Biotechnology in Food

Processing
and Preservation
• A range of technologies is applied at different levels and scale of operation in food processing
across the developing country. Low input and conventional technology includes drying,
evaporation, canning, dehydration, freezing, vacuum packing, osmotic dehydration, sugar
crystallization, etc.
• Processing assures food security by minimizing waste production and reducing the food chain
and increasing food availability and marketability.
• The purpose of food processing is to improve its quality and security.
• Food safety is a scientific discipline, which ensures that a particular food will not be the reason
of any injury to the consumer when it is manufactured and eaten according to its deliberate use.
• Biotechnology plays a pivotal role to improve the taste, flavors, color, texture, aroma of foods,
and its aesthetic and nutritional value; it is extensively used in many countries.
• Food undergoes fermentation by intentional inoculation or by natural fermentation and
eventually these desirable changes appear due to fermentation by microorganisms and/or their
enzymes, flavor, fragrance, food additives, and other value-added products.
• These high value products are used in food and nonfood use and also imported to other countries.
• Food processing involves various unit operations and techniques to convert raw, perishable, and
inedible products to consumable form with enhanced quality and shelf life.
• To produce a safe and high-quality food, the process and manufacturing protocol used in the
food processing must be of food grade, that is, free from health hazards.
• Safe food can be defined as the food that contains no harmful components that affects human
health and nutrition.
• Biotechnology is also widely employed as a tool in diagnostics to monitor food safety, prevent,
and diagnose food-borne illnesses and verify the origin of foods.
• Techniques applied in the assurance of food safety focus on the detection and monitoring of
hazards whether biological, chemical, or physical.
• Fermentation is generally used to make desirable changes in food.
• Fermentation can be carried out naturally or by intentional inoculation.
• Fermentation is the process in which carbohydrates are converted into alcohol and carbon
dioxide or organic acids when yeasts, bacteria, or a combination of them works on the food in
the absence of air.
• Fermentation is used to produce wine, beer, cider, leavening of bread, and lactic acid.
• Natural fermentation leads human history.
• The earliest evidence of fermentation dates back to 7000–6000 BC.
• It was an alcoholic beverage, made from fruits, rice, and honey in the Neolithic age in Chinese village of
Jiahu.
• Winemaking was prevalent in 6000 BC in Georgia.
• There was a jar containing traces of 7000 years old wine displayed at the University of Pennsylvania,
excavated from mountains in Iran.
• Also, the traces provided the proofs regarding the production of fermented products in Babylon c.3000 BC,
ancient Egypt c.3150 BC, pre-Hispanic Mexico c.2000 BC, and Sudan c.1500 BC.
• Louis Pasteur, French chemist was first to connect yeast to fermentation in 1856.
• He defined fermentation as respiration without air.
• Fermentation, useful for conversion of sugars and other carbohydrates into preservatives and other organic
acids, is the result of his research.
• Fermentation is generally used in food processing as it
• modifies diet by enrichment of flavors, aromas, and food texture;
• preserves food by production of acids;
• enriches food with protein, essential amino acids, and vitamins;
• removes antinutritional factors; and
• decreases process time.

Antinutritional factors are those substances generated in natural food substances

by the normal metabolism of species and by different mechanisms (e.g., inactivation
of some nutrients, diminution of the digestive process or metabolic utilization of
feed) which exert effects contrary to optimum nutrition
Methods to Improve the Quality of Microbial
Strain
• In traditional biotechnology, microbial cultures are improved for use in food processing
application by improving the quality of microorganisms and the yield of metabolites
using mutagenesis, conjugation, and hybridization (for yeast Saccharomyces cerevisiae
strain used in baking, brewing, and beverage production methods).
• Recombinant genetic engineering is the best-known technique to alter the purified
microbial strain related to food fermentations following the norms and regulations as per
customer awareness.
• Genetically modified (GM) strains are applied in the manufacture of enzymes, vitamins,
PUFA, amino acids and other fatty acids.
Applications of some food additives and processing aids derived from Genetically
modified (GM) microorganisms
Enzymes from GM microorganisms
 Genetically Modified Plants
Methods of Production of Genetically Modified Plant
• GM modified plants are generated by the biolistic method (Particle gun method) or by Agrobacterium
tumefaciens mediated transformation method.
• In biolistic or gene gun method, the gene is directly shot at the plant cell under high pressure.
• This method has been applied successfully for many crops, especially monocots, such as wheat or maize, for
which transformation using A. tumefaciens has been less successful.
• This technique is clean and safe.
• The only disadvantage of this process is that serious damage can happen to the cellular
tissue.
• In agrobacterium-mediated piece of DNA, which infects a plant is integrated into a plant chromosome,
through a tumor inducing plasmid along with the genetically engineered (GE) strain.
• Recent advancements in plant sciences and agricultural biotechnology offer new opportunities
and possibilities to improve the yield, quality, and production economics of food crops.
 Vitamin-Rich Plants

• Vitamins play a vital task on human health by varying metabolic circumstances and supporting
the biochemical processes that liberate energy from foods during digestion, making of
hormones, blood cells, nervous-system chemicals, and other genetic materials.
• Deficiency of any vitamin can cause serious health disorder. Transgenic plants can be
manufactured using knowledge of biotechnology, with increased content of vitamins in certain
crops.
 Vitamin-A
• Reduced form of vitamin A (retinal) is the source of rhodopsin.
• Rhodopsin is essential for vision and also employs to preserve epithelial and immune
cells.
• The retinoic acid is essential during the embryonic development and for homeostasis in
adult body and its scarcity develops the sign of night blindness to total blindness.
• Fruits and vegetables are principal source of β-carotene and are predecessors of vitamin-A
(Fig. 1).
• In carotenoid pathway, the extent of β-carotene formed by plants can be enhanced by
increasing the flux by raising the availability of carotenoid precursors, by communicating
enzymes in the early part of the pathway between geranyl pyrophosphate and lycopene
(Fig. 2).
Fig. 1. Biosynthesis of Vitamin A
Fig. 2. Conversion of β-
Carotene From Geranyl
Geranyl Pyrophosphate
(GGPP).
Lcy, Lycopene cyclase.
• For example, transgenic rice was developed by Swiss Federal Institute of Technology, Zurich,
Switzerland in collaboration with University of Freiburg.
• In this transgenic rice, expressing genes for β-carotene was incorporated.
• Four steps are involved in β-carotene biosynthesis of rice grain.
• This rice appeared yellow in color.
• β-Carotene (1.6 µg) is present in selected line per gram of rice endosperm and was
recognized as “golden rice.”
• The first strategy is production of carotenoid over expression in tomato by using DXP
synthase that enhances flux in the entire pathway and enhances the total carotenoid content .
 Vitamin-C
• Ascorbic acid is an important antioxidant and cofactor for various enzymes.
• It improves immunity, boost cardiovascular functions, alleviate ailment relating to connective
tissue, and it is essential for iron metabolism.
• Humans cannot synthesize ascorbic acid due to absence of l-gulono-1,4-lactone oxidoreductase,
which is needed during biosynthesis of ascorbic acid.
• Vitamin-C-rich plants are the only dietary sources of vitamin-C for humans.
• In plants, biosynthesis of vitamin-C takes place in two ways.
• First, with the conversion of d-galactouronic acid to l-galactouronic acid by d galactouronic acid
reductase enzyme followed by conversion of l-galactouronic acid to l-galactano-1,4-lactone,
immediate predecessor of ascorbic acid.
• d-galacturonic acid reductase enzyme encoding gene was isolated from strawberry and
characterized as galUR (Fig. 3) .
Fig. 3. Biosynthesis of Vitamin C.
• In alternative method vitamin-C is synthesized by recycling.
• It was hypothesized that by enhancing the expression of dehydroascorbate reductase
(DHAR) in plants, ascorbic acid synthesis also increases, and a proficient ascorbate
recovery would be accomplished.
• Following the ascorbate recycling pathway, expressing the rice dhar gene in multivitamin
maize, 6 times enhanced level of ascorbate compared to normal level had been observed
(Fig. 4).
 (glutathione)
(monodehydroascorbate
reductase)

Fig. 4. Recycling of Ascorbic Acid

 Vitamin E

• Vitamin E belongs to tocotrienol and tocopherol families and is lipid soluble.

• Mainly in plants, vitamin is produced during photosynthesis.
• Vitamin-E is important because of its therapeutic properties.
• It is best known for its activity against cancer, degenerative disorders, and cholesterol.
• Tocotrienol is more powerful antioxidant than tocopherol but not absorbed as readily.
• Researchers have taken initiative to grow vitamin-E-rich plants.
• Various methods are being used.
• Vitamin-E is not a single vitamin; actually, it describes eight fat-soluble antioxidants from
the tocotrienol and tocopherol families that are synthesized by plants photosynthetic
pathway.
• During biosynthesis of both tocopherols and tocotrienols, homogentisic acid formation from
p-hydroxyphenyl-pyruvate is the first step and it is catalyzed by the enzyme p-
hydroxyphenyl-pyruvate dioxygenase (EC 1.13.11.27).
• In another method, identification and isolation of a novel monocot gene that encodes HGGT;
(specific enzyme for tocotrienol synthesis) is done for enhancement of vitamin E.
• A 10–15-fold increase in tocotrienol synthesis is noticed in bioengineered barley with HGGT.
• The third way involves manipulation of final step in biosynthesis of vitamin-E to enhance the
vitamin-E content.
• Hereby using the enzyme γ-tocopherol methyl transferase as catalyst the conversion of γ-
tocotrienol and γ-tocopherol to α-tocotrienol and α-tocopherol is taken place.
 Essential Minerals (Iron)
• Iron insufficiency is the most common mineral malnutrition worldwide; more than 2 billion
people suffer from iron deficiency along with primary clinical symptom of anemia about half
of iron deficiency cases.
• To avoid this, several techniques, such as enrichment of food with iron and other functional
ingredients are being exploited, but the success is limited, especially in developing nations.
• So instead of supplementation, the new techniques of bioengineering are used to enhance the
essential mineral content in staple food crops.
• Strategies implied are over expression of ferritin, store large quantity of bio available iron, and
the expression of photoset, which degrade phytate, which inhibit many essential mineral, and
help to absorb easily stored iron in the human digestive system.
• Soybean ferritin cDNA was introduced into rice plants, which endorsed the accretion of iron in
rice grain endosperm 3 times more than the untransformed plants.
• Fungal (Aspergillus niger) phytase cDNA was inserted in rice and increased degradation of
phytic acid was observed.
 Essential Amino Acids
• Human beings cannot synthesize essential amino acids on their own. Out of 20 amino acids, nine
amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine tryptophan,
and valine are essential amino acids.
• Animal proteins are the complete sources of amino acids.
• Plant sources, such as cereals are deficient in lysine and threonine, legumes are deficient in
tryptophan, methionine, and/or cysteine.
• To make more nutritious plant sources, it is essential to incorporate amino acid-rich gene and to make
GE plant.
• Amino acid deficiency can be tackled by applying two GE approaches:
(1) introducing engineering plants to generate proteins including essential amino acids; and
(2) incorporating engineering design in amino acid metabolism to enhance the accessibility of
essential amino acids in the free amino acid basket.
• Lysine was chosen first in both the approaches.
• As lysine deficiency results in fatigue, lack of concentration, bad temper, pale eyes, delayed growth,
hair loss, anemia, and reproductive problems.
 Essential Phytochemicals

• Phytochemicals are precious for human nutrition.

• Indoles, isothiocyanates, and sulforaphane from vegetables, such as broccoli, alylic sulfides
from onions and garlic and isoflavonoids
from soybeans are known as plant phytochemicals.
• These are present in high concentration in raw foods, but intensities are reduced during
processing and handling.
• Enhanced amount of phytochemicals in foods can resolve this difficulty.
• Two genes IFS1/IFS2, encoding for isoflavone synthase in soybean are revealed and
expressed in Arabidopsis thaliana, to activate the synthesis of isoflavonoid genistin.
 Isoflavonoids
• Flavonoids are a group of phytochemicals responsible for the pigmentation in plant, feed
deterrence, wood protection, protection from fungi and insects, and introduction of genes for
root nodulation.
• Anthocyanins, condensed tannins, and isoflavonoids, are flavonoids, actually they are
phytochemicals.
• The major resources of isoflavonoids can be achieved by consumption of soybean-rich
products.
• Isoflavonoid levels can decrease by 50% during soya seed processing for traditional soy foods.
• Escalating isoflavonoid quantity in soybean could solve this problem.
• Alternatively, development of other isoflavonoidrich crops that can create this powerful
compound therefore, widens their consumption.
• IFS1/IFS2, two soybean genes encoding isoflavone synthase was identified, and expressed
these genes in A. thaliana, generating the synthesis of the isoflavonoid genistein.
• Approximately 2 ng/µg of fresh plant weight, genistein was produced.
 Enzymes
• Enzymes occur in all living organisms and catalyze biochemical reactions that are necessary to
support life.
• They are commonly used in food processing, preservation, and raw ingredient manufacturing.
• The use of recombinant DNA technology has made it possible to manufacture novel enzymes that
are tailored to specific food processing conditions.
• Alpha amylases with increased heat stability have been engineered for use in the production of
high-fructose corn syrups.
• These improvements were accomplished by introducing changes in the α-amylase amino acid
sequences through DNA sequence modifications of the α-amylase genes.
• Enzymes derived from recombinant microorganisms and other application of enzymes in food
processing is listed in Tables (Next and earlier).
• Application of enzymes in food preservation and manufacturing has historically been considered
nontoxic.
 Flavors, Amino Acids, and Sweeteners
• Volatile organic chemicals, such as flavors and aromas are the sensory principles of many
consumer products and govern their acceptance and market success .
• Flavors produced using microorganisms currently compete with those from traditional
agricultural sources.
• There are more than 100 commercial aroma chemicals are derived sing biotechnology either
through the screening for overproducers, the elucidation of
metabolic pathways and precursors or through the application of conventional bioengineering.

• Recombinant DNA technologies have also enhanced efficiency in the production of nonnutritive
sweeteners, such as aspartame and thaumatin.
• Market development has been particularly dynamic for the flavor enhancer glutamate, which
is produced by the fermentation of sugar sources, such as molasses, sucrose, or glucose using
high-performance strains of Corynebacterium glutamicum and Escherichia coli (see next Tables)
• Enzyme plays a vital role in oil extraction, purification, and modification.
• Microbial lipase is extensively used in oil extraction, purification, and oil modification.
• Commercial Cocoa Butter Equivalent may be produced by combination of different
processing steps, including blending, interesterification, fractionation, and refining using
lipase.

Manufacturing of Cocoa Butter

Equivalent (CBE) Using Lipase
 Bioengineered Microorganisms
• More than 5000 years, human beings have, consciously and innocently, made use of natural
fermentation of a range of food items, which comprise bread, dairy products, alcoholic
beverages, vegetable products, and meat products.
• But it was more in recent times, just in the last century, that researchers comprehend that the
method of fermentation was done by the exploitation of microorganisms and that each
microorganism accountable for a particular food processing could be isolated and identified.
• Now, with superior bioengineering practice, it is possible to characterize the important food
strains with high precision, isolation, and improvement of genes involved in the process of
fermentation and transfer desirable traits between strains or even between different
organisms.
 Elimination of Carcinogenic Compounds
• One of the most important, commonly used, and very well-known microorganisms is S.
cerevisiae, known as Brewer’s yeast in the food industry.
• In bread manufacturing, yeast is generally used as a leavening agent and during alcoholic
beverage manufacturing, yeast is used as starter culture and grain residues or molasses are
used as raw material for fermentation.
• During fermentation, occasionally undesirable products are produced. Recombinant DNA
technology is the only solution.
• With the help of recombinant DNA technology, undesirable by-products can be removed by
incorporating properties in the GM yeast strain.
• Ethylcarbamate or urethane is the unwanted by-product of foods and beverages during yeast
fermentation.
• Ethylcarbamate or urethane is the probable carcinogenic material.
• Reduction of ethylcarbamate from alcoholic beverage is the main plunge of the alcoholic
beverage industry; a large amount of investment has been done for the research .
• The spontaneous reaction between ethanol and urea produces ethylcarbamate, which is the
degradation product of grape arginine.
• In the presence of yeasts, which produces arginase and that catalyzes arginine degradation.
• If arginase is blocked, the reaction of arginine to urea can be prevented; therefore, ethanol to
ethylcarbamate conversion can be stopped.
• A transgenic yeast strain was developed by inactivating CAR1 gene, which encodes for the
enzyme arginase (EC 3.5.3.1) to reduce the formation of urea in sake.
• By incorporating an unproductive CAR1 gene in the area of the arginase gene, the scientist
developed the mutant yeast strain, flanked by DNA sequence homologous to the region of
the arginase gene.
• The useless gene was incorporated into the active CAR1 gene through homologous
recombination, in the yeast chromosome, and disturbed its function.
• As a result, urea was detached and ethylcarbamate was not produce during sake fermentation.
• The analogous tactic can be applied to remove ethylcarbamate from other alcoholic beverages
including wine.