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INTRODUCTION Vitamin C is a product that is usually found in many foods and produced by an abundance of chemical companies as dietary supplements. This vitamin is essential to the scurvy patients and used abundantly in cosmetic company due to its antioxidant properties. In the terms of its production, the fact that vitamin C has two totally and distinctly separate sides is still unknown to many people even doctors. The two sides consist of “L”-Ascorbic Acid, which is the (-) side, and DAscorbic Acid, which is the (+) side. The L side of Vitamin C is the active side, and is the side which is beneficial to mankind.

The D-side of Vitamin C is designated as useless and discarded by the body, as most research shows. People ingesting Vitamin C would only benefit from the “L” side of Vitamin C for example, in an orange, the Vitamin C is primarily the L side [1]. Therefore, during the whole process of the production of vitamin c, the desired end product is Lascorbic acid. THE PRODUCTION OF VITAMIN C Vitamin C has been industrially produced for around 70 years. Over these years, many methods has been invented and proposed to produce vitamin C efficiently such as Reichstein Process, TwoStep Fermentation Process and Single Step Fermentation Process.

The most common methods used are Reichstein Process and Two-Step Fermentation Process. The first method used is the classical Reichstein process back in the 30s. This process was introduced by Tadeus Reichstein and his collegues in 1935 [1]. The Reichstein process uses a single pre-fermentation process followed by a purely chemical route which involves five steps. REICHSTEIN PROCESS This process commonly use glucose or sugar as its raw material. Sugars can be derived from any sugar bearing plants such as corn, wheat, and many more. However, for the production of vitamin C, D-glucose is used as its raw material [1][2].

The first step is the hydrogenation of D-glucose to D-sorbitol, an organic reaction with nickel as a catalyst. In this step, D-glucose is reduced to sorbitol under high temperature (140-150°C) and high pressure (80-125 atm ). This is done by putting it in a big boiler[3]. The next step is the microbial oxidation of sorbitol to L-sorbose at pH 4 to pH 6 and temperature of 30 °C by using a bacteria. A bacteria called Acetobacter suboxydans is added to eat some hydrogens so that oxygen double bond is formed. In the protection step, acetone is used as catalyst. The four remaining hydroxyl group in sorbose are protected by acetal linkages.

The product of this step is Diacetone-L-sorbose. The forth step will be the organic oxidation with potassium permanganate followed by heating with water at temperature over 100°C[3]. The unprotected hydroxyl group is chemically oxidized to carboxylic acid. The most complicating part of this step is to make sure the right part is oxidized. At the end of this step, 2-keto-L-glucanic acid is produced [4]. The only thing left to do is to join the acid with alcohol and removes the water. This step is called Gamma Lactonisation. Hydrochloric acid and ethanol were added.

Hydrolysis with the acid removes the two acetal groups and the acid will joined to the alcohol forming a circle. The end result is Vitamin C in crystals [4]. Chemical reaction of each steps in Reichstein process : D-glucose D-sorbitol L-sorbose L-ascorbic acid (vitamin c) 2-keto-L-glucanic acid Diagram 1[5]. DiacetoneL-sorbose However, Reichstein process has many disadvantages. The process needs to go through six working procedures and is difficult to operate continuously. Therefore, many researchers are eager to find out several different processes to improve the traditional Reichstein process.

One of the most successful routes is the “two-step fermentation” process, which was already applied in industrial scale for more than 40 years [6]. Along with the development of biotechnology industry, there are further improvements in the two-step fermentation process which include the use of bacteria such as Corynebacterium sp. as the enzymatic-based approaches [7]. Corynebacteria is a rod-shaped bacteria belongs to the Corynebacteriaceae Actinobacteria group. The Corynebacteria are small, generally non-motile, non-sporulating gram positive bacteria. They form grayish colonies with a granular appearance [8].

They often lie in clusters referred to as Chinese letters. Corynebacteria grow slowly, even in enriched media. In terms of nutritional requirements, all need biotin (also known as vitamin H) to grow [9]. TWO-STEP FERMENTATION PROCESS Compared to the Reichstein Process, there are only a few chemical steps to be followed in this process. This process mainly used the fermentation techniques by using bacteria such as Corynebacterium sp. and Erwinia herbicola. The steps are shown as below: Sugar (d-glucose) fermentation Erwinia herbicola 2,5-diketo-D-gluconic acid fermentation Corynebacterium sp. Gamma Lactonisation -keto-L-gulonate (precursor of vitamin C) L-ascorbic Acid (vitamin C) Figure 2 : The flow diagram of Vitamin C production [10]. Two-step fermentation system is basically a practical method for the production of 2-keto-Lgulonate (a precursor in the synthesis of L-ascorbic acid) from D-glucose by using biotechnological approaches. The product can only be obtained after several fermentation processes of the glucose using a certain microbacteria or enzymes[11]. For the first step, D-glucose is oxidized to 2,5-diketo-D-gluconic acid by Erwinia strains via the intermediates D-gluconic acid and 2-keto-D-gluconic acid.

Firstly, D-glucose was converted to 2,5diketo-D-gluconic acid by a mutant strain of Erwinia sp. in a medium containing D-glucose, corn steep liquor, ammonium hydrogen phosphate (NH4)2HPO4, and CaCO3. After a 26 hours cultivation, 2,5-diketo-D-gluconate was obtained with a 94. 5% yield from D-glucose. This broth was used directly for the next conversion without removal of cells by treatment with sodium dodecyl sulfate. Next step, the stereospecific reduction of 2,5-diketo-D-gluconate to 2-keto-L-gulonate was performed with a mutant strain of Corynebacterium sp. This reaction is catalyzed by an enzyme which is 2,5-diketo-D-gluconate reductase .

When the cell growth reached a maximum (this takes about 16 hours) in a medium containing D-glucose, corn steep liquor, NaNO3,potassium dihydrogen phosphate KH2PO4, and trace elements, NaNO3 was added to the culture, and then the 2,5-diketo-D-gluconate broth was fed over a period of about 50 hours. Since the mutant strain requires a hydrogen donor for reduction, the calcium 2,5-diketo-D-gluconate broth was mixed with D-glucose before being fed. 2-keto-L-gulonate was stable in the broth. Neither 2-keto-Dgluconic acid (intermediates) nor 5-keto-D-gluconic acid was detected in the final broth [10].

At the end of this process, 2-leto-L-gulonate is obtained[12]. From 2-keto-L-gulonate, the L-ascorbic acid is extracted via Gamma Lactonisation process. Hydrochloric acid and ethanol were added. Hydrolysis with the acid removes the two acetal groups and the acid will joined to the alcohol forming a circle. Water is removed. The end result is Vitamin C in crystals. Ths step is similar to the last step in the Reichstein process. As the product, Lascorbic acid or Vitamin C is produced[13]. SINGLE STEP FERMENTATION PROCESS This is yet the most innovative process in producing L-ascorbic acid from d-glucose.

However, this method still under development and research before being applied in the production of vitamin C in an industry. The one-step fermentation process based on two-step fermentation process [14]. In order to develop a one-step microbial conversion of d-glucose to 2-keto-L-gluconate, the pathway of Erwina herbicola was enhanced using gene cloning techniques. The gene encoding the enzyme 2,5-diketo-D-gluconate reductase was cloned from Corynebacterium sp. and expressed in E. herbicola. With optimized culture conditions, these recombinant strains of E. herbicola yield about 60% of L-ascorbic acid product.

The production of Vitamin C using this new process was clearly lower than the two-step fermentation process. However, without doubt this process may lead to another economical processes of vitamin C production in the future[15]. CONCLUSION The Reichstein process and the two-step fermentation process are two processes used to produce Vitamin C (ascorbic acid). The two-step fermentation process is a newer process that was developed in China. This process used biological oxidation instead of chemical oxidation which is used in the Reichstein process [16]. Each processes have their own pros and cons. Reichstein process have many disadvantages. This process uses heavy machinery and it costs a lot in terms of mantainance, waste disposal and labour. It also requires many organic and inorganic solvents and reagents such as acetone, hyrochloric acid and oxidizers. Most of these reagents cannot be recycled and have high disposal costs [17]. Not only that, a large amount of poisonous gas the “three-waste” are generated during production, possessing high risk of fire and explosion [18]. Overall, this process requires high energy, high-amount of organic solvent and can cause serious environmental pollution.

In contrast of the classical Reichstein process, the two-step fermentation process have several advantages such as, reducing the cost for raw materials and grain requirements, reducing the amount of explosive, flammable and poisonous gasses. Since this process are much more simpler than the classical Reichstein process, less equipment is needed since the production processes are also shortened [19]. By looking and examining all the three methods mentioned above, the two-steps fermentation system is the most productive method.

Compared to the classical method introduced by Tadareus Reichstein which needs heavy machinery and toxic chemical reagents, the two-step system uses more safe and cheap microbiological approaches [20]. However, this modern method required an in-depth knowledge in biochemistry in order to maximise the production. References [1] http://www. xpressnet. com/bhealthy/vitaminc. html [2] Reichstein, T. und Grussner, A. (1934): Eine ergiebige Synthese der L-Ascorbinsaure (C-Vitamin), Helv. Chim. Acta 17, S. 311–328 [3] http://yr12-chem-2012. wikispaces. com/The+Reichstein+Process+-+Matt+Shaw [4] http://upload. wikimedia. rg/wikipedia/commons/c/c4/The_industrial_synthesis_ of_ascorbic_acid_from_glucose. svg [5] Expresspharmaonline. com (2004) New technology for vitamin C production may end Chinese monopoly – Cover Story – Express Pharma Pulse [6] Hubbs, J. C. (1998) Enzymatic process for the manufacture of ascorbic acid, 2-keto-LGulonic acid and esters of 2-keto-L-gulonic acid. US Patent 5817490 [7] http://books. google. com. my/books? id=u7ptVNB6i_kC&pg=PA253&dq=two+step+fermentation +process+for+vitamin+c&hl=en&sa=X&ei=H1GpUIrqAYbprQfX9oCoDQ&ved=0CDEQ6AEwAQ #v=onepage&q=two%20step%20fermentation%20process%20for%20vitamin%20c&f=false [8] Grindley, J.

F. , Payton, M. A. , van de Pol, H. , Hardy, K. G. , 1988. Conversion of glucose to 2keto-l-gulonate, an intermediate in l-ascorbate synthesis, by a recombinant strain of Erwinia citreus. Appl. Environ. Microbiol. 54, 1770–1775. [9] Chotani, G. , Dodge, T. , Hsu, A. , Kumar, M. , LaDuca, R. , Trimbur, D. ,Weyler,W. , Sanford, K. , 2000. The commercial production of chemicals using pathway engineering. Biochim. Biophys. Acta 1543, 434–455. [10] http://yr12-chem-2011. wikispaces. com/The+Reichstein+Process [11] Source: BASF. [12] Skatrud, T. J. and Huss, R. J. 1991) L-Ascorbic acid production in microorganisms. US Patent 5001059 [13]Inventors. about. com (1905) History of Vitamins. [online] Available at: http://inventors. about. com/library/inventors/bl_vitamins. htm[Accessed: 3 Apr 2012]. [14] http://books. google. com. my/books? id=JiuR_dmTIscC&pg=PA887&dq=two+step+fermentation +process+for+vitamin+c&hl=en&sa=X&ei=H1GpUIrqAYbprQfX9oCoDQ&ved=0CC4Q6AEw AA#v=onepage&q=two%20step%20fermentation%20process%20for%20vitamin%20c&f=false [15] Rosenberg, H. R. , 1942. Chemistry and Physiology of the Vitamins. Interscience, New York. [16] H. Hofmann, W. Bill, Chemie-Ing.

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