WO2023051492A1 - 一种食醋发酵人工菌群构建方法及应用 - Google Patents

一种食醋发酵人工菌群构建方法及应用 Download PDF

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WO2023051492A1
WO2023051492A1 PCT/CN2022/121559 CN2022121559W WO2023051492A1 WO 2023051492 A1 WO2023051492 A1 WO 2023051492A1 CN 2022121559 W CN2022121559 W CN 2022121559W WO 2023051492 A1 WO2023051492 A1 WO 2023051492A1
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fermentation
vinegar
flora
microorganisms
species
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French (fr)
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郑宇�
夏梦雷
王敏
赵翠梅
肖云
夏婷
李暄
刘丹彤
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天津科技大学
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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • G16B30/10—Sequence alignment; Homology search
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B50/00—ICT programming tools or database systems specially adapted for bioinformatics
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    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

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  • the invention belongs to the technical field of food fermentation, and in particular relates to a rational construction method and application of artificial bacterial flora for vinegar fermentation.
  • vinegar in my country mainly includes grain vinegar and fruit vinegar as raw materials, and is produced by traditional multi-strain solid-state fermentation or pure bacteria (acetic acid bacteria) liquid-state fermentation.
  • the former has a mellow flavor but slow fermentation, while the latter ferments faster but has a single flavor.
  • Traditional vinegar is mostly produced by open fermentation process, and the acetic acid fermentation stage is the main stage for the formation and accumulation of flavor substances.
  • seed grains provide abundant microorganisms for the acetic acid fermentation stage of vinegar, but in the actual production process, the microbial community rich in "seed grains” is prone to fluctuations due to various influences, resulting in vinegar fermentation process and product quality.
  • the food fermentation process is driven by limited microbial species. They not only produce flavor compounds, but also interact with microorganisms to make the fermentation develop towards a stable trend, thus ensuring the normal progress of food fermentation.
  • the acetic acid produced by acetic acid bacteria is the main taste substance in vinegar
  • the lactic acid produced by lactic acid bacteria can greatly reduce the sourness of vinegar, and at the same time produce various beneficial factors and bacteriostats.
  • Bacillus can interact with lactic acid bacteria Synergistically produce more flavor substances.
  • the construction and application of artificial flora based on the analysis of flora structure and composition have been preliminarily studied.
  • the flora structure obtained by this method does not necessarily have high metabolic activity, and cannot reflect the real metabolism and function of the colonies in the fermentation stage.
  • the invention provides a rational construction method and application of artificial bacterial flora for vinegar fermentation.
  • the artificial flora construction method provided by the present invention is based on the metatranscriptome sequencing technology, and adopts grouping and clustering analysis to obtain the core microbiome and construct the artificial flora.
  • This technology is applied to the fermentation process of vinegar, which can improve the flavor quality of vinegar while realizing stable production.
  • This technology solves the problem that the artificially constructed flora structure does not necessarily have high metabolic activity, and cannot reflect the real metabolism and function of the colonies in the fermentation stage.
  • the technology uses group clustering analysis to obtain the core microbiome and construct artificial flora. On this basis, based on the analysis of microbial function and activity, the metabolic network of active microorganisms is reconstructed, and vinegar fermentation is constructed.
  • the artificial flora can effectively guarantee the stability of the fermentation process and product quality.
  • a method for rationally constructing vinegar fermentation artificial flora comprising the steps of:
  • the minimum species required when the microbiome and the original microbiome of each stage are successfully clustered and the similarity is ⁇ 90% is determined as the initial core microbiome;
  • the species abundance includes microbial species and corresponding concentrations
  • the similarity is >95%
  • the core microbiome metabolic gene transcription accounts for more than 85%;
  • the main flavor substances refer to the detection of the composition of the main flavor substances in the sample at the end of fermentation by using high-performance liquid chromatography, gas chromatography-mass spectrometer and other instruments, and combining the threshold value and content of the flavor substances with the flavor intensity of the substance
  • the organic acids and amino acids with TAV>1 were defined as the main taste substances, and they made important contributions to the tastes of vinegar, such as sour, sweet, and bitter; those with ROAV>0.1 (relative aroma activity value)
  • Volatile aroma compounds are defined as the main aroma compounds, which together constitute the main flavor substances of vinegar;
  • the selective culture medium can adopt the culture medium containing different substrates such as starch, cellulose, glucose, ethanol, acetic acid, etc. to screen samples such as vinegar grains that have starch or cellulose hydrolysis, conversion of glucose or ethanol to produce organic acids, alcohols, etc. , esters, aldehydes and other characteristics of microorganisms;
  • substrates such as starch, cellulose, glucose, ethanol, acetic acid, etc.
  • screen samples such as vinegar grains that have starch or cellulose hydrolysis, conversion of glucose or ethanol to produce organic acids, alcohols, etc. , esters, aldehydes and other characteristics of microorganisms
  • the ratio between the artificial flora strains is compounded according to the ratio of the average biomass of the corresponding microorganisms in the core microbiome in the original fermentation sample at each stage;
  • the mixed culture of the artificial flora strains has no obvious inhibitory effect, and can maintain the original physiological activity characteristics under the condition of 20%-40% water.
  • the present invention also provides the application of the above-mentioned artificial flora, especially in the application of the above-mentioned artificial flora to replace the traditional "seed grains" for vinegar fermentation;
  • the strains of the artificial flora are cultivated by pure strains, they are made into pure bacterial agents or bran yeast, and the addition ratio of each strain is determined according to the average ratio of microbial composition in each fermentation stage in the macrotranscriptome sequencing results, and the proportion of each strain is determined according to the corresponding The ratio is compounded, and then applied to the acetic acid fermentation stage of vinegar:
  • the mixed microbial agent is applied to the fermentation process of liquid or solid grain vinegar or fruit vinegar;
  • mixed bran yeast is applied to the fermentation process of liquid or solid grain vinegar or fruit vinegar;
  • the inoculum amount of the artificial flora is 10 8 -10 12 cfu per 1 kg of raw material.
  • the present invention uses grouping and clustering analysis to construct an artificial flora for the first time.
  • the similarity with the active microbial flora in the vinegar in situ acetic acid fermentation process is >90%, and the metabolic characteristics of the flora are > 80%, can realize the rational construction of artificial flora.
  • the invention can improve the stability of vinegar fermentation, and can also be used as a reference for the construction and analysis of other traditional fermented food flora and for repeatable flavor synthesis technology, which has good scientific value and application value.
  • the present invention constructs the flora on the basis of metatranscriptome sequencing. This technology is accurate in analyzing the main functional microorganisms and has good theoretical significance.
  • the present invention uses artificial flora to replace traditional seed grains to ferment grain vinegar or fruit vinegar.
  • the artificial flora as a starter has a faster fermentation rate, and the fermentation period is shortened by 2 to 8 days; raw material utilization rate Increased by 6% to 13%; non-volatile acid, reducing sugar, amino acid nitrogen, and total ester increased by 30% to 150%, 10% to 40%, 10% to 25%, and 10% to 50% respectively; Acid/volatile acid increased by 50% to 180%. It not only increases the softness of vinegar, but also improves the quality of vinegar.
  • Figure 1 Dynamic changes in taxonomic composition and relative abundance of species based on metatranscriptome sequencing results
  • Figure 2 The top 10 species with average abundance in the fermentation stage based on metatranscriptome sequencing (arranged in descending order from left to right);
  • Figure 3 Cluster analysis of microbial groups, i.e. determination of initial core microbial genera
  • d represents the original fermentation sample
  • d-1 represents the clustering using the top 5 microorganisms in abundance
  • d-2 represents the clustering using the top 6 microorganisms in abundance.
  • the TAV (taste intensity) of the present invention refers to the sensory contribution rate of non-volatile flavor substances evaluated by the taste intensity value method, and the TAV value is the ratio of the content of each taste substance to its threshold value.
  • T i the sensory threshold corresponding to the taste substance.
  • the substance When the TAV value is greater than 1, the substance is considered to contribute to the taste, while when the TAV value is less than 1, the substance is considered to have no contribution to the taste.
  • ROAV relative aroma activity value
  • C i , T i the relative percentage content of each volatile substance and the corresponding sensory threshold.
  • C max , T max the relative percentage of the component that contributes the most to the overall flavor of the sample and the corresponding sensory threshold.
  • the components with ROAV ⁇ 1 are the key flavor compounds of the analyzed samples, and the components with 0.1 ⁇ ROAV ⁇ 1 have an important modification effect on the overall flavor of the samples.
  • the sensory thresholds involved in the calculation of TAV and ROVA can be determined according to the thresholds disclosed in the prior art, and can also refer to "Shanxi Mature Vinegar Flavor Analysis and Quality Improvement” [D]. Cheng Cheng. Tianjin University of Science and Technology, 2018.
  • Example 1 Analysis of active community composition in traditional fermentation process
  • Metatranscriptome sequencing Taking Shanxi aged vinegar as an example, equal amounts of vinegar fermented grain samples from the 1st, 3rd, 5th, 7th, and 9th days of acetic acid fermentation were taken to remove impurities, and the total RNA of microbial communities in samples at each stage was extracted. Metatranscriptome sequencing of total RNA at each stage;
  • RNA-targeted metatranscriptome sequencing is aimed at the functional gene transcription of the microbial community, which represents the metabolic activity of the microorganisms in the fermentation process and can better reflect the actual contribution of the microorganisms in the fermentation process. . Therefore, the present invention selects RNA as the target metatranscriptome sequencing for subsequent analysis of flora.
  • Table 1 The composition of main microorganisms in the sequencing results of metagenomics and metatranscriptomes
  • Example 2 Rational construction of artificial flora taking traditional fermented Shanxi mature vinegar as an example
  • Metatranscriptome sequencing Take the same amount of vinegar fermented grain samples from the 1st, 3rd, 5th, 7th, and 9th days (AAF1d, AAF3d, AAF5d, AAF7d, AAF9d) of the acetic acid fermentation stage of vinegar, remove impurities, and extract the total number of microbial communities.
  • RNA metatranscriptome sequencing of total RNA;
  • the main taste substances in the samples are oxalic acid, succinic acid, citric acid, lactic acid, tartaric acid, acetic acid, glutamic acid, histidine, alanine and valine; as shown in Table 4,
  • the main aroma substances in the sample are acetic acid (as both aroma and taste substances), 3-methylbutyric acid, benzaldehyde, furfural, ethyl acetate, 2,3-butanedione, 3-hydroxy-2- Butanone, Phenylethyl Acetate, Isoamyl Acetate, 2,3,5-Trimethylpyrazine, Furfuryl Alcohol.
  • citric acid Take the synthesis of citric acid as an example:
  • Transcriptome data can correspond a gene to a species one-to-one.
  • species annotation and functional annotation of each gene ID in the sequencing result have already corresponded to the corresponding gene name and species name.
  • Macro Transcriptome can be obtained: microbial community composition data, one-to-one corresponding source microorganisms of a gene, relative transcription concentration of a gene, etc.).
  • 3Statistics of key gene transcription results Calculate the ratio of the sum of the transcription of key genes for the formation of main flavor substances by microorganisms in the initial core microbiome at each stage of fermentation to the transcription of key genes for the formation of main flavor substances at each stage.
  • citric acid as an example: from step 2, it can be seen that during the acetic acid fermentation process of vinegar, the key genes for the formation of citric acid are gltA, ACLY, and acnA. and, the transcriptional expression levels of the three genes in each fermentation stage were summed and the ratio was calculated to obtain the microorganisms in the initial core microbiome (Lactobacillus acetotolerans, Acetobacter pasteurianus, Lactobacillus helveticus, Lactobacillus kunkeei, Lactobacillus fermentum, Streptococcus lactis) In the 1st, 3rd, 5th, 7th, and 9th days of fermentation, the transcription of key genes related to citric acid formation accounted for 46.15%, 48.28%, 84.06%, 98.20%, and 100% of the microorganisms in each stage, respectively.
  • the initial core microbiome Lacobacillus acetotolerans, Acetobacter pasteurianus
  • the metabolic gene transcription ratio of the initial core microbiome is less than 80% on the 1st, 5th, and 7th days of fermentation, according to the abundance of microbial species in (2) to (4), the 7th gene was added to the initial core microbiome.
  • Lactobacillus plantarum in the first place calculate the transcription ratio of the key genes for the formation of the main flavor substances in the new microbiome, and obtain the proportions of the microbiome in the 1st, 3rd, 5th, 7th, and 9th days of fermentation, respectively. 73.39%, 89.68%, 73.96%, 78.17%, 82.74%, do not meet the requirements.
  • Embodiment 3 Artificial flora makes mixed bran yeast and is applied to the process of manual production of grain vinegar acetic acid fermentation
  • Microbial re-screening Sequence the purely cultured microorganisms obtained from the above screening, and compare the fermentation and tolerance performance of the same microorganisms, and select the ones with better fermentation and tolerance (acid resistance, temperature resistance, alcohol resistance) Microorganisms are the dominant bacteria of the same kind of microorganisms. If no dominant microorganism is obtained in this step, repeat the screening and identification according to the microbial screening method.
  • fermentation performance refers to the ability of the strain to produce acetic acid and lactic acid respectively; for Bacillus, fermentation performance refers to the ability of the strain to produce amylase, protease, and cellulase (decomposing raw materials).
  • select or set screening media containing different substrates such as starch, cellulose, glucose, ethanol or acetic acid for the above-mentioned fermentation performance, and screen the samples of vinegar fermented grains with starch or cellulose hydrolysis, glucose or ethanol to produce organic Microorganisms with properties such as acids, alcohols, esters, and aldehydes.
  • tolerance mainly includes the tolerance of microorganisms to acetic acid, ethanol and temperature.
  • Different acetic acid gradients (0%, 1%, 2%, 3%, 4%), different ethanol gradients (0%, 2%, 4%, 6%, 8%) and different Temperature gradient (30°C, 35°C, 40°C, 45°C, 50°C), after the inoculated strains were cultured, the growth of microorganisms cultured for 24-36 hours were tested by plate counting method.
  • the simulated vinegar solid-state fermentation medium consists of: bran 30%, rice husk 10%, glucose 1.2%, peptone 0.6%, beef extract 0.6%, yeast extract 0.3%, anhydrous sodium acetate 0.3%, Tween 80 0.06%, triammonium citrate 0.12%, dipotassium hydrogen phosphate 0.12%, magnesium sulfate 0.035%, manganese sulfate 0.015%, and the balance is water.
  • Step (2) Screen the activated and proliferated microbial liquid, culture at 30°C to 37°C for 60 to 65 hours, crush to obtain bran koji, and then expand the culture to make shallow dish koji and count the viable bacteria ;
  • Alcoholic fermentation crush the sorghum and moisten the material with warm water for 4-8 hours.
  • 0.1% high-temperature-resistant ⁇ -amylase according to the mass of sorghum is liquefied at a temperature above 90° C. for 30 minutes.
  • the temperature drops to 60°C, add solid glucoamylase at 2% of the mass of sorghum, and saccharify at 58-60°C for 1 hour.
  • 62.5% of crushed Daqu is added according to the mass of sorghum, and activated active dry yeast is added after replenishing water.
  • enter the wine vat for alcoholic fermentation open-top fermentation for the first 3 days, and sealed fermentation for the next 4 days, a total of 7 days.
  • the fermentation temperature is maintained between 28°C and 30°C.
  • Table 6 The physical and chemical indicators of artificial flora applied to grain vinegar acetic acid fermentation (unit: g/100g vinegar unstrained spirits)
  • Embodiment 4 Artificial flora makes mixed bacterial agent and is applied to fruit vinegar liquid state fermentation process
  • 1Preparation of pure bacterial agents Inoculate the activated strains in the proliferation medium with an inoculation amount of 1% to 10% and cultivate them to the stable growth stage, and then use spray freeze-drying to prepare Lactococcus lactis, Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus respectively Paracase, Lactobacillus fermentum, Acetobacter pasteurianus pure bacteria agent;
  • 1Raw material pretreatment and alcoholic fermentation after destemming and crushing fresh grapes, add 0.2% SO 2 (potassium metabisulfite) to sterilize for 1 hour, then add 0.1% pectinase (10000U/g enzyme activity) to enzymolyze at 50°C for 4 hours, After cooling down to room temperature, the sugar content is adjusted to 18 degrees, and finally the activated active dry yeast is added to it and put into a fermenter for fermentation at 30°C. When the specific gravity and sugar content no longer change (about 3 to 5 days), the alcoholic fermentation ends.
  • SO 2 potential metabisulfite
  • 2Acetic acid fermentation Use grape juice to reduce the alcohol content of the original wine to about 8 degrees, the fermenter is filled with 75% to 80%, the ventilation rate is adjusted to 0.15vvm, and the speed is adjusted to 2000r/min. After the temperature reaches 30°C, calibrate the dissolved oxygen electrode, use saturated anhydrous sodium sulfite to calibrate zero, and insert the dissolved oxygen electrode into the fermentation broth to calibrate 100%. Insert 0.4% (v/w, 100mL raw material plus 0.4g mixed bacterial agent) mixed bacterial agent dissolved in water, follow up sampling, when the alcohol content is lower than 0.5%, the acidity no longer rises and the fermentation is stopped.
  • Three experimental groups were set up as parallel controls.
  • Grape vinegar fermented by artificial flora the appearance is purplish red, clear, transparent, and shiny; sweet and sour, with strong grape fruit flavor and no peculiar smell. It can be seen from Table 7 that compared with naturally fermented grape vinegar, the total acid content increased by 14.44%-16.14%, the non-volatile acid increased by 159.57%-176.60%, and the non-volatile acid/volatile acid increased by 155.87%-180.01%. The content of acetic acid, lactic acid, malic acid and total esters increased by 7.88%-10%, 282.61%-313.04%, 33.33%-46.67%, respectively, the fermentation time was shortened by 53.33%, and the formation rate of acetic acid increased by 70.21%-74.47%.
  • non-volatile acids especially lactic acid
  • lactic acid bacteria increases the content of total esters, increases the mellow feeling of fruit vinegar, and improves the quality of fresh grape fruit vinegar.

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Abstract

本发明属于食品发酵技术领域，涉及一种食醋发酵人工菌群理性构建方法及应用。本发明所提供的人工菌群构建方法以宏转录组测序技术为基础，首次采用分组聚类分析构建人工菌群，与食醋原位醋酸发酵过程中的活性微生物群落的相似度≥90％，菌群代谢特征≥80％，能实现人工菌群的理性构建。该发明能很好的提高食醋发酵的稳定性，也可作为其他传统发酵食品菌群构建与分析以及为可重复性风味合成技术提供参考，具有较好的科学价值和应用价值。

Description

一种食醋发酵人工菌群构建方法及应用 技术领域
本发明属于食品发酵技术领域，尤其涉及一种食醋发酵人工菌群理性构建方法及应用。
背景技术
我国食醋的生产主要包括以谷物醋和果醋为原料，采用传统的多菌种固态发酵或纯菌(醋酸菌)液态发酵生产，前者风味醇厚但发酵慢，而后者发酵更快但风味单一。传统食醋多采用开放式发酵工艺生产，醋酸发酵阶段是风味物质形成和积累的主要阶段。“种醅”作为天然发酵剂为食醋醋酸发酵阶段提供丰富的微生物，但在实际生产过程“种醅”所富含的微生物群落受多方面影响容易发生波动，导致食醋发酵过程及产品品质不稳定，成为了行业亟需解决的共性技术问题。随着科技的发展和对微生物群落研究的不断增加，从天然发酵到可处理发酵的人工菌群的转变在保证发酵食品品质上是必不可少的。
食品发酵过程依赖于有限的微生物属驱动，它们不仅产生风味化合物，而且存在微生物相互作用，使发酵朝着稳定的趋势发展，从而保障食品发酵正常进行。如醋酸菌产生的乙酸是食醋中主要的呈味物质，乳酸菌产生的乳酸能大大减轻食醋的尖酸感，同时还能产生各种有益因子和抑菌素，另外，芽孢杆菌能和乳酸菌相互协同产生更多的风味物质。一些研究者们将不同功能的微生物进行简单组合来生产食醋，但这种方式缺乏科学性，不能有效保证食醋的品质。
得益于高通量测序技术的发展，基于菌群结构组成分析进行人工菌群的构建与应用已得到初步研究。然而此方法得到的菌群结构并不一定具有较高的代谢活性，不能反映发酵阶段菌落的真实代谢情况与功能。
发明内容
本发明提供一种食醋发酵人工菌群理性构建方法及应用。本发明所提供的人工菌群构建方法是以宏转录组测序技术为基础，采用分组聚类分析得到核心微生物组并构建人工菌群。该技术应用于食醋发酵过程，在实现稳定生产的同时能提高食醋风味品质。该技术解决了人工构建的菌群结构并不一定具有较高的代谢活 性，不能反映发酵阶段菌落的真实代谢情况与功能等问题。该技术以宏转录组测序技术为基础，采用分组聚类分析得到核心微生物组并构建人工菌群，并在此基础上，基于微生物功能活性分析，重构活性微生物的代谢网络，构建食醋发酵人工菌群，可以有效保障发酵过程和产品品质的稳定性。
一种食醋发酵人工菌群理性构建方法，包括如下步骤：
(1)微生物群落功能和物种注释：取不同发酵阶段的待模拟醋醅样品，除去杂质，分别提取微生物的总RNA，进行宏转录组测序。对测序结果进行功能和物种注释；
(2)污染和致病微生物的去除：对注释到相应功能的微生物，去除其中的污染微生物和致病微生物；
(3)初始核心微生物的确定：以宏转录组结果中各发酵阶段所有微生物(N个物种)的丰度为基础，按照整个发酵过程中各个物种平均丰度逐渐降低的顺序排序，将最靠前的两个物种组合成第1组，并依次逐渐增加一个物种进行组合，最终得到(N-1)个微生物组；
将微生物组与各阶段的原始微生物组均聚类成功且相似度均≥90％时所需的最少物种确定为初始核心微生物组；
进一步地，将各微生物组物种丰度数据导入软件Minitab，采用最长距离法及Euclidean距离计算各个微生物组和各阶段原始微生物菌群的相似度并进行聚类分析；
所述物种丰度包括微生物种类及对应浓度；
优选地，相似度均＞95％；
(4)核心微生物组代谢活性验证：
①确定发酵结束时样品中的主要风味物质及对应的代谢基因；
②计算初始核心微生物组关于上述代谢基因的转录表达量在各个发酵阶段原始微生物关于上述代谢基因的转录表达量的占比，若与各阶段主要风味代谢基因转录占比均≥80％，则进行下一步；若与某一阶段主要风味物质代谢基因转录占比＜80％，则按步骤(3)中物种丰度依次增加一个物种，并重复步骤(4)，直至与每个阶段主要风味物质代谢基因转录占比均达到≥80％，至此确定出核心微生物组；
优选地，核心微生物组代谢基因转录占比达到85％以上；
进一步地，所述主要风味物质，是指采用高效液相色谱、气质联用仪等仪器对发酵结束时样品中主要风味物质组成进行检测，并结合风味物质的阈值及含量对该物质的风味强度进行分析，其中TAV＞1(滋味强度值)的有机酸、氨基酸定义为主要滋味物质，它们对食醋的酸、甜、苦等滋味有重要贡献；ROAV＞0.1(相对香气活度值)的挥发性香气化合物定义为主要香气化合物，两者共同组成食醋的主要风味物质；
进一步地，各发酵阶段主要风味物质的代谢基因的确定是通过在KEGG数据库中对比，并在宏转录组测序结果中筛选确定；
(5)人工菌群菌株的筛选：根据核心微生物组的组成，从曲、酒醪、醋醅等样品中筛选获得性能优异的菌种；
进一步地，根据需求采用不同的选择性培养基先对醋醅样品中微生物进行初筛，并以耐酸、耐温、耐醇等条件进行菌株的复筛，最后根据不同的实验菌株进行发酵功能验证，从而确定最优菌株，并最终确定核心微生物组的具体微生物组成；
进一步地，选择性培养基可以采用含有淀粉、纤维素、葡萄糖、乙醇、乙酸、等不同底物的培养基筛选醋醅等样品中具有淀粉或纤维素水解、转化葡萄糖或乙醇产生有机酸、醇、酯类、醛类等特性的微生物；
进一步地，人工菌群菌株之间的比例按照各阶段原始发酵样品中核心微生物组中相应微生物的平均生物量的比例进行复配；
进一步地，所述人工菌群菌株之间混合培养时无明显抑制作用，且在20％～40％的水分条件下能保持原有的生理活性特点。
本发明还提供上述人工菌群的应用，特别是在采用上述人工菌群替换传统“种醅”进行食醋发酵中的应用；
进一步地，所述人工菌群的菌株分别经纯种培养后，制成纯菌菌剂或者麸曲，根据宏转录组测序结果中各个发酵阶段微生物组成的平均比例确定各个菌株添加比例，按相应比例进行复配，再应用于食醋醋酸发酵阶段：
进一步地，将混合菌剂应用于液态或固态的谷物醋或果醋发酵过程；
进一步地，将混合麸曲应用于液态或固态的谷物醋或果醋发酵过程；
进一步地，人工菌群的接种量为每1kg原料添加活菌数为10 8～10 12cfu。
本发明具有的优点和积极效果是：
(1)本发明以宏转录组测序技术为基础，首次采用分组聚类分析构建人工菌群，与食醋原位醋酸发酵过程中的活性微生物群落的相似度＞90％，菌群代谢特征＞80％，能实现人工菌群的理性构建。此外，该发明能很好的提高食醋发酵的稳定性，也可作为其他传统发酵食品菌群构建与分析以及为可重复性风味合成技术提供参考，具有较好的科学价值和应用价值。
(2)本发明在宏转录组测序基础上进行菌群的构建，该技术对主要功能微生物分析准确，具有较好的理论意义。
(3)本发明采用人工菌群替代传统种醅进行谷物醋发酵或进行果醋发酵，在发酵结束时，人工菌群作为发酵剂的发酵速率更快，发酵周期缩短2～8d；原料利用率提高了6％～13％；不挥发酸、还原糖、氨基酸态氮、总酯分别增加了30％～150％、10％～40％、10％～25％、10％～50％；不挥发酸/挥发酸提高了50％～180％。不仅增加了食醋的柔和感，还使得食醋品质得以提升。
附图说明
图1：基于宏转录组测序结果中的物种分类组成及其相对丰度的动态变化；
图2：基于宏转录组测序的发酵阶段平均丰度排名前10的物种(按从左到右逐渐降低的顺序排列)；
图3：微生物分组聚类分析即初始核心微生物属的确定
其中，d代表原始发酵样品，d-1代表利用丰度排名前5的微生物进行聚类，d-2代表利用丰度排名前6的微生物进行聚类。
具体实施方式
下面通过具体的实施方案叙述本发明。除非特别说明，本发明中所用的技术手段均为本领域技术人员所公知的方法。另外，实施方案应理解为说明性的,而非限制本发明的范围,本发明的实质和范围仅由权利要求书所限定。对于本领域技术人员而言,在不背离本发明实质和范围的前提下,对这些实施方案中的物料成分和用量进行的各种改变或改动也属于本发明的保护范围。
实施例中涉及到的百分号“％”，若未特别说明，固体指质量百分比，溶液的百分比指100mL中含有溶质的克数，液体之间的百分比，是指在25℃时溶液的体积比例。
本发明所述的TAV(滋味强度)，是指采用味觉强度值法评价不挥发性风味物质的感官贡献率，TAV值为各个呈味物质的含量与其阈值的比。
TAV i＝C i/T i
式中：
C i—各呈味物质的含量；
T i—该呈味物质相对应的感觉阈值。
当TAV值大于1时，认为该物质对呈味有贡献，而TAV值小于1时认为该物质对呈味没有贡献。
本发明所述的ROAV(相对香气活度值)：特征挥发性风味化合物的确定采用相对香气活度值法，评价各挥发性成分对样品整体香气的贡献，即：
ROVA i≈100×C i/C max×T max/T i
式中：C i、T i—各挥发性物质的相对百分含量和相对应的感觉阈值。
C max、T max—对样品总体风味贡献最大的组分的相对百分含量和相对应的感觉阈值。
ROAV≥1的组分为所分析样品的关键风味化合物，0.1＜ROAV＜1的组分对样品的总体风味具有重要的修饰作用。
TAV和ROVA计算过程中涉及的感觉阈值可以根据现有技术中公开的阈值确定，也可参考《山西老陈醋风味分析及品质改良》[D].程程.天津科技大学,2018。
以下结合具体实施例对本发明作进一步地解释说明。
实施例1：传统发酵过程活性群落组成分析
(1)宏转录组测序：以山西老陈醋为例，取等量醋酸发酵阶段第1，3，5，7，9d的醋醅样品，除去杂质，提取各阶段样品微生物群落的总RNA，对各阶段的总RNA进行宏转录组测序；
(2)菌群组成分析：每个阶段样本的Unigene序列与NCBI-NT数据库中的细菌、古菌、真菌和病毒序列进行BLASTN比对，获得各样本在科、属、种各分类等级上的活性菌群组成分布。以DNA作为靶点的宏基因组测序结果的菌 群组成为对照。在整个发酵阶段平均相对丰度前10的菌群分布结果见表1。由表1可知，基于宏基因组测序结果中Lactobacillus和Acetobacter丰度较大，分别为52.32％和10.83％，且Unclassified的丰度占29.52％；基于宏转录组测序结果中Lactobacillus和Acetobacter占比分别为66.21％和11.05％，Unclassified丰度占比为5.75％。说明两种不同条件下测序结果相差较大，以RNA为靶点的宏转录组测序是针对微生物群落的功能基因转录情况，代表了发酵过程微生物的代谢活性，更能反应发酵过程微生物的实际贡献。因此，本发明将选择RNA为靶点的宏转录组测序进行后续菌群的分析。
表1宏基因组与宏转录组测序结果中主要微生物组成
Figure PCTCN2022121559-appb-000001
实施例2：以传统发酵山西老陈醋为例的人工菌群的理性构建
(1)宏转录组测序：取等量食醋醋酸发酵阶段第1，3，5，7，9d(AAF1d,AAF3d,AAF5d,AAF7d,AAF9d)的醋醅样品，除去杂质，提取微生物群落的总RNA，对总RNA进行宏转录组测序；
(2)活性菌群组成和功能注释：每个样本的Unigene序列与NCBI-NT数据库中的细菌、古菌、真菌和病毒序列进行BLAST比对，获得各样本在种等级上的活性菌群组成分布，如图1及表2显示了食醋各个发酵过程活性微生物丰度排名(排名前列部分)。利用KEGG数据库对测序结果中的Unigene序列进行功能注释。
表2物种分类组成和动态变化
Figure PCTCN2022121559-appb-000002
Figure PCTCN2022121559-appb-000003
(3)污染和致病微生物的去除：对注释结果中的微生物，去除其中Alternaria alternata、Pantoea ananatis等污染微生物和致病微生物。
(4)核心微生物确定：以整个发酵阶段宏转录组结果中活性微生物(N个物种)的丰度为基础，按各个物种丰度逐渐降低的顺序排序(图2显示了排名前10的物种，并按从左到右物种丰度逐渐降低的顺序排列)，将最靠前的两个物种组合成第1组，并依次逐渐增加一个物种进行组合，最终得到N-1个微生物组。利用软件Minitab，采用最长距离法及Euclidean距离计算各个微生物组和各阶段原始微生物组之间的相似度并进行聚类分析。将各个微生物组与各阶段原始微生物组成均聚类成功且相似度≥90％所需的最少物种的组合确定为初始核心微生物组，结果见图3。其中，丰度排名前5的微生物组合与原始微生物组合聚类结果中出现交叉现象(图3-A)，聚类不成功。而排名前6的微生物组合与原始微生物组合的聚类结果较好(图3-B)，与发酵第1、3、5、7、9d的原始发酵样品微生物的相似度分别为91.04％、98.87％、92.39％、94.37％和93.84％，各组样品的相似度均＞90％，因此，这6个微生物Lactobacillus acetotolerans、Acetobacter pasteurianus、Lactobacillus helveticus、Lactobacillus kunkeei、Lactobacillus fermentum、Streptococcus lactis确定为初始核心微生物组。
(5)核心微生物组代谢活性验证：
①主要风味物质确定：采用高效液相色谱、气质联用仪等仪器检测食醋发酵结束时样品中的风味物质组成及含量，通过风味物质的阈值和含量计算各个风味物质的风味强度，将TAV＞1的呈味物质，ROVA＞0.1的呈香物质作为食醋中的主要风味物质。
如表3所示，样品中的主要呈味物质为草酸、琥珀酸、柠檬酸、乳酸、酒石酸、乙酸、谷氨酸、组氨酸、丙氨酸和缬氨酸；如表4所示，样品中的主要呈香 物质为乙酸(同时作为呈香和呈味物质)、3-甲基丁酸、苯甲醛、糠醛、乙酸乙酯、2,3-丁二酮、3-羟基-2-丁酮、乙酸苯乙酯、乙酸异戊酯、2,3,5-三甲基吡嗪、糠醇。
表3呈味物质的阈值和味觉活度值(TAV)
Figure PCTCN2022121559-appb-000004
表4呈香物质的阈值和相对香气活性值(ROVA)
Figure PCTCN2022121559-appb-000005
②主要代谢基因的确定：利用KEGG数据库对比得到主要风味物质的形成最关键的一个或几个代谢基因，并在宏转录组测序结果中对应筛选得到相应的物种，且相同(或不同)的基因对应的不同物种分开计算；
以柠檬酸的合成为例：
1)柠檬酸形成关键基因的查找。在KEGG中搜索citric acid，点击编号为C00158(即柠檬酸)，再点击pathway，可得到一系列的柠檬酸参与的代谢途径。点击map00020(TCA循环)，得到形成柠檬酸的关键基因gltA(EC:2.3.3.1)、ACLY (EC:2.3.3.8)、EC:2.3.3.3、acnA(EC:4.2.1.3)；再点击第二个途径map00250，此时没有柠檬酸的形成(无形成柠檬酸的基因)，则跳过；点击下一个途径map00630(乙醛酸代谢)，得到形成柠檬酸的关键基因gltA和acnA。以此类推，找到所有代谢途径中形成柠檬酸的关键基因(关键基因特指合成柠檬酸最后一步的基因)。综合结果得到柠檬酸形成的关键基因为gltA、ACLY、EC:2.3.3.3、acnA。
2)在转录组测序结果中查找基因gltA、ACLY、EC:2.3.3.3、acnA，得到该基因的转录表达量以及所对应的微生物，结果见表5，其中，未搜索到相关基因EC:2.3.3.3的转录结果。(转录组数据可以将某一基因和某一物种一一对应。前面步骤中对测序结果里面的每个基因ID进行物种注释和功能注释中，就已经对应到相应的基因名称和物种名称。宏转录组可以得到：微生物群落组成数据，某个基因的一一对应的来源微生物，某个基因的相对转录浓度等。)。
表5以柠檬酸形成为例的关键酶转录结果的统计
Figure PCTCN2022121559-appb-000006
③关键基因转录结果的统计：计算初始核心微生物组中的微生物在发酵各阶段对于主要风味物质形成关键基因的转录之和占各阶段主要风味物质形成关键基因转录的比例。
以柠檬酸为例：由步骤②可知，食醋醋酸发酵过程中，柠檬酸形成关键基因为gltA、ACLY、acnA，将表5中各阶段中核心微生物对应的3个基因的转录表达量分别求和，将3个基因在各个发酵阶段的转录表达量分别求和，求比值，即可得到初始核心微生物组(Lactobacillus acetotolerans、Acetobacter pasteurianus、Lactobacillus helveticus、Lactobacillus kunkeei、Lactobacillus fermentum、Streptococcus lactis)中的微生物在发酵第1、3、5、7、9d中关于柠檬酸形成的关键基因的转录分别占各阶段微生物的46.15％、48.28％、84.06％、98.20％、100％。
按照同样的方法计算初始核心微生物组在各个发酵阶段对于其他风味物质形成的关键基因转录表达量之和，并把各个发酵阶段的涉及的风味物质关键基因表达量相加，计算初始核心微生物组的表达量在各阶段风味物质关键基因转录中的占比。
结果得到：在发酵第1、3、5、7、9d，初始核心微生物组参与主要风味物质形成基因的转录分别占各个阶段(1、3、5、7、9d)原始微生物组基因转录的72.03％、87.54％、73.15％、77.78％、82.17％。
(6)核心微生物组代谢活性验证与人工菌群的完善
由于初始核心微生物组在发酵第1、5、7d的代谢基因转录占比＜80％，因此，按照(2)～(4)中微生物物种丰度的高低，在初始核心微生物组中添加第7位的Lactobacillus plantarum，按照步骤(5)描述方法计算新的微生物组关于主要风味物质形成的关键基因的转录占比，得到该微生物组在发酵第1、3、5、7、9d的占比分别为73.39％、89.68％、73.96％、78.17％、82.74％，不符合要求。按照同样的方法继续添加第8位的Bacillus amyloliquefaciens，所得微生物组在发酵第1、3、5、7、9d关于主要风味物质形成的关键基因的转录占比分别为78.77％、94.38％、79.08％、82.23％、87.24％，不符合要求。按照同样的方法继续添加第9位的Pediococcus pentosaceus，所得微生物组关于主要风味物质形成的关键基因的转录占比在发酵第1、3、5、7、9d分别为83.51％、96.07％、83.87％、85.17％、91.47％，各组均＞80％，符合要求。因此，将Lactobacillus acetotolerans、Acetobacter pasteurianus、Lactobacillus helveticus、Lactobacillus kunkeei、Lactobacillus fermentum、Streptococcus lactis、Lactobacillus plantarum、Bacillus amyloliquefaciens、Pediococcus pentosaceus9种核心微生物作为人工菌群起始物种。
实施例3：人工菌群制成混合麸曲应用于手工生产谷物醋醋酸发酵过程
(1)人工菌群构建：按照实施例2方法确定人工菌群；
(2)人工菌群菌株筛选：
①微生物初筛：以食醋发酵过程涉及到的曲、酒醪、醋醅等为筛选样品，使用不同培养基筛选样品中的Lactobacillus acetotolerans、Acetobacter pasteurianus、Lactobacillus helveticus、Lactobacillus kunkeei、Lactobacillus fermentum、Streptococcus lactis、Lactobacillus plantarum、Bacillus amyloliquefaciens、Pediococcus pentosaceus，挑选生长较快，菌落较大，分布密度较广但形态不同的菌落，纯化后保存；
②微生物复筛：对上述筛选获得的纯培养微生物进行测序，并对同一种的微生物进行发酵和耐受性能进行比较，选择发酵和耐受性(耐酸、耐温、耐醇)能较好的微生物作为同种微生物的优势菌。若本步骤未获得优势微生物，则按照微生物筛选方法进行重复筛选和鉴定。
其中，对于醋酸菌和乳酸菌而言，发酵性能指菌株分别产乙酸和乳酸的能力；对于芽孢杆菌而言，发酵性能指菌株产淀粉酶、蛋白酶、纤维素酶(分解原料)的能力。初筛时也同样针对上述发酵性能选择或设置含有淀粉、纤维素、葡萄糖、乙醇或乙酸等不同底物的筛选培养基，筛选醋醅样品中具有淀粉或纤维素水解、转化葡萄糖或乙醇产生有机酸、醇、酯类、醛类等特性的微生物。
其中，耐受性主要包括微生物对乙酸、乙醇和温度的耐受性。在食醋固态发酵培养基中设置不同乙酸梯度(0％、1％、2％、3％、4％)、不同乙醇梯度(0％、2％、4％、6％、8％)和不同温度梯度(30℃、35℃、40℃、45℃、50℃)，接种菌株培养后，采用平板计数法分别对培养24～36h的微生物的生长进行测试。
其中，模拟食醋固态发酵培养基组成为：麸皮30％，稻壳10％，葡萄糖1.2％，蛋白胨0.6％，牛肉膏0.6％，酵母提取物0.3％，无水乙酸钠0.3％，吐温80 0.06％，柠檬酸三铵0.12％，磷酸氢二钾0.12％，硫酸镁0.035％，硫酸锰0.015％，余量为水。
(3)混合麸曲制备：
①纯菌麸曲制备：称取麸皮450g，加水360mL搅拌均匀，分装于500mL的三角烧中，每瓶100g。灭菌后接入1％～10％步骤(2)筛选活化增殖后的微 生物菌液，30℃～37℃培养60～65h，粉碎得到麸曲，再扩大培养制成浅盘曲并进行活菌计数；
②混合麸曲制备：根据实施例2中宏转录组测序结果，以菌种在整个发酵阶段中的平均比例(各阶段样品等量混合后测定菌种比例)为基础。根据①的纯菌麸曲计数结果，以Lactobacillus acetotolerans：Lactobacillus helveticus：Lactobacillus kunkeei：Lactobacillus fermentum：Acetobacter pasteurianus：Streptococcus lactis：Lactobacillus plantarum：Bacillus amyloliquefaciens：Pediococcus pentosaceus＝60：10：5：2：15：2：1：1：1的比例将纯菌麸曲混匀即得到混合麸曲。
(4)混合麸曲应用于谷物醋醋酸发酵过程
①酒精发酵：将高粱粉碎后温水润料4～8h。按高粱质量0.1％的耐高温α淀粉酶90℃以上液化30min。待温度降至60℃，按高粱质量的2％添加固体糖化酶，58～60℃糖化1h。之后按高粱质量加入62.5％粉碎后的大曲，补水后接入活化好的活性干酵母。入酒缸进行酒精发酵，前3天敞口发酵，后4天封口发酵，共7天。发酵温度维持在28℃～30℃之间。
②混合麸曲应用于醋酸发酵：酒精发酵结束后，按照酒醪：麸皮：稻壳＝5：1.1：0.6的比例添加麸皮和稻壳，混匀后分装至醋缸。按照10 9～10 10cfu/kg(原始样品中核心微生物的浓度)原料添加混合麸曲并与醅反复混匀，缸口上盖草垫，每天进行一次翻醅(倒缸)。跟踪取样，当酒精度下降至0.5％，酸度、还原糖不变时停止发酵。设置3个平行实验组。
③在相同条件下采用传统“种醅”进行食醋固态发酵作为对照组，检测发酵结束时醋醅的各项理化指标。结果见表6。
表6人工菌群应用于谷物醋醋酸发酵的理化指标(单位：g/100g醋醅)
Figure PCTCN2022121559-appb-000007
Figure PCTCN2022121559-appb-000008
由表6可知，相对于传统种醅，采用人工菌群生产食醋发酵周期缩短22.22％，发酵结束时，不挥发酸、还原糖、氨基酸态氮、总酯、原料利用率分别提高了33.33％～41.95％、35.77％～39.43％、15.79％～21.05％、20.87％～25.51％、10.46％～12.54％。且不挥发酸/挥发酸增加了49.89％～61.13％，增加了食醋的柔和感。
实施例4：人工菌群制成混合菌剂应用于果醋液态发酵过程
(1)人工菌群构建：以自然发酵葡萄醋为研究对象，按照实施例2方法构建人工菌群，确定了核心微生物菌群为：Lactococcus lactis、Lactobacillus plantarum、Lactobacillus casei、Lactobacillus paracase、Lactobacillus fermentum、Acetobacter pasteurianus。并根据宏转录组测序结果得到各菌种比例为Lactococcus lactis：Lactobacillus plantarum：Lactobacillus casei：Lactobacillus paracase：Lactobacillus fermentum：Acetobacter pasteurianus＝10：25：25：25：25：1；
(2)人工菌群菌株筛选：按照实施例3方法筛选菌株；
(3)混合菌剂制备
①纯菌菌剂制备：将活化后的菌株以1％～10％的接种量接种于增殖培养基培养至生长稳定期，然后利用喷雾冷冻干燥分别制得Lactococcus lactis、Lactobacillus plantarum、Lactobacillus casei、Lactobacillus paracase、Lactobacillus fermentum、Acetobacter pasteurianus纯菌菌剂；
②混合菌剂制备：按照纯菌菌剂活菌数Lactococcus lactis：Lactobacillus plantarum：Lactobacillus casei：Lactobacillus paracase：Lactobacillus fermentum：Acetobacter pasteurianus＝10 9：2.5×10 9：2.5×10 9：2.5×10 9：2.5×10 9：10 8的比例进行复配得到混合菌剂。
(4)混合菌剂应用于葡萄醋液态发酵过程
①原料预处理及酒精发酵：新鲜葡萄除梗破碎后，添加0.2％SO 2(偏重亚硫酸钾)杀菌1h，再添加0.1％果胶酶(10000U/g酶活)于50℃酶解4h，降至 室温后调糖度为18度，最后将活化后的活性干酵母加入其中，并入发酵罐30℃发酵。当比重和糖度不再变化时(约3～5d)，酒精发酵结束。
②醋酸发酵：用葡萄汁将原酒酒度降度为8度左右，发酵罐装料量为75％～80％、调节通气量至0.15vvm、转速调至2000r/min。待温度至30℃后，校准溶氧电极，使用饱和无水亚硫酸钠校准零，将溶氧电极插入发酵液中校准100％。接入0.4％(v/w，100mL原料添加0.4g混合菌剂)的加水溶解的混合菌剂，跟踪取样，当酒精度低于0.5％，酸度不再上升停止发酵。1g菌剂中含有微生物菌体的数量为Lactococcus lactis：Lactobacillus plantarum：Lactobacillus casei：Lactobacillus paracase：Lactobacillus fermentum：Acetobacter pasteurianus＝10 9：2.5×10 9：2.5×10 9：2.5×10 9：2.5×10 9：10 8。设置三个实验组作为平行对照。
③在上述条件下与自然发酵的葡萄醋为对照，并检测发酵结束时样品中的各项理化指标，结果见表7。
表7人工菌群应用于液体发酵葡萄醋理化指标(单位：g/100mL)
Figure PCTCN2022121559-appb-000009
采用人工菌群发酵所得的葡萄醋：外观呈紫红颜色，澄清透明，有光泽；酸甜爽口，葡萄果香味浓郁，无异味。由表7可知，同自然发酵葡萄醋相比，总酸含量增加了14.44％～16.14％，不挥发酸增加了159.57％～176.60％，不挥发酸/挥发酸提高了155.87％～180.01％。乙酸、乳酸、苹果酸、总酯含量分别增加了 7.88％～10％、282.61％～313.04％、33.33％～46.67％，发酵时间缩短了53.33％，乙酸形成速率提高了70.21％～74.47％。不挥发酸尤其是乳酸含量大大增加，使葡萄醋酸味更加柔，减少了尖酸感，同时乳酸菌的添加增加了总酯的含量，增加了果醋的醇厚感，提高了鲜葡萄果醋的品质。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本专利构思的前提下，上述各实施方式还可以做出若干变形、组合和改进，这些都属于本专利的保护范围。因此，本专利的保护范围应以权利要求为准。

Claims (10)

  1. 一种食醋发酵人工菌群构建方法，其特征在于，包括如下步骤：
    (1)微生物群落功能和物种注释：取不同发酵阶段的醋醅样品，除去杂质，分别提取微生物的总RNA，进行宏转录组测序，对测序结果进行功能和物种注释；
    (2)污染和致病微生物的去除：对注释到相应功能的微生物，去除其中的污染微生物和致病微生物；
    (3)初始核心微生物的确定：以宏转录组结果中各发酵阶段所有微生物(N个物种)的丰度为基础，按照整个发酵过程中各个物种平均丰度逐渐降低的顺序排序，将最靠前的两个物种组合成第1组，并依次逐渐增加一个物种进行组合，最终得到N-1个微生物组；
    将微生物组与各阶段的原始微生物组均聚类成功且相似度均≥90％时所需的最少物种确定为初始核心微生物组；
    (4)核心微生物组代谢活性验证：
    ①确定发酵结束时样品中的主要风味物质及对应的代谢基因；
    ②计算初始核心微生物组关于上述代谢基因的转录表达量在各个发酵阶段原始微生物关于上述代谢基因的转录表达量的占比，若与各阶段主要风味代谢基因转录占比均≥80％，则进行下一步；若与某一阶段主要风味物质代谢基因转录占比＜80％，则按步骤(3)中物种丰度依次增加一个物种，并重复步骤(4)，直至与每个阶段主要风味物质代谢基因转录占比均达到≥80％，至此确定出核心微生物组；
    (5)人工菌群菌株的筛选：根据核心微生物组的组成，从曲、酒醪、醋醅等样品中筛选获得性能优异的菌种，并按照各阶段原始发酵样品中核心微生物组中相应微生物的平均生物量的比例进行复配，获得最终的人工菌群组成。
  2. 如权利要求1所述的一种食醋发酵人工菌群构建方法，其特征在于，步骤(3)中将各微生物组物种丰度数据导入软件Minitab，采用最长距离法及Euclidean距离计算各个微生物组和各阶段原始微生物菌群的相似度并进行聚类分析。
  3. 如权利要求1所述的一种食醋发酵人工菌群构建方法，其特征在于，步骤(4)所述主要风味物质，是指对发酵结束时样品中主要风味物质组成进行检测分析，其中TAV＞1的有机酸、氨基酸定义为主要滋味物质，ROAV＞0.1的挥发性香气化合物定义为主要香气化合物，两者共同组成食醋的主要风味物质。
  4. 如权利要求1所述的一种食醋发酵人工菌群构建方法，其特征在于，步骤(4)所述主要风味物质对应的代谢基因的确定是通过在KEGG数据库中对比，并在宏转录组测序结果中筛选确定。
  5. 如权利要求1所述的一种食醋发酵人工菌群构建方法，其特征在于，步骤(5)所述菌株的筛选是指，采用不同的选择性培养基先对醋醅样品中微生物进行初筛，并以耐酸、耐温、耐醇等条件进行菌株的复筛，最后根据不同的实验菌株进行发酵功能验证，从而确定最优菌株，并最终确定人工菌群组成。
  6. 如权利要求1所述的一种食醋发酵人工菌群构建方法，其特征在于，微生物组与各阶段的原始微生物组均聚类成功且相似度均≥90％。
  7. 如权利要求1所述的一种食醋发酵人工菌群构建方法，其特征在于，核心微生物组关于主要风味物质代谢基因的转录表达量在各个发酵阶段原始微生物主要风味物质代谢基因转录表达量的占比≥85％。
  8. 由权利要求1-5任意一项构建的人工菌群。
  9. 权利要求8所述人工菌群在食醋醋酸发酵中的应用。
  10. 如权利要求9所述的应用，其特征在于，应用于液态或固态的谷物醋或果醋发酵过程。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180137243A1 (en) * 2016-11-17 2018-05-17 Resilient Biotics, Inc. Therapeutic Methods Using Metagenomic Data From Microbial Communities
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CN110564581A (zh) * 2019-09-17 2019-12-13 天津科技大学 一种传统固态食醋定向风味调控发酵技术及其应用
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Publication number Priority date Publication date Assignee Title
US20180137243A1 (en) * 2016-11-17 2018-05-17 Resilient Biotics, Inc. Therapeutic Methods Using Metagenomic Data From Microbial Communities
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