Article(id=1320882362192343606, tenantId=1146029695717560320, journalId=1260987677001138203, issueId=1320882022160126288, articleNumber=null, orderNo=null, doi=10.13386/j.issn1002-0306.2024120371, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1735488000000, receivedDateStr=2024-12-30, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1792852495953, onlineDateStr=2026-10-24, pubDate=1767196800000, pubDateStr=2026-01-01, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1792852495953, onlineIssueDateStr=2026-10-24, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1792852495953, creator=13701087609, updateTime=1792852495953, updator=13701087609, issue=Issue{id=1320882022160126288, tenantId=1146029695717560320, journalId=1260987677001138203, year='2026', volume='47', issue='1', pageStart='1', pageEnd='498', issueExtLink='null', onlineDate='null', pubDate='1767196800000', pubDateStr='2026-01-01', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1792852414884, creator='13701087609', updateTime=1792852414884, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext=null, issueFiles=null, downloadFileDto=null}, startPage=112, endPage=119, ext={EN=ArticleExt(id=1320882362385281591, articleId=1320882362192343606, tenantId=1146029695717560320, journalId=1260987677001138203, language=EN, title=Preparation of Limosilactobacillus reuteri Fermented Milk Containing Reuterin and Its Antibacterial Properties, columnId=1261336277291548795, journalTitle=Science and Technology of Food Industry, columnName=Research and Investigation, runingTitle=null, highlight=null, articleAbstract=

To prepare a probiotic fermented milk that could produce antibacterial substances in situ, Limosilactobacillus reuteri HLRE05, which had the ability to metabolize glycerol into broad-spectrum antimicrobial Reuterin, was used to investigate the optimum conditions for preparation of the fermented milk. This study aimed to evaluate the effects of glycerol addition on the Reuterin production and the properties of the fermented milk, analyze the biochemical indices and the Reuterin content of the fermented milk during storage, and assess antibacterial ability. The results showed that the optimal preparation conditions for the Reuterin-containing fermented milk was a total inoculum volume of 5%, with a 1:1 ratio of YO-MIX 495 to HLRE05, a sugar content of 6% (a 1:1 ratio of rhamnose to sucrose), and a fermentation temperature of 37 ℃. When supplemented with glycerol at a concentration of 100 mmol/L, the in situ production of Reuterin reached 0.28 mg/mL, and the content of L. reuteri and water retention in fermented milk were better. In addition, fermented milk contained Reuterin maintained good water-holding capacity, stable pH and high Reuterin concentration, and was characterized by the effective inhibition of pathogen proliferation during storage. This study could pave the way for the exploration and application of characteristic lactic acid bacteria, such as L. reuteri HLRE05, in dairy products.

, authors=Ke ZHU1, Xinrui LI1, Mengyu ZHOU1, Hua WEI1, 2, Zhihong ZHANG1, 2, 3, *, authorsList=Ke ZHU, Xinrui LI, Mengyu ZHOU, Hua WEI, Zhihong ZHANG, authorCompany=null, correspAuthors=Zhihong 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, fund=null), CN=ArticleExt(id=1320882365568758344, articleId=1320882362192343606, tenantId=1146029695717560320, journalId=1260987677001138203, language=CN, title=含Reuterin罗伊氏乳杆菌发酵乳的制备及其抗菌特性研究, columnId=1261336277849391232, journalTitle=食品工业科技, columnName=研究与探讨, runingTitle=null, highlight=null, articleAbstract=

为制备一种可原位产生抗菌物质的益生菌发酵乳,本文以能代谢甘油产广谱抗菌物质Reuterin的罗伊氏乳杆菌HLRE05为发酵菌,探究制备发酵乳的最适条件,评价甘油的添加对Reuterin产生及发酵乳特性的影响,分析发酵乳贮藏期内的生化指标和Reuterin含量变化,并对含Reuterin发酵乳的抑菌能力进行评估。结果表明:含Reuterin发酵乳的制备条件可选择接种量为5%(YO-MIX 495:HLRE05=1:1)、糖含量为6%(鼠李糖:蔗糖=1:1)和发酵温度为37 ℃;当甘油的添加量为100 mmol/L时,原位产生Reuterin的含量达0.28 mg/mL,且其罗伊氏乳杆菌含量和持水性较好;另外,含Reuterin发酵乳在贮藏期内也能够保持较好的持水性,稳定的pH以及较高的Reuterin浓度,并能有效抑制病原微生物繁殖。该研究将为特色乳酸菌(如罗伊氏乳杆菌HLRE05)资源挖掘及其在乳制品中的应用奠定基础。

, authors=朱可1, 李新瑞1, 周梦雨1, 魏华1, 2, 张志鸿1, 2, 3, *, authorsList=朱可, 李新瑞, 周梦雨, 魏华, 张志鸿, authorCompany=null, correspAuthors=张志鸿, authorNote=

朱可(2000−),女,硕士,研究方向:特色乳酸菌功能挖掘,E-mail:

, correspAuthorsNote=
张志鸿(1988−),男,博士,助理研究员,研究方向:益生菌与机体健康,E-mail:。
, copyrightStatement=版权所有 © 2026《食品工业科技》编辑部, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=eCLK4OIa+niBwB3WkptpsQ==, magXml=sQ8am3iY3ghSgznpAEYxuQ==, pdfUrl=null, pdf=8Jv8SPaQRN3x40qHh247lg==, pdfFileSize=1438286, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=IyhlyCl+1UDjR26bS4ZKrw==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=REsfxA3UeI5eEGBZdQNXwQ==, mapNumber=null, fund=null)}, authors=[Author(id=1320882366046909011, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882362192343606, orderNo=0, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=2522371533@qq.com, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1320882366118212181, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882362192343606, authorId=1320882366046909011, language=EN, stringName=Ke ZHU, firstName=Ke, middleName=null, lastName=ZHU, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=1, address=1State Key Laboratory of Food Science and Resources, Nanchang University, Nanchang 330047, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1320882366189515350, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882362192343606, authorId=1320882366046909011, language=CN, stringName=朱可, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=1, address=1南昌大学食品科学与资源挖掘全国重点实验室,江西南昌 330047, bio={"content":"

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注:不同大写字母代表组间活菌数差异显著,P<0.05;不同小写字母代表组间pH差异显著,P<0.05,图2~图3同。

, figureFileSmall=OllwOCuz6KHFg8muu58CZA==, figureFileBig=IyhlyCl+1UDjR26bS4ZKrw==, tableContent=null), ArticleFig(id=1320882371893768826, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882362192343606, language=EN, label=Fig.2, caption=Effect of sugar content on viable count of L. reuteri and pH of fermented milk, figureFileSmall=H3q0mYAQgn/wcSAxsesMVw==, figureFileBig=bHUyCFDDqNVQjJBTIiarPw==, tableContent=null), ArticleFig(id=1320882371986043515, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882362192343606, language=CN, label=图2, caption=糖含量对发酵乳中罗伊氏乳杆菌活菌数和pH的影响, figureFileSmall=H3q0mYAQgn/wcSAxsesMVw==, figureFileBig=bHUyCFDDqNVQjJBTIiarPw==, tableContent=null), ArticleFig(id=1320882372195758716, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882362192343606, language=EN, label=Fig.3, caption=Effect of temperature on viable count of L. reuteri and pH of fermented milk, figureFileSmall=kwHLCCfd37D9+f6/Dde4kQ==, figureFileBig=tFaYRzvu67lntEUQSeI/rA==, tableContent=null), ArticleFig(id=1320882372271256189, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882362192343606, language=CN, label=图3, caption=温度对发酵乳中罗伊氏乳杆菌活菌数和pH的影响, figureFileSmall=kwHLCCfd37D9+f6/Dde4kQ==, figureFileBig=tFaYRzvu67lntEUQSeI/rA==, tableContent=null), ArticleFig(id=1320882372342559358, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882362192343606, language=EN, label=Fig.4, caption=Content of Reuterin in fermented milk at different glycerol concentrations, figureFileSmall=Kg2l2hMGDJN0KQLhpvMoyA==, figureFileBig=rLbmxebSNBhZR6P/ObgboQ==, tableContent=null), ArticleFig(id=1320882372615189119, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882362192343606, language=CN, label=图4, caption=不同甘油浓度下发酵乳中Reuterin的含量

注:不同小写字母代表组间Reuterin含量差异显著,P<0.05。

, figureFileSmall=Kg2l2hMGDJN0KQLhpvMoyA==, figureFileBig=rLbmxebSNBhZR6P/ObgboQ==, tableContent=null), ArticleFig(id=1320882372686492288, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882362192343606, language=EN, label=Fig.5, caption=Effect of the addition of glycerol on fermented milk, figureFileSmall=55NTYKplfk1HGbUa3cL56g==, figureFileBig=kDln0GuEkzIhvuclwgTkgg==, tableContent=null), ArticleFig(id=1320882372770378369, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882362192343606, language=CN, label=图5, caption=甘油的添加对发酵乳的影响

注:(A)罗伊氏乳杆菌HLRE05活菌数;(B)pH。L.r:不添加甘油的罗伊氏乳杆菌发酵乳组(对照组),L.r+gly:添加甘油的罗伊氏乳杆菌发酵乳组(实验组)。L.r vs L.r+gly,*P<0.05;**P<0.01;***P<0.001;****P<0.0001;图6~图8同。

, figureFileSmall=55NTYKplfk1HGbUa3cL56g==, figureFileBig=kDln0GuEkzIhvuclwgTkgg==, tableContent=null), ArticleFig(id=1320882373043008130, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882362192343606, language=EN, label=Fig.6, caption=Characteristics of fermented milk after maturation for 24 h, figureFileSmall=bO27UjV546YShrcC+ZbpEg==, figureFileBig=/TKjS+vwRxL8iDImI5Gr8Q==, tableContent=null), ArticleFig(id=1320882374485848707, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882362192343606, language=CN, label=图6, caption=发酵乳后熟24 h后的特征

注:(A)活菌数;(B)pH;(C)持水性。

, figureFileSmall=bO27UjV546YShrcC+ZbpEg==, figureFileBig=/TKjS+vwRxL8iDImI5Gr8Q==, tableContent=null), ArticleFig(id=1320882374590706308, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882362192343606, language=EN, label=Fig.7, caption=Changes of indexes during storage of fermented milk, figureFileSmall=8oDFdDg1LJM81Bb62h3/qw==, figureFileBig=hKGq8OYysFWoIw20NoR+CQ==, tableContent=null), ArticleFig(id=1320882374691369605, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882362192343606, language=CN, label=图7, caption=发酵乳贮藏期内指标的变化

注:(A)活菌数;(B)pH;(C)持水性;(D)Reuterin含量。

, figureFileSmall=8oDFdDg1LJM81Bb62h3/qw==, figureFileBig=hKGq8OYysFWoIw20NoR+CQ==, tableContent=null), ArticleFig(id=1320882374775255686, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882362192343606, language=EN, label=Fig.8, caption=Ability of fermented milk with or without glycerol to inhibit the growth of pathogens, figureFileSmall=J6bNPq0nuR8vK4NISUOMZQ==, figureFileBig=2B6Qwxi2pFg3/7Rtuf4iNw==, tableContent=null), ArticleFig(id=1320882374850753159, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882362192343606, language=CN, label=图8, caption=添加或不添加甘油发酵乳抑制病原微生物生长的能力

注:(A)S. Typhimurium ATCC13311;(B)L. monocytogenes CMCC54007;(C)S. aureus CMCC26003;(D)E. coli O157:H7在发酵乳中的生长曲线。

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含Reuterin罗伊氏乳杆菌发酵乳的制备及其抗菌特性研究
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朱可 1 , 李新瑞 1 , 周梦雨 1 , 魏华 1, 2 , 张志鸿 1, 2, 3, *
食品工业科技 | 研究与探讨 2026,47(1): 112-119
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食品工业科技 |研究与探讨 2026 , 47 (1) : 112 -119
含Reuterin罗伊氏乳杆菌发酵乳的制备及其抗菌特性研究
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朱可1 , 李新瑞1, 周梦雨1, 魏华1, 2, 张志鸿1, 2, 3, *
作者信息
  • 1南昌大学食品科学与资源挖掘全国重点实验室,江西南昌 330047
  • 2南昌大学中德联合研究院,江西南昌 330047
  • 3南昌大学重庆研究院,重庆 402660
通讯作者:
张志鸿(1988−),男,博士,助理研究员,研究方向:益生菌与机体健康,E-mail:。
作者简介:

朱可(2000−),女,硕士,研究方向:特色乳酸菌功能挖掘,E-mail:

Preparation of Limosilactobacillus reuteri Fermented Milk Containing Reuterin and Its Antibacterial Properties
Ke ZHU1 , Xinrui LI1, Mengyu ZHOU1, Hua WEI1, 2, Zhihong ZHANG1, 2, 3, *
Affiliations
  • 1State Key Laboratory of Food Science and Resources, Nanchang University, Nanchang 330047, China
  • 2Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, China
  • 3Chongqing Research Institute, Nanchang University, Chongqing 402660, China
出版时间: 2026-01-01 doi: 10.13386/j.issn1002-0306.2024120371
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为制备一种可原位产生抗菌物质的益生菌发酵乳,本文以能代谢甘油产广谱抗菌物质Reuterin的罗伊氏乳杆菌HLRE05为发酵菌,探究制备发酵乳的最适条件,评价甘油的添加对Reuterin产生及发酵乳特性的影响,分析发酵乳贮藏期内的生化指标和Reuterin含量变化,并对含Reuterin发酵乳的抑菌能力进行评估。结果表明:含Reuterin发酵乳的制备条件可选择接种量为5%(YO-MIX 495:HLRE05=1:1)、糖含量为6%(鼠李糖:蔗糖=1:1)和发酵温度为37 ℃;当甘油的添加量为100 mmol/L时,原位产生Reuterin的含量达0.28 mg/mL,且其罗伊氏乳杆菌含量和持水性较好;另外,含Reuterin发酵乳在贮藏期内也能够保持较好的持水性,稳定的pH以及较高的Reuterin浓度,并能有效抑制病原微生物繁殖。该研究将为特色乳酸菌(如罗伊氏乳杆菌HLRE05)资源挖掘及其在乳制品中的应用奠定基础。

罗伊氏乳杆菌  /  Reuterin  /  发酵乳  /  抗菌  /  贮藏期

To prepare a probiotic fermented milk that could produce antibacterial substances in situ, Limosilactobacillus reuteri HLRE05, which had the ability to metabolize glycerol into broad-spectrum antimicrobial Reuterin, was used to investigate the optimum conditions for preparation of the fermented milk. This study aimed to evaluate the effects of glycerol addition on the Reuterin production and the properties of the fermented milk, analyze the biochemical indices and the Reuterin content of the fermented milk during storage, and assess antibacterial ability. The results showed that the optimal preparation conditions for the Reuterin-containing fermented milk was a total inoculum volume of 5%, with a 1:1 ratio of YO-MIX 495 to HLRE05, a sugar content of 6% (a 1:1 ratio of rhamnose to sucrose), and a fermentation temperature of 37 ℃. When supplemented with glycerol at a concentration of 100 mmol/L, the in situ production of Reuterin reached 0.28 mg/mL, and the content of L. reuteri and water retention in fermented milk were better. In addition, fermented milk contained Reuterin maintained good water-holding capacity, stable pH and high Reuterin concentration, and was characterized by the effective inhibition of pathogen proliferation during storage. This study could pave the way for the exploration and application of characteristic lactic acid bacteria, such as L. reuteri HLRE05, in dairy products.

Limosilactobacillus reuteri  /  Reuterin  /  fermented milk  /  antibacterial  /  storage period
朱可, 李新瑞, 周梦雨, 魏华, 张志鸿. 含Reuterin罗伊氏乳杆菌发酵乳的制备及其抗菌特性研究. 食品工业科技, 2026 , 47 (1) : 112 -119 . DOI: 10.13386/j.issn1002-0306.2024120371
Ke ZHU, Xinrui LI, Mengyu ZHOU, Hua WEI, Zhihong ZHANG. Preparation of Limosilactobacillus reuteri Fermented Milk Containing Reuterin and Its Antibacterial Properties[J]. Science and Technology of Food Industry, 2026 , 47 (1) : 112 -119 . DOI: 10.13386/j.issn1002-0306.2024120371
益生菌是功能性食品中的重要成分,在基础研究和工业应用中受到广泛关注,其产品可满足人们对某种特定健康功效食品的追求[1−2]。发酵乳是一种良好的益生菌载体[3]。研究表明,一些益生菌发酵乳具有降低血清胆固醇、改善肠道健康、促进免疫应答和改善认知障碍等功效[4−7]。目前,市面上的活菌型发酵乳,普遍保质期短,这极大限制了产品的销售。此外,一些大容量的活菌型发酵乳,开封后储存于低温环境中极易被潜在的病原微生物污染,对消费者健康造成威胁。因此,开发一种新型的能够减少发酵乳贮藏期污染或延长发酵乳保质期的产品具有重要意义。
罗伊氏乳杆菌是一种存在于大多数脊椎动物肠道的益生菌[8−9],具有降胆固醇[10]、调节肠道菌群[11]和增强肠道屏障[12]等多种益生功能,可应用于功能性食品[13]。含有pdu基因簇的罗伊氏乳杆菌能够代谢甘油产生具有广谱抑菌作用的Reuterin[14],其化学成分为3-羟基丙醛(3-HPA)[15]。3-HPA能够有效抑制细菌、酵母、真菌和病原体,被广泛用于防腐和疾病治疗[16−17]。有研究发现,当Reuterin存在于发酵乳中时,同样能够显著抑制沙门氏菌和空肠弯曲菌等病原菌的生长[18]。此外,Reuterin作为益生菌代谢产物的一种,对人体无害,被认为具有较高的安全性[19−20]。
前期研究证实健康家禽来源的罗伊氏乳杆菌HLRE05具有代谢甘油高产Reuterin的能力,并能利用多种复杂碳水化合物进行发酵[21]。另外,鼠李糖作为一种天然丰富的脱氧糖,被广泛用作食品中非营养性甜味剂,可在保证甜度的情况下,减少糖及卡路里的摄入,满足消费者对健康饮食的需求[22]。目前利用罗伊氏乳杆菌和碳水化合物制备新型发酵乳的研究鲜有报道,主要聚焦于乳制品中Reuterin抗菌能力分析[15,23]。鉴于此,本文将利用罗伊氏乳杆菌HLRE05和鼠李糖制备一款能够原位产生Reuterin,并能有效预防病原菌污染的功能型发酵乳。本研究将为挖掘特色罗伊氏乳杆菌HLRE05的应用潜力奠定坚实基础。
罗伊氏乳杆菌(Limosilactobacillus reuteri)HLRE05 分离于健康家禽(鸡)粪便;大肠埃希氏杆菌O157:H7(Escherichia coli O157:H7)、单核增生李斯特菌CMCC54007(Listeria monocytogenes CMCC54007)、金黄色葡萄球菌CMCC26003(Staphylococcus aureus CMCC26003)、鼠伤寒沙门氏菌ATCC13311(S. Typhimurium ATCC13311)、发酵剂丹尼斯克酸奶菌种YO-MIX 495 均保存于食品科学与资源挖掘全国重点实验室;胰蛋白胨、牛肉膏、酵母提取物、琼脂粉、MRS肉汤培养基 北京索莱宝科技有限公司;脱脂乳 金薄金生态科技有限公司;1,1-二苯基-2-三硝基苯肼(DPPH)、2,2’-联氮基-双-(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS) 上海源叶生物科技有限公司;邻苯二甲醛、三氯乙酸、β-巯基乙醇、邻菲啰啉均为分析纯 上海阿拉丁生化科技股份有限公司;其他有机试剂均为国产分析纯。
Anaerobox IV型厌氧培养箱 美国Gene Science公司;ZXSD-B1090型恒温培养箱 中国上海智城分析仪器有限公司;ST16R型台式高速冷冻离心机 德国Eppendorf公司;MultiskanGO型全波长酶标仪 美国Thermo Fisher Scientific公司;PHS-3E型pH计 中国上海仪电科学仪器有限公司。
从−80 ℃冰箱取出罗伊氏乳杆菌HLRE05,在MRS固体培养基上划线,于37 ℃静置厌氧培养24 h,将单菌落挑至5 mL MRS液体培养基中,37 ℃静置厌氧培养24 h,以1.0%(v/v)接种量传代两次。大肠埃希氏杆菌O157:H7、单核增生李斯特菌CMCC54007、金黄色葡萄球菌CMCC26003、鼠伤寒沙门氏菌ATCC13311均需在LB固体培养基上划线,并传代至5 mL LB培养基中,在37 ℃、180 r/min条件下振荡培养12 h。
称取适量全脂灭菌生牛乳,边搅拌边加入所需浓度的配料(蔗糖:鼠李糖=1:1,w/w),并在无菌操作下接入菌种(罗伊氏乳杆菌HLRE05:丹尼斯克酸奶菌种YO-MIX 495=1:1,v/v),搅拌5 min使物料充分混匀,于恒温条件下厌氧发酵12 h,搅拌破乳后冷却至室温,放置于4 ℃冰箱贮藏得到成品发酵乳。低温后熟:发酵结束后进行搅拌破乳,于4 ℃下后熟12 h;产品贮藏:将不添加甘油的发酵乳(对照组)与添加甘油的发酵乳(实验组)各保留三个平行样品,于4 ℃冰箱中低温冷藏28 d。
将菌液以2%、3%、4%、5%和6%(v/v)的接种量(罗伊氏乳杆菌:丹尼斯克酸奶菌种YO-MIX 495=1:1)接种于糖含量为6%的(蔗糖:鼠李糖=1:1)巴氏灭菌生牛乳中,并于37 ℃条件下厌氧发酵12 h后,取样测定发酵乳pH和罗伊氏乳杆菌HLRE05活菌数。
制备糖含量分别为4%、5%、6%、7%和8%的巴氏灭菌生牛乳(蔗糖:鼠李糖=1:1),再接种4%的重悬菌液(罗伊氏乳杆菌:丹尼斯克酸奶菌种YO-MIX 495=1:1),于37 ℃下厌氧发酵12 h后,测定发酵乳pH和罗伊氏乳杆菌HLRE05活菌数。
以4%的接种量将菌液(罗伊氏乳杆菌:丹尼斯克酸奶菌种YO-MIX 495=1:1)接种至糖含量为6%的巴氏灭菌生牛乳中,随后分别于31、34、37、40和43 ℃恒温厌氧发酵12 h,对发酵乳pH和罗伊氏乳杆菌HLRE05活菌数进行测定。
取不同发酵条件制备的发酵乳,用pH计测定其pH;用1×PBS对发酵乳样品进行10倍梯度稀释,然后在MRS固体培养基上涂板,于37 ℃条件下厌氧培养24 h,计算活菌数。
根据前期所得发酵乳制备条件,将菌液(罗伊氏乳杆菌HLRE05:丹尼斯克酸奶菌种YO-MIX 495=1:1)添加至含糖的巴氏杀菌生牛乳中,于37 ℃厌氧发酵4 h后,添加浓度分别为0、25、50、100、200和400 mmol/L的甘油于发酵乳中,相同条件下厌氧发酵12 h。制备完成后,通过测定发酵乳中Reuterin含量,确定后续制备含Reuterin发酵乳的最佳甘油浓度。
将制备完成的含Reuterin发酵乳充分搅拌3 min后离心(12000 r/min,4 ℃,10 min),收集上清后使用0.22 μm滤膜过滤。采用比色法定量Reuterin[24],吸取0.0625、0.125、0.25、0.5和1 mg/mL的丙烯醛溶液100 μL,加入75 μL色氨酸(0.01 mol/L色氨酸溶于0.05 mol/L盐酸溶液中)和300 μL浓盐酸,置于37 ℃孵育30 min后,测定OD560值。以丙烯醛质量浓度为横坐标,OD560值为纵坐标绘制标准曲线。取100 μL发酵上清代替上述不同浓度的丙烯醛溶液,测定OD560,代入标准曲线计算发酵乳中Reuterin含量。
以最佳甘油添加量制备含Reuterin的发酵乳。将成品发酵乳置于4 ℃冷藏24 h达后熟期,并于相同条件下保存28 d模拟发酵乳贮藏期,随后分别取样测定两个时期内发酵乳中罗伊氏乳杆菌HLRE05活菌数、pH和持水性。每7 d对贮藏期发酵乳取样,并且对其Reuterin含量进行测定。以不添加甘油的罗伊氏乳杆菌发酵乳为对照组。其中持水性测定方法参考文献[25],称取空的10 mL离心管质量记为W0,加入5 mL成品发酵乳(添加或者不添加甘油的组)质量记为W1。在3000 r/min,4 ℃的条件下离心10 min。弃去上清液,称取质量记为W2,平行测定3次。计算公式如下:
$ 持水性(\text{%})= \frac{{\mathrm{W}}_2-{\mathrm{W}}_0}{{\mathrm{W}}_1-{\mathrm{W}}_0}\times 100 $
为模拟发酵乳在贮藏期被污染状态,在制备完成的含Reuterin发酵乳中,分别接种食源性病原菌鼠伤寒沙门氏菌、单核增生李斯特菌、大肠杆菌和金黄色葡萄球菌,使其在发酵乳中的终浓度为102~103 CFU/mL,于4 ℃静置培养。分别在0、4、8和12 h取发酵液1.0 mL,用1×PBS进行10倍梯度稀释,在LB固体平板上进行涂布,计算活菌数。实验重复三次。
使用软件GraphPad Prism 10.0对数据进行分析处理并绘制数据图,各实验数据以平均值±标准差表示。按照双因素方差分析方法(Two-way ANOVA)或不配对的t检验(Unpaired t test)对组间差异进行检验(P<0.05,差异显著)。
接种量直接影响发酵乳中菌株生长速度,并对发酵乳的凝乳进程产生影响。本研究结果发现随着接种量增加,发酵乳中活菌数呈明显上升趋势,但当接种量达到5%后,发酵乳中罗伊氏乳杆菌HLRE05活菌数无显著变化,最终浓度为8×107 CFU/mL(图1)。而接种量对发酵乳pH的影响呈波动趋势,当接种量为2%时,发酵乳pH最高,并在5%接种量时获得最低pH为4.5。这种波动式变化可能是由于混合菌种发酵存在着共生关系,影响了发酵乳的pH,而接种量的变化会改变它们之间的共生强度,从而影响发酵特性及发酵过程中乳酸等有机酸的生成[26]。因此结合活菌数和pH变化,选取5%为最优接种量。
碳源是支持菌株生长且影响发酵乳口感风味的重要因素,为此本研究进一步探究了糖含量对发酵乳的影响。结果显示,在发酵乳中添加不同浓度的糖,发现其对罗伊氏乳杆菌活菌数有一定的影响,表现为当糖含量为6%时,活菌数最高,与糖含量为4%时有显著差异(P<0.05,图2)。这可能是由于糖浓度过低时,菌株缺乏足够的碳源而生长受限,导致活菌数减少;而当浓度升高后,菌株的代谢活动更加旺盛,导致部分代谢产物的积累速度加快,从而影响菌株的活性,降低其活菌数[27]。然而,随着含糖量的变化,pH并无明显差异,始终维持在4.30左右(图2),这说明在一定范围内含糖量的变化不足以显著改变发酵乳中有机酸的产量。综上,后续采用6%的糖含量。
发酵温度是影响乳酸菌发酵乳品质的另一个因素,适宜的发酵温度可使乳制品形成良好的风味和质地,因此评估罗伊氏乳杆菌发酵的最适温度具有重要意义。如图3所示,随着发酵温度的升高,发酵乳中罗伊氏乳杆菌的活菌数呈先上升后下降的趋势,于37 ℃达到最高,接近108 CFU/mL;同时,温度低于40 ℃时,pH维持在4.30左右,但在43 ℃时,发酵乳pH显著升高(P<0.05)。这主要是由于适宜的生长温度有利于罗伊氏乳杆菌的生长代谢,从而使发酵乳pH下降,而温度过高时,则抑制了菌株的生长和代谢,导致活菌数及发酵过程中分解产生的有机酸减少。综上,后续采用37 ℃作为发酵温度。
Reuterin是罗伊氏乳杆菌代谢甘油产生的一种非蛋白类广谱抑菌物质,其抑菌活性不会被核酸酶、蛋白酶和脂肪酶所破坏,具有较强的稳定性[28]。发酵乳中Reuterin的产生可以有效抑制贮藏期间食源性病原菌的污染,本研究采用比色法进一步探究了发酵乳中原位产生Reuterin的情况。利用标准曲线(Y=1.233X+0.08035,R2=0.9968)对发酵乳中的Reuterin进行定量,结果如图4所示,随着甘油添加量的上升,发酵乳中Reuterin的浓度呈先上升后下降的趋势,当甘油浓度为100 mmol/L时,达到最高0.28 mg/mL(3.78 mmol/L),高于此前Langa等[23]测定的5株不同罗伊氏乳杆菌在酸奶中产的最高浓度1.5 mmol/L,这种变化趋势可能是由于在一定范围内增加甘油浓度可促进Reuterin的生成,但超过某个阈值后,过高的甘油浓度会抑制甘油脱水酶的活性而降低合成效率[29],且浓度积累到一定水平的Reuterin会对罗伊氏乳杆菌的生长产生抑制作用从而限制其生产[30−31]。因此,选用100 mmol/L的甘油添加量进行后续实验。
进一步探究添加甘油对发酵乳产生的影响。结果显示,在添加100 mmol/L甘油的发酵乳中,罗伊氏乳杆菌HLRE05的活菌数显著高于未添加甘油组(P<0.01),可达1.2×108 CFU/mL(图5A),这主要是因为甘油可作为电子受体,通过磷酸乙酮醇酶途径产生额外的ATP促进菌株生长[31];与此同时,发酵乳的pH也显著提高(P<0.0001),达到4.3(图5B)。这可能是因为在不添加甘油的发酵乳中,罗伊氏乳杆菌以糖作为唯一碳源进行异型乳酸代谢,主要代谢产物为乳酸、乙酸等有机酸,使pH较低;而在发酵初期加入甘油后,甘油与糖作为共同底物使菌株代谢特征发生变化,在促进罗伊氏乳杆菌生长繁殖的同时,代谢途径的改变影响了代谢产物的生成,使乳酸等有机酸的产量下降,从而导致pH升高[32]。总之,在发酵过程中添加甘油未对发酵乳品质造成不良影响。
为促进香味物质的生成,改善发酵乳的持水性、质地及稳定性,成品发酵乳一般要经历24 h的后熟阶段。本研究制备的含Reuterin的发酵乳于4 ℃冷藏24 h后,活菌数为9.8×107 CFU/mL,显著高于不含甘油对照组的6.4×107 CFU/mL(P<0.01,图6A),pH为4.44,显著高于对照组的4.19(P<0.001,图6B),活菌数相对后熟前(图5A)有所下降,这可能是由于代谢产物的积累以及温度降低等环境因素,使得罗伊氏乳杆菌的代谢受到抑制,而pH的升高,则可能是后熟期乳糖被耗尽,菌体自溶释放的碱性物质中和了有机酸。另外,发酵乳品质与其持水性密切相关,本研究发现添加甘油后,发酵乳持水性也有所升高(图6C)。这主要是由于甘油具有很强的吸湿性和保湿性,可以吸收并保留住乳制品中的水分,此外其还可以作为一种乳化剂[33],稳定发酵乳中的乳状液。
本研究进一步评价了发酵乳贮藏28 d内的生物特性,结果显示,随着贮藏期的延长,未添加甘油的发酵乳中罗伊氏乳杆菌的活菌数呈下降趋势(图7A),这与贮藏期内低温环境的影响有关。然而,添加了甘油的发酵乳,在贮藏的第7 d已无法检出罗伊氏乳杆菌,说明罗伊氏乳杆菌长时间存在于较高浓度Reuterin中时其活性会受到较强破坏[31]。贮藏期发酵乳pH不随时间的延长而发生显著变化(图7B),说明此阶段罗伊氏乳杆菌代谢缓慢或者产酸能力不强。另外,添加甘油(L.r+gly组)与不添加甘油(L.r组)的发酵乳在贮藏期持水性均有下降趋势,但L.r+gly组持水性始终显著高于L.r组(P<0.01,图7C),这可能是由于L.r组pH显著低于L.r+gly组(P<0.0001),酪蛋白胶粒稳定性较低,更容易聚集沉降,导致持水性降低[34]。综上所述,甘油的添加有利于维持发酵乳的持水性。特别地,L.r组发酵乳中Reuterin含量始终保持在基线水平,说明不产Reuterin,而L.r+gly组发酵乳中Reuterin随贮藏时间的延长,含量逐渐下降,从0.27 mg/mL降至0.11 mg/mL,这可能是部分Reuterin被辅酶NAD+和氧化还原酶还原为1,3-丙二醇导致的[21]。
Reuterin是一种天然广谱抗菌剂,其存在于发酵乳中是否有利于预防病原菌的污染还有待更多数据支撑。本研究以典型的食源性病原菌鼠伤寒沙门氏菌、大肠杆菌、金黄色葡萄球菌和单核增生李斯特菌为指示菌,通过人工污染模拟含Reuterin发酵乳被污染的情况,结果发现四种指示菌在发酵乳中均能被有效抑制,添加了甘油的发酵乳因原位产生Reuterin,抑制污染的效果更强(图8)。其中,12 h后沙门氏菌和单增李斯特菌几乎无法检出,浓度分别从1.8×103和9.7×102 CFU/mL降为7.6×101和102 CFU/mL(图8A和B);发酵乳对金黄色葡萄球菌和大肠杆菌的抑制效果稍弱,添加了甘油的组,它们的浓度从2.7×102和4.6×102 CFU/mL持续降至9×101和2.15×102 CFU/mL(图8C和D)。整体上,大肠杆菌对Reuterin的敏感性相比其他指示菌要弱,但抑制效果依然显著高于对照组(P<0.01,图8D),说明含Reuterin发酵乳能够有效预防病原微生物的污染。
罗伊氏乳杆菌HLRE05是一株能利用复杂碳水化合物并代谢甘油产广谱抗菌物质Reuterin的乳酸菌,挖掘此“天然”抗菌成分在乳制品中作为天然防腐剂的价值具有重要意义。因此,本研究利用罗伊氏乳杆菌HLRE05为发酵剂,开发了一款可原位产生Reuterin的新型益生菌发酵乳。在甘油浓度为100 mmol/L时,Reuterin含量高达0.28 mg/mL(3.78 mmol/L);在后熟阶段,甘油的添加还显著提升了发酵乳中罗伊氏乳杆菌HLRE05的活菌数及其持水性;虽然贮藏期内发酵乳中Reuterin的含量有所下降,但仍能较稳定存在。除此之外,该发酵乳还能有效抑制沙门氏菌、单增李斯特菌和金黄色葡萄球菌的污染。综上所述,本研究将为Reuterin作为一种化学防腐剂的替代品用于乳制品开发奠定工作基础。然而,原位产生高浓度Reuterin也会损伤发酵剂的活性,如何维持罗伊氏乳杆菌在发酵乳贮藏期内的活性仍有待进一步探究。

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doi: 10.13386/j.issn1002-0306.2024120371
  • 接收时间:2024-12-30
  • 首发时间:2026-10-24
  • 出版时间:2026-01-01
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    1南昌大学食品科学与资源挖掘全国重点实验室,江西南昌 330047
    2南昌大学中德联合研究院,江西南昌 330047
    3南昌大学重庆研究院,重庆 402660

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张志鸿(1988−),男,博士,助理研究员,研究方向:益生菌与机体健康,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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