Preliminary Study On Solid Fermentation Technology Of Compound Probiotics

Probiotics can adjust the animal intestinal microecological balance,improve the body's immune ability and promote animal growth,and with the advantages of no adverse effect,no residues and noresistance,so probiotics has become a hot spotin the world.This paper try to provide technic support for production and application of compound probiotics by strains screening of compound probiotics,optimization of fermentation process of compound probiotics,security verification of strains and probiotics.To explore a more suitable production process for long-term preservation of strains of probiotics by studying on the changes in cell concentration of bacillus,lactic acid bacteria and yeastcontent in three different forms(solid vacuum,solid non-vacuum and liquid)of commercially availableprobiotics under different storage temperature.We tried to find a more suitablestorage temperature for long-term preservation of strains of probiotics by studying on the diversity of the changes of living bacteria number under different storage temperature.The results showed that the best storage temperature was 4?,followed by 25?,but the difference between the two temperatures was not sharp,and the cell concentration of these microorganisms declined significantly under37?storage.By synthesizing the result of bacteria count and the cost of processing,solid fermentation non-vacuum-packed was better for fermentation and processingtechnology of compound probiotics.It was not only cost savings but also better for keeping the stability of product efficacy for a long time.Cryogenic treatment was not necessary in the transportation and store of compound probiotics,but should be away fromhot environment and stored in the shade as possible.Based on physiological,biochemical identification and molecular biological identi-fication of partial unknown strains,some strains were picked outfor antagonistic experiments with other known strains.Then took the strains without antagonism for stress resistanceexperiments.These experiments included bile saltsresistance test, acidproof test,digestive enzymeresistance and heat resistance.After comprehensive comparison of the results,Bacillus subtilis strain R5,Lactobacillus casei M9 and Candida utilis Y5 were screened for development of compound probiotics.Multiple linear regression technique was used to optimize the fermentation medium of compound probiotics.In the first optimization step,Plackett-Burman(P-B)design was used to evaluate the effects of many related factors on the cell concentration of probioticsfermentation.After regression analysis,soluble starch,(NH4)2SO4 and ZnSO4 were found to be important for the cell concentration of three strains.Second,the path of steepest ascent experimentwas confirm to approach the optimal region of these factorsaccordingto the results of the P-B experiment.Finally,a Box-Behnken design and response surface analysis were used to optimize the three key factors,to find out the optimal concentration levels of these factors.The optimal fermentation medium was as follows:wheat bran 21 g,bean pulp 3g,powder of corn husks 6g,soluble starch 1.3g,CaCO3 0.15g,(NH4)2SO4 0.24g,ZnSO4 0.05g,KH2PO4 0.03g,water 24mL.Single factor experiment was used to optimize the fermentation conditions of compound probiotics.The results indicated that the optimized cultivation conditions were as follows:the ratio of material and water 1:0.8,fermentation temperature 34?,inoculation 16h delayed of Bacillus subtilis,inoculum levels of three strains are Bacillus subtilis5%,Lactobacillus casei 7.5%,Candida utilis5%,cultivation time 88h.To examine the safety of these three strains(Bacillus sub til is,Lactobacillus casei,Candida utilis)and the probiotics with mice feeding experiment.The results showed that these strains were non-toxic for mice with the cell concentration of 2×108CFU/mLon the apparent level.The compound probiotics were non-toxic for mice on the apparent level,cellular level and blood routine index.