Carbohydrate metabolism of starter culture

1. CARBOHYDRATE UTILIZATION BY
LACTIC
ACID BACTERIA
1

2. • Lactic acid bacteria are classified as heterotrophic
chemoorganotrophs, meaning that they require
preformed organic carbon as a source of both carbon
and energy.
• LAB chiefly rely on 2 principal pathways for catabolism
of carbohydrates-homo and heterofermentation.
• In the homofermentative pathway, hexoses are
metabolized via enzymes of the Embden-Meyerhoff
pathway (Fig. a, b & 1), yielding 2 mol of pyruvate and 2
mol of ATP per mole of hexose.
• The Embden-Meyerhof-Parnas pathway (EMP pathway),
was first discovered by Gustav Embden, Otto
Meyerhof and Jakub Karol Parnas.
2

3. Otto Meyerhof
Jakub Karol Parnas
3

4. Fig a. Stages of Glycolysis
• 2 ATP are used
• Glucose is split to form 2 glucose-3-phosphate
4

5. Fig b. Stage of Glycolysis(Contd)
• 2 glucose-3-phosphate oxidized to 2 pyruvic acid
• 4 ATP produced
• 2 NADH produced
5

6. Figure 1 The Embden-Meyerhoff pathway used by homofermentative lactic acid bacteria.
6

7. • Pyruvate is subsequently reduced to lactate by
lactate dehydrogenase, so that more than 90% of the
starting material (i.e., glucose) is converted to lactic
acid.
• The NADH formed via the glyceraldehyde-3-
phosphate dehydrogenase reaction is also reoxidized
(forming NAD) by lactate dehydrogenase, thus
maintaining the NADH/NAD balance.
• Among LAB used as dairy starter cultures, most are
homofermentative e.g. Lactococcus lactis,
Streptococcus thermophilus, Lactobacillus helveticus,
and Lb. delbrueckii subsp. bulgaricus
7

8. • In heterofermentative metabolism, hexoses are
catabolized by the phosphoketolase pathway
(Fig. 2), which results in approximate equimolar
production of lactate, acetate, ethanol, and
CO2.
• Only 1 mol of ATP is made per hexose.
• Although several species of Lactobacillus are
heterofermentative, Leuconostoc spp. are the
only heterofermentative LAB used as starter
cultures in dairy products
8

9. Figure 2 The phosphoketolase pathway used by heterofermentative lactic acid bacteria
9

10. Metabolism of Lactose by LAB
• As described earlier, LAB generally rely on either the
Embden- Meyerhoff or phosphoketolase pathway for
metabolism of sugars.
• In fact, these catabolic pathways are only a part of the
overall metabolic process used by these bacteria.
• The first, and perhaps most important, step in carbohydrate
metabolism involves transport of the sugar substrate across
the cytoplasmic membrane and its subsequent
accumulation in the cytoplasm.
• This process of transport and accumulation is important for
several reasons.
– First, active transport of sugars requires energy, and much of the
energy gained by cells as a result of catabolism must then be
used to transport more substrate.
– Second, the transport system used by a particular strain dictates,
in part, the catabolic pathway used by that organism.
10

11. Lactose Phosphotransferase System
of L. lactis
• There are, in general, two different systems used by
LAB to transport carbohydrates, and it is convenient
to group them according to the system used to
transport their primary substrate, lactose.
• The phosphoenolpyruvate (PEP)–dependent
phosphotransferase system (PTS) is used by most
mesophilic, homofermentative LAB, especially
lactococci used as starter cultures for cottage,
Cheddar, Gouda, and other common cheese
varieties.
11

12. • The Lactococcus lactose PTS, first described by
McKay et al. (1969), consists of a cascade of
cytoplasmic and membrane-associated proteins
that transfer a high-energy phosphate group
from its initial donor, PEP, to the final acceptor
molecule, lactose.
• Phosphorylation of lactose occurs concurrent
with the movement of lactose across the
cytoplasmic membrane (from out to in) and
results in intracellular accumulation of lactose
• The product of the lactose PTS, thus, is lactose-
phosphate, or more specifically, glucose-β-1,4-
galactosyl-6-phosphate .
12

13. • Hydrolysis of this compound occurs via phospho-β-
galactosidase, yielding glucose and galactose-6-
phosphate.
• Glucose is phosphorylated by hexokinase (via an ATP) to
glucose-6-phosphate, which then feeds directly into the
Embden-Meyerhoff pathway, as described earlier.
• Galactose-6-phosphate, in contrast, takes a different
route altogether, as it is first isomerized to tagatose-6-
phosphate and then phosphorylated to form tagatose-
1,6-diphosphate (Fig. 3).
• The latter is then split by tagatose-1,6-
diphosphatealdolase to form the triose phosphates,
glyceraldehyde-3-phosphate and dihydroxyacetone
phosphate, in a reaction analogous to the aldolase of the
Embden- Meyerhoff pathway.
• It is important to note that in Lc. lactis, glucose and
galactose moieties of lactose, despite taking parallel
pathways, are fermented simultaneously. 13

14. Figure 3 Tagatose pathway in lactococci. Galactose-6-phosphate is formed from
hydrolysis of lactose-phosphate, the product of the lactose PTS.
14

15. Lactobacillus casei Lactobacillus bulgaricus
Streptococcus thermophilus Lactococcus lactis subsp lactis
15

16. Lactose Transport and Hydrolysis by
S. thermophilus
• Although the PTS is widely distributed among LAB,
several important dairy species rely on a lactose
permease for transport and a β-galactosidase for
hydrolysis.
• Some species have both pathways for lactose, and
some have a PTS for one sugar and a permease for
another.
• The best example of the lactose permease/β-
galactosidase system is that which occurs in S.
thermophilus, Lb. helveticus, and Lb. delbruecki
subsp. bulgaricus
16

17. • Figure 4. Lactose
transport and hydrolysis
by S. thermophilus.
• Lactose uptake is driven
by galactose efflux; both
solutes may be
transported in symport
with a proton.
17

18. • After hydrolysis, S. thermophilus rapidly
ferments glucose to lactic acid by the Embden-
Meyerhoff pathway, yet most strains, especially
those used as dairy starter cultures, do not
ferment the galactose moiety of lactose.
Rather, galactose is
effluxed (pumped outside
the cell) and accumulates
in the extracellular
medium.
18

19. • In the manufacture of dairy products made
with an S. thermophilus–containing culture,
such as yogurt or mozzarella cheese, galactose
may appear in the finished product.
• With yogurt, accumulated galactose is of little
consequence, but for mozzarella cheese, even a
small amount of galactose can present
problems.
• This is because of the nonenzymatic browning
reaction that occurs when galactose, a reducing
sugar, is heated in the presence of free amino
acids. 19

20. • Since most mozzarella cheese is used
for pizzas, high-temperature baking
accelerates nonenzymatic browning
reactions.
• Cheese containing galactose can brown
excessively, a phenomenon considered
as a defect by many pizza
manufacturers.
• Therefore, mozzarella producers may
be asked by their customers to satisfy
specifications for ‘‘low-browning’’ or
low galactose cheese.
• Although some cheese manufacturers
can rely on their cheesemaking know-
how and simply modify the production
procedures to remove unfermented
galactose, other manufacturers have
chosen to use cultures that have low-
browning potential.
20

21. Lactose Metabolism by Lactobacillus and Other
LAB
• Most other LAB rely on one or the other of the two
pathways described earlier.
• With the exception of Lc. lactis and Lb. casei, however,
most dairy LAB do not have a lactose PTS, and instead use
a lactose permease/β-galactosidase system for
metabolism of lactose.
• Some strains have more than one system; for example, Lc.
lactis and Lb. casei have both a lactose PTS and a lactose
permease/β-galactosidase.
21

22. • Many of the lactobacilli and Leuconostoc spp. that
transport and hydrolyze lactose by a permease and a β-
galactosidase, respectively, also ferment glucose and
galactose simultaneously.
• This is important, because in almost all fermented dairy
products made with a culture containing S.
thermophilus, a galactose-fermenting Lactobacillus sp.
is also present.
• For some products, such as Swiss-style cheeses, the
galactose that is effluxed into the curd by S.
thermophilus is subsequently fermented by Lb.
helveticus.
• Otherwise, the free galactose could be fermented by
other members of the microflora, resulting in
heterofermentative end products that could contribute
to off-flavors and other product defects.
22

23. 23

24. Luis F Leloir
• Luis F. Leloir (1906 - 1987) was
awarded the 1970 Nobel Prize
in Chemistry for his work on the
metabolism of carbohydrates,
specifically glycogen [Glycogen
Synthesis].
• He discovered a key
intermediate in that pathway;
namely UDP-glucose.
• His discovery led to the
realization that sugar
nucleotides play an important
role in many different
metabolic pathways.
24

25. • During growth in milk, LAB ordinarily encounter free
galactose only after intracellular hydrolysis of lactose.
• For lactococci and those lactobacilli that transport lactose
via the PTS, galactose-6-phosphate is the actual hydrolysis
product (resulting from hydrolysis of lactose-phosphate
by phospho-β-galactosidase).
• Galactose-6-phosphate feeds directly into the tagatose
pathway, as described earlier.
• However free galactose will appear and accumulate in
fermented dairy products made with thermophilic starter
cultures containing S. thermophilus, Lb. bulgaricus, or
other galactose nonfermenting strains.
• Yogurt and mozzarella cheese, for example, can contain
up to 2.5 and 0.8% galactose, respectively.
• Therefore, metabolism of free galactose may be of
practical importance.
Galactose Metabolism
25

26. Figure 4 Leloir pathway in lactic acid bacteria. Phosphorylation of galactose may require
isomerization by mutarotase (not shown). The subsequent steps convert galactose-1-
phosphate into glucose-6-phosphate, which feeds into the EM pathway (homofermentative
bacteria) or phosphoketolase (PK) pathway (heterofermentative bacteria).
26

27. Galactosemia
• Galactosemia is a condition in which
the body is unable to use
(metabolize) the simple sugar
galactose.
• Galactosemia is an inherited
disorder. This means it is passed
down through families.
• It occurs in approximately 1 out of
every 60,000 births among
Caucasians. The rate is different for
other groups.
• There are three forms of the disease:
– Galactose-1 phosphate uridyl
transferase deficiency (classic
galactosemia, the most common
and most severe form)
– Deficiency of galactose kinase
– Deficiency of galactose-6-
phosphate epimerase
27

28. • For the lactococci and some lactobacilli, free galactose
appears to be transported by either a galactose-specific
PTS or by a galactose permease.
• The intracellular product of the galactose PTS (galactose-
6-phosphate) simply feeds into the tagatose pathway.
• When galactose accumulates via galactose permease, the
intracellular product is free galactose.
• Subsequent metabolism occurs via the Leloir pathway,
which phosphorylates galactose, and then converts
galactose-1-phosphate into glucose-6-phosphate .
• The latter then feeds into the glycolytic pathway.
Interestingly, in Lc. lactis, galactose permease may be the
primary means for transporting galactose, since it has a
much higher apparent affinity for galactose than the PTS
transporter.
• The Leloir pathway is used not only by lactococci, but it is
also the pathway used by Lb. helveticus, Leuconostoc spp.,
and galactose-fermenting strains of S. thermophilus. 28

29. Gal80
Galactose
GAL 1,Gal7,lactose
permease, beta
galactosidase gene
expression
29

30. Gal80
No gene expression
Galactose
30

31. • During growth on lactose, these bacteria rely on a
lactose isomerization by mutarotase .
• The subsequent steps convert galactose-1-
phosphate into permease/β-galactosidase system
and therefore generate free intracellular galactose.
• In some instances, they will also encounter free
extracellular galactose, especially if they are grown
in the presence of galactose-nonfermenting
strains, as described earlier.
• Subsequent galactose fermentation by Lb.
helveticus and Leuconostoc lactis occurs via the
Leloir pathway.
• Transport is mediated by a permease, 31

32. • The ability of these strains, especially
lactobacilli, to ferment galactose can be quite
variable, and strain selection is important.
• Galactose fermentation by lactobacilli has also
been used as a basis for distinguishing
between Lb. helveticus (Gal positive) and Lb.
delbrueckii subsp. bulgaricus (Gal negative).
• As noted earlier, some culture suppliers
promote ‘‘nonbrowning’’ cultures for use in
mozzarella cheese production; invariably,
these cultures contain galactose-fermenting
lactobacilli.
32

33. 33

34. Permease
PEP-PTS
Lactose
6-P
Lactose
Lactose
6-PGalactose
6-PTagatose
1,6-PTagatose
GlucoseEMP
P-β-galactosidase
Galactose-6-P isomerase
Tagatose-6-P Kinase
Galactose
1-PGalactose
UDP Glu
UDP Gal
Glucose 1-P
6-PGlucose
Galactose
Galactokinase
Galactose-1-P
uridyl transferase
Phosphoglucomutase
EMP PKP
LeloirPathwayLeloirPathway
Glucose
Galactose
Galactose
S.
thermophilus
Lb.bulgaricus
EMP
Permease
LactococciLactobacilli
β-galactosidase
Tagatose pathwayTagatose pathway
Lactococci
34

35. Alternate Routes for Pyruvate
• As described earlier, LAB are ordinarily considered as being
either homofermentative or heterofermentative, with some
species being able to metabolize sugars by both pathways.
• However, sugar metabolism, even by obligate
homofermentative strains, can result in formation of end
products other than lactic acid by a variety of pathways (Fig.
5).
• In general, these alternative fermentation products are
formed as a consequence of accumulation of excess pyruvate
and the requirement of cells to maintain a balanced
NADH/NAD ratio.
• That is, when the intracellular pyruvate concentration exceeds
the rate at which lactate can be formed via lactate
dehydrogenase, other pathways must be recruited
• to remove pyruvate
• to provide a means for oxidizing NADH.
35

36. • Several enzymes and pathways have been
identified in lactococci and other LAB that are
responsible for diverting pyruvate away from
lactic acid and toward other products.
• In anaerobic conditions, and when
carbohydrates are limiting and growth rates are
low, a mixed-acid fermentation occurs, and
ethanol, acetate, and formate are formed.
• Under these conditions, pyruvate-formate lyase
is activated, and pyruvate is split to form
formate and acetyl-CoA.
• Acetyl-CoA can be converted to ethanol and/or
acetate.
36

37. • The latter also results in formation of an ATP via
acetate kinase.
• If the environment is aerobic, pyruvate-formate
lyase is inactive, and instead pyruvate is
decarboxylated by pyruvate dehydrogenase to
form acetate and CO2.
• Finally, excess pyruvate can be diverted to α-
acetolactate via α- acetolactate synthase.
• This reaction has other important implications,
since α- acetolactate is the precursor for diacetyl
formation
37

38. • Under certain conditions, cells may divert
excess pyruvate to highly desirable products,
specifically the aroma compound diacetyl.
• Ordinarily diacetyl is made from citrate, but
even citrate-nonfermenting cells will make
diacetyl from lactose if appropriate conditions
are established or if cells are genetically
modified.
• For example, overexpression of NADH oxidase
in Lc. lactis decreases lactate formation from
pyruvate, and instead α-acetolactate, the
precursor for diacetyl, is formed.
38

39. Alternative routes of pyruvate metabolism 39

40. Overview of the different metabolic pathways in LAB that could lead to acetaldehyde
formation. Acetyl-CoA, acetyl coenzyme A. 40

41. PROTO-COOPERATION
• The complex and the specific properties of the yogurt are
determined mainly by the biological characteristics of the two
species S. thermophilus and Lb. bulgaricus, their ratio and the
biochemical and microbial activities in their mutual
development.
• The difficulties in obtaining and maintaining of the starter
culture arise from the differences in generation time and
optimal growth temperature for the two species.
• The interaction between S. thermophilus and Lb. bulgaricus in a
yogurt starter culture is described by the ecological term
“proto-cooperation”.
41

42. • Proto-cooperation between these two species LAB is the main
important interaction that determines the fermentation process
and the product quality.
• The mutual fermentation of the selected strains
of S. thermophilus and Lb. bulgaricus with proven compatibility
leads to a mutual benefit for the two thermophilic microorganisms.
• However, the association between these two species is not
obligatory and they can survive separately too.
42

43. • The positive effect of the proto-cooperation between the two species is proven by the
following characteristic of their mutual metabolism during their cultivation in milk :
• The two bacteria species coagulate separately the sterile milk at temperature 45 °С for
6-10 h. The milk coagulates for 2.0-2.5 h when is used a mixed cultures of the two
species;
• The milk coagulated with monocultures of S. thermophilus or Lb. bulgaricus is with
consistency, flavor and aroma different from those of the mixed culture coagulated milk
that is with thick consistency and well expressed lactic acid flavor and aroma ;
• During the mutual fermentation of the two LAB species more volatile aroma
compounds (acetaldehyde, diacetyl and acetone) are produced ;
• In the case of mixed culture fermentation the both species show a higher acid
resistance ;
• In the case of separate cultivation Lb. bulgaricus and S. thermophilus loose faster their
typical morphological characteristics and degrade, while in associated cultivation they
keep longer these characteristics .
43

44. 44

45. • It is known that S. thermophilus goes faster trough lag-phase
reducing the redox potential and active acidity pH from 6.7 to 5.7.
• Thus S. thermophilus supports the growth of L. bulgaricus mainly
by producing lactic and formic acid.
• S. thermophilus assimilates oxygen in the milk faster thus creating
favourable conditions for Lb. bulgaricus growth.
• СО2 produced by S. thermophilus during urea hydrolysis in the milk
stimulates growth of Lb. bulgaricus in their cultivation together.
• Lb. bulgaricus provides the necessary nutrition substances (amino-
acids) for S. thermophilus, and then the thermophilic streptococcus
produces formate, which supports the growth of lactobacillus
45