Soybean sauce crude oil quality evaluation method based on umami dipeptide

Abstract

The invention discloses a soy sauce crude oil quality evaluation method based on umami dipeptide, which comprises the following steps: accurately screening and identifying dipeptide in soy sauce crude oil by adopting a Q-TOF (Quality-Time of Flight) instrument, carrying out secondary screening on the identified dipeptide by virtue of an ion scanning (SIM) mode in a QqQ instrument, and evaluating the umami of the secondarily screened dipeptide in a sensory evaluation mode. Identifying umami dipeptides with an obvious freshness enhancing effect, and determining the umami dipeptides as GM and GE; then qualitative and quantitative detection is performed on the umami dipeptide of the soy sauce crude oil by using an MRM multi-reaction monitoring mode, so that the umami quality of the soy sauce crude oil is evaluated; the method overcomes the defect that in existing soy sauce quality physicochemical index evaluation, only the amino acid nitrogen content is used for indirectly evaluating the flavor of the soy sauce crude oil, a good reference index is provided for research of a flavor soy sauce production process, and therefore a new direction for improvement and innovation of soy sauce products is developed.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and detection of condiments, and particularly relates to a method for evaluating the quality of crude soy sauce based on umami dipeptides. Background Art

[0002] Soy sauce is a condiment widely used in cooking and food processing. With the continuous improvement of consumers' requirements for food quality, the market demand for high-umami soy sauce is increasing day by day. In the field of home cooking, high-umami soy sauce can enhance the flavor of dishes and stimulate appetite; in the field of food processing, high-umami soy sauce can be used to season meat and vegetarian products, giving them a better taste; in the field of food service, high-umami soy sauce can be used to season various dishes to meet the taste needs of different people.

[0003] The umami of soy sauce mainly comes from umami substances such as amino acids, polypeptides, and nucleotides. For example, high-umami soy sauce contains more umami substances. Taking high-salt liquid-state soy sauce as an example, the crude soy sauce uses soybeans and / or defatted soybeans, wheat flour as raw materials, which are steamed, and then mixed with brine into a thin mash after Aspergillus oryzae koji-making, and then made through microbial fermentation. The protein-rich raw materials are acted on by multi-enzyme systems such as protease, peptidase, and glutaminase secreted by microbial metabolism, making the crude soy sauce rich in protein hydrolysates of oligopeptides, and these hydrolysates contain various umami substances. These umami substances that directly affect the sensory properties are actually closely related to the quality of soy sauce.

[0004] The prior art has always used soluble solids-free, amino acid nitrogen, and total nitrogen as physical and chemical indicators to evaluate the quality of finished soy sauce. For the semi-finished product of crude soy sauce, enterprises usually use at least one of the indicators of salt content, amino acid nitrogen, protein content, and total acid for quality monitoring and evaluation; while for the umami evaluation of both finished soy sauce and crude soy sauce, only sensory characteristics are used as the evaluation standard, without qualitative and quantitative physical and chemical indicators.

[0005] This is because the protease and peptidase in the fermentation process of crude soy sauce mainly come from the microorganisms added during the koji-making process. The microbial enzyme system is complex, and the action sites of the enzymes are widely distributed, making the polypeptide composition of the protease hydrolysis products usually very complex, containing tens of thousands of polypeptides, and the peptide molecular weight distribution shows a complex dynamic change law, which brings great difficulties to the analysis and content determination of the polypeptide components of crude soy sauce. Therefore, there are still many deficiencies in the research on the potential functional components and quality of crude soy sauce in the prior art.

[0006] In the prior art, the direct objects of the determination of protein content, total amino acid nitrogen, and the distribution of amino acid nitrogen are the total protein, amino acid nitrogen, and the distribution of free amino acid nitrogen produced during the enzymatic hydrolysis of soy protein. Although these indicators can indirectly describe the characteristics of the enzymatic hydrolysis products in crude soy sauce to a certain extent, they cannot accurately describe the products directly, so it is difficult to accurately reflect the role of their umami substances (such as polypeptides) in the product process research. Therefore, the development and innovation of the current crude soy sauce production process are restricted to a certain extent.

[0007] Furthermore, in the current industry, in addition to evaluating the umami of soy sauce by sensory characteristics, the total amino acid nitrogen is used as an indirect indicator to evaluate the umami of soy sauce, that is, the higher the total amino acid nitrogen, the generally better the umami; however, since amino acids are not the only factor affecting the umami of soy sauce, the total amino acid nitrogen alone cannot fully and truly reflect the umami level of soy sauce. Because, in the research and development process of improving or innovating the soy sauce production process, the crude soy sauce produced by different processes may have comparable quality physical and chemical indicators in the national standard, especially when the total amino acid nitrogen is basically the same or similar; in this case, there are differences in the sensory characteristics of evaluating the umami of crude soy sauce, but since there is no better indicator to measure the umami of soy sauce currently, it is difficult for researchers to further judge the advantages and disadvantages of the production results of different processes, thus affecting the development and innovation of the soy sauce production process.

[0008] Although there have been literature reports in the prior art on extracting umami peptides from crude soy sauce, the prior art research on umami peptides in crude soy sauce has basically focused on extraction and synthetic applications, and there has been no report on using qualitative and quantitative data of umami peptides for umami evaluation of soy sauce. Chinese invention patent CN118294568A discloses "A method for establishing a fingerprint map of soy sauce umami peptides", in which a MALDI-TOF mass spectrometer is used to perform mass spectrometry analysis on the so-called soy sauce umami peptides to obtain the fingerprint map of the so-called umami peptides of the soy sauce to be evaluated. However, according to the extraction method of the so-called soy sauce umami peptides disclosed in this technology, which is the conventional extraction method of polypeptides, it can be known that the sample alleged to be soy sauce umami peptides used in this technology is actually a sample of mixed polypeptides extracted from soy sauce, rather than a pure soy sauce umami peptide sample, and it lacks the step of separating the umami peptide sample from the mixed polypeptide sample. Therefore, the fingerprint map established based on the mass-to-charge ratio should be the fingerprint map of soy sauce mixed polypeptides, rather than the fingerprint map of soy sauce umami peptides. The method of rating soy sauce using this fingerprint map is actually a soy sauce rating method based on soy sauce mixed polypeptides rather than on umami peptides, and this method does not propose a detection method that can qualitatively and quantitatively detect umami peptides in soy sauce (see its claim 1 and paragraphs

[0018] ,

[0019] of the specification).

[0009] In addition, so far, 34 dipeptides and tripeptides with umami taste identified from crude soy sauce have been reported in the existing literature. Among them, the umami-enhancing effect of dipeptides is obvious. However, there are significant differences in the dipeptide content produced under different production process conditions. Therefore, establishing a quality evaluation method based on rapid screening and identification of umami dipeptides in soy sauce under different production processes provides a new means for evaluating the quality of soy sauce, which will be beneficial to the research and development of improving the quality of soy sauce products and is of great significance to the research and development of umami soy sauce. Summary of the Invention

[0010] The purpose of the present invention is to provide a quality evaluation method for crude soy sauce based on umami dipeptides, and to evaluate the umami quality of soy sauce by identifying and analyzing the content of umami dipeptides.

[0011] The purpose of the present invention can be achieved by the following technical solutions.

[0012] A quality evaluation method for crude soy sauce based on umami dipeptides, characterized by including the following steps:

[0013] (1) Screening and identification of dipeptides in crude soy sauce:

[0014] Using macroporous resin to pretreat crude soy sauce to obtain a crude oil sample solution; detecting the crude oil sample solution with a high-resolution liquid chromatography quadrupole time-of-flight mass spectrometer (QTOF) to screen and identify dipeptides;

[0015] (2) Secondary screening of dipeptides in crude soy sauce:

[0016] Select the dipeptides with the top-ranked signal intensities from the dipeptides identified in step (1), that is, select the dipeptides with the top-ranked contents; then use a triple quadrupole liquid chromatography-mass spectrometry (QqQ) to perform secondary screening through the selected ion monitoring mode (SIM). Specifically, quantitatively detect the above-selected dipeptides in the crude oil sample, and screen out the dipeptides suitable for monitoring according to the average value of the response peak areas in the measured spectrum;

[0017] (3) Identification of umami dipeptides in crude soy sauce:

[0018] Obtain the dipeptides screened in step (2), conduct a sensory evaluation, and identify the umami dipeptides with umami-enhancing effects from them;

[0019] (4) Determination of the content of umami dipeptides in the crude soy sauce to be evaluated:

[0020] Using macroporous resin to pretreat the crude soy sauce to be evaluated to obtain a crude oil sample solution to be evaluated;

[0021] Take the standard product of the umami dipeptide and prepare a standard solution of the umami dipeptide;

[0022] The multi-reaction monitoring mode (MRM) of a triple quadrupole liquid chromatography-mass spectrometry instrument was used to detect the crude oil sample solution to be evaluated. Qualitative and quantitative analysis of the umami dipeptides in it was carried out according to the umami dipeptide standard solution, and then the quality of the soy sauce crude oil was evaluated based on the content of the umami dipeptides.

[0023] The working principle of the present invention takes the umami dipeptides in the soy sauce crude oil as a breakthrough point to establish a method for identifying and determining the content of umami dipeptides: a Q-TOF instrument is used to accurately screen and identify the dipeptides in the soy sauce crude oil, and then the identified dipeptides are secondarily screened by the ion scanning (SIM) mode in the QqQ instrument. The secondarily screened dipeptides are evaluated for their umami by sensory evaluation to identify the umami dipeptides with obvious umami-enhancing effects, and it is determined that the umami dipeptides are GM and GE. Then, the MRM multi-reaction monitoring mode is used to micro-determine the umami dipeptides to achieve qualitative and quantitative detection of the umami dipeptides in the soy sauce crude oil.

[0024] Preferably, the operation of the pretreatment is as follows: the soy sauce crude oil is rotary evaporated and concentrated to 10% of the original volume under low-temperature and negative-pressure conditions to obtain a concentrated solution; the concentrated solution is fully adsorbed by macroporous resin, rinsed with deionized water until the rinsing solution is colorless, eluted with a 50% ethanol solution, and the eluate is collected; the eluate is rotary evaporated and concentrated under negative-pressure conditions, and then vacuum freeze-dried and concentrated to obtain a freeze-dried sample; 0.1 - 0.2 g of the freeze-dried sample is weighed into a 10 mL volumetric flask, fixed to the scale with water, and filtered through a 0.22 μm filter membrane to remove insoluble substances, which is the crude oil sample solution. The crude oil sample solution is diluted 10 times in volume and used as the sample solution for detection.

[0025] Preferably, in step (1), the chromatographic conditions of the high-resolution liquid chromatography quadrupole time-of-flight mass spectrometer are as follows: the chromatographic column is a C18 chromatographic column; the column oven temperature is 35 - 45 °C; the mobile phase consists of mobile phase A and mobile phase B. Mobile phase A is an acetonitrile aqueous solution containing 0.1% formic acid, and mobile phase B is a 0.1% formic acid aqueous solution; the mobile phase flow rate is 0.2 - 0.3 mL / min, and the injection volume is 1 - 2 μL. The mass spectrometry conditions are as follows: the working mode is selected as the positive ion mode, the ionization mode of the mass spectrometry ion source is the ESI electrospray ion source, the interface temperature is 520 °C, the spray voltage is 5600 V, the scanning period is 0.602 s, the TOF primary scanning range is set to 138 - 390 Da with reference to the molecular weight range of glycine-glycine and tryptophan-tryptophan, the secondary scanning range is 50 - 800 Da, the working mode is DDA, the maximum number of candidate ions is 4, dynamic exclusion is turned on, and the remaining parameters use the default values of the proteomics method.

[0026] Preferably, in step (2), the triple quadrupole liquid chromatography-mass spectrometry instrument uses the same chromatographic conditions as the high-resolution liquid chromatography quadrupole time-of-flight mass spectrometry instrument in step (1); its mass spectrometry conditions are: the chromatographic column is a C18 chromatographic column; the selected working mode is the positive ion mode; the ionization mode of the mass spectrometry ion source is the ESI electrospray ion source, with an interface temperature of 300-320 °C; the desolvation temperature is 526-550 °C; the DL temperature is 250-280 °C; the heating block temperature is 400-420 °C; the atomizing gas flow rate is 3-5 L / min; the heating gas flow rate is 10-12 L / min; the drying gas flow rate is 10-12 L / min; the selected ion scanning mode is adopted, and its setting value is determined according to the ions of the dipeptides with the top-ranked signal intensities selected.

[0027] Preferably, in step (4), the triple quadrupole liquid chromatography-mass spectrometry instrument uses the same chromatographic conditions as the high-resolution liquid chromatography quadrupole time-of-flight mass spectrometry instrument in step (1); its mass spectrometry conditions are: the chromatographic column is a C18 chromatographic column; the selected working mode is the positive ion mode; the ionization mode of the mass spectrometry ion source is the ESI electrospray ion source, with an interface temperature of 300-320 °C; the desolvation temperature is 526-550 °C; the DL temperature is 250-280 °C; the heating block temperature is 400-420 °C; the atomizing gas flow rate is 3-5 L / min; the heating gas flow rate is 10-12 L / min; the drying gas flow rate is 10-12 L / min; the multiple reaction monitoring mode is adopted, and the mass spectrometry scanning rate is set to 50-300 Da / s. The specific monitoring setting parameters of the primary precursor ions and the secondary fragment ions are determined according to the umami dipeptides.

[0028] Preferably, in step (4), the monitoring setting parameters of the primary precursor ions and the secondary fragment ions in the multiple reaction monitoring mode are respectively: for GM: primary 207.2 m / z, secondary 106.7 m / z; for GE: primary 205.1 m / z, secondary 96.1 m / z.

[0029] Preferably, in step (3), the dipeptides screened in step (2) can be prepared by conventional chemical synthesis methods or purchased as commercially available standards for sensory evaluation.

[0030] Preferably, in step (3), in addition to sensory evaluation, the dipeptides screened in step (2) can also be analyzed by an electronic tongue to identify the umami dipeptides with umami-enhancing effects; the method for the electronic tongue analysis is: prepare a 1 mg / mL dipeptide solution from the dipeptides screened in step (2), and set a 1 mg / mL sodium glutamate solution as the control; use the electronic tongue to measure the taste characteristics of the dipeptide solution and the sodium glutamate solution at 25 °C, balance for 30 s, measure for 30 s, cut-off time 30 s, acquisition period 1 s, and detect the sample solution using the five-taste sensor array of sour, salty, umami, sweet, and bitter.

[0031] Preferably, in step (3), the method of sensory evaluation is as follows: Prepare a dipeptide solution with a concentration of 1 mg / mL from the dipeptides screened in step (2), and set the 1 mg / mL sodium glutamate solution as a control, and evaluate the five flavors of acid, saltiness, umami, sweetness and bitterness; The evaluation is carried out at 25 ± 2 °C; The evaluator tastes the sample solution and then rinses the mouth with water, and the tasting test interval is 1 minute.

[0032] The present invention has the following beneficial technical effects:

[0033] 1. The present invention realizes the qualitative and quantitative detection of umami dipeptides in crude soy sauce.

[0034] Taking the umami dipeptides in crude soy sauce as a breakthrough point, the present invention establishes a method for identifying and determining the content of umami dipeptides. Specifically, Q-TOF is used to accurately screen and identify soy sauce dipeptides, and the selected ion scanning (SIM) mode in QqQ is used to perform secondary screening on the identified dipeptides. The dipeptides obtained from the secondary screening are evaluated for their umami by sensory evaluation, and the umami dipeptides with obvious umami-enhancing effects are identified. Then, the MRM multiple reaction monitoring mode is used for trace determination to realize the qualitative and quantitative detection of umami dipeptides in crude soy sauce. Thus, the umami quality of crude soy sauce can also be evaluated by physical and chemical indexes.

[0035] 2. By virtue of the high-resolution characteristics of the QTOF instrument, the present invention performs primary screening on dipeptides, effectively locking the possible types of dipeptides. With the SIM mode of the QqQ instrument, the screened dipeptides are further verified and eliminated, reducing the sample size for subsequent sensory evaluation and the verification of the use of standard products in the MRM mode of the QqQ instrument, and quickly locking the types of umami-enhancing dipeptides.

[0036] 3. The present invention provides a supplementary means for evaluating the quality of crude soy sauce based on umami dipeptides.

[0037] By performing qualitative and quantitative detection of umami dipeptides in crude soy sauce, the present invention can truly reflect the umami situation of crude soy sauce, and can better evaluate the umami differences brought by different processes, which are difficult to distinguish with the existing soy sauce quality evaluation standards. It makes up for the deficiency of indirectly evaluating the umami of crude soy sauce only by using the content of amino acid nitrogen in the existing physical and chemical indexes for evaluating soy sauce quality, helps those skilled in the art to explore the differences in the content of umami dipeptides in crude oils of different production processes, provides a better reference index for the research of umami soy sauce production processes, and thus opens up a new direction for the improvement and innovation of soy sauce products.

[0038] 4. When performing qualitative and quantitative detection of umami dipeptides in crude soy sauce in production and R & D work, only step 4 is required, without the need to re-screen umami dipeptides in steps 1-3, which is simple and fast. Description of the Drawings

[0039] Figure 1 It is the mass spectrometry diagram of the umami dipeptide GM;

[0040] Figure 2 It is the mass spectrometry diagram of the umami dipeptide GE;

[0041] Figure 3 It is the standard linear graph of the peak area and concentration of the mass spectrometry diagram of the umami dipeptide GM;

[0042] Figure 4 It is the standard linear graph of the peak area and concentration of the mass spectrometry diagram of the umami dipeptide GE. Specific embodiments

[0043] The technical solutions of the present invention will be described in detail below in conjunction with specific embodiments, so that those skilled in the art can better understand and implement the technical solutions of the present invention. The following embodiments and accompanying drawings are only for illustrative purposes and should not be construed as limiting the present invention. The reagents or materials used in the embodiments, unless otherwise specified, are all from commercial channels. Unless otherwise specified, the experimental instruments used are all conventional laboratory instruments.

[0044] Example 1: Determination of umami dipeptides in crude soy sauce

[0045] (1) Screening and identification of dipeptides in crude soy sauce:

[0046] ① Pretreatment of crude soy sauce

[0047] 1000 mL of crude soy sauce was rotary evaporated and concentrated to about 100 mL under low temperature and negative pressure conditions, where the temperature was controlled at 30 - 40 °C and the pressure was controlled at 30 - 50 mbar; then the concentrated solution was fully adsorbed by macroporous resin, rinsed with deionized water until the rinsing solution was colorless, eluted with 50% ethanol solution, and the eluate was collected; the eluate was rotary evaporated and concentrated under negative pressure conditions, and then vacuum freeze-dried and concentrated to obtain a freeze-dried sample; 0.1 - 0.2 g of the freeze-dried sample was weighed into a 10 mL volumetric flask and made up to the mark with water, and passed through a 0.22 um filter membrane to remove insoluble substances, which was the crude oil sample solution.

[0048] ② Screening and identification of dipeptides in crude soy sauce

[0049] The above crude oil sample solution was diluted 10 times in volume and used as the sample solution for injection, and detected by QTOF;

[0050] The QTOF detection conditions are as follows:

[0051] QTOF chromatographic conditions: The chromatographic column is an ACQUITY UPLC HSS T3 Column produced by Waters, 1.8 μm, 2.1 mm × 100 mm; the column oven temperature is 35 - 45 °C; the mobile phase consists of mobile phase A and mobile phase B. Mobile phase A is an aqueous acetonitrile solution containing 0.1% formic acid, and mobile phase B is an aqueous solution of 0.1% formic acid; the flow rate of the mobile phase is 0.2 - 0.3 mL / min, and the injection volume is 1 - 2 μL; the set ratio of the mobile phase gradient elution is shown in Table 1.

[0052] Table 1 Set ratio of mobile phase gradient elution

[0053]

[0054] QTOF mass spectrometry conditions: The positive ion mode is selected, the ionization mode of the mass spectrometry ion source is the ESI electrospray ion source, the interface temperature is 520 °C, the spray voltage is 5600 V, the scan cycle is 0.602 s, the TOF primary scan range is set to 138 - 390 Da with reference to the molecular weight ranges of glycine - glycine and tryptophan - tryptophan, the secondary scan range is 50 - 800 Da, the working mode is DDA, the maximum number of candidate ions is 4, dynamic exclusion is enabled, and the remaining parameters use the default values of proteomics methods.

[0055] Primary screening of dipeptides: Dipeptides are formed by pairwise dehydration condensation of 20 common amino acids (see Table 2), and there are 400 combination ways of dipeptides. The QTOF off - machine data is matched with these 400 components, and when ppm is less than 0.5, a total of 125 dipeptides are screened and identified.

[0056] Table 2 Names, abbreviations, and molecular weights of each amino acid

[0057]

[0058]

[0059] (2) Secondary screening of dipeptides in crude soy sauce:

[0060] Select the top 30 dipeptides with the strongest signal intensities (see Table 3) from the 125 dipeptides identified in step (1) for secondary screening.

[0061] Quantitatively detect the above 30 dipeptides in the crude oil sample by LC - MS / MS through the selected ion scanning mode (SIM).

[0062] The LC - MS / MS detection conditions are as follows:

[0063] Chromatographic conditions: The same as QTOF chromatographic conditions:

[0064] Mass spectrometry conditions: The chromatographic column was an ACQUITY UPLC HSS T3 Column produced by Waters, 1.8 μm, 2.1 mm × 100 mm; the selected working mode was the positive ion mode; the ionization mode of the mass spectrometry ion source was the ESI electrospray ion source, the interface temperature was 300 - 320 °C; the desolvation temperature was 526 - 550 °C; the DL temperature was 250 - 280 °C; the heating block temperature was 400 - 420 °C; the atomizing gas flow rate was 3 - 5 L / min; the heating gas flow rate was 10 - 12 L / min; the drying gas flow rate was 10 - 12 L / min; the SIM mode was adopted, and the SIM / MH+ parameter values of LC-MS / MS were shown in Table 3.

[0065] Table 3 List of verification situations of TOP 30 dipeptides in QTOF under LC-MS / MS SIM conditions

[0066]

[0067]

[0068] According to the results in Table 3, through the confirmation of SIM / MH+ of LC-MS / MS, the dipeptides suitable for monitoring were screened out as FA, GM, GE, DP, and EL based on the measured average response peak area.

[0069] (3) Identification of umami dipeptides in crude soy sauce:

[0070] The above 5 dipeptides FA, GM, GE, DP, and EL suitable for monitoring and evaluation were respectively prepared into 1 mg / mL dipeptide solutions, and a 1 mg / mL sodium glutamate solution was set as the control for sensory evaluation. The results are shown in Table 4.

[0071] The sensory evaluation method was as follows: A sensory evaluation panel was composed of 20 tasters (10 males and 10 females, aged 22 to 40 years) recruited from the company without taste disorders; before the formal evaluation, the tasters were trained to be able to distinguish five basic tastes (sour, sweet, bitter, salty, and umami); the evaluation was carried out at 25 ± 2 °C; the tasters tasted the sample solution and then rinsed their mouths with water; the tasting test interval was 1 minute, and no eating or drinking was allowed 1 hour before the evaluation.

[0072] Determination of the threshold value using the taste dilution method and the triangle test method: The dipeptide solution and the sodium glutamate solution with a concentration of 1 mg / mL were diluted step by step with ultrapure water. The dipeptide solution and the sodium glutamate solution were placed in the order of low concentration to high concentration, and then the triangle test was carried out (for each concentration of the peptide solution, two blank control groups without dipeptide were prepared, that is, 1 experimental group and 2 blank groups). The subjects started tasting from the peptide solution with a low concentration until they could distinguish the experimental group among the 3 groups of samples. Referring to the requirements of GB / T 12311-2012 Sensory analysis - Triangle test, when the number of people who could distinguish was ≥ 11, it indicated a significant difference (P ≤ 0.05). Record the peptide concentration of this experimental group and the peptide concentration of the previous experimental group, and calculate the average value of the two as the taste threshold of this synthetic peptide. Similarly, in the test of the umami enhancement threshold, the dipeptide solution and the sodium glutamate solution with the umami threshold concentration were used as the blank groups, and the dipeptide solution and the sodium glutamate solution with a gradually increasing concentration were used as the experimental groups to carry out the triangle test; the subjects started tasting from the low increase amount of the dipeptide solution and the sodium glutamate solution concentration until they could distinguish the experimental group among the 3 groups of samples, and record the concentration difference between the peptide concentration of this experimental group and the blank group as the umami enhancement threshold of this dipeptide solution and the sodium glutamate solution.

[0073] Table 4 Taste attributes and threshold values of the dipeptide solution

[0074]

[0075] As can be seen from Table 4, the two dipeptides GM and GE have obvious umami, and both the umami threshold and the umami enhancement threshold are lower than that of sodium glutamate. Therefore, the umami of GM and GE is significantly stronger than that of sodium glutamate. Therefore, GM and GE were determined as the umami dipeptides in the crude soy sauce and used as the standard substances for monitoring.

[0076] Example 2: Quality evaluation of crude soy sauce with different production processes

[0077] In this example, the contents of the umami dipeptides GM glycine - methionine (primary 207.2 m / z, secondary 106.7 m / z) and GE glycine - glutamate (primary 205.1 m / z, secondary 96.1 m / z) in the crude oil were specifically determined for quality evaluation. Among them, the primary ions of GM and GE are obtained by adding hydrogen to the molecular weight, that is, adding 1 to their molecular weight; the secondary ions are the strongest ion peaks obtained by the instrument through testing the primary 207.2 m / z and primary 205.1 m / z of the GM and GE standards, which are 106.7 m / z and 96.1 m / z respectively, and are selected as the secondary ions.

[0078] The specific operation steps for the detection of crude soy sauce are as follows:

[0079] (1) Pretreatment of crude soy sauce

[0080] 1000 mL of crude soy sauce was rotary evaporated and concentrated to approximately 100 mL under low temperature and negative pressure conditions; the temperature was controlled at 30 - 40 °C, and the pressure was controlled at 30 - 50 mbar. Then the concentrated solution was fully adsorbed by macroporous resin, rinsed with deionized water until the eluent was colorless, eluted with 50% ethanol solution, and the eluate was collected; the eluate was rotary evaporated and concentrated under negative pressure conditions, and then freeze-dried under vacuum to obtain a freeze-dried sample; 0.1 - 0.2 g of the freeze-dried sample was weighed into a 10 mL volumetric flask, made up to the mark with water, and filtered through a 0.22 μm filter membrane to remove insoluble substances, which was the crude oil sample solution.

[0081] (2) Preparation of standards

[0082] The umami dipeptides GM and GE are artificially chemically synthesized standards. A certain amount of analytical standards were accurately weighed and standard solutions with concentrations of 0.5, 1.0, 2.5, 4.0, and 8.0 μg / mL were prepared using ultrapure water.

[0083] (3) Determination of umami dipeptide content

[0084] The above-prepared GM and GE standard solutions were determined by LC-MS / MS in the MRM mode to obtain the mass spectra of GM and GE, and the linear relationship between the peak area and concentration was determined.

[0085] The LC-MS / MS detection conditions are as follows:

[0086] Chromatographic conditions: The chromatographic column was an ACQUITY UPLC HSS T3 Column, 1.8 μm, 2.1 mm × 100 mm produced by Waters; the column oven temperature was 40 °C; the mobile phase flow rate was 0.3 mL / min, and the injection volume was 2 μL; mobile phase A was an acetonitrile aqueous solution containing 0.1% formic acid, and mobile phase B was a 0.1% formic acid aqueous solution. The specific setting ratio of the mobile phase gradient elution is shown in Table 5.

[0087] Table 5 Mobile phase setting ratio

[0088]

[0089]

[0090] The mass spectrometry conditions are as follows: Analyzed by triple quadrupole mass spectrometry, the ionization mode of the mass spectrometry ion source is ESI electrospray ion source, the interface temperature is 300 °C; the desolvation temperature is 526 °C; the DL temperature is 280 °C; the heating block temperature is 400 °C; the nebulizing gas flow rate is 3 L / min; the heating gas flow rate is 10 L / min; the drying gas flow rate is 10 L / min; the selected working mode is positive ion mode; the MRM multiple reaction monitoring mode is adopted, the primary precursor ion and the secondary fragmentation ion are respectively set as GM (primary 207.2 m / z, secondary 106.7 m / z), GE (primary 205.1 m / z, secondary 96.1 m / z), and the mass spectrometry scanning rate is set to 100 Da / s; when two secondary fragment ions of the above-mentioned target polypeptide are detected simultaneously, it is considered that the target dipeptide is successfully qualitatively analyzed; the external standard method is used to quantitatively analyze the target dipeptides GM (primary 207.2 m / z, secondary 106.7 m / z) and GE (primary 205.1 m / z, secondary 96.1 m / z) with the peak area. The mass spectrometry diagram of the machine test is shown in Figure 1 and Figure 2 .

[0091] As Figure 1 and Figure 2 shown, the retention times of GM and GE are 10.85 min and 9.25. The standard curve equation for quantitatively analyzing GM by the external standard method is y = 22209.9X + 675.606, r2 = 0.9987377, r = 0.9993686, as shown in Figure 3 ; the standard curve equation for GE is y = 365138X - 2979.51, r2 = 0.9983001, r = 0.9991497, as shown in Figure 4 ; the linearity of the above curves is good.

[0092] Dilute the crude oil sample solution prepared in step (1) by 10 times in volume as the solution for machine test, and detect it by LC-MS / MS through the MRM mode (the detection conditions are the same as those in the previous text). Then, according to the above Figures 1 to 4 results, qualitatively and quantitatively analyze GM and GE in it to obtain the contents of GM and GE in the soy sauce crude oil.

[0093] The contents of GM and GE in the crude soy sauce produced by different production processes A, B, and C determined according to the above steps are shown in Table 6. The specific production processes of the crude soy sauce are as follows: The soybeans are steamed at 110 - 125 °C until cooked through, and then the cooked soybeans are crushed into soybean fragments with a roller crusher, requiring each soybean to be crushed so that the soybean hull falls off or is crushed from the middle; then, 1 - 1.5 parts by mass of soybean fragments, 0.40 parts by mass of flour, and 0.5 - 1.0‰ parts by mass of strain powder are mixed in proportion, and thick-layer aerated koji-making is carried out. The koji-making temperature is 28 - 36 °C, the humidity is 70 - 95%, and the cultivation time is 44 hours to obtain the koji; brine is added to the koji, and the mass ratio of the koji to the brine is 1:3.5, and fermentation is carried out using the high-salt dilute-state process for 4 months; after fermentation is completed, oil is leached to obtain the crude soy sauce. The process differences among A, B, and C are as follows: In process A, the soybeans are crushed into 1 - 2 pieces, the addition amount of soybean fragments is 1.5 parts by mass, and the addition amount of strain powder is 1.0‰ parts by mass; the difference in process B is that the soybeans are crushed into 5 - 8 pieces, the addition amount of soybean fragments is 1 part by mass, and the addition amount of strain powder is 1.0‰ parts by mass; the difference in process C is that the soybeans are crushed into powder, the addition amount of soybean fragments is 1 part by mass, and the addition amount of strain powder is 0.5‰ parts by mass.

[0094] As can be seen from Table 6, there are obvious differences in the contents of the two umami dipeptides in different production processes. Among them, the contents of the two umami dipeptides in process A are the highest and the quality is the best. Moreover, GM in the crude oil of process A is more than 8 times that of the crude oil of process C, and GE is nearly 30 times that of the crude oil of process C. However, the differences in total acid, amino acid nitrogen, salt, etc. in the soy sauce produced by the three processes are very small.

[0095] Thus, it can be seen that the quality evaluation method based on the contents of umami dipeptides GM and GE developed in the present invention can better evaluate the umami differences that are difficult to distinguish using the existing soy sauce quality evaluation standards for different processes, make up for the deficiencies of the existing crude soy sauce quality evaluation methods, and provide a better reference index for the research on the production process of umami soy sauce.

[0096] Table 6 Test results of 5 indicators in the crude oil of three different processes

[0097]

[0098] At the same time, a sensory evaluation of the umami strength ranking of the crude oil samples of processes A, B, and C is carried out to test the evaluation method of the present invention; specifically, the sensory evaluation is carried out with reference to GB / T 12315 - 2008 "Sensory analysis methodology - Ranking method".

[0099] Fourteen assessors were selected from the company (7 males and 7 females, aged from 22 to 40 years old) to form a sensory evaluation panel; the evaluation was carried out at 25±2°C; preferably, the assessors tasted the samples of three processes A (No. 448), B (No. 362), and C (No. 188), and then rinsed their mouths with water; the tasting test interval was 1 minute, and no eating or drinking was allowed 1 hour before the evaluation; the umami intensity was ranked from strong to weak (1 = strongest, 3 = weakest), and the results are shown in Table 7.

[0100] Table 7 Ranking results of umami sensory tests for crude oils of three different processes

[0101]

[0102]

[0103] For the umami ranking data of crude oils under the three process conditions A, B, and C in Table 7, the rank sums of A, B, and C are 16, 29, and 39 respectively, and the number of assessors n = 14. The Friedman test was used for significance analysis. When the significance level α = 0.05, referring to the critical value table of the Friedman test, the critical value is 5.991; the calculated value is 18; that is, there are significant differences in the umami intensities of A, B, and C. For pairwise comparisons, the Nemenyi test was used, and the critical range of the pairwise rank sum difference is 1.335; pairwise rank sum difference: ∣R A -R B ∣ = 13 > 1.335; ∣R A -R B ∣ = 23 > 1.335; ∣R A -R B ∣ = 10 > 1.335; there are significant differences between any two pairs. Process A soy sauce was ranked first the most times, and after statistical analysis, the difference was significant. It can be concluded that the umami of the crude oil of Process A is the strongest, followed by the crude oil of Process B, and the umami of the crude oil of Process C is the weakest. The umami order of the crude oil of soy sauce in the sensory evaluation is consistent with the results of evaluating the crude oil of soy sauce using umami dipeptides GM and GE in the present invention.

[0104] Quality evaluation of crude oils of soy sauce with the same production process but different batches in Example 3

[0105] In this example, three batches of crude oils under the above Process A conditions were selected to determine the contents of umami dipeptides GM and GE. The specific operations were the same as those in Example 2, and the results are shown in Table 8.

[0106] As can be seen from Table 8, under the same production process conditions, the quality of the crude oil is relatively stable, and the five indicators of GM, GE, amino acid nitrogen, total acid, and salt content fluctuate little.

[0107] Table 8 Detection results of 5 indicators in 3 batches of crude oils under Process A conditions

[0108]

[0109]

[0110] Meanwhile, under the condition that the amino acid nitrogen, total acid, salt, GM, and GE are relatively close, the sensory evaluation method consistent with that in Example 2 is used to rank the umami of 3 batches of crude oil samples under the above A process conditions from strong to weak. Preferably, the evaluators taste the samples of three batches A1 (No. 868), A2 (No. 352), and A3 (No. 451), and then rinse their mouths with water; the tasting test interval is 1 minute, and no eating or drinking is allowed 1 hour before the evaluation; the umami intensity is ranked from strong to weak (1 = strongest, 3 = weakest), and the results are shown in Table 9.

[0111] Table 9 Ranking results of sensory tests on the umami of 3 batches of crude oil under the A process conditions

[0112]

[0113] For the umami ranking data of the three batches A1, A2, and A3, the rank sums of A1, A2, and A3 are 26, 28, and 30 respectively, and the number of evaluators n = 14. The Friedman test is used for significance analysis. When the significance level α = 0.05, referring to the critical value table of the Friedman test, the critical value is 5.991; the calculated value is 0.57. That is, there is no significant difference in the umami intensity of the three batches of samples, which is consistent with the evaluation results of GM and GE adopted in the present invention.

[0114] Example 4 Quality evaluation of crude oil with different fermentation days

[0115] In this example, the crude oil on the 60th, 90th, and 120th days of fermentation under the A process conditions is selected to measure the contents of umami dipeptides GM and GE. The specific operations are the same as those in Example 2, and the results are shown in Table 10.

[0116] As can be seen from Table 10, among the crude oils with different fermentation days, the contents of GM and GE vary greatly, and GM and GE are mainly produced in the last 100 days of fermentation. The formation of amino acid nitrogen also basically occurs in the last 100 days of fermentation. The total acid reaches 0.9 g / 100 mL in the first 80 days and reaches the highest at 120 days. The salt content shows a decreasing trend during fermentation, mainly because the salt in the crude oil gradually penetrates into the cheese. Thus, it can be seen that among the three, the quality of the crude oil after 120 days of fermentation is better.

[0117] Table 10 Monitoring results of 5 indicators in crude oil with different fermentation days under the A process conditions

[0118]

[0119] Methodological Research and Analysis on the Determination of the Contents of Umami Dipeptides GM and GE in Example 5

[0120] In Example 2, the standard curves of GM and GE of the present invention have been evaluated. Then, in this example, the methods for determining the contents of GM and GE will be evaluated from two aspects: precision and accuracy.

[0121] (1) Instrument precision test: Prepare a mixed polypeptide standard of GM and GE with a polypeptide concentration of 5 μg / mL, inject samples continuously for 6 times, and measure the RSD values of the peak areas of each component ion pair of GM (primary 207.2 m / z, secondary 106.7 m / z) and GE (primary 205.1 m / z, secondary 96.1 m / z) to be 1.8% and 2.3% respectively, both of which are less than 3% (see Table 11), indicating that the instrument precision is good.

[0122] Table 11 Results of Instrument Precision Test for GM and GE

[0123]

[0124]

[0125] (2) Precision test of sample repeatability: Prepare 6 portions of crude oil samples with the same concentration (specifically, the crude oil produced by Process A in Example 2), and measure the contents of GM (primary 207.2 m / z, secondary 106.7 m / z) and GE (primary 205.1 m / z, secondary 96.1 m / z) in the samples according to the above method. The results are shown in Table 12. As shown in Table 12, the average contents of GM and GE are 281 μg / L and 613 μg / L respectively, and the corresponding RSD values are 2.6% and 1.1% respectively, indicating good method reproducibility; in addition, the detection results of the contents of each component obtained based on each ion pair in the sample have relatively small deviations from each other, indicating that the selection of ion pairs is relatively ideal and the detection results can be obtained better.

[0126] Table 12 Results of Sample Precision Test for GM and GE

[0127]

[0128] (3) Evaluation of sample stability over different days: Prepare the same concentration of the same crude oil sample (specifically, the crude oil produced by Process A in Example 2), and according to the above detection and analysis method, refrigerate the processed sample in a refrigerator at 4 - 8 °C. Inject samples and measure the contents at 0, 6, 18, 24, and 48 h respectively. The results are shown in Table 13 to evaluate the method stability. The results show that the RSD values of the two components GM (primary 207.2 m / z, secondary 106.7 m / z) and GE (primary 205.1 m / z, secondary 96.1 m / z) are 3.4% and 1.2% respectively, both of which are less than 5.0%, indicating good stability of the sample and the target polypeptide within 48 h.

[0129] Table 13 Results of GM and GE Daytime Precision Tests

[0130]

[0131] (4) Accuracy test: Take a total of 6 crude oil samples with known contents of 2 polypeptides (specifically, the crude oil produced by Process A in Example 2), accurately add GM and GE standard substances, and measure their recovery rates. The results are shown in Table 14. It can be seen from Table 14 that the recovery rates of polypeptides GM and GE are in the range of 93-106%, and the RSDs are all less than 3%, indicating that polypeptides GM and GE have good stability and linear change relationships in the samples and have good quantitative detection effects.

[0132] Table 14 Results of GM and GE Accuracy Tests

[0133]

[0134] The above embodiments are only used to illustrate the present invention, and the protection scope of the present invention is not limited to the above embodiments. Those of ordinary skill in the art can achieve the purpose of the present invention based on the content disclosed above. Any improvements and variations made on the basis of the idea of the present invention fall within the protection scope of the present invention. The specific protection scope shall be subject to what is recorded in the claims.

Claims

1. A method for evaluating the quality of crude soy sauce based on umami dipeptides, characterized in that, The following steps are involved: (1) Screening and identification of dipeptides in crude soy sauce oil: The soy sauce crude oil was pretreated with macroporous resin to obtain a crude oil sample solution. The crude oil sample solution was detected by high-resolution liquid chromatography quadrupole time-of-flight mass spectrometry to screen and identify dipeptides. (2) Secondary screening of dipeptides in crude soy sauce oil: Selecting the dipeptides with the highest signal intensity ranking from the dipeptides identified in step (1), that is, selecting the dipeptides with the highest content ranking; then performing secondary screening using a triple quadrupole liquid chromatography-mass spectrometer in a selected ion scanning mode, specifically quantitatively detecting the above-selected dipeptides in the crude oil sample, and screening out the dipeptides suitable for monitoring based on the mean value of the response peak area in the measured spectrum; (3) Identification of umami dipeptides in crude soy sauce oil: Obtaining the dipeptides screened in step (2), performing sensory evaluation, and identifying dipeptides with umami-enhancing effects; (4) Determination of umami dipeptide content in the crude soy sauce oil to be evaluated: The soy sauce crude oil to be evaluated is pre-treated with a macroporous resin to obtain a crude oil sample solution to be evaluated; Taking the standard of the umami dipeptide to prepare an umami dipeptide standard solution; The crude oil sample solution to be evaluated was detected in the multiple reaction monitoring mode of triple quadrupole liquid chromatography-mass spectrometry. The umami dipeptides therein were qualitatively and quantitatively analyzed based on the umami dipeptide standard solution. Then, the quality of the soy sauce crude oil was evaluated based on the umami dipeptide content.

2. The method for evaluating the quality of crude soy sauce based on umami dipeptides according to claim 1, characterized in that In step (1), the chromatographic conditions of the high-resolution liquid chromatography quadrupole time-of-flight mass spectrometer are as follows: the chromatographic column is a C18 chromatographic column; the column oven temperature is 35-45° C.; the mobile phase consists of mobile phase A and mobile phase B, mobile phase A is an acetonitrile aqueous solution containing 0.1% formic acid, and mobile phase B is a 0.1% formic acid aqueous solution; the mobile phase flow rate is 0.2-0.3 mL / min, and the injection volume is 1-2 μL; the mass spectrometry conditions are as follows: the working mode is selected as the positive ion mode, the mass spectrometry ion source ionization mode is the ESI electrospray ion source, the interface temperature is 520° C., the spray voltage is 5600 V, the scanning period is 0.602 s, the TOF primary scanning range is set to 138-390 Da with reference to the molecular weight range of glycine-glycine and tryptophan-tryptophan, the secondary scanning range is 50-800 Da, the working mode is DDA, the maximum number of candidate ions is 4, dynamic exclusion is turned on, and the remaining parameters use the proteomics method default values.

3. The soy sauce crude oil quality evaluation method based on umami dipeptides according to claim 2, characterized in that, in In step (2), the triple quadrupole liquid chromatography-mass spectrometer adopts the same chromatographic conditions as the high-resolution liquid chromatography quadrupole time-of-flight mass spectrometer in step (1); its mass spectrometry conditions are: the chromatographic column is a C18 chromatographic column; the working mode is selected as the positive ion mode; the mass spectrometry ion source ionization mode is the ESI electrospray ion source, and the interface temperature is 300-320°C; the desolvation temperature is 526-550°C; the DL temperature is 250-280°C; the heating block temperature is 400-420°C; the nebulizing gas flow rate is 3-5 L / min; the heating gas flow rate is 10-12 L / min; and the drying gas flow rate is 10-12 L / min; a selected ion scanning mode is adopted, and its setting value is determined according to the ion of the dipeptide with the highest signal intensity selected.

4. The method for evaluating the quality of crude soy sauce based on umami dipeptides according to claim 3, characterized in that, The umami dipeptides are GM and GE.

5. The method for evaluating the quality of crude soy sauce based on umami dipeptides according to claim 4, characterized in that, In step (4), the triple quadrupole liquid chromatography-mass spectrometry instrument uses the same chromatographic conditions as the high-resolution liquid chromatography quadrupole time-of-flight mass spectrometry instrument in step (1); its mass spectrometry conditions are as follows: the chromatographic column is a C18 chromatographic column; the selected working mode is the positive ion mode; the ionization mode of the mass spectrometry ion source is the ESI electrospray ion source, with an interface temperature of 300 - 320 °C; the desolvation temperature is 526 - 550 °C; the DL temperature is 250 - 280 °C; the heating block temperature is 400 - 420 °C; the nebulizing gas flow rate is 3 - 5 L / min; the heating gas flow rate is 10 - 12 L / min; the drying gas flow rate is 10 - 12 L / min; the multiple reaction monitoring mode is adopted, the mass spectrometry scanning rate is set to 50 - 300 Da / s, and the monitoring setting parameters of the first-stage precursor ion and the second-stage fragmentation ion are respectively: for GM: the first stage is 207.2 m / z and the second stage is 106.7 m / z; for GE: the first stage is 205.1 m / z and the second stage is 96.1 m / z.

6. The method for evaluating the quality of crude soy sauce based on umami dipeptides according to claim 5, wherein In step (3), the method of sensory evaluation is as follows: the dipeptides screened in step (2) are formulated into a 1 mg / mL dipeptide solution, and a 1 mg / mL sodium glutamate solution is set as a control, and the evaluation is carried out for the five flavors of acid, salt, umami, sweet, and bitter; the evaluation is carried out at 25 ± 2 °C; the evaluator tastes the sample solution and then rinses the mouth with water, and the tasting test interval is 1 minute.

7. The method for evaluating the quality of crude soy sauce based on umami dipeptides according to claim 6, characterized in that, in In step (3), in addition to sensory evaluation, the dipeptides screened in step (2) are analyzed by an electronic tongue to identify the umami dipeptides with umami-enhancing effects; the method of electronic tongue analysis is as follows: the dipeptides screened in step (2) are formulated into a 1 mg / mL dipeptide solution, and a 1 mg / mL sodium glutamate solution is set as a control; the taste characteristics of the dipeptide solution and the sodium glutamate solution are measured at 25 °C by an electronic tongue, balanced for 30 s, the measurement time is 30 s, the cut-off time is 30 s, the acquisition period is 1 s, and the sample solution is detected by the five-flavor sensor array of acid, salt, umami, sweet, and bitter.

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