| Agricultural probiotics played a significant role in improving animal microecological balance, preventing disease,promoting growth and purifying aquaculture environment.At present,agents preparations are used as complex and expensive special ingredients culture medium.Our study selected yeast, Lactobacillus acidophilus,Lactobacillus bulgaricus,Streptococcus thermophilus , Bacillus subtilis and Aspergillu as research target and used fresh apple pomace with other substances as material,which is try to through microbial fermentation to increase the protein content,while cultured a high concentration of probiotics bacteria,so that the final products have dual function of feed additives and probiotics products.This method is low cost,easy to operate and need less equipment.The results were as follows:Lactic acid bacteria alone fermentation: After cultivating LAB in defferent medium,they grew best Urea medium and whey powder medium,next were sucrose medium and brown sugar medium,so selection Urea and whey powder as foreign substances, setting added to the pomace medium with different concentrations, inoculating LB, ST and LA to sterilized medium, the inoculation volume is 5%,cultivated in 37 ~ 41℃,the pH value and viable cell number growth,Cell morphology and fermentation broth pH value changes during the fermentation time were detected every day,the results showed: LA growth condition was best in 1% urea supplemented with 1.5% whey powder volume , the viable count reached the highest,was 3.81×10~8,the curd pH was 3.75; Added the urea of 1.5% and 2.5% of whey powder, the growth condition of LB was best, After cultivating 4d,the total number of viable cells reached 1.41×10~8, pH was 4.40;ST had the best growth in the urea was 2% and whey powde of 2.5%,bacteria form long chains, after 4d, reached to the maximum of 3.88×10~8 , pH was 5.23.lactic acid bacteria mixture fermentation:selecting five different inoculation ratio: 1:1:1,1:1:2,1:2:1,1:2:2,2:2:1 (LA, LB, ST),three kinds of culture temperature: 37℃,39℃,41℃, inoculation volume is 5%,bacterial counts and pH change were measured every 24h.The results showed that: at 37℃,the inoculation ratio was 1:1:2,the viable counts was 3.60×10~8 which is the highest,the overall trend of the five proportion is similar, but examination found that the distribution was dominated by Bacillus;at 39℃when the inoculation ratio was 1:2:1,the viable count reached the highest,was 4.01×10~8,pH variation trends similar,the cell number of Bacillus and coccus remained dominant; at 41℃the ratio of 1:2:2 had the largest strain concentration,which was 3.84×10~8,pH discreased gradually,examination results similer with 39℃.Selecting three of the above good again comparison fermented,the number of bacterium as the standard of appraisal.The result showed: the total number of viable cells had no significant difference,at 39℃,when the ratio was 1:2:1,the distribution of bacteria and coli is uniform.Yeast fermentation:adding different exogenous substance to the pomace, inoculating yeast in the sterilized,cultivated at 28℃.yeast grew best in urea and sucrose medium,better in whey powder medium,worst in brown sugar medium. Then cultureed yeast at different amount incubation of urea,the cell growth and the pH value of fermentation broth change were measured every 24h.the results show: yeast grew best of 2% urea addtional,there was clear broth white milky on the top,fermented flavor fragrance,and the microscopic examination results showed cell morphology better,was column, the first 4d,highest number of viable cells up to 5.62×10~8,final fermentation pH was 5.61.To provide temperatur parameters for yeast and lactic acid bacteria Mixture fermentation,and then inoculating yeast in above medium at different temperatures,measured the viable cell number.the results showed: cultured at 30 ~ 36℃,the number of yeast increased significantly,can quickly reach to 108,at 30℃,the maximum count was 7.47×10~8.Lactic acid bacteria and yeast mixture fermentation:Selecting six different inoculation ratio: 1:1:1, 1:1:2, 1:2:1, 1:2:2, 2:2:1 (LA:LB:ST:Y),four kinds of culture temperature: 35℃,37℃,39℃,41℃,inoculation volume is 5 % the total number of cultured viable cells and broth pH were measured 24h,the results showed that:In 33 ~ 39℃,after a certain time,the viable cells are up to 108 / ml at different temperatures of different proportion. At 35℃,with the ratio of 1:1:1:6,viable cells had the highest number,which is 1.99×10~8; at 37℃,the total number of viable cells reached 2.30×10~8,the ratio is 1:1:1:3; at 39℃,during the fermentation process cell growth had no significant differences;the highest number of viable cells is 1.48×10~8,which ratio is 1:1:1:2.At the end of fermentation,pH values are basically in the 3.80 or so. Microscopic examination showed yeast grew less,lactic acid bacteria was dominant.Based on the principle of Box-Benhnken experimental design and above results,using response surface methodology to optimize the process.The results showed that: fermentation temperature is 36℃,the ratio is 1:1:1:4,cultured 100h,the product is good at the viable counts and distribution.It will be the best technique.Verification of the optimum test: whether fresh or dried pimace.under the best conditions to verify the number of viable cells very similar to each other, almost no difference,the largest number of yeast strains is 1.15×10~8,lactic acid bacteria was 1.21×10~8.The results of other probiotic fermentation: Direct use of the pre-trial medium formula: apple: distilled water = 1:10,urea of 2%,whey powder was 2.5%,inoculation volume was 5%,cultuerd in 28℃and 37℃for Aspergillus and Bacillus subtilis respectively.The results showed that: Bacillus subtilis had the best growth in the determited medium,and after 2d,the viable count up to 4.73×10~8;Aspergillus can continue to growth in the culture medium.Even thecycle of growth time is similar in the optimal medium (7d),so that it is feasible to culture Bacillus subtilis and Aspergillus in the experimented medium.Preparation products and prediction shelflife :Under the best technology of respective pre-test,Preparation bacterium agents,measured indexs of products The results showed: The average crude protein content of products is 28.31%, true protein is 17.42%;reducing sugar content of 6.7%;the total number of viable cells was 6.85×10~8 at beginning,at room temperature probiotics in product was decreased with the prolongation of the time,after 5d storage the cell was decreased to 3.57×10~7 at 28℃, and it was only 2.39×10~7at 37℃. |
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