甘蔗渣发酵饲料中乳酸菌复合系的筛选及其发酵特征的研究
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摘要
甘蔗渣资源占我国生物质资源的重要组成部分,由于其自身高纤维素含量的特点,一直以来被广泛应用于建材等方面广为应用,但作为饲料资源的应用报道较少。由于甘蔗渣具有养分低,适口性差,消化率低等特点,直接饲喂动物采食量低,因此必须寻找新的途径解决该问题。如果能提高其进食率并提高其营养成分,甘蔗渣作为反刍动物的饲料便成为缓解人畜争粮问题和增加饲料品种来源的重要途径,也符合我国畜牧业长远发展的目标。
本研究从筛选接种于甘蔗渣发酵的乳酸菌复合系出发,筛选出较稳理想的甘蔗渣发酵菌剂LBC-8,并对乳酸菌复合系LBC-8的组成进行了16SrDNA系统发育分析,即菌体克隆。结果显示其主要组成菌为：乳酸杆菌(Lactobacillus sp)、发酵乳杆菌(Lactobacillus fermentum)、(Lactobacillus pantheris)、植物乳杆菌(Lactobacillus plantarum)、干酪乳杆菌(Lactobacillus casei)、副干酪乳杆菌(Lactobacillus paracasei)和醋酸菌(Acetobacter pasteurianus)等组成。
在将乳酸菌复合系LBC-8直接接种到甘蔗渣后发现,复合系菌群能够占主导地位成为优势菌株。
通过甘蔗渣起始糖浓度对甘蔗渣发酵饲料品质的影响进行研究。结果表明,甘蔗渣起始糖浓度5.0%DM(干物质)为甘蔗渣能否成功发酵的分界线,通过提高甘蔗渣糖浓度可以在一定程度上改善发酵饲料的品质；DGGE结果证明,当甘蔗渣起始糖浓度达到5.0%以上时,乳酸菌增殖明显。
在研究可溶性糖对甘蔗渣乳酸发酵影响的基础上,还进行了适合甘蔗渣发酵饲料喷施的方式及保存的方法的选择。通过相关性质分析及DGGE结果证明,在不同的喷施菌液的方式及保存发酵饲料的方法在结果上有很大的差异,以喷淋菌液并密封保存的方法效果最佳。
通过对乳酸菌复合系的筛选及性质的研究,并接种于甘蔗渣进行发酵饲料的制作,使微生物菌剂用于生物质资源的开发和利用又向前迈进了一步,并且使甘蔗渣微生物饲料领域更为丰富化。为以后的实际生产和应用打下了坚实的基础。
Bagasse resources were an important part of China's biomass resources, it has been widely used in building materials and so widely used because of the high cellulose content of its own characteristics. we must seek new ways to solve the problem that the bagasse with low nutrients, poor palatability, low digestibility and low intake by direct-fed animal. It was an important way that as a ruminant animal feed has become an indisputable human and animal food to alleviate the problems and increase the variety of sources if the consumption rate and the nutrition of bagasse can be increased, it also in line with the long-term development objectives of China's animal husbandry.
This study from the screening the lactic acid bacteria community of bagasse fermentation starting, and filter out the more appropriate lactic acid bacteria community LBC-8, and also made the analysis of bacterial composition of LBC-8 with the 16S rDNA phylogenetic analysis(bacterial clone). The results showed that the bulk of their strains as follows:Lactobacillus sp, Lactobacillus fermentum, Lactobacillus pantheris, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei and Acetobacter pasteurianus.
The lactic acid bacteria community LBC-8 inoculation into the bagasse and found that the community can be the dominant to become predominant strains.
By the reseach of effects of different soluble sugar concentration on fermentation characteristics of bagasse found that water soluble sugar concentration 5.0% DM can be success of the fermentation of bagasse and improvong the quality of bagasse fermentation. DGGE results show that lactic acid bacteria obvious proliferation when the water soluble sugar concentration 5.0% DM.
We made a choice in the spraying methods and preservation options of fermented bagasse on the basis of effects of water soluble sugar concentration on fermentation characteristics of bagasse. From the patterns of denaturing gradient gel electrophoresis (DGGE), there were different results in a different way of spraying and the preservation methods in fermented feed, the best method is sprayed sealed way.
It maked the microbial agents used in the development of biomass resources and made great progress by the screening of lactic acid bacteria and the nature studies of the community and inoculated in the bagasse for the production of fermented feed, and to make more abundant of the field of microbial feed, and also laid a solid foundation for the practical production and application.
本研究从筛选接种于甘蔗渣发酵的乳酸菌复合系出发,筛选出较稳理想的甘蔗渣发酵菌剂LBC-8,并对乳酸菌复合系LBC-8的组成进行了16SrDNA系统发育分析,即菌体克隆。结果显示其主要组成菌为：乳酸杆菌(Lactobacillus sp)、发酵乳杆菌(Lactobacillus fermentum)、(Lactobacillus pantheris)、植物乳杆菌(Lactobacillus plantarum)、干酪乳杆菌(Lactobacillus casei)、副干酪乳杆菌(Lactobacillus paracasei)和醋酸菌(Acetobacter pasteurianus)等组成。
在将乳酸菌复合系LBC-8直接接种到甘蔗渣后发现,复合系菌群能够占主导地位成为优势菌株。
通过甘蔗渣起始糖浓度对甘蔗渣发酵饲料品质的影响进行研究。结果表明,甘蔗渣起始糖浓度5.0%DM(干物质)为甘蔗渣能否成功发酵的分界线,通过提高甘蔗渣糖浓度可以在一定程度上改善发酵饲料的品质；DGGE结果证明,当甘蔗渣起始糖浓度达到5.0%以上时,乳酸菌增殖明显。
在研究可溶性糖对甘蔗渣乳酸发酵影响的基础上,还进行了适合甘蔗渣发酵饲料喷施的方式及保存的方法的选择。通过相关性质分析及DGGE结果证明,在不同的喷施菌液的方式及保存发酵饲料的方法在结果上有很大的差异,以喷淋菌液并密封保存的方法效果最佳。
通过对乳酸菌复合系的筛选及性质的研究,并接种于甘蔗渣进行发酵饲料的制作,使微生物菌剂用于生物质资源的开发和利用又向前迈进了一步,并且使甘蔗渣微生物饲料领域更为丰富化。为以后的实际生产和应用打下了坚实的基础。
Bagasse resources were an important part of China's biomass resources, it has been widely used in building materials and so widely used because of the high cellulose content of its own characteristics. we must seek new ways to solve the problem that the bagasse with low nutrients, poor palatability, low digestibility and low intake by direct-fed animal. It was an important way that as a ruminant animal feed has become an indisputable human and animal food to alleviate the problems and increase the variety of sources if the consumption rate and the nutrition of bagasse can be increased, it also in line with the long-term development objectives of China's animal husbandry.
This study from the screening the lactic acid bacteria community of bagasse fermentation starting, and filter out the more appropriate lactic acid bacteria community LBC-8, and also made the analysis of bacterial composition of LBC-8 with the 16S rDNA phylogenetic analysis(bacterial clone). The results showed that the bulk of their strains as follows:Lactobacillus sp, Lactobacillus fermentum, Lactobacillus pantheris, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei and Acetobacter pasteurianus.
The lactic acid bacteria community LBC-8 inoculation into the bagasse and found that the community can be the dominant to become predominant strains.
By the reseach of effects of different soluble sugar concentration on fermentation characteristics of bagasse found that water soluble sugar concentration 5.0% DM can be success of the fermentation of bagasse and improvong the quality of bagasse fermentation. DGGE results show that lactic acid bacteria obvious proliferation when the water soluble sugar concentration 5.0% DM.
We made a choice in the spraying methods and preservation options of fermented bagasse on the basis of effects of water soluble sugar concentration on fermentation characteristics of bagasse. From the patterns of denaturing gradient gel electrophoresis (DGGE), there were different results in a different way of spraying and the preservation methods in fermented feed, the best method is sprayed sealed way.
It maked the microbial agents used in the development of biomass resources and made great progress by the screening of lactic acid bacteria and the nature studies of the community and inoculated in the bagasse for the production of fermented feed, and to make more abundant of the field of microbial feed, and also laid a solid foundation for the practical production and application.
引文
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[3]梁坤,谭京梅,孙可伟.甘蔗渣的综合利用现状及展望[J].中国资源综合利用,2003,5：26
[4]张远平.发展蔗渣牛饲料的研究[J].广东轻工职业技术学院学报,2003,2(2)：21
[5]聂艳丽,刘永国,李娅等.甘蔗渣资源利用现状及开发前景[J].林业经济,2007,5：61
[6]高星超,盛家荣,赵星华.甘蔗渣的研究进展[J].广西师范学院学报,2007,24(4)：103
[7]白元生.饲料原料学[M].北京：中国农业出版社,1999
[8]冯定远,颜惜玲,蒲英远.产奶牛对碱化甘蔗渣消化利用的研究[J].广东农业科学,1996,2：40
[9]刘刚.甘蔗渣饲料资源的开发利用[J].饲料研究,2000,12：21
[10]陈驹声.发酵工业辞典[M].北京：轻工业出版社,1991
[11]周福娟,丁东梅,田玉兰.防止秸秆发酵饲料二次发酵的措施[J].辽宁畜牧兽医2000,(1)：26
[12]刘学剑.青贮饲料添加剂[J].饲料工业,1996,17(5)：28-29
[13]Baytok, E, Muruz, H. The effects of formic acid or formic acid plus molasses additives on the fermentation quality and DM and ADF degradabilities of grass silage[J]. Turk J Vet Anim Sci,2003,27:425-431
[14]石其伟,刘强,荣湘民,等.不同微生物菌剂对水稻秸秆发酵效果的影响[J].湖南农业大学学报,2006,32(3)：264
[15]Ring,E,Gatesoupe, F. J. Lactic acid bacteria in fish:a review[J]. Aquaculture,1998, 160：177-203
[16]蒋云霞,郑天凌,田蕴.红树林土壤微生物的研究[J].微生物学报,2006,46(5)：848-851
[17]Muyzer, G, Waal, E. C. D, Uitterlinden, A. G. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16SrRNA[J]. Appl Environ Microbiol,1993,59:695-700
[18]王大威,杨振宇,石梅,刘永建.分子生物学在石油微生物多样性研究中的应用[J].生物技术,2005,15(5)：86-89
[19]Muyzer, G. DGGE/TGGE a method for identifying genes from natural ecosystem [J].Curr Opin Microbiol,1999,2:317-322
[20]Rowan, A. K, Snape, J. R., Fearnside, D, et al. Composition and diversity of ammonia-oxidising bacterial communities in wastewater treatment reactors of different design treating identical wastewater [J]. FEMS Microbiol Ecol,2003,43: 195-206
[21]Duineveld, B. M, Kowalchuk, G. A, Keijzer, A.,et al. Analysis of bacterial communities in the rhizosphere of Chrysanthemum via denatureing gradient gel electrophoresis of PCR-amplified 16S rRNA as well as DNA fragments coding for 16S rRNA[J]. Appl Enviro Micro.biol,2001,67(1):172-178
[22]Wang, X. F, Haruta, S, Wang, P, et al. Diversity of a stable enrichment culture which is useful for silage inoculant and its sucession in alfalfa silage[J]. FEMS Microbiol Ecolog,2006,57:106-115
[23]Florez, A. B, Mayo, B. PCR-DGGE as a tool for characterizing dominant microbial populations in the Spanish blue-veined Cabrales cheese [J]. Int Dairy J, 2006,16:1205-1210
[24]Chandler, D. P, Li, S.-M, Spadoni, D. M, et al. A molecular comparison of culturable aerobic heterotrophic bacteria and 16S rDNA clones derived from a deepsubsurface sediment[J]. FEMS Microbiol Ecol,1997,23,131-144
[25]Branco, R,Chung, A. P, Verissimo, A, et al. Impact of chromium-contaminated wastewaters on the microbioal community of a river [J]. FEMS Microbiol Ecol, 2005,54:35-46
[26]Li, Z, Hu, Y, Liu, Y, et al.16S rDNA clone library-based bacterial phylogenetic diversity associated with three South China Sea sponges[J]. World J Microbiol Biotechnol,2007,23:1265-1272
[27]Brambilla, E, Hippe, H, Hagelstein, A, et al.16S rDNA diversity of cultured and uncultured prokaryotes of a mat sample from Lake Fryxell, McMurdo Dry Valleys,Antarctica.Extremophiles,2001,5:23-33
[28]Kemp, P. F, Aller, J. Y. Bacterial diversity in aquatic and other environments: what 16S rDNA libraries can tell us[J]. FEMS Microbiol Ecol,2004,47:161-177.
[29]联合国粮食及农业组织.秸秆养畜-中国的经验,粮农组织家畜生产及卫生文集2002,149
[30]曲永利,苗树君.反刍动物日粮中蛋白质和脂肪水平对粗纤维消化率的影响黄牛杂志.2003,29(1)：55
[31]杨洁彬,凌代文,郭兴华,等.,乳酸菌—生物学基础及应用[M].北京：中国轻工业出版社,1996
[32]Weinberg, Z. G, Muck, R. E. New trends and opportunities in the development and use of inoculants for silage[J]. FEMS Microbiol Rev,1996,19:53-68
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