Aspergillus oryzae with high yield of lipase, application and method for preparing fresh chili pepper beans rich in unsaturated fatty acids

Abstract

The application discloses an aspergillus oryzae with high lipase yield, application and a method for preparing fresh chili pepper and broad bean rich in unsaturated fatty acids, relates to the technical field of microorganisms and food fermentation, and the taxonomic name of the aspergillus oryzae is Aspergillus oryzae , which is preserved in the Guangdong Microbial Culture Collection Center on Nov. 14, 2025, and has a preservation number of GDMCC No: 67295. The aspergillus oryzae has balanced enzyme production capacity, high lipase yield, and good genetic stability, and does not produce aflatoxin B1. The prepared fresh chili pepper and broad bean have a high unsaturated fatty acid content of 1770.13 mg / kg, and the content of volatile flavor substances is greatly improved.

Description

Technical Field

[0001] This invention relates to the fields of microbial and food fermentation technology, and in particular to a strain of Aspergillus oryzae that produces a high amount of lipase, its application, and a method for preparing fresh chili bean paste rich in unsaturated fatty acids. Background Technology

[0002] Doubanjiang (fermented broad bean paste) is a typical representative of traditional Chinese fermented condiments, beloved by consumers for its unique flavor and quality. With the increasing demand for healthy and nutritious foods, the functionalization and nutritional upgrading of traditional fermented foods has become an inevitable trend in the industry's development. During the fermentation process of doubanjiang, free fatty acids, as key precursors in the formation of aromatic components such as esters, significantly contribute to the complexity and richness of the product's flavor. Lipases, as key enzymes catalyzing the hydrolysis of oils and releasing free fatty acids, represent a potential breakthrough for enhancing the product's flavor complexity and nutritional value.

[0003] However, in current industrial production, the Aspergillus oryzae strains used for fermenting fermented soybean paste have long focused on high-yield production of proteases and amylases, resulting in strains generally exhibiting inherent defects such as low and unstable lipase secretion capacity. Directly adding commercial lipases exogenously faces problems such as high cost and limited enzyme system availability. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a strain of Aspergillus oryzae that produces high levels of lipase, its application, and a method for preparing fresh chili bean paste rich in unsaturated fatty acids. A new strain was obtained by screening from chili bean paste, and using this new strain to prepare fresh chili bean paste can significantly increase the content of unsaturated fatty acids in the fresh chili bean paste.

[0005] In order to achieve the objective of this invention, the following solution is proposed:

[0006] This invention provides a high-lipase-producing Aspergillus oryzae strain ( Aspergillus oryzae KDX-66, deposited at Guangdong Provincial Center for Microbial Culture Collection, on November 14, 2025, with accession number GDMCC No:67295.

[0007] Aspergillus oryzae ( Aspergillus oryzae The morphological characteristics of KDX-66 are as follows: on PDA medium, the colonies of this strain are all yellow-green, velvety in texture, with short and loose hyphae that show radial wrinkles; microscopic morphology shows that it has conidiophores, the conidiophore stems are thin and long, with a spherical apical sac at the top, and radial pedicels on the surface. The conidia are arranged in chains and are round.

[0008] Aspergillus oryzae ( Aspergillus oryzaeKDX-66 does not produce aflatoxin and has a balanced enzyme production capacity, especially high lipase production, with lipase activity reaching 246.06 U / g; this strain has good genetic stability, and its lipase activity only decreased by 4.4% after 7 generations.

[0009] Aspergillus oryzae ( Aspergillus oryzae KDX-66 belongs to the genus Aspergillus ( Aspergillus Its ITS gene sequence is 574 bp in length, and the strain with high homology to it is... Aspergillus oryzae The strain (GenBank accession number: KP418788.1) and the two strains shared 99.29% sequence identity. *Aspergillus oryzae* ( Aspergillus oryzae The ITS gene sequence of KDX-66 is shown in SEQ ID No: 1.

[0010] This invention provides the Aspergillus oryzae ( Aspergillus oryzae KDX-66 is used to prepare starter cultures, which include Aspergillus oryzae spore powder.

[0011] This invention provides the Aspergillus oryzae ( Aspergillus oryzae KDX-66 is used to increase the content of unsaturated fatty acids in fermented condiments.

[0012] Specifically, the fermented condiments include one or more of soy sauce, broad bean paste, soybean paste, and fermented black beans.

[0013] Specifically, the unsaturated fatty acids include one or more of oleic acid, linoleic acid, myristoleic acid, palmitoleic acid, and linolenic acid.

[0014] This invention provides a method for preparing fresh chili bean paste rich in unsaturated fatty acids, comprising the following steps:

[0015] (1) Making koji: After blanching the shelled broad beans in boiling water for 3 to 5 minutes, immediately cool them to 35℃ to 40℃. Then mix the broad beans and wheat flour at a mass ratio of 9 to 11:2 and inoculate them with the fermentation agent. Transfer the inoculated koji material to a ventilated tank or a disc koji making machine and carry out koji making and fermentation at a temperature of 28℃ to 35℃ and a relative humidity of ≥85%. Turn the koji once every 12 hours and 24 hours. When the surface of the koji material is covered with yellow-green mycelium, you will get broad bean koji.

[0016] Specifically, the inoculation amount of the fermenting agent is 0.01% to 0.05% of the total mass of broad beans and wheat flour, with a preferred inoculation amount of 0.03%.

[0017] (2) Fermentation: Fresh red Erjingtiao chili peppers are destemmed, cleaned, and crushed into chili mash with a particle size of 2nm~4nm. Then, they are mixed evenly with the fermented broad bean paste at a certain mass ratio, and the NaCl mass concentration of the fermentation system is controlled by adding salt. Then, they are transferred to a cleaned and disinfected ceramic jar and sealed with food-grade plastic film and cotton cloth. Finally, they are placed in a closed high-cleanliness workshop for natural fermentation for 3 to 6 months to obtain fresh chili broad bean paste rich in saturated fatty acids.

[0018] Specifically, the mass ratio of the chili mash to the fermented soybean paste is 7:3 to 8:2, with a preferred mass ratio of 7:3.

[0019] Specifically, the NaCl mass concentration of the fermentation system is 10%~18%, preferably 12%.

[0020] The beneficial effects of this invention are as follows:

[0021] 1. This invention screened a new strain from fermented soybean paste, which was identified as Aspergillus oryzae (…). Aspergillus oryzae ).

[0022] 2. Aspergillus oryzae ( Aspergillus oryzae KDX-66 has a high lipase production capacity, with lipase activity reaching up to 246.06 U / g, which is more than 70% higher than that of the common industrial strain Aspergillus oryzae 3.042. It also has good genetic stability, with lipase activity decreasing by only 4.4% after 7 generations. It can be used for traditional fermentation of condiments.

[0023] 3. Aspergillus oryzae ( Aspergillus oryzae KDX-66 exhibits a more balanced enzyme production capacity, with its neutral protease, acidic protease, and amylase activities all superior to the industrial strain Hu Niang 3.042. Furthermore, Aspergillus oryzae (… Aspergillus oryzae KDX-66 does not produce aflatoxin B1 and is a safe strain.

[0024] 4. Based on Aspergillus oryzae ( Aspergillus oryzae KDX-66-prepared fresh chili bean paste efficiently hydrolyzes the oil in the raw materials, significantly increasing the content of oleic acid, linoleic acid, myristoleic acid, palmitoleic acid, etc. in the fresh chili bean paste. The content of unsaturated fatty acids is as high as 1770.13mg / kg, especially the oleic acid content is increased by more than 1 times, which has high application value.

[0025] 5. Based on Aspergillus oryzae ( Aspergillus oryzae The fresh chili bean paste prepared by KDX-66 has a significantly increased content of volatile flavor compounds, including phenolic compounds up to 65.63 μg / g, ester compounds up to 377.59 μg / g, and alcohol compounds up to 166.15 μg / g. Attached Figure Description

[0026] Figure 1 Aspergillus oryzae ( Aspergillus oryzae Colony morphology diagram of KDX-66.

[0027] Figure 2 Aspergillus oryzae ( Aspergillus oryzae Microscopic morphology of KDX-66.

[0028] Figure 3 This is a graph showing the content of unsaturated fatty acids in fresh chili bean paste.

[0029] Biological material preservation information: Aspergillus oryzae KDX-66 ( Aspergillus oryzae KDX-66 is deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 14, 2025, with accession number GDMCC No:67295. Detailed Implementation

[0030] Example 1

[0031] The screening process for high-lipase-producing strains is as follows:

[0032] 1.1 Separation and Purification

[0033] Weigh 10.0 g of fermented soybean paste sample into 90 mL of sterile physiological saline, and incubate at 30℃ with shaking at 120 r / min for 2 h to ensure thorough dispersion of microbial cells; perform serial dilutions of the supernatant, and take 100 µL of each dilution gradient. -3 ~10 -5 The diluted solution was spread onto PDA medium; the spread plates were inverted and incubated in an incubator at 30℃±1℃ for 48h; single colonies with typical Aspergillus morphology were picked according to the colony morphology and purified by streak plating on PDA plates. This process was repeated 2 to 3 times until a pure strain free of contaminants was obtained.

[0034] 1.2 Initial screening

[0035] Using a sterile inoculation loop, pick up a small amount of fungal spores from the slant culture and place them in a test tube containing 9 mL of sterile physiological saline. Shake thoroughly to disperse the spores, and then serially dilute to 10⁻⁶. -1 ~10 -8 Different concentrations of spore suspensions, 100µL of each suspension was taken. -5 ~10 -8 The spore suspension of a certain concentration was spread on a purple oil assimilation plate, inverted in an incubator at 30℃±1℃ and incubated for 72 hours. Colonies that produced a yellow discoloration zone were picked and streaked for preservation for later rescreening.

[0036] 1.3 Secondary screening

[0037] A small number of mold spores from the initial screening slant were picked up with a sterile inoculation loop and affixed to an oil assimilation plate. The plate was then incubated upside down in an incubator at 30℃±1℃ for 72 hours. The colony diameter (d) and the diameter of the discoloration zone (D) were determined, and strains with a larger D / d value were screened.

[0038] 1.4 Experimental Results

[0039] Twenty-five mold strains were screened from soybean paste samples. Initial screening identified 15 of these strains as capable of producing lipase. Further screening of these 15 strains revealed (see Table 1) that four strains exhibited strong lipase production (D / d value ≥ 1.5): KDX-8, KDX-13, KDX-66, and KDX-21; five strains showed moderate lipase production (D / d value ≥ 1.2, D / d value > 1.5): KDX-5, KDX-6, KDX-14, KDX-18, and KDX-23; and the remaining strains showed weak lipase production. Therefore, KDX-8, KDX-13, KDX-66, and KDX-21 were identified as high-lipase-producing strains.

[0040] Table 1. Results of lipase production assay for the secondary screening strains.

[0041]

[0042] Example 2

[0043] The enzyme production characteristics and safety evaluation of the strain are as follows:

[0044] 2.1 Preparation of Bran Chorchard

[0045] (1) Bran culture medium

[0046] Mix wheat bran, soybean flour, and water in a mass ratio of 4:1:4, stir well, and soak for 30 minutes. Dispense 60g portions into 1L Erlenmeyer flasks, seal with 8 layers of gauze, and sterilize at 121℃ for 20 minutes.

[0047] (2) Inoculation and culture

[0048] A small amount of spores of the above four mold strains (KDX-8, KDX-13, KDX-66, and KDX-21) were picked up with a sterile inoculation loop and inoculated into the bran culture medium. The bran culture medium inoculated with industrial strain Aspergillus oryzae 3.042 was used as a control group. The inoculated bran culture medium was cultured at 30°C. After 16 hours, the clumped bran culture medium was shaken to break up the clumps and cultured for a second time. After 24 hours, the flasks were shaken again. After 48 hours of culture, the bran koji was obtained when the surface of the bran was evenly covered with yellow-green mycelium.

[0049] 2.2 Enzyme activity assay

[0050] (1) Lipase assay

[0051] Sample extraction: Weigh 2.0g of bran koji sample into a 250mL Erlenmeyer flask, add 50mL of 0.1mol / L Tris-HCl buffer (pH 7.5), shake well and place at 37℃ for 1h of extraction; after extraction, filter with filter paper to obtain crude enzyme solution.

[0052] Sample determination: Pipette 1.0 mL of 30 mmol / L p-nitrophenyl palmitate (p-NPP) solution into a 10 mL centrifuge tube, add 2.0 mL of 0.1 mol / L Tris-HCl buffer (pH 7.5), preheat in a 40 °C water bath for 5 min, then add 1.0 mL of appropriately diluted crude enzyme solution, continue in a 40 °C water bath for 5 min, and immediately add 4 mL of ethanol to terminate the reaction. Finally, measure the absorbance at 410 nm. For the blank group, add ethanol first and then crude enzyme solution to the reaction system, and perform the other operations the same as the experimental group.

[0053] Enzyme activity calculation: Prepare p-nitrophenol solutions with concentrations of 10 mmol / mL, 20 mmol / mL, 40 mmol / mL, 60 mmol / mL, and 80 mmol / mL using ultrapure water, and then measure the absorbance at 410 nm; establish a standard curve with concentration on the x-axis and absorbance on the y-axis; calculate the p-nitrophenol concentration in the reaction solution from the standard curve, and calculate the lipase activity (U / g) of the sample using the following formula:

[0054]

[0055] X i The lipase activity in the sample is expressed in U / g.

[0056] A1 represents the concentration of p-nitrophenol in the experimental group calculated according to the standard curve, in mmol / mL.

[0057] A2 is the control group. The concentration of p-nitrophenol was calculated according to the standard curve, and the unit is mmol / mL.

[0058] V1 represents the total volume of the crude enzyme extract of the sample, in mL;

[0059] 8 represents the total volume of the reaction reagents, in mL;

[0060] V2 is the volume of crude enzyme solution added during the reaction, in mL;

[0061] m i The mass of the sample is expressed in grams.

[0062] n i This refers to the secondary dilution factor of the crude enzyme solution in the sample;

[0063] T represents the reaction time, measured in minutes.

[0064] (2) Determination of acidic and neutral proteases

[0065] The determination was performed in accordance with the national standard SB / T 10317-1999.

[0066] (3) Amylase assay

[0067] Sample extraction: Accurately weigh 5.0g of bran koji sample into a 250mL Erlenmeyer flask, add 50mL of pH 6.0 phosphate buffer, and extract by shaking in a 40℃ water bath for 1h. Filter with filter paper to obtain crude enzyme solution.

[0068] Sample determination: In the experimental group, 5 mL of 5 g / L starch solution was preheated in a water bath at 40℃ for 10 min, 0.5 mL of diluted enzyme solution was added, and the mixture was shaken in a water bath at 40℃ for 5 min. Then, 5 mL of 0.1 mol / L sulfuric acid solution was added to inactivate the enzyme. 0.5 mL of the reaction solution was then taken out, 5 mL of dilute iodine solution was added, and the absorbance was measured at 620 nm. In the control group, 5 mL of 0.1 mol / L sulfuric acid solution was added first, followed by diluted crude enzyme solution to inactivate the enzyme and prevent catalytic reaction. The remaining steps were the same as in the experimental group.

[0069] Enzyme activity calculation: Calculate the sample amylase activity (U / g) using the following formula:

[0070]

[0071] X i The amylase activity in the sample is expressed in U / g.

[0072] R1 is the absorbance value of the control group;

[0073] R2 is the absorbance value of the experimental group;

[0074] c. The concentration of the starch solution is expressed in mg / mL.

[0075] V1 is the volume of the starch solution, in mL;

[0076] V2 is the volume of enzyme solution added, in mL;

[0077] V0 represents the total volume of the sample extract, in mL;

[0078] m represents the sample mass, expressed in grams.

[0079] 2.3 Safety Evaluation

[0080] Aflatoxin B1 (AFB1) in wheat bran koji was determined using an enzyme-linked immunosorbent assay (ELISA) kit. The specific procedure is as follows:

[0081] Sample extraction: Crush and weigh 5.0g of sample and place it in a 100mL Erlenmeyer flask. Add 25mL of 60% methanol and shake vigorously on a shaker for 10min. Centrifuge the liquid at 4000r / min for 5min. Take 1mL of the supernatant and add 4mL of deionized water to mix well for subsequent analysis.

[0082] Sample color development: Add 50µL of standard or sample to the corresponding well, add 50µL of AFB1 enzyme label / well, then add 50µL of AFB1 anti-reagent reagent / well, gently shake to mix, cover with a cover film and incubate at 25℃ in the dark for 30min; carefully remove the cover film, shake off the liquid in the well, wash thoroughly with 250µL of washing buffer 4~5 times, with an interval of 10s each time, and pat dry with absorbent paper; finally, add 50µL of substrate solution A / well, then add 50µL of substrate solution B / well, gently shake to mix, cover with a cover film and incubate at 25℃ in the dark for 15min.

[0083] Assay: Add 50 µL of stop solution per well, gently shake to mix, and measure the absorbance at 450 nm using a microplate reader; calculate according to the professional analysis software of the kit.

[0084] 2.4 Results Analysis

[0085] Table 2 shows the results of the determination of lipase, neutral protease, acidic protease, amylase activity, and aflatoxin B1 content in bran koji prepared by different strains. As can be seen from the table, strain KDX-66 exhibited the strongest lipase production capacity, with an activity as high as 246.06 U / g, which is more than 70% higher than that of the common industrial strain *Aspergillus oryzae* 3.042. Furthermore, strain KDX-66 showed a more balanced enzyme production capacity, with its neutral protease, acidic protease, and amylase activities all superior to those of the industrial strain *Aspergillus oryzae* 3.042. Meanwhile, trace amounts of aflatoxin B1 were detected in bran koji prepared by strains DX-8, DX-13, and DX-21, but their content was far below the national food safety limit of 5.0 μg / kg for fermented foods; while neither KDX-66 nor *Aspergillus oryzae* 3.042 produced aflatoxin B1. Therefore, it can be concluded that strain KDX-66 produces enzymes in a balanced manner, especially high levels of lipase, and does not produce aflatoxin B1, making it an ideal strain for food fermentation.

[0086] Table 2. Analysis of enzyme production capacity of different strains

[0087]

[0088] Example 3

[0089] Identification of mold strain KDX-66 is as follows:

[0090] 3.1 Morphological Identification

[0091] Colony morphology observation: KDX-66 strain was inoculated onto PDA medium plates and streaked for activation culture. After incubation at 30℃ for 48 hours, a small amount of spore powder was dipped into sterile physiological saline with a disposable sterile inoculation loop and shaken thoroughly to disperse the spores. Then, a small amount of spore suspension dilution was dipped into a disposable sterile inoculation loop and spread onto PDA medium plates. After incubation at 30℃ for 48 hours, the colony morphology was observed.

[0092] Microscopic morphological observation: Spores and hyphae of single colonies were picked from PDA medium plates, stained with lactic acid phenol cotton blue dye, and slides were prepared. The hyphae, apical vesicles and spore landing morphology were observed using a binocular biological microscope.

[0093] 3.2 Molecular biological identification

[0094] KDX-66 strain was inoculated into YM liquid medium and cultured at 30℃ and 120 rpm for 24 h with shaking. After centrifugation at 4000 rpm for 5 min, bacterial cells were obtained. 20 mg of bacterial cells were taken, thoroughly ground in liquid nitrogen, and then mycotic DNA was extracted using a TSINGKE DNA extraction kit. Using this DNA as a template, the mycotic ITS fragment was amplified using ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') primers. The polymerase chain reaction (PCR) amplification system (50 µL): mix 45 µL, ITS1 2 µL, ITS4 2 µL, DNA template 1 µL. PCR amplification program: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 56℃ annealing for 10 s, 72℃ extension for 10 s, for a total of 35 cycles, followed by a final extension at 72℃ for 5 min. The amplified samples were sent to Beijing Qingke Xinyue Biotechnology Co., Ltd. for sequencing. After sequencing, the sequences were assembled using the basic local alignment search tool (BLAST) program in the National Center for Biotechnology Information (NCBI) and compared with data in the NCBI database to obtain the homologous sequence with the highest similarity to the target species, thus determining the species relationship of the strain.

[0095] 3.3 Results Analysis

[0096] The colony morphology and microscopic structure of strain KDX-66 are shown in the figures below. Figure 1 and Figure 2 .Depend on Figure 1It can be seen that the colonies of this strain on PDA medium are all yellow-green, velvety in texture, with short and loose hyphae exhibiting radial wrinkles. Figure 2 Microscopic examination revealed the presence of conidiophores with thin, long stem walls and a spherical apical sac bearing radiating pedicels. Conidia were found in chains and were round. Based on these morphological characteristics, strain KDX-66 was preliminarily identified as *Aspergillus oryzae*. Aspergillus oryzae Molecular biological identification results showed that the full-length ITS gene sequence of strain KDX-66 was 574 bp, and the gene sequence is shown in SEQ ID No: 1. BLAST sequence homology comparison in GenBank was used to determine its species. Strains highly homologous to strain KDX-66 are... Aspergillus oryzae The strain KDX-66 (GenBank accession number: KP418788.1) and the two strains shared a 99.29% sequence identity. Based on morphological characteristics and aflatoxin metabolic features, strain KDX-66 was identified as *Aspergillus oryzae*. Aspergillus oryzae ).

[0097] Example 4

[0098] Aspergillus oryzae ( Aspergillus oryzae The genetic stability experiment of KDX-66 is as follows:

[0099] 4.1 Passage culture

[0100] The 0th generation Aspergillus oryzae (frozen at -80℃) Aspergillus oryzae KDX-66 was inoculated into fresh PD liquid medium and cultured at 30℃ with shaking at 120r / min for 24h. Then, a small amount of culture medium was streaked onto the slant of PDA medium using a disposable sterile inoculation loop and cultured statically at 30℃ for 48h. Each subsequent subculturing was counted as one generation, and the process was repeated up to the 7th generation. Each generation of fresh PD liquid medium slant was sealed with glycerol and stored in a refrigerator at 4℃.

[0101] 4.2 Lipase activity stability

[0102] Wheat bran culture medium: Mix wheat bran, soybean flour and water in a mass ratio of 4:1:4, stir well and soak for 30 minutes, dispense 60g into 1L conical flasks, seal with 8 layers of gauze, and sterilize at 121℃ for 20 minutes.

[0103] Bran koji preparation: Select a small amount of Aspergillus oryzae from each of the above generations ( Aspergillus oryzae Spores of strain KDX-66 were inoculated into bran culture medium and cultured at 30°C. During the 16-hour period, the clumped bran culture medium was shaken to break up the clumps and cultured for another 24 hours. After 48 hours of culture, the bran koji was obtained when the surface of the bran was evenly covered with yellow-green mycelium.

[0104] Lipase activity assay: See Example 2 for specific methods.

[0105] 4.3 Analysis of Experimental Results

[0106] Aspergillus oryzae (7 generations of continuous subculturing) Aspergillus oryzae The lipase activity of Aspergillus oryzae strain KDX-66 in the preparation of bran koji is shown in Table 3. As can be seen from the table, the lipase activity of Aspergillus oryzae strain KDX-66 in the preparation of bran koji is... Aspergillus oryzae After 7 generations, the lipase activity of KDX-66 decreased slightly, but the 7th generation still had a high lipase activity of 239.10 U / g, with an enzyme activity decrease of only 4.4%. Therefore, it can be concluded that *Aspergillus oryzae* (…) Aspergillus oryzae KDX-66 exhibits good genetic stability.

[0107] Table 3. Record of lipase stability during KDX-66 strain passage.

[0108]

[0109] Example 5

[0110] Using Aspergillus oryzae ( Aspergillus oryzae The method for preparing fresh chili bean paste rich in unsaturated fatty acids using KDX-66 is as follows:

[0111] 5.1 Preparation method

[0112] (1) Koji making: After the shelled broad beans are selected and impurities are removed, they are blanched in boiling water for 3-5 minutes and immediately cooled to 37°C. Then, the broad beans and wheat flour are mixed at a mass ratio of 5:1, and Aspergillus oryzae is inoculated at an inoculation rate of 0.03% (based on the total mass of broad beans and wheat flour). Aspergillus oryzae KDX-66 spore powder was prepared; the inoculated koji material was transferred to a disc koji maker and spread evenly, with the material thickness controlled at 40cm. Koji fermentation was carried out at a temperature of 30℃ and a relative humidity of ≥85%. The koji was turned over once at 12h and 24h. When the surface of the koji material was covered with yellow-green mycelium, the fermented koji was obtained.

[0113] (2) Fermentation: After removing the stems, impurities and washing the fresh red Erjingtiao peppers, crush them into pepper mash with a particle size of 2nm~4nm. Then, mix the pepper mash and broad bean mash at a mass ratio of 7:3 and add salt to control the NaCl mass concentration of the fermentation system to 12%. Then, transfer the fresh pepper and broad bean mash to a cleaned and disinfected earthenware jar and seal it with food-grade plastic film and cotton cloth. Finally, place the earthenware jar in a sealed high-cleanliness workshop for natural fermentation at room temperature. Stir it once every 3 days with cleaning tools. After 6 months of fermentation, fresh pepper broad bean mash rich in saturated fatty acids is obtained.

[0114] 5.2 Indicator Analysis

[0115] (1) Detection of fatty acid content

[0116] The fatty acid composition of the sample was determined using gas chromatography. The specific procedure is as follows:

[0117] Sample extraction: Weigh 2.0 g (accurate to 0.1 mg) of the uniform sample and transfer it to a 250 mL Erlenmeyer flask. Add 20 mL of 95% ethanol and shake to extract. Transfer the extract from the Erlenmeyer flask to a separatory funnel. Rinse the Erlenmeyer flask with 50 mL of a mixture of diethyl ether and petroleum ether, and add the rinse solution to the separatory funnel. Cover the funnel and shake for 5 min. Let it stand for 10 min. Collect the ether extract in a 25 mL flask. Repeat the above steps 3 times. Finally, rinse the separatory funnel with a mixture of diethyl ether and petroleum ether and collect the extract in a 250 mL flask. Concentrate to dryness using a rotary evaporator. The residue is a fat extract.

[0118] Fatty acid methyl esterification: Add 8 mL of 2% sodium hydroxide and methanol solution to the fatty acid extract, connect a reflux condenser, and reflux in a water bath at 80℃±1℃ until the oil droplets disappear; add 7 mL of 15% boron trifluoride methanol solution from the top of the reflux condenser, and continue reflux in a water bath at 80℃±1℃ for 2 min; rinse the reflux condenser with a small amount of water; stop heating, remove the flask from the water bath, and quickly cool to room temperature; accurately add 20 mL of n-heptane, shake for 2 min, then add saturated sodium chloride aqueous solution, and allow to stand for separation; pipette approximately 5 mL of the upper n-heptane extract solution into a 25 mL test tube, add approximately 3 g~5 g of anhydrous sodium sulfate, shake for 1 min, let stand for 5 min, and pipette the upper solution into a sample vial for analysis.

[0119] Gas phase conditions: Inlet temperature 260℃, detection chamber temperature 280℃, flow rate 1.0 mL / min, temperature program: 100℃ held for 13 min; 10℃ / min to 180℃ held for 6 min; 1℃ / min to 200℃ held for 20 min; 4℃ / min to 230℃ held for 10.5 min.

[0120] (2) Detection of volatile flavor compounds

[0121] The volatile flavor components in fermented soybean paste were detected using headspace solid-phase microextraction combined with gas chromatography-mass spectrometry. The specific procedures are as follows:

[0122] Sample testing: Accurately weigh 2.0 g of sample into a 15 mL headspace vial, add 5 μL of internal standard (4-methyl-2-pentanol solution, concentration 0.5 μg / mL), preheat the vial in a 60 °C water bath for 2 min, then insert the aged SPME extraction head into the vial, perform headspace extraction at 60 °C for 50 min, then remove the extraction head and insert it into the GC-MS gas chromatography injection port, and parse at 250 °C for 5 min. Each sample is measured independently 3 times.

[0123] Gas chromatography conditions: DB-WAX capillary column (60m×0.25mm, 0.25μm); carrier gas: helium, flow rate: 1mL / min; injection port temperature: 250℃; splitless injection; temperature program: initial temperature 50℃, increase to 85℃ at 10℃ / min (hold for 1.5min), then increase to 100℃ at 5℃ / min (hold for 1min), increase to 175℃ at 2.5℃ / min (hold for 1.5min), and finally increase to 250℃ / min at 10℃ / min.

[0124] Mass spectrometry conditions: EI ionization source, electron bombardment energy 70 eV; ion source temperature 230℃; interface 250℃; mass scan range 35 amu~350 amu; detector voltage 0.1 kV; tuning file stuneu; scan mode scan.

[0125] Qualitative and quantitative analysis: The chromatograms obtained by GC-MS were compared and searched in the standard spectral library NIST11 by computer. Substances with similarity (SI) > 80 (maximum value 100) were selected for qualitative analysis and each volatile component was accurately identified. At the same time, 4-methyl-2-pentanol (0.5 μg / mL) was used as an internal standard for semi-quantitative analysis to obtain the mass concentration of each component.

[0126] Compare with Example 1

[0127] The only difference between Comparative Example 1 and Example 5 is that Aspergillus oryzae was not inoculated in step (1). Aspergillus oryzae The spore powder prepared by KDX-66 was inoculated with Aspergillus oryzae strain 3.042 from Huniang, and all other conditions were exactly the same.

[0128] The results of Example 5 are compared with those of Control Example 1, as follows:

[0129] The results of fatty acid detection in fresh chili bean paste prepared in Example 5 and Control Example 1 are as follows: Figure 3 As shown in Table 4. From Figure 3 It can be seen that Example 5 utilizes Aspergillus oryzae ( Aspergillus oryzae The unsaturated fatty acid content in the fresh chili bean paste made with KDX-66 was as high as 1770.13 mg / kg, far exceeding the 1128.91 mg / kg of Control Example 1; compared with Control Example 1, the unsaturated fatty acid content in Example 5 increased by 56.80%. As shown in Table 4, compared with Control Example 1, Example 5 utilized Aspergillus oryzae (… Aspergillus oryzaeIn the fresh chili bean paste prepared with KDX-66, the content of unsaturated fatty acids such as myristoleic acid, palmitoleic acid, oleic acid, and linoleic acid was significantly increased, with relative increases of 36.71%, 62.67%, 173.71%, and 7.86%, respectively. In the fresh chili bean paste prepared in Example 5, the total content of free fatty acids was also increased by 65.09% compared to Control Example 1.

[0130] Table 4. Record of Fatty Acid Composition in Fresh Chili Bean Paste

[0131]

[0132] Furthermore, the volatile flavor components of the fresh chili bean paste prepared in Example 5 and Control Example 1 were analyzed, and the results are shown in Table 5. As can be seen from the table, Example 5 utilized Aspergillus oryzae (… Aspergillus oryzae In fresh chili bean paste made with KDX-66, the content of volatile flavor compounds was significantly increased, especially phenolic compounds, which increased by 114.20%; followed by esters and alcohols, which increased by 39.63% and 26.41%, respectively. This indicates that using Aspergillus oryzae (… Aspergillus oryzae KDX-66 can improve the flavor and quality of fresh chili bean paste.

[0133] Table 5. Volatile flavor components in fresh chili bean paste.

[0134]

[0135] The above embodiments are only used to illustrate the technical concept and features of the present invention, and are not intended to be unique or to limit the present invention. Those skilled in the art should understand that various changes or equivalent substitutions made to the present invention without departing from its scope are all within the protection scope of the present invention.

Claims

1. A strain of Aspergillus oryzae that produces a high amount of lipase ( Aspergillus oryzae KDX-66, characterized in that, Preserved in Guangdong Microbial Culture Collection Center on November 14, 2025, with the preservation number GDMCC No: 67295.

2. Use of the Aspergillus oryzae according to claim 1, characterized in that For preparing a leavening agent, the leavening agent comprising Aspergillus oryzae spore powder.

3. Use of the Aspergillus oryzae according to claim 1, characterized in that, For preparing a fermented condiment.

4. Use of Aspergillus oryzae according to claim 3, characterized in that For increasing the content of unsaturated fatty acids and / or volatile flavor substances in the fermented condiment.

5. Use of Aspergillus oryzae according to claim 3, characterized in that, The fermented condiment comprises one or more of soy sauce, doubanjiang, soybean paste, and douchi.

6. Use of Aspergillus oryzae according to claim 4, characterized in that The unsaturated fatty acids comprise one or more of oleic acid, linoleic acid, myristoleic acid, palmitoleic acid, and linolenic acid.

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