复合益生菌发酵饲料发酵条件优化及品质鉴定

Fermentation Condition Optimization and Quality Evaluation of Feed Fermented by Compound Probiotics

本试验旨在探究不同发酵条件对复合益生菌发酵饲料品质的影响。选用3株乳酸菌(鼠李糖乳杆菌、乳酸片球菌、干酪乳杆菌)、2株酵母菌(酿酒酵母菌、毕赤酵母菌),采用4因素(接种比例、接种量、水料比、温度)3水平正交设计对精补料发酵条件进行优化,共设9个组,测定各组发酵饲料中氨态氮(NH3-N)、可溶性糖(SS)、淀粉(ST)含量,pH以及3种霉菌毒素——黄曲霉毒素B1(AFB1)、玉米赤霉烯酮(ZEN)、呕吐毒素(DON)含量和微生物群落结构变化,用极差分析法确定综合指标最优时的发酵条件;将精补料在最优发酵条件下发酵不同时间(0、1、3、5、7、10、15 d),通过测定发酵饲料中常规营养成分含量、酶活性、霉菌毒素含量以及有氧稳定性,确定最佳发酵时间。结果表明:1)AFB1、ZEN含量优化组为G1(接种量5%,接种比例1∶1、温度20℃、水料比0.4∶1)、G7组(接种量15%,接种比例2∶1、温度20℃、水料比1∶1),NH3-N含量优化组为G3(接种量5%,接种比例2∶1、温度30℃、水料比1∶1)、G7组,ST含量优化组为G6组(接种量10%,接种比例2∶1、温度30℃、水料比0.4∶1),DON含量优化组为G5(接种量10%,接种比例1∶2、温度25℃、水料比1∶1)、G7组,SS含量优化组为G4(接种量10%,接种比例2∶1、温度20℃、水料比0.6∶1)、G6组。2)16S测序结果显示,G3组发酵饲料中微生物的α多样性和β多样性均显著高于G1、G2(接种量5%,接种比例1∶2、温度25℃、水料比0.6∶1)、G4、G6、G7、G8(接种量15%,接种比例1∶1、温度30℃、水料比0.4∶1)、G9组(接种量15%,接种比例1∶2、温度25℃、水料比0.6∶1)(P<0.05),略高于G5组(P>0.05);G3组中植物乳杆菌(Lactobacillus plantarum)、短乳杆菌(Lactobacillus brevis)的相对丰度显著高于G1、G2、G4、G5、G6、G8、G9组(P<0.05),与G7组差异不显著(P>0.05)。3)与发酵第0天(未发酵)时相比,发酵饲料粗蛋白质(CP)含量、纤维素酶(CL)、脂肪酶(LPS)、酸性蛋白酶(ACP)活性均在发酵第5天时显著升高(P<0.05),NH3-N、中性洗涤纤维(NDF)、酸性洗涤纤维(ADF)含量均在发酵第5天时显著降低(P<0.05),β-淀粉酶(β-AL)活性、pH以及SS、ST、AFB1、ZEN、DON含量均在发酵第10天时显著降低(P<0.05)。4)发酵饲料有氧暴露20 d后,其中心温度高出环境温度2℃,同时pH异常增大。结论:1)精补料最优发酵条件为接种量5%,接种比例2∶1、温度30℃、水料比1∶1,该发酵条件能够有效提高发酵饲料中微生物的多样性,提高Lactobacillus brevis的相对丰度。2)精补料最佳发酵时间为5 d,该发酵时间可提高发酵饲料的营养成分含量,降低霉菌毒素含量。3)发酵结束开盖后,发酵饲料应在20 d内喂完。

This experiment was conducted to explore the effects of different fermentation conditions on the quality of feed fermented by compound probiotics. Chose 3 strains of lactic acid bacteria(Lactobacillus rhamnosus,Pediococcus lactis and Lactobacillus casei)and 2 strains of yeasts(Saccharomyces cerevisiae and Pichia pastoris),using 4 factors(inoculation amount,inoculation ratio,temperature and water to material ratio)and 3 levels orthogonal design to optimize the fermentation conditions of concentrate supplement. There were 9 groups,and the ammonia nitrogen(NH3-N),soluble sugar(SS),starch(ST)contents,pH,3 kinds of mycotoxins—aflatoxin B1(AFB1),zearalenone(ZEN),deoxynivalenol(DON)contents and microbial flora changes in each group of fermented feed were determined,and using range analysis method to find the optimal fermentation conditions of comprehensive indicators. The concentrate supplement was fermented for different time(0,1,3,5,7,10 and 15 d)under optimal fermentation conditions,by determining the conventional nutrient contents,enzyme activities,mycotoxin contents,and aerobic stability of the fermented feed to determine the optimal fermentation time. The results showed as follows:1)AFB1and ZEN content optimized groups were G1(inoculation amount 5%,inoculation ratio 1∶1,temperature 20 ℃ and water to material ratio0.4∶1)and G7 groups(inoculation amount 15%,inoculation ratio 2∶1,temperature 20 ℃ and water to material ratio 1∶1),NH3-N content optimized groups were G3(inoculation amount 5%,inoculation ratio 2∶1,temperature 30 ℃ and water to material ratio 0.4∶1)and G7 groups,ST content optimized group was G6 group(inoculation amount 10%,inoculation ratio 2∶1,temperature 30 ℃ and water to material ratio 1∶1),DON content optimized groups were G5(inoculation amount 10%,inoculation ratio 1∶2,temperature 25 ℃ and water to material ratio 1∶1)and G7 groups,and SS content optimized groups were G4(inoculation amount 10%,inoculation ratio 2∶1,temperature 20 ℃ and water to material ratio 0.6∶1)and G6 groups. 2)The 16S sequencing results showed that theαdiversity andβdiversity of the microorganisms in fermented feed in the G3group were significantly higher than those in the G1,G2(inoculation amount 5%,inoculation ratio 1∶2,temperature 25 ℃ and water to material ratio 0.6∶1),G4,G6,G7,G8(inoculation amount 15%,inoculation ratio 1∶1,temperature 30 ℃ and water to material ratio 0.4∶1)and G9 groups(inoculation amount 15%,inoculation ratio 1∶2,temperature 25 ℃ and water to material ratio 0.6∶1)(P<0.05),and slightly higher than those in the G5 group(P>0.05);the relative abundances of Lactobacillus plantarum and Lactobacillus brevis in the G3 group were significantly higher than those in the G1,G2,G4,G5,G6,G8 and G9 groups(P<0.05),but had no significant difference between G3 and G7 groups(P>0.05). 3)Compared with day 0 of fermentation(no fermentation),the crude protein(CP)content,cellulase(CL),lipase(LPS)and acid protease(ACP)activities were significantly increased on the day 5 of fermentation(P<0.05),the NH3-N,neutral detergent fiber(NDF)and acid detergent fiber(ADF)contents were significantly reduced on day 5 of fermentation(P<0.05),and theβ-amylase( β-AL)activity,pH,SS,ST,AFB1,ZEN and DON contents were significantly reduced on day 10 of fermentation(P<0.05). 4)After the fermented feed was exposed to oxygen for 20 days,the temperature of the center of the feed was 2 ℃ higher than the ambient temperature,and the pH increased abnormally. Conclusion:1)the optimal fermentation conditions for concentrate supplement are inoculation amount 5%,inoculation ratio 2∶1,temperature 30 ℃,water to feed ratio 1∶1,which can effectively increase the diversity of microorganisms in the fermented feed and increase the relative abundance of Lactobacillus brevis. 2)The optimal fermentation time for concentrate supplement is 5 days,which can increase the nutrient contents of the fermented feed and reduce the content of mycotoxins. 3)After the fermentation is finished and the lid is opened,the fermented feed should be fed within 20 days.[Chinese Journal of Animal Nutrition,2022,34(5):3358-3375]