Study On Isolation Of Protease-producing Microorganism And Production Of High-quality Protein Materials By Solid-state Fermentation

Now there is severely short protein resources in the world, especially in China.With the development of animal husbandry and improvement of living standard, the resources of protein feedstuffs are far beyond the requirement of feed industry. The discovery of new unconventional protein feed resources has become important in our country. The production of high-quality protein stuffs from the low-quality unconventional protein resources by microorganism fermentation is the main research direction all over the world. In this study, the low-quality protein stuffs such as cottonseed meal, blood meal and feather meal were fermented by a strain isolated from cow rumen with production of protease and other enzymes, and a nutrient metabolism experiment of broilers was used to determine the nutrient values of the fermented protein materials. Four experiments were conducted in this study.Experiment 1. Isolation of the microorganisms for protein fermentation: Six strains producing large transparent circle in the casein plate medium were isolated from the rumen fluid of a cow, which were used to ferment unconventional protein mateials. At last, strain B1 was found to produce high activity of protease, amylase and cellulase for fermenting unconventional protein resources, it was identified as Aspergillus oryzae. After it was used to ferment the unconventional protein materials, the soluble protein content and the nitrogen of free amino acids were increased by 66.24% and 2.89 times, respectively.Experiment 2. Study on optimum solid-fermentation standards for protease production and enzyme characteristics of Aspergillus oryzae: Orthogonal design and one-way ANOVA test were applied to select the optimum fermentation conditions of Aspergillus oryzae for improving the capacity of producing enzyme. The results indicated that the optimum compositions of culture medium were as follows: 8 g of wheat bran and 2 g of soybean added with 0.5% K2HPO4 and 1%CaCl2, followed by 40% moisture; and the optimal medium for production of enzyme was: 40% moisture, pH 6, 4% inoculation amount, 10g/250ml incubation volume and 48 h incubation. The optimum temperature and pH were 55℃and 7.2, respectively; the enzyme was rather stable at 40℃～50℃and at pH3.0～7.0. The enzyme was activitied by Mg²⁺, K⁺, Na⁺, Ca²⁺, Mn²⁺, while inhibited by Zn²⁺, Cu²⁺, Fe²⁺, among which Cu²⁺ had the strongest inhibition on enzyme activity. Under the optimum culture conditions, protease, amylase and cellulase activity were 4211.57 U/g, 3146.70 U/g, 7278.14 U/g, respectively.Experiment 3. Study on the optimum solid-state fermentation for production of high-quality protein materials by Aspergillus oryzae: The orthogonal experiment and single factor analysis of variance method were used to select the optimal fermentation nutrient medium and fermentation conditions. The results showed that the best medium formula of raw materials were 30% blood meal, 26% cottonseed meal, 4% feather meal, 35% wheat bran, 5% corn flour; the best fermentation conditions were initial pH 6, 40% moisture, 2% fungal culture, and 60 h fermentation. Under the best conditions of fermentation, the soluble protein content, amino nitrogen content, crude protein content, free amino acids in the fermented products were 173.72 mg/g, 34.75 mg/g, 48.97%, 100.945 mg/g, which were increased by 82.68%, 379.97%, 12.91%, 570%, compared with the unfermented product, respectively (P<0.05). The activity of protease, amylase and cellulase in the fermented products were 2341.83 U/g, 36780.8 U/g, 6835.43 U/g, respectively. It can be concluded that the unconventional protein resources can be significantly improved by using the method of solid-state fermentation of Aspergillus oryzae. The experiment result will provide an important theoretical basis for solving current shortage of protein feed resources.Experiment 4. Effect of fermentation product on apparent digestibility of nutrients, gut microbes and digestive enzyme activity of broilers: Twenty five 7-weeks-old AA broilers were divided randomly into 5 groups, 5 for each group. The experimental diets included the control group (the normal diet), fermented protein groups (90%, 60%, 30% soybean meals in the diets of treatment I, II, III were relaced by the fermented protein stuffs, respectively), unfermented protein group (60% soybean meals in the diets of treatment IV was relaced by the unfermented protein stuffs). The results showed that apparent digestibilities of nutrients (CP, EE, Ca, P) and the number of lactic acid bacteria in intestine in treatment II, III were not significant different with that in the control (P>0.05); the number of E.coli in the intestine were significantly lower, while the number of Aspergillus oryzae and the digestive enzyme activity in the intestine were significantly incerased in treatment II and III, compared with the control group(P<0.05). Apparent digestibilities of nutrients (CP, Ca, P) , the number of lactic acid bacteria and Aspergillus oryzae in intestine and the digestive enzyme activity were significantly incerased in fermented protein group, compared with the unfermented protein group(P<0.05); and the number of E.coli in intestine were significantly lower in fermented protein group than that in the unfermented protein group (treatment IV)(P<0.05).