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PRODUCTION OF VITAMINS

Introduction

Vitamins are essential micronutrients required in trace amounts/quantities and

cannot be synthesized by mammals. They are essential for the metabolism of all
living organisms and are synthesized by microorganisms or plants. Vitamins have
no calories and are not energy source but assist in metabolizing nutrients in food.
Their excess or depletion can lead to acute and chronic diseases. Apart from their
in vivo nutritional-physiological roles as growth factors for humans, animals,
plants and microorganisms, vitamins are now increasingly being introduced as
food/feed additives, as medical therapeutic agents, as health aids etc. today, many
processed foods, feeds, pharmaceuticals, cosmetics and chemicals contain
extraneously added vitamins or vitamin-related compounds.

Vitamins are divided into two classes based on their solubility. The fat- soluble
vitamins include:

- Vitamin D
- Vitamin E
- Vitamin A
- Vitamin K

The water soluble vitamins are;

- Vitamin B12 (Cyanocabalamin)

- Vitamin B9( Folate or folic acid)
- Vitamin B7 (Biotin)
- Vitamin B6 (Pyrixodine)
- Vitamin B5 (Panthothenic acid)
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- Vitamin B3 (Niacin)
- Vitamin B2 (Riboflavin)
- Vitamin B1 (Thiamine)
- Vitamin C (Ascorbic acid)

Fat soluble vitamins contain only carbon, hydrogen and oxygen, while water
soluble vitamins contain in addition to these three elements, nitrogen and
sometimes sulphur, while fat soluble vitamins can be stored in appreciable
amounts in the body, water- soluble vitamins cannot be stored in the body.

Sources of Vitamins

Table 1: Sources of Vitamins

Vitamins Sources

Vitamin A Palm oil, vegetable oil, groundnut oil etc

Vitamin B Fortified bread, cereal, whole grains, lean meats (especially
pork), fish, dried beans, soybeans etc
Vitamin B2 Organ meats (liver, kidney, heart), mushrooms, green leafy
vegetables etc
Vitamin B3 Dairy products, fish, meats, nuts and eggs
Vitamin B5 Cheese, corn, eggs, liver, meats, peanuts, soybeans, brewer’s
yeast etc
Vitamin B6 White meat, bananas, liver, whole grain breads and cereals,
soybeans, vegetables etc
Vitamin B7 Organ meats, oatmeals, egg yolk, mushroom, peanuts, banana,
brewer’s yeast etc
Vitamin B9 Beans, leafy vegetables, citrus fruits, meat etc
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Vitamin B12 Beans, soybeans, peanuts etc

Vitamin C Cabbage, dark green leafy vegetable etc
Vitamin D Exposure to sunlight, milk, fatty fish etc
Vitamin E Germ of a seed or grain
Vitamin K Cabbage, spinach, soybeans etc.

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, pharmaceutical 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, B6, B12 and vitamin C.

Fermentation technologies provide an alternative to chemical processes in the

production of wide range of 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 microorganisms used for the production of vitamin by fermentation
techniques are shown in Table 2.

Table 2: Microorganisms used for the production of vitamins by fermentation

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Vitamins Microorganisms
Riboflavin (vitamin B2) Eremothecium ashbyi
Ashbya gossypii
Bacillus subtilis
Pichia guilliermondii
Pichia pastoris
Bacillus megaterium
Enterobacter aerogenes
Corynebacterium ammoniagenes
Candida famata
Candida flaveneri
Candida guilliermondii
Lactococcus lactis

Vitamin B6 Propionibacterium shermanii

E. coli
Rhizobium meliloti
Rhizobium leguminosarum

Vitamin B12 Propionibacterium shermanii

Propionibacterium acidipropionici
Pseudomonas denitrificans
Acetobacterium sp

Vitamin C Saccharomyces cerevisiae

Pichia fermentans
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Crytococcus terreus
Clavispora lusitaniae

Vitamin B2

Vitamin B2 or riboflavin functions as part of metabolic systems concerned with the

oxidation of carbohydrates and amino acids.

Various biotechnological processes have been developed for industrial scale

riboflavin biosynthesis using different mciroorganisms, including filamentous
fungi as Ashbya gossypii, Candida famata and Candida flaveri as well as the
bacteria Corynebacterium ammoniagenes and Bacillus subtilis.

Majority of the microorganism can synthesize riboflavin from simple medium

components. Some bacteria, yeast and yeast-like fungi are able to over produce
riboflavin under specified cultivation condition in quantities which exceed their
physiological requirements. This characteristic makes it possible to obtain the
vitamin on an industrial scale.

There are reports on the production of riboflavin using substrates such as activated
bleaching earth discharged from an oil refinery containing either rapeseed oil or
palm oil; molasses, peanut seed cake, Carica papaya seeds, rice bran oil, whey etc.

Vitamin B6

Vitamin B6 is a water-soluble vitamin. It is involved in macronutrient metabolism,

neurotransmitter synthesis, histamine synthesis, haemoglobin synthesis and gene
expression.
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Several strains of Klebsiella, Achromobacter cycloclastes, Flavobacterium,

Bacillus and Rhizobuim have been reported to produce vitamin B6.

The production media for flask fermentation by Rhizobium meliloti IFO14872 of

vitamin B6 consists of:

Glucose 1%

Polypeptone 0.5%

Yeast extract 0.2%

KH2PO4 0.1%

MgSO4.7H2O 0.05%

MnSO4.7H2O 0.001%

FeSO4.7H2O 0.001%

pH 6.8

Vitamin B12

Vitamin B12 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 and fatty acid synthesis.

Vitamin B12 can be produced by fermentation employing microorganisms such as

Propionibacterium shermanii and Pseudomonas denitrificans. Also, a mixed
culture of Bacillus firmus AZ-78B and Streptomyces halstedii AZ-8A has been
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reported for vitamin B12 production using agricultural wastes supplemented with
minerals salts (solid state fermentation).

Vitamin C

Vitamin C or L – ascorbic acid is an important metabolite for most living

organisms. In human, it is necessary for different physiological functions and thus
is an essential nutrient. It is widely used as a food additive.

Microorganisms such as Sacharomyces cerevisiae, Acetobacter suboxydans,

Xanthomonas campestris have been reported to aid in the production of vitamin C.

Parameters Controlling Vitamin Production by Fermentation

Several physical and nutritional parameters influence vitamin production.

Physical Parameters

pH. Highest yields of vitamin B 2 has been reported by Eremothecium ashbyi at

constant pH of 4.5 and 5.5, while little or no riboflavin production at either pH 3.5
or 8.5.

Temperature. On a study conducted on the influence of temperature on vitamin

B12, strains of Citrobacter freundii and Klebsiella pneumoniae isolated from
Indonesian tempeh, it was found out that a decrease in temperature from 32-24°C
led to a decrease in vitamin B12 production.

Nutritional Parameters

This include the influence of the initial concentrations of carbon and nitrogen
sources and also other flavinogenic stimulants or metals or even amino acids.
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It has been reported that L-galactonic acid and γ-lactone stimulate ascorbic acid
production in strain of Sacharomyces cerevisiae, Clavispora lusitaniae,
Cryptococcus terreus, Pichia fermentans in which this is undetected whenever
glucose represents the sole carbon source.

Down Stream Processing

This refers to the recovery and purification of products from natural sources such
as animals or plant tissue or fermentation broth. The study of separation and
purification processes of fermentation products is most important for their
commercial success.

Recovery and purification of these bioproducts from their crude sources include
various steps such as precipitation, centrifugation, extraction, membrane filteration
and sorption.

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 the 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

interaction, 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. A large scale cyclic operation involves
three steps: adsorption, desorption and washing.
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For vitamin B2, recovery from the fermentation broth is by centrifugation after
inactivation of the microorganisms by heat. Differential centrifugation leads to
separation of cells and riboflavin crystals because of differences in size and
sedimentation behavior. Riboflavin is then recovered from cell-free broth using
evaporation and vacuum drying.

For the extraction of vitamin B12 after fermentation, the whole broth or an aqueous
suspension of harvested cells is heated at 80-120°C for 10-30min at pH 6.5-8.5.
The conversion to cyanocobalamin is obtained by treating the heated broth or cell
suspension with cyanide or thiocyanate. The whole solution is clarified by
filteration 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 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,
aluminum oxide, or activated carbon can be used. Pure vitamin B 12 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 B 12 are
summarized in Fig. 1.
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Fermentation broth

Harvesting cells by centrifugation

Extraction (in alcohol) or heat

Purification by adsorption, clarification

of filteration
Conversion to cyano form with 0.1%KCN

Feed quality
vitamin B12
Chromatography (e.g. Al2O3)

Crystallization

Food quality vitamin B12

Fig. 1: Downstream processing of vitamin B12

Quantitative Estimation of Vitamins

Vitamins can be quantitatively estimated by various method. Of these,

microbiological assay is most widely used. HPLC determination is also carried out
for the estimation of vitamins.

Estimation of Vitamin B12

Estimation of vitamin B12 can be done by various processes

Microbiological assay

Although vitamin B12 can be assayed biologically with mice, chicks or rats,
microbiological method of assay is preferred since it is more rapid and economical.
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Certain species of bacteria and some yeast can only grow in the presence of certain
vitamins. If these test organisms’ are transferred to a defined culture media which
contains all the compounds essential for their growth apart from the vitamin in
question, proliferation of the test organism 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. Lactobacillus
leichmannii and L. lactis have are widely used to assay for vitamin A.

Other methods include HPLC and spectroscopic determination.

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 filterated, and 20ml of
NaCN 10% (w/v) is added to 1ml of aqueous phase. The resulting upper aqueous
phase is injected into the HPLC system with NH2 column under a wavelength of
360nm. The mobile phase is 250mM of phosphoric acid/acetonitrile (30/70, v/v).

Spectroscopic Estimation

For the determination of vitamin B12 concentration, microorganism is disrupted in a

O.1N phosphate buffer solution containing 0.01% KCN at pH 5.5 for 20min at
1000C. Vitamin B12 formed intracellularly is determined spectrophotometrically in
the dicyano-form at wavelength of 367nm.