角蛋白酶水解豆粕的微生物多样性分析

Microbial diversity of soybean meal hydrolysate treated by keratinase

摘要:

【背景】酶解饲料底物在酶解至干燥过程中微生物的变化影响着酶解饲料的营养成分以及风味的改变，从而影响动物对其进行采食、消化和吸收，最终影响动物肠道健康。【目的】探究两种酶解物料(纯豆粕和豆粕麦麸混合物)在酶解至干燥过程中的微生物多样性变化。【方法】实验室条件下，采用平板计数法测定物料总细菌数、总霉菌和酵母菌数变化。中试条件下，采用16S rRNA基因和ITS rDNA的高通量测序，检测物料中细菌和真菌多样性随酶解时间(0?36 h)的变化。【结果】酶解前后的平板计数结果显示，加入角蛋白酶处理24 h后，两种物料中的细菌数量相对于原料提升了1?000?10 000倍。而经过风干过后，细菌和真菌的数量较风干前下降90%?99%。通过MiSeq平台的16S rRNA基因和ITS rDNA测序结果表明，两种酶解豆粕均具有相似的α多样性指标，微生物丰富度水平相近。然而，16S rRNA基因测定的β多样性结果显示，Fructobacillus属和魏斯属(Weissell)在36 h的酶解进程中成为优势菌属；纯豆粕酶解物中的Fructobacillus属在数量上更占优势，豆粕麦麸混合酶解物中则魏斯属更具数量上的优势。ITS rDNA测定的β多样性结果显示，纯豆粕酶解物中曲霉属(Aspergillus)一直占据着数量上的绝对优势，而豆粕麦麸混合酶解物中链格孢属(Alternaria)、赤霉属(Gibberella)和曲霉属(Aspergillus)则在前18 h相对含量较高，但随着时间的延长相对数量减少；毕赤酵母属(Pichia)和酵母属(Remersonia)逐渐占据数量上的优势。【结论】小麦麸作为酶解豆粕辅料能够改变酶解豆粕中的微生物多样性，并使其中的微生物生长更偏向于魏斯属细菌和酵母类真菌。

Abstract:

[Background] The nutritional value and flavor of feed ingredient was improved after fermentation and keratinase hydrolysis because microbiota compositions in the enzymolytic feed can be altered. In addition, enzymolytic soybean meal has significant improvements in growth performance and intestinal health. [Objective] Therefore, in this study, enzymolytic soybean meal (ESBM) was produced by fermentation and keratinase hydrolysis. The aim of this study was to explore the effect of two kinds of ESBM (keratinase hydrolyzed soybean meal (KHS) and keratinase hydrolyzed SBM with 30% inclusion of wheat bran (KHS-WB) on the microbiota diversity in the process of hydrolysis to drying. [Methods] First of all, the numbers of total bacteria, total mildew and yeast were measured by plate counting in laboratory. Additionally, Illumina MiSeq sequencing of 16S rRNA gene and ITS rDNA were employed to test the bacterial and fungal diversity dynamics of hydrolysis samples (0?36 h) in the pilot experiment. [Results] The plate counting result showed that the numbers of bacteria of two kinds of ESBM were 1 000?10 000 times higher than that of raw materials after treated with keratinase for 24 h. However, after drying, the counts of bacteria and fungi of ESBM were significantly decreased by 90%?99% than that of before drying. Moreover, the Illumina MiSeq sequencing results of 16S rRNA gene and ITS rDNA indicated that two kinds of ESBM had a similar alpha diversity index and microflora richness level. Additionally, the beta diversity of 16S rRNA sequencing result showed that Fructobacillus in KHS and Weissella in KHS-WB kept sustainable growth, occupied the privilege and became dominant bacterial genus (36 h), respectively. The results of ITS rDNA sequencing demonstrated that Aspergillus occupied the privilege in all genus of KHS while the diversity of dominant fungi was more abundant of KHS-WB in the process of hydrolysis. [Conclusion] Wheat bran as ESBM substrate can influence the microbiota diversity of ESBM, as well as the Weissella and yeast fungi will be the diversity of dominant microbes in the ESBM.