FOOD BIOTECHNOLOGY

Sheikh Mahatabuddin, Ph. D.

Associate Professor
Department of NFE
Daffodil International University
smuddin.nfe@diu.edu.bd

1
 Plant Biotechnology: Secondary metabolites from
plants, Plant Tissue culture, Modern hybridization,
vegetable and crop fermentation.

©DSM
Present definition of biotechnology
“Any technological application that uses biological
systems, living organisms, or derivatives theory, to make
or modify products or processes for specific use’’
(According to the Convention on Biological Diversity) ©DSM

“Plant biotechnology describes a precise process in

which scientific techniques are used to develop useful
and beneficial plants’’
(According to the Council for Biotechnology Information )
Plant biotechnology is a set of techniques used to
adapt plants for specific needs or opportunities.
Situations that combine multiple needs and
opportunities are common. For example, a single crop
may be required to provide sustainable food and
healthful nutrition, protection of the environment, and
opportunities for jobs and income. Finding or developing
suitable plants is typically a highly complex challenge.
©DSM
Ancient Plant Biotechnology
❑Humans domesticate crops.
❑Breed plants to further improve desirable characteristics
Plant breeding 12,000 years ago ...
Classical Biotechnology
 Hybrid breeding
❑ Two parental lines of normally outbreeding species are inbred through several self-
pollinations.
❑ When crossing such lines the first generation has hybrid vigour.
❑ The vigor gradually disappears over the next generations so new sowing seeds have
to be purchased every year
❑ Selection operates on desirable traits, not on survival in the wild.

1923
Russet Burbank hybrid
potato launched
1933
First hybrid maize variety
launched in USA
 HISTORY OF PLANT TISSUE CULTURE
1838-39 cellular theory (Cell is autonom Schleiden-
and totipotent) Schwann
1902 First attempt of plant tissue Harberlandt
culture
1939 Continuously growing callus White
culture
1946 Whole plant developed from Ball
shoot tip
1950 Organs regenerated on callus Ball
1954 Plant from single cell Muir
1960 Protoplast isolation Cocking
 HISTORY OF PLANT TISSUE CULTURE

1962cam MS media Murashige -

Skoog
1964 Clonal propagation of orchids Morel
1964 Haploids from pollen Guha
1970 Fusion of protoplasts Power
1971 Plants from protoplasts Takebe
1981 Somaclonal variation Larkin
Plant biotechnologies that assist in developing new varieties and traits include genetics and
genomics, marker-assisted selection (MAS), and transgenic (genetic engineered) crops.
These biotechnologies allow researchers to detect and map genes, discover their functions,
select for specific genes in genetic resources and breeding, and transfer genes for specific
traits into plants where they are needed. NIFA funds research, training, and extension for
developing and using biotechnologies for food and agriculture. Areas of work include, but
not limited to:

•Genetic structures and mechanisms

•Methods for transgenic biotechnology (also known as genetic engineering)
•Identification of traits and genes that can contribute to national and global goals for
agriculture
•Plant genome sequences; molecular markers, and bioinformatics
•Gene Editing/Genome Editing
•Synthetic Biology
Most public research on transgenic crops focuses in some way on two general objectives:
•Better understanding of all aspects of the transgenic/genetic engineering process, for
enhancing efficiency, precision, and proper expression of the added genes or nucleic acid
molecules.
•A wider range of useful and valuable traits, including complex traits.
Modern Plant breeding
A basic type of modern plant breeding:
1. Mutation breeding
2. Green revolution
3. Plant tissue culture breeding

1. Mutation breeding
o Seeds are treated with either radiation or
mutagenic chemicals to induce larger or
smaller lesions in the genes.

o The mutations are at random over the

genome.

o Usually mutation results in a loss of

function of genes
1928
Stadler produced mutations in barley
Dr. Norman Borlaug Nobel peace prize 1970 Father of Green Revolution
Plant tissue culture breeding

The process of selectively mating plants in aseptic culture.

❑ Embryo rescue
❑ Somaclonal variation selection
❑ Somatic hybrid (i.e. fusion protoplast)
❑ Generation of haploid (i.e. anther/microspore culture)
Highlights of Plant tissue culture
1902 Gottlieb Haberlandt proposed that all cells are totipotent.

1904
Hanning isolated nearly mature zygotic
embryosfrom seeds of Crucifers and successfully
grew them to maturityin a defined medium.

1925
Laibach isolated and grew embryos of interspecific
cross Linum perenneand L. austriacumthat
aborted in vivo.

1948
Folke Skoog discovered that kinetin could induce
organogenesis in callus culture of tobacco
1957
Skoog and Miller demonstrated the effects and interaction of
phytohormones
Auxin : cytokin > 1 Root formation
Auxin : cytokin <1 Shoot formation
Auxin : cytokin = 1 Callus formation
 Plant cell

Tissue Callus

Plant organ Embryo

New plant
 Factors Affecting Plant Tissue Culture
Growth Media
Minerals, Growth factors, Carbon source, Hormones
Environmental Factors
Light, Temperature, Photoperiod, Sterility, Media
Explant Source
Usually, the younger, less differentiated the explant, the
better for tissue culture
Genetics
Different species show differences in amenability to tissue
culture
In many cases, different genotypes within a species will
have variable responses to tissue culture; response to
somatic embryogenesis has been transferred between
melon cultivars through sexual hybridization
 Three Fundamental Abilities of Plants
✓Totipotency
the potential or inherent capacity of a plant cell to
develop into an entire plant if suitably stimulated.
It implies that all the information necessary for growth
and reproduction of the organism is contained in the cell
✓Dedifferentiation
Capacity of mature cells to return to meristematic
condition and development of a new growing point, follow by
redifferentiation which is the ability to reorganise into new
organ
✓Competency
the endogenous potential of a given cells or tissue to
develop in a particular way
 Types of In Vitro Culture
✓ Culture of intact plants (seed and seedling culture)
✓ Embryo culture (immature embryo culture)
✓Organ culture
1. shoot tip culture
2. root culture
3. leaf culture
4. anther culture
✓Callus culture
✓Cell suspension culture
✓Protoplast culture
Tissue Culture Applications
✓Micropropagation
✓Germplasm preservation
✓Somaclonal variation
✓dihaploid production
✓Protoplast fusion
✓Secondary metabolites production
✓Genetic engineering
 Micropropagation

• Embryogenesis
– Direct embryogenesis
– Indirect embryogenesis
• Organogenesis
– Organogenesis via callus formation
– Direct adventitious organ formation
• Microcutting
– Meristem and shoot tip culture
– Bud culture
Somatic Embryogenesis
The production of embryos
from somatic or “non-germ”
cells.
Usually involves a callus
intermediate stage which can
result in variation among
seedlings
Peanut somatic embryogenesis
Organogenesis
• The production of roots,
shoots or leaves.

• These organs may arise out

of pre-existing meristems
or out of differentiated
cells.

• This, like embryogenesis,

may involve a callus
intermediate but often
occurs without callus.
Somatic Embryogenesis and Organogenesis

Both of these technologies can be used as methods of

micropropagation.
Not always desirable because they may not always result in
populations of identical plants.
The most beneficial use of somatic embryogenesis and
organogenesis is in the production of whole plants from a
single cell (or a few cells).
Methods Used in Plant Transgenesis
• Plant secondary metabolites are a diverse group of
molecules that are involved in the adaptation of
plants to their environment but are not part of the
primary biochemical pathways of cell growth and
reproduction.
 These compounds are played many important
rules in plant life such as involved in defense
against herbivores and pathogens, regulation
of symbiosis, control of seed germination,
and chemical inhibition of competing plant
species (allelopathy), and therefore are an
integral part of the interactions of species in
plant and animal communities and the
adaptation of plants to their environment.

▪ Terpenes

▪ Alkaloids
 CO2

Photosynthesis

Primary carbon metabolism

Pentose phosphate pyruvate

Phosphoenol pyruvate
pathway

TCA Cycle Acetyl Co-A

Erythrose-4-
phosphate
Aliphatic amino acid

Shikimic acid Nitrogen Malonic acid Mevalonic acid

containing Pathway Pathway
compounds
Aromati
c amino
acids Phenolic
compounds Terpenes
Alkaloids
Applications of Alkaloids
Terpenoids are the largest class of all known natural products. Plants produce a variety of terpenoid
compounds that number in the thousands. Some terpenoids are involved in plant growth and development
directly (i.e., in primary metabolism), but most plant terpenoids are thought to function in interactions of
plants with their biotic and abiotic environment and have traditionally been referred to as secondary
metabolites. In addition to the isolation and identification of plant terpenoids, research has concentrated on
the biosynthesis, the biological function, and the exploitation of plant terpenoids for human use as
biomaterials and pharmaceuticals. Plant terpenoids are biosynthesized from C5 precursors by the action of
prenyl transferases and terpenoid synthases. Often, terpenes are acted on by cytochromes P450 and other
enzymes to increase their functionalization. Terpenoid biosynthesis in plants involves several subcellular
compartments. The accumulation of terpenoids requires efficient transport systems and specialized
anatomical structures. Using isoprene (a hemiterpene), menthol (a monoterpene), artemisinin (a
sesquiterpene), and paclitaxel [better known under the registered trademark Taxol (Bristol Myers Squibb,
New York)] and diterpene resin acids (diterpenes) as examples, we highlight some strategies, techniques, and
results of plant terpenoid research with a strict focus on the low-molecular-weight (C5-C20) terpenoids of
specialized plant metabolism.

All plant terpenoids are derived from C5 precursors, and most plant terpenoids can be grouped according to
their number of C5 building blocks as hemiterpenoids (C5), monoterpenoids (C10), sesquiterpenoids (C15),
diterpenoids (C20), or polyterpenoids (C5xn). In addition, condensation of C15- and C20-intermediates give rise
to triterpenoids (C30) and tetraterpenoids (C40), respectively. Many irregular terpenoids or terpenoid derivates
are also found in plants. Terpenoids are involved in all forms of plant interactions with other organisms
including plant reproduction, defense, and signaling. They are the most diverse group of plant chemicals and
have been used by humans for centuries in both traditional and modern industrial applications. As such, plant
terpenoids are used widely as pharmaceuticals, flavor and aroma chemicals, vitamins, pigments, and large-
volume biological feedstock for the production of a suite of industrial materials such as, for example, industrial
resins and print inks. Plant terpenoids are an important group of natural product chemicals that are actively
being explored as alternatives for petroleum-based materials.
 linalool geraniol

dioxabicycloheptane

Terpenoids

nerol myrcene ocimenone

Isoprene Rule
Compounds classified as terpenes constitute what is arguably the largest and
most diverse class of natural products. A majority of these compounds are found
only in plants, but some of the larger and more complex terpenes (e.g. squalene
& lanosterol) occur in animals. Terpenes incorporating most of the common
functional groups are known, so this does not provide a useful means of
classification. Instead, the number and structural organization of carbons is a
definitive characteristic. Terpenes may be considered to be made up of isoprene
(more accurately isopentane) units, an empirical feature known as the isoprene
rule. Because of this, terpenes usually have 5n carbon atoms (n is an integer),
and are subdivided as follows:
Classification of the terpenoids:
Classification Isoprene Units Carbon Atoms
Monoterpenes 2 C10
Sesquiterpenes 3 C15
Diterpenes 4 C20
Sesterterpenes 5 C25
Triterpenes 6 C30
Extraction and separation
Isolation of the plant terpenoids usually begins with some form of extraction from the
plant source. Certain tissue types such as roots, leaves, or flowers are often extracted.
Often, the terpenoids of interest are produced in very specialized tissues on plant
surfaces or within the plant such as the glandular trichomes of Mentha spp. or the resin
ducts of conifer species. If known, the increased concentration and reduced complexity
gained by selectively extracting these specific cell types may outweigh the increased
difficulty of isolating them. Homogenization of the tissue in an appropriate solvent
usually is sufficient to extract the terpenoids. If the terpenoids of interest are relatively
volatile, then vapor-phase collection can reduce sample complexity; this can be done
without the use of solvent in some instances. Recently, a group reviewed the analytical
methods of headspace sampling, solid phase micro-extraction (SPME), and the capture of
plant volatiles onto absorbent media for subsequent analysis by gas chromatography
(Tholl et al.).
Biosynthesis of Terpenes
 Vegetable and Crop Fermentation
Fermentation is an anaerobic (non-oxygen-requiring) pathway for breaking down
the carbohydrates, one that's performed by many types of organisms and cells.
In fermentation, the only energy extraction pathway is glycolysis, with one or two extra
reactions tacked on at the end.

Fermentation: Three Main Different Types

There are three basic forms of fermentation:
1. Lactic acid fermentation: when yeasts and bacteria convert starches or sugars into
lactic acid in foods like sauerkraut, kimchi, pickles, yoghurt and sourdough bread.

2. Ethyl alcohol fermentation: where the pyruvate molecules in starches or sugars are
broken down by yeasts into alcohol and carbon dioxide molecules to produce wine and
beer.

3. Acetic acid fermentation of starches or sugars from grains or fruit into sour tasting
vinegar and condiments. This is the difference, for example, between apple cider vinegar
and apple cider.
Each of these kinds of fermentation is down to the work of microbes specialized at
converting certain substances into others.
The diversity of fermented foods
Numerous fermented foods are consumed The common fermentation process
around the world. Each nation has its own
types of fermented food, representing the
staple diet and the raw ingredients available in
that particular place. Although the products
are well know to the individual, they may not
be associated with fermentation. Indeed, it is
likely that the methods of producing many of
the worlds fermented foods are unknown and
came about by chance. Some of the more
obvious fermented fruit and vegetable
products are the alcoholic beverages - beers
and wines. However, several more fermented
fruit and vegetable products arise from lactic
acid fermentation and are extremely
important in meeting the nutritional
requirements of a large proportion of the
worlds population. Table 2.1 contains
examples of fermented fruit and vegetable
products from around the world.
The most common groups of micro-organisms involved in food fermentations are:
1. Bacteria 2. Yeasts 3. Moulds
Bacteria
Several bacterial families are present in foods, the majority of which are concerned with food spoilage.
As a result, the important role of bacteria in the fermentation of foods is often overlooked. The most
important bacteria in desirable food fermentations are the lactobacillaceae which have the ability to
produce lactic acid from carbohydrates. Other important bacteria, especially in the fermentation of
fruits and vegetables, are the acetic acid producing acetobacter species.
Yeasts
Yeasts and yeast-like fungi are widely distributed in nature. They are present in orchards and
vineyards, in the air, the soil and in the intestinal tract of animals. Like bacteria and moulds, yeasts can
have beneficial and non-beneficial effects in foods. The most beneficial yeasts in terms of desirable
food fermentation are from the Saccharomyces family, especially S. cerevisiae. Yeasts are unicellular
organisms that reproduce asexually by budding. In general, yeasts are larger than most bacteria.
Yeasts play an important role in the food industry as they produce enzymes that favour desirable
chemical reactions such as the leavening of bread and the production of alcohol and invert sugar.
Moulds
Moulds are also important organisms in the food industry, both as spoilers and preservers of foods.
Certain moulds produce undesirable toxins and contribute to the spoilage of foods. The Aspergillus
species are often responsible for undesirable changes in foods. These moulds are frequently found in
foods and can tolerate high concentrations of salt and sugar. However, others impart characteristic
flavours to foods and others produce enzymes, such as amylase for bread making. Moulds from the
genus Penicillium are associated with the ripening and flavour of cheeses. Moulds are aerobic and
therefore require oxygen for growth. They also have the greatest array of enzymes, and can colonise
and grow on most types of food. Moulds do not play a significant role in the desirable fermentation of
fruit and vegetable products.
Enzymes
The changes that occur during fermentation of foods are the result of enzymic activity. Enzymes are
complex proteins produced by living cells to carry out specific biochemical reactions. They are known
as catalysts since their role is to initiate and control reactions, rather than being used in a reaction.
Because they are proteinaceous in nature, they are sensitive to fluctuations in temperature, pH,
moisture content, ionic strength and concentrations of substrate and inhibitors. Each enzyme has
requirements at which it will operate most efficiently. Extremes of temperature and pH will denature
the protein and destroy enzyme activity. Because they are so sensitive, enzymic reactions can easily be
controlled by slight adjustments to temperature, pH or other reaction conditions. In the food industry,
enzymes have several roles - the liquefaction and saccharification of starch, the conversion of sugars
and the modification of proteins. Microbial enzymes play a role in the fermentation of fruits and
vegetables.
Nearly all food fermentations are the result of more than one micro-organism, either working together
or in a sequence. For example, vinegar production is a joint effort between yeast and acetic acid
forming bacteria. The yeast convert sugars to alcohol, which is the substrate required by the
acetobacter to produce acetic acid. Bacteria from different species and the various micro-organisms -
yeast and moulds -all have their own preferences for growing conditions, which are set within narrow
limits. There are very few pure culture fermentations. An organism that initiates fermentation will
grow there until it’s by-products inhibit further growth and activity. During this initial growth period,
other organisms develop which are ready to take over when the conditions become intolerable for the
former ones.
In general, growth will be initiated by bacteria, followed by yeasts and then moulds. There are definite
reasons for this type of sequence. The smaller micro-organisms are the ones that multiply and take up
nutrients from the surrounding area most rapidly. Bacteria are the smallest of micro-organisms,
followed by yeasts and moulds. The smaller bacteria, such as Leuconostoc and Streptococcus grow and
ferment more rapidly than their close relations and are therefore often the first species to colonise a
substrate (Mountney and Gould, 1988). http://www.fao.org/3/x0560e/x0560e07.htm
Manipulation of microbial growth and activity
Moisture: The amount of water available for micro-organisms is referred to as the water
activity (aw). Pure water has a water activity of 1.0. Bacteria require more water than yeasts,
which require more water than moulds to carry out their metabolic activities. Almost all
microbial activity is inhibited below aw of 0.6. Most fungi are inhibited below aw of 0.7, most
yeasts are inhibited below aw of 0.8 and most bacteria below aw 0.9.

Oxidation-Reduction potential: Oxygen is essential to carry out metabolic activities that

support all forms of life. Free atmospheric oxygen is utilised by some groups of micro-
organisms, while others are able to metabolise the oxygen which is bound to other compounds
such as carbohydrates. This bound oxygen is in a reduced form. Moulds do not grow well in
anaerobic conditions, therefore they are not important in terms of food spoilage or beneficial
fermentation, in conditions of low oxygen availability.
Temperature: Temperature affects the growth and activity of all living cells. At high
temperatures, organisms are destroyed, while at low temperatures, their rate of activity is
decreased or suspended. Micro-organisms can be classified into three distinct categories
according to their temperature preference.

Nutritional requirements Hydrogen ion concentration (pH) Inhibitors

Controlled fermentation
Controlled fermentations are used to produce a range of fermented foods, including
sauerkraut, pickles, olives, vinegar, dairy and other products. Controlled fermentation is
a form of food preservation since it generally results in a reduction of acidity of the food,
thus preventing the growth of spoilage micro-organisms. The two most common acids
produced are lactic acid and acetic acid, although small amounts of other acids such as
propionic, fumaric and malic acid are also formed during fermentation.

It is highly probable that the first controlled food fermentations came into existence
through trial and error and a need to preserve foods for consumption later in the season.
It is possible that the initial attempts at preservation involved the addition of salt or
seawater. During the removal of the salt prior to consumption, the foods would pass
through stages favourable to acid fermentation. Although the process worked, it is likely
that the causative agents were unknown. Today, there are numerous examples of
controlled fermentation for the preservation and processing of foods. However, only a
few of these have been studied in any detail - these include sauerkraut, pickles, kimchi,
beer, wine and vinegar production. Although the general principles and processes for
many of the fermented fruit and vegetable products are the same -relying mainly on
lactic acid and acetic acid forming bacteria, yeasts and moulds, the reactions have not
been quantified for each product. The reactions are usually very complex and involve a
series of micro-organisms, either working together or in succession to achieve the final
product.
Biotechnology Allows for More Judicious
Use of Insecticides

Important tools for protecting crops,

the environment:
• Responsible use of biotech seeds
• Responsible use of crop
protection products
• Integrated weed and pest
management
practices
 Biotechnology Reduces Carbon Footprint

• No-till / Conservation tillage:

• Agriculture’s “carbon
footprint” decreased by:
46.5 billion pounds
• Carbon emissions are
lower on farms that use
biotechnology
• 2011: Estimated carbon
dioxide reductions:
4.19 billion pounds
 Biotechnology Makes it Possible to Produce
More Food Per Acre and Per Animal

• Crops thrive with better weed and insect control.

• Less land, insecticides, fertilizers, fuel, animals, and
feed needed to produce same amount of food.
• With rbST and proper management,
5 cows can produce as much milk as previously
took 6 cows = More Sustainable
 Biotechnology Improves Economic
Sustainability for Family Farms Worldwide

• We can help poor farmers sustainably

increase their productivity so they can feed
themselves and their families. By doing so,
they will contribute to global food security.
But that will happen only if we prioritize
agricultural innovation.”
• - Bill Gates, co-founder,
The Bill & Melinda Gates
Foundation, 2012
 More Food, Better Nutrition Needed for a Growing
Global Population

• “The past 50
years have
been the most
productive
By 2050, the global population is expected period in
global
to reach agricultural
history, leading
9 billion people, to the greatest
reduction in
requiring 70% hunger the
world has ever
seen.”
more food than
is produced • Former President
Jimmy Carter.
today. • Wall Street
Journal, October
14, 2005.
 Biotechnology Improves Harvest Per Acre

• Increasing yield in
developing nations,
ensuring greater access
to food.

• Strengthening crops against

extreme temperatures,
drought, poor soil
conditions – critical in
developing nations
Biotechnology Offers Solutions for Reversing Malnutrition

Where malnutrition is rampant,

nutritionally improving staple food
crops and native foods has great
potential to improve the health of
entire communities

In development:
• Golden Rice
• beta-carotene →vitamin A

• Biofortified sorghum
• vitamin A, iron, zinc
Biotechnology Applications in the U.S. Today
In Crops:
• Insect protection
• Herbicide tolerance
• Virus resistance
• Stacked traits, tailored to agricultural needs

In Dairy Cows:
• Protein hormones for increased milk production efficiency
 Foods From Crops & Animals Raised Using
Biotechnology

• Sweet Corn
• Papaya
• Dairy Products
• Food ingredients
• Sweeteners
(e.g. corn syrup, sugar)
• Vegetable oils
• Corn starch
• Soy protein
• And more
 Future Biotechnology Benefits
• Foods higher in omega-3s and
other nutrients.
• Foods with better taste,
freshness.
• Ability to grow crops in
difficult climates and poor
soil.
• Further improvements in
yield and disease protection.
 Gene
Segment of DNA that has the information (the code) for a protein
or RNA.
A single molecule of DNA has thousands of genes on the molecule.

Remember: DNA to RNA to Protein

Chromosomes
DNA and the proteins associated with the DNA.
Histone proteins help DNA coil up and form its shape.
Non-histone proteins regulate turning on and turning off
genes.
Found in the nucleus.
Food Biotechnology