| The total production of grain in China is 200 million tons (2012-2013), which rank the highest in the world. Every year, a large number of broken rice, rice dregs and rice bran, as the by-products, are produced. Rice protein has a proper amino acid pattern and is a kind of important resource of protein with low sensitization, compared with other plant protein which contains anti-nutritional factors and allergen. So it is used as ingredient in infant food and special food.Obesity, the sleep time or quality, bad living habits such as smoking and drinking can lead to loss of immunity and cause all sorts of diseases, to seek appropriate immune regulator to improve the body’s immune ability, become the human society urgently needs.Therefore, this study intends to prepare rice immuoactivity peptides (RIAP) from rice by-produce processing, as well as launch an in-depth study of the immuoactivity. The aim of this study is to provide a theoretical basis for the effective and rational use of RIAP and improvement of social benefit and economic benefit.Here, according to degree of hydrolysis (DH) as examining index, time, temperature, pH, amount of enzyme and substrate concentration was researched. By single factor experiment methods, the optimal enzymatic hydrolysis conditions were determined as:the substrate concentration 5%, enzyme amount 2800 U/g, pH 8.0, enzymolysis temperature 60℃, enzymolysis time 3 h. Under the condition, DH was 23.21%. According to DH and macrophage proliferation index (SI) as examining index, the optimal enzymatic hydrolysis conditions was determined by orthogonal experiment methods as:the substrate concentration 6%, enzyme amount 2600 U/g, pH 7.8, enzymolysis temperature 60℃, enzymolysis time 3 h. Under the optimal enzymatic hydrolysis condition, DH and SI values were 22.04% and 1.453 respectively; peptides with relative molecular mass between 180-500 Da was 52.32%; peptides with relative molecular mass<180 Da was only 3.36%. It was clear from the results of amino acid composition analysis that the RIAP hydrophobic amino acids, Ala, Val, Met, Phe, Ile, Leu, Pro, the content of Trp were 30.12%; the proportion of basic amino acid reached to 11.84%; the hydrophobic Q value was 4.94 KJ/mol.Macroporous adsorption resins were used to purify rice immune active peptide and explored its immuno-activity. Through static adsorption and desorption experiment, the desorption rate was 79.78%; the adsorption quantity sample was 317.92 mg/mL when the concentration of the sample was 30 mg/mL. At the condition that dynamic sample flow rate was 0.5 BV/h, the sample concentration of 30 mg/mL, ionic strength was 0.4 mol/L NaCl and 70% ethanol was used to desorb rice immuno-active peptide, desalination rate of 74.47%, the desorption rate was 86.00% and rice immuno-activity peptide purity increased by 13.55%. Different concentrations of ethanol were used to purify rice immune active peptide. Hydrophobic Q peak value was 5.78 KJ/mol. With the increased ethanol concentration, the hydrophobic Q value also increased; content of peptide with molecular weight ranging from 1000 to 2000 D was significantly increased by 63.89%. SI value also increased to 1.551. This was suggested that an unknown relationship existed between the polarity of the rice immuno-activity peptide and SI value.According to different charge property, the type 201×7 strongly basic anion resin and DEAE Sepharose Fast Flow weak anion exchange resin were used to purify rice immuno-activity peptide. With 201×7 strong anion exchange resin, SI value of elution peak, using 0.1 N HCl, was 1.649, which was greater than the SI value of 55% elution fractions. The purified fractions by DEAE Sepharose Fast Flow weak anion exchange resin were of higher immno-activity, but the yield was only 44.50%. As a result,201×7 strong anion exchange resin was more suitable for purification of rice immuno-activity peptide. |
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