Antioxidant Peptide And ACE Inhibitory Peptide Derived From Cottonseed Protein

Cotton is not only the most important fiber crop in the world but also one of the best potential sources for plant proteins after soybean. Cottonseed protein is generally regarded as potential sources of nutrients for human and animals due to their good balance in essential amino acids composition, and low level of antinutritional factors. It has also been a subject for numerous investigations. To our knowledge, however, few investigations have been done on the antioxidant and ACE inhibitory activities of cottonseed protein hydrolysate (CPH). Therefore, the antioxidant and ACE inhibitory activities of cottonseed protein hydrolysates were investigated in this study. The antioxidant peptide and ACE inhibitory peptide were separated and analyzed by using ultrafiltration, gel filtration chromatography, RP-HPLC and MALDI-TOF-TOF. The main results are as follows.1. The cottonseed protein isolates was prepared according to the alkaline method, and the extraction conditions were optimized by orthogonal test. The molecular weight, physical properties of cottonseed protein was investigated. Cottonseed protein has better function, including water absorption, oil absorption, emulsifying properties and emulsion stability. So cottonseed protein can be applied to the food industry.2. Six proteolytic enzymes, including Alcalase, Flavourzyme, Trypsin, Neutrase, Papain and Pepsin, were employed to hydrolyze cottonseed protein to produce the hydrolysates with antioxidant activity and ACE inhibitory activity. The result indicated that the cottonseed protein hydrolysate (CPH) produced by Neutrase presented stronger scavenging activity against free radicals. To achieve the maximum hydrolysis degree in the enzymolysis of cottonseed, response surface methodology was applied to determine the optimum conditions based on the single factor test. The optimum conditions were:enzymolysis temperature,39.5℃; time,5.5h; pH,7.5. Under these conditions, the hydrolysis degree reached 38.09%.3. Cottonseed protein hydrolysate (CPH) was prepared by Neutrase and was fractionated into four fractions (Fra.Ⅰ, Fra.Ⅱ, Fra.Ⅲ, and Fra.Ⅳ) by gel filtration on Sephadex G-25, the antioxidant potential of the CPH fractions and the CPH were investigated by using the classical methods, including the inhibiting effect on the autoxidation of linoleic acid, the scavenging ability to DPPH, hydroxyl radical, and superoxide radical. The results revealed that fractionⅢhas the highest antioxidant and free radicals scavenging activities. The peptide purified by using consecutive chromatographic methods had a molecular mass of 857.4 Da and amino acid sequence was identified as HDDAPRF (His-Asp-Asp-Ala-Pro-Arg-Phe) by MALDI-TOF-TOF analysis.4. Six proteolytic enzymes, including Alcalase, Flavourzyme, Trypsin, Neutrase, Papain and Pepsin, were employed to hydrolyze cottonseed protein to produce the hydrolysates of ACE inhibitory activity. Papain was the best one among six enzymes according to the ACE inhibitory activity. On the basis of single factor test, the optimum conditions for Papain reaction were determined by response surface methodology. Temperature 38.9℃, E/S ratio 1.04%, and pH 7.5 were found to be the optimal conditions to obtain high ACE inhibitory activity close to 88.15% and DH of the cottonseed protein was 25.69%. The result indicated that the cottonseed protein hydrolysate (CPH) produced by Papain had the highest ACE inhibitory activity (85.61%), and the CPH was separated into four ranges of molecular weight (UF-Ⅰ,> 30 kDa; UF-Ⅱ,30-10 kDa; UF-Ⅲ,10-5 kDa; UF-Ⅳ,< 5 kDa) by using an ultrafiltration (UF) membrane bioreactor system. Among them, UF-Ⅳshowed the highest ACE inhibitory activity (IC50=0.792 mg/mL). UF-Ⅳwas further fractionated with Sephadex G-25 gel filtration chromatography into four fractions (Fra.Ⅰ, Fra.Ⅱ, Fra.Ⅲand Fra.Ⅳ) that were composed of peptides of >2.43 kDa,2.43～0.82 kDa, 0.82 -0.35 kDa, and <0.35 kDa, respectively. Fra.Ⅱexhibited the strongest ACE inhibitory ability (IC50=0.159 mg/mL) with the yield of 41.63%. FPAIGMK (Phe-Pro-Ala-Ile-Gly-Met-Lys) had a molecular mass of 763.4 Da was separated by two step RP-HPLC from Fra.Ⅱ, and was identified by MALDI-TOF-TOF spectrometry.5. A modified spectrophotometric assay was developed for determination of angiotensinⅠ-converting enzyme (ACE) inhibitory activity of peptides derived from plant protein. This method relies on classical paper chromatography determination of hippuric acid (HA) content in the urine, which was based on the specific colorimetric reaction of HA with 4-(dimethylamino) benzaldehyde in the presence of pyridine and acetic anhydride. In this study, the maximum absorbance of HA was measured at 459 nm according to the modified method. The absorbance is reduced when the angiotensinⅠ-converting enzyme inhibitor is added, and the change of color in the reaction solution was measured by spectrophotometer for analyzing the ACE inhibitory activity. The method was more sensitive, more accurate and reproducible than the classical method, and it could be used for the screening of ACE inhibitory peptides derived from food proteins.