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文章信息
- 高传强, 阳飞, 张华山
- GAO Chuan-Qiang, YANG Fei, ZHANG Hua-Shan
- 芝麻香型白酒微生物菌群及风味物质研究进展
- Advances in microbial flora and flavor components of sesame flavor liquor
- 微生物学通报, 2017, 44(4): 940-948
- Microbiology China, 2017, 44(4): 940-948
- DOI: 10.13344/j.microbiol.china.160388
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文章历史
- 收稿日期: 2016-05-15
- 接受日期: 2016-09-27
- 优先数字出版日期(www.cnki.net): 2016-09-29
2. 工业发酵湖北省协同创新中心 湖北 武汉 430068;
3. 安徽宣酒集团股份有限公司 安徽 宣城 242000
2. Hubei Collaborative Innovation Center for Industrial Fermentation, Wuhan, Hubei 430068, China;
3. Xuanjiu Group Co. Ltd., Xuancheng, Anhui 242000, China
芝麻香型白酒以高粱、小麦、麦麸等为原料经过传统固态发酵、蒸馏、陈酿勾兑而成[1]。芝麻香型白酒是建国后通过学创结合、自主创新的两个香型之一,兼具浓香型的醇厚绵柔、清香型的典雅清新、酱香型的细腻幽雅三大风格特点,但在风味上又别具一格,有着炒芝麻的特有香气。芝麻香型白酒还可以作为其他白酒的调味酒,通过增加杂环类物质,令酒体香气更加丰富[2-3]。近年来,随着芝麻香技术研究的不断深入,其工艺愈来愈成熟可靠,产品的风格质量也由原来的飘乎不定变得越来越稳定,其典型风格质量同样也在发生一系列的变化,在保持其香气幽雅的基础上,克服最初的“苦、涩、酸、杂”等不足,向着绵甜、细腻、净爽的风格方向转变[4]。芝麻香因特有的幽雅复合香,在现代白酒香型融合的背景下,发挥着越来越大的功能与作用,深受广大消费者的青睐,市场发展前景广阔[1]。芝麻香型白酒的典型代表生产企业有山东扳倒井股份有限公司、山东泰山酒业集团有限公司、山东济南趵突泉酿酒有限责任公司、山东景芝酒业集团有限公司、山东泰山生力源集团股份有限公司、河南仰韶酒业有限公司、江苏今世缘酒业股份有限公司、江苏泰州梅兰春酒厂有限公司以及安徽宣酒集团股份有限公司等。虽然芝麻香型白酒酿造工艺日益成熟,但芝麻香味酒的风格特征和批次质量并不稳定,此外,芝麻香型白酒酿造机理和呈香呈味机理目前还不是很清楚[1]。
1 芝麻香型白酒酿造工艺与特点芝麻香型白酒工艺具有泥底砖窖、清蒸续渣、高氮配料、高温堆积、高温发酵、多微共酵、分层蒸馏、长期贮存以及科学勾兑等特点[1, 4]。泥底可增加浓香型的风味成分,使其在一定程度上兼具浓香型的特点,窖壁采用砖或石材,有利于控制己酸菌等微生物的生长与代谢,从而突出芝麻香风格;清蒸有利于排除杂味,保证芝麻香香气的清雅与纯正,续渣不仅利于促进香味成分的生成与积累,还可以调整糟醅酸度,控制适宜的发酵速度;高氮配料为特征香气以及美拉德反应和细菌的蛋白质发酵提供重要的前体物质;高温堆积为耐高温的微生物提供合适的生长环境,调整微生物种群间的分布,并代谢产生一定的芝麻香风味物质及风味前体物质;高温发酵抑制有害微生物的同时保证糖化酶和蛋白酶的高效率,促进蛋白质的分解代谢,是芝麻香典型风味物质形成的重要环节;多微共酵通过细菌、酵母菌以及霉菌生长发酵产酶协同作用,在产酒的同时形成大量的风味物质;分层蒸馏可以实现分级贮存、合理勾兑以及分型勾兑;入库后的原酒通过3年以上的长期贮存,发生一系列的物理及化学变化,特别是分子间缓慢的化学反应把发酵与蒸馏过程所形成的风味物质进一步转化为芝麻香特征风味成分。
2 芝麻香型白酒酿造微生物菌群芝麻香型白酒酿造采用“大麸结合”,使用传统高温大曲与纯培养的麸曲结合作为糖化发酵剂。酿造芝麻香型白酒的重要微生物来源于高温大曲、纯培养的麸曲,并与原料和高温环境密切相关,因此微生物群落组成复杂。鉴于此,对芝麻香型白酒酿造过程微生物的研究主要基于现代生物化学、生物标记以及分子生物学技术手段,从而克服传统的微生物生态学研究技术的局限性,主要分析方法包括群落水平生理指纹法 (CLPP)[5]、磷脂酸图谱法 (PLFA)[6]、Biolog微平板技术[7]、脂肪酸甲酯图谱分析法 (FAME)[8]、醌指纹图谱法 (QP)[9]、细菌总DNA提取法[10]、变形梯度凝胶电泳法 (DGGE)[11-12]、温度梯度凝胶电泳法 (TGGE)[8]、限制性片段长度多态性分析法 (RFLP)[13]、单链构象多态性分析法 (SSCP)[14]、16S rRNA基因文库构建[15]、核糖体DNA扩增片段限制性内切酶分析法 (ARDRA)[16]以及系统法与分析法[17]等。酿造芝麻香型白酒的重要微生物由以高温细菌为主的特殊微生物菌群构成,优势菌群[18-21]包括细菌、酵母菌和霉菌,细菌菌落较小,繁殖速度快;酵母菌具有产酒精、产酸和产酯能力;霉菌菌落较大,主要产生复合酶系。
2.1 细菌研究表明,芝麻香型白酒酿造过程细菌[8, 12, 15, 22]有Acetobacter、Acinetobacter、Bacillus、Brevibacterium、Corynebacterium、Lactobacillus、Pseudofulvimonas、Propionibacterium、Pediococcus、Solitalea、Saccharothrix、Streptococcus、Staphylococcu、Thermoactinomyces、Virgibacillus、Weissella,在堆积过程中的优势菌为Bacillus、Thermoactinomyces,堆积后期的次优势菌为Acetobacterium、Lactobacillus。徐泽江[8]研究发现酒曲中存在约翰不动杆菌 (Actinotobacter johnsonii)、琼氏不动杆菌 (Actinotobacter junii)、葡萄球菌属以及片球菌等,其中葡萄球菌属主要分解蛋白质和脂肪,片球菌能在高温、高酒精度和低pH条件下生长。
通过对芝麻香型白酒酿造过程相关细菌的研究,对某些细菌的代谢作用也有一定的了解 (表 1)。芝麻香酿造用曲中一般采用一定比例的高温大曲和细菌麸曲,而细菌麸曲所使用的菌种一般也是从高温大曲中分离纯化出来,细菌在芝麻香型白酒酿造过程中代谢产物包括丁酸、乳酸、醋酸、正丁酸、酪氨酸、酮类、缩醛类等,以及种类繁多的杂环化合物,主要包括烷基吡嗪类、呋喃类、吡喃类、吡啶类等[4]。
细菌种类 Bacterial species |
代谢酶系及生化作用 Metabolic enzyme systems and biochemical function |
Bacillus licheniformis | Producing enzymes of acid protease and amylase; hydrolysis of starch and protein with the products of glycerol, 2, 3-butanediol, acyl-polypeptide and fructan, in order to promote Maillard Reaction[16, 20, 23-25] |
Bacillus subtilis | Producing protease decomposing wheat ban to 2, 3-butanediol, 4-methylpyrazine and acetylmethylcarbinol in order to promote Maillard Reaction and generate flavor components[23, 26-28] |
Thermophilic actinomycetes | Addicted to high temperature properties; producing cytase, thermophilic amylase, neopullulanase, alkaline phosphate enzyme, esterase, naphthol-AS-BI-phosphoric acid hydrolase and lipid esterase[13, 16, 29] |
Bacillus stearothermophilus | Strong thermostability of enzyme producing, decomposing carbonhydrates to L-lactic acid, methane acid and small amount of ethanol[13, 23] |
Schlegelella aquatica | Producing alkaline phosphate enzyme, esterase, lipid esterase, leucine aromatic amine enzyme, valerian aromatic amine enzyme, naphthol-AS-BI-phosphoric acid hydrolase and α-glucosidase[16] |
Lactobacillus | Promoting and maintaining the liquor-making microbes ecological environment, producing catalase and generating the precursor components of lactic acid and amino acids for esterification reaction in order to promote Maillard Reaction[13, 28] |
Klebsiellosi | Producing lipase; improving the content of ester substances, balance of acids, alcohols and esters; shortening the time of Aging of liquor wine; giving fullness and soft aroma[30] |
酵母菌除生成酒精外,还具有较强的产酸、酯、高级醇、含硫化合物以及醛类等能力,从而增进酒体香味,提高酒的品质[18]。研究表明,酵母菌[8, 15, 23]包括Pichia、Hansenula、Saccharomyces、Candida、Torulaspora、Torulopsis、Wickerhamomyces,其中在堆积前期表层优势菌为Saccharomyces。酿酒酵母是主要的产酒酵母,发酵速度快、繁殖能力强、耐酸耐酒精、适应性强、出酒率高[8]。生香酵母主要包括汉逊酵母、假丝酵母、意大利酵母、地衣酵母、球拟酵母、异常汉逊氏酵母、Wickerhamomyces等[20, 28, 31],产酯生香能力强、耐高温,其中汉逊酵母酱香明显,假丝酵母酯香浓,球拟酵母焦香大,意大利酵母和地衣酵母主要产有机酸、酯类、醇以及硫甲基丙醇等,异常汉逊氏酵母主要同化硝酸盐、氧化烃类,在高温条件下具有较强的发酵能力和酯化能力。张彬等[32]对泰山酒业集团芝麻香酒研究表明东方伊萨酵母、酿酒酵母、异常毕赤酵母以及假丝酵母等通过新陈代谢和死亡自溶为美拉德反应提供单细胞蛋白,从而赋予白酒独特的鲜香味。朱双良等认为,存在着与高温环境相适应的耐受高温环境的酵母,与高温细菌相互影响、互为补充、交替作用、相互促进、协同配合,同时相互制约、互为牵制,产生许多新的风味物质,说明酵母在高温堆积过程中对芝麻香风味物质的形成有重要作用,而非一直以来只强调高温细菌的作用[1, 4, 33]。
2.3 霉菌霉菌是芝麻香型白酒生产过程中必不可少的糖化菌株[34],产酸产香能力高,通过产生丰富的酶系降解淀粉、蛋白质等大分子物质,产生醇类、高级酯类以及多碳酸类等。但霉菌一旦过多会出现大范围板结现象,使酒醅产生霉味,最终导致白酒具有霉味与苦味[35](表 2)。霉菌包括Aspergillus、Mucor、Rhizopus、Monascus、Trichoderma,其中优势菌为Aspergillus。
霉菌种类 Mould species |
代谢酶系及生化作用 Metabolic enzyme systems and biochemical function |
Hanoi baiqu | Producing glucolase, α-amylase, carboxypeptidase, hydroxyl-peptidase and acid protease, with properties of high yield acid producing and acid resistant, hydrolysis the starch and protein of fermenting materials, and generate flavor components[28] |
Monascus | Producing α-amylase, glucoamylase, esterification synthase and proteolytic enzymewith the function of additive acid and esterification reaction; producing the precursor components of flavor such as amino acid, polyunsaturated fatty acids, γ-aminobutyric acid, succinic acid and so on[13, 36] |
Aspergillus orzyae | Producing acid protease and carboxypeptidase; controling the growth of mixed bacterium in the brewing process; inhibiting bacterium and yeast with over much acid producing[34, 37] |
Aspergillus niger | Producing acid protease, aminopeptidase, neutral protease, cellulose, hemicellulose, lipase, proteolytic enzymeand lignocellulose; catalysising ester bond formation and producing amino acid, peptide and ester aroma substances[20] |
Aspergillus flavus | With strong ability of protein decomposition and liquefaction power; the ability of saccharification is lower than Aspergillus niger |
Rhizopus | Breeding fast; with strong ability of saccharification and acid resistant; hydrolysis of starch to acetoin, n-pentanol, biphenyl, dimethyl phthalateand 2, 3, 4, 5-tetramethylpyrazine[20] |
Cladosporium herbarum | Producing ethyl acetate, pyridine, n-pentanol, 2, 3-butanediol, dimethyl cyolohexlemine, dimethyl phthalate, and small amount of tetramethylpyrazine[20] |
高传强[1, 4]认为,河内白曲作为芝麻香酿造糖化发酵剂的重要组成部分,具有酸性蛋白酶含量高、生酸能力强、耐酸耐酒精能力强及糖化力相对较高等特点。酒中的高级醇主要来源于酸性蛋白酶分解蛋白质所生成的氨基酸、高级醇及其派生出来的酸、醛、酮、酯等,都是白酒香味的重要组分;芝麻香型白酒的重要香气物质如吡嗪类更离不开酸性蛋白酶的作用;较强的产酸能力,对芝麻香在高温发酵时有效地控制杂菌的生长,保证酒体的清雅纯正也起着重要的作用。
2.4 微生物种群间的相互作用与影响种群是微生物的基本生存单位,执行相关代谢功能的种群组合在一起形成了微生物系统。芝麻香酒采用的是“大麸结合、多微共酵”工艺,所采用的糖化发酵剂由高温大曲、中温大曲、细菌麸曲、酵母麸曲、霉菌麸曲等组成,不同的曲所带入的微生物种群不同,加之开放式的生产工艺,酿造环境中微生物必将进入发酵体系,从而形成了酿造微生物的多样性与复杂性。经过多年的实践与探索,高传强[4]认为,芝麻香的工艺特征与酱香型有着很大的相似性,因此对酱香型酿造微生物区系平衡的研究成果同样对芝麻香型酿造工艺有着指导意义。在芝麻香的堆积及发酵过程中微生物种群间相互影响,导致代谢产物的多样性与复杂性,也给研究带来很大的困难;不依赖于传统的分子生态学技术,能实现系统中微生物的原位分析,认识微生物结构与功能的关系,通过调控种群结构达到系统功能的最佳状态,从而建立适用于中国白酒微生物菌群研究的新方法。
3 芝麻香型白酒风味物质芝麻香型白酒中的风味成分相当复杂,它们的含量和量比关系决定了白酒的风格特点。随着分析检测方法的改进,从纸层分析、柱色谱、气相色谱与质谱联用到多维气相色谱-质谱,通过GC法、GC-O法、GC-NPD法、GC-MS法、GC-MS/MS法、GC-FPD法、GC-TOF-MS法、UA-DLLME-GC-FPD、HPLC法、HPLC-MS/MS法、HS-SPME-GC-MS法等[20, 38-48],对芝麻香型白酒风味物质成分的报道越来越多 (表 3)。研究表明,景芝白干芝麻型白酒的风味成分检出达163种,生力源芝麻香型白酒风味成分检出达51种,梅兰春芝麻香型白酒风味成分检出达121种。刘传贺等[46]在白酒“风味轮”感官术语的基础上将感官定量描述分析方法与传统评价方法相结合对12款具代表性芝麻香型白酒进行感官评价,统计筛选出芝麻香型白酒的感官特征描述语,包括8个香气描述语、3个口味描述语和5个口感描述语,以评分较高的产品为标准构建风味剖面图,建立芝麻香型白酒可视化感官特征标准,为建立白酒感官质量标准提供了理论和研究基础。
风味物质 Flavor components |
成分 Composition |
作用 Function |
Acids[8, 41] | Acetic acid, Caproic acid, Lactic acid, Butyric acid, Propionic acid, Isobutyric acid, Isovaleric acid, Octanic acid, Linoleic acid, Linolenic acid, Arachidonic acid, Vanillic acid, 3-methybutanoic acid | Increasing alcohol sweet taste, acid feeling, soft sense and after taste |
Esters[18, 41, 44] | Ethyl acetate, Ethyl lactate, Ethyl caproate, Ethyl butyrate, Ethyloctanoate, Ethyl decanoate, Ethyl oenanthate, Ethyl valerate, Vanillic acid ester, γ-nonalactone, Propionate, Tetradecanoic acid esters, Palmitic acid esters, Lauric acid esters, Linoleate, Benzoic acid esters, Phthalate esters | Aroma composition of skeleton, releasing complex smell with fruits, flowers and sweet |
Alcohols[41] | n-propyl alcohol, Isoamyl alcohol, Isobutanol, 2, 3-butanediol, Glycerol, Mannitol, β-phenethyl alcohol, 2-heptanol, 3-methylbutanol, 3-octanol, 1-butanol, Acetal ethylene glycol, polyhydric alcohols | Adding full-bodied taste, aftertaste and soft-sweet sense |
Heterocyclic[28, 41, 49-51] | 2-methylpyrazine, 3-methylpyrazine, 4-methylpyrazine, 2, 6-dimethylpyrazine, 2, 3, 5, 6-tetramethylpyrazine, alkylpyrazine, Acetylpyrazine | With unique coke-aroma, gives fullness and soft aroma |
Aldoketones[18, 20] | Ethanal, Aldehyde acetal, Propaldehyde, Furaldehyde, Isovaleraldehyde, Vanillic aldehyde, Nonanal, 3-methylbutyraldehyde, 2, 3-dimethylvaleraldehyde, 2, 3-butanedione, 3-hydroxylbutanone | Enhancing full-bodied taste |
Sulfides[18, 43, 52-53] | Methanthiol, Ethanethiol, Methylthiopentanol, 3-methylthiopropanol, 3-methylthiopropyl aldehyde, Methylthiopropionate, Dimethyl disulfides, Dimethyl trisulfides, Methylthio acetate, Furfurymercaptan | With onion aroma |
Else[41, 44] | 4-ethylguaiacol, 4-methylguaiacol, 4-ethenylguaiacol, 4-methylphenol, 4-ethylphenol, 4-ethenyl-2-metoxyphenol, Metoxyphenol, α-cedrene, α-terpineol | Contribute to the formation of coke-aroma, elegant aroma with sandal wood and flowers |
由于芝麻香型白酒酿造一般采用大麸结合、多微生物协同发酵工艺,因此芝麻香香味成分相对复杂,主体香可能是在醇、醛、酸、酯达到一定比例后再辅以吡嗪类、呋喃类、酚类等杂环化合物以及含硫化合物而形成[20, 33, 41, 44, 54-55]。因此,芝麻香白酒具有“芝麻香突出,优雅丰满,圆润细腻,香味协调,回味悠长,空杯留香持久”的特点。酸类物质主要通过微生物糖酵解途径或者氨基酸脱氨作用产生,主要的呈味物质在酒体中起平衡协调作用[4];酯类主要通过酸与醇类酯化反应产生,是形成酒体香气浓郁的关键因素,高级酯属于发酵后期的次生代谢产物,为芝麻香型白酒提供水果香味以及类玫瑰香味;醇类也是呈味物质,主要由蛋白质的代谢产生且在芝麻香中醇类物质总的含量也较高[1],同时也是酯类前体物质,芳香成分之一,含量过低会导致白酒风味不足;杂环类主要由美拉德反应、蛋白质热分解、氨基酸热分解及微生物代谢等多种途径生成,大幅度提高芝麻香型复杂香味成分,对其它香味具有显著的烘托和叠加作用;醛酮类主要通过氨基酸脱氨、脱羧和醇氧化、酮酸脱羧等反应产生,对香味起辅助作用;含硫类化合物主要通过美拉德反应以及甲硫氨酸酵母菌转化,具有强烈的风味。高传强等[51-52, 56-58]研究表明,适量的己酸乙酯对芝麻香的放香具有较好的烘托效果,同时对酒体的绵甜感、细腻度和适口性也起到了十分重要的作用,四甲基吡嗪在芝麻香酒的含量仅次于酱香型酒和兼香型酒,不仅是中国白酒风味相关的重要化合物,同时具有特殊的药理作用,赋予中国白酒有益健康的功能,对芝麻香典型风格的形成具有重要的影响。
4 展望通过研究芝麻香型白酒酒醅微生物群落组成变化,利用分析化学结合感官品评的现代风味化学研究手段全面深入剖析风味成分以及对酒体风味贡献作用大小,深层探索微生物菌群生长环境理化因子与芝麻型白酒质量关键风味物质变化的动态关系,揭示酿造过程动力学、代谢途径以及风味物质的形成与积累机理,可为深入揭示和阐明芝麻香型白酒的发酵机理奠定理论基础,从而更好地指导生产,服务企业。
今后芝麻香型白酒研究主要应从以下三个方面着手:(1) 应用更先进的分析仪器和方法,以风味化学为指导,进一步研究芝麻香特征相关风味物质,明确典型或特征风味物质成分;(2) 深入研究典型或特征风味物质成分与微生物菌群的关系,应用风味导向技术筛选及采用基因工程定向优选特定功能的微生物,既具有产风味物质的功能,同时又具有产健康功能的活性因子,应用纯培养技术及现代化的设备提高其产量与质量;(3) 开展芝麻香在香型融合方面应用研究,发挥其与其它香型白酒融合方面的作用与功能。
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