Study On Clostridium Butyricum SH-2 And Lactococcus Lactis LY-3 Combined Fermentation Of Soybean Meal

In order to address the growing problem of the abuse of fodder antibiotics,reduce the cost and risk of farmers.This study aimed to develop a new kind of"green"feed which is rich in probiotics,bioactive peptide and without anti nutritional factors.The soybean meal,as a by-product of the oil industry,is also one of the most readily available plant-derived protein.Through the research and optimization of the co-culture of Clostridium butyricum and Lactococcus lactis are used in biological fermentation soybean to improve its physicochemical properties and total viable count.Its final fermentation product can be directly used as forage to decrease even alternate the use of antibiotics,enhanced the immunity of animals,and augment the bioavailability of feed.Besides,through the study of nitrogen flushing,extrusion,pad tightening,tamping and other deoxidation pretreatment of these two bacteria in the solid,liquid culture successfully get rid of dependence on anaerobic equipment.This study not only provides data support and theoretical basis for the development and research of soybean meal mixed fermentation and non-anti-nutritional factor feed,but also lay the foundation of the fermentation processes of obligate anaerobic and facultative anaerobic bacteria under non-strict anaerobic conditions practice the foundation to open up new research ideas and directions.The research work of this paper mainly includes four aspects:Firstly,the cultivation conditions of these two bacteria,the conditions of co-culture and the fermentation process under non-strict anaerobic circumstances;Secondly optimization of the optimum medium composition and mixed medium composition of these two bacteria;Thirdly,single bacterium solid-state fermentation of two bacteria,the fermentation process of mixed bacteria or under non-strict anaerobic circumstances.Finally,analysis of the number of viable cells and other physicochemical properties analysis of multi-modulater prebiotic.The main approaches,results and conclusions of the study are as follows:The research work of this paper mainly includes four aspects:Firstly,the technology of C.Butyricum suspension culture under strict or non-strict anaerobic circumstances,liquid medium components and optimization of solid-state fermented soybean meal process.Secondly,the technology of L.lactis suspension culture under strict or non-strict anaerobic circumstances,liquid medium components and optimization of solid-state fermented soybean meal process.Thirdly,the optimum conditions of mixed bacterium co-culture,optimization of its medium components and study on mixed bacterium solid-state fermented soybean meal process.Finally,the analysis of physicochemical properties of fermented soybean meal under varying fermentation process.The main approaches,results and conclusions of the study are as follows:1.Through the analysis of single factor and orthogonal test conditions for C.Butyricum to determine and optimize the technology of its single suspension culture under strict anaerobic circumstances,liquid medium components and process of solid-state fermented soybean meal are as follows:add urea 3.0g,NH₄NO₃ 0.8g,K₂HPO₄ 0.2g and MnSO₄ 1.2g into each 1 litre Reinforced Medium of Clostridia.The initial pH value is 6.2,the time of continuous cultivation was 26 hours,the culture temperature was 37?,where the viable cell counts of the group,which was cultured in anaerobic incubator,were 5.03±0.81×10¹⁰CFU/g and the rate of maturing gemma was 90.06%,whereas the viable cell counts of the group,which was cultured in Hungate tubes with nitrogen flushing,were 6.09±0.031×10¹⁰CFU/g and the rate of maturing gemma was 91.27%.The optimal solid state fermentation conditions were as follows:the culture temperature was 37?,the volume of inoculation was10%?v/w?,60%water content of solid substrate,the time of continuous cultivation was 36 hours,under above conditions,the viable cell counts of C.Butyricum of solid-state fermented soybean meal were 4.99±0.078×10⁹CFU/g and the rate of maturing gemma was 89.66%±0.22%.The viable cell counts of the non-strict anaerobic group,which was cultured in Hungate tubes without nitrogen flushing,were 5.40±0.27×10¹⁰CFU/g and the rate of maturing gemma was 92.13%.Using 100mL centrifuge tube as a culture vessel,in addition to the extrusion,pad,compaction of oxygen removal operations the rest are all consistent with anaerobic fermentation process under solid state conditions,and then obtained the viable cell counts of C.Butyricum of solid-state fermented soybean meal were4.85±0.013×10⁹CFU/g and the rate of maturing gemma was 96.47±0.15%.2.Through the analysis of single factor and orthogonal test conditions for L.lactis to determine and optimize the technology of its single suspension culture under strict anaerobic circumstances,liquid medium components and process of solid-state fermented soybean meal are as follows:add CaCO₃ 1.0g,NH₄NO₃ 0.4g,KH₂PO₄0.5g,K₂HPO₄ 0.4g and urea 1.2g into each 1 litre MRS broth.The initial pH value is6.2,the time of continuous cultivation was 20 hours,the culture temperature was37?,and the viable cell counts of L.lactis were 2.15±0.027×10¹11 CFU/g.Using this suspension as seed and inoculating 10%?v/w?of it into soybean meal,which was content 60%water,under 37?24 hours continuous cultivation.Then obtained fermented soybean meal with the viable cell counts were 8.58±0.026×10¹⁰CFU/g3.The single factor tests were used to determine and optimize medium components and co-cultured conditions of C.Butyricum and L.lactis were as follows:Inoculating C.Butyricum first,then L.lactis 8 hours later,its inoculation ratio of C.Butyricum and L.lactis was 1:3,put this inoculated both two bacteria suspension into constant temperature incubator at 37?during 22 hours continuous cultivation,obtained the viable cell counts of C.Butyricum and L.lactis were 12.88×10¹00 CFU/g and 15.37×10¹00 CFU/g,total number were 28.25×10¹00 CFU/g.Solid-state fermented soybean meal process under strict anaerobic circumstances were as follows:Using co-cultured suspension to inoculate soybean meal at 37?during 24 hours continuous cultivation.Then obtained fermented soybean meal with the viable cell total counts were 5.96±0.62×10¹00 CFU/g.4.With different fermentation processes,there's different physicochemical properties of soybean meal.Single L.lactis fermented group's properties are as follows:crude protein 53.70%,acid soluble protein 34.58±0.36%,,urease activity0.087±0.0055U/g;Single C.Butyricum fermented group's physicochemical properties are as follows:crude protein 62.50%,acid soluble protein 45.37±0.35%,urease activity 0.045±0.0052U/g;Co-cultured group's are as follows:crude protein65.58%,acid soluble protein 60.44±0.39%,urease activity 0.0075±0.0041U/g;Control group?'s are as follows:crude protein 63.47%,acid soluble protein61.32±0.46%,urease activity 0.039±0.0015U/g;Control group?'s are as follows:crude protein 65.32%,acid soluble protein 60.68±0.45%,urease activity0.0068±0.00075U/g.The physicochemical properties and the viable cell total counts of these fermented soybean meal above are better than the industry standards,where the co-cultured were better than the single one.