Fermentative Production of Vitamins: Indian and Global Scenario Irfan Ahmad M. Tech. Pharm. Tech. (Biotech.)National Institute of Pharmaceutical Education and Research, S.A.S. Nagar
CONTENTS 1. Introduction to Vitamins. 2. Industrial Production of Vitamins. 3. Vitamins Market Scenario. 4. Fermentative Production of Water Soluble Vitamins. i. Vitamin B₁₂ ii. Vitamin B₂ iii. Vitamin C iv. Vitamin H 5. Fermentative Production of Fat Soluble Vitamins. i. Vitamin E ii. Vitamin K 6. Summary.
VitaminsVitamins are defined as essential micronutrients that arerequired in trace quantity and cannot be synthesized bymammals but are essential for metabolism of all livingorganisms VITAMINSFat Soluble Vitamins Water Soluble VitaminsVitamin A Vitamins BVitamin D Vitamin CVitamin E Vitamin HVitamin K Vitamin P, etc.
Role of Vitamins1 Nutritional and Physiologic Role .2 Food/Feed Additives3 Therapeutic Agents4 Health Aids5 Technical Aids
Industrial Production Of Vitamins Extraction Chemical Fermentation
Industrial Production of Vitamins Production Method World Production (tonne) Biotech. Chem. Extr. 1980 1990 2000 2010Thiamine (B1) + 1700 4200 3700Riboflavin (B2) + 2000 2400 4400Niacin + + 8500 22000 2800Pantothenic acid + + 5000 7000Pyridoxine (B6) + 1600 2550 3800Biotin + + 2.7 2.5 88 112Folic acid + 100 400 534Vitamin B12 + 2 10 25 175Vitamin C + + 40000 60000 10700Vitamin A + + 16800 2500 2700Vitamin D + + 5000Tocopherol (E) + + + 6800 22000 30000Vitamin K + 500 Survase SA, Bajaj IB, Singhal RS: Biotechnological production of vitamins
Microbial Production of VitaminsVitamin Enzyme (Microorganism)Vitamin C 2,5-diketo-D-gluconic acid reductase (Corynebacterium sp.). ucuBiotin Fermentation (Serratia marcescens); Multiple enzyme system (Bacillus sphaericus).Riboflavin Fermentation (Eremothecium ashbyii, Ashbya gossypii, Bacillus sp.). Fermentation (Propionibacterium shermanii, PseudomonasVitamin B12 denitrificans)Vitamin E Freshwater microalgae, Euglena gracilis. Shimizu S: Vitamins and related compounds: microbial production
Fermentative Production of VitaminsAdvantages Disadvantages i. Efficient, i. Complex, ii. Less Energy Intensive, iii. Carried out at ambient ii. Require Careful Monitoring, temperature and pressure, iii. Limited Operations Region, iv. Produce Renewable iv. Susceptible to Substrate and byproducts (e.g., Biomass), Product Inhibition, v. Low Cost of Waste Disposal, v. High Cost of Downstream vi. Highly enantioselective and Processing, regioselective, vii. Don’t suffer from consumer vi. High Risk of Contamination, consciousness with regard to vii. Tendency to Elicit Allergic safety. Manifestations.
Vitamin Market Scenario• Europe represents the largest regional market; the US constitutes the single largest market globally.• Asia-Pacific is likely to emerge as the fastest growing market, with a CAGR of about 4.0% over the analysis period.• Vitamin E represents the largest segment, owing to the extensive use of these vitamins in cosmetics, pharmaceuticals, and food end-use applications• The cosmetics industry, though relatively small in terms of the percentage share, is emerging as a key end-user with a CAGR of 4.6% over the analysis period.• The global vitamin market was $2.3 billion in 2000. However, the vitamin market is expected to reach US$3.2 Billion by 2017.
Top Five Sources of US Vitamin Imports China Belgium Other Germany India NetherlandThe vitamin industry was oncedominated by a core group of 3% 3%producers in the developed economies 7%but is now far more open and 13%competitive as an increasing numberof manufacturers in emerging 57%economies such as China and India 17%weigh in.
Hoffmann La Roche(Switzerland), BASF(Germany), ADM(USA), Hubei Guangji(China), Merck(Germany) and Takeda(Japan) are some of theglobal leaders in vitaminproduction.
Sales Single vitaminIn 2011, the Indian vitamin segment 0.3%grew 7.5 per cent in volumes (against Single0.1 decline in the year 2010) and 10.3 Mineralper cent in value (against 5.4 per cent 9% Tonicsgrowth in 2010).By the end of 12%2014, the vitamins and mineralscategory will be worth INR21,174.6m ($486.7m), with anexpected CAGR of 3% between 2009and 2014. Multivitamins 79%Five of the top 10 players are foreignmultinational firms. Among Indiancompanies, the largest vitaminproducers are Wockhardt, RaptakosBrett, Piramal Healthcare and Alkem.
WATER SOLUBLE VITAMINS Vitamin B₁₂ Vitamin B₂ Vitamin C Vitamin H
Vitamin B₁₂( Cyanocobalamin) Cyanocobalamin is the industrially produced stable cobalamin form which is not found in nature. It is obtained exclusively by fermentation process. Merck began production of vitamin B₁₂ by Pseudomonas denitrificans in 1952 and have improved the efficiency of culture more than 30- fold relative to the performance of the original soil isolates by genetic manipulations and microbial screening. Mutagenic treatments have resulted in improved activity. Sumi: Microbial Production of Vitamin B12
Biosynthesis of Vitamin B₁₂
Downstream Process of Vitamin B₁₂
Vitamin B2 (Riboflavin)• Riboflavin has been produced commercially by chemical synthesis, by fermentation and by a combination of fermentation and chemical synthesis.• Recently, fermentation route has been widely used as it produces the vitamin in a single step, resulting in substantial cost savings. In contrast, chemical processes are multistage, and incur a lot of cost.• Most of the producers like BASF, Roche, ADM/Aventis, Hubei Guangji prefer fermentative production of riboflavin over chemical process.
Vitamin 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 40000 times more vitamin than it needs for its own growth.• Recently, Nippon Roche, Japan, has developed and commercialized a single step fermentative riboflavin production using a recombinant Bacillus subtilis strain, which effectively produces riboflavin directly from glucose in fed-batch operation.• Improved strains for the production of riboflavin were constructed through metabolic engineering using recombinant DNA techniques in Corynebacterium ammoniagenes.• Sybesma et al. developed Lactococcus lactis strain using both direct mutagenesis and metabolic engineering for simultaneous overproduction of both folate and riboflavin.
Vitamin C (Ascorbic Acid)• L-ascorbic acid finds its use mainly in food industry, being a vitamin as well as an antioxidant.• Majority of commercially manufactured L-ascorbic acid is synthesized via Reichstein process using D-glucose as a starting material• Approximately 50 % of synthetic ascorbic acid is used in vitamins supplements and pharmaceutical preparations.• Because of its antioxidant properties and its potential to stimulate collagen production, it is also widely used as an additive to cosmetics.• The current global market of L-ascorbic acid is in excess of US$ 585 million with an annual growth rate of 3 %.
Vitamin C (Ascorbic Acid). D-Glu D-Sorbitol Reichestein Process L-Sorbose L- Ascorbic Diacetone-L- 2-Keto-L- acid Sorbose Gluconic acid methyl ester
Bacterial Fermentation of Vitamin C 2-Keto-D-gluconic acidSorbitol Pathway Pathway
Yeast Based Fermentation Process• Saccharomyces cerevisiae and Zygosaccharomyces bailiiaccumulate 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.
Algae Based Fermentation Process• Skatrud and Huss described a method that involved initial growth of Chlorella pyrenoidosa ATCC53170 in a fermentor with a carbon source that is sufficient for the cells to grow to an intermediate density. At the depleted stage, additional carbon source was added sequentially or continuously to maintain the carbon source concentration below a predetermined level until the addition is terminated. This resulted in the production of 1.45 g/L of L-ascorbic acid.• Euglena gracilis Z. is one of the few microorganisms which simultaneously produce antioxidant vitamins such as carotene (71 mg/L), vitamin C (86.5 mg/L) and vitamin E (30.1 mg/L).
Vitamin H (Biotin) 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, creating a world market of 10–30 t/year. Majority of the biotin sold is synthesized chemically via Goldberg and Sternbach syntheses. 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 bioF gene bioA gene bioD geneThe conversion of bioB genedethiobiotin to biotinhas not been resolved.
Fermentative Production of Biotin• 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.• Brown et al. studied the production of biotin by recombinant strains of E. coli. The strain used was E. coli C268 having a bio A genotype and plasmid pTG3410 with two expression cassettes .• Biotin production under limiting growth conditions by Agrobacterium/Rhizobium HK4 transformed with a modified Escherichia coli bio operon.
FAT SOLUBLE VITAMINS 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 are capable of producing α-tocopherol. In humans, ∞-tocopherol is believed to play a major role in prevention of light induced pathologies of the skin, eyes and degenerative disorders such as atherosclerosis, cardiovascular diseases and cancer. Industrial application of ∞-tocopherol includes its use in preservation of food, in cosmetics and sunscreens.
Vitamin E Extraction The synthetic form is a mixture of eight stereoisomersExtraction from oils is not collectively known as all-rac-efficient, as these typically alpha-tocopherol.contain low levels of Vitamin E∞-tocopherol when administered in equal amounts, the bioavailability of natural with respect to synthetic ∞-tocopherol is 2:1. Chemical Synthesis Chemical Synthesis
Vitamin EFermentative Production of Vitamin K₂ Microalgae Euglena gracilis Z and marine microalgae Dunaliella tertiolecta produce ∞-tocopherol in concentrations higher than conventional foods. Carballo-Cardenas et al. studied ∞-tocopherol production with Dunaliella tertiolecta and Tetraselmis suecica, demonstrating that nutrient composition can be used as a tool to improve α-tocopherol productivity.
Vitamin K₂ Vitamin K₁ . Vitamin K Vitamin K₂
Vitamin KFermentative 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.
Summary Fermentative productions of vitamins have many advantages compared to conventional chemical synthesis processes. Different methods like media optimization, mutation and screening, genetic engineering and biocatalyst conversion have been used for improvement of the production of vitamins.