CN113707221A - Fish sauce flavor forming functional microbial exoenzyme mining method based on multi-dimensional data - Google Patents
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Abstract
The invention provides a traditional fermented fish sauce flavor formation functional microbial exoenzyme mining method based on multi-dimensional data drive, which is characterized by respectively constructing a flavor substance change data set and a fermented fish sauce microbial function gene database in a fish sauce fermentation process, performing species annotation analysis on unigene translated protein and identified protein, inferring an amino acid sequence through reverse genetics to obtain corresponding encoding gene information, tracking the functions of microbial communities in a complex environment, mining microbial exoenzyme influencing flavor formation, and providing technical support for realizing traditional fermented marine food quality targeted regulation and control.
Description
Technical Field
The invention relates to the field of food biological manufacturing, in particular to a fish sauce flavor forming functional microorganism ectoenzyme excavation method based on multi-dimensional data.
Background
The fish sauce is also called 'fish sauce', and is an aquatic seasoning with unique flavor prepared by taking low-value fish or aquatic product processing byproducts as raw materials and performing natural fermentation. The fish sauce is amber in color and delicious in taste, is rich in taurine, essential amino acids for human bodies, organic acids and trace elements, and is popular with people. The production of the traditional fish sauce in China is mostly distributed in the southeast coastal areas, and the traditional fish sauce is produced in Shandong, Jiangsu, Zhejiang, Fujian, Guangdong and the like. The annual output of the fish sauce in Guangdong province is about 2 million tons, which accounts for more than 60 percent of the total annual output in China, wherein the fish sauce in the Chaoshan region in Guangdong is famous and has a large production scale. Fish sauce is also an important catering condiment in asian countries such as thailand, malaysia, japan and korea.
The traditional fish sauce fermentation process generally adopts a natural fermentation mode of high-salt salting (the salt adding amount is generally 25% -30%), under the synergistic action of microorganisms and endogenous enzymes in fish bodies, nutritional ingredients such as protein, lipid and the like in the fish bodies are decomposed and fermented, and then the fish sauce is brewed after ageing. The traditional fish sauce industry has the main advantages of long history, unique flavor and the like, but the development of the traditional fish sauce industry is far lagged behind other traditional fermented foods. The production period of the traditional fish sauce is long, the product quality is unstable, the production cost is increased, and the large-scale production degree is reduced. The reason for this is mainly that the research level of the fish gravy production is low at present, the theoretical system support of the system is lacked, and the actual production activity cannot be well guided. The fermentation process of the fish gravy is a key process for forming the quality of the fish gravy, and a complex fermentation system is formed among microorganisms, enzymes and metabolites through a series of exchanges of substances, energy and the like in the fermentation process of the fish gravy. The rapid fermentation of fish sauce is a research hotspot of the traditional fish sauce industry, but the research work of the rapid fermentation of fish sauce at present mainly stays at the stage of preliminary research and pilot-scale demonstration in a laboratory, and the flavor of the product is poor, so that the industrial production and application cannot be carried out. In the current stage, a link of urgent need to be overcome is to overcome the problems of long fermentation period, high salinity and the like of the traditional fish gravy, strive to directionally excavate an excellent microbial enzyme preparation and promote the development of flavor improvement and rapid fermentation of the traditional fish gravy.
Disclosure of Invention
The invention provides a fish sauce flavor forming functional microorganism ectoenzyme excavation method based on multi-dimensional data, aiming at the problems that most of the prior technical researches are mainly based on microorganism and ectoenzyme screening of the traditional pure culture mode, lack of accurate identification on types, functions and sources in a fish sauce fermentation system and cannot obtain detailed microorganism ectoenzyme information in the fish sauce fermentation system on the whole.
The purpose of the invention is realized by adopting the following technical scheme:
a method for excavating functional microbial exoenzymes for forming fish sauce flavor based on multidimensional data comprises the following steps:
(1) measuring the dynamic change of the content of flavor metabolites including free amino acids and flavor nucleotides in the traditional fish sauce fermentation process, and constructing a flavor substance change data set;
(2) performing metagenome sequencing on the traditional fish gravy microbial communities in different fermentation stages; performing optimization treatment including joint removal, quality shearing and pollution removal on the original sequence, predicting spliced genome by using MetaGene, and constructing a traditional fermented fish sauce microbial functional gene database;
(3) desalting the fermentation liquor, collecting protein component effluent, and performing quantitative analysis on the extracted protein;
(4) carrying out reductive alkylation and Trypsin enzymolysis on the extracted qualified protein in sequence, and identifying and quantitatively analyzing a product obtained after the Trypsin enzymolysis by adopting liquid chromatography-mass spectrometry according to a quantitative result;
(5) comparing macro-gene unigene translated protein data sets of the traditional fish gravy samples in different fermentation stages constructed in the step (2) as a database, submitting an RAW file to a Mascot server through a Proteome resolver, performing species annotation analysis on unigene translated proteins, and performing taxonomic species annotation analysis on identified proteins;
(6) adopting Simca-14.1 software to introduce a traditional microbial extracellular enzyme database and flavor metabolites to carry out O2PLS latent variable regression analysis, defining the property of a variable, defining a microbial extracellular enzyme data set as an independent variable X and a flavor data set as a dependent variable Y, and defining and calculating an independent variable projection Importance index VIP (variable immunity in project), wherein the calculation formula is as follows:
rd (Y; t) in the formulai) Denotes the interpretability of the dependent variable Y by ti, wik2Representing the degree of component marginal contribution of xk to ti;
and (3) digging the flavor of the traditional fish gravy fermentation process to form microbial exoenzymes on the basis of the constructed O2PLS model under the conditions that VIP >1 and p are less than 0.05, and performing bioinformatics analysis on the identified protein.
Preferably, the content of the taste metabolites is determined by a derivatization method, after a primary amino acid is derivatized with o-phthalaldehyde (OPA) and a secondary amino acid is derivatized with fluorenylmethoxycarbonyl chloride (FMOC), a sample is separated by a ZORBAX eclipseAAA chromatographic column (150mm multiplied by 4.6mm), and the flow rate of the column is 1mL/min at 40 ℃; proline was detected using a fluorescence detector at 266nm and 305nm excitation and emission wavelengths, respectively, while the other amino acids were detected using an ultraviolet detector at 338nm, mobile phase a was sodium dihydrogen phosphate buffer at pH 7.8 and 0.04mol/L concentration, mobile phase B was acetonitrile: methanol: the water volume ratio is 45: 45: 10, and (b) a mixed solution.
Preferably, the IlluminaHiSeq technology is adopted to carry out the metagenomic sequencing on the traditional fish dew microbial communities in different fermentation stages, a nucleic acid cutting instrument is firstly used for breaking the genomic DNA into short DNA fragments, the TruSeq DNA Sample Prep Kit is used for constructing a PE library, then the cBot Truseq PE Cluster Kit is used for carrying out bridge PCR, and then Illumina Hiseq 2500 is used for carrying out the metagenomic sequencing on the traditional fish dew microbial communities in different fermentation stages.
Preferably, the desalting treatment in the step (3) adopts a PD-10 purification desalting column for purification desalting.
Preferably, the reductive alkylation in the step (4) comprises the following steps:
s1, adding a TAC/acetone solution with the volume 4 times that of the protein component effluent to precipitate the protein for 12 hours, centrifugally collecting the protein precipitate, washing with acetone, adding 8mol/L urea lysis solution to carry out heavy suspension, and carrying out ultrasonic crushing after heavy suspension to obtain a protein solution;
s2, adding 1mol/L dithiothreitol solution into the protein solution, mixing uniformly, keeping the temperature at 37 ℃ for 1h, adding 1mol/L iodoacetamide solution, mixing uniformly, standing in the dark at room temperature, and reacting for 1h to obtain a reduction alkylation product;
wherein the volume ratio of the protein solution to the dithiothreitol solution to the iodoacetamide solution is 12: 1: 4.
preferably, the separation mobile phase A of the liquid chromatography-mass spectrometry combined in the step (4) is 0.1 wt% formic acid solution, the mobile phase B is 80 vol% acetonitrile solution and 0.08 wt% formic acid solution, and the sample is loaded at the flow rate of 600nL/min and sequentially passes through a 3 μm × 100 μm × 20mm C18 mass spectrometry pre-column and a 1.9 μm × 50 μm × 120mm C18 chromatographic column for separation.
The invention has the beneficial effects that:
the invention provides a traditional fermented fish sauce flavor forming functional microbial exoenzyme excavation method based on multi-dimensional data driving, corresponding coding gene information is obtained through reverse genetics to speculate an amino acid sequence, the function of microbial communities in a complex environment is tracked, the dynamic change of flavor components in the traditional fish sauce fermentation process is integrated, the flavor is directionally excavated to form the functional microbial exoenzyme, an effective multi-dimensional fish sauce fermentation flavor excavating method based on a multi-dimensional data driving model is established, compared with the traditional pure culture blind screening, the method has the advantages of high flux, strong purposiveness, high predictability and the like, and technical support is provided for achieving the targeted regulation and control of the traditional fermented marine food quality.
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The invention is further illustrated by means of the attached drawings, but the embodiments in the drawings do not constitute any limitation to the invention, and for a person skilled in the art, other drawings can be obtained on the basis of the following drawings without inventive effort.
FIG. 1 is a functional annotation statistical chart of conventional fish gravy samples at different stages of fermentation based on the KEGG database;
FIG. 2 is a Bradford quantitative standard curve;
FIG. 3 is a graph of relative molecular mass distribution of identified proteases;
FIG. 4 is the annotation information of the extracellular enzyme species of the conventional fish gravy microorganism.
Detailed Description
The invention is further described with reference to the following examples.
The embodiment of the invention relates to a method for excavating functional microbial exoenzymes, which is applied to a Chinese traditional fish gravy fermentation system, in particular to a method for excavating functional microbial exoenzymes for forming the taste of traditional fermented fish gravy based on multi-dimensional data drive, and aims to excavate the taste of the functional microbial exoenzymes for forming the taste by synthesizing the dynamic change of taste components in the traditional fish gravy fermentation process.
The traditional fish sauce used in this example was obtained from Chaoshan region of Guangdong province, the fermentation time was 1, 3, 6, 9, 12 months, and 5 samples of different fermentation points were taken at each fermentation stage, mixed, and filtered to obtain a fish sauce sample.
1. Weighing 5.00g of a filtered fish gravy sample in a 50mL centrifuge tube, adding 5mL of 0.02mol/L hydrochloric acid solution into the centrifuge tube, performing ultrasonic extraction for 10min at 25 ℃, then centrifuging for 10min at 10000rpm at 4 ℃, taking supernatant, fixing the volume to 10mL by using ultrapure water, filtering by using a 0.22 mu m filter membrane, and analyzing by using Agilent 1100 HPLC; adopting an automatic online derivatization method of Agilent company, after a primary amino acid is derivatized with o-phthalaldehyde (OPA) and a secondary amino acid is derivatized with fluorenylmethoxycarbonyl chloride (FMOC), separating a sample by a ZORBAX eclipse AAA chromatographic column (150mm multiplied by 4.6mm), wherein the flow rate of the column is 1mL/min at 40 ℃; proline was detected using a fluorescence detector at 266nm and 305nm excitation and emission wavelengths, respectively, while the other amino acids were detected using an ultraviolet detector at 338nm, mobile phase a was 0.04mol/L sodium dihydrogen phosphate buffer (pH 7.8), mobile phase B was acetonitrile: methanol: water 45%: 45%: 10 percent;
2. extracting the microbial metagenome DNA in the fish gravy sample by adopting a metagenome DNA extraction kit (DP326) of Tiangen biochemical technology; the purity and the concentration of a DNA sample are detected by using NanoDrop2000, and the microbial metagenome DNA meeting the subsequent test requirements is placed in a refrigerator at the temperature of-20 ℃ for later use;
3. the genomic DNA was fragmented into short DNA fragments of about 300bp in length using a Covaris M220 nucleic acid cutter, and TruSeq was usedTMConstructing a PE library by using the DNA Sample Prep Kit, performing bridge PCR by using a cBot Truseq PE Cluster Kit, and then performing macro-genome sequencing on the traditional fish gravy microbial community at different fermentation stages by using Illumina Hiseq 2500 to construct a traditional fermented fish gravy microbial functional gene database;
the functional annotation results of the traditional fish gravy samples at different fermentation stages based on the KEGG database are shown in FIG. 1;
4. measuring 8mL of filtered fish sauce sample, centrifuging for 5min under the condition of 15000r/min, taking the centrifuged supernatant, manually purifying and desalting by a PD-10 purification desalting column, collecting protein component effluent after purification and desalting, adding 4 times of volume of TAC/acetone solution, standing overnight, precipitating the protein for 12h, then centrifuging the overnight protein solution for 5min under the condition of 10000r/min, collecting protein precipitate, washing the protein precipitate by acetone, adding 8mol/L of urea lysate for re-suspension, and ultrasonically crushing the re-suspended protein solution (the power is 900W x 30%, the interval is 4s per 2s of ultrasound, and 10 cycles of ultrasonic treatment are carried out);
5. accurately weighing 2 mug, 4 mug, 6 mug, 8 mug and 10 mug of standard substance respectively, and making a Bradford protein quantitative standard curve; quantifying the Bradford protein by adopting an H4MFPTAD enzyme-labeling instrument;
the Bradford protein quantitative standard curve is shown in figure 2, and the linearity of the standard curve is good;
6. respectively measuring 60 mu L of fish sauce protein solution in five different fermentation stages into a centrifuge tube, adding 5 mu L of 1mol/L dithiothreitol solution into the centrifuge tube, uniformly mixing by using an oscillator, carrying out water bath heat preservation for 1h at 37 ℃, adding 20 mu L of 1mol/L iodoacetamide solution, carrying out oscillation and uniform mixing, standing in the dark at room temperature for reaction for 1h, transferring the treated protein sample into an ultrafiltration tube, centrifuging to discard a collection solution, adding 100 mu L of UA solution (8mol/L urea, 0.1 mol/L-HCl, pH8.0) into the ultrafiltration tube, centrifuging to remove the collection solution, repeating the operation twice, adding 100 mu L of 0.05mol/L ammonium bicarbonate solution, centrifuging to remove the collection solution, repeating the operation three times, and replacing a new collection tube according to the protein: trypsin 50: 1, adding Trypsin according to the weight ratio, and then carrying out enzymolysis for 12-16h at 37 ℃;
7. taking a product obtained after the Trypsin enzymolysis according to a quantitative result, carrying out liquid phase-mass spectrum combined analysis, carrying out three biological repetitions on each sample, and separating by adopting Easy nLC/Ultimate 3000 liquid phase, wherein a mobile phase buffer solution A is 0.1 wt.% formic acid, a buffer solution B consists of 80 vol.% acetonitrile and 0.08 wt.% formic acid, the sample is loaded to a 3 mu m 100 mu m 20mm C18 mass spectrum pre-column at the flow rate of 600nL/min by an automatic sample injector, and is separated by a 1.9 mu m 50 mu m 120mm C18 chromatographic column;
8. the original data of mass spectrometry is RAW file, and the library checking identification and quantitative analysis are carried out by using Mascot 2.1 software and Proteome resolver (thermo), and the relative molecular mass of the identified microbial extracellular enzyme is mainly concentrated between 10 and 50kDa, as shown in figure 3; performing species annotation analysis on unigene translated protein by using the fish gravy microorganism functional gene database constructed in the step 3, performing taxonomic species annotation analysis on 571 proteins identified after filtration, and accurately identifying the type, function and source of the biological extracellular enzyme in the fish gravy fermentation system; when in comparison, submitting the RAW file to a Mascot server through a Proteome discover machine;
the annotation information of the extracellular enzyme species of the traditional fish gravy microorganisms is shown in FIG. 4;
9. the method comprises the following steps of constructing a correlation matrix between microbial exoenzymes and flavor substances in the traditional fish gravy fermentation process by adopting an orthogonal partial least squares (O2PLS) method in Simca-P14.1, mining the microbial exoenzymes influencing flavor formation by comparing independent variable VIP values and taking VIP >1 and P < 0.05 as conditions, wherein the specifically obtained key microbial exoenzyme information is shown in the following table:
finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (6)
1. A method for excavating functional microbial exoenzymes for forming fish sauce flavor based on multidimensional data is characterized by comprising the following steps:
(1) measuring the dynamic change of the content of flavor metabolites including free amino acids and flavor nucleotides in the traditional fish sauce fermentation process, and constructing a flavor substance change data set;
(2) performing metagenome sequencing on the traditional fish gravy microbial communities in different fermentation stages; performing optimization treatment including joint removal, quality shearing and pollution removal on the original sequence, predicting spliced genome by using MetaGene, and constructing a traditional fermented fish sauce microbial functional gene database;
(3) desalting the fermentation liquor, collecting protein component effluent, and performing quantitative analysis on the extracted protein;
(4) carrying out reductive alkylation and Trypsin enzymolysis on the extracted qualified protein in sequence, and identifying and quantitatively analyzing a product obtained after the Trypsin enzymolysis by adopting liquid chromatography-mass spectrometry according to a quantitative result;
(5) comparing macro-gene unigene translated protein data sets of the traditional fish gravy samples in different fermentation stages constructed in the step (2) as a database, submitting an RAW file to a Mascot server through a Proteome resolver, performing species annotation analysis on unigene translated proteins, and performing taxonomic species annotation analysis on identified proteins;
(6) introducing Simca-14.1 software into a traditional microbial extracellular enzyme database and flavor metabolites to perform O2PLS latent variable regression analysis, defining the property of a variable, defining a microbial extracellular enzyme data set as an independent variable X and a flavor data set as a dependent variable Y, and performing defined calculation on an independent variable projection importance index VIP, wherein the calculation formula is as follows:
rd (Y; t) in the formulai) Denotes the interpretability of the dependent variable Y by ti, wik2Representing the degree of component marginal contribution of xk to ti;
and (3) digging the flavor of the traditional fish gravy fermentation process to form microbial exoenzymes on the basis of the constructed O2PLS model under the conditions that VIP >1 and p are less than 0.05, and performing bioinformatics analysis on the identified protein.
2. The method for excavating the functional microbial extracellular enzymes for forming the fish sauce flavor based on the multidimensional data as claimed in claim 1, wherein the content of the flavor metabolites is measured by a derivatization method, and after a primary amino acid is derivatized with o-phthalaldehyde and a secondary amino acid is derivatized with fluorenylmethoxycarbonyl chloride, a sample is separated by a ZORBAX Eclipse AAA chromatographic column, wherein the column flow rate is 1mL/min at 40 ℃; proline was detected using a fluorescence detector at 266nm and 305nm excitation and emission wavelengths, respectively, while the other amino acids were detected using an ultraviolet detector at 338nm, mobile phase a was sodium dihydrogen phosphate buffer at pH 7.8 and 0.04mol/L concentration, mobile phase B was acetonitrile: methanol: the water volume ratio is 45: 45: 10, and (b) a mixed solution.
3. The method for mining functional microbial exoenzymes for fish sauce flavor formation based on multidimensional data as claimed in claim 1, wherein Illumina HiSeq technology is adopted to perform metagenomic sequencing on traditional fish sauce microbial communities in different fermentation stages, a nucleic acid cutting instrument is firstly used to break genomic DNA into short DNA fragments, TruSeq DNA Sample Prep Kit is used to construct PE library, then cBot Truseq PE Cluster Kit is used to perform bridge PCR, and Illumina Hiseq 2500 is then used to perform metagenomic sequencing on traditional fish sauce microbial communities in different fermentation stages.
4. The method for mining the functional microbial exoenzyme for fish sauce flavor development based on multidimensional data as claimed in claim 1, wherein the desalting treatment in step (3) is carried out by using a PD-10 purification desalting column for purification and desalting.
5. The method for mining fish sauce flavor-forming functional microbial exoenzymes according to claim 1, wherein the reductive alkylation in the step (4) comprises the following steps:
s1, adding a TAC/acetone solution with the volume 4 times that of the protein component effluent to precipitate the protein for 12 hours, centrifugally collecting the protein precipitate, washing with acetone, adding 8mol/L urea lysis solution to carry out heavy suspension, and carrying out ultrasonic crushing after heavy suspension to obtain a protein solution;
s2, adding 1mol/L dithiothreitol solution into the protein solution, mixing uniformly, keeping the temperature at 37 ℃ for 1h, adding 1mol/L iodoacetamide solution, mixing uniformly, standing in the dark at room temperature, and reacting for 1h to obtain a reduction alkylation product;
wherein the volume ratio of the protein solution to the dithiothreitol solution to the iodoacetamide solution is 12: 1: 4.
6. the method for digging functional microbial extracellular enzymes for fish sauce flavor formation based on multidimensional data, as claimed in claim 1, wherein the liquid chromatography-mass spectrometry is used in the step (4) to separate the formic acid solution with a mobile phase A of 0.1 wt% and the acetonitrile solution with a mobile phase B of 80 vol% and the formic acid solution of 0.08 wt%, and the sample is loaded at a flow rate of 600nL/min and sequentially separated by a mass spectrum pre-column of 3 μm x 100 μm x 20mm C18 and a mass spectrum column of 1.9 μm x 50 μm x 120mm C18.
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| CN109979527A (en) * | 2019-03-08 | 2019-07-05 | 广州基迪奥生物科技有限公司 | A kind of transcript profile and metabolism group data relation analysis method and system |
| CN112342284A (en) * | 2020-10-19 | 2021-02-09 | 北京大学 | Method for analyzing microbial community functional gene transcription and translation activity |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109979527A (en) * | 2019-03-08 | 2019-07-05 | 广州基迪奥生物科技有限公司 | A kind of transcript profile and metabolism group data relation analysis method and system |
| CN112342284A (en) * | 2020-10-19 | 2021-02-09 | 北京大学 | Method for analyzing microbial community functional gene transcription and translation activity |
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| Title |
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| SHINYA YAMAMOTO 等: "Analysis of the correlation between dipeptides and taste differences among soy sauces by using metabolomics-based component profiling", 《JOURNAL OF BIOSCIENCE AND BIOENGINEERING》 * |
| WENHUI ZHU等: "Changes in taste substances during fermentation of fish sauce and the correlation with protease activity", 《FOOD RESEARCHINTERNATIONAL》 * |
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