Article(id=1280817668580159683, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1280817479555462000, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20260083, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1769529600000, receivedDateStr=2026-01-28, revisedDate=null, revisedDateStr=null, acceptedDate=1772121600000, acceptedDateStr=2026-02-27, onlineDate=1783300328631, onlineDateStr=2026-07-06, pubDate=1783094400000, pubDateStr=2026-07-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1783300328631, onlineIssueDateStr=2026-07-06, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1783300328631, creator=13701087609, updateTime=1783300328631, updator=13701087609, issue=Issue{id=1280817479555462000, tenantId=1146029695717560320, journalId=1192105938417971205, year='2026', volume='66', issue='7', pageStart='3121', pageEnd='3677', issueExtLink='null', onlineDate='null', pubDate='1783094400000', pubDateStr='2026-07-04', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=1, specialIssue=null, createTime=1783300283564, creator='13701087609', updateTime=1783326087324, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1280925708813832745, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1280817479555462000, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1280925708813832746, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1280817479555462000, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=3425, endPage=3437, ext={EN=ArticleExt(id=1280817668953452740, articleId=1280817668580159683, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Lactobacillus paragasseri CCFM1526 promotes the fermentative biotransformation of cyanocobalamin against chronic sleep deprivation-induced nerve injury via the corresponding mechanism, columnId=1192149543992045670, journalTitle=Acta Microbiologica Sinica, columnName=Research Article, runingTitle=null, highlight=null, articleAbstract=

Objective Cyanocobalamin (CN-CbI) requires to be converted into adenosylcobalamin (Ado-CbI) to exert neuroprotective effects, yet its conversion efficiency is impaired under conditions such as chronic sleep deprivation (CSD). This study aimed to obtain a bacterial strain with high efficiency in converting CN-CbI to Ado-CbI that could enhance neuroprotective effects. Methods Lactobacillus paragasseri CCFM1526, a strain capable of converting CN-CbI to Ado-CbI, was isolated via UPLC. A mouse model of CSD was established, and the cognitive functions of mice were evaluated by the novel object recognition and Morris water maze tests. The neuronal structure in the hippocampal dentate gyrus (DG) region was observed via histological staining. Tissue vitamin B12 levels were measured, and the ERK/mTOR signaling pathway along with related neural protein expression was analyzed to assess the neuroprotective mechanism of the fermentation broth. Results Compared with CN-CbI supplementation alone, the fermentation broth of L. paragasseri CCFM1526 significantly improved the cognitive function and alleviated the structural damage in the hippocampal DG region of CSD mice. Intervention with the fermentation broth increased the total vitamin B12 content in the liver, serum, and brain by 10.2%, 16.3%, and 29.0%, respectively (P<0.05). Meanwhile, it activated the ERK/mTOR signaling pathway, leading to increases of 21.5%, 52.4%, 17.3%, and 19.7% in the content of myelin basic protein, postsynaptic density protein 95, brain-derived neurotrophic factor, and nerve growth factor, respectively (P<0.05). Conclusion L. paragasseri CCFM1526 converts CN-CbI into Ado-CbI through fermentation, subsequently activating the ERK/mTOR signaling pathway and upregulating the expression of neurotrophic and myelin repair-related proteins, thereby alleviating CSD-induced nerve injury.

, authors=Xingru CHEN, Xin TANG, Bingyong MAO, Jianxin ZHAO, Qiuxiang ZHANG, Shumao CUI, authorsList=Xingru CHEN, Xin TANG, Bingyong MAO, Jianxin ZHAO, Qiuxiang ZHANG, Shumao CUI, authorCompany=null, correspAuthors=Shumao CUI, authorNote=null, correspAuthorsNote=
E-mail:
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目的 氰钴胺(cyanocobalamin, CN-CbI)需转化为腺苷钴胺(adenosylcobalamin, Ado-CbI)才能发挥神经保护作用,但在慢性睡眠剥夺(chronic sleep deprivation, CSD)等状态下其转化效率受限。本研究旨在筛选出能够高效转化CN-CbI为Ado-CbI,并协同发挥神经保护作用的菌株。 方法 通过超高效液相色谱(UPLC)筛选获得能将CN-CbI转化为Ado-CbI的菌株——副格氏乳杆菌CCFM1526;建立CSD小鼠模型,采用新物体识别实验和Morris水迷宫实验评价小鼠的认知功能,通过组织病理学观察海马DG区神经元结构;测定组织中维生素B12 (vitamin B12, VB12)含量,并检测ERK/mTOR信号通路及相关神经蛋白的表达,以评价发酵液的神经保护机制。 结果 与单纯补充CN-CbI相比,CCFM1526发酵液显著提升了小鼠的认知能力,减轻了海马DG区神经元结构损伤。发酵液干预使小鼠肝脏、血清和脑中的总维生素B12含量分别提升10.2%、16.3%和29.0% (P<0.05),并激活了ERK/mTOR信号通路,使髓鞘碱性蛋白、突触后致密蛋白95、脑源性神经营养因子及神经生长因子的含量分别增加21.5%、52.4%、17.3%和19.7% (P<0.05)。 结论 副格氏乳杆菌CCFM1526通过发酵将CN-CbI转化为Ado-CbI,进而通过激活ERK/mTOR信号通路,上调神经营养与髓鞘修复相关蛋白的表达,从而缓解CSD诱导的神经损伤。

, authors=陈星茹, 唐鑫, 毛丙永, 赵建新, 张秋香, 崔树茂, authorsList=陈星茹, 唐鑫, 毛丙永, 赵建新, 张秋香, 崔树茂, authorCompany=null, correspAuthors=崔树茂, authorNote=

作者贡献声明

陈星茹:方案设计、实验操作、数据分析、初稿写作;唐鑫:方案设计、软件程序;毛丙永:实验操作、软件程序;赵建新:实验指导、提供材料、稿件润色修改;张秋香:实验指导、稿件润色修改、经费支持;崔树茂:研究构思和设计、监督指导、经费支持。

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British Journal of Pharmacology, 2019, 176(9): 1282-1297., articleTitle=Modafinil protects hippocampal neurons by suppressing excessive autophagy and apoptosis in mice with sleep deprivation, refAbstract=null)], funds=[Fund(id=1280925242520474565, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817668580159683, awardId=null, language=EN, fundingSource=the Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, fundOrder=null, country=null)], companyList=[AuthorCompany(id=1280925237235651466, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817668580159683, xref=null, ext=[AuthorCompanyExt(id=1280925237244040075, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817668580159683, companyId=1280925237235651466, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu, China), AuthorCompanyExt(id=1280925237252428684, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817668580159683, companyId=1280925237235651466, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=江南大学,食品科学与资源挖掘全国重点实验室,江苏 无锡)])], figs=[ArticleFig(id=1280925241211851701, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817668580159683, language=EN, label=Figure 1, caption=UPLC analysis of CN-CbI fermentation conversion by Lactobacillus paragasseri CCFM1526. Slight retention time shift due to matrix complexity; The peak was confirmed as Ado-CbI by LC-MS/MS., figureFileSmall=BRDHylkLWEiTTW4J5EjcgQ==, figureFileBig=VmRsFpHgYtWzHo9ogppFpw==, tableContent=null), ArticleFig(id=1280925241274766262, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817668580159683, language=CN, label=图1, caption=副格氏乳杆菌CCFM1526发酵转化CN-CbI UPLC图谱, figureFileSmall=BRDHylkLWEiTTW4J5EjcgQ==, figureFileBig=VmRsFpHgYtWzHo9ogppFpw==, tableContent=null), ArticleFig(id=1280925241396401079, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817668580159683, language=EN, label=Figure 2, caption=Effects of different intragastric treatments on the behaviors of chronic sleep-deprived mice. A: NOR discrimination index; B: NOR recognition index; C: Number of platform crossings in the MWM; D: NOR trajectory map; E: MWM trajectory map. H-1526: Live 1526 group; H-1526+CN-CbI: Live 1526+CN-CbI group; F-1526: 1526 fermentation broth. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.000 1 vs. the Model group; #P<0.05, ##P<0.01, ###P<0.001 vs. the CN-CbI group., figureFileSmall=71TgQK7NkvZvioGvxMEhaQ==, figureFileBig=tqKEFWUUnwwfOlw3nCjXOw==, tableContent=null), ArticleFig(id=1280925241455121336, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817668580159683, language=CN, label=图2, caption=不同灌胃物质对慢性睡眠剥夺小鼠行为学的影响, figureFileSmall=71TgQK7NkvZvioGvxMEhaQ==, figureFileBig=tqKEFWUUnwwfOlw3nCjXOw==, tableContent=null), ArticleFig(id=1280925241522230201, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817668580159683, language=EN, label=Figure 3, caption=HE staining of hippocampal DG region in mice. H-1526: Live 1526 group; H-1526+CN-CbI: Live 1526+CN-CbI group; F-1526: 1526 fermentation broth. Green arrows: Normal neurons; Yellow arrows: Damaged neurons. Scale bar=200 μm., figureFileSmall=1mztuNr9DzTD8ea+a/IsjA==, figureFileBig=e4sM27HsFo2yAAq5yAJCeA==, tableContent=null), ArticleFig(id=1280925241593533370, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817668580159683, language=CN, label=图3, caption=小鼠海马DGHE染色结果, figureFileSmall=1mztuNr9DzTD8ea+a/IsjA==, figureFileBig=e4sM27HsFo2yAAq5yAJCeA==, tableContent=null), ArticleFig(id=1280925241677419451, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817668580159683, language=EN, label=Figure 4, caption=Effects of different intragastric treatments on VB12 levels in mice. A:VB12 content in liver; B: VB12 content in serum; C: VB12 content in brain. H-1526: Live 1526 group; H-1526+CN-CbI: Live 1526+CN-CbI group; F-1526: 1526 fermentation broth. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.000 1 vs. the Model group; #P<0.05, ##P<0.01, ###P<0.001 vs. the CN-CbI group., figureFileSmall=tGbDFFojkyNYJxbvsjRjsQ==, figureFileBig=+94EF+eP9jQMifIgDAD8GQ==, tableContent=null), ArticleFig(id=1280925241748722620, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817668580159683, language=CN, label=图4, caption=不同灌胃物质对小鼠体内VB12 含量的影响, figureFileSmall=tGbDFFojkyNYJxbvsjRjsQ==, figureFileBig=+94EF+eP9jQMifIgDAD8GQ==, tableContent=null), ArticleFig(id=1280925241820025789, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817668580159683, language=EN, label=Figure 5, caption=Effects of different intragastric treatments on the central nervous system of mice. A: MBP; B: PSD-95; C: BDNF; D: NGF. H-1526: Live 1526 group; H-1526+CN-CbI: Live 1526+CN-CbI group; F-1526: 1526 fermentation broth. * P<0.05, ** P<0.01, *** P<0.001; **** P<0.000 1 vs. the Model group; #P<0.05, ##P<0.01, ###P<0.001 vs. the CN-CbI group., figureFileSmall=t+oOtO5jV1Hr/p7PtNI/AA==, figureFileBig=yb6xoRHQF+KW7v/YnWMHlw==, tableContent=null), ArticleFig(id=1280925241916494782, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817668580159683, language=CN, label=图5, caption=不同灌胃物质对小鼠中枢神经系统的影响, figureFileSmall=t+oOtO5jV1Hr/p7PtNI/AA==, figureFileBig=yb6xoRHQF+KW7v/YnWMHlw==, tableContent=null), ArticleFig(id=1280925241987797951, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817668580159683, language=EN, label=Figure 6, caption=Effects of different intragastric treatments on ERK and mTOR signaling pathways in mouse brain tissue. A: p-ERK; B: p-mTOR. H-1526: Live 1526 group; H-1526+CN-CbI: Live 1526+CN-CbI group; F-1526: 1526 fermentation broth. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.000 1 vs. the Model group; #P<0.05, ##P<0.01, ###P<0.001 vs. the CN-CbI group., figureFileSmall=Ic6sW/P3mEcNg95+sgzPJQ==, figureFileBig=ibRaqxVVz8cBq+9D0rg0Jg==, tableContent=null), ArticleFig(id=1280925242059101120, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817668580159683, language=CN, label=图6, caption=不同灌胃物质对小鼠脑组织ERKmTOR信号通路的影响, figureFileSmall=Ic6sW/P3mEcNg95+sgzPJQ==, figureFileBig=ibRaqxVVz8cBq+9D0rg0Jg==, tableContent=null), ArticleFig(id=1280925242159764417, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817668580159683, language=EN, label=Table 1, caption=

Animal experiment design

, figureFileSmall=null, figureFileBig=null, tableContent=
GroupTreatment
ControlPhysiological saline
ModelCSD+Physiological saline
CN-CbICSD+70 μg/(kg·d) CN-CbI
PositiveCSD+70 μg/(kg·d) Me-CbI
H-1526CSD+1×109 CFU live CCFM1526
H-1526+CN-CbICSD+70 μg/(kg·d) CN-CbI+1×109 CFU live CCFM1526
F-1526CSD+Fermentation broth [70 μg/(kg·d) CN-CbI]
), ArticleFig(id=1280925242231067586, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817668580159683, language=CN, label=表1, caption=

动物实验方案

, figureFileSmall=null, figureFileBig=null, tableContent=
GroupTreatment
ControlPhysiological saline
ModelCSD+Physiological saline
CN-CbICSD+70 μg/(kg·d) CN-CbI
PositiveCSD+70 μg/(kg·d) Me-CbI
H-1526CSD+1×109 CFU live CCFM1526
H-1526+CN-CbICSD+70 μg/(kg·d) CN-CbI+1×109 CFU live CCFM1526
F-1526CSD+Fermentation broth [70 μg/(kg·d) CN-CbI]
), ArticleFig(id=1280925242310759363, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817668580159683, language=EN, label=Table 2, caption=

Test results of CN-CbI fermentation by CCFM1526

, figureFileSmall=null, figureFileBig=null, tableContent=
ItemsParameter at 0 hParameter at 48 h
CN-CbI concentration/(μg/mL)500.0364.3
Ado-CbI concentration/(μg/mL)0.052.1
CN-CbI conversion/%-27.1
), ArticleFig(id=1280925242386256836, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817668580159683, language=CN, label=表2, caption=

CCFM1526发酵CN-CbI测定结果

, figureFileSmall=null, figureFileBig=null, tableContent=
ItemsParameter at 0 hParameter at 48 h
CN-CbI concentration/(μg/mL)500.0364.3
Ado-CbI concentration/(μg/mL)0.052.1
CN-CbI conversion/%-27.1
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副格氏乳杆菌CCFM1526促进氰钴胺发酵转化并缓解慢性睡眠剥夺诱导的神经损伤及机制
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陈星茹 , 唐鑫 , 毛丙永 , 赵建新 , 张秋香 , 崔树茂
微生物学报 | 研究报告 2026,66(7): 3425-3437
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微生物学报 |研究报告 2026 , 66 (7) : 3425 -3437
副格氏乳杆菌CCFM1526促进氰钴胺发酵转化并缓解慢性睡眠剥夺诱导的神经损伤及机制
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陈星茹, 唐鑫, 毛丙永, 赵建新, 张秋香, 崔树茂
作者信息
  • 江南大学,食品科学与资源挖掘全国重点实验室,江苏 无锡
作者简介:

作者贡献声明

陈星茹:方案设计、实验操作、数据分析、初稿写作;唐鑫:方案设计、软件程序;毛丙永:实验操作、软件程序;赵建新:实验指导、提供材料、稿件润色修改;张秋香:实验指导、稿件润色修改、经费支持;崔树茂:研究构思和设计、监督指导、经费支持。

Lactobacillus paragasseri CCFM1526 promotes the fermentative biotransformation of cyanocobalamin against chronic sleep deprivation-induced nerve injury via the corresponding mechanism
Xingru CHEN, Xin TANG, Bingyong MAO, Jianxin ZHAO, Qiuxiang ZHANG, Shumao CUI
Affiliations
  • State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu, China
出版时间: 2026-07-04 doi: 10.13343/j.cnki.wsxb.20260083
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目的 氰钴胺(cyanocobalamin, CN-CbI)需转化为腺苷钴胺(adenosylcobalamin, Ado-CbI)才能发挥神经保护作用,但在慢性睡眠剥夺(chronic sleep deprivation, CSD)等状态下其转化效率受限。本研究旨在筛选出能够高效转化CN-CbI为Ado-CbI,并协同发挥神经保护作用的菌株。 方法 通过超高效液相色谱(UPLC)筛选获得能将CN-CbI转化为Ado-CbI的菌株——副格氏乳杆菌CCFM1526;建立CSD小鼠模型,采用新物体识别实验和Morris水迷宫实验评价小鼠的认知功能,通过组织病理学观察海马DG区神经元结构;测定组织中维生素B12 (vitamin B12, VB12)含量,并检测ERK/mTOR信号通路及相关神经蛋白的表达,以评价发酵液的神经保护机制。 结果 与单纯补充CN-CbI相比,CCFM1526发酵液显著提升了小鼠的认知能力,减轻了海马DG区神经元结构损伤。发酵液干预使小鼠肝脏、血清和脑中的总维生素B12含量分别提升10.2%、16.3%和29.0% (P<0.05),并激活了ERK/mTOR信号通路,使髓鞘碱性蛋白、突触后致密蛋白95、脑源性神经营养因子及神经生长因子的含量分别增加21.5%、52.4%、17.3%和19.7% (P<0.05)。 结论 副格氏乳杆菌CCFM1526通过发酵将CN-CbI转化为Ado-CbI,进而通过激活ERK/mTOR信号通路,上调神经营养与髓鞘修复相关蛋白的表达,从而缓解CSD诱导的神经损伤。

副格氏乳杆菌CCFM1526  /  氰钴胺  /  腺苷钴胺  /  神经损伤  /  ERK/mTOR信号通路

Objective Cyanocobalamin (CN-CbI) requires to be converted into adenosylcobalamin (Ado-CbI) to exert neuroprotective effects, yet its conversion efficiency is impaired under conditions such as chronic sleep deprivation (CSD). This study aimed to obtain a bacterial strain with high efficiency in converting CN-CbI to Ado-CbI that could enhance neuroprotective effects. Methods Lactobacillus paragasseri CCFM1526, a strain capable of converting CN-CbI to Ado-CbI, was isolated via UPLC. A mouse model of CSD was established, and the cognitive functions of mice were evaluated by the novel object recognition and Morris water maze tests. The neuronal structure in the hippocampal dentate gyrus (DG) region was observed via histological staining. Tissue vitamin B12 levels were measured, and the ERK/mTOR signaling pathway along with related neural protein expression was analyzed to assess the neuroprotective mechanism of the fermentation broth. Results Compared with CN-CbI supplementation alone, the fermentation broth of L. paragasseri CCFM1526 significantly improved the cognitive function and alleviated the structural damage in the hippocampal DG region of CSD mice. Intervention with the fermentation broth increased the total vitamin B12 content in the liver, serum, and brain by 10.2%, 16.3%, and 29.0%, respectively (P<0.05). Meanwhile, it activated the ERK/mTOR signaling pathway, leading to increases of 21.5%, 52.4%, 17.3%, and 19.7% in the content of myelin basic protein, postsynaptic density protein 95, brain-derived neurotrophic factor, and nerve growth factor, respectively (P<0.05). Conclusion L. paragasseri CCFM1526 converts CN-CbI into Ado-CbI through fermentation, subsequently activating the ERK/mTOR signaling pathway and upregulating the expression of neurotrophic and myelin repair-related proteins, thereby alleviating CSD-induced nerve injury.

Lactobacillus paragasseri CCFM1526  /  cyanocobalamin  /  adenosylcobalamin  /  nerve injury  /  ERK/mTOR signaling pathway
陈星茹, 唐鑫, 毛丙永, 赵建新, 张秋香, 崔树茂. 副格氏乳杆菌CCFM1526促进氰钴胺发酵转化并缓解慢性睡眠剥夺诱导的神经损伤及机制. 微生物学报, 2026 , 66 (7) : 3425 -3437 . DOI: 10.13343/j.cnki.wsxb.20260083
Xingru CHEN, Xin TANG, Bingyong MAO, Jianxin ZHAO, Qiuxiang ZHANG, Shumao CUI. Lactobacillus paragasseri CCFM1526 promotes the fermentative biotransformation of cyanocobalamin against chronic sleep deprivation-induced nerve injury via the corresponding mechanism[J]. Acta Microbiologica Sinica, 2026 , 66 (7) : 3425 -3437 . DOI: 10.13343/j.cnki.wsxb.20260083
维生素B12 (vitamin B12, VB12)是维持神经系统发育和功能完整性的必需微量营养素,其在线粒体中转化为具有生物活性的腺苷钴胺(adenosylcobalamin, Ado-CbI),该活性形式是甲基丙二酰辅酶A变位酶的必需辅因子,可催化甲基丙二酰辅酶A转化为琥珀酰辅酶A[1]。这一反应不仅能减少神经毒性代谢物甲基丙二酸的积累,也能为髓鞘脂质合成与轴突结构的维持提供必要底物,从而有助于维持神经信号传导效率与神经网络稳定性,在神经保护中发挥不可替代的作用[2-3]。日常广泛使用的VB12补充剂多为化学性质稳定的氰钴胺(cyanocobalamin, CN-CbI),其必须在体内经多步酶促反应转化为Ado-CbI才能发挥生物活性,该转化过程高度依赖于细胞的能量代谢与氧化还原稳态[4]。然而,在慢性应激、衰老及神经退行性疾病等状态下,细胞线粒体功能易受损,相关酶活性下降,导致CN-CbI向Ado-CbI的转化效率显著降低,从而严重限制了其实际神经保护效果[5]。因此,克服CN-CbI的体内转化瓶颈,提高其在神经系统的Ado-CbI生物利用度,对于开发针对神经损伤的新型干预手段具有重要意义。
与直接补充化学性质不稳定、易降解的Ado-CbI相比,基于益生菌的转化策略有望提供更稳定且可控的活性VB12来源。肠道益生菌可通过其特有的酶系统将膳食中一些难以被宿主直接利用的复杂成分(如黄酮类、酚苷类和槲皮素等)转化为高生物活性的小分子物质,从而调节宿主的生理功能[6]。这提示,利用特定益生菌株对CN-CbI进行发酵转化,或可绕过体内转化的限速步骤,为克服CN-CbI的生物利用瓶颈提供新的递送策略。然而,目前关于具有高效CN-CbI转化能力的益生菌株筛选及其在体内协同CN-CbI发挥神经保护作用的系统性研究仍较为缺乏。
慢性睡眠剥夺(chronic sleep deprivation, CSD)是一种可导致中枢神经损伤、认知功能衰退及情绪行为异常的经典实验模型。研究表明,CSD可导致海马等关键脑区神经元线粒体功能障碍、氧化应激与能量代谢失衡,干扰内源性Ado-CbI转化,同时引发神经炎症、髓鞘结构损伤与突触可塑性下降等一系列病理变化[7-9]。因此,CSD模型为在模拟体内CN-CbI转化受限的环境下,评估外源性或菌源性Ado-CbI的神经保护效能提供了理想的研究模型。
本研究通过超高效液相色谱筛选,获得了一株能将CN-CbI转化为Ado-CbI的益生菌——副格氏乳杆菌CCFM1526。通过CSD诱导的C57BL/6J小鼠中枢神经损伤模型,系统比较CCFM1526发酵CN‑CbI与单纯补充CN‑CbI对小鼠认知功能、脑组织病理、VB12分布以及相关神经营养和髓鞘修复蛋白表达的影响,并深入探讨其潜在作用机制。本研究旨在阐明益生菌发酵转化CN-CbI在缓解神经损伤中的作用与机制,为开发基于益生菌与微量营养素协同作用的神经保护性功能食品提供理论支持。
副格氏乳杆菌CCFM1526由江南大学食品微生物菌种保藏中心(Culture Collection of Food Microorganisms, CCFM)提供。
MRS培养基,青岛海博生物技术有限公司;氰钴胺,上海阿拉丁生化科技股份有限公司;腺苷钴胺,Sigma-Aldrich Co. LLC公司;小鼠VB12、突触后密度蛋白-95 (postsynaptic density protein-95, PSD-95)、髓鞘碱性蛋白(myelin basic protein, MBP)、脑源性神经营养因子(brain-derived neurotrophic factor, BDNF)、神经生长因子(nerve growth factor, NGF)测定试剂盒,上海酶联生物科技有限公司;BCA蛋白浓度测定试剂盒,上海碧云天生物技术股份有限公司。
多功能酶标仪,上海智城分析仪器制造有限公司;超高效液相系统、质谱仪,赛默飞世尔科技公司;高速离心机,艾本德中国有限公司;高通量组织研磨机,宁波新芝生物科技股份有限公司;切片电子扫描仪,3DHISTECH公司。
将副格氏乳杆菌CCFM1526冻存菌种在MRS平板上划线,于37 ℃有氧条件下倒置培养48 h。挑取单菌落接种于5 mL的MRS液体培养基中,37 ℃、200 r/min培养16-18 h,重复该操作3-4次,制备得到种子液。
CN-CbI发酵培养基(g/L):氰钴胺0.5,酵母浸粉5.0,葡萄糖10.0,柠檬酸氢二胺2.0,用纯净水混合,将pH调至6.0-6.2,于115 ℃灭菌20 min。
将上述种子液以体积分数5%的接种量接种至CN‑CbI发酵培养基中,37 ℃、200 r/min培养48 h。发酵完成后将发酵液12 000 r/min离心20 min,收集上清液,用0.22 μm水系过滤器过滤,置于4 ℃备用。
使用配备Phenomenex C18 (4.6 mm×250 mm, 5 µm)色谱柱的超高效液相系统进行分析。流动相A为含0.1%甲酸的水溶液,流动相B为乙腈。柱温35 ℃,样品盘温度4 ℃,进样体积2 μL。通过对CN-CbI与Ado-CbI标准品的保留时间比对进行定性,并采用(峰面积-浓度标准曲线)对发酵液中Ado-CbI含量进行半定量估算。
将CCFM1526菌液于4 ℃、6 000 r/min离心5 min收集菌体,用无菌PBS (pH 7.4)清洗2次,随后用CN-CbI溶液重悬菌体至终浓度为1×109 CFU/mL,于灌胃前新鲜制备。
将CCFM1526种子液以体积分数5%的接种量接种至CN-CbI发酵培养基(同1.3节)中,37 ℃、200 r/min培养48 h。发酵液经高压均质(4 ℃、1.2×108 Pa,循环3次)后,通过冷冻干燥得到冻干粉。灌胃前用生理盐水复溶至所需浓度。
42只8周龄雄性C57BL/6J小鼠购自斯贝福(北京)生物技术有限公司,饲养于江南大学动物实验中心SPF级别屏障环境(温度20-26 ℃、相对湿度40%-60%)。本研究所有动物实验获得江南大学实验动物伦理委员会批准,编号为No. 20250415c1800609[177]。适应1周后,随机分为7组,每组6只小鼠,分别为:空白组(Control组)、模型组(Model组)、CN-CbI组、阳性对照组(Positive组)、1526活菌组(H-1526)、1526活菌+CN-CbI组(H-1526+CbI)、1526发酵组(F-1526)。具体分组及干预措施见表1
采用改良多平台水环境法建立CSD模型[10]。将除Control组外的小鼠置于睡眠剥夺箱,每日于光周期中期开始,连续剥夺睡眠18 h (15:00至次日9:00),共持续3周。Control组置于大平台空白组实验箱中,正常饮食饮水与睡眠。干预期各实验组小鼠以0.2 mL/d的剂量灌胃给药。
于睡眠剥夺及干预的第15天开始行为学实验,包括新物体识别实验(novel object recognition test, NOR)和Morris水迷宫实验(Morris water maze test, MWM)。
NOR实验包括习惯化、训练和测试3个阶段[11]。测试阶段,记录小鼠在6 min内探索新物体时间(time of new object, TN)与旧物体时间(time of familiar object, TF),并按公式(1)、(2)分别计算辨别指数(discrimination index, DI)和认知指数(recognition index, RI)。
DI=(TN-TF)/(TN+TF)
RI=TN/(TN+TF)
MWM实验包括4 d的定位航行训练和1 d的空间探索测试[12]。训练期间记录小鼠找到隐藏平台的潜伏期,测试期间通过计算机软件记录小鼠在60 s内穿越原平台位置的次数。
行为学测试结束后,采集小鼠血清、全脑和肝脏组织。部分脑组织与肝脏组织经液氮速冻后保存于-80 ℃;另取部分脑组织置于4%多聚甲醛中固定,用于病理学分析。
脑组织在体积分数为4%的多聚甲醛溶液中固定48 h后,委托武汉赛维尔生物科技有限公司进行石蜡包埋、切片制作、苏木精-伊红染色(hematoxylin-eosin, HE),然后进行自动扫描切片,观察脑组织病理损伤情况。
使用ELISA试剂盒检测小鼠肝脏、血清和脑组织中的VB12总含量、脑部MBP、PSD-95、BDNF、NGF、磷酸化细胞外信号调节激酶(phosphorylated extracellular signal-regulated kinase, p-ERK)、磷酸化哺乳动物雷帕霉素靶蛋白(phosphorylated mammalian target of rapamycin, p-mTOR)的含量。蛋白浓度的测定参照BCA蛋白浓度试剂盒说明书进行。
所得数据使用GraphPad Prism 10.0软件进行分析,对正态分布的实验数据进行t检验(t test)及单因素方差分析(one-way ANOVA),比较组间差异,数据以平均值±标准差(mean±SD)表示(n=6)。P<0.05认为有统计学意义。*表示与Model组进行差异性的比较,#表示与CN-CbI组进行差异性的比较。
CN-CbI向Ado-CbI的转化通常涉及2类关键酶:脱氰酶与腺苷转移酶。脱氰酶负责将CN-CbI转化为羟钴胺,该过程依赖FAD和NADPH作为辅因子;随后,钴胺素腺苷转移酶(CobA)催化腺苷基团转移生成具有生物活性的Ado-CbI[13]。如图1所示,在未接种菌株的CN-CbI培养基中,仅在约5.40 min处检测到CN-CbI的特征峰。经CCFM1526发酵48 h后,在约5.71 min处出现一个新的色谱峰,其保留时间接近Ado-CbI标准品(5.82 min)。由于复杂发酵基质的基质效应常导致色谱峰整体保留时间发生漂移[14-15],本研究中的轻微偏移也与此有关。然而,仅依靠保留时间进行定性存在一定局限,因此本研究采用LC-MS/MS技术,通过比对二级特征碎片离子,确认该峰中含有Ado-CbI,相关质谱数据已上传至ScienceDB数据库(http://www.scidb.cn),CSTR编号为31253.11.sciencedb.j00231.00042。半定量分析显示,CCFM1526发酵消耗了约27.1%的CN-CbI,并生成约52.1 μg/mL的Ado-CbI (表2)。
为进一步探究CCFM1526转化CN-CbI的能力,对CCFM1526全基因组进行了分析。菌株中含有一个编码咕啉类腺苷转移酶(corrinoid adenosyltransferase)的基因(yvqk),其编码蛋白与CobA家族高度同源。BLASTp比对结果显示,该蛋白与副格氏乳杆菌JCM 5343的CobA具有高度同源性,证实其为负责腺苷化步骤的CobA同源蛋白(编号为31253.11.sciencedb.j00231.00042)。此外,基因组中存在多个编码VB12转运ATP结合蛋白的BtuD同源基因,提示菌株具备主动摄取外源性CN-CbI的能力[16]。尽管乳杆菌中尚未发现特异性钴胺素还原酶,但CCFM1526含有黄素还原酶家族基因及黄素氧还蛋白等电子传递相关基因,可能通过提供还原力参与脱氰过程[13](编号为31253.11.sciencedb.j00231.00042)。因此,CCFM1526可能通过BtuD同源蛋白摄取外源CN-CbI,经黄素还原酶等介导的脱氰作用转化为羟钴胺,最终由CobA同源蛋白催化生成Ado-CbI。该机制与发酵结果一致,证实副格氏乳杆菌CCFM1526可将CN-CbI转化为Ado-CbI。
本研究以临床上常用的神经营养药物——甲钴胺作为阳性对照以评估其相对效力[17]。啮齿动物天生具备探索新奇物体的本能,若小鼠认知能力正常,其对新物体的探索时间较旧物体更长;反之,对新旧物体的探索时间无差异[18]。NOR实验结果如图2A2B2D所示,相较于Control组,Model组小鼠对新物体的探索时间显著减少,辨别指数(DI)从0.14下降至-0.56,认知指数(RI)由0.53下降至0.24 (P<0.001),表明CSD成功损伤了小鼠的物体识别记忆。与此相比,Positive组的DI和RI分别为-0.08和0.46 (P<0.01)。补充未发酵CN-CbI后,DI和RI分别上升至-0.34和0.31 (P<0.05),虽较Model组有所改善,但效果有限。CCFM1526活菌单独干预或与CN-CbI协同干预均未观察到对小鼠认知能力的显著影响。在所有实验干预组中,CCFM1526发酵CN-CbI干预效果最优,较CN-CbI组,其DI和RI分别提高约71.1%和49.3% (P<0.05),恢复至接近Positive组水平。
在评估空间学习与记忆的MWM实验中,正常小鼠由于工作记忆完好,能准确记住原平台的位置,穿过平台的频率较高[19]。实验结果如图2C2E所示,Model组小鼠穿越平台次数较Control组显著减少了46.7% (P<0.01),提示空间记忆受损。与NOR结果一致,CCFM1526活菌单独干预或与CN-CbI简单混合干预均未有效提升小鼠记忆。CCFM1526发酵CN-CbI干预则使小鼠穿越平台次数较CN-CbI组提升了94.1% (P<0.05)。两项行为学测试结果共同表明,经CCFM1526发酵处理后的CN-CbI,在改善CSD诱导的认知功能障碍方面,效果显著优于未发酵的CN-CbI,且接近临床阳性药物甲钴胺的干预水平。
长期睡眠剥夺可引起中枢神经系统结构性改变,其中海马齿状回区(dentate gyrus, DG)作为学习记忆的关键区域,参与记忆的形成与巩固过程,其神经元形态易受睡眠缺失影响[19]。如图3A3B所示,Control组小鼠海马DG区细胞排列整齐,细胞核形态结构完整,细胞间隙相对较小;而Model组小鼠神经细胞排列散乱且不规则,神经元萎缩,胞浆染色加深,胞浆空泡化,提示CSD成功诱导了海马组织的形态学损伤。各干预组均表现出一定程度的保护作用(图3C-3G):CN-CbI和甲钴胺干预后萎缩的神经元数量减少,甲钴胺效果更显著;CCFM1526活菌单独干预的改善效果较弱,其与CN-CbI联合干预效果略优于CN-CbI组。其中,经CCFM1526发酵液干预的小鼠,其海马DG区神经元排列更为紧密,固缩现象明显减少,病理形态恢复接近Control组水平。
以上组织形态学结果证实,CCFM1526发酵CN-CbI在缓解CSD所致海马DG区的神经元结构损伤方面具有一定作用,其保护效果优于未发酵的CN-CbI及其他干预形式。该现象与先前研究一致,如夏枯草活性成分被证实可减轻睡眠剥夺所致下丘脑、大脑皮层及海马区神经元损伤[20],进一步印证了睡眠剥夺对中枢神经系统的影响及有效干预的潜在价值。
为探究发酵处理对CN-CbI体内代谢的影响,本研究检测了在等量CN-CbI摄入下各组小鼠肝脏、血清及脑组织的总VB12含量。
图4所示,直接补充CN-CbI或甲钴胺均可提高小鼠体内VB12水平,但单纯补充CCFM1526活菌无法有效提升小鼠体内VB12含量。与直接补充CN-CbI相比,补充CCFM1526发酵液(F-1526组)能显著提升小鼠关键组织中的总VB12水平。其中,肝脏中总VB12含量提升了10.2% (P<0.05),血清中提升了16.3% (P<0.05),脑组织中提升最为显著,达到29.0% (P<0.05)。
CN-CbI作为外源性VB12的主要形式,需在体内(主要在肝脏)经多步酶促反应转化为Ado-CbI等活性形式后方能发挥生理功能。本研究中F-1526组小鼠脑内VB12累积幅度显著高于肝脏,这一结果提示,发酵预处理可能改变了CN-CbI的代谢路径。我们推测,CCFM1526在发酵过程中将CN-CbI预先转化为Ado-CbI,该活性形式可能绕过了肝脏的首过代谢限制与细胞内的转化瓶颈,从而更易被吸收入血并跨越血脑屏障,最终在脑内实现更高的生物利用度[21]。这也与F-1526组在行为学与组织形态学中表现出的最优神经保护效果相互印证。
中枢神经系统的功能维系依赖于其结构完整、神经网络连接及神经营养支持三大核心层面的紧密协同。因此,本研究选取MBP、PSD-95以及BDNF/NGF三类关键蛋白,分别从髓鞘结构、突触功能及神经营养调控角度系统探究中枢神经损伤与修复的分子机制。其中,MBP是维持髓鞘结构与电生理功能完整性的基础蛋白,保障神经网络信息传导的基础效率,为物体识别、空间探索等高级认知活动创造必要生理条件;PSD-95作为突触后支架蛋白,直接参与突触结构稳定性与信号传递,是NOR记忆形成、MWM所检测的空间记忆巩固的重要细胞分子基础;BDNF和NGF作为重要的神经营养因子,在促进神经元存活、轴突生长及突触重塑中发挥核心营养支持作用,其表达水平会影响海马区神经元的结构与功能完整性,进而调控小鼠的学习记忆能力[22-23]
图5所示,CSD导致Model组小鼠脑组织中4种蛋白的表达均较Control组显著下降(P<0.001),提示CSD造成了一定程度上的中枢神经损伤,与文献报道结果[24-26]一致。CN-CbI和甲钴胺干预可不同程度提升上述蛋白的表达,而单纯CCFM1526活菌干预未表现明显改善效果。与CN-CbI组相比,F-1526组小鼠MBP、PSD-95、BDNF和NGF水平分别提升了21.5%、52.4%、17.3%和19.7% (P<0.05)。CCFM1526活菌与CN-CbI混合干预在提升MBP和NGF水平方面的效力与F-1526组相当,但对PSD-95和BDNF的调节作用则显著弱于F-1526组(图5A-5D)。这一结果提示,CCFM1526在神经修复过程中发挥一定作用,但效果受干预形式影响。我们推测,口服的CCFM1526活菌可能在肠道内对CN-CbI进行了初步转化,生成更易被细胞利用的中间体,但该过程受限于肠道通过时间以及宿主消化环境的复杂性,导致其转化过程不充分、不稳定,效率远低于发酵[6],进而在PSD-95、BDNF的调控上表现不足。F-1526组通过高效、彻底、稳定的生物转化,直接提供了充足的Ado-CbI,从而能够更全面地修复神经损伤。
ERK/mTOR信号通路在维持神经元存活及认知功能中发挥关键作用。在CSD状态下,小鼠海马组织内该信号通路受到显著抑制,同时伴随自噬和凋亡过程的异常激活,进而影响下游神经营养因子表达与神经结构的完整性[24,27]。本研究结果显示(图6A6B),CSD显著抑制了脑部ERK和mTOR信号通路的激活,与Control组相比,Model组p-ERK及p-mTOR表达分别降低40.6%与45.6% (P<0.001)。CN-CbI与甲钴胺干预不同程度上缓解上述信号通路的抑制。CCFM1526发酵液(F-1526组)干预有效恢复了小鼠脑部ERK与mTOR的磷酸化水平,其表达显著高于CN-CbI组(分别提高了11.8%和14.8%,P<0.05),并恢复至接近Control组水平。CCFM1526活菌单独干预或与CN-CbI混合干预则未观察到明显改善作用。上述结果提示,CCFM1526发酵液中的Ado-CbI介导的神经保护作用与ERK/mTOR信号通路的激活密切相关。发酵液可能通过支持线粒体能量代谢改善细胞能量状态,从而激活了mTOR信号,并协同调控了下游的ERK等通路。激活的ERK/mTOR通路可能通过促进BDNF、NGF等神经营养因子的合成[24],并可能通过抑制过度自噬或凋亡来保护神经元[27],进而支持PSD-95、MBP等结构蛋白生成,增强突触可塑性与修复髓鞘,共同缓解CSD导致的神经损伤。这些结果提示,ERK/mTOR通路可能是CCFM1526发酵液发挥神经保护作用的重要介导途径之一,但其具体机制仍有待通过通路抑制剂或基因敲除等干预手段进一步验证。
本研究基于菌株发酵筛选得到了一株能够将CN-CbI转化为Ado-CbI的副格氏乳杆菌CCFM1526。在CSD模型中,CCFM1526发酵CN-CbI能够显著提升小鼠体内VB12的含量、改善中枢神经细胞及中枢神经系统结构的损害、缓解认知能力的下降,其作用机制与激活ERK/mTOR信号通路进而促进神经营养因子表达及髓鞘修复相关蛋白合成密切相关。该研究为开发基于益生菌发酵转化的功能性VB12制品或合生制剂提供了理论依据与菌种资源,在功能性食品与神经营养干预领域具有潜在应用价值。
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doi: 10.13343/j.cnki.wsxb.20260083
  • 接收时间:2026-01-28
  • 首发时间:2026-07-06
  • 出版时间:2026-07-04
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  • 收稿日期:2026-01-28
  • 录用日期:2026-02-27
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the Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province
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    江南大学,食品科学与资源挖掘全国重点实验室,江苏 无锡

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2种不同金属材料的力学参数

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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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