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With the material of silkworm pupae, this thesis made researches in three aspects: withsilkworm pupae as the major components of culture medium, the solid and liquidfermentation of nattokinase（NK） production by Bacillus natto were studied respectively.The antihypertensive activity of silkworm pupae hydrolysate was preliminarily studied.The activity of NK was measured by fibrin plate method. With silkworm pupae as themajor components of solid fermentation culture medium, using JN-14stored in thelaboratory, both the components of solid fermentation culture medium and the fermentedconditions were optimized by study on the effect on solid fermentation of NK productionof steam temperature and steam time of silkworm pupae, moisture content, thickness ofculture medium, carbon source, additive, inoculation quantity, incubation temperature,incubation time. The fermentation products were freeze-dried. Components contained infreeze-dried powder and fermentation products were determined.The activity of NK was measured by fibrin plate method. With silkworm pupae as themajor components of liquid fermentation culture medium, using JN-14stored in thelaboratory, both the components of liquid fermentation culture medium and the fermentedconditions were optimized by study on the effect on liquid fermentation of NK productionof additive amount of silkworm pupae, carbon source, additive, initial pH, mediumvolume, inoculation quantity, shaking speed, incubation temperature, incubation time.The fermentation products were freeze-dried. Components contained in freeze-driedpowder and fermentation products were determined.Silkworm pupae were hydrolyzed by Alcalase, As1.398, Papain, Protemix, Neutraseand Flavourzyme respectively. The enzyme with the highest hydrolysis ability wasobtained by study on ACE inhibitory activities of silkworm pupae hydrolysates. ACEinhibitory activities of silkworm pupae hydrolysates hydrolyzed by complex enzymeswere studied. The antihypertensive activities of silkworm pupae hydrolysates were studiedthrough animal experiments.The experimental results indicate that:The optimized conditions of the solid fermentation of nattokinase（NK） production were confirmed: silkworm pupae steamed by121℃under high pressure for20min, moisturecontent20%, thickness of culture medium1cm, Gelcose0.6%, Geltin1%, inoculationquantity3%, incubation temperature37℃, incubation time24h. On this condition, theactivity of NK was2147.08IU/g. The total protein, soluble protein, total Gelcide andpolysaccharide of the fermentation products were:65.28%,16.85%,9.03%,4.02%. Theactivity of NK of the freeze-dried powder was:26563.80IU/g. The total protein, solubleprotein, total Gelcide and polysaccharide of the freeze-dried powder were:85.96%,27.12%,16.38%,7.14%.The optimized conditions of the liquid fermentation of nattokinase（NK） productionwere confirmed: silkworm pupae0.9%, Gelcose1%, Geltin0.5%, NaCl0.5%, K2HPO40.3%, KH2PO40.1%, MgSO40.05%, initial pH7.0, medium volume20ml, inoculationquantity2%, shaking speed150r/min, incubation temperature37℃, incubation time48h.On this condition, the activity of NK was1559.08IU/ml. The total protein, solubleprotein, total Gelcide and polysaccharide of the fermentation products were:32.08mg/mL,12.21mg/mL,5.23%,2.01%. The activity of NK of the freeze-dried powder was43270.13IU/g. The total protein, soluble protein, total Gelcide and polysaccharide of thefreeze-dried powder were:74.04%,24.26%,21.71%,14.43%.The enzyme with the highest hydrolysis ability was Alcalase. The optimum conditionswere confirmed: concentration of substrate15%, ratio of enzyme to substrate750U/gprotein, hydrolysis temperature55℃, pH7.0, hydrolysis time4h. On this condition, theACE inhibitory activity of silkworm pupae hydrolysates was89.50%.Compared to singleenzyme, the ACE inhibitory activities of silkworm pupae hydrolysates hydrolyzed bycomplex enzymes were lower. Animal experimental results indicate that: theantihypertensive activity of silkworm pupae hydrolysates was the highest, which washydrolyzed by Alcalase.
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