Article(id=1261336280806339041, tenantId=1146029695717560320, journalId=1260987677001138203, issueId=1261336272929472630, articleNumber=null, orderNo=null, doi=10.13386/j.issn1002-0306.2025040065, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1743955200000, receivedDateStr=2025-04-07, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1778655603838, onlineDateStr=2026-05-13, pubDate=1777564800000, pubDateStr=2026-05-01, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1778655603838, onlineIssueDateStr=2026-05-13, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1778655603838, creator=13701087609, updateTime=1778655603838, updator=13701087609, issue=Issue{id=1261336272929472630, tenantId=1146029695717560320, journalId=1260987677001138203, year='2026', volume='47', issue='9', pageStart='1', pageEnd='504', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=1, specialIssue=null, createTime=1778655601961, creator=13701087609, updateTime=1778657530282, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1261344361019728695, tenantId=1146029695717560320, journalId=1260987677001138203, issueId=1261336272929472630, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1261344361019728696, tenantId=1146029695717560320, journalId=1260987677001138203, issueId=1261336272929472630, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=113, endPage=122, ext={EN=ArticleExt(id=1261336284182753785, articleId=1261336280806339041, tenantId=1146029695717560320, journalId=1260987677001138203, language=EN, title=Magnetically Induced Electric Field-Enzymatic Hydrolysis Coupled Extraction, Purification and Characterization of Taurine from Abalone Viscera, columnId=1261336277291548795, journalTitle=Science and Technology of Food Industry, columnName=Research and Investigation, runingTitle=null, highlight=null, articleAbstract=

This study established a novel magnetically induced electric field-enzymatic hydrolysis (MIEF-EH) coupled technology for the sustainable taurine extraction from abalone viscera byproducts. The results of the study indicated that the optimal extraction conditions were as follows: an excitation voltage of 1400 V, an electric field frequency of 50 kHz, a flow rate of 25 L/h, and a reaction time of 1 h. Under the optimal conditions, MIEF-EH achieved a taurine yield of 12.36 mg/g, significantly (P<0.05) outperforming both individual methods (such as MIEFE, microwave, enzymatic hydrolysis and hydrothermal) as well as other coupled approaches (microwave-enzymatic hydrolysis and hydrothermal-enzymatic hydrolysis). SEM revealed pronounced particle-size reduction and the formation of a loose, porous microstructure, correlating with improved taurine extraction. Subsequent purification via ion-exchange resin yielded taurine with 92.29% recovery and 96.78% purity, while ethanol precipitation achieved 95.30% recovery and 94.43% purity. The chemical structure and molecular mass of the purified taurine were confirmed through multi-technique validation (FT-IR, MS, and 1H NMR). All physicochemical properties complied with the specifications of the Chinese National Standard for Food Additive Taurine (GB 14759-2010). In summary, the MIEF-EH coupled technology developed in this study provides a feasible technological pathway for green conversion of abalone viscera waste into high-purity natural taurine.

, correspAuthors=Meng LI, Wuyin WENG, Yucang ZHANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright © 2026 Science and Technology of Food Industry. All rights reserved., 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=Yujie CHEN, Meng LI, Qun LIU, Wuyin WENG, Yucang ZHANG), CN=ArticleExt(id=1261336300980941458, articleId=1261336280806339041, tenantId=1146029695717560320, journalId=1260987677001138203, language=CN, title=基于磁感应电场-酶解耦合技术的鲍鱼内脏牛磺酸提取、纯化及表征, columnId=1261336277849391232, journalTitle=食品工业科技, columnName=研究与探讨, runingTitle=null, highlight=null, articleAbstract=

以鲍鱼内脏废弃物为原料,通过优化磁感应电场(MIEFE)关键参数(激励电压、电场频率、流量及反应时间),建立了磁感应电场-酶解耦合提取技术(MIEF-EH),并系统完成产物纯化与结构表征,旨在实现牛磺酸的绿色、高效提取与纯化。研究结果表明:最优提取条件为激励电压1400 V、电场频率50 kHz、流量25 L/h、反应时间1 h;在优化条件下,MIEF-EH法提取量达到12.36 mg/g,显著(P<0.05)高于单一方法(MIEFE、微波、酶解及水热法)及其他耦合方法(微波-酶解、水热-酶解);SEM表征显示,经过处理后的鲍鱼内脏粒径显著减小,形成疏松多孔微结构,该变化与牛磺酸提取量提升相关;牛磺酸粗提物经离子交换树脂纯化,回收率92.29%、纯度96.78%;乙醇沉淀法回收率95.30%、纯度94.43%;FT-IR、MS及1H NMR联合分析证实纯化产物分子量与牛磺酸理论值高度吻合,化学结构与标准品一致;获得的牛磺酸产物各项理化指标符合GB 14759-2010要求。综上,本研究构建的MIEF-EH耦合技术为鲍鱼内脏废弃物向高纯度天然牛磺酸的绿色转化提供了可行技术路径。

, correspAuthors=李蒙, 翁武银, 张玉苍, authorNote=null, correspAuthorsNote=
李蒙(1990−),女,博士,讲师,研究方向:生物质资源高值化利用,E-mail:
张玉苍(1964−),男,博士,教授,研究方向:生物质资源高值化利用,E-mail:
, copyrightStatement=版权所有 © 2026《食品工业科技》编辑部, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=cJBIM+yTjEsWtnMIdZPZrw==, magXml=IpLePL+tWnSlmOeCVul2Cw==, pdfUrl=null, pdf=OF4zFZD5heruTrh0Ph+X6Q==, pdfFileSize=7585359, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=LK+EwFBv4Ph7sscNj0RXHg==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=iWHTJx30eDwD9HcXVMktSQ==, mapNumber=null, authorCompany=null, fund=null, authors=

陈钰洁(1999−),女,硕士研究生,研究方向:食品加工副产物的高值化利用,E-mail:

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陈钰洁(1999−),女,硕士研究生,研究方向:食品加工副产物的高值化利用,E-mail:

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陈钰洁(1999−),女,硕士研究生,研究方向:食品加工副产物的高值化利用,E-mail:

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Ultrasonics Sonochemistry, 2015, 24: 36−42., articleTitle=null, refAbstract=null)], funds=null, companyList=[AuthorCompany(id=1261336303736599206, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1261336280806339041, xref=null, ext=[AuthorCompanyExt(id=1261336303883399847, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1261336280806339041, companyId=1261336303736599206, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, China), AuthorCompanyExt(id=1261336303971480232, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1261336280806339041, companyId=1261336303736599206, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=集美大学海洋食品与生物工程学院,福建厦门 361021)])], figs=[ArticleFig(id=1261336337521718137, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1261336280806339041, language=EN, label=Fig.1, caption=Effects of magnetic induction electric field extraction conditions on taurine yield from abalone viscera, figureFileSmall=gkjuXwlNYnWzlZBENhtWaQ==, figureFileBig=LK+EwFBv4Ph7sscNj0RXHg==, tableContent=null), ArticleFig(id=1261336338691928959, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1261336280806339041, language=CN, label=图1, caption=磁感应电场条件对鲍鱼内脏牛磺酸提取量的影响

注:不同小写字母表示差异显著(P<0.05),图2~图3同。

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注:A,a:乙醇沉淀法,B,b:离子交换树脂法。

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Comparison of different methods for extraction of natural taurine

, figureFileSmall=null, figureFileBig=null, tableContent=
提取方法原料提取时间牛磺酸提取量(mg/g)参考文献
水热法鲍鱼内脏140 min/次×4次12.82[31]
超高压破碎牡蛎25 min22.96 [38]
高压脉冲电场辅助酶法河蚌肉脉冲数10个(电场强度25 kV/cm)+酶解时间2.95 h13.77[39]
超声辅助法牛肝10 min6.20[40]
超声-水热提取法马氏珠母贝超声时间70 min+水热时间2 h4.25[41]
MIEF-EH耦合法鲍鱼内脏磁感应电场处理时间1 h+酶解时间4 h12.36本工作
), ArticleFig(id=1261336351354532826, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1261336280806339041, language=CN, label=表1, caption=

天然牛磺酸提取方法的比较

, figureFileSmall=null, figureFileBig=null, tableContent=
提取方法原料提取时间牛磺酸提取量(mg/g)参考文献
水热法鲍鱼内脏140 min/次×4次12.82[31]
超高压破碎牡蛎25 min22.96 [38]
高压脉冲电场辅助酶法河蚌肉脉冲数10个(电场强度25 kV/cm)+酶解时间2.95 h13.77[39]
超声辅助法牛肝10 min6.20[40]
超声-水热提取法马氏珠母贝超声时间70 min+水热时间2 h4.25[41]
MIEF-EH耦合法鲍鱼内脏磁感应电场处理时间1 h+酶解时间4 h12.36本工作
), ArticleFig(id=1261336353363604444, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1261336280806339041, language=EN, label=Table 2, caption=

Effects of different purification methods on taurine

, figureFileSmall=null, figureFileBig=null, tableContent=
纯化方法MIEF-EH
纯化回收率(%)纯度(%)累计提取量(mg牛磺酸/g鲍鱼内脏冻干粉)
注:同列不同小写字母表示差异显著(P<0.05)。
乙醇沉淀法95.30±1.64a94.43±0.57b11.78a
离子交换树脂法92.29±1.90b96.78±0.27a11.41a
), ArticleFig(id=1261336353812394975, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1261336280806339041, language=CN, label=表2, caption=

不同纯化方法对牛磺酸的影响

, figureFileSmall=null, figureFileBig=null, tableContent=
纯化方法MIEF-EH
纯化回收率(%)纯度(%)累计提取量(mg牛磺酸/g鲍鱼内脏冻干粉)
注:同列不同小写字母表示差异显著(P<0.05)。
乙醇沉淀法95.30±1.64a94.43±0.57b11.78a
离子交换树脂法92.29±1.90b96.78±0.27a11.41a
), ArticleFig(id=1261336354219242469, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1261336280806339041, language=EN, label=Table 3, caption=

Physical and chemical indicators of taurine samples from abalone viscera

, figureFileSmall=null, figureFileBig=null, tableContent=
项目指标检测结果
乙醇沉淀法离子交换树脂法
电导率(μs·cm−1≤15066.6763.15
pH4.1~5.65.45.0
砷(As)(mg/kg)≤20.0023≤0.001
氯化物(以Cl计) (w/%)≤0.020.010.001
硫酸盐(以SO42−计)(w/%)≤0.02≤0.01≤0.01
铵盐(以NH4+计) (w/%)≤0.020.00030.002
重金属(以Hg计)(mg/kg)≤100.0121≤0.001
重金属(以Pb计)(mg/kg)≤100.1380≤0.001
重金属(以Sn计)(mg/kg)≤100.0888≤0.001
重金属(以Cd计)(mg/kg)≤100.0149≤0.001
干燥减量(w/%)≤0.2≤0.01≤0.01
灼烧残渣(w/%)≤0.1≤0.1≤0.1
澄清度试验通过试验合格合格
), ArticleFig(id=1261336354714170350, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1261336280806339041, language=CN, label=表3, caption=

鲍鱼内脏牛磺酸样品的理化指标

, figureFileSmall=null, figureFileBig=null, tableContent=
项目指标检测结果
乙醇沉淀法离子交换树脂法
电导率(μs·cm−1≤15066.6763.15
pH4.1~5.65.45.0
砷(As)(mg/kg)≤20.0023≤0.001
氯化物(以Cl计) (w/%)≤0.020.010.001
硫酸盐(以SO42−计)(w/%)≤0.02≤0.01≤0.01
铵盐(以NH4+计) (w/%)≤0.020.00030.002
重金属(以Hg计)(mg/kg)≤100.0121≤0.001
重金属(以Pb计)(mg/kg)≤100.1380≤0.001
重金属(以Sn计)(mg/kg)≤100.0888≤0.001
重金属(以Cd计)(mg/kg)≤100.0149≤0.001
干燥减量(w/%)≤0.2≤0.01≤0.01
灼烧残渣(w/%)≤0.1≤0.1≤0.1
澄清度试验通过试验合格合格
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基于磁感应电场-酶解耦合技术的鲍鱼内脏牛磺酸提取、纯化及表征
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陈钰洁 , 李蒙 * , 刘群 , 翁武银 * , 张玉苍 *
食品工业科技 | 研究与探讨 2026,47(9): 113-122
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食品工业科技 | 研究与探讨 2026, 47(9): 113-122
基于磁感应电场-酶解耦合技术的鲍鱼内脏牛磺酸提取、纯化及表征
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陈钰洁 , 李蒙* , 刘群, 翁武银*, 张玉苍*
作者信息
  • 集美大学海洋食品与生物工程学院,福建厦门 361021
  • 陈钰洁(1999−),女,硕士研究生,研究方向:食品加工副产物的高值化利用,E-mail:

通讯作者:

李蒙(1990−),女,博士,讲师,研究方向:生物质资源高值化利用,E-mail:
张玉苍(1964−),男,博士,教授,研究方向:生物质资源高值化利用,E-mail:
Magnetically Induced Electric Field-Enzymatic Hydrolysis Coupled Extraction, Purification and Characterization of Taurine from Abalone Viscera
Yujie CHEN , Meng LI* , Qun LIU, Wuyin WENG*, Yucang ZHANG*
Affiliations
  • College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, China
出版时间: 2026-05-01 doi: 10.13386/j.issn1002-0306.2025040065
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以鲍鱼内脏废弃物为原料,通过优化磁感应电场(MIEFE)关键参数(激励电压、电场频率、流量及反应时间),建立了磁感应电场-酶解耦合提取技术(MIEF-EH),并系统完成产物纯化与结构表征,旨在实现牛磺酸的绿色、高效提取与纯化。研究结果表明:最优提取条件为激励电压1400 V、电场频率50 kHz、流量25 L/h、反应时间1 h;在优化条件下,MIEF-EH法提取量达到12.36 mg/g,显著(P<0.05)高于单一方法(MIEFE、微波、酶解及水热法)及其他耦合方法(微波-酶解、水热-酶解);SEM表征显示,经过处理后的鲍鱼内脏粒径显著减小,形成疏松多孔微结构,该变化与牛磺酸提取量提升相关;牛磺酸粗提物经离子交换树脂纯化,回收率92.29%、纯度96.78%;乙醇沉淀法回收率95.30%、纯度94.43%;FT-IR、MS及1H NMR联合分析证实纯化产物分子量与牛磺酸理论值高度吻合,化学结构与标准品一致;获得的牛磺酸产物各项理化指标符合GB 14759-2010要求。综上,本研究构建的MIEF-EH耦合技术为鲍鱼内脏废弃物向高纯度天然牛磺酸的绿色转化提供了可行技术路径。

牛磺酸  /  鲍鱼内脏  /  磁感应电场-酶解耦合提取  /  纯化  /  结构分析

This study established a novel magnetically induced electric field-enzymatic hydrolysis (MIEF-EH) coupled technology for the sustainable taurine extraction from abalone viscera byproducts. The results of the study indicated that the optimal extraction conditions were as follows: an excitation voltage of 1400 V, an electric field frequency of 50 kHz, a flow rate of 25 L/h, and a reaction time of 1 h. Under the optimal conditions, MIEF-EH achieved a taurine yield of 12.36 mg/g, significantly (P<0.05) outperforming both individual methods (such as MIEFE, microwave, enzymatic hydrolysis and hydrothermal) as well as other coupled approaches (microwave-enzymatic hydrolysis and hydrothermal-enzymatic hydrolysis). SEM revealed pronounced particle-size reduction and the formation of a loose, porous microstructure, correlating with improved taurine extraction. Subsequent purification via ion-exchange resin yielded taurine with 92.29% recovery and 96.78% purity, while ethanol precipitation achieved 95.30% recovery and 94.43% purity. The chemical structure and molecular mass of the purified taurine were confirmed through multi-technique validation (FT-IR, MS, and 1H NMR). All physicochemical properties complied with the specifications of the Chinese National Standard for Food Additive Taurine (GB 14759-2010). In summary, the MIEF-EH coupled technology developed in this study provides a feasible technological pathway for green conversion of abalone viscera waste into high-purity natural taurine.

taurine  /  abalone viscera  /  magnetically induced electric field-enzymatic hydrolysis coupled extraction  /  purification  /  structure analysis
陈钰洁, 李蒙, 刘群, 翁武银, 张玉苍. 基于磁感应电场-酶解耦合技术的鲍鱼内脏牛磺酸提取、纯化及表征. 食品工业科技, 2026 , 47 (9) : 113 -122 . DOI: 10.13386/j.issn1002-0306.2025040065
Yujie CHEN, Meng LI, Qun LIU, Wuyin WENG, Yucang ZHANG. Magnetically Induced Electric Field-Enzymatic Hydrolysis Coupled Extraction, Purification and Characterization of Taurine from Abalone Viscera[J]. Science and Technology of Food Industry, 2026 , 47 (9) : 113 -122 . DOI: 10.13386/j.issn1002-0306.2025040065
牛磺酸(2-氨基乙磺酸,Taurine)是一种非蛋白氨基酸,纯品为无色或白色结晶,无臭无味,化学性质稳定,易溶于水,不溶于乙醇、乙醚等有机溶剂[1]。牛磺酸具有促进代谢、增强机体免疫力、参与调节神经内分泌功能、保护视网膜等生理作用[26],可以有效地降低心血管疾病、脂肪肝、糖尿病和糖尿病并发症等疾病的风险[710],因此被广泛应用于药品和保健食品中[1112]。由于人体内牛磺酸生物合成中的限速酶(半胱氨酸亚磺酸脱羧酶)活性较低,人体所需的牛磺酸主要从外部获得[1314]。牛磺酸的主要生产方法有两种:提取法和化学合成法。化学合成法往往以环氧乙烷等剧毒物质为原料,有毒试剂残留、环境污染,以及设备昂贵等问题制约化学法合成牛磺酸的可持续发展。因此,绿色、高效地从天然动植物中提取牛磺酸受到广泛关注[1]
海洋生物中含有丰富的牛磺酸,是理想的天然牛磺酸来源。鲍鱼具有重要的营养价值和药用价值,根据中国渔业统计年鉴数据显示,到2023年我国鲍鱼海水养殖产量达24.81万吨,同比增长4.76%。内脏组织占鲍鱼总重量的15%~25%,只有极少部分用于饲料加工[15]。随着鲍鱼养殖规模的扩大和鲍鱼加工业的发展,鲍鱼内脏等废弃物造成的环境污染和资源浪费问题日益加剧[16]。以鲍鱼内脏废弃物为原料提取高附加值牛磺酸是缓解这一现状的重要途径。近年来,牛磺酸等天然产物的提取研究取得了显著进展,多种新型提取技术被相继开发。这些方法在提取原理、工艺条件和提取效率等方面各具特点。物理辅助提取是指通过利用各种物理场效应改变蛋白质的高级结构和分子间作用力等,或者根据分子大小进行定向的分离,从而达到破坏鲍鱼内脏组织的效果,促进游离牛磺酸的释放[1719]。微波、超声是常见的物理辅助方法,这些方法具有无毒副作用、周期短等优点,通常只改变蛋白的二、三、四级结构,不涉及蛋白质一级结构的改变[2021]。然而,当前的物理辅助提取技术需要探头或反应器壁直接接触或近距离作用于物料,存在器械污染风险,而且能量在物料中传递存在衰减(尤其是深层物料),作用深度和均匀性可能受限。磁感应电场(Magnetic induction electric field,MIEF)作为一种新兴物理技术,其独特之处在于通过交变磁场在导电物料内部非接触式地诱导产生感应电场与涡电流。该过程不仅能通过焦耳热效应(热效应)促使蛋白质展开,更重要的是能通过非热效应(如影响分子极性、离子迁移等),改变蛋白质构象与分子间作用力,暴露更多作用位点,能量传递效率高且作用深入均匀,从而显著促进目标成分的释放[2224]。生物酶解技术是一种通过蛋白酶水解蛋白质的肽键,破坏其分子结构,提高提取率的方法。酶法处理反应条件温和,操作简单,可以在最大程度上保留天然产物原有的营养价值,常常用于活性物质的提取[2526]。前期研究表明,物理预处理结合酶解可有效提高牛磺酸得率[2627]。然而,磁感应电场(MIEF)及其耦合酶解技术应用于牛磺酸提取,特别是针对鲍鱼内脏原料的研究,目前尚缺乏系统报道。
本研究以鲍鱼内脏为原料,提出利用磁感应电场-酶解耦合提取牛磺酸的方法,优化提取工艺,对比分析不同提取方法对牛磺酸的影响,并通过纯化得到高纯度的牛磺酸产品,为鲍鱼内脏中天然牛磺酸的高效、绿色提取提供思路。
鲍鱼内脏 福州深水食品有限公司;木瓜蛋白酶(800 U/mg) 上海源叶生物科技有限公司;甲醛、醋酸钠、氢氧化钠(以上为分析纯)、无水乙腈(色谱纯) 国药集团化学试剂有限公司;无水乙醇、冰醋酸 分析纯,西陇科学股份有限公司;碳酸钠、乙酰丙酮、丹酰氯、盐酸甲胺、盐酸(以上为分析纯)、732强酸苯乙烯阳离子交换树脂(钠型) 上海阿拉丁生化科技有限公司。
FDU-1200冷冻干燥机、CA-1330旋转蒸发仪、GPS-11000C-CE 微波反应装置 日本东京理化有限公司;HWCL-3恒温水浴锅 郑州长城科工贸有限公司;Velocity14R高速台式离心机 上海莱睿科学仪器有限公司;UH5300紫外分光光度计、Chromaster5000高效液相色谱仪 日本HITACHI有限公司;MIH-RT 磁感应热反应器 无锡英都斯特仪器设备有限公司;ALPHAⅡ FT-IR傅立叶变换红外光谱仪、Bruker AVANCE Ⅲ 400M核磁共振仪、MALDI-TOF质谱仪 德国布鲁克公司。
鲍鱼内脏摘取肝脏部分,匀浆后经冷冻干燥、粉碎、过筛(40目),收集鲍鱼内脏冻干粉备用。
a.磁感应电场提取法(Magnetic induction electric field extraction,MIEFE):根据文献[18]的方法进行,并作一定修改,具体提取过程为:将鲍鱼内脏冻干粉称重,按照料液比1:50(g/mL)加入去离子水,混匀。在激励电压为1400 V、电场频率50 kHz、流量25 L/h的条件下,比较不同磁感应电场处理时间(0.5、1.0、1.5、2.0、2.5 h)对鲍鱼内脏牛磺酸提取量的影响;在处理时间1 h、电场频率50 kHz、流量25 L/h的条件下,比较不同激励电压(600、800、1000、1200、1400 V)对鲍鱼内脏牛磺酸提取量的影响;在处理时间1 h、激励电压为1400 V、流量25 L/h的条件下,比较不同电场频率(40、50、60、70、80 kHz)对鲍鱼内脏牛磺酸提取量的影响;在处理时间1 h、激励电压为1400 V、电场频率50 kHz的条件下,比较不同进样流量(10、15、20、25、30 L/h)对鲍鱼内脏牛磺酸提取量的影响。通过以上方法得到的鲍鱼内脏原液(Abalone visceral extract,AVE)记为AVE-MIEF。
b.磁感应电场-酶解耦合提取法(Magnetic induction electric field-Enzyme hydrolysis extraction,MIEF-EH):将磁感应电场作为预处理手段,与生物酶法进行耦合,提取鲍鱼内脏牛磺酸。磁感应电场预处理条件同上,不同之处在于,在磁感应电场处理后的料液中加入2000 U/g木瓜蛋白酶,放入恒温水浴锅中在60 ℃下酶解4 h,在100 ℃恒温水浴锅中水浴10 min灭酶,得到的鲍鱼内脏原液记为AVE-MIEF-EH。
生物酶法[26]、水热提取法[25]以及微波提取法[28]均根据文献方法进行,在前期单因素优化结果基础上(数据未在本文中呈现),固定料液比1:50(g/mL),选取各自较优的固定条件进行下述实验。
a.生物酶法(Enzymatic hydrolysis,EH):将鲍鱼内脏冻干粉称重,按照料液比1:50(g/mL)加入去离子水,自然pH条件下,加入2000 U/g木瓜蛋白酶,放入恒温水浴锅中在60 ℃下酶解4 h,在100 ℃恒温水浴锅中水浴10 min灭酶,得到的鲍鱼内脏原液记为AVE-EH。
b.水热提取法(Hydrothermal extraction,HE):将鲍鱼内脏冻干粉称重,按照料液比1:50(g/mL)加入去离子水,放入恒温水浴锅,在100 ℃下反应0.5 h,得到的鲍鱼内脏酶解原液记为AVE-HE。
c.微波提取法(Microwave extraction,ME):将鲍鱼内脏冻干粉称重,按照料液比1:50(g/mL)加入去离子水,在微波功率60 W、微波夹套温度70 ℃的条件下微波反应25 min,得到的鲍鱼内脏原液记为AVE-ME。
d.水热-酶解耦合提取法(Hydrothermal-Enzyme hydrolysis extraction,H-EH):将鲍鱼内脏冻干粉称重,按照料液比1:50(g/mL)加入去离子水,放入恒温水浴锅中在100 ℃下水热0.5 h,加入2000 U/g木瓜蛋白酶,放入恒温水浴锅中在60 ℃下酶解4 h,在100 ℃恒温水浴锅中水浴10 min灭酶,得到的鲍鱼内脏原液记为AVE-H-EH。
e.微波-酶解耦合提取法(Microwave-Enzyme hydrolysis extraction,M-EH):将鲍鱼内脏冻干粉称重,按照料液比1:50(g/mL)加入去离子水,在微波功率60 W、微波夹套温度70 ℃的条件下微波辅助反应25 min后,加入2000 U/g木瓜蛋白酶,放入恒温水浴锅中在60 ℃下酶解4 h,在100 ℃恒温水浴锅中水浴10 min灭酶,得到的鲍鱼内脏原液记为AVE-M-EH。
使用紫外分光光度法检测AVE中的牛磺酸。在醋酸钠环境中,牛磺酸与乙酰丙酮和甲醛进行加热反应,生成黄色的N-取代基-2,6-二甲基-3,5-二乙酰基-1,4-二氢吡啶[29]。取1 mL AVE,加入显色剂后加入蒸馏水稀释,在60 ℃条件下加热10 min,冷却至室温后,测定样品在波长330 nm处的吸光度。根据公式(1)计算不同方法的牛磺酸提取量:
$ \rm 牛磺酸提取量\left({mg}/{g}\right)=\frac{(A-0.0043)\times a}{471.24}\times\frac{f}{m} $
式中:A为样品吸光度,a为稀释倍数,m为鲍鱼内脏冻干粉(g),f为鲍鱼内脏酶解液的初始滤液体积(mL)。
取少量处理前后的鲍鱼内脏样品,冷冻干燥后,在4~6 kV的加速电压下,对样品喷金处理30 s,喷金电流为10 mA。用SEM观察其微观形貌结构。
分别采用乙醇沉淀法和离子交换树脂法对磁感应电场-酶解耦合法提取的鲍鱼内脏牛磺酸粗品进行分离纯化。
将收集到的AVE-MIEF-EH冷却至室温,通过纱布过滤,在10000 r/min条件下离心10 min,收集上清液并添加4倍体积的无水乙醇,静置后取上清液在10000 r/min条件下离心10 min,取上清液,经旋转蒸发仪浓缩至可溶性固形物含量为50%,此时浓缩液体积约50 mL。向浓缩液中加入250 mL无水乙醇后置于4 ℃下静置12 h,收集沉淀。向沉淀中加入5~6倍80 ℃热水,溶解后加入活性炭(5 g/100 mL)处理,抽滤除去活性炭,得到滤液。向上述滤液中加入3倍体积的无水乙醇后立即抽滤除杂,滤液置于4 ℃下静置结晶24 h以上,所得固形物即为牛磺酸粗结晶。将牛磺酸粗结晶反复用水溶解、乙醇结晶。直至获得纯度较高的牛磺酸[3031],记为乙醇沉淀法牛磺酸样品。
将收集到的AVE-MIEF-EH冷却至室温,通过纱布过滤,10000 r/min离心10 min收集上清液。将上清液经旋转蒸发仪浓缩至体积为AVE的10%,得浓缩液。使用0.2 mol/L的NaOH溶液调节浓缩液pH至10.5,10000 r/min离心10 min去除酸性蛋白质;使用0.2 mol/L的HCl溶液调节浓缩液pH至3.5,10000 r/min离心10 min去除碱性蛋白质。将酸碱除杂后的浓缩液再次经旋转蒸发仪浓缩至可溶性固形物含量为49%以下后,使浓缩液在室温下经过732强酸苯乙烯阳离子交换树脂(钠型)柱洗脱,洗脱速度为2 mL/min,收集牛磺酸高流区间,得到洗脱液。往所述洗脱液中添加4~5倍体积无水乙醇,室温静置取上清液浓缩,将上清液用常压浓缩或减压浓缩至可溶性固形物含量为50%,加入4倍体积无水乙醇置于4 ℃静置12 h以上,收集沉淀。往沉淀中加入5~6倍80 ℃热水,溶解后加入活性炭(5 g/100 mL)处理,抽滤除去活性炭,得到滤液。向上述滤液中加入3倍体积的无水乙醇后立即抽滤除杂,滤液置于4 ℃下静置结晶24 h以上,所得固形物即为牛磺酸粗结晶。将牛磺酸粗结晶反复用水溶解、乙醇结晶,直至获得纯度较高的牛磺酸[32],记为离子交换树脂法牛磺酸样品。
将上述2种纯化方法获得的牛磺酸产品分别在105±2 ℃干燥3 h,称量记录鲍鱼内脏牛磺酸的质量m。根据公式(2)计算鲍鱼内脏原液中牛磺酸的纯化回收率:
$\rm 牛磺酸回收率(\text{%})=\frac{m}{A \times M}\times 100 $
式中:m为鲍鱼内脏牛磺酸的质量(mg),M为鲍鱼内脏冻干粉原料的质量(g),A为鲍鱼内脏原液中的牛磺酸提取量(mg/g)。
根据GB 5009.169-2016高效液相色谱法检测鲍鱼内脏提取的牛磺酸产品含量,并通过峰面积归一法计算牛磺酸产品的纯度。检测条件:C18色谱柱(250 mm×4.6 mm,5 μL);柱温:室温;进样量:20 μL:流动相:乙酸钠缓冲溶液(10 mmol/L,pH4.2)与乙腈(体积比70:30),等度洗脱,流速1 mL/min。
将纯化后的牛磺酸样品与溴化钾粉末混合,研磨,然后压成薄片,使用FT-IR光谱仪在400~4000 cm−1的波数范围内进行检测。
采用配备电喷雾离子源(ESI)的质谱系统,在质谱正负模式下获得离子质谱,质谱条件:正离子模式(ESI+)电压5.5 kV;负离子模式(ESI)电压4.5 kV;雾化气温度450 ℃;气帘气40 psi;气体离子源130 psi;气体离子源230 psi;流动相A:0.1%甲酸水溶液;流动相B:乙腈溶液;流速:0.2 mL/min,进样量:5 μL,质谱扫描范围:100~1500 m/z。
纯化牛磺酸的1H NMR谱图在400 MHz核磁共振波谱仪上记录,使用D2O作为溶剂。
对纯化牛磺酸理化指标进行测定。电导率、pH、硫酸盐(以SO42−计)、灼烧残渣(w/%)、干燥减量(w/%)、澄清度的检测参考GB 14759-2010《食品安全国家标准 食品添加剂 牛磺酸》,重金属(As、Hg、Pb、Sn、Cd)含量采用电感耦合等离子体质谱仪(ICP-MS)检测,氯化物(以Cl计)、铵盐(以NH4+计)采用离子色谱(IC)检测。
除特别说明外,所有实验均重复三次;数据以平均值±标准差表示。采用Excel 2017、SPSS24.0(IBM,Armonk,NY,USA)、minitab19对数据进行分析,并采用Duncan多重范围检验(P<0.05)。
磁感应电场(MIEFE)作为一种新兴的非接触式绿色处理技术,将其直接用于鲍鱼内脏中牛磺酸的提取。不同磁感应电场条件下牛磺酸的提取效果如图1所示。随着磁感应电场处理时间的延长,牛磺酸提取量先升高再下降(图1A)。当处理时间为1 h时,鲍鱼内脏牛磺酸提取量(7.79 mg/g)最高,与其他时间下提取量差异显著(P<0.05)。磁感应电场技术通过磁路将磁能高效地转换为感应电场,感应电流流经具有一定电阻的料液时,产生的热量导致介质温度升高,有利于牛磺酸的释放[23,33]。然而,长时间的作用可能导致鲍鱼内脏中的大分子蛋白机械旋转内能增加,引起蛋白质变性,反而不利于牛磺酸的提取[34]。因此,选择1 h为最佳磁感应电场处理时间。
图1B可知,随着激励电压的增加,牛磺酸提取量呈现出逐渐升高的趋势。在仪器最大激励电压1400 V时,牛磺酸提取量达到8.12 mg/g,与1200 V的提取量相当,但显著高于低激励电压(600~1000 V)条件下的提取量(P<0.05)。磁感应电场处理过程中,交变磁场穿过特制的磁路结构,在具有导电性的处理介质(料液)中诱导产生强感应涡旋电场,使膜磷脂双分子层发生极化,膜电位失衡,导致膜上形成微孔等结构缺陷,破坏细胞结构完整性,促进了胞内物质(如牛磺酸)向外扩散[3336];此外,感应电场驱动料液及细胞内外的带电离子和极性分子进行定向迁移和高频振荡,产生的机械应力能进一步扰动和破坏细胞膜结构,加速了牛磺酸从胞内向胞外介质的传质过程[24]。提高激励电压有利于破坏细胞结构完整性、定向迁移与热效应的多重作用,进而促进牛磺酸自鲍鱼内脏中的溶出过程;然而,当电压达到1200 V后,激励电压的影响不显著。因此,激励电压控制在1200~1400 V较为适宜。
图1C可知,随着电场频率的增加,牛磺酸提取量呈现出先升高后降低的趋势。当电场频率为50 kHz时,牛磺酸提取量与60 kHz相当,但显著高于其他电场频率(如40、70和80 kHz)下的提取量(P<0.05)。根据电磁感应原理,提高频率而感应电压保持不变时,系统中的感应电流密度下降,初级线圈的阻抗则会增加,最终造成系统的总磁通量和输出功率降低。因此,特定的料液系统具有最适的工作频率,在该工作频率时系统磁损耗最小,能量输入水平最高[22,33]。当前系统的最适频率为50~60 kHz。
图1D可知,当流量处于25 L/h及以下区间时,流量的波动对牛磺酸提取量的影响相对较小;在流量为25 L/h时,牛磺酸提取量达到峰值(8.17 mg/g);然而,当流量达到30 L/h时,牛磺酸提取量显著降低(P<0.05)。此外,考虑到磁场反应器为循环管路式反应器,提高流速有利于物料及时散热,可能减少副反应发生。因此,流量25 L/h较为适宜。
以上研究结果表明,磁感应电场技术对鲍鱼内脏中牛磺酸的释放具有积极作用,较优的磁感应电场处理条件为:激励电压1200~1400 V,电场频率50~60 kHz,流量控制在25 L/h,反应时间1 h。
生物酶法(EH)是一种绿色、安全且无需特殊设备的牛磺酸提取方法。为进一步提升鲍鱼内脏中牛磺酸的提取效率,本研究将磁感应电场(MIEFE)技术与温和的EH技术进行耦合处理。如图2所示,耦合方法(MIEF-EH)的牛磺酸提取量随磁感应电场条件的变化规律与单一MIEFE处理(图1)类似。不同之处在于,相较于MIEFE法,MIEF-EH耦合法对激励电压、电场频率及流量参数更为敏感。
在处理时间方面,磁感应电场处理1 h时,耦合方法的牛磺酸提取量显著高于其他处理时间点(P<0.05),且明显高于MIEFE法。在电压参数方面,在最高电压(1400 V)下的提取量最高(P<0.05)。此外,最佳电场频率为50 kHz(P<0.05),最佳流量为25 L/h(P<0.05)。因此,较优的MIEFE预处理条件为:激励电压1400 V,电场频率50 kHz,流量25 L/h,反应时间1 h,此时最高牛磺酸提取量达到12.36 mg/g。
以上研究结果表明,MIEFE技术可作为EH的有效预处理手段,即MIEF-EH耦合法显著提升鲍鱼内脏牛磺酸的提取水平。
不同方法对鲍鱼内脏牛磺酸提取效果如图3所示,与水热提取法(HE)、生物酶法(EH)和微波提取法(ME)这3种常见的绿色提取方法比较而言,MIEFE法对鲍鱼内脏中牛磺酸的提取量明显更高,差异显著(P<0.05)。
在此基础上,进一步将 MIEFE、HE、ME 分别与温和的EH耦合并评估其协同效应(图3)。其中,水热-酶解耦合法(H-EH)提取量最低,仅与单一MIEFE法相当;微波-酶解耦合法(M-EH)提取量为10.97 mg/g;磁感应电场-酶解耦合法(MIEF-EH)提取量高达12.36 mg/g,显著优于所有对照组(P<0.05),较4种单一方法提升0.51~1.19倍,为M-EH的112.7%。若改变耦合顺序,即先酶解后施加MIEFE(即EH-MIEF耦合法),则提取量降至10.65 mg/g(P<0.05)。在MIEF-EH耦合方法中,MIEFE作为预处理步骤时,可通过磁感应电场瞬时增压效应破坏鲍鱼内脏细胞结构,提高后续酶的可及性,进而协同提升水解度与牛磺酸释放[29,37]。而当酶解先行时(EH-MIEF),底物结构未被充分破坏,限制了酶-底物有效结合,导致协同效应减弱。
与文献报道相比(表1),传统水热法需反复提取4次方可获得12.82 mg/g的提取量[31],工艺冗长;以牡蛎或河蚌肉为原料的提取虽可达22.96 mg/g与13.77 mg/g提取量[3839],但原料成本较高,与鲍鱼加工副产物相比缺乏经济优势。若以牛肝及马氏珠母贝为来源,牛磺酸提取量仅分别为本研究MIEF-EH法的50.2%与34.4%[4041]
综上,本研究创新性地采用磁感应电场技术耦合酶解法(MIEF-EH),以鲍鱼加工副产物为原料提取牛磺酸。该方法反应条件温和、操作过程简便、全程无化学试剂引入,兼顾高效与绿色提取优势,为贝类副产物高值化利用提供了思路。
通过扫描电子显微镜(SEM)对不同提取方法处理的鲍鱼内脏残渣冻干样品微观形貌进行表征。如图4所示,未经处理的鲍鱼内脏表面相对光滑且结构密实。经不同方法处理后,鲍鱼内脏结构被破坏,颗粒尺寸变小,整体呈现不规则的疏松多孔特征。与4种单一提取方法比较而言,3种耦合方法处理后的鲍鱼内脏残渣颗粒更小,分布更均匀,具有更明显的孔洞结构。这些微观结构的改变表明,提取过程有效破坏了细胞组织结构,导致比表面积增大,暴露出更多作用位点。这有利于牛磺酸的游离释放,从而提高提取效率[20,42]。上述观察结果与2.3节中牛磺酸提取量的数据相吻合。
分别通过乙醇沉淀法和离子交换树脂法对磁感应电场-酶解耦合法提取的牛磺酸进行分离纯化,得到的样品均为白色结晶状粉末。根据GB 5009.169-2016高效液相色谱法第二法(丹磺酰氯柱前衍生法)进行牛磺酸检测时,色谱图中出现两个色谱峰,其中3 min左右为丹磺酰氯水解产物,3.77 min左右为牛磺酸,通过峰面积归一法计算得到牛磺酸样品的纯度。由图5表2可知,离子交换树脂法展现出优异的纯化性能,所得鲍鱼内脏牛磺酸样品纯度高达96.78%,在杂质去除和产品质量控制方面具有显著优势,特别适用于高纯度牛磺酸的制备需求。相比之下,乙醇沉淀法表现出更高的回收率(95.30%),在目标成分富集方面更具效率,更适合大规模工业化生产应用。
本研究开发的磁感应电场-酶解耦合(MIEF-EH)提取方法与传统水热法相比具有显著优势。虽然传统水热法[31]初始提取量与本研究所获结果相当,但前者处理过程繁琐且周期较长(表1),且经过后续纯化处理后,其累计提取量(3.02 mg/g)明显低于MIEF-EH法(表2)。这一差异可能源于MIEF-EH法温和的处理温度,副反应减少,产物中杂质少,实现对目标产物的精准提取,有效提升了纯化回收率[22,4345]
综上所述,磁感应电场耦合酶解法(MIEF-EH)作为一种绿色、高效的提取方法,在天然活性成分的提取领域具有广阔的应用前景。
利用傅里叶变换红外光谱(FT-IR)对上述纯化的牛磺酸(NH2CH2CH2SO3H)样品的化学结构进行表征,如图6所示。结合牛磺酸标准品谱图及文献[46]的报道,对特征峰归属如下:在波数3207、3043、1610 cm−1处显示氨基(-NH2)与磺酸基(-SO3H)特征峰;在波数2969、1510、1306、1107 cm−1处显示亚甲基(-CH2-)与碳硫键(C-S)特征峰;在1211、958 cm−1处显示硫氧键(S=O)与碳硫氧(C-S-O)特征峰。乙醇沉淀法牛磺酸样品和离子交换树脂法牛磺酸样品谱图特征峰位置与上述标准品一致,初步证实所得产物为牛磺酸。然而,两种纯化方法所得样品的FT-IR谱图相比仍然存在微小差异。离子交换树脂法牛磺酸样品在部分波数(如1306 cm−1)显示出更强的吸收峰强度。这表明离子交换树脂法可能在保留特定官能团方面更具优势。这种更完整的官能团保留有利于维持牛磺酸的两性离子结构,与表2中该样品更高的纯度结果相吻合。
进一步利用质谱对上述纯化牛磺酸样品分子量进行评估,样品在正负离子模式下的离子质谱图如图7所示。在负离子模式下,乙醇沉淀法牛磺酸样品和离子交换树脂法牛磺酸样品检测到m/z分别为124.13([M-H])和124.18([M-H])的离子峰,对应去质子化分子离子(理论值124.14 Da);在正离子模式下,两种样品检测到m/z分别为125.81([M+H]+)和125.93([M+H]+)的离子峰,对应质子化分子离子(理论值126.02 Da)[41]。上述实测值与理论分子量的偏差均小于0.6 Da,处于仪器检测精度范围内。以上结果进一步验证两种纯化的样品分子量与牛磺酸理论值一致。
图8所示,两种纯化牛磺酸样品的1H NMR谱图均在3.34和3.16 ppm处出现SCH2和NCH2的质子化峰,此结果与牛磺酸标准品一致[4041],表明牛磺酸的化学结构得到完整保留。综上,核磁共振数据(图8)与前期的质谱(图7)、FT-IR 结构表征(图6)及HPLC分析(图5)结果相互印证,表明两种纯化方法所得产物均为高纯度牛磺酸。
分别对乙醇沉淀法和离子交换树脂法纯化获得的牛磺酸样品进行理化指标检测,结果如表3所示。根据GB 14759-2010《食品安全国家标准 食品添加剂 牛磺酸》的技术要求,两种方法纯化所得的牛磺酸产品在重金属残留等关键指标上均符合国家标准,可作为食品添加剂安全应用于食品工业。进一步地,离子交换树脂法在砷、氯化物等杂质去除方面优于乙醇沉淀法,适合需要更高纯度产品的场景。以上研究结果表明,常见的离子交换树脂法与乙醇沉淀法均可有效地从磁感应电场-酶解耦合法提取的鲍鱼内脏原液中分离、纯化出符合国家标准的牛磺酸产品,间接说明该提取方法产生的杂质和副产物较少,验证了该提取原液具有良好的纯化适配性,为高纯度牛磺酸的规模化生产提供依据。
以上研究结果表明,磁感应电场-酶解耦合提取技术结合乙醇沉淀法或离子交换树脂法,能够建立一套高效的鲍鱼内脏牛磺酸提取、纯化工艺体系。该体系不仅保证了提取效率,同时可根据不同应用需求灵活选择适宜的纯化方法,为牛磺酸的工业化生产提供了新的技术方案。
本研究以鲍鱼加工副产物(鲍鱼内脏)为原料,成功应用非接触式磁感应电场技术耦合生物酶解技术(MIEF-EH)实现了牛磺酸的高效绿色提取。在最优磁感应电场预处理条件下(激励电压1400 V,电场频率50 kHz,流量25 L/h,处理时间1 h),牛磺酸提取量达到12.36 mg/g。 提取液经乙醇沉淀或离子交换树脂纯化后,可获得高纯度牛磺酸(>94%),累计提取量稳定在11 mg/g以上,显著优于传统水热法。经HPLC、FT-IR、MS及1H NMR多谱学技术确证,纯化产物的分子量、化学结构及理化指标均与牛磺酸标准品一致。本研究所建立的MIEF-EH耦合工艺具有显著优势:全程无需添加化学试剂,反应条件温和,工艺流程简洁,为天然牛磺酸的绿色高效制备提供了创新性技术途径。同时,该工艺以鲍鱼内脏等低值水产副产物为原料,契合水产加工业循环经济与可持续发展需求。未来研究需进一步深入探讨磁场与酶分子的作用机制,建立关键参数优化模型,并验证该技术在其他水产副产物活性成分提取中的普适性。
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2026年第47卷第9期
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doi: 10.13386/j.issn1002-0306.2025040065
  • 接收时间:2025-04-07
  • 首发时间:2026-05-13
  • 出版时间:2026-05-01
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    集美大学海洋食品与生物工程学院,福建厦门 361021

通讯作者:

李蒙(1990−),女,博士,讲师,研究方向:生物质资源高值化利用,E-mail:
张玉苍(1964−),男,博士,教授,研究方向:生物质资源高值化利用,E-mail:
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https://castjournals.cast.org.cn/joweb/spgykj/CN/10.13386/j.issn1002-0306.2025040065
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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

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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