Pharmacognosy Note

1. Fermentation

2. Fermentation is the term used by microbiologists to describe any process for the
production of a product by means of the mass culture of a microorganism.
The product can either be:
1. The cell itself: referred to as biomass production.
2. A microorganisms own metabolite: referred to as a product from a natural or
genetically improved strain. (Amino acids Lipids Antibacterial agents Antifungal
agents Organic synthesis intermediates Antiprotozoal agents Pharmaceutical
significant compounds Carbohydrates Plant growth factors Steroids Enzymes
Toxins Foods Vitamins and coenzymes)
3. A microorganisms foreign product: referred to as a product from recombinant
DNA technology or genetically engineered strain, i.e. recombinant strain. (Human
therapeutics, Enzymes, Amino acids)

3. Fermentation refers to the metabolic process by which organic molecules
(normally glucose) are converted into acids, gases, or alcohol in the absence
of oxygen or any electron transport chain.
Fermentation pathways regenerate the coenzyme nicotinamide adenine
dinucleotide (NAD+), which is used in glycolysis to release energy in the form
of adenosine triphosphate (ATP).
Fermentation only yields a net of 2 ATP per glucose molecule (through
glycolysis), while aerobic respiration yields as many as 32 molecules of ATP per
glucose molecule with the aid of the electron transport chain.

4. Alcoholic Fermentation:
Generally A F refers to the production of Ethanol from glucose by yeasts.
● occurs in yeast cells & some other microorganisms
● pyruvic acid is broken down into a 2-Carbon alcohol plus carbon dioxide
Alcoholic fermentation is important in food
and industrial microbiology and is used to
produce beer, wine, distilled sprits etc.
It is also used in production of fermented
food products.
Important to brewers & bakers! – When
yeast in dough runs out of oxygen it
ferments, giving off bubbles of carbon
dioxide gas —which forms air space in
bread – Alcohol produced in the dough
evaporates when bread is baked
*(when the level of alcohol reaches 12%,
yeast cells die)

5. Lactic acid fermentation:
In this pathway pyruvate is reduced to lactic acid. This is a single step reaction
carried out by Lactic acid bacteria (LAB)
● lactic acid is produced in muscles during rapid exercise (body can't supply
enough O2 to the tissues) ● the build-up of lactic acid causes a burning, painful
sensation in your muscles
There are two types of lactic acid fermentation.
i. Homo lactic fermentation
ii. Hetero lactic fermentation

6. Homolactic fermentation:
In homolactic fermentation, end product is lactic acid.
Pyruvate is reduced to lactate or lactic acid by the enzyme lactate
dehydrogenase (Pyruvate reductase).
Homolactic bacteria: Streptococcus thermophiles, Streptococcus lactis,
lactobacillus lactis, Lactobacillus bulgarius, Pediococcus, Enterococcus
Application:
Homolactic fermentation is important in dairy industry for souring of milk to
produce various fermented products.
Streptococcus mutant, a bacteria responsible for dental caries is a homolactic
fermenting bacteria.
Lactobacillus spp in the digestive tract of human helps in digestion of lactose
present in milk.

7. Heterolactic fermentation:
In hetero lactic fermentation, end product is ethanol and CO2 in addition to
lactic acid.
In this reaction glucose is first metabolized to pyruvate and acetaldehyde by
Pentose phosphate pathway.
Pyruvate is then reduced
to lactic acid whereas
Acetaldehyde is reduced to
ethanol and CO2.
Heterolactic bacteria:
Leuconostoc mesenteroides,
Lactobacillus bifermentous,
Leconostoc lactis

8. Mixed acid fermentation:
In mixed acid fermentation, mixture of acids such as lactic acid, acetic acid,
ethanol, formic acid etc are produced as the end product.
At first pyruvate is cleaved by the enzyme Pyruvate formate lyase to yield
formic acid and Acetyl coA.
From formic acid various other end products such as acetic acid, lactic acid,
succinic acid, ethanol or CO2 and water are formed according to types of
pathway and types of bacteria. However formic acid is always the
intermediate product in this pathway.
This pathway is followed by member of Enterobacteriaceae family such
as E. coli, Salmonella, Klebsiella etc.

10. Antibiotics Production by Fermentation
Production of penicillin by penicillium chrysogenum.
Production of tetracycline's by Streptomyces aureginosa and S. ramosus
Others……,.

11. BATCH FERMENTATION
A batch fermentation can be considered to be a closed system. At time t=0
the sterilized nutrient solution in the fermentor is inoculated with
microorganisms and incubation is allowed to proceed.
In the course of the entire fermentation, nothing is added, except
oxygen (in case of aerobic microorganisms), an antifoam agent, and acid or
base to control the pH.
The composition of the culture medium, the biomass concentration, and the
metabolite concentration generally change constantly as a result of the
metabolism of the cells.
After the inoculation of a sterile nutrient solution with microorganisms and
cultivation under physiological conditions, four typical phases of growth are
observed.

12. Lag phase
Physicochemical equilibration between microorganism and the environment
following inoculation with very little growth.
Log phase
By the end of the lag phase cells have adapted to the new conditions of growth.
Growth of the cell mass can now be described quantitatively as a doubling of cell
number per unit time for bacteria and yeast’s, or a doubling of biomass per unit time
for filamentous organisms as fungi. By plotting the number of cells or biomass against
time on a semilogarithmic graph, a straight line results, hence the term log phase.
Although the cells alter the medium through uptake of substrates and excretion of
metabolic products, the growth rate remains constant during the log phase. Growth
rate is independent of substrate concentration as long as excess substrate is present.

13. Stationary phase
As soon as the substrate is metabolized or toxic substances have been formed,
growth slows down or is completely stopped. The biomass increases only
gradually or remains constant during this stationary phase, although the
composition of the cells may change. Due to lysis, new substrates are released
which then may serve as energy sources for the slow growth of survivors.
Various metabolites are formed in the stationary phase
Death phase
In this phase the energy reserves of the cells are exhausted. A straight line may
be obtained when a semilogarithmic plot is made of survivors versus time,
indicating that the cells are dying at an exponential rate. The length of time
between the stationary phase and the death phase is dependent on the
microorganism and the process used.

14. FED BATCH FERMENTATION
In the conventional batch process just described, all of the substrate is added
at the beginning of the fermentation.
An enhancement of the closed batch process is the fed batch fermentation.
In the fed-batch process, substrate is added in increments as the
fermentation progresses.
In the fed-batch method the critical elements of the nutrient solution are
added in small concentrations at the beginning of the fermentation and these
substances continue to be added in small doses during the production phase.

15. CONTINUOUS FERMENTATION
In continuous fermentation, an open system is set up.
Sterile nutrient solution is added to the bioreactor continuously and an
equivalent amount of converted nutrient solution with microorganisms is
simultaneously taken out of the system.
In the case of a homogeneously mixed bioreactor we refer to a chemostat or
a turbidistat.
In the chemostat, cell growth is controlled by adjusting the concentration of
one substrate.
In the turbidistat, cell growth is kept constant by using turbidity to monitor
the biomass concentration and the rate of feed of nutrient solution is
appropriately adjusted.

16. Penicillin

17. Penicillin is the most widely used antibiotic for general therapeutic purpose.
The first antibiotic was discovered in 1896 by Ernest Duchesne and
"rediscovered" by Alexander Flemming in 1928 from the filamentous fungus
Penicilium notatum.
When penicillin was first made at the end of the second world war using the
fungus Penicilium notatum, the process made 1 mg dm‐3.
• Today, using a different species (P. chrysogenum) and a better extraction
procedures the yield is 50 g dm‐3.
• There is a constant search to improve the yield.

18. Mechanism of action:
• penicillin, ampicillin, amoxycillin, methicillin • Inhibits enzymes involved in
synthesis of peptidoglycan for bacterial cell wall, causing cell lysis. • Bacteriocidal •
Narrow spectrum‐ little effect on Gram negative cells.
Structure:
The basic structure of penicillin consists of a thiozolidine ring condensed with a B-
lactum ring.
Natural penicillin is 6-amino-penicillinic acid (6APA).
The penicillin's differ from one another in the side chain attached to its amino gp.

19. Fermentation Process for Penicillin production:
Commercial production is carried out by fed batch process.
Strain Development:
The variety of molds which yield greater amount of penicillin is called as high
yielding strain.
They are generally developed from the wild P. chrysogenum by a process called
sequential genetic selection.
This process consists of stepwise development of improved mutant by treating
the wild strain of P. chrysogenum with a series of mutagenic agents or exposing to
ultraviolet radiation either individually or in combination, such as X-rays and
chemical mutagens, is called as strain improvement.

20. Inoculum preparation:
The microorganism which is used in a fermentation process is called as the
inoculum.
For inoculum preparation, spore from heavily sporulated working stocks are
suspended in water or non-toxic wetting agents (sodium sulfonate 1: 10000).
Theses spore are then added to flask containing wheat bran and nutrient
solution for heavy sporulation.
Incubate for 5-7 days at 24C.
Spore are then transferred to seed tank and incubated for 24-48 hours at 24C
with aeration and agitation for sufficient mycelial growth.
These mycelia can be used for production fermenter.

21. Production fermentation:
Method: fed-batch or batch
Substrate: corn steep liquor 8.5 % ● Glucose 1% ● 80% ethanol ● phenylacetic acid
● Probenecid ● Lactose 1% ● Calcium Carbonate 1% ● Sodium hydrogen phosphate
0.4% ● Antifoaming agent: vegetable oil, inorganic ions, growth factors.
If a particular penicillin is produced, specific precursor is added in the medium. E.g.
phenyl acetic acid to get Penicillin –G.
Fermenter: stirred tank or air lift tank
pH: set at 5.5 to 6.0 which may increase upto 7-7.5 (optimum) due to liberation of
NH3 gas and consumption of lactic acid. If pH is 8 or more, CaCO3 or MgCO3 or
phosphate buffer is added
temperature: 25-27 C
aeration: 0.5-1 vvm (initially more, latter less O2 )
agitation: 120-150 rpm
time: 3-5 days
antifoam: edible oil (0.25%) corn oil, soyabean oil etc.

22. Process Description:
The production fermenter is run in a fed batch mode in which a nutrient e.g.,
glucose is added continuously throughout the fermentation to enhance the duration
of antibiotic production.
This is accompanied by withdrawal of small volumes of broth to check increase in
volume of the broth in the fermenter.
Antifoaming agents are added at the appropriate stage of fermentation.
3 phases of growth can be differentiated:
Ist phase: growth of mycelium occur and yield of antibiotic is quite low. Lactose is
used slowly and ammonia is liberated in the system causing increase in pH.
2nd phase: rapid synthesis of penicillin occur, due to rapid utilization of lactose and
amonium nitrogen. The mycelial mass increases.
3rd phase: the conc: of antibiotic decrease. Autolysis of mycelium start liberation of
ammonia and slight rise in pH.

24. Product recovery:
Separation of Mycelium:
The first step is the recovery process is the removal of mycelium or cells by
filtration or centrifuging.
Mycelium is separated from the medium by employing rotatory vacuum filter.
This process should be performed carefully in order to avoid contaminating
microorganisms which produce penicillinase enzyme, degrading the penicillin.

25. Extraction of Penicillin:
Second step is to remove the antibiotic from the spent production medium by solvent
extraction, adsorption or precipitation.
Additional solvent extraction, distillation, sublimation, column chromatography or
other methods accomplish purification.
The penicillin is excreted into the medium and less than 1% remains as mycelium
bound.
Extraction of penicillin is carried out by employing counter current extraction method.
The pH of the liquid after separation of the mycelium is adjusted to 2.0 to 2.5 by adding
phosphoric or sulphuric acid.
This treatment converts penicillin into anionic form.

26. The liquid is immediately extracted with an organic solvent such as amyl acetate or
butyl acetate or methyl isobutyl ketone.
This step has to be carried out quickly because penicillin is quite unstable at low pH
values.
The penicillin is then back extracted into water from the organic solvent by adding
enough potassium or sodium hydroxide which also results in the elevation of pH to 7.0
to 7.5.
The resulting aqueous solution is again acidified and re-extracted with organic solvent.
These shifts between the water and the solvent help in the purification of the
penicillin.
Finally, the penicillin is obtained in the form of sodium penicillin. The spent solvent is
recovered by distillation for reuse.

27. Treatment of Crude Extract:
The resulted sodium penicillin is treated with charcoal to remove pyrogens
(fever causing substances).
It is also, sometimes, sterilized to remove bacteria by using Seitz filter.
Then, the sodium penicillin is prepared in crystalline form by crystallization.
The basic product may be then modified by chemical procedures to yeild a
variety of semisynthetic penicillins such as ampicillin, amoxycillin, etc.
It may be packed as powder in sterile vials or prepared in the form of tablets
or in the form of syrups for oral usage.

29. Uses of Penicillin:
1. Most of the penicillin’s are active against Gram-positive bacteria, in which
they inhibit the cell wall synthesis leading to the death of bacteria.
2. Used therapeutically in the treatment of infectious diseases of humans
caused by Gram (+) positive bacteria.
3. G positive cocci infections, syphlis, gonorrhea, meningococcal meningitis
etc.

30. http://www.biotechnologynotes.com/antibioti
cs/production/production-of-antibiotics-by-
fermentation-bacteria-fungi-and-
penicillin/13886