Aspergillus oryzae strain ZA305 and application thereof

Abstract

This invention relates to the field of microbial technology, specifically to a strain of Aspergillus oryzae (… Aspergillus oryzae ZA305 and its applications. This invention uses Aspergillus oryzae strain Hu Niang 3.042 as the starting strain for ARTP mutagenesis. Through a multi-level directional screening method guided by flavor compounds, a strain of Aspergillus oryzae, ZA305, was obtained. This strain, when used in the fermentation of fermented black beans and broad bean paste, can significantly enhance the activity of key enzymes in the fermentation starter, increase the content of free amino acids for sweetness, soy sauce aroma, and fruit aroma key flavor substances in the product, and significantly reduce the content of undesirable flavor substances. It has a dual effect of enhancing aroma and suppressing defects, and can significantly improve the flavor, taste, and aroma harmony of fermented black beans and broad bean paste, significantly enhancing their soy sauce aroma, ester aroma, and umami flavor, and effectively reducing off-flavors, thereby comprehensively improving the quality of the product.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Aspergillus oryzae ZA305 and its applications. Background Technology

[0002] my country has a long history of traditional fermented soybean products, with fermented black beans (douchi) and broad bean paste (doubanjiang) being representative examples. Due to their unique flavors and rich nutritional value, they hold an important place in Chinese culinary culture. Volatile flavor is one of the most direct attributes influencing product quality and consumer acceptance of fermented black beans and broad bean paste. The volatile flavor compounds in fermented black beans and broad bean paste mainly include esters, alcohols, aldehydes, pyrazines, and acids, exhibiting excellent flavors such as soy sauce aroma, fermented black bean aroma, wine aroma, sweet aroma, and smoky flavor, as well as off-flavors such as musty and fishy odors. The aroma profile of traditional fermented products largely depends on the growth and metabolism of microorganisms during fermentation.

[0003] Aspergillus oryzae plays a crucial role in the fermentation of fermented black beans and broad bean paste, serving as the starting strain. During the koji-making process, Aspergillus oryzae secretes abundant proteases, peptidases, glutaminases, and amylases, breaking down large protein and starch molecules in the raw materials into smaller molecules such as polypeptides, amino acids, and glucose, providing essential nutrients for the growth of other microorganisms. Simultaneously, these products, through the combined action of various microorganisms and environmental factors, produce flavor compounds such as alcohols, aldehydes, esters, and phenols during fermentation, becoming important components of the flavor of fermented black beans and broad bean paste. Currently, the Aspergillus oryzae strain Hu Niang 3.042 is widely used in the industrial production of fermented black beans and broad bean paste due to its stable protease and amylase system. However, long-term reliance on a single strain has directly led to the problem of product flavor convergence and severe homogenization, making it difficult to meet consumers' growing demand for flavor diversity.

[0004] In recent years, some studies have also focused on enhancing the flavor of fermented black beans or broad bean paste by increasing the protease activity of Aspergillus oryzae. For example, patent CN201810134200.4 discloses an Aspergillus oryzae strain PCSM002 and its application. This strain has a fast growth rate and strong protease production capacity. When used to make broad bean paste, it significantly improves the koji-making speed and amino nitrogen content, thus improving the quality and flavor of the product. However, this study only focuses on protease activity and growth characteristics, neglecting the activity of enzymes directly related to flavor, such as glutaminase and aminopeptidase. Furthermore, while amino nitrogen content is highly positively correlated with the total content of various amino acids, a high content does not necessarily mean excellent umami, sweetness, or other flavor indicators. Breeding a new Aspergillus oryzae strain that can simultaneously enhance the activity of multiple key enzymes and significantly optimize the composition of terminal flavor compounds will provide core germplasm resources for breaking through the bottleneck of flavor homogenization in fermented soybean products. 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. By using a multi-level targeted screening method guided by flavor compounds, a strain of Aspergillus oryzae, ZA305, was obtained. This strain has high activity of key enzymes such as glutaminase and aminopeptidase in koji making. In the fermentation of fermented black beans and broad bean paste, it can significantly increase the content of umami-sweet free amino acids, soy sauce aroma, and fruit aroma key flavor substances, while significantly reducing the content of undesirable flavor substances. It has a dual effect of enhancing aroma and suppressing defects, and significantly improves product quality.

[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 strain of Aspergillus oryzae ZA305, the preservation number of which is GDMCC No: 65503.

[0008] The strain was deposited on November 19, 2024 at the Guangdong Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0009] Secondly, the present invention provides a fermentation agent comprising Aspergillus oryzae ZA305.

[0010] Thirdly, the present invention provides the application of the above-mentioned Aspergillus oryzae ZA305 or fermentation agent in fermented condiments. The condiments mainly refer to fermented condiments that produce flavor by fermentation using microorganisms and their enzymes, such as soy sauce, sauce, vinegar, fermented black beans or fermented bean curd, especially fermented bean products, preferably fermented black beans or broad bean paste.

[0011] Fourthly, the present invention provides a method for preparing fermented black beans or broad bean paste, the method comprising the step of fermentation using the above-mentioned Aspergillus oryzae ZA305 or fermentation agent, preferably the Aspergillus oryzae ZA305 or fermentation agent is used in the koji-making step.

[0012] 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.

[0013] Furthermore, the amount of Aspergillus oryzae ZA305 added to the fermented soybeans is 2-4% of the weight of dried soybeans, and the amount of Aspergillus oryzae ZA305 added to the broad bean paste is 0.5-2‰ of the weight of dried broad beans.

[0014] Furthermore, the fermentation time is 25-35 days.

[0015] Fifthly, the present invention provides a fermented black bean paste or broad bean sauce, wherein the fermented black bean paste or broad bean sauce is prepared by any of the preparation methods 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. This application uses Aspergillus oryzae Hu Niang 3.042 as the starting strain for ARTP mutagenesis, and obtains the Aspergillus oryzae enzyme mutant strain ZA305 through flavor-oriented multi-level directional screening. It produces high levels of glutaminase and can be used for fermentation of fermented black beans and broad bean paste. It can significantly improve the activity of key enzymes in the fermentation process, increase the content of key flavor compounds such as free amino acids for sweetness, soy sauce aroma, and fruit aroma, and significantly reduce the content of undesirable flavor compounds. It has a dual effect of enhancing aroma and suppressing defects, and can significantly improve the flavor, taste, and aroma harmony of fermented black beans and broad bean paste, significantly enhancing their soy sauce aroma, ester aroma, and umami flavor, and effectively reducing off-flavors, thereby comprehensively improving the quality of the product.

[0018] 2. The Aspergillus oryzae strain ZA305 provided by this invention produces fermented black beans and broad bean paste with glutaminase activity increased by more than 20% and aminopeptidase activity increased by more than 70% compared with Hu Niang 3.042, thus ensuring the efficient generation of umami-sweet amino acids from a mechanistic perspective. The fermented fermented black beans and broad bean paste products have increased content of umami-sweet substances, with umami amino acid content increased by 9.8% and 14.9% respectively compared with the Aspergillus oryzae Hu Niang 3.042 group, and sweet amino acid content increased by 13.0% and 10.5% respectively compared with the Aspergillus oryzae Hu Niang 3.042 group, thus enhancing the umami-sweetness of the products.

[0019] 3. The Aspergillus oryzae strain ZA305 provided by this invention can significantly improve the flavor profile of fermented black beans and broad bean paste. Compared with the Aspergillus oryzae Hu Niang 3.042 fermentation group, the content of 2,6-diethylpyrazine, a key flavor compound for soy sauce aroma, is increased by 84.6% and 146.9%, respectively; the content of 2-methylpyrazine is increased by 68.3% and 91.7%, respectively; the content of ethyl acetate, a key flavor compound for fruit aroma, is increased by 140.1% and 42.1%, respectively; the content of ethyl phenylacetate, a key flavor compound for floral and honey aroma, is increased by 75.0% and 62.6%, respectively; and the content of 4-vinylguaiacol, a key flavor compound for smoky aroma, is increased by more than 72.7%, with an increase of up to 295.1% in broad bean paste products. This makes the overall aroma more complex and avoids the monotonous and dull aroma of traditional fermented products.

[0020] 4. The Aspergillus oryzae strain ZA305 provided by this invention can effectively inhibit the generation of undesirable flavors in fermented black beans and broad bean paste. The hexanal content in fermented black beans and broad bean paste fermented with ZA305 decreased by 61.7% and 70.0% respectively compared with the Aspergillus oryzae Hu Niang 3.042 group, effectively reducing the raw and stale smell of the product and ensuring a pure and mellow flavor.

[0021] The Aspergillus oryzae ZA305 strain provided by this invention was deposited at the Guangdong Microbial Culture Collection Center on November 19, 2024, at the address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC NO: 65503. The strain was received and registered by the collection center on November 19, 2024, and was confirmed to be a viable strain by the collection center on November 19, 2024. Detailed Implementation

[0022] 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.

[0023] 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.

[0024] Unless otherwise specified, the reagents, methods and equipment used in this invention are conventional food-grade reagents, methods and equipment in the art.

[0025] 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.

[0026] Example 1: Screening of the target strain

[0027] 1. Aspergillus oryzae in Shanghai brewing 3.042 ARTP mutagenesis

[0028] Aspergillus oryzae var. oryzae 3.042 was inoculated onto YPD agar plates using the spot inoculation method and cultured at 30°C for 48 h until spores matured. 2 mL of physiological saline was added to the medium, and spores were scraped off using an L-shaped spreader to suspend them and collected in 2 mL centrifuge tubes. The spore suspension was diluted to an appropriate concentration, and after counting using a hemocytometer, the spore concentration in the original solution was diluted to 2 × 10⁻⁶. 8 CFU / mL. Prepare a sterile 10% (V / V) glycerol and spore suspension by mixing thoroughly at a 1:1 ratio. Take 10 μL of the mixture and spread it onto a sterile slide, then transfer the slide to a sterile stage.

[0029] ARPT mutagenesis conditions: Helium was used as the working gas, the distance between the slide and the plasma emitter port was 2 mm, the power was 120 W, the gas flow rate was 10 SLM, and the mutagenesis time was 150 s.

[0030] 2. Preliminary screening of bacterial strains

[0031] After ARTP mutagenesis, the spore suspension was diluted to an appropriate concentration and placed in a Miss Cell device to form 4500 droplets in a culture tube. After 36 h of incubation, the droplets were sorted by OD and transferred to a sterile 96-well plate for the next round of screening. The first stage of droplet sorting yielded 434 monoclonal droplets.

[0032] 100 μL of YPD medium was added to a 96-well microplate and cultured with shaking at 30℃ and 220 rpm for 48 h. Then, iGluSnFR (final concentration 0.2 μM, reaction at room temperature for 2 h) and glutamine solution (final concentration 2 mM) were added to detect glutaminase activity. The results showed that 21 mutant strains with high fluorescence signals were obtained.

[0033] 3. Secondary screening of strains

[0034] A secondary screening was conducted by determining the glutamate content and glutaminase activity in the finished koji samples using shallow tray koji preparation. The 21 mutant strains obtained from the initial screening, along with Hu Niang 3.042, were prepared in shallow trays (soybean:flour = 10:1) and incubated at 35℃ and 80% humidity for 72 h. The glutamate content and glutaminase activity of the finished koji samples were then measured, and mutant strains with glutaminase activity exceeding 10% compared to Hu Niang 3.042 were selected.

[0035] Table 1. Results of simulated fermentation of fermented soybeans using Aspergillus oryzae mutant strain.

[0036]

[0037] As shown in Table 1, three mutant strains exhibited glutaminase activity exceeding 10% in their fermented soybean paste compared to the Hu Niang 3.042 group, and also showed a certain degree of increase in glutamate content. Among them, mutant strain HT-456 showed a 20.4% increase in glutaminase activity and the highest corresponding glutamate content (3.83 g / kg), a 9.3% increase compared to the Hu Niang 3.042 group. The results indicate that these three mutant strains have the potential to enhance the umami flavor of fermented soybean products. However, functional improvements in the strains do not necessarily translate to improved flavor quality in traditional fermented sauces, especially the "soulful aroma" of soy sauce. Therefore, further flavor screening of these three mutant strains is necessary.

[0038] Studies have shown that pyrazine compounds are considered the main components of the aroma in traditional fermented sauces such as fermented black beans and broad bean paste. Among them, 2,6-diethylpyrazine has a low threshold (6.0 μg / kg in water), and even slight changes in its content can lead to different sensory experiences. Ester compounds play an important role in contributing pleasant aromas such as ester aromas and fruity aromas, effectively enhancing the complexity and freshness of the product's aroma. Ethyl acetate is a typical ester compound representing fruity aromas. In addition, hexanal is considered a product of lipid oxidation, which brings unpleasant raw and stale flavors and is an undesirable flavor compound that compromises the purity of the product. Therefore, the changes in the content of 2,6-diethylpyrazine, ethyl acetate, and hexanal in fermented samples are the focus of this study, and these changes are used as standards for flavor-oriented rescreening.

[0039] Take the fermented soybean starter prepared in the previous step, add salt water to the starter in a certain proportion, then package the sample into sealed bags and carry out simulated fermentation in an environment of 30℃ for 5 days. Take the fermentation sample for headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) analysis to determine the contents of 2,6-diethylpyrazine, ethyl acetate and hexanal.

[0040] Table 2. Flavor screening results of Aspergillus oryzae mutant strains

[0041]

[0042] As shown in Table 2, in the fermentation samples of the HT-456 group, the contents of 2,6-diethylpyrazine (16.74 μg / kg) and ethyl acetate (67.76 μg / kg) were increased by 138.4% and 140.1% respectively compared with the Hu Niang 3.042 group, while the content of hexanal (13.47 μg / kg) was decreased by 61.7% compared with the Hu Niang 3.042 group. In summary, the mutant strain HT-456 obtained by screening has the potential to enhance freshness and aroma and suppress undesirable flavors.

[0043] The strain HT-456 obtained from the above screening was named Aspergillus oryzae ZA305 and was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 19, 2024, with the accession number GDMCC NO: 65503.

[0044] Example 2: Test on the fermentation effect of Aspergillus oryzae ZA305 on fermented soybeans

[0045] Aspergillus oryzae ZA305 and Hu Niang 3.042 were used for fermenting soybeans, with Aspergillus oryzae ZA305 serving as the experimental group and Hu Niang 3.042 as the control group. The fermentation process was as follows: Selected soybeans were cleaned and soaked in twice their weight of purified water for 3 hours. After removing excess water, the soybeans were steamed at 115℃ for 20 minutes. After cooling the soybeans to room temperature, the two materials were mixed evenly at a ratio of 10:1 (w / w) of dry soybeans to flour. Then, 3% of the dry soybean weight of Aspergillus oryzae starter was added and mixed evenly. The mixture was then placed in an environment with a constant temperature and humidity of 35℃ and 80%, and the starter was turned over every 12 hours until 48 hours when the soybean surface was covered with yellow-green spores that flew up when gently turned. This indicated the end of the starter-making process. Then, according to the Taihe fermented soybean production method, salt, spices, water, and other auxiliary materials were added to the starter in proportion, stirred evenly, and allowed to stand for 24 hours. The starter was stirred every 12 hours during this period. After the fermentation process is completed, the samples are packaged into sealed bags and left to ferment at 30°C for 30 days.

[0046] 1. Analysis of the activity of key enzymes in the fermentation process

[0047] After the koji-making process, the fermented koji from both strains was extracted with a phosphate buffer solution at pH 7.2 for 1 h, and the extracts were used to determine the activity of key enzymes. The results are shown in Table 3. The data show that the glutamine enzyme activity of the Aspergillus oryzae ZA305 group was significantly increased by 14.0% compared with the Hu Niang 3.042 group, which is directly related to the potential for the conversion of glutamine to glutamic acid (umami) in the koji. At the same time, its aminopeptidase activity reached 268.28 U / g, which was 71.0% higher than that of the Hu Niang 3.042 group. This enzyme is responsible for releasing free amino acids from the ends of polypeptide chains, especially hydrophobic and sweet amino acids. The enhancement of its activity is crucial for reducing bitter peptides and increasing the content of sweet amino acids (such as alanine and serine). Furthermore, it is worth noting that the protease and amylase activities of the Aspergillus oryzae ZA305 group were increased by 51.0% and 28.1% respectively compared with the Hu Niang 3.042 group. This indicates that while the mutant strain acquired new characteristics, its core basic metabolic enzyme system was not negatively affected, but rather improved to a certain extent. This can achieve a synergistic effect of multiple enzyme systems, ensuring the stability of its koji-making efficiency.

[0048] Table 3 Comparison of key enzyme activities in fermented soybean koji production

[0049]

[0050] 2. Analysis of the content of key flavor compounds in fermented black soybeans

[0051] After fermentation, the fermentation products of the two strains of fermented soybeans were taken to determine the content of free amino acids with umami flavor, and the content of volatile flavor substances in the products was measured by GC-MS. The results are recorded in Table 4-5.

[0052] Table 4 Comparison of the content of amino acids that enhance the sweetness of fermented soybeans

[0053]

[0054] As shown in Table 4, the Aspergillus oryzae ZA305 fermentation group exhibited certain advantages in the accumulation of flavor precursors. In its fermentation products, the content of the core umami amino acid glutamic acid increased by 11.5% compared to the Hu Niang 3.042 group, which is consistent with its high glutaminase activity. The aspartic acid content also increased to some extent, resulting in a 9.8% increase in the total umami amino acid content. Simultaneously, the contents of alanine, glycine, and serine, all sweet amino acids, were also increased, with a total increase of 13.0%. This may be attributed to the enhanced aminopeptidase activity and optimized amino acid metabolic network.

[0055] Table 5 Comparison of Key Volatile Flavor Compound Contents

[0056]

[0057] As shown in Table 5, the determination results of key volatile flavor compounds revealed that strain ZA305 has a strong ability to reshape the aroma profile of fermented black beans. First, the content of volatile compounds that positively enhance the aroma of black beans increased significantly compared with the Hu Niang 3.042 group. The content of pyrazine compounds such as 2,6-diethylpyrazine and 2-methylpyrazine increased by 84.6% and 68.3%, respectively; the content of ester compounds such as ethyl acetate and ethyl phenylacetate increased by 140.1% and 75.0%, respectively; and the content of 4-vinylguaiacol reached 115.92 μg / kg, an increase of 72.7% compared with the previous group. Pyrazines and phenolic compounds constitute the "soul aroma" of traditional sauce products—soy sauce aroma and roasted aroma; while the doubling of ester compounds introduces fresh fruity and honey aromas. The significant increase in the content of these substances makes the overall aroma of the product richer and avoids the monotony and dullness of the aroma of traditional black bean products. Secondly, undesirable flavor compounds were effectively suppressed. Hexanal, a hallmark product of lipid oxidation, brings a raw, grassy taste and an oxidized, stale aroma. Its content in the ZA305 group was only 13.47 μg / kg, a 61.7% reduction compared to the Shanghai Brewing 3.042 group. This may be because ZA305 effectively inhibited the oxidative rancidity pathway by regulating its own lipid metabolism or antioxidant enzyme system. This directly translates into a reduction in the raw, grassy taste and an enhancement of the "mellow" flavor in the product's sensory presentation. This dual effect of "enhancing aroma and suppressing defects" is the core mechanism by which the mutant strain of this invention achieves improved flavor and quality.

[0058] 3. Sensory evaluation

[0059] A panel of 24 professionals (12 men and 12 women, aged 24-35) specializing in sensory evaluation of fermented foods was formed to score the umami, sweetness, savory aroma, and overall liking of the fermented soybeans. The scoring range was 1-5 points, with higher scores indicating stronger sensory attributes. The average score of each sensory attribute was taken as the final score, and the results are recorded in Table 6.

[0060] Table 6 Sensory evaluation results of fermented soybeans

[0061]

[0062] Sensory evaluation is the ultimate reflection of the above physicochemical indicators and volatile flavor compound detection data. As shown in Table 6, the fermented black beans of group ZA305 are superior to those of group Hu Niang 3.042 in terms of umami, sweetness, and soy sauce aroma, especially in umami and soy sauce aroma, which is consistent with the detection results of flavor compound content. The overall preference is also significantly better than that of group Hu Niang 3.042, indicating that Aspergillus oryzae ZA305 has a great improvement on the overall flavor of fermented black beans.

[0063] Example 3: Test on the fermentation effect of Aspergillus oryzae ZA305 on fermented broad bean paste

[0064] Doubanjiang (fermented broad bean paste) was brewed using Aspergillus oryzae strain ZA305 and Hu Niang 3.042. First, broad beans were thoroughly washed and soaked in water for 3 hours. After soaking, the broad beans were removed and drained. Flour was then mixed in according to a ratio of dry broad beans to flour (dry broad beans: flour = 3:1, calculated by weight), and the mixture was thoroughly combined. Then, Aspergillus oryzae strain ZA305 and Hu Niang 3.042 were inoculated separately according to a ratio of dry broad beans to mold (1000:1, calculated by weight). The thoroughly mixed koji was placed in a constant temperature and humidity incubator, with the temperature controlled within the range of 28-32℃ and the relative humidity maintained at 90%, and cultured for 40-48 hours. Once the surface of the broad beans was covered with yellow-green spores, the koji was ready. The glutaminase activity in the starter culture was measured by sampling. Then, the prepared starter culture was mixed with brine at a volume ratio of 1:1.5 and fermented for 30 days. The free amino acid and volatile flavor compound content of the sample after fermentation was measured and sensory evaluation was performed according to the method described in Example 2. The results are recorded in Tables 7-9.

[0065] Table 7 Comparative Analysis of Glutaminase Activity and Free Amino Acids

[0066]

[0067] As shown in Table 7, during the fermentation process, the glutaminase activity of the ZA305 group reached 4.05 U / g, which was 22.1% higher than that of the Hu Niang 3.042 group. This indicates that the mutant strain ZA305 also has the potential to enhance the umami flavor in fermented broad bean paste. The measurement results of core umami amino acids in the fermented broad bean paste samples also showed that the glutamate content increased by 22.6% compared to the Hu Niang 3.042 group, which is completely consistent with the characterization of high glutaminase activity in ZA305. In addition, the contents of various sweet amino acids increased by 4.3%-14.9% compared to the Hu Niang 3.042 group, with a total increase of 10.5%.

[0068] Table 8 Comparison of Key Volatile Flavor Compound Contents

[0069]

[0070] As shown in Table 8, the results of the determination of volatile flavor compounds revealed that strain ZA305 can also play a role in enhancing aroma and suppressing defects during the fermentation of fermented soybean paste. First, the contents of pyrazine compounds, such as 2,6-diethylpyrazine and 2-methylpyrazine, which are key substances representing the aroma of fermented soybean paste, increased by 146.9% and 91.7% respectively compared with the Hu Niang 3.042 group; the content of ethyl acetate, which imparts fruity and sweet aromas to the product, reached 127.38 μg / kg, an increase of 42.1%; the content of ethyl phenylacetate, which imparts floral and honey aromas to the product, reached 25.69 μg / kg, an increase of 62.6%; 4-vinylguaiacol, which imparts a smoky aroma to traditional fermented soybean paste, reached 171.03 μg / kg in the ZA305 fermented sample, an increase of 295.1%. The significant increase and synergistic effect of these substances can give fermented soybean paste a richer and more complex aroma. Secondly, the content of hexanal, which brings a raw, grassy, ​​oxidized, and stale odor, is reduced by 70.0%, which can effectively ensure the purity, richness, and stability of the flavor of fermented soybean paste products.

[0071] Table 9 Sensory evaluation results of fermented broad bean paste

[0072]

[0073] As shown in Table 9, the sensory evaluation results show that the fermented broad bean paste of the ZA305 group is significantly better than that of the Hu Niang 3.042 fermentation group in terms of umami, sweetness, soy sauce aroma and overall liking. This indicates that the mutant strain ZA305 has a great improvement on the flavor of the fermented broad bean paste product. The use of this strain in fermenting broad bean paste can significantly improve the flavor and overall quality of the broad bean paste.

[0074] In summary, fermenting fermented black beans or broad bean paste with Aspergillus oryzae ZA305 can significantly enhance the activity of key enzymes in the prepared koji, thereby significantly increasing the content of sweet and umami amino acids and key volatile flavor compounds in the fermented black beans or broad bean paste. In particular, the content of 2,6-diethylpyrazine, 2-methylpyrazine, ethyl acetate, ethyl phenylacetate, and 4-ethyl-guaiacol is significantly increased, while the content of hexanal, which has a raw, grassy, ​​and oxidized stale odor, is significantly reduced. The changes in the content of these compounds can significantly improve the flavor, texture, and aroma harmony of fermented black beans and broad bean paste, significantly enhancing their soy sauce aroma, ester aroma, and umami flavor, and effectively reducing off-flavors, thus comprehensively improving the quality of the product.

[0075] 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 strain of Aspergillus oryzae ( Aspergillus oryzae ZA305, characterized in that, The preservation number of Aspergillus oryzae ZA305 is GDMCC No: 65503.

2. A fermentation inoculant characterized in that, The fermentation agent comprises Aspergillus oryzae ZA305 as described in claim 1.

3. The application of Aspergillus oryzae ZA305 as described in claim 1 or the fermentation agent as described in claim 2 in fermented condiments, wherein the fermented condiment is fermented black beans or broad bean paste.

4. A method of preparing tempeh or kecap, characterized by, The method includes the step of fermentation using Aspergillus oryzae ZA305 as described in claim 1 or the fermentation agent as described in claim 2.

5. The preparation method according to claim 4, characterized in that, The Aspergillus oryzae ZA305 or fermentation agent is used in the koji-making process.

6. The preparation method according to claim 4, characterized in that, The amount of Aspergillus oryzae ZA305 added to the fermented soybeans is 2-4% of the weight of the dried soybeans.

7. The preparation method according to claim 4, characterized in that, The amount of Aspergillus oryzae ZA305 added to the fermented broad bean paste is 0.5-2‰ of the weight of the dried broad beans.

8. The preparation method according to claims 4-7, characterized in that, The fermentation time is 25-35 days.

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