Aspergillus oryzae and application thereof

Abstract

The invention provides Aspergillus oryzae and application thereof, the strain name of the Aspergillus oryzae is Aspergillus oryzae ZD01, the Aspergillus oryzae is preserved in China Center for Type Culture Collection on April 11, 2025, the preservation address is Wuhan University, Wuhan, Hubei, China, and the preservation number is CCTCC NO: M 2025761. The thick broad-bean sauce prepared by the strain does not generate white spots, and the thick broad-bean sauce fermented by the aspergillus oryzae has obvious advantages in flavor and color. The prepared thick broad-bean sauce does not generate white spots, and the thick broad-bean sauce fermented by the aspergillus oryzae has obvious advantages in flavor and color.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and particularly relates to Aspergillus oryzae and application thereof. Background Art

[0002] Doubanjiang has won the favor of consumers with its unique flavor. Aspergillus oryzae ) Shanghai Brewing 3.042 was artificially inoculated and fermented. This strain is prone to produce white spots during the fermentation process, and the white spots are mainly caused by the precipitation of tyrosine crystals. Since the existing strains have the defect of producing white spots, screening a strain of Aspergillus oryzae that does not produce white spots is of great value in improving the quality of bean paste. In the production process of Hubei bean paste, natural materials such as yellow vitex leaves and rice straw are usually used for covering and paving to achieve natural koji fermentation. The mold substances in the koji making process mainly come from yellow vitex leaves and rice straw, and no "white spots" appear in the final product. However, the traditional bean paste production process is easily affected by a variety of uncertain factors, making it difficult to stably control the product quality. As the heat preservation fermentation process gradually replaces the traditional natural fermentation process, in order to overcome the defects of the existing process, it is urgent to screen and obtain a high-quality Aspergillus oryzae strain that does not produce white spots to meet the needs of artificial inoculation heat preservation fermentation process, thereby improving the stability of product quality. Summary of the Invention

[0003] In view of this, the present invention provides an Aspergillus oryzae and an application thereof, wherein the prepared broad bean paste does not produce white spots, and the broad bean paste fermented by Aspergillus oryzae has obvious advantages in flavor and color.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a strain of Aspergillus oryzae, the strain of which is named Aspergillus oryzae ZD01 was deposited in the China Center for Type Culture Collection on April 11, 2025, with the deposit address being Wuhan University, Wuhan, Hubei Province, China, with the deposit number being CCTCC NO: M 2025761.

[0005] In a second aspect, the present invention provides a method for preparing the Aspergillus oryzae, wherein the broad bean koji used in the fermentation process of Hubei bean paste is placed on the surface of a culture medium for culture, purification and screening to obtain a single strain of bacteria.

[0006] Preferably, the culture medium is potato dextrose agar medium.

[0007] Preferably, it is placed on the surface of the culture medium for culture, purification and screening, and the culture temperature is 28°C.

[0008] Preferably, the culture time is 72 hours.

[0009] In a third aspect, the present invention provides a fermentation agent, which comprises the Aspergillus oryzae or the Aspergillus oryzae prepared by the preparation method.

[0010] In a fourth aspect, the present invention provides a use of the Aspergillus oryzae or the fermentation agent in the preparation of Hubei bean paste.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a kind of Aspergillus oryzae, the strain is named Aspergillus oryzae ZD01 was deposited with the China Center for Type Culture Collection at Wuhan University, Wuhan, Hubei Province, China, on April 11, 2025, with the accession number CCTCC NO: M 2025761. The fermented broad bean paste produced by this strain does not produce white spots, and the fermented broad bean paste produced by Aspergillus oryzae has significant advantages in flavor and color. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Figure 7 of Aspergillus oryzae colony morphology provided by the present invention; Figure 2 A diagram showing the white spot production in fermented mash using seven strains of Aspergillus oryzae screened by the present invention and Huniang 3.042; Figure 3 This is a diagram showing the white spot situation of fermented broad bean paste produced by seven strains of Aspergillus oryzae screened out by the present invention and Huniang 3.042; Figure 4 The present invention provides Figure 3 The strain morphology of strain No. 3 in ; Figure 5 The present invention provides Figure 3 The strain morphology of strain No. 6 in ; Figure 6 The present invention provides Figure 3 The strain morphology of strain No. 7 in ; Figure 7 The present invention provides Figure 3 Phylogenetic tree diagram of strain No. 3; Figure 8 The present invention provides Figure 3 Phylogenetic tree of strain No. 6; Figure 9 The present invention provides Figure 3 Phylogenetic tree of strain No. 7; Figure 10 A comparison chart of the growth curves of Aspergillus oryzae Nos. 3, 6, 7 and 3.042 provided by the present invention; Figure 11This is a graph showing the effect of different salinities on the growth of Aspergillus oryzae strains 3 (a), 6 (b), 7 (c) and Shanghai Brewing 3.042 (d) provided by the present invention; Figure 12 This is a graph showing the effect of different pH values ​​on the growth of Aspergillus oryzae strains 3 (a), 6 (b), 7 (c) and Shanghai Niang 3.042 (d) provided by the present invention; Figure 13 This is a diagram showing the effect of different temperatures on the growth of Aspergillus oryzae No. 3 (a), 6 (b), 7 (c) and Shanghai Brewing 3.042 (d) provided by the present invention; Figure 14 This is a graph showing changes in the amino acid nitrogen content during the mixed fermentation process of broad bean paste provided by the present invention; Figure 15 This is a graph showing the changes in total acid content (a) and pH (b) during the mixed fermentation process of broad bean paste provided by the present invention; Figure 16 This is a graph showing changes in reducing sugar content (a) and salinity (b) during the mixed fermentation process of broad bean paste provided by the present invention; Figure 17 This is a statistical chart of the number of different types of volatile flavor substances fermented by four types of Aspergillus oryzae provided by the present invention; Figure 18 This is a statistical chart of the relative contents of different types of volatile flavor substances fermented by four types of Aspergillus oryzae provided by the present invention; Figure 19 This is a clustering heat map of the volatile flavor substances in four types of bean paste provided by the present invention; Figure 20 The present invention provides the composition and relative content of various flavor amino acids in four different bean pastes.

[0013] Note: Figure 1 The 7 kinds of Aspergillus oryzae powder correspond to the 7 numbers in the figure; Figure 2 Among them, the 7 species of Aspergillus oryzae correspond to the 7 numbers in the figure, number 8 is Shanghai Niang 3.042; Figure 4-6 In the figure, (a) is the colony morphology, (b) is the microstructure 100X image, and (c) is the microstructure 400X image; Figure 10 、 14 , 15, 16, 17, and 18, 3 is strain 3, 6 is strain 6, 7 is strain 7, and 8 is Shanghai Brewing 3.042; Figure 20 Figures (a), (b), (c), and (d) are bean pastes containing strains 3, 6, 7, and Shanghai Brewing 3.042, respectively. DETAILED DESCRIPTION

[0014] The present invention will be further described in detail below with reference to specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0015] Experimental Materials Broad bean koji: provided by Hubei Lianshi Food Co., Ltd. Aspergillus oryzae Shanghai brewing 3.042 koji essence: purchased from Yiyuan Kangyuan Biotechnology Co., Ltd.; Douban: Kemi Manor Trading Co., Ltd. Bran: purchased from Taobao; Fermented chili mash: provided by Hubei Lianshi Food Co., Ltd.

[0016] Example 1 Isolation and purification of strains Following the experimental method of Zhang Wenping (2019) and improving upon it, sterilized potato dextrose agar (PDA) medium was first poured into a disposable Petri dish in a clean bench and allowed to cool naturally and solidify. Subsequently, sterilized tweezers were used to precisely pick the broad bean koji used in the fermentation of Hubei doubanjiang (fermented bean paste) and place it on the surface of the PDA medium. After completing these steps, the Petri dish was placed in a constant-temperature biochemical incubator set at 28°C for 72 hours of constant-temperature incubation. After the incubation period, the colony morphology on the culture medium was carefully observed, and mold strains of varying morphology were selected for further purification until only a single species remained on the PDA medium.

[0017] Example 2 Pure strain back-inoculation fermentation test 1. Making koji To prepare seed koji, bran and water were mixed at a ratio of 1.0:1.0 (g:mL). The mixture was placed in a conical flask and sealed with parafilm and newspaper. The flask was then sterilized in a vertical steam autoclave at 121°C for 20 minutes. After cooling to room temperature, the isolated mold strain was picked using a sterilized inoculating loop, with Shanghai Niang 3.042 as a control, and inoculated into the bran mixture. The inoculated flask was incubated in a 30°C incubator for 72 hours. After the incubation period, the mature seed koji was collected. To prepare broad bean koji, broad bean petals were blanched in boiling water for 2 minutes, then quickly removed and drained. The petals were then allowed to cool naturally to approximately 40°C. 15% flour (based on dry bean petals) was added to the cooled broad bean petals, mixed, and 3% seed koji was added. The mixture was then spread onto a koji plate to a suitable thickness to facilitate ventilation and mycelial growth. The koji tray is placed in a constant-temperature incubator set at 30°C for 72 hours. During the incubation process, the koji material is turned over every 24 hours to ensure uniform fermentation and promote mycelial growth and metabolism. After 72 hours of incubation, the mature broad bean koji will have lush yellow-green spores on its surface, indicating that the koji preparation process is complete and the koji is ready for subsequent fermentation processes (Li Feng et al. 2012).

[0018] 2. Fermentation Fermentation of the fermented mash: Wash the prepared broad bean koji with clean water, dry it, and place it in a fermentation tank. Add an appropriate amount of brine to the fermentation tank, adjusting the salt concentration to 13%. Seal the fermentation tank and place it in a constant-temperature incubator at 45°C for fermentation. Stir the mixture every five days for a total of 25 days. Throughout the fermentation process, regularly observe changes in the surface of the mash, especially observing for the formation of white spots. After 25 days of fermentation, the mature mash will have a unique flavor and texture, and the fermentation process is complete.

[0019] Mixed Fermentation: The fermented fermented soybean paste and fermented chili paste are mixed in a ratio of 10:7. Appropriate amounts of monosodium glutamate, spices, and sugar are then added and mixed thoroughly. The mixed ingredients are then placed in a fermentation tank and placed in a 30°C incubator for 25 days. Stir regularly and monitor the temperature and condition of the ingredients. The fermentation is complete, marking the finished bean paste as mature. The finished sauce is inspected for white spots.

[0020] Performance Testing 1. Fungal morphological observation Referencing the method of Zhao Ying et al. (2018) with appropriate adjustments, use a sterilized inoculating loop to select mold colonies of varying morphology and inoculate them onto PDA culture medium. After incubating the culture dish in a constant-temperature incubator for 72 hours, observe the mold colony morphology. Once the colony matures, use an inoculating loop to remove a piece of the colony from the culture medium and transfer it to a PDA plate. Subsequently, sterilized coverslips are inserted obliquely into the PDA culture medium, with 3-4 coverslips inserted per plate. The plates are then placed in a constant-temperature incubator at 28°C and incubated for approximately 72 hours. After incubation, observe the morphology and characteristics of the spores and hyphae on the coverslips under a microscope to determine the mold species, and record the observations in detail.

[0021] 2. Molecular biological identification of mold Genomic DNA from the isolated fungus was extracted using a Meiji magnetic bead DNA extraction kit. The ITS fragment of the fungus was amplified using universal primers ITS1 / ITS4 using this genomic DNA as a template for polymerase chain reaction (PCR). The total PCR reaction volume was 30 μL and consisted of 10 μL of ddH2O, 2 μL of each primer, 1 μL of DNA template, and 15 μL of 2× Taq PCR Master Mix. Amplification conditions were as follows: initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec, followed by a final extension at 72°C for 5 min. To ensure the specificity of the PCR amplification, 2 μL of the PCR product was analyzed by electrophoresis on a 1% agarose gel. The specificity of the amplified product was determined by observing the electrophoretic band pattern of the PCR product. Good PCR products with electrophoretic bands that met the target size were sent for Sanger sequencing. The experimental procedures and identification work were performed by Wuhan Tianyi Huiyuan Biotechnology Co., Ltd. Sequences were analyzed using BLAST comparisons in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). A phylogenetic tree was constructed using Mega 7.0.26 to identify the closest species with high homology.

[0022] 3. Strain growth curve drawing The spore solution of Aspergillus oryzae was diluted to a concentration of 1×10 8 The cells were collected at 4% CO2 / mL and then inoculated into a flask containing 150 mL of PDB medium. The flask was placed in a 30°C incubator for static culture. Samples were taken every 12 hours, and the mycelium was separated by filtration and dried in a drying oven at 105°C to constant weight. The growth of Aspergillus oryzae was assessed by measuring the dry weight of the cells. In the experiment, Aspergillus oryzae Shanghai Niang 3.042 strain was used as the control group to compare the growth characteristics of different strains (Kang Le et al. 2022).

[0023] 4. Effects of environment on the growth of Aspergillus oryzae strains 1) Effects of different salinities on the growth of Aspergillus oryzae PDA culture medium containing 0%, 3%, 6%, 9%, and 12% NaCl was prepared. Aspergillus oryzae spores were picked with a sterile inoculating loop and spotted in the center of a disposable plate. The plates were incubated at 30°C. Colony diameters were measured every 24 hours, and growth was recorded. The Aspergillus oryzae strain, Shanghai Niang 3.042, was used as a control to evaluate the effects of different salinity levels on the growth of Aspergillus oryzae.

[0024] 2) Effects of different pH values ​​on the growth of Aspergillus oryzae PDA culture medium was prepared at pH values ​​of 4, 6, 7, 8, and 10. Aspergillus oryzae spores were picked with a sterile inoculating loop and spotted in the center of a disposable plate. The plates were incubated at 30°C. Colony diameters were measured every 24 hours, and growth was recorded. The strain A. oryzae Shanghai Niang 3.042 was used as a control to evaluate the effects of different pH values ​​on the growth of A. oryzae.

[0025] 3) Effects of different temperatures on the growth of Aspergillus oryzae Aspergillus oryzae spores were spotted in the center of a disposable plate using a sterile inoculating loop and incubated in a constant-temperature incubator at 25°C, 30°C, 35°C, 40°C, and 45°C. Colony diameters were measured every 24 hours, and growth was recorded. The Aspergillus oryzae strain, Shanghai Niang 3.042, was used as a control to evaluate the effects of different temperatures on the growth of Aspergillus oryzae.

[0026] 5. Determination of amino acid nitrogen and total acid content The determination of amino acid nitrogen content follows GB 5009.235, "Determination of Amino Acid Nitrogen in Foods," using the acidometer method. Grind the sample thoroughly in a mortar until it becomes a powder. Weigh 1 g of the ground sample and place it in a clean beaker. Add 20 mL of 80°C distilled water and stir thoroughly to dissolve the sample components. After stirring, filter the solution. Take 10 mL of the filtrate as the test solution and add 60 mL of distilled water. Titrate the solution with 0.05 mol / L sodium hydroxide standard solution. During the titration, stir the solution continuously and monitor the pH in real time with a pH meter until the pH reaches 8.2. At this point, record the volume of sodium hydroxide solution consumed. Subsequently, add 10 mL of formaldehyde solution to the solution, stir thoroughly to mix, and continue titrating with 0.05 mol / L sodium hydroxide standard solution until the pH reaches 9.2. Record the volume of sodium hydroxide solution consumed again. In this experiment, 70 mL of distilled water was used as the control solution. The titration procedure was repeated using the same steps as for the sample. The volume of sodium hydroxide solution consumed by the blank control was recorded. By comparing the titration data for the blank control and sample groups, the amino acid nitrogen content in the sample could be calculated. Total acid content was determined using the pH meter potentiometric titration method specified in GB 12456, "Determination of Total Acidity in Foods."

[0027] 6. Determination of reducing sugar content The reducing sugar content was determined according to the method of Yu Dan (2019) with slight modifications.

[0028] Preparation of glucose standard curve: First, prepare a glucose standard solution with a concentration of 1 mg / mL. Take different volumes of glucose standard solution and distilled water and place them in a series of clearly marked colorimetric tubes. Then, add 1.5 mL of DNS reagent to each colorimetric tube to ensure that the reagent and glucose solution are fully mixed. Place the colorimetric tube in a boiling water bath and heat for 10 minutes. After heating is completed, quickly remove the colorimetric tube and cool to room temperature. Then, add distilled water to each colorimetric tube to the scale line, mix thoroughly, and measure the absorbance of each tube solution at a wavelength of 540 nm. The absorbance (OD 540 ) as the vertical axis and glucose content (mg / mL) as the horizontal axis to draw a standard curve.

[0029] Determination of reducing sugar content: Weigh 0.5g of the crushed sample, add 20mL of distilled water, place in a 40℃ water bath, and stir at 150r / min for 1h. After standing for 30min, filter the sample and dilute with an appropriate amount of distilled water to the appropriate concentration. Take 2mL of the diluted solution and add it to a colorimetric tube containing 1.5mL of DNS solution. Heat in a boiling water bath for 10min. After cooling, dilute to the mark with distilled water. Mix thoroughly and measure the absorbance (OD) at a wavelength of 540nm. 540 ).

[0030] 7. pH determination Weigh 2.5g of a thoroughly ground sample. Place the ground sample in a beaker and add an appropriate amount of distilled water. Transfer the solution to a 50mL volumetric flask and dilute to the mark with distilled water. After allowing the solution to stand for a while, remove the supernatant and measure the pH using a precision pH meter.

[0031] 8. Determination of salinity Salinity (edible salt) was determined according to GB / T 5009.39, "Analytical Methods for Hygienic Standards of Soy Sauce." The specific steps are as follows: Accurately weigh 5 g of thoroughly ground sample, add distilled water to 100 mL, and centrifuge at 4000 rpm for 10 minutes to separate the supernatant. Take 2 mL of the supernatant, add 100 mL of distilled water and 1 mL of 50 g / L potassium chromate solution, and titrate with 0.100 mol / L silver nitrate (AgNO3) standard titrant until the solution begins to turn orange-yellow, which is the titration endpoint. Record the volume of AgNO3 standard titrant consumed for salinity calculation.

[0032] 9. Determination of total colony count and total mold count The total bacterial count (TC) was determined in accordance with GB 4789.2, "National Food Safety Standard for Determination of Total Apoptotic Count," using plate count agar for culture and enumeration. First, thoroughly stir the sample. Then, weigh 25 g of sample and place it in a sterile homogenizing bag. Add 225 mL of sterile saline and mix thoroughly by hand-patting. Next, use a sterile pipette to draw 1 mL of the homogenized solution into a test tube containing 9 mL of sterile saline and mix thoroughly to prepare a 1:100 sample dilution. Continue serially diluting the sample dilution according to the above steps until the appropriate concentration range is reached. Select 2-3 sample dilutions of varying dilutions and, using a sterile pipette, draw 1 mL of each sample dilution into a sterile disposable Petri dish. Then, pour 20-25 mL of plate count agar (PCA) cooled to approximately 50°C into the dish, ensuring that the medium and sample dilution are thoroughly mixed and evenly spread. Two replicates are performed for each dilution gradient. A 1 mL blank dilution is also used as a control. After the culture medium has cooled and solidified, invert the culture dish in a constant-temperature incubator at 37 ± 1°C and incubate for 48 ± 2 hours. After incubation, count the colonies on the plate to determine the total number of colonies in the sample. Observe visually or with a magnifying glass, recording the dilution factor and the corresponding colony count. Results are expressed as logarithm of the total colony count (CFU / g).

[0033] The total mold count is determined in accordance with GB 4789.15, "National Food Safety Standard for Mold and Yeast Count." This method is similar to the total colony count method. First, prepare sample dilutions of appropriate concentrations according to the method for total colony count. Next, select two or three sample dilutions at different dilution gradients and use a sterile pipette to aspirate 1 mL of each sample dilution into a sterile disposable Petri dish. Next, pour 20-25 mL of Bengal rose agar, cooled to approximately 46°C, into the dish. Gently shake the dish to spread the medium evenly and allow it to cool and solidify. After solidification, invert the dish and incubate it in a constant temperature incubator at 27±1°C for 120±2 hours. After incubation, observe colony formation and visually count the total mold colonies. The final result is expressed as the logarithm of the total colony count, i.e., log (CFU / g).

[0034] 10. Determination of volatile flavor substances The volatile flavor compounds in doubanjiang were detected using a headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) technique slightly modified from that used by Li Xiongbo (2020). The specific method is as follows: 2.0 g of ground sample was accurately weighed and placed in a 15 mL headspace vial. The vial was preheated in a 60°C water bath for 2 minutes. An aging SPME tip was then inserted into the vial, and headspace extraction was performed at 60°C for 50 minutes. After extraction, the tip was transferred to the GC-MS inlet and desorbed at 250°C for 5 minutes.

[0035] Chromatographic conditions: A DB-WAX capillary column (60 m × 0.25 mm, 0.25 μm) was used, with helium as the carrier gas at a flow rate of 1.0 mL / min. The inlet temperature was set at 250°C, and splitless injection mode was used. The temperature program was as follows: initial temperature 50°C, ramped to 85°C at 10°C / min (hold for 1.5 min), then to 100°C at 5°C / min (hold for 1 min), then to 175°C at 2.5°C / min (hold for 1.5 min), and finally to 250°C at 10°C / min.

[0036] The mass spectrometry conditions were as follows: EI ionization source, electron impact energy 70 eV; ion source temperature 230°C; interface 250°C; mass scan range 35-350 amu; detector voltage 0.1 kV; tuning file Stuneu; and data acquisition in full scan mode.

[0037] The chromatograms were searched and matched, and substances with a similarity of 70 or greater were screened for analysis. The types of volatile compounds were determined based on the matching results. The relative contents of volatile substances in bean paste were calculated using the peak area normalization method.

[0038] 11. Determination of free amino acids Free amino acids were determined according to the method of Bao Yida (2020). Sample extraction: Accurately weigh 4 g of sample, add 20 mL of ultrapure water, and sonicate in an ice bath for 30 minutes to fully extract the free amino acids in the sample. Initial centrifugation: Centrifuge the sonicated sample at 10,000 rpm for 5 minutes at 4°C to separate the supernatant. Protein precipitation: Take 400 μL of the supernatant and add 100 μL of 10% sulfosalicylic acid solution to precipitate the protein and further purify the sample. Filtration: Filter the mixture using a 0.45 μm filter to remove insoluble impurities. Standing and secondary centrifugation: Place the filtered solution in a 4°C refrigerator for 1 hour, then centrifuge at 14,500 rpm for 15 minutes to further separate impurities. Purification: Take the supernatant and centrifuge it again at 14,500 rpm for 5 minutes to ensure sample purity. Dilution and preparation: Dilute the sample appropriately using a lithium salt system sample diluent based on the approximate amino acid content. Final filtration and analysis: The diluted sample was filtered through a 0.22 μm filter to remove any possible tiny particles, and finally the sample was injected into a fully automatic amino acid analyzer for quantitative analysis.

[0039] 12. Colorimetry The Lab three-color method was used to analyze the color of the fermented bean paste samples (Zhang et al 2020). The four fermented bean paste samples were placed in transparent glass bottles and the color of the fermented bean paste samples was measured using a spectrophotometer. 、a 、b Each sample was measured three times, and the average value was taken as the result.

[0040] 13. Data Processing Data were analyzed using Microsoft Excel 2016 for basic statistical calculations, and the results are presented as mean ± standard error. Significance tests (p < 0.05) were performed using the Duncan test and one-way analysis of variance (ANOVA) using SPSS 26.0 statistical software. GraphPad Prism 6.02 was used for plotting and export.

[0041] result 1. Isolation and purification of strains Seven fungi strains (numbered 1-7) were isolated from the broad bean koji used in the fermentation of Hubei Doubanjiang. The colony morphology of each strain was Figure 1 The detailed morphological characteristics are shown in Table 1. The isolated fungi were purified and stored at 4 °C for subsequent experiments.

[0042] Table 1 Fungal colony morphology

[0043] 2. Pure strain back-inoculation fermentation The pure fermentation experiment of bean paste was carried out using the 7 isolated molds and the control group Aspergillus oryzae Shanghai Niang 3.042. After 25 days of fermentation at 42℃, the Figure 2 The results showed that white spots appeared in the fermented mash of the strains No. 1, 2, 4 and 5 and the control group, Aspergillus oryzae 3.042. In contrast, the fermented mash of the strains No. 3, 6 and 7 did not produce white spots. Figure 3 As shown, the fermented bean pastes with strains 3, 6, and 7 still did not produce white spots.

[0044] 3. Morphological identification The three strains of Aspergillus oryzae that did not produce white spots (No. 3, 6, and 7) that were initially screened were morphologically observed. Figure 4 、 Figure 5 、 Figure 6 As shown. Strain 3: The colony is yellow-green, with a relatively smooth surface. The green spores are mainly attached to the middle ring, the outer circle is white hyphae, and the reverse side is light yellow-green. Under microscopic observation, the conidial heads of the strain are radiant, and the conidia are nearly spherical. Strain 6: The colony is yellow-green, with a smooth surface, densely distributed spores, and white hyphae on the edge. Under microscopic observation, the conidia of the strain are nearly spherical. Strain 7: The colony is yellow-green; the middle is velvety, the edges are relatively smooth, the spores are concentrated in the middle part, the edges are white hyphae, and the reverse side is light yellow. Under microscopic observation, the conidia are nearly spherical. The plate growth morphology and microscopic spore morphology of the three strains are consistent with the description of Aspergillus oryzae by Lu Huaijin (2021) and others, and the morphological characteristics are consistent with the typical morphological characteristics of Aspergillus oryzae (Qi Zutong 1997). It should be noted that in Figure 4-6 In the figure, (a) is the colony morphology, (b) is the microstructure 100X, and (c) is the microstructure 400X.

[0045] 4. Molecular Biology Identification The non-white spot mold fungi that were initially screened out were subjected to molecular biological identification. The sequencing data of the three strains numbered 3, 6, and 7 were uploaded to the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). Blast alignment was performed to screen out highly matched sequences. Subsequently, a phylogenetic tree was constructed using MEGA 7.0.26 software (e.g. Figure 7-9The results showed that strain 3 and Aspergillus oryzae isolate 2011F18 were clustered in the same branch, with the highest similarity; strain 6 was most similar to Aspergillus oryzae strain QRF378; and strain 7 was most similar to Aspergillus oryzae isolate RP-1. The morphological identification results were highly consistent with the phylogenetic tree analysis results. Combined with the colony morphology, spore structure and hyphae characteristics (such as Figure 4 、 Figure 5 、 Figure 6 As shown in Figure 3 ), all three strains exhibited typical characteristics of Aspergillus oryzae. Therefore, based on the combined molecular biology and morphological identification results, it was determined that all three strains were Aspergillus oryzae.

[0046] 5. Growth curve of Aspergillus oryzae In this study, Aspergillus oryzae strains 3, 6, 7 and Shanghai Niang 3.042 were selected as research objects. PDB medium was used for liquid culture at a constant temperature of 30℃ for 72 hours, and the dry weight of the bacteria was determined at an interval of 12 hours. Figure 10 ) Analysis found that after 24 hours of culture, the biomass accumulation rates of strains 3, 6, and 7 in the experimental groups were all higher than those of the control group (3.042). After 72 hours of culture, the dry weights of the four strains tended to be consistent.

[0047] 6. Effects of salinity, pH, and temperature on the growth of Aspergillus oryzae The present invention conducted experiments on the effects of factors such as salinity, pH value and temperature on the growth characteristics of Aspergillus oryzae strains No. 3, No. 6, No. 7 and Shanghai Niang 3.042. The results are as follows Figure 11 , within the same culture time, Aspergillus oryzae strain No. 3 showed the fastest growth rate and the largest colony diameter when the salt concentration of the culture medium was 6%. In contrast, the other three Aspergillus oryzae strains (No. 6, No. 7 and Shanghai Brewing 3.042) grew fastest at a salt concentration of 3%. When the salt concentration increased to 9%, the growth of all four Aspergillus oryzae strains was inhibited. Furthermore, when the salt concentration reached 12%, the inhibitory effect was more obvious, indicating that the high-salt environment had a strong inhibitory effect on the growth of Aspergillus oryzae strains, which shows that Aspergillus oryzae plays an important role in the early stage of the fermentation process. As the salt concentration increases, the growth of Aspergillus oryzae is inhibited, and other microorganisms play a role. Figure 12As shown in the figure, pH value has a certain effect on the growth of four strains of Aspergillus oryzae. Specifically, when the pH value of the culture medium is 4, the strong acidic environment has a significant inhibitory effect on the growth of Aspergillus oryzae strains, and the colony grows slowly and has a small diameter. However, when the pH value is adjusted to 6, the growth state of the four strains of Aspergillus oryzae reaches the best, and the colony diameter increases. As the pH value continues to increase, the culture medium gradually tends to be alkaline, the growth rate of Aspergillus oryzae begins to slow down, and the colony diameter also decreases accordingly, which shows that excessively high pH values ​​will also have an adverse effect on the growth of Aspergillus oryzae. Temperature has a significant effect on the growth of the four strains of Aspergillus oryzae. As Figure 13 As shown in the figure, the optimal growth temperature of the four Aspergillus oryzae strains is about 30℃. At this temperature, the strains grow vigorously and the colony diameter reaches the maximum. However, as the temperature rises, the growth rate of Aspergillus oryzae gradually decreases. When the temperature rises to 45℃, the growth of Aspergillus oryzae strains almost stagnates, indicating that high temperature has a strong inhibitory effect on the growth of strains. It should be noted that at Figure 11-13 In the figure, (a) shows the curve graph of strain No. 3, (b) shows the curve graph of strain No. 6, (c) shows the curve graph of strain No. 7, and (d) shows the curve graph of Shanghai Brewing 3.042.

[0048] 7. Tracking determination of amino acid nitrogen content Amino acid nitrogen is another important parameter for measuring the quality of doubanjiang (fermented bean paste). It reflects the degree of hydrolysis of broad bean protein during the fermentation process (Zhang 2019). As an important flavoring substance, it is closely related to the flavor of doubanjiang and is the key source of its unique umami flavor (Kang Lei et al. 2014). The amino acid nitrogen content of doubanjiang during the mixed fermentation process was tracked and measured. The results are as follows: Figure 14As shown, the amino acid nitrogen content of the four types of fermented bean pastes fermented at 30°C for 25 days initially increased and then decreased. The amino acid nitrogen content trends reported by Sun et al. (2019) using a single bacterial strain in fermented bean pastes are consistent with the above trend. This phenomenon may be closely related to the metabolic behavior of microorganisms during fermentation. In the early stages of fermentation, proteases secreted by microorganisms (such as Aspergillus oryzae and yeast) break down proteins in the bean paste into amino acids, leading to an increase in amino acid nitrogen content (Chou and Ling 1998, Yun et al. 2020). However, as fermentation progresses, some amino acids may be further metabolized by microorganisms or participate in other biochemical reactions (such as the Maillard reaction), causing the amino acid nitrogen content to gradually decrease in the later stages (Yu et al. 2022). Among the four types of fermented bean pastes, the one fermented for 25 days using the Shanghai Niang 3.042 Aspergillus oryzae strain (No. 8) showed an advantage in amino acid nitrogen accumulation. Its amino acid nitrogen content steadily increased from 0.50 g / 100 g on day 0 of initial mixed fermentation to 1.01 g / 100 g, consistently ranking highest among the four fermented bean pastes. Notably, the finished product also exhibited significant white specks. In contrast, the fermented bean paste with strain 7, while boasting a final amino acid nitrogen content (1.01 g / 100 g) comparable to that of strain 8, initially accumulated more slowly. Strain 6 ranked second, with a final amino acid nitrogen content of 0.96 g / 100 g, slightly lower than strains 7 and 8. The fermented bean paste with strain 3 exhibited the lowest amino acid nitrogen content after fermentation, at only 0.89 g / 100 g, consistent with the protease activity assay results for strain 3. This difference may be due to the specificity of the different strains in protein degradation efficiency and metabolic pathways, such as greater protease secretion and more efficient amino acid conversion mechanisms.

[0049] 8. Total acid content and pH value tracking determination The present invention dynamically monitored the changes in total acid content during the mixed fermentation of broad bean paste. The results showed that as the fermentation time increased, the total acid content showed a trend of first increasing and then decreasing. This pattern of change may be closely related to the metabolic activity of microorganisms and the accumulation of their products during the fermentation process. In the early stage of fermentation, microbial metabolism is active, and a large amount of organic acids are produced, resulting in an increase in total acid content; in the later stage of fermentation, some organic acids may be further converted or degraded, resulting in a decrease in total acid content. In actual production, national standards have strict restrictions on total acid content, because excessively high acid content may indicate an imbalance in the microbial community during the fermentation process, increased accumulation of putrefactive acids, and thus affect product quality (Xie Si et al. 2021), and may also cause the product to have a pungent sour taste (Lee et al. 2014). After fermentation of chili mash with four kinds of sauce mash at 30℃ for 25 days, the total acid content of the obtained bean paste was 1.08g / 100g (No. 3), 1.06g / 100g (No. 6), 1.07g / 100g (No. 7) and 1.04g / 100g (No. 8), respectively.

[0050] from Figure 15 As can be seen, the pH value decreases during the early stages of fermentation and then stabilizes. This trend is closely linked to the metabolic behavior of the microorganisms during fermentation. In the early stages of fermentation, microorganisms such as Aspergillus oryzae are extremely metabolically active, secreting large amounts of organic acids such as lactic acid and malic acid, causing the pH value to drop sharply. As the fermentation process progresses, microbial metabolism gradually reaches equilibrium, and the production and consumption of organic acids enter a dynamic equilibrium, resulting in a stable pH value (Zheng Rong et al. 2024). At the end of fermentation, the pH values ​​of the fermented bean pastes produced by different strains varied. The bean paste fermented with Aspergillus oryzae No. 3 had the highest pH value of 5.43, followed by strain No. 7 (5.38), while the bean paste fermented with strain No. 8 had the lowest pH value of 5.25. This difference may be related to the metabolic characteristics of the different strains and their adaptability to the fermentation environment. Furthermore, changes in the microbial community structure during the fermentation process can also affect the stability of the pH value (Li et al. 2023).

[0051] 9. Tracking determination of reducing sugar content and salinity Reducing sugar is an important carbon source for microbial metabolism during the fermentation of bean paste, and changes in its content have a significant impact on the fermentation rate and the production of flavor substances. Figure 16As shown, the reducing sugar content of the four fermented bean pastes showed a trend of initially increasing, then decreasing, and then increasing again. This phenomenon may be due to the combined action of multiple microorganisms during the mixed fermentation process, leading to the simultaneous saccharification of carbohydrates and the formation of organic acids (Jin et al. 2017, Liu et al. 2018). Specifically, in the initial fermentation stage, polysaccharides such as starch are hydrolyzed into reducing sugars by enzymes, a process that increases the reducing sugar content. As fermentation progresses, microorganisms consume large amounts of reducing sugars for their own growth and metabolic activities, causing the reducing sugar content to gradually decrease. After 20 days of mixed fermentation of the chili and soybean fermented mash, microbial metabolic activity weakens, reducing sugar consumption slows, and new reducing sugars may be generated, causing the reducing sugar content to rise again. After fermentation, there was no significant difference in the reducing sugar content of the bean paste fermented by strains 3, 6, and 7, which were 4.20%, 4.25%, and 4.60%, respectively. They were all significantly higher than the reducing sugar content of the bean paste fermented by strain 8 (Shanghai Brewing 3.042) (3.28%) (p<0.05), which may be related to the combined action of the strains' amylase, saccharifying enzyme, etc.

[0052] Generally speaking, sodium chloride is a key factor in preventing soy sauce from spoiling and enhancing its flavor through its synergistic effect with other umami compounds such as glutamate (Shah Syifaa et al. 2016). In this study, the salinity of the four fermented bean pastes gradually increased with increasing mixed fermentation time, ultimately stabilizing between 11.1% and 13.5%, meeting the typical salt content requirement for bean paste.

[0053] 10. Determination of volatile flavor substances Volatile flavor compounds play a crucial role in determining the flavor and texture of fermented foods such as doubanjiang, and are crucial to their quality and consumer acceptance (Zhao et al 2021). To further elucidate the differences in flavor compounds in doubanjiang fermented with four different Aspergillus oryzae strains (3, 6, 7, and Shanghai Niang 3.042), this study used HS-SPME-GC-MS for detection and analysis. Figure 17 As shown in the figure, a total of 82 volatile compounds were detected, including esters, alcohols, and other categories. Specifically, there were 24 esters, 12 alcohols, 2 aldehydes, 1 acid, 1 ketone, 3 phenols, 6 ethers, and 33 other categories. The volatile flavor compounds detected in the fermented bean paste inoculated with the four strains were 8, 40, 50, and 17, respectively. The bean paste fermented with Aspergillus oryzae strains 6 and 7 had richer volatile flavors than those with strains 3 and 8. The bean paste fermented with strain 7 had an advantage in the types of esters and other substances, followed by strain 6.

[0054] From the perspective of relative percentage (see Figure 18 ), compared with other fermented bean pastes, the fermented bean paste produced by strain No. 6 not only ranked first in the relative content of esters and alcohols, but also showed a higher relative content of other types of volatile flavor substances.

[0055] Through heat map cluster analysis, the composition differences of volatile flavor compounds in different bean paste products were deeply compared. Figure 19 As shown in the figure, the relative contents of ethanol, phenylethanol, 2,3-butanediol, linalool, ethyl phenylacetate, ethyl hexanoate, and ethyl hexadecanoate in fermented doubanjiang (fermented bean paste) from Aspergillus oryzae strain 6 were higher than those in the other three varieties. Tu Dawei et al. (2025) found that the main flavor compounds in Sichuan and Chongqing doubanjiang are alcohols and esters. Esters are one of the most important flavor compounds. Due to their volatility, they impart honey and fruity flavors to fermented foods. Esters also promote the formation of various flavor compounds, providing fermented doubanjiang with its unique sauce-flavored aroma. Alcohols in fermented foods originate from the metabolism of sugars and amino acids during microbial fermentation. They are important components that determine the flavor profile of doubanjiang (Yun et al. 2020), imparting its pleasant aroma and sweetness (Steinhaus et al. 2009). In fermented bean paste No. 6, phenylethanol and ethanol are the top two alcohol components in terms of content. This aligns with the findings of Luo Jing et al. (2018) on volatile aroma compounds in the post-fermentation stage of Pixian bean paste. Feng et al. (2014) found that ethanol plays a significant role in enhancing the quality and flavor of soy sauce. Zhao et al. (2021) identified five alcohols in their characterization of key aroma compounds in Pixian bean paste, of which 2,3-butanediol was the most important, exhibiting a creamy aroma. Pang Weiqiao et al. (2016) found a high level of 2,3-butanediol in their analysis of the flavor compounds in Heilongjiang bean paste. These alcohols were found in the highest relative concentrations in fermented bean paste fermented with strain No. 6. Other volatile flavor compounds, such as aldehydes, are also important in producing various odors and, as substrates in food, are implicated in flavor production.

[0056] In summary, during the fermentation process, strain No. 6 can promote the production of flavor substances, thereby giving the bean paste a more complex and rich flavor profile.

[0057] 11. Determination of free amino acids Free amino acids, through the Maillard reaction and Strecker amino acid degradation, promote the production of volatile flavor compounds such as aldehydes, ketones, esters, and pyrazines, which are crucial for the unique flavor profile of fermented products (Chen et al. 2020). The amino acid profiles of the samples were compared with the amino acid profiles of the standard samples, and a total of 16 free amino acids were detected (Table 2). The total free amino acid content of fermented bean pastes fermented with strains 6 and 7 was lower than that of the other two fermented bean pastes. This may be because certain microorganisms can produce large amounts of volatile compounds, such as alcohols and esters, through certain reactions (Jin Manqin et al. 2023). These reactions may be preferentially activated during fermentation, leading to the accumulation of volatile flavor compounds at the expense of free amino acids. Considering that the formation of white spots is closely related to tyrosine content, the formation of white spots is closely related to tyrosine content. Tyrosine has an extremely low solubility (0.45 g / L at 25°C) (Min Shihao et al. 2020), and its solubility is affected by factors such as pH and salinity (Lei Hongjie et al. 2008). Therefore, it easily crystallizes and precipitates during fermentation, forming white spots. Experimental results showed that the tyrosine content in fermented broad bean pastes from different strains varied. Among them, the tyrosine content in broad bean paste fermented by strain 8 was higher than that of other strains, a result consistent with the observation that its fermented products exhibited severe white spotting. In contrast, the tyrosine content in broad bean paste fermented by strain 6 was the lowest (0.60 ± 0.14 mg / g), and the probability of white spots in its products was also correspondingly minimal. These findings further confirm the direct correlation between tyrosine content and white spot formation, providing an important basis for strain screening to reduce white spot formation.

[0058] Table 2 Amino acid composition and content of four kinds of bean paste

[0059] Note: ND means not detected. Different letters in the same row indicate significant differences (p<0.05).

[0060] Based on their taste properties, free amino acids can be systematically divided into four categories: umami, bitter, sweet, and odorless. Umami and bitter amino acids are the primary flavor components that contribute to the characteristic flavor of doubanjiang (Wu et al. 2024). Ding et al. (2022) conducted in-depth research and confirmed that umami amino acids, particularly glutamate, significantly contribute to the overall flavor profile of Pixian doubanjiang. Figure 20 The composition and relative content of various flavor amino acids in four different fermented bean paste samples are shown. Notably, the umami amino acid content in fermented bean paste fermented with Aspergillus oryzae strain 6 (45.08%) was second only to that of the sample fermented with strain 3 (46.36%).

[0061] 12. Colorimetry h The value is an important indicator of the hue parameter, and its calculation formula is h =a / b When h When the value is positive, the larger the value, the darker the red. The change in the value can accurately capture the subtle differences in color between different varieties (Yi Sibing 2021). The chromaticity values ​​of the four types of bean paste are shown in the table above. As can be seen from Table 3, the brightness (L The value of the doubanjiang fermented with strain 6 was significantly higher than that of the other three samples (p < 0.05), indicating a brighter color. Furthermore, the h* value of the doubanjiang fermented with strain 6 was 1.21, higher than that of the other three samples, indicating a more vivid red color. Overall, the doubanjiang fermented with strain 6 exhibited the greatest color vividness.

[0062] Table 3 Colorimetric determination of broad bean paste

[0063] Note: Different letters in the same column indicate significant differences (p<0.05).

[0064] In summary, the present invention selected the broad bean koji used in the fermentation process of Hubei broad bean paste as the research object. Seven strains of mold were separated and purified from the broad bean koji using PDA culture medium. Subsequently, these seven strains of mold and Aspergillus oryzae Shanghai Niang 3.042 strain were respectively inoculated back into the fermentation system for koji making and fermentation experiments. After 25 days of fermentation of the mash at 42°C and 25 days of mixed fermentation at 30°C, it was observed that only the mash and broad bean paste fermented by the three strains of mold numbered 3, 6, and 7 did not produce white spots. Morphological observation and molecular biological identification were carried out on the above strains, and it was finally determined that these three strains belonged to Aspergillus oryzae ( Aspergillus oryzae The selected A. oryzae strains, along with the Shanghai Brewing 3.042 strain, were further analyzed for their growth characteristics. This included plotting growth curves and determining optimal salinity, pH, and temperature for growth. The results showed that all four strains exhibited similar growth characteristics: they grew most vigorously in environments with lower salt concentrations, with an optimal pH of approximately 6 and an optimal temperature of around 30°C.

[0065] After fermentation, we conducted a comprehensive analysis of the finished fermented bean paste for volatile flavor compounds and free amino acid content, and measured its colorimetric value. The results showed that fermented bean paste with strain 6 exhibited a distinct advantage in flavor profile. Of particular note, regarding the critical issue of white spot formation, the samples fermented with strain 6 had the lowest tyrosine content, at 0.60 ± 0.14 mg / g. This indicates a minimal risk of white spot formation. Furthermore, fermented bean paste with strain 6 exhibited the brightest color. Based on these indicators, strain 6 was identified as a high-quality, white spot-free Aspergillus oryzae strain.

[0066] The strain No. 6 is named Aspergillus oryzae ZD01 was deposited in the China Center for Type Culture Collection on April 11, 2025, with the deposit address being Wuhan University, Wuhan, Hubei Province, China, with the deposit number being CCTCC NO: M2025761.

[0067] The raw materials not specifically described in the present invention are all existing materials that can be directly purchased from the market.

[0068] The above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An Aspergillus oryzae, characterized in that The strain is named Aspergillus oryzae ZD01 was deposited in the China Center for Type Culture Collection on April 11, 2025, with the deposit address being Wuhan University, Wuhan, Hubei Province, China, with the deposit number being CCTCC NO: M 2025761.

2. The preparation method of Aspergillus oryzae according to claim 1, wherein The broad bean koji used in the fermentation process of Hubei bean paste is placed on the surface of the culture medium for cultivation, purification and screening to obtain a single strain.

3. The preparation method according to claim 2, characterized in that The culture medium was potato dextrose agar.

4. The preparation method according to claim 2, characterized in that It was placed on the surface of the culture medium for culture, purification and screening, and the culture temperature was 28°C.

5. The preparation method according to claim 4, characterized in that The culture time is 72h.

6. A fermentation agent, characterized in that: The fermentation agent comprises the Aspergillus oryzae according to claim 1, or the Aspergillus oryzae prepared by the preparation method according to any one of claims 2 to 5.

7. Use of the Aspergillus oryzae according to claim 1 or the fermentation agent according to claim 6 in preparing Hubei broad bean paste.

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