A method for evaluating the bitterness of soy sauce during shelf life

Abstract

The present invention discloses a method for evaluating the bitterness of soy sauce during its shelf life, and belongs to the field of analytical detection technology. The method for evaluating the bitterness of soy sauce during its shelf life provided by the present invention comprises the following steps: S1: collecting bitter substances in a soy sauce sample by separation using a macroporous resin; S2: detecting the fluorescence intensity X of an aqueous solution of the bitter substances using fluorescence spectroscopy, and substituting X into an evaluation formula Y=(X-5273.3) / 589.44 to calculate a bitterness value Y. The method for evaluating the bitterness of soy sauce during its shelf life provided by the present invention uses macroporous resin separation and purification combined with a fluorescence testing method for the first time to evaluate changes in the emphasis of the bitterness of soy sauce during its shelf life. As a whole, the method for evaluating the bitterness of soy sauce during its shelf life has good correspondence with traditional sensory evaluation and can replace sensory evaluation. Moreover, the evaluation method provided by the present invention has the advantages of being fast, accurate, and low-cost compared to sensory evaluation, and can be actually applied to evaluating the bitterness of soy sauce during its shelf life.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and in particular relates to a method for evaluating the bitterness of soy sauce during its shelf life. Background Art

[0002] Soy sauce production has a long history in my country and is a daily necessity. However, shelf stability has long been a problem, with issues such as color control, flavor variations, and precipitation significantly impacting the quality and sensory experience of soy sauce. Consequently, the shelf stability of soy sauce is receiving increasing attention.

[0003] The Maillard reaction is a widespread phenomenon in soy sauce during thermal processing and storage. This reaction involves reversible or irreversible chemical recombination reactions, such as oxidation, cyclization, and polymerization, between the hemiacetal groups of reducing sugars and proteins, peptides, amino acids, or free amines. Due to its complex mechanism and numerous products, this reaction can impart a rich color, aroma, and flavor to soy sauce, but it can also produce compounds with negative effects, such as the accumulation of browning substances and the production of bitter compounds. Existing research suggests that shelf life changes in soy sauce and the Maillard reaction share many similarities, suggesting that shelf-life degradation may be related to the Maillard reaction. However, due to its complexity, research on the Maillard reaction in both industry and academia is currently limited. Furthermore, the complex flavor profiles in soy sauce are primarily salty and umami, supplemented by sweet and sour, with a slight bitterness. The interactions between these flavor components significantly impact the taste perception of specific components and significantly interfere with their detection and identification. Therefore, the Maillard reaction, particularly in complex systems like soy sauce, has long been a challenge to study. How to extract key shelf life evaluation indicators from a complex system is an important prerequisite for guiding the development of shelf life stabilization technology and promoting the research on the Maillard reaction mechanism of soy sauce shelf life.

[0004] Currently, research on the technologies and mechanisms for controlling the shelf life of soy sauce is very limited, focusing primarily on correlation analysis of conventional physical and chemical indicators, lacking the refined separation, identification, and analysis of key flavor compounds. Furthermore, flavor evaluation relies on traditional sensory evaluation, which is subject to significant subjectivity and variability. The main drawbacks of sensory evaluation are that, first, even subtle changes in the intensity of soy sauce's bitterness during its shelf life are difficult for human senses to detect, and the sensory process is easily influenced by other flavor compounds, making it difficult to objectively assess changes in bitterness during shelf life. Second, sensory results are susceptible to both internal and external factors, including the individual's state, environment, and the different evaluators. Furthermore, organizing the evaluation requires significant human and material resources, and reproducibility of experimental results is difficult to guarantee. Electronic tongues, as a substitute and extension of human taste perception, are already being increasingly used in food testing. However, these applications are generally limited to testing the relationship between electronic tongue responses and concentrations of known flavor components. Alternatively, they employ principal component analysis (PCA), discriminant factor analysis (DFA), and artificial neural networks (ANN) to identify models of different soy sauce samples, using partial least squares (PLS) to establish quantitative models linking basic physicochemical components to electronic tongue outputs of individual flavor components. This means that electronic tongues are primarily used to analyze overall differences between samples, but are difficult to quantify for specific flavors. Furthermore, bitterness changes over shelf life due to its complex composition, making it difficult to pinpoint specific components. Furthermore, bitterness is easily influenced by other flavor components, making it difficult to single out the intensity of changes in bitterness over shelf life. Furthermore, among existing separation methods, ultrafiltration primarily utilizes molecular weight cutoff to fractionate samples, but this struggles to selectively isolate specific flavor components. Ion exchange utilizes the interaction between the charge of the substance and the charge of the chromatographic carrier to achieve separation and purification, but this often introduces other ions during the separation process, impacting subsequent sensory evaluation. Consequently, no optimal specific separation method currently exists. Therefore, combining modern material separation technology, precision analysis technology, and electronic sensory technology for auxiliary research is of great significance for studying and analyzing the causes, degree, and mechanism of changes in the shelf life of soy sauce. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a rapid, accurate and low-cost method for evaluating the bitterness of soy sauce during its shelf life.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a method for evaluating the bitterness of soy sauce during shelf life, the evaluation method comprising the following steps:

[0007] S1: Bitter substances in soy sauce samples were separated and collected by macroporous resin;

[0008] S2: Detect the fluorescence intensity X of the aqueous solution of the bitter substance using fluorescence spectroscopy, and substitute X into the evaluation formula Y=(X-5273.3) / 589.44 to calculate the bitterness value Y.

[0009] The present invention provides a method for evaluating the bitterness of soy sauce during its shelf life. The method first separates and collects the main bitter substances in the soy sauce using a macroporous resin, then detects the fluorescence intensity using fluorescence spectroscopy, and then converts the detected fluorescence intensity value into a bitterness value using an evaluation formula. This allows for rapid, accurate, and low-cost evaluation of the bitterness of the soy sauce. Specifically, on the one hand, the flavor substances in soy sauce are complex, and the various flavor substances interact with each other, and bitter substances are mostly substances with strong hydrophobicity and weak polarity. Therefore, the present invention adopts macroporous resin separation to utilize the interaction force between molecules to separate bitter substances and other substances, thereby eliminating the interference of most non-target components and improving detection sensitivity; on the other hand, the currently known bitter substances are mainly divided into alkaloids, terpenoids, glycosides and flavan-3-ols, and their structures generally contain heteroatoms, conjugated double bonds or cyclic structures. At the same time, fluorescence analysis is a spectral analysis between the emission spectrum and the absorption spectrum with light as the excitation source. Therefore, the present invention performs fluorescence analysis on the separated bitter substances, and can simultaneously perform qualitative or quantitative analysis on multiple components in the substance, thereby further improving the accuracy of the evaluation method.

[0010] As a preferred embodiment of the evaluation method of the present invention, step S1 specifically includes the following steps:

[0011] S11: Activate the macroporous resin and fill it into the chromatography column;

[0012] S12: adding the soy sauce sample to the chromatography column for adsorption;

[0013] S13: Gradient elution and collection of the eluate, concentration and freeze-drying to obtain a bitter substance.

[0014] As a preferred embodiment of the evaluation method of the present invention, in step S11, the method for activating the macroporous resin is: soaking the macroporous resin in 95% ethanol for more than 24 hours.

[0015] As a preferred embodiment of the evaluation method of the present invention, after the activated macroporous resin is filled into the chromatography column, a step of rinsing with pure water is further included, and the end point of the rinsing is that the rinsing liquid has no alcohol taste.

[0016] As a preferred embodiment of the evaluation method of the present invention, the macroporous resin is a medium-polar macroporous resin, specifically, any one of DM130 and AB-8.

[0017] As a preferred embodiment of the evaluation method of the present invention, the mass ratio of the macroporous resin to the soy sauce sample is macroporous resin:soy sauce sample=(15-30) g:(5-25) g.

[0018] When the mass ratio of the macroporous resin to the soy sauce sample is within the above range, it can be ensured that the resin mass is sufficient and the usage amount is appropriate. In actual use, it is sufficient to visually observe that there is unadsorbed resin on the upper layer.

[0019] The inventors have found that if the soy sauce is first subjected to ultrafiltration separation before macroporous resin separation to obtain a fraction with a molecular weight less than 1 kDa, and then subjected to macroporous resin separation, bitter substances can also be obtained. However, ultrafiltration has the problem of difficult to control separation stability. Therefore, it is preferred to directly use macroporous resin for separation.

[0020] As a preferred embodiment of the evaluation method of the present invention, in step S12, the adsorption time is 10-30 minutes.

[0021] As a preferred embodiment of the evaluation method of the present invention, in step S13, the gradient elution and collection of the eluate for concentration and freeze-drying specifically include the following steps: first, elution is performed three times with the liquid in the chromatography column, followed by elution with pure water and 10-20% ethanol in sequence, and the 10-20% ethanol eluate is collected, the 10-20% ethanol eluate is concentrated at 45-55°C to remove ethanol, and then freeze-dried to obtain the bitter substance.

[0022] The inventors have found that by adopting the above-mentioned gradient elution procedure for elution, the fluorescence intensity value of the bitter substance obtained can be well correlated with the bitterness value, thereby improving the accuracy and sensitivity of detection.

[0023] As a preferred embodiment of the evaluation method of the present invention, the elution rate is 5-15 mL / min.

[0024] As a preferred embodiment of the evaluation method of the present invention, the method for determining the end point of the elution with pure water and 10-20% ethanol is: the absorbance value of the eluate at 230 nm is less than 0.35.

[0025] As a preferred embodiment of the evaluation method of the present invention, the mass ratio of the bitter substance aqueous solution to the soy sauce sample is (250-1500):1.

[0026] As a preferred embodiment of the evaluation method of the present invention, in step S2, in the fluorescence spectrum detection, the excitation wavelength is 300-360 nm, and the emission wavelength is 400-440 nm.

[0027] The inventors have found that the sensitivity of the evaluation method obtained at the above excitation wavelength and emission wavelength is excellent.

[0028] In addition, the present invention also provides an application of the evaluation method in evaluating the bitterness of soy sauce during its shelf life.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention provides a method for evaluating the bitterness of soy sauce during its shelf life, which uses a macroporous resin separation and purification method combined with a fluorescence testing method for the first time to evaluate changes in the bitterness emphasis of soy sauce during its shelf life. As a whole, the shelf life bitterness evaluation method has good correspondence with traditional sensory evaluation and can replace sensory evaluation. In addition, compared with sensory evaluation, the evaluation method provided by the present invention has the advantages of being fast, accurate, and low-cost, and can be actually applied to the evaluation of the bitterness of soy sauce during its shelf life. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a graph showing the relationship between the fluorescence intensity and the mass ratio of the bitter substance aqueous solution to the soy sauce sample in Example 2;

[0032] Figure 2 This is a graph showing the relationship between fluorescence intensity and bitterness value in Example 3. DETAILED DESCRIPTION

[0033] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0034] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0035] Example 1

[0036] The embodiment of the present invention utilizes macroporous resin to separate and evaluate bitter substances, specifically comprising the following steps:

[0037] 1. Separation of bitter substances

[0038] 25 g of DM130 macroporous resin was accurately weighed, soaked in 95% ethanol at room temperature for 30 h, and then filled into a chromatography column. The column was rinsed with distilled water until no alcohol smell was found (approximately 500 g of pure water was used for rinsing). Subsequently, 10 g of soy sauce A1 was placed in the chromatography column for static adsorption. After adsorption for 15 min, the column was repeatedly eluted three times with the liquid in the column. Subsequently, pure water, 10-20% ethanol, 30-40% ethanol, and 50-70% ethanol were used for elution in sequence. The endpoint of each elution process was an absorbance value of the eluent at 230 nm of less than 0.35. The eluents of each process were collected, concentrated by rotary evaporation at 45-55° C., and freeze-dried. The product of the pure water eluent was component I, the product of the 10-20% ethanol eluent was component II, the product of the 30-40% ethanol eluent was component III, and the product of the 50-70% ethanol eluent was component IV.

[0039] 2. Sensory evaluation

[0040] Six experimenters with experience in sensory analysis (3 males and 3 females, aged 25-35 years) conducted a sensory evaluation on the four components separated from soy sauce A1; the evaluation items were, on the one hand, the aqueous solution of the four components in soy sauce A1 (the mass ratio of the aqueous solution of the four components to the soy sauce sample providing the four components was 300:1), and on the other hand, the four components in soy sauce A1 were dissolved in soy sauce A2 (the total mass ratio of the components dissolved in soy sauce A2 and the soy sauce sample providing the four components was 300:1, where soy sauce A1 and soy sauce A2 were soy sauces produced by the same production line, but with different production times, where soy sauce A1 was a sample from January 2021 and soy sauce A2 was a sample from November 2021); the specific evaluation results are shown in Table 1;

[0041] Table 1

[0042]

[0043] At the same time, further sensory description and comparison were conducted on the sample obtained by dissolving component II in soy sauce A1 in soy sauce A2. The bitterness of the sample was enhanced and close to that of soy sauce A1, the astringency was significantly weaker than that of soy sauce A1, and the stale aroma was significantly weaker than that of soy sauce A1.

[0044] Therefore, as shown in Table 1 and further sensory descriptions, macroporous resins are capable of separating and purifying bitter substances in soy sauce. The resulting fraction II exhibits a distinct bitterness, fraction III exhibits a distinct astringency, and fractions I and IV exhibit no bitterness. When fraction II from soy sauce A1 is added to soy sauce A2, the bitterness is significantly enhanced and approaches that of soy sauce A1. This suggests that fraction II represents the component that causes soy sauce to become bitter during shelf life and is therefore defined as the bitter substance in soy sauce.

[0045] Example 2

[0046] The embodiment of the present invention explores the relationship between the ratio of the mass ratio of the bitter substance aqueous solution to the soy sauce sample and the fluorescence intensity. According to the method in Example 1, soy sauce B1 and soy sauce B2 (wherein soy sauce B1 and soy sauce B2 are soy sauces produced by the same production process line, but there is a difference in production time, wherein soy sauce B1 is a sample in November 2021, and soy sauce B2 is a sample in July 2020) are separated from the bitter substances to obtain components II of the two, respectively. The bitter substances of the two are then redissolved in water, wherein the ratio gradient of the mass ratio of the bitter substance aqueous solution to the soy sauce sample is set to 1500:1, 750:1, 500:1, 375:1, 300:1, 250:1, and 214:1, and then detected under the same fluorescence detection conditions (excitation wavelength of 360 nm, emission wavelength of 440 nm). The detection results are as follows. Figure 1 shown.

[0047] from Figure 1 It can be seen that when the mass ratio of bitter substance aqueous solution to soy sauce sample is in the range of (250-1500):1, the fluorescence intensity and concentration have a good linear relationship, and the correlation coefficient R 2 Above 0.99; in subsequent experiments for the detection of fluorescence emphasis, the mass ratio of the bitter substance aqueous solution to the soy sauce sample was maintained at 300:1.

[0048] Example 3

[0049] The embodiment of the present invention protects the process of establishing an evaluation formula: taking soy sauce A products with different shelf lives, subjecting them to macroporous resin separation according to the method in Example 1 to obtain corresponding fraction II, and then re-dissolving fraction II in water until the mass ratio of the aqueous solution to the soy sauce sample is 300:1, and then detecting under the fluorescence detection conditions in Example 2 to obtain the corresponding fluorescence intensity;

[0050] In addition, the bitterness value of the corresponding component II obtained above was obtained using the taste dilution analysis (TDA) method. Specifically, the alcohol-washed freeze-dried component was diluted with water until the bitterness was just no longer tasted. The dilution factor was the bitterness value of the sample. The sensory test was conducted at room temperature (25±2)°C by 6 experimentalists with sensory analysis experience (3 males and 3 females, aged 25-35 years). When 3 or more tasters had the same bitter and astringent taste stimulus response to a certain taste solution S, and at least 4 tasters had a bitter and astringent taste stimulus response to the highest concentration taste solution, S was considered the bitterness value. The bitterness values ​​and fluorescence intensities of samples with different shelf lives are shown in Table 2.

[0051] Table 2

[0052]

[0053] The fluorescence intensity and bitterness value in Table 2 are plotted and dashed to obtain Figure 2 ,from Figure 2 It can be seen that the bitterness value has a strong correlation with the fluorescence intensity, with a correlation coefficient of 0.98. Further conversion yields the relationship between the bitterness value Y and the fluorescence intensity X, namely, Y = (X-5273.3) / 589.44; and from Figure 2 As can be seen in the figure, when the bitterness values ​​are the same, the sensory organs cannot distinguish the differences between the samples, but the fluorescence intensity can; therefore, this also demonstrates the high sensitivity of the evaluation method provided by the present invention. An electronic tongue can also be used to determine a specific bitterness value, but its sensitivity is not as high as that obtained using the taste dilution analysis method.

[0054] Example 4

[0055] The accuracy of the evaluation formula obtained in Example 3 was verified in the example of the present invention. Soy sauce D and soy sauce E with different shelf lives were taken and subjected to macroporous resin separation according to the method in Example 1 to obtain the corresponding component II. Component II was then redissolved in water until the mass ratio of the aqueous solution to the soy sauce sample was 300:1. Subsequently, the fluorescence intensity was detected under the fluorescence detection conditions in Example 2 to obtain the corresponding fluorescence intensity, and the evaluation formula in Example 3 was used for calculation to obtain the calculated bitterness value. At the same time, the taste dilution analysis (TDA) method in Example 3 was used to obtain the TDA bitterness value. The results are shown in Table 3. The deviation was calculated as follows: Deviation = (calculated bitterness value - TDA bitterness value) / TDA bitterness value * 100%.

[0056] Table 3

[0057]

[0058]

[0059] As can be seen from Table 3, the evaluation method of the present invention can accurately detect the bitterness values ​​of soy sauces with different shelf lives.

[0060] The evaluation method of the present invention was compared with the taste dilution analysis (TDA) method. The comparison results are shown in Table 4.

[0061] Table 4

[0062]

[0063] It can be further seen from Table 4 that the technical solution provided by the present invention is not only highly efficient and low-cost, but also does not require professional evaluators and can achieve excellent accuracy.

[0064] Example 5

[0065] The present invention explores the use of the evaluation method of the present invention for improving the bitterness of soy sauce E during shelf life. The control group and the control group were subjected to shelf life inventory tests for 6 months (production date 202111) and 12 months (production date 202105), and the bitterness values ​​of the two methods of calculation and sensory analysis were evaluated respectively. The test results are shown in Table 5;

[0066] Table 5

[0067]

[0068] It can be seen from Table 5 that the evaluation method of the present invention can be applied to improve and regulate the bitterness of soy sauce during its shelf life.

[0069] Finally, it should be noted that the above embodiments are intended to illustrate the technical solutions of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for evaluating the bitterness of soy sauce during its shelf life, characterized in that: The evaluation method comprises the following steps: S1: Bitter substances in soy sauce samples were separated and collected by macroporous resin; S2: Using fluorescence spectroscopy to detect the fluorescence intensity X of the aqueous solution of the bitter substance, and substituting X into the evaluation formula Y = (X - 5273.3) / 589.44 to calculate the bitterness value Y; The step S1 specifically includes the following steps: S11: Activate the macroporous resin and fill it into the chromatography column; S12: adding the soy sauce sample to the chromatography column for adsorption; S13: gradient elution and collection of the eluate, concentration and freeze-drying to obtain a bitter substance; In step S13, gradient elution and collection of the eluate for concentration and freeze-drying specifically include the following steps: first eluting three times with the liquid in the chromatography column, then eluting with pure water and 10-20% ethanol in sequence, collecting the 10-20% ethanol eluate, concentrating the 10-20% ethanol eluate at 45-55° C. to remove ethanol, and then freeze-drying to obtain the bitter substance; The elution rate is 5-15 mL / min; The mass ratio of bitter substance aqueous solution to soy sauce sample is (250-1500):1; In step S2, in the fluorescence spectrum detection, the excitation wavelength is 300-360 nm, and the emission wavelength is 400-440 nm; The macroporous resin includes any one of DM130 and AB-8.

2. The evaluation method according to claim 1, wherein: In step S11, the method for activating the macroporous resin is: soaking the macroporous resin in 95% ethanol for more than 24 hours.

3. The evaluation method according to claim 1, wherein: In step S12, the adsorption time is 10-30 minutes.

4. The evaluation method according to claim 1, wherein: The method for judging the end point of elution with pure water and 10-20% ethanol is: the absorbance value of the eluate at 230 nm is less than 0.

35.

5. Application of the evaluation method according to any one of claims 1 to 4 in evaluating the bitterness of soy sauce during its shelf life.

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