Preparation Of ACE Inhibitory Peptide By Fermentation Of Cottonseed Protein

Cottonseed protein is an important and high quality plant protein resources due to its complete range of amino acids and rich in nature. However, only is a small amount of cottonseed protein used for the feed additive because the gossypol in cottonseed is harmful to the health of mankind. In recent years, the development of de-gossypol methods and the cultivation of non-toxic cotton provides a prospect for the applicant of cottonseed protein. The detoxified cottonseed protein can be used for food and feed additives, preparation of bioactive peptides like soybean protein. Bioactive peptides is a kind of peptides that have many specific bioactiveities such as antioxidation, antifatigue, reducing blood fat and resisting arteriosclerosis. Especially, the hypertension can be reduced by angiotensin-I-converting enzyme inhibitory (ACEI) peptides that derived from food proteins, which showed safe and effective for controlling hypertension. Though many kinds of plant proteins have been reported to produce the ACEI peptides, there is no report regarding to the ACEI through fermentation of cottonseed protein. In this thesis, ACEI peptide was produced by cottonseed protein fermentation, the conditions of fermentation were optimized with response surface methodology, and the ACEI peptide was separated and purified by ultrafiltration, gel chromatography and reversed-phase high performance liquid chromatography (RP-HPLC). The amino acid sequences of the ACEI peptide was identified by mass spectrum (MS). The main results are as follows:1. The bacterium strain employed for fermentation was screened according to the degree of hydrolysis and the ACE inhibitory ratio of the fermented liquid from cottonseed protein. Three strains with protease activity were lactobacilli isolated from the pickle juice, and the other strain Bacillus Subtilis NCT314 was preserved in our lab. Bacillus Subtilis NCT314 presented the highest hydrolysis degree and the strongest ACE inhibitory activity, which were 18.36% and 64.86%, respectively, it was selected as the fermentation strain in this research.2. Single factor experiments were designed to evaluate the yield of peptides and ACE inhibitory ratio under different time (0,6,12,20,22,24,26,30,36,48,60 h), temperature (27,30,32,35,37,40,42,47℃), pH (6.0,6.5,7.0,7.5,8.0) and inoculum size (1%,3%,5%,7%,9%). The result indicated that the fermented liquid presented the strongest ACE inhibitory activity under time 24 h, temperature 32 ℃, pH 7.0 and inoculum size 5%. Based on the single factor experiments, response surface methodology was applied to optimize fermentation condition. The result showed the optimum fermentation condition was time 22.6 h, temperature 32.08℃and pH 7.2. Under this condition, the ACE inhibitory ratio of fermented liquid reached to 78.06%.3. The ACEI peptide from fermented cottonseed protein was separated by ultrafiltration, gel column chromatography and two step RP-HPLC. The fermented liquid was separated into four fractions (U1, U2, U3 and U4) after ultrafiltrated through 30 K Da,10K Da and 5K Da membrane respectively. U4 (<5K Da) presented the highest ACE inhibitory activity (IC50=0.94 mg/mL) and was divided into three elution peaks (marked with S1, S2 and S3 respectively) by Sephadex G-25 gel chromatography. The component S2 exhibiting the strongest ACE inhibitory ability (IC50=0.19 mg/mL) was separated into five main elution peaks (marked as NO.1 to NO.5) by RP-HPLC. The NO.4 elution peak presented the highest ACE inhibitory activity and contained the single peptide. The amino acid sequence of the ACEI peptide from NO.4 was identified as LPGVY (Leu-Pro-Gly-Val-Tyr) and the molecular weight was 547.66.