Lesson 19. APPLICATION OF GENETIC ENGINEERING IN DAIRY STARTER CULTURES

Module 5. Application of biotechnology in dairying

Lesson 19
APPLICATION OF GENETIC ENGINEERING IN DAIRY STARTER CULTURES

19.1 Introduction

Currently genetic manipulation techniques are in extensive use and are playing an important role in enhancing the performance of microorganisms in dairy products processing. The two important variables that can be manipulated through genetic engineering for use in dairy field industry include

i) Bacteria, used as starter cultures for the production of lactic acid, flavors, proteolysis and also in the by-product utilization and

ii) Enzymes, used for various metabolic changes in dairy foods for bringing desirable changes.

Genetic manipulation of microorganisms with significant applications to foods was initiated by taking Escherichia coli as a microbial model because of its ease of growing and manageability. A number of genetic modifications can be made in industrial as well as laboratory strains of lactic acid bacteria. These include deletion of a gene from a strain; replacing a gene with a similar gene from another strain; replacing a gene in a strain with the same gene that has been modified in vitro; increasing the number of copies of a gene; introducing a new gene and using recombinant DNA as selection principle for the isolation of mutants with target DNA.

19.2 Target Areas for Biotechnological Applications in Dairying

Modification of genetic components of bacteria and manipulation of enzymatic action offer dramatic prospects in dairy industry. A potential application of genetic engineering for dairy industry is in the generation of enhanced starter cultures for yoghurt and cheese manufacture. Some of the target areas of genetic manipulation for dairy industry are given in Table 19.1

Table 19.1 Target areas for genetic manipulation in dairying

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19.2.1 Metabolic properties of dairy starter cultures

Lactose utilization, proteolysis and citrate fermentation are the key industrially important metabolic functions of dairy starter cultures. The pioneering work on genetic engineering in dairy starter cultures was initiated by Larry McKay and co-workers in early 70’s. Because of industrial relevance, L. lactis has been regarded as a model organism.

19.2.1.1 Lactose utilization

The primary function of starter cultures is the production of lactic acid from lactose which is mediated mostly by plasmid DNA in several lactic acid bacteria. There are ample chances for the loss of lac+ plasmid during the multiplication of starter cultures and hence lactic acid production has been reported to be unstable. The main biotechnological emphasis would be the stabilization lac+ in chromosome of LAB for sustained production of lactic acid from lactose. The first notable success was reported by McKay and Baldwin in 1978 by improving a strain by the stabilization of the lac/prt plasmid in chromosome. In the last two decades, significant effort was put into the genetic investigations of LAB and the complete genome sequences have been published for four strains i.e. Lactococcus lactis IL1403 and MG1363, SK11 and KF147. Recently a nisin-producing starter culture that produced acid at rates suitable for Cheddar cheese manufacture was developed by combining the naturally occurring lactose-fermenting, nisin-producing, and proteinase-positive strain Lactococcus lactis ssp. lactis NCDO 1404 and the lactose-positive, nisin-positive, proteinase-positive transconjugant Lactococcus lactis ssp. cremoris JS102.

19.2.1.2 Proteolysis

Caseins are the most significant substrates for proteolysis in the preparation of fermented milks and in cheese ripening. Increasing proteolytic activity and improving starter culture’s amino acid converting activities are the major aspects taken into consideration for cheese falvor improvement.

The enzymes involved are proteinases, either endopeptidases (with active sites in the interior of the polypeptide chain, yielding peptides as the primary product) or exopeptidases (with active sites at the ends of the polypeptide chain, yielding amino acids as the primary product). To stabilize these enzyme genes, food-grade approaches to genetic engineering of lactic acid bacteria rely either on a “chromosomal integration of a target gene” approach, or on the construction of food-grade vectors fulfilling the self-cloning definition. Overexpression of genes encoding components of proteolytic system has been achieved in many species of lactic acid bacteria under industrial conditions of cheese making.

19.2.1.3 Falvor production

The two important organisms used in dairy industry for citrate utilization for falvor development are Leuconostoc spp. and L. lactis subsp. lactis biovar diacetylactis. Citrate permease the key enzyme in citrate fermentation is linked with an 8-kb plasmid in Lactococcus lactis subsp. lactis biovar diacetylactis, whereas in Leuconostoc the citrate genes are associated with plasmids as large as 22 kb. This metabolic property is also not stable since they are coded by plasmids, the unstable DNA entities. Two promising approaches are visualized for stabilization of this metabolic property i.e. stabilization of plasmid DNA in LAB or inactivation of the gene encoding α-acetolactate decarboxylase (aldB), the enzyme that converts α-acetolactate to acetoin. The falvor and falvor stability of buttermilk was improved by inactivation of the aldB gene encoding α-acetolactate decarboxylase (Swindell et al. 1996). This later approach results in accumulation of α-acetolactate, the immediate precursor to diacetyl, which in turn leads to an increased concentration of diacetyl in the growth medium.

19.2.2 Bacteriophage resistance

Bacteriophage infection during the manufacture of fermented milk products is an ever-present danger leading to significant economic losses to fermented milk industry. To address this problem, development of bacteriophage resistance in starter cultures has attracted extensive scientific interest. Many phage-resistance plasmids are conjugative, and this factor has been exploited to improve the phage resistance of phage-sensitive commercial cultures. The technique is relatively simple and involves the isolation of the strain harboring the phage-resistance plasmid and mixed with lac+ phages recipients. Lac+ phager transconjugants are selected in the presence of excess virulent phage on lactose agar, which contains a dye to indicate acid production and checked for their ability to produce acid and for the presence of the phage-resistance plasmid. Bacteriophage resistant strains by conjugal transfer of plasmid encoding phage resistant determinants have been developed and successfully used at industrial level.

19.2.3 Natural biological inhibitors

Another starter enhancement that has been extensively studied using genetic manipulation in LAB is the production of small proteins called bacteriocins that inhibit related bacteria.

Some of the well characterized bacteriocins produced by starter bacteria have been shown in Table 19.2

Table 19.2 Examples of well characterized bacteriocins

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One such important antimicrobial substance, nisin, is produced by the "food grade" starter strain, Lactococcus lactis ssp. lactis and is used as a natural preservative in food systems. It has activity against a wide variety of Gram-positive bacteria, including food-borne pathogens such as Listeria, Staphylococcus and Clostridium spp. Genetically engineered Lactococcus spp. showed enhanced production of Nisin due to the introduction of multiple copy genes like nisZ, nisRK, or nisFEG that are responsible for Nisin production. María et al. (2010) developed a Lactococcus lactis UQ2 strain that performed well in milk and synthesized 200 IU/mL nisin, 40 times more than the original strain by conjugal mating.

19.2.4 Rennet substitutes

Rennet is the key enzyme used as a milk-clotting agent during the production of cheese. Due to acute shortage of calf rennet and or socio-cultural reasons, there is a great emphasis for the use of rennet substitutes due to reduction or elimination of the usage of conventional rennet obtained from calf stomach.

One of the most success stories of commercial genetic engineering is the manufacture of recombinant chymosin. The gene coding for the chymosin enzyme has been cloned into several microorganisms and commercially exploited by successful expression in bacteria (Escherichia coli, and Bacillus spp,), yeast (Kluyveromyces lactis) and moulds (Mucor miehei, Mucor pumilus, Aspergillus niger). Manufacturers claim that these enzymes cause more consistent milk clotting and have better proteolytic activities, offering a more standardized product. This rennet substitute has more predictable properties than animal-derived chymosin and has fewer impurities. In future, genetic manipulation of enzyme together with scientific advances in understanding the protein structure, allow the possibility of improving the properties and making specific changes in the structure of the enzyme.

19.2.5 Accelerated cheese ripening

Traditional ripening of cheese takes 6 to 12 months depending on the type of cheese under rigorously controlled conditions. This enormously increases the cost of cheese production due to maintenance of refrigeration of ripening rooms and blockage of capital cost. Now the enzymes synthesized from genetically modified bacteria like proteinases, lipase and β-D-galactosidase are used for enhancing the ripening of cheese. So far, the most effective example of accelerating the cheese ripening process by metabolic engineering has been the nisin-induced expression of bacteriophage lysin and holin in L. lactis, resulting in complete lysis of the cells, complete release of peptidases and of other enzymes and a sharp increase in production of free amino acids and falvor compounds in cheese. Yet another example for commercially successful enzyme is Neutrase produced from Bacillus subtilis which is reported to be helpful in decreasing the ripening period by 50%.

19.2.6 Tackling lactose intolerance

Lactose intolerance problem has gained worldwide dimensions as a vast majority of population is not able to digest the lactose due to inability of synthesizing lactase enzyme in their intestine. Biotechnology has offered a solution by utilizing β -D-galctosidase to hydrolyse lactose into glucose and galactose. This enzyme is commercially obtained from yeast (Kluyveromyces lactis), fungi (Asperigillus niger) Bacteria (E. coli, Bacillus stearothermophilus, lactic acid bacteria used as dairy starters)

Ex: In USA Lactacid, a brand of modified milk is available in which 70% of lactose is hydrolysed.

19.2.7 Microbial epoxy polysaccharides

The conventional hydrocolloids of polysaccharides are derived from gums, mucilages and extracts of plant origin. These are having different functional properties owing to their molecular structural diversity and are useful in providing stability and preventing wheying off of fermented products. Due to high cost and increasing demand for hydrocolloids, attention has been shifted for the development of bacterial cultures capable of producing polysaccharides and having synergistic growth capability with other dairy cultures. FAO has already permitted many microbial polysaccharides and some are under toxicological safety testing.

EPS synthesis might be plasmid mediated as seen in L. lactis and Lactobacillus casei, or located on the chromosome, as the case may be in all the thermophilic LAB. Enhanced EPS synthesis can be achieved by

a. Complete gene clusters, encoding exopolysaccharide producing enzymes can be transformed from one LAB strain to another one and the newly generated strains could influence viscosity and texture of the fermented product (Germond et al. 2001)

b. In S. thermophilus, the phosphoglucomutase gene was inactivated resulting in improved exopolysaccharide production enhancing the viscosity of the fermented food product (Levander et al. 2002).

c. Using a self-cloning strategy also, exopolysaccharide production in L. lactis can be enhanced

19.2.8 Vaccine delivery

Recently Lactococcus lactis has been shown to deliver antigens that stimulate mucosal immunity to non respiratory pathogens as well as Human papilloma virus and the malarial parasite. Lactic starter cultures also possess strong adjuvant properties that can also be explored during oral vaccine development for boosting antibody production in the host. L. lactis being the non-pathogenic food grade bacteria shows much efficacy as live antigen and enzyme carriers, thus it proves beneficial for the oral administration than the attenuated pathogenic microorganisms like Salmonella typhi and Vibrio chlorella. The bacterium can be genetically engineered to produce proteins from pathogenic species on their cell surfaces. Intra nasal inoculation with the modified strain will elicit an immune response to the cloned protein and provide immunity to the pathogen.

As per the reports available, Lactococcus spp., could be engineered to present the conserved portion of the streptococcal M protein required for streptococcal adherence and colonization to the nasopharyngeal mucosa, in order to provide immunity to Streptococcus pyogenes. Consequently, the resulting local immune response could protect the individual from strep throat caused by the streptococcus. Similar approach can be used for other human pathogens such as Streptococcus pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis and Neisseria meningitides. Moreover, L. lactis is non-invasive and does not colonize the gut and thus has less potential to trigger immune-tolerance or side effects upon prolonged use

19.2.9 Other areas of importance

a. Single cell protein (SCP) represents microbial cells (primary) grown in mass culture and harvested for use as protein sources in foods or animal feeds. Synthesis of SCP by employing genetically engineered yeast cultures of Saccharomyces spp. and Kluyeromyces sp. has been successful and is in wide use.

b. Many health benefits have been identified with probiotic use and it is possible to identify several desirable enhancements that might be made to existing probiotic bacteria. The combination of a probiotic and a prebiotic is known as a ‘synbiotic’ and the prebiotic is thought to enhance the survival of the probiotic. The desirable property could be the generation of probiotics that are genetically modified to digest speciality ‘prebiotic’ carbohydrates. There is considerable interest at the present time in identifying novel prebiotic oligosaccharides and synbiotic combinations and many new possibilities would be offered if this activity were to proceed in parallel with the development of probiotic strains designed to metabolize them.

Books:

Biotechnology of lactic acid bacteria-Novel applications: Fernanda Mozzi, Raul R. Raya and Graciela M. Vignolo (Eds) 2010 : Wiley-Blackwell Publications

References

Germond, Jacques-Edouard; Delley, Michèle; D'amico, Nicola and Vincent, Sébastien (2001) Heterologous expression and characterization of the exopolysaccharide from Streptococcus thermophilus Sfi39. European Journal of Biochemistry, Vol. 268 : 5149-5156.

Levander, Fredrik; Svensson, Malin and Radstrom, Peter (2002). Enhanced exopolysaccharide production by metabolic engineering of Streptococcus thermophilus. Applied and Environmental Microbiology, Vol. 68: 784-790.

McKay L. L. and K. A. Baldwin (1978) Stabilization of Lactose Metabolism in Streptococcus lactis C2., Appl Environ Microbiol Vol 36(2): 360–367.

Sanders, M.E., (1988) Phage resistance in lactic acid bacteria, Biochimie Vol 70: 411-422.

Second Symposium on Lactic Acid Bacteria: Genetics, Metabolism and Applications. (1987). FEMS Microbiology Reviews, 46, 201-379.

Swindell, S.R.; Benson, K.H.; Griffin, H.G.; Renault, P.; Ehrlich, S.D. and Gasson, M.J. (1996) Genetic manipulation of the pathway for diacetyl metabolism in Lactococcus lactis. Applied and Environmental Microbiology, Vol. 62: 2641-2643.

María D García-Parra, Ana B Campelo, Blanca e García-Almendárez, Carlos Regalado, Ana Rodríguez, Beatriz Martínez (2010) Enhancement of nisin production in milk by conjugal transfer of the protease-lactose plasmid pLP712 to the wild strain Lactococcus lactis UQ2, International Journal of Dairy Technology 63: 523–529

Last modified: Tuesday, 23 October 2012, 6:58 AM