1. Distribution of Trypsin Inhibitor in Soybean Trypsin inhibitors are distributed in various parts of soybean, but are mainly found in soybean seeds. The content of trypsin inhibitors in soybean seeds can reach 6% to 8% of the total protein.
2. Biochemical characteristics of trypsin inhibitors in soybean
2.1 Structure and Function of Trypsin Inhibitors Trypsin inhibitors are functional proteins with small molecular weight and physiological activity. Trypsin inhibitors in soybeans can be divided into the following two categories:
2.1.1 kunitz inhibitors. Directly and exclusively on trypsin, the binding of this type of inhibitor to trypsin proceeds quantitatively, ie 1 g of the molecule's inhibitor can bind 1 g of the molecule's trypsin.
2.1.2 Bowman Birk inhibitors. Can be combined with trypsin and chymotrypsin, respectively, because the inhibitor has two active centers within the molecule, it is known as double-headed inhibitor.
Kunitz-like and BowmanBirk-like inhibitors are the two most important trypsin inhibitors in soybeans, and their levels in soybeans are 1.4% and 0.6%, respectively. These two kinds of trypsin inhibitors have different stability to heat, acid and alkali. The purified kunitz inhibitors maintain their activity at a temperature below 30°C and a pH range of 1-12. Heating them at 80°C for a short time can make them. Reversible denaturation, while heating at 90 °C makes it irreversibly inactivated. BowmanBirk inhibitors are more stable than kunitz inhibitors against heat, acids and bases and retain their activity when heated in the dry state at 105°C or with their 0.02% water solubility at 100°C for 10 minutes (Fennema 1985). ). Contrary to their thermal stability, it is also reported that BowmanBirk inhibitors are more thermally unstable than kunitz inhibitors (Dilietroh and Liener 211989).
2.2 Reaction Mechanism of Trypsin Inhibitor and Target Enzyme The trypsin inhibitor in soybean is a serine protease inhibitor that reacts with serine proteases secreted by the pancreas. The trypsin inhibitor interacts with the target enzyme, usually enzymes and Like the interactions between substrates, they are complementary mechanisms of action. When the two react, the active center where the inhibitor is exposed and the active center of the target enzyme are connected by hydrogen bonds to form a stable covalent complex, which results in the blocking of the enzyme activity center and the loss of activity of the target enzyme. Compared with the enzyme-catalyzed reaction, the Michaelis constant of the reaction between the protease and the inhibitor is very low, so the affinity of the protease and the inhibitor is large, and the two can be quickly combined to form a complex. Unlike the substrates of general enzymes, the peptides at their active centers do not cleave or cleave very slowly after the inhibitor binds to the enzyme. Therefore, although the complex can be decomposed into free enzymes and denatured or undenatured inhibitors, the dissociation rate is very slow.
The above description indicates that the inhibitor can be regarded as a substrate for the enzyme in a certain sense, and the contact site with the enzyme mainly concentrates on the amino acid residues near the center of the inhibitor activity.
3. Toxicology of Trypsin Inhibitors in Soybeans The detrimental effects of trypsin inhibitors on animals are mainly to cause growth inhibition and to cause pancreatic hypertrophy in some animals.
Trypsin inhibitors have an inhibitory effect on animal growth and are generally considered to have the following two causes:
3.1 Trypsin inhibitors can bind to trypsin and chymotrypsin in the small intestine to form a stable complex that inactivates enzymes, leading to reduced digestibility of food proteins and loss of exogenous nitrogen.
3.2 Trypsin inhibitors can cause enhanced pancreatic secretion, resulting in excessive secretion of trypsin and chymotrypsin. Since these proteases contain very rich sulfur-containing amino acids, these amino acids used in the synthesis of body tissue proteins are used to synthesize proteases, form complexes with inhibitors, and eventually excreted through feces, resulting in endogenous nitrogen and organisms. Sulfurous amino acid loss. Soy protein is inherently deficient in sulfur-containing amino acids, and the additional loss of sulphur-containing amino acids caused by inhibitors leads to imbalances in amino acid metabolism in the body, thus impeding animal growth. Studies have suggested that when diets containing protease inhibitors are ingested, the endogenous loss of sulfur-containing amino acids has an effect on the body's nitrogen balance more than the effect of amino acid losses from the diet (exogenous losses). Great (Barth et al., 1994).
4. Method for removing trypsin inhibitor in soybean
4.1 Heat treatment method Soybean trypsin inhibitor itself is a combination of protein or protein, is not stable to heat, and can be fully heated to denature and inactivate its harmful effects.
Soybean heat treatment methods include boiling, steam treatment (atmospheric or high pressure steam), baking, infrared radiation treatment, microwave radiation treatment, extrusion (dry or wet extrusion). Wet heating (steam, cooking, etc.). Trypsin inhibitors can be inactivated by steam treatment with atmospheric steam heating for 30 minutes or 98 kPa pressure for 15-20 minutes. In addition, the effect of extrusion is also better.
4.2 Chemical treatment Some studies have shown that trypsin inhibitors in soybeans can be inactivated by chemical treatment. The mechanism of action of such methods is generally to use chemical substances to destroy the disulfide bonds of trypsin inhibitors, thereby changing the molecular structure of trypsin inhibitors to achieve the purpose of inactivation. Chemicals that have been used include sodium sulfite, sodium bisulfite, copper sulfate, ferrous sulfate, sodium thiosulfate, glutaraldehyde, and some compounds having a thiol group (cystine), N-acetylcystine, and the like. Hou Shuisheng et al. (1994) reported that using 0.5% sodium sulfite to treat raw soybeans for more than one week can reduce the trypsin inhibitor by 59.83%. Zhang Jian et al. (1999) reported that treatment of raw soybeans with 5% urea and 20% water for 30 days resulted in a 78.55% inactivation rate of trypsin inhibitor.
4.3 Enzyme treatment Some people in France have studied the use of specific enzymes produced by strains of certain fungi and bacteria to inactivate trypsin inhibitors in soybeans (Meijer and Spekking 1993, Huo et al., 1993).
4.4 Crop Breeding The study of the soybean Ti gene (the recessive gene without trypsin inhibitor) and its genetic regularity in France and abroad provide the basis for reducing the activity of soybean trypsin inhibitor through crop breeding. Hymowitz successfully cultivated a new soybean variety with low trypsin inhibitors in 1986. Its trypsin inhibitor activity is 50% lower than that of normal soybean.

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