenous lactobacilli are able to overcome other contaminating microflora
largely by thriving under the fermentation conditions (C. A. Batt, 2014).

18. Lactobacillus
– During the fermentation process, lactic acid builds up levels approaching 1%
and a small amount of acetic acid is also produced. The number of lactic acid
bacteria can reach 107 cfu g-1
– Another property of lactobacilli that has become more appreciated is their
ability to produce bacteriocins. The bacteriocins produced by lactobacilli are
presented in the next table. Bacteriocins probably evolved to provide the
producing organism with a selective advantage in a complex microbial niche.
Incorporation of Lactobacillus spp. as starters or the inclusion of a purified or
semi purified bacteriocin preparation as an ingredient in a food product may
provide a margin of safety in preventing pathogen growth (C. A. Batt, 2014).

19. Streptococcus
– The genus Streptococcus consists of Gram-positive, spherical ovoid, or
coccobacillary cells, with a diameter less than 2 mm, that form chains or pairs.
Cells in older cultures may appear Gram variable, and some strains are
pleomorphic. Streptococcus spp. are nonsporing and nonmotile. Streptococci
are catalase negative, with the exception of the recently described species
Streptococcus didelphis, which, on initial isolation on blood agar, gives vigorous
catalase activity that is lost after several passages. They ferment carbohydrates
to produce mainly lactic acid, but no gas, and have complex nutritional
requirements. Under glucoselimiting conditions, formate, acetate, and ethanol
are also produced. Most are facultatively anaerobic or aerotolerant anaerobes;
some are capnophilic (CO2-requiring) (Gobbetti & Calasso, 2014).

20. Streptococcus
– Except for S. thermophilus, streptococci sensu stricto are not currently used in
food fermentation. The naturally occurring S. gallolyticus subsp. macedonicus is
another promising multifunctional Streptococcus starter culture. Some
pathogens, however, are introduced into humans and animals by foods
(Gobbetti & Calasso, 2014).
– Streptococcus thermophilus belongs to the thermophilic group of lactic acid
bacteria. It is traditionally used in association with one or several Lactobacillus
species as a starter culture in the production of yogurt and the manufacture of
Swiss and Italian cheeses such as Emmentaler and Mozzarella.

21. Streptococcus
– Streptococcus thermophilus is a homofermentative bacterium, fermenting lactose
via the Embden–Meyerhof pathway (EMP) to L(+) lactate, Gram-positive spherical
to ovoid nonmotile coccus, 0.7–0.9 mm in diameter, occurring in pairs and chains,
some of which can be very long. The bacterium has an optimum growth
temperature of 40–45 °C, a minimum of 20–25 °C, and a maximum near 47–50
°C. Streptococcus thermophilus does not hydrolyze arginine. It ferments a limited
number of sugars including lactose, fructose, sucrose, and glucose. Streptococcus
thermophilus does not ferment galactose during lactose metabolism. It is also
characterized by being relatively sensitive to antibiotics and sanitizers and having
low proteolytic activity. It is unique among the streptococci in having no group-
specific antigen. Strains of S. thermophilus that produce bacteriocins are rare, and
those that have been isolated have not been well characterized (Harnett, Davey,
Patrick, Caddick, & Pearce, 2011).

22. Pediococcus
– Pediococcal colonies vary in size (1.0–2.5 mm in diameter), and they are smooth,
round, and greyish white. All species grow at 30 °C, but the optimum temperature
range is 25–40 °C. Pediococcus pentosaceus has a lower optimum temperature for
growth (28–32 °C) than P. acidilactici (40 °C), but the latter grows at 50 °C. The
optimum pH for growth is 6.0–6.5. Half of the species grow at pH 4.2, and most of
them (except P. damnosus) grow at pH 7.0. Most Pediococcus species (except for P.
damnosus) can grow in the presence of 4.0 and 6.5% NaCl but not in the presence of
10% NaCl.
– Lactic acid production by P. pentosaceus, in a bacteriological medium at 27 °C, is
inhibited 36.0–51.0% by concentrations of NaCl from 3.0 to 3.9% (w/v), respectively.
Some strains of P. acidilactici and P. pentosaceus have proteolytic enzymes, such as
protease, di-peptidase, dipeptidyl aminopeptidase, and amino-peptidase. Pediococcus
pentosaceus shows strong leucine and valine arylamidase activities (Carl A. Batt &
Tortorello, 2014).

23. Pediococcus
– Pediococci are gram-positive, nonmotile oxidase-negative, and catalase-negative
organisms occurring as spherical cells uniform in size that form tetrads via alternate
division in two perpendicular directions. Pediococci are facultatively aerobic
homofermenters that produce lactic acid as the major end product of glucose
fermentation by the EMP to dl-lactic acid except for strains of Pediococcus claussenii,
which convert glucose to l (+)-lactic acid. Fructose, mannose, and cellobiose are
fermented by all species. Most species are also able to ferment galactose and
maltose, although it has been reported that some strains of Pediococcus damnosus,
Pediococcus Pparvulus, and . claussenii lack this ability. Sucrose is also fermented by
all species except Pediococcus inopinatus, P. parvulus, Pediococcus pentosaceus, and
P. claussenii. In contrast, rhamnose, melibiose, melezitose, raffinose, inulin, and α-
methyl glucoside D are not fermented by most pediococci. Like many other LAB
pediococci also produce bacteriocins (i.e., pediocins).

24. Pediococcus
– Producer strains of pediocins have mainly been found in the phylogenetically and
biochemically related species Pediococcus acidilactici and P. pentosaceus and, more
recently, also in P. damnosus (LAHTINEN et al., 2011).
– The pediococci are used in the commercial fermentation of meats, vegetables, and
sour wheat flour with no added sugar. Lactose-positive pediococci may replace
Streptococcus thermophilus in Italian cheese starter blends to combat S.
thermophilus bacteriophage problems in mozzarella cheese plants. Pediococcus
acidilactici and P. pentosaceus are used in the fermentation of meats. Manganese
enhances the fermentation of meats at a suboptimal incubation temperature for
the culture. Pediococci were inhibited by KCl, as a salt substitute. The use of mix
starter cultures could be a problem as some strains of pediococci may inhibit the
growth of other strains of pediococci, L. plantarum, and Leuconostoc mesenteroides
(Carl A. Batt & Tortorello, 2014).

25. Pediococcus
– The pediococci may be useful as biopreservatives to control the growth of
Salmonella typhimurium and Pseudomonas sp. (in pasteurized liquid whole
eggs and cooked mechanically deboned poultry meat), Staphylococcus aureus
(cooked mechanically deboned poultry meat), and Listeria (milk). Pediococci
also increase the shelf life of refrigerated mechanically deboned poultry meat,
ground beef, and ground poultry breast. There are conflicting reports as to the
inhibition of Clostridium botulinum by pediococcal bacteriocin. Pediocin may be
effective in controlling Listeria in milk and during the fermentation of turkey
summer sausage. Both P. acidilactici and P. pentosaceus may control the growth
of Yersinia enterocolitica serotype 0:3 and 0:8 in fermenting meat (Carl A. Batt
& Tortorello, 2014).

26. Tetragenococcus
– Phylogenetically, the genus Tetragenococcus is a recognized member of the
family Enterococcaceae within the order Lactobacillales. Morphologically,
tetragenococci cannot be readily distinguished from members of the genus
Pediocccus. Both genera also share a facultative aerobic homofermentative
metabolism, the ability to ferment a relatively wide range of sugars.
– T. halophilus and T. muriaticus have primarily been associated with habitats rich
in salt and protein. Both salt-tolerant species are known to play an important
role in halophilic fermentation processes such as the production of soy sauce,
soy paste, brined anchovies, fish sauce, Japanese fermented puffer fish ovaries,
Indonesian “terasi” shrimp paste, and fermented mustard.

27. Tetragenococcus
– . However, T. halophilus also constitutes the dominant microbiota in
concentrated sugar thick juice, a sugar-rich intermediate in the production of
beet sugar. In fact, strains of this species have been associated with thick juice
degradation, a process characterized by a pH shift from pH 9 to 5–6 and by an
increase in reducing sugar content resulting in economic losses. Tetragenococci
can be readily distinguished from pediococci mainly by their high salt tolerance
(depending on the species, from 6.5% to 25% NaCl [w/v]) and ability to grow at
high pH values up to 9.0 but not at pH 5.0 (LAHTINEN et al., 2011).

28. Leuconostoc
– Leuconostocs are mesophilic, Gram-positive, catalase-negative, nonmotile,
aerotolerant, obligately heterofermentative cocci, often ellipsoidal. They usually
occur in pairs and chains (Holland & Liu, 2011; LAHTINEN et al., 2011). When
grown on a solid medium, cells are elongated and can be mistaken for rods.
True cellular capsules are not formed, but many leuconostocs produce
extracellular dextran that forms an electron-dense coat on the cell surface.
Growth may occur at pH 4.5, leuconostocs prefer an initial medium pH of 6.5.
The optimal growth temperature is between 20°C and 30°C (LAHTINEN et al.,
2011). Leuconostoc spp. are widespread in the environment, and have been
isolated from plant matter, human clinical sources, and foods such as chill-
stored and fermented meats, fermented vegetables (e.g., sauerkraut, kimchi),
and fermented dairy products (e.g., cheese, kefir, yogurt).

29. Leuconostoc
– The exact species of Leuconostoc occurring in dairy starter cultures are not
always defined, but generally there are only two: Ln. lactis and Ln.
mesenteroides (subsp. mesenteroides, subsp. cremoris, and subsp. dextranicum).
Leuconostoc mesenteroides subsp. cremoris is the subspecies most frequently
isolated from mesophilic mixed-strain dairy starter cultures and from fermented
dairy products. In addition, Ln. citreum has been reported as an isolate from
cheese. A number of bacteriophages that attack some dairy leuconostocs have
been isolated. However, most leuconostocs are insensitive to bacteriophages.
There are no reports of bacteriophage attack in dairy fermentations associated
with leuconostocs. Dairy leuconostocs are known to produce antimicrobial
compounds against pathogenic and spoilage microorganisms.

30. Leuconostoc
– However, the inhibitory activity is generally attributed to the action of organic
acids such as acetic acid, CO2, and H2O2 that they produce. In general,
bacteriocins produced by leuconostocs may not necessarily be active against
lactic acid bacteria, but are active against Listeria monocytogenes, a major food
pathogen (Holland & Liu, 2011).

31. Characteristics of the dairy
leuconostocs adapted from (Holland & Liu, 2011)

32. Weissella
– Weissellas are gram-positive, nonmotile, and asporogenous short rods with
rounded tapered ends, or ovoid. They occur in pairs or in short chains, and
there is tendency toward pleomorphism in some of the species. They are
catalase negative, facultatively anaerobic chemo-organotrophs, and were
originally considered not to contain cytochromes. Weissellas ferment glucose
heterofermentatively. Carbohydrates are fermented via the hexose
monophosphate and phosphoketolase pathways. End products of glucose
fermentation are CO2, ethanol, and/or acetate. Depending on the species, the
configuration of the lactic acid produced is either dl- or d (–). Weissellas have
complex nutritional requirements as amino acids, peptides, fermentable
carbohydrates, fatty acids, nucleic acids, and vitamins are generally required for
growth. Biotin, nicotine, thiamine, and panthotenic acid or its derivatives are
required.

33. Weissella
– Arginine is not hydrolyzed by all species. Growth occurs at 15°C; some species
grow at 42–45°C.
– Members of W. cibaria, W. confusa, and W. koreensis have been detected in
fermented foods of vegetable origin, whereas W. confusa has been associated
with Greek salami, Mexican pozol, and Malaysian chili bo. W. cibaria and W.
confusa have also been associated with various types of sourdoughs. W.
ghanensis and W. fabaria were detected in traditional heap fermentations of
Ghanaian cocoa bean. W. beninensis originates from submerged fermenting
cassava (LAHTINEN et al., 2011).

34. Oenococcus
– The name Oenococcus refers to ‘a little round berry from wine’ and only one
species is found in wine; Oenococcus oeni. This species is well adapted to the
harsh wine environment: low nutrients, high acidity, and high ethanol
concentration. When viewed under a microscope, the cells are ellipsoidal to
spherical in shape, usually present in pairs, but they will form chains in the
presence of ethanol. They form small colonies on solid media (agar), and
growth is slow, usually 7–10 days at 22 C. O. oeni will grow better, and even be
stimulated, in the presence of lower oxygen concentrations (micro-aerophilic)
(Carl A. Batt & Tortorello, 2014)

35. Oenococcus
– Next to the type species O. oeni, the genus Oenococcus also comprises the
nonacidophilic and non-malolactic-fermenting species Oenococcus kitaharae,
which was proposed to accommodate isolates from a composting distilled
shochu residue in Japan (LAHTINEN et al., 2011).

36. References
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microbiology Encyclopedia of food microbiology.
– Björkroth, J., & Koort, J. (2016). Lactic Acid Bacteria: Taxonomy
and Biodiversity Reference Module in Food Science: Elsevier.
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Lactobacillus spp.: Other Species A2 - Fuquay, John W
Encyclopedia of Dairy Sciences (Second Edition) (pp. 125-131).
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– De Angelis, M., & Gobbetti, M. (2016). Lactobacillus SPP.:
General Characteristics Reference Module in Food Science:
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lactic acid bacteria. University of Çukurova, Faculty of
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(2011). Lactic Acid Bacteria | Streptococcus thermophilus A2 -
Fuquay, John W Encyclopedia of Dairy Sciences (Second Edition)
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Leuconostoc spp. A2 - Fuquay, John W Encyclopedia of Dairy
Sciences (Second Edition) (pp. 138-142). San Diego: Academic
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– LAHTINEN, S., OUWEHAND, A. C., SALMINEN, S., & WRIGHT, A.
V. (2011). Lactic acid bacteria : Microbiological and functional
aspects (pp. 779).
– O'Bryan, C. A., Crandall, P. G., Ricke, S. C., & Ndahetuye, J. B.
(2015). Lactic acid bacteria (LAB) as antimicrobials in food
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