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Article(id=1276204533445431891, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276204178091413862, articleNumber=null, orderNo=null, doi=10.3969/j.issn.1000-2561.2024.12.021, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1716307200000, receivedDateStr=2024-05-22, revisedDate=1717603200000, revisedDateStr=2024-06-06, acceptedDate=null, acceptedDateStr=null, onlineDate=1782200471507, onlineDateStr=2026-06-23, pubDate=1735056000000, pubDateStr=2024-12-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1782200471507, onlineIssueDateStr=2026-06-23, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1782200471507, creator=13701087609, updateTime=1782200471507, updator=13701087609, issue=Issue{id=1276204178091413862, tenantId=1146029695717560320, journalId=1235980609244409860, year='2024', volume='45', issue='12', pageStart='2487', pageEnd='2737', issueExtLink='null', onlineDate='null', pubDate='1735056000000', pubDateStr='2024-12-25', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1782200386783, creator='13701087609', updateTime=1782200456354, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1276204470308565242, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276204178091413862, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1276204470308565243, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276204178091413862, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=2689, endPage=2696, ext={EN=ArticleExt(id=1276204533730644565, articleId=1276204533445431891, tenantId=1146029695717560320, journalId=1235980609244409860, language=EN, title=Optimization of Fermentation Formula and Stability of Cultural Filtrate Produced by Streptomyces samsunensi 17-7, columnId=1236292524264968282, journalTitle=Chinese Journal of Tropical Crops, columnName=Plant Protection & Bio-safety, runingTitle=null, highlight=null, articleAbstract=

In order to enhance the antifungal activity of Streptomyces samsunensis 17-7 strain and effectively utilize its secondary metabolites, this study focused on optimizing the fermentation medium formula. Orthogonal and single factor experiments were conducted based on the original medium, with Phellinus noxious as the indicator. The optimal formulation for producing active substances from the strain was determined to include 25 g/L soy powder, 10 g/L corn meal,10 g/L glucose, 2 g/L yeast extract, 1 g/L K2HPO4, 0.5 g/L NaCl, and 0.5 g/L CaCO3. Following a 500 times dilution, the relative antifungal activity of the fermentation solution increased to 51.37%, a 17.66% improvement compared to the previous optimization, demonstrating a broad spectrum of antifungal activity. The stability of the antifungal substance was evaluated in response to various conditions such as temperature, pH, protease, UV-irradiation, and storage. Results indicated that the fermentation broth of strain 17-7 exhibited high temperature resistance, a significant decrease in antifungal activity after one hour of UV irradiation, but retained its activity after long-term exposure to UV light. Furthermore, the broth demonstrated good acid-base tolerance, effective antibacterial activity in the pH range of 3-10, and resistance to trypsin and pepsin. The fermentation broth of strain 17-7 maintained its antifungal activity for 120 days at both 4 ℃ and room temperature (25 ℃). This study on the stability and storage conditions of strain 17-7’s fermentation broth offers valuable data for potential future applications of the strain as a biocontrol agent.

, authors=null, authorsList=Yixian LIU, Yuping SHI, Lanlan LI, Liming DAI, Zhiying CAI, authorCompany=null, correspAuthors=Zhiying CAI, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1276204535131542115, articleId=1276204533445431891, tenantId=1146029695717560320, journalId=1235980609244409860, language=CN, title=生防链霉菌17-7菌株发酵配方优化及发酵滤液稳定性研究, columnId=1236292524520820846, journalTitle=热带作物学报, columnName=植物保护与生物安全, runingTitle=null, highlight=null, articleAbstract=

为了提升链霉菌17-7菌株的抑菌活性，合理利用菌株的代谢产物，本研究以橡胶树褐根病菌为靶标菌，采用正交试验和单因子试验相结合，在原培养基的基础上对链霉菌17-7菌株的发酵培养基配方进一步优化，获得该菌株最适宜产活性物质的配方为：大豆粉25 g/L、玉米粉10 g/L、葡萄糖10 g/L、酵母粉2 g/L、K2HPO4 1 g/L、NaCl 0.5 g/L、CaCO3 0.5 g/L。发酵滤液稀释500倍后对橡胶树褐根病菌的相对抑菌活性为51.37%，较优化前提高了17.66%，并且具有广谱的抑菌活性。在此基础上测定了温度、紫外线辐射、蛋白酶、酸碱度和贮存条件对17-7菌株发酵滤液抗真菌物质稳定性的影响。结果表明：17-7菌株发酵滤液对高温不敏感；紫外线照射1 h后发酵滤液的抑菌活性开始显著下降，长时间紫外照射并不会丧失抑菌活性；17-7菌株发酵滤液还具有较好的酸碱耐受性，pH为3~10时其发酵滤液均能发挥较好的抑菌活性；并且对胰蛋白酶和胃蛋白酶不敏感；菌株发酵滤液在低温（4 ℃）和室温（25 ℃）条件下贮存120 d仍能保持较强生物活性。对17-7菌株无菌发酵滤液稳定性和贮存条件的研究可为后续该菌株的开发利用提供理论依据和数据支持。

, authors=

刘一贤（1988—），女，硕士，副研究员，研究方向：热带作物病害防治。

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* 蔡志英（CAI Zhiying），E-mail：。
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刘一贤（1988—），女，硕士，副研究员，研究方向：热带作物病害防治。

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刘一贤（1988—），女，硕士，副研究员，研究方向：热带作物病害防治。

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Molecules,2020, 25(14): 3221., articleTitle=Streptomyces-derived metabolites with potential photoprotective properties: a systematic literature review and meta-analysis on the reported chemodiversity, refAbstract=null)], funds=[Fund(id=1276204541435581084, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, awardId=QNCZ2021-2; QNCZ2022-3, language=CN, fundingSource=云南省热带作物科学研究所青年成长基金项目(QNCZ2021-2; QNCZ2022-3), fundOrder=null, country=null), Fund(id=1276204541498495645, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, awardId=RF2024-4-1, language=CN, fundingSource=云南省热带作物科技创新专项资金项目(RF2024-4-1), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1276204535349645925, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, xref=null, ext=[AuthorCompanyExt(id=1276204535358034534, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, companyId=1276204535349645925, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Yunnan Key Laboratory of Sustainable Utilization Research on Rubber Tree / Yunnan Institute of Tropical Crops, Jinghong, Yunnan 666100, China), AuthorCompanyExt(id=1276204535366423143, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, companyId=1276204535349645925, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=云南省天然橡胶可持续利用研究重点实验室/云南省热带作物科学研究所，云南景洪　666100)])], figs=[ArticleFig(id=1276204539900465802, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, language=EN, label=Fig. 1, caption=Effect of different inorganic salts on inhibition activity of submerged cultured strain 17-7, figureFileSmall=LfUyOiCLHEeW4R6Sp7gJ4w==, figureFileBig=FtsC2J/cWv9zWhUYTfaFAw==, tableContent=null), ArticleFig(id=1276204539988546187, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, language=CN, label=图1, caption=不同无机盐对17-7菌株发酵滤液抑菌活性的影响

不同小写字母表示差异显著（P<0.05）。

, figureFileSmall=LfUyOiCLHEeW4R6Sp7gJ4w==, figureFileBig=FtsC2J/cWv9zWhUYTfaFAw==, tableContent=null), ArticleFig(id=1276204540160512652, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, language=EN, label=Fig. 2, caption=Effect of temperature on antifungal activity of cultural filtrate from strain 17-7, figureFileSmall=IG1+dDefpRymQ/ZOLhkmLw==, figureFileBig=wb4hsXz/h/r7/INQj+vchA==, tableContent=null), ArticleFig(id=1276204540219232909, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, language=CN, label=图2, caption=温度对发酵滤液抑菌活性的影响

不同小写字母表示差异显著（P<0.05）。

, figureFileSmall=IG1+dDefpRymQ/ZOLhkmLw==, figureFileBig=wb4hsXz/h/r7/INQj+vchA==, tableContent=null), ArticleFig(id=1276204540328284814, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, language=EN, label=Fig. 3, caption=Effect of UV irradiation on antifungal activity of cultural filtrate from strain 17-7, figureFileSmall=6D5xds+wDbVJhGu6nYWwJw==, figureFileBig=BRaBngtcAsWe+K51C/K6wQ==, tableContent=null), ArticleFig(id=1276204540420559503, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, language=CN, label=图3, caption=紫外照射时间对发酵滤液抑菌活性的影响

不同小写字母表示差异显著（P<0.05）。

, figureFileSmall=6D5xds+wDbVJhGu6nYWwJw==, figureFileBig=BRaBngtcAsWe+K51C/K6wQ==, tableContent=null), ArticleFig(id=1276204540483474064, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, language=EN, label=Fig. 4, caption=Effect of protease irradiation on antifungal activity of cultural filtrate from strain 17-7, figureFileSmall=fVFVRTCnqv/li3SJkKYIRA==, figureFileBig=RBHdw2pb/2XTzysskTTIDA==, tableContent=null), ArticleFig(id=1276204540554777233, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, language=CN, label=图4, caption=蛋白酶对发酵滤液抑菌活性的影响

不同小写字母表示差异显著（P<0.05）。

, figureFileSmall=fVFVRTCnqv/li3SJkKYIRA==, figureFileBig=RBHdw2pb/2XTzysskTTIDA==, tableContent=null), ArticleFig(id=1276204540621886098, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, language=EN, label=Fig. 5, caption=Effect of pH irradiation on antifungal activity of cultural filtrate from strain 17-7, figureFileSmall=0uZWqmH84epPpIPdaKbUZw==, figureFileBig=kp4O8Y4Jt2bWXgzlyJY2RQ==, tableContent=null), ArticleFig(id=1276204540693189267, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, language=CN, label=图5, caption=pH对发酵滤液抑菌活性的影响

不同小写字母表示差异显著（P<0.05）。

, figureFileSmall=0uZWqmH84epPpIPdaKbUZw==, figureFileBig=kp4O8Y4Jt2bWXgzlyJY2RQ==, tableContent=null), ArticleFig(id=1276204540764492436, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, language=EN, label=Fig. 6, caption=Effect of storage irradiation on antifungal activity of cultural filtrate from strain 17-7, figureFileSmall=MbllqpAoPstmm7gurCAkTg==, figureFileBig=CeEPjhMo8ZE9qj67/ZytAg==, tableContent=null), ArticleFig(id=1276204540844184213, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, language=CN, label=图6, caption=储藏时间对发酵滤液抑菌活性的影响, figureFileSmall=MbllqpAoPstmm7gurCAkTg==, figureFileBig=CeEPjhMo8ZE9qj67/ZytAg==, tableContent=null), ArticleFig(id=1276204540915487382, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, language=EN, label=Tab. 1, caption=

Factors and levels design of orthogonal test for fermentation conditions

, figureFileSmall=null, figureFileBig=null, tableContent=
水平Level因素Factor
A大豆粉Soy powder/(g·L-1)B玉米粉Corn meal/(g·L-1)C葡萄糖Glucose/(g·L-1)D酵母粉Yeast extract/(g·L-1)
110552
21510104
32015156
42520208
), ArticleFig(id=1276204540982596247, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, language=CN, label=表1, caption=

发酵条件正交试验的因素和水平设计

, figureFileSmall=null, figureFileBig=null, tableContent=
水平Level因素Factor
A大豆粉Soy powder/(g·L-1)B玉米粉Corn meal/(g·L-1)C葡萄糖Glucose/(g·L-1)D酵母粉Yeast extract/(g·L-1)
110552
21510104
32015156
42520208
), ArticleFig(id=1276204541049705112, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, language=EN, label=Tab. 2, caption=

Results of orthogonal test with carbon sources and nitrogen source on antifungal activity of submerged cultured 17-7

, figureFileSmall=null, figureFileBig=null, tableContent=
编号No.因素Factor抑菌率Inhibition ratio/%
ABCD
111114.62±1.07e
2122227.64±2.72cd
3133325.68±4.33cd
4144425.68±4.33cd
5212312.00±6.59e
6221426.27±4.20cd
7234141.25±3.28b
8243236.61±7.23bc
9313425.19±1.19cd
10324321.28±2.50d
11331211.76±1.82e
12342159.58±4.46a
13414235.72±2.72bc
14423141.20±2.56b
15432440.15±3.35b
16441328.39±4.70cd
K120.9119.3817.7636.66
K229.0329.1034.8427.93
K329.4529.7132.1721.84
K436.3737.5730.9829.32
R15.4618.1817.0814.83
), ArticleFig(id=1276204541146174105, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, language=CN, label=表2, caption=

17-7菌株发酵培养基中碳源、氮源正交试验结果

, figureFileSmall=null, figureFileBig=null, tableContent=
编号No.因素Factor抑菌率Inhibition ratio/%
ABCD
111114.62±1.07e
2122227.64±2.72cd
3133325.68±4.33cd
4144425.68±4.33cd
5212312.00±6.59e
6221426.27±4.20cd
7234141.25±3.28b
8243236.61±7.23bc
9313425.19±1.19cd
10324321.28±2.50d
11331211.76±1.82e
12342159.58±4.46a
13414235.72±2.72bc
14423141.20±2.56b
15432440.15±3.35b
16441328.39±4.70cd
K120.9119.3817.7636.66
K229.0329.1034.8427.93
K329.4529.7132.1721.84
K436.3737.5730.9829.32
R15.4618.1817.0814.83
), ArticleFig(id=1276204541238448794, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, language=EN, label=Tab. 3, caption=

Inhibition of 11 pathogenic fungi by fermentation filtrate of strain 17-7 at different dilution ratios

, figureFileSmall=null, figureFileBig=null, tableContent=
编号No.病原菌Pathogen抑菌率Inhibition ratio/%
稀释100倍Diluted 100 times稀释500倍Diluted 500 times
1云南链格孢(A. yunnanensis)87.08±0.66b64.96±0.15e
2橡胶树炭疽病菌(C. bannaense)78.68±0.65ef68.33±1.22d
3橡胶树炭疽病菌(C. brevisporum)84.29±0.22c77.94±0.52ab
4橡胶树炭疽病菌(C. fructicola)86.77±1.84b75.71±0.37b
5橡胶树炭疽病菌(C. karstii)80.51±1.30de72.20±3.56c
6橡胶树炭疽病菌(C. plurivorum)80.37±1.13def78.89±1.11a
7橡胶树炭疽病菌(C. siamense)88.94±0.32a69.03±0.52d
8橡胶树炭疽病菌(C. wanningense)81.08±0.58d69.40±0.98d
9橡胶树棒孢霉落叶病菌(C. cassiicola)78.54±1.27f56.72±0.76f
10橡胶树褐根病菌(P. noxius)83.93±1.49c51.37±1.66g
11橡胶树白根病菌(R. lignosus)57.45±0.44g42.57±0.84h
), ArticleFig(id=1276204541334917787, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276204533445431891, language=CN, label=表3, caption=

17-7菌株不同稀释倍数发酵滤液对11种病原真菌的抑制作用

, figureFileSmall=null, figureFileBig=null, tableContent=
编号No.病原菌Pathogen抑菌率Inhibition ratio/%
稀释100倍Diluted 100 times稀释500倍Diluted 500 times
1云南链格孢(A. yunnanensis)87.08±0.66b64.96±0.15e
2橡胶树炭疽病菌(C. bannaense)78.68±0.65ef68.33±1.22d
3橡胶树炭疽病菌(C. brevisporum)84.29±0.22c77.94±0.52ab
4橡胶树炭疽病菌(C. fructicola)86.77±1.84b75.71±0.37b
5橡胶树炭疽病菌(C. karstii)80.51±1.30de72.20±3.56c
6橡胶树炭疽病菌(C. plurivorum)80.37±1.13def78.89±1.11a
7橡胶树炭疽病菌(C. siamense)88.94±0.32a69.03±0.52d
8橡胶树炭疽病菌(C. wanningense)81.08±0.58d69.40±0.98d
9橡胶树棒孢霉落叶病菌(C. cassiicola)78.54±1.27f56.72±0.76f
10橡胶树褐根病菌(P. noxius)83.93±1.49c51.37±1.66g
11橡胶树白根病菌(R. lignosus)57.45±0.44g42.57±0.84h
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生防链霉菌17-7菌株发酵配方优化及发酵滤液稳定性研究
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刘一贤 , 施玉萍 , 李岚岚 , 戴利铭 , 蔡志英 *
热带作物学报 | 植物保护与生物安全 2024,45(12): 2689-2696
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热带作物学报 |植物保护与生物安全 2024 , 45 (12) : 2689 -2696
生防链霉菌17-7菌株发酵配方优化及发酵滤液稳定性研究
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刘一贤, 施玉萍, 李岚岚, 戴利铭, 蔡志英*
作者信息
  • 云南省天然橡胶可持续利用研究重点实验室/云南省热带作物科学研究所，云南景洪　666100
通讯作者:
* 蔡志英（CAI Zhiying），E-mail：。
Optimization of Fermentation Formula and Stability of Cultural Filtrate Produced by Streptomyces samsunensi 17-7
Yixian LIU, Yuping SHI, Lanlan LI, Liming DAI, Zhiying CAI*
Affiliations
  • Yunnan Key Laboratory of Sustainable Utilization Research on Rubber Tree / Yunnan Institute of Tropical Crops, Jinghong, Yunnan 666100, China
出版时间: 2024-12-25 doi: 10.3969/j.issn.1000-2561.2024.12.021
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摘要
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为了提升链霉菌17-7菌株的抑菌活性，合理利用菌株的代谢产物，本研究以橡胶树褐根病菌为靶标菌，采用正交试验和单因子试验相结合，在原培养基的基础上对链霉菌17-7菌株的发酵培养基配方进一步优化，获得该菌株最适宜产活性物质的配方为：大豆粉25 g/L、玉米粉10 g/L、葡萄糖10 g/L、酵母粉2 g/L、K2HPO4 1 g/L、NaCl 0.5 g/L、CaCO3 0.5 g/L。发酵滤液稀释500倍后对橡胶树褐根病菌的相对抑菌活性为51.37%，较优化前提高了17.66%，并且具有广谱的抑菌活性。在此基础上测定了温度、紫外线辐射、蛋白酶、酸碱度和贮存条件对17-7菌株发酵滤液抗真菌物质稳定性的影响。结果表明：17-7菌株发酵滤液对高温不敏感；紫外线照射1 h后发酵滤液的抑菌活性开始显著下降，长时间紫外照射并不会丧失抑菌活性；17-7菌株发酵滤液还具有较好的酸碱耐受性，pH为3~10时其发酵滤液均能发挥较好的抑菌活性；并且对胰蛋白酶和胃蛋白酶不敏感；菌株发酵滤液在低温（4 ℃）和室温（25 ℃）条件下贮存120 d仍能保持较强生物活性。对17-7菌株无菌发酵滤液稳定性和贮存条件的研究可为后续该菌株的开发利用提供理论依据和数据支持。

链霉菌17-7  /  发酵配方优化  /  稳定性  /  抑菌活性

In order to enhance the antifungal activity of Streptomyces samsunensis 17-7 strain and effectively utilize its secondary metabolites, this study focused on optimizing the fermentation medium formula. Orthogonal and single factor experiments were conducted based on the original medium, with Phellinus noxious as the indicator. The optimal formulation for producing active substances from the strain was determined to include 25 g/L soy powder, 10 g/L corn meal,10 g/L glucose, 2 g/L yeast extract, 1 g/L K2HPO4, 0.5 g/L NaCl, and 0.5 g/L CaCO3. Following a 500 times dilution, the relative antifungal activity of the fermentation solution increased to 51.37%, a 17.66% improvement compared to the previous optimization, demonstrating a broad spectrum of antifungal activity. The stability of the antifungal substance was evaluated in response to various conditions such as temperature, pH, protease, UV-irradiation, and storage. Results indicated that the fermentation broth of strain 17-7 exhibited high temperature resistance, a significant decrease in antifungal activity after one hour of UV irradiation, but retained its activity after long-term exposure to UV light. Furthermore, the broth demonstrated good acid-base tolerance, effective antibacterial activity in the pH range of 3-10, and resistance to trypsin and pepsin. The fermentation broth of strain 17-7 maintained its antifungal activity for 120 days at both 4 ℃ and room temperature (25 ℃). This study on the stability and storage conditions of strain 17-7’s fermentation broth offers valuable data for potential future applications of the strain as a biocontrol agent.

Streptomyces samsunensi 17-7  /  fermentation medium optimization  /  stability  /  antifungal activity
刘一贤, 施玉萍, 李岚岚, 戴利铭, 蔡志英. 生防链霉菌17-7菌株发酵配方优化及发酵滤液稳定性研究. 热带作物学报, 2024 , 45 (12) : 2689 -2696 . DOI: 10.3969/j.issn.1000-2561.2024.12.021
Yixian LIU, Yuping SHI, Lanlan LI, Liming DAI, Zhiying CAI. Optimization of Fermentation Formula and Stability of Cultural Filtrate Produced by Streptomyces samsunensi 17-7[J]. Chinese Journal of Tropical Crops, 2024 , 45 (12) : 2689 -2696 . DOI: 10.3969/j.issn.1000-2561.2024.12.021
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链霉菌（Streptomyces）是放线菌门中最为庞大且极具代表性的分支，具有强大的生态适应能力，广泛分布于各种环境中[1]。并且链霉菌也是目前最大的新型次生代谢产物资源菌，是天然产物的重要工业生产菌[2]。链霉菌产生的活性物质可用作农用杀菌剂、杀虫剂、除草剂和植物生长调节剂，因其具有高效、低毒和环境友好等特点在农作物病虫草害绿色防控中占据重要地位[3]。
链霉菌具有大量编码天然产物的生物合成基因簇，然而大部分基因簇在常规条件下处于沉默状态[4]。为了增强菌株代谢产物活性，激活链霉菌沉默基因表达，以获得更多的活性天然产物，非定向激活策略是普遍使用的一种方法，即通过改变培养基的组成（碳源、氮源、无机盐）等方式，诱导菌株产生更多的代谢产物[5-7]。
天然橡胶是我国热区重要的农业产业，也是国家重要的战略物资。我国橡胶树上已报道的病害有91种，局部或全面严重危害的有9种[8]，已成为制约橡胶产业发展的重要因素。运用微生物资源或其代谢产物进行生物防治已成为绿色农业潮流下的主要趋势。Streptomyces samsunensi 17-7菌株是由本实验室从橡胶树根际土壤中分离到的一株具有巨大生防潜力的链霉菌，对橡胶树褐根病菌具有较强抑制作用[9]。但近期试验发现17-7菌株发酵滤液的抑菌活性有所下降，稀释500倍后对橡胶褐根病菌的相对抑菌活性为33.71%，并且该菌株发酵滤液对外界环境条件的稳定性仍不明确。为了提升17-7菌株的抑菌活性，获得稳定的活性物质，并进一步研究其发酵滤液的稳定性。本研究通过改变培养基组成，在原培养基的基础上添加玉米粉和酵母粉，采用正交试验和单因子试验相结合，筛选出对橡胶褐根病菌具有最佳抑菌活性的培养基组成；并进一步通过单因子变量试验研究外界环境对17-7菌株发酵滤液抑菌活性的影响，为更好地发挥菌株的生防潜力提供理论依据和数据支持。
1　材料与方法
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1.1　材料
1.1.1　供试菌株
17-7菌株（S. samsunensi）、橡胶树白根病菌（Rigidoporus lignosus）、橡胶树褐根病菌（Phellinus noxius）、云南链格孢（Alternaria yunnanensis）、橡胶树棒孢霉落叶病菌（Corynespora cassiicola）、橡胶树炭疽病菌（Colletotrichum siamense、C. karstii、C. wanningense、C. plurivorum、C. fructicola、C. bannaense、C. brevisporum）均由云南省热带作物科学研究所植物保护与微生物利用研究中心分离保存。
1.1.2　培养基
种子液培养为液体高氏一号培养基（每升含可溶性淀粉20.0 g，KNO3 1.0 g，K2HPO4 0.5 g，MgSO4·7H2O 0.5 g，NaCl 0.5 g，FeSO4·7H2O 0.01 g，琼脂20 g，pH为7.4~7.6）；发酵基础培养基[9]（每升含葡萄糖20 g、大豆粉25 g、K2HPO4 1 g、NaCl 0.5 g、CaCO3 0.5 g）；马铃薯葡萄糖琼脂培养基（PDA）。
1.1.3　主要仪器
CLIMACELL培养箱（美国3M）；BSD-400博迅振荡培养箱。
1.2　方法
1.2.1　放线菌发酵滤液的制备
种子液：挑取单个菌落接种于100 mL的液体高氏一号培养基中，于28 ℃、140 r/min振荡培养5 d，制成种子液。
发酵：发酵基础培养基初始pH为8，培养基装瓶量为150 mL/500 mL，摇床转速为140 r/min，种子液的接种量为10%，在28 ℃条件下培养5 d后，12 000 r/min离心15 min，经0.22 µm滤膜过滤，获得无菌发酵滤液。
1.2.2　菌丝生长速率法
以橡胶树褐根病菌为指示菌，采用内径为5 mm的打孔器在活化的橡胶树褐根病菌菌落周围打取菌饼。将PDA培养基融化冷却至50 ℃左右，取1 mL滤液与99 mL PDA培养基混匀，倒入无菌培养皿中制成带毒培养基平板。在每个平板中接入1个菌饼，带菌丝的一面贴在培养基表面，以无菌水与培养基混合的平板为对照。28 ℃下暗培养4 d后，用十字交叉法测定供试菌生长直径，计算抑制率，抑制率=[(对照菌直径-处理菌直径)/(对照菌直径-菌饼直径)]×100%。
1.2.3　双碳源、双氮源对17-7菌株抑菌物质产生的影响
双碳源和双氮源正交试验：基于发酵基础培养基，选择双碳源（葡萄糖、酵母粉）和双氮源（大豆粉、玉米粉）共4个因素，设置4个水平（表1），选用L16(44)正交表进行正交试验，发酵试验重复3次。将菌株发酵滤液稀释5倍后取1 mL与99 mL PDA培养基混匀，制成含毒平板。根据菌丝生长速率法测定抑菌率。
1.2.4　无机盐筛选
以1.2.3中筛选出的双碳源、双氮源培养基为基础，选择CuSO4、ZnSO4、MnCl2、NaCl、FeSO4、K2HPO4、CaCO3、MgSO4、KCl等9种无机盐，按质量体积分数为0.1%加入双碳氮源培养基，配成含不同无机盐离子的培养基。测定不同无机盐对17-7菌株产生抑菌物质的影响，每个处理3次重复，选择抑菌活性较好的3种无机盐。根据菌丝生长速率法测定抑菌率。
1.2.5　优化后培养基发酵滤液抑菌谱测定
分别取优化后的菌株发酵滤液原液1 mL或稀释5倍后取1 mL与99 mL PDA培养基混匀，倒入无菌培养皿中分别制成含稀释100倍与稀释500倍发酵滤液的PDA平板，按照1.2.2的方法接种11株病原菌，并测定抑菌率，对比优化后培养基不同稀释倍数对供试病原菌的抑菌活性。
1.2.6　发酵滤液稳定性测试
（1）温度稳定性。将发酵滤液分别于20、40、60、80、100 ℃水浴处理30 min，取菌株发酵滤液1 mL与99 mL PDA培养基混匀。每处理重复3次，根据菌丝生长速率法测定抑菌率。
（2）紫外稳定性。将发酵滤液置于15 W紫外灯下约20 cm处，分别照射1、2、3、4、5、6 h。每处理重复3次，根据菌丝生长速率法测定抑菌率。
（3）蛋白酶稳定性。在发酵滤液中分别加入终浓度为1 mg/mL的胃蛋白酶、胰蛋白酶和蛋白酶K，于37 ℃处理2 h，以未添加蛋白酶处理的发酵滤液为对照。每处理重复3次，根据菌丝生长速率法测定抑菌率。
（4）酸碱稳定性。采用1 mol/L NaOH和1 mol/L HCl调节发酵滤液pH为1.0、2.0、3.0、4.0、5.0、6.0、7.0、8.0、9.0、10.0、11.0、12.0、13.0，放置24 h后再次将pH调回中性。每处理重复3次，根据菌丝生长速率法测定抑菌率。
（5）储藏稳定性。将发酵滤液分别置于4 ℃和25 ℃下放置3、6、9、15、30、60、120 d。每处理重复3次，根据菌丝生长速率法测定抑菌率。
1.3　数据处理
利用Excel 2003和SAS 9.4M6软件对试验数据进行统计分析，利用GraphPad Prism 8.0软件作图，并应用Duncan’s新复极差法进行差异显著性检验。
2　结果与分析
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2.1　17-7菌株碳、氮源正交试验
选择葡萄糖、酵母粉、大豆粉、玉米粉4个因素，进行4因素4水平正交试验（表2），根据抑菌活性结果，确定最终发酵配方。正交试验结果表明，4个因素对橡胶褐根病菌抑制率的影响顺序为B>C>A>D，最佳水平组合为A4B4C2D1。根据方差分析的结果，主效因子B在2、3、4水平差异不显著，出于成本考虑，最佳组合为A4B2C2D1，即大豆粉25 g，玉米粉10 g，葡萄糖10 g，酵母粉2 g。
2.2　不同无机盐对17-7菌株抑菌活性的影响
供试的9种无机盐对17-7菌株抑菌活性的影响具有显著差异（图1）。添加K2HPO4和CaCO3的抑菌活性显著高于其他无机盐离子，其次为NaCl，选取抑菌活性较高的3种无机盐离子进行后续试验。根据前期研究结果[9]，K2HPO4选择添加量为1 g时，17-7菌株的抑菌活性最好，NaCl和CaCO3不同水平添加量的抑菌率无显著差异，因此选择NaCl和CaCO3添加量为0.05%，即1 L发酵培养基中含大豆粉25 g、玉米粉10 g、葡萄糖10 g、酵母粉2 g、K2HPO4 1 g、NaCl 0.5 g、CaCO3 0.5 g。
2.3　17-7菌株发酵滤液抑菌谱测定
采用优化后17-7菌株的发酵滤液，对供试的11种橡胶树病原菌进行抑菌活性测定，发现17-7菌株具有广谱的抑菌活性。发酵滤液在稀释100倍和500倍时均具有较强的抑菌活性，对橡胶褐根病菌的抑菌率分别为83.93%和51.37%，稀释500倍时抑菌率较优化前提高了17.66%。17-7菌株发酵滤液对炭疽病菌的抑菌效果较突出，稀释500倍时其抑菌率仍能达到68.33%~78.89%（表3）。
2.4　发酵滤液的温度稳定性
由图2可以看出，在20~100 ℃范围内，17-7菌株发酵滤液热处理30 min均具有较好的抑菌活性，抑菌率均在80%以上，其中，20~80 ℃范围内的抑菌活性最佳，与CK发酵滤液抑菌活性无显著性差异；100 ℃时，17-7菌株发酵滤液的抑菌活性略有下降，其抑菌率为84.31%。发酵滤液在100 ℃处理30 min后仍具有较好的抑菌活性，说明发酵滤液中的抑菌活性物质对高温不敏感，具有较好的热稳定性。121 ℃处理30 min后，抑菌活性为56.53%，说明发酵滤液中存在部分抑菌活性物质在高温高压条件下易失活。
2.5　发酵滤液的紫外稳定性
不同紫外照射时间对17-7菌株发酵滤液抑菌活性的影响具有明显差异（图3）。紫外照射时间越长其抑菌活性越低，照射1 h后其抑菌率为87.81%，与CK无显著差异，仍具有较好的抑菌活性。紫外照射2 h后其抑菌率下降至73.79%，照射4 h后抑菌率降至46.75%，并逐渐趋于稳定。说明17-7菌株发酵滤液中可能存在对紫外敏感的活性物质，或者由于发酵滤液中存在某种紫外保护剂延缓了紫外线对发酵滤液抑菌活性的影响。
2.6　发酵滤液的蛋白酶稳定性
经胃蛋白酶和胰蛋白酶处理后，17-7菌株发酵滤液的抑菌活性与CK发酵滤液无显著差异，抑菌率均在80%以上，经蛋白酶K处理的菌株发酵滤液抑菌率为78.98%，略有下降（图4）。说明17-7菌株发酵滤液中，主要的抑菌活性物质对蛋白酶不敏感，代谢产物中可能存在少量的抑菌蛋白，经蛋白酶K处理后失活。
2.7　发酵滤液的酸碱稳定性
在酸碱稳定性试验中，17-7菌株发酵滤液在pH为4~7时均具有较好的抑菌活性，其抑菌率与CK无显著差异，发酵滤液对酸性环境更为耐受，pH为4时其抑菌率最大为78.98%。pH为2~12时，发酵滤液的抑菌活性变化趋缓，抑菌率仍能保持在57.00%以上，但强碱性环境下更容易使发酵滤液中的活性物质失活。在强酸或强碱环境下发酵滤液抑菌活性下降趋势明显，pH为1和13时，抑菌率分别降至31.02%和21.16%（图5）。
2.8　发酵滤液的储藏稳定性
在不同的储藏温度下，17-7菌株发酵滤液的抑菌活性具有一定差异（图6）。储藏30 d内，4 ℃环境下发酵滤液抑菌活性明显高于25 ℃环境下的抑菌活性，4 ℃储藏3 d时抑菌率为91.99%，而25 ℃储藏3 d时抑菌率降至53.36%，说明17-7菌株发酵滤液中存在一类活性物质在水相中极不稳定，低温环境可以延缓其反应速度。值得注意的是，30 d后不同储存温度下的发酵滤液抑菌活性相当，抑菌率分别为49.18%、48.01%，到120 d时呈现逐渐上升趋势，抑菌率分别上升至62.58%、61.91%，均显著高于30 d时的抑菌率（P<0.05）。
3　讨论
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链霉菌能产生约10万种抗生素化合物，占所有农化应用天然产物的70%~80%，被称为天然产物的储存库[10-12]。培养基组成（即碳源、氮源、无机盐等）对次级代谢产物的影响非常复杂，且对不同微生物产生明显差异[7,13]。研究表明，营养组成的变化可激发链霉菌产生不同类型的活性物质[14]。本研究以橡胶树褐根病菌为靶标菌，在原培养基的基础上添加了玉米粉和酵母粉，采用正交试验和单因子试验相结合，对链霉菌17-7菌株的发酵培养基进行进一步优化，获得该菌株最适宜产活性物质的配方为：大豆粉25 g/L、玉米粉10 g/L、葡萄糖10 g/L、酵母粉2 g/L、K2HPO4 1 g/L、NaCl 0.5 g/L、CaCO3 0.5 g/L，发酵滤液稀释500倍后对橡胶树褐根病菌的抑菌率为51.37%，较优化前提高了17.66%。
微生物生物合成基因簇的激活通常与营养类型或浓度的变化有关，特别是碳、氮或无机盐的变化能成功诱导微生物次生代谢产物的产生[15]。SCHWARZ等[14]选择5株具有丰富次生代谢产物基因簇背景的菌株，通过改变培养基组成和培养条件，在30种不同培养条件下，从发酵液中共检测到590个新的化合物，编码这些化合物的基因簇数量远远超过了所预测的次生代谢产物基因簇数量。大豆粉、玉米粉等农副产品具有高蛋白质含量，利用其作为氮源的放线菌能产生更多的活性物质[16-17]。葡萄糖作为一种优质碳源，被广泛应用于一些工业化生产的抗生素中，酵母粉也是卡那霉素链霉菌（S. kanamyceticus）等链霉菌产抗菌素的理想碳源[18]。本研究通过改变培养基组成和添加量，有效提升了菌株的抑菌活性，推测可能是由于营养成分的改变激活了一些沉默的次生代谢产物生物合成基因簇，增加了活性物质的种类。
本研究对17-7菌株抑菌物质稳定性分析显示，17-7菌株发酵滤液具有较好的热稳定性，100 ℃处理30 min后的抑菌效果与CK无显著差异，但121 ℃处理30 min后抑菌率降至56.53%，说明17-7菌株发酵滤液中存在一部分耐高温高压的活性物质。链霉菌发酵液普遍具有热稳定性，如黄麻链霉菌AUH-1菌株发酵液在60~80 ℃热处理下对水稻纹枯病菌的抑制率无显著差异[19]，黄三素链霉菌15-6菌株发酵液在60 ℃和80 ℃不同时间处理后的抗菌效果也无明显改变[20]，但是当温度明显高于80 ℃时，其发酵液的抗菌活性均明显降低。紫外线长时间照射会破坏17-7菌株发酵滤液的抑菌活性，随着紫外线照射时间的延长抑菌活性逐渐降低，但紫外照射时间在1 h以内的抑菌活性与CK无显著差异。链霉菌能产生一些具有吸收紫外线、抗氧化活性的代谢产物，可以通过减轻紫外线辐射的有害影响来实现光保护作用[21]。SÁNCHEZ-SUÁREZ等[22]发现链霉菌代谢产生的生物碱是抗氧化化合物中最具代表性的一类，17-7菌株可能通过代谢产生该类物质来实现光保护特性。17-7菌株发酵滤液还具有较好的酸碱耐受性，并且对胰蛋白酶和胃蛋白酶不敏感，具有较好的抑菌稳定性。pH为3~10时发酵滤液均能发挥较好的抑菌活性，只有在强酸强碱（pH为1或13）环境下会显著降低发酵滤液的抑菌活性。此外，值得注意的是17-7菌株发酵滤液在4 ℃和25 ℃的抑菌活性均呈先降后升的趋势，储藏120 d后其抑菌活性分别上升至62.58%、61.91%，显示17-7菌株具有更广泛的应用价值。
本研究以橡胶树褐根病菌为生物活性指示菌，通过改变培养基组成提升17-7菌株发酵滤液的抑菌活性，并明确了菌株发酵滤液在不同温度、酸碱度、紫外线照射、酶液处理等条件下的稳定性，为17-7菌株的田间应用提供理论基础。
基金
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  • 云南省热带作物科学研究所青年成长基金项目(QNCZ2021-2; QNCZ2022-3)
  • 云南省热带作物科技创新专项资金项目(RF2024-4-1)
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2024年第45卷第12期
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doi: 10.3969/j.issn.1000-2561.2024.12.021
  • 接收时间：2024-05-22
  • 首发时间：2026-06-23
  • 出版时间：2024-12-25
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  • 收稿日期：2024-05-22
  • 修回日期：2024-06-06
基金
云南省热带作物科学研究所青年成长基金项目(QNCZ2021-2; QNCZ2022-3)
云南省热带作物科技创新专项资金项目(RF2024-4-1)
作者信息
    云南省天然橡胶可持续利用研究重点实验室/云南省热带作物科学研究所，云南景洪　666100

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* 蔡志英（CAI Zhiying），E-mail：。
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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