乳及乳制品是居民膳食结构的重要组成部分。乳是一种水包油的乳状液,其中的脂类以乳脂肪球的形式存在,98%的乳脂肪球核是以1个碳骨架和3个脂肪酸分子组成的非极性脂质甘油三酯的形式存在[1]。乳脂中脂肪酸的组成与其营养品质和理化性质密切相关[2]。据报道,乳脂约有大于400种脂肪酸组成[3],其中短链脂肪酸(short-chain fatty acids,SCFAs)不足3%。SCFAs是一组碳原子数在2~6之间的有机酸,通常包括直链的乙酸、丙酸、丁酸、戊酸、己酸,以及支链的异丁酸、2-甲基丁酸、异戊酸、2-甲基戊酸、3-甲基戊酸等[4-6](表 1)。乳脂中SCFAs研究较多的是丁酸和己酸。本文就乳中SCFAs的来源、分布、组成、生理功能及检测技术进行综述,为进一步检测乳中短链脂肪存在及应用提供科学参考。
乳中SCFAs主要来源于肠道微生物群发酵副产物,并通过循环分配到乳腺以及乳腺的从头合成,涉及乙酰辅酶A羧化酶和脂肪酸合酶等酶[8-9]。动物机体胃肠道微生物将复杂的碳水化合物分解为SCFAs。牛、羊和骆驼等反刍动物利用瘤胃内微生物发酵饲粮纤维产生乙酸、丙酸、丁酸等直链SCFAs,而人和猪等单胃动物则主要通过肠道微生物发酵抗性淀粉、低聚糖等产生[6, 10]。SCFAs在胃肠道的产生路径涉及到乙酰辅酶A、β-羟基丁酰辅酶A、丙酮酸、磷酸烯醇式丙酮酸、琥珀酸、岩藻糖、鼠李糖等(图 1)[11-12]。而支链SCFAs,如异丁酸、2-甲基丁酸、异戊酸、2-甲基戊酸、3-甲基戊酸则主要来源于蛋白质发酵,如缬氨酸、亮氨酸、异亮氨酸,其主要发生于饲粮纤维发酵供不应求的情况下[9-11]。绝大部分SCFAs在瘤胃和肠腔中以游离态SCFAs的形式存在,只有少数以未解离SCFAs的形式存在[13]。随后,SCFAs通过反刍动物瘤胃上皮或单胃动物肠道上皮的亲脂性扩散和单羧酸转运蛋白(MCT)、游离态SCFAs-碳酸氢根(HCO3-)交换、钠离子/氢离子(Na+/H+)交换等特异性载体的运输吸收入血[13-15],血液中的脂类则被乳腺泡毛细血管内脂蛋白酶水解后经基底细胞膜运输至乳腺组织进一步被利用。乳腺摄取血液中游离态SCFAs的能力有限,进入反刍动物血液中的乙酸、β-羟基丁酸等经血液运输至乳腺参与乳中SCFAs的合成,单胃动物则利用血液中的葡萄糖作为乳脂合成的前体物。
SCFAs在乳中主要以酯化形式存在于甘油三酯Sn-1和Sn-3位,部分以游离态形式存在。SCFAs在乳脂中含量较低,动物乳中含量略高于人乳。目前文献对乳中SCFAs的研究主要集中于人乳和动物乳中丁酸和己酸2种含量较高的SCFAs。其中,丁酸和己酸在人乳中含量分别为0.06%~0.60%、0.07%~0.43%[16-17],驴乳中含量分别为0.03%~0.05%、0.20%~0.27%[18],水牛乳中含量分别为1.62%~3.80%、2.09%~2.31%[19],牛乳中含量分别为1.23%~3.45%、1.75%~2.15%[19-20],牦牛乳中含量分别为2.05%~3.19%、2.20%~3.42%[20-21],骆驼乳中含量分别为0.04%~0.06%、0.11%~0.17%[22],山羊乳中含量分别为0.93%~2.09%、2.51%~3.64%[20, 23]。由于现有检测技术的局限,极少数研究对乳中乙酸、丙酸、戊酸和支链SCFAs含量进行了检测。Stinson等[9]对不同国家(美国、澳大利亚、日本、南非、挪威)母乳中SCFAs含量的研究发现,母乳中SCFAs含量与地域有关,不同国家母乳中乙酸、丁酸含量分别为20.4~651.7 μmol/L、32.4~232.1 μmol/L,丙酸、异丁酸、戊酸、异戊酸均未在人乳中检测到。Jiang等[24]测定4种不同类型乳(母乳、婴幼儿配方奶粉、纯牛奶乳、发酵乳)SCFAs的组成并建立指纹图谱,研究发现不同类型乳中乙酸含量较高,为总SCFAs的17.54%~53.36%,丁酸和己酸含量较乙酸低,而丙酸含量极低甚至没有。
2 SCFAs的功能近年来,SCFAs越来越受到人们的关注,尤其在生理功能和生理疾病方面取得了广泛的研究,尽管乳中SCFAs功能的研究仍相对较少。
2.1 生理功能SCFAs是动物机体重要的能量来源,乙酸、丙酸、丁酸是机体重要的供能物质[25],参与宿主能量代谢(脂质、糖类、胆固醇)的调节。SCFAs在被结肠细胞吸收之后,会进入线粒体的三羧酸循环中,为细胞产生ATP和能量,同时促进动物机体水、钠吸收,维持动物机体的电解质平衡[26]。此外,SCFAs充当各种组织中的底物或信号分子,乙酸、丙酸和丁酸被认为是肠道微生物和宿主细胞之间传递信息的纽带,起到调节宿主代谢和免疫功能的作用,是微生物群-肠-脑轴中重要的功能性和生命性成分[27-29],与老年人的认知评分、长期记忆等有关。SCFAs可以通过血脑屏障起到保护作用。丁酸能够通过激活一磷酸腺苷(AMP)激活蛋白激酶的表达,来改变紧密连接蛋白的表达,从而达到增强肠道屏障功能、降低肠道炎症的效果,以此保持肠道的完整性[12]。乳中SCFAs提供了许多乳制品特有的风味和气味,特别是发酵乳制品和乳酪的风味[30]。游离态SCFAs,特别是丁酸,也是牛乳和其他乳制品酸败的原因[1]。
2.2 生理疾病SCFAs在维持健康稳态和各种病理条件的发展都具有重要意义。SCFAs在代谢性疾病、炎症反应、心脑血管疾病方面的作用被逐一揭示。SCFAs表现出广谱的抗菌活性,包括抗炎、抑菌以及抑制结肠炎症和癌类细胞系的生长[31-32];丁酸在维持肠道上皮健康方面具有积极作用,包括对肠道黏膜的保护作用,提高肠道健康和免疫力[31, 33]。SCFAs还与许多代谢疾病相关,如婴幼儿肥胖、高血脂、糖尿病、心脑血管疾病[10, 34]等。SCFAs参与各种体内代谢通路,包括微生物群-肠-脑轴、肠道菌群-SCFAs-免疫轴等,被证实与抑郁症、自闭症、帕金森症、精神分裂症、阿尔兹海默症等一系列精神疾病相关联。乳中SCFAs在生理疾病方面也表现出积极的作用,研究表明婴幼儿早期从母乳摄入的SCFAs可以降低特应性疾病(食物过敏、哮喘、特应性鼻炎等)或肥胖的风险[9],母乳与早产儿定殖的肠道细菌相互作用产生SCFAs,如乙酸、丙酸、丁酸等,在早产儿未成熟的肠道细胞中起到预防过度炎症引起的坏死性小肠结肠炎等作用[35]。综上所述,SCFAs的生理功能和疾病预防方面意义的发现加强了对有效和精确定量方法的需求。
3 乳中SCFAs检测技术SCFAs具有极性大、易挥发、水溶性强、相对分子质量小等特性,对其准确定量提出了挑战。此外,乳中SCFAs存在形式包括结合态和游离态2种[16],进一步增加了其准确定量的困难性。目前,乳中SCFAs的检测方法主要包括气相色谱(gas chromatography,GC)法、液相色谱法、核磁共振等。
3.1 GC法GC法是一种利用气体作流动相的色谱分离分析方法,具有分离能力强、检测器选择简单、定量准确、灵敏度高以及仪器成本相对低廉等优点。由于SCFAs的挥发性,GC法广泛地应用于SCFAs分析。根据GC法连接的检测器的不同,GC法主要分为气相色谱-氢火焰离子检测器(gas chromatography-flame ionization detector,GC-FID)和气相色谱-质谱联用(gas chromatography-mass spectrometry,GC-MS)2种。与GC-FID相比,GC-MS提供了更好的灵敏性和选择性。
Zebari等[36]对脂质进行水提后使用GC-FID测定了乙酸、丙酸、丁酸、异戊酸、戊酸、己酸6种SCFAs含量,研究了乳中脂肪酸组成与泌乳荷斯坦奶牛发情期的关系。然而通过水提法进行GC法分析时可能因SCFAs部分水解而导致回收率降低[37],因此通常在脂质提取后加入盐酸、硫酸、偏磷酸等抑制其水解以达到提高提取效率和SCFAs峰形的效果。Jiang等[24]通过生乳中添加5%盐酸乙醇溶液涡旋振荡离心后上机,建立了检测乳中乙酸、丙酸、丁酸、异丁酸、己酸5种SCFAs含量的方法,并通过检出限、定量限、日间差异、日内差异等指标对该方法的灵敏度、精密度进行方法学验证。该方法优势在于反应快速,前处理简单,结果显示出良好的精密度和灵敏度,表明在不使用有机溶剂的情况下使用酸化水法仍能取得较好的结果。然而由于SCFAs羧基基团极性较强,只进行酸化步骤在进行GC法分析时会出现吸附作用而导致结果重复性较差,特别含量较低(<1 mmol/L)时很容易产生这种误差[38]。乳中SCFAs含量较低,因此在进行GC法分析前必须对酸进行衍生化。衍生化目的在提高目标分析物的热稳定性,降低挥发性脂肪酸的蒸发损失,提高分析分离度和灵敏度,改善色谱保留时间、峰形等,减少样品与色谱柱涂层间的反应。Dai等[16]以甲醇为衍生化试剂,三氟化硼为催化剂,70 ℃水浴5 min来检测不同胎龄婴儿摄入的人乳中SCFAs含量及其在哺乳期的变化。然而,Mannion等[39]以庚烷/乙醚提取脂质,以丁醇作为衍生化试剂,通过三氟化硼80 ℃水浴加热1 h来催化丁酯化反应,测定乳中游离态的丁酸、己酸含量,通过丁酯化与甲酯化的对比,发现SCFAs丁酯具有高分子质量、低挥发性等优点,对易挥发的SCFAs含量测定更准确。丁岩等[40]通过GC法测定发酵乳中7种SCFAs含量,发酵乳振荡离心后调节pH至弱碱性,上清液中依次加入磷酸盐缓冲液、衍生化试剂和丙酮后水浴加热,加入正己烷后通过GC法测定。然而,由于乳中SCFAs存在形式的特殊性以及基质的复杂性,关于乳中SCFAs含量的GC法分析的研究是相对有限的。
3.2 液相色谱法液相色谱法是以液体为流动相通过高压输液系统实现两相分离的监测技术,具有高效、灵敏、分离效率高、选择性好、同时定量多组分、适用范围广等优点。液相色谱法也被应用于各种生物样品中SCFAs含量的检测。
Contarini等[41]通过液相色谱-质谱联用/质谱联用定量驴乳中性脂质和磷脂部分存在的丁酸、己酸,其中丁酸、己酸在中性脂质部分被准确定量,磷脂部分未检出。Liu等[42]通过乳样中加入丁醇、甲醇、氯仿脂肪混提物(3 ∶ 5 ∶ 4),涡旋振荡离心后直接进行液相色谱-质谱联用分析热应激对乳中以甘油三酯形式存在的脂肪酸组成的影响,结果表明,热应激改变牛乳的甘油三酯谱,其中SCFAs-甘油三酯含量下降。通过液相色谱分析乳中SCFAs含量主要集中于甘油三酯含量的分析,是相对有限的,液相色谱也被广泛应用于其他生物基质中SCFAs含量的分析[43-44]。
3.3 核磁共振核磁共振是根据核磁共振效应来确定分子结构的高新技术,具有无损、快速、重现性高、覆盖范围广、制备工艺简单等优点,被广泛应用于代谢组学分析。核磁共振已用于粪便和肠道内容物SCFAs含量的分析,乳中SCFAs含量的分析也有涉及。
Wiking等[45]通过1H核磁共振测定了乳中与风味和气味相关的SCFAs含量,结果表现出良好的相关性。Jensen等[3]和Picariello等[46]通过13C核磁共振分别鉴别不同品种动物乳中和建立高分辨的乳脂掺假合成的中短链甘油三酯含量。Prentice等[10]通过乳样与氘代氯仿混合后振荡离心,利用1H核磁共振测定母乳中结合和游离态SCFAs含量,通过母乳中SCFAs含量来研究其与婴幼儿生长发育的关系。Stinson等[9]通过1H核磁共振对109位母亲产后1个月母乳样品中SCFAs含量进行分析来研究SCFAs含量与地域、特应性状态的联系。
3.4 其他方法除此以外,SCFAs的定量方法也包括毛细管电泳[34, 47]、微生物检测器[48]、红外或近红外检测[49]等。曹玉梅[48]通过微生物检测器测定牛乳样品中的SCFAs含量。Ferrand-Calmels等[49]通过中红外光谱中测算得出牛乳和羊乳中SCFAs组成。
4 小结与展望综上所述,SCFAs在机体生理功能和生理疾病方面具有重要意义。然而,由于乳中SCFAs存在形式的复杂性和不确定性,以及现有检测方法的局限性,乳中SCFAs的准确定量仍然是最复杂和极具挑战性的工作之一。建立简便、快捷、定性和定量准确的SCFAs含量检测方法亟待完成,以此提高我们对乳中SCFAs组成、存在形式、生理功能等方面的认知。
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