Transcript
ASHIKA RAVEENDRAN
2ND MSC BIOTECHNOLOGY
VITAMINS
Vitamins are essential micronutrients required
in trace quantities that cannot be synthesized
by mammals
They are essential for metabolism for all living
organisms
Apart from their nutritional –physiological roles
as growth factor ,vitamins are increasingly
being introduced as food/feed additives ,as
medical therapeutic agents .
Today many processed foods, feeds
pharmaceuticals, cosmetics and chemical
contain extraneously added vitamins or vitamin
related compounds.
Presently few of the vitamins are chemically
synthesized or via extraction processes
With growing consumer consciousness led to
substituting with biotechnological processes.
FAT SOLUBLE VITAMINS
Vitamin A
Vitamin D
Vitamin E
Vitamin K
Vitamin E Most abundant among fat soluble vitamins and has
the highest antioxidant activity in vivo.
In nature, only photosynthetic organisms arecapable of producing α-tocopherol.
In humans, ∞-tocopherol is believed to play amajor role in prevention of light inducedpathologies of the skin, eyes and degenerativedisorders such as atherosclerosis, cardiovasculardiseases and cancer.
Industrial application of ∞-tocopherol includes itsuse in preservation of food, in cosmetics andsunscreens
Currently ∞-tocopherol is obtained by chemical
synthesis and by extraction from vegetable oils
Extraction from oil is not efficient ,as these typically
contains low levels of ∞-tocopherol.
Several strains of freshwater microalgae Euglena
gracilis Z and marine microalgae Dunaliella
tertiolecta produce
∞-tocopherol in concentrations higher than
conventional foods.
production of high amounts of vitamin E has been successfully demonstrated by E. gracilis Z. using two-step culture.
In the first step of the batch culture, E. gracilis Z. was photo- heterotrophically cultivated in modified Oda and modified Hunter media at high light intensity.
When the cells reached late exponential phase, they were separated, washed and resuspended in the same volume of Cramer and Mayers (CM) medium for the second step of cultivation.
The two-step cultures using high cell densities gave high productivity of antioxidant vitamin
.Vitamin KVitamin K₂ Vitamin K₂Vitamin K₁
Fermentative Production of
Vitamin K₂ Tani and Taguchi have reported that as much
as 182 mg/L MK was produced using
detergent supplement culture and a mutant of
Flavabacterium.
Lactic acid bacteria are reported to produce
MK with the yield of 29–123 g/L MK-7, MK-8,
MK-9 and MK-10.
In fermented soybeans, Bacillus subtilis
produces menaquinones, the major component
being MK-7 and the minor one being MK-6.
Sumi studied production of MKs by the
fermentation of okara with seven different
natto bacilli.
The highest production rate of 36.6 mg/g was seen in the Chinese natto strain followed by (in mg/g of okara-natto wet mass): 14.2 in Naruse, 11.9 in Asahi, 6.8 in Takahashi, 1.9 in Miyagino (natto bacilli for food production), and 5.2 in Nitto and 1.9 in Meguro (natto bacilli for medicine) after incubation for 4 days at 37 °C.
The water-soluble vitamin K was isolated as a dark yellow powder by DEAE Sepharosechromatography and membrane filter fractionations.
WATER SOLUBLE VITAMINS
Biotin
Riboflavin
cyanocobalamin
VITAMIN B 12
Cyanocobalamin, by definition vitamin B12, is the industrially produced stable cobalamin form which is not found in nature.
Vitamin B12 is obtained exclusively by fermentation process.
Important dietary component, requirement 0.001 mg/day.
Cyanocobalamin consist of a cobinamidelinked to a nucleotide.
Cobinamide – cobalt linked to cyanide grp, surrounded by 4 reduced pyrrole ring.
Nucleotide – 5 , 6 – dimethylbenziminazole
VITAMIN B 12
BIOSYNTHESIS
MICROORGANISMS IN
INDUSTRIAL PRODUCTION OF
VIT. B12 Streptomyces griseus , S. olivaceus , Bacillus
megaterium ,
B. coagulans , Pseudomonas denitrificans ,
Propionibacterium freudenreichii , P.
shermanii and
a mixed fermentation of a Proteus spp and a
Pseudomonas sp.
Manufactured by submerged fermentation
Aeration and agitation of medium essential
Fermentation process completed in 3 to 5 days
VIT.B12 PRODUCTION USING Streptomyces olivaceus NRRL B-1125
PREPARATION OF INOCULUM
Pure slant culture of Streptomyces olivaceus
NRRL B-1125 is inoculated and grown in 100
to 250 ml of inoculum medium.
Seeded flask are kept on shaker for incubation
.
Flask cultures are used to inoculate large
amount of inoculum media arranged in series
of tank .
2 or 3 successive transfers are made to obtain
required amount of inoculum cultures.
Inoculum of production tank must be 5% of the
volume of production medium
PRODUCTION MEDIUM
Consist of carbohydrate ,proteinaceous material ,
and source of cobalt and other salts .
Sterilization of medium batch wise or continuously .
Batch – medium heated at 250°F for 1 hr
Continuous – 330°F for 13 min by mixing with live
steam.
COMPONENTS AMOUNT (%)
Distillers solubles 4.0
Dextrose 0.5 to 1
CaCO3 0.5
COCl2.6H2O 1.5 to 10 p.p.m.
TEMPERATURE , PH ,
AERATION AND AGITATION Temperature : 80°F
pH: At starting of process pH falls due to rapidconsumption of sugar, then rises after 2 to 4due to lysis of mycelium
pH 5 is maintained with H2SO4 and reducingagent Na2SO4 .
Aeration and agitation : Optimum rate ofaeration is
0.5 vol air/vol medium/min. Excess aerationcause foaming.
ANTIFOAM AGENT ,
PREVENTION OF
CONTAMINATION Antifoam agent : soya bean oil , corn oil,
lard oil and silicones (sterilized before adding) .
Prevention of contamination : essential to maintain sterility ,
contamination results in reduced yields , equipments must be sterile and all transfers are carried out under aseptic conditions
Yield : yield of cobalamin are usually in the range of 1 to 2 mg. per litre in the fermented broth
DOWNSTREAM PROCESS OF
VITAMIN B12DISSOLVED VITAMIN B
12
CONCENTRATION OF CELL MASS
CREAM CONCENTRATION OF CELL MASS
EX(TRACTION Eg :alcohol such as
methanol
Dissolved vitamin B12
Chromatography
Pure vitamin B12
Crystallisation from organic
solvents
RIBOFLAVIN
Riboflavin, or vitamin B2, is used for human
nutri- tion and therapy and as an animal feed
additive.
Its deficiency in humans is correlated with loss
of hair, inflammation of skin, vision deterioration,
and growth failure.
This vitamin has also been found to be
successful in treatment of migraine and malaria .
Riboflavin has been produced commercially by
chemical synthesis, by fermentation and by a
combination of fermentation and chemical
synthesis.
MICRO –ORGANISMS IN
INDUSTRIAL PRODUCTION OF
VIT. B2 Although bacteria (Clostridium sp.) and yeasts
(Candida sp.) are good producers, two closely
related ascomycete fungi, Eremothecium
ashbyii and Ashbya gossypii, are considered
the best riboflavin producers.
Ashbya gossypii produces 40 000 times more
vitamin than it needs for its own growth.
OTHER MICRO -ORGANISMS
PRODUCING VIT. B 2 Gene amplification and substitution of wild type
promoters and the regulatory regions with strong constitutive promoter from Bacillus subtilis have resulted in increased riboflavin production .
Lactococcus lactis MG 1363 strain using both direct mutagenesis and metabolic engineering for simultaneous overproduction of both folate and riboflavin
Improved strains for the production of riboflavin were constructed through metabolic engineering using recombinant DNA techniques in Corynebacterium ammoniagenes
PREPARATION OF INOCULUM
Starts from slants or spores dried on sand.
After 1 or 2 stages, further propagation is
carried on 1 or 2 tank inoculum stages
PRODUCTION MEDIUM Fermentor 10,000 to 1,00,000 gals range.
Production medium designed according to type of micro- organism.
Ashbya gossypii : sources - palm oil ,corn steep liquor, glucose,
molasses , whey, collagen , soya oil , glycine.
Stahmann et al. reported riboflavin yields in excess of 15 g/L of culture broth in a sterile aerobic submerged fermentation of Ashbya gossypii with a nutrient medium containing molasses or plant oil as
major carbon source.
Ertrk et al. studied fermentative production of riboflavin by
Ashbya gossypii in a medium containing whey.
The quantities of riboflavin produced by Ashbya gossypii in whey
with different supplements.
Supplement Quantity of riboflavin
(mg./L)
Bran 389.5
Glycine + peptone 120
Sucrose 87.5
Glycine 78.3
Yeast extract 68.4
Peptone 23.2
Soyabean oil 17.5
For Eremothecium ashbyii - still slops from alcohol industry with skim milk ,
soya bean meal or casein(protein source),maltose/ sucrose/glucose (carbohydrate
source).
low cost organic wastes as flavinogenic factors and the various concentrations at
which they induced flavinogenecity resulting in higher yields of riboflavin
Organic wastes like beef extract, hog casings, blood meal or fish meal supported
the production of riboflavin from Eremothecium ashbyii NRRL 1363.
Recent studies with wild type of E. ashbyii have yielded 3.3 g/L of riboflavin
using molasses and peanut seed cake as carbon and nitrogen source, respectively
CONDITIONS
pH : 6 to 7.5
Temperature : 26 to 28 °C
Fermentation : submerged aerated fermentation
Fermentation time : 96 to 120 hrs
Aeration & agitation required.
Yield : 3 to 6 g or more / litre
DOWNSTREAM
PROSSESSING OF
RIBOFLAVIN Riboflavin is recovered from the broth by
centrifu- gation after inactivation of the microorganisms by heat.
Pasteurization of the broth ensures that no viable cells of the production organism are present in the final product.
After heating, the cell mass is separated from fermentation broth by centrifugation.
Differential centrifugation leads to separation of cells and riboflavin crystals because of differences in size and sedimentation behaviour.
Riboflavin is then recovered from cell-free broth by using evaporation and vacuum drying.
VITAMIN C
L-ascorbic acid finds its use mainly in foodindustry, being a vitamin as well as anantioxidant.
Majority of commercially manufactured L-ascorbic acid is synthesized via Reichsteinprocess using D-glucose as a startingmaterial
Approximately 50 % of synthetic ascorbic acidis used in vitamins supplements andpharmaceutical preparations.
Because of its antioxidant properties and itspotential to stimulate collagen production, it isalso widely used as an additive to cosmetics.
REICHESTEIN PROCESS
.
Reichestein Process
Diacetone-L-
Sorbose
L-Sorbose
D-Sorbitol
D-Glu
2-Keto-L-
Gluconic
acid methyl
ester
L-
Ascorbic
acid
Sorbitol
pathway
2-keto-D-
gluconic
acid
pathway
Yeast Based Fermentative
Processes Saccharomyces cerevisiae and
Zygosaccharomyces sp. produce L-ascorbic
acid intracellularly when incubated with L-
galactose.
Over-expression of the D-arabinose
dehydrogenase and D-arabinono-1,4-lactone
oxidase in Saccharomyces cerevisiae
enhances this ability significantly.
FERMENTATION BY ALGAE
Skatrud and Huss described a method thatinvolved initial growth of Chlorella pyrenoidosaATCC53170 in a fermentor with a carbon sourcethat is sufficient for the cells to grow to anintermediate density. At the depleted stage,additional carbon source was added sequentiallyor continuously to maintain the carbon sourceconcentration below a predetermined level untilthe addition is terminated. This resulted in theproduction of 1.45 g/L of L-ascorbic acid.
Euglena gracilis Z. is one of the fewmicroorganisms which simultaneously produceantioxidant vitamins such as carotene (71mg/L), vitamin C (86.5 mg/L) and vitamin E (30.1mg/L).
BIOTIN (VITAMIN H) Biotin (vitamin H) is one of the most fascinating
cofactors involved in central pathways in pro- and
eukaryotic cell metabolism.
While humans and animals require several hundred
micrograms of biotin per day, most microbes, plants
and fungi appear to be able to synthesize the cofactor
themselves.
Biotin is added to many food, feed and cosmetic
products.
Majority of the biotin sold is synthesized chemically.
The chemical synthesis is linked with a high
environmental burden, much effort has been put into
the development of biotin-overproducing microbes
Biosynthesis of Biotin
The conversion of
dethiobiotin to biotin
has not been
resolved.
bioF gene
bioD gene
bioB gene
bioA gene
Ogata et al. screened microorganisms and
demonstrated that the bacterium B.
sphaericus can excrete significant quantities
of biotin synthetic pathway intermediates from
precursor, Pimelic acid.
Microbial fermentation of amino acids
Glutamic acid
Microbial production of l-glutamic acid has been
extensively studied by a large number of research
investigators.
The most popular Coryneform species include
C.glutamicum, Corynebacterium, Brevibacterium
flavum, Brevibacterium lactofermentum,
Brevibacterium divarticum, Brevibacterium
ammoniagenes, Brevibacterium thio- genetalis,
Brevibacterium saccharoliticum, and
Brevibacterium roseum .
Other glutamic acid-producing organisms
include Escherichia coli, Bacillus
megaterium, Bacillus circulans, Bacillus
cereus, and Sarcina lutea.
Industrially, glutamic acid is usually
manufactured by batch/fed-batch
submerged fermentation processes using
genetically modified strains of
Corynebacterium or Brevibacterium.
MEDIA COMPOSITION
The seed medium composition can be used:
glucose (8%), NH4Cl (0.5%), corn steep liquor
(0.3%), K2HPO4 (0.5%), KH2PO4 (0.5%),
MgSO4·7H2O (0.03%), CaCO3 (1.0%), and
deionized water to make 100%.
The pH of the medium -7.2
The inoculated flasks are grown in an orbital
shaker incubator maintained at 30°C and 230
rpm for 15 h.
The entire contents of the one flask is then
transferred to a 2.0 L capacity fermenter with
500 mL of sterile nutrient medium containing
molasses (20%), KH2PO4 (0.5%), KH2PO4
(0.5%), MgSO4·7H2O (0.3%), urea (0.8%),
CaCO3 (1.0%), and deionized water to make
100%.
In most cases, the optimum pH of the medium
was recorded as 7.0.
The fermentation is usually initiated with
continuous agitation and aeration for 48 h at
30°C
INDUSTRIAL APPLICATIONS
AND THERAPEUTIC ROLE The greatest application of glutamic acid and
its salt is in the food industry as a flavorenhancer.
To aid in peptic ulcer healing
One of the leading roles of glutamic acid in pharmaceuticals is that of a neurotransmitter.
The blockage of NMDA receptors can greatly affect the memory and overall mental performance of an individual.
Glutamic acid and aspartic acid have the capability to combine with NMDA receptors thus increasing cation conductance, depolarizing the cell membrane, and deblocking the NMDA receptors.
LYSINE
l-Lysine is one of the leading and most
exploited amino acid among the
essential amino acids list.
l-lysine can be synthesized from α-
aminoadipic acid by yeast and
Neurospora mold, or from
diaminopimelic acid (DAP) by E. coli
FERMENTATIVE PRODUCTION
OF LYSINE Organism: mutant strain of C.glutamicum
In commercial-scale starches, molasses and glucose are mostly used as the carbon source.
Care must be taken to create a balance between carbon and nitrogen sources such as corn steep liquor, soybean cake acid hydrolysate, yeast extract, peptone, and the like
Inorganic salts such as KH2PO4, K2HPO4, MgSO4·7H2O,FeSO4·7H2O, ZnSO4·7H2O, MnSO4·7H2O, (NH4)2 SO4.
Other nutrients are biotin and vitamin B1.
In most cases, the optimum pH of the
medium has been 7.2 and
temperature at 30°C.
The seed stage cultivation requires
around 24 h, whereas the
fermentation stage is complete by
approximately 96 h.
After this, harvesting is done and the
product l-lysine is recovered using
some suitable and economical method
INDUSTRIAL APPLICATIONS
AND THERAPEUTIC ROLE It is an important additive to animal feed for
optimizing the growth of pigs and chickens.
In the food industry, l-lysine is used in a number of dietary or nutritional supplements that are popularly used by athletes, weight lifters, bodybuilders, and even some individuals to boost their energy level and protect their muscles from deterioration.
l-lysine is also recommended for the treatment of some viral infections, for example, herpes simplex, cold sores, shingles, and human papillomavirusinfections such as genital warts and genital herpes
TRYPTOPHAN
No scientific reports were available relating to the microbial direct production of tryptophan. During this period, more attention was given by researchers looking into the possibility of tryptophan production
With the introduction of efficient strains of Corynebacterium and E. coli, now tryptophan is largely produced by fermentation
FERMENTATIVE PRODUCTION
Genetically modified strain of C. glutamicumthat is capable of producing tryptophan.
Fermentation medium may be prepared from molasses (30%), corn-steep liquor (0.7%), KH2PO4 (0.05%), K2HPO4 (0.15%), MgSO4·7H2O (0.025%), (NH4)2SO4 (1.5%), and calcium carbonate (1%).
In addition vitamin B1, biotin, l-phenylalanine, and l-tyrosine.
pH adjusted to 6.8
1.0 mL of 20% silicon RD in deionized water is added as antifoam.
The fermenters can be harvested after 72 h.
Product recovery is usually done using ultracentrifugation at around 10,000 rpm, followed by treatment with cationexchange resin and decolorizationwith activated carbon.
After further centrifugation, the mixture can be subjected to drying under a vacuum dryer.
INDUSTRIAL APPLICATIONS
AND THERAPEUTIC ROLE Tryptophan has a wide range of
applications in the feed and
pharmaceutical industries.
As an essential amino acid with a
unique indole side chain, which
indicates its use as a precursor for a
number of neurotransmitters in the
brain.
Its application in the chemical
synthesis of some antidepressant
drugs
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