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Production of Vitamins 959 Production of Vitamins: January 2010
Production of Vitamins 959 Production of Vitamins: January 2010
Production of Vitamins 959 Production of Vitamins: January 2010
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31
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. Fat-
soluble 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, whole-
grain 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.
3.1. 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
N
NH
N N O
OH
OH
HO
OH
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 O
HO OH
HO P
O O
HO N
N
CONH2
CH3 H3C
CONH2
H2NOC CONH2
H 3C A B
X N
H 3C N
Co
H
H2NOC N N
D CH3
C
CH3
CH3 CH
3
CONH2
HN O N CH3
H 3C
N CH3
HO
O
O
P O
O
OH
O
COOH
Hooc HOOC
oooH OOOH
H2N
0 Hooc oooH HOOC OH2 OOOH
0
COON NH HN H 2O NH N
O NH HN NH HN
H2N Hooc oooH HOOC OOOH
5-Aminolevulinic aciu
Glutamyl-tRNA Hooc oooH HOOC OOOH
Uroporphyninogen III Precomin-2
2*
Co O2
A
Acatatcohydo N A
A Acetic acid
E
E R
R O
O B
B I
I C
CONH2 C
CONH2
CH2 2*
H2NOC CH2 CONH2 Co
H 2C CONH2 CONH2 CONH2
Ado OONH2
H 2C N N CH2 CO2
Co CH2 CH2
H H2NOC OONH2 H2NOC OONH2
H2NOC N N CH2 H 2C Ado H 2C Ado
H 2C N N H 2C
O CH2 Co O
CH2 H H
HN H2NOC N N CH2 H2NOC 2 N N CH2
H CH2
CONH2
CH2 O CH2 CH2
O O CH2 CH2 CH2 CH2CH2
O OH HN
OH H CH2 H CONH2 COOH CONH2
CH2
HO O H OH
Adenosylcobynlc acid
Production of Vitamins
Adenosylcobsismin Adenosylcobinamide
965
Fig. 4. Schematic representation of the aerobic and anaerobic cobalamin biosynthetic pathways
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 precorrin-
3A while, in the anaerobic pathway, precorrin-2 is chelated with cobalt to give cobalt-precorrin-
2. 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 two-
step 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 AZ-
78B 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
O
O
H
HO OH
Fig. 5. Chemical structure of vitamin C.
Production of Vitamins 967
H CH2OH CH2OH
O H OH O H OH O
H H L-Gal-DH O L-GL-DH
CH2OH O
Plants
H OH
OH H
OH OH cytCON cytCrtd H
NAD NADH
H H O H OH OH
L-Galactose L-Galactono-1,4-lactone L-Ascorbic acid
D-Glucose
H
O CH2OH O CH2OH O
H H O
H D-Ara-DH OH D-AL-Ox O
H
H OH H H
Yeast
OH OH
NADP NADPH O2 H2O2
O H O H OH OH
D-Arabinose D-Arabinono-1,4-lactone D-Erythroascorbic acid
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
Lactonization/isolation
Ascorbic acid
1969. In the two-stage process, the chemical reactions of the Reichstein process to produce di-
acetone-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-
Production of Vitamins 969
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).
4.1.1. 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 24°C
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 40°C and aerobic conditions (0.5 vvm
aeration at 100 rpm) with a pH value of 6.5.
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 µg/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 polypepton-
S, 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 γ-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 γ-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 γ-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
Production of Vitamins 971
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.
Fermentation Broth
Crystallization
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).
(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 25°C). 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 115°C). After cooling inoculate the test tubes (apart from sterile controls) with 1 drop
of inoculation culture. Incubate for 24 hours at 37°C. 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 tube-
assay technique is more sensitive than the plate method, which is best suited to the assay of higher-
potency 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.
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).
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).
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Aiguo J & Peiji G, 1998, Synthesis of 2-keto-l-gulonic acid from gluconic acid by co-immobilized
Gluconobacter oxydans and Corynebacterium sp., Biotechnology Letters, 20, 939-942.
Alberto J, María AS & José LR, 2008, Phosphoribosyl pyrophosphate synthetase activity affects
growth and riboflavin production in Ashbya gossypii, BMC Biotechnology, 8, 67.
Alberto EB, Belén E, Hisashi N & Naomichi N, 2000, Production of vitamin B12 in an upflow
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