FERMENTATION.pptx

MODULE- 5
FERMENTA
TIONAND
BLOOD PRODUCTS
BALASUNDARESAN M
B Pharmacy 6th sem
Subject: Pharmaceutical Biotechnology
SubjectCode: BP605T

Objectives of Course
•Appreciate the use of microorganisms
technology.
• Genetic engineering applications in relation
in fermentation
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 A
TP
, which means it is a form
of substrate-level phosphorylation. This contrasts with
oxidative phosphorylation, which uses energy from an
established proton gradient to produceA
TP
.

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
where pyruvate (from glucose
fermentation
metabolism) is broken into ethanol and
carbon dioxide. The net chemical
for the production of ethanol from
equation
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.

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 (lacticacid)
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 OFFERMENTATION
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 generateA
TPin respiration. This iscalled

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 + 2A
TP+ 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 A
TP 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
Y
ogurts
 cheese
 Sauerkraut
 Kimchi
 Pepperoni
ETC………..

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 ATPgain from onemolecule
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.

Introduction
Blood: It consistsof
1. Cells i.e. RBC, WBC, Platelets
2. Plasma i.e. Proteins, Coagulation factors
Blood Substitutes: It consistsof
1. Volume Expanders
2. Synthetic oxygen carriers
Blood Products: : Any therapeutic substance prepared from the blood. It consists of
Blood Components
Constituent separated from whole
blood
Plasma Derivatives
1. Red cell concentrate
2. Leuko-reduced RBC
3. Plasma
4. Plasma derivatives
5. Granulocyte concentrate
6. Platelet concentrate
1. Fresh frozen plasma
2. Cryoprecipitate
3. Albumin
4. Coagulation factors concentrate
5. Immunoglobulins

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.
“ “ “ 350 ml
OR, in 49 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)
Pediatric pts
by 0.8 gm/dl
by 1gm/dl

• Indication :
• Exchange transfusions.
• Hemorrhage (>= 20%blood loss):
-To ↑O₂ carrying capacity
-volume replacement
-stabilize coagulation factors
Advantages :
• simple and inexpensive
• no special equipment required for
processing
Disadvantages :
•Risk of circulatory overload
•Maintaining at ~4oC leads to:
-platelet dysfunction
-degradation of coagulation
factors
-decreased 2,3 DPG levels
• Febrile reactions
• Narrow Shelf life- 5wks

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
(+adenine)-
ATP production
ADP levels- glycolysis-
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 cel 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
 Stored at -40 to -50oC
 Composed of plasma, all coagulation factors, albumin & Ig
 1 unit= 200-250ml
 Plasma removed from a unit of whole blood & frozen (by
immersing in a solid carbondioxide and ethyl alcohol
 mixture) at/ below -25ºC within 4 hrs. ofcollection.
 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.
at -40oC
▶Shelf life:2 years , if frozen
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

Immunoglobulin
▶ It is a concentrated solution of IgG
antibody component of plasma
▶ Prepared from large pools of donors
▶ Uses
1. To reduce infective complications in
patients with Ab deficiencies
2. Immunological d/o- Immune
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
Fibrinogen
▶ Prepared by organic liquid fractionation of plasma
▶ Stored in dried form
▶ Can be reconstituted with distilled water
▶ Used in cases of severe fibrinogendepletion
eg DIC, congenital afibrinogenaemia
▶ Carry high risk of hepatitis

Blood Substitutes
1. volume expanders
2. synthetic oxygen carriers
1.Volume expanders: inert compounds
• crystalloid-based • colloid-based
Ringer's lactate
Normal saline
D5W (dextrose 5% in water)
Dextrose with normal saline
Hypertonic saline
• Dextrans
• Albumin
• Gelofusin
• Hydroxyethyl starch

Crystalloids
a) Is isotonic, but with the
metabolism of glucose
 inside the body, becomes
hypotonic.
b) Blood cannot be given through
the same drip set otherwise
rouleaux formation will cause
clumping of RBCs
4) Dextrose Normal Saline (DNS)
a) Is hypertonic
b) But 1/5 NS +4.3% dextose and ¼ NS +5%
dextrose are isotonic and are best used as
maintenance fluids.
1) ) Ringer’s Lactate Solution(Hartman
Solution)
a) Consists of Na, Cl, K, Ca, Lactate. pH=6.5,
Osmolarity=273mmol/lt (slightly hypotonic)
b) Blood should not be given through the same drip set as
it contains Ca.
c) Crystalloid of choice for blood loss replacement.
2) ) Normal Saline {0.9% NaCl (isotonic)}
a) Preferred over RL for treating
• Hypochloremic metabolic alkalosis
• Brain injury (Ca2+ and lactate can increase the
neuronal injury)
• Hyponatremia
▶ 3)5% Dextose

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
▶ High O₂ carryingcapacity
▶ Emulsified and transfused
▶ Short survival in the circulation
▶ Adverse effects : Flu like symptoms.
Immunologic effects.
HBOC
▶ Hb synthesised by controlled lysis of Red
Cells
▶ Short half life as compared to RBCs
▶ Side effects :
GI distress, neurotoxicity,
vasoconstriction,
interfere with macrophage system.

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