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圈养大熊猫肠道乳酸菌分离及益生特性
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北京动物园圈养野生动物技术北京市重点实验室开放课题(ZDK202213)


Isolation and probiotic characteristics of lactic acid bacteria from intestinal tract of giant panda in captivity
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    摘要:

    【背景】大熊猫数量稀少、繁殖困难,在其生长过程中极易感染消化系统疾病甚至死亡。【目的】分析圈养大熊猫肠道可培养乳酸菌的群落结构与功能,筛选具有益生特性的乳酸菌菌株,为大熊猫消化系统疾病的预防和肠道微生物研究提供理论参考及菌种资源。【方法】采用3种培养基分离大熊猫粪便中的乳酸菌,通过革兰氏染色镜检和过氧化氢试验对分离的菌株进行初步鉴定,基于BOXA1R-PCR图谱遗传多样性选取代表菌株进行16S rRNA基因测序分析并进行主成分分析(principal component analysis, PCA),同时分析乳酸菌菌株安全性和益生特性。【结果】通过初步鉴定共分离获得58株乳酸菌,根据BOXA1R-PCR结果挑选20株菌进行测序,结果显示20株菌分属于明串珠菌属(Leuconostoc)、肠球菌属(Enterococcus)、魏斯氏菌属(Weissella)和链球菌属(Streptococcus)这4个属,主成分分析结果表明不同年龄段的大熊猫肠道乳酸菌群落结构存在差异。20株乳酸菌均不溶血,17株乳酸菌对11种抗生素均敏感;11株菌耐pH值2.0酸性条件,14株菌对0.3%的胆盐具有良好的耐受性。5株菌SW-51、SW-48、MQ-41、SW-58和MX-23对3种指示菌均具有抑菌效果,50%以上的供试菌株具有自凝聚和共凝聚能力,3株菌的产酸和生长速率较一致,其中乳酸明串珠菌(Leuconostoc lactis) MX-23生长最快且产酸能力最强。【结论】大熊猫肠道内蕴藏着丰富的乳酸菌资源,其结构与组成受其年龄影响,具有较好的益生特性和应用前景。

    Abstract:

    [Background] The giant pandas are rare, difficult to be bred, and susceptible to digestive system diseases during the growth process. [Objective] To reveal the community structure features and the roles of lactic acid bacteria (LAB) in the intestinal tract of the giant panda and screen out the LAB strains with probiotic characteristics for the prevention of digestive system diseases and the research on intestinal flora in the giant panda. [Methods] Three culture media were employed to isolate the LAB from the intestines of giant pandas in captivity. The isolates were preliminarily identified by Gram staining microscopy and hydrogen peroxide test. The representative strains were selected for 16S rRNA gene sequencing and Principal component analysis (PCA) based on the genetic diversity of BOXA1R-PCR spectrum, and the safety and probiotics of LAB were further analyzed. [Results] A total of 58 LAB strains were isolated, from which 20 representative strains were selected for 16S rRNA gene sequencing according to BOXA1R-PCR results. The 20 strains belonged to Streptococcus, Leuconostoc, Weissella, and Enterococcus, respectively. The principal component analysis showed that the community structure of LAB varied in the giant pandas at different ages. All the 20 strains showed negative results in hemolysis, and 17, 11, and 14 strains were susceptible to 11 antibiotics, tolerant to acidic conditions of pH 2.0, and tolerant to 0.3% bile salts, respectively. Strains SW-51, SW-48, MQ-41, SW-58, and MX-23 demonstrated strong inhibitory activities to 3 pathogenic bacterial species. Over 50% of the strains showed high auto-aggregation and co-aggregation capacities. Three strains showed consistent acid production and growth rate, among which Leuconostoc lactis MX-23 showed the best performance in growth and acid production. [Conclusion] The intestines of giant pandas harbor abundant LAB with valuable probiotic characteristics and application prospects, and the structure and composition of the LAB were influenced by age.

    参考文献
    [1] 郭本恒. 益生菌[M]. 北京: 化学工业出版社, 2015: 37-157. GUO BH. Probiotics[M]. Beijing: Chemical Industry Press, 2015: 37-157 (in Chinese).
    [2] 华鹤良. 乳酸菌的分离鉴定及其抗菌肽与发酵性能研究[D]. 扬州: 扬州大学博士学位论文, 2013. HUA HL. Isolation, identification, antibacterial peptide activities and fermentation performance of lactic acid bacteria[D]. Yangzhou: Doctoral Dissertation of Yangzhou University, 2013 (in Chinese).
    [3] 杨洁彬. 乳酸菌: 生物学基础及应用[M]. 北京: 中国轻工业出版社, 1996. YANG JB. Lactic Acid Bacteria: Biological Basis and Application[M]. Beijing: China Light Industry Press, 1996 (in Chinese).
    [4] 侯成立. 罗伊氏乳杆菌全基因组序列分析及其调节仔猪肠黏膜免疫功能的研究[D]. 北京: 中国农业大学博士学位论文, 2015. HOU CL. Whole genome sequence analysis of Lactobacillus reuteri and its regulation in intestinal mucosal immune of piglets[D]. Beijing: Doctoral Dissertation of China Agricultural University, 2015 (in Chinese).
    [5] 崔明全, 何廷美, 钟志军, 王承东, 张婷婷, 周潇潇, 周紫峣, 李德生, 张和民, 李才武, 何永果, 彭广能. 大熊猫粪便菌群ERIC-PCR指纹图谱的分析及优势菌群的鉴定[J].畜牧与兽医, 2013, 45(9): 6-11. CUI MQ, HE TM, ZHONG ZJ, WANG CD, ZHANG TT, ZHOU XX, ZHOU ZY, LI DS, ZHANG HM, LI CW, HE YG, PENG GN. Analysis of characteristics of faecal flora pandas by ERIC-PCR fingerprinting and identification of the dominant flora[J]. Animal Husbandry & Veterinary Medicine, 2013, 45(9): 6-11 (in Chinese).
    [6] 凌代文, 东秀珠. 乳酸细菌分类鉴定及实验方法[M].北京:中国轻工业出版社, 1999: 85-124. LIN DW, DONG XZ. Classification and Identification of Lactic Acid Bacteria and Experimental Methods[M]. Beijing: China Light Industry Press, 1999: 85-124 (in Chinese).
    [7] 夏雪娟, 陈芝兰, 陈宗道, 阚建全, 杨吉霞. 16S rDNA序列分析法快速鉴定西藏地区传统乳制品中的乳酸菌[J]. 食品科学, 2013, 34(14): 245-249. XIA XJ, CHEN ZL, CHEN ZD, KAN JQ, YANG JX. Rapid identification of lactic acid bacteria from traditional dairy products in Tibet area by 16S rDNA sequence analysis[J]. Food Science, 2013, 34(14): 245-249 (in Chinese).
    [8] WAITZ JA. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard—Ninth Edition[M]. Pennsylvania: Clinical & Laboratory Standards Institute, 2012.
    [9] 何杉杉, 王晓蕊, 彭禹熙, 马立娟, 杜丽平. 雪莲菌中乳酸菌的益生特性[J].食品科学, 2022, 43(2): 210-216. HE SS, WANG XR, PENG YX, MA LJ, DU LP. Probiotic properties of lactic acid bacteria isolated from Tibetan kefir grain[J]. Food Science, 2022, 43(2): 210-216 (in Chinese).
    [10] ZHANG Y, WU YT, ZHENG W, HAN XX, JIANG YH, HU PL, TANG ZX, SHI LE. The antibacterial activity and antibacterial mechanism of a polysaccharide from Cordyceps cicadae[J]. Journal of Functional Foods, 2017, 38: 273-279.
    [11] WANG J, PU Y, ZENG Y, CHEN YY, ZHAO W, NIU LL, CHEN BH, YANG ZH, WU LQ, PAN KC, JING B, ZENG D, NI XQ. Multi-functional potential of five lactic acid bacteria strains derived from giant panda (Ailuropoda melanoleuca)[J]. Probiotics and Antimicrobial Proteins, 2022: 1-14.
    [12] 纪绍梅. 微生物培养基质控与图解[M].北京:北京科学技术出版社, 2006. JI SM. Quality Control and Illustration of Microbiological Medium[M]. Beijing: Beijing Science & Technology Press, 2006 (in Chinese).
    [13] LEE J, MO JH, KATAKURA K, ALKALAY I, RUCKER AN, LIU YT, LEE HK, SHEN C, COJOCARU G, SHENOUDA S, KAGNOFF M, ECKMANN L, BEN-NERIAH Y, RAZ E. Maintenance of colonic homeostasis by distinctive apical TLR9 signalling in intestinal epithelial cells[J]. Nature Cell Biology, 2006, 8(12): 1327-1336.
    [14] 李永儒, 丁晓贝, 左浩江, 王嵬, 黄玮, 裴晓方. 肠道菌群与粪便性状关系初探[J].现代预防医学, 2010, 37(9): 1623-1625. LI YR, DING XB, ZUO HJ,WANG W, HUANG W, PEI XF. Preliminary Investigation of the Relationship between enteric bacteria and character of feces[J]. Modern Preventive Medicine, 2010, 37(9): 1623-1625 (in Chinese).
    [15] 张志和, 何光昕, 王行亮, 钟顺隆, 张安居, 李光汉. 大熊猫肠道正常菌群的研究[J]. 兽类学报, 1995, 15(3): 170-175. ZHANG ZH, HE GT, ZHANG AJ, WANG HL, ZHONG SL, ZHANG AJ, LI GH. Study on the normal flora of the giant panda’s intestines[J]. Acta Theriologica Sinica, 1995, 15(3): 170-175 (in Chinese).
    [16] HIRAYAMA K, KAWAMURA S, MITSUOKA T, TASHIRO K. The faecal flora of the giant panda (Ailuropoda melanoleuca)[J]. The Journal of Applied Bacteriology, 1989, 67(4): 411-415.
    [17] 王晓艳. 成年与老年大熊猫肠道菌群16S rDNA-RFLP技术分析[D]. 雅安: 四川农业大学硕士学位论文, 2013. WANG XY. Analyze the intestinal flora of adult and elderly pandas by 16S rDNA-RFLP technolgy[D]. Yaan: Master’s Thesis of Sichuan Agricultural University, 2013 (in Chinese).
    [18] 简平, 王强, 王剑, 牛李丽, 祝辉, 曾燕, 倪学勤, 曾东. 不同年龄段川金丝猴肠道菌群结构差异分析[J]. 动物营养学报, 2015, 27(4): 1302-1309. JIAN P, WANG Q, WANG J, NIU LL, ZHU H, ZENG Y, NI XQ, ZENG D. Difference analysis of gut microbiome of Rhinopithecus roxellana in different ages[J]. Chinese Journal of Animal Nutrition, 2015, 27(4): 1302-1309 (in Chinese).
    [19] 郭壮. 应用焦磷酸测序技术对不同人群肠道微生物群落结构的研究[D]. 无锡: 江南大学博士学位论文, 2013. GUO Z. Study on intestinal microbial community structure of different populations by pyrophosphate sequencing technology[D]. Wuxi: Doctoral Dissertation of Jiangnan University, 2013 (in Chinese).
    [20] CLAESSON MJ, CUSACK S, OSULLIVAN O, GREENE-DINIZ R, WEERD H, FLANNERY, E, MARCHESI, JR, FALUSH D, DINAN T, FITZGERALD G, STANTON C, SINDEREN D, O’CONNOR M, HARNEDY N, O’CONNOR K, HENRY C, O'MAHONY D, FITZGERALD AP, SHANAHAN F, TWOMEY C, HILL C, ROSS RP, O’TOOLE PW. Composition, variability, and temporal stability of the intestinal microbiota of the elderly[J]. Proceedings of the National Academy of Sciences, 2011, 108(supplement_1): 4586-4591.
    [21] 郭飞翔. 广西巴马长寿地区不同年龄人群肠道菌群分析[D]. 扬州: 扬州大学硕士学位论文, 2015. GUO FX. Analysis of intestinal flora of people of different ages in Changshou area of Bama, Guangxi[D]. Yangzhou: Master’s Thesis of Yangzhou University, 2015 (in Chinese).
    [22] IBRAHIM SA, AYIVI RD, ZIMMERMAN T, SIDDIQUI SA, ALTEMIMI AB, FIDAN H, ESATBEYOGLU T, BAKHSHAYESH RV. Lactic acid bacteria as antimicrobial agents: food safety and microbial food spoilage prevention[J]. Foods (Basel, Switzerland), 2021, 10(12): 3131.
    [23] TEUBER M, MEILE L, SCHWARZ F. Acquired antibiotic resistance in lactic acid bacteria from food[J]. Antonie Van Leeuwenhoek, 1999, 76(1-4): 115-137.
    [24] GEVERS D, HUYS G, SWINGS J. In vitro conjugal transfer of tetracycline resistance from Lactobacillus isolates to other Gram-positive bacteria[J]. FEMS Microbiology Letters, 2003, 225(1): 125-130.
    [25] ARGYRI AA, ZOUMPOPOULOU G, KARATZAS KA G, TSAKALIDOU E, NYCHAS GJ E, PANAGOU EZ, TASSOU CC. Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests[J]. Food Microbiology, 2013, 33(2): 282-291.
    [26] 聂紫玉, 吴艳阳, 王增光, 李子晗, 康文丽, 潘丽娜, 汪家琦, 戴智勇, 赵玲艳, 邓放明. 植物源益生乳酸菌的筛选及其特性[J].食品科学, 2022, 43(18): 143-151. NIE ZY, WU YY, WANG ZG, LI ZH, KANG WL, PAN LN, WANG JQ, DAI ZY, ZHAO LY, DENG FM. Screening and characterization of plant-derived probiotic lactic acid bacteria[J]. Food Science, 2022, 43(18): 143-151 (in Chinese).
    [27] 胡爱华, 敖晓琳, 陈岑, 蒲彪, 陈安均, 姜欢笑. 乳酸菌耐酸耐胆盐机制的研究进展[J].食品工业科技, 2015, 36(8): 380-383, 389. HU AH, AO XL, CHEN C, PU B, CHEN AJ, JIANG HX. Research progress on mechanism of lactic acid bacteria acid and bile salt resistance[J]. Science and Technology of Food Industry, 2015, 36(8): 380-383, 389 (in Chinese).
    [28] GRIGORYAN S, BAZUKYAN I, TRCHOUNIAN A. Aggregation and adhesion activity of lactobacilli isolated from fermented products in vitro and in vivo: a potential probiotic strain[J]. Probiotics and Antimicrobial Proteins, 2018, 10(2): 269-276.
    [29] 麦热姆妮萨·艾麦尔, 乌斯满·依米提, 阿迪拉·多力坤, 多力坤·麦麦提优素莆. 具有高效抑菌活性的肠道乳酸菌的筛选[J]. 食品工业科技, 2012, 33(24): 210-214. AMAR M, YIMIT W, DOLKUN A, MAMATYUSUF D. Selection of high-efficient antibacterial intestinal lactic acid bacteria[J]. Science and Technology of Food Industry, 2012, 33(24): 210-214 (in Chinese).
    [30] 万荣峰, 江善祥. 3株乳酸菌体外拮抗致病性大肠杆菌试验[J]. 畜牧与兽医, 2007, 39(3): 50-52. WAN RF, JIANG SX. Antagonism of three lactic acid bacteria against pathogenic Escherichia coli in vitro[J]. Animal Husbandry & Veterinary Medicine, 2007, 39(3): 50-52 (in Chinese).
    [31] 周罗雄. 乳酸菌对肉鸡肠道中肠炎沙门氏菌的抑菌作用研究[D]. 杨凌: 西北农林科技大学硕士学位论文, 2014. ZHOU LX. Study on bacteriostasis of lactic acid bacteria against Salmonella enteritidis in broiler intestine[D]. Yangling: Master’s Thesis of Northwest A&F University, 2014 (in Chinese).
    [32] 崔美岩, 吕好新, 张志霞, 张淼, 张蓓, 陈骏, 谈重芳. 青海湖裸鲤肠道乳酸菌多样性与抑菌活性[J]. 微生物学通报, 2016, 43(9): 2028-2039. ZHANG ZX, ZHANG M, ZHANG B, CHEN J, TAN ZF. Diversity and antimicrobial activity of intestinal lactic acid bacteria isolated from Gymnocypris przewalskii[J]. Microbiology China, 2016, 43(9): 2028-2039 (in Chinese).
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李倜,胡昕,张潇月,杨晨曦,李静,曹雪笛,石雪雪,邹立扣,赵珂. 圈养大熊猫肠道乳酸菌分离及益生特性[J]. 微生物学通报, 2023, 50(9): 4063-4077

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  • 收稿日期:2023-02-14
  • 录用日期:2023-04-10
  • 在线发布日期: 2023-09-04
  • 出版日期: 2023-09-20
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