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Article(id=1217779725020348437, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1217779717386715826, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20250313005, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1741795200000, receivedDateStr=2025-03-13, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1768270911697, onlineDateStr=2026-01-13, pubDate=1750780800000, pubDateStr=2025-06-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1768270911697, onlineIssueDateStr=2026-01-13, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1768270911697, creator=13701087609, updateTime=1768270911697, updator=13701087609, issue=Issue{id=1217779717386715826, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='12', pageStart='1', pageEnd='320', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1768270909877, creator=13701087609, updateTime=1768299620707, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1217900139386163208, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1217779717386715826, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1217900139386163209, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1217779717386715826, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=88, endPage=96, ext={EN=ArticleExt(id=1217779725460750379, articleId=1217779725020348437, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Study on the regulation and mechanism of carboxymethyl pachymaran on pH stability and bioactivity of casein, columnId=1217529311867883548, journalTitle=Journal of Food Safety & Quality, columnName=Highlight: Analysis and Monitoring of Toxic and Harmful Substances in Food, runingTitle=null, highlight=null, articleAbstract=

Objective To investigate the effects of carboxymethyl pachymaran on the stability, structure, physicochemical properties and biological activity of casein at different ratios. Methods Pachymaran was derivatized with carboxymethyl groups to enhance its solubility, and its structural features were studied; carboxymethyl pachymaran was mixed with casein in different ratios, and the turbidity, zeta potential, protein solubility, and phase diagram of the complex were evaluated at different pH values; the aggregation degree and physicochemical properties of different casein-based samples were studied under acidic conditions using confocal laser microscopy and rotational rheometer; free radical scavenging ability and non-enzymatic glycation product inhibition ability of different casein-based samples were compared. Results The molecular weight of carboxymethyl pachymaran obtained in this study was 2.43×105 g/mol. The successful preparation of carboxymethyl pachymaran was determined by the degree of substitution (0.84) and the characteristic absorption peak of carboxymethyl group. When carboxymethyl pachymaran was mixed with casein, it could effectively affect the pH stability and reduce the turbidity of casein under acidic conditions, and its solubility was also enhanced. This might be due to the electrostatic interactions between polysaccharides and proteins affecting the surface charge distribution of the molecules. Among them, the best stabilizing effect was observed when carboxymethyl pachymaran was mixed with casein at 5:1 (w/w). Subsequent studies on the structure and physicochemical properties of the complex showed that with the introduction of polysaccharides, especially when they were mixed at a ratio of 5:1 (w/w), the aggregation degree, apparent viscosity and modulus of the complex were lower, indicating that its gel network structure had better stability. The biological activity results showed that the complex, especially when they were mixed at a ratio of 5:1 (w/w), exhibited the strongest antioxidant activity and non-enzymatic glycation product inhibition ability. Conclusion The carboxymethyl pachymaran-casein complex obtained in this study has better structural stability and biological activity in acidic environments, providing a theoretical basis and technical support for the development and processing of stable acidic dairy products.

, correspAuthors=Li FENG, 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=Yi-Na CHENG, Wen LI, Ji-Ying LI, Jing-Yi WANG, Li FENG), CN=ArticleExt(id=1217779727033614470, articleId=1217779725020348437, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=羧甲基茯苓多糖对酪蛋白pH稳定性与生物活性的调控与机制研究, columnId=1217529312056627244, journalTitle=食品安全质量检测学报, columnName=本期重点：食品中有毒有害物质分析与监测, runingTitle=null, highlight=null, articleAbstract=

目的 探究羧甲基茯苓多糖以不同比例与酪蛋白复配时对酪蛋白稳定性、结构与理化性质以及生物活性的影响。方法 通过羧甲基衍生化, 提升茯苓多糖的溶解性并表征其结构; 以不同比例将羧甲基茯苓多糖与酪蛋白进行物理共混, 测定复合物在不同pH下的浊度、zeta电位、蛋白质溶解性、相图; 通过激光共聚焦显微镜和旋转流变仪比较不同酪蛋白样品在酸性条件下的聚集特性与理化特性; 通过自由基清除能力与非酶糖基化产物的抑制能力比较不同酪蛋白样品的生物活性。结果 本研究所得羧甲基茯苓多糖的分子量为2.43×105 g/mol, 通过取代度(0.84)和红外光谱中的羧甲基特征吸收峰可以确定羧甲基多糖的成功制备。当羧甲基茯苓多糖与酪蛋白共混后, 能够有效提高酪蛋白的pH稳定性, 显著影响了酪蛋白在酸性条件下的浊度、提高蛋白溶解性, 这可能是由于多糖-蛋白质间静电互作力影响了复合物分子的表面电荷分布。其中, 当二者以5:1 (w/w)复合时, 羧甲基茯苓多糖表现出最好的稳定效果。随后的结构与理化性质研究表明, 引入多糖, 特别当二者以5:1 (w/w)复合时, 复合物具有较低的聚集程度、表观黏度与模量, 表明此时复合物的凝胶网络结构具有更好的结构稳定性。生物活性结果表明, 复合物, 特别以5:1 (w/w)复合时, 表现出最强的抗氧化活性和非酶糖基化产物的抑制能力。结论 本研究所得羧甲基茯苓多糖-酪蛋白复合物在酸性环境下具有更好的结构稳定性与生物活性, 为稳定的酸性乳制品的开发与加工提供理论基础和技术支持。

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*冯利(1981—), 女, 博士, 教授, 主要研究方向为医药职业教育。E-mail:
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程熠娜(1992—), 女, 硕士, 主要研究方向为中药加工与安全。E-mail:

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程熠娜(1992—), 女, 硕士, 主要研究方向为中药加工与安全。E-mail:

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Synchronous generation pattern of heterocyclic amines and advanced glycation end products in plant hamburger meat under different thermal processing conditions[J]. Journal of Food Safety & Quality, 2025, 16(7): 45-54., articleTitle=Synchronous generation pattern of heterocyclic amines and advanced glycation end products in plant hamburger meat under different thermal processing conditions, refAbstract=null), Reference(id=1217895183476244945, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, doi=null, pmid=null, pmcid=null, year=2024, volume=267, issue=null, pageStart=131387, pageEnd=null, url=null, language=null, rfNumber=[34], rfOrder=34, authorNames=LIU D, MEI XY, MAO YT, journalName=International Journal of Biological Macromolecules, refType=null, unstructuredReference=LIU D, MEI XY, MAO YT,et al. Lentinus edodes mycelium polysaccharide inhibits AGEs-induced HUVECs pyroptosis by regulating LncRNA MALAT1/miR-199b/mTOR axis and NLRP3/Caspase-1/ GSDMD pathway[J]. International Journal of Biological Macromolecules, 2024, 267: 131387., articleTitle=Lentinus edodes mycelium polysaccharide inhibits AGEs-induced HUVECs pyroptosis by regulating LncRNA MALAT1/miR-199b/mTOR axis and NLRP3/Caspase-1/ GSDMD pathway, refAbstract=null)], funds=[Fund(id=1217895179860754862, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, awardId=32301996, language=CN, fundingSource=国家自然科学基金项目(32301996), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1217895175821640057, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, xref=1, ext=[AuthorCompanyExt(id=1217895175825834362, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, companyId=1217895175821640057, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1 Department of Medical Nursing, Changjiang Polytechnic, Ezhou 436032, China), AuthorCompanyExt(id=1217895175830028667, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, companyId=1217895175821640057, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1 长江职业学院医药护理学院, 鄂州 436032)]), AuthorCompany(id=1217895175897137532, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, xref=2, ext=[AuthorCompanyExt(id=1217895175905526141, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, companyId=1217895175897137532, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2 School of Life and Health Sciences, Hubei University of Technology, Wuhan 430068, China), AuthorCompanyExt(id=1217895175909720446, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, companyId=1217895175897137532, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2 湖北工业大学生命科学与健康工程学院, 武汉 430068)])], figs=[ArticleFig(id=1217895178787013024, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, language=EN, label=Fig.1, caption=UV visible absorption spectrum and Fourier transform infrared spectrum of CMP, figureFileSmall=6kiezAMud5JWHXYlzPoblg==, figureFileBig=RuWvYqhoW+jrphOCFvSoiw==, tableContent=null), ArticleFig(id=1217895178849927585, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, language=CN, label=图1, caption=CMP的紫外和红外谱图, figureFileSmall=6kiezAMud5JWHXYlzPoblg==, figureFileBig=RuWvYqhoW+jrphOCFvSoiw==, tableContent=null), ArticleFig(id=1217895178963173794, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, language=EN, label=Fig.2, caption=Variation of physicochemical properties of complex solutions with pH value under different CMP:CS composite ratios (n=3), figureFileSmall=VtjW9B127wARSD930Eehtw==, figureFileBig=rqKVTVvkwIunjFDM8uRObQ==, tableContent=null), ArticleFig(id=1217895179093197219, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, language=CN, label=图2, caption=不同CMP:CS复合比例下混合溶液的理化性质随pH变化(n=3), figureFileSmall=VtjW9B127wARSD930Eehtw==, figureFileBig=rqKVTVvkwIunjFDM8uRObQ==, tableContent=null), ArticleFig(id=1217895179156111780, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, language=EN, label=Fig.3, caption=Laser confocal microscopy images of different CS samples, figureFileSmall=4m6Gb5duY7sjZxvzqs37NQ==, figureFileBig=yuJt8vZ4kHB+PtU6KMrexQ==, tableContent=null), ArticleFig(id=1217895179210637733, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, language=CN, label=图3, caption=不同CS样品的激光共聚焦显微镜图片

注: A. CS; B. CMP-CS (5:1); C. CMP-CS (1:1); D. CMP-CS (1:5); 所有样品均在pH=4的溶液中制备, 并在500倍下进行观察。

, figureFileSmall=4m6Gb5duY7sjZxvzqs37NQ==, figureFileBig=yuJt8vZ4kHB+PtU6KMrexQ==, tableContent=null), ArticleFig(id=1217895179265163686, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, language=EN, label=Fig.4, caption=Characterization of rheological properties of different CS samples, figureFileSmall=swddAcnISj9+AiOr7GODMA==, figureFileBig=l5NAzdbR8cMrZOrFH8Hm+g==, tableContent=null), ArticleFig(id=1217895179332272551, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, language=CN, label=图4, caption=不同CS样品流变学特性的表征

注: A. 表观黏度图; B. 模量图; 所有样品均在pH=4的溶液中制备。

, figureFileSmall=swddAcnISj9+AiOr7GODMA==, figureFileBig=l5NAzdbR8cMrZOrFH8Hm+g==, tableContent=null), ArticleFig(id=1217895179395187112, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, language=EN, label=Fig.5, caption=Comparison of antioxidant activity of different CS samples (n=3), figureFileSmall=b4Fq+fkmUBHI4TabDAAVlQ==, figureFileBig=sO+glio7GpwsIcqgf6J1VA==, tableContent=null), ArticleFig(id=1217895179466490281, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, language=CN, label=图5, caption=不同CS样品的抗氧化活性比较(n=3)

注: 不同字母表示组间具有显著性差异, P<0.05, 下同。

, figureFileSmall=b4Fq+fkmUBHI4TabDAAVlQ==, figureFileBig=sO+glio7GpwsIcqgf6J1VA==, tableContent=null), ArticleFig(id=1217895179525210538, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, language=EN, label=Fig.6, caption=Comparison of inhibitory effects on non-enzymatic glycation product generation of different CS samples (n=3), figureFileSmall=gcA5cV+meJkWB436PwCQkg==, figureFileBig=chIKlCI8DkaQ4qPEyv7/cg==, tableContent=null), ArticleFig(id=1217895179579736491, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, language=CN, label=图6, caption=不同CS样品的非酶糖基化产物生成的抑制效果比较(n=3), figureFileSmall=gcA5cV+meJkWB436PwCQkg==, figureFileBig=chIKlCI8DkaQ4qPEyv7/cg==, tableContent=null), ArticleFig(id=1217895179646845356, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, language=EN, label=Table 1, caption=

Chemical composition and basic structure characterization of CMP

, figureFileSmall=null, figureFileBig=null, tableContent=
化学组成 CMP
总糖含量/% 87.37±1.35
蛋白质含量/% 0.19±0.07
糖醛酸含量/% 7.43±0.36
还原糖含量/% 5.21±0.63
取代度 0.84±0.02
分子量/(g/mol) 2.43×105 (±2.16%)
葡萄糖含量/mol 9.43
甘露糖含量/mol 1.24
), ArticleFig(id=1217895179718148525, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217779725020348437, language=CN, label=表1, caption=

CMP的化学组成与基本结构表征

, figureFileSmall=null, figureFileBig=null, tableContent=
化学组成 CMP
总糖含量/% 87.37±1.35
蛋白质含量/% 0.19±0.07
糖醛酸含量/% 7.43±0.36
还原糖含量/% 5.21±0.63
取代度 0.84±0.02
分子量/(g/mol) 2.43×105 (±2.16%)
葡萄糖含量/mol 9.43
甘露糖含量/mol 1.24
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羧甲基茯苓多糖对酪蛋白pH稳定性与生物活性的调控与机制研究
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程熠娜 1 , 李雯 1 , 李吉莹 1 , 王静祎 2 , 冯利 1, *
食品安全质量检测学报 | 本期重点：食品中有毒有害物质分析与监测 2025,16(12): 88-96
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食品安全质量检测学报 | 本期重点：食品中有毒有害物质分析与监测 2025, 16(12): 88-96
羧甲基茯苓多糖对酪蛋白pH稳定性与生物活性的调控与机制研究
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程熠娜1 , 李雯1, 李吉莹1, 王静祎2, 冯利1, *
作者信息
  • 1 长江职业学院医药护理学院, 鄂州 436032
  • 2 湖北工业大学生命科学与健康工程学院, 武汉 430068

    • 程熠娜(1992—), 女, 硕士, 主要研究方向为中药加工与安全。E-mail:

通讯作者:

*冯利(1981—), 女, 博士, 教授, 主要研究方向为医药职业教育。E-mail:
Study on the regulation and mechanism of carboxymethyl pachymaran on pH stability and bioactivity of casein
Yi-Na CHENG1 , Wen LI1, Ji-Ying LI1, Jing-Yi WANG2, Li FENG1, *
Affiliations
  • 1 Department of Medical Nursing, Changjiang Polytechnic, Ezhou 436032, China
  • 2 School of Life and Health Sciences, Hubei University of Technology, Wuhan 430068, China
出版时间: 2025-06-25 doi: 10.19812/j.cnki.jfsq11-5956/ts.20250313005
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目的 探究羧甲基茯苓多糖以不同比例与酪蛋白复配时对酪蛋白稳定性、结构与理化性质以及生物活性的影响。方法 通过羧甲基衍生化, 提升茯苓多糖的溶解性并表征其结构; 以不同比例将羧甲基茯苓多糖与酪蛋白进行物理共混, 测定复合物在不同pH下的浊度、zeta电位、蛋白质溶解性、相图; 通过激光共聚焦显微镜和旋转流变仪比较不同酪蛋白样品在酸性条件下的聚集特性与理化特性; 通过自由基清除能力与非酶糖基化产物的抑制能力比较不同酪蛋白样品的生物活性。结果 本研究所得羧甲基茯苓多糖的分子量为2.43×105 g/mol, 通过取代度(0.84)和红外光谱中的羧甲基特征吸收峰可以确定羧甲基多糖的成功制备。当羧甲基茯苓多糖与酪蛋白共混后, 能够有效提高酪蛋白的pH稳定性, 显著影响了酪蛋白在酸性条件下的浊度、提高蛋白溶解性, 这可能是由于多糖-蛋白质间静电互作力影响了复合物分子的表面电荷分布。其中, 当二者以5:1 (w/w)复合时, 羧甲基茯苓多糖表现出最好的稳定效果。随后的结构与理化性质研究表明, 引入多糖, 特别当二者以5:1 (w/w)复合时, 复合物具有较低的聚集程度、表观黏度与模量, 表明此时复合物的凝胶网络结构具有更好的结构稳定性。生物活性结果表明, 复合物, 特别以5:1 (w/w)复合时, 表现出最强的抗氧化活性和非酶糖基化产物的抑制能力。结论 本研究所得羧甲基茯苓多糖-酪蛋白复合物在酸性环境下具有更好的结构稳定性与生物活性, 为稳定的酸性乳制品的开发与加工提供理论基础和技术支持。

茯苓多糖  /  酪蛋白  /  聚集程度  /  抗糖化活性

Objective To investigate the effects of carboxymethyl pachymaran on the stability, structure, physicochemical properties and biological activity of casein at different ratios. Methods Pachymaran was derivatized with carboxymethyl groups to enhance its solubility, and its structural features were studied; carboxymethyl pachymaran was mixed with casein in different ratios, and the turbidity, zeta potential, protein solubility, and phase diagram of the complex were evaluated at different pH values; the aggregation degree and physicochemical properties of different casein-based samples were studied under acidic conditions using confocal laser microscopy and rotational rheometer; free radical scavenging ability and non-enzymatic glycation product inhibition ability of different casein-based samples were compared. Results The molecular weight of carboxymethyl pachymaran obtained in this study was 2.43×105 g/mol. The successful preparation of carboxymethyl pachymaran was determined by the degree of substitution (0.84) and the characteristic absorption peak of carboxymethyl group. When carboxymethyl pachymaran was mixed with casein, it could effectively affect the pH stability and reduce the turbidity of casein under acidic conditions, and its solubility was also enhanced. This might be due to the electrostatic interactions between polysaccharides and proteins affecting the surface charge distribution of the molecules. Among them, the best stabilizing effect was observed when carboxymethyl pachymaran was mixed with casein at 5:1 (w/w). Subsequent studies on the structure and physicochemical properties of the complex showed that with the introduction of polysaccharides, especially when they were mixed at a ratio of 5:1 (w/w), the aggregation degree, apparent viscosity and modulus of the complex were lower, indicating that its gel network structure had better stability. The biological activity results showed that the complex, especially when they were mixed at a ratio of 5:1 (w/w), exhibited the strongest antioxidant activity and non-enzymatic glycation product inhibition ability. Conclusion The carboxymethyl pachymaran-casein complex obtained in this study has better structural stability and biological activity in acidic environments, providing a theoretical basis and technical support for the development and processing of stable acidic dairy products.

pachymaran  /  casein  /  aggregation degree  /  anti-glycation
程熠娜, 李雯, 李吉莹, 王静祎, 冯利. 羧甲基茯苓多糖对酪蛋白pH稳定性与生物活性的调控与机制研究. 食品安全质量检测学报, 2025 , 16 (12) : 88 -96 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20250313005
Yi-Na CHENG, Wen LI, Ji-Ying LI, Jing-Yi WANG, Li FENG. Study on the regulation and mechanism of carboxymethyl pachymaran on pH stability and bioactivity of casein[J]. Journal of Food Safety & Quality, 2025 , 16 (12) : 88 -96 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20250313005
正文
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0 引言
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酸化乳饮料通常指一大类pH范围约为3.4~4.6的酸性含乳饮料, 具有口感清爽、营养丰富、风味独特等优点。近年来, 由于蛋白质基饮料的健康益处, 其需求激增的同时也使得酸化乳饮料逐渐受到重视[1]。然而, 降低pH会导致乳饮料的稳定性显著下降并影响产品的感官质量, 其主要原因是酪蛋白(casein, CS)在酸性条件下的聚集[2]。CS是牛乳中的主要蛋白质, 占牛乳蛋白总量的80%左右, 通常以胶束结构稳定存在于牛乳中。在牛乳的正常pH (~6.7)时, CS胶束之间具备充分的空间位阻与静电斥力, 使CS胶束稳定分散。当体系pH接近CS的等电点(~4.6)时, 其分子表面呈电中性, CS亚组分构象崩溃, CS分子间空间位阻和静电斥力降低, 导致CS胶束的聚集和沉淀[3]。因此, 提高CS的pH稳定性对于开发符合消费者需求的酸化乳饮料十分重要。
目前, 已有研究表明向酸性乳中添加阴离子多糖, 例如卡拉胶、果胶、羧甲基纤维素等, 能够显著提高酸性乳的整体稳定性, 并且这一手段在科学研究和工业生产中已经较常见[4]。阴离子多糖吸附在CS表面, 提供一定的空间稳定作用; 此外, 阴离子多糖能够通过非共价相互作用与CS形成凝胶网络结构, 使其在低pH下恢复空间位阻和静电排斥力[5]。然而, 上述阴离子多糖主要因较好的稳定效果被应用于酸性乳中, 尚未考虑到所选多糖的生物活性。即筛选出既能够有效稳定酸性乳, 同时也可以赋予酸性乳显著生物活性的酸性多糖, 这对于以目前大健康饮食为趋势的消费与政策导向十分关键, 但综合二者的相关研究极少。茯苓是一种药食同源的中药材, 其主要活性成分茯苓多糖已被证实具有抗氧化、抗炎等生物活性, 在许多药方、饮食中均得到应用。然而, 茯苓多糖主要由β-(1,3)-葡聚糖组成, 水溶性极差, 故常通过羧甲基衍生化提高其溶解性与生物利用率, 羧甲基的引入无疑为茯苓多糖在酸性乳中的应用提供了可能性[6]。然而, 目前尚未有关于羧甲基茯苓多糖(carboxymethyl pachymaran, CMP)在调控CSpH稳定性中的应用效果与机制研究。
同时, 许多因素被认为会影响多糖-CS相互作用和体系的物理稳定性, 例如多糖类型、结构特征、环境条件等。其中, 多糖的浓度(多糖-蛋白复合比例)以及环境pH受到最多的研究[7]。因此, 本研究利用浊度、zeta电位、激光共聚焦显微镜、流变等手段探究CMP对CS溶液稳定性的调控作用, 着重关注多糖-蛋白复合比例在不同pH下的影响。此外, 通过抗氧化活性与非酶糖基化产物的生成抑制作用共同探究CMP在酸性条件下对CS生物活性的促进效果, 为新型、高品质酸性乳饮料的开发提供理论依据与指导。
1 材料与方法
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1.1 材料与试剂
白茯苓粉(湖北罗田九资河); CS、牛血清蛋白(美国Sigma-Aldrich公司); 盐酸、冰醋酸、氢氧化钠、乙酸、乙醇、氯化钠(分析纯, 国药集团化学试剂有限公司); 1,1-二苯基-2-三硝基苯肼自由基(1,1-diphenyl-2-picrylhydrazyl radical, DPPH)、8-苯胺基-1-萘磺酸铵(分析纯, 源叶生物有限公司); 鼠李糖、木糖、甘露糖、阿拉伯糖、半乳糖和葡萄糖标准品(纯度≥99%, 上海阿拉丁生物有限公司)。
1.2 仪器与设备
HH-W600恒温水浴锅(东鹏仪器有限公司); UV-2600i紫外分光光度计(上海美析仪器有限公司); DAWN HELEOS凝胶渗透色谱-多角激光散射仪(美国沃特世科技公司); CY-3000磁力搅拌器(常州聚辉仪器制造有限公司); CR21N高速冷冻离心机(德国Eppendorf有限公司); Labconco FreeZon真空冷冻干燥机(宁波新芝生物科技股份有限公司); Thermo Scientific Nicolet iS10傅里叶红外光谱仪(中科瑞捷科技有限公司); Nano ZS纳米粒度及电位分析仪(荷兰弗尔德仪器设备公司); LEICA SP8激光扫描共聚焦显微镜(德国徕卡显微系统贸易公司); Mars60旋转流变仪(奥地利安东帕有限公司)。
1.3 实验方法
1.3.1 CMP的制备与表征
(1) CMP的制备与纯化
参考WANG[8]的方法, 将茯苓粉末以料液比1:50 (w/V)加至0.6 mol/L氢氧化钠溶液中, 4 ℃下静置提取8 h, 经离心(8000 r/min, 15 min)得到上清液。利用冰醋酸溶液中和上清液至pH=6, 沉淀经流水透析(3000 D) 3 d、冷冻干燥后即可得碱溶性茯苓多糖。
随后, 向碱溶性茯苓多糖中加入20% (V/V)氢氧化钠溶液, 冰浴搅拌3 h使其完全溶解; 然后加入氯乙酸(24 g氯乙酸溶解于100 mL异丙醇溶液中)和20% (V/V)氢氧化钠混合溶液, 常温搅拌3 h; 再次加入等量该混合溶液, 在60 ℃环境下反应1 h, 用冰醋酸溶液中和反应溶液至pH=7, 旋蒸法回收异丙醇, 剩余溶液用透析袋(3000 D)流水透析3 d, 透析液经浓缩后用95%乙醇沉淀过夜, 离心收集沉淀, 冷冻干燥即得到CMP。
(2) CMP的化学组成与结构表征
参考LI等[9]的方法, 以葡萄糖为标准品, 采用苯酚-硫酸法测定CMP的总糖含量; 以牛血清白蛋白为标准品, 按照考马斯亮蓝法测定蛋白质含量; 使用3,5-二硝基水杨酸法测定还原糖含量; 以半乳糖醛酸为标准品, 采用咔唑-硫酸法测定半乳糖醛酸含量; 通过铜盐沉淀法测定了CMP的取代度值, 10 mL CMP (10 mg/mL)与4 mL NH4Cl缓冲溶液(0.1 mol/L)混合后调节pH至6.5并加入10 mL CuSO4标准溶液(0.05 mol/L), 摇匀后过滤并将上清液pH调至8.0, 加入0.05 g紫脲酸铵指示剂, 以乙二胺四乙酸(ethylene diamine tetraacetic acid, EDTA)标准溶液(0.05 mol/L)滴定该溶液至呈紫蓝色。记录EDTA消耗量并计算取代度。
将CMP使用超纯水溶解, 配置成质量浓度1 mg/mL的溶液, 用紫外可见分光光度计扫描得到图谱, 设置200~800 nm的波长扫描范围; 采用Nexus 470红外光谱仪测定CMP的红外光谱(400~4000 cm-1), 并采用Ominic7.3软件进行分析; 采用配备HP-5硅胶毛细管柱(30 m× 0.32 mm, 0.25 μm)的GC-6890N气相色谱分析CMP的单糖组成, CMP以3 mol/L三氟乙酸溶解后密封并于110 ℃度下水解6 h, 除酸后向水解物分别加入10 mg盐酸羟胺、1.5 mg肌醇和1.0 mL吡啶并混合均匀, 密封后于90 ℃下水浴30 min, 冷却后加入乙酸酐0.5 mL并再次于90 ℃下反应30 min后将样液过膜进样, 以鼠李糖、木糖、甘露糖、阿拉伯糖、半乳糖和葡萄糖为单糖标准品; 通过配备He-Ne激光器(λ=658 nm)和Styragel HMW 6E色谱柱(7.8 mm×300 mm)的凝胶渗透色谱-多角激光散射仪测定CMP的重均分子量(Mw)和均方根旋转半径(Rg), 其中1.0 mg/mL CMP溶于0.1% NaCl水溶液[10]。
1.3.2 CMP对CS溶液pH响应特性的调控作用
(1)浊度的测定
将CS粉末分散于0.01 moL/L磷酸盐溶液中过夜搅拌, 得到CS溶液。随后, CS溶液质量浓度固定为0.1 mg/mL, 分别向其中添加等体积的0.50、0.10与0.02 mg/mL的CMP溶液(CMP:CS=5:1, 1:1, 1:5, m/m, 通过预实验确定这些比例下复合物稳定性具有显著性差异)。最后, 将这些混合物用0.1~1.0 moL/L盐酸溶液从pH=7酸化逐渐至pH=1.00±0.02, 并在25 ℃下测定不同pH时CMP, CS, CMP-CS复合溶液的OD600值, 重复3次[11]。
(2) zeta电位的测定
在25 ℃下, 使用Nano-ZS-90仪器测量上述CMP-CS复合物的zeta电位, 重复3次。
(3)溶解度的测定
将CS溶液以及CMP-CS混合物溶液调至不同pH后, 在3000 r/min下离心10 min。上清液采用考马斯亮蓝法检测蛋白质含量, 重复3次。分别取1 mL、质量浓度为0~0.8 mg/mL的标准牛血清白蛋白溶液与5 mL考马斯亮蓝G250试剂混匀并在室温下避光反应5 min, 随后测定595 nm处的吸光度以绘制标准曲线。样品按照上述方法测定并计算蛋白质含量。
(4)相图与外观变化
在将CS溶液(2 mg/mL)与CMP溶液(2 mg/mL)等体积混合, 并用0.1~1.0 moL/L盐酸酸化至不同pH。随后将样品于4 ℃下静置24 h, 记录溶液相分离状态, 包括浑浊悬浮液沉淀()、无沉淀澄清溶液(〇)、浑浊悬浮液(□)、有沉淀澄清溶液(■)[12]。
1.3.3 激光共聚焦显微镜观察
在pH 4.0下使用激光扫共聚焦描显微镜在室温下记录CS-CMP混合物的激光共聚焦显微图像。将2 mL不同样品溶液和20 μL罗丹明B (0.05 g/100 mL乙醇)共混并旋涡2 min。随后将200 μL染色液在555 nm的激发波长和630 nm的发射波长下观察并记录图像[13]。
1.3.4 流变特性测定
在pH 4.0下采用旋转流变仪测量CS以及CS-CMP混合溶液的表观黏度与模量。取1 mL样品溶液于流变仪平板上。在稳态剪切测量中, 在0.1~500.0 1/s的剪切速率范围内测定CS溶液的黏度。在动力振荡实验中, 首先通过动态应变扫描试验确定试样的线性黏弹性区, 随后在1%的固定振幅下对样品溶液进行频扫测量, 记录存储模量(G′/G′′)随角频率(0.1~100 rad/s)的函数关系[14]。
1.3.5 体外抗氧化活性测定
参考LIU等[15]的方法, 室温下取2 mL不同样品与2 mL 2×10-4 mol/L DPPH共混并避光反应30 min后于517 nm测定其吸光度A0。同时, 测定2 mL 2×10-4 mol/L DPPH溶液与2 mL无水乙醇混合液的吸光度A1, 以及2 mL样品溶液与2 mL无水乙醇混合液的吸光度A2, 重复3次。计算见公式(1):
DPPH抑制率/%=$(1-\frac{{A}_{0}-{A}_{2}}{{A}_{1}})$×100%
式中: A0为样品溶液加DPPH溶液的吸光度; A2为样品溶液加无水乙醇的吸光度; A1为DPPH溶液加无水乙醇的吸光度。
此外, 将7 mmol/L的2,2’-联氮-双-3-乙基苯并噻唑啉-6-磺酸(2,2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid, ABTS)水溶液与2.45 mmol/mL的K2S2O8水溶液等体积混合并用无水乙醇将其稀释至734 nm处的吸光值为0.7, 即得ABTS工作液。随后, 将0.1 mL多糖溶液与1.9 mL ABTS工作液混匀、避光反应6 min后测定734 nm处吸光值, 以无水乙醇代替样品溶液作为空白。根据下式计算消化液对ABTS阳离子自由基的清除率, 重复3次。计算见公式(2):
ABTS阳离子抑制率/%=$(1-\frac{{A}_{0}-{A}_{2}}{{A}_{1}})$×100%
式中: A1为样品溶液加DPPH溶液的吸光度; A0为ABTS工作液加无水乙醇的吸光度。
1.3.6 体外对非酶糖基化的抑制作用
参照FENG等[16]的实验方法并稍做改动, 建立甲基乙二醛-牛血清白蛋白非酶糖基化模型。牛血清白蛋白(终浓度为5 mg/mL)与甲基乙二醛(终浓度为10 mmol/L)溶液等体积均匀混合后, 添加5 mg/mL样品(2 mL)并用磷酸盐缓冲液(pH 7.4)定容至10 mL, 并在37 ℃下避光反应10 d, 分别于0、5、10 d取样进行测定。其中, 取0.4 mL样品溶液, 分别加入吉腊德-T储备溶液(500 mmol/L, 0.2 mL)和甲酸钠(500 mmol/L, pH 2.9, 3.4 mL), 均匀混合并于37 ℃孵育1 h, 测定294 nm处吸光值以计算二羰基化合物的生成抑制率。而对于荧光晚期糖基化终末产物(advanced glycation end products, AGEs)抑制率, 样品通过荧光酶标仪在激发波长370 nm, 发射波长440 nm条件下检测荧光值并计算抑制率, 重复3次, 以磷酸盐缓冲液作为空白对照。
1.4 数据处理
所有实验均为3次重复, 结果以平均值±标准偏差表示。采用SPSS 18.0统计软件对数据进行单因素方差分析和多重比较(Duncan), P<0.05表示具有显著性差异。
2 结果与分析
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2.1 CMP的化学组成与结构特征
由表1可知, CMP总糖含量达到87.37%, 同时蛋白质含量极低, 仅为0.19%, 表明其是一种纯度较高的多糖。单糖组成结果表明, CMP主要由葡萄糖和甘露糖组成, 其中葡萄糖为主要的结构单糖, 表明CMP是以葡聚糖为主要成分的多糖。同时, CMP的取代度经测试高达0.84, 不仅表明羧甲基衍生化的成功, 同时意味着茯苓多糖的水溶性以及其他生物活性可能得到显著提升。
通过CMP的紫外光谱图(图1A)可以发现, CMP在200~500 nm范围内无明显吸收峰, 表明其纯度较高。此外, 通过红外光谱图(图1B)对其特征吸收峰进行判定, 其中3277 cm-1处归属于CMP的O-H键拉伸振动, 而1600 cm-1处吸收峰归属于CMP的C=O键, 是羧甲基的特征吸收峰[17], 而未衍生化的茯苓多糖在此处没有观察到明显的吸收峰。因此, CMP是一种具有丰富羧甲基的多糖。
2.2 CMP对CS溶液pH响应特性的调控作用理化性质的影响
浊度是分散体中分散颗粒大小和数量的敏感指标, 在复杂的初始阶段很容易发现细微的变化。由浊度变化(图2A)可知, pH从7.0降至6.0时, 不同复配比例的CMP-CS复合物溶液的浊度变化并不明显, 说明CMP与CS之间不存在强相互作用。当pH由6.0接近4.0时, CS样品的浊度显著上升, 并在pH 4.8处达到最大, 这是由于pH逐渐降至CS等电点所导致的蛋白分子聚集[18]。由于CMP的加入, CMP-CS复合物的浊度在该阶段同样显著增加, 但二者以5:1比例复配的复合物的最大浊度均显著高于CS, 但1:1和1:5的比例下较CS低。在之前的报道中, 壳聚糖越高, 其对CS的pH稳定性提升越显著[19]。通过它们的zeta电位(图2B)与CS溶解性(图2C)可以看出, 此时带负电荷的CMP通过静电作用力与带正电荷的CS结合, 表明当CMP含量高于CS时, 二者间在该pH范围内通过相互作用形成了复杂聚集体, 浊度较CS或其它复合比例复合物显著增加。最后, 当pH从4.0降低到3.0时, 由于CMP和CS携带足够的相反净电荷, 导致电荷中和并且相分离, 稳定效果受到限制; 此外, 在非常低的pH (<3.0)下, 即pH接近或低于羧基的解离常数(4~5)值时, 羧基的质子化使CMP具有净零表面电荷[20], 削弱了它与CS的静电相互作用, 从而降低了复合物的浊度与蛋白溶解度, 但其浊度稳定性仍旧高于CS。根据HOU等[21]的研究, 卵黄蛋白在不同pH下的稳定性能够受到羧甲基纤维素的浓度影响, 且与多糖的添加浓度紧密相关。因此, 在一定pH范围内, 适量CMP的引入能够提高CS的pH稳定性, 且随着CMP含量的增加而更加显著。
图2D记录了CMP-CS混合物溶液的相行为随pH、混合比的变化规律。从图2D中可以看出, 在偏中性环境(pH 5和6)时, 观察到CMP与CS的共溶性, 可能是由于羧基(-COO)增强了与水相的离子偶极矩[22-23]。当pH调整到酸性时, 在pH 4~5以及5:1比例下的复合物具有更好的稳定性, 能观察到无沉淀的混浊溶液。然而, 在1:1和1:5比例下观察到与CS溶液类似的大量沉淀, 这表明在该pH范围内CMP与CS之间的静电相互作用较强。而pH的进一步降低触发了具有不同相行为的不溶性复合物的逐步形成, CMP-CS复合物在pH为2~4范围内形成浑浊悬浮液沉淀。此外, 随着pH降低到2时, 5:1比例下的复合物开始聚集, 出现明显的沉淀。因此, 高浓度CMP能在更宽的pH范围内维持CS悬浮液的稳定, 具有明显的稳定促进效果。
总的来说, 由图2可知, CMP对CS对pH稳定性具有显著的积极作用, 特别是在等电点附近。因此, 后续实验研究选取pH=4来探究CMP对CS溶液pH响应特性的调控作用以及理化性质的影响。
2.3 激光共聚焦分析
为了更好地了解pH=4下CMP-CS的微观结构特性, 本研究通过激光共聚焦显微镜对它们的微观结构与聚集情况进行直接观察。如图3所示, 在pH=4条件下, 能够观察CS溶液均匀且成片状的大面积聚集体(图3A), 类似的微观结构在大豆乳清蛋白-阿拉伯胶的可溶性配合物中也有报道[24]。这再次证实了CS分子在pH=4时具有较强的聚集性。然而, 在图3B~D中, 能观察到不均匀分布的聚集体, 即对于不同比例下的CMP-CS复合物(CS浓度始终保持一致), 它们的聚集程度较CS发生了显著降低, 这可归因于两种带相反电荷的生物聚合物之间较弱的相互作用[25], 表明CMP通过静电相互作用能够显著提升CS的pH稳定性。对于不同CMP-CS复合比例而言, 结果与图2类似, 5:1时复合物具有最低的聚集程度, 代表着较高的稳定性, 而1:1和1:5下的复合物则相对较差。这表明, 在pH 4的情况下, CMP-CS的静电相互作用及其引发的复合物稳定性可能会随着CMP含量的增加而逐渐变强。
2.4 流变特性
在pH=4情况下, CMP-CS混合比例对复合物流变性能的影响如图4A和B所示。表观黏度代表着流体对样品溶液流动的阻力, 这取决于蛋白质分子之间的聚集状态和相互作用。由图4A可以看出, 所有测试样品的表观黏度均随着剪切速率的增加而持续下降, 表现出显著的剪切稀释行为, 这归因于样品分子结构破坏和重排或相邻聚合物链之间相互作用的减少[26]。特别的, 与单独CS相比, 不同比例CMP-CS复合物表观黏度相对较低, 这是由于小颗粒聚集体的形成, 导致分子间的流动阻力降低, 相似的结果也在乳蛋白-壳聚糖相互作用中被观察到[27]。此外, 这种黏度变化随多糖含量的增加而减小, 这有可能是因为黏度较高的CS的相对含量在降低, 也有可能是由于5:1时CMP-CS复合物的粒径与聚合度最小, 使得分子间的摩擦力较低, 黏度下降。
为了进一步研究复合物结构特性的变化, 图4B显示了不同混合比例的复合物的G°(储能模量)和G(损耗模量)随频率的变化。结果表明, 在0.1~100.0 rad/s频率范围内, 各个样品的G°和G随频率的变化而增大。此外, 由于G°始终高于G, 表明基于CS形成的络合物呈凝胶状网络结构, 且主要依靠非共价相互作用维持[28]。对于不同复合比例, 复合物的模量随着CMP含量的增大而减少。这表明, CMP的加入会导致复合物絮凝状态或CS之间的已经形成的网络结构被破坏。这与CLSM、浊度、相图等实验结果相一致。即CMP, 特别是高浓度CMP, 通过静电相互作用与CS发生非共价相互作用, 破坏了酸性条件下CS已经形成的聚集体, 导致CMP-CS复合物的聚集程度大大下降, 表现为该复合物溶液更加清澈、均匀。此外, 高浓度CMP具有的高黏度同样会阻止CS在酸性环境下的聚集, 提高其稳定性。
2.5 体外抗氧化能力
由图5可以看出, 不同CS样品均具有一定的抗氧化活性。其中, CS表现出极为有限的效果, 其对DPPH自由基和ABTS阳离子自由基的清除率仅为19.5%和6.5%, 这表明CS本身可能不具备显著的生物活性。当引入CMP后, 复合物(pH=4下制备)的抗氧化活性较CS具有显著的提高, 其原因可能是由于CMP具有较CS更为明显的抗氧化活性, 因为多糖是一种多羟基化合物, 能够在一定程度上发挥猝灭自由基的作用, 这与其它研究是一致的[29]。而对于3种比例下的复合物, 可以发现复合物中CMP含量越高, 其抗氧化活性越强, 特别是5:1复合比例时的化合物。原因可能有以下两点: 首先, 此时复合物中具有抗氧化活性的CMP含量较高, 导致整体抗氧化能力较强; 其次, 通过结构表征结果可知, 5:1复合比例下的复合物具有较为分散的结构, 聚集程度较低, 这有利于大分子化合物捕获自由基, 而其它比例下复合物分子间聚集程度较高, 难以发挥抗氧化活性[30]。而与CMP相比, 复合物(5:1)具有较高的抗氧化能力, 原因可能为复合物的结构较CMP更加舒展或CMP与CS的非共价相互作用影响了CS的构象并导致CS的抗氧化能力增强。因此, 合适的复配比例对于CMP-CS复合物的生物活性具有重要意义。
2.6 体外非酶糖基化产物生成的抑制效果
AGEs是美拉德反应的产物之一, 是还原糖中的羰基与蛋白质中的氨基之间经过非酶催化的缩合、重排、裂解即氧化修饰等一系列反应所形成的复合物[31]。研究表明, AGEs在体内长期过度积累会诱导炎症、氧化应激, 从而导致糖尿病、肠炎等病症, 寻求天然、安全的AGEs抑制剂具有重要意义[32-33]。
如图6所示, 不同CS样品对于荧光AGEs和二羰基化合物的形成具有不同程度的抑制作用, 并且具有明显规律。首先, 在反应体系储藏10 d期间, 样品的抑制率随着储藏时间的增加而下降, 表明CS样品的抑制能力被逐渐消耗。其次, 与CMP相比, CS具有较低的抑制作用。研究表明, 活性样品的抗氧化能力直接影响美拉德反应中的氧化反应, 并能决定反应的中间产物与最终产物的生成效率[34]。在本研究的实验结果中, 不难发现不同样品的AGEs抑制率与它们的抗氧化活性成正比。其中, 复合物(5:1)具有最强的抑制活性, 这不仅是由于它在所有样品中具有最为显著的抗氧化活性, 同时可能由于它能够干预美拉德反应原料分子间的结合。其中, 复合物(5:1)较CMP具有更好的抑制能力, 可能与复合物具有最佳的抗氧化活性以及与干预美拉德反应中间产物生成紧密相关。因此, 本研究可以推测, 当向酸性乳中添加合适剂量的CMP, 能够使复合物具有极好的AGEs抑制能力。
3 结论
收起
本研究探究了CMP对CS溶液pH响应特性的调控以及生物活性的影响。根据浊度、zeta电位、蛋白质溶解性、相图结果的综合分析, CMP:CS=5:1时, CMP对CS在酸性条件下的聚集具有显著的改善作用, 增加了CS的稳定性与溶解性, 但CMP含量下降时这种效果得到削弱。激光共聚焦显微镜结果也显示出, pH=4下CS聚集程度会随着高浓度CMP的引入而降低, CS主要以碎片化形态均一分布。而流变结果表明, CMP能够降低酸性环境下CS的黏度与模量, 且随着多糖含量的增加而愈加显著, 表明CS的粒径减小、网络结构被破坏, 意味着在酸性环境下CMP在一定程度上阻止了CS的聚集。最后, 通过抗氧化活性与非酶糖基化产物抑制能力, 发现高浓度CMP能够使多糖-CS复合物在酸性环境下表现出显著的生物活性。综上所述, 通过控制CMP的添加量与体系pH可以实现显著提高CS体系稳定性的目的, 为CMP作为新的功能性与生物活性多糖在酸性乳饮料中的应用提供理论基础。
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  • 国家自然科学基金项目(32301996)
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20250313005
  • 接收时间：2025-03-13
  • 首发时间：2026-01-13
  • 出版时间：2025-06-25
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  • 收稿日期：2025-03-13
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国家自然科学基金项目(32301996)
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    1 长江职业学院医药护理学院, 鄂州 436032
    2 湖北工业大学生命科学与健康工程学院, 武汉 430068

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*冯利(1981—), 女, 博士, 教授, 主要研究方向为医药职业教育。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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