| Mil-brone bioactive peptides(BPs) consisted of 2-20 amino acid(aa) do positive effect on human health, such as antihypertensive, immunomodulation, antioxcidant,etc. The BPs encrypted in matrix proteins, which were usually released by digestion with protein ezymes or by fermentation with probiotics to to play a physiological function. Due to the complexity of the products obtained by the two methods mentioned above, it was very difficult to isolate and purify the single BP. So, it is very significant to fully evaluate the physiological activities of the peptide fractions in the fermented products with probiotics in vitro for measurement of the nutrition promotion of them.In this study, Lactobacillus delbrueckii subsp.bulgaricus LB340 LYO was applied to ferment the skimmed milk, and the fermentation process was opitimized. Moreover, the isolation, purification and phisicological activities in vitro were investigated, which was necessary to produce functional fermented dairy products containing BPs with LB340 LYO. The results obtained were listed as follows:(1), The peptone, the products of casein degisted with pepsin, was choosed as standard reference based on the peptide resources and molecular weight distribution, and the peptone-Folin phenol method was established to measure the concentration of the milk-brone peptides. The standard curve equation was obtained as follows: Y= 0.48158X, where Y is absorbance value at 215nm, X represents the concentration of small peptides mixture. Linear correlation coefficient r is 0.99936, illustrating that the results was well correlated and could be used for the determination of sample concentration.(2), The amount of produced peptides and the abundance of objectives the peptide fraction was used as an indicator, and the fermentation process was optimized from the three factors, such as the strain ratio, fermentation time and after-ripening process. Finally, the optimization conditions for producing peptide was determined as follows: a single LB 340 LYO lactobacillus strain, 42°C for 16 hours and without after-ripening process.(3), The small peptide fractions were isolated and purified from the fermentation product using the method combined of centrifugation, ultrafiltration, gel chromatography and reversed-phase high performance liquid chromatography (RP-HPLC). The concentration of the obtained peptide fraction with the molecular less than 1kDa was about 3.655μg/μL. The samples less than 1kDa were further isolated by gel chromatography, and six main peaks, named F1-F6, were detected, among which collected peptides in F2 fraction had the highest ACE inhibitory activity with IC50 of 67.79μg/μL. By the RP-HPLC for further separation of F2 peak fraction, four main peaks, named F2-1, F2-2, F2-3 and F2-4, were detected, and peptide samples in them were collected, respectively. After being lyophilized, the concentration of them was determined to 2μg/mL. All collected samples showed good ACE inhibitory activity with inhibition rate of 22.19%, 24.35%, 21.66% and 18.77%, respectivelu. The results of MTT described that the collected peptides from the fraction less than 1kDa sperated by ultrafiltrate had a positive stimulating effect on mice lymphocyte proliferation, and the results of further analysis showed that some fractions, such as F1, F3, F4 and F6, could stimulate the proliferation of mouse spleen lymphocytes with stimulation index (SI) were 0.014, 0.096, 0.646 and 0.729, respectively. However, the coolected samples from F5 peak fraction had obvious negative regulation effect, indicating that the sample had a certain anti-hypersensitivity effects probably. In addition, these samples collected according to molecular weight classified and isolated by ultrafiltration all presented the scavenging activity of the [dpph *] free radicals, in which the samples with molecular weight between 5kDa and 10kDa had the highest free radical scavenging rate of 65.98% and was significantly different from others(p<0.05). the results of FRAP analysis showed that these samples also had a certain degree of reducing power, but not present a obvious correlation to their antioxidant capacity, which may due to the differences in reaction mechanism of the two detection methods. |
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