Yin sauce and preparation method thereof

Abstract

The invention provides a yin sauce and a preparation method thereof, and the method is characterized in that aspergillus oryzae and aspergillus niger are respectively subjected to independent starter propagation with soybeans, the two obtained pure starter strains are mixed, and then the mixture, saccharomycopsis fibuligera and auxiliary materials are synchronously subjected to integrated fermentation. According to the process, the problem that aspergillus niger produces acid to inhibit the activity of aspergillus oryzae neutral protease in mixed koji making is solved through physical separation culture, and the total activity of protease of the sauce koji is improved to the maximum extent; and the saccharomycopsis fibuligera with efficient ester synthesis capability is inoculated at the initial stage of fermentation, so that real-time collaboration of mould enzymolysis and yeast flavor metabolism is realized. According to the method, the amino acid nitrogen content and the total ester content of the final product are remarkably improved, so that the product has mellow delicate flavor and unique and rich ester flavor, and the technical problems of single flavor and insufficient synergy in the traditional process are solved.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a type of fermented bean paste and its preparation method. Background Technology

[0002] Sauces (such as soy sauce, bean paste, and fermented bean paste) are traditional fermented condiments with a history of thousands of years in China and even East Asia. They have unique flavors and are rich in nutrients, occupying a central position in food culture. The production of traditional sauces is essentially a complex relay fermentation process by a microbial community. The core step is the "koji-making" stage, which uses mold as the main agent to hydrolyze macromolecules such as proteins and starches in the raw materials into flavor precursors such as amino acids, peptides, and sugars. Subsequently, in the "sauce mash fermentation" stage, microorganisms such as yeast and lactic acid bacteria convert these precursors into alcohols, esters, acids, and other key compounds that constitute the final sauce aroma.

[0003] With the development of modern microbiology and food engineering, the traditional "wild koji-making" method, which relied on inoculation by microorganisms from the natural environment, has gradually evolved into modern fermentation technology using pure culture inoculation. This has greatly improved production efficiency and product hygiene and safety. In pure culture fermentation technology, *Aspergillus oryzae*, due to its excellent protease and amylase systems, has become the undisputed core strain in soy sauce production. Its secreted neutral protease can efficiently decompose soybean protein to produce flavor-enhancing amino acids, which are the main source of the umami flavor of soy sauce (indicated by amino acid nitrogen). Therefore, most modern brewing processes use *Aspergillus oryzae* as a single or dominant koji-making strain.

[0004] To further optimize product flavor and raw material utilization, the industry has begun exploring multi-strain synergistic fermentation pathways. Among these, *Aspergillus niger*, an important food industry microorganism, has attracted attention due to its ability to secrete abundant acidic proteases, saccharifying enzymes, pectinases, and cellulases. Studies have shown that introducing *Aspergillus niger* into sauce fermentation can compensate for the insufficient enzyme activity of *Aspergillus oryzae* in acidic environments, allowing for more complete decomposition of plant cell walls and complex carbohydrates, thereby improving the utilization rate of total nitrogen in raw materials and the richness of flavor compounds. Existing technologies include numerous patents and publications disclosing schemes combining *Aspergillus oryzae* and *Aspergillus niger*, as well as processes that combine these two molds with yeasts, lactic acid bacteria, and other microorganisms for fermentation. Although existing sauce fermentation technologies have evolved from single-strain to multi-strain combinations, the mechanical combination methods, limited microbial functions, and fragmented process stages result in core defects such as unmaximized enzyme activity potential, mismatch between flavor precursors and aroma metabolism, and insufficient generation of characteristic aroma substances. Therefore, there is an urgent need in this field for a novel strain combination strategy and fermentation process that can fundamentally break down the metabolic barriers between fermentation strains. Through ingenious process design, it can ensure efficient protein hydrolysis and improve umami indicators while revolutionarily enhancing the ester aroma and flavor complexity of the product, providing a core technology solution for the development of high-end sauces and innovative flavor sauces. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a fermented soybean paste and its preparation method. First, *Aspergillus oryzae* and *Aspergillus niger* are fermented separately to prepare a starter culture. Then, the starter culture is mixed with *Saccharomyces cerevisiae* and auxiliary materials for fermentation to prepare soybean paste. Separate starter culture preparation solves the problem of enzyme activity inhibition during mixed fermentation, while the synergistic effect of the molds improves the flavor and quality of the soybean paste. One objective of this invention is to provide a method for enhancing the umami and flavor of fermented soybean paste. Another objective is to provide a fermented soybean paste with a rich umami flavor, a fragrant aroma, and excellent quality.

[0006] To achieve the above objectives, the present invention employs the following technical solution: On one hand, the present invention provides a method for preparing Yinjiang (a type of fermented bean curd), comprising the following steps: S1: Soybeans are soaked, rinsed, steamed, and cooled before being mixed with rice flour or wheat flour. S2: Divide the soybeans after mixing with flour into two portions, inoculate one portion with Aspergillus oryzae and the other portion with Aspergillus niger, and ferment them separately to prepare koji. S3: The koji is mixed with auxiliary materials and yeast and then fermented to obtain Yinjiang sauce.

[0007] While traditional fermentation processes have evolved from single-strain fermentation to multi-strain compound applications in pursuit of flavor and efficiency, significant technical bottlenecks remain in achieving truly efficient synergy among strains, avoiding mutual inhibition, and specifically shaping unique flavor compounds. The ingenuity of this invention lies in constructing a novel microbial co-fermentation system by separately preparing koji (fermentation starter culture) with *Aspergillus oryzae* and *Aspergillus niger*, and then inoculating the koji with a specific yeast and raw materials for fermentation. This system not only breaks through the limitations of traditional models in principle but also achieves synergistic enhancement and breakthrough improvement in key quality indicators, especially the content of amino acid nitrogen and characteristic esters.

[0008] In existing technologies, combining *Aspergillus oryzae* and *Aspergillus niger* to enrich the enzyme system has become an improved approach. However, common mixed koji-making methods have fundamental flaws. *Aspergillus oryzae* and *Aspergillus niger* have essential differences in their growth and metabolic characteristics, the most critical being the difference in their optimal pH environments for enzyme production. *Aspergillus oryzae* synthesizes its neutral and alkaline proteases within a pH range of 6.0 to 8.0; these proteases are key enzymes for efficiently degrading soybean globulins and generating umami amino acids. *Aspergillus niger*, on the other hand, rapidly metabolizes and produces acid during its growth, causing the pH of the fermentation substrate environment to drop rapidly to the optimal range (pH 3.0-5.5) for its own enzyme production (such as acidic proteases). When the two bacteria are cultured together, the acid-producing activity of *Aspergillus niger* causes the environmental pH to deviate prematurely from the optimal range for the synthesis and activity of neutral proteases in *Aspergillus oryzae*, thus severely inhibiting their activity. Therefore, this invention first addresses this problem by proposing a solution for separate koji-making. Through physical separation, *Aspergillus oryzae* was able to synthesize neutral and alkaline proteases under stable conditions; simultaneously, *Aspergillus niger* independently and efficiently synthesized its unique acidic protease and various carbohydrate hydrolases. Finally, the "pure culture" of both, matured under their respective optimal conditions, was mixed to obtain a composite enzyme system with intact enzyme activity and complementary functions. This step fundamentally eliminated interspecific inhibition in mixed culture, ensuring that the protease system, as the basis for flavor precursor production, reached its maximum potential. This laid a solid enzymatic foundation for the efficient accumulation of amino acid nitrogen in subsequent fermentation.

[0009] To enrich the flavor of fermented soybean paste, traditional methods often involve co-fermenting Aspergillus oryzae and Aspergillus niger with other microorganisms such as yeast and lactic acid bacteria. However, the results are often unsatisfactory. The key technical flaw lies in the simultaneous fermentation of multiple microorganisms. In the initial stage of fermentation, with ample nutrients, all three microorganisms grow rapidly. Aspergillus niger rapidly produces acid, causing the pH value to drop prematurely, severely inhibiting the synthesis and accumulation of neutral protease, a crucial umami enzyme system, in Aspergillus oryzae. The early proliferation of yeast or lactic acid bacteria fiercely competes with the two molds for carbon, nitrogen, and oxygen, preventing any one from forming a dominant population or reaching optimal metabolic state. Therefore, this invention abandons the traditional multi-strain co-fermentation model and innovatively adopts a method of preparing the koji first and then inoculating yeast. That is, after separately preparing and mixing the koji to obtain the composite koji, a capsule-coated yeast is introduced during the fermentation of the koji and raw materials for integrated fermentation. In this mode, the enzymatic hydrolysis of mold and the metabolic activity of yeast occur simultaneously. Yeast can rapidly utilize amino acids and other substances produced by the hydrolysis of various proteases, greatly improving the utilization efficiency of precursor substances and providing optimal conditions for the rapid initiation and efficient operation of yeast metabolism. More importantly, it establishes a dynamic microecological balance: the continuous consumption of amino acids by yeast effectively reduces the concentration of free amino acids in the fermentation system, thereby mitigating the feedback inhibition of protease activity by the products. This allows the proteases from the koji to maintain high activity for a longer period, further promoting the deep hydrolysis of proteins, thus synergistically promoting the fermentation effect and enhancing the umami and flavor of the fermented soybean paste.

[0010] Furthermore, the yeast is *Saccharomyces cerevisiae*.

[0011] The choice of yeast strain is another decisive factor in determining whether mixed fermentation can achieve a breakthrough in flavor enhancement. Traditional brewing methods, such as *Saccharomyces rouxii*, are primarily metabolically suited to tolerate hyperosmolar environments and alcoholic fermentation, but have relatively limited ability to synthesize flavor compounds like esters. Through screening and comparison, this invention found that *Saccharomyces cerevisiae* has a significant advantage over some commonly used brewing yeasts in synthesizing esters and promoting the synthesis of umami substances. The core advantage of *Saccharomyces cerevisiae* lies in its exceptionally active ester synthesis enzyme system. This enzyme efficiently catalyzes the esterification reaction between acetyl-CoA and alcohols (such as isoamyl alcohol and phenylethanol), thereby generating large amounts of short-chain fatty acid esters such as isoamyl acetate and phenylethyl acetate, increasing the total ester content in the sauce. These esters possess distinct fruity and floral aromas, enriching the flavor of the sauce. Meanwhile, *Saccharomyces cerevisiae* also significantly outperforms other yeasts in promoting amino acid nitrogen synthesis. This is because *Saccharomyces cerevisiae* possesses extremely active amino acid assimilation and metabolic pathways, enabling it to rapidly absorb and consume free amino acids accumulated from protein hydrolysis by proteases. It efficiently overcomes product feedback inhibition, allowing proteases from *Aspergillus oryzae* and *Aspergillus niger* to work more persistently and efficiently, driving the protein hydrolysis reaction forward and ultimately accumulating more amino acid nitrogen. In conclusion, the introduction of *Saccharomyces cerevisiae* not only significantly enhances the umami flavor of fermented bean curd but also enriches its flavor profile, resulting in a more complex taste profile and satisfying the needs of a wider range of consumers.

[0012] Further, in step S1, the soybeans are 1 to 100 parts by weight, and the rice flour or wheat flour is 1 to 50 parts by weight.

[0013] Further, in step S2, the inoculation amount of Aspergillus oryzae and Aspergillus niger is 0.01~1%, the fermentation temperature is 20~40℃, and the fermentation time is 12~72 h.

[0014] Further, in step S3, the inoculation amount of the capsule-coated yeast is 0.5-2% of the mass of the fermentation starter, the fermentation temperature is 25-30℃, and the fermentation time is 10-120 days.

[0015] Furthermore, in step S3, the auxiliary ingredients are selected from at least one of dried chili peppers, salt, sugar, ginger, garlic, white wine, soy sauce, and Sichuan peppercorns.

[0016] Further, in step S3, the auxiliary materials, by weight, include 1-150 parts dried chili peppers, 1-100 parts salt, 1-20 parts sugar, 1-20 parts ginger, 1-20 parts garlic, 1-20 parts white wine, 1-20 parts soy sauce, and 0.1-2 parts Sichuan peppercorns.

[0017] Furthermore, the dried chili peppers are made from locally grown white chili peppers from Longyou.

[0018] The Yinjiang product provided by this invention uses dried chili peppers made from locally grown white chili peppers from Longyou. Compared to ordinary commercially available dried chili peppers and fresh Longyou white chili peppers, the Yinjiang made from locally grown dried chili peppers from Longyou has a more moderate sour taste and a richer umami and flavor, providing an optimal raw material for the preparation of high-quality Yinjiang products.

[0019] On the other hand, the present invention provides a type of fermented bean paste prepared according to the method described above.

[0020] In another aspect, the present invention provides the use of cladomycin in the preparation of reagents for enhancing the umami and flavor of savory sauce.

[0021] The present invention has the following beneficial effects: 1. Pure koji is prepared by separately and independently culturing Aspergillus oryzae and Aspergillus niger, which avoids the inhibition of neutral protease activity of Aspergillus oryzae caused by pH drop during mixed koji preparation. This significantly improves the total protease activity of the koji, thereby more thoroughly hydrolyzing soybean protein and bringing higher amino acid nitrogen content and rich umami flavor to the product.

[0022] 2. Introducing taenia-coated yeast during the fermentation stage utilizes its unique synthetic enzyme system to generate a large amount of esters, enriching the flavor and quality of the product. On the other hand, the yeast's efficient consumption of amino acids effectively relieves the feedback inhibition of amino acids on proteases, driving the protein hydrolysis reaction to proceed in the positive direction, ultimately accumulating more amino acid nitrogen and enhancing the umami flavor of the product.

[0023] 3. Locally grown dried chili peppers from Longyou are specially selected as the raw material for Yinjiang sauce, further enriching the flavor and quality of the product; This invention provides a complete and efficient technical solution for producing Yinjiang sauce products with unique regional flavor and high quality, which is suitable for standardized and industrialized production. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to embodiments. It should be noted that the following embodiments are only used to explain and illustrate this invention and are not intended to limit this invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] Example 1: A method for preparing Yinjiang (a type of fermented soybean paste) provided by the present invention. The preparation process of Yinjiang provided in this embodiment is as follows: (1) Preparation of strains and raw materials: a) Strain: Aspergillus oryzae ( Aspergillus oryzae Shanghai-brewed 3.042, Aspergillus niger ( Aspergillus black AS 3.350, capsule-forming yeast ( Saccharomycopsis fibuligera CGMCC 2.0354; b) Main ingredients (by weight): 50 parts soybeans, 25 parts glutinous rice flour; c) Auxiliary ingredients (by weight): 100 parts dried chili peppers, 200 parts water, 50 parts salt, 10 parts sugar, 5 parts ginger, 5 parts garlic, 5 parts white wine, 5 parts soy sauce, and 1 part Sichuan peppercorns. The dried chili peppers are made from local Longyou white chili peppers dried at 40–80℃.

[0028] (2) Seed liquid preparation a) Aspergillus oryzae and Aspergillus niger spore suspensions: Frozen Aspergillus oryzae and Aspergillus niger strains were inoculated separately onto PDA solid medium plates (potato 200 g / L, glucose 20 g / L, agar 15-20 g / L). Aspergillus oryzae was incubated at 30℃ for 72 hours, and Aspergillus niger was incubated at 33℃ for 60 hours until spores were abundant. The plates were washed with sterile saline containing 0.05% Tween-80, and the spores were collected and counted using a hemocytometer to prepare spore concentrations of approximately 1×10⁻⁶. 7 ~1×10 8 Prepare a spore suspension of 1 spore per mL for later use.

[0029] b) Seed culture of *Saccharomyces cerevisiae*: Reconstitute the frozen *Saccharomyces cerevisiae* strain under aseptic conditions (cryopreservation tubes should be thawed in a low-temperature, dry environment to prevent strain degradation), then inoculate into YPD liquid medium (10 g / L peptone, 20 g / L glucose, 5 g / L yeast extract). Activate the culture by incubating at 28°C and 180 r / min for 12 hours on a shaker. After incubation, check the bacterial concentration to ensure the seed culture concentration is ≥10⁶. 8 A qualified yeast seed culture is obtained when the concentration is CFU / mL.

[0030] (3) Separate fermentation to prepare soy sauce starter a) Raw material processing: Soak soybeans for 4-6 hours until they are fully hydrated, rinse to remove impurities, and then steam until cooked through, until soft but not mushy. Drain and spread out to cool to room temperature. Mix the steamed soybeans with glutinous rice flour, ensuring the glutinous rice flour adheres evenly to the surface of the soybeans, as the base material for making koji.

[0031] b) Preparation of Aspergillus oryzae koji: Take half of the above basic materials, inoculate with Aspergillus oryzae spores (inoculation amount 0.01~1% (v / w), preferably 0.5% (v / w)), ferment at 35℃ for 64 hours, turning the koji once every 12 hours during the period to dissipate heat and ventilate, to obtain Aspergillus oryzae koji.

[0032] c) Preparation of Aspergillus niger koji: Take the remaining half of the base material, inoculate with Aspergillus niger spores (inoculation amount 0.01~1% (v / w), preferably 0.3% (v / w)), ferment at 35℃ for 48 hours, turning the koji once every 12 hours during the period to dissipate heat and ventilate, to obtain Aspergillus niger koji.

[0033] (4) Mixed fermentation Mix the above-mentioned Aspergillus oryzae koji and Aspergillus niger koji evenly to obtain soy sauce koji. Then, inoculate it with 0.5~2% (v / w) (preferably 1% (v / w)) of saccharin seed liquid and add the above-mentioned auxiliary materials: 100 parts by weight of dried chili peppers, 200 parts by weight of water, 50 parts by weight of salt, 10 parts by weight of sugar, 5 parts by weight of ginger, 5 parts by weight of garlic, 5 parts by weight of white wine, 5 parts by weight of soy sauce, and 1 part by weight of Sichuan peppercorns. After stirring evenly, place it in an environment of 28~35℃ (preferably 35℃) and seal it for fermentation for 10~120 days (preferably 30 days). After fermentation is completed, you will get the fermented soy sauce.

[0034] Example 2: Verification of the necessity of "separate koji making" followed by "mixed fermentation" To verify that the technical solution of this invention (i.e., preparing pure koji separately from Aspergillus oryzae and Aspergillus niger, and then simultaneously fermenting it with sac-forming yeast and auxiliary materials) has significant synergistic advantages over existing conventional processes in improving the umami and flavor of fermented soybean paste, the following four sets of comparative experiments were designed in this embodiment: Experimental group (technical solution of this invention): strictly followed the preparation method of Example 1; Control group 1 (no yeast inoculation): Except for not inoculating any yeast, the koji-making, fermentation and other processes were exactly the same as in Example 1; Control group 2 (mixed koji preparation followed by fermentation): The preparation method is basically the same as that of Example 1, except that the spore suspensions of Aspergillus oryzae and Aspergillus niger are mixed and inoculated into the same batch of raw materials, fermented at 35°C for 56 hours to obtain mixed koji, and then fermented again. Control group 3 (compound fermentation): The preparation method is basically the same as that of Example 1, except that the Aspergillus oryzae and Aspergillus niger spore suspensions and the seed liquid of the capsule-coated yeast were mixed and inoculated into the same batch of raw materials. After fermentation at 35°C for 56 hours, auxiliary materials were added to continue fermentation.

[0035] Samples were taken from all groups at the time of preparation of the koji and at the time of completion of the finished product preparation. The protease activity in the koji, the amino acid nitrogen content, total acid and total ester content of the finished product were measured. Twenty volunteers were randomly recruited to conduct sensory evaluation. The evaluation criteria are shown in Table 1. The test results for each group are shown in Table 2.

[0036] Table 1 Sensory Evaluation Criteria Table 2. Detection results of quality indicators of fermented soybean paste prepared by different methods As shown in Table 2, comparing the experimental group and control group 1, it can be found that the protease activity, amino acid nitrogen content, and total ester content of the fermented soybean paste prepared in the experimental group were significantly higher than those in control group 1. In particular, the total ester content of the experimental group was significantly higher, while the total acid content was slightly lower than that of control group 1, with a score of 92. This is because, although both groups synthesized proteases with comparable activity using separate fermentation methods, the method of this invention, after inoculating with *Aspergillus oryzae*, allows the yeast to further metabolize and transform the amino acids and peptides after protease hydrolysis into esters, alcohols, and other substances, increasing the ester flavor of the fermented soybean paste. At the same time, the continuous consumption of amino acids by the yeast effectively reduces the feedback inhibition of free amino acid concentration on protease activity, allowing the proteases synthesized by *Aspergillus oryzae* and *Aspergillus niger* to work continuously and efficiently, thereby continuously increasing the amino acid nitrogen content and comprehensively improving the umami flavor of the fermented soybean paste. The absence of yeast also leads to the ineffective accumulation of organic acids produced by *Aspergillus niger*, and the relatively lower levels of alkaline substances such as ammonia produced by protein decomposition due to different metabolic pathways, ultimately resulting in significantly higher acidity. Excessive acidity can lead to an overly sour and pungent taste, masking other flavors and reducing the quality of the fermented bean curd. Therefore, it is essential to introduce a sac-coated yeast after the fermentation process is complete.

[0037] Comparing the experimental group and control group 2, it can be seen that the experimental group had significantly higher protease activity, amino acid nitrogen content, and total ester content than control group 2, while the total acid content was slightly lower. The resulting product scored higher and was of better quality. This result indicates that the method of preparing pure koji separately from *Aspergillus oryzae* and *Aspergillus niger* before mixing is significantly superior to the method of mixing the two. The reason for this is that in traditional mixed koji preparation, the acid production by *Aspergillus niger* leads to a decrease in the environmental pH, severely inhibiting the synthesis and activity of neutral and alkaline proteases in *Aspergillus oryzae*. These two proteases are the main contributors to the decomposition of soybean globulin into soluble peptides and free amino acids. However, through physical separation, *Aspergillus oryzae* and *Aspergillus niger* can efficiently synthesize neutral, alkaline, and acidic proteases under suitable conditions, increasing protease activity. The mixing of the two systems enables complete hydrolysis of soybean protein, thereby significantly increasing the amino acid peptide nitrogen content. Based on this, the introduction of yeast for vigorous metabolic activity results in a greater amount of esters generated from the conversion of amino acids and peptides compared to the traditional mixed koji preparation method.

[0038] Comparing the experimental group and control group 3, it can be seen that the quality of the fermented sauce prepared by the combined fermentation of *Aspergillus oryzae*, *Aspergillus niger*, and *Saccharomyces cerevisiae* was significantly lower than that of the present invention. Although the acidity of the fermented sauce in control group 3 was similar to that of the experimental group, the protease activity, amino acid nitrogen content, and total ester content were significantly reduced. It is speculated that this is because the combined fermentation method is equivalent to operating in a nutrient-poor and highly competitive environment, which severely inhibits the growth of *Saccharomyces cerevisiae*, preventing it from effectively proliferating and expressing its ester-producing function. Simultaneously, resource competition may also affect the enzyme production efficiency of *Aspergillus oryzae* and *Aspergillus niger*, thus impacting protease activity and amino acid peptide nitrogen content. Conversely, when the fermentation starter is prepared first and then *Saccharomyces cerevisiae* is inoculated, the nutrients are abundant, allowing the yeast to ferment efficiently and continuously in the early stages, synergistically improving the fermentation effect.

[0039] In summary, the Yinjiang preparation method of the present invention, which involves "separate koji making" followed by "mixed fermentation", has a significant synergistic effect in improving the umami and soy sauce aroma of Yinjiang, transforming the product from a traditional soy sauce aroma to a complex ester aroma of fruit and soy sauce. This provides a brand-new technical means for developing new Yinjiang products with unique flavors.

[0040] Example 3: Screening and Optimization of Yeast As mentioned earlier, one of the core innovations of this invention lies in the simultaneous inoculation of yeast at the early stage of fermentation, based on the highly efficient enzymatic hydrolysis constructed by separately preparing Aspergillus oryzae and Aspergillus niger, to drive the targeted synthesis of flavor substances. The metabolic characteristics of the yeast strain directly determine the direction and intensity of the synergistic effect. To verify the effectiveness of this invention, the selected encapsulated yeast (… Saccharomycopsis fibuligera To leverage the unique advantages of the system in this invention, this embodiment includes a comparative screening experiment.

[0041] The preparation method of the fermented soybean paste was basically the same as in Example 1, using the same batch of raw materials, *Aspergillus oryzae* Hu Niang 3.042, and *Aspergillus niger* AS 3.350. The difference was that the yeast seed cultures inoculated during the mixed fermentation stage were *Saccharomyces cerevisiae* CGMCC2.0354 (SF group), *Saccharomyces rouxii* CGMCC 2.0389 (ZR group), *Saccharomyces cerevisiae* CGMCC 2.2020 (TS group), and *Saccharomyces cerevisiae* CGMCC 2.0087 (SC group), respectively. The comprehensive impact of different yeast inoculations on the final state indicators (amino acid nitrogen, total acid, and total esters) of the entire fermentation system was then evaluated. The test results are shown in Table 3.

[0042] Table 3. Effects of different yeast strains on the quality of fermented soybean paste. As shown in Table 3, the detection data of each group showed significant differences after fermentation. In terms of total ester content, the SF group was 2.8 times, 2.3 times, and 4.9 times higher than the ZR, TS, and SC groups, respectively, directly demonstrating that *Saccharomyces cerevisiae* had a significantly better ability to convert mold hydrolysis products into esters than other yeast strains. Regarding amino acid nitrogen content, the SF group's level was still significantly higher than the other three groups. This result significantly indicates that inoculating *Saccharomyces cerevisiae* did not lower the final level of flammable substances due to the yeast's vigorous amino acid consumption; on the contrary, it may have delayed the inactivation rate of proteases in the later stages of fermentation by maintaining a more suitable microenvironment (such as pH dynamic balance) or by reducing protease product inhibition due to its metabolites, thus allowing it to exert its effect for a longer period. In contrast, while traditional *Saccharomyces rouxii* and *Saccharomyces globosa* showed some synergistic effect, their effect was not as good as *Saccharomyces cerevisiae*, and *Saccharomyces cerevisiae* could not adapt to this system, showing the lowest results in all aspects and therefore not suitable. Therefore, mixed fermentation of *Saccharomyces cerevisiae* with *Aspergillus oryzae* and *Aspergillus niger* was chosen as the preferred scheme.

[0043] Example 4: The effect of chili peppers from different sources on the quality of fermented soybean paste In flavored fermented sauces, auxiliary ingredients are not merely flavor enhancers; their bioactive components and physicochemical properties can deeply influence the fermentation process, affecting microbial metabolism and the formation of end products. Chili peppers, as a key flavoring auxiliary ingredient in the fermented sauce of this invention, may have their fermentation process influenced by their variety, origin, and processing state (fresh / dried) through factors such as sugar and organic acids. To determine the most suitable chili pepper raw material for the process system of this invention and ensure the typicality of product flavor and quality stability, this embodiment aims to systematically evaluate and compare the impact of three representative chili pepper sources—commonly available dried chili peppers, dried chili peppers from Longyou, and fresh chili peppers from Longyou—on key quality indicators of the fermented sauce, thereby providing a scientific basis for the selection of auxiliary ingredients.

[0044] The preparation process was strictly followed as in Example 1, using the same batch of main ingredients, *Aspergillus oryzae* (Hu Niang 3.042), *Aspergillus niger* (AS 3.350), and *C. GMCC* (CGMCC 2.0354). The difference was that the chili peppers added to the auxiliary ingredients were commercially available dried chili peppers, Longyou dried chili peppers (white chili pepper variety), and Longyou fresh chili peppers (white chili pepper variety), respectively. No chili peppers were added to the blank control group. After fermentation, samples of the fermented sauce from each group were taken to test the amino acid nitrogen content, total acid content, and total ester content. The results are shown in Table 4.

[0045] Table 4. Effects of chili peppers from different sources on the quality of fermented soybean paste. As shown in Table 4, the control group had the lowest overall indicators, indicating that the addition of chili peppers had a positive impact on the fermentation of the fermented soybean paste. Specifically, in terms of amino acid nitrogen content, group B (Longyou dried chili peppers) had the highest content, significantly better than groups A and C. This suggests that local Longyou dried chili peppers may contain substances that are more conducive to the maintenance of protease activity or microbial growth, thereby indirectly promoting the final hydrolysis efficiency of proteins. Regarding total acidity, group C (Longyou fresh chili peppers) had the highest acidity. This may be due to the participation of acid-producing bacteria such as lactic acid bacteria carried by the fresh chili peppers in acid production during the early stage of fermentation, as well as the contribution of organic acids contained in the chili peppers themselves. Group B had the most ideal total acidity control, lower than groups A and C, indicating that it introduced the least acidity interference, resulting in a more mellow and harmonious sour taste in the product. Regarding total ester content, group B had a significantly higher total ester content than all other groups. This result indicates that during the drying process of local Longyou chili peppers, the internal sugars and amino acids may undergo a certain degree of Maillard reaction, generating aromatic precursors. Simultaneously, the cellular structural changes caused by drying may facilitate the slow release and migration of flavor compounds in the chili peppers during fermentation. These precursors and flavor compounds provide unique and efficient substrates for the ester synthesis metabolism of *Saccharomyces cerevisiae*, thereby driving the formation of higher-level esters.

[0046] In summary, considering the three core indicators of amino acid nitrogen, total acid, and total esters, Longyou locally grown dried chili peppers exhibit comprehensive adaptability and enhancement effects that cannot be replicated by ordinary commercially available dried chili peppers or fresh chili peppers of the same origin. Therefore, Longyou locally grown dried chili peppers are preferred as a specific auxiliary ingredient in the sauce of this invention.

[0047] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any modifications and alterations made by those skilled in the art without departing from the spirit and scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing a type of fermented bean paste, characterized in that, Includes the following steps: S1: Soybeans are soaked, rinsed, steamed, and cooled before being mixed with rice flour or wheat flour. S2: Divide the soybeans after mixing with flour into two portions, inoculate one portion with Aspergillus oryzae and the other portion with Aspergillus niger, and ferment them separately to prepare koji. S3: The koji is mixed with auxiliary materials and yeast and then fermented to obtain Yinjiang sauce.

2. The preparation method according to claim 1, characterized in that, in, The yeast strain is *Saccharomyces cerevisiae*.

3. The preparation method according to claim 2, characterized in that, In step S1, by weight, the soybeans are 1 to 100 parts and the rice flour or wheat flour is 1 to 50 parts.

4. The preparation method according to claim 3, characterized in that, In step S2, the inoculum amount of Aspergillus oryzae and Aspergillus niger is 0.01~1%, the fermentation temperature is 20~40℃, and the fermentation time is 12~72 h.

5. The preparation method according to claim 4, characterized in that, In step S3, the inoculation amount of the capsule-coated yeast is 0.5-2% of the mass of the koji, the fermentation temperature is 25-30℃, and the fermentation time is 10-120 days.

6. The preparation method according to claim 5, characterized in that, In step S3, the auxiliary ingredients are selected from at least one of dried chili peppers, salt, sugar, ginger, garlic, white wine, soy sauce, and Sichuan peppercorns.

7. The preparation method according to claim 6, characterized in that, In step S3, the auxiliary materials, by weight, include 1-150 parts dried chili peppers, 1-100 parts salt, 1-20 parts sugar, 1-20 parts ginger, 1-20 parts garlic, 1-20 parts white wine, 1-20 parts soy sauce, and 0.1-2 parts Sichuan peppercorns.

8. The preparation method according to claim 7, characterized in that, The dried chili peppers were made from locally grown white chili peppers from Longyou.

9. Yinjiang prepared by the method of any one of claims 1-8.

10. Use of sac-forming yeast in the preparation of reagents for enhancing the umami and flavor of savory sauce.

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