LACTIC ACID BACTERIALecturer: HUL Seingheng Students: I3-B GCA, Group10

TEV KuntheaTHAING SreythuokYAM KoemfyYANN SokhounYUN Pheakdey

2010-2011

INTRODUCTION Lactic acid bacteria are present in all grape musts and wines.

It play an important role in transformation of grape must into wine.

THE DIFFERENT COMPONENTS OF THE BACTERIA CELL

Prokaryotic cell with an extremely simple organization.

1. Cellular envelopA. Cell wall

Composed of a peptidoglycan Contian ribitol phosphate or

glycerol phosphate polymers

THE DIFFERENT COMPONENTS OF THE BACTERIA CELL

B. Plasmic Membrane A lipid bilayer membrane lipid

• Phospholipid• glycolipids .

THE DIFFERENT COMPONENTS OF THE BACTERIA CELL

B. Plasmic Membrane Phospholipids consist of a glycerol

molecule which has a primary alcohol and secondary alcohol function esterified by fatty acids.

THE DIFFERENT COMPONENTS OF THE BACTERIA CELL

B. Plasmic Membrane Glycolipids

Generally glycosides of glycerides

Are formed by glycosidic bonds between a fatty acids(possess a long hydrocarbon chain and a terminal carboxylic acid function)

THE DIFFERENT COMPONENTS OF THE BACTERIA CELL

It also contain a characteristic cyclopropanic acid : lactobacillic acid.

THE DIFFERENT COMPONENTS OF THE BACTERIA CELL

2. The cytoplasm The cytoplasm contains the main

elements for cell operation : enzyme , nuclear material and sometimes reserve substance.

THE DIFFERENT COMPONENTS OF THE BACTERIA CELL

3. The nucleus The bacteria nucleus consist of a

single circular chromosome of double straned DNA suspended in in the cytoplasm without any separation .

Its size varies depending on the species and strain of bacteria.

THE DIFFERENT COMPONENTS OF THE BACTERIA CELL

3.The nucleus The chromosome carries the

essential genetic information of cell.

THE DIFFERENT COMPONENTS OF THE BACTERIA CELL

4. Multiplication of Bacteria

All bacteria multiply by binary division(a cell gives two completely identical daughter cells)

TAXONOMY The objective of taxonomy is to

identify , describe and class microorganism.

Highest level is prokaryote Lowest level is species

Lactic acid bacteria belong to Gram-positive group(violet coloration).

It is the primary production of metabolism of glucose.

PHENOTYPIC TAXONOMY, MOLECULAR TAXONOMY AND PHYLOGENY

Phenotypic TaxonomicInclude morphological, physiological, biochemical and immunological characters.

Molecular Taxonomy Classification according to similarities

in their genome. Among Gram-positives, lactic acid bacteria belong

to the Phylum Clostridium.

MOLECULAR TAXONOMY

PHENOTYPIC TAXONOMIC, MOLECULAR TAXONOMY AND PHYLOGENY

PhylogenyThe evolutionary development

of a species or of a taxonomic group of organisms.

Another type of analysis consist of sequencing the 16 S , 23 S and 5 S RNA (ribosomals).

CLASSIFICATION OF WINE LACTIC ACID BACTERIA. DESCRIPTION OF GENERA Sequencing of the 16 S rRNA devide lactic acid

bacteria into three phylogenetic groups: The group Lactobacillus

delbruekii The group Leuconostoc

One containing L.paramesenteroides and heterofermentative lactobacilli.

Two containing Leuconostoc sensu stricto

The group Lactobacillus casei

CLASSIFICATION OF WINE LACTIC ACID BACTERIA. DESCRIPTION OF GENERAGroup I: strict homofermenters

(has never been identified in wine) ,do not ferment pentose.

Group II: facultative herterofermenters (pentose are fermented to lactic and acetic acids )

Group III: strict herterofermenters(ferment glucose to , lactic and acetic acids, ethanol; pentose to lactic and acetic acids )

DESCRIPTION OF GENERA

In general, there are three genera of lactic acid bacteria:

Genus Leuconostoc Oenococcus

Genus Pediococcus Genus Lactobacillus

IDENTIFICATION OF LACTIC ACID BACTERIA General Principle

Since the beginning of microbiology, the identification of bacteria has been based on their phenotypic characters.

For long time, lactic acid bacteria of wine were identified by their phenotypes but now with DNA analysis.

IDENTIFICATION OF LACTIC ACID BACTERIA Phenotypic Analysis

Microscopic observation is couple with the Gram coloration test, which is used to verify that bacteria are Gram-positive.

Determine the character of homofermentative or heterofermentative.

IDENTIFICATION OF LACTIC ACID BACTERIA Phenotypic Analysis

A release of CO2 manifests the heterofermentative character of the strain.

Conversely, the exclusive formation of lactic acid attests to acid attests to a homofermentative character.

Determine the optical nature of lactic acid formed from glucose.

IDENTIFICATION OF LACTIC ACID BACTERIA Phenotypic Analysis• Two stereoisomers of lactic acid(L

and D) identification of heterofermentative cocci (Oenococcus oeni, Leuconostoc mesenteroides) form the D isomer, and of Lactobacillus casei forms L-lactic acid.

PHENOTYPIC ANALYSIS

EXTRACTION AND VISUALIZATION OF DNA FOR GENOMIC STUDY

Centrifugation Electrophoresis

IDENTIFICATION BASED ON RESTRICTION FRAGMENT LENGTH POLYMORPHISM

This method consists of hydrolyzing the DNA with the help of restriction enzymes. Restriction have 3 form

Restriction productsRestriction polymorphismRestriction profile

IDENTIFICATION BY SPECIFIC PROBE DNA/DNA HYBRIDIZATIONHybridization is a technique often

used in molecular genetics and it is very well adapted for the identification of species and even strains.

DNA/DNA hybridization is also an excellent tool for identifying strains that differ in phenotype but belong to the same species.

IDENTIFICATION BY POLYMERIZATION CHAIN REACTION(PCR)

PCR consists of using polymerization to amplify one or more DNA fragments, located by specific sequences.

The PCR technique uses two oligonucleotide primers, chosen for their complementary sequences: each one is complementary to a single strand of the DNA target.

IDENTIFICATION BY POLYMERIZATION CHAIN REACTION(PCR)

The products of PCR are analyzed

by electrophoresis. The specificity of PCR is based on

the level of hybridization between the oligonucleotides and the template.

IDENTIFICATION BY FATTY ACID AND PROTEIN COMPOSITION Fatty acid

The total fatty acids are dosed in the form of esters after sapomification.

Bacteria can only be identified by their composition in total fatty acids. when the culture of the cells to be analyzed is standardized .

IDENTIFICATION BY FATTY ACID AND PROTEIN COMPOSITION

Protein Composition: Bacteria cell protein constitute another level of genomic expression .

CONCLUSION This presentation is based on lactic

acid bacteria . It mention about the different

methods to identify lactic acid bacteria.

Lactic acid bacteria play an important role in the transformation of grape musts into wine.