Article(id=1156967371794632821, tenantId=1146029695717560320, journalId=1146119944283992078, issueId=1156967360193192910, articleNumber=null, orderNo=null, doi=null, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=null, receivedDateStr=null, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1753772116850, onlineDateStr=2025-07-29, pubDate=null, pubDateStr=null, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1753772116850, onlineIssueDateStr=2025-07-29, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1753772116850, creator=13701087609, updateTime=1753772116850, updator=13701087609, issue=Issue{id=1156967360193192910, tenantId=1146029695717560320, journalId=1146119944283992078, year='2024', volume='2', issue='8', pageStart='1', pageEnd='160', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=0, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1753772114084, creator=13701087609, updateTime=1753778008087, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1156992081479689064, tenantId=1146029695717560320, journalId=1146119944283992078, issueId=1156967360193192910, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1156992081479689065, tenantId=1146029695717560320, journalId=1146119944283992078, issueId=1156967360193192910, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=140, endPage=145, ext={EN=ArticleExt(id=1156967372780294274, articleId=1156967371794632821, tenantId=1146029695717560320, journalId=1146119944283992078, language=EN, title=Transformation issue in using GB 5009.86 high performance liquid chromatography method to determine vitamin B3 for milk powder containing Lactic acid bacteria, columnId=1156641066674676444, journalTitle=Laboratory Testing, columnName=Evaluation and Analysis, runingTitle=null, highlight=null, articleAbstract=

Objective Study why nicotinamide is transformed tonicotinic acid occurred when detect vitamin ${\mathrm{B}}_{3}$ in milk powdercontaining lactic acid bacteria by GB 5009.89-2016 high performanceliquid chromatography method, and its mechanism was discussed. MethodsThe samples were pretreated according to standard procedures, and theeffects of the placement time of the sample extraction solution, theplacement time of the sample solution before acid hydrolysis and the $\mathrm{{pH}}$ value of acidhydrolysis were investigated by single factor test. Results Thetransformation of nicotinamide to nicotinic acid occurred in theplacement step after the sample was dissolved and before acidhydrolysis, but after adjusting the pH value to 1.7 specified in thestandard for acid hydrolysis, the transformation was completelyinhibited. Conclusion If you want to detect the content of nicotinicacid or nicotinamide separately in samples containing lactic acidbacteria, you need to adjust the $\mathrm{{pH}}$ value to 1.7 when thesample is dissolved to inhibit the transformation of nicotinamide tonicotinic acid.

, correspAuthors=Zhi-Wen LEI, authorNote=null, correspAuthorsNote=
*LEI Zhi-Wen, Researcher, Qingdao Customs Technology Center, Qingdao 266109, China. E-mail:
, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Yong JIANG, Yuan-Jiao GAO, Xiu-Yu ZHANG, Feng-Yan ZHANG, Lin-Lin JIN, Man-Xia WANG, Fei HE, Zhi-Wen LEI), CN=ArticleExt(id=1156967429613114209, articleId=1156967371794632821, tenantId=1146029695717560320, journalId=1146119944283992078, language=CN, title=GB5009.89 高效液相色谱法检测含乳酸菌奶粉中 维生素 ${\mathbf{B}}_{3}$ 的问题研究, columnId=1152194691782505406, journalTitle=实验室检测, columnName=评价与分析, runingTitle=null, highlight=null, articleAbstract=

目的 研究使用 GB 5009.89-2016第二法高效液相色谱法检测含乳酸菌的奶粉中维生素 ${\mathrm{B}}_{3}$ 含量时,发生的烟酰胺转化为烟酸的现象,探讨其机理。方法 样品按照标准方法程序进行前处理,通过单因素实验分别考察了试样测定液静置时间、溶液酸水解前静置时间以及酸水解 $\mathrm{{pH}}$ 值对结果的影响。结果 烟酰胺向烟酸的转化发生在样品溶解后、酸水解之前的静置步骤,但调节 $\mathrm{{pH}}$ 值至标准规定的 1.7进行酸水解以后,转化作用被完全抑制。 结论如果要检测含乳酸菌样品中烟酸或烟酰胺的含量,需要在样品溶解时立即将 $\mathrm{{pH}}$ 值调整到1.7,以抑制烟酰胺向烟酸的转化。

, correspAuthors=雷质文, authorNote=null, correspAuthorsNote=
*雷质文,三级研究员,研究方向为食品微生物检测和质量控制。E-mail:
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姜勇,硕士,高级工程师,研究方向为食品检测。

雷质文,三级研究员,研究方向为食品微生物检测和质量控制。

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figureFileBig=eZkD9YjLcvDpIsVfJWCi7A==, tableContent=null), ArticleFig(id=1156967434604336048, tenantId=1146029695717560320, journalId=1146119944283992078, articleId=1156967371794632821, language=CN, label=图 8, caption=酸溶解试制样品 A 的烟酸和烟酰胺检测色谱图, figureFileSmall=kln0lIWETyEmf9FG3ietNQ==, figureFileBig=eZkD9YjLcvDpIsVfJWCi7A==, tableContent=null), ArticleFig(id=1156967434663056305, tenantId=1146029695717560320, journalId=1146119944283992078, articleId=1156967371794632821, language=EN, label=Table 1, caption=Results of nicotinic acid and nicotinamide for trial Sample A and trial Sample B, figureFileSmall=null, figureFileBig=null, tableContent=
样品 烟酸 (μg/100 g) 烟酰胺 (μg/100 g) 维生素 ${\mathrm{B}}_{3}$(μg/100 g)
试制样品 $\mathrm{A}$ 787 3529 4344
试制样品 $\mathrm{B}$ 0 4254 4288
), ArticleFig(id=1156967434717582258, tenantId=1146029695717560320, journalId=1146119944283992078, articleId=1156967371794632821, language=CN, label=表 1, caption=试制样品 A 与试制样品 B 烟酸和烟酰胺检测结果, figureFileSmall=null, figureFileBig=null, tableContent=
样品 烟酸 (μg/100 g) 烟酰胺 (μg/100 g) 维生素 ${\mathrm{B}}_{3}$(μg/100 g)
试制样品 $\mathrm{A}$ 787 3529 4344
试制样品 $\mathrm{B}$ 0 4254 4288
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放置时间 (h) 烟酸 $\left({\mu \mathrm{g}/{100}\mathrm{\;g}}\right)$ 烟酰胺 (μg/100 g) 维生素 ${\mathrm{B}}_{3}$(μg/100 g)
0 787 3529 4344
1 787 3532 4347
2 789 3536 4354
3 785 3539 4352
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放置时间 (h) 烟酸 $\left({\mu \mathrm{g}/{100}\mathrm{\;g}}\right)$ 烟酰胺 (μg/100 g) 维生素 ${\mathrm{B}}_{3}$(μg/100 g)
0 787 3529 4344
1 787 3532 4347
2 789 3536 4354
3 785 3539 4352
), ArticleFig(id=1156967434914714550, tenantId=1146029695717560320, journalId=1146119944283992078, articleId=1156967371794632821, language=EN, label=Table 3, caption=Results of nicotinic acid and nicotinamide after the trial Sample A solution placed for different time, figureFileSmall=null, figureFileBig=null, tableContent=
静置时间 (min) 烟酸 (μg/100 g) 烟酰胺 (μg/100 g) 维生素 ${\mathrm{B}}_{3}$(μg/100 g)
5 950 3303 4279
10 1910 2195 4122
30 2803 1435 4249
60 3617 566 4188
120 4147 0 4147
), ArticleFig(id=1156967434986017719, tenantId=1146029695717560320, journalId=1146119944283992078, articleId=1156967371794632821, language=CN, label=表 3, caption=试制样品 A 溶液静置不同时间后烟酸和烟酰胺检测结果, figureFileSmall=null, figureFileBig=null, tableContent=
静置时间 (min) 烟酸 (μg/100 g) 烟酰胺 (μg/100 g) 维生素 ${\mathrm{B}}_{3}$(μg/100 g)
5 950 3303 4279
10 1910 2195 4122
30 2803 1435 4249
60 3617 566 4188
120 4147 0 4147
), ArticleFig(id=1156967435040543672, tenantId=1146029695717560320, journalId=1146119944283992078, articleId=1156967371794632821, language=EN, label=Table 4, caption=Results of nicotinic acid and nicotinamide for different $\mathrm{{pH}}$ values of acid hydrolysis of trial Sample A, figureFileSmall=null, figureFileBig=null, tableContent=
酸水解 pH 烟酸 $\left({\mu \mathrm{g}/{100}\mathrm{\;g}}\right)$ 烟酰胺 (μg/100 g) 维生素 ${\mathrm{B}}_{3}$(μg/100 g)
1.7 225 3980 4237
3.0 1061 3244 4330
4.0 3665 553 4222
6.8 4073 0 4073
), ArticleFig(id=1156967435107652537, tenantId=1146029695717560320, journalId=1146119944283992078, articleId=1156967371794632821, language=CN, label=表 4, caption=试制样品 A 不同酸水解 pH 值的烟酸和烟酰胺检测结果, figureFileSmall=null, figureFileBig=null, tableContent=
酸水解 pH 烟酸 $\left({\mu \mathrm{g}/{100}\mathrm{\;g}}\right)$ 烟酰胺 (μg/100 g) 维生素 ${\mathrm{B}}_{3}$(μg/100 g)
1.7 225 3980 4237
3.0 1061 3244 4330
4.0 3665 553 4222
6.8 4073 0 4073
), ArticleFig(id=1156967435162178490, tenantId=1146029695717560320, journalId=1146119944283992078, articleId=1156967371794632821, language=EN, label=Table 5, caption=Results of nicotinic acid and nicotinamide after acid-dissolved trial Sample A, figureFileSmall=null, figureFileBig=null, tableContent=
试制样品 A 烟酸 (μg/100 g) 烟酰胺 (μg/100 g) 维生素 ${\mathrm{B}}_{3}$(μg/100 g)
酸溶解 pH1.7 0 4368 4403
), ArticleFig(id=1156967435220898747, tenantId=1146029695717560320, journalId=1146119944283992078, articleId=1156967371794632821, language=CN, label=表 5, caption=酸溶解试制样品 A 的烟酸和烟酰胺检测结果, figureFileSmall=null, figureFileBig=null, tableContent=
试制样品 A 烟酸 (μg/100 g) 烟酰胺 (μg/100 g) 维生素 ${\mathrm{B}}_{3}$(μg/100 g)
酸溶解 pH1.7 0 4368 4403
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GB5009.89 高效液相色谱法检测含乳酸菌奶粉中 维生素 ${\mathbf{B}}_{3}$ 的问题研究
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姜勇 1, 2 , 高元娇 2 , 张秀宇 3 , 张凤艳 4 , 靳琳琳 2 , 王曼霞 2 , 何飞 2 , 雷质文 2, *
实验室检测 | 评价与分析 2024,2(8): 140-145
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实验室检测 | 评价与分析 2024, 2(8): 140-145
GB5009.89 高效液相色谱法检测含乳酸菌奶粉中 维生素 ${\mathbf{B}}_{3}$ 的问题研究
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姜勇1, 2, 高元娇2, 张秀宇3, 张凤艳4, 靳琳琳2, 王曼霞2, 何飞2, 雷质文2, *
作者信息
  • 1 达能特殊营养品(青岛)有限公司 青岛 266113
  • 2 青岛海关技术中心 青岛 266109
  • 3 北京信睿浩扬科技有限公司 北京 100070
  • 4 青岛市食品药品检验研究院 青岛 266071
  • 姜勇,硕士,高级工程师,研究方向为食品检测。

    雷质文,三级研究员,研究方向为食品微生物检测和质量控制。

通讯作者:

*雷质文,三级研究员,研究方向为食品微生物检测和质量控制。E-mail:
Transformation issue in using GB 5009.86 high performance liquid chromatography method to determine vitamin B3 for milk powder containing Lactic acid bacteria
Yong JIANG1, 2, Yuan-Jiao GAO2, Xiu-Yu ZHANG3, Feng-Yan ZHANG4, Lin-Lin JIN2, Man-Xia WANG2, Fei HE2, Zhi-Wen LEI2, *
Affiliations
  • 1 Danone Specialized Nutrition (Qingdao) Co., Ltd. Qingdao 266113 China
  • 2 Qingdao Customs Technology Center Qingdao 266109 China
  • 3 Beijing Xinrui Haoyang Technology Co., Ltd. Beijing 100070 China
  • 4 Qingdao Food and Drug Inspection Institute Qingdao 266071 China
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目的 研究使用 GB 5009.89-2016第二法高效液相色谱法检测含乳酸菌的奶粉中维生素 ${\mathrm{B}}_{3}$ 含量时,发生的烟酰胺转化为烟酸的现象,探讨其机理。方法 样品按照标准方法程序进行前处理,通过单因素实验分别考察了试样测定液静置时间、溶液酸水解前静置时间以及酸水解 $\mathrm{{pH}}$ 值对结果的影响。结果 烟酰胺向烟酸的转化发生在样品溶解后、酸水解之前的静置步骤,但调节 $\mathrm{{pH}}$ 值至标准规定的 1.7进行酸水解以后,转化作用被完全抑制。 结论如果要检测含乳酸菌样品中烟酸或烟酰胺的含量,需要在样品溶解时立即将 $\mathrm{{pH}}$ 值调整到1.7,以抑制烟酰胺向烟酸的转化。

烟酸  /  烟酰胺  /  GB 5009.89  /  乳酸菌

Objective Study why nicotinamide is transformed tonicotinic acid occurred when detect vitamin ${\mathrm{B}}_{3}$ in milk powdercontaining lactic acid bacteria by GB 5009.89-2016 high performanceliquid chromatography method, and its mechanism was discussed. MethodsThe samples were pretreated according to standard procedures, and theeffects of the placement time of the sample extraction solution, theplacement time of the sample solution before acid hydrolysis and the $\mathrm{{pH}}$ value of acidhydrolysis were investigated by single factor test. Results Thetransformation of nicotinamide to nicotinic acid occurred in theplacement step after the sample was dissolved and before acidhydrolysis, but after adjusting the pH value to 1.7 specified in thestandard for acid hydrolysis, the transformation was completelyinhibited. Conclusion If you want to detect the content of nicotinicacid or nicotinamide separately in samples containing lactic acidbacteria, you need to adjust the $\mathrm{{pH}}$ value to 1.7 when thesample is dissolved to inhibit the transformation of nicotinamide tonicotinic acid.

nicotinic acid  /  nicotinamide  /  GB 5009.89  /  Lactic acid bacteria
姜勇, 高元娇, 张秀宇, 张凤艳, 靳琳琳, 王曼霞, 何飞, 雷质文. GB5009.89 高效液相色谱法检测含乳酸菌奶粉中 维生素 ${\mathbf{B}}_{3}$ 的问题研究. 实验室检测, 2024 , 2 (8) : 140 -145 .
Yong JIANG, Yuan-Jiao GAO, Xiu-Yu ZHANG, Feng-Yan ZHANG, Lin-Lin JIN, Man-Xia WANG, Fei HE, Zhi-Wen LEI. Transformation issue in using GB 5009.86 high performance liquid chromatography method to determine vitamin B3 for milk powder containing Lactic acid bacteria[J]. Laboratory Testing, 2024 , 2 (8) : 140 -145 .
维生素 ${\mathrm{B}}_{3}$ (Niacin) 又称维生素 $\mathrm{{PP}}$ 、尼克酸,有烟酸 (Nicotinic acid) 和烟酰胺 (Nicotinamide) 两种形式 [ 1 ] ,它们的结构中都存在吡啶环, 分别与羧基和甲酰胺基相结合 ( 图 1 )。但通常人们把维生素 ${\mathrm{B}}_{3}$ 也称作烟酸,这很容易造成混淆,因此在实际工作中应注意区分。烟酰胺是维生素 ${\mathrm{B}}_{3}$ 最常用的强化形式,具有与烟酸相同的生理活性 [ 2 ] 。正常情况下该类维生素很稳定。
维生素 ${\mathrm{B}}_{3}$ 在食品中广泛存在 [ 3 ] ,对维持人体皮肤、消化和神经系统正常功能起关键作用 [ 4 ] ,对婴幼儿的生长和发育尤为重要 [ 5 ] ,也是许多类别食品中的重要检测指标 [ 6 ]
维生素 ${\mathrm{B}}_{3}$ 的检测方法主要有比色法 [ 7 ] 、微生物法 [ 7 - 8 ] 和高效液相色谱法 [ 9 - 11 ] 。GB 5009.89-2016《食品安全国家标准 食品中烟酸和烟酰胺的测定》 [ 12 ] 中规定了微生物法和高效液相色谱法,其中微生物法检测的是以烟酸为维生素 ${\mathrm{B}}_{3}$ 当量的烟酸和烟酰胺的总和, 但因操作步骤复杂、精密度差、效率低, 应用较为局限 [ 13 ] ; 而高效液相色谱法分别检测烟酸和烟酰胺的含量, 再折算成以烟酸表示的维生素 ${\mathrm{B}}_{3}$ 总量,这种方法操作步骤简便、 精密度高、效率高,通常被实验室作为首选检测方法。
需要检测维生素 ${\mathrm{B}}_{3}$ 的食品类别有很多,有些食品既强化了维生素 ${\mathrm{B}}_{3}$ ,又强化了乳酸菌,比如婴幼儿配方食品、调制乳粉等, 而在维生素 ${\mathrm{B}}_{3}$ 检测的样品前处理过程中,高含量的乳酸菌会把烟酰胺 (Nicotinamide) 转化为烟酸 (Nicotinic acid), 从而对检测产生干扰。本研究对 GB 5009.89-2016 的样品前处理过程进行了改进以避免此问题,为其在实际工作中的应用提供参考。
烟酸 $\left({{\mathrm{C}}_{6}{\mathrm{H}}_{5}{\mathrm{{NO}}}_{2}}\right)$ 标准品: 纯度 99.5%,德国默克公司; 烟酰胺 $\left({{\mathrm{C}}_{6}{\mathrm{H}}_{6}{\mathrm{\;N}}_{2}\mathrm{O}}\right)$ 标准品: 纯度 99.6%,德国默克公司; 盐酸 (HCl): 优级纯, 国药集团化学试剂有限公司。氢氧化钠 (NaOH): 优级纯,国药集团化学试剂有限公司。高氯酸 $\left({\mathrm{{HClO}}}_{4}\right)$ : 质量分数为 70%~72%,优级纯,天津市科密欧化学试剂有限公司。甲醇 $\left({{\mathrm{{CH}}}_{3}\mathrm{{OH}}}\right)$ : 色谱纯,德国默克公司。异丙醇 $\left({{\mathrm{C}}_{3}{\mathrm{H}}_{8}\mathrm{O}}\right)$ : 色谱纯, 德国默克公司。庚烷磺酸钠 $\left({{\mathrm{C}}_{7}{\mathrm{H}}_{15}{\mathrm{{NaO}}}_{3}\mathrm{\;S}}\right)$ : 色谱纯, J.T.Baker。 水为符合 GB/T 6682 [ 14 ] 规定的一级水。
高效液相色谱仪: Agilent 1260 带二极管阵列检测器(即 DAD 检测器)。GENESYS 150 紫外可见分光光度计:赛默飞世尔科技。MS205DU 电子天平: 感量 0.1mg,梅特勒 - 托利多仪器(上海)有限公司。XT 5018P-GP 18 恒温水浴: ${37}^{\circ }\mathrm{C}\sim {70}^{\circ }\mathrm{C}$ , 杭州雪中炭技术有限公司。SCQ-7201 超声波振荡器:上海声彦超声波仪器有限公司。FE28 pH 计: 精度 ±0.01,梅特勒 - 托利多仪器 (上海) 有限公司。HY-2 涡旋混合仪: 上海仪电科学仪器股份有限公司。
试制样品 A (奶粉基质,强化烟酰胺,唾液乳杆菌 Lactobacillus salivarius 添加量约 ${10}^{7}\sim {10}^{8}\mathrm{{CFU}}/\mathrm{g}$ )。
试制样品 B(除未添加唾液乳杆菌 Lactobacillus salivarius 外, 其余与试制样品 A 一致)。
称取混合均匀固体试样约 ${5.0}\mathrm{\;g}$ (精确到 ${0.01}\mathrm{\;g}$ ),加入约 ${25}\mathrm{\;{mL}}{45}\sim {50}^{\circ }\mathrm{C}$ 的水,称取混合均匀液体试样约 ${20.0}\mathrm{\;g}$ (精确到 ${0.01}\mathrm{\;g}$ )于 ${150}\mathrm{\;{mL}}$ 锥形瓶中,置于超声波振荡器中振荡约 ${10}\mathrm{\;{min}}$ 以上充分溶解,静置 $5 \sim {10}\mathrm{\;{min}}$ ,并冷却至室温。
待试样溶液降至室温后,用 ${5.0}\mathrm{\;{mol}}/\mathrm{L}$ 盐酸溶液和 ${0.1}\mathrm{\;{mol}}/\mathrm{L}$ 盐酸溶液调节试样溶液的 $\mathrm{{pH}}$${1.7}\pm {0.1}$ ,放置约 $2\mathrm{\;{min}}$ 后,再用 ${5.0}\mathrm{\;{mol}}/\mathrm{L}$ 氢氧化钠溶液和 ${0.1}\mathrm{\;{mol}}/\mathrm{L}$ 氢氧化钠溶液调节试样溶液的 $\mathrm{{pH}}$${4.5}\pm {0.1}$ ,置于 ${50}^{\circ }\mathrm{C}$ 水浴超声波振荡器中振荡 ${10}\mathrm{\;{min}}$ 以上充分提取,冷却至室温后转至 ${100}\mathrm{\;{mL}}$ 容量瓶中, 用水反复冲洗锥形瓶,洗液合并于 ${100}\mathrm{\;{mL}}$ 容量瓶中,用水定容至刻度后混匀,经滤纸过滤。滤液再经 ${0.45\mu }\mathrm{m}$ 微孔滤膜加压过滤,用样品瓶收集,即为试样测定液。
色谱柱: ${\mathrm{C}}_{18}$ 色谱柱 $\left({{150}\mathrm{\;{mm}}\times {4.6}\mathrm{\;{mm}}\text{,粒径}{5\mu }\mathrm{m}}\right)$ ; 柱温: ${\left({25}\pm {0.5}\right)}^{\circ }\mathrm{C}$ ;DAD 检测器:检测波长为 ${261}\mathrm{\;{nm}}$ ;流动相:甲醇 70 mL、异丙醇 20 mL、庚烷磺酸钠 $1\mathrm{\;g}$ ,用 ${910}\mathrm{\;{mL}}$ 水溶解并混匀后,用高氯酸调 $\mathrm{{pH}}$${2.1}\pm {0.1}$ ,经 ${0.45\mu }\mathrm{m}$ 有机微孔滤膜过滤;流速:1.0 mL/min;进样量:20 μL。
同时前处理试制样品 $\mathrm{A}$ 与试制样品 $\mathrm{B}$ ,然后上机分析,检测结果如 表 1 所示, 色谱图见 图 2 。由 表 1图 2 可知, 试制样品 $\mathrm{A}$ 中检出了烟酸,其含量为 ${787\mu }\mathrm{g}/{100}\mathrm{g}$ ,而试制样品 $\mathrm{B}$ 中未检出烟酸,而两个样品的维生素 ${\mathrm{B}}_{3}$ 总量检测结果基本一致。 因为两个试制样品中均强化了烟酰胺而未强化烟酸, 且配方唯一的差异在于试制样品 $\mathrm{A}$ 中添加了 ${10}^{7}\sim {10}^{8}\mathrm{{CFU}}/\mathrm{g}$ 的唾液乳杆菌 Lactobacillus salivarius,而试制样品 $\mathrm{B}$ 中未添加菌种; 故推测试制样品 $\mathrm{A}$ 中检出的烟酸是在样品分析过程中,由于唾液乳杆菌的作用将烟酰胺转化为烟酸。因为在很多乳酸菌中都含有烟酰胺酶,在该酶的催化作用下,烟酰胺脱氨基转化为烟酸 [ 15 ] ( 图 3 )。
试制样品 $\mathrm{B}$
试制样品 $\mathrm{A}$ 经前处理获得的试样测定液静置 $0\text{、}1\text{、}2\text{、}3\mathrm{\;h}$ 后再进样分析。检测结果如 表 2 所示, 色谱图见 图 4 。由 表 2 数据可知,烟酸、烟酰胺和维生素 ${\mathrm{B}}_{3}$ 的含量基本不随时间发生变化, 故烟酰胺向烟酸的转化发生在前面的步骤。因此根据样品前处理程序, 分别考察了酸水解前样品溶液静置时间和酸水解 $\mathrm{{pH}}$ 值的影响,以确定转化发生的步骤。
在样品酸水解前,将混合均匀的试制样品 $\mathrm{A}$ 溶液静置 5、 ${10}\text{、}{30}\text{、}{60}\text{、}{120}\mathrm{\;{min}}$ 后再进行后续分析。检测结果如 表 3 所示, 色谱图见 图 5 , 由 表 3 数据可知, 随静置时间延长烟酰胺含量逐渐减少,烟酸含量逐渐增加。以 表 3 中烟酸、烟酰胺和维生素 ${\mathrm{B}}_{3}$ 含量对静置时间作图,如 图 6 所示,明显可以看出烟酰胺逐渐转化为烟酸,而维生素 ${\mathrm{B}}_{3}$ 总量基本维持不变。而且在最初的 ${10}\mathrm{\;{min}}$ 内,即标准方法规定的最大静置时间,已有约 ${45}\%$ 的烟酰胺转化为烟酸;推测这与样品中高浓度的菌种添加量和标准方法规定的 ${45}\sim {50}^{\circ }\mathrm{C}$ 溶解水温有关,随着静置时间的延长, 溶解后的样品温度逐渐恢复至室温, 烟酰胺转化为烟酸的速度变慢;到 ${120}\mathrm{\;{min}}$ 以后烟酰胺完全转化为烟酸。
试制样品 $\mathrm{A}$ 溶液静置 ${120}\mathrm{\;{min}}$
为了确定酸水解 $\mathrm{{pH}}$ 对烟酰胺的转化是否有影响,将混合均匀的试制样品 $\mathrm{A}$ 溶液立即使用盐酸调节不同的 $\mathrm{{pH}}: {1.7}$ 、3.0、 4.0、6.8 (未加盐酸调节),水解 $2\mathrm{\;{min}}$ 后,然后再用氢氧化钠溶液调节 $\mathrm{{pH}}$ 至4.5(最后一个样品用盐酸调节),放置 ${120}\mathrm{\;{min}}$ 后, 再按后续步骤处理并上机分析, 检测结果如 表 4 所示, 色谱图见 图 7 。由 表 4 数据可知,酸水解 $\mathrm{{pH}}$ 值越低,烟酰胺向烟酸的转化得越少; 推测在低 $\mathrm{{pH}}$ 值下,唾液乳杆菌的作用受到抑制。
在标准方法规定的酸水解 $\mathrm{{pH}}$ 值 1.7 条件下,仅有约 $5\%$ 的烟酰胺转化为烟酸,这可能是在样品溶解过程中发生的;为了验证这一猜想,直接使用盐酸调节后 $\mathrm{{pH}}$ 值为 1.7 的水溶解试制样品 $\mathrm{A}$ ,并迅速调节溶液 $\mathrm{{pH}}$ 值至1.7,然后按照标准步骤操作, 检测结果如 表 5 所示,色谱图见 图 8 ; 并且对调节 $\mathrm{{pH}}$ 前后的样品溶液进行了乳酸菌计数,发现菌数下降了约 ${0.6}\log \mathrm{{CFU}}/\mathrm{{mL}}$ 。 由此证明在 $\mathrm{{pH}}$ 值为 1.7 时,唾液乳杆菌的作用完全受到抑制, 在此情况下不会有烟酰胺转化为烟酸。
本实验探讨了使用 GB 5009.89-2016 食品安全国家标准 食品中烟酸和烟酰胺的测定中第二法高效液相色谱法检测含乳酸菌的奶粉中维生素 ${\mathrm{B}}_{3}$ 时,由于高浓度乳酸菌的存在,导致的烟酰胺被乳酸菌所具有的烟酰胺酶转化为烟酸的现象。通过单因素实验确认了转化发生在样品溶解后、酸水解之前的静置步骤, 但按照标准方法所规定的酸水解条件调节 $\mathrm{{pH}}$ 值至1.7以后,转化作用被完全抑制。虽然该转化作用不会影响总维生素 ${\mathrm{B}}_{3}$ 的检测结果, 但如果单独检测烟酸或烟酰胺含量时, 或研究方法性能时会对检验者造成困扰。因此如果要单独检测此类样品中烟酸或烟酰胺的含量,需要在样品溶解时将 $\mathrm{{pH}}$ 值调整到 1.7,以抑制该转化作用。
关于乳酸菌添加量、样品溶解水温以及不同乳酸菌菌种对方法的影响尚待进一步研究; 而且有文献表示, 某些菌株可利用烟酰胺作为唯一的碳源、氮源和能量来源 [ 16 ] ,这意味着如果样品中存在这些菌株,无论使用 GB 5009.89-2016 中微生物法还是高效液相色谱法检测维生素 ${\mathrm{B}}_{3}$ ,检测结果均有可能低于实际值。当前, 出于健康的需求越来越多的食品中添加各种各样的益生菌, 然而恰恰是这些具有活性的微生物会对检测造成干扰, 这是未来食品安全国家标准制修订需要重点考虑的一个方面。
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    1 达能特殊营养品(青岛)有限公司 青岛 266113
    2 青岛海关技术中心 青岛 266109
    3 北京信睿浩扬科技有限公司 北京 100070
    4 青岛市食品药品检验研究院 青岛 266071

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*雷质文,三级研究员,研究方向为食品微生物检测和质量控制。E-mail:
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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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