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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5714_Библиотеки_им_академика_М_И_Перельмана.pdf
Medicinal Plant Biotechnology
95
including vinblastine and vincristine, and possible complex mixture such as rose and jasmine oil.
Mitsui Petrochemical Industries of Japan is producing red-coloured phenolic naphthoquinone
compound— shikonin from cell cultures of Lithospermum erythrorhizon—which is used as red
dye and as an astringent. A German pharmaceutical company is producing digitalis glycosides by
biotransformation in cell cultures. The anticancer alkaloids of Catharanthus roseus are produced
on industrial scale using tissue culture technique based on selection of high-yielding cell lines.
The selection of a cell line with a high content of desired metabolite and use of specific precursor
are prerequisites to the development of cell cultures with high yields. The applicability of enzyme
immunoassay and radioimmunoassay techniques for quantitative analysis of bioconstituents has
revolutionized the selection of plants and cell clones with high yields. The techniques have been
successfully applied in the bioproduction of vindoline, serpentine and ajmalicine. The unusual
ether lipids, 1(3),2-diacylglyceryl-3(1)-O-4'-(N,N,N-trimethyl)homoserine were isolated from
algae cultures of Chlorella fusca.
The biomedicinals of pharmaceutical significance produced at high levels in plant cell cultures
are indicated in Table 6.2.
BIOPRODUCTION OF USEFUL METABOLITES IN HAIRY ROOT AND
MULTIPLE SHOOT CULTURES
Hairy cultures are developed by way of incorporating a segment of Ri-DNA from
Agrobacterium
rhizogenes
into the plant genome that brings out a phenotypic character called
hairy roots
(Fig. 6.13).
These are able to grow rapidly and indefinitely with profuse lateral roots. They grow on a medium
Table 6.2 High levels of biomedicinals in plant cell cultures
Compounds Species Yield (% dry wt)
Acetonide
Syringa vulgaris
5.0
Ajmalicine
Catharanthus r
oseus
1.8
Anthraquinones
Morinda citrifolia
18.0
Benzylisoquinolines
Coptis japonica
15.0
Berberine alkaloids
Berberis wilsoniae
10.0
Diosgenin
Dioscorea deltoidea
7.8
Ginsengoside
Panax ginseng
17.0
Nicotine
Nicotiana tabacum
5.0
Rosmarinic acid
Coleus blumei
21.4
Serpentine
Catharanthus roseus
2.2
Shikimic acid
Galium mollugo
10.0
Shikonin
Lithospermum erythrorhizon
12.4
Textbook of Pharmaceutical Biotechnology
96
without plant hormones and can accumulate levels of secondary metabolites similar to those
occurring in natural roots. Many plants have been subjected to these studies. This technique has
yielded positive results mostly in nonwoody dicotyledonous plants. It is too early to envisage the
commercial impact of genetic manipulation on secondary metabolite production in tissue cultures.
The hairy root cultures of Catharanthus roseus capable of producing appreciable quantities of
ajmalicine, serpentine and catharanthine have been successfully established.
The bioproduction of scopolamine and hyoscyamine in hairy roots developed from Scopolia
japonica has in some cases found to be lower or greater as compared to the entire plant. The
bioproduction of shikonin and betacynin in appreciable quantities has been reported in hairy root
cultures of Lithospermum erythrorhizon and Beta vulgaris, respectively. The inherent ability of a
large number of medicinal plants to generate multiple shoots in culture from axillary and shoot
tip meristem has opened up new avenues for bioproduction of medicinal agents and preservation/
propagation of elite genotype. The axillary meristem and shoot tip develop into shoots on a minimal
inorganic basal MS medium both on agar surface and in liquid suspension. They can be propagated
by routine periodic subcultures in liquid medium. The
type of explant, growth conditions and
combination of hormone used play an important role in maintenance of cultures. The bioactive
shoot cultures have been established for belladonna, dioscorea, solanum and vinca.
Immobilized Cell Systems
The technique of immobilizing plant cells or enzymes has facilitated the utility of plant cell
biotechnology for production of pharmaceuticals. Immobilization by use of matrices such as calcium
alginate, agarose, gelatin, carrageenan or polyacrylamide has number of advantages, including
reuse of expensive biocatalysts, simplified control with automatic separation of the catalyst from
the product and stabilization of the biocatalyst. The plant tissue culture techniques have wide
applications in the study of fundamental problems of plant breeding, plant physiology, cytology,
genetics, as well as viral, bacterial and nematode plant relationships. The use of these techniques
Young plant tissue
Agrobacterium
Hairy root culture
Bioactive substances
Transformation
Increased production of
bioactive substances
Figure 6.13 Hairy root culture.
Medicinal Plant Biotechnology
97
offers a unique approach to the analysis of the metabolic potentialities of higher plant cells, for
studies of physiological interrelationships amongst cells, tissues and organs within the multicellular
plant; for controlling the expression and differentiation and morphogenesis of the totipotency of
such cells. Some of the medicinal compounds localized in morphologically specialized organs or
tissues have been produced in culture systems, not only by inducing specific organized structures
in submerged cultures, but also by certain culture strains in the absence of organization.
The recent advances in the field of plant tissue cultures have strengthened our hopes towards
the realization of full-scale industrial production of biomedicinals in the near future. The increasing
awareness of pharmacists in the biotechnological applications of plant tissue culture promises
an ever-increasing involvement in a branch of bioscience that could lead to production of new
pharmaceutical products to be used in service of the mankind.
QUESTIONS
1. Write a note on mutation in plants.
2. Write a short note on polyploidy, haploid culture, transgenic plants, organogenesis and
embryogenesis, callus culture and chemical mutagens.
3. Describe application of transgenic plants.
4. Explain role of various factors in the production of secondary metabolites.
5. Explain role of mutation and polyploidy in improving quality of plants.
6. What is mutation? Write the different types of mutation with its application.
7. Write methods of culturing cells in suspension.
8. Write a short note on embryo culture.
9. Write a short note on factors affecting rate of mutation.
10. Write a note on nutrients incorporated in culture medium of plant tissue culture.
11. Write in brief application of transgenic plants.
12. Explain role of growth regulators in production of secondary metabolites.
13. Explain role of mutation and polyploidy in improving quality of crops.
14. Give an account of pharmaceuticals produced in tissue cultures.
7KLVSDJHLQWHQWLRQDOO\OHIWEODQN
INTRODUCTION
Microbial population dominates the biospheres in terms of number and metabolic impact. Amongst
the various stages of microbes, prokaryotes have a wide spread on the planet, often tolerating
extremes of pH, temperature, salts, concentration, etc. Metabolic diversity is greater amongst
prokaryotes than all eukaryotes. The bacterial, yeast and fungal populations are systematically
utilised for the manufacture of various chemicals, biochemicals, beverages, antibiotics, etc. Microbes
have been involved in the daily affairs of human beings who have been taking their help from time
immemorial. The production of alcohol, vinegar, amino acids, vitamins, antibiotics, therapeutics
antibodies, acetone and other solvents and recombinant proteins is accomplished by the large-scale
cultivation of microbial cell lines of bacteria, algae, yeast and fungi on industrial scale.
The microbiological culture refers to the multiplication of microbial organisms in predetermined
culture medium under controlled laboratory conditions. Microbial cultures are used to determine
the type of organism, its abundance in the sample being tested, or both. The culture is used for
diagnosis of the cause of infectious disease by letting the agent multiply in a predetermined medium.
For example, a throat culture is prepared by scraping the lining of tissue in the back of the throat
and blotting the sample into a medium for enabling screening for harmful microorganism, such as
Streptococcus pyogenes, the causative microorganism of strep throat. Implementation of quality
measures, compliance with the recommendations of Convention on Biological Diversity (CBD) and
adoption of latest bioinformatics tools are essential prerequisites for microbial culture collections
to provide resources for the emerging area of the knowledge-based bioeconomy. It should be in
association with the deposition of phylogenetically and physiologically diverse microbiological
organisms. In view of the fact that the vast majority of microbial isolates do not find their way
into public collections, a strategy should be devised to encourage researchers to deposit a higher
fraction of strains.
Microbial Culture and
Fermentation Process
7
CHAPTER
Introduction
Microorganisms
Microbial Culture
Quality Control of Microbial Culture Media
Questions
Textbook of Pharmaceutical Biotechnology
100
MICROORGANISMS
Types
Out of more than one million microorganisms present in nature, about a few hundred species
synthesize the useful products. The examples of some of these important microorganisms are given
below.
1. Algae: Chlorella sorokiniana, Spirulina maxima, Gephyrocapsa oceanica, Haematococcus
pluvialis.
2. Bacteria: Acetobacter woodii, Bacillus polymyxa, Bacillus subtilis, Bacillus thuringiensis,
Clostridium aceticum, Pseudomonas denitrifi cans, etc.
3. Actinomycetes: Micromonospora purpurea, Nocardia mediterranei, Streptomyces
aureofaciens, Streptomyces griseus, Streptomyces noursei, etc.
4. Fungi: Aspergillus niger, Aspergillus oryzae, Aspergillus terreus, Candida utilis,
Cephalosporium acremonium, Fusarium moniliforme, Gibberella fujikuroi, Morchella
esculenta, Paecilomyces lilacinus, Penicillium chrysogenum, Penicillium notatum, Rhizopus
nigricans, Saccharomyces cerevisiae, Saccharomycopsis lipolytica, Sardinella rouxi,
Trichoderma harzianum, Trichoderma reesei, Trichoderma viride, etc.
MICROBIAL CULTURE
The microorganism isolated from different sources (soil, water and air) or obtained through genetic
manipulations are cultured on sterilized growth medium supplemented with sources of carbon,
nitrogen, phosphorus, amino acid, trace elements, etc.
Principle of Microbial Growth and Culture System
The growth of microorganism is a highly complex process and is usually expressed in terms of
increase in cell number or cell mass. The process of growth depends on the availability of requisite
nutrients and their transport into the cells, and the environmental factor, such as aeration, O
2
supply,
temperature and pH. Doubling time normally increases with increasing cell size and its complexity.
It normally presents in the following ranges for different types of microorganisms: bacteria, 0.30–1
h; yeast, 1–2 h; animal cells, 25–48 h and plant cells, 20–70 h.
Culture Ingredients and Preparation
1. Carbon source: The main carbon source used in the culture medium is carbohydrate (sugar). It
is required to provide energy and carbon skeleton for the synthesis of various other biological
compounds.
2. Nitrogen source: Commonly used nitrogen sources in culture medium are ammonium salts,
amino acid mixture, urea, animal tissue extracts and plant tissue extracts.
Microbial Culture and Fermentation Process
101
3. Microelements: Microelements required in small amounts are Ca, Co, Fe, Mg, Mn, Zn, etc.
4. Growth factors: Growth factors are certain organic compounds that are essential for the growth
and multiplication of cell, but cannot be synthesized by the cells, e.g. adrenomedullin (AM),
erythropoietin (EPO), bone morphogenetic proteins (BMPs), insulin-like growth factor (IGF),
etc.
5. Antifoams: This is not a nutritive component of the medium. The culture medium rich in
nutritive compounds, such as starch, proteins and other organic materials on agitation for
aeration can result in excessive foaming. In the production of useful substances by deep-
aerobic culture, a large quantity of foam is formed, which causes various problems. In such
cases, a fermenter is filled with foam, which lowers a culturing capacity per unit volume
and causes the culture solution to overflow from the fermenter. It is necessary to overcome
this problem of foaming. This may be arrested by using a suitable antifoaming agent (e.g.
Acepol™ 20, Acepol™ 7203, etc.).
6. Water: Whether it is liquid or solid, water is the base of any culture medium. In the preparation
of the agar nutrient medium, water is comparatively required less than that of liquid nutrient
medium. Water is also required for a large number of other services in the laboratory, such as
cooling, heating, steaming, etc.
Types of Cultures
The culture vessels and culture medium are sterilized by autoclaving at 120°C for 15–20 min to
prevent the growth of unwanted microorganism and possible contamination. If the laboratory-scale
experiments are carried out in 100–1000 mL flask, or in lesser volumes of 50 mL or 10 mL, the
sterilization may be carried out alternatively in a pressure cooker of convenient size.
Solid or Semisolid Culture
The nutrient medium is supplemented with different concentrations of agar to give solid or
semisolid medium. For research purpose, these types of media are regularly used. They are usually
not preferred for large-scale production of microbial products, because such media occupy space
and are difficult to harvest. However, for the production of amylase from A. oryzae, it is used. After
the growth of A. oryzae for several days at 30°C, mycelia are harvested, dried and ground, which
result in crude preparation of amylase.
Batch Culture
Batch fermentation is regarded as a closed system. The sterile nutrient culture medium in the
bioreactor is inoculated with microorganism. The incubation is carried out under optimal condition
of pH, temperature, oxygen supply, agitation duration, etc.
The following six typical phases of growth are observed in batch fermentation (Fig. 7.1):
1. Lag phase (a)
2. Acceleration phase (b)
3. Logarithmic phase (c)
Textbook of Pharmaceutical Biotechnology
102
4. Deceleration phase (d)
5. Stationary phase (e)
6. Death phase (f)
Lag phase
The initial brief period of culturing after inoculation is referred to as lag phase. During this phase of
growth, the microorganism adapts to the new environment. There is no increase in the cell number,
although the cellular weight may slightly increase. The length of the lag phase is variable and is
mostly determined by the new set of physical and physiological conditions.
Acceleration phase
This is a brief transient period during which cells start growing slowly. In fact, acceleration phase
connects the lag phase and log phase.
Log phase
This is the most active phase for growth of microorganism, wherein multiplication occurs rapidly.
When the number of cells or biomass is plotted against time on semilogarithmic graph, a straight
line is obtained, hence the term log phase. As long as excess substrate is present, growth rate of
microbes in log phase is independent of substrate concentration, and there are no growth inhibitors
in the medium.
Deceleration phase
With the decline in growth rate of microorganism at the end of log phase, commencement of
declaration phase take place.
Stationary phase
As the substrate in the growth medium gets depleted, and the metabolic end products that are
formed inhibit the growth, the cells enter into the stationary phase. The microbial growth may
either slow down or completely stop during this phase of growth.
Figure 7.1 Typical growth phases.
a
b
c
d
e
f
Optical density
O
Time (h)
Microbial Culture and Fermentation Process
103
Death phase
This phase is associated with cessation of metabolic activity and depletion of energy reserves. The
cell dies at an exponential rate.
Fed- Batch Culture
In this type of culture, the limiting substrate is fed without diluting the culture. The volume of culture
can also be practically maintained constant by feeding the growth-limiting substrate in undiluted
form, as a very concentrated liquid or gas (e.g. oxygen). Alternatively, the limiting substrate can be
added by dialysis. In a photosynthetic culture, radiation may serve the purpose of growth-limiting
factor without affecting the culture volume. A certain type of extended fed-batch—the cyclic fed-
batch culture for fi xed volume systems—refers to a periodic withdrawal of a portion of the culture
and use of the residual culture as the starting point for a further fed-batch process. Basically, once the
fermentation reaches a stage, when aerobic conditions cannot be maintained anymore, the culture is
removed and the biomass is diluted to the original volume with sterile water or medium containing
the feed substrate. The dilution decreases the biomass concentration and results in an increase in
the specific growth rate (Fig. 7.2). Subsequently, as feeding continues, the growth rate declines
gradually as biomass increases and approaches the maximum sustainable level in the vessel once
more. At this stage, the culture may be diluted again to optimize the process.
Variable volume fed-batch
As the name implies, a variable volume fed-batch is one in which the volume changes with the
fermentation time due to the quality of substrate feed.
The feed can be provided according to one of the following options.
1. The same medium used in the batch mode is added.
2. A solution of the limiting substrate at the same concentration as that in the initial medium is
added.
3. A very concentrated solution of the limiting substrate is added.
Figure 7.2 Fed-batch culture.
Variable-speed pump
ADDA
converter
Glucose
Rs232/power cable
Analyser
Carrier/standard solutions
Textbook of Pharmaceutical Biotechnology
104
Advantages of fed-batch reactors
1. Production of high cell densities due to extension of working time (particularly, important in
the production of growth-associated products).
2. The mode of operation can overcome and control deviations in the organism’s growth pattern
as found in batch fermentation.
3. Alternative mode of operation for fermentations dealing with toxic substrates or low-solubility
compounds.
4. Allows the replacement of water loss by evaporation.
5. Control over the production of by-products or catabolite repression effects due to limited
provision of substrates solely required for product formation.
6. Controlled conditions in the provision of substrates during fermentation, particularly regarding
the concentration of specific substrates (e.g. carbon source).
7. Increase of antibiotic-marked plasmid stability by providing the correspondent antibiotic
during the time span of the fermentation.
8. No additional special piece of equipment is required as compared with the batch fermentation
mode of operation.
Limitations of fed-batch reactors
1. Special operational skills are required for development and setting up of the process.
2. It requires previous analysis of the microorganism, its requirements and the understanding
of its physiology with the profile of productivity.
3. The special care is required to be taken in the design of the process to ensure that toxins do
not accumulate to inhibitory levels.
4. The quantities of the components to control must be above the detection limits of the
available measuring equipment.
Continuous Culture
The culture medium is continuous in bioreactor, and it is replaced with a fresh sterile medium.
Homogeneously mixed bioreactor
The culture solution is homogeneously mixed in this type of bioreactor. The bioreactors are of the
following two types:
1. Chemostat bioreactor: In this case, the concentration of any one of the substrates is adjusted
to control the cell growth and maintain a steady state. An important feature of chemostat
is that microorganism can be grown in a physiological steady state, wherein growth occurs
at a constant rate and all culture parameters—such as culture volume, dissolved oxygen
concentration, nutrient and product concentrations, cell density, pH, etc.—remain constant.
The microorganisms grown in chemostats naturally strive in the steady state. If a low
amount of cells are present in the bioreactor, the cells can grow at growth rates higher
than the dilution rate, as growth is not limited by the addition of the limiting nutrient.
The limiting nutrient is a nutrient that is essential for growth and present in the media at
a limiting concentration (all other nutrients are usually supplied in surplus). If the cell