| Acremonium terricola is a cordyceps fungus isolated from Cordyceps gunnii.Its mycelium contains a variety of active substances such as entomolic acid,cordycepin,cordyceps polysaccharide,ergosterol,amino acids and alkaloids.Therefore,Acremonium terricola culture can play a similar role to antibiotics in feed additive.Camellia oleifera meal is a by-product of Camellia oleifera seed after extracting oil and contains a variety of nutrients and bioactive substances for feed.However,there are still few reports on the use of Camellia oleifera meal fermented by Acremonium terricola as feed.This study was aim to prepare antibiotic free feed by solid-state fermentation of Camellia oleifera meal by Acremonium terricola.The antimicrobial activity and mechanism of tea saponin against Acremonium terricola were researched,and the tolerance concentration of Acremonium terricola to tea saponin was detected and used to guide the desaponification of Camellia oleifera meal.Finally,the feasibility analysis of feed preparation was carried out with Acremonium terricola as fermentation strain and desoapated Camellia oleifera meal,rice bran and wheat bran complex as fermentation substrate.The main results were as follows:(1)We analyzed the effect of tea saponin on the growth of Acremonium terricola,and found that the minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of tea saponin to Acremonium terricola were 1.0%and 1.5%,respectively.When Acremonium terricola was cultured under 1 MIC tea saponin,the cell wall structure of mycelium and spore cells was loose,the boundary of cell membrane of mycelium and spore cells was blurred,the structure of internal organelles of mycelium and spore cells was significantly damaged,and the relative conductivity of Acremonium terricola was increased by 3.63 times.Moreover,the activities of catalase(CAT),glutathione peroxidase(GSH-Px)and superoxide dismutase(T-SOD)decreased by 50%,58.85%and 64.00%,respectively;the activities of mitochondrial complex enzymes Ⅰ,Ⅱ,Ⅲ and Ⅳ decreased by 67.80%,42.22%,83.55%,and 24.44%,respectively;the ATP content decreased by 62.10%and reactive oxygen species(ROS)content increased by 1.52 times.However,the content of cordycepic acid was not affected at low concentration of tea saponin(in the range of 1/8 MIC to 1/2 MIC)compared with control group without tea saponin treatment,and the contents of ergosterol and chitin were also increased by 1.53 times and 1.42 times,respectively.Therefore,the tolerance concentration of Acremonium terricola to tea saponin was 1.0%,and the optimal concentration of tea saponin in fermentation substrate was between 1/8 MIC and 1/2 MIC.(2)In order to increase the content of Camellia oleifera meal in the compound substrate,the Camellia oleifera meal was de-soaped and the optimal de-soap process was obtained.The best de-soap process for Camellia oleifera meal was as follows:the extraction time was 3 h,the extraction temperature was 80℃,the ratio of solid to liquid was 1:12,the concentration of ethanol was 60%,and the extraction times were 3 times.Under this de-soap process,the tea saponin content in Camellia oleifera meal was decreased from 21.18%to 2.60%,the tannin content was decreased from 7.00%to 1.46%,and the caffeine content was decreased from 0.03%to undetectable.Furthermore,the content of anti-nutritional factors in Camellia oleifera meal was greatly reduced.We also analyzed the fermentation characteristics of Acremonium terricola using desoapated Camellia oleifera meal,rice bran or wheat bran as the only substrate.The Acremonium terricola was grown slowly in desoapated Camellia oleifera meal,and the nutrient content in the fermentation product only increased gradually after 9～15 d.However,when the Acremonium terricola was grown in a single substrate of rice bran or wheat bran,the mycelium could grow full within 2～3 d,and the nutrient content in fermentation product was also significantly increased during this period.(3)The contents of total protein,water-soluble protein,total sugar,reducing sugar,cordyceps acid and ergosterol in fermentation products were taken as optimization indexes,and the optimal culture conditions of Acremonium terricola were obtained through single factor experiment.It was concluded that the best culture conditions as follows:the fermentation time was 9 d,the inoculation amount was ranged from 5%to 10%,the content of desoapated Camellia oleifera meal was 15%,the ratio of rice bran to wheat bran was 2:1(that is,56.67%rice bran and 28.33%wheat bran),the content of urea was 1.0%,and the content of magnesium sulfate was 0.3%.Uniform test was carried out according to U10(56)design table.SPSS software was used to conduct multiple stepwise linear regression analysis on the test data,and 6 regression equations were obtained about the content of total protein,water-soluble protein,total sugar,reducing sugar,cordyceps acid and ergosterol in the product.Excel software was used to plan and solve each regression equation.It was predicted that the content of water-soluble protein and ergosterol could reach the maximum value,which was 48.87mg/g and 1.67mg/g,respectively,when the fermentation time was 7d,the inoculation amount was 10%,the content of desoapated Camellia oleifera meal was 15%,the ratio of rice bran to wheat bran was 3:1(that is,56.67%rice bran and 28.33%wheat bran),the urea content was 0.5%and the magnesium sulfate content was 0.3%.At this time,the contents of total protein,total sugar,reducing sugar and cordycepic acid were 112.91 mg/g,175.20 mg/g,41.00 mg/g and 58.10 mg/g,respectively.These results showed that the use of Acremonium terricola to prepare feed additive under above fermentation conditions could significantly improve the feed value of Camellia oleifera meal. |
