Study On The Peanut Peptides’content Determination And Anti-oxidation

As a kind of oil economic crop, widely planted peanut has a huge demand in production and consumption. They are rich in nutrients, contained50%fat、24-36%protein、3%minernals、little vitamins and plant sterols. At present, the edible rate of domestic peanuts is far below the foreign rate, just only27%. The records show that the exports of peanut primary products account for92.7%of total exports, but the deep processed products only account for7.3%. In fact, the deep processed products can increase the value in2-10times, however the primary products just only in0.5-1.0times. So we should strive to develop the deep processed product, improving the economic benefits. As the hydrolyzate of protein, the peanut peptides belong to functional material.This paper mainly studied the application of Amino Acid Analyzer method, Biuret method, Kjeldahl method and UV Spetrophotometry method in the content determination of peanut peptides, and prepared the specific peanut peptides after analyzing its characteristic.(1) The selection of the protein precipitation agent and its application conditionsThrough comparing the precipitation effect of three chloroacetic acid、oxalic acid、 sulfosalicylic acid, sulfuric acid、hydrochloric acid, phosphoric acid、alcohol and acetone, it was found that when using Biuret method, TCA was the best choice; when using the Kjeldahl method, TCA、oxalic acid、phosphoric acid and alcohol could be slected to precipitate the protein. So TCA could be as the precipitating agent for the two methods.When using TCA to remove the protein, the concentration needed be in the range of0.66-1.32mol/L. When the concentration was0.66mol/L, it could react with sample in1:1, and the determination should be finished in4h. When the concentration was1.32mol/L, it could react with sample in1:1, precipitated30min, and the determination should be finished in2h. After precipating, the sample could keep in low temperature for a short time.(2) The determination of acid souble peanut peptides’content Using amino acid analyzer to determine the free amino acid and hydrolyzed amino acid of the sample precipitated by TCA, the content of amino acid residues was the peptides content, the value was0.49g/100g.The measuring condition of Biuret method:kept the temperature stable; when TCA concentration was1.32mol/L, the sample would react with biuret reagent at least20min, when below the concentrate, at least30min; kept the reaction system pH stable; the optimum volume ratio between sample and biuret reagent was1:3. The acid souble peanut peptides’content determinated by the method was0.69g/100g, lower than the content of amino acid residues.According to the five kinds of calculation methods of nitrogen-to-peptide conversion factor, the value KA、KA’、KP, K、K’was separately4.90、4.70、3.55、4.22、4.13, and the relevant peptides’content was0.57g/100g、0.54g/100g、0.38g/100g、0.47g/100g、0.46g/100g. Comparing with the result value measured by Amino acid analyzer method, it was found that the content0.47g/100g calculated by value K was the closet. So the nitrogen-to-peptide conversion factor was4.22, which was far below the peanut protein conversion factor5.46.(3) The purification of peanut peptidesThe peanut protein hydrolysates needed to de-fat and used macroporous resin to purify. The degreasing process:each5.00g sample dried3h under60℃, then added30.00mL n-hexane, skimed6h under70℃. Macroporous resin purified conditions:at a speed of0.5mL/min, added50.00mL30.00mg/mL defated sample solution in the column filled by DA201-C macroporous resin, eluted by75%ethanol at a speed of2.0mL/min, and then collected the purification product under the250nm ultraviolet wavelength, and the by-product under278nm.(4) The determination of purified peanut peptides’contentMeasured by UV Spetrophotometry method and Fluorescence proble method, the content and the hydrophobic value of10.00mg/mL peanut peptides which collected under250nm and278nm was separately (28.13±0.13mg/mL,8146.00) and (9.47±0.086mg/mL,1328.70). It was suggested that high hydrophobicuty would lead the result value bigger.Measured by Biuret method, the content value of10.00mg/mL peanut peptides which collected under250nm and278nm was respectively0.68±0.052mg/mL and1.40±0.065mg/mL, much lower than the content value measured by UV Spetrophotometry method.(5) The characteristic of purified peanut peptidesThe scavenging hydroxyl free radical capability of peptides collected under250nm and278nm was separately35.97%and4.32%. So the peptides collected under250nm was got to purified by Sephadex G-25. According to the elution order, it could be known that the molecular weight size order was constituent1>constituent2>constituent3, thus their scavenging hydroxyl free radical capability were separately32.01%、42.09%and19.42%. It was suggested that the smaller molecular weight didn’t mean that the scavenging hydroxyl free radical capability was stronger.Trough comparing the scavenging hydroxyl free radical capability of peptides with different pI, it was found that pI3peptides had the strongest scavenging capacity.(6) The preparation of antioxidant peanut peptidesThe pI3peptides’ capability to scaveng the hydroxyl free radical was took as the index. Slected the orthogonal level of the enzyme concentration、enzymolysis time temperature and pH.The orthogonal experimental results showed that, the optimal experimental conditons:added90.00mg peanut compound protease to80.00mL peanut protein liquid, hydrolyzed2h under pH8.0and55℃.(7) The new peptides’ conversion factor and the antioxidant amino acids analysisThrough caculating, the new peptides’ conversion factor was4.11. By amino acid analysis, the content of acid souble peanut peptides’ antioxidant amino acids in hydrolyzate was28.20%, but the content of the specific peptides in the hydrolyzate was36.25%.