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Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
Consumption and production
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Table 1 - Major uses of glutamic acid and its derivatives in research
PROTEIN ENGINEERING Peptide synthesis Protein modification Polymer supports
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Therapeutic applications of glutamic acid
ENTERAL NUTRITION
Crystalline glutamic acid in solution: It is the source of protein precursors for
hospitalized patients unable to eat or eat enough to get or stay healthy.
PARENTERAL NUTRITION
Crystalline glutamic acid in solution: Given to the patient through circulatory system via
a vein
Tablet, capsule or powder protein precursors: For people able to eat but who need very
highly concentrated source to recover from illness or surgery
O O
HO HO
NH2
Contd…..
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Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
Final purification is performed to ensure the required quality for the
intended use
The product is released only after quality tests have verified that the product
meets specific requirements, and the normal functioning of each process
step has been verified
All manufacturing processes for the production of amino acids for medical
use must comply with current good manufacturing practice requirements
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Manufacturing of L-Gln
It is essential to the outcome of the fermentation process to maintain a clean
and sterile fermentation tank
Compared with wild-type strains, L-Gln-producing strains are weak and are
compromised in a contaminated environment
Furthermore, it is important to maintain the tank under positive pressure by
aeration during fermentation to prevent contamination by other
microorganisms and external materials
The fermentation medium consists of glucose as a carbon source, ammonia
as a nitrogen source, a small amount of minerals and vitamins as growth
factors
Control factors during fermentation are pH, temperature and dissolved
oxygen
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Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
Schematic representation of glutamic acid production
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Strains producing glutamic acid
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Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
Biosynthesis of glutamic acid
Glutamic Acid
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Conditions of manufacture
Carbon sources: glucose, sucrose, maltose, xylose, sugarcane and
sugarbeet, molasses, strach, hydrolysates
Nitrogen sources: ammonium salts, ammonia, urea
Oxygen supply:
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Overview of fermentation
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Fermented broth
Centifugation
Collect supernatant
Column packing
Separation of fluid
(acidified to pH 3.2, with 1 N HCl. Storage at 20°C for 48 h)
Crystallization
Downstream Processing
K. Madhavan Nampoothiri et al; Revista de
microbiologta.1999; 30. 17
Preparation of resin
Spherical particles of cation exchange resin, Amberlite is used
After two washes with distilled water, the resin is then washed with
2 N NaOH until the filtrate was alkaline
The procedure was repeated two times. The resin was filtered and
washed with distilled water to make it free of alkali
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Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
Effect of temperature on solubility of glutamate
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Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
Crystallization
Purification of amino acids by crystallization is an effective means to produce
polymorphism, two crystal forms can be used
After crystallization of amino acid in the one form, the crystals are dissolved, and
then recrystallized in the other form
Therefore, to use crystallization for purification, there is no way other than the
inefficient simple repetitive crystallization of the one crystal form of
L-Gln
By changing the pH to the isoelectric point (3.2) and by the subsequent cooling
of the eluent, glutamic acid was crystallized out.
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Simplified production flow chart of the L-Gln manufacturing
process
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Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
On-line optimization
Higher glutamate concentration could be achieved by constantly
controlling dissolved oxygen concentration (DO) at a lower level; however,
by-product lactate also severely accumulated
The entire metabolic network flux analysis showed that the lactate
overproduction was because the metabolic flux in TCA cycle was too low
to balance the glucose glycolysis rate
The concentrations of cells, glucose, glutamate, and lactate are measured during
the course of fermentation
The CO2 and O2 concentrations (partial pressure) in the inlet and exhaust gas were
on-line measured by a gas analyzer
The collected on-line data were smoothly filtered, and then oxygen uptake rate
(OUR) and CO2 evolution rate (CER) were on-line calculated
Respiratory quotient (RQ) was determined by its definition (RQ = CER/OUR) using
the on-line measured OUR and CER data.
Based on the RQ set-points and the measured RQ, the PC on-line regulated
the agitation rate of the fermentor (AGT) with the equation below
• A higher glutamate production rate could be achieved when the DO was controlled
at a lower level of 10% and the final glutamate concentration reached about
91.5 g/L at 34 h
• Final glutamate concentration stopped at a lower level of 72.7 g/L (30 h) when
controlling DO at 50%
Contd…..
Xiao J et al; Bioprocess Biosystem Engineering. 2006;29(2):109–117. 26
•These results suggested that the enzymatic activities of GDH and LDH under lower and higher
DO level might be quite different
•To verify the above speculation, an experiment under extremely low DO level was conducted.
In the fermentation, DO was initially controlled at 30%, and the agitation rate was manually
reduced to bring DO down to 0% instantly at 12 h
•Then, the same agitation rate was kept for the next 6 h.
•During this period, the fermentation could be considered as implemented under anaerobic
condition, glutamate production stopped and lactate overflowed
•At 18 hr, the automatic control of DO was resumed to quickly bring DO back to 30%, a partial
recovery of glutamate production was observed
•However, the final glutamate concentration ended at a very low level of about 45 g/L
•The results indicated that the occurrence of anaerobic condition even for a short period
would be both fatal and irretrievable to glutamate fermentation
Contd…..
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Effect of different DO levels on glucose consumptions during aerobic
conditions
Contd…..
29
● Under the lower DO level, even though GDH activity was higher, the
higher glutamate synthesis rate (r6) still could not completely balance
with the glycolysis rate (r1), as TCA cycle was almost closed completely
and the TCA metabolic flux (r4) was very low
● On the other hand, under the higher DO level, GDH activity was
relatively low, but the TCA cycle was nearly open for a complete
oxidation
● Under this condition, lactate was not necessarily overflowed, as the
glutamate synthesis rate (r6) plus the higher TCA metabolic flux (r4)
were big enough to balance with the glycolysis rate (r1), even though
LDH exhibited almost equivalent activity as compared with that of the
lower DO case
Contd…..
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Effect of DO Levels on various parameters
Glutamate concentration: (filled circle) DO 10%, (filled triangle) DO 50%; lactate concentration:
(open circle) DO 10%, (open triangle) DO 50%. b GDH activity: (filled circle) DO 10%, (open circle)
DO 50%; LDH activity: (filled triangle) DO 10%, (open triangle) DO 50%. c Cells concentration:
(filled circle) DO 10%, (filled triangle) DO 50%. d Glucose concentration: (filled circle) DO 10%,
(filled triangle) DO 50%
• Initial studies were carried out in shake flasks, which showed that even
though the yield was high with 85-90 DE (Dextrose Equivalent value), the
maximum conversion yield (~34%) was obtained by using only partially
digested starch hydrolysate, i.e. 45-50 DE
●Cassava starch hydrolysate (85-90 DE) was diluted to 5% initial sugar concentration
and was supplemented with 1 ml mineral solution, 100 µl corn steep liquor and one
drop of Tween 80 in 100 ml starch hydrolysate (pH 7.2)
● The initial concentration of reducing sugars in the medium was 5%, and at the
stages, where the concentration fell to 2%, starch hydrolyzate solution containing
10% reducing sugars, was added to bring the sugar concentration of fermenting
medium as 5%
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K. Madhavan Nampoothiri et al; Revista de microbiologia. 1999;30.
Growth and glutamic acid production based on the
hydrolysate having different DE values
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Consumption of reducing sugars by Brevibacterium sp. at
different DE values
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Yields of L-glutamic acid at different DE value
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Percentage conversion at different DE values
A= 15-20% DE
B= 30-35% DE
C= 45-50% DE
D= 60-65% DE
E= 85-90% DE
With 85-90 DE hydrolysate, the conversion was lowest (~27%). Thus, if conversion
factor has to be considered as a major criterion, a low DE value hydrolysate, i.e. 45-50
DE would be sufficient for L-glutamic acid production.
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Recovery of glutamic acid
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Suppliers
Raj Enterprises
BUSINESS PROFILE
Manufacturing and supplying food ingredients such as xanthan gum, sodium gluconate, silicon di-
oxide, potassium sorbate, monosodium glutamate, ascorbic acid use as a chemical preservatives for
sauces and homemade products.
CONTACT DETAILS
Street Address: 236/238, 4th Floor, Samuel Street
City: Mumbai State: Maharashtra PIN: 400 003 Country: India
Phone: +(91)-(22)-23402742 Fax: +(91)-(22)-66315361
Eurofine Chemicals
BUSINESS PROFILE
Manufacturers and exporters of zinc stearate, zinc cyanide, sodium acetate, mono sodium
glutamate, magnesium turnings etc.
CONTACT DETAILS
Street Address: 212, Maker Bhavan No. 3, 21, New Marine Lines
City: Mumbai State: Maharashtra PIN: 400 009 Country: India
Phone: +(91)-(22)-23455101/23475416/22074206 Fax: +(91)-(22)-23475049
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P.D. Udyog
BUSINESS PROFILE
Manufacturer of citric acid, dried yeast and mono-sodium-glutamate.
CONTACT DETAILS
Street Address: No. 7, 1st Floor, G. S. Market, Patnoolpet O. T. Pet Cross
City: Bangalore State: Karnataka PIN: 560 053 Country: India
Phone: +(91)-(80)-23381195
Website: http://www.indiamart.com/company/1049219/
R. M. Chemicals
BUSINESS PROFILE
Suppliers of all kinds of mono sodium glutamate, citric acid, ascorbic acid etc.
CONTACT DETAILS
Street Address: 47, Nattu Pilliar, Koil Street
City: Chennai State: Tamil Nadu PIN: 600 001 Country: India
Phone: +(91)-(44)-25221743
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