Article(id=1312006333143015885, tenantId=1146029695717560320, journalId=1311385739772133495, issueId=1311999647825101743, articleNumber=null, orderNo=null, doi=10.6041/j.issn.1000-1298.2025.12.071, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1726243200000, receivedDateStr=2024-09-14, revisedDate=1730476800000, revisedDateStr=2024-11-02, acceptedDate=null, acceptedDateStr=null, onlineDate=1790736285719, onlineDateStr=2026-09-30, pubDate=1766592000000, pubDateStr=2025-12-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1790736285719, onlineIssueDateStr=2026-09-30, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1790736285719, creator=13701087609, updateTime=1790736285719, updator=13701087609, issue=Issue{id=1311999647825101743, tenantId=1146029695717560320, journalId=1311385739772133495, year='2025', volume='56', issue='12', pageStart='1', pageEnd='818', issueExtLink='null', onlineDate='null', pubDate='1766592000000', pubDateStr='2025-12-25', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=1, specialIssue=null, createTime=1790734691815, creator='13701087609', updateTime=1790738411040, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1312015247465083866, tenantId=1146029695717560320, journalId=1311385739772133495, issueId=1311999647825101743, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1312015247465083867, tenantId=1146029695717560320, journalId=1311385739772133495, issueId=1311999647825101743, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=769, endPage=776, ext={EN=ArticleExt(id=1312006333373702607, articleId=1312006333143015885, tenantId=1146029695717560320, journalId=1311385739772133495, language=EN, title=Effects of Milk Fat Content and Fat Globule Structure on Perception of Astringency in Milk Tea System, columnId=1312006333306593742, journalTitle=Transactions of the Chinese Society for Agricultural Machinery, columnName=Agricultural Products Processing, runingTitle=null, highlight=null, articleAbstract=

Milk tea beverage is a drink widely loved by consumers. The astringency caused by the rich tea polyphenols in the tea soup is one of the key factors affecting the sensory quality of milk tea beverage. Existing research has shown that milk components, such as milk proteins, can reduce astringency. Milk fat, an important component in cow's milk, is composed of milk fat globules. However, the effect of its content and structure on astringency in milk tea systems remained unclear. It was aimed to investigate the impact of milk fat content and fat globule structure on astringency in milk tea systems. Firstly, it used descriptive sensory evaluation to explore whether milk fat and fat content affected the astringency of milk tea system. Next, three types of milk fat droplets with the same fat content but different structural characteristics were constructed by adjusting the heating intensity and homogenization pressure: milk fat droplet C, milk fat droplet T and milk fat droplet H, and the particle size,ζ-potential, microscopic structure, milk fat globule membrane protein content and composition, and milk fat globule membrane phospholipid content were measured, which characterized the characteristics of milk fat globules. Then different milk fat droplets were added to the simulated milk tea system to conduct sensory evaluation of astringency intensity, and quantify astringency through fluorescence spectroscopy analysis. The results showed that when the milk fat content reached above 4 g/(100 mL), the addition of milk fat can significantly reduce the astringency level in milk tea. In addition, changing the particle size and membrane structure of milk fat globules also had a significant impact on the astringency of the milk tea system. Compared with the other two milk fat droplets, the fat globule size in milk fat droplet H was the smallest (0.49 μm), the protein content per gram fat globule membrane was the highest (12.60 mg), and the astringency was the weakest. Milk fat droplet T had the largest particle size (4.20 μm), the lowest protein content per gram fat globule membrane (4.12 mg), and a strong astringency. Therefore, milk fat content, particle size and membrane structure would affect the astringency intensity of milk tea system. Smaller milk fat globule size and more milk fat globule membrane interface proteins had the greatest effect on reducing the astringency intensity of milk tea system. It was confirmed the impact of milk fat on the perception of astringency in the milk tea system, laying a theoretical foundation for the development of green and healthy milk tea drinks with a smoother taste.

, authors=Xingyi LI, Wei MAO, Yajuan LI, Hui ZHOU, Xiankang FAN, Jie LUO, authorsList=Xingyi LI, Wei MAO, Yajuan LI, Hui ZHOU, Xiankang FAN, Jie LUO, authorCompany=null, correspAuthors=Jie LUO, authorNote=null, correspAuthorsNote=null, 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, fund=null), CN=ArticleExt(id=1312006334929789405, articleId=1312006333143015885, tenantId=1146029695717560320, journalId=1311385739772133495, language=CN, title=乳脂肪含量与脂肪球结构对乳茶体系涩感感知的影响研究, columnId=1312006333474365904, journalTitle=农业机械学报, columnName=农产品加工工程, runingTitle=null, highlight=null, articleAbstract=

乳茶饮料是广受消费者喜爱的一种饮品,茶汤中丰富的茶多酚导致的涩感是影响乳茶饮料感官质量的关键因素之一。已有研究显示乳组分如乳蛋白等能够降低涩感,乳脂肪由乳脂肪球组成,作为牛乳中的一种重要组成成分,其含量和结构对乳茶体系涩感的影响尚不明确。为探究牛乳脂肪含量及脂肪球结构对乳茶体系中涩感的影响,首先通过描述型感官评价探究乳脂肪及其含量对乳茶体系涩感是否有影响。然后,研究通过调控加热强度和均质压力,构建了3种脂肪含量一致但结构特征不同的乳脂滴:乳脂滴C、乳脂滴T和乳脂滴H,并通过测定粒径、ζ-电势、微观结构、乳脂肪球膜蛋白含量和组成、乳脂肪球膜磷脂含量表征乳脂肪球特性,再将不同乳脂滴添加至模拟乳茶体系,进行涩感强度的感官评价,并通过荧光光谱分析量化涩感差异。结果表明,当乳脂肪质量浓度达到4 g/(100 mL)以上时,乳脂肪的添加可显著降低乳茶中的涩感水平。此外,改变乳脂肪球的粒径及膜结构也对乳茶体系的涩感有显著影响。与其他两种乳脂滴相比,乳脂滴H中的脂肪球粒径最小(0.49 μm),每克脂质的蛋白质量最高(12.60 mg),而涩感最弱。乳脂滴T粒径最大(4.20 μm),每克脂质的蛋白质量最低(4.12 mg),涩感较强。因此,乳脂肪含量、粒径和膜结构会影响乳茶体系的涩感强度,更小的乳脂肪球尺寸和更多的乳脂肪球膜界面蛋白对乳茶体系的涩感强度降低作用更大。研究探究了乳脂肪对乳茶体系涩感感知的影响,为口感更顺滑的绿色、健康乳茶饮料的开发奠定了理论基础。

, authors=李星漪, 毛薇, 黎雅娟, 周辉, 范贤康, 罗洁, authorsList=李星漪, 毛薇, 黎雅娟, 周辉, 范贤康, 罗洁, authorCompany=null, correspAuthors=罗洁, authorNote=

李星漪(2000—),女,博士生,主要从事食品加工与安全研究,E-mail:

, correspAuthorsNote=
罗洁(1987—),女,教授,博士生导师,主要从事乳品科学研究,E-mail:
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李星漪(2000—),女,博士生,主要从事食品加工与安全研究,E-mail:

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李星漪(2000—),女,博士生,主要从事食品加工与安全研究,E-mail:

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Protein and phospholipid content,coverage and specific surface area of milk fat globule membrane under different processing methods

, figureFileSmall=null, figureFileBig=null, tableContent=
参数乳脂滴C乳脂滴T乳脂滴H
比表面积/(m3·mg-1)(1.95±0.01)c(2.21±0.05)b(12.62±0.04)a
每克脂质的蛋白质量/mg(4.81±0.07)b(4.12±0.01)c(12.60±0.26)a
蛋白质覆盖率/(mg·m-2)(2.47±0.04)a(1.87±0.01)b(1.00±0.02)c
每克脂质的磷脂质量/mg(22.63±5.73)a(21.78±4.26)a(18.33±5.08)a
磷脂覆盖率/(mg·m-2)(11.61±2.94)a(9.86±2.18)a(1.45±2.61)b
), ArticleFig(id=1312006338029380112, tenantId=1146029695717560320, journalId=1311385739772133495, articleId=1312006333143015885, language=CN, label=表1, caption=

不同加工方式乳脂肪球膜蛋白质、磷脂含量和覆盖率及比表面积

, figureFileSmall=null, figureFileBig=null, tableContent=
参数乳脂滴C乳脂滴T乳脂滴H
比表面积/(m3·mg-1)(1.95±0.01)c(2.21±0.05)b(12.62±0.04)a
每克脂质的蛋白质量/mg(4.81±0.07)b(4.12±0.01)c(12.60±0.26)a
蛋白质覆盖率/(mg·m-2)(2.47±0.04)a(1.87±0.01)b(1.00±0.02)c
每克脂质的磷脂质量/mg(22.63±5.73)a(21.78±4.26)a(18.33±5.08)a
磷脂覆盖率/(mg·m-2)(11.61±2.94)a(9.86±2.18)a(1.45±2.61)b
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乳脂肪含量与脂肪球结构对乳茶体系涩感感知的影响研究
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李星漪 , 毛薇 , 黎雅娟 , 周辉 , 范贤康 , 罗洁
农业机械学报 | 农产品加工工程 2025,56(12): 769-776
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农业机械学报 |农产品加工工程 2025 , 56 (12) : 769 -776
乳脂肪含量与脂肪球结构对乳茶体系涩感感知的影响研究
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李星漪 , 毛薇, 黎雅娟, 周辉, 范贤康, 罗洁
作者信息
  • 湖南农业大学食品科学技术学院,长沙 410128
通讯作者:
罗洁(1987—),女,教授,博士生导师,主要从事乳品科学研究,E-mail:
作者简介:

李星漪(2000—),女,博士生,主要从事食品加工与安全研究,E-mail:

Effects of Milk Fat Content and Fat Globule Structure on Perception of Astringency in Milk Tea System
Xingyi LI , Wei MAO, Yajuan LI, Hui ZHOU, Xiankang FAN, Jie LUO
Affiliations
  • College of Food Science and Engineering, Hunan Agricultural University, Changsha 410128, China
出版时间: 2025-12-25 doi: 10.6041/j.issn.1000-1298.2025.12.071
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乳茶饮料是广受消费者喜爱的一种饮品,茶汤中丰富的茶多酚导致的涩感是影响乳茶饮料感官质量的关键因素之一。已有研究显示乳组分如乳蛋白等能够降低涩感,乳脂肪由乳脂肪球组成,作为牛乳中的一种重要组成成分,其含量和结构对乳茶体系涩感的影响尚不明确。为探究牛乳脂肪含量及脂肪球结构对乳茶体系中涩感的影响,首先通过描述型感官评价探究乳脂肪及其含量对乳茶体系涩感是否有影响。然后,研究通过调控加热强度和均质压力,构建了3种脂肪含量一致但结构特征不同的乳脂滴:乳脂滴C、乳脂滴T和乳脂滴H,并通过测定粒径、ζ-电势、微观结构、乳脂肪球膜蛋白含量和组成、乳脂肪球膜磷脂含量表征乳脂肪球特性,再将不同乳脂滴添加至模拟乳茶体系,进行涩感强度的感官评价,并通过荧光光谱分析量化涩感差异。结果表明,当乳脂肪质量浓度达到4 g/(100 mL)以上时,乳脂肪的添加可显著降低乳茶中的涩感水平。此外,改变乳脂肪球的粒径及膜结构也对乳茶体系的涩感有显著影响。与其他两种乳脂滴相比,乳脂滴H中的脂肪球粒径最小(0.49 μm),每克脂质的蛋白质量最高(12.60 mg),而涩感最弱。乳脂滴T粒径最大(4.20 μm),每克脂质的蛋白质量最低(4.12 mg),涩感较强。因此,乳脂肪含量、粒径和膜结构会影响乳茶体系的涩感强度,更小的乳脂肪球尺寸和更多的乳脂肪球膜界面蛋白对乳茶体系的涩感强度降低作用更大。研究探究了乳脂肪对乳茶体系涩感感知的影响,为口感更顺滑的绿色、健康乳茶饮料的开发奠定了理论基础。

乳脂肪球  /  涩感  /  乳茶体系

Milk tea beverage is a drink widely loved by consumers. The astringency caused by the rich tea polyphenols in the tea soup is one of the key factors affecting the sensory quality of milk tea beverage. Existing research has shown that milk components, such as milk proteins, can reduce astringency. Milk fat, an important component in cow's milk, is composed of milk fat globules. However, the effect of its content and structure on astringency in milk tea systems remained unclear. It was aimed to investigate the impact of milk fat content and fat globule structure on astringency in milk tea systems. Firstly, it used descriptive sensory evaluation to explore whether milk fat and fat content affected the astringency of milk tea system. Next, three types of milk fat droplets with the same fat content but different structural characteristics were constructed by adjusting the heating intensity and homogenization pressure: milk fat droplet C, milk fat droplet T and milk fat droplet H, and the particle size,ζ-potential, microscopic structure, milk fat globule membrane protein content and composition, and milk fat globule membrane phospholipid content were measured, which characterized the characteristics of milk fat globules. Then different milk fat droplets were added to the simulated milk tea system to conduct sensory evaluation of astringency intensity, and quantify astringency through fluorescence spectroscopy analysis. The results showed that when the milk fat content reached above 4 g/(100 mL), the addition of milk fat can significantly reduce the astringency level in milk tea. In addition, changing the particle size and membrane structure of milk fat globules also had a significant impact on the astringency of the milk tea system. Compared with the other two milk fat droplets, the fat globule size in milk fat droplet H was the smallest (0.49 μm), the protein content per gram fat globule membrane was the highest (12.60 mg), and the astringency was the weakest. Milk fat droplet T had the largest particle size (4.20 μm), the lowest protein content per gram fat globule membrane (4.12 mg), and a strong astringency. Therefore, milk fat content, particle size and membrane structure would affect the astringency intensity of milk tea system. Smaller milk fat globule size and more milk fat globule membrane interface proteins had the greatest effect on reducing the astringency intensity of milk tea system. It was confirmed the impact of milk fat on the perception of astringency in the milk tea system, laying a theoretical foundation for the development of green and healthy milk tea drinks with a smoother taste.

milk fat globules  /  astringency  /  milk tea system
李星漪, 毛薇, 黎雅娟, 周辉, 范贤康, 罗洁. 乳脂肪含量与脂肪球结构对乳茶体系涩感感知的影响研究. 农业机械学报, 2025 , 56 (12) : 769 -776 . DOI: 10.6041/j.issn.1000-1298.2025.12.071
Xingyi LI, Wei MAO, Yajuan LI, Hui ZHOU, Xiankang FAN, Jie LUO. Effects of Milk Fat Content and Fat Globule Structure on Perception of Astringency in Milk Tea System[J]. Transactions of the Chinese Society for Agricultural Machinery, 2025 , 56 (12) : 769 -776 . DOI: 10.6041/j.issn.1000-1298.2025.12.071
乳茶饮料是将茶和液态奶按一定比例调配而成的休闲饮品,广受消费者欢迎。其中茶汤中含有大量的茶多酚等天然活性成分,具有许多药理和生理作用[1],然而,这些活性成分也会带来一些特殊的风味,如涩感。牛乳中含有大量的蛋白质、钙等成分,营养价值高。现有研究表明,牛乳能够有效降低茶汤中的涩感[2-3],将茶和牛乳拼配而成的乳茶既有茶和牛乳的双重营养价值,又有顺滑可口的风味,因此乳茶饮料深受消费者喜爱。
“涩”是一种重要的感官特性,可以极大地影响食品和饮料的整体感官,如葡萄酒、咖啡以及茶[4-6]。涩感被定义为皱褶、干燥或粗糙的复杂三叉神经感觉[7],它是由涩感物质接触口腔上皮细胞从而刺激神经末梢产生的[8],其中多酚类物质是引起口腔涩感的一大重要因素,其中以表没食子酸儿茶素没食子酸酯(Epigallocatechin gallate,EGCG)为代表的儿茶素类物质能够与唾液蛋白聚集沉淀导致涩感,进而影响食物的口感[9-10]。大量研究表明,牛乳中的组分可有效降低涩感,如乳蛋白可通过与口腔环境蛋白质竞争儿茶素类物质,降低涩感的感知[11]。除此以外,乳脂肪或有类似的效果,如奶酪与红酒同时食用,有利于降低红酒涩感的持续时间[12]。然而,乳脂肪的特性对乳茶体系涩感是否有影响等问题尚不明确[13-15]。
乳脂大部分以脂肪球的形式存在于牛奶中,由于其具有独特的风味、质地和物理化学及营养特性,对乳制品品质至关重要。乳脂肪球膜(MFGM)是由蛋白质、糖蛋白、酶、中性脂质、极性脂质和胆固醇等组成的复杂体系,表面包裹着3层磷脂[16]。乳脂肪通过其风味和质地等特征在口腔感知的过程中起到至关重要的作用;脂肪感知本身是一种多模态感觉,涉及味觉、嗅觉和质地感知等。乳脂肪的味觉感知主要通过游离脂肪酸(FFA)体现[17];在触觉方面,乳脂肪在体温下会出现更多融化的短链脂肪酸,影响其质地感知[18]。因此,乳脂肪的含量、乳脂液滴的大小、乳脂肪球的粒径和脂肪球膜的界面特性都是影响口腔感知和消费者脂肪喜好的关键驱动因素。然而,很少有研究报道以上乳脂肪的特征对乳茶饮料中涩味感知的影响。
因此,本文使用不同脂肪含量的牛乳与EGCG溶液混合,以加入脱脂乳的EGCG溶液作为对照,通过描述型感官评价法探究乳脂肪对乳茶饮料涩味感知的影响以及这种影响与乳脂肪浓度的相关性。并构建脂肪含量相同而结构特征不同的乳脂滴,与天然未经处理的牛乳脂滴对比,随后将3种乳脂滴添加至模拟乳茶体系中,通过描述型感官评价和荧光光谱法探究乳脂滴粒径和脂肪球膜结构与乳茶饮料涩味感知之间的关系。
鲜牛乳采样及加工均来自湖南新希望南山液态乳业有限公司,并将样品转移至密封瓶中,并在2 h之内运回实验室置于4℃冰箱保存;表没食子酸儿茶素没食子酸酯(分析纯),浙江天草生物科技股份有限公司;磷酸二氢钾、柠檬酸三钾、柠檬酸三钠、硫酸钾、氯化钙、六水合氯化镁、碳酸钾、氯化钾、氢氧化钾(分析纯),国药集团化学试剂有限公司;Tris缓冲液、PBS缓冲液,北京雷根生物技术有限公司。
Master sizer 3000型激光粒度仪,Malvern仪器有限公司,英国;Zetasizer Nano ZS型纳米粒度仪,Malvern仪器有限公司,英国;LSM 710型激光扫描共聚焦显微镜,蔡司公司,德国;GBox F3型凝胶成像仪,Syngene公司,英国;TP-3000E型分析天平,湘仪天平仪器设备有限公司;HH-2型数显恒温水浴锅,常州市金坛区环宇科学仪器厂;F-7000型荧光分光光度计,日立高新技术公司,日本;Vortex-2型高速冷冻离心机,湖南迈克尔实验仪器有限公司;SP60/SLP型乳成分分析仪,Lactoscan公司,保加利亚。
不同脂肪含量样品的制备参考迟雪露等[19]的方法并加以修改,将未经过加工处理的鲜牛乳通过乳成分分析仪测量脂肪含量,然后通过4 200 r/min、10 min离心获取上层稀奶油,计算上层乳脂肪含量。下层为不含脂肪的脱脂牛乳。通过计算在脱脂乳基底中加入不同质量的稀奶油,以达到脂肪质量浓度为3、4、5 g/(100 mL)的牛乳样品,通过磁力搅拌器搅拌20 min,使得乳脂肪在牛乳中均匀分散。制备所得样品置于(4±1)℃冰箱保存,备用。
将EGCG配制为3 g/L的水溶液,与不同脂肪含量的牛乳以3∶1的体积比均匀混合,制备成不同乳脂肪含量的模拟乳茶,并选择脱脂牛乳为空白对照。
招募感官评价小组志愿者,并根据其感官能力、兴趣进行选拔。小组人员通过培训熟悉测试样品和过程。利用线性标度法(即在一条100 mm的带刻度线段上,规定不添加物质的溶液涩感评分为最小值0,2 g/L EGCG溶液涩感评分为最大值10.0,根据品尝感受标出强度,根据所标记值定义强度大小,精确到0.1),对成员进行涩感强度的排序训练。对质量浓度为0、0.4、0.8、1.2、1.6、2.0 g/L的EGCG溶液进行反复定位。培训环境为室温((18±2)℃),小组成员将全部溶液(30 mL)倒入口腔,保持10 s后吐出,根据感受标记涩感强度。之后用蒸馏水清洁口腔,并咀嚼苏打饼干使味觉得到恢复。
利用上述质量浓度的标准溶液作为参照。在室温条件下,每次评价4组模拟乳茶样品(含3、4、5 g/(100 mL)脂肪牛乳以及脱脂牛乳),感官评价环节与培训环节的过程相同。
牛乳样品分为4组,分别为原料牛乳(未经加工)、高温牛乳(85℃,5 min)、均质牛乳(压力18~20 MPa,温度65~70℃)及脱脂牛乳。均质牛乳及脱脂牛乳均在湖南新希望南山液态乳业有限公司加工得到,所有加工后的牛乳样品都保存在4℃下进行进一步分析。接着,在不同加工方式的牛乳中提取奶油,原料及高温组是通过4 200 r/min、10 min离心得到的,而均质组通过10 000 r/min、20 min得到,离心温度保持在4℃。将未处理组、高温处理组和均质处理组离心后收集顶层,在模拟乳超滤液中重新分散,形成脂肪质量浓度为0.04 g/mL的分散液[18],得到具有不同特性的乳脂滴,分别命名为:乳脂滴C、乳脂滴T和乳脂滴H,用于后续模拟乳茶体系涩感强度评价。
使用激光粒度分析仪测量不同乳脂滴样品的粒度分布。水和脂滴在466 nm处的折射率分别为1.333和1.460。将样品加入仪器测量池中的500 mL超纯水中,以达到10%~20%的遮盖度,乳脂肪球的比表面积(m2/g),通过仪器软件计算[20]。
用纳米粒度仪在25℃和pH值7.0时测量了乳脂滴样品中脂肪球表面电势。乳脂滴样品在测定前用0.01 mol/L磷酸盐缓冲液(PBS,pH值7.0)稀释1 000倍[21]。
将20 μL丽丝胺罗丹明B染料和20 μL快绿染料加入到0.5 mL脂滴乳液样品中,在25℃避光染色1 h。质量分数1%的低熔点琼脂糖与乳液以料液比2 g/mL混合,在激光扫描共聚焦显微镜上用63×油镜采集脂滴图像,选取合适的放大倍数观察,并使用Zen软件进行分析[22-23]。
使用WANG等[24]的方法从乳脂滴上层脂质中分离出乳脂肪球界面蛋白。通过在4℃下以10 000 g离心30 min来分离样品。收集上层乳状物,并与3倍体积的Tris-SDS缓冲液(50 mM Tris-HCl(pH值7.4),4% SDS)混合,并在60℃下孵育30 min。然后,将混合物在10 000 g离心30 min,弃去上层,剩余的液体中溶解的蛋白即为界面蛋白。
通过1.3.7.1节得到乳脂肪球界面蛋白,使用BCA蛋白检测试剂盒测定蛋白浓度。将各个稀释浓度的蛋白质标品和待测蛋白质样品加入到96孔板中,加入BCA工作液。37℃孵育30 min后,以空白为对照,测量样品在562 nm处吸光度。通过吸光度得到样品蛋白质浓度。通过将每克分离脂质的界面蛋白量除以脂肪球的比表面积来计算界面蛋白覆盖率,每个测量重复3次。
得到乳脂肪球界面蛋白后,利用SDS-PAGE凝胶电泳对界面蛋白进行分离和鉴定,经凝胶成像获得蛋白的凝胶电泳图像,鉴定乳脂肪球界面蛋白的类别。样品与上样缓冲液混合,煮沸5 min,确保蛋白质完全变性。上样量为15 μL,分离胶质量分数为12%,浓缩胶质量分数为5%。电泳操作电压为:浓缩胶80 V;分离胶120 V。用0.25%考马斯蓝R250溶液(考马斯蓝R250 1.25 g,加入50%甲醇溶液454 mL和冰醋酸46 mL,混合均匀)染色5 min,静置12 h脱色。使用Image J软件对界面蛋白进行灰度分析。
将10 mL样品溶于30 mL三氯甲烷/甲醇(体积比2∶1)中,涡旋振荡3 min,然后加入9 mL氯化钾,再次涡旋振荡3 min,以8 000 r/min离心10 min,吸出氯仿相。重复上述萃取操作,将两次氯仿相合并。除去氯仿,并用氮气吹干[25]。用固相萃取除去非极性脂质,氮吹至质量恒定,减去空管质量得到磷脂的质量。磷脂质量除以总脂质质量再除以脂肪球的比表面积,得出乳脂肪球界面磷脂覆盖率。
将通过1.3.4节得到的3组乳脂滴与EGCG混合,比例及浓度同1.3.2节,另外分别用超纯水(感官评价)和模拟乳超滤液(荧光光谱法)作为对照组。
感官训练参考1.3.3.1节。正式感官评价时,要求感官评价小组针对乳脂滴C、乳脂滴T、乳脂滴H、超纯水对照组4组样品进行涩感评分。
在每次荧光光谱测量之前,配制25 mmol/L黏蛋白溶液,将不同的乳脂滴溶液与EGCG溶液以体积比3∶5均匀混合,随后加入与EGCG等比例的黏蛋白。荧光发射光谱是在荧光分光光度计上使用1 cm比色皿记录的。激发和发射狭缝宽度均为5.0 nm,扫描速度为600 nm/min。
所有试验样品一式三份,数据以平均值±标准差表示。统计分析用SPSS软件,采用单因素方差分析和邓肯多因素检验判断两组间差异是否有统计学意义(P<0.05)。利用Image J对蛋白质数据进行背景识别、背景去除和灰度计算。
通过感官分析方法(QDA)对不同脂肪含量的乳茶体系进行比较,结果如图1所示,图中字母A~J代表感官评价小组成员个人涩感评分,误差棒代表10位感官评价小组成员涩感评分标准差。未经处理的牛乳中乳脂肪质量浓度约为4 g/(100 mL),其与乳脂肪含量为零的脱脂乳相比,涩感评分出现了显著下降,因此,乳脂肪的存在,会对感官评价中涩味感知产生影响。接着,探索了不同乳脂肪含量的牛乳对于乳茶体系的涩感影响,随着乳脂肪添加量的增加,乳茶体系涩感评分呈先升高再降低的趋势,当脂肪质量浓度在3 g/(100 mL)时,对涩感几乎无影响,且评分个体差异大,随机性强;当添加量为4 g/(100 mL)时,乳茶体系涩感显著降低,此时涩感评分为3.25分,评分较为集中,当添加量超过4 g/(100 mL),乳茶体系的涩感强度变化较小,评分分化较大。综上,说明乳脂肪降低乳茶中涩感的功能有一定的浓度要求,这可能是因为乳脂肪对口腔存在润滑效果或对茶中的多酚类物质存在包裹或吸附效果,降低了茶多酚所引起的口腔干燥、皱缩的涩味感知。
粒度和ζ-电势是乳脂肪球至关重要的物理性质,这些特征很大程度影响乳脂肪在口腔中的感官特性。对乳脂肪球粒径和ζ-电势的测试结果如图2所示,与乳脂滴C中的脂肪球相比,乳脂滴T中乳脂肪球粒径略微变大,从3.56 μm增大到4.20 μm,这是因为高温加剧了乳脂肪球的布朗运动或奥斯特瓦尔德成熟,使得其出现略微聚集,脂肪球粒径随之变大,但在乳脂滴T中,粒径的变化程度较小,作用有限。与之相反的是,在均质机的高速搅拌和剪切下,乳脂滴H中的脂肪球粒径与乳脂滴C相比显著下降,从3.56 μm降低到0.49 μm。因此,相较于乳脂滴C与H中脂肪球的粒径差异,乳脂滴T与C中脂肪球的粒径差异较小,可近似忽略不计。在ζ-电势方面,乳脂滴的ζ-电势差异主要与脂肪球比表面积和界面蛋白的吸附有关,且3种乳脂滴ζ-电势差异较小。
此外,通过共聚焦激光显微镜(CLSM)对3种乳脂滴进行微观结构观察,结果如图3所示,绿色区域是乳脂滴中的蛋白质,红色区域是乳脂滴中的磷脂,黄色区域是二者重叠区域。乳脂滴C与乳脂滴T中,左侧3幅图标尺为20 μm,右侧3幅图为100 μm,乳脂滴H中,左侧3幅图标尺为100 μm,右侧3幅图为200 μm。可以发现,无论哪一种乳脂滴,其中脂肪球核心的中性脂质都呈球形,这与王霁月[26]的研究发现一致,乳脂滴T相较于C脂肪球出现了轻微的聚集,但体积差异不大,直径在20 μm到100 μm不等;而乳脂滴H中的脂肪球与其余两种乳脂滴差异明显,小脂肪球数量大幅增加,大多数单个的脂肪球直径小于1 μm。综上,通过对粒径、ζ-电势的测定和微观结构的观察,结果证明成功构建了粒径显著不同于乳脂滴C和T的乳脂滴H。
BCA结果如表1所示,乳脂滴T与乳脂滴C相比,每克脂质的蛋白质量显著下降,从每克脂质(4.81±0.07)mg降低到(4.12±0.01)mg。与之相反,乳脂滴H中脂肪球的界面蛋白含量显著提高,达到每克脂质(12.60±0.26)mg。虽然乳脂滴H中的脂肪球有更多的乳蛋白吸附或共价结合在乳脂肪球膜上,但其乳脂肪球的直径大幅减小,数量大幅增加,比表面积显著增加,所以在分析蛋白质在乳脂肪球膜上的锚定比率时,乳脂滴H中的脂肪球蛋白的覆盖率低于乳脂滴T与C中的脂肪球。
乳脂肪球的界面蛋白主要由MFGM蛋白组成,包括黏蛋白1(MUC1)、黄嘌呤氧化酶(XO)、分化簇36(CD36)、嗜乳脂蛋白(BTN)和高碘酸希夫糖蛋白6/7(PAS6/7),还有少量的酪蛋白和β-乳球蛋白(β-Lactoglobulin,β-LG)吸附在乳脂肪球的界面上[27]。SDS-PAGE结果如图4所示,显示了MFGM蛋白的变化。乳脂滴T的脂肪球中,XO和CD36条带几乎看不到,此外,与乳脂滴C的脂肪球界面蛋白相比,MUC1条带颜色变浅,蛋白含量降低,这是因为这些蛋白在较高温度下与脂肪球膜结合力最弱,在提取过程中损失最多,因此,无论在蛋白质含量和丰度上,乳脂滴T中的脂肪球都比乳脂滴C中的脂肪球界面蛋白低。反之,乳脂滴H中的脂肪球与乳脂滴C相比,酪蛋白和β-乳球蛋白的条带变得更加明显,而MUC1、XO、CD36、PAS7和PAS6条带几乎消失,这些蛋白与膜的结合更为松散,更容易与膜分离,这与徐婉莉[28]的研究结果类似。因此,乳脂滴H中的脂肪球膜蛋白丰度低于乳脂滴C中的脂肪球膜蛋白,但数量显著增大。
综上,通过蛋白含量、锚定比率的测定和种类的分析,结果证明成功构建了3种不同脂肪球膜组成和结构的乳脂滴。
如表1所示,乳脂滴C中的脂肪球表面磷脂含量为每克磷脂22.63 mg,与乳脂滴T中的脂肪球表面磷脂含量每克磷脂21.78 mg和乳脂滴H中的脂肪球表面磷脂含量18.33 mg相比并无显著差异。因此,3种乳脂滴的脂肪球在磷脂方面无显著差异,可能不会显著影响乳脂滴在乳茶体系涩感降低中的作用。
通过感官分析方法(QDA)对加入不同乳脂滴的乳茶体系进行涩感评分比较。对照组添加纯净水的乳茶体系涩感评分达到9.38±0.77,而无论添加何种乳脂滴,涩感评分均出现了显著下降,因此,乳脂肪对乳茶体系的涩感降低有明确作用,这种作用效果可能来源于乳脂肪的润滑和对EGCG的包裹及吸附作用。接着,对比添加3种不同乳脂滴的乳茶体系涩感评分,添加了乳脂滴H的乳茶体系获得了最低的涩感评分5.95±1.52,而添加了乳脂滴C和乳脂滴T的乳茶体系涩感评分分别为7.10±1.58和8.00±1.28。添加了乳脂滴C的乳茶体系涩感评分低于添加了乳脂滴T的乳茶体系,这可能是因为乳脂滴C中的脂肪球表面拥有数量和丰度都更高的蛋白,这一点在添加乳脂滴H的乳茶体系中体现得更为明显,乳脂滴H中的脂肪球蛋白含量达到乳脂滴T中脂肪球蛋白含量的3倍。先前的研究表明,MUC1、CD36、酪蛋白和β-LG等蛋白会与EGCG通过氢键和疏水作用力发生非共价结合,这些蛋白会和与涩感产生密切相关的主要蛋白——口腔黏蛋白竞争EGCG,减少黏蛋白的沉淀从而降低涩感[29-30]。此外,乳脂肪球粒径也可能对其涩感评分的降低具有重要影响,乳脂滴H中的脂肪球粒径减小到其他两种乳脂滴的1/9,更小的脂肪球粒径拥有更大的比表面积,细小的脂肪球对口腔的润滑效果更好,更重要的是,更大的比表面积使得脂肪球膜蛋白有更多的氨基酸残基裸露出来,其与EGCG相互作用更高效,作用程度更深。由于造膜过程中乳脂肪球膜中的另一个重要功能组分磷脂含量变化不显著,因此无法直接证明磷脂含量对乳茶体系涩感是否有影响,在后续可以进行更具针对性的探究。综上,通过感官分析,证明了乳脂肪球对乳茶体系具有涩感降低效果,并且,更小的粒径和更多的乳脂肪球膜界面蛋白可能对降低涩感具有积极影响。
大量研究表明,荧光光谱法可以用于研究黄烷醇等酚类物质与蛋白质的结合,荧光猝灭可以提供有关酚类物质和蛋白质相互作用引起的蛋白质结构修饰信息,研究相互作用的结合机制[31-32]。添加不同乳脂滴的乳茶体系的黏蛋白荧光光谱如图5所示,无论何种乳茶体系,黏蛋白荧光光谱都在380 nm处出现特征吸收峰。未添加乳脂滴的模拟乳茶体系在380 nm处荧光强度最低,证明了乳脂滴对黏蛋白的保护作用,这与感官分析的结果相符。乳脂滴对黏蛋白的保护作用程度从大到小依次为:乳脂滴H、乳脂滴C与乳脂滴T,从作用程度上来看,添加了乳脂滴H的模拟乳茶体系黏蛋白荧光强度远大于添加其他两种乳脂滴的乳茶体系,达到两倍以上,这与乳脂滴H中脂肪球更小的粒径和膜表面大量的酪蛋白和β-LG相关,这与感官分析得到的结果相同。
本研究探究了乳脂肪含量和乳脂肪球结构在乳茶体系中对涩感的影响作用。首先通过感官分析明确了乳脂肪在乳茶体系的涩感评分降低方面具有明显作用,且该作用效果受乳脂肪浓度影响,只有在乳脂肪质量浓度大于4 g/(100 mL)时,涩感评分才出现显著下降。接着,构建了3种具有不同乳脂肪球特征的乳脂滴,并通过粒径、ζ-电势的测定、微观结构观察、乳脂肪球膜界面蛋白含量和种类分析、乳脂肪球膜界面磷脂含量分析等方式验证,成功构建了天然未处理的乳脂肪球C,粒径与乳脂肪球C相似而乳脂肪球膜结构不同的乳脂肪球T,以及粒径与乳脂肪球膜结构与乳脂肪球C都不同的乳脂肪球H。最后,通过感官分析和荧光光谱分析,发现了乳脂滴粒径、脂肪球表面组成和结构等特征都对乳茶体系的涩感有影响。结果表明更小的乳脂肪球尺寸和更多的乳脂肪球膜界面蛋白,特别是酪蛋白和β-乳球蛋白对乳茶体系的涩感强度降低作用最佳。本研究探索了乳脂肪特性与其降低乳茶饮料涩感这一功能之间的联系,为未来开发具有更顺滑口感的绿色、健康乳茶饮料产品提供了理论基础。
  • 湖南省自然科学优秀青年基金项目(2022JJ20026)
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2025年第56卷第12期
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doi: 10.6041/j.issn.1000-1298.2025.12.071
  • 接收时间:2024-09-14
  • 首发时间:2026-09-30
  • 出版时间:2025-12-25
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  • 收稿日期:2024-09-14
  • 修回日期:2024-11-02
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湖南省自然科学优秀青年基金项目(2022JJ20026)
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    湖南农业大学食品科学技术学院,长沙 410128

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罗洁(1987—),女,教授,博士生导师,主要从事乳品科学研究,E-mail:
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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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