Article(id=1226956556521096082, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226956547847275311, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20250153, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1740585600000, receivedDateStr=2025-02-27, revisedDate=null, revisedDateStr=null, acceptedDate=1743091200000, acceptedDateStr=2025-03-28, onlineDate=1770458838825, onlineDateStr=2026-02-07, pubDate=1756915200000, pubDateStr=2025-09-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1770458838825, onlineIssueDateStr=2026-02-07, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1770458838825, creator=13701087609, updateTime=1770458838825, updator=13701087609, issue=Issue{id=1226956547847275311, tenantId=1146029695717560320, journalId=1192105938417971205, year='2025', volume='65', issue='9', pageStart='3821', pageEnd='4232', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1770458836757, creator=13701087609, updateTime=1770459153781, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1226957877613605816, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226956547847275311, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1226957877613605817, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226956547847275311, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=4075, endPage=4087, ext={EN=ArticleExt(id=1226956556948915109, articleId=1226956556521096082, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Effects of sialic acid and 6′-sialyllactose on in vitro fermentation characteristics of gut microbiota in weaned piglets, columnId=1192149543992045670, journalTitle=Acta Microbiologica Sinica, columnName=Research Article, runingTitle=null, highlight=null, articleAbstract=

6′-sialyllactose (6′-SL) is an important component of porcine breast milk oligosaccharides, and sialic acid (SIA) is the monomer of sialylated breast milk oligosaccharides. Both can regulate the structure of human colonic microbiota. However, there are few reports regarding the effects of SIA and 6′-SL on the composition and fermentation characteristics of piglet colonic microbiota in vitro. [Objective] To investigate the effects of SIA and 6′-SL on the composition and fermentation characteristics of colonic microbiota in suckling piglets, aiming to provide a reference for using these substances to regulate gut health in piglets. [Methods] We used mixed colonic chyme from three suckling piglets as the microbial inoculum. The experiment was conducted with four groups: no carbon (NCB), lactose (LAC), SIA, and 6′-SL, each with four replicates. [Results] After 24 h of fermentation, the pH in the 6′-SL group was higher than that in the LAC group (P<0.05). After fermentation for 12 h and 24 h, the gas production in the SIA and 6′-SL groups was lower than that in the LAC group (P<0.05). At the time point of 24 h, the SIA and 6′-SL groups had higher acetic acid content and lower propionic acid and butyric acid content than the LAC group (P<0.05). Moreover, the SIA and 6′-SL groups had higher Chao1 and ACE indices than the LAC group (P<0.05). Beta diversity of the microbial community in the LAC, SIA, and 6′-SL groups changed (P<0.05). As fermentation progressed, compared with LAC, SIA increased the relative abundance of Bacillota, Anaerovibrio, and Faecalibacterium and decreased that of Pseudomonadota, Escherichia-Shigella, Mitsuokella, and Streptococcus (P<0.05); 6′-SL increased the relative abundance of Bacteroidetes, Prevotella, Bacteroides, and Roseburia and decreased that of Pseudomonadota, Bacillota, Megamonas, Escherichia-Shigella, Mitsuokella, and Prevotellaceae_NK3B31_group (P<0.05). [Conclusion] Both 6′-SL and SIA can effectively regulate the gut microbiota structure in piglets. Specifically, 6′-SL increases the relative abundance of Bacteroidetes, Prevotella, and Bacteroides, while SIA increases that of Bacillota and Anaerovibrio. Both substances promote colonic microbial fermentation in piglets to increase the production of short-chain fatty acids (SCFAs), especially acetic acid.

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*E-mail:
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6′-唾液酸乳糖(6′-sialyllactose, 6′-SL)是猪母乳寡糖中的重要组分,唾液酸(sialic acid, SIA)是唾液酸化乳寡糖的单体,它们均可调控人的结肠微生物结构。目前关于SIA和6′-SL在体外对仔猪结肠微生物的组成和发酵特性的影响鲜有报道。 【目的】 探究SIA和6′-SL对哺乳仔猪结肠微生物组成及发酵特性的影响,为SIA和6′-SL调控哺乳仔猪肠道健康提供参考。 【方法】 以3只哺乳仔猪的结肠混合食糜为微生物接种物,探究SIA和6′-SL对哺乳仔猪结肠微生物组成及发酵特性的影响。试验分为4组:无碳源(no carbon, NCB)、乳糖(lactose, LAC)、SIA和6′-SL,各组均为4个重复。 【结果】 发酵至24 h时,6′-SL组的pH显著高于LAC组(P<0.05);发酵至12 h和24 h时,SIA和6′-SL组的产气量均显著低于LAC组(P<0.05);发酵至24 h时,SIA和6′-SL组的乙酸含量均显著高于LAC组(P<0.05),丙酸和丁酸含量均显著低于LAC组(P<0.05),显著提高了Chao1指数和ACE指数(P<0.05);LAC组、SIA组和6′-SL组菌群的β多样性发生显著改变(P<0.05);随着发酵时间延长,与LAC组相比,SIA组显著提高了芽孢杆菌门(Bacillota)、厌氧弧菌属(Anaerovibrio)和普氏栖粪杆菌属(Faecalibacterium)的相对丰度,降低了假单胞菌门(Pseudomonadota)、埃希氏菌-志贺氏菌(Escherichia-Shigella)、光冈菌属(Mitsuokella)和链球菌属(Streptococcus)的相对丰度(P<0.05);6′-SL组显著提高了拟杆菌门(Bacteroidetes)普雷沃氏菌属Prevotella、拟杆菌属Bacteroides和罗氏菌属Roseburia,降低了假单胞菌门、芽孢杆菌门、巨单胞菌属(Megamonas)、埃希氏菌-志贺氏菌、光冈菌属和普雷沃氏菌科NK3B31组(Prevotellaceae_NK3B31_group)的相对丰度(P<0.05)。 【结论】 6′-SL和SIA均可有效调控仔猪肠道菌群结构,6′-SL能提高拟杆菌门以及普雷沃氏菌属和拟杆菌属的相对丰度,而SIA能提高芽孢杆菌门以及厌氧弧菌属的相对丰度,且2种底物都明显促进仔猪结肠微生物发酵,提高短链脂肪酸(short chain fatty acids, SCFAs)产量,尤其是乙酸。

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作者贡献声明

刘晓英:参与试验设计,试验数据收集和分析,论文撰写等;李轩:参与试验方案设计,试验和数据分析;吴海琴:参与数据分析和论文修改;许来鹏:参与试验和数据分析;赵德辉:参与试验指导和论文修改;马季祥:参与论文修改;慕春龙:试验方案设计,试验数据收集和协助分析以及论文修改;朱伟云:试验指导,论文审核修改;潘龙:论文撰写,论文修改和审核等。

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A: pH; B: Gas production. All values are presented as mean±SEM, and one-way ANOVA followed by Duncan’s test for multiple comparisons was performed. Letters indicate significant differences among groups (P<0.05), the same below., figureFileSmall=YMdTJ4i4Gzuz/N9GaMgyVg==, figureFileBig=U0YXUU5YW94g6hNyRcHpqQ==, tableContent=null), ArticleFig(id=1226964059698934751, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956556521096082, language=CN, label=图1, caption=结肠微生物发酵液中不同时间点pH和产气量的变化。A:pH;B:产气量。所有数值均为平均值±SEM,进行单因素方差分析,邓肯检验的多重比较。标识不同字母的表示组间差异显著(P<0.05),下同。, figureFileSmall=YMdTJ4i4Gzuz/N9GaMgyVg==, figureFileBig=U0YXUU5YW94g6hNyRcHpqQ==, tableContent=null), ArticleFig(id=1226964059845735401, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956556521096082, language=EN, label=Figure 2, caption=Changes in SCFAs and lactic acid in colonic microbial fermentation broth at different time points. A: Acetate; B: Propionate; C: Butyrate; D: Total SCFAs; E: Lactic acid., figureFileSmall=tnzy+XOrdYQ5m50aRUDXMg==, figureFileBig=gPoR4EIryi58pPg1KJPyAA==, tableContent=null), ArticleFig(id=1226964059954787315, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956556521096082, language=CN, label=图2, caption=不同时间点结肠微生物发酵液SCFAs和乳酸的变化。A:乙酸;B:丙酸;C:丁酸;D:总短链脂肪酸;E:乳酸。, figureFileSmall=tnzy+XOrdYQ5m50aRUDXMg==, figureFileBig=gPoR4EIryi58pPg1KJPyAA==, tableContent=null), ArticleFig(id=1226964060076422135, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956556521096082, language=EN, label=Figure 3, caption=Alpha diversity of colonic microbiota in SIA and 6′-SL groups at different fermentation time points. A: Chao1 index; B: ACE index; C: Shannon index; D: Simpson index. BLA group represented 0, 6, 12, and 24 h in the NCB group, 0 h in the LAC group, 0 h in the SIA group, and 0 h in the 6′-SL group; LAC-6, LAC-12, LAC-24 represent LAC group at 6 h, 12 h, and 24 h, respectively; SIA-6, SIA-12, SIA-24 represent the SIA group at 6 h, 12 h, and 24 h, respectively; 6′-SL-6, 6′-SL-12, 6′-SL-24 represent the 6′-SL group at 6 h, 12 h, and 24 h, respectively. The same below., figureFileSmall=3/bWIH/Dv4QaSumL0mE4Ig==, figureFileBig=NyvJkYqa8yMM8Ffw6sjAKQ==, tableContent=null), ArticleFig(id=1226964060214834175, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956556521096082, language=CN, label=图3, caption=不同发酵时间点SIA6′-SL组结肠微生物α多样性。A:Chao1指数;B:ACE指数;C:Shannon指数;D:Simpson指数。BLA组代表NCB组0、6、12和24 h、LAC组0 h、SIA组0 h及6′-SL组0 h;LAC-6、LAC-12、LAC-24分别代表LAC组6、12和24 h;SIA-6、SIA-12、SIA-24分别代表SIA组6、12和24 h;6′-SL-6、6′-SL-12、6′-SL-24分别代表6′-SL组6、12和24 h,下同。, figureFileSmall=3/bWIH/Dv4QaSumL0mE4Ig==, figureFileBig=NyvJkYqa8yMM8Ffw6sjAKQ==, tableContent=null), ArticleFig(id=1226964060294524932, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956556521096082, language=EN, label=Figure 4, caption=Effects of SIA and 6′-SL on beta diversity of colonic microbiota. A: Beta diversity of microbiota at 0 h; B: Beta diversity of microbiota at 6 h; C: Beta diversity of microbiota at 12 h; D: Beta diversity of microbiota at 24 h., figureFileSmall=fOaX7vHXloDjqVwG2YXcKw==, figureFileBig=4NS4oYoyoAs9MLiJIf8zQQ==, tableContent=null), ArticleFig(id=1226964060453908497, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956556521096082, language=CN, label=图4, caption=SIA6′-SL对结肠微生物β多样性的影响。A:0 h菌群β多样性;B:6 h菌群β多样性;C:12 h菌群β多样性;D:24 h菌群β多样性。, figureFileSmall=fOaX7vHXloDjqVwG2YXcKw==, figureFileBig=4NS4oYoyoAs9MLiJIf8zQQ==, tableContent=null), ArticleFig(id=1226964060579737629, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956556521096082, language=EN, label=Figure 5, caption=Differences in relative abundance at the phylum level between SIA and 6′-SL groups at different fermentation time points. A: Top 5 dominant phyla in colonic chyme fermentation broth at different fermentation time points; B: Relative abundance of Bacillota; C: Relative abundance of Bacteroidota; D: Relative abundance of Pseudomonadota; E: Relative abundance of Desulfobacterota., figureFileSmall=2ljxgD7ZM6m4uo61e3S/YQ==, figureFileBig=5TK23lEKqOJcp647aDo06g==, tableContent=null), ArticleFig(id=1226964060705566760, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956556521096082, language=CN, label=图5, caption=不同发酵时间点SIA6′-SL组微生物门水平相对丰度的差异。A:不同发酵时间点结肠食糜发酵液中的top 5优势门;B:Bacillota的相对丰度;C:Bacteroidota的相对丰度;D:Pseudomonadota的相对丰度;E:Desulfobacterota的相对丰度。, figureFileSmall=2ljxgD7ZM6m4uo61e3S/YQ==, figureFileBig=5TK23lEKqOJcp647aDo06g==, tableContent=null), ArticleFig(id=1226964060818812976, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956556521096082, language=EN, label=Figure 6, caption=Differences in relative abundance at the genus level between SIA and 6′-SL groups at different fermentation time points. A: Top 15 dominant genera in colonic chyme fermentation broth at different fermentation time points; B: Relative abundance of Anaerovibrio; C: Relative abundance of Megasphaera; D: Relative abundance of Prevotella; E: Relative abundance of Bacteroides; F: Relative abundance of Prevotella_9; G: Relative abundance of Succinivibrio; H: Relative abundance of Escherichia-Shigella; I: Relative abundance of Roseburia; J: Relative abundance of Phascolarctobacterium; K: Relative abundance of Mitsuokella; L: Relative abundance of Streptococcus; M: Relative abundance of Prevotellaceae NK3B31 group; N: Relative abundance of Faecalibacterium; O: Relative abundance of Alloprevotella; P: Relative abundance of UCG-005. The relative abundance of any one group is greater than 1%., figureFileSmall=vNX8Or1IFgW5CRELrwaXjw==, figureFileBig=l4P78JNs/8iIA+V6oD/4eg==, tableContent=null), ArticleFig(id=1226964060990779451, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956556521096082, language=CN, label=图6, caption=不同发酵时间点SIA6′-SL组微生物属水平相对丰度的差异。A:不同发酵时间点结肠食糜发酵液中的top 15优势属;B:Anaerovibrio的相对丰度;C:Megasphaera的相对丰度;D:Prevotella的相对丰度;E:Bacteroides的相对丰度;F:Prevotella_9的相对丰度;G:Succinivibrio的相对丰度;H:Escherichia-Shigella的相对丰度;I:Roseburia的相对丰度;J:Phascolarctobacterium的相对丰度;K:Mitsuokella的相对丰度;L:Streptococcus的相对丰度;M:Prevotellaceae NK3B31 group的相对丰度;N:Faecalibacterium的相对丰度;O:Alloprevotella的相对丰度;P:UCG-005的相对丰度。任意一组的相对丰度大于1%。, figureFileSmall=vNX8Or1IFgW5CRELrwaXjw==, figureFileBig=l4P78JNs/8iIA+V6oD/4eg==, tableContent=null), ArticleFig(id=1226964061116608580, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956556521096082, language=EN, label=Figure 7, caption=Correlation 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唾液酸和6′-唾液酸乳糖对仔猪肠道微生物体外发酵特性的影响
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刘晓英 , 李轩 , 吴海琴 , 许来鹏 , 赵德辉 , 马季祥 , 慕春龙 , 朱伟云 , 潘龙
微生物学报 | 研究报告 2025,65(9): 4075-4087
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微生物学报 | 研究报告 2025, 65(9): 4075-4087
唾液酸和6′-唾液酸乳糖对仔猪肠道微生物体外发酵特性的影响
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刘晓英, 李轩, 吴海琴, 许来鹏, 赵德辉, 马季祥, 慕春龙, 朱伟云, 潘龙
作者信息
  • 南京农业大学 动物科技学院,消化道微生物研究室,江苏省消化道营养与动物健康重点实验室,动物消化道营养国际联合研究中心,江苏 南京
Effects of sialic acid and 6′-sialyllactose on in vitro fermentation characteristics of gut microbiota in weaned piglets
Xiaoying LIU, Xuan LI, Haiqin WU, Laipeng XU, Dehui ZHAO, Jixiang MA, Chunlong MU, Weiyun ZHU, Long PAN
Affiliations
  • Laboratory of Gastrointestinal Microbiology, Jiangsu Key Laboratory of Gastrointestinal Nutrition and Animal Health, National Center for International Research on Animal Gut Nutrition, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu, China
出版时间: 2025-09-04 doi: 10.13343/j.cnki.wsxb.20250153
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6′-唾液酸乳糖(6′-sialyllactose, 6′-SL)是猪母乳寡糖中的重要组分,唾液酸(sialic acid, SIA)是唾液酸化乳寡糖的单体,它们均可调控人的结肠微生物结构。目前关于SIA和6′-SL在体外对仔猪结肠微生物的组成和发酵特性的影响鲜有报道。 【目的】 探究SIA和6′-SL对哺乳仔猪结肠微生物组成及发酵特性的影响,为SIA和6′-SL调控哺乳仔猪肠道健康提供参考。 【方法】 以3只哺乳仔猪的结肠混合食糜为微生物接种物,探究SIA和6′-SL对哺乳仔猪结肠微生物组成及发酵特性的影响。试验分为4组:无碳源(no carbon, NCB)、乳糖(lactose, LAC)、SIA和6′-SL,各组均为4个重复。 【结果】 发酵至24 h时,6′-SL组的pH显著高于LAC组(P<0.05);发酵至12 h和24 h时,SIA和6′-SL组的产气量均显著低于LAC组(P<0.05);发酵至24 h时,SIA和6′-SL组的乙酸含量均显著高于LAC组(P<0.05),丙酸和丁酸含量均显著低于LAC组(P<0.05),显著提高了Chao1指数和ACE指数(P<0.05);LAC组、SIA组和6′-SL组菌群的β多样性发生显著改变(P<0.05);随着发酵时间延长,与LAC组相比,SIA组显著提高了芽孢杆菌门(Bacillota)、厌氧弧菌属(Anaerovibrio)和普氏栖粪杆菌属(Faecalibacterium)的相对丰度,降低了假单胞菌门(Pseudomonadota)、埃希氏菌-志贺氏菌(Escherichia-Shigella)、光冈菌属(Mitsuokella)和链球菌属(Streptococcus)的相对丰度(P<0.05);6′-SL组显著提高了拟杆菌门(Bacteroidetes)普雷沃氏菌属Prevotella、拟杆菌属Bacteroides和罗氏菌属Roseburia,降低了假单胞菌门、芽孢杆菌门、巨单胞菌属(Megamonas)、埃希氏菌-志贺氏菌、光冈菌属和普雷沃氏菌科NK3B31组(Prevotellaceae_NK3B31_group)的相对丰度(P<0.05)。 【结论】 6′-SL和SIA均可有效调控仔猪肠道菌群结构,6′-SL能提高拟杆菌门以及普雷沃氏菌属和拟杆菌属的相对丰度,而SIA能提高芽孢杆菌门以及厌氧弧菌属的相对丰度,且2种底物都明显促进仔猪结肠微生物发酵,提高短链脂肪酸(short chain fatty acids, SCFAs)产量,尤其是乙酸。

唾液酸  /  6′-唾液酸乳糖  /  体外发酵  /  肠道微生物

6′-sialyllactose (6′-SL) is an important component of porcine breast milk oligosaccharides, and sialic acid (SIA) is the monomer of sialylated breast milk oligosaccharides. Both can regulate the structure of human colonic microbiota. However, there are few reports regarding the effects of SIA and 6′-SL on the composition and fermentation characteristics of piglet colonic microbiota in vitro. [Objective] To investigate the effects of SIA and 6′-SL on the composition and fermentation characteristics of colonic microbiota in suckling piglets, aiming to provide a reference for using these substances to regulate gut health in piglets. [Methods] We used mixed colonic chyme from three suckling piglets as the microbial inoculum. The experiment was conducted with four groups: no carbon (NCB), lactose (LAC), SIA, and 6′-SL, each with four replicates. [Results] After 24 h of fermentation, the pH in the 6′-SL group was higher than that in the LAC group (P<0.05). After fermentation for 12 h and 24 h, the gas production in the SIA and 6′-SL groups was lower than that in the LAC group (P<0.05). At the time point of 24 h, the SIA and 6′-SL groups had higher acetic acid content and lower propionic acid and butyric acid content than the LAC group (P<0.05). Moreover, the SIA and 6′-SL groups had higher Chao1 and ACE indices than the LAC group (P<0.05). Beta diversity of the microbial community in the LAC, SIA, and 6′-SL groups changed (P<0.05). As fermentation progressed, compared with LAC, SIA increased the relative abundance of Bacillota, Anaerovibrio, and Faecalibacterium and decreased that of Pseudomonadota, Escherichia-Shigella, Mitsuokella, and Streptococcus (P<0.05); 6′-SL increased the relative abundance of Bacteroidetes, Prevotella, Bacteroides, and Roseburia and decreased that of Pseudomonadota, Bacillota, Megamonas, Escherichia-Shigella, Mitsuokella, and Prevotellaceae_NK3B31_group (P<0.05). [Conclusion] Both 6′-SL and SIA can effectively regulate the gut microbiota structure in piglets. Specifically, 6′-SL increases the relative abundance of Bacteroidetes, Prevotella, and Bacteroides, while SIA increases that of Bacillota and Anaerovibrio. Both substances promote colonic microbial fermentation in piglets to increase the production of short-chain fatty acids (SCFAs), especially acetic acid.

sialic acid  /  6′-sialyllactose  /  in vitro fermentation  /  gut microbiota
刘晓英, 李轩, 吴海琴, 许来鹏, 赵德辉, 马季祥, 慕春龙, 朱伟云, 潘龙. 唾液酸和6′-唾液酸乳糖对仔猪肠道微生物体外发酵特性的影响. 微生物学报, 2025 , 65 (9) : 4075 -4087 . DOI: 10.13343/j.cnki.wsxb.20250153
Xiaoying LIU, Xuan LI, Haiqin WU, Laipeng XU, Dehui ZHAO, Jixiang MA, Chunlong MU, Weiyun ZHU, Long PAN. Effects of sialic acid and 6′-sialyllactose on in vitro fermentation characteristics of gut microbiota in weaned piglets[J]. Acta Microbiologica Sinica, 2025 , 65 (9) : 4075 -4087 . DOI: 10.13343/j.cnki.wsxb.20250153
猪乳中含有仔猪生长发育所必需的多种有效成分,包括碳水化合物、蛋白质、脂质、免疫细胞和生物活性因子等[1-2]。猪乳寡糖是母乳中第三大固体成分,一般由3-10个单糖通过糖苷键连接而成,组成母乳寡糖的单糖主要有d-葡萄糖、d-半乳糖、N-乙酰氨基葡萄糖、l-岩藻糖和唾液酸等[3-4]。近年来,母乳寡糖作为益生元的作用备受关注,研究表明猪母乳寡糖可调控仔猪肠道微生物组成、增强黏膜免疫系统以及维护肠道上皮屏障功能[5]
唾液酸(sialic acid, SIA)是一种包含N-乙酰神经氨酸(N-Acetylneuraminic acid, NeuAc)在内的九碳酸性单糖的总称,具有多种生物学功能,如神经传递、抗病毒活性、促进肠道健康和营养吸收以及加速大脑的认知发展[6-7]。6′-唾液酸乳糖(6′-sialyllactose, 6′-SL)是母乳寡糖的重要组分,约占母乳寡糖的5%,由NeuAc通过α-(2,6)糖苷键连接到乳糖上[8-9]。SIA和6′-SL作为益生元调节肠道微生物群的作用备受关注。在小鼠研究中口服SIA改变了结肠微生物组成,增加了拟杆菌门和芽孢杆菌门的丰度等[10]。使用仿生胃肠道反应器研究SIA对健康人体肠道微生物群落组成和代谢物的影响中发现,SIA改变了结肠肠道菌群和代谢物组成[11]。此外,在小鼠的饮食中加入益生元6′-SL会影响应激源诱导的结肠微生物群落组成的变化并维持正常的微生物群落结构[12]。在婴儿的体外结肠模型中发现,6′-SL促进了婴儿粪便中双歧杆菌的生成[13]。在配方奶粉中补充6′-SL可以调节新生仔猪的结肠微生物群[14]。这些结果说明SIA和6′-SL可以抵抗消化并到达结肠供肠道微生物群使用,进而调节肠道微生物的组成结构。然而,目前关于SIA和6′-SL对哺乳仔猪结肠微生物及其代谢的影响还鲜有报道。
本研究以乳糖(lactose, LAC)作为对照,采用体外发酵的方法探究SIA和6′-SL对哺乳仔猪结肠微生物组成及发酵参数的影响,以期为SIA和6′-SL调控哺乳仔猪肠道健康提供参考。
乳糖,上海麦克林生化科技股份有限公司;唾液酸,上海源叶生物科技有限公司;6′-唾液酸乳糖,上海毕得医药科技有限公司;乳酸测试盒,南京建成生物工程研究所。
气相色谱仪,岛津公司。
以10 g/L的LAC、SIA或6′-SL为唯一碳源,浓度参考叶艳新[15]、Katoh等[16]和Lou等[17]。基础培养基的配方参考Williams等[18]和叶艳新[15],同时参考Li等[19]去掉了配方中的脂肪酸溶液。具体操作步骤如下:(1) 将配制好的培养基在微波炉中加热至沸腾(多次沸腾),通入CO2至常温;(2) 分装前加入1 g半胱氨酸盐酸盐,并用0.2 mol/L HCl和0.2 mol/L NaOH溶液调整pH值至6.8左右;(3) 按培养基和菌液9:1的比例将5.4 mL的基础培养基分装至血清瓶中,盖好橡胶塞并打好铝盖;(4) 120 ℃灭菌15 min;(5) 灭菌完成后将培养基放置于常温,然后用注射器向每瓶血清瓶中注射0.06 mL维他命磷酸盐溶液(过夜)。
将3只哺乳仔猪屠宰后,将肠道食糜置于厌氧自封袋中,放在冰上,快速运回实验室。收集3只哺乳仔猪结肠内容物并充分混匀,用无菌的PBS以9:1的比例稀释。然后参照叶艳新[15]的方法,经四层无菌纱布过滤制备接种物。用注射器吸取0.6 mL接种物,注入用37 ℃摇床提前预热的装有培养基的血清瓶中,全程通入CO2以保持厌氧环境。接种完成后,使用注射器和气压转换器将血清瓶内气压调整至0,然后将样品置于37 ℃恒温振荡器中发酵。本研究所有动物实验获得南京农业大学机构动物护理和使用委员会批准,编号为NJAU.No20240401N06。
在厌氧培养基中分别添加10 g/L的LAC、SIA和6′-SL作为唯一碳源,然后分别接种上述制备的结肠食糜接种物,每组4个重复。试验共有4个处理组:无碳源(no carbon, NCB)、LAC、SIA和6′-SL组。接种后将发酵瓶在37 ℃、100 r/min培养24 h。
样品发酵至0、6、12和24 h时,将需要采样的瓶子取出放置于冰盒上,用注射器及气压转换器测定产气量。随后将样品摇匀,每个时间点的发酵瓶均取3 mL发酵液,用于短链脂肪酸(short chain fatty acids, SCFAs)、乳酸的检测以及发酵液DNA的提取;剩余样品用于测pH值,并装于15 mL无酶离心管中备用。采集完毕后将发酵液置于-20 ℃冰箱保存。
参照Zhang等[20]的方法,使用气相色谱仪测定和计算SCFAs浓度。详细步骤如下:取1 mL发酵液样品,加入200 μL 25%的偏磷酸/巴豆酸混匀,于-20 ℃冰箱过夜。测定前室温解冻,12 000 r/min离心10 min,取上清液用0.22 μm的无菌滤膜过滤。用1 μL微量进样器取0.6 μL过滤液上机(气相色谱仪SPL1、色谱柱和FID1温度分别设置为210、140和210 ℃;测定完成后将SPL1、色谱柱和FID1温度均设置为50 ℃进行降温)进行测定。
发酵液细菌总DNA提取采用粪便基因组DNA提取试剂盒进行提取。使用上游引物341F (5′-CCTAYGGGRBGCASCAG-3′)和下游引物806R (5′-GGACTACNNGGGTATCTAAT-3′)扩增细菌16S rRNA基因的V3-V4区。PCR反应由上海凌恩生物科技有限公司完成。扩增纯化结束后,在PE250平台进行测序。得到原始数据后进行筛选和质量控制,以获得高质量的功能分类单元。原始数据已上传至NCBI (登录号:PRJNA1229144)。
差异菌属和短链脂肪酸数据的相关性分析在生科云平台完成(www.bioincloud.tech)。其余数据经Excel计算整理后,使用SPSS 26.0软件进行统计分析。同一时间点内不同处理采用单因素方差分析,邓肯法进行多重比较,使用GraphPad Prism 9进行绘图。P<0.05表示差异显著。
发酵液pH用于指示发酵环境中的产酸情况。在哺乳仔猪结肠微生物发酵过程中,NCB、LAC、SIA和6′-SL组的pH值在6 h时无显著差异;发酵至12 h时,SIA和6′-SL组的pH值无显著差异,均显著高于LAC组(P<0.05);发酵至24 h时,6′-SL组的pH值显著高于LAC组且显著低于NCB组(P<0.05),而6′-SL与SIA组无显著差异(图1A)。产气是体外发酵过程的主要特征之一。发酵至12 h和24 h时,SIA和6′-SL组的产气量无显著差异,均显著高于NCB组且显著低于LAC组(P<0.05) (图1B)。
发酵至12 h时,SIA和LAC组的乙酸含量无显著差异,均显著低于6′-SL组且显著高于NCB组(P<0.05),发酵至24 h时,SIA和6′-SL组的乙酸含量无显著差异,均显著高于LAC和NCB组(P<0.05) (图2A)。发酵至12 h时,SIA和NCB组的丙酸含量无显著差异,均显著低于LAC和6′-SL组(P<0.05);发酵至24 h时,SIA和6′-SL组的丙酸和丁酸含量无显著差异,均显著低于LAC组且高于NCB组(P<0.05) (图2B2C)。发酵至12 h时,SIA组的总短链脂肪酸含量显著低于LAC组和6′-SL组且显著高于NCB组(P<0.05),LAC和6′-SL组的总短链脂肪酸含量无显著差异;发酵至24 h时,LAC、SIA和6′-SL组的总短链脂肪酸含量无显著差异,均显著高于NCB组(P<0.05) (图2D)。发酵至6 h时,6′-SL组的乳酸含量显著低于LAC组且显著高于SIA和NCB组,发酵至12 h时,LAC组的乳酸含量显著高于其他3组(P<0.05);发酵至24 h时,4组的乳酸含量无显著差异(图2E)。
发酵液中菌群Chao1、ACE、Shannon和Simpson指数随发酵时间发生的变化如图3所示。样品发酵至6 h时,与LAC和SIA组相比,6′-SL组显著提高了Shannon指数(P<0.05);样品发酵至24 h时,与LAC组相比,SIA组和6′-SL组显著提高了Chao1指数和ACE指数(P<0.05),且SIA组的Chao1指数和ACE指数均显著低于6′-SL组(P<0.05);其他时间点各组之间的菌群α多样性均无显著差异。这些结果说明SIA和6′-SL改变了结肠微生物的结构。
SIA和6′-SL对哺乳仔猪结肠微生物发酵的β多样性分析结果如图4所示。随着发酵时间的延长,LAC、SIA和6′-SL组菌群的β多样性发生了显著改变,且SIA组的菌群组成与其他两组差异明显(P<0.05)。
发酵0、6、12和24 h时各组的菌群门水平相对丰度如图5所示。在门水平上,结肠食糜发酵液微生物主要由芽孢杆菌门(Bacillota)、拟杆菌门(Bacteroidota)、梭杆菌门(Fusobacteriota)、假单胞菌门(Pseudomonadota)和脱硫弧菌门(Desulfobacterota)组成。在0 h时,各组的最优势菌门均为芽孢杆菌门,其次为拟杆菌门。在6、12和24 h时,LAC和SIA组的最优势菌门均为芽孢杆菌门,其次为拟杆菌门。在6 h和12 h,6′-SL组的最优势菌门为芽孢杆菌门,其次为拟杆菌门,而在24 h时,6′-SL组的最优势菌门为拟杆菌门,其次为芽孢杆菌门。随着发酵时间的延长,与LAC相比SIA组显著提高了芽孢杆菌门的相对丰度(P<0.05),而6′-SL组在发酵24 h时显著降低了芽孢杆菌门的相对丰度(P<0.05)。此外,与LAC相比,6′-SL组显著提高了拟杆菌门的相对丰度(P<0.05),SIA组和6′-SL组均显著降低了假单胞菌门的相对丰度(P<0.05),各组脱硫弧菌门的相对丰度均在不断减少。
图6所示,在0 h各组的最优势菌属均为普雷沃氏菌属(Prevotella)。随着发酵时间的延长,与LAC相比,SIA组显著增加了厌氧弧菌属(Anaerovibrio)、普氏栖粪杆菌属(Faecalibacterium)和拟普雷沃氏菌属(Alloprevotella)的相对丰度,降低了巨球型菌属(Megasphaera)、琥珀酸弧菌属(Succinivibrio)、埃希氏菌-志贺氏菌(Escherichia- Shigella)、光冈菌属(Mitsuokella)和链球菌属(Streptococcus)的相对丰度(P<0.05);6′-SL组显著增加了普雷沃氏菌属(Prevotella)、拟杆菌属(Bacteroides)和普雷沃氏菌属(Prevotella_9)、罗氏菌属(Roseburia)、考拉杆菌属(Phascolarctobacterium)和UGG-005的相对丰度,降低了巨单胞菌属(Megasphaera)、埃希氏菌-志贺氏菌(Escherichia-Shigella)、光冈菌属(Mitsuokella)、普雷沃氏菌科NK3B31组(Prevotellaceae NK3B31 group)的相对丰度(P<0.05)。
图7所示,链球菌属与丙酸和丁酸含量呈现显著正相关(P<0.05),普雷沃氏菌科NK3B31组、UGG-005、拟普雷沃氏菌属和考拉杆菌属与乙酸、丙酸和丁酸含量呈现显著负相关(P<0.05),普氏栖粪杆菌属和普雷沃氏菌属与丙酸和丁酸含量呈现显著负相关(P<0.05),埃希氏菌-志贺氏菌和光冈菌属与乙酸含量呈现显著负相关(P<0.05)。
6′-SL作为猪乳寡糖中的重要组分,可促进仔猪结肠有益菌的定植,改善肠道健康[14]。然而,目前尚不清楚6′-SL及其重要组成成分SIA如何影响仔猪肠道微生物的组成和结构以及代谢功能。本研究使用体外发酵模型,以LAC作为对照,探究SIA和6′-SL对哺乳仔猪结肠中微生物群落和代谢物的影响,为猪乳寡糖调控仔猪肠道健康提供理论依据。
产气量是评价细菌发酵底物效率的指标之一。本研究发现,NCB组的气体和SCFAs含量未明显增加,而其他各组的气体和总短链脂肪酸含量随着发酵时间的进行明显增加,表明纤维或碳水化合物是驱动发酵的主要底物,碳源类型的差异可能是影响发酵特性的主要因素[21]。随着发酵时间的延长,SIA和6′-SL组的产气量无显著差异,均显著低于LAC组,表明由于几种碳源的化学成分和结构不同,SIA和6′-SL不易被细菌利用。
SCFAs和乳酸是肠道菌群的重要代谢产物,在维持肠道稳态、免疫调节和能量供应方面发挥着不可替代的作用[11]。本研究中随着发酵时间的延长,LAC、SIA和6′-SL组的总短链脂肪酸含量明显增加,而pH明显降低。研究表明后肠发酵过程中的总短链脂肪酸含量与产气量呈正相关,而与pH呈负相关[22],这与本研究相一致。此外,研究表明SIA体外发酵成人粪便以及6′-SL体外发酵婴儿粪便的主要有机酸代谢产物是乙酸,其次是丙酸和丁酸[11,23]。本研究发现,发酵至24 h时LAC、SIA和6′-SL组的主要有机酸代谢产物是乙酸,其次是丙酸和丁酸,这与其他研究相一致,表明哺乳仔猪结肠微生物的乙酸代谢途径非常活跃。据报道,乙酸和丁酸是通过醛单糖(如葡萄糖、半乳糖和甘露糖)的发酵产生的,而丙酸主要是通过酮单糖(如果糖和阿拉伯糖)的发酵产生的[24]。发酵至24 h时,SIA和6′-SL组的乙酸含量显著高于LAC组,而丙酸和丁酸含量显著低于LAC组,这可归因于LAC、SIA和6′-SL的单糖成分组成不同。本研究发现SIA和6′-SL组的乳酸含量在整个发酵期间都比较低。乳酸作为中间体,可不断被微生物转化为丙酸和丁酸。因此SIA和6′-SL组丙酸和丁酸的产生可分别归因于乳酸生成细菌和乳酸利用细菌之间交叉摄食的乳酸转化[25]。这些结果表明,SIA和6′-SL可以像其他益生元一样改善肠道健康。
本研究发现SIA和6′-SL改变了发酵液的菌群组成和结构。本研究中,与LAC相比SIA组显著提高了芽孢杆菌门以及降低了假单胞菌门的相对丰度,这可能与普氏栖粪杆菌属的显著增加和埃希氏菌-志贺氏菌的显著降低有关。普氏栖粪杆菌属是肠道中的共生菌,作为益生菌发挥作用[26]。埃希氏菌-志贺氏菌属是条件致病菌,被认为是诱发仔猪腹泻的主要因素之一[27]。这说明SIA可能具有促进肠道中有益菌的增殖以及抑制有害细菌繁殖的潜力。此外,研究表明SIA可以显著提高芽孢杆菌门以及降低假单胞菌门的丰度[28],这与本研究结果相一致。厌氧弧菌属是肠道中常见的有益细菌,它可以发酵多糖并产生SCFAs,是在肠道中产生乙酸和丙酸的细菌的重要成员[29]。本研究发现SIA组显著增加了厌氧弧菌属的相对丰度,这提示厌氧弧菌属具有代谢SIA产生乙酸和丙酸的潜力,但是具体代谢机制还有待研究。链球菌能够发酵碳水化合物,以乳酸作为主要的末端代谢产物[30]。本研究发现SIA显著降低了链球菌属的相对丰度,推测SIA组中乳酸含量较低可能是与链球菌属的减少有关。此外,研究表明SIA抑制链球菌属的生长并促进猪肠道中有益细菌的生长,这与本研究相一致[31]。6′-SL组显著提高了拟杆菌门但降低了芽孢杆菌门的相对丰度,这可能取决于普雷沃氏菌属和拟杆菌属的增加以及巨单胞菌属和光冈菌属的降低。普雷沃氏菌属和拟杆菌属是肠道中主要的复合多糖降解剂,可释放各种碳水化合物活性酶来降解6′-SL,这些细菌也被认为是肠道中丙酸主要的生产者[32]。这提示普雷沃氏菌属和拟杆菌属具有代谢6′-SL产生丙酸的潜力。有研究发现在配方奶粉中补充6′-SL能增加仔猪结肠中普雷沃氏菌属和拟杆菌属的丰度,从而改善肠道健康[14],这与本研究结果相一致。巨单胞菌属可以发酵各种碳水化合物产生乳酸[33]。这说明6′-SL组中乳酸含量较低可能与该细菌的减少有关。本研究发现光冈菌属与乙酸含量呈显著负相关,说明SIA和6′-SL组中乙酸的产生可能与该菌属的减少有关。罗氏菌属是丁酸的主要生产者[34]。本研究中6′-SL组显著增加了罗氏菌属的相对丰度,提示罗氏菌属具有代谢6′-SL产生丁酸的潜力。6′-SL组可以显著降低埃希氏菌-志贺氏菌的相对丰度,且埃希氏菌-志贺氏菌属与乙酸含量呈显著负相关,这说明6′-SL可能抑制肠道中潜在致病菌的定殖以及乙酸的产生可能与该菌属的减少有关。总之,SIA和6′-SL可以通过不同方式调节肠道菌群的群落组成,抑制有害病原体的生长,促进不同种类益生菌的增殖,改善肠道健康。
综上所述,在体外发酵模型中,SIA和6′-SL对哺乳仔猪结肠微生物群落的组成和结构的影响存在显著差异。然而,由于肠道微生物群的多样性和复杂性,微生物对SIA和6′-SL的利用机制仍不清楚,后续可深入结合宏基因组、代谢组学以及交互喂养等试验进一步探究特定细菌对SIA和6′-SL的降解机制。
本研究发现6′-SL和SIA均可有效调控仔猪肠道菌群结构,6′-SL能提高拟杆菌门以及普雷沃氏菌属和拟杆菌属的相对丰度,而SIA能提高芽孢杆菌门以及厌氧弧菌属的相对丰度,且2种底物都明显促进仔猪结肠微生物发酵,提高SCFAs产量,尤其是乙酸产量。
  • 国家重点研发计划(2022YFD1300403)
  • 国家自然科学基金(31902166)
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2025年第65卷第9期
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doi: 10.13343/j.cnki.wsxb.20250153
  • 接收时间:2025-02-27
  • 首发时间:2026-02-07
  • 出版时间:2025-09-04
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  • 收稿日期:2025-02-27
  • 录用日期:2025-03-28
基金
National Key Research and Development Program of China(2022YFD1300403)
国家重点研发计划(2022YFD1300403)
National Natural Science Foundation of China(31902166)
国家自然科学基金(31902166)
作者信息
    南京农业大学 动物科技学院,消化道微生物研究室,江苏省消化道营养与动物健康重点实验室,动物消化道营养国际联合研究中心,江苏 南京
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https://castjournals.cast.org.cn/joweb/wswxb/CN/10.13343/j.cnki.wsxb.20250153
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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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