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Production of Vitamins

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Production of Vitamins
Parameswaran Binod, Raveendran Sindhu and Ashok Pandey

1. INTRODUCTION
Vitamins are a group of organic food substances or nutrients found only in living things, plants
and animals. Vitamins were discovered by Dutch physician, Christian Eijkmann, who won the
Nobel Prize in physiology and medicine in 1929. Vitamins are necessary in small amounts for
normal metabolism and good health. Vitamins has no calories and are not an energy source, but
assist in metabolizing nutrients in food and are invaluable in keeping body running smoothly.
Vitamins make it possible for other nutrients to be digested, absorbed and metabolized by the
body. They are required to do many things and their excess or depletion can lead to acute and
chronic diseases.
Vitamins are divided into two classes based on their solubility. The fat-soluble vitamins include
vitamin D, vitamin E, vitamin A and vitamin K. The water-soluble vitamins are vitamin B12
(cyanocobalamin), vitamin B9 (folate or folic acid), vitamin B7 (biotin), vitamin B6 (pyridoxine),
Vitamin B5 (pantothenic acid), vitamin B3 (niacin), vitamin B2 (riboflavin), vitamin B1 (thiamin),
and vitamin C (ascorbic acid). Fat-soluble vitamins contain only carbon, hydrogen and oxygen,
while water-soluble vitamins contain these three elements plus nitrogen and sometimes sulfur. Fatsoluble vitamins can be stored in appreciable amounts in the body and the water-soluble vitamins
cannot be stored in the body.
Vitamins have diverse biochemical functions, including function as hormones (e.g. vitamin D),
antioxidants (e.g. vitamin E), and mediators of cell signaling and regulators of cell and tissue
growth and differentiation (e.g. vitamin A). The largest number of vitamins (e.g. B complex
vitamins) functions as precursors for enzyme cofactor bio-molecules (coenzymes) that act as
catalysts and substrates in metabolism. When acting as part of a catalyst, vitamins are bound to
enzymes and are called prosthetic groups. Vitamins also act as coenzymes to carry chemical groups

960 Food Fermentation Biotechnology

between enzymes. For example, folic acid carries various forms of carbon group - methyl, formyl
and methylene - in the cell.
2. SOURCES OF VITAMINS
Most of the vitamins can be found in plant and animal sources. They can also be chemically
synthesized. Vitamin A occurs in nature in two forms: preformed vitamin A and provitamin A,
or carotene. The vegetable sources of beta-carotene are fat and cholesterol free. Thiamine (vitamin
B1) is found in fortified breads, cereals, pasta, whole grains, lean meats (especially pork), fish,
dried beans, peas and soybeans. Sources of riboflavin include organ meats (liver, kidney and heart)
and certain plants such as almonds, mushrooms, whole grain, soybeans and green leafy vegetables.
Niacin is found in dairy products, poultry, fish, lean meats, nuts and eggs. Common sources of
pantothenic acid are cheese, corn, eggs, liver, meats, peanuts, peas, soybeans, brewers yeast and
wheat germ. Foods rich in vitamin B6 include white meat (poultry and fish), bananas, liver, wholegrain breads and cereals, soyabeans and vegetables. Beans, leafy green vegetables, citrus fruits,
beets, wheat germ, and meat are good sources of folic acid. Vitamin B12 is found naturally in
food sources in protein-bound forms. Good dietary sources of biotin include organ meats, oatmeal,
egg yolk, soy, mushrooms, bananas, peanuts, and brewers yeast. Cabbage and many dark green
leafy vegetables are all good sources of vitamin C. Exposure to sunlight is an important source
of vitamin D. Good food sources of vitamin D include milk, fatty fish. Vitamin E is found in
the germ of a seed or grain. Rich sources of vitamin K include broccoli, Brussels sprouts, cabbage,
cauliflower, kale, spinach and soybeans. Bioflavonoids are abundant in the pulp and rinds of citrus
fruits and other foods containing vitamin C.
3. MICROBIAL PRODUCTION OF VITAMINS
All vitamins can be extracted from natural sources, but fat-soluble vitamins are often produced
commercially by synthetic processes. Several vitamins are now industrially produced and are used
in foods, pharmaceuticals and cosmetics. Presently, few of the vitamins are exclusively produced
via chemical synthesis, while a few others are produced either by chemical synthesis or via
extraction processes. These processes are energy-intensive, and also suffer from high cost of waste
disposal. This has led to increased interest in substituting these processes with biotechnological
processes. Microbial production is commercially feasible for some vitamins, such as vitamin B2,
vitamin B6, vitamin B12 and vitamin C.
Fermentation technologies provide an alternative to chemical processes in the production of wide
range vitamins from microbes. Different methods like media optimization, mutation and screening,
genetic engineering and biocatalyst conversion have been used for improvement of the production
of vitamins. List of various microorganism used for the production of vitamins by fermentation
techniques are shown in Table 1.

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Table 1. Microorganism used for the production of vitamins by fermentation


Vitamins

Source
Eremothecium ashbyii
Ashbya gossypii
NRRL 1363
Pichia guilliermondii
Bacillus subtilis

Riboflavin (vitamin B2)

Reference
Kalingan (1998)
Park et al (2007);
Alberto et al (2008);
Sugimoto et al (2009)
Fayura et al (2007)
Mironov et al (2008)

Eremothecium ashbyii
Lactococcus lactis
Bacillus subtilis RH44
Pichia pastoris
Bacillus megaterium
ATCC 13639
Enterobacter aerogenes
Candida tropicalis
Corynebacterium ammoniagenes
Candida famata
Candida guilliermondii

Pujari & Chandra (2000)


Jeroen et al (2002)
Qiu-Li et al (2007)
Hans et al (2008)
Chung & Fields (2006)

Vitamin B6

Rhizobium leguminosarum
Propionibacterium shermanii
E.coli

Massaki et al (1999)
Quesada-Chanto et al (1998)
Sakai et al (2004)

Vitamin B12

Pseudomonas denitrificans
Propionibacterium acidipropionici
Acetobacterium sp.
Citrobacter freundii
Lentinula edodes
Bacillus megatherium
Propionibacterium freudenreichii
XanthomonasCampestris

Li et al (2008)
Quesada-Chanto et al (1994)
Alberto et al (2000)
Sylvia and Bernward (1994)
Turlo et al (2008)
Halbrook et al (1950)
Ye et al (1996)
Manjula & Sureshkumar (2000)

Ascorbic acid (Vitamin C)

Ochromonas danica
Ketogulonicigenium vulgare
DSM 4025
Saccharomyces cerevisiae
Clavispora lusitaniae
Cryptococcus terreus
Pichia fermentans
Euglena gracilis

Johaness et al (1982)
Sugisawa et al (2005)

Chung & Fields (2006)


Bussini & Rossi (1998)
Koizumi et al (2000)
Stahmann et al (2000)
Sabry et al (1988)

Robert et
Onofri et
Onofri et
Onofri et
Takeyama

al
al
al
al
et

(2000)
(1997)
(1997)
(1997)
al (1997)

962 Food Fermentation Biotechnology

3.1. Vitamin B2
Vitamin B2 or riboflavin functions as part of metabolic systems concerned with the oxidation of
carbohydrates and amino acids. It is active not in the free form but in more complex compounds
known as coenzymes, such as flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD),
or flavoprotein. It plays a key role in energy metabolism, and is required for the metabolism of
fats, ketone bodies, carbohydrates and proteins. Vitamin B2 was isolated in pure form in 1933
and was first synthesized in 1935. Chemical structure of vitamin B2 is shown in Fig. 1.
Various biotechnological processes have been developed for industrial scale riboflavin biosynthesis
using different microorganisms, including filamentous fungi such as Ashbya gossypii, Candida
famata and Candida flaveri as well as the bacteria Corynebacterium ammoniagenes and Bacillus
subtilis. The latter organism has been genetically modified to increase the bacterias production
of riboflavin and to introduce an antibiotic (ampicillin) resistance marker, and is now successfully
employed at a commercial scale to produce riboflavin for feed and food fortification purposes.
The chemical company BASF has installed a plant in South Korea, which is specialized on riboflavin
production using Ashbya gossypii.
Majority of the microorganisms can synthesize riboflavin from simple medium components. Some
bacteria, yeast and yeast-like fungi are able to overproduce riboflavin under specified cultivation
conditions in quantities which exceed their physiological requirements. This characteristic makes
it possible to obtain the vitamin on the industrial scale. Ming et al (2003) reported riboflavin
production from Ashbya gossypii using waste activated bleaching earth discharged from an oil
refinery containing 40% rapeseed oil. When 125 g/L waste activated bleaching earth that contained
50 g/L rapeseed oil was added into the culture, the riboflavin concentration was 1.12 g/L, which
was almost 1.6-fold as high as that of pure rapeseed oil. Waste activated bleaching earth contained
the rapeseed oil discharged from oil refinery factories was a good carbon source in riboflavin
production by A. gossypii. A similar study by Enoch et al (2004) also shows an increased riboflavin
O
N

NH
O

N
OH

HO

OH

OH
Fig. 1. Chemical structure of vitamin B2

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963

production from A. gossypii using waste activated bleaching earth discharged from an oil refinery
containing palm oil. The riboflavin production was almost 1.5 times higher than for cultures grown
on pure palm oil. Kalingan & Krishnan (1997) reported riboflavin production using molasses and
peanut seed cake as carbon and nitrogen sources. They used certain seeds as flavinogenic stimulators
like Achras sapota, Cucumis melo, Carica papaya and Anona squamosa. They reported that rice
bran oil act as a stimulant for the riboflavin production in a medium containing molasses and
peanut seed cake. Rice bran oil contains high percentage of oleic acid (46.1%) linoleic acid (31.5%)
and palmitic acid (18.9%) which might have induced growth and flavinogenesis. Individual effects
of these fatty acids were not known. Ersin et al (1998) carried out fermentation studies on the
production of riboflavin with whey and whey supplemented with various supplements in Erlenmayer
flasks and a laboratory fermenter using Ashbya gossypii. Riboflavin production started to increase
most rapidly after 2 days of fermentation and continued to increase until the 5th day. The quantities
of riboflavin produced by A. gossypii in whey after 8 days of flask fermentation with bran as
supplement was 389.5 mg/L. Riboflavin produced in whey by A. gossypii during 8 days of
fermentation in a fermenter were found to be four times higher than in flask studies.
3.2. Vitamin B6
Vitamin B6 is a water-soluble vitamin and includes a group of closely related compounds pyridoxine
(PN), pyridoxal (PL), and pyridoxamine (PM). Pyridoxal phosphate (PLP) (Fig. 2) is the active
form and function as cofactor in many reactions of amino acid metabolism, including transamination,
deamination and decarboxylation. Pyridoxal phosphate is involved in macronutrient metabolism,
neurotransmitter synthesis, histamine synthesis, hemoglobin synthesis and gene expression. Major
sources of vitamin B6 include cereal grains, legumes, vegetables, potatoes, milk, cheese, eggs, fish,
liver, meat and flour.
In the past several decades, screening for vitamin B6 producing microbes has been done extensively.
Several strains such as Klebsiella, Achromobacter cycloclastes, Flavobacterium, Bacillus, Pichia
guilliermondii and Rhizobium were reported to produce vitamin B6. The production media for flask
fermentation of vitamin B6 consists of 1% glucose, 0.5% polypeptone,0.2% yeast extract,0.1%
KH2PO4, 0.05% MgSO4.7H2O, 0.001% MnSO4.7H2O and 0.001% FeSO4.7H2O (pH 6.8)
(Masaaki et al 1999). The best producer for vitamin B6 was R.meliloti IFO14872 which produces
51 mg/L.
HO

OH

HO

HO

OH

HO
O

Fig. 2. Chemical structure of Vitamin B6

964 Food Fermentation Biotechnology

3.3. Vitamin B12


Vitamin B12 (Fig. 3) is a water soluble vitamin and plays an important role in the normal functioning
of the brain and nervous system, and for the formation of blood. It plays an important role in
cell metabolism like DNA synthesis and fatty acid synthesis.
The first step in the biosynthetic pathway of vitamin B12 involves tRNA bound glutamate is reduced
to glutamate-1-semialdehyde by glutamyl-tRNA reductase. It is converted to 5-aminolevulinic acid.
Two molecules of 5-aminolevulinic acid are condensed to porphobilinogen. Four pyrrole molecules
are polymerized to form uroporphyrinogen III. Decarboxylation of uroporphyrinogen III leads to
CONH2
CH3 H3C

CONH2

H2NOC

CONH2
H 3C

A
N

H 3C

Co
H
N

H2NOC

CH3
CH3

CH3 CH
3
CONH2

HN
H 3C

CH3

CH3

HO
O
O

P
O
O

OH

Fig. 3. Chemical structure of vitamin B12

Glutamyl-tRNA

H2N

COOH

CH2
CH2

CONH2

CH2
CH2
H
CONH2
CH2
CH2
O
O
OH
O
OH
H
CH2

Ado

N
N
Co
N N

CH2

CONH2

oooH

oooH

OONH2

CH2
CH2

CH2

CONH2

Adenosylcobinamide

Co

2*

NH HN

NH N

OOOH

CONH2

A
N
A
E
R
O
B
I
C

A
E
R
O
B
I
C
Co

O2

2*

Acetic acid

OOOH

OOOH
OH2 OOOH

Precomin-2

HOOC

HOOC

H 2O

HOOC

HOOC

Siroheme

Adenosylcobynlc acid

OONH2
CO2
CH2
H2NOC
OONH2
H 2C
Ado
H 2C
O
H
H2NOC 2 N N
CH2
CH2
CH2CH2
COOH CONH2

Acatatcohydo

CH2 CH2
H
CONH2
CH2
OH

Ado

N N
Co
N N

CH2

CONH2
H2NOC
H 2C
H 2C
H
H2NOC
O
HN

NH HN

Uroporphyninogen III

Hooc

Hooc

NH HN

oooH
oooH

Heme

Fig. 4. Schematic representation of the aerobic and anaerobic cobalamin biosynthetic pathways

O H
HO
Adenosylcobsismin

HN

H2NOC
H 2C
H 2C
H
H2NOC

CH2

CONH2

O
H2N
5-Aminolevulinic aciu

COON

Hooc

Hooc

Chlorophyll
Bacteriochlorophyll

Production of Vitamins
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966 Food Fermentation Biotechnology

the biosynthesis of hemes and chlorophylls, methylation of uroporphyrinogen III results in the
synthesis of precorrin-2. At precorrin-2, the two pathways for cobalamin biosynthesis diverge, in
the aerobic pathway, precorrin-2 is methylated by a further methyltransferase to give precorrin3A while, in the anaerobic pathway, precorrin-2 is chelated with cobalt to give cobalt-precorrin2. Thus, the oxygen-dependent and independent pathways for B12 synthesis are quite distinct: the
oxygen-independent part of the pathway starts with the insertion of cobalt into precorrin-2, while
this chelation reaction in the oxygen-dependent part occurs only after nine further reaction steps.
The oxygen-dependent pathway requires ATP, in contrast to its anaerobic counterpart which requires
no high-energy equivalents. The biosynthetic pathway of vitamin B12 is represented in Fig. 4.
The cost of production of vitamin B12 by chemical synthesis are complicated and expensive, it
is now exclusively produced by fermentation processes. Propionibacterium shermanii and
Pseudomonas denitrificans are the commonly used bacteria for the commercial production of
vitamin B12. These organisms have been successfully applied to the commercial production of
vitamin B12 because of its rapid growth and high productivity. This organism produces about 50,000
to 100,000 times more vitamin B12 than needed for their own growth. With P. shermanii twostep fermentation is carried out, initially under anaerobic conditions in the absence of precursor,
5,6-dimethylbenzimidazole and subsequently the culture is aerated during which the cobinamide
is converted to vitamin B12. This two-step fermentation not only reduces the feedback inhibition
but it reduces the time of fermentation as well. Fermentation with Pseudomonas denitrificans is
carried out under low oxygen conditions because higher levels of oxygen oxidize the intracellular
environment, which represses formation of early enzymes in the pathway.
Atta et al (2008) carried out solid state fermentation using mixed cultures of Bacillus firmus AZ78B and Streptomyces halstedii, AZ- 8A on agricultural wastes supplemented with mineral salts
for vitamin B12 production. A continuous fermentation of extracellular vitamin B compounds was
attempted by Mazumder et al (1987) using a diatomaceous clay fixedbed reactor. They used
Methanosarcina bakeri for the production of vitamin B12 using methanol as the substrate.
3.4. Vitamin C
Vitamin C or L-ascorbic acid (Fig. 31.5) is an important metabolite for most living organisms.
HO

HO

H
HO

OH

Fig. 5. Chemical structure of vitamin C.

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CH2OH
CH2OH
OH O
O
H
OH O
H
H
H
O
L-GL-DH
L-Gal-DH
CH2OH
O
OH
H
OH
H
OH
OH
cytCON cytCrtd H
NAD NADH
H
H
OH
OH
H
O
L-Ascorbic acid
L-Galactono-1,4-lactone
L-Galactose

Plants

D-Glucose
H

Yeast

H
OH

O
H
H
O

OH

H
OH

D-Arabinose

CH2OH O
D-Ara-DH
H

O
OH

CH2OH O
D-AL-Ox
O2

NADP NADPH
O

H2O2

D-Arabinono-1,4-lactone

OH

OH

D-Erythroascorbic acid

Fig. 6. Biosynthetic pathway of Vitamin C in plants and yeast.

In humans, it is necessary for different physiological functions and thus is an essential nutrient.
It is widely used as food additive.
Novel biotechnological processes that convert glucose to vitamin C in one step would be desirable.
In this respect, yeasts such as Saccharomyces cerevisiae, offer themselves as biocatalysts due to
their generally recognized as safe (GRAS) status. However, yeast cells naturally lack the ability
to produce L-ascorbic acid. Instead, erythroascorbic acid, a structurally related compound with
chemical properties very similar to those of L-ascorbic acid, is the molecule occurring in yeast
cells while in plants glucose is epimerized to D-mannose and then to L-galactose, and finally
L-galactono-1, 4-lactone is converted into the ascorbic acid (Fig. 6).
Vitamin C is manufactured commercially by Reichstein-Grussner synthesis in which D-glucose
is hydrogenated catalytically to D-sorbitol at elevated temperature and pressure using a nickel
catalyst and then fermented with Acetobacter suboxydans. The fermentation oxidizes D-sorbitol
to L-sorbose, which subsequently isolated by crystallization, filtration and drying. L-sorbose is
then converted to bis-O-isopropylidene-a-L-sorbofuramose on reaction with acetone and excess
sulfuric acid at low temperatures in the presence of ferric chloride or perchloric acid as catalysts.
This substance is then oxidized at elevated temperature in dilute sodium hydroxide in presence
of nickel chloride or palladium-carbon as catalyst. After completeion of the reaction, the mixture
is acidified to bis-O-isopropylidene-2-oxo-L-gluconic acid, which on treatment with hydrochloric
acid gas in water-free chloroform-ethanol mixture gives ascorbic acid. The schematic representation
of this process is shown Fig. 7.
The first process competing with the Reichstein process was developed by Chinese scientists in

968 Food Fermentation Biotechnology


D-Sorbitol

Fermentation
L-Sorbose
Acetonization

Diacctone-L-sorbose

Oxidation/hydrolysis

2-kcto-L-gluconic acid

Esterification

2-keto-L-gluconic acid methyl ester

Lactonization/isolation

Ascorbic acid
Fig. 7. Schematic representation of vitamin C production

1969. In the two-stage process, the chemical reactions of the Reichstein process to produce diacetone-ketogulonic acid are replaced by a second fermentation step, which results in the formation
of 2-keto-L-gluconic acid (Yin et al 2001). The 2-keto-L-gluconic acid is then chemically processed
to produce L-ascorbic acid.
Sugisawa et al (2005) reported L-ascorbic acid production from Ketogulonicigenium vulgare DSM
4025 using L-sorbosone as substrate. The strain produced 1.37g/l of L-ascorbic acid from 5g/
l of L -sorbosone. In another approach, Rao & Sureshkumar (2000) were able to show direct
synthesis of L -ascorbic acid from D-glucose by Xanthomonas campestris by a free-radical inducing
treatment. X. campestris probably lactonizes 2-keto-L-gluconic acid under oxidative stress to form
L-ascorbic acid. Using this method 20.4 g/l L-ascorbic acid were found in the extracellular broth
which corresponds to the 14-fold amount of that detected in yeast cells directly synthesizing L-

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ascorbic acid from D-glucose (Rao & Sureshkumar, 2000). Sonoyama et al (1982) described the
second two stage fermentation process for 2-keto-L-gluconic acid production. In the first step,
D-glucose is oxidized to 2,5-diketo-D- gluconate by Erwinia or Acetobacter strains via the
intermediates D-gluconate and 2-keto-D-gluconate. In the second step, a Corynebacterium strain
converts 2, 5-diketo-D-gluconate to 2-keto-L-gluconic acid. This reaction is catalyzed by an
NADPH-dependent 2, 5-diketo-D-gluconate reductase. In a similar approach, combinations of
different microorganisms such as Pseudomonas striata, G. oxydans, and Corynebacterium sp were
used for the direct production of 2-keto-L-gluconic acid from D-gluconate (Aiguo & Peiji, 1998).
4. PARAMETERS CONTROLLING VITAMIN PRODUCTION BY FERMENTATION
Several physical and nutritional parameters influence vitamin production.
4.1. Physical parameters
4.1.1. pH
Kolonne et al (1994) reported pH of the medium influence riboflavin production by Eremothecium
ashbyii grown in a chemically defined medium in batch culture. Highest yields were obtained
at constant pH of 4.5 and 5.5, while little or no riboflavin was detected at either pH 3.5 or 8.5.
Kalingan & Krishnan (1997) observed that in Eremothecium ashbyii NRRL 1363 when the pH
was raised from 6.0 to 6.5, the biomass as well as the riboflavin production was enhanced. An
increase in pH from 6.5 to 7.0 did not repress riboflavin production but inhibited biomass production
drastically, indicating pH 6.5 to be more conducive for all the enzyme activities involved in the
growth of the fungus. The pH 7.0 did not promote growth or repress or derepress riboflavin
production, indicating that the enzymes involved in flavinogenesis were stable and active in a
broader pH range (6.5 7.0).
4.1.2. Temperature
Sylvia et al (1994) studied the influence of temperature on vitamin B12 formation by strains of
Citrobacter freundii and Klebsiella pneumoniae isolated from Indonesian tempeh samples during
tempeh fermentation was investigated. A decrease in fermentation temperature from 32 to 24C
led to a decrease in vitamin B12 formation. Quesada-Chanto et al (1994) optimized the production
of vitamin B12 by using Propionibacterium acidipropionici NRRL B3569 in continuous culture.
Optimal vitamin B12 production required a temperature of 40C and aerobic conditions (0.5 vvm
aeration at 100 rpm) with a pH value of 6.5.
4.1.3. Nutritional parameters
Riboflavin production is significantly determined by the type and initial concentration of the carbon

970 Food Fermentation Biotechnology

and nitrogen sources and also by other flavinogenic stimulants (Kalingan & Krishnan 1997). Ersin
et al (1998) carried fermentation studies on the production of riboflavin with whey and whey
supplemented with various supplements. Riboflavin biosynthesis rate in many microorganism species
can be modulated by metals. Shavlovsky & Logvinenko (1988) reported that iron deficient media
cause the derepression of enzymes of the riboflavin biosynthesis pathway, which in turn leads
to riboflavin overproduction. The chromium induced riboflavin synthesis might involve the
derepression of flavinogenesis enzymes by some interaction of Cr with the intracellular pool of
iron. Chromium causes a temporary growth inhibition characterized by a prolonged lag phase during
which riboflavin is over synthesized (Daria et al 2001). Li et al 2008 studied the effects of Zn2+,
Co2+ and dimethylbenzimidazole (DMBI) on vitamin B12 production by Pseudomonas denitrificans
in shake flasks. The results demonstrated that the addition of the three components to the
fermentation medium could significantly stimulate the biosynthesis of vitamin B12. As a result,
vitamin B12 production was increased from 69.36 to 78.23 g/ml.
Masaaki et al (1999) studied the effect of peptone on the production of vitamin B6. Pancreatic
digest of vegetable casein was found to be more effective when compared to that of polypeptonS, polypepton-P1 and polypepton-Y. Polypeptone-S gave maximum production of riboflavin (60mg/
l) and when supplemented with 0.8% yeast extracts (84mg/l). Marwaha et al (1983) have studied
the role of amino acids, betaine and choline in vitamin B12 biosynthesis by the three strains of
Propionibacterium, viz. P. shermanii 566, P. shermanii and Propionibacterium AKU 1251. They
supplemented whey permeate medium with eleven amino acids (0.05 %, by mass per volume).
Of the eleven amino acids tested, L-glutamic acid promoted both growth and product formation
to a maximum in all three strains.
Ketogulonicigenium vulgare DSM 4025 produced L-ascorbic acid from D-sorbitol, L-sorbose, and
L-sorbosone as the substrate under a growing or resting condition. K. vulgare DSM 4025 produced
1.37 g per liter of L-ascorbic acid from 5.00 g per liter of L-sorbosone during 4 h incubation
time at 30oC under the resting cell condition having 5.70 g per liter of wet cells. Onofri et al
(1997) observed that L-galactonic acid and -lactone stimulate ascorbic acid production in strains
of Saccharomyces cerevisiae, Clavispora lusitaniae, Cryptococcus terreus, Pichia fermentans in
which this is undetected whenever glucose represents the sole carbon source. Cryptococcus terreus
(strains DBVP 6012 and 6242) does not show ascorbic acid production either in presence or in
the absence of L-galactonic acid -lactone. This feature is probably connected to the insensibility
of the strain to the lycorine, an alkaloid which commonly inhibits cell division probably by blocking
L-galactonic acid -lactone convertion into ascorbate.
5. DOWN-STREAM PROCESSING
Down-stream processing refers to the recovery and purification of products from natural sources
such as animal or plant tissue or fermentation broth. The study of separation and purification
processes of the fermentation products is most important for their commercial success. Recovery

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and purification of these bioproducts from their crude sources involve various steps (precipitation,
centrifugation, extraction, membrane filtration and sorption) which lead to low overall yields.
Conventional chromatographic and chemical processes for recovery of the desired product from
fermentation broth are capital and energy intensive due to a number of post- and pre-treatment
steps involved in a processing scheme. A rapid and selective mode of recovery of the target molecule
from the crude feedstock can prove highly advantageous in improving the product yields and thereby
reduce the overall cost of downstream processing.
Adsorptive separations are often used in downstream processing using various interactions, e.g.,
ionic, hydrophobic, affinity, etc. for the recovery of biomolecules. The adsorption-based separation
technique is one of the most promising methods for separation processes since it is a non-denaturing,
highly selective, energy efficient and relatively inexpensive process. On the other hand, in many
fermentation processes, the bioconversions are regulated by product inhibition and product
degradation. The in situ removal of product during bioconversions is a desirable concept to obtain
high productivity and it also reduces the product costs and can be achieved by means of sorption
processes. A large-scale cyclic operation involves three steps: adsorption, desorption and washing.
Design and scale-up of such large-scale separation process require basic information to simulate
the dynamic behavior both in adsorption and desorption steps. Measurement of the mass transport
in solid-liquid systems helps the design of more efficient separations; batch adsorption has been
a method of estimating the rates of mass transfer in the adsorption process. Diffusion coefficients
for the adsorbent particle may be estimated and used in the design of fixed bed adsorbers. The
screening of adsorbents is the first step for the development of a purification adsorptive process.
Non-ionic polymeric resins, ion-exchange resins, activated carbon, molecular sieves, etc. have been
suggested as adsorbents for the isolation of hydrophilic fermentation products in general. Polymeric
adsorbents have been used for purification and bulk separations in the food, pharmaceutical and
chemical industries; they are attractive due to their elution and regeneration characteristics although
they may have lower adsorption capacities than ion exchangers or activated carbons. The optimum
resin having to be experimentally selected depending on the type of the fermentation medium
and the nature of the impurities present.
5.1. Extraction of vitamin B2
Riboflavin is recovered from the broth by centrifugation 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.

972 Food Fermentation Biotechnology


Fermentation Broth

Harvesting cells by centrifugation

Extraction (in alcohol) or heat

Purification by adsorption, clarification of filtration

Conversion to cyano form with 0.1% KCN


Feed quality Vitamin B12
Chromatography (eg: Al2O3)

Crystallization

Food quality Vitamin B12


Fig. 8. Downstream processing of vitamin B12

5.2. Extraction of vitamin B12


For the extraction of Vitamin B12 after fermentation, the whole broth or an aqueous suspension
of harvested cells is heated at 80120C for 1030 min at pH 6.58.5. The conversion to
cyanocobalamin is obtained by treating the heated broth or cell suspension with cyanide or
thiocyanate. The whole solution is clarified by filtration or treatment with zinc hydroxide and then
precipitated out by the addition of tannic acid or cresol to give the product of 80% purity, which
is suitable for use as animal food additive. For greater purity, which is required for pharmaceutical

Production of Vitamins

973

use, the clarified solution is extracted with organic solvents, such as carbon tetrachloride, and
then with water and butanol, followed again by organic solvents. In addition, adsorption processes
such as on ion exchangers, aluminium oxide, or activated carbon can be used. Pure vitamin B12
can be obtained by crystallization after the addition of organic solvents, such as phenol and water.
The steps in the downstream processing for the recovery of vitamin B12 are summarized in
Fig. 8.
A method of purifying vitamin B12, methylcobalamine, hydroxocobalamine, or coenzyme-type
vitamin B12 which comprises the steps of heating an aqueous solution containing vitamin B12,
methylcobalamine, hydroxocobalamine, or coenzyme-type vitamin B12, in an amount of 50-300g/
L at a temperature of 50 80oC; adding thereto a water soluble ionic compound selected from
the group consisting of sodium phthalate, sodium acetate, potassium acetate, sodium chloride, and
calcium butyrate in an amount of 20 to 70 mass % based on the mass of said solution provided
that in the case where the solution is strongly alkalized after the addition of said ionic compound,
it is maintained around neutral by adding an acid selected from the group consisting of hydrochloric
acid, sulfuric acid, acetic acid, oxalic acid, and formic acid; and cooling the solution to a temperature
of 5 25oC such that vitamin B12, methylcobalamine, hydroxocobalamine, or coenzyme-type vitamin
B12 is crystallized (Hirayama et al 1999).
6. QUANTITATIVE ESTIMATION OF VITAMINS
Vitamins can be quantitatively estimated by various methods, of these microbiological assay is
most widely used. HPLC determination is also carried out for the estimation of vitamins.
6.1. Estimation of Vitamin B12
Estimation of Vitamin B12 can be estimated by various processes.
6.1.1. Microbiological assay
Although vitamin B12 can be assayed biologically with mice, chicks or rats, the microbiological
method of assay is preferred since it is more rapid and economical. Certain species of bacteria
and some yeast can only grow in the presence of certain vitamins. If these test organisms are
transferred to defined culture media which contain all the compounds essential for their growth
apart from the vitamin in question, proliferation of the test organisms is totally inhibited or at
least drastically reduced. After adding the vitamin the organism can then grow, its growth being
dependent on the concentration of the vitamin. The amount of vitamin present can be determined
by measuring the turbidity produced as a result of microbial growth or by quantitative assay of
a metabolite (e.g. lactic acid). Parallel assays with a pure vitamin preparation of known activity
serve as standards. The test organisms is inoculated in micro-Inoculum-Broth and incubated for
20 hours at 37C. Then the culture is centrifuged and rinsed three times with physiological saline
and adjusted to a microbial count of 108 bacteria/ml. Suspend 100 mg of dried cyanocobalamin

974 Food Fermentation Biotechnology

(Vitamin B12) in 1 liter of Double distilled water (content: 100 mcg/ml). Before use, this stock
solution is diluted to 100 pg/ml to give the reference solution. For calibration different concentration
series of cyanocobalamin is made by pipetting reference solution into test tubes and filling up
to 5.0 ml with double distilled water. By briefly boiling, dissolve 83 g of dehydrated Vitamin
B12 (Lactobacillus) Assay Broth together with 2 ml Tween 80 in 1 liter double distilled water.
Check the pH and correct if necessary (6.0 at 25C). Add 5 ml of culture medium to all test
tubes with control, sample or reference solution, close with caps and sterilize by autoclaving (10
min at 115C). After cooling inoculate the test tubes (apart from sterile controls) with 1 drop
of inoculation culture. Incubate for 24 hours at 37C. The optical density (OD) of the reference
and sample batches is measured photometrically at 546 nm against the culture control. A calibration
curve is made by applying the turbidity values on the linear ordinate to the corresponding active
substance amounts on the logarithmic abscissa.
Under specified conditions, several of the lactic-acid bacteria require vitamin B12. Of these, strains
of Lactobacillus leichmannii and L. lactis are widely used to assay the vitamin. A mutant of E.
coli requiring vitamin B12 is now increasingly used in a plate method. The method is speedy and
simple in operation and allows large numbers of assays to be carried out on a routine basis. It
is subject to interference by methionine and to a lesser extent by other substances, but in general
such interference is readily detected from the appearance of the zones of exhibition.
Burkholder (1951) has described the use of this organism in a tube assay and reported good
agreement between assays of a number of substances with it and with Euglena gracilis. This tubeassay technique is more sensitive than the plate method, which is best suited to the assay of higherpotency materials and extracts.
One serious shortcoming of many microbiological B12 assay procedures is the lack of specificity
when crude materials are assayed. Crude materials can contain inhibitory or stimulatory factors
which cause serious errors in assay results. One of the interfering substances in the microbiological
assay of vitamin B12 with Lactobacillus leichmannii is pseudovitamin B12.
6.1.2. HPLC determination
Vitamin B12 concentration in fermentation broth can be determined by high-performance liquid
chromatography (HPLC). To the fermented sample 8% (w/v) NaNO2 is added. The pH is adjusted
to 3.0 to 4.0 with glacial acetic acid and it is then boiled for 30 minutes. After boiling, the mixture
is filtrated, and 20 ml of NaCN 10% (w/v) is added to 1 ml of aqueous phase. The resulting
upper aqueous phase is injected into the HPLC system with NH2 column under a wavelength
of 360 nm. The mobile phase was 250 mM of phosphoric acid/acetonitrile (30/70, v/v) (Li et
al 2008).
6.1.3 Spectroscopic estimation
For determination of vitamin B12 concentration, microorganism is disrupted in a 0.1N phosphate

Production of Vitamins

975

buffer solution containing 0.01% KCN at pH 5.5 for 20 min at 100oC. Vitamin B12 formed
intracellularly is determined spectrophotometrically in the dicyano-form at wavelength of 367 nm
(Nakano, 1996).
7. STRAIN IMPROVEMENT FOR VITAMIN PRODUCTION
Recent advances in the cloning of riboflavin, cobalamin and biotin biosynthesis genes in Bacillus
species provide potential for the use of Bacillus strains in vitamin production. It was observed
that mutations in the ribO operator site of the riboflavin operon, which contains six structural
genes, resulted in the development of a number of overproducers (Stepanov et al 1977; Debabov
1982). The strategy to develop a commercial riboflavin production strain of Bacillus subtilis was
to combine classical genetic mutant selection and fermentation improvement with genetic
engineering of the riboflavin biosynthetic genes. Perkins et al (1999) developed a recombinant
strain of Bacillus subtilis that produces commercially attractive levels of riboflavin. The B. subtilis
riboflavin production strain contains multiple copies of a modified B. subtilis riboflavin biosynthetic
operon.
Hans et al (2008) reported overexpression of riboflavin biosynthetic pathway by sequential
deregulation of all the genes, by exchange of their native promoter with the strong and constitutive
glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP). Park et al (2007) mutated Ashbya
gossypii spores by exposure to UV light. The mutant ZP4 strain, produced threefold riboflavin
than that of the wild-type. Proteomic analysis of ZP4 strain showed the expression patterns of
eight types of genes related to riboflavin biosynthesis different from those of the wild-type strain
and those enzyme activities were investigated.
Koizumi et al (2000) carried out strain improvement studies for the production of riboflavin (vitamin
B2) through metabolic engineering using recombinant DNA techniques in Corynebacterium
ammoniagenes. The recombinant strain produced 17-fold as much riboflavin as the host strain.
Saito et al (1997) transformed the L-sorbose accumulating strain G. oxydans G624, with the genes
encoding L-sorbose dehydrogenase and L-sorbosone dehydrogenase of G. oxydans T-100. With this
recombinant strain, high level production of 2-keto-L-gluconic acid (130 g/l) was achieved. The
2-keto-L-gluconic acid production was improved by a promoter exchange and expression
optimization of the various genes to around 230% compared to that of the original strain. Further
improvement of the strain was possible by suppressing the L-idonate pathway.
Shibata et al (2000) generated a recombinant Pseudomonas putida strain in which the genes
encoding D-sorbitol dehydrogenase, L-sorbose dehydrogenase, and L-sorbosone dehydrogenase
from G. oxydans were introduced and expressed. After optimization of culture conditions, the
recombinant strain produced 16 g/l 2-keto-L-gluconic acid from 50 g/l d-sorbitol. Running et al
(1994) described a one-step fermentation process for the production of L-ascorbic acid using the
heterotrophic green microalga Chlorella pyrenoidosa. Whereas low amounts of up to 40 mg/l Lascorbic acid were obtained by culturing wild-type cells, after repeated rounds of chemical

976 Food Fermentation Biotechnology

mutagenesis and fermentation optimization, improved amounts of up to 2 g/l L-ascorbic acid were
achieved. The produced amounts correspond to a 70-fold improvement of L-ascorbic acid formation
as compared to the wild-type. Because the bulk of L-ascorbic acid produced by the microalgae
remained intracellular, the process was patented as a method for the production of L-ascorbic acid
enriched biomass for animal feed or as dietary supplement (Doncheck et al 1996).
8. CONCLUSION AND PERSPECTIVES
Several vitamins are now industrially produced and are used in food, feed, pharmaceuticals and
cosmetics industries. Presently, few of the vitamins are exclusively produced via chemical synthesis,
while a few others are produced either by chemical synthesis or via extraction processes. These
processes are energy-intensive, and also suffer from high cost of waste disposal. This has led
to increased interest in substituting these processes with biotechnological processes. Microbial
production is commercially feasible for some vitamins, such as vitamin B2, vitamin B6, vitamin
B12 and vitamin C but these are produced in low concentrations, even in optimized fermentation
methods. In addition recovery and purification is a complex process and it involves several
operational steps. Therefore a wide scope exists for extensive research in recovering vitamins from
fermentation broth with minimum number of steps.
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