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Article(id=1195009886493852257, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1195009883369091469, articleNumber=1001-2494(2025)09-0949-07, orderNo=null, doi=10.11669/cpj.2025.09.007, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1733414400000, receivedDateStr=2024-12-06, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1762842159149, onlineDateStr=2025-11-11, pubDate=1746028800000, pubDateStr=2025-05-01, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1762842159149, onlineIssueDateStr=2025-11-11, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1762842159149, creator=13701087609, updateTime=1762842159149, updator=13701087609, issue=Issue{id=1195009883369091469, tenantId=1146029695717560320, journalId=1190317699101192196, year='2025', volume='60', issue='9', pageStart='893', pageEnd='1004', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1762842158405, creator=13701087609, updateTime=1762846632399, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1195028649066893312, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1195009883369091469, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1195028649071087617, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1195009883369091469, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=949, endPage=955, ext={EN=ArticleExt(id=1195009886720344679, articleId=1195009886493852257, tenantId=1146029695717560320, journalId=1190317699101192196, language=EN, title=Optimization of Polysaccharide Extracted from Ganoderma Using Response Surface Methodology, and Evaluation of Antioxidant Activity, columnId=null, journalTitle=Chinese Pharmaceutical Journal, columnName=null, runingTitle=null, highlight=null, articleAbstract=

OBJECTIVE To optimized the extraction process of Ganoderma lucidum crude polysaccharide using Ganoderma lucidum fruiting body as experimental material, and discuss its antioxidant activity. METHODS In this study, the extraction conditions of crude polysaccharide from Ganoderma lucidum were optimized by single factor experiment and response surface methodology. The polysaccharide was extracted by hot water extraction method, protein was removed by Sevag method, and purified by anion exchange column chromatography and gel column chromatography. Its structure was preliminarily determined by UV spectrum, FT-IR spectrum and atomic force microscope. The antioxidant activity of Ganoderma lucidum polysaccharide was evaluated by detecting DPPH radical, ABTS radical, hydroxyl radical and superoxide anion scavenging ability. RESULTS The results showed that the best extraction conditions of Ganoderma lucidum crude polysaccharide was extraction temperature 92.4 ℃, extraction time 5 h 5 min, liquid-solid ratio 21∶1, and the yield was 2.967±0.228%. Ganoderma lucidum polysaccharide obtained by extraction and purification conform to the structural characteristics of polysaccharide, and did not contain nucleic acids and proteins. Atomic force microscopy showed that most of them presented a chain like conformation. Ganoderma lucidum polysaccharide can significantly scavenge DPPH radical, ABTS radical, hydroxyl radical and superoxide anion. CONCLUSION Ganoderma lucidum polysaccharide has good antioxidant ability, this study provides a basis for further development and application of Ganoderma lucidum.

, correspAuthors=Lu FU, Linbo Zhang, 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=Xiaohan LI, Wenjuan BIAN, Xueying QIAO, Kun WANG, Lu FU, Linbo Zhang), CN=ArticleExt(id=1195010227448823880, articleId=1195009886493852257, tenantId=1146029695717560320, journalId=1190317699101192196, language=CN, title=响应面法优化赤灵芝多糖的提取工艺及其抗氧化活性研究, columnId=1190352405612040510, journalTitle=中国药学杂志, columnName=论著, runingTitle=null, highlight=null, articleAbstract=

目的 以赤灵芝(Ganoderma lucidum)子实体为实验材料,优化赤灵芝粗多糖的提取工艺,探讨其抗氧化活性。方法 通过单因素实验和响应面法优化赤灵芝粗多糖的提取条件,用热水浸提法提取多糖,Sevag法除蛋白,阴离子交换柱层析和凝胶柱层析纯化,通过紫外光谱、傅里叶红外光谱和原子力显微镜对其结构进行初步测定,通过检测1,1-二苯基-2-苦基肼(DPPH)自由基、2,2'-联氨-双(3-乙基苯并噻唑啉-6-磺酸)二胺盐(ABTS)自由基、羟自由基和超氧阴离子清除能力,评价赤灵芝多糖的抗氧化活性。结果 赤灵芝粗多糖的最佳提取条件为提取温度92.4 ℃,提取时间5 h 5 min,液料比21∶1,得率为(2.967±0.228)%。提取纯化获得的赤灵芝多糖符合多糖的结构特征,不含有核酸和蛋白质,原子力显微镜表明其大多数呈现链状构象。赤灵芝多糖可以显著清除DPPH自由基、ABTS自由基、羟自由基和超氧阴离子。结论 赤灵芝多糖具有较好的抗氧化能力,本研究为赤灵芝的进一步开发应用提供依据。

, correspAuthors=付璐, 张林波, authorNote=null, correspAuthorsNote=
*付璐,女,博士,讲师 研究方向:微生物与免疫学 Tel:(0431)84532886;
张林波,男,博士,教授 研究方向:微生物与免疫学 Tel:(0632)3786399
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李晓涵,女,硕士研究生 研究方向:微生物与免疫学

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李晓涵,女,硕士研究生 研究方向:微生物与免疫学

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李晓涵,女,硕士研究生 研究方向:微生物与免疫学

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tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1195009886493852257, language=CN, label=图10, caption=GLP的超氧阴离子清除能力。n=5, $\overline{x}$±s, figureFileSmall=2QS/j7OE0iYUllgBrv3aWA==, figureFileBig=6m5QDOfHPZe1NoCF3V24Xg==, tableContent=null), ArticleFig(id=1195061511728141128, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1195009886493852257, language=EN, label=Tab.1, caption=

Factors and levels of response surface analysis of crude polysaccharides from Ganoderma lucidum

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Factor Level
-1 0 1
A: extraction temperature/℃ 80 90 100
B: extraction time/h 4 5 6
C: liquid to material ratios 15∶1 20∶1 25∶1
), ArticleFig(id=1195061511786861385, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1195009886493852257, language=CN, label=表1, caption=

赤灵芝粗多糖的响应面分析因素及水平

, figureFileSmall=null, figureFileBig=null, tableContent=
Factor Level
-1 0 1
A: extraction temperature/℃ 80 90 100
B: extraction time/h 4 5 6
C: liquid to material ratios 15∶1 20∶1 25∶1
), ArticleFig(id=1195061511853970250, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1195009886493852257, language=EN, label=Tab.2, caption=

ANOVA for response surface quadratic model for the yield of Ganoderma lucidum crude polysaccharide

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Source Sun of squares df Mean Squares F P
Model 4.12 9 0.46 98.32 <0.000 1
A 0.35 1 0.35 75.91 <0.000 1
B 0.040 1 0.040 8.50 0.022 5
C 0.19 1 0.19 40.40 0.000 4
AB 0.083 1 0.083 17.87 0.003 9
AC 0.099 1 0.099 21.23 0.002 5
BC 0.001 1 0.001 0.26 0.062 39
A2 0.75 1 0.75 160.38 <0.000 1
B2 1.58 1 1.58 339.07 <0.000 1
C2 0.69 1 0.69 148.98 <0.0001
Residual 0.033 7 0.005
Lack of fit 0.017 3 0.006 1.44 0.3560
Pure error 0.016 4 0.004
Cor total 4.15 16
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赤灵芝粗多糖的响应面模型的方差分析

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Source Sun of squares df Mean Squares F P
Model 4.12 9 0.46 98.32 <0.000 1
A 0.35 1 0.35 75.91 <0.000 1
B 0.040 1 0.040 8.50 0.022 5
C 0.19 1 0.19 40.40 0.000 4
AB 0.083 1 0.083 17.87 0.003 9
AC 0.099 1 0.099 21.23 0.002 5
BC 0.001 1 0.001 0.26 0.062 39
A2 0.75 1 0.75 160.38 <0.000 1
B2 1.58 1 1.58 339.07 <0.000 1
C2 0.69 1 0.69 148.98 <0.0001
Residual 0.033 7 0.005
Lack of fit 0.017 3 0.006 1.44 0.3560
Pure error 0.016 4 0.004
Cor total 4.15 16
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响应面法优化赤灵芝多糖的提取工艺及其抗氧化活性研究
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李晓涵 1, 2, 3 , 边文娟 1, 2, 3 , 乔雪莹 2 , 王昆 2, 3 , 付璐 1, * , 张林波 2, 3, *
中国药学杂志 | 论著 2025,60(9): 949-955
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中国药学杂志 | 论著 2025, 60(9): 949-955
响应面法优化赤灵芝多糖的提取工艺及其抗氧化活性研究
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李晓涵1, 2, 3, 边文娟1, 2, 3, 乔雪莹2, 王昆2, 3, 付璐1, *, 张林波2, 3, *
作者信息
  • 1 吉林农业大学生命科学学院, 长春 130118
  • 2 枣庄学院食品科学与制药工程学院, 山东 枣庄 277160
  • 3 鲁南智慧生物医药工程研究院, 山东 枣庄 277160

    • 李晓涵,女,硕士研究生 研究方向:微生物与免疫学

通讯作者:

*付璐,女,博士,讲师 研究方向:微生物与免疫学 Tel:(0431)84532886;
张林波,男,博士,教授 研究方向:微生物与免疫学 Tel:(0632)3786399
Optimization of Polysaccharide Extracted from Ganoderma Using Response Surface Methodology, and Evaluation of Antioxidant Activity
Xiaohan LI1, 2, 3, Wenjuan BIAN1, 2, 3, Xueying QIAO2, Kun WANG2, 3, Lu FU1, *, Linbo Zhang2, 3, *
Affiliations
  • 1 College of Life Science, Jilin Agricultural University, Changchun 130118, China
  • 2 College of Food Science and Pharmaceutical Engineering, Zaozhuang University, Zaozhuang 277160, China
  • 3 Lunan Institute of Intelligent Biomedical Engineering, Zaozhuang 277160, China
出版时间: 2025-05-01 doi: 10.11669/cpj.2025.09.007
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摘要
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目的 以赤灵芝(Ganoderma lucidum)子实体为实验材料,优化赤灵芝粗多糖的提取工艺,探讨其抗氧化活性。方法 通过单因素实验和响应面法优化赤灵芝粗多糖的提取条件,用热水浸提法提取多糖,Sevag法除蛋白,阴离子交换柱层析和凝胶柱层析纯化,通过紫外光谱、傅里叶红外光谱和原子力显微镜对其结构进行初步测定,通过检测1,1-二苯基-2-苦基肼(DPPH)自由基、2,2'-联氨-双(3-乙基苯并噻唑啉-6-磺酸)二胺盐(ABTS)自由基、羟自由基和超氧阴离子清除能力,评价赤灵芝多糖的抗氧化活性。结果 赤灵芝粗多糖的最佳提取条件为提取温度92.4 ℃,提取时间5 h 5 min,液料比21∶1,得率为(2.967±0.228)%。提取纯化获得的赤灵芝多糖符合多糖的结构特征,不含有核酸和蛋白质,原子力显微镜表明其大多数呈现链状构象。赤灵芝多糖可以显著清除DPPH自由基、ABTS自由基、羟自由基和超氧阴离子。结论 赤灵芝多糖具有较好的抗氧化能力,本研究为赤灵芝的进一步开发应用提供依据。

赤灵芝  /  多糖  /  响应面法  /  抗氧化活性

OBJECTIVE To optimized the extraction process of Ganoderma lucidum crude polysaccharide using Ganoderma lucidum fruiting body as experimental material, and discuss its antioxidant activity. METHODS In this study, the extraction conditions of crude polysaccharide from Ganoderma lucidum were optimized by single factor experiment and response surface methodology. The polysaccharide was extracted by hot water extraction method, protein was removed by Sevag method, and purified by anion exchange column chromatography and gel column chromatography. Its structure was preliminarily determined by UV spectrum, FT-IR spectrum and atomic force microscope. The antioxidant activity of Ganoderma lucidum polysaccharide was evaluated by detecting DPPH radical, ABTS radical, hydroxyl radical and superoxide anion scavenging ability. RESULTS The results showed that the best extraction conditions of Ganoderma lucidum crude polysaccharide was extraction temperature 92.4 ℃, extraction time 5 h 5 min, liquid-solid ratio 21∶1, and the yield was 2.967±0.228%. Ganoderma lucidum polysaccharide obtained by extraction and purification conform to the structural characteristics of polysaccharide, and did not contain nucleic acids and proteins. Atomic force microscopy showed that most of them presented a chain like conformation. Ganoderma lucidum polysaccharide can significantly scavenge DPPH radical, ABTS radical, hydroxyl radical and superoxide anion. CONCLUSION Ganoderma lucidum polysaccharide has good antioxidant ability, this study provides a basis for further development and application of Ganoderma lucidum.

Ganoderma lucidum  /  polysaccharide  /  response surface methodology  /  antioxidant activity
李晓涵, 边文娟, 乔雪莹, 王昆, 付璐, 张林波. 响应面法优化赤灵芝多糖的提取工艺及其抗氧化活性研究. 中国药学杂志, 2025 , 60 (9) : 949 -955 . DOI: 10.11669/cpj.2025.09.007
Xiaohan LI, Wenjuan BIAN, Xueying QIAO, Kun WANG, Lu FU, Linbo Zhang. Optimization of Polysaccharide Extracted from Ganoderma Using Response Surface Methodology, and Evaluation of Antioxidant Activity[J]. Chinese Pharmaceutical Journal, 2025 , 60 (9) : 949 -955 . DOI: 10.11669/cpj.2025.09.007
正文
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赤灵芝(Ganoderma lucidum)为担子菌门(Basidiomycota)、多孔菌目(Polyporales)、灵芝科(Ganodermataceae)、灵芝属(Ganoderma)真菌,别名丹芝、血灵芝、万年蕈等[1]。赤灵芝在中国已有超过2 000年的药用历史,它的药用价值最早在东汉时期的《神农本草经》一书中被描述[2]。赤灵芝作为一种潜在的保健产品和食物来源,越来越受到关注[3]。2020年被纳入“药食同源”试点目录[4],赤灵芝含有多种活性成分,包括多糖、蛋白质、生物碱、萜类等[5],其中多糖是主要的活性成分之一。多糖是一类天然高分子化合物,因具备良好的生理、药理活性以及低毒性的特点,在药品、食品乃至化妆品等多个领域均展现出广阔的应用前景[6]。近年来,研究表明赤灵芝多糖具有抗氧化[7-9]、免疫调节[10-12]、抗肿瘤[13-15]、抗炎[16-17]、降糖降脂[18]、保肝[19]等生物活性。已有研究表明,多糖的抗氧化活性主要体现在其能够增强抗氧化酶的作用,清除自由基,减少脂质过氧化,并保护人体免受自由基相关的健康危害[20]。
本研究以赤灵芝子实体为实验材料,用热水浸提法提取多糖,通过响应面法优化赤灵芝粗多糖的提取条件,对其结构进行初步测定;在此基础上通过检测1,1-二苯基-2-苦基肼(DPPH)自由基清除能力、2,2'-联氨-双(3-乙基苯并噻唑啉-6-磺酸)二胺盐(ABTS)自由基清除能力、羟自由基清除能力和超氧阴离子清除能力,评价赤灵芝多糖的抗氧化活性。本研究旨在优化赤灵芝粗多糖提取条件,探究赤灵芝多糖的抗氧化活性,为赤灵芝的进一步开发应用提供依据。
1 材料与方法
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1.1 供试材料
赤灵芝子实体,产自吉林省长春市,经吉林农业大学张林波教授鉴定为赤灵芝(Ganoderma lucidum)。
1.2 试剂与仪器
DPPH自由基清除能力检测试剂盒、ABTS自由基清除能力检测试剂盒、羟自由基清除能力检测试剂盒、超氧阴离子清除能力检测试剂盒(北京索莱宝科技有限公司),旋转蒸发仪(RE-52AA,上海亚荣生化仪器厂),低温高速离心机(PK-16A,湖南平科科学仪器有限公司),冷冻干燥机(SCIENTZ-10N/A,宁波新芝生物科技股份有限公司),紫外可见分光光度计(UV-26001,岛津仪器有限公司),FTIR-650傅里叶变换红外光谱仪(FTIR-650,天津港东科技股份有限公司),原子力显微镜(Ⅸ73,OLYMPUS),智能恒温金属浴(HX-20S,上海沪析实业有限公司),微孔板检测系统(SpectraMax ABS Plus/ABS,美谷分子仪器有限公司)
1.3 赤灵芝多糖的提取及纯化
1.3.1 赤灵芝粗多糖的提取
准确称取赤灵芝子实体干品1 000 g,用粉碎机将其制成均匀的粉末,按液料比15∶1的比例加入蒸馏水,80 ℃浸提4 h,重复两次,收集浸提液,抽滤后旋转蒸发,在收集的浓缩液中加入3倍体积的无水乙醇静置过夜,4 500 r·min-1离心15 min,弃上清,收集沉淀。冷冻干燥后获得赤灵芝粗多糖。使用苯酚-硫酸法测定赤灵芝粗多糖的多糖含量,考马斯亮蓝法测定赤灵芝粗多糖的蛋白质含量。
1.3.2 赤灵芝粗多糖的纯化
称取适量的赤灵芝粗多糖溶于蒸馏水中与Sevag试剂(氯仿-正丁醇=4∶1)按4∶1的比例进行混合,置于摇床内25 ℃处理0.5 h,8 000 r·min-1离心10 min,重复上述步骤直到蛋白层稀薄或消失,回收合并上层多糖溶液,置于截留相对分子质量3×106的透析袋中,流水透析24 h,蒸馏水透析24 h,收集透析后的多糖溶液,加入3倍体积的无水乙醇静置沉淀,离心收集沉淀,冷冻干燥后获得赤灵芝纯化多糖(GLP),测定GLP的多糖含量和蛋白质含量。将GLP通过纤维素DE-52用超纯水、0.3 mol·L-1 NaCl和0.5 mol·L-1 NaCl进行洗脱,合并相同洗脱液,透析醇沉,冷冻干燥后得到不同的多糖组分:GLP-A、GLP-B、GLP-C。将GLP-B溶于超纯水中,用葡聚糖凝胶G-100进一步纯化,收集后透析醇沉并冷冻干燥,最终得到纯化多糖GLP-B-1。
1.4 单因素实验设计
1.4.1 不同提取温度对赤灵芝粗多糖得率的影响
以提取温度为实验变量,固定提取时间为4 h,液料比为15∶1,探究不同提取温度(60、70、80、90、100 ℃)对赤灵芝粗多糖得率的影响。
1.4.2 不同提取时间对赤灵芝粗多糖得率的影响
以提取时间为实验变量,固定提取温度为80 ℃,液料比为15∶1,探究不同提取时间(2、3、4、5、6 h)对赤灵芝粗多糖得率的影响。
1.4.3 不同液料比对赤灵芝粗多糖得率的影响
以液料比为实验变量,固定提取时间为4 h,提取温度为80 ℃,探究不同液料比(5∶1,10∶1,15∶1,20∶1,25∶1)对赤灵芝粗多糖得率的影响。
1.5 响应面优化设计
根据单因素实验的结果,设计响应面优化实验,使用软件Design-expert 8.0.6进行Box-Behnken实验设计,设计三因素三水平的响应面优化实验,表1所示三个变量因素为A:提取温度(℃)、B:提取时间(h)和C:液料比(mL∶g),3个变量水平用-1、0、1来表示。
1.6 赤灵芝多糖的结构测定
1.6.1 紫外光谱分析
GLP-B-1配置成0.2 mg·mL-1的溶液,用紫外分光光度计在200~800 nm扫描。
1.6.2 傅里叶红外光谱分析
将1 mg GLP-B-1与100 mg溴化钾在研钵中混合均匀后压片,用傅里叶红外光谱仪扫描溴化钾空白压片和样品压片,扫描范围为4 000~400 cm-1,扫描次数为32次。
1.6.3 原子力显微镜分析
配置20 μg·mL-1的GLP-B-1溶液,将少量溶液滴在解离后的云母片上,干燥后通过原子力显微镜观察样本,用Gwyddion软件分析结果。
1.7 赤灵芝多糖的抗氧化活性分析
1.7.1 DPPH自由基清除能力
用提取液将GLP配置成0.5、1、2、3、4、5 mg·mL-1的溶液,按照试剂盒说明书检测GLP的DPPH自由基清除能力。
1.7.2 ABTS自由基清除能力
用提取液将GLP配置成0.3、0.4、0.5、0.6、0.7、0.8 mg·mL-1的溶液,按照试剂盒说明书检测GLP的ABTS自由基清除能力。
1.7.3 羟自由基清除能力
用提取液将GLP配置成0.3、0.4、0.5、0.6、0.7、0.8 mg·mL-1的溶液,按照试剂盒说明书检测GLP的羟自由基清除能力。
1.7.4 超氧阴离子清除能力
用提取液将GLP配置成0.3、0.4、0.5、0.6、0.7、0.8 mg·mL-1的溶液,按照试剂盒说明书检测GLP的超氧阴离子清除能力。
1.8 数据处理
试验数据经Excel记录后用SPSS 27.0进行统计学分析,试验数据用平均数±标准差( $\overline{x}$±s)来表示。
2 结果
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2.1 赤灵芝多糖的提取纯化结果
热水浸提法获得的赤灵芝粗多糖中多糖含量为27.64%,蛋白质含量为7.73%。Sevag法除蛋白后,测定GLP的多糖含量为65.088%,蛋白含量为0.12%。GLP经纤维素DE-52阴离子交换柱层析进一步纯化后,得到3种不同的多糖组分:GLP-A(超纯水洗脱),GLP-B(0.3 mol·L-1 NaCl洗脱),GLP-C(0.5 mol·L-1 NaCl洗脱),洗脱曲线见图1A。GLP-B经葡聚糖凝胶G-100纯化后,得到GLP-B-1,洗脱曲线见图1B。
2.2 赤灵芝粗多糖得率的单因素实验结果
通过单因素实验探究不同提取温度、提取时间、液料比对赤灵芝粗多糖得率的影响。结果表明,随着提取温度的上升,得率逐渐上升,在90 ℃时得率最大(图2A);随着提取时间的增加,得率逐渐上升,在5 h时达到顶峰(图2B);随着液料比增大,多糖的得率增加,当液料比为20∶1时得率最高(图2C)。
2.3 赤灵芝粗多糖得率的响应面优化实验结果
响应面优化实验结果(表2)显示,赤灵芝粗多糖得率的回归模型极具显著性(P<0.001),失拟项不具有显著性(P>0.05),说明该回归模型有效,回归拟合方程为:得率(%)=2.81+0.21A+0.070B+0.15C+0.14AB-0.16AC-0.017BC-0.42A2-0.61B2-0.41C2,模型预测的赤灵芝粗多糖最佳提取条件为:提取温度92.38 ℃,提取时间5.08 h,液料比20.71∶1,预测最高提取率为2.844%,根据实际条件以提取温度92.4 ℃,提取时间5 h 5 min,液料比21∶1进行GLP的提取,验证实验中,赤灵芝粗多糖的得率为(2.967±0.228)%,接近模型的预测值,说明该模型计算的最佳提取条件具有可信度(图3)。
2.4 赤灵芝多糖的结构测定
2.4.1 紫外光谱分析
260 nm处没有出现紫外吸收峰,说明GLP-B-1中不含有核酸(图4);280 nm处没有出现紫外吸收峰,说明GLP-B-1中不含有蛋白质。
2.4.2 傅里叶红外光谱分析
在3 405 cm-1处出现的强吸收峰是由糖链上的O-H伸缩振动引起的,也是糖类的特征峰(图5)。2 940 cm-1处出现的吸收峰是由于糖链上C-H伸缩振动,1 421 cm-1处出现的吸收峰归因于糖链上C-H的剪式振动峰,在1 056 cm-1处显示了C-O的吸收峰,在927 cm-1处的吸收峰表明多糖中具有α构型部分,在549 cm-1处的吸收峰是吡喃糖链骨架特有的,这些信号均符合多糖的结构特征,1 740 cm-1附近没有吸收峰,表明GLP-B-1糖链中没有糖醛酸。
2.4.3 原子力显微镜分析
赤灵芝多糖在中性水溶液中呈现链状构象,具有高度的分支结构,链间重叠交织。赤灵芝多糖单链直径在26.2~64.6 nm,链高在0.621~1.269 nm;链间交叠最高处直径为55.02~152.8 nm,链高为1.273~4.067 nm,见图6。
2.5 赤灵芝多糖的抗氧化活性分析结果
2.5.1 DPPH自由基清除能力
GLP有清除DPPH自由基的能力,质量浓度在(0.5~4.0) mg·mL-1时,GLP的DPPH自由基清除率随浓度增大而提高,当质量浓度为4 mg·mL-1时GLP对DPPH自由基的清除率最高,为11.118%,见图7。
2.5.2 ABTS自由基清除能力
GLP有清除ABTS自由基的能力,质量浓度在(0.3~0.7)mg·mL-1时,GLP的ABTS自由基清除率随浓度增大而提高,当质量浓度为0.7 mg·mL-1时GLP对ABTS自由基的清除率最高,为57.529%,见图8。
2.5.3 羟自由基清除能力
GLP有清除羟自由基的能力,质量浓度在(0.3~0.8) mg·mL-1时,GLP的羟自由基清除率随浓度增大而提高,当质量浓度为0.8 mg·mL-1时GLP对羟自由基的清除率最高,为5.116%,见图9。
2.5.4 超氧阴离子清除能力
GLP能清除超氧阴离子自由基,质量浓度在(0.3~0.7) mg·mL-1时,GLP的超氧阴离子自由基清除率随浓度增大而提高,当质量浓度为0.7 mg·mL-1时GLP对超氧阴离子自由基的清除率最高,为50.311%,见图10。
3 讨论
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响应面优化实验是用于拟合包含多个连续变量的预测模型的实验[21],是20世纪50年代开发的一组统计工具,用于优化实验设计和数据分析,以改善产品或过程的质量[22],响应面图能够直观地反映各因素之间的交互关系[23],运用图形技术将其中的函数关系显示出来,以便选择试验设计中最优实验条件[24]。已有报道,赤灵芝子实体热浸提得率为1.71%~2.67%[25]。Kan等[26]在提取时间137 min、提取温度66 ℃和液料比35∶1的条件提取,得率为 2.44%。Li等[27]得到赤灵芝多糖的最佳提取条件为:采用90 ℃热水提取90 min,料液比1∶20,提取2次,提取率为2.67%。本研究通过响应面优化实验得到的赤灵芝粗多糖最佳提取温度为92.4 ℃,提取时间5 h 5 min,液料比21∶1,在此条件下,多糖得率为(2.967±0.228)%,接近模型预测值2.844%,优化了赤灵芝粗多糖的提取工艺,提高多糖得率。
水提取法是目前应用最广泛的多糖提取技术,水是目前最常见的提取溶剂,也是其他提取方法的基础溶剂[28],基于多糖分子与水的极性相容的原理,在提取过程中多糖分子更易于被溶解[29]。本研究使用热水浸提法提取,Sevag法除蛋白,阴离子交换柱层析和凝胶柱层析纯化,通过紫外光谱、傅里叶红外光谱和原子力显微镜分析赤灵芝多糖,结果证实纯化分离的赤灵芝多糖符合多糖的化学特性,在中性水溶液中呈现链状构象,与已有报道基本一致,目前有关多糖提取纯化及活性研究方面报道较多,但应用原子力显微镜对多糖表面形态的研究报道较少。
已有研究表明,赤灵芝提取物及其生物活性化合物已被用作抗氧化剂和抗菌剂的替代品[20],Kan等[26]检测发现赤灵芝多糖具有较好的还原力、DPPH自由基清除能力和超氧阴离子清除能力,这说明灵芝可以作为天然抗氧化剂的来源。Fan等[30]发现多糖抗氧化活性与糖醛酸含量正相关,糖醛酸可以通过与自由基反应来清除自由基。本研究表明GLP的DPPH自由基清除能力、ABTS自由基清除能力、羟自由基清除能力和超氧阴离子清除能力的清除率分别为11.118%、57.529%、5.116%和50.311%,说明GLP具有较好的抗氧化能力。
综上,本研究优化了赤灵芝粗多糖的提取工艺,进一步证实GLP具有较好的抗氧化活性,这为赤灵芝的进一步开发应用提供了依据。
基金
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  • 贵州省食用菌育种重点实验室开放课题项目资助(黔科合平台人才[2019]5105-2008号)
  • 枣庄学院博士科研启动基金资助(1020737)
  • 枣庄学院(自然科学类)校级科研基金项目联合培养研究专项资助(102062206)
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2025年第60卷第9期
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doi: 10.11669/cpj.2025.09.007
  • 接收时间：2024-12-06
  • 首发时间：2025-11-11
  • 出版时间：2025-05-01
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  • 收稿日期：2024-12-06
基金
贵州省食用菌育种重点实验室开放课题项目资助(黔科合平台人才[2019]5105-2008号)
枣庄学院博士科研启动基金资助(1020737)
枣庄学院(自然科学类)校级科研基金项目联合培养研究专项资助(102062206)
作者信息
    1 吉林农业大学生命科学学院, 长春 130118
    2 枣庄学院食品科学与制药工程学院, 山东 枣庄 277160
    3 鲁南智慧生物医药工程研究院, 山东 枣庄 277160

通讯作者:

*付璐,女,博士,讲师 研究方向:微生物与免疫学 Tel:(0431)84532886;
张林波,男,博士,教授 研究方向:微生物与免疫学 Tel:(0632)3786399
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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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