1 Background • Industrial production of amino acids have started with availability of monosodium glutamate (MSG) in 1909 • Discovered by Dr. Kikunae Ikeda in 1908 • Originally manufactured by extraction from acid hydrolysis of plant protein • In late 1950 fermentation technology was established and commercially exploited for other amino acids • L-Glutamine fermentation started in late 1960 Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
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Background - gpat victory€¦ · • Industrial production of amino acids have started with availability of monosodium glutamate(MSG) in 1909 • Discovered by Dr. Kikunae Ikedain
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1
Background
• Industrial production of amino acids have started with availability ofmonosodium glutamate (MSG) in 1909
• Discovered by Dr. Kikunae Ikeda in 1908
• Originally manufactured by extraction from acid hydrolysis of plant protein
• In late 1950 fermentation technology was established and commerciallyexploited for other amino acids
• L-Glutamine fermentation started in late 1960
Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
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Consumption and production
• Worldwide consumption of amino acids is about 2 million tonnes
• About 1.5 million tonnes was sold in 2001
• 4% annual growth in sale is observed
• The annual demand of amino acids in food and pharmaceutical industry is 4,60,000 tonnes
• The annual worldwide production of L-glutamine is 3,70,000 tonnes
Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
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Uses
MSG is used commercially as a flavour enhancer.
Although once stereotypically associated with foods in Chineserestaurants; it is now found in many common food items, particularlyprocessed foods
Examples include:
Canned soups
Pre-prepared stocks
Common snack foods
Most fast foods
Instant meals such as the seasoning mixtures for instant noodles
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Table 1 - Major uses of glutamic acid and its derivatives in research
PROTEIN ENGINEERING Peptide synthesis Protein modification Polymer supports
BIOCHEMICAL/CELL BIOLOGY Biochemical experiments Cell biology research
ANALYTICAL APPLICATIONS Analytical standards Diagnostic products and procedures
Table 2 - Analytical uses of glutamic acid and its derivatives
DIAGNOSING DISEASE Congenital metabolic diseases, Disorders or malfunction of brain/nervous system
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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
PRESCRIPTION DIETARY SUPPLEMENTS
Tablet, capsule or powder protein precursors: For people able to eat but who need veryhighly concentrated source to recover from illness or surgery
CONGENITAL METABOLIC DISEASES
Powder Prescription diets: For newborn babies and others who need a diet devoid of or with highly reduced content of specific amino acid
HYPERTENSION (Capsules or injection)
Prescription drugs that reduce blood pressure
NEURO-REGULATORS (Capsules or injection)
Therapeutic applications of glutamic acid
Structure
HO HO
O O
NH2
●Glutamic acid is a dicarboxylic monoamino acid●Non-essential or dispensible amino acid
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General manufacturing process
The manufacturing methods of amino acids are-
Extraction from acid hyrolysates
Chemical synthesis
Fermentation
Enzymatic
Leucine, proline, tyrosine, cystine are manufactured by extraction, fermentationand chemical synthesis
L-Glutamic acid is manufactured world wide using fermentation
The manufacturing process of an amino acid by fermentation comprisesfermentation, crude isolation and purification processes.
In the crude isolation process, most impurities contained in the fermentationbroth are removed by combining various technologies
Contd…..
Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
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Final purification is performed to ensure the required quality for theintended use
The final product is obtained as a crystalline powder
The product is released only after quality tests have verified that the productmeets specific requirements, and the normal functioning of each processstep has been verified
All manufacturing processes for the production of amino acids for medicaluse 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 arecompromised in a contaminated environment
Furthermore, it is important to maintain the tank under positive pressure byaeration during fermentation to prevent contamination by othermicroorganisms and external materials
The fermentation medium consists of glucose as a carbon source, ammoniaas a nitrogen source, a small amount of minerals and vitamins as growthfactors
Control factors during fermentation are pH, temperature and dissolvedoxygen
Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
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Schematic representation of glutamic acid production
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Strains producing glutamic acid
Most exploited is Corynebacterium glutamicum
Other genera of Corynebacterium is also used
Brevibacterium sp., Microbacterium sp., Arthrobacter sp. are
also used
All glutamic acid producers require biotin for their activity
All the strains show little activity of α-ketoglutarate
dehydrogenase
Increased activity of glutamate dehydrogenase
Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
Separation of fluid(acidified to pH 3.2, with 1 N HCl. Storage at 20°C for 48 h)
Crystallization
Downstream ProcessingK. Madhavan Nampoothiri et al; Revista de
microbiologta.1999; 30.
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Spherical particles of cation exchange resin, Amberlite is used
The resin is washed thoroughly two times with 4 N HCI
After two washes with distilled water, the resin is then washed with2 N NaOH until the filtrate was alkaline
The resulting material (sodium salt of the resin) is suspended in 3-timesits volume of 1 N NaOH and heated over a steam bath for 2 h withoccasional mixing
The supernatant fluid was decanted after 30 minutes of settling andreplaced with fresh hot 1 N NaOH
The procedure was repeated two times. The resin was filtered andwashed with distilled water to make it free of alkali
The resin was finally stored as the moist sodium salt
Preparation of resin
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Flow diagram of the isolation process.
Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
• The solubility of L-Gln is barely affected by temperature as shown by the flat
solubility curve.
• Consequently, cooling crystallization is not applicable for harvesting L-Gln.
Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
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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
In this manner, it is possible to remove impurities based on their different affinities for the two crystal forms
Unfortunately, L-Gln occurs only as one crystal 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.
Crystallization
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Simplified production flow chart of the L-Gln manufacturing process
Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
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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
Activities of glutamate and lactate dehydrogenases changed during thethe fermentation
The entire metabolic network flux analysis showed that the lactateoverproduction was because the metabolic flux in TCA cycle was too lowto balance the glucose glycolysis rate
As a result, the respiratory quotient (RQ) adaptive control based“balanced metabolic control” (BMC) strategy was proposed and used toregulate the TCA metabolic flux rate at an appropriate level to achieve themetabolic balance among glycolysis, glutamate synthesis, and TCAmetabolic flux
Xiao J et al; Bioprocess Biosystem Engineering. 2006; 29(2);109–117.
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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.
Xiao J et al; Bioprocess Biosystem Engineering. 2006;29(2):109–117.
Analytical methods
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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
• k represented the current control instant
• RQset was the RQ set-point which might be subject to changes during thecontrol
• KC and τI were proportional and integral constants of the feedbackcontroller, respectively
• KC and τI were determined by observing the RQ response to a step changein the input (agitation rate) during a certain period of the glutamateproduction phase.
On-line control system
Xiao J et al; Bioprocess Biosystem Engineering. 2006;29(2):109–117.
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• Glutamate and lactate formation pattern strongly depended on the DO control level
• 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%
• On the other hand, lactate severely accumulated up to 28 g/L when DO was controlled at a lower level of 10%
• Almost no lactate accumulation occurred when controlling DO at 50%
Observations after setting a particular DO level
Contd…..Xiao J et al; Bioprocess Biosystem Engineering. 2006;29(2):109–117.
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•These results suggested that the enzymatic activities of GDH and LDH under lower and higher DO level might be quite different
•Generally, it is considered that the anaerobic condition is extremely harmful to glutamate production
•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
Open circle: DO = 10%
Open triangle: DO = 50%
Xiao J et al; Bioprocess Biosystem Engineering. 2006;29(2):109–117.
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Balanced metabolic control
● In glutamate fermentation glycolysis rate should balance withglutamate synthesis, lactate formation and TCA metabolicflux
● Severe lactate accumulation at lower DO control (DO = 10%)was due to the carbon metabolic balance rather than thehigher LDH activity by the following facts-
1) The changing patterns of glycolysis rate (r1) at different DO levels were almost the same
2) No significant differences in LDH activities were shown under different DO level
3) The metabolic flux of TCA significantly decreased with the decrease in DO control level
Contd…..
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● 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
● Under this circumstance, lactate had to be overflowed or excreted (r5) into the broth to achieve the entire intracellular carbon balance
● 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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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%
Effect of DO Levels on various parameters
Xiao J et al; Bioprocess Biosystem Engineering. 2006;29(2):109–117.
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• Carried by K. Nampoothiri and Ashok Pandey, Biotechnology Division, Regional Research Laboratory, CSIR, Trivandrum
• Brevibacterium sp. was used
• 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
K. Madhavan Nampoothiri et al;Revista de microbiologia. 1999;30
Fermentation and recovery of L-glutamic acid from cassava starch hydrolysate
●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)
●Fermentation was carried out with a working volume of 2.5 L in a 5 L fermenter
●Dissolved oxygen was maintained at 60% of air saturated medium
K. Madhavan Nampoothiri et al; Revista de microbiologia.1999;30.
Batch process for Casava starch hydrolysate
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● Fed-batch process was also carried out in the fermenter
● 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%
● Fermentation conditions were the same as for batch process
Contd…..K. Madhavan Nampoothiri et al; Revista de microbiologia.1999;30
Fed-batch process for Casava starch hydrolysate
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●Fermentations were carried out in batch mode in a 5 L fermenter, usingsuitably diluted cassava starch hydrolysate, using a 85-90 DE value hydrolysate
●Media supplemented with nutrients resulted in an accumulation of 21 g/Lglutamic acid with a fairly high (66.3%) conversation yield of glucose toglutamic acid (based on glucose consumed and on 81.74% theoreticalconversion rate)
●The bioreactor conditions most conducive for maximum production were pH7.5, temperature 30°C and an agitation of 180 rpm
●When fermentation was conducted in fed-batch mode by keeping the residualreducing sugar concentration at 5% w/v, 25.0 g/L of glutamate was obtainedafter 40 h fermentation (16% more the batch mode)
K. Madhavan Nampoothiri et al; Revista de microbiologia. 1999;30.
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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
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.
K. Madhavan Nampoothiri et al; Revista de microbiologia. 1999;30.
Comparison of bacterial growth and L-glutamic acid production in batch and fed-batch process
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● The fermented broth contained various impurities such as bacterial cells,
macromolecules, pigments, inorganic substances, organic substances etc., which
were removed by filtration and centrifugation
● Glutamic acid was purified from cation exchange resin
K. Madhavan Nampoothiri et al; Revista de microbiologia.1999;30.
Recovery of glutamic acid
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Glutamic acid recovered at different elution volumes through ion-exchange resin column
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Raj EnterprisesBUSINESS PROFILEManufacturing 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.
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P.D. UdyogBUSINESS PROFILEManufacturer of citric acid, dried yeast and mono-sodium-glutamate.CONTACT DETAILSStreet Address: No. 7, 1st Floor, G. S. Market, Patnoolpet O. T. Pet Cross City: Bangalore State: Karnataka PIN: 560 053 Country: IndiaPhone: +(91)-(80)-23381195Website: http://www.indiamart.com/company/1049219/
Dudley & Brother Private Limited, KolkataBUSINESS PROFILESellers of various types of mono sodium glutamate.CONTACT DETAILSStreet Address: 13, Ganesh Chandra Avenue City: Kolkata State: West Bengal PIN: 700 013 Country: IndiaPhone: +(91)-(33)-22368899/22367322 Fax: +(91)-(33)-22252714
R. M. ChemicalsBUSINESS PROFILESuppliers of all kinds of mono sodium glutamate, citric acid, ascorbic acid etc.CONTACT DETAILSStreet Address: 47, Nattu Pilliar, Koil Street City: Chennai State: Tamil Nadu PIN: 600 001 Country: IndiaPhone: +(91)-(44)-25221743