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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5406_Библиотеки_им_академика_М_И_Перельмана.pdf
232 Pharmaceutical Chemistry
11. Describe the synthesis of artemisinin.
12. How similar pharmacophores of artemisinin are useful?
13. Explain the metabolite and mode of action of artemisinin.
14. Describe the synthesis of primaquine.
15. What are the metabolic products of premaquine?
16. What is the mode of action of premaquine? Explain in detail.
17. Describe the structure of antimalarial drugs.
18. What are the WHO guidelines for the treatment of malaria?
19. What are the side effects of antimalarial agents?
20. How does drug policy work? Explain with an example.
10
Fermentation
10.1 INTRODUCTION
Since ancient times, use of nonsystematic process (i.e., fermentation technique in modern
science) has been applied to alter and conserve the food materials that had been carried out
without understanding the involvement of microbial mechanisms. Majorly this technique
has been applied to milk for preparing variety of milk products like curd, chesse, etc., and
simultaneously for preparing alcohol beverages form cereals and plant extracts. The term
“fermentation” comes from a Latin word fermentum (means ferment) and it can be described
by the process in which organic substrate goes through chemical changes due to the action
of microbial enzymes in the absence of air. Zymology is the common name where we study
about the fermentation and later in the 19
reported that yeast is responsible for the fermentation. Fermentation is the natural process
of decomposition or rotting of substances, whereas the present explanation, fermentation
is a metabolic process where carbohydrates and related fibrous carbohydrate derivatives
are partially oxidized with the release of energy in the absence of any external electronacceptors organic components are produced by breaking down carbohydrates. In anaerobic
fermentation, less energy is obtained as compared to aerobic fermentation due to incomplete
oxidation of organic compounds. Fermentation of products releases less energy which
means products are not fully oxidized, thus require more oxygen for complete
mineralization. Anaerobic fermentation of glucose (one molecule) yielded two ATP
molecules, whereas aerobic respiration resulted in 36 ATP molecules.
Ethanol and acetone-butane are the only chemicals where the fermentation process is
applied and were established during the World War I, while the fermentation method was
started and applied for food production just before World War II. During World War II,
the conventional fermentation of some products (viz., antibiotics and germ warfare, etc.)
were established and thus increased the interest in industrial utilization of microorganisms.
Types of Fermentation
There are various types of fermentation which are as follows:
1. Solid State Fermentation
2. Submerged Fermentation
3. Anaerobic Fermentation
4. Aerobic Fermentation
th
century Louis Pasteur (first zymologist)
234 Pharmaceutical Chemistry
5. Immobilized Cell Bioreactors
6. Immobilized Enzyme Bioreactors
1. Solid state fermentation (SSF): SSF is the process where microbial growth and
product formation occur at the surface of solid substrates, viz., starter cultures,
mushroom cultivation and mold ripened cheeses, etc. Recently, this process has
been applied for the production of certain valuable chemicals, extracellular enzymes,
fungal spores (used for biotransformation) and fungal toxins. Traditional substrates,
viz., rice, wheat, maize and soybean, etc., are the major and rich sources of nutrients.
These substrates support the development of mycelial organisms which leads to
extracellular enzyme production, viz., numerous filamentous fungi, and bacteria
(strain of Bacillus named Actinomycetes). Solid state fermentation is divided into the
following groups on the basis of their physical states:
(i) Low moisture solids fermented without/with occasional or continuous agitation.
(ii) Suspended solids fermented in packed columns.
Rotary drum fermenters are less used for solid state fermentation. For large-scale
solid state fermentation, stationary or rotary trays have been used frequently.
2. Submerged fermentation
Batch culture: It is used for limited amount of nutrient medium in a closed culture
system where after inoculation, the cells of nutrient medium increase in size. Then
the culture enters the lag phase (or so-called exponential growth phase) where
maximum rate of the cell division and minimum rate of the cell generation take
place. Thus, increased bacterial cell population starts depletion of the nutrients and
thus accumulates as inhibitory end products in the medium and finally enters the
stationary phase. Finally, the bacterial cell population in stationary phase terminates
into death phase.
Fed-batch culture: In this process, fresh nutrient medium (without removing the
growing microbial culture) is used. It is used with such nutrients which are basically
needed for the terminal stages of the culture (viz., secondary metabolites production);
thus it is observed that culture volume increases with increase in time in fed-batch
culture. Finally, fed-batch cultures lead to higher cell concentration than the batch
culture. This culture is specially used when high concentration of substrate causes
growth inhibition and allows substrate to be used at lower non toxic levels, followed
by subsequent feeding. Using this culture, maximum production of cellular
metabolites may be achieved.
Continuous culture: It is an industrial process used to maintain exponential growth
of microbial population over a long period. In this process, growing microbes in a
culture results into end products using continuous supply of nutrients and finally
end products are removed.
3. Anaerobic fermentation: Using this method, fermented cell or molecules are used
to extract energy from carbohydrate in the absence of oxygen or other electronacceptors which actually differentiate from anaerobic respiration which is carried
out in the presence of oxygen.
Fermentation 235
Fermenter headspaces are generally filled with air and should be replaced by
suitable mixture of CO
, H2, N
2
. The fermentation liberates CO2 and H
2
which are
2
collected in a special manner due to environmental causes. For example, 400
fermenters have been used to culture Acetogens by bubbling sterile CO
. By
2
applying this method, 3 kg cells of acetogens could be harvested in each run. Most
of enzymes present in the organisms are highly O
sensitive and thus it’s very
2
difficult to recover.
4. Aerobic fermentation: Adequate aeration is the primary consent of aerobic
fermentation and in some cases, 60 times air is needed per medium volume per
hour. Thus, aerobic fermentation bioreactors are designed in a specific manner so
that adequate supply of sterile air or air spurge into the medium and mix cells very
well. Thus, batch or closed type bioreactors and sometimes continuous flow reactors
have been used for this process. Hence, designed aerobic fermenters must contain
one of the following: i) mechanical motor driven stirred tank or, ii) air-lift type
fermenter where agitation is achieved by the air bubbles through air supply while
there is no mechanical stirrer used.
5. Immobilized cell bioreactors: These bioreactors are based on immobilized cells
due to their advantages when, i) the intracellular enzymes, ii) unstable extracted
enzymes, iii) no interfering enzymes in cells, and iv) release of low molecular weight
components in the medium.
Thus, these advantages over enzyme immobilization may lead to the following
outputs: i) no need of enzyme purification, ii) high activity even in the presence of
unstable enzymes, iii) high operational stability and low cost, and iv) reactor volume
and pollution may be reduced.
One of the following routes may be applied to achieve cell immobilization:
(i) Cells may be directly bound to water-insoluble carriers (viz., cellulose, brick,
dextran, sand, ion-exchange resins, porous glass, etc.).
(ii) Cells can be cross-linked to bi- or multi-functional reagents (viz., glutaraldehyde).
(iii) Cells may be trapped inside the polymer matrices (viz., K-Carrageenan,
polyacylamide gel, calcium alginate (used with very sensitive cells), polyglycol
oligomers, etc.
The commercial production of some organic acids and amino acids are generally
carried out using cell immobilization, viz., production of organic acids such as
fumaric acid results in L-malic acid using Brevibacterium ammoniagenes (or B.
ammoniagenes may be used) cells immobilized in polyacrylamide gel/K-Carrageenan,
production of NADP by yeast (Saccharomyces cerevisiae) and B. ammoniagenes cells
immobilized together in polyacrylamide gel; and the production of L-aspartic acid
using E. coli, production of L-alanine using a mixture of Pseudomonas dacunhae and
E. coli.
236 Pharmaceutical Chemistry
6. Immobilized enzyme bioreactors: Continuous-flow reactors are mostly immobilized enzyme based reactors. They may have the following advantages:
(i) Used enzymes may lead to greater productivity
(ii) Substrates having low solubility may be used
(iii) Uniformity in the product quality (depends on batch used)
These reactors may be of the following three types:
(i) Continuous-flow stirred tank reactor
(ii) Packed-bed reactor
(iii) Fluidized-bed reactor
10.2 PRODUCTION OF ETHYL ALCOHOL AND CITRIC ACID
10.2.1 Ethanol Fermentation
Fermentation is a process where ethyl alcohol may be produced by fermentation of any
carbohy drate or sugar. The alcoholic fermentation by yeast is described by the equation:
C
Yeasts ferment glucose by glycolysis synthesis (Kreb’s cycle) to pyruvate in the presence
of pyruvate decarboxylase enzyme, which further decarboxylated to yield CH
. Later NADH2 helps in reduction of acetaldehyde to produce ethanol. In glycolysis,
CO
2
two ATP molecules per mol glucose was synthesized (anaerobic respiration) as compared
to 36 ATP molecules from aerobic (aerobic respiration).
Zymomonas mobilis, used in ethanol fermentation, was isolated from Mexican pulque.
Entner–Doudoroff pathway is used in the alcoholic fermentation of Zymomonas species
that lead to two pyruvate mol/glucose which further decarboxylated in presence of
pyruvate decarboxylase enzyme that yields into acetaldehyde. Then, NAD(P)H
during glycolysis synthesis again used in the reduction of the two acetaldehyde molecules
into ethanol. Thus, it is a better method than glycolysis in respect to ATP synthesis.
Glycolysis generally synthesized one ATP per glucose molecule while using Entner–
Doudoroff pathway, two ATPs per glucose have been synthesized. The environmental
energy problem can be balanced by using glucose transporter catalysts in high sugar
containing Zymomonas species.
Ethanol is a by-product of many fermentation reactions, for example, ethanol may, be
reduced to end product of many lactic acid bacteria (viz., clostridia and enterobacteria) to
maintain their redox balance. The reduction of all the conversions starts from acetyl-CoA
to acetaldehyde and finally into ethanol by using acetaldehyde dehydrogenase and ethanol
dehydrogenase enzymes, respectively. Since ancient times humans are using yeasts for
ethanol fermentation process to produce various alcoholic beverages. Spontaneous
fermentation is also observed in fruit juices which is usually caused by wild yeasts present
in the fruits. The wine (produced from fruits) and beer (produced from malted grains) are
the most common alcoholic beverages in the world. The impure alcohol under distillation
o 2 C2H5OH + 2 CO
6H12O6
2
CHO and
3
formed
2
Fermentation 237
produced various types of spirits, for example, whisky may be produced using malt brews
distillation, and vodka by using distillation of fermented grain or potato.
In industry, ethanol has various roles, viz., an additive to fuel, thus can be produced
from various sources. Yeasts are susceptible to ethanol inhibition, thus many industrial
strains have been used to produce approximately 150 g/L of ethanol from raw materials,
viz., sugar crops, industrial, food processing wastes (starches, whey, lignocelluloses and
sulfite liquors), etc.
10.2.2 Citric Acid Production
Karls Scheels isolated citric acid in 1874. Later, Wehmer (1923) found that citric acid is a
by-product of Ca-oxalate produced by Penicillium glaucum culture. Currie (1917) also
found that citric acid has been isolated in sugar medium from the Aspergillus niger. Some
fungi from the genus Citromyces (namely, Penicillium) are also used as a citric acid source.
Microorganisms used for citric acid production
Most of microorganisms such as fungi (Aspergillus niger, A. awamori, A. aculeatus, A.
carbonarius, A. wentii, A. foetidus, and Penicillium janthinelum), bacteria (Bacillus licheniformis,
Corynebacterium sp. and Arthrobacter paraffinens) and yeasts (Saccahromicopsis lipolytica,
Candida tropicalis, C. parapsilosis, C. oleophila, C. citroformans, C. guilliermondii, and Hansenula
anamola) may be used for the citric acid production.
Commercial production of citric acid is mostly carried out using fungi (A. niger) and
certain yeasts (Saccharomycopsis sp.). Mostly, A. niger has been used for commercial
production among all which has some advantages, like:
Cheap raw materials and better ability to ferment
Ease of handling
Produced high yield citric acid.
Production techniques and raw materials
Liquid fermentation of molasses, starch or sucrose based media yields citric acid. In 1983,
Rohr et al., classified citric acid production from raw materials into two groups:
(i) using standard procedures, cations may be removed from low ash content (viz.,
beet sugar, cane sugar, crystallized dextrose and dextrose syrups); and
(ii) raw materials and high amount of non-sugar substances with a high ash content
(viz., crude unfiltered starch hydrolysates and cane & beet molasses).
Low cost molasses (both cane and beet) is high in sugar content (40–55%) is generally
preferred in citric acid production. Beet molasses preferred over cane molasses due to less
trace metals available, viz., Ca, Mg, Mn, Fe and Zn. These metals are a setback to the citric
acid production due to interference in the production. Sugar content in molasses depends
on various factors, viz., type of beet and cane molasses, crops production and cultivation
methods, pesticides, fertilizers used during cultivation, and physiological conditions (viz.,
temperature variations, transport).
238 Pharmaceutical Chemistry
Citric acid production from solid-state fermentation techniques using various agroindustrial residues are also in use, such as apple pomace, wheat straw, sugar beet cosset,
pineapple waste, kiwi fruit peel, cassava bagasse, etc. These substrates are full of cellulosic
and starchy substances and also have rich nutrients which help to make preferred
microorganisms.
Liquid fermentation
(a) Submerged fermentation (SmF): It is a common technique for the production of
citric acid and covers more than 80% of world production due to following
advantages:
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/RZODERXUFRVWV
Conventional stirred and tower fermenters are employed for SmF due to its easy
operation, low price and size. Preferentially high grade steel fermenters are used
which can have proper aeration system, able to maintain a high dissolved oxygen
level and external water cooling system on the entire outside wall of the fermenter.
Sugar and starch based media is generally employed in SmF; however molasses
and other raw material needs pretreatment. This fermentation can also be performed
in batch mode frequently, while continuous or fed batch systems can be used less
frequently and it may take 5–10 days to complete the process generally depending
on the process conditions.
(b) Surface fermentation: In 1919, the first individual liquid surface culture technique
was introduced for citric acid production in Belgium. It was a more sophisticated
surface method of fermentation which required less effort in installation, operation
and an energy shaver.
Citric acid production is carried through classical process where the culture solution
is held in shallow trays (high purity aluminium trays with 50 to 100 litre. capacity)
and the fungus develops on the surface of the medium as a mycelial mat. Mostly,
Aspergilli, Penicilia, yeast and lactic bacteria are responsible to contaminate the
culture. The fermentation chambers are designed in proper air circulation to control
the humidity and temperature.
(c) Solid-state fermentation (SSF) or Koji process: Japan has developed the simplest
and an alternative method for the production of citric acid from the agro-industrial
residues. Some common equipment, viz., column reactors, conical flasks, glass
incubators, Erlen-Meyer flasks, glass columns, trays, etc., are used in this
fermentation process. A. niger strain and yeasts can be used in SSF using several
raw materials, viz., sugar cane, sugar cane bagasse, cellulose, grape pomace, apple
pomace, beet molasses, peel of kiwi fruit and cassava bagasse, fibrous residues
from grape pommace, apple, sweet potato, rice starch, wheat bran, potato, various
residual pulp containing items, etc. Usually, on the basis of substrate absorption
Fermentation 239
capacity the substrate can be moistened to about 70% and pH adjusted between
4.5–6.0, however temperature of incubation may vary between 28 and 30°C. SSF has
some advantages such as trace elements don’t interfere in the production of citric
acid and also substrate pretreatment is not required. Thus, SSF has many advantages
over SmF.
10.3 PRODUCTION OF ANTIBIOTICS
Antibiotics are used to inhibit the growth of many harmful microorganisms or sometimes
kill the microorganism. They can be classified on the basis of their mode of action, microbial
origin and structural characteristics, viz., the broad spectrum antibiotics are tetracyclines,
which has potent activity against Gram-positive and Gram-negative bacteria, as well as it
has potency against psittacosis causing organisms and rickettsias. Another broad spectrum
antibiotic is ciprofloxacin which is used in the treatment of urinary tract infections.
Bacitracin, erythromycins, penicillin and cephalosporins are medium spectrum antibiotics
and have potency against Gram-positive bacteria, however streptomycins have potency
against both Gram-negative and Gram-positive bacteria. Polymixins are narrow spectrum
antibiotics and exhibit potency against a few bacterial species. The use of these antibiotics
causes toxicity in the living organisms.
Scientists have worked since ancient times to understand how to destroy the infection.
First time China used antibiotic containing drug 2500 years ago. In early 350 AD, tetracycline
type of constituents had been used as an antibiotic by Sudanese-Nubian civilization. Louis
Pasteur (1877) had found an antagonistic effect of some bacteria (saprophytic bacteria) on
the anthrax organism and he suggested these bacteria may be used as therapeutics. Later
Paul Ehrlich (German chemist) also developed an idea of selective toxicity: toxicity in some
of the organisms is especially susceptive due to certain chemicals, viz., harmful infectious
bacteria to the organisms and humans. Alexander Fleming (1928) also observed that
staphylococcus bacterium in culture was completely destroyed by a mold called
notatum. Rene Dubos (1939, American microbiologist) reported that the starch-like capsule
of Pneumococcus bacterium was completely decomposed by soil bacterium. Later, Dubos
reported a highly toxic chemical antibacterial tyrothricin from the microbe called Bacillus
brevis which was present in the soil. Tyrothricin is a mixture of the two peptides
gramicidin and tyrocidine. It has toxicity towards reproductive cells and red blood cells
(RBCs) in humans. Later, penicillin,
streptomycin and a number of other antibiotics were
introduced from a specific species.
The mass production antibiotics, viz., streptomycin and penicillin started during World
War II. Presently, staged fermentation is used in the mass production of most of the
antibiotics. By this method, antibiotics are obtained along with the broth and thus antibiotics
may be removed using broth filtration, precipitation, and other separation techniques.
Some antibiotics are synthesized using chemical modification of natural substances,
laboratory synthesis and semi-synthetic way. Many of these synthesized antibiotics are
more effective against infecting organisms as compared to the natural ones. Usually, very
Penicillium
known as
240 Pharmaceutical Chemistry
small quantity of antibiotics occurs in the nature, thus fermentation process was developed
to synthesize on large scale. It is necessary to maintain fermenters from unwanted microbial
contamination which can ruin fermentation. Generally, the following trends are followed
for the production of antibiotics:
Starting the culture
1. Desired antibiotic producing organism must be isolated from the culture before the
fermentation process starts. For starting this process, a starter sample is taken from
the culture and transferred to agar-containing plate at cold desirable conditions.
This initial culture is then transferred to shaking flasks containing food and other
growth nutrients responsible for the organism. This process finally leads to a
suspension of desired antibiotic producing organism which is ultimately used for
further growth by transferring to the seed tanks.
2. Steel tanks or seed tanks with adaptable environment can be designed for growing
microorganisms. They are filled with warm water, specified microorganisms,
carbohydrate containing foods (viz., lactose or glucose), and other necessary
C-sources (viz., hydrocarbons, alcohols, acetic acid and nitrogen sources, etc). After
~24-28 hours, the seed tank mixture is transferred to the primary fermentation
tanks for further production.
Fermentation
3. Specially designed fermentation steel tank with capacity of 30,000 gallons is used
for further fermentation processes. It is then filled with seed tank mixture as it is at
optimal conditions, viz., temperature, air flow and pH. Thus, the microorganisms
are allowed to grow and multiply and thereby excrete a huge amount of desired
antibiotics. Then the tanks are allowed to cool between 23 and 27.2°C in the presence
of continuous stream of sterilized air (anti-foaming agents can be added periodically).
Isolation and purification
4. Isolation usually starts after 3 to 5 days when the production amount of antibiotic
is maximum. Various purification methods can be used for fermentation broth
when specific antibiotic is produced. For example, ion-exchange method is used for
purification of water-soluble antibiotic, solvent extraction method is used for
purification of an oil-soluble antibiotic (viz., penicillin).
Refining
5. Antibiotics are found in different forms, viz., pill or gel capsule form, syringes or
solutions for intravenous bags, powders, and also can be used with topical
ointments. After the initial isolation, various refining procedures may be explored
and generally it depends on the purity level of final antibiotic product produced.
For example, crystalline antibiotic may be dissolved in a solution for intravenous
bags and an antibiotic may be mixed into the ointment gel capsules before use in
topical ointments.
6. Then these pure antibiotics are transported to the final packing and to the
distributors, pharmacies and hospitals (Fig. 10.1).
10.3.1 Penicillin
Fermentation 241
Fig. 10.1: Outline of fermentation process of drugs
Penicillin was isolated in the 1940s which had a significant
contribution in the history of antibiotics. In World War-II, Penicillin
got attention and was prominently used as an antibiotic. Thus, major
production of penicillin started in the 1970s by deep fermentation
methods. There are two industrial processes for the production of
penicillin; first, upstream processing used for the synthesis of
R
O
H
N
N
O
Penicillin
S
COOH
CH
CH
3
3
penicillin by exploration, development and finally production, and the second is
downstream processing which includes extraction and purification of penicillin from
fermentation. Regular or excess use of penicillins is associated with the allergic reactions
in the patients or individual using penicillin.
Fermentation process of penicillin
Commercial production of penicillin can be performed using fed-batch process that
consists of stainless steel tank reactors having 30000-100000 gallons capacity. Usually, this
process takes 120-200 hours to complete through various steps, first 2-3 initial seed growth
phases and then the second, a fermentation production phase.
This process includes crude sugars, glucose and sucrose as major carbon sources where
10% carbon source is used for penicillin production, however ~65% is used for the cellular
maintenance and 25% for the growth. During penicillin production, sugar can be used to
regulate the pH value of the phase. Mini-harvest protocols may be applied to this process
which helps in the removal fermenter contents (~20-40%) and this procedure may be
repeated several times which leads to increasing yield of penicillin. This process may be
performed at acidic pH for better extraction efficiency, however chilled acidified broth