Aspergillus niger MQM20136 and application thereof

Abstract

The invention discloses an Aspergillus niger MQM20136 and an application of the Aspergillus niger MQM20136. The strain is a strain MQM20136, the classification name of the strain is Aspergillus niger, the strain is preserved in the China Center for Type Culture Collection in Wuhan, the preservation time is September 25, 2025, and the preservation number is CCTCC NO: M 20252120. The Aspergillus niger MQM20136 has the brewing characteristics of high esterification level and strong amylase activity, can be used as a special native strain for preparing the monascus purpureus, and plays an important role in improving the fermentation performance and stability of the monascus purpureus.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to a strain of Aspergillus niger MQM20136 and its applications. Background Technology

[0002] Wuyi Red Yeast Rice is a unique saccharifying agent, fermenting agent, and aroma enhancer in red yeast rice wine brewing. It is produced by inoculating rice as the fermentation substrate with koji (mainly Aspergillus niger) and koji mother (mainly Aspergillus rubrum) in an open environment. Compared to Gutian Red Yeast Rice, which is only inoculated with Aspergillus rubrum, Wuyi Red Yeast Rice, with the addition of Aspergillus niger, has higher saccharification and liquefaction capabilities. This demonstrates the synergistic effect of the combination of Aspergillus niger and Aspergillus rubrum. However, in the traditional preparation process of Wuyi Red Yeast Rice, Aspergillus niger and Aspergillus niger are mainly inoculated using a successive generation method, which has drawbacks such as susceptibility to contamination, degeneration, or mutation, leading to a decrease in the saccharification and liquefaction capabilities of the red yeast rice, thus reducing the utilization rate of brewing raw materials. Yeasts, on the other hand, mainly originate from free-falling bacteria in the open koji-making process, resulting in significant uncertainty in strain type and abundance, making the quality of the red yeast rice highly unstable and difficult to control. Furthermore, esters are one of the main aroma components of red yeast rice wine, and high-ester-producing strains play a crucial role in the esterification reaction during the brewing process.

[0003] Therefore, based on the microbial community structure and functional characteristics of *Aspergillus oryzae*, identifying strains with excellent brewing properties and then using pure-culture compound koji production could potentially improve the fermentation performance and stability of *Aspergillus oryzae*. Existing research has focused on screening *Aspergillus oryzae* and yeasts that produce high color and high alcohol yields for brewing *Aspergillus oryzae*, but isolation and screening targeting high-ester-producing native *Aspergillus niger* is relatively lacking. Currently, there are patents related to *Aspergillus niger* ester production, such as its application in the catalytic synthesis of flavor esters in baijiu (CN201910935019.8) and its application in the preparation of quick koji and its use in aged vinegar production (CN201910136401.2), but research on its application in the preparation of *Aspergillus niger* fermentation agents is scarce. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Aspergillus niger MQM20136 and its applications.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention provides a strain MQM20136, classified as Aspergillus niger, which has been deposited at the China Center for Type Culture Collection in Wuhan on September 25, 2025, with accession number CCTCC NO: M 20252120.

[0007] This invention also provides the application of Aspergillus niger MQM20136 in the preparation of black yeast rice.

[0008] The present invention also provides a black-coated red yeast rice prepared using Aspergillus niger MQM20136.

[0009] Further, the preparation method of the black-coated red yeast rice is as follows: A compound microbial agent consisting of Aspergillus niger MQM20136, Monascus purpureus 40269, and Saccharomyces cerevisiae JH301 is inoculated into indica rice substrate. The inoculation amount of the compound microbial agent is 1.0-1.1% of the mass of the indica rice substrate. After thorough mixing, fermentation is carried out using conventional preparation processes to obtain black-coated red yeast rice. The preparation method of the compound microbial agent is as follows:

[0010] 1) Preparation of Aspergillus niger MQM20136 seeds: After activating Aspergillus niger MQM20136, seeds were prepared into 1-3×10⁻⁶ seeds. 6 Prepare Aspergillus niger MQM20136 spores per mL; dispense sterilized indica rice culture medium into sterile white porcelain dishes, inoculate with Aspergillus niger MQM20136 spores at an inoculum rate of 4.8-5.2% (v / m), and mix thoroughly; pile the material in the center of the dish, cover the dish with eight layers of sterile gauze, and incubate at 30 ℃ and 95% humidity; after 23-25 ​​h, spread the material evenly to a thickness of 2.0-2.5 cm, and continue incubation for 4-5 days; remove the sample, dry it, and obtain Aspergillus niger MQM20136 seeds.

[0011] 2) Preparation of purple red Monascus 40269 seeds: After activating purple red Monascus 40269, seeds were prepared in the form of 1-3 × 10⁻⁶ seeds. 6 The sample was prepared by inoculating a solution of *Monascus purpureus* 40269 spores at a concentration of 4.8-5.2% (v / m) into sterile white porcelain dishes with sterilized rice culture medium. The mixture was thoroughly mixed. The material was piled in the center of the dish and covered with eight layers of sterile gauze. The dish was then incubated at 30°C and 95% humidity. After 23-25 ​​hours, the material was spread evenly to a thickness of 2.0-2.5 cm and incubated for another 23-25 ​​hours, turning the sample every 12.0-12.5 hours. After 6-7 days, the sample was removed, dried, and *Monascus purpureus* 40269 seeds were obtained.

[0012] 3) Preparation of compound microbial strains: Aspergillus niger MQM20136 seeds, Monascus purpureus 40269 seeds, and Saccharomyces cerevisiae JH301 direct-inoculation inoculum were added to a 2.4-2.6 g / L acetic acid solution, so that the inoculum quantity of each strain was 1-3 × 10⁻⁶. 6 pcs / g, 1-3×10 6 pcs / g and 1-3×10 7 CFU / g, let stand for 6-8 h, homogenize into a paste to obtain a compound bacterial strain.

[0013] The Aspergillus niger MQM20136 of this invention has brewing characteristics of high esterification level and strong amylase activity. It can be used as a local strain for the preparation of Wuyi red yeast rice and plays an important role in improving the fermentation performance and stability of Wuyi red yeast rice. Attached Figure Description

[0014] Figure 1 The ester production capacity of the isolated strains; (a) the distribution of strains with different ester production amounts; (b) the difference in ester production amounts of strains with higher than 140 g / L; p < 0.05.

[0015] Figure 2 The values ​​represent the amylase and saccharifying enzyme activities of the isolated strains; uppercase letters indicate differences in liquefaction capacity among strains, and lowercase letters indicate differences in saccharifying capacity among strains; p < 0.05.

[0016] Figure 3 Morphological characteristics of strain MQM20136; (a) colony morphology; (b) scanning electron micrograph (×250).

[0017] Figure 4 This is the phylogenetic tree of strain MQM20136. Detailed Implementation

[0018] Example 1: Isolation, screening and identification of Aspergillus niger MQM20136

[0019] 1. Materials and Methods

[0020] 1.1 Materials

[0021] 1.1.1 Sample

[0022] Thirteen portions of red yeast rice wine fermentation agent were collected by laboratory staff from typical production areas such as Fujian and Zhejiang.

[0023] 1.1.2 Culture medium

[0024] (1) Bengal red agar medium and malt extract agar medium: finished products, Guangdong Huankai Microbial Technology Co., Ltd. (2) Seed culture medium: NaNO3 0.2%, KH2PO4 0.2%, MgSO4 0.1%, glucose 6%, peptone 2%, pH natural. (3) Selective medium for amylase and saccharifying enzyme: peptone 0.5%, yeast extract 0.5%, soluble starch 3.0%, CaCl2·2H2O 0.05%, MnCl2·4H2O 0.05%, MgSO4·7H2O 0.05%, KH2PO4 0.1%, pH 7.0-7.2. (4) Glutinous rice culture medium: glutinous rice flour: water = 1:1.5, adjust pH to 5.0 with lactic acid, sterilize at 105 ℃ for 20 min.

[0025] 1.2 Methods

[0026] 1.2.1 Isolation of filamentous fungi and preparation of spore suspensions

[0027] (1) Sample preparation: The collected sample was ground into fine powder under aseptic conditions. 5.0 g was weighed into 45 mL / 250 mL of sterile water (45 mL / 250 mL means 45 mL of solution and 250 mL of container volume, the same below), shaken at 150 r / min for 2.0 h, and then refrigerated at 4 ℃ for later use.

[0028] (2) Single strain isolation and purification: strains were isolated and purified by dilution and plating method, and Bengal Red medium was used to prepare plates; malt extract agar slants were used for strain activation and preservation.

[0029] (3) Preparation of spore suspension for screening: Take the isolated bacteria activated on malt extract agar slant and an appropriate amount of sterile water. Wash off the spores from the slant bacteria at a ratio of 1 tube (18×150) of isolated bacteria to 20 mL of sterile water. Transfer the spores to a sterile Erlenmeyer flask containing 4-6 glass beads, shake well, and adjust the spore count in the spore suspension to 1-5×10 using the hemocytometer method. 6 Quantity / mL, for later use.

[0030] 1.2.2 Evaluation of the ester production capacity of the strain

[0031] Seed culture preparation: The spore suspension to be screened was inoculated into 50 mL / 250 mL seed culture medium at an inoculation rate of 10% (v / v). The culture was incubated at 30 ℃ and 150 r / min with constant temperature shaking for 2-3 days. The mycelia were filtered out using four layers of sterile gauze, and the filtrate was the seed culture, which was then used for later use. Using commonly used production strains as controls, the seed culture of each isolated strain was inoculated into glutinous rice culture medium at an inoculation rate of 10% (v / m). The culture was incubated at 30 ℃ and 150 r / min for 7 days. The culture was centrifuged at 6000 r / min for 10 min, and the supernatant was used for total ester determination.

[0032] 1.2.3 Evaluation of the strain's ability to produce amylase and saccharifying enzyme

[0033] The seed culture of the bacteria to be screened (prepared in the same way as above) was inoculated in equal amounts into 150 mL / 500 mL selective medium for amylase and saccharifying enzyme. The treatment with commonly used production strain, Monascus purpureus FM23, was used as the control (CK). The culture was carried out in a shaker at 30 ℃ and 180 r / min. After fermentation, the mycelium was separated from the fermentation broth by ultrafiltration. The filtrate was the crude enzyme solution, which was used to determine the activities of saccharifying enzyme and amylase. The mycelium was washed three times to remove the residue, dried to constant weight, and then the biomass was determined.

[0034] 1.2.4 Methods for identifying fungal strains

[0035] (1) Morphological observation: The activated strain was inoculated on Bengal Red or malt extract agar plates using the spot inoculation method and placed in a 30 ℃ constant temperature incubator. The colony morphology and growth were observed and recorded daily for 3-5 days.

[0036] (2) Gene sequence analysis: DNA was extracted from the test strains using a fungal genomic DNA extraction kit. PCR amplification was performed using universal primers ITS4-TCCTCCGCTTATTGATATGC / ITS5-GGAAGTAAAAGTCGTAACAAGG and Bt2a-GGTAACCAAATCGGTGCTGCTTTC / Bt2b-ACCCTCAGTGTAGTGACCCTTGGC, followed by sequencing. The sequencing results were compared using the BLAST program in the NCBI database. Sequences of strains with homology higher than 98% were downloaded. A phylogenetic tree was constructed using MEGA 4.0 software based on the neighbor-joining method. The reliability of the branching was assessed using the Bootstrap test, and the taxonomic position of the strains was finally determined.

[0037] 1.2.5 Determination Method

[0038] (1) Determination of bacterial count: The spore count was determined using the hemocytometer method; the mycelial count was determined using the dry weight method. (2) Determination of total esters: The determination was performed according to GB / T 10345-2022 "Analytical Methods for Baijiu". (3) Determination of amylase activity: The determination was performed using the kit method, and the specific operation was performed according to the product instructions. The definition of amylase activity unit is: 1 g of amylase produced by mycelium liquefies 1 mg of soluble starch in 1 h at 40℃ and pH 6.0, which is one unit, symbolized by U, and expressed as U / g. (4) Determination of saccharifying enzyme activity: The determination was performed according to the method of Ren et al. The definition of saccharifying enzyme activity unit is: 1 g of saccharifying enzyme produced by mycelium hydrolyzes soluble starch to produce 1 mg of glucose in 1 h at 40℃ and pH 4.6, which is one unit, symbolized by U, and expressed as U / g.

[0039] 2 Results and Analysis

[0040] 2.1 Isolation and ester-producing capacity evaluation of filamentous fungi

[0041] A total of 67 filamentous fungi with different growth characteristics were isolated and purified from the collected samples. The ester production capacity of these strains was as follows: Figure 1As shown, most strains (including the control strain) produced less than 140 g / L of esters, with only 13 strains producing more than 140 g / L, and the differences between strains were significant. Among them, strain W3 produced the highest ester, reaching 178.3 g / L, which was significantly better than other tested strains (p < 0.05) and 2.43 times that of the control strain; strain B12 produced slightly less ester than W3, reaching 171.1 g / L, which was 2.33 times that of the control strain; both strains also exhibited a prominent ester aroma during fermentation.

[0042] 2.2 Liquefaction and saccharification capabilities of the isolated strains

[0043] As a microorganism used in red yeast rice wine brewing, its ability to saccharify and liquefy starch is also one of the key indicators for evaluating the fermentation performance of the strain. Figure 2 It was found that there were significant differences in the amylase and saccharifying enzyme activities between strains W3 and B12. The enzyme activity of W3 was significantly lower than that of the control strain CK and B12; the saccharifying enzyme activity of B12 was comparable to that of the control strain, while the amylase activity was 8.90% higher than that of the control strain. Therefore, B12 should be selected as the target strain for further research such as strain identification and characteristic analysis, and it should be renumbered as MQM20136.

[0044] 2.3 Species identification of strain MQM20136

[0045] (1) Macroscopic and microscopic morphological characteristics of MQM20136

[0046] MQM20136 was inoculated onto malt extract agar plates and incubated at 28 °C for 3-5 days. The colony morphology was as follows: Figure 3 As shown in (a). The colonies are round and opaque; the hyphae are fluffy, radiating outward from the dark brown center to brown and then to yellow on the periphery, forming several rings of fungal clusters. Under a scanning electron microscope, the hyphae are slender, branched, and septate; spores are attached to the tips of the hyphae, and the sporangia are spherical or nearly spherical.

[0047] (2) Molecular biological characteristics of MQM20136

[0048] The ITS rDNA and BenA sequencing lengths of MQM20136 were 623 bp (SEQ ID NO.1) and 444 bp (SEQ ID NO.2), respectively. Alignment and phylogenetic tree construction were performed using NCBI, and the results are as follows: Figure 4 Strain MQM20136 and Aspergillus niger ATCC 16888 (AY373852.1) Figure 4 a), Aspergillus niger NRRL 236T (LC589349.1) ( Figure 4b) The bacteria clustered on the same branch and showed 100% homology. Based on morphological characteristics, the bacteria were identified as Aspergillus niger and deposited on September 25, 2025, at the China Center for Type Culture Collection in Wuhan, with accession number CCTCC NO: M 20252120.

[0049] Example 2: Application of Aspergillus niger MQM20136 in the preparation of black-coated red yeast rice

[0050] 1. Materials and Methods

[0051] 1.1 Materials

[0052] 1.1.1 Microbial Strains and Sources: * *Monascus purpureus* 40269: purchased from the China Industrial Microbial Culture Collection Center. * *Aspergillus niger* MQM20136: self-isolated, preservation number CCTCC NO: M20252120. * *Saccharomyces cerevisiae* JH301 direct-inoculation inoculum: prepared in the laboratory, with a bacterial count of 1.18 × 10⁻⁶. 10 CFU / g, of which the preservation number of Saccharomyces cerevisiae JH301 is CCTCC NO: M 2015226, which has been disclosed in Chinese Patent CN105176854B, and the preparation method of direct inoculation agent of Saccharomyces cerevisiae JH301 has been disclosed in Chinese Patent CN112779171B.

[0053] 1.1.2 Indica rice culture medium: Soak commercially available indica rice in water for 10-12 hours (the amount of water should just cover the rice), drain the water, put it into a beaker (4 / 5 of the beaker's capacity), seal it with 8 layers of gauze, sterilize it at 105 ℃ for 20 minutes, spread it out and cool it under aseptic conditions, and set it aside for later use.

[0054] 1.2 Methods

[0055] 1.2.1 Seed preparation

[0056] (1) Preparation of Aspergillus niger solid culture: After activating Aspergillus niger MQM20136, 1-3×10 6The spore count was calculated as follows: Sterilized indica rice culture medium was dispensed into sterile white porcelain dishes. Aspergillus niger MQM20136 spores were inoculated at a rate of 5% (v / m) and thoroughly mixed. The material was piled in the center of the dish, covered with eight layers of sterile gauze, and incubated at 30 ℃ and 95% humidity. After 24 h, the material was evenly spread to a thickness of 2.0-2.5 cm, and incubation continued, with the addition of appropriate amounts of sterile water depending on the dryness of the rice grain surface. After 4-5 days, the samples were removed and dried in a 40 ℃ oven until the moisture content was below 10%, and the spore count was measured.

[0057] (2) Preparation of solid strain of Monascus purpureus, i.e., Monascus purpureus purpleis: Monascus purpureus 40269 was activated and then prepared into 1-3×10⁻⁶ solid strains. 6 The spores were prepared at a concentration of 5 spores per mL. The sterilized rice culture medium was dispensed into sterile white porcelain dishes, and Monascus purpureus 40269 spores were inoculated at a rate of 5% (v / m), mixing thoroughly. The material was piled in the center of the dish, covered with eight layers of sterile gauze, and incubated at 30 ℃ and 95% humidity. After 24 h, the material was evenly spread to a thickness of 2.0-2.5 cm and incubated further. The mixture was then turned over every 12.0-12.5 h, and sterile water was added as needed based on the dryness of the rice grains. After 6-7 days, the samples were removed and dried in a 40 ℃ oven until the moisture content was below 10%.

[0058] 1.2.2 Preparation of pure-strain compound black yeast rice

[0059] The preparation of black-coated red yeast rice follows the traditional preparation process, with the following adjustments made to the inoculation step:

[0060] Preparation and inoculation of the compound microbial strain: Take appropriate amounts of *Aspergillus niger* (MQM20136) seeds, *Monascus purpureus* (40269) seeds, and *Saccharomyces cerevisiae* (JH301) direct-inoculation inoculum, and add them to a 2.4-2.6 g / L acetic acid solution to make the inoculum quantity of each strain 2×10⁻⁶. 6 Units / g, 2×10 6 pcs / g and 2×10 7 CFU / g, let stand for 6-8 hours, homogenize into a paste, and set aside for use.

[0061] Inoculation and fermentation: The compound strain was inoculated into the indica rice substrate at a mass ratio of 1%, and thoroughly mixed. Fermentation was carried out using the conventional preparation process of *Zingiber officinale*. The specific operation steps can be referred to the literature "Study on the regulatory effect of alkali stimulation treatment on fungal flora and fermentation performance of *Zingiber officinale* (Food and Fermentation Industries, DOI: 10.13995 / j.cnki.11-1802 / ts.043811)".

[0062] 1.2.3 Performance Evaluation of Black-coated Red Yeast Rice

[0063] Using commercially available Wuyi red yeast as a control (CK), and the above-prepared compound Wuyi red yeast as a fermenting agent, a red yeast wine brewing experiment was conducted. After fermentation, the basic physicochemical indicators such as alcohol content and total acidity of the brewed red yeast wine, as well as indicators such as raw material utilization rate and alcohol yield, were measured to comprehensively evaluate the fermentation performance of the compound Wuyi red yeast.

[0064] Red yeast rice wine brewing process: Take 5 kg of earthenware jars, add 2.0 kg of purified water, 100 g of black yeast rice fermentation agent, and 2.0 kg of glutinous rice that has been soaked, steamed, and cooled. Stir well and place at 20-22 ℃ until fermentation is complete. Press out the raw wine and sterilize it in an 80 ℃ water bath for 30 minutes. After cooling, seal and age at 20-25 ℃ to become the base wine of red yeast rice wine. Samples are taken to test the physicochemical indicators such as alcohol content of each base wine. After aging for 10 months, a fuzzy comprehensive sensory evaluation is conducted on appearance, aroma, and taste.

[0065] 1.2.4 Determination Method

[0066] (1) Determination of microbial count: The spore count was determined by hemocytometer method; the yeast count was determined by plate count method. (2) Moisture content: The moisture content was determined according to the direct drying method in GB / T 5188-2017 "Determination of moisture content in brewing red yeast". (3) Fermentation power: The fermentation power was determined according to QB / T 4257-2011 "General analytical method for brewing koji". (4) Saccharification power: The iodometric method in QB / T 5188-2017 "Brewing red yeast" was used. (5) Liquefaction power: The liquefaction power was determined according to QB / T 4257-2011 "General analytical method for brewing koji". (6) Esterification power: The esterification power was determined according to the method in QB / T 5188-2017 "Brewing red yeast". (7) Determination of alcohol content: The alcohol content was determined according to GB / T13662-2018 "Huangjiu" (yellow wine). (8) Raw material utilization rate: Raw material utilization rate = alcohol yield / rice consumption × 100. (9) Brewing alcohol yield: Standard atmospheric pressure, 20 ℃, the alcohol yield of one unit with a content of 15%. The formula for calculating the alcohol yield is: Alcohol yield (%, based on 15% alcohol content) = (Alcohol content % × alcohol yield kg / rice consumption kg / 15% alcohol).

[0067] 2 Results and Analysis

[0068] As shown in Table 1, all relevant indicators of the Wuyi Red Yeast Rice tested met the relevant standard requirements. Compared with commonly used commercially available Wuyi Red Yeast Rice, the Wuyi Red Yeast Rice prepared using pure-strain compound koji showed increases in liquefaction power, saccharification power, fermentation power, and esterification power of 16.5%, 11.2%, 20.2%, and 26.0%, respectively. Specifically, the saccharification power and fermentation power met the first-grade brewing red yeast rice standard in DBS 35 / 002-2017 "Local Food Safety Standard for Brewing Red Yeast Rice"; the esterification power met the standard for esterified red yeast rice in QB / T 5188-2017 "Brewing Red Yeast Rice". Therefore, Wuyi Red Yeast Rice prepared using high-quality pure-strain compound koji can comprehensively improve its fermentation performance.

[0069] As shown in Table 2, compound koji also performed exceptionally well in the brewing of red yeast rice wine; the raw material utilization rate and brewing yield increased by 8.79% and 18.80%, respectively. In addition, red yeast rice wine brewed using compound koji had high sensory scores and a rich ester aroma.

[0070] Table 1 Fermentation performance indicators of *Ziziphus jujuba*

[0071]

[0072] Table 2 Physicochemical Indicators of Red Yeast Rice Wine Brewing

[0073]

[0074] The specific embodiments of the present invention have been described above. However, those skilled in the art should understand that the specific embodiments are merely illustrative and not intended to limit the scope of protection of the present invention. Any equivalent modifications or variations made by those skilled in the art without departing from the spirit of the present invention should be included within the scope of protection defined by the claims of the present invention.

Claims

1. Aspergillus niger MQM20136, characterized in that, Its classification name is Aspergillus niger, and it has been deposited at the China Center for Type Culture Collection in Wuhan, on September 25, 2025, with accession number CCTCCNO: M 20252120.

2. The application of Aspergillus niger MQM20136 as described in claim 1 in the preparation of black yeast rice.

3. A type of black yeast prepared using Aspergillus niger MQM20136 as described in claim 1.

Images