Lactic Acid Bacteria

LAB or the lactic acid bacteria is a group of gram positive bacteria and produce lactic acid by different pathway i.e.; homofermentative or heterofermentative .They are found in human digestive system.They are beneficial because they have ability to break down proteins,fats ,carbohydrates present in food.They also help in absorption of necessary minerals ,vitamins ,amino acids and other nutrients necessary for the growth and survival.
Applications of LAB
LAB are widely used as probiotics in various fermentative processes. They exert a strong activity against many food-contaminating microorganisms as a result of the production of organic acids, hydrogen peroxide, diacetyl, inhibitory enzymes and bacteriocins. Lactobacillus exerts a positive role in the prevention and treatment of gastrointestinal disorders. Certain Lactobacilli synthesize antimicrobial compounds that are related to the bacteriocin family. Antimicrobial substances produced by LAB are used in association with selective insensitive starter to inhibit competitive microflora. Lactobacilli produce many different bacteriocins of similar activity and are usually predominant species
Bacteriocin
Bacteriocins are the proteinaceous antibacterial compounds .They exhibit bacteriocidal activity against closely related species or strains. Many of them are active against food borne pathogens.Many types of bacteriocins from food-associated LAB have been identified and characterized. The important Bacteriocins found are nisin, diplococcin, acidophilin, bulgarican, helveticins, lactacins, and plantaricins .Nisin is the only bacteriocin commercially available and marketed. Nisin is very active against Gram-positive bacteria, particularly the Spore-formers. Other bacteriocin of Lactobacilli is effective against closely related species of mesophilic Lactobacillus and therefore considered as potential natural food preservatives.According to the original definition, the term bacteriocin refers to proteins of the colicin type, characterized by lethal biosynthesis, intraspecific activity, and adsorption to specific receptors. Those produced by gram-positive bacteria fit closely to the classical colicin model. The bacteriocin are relatively hydrophobic and heat stable.

Applications of bacteriocins
There are many applications of bacteriocins in various industries.Bacteriocins are widely used in pharmaceutical,food, cattle and poultry feed industry etc. They can be used as antimicrobial film for packaging of food items. The antimicrobial film is prepared by incorporation of antimicrobial substances into the extruder when the film or the co ' extruded film is produced.

OBJECTIVE
The main objective of this project is to isolate mainly lactobacillus from milk sample and its strains which produce bacteriocin and show bacteriocin activity. The bacteriocin may be used as antibiotics, for food preservation and providing aseptic conditions in environment.

REVIEW OF LITERATURE
Earlier it was said that the bacteriocin is an antibiotic like compound with specificity primarily restricted to bacterial strains, but later their specificity and chemical composition was found to be different from so called classical antibiotics.

It was observed that the production of a highly specific antibiotic substances by one strain of E.coli was highly active against another strain of the same species. These substances produced by E. coli were called as colicin and other members of the family as Enterobactriaceae. This bacteriocin definition was based on the properties of the colicins, that is to say, a lethal biosynthesis, a very narrow spectrum of activity limited to the same species as the producer bacteria and a receptor mediated mechanism of action.
Later the bacteriocin is also defined as, ribosomally synthesized biologically active proteins or protein complexes having bactericidal mode of action and showing antimicrobial activity spectrum towards Gram positive bacteria and particularly towards closely related species of producers strain. .Many types of bacteriocins from food-associated LAB have been identified and characterized. The important Bacteriocins found are nisin, diplococcin, acidophilin, bulgarican, helveticins, lactacins, and plantaricins .Nisin is the only bacteriocin commercially available and marketed. Nisin is very active against Gram-positive bacteria, particularly the Spore-formers. Other bacteriocin of Lactobacilli is effective against closely related species of mesophilic Lactobacillus and therefore considered as potential natural food preservatives.According to the original definition, the term bacteriocin refers to proteins of the colicin type, characterized by lethal biosynthesis, intraspecific activity, and adsorption to specific receptors. Those produced by gram-positive bacteria fit closely to the classical colicin model. The bacteriocin are relatively hydrophobic and heat stable

Classification of bacteriocins:
Bacteriocins are generally classified from the producing genus or species. For example Colicin from E.coli,Megacins from Bacillus megaterium,Dipthericins from Corynebacterium diphtheria and Pyocin from Pseudomonas pyocynea .
Bacteriocins produced by lactic acid bacteria (LAB) are classified in to four main groups or classes. Out of which lantibiotics or Class I being the most documented and industrially exploited group. .

Bacteriocins produced by LAB
LAB include the genera Lactococcus,Streptococcus,Lactobacillus,Pediococcus, Leuconostoc, Enterococcus, Carnobacterium and Propnibacterium. Lactobacilli are divided in to homo-fermentative and hetero-fermentative. LAB in particular produce a wide variety of antimicrobial substances including organic acids, hydrogen peroxide, secretary reactions products , diacetyl and bacteriocins which have potential to inhibit a variety of other microorganisms or strains.

MODE OF ACTION:

The antibiotic activity of bacteriocin from Gram positive bacteria is based on interaction with bacterial membrane. Some of the bacteriocins workout amphiphilic property generalized membrane disruption by pore formation. LAB produce different types of pore forming peptides. Most of bacteriocins are found from LAB contains bactericidal peptides which act primarily by creating pores in the membrane of their target cells. The formation of pore differ by the size, stability and conductivity from bacteriocins to bacteriocins. The formation of pore complexes causes an ionic imbalance and leakage of inorganic phosphate. These mechanisms based upon stabilizing interactions between membrane phospholipids and the cationic residues of the peptides allowing the insertion of hydrophobic regions into the outer leaflet of the membrane. When one is associated with the membrane surface, a number of the ordered bacteriocins could potentially aggregate.

The bacteriocin complex completely spans the membrane resulting in formation of a transient pore wherein the dissipation of proton motive force (PMF) and involves the partial or total dissipation of either or both the transmembrane potential and a pH gradient. In this way most bacteriocin interacts with anionic lipids which are abundantly present in the membrane of Gram Positive Bacteria. Theses anionic lipids normally enhance the conductivity and stability of antibiotic pores as docking molecule or acts as receptors in different bacteriocins.

ANTIMICROBIAL FOOD PACKAGING:

Antimicrobial packaging is an active packaging, which kills or inhibits spoilage and pathogenic microorganisms which contaminates foods. The new antimicrobial function can also be achieved by adding antimicrobial agents in the packaging system or by using antimicrobial polymer which can satisfy conventional packaging materials to extend the lag phase and reduce growth rate of microorganisms so that it could maintain the quality and safety of food for a long time.
Antimicrobial agents are incorporated into the packaging materials initially which are migrated into the food through diffusion and partitioning. Besides diffusion and equilibrated sorption, some antimicrobial or fungicide or active moieties such as amine groups are also used. In this way it utilizes surface inhibition of microbial growth by immobilization of the non-food grade antimicrobial substances without diffusion mass transfer. The incorporation of antimicrobial substances into a food packaging system is done by adding it in the extruder when the film or the co-extruded film is produced.

APPLICATION OF BACTERIOCIN

As preservatives:
Application of bacteriocin in food preservation and hygiene.
Many physical, chemical, enzymatic and microbiological reactions are responsible for food deterioration. So many preservation techniques are used to avoid different forms of spoilage, food poisoning, reduction in temp, water activity and pH as well as addition of preservatives, such as antimycotic, inorganic and organic compounds may be used to slow or prevent growth of microorganisms. Nisin is the important bacteriocin produced by Lactococcus lactis subsp. Lactis is widely used as food preservatives . It may be used to control some food borne pathogens, especially some species of the genera, Staphylococcus Bacillus, Enterococcus, Listeria, Clostridium, Micrococcus, and Aeromonas. Biopreservatives in the form of protective culture or additives is significant having less potentiality of toxins or carcinogens than current antimicrobial agents, LAB and their by products are more effective and flexible in several applications. In addition of that, functional properties in LAB improve preservatives effect and add flavour and taste.

As Packaging Film:
Bacteriocins are used for packaging films to control food spoilage and pathogenic organisms. Antimicrobial packaging film prevents microbial growth on food surface by direct contact of the package with the surface of foods, such as meat and cheese. For this reason, for it to work, the antimicrobial packaging film must contact the surface of the food, so that bacteriocins can diffuse to the surface. The gradual release of bacteriocins from a packaging film to the food surface may have an advantage over dipping and spraying foods with bacteriocins.Two methods have been commonly used to prepare packaging films with bacteriocins .One is to incorporate bacteriocins directly into polymers. Example includes incorporation of nisin into biodegradable protein films. And other two packaging film ' forming methods, casting and heat press were used to incorporate nisin into films made from soy protein .Polyetelene based plastic film incorporated with nisin was used to vacuum pack bed carcasses.

Another method to incorporate bacteriocin into packaging film is to coat or adsorb bacteriocins to polymer surface. Examples include nisin / methylcellulose coatings for polyetelene films and nisin coatings for poultry. It was demonstrated that, nisin absorbed onto salinized silica surfaces inhibited the growth of Listeria monocytogenes. Nisin films were exposed to medium containing Listeria monocytogenes and the contacting surfaces were evaluated at 4 hours intervals for 12 hours. Cells on surface that had been in contact with a high concentration of nisin (40000 IU/ml) exhibited no signs of growth and many displayed evidence of cellular deterioration. The efficacy of bacteriocins coating on the inhibition of pathogens has also been demonstrated in other studies. For example, coating of pediocin onto cellulose casing and plastic bags has been found to completely inhibit growth on inoculated Listeria monocytogenes in meat and poultry through 12 week storage at 4 degree centigrade. Among the emerging conservation technologies is the use of natural additives such as egg white lysozyme and bacteriocins of LAB to control the growth or survival of undesirable microorganisms. Nisin is currently approved in more than 60 countries for use in cheese, liquid egg products, canned vegetables, diverse pasteurized dairy and salad dressing.

MATERIAL AND METHODS
COLLECTION OF SAMPLES
Sample (1) Curd, Sample (2) Raw milk, were collected from sector- 31, Noida. The samples were collected in aseptic conditions and in sterile containers and transported to the laboratory within 24 hours for experiments.
Procedure for isolation of bacteria from the samples
To prepare 150 ml MRS agar, material required:
Peptone: 1.5gm, beef extract: 1.5gm, yeast extract: 0.75g, glucose: 3 gm, tween 80: 0.15ml, Na2HPO4:0.3 gm, Sodium Acetate: 0.75 gm, Triammonium Citrate: 0.3gm, MgSO4.7H2O:0.03gm, MnSO4. 4H2O: 0.03 gm, distilled water: 150 ml, Agar: 2.25 gm
To prepare 50 ml MRS broth material required:
Peptone: 0.5gm, beef extract: 0.5 gm, yeast extract: 0.25 gm, glucose: 1.0 gm, tween 80:0.05ml, Na2HPO4:0.1 gm, Sodium Acetate: 0.25 gm Sodium Acetate: 0.75 gm Triammonium Citrate: 0.1 gm, MgSO4.7H2O:0.01gm, MnSO4. 4H2O: 0.1 gm, distilled water: 50 ml
PROCEDURE
Bacteria were isolated by serial dilution. 500 micro litres each of samples were serially diluted in 4.5ml of sterile distilled water and homogenized. Serial dilution was carried out on respective samples to obtain dilution factor of 10-3. 0.1 ml of appropriate serial dilution were plated with molten MRS agar plates and incubated an aerobically at 37??C for 48 hours. After incubation, plates were observed for bacterial growth and distinct colonies were randomly selected. Selected colonies were picked and repeatedly streaked on MRS agar plates and incubated for 48 hrs at 35??C until pure cultures were obtained. The pure cultures were maintained on MRS agar plate at 5??C after visible growth on the plate. Randomly from each sample one culture was picked as the desired culture. The colony on the MRS agar plates were sub-cultured in MRS broth for biochemical tests and bacteriocin assay.

Bacteriocin Assay
All the isolates were tested for bacteriocin production by centrifuging 1 ml of broth culture at 10000 rpm for 10 minutes and the supernatant is transferred into a new eppendorf tube followed by another centrifugation to get a pure supernatant.
Escherichia coli, Pseudomonas and Bacillus were taken as the test organism. Bacteriocin activity was tested against these test organisms got from the stored cultures of lab. To check bacteriocin activity, three petriplates with already set nutrient agar were taken. About 100 micro litres of the broth culture of each test organism were spread on nutrient agar plates. Wells were cut in the agar using sterile 1 ml pipette tips.

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