This document discusses various lactic acid bacteria used in food fermentations. It describes the classification and characteristics of important genera used as starter cultures including Lactococcus, Streptococcus, Leuconostoc, Pediococcus, and Lactobacillus. It also provides background on the taxonomy and phylogeny of these microorganisms based on 16S rRNA sequencing and their roles in important fermented foods like cheese, yogurt, sausages and vegetables.

1. CLASSIFICATION OF LACTIC ACID
BATERIA

2. Microorganisms Used in Food
Fermentations
Lactic acid producing bacteria
– Homofermentative LAB
– Heterofermentative LAB
Ethanol producing yeast
– Saccharomyces cerevisiae
Flavor producing microorganisms
– Bacteria, yeasts, molds
Acetic acid producing bacteria
– Acetobacter

3. 3
We can readily see that fermentations occupy
a special place in the series of chemical and
physical phenomena. What gives to
fermentations certain exceptional characters,
of which we are only now beginning to
suspect the causes, is the mode of life in the
minute plants designated under the generic
name of ferments, a mode of life which is
essentially different from that of other
vegetables, and from which result phenomena
equally exceptional throughout the whole
range of the chemistry of living beings.”
From The Physiological Theory of
Fermentation by Louis Pasteur, 1879

4. 4

5. Milk Fermentation
Joseph Lister IN 1873
published his study of
lactic fermentation of
milk, demonstrating the
specific cause of milk
souring.
His research was
conducted using the first
method developed for
isolating a pure culture
of a bacterium, which he
named Bacterium
lactis(Lactococcus lactis).

6. LAB have a long history of use by man for food
production and food preservation.
LAB are gram-positive, non-spore forming
bacteria and naturally present in raw food
material and in the human gastro-intestinal tract.
The heterogenous group of LAB include the
rod-shaped bacteria like lactobacilli and cocci,
such as streptococci, lactococci, pediococci and
leuconostocs.

7. LAB are widely used as starter cultures for
fermentation in the dairy, meat and other food
industries.
These food-grade bacteria can also improve the
safety, shelf life, nutritional value, flavor and
quality of the product.
Moreover, LAB can be used as cell factories for
the production of food additives and aroma
compounds.

8. E, Lactococcus lactis;
F,Brevibacterium
linens;
G,Lactobacillus
helveticus;
H,Streptococcus
thermophilus;
I,Bifidobacterium
longum.
8
A, Lactobacillus
delbrueckkii subsp.
bulgaricus;
B, Lactobacillus
brevis;
C, Pediococcus
pentosaceus;
D, Lactobacillus
casei;
Electron micrographs of lactic acid and related bacteria

9. Worldwide total
turnover
for cheese alone in
2007 was estimated to
be worth 74.4 billion
US $ while fermented
fresh dairy products
represent a total
economic value of
54.2 billion US
$ annually (Sieuwerts
et al. 2008).

11. 11
Sherman scheme for classification of
streptococci.

12. Homofermentative LAB
Anaerobic glycolysis for glucose utilization
Glucose … 2 Pyruvate … 2 Lactate
Actual yield of 1.8 mole lactate per mole
glucose (~90% conversion)
Net energy yield: 2 mole ATP/mole glucose
Lactococcus, Streptococcus, Pediococcus,
Lactobacillus (some)

13. Heterofermentative LAB
 Hexose monophosphate or pentose phosphate
pathway
 Glucose … Lactic acid (~50%) + Ethanol + CO2
 Can also form acetate
 Net energy yield: 1 mole ATP/mole glucose
 Leuconostoc, Lactobacillus (some)
 Some heterofermentative Lactobacillus
– Additional biochemical pathways to obtain energy
• Arginine fermentation
• Proton motive force

14. Classification of LAB
 According to current taxonomy, the lactic acid bacteria
group consists of twelve genera .
 All are in the phylum Firmicutes, Order, Lactobacillales.
 Based on 16S rRNA sequencing and other molecular
techniques, the lactic acid bacteria can be grouped into a
broad phylogenetic cluster, positioned not far from other
low G C Gram positive bacteria .
 Five sub-clusters are evident from this tree, including:
(1) a Streptococcus-Lactococcus branch (Family
Streptococcaceae),
(2) a Lactobacillus branch (Family Lactobacillaceae),
(3)a separate Lactobacillus-Pediococcus branch (Family
Lactobacillaceae);
(4) an Oenococcus- Leuconostoc-Weisella branch (Family
Leuconostocaceae),
(5) a Carnobacterium- Aerococcus-Enterococcus-
Tetragenococcus- Vagococcus branch (Families
Carnobacteriaceae, Aerococcaceae, and Enterococcaceae).
14

15. Seven of the twelve genera of lactic acid bacteria,
Lactobacillus, Lactococcus, Leuconostoc,
Oenococcus,Pediococcus, Streptococcus, and
Tetragenococcus, are used directly in food
fermentations.
Although Enterococcus sp. are often found in
fermented foods (e.g., cheese, sausage,
fermented vegetables), except for a few
occasions, they are not added directly.
In fact, their presence is often undesirable, in
part, because they are sometimes used as
indicators of fecal contamination and also
because some strains may harbor mobile
antibiotic resistance genes.
15

16. Phylogeny of lactic acid and other Gram
positive bacteria (based on 16s rRNA).
16

17. Complete Sequencing of Genome
Nowadays, the availability of whole genome
sequences for these microorganisms is
opening up a whole new platform for the
production of superior fermented foods
which are tastier, healthier and more
convenient to produce.
Indeed, the availability of a complete list of
genes for an organism is a powerful tool,
ultimately enabling a thorough estimation of
the metabolic pathways and how they may
be manipulated.
For example, genome sequence analysis has
already been exploited for LAB to
1. predict flavour formation from amino acids
2. assist in the reconstruction of genome-scale
metabolic pathways .

18. These pathways provide an overview of all
metabolic conversions in an organism based
on its genome sequence, making it feasible to
visualise different metabolic pathways, such
as amino acid metabolism and have been
constructed for Lactococcus lactis subsp.
lactis, Lactobacillus plantarum and
Streptococcus thermophilus.
For example reconstruction of the metabolic
network of L. lactis subsp. lactis IL1403 based
on the annotated genome sequence
established a total of 621 reactions and 509
metabolites, representing the overall
metabolism of Lac. lactis subsp. Lactis
(Oliveira et al. 2005).

19. In terms of the LAB, 25 genomes have
been sequenced and annotated (15
Lactobacillus, three Lactococcus,
three Streptococcus, two
Leuconostoc, one Pediococcus and
one Oenococcus), while 67 projects
are in progress (59 Lactobacillus,
three Lactococcus, three
Leuconostoc, one Streptococcus, one
Oenococcus) (Zhu et al. 2009)

21. Lactococcus
 L. lactis subsp. lactis (formerly Streptococcus lactis[11]
) is used in
the early stages for the production of many cheeses,
including Brie, Camembert, Cheddar, Colby, Gruyère,Parmesan,
and Roquefort.
 Wisconsin, the number one cheese-producing state in theUnited
States, named this bacterium in 2010 as the official state
microbe, the first and only such designation by a state legislature
in the nation
 L. lactis strains form one of the main constituents in both
industrial and artisanal starter cultures where their most
important role lies in their ability to produce acid in milk and
to convert milk protein into flavour compounds
 L. lactis, is among the most important of all lactic acid
bacteria (and perhaps one of the most important organisms
involved in food fermentations.
 L. lactis is the “work horse” of the dairy products industry.

22. The genus Lactococcus consists of five
phylogenetically- distinct species: Lactococcus
lactis, L. garviae, L. piscium, L. plantarum, and
L. raffinolactis.
They are all non-motile, obligately
homofermentative, facultative anaerobes, with
an optimum growth temperature near 30°C.
They have a distinctive microscopic
morphology, usually appearing as cocci in pairs
or short chains.
L. lactis cultures found in dairy fermentations
are classified as the subspecies cremoris,
subspecies lactis and subspecies lactis biovar
diacetylactis.

23. Phylogeny of Lactococcus based on 16S rRNA sequence
analysis. 23

24. Differentiation of lactococci at subspecies level
Test subsp.
cremoris
subsp.
lactis
Growth at 45°C – (+)
Growth with 4% NaCl – +
Arginine hydrolysis – +
Acid from lactose + +
Acid from mannitol – (–)
Acid from raffinose – –
Pyrrolidonyarylamidase
Glutamamte Decarboxylase Activity
–
+
(–)
-

25. Streptococcus
thermophilus
The genus Streptococcus contains many diverse
species with a wide array of habitats.
Included in this genus are human and animal
pathogens, oral commensals, intestinal
commensals, and one (and only one) species,
Streptococcus thermophilus, that is used in the
manufacture of fermented foods.
 In general, streptococci are non-motile,
facultative anaerobes, with an obligate
homofermentative metabolism.

26. S. thermophilus is considered the second
mostimportant industrial starter after lactococci.
 It is a member of the thermophilic LAB and has
been traditionally used in combination with
Lactobacillus delbreuckii subsp. bulgaricus or
Lactobacillus helveticus at a high process
temperature (45C) for the manufacture of yogurt
and so-called hard ‘cooked’ cheeses (e.g.
Emmental, Gruyere, Grana).
This bacterium is also used alone or in
combination with lactobacilli for the production
of Mozzarella and Cheddar cheeses.
In addition, this strain also has some reputation
as a probiotic, alleviating symptoms of lactose
intolerance and other gastrointestinal (GI)
disorders.

27. Leuconostoc
 The Leuconostoc belong to the Leuconostocaceae Family,
which also contains the closely related genera Weissella and
Oenococcus.
 Leuconostocs are mesophilic, with optimum growth
temperatures ranging from 18°C to 25°C.
 Microscopically, they appear coccoid or even somewhat rod-
like, depending on the composition and form of the growth
medium (liquid versus solid).
 The leuconostocs, in contrast to the obligate
homofermenting lactococci and streptococci, are obligately
heterofermentative

28. Most species are associated with particular
habitats, including plant and vegetable material,
milk and dairy environments, and meat products.
In addition, some species are involved in food
spoilage (e.g., Leuconostoc gasicomitatum)
whereas others are used in food fermentations.
The latter include Leuconostoc mesenteroides
subsp. cremoris, Leuconostoc mesenteroides
subsp. mesenteroides and Leuconostoc lactis,
which are used in dairy fermentations, while
Leuconostoc mesenteroides subsp.
mesenteroides, Leuconostoc kimchii, and
Leuconostoc fallax, that are used in vegetable
fermentations.

29. Phylogeny of Leuconostoc based on 16S
rRNA sequence analysis. 29

30. Pediococcus
 The pediococci are similar, in many respects, to other
coccoid-shaped, obligate homofermentative LAB, with one
main exception.
 When these bacteria divide, they do so in two “planes” (and
in right angles).
 Thus, tetrads are formed, which can be observed visually.
 Cells may appear as pairs (and always spherical in shape),
but chains are not formed, as they are for lactococci,
streptococci, and leuconostocs.
 Pediococci, like other LAB, are facultative anaerobes, with
complex nutritional requirements.
 They have optimum growth temperatures ranging from
25°C to 40°C, but some species can grow at temperatures
as high as 50°C.
 Several of pediococci are also distinguished from other
LAB by their ability to tolerate high acid (growth at pH 4.2)
and high salt (growth at 6.5% NaCl) environnements.

31. Phylogeny of Pediococcus based on 16S rRNA sequence analysis.
31

32.  The pediococci can be found in diverse habitats,
including plant material, milk, brines, animal urine, and
beer.
 There are six recognized species of Pediococcus;
several are important in food fermentations.
 Two species, Pediococcus acidilactici and P.
pentosaceus, are naturally present in raw vegetables
where, under suitable conditions, they play a key role in
the manufacture of sauerkraut and other fermented
vegetables.
 These same species may also be added to meat to
produce fermented sausages.
 Despite their inability to ferment lactose, P. acidilactici
and P. pentosaceus are frequently found in cheese,
where they may participate in the ripening process.
 Pediococci are also important as spoilage organisms in
fermented foods, in particular, beer, wine, and cider.
 One species, P. damnosus, is especially a problem in
beer, where it produces diacetyl, which in beer is a
serious defect

33. Lactobacillus
Lactobacilli occupy a wide range of habitats.
often described as being ubiquitous in nature:
plant and vegetable material, dairy and meat
environments, juice and fermented beverages,
grains and cereal products, the animal and
human gastrointestinal tract, stomach, mouth
etc(probiotic activity).
In foods, they are involved not only in many
important fermentations, but are also frequently
implicated in spoilage of fermented and non-
fermented foods.

34. Phylogeny of
Lactobacillus
based on 16S
rRNA sequence
analysis.
Not all of the
species
are listed.
34

35. Optimum temperature varies widely, from 30°C
to 45°C. Although
Most species are mesophilic, the genus also
contains species that are psychrotrophic,
thermoduric, or thermophilic.
 Some species show high tolerance to
– salt,
– osmotic
– pressure
– oxygen
– low water activity
– low pH

36. Lactobacilli can be divided into 3 groups.
– Group I -obligate homofermenting species,
– Group II-facultative heterofermenting
species,
– Group III contains obligate heterofermenting
species
Starter culture lactobacilli are used primarily in
dairy and sausage applications.
There are two main species used as dairy
starter cultures (mainly for cheese and yogurt),
L. helveticus and L. delbrueckii subsp.
bulgaricus.
Other common dairy-related species include L.
casei and L.acidophilus (both used frequently
as probiotics).

38. Other Bacteria Important
in Food Fermentations
In addition to LAB several other
genera are involved in fermented
foods.
These non-LAB represent several
different genera e.g.
– Bifidobacterium
– Brevibacterium
– Propionibacterium

39. Bifidobacterium
Bifidobacterium is a Gram-positive, non-motile, often
branched anaerobic bacteria which produce lactic and
acetic acids in the ratio of 2: 3.
These bacteria are not used in the manufacture of
any fermented food, nor are they even found in most
raw food materials.
Rather, they are added to certain foods, mostly milk
and fermented dairy products, strictly for their
probiotic functions.
The intestinal tract is their primary habitat.
Common species are:
– Bifidobacterium bifidum,
– Bifidobacterium adolescentis,
– Bifidobacterium breve,
– Bifidobacterium infantis,
– Bifidobacterium lactis,
– Bifidobacterium longum.

40. Brevibacterium
 The genus Brevibacterium are non-motile, non-sporing,,non-
acid-fast, irregular-shaped Gram positive rods, strictly
aerobic, catalase-positive, mesophiles, with an optimum
growth temperatures between 20°C and 35°C.
 Most species are salt-tolerant (10%) and able to grow over a
wide pH range.
 One species, Brevibacterium linens, is important in fermented
foods, mainly because it is involved in the manufacture of
bacterial, surface ripened cheeses, such as Limburger and
Muenster.
 In these products, B. linens produces a yellow-orange-red
pigment on the cheese surface that gives these cheeses their
characteristic appearance.

42. Propionibacterium
 The propionibacteria are in the phylum Actinobacteria.
 They are non-sporing, Gram positive, non-motile rods,
catalase positive, anaerobic to aerotolerant mesophiles.
 In general, the propionibacteria are associated with two quite
different habitats, the human skin and dairy products.
 The dairy group consists of several species that are important
in food fermentations, due to their use in the manufacture of
Swiss-type cheeses.
 The most frequently used dairy species include
Propionibacterium freudenreichii subsp. shermanii,
Propionibacterium freudenreichii subsp. freudenreichii,
Propionibacterium acidopropionici,
Propionibacterium jensenii.

43. Yeasts and Molds Used in the
Manufacture of Fermented Foods
Most of the fungi relevant to fermented foods are
classified as Zygomycota, Ascomycota, or as
deuteromycetes.
These groups contain several important genera
of yeasts, including Saccharomyces,
Kluyveromyces, and Zygosaccharomyces, as
well as the mold genera Aspergillus, Penicillium,
and Rhizopus.

44. Some selected characteristics of micro-organisms and their principal
metabolic products used in fermented milks.