一株解淀粉芽胞杆菌的筛选及其益生特性研究

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Abstract Abstract Bacillus strains are widely used as animal probiotics. Cellulase produced by Bacillus strains could be used to increase efficiency of feed utilization and improve the intestinal microflora. The aim of this study was to screen the Bacillus strains with high celluloytic activities from the rhizosphere soil of plants (sugarcane and hemp, and so on) to provide strain resources for development of animal probiotics. Firstly, culturable Bacillus strains were isolated from the soil samples through dilution seperation method on LB plate. Eight celluloytic Bacillus strains were obtained by screening with the sodium carboxymethyl cellulose (CMC-Na) medium. Among them, the strain FJAT-29941 exhibited the strongest cellulase activities and was identified as Bacillus amyloliquefaciens according to the morphology, physical and biochemical characteristics and 16S rRNA gene sequences analysis. The cellulase production conditions, gastrointestinal tolerance, filter paper degradation and hemolytic activity of strain FJAT-29941 were studied furtherly. The fermentative conditions for cellulase-producing of the strain FJAT-29941 were as follows: pH 4.0~9.0 (optimum pH 9.0), 20~50 ℃ (optimum 30 ℃ ), 18~72 h (optimum 42 h) and the optical fermentative medium was NA (nutrient agar) containing 0.5% NaCl. Moreover, the cellulases displayed excellent tolerance to pH and temperature. The survival rate of the strain FJAT-29941 in the artificial intestinal fluid containing 1% typsin and the artificial gastric fluid containing 1% pepsin was 27.98% and 64.91%, respectively, suggesting a great tolerance to gastrointestinal environments. It was showed that the cultures could rapidly degrade intact long filter paper scrips into fragments within 48 hours of incubation, showing strong ability of degradation on cellulose substrates. Finally, no hemolysis of the strain FJAT-29941 was observed on the goat blood agar plates, indicating its safety toward use of this isolate as a potential probiotics. Taken together, the strain B. amyloliquefaciens FJAT-29941 exhibited both excellent cellulose degrading ability and probiotic potential, which would provide a strain resource and scientific basis for the probiotics development.

Key words： Bacillus amyloliquefaciens Cellulose Gastrointestinal tolerance Probiotic

Received: 13 November 2017 Published: 21 May 2018

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	Articles by authors
	LIU Guo-Hong
	LIU Bei
	CHE Jian-Mei
	YU Jie-Beng
	CHEN Qian-Qian
	YAO Yun-Fa
	LIU Qin-Yang

Cite this article:

LIU Guo-Hong,LIU Bei,CHE Jian-Mei, et al. Screening of a Bacillus amyloliquefaciens Strain and Its Probiotic Characteristics[J]. , 2018, 26(6): 1043-1055.

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http://journal05.magtech.org.cn/Jwk_ny/EN/ OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2018/V26/I6/1043

1. 曹晶晶, 杨海艳, 谢咸升, 等. 2016. 解淀粉芽胞杆菌对小麦黄花叶病的生物防治[J]. 植物保护学报, 43(4):588-593. (Cao J J, Yang H Y, Xie X S, et al. 2016. Control effect of Bacillus amyloliquefaciens on wheat yellow mosaic disease [J]. Acta Phytophylacica Sinica, 43(4):588-593.)
2. 崔海洋, 程仕伟, 黄田红, 等. 2014. 产纤维素酶的解淀粉芽孢杆菌分离鉴定及酶学性质研究[J]. 食品科学技术学报, 32(3):43-47. (Cui H Y, Chen S W, Huang T H, et al. 2014. Screening and Identifying of Bacillus amyloliquefaciens for Cellulase Production and its Enzymatic Characters [J]. Journal of Food Science and Technology, 32(3):43-47.)
3. 东秀珠, 蔡妙英. 2001. 常见细菌系统鉴定手册[M]. 北京: 科学出版社. (Dong X Z, Cai M Y. 2001. (Systematic identification Mannual of Commen Bacteria). Beijing: Science Press.)
4. 董丹, 车振明, 关统伟. 2016. 白酒酒糟中产纤维素酶菌株的筛选及其酶活力特性检测[J]. 中国酿造, 34(8): 44-48. (Dong D, Che Z M, Guan T W. 2016. Screening of cellulose-producing strains and enzyme activity characteristic detection in distiller's grains [J]. China Brewing, 34(8): 44-48.)
5. 樊程, 李双江, 李成磊, 等. 2012. 大熊猫肠道纤维素分解菌的分离鉴定及产酶性质[J]. 微生物学报, 52(9): 1113-1121. (Fan C, Li S J, Li C L, et al. 2012. Isolation, identification and cellulase production of a cellulolytic bacterium from intestines of giant panda [J]. Acta Microbiologica Sinica, 52(9): 1113-1121.)
6. 黄玲玲, 裘纪莹, 唐琳, 等. 2015. 解淀粉芽胞杆菌NCPSJ7对采后苹果轮纹病的生物防治作用[J]. 中国食物与营养, 21(2):20-24. (Huang L L, Qiu J Y, Tang L, et al. 2015. Postharvest biological control of Bacillus amyloliquefaciens NCPSJ7 on apple ring rot [J]. Food and Nutrition in China, 21(2):20-24.)
7. 李红亚, 李术娜, 王树香, 等. 2015. 解淀粉芽孢杆菌MN-8对玉米秸秆木质纤维素的降解[J]. 应用生态学报, 26(5):1404-1410. (Li H Y, Li S N, Wang S X, et al. 2015. Degradation of lignocellulose in the corn straw by Bacillus amyloliquefaciens MN-8 [J]. Chinese Journal of Applied Ecology, 26(5):1404-1410.)
8. 刘国红, 朱育菁, 刘波, 等. 2014. 玉米根际土壤芽胞杆菌的多样性[J]. 农业生物技术学报, 22(11):1367-1379. (Liu G H, Zhu Y J, Liu B, et al. 2014. Diversity of culturable Bacillus Species from Maize (Zea mays) rhizosphere soil [J]. Journal of Agricultural Biotechnology, 22(11):1367-1379.)
9. 刘锁珠, 李龙, 付冠华, 等. 2017. 藏猪源高产纤维素酶菌株的筛选及鉴定[J]. 西北农林科技大学学报:自然科学版, 45(3):43-50. (Liu S Z, Li L, Fu G H, et al. 2017. Screening and identification of high-yield cellulase degrading bacteria strain from Tibetan pigs [J]. Journal of Northwest A&F University (Natural Science Edition), 45(3):43-50.)
10. 罗晶, 张鑫, 黄海, 翟枫, 等. 2013. 解淀粉芽胞杆菌Ba-168菌株对小麦全蚀病的防治及其机制. 植物保护学报, 40(5):475-476. (Luo J, Zhang X, Huang H, et al. 2013. Control effect and mechanism of Bacillus amyloliquefaciens Ba-168 on wheat take-all. Acta Phytophylacica Sinica, 40(5):475-476.)
11. 吕钊, 张丽萍, 程辉彩, 等. 2013. 防治蔬菜灰霉病解淀粉芽胞杆菌的分离鉴定及抑菌特性[J]. 农药, 52(7):490-493. (Lv Z, Zhang L P, Cheng H C, et al. 2013. Screening and Identification of Bacillus amyloliquefaciens against vegetable Botrytis cinerea and its antimicrobial characteristics [J]. Pesticides, 52(7):490-493.)
12. 马军, 王耀嵘, 侯萍, 等. 2016. 一株源于红树林根际土壤产纤维素酶芽孢杆菌的鉴定及其酶学性质分析. 海洋与湖沼, 47(5): 997-1004. (Ma J, Wang Y R, Hou P, et al. 2016. Identification and enzymatic property of cellulase-producing Bacillus subtilis isolated from mangrove rhizosphere soil. Oceanologia et Limnologia Sinica, 47(5): 997-1004.)
13. 杨敬辉，王敬根, 张玉军, 等. 2017. 草鱼肠道细菌CD-17的鉴定和生防活性研究. 西北农业学报, 26(6)：932-938. (Yang J H, Wang J G, Zhang Y J, et al. 2017. Biocontrol Activity and Taxonomy of Grass Carp Intestine Bacteria CD-17. Acta Agriculturae Boreali-occidentalis Sinica, 26(6):932-938.)
14. 易旻, 杨玉婷, 李梦霖, 等. 2017. 一株耐高温纤维素降解菌的分离筛选及鉴定. 吉首大学学报(自然科学版), 38(4): 72-77. (Yi M, Yang Y T, Li M L, et al. 2017. Screening and identification of a high-temperature resistant cellulose-degrading bacterium. Journal of Jishou University (Natural Science Edition), 38(4): 72-77.)
15. 朱晓飞, 张晓霞, 牛永春, 等. 2011. 一株抗水稻纹枯病菌的解淀粉芽胞杆菌分离与鉴定. 微生物学报, 51(8):1128-1133. （Zhu X F, Zhang X X, Niu Y C, et al. 2011. Isolation and identification of a Bacillus amyloliquefaciens YB-3 against Rhizoctonia solani. Acta Microbiologica Sinica, 51(8):1128-1133.）
16. Cao H, He S, Wei R., et al. 2011. Bacillus amyloliquefaciens G1: a potential antagonistic bacterium against eel-pathogenic Aeromonas hydrophila [J]. Evidence-Based Complementary and Alternative Medicine, 824104.
17. Chen X H, Koumoutsi A, Scholz R, et al. 2007. Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42 [J]. Nat Biotechnol, 25(9):1007-1014.
18. Cheng H R, Jiang N. 2006. Extremely rapid extraction of DNA from bacteria and yeasts [J]. Biotechnology letters, 28(1): 55-59.
19. Elshaghabee F M F, Rokana N, Gulhane R D, et al. 2017. Bacillus as potential probiotics: status, concerns, and future perspectives [J]. Front Microbiol, 8:1490.
20. Farhat-Khemakhem A, Blibech M, Boukhris I, et al. 2017. Assessment of the potential of the multi-enzyme producer Bacillus amyloliquefaciens US573 as alternative feed additive [J]. Journal of Science of Food and Agriculture, doi: 10.1002/jsfa.8574.
21. Felsenstein J. 1985. Confidence limits on phylogenies: An approach using the bootstrap [J]. Evolution, 39:783-791.
22. Ghani M, Ansari A, Aman A, et al. 2013. Isolation and characterization of different strains of Bacillus licheniformis for the production of commercially significant enzymes [J]. Pak. J. Pharm. Sci., 26: 691–697.
23. Ghose T K. 1987. Measurement of cellulose activities [J]. Pure and Applied Chemistry, 59(2): 257-268.
24. Hoa N T, Baccigalupi L, Huxham A, et al. 2000. Characterization of Bacillus species used for oral bacteriotherapy and bacterioprophylaxis of gastrointestinal disorders [J]. Applied and Environmental Microbiology, 66: 5241–5247.
25. Jukes T H, Cantor C R. 1969. Evolution of protein molecules [M]. In Mammalian Protein Metabolism, vol. 3, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
26. Kim O S, Cho Y J, Lee K, et al. 2012. Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species [J]. Int J Syst Evol Microbiol, 62: 716-721.
27. Kumar S, Stecher G, Tamura K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33:1870-1874.
28. Lee A, Cheng KC, Liu JR. 2017. Isolation and characterization of a Bacillus amyloliquefaciens strain with zearalenone removal ability and its probiotic potential [J]. PLoS One, 12(8):e0182220.
29. Lee Y, Kim B, Lee B, et al. 2008. Purification and characterization of cellulase produced by Bacillus amyloliquefaciens DL–3 utilizing rice hull [J]. Bioresource Technology, 99:378–386.
30. Liu B, Liu G H, Cetin S, et al. 2014. Bacillus cihuensis sp. nov., isolated from rhizosphere soil of a plant in the Cihu area of Taiwan [J]. Antonie van Leeuwenhoek, 106:1147–1155.
31. Majeed M, Majeed S, Nagabhushanam K, et al. 2016, Evaluation of the stability of Bacillus coagulans MTCC 5856 during processing and storage of functional foods. Int. J. Food Sci. Technol, 51: 894–901.
32. Manhar AK, Saikia D, Bashir Y, et al. 2015. In vitro evaluation of celluloytic Bacillus amyloliquefaciens AMS1 isolated from traditional fermented soybean (Churpi) as an animal probiotic [J]. Research in Veterinary Science, 99:149–156.
33. Mohammed Y, Lee B, Kang Z, et al. 2014. Development of a two-step cultivation strategy for the production of vitamin B12 by Bacillus megaterium [J]. Microb. Cell Fact, 13:102.
34. Ouattara H G, Reverchon S, Niamke S L, et al. 2017. Regulation of the synthesis of pulp degrading enzymes in Bacillus isolated from cocoa fermentation [J]. Food Microbiol, 63: 255–262.
35. Saitou N, Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees [J]. Molecular Biology and Evolution, 1987, 4:406-425.
36. Singh S, Dikshit PK, Moholkar VS, et al. 2014. Purification and characterization of acidic cellulase from Bacillus amyloliquefaciens SS35 for hydrolyzing Parthenium hysterophorus biomass [J]. Environmental Progress & Sustainable Energy, 34(3):810-818.
37. Sorokulova I B, Pinchuk I V, Denayrolles M, et al. 2008. The safety of two Bacillus probiotic strains for human use [J]. Digestive Diseases and Sciences, 53: 954–963.
38. Takano H. 2016. The regulatory mechanism underlying light-inducible production of carotenoids in non phototrophic bacteria [J]. Biosci. Biotechnol. Biochem, 80:1264–1273.
39. Tanaka K, Takanaka S, Yoshida K I. 2014. A second-generation Bacillus cell factory for rare inositol production. Bioengineered, 2014, 5:331–334.
40. Thompson J D, Gibson T J, Plewniak F, et al. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools [J]. Nucleic Acids Res, 25:4876–4882.
41. Vilanova L, Teixidó N, Usall J, et al. 2017. Environmental fate and behaviour of the biocontrol agent Bacillus amyloliquefaciens CPA-8 after preharvest application to stone fruit [J]. Pest Manag Sci, doi: 10.1002/ps.4716.
42. Wang Y, Zhang H, Zhang L, et al. 2010. In vitro assessment of probiotic properties of Bacillus isolated from naturally fermented congee from Inner Mongolia of China [J]. World Journal of Microbiology and Biotechnology, 26:1369–1377.