Method for improving strong taste of high-salt liquid-state fermented soy sauce and obtained product

Abstract

The invention discloses a method for improving the strong taste of high-salt liquid-state fermented soy sauce and an obtained product, and belongs to the technical field of food seasoning processing. The method comprises the following steps: soaking soybeans, cooking the soaked soybeans, and filtering the cooked soybeans to obtain cooked soybeans; mixing the cooked beans with wheat flour and aspergillus oryzae koji essence in proportion, and performing koji-making and fermentation to obtain finished koji; mixing the finished koji with saline water and enzyme preparations (xylanase and glutaminase), and carrying out high-salt liquid-state fermentation; after fermentation is completed, squeezing, sterilizing and filtering soy sauce mash to obtain a clarified finished soy sauce product. The relative abundance of gamma-glutamyl peptide in the soy sauce prepared by the method disclosed by the invention is remarkably improved, the strong taste is obviously improved compared with that of the traditional soy sauce, and the flavor quality of the soy sauce is favorably enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of food seasoning processing, and specifically relates to a method for enhancing the rich flavor of high-salt dilute fermented soy sauce by increasing the relative abundance of γ-glutamyl peptides (γ-GPs) and the resulting soy sauce product, belonging to a fermentation process improvement technology for enhancing the flavor function of condiments. Background Art

[0002] Soy sauce is a typical microbially fermented condiment. Its unique flavor derives from the synergistic effect of multiple flavoring substances, including saltiness, umami, and the "kokumi" flavor, which has garnered widespread attention in recent years. Saltiness is primarily provided by the high salt environment, while umami originates primarily from glutamate and flavor nucleotides (such as IMP and GMP). Kokumi, on the other hand, manifests as a rich, persistent, and coordinated taste. It is not directly perceived by a single taste receptor and can enhance the intensity and duration of umami, sweetness, and saltiness, giving the soy sauce a richer, fuller flavor. Research has shown that the production of kokumi is closely linked to the activation of the tongue's calcium-sensing receptor (CaSR), with γ-GPs being one of the key activators.

[0003] γ-GPs are a class of small peptides formed by glutamic acid and other amino acids linked by γ-amide bonds. They are widely present in various fermented foods, especially in traditional fermented soybean products such as soy sauce, miso, and natto. Typical γ-GPs include γ-Glu-Gly, γ-Glu-Ala, γ-Glu-Val, γ-Glu-Phe, γ-Glu-Leu, γ-Glu-Ile, γ-Glu-Met, γ-Glu-Ser, γ-Glu-Trp, γ-Glu-Cys, γ-Glu-Tyr, γ-Glu-His, γ-Glu-Gln, γ-Glu-Asp, γ-Glu-Thr, γ-Glu-Pro, γ-Glu-Asn, γ-Glu-Glu, as well as tripeptides such as γ-Glu-Val-Gly and γ-Glu-Cys-Gly. These small peptides don't inherently possess a strong umami flavor, but they can enhance the depth and duration of umami through synergistic interactions with free glutamate, flavor nucleotides, and other amino acids. Furthermore, γ-GPs activate the CaSR, enhancing the taste system's overall perception of umami, sweetness, and saltiness, thereby playing a key role in enhancing the "kokumi" flavor of soy sauce. Therefore, the type and content of γ-GPs are widely considered important indicators for evaluating soy sauce flavor quality.

[0004] In recent years, industrial soy sauce production has gradually shifted away from the traditional "high-salt, dilute fermentation" process. This refers to a soy sauce system using soybeans, wheat, and other raw materials, fermented in liquid form at a salt concentration of 16%-20% after koji preparation. This system offers excellent fluidity, low solids content, and facilitates temperature control and staged inoculation. It is well-suited to modern industrial fermentation tank control and the timed addition of complex enzymes, and has become a key development direction for high-salt fermented soy sauce.

[0005] However, the natural production of γ-GPs in soy sauce is low under the high-salt, dilute soy sauce fermentation process, primarily due to the following limitations: First, the synthesis of γ-GPs relies on the key enzyme γ-glutamyltransferase (γ-GGT), but the expression or activity of this enzyme in traditional brewing strains is low, limiting synthesis efficiency. Second, the accumulation of substrates required for γ-GP synthesis, such as glutamate and glutathione, is limited during the fermentation process, further restricting γ-GP production. Third, factors such as the high salt environment and fermentation temperature can inhibit the catalytic activity of γ-GGT and related enzymes, thereby affecting the synthesis reaction of γ-GPs. The variety and content of γ-GPs in traditional soy sauce are generally low, and the "kokumi" flavor is insufficiently expressed, affecting its overall flavor and market competitiveness.

[0006] Amidst the current trend of upgrading condiment consumption and fostering a health-conscious consumer, the depth of flavor and layered texture of soy sauce have become crucial factors influencing product competitiveness. To enhance the "kokumi" flavor of soy sauce, recent research has focused on the introduction and application of enzymes, particularly proteases and glutaminases. For example, patent applications CN201910736869.5, "Method for the Preparation and Application of Kokumi Peptides in Soy Sauce," CN201710511184.1, "Kokumi Peptides, Kokumi-Imparting Agents, Seasonings, and Preparation Methods," CN201510416942.2, "Method for the Preparation and Application of Kokumi Peptides in White Soup Sauce," and CN201410847014.7, "Kokumi Peptides and Their Preparation and Applications," respectively propose catalyzing the synthesis of specific kokumi peptides and improving flavor profile by adding glutaminase, γ-glutamyl transpeptidase, or plant proteases. Although the above methods can improve the production of specific flavor peptides to a certain extent, the following problems are common: the types of substrates used are limited, the types of peptides produced are single, and there is a lack of synergistic optimization with other enzymes. In particular, the potential of auxiliary enzymes (such as xylanases) in substrate structure remodeling and protein release has not been fully explored in existing technologies, resulting in low efficiency of the overall enzymatic system and limited increase in the types and total amount of γ-GPs.

[0007] Therefore, there is an urgent need for a scientific, efficient and controllable fermentation process that can fully utilize the synergistic effect of complex enzyme preparations while maintaining the stability of the high-salt dilute fermentation system, thereby improving the production efficiency of γ-GPs and enhancing the "rich flavor" of soy sauce to meet the needs of modern consumers for high-quality flavor condiments. Summary of the Invention

[0008] The present invention aims to solve the problems of low γ-GPs synthesis efficiency, insufficient variety and content, and insufficient expression of the "rich flavor" of soy sauce in the existing technology. The present invention provides a fermentation process method that introduces a composite enzyme preparation system of glutaminase and xylanase during the soy sauce production process to synergistically catalyze the production of γ-GPs, thereby significantly improving the relative abundance of γ-GPs, thereby preparing a high-quality soy sauce product with a mellow flavor and rich taste.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] A method for improving the kokumi flavor of high-salt, dilute fermented soy sauce is achieved by increasing the γ-GPs content during the fermentation process of the soy sauce. The preparation of the high-salt, dilute fermented soy sauce with kokumi flavor comprises the following preparation steps:

[0011] (1) Raw material pretreatment: soaking, steaming, and cooling soybeans to obtain steamed soybeans;

[0012] (2) Koji making: Mix the steamed soybeans, wheat flour and koji extract evenly, and stir the koji evenly to obtain koji material;

[0013] (3) koji formation: subjecting the koji material described in step (2) to solid-state fermentation to obtain koji; the solid-state fermentation comprises: fermenting at 28-36° C. for 16-24 hours, and then fermenting at 26-32° C. for 16-44 hours; completing the first turning over 12-16 hours after the start of the solid-state fermentation, and completing the second turning over 20-24 hours after the start of the solid-state fermentation, to obtain koji;

[0014] (4) Fermentation: The koji prepared in step (3) is mixed with brine at a ratio of 1:1.8 to 3 (w / v) of koji mass to brine volume, and the fermentation is continued for 60-180 days.

[0015] (5) Sterilization by squeezing: the fermented soy sauce mash in step (4) is squeezed, and the resulting liquid is sterilized at 80-130° C. for 1 second to 30 minutes to obtain a high-salt dilute soy sauce product rich in γ-GPs.

[0016] Furthermore, in step (1), the soybeans are soaked in water for 5-10 hours, steamed for 14-18 minutes, and steamed at a temperature of 116-130°C, preferably soaked for 6 hours and steamed at 125°C for 15 minutes.

[0017] As a preferred implementation method of the preparation method of the present invention, in step (2), the mass ratio of steamed soybeans to wheat flour is 1:1-1:4 (w / w), preferably 1:4.

[0018] As a preferred implementation method of the preparation method of the present invention, in step (2), the amount of koji added is 0.02%-0.08% of the total mass of wheat flour and steamed soybeans, preferably 0.05%.

[0019] As a preferred implementation method of the preparation method of the present invention, in step (4), the mass percentage concentration of the brine is 17-23%, preferably 17%.

[0020] As a preferred embodiment of the preparation method of the present invention, in step (4), the enzyme preparation is one of glutaminase and xylanase, or a mixture of the two, with a mass ratio of glutaminase to xylanase of 1 to 6:1 to 6, and the total addition amount is 0.02% to 0.12% of the mass of the finished koji. As a preferred embodiment of the preparation method of the present invention, in step (4), the fermentation process temperature is 30-38°C, the humidity is 85%-95%, and the koji making time is 60-120 days, preferably the fermentation temperature is 30°C, the humidity is 86%, and the fermentation time is 90 days.

[0021] As a preferred implementation method of the preparation method of the present invention, in the step (5), the sterilization temperature is 80-110°C, the sterilization time is 12-30 minutes, preferably sterilized at 95°C for 15 minutes.

[0022] The present invention provides a preparation method for improving the "rich taste" of high-salt dilute fermented soy sauce and a soy sauce product obtained by the method, wherein the enhancement of the "rich taste" is achieved by significantly increasing the relative abundance of γ-GPs during the fermentation process. Specifically, the present invention adds a specific proportion of a composite enzyme preparation (compounded by glutaminase and xylanase in a mass ratio of 1 to 6: 1 to 6) during the high-salt dilute fermentation process, promotes the decomposition and remodeling of the substrate structure through the synergistic action of the enzyme system, enhances the generation efficiency of γ-GPs, thereby improving its relative abundance in the final product, and significantly enhancing the flavor level and "rich taste" of the soy sauce. Compared with the prior art, the present invention achieves the efficient generation of γ-GPs and the significant improvement of the "rich taste" through the combination of specific enzymes and the optimization of fermentation process parameters without introducing exogenous chemical additives, and has obvious technical progress and practical application value.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention adds a composite enzyme preparation composed of glutaminase and xylanase during the main fermentation stage of soy sauce. Under the premise of maintaining the stability of the high-salt dilute fermentation system, it fully utilizes the synergistic effect of the two and effectively improves the synthesis efficiency of γ-GPs in the fermentation system. This enhances the "rich taste" characteristic of soy sauce and improves the richness, layering and persistence of its flavor. At the same time, the preparation process of the present invention is simple, does not require the use of exogenous chemical additives or special reaction devices, is easy to operate, has low cost and high process stability, is suitable for promotion and application in the soy sauce industrial production, and has broad market prospects and industrial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The present invention provides a flow chart for preparing high-salt dilute fermented soy sauce rich in γ-GPs.

[0026] Figure 2 is the structural formula of γ-GPs.

[0027] Figure 3 This is the identification spectrum of the secondary mass spectrometry fragment ions of γ-Glu-Pro.

[0028] Figure 4 This is the identification spectrum of the secondary mass spectrometry fragment ions of γ-Glu-Ala.

[0029] Figure 5 This is the identification spectrum of the secondary mass spectrometry fragment ions of γ-Glu-Val.

[0030] Figure 6 This is the identification spectrum of γ-Glu-IIe secondary mass spectrometry fragment ions.

[0031] Figure 7 This is the identification spectrum of γ-Glu-Leu secondary mass spectrometry fragment ions.

[0032] Figure 8 This is the identification spectrum of γ-Glu-Glu secondary mass spectrometry fragment ions.

[0033] Figure 9 This is the identification spectrum of the secondary mass spectrometry fragment ions of γ-Glu-Met.

[0034] Figure 10 This is the identification spectrum of γ-Glu-Phe secondary mass spectrometry fragment ions.

[0035] Figure 11 This is the identification spectrum of γ-Glu-Thr secondary mass spectrometry fragment ions.

[0036] Figure 12 This is the identification spectrum of γ-Glu-Trp secondary mass spectrometry fragment ions.

[0037] Figure 13This is the identification spectrum of γ-Glu-Tyr secondary mass spectrometry fragment ions.

[0038] Figure 14 The Kokumi sensory score results of the high-salt dilute fermented soy sauce prepared in Examples and Comparative Examples are shown.

[0039] Figure 15 The relative abundance of γ-GPs in the high-salt dilute fermented soy sauce prepared in Examples and Comparative Examples. DETAILED DESCRIPTION

[0040] To better illustrate the technical principles and innovative features of the present invention, the high-salt, dilute soy sauce for increasing γ-GPs content and its preparation method are further described with reference to several specific examples. However, it should be understood that these examples are merely illustrative of the technical solutions of the present invention and do not constitute a limitation on the scope of protection of the present invention. Any equivalent substitutions or modifications made in accordance with the spirit of the present invention are intended to be encompassed within the scope of protection of the present invention.

[0041] Unless otherwise specified, the raw materials, reagents, process parameters and instruments and equipment involved in the present invention are conventional materials and technical means known to those skilled in the art.

[0042] Comparative Example 1

[0043] A method for preparing high-salt dilute soy sauce without using enzyme preparations, determining the type and content of γ-GPs in the soy sauce, and performing sensory evaluation on the soy sauce, comprising the following steps:

[0044] (1) Raw material pretreatment: Select soybeans with full grains, soak them in clean water for 6 hours, and after sufficient soaking, steam them at 125°C for 15 minutes. Filter the residue to obtain the steamed soybeans.

[0045] (2) Koji making: the steamed soybeans obtained in step (1) are cooled to 37° C., and wheat flour and koji spirit are added and mixed evenly. The mass ratio of wheat flour to steamed soybeans is 1:4 based on dry weight. The koji spirit used is Aspergillus oryzae, and the inoculation amount of the koji spirit is 0.05% of the amount of the mixture of wheat flour and steamed soybeans based on dry weight.

[0046] (3) Finishing the koji: The koji obtained in step (2) is subjected to solid-state fermentation, and is fermented at 30°C for 16 hours, and then cultured at 28°C for 26 hours. The koji is turned over once at 16 hours, and the culture is continued for 5 hours before being turned over again to obtain finished koji.

[0047] (4) Mash fermentation: The koji prepared in step (3) was mixed with 17% saline in a volume ratio of 1:2.5 (w / v), without adding any enzyme preparation, and stirred evenly to form a mash, which was then naturally fermented in an environment at 30±1°C and a relative humidity of 86±1% for 90 days.

[0048] (5) Sterilization by squeezing: After fermentation for 90 days, the mash of step (4) is squeezed and filtered to obtain raw soy sauce; then sterilized at 95°C for 15 minutes, cooled and filtered to obtain finished soy sauce.

[0049] (6) γ-GPs determination: 1 mL of soy sauce sample was diluted 10-fold, 3 volumes of acetonitrile were added, and the mixture was centrifuged at 8000 rpm and 4°C for 15 min. The supernatant was taken and diluted 4-fold again, and the type and content of γ-GPs were analyzed by UPLC-MS / MS system.

[0050] (7) Sensory evaluation: A panel of six well-trained evaluators (three men and three women) used quantitative sensory descriptive analysis (QDA) to score the "rich flavor" of fermented soy sauce (including the comprehensive perception of the three attributes of richness, persistence, and flavor harmony) using a 10-point scale (0 for no perception and 10 for extremely strong perception). Each evaluator blindly tested the samples in turn and independently scored them. The average score was used as the basis for the sensory score of the samples. During all evaluation processes, the samples were randomly coded. After evaluating each sample, the evaluators were required to thoroughly clean their mouths between samples before evaluating the next sample to ensure the objectivity and accuracy of the evaluation results.

[0051] Comparative Example 2

[0052] A method for preparing high-salt dilute soy sauce using glutaminase fermentation, determining the type and content of γ-GPs in the soy sauce, and performing sensory evaluation on the soy sauce comprises the following steps:

[0053] (1) Raw material pretreatment: same as Comparative Example 1.

[0054] (2) Koji making: same as Comparative Example 1.

[0055] (3) Finished song: Same as comparative example 1.

[0056] (4) Mash fermentation and enzyme addition: The koji described in step (3) was mixed with 17% brine in a volume ratio of 1:2.5 (w / v), glutaminase was added (the addition amount was 0.05% of the mass of the koji), and the mash was formed after stirring evenly. The mash was naturally fermented in an environment of 30±1°C and relative humidity of 86±1% for 90 days.

[0057] (5) Sterilization by squeezing: same as in Comparative Example 1.

[0058] (6) γ-GPs determination: same as Comparative Example 1.

[0059] (7) Sensory evaluation: Same as Comparative Example 1.

[0060] Comparative Example 3

[0061] A method for preparing high-salt dilute soy sauce by fermenting with xylanase, determining the type and content of γ-GPs in the soy sauce, and performing sensory evaluation on the soy sauce comprises the following steps:

[0062] (1) Raw material pretreatment: same as Comparative Example 1.

[0063] (2) Koji making: same as Comparative Example 1.

[0064] (3) Finished song: Same as comparative example 1.

[0065] (4) Mash fermentation and enzyme addition: the koji was mixed with 17% brine at a volume ratio of 1:2.5 (w / v), xylanase was added (the addition amount was 0.05% of the mass of the koji), and the mixture was stirred evenly to form a mash, which was naturally fermented in an environment of 30±1°C and relative humidity of 86±1% for 90 days.

[0066] (5) Sterilization by squeezing: same as in Comparative Example 1.

[0067] (6) γ-GPs determination: same as Comparative Example 1.

[0068] (7) Sensory evaluation: Same as Comparative Example 1.

[0069] Comparative Example 4

[0070] A method for preparing high-salt dilute soy sauce by fermentation using a composite enzyme preparation, determining the type and content of γ-GPs in the soy sauce, and performing sensory evaluation on the soy sauce, comprising the following steps:

[0071] (1) Raw material pretreatment: same as Comparative Example 1.

[0072] (2) Koji making: same as Comparative Example 1.

[0073] (3) Finished song: Same as comparative example 1.

[0074] (4) Fermentation of mash and addition of enzyme preparation: the finished koji was mixed with 17% brine in a volume ratio of 1:2.5 (w / v), and a composite enzyme preparation was added (the mass ratio of glutaminase to xylanase was 8:1, and the total addition amount was 0.05% of the mass of the finished koji). After stirring evenly, the mash was formed and naturally fermented in an environment of 30±1°C and relative humidity of 86±1% for 90 days.

[0075] (5) Sterilization by squeezing: Same as in Example 1.

[0076] (6) γ-GPs determination: same as Example 1.

[0077] (7) Sensory evaluation: Same as Example 1.

[0078] Comparative Example 5

[0079] A method for preparing high-salt dilute soy sauce by fermentation using a composite enzyme preparation, determining the type and content of γ-GPs in the soy sauce, and performing sensory evaluation on the soy sauce, comprising the following steps:

[0080] (1) Raw material pretreatment: same as Comparative Example 1.

[0081] (2) Koji making: same as Comparative Example 1.

[0082] (3) Finished song: Same as comparative example 1.

[0083] (4) Fermentation of mash and addition of enzyme preparation: the finished koji was mixed with 17% brine in a volume ratio of 1:2.5 (w / v), and a composite enzyme preparation was added (the mass ratio of glutaminase to xylanase was 1:8, and the total addition amount was 0.05% of the mass of the finished koji). After stirring evenly, the mash was formed and naturally fermented in an environment of 30±1°C and relative humidity of 86±1% for 90 days.

[0084] (5) Sterilization by squeezing: Same as in Example 1.

[0085] (6) γ-GPs determination: same as Example 1.

[0086] (7) Sensory evaluation: Same as Example 1.

[0087] Comparative Example 6

[0088] A method for preparing high-salt dilute soy sauce by fermentation using a composite enzyme preparation, determining the type and content of γ-GPs in the soy sauce, and performing sensory evaluation on the soy sauce, comprising the following steps:

[0089] (1) Raw material pretreatment: same as Comparative Example 1.

[0090] (2) Koji making: same as Comparative Example 1.

[0091] (3) Finished song: Same as comparative example 1.

[0092] (4) Fermentation of mash and addition of enzyme preparation: the finished koji was mixed with 17% brine in a volume ratio of 1:2.5 (w / v), and a composite enzyme preparation (glutaminase and protease, with a mass ratio of 1:2.5, and a total addition amount of 0.05% of the mass of the finished koji) was added, and the mash was formed after stirring evenly, and naturally fermented in an environment of 30±1°C and a relative humidity of 86±1% for 90 days.

[0093] (5) Sterilization by squeezing: Same as in Example 1.

[0094] (6) γ-GPs determination: same as Example 1.

[0095] (7) Sensory evaluation: Same as Example 1.

[0096] Comparative Example 7

[0097] A method for preparing high-salt dilute soy sauce by fermentation using a composite enzyme preparation, determining the type and content of γ-GPs in the soy sauce, and performing sensory evaluation on the soy sauce, comprising the following steps:

[0098] (1) Raw material pretreatment: same as Comparative Example 1.

[0099] (2) Koji making: same as Comparative Example 1.

[0100] (3) Finished song: Same as comparative example 1.

[0101] (4) Fermentation of mash and addition of enzyme preparation: the finished koji was mixed with 17% brine in a volume ratio of 1:2.5 (w / v), and a composite enzyme preparation (protease and xylanase, with a mass ratio of 1:2.5, and a total addition amount of 0.05% of the mass of the finished koji) was added, and the mash was formed after stirring evenly, and naturally fermented in an environment of 30±1°C and a relative humidity of 86±1% for 90 days.

[0102] (5) Sterilization by squeezing: Same as in Example 1.

[0103] (6) γ-GPs determination: same as Example 1.

[0104] (7) Sensory evaluation: Same as Example 1.

[0105] Example 1

[0106] A method for preparing high-salt dilute soy sauce by fermentation using a composite enzyme preparation, determining the type and content of γ-GPs in the soy sauce, and performing sensory evaluation on the soy sauce, comprising the following steps:

[0107] (1) Raw material pretreatment: Select soybeans with full grains, soak them in clean water for 6 hours, and after sufficient soaking, steam them at 125°C for 15 minutes. Filter the residue to obtain the steamed soybeans.

[0108] (2) making koji: cooling the steamed soybeans obtained in step (1) to 37° C., adding wheat flour and koji extract and mixing uniformly, wherein the mass ratio of the steamed soybeans to the wheat flour is 1:4 based on dry weight; the koji extract used is Aspergillus oryzae, and the inoculation amount of the koji extract is controlled to be 0.05% of the total weight of the steamed soybeans and wheat flour based on dry weight, and the three are mixed uniformly to obtain Daqu;

[0109] (3) Finishing the koji: The koji obtained in step (2) is subjected to solid-state fermentation, first at 30°C for 16 hours, and then at 28°C for 26 hours. The koji is turned over once at 16 hours, and after a further 5 hours of incubation, the koji is turned over again to obtain finished koji.

[0110] (4) Fermentation of mash and addition of enzyme preparation: the finished koji was mixed with 17% brine in a volume ratio of 1:2.5 (w / v), and a composite enzyme preparation was added (the mass ratio of glutaminase to xylanase was 1:2.5, and the total addition amount was 0.05% of the mass of the finished koji). After stirring evenly, the mash was formed and naturally fermented in an environment of 30±1°C and relative humidity of 86±1% for 90 days.

[0111] (5) Sterilization by squeezing: After fermentation in step (3) for 90 days, the mash is squeezed and filtered to obtain raw soy sauce; then sterilized at 95°C for 15 minutes, cooled and filtered to obtain finished soy sauce.

[0112] (6) γ-GPs determination: 1 mL of soy sauce sample was diluted 10-fold, 3 volumes of acetonitrile were added, and the mixture was centrifuged at 8000 rpm and 4°C for 15 min. The supernatant was taken and diluted 4-fold again, and the type and content of γ-GPs were analyzed by UPLC-MS / MS system.

[0113] (7) Sensory evaluation: A panel of six well-trained evaluators (three men and three women) used quantitative sensory descriptive analysis (QDA) to score the "rich flavor" of fermented soy sauce (including the comprehensive perception of the three attributes of richness, persistence, and flavor harmony) using a 10-point scale (0 for no perception and 10 for extremely strong perception). Each evaluator blindly tested the samples in turn and independently scored them. The average score was used as the basis for the sensory score of the samples. During all evaluation processes, the samples were randomly coded. After evaluating each sample, the evaluators were required to thoroughly clean their mouths between samples before evaluating the next sample to ensure the objectivity and accuracy of the evaluation results.

[0114] The process flow of Example 1 is basically the same as that of Comparative Examples 1-4, except for the addition of the enzyme preparation in step (4). Other conditions are the same to obtain a soy sauce.

[0115] Result Analysis

[0116] Compared with Comparative Example 1, after adding 0.05% complex enzyme preparation, the contents of γ-Glu-Pro, γ-Glu-Val, γ-Glu-Ile, γ-Glu-Leu, γ-Glu-Glu, γ-Glu-Met, γ-Glu-Phe, γ-Glu-Trp and γ-Glu-Thr in the fermented soy sauce in Example 1 increased by 40.46%, 28.83%, 109.90%, 66.25%, 113.66%, 22.17%, 45.75%, 20.46% and 172.43%, respectively, and the overall γ-GPs content increased by 65.54%. Compared with Comparative Examples 2 and 3, the γ-GPs content of Example 1 increased by 23.34% and 22.25%, respectively. In addition, by Figure 14 As can be seen, Example 1 outperformed Comparative Examples 1, 2, and 3 in the sensory score for "kokumi" by 1.51, 1.32, and 1.18 times, respectively. This suggests that adding 0.05% of the complex enzyme preparation (glutaminase to xylanase ratio of 1:2.5) significantly increases the types and content of γ-GPs in soy sauce, significantly enhancing the "kokumi" characteristic.

[0117] Example 2

[0118] A method for preparing high-salt dilute soy sauce by fermentation using a composite enzyme preparation, determining the type and content of γ-GPs in the soy sauce, and performing sensory evaluation on the soy sauce, comprising the following steps:

[0119] (1) Raw material pretreatment: same as in Example 1.

[0120] (2) Koji making: same as in Example 1.

[0121] (3) Completion of the song: Same as in Example 1.

[0122] (4) Fermentation of mash and addition of enzyme preparation: the finished koji was mixed with 17% brine in a volume ratio of 1:2.5 (w / v), and a composite enzyme preparation was added (the mass ratio of glutaminase to xylanase was 3:1, and the total addition amount was 0.08% of the mass of the finished koji). After stirring evenly, the mash was formed and naturally fermented in an environment of 30±1°C and relative humidity of 86±1% for 90 days.

[0123] (5) Sterilization by squeezing: Same as in Example 1.

[0124] (6) γ-GPs determination: same as Example 1.

[0125] (7) Sensory evaluation: Same as Example 1.

[0126] Result Analysis

[0127] Compared with Comparative Example 1, after adding 0.08% complex enzyme preparation, the contents of γ-Glu-Pro, γ-Glu-Val, γ-Glu-Ile, γ-Glu-Leu, γ-Glu-Glu, γ-Glu-Met, γ-Glu-Phe, γ-Glu-Trp and γ-Glu-Thr in fermented soy sauce increased by 54.60%, 28.83%, 70.11%, 64.14%, 112.63%, 23.01%, 37.14%, 11.13% and 182.44%, respectively, and the overall γ-GPs content increased by 55.08%. Compared with Comparative Examples 2 and 3, the γ-GPs content of Example 2 increased by 15.54% and 14.77%, respectively. In addition, by Figure 14 As can be seen, Example 2 outperformed Comparative Examples 1, 2, and 3 in the sensory score for "kokumi" by 1.43, 1.25, and 1.12 times, respectively. This suggests that adding 0.08% of the complex enzyme preparation (glutaminase to xylanase ratio of 3:1) significantly increased the types and content of γ-GPs in soy sauce, significantly enhancing the "kokumi" characteristic.

[0128] Example 3

[0129] A method for preparing high-salt dilute soy sauce by fermentation using a composite enzyme preparation, determining the type and content of γ-GPs in the soy sauce, and performing sensory evaluation on the soy sauce, comprising the following steps:

[0130] (1) Raw material pretreatment: same as in Example 1.

[0131] (2) Koji making: same as in Example 1.

[0132] (3) Completion of the song: Same as in Example 1.

[0133] (4) Fermentation of mash and addition of enzyme preparation: the finished koji was mixed with 17% brine in a volume ratio of 1:2.5 (w / v), and a composite enzyme preparation was added (the mass ratio of glutaminase to xylanase was 4:3, and the total addition amount was 0.06% of the mass of the finished koji). After stirring evenly, the mash was formed and naturally fermented in an environment of 30-32°C and a relative humidity of 86±1% for 90 days.

[0134] (5) Sterilization by squeezing: Same as in Example 1.

[0135] (6) γ-GPs determination: same as Example 1.

[0136] (7) Sensory evaluation: Same as Example 1.

[0137] Result Analysis

[0138] Compared with Comparative Example 1, after adding 0.06% complex enzyme preparation, the contents of γ-Glu-Pro, γ-Glu-Val, γ-Glu-Ile, γ-Glu-Leu, γ-Glu-Glu, γ-Glu-Met, γ-Glu-Phe, γ-Glu-Trp and γ-Glu-Thr in the fermented soy sauce increased by 56.92%, 11.11%, 65.89%, 66.72%, 112.27%, 31.60%, 44.76%, 29.89% and 213.98%, respectively, and the overall γ-GPs content increased by 57.70%. Compared with Comparative Examples 2 and 3, the γ-GPs content of Example 3 increased by 17.49% and 16.71%, respectively. In addition, by Figure 14 As can be seen, Example 3 outperformed Comparative Examples 1, 2, and 3 in the sensory score for "kokumi" by 1.47, 1.28, and 1.15 times, respectively. This suggests that adding 0.06% of the complex enzyme preparation (glutaminase to xylanase ratio of 4:3) significantly increased the types and content of γ-GPs in soy sauce, significantly enhancing the "kokumi" characteristic.

[0139] Example 4

[0140] A method for preparing high-salt dilute soy sauce by fermentation using a composite enzyme preparation, determining the type and content of γ-GPs in the soy sauce, and performing sensory evaluation on the soy sauce, comprising the following steps:

[0141] (1) Raw material pretreatment: same as in Example 1.

[0142] (2) Koji making: same as in Example 1.

[0143] (3) Completion of the song: Same as in Example 1.

[0144] (4) Fermentation of mash and addition of enzyme preparation: the finished koji was mixed with 17% brine in a volume ratio of 1:2.5 (w / v), and a composite enzyme preparation was added (the mass ratio of glutaminase to xylanase was 1:6, and the total addition amount was 0.1% of the mass of the finished koji). After stirring evenly, the mash was formed and naturally fermented in an environment of 30±1°C and relative humidity of 86±1% for 90 days.

[0145] (5) Sterilization by squeezing: Same as in Example 1.

[0146] (6) γ-GPs determination: same as Example 1.

[0147] (7) Sensory evaluation: Same as Example 1.

[0148] Result Analysis

[0149] Compared with Comparative Example 1, after adding 0.1% complex enzyme preparation, the contents of γ-Glu-Pro, γ-Glu-Val, γ-Glu-Ile, γ-Glu-Leu, γ-Glu-Glu, γ-Glu-Met, γ-Glu-Phe, γ-Glu-Trp and γ-Glu-Thr in fermented soy sauce in Example 4 increased by 547.49%, 19.76%, 71.94%, 64.14%, 105.20%, 36.30%, 52.24%, 40.69% and 179.16%, respectively, and the overall γ-GPs content increased by 59.31%. Compared with Comparative Examples 2 and 3, the γ-GPs content of Example 4 increased by 17.49% and 16.71%, respectively. In addition, by Figure 14 As can be seen, Example 4 outperformed Comparative Examples 1, 2, and 3 in the sensory score for "kokumi" by 1.49, 1.30, and 1.17 times, respectively. This suggests that adding 0.1% of the complex enzyme preparation (glutaminase to xylanase ratio of 1:6) significantly increases the types and content of γ-GPs in soy sauce, significantly enhancing the "kokumi" characteristic.

[0150] Figure 1 The present invention provides a flow chart for preparing high-salt dilute fermented soy sauce rich in γ-GPs.

[0151] Figure 2 This is the structural formula of γ-GPs.

[0152] Figures 3 to 13 The secondary mass spectrometry fragment ion identification patterns of γ-Glu-Pro, γ-Glu-Ala, γ-Glu-Val, γ-Glu-Iie, γ-Glu-Leu, γ-Glu-Glu, γ-Glu-Met, γ-Glu-Phe, γ-Glu-Thr, γ-Glu-Trp, and γ-Glu-Tyr respectively.

[0153] Combine Figure 14 and Figure 15 The results showed that Example 1 achieved the best effect of increasing the total amount of γ-GPs under the conditions of a composite enzyme ratio (glutaminase: xylanase = 1:2.5) and a total addition amount of 0.05%, which was better than Examples 2, 3, 4 and each comparative example. When the ratio of xylanase to glutaminase was moderate, the release of the second substance was appropriate and rich in variety, and glutaminase was able to efficiently catalyze the synthesis of γ-GPs, synergistically promote the accumulation of γ-GPs, and effectively avoid substrate competition and side reactions caused by excessive enzyme dosage. On the contrary, when the ratio of xylanase was too high, excessive substrate release easily led to limited glutaminase catalysis, resulting in suppressed growth of the total amount of γ-GPs.

[0154] Figure 14The Kokumi sensory evaluation results of the high-salt, dilute fermented soy sauces prepared in Examples 1-4 and Comparative Examples 1-7 are shown. The sensory evaluation of "rich flavor" was performed using QDA, with six trained sensory evaluators (3 men and 3 women) giving a comprehensive score based on the "richness, persistence, and flavor coordination" of the soy sauce in the mouth. The score range was 0-10 (0 means no sensation at all, 10 means a very strong, rich, and long-lasting rich flavor). Figure 14 It can be seen that the kokumi scores of Examples 1-4 are significantly higher than those of Comparative Examples 1-5, indicating that the fermented soy sauce treated with the composite enzyme preparation (a combination of glutaminase and xylanase) can significantly enhance the "kokumi" characteristic, and the effect is significantly better than the control group to which the enzyme preparation is added alone or without the enzyme preparation. In particular, Example 1 performs best in both γ-GPs content and sensory kokumi score, reflecting the representative technical advantages of the optimal enzyme ratio and dosage combination. Example 1 uses glutaminase and xylanase in a mass ratio of 1:2.5, with a total addition amount of 0.05% of the mass of the finished koji. While ensuring an adequate supply of substrate, it promotes the efficient synthesis of γ-GPs. Glutaminase promotes the formation of γ-glutamyl bonds, and xylanase decomposes macromolecular substrates to release small peptides. The synergistic effect of the two optimizes the fermentation environment. At the same time, the appropriate addition avoids substrate competition and side reactions caused by excessive enzyme dosage, thereby achieving a significant increase in γ-GPs content and the optimal expression of the "kokumi" characteristic. Therefore, the fermentation process adopted in Example 1 demonstrates the best technical effect of the present invention in terms of improving the flavor mellowness, richness and persistence of high-salt dilute soy sauce.

[0155] Although both Comparative Examples 4 and 5 formally used a combination of glutaminase and xylanase, due to the serious imbalance in the ratio of the enzyme species (Comparative Example 4 is 8:1; Comparative Example 5 is 1:8), an effective synergistic effect was not achieved, resulting in a significantly lower total relative abundance of γ-GPs than that of the Example group, and the soy sauce produced had a weaker "rich taste" in terms of sensory perception and an uncoordinated flavor structure. Specifically, the xylanase ratio in Comparative Example 4 was too low, making it difficult to fully release substrate resources, limiting the substrate accessibility for γ-GPs synthesis; and the insufficient glutaminase ratio in Comparative Example 5 hindered the key γ-GPs synthesis reaction. Even if the substrate was fully released, it was difficult to achieve effective conversion to γ-GPs. In contrast, Examples 1-4, with the optimized combination of the complex enzyme ratio (1-6:1-6) and the addition amount (0.05%-0.10%), not only significantly improved the relative abundance of γ-GPs, but also exhibited a more prominent "rich taste" feature in the sensory evaluation. The above structure fully verifies the criticality of the ratio and addition amount of the complex enzyme to the technical effect, and further illustrates that the parameter range defined by the present invention has significant technical progress and rationality. In addition, Comparative Examples 6 and 7 further demonstrate the species specificity of the complex enzyme. Although both use a combination of glutaminase: protease and xylanase: protease, and adopt the same enzyme ratio as Example 1 (1:2.5), the protease cannot effectively release a specific substrate or promote the specific synthesis path of γ-GPs, resulting in a total amount of γ-GPs of 15,440,630.47 and 13,038,965.21, respectively, and the sensory scores also drop to 6.55 and 6.15, respectively, which are significantly lower than the embodiment group. It can be seen from this that even if the composite ratio is reasonable, if a specific synergistic enzyme combination (glutaminase + xylanase) is not adopted, the ideal γ-GPs generation and flavor enhancement effect cannot be obtained.

[0156] The above embodiments are only preferred embodiments of the present invention and are only used to illustrate the technical solutions of the present invention, rather than to limit the present invention. Any changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit and essence of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for improving the rich taste of high-salt dilute fermented soy sauce, characterized in that: The steps include: (1) Raw material pretreatment: soaking, steaming and cooling soybeans to obtain steamed soybeans; (2) Koji making: Mix the steamed soybeans, wheat flour and koji extract evenly, and stir the koji evenly to obtain koji material; (3) koji formation: subjecting the koji material described in step (2) to solid-state fermentation to obtain koji; (4) Fermentation: mixing the koji prepared in step (3) with brine in a ratio of koji mass to brine volume; adding an enzyme preparation to the mixture for fermentation; (5) Squeezing and sterilizing: The fermented soy sauce mash in step (4) is squeezed and sterilized to obtain a high-salt dilute soy sauce product rich in γ-glutamyl peptides.

2. The method according to claim 1, characterized in that In step (1), the soybeans are soaked in water for 5-10 hours, steamed for 14-18 minutes, and at a temperature of 116-130°C.

3. The method according to claim 1, characterized in that The mass ratio of the steamed soybeans to the wheat flour in step (2) is 1:1-1:4 (w / w); the inoculation amount of the koji in step (2) is 0.02%-0.08% of the mass of the mixture of wheat flour and steamed soybeans.

4. The method according to claim 1, wherein The solid-state fermentation in step (3) is carried out at 28-36° C. for 16-24 hours, and then at 26-32° C. for 16-44 hours; the first stirring is carried out 12-16 hours after the start of fermentation, and the second stirring is carried out 20-24 hours after the start of fermentation, and the finished koji is obtained after the fermentation is completed.

5. The method according to claim 1, characterized in that The mass percentage concentration of the brine in step (4) is 17%-23%; the volume ratio of the finished koji mass to the brine is 1:1.8~3.0 (w / v).

6. The method according to claim 1, characterized in that The enzyme preparation in step (4) is glutaminase and xylanase, which are mixed and added in a mass ratio of 1-6:1-6, and the total addition amount is 0.02%-0.12% of the mass of the finished koji.

7. The method according to claim 1, characterized in that In step (4), the temperature of the fermentation process is 30-38° C., the humidity is 85%-95%, and the fermentation days are 60-180 days.

8. The method according to claim 1, characterized in that In step (5), the sterilization temperature is 80-130°C and the time is 1s-30min.

9. A high-salt dilute soy sauce product with rich flavor prepared by the method according to any one of claims 1 to 8.

10. The high-salt, dilute soy sauce product with rich flavor according to claim 9, characterized in that The product enhances the rich flavor of soy sauce by increasing the relative abundance of γ-glutamyl peptides in soy sauce; the γ-glutamyl peptides include γ-Glu-Pro, γ-Glu-Ala, γ-Glu-Val, γ-Glu-Ile, γ-Glu-Leu, γ-Glu-Glu, γ-Glu-Met, γ-Glu-Phe, γ-Glu-Thr, γ-Glu-Trp, and γ-Glu-Tyr.

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