Abstract: 【Objective】 In order to provide a theoretical basis and technical support for efficient utilization of cottonseed protein, two methods were used to prepare cottonseed protein, then the isolated proteins were compared and digested in vitro by pepsin and trypsin, lastly the hydrolysates were detected and selected in antibacterial activity. 【Method】 In this paper, cottonseed protein isolate was prepared by the traditional method which is alkali extraction and acid precipitation and Osborne fractionation method. The protein content and molecular weight of the obtained proteins were determined by the Kjeldahl method and Tricine-SDS-PAGE respectively, microstructure of the proteins were examined by scanning electron microscopy. Amino acid composition of cottonseed proteins were analyzed by automatic amino acid analyzer (L-8900). And then proteins were digested by pepsin and trypsin in vitro in the process of digestination, the degree of hydrolysis (DH) of cottonseed protein isolate (CPI) and cottonseed globulin (CPG) hydrolysates were determined by ortho-phthalaldehyde (OPA), and then kanamycin (Kan) and protein solution without hydrolysis were used as control to determine the antibacterial activity of protein hydrolysates with Staphylococcus aureus and Escherichia coli bacteria. Meanwhile, the molecular weight distribution of the hydrolysis products were detected by high performance liquid chromatography (HPLC). 【Result】The extraction rate of CPI by the method of alkali extraction and acid precipitation was (70.52±2.40)% and its protein content reached (89.53±0.66)%. While the albumin, globulin, prolamin and glutelin were classified by Osborne fractionation method, and compared with the other three proteins, cottonseed globulin got the highest protein content that was (82.57±1.02)%. And there was no significant difference (P＞0.05) in protein extraction rate between the two preparation methods for the proteins extraction rate of Osborne fractionation method was (67.55±1.16)%. The Tricine-SDS-PAGE pattern showed that the subunits composition of CPG was more closely to CPI, compared to the other obtained proteins by Osborne fractionation method. The microstructures of the obtained proteins were different from each other. The structure of CPI showed a neat honeycomb structure, which was similar to glutenins’ microstructure, while the CPG showed a uneven granulated one. Amino acid composition analysis indicated that the amino acids of CPG were well maintained by Osborne method, compared to CPI. Cottonseed proteins were hydrolyzed by pepsin and trypsin simulated the digestive system in vitro. The hydrolysis degree of cottonseed protein hydrolysates was determined by microplate reader, and the result showed that the degree of hydrolysis of CPI hydrolysates was higher than CPG at the same amount of added enzyme, which indicated that CPI was easier to be hydrolysed than CPG. The molecular weight of cottonseed protein hydrolysates was measured by HPLC, and the result showed that the content of peptides (MW≤0.8 kDa) of CPI hydrolysates was significantly higher than CPG with the content of 70%-85% and 40%-60%, respectively. Results of antibacterial activity analysis showed that the antibacterial ability of these protein hydrolysates were strongest when the amount of pepsin-trypsin in digestion simulation system was 5 000-5 000 U (2-2), and the degree of hydrolysis of these protein hydrolysates were (24.72±1.07)% and (19.26±0.39)%, respectively, under such digested conditions, and the ability of both digestions to against E. coli were higher than S. aureus. CPI was higher than CPG in antibacterial activity, while both of the original protein without digestion treatment showed no antibacterial ability. The molecular weight determination results showed that the molecular weight of antibacterial activity peptides of cottonseed protein isolate should be in 0.57-0.75 kDa while cottonseed globulins’ were in 0.66-0.78 kDa.【Conclusion】 There was no significant difference between the two methods in the extracted yield of protein (P＞0.05), proteins classified by Osborne fractionation method showed an obvious difference in subunit composition, but the CPG and CPI showed a similarity. CPI is easier to be digested compared to the CPG and both of these two proteins’ hydrolysates showed an antibacterial activity, the hydrolysates of CPI is higher than CPGs’ in antibacterial activity.

Key words: cottonseed protein isolate, cottonseed globulin, in vitro digestion, antibacterial peptide, molecular weight distribution