Composite fermentation inoculant and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a zygosaccharomyces rouxii ZB445 and Weissella fusiformis ZF629 composite zymophyte agent and application of the zygosaccharomyces rouxii ZB445 and Weissella fusiformis ZF629 composite zymophyte agent. The compound fermentation inoculant provided by the invention has high stress resistance and excellent capability of producing alcohol and ester volatile flavor substances, is used for fermenting the thick broad-bean sauce, and can obviously increase the content of various key volatile compounds in the thick broad-bean sauce, including the total content of flavor substances such as alcohol and ester, so that the yield of the thick broad-bean sauce is increased. The content of key flavor compounds, especially phenethyl alcohol, 3-methylthio propyl alcohol, eugenol, ethyl caprylate and the like, is reduced, and meanwhile, excellent flavor amino acid accumulation capacity is also shown. The composite fermentation inoculant is used for fermenting the thick broad-bean sauce, so that the mellow fragrance and the fruit ester fragrance of the thick broad-bean sauce can be remarkably improved, the fresh, sweet and mellow feeling is enhanced, the overall fragrance and flavor of the thick broad-bean sauce are improved, the flavor and taste of the thick broad-bean sauce are richer, more complex, more harmonious and layered, and the overall flavor and quality of the thick broad-bean sauce are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a combined fermentation agent of a strain of Zygomyces rouxii ZB445 and a strain of Fusionia weissii ZF629 and its application. Background Technology

[0002] my country has a long history of traditional fermented soybean products, with broad bean paste (doubanjiang) being a representative product, holding an important place in culinary culture due to its unique and rich flavor. The formation of its flavor is a complex biochemical process, essentially involving microorganisms utilizing macromolecules such as proteins and starches in raw materials to produce volatile flavor compounds such as esters, alcohols, acids, and aldehydes through metabolism. The composition and proportion of these substances collectively constitute the typical "soy sauce aroma," "alcoholic aroma," and "ester aroma" characteristics of broad bean paste, which are core attributes determining product quality and consumer acceptance.

[0003] In the modern industrial production of fermented soybean paste, the use of microbial agents with clearly defined functions for enhanced fermentation has become a key technology for improving product quality and stability. Currently, research and practice in this field mainly focus on using functional yeasts and lactic acid bacteria to optimize the fermentation process. However, existing technologies still have significant limitations in terms of synergistic effects and precise flavor control. For example, patent CN118716566A discloses a method for fermenting fermented soybean paste using a staged inoculation of *Zygosacchariformis* and *Staphylococcus aureus*, which to some extent increases the content of alcohols and esters and shortens the fermentation cycle. However, the staged inoculation strategy increases the complexity of the process, which is not conducive to large-scale production. Patent CN119366605A discloses a method for co-fermenting low-salt fermented soybean paste using a fusion of *Weissella fussima* and *Candida escherichia*. This technology focuses on maintaining flavor under low-salt conditions. Its invention purpose and technical effects mainly revolve around overcoming the flavor loss and safety issues caused by salt reduction. The functional design of its strain combination leans towards acid production and colonization and the construction of basic flavors. Patent CN120484993A starts with red yeast and screens out a strain that can produce high levels of ethyl phenylacetate, but its flavor contribution is relatively singular and it is difficult to independently support the complex and harmonious aroma profile required for fermented bean paste.

[0004] Existing technologies largely focus on the application of single-functional strains, or while employing combinations of strains, the synergistic effect is insufficient, generally resulting in problems such as limited flavor enhancement, imbalanced alcohol-ester ratios, and poor aroma fullness and complexity. Therefore, breaking through existing technological thinking and starting from the perspective of complementary microbial metabolic networks, scientifically screening and constructing key microbial combinations that can produce significant synergistic effects during fermentation, especially those that can directionally and efficiently enhance alcohol and ester aromas, is of vital importance for developing high-end fermented soybean paste products with superior flavor characteristics and breaking the current homogeneous competitive landscape. Summary of the Invention

[0005] Based on the above technical problems, the main objective of this invention is to overcome the shortcomings of the aforementioned background technology and provide a compound fermentation agent containing Zygosaccharomyces rouxii ZB445 and Weissella confossa ZF629. This fermentation agent has high stress resistance and excellent ability to produce volatile flavor compounds such as alcohols and esters. When used in fermentation of broad bean paste, it can synergistically optimize the aroma profile of the product, effectively suppress unpleasant odors while enhancing pleasant flavors, significantly improve the flavor, mouthfeel, and aroma harmony of broad bean paste, and enhance the overall aroma, flavor, and quality of broad bean paste.

[0006] To achieve the above objectives, the inventors conducted in-depth research and, through repeated studies and demonstrations, obtained the solution of this invention, as detailed below:

[0007] In a first aspect, the present invention provides a compound fermentation agent comprising ZB445 Zygomyces rouxii and ZF629 Fusion Weissella.

[0008] Secondly, the present invention provides a compound fermentation method, wherein the bacteria inoculated by the fermentation method include the above-mentioned Zygosaccharomyces rouxii ZB445 and Zygosaccharomyces fusionis ZF629.

[0009] Thirdly, the present invention provides the application of the above-mentioned fermentation agents or compound fermentation methods in the processing of fermented foods. The fermented foods mainly refer to fermented foods that produce flavor by using microorganisms and their enzymes for fermentation, such as soy sauce, sauce, vinegar, fermented black beans or fermented bean curd, especially fermented bean products, preferably fermented soybean paste.

[0010] Fourthly, the present invention provides a method for preparing broad bean paste, wherein the method involves adding the above-mentioned fermentation agent during the fermentation process of broad bean paste.

[0011] The preparation method described herein can employ fermentation methods commonly used in the field, and the process parameters of the fermentation method can be adjusted according to actual production conditions.

[0012] Furthermore, the fermentation agent is added during the early stage of fermentation of the soy sauce mash.

[0013] Furthermore, the amount of the fermentation agent added is 10. 5 ~10 7 CFU / mL.

[0014] Furthermore, the ratio of Zygosaccharomyces rouxii ZB445 to Zygosaccharomyces fusionis ZF629 in the fermentation agent is 1:1.

[0015] Fifthly, the present invention provides a fermented soybean paste, which is prepared by the preparation method described in the present invention.

[0016] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages:

[0017] 1. The compound fermentation agent provided by this invention has high stress resistance and excellent ability to produce volatile flavor substances such as alcohols and esters; the two can also play a good synergistic role. Among them, *Zygosacchariformis* acts as the aroma-producing core, responsible for the efficient synthesis of alcohols and esters, while *Weissella* provides esterification precursors and enriches fruit aroma. Through synergistic metabolism, it effectively drives alcohol synthesis and esterification reactions, achieving a breakthrough improvement in the intensity of alcohol and ester aromas; it can be used in the production of high-salt fermented foods to enhance product flavor and thus improve food quality.

[0018] 2. The compound fermentation agent provided by this invention, when used for fermenting broad bean paste, greatly improves the aroma profile of the broad bean paste; its total ester content reaches 11.32 mg / kg, an increase of 248.3% compared with the control group (3.25 mg / kg); at the same time, the total alcohol content also increases by 170.9%, reaching 13.87 mg / kg; the contents of key aroma compounds phenethyl alcohol, 1-octen-3-ol, 3-methylthiopropanol, phenylacetaldehyde, 2-methoxy-4-vinylphenol, eugenol, ethyl octanoate, ethyl isovalerate, and ethyl acetate are increased by 48.6%-200.2% compared with the control group, among which the contents of phenethyl alcohol (rose aroma) and ethyl octanoate (sweet orange aroma) are increased by 155.9% and 200.2% respectively. The increase in the content of these flavor compounds can significantly enhance the "alcoholic aroma" and "fruit ester aroma" of broad bean paste.

[0019] 3. The compound fermentation agent provided by this invention is used for fermentation of broad bean paste. While enhancing the aroma, the taste of the product is improved simultaneously. The mixed fermentation group of Zygosacchariformis ZB445 and Zygosacchariformis ZF629 showed excellent accumulation ability of flavor amino acids, which increased the total free amino acids, umami amino acids and sweet amino acids by 15.2%, 16.1% and 15.4% respectively, enhancing the fresh, sweet and mellow taste of the product.

[0020] 4. The compound fermentation agent provided by this invention is used for fermentation of broad bean paste. The total acid content of its fermentation system (1.50 g / 100g) is 26.1% higher than that of the control group (1.19 g / 100g), accompanied by a decrease in pH value, which reduces the possibility of the growth of miscellaneous bacteria. It can provide a more suitable fermentation environment for the formation and preservation of flavor in broad bean paste, and help improve the stability and safety of the product.

[0021] 5. The present invention provides a compound fermentation method for fermented broad bean paste, which is simple in process and can be used for large-scale production. The fermented broad bean paste is significantly better than naturally fermented broad bean paste in terms of overall flavor, taste and aroma coordination. While enhancing the pleasant flavor, it effectively suppresses unpleasant odors, improves the overall aroma and flavor of the broad bean paste, and makes its flavor and taste richer, more harmonious and layered, significantly improving the overall flavor and quality of the broad bean paste. Attached Figure Description

[0022] Figure 1 This is a colony morphology diagram of ZB445 *Zygosaccharomyces rouxii*.

[0023] Figure 2 To integrate the colony morphology of Weissella ZF629;

[0024] The Zygosaccharomyces rouxii ZB445 strain provided by this invention was deposited at the Guangdong Microbial Culture Collection Center on May 19, 2025, at the address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC NO: 66356. This strain was received and registered by the collection center on May 19, 2025, and was confirmed to be a viable strain by the collection center on May 19, 2025.

[0025] The *Weissella confusa* ZF629 provided by this invention was deposited on May 19, 2025, at the Guangdong Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC NO: 66357. This strain was received and registered by the collection center on May 19, 2025, and was confirmed to be a viable strain by the collection center on May 19, 2025. Detailed Implementation

[0026] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] Unless otherwise specified, the reagents, methods and equipment used in this invention are conventional food-grade reagents, methods and equipment in the art.

[0029] The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0030] Example 1: Screening of the target strain

[0031] 1. Microbial isolation and purification

[0032] Using raw soybean paste mash from different fermentation stages as samples, 10.0 g of mash was accurately weighed and added to 90 mL of sterile physiological saline. After thorough shaking and mixing, a series of serial dilutions (10 g / mL) were performed using sterile physiological saline. -1 Up to 10 -5 100 μL of bacterial suspensions at different dilution gradients were evenly spread onto the surface of LB agar plates and incubated at 30°C for 72 hours. Based on differences in colony morphology, the plate streak method was used for three-stage purification to obtain single colonies. After preliminary identification by colony morphology observation, typical single colonies were selected and inoculated into liquid culture tubes, and cultured with shaking until bacterial precipitation. Finally, the cultured bacterial suspension was mixed with 50% glycerol preservation solution at a 1:1 (v / v) ratio, aliquoted, and stored in an ultra-low temperature freezer at -80°C.

[0033] The purified microorganisms were subjected to molecular biological identification, and a total of 153 strains belonging to 7 genera were successfully isolated and identified from the fermented soybean paste samples, as shown in Table 1. To obtain functional strains producing alcohol and ester aromas, these 153 microorganisms need to be screened.

[0034] Table 1 Summary of bacterial strains isolated from fermented soybean paste

[0035]

[0036] 2. Preliminary screening of bacterial strains

[0037] The purified strains were inoculated into the corresponding LB, MRS, and PDA liquid media. Bacteria were cultured at 37°C and 220 rpm / min, while fungi were cultured at 30°C and 220 rpm / min. After reaching the stationary phase, the fermentation broth was collected. Trained aroma evaluators conducted a blind odor assessment of the fermentation broth, focusing on strains exhibiting pleasant aromas such as alcoholic (wine-like) and fruity (ester-based) notes.

[0038] After initial screening, 32 strains with distinctly pleasant aromas were preliminarily selected from 153 isolates to proceed to the next round of secondary screening.

[0039] 3. Quantitative rescreening of aroma production capacity (GC-MS analysis)

[0040] The initially screened strains were inoculated into a simulated fermentation medium (containing 5% soybean meal, 0.5% wheat flour, 14% NaCl, pH 5.5) and cultured at 30°C for 7 days. A control without inoculation was also included. The contents of esters and alcohols in the fermentation broth were quantitatively analyzed using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). Nine strains were screened that showed an advantage in alcohol and ester synthesis, and the results are recorded in Table 2.

[0041] Table 2. Quantitative rescreening strains for aroma production capacity

[0042]

[0043] As shown in Table 2, *Zygosaccharomyces rouxii* CX350 and CX353 exhibited a clear advantage in alcohol and ester synthesis capabilities. These two strains significantly outperformed other strains in the accumulation of total alcohols and esters, while the accumulation of the key alcohol compound phenylethanol was similar. However, under the same conditions, strain CX350 showed a greater advantage than CX353 in accumulating esters and the key ester compound isoamyl acetate, with total ester content of 215.8 μg / L and isoamyl acetate content of 62.5 μg / L, representing increases of 102.8% and 189.4% respectively compared to the CX353 group. Furthermore, among the representative strains of other genera, *Westernella fusionis* CX121 also demonstrated good alcohol and ester production characteristics, with total alcohol and total ester content (128.4 μg / L and 78.9 μg / L, respectively) higher than other screened strains, and approximately 1.50 times and 1.75 times that of another *Westernella fusionis* strain, CX125. The above data indicate that *Zygosacchariformis* CX350 and *Westernella fusionis* CX121 not only possess excellent capabilities in producing volatile flavor compounds such as alcohols and esters in the isolated microbial community of fermented soybean paste, but also maintain their advantages in producing alcohol and ester compounds among the same strains. In existing literature, *Westernella fusionis* is described as playing a role in the early stages of fermentation of soybean products, while *Zygosacchariformis* typically plays a role in the later stages. Therefore, *Zygosacchariformis* CX350 and *Westernella fusionis* CX121, which exhibit superior performance in producing alcohol and ester compounds, were selected for the backfill fermentation of fermented soybean paste.

[0044] The CX350 and CX121 obtained from the above screening were named ZB445 and ZF629 respectively, and were deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 19, 2025, with accession numbers GDMCC NO: 66356 and GDMCC NO: 66357 respectively.

[0045] Example 2 Performance testing of fused strains of *Weissella vesicatoria* ZF629 and *Zygosaccharomyces rouxii* ZB445

[0046] To ensure that *Zygosacchariformis* CX350 and *Weissella fusionis* CX121 can survive and function in the high-salt, slightly acidic environment of real fermented soybean paste, their stress resistance was verified.

[0047] 1. Salt tolerance verification: ZB445 *Zygosacchariformis* and ZF629 *Weissella fusionis*, both in late logarithmic growth phase, were inoculated at 2% (v / v) into MRS and PDA broths containing different NaCl concentration gradients (0%, 4%, 8%, 10%, 12%, 16%, 20%). Bacteria were cultured at 37°C and 220 rpm / min, while fungi were cultured at 30°C and 220 rpm / min. After 72 h of culture, the absorbance (OD) of the bacterial suspensions in each experimental group was measured at 600 nm using a microplate reader. 600 Each experiment was conducted in triplicate, and the average results were recorded in Table 3.

[0048] 2. Acid resistance verification: ZB445 *Zygosacchariformis* and ZF629 *Weissella fusionis* strains in late logarithmic growth were inoculated at 2% (v / v) into MRS and PDA liquid media with pH gradients (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0), respectively. Bacteria were cultured at 37°C and 220 rpm / min, while fungi were cultured at 30°C and 220 rpm / min. After 72 h of incubation, the absorbance (OD) of the bacterial suspensions in each experimental group was measured at 600 nm using a microplate reader. 600 Each experiment was conducted in triplicate, and the average results were recorded in Table 3.

[0049] Table 3 Results of Salt and Acid Resistance Tests

[0050]

[0051] The results are shown in Table 3. Both strains grew well in the typical environment of fermented soybean paste (salinity 14%, pH 4.5-6.0). *Weissella fusionis* ZF629 grew well in environments with salinity below 16% and pH 5.0-7.0, adapting to the environment in the early and middle stages of fermentation. *Zygosaccharomyces rouxii* ZB445 exhibited excellent salt and acid tolerance, growing well in environments with 0-20% salinity and pH 4.0-7.0, indicating that this strain may continue to proliferate and produce aroma in the harsh environment of the later stages of fermentation, which is key to its core role.

[0052] 3. Antibacterial activity test: The antibacterial activity of the target strains was evaluated using the disk diffusion method. *Escherichia coli* ATCC 25922 and *Staphylococcus aureus* ATCC6538 were selected as indicator strains. The activated indicator bacterial solution (concentration adjusted to 10⁻⁶) was used... 6 -10 7 Take 0.1 mL of (CFU / mL) and spread it evenly on the surface of a nutrient agar plate using a sterile cotton swab. Place four sterile filter paper discs (6 mm in diameter) at equal intervals on each plate. Culture *Zygosaccharidosis rouxii* ZB445 and *Weissella fusionis* ZF629 strains to mid-logarithmic growth, centrifuge the culture for 10 minutes, and collect the supernatant. Add 15 μL of the sterile filtered supernatant to the filter paper discs and incubate at 37°C for 48 hours.

[0053] Table 4 Results of antibacterial test

[0054]

[0055] As shown in Table 4, Fusion Weissella ZF629 showed significant inhibitory effects on both Escherichia coli and Staphylococcus aureus, while ZB445 of Zygosacchariformis also showed some inhibitory ability against Staphylococcus aureus, thus ensuring the safety of fermented soybean paste for consumption to a certain extent.

[0056] Example 3: Test of fermentation of broad bean paste using fusion strains of Weissella ZF629 and Zygomyces rouxii ZB445.

[0057] 1. Fermentation of fermented soybean paste

[0058] Thoroughly wash the broad beans and soak them in water for 3 hours. After soaking, remove the broad beans and drain them. Mix them with flour in a ratio of 3:1 (dry broad beans: flour, w:w), and inoculate with Aspergillus oryzae at a ratio of 1000:1 (w:w). After mixing thoroughly, place the mixture in a constant temperature and humidity incubator, controlling the temperature between 28 and 32°C and maintaining a relative humidity of 90%, and continue incubating for 40 to 48 hours. During this period, the koji material needs to be turned over periodically to prevent "koji burning" due to excessive temperature. Once the surface of the broad beans is covered with yellow-green spores, the koji is ready. Then, mix the prepared koji with 22% saline solution at a volume ratio of 1:1.5, mark it as day 0, and set the entire fermentation cycle to 30 days.

[0059] The fermentation experiment included one blank control group (A) and three fermentation groups (B, C, and D). The specific strain combinations and sequencing design are shown in Table 5. The screened *Zygosacchariformis* ZB445 and *Weissella fusionis* ZF629 were activated and cultured for two generations to prepare a 10⁻⁶ concentration...7 At the start of fermentation (0 day), the bacterial suspension of CFU / mL was inoculated into the fermented soybean paste at a 2% (v / v) inoculation rate for the single-strain fermentation group. For the mixed-strain fermentation group, the seed cultures of ZB445 and ZF629 were first mixed at a 1:1 (v / v) volume ratio before being inoculated at a 2% inoculation rate. All experimental groups fermented at 30°C for 30 days, with two parallel fermenters set up for each group to ensure the reliability of the experimental results.

[0060] Table 5 Fermentation Experiment Groups

[0061]

[0062] 2. Physicochemical index testing

[0063] Accurately weigh 5.0 g of fermentation sample, add 45 mL of ultrapure water and mix thoroughly. Homogenize at 180 rpm for 1 h at 30°C. After removing soybean residue with filter paper, centrifuge the filtrate at 8000 rpm for 5 min. Use the supernatant for the following determinations: Total acid content was determined by acid-base titration; pH value was directly measured using a pH meter; amino acid nitrogen content was determined by formaldehyde titration; and reducing sugar content was determined by the 3,5-dinitrosalicylic acid method. The results are recorded in Table 6.

[0064] Table 6. Results of Physicochemical Indicators

[0065]

[0066] As shown in Table 6, the amino acid nitrogen content of the backfilled fermentation group showed a certain advantage over the control group, with an increase of 4.2%-11.3%. The amino acid nitrogen content of the two fermentation groups with backfilled ZB445 (0.79 g / 100 g and 0.78 g / 100 g) was higher than that of fermentation group A and fermentation group B. This indicates that strain ZB445 may promote the degradation of protein or the synthesis and accumulation of amino acids in the fermented mash through its metabolic activities. Moreover, this metabolic activity was not weakened when it was combined with Weissella fusion for backfilled fermentation.

[0067] In terms of reducing sugar and total acid content, the reducing sugar content in the mixed fermentation group (Group D) (2.36 g / 100g) was significantly lower than that in the control group (3.25 g / 100g) and the single-strain fermentation group (4.12 g / 100g and 3.15 g / 100g), indicating a higher utilization rate of reducing sugar. Simultaneously, the total acid content (1.50 g / 100g) was also higher than that in the single-strain fermentation group (1.43 g / 100g and 1.37 g / 100g) and the control group (1.19 g / 100g). This data suggests that the combination of *Weissella fusionis* and *Zygosacchariformis* may accelerate the utilization of reducing sugar and effectively convert it into organic acids. The increased total acid content in Group D (26.1% higher than the control group A) also directly resulted in a significantly lower pH value (5.67) compared to other groups.

[0068] Therefore, by applying ZB445 of Zygosacchariformis and ZF629 of Zygosacchariformis to the fermentation stage of soybean paste, the physicochemical microenvironment of the mash can be significantly changed, resulting in higher levels of amino acid nitrogen and total acid, as well as more efficient conversion of reducing sugars. This provides important precursors and suitable acidic conditions for the formation of flavor compounds.

[0069] 3. Analysis of free amino acid composition

[0070] Accurately transfer 2 mL of the supernatant into a centrifuge tube and add 8 mL of 5% (w / v) trichloroacetic acid solution. Mix well and incubate at 4°C for 60 min. Centrifuge the sample solution at 4°C for 5 min, collect the supernatant, and repeat the above steps twice. Dilute the final centrifuged solution 5 times with deionized water, filter the diluted solution through a 0.22 μm microporous membrane, collect the filtrate in a liquid chromatography vial, and detect the amino acid content in the sample using an automated amino acid analyzer. The results are recorded in Table 7.

[0071] Table 7. Free amino acid content at the end of fermentation of fermented broad bean paste

[0072]

[0073] As shown in Table 7, the contents of total free amino acids, umami amino acids (Asp+Glu), and sweet amino acids (Thr+Ser+Gly+Ala+Lys) in all backfill fermentation groups were significantly higher than those in the control group. In particular, the combined fermentation group (Group D) of *Zygosacchariformis* ZB445 and *Westernella fusion* ZF629 achieved the highest values ​​in all indicators. Its total free amino acids, umami amino acids, and sweet amino acids were 15.2%, 16.1%, and 15.4% higher than the control group, respectively, and also significantly higher than the single-strain backfill fermentation group. This indicates that simultaneous inoculation of *Zygosacchariformis* ZB445 and *Westernella fusion* ZF629 exhibited a good synergistic effect, promoting the accumulation of flavor amino acids in fermented soybean paste, with better results than single-strain inoculation. Therefore, applying *Zygosacchariformis* ZB445 and *Westernella fusion* ZF629 to the fermentation stage of fermented soybean paste can more effectively drive protein hydrolysis and the enrichment of specific flavor amino acids, directly laying the foundation for the product's fresh, sweet, and mellow flavor.

[0074] 4. Analysis of volatile flavor components

[0075] The volatile matter content of fermented soybean paste samples at the end of fermentation was determined by headspace solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS). 2.0 g of the ground sample and 3 mL of 15% (w / v) saline solution were weighed, followed by the addition of 0.01 mL of 4.85 ppm 2-octanol internal standard solution to a 20 mL headspace vial. The vial was placed in an incubator and equilibrated at 60°C for 20 min. Headspace adsorption was then performed at 60°C for 30 min using a solid-phase microextraction head. Subsequently, the extraction head was transferred to the gas chromatograph injection port, and thermal desorption was performed at 250°C for 5 min to complete the injection. GC conditions: HP-INNOWax capillary column (60 m × 250 μm × 0.25 μm); injection port temperature set at 250°C; helium as carrier gas; constant flow rate of 1.2 mL / min. The temperature program was set as follows: initial column temperature 40°C, held for 3 min, then increased to 230°C at a rate of 6°C / min and held for 10 min. MS conditions: ion energy 70 eV; electron ionization source; ion source temperature 250°C; interface temperature 250°C; full scan mode. The detection results are recorded in Tables 8 and 9.

[0076] Table 8. Contents of alcohols, esters, and total volatile substances in samples of fermented broad bean paste at the end of fermentation.

[0077]

[0078] As shown in Table 8, inoculation with Zygosacchari rouxii ZB445 alone (Group C) resulted in total alcohol and total ester contents of 11.24 mg / kg and 7.85 mg / kg, respectively, which were 2.2 times and 2.4 times higher than the control group (5.12 mg / kg and 3.25 mg / kg). This indicates that Zygosacchari rouxii ZB445 can significantly promote the biosynthesis of alcohols and esters during the fermentation of fermented soybean paste. When strain ZB445 was fermented in combination with Fusionia weissori ZF629 (Group D), the aroma-producing ability was further enhanced, and the total alcohol and total ester contents increased further to 13.87 mg / kg and 11.32 mg / kg, respectively. Compared with Group C, the total ester content increased by an additional 44.2%, and the total volatile matter content increased by 32.6%.

[0079] The superior performance of Group D in total ester synthesis is likely due to the "metabolic division of labor" among microorganisms. *Westernella fusionis* ZF629, as a lactic acid bacterium, produces various organic acids through metabolism during the fermentation of fermented soybean paste; these organic acids can serve as acyl donors. Meanwhile, *Zygosacchariformis* ZB445 possesses a strong alcohol-producing capacity and esterification enzyme system, capable of catalyzing the synthesis of esters from alcohols and acids. This symbiotic relationship of "acid-alcohol esterification" is the fundamental reason why the ester content of Group D (11.32 mg / kg) is significantly higher than that of Group C (7.85 mg / kg).

[0080] Table 9 Content of Key Volatile Compounds

[0081]

[0082] As shown in Table 9, firstly, the contribution of alcohols and esters to the flavor of fermented broad bean paste in group D was significantly enhanced. The content of key substances was significantly higher than that of group A, and also significantly higher than that of groups B and C, showing a significant synergistic effect. Alcohols and esters are the core components constituting the "alcoholic aroma," "fruity aroma," and "floral aroma." Phenylene alcohol (with rose and honey aroma) is one of the key compounds constituting the "soy sauce aroma" and "floral aroma" of broad bean paste. The phenylene alcohol content in fermented broad bean paste in group D reached 3.056 mg / kg, and the absolute content far exceeded its odor threshold (0.39 mg / kg), which was 155.9% higher than that of group A and 41.2% higher than that of group C. The content of ethyl octanoate (with sweet orange and brandy aroma) (0.148 mg / kg) increased by 200.2% compared with group A (0.049 mg / kg), and the increase was far greater than that of group C (73.9%). Furthermore, the content of 1-octen-3-ol (with an extremely low threshold of only 0.0015 mg / kg), which imparts a savory aroma to mushrooms, was significantly higher in group D than in other groups, increasing by 99.5%, 102.7%, and 19.7% compared to groups A, B, and C, respectively, thus significantly enhancing the complexity of the aroma. This indirectly confirms the highly efficient symbiotic relationship between the acidic precursor produced by the fusion of Weissler ZF629 metabolism and the esterification ability of Zygomyces rouxii ZB445, thereby directionally and efficiently enriching the fruity notes of the product.

[0083] Secondly, the characteristic flavor compounds of aldehydes and phenols in the fermented broad bean paste of group D were simultaneously enriched. The content of phenylacetaldehyde (with hyacinth and almond aromas) was significantly increased, and the contents of eugenol, which imparts spicy and clove flavors, and 2-methoxy-4-vinylphenol, which presents smoky and spice aromas, also increased significantly in the backfill fermentation group. Specifically, the eugenol content in group D reached 0.922 mg / kg, which was 145.5%, 57.6%, and 21.2% higher than that in groups A, B, and C, respectively; while the 2-methoxy-4-vinylphenol content reached 0.074 mg / kg, which was 107.7%, 13.8%, and 39.6% higher than that in groups A, B, and C, respectively. The increase of these compounds, along with the significant increase in alcohols and esters, can significantly enhance the flavor, texture, and aroma harmony of fermented soybean paste. It not only improves the overall aroma and flavor of fermented soybean paste, but also makes its flavor and texture richer, more complex, layered, and more distinctive, thus significantly improving the overall flavor and quality of fermented soybean paste.

[0084] 5. Sensory evaluation

[0085] A panel of 15 professionals with expertise in sensory evaluation was selected to assess the key aroma characteristics of fermented soybean paste. The evaluation criteria included four dimensions: soy sauce aroma, fruity aroma, mellow aroma, and unpleasant odors, ranging from 0 (no intensity) to 5 (high intensity). The average score across all criteria was used as the final score. Detailed scoring standards are shown in Table 10, and the evaluation results are recorded in Table 11.

[0086] Table 10 Sensory Evaluation Criteria

[0087]

[0088] Table 11 Sensory Evaluation Results

[0089]

[0090] As shown in Table 11, the sensory evaluation results of the fermentation endpoint samples indicate that the co-fermentation of Zygosacchariformis ZB445 and ZF629 Fusion Weissella exhibited a significant synergistic effect in fermenting broad bean paste. The scores for aroma and fruitiness reached 4.8 and 4.7, respectively, while the score for "unpleasant odors" was reduced to a minimum (1.8). Furthermore, the "soy sauce aroma" score remained stable at 4.0, indicating that the newly introduced aromas blended well with the inherent flavor of the product. The sensory evaluation data confirms that the co-fermentation of Zygosacchariformis ZB445 and ZF629 in broad bean paste can synergistically optimize the aroma profile of the product to a greater extent, effectively suppressing unpleasant odors while enhancing pleasant flavors, ultimately resulting in a rich and harmonious high-quality broad bean paste flavor profile.

[0091] In summary, by inoculating the fermented soybean paste mash with ZB445 *Zygosacchariformis* and ZF629 *Weissella fusionis*, the content of several key volatile compounds in the fermented soybean paste was significantly increased, including the total content of flavor substances such as alcohols, esters, and phenols. In particular, the contents of phenylethyl alcohol, 1-octen-3-ol, 3-methylthiopropanol, phenylacetaldehyde, 2-methoxy-4-vinylphenol, eugenol, ethyl octanoate, ethyl isovalerate, and ethyl acetate were significantly increased. At the same time, the contents of total free amino acids, umami amino acids, and sweet amino acids were also significantly increased. The increase of these compounds can significantly enhance the aroma and fruit ester aroma of the fermented soybean paste, enhance its fresh, sweet, and mellow taste, not only improve the overall aroma and flavor of the fermented soybean paste, but also make its flavor and taste more rich, complex, harmonious, and layered, significantly improving the overall flavor and quality of the fermented soybean paste.

[0092] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A compound fermentation agent, characterized in that, The fermentation agent comprises ZB445 of *Zygosaccharomyces rouxii* with accession number GDMCC NO: 66356 and ZF629 of *Westernia fusionis* with accession number GDMCC NO: 66357.

2. A compound fermentation method, characterized in that, The bacteria inoculated by the fermentation method include the *Zygosaccharomyces rouxii* ZB445 and *Weissella fusionis* ZF629 as described in claim 1.

3. The application of the fermentation agent according to claim 1 or the compound fermentation method according to claim 2 in fermented food processing.

4. The application as described in claim 3, characterized in that, The fermented food is any one or more fermented legume foods.

5. The application as described in claim 4, characterized in that, The fermented soybean product is broad bean paste.

6. A method for preparing fermented soybean paste, characterized in that, The method involves adding the fermentation agent described in claim 1 during the fermentation process of fermented soybean paste.

7. The preparation method according to claim 6, characterized in that, The fermentation agent is added during the early stage of fermentation of the soy sauce mash.

8. The preparation method according to claim 6, characterized in that, The amount of fermentation agent added is 10. 5 ~10 7 CFU / mL.

9. The preparation method according to claim 6, characterized in that, The ratio of Zygosaccharomyces rouxii ZB445 to Zygosaccharomyces fusionis ZF629 in the fermentation agent is 1:

1.

10. A type of fermented soybean paste, characterized in that, The fermented soybean paste is prepared by any one of the preparation methods described in claims 6-9.

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