B Pharmacy 6th sem

Subject: Pharmaceutical Biotechnology

Subject Code: BP605T

MODULE- 5
FERMENTATION AND
BLOOD PRODUCTS

Chamanpreet Kaur ( Assistant Professor)

Pharmaceutics
ASBASJSM COLLEGE OF PHARMACY, BELA
 Objectives of Course

• Appreciate the use of microorganisms in fermentation

technology.
• Genetic engineering applications in relation to production of
pharmaceuticals.
Learning Outcomes

• Students will learn about various fermentation methods

including their sterlization methods, aeration and stirring methods.
• They will also learn about various types of fermentor design and
parameters used to control fermentation process.
Definition:
Fermentation is the chemical transformation of organic
substances into simpler compounds by the action of
enzymes, complex organic catalysts, which are produced by
microorganisms such as molds, yeasts, or bacteria.
Enzymes act by hydrolysis, a process of breaking down or
predigesting complex organic molecules to form smaller
(and in the case of foods, more easily digestible)
compounds and nutrients.
HISTORY OF FERMENTATION
Fermentation is a natural process. People applied
fermentation to make products such as wine, mead (Made of

Fermented honey and water), cheese and beer long before the
biochemical process was understood. In the 1850s and
1860s Louis Pasteur became the first zymurgist (One who
expert in the field of fermentation) or scientist to study
fermentation when he demonstrated fermentation was
caused by living cells.
The first solid evidence of the living nature of yeast
appeared between 1837 and 1838 when three publications
appeared by C. Cagniard de la Tour, T. Swann, and F.
Kuetzing, each of whom independently concluded as a
result of microscopic investigations that yeast was a living
organism that reproduced by budding. The word "yeast," it
should be noted, traces its origins back to the Sanskrit word
meaning "boiling." It was perhaps because wine, beer, and
bread were each basic foods in Europe, that most of the early
studies on fermentation were done on yeasts, with which
they were made. Soon bacteria were also discovered; the
term was first used in English in the late 1840s, but it did not
come into general use until the 1870s, and then largely in
connection with the new germ theory of disease.
The view that fermentation was a process initiated by living
organisms soon aroused fierce criticism from the finest
chemists of the day, especially Justus von Liebig, J.J.
Berzelius, and Friedrich Woehler. This view seemed to give
new life to the waning mystical philosophy of vitalism, which
they had worked so hard to defeat. Proponents of vitalism held
that the functions of living organisms were due to a vital
principal (life force, chi, ki, prana , etc.) distinct from physico-
chemical forces, that the processes of life were not explicable
by the laws of physics and chemistry alone, and that life was in
some part self determining. As we shall soon see, the vitalists
played a key role in debate on the nature of
fermentation. A long battle ensued, and while it was
gradually recognized that yeast was a living organism, its
exact function in fermentations remained a matter of
controversy. The chemists still maintained that fermentation
was due to catalytic action or molecular vibrations.
The debate was finally brought to an end by the great French
chemist Louis Pasteur (1822-1895) who, during the 1850s
and 1860s, in a series of classic investigations, proved
conclusively that fermentation was initiated by living
organisms. In 1857 Pasteur showed that lactic acid
fermentation is caused by living organisms. In 1860 he
demonstrated that bacteria cause souring in milk, a process
formerly thought to be merely a chemical change, and his
work in identifying the role of microorganisms in food
spoilage led to the process of pasteurization. In 1877,
working to improve the French brewing industry, Pasteur
published his famous paper on fermentation, Etudes sur la
Biere , which was translated into English in 1879 as Studies
on Fermentation . He defined fermentation (incorrectly) as
"Life without air," but correctly showed specific types of
microorganisms cause specific types of fermentations and
specific end products. In 1877 the era of modern medical
bacteriology began when Koch (a German physician; 1843-
1910) and Pasteur showed that the anthrax bacillus caused
the infectious disease anthrax. This epic discovery led in
1880 to Pasteur's general germ theory of infectious disease,
which postulated for the first time that each such disease
was caused by a specific microorganism. Koch also made
the very significant discovery of a method for isolating
microorganisms in pure culture.
TYPES BASED ON RESPIRATION (AEROBIC AND
AN-AEROBIC)
Aerobic Fermentation: Aerobic fermentation means that
oxygen is present. Wine, beer and acetic acid vinegar (such
as apple cider vinegar), need oxygen in the “primary” or
first stage of fermentation.
When creating acetic vinegar, for example, exposing the
surface of the vinegar to as much oxygen as possible,
creates a healthy, flavorful vinegar with the correct pH.
Anaerobic Fermentation: Anaerobic fermentation is a
method cells use to extract energy from carbohydrates
when oxygen or other electron acceptors are not available
in the surrounding environment. This differentiates it from
anaerobic respiration, which doesn’t use oxygen but does
use electron-accepting molecules that come from outside
of the cell. The process can follow glycolysis as the next
step in the breakdown of glucose and other sugars to
produce molecules of adenosine triphosphate (ATP) that
create an energy source for the cell.
Through this method, a cell is able to regenerate
nicotinamide adenine dinucleotide (NAD+) from the
reduced form of nicotinamide adenine dinucleotide
(NADH), a molecule necessary to continue glycolysis.
Anaerobic fermentation relies on enzymes to add a
phosphate group to an individual adenosine diphosphate
(ADP) molecule to produce ATP, which means it is a form
of substrate-level phosphorylation. This contrasts with
oxidative phosphorylation, which uses energy from an
established proton gradient to produce ATP.
There are two major types of anaerobic fermentation:
ethanol fermentation and lactic acid fermentation. Both
restore NAD+ to allow a cell to continue generating ATP
through glycolysis.
Ethanol fermentation: Ethanol fermentation converts two
pyruvate molecules, the products of glycolysis, to two
molecules of ethanol and two molecules of carbon dioxide.
The reaction is a two-step process in which pyruvate is
converted to acetaldehyde and carbon dioxide first, by the
enzyme pyruvate decarboxylase.
Yeast and certain bacteria perform ethanol
fermentation where pyruvate (from glucose
metabolism) is broken into ethanol and
carbon dioxide. The net chemical equation
for the production of ethanol from glucose
is:
C6H12O6 (glucose) → 2 C2H5OH (ethanol) + 2 CO2 (carbon dioxide)
Ethanol fermentation is used the production of beer, wine
and bread. It's worth noting that fermentation in the presence
of high levels of pectin result in the production of small
amounts of methanol, which is toxic when consumed.
Anaerobic fermentation -1
Lactic acid fermentation: Lactic acid fermentation is a
biological process by which glucose and other six-carbon
sugars (also, disaccharides of six-carbon sugars, e.g.
sucrose or lactose) are converted into cellular energy and
the metabolite lactate.
The pyruvate molecules from glucose metabolism
(glycolysis) may be fermented into lactic acid. Lactic acid
fermentation is used to convert lactose into lactic acid in
yogurt production. It also occurs in animal muscles when
the tissue requires energy at a faster rate than oxygen can be
supplied. The next equation for lactic acid production from
glucose is:
C6H12O6 (glucose) → 2 CH3CHOHCOOH (lactic acid)
The production of lactic acid from lactose and water may be
summarized as:
C12H22O11 (lactose) + H2O (water) → 4 CH3CHOHCOOH
(lactic acid)
Yogurt is made by fermenting milk. It's high in protein,
calcium, and probiotics ("good" bacteria). Here's how to
make yogurt and a look at the chemistry of yogurt.
TYPES OF FERMENTATION
Homo Lactic fermentation: The fermentation in which only
the lactic acid is produced. There is no any side product
formed after the reaction.
Hetero-Lactic Fermentation: The Fermentation in which the
lactic acid is produced along with some by products like
gases.
MECHANISM OF FERMENTATION
Fermentation takes place when the electron transport chain is
unusable (often due to lack of a final electron receptor, such
as oxygen), and becomes the cell’s primary means of ATP
(energy) production.[1] It turns NADH and pyruvate
produced in glycolysis into NAD+ and an organic molecule
(which varies depending on the type of fermentation; see
examples below). In the presence of O2, NADH and pyruvate
are used to generate ATP in respiration. This is called
oxidative phosphorylation, and it generates much more ATP than
glycolysis alone. For that reason, cells generally benefit from
avoiding fermentation when oxygen is available, the exception
being obligate anaerobes which cannot tolerate oxygen.
The first step, glycolysis, is common to all fermentation
pathways this is the cause of fermentation:
C6H12O6 + 2 NAD+ + 2 ADP + 2 Pi → 2 CH3COCOO− + 2
NADH + 2 ATP + 2 H2O + 2H+
Pyruvate is CH3COCOO−. Pi is inorganic phosphate. Two ADP
molecules and two Pi are converted to two ATP and two water
molecules via substrate-level phosphorylation. Two molecules of
NAD+ are also reduced to NADH.
In oxidative phosphorylation the energy for ATP formation
is derived from an electrochemical proton gradient
generated across the inner mitochondrial membrane (or, in
the case of bacteria, the plasma membrane) via the
electron transport chain. Glycolysis has substrate-level
phosphorylation (ATP generated directly at the point of
reaction).

Humans have used fermentation to produce food and

beverages since the Neolithic age. For example,
fermentation is used for preservation in a process that
produces lactic acid as found in such sour foods as
pickled cucumbers, kimchi and yogurt (see fermentation
in food processing), as well as for producing alcoholic
beverages such as wine (see fermentation in
winemaking) and beer. Fermentation can even occur
within the stomachs of animals, such as humans.
PRODUCTS OF FERMENTATION
Wine
Beer
Lactic acid
Vinegar
 Yogurts
 cheese
 Sauerkraut
 Kimchi
 Pepperoni
ETC………..
FERMENTER DESIGN
PRODUCTION OF PENCILLIN
PRODUCTION OF GLUMATIC ACID
PRODUCTION OF CITRIC ACID
PRODUCTION GRISEOFULVIN
FERMENTATION AS A FOOD PRESERVATION
TECHNIQUE
Fermented foods are foods that have been prepared in a way so
that the bacteria naturally found within them starts to ferment.
Fermentation, also known as lacto-fermentation, is a chemical
process in which bacteria and other micro-organisms break
down starches and sugars within the foods, possibly making
them easier to digest, and resulting in a product that is filled
with helpful organisms and enzymes.
This process of fermentation is a natural preservative, which
means that fermented foods can last a long time.
ADVANTAGES AND DIS ADVANTAGES OF
FERMENTATION
 Advantages of fermentation are that lactic acid can be
produced and it can produce energy forATPs.
 Disadvantages of fermentation are that production can be
slow, the product is impure and needs to have further treatment
and the production carries a high cost and more energy.
IMPORTANCE OF FERMENTATION
Fermentation is important to cells that
don't have oxygen or cells that don't use
oxygen because:
1.It allows the cells to get 2 ATP gain from one molecule
of glucose, even without oxygen.
2.Fermentation takes away the end products of glycolysis
so glycolysis can continue ... freeing up the electron
carriers, and so on.
3.Fermentation is important to the baking industry because
it is the process that yeast uses to produce the bubbles of
carbon dioxide that make the dough rise.
4.Fermentation is important in wineries and breweries
because yeast uses fermentation to produce alcohol.
5.Fermentation is important in muscles because it allows the
muscles to keep getting a little energy from glucose even
when the oxygen supply can't keep up with the demand.
Blood Products /
Substitutes
Introduction
Blood Products: : Any therapeutic substance prepared from the blood. It consists of

Blood Components Plasma Derivatives

Constituent separated from whole
Blood: It consists of
blood
1. Cells i.e. RBC, WBC, Platelets
Red1. cell concentrate 1. Fresh frozen plasma
2. Leuko-reduced RBC 2. Cryoprecipitate
3. 2.
Plasma Plasma i.e. Proteins, Coagulatio n factors
3. Albumin
4. Plasma derivatives 4. Coagulation factors concentrate
5. Granulocyte concentrate 5. Immunoglobulins
6. Platelet concentrate

Blood Substitutes: It consists of

1. Volume Expanders
2. Synthetic oxygen carriers
Collection
 350ml{301ml blood+49ml anticoagulant} is taken from previously
screened person .

 Tested for- syphilis, HBsAg, HCV, HIV 1&2

 Platelet concentrates for bacterial contamination

 Group determination (ABO & Rh) & presence of any RBC

antibody

 Processed into sub-components

Criteria for donor
 Normal body temperature, Blood pressure

 Weight above 50-55 kgs

 Normal Hb levels..

 Free from RTI, skin dz or blood dz

 No h/o drug addiction

 No h/o viral hepatitis

 No HIV infection or risk for it

 No h/o blood transfusion (<=6mo)

 Whole Blood
 It is donor blood mixed with an anticoagulant

 Collected by venesection

 Collected in 63 ml of anticoagulant to form 450 ml.

OR, in 49 ml “ “ “ “ “ “ 350 ml

 Stored at 1-6º C
 Shelf Life up to 5 weeks

 1 unit{350ml} will increase the Hb level of an

Adult (60-70Kg) by 0.8 gm/dl
Pediatric pts by 1gm/dl
• Indication :
Disadvantages :
• Exchange transfusions.
•Risk of circulatory overload
• Hemorrhage (>= 20%blood loss): •Maintaining at ~4oC leads to:
-To ↑O₂ carrying capacity -platelet dysfunction
-degradation of coagulation
-volume replacement
factors
-stabilize coagulation factors -decreased 2,3 DPG levels
• Febrile reactions
Advantages : • Narrow Shelf life- 5wks
• simple and inexpensive
• no special equipment required for
processing
Preservation techniques
 Chemical incorporation

 Rejuvenation solutions

 Additive solutions

 Red cell freezing

 Addition of buffers
 Anticoagulants
Citrate -chief component of almost all anticoagulants used

chelates Ca2+ ... stops coagulation

Dextrose- energy source.
Phosphate- buffer

1. CG (sod.citrate+glucose)- 1st soln ever used

2. ACD (acid+citrate+dextrose)

3. CPD (citrate+phosphate+dextrose)- Higher pH & 2,3 DPG level is maintained.

SHELF-LIFE is 21 days.
 4.CPDA ADP levels- glycolysis- ATP production
(+adenine)- increases RBC viability to 35 days

5.SAG-M (saline+adenine+glucose+mannitol)-

maintains cell nutrition

increases viability to 42 days,
M-prevents spontaneous hemolysis
 Red cell concentrate
Blood to separate under gravity (sedimentation method)
Or,Centrifugation done in special refrigerated centrifuge

Most of the plasma is removed and replaced with a solution of

glucose and adenine in saline to maintain viability of red cells.

 Maintained at temp : 2ºC to 6ºC

 High O₂ carrying capacity

 Optimal target for infusion- 7g/dl

 Indications:
- replacement of red cells in anemic patients
- acute massive blood loss
Advantages :
 Easy to prepare
 To avoid volume overload in c/o CCF
 Less chances of infection or alloimmunization
 Less immunosuppressant
 Dec. allergic reaction if plasma is also removed

Disadvantages :
 High Red cells to plasma ratio ↑viscosity
.·. ↑ time required for passing
through cannula & vessels
thus Hct should not exceed 80%
 Platelet concentrates
 Platelets separated from plasma obtained after 4-6
donations are pooled
or,
from a single donor by plateletapheresis

 Composed of mainly platelets, some nonfunctional

WBCs, few RBCs & plasma[maintains pH].

volume= 50 ml contains 5.5x10^9/lt plts

Stored at 20-24oC
Shelf life- 5 days.. Once opened, transfuse within 6 hrs
1 unit of PC increases platelet count by: 5000-10000(adults)
20,000 (children)
75,000-1,00,000(infants)
If single donor- 1 unit of PC should contain >5.5 х 10 9platelets

If pooled then, > 250х 109 platelets

• Indications:
- Thrombocytopenia
- Platelet dysfunction
- Complication of anti-platelet
therapy(Clopidogrel)
- DIC
 Fresh Frozen Plasma
 Plasma removed from a unit of whole blood & frozen (by
immersing in a solid carbondioxide and ethyl alcohol
 mixture) at/ below -25º C within 4 hrs. ofcollection.

 Stored at -40 to -50oC

 Composed of plasma, all coagulation factors, albumin & Ig

 1 unit= 200-250ml
 Each unit of FFP increases the level of each clotting
factor by 2-3 %

 Shelf life: Frozen—1 year(<-30 degree centigrade)

Indications:

-active bleeding in pts with multiple factor

deficiencies

-surgery in patient with liver failure(treatment of choice)

-after massive transfusion
-DIC

-rapid reversal of warfarin(Prothrombim complex

concentrates,with factors II,IX,X can also be given)
-TTP

Advantage:

it is a acellular component so no chance of transmission of

intracellular infection
Cryoprecipitate

 When FFP is allowed to Thaw at 4oC, glutinous precipitate remains and,

if the suprenatant plasma is removed, cryoprecipitate
 contains factors VIII (very rich) , fibrinogen as well as factor XIII &
vwf.
 Shelf life:2 years , if frozen at -40oC
Used
1. If fibrinogen <1g/dl due to dilution
2. DIC
3. Von Willebrand disease and hemophilia VIII deficiency
 Coagulation Factors
Factor VIII concentrate
 Prepared from large pools of donor plasma by fractionation process.
 Commercially prepared, lyophilized powder
 Hemophilia A & von Willebrand’s disease.
 Storage : 2 to 6°C

Factor IX
 Commercially prepared, lyophilized powder
 Hemophilia B
 Contains Factor II (prothrombin), VII,IX,X.
 Purified Factor IX contains only IX.
 Refrigerated at 35-45 °F
 Fibrinogen
Immunoglobulin
 It is a concentrated solution of IgG  Prepared by organic liquid fractionation of plasma
antibody component of plasma
 Stored in dried form
 Prepared from large pools of donors
 Can be reconstituted with distilled water
 Uses
 Used in cases of severe fibrinogen depletion
1. To reduce infective complications in
patients with Ab deficiencies eg DIC, congenital afibrinogenaemia
2. Immunological d/o- Immune
 Carry high risk of hepatitis
thrombocytopenia, GBS
3. Anti-zoster Ig in varicella zoster
prophylaxis
4. Anti-Rhesus D Ig in pregnancy to
prevent hemolytic dsz in newborn
 Can Cause
 Acute renal failure ( in elderly)
 Acute reactions
 Blood Substitutes
1. volume expanders
2. synthetic oxygen carriers

1.Volume expanders: inert compounds

• crystalloid-based • colloid-based
 Ringer's lactate • Dextrans
 Normal saline • Albumin
 D5W (dextrose 5% in water) • Gelofusin
 Dextrose with normal saline • Hydroxyethyl starch
 Hypertonic saline
 Crystalloids
 3)5% Dextose
1) ) Ringer’s Lactate Solution(Hartman a) Is isotonic, but with the
Solution) metabolism of glucose
 inside the body, becomes
a) Consists of Na, Cl, K, Ca, Lactate. pH=6.5,
Osmolarity=273mmol/lt (slightly hypotonic)
hypotonic.
b) Blood should not be given through the same drip set as b) Blood cannot be given through
it contains Ca.
the same drip set otherwise
Crystalloid of choice for blood loss replacement.
c)
rouleaux formation will cause
2) ) Normal Saline {0.9% NaCl (isotonic)}
clumping of RBCs
a) Preferred over RL for treating
• Hypochloremic metabolic alkalosis

• Brain injury (Ca2+ and lactate can increase the 4) Dextrose Normal Saline (DNS)
neuronal injury) a) Is hypertonic
• Hyponatremia b) But 1/5 NS +4.3% dextose and ¼ NS +5%
dextrose are isotonic and are best used as
maintenance fluids.

Colloids
 1) Dextrans (Lomodex)
 Polysaccharides, can be stored for 10 years, half life=2-8 hours
 Advantages
a) Non toxic, neutral and chemically inert
b) Low molecular wt dextran improves microcirculation
 Drawbacks

a) Interfere with blood grouping and cross matching (by causing RBC aggregation, so a
blood sample should be taken before-hand)
b) Interferes with platelet function and is a/w abnormal bleeding (so total volume given
should not exceed 1000ml)
c) Can cause severe anaphylaxis
d) Large molecular weight dextrans can block renal tubules
e) ARDS (rarely) because of direct toxic effect on pulmonary capillaries
 2) Albumin(available as 5% and 20% solution)
 Very expensive, intravascular half-life= 10-15days
 Used in protein loss like, peritonitis, liver failure, burns, protein losing enterepathies.
 20% is hypertonic and expands plasma volume by more than the amount infused.
 Stored for several months in liquid form at 4degree

 3)Gelatins (Haemaccel)(available as 3.5% solution)

 Consists of Gelatin, Na, Cl, K, Ca
 Expand plasma effectively for 2 hours
 At clinically used doses, these do not interfere with blood grouping, plt fxn and clotting
but at high doses can interfere clotting.
 As it contains high Calcium, citrated blood should not be mixed.
Synthetic oxygen carriers:
mimic blood's O₂ transport ability
Types
1. Abiotic - perfluorocarbons, perfluoroctyl bromide
2. Biomimetic - HBOC( Hb based oxygen carriers)

- recombinant erythropoietin, hemoglobin under clinical trials

some available products:

Hemopure
Oxygent
PolyHeme
 Perfluorocarbons HBOC
 Hb synthesised by controlled lysis of Red
 High O₂ carrying capacity Cells

 Emulsified and transfused  Short half life as compared to RBCs

 Short survival in the circulation  Side effects :

GI distress, neurotoxicity,
 Adverse effects : Flu like symptoms. vasoconstriction,
Immunologic effects. interfere with macrophage system.