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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