角蛋白酶水解豆粕的微生物多样性分析
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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24h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36h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。
[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.
[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.
引文
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[12]Wei JT,Li SZ,Yang XH,et al.Effect of liquid enzymolysis and fermented soybean meal on growth performance and partial nutriment in feces excreta of growing pigs[J].Chinese Journal of Veterinary Science,2012,32(3):478-482(in Chinese)魏金涛,李绍章,杨雪海,等.液态酶解发酵豆粕对生长育肥猪生产性能及部分营养物质粪排泄量的影响[J].中国兽医学报,2012,32(3):478-482
[13]Yang XH,Zhao N,Wei JT,et al.Effects of the liquid enzymolysis and fermented soybean meal on growth performance and physiological-biochemical parameters of Gushi broiler[J].China Feed,2015(20):22-24(in Chinese)杨雪海,赵娜,魏金涛,等.豆粕液态酶解发酵物对育雏期固始鸡生长性能及血清生化指标的影响[J].中国饲料,2015(20):22-24
[14]Degnan PH,Ochman H.Illumina-based analysis of microbial community diversity[J].The ISME Journal,2012,6(1):183-194
[15]Polka J,Rebecchi A,Pisacane V,et al.Bacterial diversity in typical Italian salami at different ripening stages as revealed by high-throughput sequencing of 16S r RNA amplicons[J].Food Microbiology,2015,46:342-356
[16]Wang WQ,Zhao SG,Ma LQ,et al.The research progress and application of resequencing based on animal genomics[J].Acta Veterinaria et Zootechnica Sinica,2016,47(10):1947-1953(in Chinese)汪文强,赵生国,马利青,等.动物基因组学重测序的应用研究进展[J].畜牧兽医学报,2016,47(10):1947-1953
[17]Endo A,Tanaka N,Oikawa Y,et al.Fructophilic characteristics of Fructobacillus spp.may be due to the absence of an alcohol/acetaldehyde dehydrogenase gene(adhE)[J].Current Microbiology,2014,68(4):531-535
[18]Li ZH,Duan XB.New progress in the classification and identification of bacteria of the genus Weiss[J].International Journal of Laboratory Medicine,2006,27(8):721-723(in Chinese)李中华,段雄波.魏斯菌属细菌的分类与鉴定新进展[J].国际检验医学杂志,2006,27(8):721-723
[19]Formenti LR,N?rregaard A,Bolic A,et al.Challenges in industrial fermentation technology research[J].Biotechnology Journal,2014,9(6):727-738
[20]Hong SB,Lee M,Kim DH,et al.Aspergillus cibarius sp.nov.,from traditional meju in Korea[J].Journal of Microbiology,2012,50(4):712-714
[21]Ellis WO,Smith JP,Simpson BK,et al.Aflatoxins in food:occurrence,biosynthesis,effects on organisms,detection,and methods of control[J].Critical Reviews in Food Science and Nutrition,1991,30(4):403-439
[2]Wang D,Piao XS,Zeng ZK,et al.Effects of keratinase on performance,nutrient utilization,intestinal morphology,intestinal ecology and inflammatory response of weaned piglets fed diets with different levels of crude protein[J].Asian-Australasian Journal of Animal Sciences,2011,24(12):1718-1728
[3]Panda R,Tetteh AO,Pramod SN,et al.Enzymatic hydrolysis does not reduce the biological reactivity of soybean proteins for all allergic subjects[J].Journal of Agricultural and Food Chemistry,2015,63(43):9629-9639
[4]Wang Y,Liu XT,Wang HL,et al.Optimization of processing conditions for solid-state fermented soybean meal and its effects on growth performance and nutrient digestibility of weanling pigs[J].Livestock Science,2014,170:91-99
[5]Lu WQ,Zhou ZX,Yu HT,et al.Method for eliminating beta-conglycinin in fermented soybean meal:CN,CN201610890870.X[P].2016-10-12(in Chinese)陆文清,周智旋,于海涛,等.一种消除发酵豆粕中β-伴大豆球蛋白的方法:中国,CN201610890870.X[P].2016-10-12
[6]Yu HT.Study on solid-state fermented soybean meal combined with wheat bran and its application on growing pigs[D].Beijing:Master’s Thesis of China Agricultural University,2016(in Chinese)于海涛.豆粕麦麸混合固态发酵及其仔猪养殖中的应用研究[D].北京:中国农业大学硕士学位论文,2016
[7]Williams CM,Richter CS,Mackenzie JM,et al.Isolation,identification,and characterization of a feather-degrading bacterium[J].Applied and Environmental Microbiology,1990,56(6):1509-1515
[8]Odetallah NH,Wang JJ,Garlich JD,et al.Keratinase in starter diets improves growth of broiler chicks[J].Poultry Science,2003,82(4):664-670
[9]Ma HH.The study on difference and mechanisim of different feedstuff on aflatoxin B1 production by Aspergillus parasiticus[D].Wuhan:Doctoral Dissertation of Huazhong Agricultural University,2015(in Chinese)马慧慧.寄生曲霉菌在不同饲料原料中产生黄曲霉毒素B1的差异性及其机制研究[D].武汉:华中农业大学博士学位论文,2015
[10]Chen NS,Ai QH,Wang DZ.Studies on soybean protein as a substitute for fish meal in formulated diets for Anguilla anguilla[J].Journal of Fisheries of China,1998,22(3):283-287(in Chinese)陈乃松,艾庆辉,王道尊.欧洲鳗配合饲料中大豆蛋白替代鱼粉的研究[J].水产学报,1998,22(3):283-287
[11]Wang ZS,Kuang YG,Liu HF,et al.The effects of the compound-enzyme degraded antigen-protein feed on piglet growth performance[J].Journal of Sichuan Agricultural University,2003,21(4):338-342(in Chinese)王之盛,况应谷,刘惠芳,等.酶解去除抗原蛋白饲料对仔猪生产性能的影响[J].四川农业大学学报,2003,21(4):338-342
[12]Wei JT,Li SZ,Yang XH,et al.Effect of liquid enzymolysis and fermented soybean meal on growth performance and partial nutriment in feces excreta of growing pigs[J].Chinese Journal of Veterinary Science,2012,32(3):478-482(in Chinese)魏金涛,李绍章,杨雪海,等.液态酶解发酵豆粕对生长育肥猪生产性能及部分营养物质粪排泄量的影响[J].中国兽医学报,2012,32(3):478-482
[13]Yang XH,Zhao N,Wei JT,et al.Effects of the liquid enzymolysis and fermented soybean meal on growth performance and physiological-biochemical parameters of Gushi broiler[J].China Feed,2015(20):22-24(in Chinese)杨雪海,赵娜,魏金涛,等.豆粕液态酶解发酵物对育雏期固始鸡生长性能及血清生化指标的影响[J].中国饲料,2015(20):22-24
[14]Degnan PH,Ochman H.Illumina-based analysis of microbial community diversity[J].The ISME Journal,2012,6(1):183-194
[15]Polka J,Rebecchi A,Pisacane V,et al.Bacterial diversity in typical Italian salami at different ripening stages as revealed by high-throughput sequencing of 16S r RNA amplicons[J].Food Microbiology,2015,46:342-356
[16]Wang WQ,Zhao SG,Ma LQ,et al.The research progress and application of resequencing based on animal genomics[J].Acta Veterinaria et Zootechnica Sinica,2016,47(10):1947-1953(in Chinese)汪文强,赵生国,马利青,等.动物基因组学重测序的应用研究进展[J].畜牧兽医学报,2016,47(10):1947-1953
[17]Endo A,Tanaka N,Oikawa Y,et al.Fructophilic characteristics of Fructobacillus spp.may be due to the absence of an alcohol/acetaldehyde dehydrogenase gene(adhE)[J].Current Microbiology,2014,68(4):531-535
[18]Li ZH,Duan XB.New progress in the classification and identification of bacteria of the genus Weiss[J].International Journal of Laboratory Medicine,2006,27(8):721-723(in Chinese)李中华,段雄波.魏斯菌属细菌的分类与鉴定新进展[J].国际检验医学杂志,2006,27(8):721-723
[19]Formenti LR,N?rregaard A,Bolic A,et al.Challenges in industrial fermentation technology research[J].Biotechnology Journal,2014,9(6):727-738
[20]Hong SB,Lee M,Kim DH,et al.Aspergillus cibarius sp.nov.,from traditional meju in Korea[J].Journal of Microbiology,2012,50(4):712-714
[21]Ellis WO,Smith JP,Simpson BK,et al.Aflatoxins in food:occurrence,biosynthesis,effects on organisms,detection,and methods of control[J].Critical Reviews in Food Science and Nutrition,1991,30(4):403-439