Microscopic View

LACTIC ACID BACTERIA:

Normal flora of humans in the oral cavity
and the intestinal tract.
Gram-positive, non-sporeforming
coccobacilli or rods

cocci,

Non sporulating and non respiratory and

lack catalase.
Low G+C.
Lactic acid as the major metabolite
Grow in the range of 10C -45C with pH
from 4.4-9.6.
Each genera comprises a well described
species ranging from 10 100 numbers
forms an ideal candidate for diversity study.

Catalase Test

MEMBERS OF LACTIC ACID BACTERIA

Sugar fermentation
by LAB
Homofermentation
Homofermentation
11Hexose+
Hexose+22ADP+2Pi
ADP+2Pi

2Lactate
2Lactate++22ATP
ATP

The members of Pediococcus, Lactococcus, Streptococcus, Vagococcus, along

with some lactobacilli are homofermenters.

Heterofermentation
Heterofermentation
11Hexose+
Hexose+11ADP+Pi
ADP+Pi
11Hexose+
2
ADP
Hexose+ 2 ADP++PiPi

Lactate
Lactate++Ethanol
Ethanol+CO2+
+CO2+1ATP
1ATP
Lactate
+
Acetate
+CO2+
2ATP
Lactate + Acetate +CO2+ 2ATP

The members of Carnobacterium, Oenococcus, Enterococcus, Lactosphaera,

Weissells and Lecconostoc and some Lactobacilli are heterofermenters

The heterolactics are more important than the homolactics in

producing flavour and aroma components such as acetylaldehyde
and diacetyl.

Functional properties of lactic acid bacteria

Properties

Description

Acidification

Production of lactic acid that reduces pH

Coagulation

Proteolytic activity causes proteolysis of milk

proteins

Vitamin synthesis

Biosynthesis of folate, Riboflavin

Phytic acid degradation

Phytase production

Oligosaccharide synthesis

Transgalactosylation activity of -galactosidase

Diacetyl production

Production of aroma in Butter

Exopolysaccharide production

Produced by glucansucrase and fructansucrase

activity that contribute to rheology and texture
of product

Antimicrobial activity

Bacteriocin production, biopreservation.

Antioxidant activity

Superoxide
peroxidase

10

Alanine

Flavor production

SN.

dismutase,

cinnamyl

esterase,

Metabolites produced by Lactic Acid Bacteria

CH3hydantoin

MA-lactone
Bacteriocins

Alcohol
H2O2

CO2

LAB
Organic Acid

Diacetyl

Reuterin

Benzoic Acid

Industrial applications of LAB

Pharmaceutical

Food Industry

Chemical industry
(Biodegradable plastics)

Lactic acid
Biopreservative
(Nisin Bacteriocin)
Exopolysaccharide
(EPS)
Therapeutic Proteins
(Interleukin-10
Tetanus toxin fragment C)

Streptococcus
Lactobacillus

Lactococcus
Leuconostoc
Pediococcus

Flavour compounds
(Diacetyl. Acetoin,
Aldehydes)
Starter culture
(Fermented food
with functional
properties)

Biomass
Probiotic with functional properties

Yogurt
Cheese
Buttermilk
Cream
Fermented
Meat
Fermented
Vegetables

Diversity
The number and variety of species present in an area
in accord with their spatial distribution.

Species
Diversity

Ecosystem
Diversity

Genetic
Diversity

Species Diversity: Is the effective number of different

species that are represented in a collection of
individuals.
Ecosystem Diversity: Is the variety of species and
ecological processes that occur in different physical
settings.

Group

Global species diversity

No. of described Species

Bacteria and blue-green algae

Fungi
Algae
Bryophytes (Mosses and Liverworts)
Gymnosperms / Angiosperms (flow plants)
Protozoans / Sponges
Corals and Jellyfish

4,760
46,983
26,900
17,000
750 // 250,000
30,800 // 5,000
9,000

Genetic diversity:
It is defined as any variation in the nucleotides of DNA, genes, chromosomes, or
whole genomes of organisms and serves as a way to adapt to changing environments
Greater the genetic diversity within a species, higher the chances of long-term
survival.
In biodiversity, genetic diversity comprises total number of genetic characteristics in
the genetic makeup of a species. On the other hand genetic variability describes the
tendency of genetic characteristics.
Measures of genetic diversity:
Genetic diversity can be measured directly at genes level that are represented by
genetic code.
Gene diversity and heterozygosity are the important tools to assess genetic
diversity.
In gene diversity, we calculate the proportion of polymorphic loci across the genome,
where as in case of heterozygosity, mean number of individuals with polymorphic
loci is being considered.

Importance of genetic diversity:

Genetic diversity protects a species against extinction by
providing multiple phenotypes.
The phenotypic differences may provide disease resistance,
adaptability to a changing climate, or some other trait
necessary for the species to survive in the ever-changing
world.
Generally, multicellular organisms tend to have more DNA than
single-celled organisms. Similarly, there appears to be an
overall trend of increasing amount of DNA with increasing
complexity of organisms.
Genetic variation is attributable not to differences in the
number of functional genes, but in the amounts of non-coding
DNA.

Genetic Fingerprinting v/s

Genetic diversity
Fingerprinting: Unambiguous identification
of an individual (cultivar, inbred line or
individual from a population)
Microsatellites, the parts of the DNA are used
in genetic fingerprinting.
In a species with low diversity, a large number
of microsatellites ( for eg: with a lot of
different bead strings) have to be identified
in order to tell individuals apart.
In a high-diversity species, it would probably
take 5 or 6 microsatellites to tell individuals
apart.

Cluster Analysis
ClusterAnalysisreferstoagroupofmultivariatetechniqueswhoseprimarypurposeistogroup
individualsorobjectsbasedonthecharacteristicstheypossess,sothatindividualswithsimilar
descriptionsaremathematicallygatheredintothesamecluster(Hairetal.,1995).

Clustersshouldexhibithighinternal(withincluster)homogeneityandhighexternal(betweencluster)
heterogeneity
3. Analysis of gene
copy number for
the specific trait

4. Signature pattern
on agarose gel

Analysis of genotypes
Generation of data set
Genetic
Similarity/Distance
matrix
Analysis of Gs/GD
matrix using specific
clustering algorithm

2. Extraction of gDNA
from isolated
bacterial cultures

Graphical presentation

1. Fermented food with

bacterial entities

Interpretation of genetic
relationships

Molecular techniques employed for bacterial Diversity

Nature of study

Mol technique/s

Application

Reference

Genomic diversity

RAPD-PCR

Identification of pediococcal
species

Simpson et al. 2002

Microbial succession

Genus specific PCR

LAB dynamics in fermenting

cucumber

Singh and Ramesh, 2008

Population dynamics

PCR DGGE

Sourdough fermentation

Van der Meulen et al.

2007.

Genetic diversity

repPCR, 16S rDNA sequencing

LAB diversity in cassava

fermentation

Kostinek et al. 2005

Microbial community
evaluation

PCR-RFLP

Dominant species identification

Ruiz et al. 2010

Bacterial community structure

ARDRA

Microflora of kimchi

Kim and Chun 2005

Community dynamics and

pattern of succession

DGGE

LAB colonization in vacuum

packed food

Fontana et al. 2006.

Genetic diversity

16S rDNA sequencing

LAB diversity in fermented tofu

Chao et al. 2008

Relative resolution of various fingerprinting and DNA

Family

genus

species

DNA sequencing
16S rDNA sequencing
ARDRA
DNA-DNA
reassociation

tRNA PCR

ITS - PCR
RFLP LFRFA PFGE
Multilocus isozyme
Whole-cell protein profiling
AFLP
RAPDs APPCR
rep- PCR

subspecies

Hierarchical clustering Algorithms

Single linkage (nearest neighbour)

Complete linkage (further neighbour)

Unweighted pair-group average (UPGMA)
Weighted pair-group average (WPGMA)
Unweighted pair-group centroid (UPGMC)
Weighted pair-group centroid (WPGMC)
Wards method of minimum variance

A
B
C

4
3.84

Diversity

3.68
3.52
3.36
3.2
3.04
2.88
0

0.5

1.5

2.5

3.5

RAPD profile of EryR LAB carrying

TetR

I. Enterococcus

II. Pediococcus &

Lactobacillus
III. Lactobacillus

Rao SC & Halami PM (2011) Syst Appl Microbio

RAPD dendrogram of LAB cultures isolated from marine source

Gene Organisation

Pediocin Distribution

Food Fermentation

Foods that have been subjected to the action of micro-organisms or enzymes, in order to bring
about a desirable change.

Numerous food products owe their production and characteristics to the fermentative activities of
microorganisms.

Fermented foods originated many thousands of years ago when presumably micro-organism
contaminated local foods.
Micro-organisms cause changes in the foods which:
Help to preserve the food,
Extend shelf-life considerably over that of the raw materials from which they are made,
Improve aroma and flavour characteristics,
Increase its vitamin content or its digestibility compared to the raw materials.

Benefits of fermentation
Benefit
Preservation
Enhancement of safety
Acid production
Acid and alcohol production
Production of bacteriocins
Removal of toxic components

Raw
material

Fermented
food

Milk
Yoghurt, cheese
(Most materials)

Fruit
Barley
Grapes
Meat
Cassava
Soybean

Enhancement of nutritional value

Improved digestibility
Retention of micronutrients
Increased fibre content
Synthesis of probiotic compounds

Wheat
Leafy vegetables.
Coconut
Milk

Improvement of flavour

Grapes

Vinegar
Beer
Wine
Salami
Gari, polviho azedo
Soy sauce

Bread
Kimchi, sauerkraut
Nata de coco
Bifidus milk, Yakult,
Acidophilus yoghurt
Wine

Starter Cultures

Traditionally the fermenting organisms came from the natural microflora or a portion of the
previous fermentation.

In many cases the natural microflora is either inefficient, uncontrollable, and unpredictable, or is
destroyed during preparation of the sample prior to fermentation (eg pasteurisation).

A starter culture can provide particular characteristics in a more controlled and predictable
fermentation.

Lactic starters always include bacteria that convert sugars to lactic acid, usually:
Lactococcus lactis subsp. lactis,
Lactococcus lactis subsp. cremoris or
Lactococccus lactis subsp. lactis biovar diacetylactis.

Where flavour and aroma compounds such as diacetyl are desired the lactic acid starter will
include heterofermentative organisms such as:
Leuconostoc citrovorum or
Leuconostoc dextranicum.
The primary function of lactic starters is the production of lactic acid from sugars
Other functions of starter cultures may include the following: flavour, aroma, and alcohol production
proteolytic and lipolytic activities, inhibition of undesirable organisms

A good Starter culture will:

Convert most of the sugars to lactic acid

Increase the lactic acid concentration to 0.8 to 1.2 %

(Titratable acidity)

Drop the pH to between 4.3 to 4.5

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

Kimchi Saurkraut Yoghurt