风味是传统发酵饮料和调味品的灵魂。同样是白酒,不同产地的白酒风味截然不同,如产于茅台镇的酱香型白酒和产于山西杏花村的汾酒;即使是同一香型白酒,如产于四川的浓香型白酒和产于江苏北部的浓香型白酒,区别也十分明显。食品风味的研究特别是现代风味化学兴起后,主要的研究思路是“分子感官科学”(molecular sensory science)[1]或称“感官组学”(sensomics)[2-4],其路径是风味物的提取(isolation)、浓缩(concentration)、分级(fractionation)(必要时)、色谱分离(separation)、闻香(sniffing)(嗅觉)或口尝(tasting)(味觉)、鉴定(identification)、定量(quantitation)(需绝对定量,非相对定量)、阈值测定(需考察被研究对象的体系或模拟体系)、气味活力值(odor activity value,OAV)或味觉剂量阈值比(dose-over-threshold,DoT)或味觉活力值(taste active value,TAV)计算、气味或味觉或口感重构(recombinate),以及缺失实验(omission experiment)[5-7]。最终通过缺失实验来确定关键香气成分。

在风味化合物研究的系列操作中,最重要的操作之一是风味化合物的提取或萃取,将风味物干扰物或基质进行分离。如果一开始就没有将需要研究的风味化合物提取出来,则后面的所有研究可能不会获得正确的结果甚至是错误的结果。大部分的分离提取技术均可以用于此过程,但更多的研究发现,液液萃取技术可能是传统发酵饮料与调味品风味提取与分离的最佳方法。基于此,本研究针对传统发酵饮品与调味品风味研究时液液萃取技术进行回顾与展望,以期对风味研究人员有所启发。

1 原料预处理技术

在对传统发酵饮料和调味品及其原料和发酵过程进行分析时,其原料形态可能为固态、液态、半流质等状态和含生物活性材料。尽管它们样品的预处理方法是不一样的,但是萃取溶剂选择的原则均是原样品的特征风味化合物、全部风味化合物、目标待测风味化合物必须被萃取出来。

粮食、中药材、曲(大曲、小曲、麸曲)、橡木片、酒醅等固态原料,在进行固态基质分析前,一般要将固态基质中的成分转移到液态基质中,以便于分析。通常情况下,将水、酒精-水溶液、酒精或模拟葡萄汁等作为液态基质。如小曲制作时使用的大青叶和肉桂叶,其香气化合物的浸提溶剂宜选择煮沸5 min后冷却至室温的超纯水[8]。橡木片通常用添加酒石酸(质量浓度为0.7 g/L)和酒石酸氢钾(质量浓度为1.11 g/L)的体积分数为12%的乙醇水溶液浸提[9-10]。在样品浸泡时,通常会使用声波加速浸提[11-13]。因超声波会造成温度上升,故要加冰块维持低温,即冰浴超声[12,14]。如果是胶囊状样品,可用水释放风味物质,再加入溶剂萃取。

醋、酱油、咖啡等液体原料,可以直接使用液液萃取,但直接萃取可能会出现乳化现象或严重乳化现象,需要破乳处理。另外,也常用超滤处理技术分离大分子与小分子化合物[4,15-16]。研究醋中的不挥发性有机酸等风味物质时,应使用先添加乙腈再离心的方法脱蛋白[17]。

酱(豆瓣酱、面酱、酱醅)等半流质原料,需要使用溶剂进行萃取。研究豆瓣酱的风味化合物时发现,用水作为溶剂萃取,其萃取液并不具有原豆瓣酱的风味;进一步研究发现,乙醚萃取液GC-MS图谱中峰的数量最多,且丰度最高,乙醚萃取液与原豆瓣酱样品的整体香气轮廓最接近(图1)[18]。

葡萄、酒曲等含生物活性物质的原料,使用溶剂萃取前应进行冷冻、灭酶等处理。如在研究鲜果葡萄时,需要将果皮与果肉分离,然后用液氮冷冻[12];冷冻的果皮或果肉在液氮中用球形磨粉碎,-80 ℃贮存待分析[19]。含有生物活性酶的曲[14,20]或果蔬[12,21],可通过添加质量分数为1%的无水CaCl2进行灭酶处理。

所有酒类原料和使用酒精-水溶液及纯酒精萃取得到的原料,在进行液液萃取前,需要将酒精度稀释到体积分数为10%～12%,此时,能萃取得到更多的风味化合物[22-24]。

液液萃取目标风味化合物前,需要去除干扰物质。如研究咖啡液的味觉成分时,要先用戊烷萃取去除干扰物质,再用乙酸乙酯萃取。将乙酸乙酯萃取液去除溶剂、冻干,冻干后残留物溶解于水中,然后再用超滤方法依据分子质量分成不同组分[15]。

在研究味觉成分时,应该注意分辨呈味成分是否为挥发性组分。如白酒属于蒸馏酒,白酒的呈味或口感类物质更多的是来源于其挥发性成分,而不是非挥发性化合物[25]。在研究白酒苦味、涩味时,若先用旋转蒸发仪将挥发性与非挥发性物质分开,即可发现白酒的苦味、涩味存在于挥发性馏分中,而不是非挥发性馏分中。

液液萃取研究过程中使用的所有玻璃器皿洗涤时,必须使用无气味洗涤剂浸泡,然后冲洗;必要时,需要超声波洗涤30 min以上。洗净后,用自来水冲洗,再用纯净水冲洗,然后用加热(5～10 min)冷却后的纯净水冲洗,最后高温烘干[26]。

2 风味化合物的液液萃取技术

通常待测特征风味化合物在样品中的含量较低,大部分是mg/L、μg/L甚至是ng/L级的;含量与浓香型白酒中关键香气物质——己酸乙酯(g/L级)相当的化合物是极少数[27]。因此,必须采用适当的技术将目标研究物从样品基质中分离出来,并进行浓缩处理,以使目标研究物含量在仪器(包括鼻腔或口腔测量)检出限范围之内。

液液萃取技术是常用的风味化合物提取分离方法,已经在白酒[28-29]、大曲[14,30]、黄酒[31-32]、葡萄酒香气[33]、葡萄酒苦味[34]、蔬菜[35]等风味研究中得到广泛应用。

2.1 萃取分离原则

样品中挥发性风味化合物液液萃取方法的选择应该考虑以下主要特征[36]。1)待测风味化合物的挥发性和沸点。如白酒中呈现涩味的化合物是挥发性的,而不是非挥发性的。2)待测风味化合物的极性。对于极性化合物需要选择极性萃取剂;反之,则使用非极性萃取剂。3)气味化合物的高温稳定性、氧的影响。4)待测化合物在产品中的浓度以及由此决定的要处理的样品量。5)分析的目的。如定性、定量、特征风味等研究目的。6)待测物质在整个样品中的分布。7)产品的自然状态和总的组成等。

液液萃取目标化合物较理想的结果：1)极性和非极性化合物应该同时萃取,如极性的乙酸能够与非极性的碳氢类化合物同时萃取[26],但要注意到不同的萃取剂对同一待测物的萃取回收率并不一样。如萃取梅鹿辄葡萄酒中β-紫罗兰酮时,二氯甲烷萃取的回收率为91%,乙醚的回收率为93%,V(戊烷)∶V(二氯甲烷)=1∶2时的回收率为73%,V(乙醚)∶V(己烷)=1∶1时的回收率为86%[37]。当不能确定使用何种萃取剂时,可以进行萃取剂选择的优化[14]。2)不会引起风味化合物的热降解、氧化和还原。如饮料酒中的甲硫醇极易氧化为二甲基二硫和二甲基三硫[38]。3)不会引起pH值变化。如呋喃扭尔在pH值为4～5时,没有出现互变异构现象,但超出此pH范围时,会发生互变异构现象[38]。4)不会产生高挥发性化合物的损失。如饮料酒中的乙醛沸点为20.2 ℃,甲硫醇沸点为6 ℃,硫化氢沸点则更低(-60.28 ℃)[26]。这就要求使用的萃取剂应该是低沸点的,才可以很容易地从萃取液中去掉溶剂,且保证样品中的目标化合物没有明显损失。

假如已经知道要提取的样品中有大分子质量或不挥发性物质(如葡萄酒中色素、黄酒中糖和焦糖色素、醋和酱中的呈色物质),就应该选择使用如溶剂辅助风味蒸发(solvent-assisted flavor evaporation,SAFE)的方法,以降低这些物质的干扰及对色谱进样口和色谱柱的污染[12,13,21,33,39-41]。

2.2 萃取液乳化的消除

在液液萃取时,往往会出现乳化现象,可能是由于基质中含有蛋白质、糖、脂肪等化合物。消除乳化现象的方法主要有：1)加盐。用饱和氯化钠溶液代替纯蒸馏水稀释样品[22-23]。2)超声波破乳化。3)高速冷冻离心[8,42]。4)SAFE技术[40]。5)通过玻璃棉过滤乳化液样品。6)通过相过滤法过滤样品。7)加入少量的不同极性有机溶剂。8)固相萃取(solid phase extraction,SPE)和固相盘萃取(solid phase disc extraction,SPDE)阻止乳化的形成。SPE和SPDE还有利于样品的预浓缩和去除样品中的不挥发性物质。9)在样品萃取前,加入硫酸铵可以去除脂肪[42],分析醋时,K4Fe(CN)6·3H2O 和 ZnSO4·H2O 可作为除蛋白的沉降剂(图2)[43]。

3 萃取液的浓缩技术

萃取物通常使用无水硫酸钠或硫酸镁干燥,以除去水分,然后过滤,再去除绝大部分溶剂。可以使用维格勒(Vigreaux)柱蒸馏、旋转蒸发或使用康德尔纳-戴立喜(Kuderna-Danish)浓缩器(KD浓缩器)将其浓缩到10～15 mL,接着使用氮吹仪浓缩到 1～2 mL,再进一步浓缩到250 μL[22,44]。

对于非挥发性的组分主要采用真空旋转蒸发[45]或冻干的方法进行浓缩[46]。冻干适合于对温度敏感的化合物。

4 萃取液的分级技术

大部分情况下,萃取液成分众多,甚至可达上千种,直接进行GC-MS或GC-O分析时,会出现较多重叠峰,且香气互相干扰(如无气味的棕榈酸乙酯与烟熏气味的2,6-二甲氧基苯酚重叠[43])。为此,需要对萃取液进行分级(fractionation)。通常根据样品的复杂程度,可将萃取液分级为酸性组分、碱性组分、水溶性组分和中性组分[20,47-48](图3),或分级为酸性-水溶性组分、中性组分和碱性组分[27,31,44,49-53],或分级为酸性组分、水溶性组分和中碱性组分[54],或分级为酸性-水溶性组分和中碱性组分[5,18,22,28,33,43,55-56]。

如果中性组分比较复杂,可以再去除如羰基化合物等的干扰,或直接按组分极性进行柱色谱分离(通常是硅胶柱、正相柱色谱分离)[22,28,47,52,56-57],然后用GC-O闻香分析或GC-MS分析(图 4)。复杂的酸性组分也可以使用硅胶柱分级[41],具备条件的,可以使用正相液相色谱(normal phase liquid chromatography,NP-LC)结合紫外可见光检测器进行分离[58]。凝胶吸附色谱(gel adsorption chromatography)可以用于多种产物的分离,通常使用葡聚糖凝胶分离,如橡木乙醇提取物[59]、食醋低分子量化合物等[16,60-61]。2,4-二硝基苯肼(2,4-dinitrophenylhydrazine,2,4-DNPH)、丹磺酰肼(dansylhydrazine)、O-(2,3,4,5,6-五氟苯)羟胺 [O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine,PFBHA] 常被用来减少萃取物中羰基化合物干扰。

如果呈味或口感类物质是挥发性的,就可以采用图 5方法进行提取与分级[62]。首先判断这些化合物的分布,然后采用半制备或制备液相色谱分离。水相除溶剂,采用真空旋转蒸发的方法。

5 结束语

风味研究的前提是将欲研究的目标物与干扰物分离,在精准获得目标物后,才能实施香气或味觉鉴定(包括定性、定量)。虽然用于提取、分离香气或味觉及口感类化合物的方法很多,但是无论采用何种分离方法与手段,提取或分离后均需要进行确认,通过人的感觉器官来进行判别。在几乎所有的分离与提取方法中,液液萃取是最常用的、最有效的方法。液液萃取前的原料预处理、液液萃取方法的选择原则、萃取液乳化的消除、萃取液浓缩技术、萃取液的分级都是传统发酵饮料与调味品中风味化合物分析的影响因素。通过本研究对传统发酵饮料与调味品中风味化合物液液萃取技术的回顾,希望对风味研究人员的研究思路有一定的启发。

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Liquid-Liquid Extraction Technology for Aroma and Flavor Compounds in Traditional Fermented Beverages and Seasonings

FAN Wenlai

(School of Biotechnology,Jiangnan University,Wuxi 214122,China)

Abstract：The aroma and flavor of traditional fermented beverages and seasonings is an important aspect of determining their quality.The use of modern flavor chemistry theory and methods to study their aroma and flavor has been recognized by the industry.One of the most important operations in the series of studies on aroma and flavor compounds is the isolation or extraction of flavor compounds,which involves the isolation of flavor interfering substances or matrices.If the target flavor compounds could not be extracted at the sampling,the correct results could not be obtained.Most isolation and extraction techniques can be used for this process,but some researchers have found that liquid-liquid extraction technology could be the best method for extracting and isolating the aroma and flavor of traditional fermented beverages and seasonings.Before liquid-liquid extraction,the pre-treatment methods of solid,liquid,semi-liquid,and bioactive raw materials are different.The principle for selecting the extraction solvent is that the characteristic aroma and flavor compounds,all flavor compounds,and target flavor compounds of the original sample must be extracted.The selection of liquid-liquid extraction methods should consider the volatility,boiling point,polarity,stability,concentration,distribution in the sample,and other characteristics of the flavor compounds to be tested.The ideal liquid-liquid extraction result is the simultaneous extraction of polar and non-polar compounds,without causing thermal degradation and redox of flavor compounds,without changing pH value,and without significant loss of high volatile compounds.Due to the presence of compounds,such as proteins,sugars,and fats in the matrix,emulsification often occurs during liquid-liquid extraction.Demulsification could be achieved through methods such as salt addition,ultrasound,freeze-centrifugation,solvent assisted flavor evaporation (SAFE),glass wool filtration,phase filtration,addition of solvents with different polarity,and addition of precipitant.Due to the complex composition of aroma and flavor compounds in the extraction solution,direct GC-MS or GC-O analysis could result in overlapping peaks of compound coeluting and interference of aromas.The extraction solution could be fractioned into acidic,alkaline,and neutral fractions,and further analysis could be carried out by combining chromatographic separation techniques such as silica gel column or normal phase column.The systematic reviews of the liquid-liquid extraction technology of traditional fermented beverages and seasonings flavors could inspire the research ideas of flavor researchers.

Keywords：preprocessing;liquid-liquid extraction;emulsion;fractionation;aroma compounds

中图分类号：TS264.2

文献标志码：A

doi：10.12301/spxb202300257

文章编号：2095-6002(2023)03-0019-09

引用格式：范文来.传统发酵饮料与调味品中风味物质的液液萃取技术[J].食品科学技术学报,2023,41(3)：19-27.

FAN Wenlai.Liquid-liquid extraction technology for aroma and flavor compounds in traditional fermented beverages and seasonings[J].Journal of Food Science and Technology,2023,41(3)：19-27.

收稿日期：2023-05-01

基金项目：国家重点研发计划项目(2022YFD2101201)。

Foundation：National Key Research and Development Program of China (2022YFD2101201).

作者简介：范文来,男,研究员,主要从事风味化学、品质安全和副产物再利用等方面的研究。

(责任编辑：李 宁)