WO2023173756A1 - Metabolism and metagenomics combined research method for changes in cantonese soy sauce fermentation process - Google Patents

Metabolism and metagenomics combined research method for changes in cantonese soy sauce fermentation process Download PDF

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WO2023173756A1
WO2023173756A1 PCT/CN2022/127929 CN2022127929W WO2023173756A1 WO 2023173756 A1 WO2023173756 A1 WO 2023173756A1 CN 2022127929 W CN2022127929 W CN 2022127929W WO 2023173756 A1 WO2023173756 A1 WO 2023173756A1
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soy sauce
metagenomics
cantonese
fermentation process
metabolism
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李俊
曹庸
温林凤
陈聪
刘果
王培培
徐婷
林虹
扈圆舒
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广东美味鲜调味食品有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation

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  • the present invention relates to the field of food fermentation mechanism research, specifically a method for jointly studying changes in the Cantonese soy sauce fermentation process using metabolism and metagenomics.
  • Cantonese-style soy sauce adopts a high-salt dilute fermentation method.
  • the high-salt dilute fermentation process refers to adding 18-20% salt water (generally 2-2.5 times of the finished koji) to the finished koji to ferment the mash into a flowing state. This method has the characteristics of high salt content, high moisture content, and long fermentation cycle (3-6 months).
  • soy sauce brewing The two most important processes of soy sauce brewing are koji making and mash fermentation.
  • koji making and fermentation process of soy sauce production the population structure of Aspergillus oryzae, yeast, Streptococcus halophilus and other microorganisms and the regulation of their metabolites play a key role in the formation and quality of the color, aroma, taste and body of soy sauce. Therefore, the various metabolic substances of microorganisms during the fermentation process and their abundance changes are closely related to the flavor substances of soy sauce.
  • Cantonese-style soy sauce is mostly based on the normal-temperature high-salt dilute fermentation process, combined with bacterial strain making, natural sun-drying process, and cooking with oil.
  • the final product has a strong sauce aroma and good flavor. Due to natural fermentation at room temperature, the cycle is long, it is greatly affected by the natural environment, and process changes are difficult to control. There is a problem that the flavor of fermented products in different seasons has certain differences.
  • the purpose of the present invention is to provide a method for jointly studying changes in the fermentation process of Cantonese soy sauce using metabolism and metagenomics, so as to solve the technical problems raised by the above background technology.
  • the present invention provides the following technical solution: a method for jointly studying changes in the fermentation process of Cantonese soy sauce using metabolism and metagenomics. Combining the characteristics of the fermentation process, a sampling plan is established to analyze the soy sauce base and water at different fermentation time points. Huang conducts production sampling, taking at least 3 tanks at each time node. After sampling, the samples are frozen and stored at -20°C or -80°C for later use.
  • the specific experimental steps include the following:
  • Metabolomics samples were extracted step by step using solvents such as 5-10mL petroleum ether, ethyl acetate, absolute ethanol, 50-90% ethanol and deionized water at room temperature or ultrasonic for 0.5-5 minutes. After extraction, centrifuge at 3000-5000rpm/min for 10-15min, collect the supernatant, and use the lower precipitate as the next level of extraction. The final extracts of each section are removed by nitrogen blowing, vacuum drying, freeze-drying, and freeze-centrifugation to remove the solvent accurately. Weigh and peel to obtain the weight of dry matter extracted under different solvents. The SPE solid phase extraction column is activated with 2-8 mL of methanol, and then equilibrated with 2-5 mL of deionized water.
  • solvents such as 5-10mL petroleum ether, ethyl acetate, absolute ethanol, 50-90% ethanol and deionized water at room temperature or ultrasonic for 0.5-5 minutes. After extraction, centrifuge at 3000-5000rpm
  • the extract is prepared into a 0.5-2 mL aqueous solution with a concentration of 0.5-2 mg/mL for loading.
  • the sample is desalted through a desalting column. Afterwards, it is dried, dissolved, and passed through a membrane, and the loading conditions of the liquid chromatography mass spectrometer are set for detection.
  • Metagenomics samples are frozen and stored at -80°C. Each sample is set with 3 biological replicates. Lysis solution and enzyme solution are added to the centrifuge tube to ultrasonically extract DNA samples from the sample tissue cells.
  • the lysis solution contains Tris-HCl buffer. solution, EDTA, NaCl, SDS and deionized water, etc.
  • the enzymes used mainly include lysozyme, protease, etc.
  • the extracted DNA sample is tested for concentration through QubitFluorometer, and its purity and integrity are tested using agarose gel electrophoresis. .
  • Metagenomics analyzes differential species, differential functions, corresponding enzyme system characteristics, and microbial communities through gene Venn analysis, differential gene analysis, and gene function annotation to find dominant and differential bacterial groups, and combine the results of the two omics analyze.
  • the present invention mainly has the following beneficial effects:
  • the present invention screens out the differential metabolites between each group. Based on the sources of the differential metabolites, circulation metabolic pathways, etc., the microbial changes and metabolic pathways are inferred to determine The iconic metabolites between groups reveal the metabolic changes of the aroma components and taste components of soy sauce during the fermentation process and their formation mechanisms, thereby achieving control of the fermentation process.
  • the soy sauce fermentation process can be controlled and the problem of seasonal fluctuations in flavor and output value of Cantonese-style high-salt dilute fermentation soy sauce can be solved.
  • Figure 1 is a DNA electrophoresis diagram of a sample in a specific embodiment of the present invention.
  • Figure 2 is a species cluster analysis diagram of the present invention
  • Figure 3 is a diagram showing the change pattern of the top 5 bacterial groups in the present invention.
  • Figure 4 is a correlation analysis of the microbial community of the present invention and the physical and chemical indicators of soy sauce.
  • a method for jointly studying changes in Cantonese soy sauce fermentation process using metabolism and metagenomics including the following steps:
  • the soy sauce fermentation base material and water yellow sample are fermented in the Cantonese high-salt dilute state method, including fermentation base materials at different stages within the different fermentation days of 0-180 days for samples that started fermentation in spring, summer, autumn and winter.
  • the low-temperature freeze-drying methods used include vacuum freeze-drying, liquid nitrogen drying, and freeze-spray drying.
  • tissue cells of the sample use a cell disruptor, ball mill, or bead mill to disrupt the tissue cells of the sample.
  • Part 1 extracts and processes the raw materials through different solvents and then performs liquid chromatography mass spectrometry detection.
  • the solvents include petroleum ether, ethyl acetate, ethanol of different concentrations and deionized water.
  • the extracted sample processing methods include concentration and desalination. , filtering, etc. for computer testing.
  • Part 2 Add lysis solution and enzyme solution to extract DNA samples from tissue cells.
  • the lysis solution includes Tris-HCl buffer, EDTA, NaCl, SDS and deionized water.
  • the enzymes used mainly include lysozyme, protease, etc.
  • the extracted DNA samples were sequenced, filtered and assembled, followed by metagenomics analysis.
  • Methods used to establish the correlation between flavor substances and microbial diversity, population structure or functional activity include establishing a flavor substance database, cluster analysis, species abundance analysis, significant difference analysis and other methods.
  • the metagenomic samples were selected at the same time point, and there were 3 biological replicates at each time point. After accurately weighing the samples, the sample tissues were broken according to the method of metabolomics processing samples. The samples were extracted with a DNA extraction kit and the concentration was determined using Agarose gel electrophoresis was used to analyze the quality of extracted DNA. The electrophoresis conditions used were: sample volume: 1 ⁇ L, gel concentration: 1%, voltage: 120V, time: 20 min.
  • Tetracoccus halophilus Lactobacillus weissenii, Weissella mesenteroides, Enterobacter horsi and Aspergillus oryzae .
  • Halophilic Tetracoccus increased significantly from the 15th day of fermentation, while Aspergillus oryzae decreased significantly.
  • Metabolic pathway analysis was conducted on the results of different fermentation stages.
  • the metabolic pathways involved in metabolites in different fermentation stages have the same pathways, which mainly include: degradation of aromatic compounds, coenzyme A, amino acid formation, metabolism of salts and amines, and indole substances. Formation, etc.; in the 30- to 45-day period, the formation of bitter substances and the formation of some free amino acids increased.
  • Streptococcus, Aspergillus oryzae, and Enterobacter sakazakii were positively correlated, and were positively correlated with pH value and salt content
  • Leuconostoc pseudoenteros, Leuconostoc mesenteric, Tetradococcus, and Leuconostoc citron were positively correlated, and were positively correlated with pH value and salt content are negatively correlated.

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Abstract

A metabolism and metagenomics combined research method for changes in a Cantonese soy sauce fermentation process. The soy sauce metabolism and metagenomics combined method comprises: performing liquid chromatography-mass spectrometry analysis on soy sauce samples in different seasons and different fermentation stages, combining sequencing, filtering and assembling, and then performing metagenomic gene prediction, gene function annotation, species analysis, function analysis, diversity analysis, and the like. Flavor substance change, microbial diversity, population structure, evolutionary relationship, functional activity, and mutual cooperation relationship in the soy sauce fermentation process are researched by means of a liquid chromatography-mass spectrometry and metagenomics analysis method, so that the purpose of regulating and controlling the soy sauce fermentation process is achieved; the problem that the flavor and output value of the Cantonese high-salt diluted-state fermented soy sauce fluctuate along with seasons is solved, and the product quality is kept at a high level throughout the year.

Description

一种代谢与宏基因组学联合研究广式酱油发酵过程变化的方法A method to jointly study changes in Cantonese soy sauce fermentation process using metabolism and metagenomics 技术领域Technical field
本发明涉及食品发酵机理研究领域，具体为一种代谢与宏基因组学联合研究广式酱油发酵过程变化的方法。The present invention relates to the field of food fermentation mechanism research, specifically a method for jointly studying changes in the Cantonese soy sauce fermentation process using metabolism and metagenomics.
背景技术Background technique
广式酱油采用高盐稀态发酵方式，高盐稀态发酵工艺是指在成曲中加入18-20％的盐水(一般为成曲的2-2.5倍)，使酱醪成流动状态发酵，该法具有含盐量高、水份多、发酵周期长(3-6个月)等特点。Cantonese-style soy sauce adopts a high-salt dilute fermentation method. The high-salt dilute fermentation process refers to adding 18-20% salt water (generally 2-2.5 times of the finished koji) to the finished koji to ferment the mash into a flowing state. This method has the characteristics of high salt content, high moisture content, and long fermentation cycle (3-6 months).
酱油酿造最重要的两个工艺环节是制曲和酱醪发酵。其中，在酱油生产的制曲和发酵过程中，米曲霉、酵母菌、嗜盐链球菌等微生物的种群结构及其代谢产物的调控对酱油色、香、味和体态的形成和品质起到了关键的作用，因此，微生物在发酵过程的各种代谢物质及其丰度变化与酱油的风味物质密切相关。The two most important processes of soy sauce brewing are koji making and mash fermentation. Among them, in the koji making and fermentation process of soy sauce production, the population structure of Aspergillus oryzae, yeast, Streptococcus halophilus and other microorganisms and the regulation of their metabolites play a key role in the formation and quality of the color, aroma, taste and body of soy sauce. Therefore, the various metabolic substances of microorganisms during the fermentation process and their abundance changes are closely related to the flavor substances of soy sauce.
广式酱油多以常温的高盐稀态发酵工艺为主，结合菌种制曲、天然晒露工艺，抽油的调配煮制，最终其产品酱香浓郁、风味好。由于常温自然发酵，周期长，受自然环境影响较大，过程变化难以控制，存在不同季节发酵产品风味有一定差异的问题。Cantonese-style soy sauce is mostly based on the normal-temperature high-salt dilute fermentation process, combined with bacterial strain making, natural sun-drying process, and cooking with oil. The final product has a strong sauce aroma and good flavor. Due to natural fermentation at room temperature, the cycle is long, it is greatly affected by the natural environment, and process changes are difficult to control. There is a problem that the flavor of fermented products in different seasons has certain differences.
发明内容Contents of the invention
基于此，本发明的目的是提供一种代谢与宏基因组学联合研究广式酱油发酵过程变化的方法，以解决上述背景技术提出的技术问题。Based on this, the purpose of the present invention is to provide a method for jointly studying changes in the fermentation process of Cantonese soy sauce using metabolism and metagenomics, so as to solve the technical problems raised by the above background technology.
为实现上述目的，本发明提供如下技术方案：一种代谢与宏基因组学联合研究广式酱油发酵过程变化的方法，结合发酵过程特点，建立取样计划，对发酵不同时间点的酱油基料和水黄进行生产取样，每个时间节点至少取3个罐，取样后样品-20℃或-80℃冷冻保存备用，具体包括以下实验步骤：In order to achieve the above purpose, the present invention provides the following technical solution: a method for jointly studying changes in the fermentation process of Cantonese soy sauce using metabolism and metagenomics. Combining the characteristics of the fermentation process, a sampling plan is established to analyze the soy sauce base and water at different fermentation time points. Huang conducts production sampling, taking at least 3 tanks at each time node. After sampling, the samples are frozen and stored at -20°C or -80°C for later use. The specific experimental steps include the following:
(1)将每个样品采用液氮和冷冻干燥等方法进行低温冻干，准确称取0.1-5g酱油发酵基料或水黄样品至离心管中，空离心管提前称重，样品量的多少根据物质的检测限及样品的固形物含量进行调整。(1) Freeze-dry each sample at low temperature using methods such as liquid nitrogen and freeze-drying. Accurately weigh 0.1-5g of soy sauce fermentation base or water yellow sample into a centrifuge tube. Weigh the empty centrifuge tube in advance to determine the amount of the sample. Adjust according to the detection limit of the substance and the solid content of the sample.
(2)将装有样品的离心管中加入研磨用的不同1-3mm直径的珠子对样品进行组织破碎，研磨时间为1-5min，每20-30s暂停5-10s。(2) Add beads of different diameters of 1-3mm for grinding into the centrifuge tube containing the sample to disrupt the sample. The grinding time is 1-5min, pausing for 5-10s every 20-30s.
(3)代谢组学样品采分别用5-10mL石油醚、乙酸乙酯、无水乙醇、50-90％乙醇和去离子水等溶剂室温下震荡或超声0.5-5min进行逐级提取，每次提取后采用3000-5000rpm/min离心10-15min，取上清收集，下层沉淀作为下一级提取，最后各段提取液分别采用氮吹、真空干燥、冷冻干燥和冷冻离心等方法除去溶剂后准确称重，去皮得到不同溶剂下提取得到的干物质重量。将SPE固相萃取柱采用2-8mL甲醇活化，再用2-5mL去离子水平衡，将提取液配成0.5-2mg/mL浓度水溶液0.5-2mL进行上样，样品通过脱盐柱脱除样品盐分后，干燥、溶解、过膜，设定液相色谱质谱的上样条件，进行检测。(3) Metabolomics samples were extracted step by step using solvents such as 5-10mL petroleum ether, ethyl acetate, absolute ethanol, 50-90% ethanol and deionized water at room temperature or ultrasonic for 0.5-5 minutes. After extraction, centrifuge at 3000-5000rpm/min for 10-15min, collect the supernatant, and use the lower precipitate as the next level of extraction. The final extracts of each section are removed by nitrogen blowing, vacuum drying, freeze-drying, and freeze-centrifugation to remove the solvent accurately. Weigh and peel to obtain the weight of dry matter extracted under different solvents. The SPE solid phase extraction column is activated with 2-8 mL of methanol, and then equilibrated with 2-5 mL of deionized water. The extract is prepared into a 0.5-2 mL aqueous solution with a concentration of 0.5-2 mg/mL for loading. The sample is desalted through a desalting column. Afterwards, it is dried, dissolved, and passed through a membrane, and the loading conditions of the liquid chromatography mass spectrometer are set for detection.
(4)宏基因组学样品-80℃冷冻保存，每个样品设置3个生物学重复，离心管中加入裂解液、酶液超声提取样品组织细胞的DNA样本，其中的裂解液包含Tris-HCl缓冲液、EDTA、NaCl、SDS和去离子水等，所使用的酶主要有溶菌酶、蛋白酶等，将所提取的DNA样本通过QubitFluorometer检测浓度，并采用琼脂糖凝胶电泳方法检测其纯度和完整性。(4) Metagenomics samples are frozen and stored at -80°C. Each sample is set with 3 biological replicates. Lysis solution and enzyme solution are added to the centrifuge tube to ultrasonically extract DNA samples from the sample tissue cells. The lysis solution contains Tris-HCl buffer. solution, EDTA, NaCl, SDS and deionized water, etc. The enzymes used mainly include lysozyme, protease, etc. The extracted DNA sample is tested for concentration through QubitFluorometer, and its purity and integrity are tested using agarose gel electrophoresis. .
(5)对代谢组学样品的检测结果导出后采用软件和平台进行代谢物层次聚类分析、样本树状图分析和差异代谢物筛选，寻找差异代谢物。宏基因组学通过基因Venn分析、差异基因分析、基因功能注释分析差异物种、差异功能及相应的酶系特点、微生物群落，找出优势菌群和差异菌群，将两种组学的结果进行联合分析。(5) After exporting the test results of metabolomics samples, use software and platforms to perform metabolite hierarchical clustering analysis, sample dendrogram analysis and differential metabolite screening to find differential metabolites. Metagenomics analyzes differential species, differential functions, corresponding enzyme system characteristics, and microbial communities through gene Venn analysis, differential gene analysis, and gene function annotation to find dominant and differential bacterial groups, and combine the results of the two omics analyze.
综上所述，本发明主要具有以下有益效果：To sum up, the present invention mainly has the following beneficial effects:
本发明通过对不同发酵阶段酱油的代谢物和宏基因联合分析，筛选到各 组间的差异性代谢物，根据差异性代谢物的来源、循环代谢途径等，推测其微生物的变化、代谢路径，确定组间的标志性代谢物，揭示酱油的香气成分和滋味成分在发酵过程的代谢变化规律及其形成的机理，实现发酵过程控制。通过对风味形成代谢机理的研究，实现酱油发酵过程可控化，解决广式高盐稀态发酵酱油风味及产值数随季节波动的问题。By jointly analyzing the metabolites and metagenes of soy sauce at different fermentation stages, the present invention screens out the differential metabolites between each group. Based on the sources of the differential metabolites, circulation metabolic pathways, etc., the microbial changes and metabolic pathways are inferred to determine The iconic metabolites between groups reveal the metabolic changes of the aroma components and taste components of soy sauce during the fermentation process and their formation mechanisms, thereby achieving control of the fermentation process. Through the study of the metabolic mechanism of flavor formation, the soy sauce fermentation process can be controlled and the problem of seasonal fluctuations in flavor and output value of Cantonese-style high-salt dilute fermentation soy sauce can be solved.
附图说明Description of the drawings
图1为本发明具体实施方式中样品的DNA电泳图；Figure 1 is a DNA electrophoresis diagram of a sample in a specific embodiment of the present invention;
图2为本发明的物种聚类分析图；Figure 2 is a species cluster analysis diagram of the present invention;
图3为本发明排名前5位的菌群变化规律图；Figure 3 is a diagram showing the change pattern of the top 5 bacterial groups in the present invention;
图4为本发明的微生物群落与酱油理化指标相关性分析。Figure 4 is a correlation analysis of the microbial community of the present invention and the physical and chemical indicators of soy sauce.
具体实施方式Detailed ways
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述。下面通过参考附图描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. The embodiments described below with reference to the drawings are exemplary and are only used to explain the present invention and cannot be understood as limiting the present invention.
一种代谢与宏基因组学联合研究广式酱油发酵过程变化的方法，包括以下步骤：A method for jointly studying changes in Cantonese soy sauce fermentation process using metabolism and metagenomics, including the following steps:
S1、取不同发酵阶段酱油发酵基料及水黄，采用低温冷冻干燥方法去除水分；S2、对样品的组织细胞进行破碎，部分一通过不同溶剂对原料进行提取并处理后进行液相色谱质谱检测并对谱图结果物质进行分析鉴定，部分二加入裂解液、酶液提取组织细胞的DNA样本；S3、分析酱油发酵过程物质变化规律，建立风味物质与微生物多样性、种群结构或功能活性之间的关联。S1. Take the soy sauce fermentation base and water yellow at different fermentation stages, and use low-temperature freeze-drying to remove water; S2. Fragment the tissue cells of the sample, extract and process the raw materials with different solvents, and then conduct liquid chromatography mass spectrometry detection and Analyze and identify the substances from the spectrum results. In Part 2, add lysis solution and enzyme solution to extract DNA samples from tissue cells; S3. Analyze the changes in substances during the soy sauce fermentation process and establish the relationship between flavor substances and microbial diversity, population structure or functional activity. association.
酱油发酵基料及水黄样品为广式高盐稀态方式发酵的，包括春、夏、秋、冬开始发酵的样品的不同发酵天数0-180天内不同阶段的发酵基料。The soy sauce fermentation base material and water yellow sample are fermented in the Cantonese high-salt dilute state method, including fermentation base materials at different stages within the different fermentation days of 0-180 days for samples that started fermentation in spring, summer, autumn and winter.
采用的低温冷冻干燥方法包括真空冷冻干燥、液氮干燥、冷冻喷雾干燥。The low-temperature freeze-drying methods used include vacuum freeze-drying, liquid nitrogen drying, and freeze-spray drying.
对样品的组织细胞进行破碎中通过细胞破碎仪、球磨仪、珠磨仪对样品 的组织细胞进行破碎。During the disruption of the tissue cells of the sample, use a cell disruptor, ball mill, or bead mill to disrupt the tissue cells of the sample.
部分一通过不同溶剂对原料进行提取并处理后进行液相色谱质谱检测，其中的溶剂包括石油醚、乙酸乙酯、不同浓度乙醇和去离子水等，萃取后的样品的处理方法包括浓缩、脱盐、过滤等进行上机测试。 Part 1 extracts and processes the raw materials through different solvents and then performs liquid chromatography mass spectrometry detection. The solvents include petroleum ether, ethyl acetate, ethanol of different concentrations and deionized water. The extracted sample processing methods include concentration and desalination. , filtering, etc. for computer testing.
部分二加入裂解液、酶液提取组织细胞的DNA样本，其中的裂解液包含Tris-HCl缓冲液、EDTA、NaCl、SDS和去离子水等，所使用的酶主要有溶菌酶、蛋白酶等，将所提取的DNA样本进行测序、过滤及组装，之后进行宏基因组学分析。Part 2: Add lysis solution and enzyme solution to extract DNA samples from tissue cells. The lysis solution includes Tris-HCl buffer, EDTA, NaCl, SDS and deionized water. The enzymes used mainly include lysozyme, protease, etc. The extracted DNA samples were sequenced, filtered and assembled, followed by metagenomics analysis.
建立风味物质与微生物多样性、种群结构或功能活性之间的关联所采用方法包括建立风味物质数据库、聚类分析、物种丰度分析、显著差异分析等方法。Methods used to establish the correlation between flavor substances and microbial diversity, population structure or functional activity include establishing a flavor substance database, cluster analysis, species abundance analysis, significant difference analysis and other methods.
具体实施方式如下：The specific implementation is as follows:
首先选取7个时间点(分为A、B、C、D、E、F和G组)取酱油发酵基料样品，每个时间点3个重复，将样品进行冻干，每种样品各取3g(精确至小数点后三位小数)至15mL离心管中，加入1mm和3mm珠子用珠磨仪研磨2min，每隔20s暂停5s，加入相应试剂，用石油醚、乙酸乙酯、无水乙醇、90％乙醇逐级提取，具体操作如下：First, select 7 time points (divided into groups A, B, C, D, E, F and G) to take samples of soy sauce fermentation base material. Each time point is repeated 3 times. The samples are freeze-dried and each sample is taken. 3g (accurate to three decimal places after the decimal point) into a 15mL centrifuge tube, add 1mm and 3mm beads and grind with a bead mill for 2 minutes, pause for 5s every 20s, add the corresponding reagents, use petroleum ether, ethyl acetate, absolute ethanol, 90% ethanol is extracted step by step. The specific operations are as follows:
(1)在已取样的15mL离心管中加入9mL石油醚，旋涡震荡30s，再将上清倒入2个5mL离心管中；(1) Add 9 mL petroleum ether to the sampled 15 mL centrifuge tube, vortex for 30 seconds, and then pour the supernatant into two 5 mL centrifuge tubes;
(2)(2)加入9mL乙酸乙酯，旋涡震荡20s，再将上清倒入2个5mL离心管中；(2) (2) Add 9mL of ethyl acetate, vortex for 20s, and then pour the supernatant into two 5mL centrifuge tubes;
(3)加入9mL无水乙醇，旋涡震荡20s，再将上清倒入2个5mL离心管中；(3) Add 9 mL of absolute ethanol, vortex for 20 seconds, and then pour the supernatant into two 5 mL centrifuge tubes;
(4)加入9mL90％乙醇，旋涡震荡20s，再将上清倒入2个5mL离心管中；(4) Add 9 mL of 90% ethanol, vortex for 20 seconds, and then pour the supernatant into two 5 mL centrifuge tubes;
(5)加入9mL蒸馏水，旋涡震荡20s，再将上清倒入2个5mL离心管中，将以上5mL离心管放入真空干燥器中，干燥至无液体后称重，(5) Add 9mL of distilled water, vortex for 20 seconds, then pour the supernatant into two 5mL centrifuge tubes, put the above 5mL centrifuge tubes into a vacuum desiccator, dry until there is no liquid and then weigh.
(6)用2mL纯甲醇活化柱子，再用2mL纯水平衡柱子，然后上样(体积：1mL，浓度：1mg/mL浓度)，同时用2mL离心管收集过柱液；再1mL 95％甲醇洗柱，同时用2mL离心管收集过柱液；再用3mL甲醇洗脱，同时用5mL离心管收集过柱液，用真空干燥仪干燥，溶解，待测；(7)采用超高压色谱柱，设置液相条件和流动相参数，以及一级和二级质谱条件上样检测。(6) Use 2mL of pure methanol to activate the column, then use 2mL of pure water to balance the column, then load the sample (volume: 1mL, concentration: 1mg/mL concentration), and use a 2mL centrifuge tube to collect the column liquid; then wash with 1mL of 95% methanol column, and use a 2 mL centrifuge tube to collect the liquid that has passed through the column; then use 3 mL of methanol to elute, and use a 5 mL centrifuge tube to collect the liquid that has passed through the column, dry it with a vacuum dryer, dissolve it, and wait for testing; (7) Use an ultra-high-pressure chromatographic column and set it Liquid phase conditions and mobile phase parameters, as well as primary and secondary mass spectrometry conditions for sample loading and detection.
宏基因组学样品选取同样的时间点，每个时间点3个生物学重复，准确称取样品后按照代谢组学处理样品的方式将样品组织破碎，样品采用DNA提取试剂盒提取后鉴定浓度，采用琼脂糖凝胶电泳分析提取DNA的质量，采用的电泳条件为：点样量为1μL，胶浓度：1％，电压：120V，时间：20min。The metagenomic samples were selected at the same time point, and there were 3 biological replicates at each time point. After accurately weighing the samples, the sample tissues were broken according to the method of metabolomics processing samples. The samples were extracted with a DNA extraction kit and the concentration was determined using Agarose gel electrophoresis was used to analyze the quality of extracted DNA. The electrophoresis conditions used were: sample volume: 1 μL, gel concentration: 1%, voltage: 120V, time: 20 min.
得到的相关数据如下相关的附表如下所示：The relevant data obtained are as follows and the relevant appendices are as follows:
Figure PCTCN2022127929-appb-000001
Figure PCTCN2022127929-appb-000001
Figure PCTCN2022127929-appb-000002
Figure PCTCN2022127929-appb-000002
Figure PCTCN2022127929-appb-000003
Figure PCTCN2022127929-appb-000003
DNA样品质检信息表DNA sample quality inspection information sheet
通过代谢组学和宏基因组学的联合分析，菌群物种丰度排在前五的分别为：嗜盐四联球菌、魏斯氏乳酸菌、类肠膜魏斯氏菌、霍氏肠杆菌和米曲霉。嗜盐四联球菌从发酵开始15天起显著增加，米曲霉则显著降低。Through joint analysis of metabolomics and metagenomics, the top five bacterial species in terms of abundance are: Tetracoccus halophilus, Lactobacillus weissenii, Weissella mesenteroides, Enterobacter horsi and Aspergillus oryzae . Halophilic Tetracoccus increased significantly from the 15th day of fermentation, while Aspergillus oryzae decreased significantly.
对不同发酵阶段的结果进行代谢通路分析，不同发酵阶段代谢物参与的代谢通路存在相同的路径，主要包括：芳香族化合物的降解、辅酶A、氨基酸形成、盐、胺的代谢、吲哚类物质形成等；30天至45天阶段内，增加了苦味物质形成以及一些游离氨基酸形成路径。物质的变化与菌群的变化存在一定程度的关联性。其中，链球菌、米曲霉、阪崎肠杆菌呈正相关，且与pH值、盐含量呈正相关；假肠膜明串珠菌、肠系膜明串珠菌、四联球菌、柠檬明串珠菌呈正相关，与pH值、盐含量呈负相关。Metabolic pathway analysis was conducted on the results of different fermentation stages. The metabolic pathways involved in metabolites in different fermentation stages have the same pathways, which mainly include: degradation of aromatic compounds, coenzyme A, amino acid formation, metabolism of salts and amines, and indole substances. Formation, etc.; in the 30- to 45-day period, the formation of bitter substances and the formation of some free amino acids increased. There is a certain degree of correlation between changes in substances and changes in bacterial flora. Among them, Streptococcus, Aspergillus oryzae, and Enterobacter sakazakii were positively correlated, and were positively correlated with pH value and salt content; Leuconostoc pseudoenteros, Leuconostoc mesenteric, Tetradococcus, and Leuconostoc citron were positively correlated, and were positively correlated with pH value and salt content are negatively correlated.
尽管已经示出和描述了本发明的实施例，但本具体实施例仅仅是对本发明的解释，其并不是对发明的限制，描述的具体特征、结构、材料或者特点 可以在任何一个或多个实施例或示例中以合适的方式结合，本领域技术人员在阅读完本说明书后可在不脱离本发明的原理和宗旨的情况下，可以根据需要对实施例做出没有创造性贡献的修改、替换和变型等，但只要在本发明的权利要求范围内都受到专利法的保护。Although embodiments of the invention have been shown and described, these specific embodiments are merely illustrative of the invention and are not intended to limit the invention. The specific features, structures, materials or characteristics described may be used in any one or more The embodiments or examples are combined in a suitable manner. After reading this description, those skilled in the art can make modifications and substitutions to the embodiments as needed without inventive contribution without departing from the principles and purposes of the present invention. and modifications, etc., as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims (7)

  1. 一种代谢与宏基因组学联合研究广式酱油发酵过程变化的方法，其特征在于，包括以下步骤：A method for jointly studying changes in Cantonese soy sauce fermentation process using metabolism and metagenomics, which is characterized by including the following steps:
    S1、取不同发酵阶段酱油发酵基料及水黄，采用低温冷冻干燥方法去除水分；S1. Take soy sauce fermentation base material and water yellow at different fermentation stages, and use low-temperature freeze-drying method to remove moisture;
    S2、对样品的组织细胞进行破碎，部分一通过不同溶剂对原料进行提取并处理后进行液相色谱质谱检测并对谱图结果物质进行分析鉴定，部分二加入裂解液、酶液提取组织细胞的DNA样本；S2. Fragment the tissue cells of the sample. Part 1 extracts and processes the raw materials through different solvents and then performs liquid chromatography mass spectrometry detection and analyzes and identifies the spectral results. Part 2 adds lysis solution and enzyme solution to extract the tissue cells. DNA sample;
    S3、分析酱油发酵过程物质变化规律，建立风味物质与微生物多样性、种群结构或功能活性之间的关联。S3. Analyze the changes in substances during the fermentation process of soy sauce, and establish the relationship between flavor substances and microbial diversity, population structure or functional activity.
  2. 根据权利要求1所述的一种代谢与宏基因组学联合研究广式酱油发酵过程变化的方法，其特征在于：酱油发酵基料及水黄样品为广式高盐稀态方式发酵的，包括春、夏、秋、冬开始发酵的样品的不同发酵天数0-180天内不同阶段的发酵基料。A method for jointly studying changes in the Cantonese-style soy sauce fermentation process by metabolism and metagenomics according to claim 1, characterized in that: the soy sauce fermentation base material and the water yellow sample are Cantonese-style high-salt dilute state fermentation, including spring, Fermentation base materials at different stages within 0-180 days of different fermentation days for samples that started fermenting in summer, autumn and winter.
  3. 根据权利要求1所述的一种代谢与宏基因组学联合研究广式酱油发酵过程变化的方法，其特征在于：采用的低温冷冻干燥方法包括真空冷冻干燥、液氮干燥、冷冻喷雾干燥。A method for jointly studying changes in Cantonese soy sauce fermentation process by metabolism and metagenomics according to claim 1, characterized in that: the low-temperature freeze-drying method used includes vacuum freeze-drying, liquid nitrogen drying, and freeze-spray drying.
  4. 根据权利要求1所述的一种代谢与宏基因组学联合研究广式酱油发酵过程变化的方法，其特征在于：对样品的组织细胞进行破碎中通过细胞破碎仪、球磨仪、珠磨仪对样品的组织细胞进行破碎。A method for jointly studying changes in Cantonese soy sauce fermentation process by metabolism and metagenomics according to claim 1, which is characterized in that: when the tissue cells of the sample are broken, the sample is processed by a cell disruptor, a ball mill, and a bead mill. The tissue cells are broken.
  5. 根据权利要求1所述的一种代谢与宏基因组学联合研究广式酱油发酵过程变化的方法，其特征在于：部分一通过不同溶剂对原料进行提取并处理后进行液相色谱质谱检测，其中的溶剂包括石油醚、乙酸乙酯、不同浓度乙醇和去离子水，萃取后的样品的处理方法包括浓缩、脱盐、过滤等进行上机测试。A method for jointly studying changes in Cantonese soy sauce fermentation process by metabolism and metagenomics according to claim 1, characterized in that: Part 1 extracts and processes the raw materials through different solvents and then performs liquid chromatography mass spectrometry detection, wherein The solvents include petroleum ether, ethyl acetate, ethanol of different concentrations and deionized water. The processing methods of the extracted samples include concentration, desalting, filtration, etc. for on-machine testing.
  6. 根据权利要求1所述的一种代谢与宏基因组学联合研究广式酱油发酵 过程变化的方法，其特征在于：部分二加入裂解液、酶液提取组织细胞的DNA样本，其中的裂解液包含Tris-HCl缓冲液、EDTA、NaCl、SDS和去离子水，所使用的酶主要有溶菌酶、蛋白酶等，将所提取的DNA样本进行测序、过滤及组装，之后进行宏基因组学分析。A method for jointly studying changes in Cantonese soy sauce fermentation process by metabolism and metagenomics according to claim 1, characterized in that: in part two, a lysis solution and an enzyme solution are added to extract DNA samples of tissue cells, and the lysis solution contains Tris -HCl buffer, EDTA, NaCl, SDS and deionized water. The enzymes used mainly include lysozyme, protease, etc. The extracted DNA samples are sequenced, filtered and assembled, and then metagenomic analysis is performed.
  7. 根据权利要求1所述的一种代谢与宏基因组学联合研究广式酱油发酵过程变化的方法，其特征在于：建立风味物质与微生物多样性、种群结构或功能活性之间的关联所采用方法包括建立风味物质数据库、聚类分析、物种丰度分析、显著差异分析。A method for jointly studying changes in Cantonese soy sauce fermentation process by metabolism and metagenomics according to claim 1, characterized in that: the method used to establish the correlation between flavor substances and microbial diversity, population structure or functional activity includes Establish flavor substance database, cluster analysis, species abundance analysis, and significant difference analysis.
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