酱香型白酒是中国传统白酒的四大香型之一,以其独特的酱香风味和复杂的口感著称,其风味特征表现为酱香突出、幽雅细腻、酒体醇厚、回味悠长,香气复杂,包含酱香、焦香、糊香、花果香等多种风味[1]。酱香型白酒的发酵过程依赖于复杂的微生物群落,主要包括霉菌、酵母菌和细菌,其菌群组成和相互作用与酱香型白酒的风味特征密不可分[2]。堆积发酵是酱香型白酒生产中的关键工序之一,具有网络酿造环境中微生物的作用,被称作为“二次制曲”[3]。其中酿造3～5轮次基酒产量占比大,是酱香型白酒生产的黄金轮次,也称为“大回酒”轮次,其产质量的好坏直接决定了一年的生产效果[4]。已有研究表明,“大回酒”轮次酒醅中优势微生物群落组成存在差异,并且其演替规律存在时空效应,对不同轮次基酒风格的形成具有重要作用[5-6]。

传统酱香制酒生产劳动强度大、人工成本高,通过采用机械化、自动化方式不仅可以减少人力成本,而且还可以提高生产效率。目前一些酒企如黄酒、米酒等已基本实现机械化、自动化酿造[6-7],对于白酒酒企而言,浓香型、清香型、酱香型酒企均已在开展机械化、自动化研究,但由于酿造工艺的差异和复杂程度不同,不同香型酒企间机械化、自动化生产方式存在差异[5,8-9]。研究表明机械化生产由于引进大量设备,在工艺上对发酵物料的理化特性具有重要影响,造成水分、酸度以及含氧量的变化不同,从而导致微生物数量以及种类的不同,最终可能影响风味化合物的生成[10-12]。因此,研究机械化与传统酿造在酒醅堆积发酵过程中酒醅微生物的群落演替差异,对酱香型白酒的数字化、智能化转型具有重要的意义。课题组前期研究表明,机械化和传统生产班组三轮次堆积发酵过程中,优势细菌和真菌不存在显著差异,但机械化生产对优势真菌群落组成具有显著影响,并且理化因子与优势微生物的关联作用也存在差异[12]。“大回酒”轮次在生产时间上存在差异,但目前尚未见关于酱香型白酒机械化与传统“大回酒”轮次微生物多样性差异比较分析的系统研究报道,未对轮次生产带来的时间差异进行研究。

基于此,本研究以酱香型白酒机械化和传统酿造“大回酒”轮次酒醅为样品,采用高通量测序技术,并结合多元统计分析对微生物多样性差异进行比较分析,以期阐明2种生产方式下“大回酒”不同生产轮次微生物群落结构和主要优势菌群的变化和差异,并解析酒醅理化指标变化与优势微生物菌群的相关性异同,旨在为持续优化酱香型白酒的机械化酿造工艺及基酒品质提升提供理论依据。

1 材料与方法

1.1 样品与试剂

酒醅样品取自贵州省遵义市某酱香型白酒酒厂生产车间(CT:传统车间,FJ:机械化车间)的3、4、5生产轮次堆积过程发酵酒醅。为保证采样的对应性、均一性和代表性,每个轮次按堆积完堆(I)、堆积中期(M)及入窖(L)3个阶段进行采集,并按图1方式,将6个点位(A、B、C、D、E、F)的样品均匀混合后视为一份综合样。

1.2 仪器与设备

SW-CJ-2F无菌操作台,苏州净化设备有限公司;MIKRO 220R台式高速冷冻离心机,德国Hettich公司;AR2130/C电子天平,奥豪斯上海公司。

1.3 实验方法

理化指标分析参照T/CBJ 004—2018《固态发酵酒醅通用分析方法》对酒醅水分、总酸、还原糖含量和pH值进行测定[13]。

基因组提取采用间接提取法,首先称取10.0 g酒醅样品于50 mL无菌离心管中,后加入15 mL已灭菌且冷却后的0.1 mol/L PBS(pH值=7.3),并加入3颗已灭菌玻璃珠,涡旋振荡5 min,于300×g,4 ℃离心5 min,收集上清液,重复3次收集全部的上清液。将收集的上清液于9 000×g,4 ℃离心3 min,弃上清液,收集细胞沉淀,并用5 mL PBS洗涤细胞沉淀3次,最后使用1 mL PBS重悬菌体,微生物基因组采用Ezup柱式基因组DNA试剂盒对菌株DNA进行提取[14],具体操作流程见试剂盒说明书。

高通量测序采用Illumina MiSeq测序平台,分别对细菌16S rRNA基因V3～V4区和真菌ITS3～ITS4区进行高通量测序分析。参考本课题组报道的方法[15],细菌扩增测序采用通用引物341F(CCTACGGGNGGCWGCAG)和805R(GACTACHVGGGTATCTAATCC);真菌扩增测序采用通用引物ITS3(GCATCGATGAAGAACGCAGC)和引物ITS4(TCCTCCGCTTATTGATATGC)。PCR扩增条件为:95 ℃预变性3 min;94 ℃变性20 s,55 ℃退火20 s,72 ℃延伸30 s,25次循环;72 ℃终延伸5 min降至4 ℃。PCR体系为:2×Hieff®Robust PCR Master Mix 15 μL、正向及反向引物各1 μL、DNA模板20～30 ng,使用ddH2O补齐体系至30 μL。高通量测序分析由生工生物工程(上海)股份有限公司完成。

1.4 数据处理

通过16S rRNA和内转录间隔区(internal transcribed spacer,ITS)测序获取酒醅样本的微生物群落数据(属水平),计算相对丰度矩阵,利用Origin 2021软件对微生物相对丰度进行绘图。通过Kruskal-Wallis和Wilcoxon检验(P<0.05),对线性判别分析(linear discriminant analysis,LDA)阈值评分大于3的组分进行线性判别分析效应大小(LDA effect size,LEfSe)分析。使用联川生物平台进行Mantel-test以及冗余分析(redundancy analysis,RDA)的绘制。

2 结果与分析

2.1 理化指标分析

堆积酒醅各理化指标含量变化如图2所示,通过对酱香型白酒3～5轮次堆积发酵阶段理化指标分析发现,随着轮次生产推进,酒醅水分整体呈上升趋势,且传统车间酒醅水分(46.64%～55.76%,平均50.94%)整体高于机械化车间(42.56%～52.96%,平均47.39%),而同一发酵轮次出窖酒醅水分含量低于入窖酒醅水分,与王贵军等[16]的研究结果一致。传统车间酒醅酸度3轮次最低(21.78～24.10 mmol/100 g),4轮次开始上升(21.28～32.63 mmol/100 g),5轮次达到最大(34.05～35.47 mmol/100 g),机械化车间酒醅酸度在3轮次时最低(17.6～24.56 mmol/100 g),4轮次时上升至最大值(36.89～38.78 mmol/100 g),5轮次下降至26.95 mmol/100 g。酒醅发酵过程中还原糖呈现“堆积积累”的现象,堆积发酵后期酒醅还原糖含量上升,这与梁泺等[17]的研究一致,传统车间(1.35%～1.68%)酒醅还原糖含量低于机械化车间(1.48%～2.40%)。3～5轮次传统和机械化车间酒醅pH值维持在3.77～3.95,处于较为稳定的pH环境。

2.2 测序质量与微生物多样性分析

通过高通量测序技术分析,从传统车间酒醅样品细菌16S rRNA V3～V4序列共获得606 471条有效读段,机械化车间共获得568 281条有效读段;传统车间酒醅真菌ITS序列获得892 903条高质量读段,机械化车间酒醅真菌获得688 666读段,结果表明,整体上传统车间微生物的种类多样性高于机械化车间,符长彪等[18]对浓香型白酒机械化酿造的研究也表明传统车间微生物种类比机械化车间更加丰富。在3～5轮次堆积发酵过程中,Shannon指数和Chao 1指数也存在差异,如表1所示,不同轮次不同阶段堆积发酵过程中微生物群落结构发生动态变化,此外,所有样品测序细菌和真菌操作分类单元(operational taxonomic unit,OTU)覆盖率均在0.99以上,说明测序可覆盖样本中绝大多数的微生物种群信息,测序深度适合。

2.3 机械化与传统细菌群落组成和差异分析

如图3-A、图3-C、图3-E所示,传统和机械化车间3～5轮次发酵过程中未分类_芽孢杆菌属_2(unclassified_Bacillaceae_2)占据优势地位,其次是未分类_高温放线菌属_1(unclassified_Thermoactinomycetaceae_1)。传统车间3轮次发酵前中期相对丰度前3的细菌为unclassified_Bacillaceae_2(41.8%～58.5%)、unclassified_Thermoactinomycetaceae_1(14.9%～22.6%)和大洋孢杆菌属(Oceanobacillus)(4.1%～12.1%),而后期则转变为未分类_乳杆菌属(unclassified_Lactobacillales)(40.8%)和unclassified_Bacillaceae_2(35.3%)。机械化车间在整个3轮次相对丰度前3的细菌始终是unclassified_Bacillaceae_2(43.1%～62.9%)、unclassified_Thermoactinomycetaceae_1(15.8%～19.1%)和红球菌属(Rhodococcus)(4.3%～18.8%)。通过LEfSe分析差异微生物表明(图3-B),传统与机械化车间3轮次的差异细菌有未分类_卟啉单胞菌属(unclassified_Porphyromonadaceae)(CT)、肠球菌属(Enterococcus)(CT)、unclassified_Lactobacillales(CT)、假苯基杆菌属(Phenylobacterium)(FJ)、魏斯氏菌属(Weissella)(FJ)、中慢生根瘤菌属(Mesorhizobium)(FJ)、鞘脂菌属(Sphingobium)(FJ)、不动杆菌属(Acinetobacter)(FJ)、球形发丝菌属(Sphaerochaeta)(FJ)、阿克曼氏菌属(Akkermansia)(CT)。

传统车间4轮次发酵优势细菌与3轮次相似,前中期主要是Unclassified_Bacillaceae_2(34.8%～44.2%)、unclassified_Thermoactinomycetaceae_1(14.8%～15.4%)和Oceanobacillus(8.3%～10.8%),后期为unclassified_Thermoactinomycetaceae_1(13.7%)、Unclassified_Lactobacillales(35.9%)和Rhodococcus(12.4%)。机械化车间4轮次发酵前中期主要优势细菌为Unclassified_Bacillaceae_2(43.1%～50.3%)、unclassified_Thermoactinomycetaceae_1(17.4%～17.9%)和Oceanobacillus(13.4%～15.6%),后期为unclassified_Bacillaceae_2(27.5%)、unclassified_Thermoactinomycetaceae_1(14.6%)和unclassified_Lactobacillales(16.7%),另外Rhodococcus的相对丰度也达到9.8%(图3-C)。4轮次传统与机械化车间的差异细菌为栖水菌属(Enhydrobacter)(FJ),Enhydrobacter可能通过类似的代谢途径影响酸类物质积累,胡小霞等[19]的研究表明,Enhydrobacter常与假单胞菌属(Pseudomonas)共同出现。

传统与机械化车间5轮次优势细菌属如图3-E所示,传统车间发酵前中期优势细菌均为unclassified_Bacillaceae_2(29.3%～41.3%)、unclassified_Thermoactinomycetaceae_1(8.7%～13.3%)和Pseudomonas(18.3%～26.2%),发酵后期unclassified_Lactobacillales(17.9%)、Pseudomonas(36.1%)和Unclassified_Bacillaceae_2(7.47%)则为主要优势细菌。5轮次发酵前期Oceanobacillus(7.9%)、unclassified_Thermoactinomycetaceae_1(33.2%)、Pseudomonas(22.3%)为机械化车间的优势细菌,中期优势细菌为Unclassified_Bacillaceae_2(32.9%)、Pseudomonas(18.6%)、醋酸杆菌属(Acetobacter)(11.8%),后期unclassified_Lactobacillales(27.3%)、乳杆菌属(Lactobacillus)(17.3%)、Acetobacter(27.7%)则为优势细菌。5轮次传统与机械化车间的差异细菌为发酵杆菌属(Macellibacteroides)(FJ)、冰冷杆菌属(Gelidibacter)(CT)、未分类_绿弯菌属(unclassified_Chloroflexi)(FJ)、嗜碱菌(Alkaliphilus)(CT)、Acetobacter(FJ)、鞘氨醇单胞菌属(Sphingomonas)(CT)、硫卵菌属(Sulfurovum)(CT)、砂单胞菌属(Arenimonas)(FJ)。之前的研究表明,酱香型白酒机械化堆积发酵过程中Acetobacter相对丰度更高,有检出Sphingomonas,这与传统工艺显著不同[20-21]。

2.4 机械化与传统真菌群落组成和差异分析

糖化堆堆积发酵过程中在属水平上相对丰度大于1%的优势真菌如图4-A、图4-C、图4-E所示,传统车间3轮次发酵前期优势真菌为伊萨酵母属(Issatchenkia)(36.3%)、曲霉属(Aspergillus)(27.1%)、嗜热丝孢菌属(Thermomyces)(27.0%),发酵中后期Issatchenkia(79.8%～89.8%)成为绝对优势真菌。机械化车间3轮次发酵过程中Issatchenkia、Thermomyces、Aspergillus和红曲霉属(Monascus)是主要的优势真菌,Monascus(3.6%～9.8%～39.8%)和Issatchenkia(7.2%～19.7%～43.5%)的相对丰度在发酵过程中呈上升趋势,Thermomyces(32.9%～25.3%～4.4%)和Aspergillus(48.4%～31.5%～5.4%)则呈下降趋势,Thermomyces在发酵前中期占优势,这与张红霞[22]的研究一致。差异微生物分析表明(图4-B),传统与机械化车间在3轮次差异真菌为Monascus(FJ)、丝衣霉属(Byssochlamys)(FJ)、假丝酵母属(Candida)(FJ)、红酵母属(Rhodotorula)(CT)、毛霉属(Mucor)(FJ)。

4轮次发酵过程中Issatchenkia(77.7%～89.8%)是传统车间的绝对优势真菌,哈萨克酵母属(Kazachstania)(1.9%～2.9%～13.4%)相对丰度在4轮次呈上升趋势(图4-C)。机械化车间4轮次发酵前中期主要优势真菌为Issatchenkia、Thermomyces、Aspergillus和Monascus,与传统车间3轮次相同Monascus(5.6%～12.6%)和Issatchenkia(6.7%～9.8%)的相对丰度在发酵过程中呈上升趋势,Thermomyces(51.1%～40.3%)和Aspergillus(28.5%～27.3%)则呈下降趋势,发酵后期Issatchenkia(52.4%)成为绝对优势真菌,Kazachstania的相对丰度上升到了13.9%。4轮次传统与机械化车间主要的差异真菌为Aspergillus(FJ)、Monascus(FJ)、耐干霉菌属(Xeromyces)(FJ)、Byssochlamys(CT)、Thermomyces(FJ)、丝孢酵母属(Hyphopichia)(FJ)、Issatchenkia(CT)、Candida(FJ)、多糖球菌(Plectosphaerella)(FJ)、单端孢霉属(Trichothecium)(FJ)、土赤壳属(Ilyonectria)(FJ)、根毛霉属(Rhizomucor)(FJ)、嗜热毛霉属(Thermomucor)(FJ)、根霉属(Rhizopus)(FJ)。

传统车间5轮次发酵前期优势真菌为青霉属(Penicillium)(61.9%)、Issatchenkia(28.4%),中后期Issatchenkia(93.9%～94.5%)为绝对优势真菌(图4-E)。机械化车间5轮次发酵前期优势真菌为Issatchenkia(44.0%)、Penicillium(34.2%)、Monascus(11.0%),中后期Issatchenkia(77.4%～89.1%)为绝对优势真菌,Pichia的相对丰度在中期升高(14.4%),后期下降到3.4%。5轮次发酵阶段差异真菌如图4-F所示,主要有棒孢霉属(Corynespora)(CT)、Pichia(CT)、Candida(CT)。程伟等[23]的研究表明Pichia、Candida在传统发酵酒醅中是主要的优势真菌,另外Pichia和Candida与Lactobacillus、酿酒酵母属(Saccharomyces)等协同主导风味代谢(如酯类、高级醇类),通过产酯、降解酸类、分泌功能酶提升酒醅风味复杂性和稳定性[24-26]。

2.5 核心细菌群落及差异细菌群落与理化因子的关联性分析

为了进一步分析传统与机械化车间不同酒醅中理化因子对细菌群落演替的影响,本研究通过RDA解释核心微生物群落(堆积发酵过程中的优势微生物)之间与理化因子之间的相互关系。理化因子对核心细菌群落的影响如图5所示,RDA1可以解释27.74%,RDA2可以解释8.28%,两坐标轴累计可解释36.02%细菌属与理化因子间的差异比例。图中3CT-I和3FJ-I相距较近,表明传统和机械化车间在3轮次堆积发酵前期核心细菌群落差异较小,随着4～5轮次进行,4CT-I与4FJ-I和5CT-I与5FJ-I之间的距离逐渐变大,表明传统和机械化车间发酵前期核心细菌群落差异越来越大,3～5轮次发酵后期同样也是核心细菌群落差异逐渐变大。

如图5所示酸度和水分呈正相关,还原糖和水分则呈负相关,酸度、水分、还原糖对细菌群落的影响较大。红色箭头代表理化因子,水分的箭头最长,酸度的箭头与多种细菌属(灰色箭头)的夹角最小,酒醅理化结果也表明传统与机械化车间酒醅的水分与酸度存在差异,这说明水分和酸度可能是影响酒醅细菌群落演替的主要驱动因子,YANG等[27]的研究也发现水分是驱动细菌群落演化的主要环境因子。Lactobacillus与酸度的夹角较小,说明酸度与其呈正相关,赵皓静等[28]的研究表明,Lactobacillus有较强的代谢产生乙酸和乳酸的能力。Kroppenstedtia、unclassified_Bacillaceae_2、Sphaerochaeta、Bacillus和Oceanobacillus与还原糖呈正相关性,与酸度、水分呈负相关性,Rhodococcus也与酸度、水分呈负相关性;Lactobacillus、Acetobacter与还原糖有负相关性。

2.6 核心真菌群落及差异真菌群落与理化因子的关联性分析

理化因子对核心真菌群落和差异真菌的影响如图6所示,RDA1可以解释23.36%,RDA2可以解释16.51%,两坐标轴累计可解释39.87%真菌属与理化因子间的差异比例。图中4CT-I和4FJ-I距离较远,3CT-I和3FJ-I、5CT-I和5FJ-I距离相对较近,说明3～5轮次堆积发酵前期,传统和机械化车间在4轮次的核心真菌群落差异较大,在堆积发酵后期,同样是4轮次真菌群落差异较大。与细菌群落的差异相反,5CT-L和5FJ-L距离最近,表明5轮次堆积后期传统和机械化车间真菌群落差异最小。

由图6可知,水分与大部分真菌的夹角均大于90°,表明水分与大部分真菌有着负相关性,如Thermomyces、Aspergillus、Xeromyces和Rhizomucor与水分均呈负相关,课题组前期研究结果也表明堆积发酵过程中水分与多种真菌呈现负相关[29]。还原糖与Kazachstania、Rhizopus、Hyphopichia、Candida均呈正相关性,传统与机械化车间酒醅还原糖含量的差异可能是导致真菌群落差异的原因,这与曾波等[30]的研究一致,另外,Saccharomyces和Kazachstania与酸度和水分也有正相关性。

3 结论与讨论

本文系统性研究了酱香型白酒3～5轮次传统和机械化酿造酒醅堆积发酵过程中的理化指标和微生物群落差异。理化指标方面,传统车间(46.64%～55.76%)酒醅水分整体上高于机械化车间(42.56%～52.96%);传统车间酒醅酸度5轮次时最高(34.05～35.47 mmol/100 g),机械化车间4轮次时酸度最高(36.89%～38.78 mmol/100 g);机械化车间(1.48%～2.40%)还原糖含量高于传统车间(1.35～1.68%);3～5轮次传统与机械化车间酒醅pH值维持在3.77～3.95。

微生物多样性方面,传统车间微生物的种类多样性高于机械化车间,不同轮次不同阶段堆积发酵过程中微生物群落结构发生动态变化。传统车间3轮次发酵前中期主要的优势细菌为unclassified_Bacillaceae_2(41.8%～58.5%)、unclassified_Thermoactinomycetaceae_1(14.9%～22.6%)和Oceanobacillus(4.1%～12.1%),而后期则转变为unclassified_Lactobacillales(40.8%)和unclassified_Bacillaceae_2(35.3%)。机械化车间在3轮次期间的主要优势细菌与传统车间相似,但Rhodococcus的相对丰度较高(4.3%～18.8%)。差异微生物分析表明,传统与机械化车间3轮次的主要差异细菌有Weissella、unclassified_Lactobacillales、Sphingobium、Acinetobacter。传统与机械化车间4轮次发酵前中期优势细菌与传统车间3轮次前中期相同,均为unclassified_Bacillaceae_2、unclassified_Thermoactinomycetaceae_1和Oceanobacillus,4轮次发酵后期unclassified_Lactobacillales(35.9%)和Rhodococcus(12.4%)变为两车间的优势细菌,4轮次的差异细菌为Enhydrobacter。5轮次发酵阶段,传统和机械化车间的Pseudomonas均为优势细菌,相对丰度较高(12.1%～36.1%);发酵后期机械化车间Acetobacter(27.7%)相对丰度显著高于传统车间(0.13%),5轮次的主要差异细菌为Acetobacter、Sphingomonas。

3轮次发酵前期,Issatchenkia、Aspergillus、Thermomyces均为传统与机械化车间的优势真菌,但发酵中后期传统车间(79.8%～89.8%)的Issatchenkia相对丰度远高于机械化车间(7.2%～19.7%～43.5%)。且机械化车间Monascus(3.6%～9.8%～39.8%)的相对丰度在发酵过程中呈上升趋势,3轮次主要的差异真菌为Monascus、Byssochlamys、Candida。4轮次发酵过程中Issatchenkia是传统和机械化车间发酵后期的绝对优势真菌,传统车间(89.8%)的Issatchenkia相对丰度远高于机械化车间(52.4%),Kazachstania均为传统(13.4%)和机械化车间(13.9%)的优势细菌,4轮次主要的差异真菌为Aspergillus、Monascus、Thermomyces、Issatchenkia、Candida。传统和机械化车间5轮次发酵前期优势真菌均为Penicillium、Issatchenkia,中后期Issatchenkia为绝对优势真菌,但传统车间(93.9-94.5%)的Issatchenkia相对丰度依然高于机械化车间(77.4%～89.1%),但机械化车间的Monascus相对丰度始终高于传统车间,5轮次发酵阶段差异真菌主要有Pichia、Candida。

RDA表明,传统和机械化车间在3轮次发酵前期核心细菌群落差异较小,随着轮次进行,传统和机械化车间核心细菌群落的差异逐渐变大;水分和酸度可能是影响酒醅细菌群落演替的主要驱动因子。4轮次时传统与机械化车间的真菌群落差异最大,5轮次时真菌群落差异变小,水分与大部分真菌有着负相关性,还原糖与Kazachstania和Candida呈正相关。本研究系统分析了酱香型白酒机械化与传统车间“大回酒”轮次堆积发酵过程中酒醅的理化指标和微生物组成、演替规律及差异,为酱香型白酒数字化、智能化转型提供了理论数据。

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Comparative analysis of microbial diversity differences between mechanized and traditional processes during the Dahui-jiu round stacking fermentation of Jiangxiangxing Baijiu

SHEN Ji1,2,3,4,5, WU Cheng1,2,3,4,5*, XIAO Shizheng1, CAI Yong1,4, CHEN Wanwen1,4, LU Hu1,2,3,4,5, HU Jianfeng1,2,3,4,5, HU Feng1,2,3,4,5

1(Guizhou Xijiu Co.Ltd., Xishui 564622, China)

2(Key Laboratory of Quality and Safety of Jiangxiangxing Baijiu, State Administration for Market Regulation, Guiyang 550000, China)

3(Guizhou Province Technology Innovation Center for Jiangxiangxing Baijiu, Xishui 564622, China)

4(Engineering Technology Research Center for Intelligent Brewing of Jiangxiangxing Baijiu, China, Xishui 564622, China)

5(Guizhou Province Manufacturing Innovation Center for Baijiu, Renhuai 564500, China)

ABSTRACT The base liquor of the Dahui-Jiu round (rounds 3-5) in Jiangxiangxing Baijiu accounts for a large proportion of the total output, making it the “golden round” in Jiangxiangxing Baijiu production.This study took the fermented grains from the stacked fermentation process of mechanized and traditional brewing for rounds 3-5 of Jiangxiangxing Baijiu as the research objects, and analyzed the differences in physicochemical factors and microbial communities of fermented grains between the mechanized and traditional during the stacked fermentation.The moisture content of fermented grains in the traditional workshop ranged from 46.64% to 55.76%, which was higher than that in the mechanized workshop (42.56%-52.96%).In contrast, the reducing sugar content in the mechanized workshop (1.48%-2.40%) was higher than that in the traditional workshop (1.35%-1.68%).In terms of microbial diversity, the species diversity of microorganisms in the traditional workshop was higher than that in the mechanized workshop.For bacteria, in round 3, the relative abundance of Rhodococcus in the mechanized workshop was 4.3%-18.8%, significantly higher than that in the traditional workshop (2.3%-5.3%).In the late fermentation stage of round 4, the relative abundance of Unclassified_Lactobacillales in the traditional workshop was 35.9%, which was higher than that in the mechanized workshop (16.7%).In round 5, Pseudomonas was the dominant bacterium, with a relative abundance of 12.1%-36.1%.In the late fermentation stage of round 5, the relative abundance of Acetobacter in the mechanized workshop was 27.7%, significantly higher than that in the traditional workshop (0.13%).LEfSe analysis showed that the main differential bacteria between the traditional and mechanized workshops included Weissella, Sphingobium, unclassified_Lactobacillales, Enhydrobacter, Acetobacter, and Sphingomonas.For fungi, Issatchenkia was the absolute dominant fungus in the late fermentation stage, and its relative abundance in the traditional workshop was consistently higher than that in the mechanized workshop.The relative abundance of Monascus in the mechanized workshop was significantly higher than that in the traditional workshop.During the fermentation of rounds 3-5, the main differential fungi between the two workshops were Monascus, Byssochlamys, Candida, Aspergillus, Thermomyces, Issatchenkia, and Pichia.Redundancy analysis (RDA) indicated that with the progression of rounds, the difference in the core bacterial communities between the traditional and mechanized workshops gradually increased, and the difference in fungal communities reached the maximum in round 4.Moisture and acidity might be the main driving factors affecting the succession of the bacterial community in fermented grains, and moisture showed a negative correlation with most fungi.This study conducted an in-depth analysis of the physicochemical and microbial differences in the stacked fermentation grains between the traditional and mechanized workshops during rounds 3-5, providing basic theoretical and disciplinary support for promoting the development of mechanized brewing of Jiangxiangxing Baijiu.

Key words Jiangxiangxing Baijiu; stacking fermentation; traditional and mechanized; Dahui-Jiu round; differential microbial; physicochemical factors

DOI：10.13995/j.cnki.11-1802/ts.044316

引用格式：沈记,吴成,肖世政,等.酱香型白酒机械化与传统“大回酒”轮次堆积发酵微生物多样性差异比较分析[J].食品与发酵工业,2026,52(10):201-210.SHEN Ji,WU Cheng,XIAO Shizheng, et al.Comparative analysis of microbial diversity differences between mechanized and traditional processes during the Dahui-jiu round stacking fermentation of Jiangxiangxing Baijiu[J].Food and Fermentation Industries,2026,52(10):201-210.

第一作者：硕士(吴成工程师为通信作者,E-mail:742990507@qq.com)

基金项目：贵州省科技支撑计划项目(黔科合支撑[2023]一般342,黔科合支撑[2023]一般343,黔科合支撑[2024]一般030);贵州省市场监管局质量发展项目(黔质量发展项目(2024)3号(重点));遵义市科技计划项目(遵市科合支撑[2024]20号);贵州省酱香型白酒技术创新中心(黔科合平台JSZX〔2025〕002);遵义市科技创新团队建设项目(遵KCTD074)

收稿日期：2025-08-24,改回日期:2025-10-24