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rDNA Technology
and Food Uses
Adeena Shafique
Sana Javed
Tehreem Tanveer
Uzair Hashmi
Muhammad Nasir Younis
Class: UG-3
Submitted to: Sir Zaffar Mehboob
Submission date: November 12th, 2012
TABLE OF CONTENTS:
INTRODUCTION TO rDNA TECHNOLOGY
THE TECHNOLOGY
FOOD AND rDNA TECHNOLOGY USES
RECOMBINANT FOOD CONTROVERSIES
REGULATIONS FOR rDNA-DERIVED FOOD
REGULATIONS FOR rDNA-DERIVED FOOD
APPLICATION OF RECOMBINANT DNA TECHNOLOGY IN AGRICULTURE
DEVELOPMENT OF STRESS TOLERANT PLANTS
DEVELOPMENT OF PLANTS HAVING INCREASE IN QUALITY OF PLANT PRODUCTS
TRANSGENIC PLANTS AS A SOURCE OF BIO PHARMACEUTICALS
RECOMBINANT ENZYMES USED IN FOOD PROCESSING INDUSTRIES
FOOD INGREDIENTS
CONCLUSION
USES OF RECOMBINANT DNA TECHNOLOGY IN WINE INDUSTRY
GENETICALLY MODIFIED GRAPEVINES
CONTROVERSIES AGAINST GM GRAPEVINES
WINE YEAST IMPROVEMENT
WINE BACTERIA IMPROVEMENT
BUSINESS PLAN
INTRODUCTION TO rDNA TECHNOLOGY
The birth of Recombinant DNA technology is accredited to Paul Berg, Herbert Boyer and
Stanley Cohen. In his goal to insert new genes into living cells, Berg was the first scientist to
splice the DNA segments from bacteriophage Lambda using restriction enzyme ECoR1, and
inserting them into the Simian Virus 40 DNA- thus producing the very first recombinant DNA
molecule. Later in 1973, Herbert Boyer and Stanley Cohen made further advances in the
technology by producing a recombinant plasmid using the same method as Berg, and then
introducing this plasmid into E.Coli cells by transformation. This resulted in the production of a
recombinant organism for the first time in history.
Further developments which lead to the true advent of rDNA technology included the production
of the first human protein, Somatostatin-a growth hormone regulator, using rDNA Technology
by Genentech in 1978. The technology has been progressing ever since.
Recombinant DNA Technology modifies the genome of an organism through transformation, by
inserting DNA from another organism with desired genes. Also known as genetic engineering,
the rDNA technology is now widely used in the production of human proteins and drugs,
biotechnological products, research and the food industry to create genetically-modified
organisms (GMOs) that produce genetically-modified (GM) products. The first GM food was
the Flavr Savr tomato, produced in 1994, which had a longer shelf life and an enhanced flavor.
Since then, the number of GMOs has exploded as producers prefer them over traditional crops
because they yield more and require less care.
THE TECHNOLOGY
The basic mechanism used in rDNA technology involves selecting the desired gene and the
cloning vector, their ligation and formation of the rDNA, the transformation of this rDNA into a
suitable host, which would then produce the desired gene product as a result of transformation.
Once this gene product is produced, it can result in improved enzymes, crops, vitamins and
amino acids, optimized microorganisms for beer and wine industry and several other food uses,
depending upon the purpose the rDNA was synthesized. The major steps involved in the
technology are described below.
Isolation and Preparation of Gene of Interest: The desired gene which is to be cloned is
extracted from the organism which produces it naturally or is obtained from the gene library.
This purified donor DNA segment is then treated with restriction endonucleases; these are the
enzymes which cleave DNA strands at specific sites and result in the formation of sticky ends.
Preparation of the Cloning Vector: The vector used for cloning is a plasmid in most cases,
which is a circular DNA molecule present in bacteria such as E.Coli. The plasmid has
independent, self-replicative properties and is manipulated for biotechnological purposes. It also
possesses selectable markers and insertion sites, where the foreign donor gene is inserted. Other
than bacterial plasmids, Ti plasmids, bacteriophages, cosmids and yeast artificial chromosomes
may also serve as the cloning vector, which require essentially the same procedure as the
plasmids
for
foreign
gene
expression. The plasmid is also
treated with the same endonuclease
enzyme as the one used for
cleaving the gene of interest. Thus
the plasmid DNA is also cleaved at
same specific sites, producing
sticky ends in it.
rDNA Formation: The use of the
same restriction enzyme for
cleaving the foreign gene and the
vector results in the formation of
sticky ends in both, which are
complementary to eachother. This
complementarity helps the gene of
interest and the plasmid vector to
ligate together in the presence of
ligase enzyme- thus forming the
recombinant DNA (rDNA).
Transformation: The rDNA is
introduced into the host organism,
where it would be replicated,
transcribed and translated to produce the desired gene product. The host organisms are usually
bacteria such as E.coli, which take up the recombinant vector, and help it to replicate and
translate, producing copies of the desired recombinant protein. Methods such as electroporation
(DNA translocation across the cell membrane using high voltage pulses) and packaging the DNA
inside phages may be used for transforming the host organism.
Screening rDNA Clones: The bacteria and other host organisms which are successfully
transformed are selected and screened using various methods such as selectable markers present
in plasmid vectors, DNA hybridization methods, and antibody probes etc. The selected
organisms producing the desired gene products are then utilized for food uses.
FOOD AND rDNA TEECHNOLOGY USES
The recombinant gene products produced through the process above may directly be used for
food purposes; for example, these gene products could be vitamins (Vitamin C, riboflavin etc.),
which would be taken as supplements. They could also be food additives, artificial sweeteners
(aspartate and other amino acids) and flavor enhancers (monosodium glutamate).
Else, these gene products may optimize the bacteria and other hosts to function efficiently; for
example, these products could be more efficient enzymes, which are more stable, more specific
or have a better productivity. Thus these enzymes (chymosin, rennin etc.) would be used to give
better results in industries such as wine, beer and cheese industries. The productivity of these
industries may also be enhanced due to the optimization of fermenting bacteria in terms of better
ethanol production.
rDNA Technology can also be used to produce genetically modified plants with characteristics
such as better productivity, resistance to pesticides, herbicides, insecticides, and diseases, and
imparting qualities such as tolerance to environmental stress. The food quality may also be
enhanced by improved nutritional density, prolonged shelf life by delayed ripening and better
flavors.
RECOMBINANT FOOD CONTROVERSIES
Although the use of rDNA technology in food industry has many benefits of improved food
quality and quantity, this area is still under tremendous amount of scrutiny from the public. This
is the reason that genetically modified foods have still not revolutionized the food industries
particularly in Europe and Asia. Recombinant food safety is a topic of heated debates, and the
public is concerned about various issues regarding the safety, ethics and environmental aspects
of these foods. Major issues are mentioned below.
Food safety
In rDNA technology, the genetic makeup of an organism is modified by the introduction of
genes from another organism. This DNA insertion is an uncontrolled event, which can possibly
result in genetic alterations in different ways, giving rise to the potential of creating unintended
effects. For example, the inserted DNA may interfere with the regulation and expression of
various genes which exert a positive influence on the required GM product. This can produce
health hazards in the modified products such as
1. Toxins
2. Allergens
3. Nutritional loss
Such faulty insertions are unique to each insertional event, and thus it is practically impossible to
search for every possible effect that a gene insertion might have.
Environmental Concerns
The genetically modified crops can impact the environment by escaping into the natural process
of gene flow. The pollens or seeds of the modified agricultural crops can escape into the
environment, and can thus spread the engineered gene into natural strains and species of crops
thus threatening the wildtype crop species for competition. Another concern is associated with
the insect resistant crops; the resistance mechanism of these crops is non-specific, thus they have
the potential of threatening the beneficial insects too, which could disturb the ecosystem, posing
a serious concern for the integrity of biodiversity.
Ethical and Social Issues
There are many concerns over the social and bioethical aspects of the rDNA derived foods.
Many people are opposed to the idea of altering the natural genetic makeup of a species in the
first place, terming it as an ‘unnatural abomination’. Others are opposed to the idea of
transferring genes from animals to plant foods due to their religious beliefs which prohibit the
diet of some or all of the animals. Yet others have concerns about the disruption of the ecosystem
and thus consider rDNA food technology as unethical.
All these controversies associated with genetically modified food are valid, and are addressed by
the regulatory authorities who assess the food safety and quality. These authorities are also
responsible for assuring that the environment and biodiversity isn’t affected to a large extent, and
are also accountable for labeling the foods which may contain prohibited animal derived
products to counter the ethical and social liabilities.
REGULATIONS FOR rDNA-DERIVED FOOD
The safety assessment of the genetically engineered foods is conducted under the ground rules
provided by the consensus among WHO (World Health Organization), FAO ( Food and
Agricultural Organization) and OECD (Organization for Economic Co-operation and
Development) and follows the basic concepts of risk assessment, risk management and risk
communication.
The safety testing involves nutritional analysis and compositional studies, toxicological studies
as well as the allergenicity studies. These analyses follow the ‘substantial equivalence’ concept,
according to which the engineered products are compared to the natural products-which are set
as standard- and the engineered foods are evaluated according to their natural counterparts. Any
health hazards, if suspected under any circumstances in the finished product are labeled so that
the consumer is aware of any potential risks of allergies and anaphylaxis, and of the nutritional
charecteristics if they vary from the unengineered product.
Environmental concerns which exist in the case of modified crops are controlled by preventing
horizontal gene transfer within and among crop species. This can be done by isolating the growth
of genetically modified crops from the wild type species. Special fields are allocated to plant
rDNA derived crops, which reduces the chances of the fleeing of seeds and pollen of these crops
to areas where natural crops are grown.
Dealing with the ethical concerns is tough, and the most accepted regulatory rule is to label the
foods which contain animal-derived genes in them so that the believers of certain religions or
school of thoughts can differentiate between labeled and unlabeled products.
rDNA technology is a field of tremendous potential for food uses in today’s world of everincreasing population size and depleting food and nutrients. If managed properly under the
regulatory guidelines, this technology can possibly alleviate food, hunger and malnutrition
conditions due to the sufficient availability of rDNA foods such as crops, fermentation
industries, enzymes and other nutrients such as amino acids and vitamins.
APPLICATION OF RECOMBINANT DNA TECHNOLOGY IN AGRICULTURE
The genetic manipulation of plants has been going on since prehistoric times, when early farmers
began carefully selecting and maintaining seeds from their best sow for the next season. Plant
breeders have cross fertilized related plants to provide next generation plants with new
characteristics such as higher yield, resistance to diseases and better nutrient content long before
the science of genetics was developed. Recombinant DNA technology can be used for insertion
of genes in plants not only from related plant species, but also from unrelated species such as
microorganisms. This process of creation of transgenic plants is far more precise and selective
than traditional breeding. Application of recombinant technology is primarily for the production
of transgenic plants with higher yield and nutritional values, increased resistance to stress and
pests. Several commercially important transgenic crops such as maize, soybean, tomato, cotton,
potato, mustard, rice etc. have been genetically modified. During the last couple of decades,
considerable progress has been made to understand the function of genes, isolation of novel
genes and promoters as well as the utilization of these genes for the development of transgenic
crops with improved and new characters. There are many potential applications of plant genetic
engineering. In fact, in 2002, more than 5.5 million farmers worldwide cultivated about 58.7
million hectares (about 148 million acres) crops that were genetically manipulated for herbicide
tolerance, insect resistance, delayed fruit ripening and improved oil quality. Application of
recombinant DNA technology has primarily helped in producing three major types of transgenic
plant having improved performances. These are:
1. Development of stress tolerant plants
2. Development of plants having improved yield
3. Transgenic plants as a source of biopharmaceuticals
DEVELOPMENT OF STRESS TOLERANT PLANTS
(a) Plant resistant to environmental stress: Plants need to cope up with abiotic stresses such as
drought, cold, heat and soils that are too acidic or salty to support plant growth. While plant
breeders have successfully incorporated genetic resistance to biotic stresses into many crop
plants through crossbreeding, their success at creating crops resistant to abiotic stresses has been
more limited, largely because few crops have close relatives with genes for resistance to these
stresses. Therefore rDNA technology is being increasingly used to develop crops that can
tolerate difficult growing conditions.
(b) Herbicide Resistant plant:
Many effective broad spectrum herbicides do not distinguish between weeds and crops, but crop
plants can be modified to make them resistant to herbicides, so as to eliminate weeds more
TM
selectively. For example, the herbicide Roundup contains the active ingredient glyphosate,
which kills plants by binding to the active site of enzymes called enolpyruvalshikimate
phosphate synthase (EPSP synthase). This enzyme is critical for the synthesis of aromatic amino
acids. Roundup is an extremely effective herbicide but it kills almost all species of plants,
including most crop plants. On the other hand, it is very safe for humans and animals because
they do not have EPSP synthase. By using rDNA technology, modified EPSP synthase gene (that
produced enzymes that were still functional but were not inhibited by glyphosate) have been
introduced into crop plants such as cotton and soyabean. These genetically modified plants were
found to be highly resistant to treatment with Roundup. Genes that provide resistance to other
herbicides such as sulfonyl ureas, gluphosinates etc. have also been developed and transferred to
produce various transgenic plants.
(c) Insect resistant plant:
To minimize crop damage by insects, mites and nematodes, farmers use synthetic pesticides
extensively which cause severe effects on human health and environment. The transgenic
technology provides an alternative and innovative method to improve pest control management
which is eco friendly, effective, sustainable and beneficial in term of yield. This involves genetic
incorporation of toxic gene (product of which is lethal to insect) into the plant. The first genes
available for genetic engineering of crop plants for pest resistance were Cry genes (popularly
known as Bt genes) from a bacterium Bacillus thuringiensis. These are specific to particular
group of insect pests, and are not harmful to other useful insects like butter flies and silk worms.
Transgenic crops (e.g. cotton, rice, maize, potato, brinjal, cauliflower, cabbage etc.) with Bt
genes have been developed and such transgenic varieties proved effective in controlling the
insect pests and it has been claimed worldwide that it has led to significant increase in yield
along with dramatic reduction in pesticides use. The most notable example is Bt cotton (which
contains Cry/Ac gene) that is resistant to a notorious insect pest Bollworm (Hellicoperpa
armigera) and only last year (2002) Bt cotton was adopted in India.
Biotechnology has opened up new avenues for natural protection for plants by providing new
biopesticides, such as microorganisms, that are toxic to targeted crop pests but do not harm
humans, animals, fish, birds or beneficial insects. As biopesticides act in unique ways, they can
even control pest population that has developed resistance to conventional pesticides. Using
recombinant DNA technology, the gene that makes these microorganisms lethal to certain insects
can be transplanted into the plants on which that insect feeds. The plant that once was a food
source for the insect now kills it, lessening the need to spray crops with chemical pesticides to
control infestation. One such microorganism is commonly found soil bacterium Bacillus
thuringiensis. The spores of Bacillus thuringiensis (Bt) contain a crystalline protein (Cry) which
breaks down to release a toxin, known as delta-endotoxin which is highly toxic to lepidopteran
larvae. This toxin binds the intestinal lining and creates pores resulting in an ion imbalance,
paralysis of the digestive system, and consequent death of the insect. Bt toxin sprays and
powders have been in use for many years.
Different Cry genes, also known as Bt genes have been identified, cloned and characterized.
Effective gene constructs have made it possible to deliver these genes into plant tissues so that
they are expressed at levels high enough to kill the insects. The Bt genes are effective against
different orders of insects. Bt cotton and maize which have increased resistance to boll worms
have been developed and cultivated since 1996. Farmers get benefited by saving costs by using
less of traditional pesticides. However, one of the major concerns about Bt based transgenics is
the possibility of development of toxin resistant insects. Efforts are also underway to identify and
transfer other genes from Bt, which can impart insecticidal properties to the plants. One example
in this is transfer of vip gene i.e. vegetative insecticidal proteins, for which the trials are being
conducted in some countries.
(d) Disease resistant plant:
Plants are susceptible to viral, bacterial and fungal diseases. Much progress has been made in
evolving transgenic plants resistant to viruses. For example, expression of a gene that encodes
the coat protein of tobacco mosaic virus (TMV) in transgenic tobacco plants has been shown to
cause the plants to resist TMV infection. A number of other viral resistant plants species have
been developed including squash and potatoes. Genetic engineering of crop plants for resistance
to fungal and bacterial infections has been more difficult. However, by studying the protective
genes that are expressed in naturally disease-resistant plants, an encouraging progress has been
made. The proteins encoded by these so called pathogenesis related proteins (PR proteins) can, in
some cases, provide limited disease protection in transgenic plants. There are several strategies
for engineering plants for viral resistance and these utilizes the genes from virus itself (e.g. the
viral coat protein gene). The virus-derived resistance has given promising results in number of
crop plants such as tobacco, tomato, potato, alfalfa, and papaya. Some viral resistant transgenic
plants like papaya resistance to papaya ring spot virus have been commercialized in some
countries. Plants respond to pathogens by inducing a variety of defense responses like
pathogenesis-related proteins (PR proteins), enzymes that degrade/destroy fungal cell wall
(chitinase), antifungal proteins and compounds, phytoalexins, etc. Several transgenic crop plants
showing increased resistance to fungal pathogens are being raised with genes coding for the
different compounds mentioned above.
DEVELOPMENT OF PLANTS HAVING INCREASE IN QUALITY OF PLANT
PRODUCTS
One of the most successful research efforts to change the characteristics of a plant produce was
carried out with tomatoes. Tomatoes need to be picked while still green so that they are firm
enough to withstand mechanical handling and transport. Unfortunately, they do not develop the
same flavor and texture of vine-ripened tomatoes. Softening of tomatoes and many other fruits is
caused by the enzyme pectinase or polygalacturonase (PGA). This enzyme digests the pectin
polysaccharide that cements the plant cells together. Softening of the fruit is caused, in part by
this breakdown of pectin. In order to reduce the levels of PGA in ripening tomatoes, researchers
placed the PGA gene in reverse orientation relative to the CaMV 35S promoter. This results in
transcription of an antisense RNA that is complementary to the normal sense PGA mRNA.
Although the exact mechanism is unknown, antisense RNA is able to arrest the translation of the
endogenous PGA mRNA in the tomato fruit. Transgenic tomato plants that express an antisense
PGA gene only have about 5 to 10% of normal PGA levels. Fruits of these plants have normal
color and flavor but they soften more slowly and can be picked and processed after they are ripe.
They also have a higher content of soluble solids and are therefore better than normal tomatoes
for processed tomato products. Transgenic lines of potato having increased levels of starch also
have been developed by introducing a gene construct that expresses a gene from bacteria that
produce an enzyme that enhances starch biosynthesis. A promoter from a potato gene that
encodes the major protein in potato tubers has been used, so that the expression of the introduced
gene is limited to the tuber. Tubers accumulate approximately 3 to 5% more starch than normal
potatoes and when they are deep fried absorb less oil and yield chips having fewer calories.
Some of the other value added transgenic crops include:
1. Golden rice: containing beta carotene to overcome vitamin A deficiency in regions where
rice is the staple food
2. Canola containing high levels of oleic acids and laurate
3. Barley containing feed enzymes
4. Tomatoes which does not rot in room temperature
5. Other vegetables and fruits with delayed ripening as well as modified flavour
characteristics
TRANSGENIC PLANT AS A SOURCE OF BIO PHARMACEUTICALS
Plants are among the most efficient bioreactors which produce quantities of material with
sunlight and soil based nutrients as inputs. Attempts are being made to replace the traditional
fermentation procedure for the production of biopharmaceuticals to plant based production. The
benefits of using plants are the ability to increase production at low cost by planting more acres,
rather than building fermentation capacity, lower capital and operating cost, simplified
downstream processing etc. Therapeutic drugs to treat cancer, infectious diseases, autoimmune
diseases, cardiovascular diseases and other conditions and several vaccines can potentially be
grown in plants. Plant transgenic technology is being used to produce a plant that will generate a
seed that expresses a desired therapeutic protein. This seed can propagate under the right
growing conditions to yield plants and seed stock for producing the desired protein. The desired
protein can be extracted from the seed to make a biopharmaceutical. Plant based therapeutics are
expected to be much more cost effective. For example, Dow Plant Pharmaceuticals is using corn
to grow pharmaceuticals by designing and selecting the plant which will contain the active
pharmaceutical within the endosperm seed compartment. Benefits of producing the
pharmaceuticals in the corn include long term storage advantage, easier purification in view of
limited number of soluble seed proteins in a corn seeds, low microbial load, low proteolytic
activity and specialized promoters to enable expression of the protein in specific parts of the
plants.
Edible vaccines:
Crop plants offer cost-effective bioreactors to express antigens which can be used as edible
vaccines. The genes encoding antigenic proteins can be isolated from the pathogens and
expressed in plants and such transgenic plants or their tissues producing antigens can be eaten for
vaccination/immunization (edible vaccines). The expression of such antigenic proteins in crops
like banana and tomato are useful for immunization of humans since banana and tomato fruits
can be eaten raw. The edible vaccines that are produced in transgenic plants have great
advantages like the alleviation of storage problems, easy delivery system by feeding and low cost
as compared to recombinant vaccines produced by bacterial fermentation. Vaccinating people
against dreadful diseases like cholera and hepatitis B by feeding them banana/ tomato, and
vaccinating animals against important diseases such as foot and mouth disease by feeding them
sugar beets could be a reality in the near future.
RECOMBINANT ENZYMES USED IN FOOD PROCESSING INDUSTRIES
Food industries commonly use enzymes for processing, as they use them in producing food
ingredients. As traditional methods of isolation of enzymes, from different organismal sources,
often do not adapt to conditions used in modern methods of food production, hence, rDNA
technology is used to manufacture such enzymes. These recombinant enzymes are suitable for
the conditions that are particular for food processing.
The use of recombinant technology has shown an immense increase in the yield of desired
enzymes by introducing gene into production organism thereby influencing the regulator
sequences. Among these, the main strategy involves introducing the gene that encodes the
enzyme in safe and efficient microorganism. For this purpose, yeasts have been proved to be
ideal system for the expression of heterologous proteins.
FDA has received, on food processing enzymes that are derived from recombinant
microorganism, number of petitions. Following is the table that includes list of these enzymes
along with their source of organisms while the complete list can be found on Enzyme Technical
Association website.
FOOD INGREDIENTS
Biotechnological fermentation and biotransformation are the two processes that have been used
for producing food products including organic acids, sweeteners, vitamins etc. rDNA technology
is applied to improve:
1. The recovery
2. Increase the yield
3. Increase in the purification of compounds
Amongst these products flavoring agents and enhancers are the ideal examples of food
ingredients, which are best, suited for recombinant DNA techniques’ application for food
production.
PL and PG from Recombinant Penicillium
Pectinase is an enzyme that is used for clarification of juices, their filtration and extraction of
fruit juices and wines.
Polygalacturonase is the enzyme that is used frequently as a sweetener. Experiments have done
to obtain a recombinant organism that will be having the ability to obtain pectin lyase (PL) and
polygalacturonase (PG) and for that penicillium griseoroseum that produced both PL &PG
simultaneously. Major steps of the experiment are as follows
1. Firstly a strain that was reported to produce high concentration of PL was taken.
2. It was then transformed using pAN52pgg2 plasmid which was having a foreign gene of
PG of P. grieoroseum and it was having a promoter from Aspergillus nidulans
3. The newly transformed P. grieoroseum T20 when checked was producing higher
concentrations of both PG and PL, around 143 folds higher PL, and 15 folds greater PG.
4. This recombinant strain uses carbon sources of low costs that is very economical
5. The enzyme preparation commercially available is free of cellulolytic and proteolytic
activities.
6. This is an efficient system that uses P. griseoroseum to express and secrete proteins.
Chymosin:
Chymosin is an example of one of pioneer enzymes that are obtained by modifying
microorganism genetically. Chymosin is a milk clotting protease that is produced via
recombinant DNA techniques and has been approved to be use in food.
Chymosin or rennin is an enzyme found in rennet. It is produced by ruminant animals in the
lining of the abomasum. Chymosin is produced by gastric chief cells in infants to curdle the milk
they ingest, allowing a longer residence in the bowels and better absorption. Bovine Chymosin is
now made in E. coli, Aspergillus Niger recombinantly. Its gene is also found in humans (on
chromosome 1), but it is not expressed.
Commercial preparation of 100 % pure Chymosin has been done through genetic technology.
And the produced Chymosin is frequently referred to as fermentation produced Chymosin. The
microorganisms are usually non pathogenic that are used to produce it, including E.coli K-12, A.
Niger etc. prochymosin genes are isolated from young calves and then they are transferred to
plasmid and this plasmid is then introduced into microorganisms. On expression prochymosin is
activated into Chymosin. Producing 100 % pure Chymosin.
The first step is the milk clotting process in cheese making in which k-caeinolytic enzymes
contribute to micelle precipitation and because of its specificity toward k –casein it the best
enzyme for this purpose.RBC (recombinant bovine Chymosin ) is commonly used but other
sources are now available that includes goat ,camel ,or buffalo. The studies have shown that
recombinant goat Chymosin exhibits
1. Best catalytic activity when compared with all the above mentioned Chymosin sources as
well as
2. The best proteolytic activity
3. Lower glycosylation degree
4. Wider pH range of action
5. Proposed as an alternative to recombinant bovine Chymosin
The whole process is shown by the following figure
CONCLUSION
So overall the recombinant enzymes in food processing have improvements as microbial strains
are developed to increase the enzyme yield which is done by deletion of genes for proteases.
Fungal strains have been modified by reducing or possibly eliminating their potential for toxic
secondary metabolites. Scientists are still finding alternatives to make the production cost
efficient, with low cost in the preparation, highly specific activity, working efficiently at wider
conditions range, to overcome all the loses that industries are still facing. Somehow scientists are
also aiming to develop ways leading to better and safer development procedures for enzyme used
in food processing industries.
USES OF RECOMBINANT DNA TECHNOLOGY IN WINE INDUSTRY
If we look back in time during the early days of winemaking, different techniques were used to
produce wine of different quality, taste and styles. But during the last 150 years, scientific basis
of winemaking has become clearer to us, and many practices which were once impossible to be
done, are now done in routine.
One of the latest technologies used in the winemaking process is recombinant DNA technology.
During the last decade, it has been identified as a most promising technology and it has widened
the possibilities to introduce new properties in wine Recombinant DNA technology is used in the
wine fermentation i.e primary and secondary--also sometimes described as aerobic and anaerobic
fermentations.
In the process of winemaking, rDNA technology is used to produce genetically modified
grapevines with improved qualities, genetically modified yeast which is used in the primary
fermentation of wine, genetically modified bacteria that works during secondary fermentation of
wine.
GENETICALLY MODIFIED GRAPEVINES
Recombinant DNA technology is used to introduce new and improved variety of grapevine
which are used in winemaking process New initiatives are being seized from recombinant DNA
technology, extensive efforts are underway to characterize the genomes of agriculturally
important species. Target for the grapevine improvement is to produce high quality fruit with
reduced susceptibility to disease and pests and other stress conditions, as well as the enhanced
nutritional value. They also look at the optimal ripening in the grapevine. Transgenic approaches
have also speeded up the development of plant lines able to adapt to adverse climatic conditions,
among them drought and salt stress, photo-damage and freezing tolerance.
Following are the potential targets which are adapted to include aspects that become important to
the industry in future.
1.
2.
3.
4.
5.
The improvement of disease and pest resistance
The improvement of virus resistance and diagnosis
The improvement of stress tolerance, including drought resistance
The improvement of plant/fruit metabolism and quality characteristics
The improvement of nutritional value
Grapevine health is threatened by different viral, fungal and bacterial diseases. To cope with this,
resistant genetically modified grapevine lines have been developed. They have increased
tolerance to pathogens, so it has a positive impact on the conservation of environment and
production cost as it has resulted in the reduced use of agrochemicals and fungicides.
CONTROVERSIES AGAINST GM GRAPEVINES:
There are several controversies about the use of GM grapevines, one of them is that the DNA
from a GM grape persists one year after wine fermentation, thus contradicting claims that wine
fermentation eliminates DNA. This GM DNA is the carrier of all the risks of recombination and
horizontal gene transfer which results in the creation of new viruses and bacteria that cause
diseases and cancer in case of human cells. Another hazard includes the toxins and allergens
from transgenic products or from unexpected metabolic disturbances to the host plant.
These are the uncertainties that act as a hurdle in the wine industry evaluation of transgenic
grapevines. There is a consequent fear because of its strong identities and deep cultural roots,
that this technology will accelerate the tendency to standardize wines, leading to loss of local
identity, variety and uniqueness.
Wine yeast improvement: In 1863, Louis Pasteur, proved that yeast is the primary catalyst in
wine fermentation. The knowledge that yeast is responsible for the biotransformation of grape
sugars into alcohol and carbondioxide has helped the winemakers to control the process from
vineyard to bottling plant.
A lot of progress has been made in the development of genetically modified yeast strains from
rDNA technology which are used in wine, brewing and baking industries. These genetically
modified yeast are used in the primary fermentation during wine production.
Commercially available yeast strains used in winemaking are;
MLO1: This GM yeast strain is commercialized and authorized by USA. It has been made by
Springer Oenologie. This yeast strain is the recipient of two transgenes. First is malate
transporter gene, this gene has been taken from another yeast named Schizosaccharomyces
pombe. Second is a malolactic enzyme gene from Oenococcus oeni, a bacteria which is
responsible for secondary fermentation i.e. malolactic fermentation after alcoholic fermentation.
This yeast is therefore able to carry out malolactic fermentation that is normally done by bacteria
at the same time as alcoholic fermentation. So this yeast strain is designed to allow malolactic
fermentation to proceed more efficiently, thereby producing fewer biogenic amines, such as
histamines, which cause headaches and asthmatic-type reactions in some people.
ECMo01: It is a strain of Saccharomyces cerevisiae, it has been genetically manipulated to
better degrade urea during the wine making process. The benefit of such a characteristic is that
the wine contains less ethyl carbamate, a chemical considered by some regulatory bodies to be a
human health risk.
In general, five major targets for the genetic improvement of wine yeast strains have been
identified which could add benefit to the wine industry:
There are five major targets for the genetic improvement of wine yeast that has been identified to
add benefit to the wine industry.
1. Efficiency of the fermentation process can be improved by better sugar utilization, increase
tolerance to ethanol, resistance to heavy metals and reduced foam and floculance
2. Processing of wine: Yeast secreting pectinase, glucanases, xylaneses and proteases has ability
to clarify and filter efficiently.
3. Wholesomeness
4. Sensorial quality: Secretion of glucanases and glucosidases may also enhance wine flavor by
hydrolysis of flavor precursor glycosides
5. Control of microbial spoilage.
Wine bacteria improvement:
Lactic acid bacteria is used in fermentation processes of many food and beverages. In
winemaking process it is a must component and performs secondary fermentation called as
malolactic fermentation. Considerable progress has been made in the last decade for the
development of tools for the genetic modification of lactic acid bacteria. Oenococcus oeni is the
lactic acid bacteria most widely used in the wine industry.
Oenococcus oeni: It is a lactic acid bacteria used commercially in the production of wine. Their
function is to do the malolactic fermentation of wine. It also has the quality to create a
characteristic aroma profile and deaccidification to the wine. Lactic acid bacteria have been
found to produce antimicrobial agents that can inhibit the growth of spoilage lactic acid bacteria,
and thus might decrease the levels of sulphur dioxide used in wine.
There are several controversies about the application of recombinant DNA technology in wine
industry, there are certain questions which are not answered satisfactorily yet. In order to assure
any future possibility of the use of recombinant technology in wine industry, there are things
about which we have to be assure about, including that the existing desirable characteristics will
not be damaged, engineered culture will be stable in practice, requirements of beverage
legislation should be met and there will be no adverse effects to the human health. So in order to
have a useful approach, two traditional wine sciences, viticulture and oenology, need to be
combined, in this way we will be able to have a histolic approach.
BUSINESS PLAN:
EXECUTIVE SUMMARY:
This report is to make a business plan to commercialize products produced by help of
recombinant DNA technology. The purpose of this report is to provide a possible solution to
enhance the quality of cheese available in market, to bridge gap between supply and demand gap
between consumers while providing economic stability to poor people of the villages. Business
plan is made using Chymosin as a product to be commercialized.
This Business plan is made for a presentation to students of Applied Biosciences. Our aim is to
make students of applied biosciences to think about the economic aspects and communal impact
of their research. Online data was searched on the business planning. Various Business models
were searched and applied on our product and best one was applied.
A member of the group participated in Discover NUST business plan competition/Workshop
held on 10-11-Nov-2012 in order to get the task of making Business Plan accomplished. This
report is presented under the guidance and experiences of participant of Discover.
Our aim is to make the cheese of high quality readily available to all categories of customers in a
affordable price while providing maximum benefit to the poor farmers and producers of the
villages.
We have focused on the market size, needs, quality of cheese and also the supply chain of the
cheese from the producer to the consumer at market. We have planned our organizational
structure, Business Model and our financial strategy in accordance with the market size, target
group and market needs. Interest of biology students towards the economical impact of their
research requires the inculcation of entrepreneurial trends, business planning and team building
strategies among the students of ASAB.
1. Background:
An estimate of livestock population and milk production in Pakistan is given in following tables.
Following data for year 2011-2012 is generated according to the 2005-2006 livestock consensus
Province wise distribution of live stock (http://www.pakdairyinfo.com/introduction.htm)
Total milk consumption of Pakistani Population (source
http://www.pakdairyinfo.com/introduction.htm)
According to FAO the prices of dairy products are on the rise throughout the year 2012
especially the cheese.
(Source http://www.fao.org/index_en.htm)
OUR RECOMMENDATIONS:
we have devised a business model in order as solution
Go to market strategy
1. Installment of mini Industrial units in villages:
According to our proposed business model we will establish mini industries in
villages where the supply of raw materials is very easy and also install mini industrial
units for the production of cheese in small villages. For every 25 houses there will be
1 industrial unit. Research department will make sure that the all the different
enzymes are available to these industrial units.
2. Goods export to market:
Cheese produced there will be exported nearby markets via vehicles and then from
these cheese collection centers it will be sent to the mega cities like Lahore,
Islamabad, Karachi etc.
3. Sales/profit:
Dairy shops, Pizza shops, 5 star hotels and Restaurants will be our intended target
places for cheese to be sold. At least 10 of profit will be used for R and D purposes.
4. Product export:
In cities additional measures will be taken to convert cheese into export quality
product. Also the enzymes produced in Research wing will be exported to the foreign
trade market.
5. Villagers:
It is planned to ensure that the standard of living and working conditions of villagers
living in villages is enhanced through revenue generated in local or foreign markets. 5
% will be used for the economic development of villagers who will work in our
cheese industry.
6. Exit Strategy:
Initial Public Offering will be the exit strategy for our business.
ORGANIZATIONAL STRUCTURE:
The organization will work under the supervision of central executive office who will make sure
that all the heads of different sections are working in a collaboration with each other. There will
wings or post which include HR section for human resource, Finance will look over all the
financial matters, sales/marketing will be ensured by people of marketing department. Pr and
communication will keep updating about market variables and market trends, all the legal
recommendations will be made by the legal sections and taxes sections.
FINANCE:
Finance
Year 1
Year 2
Year 3
Total Revenue
No profit loss
10 lacks
15 lacks
Gross Profit
0
5 lacks
10 lack
Net Income
0
2 lacks
5 lacks
Investment Needed
5 lacks
0
0
There will be no profit in the first year with a total investment of 5 lacks. Then in 2nd year gross
profit after paying all the cost of process, Vages and marketing cost will be 2 5 lacks and net
income after paying off taxes will be 2 lacks. No investment needed for 2nd and 3rd year. Gross
profit will increase to 10 lacks and net profit will be 5 lacks according to proposed plan.
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