Enzymatic Preparation And Antioxidant Activity Of Wheat Germ Protein Hydrolysates

As one of the main cereal crops, the annual output of wheat has been beyond 100,000,000 tons in China. The wheat germ, an kind of important by-product of flour milling, is produced with annual yield about 2,000,000 tons. It is the life source and the most nutritious part of wheat kernel, which contains about 30% protein and is a high quality source of vegetable protein. Some competent peptide fragments with antioxidant activity will be obtained through specific enzymatic hydrolysis. It is shown that chronic diseases and senility were related to the imbalance of free radicals in our body. Excessive free radicals could cause the organism being oxidized damage. If the damage could not be repaired in time and be accumulated to certain degree, it would result in some diseases. Some natural antioxidants from food were helpful to eliminate excessive radicals and keep the balance of free radicals. The research on investigating and developing functional food and/or food ingredients with anti-aging and antioxidant activity from wheat germ protein hydrolysates can not only provide the theoretical basis for further utilization of agricultural byproducts such as wheat germ rich in protein, but also achieve comprehensive utilization and improve added value of wheat.In this dissertation, serial researches have been carried out concerning the processing of wheat germ protein by micronization and alkali extraction, the preparation and the action mechanism of antioxidant hydrolysates from wheat germ proteins. The results are as follows:Defatted wheat germ was micronized useing both hammer mill and colloid mill to increase the extraction rate of protein, followed by alkali extraction and ultrafiltration, respectively. A quadratic mathematical model was statistically constructed by using of Box-Behnken central composite design (CCD) and response surface methodology (RSM). Under the optimized processing parameters of 23μm particle sizes, pH 9.5 and 51℃, a maximum extraction rate, 68.6%, was achieved, which is 30.63% higher than that of non- micronized wheat germ (passing through 80 mesh sieve).The SDS-PAGE result showed that the molecular mass of wheat germ proteins was in the range of 11.6 kDa～93.8 kDa. The results of function determined indicated that in many ways such as the oil absorption, emulsifying activity and foaming activity, wheat germ protein was superior to those of soybean protein isolate (SPI), but it was not as good as SPI on the water absorption, emulsifying stability and foaming stability. In addition, the solubility of wheat germ protein was very poor when pH was at the range of 4 and 5.Five kinds of proteases were selected by comparing the degree of hydrolysis (DH) and the scavenging activity of wheat germ protein hydrolysates against DPPH radical. Alcalase showed the optimal protease activity. The pretreatment, shearing and emulsifying for 5 min, can significantly improve hydrolysis degree and the yield of peptides, up to 29.85% and 12.53%, respectivly. After pretreated by shearing and emulsifying at 22000r/min for 5min, the optimized hydrolysing conditions were determined through single factor tests, Alcalase dosage is [E]/[S] 4000U/g, substrate concentration [S]4%, pH value 8.5, temperature 55?, time 240min. Under these conditions, DH was 18.61±0.23%, TCA-PSI, 71.02±0.45%, PCL, 5.37 and average molecular weight, 555.3.To better understand the antioxidant activity of peptides with different molecular weight, wheat germ protein hydrolysates were ultrafiltrated consecutively by upgrade cellulose FP membrane with the molecular cut off of 5 kDa, 3 kDa and 1 kkDa. The optimum conditions of the first-degree ultrafiltration were: pressure 0.10 MPa, material concentration 2%～2.5% and temperature 35℃. Graded hydrolysates were desalted by cation ion- exchange resins and anion ion- exchange resins. By comparing the desalting rate and peptide recovery rate, the optimum hydrolyte flow rate was 10 CV/h. The antioxidant experiment results showed that refined wheat germ peptides possess of noticeable antioxidant activity, except for peptides I with the largest relative molecular mass.The antioxidant effect of 3 wheat germ peptides evaluated using five different chemical models showed that at the concentration of 1.7 mg.mL^-1, peptides IV had the strongest reducing power, the same reducing power to that of 1.0 mg.mL^-1 GSH. The scavenging activity of 3 peptides against DPPH·were all dose independent. Their IC₅₀ were: peptides IV 0.578 mg.mL^-1, peptides HI 0.769 mg.mL^, peptidesâ…¡1.661 mg.mL^-1, and the IC₅₀ of the control GSH was 0.28 mg.mL^-1. In the system of pyrogallic acid, peptidesâ…£had no significantly scavenging activity against O₂^-·with GSH. At the same concentration, the scavenging ability against·OH and the chelating activity on Fe²⁺ of peptidesâ…¢were stronger than that of peptidesâ…¡, which indicated that the action mechanisms of peptidesâ…¢and peptidesâ…¡were somewhat different. In powder food, peptides III and peptides II with good antioxidant activity, comparing with that of 1% BHT and 1% GSH, were superior to the former and inferior to the latter when the concentration was 5% lipid content.Taken isolated rat heart mitochondria as targets, an oxidative damage pattern with high performance induced by Ca²⁺ and Fe²⁺-Vc was established on a subcellsular level. Wheat germ peptides could inhibit oxidative stress induced increase of MDA formation and carbonyl content of mitochondria as well as the lose of ATPase activity of mitochondria. The swelling of mitochondria and the reduction of membrane potential of mitochondria induced by Ca²⁺ and Fe²⁺-Vc were also prevented significantly by wheat germ peptides. The transmission electron micrographs of mitochondria showed that wheat germ peptides could release the injury to mitochondria induced by oxidative stress and help to maintain the integrity structure of mitochondria.The two peptides, SEC-5 and RP-2, showing strong antioxidant activity, were isolated using consecutive chromatographic methods including strong anion ion-exchange chromatography, gel-filtration chromatography and RP-HPLC. The molecular mass and the amino acid sequence of the two purified peptides were determined using electrospray ionization-mass spectrometry. For SEC-5, the molecular mass was 296.2, the amino acid sequence was Phe-Met; for RP-2, they were 246.1 and Leu-Asp, repectively.The amino acid composition and/or sequence of the two peptides agreed with the reported characteristics of antioxidant peptides.