畜牧与饲料科学 ›› 2023, Vol. 44 ›› Issue (6): 31-40.doi: 10.12160/j.issn.1672-5190.2023.06.004

• 动物营养与饲料科学 • 上一篇    下一篇

金针菇菌糠纤维素降解菌的分离、生物学特性及酶活性分析

周云昊1,2, 李小冬1,3, 宋莉1,2, 尚以顺1, 刘凤丹1, 裴成江1, 李世歌1, 刘军林4   

  1. 1.贵州省农业科学院/贵州省草业研究所,贵州 贵阳 550006;
    2.贵州大学生命科学学院/贵州大学农业生物工程研究院/山地植物资源保护与种质创新教育部重点实验室/贵州省农业生物工程重点实验室,贵州 贵阳 550025;
    3.贵阳康养职业技术大学,贵州 贵阳 550000;
    4.威宁彝族回族苗族自治县山地特色农业科学研究所,贵州 威宁 553100
  • 收稿日期:2023-09-26 出版日期:2023-11-30 发布日期:2024-01-11
  • 通讯作者: 李小冬(1984—),男,研究员,博士,硕士生导师,主要研究方向为饲草料加工与反刍动物营养。宋莉(1970—),女,教授,博士,硕士生导师,主要研究方向为发酵工程。
  • 作者简介:周云昊(1999—),男,硕士,主要研究方向为饲料发酵工程。
  • 基金资助:
    贵州省科技支撑计划项目(黔科合支撑[2020]1Y080); 贵州省农科院高价值专利培育项目(黔农科院专利培计[2021]04); 贵州省科研机构创新能力建设项目(黔科合服企[2022]004)

Isolation,Biological Characteristics and Enzyme Activity Analysis of Cellulolytic Bacteria from Spent Flammulina velutipes Substrate

ZHOU Yunhao1,2, LI Xiaodong1,3, SONG Li1,2, SHANG Yishun1, LIU Fengdan1, PEI Chengjiang1, LI Shige1, LIU Junlin4   

  1. 1. Guizhou Academy of Agricultural Sciences/Guizhou Institute of Prataculture,Guiyang 550006,China;
    2. College of Life Sciences/Institute of Agricultural Bioengineering/Key Laboratory of Mountain Plant Resources Conservation and Germplasm Innovation,Ministry of Education/Guizhou Provincial Key Laboratory of Agricultural Bioengineering,Guizhou University,Guiyang 550025,China;
    3. Guiyang Healthcare Vocational University,Guiyang 550000,China;
    4. Mountain Characteristic Agricultural Science Research Institute of Weining Yi,Hui and Miao Autonomous County,Weining 553100,China
  • Received:2023-09-26 Online:2023-11-30 Published:2024-01-11

摘要: [目的]分离筛选金针菇菌糠来源的纤维素降解菌,研究其生物学特性和所产纤维素降解酶活性,为菌糠资源的饲料化利用提供技术支撑。[方法]采用稀释涂布法接种金针菇菌糠样品,采用硝酸纤维素钠培养基进行纤维素降解菌的分离培养,利用刚果红培养基初步分析分离菌株的纤维素降解能力;通过16S rDNA测序分析和生物安全性预测挑选候选菌株,然后进行生理生化特性分析、不同温度下生长曲线测定以及纤维素降解酶活性动态分析。[结果]样品经硝酸纤维素钠培养基筛选培养后获得30个独立菌落,所有菌株在刚果红培养基上都能形成明显的透明圈,部分菌株具有较强的纤维素降解能力。16S rDNA测序分析显示,30株分离菌被鉴定为韩国假单胞杆菌、明斯特小陌生菌、深红沙雷菌等9个菌种;结合生物安全性预测及纤维素降解能力初筛结果,选取韩国假单胞菌(Pseudomonas koreensis,编号:JK-32)、明斯特小陌生菌(Advenella mimigardefordensis,编号:JK-52)、深红沙雷菌(Serratia rubidaea,编号:JK-56)、马葡萄球菌(Staphylococcus equorum,编号:JK-57)、腐生葡萄球菌(Staphylococcus saprophytics,编号:JK-62)、黏质沙雷菌(Serratia marcescens,编号:JK-66)作为候选菌株开展后续试验。生理生化特性分析结果显示,6株候选菌株接触酶活性试验均为阳性,甲基红试验和二乙酰试验均为阴性。JK-62株的生长受温度影响较小,细菌生长速率和最终细菌浓度在所有候选菌株中表现较好;JK-56株在温度较低(28~34 ℃)的培养条件下,生长速率和最终细菌浓度均低于其他候选菌株,而当培养温度上升到40 ℃时,其生长速率与其他菌株相近,并且最终细菌浓度较高。在培养第1~7天,JK-56株的内切葡聚糖酶活性在所有菌株中均最高;在培养第2、4、5天,JK-66株的内切葡聚糖酶活性显著(P<0.05)高于CK菌株。JK-66株的外切葡聚糖酶活性较高,在不同检测时间点均显著(P<0.05)高于CK菌株;在培养第2~4天,JK-56株的外切葡聚糖酶活性显著(P<0.05)高于CK菌株。JK-66株的β-葡萄糖苷酶活性在候选菌株中表现较好;在培养第3~7天,JK-56株的β-葡萄糖苷酶活性在候选菌株中仅次于JK-62株和JK-66株。在培养第2~5天,JK-56株的滤纸酶活性在候选菌株中最高,在培养第5天时显著(P<0.05)高于CK菌株,在此期间,JK-66株的滤纸酶活性也处于较高水平。[结论]深红沙雷菌JK-56株和黏质沙雷菌JK-66株纤维素降解酶活性整体水平较高,且内切葡聚糖酶和外切葡聚糖酶活性表现突出,可以与具有较强β-葡萄糖苷酶活性的菌种配合成复合菌系应用于金针菇菌糠饲料化开发利用。

关键词: 纤维素降解菌, 金针菇菌糠, 分离, 纤维素降解酶活性, 分析

Abstract: [Objective] The aims of the present study were to isolate and screen cellulolytic bacteria derived from spent Flammulina velutipes substrate, to assess their biological characteristics and cellulolytic enzyme activities, so as to provide technical supports for the feed utilization of spent mushroom substrate resources. [Method] Spent Flammulina velutipes substrate samples were inoculated using plate dilution coating method. The cellulolytic bacteria were then isolated and cultured on nitrate cellulose sodium medium. The cellulolytic abilities of the isolated strains were preliminarily analyzed using Congo red medium. Candidate strains were selected through 16S rDNA sequencing analysis and biosafety prediction, followed by physiological and biochemical characteristics analysis, growth curve determination at different temperatures, and dynamic analysis of cellulolytic enzyme activities. [Result] A total of 30 independent colonies were isolated after screening culture with nitrate cellulose sodium medium. All of the strains formed clear transparent circles on Congo red medium, and some strains had strong cellulolytic abilities. The 16S rDNA sequencing analysis showed that the 30 isolated strains were identified as belonging to 9 bacterial species, including Pseudomonas koreensis, Advenella mimigardefordensis, Serratia rubidaea, and so on. Taking into account both biosafety prediction and the preliminary cellulolytic abilities screening results, Pseudomonas koreensis (JK-32), Adrenella mimigardefordensis (JK-52), Serratia rubidaea (JK-56), Staphylococcus equiorum (JK-57), Staphylococcus saprophytics (JK-62) and Serratia marcescens (JK-66) were selected as candidate strains for subsequent experiments. Physiological and biochemical characteristics analysis showed that all of the 6 candidate strains were determined as positive for contact enzyme activity test, and negative for methyl red test and diacetyl test. The growth of JK-62 strain was less affected by temperature, and the growth rate and final bacterial concentration performed better among all the candidate strains. Under the low cultivation temperatures (28-34 ℃), the growth rate and final bacterial concentration of JK-56 strain were lower than the other candidate strains, while when the cultivation temperature increased to 40 ℃, its growth rate was similar to that of the other candidate strains and the final bacterial concentration was higher. On the 1st to 7th days of cultivation, the endoglucanase activity of JK-56 strain was the highest among all the candidate strains. On the 2nd, 4th, and 5th days of cultivation, the endoglucanase activity of JK-66 strain was significantly (P<0.05) higher than that of the CK. The exoglucanase activity of JK-66 strain was higher among the candidate strains and was significantly (P<0.05) higher than that of the CK at different detection time points. On the 2nd to 4th days of cultivation, the exoglucanase activity of JK-56 strain was significantly (P<0.05) higher than that of the CK. The β-glucosidase activity of JK-66 strain was higher among the candidate strains. On the 3rd to 7th days of cultivation, the β-glucosidase activity of JK-56 strain was next to JK-62 and JK-66 strains among the candidate strains. On the 2nd to 5th days of cultivation, the filter paper enzyme activity of JK-56 strain was the highest among the candidate strains. On the 5th day of cultivation, it was significantly (P<0.05) higher than that of the CK. During this period, the filter paper enzyme activity of JK-66 strain was also at a high level. [Conclusion] Serratia rubidaea JK-56 and Serratia marcescens JK-66 strains had high overall levels of cellulolytic enzyme activities, and prominent endoglucanase and exoglucanase activities, which could be used in combination with strains exhibiting strong β-glucosidase activity for the development and utilization of spent Flammulina velutipes substrate.

Key words: cellulolytic bacterium, spent Flammulina velutipes substrate, isolation, cellulolytic enzyme activity, analysis

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