| The animal husbandry in our country had made remarkable achievements with the continuous popularization of modern livestock breeding mode, but the large number of dead livestock during the feeding process was still inevitable. So it was of great significance on the ensurance of food safety and maintenance of green environment that to do a good job of livestock harmless treatment from the source by seeking a scientific and reasonable method for the disposal of animal carcasses.In addition, the rapid development of animal husbandry had put forward more requirements on the development and utilization of feed resources, especially for the increasing demand of protein resources while the protein feed supply in our country was already not adequate to the demand. Therefore, it had an important role in the alleviation of feed protein shortage by the way of improving the protein utilization ratio.Bacillus subtilis, as the important producer of protease, could degrade the organic matter like protein in the animals and realize the harmless, reduction and resource disposal of dead livestock. Additionally, the protein digestibility of fermented products could be improved through utilizing the protease secreted by the bacterium to prepare high protease activity fermented feed and to adjust the protein structure and degrade the toxic and harmful substances. In view of this, the protease production process and properties, the biological degradation ability to the protein in dead pigs and the improvement effect on the protein quality in soybean meal were investigated in this article by using Bacillus subtilis JNB001 as the starting strain. The research contents and results were as follows:1. Optimization the protease-producing conditions of Bacillus subtilis JNB001.The best protease-producing medium was obtained via single factor test, response surface analysis, etc. and they were:soluble starch 2.18 g, soybean meal 12 g, pig hair 7.06 g, K2HPO42.5 g, NaH2PO4 0.5 g, MgCl2 0.4g,CaCl2 0.31g,H201 L.Based on the fermentation medium mentioned above, the optimum fermentation conditions of the bacteria to produce protease was gained by using the single factor and orthogonal test, and they were:initial pH 6.75, inoculation volume of 7%, bottling volume of 35 mL in the 250 mL flask, cell age was 18 h, and cultivated for 36 h at 40 ℃. Under these conditions, the protease activity secreted by Bacillus subtilis JNB001 reached to 467.28 U/mL.2. Study on the protease properties of Bacillus subtilis JNB001.Through the research of enzymatic properties showed that the most suitable reaction pH was 8 and temperature at 45 ℃; The crude enzyme solution was relatively stable in the buffer solution of pH from 6.5 to 8.5; It had over 80% residual enzyme activity after insulating at 45℃ for 90 min and gradually lost the activity when the holding temperature increasing. The enzyme activity could be enhanced to a certain degree by the metal ions and chemical materials such as Ca2+, Mg2+, Na+, K+, DTT, Na2SO3, Tween-80, etc. and be inhibited by Zn2+, Fe2+,Cu2+, EDTA, SDS and other materials. The enzymatic reaction kinetic parameters Km and Vmax was 6.91 mg/mL and 47.75μg/min respectively.Animal protein substrates (pig hair, cowhide epilation) and plant protein substrates (soybean meal, microalgae meal) could be hydrolyzed at a certain level by the crude enzyme liquid. The chromatographic analysis showed that there were mainly small molecule protein and polypeptide materials whose relative molecular mass at 1～10 kDa in the enzymatic hydrolysis supernatant of animal protein while there were nearly no macromolecular protein but mostly molecular below 6000 Da materials containing lots of oligopeptides and amino acids ingredients that molecular under 1000 Da in the plant protein hydrolysis products.The mixed animal protein containing pig hair, pig nail and pig blood powder could be degradated by the protease secreted from strain JNB001 via solid state fermentation. And the acid soluble protein content in the fermentation products was influenced by the fermentation conditions such as the ratio of material to liquid, etc.3. Study on the degradation of animal protein (dead pigs).The fermentation medium of Bacillus subtilis JNB001 optimized before was expanded to culture through the fermentation tank for the preparation of microbial fermentation agents. The final fermentation conditions were determined via orthogonal test, etc. using protease activity as the index and they were:initial pH 6.75, liquid filling factor 0.7, inoculum volume 5%, ventilation volume 2 L/min with time of 30 h and temperature at 35℃. Under these optimized conditions, the bacteria number was 3.29×1011 CFU/mL and the protease activity was 662.89 U/mL in the fermentation liquid.The effects of different microbial fermentation agents adding amount to the degradation of dead pigs was studied and the result indicated that 4 kg/1000 kg of dead pigs was appropriate. Through the analysis of fermentation products showed that the degradation efficiency of the fermentation agents was good and there were nearly no high molecular protein while more than 80% materials in the water soluble protein of fermentation products was polypeptide that molecular weight less than 6 kDa. The sensory index and composition determination of the degradation products were in accordance with the technical requirements of organic fertilizer.4. The fermentation degradation of plant protein (soybean meal).By single factor and orthogonal test based on the protease activity to optimize the fementation conditions of soybean meal, and the optimal conditions were:material to liquid ratio 1:1, inoculation volume 6%, fementation temperature 37℃ and fementation time 66 h. The protease activity of fermented soybean meal was 235.43 U/g.After the fermentation of soybean meal, the crude protein and acid soluble protein content was 53.85% and 22.37% respectively; the pepsin digestion rate increased from 73.43% to 83.19%; the total amino acids contents and each amino acid composition all had a raise. In addition, the soybean antigen protein was degraded basically; the trypsin inhibitor decreased about 73.96% and the activity of urease was dropped from 0.1271 mg/g·min to 0.036 mg/g·min; the lipoxygenase activity had disappeared already. |
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