Preparation Of ACE-inhibitory Peptides On Enzymatic From Wheat Germ Under Ultrasonic Pretreatment

Wheat germ protein （WGP） is a by-product of the flour milling industry. This paper intends to enhance WGP vale by preparing ACE-inhibitory peptides （ACEIP） under ultrasonic-assisted enzymatic hydrolysis. The impactions of ultrasonic pretreatment on structure of WGP were studied by the methods of solubility, ultraviolet spectrometry, fluorescence spectrometry and free sulfhydryl, disulfide bond contents. They were related to release high activity of ACEIP. The relationship between structure of ultrasound-pretreated WGP and ACE-inhibitory activity of its hydrolysates was invested by FT-IR curve fitting. Thermodynamic parameters of WGP changed with different ultrasonic treatments. It proved that ultrasonic could promote enzymatic hydrolysis. The optimum hydrolysis condition for probe-type ultrasonic depends on the response surface methodology, and the enzyme membrane coupling system was applied to prepare ACEIP; the ACEIP were tested in vitro and in vivo to determinate effectives. The main achievements are summarized as follows:（1） In general, the solubility of WGP was reduced by probe-type and plate-type ultrasonic. It can be concluded that there was no directly relationship between solubility and digestion.（2） Changes of UV and fluorescence emission spectra indicated that the structure of WPG changed under ultrasonic treatment. The phenomena of fluorescence quenching of ultrasonic indicated the peptide chains of WPG extended. ANS fluorescence spectra of WPG changes proved protein internal hydrophobic amino acids exposed under ultrasonic treatment. The contents of free disulfide reduction and disulfuram increase by ultrasonic treatment. It was conducive to the internal hydrophobic groups contact with the disulfide bones, and the hydrolysate ACE inhibition rest with the change of content.（3） Curve fitting method of FT-IR was used to the determination of amide I changes for quantitative analysis of secondary structure of WGP. Results showed that there was negative correlation relationship between a-helical content and hydrolysate activity, and ultrasonic treatment resulted decrease of relative content ofβ-sheet and increase ofβ-turn, which means thatβ-sheet has been partially transformed into a more stretch ofβ-turn, so the protein is conducive to digestion.（4） The degree of hydrolysates of WGP was not changed under the probe-type ultrasonic and flat-type ultrasonic pretreatment, but hydrolysates of ACE inhibition were different. Compared to the control group, ACE inhibition of the hydrolysate increased by 1.95 times after 10min treatment with probe-type ultrasonic of 600W, but the results of flat-type ultrasonic were complex. In the pretreatment of single flat-type ultrasonic, fixed frequency could increase hydrolysate inhibition rate at the lower frequencies （<30kHz）. However, the sweeping ultrasonic made WPG hydrolysate release more ACE inhibitory peptides at the higher frequency （≥40kHz）. In the pretreatment of double flat-type ultrasonic, the AEC inhibition of hydrolysate was 65.5%when the two-plate frequency were fixed on 24 kHz and 68 kHz, which was close to the effect of probe-type ultrasonic. However, ACE inhibitions were less than control group when the frequency of double-plates were sweeping.（5） Analyzing results of thermodynamic parameters for WPG showed that the values of Ea, AH, AS and AG could be decreased after ultrasonic treatment. It indicated the digestion of WPG become more facility and benefit to release more activity ACEIP. In addition, the probe-type was more easily to change the thermodynamic parameters and got higher ACE inhibition hydrolysate.（6） Based on the single factor research, response surface was employed for experimental methods. WGP hydrolysate after probe-type ultrasonic treatment was optimized. The optimal conditions were as follows:substrate concentration was 1,2%, [E/S] was 2.2%and hydrolysis time was 88min. The value of IC₅₀ was 0.72mg/ml.（7） ACEIP of WGP was prepared using 5kDa membrane device for enzyme membrane coupling. By continuous feeding test, the conversion rate of substrate proteins of ACE inhibitory peptide prepared enzyme membrane reactor was 55.3% and with 36.17%increased compared with tradition methods, IC₅₀ value was 0.50mg/ml, and the working time extended two times.（8） The performance showed that antihypertensive peptides obtained from WGP had good acidic, alkaline, and thermal stability and ability of anti-intestinal digestion, and had clear decreases in systolic blood pressure after being stomach tube to spontaneously hypertensive rats. But high temperature and alkali conditions should be avoided in the process of production and storage.