| Spent mushroom substrate(SMS) is the abandoned culture medium that cultivate edible mushrooms,containing a certain amount of substances such as protein、 fat、mushroom silk、a variety of sugar、organic acids and active substances. Our country annually produce SMS about16million tons,and the SMSis discarded or burned. It not only cause the waste of resources but also give rise to the seriousenvironmental pollution. The moisture content of SMS is approximately60%,so the SMS is perishable. Tothis end,the bacillus coagulans were inoculated into the SMS Through the nutrient competition or thesecretion of lactic acid to reduce the pH,the number of mold is reduced and the storage period is extend.TheSMS is scattered and low yield,so it can be used to cultivate certain probiotics and make preparations forprobiotic agent. Inoculated with bacillus and added a little nutrients to the SMS, this study was carried out tomake preparations for probiotic agent and provide technical parameters and theoretical basis for therecycling of SMS. The test is divided into three main parts:Easier to rot and denatured，Test1was carried out to inoculate bacillus coagulans into the SMS. By theway of nutrient competition or the secretion of lactic acid to reduce the pH,the number of mold can bereduced. The experiment was due to inoculate bacillus coagulans(the number of bacillus6.6×107cfu/ml) indifferent dosages of0‰、2.5‰、5‰、7.5‰in the just crushing SMS(Diameter10cm,Height13cm),sealed and excluded all air,and placed the SMS in the incubator of40℃、50℃each. Finally we determinedthe number of molds the first day、the10th day、20th day and30th day.The results showed that with theextended storage time the mold gradually reduced, the number of mold in each treatment group wassignificantly lower than that in the control group(P<0.01), the number of mold under the conditions of50℃is lower than that in40℃,but the difference was not significant with the control group under50℃. Itshows that50℃surpasse40℃in the inhibition of mold,the best inoculum density of bacillus coagulans is5‰, the optimal temperature is50℃.Orthogonal experiment L16(45) was carried out in Test2to investigate the optimized conditions andinfluence factor that the SMS cultivate bacillus subtilis.In the process of growth,the influence ofpretreatment temperature (A),the nitrogen source(B)、 the initial pH(C)、 fermentation time(D) andinoculation size(E) on the number of spore in the fermentation products.It turned out that the optimalcondition of bacillus subtilis was pretreatment temperature35℃、, cottonseed meal3%、the initial pH=7.5、fermentation time48h and bacteria liquid inoculated10%. The optimal sequence was thenitrogen source> the initial pH> fermentation time> inoculation amount>fermentation temperature. In theoptimum conditions，the living bacteria number was up to8×10~9cfu/g.Test3use the same method to reaearch the optimized conditions and influence factor that the SMScultivate bacillus licheniformis. The optimal condition for cultivating bacillus licheniformis waspretreatment temperature40℃、, wheat bran4.5%、the initial pH=6.5、fermentation time72h andbacteria liquid inoculated10%. The optimal sequence was fermentation temperature> inoculation amount>fermentation time>the initial pH>the nitrogen source. In the optimum conditions，the living bacterianumber was up to9×10~9cfu/g.The optimum conditions for compound cultivating was researched by orthogonal experiment L4(23).Thefinal result is pretreatment temperature37℃, wheat bran4.5%、the initial pH=7.5、fermentation time72h、 BS5%(the number of Bacillus3.85×10~9cfu/ml) and BL5%((the number of Bacillus3.30×10~9cfu/ml).In the optimum conditions, the total living bacteria number was up to8.39×10~9cfu/g,andBS/BL=1/2. The fermented products was dried in45℃to make probiotics. The contents of fluorine、Aflatoxin B1and heavy metal such as As, Pb, Hg,Cd in the microbiological preparation were analyzed, andthese index is lower than the national feed additive sanitation standard. |
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