Aspergillus oryzae with high yield of lipase, application of aspergillus oryzae and method for preparing fresh pepper bean halves rich in unsaturated fatty acid

Abstract

The invention discloses Aspergillus oryzae with high yield of lipase, application and a method for preparing fresh pepper bean halves rich in unsaturated fatty acid, and relates to the technical field of microorganisms and food fermentation, the Aspergillus oryzae has the taxonomic name of Aspergillus oryzae, is preserved in Guangdong Microbial Culture Collection Center on November 14, 2025, and has the preservation number of GDMCC No: 67295. Aspergillus oryzae is relatively balanced in enzyme production capacity and high in lipase yield, the lipase activity can be up to 246.06 U / g, the genetic stability is good, and aflatoxin B1 is not produced; in the prepared fresh chili bean halves, the content of unsaturated fatty acid reaches up to 1770.13 mg / kg, and the content of volatile flavor substances is greatly increased.

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: 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.

[0006] 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.

[0007] 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.

[0008] 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.

[0009] This invention provides the aforementioned Aspergillus oryzae ( Aspergillus oryzae KDX-66 is used to prepare starter cultures, which include Aspergillus oryzae spore powder.

[0010] This invention provides the aforementioned Aspergillus oryzae ( Aspergillus oryzae KDX-66 is used to increase the content of unsaturated fatty acids in fermented condiments.

[0011] Specifically, the fermented condiments include one or more of soy sauce, broad bean paste, soybean paste, and fermented black beans.

[0012] Specifically, the unsaturated fatty acids include one or more of oleic acid, linoleic acid, myristoleic acid, palmitoleic acid, and linolenic acid.

[0013] This invention provides a method for preparing fresh chili bean paste rich in unsaturated fatty acids, comprising the following steps: (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.

[0014] 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%.

[0015] (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.

[0016] 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.

[0017] Specifically, the NaCl mass concentration of the fermentation system is 10%~18%, preferably 12%.

[0018] The beneficial effects of this invention are as follows: 1. This invention screened a new strain from fermented soybean paste, which was identified as Aspergillus oryzae (…). Aspergillus oryzae ).

[0019] 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.

[0020] 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 considered a safe strain.

[0021] 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.

[0022] 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

[0023] Figure 1 Aspergillus oryzae ( Aspergillus oryzae Colony morphology diagram of KDX-66.

[0024] Figure 2 Aspergillus oryzae ( Aspergillus oryzae Microscopic morphology of KDX-66.

[0025] Figure 3 This is a graph showing the content of unsaturated fatty acids in fresh chili bean paste.

[0026] 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

[0027] Example 1 The screening process for high-lipase-producing strains is as follows: 1.1 Separation and Purification 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.

[0028] 1.2 Initial screening Using a sterile inoculation loop, pick a small amount of fungal spores from the slant culture into 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.

[0029] 1.3 Secondary screening 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.

[0030] 1.4 Experimental Results 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.

[0031] Table 1. Results of lipase production assay for the secondary screening strains. Example 2 The enzyme production characteristics and safety evaluation of the strain are as follows: 2.1 Preparation of Bran Chorchard (1) 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 portions into 1L Erlenmeyer flasks, seal with 8 layers of gauze, and sterilize at 121℃ for 20 minutes.

[0032] (2) Inoculation and culture 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.

[0033] 2.2 Enzyme activity assay (1) Lipase assay 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.

[0034] Sample determination: 1.0 mL of 30 mmol / L p-nitrophenyl palmitate (p-NPP) solution was taken in a 10 mL centrifuge tube, 2.0 mL of 0.1 mol / L Tris-HCl buffer (pH 7.5) was added, and after preheating in a 40°C water bath for 5 min, 1.0 mL of appropriately diluted crude enzyme solution was added in turn, and after continuing to heat in a 40°C water bath for 5 min, 4 mL of ethanol was immediately added to terminate the reaction, and finally the absorbance was determined at 410 nm; the blank group was that ethanol was added first in the reaction system, and the rest of the operation was the same as the experimental group.

[0035] Enzyme activity calculation: 10 mmol / mL, 20 mmol / mL, 40 mmol / mL, 60 mmol / mL, and 80 mmol / mL of p-nitrophenol solution were prepared with ultrapure water, and then the absorbance was determined at 410 nm; a standard curve was established with the concentration as the abscissa and the absorbance as the ordinate; the concentration of p-nitrophenol in the reaction solution was calculated from the standard curve, and the sample lipase activity (U / g) was calculated according to the following formula: X i is the lipase activity in the sample, with the unit of U / g; A1 is the p-nitrophenol concentration calculated from the standard curve for the experimental group, with the unit of mmol / mL; A2 is the p-nitrophenol concentration calculated from the standard curve for the control group, with the unit of mmol / mL; V1 is the total volume of the sample crude enzyme extract, with the unit of mL; 8 is the total volume of the reaction reagent, with the unit of mL; V2 is the volume of crude enzyme solution added in the reaction, with the unit of mL; m i is the mass of the sample, with the unit of g; n i is the second dilution multiple of the sample crude enzyme solution; T is the reaction time, with the unit of min.

[0036] (2) Acid and neutral protease determination Determined according to the method of national standard SB / T 10317-1999.

[0037] (3) Amylase determination Sample extraction: 5.0 g of bran koji sample was accurately weighed into a 250 mL conical flask, 50 mL of pH 6.0 phosphate buffer was added, and extraction was carried out in a 40°C water bath for 1 h, and then the crude enzyme solution was obtained by filtering with filter paper.

[0038] Sample determination: the experimental group took 5 mL of 5 g / L starch solution and preheated it in a water bath at 40°C for 10 min, then added 0.5 mL of diluted enzyme solution, and reacted in a water bath at 40°C for 5 min, and then added 5 mL of 0.1 mol / L sulfuric acid solution to inactivate it; then 0.5 mL of reaction solution was taken out, 5 mL of dilute iodine solution was added and mixed, and its absorbance value was detected at 620 nm; the control group first added 5 mL of 0.1 mol / L sulfuric acid solution, then added the diluted crude enzyme solution, and inactivated the enzyme, so it could not catalyze the reaction, and the rest of the steps were the same as the experimental group.

[0039] Enzyme activity calculation: the sample amylase activity (U / g) was calculated according to the following formula: X i is the amylase activity in the sample, with the unit of U / g; R1 is the absorbance value of the control group; R2 is the absorbance value of the experimental group; c is the concentration of the starch solution, with the unit of mg / mL; V1 is the volume of the starch solution, with the unit of mL; V2 is the volume of the enzyme solution, with the unit of mL; V0 is the total volume of the sample extraction solution, with the unit of mL; m is the sample mass, with the unit of g.

[0040] 2.3 Safety evaluation The enzyme-linked immunoassay kit was used to determine the aflatoxin B1 (AFB1) in bran koji, and the specific operation was as follows: Sample extraction: 5.0 g of sample was ground and weighed into a 100 mL conical flask, 25 mL of 60% methanol was added, and it was vigorously shaken on a shaker for 10 min; the liquid was centrifuged at 4000 r / min for 5 min, 1 mL of supernatant was taken and 4 mL of deionized water was added and mixed for subsequent analysis.

[0041] Sample color development: 50 µL of standard or sample was added to the corresponding microwell, 50 µL of AFB1 enzyme label was added per well, 50 µL of AFB1 anti-reagent was added per well, and it was mixed gently, covered with a cover film, and placed in a 25°C light-proof environment for 30 min; the cover film was carefully removed, the liquid in the well was spun dry, 250 µL of washing solution was added per well, and it was washed thoroughly 4-5 times, with an interval of 10 s each time, and then it was patted dry with a water-absorbing paper; finally, 50 µL of substrate solution A was added per well, and 50 µL of substrate solution B was added per well, and it was mixed gently, covered with a cover film, and placed in a 25°C light-proof environment for 15 min.

[0042] Determination: add 50 μL / well of termination solution, shake gently to mix, and measure the absorbance at 450 nm using a microplate reader; calculate according to the professional analysis software of the kit.

[0043] 2.4 Result analysis The determination results of the lipase, neutral protease, acid protease, amylase activities and aflatoxin B1 content in bran koji prepared by different strains are shown in Table 2. As can be seen from the table, the KDX-66 strain has the strongest ability to produce lipase, with a lipase activity of up to 246.06 U / g, which is more than 70% higher than that of the ordinary industrial strain Aspergillus oryzae Shangnian 3.042. In addition, the KDX-66 strain has balanced enzyme production capacity, and the activities of neutral protease, acid protease and amylase are also better than those of the industrial strain Aspergillus oryzae Shangnian 3.042. At the same time, a small amount of aflatoxin B1 was detected in the bran koji prepared by the DX-8, DX-13 and DX-21 strains, but the content was far lower than the limit value of 5.0 μg / kg in the national standard for safety of fermented food; and neither KDX-66 nor Aspergillus oryzae Shangnian 3.042 produced aflatoxin B1. Therefore, the KDX-66 strain has balanced enzyme production, especially high lipase production, and does not produce aflatoxin B1, and is an ideal strain for food fermentation.

[0044] Table 2 Analysis of enzyme production capacity of different strains Example 3 The mold KDX-66 strain was identified as follows: 3.1 Morphological characteristic identification Colony morphology observation: the KDX-66 strain was inoculated on PDA medium plates for activation culture, and after 48 h of inverted culture at 30°C, a small amount of spore powder was taken with a disposable sterile inoculation loop and added to sterile normal saline, and the spores were dispersed by shaking. Then, a small amount of spore suspension diluent was taken with a disposable sterile inoculation loop and spread on PDA medium plates, and the plates were incubated at 30°C for 48 h, and the colony morphology was observed.

[0045] Microscopic morphology observation: spores and hyphae of single colonies on PDA medium plates were picked and stained with lactic acid phenol cotton blue staining solution to prepare slides, and the landing morphology of hyphae, pycnidia and spores was observed under a binocular biological microscope.

[0046] 3.2 Molecular biology identification KDX-66 strain was inoculated into YM liquid medium and cultured at 30°C with 120 r / min for 24 h, then centrifuged at 4000 r / min for 5 min to obtain the bacterial body; 20 mg of the bacterial body was ground in liquid nitrogen, and then DNA extraction was performed using a TSINGKE DNA extraction kit; the ITS fragment of the mold was amplified using ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') as primers. The PCR amplification system (50 µL) was as follows: mix 45 µL, ITS 12 µL, ITS4 2 µL, and DNA template 1 µL. The PCR amplification program was as follows: 98°C pre-denaturation for 2 min; 98°C denaturation for 10 s, 56°C annealing for 10 s, 72°C extension for 10 s, a total of 35 cycles, and 72°C re-extension for 5 min. The amplified sample was sent to Beijing Qianke New Industry Biotechnology Co., Ltd. for sequencing. After sequencing, the basic local alignment search tool (BLAST) program in the national center for biotechnology information (NCBI) was used for splicing, and the data in the NCBI database were compared to obtain the homologous sequence with the highest similarity to the test species sequence, and the strain species relationship was determined.

[0047] 3.3 Result analysis The colony morphology and microscopic morphological structure of KDX-66 strain are shown in Figure 1 and Figure 2 , respectively. As shown in Figure 1 , the colonies of the strain on PDA medium are yellow-green in color, with a silky texture, short and loose mycelium, and a radial fold. As shown in Figure 2 , the microscopic morphology shows that the strain has conidial phialides, thin and long phialide stems, spherical apical cysts, radial small stems on the surface, and conidial phialides, and the conidia are round. According to the morphological characteristics, KDX-66 strain is preliminarily determined to be Aspergillus oryzae. Aspergillus oryzae Molecular biology identification results show that the full-length ITS gene sequence of KDX-66 strain is 574 bp, and the gene sequence is shown in SEQ ID No: 1. The species was determined by BLAST sequence homology comparison in GenBank. The strain with high homology with KDX-66 strain is Aspergillus oryzaeStrain (GenBank Accession No: KP418788.1), and the sequence identity was 99.29%. Combined with morphological characteristics and aflatoxin metabolism characteristics, the KDX-66 strain was determined to be Aspergillus oryzae Aspergillus oryzae ).

[0048] Example 4 Aspergillus oryzae Aspergillus oryzae ) KDX-66 genetic stability experiment, as follows: 4.1 Subculture The 0th generation Aspergillus oryzae (KDX-66) in the -80°C freezer was inoculated in fresh PD liquid medium, and after 24h of 30°C 120r / min shaking culture, a small amount of culture was taken with a sterile inoculation loop to streak on a PDA medium slope, and the culture was placed at 30°C for 48h. After that, the transfer was counted as one generation, and the strain was subcultured to the 7th generation. The fresh PD liquid medium slope was sealed and stored in a 4°C refrigerator with glycerol. Aspergillus oryzae

[0049] 4.2 Lipase activity stability Bran medium: bran, soybean meal and water were mixed according to the mass ratio of 4:1:4, and after being mixed evenly, they were soaked for 30min. Then, 60g of the mixture was packed in a 1L conical flask, and the flask was sealed with 8 layers of gauze and sterilized at 121°C for 20min.

[0050] Bran koji preparation: a small amount of spores of the Aspergillus oryzae (KDX-66) strain of each generation described above was inoculated into bran medium, and the medium was cultured at 30°C. During the culture, the bran medium was shaken to disperse the clumps at 16h, and the shaking was repeated at 24h. After 48h of culture, when the bran surface was uniformly covered with yellow-green mycelium, bran koji was obtained. Aspergillus oryzae Lipase activity determination: the specific method is shown in Example 2.

[0051] 4.3 Analysis of experimental results

[0052] The lipase activity of the bran koji prepared from the Aspergillus oryzae (KDX-66) strain subcultured for 7 generations is shown in Table 3. As shown in the table, the lipase activity of the Aspergillus oryzae (KDX-66) strain decreased slightly after subculture for 7 generations, but the lipase activity of the 7th generation strain was still as high as 239.10U / g, and the enzyme activity only decreased by 4.4%. Therefore, the Aspergillus oryzae (KDX-66) strain has good genetic stability. Aspergillus oryzae The lipase activity of the bran koji prepared from the Aspergillus oryzae (KDX-66) strain subcultured for 7 generations is shown in Table 3. As shown in the table, the lipase activity of the Aspergillus oryzae (KDX-66) strain decreased slightly after subculture for 7 generations, but the lipase activity of the 7th generation strain was still as high as 239.10U / g, and the enzyme activity only decreased by 4.4%. Therefore, the Aspergillus oryzae (KDX-66) strain has good genetic stability. Aspergillus oryzae Aspergillus oryzae Table 3 Lipase stability record table of KDX-66 strain during subculture

[0053] ​​​​Example 5 A method for preparing fresh chili bean with high unsaturated fatty acid content using Aspergillus oryzae (KDX-66) is as follows: Aspergillus oryzae 5.1 Preparation method (1) Koji preparation: unhulled broad bean is selected and impurities are removed, then boiled in water for 3-5 min, immediately cooled to 37°C, then mixed with wheat flour at a mass ratio of 5:1, inoculated with spore powder of Aspergillus oryzae (KDX-66) at an inoculation amount of 0.03% (based on the total mass of broad bean and wheat flour); the inoculated koji material is transferred to a disc koji maker and flattened, with a material thickness of 40 cm, and fermented at a temperature of 30°C and a relative humidity of ≥85%, with each turning of the koji material at 12 h and 24 h, until the surface of the koji material is covered with yellow-green mycelium, to obtain broad bean koji. Aspergillus oryzae

[0054] (2) Fermentation: fresh red pepper is de-seeded, impurities are removed, and then washed, and then crushed into 2-4 mm pepper mash, then mixed with broad bean koji at a mass ratio of 7:3, and supplemented with salt to control the NaCl mass concentration of the fermentation system to 12%; then, the fresh chili bean mash is transferred to a ceramic jar that has been cleaned and disinfected, sealed with food-grade plastic film and cotton cloth; finally, the ceramic jar is placed in a closed high-cleanliness workshop for natural fermentation at room temperature, and stirred with clean utensils every 3 days during the fermentation period, and after 6 months of fermentation, fresh chili bean with high saturated fatty acid content is obtained.

[0055] 5.2 Index analysis (1) Fatty acid content detection The fatty acid components in the sample are determined by gas chromatography, and the specific operation is as follows: Sample extraction: 2.0 g (accurate to 0.1 mg) of uniform sample is weighed into a 250 mL conical flask, 20 mL of 95% ethanol is added, and the sample is extracted by shaking; the extraction liquid in the conical flask is transferred to a separatory funnel, the conical flask is washed with 50 mL of ethyl ether petroleum ether mixture, the washing liquid is added to the separatory funnel, and the lid is closed; shake for 5 min, stand for 10 min; collect the ether layer extract into a 25 mL flask; repeat the extraction 3 times according to the above steps, and finally wash the separatory funnel with ethyl ether petroleum ether mixture and collect it into a 250 mL flask; concentrate to dryness on a rotary evaporator, and the residue is the fat extract.

[0056] ​​Fatty acid methyl esterification: add 2% sodium hydroxide and methanol solution 8 mL in the fatty extract, connect the reflux condenser, reflux in 80℃±1℃ water bath until the oil drops disappear; add 7 mL 15% boron trifluoride methanol solution from the upper end of the reflux condenser, continue to reflux in 80℃±1℃ water bath for 2 min; rinse the reflux condenser with a small amount of water; stop heating, remove the flask from the water bath, cool quickly to room temperature; accurately add 20 mL of n-heptane, shake for 2 min, then add saturated sodium chloride aqueous solution, stand for separation; take about 5 mL of the upper n-heptane extract solution into a 25 mL test tube, add about 3g~5g of anhydrous sodium sulfate, shake for 1 min, stand for 5 min, and then take the upper solution into a sample bottle for determination.

[0057] Gas phase conditions: the injection port temperature is 260℃, the detection chamber temperature is 280℃, the flow rate is 1.0 mL / min, and the temperature rising program is 100℃ for 13 min; increased to 180℃ at 10℃ / min, kept for 6 min; increased to 200℃ at 1℃ / min, kept for 20 min; increased to 230℃ at 4℃ / min, kept for 10.5 min.

[0058] (2) Volatile flavor substance detection The volatile flavor components in the bean paste were detected by headspace solid phase microextraction combined with gas chromatography-mass spectrometry, and the specific operation was as follows: Sample detection: accurately take 2.0 g of sample into a 15 mL headspace sample bottle, add 5 μL of internal standard (4-methyl-2-pentanol solution, concentration is 0.5 μg / mL), put the sample bottle into a 60℃ water bath for preheating for 2 min, then insert the aged SPME extraction head into the sample bottle, extract at 60℃ constant temperature for 50 min, then pull out the extraction head and insert it into the GC-MS gas chromatography injection port, analyze at 250℃ for 5 min, and each sample is independently determined for 3 times.

[0059] Gas chromatography conditions: DB-WAX capillary column (60 m x 0.25 mm, 0.25 μm); carrier gas is helium, flow rate is 1 mL / min; injection port temperature is 250℃; no split injection; temperature rising program: initial temperature is 50℃, increased to 85℃ at 10℃ / min (kept for 1.5 min), then increased to 100℃ at 5℃ / min (kept for 1 min), increased to 175℃ at 2.5℃ / min (kept for 1.5 min), and finally increased to 250℃ / min at 10℃ / min.

[0060] Mass spectrometry conditions: EI ion source, electron impact 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.

[0061] Qualitative and quantitative analysis: the chromatogram obtained by GC-MS was compared and searched in the standard spectrum library NIST11 by computer, and the substances with similarity (SI) > 80 (the maximum value is 100) were selected for qualitative analysis, and each volatile component was accurately identified, and 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.

[0062] Comparative Example 1 The only difference between Comparative Example 1 and Example 5 is that in step (1), Aspergillus oryzae is not inoculated Aspergillus oryzae ) KDX-66 spore powder is inoculated with Aspergillus oryzae strain Shu Nian 3.042, and the rest of the conditions are exactly the same.

[0063] The results of Example 5 and Comparative Example 1 are compared as follows: The detection results of fatty acids in the fresh chili bean paste prepared in Example 5 and Comparative Example 1 are shown in Table 4. Figure 3 As can be seen from Table 4, compared with Comparative Example 1, the content of unsaturated fatty acids in the fresh chili bean paste prepared by Aspergillus oryzae (KDX-66) in Example 5 is as high as 1770.13 mg / kg, which is much higher than 1128.91 mg / kg in Comparative Example 1; the content of unsaturated fatty acids in Example 5 is increased by 56.80% compared with Comparative Example 1. Figure 3 Aspergillus oryzae As can be seen from Table 4, compared with Comparative Example 1, the content of unsaturated fatty acids in the fresh chili bean paste prepared by Aspergillus oryzae (KDX-66) in Example 5 is as high as 1770.13 mg / kg, which is much higher than 1128.91 mg / kg in Comparative Example 1; the content of unsaturated fatty acids in Example 5 is increased by 56.80% compared with Comparative Example 1. Aspergillus oryzae As can be seen from Table 4, compared with Comparative Example 1, the content of unsaturated fatty acids in the fresh chili bean paste prepared by Aspergillus oryzae (KDX-66) in Example 5 is as high as 1770.13 mg / kg, which is much higher than 1128.91 mg / kg in Comparative Example 1; the content of unsaturated fatty acids in Example 5 is increased by 56.80% compared with Comparative Example 1.

[0064] Table 4 Record Table of Fatty Acid Composition in Fresh Chili Bean Paste Further, the volatile flavor components of the fresh chili bean paste prepared in Example 5 and Comparative Example 1 were analyzed, and the results are shown in Table 5. As can be seen from the table, the content of volatile flavor substances in the fresh chili bean paste prepared by Aspergillus oryzae (KDX-66) in Example 5 is greatly improved, especially the content of phenolic compounds is increased by 114.20%; followed by ester compounds and alcohol compounds, the contents are increased by 39.63% and 26.41% respectively. Therefore, the fresh chili bean paste prepared by Aspergillus oryzae (KDX-66) can improve the flavor of the product and improve the quality of the product. Aspergillus oryzae Aspergillus oryzae Therefore, the fresh chili bean paste prepared by Aspergillus oryzae (KDX-66) can improve the flavor of the product and improve the quality of the product.​​

[0065] Table 5 Table of volatile flavor components in chili bean The above examples are only used to illustrate the technical ideas and characteristics of the present application, and do not mean to be the only or limit the present application. Those skilled in the art should understand that various changes or equivalent replacements made to the present application without departing from the scope of the present application, all belong to the scope of protection of the present application.

Claims

1. A strain of Aspergillus oryzae KDX-66, which is characterized in that, The taxonomic name is Aspergillus oryzae , and was 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 ferment, the ferment 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 acid and / or volatile flavor in a fermented condiment.

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

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

7. A method of preparing unsaturated fatty acid-rich fresh chilli bean in a paste, characterized by, The method comprises the following steps: (1) koji making: after boiling water blanching the unhulled broad bean for 3 min to 5 min, immediately cool to 35°C to 40°C; mix the broad bean with wheat flour at a mass ratio of 9 to 11:2, inoculate the ferment of claim 2, and the inoculation amount of the ferment is 0.01% to 0.05% of the total mass of the broad bean and the wheat flour; transfer the inoculated koji material to a koji making device, and perform koji making fermentation under the condition of a temperature of 28°C to 35°C and a relative humidity of ≥85%, and turn the koji at regular intervals during the process; when the koji material surface is covered with yellow-green mycelium, obtain the broad bean koji; (2) fermentation: after removing the stem, impurities, and washing the fresh red pepper, grind it into 2nm to 4nm pepper mash, mix the pepper mash with the broad bean koji at a mass ratio of 7:3 to 8:2, and control the mass concentration of NaCl in the fermentation system by adding salt; then transfer to a cleaned and disinfected container, seal, and finally place in a closed high-cleanliness workshop for natural fermentation for 3 months to 6 months to obtain fresh pepper broad bean rich in saturated fatty acid.

8. The method for preparing fresh chili bean paste rich in unsaturated fatty acids according to claim 7, characterized in that, In step (1), during the koji making fermentation process, turn the koji once at 12h and 24h.

9. The method of claim 7, wherein the unsaturated fatty acid-rich fresh chili bean is prepared by the steps of: In step (2), the mass concentration of NaCl in the fermentation system is 10% to 18%. ​

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