CN111380965A - Method for detecting metabolite in microbial fermentation liquid - Google Patents
Method for detecting metabolite in microbial fermentation liquid Download PDFInfo
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- 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
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
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Abstract
The invention relates to a method for detecting metabolites in microbial fermentation broth, which comprises the following steps: (1) taking a microbial fermentation liquid to be detected, placing the microbial fermentation liquid at the temperature of between 70 ℃ below zero and 90 ℃ below zero for 4 to 8 hours, carrying out vacuum freeze drying at night, dissolving the microbial fermentation liquid by using N, N-dimethylformamide, and carrying out vortex oscillation and centrifugation to obtain a supernatant which is a metabolite solution; (2) adding the metabolite solution into the internal standard solution, uniformly mixing, and adding a derivatization agent for silanization derivatization treatment; (3) and taking the derivative treated metabolite solution for GC-MS detection. The invention can reserve the metabolites as much as possible and extract the metabolites as many as possible by processing the fermentation liquor of the microorganism to be detected, and has high extraction efficiency.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, and relates to a method for detecting metabolites in microbial fermentation liquor.
Background
The fermentation technology refers to a technology for controlling the fermentation process by using the fermentation action of microorganisms and applying some technical means so as to produce fermentation products in a large scale. The raw materials produce foods desired by people in such a fermentation environment using the metabolic activity of microorganisms, and new compounds capable of promoting human health are generated in the foods while substances having adverse health potential are removed, so that the microorganisms are considered as a key factor for determining the quality of fermented foods. The fermentation converts organic substances into food, so that the food can inhibit the growth of putrefying bacteria and common pathogenic bacteria, improve the nutritive value of the original unfermented food, and change the original color, shape and flavor according to the will of people, and is increasingly accepted and favored by people. The fermented food has unique flavor and rich nutrition, and has great relation with various metabolites generated by fermentation raw materials of fermentation strains. The unique flavor and nutrient components in some fermented foods are mainly generated by converting raw materials into metabolites under the action of microorganisms and enzymes. These metabolites interact to produce a unique fermentation product flavor.
There are many metabolites of microorganisms, and 37 broad classes are known, of which 16 belong to drugs. Products produced in the logarithmic growth phase of the thalli, such as amino acid, nucleotide, polysaccharide, fatty acid, vitamin and the like, are necessary for the growth and the propagation of the thalli. These products are called primary metabolites, many of which form a variety of fermentation industries, such as food fermentation industry, respectively, and the metabolites produced by microbial fermentation produce unique and nutritious fermentation flavors. Raw materials which are not beneficial to being directly digested and absorbed by human bodies are decomposed and absorbed by microorganisms to form substances with higher nutritive value, and the metabolites interact with each other to form rich fermentation substances and flavors. The metabolites of the microorganisms play a key role in bioactive substances, food characteristics, flavor and the like in the fermented food in the fermentation process, so that the main role of the microorganisms in the fermentation process can be better revealed by the research on the metabolites of the microorganisms in the fermented food, the fermentation mechanism of the microorganisms can be better mastered by analyzing the relationship between the microorganisms and the metabolites through the qualitative and quantitative analysis on the fermentation metabolites, and the theoretical basis is provided for the quality control and industrialization of the products.
At present, the research on metabolites is mainly carried out by extracting substances to be detected, detecting the substances by a large instrument through derivatization and utilizing the support of technologies such as GC-MS and HPLC, or determining the substances according to the physicochemical properties of various substances according to the national standard method. Metabolites generated by general fermentation mainly comprise amino acids, organic acids, fatty acids, saccharides, alcohols, esters, phenols, ketones, amines and other organic matters, each substance has a unique detection method, so that each detection method has limitations, including extraction of a substance to be detected and derivatization, the types of the metabolites can be rarely detected as much as possible by one extraction method and a derivatization reagent, and the general detection time is long and the detection efficiency is low. In addition, the method according to the national standard can only roughly detect the content of certain metabolites, and can not detect specific metabolites.
Therefore, it is desired to provide a method capable of simultaneously extracting and detecting a plurality of metabolites from a microbial fermentation broth.
Disclosure of Invention
Based on the above, the main object of the present invention is to provide a method for detecting metabolites in a microbial fermentation broth. The detection method adopts proper extraction conditions, and can extract metabolites in the microbial fermentation broth as far as possible for subsequent detection.
The main purpose of the invention is realized by the following technical scheme:
a method for detecting metabolites in a microbial fermentation broth, the method comprising the steps of:
(1) taking a microbial fermentation liquid to be detected, placing the microbial fermentation liquid at the temperature of minus 250 ℃ to minus 50 ℃ for 4h to 8h, then carrying out vacuum freeze drying at night, dissolving the microbial fermentation liquid by using N, N-dimethylformamide, and centrifuging to obtain a supernatant, namely a metabolite solution;
(2) adding the metabolite solution into the internal standard solution, uniformly mixing, and adding a derivatization agent for silanization derivatization treatment;
(3) and taking the derivative treated metabolite solution for GC-MS detection.
In some embodiments, in step (2), the derivatization with a derivatization agent comprises: adding a derivatization agent, uniformly mixing, sealing, and then carrying out water bath for 1-5 h at the temperature of 37-90 ℃; the derivatization agent is bis (trimethylsilyl) trifluoroacetamide and trimethylchlorosilane in a volume ratio of (95-99.5): (0.5-5).
In some embodiments, in step (2), the derivatizing agent is a mixed solution of bis (trimethylsilyl) trifluoroacetamide and chlorotrimethylsilane in a volume ratio of 99: 1.
In some embodiments, the temperature of the water bath is 78-82 ℃, the time of the water bath is 1.5-2.5 h, and the derivatization agent is bis (trimethylsilyl) trifluoroacetamide and trimethylchlorosilane in a volume ratio of (98.5-99.5): (0.5-1.5).
In some of these embodiments, the GC-MS detection employs chromatographic conditions comprising:
a chromatographic column: a weakly polar capillary column with 5% diphenyl, 1% vinyl, 94% dimethyl polysiloxane as the filler; carrier gas: helium gas; the split ratio is as follows: 4.5-5.5; the temperature rising procedure is as follows: maintaining at 70 deg.C for 8 min; heating to 190 ℃ at the speed of 2.5-3.5 ℃ per minute for 8-48 min; 48-59 min, and heating to 300 ℃ at the speed of 9.5-10.5 ℃ per minute; the mixture was kept at 300 ℃ for 31 min.
In some of these embodiments, the GC-MS detection employs chromatographic conditions comprising:
the chromatographic column comprises HP-5(0.25 mu m × 30m × 0.25.25 mm), carrier gas and helium, the split ratio is 5.0, the temperature rise program is that the temperature is kept at 70 ℃ for 8min, the temperature rises to 190 ℃ at the speed of 3 ℃ per minute for 8min to 48min, the temperature rises to 300 ℃ at the speed of 10 ℃ per minute for 48min to 59min, and the temperature is kept at 300 ℃ for 31 min.
In some of these embodiments, the mass spectrometry conditions employed for the GC-MS detection include: ion source temperature: 215 ℃ to 225 ℃, interface temperature: 245 ℃ to 255 ℃, solvent delay time: 3.5-4.5 min, threshold: 100-1000, start time: 2.5 min-3.5 min, end time: 56.5 min-57.5 min, interval: 0.3sec to 0.5sec, scanning speed: 500-1000, range: 40m/Z to 500 m/Z.
In some of these embodiments, the mass spectrometry conditions employed for the GC-MS detection include: ion source temperature: 220 ℃, interface temperature: 250 ℃, solvent delay time: 4min, threshold: 1000, start time: 3min, end time: 57min, interval: 0.5sec, scanning speed: 1000, range: 40m/Z to 500 m/Z.
In some embodiments, the internal standard solution is prepared by adding 80mg to 100mg heptadecanoic acid particles per 10mL of N, N-dimethylformamide.
In some embodiments, the volume ratio of the fermentation broth of the microorganism to be tested in step (1), N-dimethylformamide, and the internal standard solution and the derivatizing agent in step (2) is (8-12): (18-22): 1: (18-22).
In some embodiments, the microbial fermentation broth to be tested is first soy sauce, light soy sauce, very delicious soy sauce or red Zhejiang vinegar.
The microorganism fermentation broth of the present invention includes a product obtained by liquid culture of a microorganism, a solution, suspension, and the like obtained after dispersing a product obtained by non-liquid culture of a microorganism with a solvent.
Compared with the prior art, the invention has the following beneficial effects:
in the detection method, firstly, the fermentation liquor of the microorganism to be detected is placed for 4 to 8 hours at the temperature of between 250 ℃ below zero and 50 ℃ below zero, and then the fermentation liquor is subjected to vacuum freeze drying overnight, dissolved by N, N-dimethylformamide and centrifuged to obtain metabolite solution. The invention can reserve the metabolites as much as possible and extract the metabolites as many as possible by processing the fermentation liquor of the microorganism to be detected, and has high extraction efficiency.
Furthermore, after the abundant metabolites are extracted from the microbial fermentation liquor, the invention is matched with proper silane derivatization treatment, acidic hydrogen in the molecule of the silane-substituted metabolite in the derivatization agent, namely active hydrogen in groups such as carboxyl, hydroxyl, amino, imino, sulfydryl and the like, and the obtained derivative has low polarity and good volatility and thermal stability, and the treated derivative has high derivatization degree, and can also convert some nonvolatile flavor substances into volatile substances to be detected. Meanwhile, the invention is matched with proper GC-MS detection conditions, ensures the chromatographic separation effect of the fermentation metabolites subjected to derivative treatment, improves the ionization efficiency of the substances to be detected in the mass spectrum ion source, realizes the simultaneous analysis of the fermentation metabolites in one sample injection and improves the analysis efficiency.
Drawings
FIG. 1 is a schematic diagram of an experimental process in embodiment 1 of the present invention.
FIG. 2, example 1 is a gas chromatogram of the first extract metabolite.
Figure 3, example 2 gas chromatogram of soy sauce metabolites.
FIG. 4, example 3 gas chromatogram of metabolite of very fresh soy sauce.
FIG. 5, example 4 is a gas chromatogram of the metabolite of DAHONGZHEYU Vinegar.
Fig. 6, examples 1 to 4 are schematic diagrams illustrating qualitative determination by comparing the value of a specific ion fragment of a certain substance in a sample with the value of the ion fragment in a mass spectrum library and selecting a substance with a similarity of more than 75%.
FIG. 7, comparative example 1 mass spectrum obtained by metabolite extraction method different from that of example 1.
FIG. 8 and comparative example 2 show the mass spectrum obtained by the silylation method different from that of example 1.
FIG. 9 and comparative example 3 show mass spectra obtained by a temperature raising program different from that of example 1.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Example 1: rapid analysis of the metabolite of soy sauce from the head (see FIG. 1)
1. Materials and reagents
A commercially available version of Guangdong soy sauce; n, N-Dimethylformamide (DMF); bis (trimethylsilyl) trifluoroacetamide + trimethylchlorosilane (BSTFA + TMCS, volume ratio 99: 1); heptadecanoic acid; heptadecanoic acid DMF solution (100 mg of heptadecanoic acid particles were dissolved in 10mL of DMF solvent to prepare 10mg/mL of internal standard solution).
2. Metabolite extraction method
Taking 50 mu L of soy sauce sample, putting the soy sauce sample into a 1.5mL EP tube, sealing the tube with a sealing film, tying a small hole opening, putting the tube into a refrigerator at minus 80 ℃ for 6h, and then carrying out vacuum freeze drying overnight. And (3) taking 100 mu L of LDMF to be subjected to ultrasonic dissolution in a freeze-dried EP tube in a sealing way for 30min, centrifuging at 12000r for 3min after vortex oscillation, and taking 50 mu L of supernatant to be used in another EP tube.
3. Silanization method
Taking 5 mu L of heptadecanoic acid DMF internal standard solution to the supernatant, adding 100 mu L of BSTFA + TMCS (99:1), mixing uniformly, sealing, and heating in 80 ℃ water bath for 2 h. Sample 1 μ L. The existence of water is avoided in the whole experiment process, otherwise, the silanization efficiency is influenced.
4. GC-MS conditions
Gas chromatography-mass spectrometer detector model: VARIAN 4000 GC/MC.
A chromatographic column: HP-5, film thickness: 0.25 μm, length 30m, inner diameter 0.25 mm; carrier gas: he gas, split ratio: 5.0.
mass spectrum conditions: ion source temperature: 220 ℃, interface temperature: 250 ℃, solvent delay time: 4min, threshold: 1000, start time: 3min, end time: 57min, interval: 0.5sec, scanning speed: 1000, range: 40m/Z to 500 m/Z.
The GC-MS temperature-raising program is as follows: keeping at 70 deg.C for 8min, heating to 190 deg.C at a rate of 3 deg.C per minute, heating to 300 deg.C at a rate of 10 deg.C per minute, and keeping for 31 min.
See fig. 2 and fig. 6 for results. The samples were tested using a VARIAN 4000GC/MC instrument, the NIST05 mass spectral library was retrieved for qualitative, and the relative content was quantified using the internal standard method. As shown in fig. 6, the values of the specific ion fragments of a certain kind of substances in the sample are compared with the values of the ion fragments in the mass spectrum library, and the substances with similarity degree greater than 75% are selected for characterization. The qualitative substance is the silanized result of the original flavor substance in the sample (the structure that the active hydrogen in the flavor substance is replaced by trimethyl silicon), so the original flavor substance in the sample is deduced reversely according to the structure and English name of the silanized substance. The peak of the substances in FIG. 2 reached 123, that is, 123 substances (including the internal standard substance: heptadecanoic acid). The 123 substances include 8 major substances such as amino acids, organic acids, saccharides, esters, alcohols, phenols, ketones, amines and the like. Various substances and contents of the fermented soy sauce can be detected by a rapid method. As shown in the following table: TABLE 1
Example 2: rapid analysis of soy sauce metabolites
1. Materials and reagents
A commercially available version of Guangdong soy sauce; n, N-Dimethylformamide (DMF); bis (trimethylsilyl) trifluoroacetamide + trimethylchlorosilane (BSTFA + TMCS, volume ratio 95: 5); heptadecanoic acid; heptadecanoic acid DMF solution (100 mg of heptadecanoic acid particles were dissolved in 10mL of DMF solvent to prepare 10mg/mL of internal standard solution).
2. Metabolite extraction method
Placing soy sauce sample in 1.5ml LEP tube, sealing with sealing film, sealing with small hole, freezing at-50 deg.C for 4 hr, and vacuum freeze drying overnight. Taking DMF to be subjected to ultrasonic dissolution in a freeze-dried EP tube for 30min through sealing, centrifuging at 12000r for 3min after vortex oscillation, and taking supernatant to be used in another EP tube.
3. Silanization method
Taking an internal standard solution of heptadecanoic acid DMF to be added into the supernatant, adding BSTFA + TMCS, uniformly mixing, sealing, and heating in a water bath at 37 ℃ for 1 h. Sample 1 μ L. The existence of water is avoided in the whole experiment process, otherwise, the silanization efficiency is influenced.
The volume ratio of the soy sauce to the N, N-dimethylformamide to the internal standard solution to the derivative is 8: 18: 1: 18.
4. GC-MS conditions
Gas chromatography-mass spectrometer detector model: VARIAN 4000 GC/MC.
A chromatographic column: HP-5, film thickness: 0.25 μm, length 30m, inner diameter 0.25 mm; carrier gas: he gas, split ratio: 5.0.
mass spectrum conditions: ion source temperature: 220 ℃, interface temperature: 250 ℃, solvent delay time: 4min, threshold: 1000, start time: 3min, end time: 57min, interval: 0.5sec, scanning speed: 1000, range: 40m/Z to 500 m/Z.
The GC-MS temperature-raising program is as follows: after keeping at 70 ℃ for 8min, the temperature is raised to 190 ℃ at a rate of 2.5 ℃ per minute, and then raised to 300 ℃ at a rate of 9.5 ℃ per minute, and then kept for 31 min.
See fig. 3 and 6 for results. According to the method, a VARIAN 4000GC/MC instrument is used for detecting the sample, a mass spectrum library of NIST05 is used for searching and determining the quality, and the relative content is determined by an internal standard method. As shown in fig. 6, the values of the specific ion fragments of a certain kind of substances in the sample are compared with the values of the ion fragments in the mass spectrum library, and the substances with similarity degree greater than 75% are selected for characterization. The qualitative substance is the silanized result of the original flavor substance in the sample (the structure that the active hydrogen in the flavor substance is replaced by trimethyl silicon), so the original flavor substance in the sample is deduced reversely according to the structure and English name of the silanized substance. The peak of the substances in FIG. 3 reaches 110 (including the internal standard substance: heptadecanoic acid), namely 110 substances. The 110 substances include 8 major substances such as amino acids, organic acids, saccharides, esters, alcohols, phenols, ketones, amines and the like. Various substances and contents extracted from the soy sauce fermentation product can be detected by a rapid method. As shown in the following table:
TABLE 2
Example 3: rapid analysis of extremely delicious soy sauce metabolites
1. Materials and reagents
A commercially available version of Guangdong soy sauce; n, N-Dimethylformamide (DMF); bis (trimethylsilyl) trifluoroacetamide + trimethylchlorosilane (BSTFA + TMCS, volume ratio 99.5: 0.5); heptadecanoic acid; heptadecanoic acid DMF solution (100 mg of heptadecanoic acid particles were dissolved in 10mL of DMF solvent to prepare 10mg/mL of internal standard solution).
2. Metabolite extraction method
Taking a soy sauce sample, putting the soy sauce sample into a 1.5mL EP tube, sealing the tube by using a sealing film, tying a small hole opening, freezing the tube at the temperature of minus 250 ℃ for 8 hours, and then carrying out vacuum freeze drying overnight. Taking DMF to be subjected to ultrasonic dissolution in a freeze-dried EP tube for 30min through sealing, centrifuging at 12000r for 3min after vortex oscillation, and taking supernatant to be used in another EP tube.
3. Silanization method
Taking an internal standard solution of heptadecanoic acid DMF to be added into the supernatant, adding BSTFA + TMCS, uniformly mixing, sealing, and heating in a water bath at 90 ℃ for 3 h. Sample 1 μ L. The existence of water is avoided in the whole experiment process, otherwise, the silanization efficiency is influenced.
The volume ratio of the soy sauce to the N, N-dimethylformamide to the internal standard solution to the derivative is 12: 22: 1: 22.
4. GC-MS conditions
Gas chromatography-mass spectrometer detector model: VARIAN 4000 GC/MC.
A chromatographic column: HP-5, film thickness: 0.25 μm, length 30m, inner diameter 0.25 mm; carrier gas: he gas, split ratio: 5.0.
mass spectrum conditions: ion source temperature: 220 ℃, interface temperature: 250 ℃, solvent delay time: 4min, threshold: 1000, start time: 3min, end time: 57min, interval: 0.5sec, scanning speed: 1000, range: 40m/Z to 500 m/Z.
The GC-MS temperature-raising program is as follows: after keeping at 70 ℃ for 8min, the temperature is raised to 190 ℃ at a rate of 3.5 ℃ per minute, then to 300 ℃ at a rate of 10.5 ℃ per minute, and then kept for 31 min.
The results are shown in FIGS. 4 and 6. According to the method, a VARIAN 4000GC/MC instrument is used for detecting the sample, a mass spectrum library of NIST05 is searched for qualitative analysis, and the relative content of the NIST05 is quantified by an internal standard method. As shown in fig. 6, the values of the specific ion fragments of a certain kind of substances in the sample are compared with the values of the ion fragments in the mass spectrum library, and the substances with similarity degree greater than 75% are selected for characterization. The qualitative substance is the silanized result of the original flavor substance in the sample (the structure that the active hydrogen in the flavor substance is replaced by trimethyl silicon), so the original flavor substance in the sample is deduced reversely according to the structure and English name of the silanized substance. The peak of the substances in FIG. 4 reaches 86 (including the internal standard substance: heptadecanoic acid), namely 86 substances. The 86 substances include 6 major substances such as amino acids, organic acids, saccharides, esters, alcohols, ketones, and the like. Various substances and contents extracted from the soy sauce fermentation product can be detected by a rapid method. As shown in the following table:
TABLE 3
Example 4: rapid analysis of red vinegar metabolite
1. Materials and reagents
A commercially available red vinegar; n, N-Dimethylformamide (DMF); bis (trimethylsilyl) trifluoroacetamide + trimethylchlorosilane (BSTFA + TMCS, volume ratio 99.5: 0.5); heptadecanoic acid; heptadecanoic acid DMF solution (100 mg of heptadecanoic acid particles were dissolved in 10mL of DMF solvent to prepare 10mg/mL of internal standard solution).
2. Metabolite extraction method
Putting a red Zhejiang vinegar sample into a 2mL EP tube, sealing the tube with a sealing film, tying a small hole opening, freezing at-80 ℃ for 8h, and performing vacuum freeze drying overnight. Taking DMF to be subjected to ultrasonic dissolution in a freeze-dried EP tube for 30min through sealing, centrifuging at 12000r for 3min after vortex oscillation, and taking supernatant to be used in another EP tube.
3. Silanization method
Taking an internal standard solution of heptadecanoic acid DMF to be added into the supernatant, adding BSTFA + TMCS, uniformly mixing, sealing, and heating in a water bath at 90 ℃ for 3 h. Sample 1 μ L. The existence of water is avoided in the whole experiment process, otherwise, the silanization efficiency is influenced.
The volume ratio of the bright red Zhejiang vinegar to the N, N-dimethylformamide to the internal standard solution to the derivative is 12: 22: 1: 22.
4. GC-MS conditions
Gas chromatography-mass spectrometer model detector: VARIAN 4000 GC/MC.
A chromatographic column: HP-5, film thickness: 0.25 μm, length 30m, inner diameter 0.25 mm; carrier gas: he gas, split ratio: 5.0.
mass spectrum conditions: ion source temperature: 220 ℃, interface temperature: 250 ℃, solvent delay time: 4min, threshold: 1000, start time: 3min, end time: 57min, interval: 0.5sec, scanning speed: 1000, range: 40m/Z to 500 m/Z.
The GC-MS temperature-raising program is as follows: after keeping at 70 ℃ for 8min, the temperature is raised to 190 ℃ at a rate of 3.5 ℃ per minute, then to 300 ℃ at a rate of 10.5 ℃ per minute, and then kept for 31 min.
The results are shown in FIGS. 5 and 6. According to the method, a VARIAN 4000GC/MC instrument is used for detecting the sample, a mass spectrum library of NIST05 is searched for qualitative analysis, and the relative content of the NIST05 is quantified by an internal standard method. As shown in fig. 6, the values of the specific ion fragments of a certain kind of substances in the sample are compared with the values of the ion fragments in the mass spectrum library, and the substances with similarity degree greater than 75% are selected for characterization. The qualitative substance is the silanized result of the original flavor substance in the sample (the structure that the active hydrogen in the flavor substance is replaced by trimethyl silicon), so the original flavor substance in the sample is deduced reversely according to the structure and English name of the silanized substance. The substance peak of fig. 5 reached 81, i.e., 81 substances. The 81 substances include 6 major substances such as amino acids, organic acids, saccharides, esters, alcohols, ketones, and the like. Various substances and contents of the fermented product of the red Zhejiang vinegar can be detected by a rapid method. As shown in the following table:
TABLE 4
According to analysis of metabolites of the three soy sauce products and the vinegar product obtained in the embodiment, the method can realize one-time sample injection and qualitative and quantitative determination of various metabolites, and is simple and convenient in experimental operation steps and high in detection efficiency. Through four examples, metabolic flavor substances in the first soy sauce, the raw soy sauce, the delicious soy sauce and the red Zhejiang vinegar are respectively detected, and from a gas chromatogram, the efficiency of extracting metabolites is high, for example, the substance peak in figure 2 reaches 123, namely 123 substances. The method for extracting fermentation metabolites keeps the original substances as much as possible, and has high extraction efficiency. And the silanization derivatization method has large derivatization degree, so that some nonvolatile flavor substances are converted into volatile substances to be detected. In the invention, DMF is used as a solvent to dissolve a heptadecanoic acid internal standard substance in order to increase the sample injection concentration as much as possible, reduce the reaction system and reduce the influence of methanol on metabolic substances in the system. This approach has proven to be feasible. The heating procedure of GC-MS is also a key point, amino acids have more peaks after 10 minutes, so that the temperature is increased to 190 ℃ at 3 ℃ per minute during 8-48 minutes, and the temperature is increased slowly. At this stage, amino acids, organic acids, alcohols, esters, phenols, ketones, and amines show many peaks. And in the period of 48-59 minutes, the temperature is increased to 300 ℃ per minute at 10 ℃, and the temperature is increased rapidly. The carbohydrate peaks more at this stage. Then maintaining the high temperature of 300 ℃ is beneficial to the slow peak emergence of some substances which are not easy to volatilize. The temperature raising program can ensure that the substance is completely subjected to peak generation as much as possible, so that the peak type overlapping is reduced, and the qualitative and quantitative effects are more accurate.
Comparative example 1
This comparative example is that of example 1, the main differences with respect to example 1 including the different metabolite extraction methods, in particular: freezing and coagulating 50 μ l sample at-80 deg.C, vacuum freeze drying (-80 deg.C, 0.37 Pa) overnight, adding 330 μ l methanol to remove protein, ultrasonic dissolving, centrifuging, collecting supernatant, placing in EP tube, and heating in 80 deg.C water bath for 30min to remove methanol (methanol boiling point 67.5 deg.C).
The upper part of FIG. 7 shows the mass spectrum of example 1, and the lower part shows the mass spectrum of comparative example 1. As a result, it has been found that the method for removing proteins from methanol reduces the overall peak shape, reduces the number of species, and reduces organic acids, alcohols, and esters to some extent by determining a smaller amount of saccharides.
Comparative example 2
This comparative example is a comparative example to example 1, the main differences with respect to example 1 comprising the difference in the silanization process, in particular the silanization process employed by this comparative example comprises: taking 5 mu L of heptadecanoic acid DMF internal standard solution to the supernatant, adding 100 mu L of BSTFA + TMCS (90:10), mixing uniformly, sealing, and heating in a water bath at 65 ℃ for 5 h.
The results are shown in fig. 8, with the upper part being comparative example 2 and the lower part being example 1. It can be found that many flavor components in comparative example 2 can not be detected, the complete identification of the substance types is influenced, and the overall peak type of the mass spectrogram is low and the substances are few.
Comparative example 3
This comparative example is that of example 1, the main differences with respect to example 1 including the gas chromatography conditions employed being different, and specifically, this comparative example attempts to employ the following two gas chromatography temperature-raising procedures, respectively:
a first procedure: keeping at 70 deg.C for 5min, heating to 150 deg.C at a rate of 5 deg.C per minute, keeping at 5min, heating to 280 deg.C at a rate of 10 deg.C per minute, and keeping at 10 min. The whole temperature programming takes 49 min.
And a second procedure: keeping at 70 deg.C for 8min, heating to 190 deg.C at a rate of 3 deg.C per minute, heating to 300 deg.C at a rate of 10 deg.C per minute, and keeping for 5 min. The whole temperature programming takes 64 min.
The results are shown in FIG. 9, which are mass spectra after the temperature-raising program of comparative example 3, program two and example 1, in this order from top to bottom. Obviously, along with the prolonging of the temperature-raising program time, the temperature-raising speed is slow, the maintaining time of the high-temperature part is long, so that metabolites which are not easy to volatilize and have high boiling points can be detected, the peak separation degree is relatively good, each substance can completely generate a peak, and the content calculation is relatively accurate.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A method for detecting metabolites in a microbial fermentation broth, comprising the steps of:
(1) taking a microbial fermentation liquid to be detected, placing the microbial fermentation liquid at the temperature of minus 250 ℃ to minus 50 ℃ for 4h to 8h, performing vacuum freeze drying at night, dissolving the microbial fermentation liquid by using N, N-dimethylformamide, performing vortex oscillation and centrifuging to obtain a supernatant, namely a metabolite solution;
(2) adding the metabolite solution into the internal standard solution, uniformly mixing, and adding a derivatization agent for silanization derivatization treatment;
(3) and taking the derivative treated metabolite solution for GC-MS detection.
2. The method according to claim 1, wherein the step (2) of performing silanization derivatization treatment with the derivatization agent comprises: adding a derivatization agent, uniformly mixing, sealing, and then carrying out water bath for 1-5 h at the temperature of 37-90 ℃; the derivatization agent is bis (trimethylsilyl) trifluoroacetamide and trimethylchlorosilane in a volume ratio of (95-99.5): (0.5-5).
3. The method for detecting the metabolites in the microbial fermentation broth according to claim 2, wherein the temperature of the water bath is 78-82 ℃, the time of the water bath is 1.5-2.5 h, the derivatizing agent is bis (trimethylsilyl) trifluoroacetamide and trimethylchlorosilane in a volume ratio of (98.5-99.5): (0.5-1.5).
4. The method for detecting metabolites in a microbial fermentation broth according to any one of claims 1 to 3, wherein the GC-MS detection uses chromatographic conditions comprising:
a chromatographic column: a weakly polar capillary column with 5% diphenyl, 1% vinyl, 94% dimethyl polysiloxane as the filler; carrier gas: helium gas; the split ratio is as follows: 4.5-5.5; the temperature rising procedure is as follows: maintaining at 70 deg.C for 8 min; heating to 190 ℃ at the speed of 2.5-3.5 ℃ per minute for 8-48 min; 48-59 min, and heating to 300 ℃ at the speed of 9.5-10.5 ℃ per minute; the mixture was kept at 300 ℃ for 31 min.
5. The method for detecting metabolites in a microbial fermentation broth as claimed in claim 4, wherein the GC-MS detection adopts chromatographic conditions comprising:
the chromatographic column comprises HP-5(0.25 mu m × 30m × 0.25.25 mm), carrier gas and helium, the split ratio is 5.0, the temperature rise program is that the temperature is kept at 70 ℃ for 8min, the temperature rises to 190 ℃ at the speed of 3 ℃ per minute for 8min to 48min, the temperature rises to 300 ℃ at the speed of 10 ℃ per minute for 48min to 59min, and the temperature is kept at 300 ℃ for 31 min.
6. The method for detecting metabolites in a microbial fermentation broth according to any one of claims 1 to 3, wherein the GC-MS detection is performed under mass spectrometry conditions comprising: ion source temperature: 215 ℃ to 225 ℃, interface temperature: 245 ℃ to 255 ℃, solvent delay time: 3.5-4.5 min, threshold: 100-1000, start time: 2.5 min-3.5 min, end time: 56.5 min-57.5 min, interval: 0.3sec to 0.5sec, scanning speed: 500-1000, range: 40m/Z to 500 m/Z.
7. The method for detecting metabolites in a microbial fermentation broth as claimed in claim 6, wherein the GC-MS detection uses mass spectrometry conditions comprising: ion source temperature: 220 ℃, interface temperature: 250 ℃, solvent delay time: 4min, threshold: 1000, start time: 3min, end time: 57min, interval: 0.5sec, scanning speed: 1000, range: 40m/Z to 500 m/Z.
8. The method according to any one of claims 1 to 3, wherein the internal standard solution is prepared by adding 80mg to 100mg heptadecanoic acid particles per 10mL of N, N-dimethylformamide.
9. The method for detecting the metabolites in the microbial fermentation broth according to any one of claims 1 to 3, wherein the volume ratio of the microbial fermentation broth to be detected in the step (1), N-dimethylformamide and the internal standard solution and the derivatizing agent in the step (2) is (8-12): (18-22): 1: (18-22).
10. The method according to any one of claims 1 to 3, wherein the microbial fermentation broth to be tested is soy sauce, light soy sauce, very delicious soy sauce or red Zhejiang vinegar.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113533610A (en) * | 2021-08-03 | 2021-10-22 | 山西新宝源制药有限公司 | Detection method for determining piperidine residue in bulk drug |
| CN114720589A (en) * | 2022-03-14 | 2022-07-08 | 广东美味鲜调味食品有限公司 | Method for researching Cantonese soy sauce fermentation process change by combining metabolism and metagenome |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4565699A (en) * | 1983-07-18 | 1986-01-21 | The Upjohn Company | Composition of matter and process |
| CN102621247A (en) * | 2012-04-18 | 2012-08-01 | 王益超 | Method for synchronously analyzing base, nucleotide, organic acid, fatty acid, amino acid and saccharide metabolic product with two-step derivation method |
| CN103675178A (en) * | 2013-12-05 | 2014-03-26 | 柳州联海科技有限公司 | Gas chromatography-mass spectrometry detection method for organic acids and sugar in fermentation liquid |
| US20170234843A1 (en) * | 2014-08-12 | 2017-08-17 | Aisti Science Co., Ltd. | Pre-analysis treatment device usable for amino acid, organic acid, and glucide and pre-analysis treatment method |
| CN108414660A (en) * | 2018-03-08 | 2018-08-17 | 中国药科大学 | One group early diagnoses relevant blood plasma metabolism small molecule marker and its application with lung cancer |
-
2018
- 2018-12-28 CN CN201811620247.8A patent/CN111380965A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4565699A (en) * | 1983-07-18 | 1986-01-21 | The Upjohn Company | Composition of matter and process |
| CN102621247A (en) * | 2012-04-18 | 2012-08-01 | 王益超 | Method for synchronously analyzing base, nucleotide, organic acid, fatty acid, amino acid and saccharide metabolic product with two-step derivation method |
| CN103675178A (en) * | 2013-12-05 | 2014-03-26 | 柳州联海科技有限公司 | Gas chromatography-mass spectrometry detection method for organic acids and sugar in fermentation liquid |
| US20170234843A1 (en) * | 2014-08-12 | 2017-08-17 | Aisti Science Co., Ltd. | Pre-analysis treatment device usable for amino acid, organic acid, and glucide and pre-analysis treatment method |
| CN108414660A (en) * | 2018-03-08 | 2018-08-17 | 中国药科大学 | One group early diagnoses relevant blood plasma metabolism small molecule marker and its application with lung cancer |
Non-Patent Citations (2)
| Title |
|---|
| 刘非: "基于GC-MS的酱油风味物质分析", 《中国优秀博硕士学位论文全文数据库(硕士) 工程科技Ⅰ辑》 * |
| 夏继东等: "气相色谱-质谱法同时测定人血浆中多种内源性代谢物", 《分析科学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113533610A (en) * | 2021-08-03 | 2021-10-22 | 山西新宝源制药有限公司 | Detection method for determining piperidine residue in bulk drug |
| CN114720589A (en) * | 2022-03-14 | 2022-07-08 | 广东美味鲜调味食品有限公司 | Method for researching Cantonese soy sauce fermentation process change by combining metabolism and metagenome |
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