Biotin is a cofactor of many enzymes in the body, most of which are acetyl-Coa carboxylase, pyruvate carboxylase, propionyl-Coa carboxylase , β-methylbutene-Coa carboxylase and so on in animals. The main functions of biotin in the body are as follows:
1. Involve in the body's carbohydrate metabolism
In carbohydrate metabolism, biotin catalyzes decarboxylation and carboxylation reaction, involves in glucose metabolism and neoglycogenesis, and maintains stable blood sugar. Biotin, as a coenzyme of pyruvic carboxylase, catalyzes the carboxylation of pyruvate to oxaloacetic acid, and promotes the conversion of oxalosuccinic acid to a-ketoglutaric acid, malic acid to pyruvic acid, and succinic acid to pyruvic acid.
2. Involve in the body's lipid metabolism
Biotin involves in fatty acid synthesis as a coenzyme of acetyl-CoA carboxylase, and also involves in β-oxidation of odd-carbon fatty acids as a coenzyme of propionyl-Coa carboxylase. In addition, biotin is also associated with acetylcholine synthesis and cholesterol metabolism.
3. Involve in the body's protein and nucleic acid metabolism
Biotin plays an important role in protein synthesis, amino acid deamination, purine synthesis and metabolism of leucine and tryptophan, and it is essential for the transfer and decarboxylation of many amino acids.
4. Involve in the metabolism of other substances
Biotin, as a coenzyme component, involves in the metabolism of nutrients such as lysozyme-activated acetyl-coenzyme A, vitamins B1, B2, C, folic acid and pantothenic acid. It can be seen that biotin plays an irreplaceable role in maintaining metabolic homeostasis.
1. Biosynthetic pathways of glutamic acid
The main pathways of glutamic acid biosynthesis: Glucose is then oxidized to acetyl COA (acetyl COA) by glycolysis (EMP) and hexose phosphate (HMP) pathway, and then enters the tricarboxylic acid cycle to produce a-ketoglutaric acid, which is catalyzed by glutamate dehydrogenase and in the presence of NH4+, and then restored amination reaction to produce glutamic acid. When glucose was used as raw material to produce glutamic acid, the isocitrate lyase had almost no activity under the condition of submoderate biotin. The reason is that the oxidation capacity of pyruvic acid decreases, the formation rate of acetic acid slows down, and the formation of isocitrate lyase induced by acetic acid is little. Moreover, because the enzyme is inhibited by succinic acid, under the condition of submoderate biotin, the accumulated succinic acid due to reduced oxidation capacity will inhibit the enzyme activity and inhibit the formation of the enzyme, and the glyoxylic acid cycle is basically closed, and the metabolic flow rate moves efficiently along the direction of isocitric acid → a-ketoglutaric acid → glutamic acid.
2. Effects of biotin on nitrogen metabolism
When biotin is limited, there is almost no isocitrate lyase, the oxidation of succinic acid is weak, and the decarboxylation of malic acid and oxaloacetic acid is stagnant. At the same time, due to the decrease of complete oxidation, the formation of ATP is reduced, and the protein synthesis activity is stopped. Under the condition of appropriate amount of ammonium ion, glutamic acid is formed and accumulated, and the generated glutamic acid will not form other amino acids through transamination. Under sufficient biotin conditions, isocitrate lyase, succinic acid oxidation, pyruvate oxidation, protein synthesis, glyoxylic acid cycle ratio, oxaloacetic acid and malic acid decarboxylation reactions all increase, resulting in a decrease in glutamic acid and an increase in other amino acids produced by transamination.
3. Effects of biotin on the regulatory mechanism of glutamate biosynthesis pathway
In the case of abundant biotin, the membrane synthesis of glutamate bacteria is complete, glutamic acid cannot penetrate from the inside membrane to the outside membrane, and the accumulation of glutamic acid in the cell reach up to the certain extent, and the feedback control of glutamate dehydrogenase is carried out, thus stopping the biosynthesis of glutamic acid. When biotin is limited , glutamic acid can penetrate from intracellular to extracellular due to incomplete cell membrane synthesis, which reduces the content of intracellular glutamic acid, and the feedback control of glutamic acid to glutamate dehydrogenase is dysfunctional, and glutamic acid is continuously preferenced for synthesis.
4. Effect of biotin on membrane permeability of glutamate-producing bacteria
Biotin has an important effect on glutamic acid biosynthesis pathway, but the more essential role of biotin is to affect the permeability of cell membranes. As a coenzyme of acetylcoa carboxylase, a key enzyme in the initial reaction of fatty acid biosynthesis, biotin participates in the biosynthesis of fatty acids and influences the synthesis of phospholipids. When biotin is controlled at the sub-appropriate level, fatty acid synthesis is incomplete, resulting in incomplete phospholipid synthesis. Since the cell membrane is composed of phospholipid bilayer, when the phospholipid content is reduced to half of the normal amount, the cell is deformed, and glutamic acid exudes from the cell and accumulates in the fermentation liquid. When biotin is excessive, due to the large amount of phospholipids in the cell, the cell wall and membrane are thicken, which is not conducive to the secretion of glutamic acid, resulting in the decrease in acid production rate and affecting the economic benefits of fermentation production.
Glutamate-producing bacteria are nutrient-deficient type, so biotin is very important for the growth and reproduction of glutamate-producing bacteria, and its effects on metabolites are also very obvious. When biotin is excessive, pyruvate in the fermentation pathway is transformed into lactic acid, and isocitric acid is also transformed into succinic acid, which results in rapid bacterial growth and reproduction. At the same time, biotin also promotes the biosynthesis of obstacles to the permeability of bacterial cell membrane. Biotin control directly affects the growth, reproduction and metabolism of bacterial cells, as well as the permeability and acid production rate of cell wall and membrane. Controlling the amount of biotin is the key to glutamic acid fermentation.
At present, most biotin-deficient strains are used in fermentation production, which are highly dependent on biotin during the growth and reproduction period. When biotin is low, the growth and reproduction are slow, the glycolysis of sugar is slow, and the stock of biotin as a coenzyme in cells is small, which affects the metabolism of sugar and the synthesis of glutamic acid after entering the synthesis period. However, excessive biotin leads to long growth and reproduction time of bacteria, large bacteria volume, microscopically detected bacteria with short, round heads and more eight-figure shapes. After entering the synthesis stage, sugar oxidation consumption takes a large proportion, synthesis amount of glutamic acid is low, and cell membrane permeability of bacteria is poor, which prevents bacteria from timely excreting glutamic acid in cells, and glutamate synthesis pathway is blocked. The glutamic acid excreted by the strain cells in the fermentation solution can only account for 12% of the total amino acid. When biotin is subappropriate, the metabolism of bacteria is disordered, the permeability of cell membrane is enhanced, the glutamic acid in cells can be excreted in time, which is conducive to the accumulation of glutamic acid, and the elimination of glutamic acid by bacteria cells in the fermentation solution can reach about 92% of the total amino acids.
The dosage of biotin should be determined according to the characteristics of the strains used, the inoculation amount of fermentation, the volume of wet bacteria in the large tank of proliferating cells, the type of media components, and the size of the oxygen supply capacity. With the improvement of glutamic acid fermentation control technology and accumulation of experience, and the improvement of oxygen supply capacity, the fermentation process with appropriate amount of biotin has become an important way to increase acid production. In the control of biotin dosage, not only the total biotin of raw corn pulp , molasses and pure biotin in the medium should be taken into account, but also the changes in nutrients (biotin and vitamins) caused by changes in the sugar solution due to changes in corn starch and production technology, and the ratio of biotin in the medium should be adjusted according to the fermentation sugar consumption, acid production and fermentation oxygen capacity, the amount of biotin should be controlled, fully meet the needs of growth, sugar consumption, synthesis of glutamic acid, and negative effects is avoided.
At present, most glutamate-producing bacteria in China are biotin-deficient type. Biotin is an important growth factor, biotin subappropriate process was adopted,and the control of the biotin dosage directly affects the growth, proliferation and metabolism of the producing bacteria cells, the permeability of the cell wall membrane and the acid production rate. Therefore, strict control of biotin dosage is an important key to do a good job in glutamic acid fermentation. However, the current biotin detection method still has some limitations, and can not accurately detect the biotin content of various raw materials in the fermentation process in time, so the further research and development of analytical methods with higher sensitivity, simpler methods and wider application range is a primary task of biotin analysis research in the future.