| Probiotics play an important role in health improving and disease preventing. The purpose of this research firstly is to screen the antihypertensive probiotic bacteria from a great deal of acid-producing bacteria, and identify them so as to lay a foundation for studying and developing probiotic products.(1)126 strains are received by improving MRS, NPNL, TPY medium from 9 samples,including 32 coccus and 94 bacilus.At last, 47 good performance of acid-producing bacteria are obtained .(2)The resistance to acid and bile salts are tested for all provided strains.The results are:7 strains can survive 6h in the MRS which is adjusted to pH2, 0.3% bile salts.The tolerance of strains C3 and W4 are stronger than the others.(3)Antibacterial experiments is done on pathogenic E.coli and S.enteritidis.The inhibiting circle of two strains of them exceed 15mm,7 strains of them are between 12-15mm,the remaining are all below 12mm.The circles of C3 and W4 are the biggest,they are respectively: 18.9±1.4 mm,17.9±1.6mm of C3; 17.9±1.6mm 16.6±1.2mm of W4.(4)The inhibitory activity of angiotensin-converting enzyme-I (ACE) for different acid-producing bacteria are studied. Six of them are higher than 50%. The activity of ACEI of C3 is the highest among them.(5)The physiological and biochemical examination and identification of the API series and sequence analysis on 16 SrDNA are studied about the bacteria which can endure high acid and bile salt and have high activity of ACEI. The results show that they are respecively Bifidobacterium.adolescentis and Enterococcus.faecium .(6)The characteristics of fermentation of Bifidobacterium C3 is studied. The relationships of biomass, protein hydrolysis index and the ACEI activity are inspected. With the increasing in biomass, the activity of ACEI of fermented milk also increase. The activity of ACEI of fermented milk is the biggest at 24h. With the extension of the fermentation time, the O-phthalaldehyde(OPA)index of strain C3 achieve the highest point about 0.43.(7)In order to detect the distribution of molecual weight of angiotensin I-converting enzyme inhibitory peptide ( ACEIP ) of strain C3, gel filtration chromatography is used. The Result shows that the distribution is between 385-1782Da, with the highest activity 84.07% at the eluting peak of 408Da.(8)Process of separation on ACEIP of C3 derived from fermented milk are studied. The wheys C3 are separated by 5KDa-ultrafiltration membrane in 30℃, 0.1MPa, without adjusting the pH value. The activity of the ACEI about the filtrate rises 19.47%. There are three fractions separated by SephadexG-25, among them, fraction II~III have the highest activity (p<0.01).Results are consistent about the results of SephadexG-25 and SephadexG-15.By ananlisysing SephadexG-15, we find that the molcular weight of ACEIP is bigger than captopril(217.29).(9)The effects on the stability about the activity of ACEI of fermented milk are studied. The activity of ACEI decrease by 11.21% and 19.28% after being stored about 24h and 48h at 4℃in refrigerator; It falls 1.7% when frozen at -20℃about 60 days. Under the conditions of heating treatment about 20 miniutes at the temperature of 90℃, the effect is negligible(P﹤0.05),it falls only 1.32% .( 10 ) This strain of microbial strains in China have the Stockpiling and Management Committee general microbial Preservation Center (CGMCC), for the Preservation of CGMCC No.1695, China's patent application, as follows: 200805011588.9, B.adolescentis grx066. |
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