LACTIC ACID BACTERIA

NAME : T.L.V.PEIRIS

DEPARTMENT:FOOD SCIENCE

UNIVERSITY: UNIVERSITY OF SRI JAYAWARDENAPURA

YEAR : AUGUST 2009

STUDENT NUMBER: GS/MSc/Food/3630/08

Summery

Lactic acid bacteria in food are considered as GRAS. This report contents various aspects of
lactic acid bacteria. This report gives where these microorganisms thrive and their physiological
characteristics. It further explains in very descriptive manner the major metabolic pathways
which they use to metabolize sugar. Homofermentive metabolic pathway, Heterofermentative
metabolic pathway (pentose phosphate pathway) and EMP pathways are given in detail.
Furthermore this report gives the usage of Lactic acid bacteria in food and furthermore
importance of Lactic acid bacteria as probiotics also briefly explained.

Key words: Lactic acid , Heterofermentative pathway, Homofermetative Pathway, EMP

pathway, probiotics

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Contents Page number

Introduction 04

Characteristics 04-05

Bio chemistry of Lactic acid bacteria 06-14

i. metabolism of sugars

ii. Homfermentive metabolism of hexose

iii. Heterofermentative metabolism of hexoses

iv. Metabolism of malic acid

v. Embden-Meyerhoff-Parnas pathway (EMP)

i. Activation of Glucose

ii. Hexose Splitting

iii Energy Extraction

iv. End Product Formation

Lactic acid bacteria - their uses in food 15

Probiotics and LAB 16

Reference

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Introduction

The Lactic Acid Bacteria (LAB) comprise a clade of Gram-positive, low-GC, acid-tolerant,
generally non-sporulating, non-respiring rod or cocci that are associated by their common
metabolic and physiological characteristics. These bacteria, usually found in decomposing plants
and lactic products, produce lactic acid as the major metabolic end-product of carbohydrate
fermentation. This trait has, throughout history, linked LAB with food fermentations, as
acidification inhibits the growth of spoilage agents. Proteinaceous bacteriocins are produced by
several LAB strains and provide an additional hurdle for spoilage and pathogenic
microorganisms. Furthermore, lactic acid and other metabolic products contribute to the
organoleptic and textural profile of a food item. The industrial importance of the LAB is further
evidenced by their reputed safe (GRAS) status, due to their ubiquitous appearance in food and
their contribution to the healthy microflora of human mucosal surfaces. The genera that comprise
the LAB are at its core Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and
Streptococcus as well as the more peripheral Aerococcus, Carnobacterium, Enterococcus,
Oenococcus, Sporolactobacillus, Teragenococcus, Vagococcus, and Weisella; these belong to
the order Lactobacillales.

Characteristics

The Lactic Acid Bacteria (LAB) are rod-shaped bacilli or coccus. LAB are characterized by an
increased tolerance to a lower pH range. This aspect partially enables LAB to outcompete other
bacteria in a natural fermentation, as they can withstand the increased acidity from organic acid
production (e.g., lactic acid). Laboratory media used for LAB typically includes a carbohydrate
source as most species are incapable of respiration. LAB are catalase negative.

There are two main hexose fermentation pathways that are used to classify LAB genera. Under
conditions of excess glucose and limited oxygen, homolactic LAB catabolize one mole of
glucose in the Embden-Meyerhof-Parnas (EMP) pathway to yield two moles of pyruvate.
Intracellular redox balance is maintained through the oxidation of NADH, concomitant with
pyruvate reduction to lactic acid. This process yields two moles ATP per glucose consumed.
Representative homolactic LAB genera include Lactococcus, Enterococcus, Streptococcus,
Pediococcus, and group I lactobacilli.

Heterofermentative LAB use the pentose phosphate pathway, alternatively referred to as the
pentose phosphoketolase pathway. One mole Glucose-6-phosphate is initially dehydrogenated to
6-phosphogluconate and subsequently decarboxylated to yield one mole of CO2. The resulting
pentose-5-phosphate is cleaved into one mole glyceraldehyde phosphate (GAP) and one mole
acetyl phosphate. GAP is further metabolized to lactate as in homofermentation, with the acetyl
phosphate reduced to ethanol via acetyl-CoA and acetaldehyde intermediates. In theory, end-
products (including ATP) are produced in equimolar quantities from the catabolism of one mole
of glucose. Obligate heterofermentative LAB include Leuconostoc, Oenococcus, Weissella, and
group III lactobacilli.

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Lactic acid bacteria

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Metabolism of lactic acid bacteria
Metabolism of Sugars

Lactic acid bacteria are chemotrophic, they find the energy required for their entire metabolism
from the oxidation of chemical compounds. The oxidation of sugars constitutes the principle
energy producing pathway.
Lactic acid bacteria of the genera Lactobacillus, Leuconostoc and Pediococcus, the important
bacteria to winemaking, assimilate sugars by either a homofermentative or heterofermentative
pathway.
Homofermentative Metabolism of Hexoses
Homofermentative bacteria transform nearly all of the sugars they use, especially glucose into
lactic acid. The homofermentative pathway includes a first phase of all the reactions of
glycolysis that lead from hexose to pyruvate. The terminal electron acceptor in this pathway is
pyruvate which is reduced to lactic acid. See Figure 1. In fermentation, pyruvate is
decarboxylated to ethanal, which is the terminal electron acceptor, being reduced to ethanol.

Heterofermentative Metabolism of Hexoses

Bacteria using the heterofermentative pathway, which includes Leuconostoc (the most important
bacterium in enology) use the pentose phosphate pathway. In this pathway, NADPH is generated
as glucose is oxidized to ribose 5-phosphate. This five-carbon sugar and its derivatives are
components of important biomolecules such as ATP, CoA, NAD+, FAD, RNA and
DNA. NADPH is the currency of readily available reducing power in cells (NADH is used in the
respiratory chain). This pathway occurs in the cytosol.

After being transported into the cell, a glucokinase phosphorylates the glucose into glucose 6-P
(glucose 6-phosphate). Its destination is completely different from the glucose 6-P in the
homofermentative pathway. Two oxidation reactions occur: the first leads to gluconate 6-P and
the second, accompanied by a decarboxylation, forms ribulose 5-P. See Figure 2. In each
of these reactions a molecule of NADP+ is reduced. Ribulose 5-P can then be epimerized either
to ribose 5-P or to xylulose5-P.

Xylulose 5-P is then cleaved into acetyl-phosphate and glyceraldehydes 3-phosphate. See Figure
3. The glyceraldehyde 3-phosphate is metabolized into lactic acid by following the same
pathway as in the homofermentative pathway. The acetylphosphate has two possible
destinations, depending on environmental conditions.
This molecule can be successively reduced into ethanal and ethanol, in which case the molecules
of the coenzyme NADPH formed during the two oxidation reactions of glucose at the beginning
of the heterofermentative pathway, are reoxidized.
This reoxidation is essential for regenerating the coenzymes necessary for this pathway. The
final products are then lactate and ethanol.
Or the acetyl-phosphate can produce acetate (acetic acid) through the enzyme acetate kinase.
This reaction also yields a molecule of ATP. The final products of this pathway are then lactate
and acetate.
Bacteria of the genus Leuconostoc preferentially produce lactate and ethanol in a slightly aerated
environment and lactate and acetate in an aerated environment.

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Metabolism of Malic Acid
The majority of bacterial species preponderant in wine after alcoholic fermentation break down
malic acid.
The most important bacterium in enology is the heterofermentative bacteria, Leuconostoc oenos.
This bacterium forms Dlactic acid from glucose and L-lactic acid from L-malic acid.

The major distinction between wine and vinegar is the amount of acetic acid. The amount of
acetic acid is estimated from the volatile acidity or VA (the wine is steam distilled and the
distillate titrated with sodium hydroxide using phenolphalein). A small amount of acetic acid is
produced by yeasts, in particular Saccharomyces cerevisiae to the extent of 100-300 mg.L-1
(see Figure 5, Yeast Biochemistry, Sugars).
Bacteria degrade must and wine sugars with a different affinity depending on the species. In
general, bacterial development occurs after yeast development. Since the yeast has consumed the
sugars, the lactic acid formed from them is small compared to the amount produced from malic
acid.
L. oenos can and does produce acetic acid from glucose (see figures 3 & 4), but since the lactic
acid formed from them is low, so is the acetic acid.
An increase in the VA (acetic acid) of a wine coupled with an abnormal amount of lactic acid
(>300 mg.L-1) indicates lactic disease and suggests the L. oenos fermented a significant quantity
of sugars.

Glycolysis - Embden-Meyerhoff-Parnas pathway (EMP)

EMP is the most commonly used series of reactions for oxidizing glucose to pyruvate and many
bacteria, animals and plants employ this pathway in their catabolism. EMP is so ubiquitous that
is worthwhile to use it as an example of a typical fermentation. It is an essential part of many
organisms catabolism, even yours! However, it is not the only method for the fermentation of
glucose. Remember that bacteria are remarkably creative and other pathways are present in
different species.

EMP can be divided into 3 stages, activation of glucose, hexose splitting and energy
extraction

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Activation of Glucose

Glucose is a relatively stable molecule and in order to degrade it, it must first be destabilized by
adding high energy phosphates. In the first step a phosphate is donated from ATP (or
phosphoenolpyruvate - the source of the phosphate depends on the species of microbe you look
at) to glucose to form glucose-6-phosphate. The molecule is isomerized to fructose-6-phosphate
(another sugar) and a second phosphate is added. Fructose-1,6-bisphosphate is easier to attack
than glucose and is ready to be split.

Figure 3 - Activation of glucose by phosphorylation with ATP.

Hexose Splitting

Fructose bisphosphate aldolase then breaks the phosphate loaded fructose into two 3 carbon
compounds, glyceraldehyde-3-phosphate (GAP) and dihydroxyacetonephosphate (DAP). This is
the crucial step in the EMP pathway, converting the 6 carbon glucose molecule to two 3 carbon
molecules that will eventually become pyruvate.

Figure 4 - Splitting of Fructose by aldolase.

Energy Extraction

In the next reaction, DAP is converted into GAP, which can be acted on by the rest of the EMP.
The next step is a very important one. Inorganic phosphate is added to GAP to make 1,3-
bisphosphoglycerate (BPG). No energy is required and in fact electrons are transferred from
GAP to NAD+. This reaction is the payback for running the pathway and the phosphates added
here are later transferred to ADP to make ATP. After several enzymatic rearrangements, the final
product of the EMP pathway is pyruvate.

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Figure 5 - Extraction of Energy. Note the two reactions highlighted in blue that yield energy.
Remember that each glucose molecule that comes into glycolysis generates two GAP molecules
that can then proceed down the latter half of the pathway.

The total reaction can be summarized as follows

2 ATP + glucose + 4 ADP + 2 Pi + 2 NAD+ 2 ADP + 2 pyruvate + 4 ATP + 2

NADH

The blue highlight denotes energy put into the reaction. Subtracting this from the energy
extracted, the net energy gain is 2 ATP per glucose. That is a lot of work for just 2 ATP.
Fermentations do not yield large amounts of energy and this explains why fermenting microbes
go through so much substrate without much growth.

Some of the NADH that is generated can be used for cell biosynthesis, but there is a large excess
or reducing power. Fermenting bacteria must find a way to get rid of these extra electrons and
they do it by adding them to pyruvate to form end products.

End Product Formation

One of the more familiar fermentations is conversion of glucose to ethanol to form alcoholic
beverages. After the formation of pyruvate, ethanol is formed by two simple reactions. First, CO2
is removed from pyruvate to form acetaldehyde. Then acetaldehyde is reduced by, you guess it,
NADH

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Figure 6 - Oxidation of NADH. Acetaldehyde is reduced to ethanol (the active ingredient in
alcoholic beverages). This is the final step in yeast fermentation of glucose to ethanol.

Another favorite microbial fermentation is the formation of lactic acid. This is performed by the
lactic acid bacteria. Homofermentative lactic acid bacteria use the EMP pathway to make
pyruvate and then reduce it to lactate using up their excess NADH in the process. Other bacteria
use alternative pathways to generate lactate from glucose. Close examination of the
heterofermentative pathway reveals that it does not use EMP at all. The take home message is,
EMP is common, but there are many other ways of doing business.

figure 7 - Formation of lactate by homofermentative bacteria. The pathway used is identical to

glycolysis. The final end product is lactate which is excreted by the cells into their environment.

Figure 8 - Fermentation of glucose by heterofermentative bacteria. In this pathway the top part of
the glycolytic pathway is not used. Note the recycling of NADH and the low yield. Only one
ATP is generated per glucose fermented.

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Lactic acid bacteria - their uses in food

Lactic acid bacteria have been used to ferment or culture foods for at least 4000 years. They are
used in particular in fermented milk products from all over the world, including yoghurt, cheese,
butter, buttermilk, kefir and koumiss.

Although they are best known for their role in the preparation of fermented dairy products, they
are also used for pickling of vegetables, baking, winemaking, curing fish, meats and sausages.

Without understanding the scientific basis, people thousands of years ago used lactic acid
bacteria to produce cultured foods with improved preservation properties and with characteristic
flavours and textures different from the original food.

Similarly today, a wide variety of fermented milk products including liquid drinks such as kefir
and semi-solid or firm products like yoghurt and cheese respectively, make good use of these
illustrious microbial allies.

The manufacture involves a microbial process by which the milk sugar, lactose is converted to
lactic acid. As the acid accumulates, the structure of the milk protein changes (curdling) and thus
the texture of the product. Other variables such as temperature and the composition of the milk,
also contribute to the particular features of different products.

Lactic acid also gives fermented milks their slightly tart taste. Additional characteristic flavours
and aromas are often the result of other products of lactic acid bacteria. For example
acetaldehyde, provides the characteristic aroma of yoghurt, while diacetyl imparts a buttery taste
to other fermented milks. Additional micro-organisms such as yeasts can also be included in the
culture to provide unique tastes. For example, alcohol and carbon dioxide produced by yeasts
contribute to the refreshing, frothy taste of kefir, koumiss and leben. Other manufacturing
techniques such as removing the whey or adding flavours, also contribute to the large variety of
available products.

For yoghurt, the manufacture depends on a symbiotic relationship between two bacteria,
Streptococcus thermophilus and Lactobacillus bulgaricus, where each species of bacterium
stimulates the growth of the other. This interaction results in a shortened fermentation time and a
product with different characteristics than one fermented with a single species.

With yoghurt and other fermented milks there are considerable opportunities for exploiting lactic
acid bacteria as probiotic cultures. These supplement and help our normal gut bacteria to
function more efficiently. The world-wide market for these products continues to increase in
response to the demands of an increasingly health-conscious public.

Lactic acid bacteria are therefore excellent ambassadors for an often maligned microbial world.
They are not only of major economic significance, but are also of value in maintaining and
promoting human health.

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Probiotics and LAB

Probiotics are products aimed at delivering living, potentially beneficial, bacterial cells to the gut
ecosystem of humans and other animals, whereas prebiotics are non-digestible carbohydrates
delivered in food to the large bowel to provide fermentable substrates for selected bacteria.
Strains of LAB are the most common microbes employed as probiotics. Two principal kinds of
probiotic bacteria, members of the genera Lactobacillus and Bifidobacterium, have been studied
in detail.

Most probiotic strains belong to the genus Lactobacillus. Probiotics have been evaluated in
research studies in animals and humans with respect to antibiotic-associated diarrhoea, travellers'
diarrhoea, pediatric diarrhoea, inflammatory bowel disease, and irritable bowel syndrome. It is
possible that, in the future, probiotics will be used for different gastrointestinal diseases,
vaginosis, or as delivery systems for vaccines, immunoglobulins, and other therapies.

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