Applications of genetically modified microorganisms: a. Starter cultures b. Genetically modified foods i. Food grade Bio-preservatives ii. Recombinant Dairy enzymes / Proteins
Applications of genetically modified microorganisms:
a. Starter cultures
b. Genetically modified foods
i. Food grade Bio-preservatives
ii. Recombinant Dairy enzymes /
a. Starter cultures
Introduction and History
Starter cultures are an essential component of nearly all commercially produced fermented foods. Simply defined, starter cultures consist of microorganisms that are inoculated directly into food materials in order to bring about desired and predictable changes in the finished product. These changes may include enhanced preservation, improved nutritional value, modified sensory qualities, and increased economic value. Although many fermented foods can be made without a starter culture, the addition of concentrated microorganisms, in the form of a starter culture, provides a basis for insuring that products are manufactured on a consistent schedule, with consistent product qualities.
Fermented foods and beverages have long been manufactured without the use of commercial starter cultures. Traditional methods of production include backslopping, or using a small amount of the finished specifically preserved product to inoculate a new batch, the use of microorganisms found naturally on the product, and the use of special containers that allow for the survival of the starter culture microorganisms within cracks and pores. These traditional methods allow for the development of individual varieties of fermented foods and beverages, and they are still practiced today for small- to mid-scale production facilities, as well as in less developed countries and in homemade-type products. Traditional methods, however, are prone to slow or failed fermentations, contamination, and inconsistent quality. In contrast, modern large-scale industrial production of fermented foods and beverages demands consistent product quality and predictable production schedules, as well as stringent quality control to insure food safety.
Starter culture preservation
Starter cultures produced for the inoculation of bulk starter culture vessels or that have been concentrated for the direct inoculation of cheese vats need to be preserved and stored in a state which does not restrict their viability or activity. This can be achieved by a number of means, including chilling of liquid starter cultures, air drying, vacuum drying or spray drying, although in most cases starter viability and activity are affected. The most effective method of preservation of the starter culture is by adding a cryoprotectant, such as reconstituted skim milk plus lactose, followed by rapidly freezing to below −60°C using liquid nitrogen before storing at between −20°C and −40°C. The disadvantage of this method is the necessity to keep the starter cultures frozen, which can be a problem during shipment to the cheese plant, particularly over long distances. An alternative procedure is freeze-drying where the water is removed from frozen starter cultures by sublimation under vacuum. The dried starter cultures are then stored under an inert atmosphere, which allows them to be transported and stored at ambient temperature, although viability and activity are improved if the freeze-dried starter cultures are stored at lower temperatures.
The effect of the preservation treatment on starter culture proteolytic activity is not well documented. It is possible that the treatment has little effect and it could be that starter strain variation in viability during storage has the major impact on flavour development in maturing cheese.
STARTER CULTURES | Uses in the Food Industry
Starter cultures are preparations of microorganisms serving as inoculants for the production of fermented foods. The production of cheese, yogurt, fermented milk, wine, sauerkraut, hams, and sausages occurs through the use of starter cultures that are consistent, predictable, and safe. The cultures provide the food products with a multitude of properties. Acidification of the food matrix is a primary property in a large number of food fermentations. Acidification activity often will be used to define packaging size and the unit of activity, whereas other characteristics differentiate a culture from the range of other available starter cultures. Starter cultures are commercially available in liquid, frozen, or lyophilized form from several companies serving regional or global markets.
b. Genetically modified foods
i. Food grade Bio-preservatives
Genetically modified foods (GM foods), also known as genetically engineered foods (GE foods), or bioengineered foods are foods produced from organisms that have had changes introduced into their DNA using the methods of genetic engineering. Genetic engineering techniques allow for the introduction of new traits as well as greater control over traits when compared to previous methods, such as selective breeding and mutation breeding
Genetically modified foods are foods produced from organisms that have had changes introduced into their DNA using the methods of genetic engineering as opposed to traditional cross breeding.[26][27] In the U.S., the Department of Agriculture (USDA) and the Food and Drug Administration (FDA) favor the use of the term genetic engineering over genetic modification as being more precise; the USDA defines genetic modification to include "genetic engineering or other more traditional methods"
Genetically modified crops (GM crops) are genetically modified plants that are used in agriculture
Fruits and vegetables
Corn[edit]
Corn used for food and ethanol has been genetically modified to tolerate various herbicides and to express a protein from Bacillus thuringiensis (Bt) that kills certain insects.[98] About 90% of the corn grown in the US was genetically modified in 2010.[99] In the US in 2015, 81% of corn acreage contained the Bt trait and 89% of corn acreage contained the glyphosate-tolerant trait.[42] Corn can be processed into grits, meal and flour as an ingredient in pancakes, muffins, doughnuts, breadings and batters, as well as baby foods, meat products, cereals and some fermented products. Corn-based masa flour and masa dough are used in the production of taco shells, corn chips and tortillas.[100]
Soy[edit]
Soybeans accounted for half of all genetically modified crops planted in 2014.[73] Genetically modified soybean has been modified to tolerate herbicides and produce healthier oils.[101] In 2015, 94% of soybean acreage in the U.S. was genetically modified to be glyphosate-tolerant.[42]
Rice[edit]
Golden rice is the most well known GM crop that is aimed at increasing nutrient value. It has been engineered with three genes that biosynthesise beta-carotene, a precursor of vitamin A, in the edible parts of rice.[102] It is intended to produce a fortified food to be grown and consumed in areas with a shortage of dietary vitamin A,[103] a deficiency which each year is estimated to kill 670,000 children under the age of 5[104] and cause an additional 500,000 cases of irreversible childhood blindness.[105] The original golden rice produced 1.6μg/g of the carotenoids, with further development increasing this 23 times.[106] In 2018 it gained its first approvals for use as food.[107]
Wheat[edit]
As of December 2017, genetically modified wheat has been evaluated in field trials, but has not been released commercially
Biopreservation exploits the antimicrobial activities of some microorganisms to inhibit the growth of spoilage and pathogenic microbes in foods. This biological approach seeks to minimize the addition of chemical additives to foods, such as nitrite, sodium chloride, and organic acids. Research in the field of biopreservation for meat products remains active because it is perceived that natural methods of preservation are desirable. Most research on biopreservation has focused on the antagonistic activities of lactic acid bacteria against spoilage and pathogenic bacteria. However, in the past decade, the use of bacterial viruses (bacteriophages) to eliminate pathogenic bacteria from foods has gained considerable attention.
ii. Recombinant Dairy enzymes
Introduction
The increasing use of enzymes to produce specific products with characteristic attributes can be emphasized by the world-wide sale of industrial enzymes which was about US $ 3.0 billion in 2008. Principal among some enzymes that have important and growing applications are lipases and β-D-galactosidases. Enzymes with limited applications include glucose oxidase, superoxide dismutase, sulphydryl oxidase, etc. The usage of microbial enzymes is important for the development of milk and milk products with new physical and functional properties in the food industry. The native milk enzymes can be exploited in several ways during processing including an index of thermal treatment of milk and for consumer health and food safety, e.g., to combat bacterial invasion and growth.
Enzymes native to milk
There are over 70 enzymes in milk, encompassing a wide range of activities including lipases, proteinases, alkaline phosphatase, lactoperoxidase, lysozyme, cathepsin D, lysosomal enzymes, etc. The most highly characterized enzymes include lactoperoxidase, lysozyme, plasmin, lipoprotein lipase and xanthine oxido-reductase. Many processing technologies including heat treatment destroy many of these enzymes in milk. The enzymes are predominately associated with the milk fat globule membrane (MFGM) and vesicle membranes in milk. Use of enzymes as food additives Several enzymes are used to improve the quality of food products or to enhance yield or even to produce ‘value-added ingredients’ for use in food industry. Some of the more frequently used enzyme in dairy industry is discussed herein.
I. Rennet
Technically rennet is the term for the lining of a calf's fourth stomach. The most common enzyme isolated from rennet is chymosin. Chymosin can also be obtained from several other animal, microbial or vegetable sources, but indigenous microbial chymosin (from fungi or bacteria) is ineffective for making Cheddar and other hard cheeses. The use of rennet in cheese manufacture was among the earliest applications of exogenous enzymes in food processing, dating back to approximately 6000 BC. Animal rennet (bovine chymosin) is conventionally used as a milk-clotting agent in dairy industry for the manufacture of cheeses with desired flavour and texture. Owing to an increase in demand for cheese production world wide ( i.e. 4% per annum over the past two decades), coupled with reduced supply of calf rennet, has led to search for rennet substitutes, such as microbial rennets. At present, microbial rennet is used for one-third of the cheeses produced world wide.
Rennet action in cheese making
Chymosin has low general proteolytic activity, but high milk-clotting activity. The rennet coagulation of milk is a two-stage process. The first (primary) phase involves the enzymatic production of ‘para-casein’ and ‘glycomacropeptides,’ while the second (secondary) phase involves the calcium-induced gelation of para-casein at a temperature of 30–35oC. Proteolysis is essentially complete before the onset of coagulation. k-casein is cleaved by chymosin and for most of the other proteinases used as rennets, at the bond Phe105-Met106, which is many times more susceptible to hydrolysis by acid proteinases (which include all commercial rennets) than any other bond in milk protein system. Pepsins and most other acid proteinases used as rennets hydrolyze -casein at Phe105-Met106, but the acid proteinase of Cryphonectria parasitica hydrolyzes Ser104-Phe105.
Rennet substitutes
Many microorganisms are known to produce rennet-like proteinases which can substitute calf rennet. Microorganisms like Rhizomucor pusillus, Rhizomucor miehei, Endothia parasitica, Aspergillus oryzae and Irpex lactis are used extensively for rennet production in cheese manufacture. Only six rennet substitutes have been found to be more or less acceptable: bovine, porcine and chicken pepsins and the acid proteinases from Rhizomucor miehei, Rhizomucor pusillus and C. parasitica. Microbial rennets from various microorganisms (marketed under the trade names viz., Rennilase, Fromase, Marzyme, Hanilase, etc.) are being marketed since 1970s and have proved satisfactory for the production of different kinds of cheese. Recently Novo Nordisk has succeeded in expressing one proteolytic enzyme from the fungus Rhizomucor miehei in organism Aspergillus oryzae. This host organism is able to produce the single protease that cleaves -casein at phe105–met106 peptide bond (as for Chymosin). This mono component enzyme product is being sold under trade name ‘Novoren’. Limited supplies of calf rennet have prompted genetic engineering of microbial chymosin by cloning calf prochymosin genes into bacteria. Bioengineered chymosin is reported to be involved in production of up to 70% of cheese products. Properties of rennet substitutes vis-à-vis chymosin Among the microbial milk clotting enzymes, the ones produced by Rhizomucor miehei and Rhizomucor pusillus have gained wide industrial acceptance. However, the proteolytic specificities of these fungal coagulants are different from those of calf chymosin. Fungal rennet preparations have high milk clotting activity but they exhibit a degree of tertiary (or residual) proteolytic activity resulting in the production of bitter peptides during cheese ripening. Therefore, cleavage specificity as opposed to extended proteolysis (ratio of milk clotting activity/ proteolytic activity) defines a good milk coagulant. Thermal stability of the coagulant is another important issue for being considered to be a good milk coagulant, depending on the cheese type. If the coagulant used is thermally labile, extensive unspecific proteolytic activities during cheese maturation could be prevented for the cheese varieties subjected to cooking process.
Recombinant rennet
One major drawback of using microbial rennet in cheese manufacture is the development of off flavour and bitter taste in the unripened as well as ripened cheeses. Hence, attempts have been made to clone the gene for calf chymosin, and to express it in selected bacteria, yeasts and molds. Due to shortage of calf stomachs and the economic value of cheese rennet, gene for calf chymosin was cloned and expressed in microorganisms. Several workers have cloned the gene for calf prochymosin in Escherichia coli. The enzymatic properties of recombinant E. coli chymosin are indistinguishable from those of native calf chymosin. The gene for calf chymosin has been cloned in selected bacteria, yeasts, and molds. Chymosin from genetically engineered Kluyveromyces marxianus var. lactis (Gist-brocades), Escherichia coli (Pfizer), and Aspergillus nidulans (Hansens) is commercially available and used extensively; however, these products are not yet permitted in all countries. The yeasts Saccharomyces cerevisiae and Kluyveromyces lactis and the filamentous fungi Aspergillus niger var awamori and Trichoderma reesei have been successfully used as hosts for the expression of recombinant calf chymosin which are now marketed commercially such as Maxiren (DSM Food Specialities, Netherlands) and Chymax (Chr. Hansen, Denmark). The gene for prochymosin has been cloned in Saccharomyces cerevisiae; the levels of expression are reported to be between 0.5 to 2.0% of total yeast protein. Microbial recombinant chymosin preparations do not contain any pepsin, whereas 5–50% of the milk-clotting activity of calf rennets may be due to pepsin. No major differences have been detected between cheeses made with recombinant chymosin or natural enzymes with regard to cheese yield, texture, smell, taste and ripening.
II. Proteinases/Proteases Proteinases have found additional applications in dairy technology, for example in acceleration of cheese ripening, modification of functional properties and preparation of dietetic products.
III. Lipases Lipases are extensively used in the dairy industry for hydrolysis of milk fat. The dairy industry uses lipases to modify the fatty acid chain lengths, to enhance the flavour of various cheeses. Current applications also include the acceleration of cheese ripening and the lipolysis of butterfat and cream
IV. Lactase Lactase is a glycoside hydrolase enzyme that cuts lactose into its constituent sugars viz., galactose and glucose. Lactase can be obtained from various sources like plants, animal organs, bacteria, yeasts (intracellular enzyme), or molds. Some of these sources are used for commercial enzyme preparations. Lactase preparations from A. niger, A. oryzae and Kluyveromyces lactis are considered to be safe
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