Starter Cultures and Fermented Milk Products

Module 9. Genetic improvement of starters

Lesson 14
PLASMIDS, VALUE OF IMPROVED STRAIN

14.1 Introduction

Genes of bacteria are arranged in two ways; as a major long DNA molecule the bacterial chromosomes and additional smaller non essential units of DNA, the plasmids. Plasmids are extra chromosomal covalently closed circular DNA molecules existing independently of main genetic machinery of cell i.e. chromosomal DNA and exhibiting autonomously replicating nature. Under normal conditions a plasmid is dispensable. However plasmids are reported to contain genes that are essential or confer a selective advantage to cells in certain environments. Plasmids are characteristically unstable entities. When a bacterial cell divides, each daughter cell receives a copy of chromosomal DNA along with a copy or copies of plasmids in parent. Though plasmid replicated independently of the chromosome, any mutation resulting in failure of plasmid replication results in daughter cell that does not receive a copy of that plasmid and thus is unable to perform the functions dictated by the plasmid. For this reason plasmid associated traits may be more unstable than functions controlled by chromosomal genes.

14.2 Lactic Acid Bacteria

Lactic Acid Bacteria (LAB)are gram positive non spore forming micro aerophilic bacteria whose main fermentation product from carbohydrate is lactate and comprises of cocci such as streptococus, pediacoccus, leuconostoc and rods such as lactobacillus and bifidobacterium. These bacteria are exhibiting different pathways of carbohydrate fermentation with end products that are exclusively lactates termed as homofermentative and if produces at least 50% lactate they are termed as heterofementative. With the exception of bifidobacteria all genera of lactic acid bacteria are phylogenetically related as evidenced by serological and 16s rRNA homology studies.

LAB play an important role in the manufacture of a large variety of fermented dairy products viz,yoghurt, dahi, cheese, acidophilus milk, cultured cream and cultured buttermilk. These fermentations are initiated with starter cultures for which over the years specific strains and combinations of strains have been used. The main function which have been selected are efficient and reproducible fermentative conversion of lactose into lactate, formation of desired aroma compounds or proteolytic degradation products of milk proteins so it is essential that dairy industry have its disposal microorganisms capable of fermenting milk satisfactorily.

Now it has been established that those fermentation of the lactic acid bacteria are controlled by plasmids. In addition to the wide spread use of lactic acid bacteria for biotechnological processes particularly dairy and other food fermentations has undoubtedly provided a major stimulus for the recent advances in genetic analysis of these bacteria

Table 14.1 Main products of fermentation

14.3 Plasmid Mediated Functions

a) Lactose utilization

b) Proteinase activity

c) Metabolism of citrate

d) Production of bacteriocin like substance

e) Resistance to nisin

f) Resistant to certain antibiotics

g) Utilization of sucrose &other sugars

h) Resistance to bacteriophage

i) Bacteriophage adsorption

j) Restriction – modification system.

Table14.2 Plasmids in lactic acid bacterai

14.4 Important of Plasmids

• Plasmids are unstable and this explains the irreversible loss of properties which are required for successful dairy fermentation.
• Bacteria also gain plasmids and acquire new properties via gene transfer systems.
• Provide a useful fingerprint which can be exploited in a sample way to investigate strain identities and interrelationships.
• Developing the recombinant DNA technology, to develop new strains or super strains.

The most definite studies on plasmid biology and their molecular genetics involving various gene transfer systems have been made with lactococci but to some considerable extent lactobacilli were also studied with the emphasis on their plasmids.

Lactococci are reported to contain remarkably large and complex plasmid complements with observed number varying between 2 to 11 with molecular weights ranging between 1.0 and80.0 Mdal. Most of the plasmids in lactococci are cryptic i.e. no known function is associated with particular plasmid. The large no of plasmids of cryptic nature are formed be due to coexistence of deletion derivatives from aparental plasmid. Usually the higher molecular plasmids are known to code important dairy fermentative properties like lactose utilization and Proteinase activity.

14.5 Value of Improved Strains

Starter cultures for fermented foods are today developed mainly by design rather than by screening. The design principles are based on knowledge of bacterial metabolism and physiology as well as on the interaction with the food product. In the genomics era, we will obtain a wealth of data making design on a rational basis even simpler. The design tools available are food grade tools for genetic,metabolic and protein engineering and an increased use of laboratory automation and high throughput screening methods. Successful cultures based on modern technology is expected to be launched in the areas of: probiotics, bioprotection, general improvement of yield and performance for the existing culture market and probably the introduction of cultures for fermenting other food products.

14.6 Some Notable Research Achievements

1. Peptidolytic activity can be increased by overproduction of peptide degrading enzymes originating from the same species or from other species. An example of the self-cloning strategy is the overproduction of an aminopeptidase (PepN) by L. lactis. GM-LAB with increased proteolytic properties are generated by the over expression of the genes encoding PepN, PepX, PepC and PepI peptidases from a highly proteolytic L.helveticus strain

2. The transformation of industrially important strains with phage resistance genes from other LAB could generate new phage resistant strains. Engineering of starter strains aimed at inhibiting phage development is possible by creating a system of lethal gene induction upon phage infection or by production of antisense RNA inhibiting translation of phage RNA.

3. Another important feature of food products is texture. Complete gene clusters, encoding exopolysaccharide producing enzymes could be transformed from one LAB strain to another one. The newly generated strains could influence viscosity and texture of the fermented product. In S. thermophilus the phosphoglucomutase gene was inactivated resulting in improved exopolysaccharide production enhancing the viscosity of the fermented food product. Engineering of exopolysaccharide production in L.lactis was also achieved by using a self-cloning strategy.

4. The flavour and flavour stability of buttermilk could be improved by inactivation of the aldB gene encoding α-acetolactate decarboxylase. The over expression of peptidasegenes in LAB via self-replicating plasmids could also enhance flavour formation and cheese ripening.

5. The quality of food fermentation processes could be further improved when they are protected from spoilage by other bacteria. The expression of the food-grade antibiotics in dairy starter strains could prevent spoilage from Gram positive bacteria during the process of cheese ripening.

6. The nutritional value of fermented foods can be improved via use of GM-LAB. It was shown that by over expressing the complete riboflavin gene cluster (four genes) via a multi copy plasmid and strong promoter, riboflavin production reached levels as high as 8mg/L. With this production level in fermented dairy products, average servings would already contain the DRI of riboflavin. Similarly by the over expression of genes encoding for the folate biosynthesis pathway in L. lactis,folate levels ranging from 5 - 8 mg/L can be achieved

Engineering of a L. lactis strain that contributes to the formation of lactose-free and naturally sweetened foods is now a reality. By the deletion of three genes involved in glucose uptake and degradation, and introduction of the genes for lactose-PTS and tagatose-6P pathway, the strain can grow on lactose. The galactose-moiety of lactose is used as a C-source,whereas the glucose moiety is completely excreted.