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Article(id=1217845638830670429, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1217845635080962613, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20250228008, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1740672000000, receivedDateStr=2025-02-28, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1768286626775, onlineDateStr=2026-01-13, pubDate=1756051200000, pubDateStr=2025-08-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1768286626775, onlineIssueDateStr=2026-01-13, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1768286626775, creator=13701087609, updateTime=1768286626775, updator=13701087609, issue=Issue{id=1217845635080962613, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='16', pageStart='1', pageEnd='324', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=1, specialIssue=null, createTime=1768286625881, creator=13701087609, updateTime=1768287480278, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1217849218753024879, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1217845635080962613, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1217849218753024880, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1217845635080962613, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=279, endPage=287, ext={EN=ArticleExt(id=1217845639350764146, articleId=1217845638830670429, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Extraction and antioxidant activity evaluation of exopolysaccharides from lactobacillus in kefir grains, columnId=1151895321719223288, journalTitle=Journal of Food Safety & Quality, columnName=Food Processing and Technology, runingTitle=null, highlight=null, articleAbstract=

Objective To increase the production of exopolysaccharides (EPS) from lactobacillus by optimizing fermentation conditions and evaluate the antioxidant activity of the extracted EPS. Methods P1-10 was isolated as a high yield EPS producing lactobacillus from kefir grains and identified by 16S rDNA sequencing. Meanwhile, the effects of carbon source composition and content in the medium and fermentation time on the EPS yield of P1-10 were investigated and compared. Additionally, scanning electron microscopy and in vitro antioxidant study was studied from the obtained EPS as well. Results Among the 12 kinds of isolated strains from kefir grains, P1-10 formed gram-positive colonies under microscopic examination after Gram staining, could coagulate milk, and had a string length greater than 15 mm. It was identified as Pediococcus acidilactici strain (MH143596.1 Pediocuccus acidilactici strain D15) by 16S rDNA sequencing and the maximum content of EPS was 6.52 mg/mL. In addition, the optimal conditions for P1-10 were obtained under the condition of MRS (DeMan-Rogosa-Sharpe medium) medium with the carbon source replaced by 40 g/L sucrose and fermenting at 35 ℃, pH 6.0 for 60 hours, while the highest yield of EPS was 29.09 mg/mL. Furthermore, the surface of EPS under the scanning electron microscopy was relatively rough, with a sense of layering and different sizes voids. Moreover, the elimination rates of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) free radicals in the 1.0 mg/mL vitamin C (vitamin C, VC), EPS and fermentation supernatant were 86.21%, 95.03%; 81.32%, 93.91% and 58.54%, 65.73%, respectively. Conclusion P1-10 can effectively increase the extraction efficiency of EPS compared to the normal culture with the optimizing of the culture conditions. As for the in vitro antioxidant experiments, EPS is indicated a strong ability to scavenge free radicals and antioxidant capacity due to the highly DPPH and ABTS radical scavenging rates. This study may provide a theoretical basis for the screening, extraction and functional activity research of EPS from lactic acid bacteria.

, correspAuthors=Ji XIA, 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, authorCompany=null, fund=null, authors=null, authorsList=Ji XIA, Qin YANG, Hui LI), CN=ArticleExt(id=1217845641951232710, articleId=1217845638830670429, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=开菲尔粒中乳酸菌胞外多糖的提取及其抗氧化活性评价, columnId=1151895322281734685, journalTitle=食品安全质量检测学报, columnName=食品加工与工艺, runingTitle=null, highlight=null, articleAbstract=

目的 通过优化发酵条件提升乳酸菌胞外多糖(exopolysaccharides, EPS)的产量并对提取得到的EPS进行抗氧化活性评价。方法 本研究从开菲尔粒中筛选并用16S rDNA测序法分离鉴定得到一种高产EPS乳酸菌P1-10, 同时研究并比较培养基碳源组成、含量及发酵时间对P1-10乳酸菌EPS产量的影响, 并将得到的EPS进行扫描电镜观察和体外抗氧化评价研究。结果 在从开菲尔粒中分离得到的12种菌株中, P1-10菌株形成的菌落格兰氏染色镜检呈阳性, 能凝乳, 拉丝长度大于15 mm, EPS含量最高为6.52 mg/mL; 经16S rDNA测序法鉴定为乳酸片球菌(MH143596.1 Pediocuccus acidilactici strain D15)。P1-10乳酸菌发酵的最适条件为: MRS (DeMan-Rogosa-Sharpe medium)培养基且将培养基中的碳源更换为40 g/L的蔗糖, 培养温度35 ℃, pH 6.0发酵60 h, 此时EPS的产量最高, 为29.09 mg/mL。同时在扫描电镜下观察, EPS表面比较粗糙, 具有层叠感, 且存在大小不一的空洞。1.0 mg/mL的维生素C (vitamin C, VC)、EPS和P1-10菌株发酵上清液的1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl, DPPH)自由基和2,2’-联氮-二(3-乙基-苯并噻唑啉-6-磺酸)二铵盐[2,2’-azinobis-(3-ethylbenzthiazoline-6-sulphonate), ABTS]阳离子自由基消除率分别为86.21%、95.03%; 81.32%、93.91%和58.54%、65.73%。结论 本研究得到的乳酸菌P1-10经优化培养条件后, 可有效提高EPS的提取效率, 且在体外抗氧化实验中, EPS的DPPH自由基和ABTS阳离子自由基消除率接近于VC, 表明其具有较强的自由基清除能力和抗氧化能力。该研究可为乳酸菌高产EPS的筛选、提取及功能活性研究提供一定的理论依据。

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* 夏季(1992—), 男, 硕士, 实验师, 主要研究方向为食品质量与安全。E-mail:
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夏季(1992—), 男, 硕士, 实验师, 主要研究方向为食品质量与安全。E-mail:

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articleId=1217845638830670429, language=EN, orderNo=1, keyword=kefir grains), Keyword(id=1217864258323398832, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217845638830670429, language=EN, orderNo=2, keyword=lactobacillus), Keyword(id=1217864258503753918, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217845638830670429, language=EN, orderNo=3, keyword=exopolysaccharides), Keyword(id=1217864258692497612, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217845638830670429, language=EN, orderNo=4, keyword=antioxidant activity), Keyword(id=1217864258788966611, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217845638830670429, language=CN, orderNo=1, keyword=开菲尔粒), Keyword(id=1217864258956738781, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217845638830670429, language=CN, orderNo=2, keyword=乳酸菌), Keyword(id=1217864260349247734, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217845638830670429, language=CN, orderNo=3, keyword=胞外多糖), Keyword(id=1217864260537991425, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217845638830670429, language=CN, orderNo=4, keyword=抗氧化活性)], refs=[Reference(id=1217864263348175380, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217845638830670429, doi=null, pmid=null, pmcid=null, year=2017, volume=30, issue=1, pageStart=82, pageEnd=96, url=null, language=null, rfNumber=[1], rfOrder=0, authorNames=ROSA DD, DIAS MMS, REIS SA, journalName=Nutrition Research Reviews, refType=null, unstructuredReference=ROSA DD, DIAS MMS, REIS SA, et al. 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注: 不同小写字母表示具有显著性差异(P<0.05), 图3、5同。

, figureFileSmall=ZWFRREIo/WDUMgUkSWqO7Q==, figureFileBig=b78BVJR/Vv0vMkWzqC9iZg==, tableContent=null), ArticleFig(id=1217864261427183970, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217845638830670429, language=EN, label=Fig.3, caption=Effects of medium type (a) and fermentation time (b) on lactobacillus EPS of lactic acid bacteria, figureFileSmall=/bkUwJGd5wiuGPkgriXxOw==, figureFileBig=W5Edbw0zaX5GWjbBooXypg==, tableContent=null), ArticleFig(id=1217864261599150455, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217845638830670429, language=CN, label=图3, caption=培养基类型(a)及发酵时间(b)对乳酸菌EPS产量的影响, figureFileSmall=/bkUwJGd5wiuGPkgriXxOw==, figureFileBig=W5Edbw0zaX5GWjbBooXypg==, tableContent=null), ArticleFig(id=1217864261737562501, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217845638830670429, language=EN, label=Fig.4, caption=SEM image of EPS at 1500× multiples, figureFileSmall=0KxIV00QfaPk0xpkrpWodg==, figureFileBig=1MPsYOZ6aI52LeYzHNmSHg==, tableContent=null), ArticleFig(id=1217864261880168849, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217845638830670429, language=CN, label=图4, caption=EPS在1500×倍数下的SEM图, figureFileSmall=0KxIV00QfaPk0xpkrpWodg==, figureFileBig=1MPsYOZ6aI52LeYzHNmSHg==, tableContent=null), ArticleFig(id=1217864262018580900, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217845638830670429, language=EN, label=Fig.5, caption=Effects of VC, EPS and fermentation supernatant on DPPH radical elimination (a) and ABTS radical elimination (b), figureFileSmall=5FqxB2I+KwDinxc1f09odg==, figureFileBig=rSuuMILKbM0GHrVWpVkH4g==, tableContent=null), ArticleFig(id=1217864262144410033, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217845638830670429, language=CN, label=图5, caption=VC、EPS和发酵上清液对DPPH自由基消除率(a)和ABTS阳离子自由基消除率(b)的影响, figureFileSmall=5FqxB2I+KwDinxc1f09odg==, figureFileBig=rSuuMILKbM0GHrVWpVkH4g==, tableContent=null), ArticleFig(id=1217864262240879035, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217845638830670429, language=EN, label=Table 1, caption=

Composition of different liquid culture media

, figureFileSmall=null, figureFileBig=null, tableContent=
培养基名称 培养基组成
碳源 其他营养成分
MRS液体培养基 葡萄糖20 g/L 蛋白胨10 g/L、
牛肉膏10 g/L、
酵母膏5 g/L、
柠檬酸氢二铵2 g/L、
吐温-80 1 mL/L、
乙酸钠5 g/L、
磷酸氢二钾2 g/L、
硫酸0.58 g/L、
硫酸锰0.25 g/L;
pH 6.2~6.6, 高压蒸汽灭菌(121 ℃, 20 min)
mM1 乳糖20 g/L
mM2 乳糖40 g/L
mM3 乳糖60 g/L
mM4 蔗糖20 g/L
mM5 蔗糖40 g/L
mM6 蔗糖60 g/L
mM7 /
mM8 葡萄糖40 g/L
mM9 葡萄糖60 g/L
), ArticleFig(id=1217864262354125248, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217845638830670429, language=CN, label=表1, caption=

不同液体培养基的成分组成

, figureFileSmall=null, figureFileBig=null, tableContent=
培养基名称 培养基组成
碳源 其他营养成分
MRS液体培养基 葡萄糖20 g/L 蛋白胨10 g/L、
牛肉膏10 g/L、
酵母膏5 g/L、
柠檬酸氢二铵2 g/L、
吐温-80 1 mL/L、
乙酸钠5 g/L、
磷酸氢二钾2 g/L、
硫酸0.58 g/L、
硫酸锰0.25 g/L;
pH 6.2~6.6, 高压蒸汽灭菌(121 ℃, 20 min)
mM1 乳糖20 g/L
mM2 乳糖40 g/L
mM3 乳糖60 g/L
mM4 蔗糖20 g/L
mM5 蔗糖40 g/L
mM6 蔗糖60 g/L
mM7 /
mM8 葡萄糖40 g/L
mM9 葡萄糖60 g/L
), ArticleFig(id=1217864262479954382, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217845638830670429, language=EN, label=Table 2, caption=

Comparison of color, texture, drawing length, gram staining and curdling ability of strains in kefir grain

, figureFileSmall=null, figureFileBig=null, tableContent=
编号 形状、大小 颜色 表面质地 拉丝长度 革兰氏染色 凝乳实验 胞外多糖含量/(mg/mL)
P1-1 圆、小 白、亮 黏稠湿润 + 阴性 能 1.32
P1-2 圆、小 白、亮 干燥 - 阴性 否 /
P1-3 圆、小 乳白、亮 黏稠湿润 + 阳性 否 1.76
P1-4 圆、小 白、亮 黏稠湿润 + 阴性 否 0.82
P1-5 圆、小 白、亮 黏稠湿润 +++ 阳性 否 6.07
P1-6 圆、大 乳白、亮 黏稠湿润 ++ 阳性 能 3.67
P1-7 点、小 透明 黏稠湿润 - 阴性 否 /
P1-8 圆、大 灰白、不亮 干燥 - 阴性 否 /
P1-9 圆、大 乳白、亮 黏稠湿润 ++ 阳性 能 4.47
P1-10 圆、小 乳白、亮 黏稠湿润 +++ 阳性 能 6.52
P1-11 圆、中 灰白、不亮 干燥 - 阳性 否 /
P1-12 圆、小 白、亮 黏稠湿润 ++ 阴性 能 3.37
), ArticleFig(id=1217864262664503776, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217845638830670429, language=CN, label=表2, caption=

开菲尔粒中菌株的颜色、质地、拉丝长度、革兰氏染色及凝乳能力对比

, figureFileSmall=null, figureFileBig=null, tableContent=
编号 形状、大小 颜色 表面质地 拉丝长度 革兰氏染色 凝乳实验 胞外多糖含量/(mg/mL)
P1-1 圆、小 白、亮 黏稠湿润 + 阴性 能 1.32
P1-2 圆、小 白、亮 干燥 - 阴性 否 /
P1-3 圆、小 乳白、亮 黏稠湿润 + 阳性 否 1.76
P1-4 圆、小 白、亮 黏稠湿润 + 阴性 否 0.82
P1-5 圆、小 白、亮 黏稠湿润 +++ 阳性 否 6.07
P1-6 圆、大 乳白、亮 黏稠湿润 ++ 阳性 能 3.67
P1-7 点、小 透明 黏稠湿润 - 阴性 否 /
P1-8 圆、大 灰白、不亮 干燥 - 阴性 否 /
P1-9 圆、大 乳白、亮 黏稠湿润 ++ 阳性 能 4.47
P1-10 圆、小 乳白、亮 黏稠湿润 +++ 阳性 能 6.52
P1-11 圆、中 灰白、不亮 干燥 - 阳性 否 /
P1-12 圆、小 白、亮 黏稠湿润 ++ 阴性 能 3.37
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开菲尔粒中乳酸菌胞外多糖的提取及其抗氧化活性评价
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夏季 1, * , 杨琴 1 , 李慧 2
食品安全质量检测学报 | 食品加工与工艺 2025,16(16): 279-287
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食品安全质量检测学报 | 食品加工与工艺 2025, 16(16): 279-287
开菲尔粒中乳酸菌胞外多糖的提取及其抗氧化活性评价
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夏季1, * , 杨琴1, 李慧2
作者信息
  • 1 南京财经大学食品科学与工程学院/江苏省粮油品质控制及加工技术重点实验室/江苏省现代粮食流通与安全协同创新中心, 南京 210023
  • 2 上海微谱检测认证有限公司南京分公司, 南京 210023

    • 夏季(1992—), 男, 硕士, 实验师, 主要研究方向为食品质量与安全。E-mail:

通讯作者:

* 夏季(1992—), 男, 硕士, 实验师, 主要研究方向为食品质量与安全。E-mail:
Extraction and antioxidant activity evaluation of exopolysaccharides from lactobacillus in kefir grains
Ji XIA1, * , Qin YANG1, Hui LI2
Affiliations
  • 1 College of Food Science and Engineering, Nanjing University of Finance and Economic/Key Laboratory of Grains and Oils Quality Control and Processing/Cooperative Innovation Center for Modern Grain Circulation and Security of Jiangsu Province, Nanjing 210023, China
  • 2 Nanjing Branch of Shanghai WEIPu Testing Technology Group Co., Ltd., Nanjing 210023, China
出版时间: 2025-08-25 doi: 10.19812/j.cnki.jfsq11-5956/ts.20250228008
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摘要
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目的 通过优化发酵条件提升乳酸菌胞外多糖(exopolysaccharides, EPS)的产量并对提取得到的EPS进行抗氧化活性评价。方法 本研究从开菲尔粒中筛选并用16S rDNA测序法分离鉴定得到一种高产EPS乳酸菌P1-10, 同时研究并比较培养基碳源组成、含量及发酵时间对P1-10乳酸菌EPS产量的影响, 并将得到的EPS进行扫描电镜观察和体外抗氧化评价研究。结果 在从开菲尔粒中分离得到的12种菌株中, P1-10菌株形成的菌落格兰氏染色镜检呈阳性, 能凝乳, 拉丝长度大于15 mm, EPS含量最高为6.52 mg/mL; 经16S rDNA测序法鉴定为乳酸片球菌(MH143596.1 Pediocuccus acidilactici strain D15)。P1-10乳酸菌发酵的最适条件为: MRS (DeMan-Rogosa-Sharpe medium)培养基且将培养基中的碳源更换为40 g/L的蔗糖, 培养温度35 ℃, pH 6.0发酵60 h, 此时EPS的产量最高, 为29.09 mg/mL。同时在扫描电镜下观察, EPS表面比较粗糙, 具有层叠感, 且存在大小不一的空洞。1.0 mg/mL的维生素C (vitamin C, VC)、EPS和P1-10菌株发酵上清液的1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl, DPPH)自由基和2,2’-联氮-二(3-乙基-苯并噻唑啉-6-磺酸)二铵盐[2,2’-azinobis-(3-ethylbenzthiazoline-6-sulphonate), ABTS]阳离子自由基消除率分别为86.21%、95.03%; 81.32%、93.91%和58.54%、65.73%。结论 本研究得到的乳酸菌P1-10经优化培养条件后, 可有效提高EPS的提取效率, 且在体外抗氧化实验中, EPS的DPPH自由基和ABTS阳离子自由基消除率接近于VC, 表明其具有较强的自由基清除能力和抗氧化能力。该研究可为乳酸菌高产EPS的筛选、提取及功能活性研究提供一定的理论依据。

开菲尔粒  /  乳酸菌  /  胞外多糖  /  抗氧化活性

Objective To increase the production of exopolysaccharides (EPS) from lactobacillus by optimizing fermentation conditions and evaluate the antioxidant activity of the extracted EPS. Methods P1-10 was isolated as a high yield EPS producing lactobacillus from kefir grains and identified by 16S rDNA sequencing. Meanwhile, the effects of carbon source composition and content in the medium and fermentation time on the EPS yield of P1-10 were investigated and compared. Additionally, scanning electron microscopy and in vitro antioxidant study was studied from the obtained EPS as well. Results Among the 12 kinds of isolated strains from kefir grains, P1-10 formed gram-positive colonies under microscopic examination after Gram staining, could coagulate milk, and had a string length greater than 15 mm. It was identified as Pediococcus acidilactici strain (MH143596.1 Pediocuccus acidilactici strain D15) by 16S rDNA sequencing and the maximum content of EPS was 6.52 mg/mL. In addition, the optimal conditions for P1-10 were obtained under the condition of MRS (DeMan-Rogosa-Sharpe medium) medium with the carbon source replaced by 40 g/L sucrose and fermenting at 35 ℃, pH 6.0 for 60 hours, while the highest yield of EPS was 29.09 mg/mL. Furthermore, the surface of EPS under the scanning electron microscopy was relatively rough, with a sense of layering and different sizes voids. Moreover, the elimination rates of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) free radicals in the 1.0 mg/mL vitamin C (vitamin C, VC), EPS and fermentation supernatant were 86.21%, 95.03%; 81.32%, 93.91% and 58.54%, 65.73%, respectively. Conclusion P1-10 can effectively increase the extraction efficiency of EPS compared to the normal culture with the optimizing of the culture conditions. As for the in vitro antioxidant experiments, EPS is indicated a strong ability to scavenge free radicals and antioxidant capacity due to the highly DPPH and ABTS radical scavenging rates. This study may provide a theoretical basis for the screening, extraction and functional activity research of EPS from lactic acid bacteria.

kefir grains  /  lactobacillus  /  exopolysaccharides  /  antioxidant activity
夏季, 杨琴, 李慧. 开菲尔粒中乳酸菌胞外多糖的提取及其抗氧化活性评价. 食品安全质量检测学报, 2025 , 16 (16) : 279 -287 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20250228008
Ji XIA, Qin YANG, Hui LI. Extraction and antioxidant activity evaluation of exopolysaccharides from lactobacillus in kefir grains[J]. Journal of Food Safety & Quality, 2025 , 16 (16) : 279 -287 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20250228008
正文
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0 引言
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开菲尔是一种传统发酵酸乳制品, 具有一般的乳制品饮料所不具备的酸甜、清爽的口感与风味, 在我国西北地区深受当地人民喜爱[1-2]。用于开菲尔酸奶制作的开菲尔粒是一个复杂的微生物混合物, 其主要是由乳酸菌、酵母菌及醋酸菌构成; 乳酸菌作为其中占比最高的构成菌种, 在开菲尔发酵制品的风味及组织形态形成中发挥了重要作用[3-5]。开菲尔粒酸乳发酵过程中产生的主要代谢产物有乳酸、核酸、胞外多糖(exopolysaccharides, EPS)、游离氨基酸等物质, 其中EPS由于其独特的黏弹性、吸水性、生物亲和性及存在的多种生物活性而备受专家学者关注[6-7]。EPS是指一些细菌在生长代谢中产生分泌并转运到细胞壁外的一类糖类化合物, 根据其分布位置及化学组成的不同, 可将其分为荚膜多糖、黏液多糖和同型多糖、异型多糖两类[8]。乳酸菌EPS作为微生物产EPS中最多的一种, 具有生物活性强、易生产、食用安全性高等优点, 具有巨大的研究开发价值[9-10]。
目前乳酸菌EPS的生产与提取工艺还存在着诸多问题, 乳酸菌EPS的产量普遍较低, 不同种类的菌种在发酵时产生的EPS差异很大, 因此, 筛选、分离并鉴定可以稳定发酵且产EPS较多的乳酸菌株是实现工业化开发及应用的第一步。相比于传统的检验方法, 荧光定量聚合酶链式反应(polymerase chain reaction, PCR)技术、高通量测序、16S rDNA等分子生物学的技术手段能够快速有效地进行菌种鉴定, 且人为误差较小, 已被广泛应用于纯种菌种的筛选分离与鉴定[11-12]。此外, 对于相同的乳酸菌菌株, 在不同的发酵条件下其EPS的产量也不同, 有研究报道培养基中添加碳源、氮源、金属离子的不同会影响乳酸菌EPS的产量[13]。WANG等[14]从不同地区的6种开菲尔粒中筛选分离得到一株乳酸菌菌株XLM1, 在以硫铵蔗糖培养基为原料, 30 ℃条件下发酵时间96 h EPS的产量最高可达到25.01 mg/mL。HAJ-MUSTAFA等[15]研究了不同碳源、氮源、发酵时间、pH、蔗糖浓度对鼠李糖乳杆菌519的EPS产量的影响, 结果发现, 在pH为5.7时发酵培养49 h EPS的产量最高可达到256 mg/L, pH、培养时间和蔗糖浓度对EPS产量有显著影响。与其他多糖相比, 乳酸菌EPS不仅具有发酵食品特有的优良功能特性, 还有抗氧化、抗真菌、抗炎症、抗肿瘤等一系列生物活性, 不同种类的乳酸菌EPS的功能特性也不完全相同; WANG等[16]发现植物乳杆菌70810产EPS具有多种功能特性, 可以显著抑制HepG-2、BGC-823和HT-29肿瘤细胞的增殖, 是一种天然的抗肿瘤药物; 刘冰等[17]对三地羊肚菌菌丝体EPS进行了提取与纯化, 发现其中MEP-H和MEP-N两种EPS均具有降血糖和抗氧化活性; 张菡等[18]对甜菜汁中耐高糖乳酸菌进行了筛选、分离和功能活性研究, 发现得到的COFL4菌株发酵获得的EPS具有良好的抗氧化活性。因此分离鉴定高产EPS乳酸菌菌株, 研究其EPS生产的最适条件及功能特性具有重要的意义。
本研究从开菲尔粒发酵酸乳中分离纯化产EPS的乳酸菌, 运用16S rDNA测序法对得到的乳酸菌进行菌种鉴定, 筛选鉴定产EPS能力较高的菌株; 并对其产EPS含量进行测定, 通过优化发酵时间及培养液碳源组成, 探究乳酸菌EPS的最适生产条件; 同时, 通过研究EPS对1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl, DPPH)自由基和2,2’-联氮-二(3-乙基-苯并噻唑啉-6-磺酸)二铵盐[ABTS]阳离子自由基消除率的影响评价其抗氧化能力, 以期为乳酸菌发酵产EPS产品的工业化生产工艺及生物活性产品开发应用提供理论依据。
1 材料与方法
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1.1 材料与试剂
开菲尔粒(直径约为2~6 mm), 采自新疆喀什农牧民家庭; 生牛乳购于南京市栖霞区农贸市场; MRS (DeMan-Rogosa-Sharpe medium)选择性固体培养基、MRS琼脂培养基(上海阿拉丁试剂有限公司); 维生素C (vitamin C, VC)、三氯乙酸、无水乙醇、蔗糖、乳糖、蛋白胨、牛肉膏、酵母膏、柠檬酸氢二铵、吐温-80、乙酸钠、磷酸氢二钾、硫酸镁(分析纯, 国药集团化学试剂有限公司); 快流速DEAE-琼脂糖凝胶、琼脂糖CL-6B凝胶、透析袋(截留分子量8~14 kDa)、葡萄糖醛酸、牛血清白蛋白(北京索莱宝科技有限公司); 革兰氏染液试剂盒、DNA提取试剂盒、引物、DPPH自由基清除能力试剂盒、总抗氧化能力(total antioxidant capacity, T-AOC)检测试剂盒(南京建成生物工程研究所)。9种液体培养基及其成分见表1。
1.2 仪器与设备
COY-8375250厌氧培养箱、NanoDrop One微量紫外分光光度计、QuantStudio 3荧光定量PCR仪(美国赛默飞世尔科技公司); YP2001 N分析天平(精度0.1 g, 上海精密科学仪器有限公司); MQD-S3R恒温振荡箱(上海旻泉仪器有限公司); SF-CF-1B超净工作台(上海三发科学仪器有限公司); Axio Vert.A1倒置显微镜(卡尔·蔡司公司股份公司); Tadvanced 96G梯度PCR仪(德国耶拿分析仪器有限公司); YX-280A*高压灭菌锅(上海三申医疗器械有限公司); TM 3000台式扫描电镜(日本日立集团); SCIENTZ-10N真空冷冻干燥机(宁波新芝生物科技股份有限公司)。
1.3 方法
1.3.1 开菲尔粒的活化
称取开菲尔粒5.0 g, 用无菌水冲洗干净并接种于灭菌过的生牛乳中, 接种量为5%; 然后置于震荡培养箱中在160 r/min、30 ℃的条件下培养24 h, 连续培养3次至开菲尔粒的活性恢复。
1.3.2 菌种的分离
取1.3.1中的菌悬液1 mL, 加入9 mL无菌水进行稀释并振荡混匀, 同时继续加入无菌水稀释至10-4、10-5、10-6 3个梯度。在厌氧培氧箱中, 取0.1 mL最终稀释后的菌液分别涂布于MRS固体培养基中, 37 ℃恒温厌氧培养72~96 h, 随后观察菌落形态特征, 并反复使用平板划线法进行分离纯化, 直至形成单个菌落。
1.3.3 产EPS菌株的筛选
根据田丰伟等[19]的方法进行实验, 将1.3.2中的单个菌落进行编号并分别接种于营养琼脂平板上, 37 ℃厌氧培养观察48 h。选择光滑黏稠的菌落, 用接种环检查是否能拉丝。对于分离的菌落进行革兰氏染色及凝乳实验, 并进行显微镜观察, 同时分别收集各个分离的菌落, 利用苯酚硫酸法测定EPS含量。通过评估包括形状、质地、颜色、拉丝长度、革兰氏染色、凝乳能力及EPS含量等因素, 初步筛选出产EPS乳酸菌菌株, 并将分离的菌株存储于保菌管中。
1.3.4 产EPS菌株的16S rDNA序列鉴定分析
将筛选出的菌株于MRS液体培养基中37 ℃扩大培养48 h后, 按照细菌DNA提取试剂盒说明书进行操作并提取菌株的DNA。以通用引物27F (5'-AGAGT TTGATCCTGGCTCAG-3')和1492R (5'-GGTTACCTTGT TACGACTT-3')对提取的DNA进行PCR扩增, 具体条件如下: 扩增条件为95 ℃预变性5 min; 95 ℃变性1 min, 55 ℃退火1 min, 72 ℃延伸1.5 min, 30个循环; 72 ℃再延伸10 min。PCR扩增结束后使用1.5%的琼脂糖凝胶检测。PCR产物送至江苏赛索飞生物科技有限公司进行测序, 测序得到的DNA序列通过在美国国家生物技术信息中心(natioanal center for biotechnology information, NCBI)数据库中利用基于局部比对算法的搜索工具(basic local alignment search tool, BLAST)上进行同源性序列对比, 采用Mega 7软件绘制系统发育树。
1.3.5 菌株生长曲线及最适培养条件的确定
(1)生长曲线绘制
参照石兴民[20]的方法, 将菌株按3%接种量接种至MRS液体每隔2 h取样测定在600 nm的OD吸光值至72 h结束, 以培养时间为横坐标, 对应时间段的OD吸光值为纵坐标, 绘制生长曲线。
(2)最适生长温度确定
将菌株按3%的接种量接入MRS液体培养基中, 分别于25、30、35、40和45 ℃培养24 h, 测定菌液在600 nm的OD值。
(3)最适生长pH确定
将菌株按3%的接种量接入初始pH分别为4.0、4.5、5.0、5.5、6.0、6.5、7.0、7.5、8.0的MRS液体培养基中, 37 ℃培养24 h, 测定菌液在600 nm的OD值。
1.3.6 EPS的提取及条件优化
(1) EPS的提取
按薛艳蓉等[21]的方法提取, 并稍加修改。取2份1.3.3中初筛菌液3%于15 mL MRS液体培养基中培养, 发酵完成后, 将一份发酵液于10000 r/min、4 ℃离心10 min取上清, 过0.22 μm滤膜后冷冻干燥, 在-20 ℃冰箱中保存备用。另一份发酵液于10000 r/min、4 ℃离心10 min取上清, 在上清液中加入80%三氯乙酸搅拌均匀; 再次离心除去沉淀蛋白后, 取上清液缓慢加入3倍体积预冷无水乙醇, 于4 ℃静置12 h后离心取沉淀溶解于蒸馏水中, 透析48 h后冷冻干燥, 并将得到的冻干粉末保存在-20 ℃冰箱中保存备用。
(2) EPS的含量测定
以葡萄糖为标准单糖, 利用苯酚硫酸法测定EPS含量。
(3)培养基的筛选
将筛选出菌株分别接种到MRS培养基、mM培养基1~9上, 140 r/min振荡培养24 h, 对发酵液进行EPS的提取及含量测定。
(4)发酵时间的筛选
将筛选出菌株分别以3%的接种量接种到MRS培养基中, 振荡培养24、36、48、60、72 h, 对发酵液进行EPS的提取及含量测定。
1.3.7 EPS的扫描电镜观察
将1.3.5(1)中的EPS冻干样品固定在双面胶带上, 用溅射镀膜机溅射金粉。然后, 通过扫描电子显微镜(scanning electron microscope, SEM)系统在真空条件下进行扫描, 记录EPS表面的微观结构。
1.3.8 EPS的抗氧化活性研究
(1) DPPH自由基清除率的测定
分别称取冻干EPS、1.3.6(1)中发酵上清液冻干粉各0.1 g, 加入1 mL提取液, 按照DPPH试剂盒说明书步骤进行操作测定吸光度。DPPH清除率计算见公式(1):
D/%=$\frac{{{C}_{空白}}-{{C}_{测定}}}{{{C}_{空白}}}$
式中: C空白表示空白组的吸光度; C测定表示样品组的吸光度。
(2) ABTS阳离子自由基清除率的测定
分别称取冻干EPS、1.3.6(1)中发酵上清液冻干粉各0.1 g, 加入1 mL提取液, 按照T-AOC试剂盒说明书步骤进行操作测定吸光度。ABTS阳离子自由基清除率计算计算见公式(2):
D1/%=$\frac{C{{,}_{空白}}-C{{,}_{测定}}}{C{{,}_{空白}}}$×100%
式中: C’空白表示空白组的吸光度; C’测定表示样品组的吸光度。
1.4 数据处理
实验均重复3次, 实验数据采用软件SAS (version V8)进行统计分析, 结果以均值±标准偏差表示; 多重数据采用SPSS (version 17.0) Duncan’s新复极差法检验数据的差异性, 分析水平为P<0.05。
2 结果与分析
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2.1 高产EPS乳酸菌菌株的筛选
将开菲尔粒活化后进行培养, 通过平板划线的方式筛选出12种菌株, 观察评估12种菌株的菌落形状、大小、颜色、质地, 并使用接种环检查拉丝长度情况, 结果如表2所示。其中编号P1-1、P1-3、P1-4、P1-5、P1-6、P1-9、P1-10、P1-12菌株菌落均为圆形, 颜色为白色或乳白色, 表面质地黏稠湿润。P1-1、P1-2、P1-4、P1-7、P1-8、P1-12菌株革兰氏染色为阴性, P1-3、P1-5、P1-6、P1-9、P1-10、P1-11菌株革兰氏染色为阳性, 且P1-1、P1-6、P1-9、P1-10、P1-12菌落能够凝乳。P1-5、P1-6、P1-9、P1-10、P1-12拉丝长度超过5 mm, 且P1-5和P1-10拉丝长度超过15 mm, 分别为15.7 mm和15.9 mm。
菌落是菌株鉴定的一项重要标志之一, 不同的微生物产生的菌落各不相同, 且菌落特征与微生物的菌体形态结构也密切相关。在本研究中, P1-1、P1-6、P1-9、P1-10、P1-12菌落呈圆形、白色或乳白色、表面黏稠湿润、能凝乳, 是乳酸菌的典型特征[22]。同时, 在光学显微镜下观察, 呈短杆状或圆球状, 单独或成对存在, 无芽胞, 可初步判定它们均为乳酸菌。菌落的黏稠程度、拉丝长度的多少反映了乳酸菌代谢产物的高低, 也在一定程度上反映其EPS产量的高低[23-24], 为了进一步验证12种菌株的EPS产量, 采用苯酚硫酸法测定了12种菌株的EPS含量, 其中P1-10菌株EPS产量最高, 为6.52 mg/mL; 因此选用P1-10作为高产EPS乳酸菌菌株进行后续研究。
2.2 EPS菌株16S rDNA序列分析
开菲尔粒中乳酸菌的系统发育进化树如图1所示, 根据对P1-10菌种的基因测序结果进行分析显示, 其DNA基因组经PCR扩增后测序, 与乳酸片球菌(MH143596.1 Pediocuccus acidilactici strain D15)的同源性达到100%, 可将其鉴定为乳酸片球菌。乳酸片球菌是乳酸菌的一种, 细胞呈球形, 属于片球菌属, 可作为乳制品发酵过程中的发酵剂, 具有改善风味、延长货架期的作用。同时有文献报道, 乳酸片球菌产生的片球菌素能够破坏细菌的表面结构及其能量代谢途径, 具有抑制其他细菌生长等功效[25-26]。
2.3 EPS发酵条件优化分析
EPS的产量与多种因素密切相关, 如发酵条件(温度、pH等)、培养基组成及菌株类型等。结合目前研究报道中的高产EPS产量[27-28], 可以认为当菌株的EPS含量在20 g/L时以上具有高产EPS的潜力。菌株P1-10的生长曲线如图2(a)所示, 其在培养4 h后进入指数生长期, 并于18 h后到达稳定期, 在稳定期可以持续到70 h。菌株P1-10的最适温度和初始pH如图2(b)和2(c)所示, 在30~40 ℃发酵温度范围内生长良好, 且最适温度为35 ℃; 但在超过 40 ℃及低于25 时生长缓慢。菌株P1-10在pH 5.5~6.5的酸性环境下生长繁殖迅速, 且最适pH为6.0, 这与大多数的乳酸菌的最适温度为25~35 ℃, 最适pH为5.5~6.4的研究结果一致[29]。在后续优化EPS发酵条件时, 均采用菌株P1-10的最适温度35 ℃和最适pH 6.0。
碳源作为乳酸菌生长繁殖过程中必不可少的营养物质, 其种类和含量对于EPS的产量有直接的影响。培养基中碳源种类和添加量对乳酸菌EPS产量的影响如图3(a)所示。由图3(a)可知, 与空白对照组相比, 加入葡萄糖、蔗糖和乳糖作为碳源的培养基进行培养后, 发酵后产生的EPS的产量含量均有所增加。P1-10菌株在mM5、mM6培养基中产生的EPS最多, 分别为15.27 mg/mL和15.36 mg/mL; 这表明在葡萄糖、蔗糖和乳糖三者中, 以蔗糖为培养基碳源进行发酵时, EPS的产量最高。这可能与P1-10菌株对蔗糖的利用效率较高有关, 以蔗糖作为碳源能够提高碳源底物向产物转化的速率, 并能提供给菌体更高效率的腺苷三磷酸(adenosine triphosphate, ATP), 从而促提升菌株EPS的产量[30]。由于加入40 g/L和60 g/L蔗糖浓度的培养基中, EPS的产量没有显著变化(P>0.05), 因此为了节约成本考虑, 本研究选取mM5即40 g/L蔗糖浓度作为碳源的培养基用于后续实验。
发酵时间对乳酸菌EPS产量的影响如图3(b)所示。发酵时间为24、36、48、60、72 h时, 乳酸菌EPS产量分别为15.27、19.25、23.34、29.09和25.13 mg/mL, 随着发酵时间的增加, 乳酸菌EPS产量呈现先增加后降低的趋势, 在培养60 h的时候EPS的产量达到最大。根据图2(a)可知P1-10菌株在培养60 h时仍处于稳定期, 可高效积累代谢产物, 因此本研究选取培养60 h作为最适宜的发酵培养时间用于后续实验。当发酵培养时间过长后EPS含量不再增加, 这可能与培养基内营养物质消耗过多, 产生的代谢废物增多且乳酸菌产酸导致发酵pH变化有关。随着产酸增加, 培养液的pH不断降低, 菌株不再生产EPS并分解EPS来维持自身的营养生长[31]。在本研究中使用P1-10菌株作为菌种, 以mM5培养基在温度35 ℃、pH为6.0发酵60 h的条件下, 得到EPS的产量最大为29.09 mg/mL, 相比于普通条件下的发酵培养, 有效提高了EPS的生产效率。
2.4 EPS的SEM分析结果
近年来, SEM技术凭借其高分辨率、多模态分析能力, 已成为生物大分子表面形态微观结构表征的核心工具。根据观察到的表面形态微观结构可以推断出乳酸菌EPS的某些理化特性, 更能推断出乳酸菌EPS可能存在的一些生物活性。乳酸菌EPS放大倍数为1500×下的SEM照片如图4所示, 图4中显示EPS冻干粉末在SEM下外形呈现细致的、不规则碎片状和碎屑状结构, 放大后观察发现其表面比较粗糙, 具有层叠感, 且存在大小不一的空洞, 这可能与多糖分子之间结构较为紧密, 交联度相对较高有关。具备这种表面形态特性的EPS可以通过形成水化聚合物的方式改善产物的稳定性、溶解度、持水力等特性, 并可以将其广泛应用于发酵食品等相关领域[32-33]。
2.5 EPS的抗氧化功能特性评价分析
EPS对DPPH自由基消除率(a)和ABTS阳离子自由基消除率(b)的影响如图5所示。DPPH自由基和ABTS阳离子自由基消除率是研究活性物质抗氧化能力和自由基清除能力的重要组成部分[34]。以VC为代表的活性物质能够清除自由基, 防止过氧化、减轻氧化损伤, 因此以VC作为阳性对照可以直观地反映其抗氧化能力大小。在本研究中, 当EPS的质量浓度达到1.0 mg/mL时, VC、EPS、菌株发酵上清液的DPPH自由基和ABTS阳离子自由基的消除率分别为86.21%、95.03%; 81.32%、93.91%和58.54%、65.73%。由图5可知, 乳酸菌EPS对DPPH自由基和ABTS阳离子自由基均具有一定的消除能力, 但整体上强于发酵上清液而弱于VC。随着VC、EPS及发酵上清液的浓度逐渐增加, DPPH自由基和ABTS阳离子自由基的消除率也呈现递增的趋势, 具有明显的质量浓度依赖性。EPS的DPPH自由基消除率和ABTS阳离子自由基消除率与VC接近, 表明其具有较强的自由基清除能力和抗氧化能力; 发酵上清液的DPPH自由基消除率和ABTS阳离子自由基消除率明显低于EPS, 表明EPS是乳酸菌P1-10菌株发酵后能够起到抗氧化的一类活性物质。本研究结果与张菡等[35]研究高产乳酸菌EPS的提取, 并发现其对DPPH自由基和ABTS阳离子自由基均具有一定的消除能力的研究结果一致。
3 结论
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本研究从开菲尔粒发酵酸乳中分离纯化出了一株高产EPS的乳酸菌P1-10, 并运用16S rDNA测序法鉴定为乳酸片球菌。通过改良MRS培养基, 将培养基碳源替换为40 g/L蔗糖, 培养温度为35 ℃, pH为6.0, 发酵60 h后EPS的含量最高, 为29.09 mg/mL, 很大程度上提高了乳酸菌EPS的提取量。乳酸菌P1-10所产EPS对DPPH自由基和ABTS阳离子自由基均具有较强的清除能力, EPS是该菌株发酵后能够产生抗氧化活性的一类功能性物质。本研究通过对菌株的筛选及发酵条件的优化, 为乳酸菌发酵产EPS产品的工业化生产工艺提供了一定的理论依据。在后续实验中, 将开展EPS的分离纯化、结构鉴定及表征, 同时完善EPS的抗氧化机制研究。
基金
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  • 江苏省高校优势学科建设工程资助项目(PAPD)
文献
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2025年第16卷第16期
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20250228008
  • 接收时间：2025-02-28
  • 首发时间：2026-01-13
  • 出版时间：2025-08-25
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  • 收稿日期：2025-02-28
基金
江苏省高校优势学科建设工程资助项目(PAPD)
作者信息
    1 南京财经大学食品科学与工程学院/江苏省粮油品质控制及加工技术重点实验室/江苏省现代粮食流通与安全协同创新中心, 南京 210023
    2 上海微谱检测认证有限公司南京分公司, 南京 210023

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* 夏季(1992—), 男, 硕士, 实验师, 主要研究方向为食品质量与安全。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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