A mixta calida strain, its preparation and use

Abstract

The application provides a method for preparing a yeast culture Mixta calida The bacteria are Mixta calida The bacteria are Mixta calida The AMCC 10426 strain is preserved in the China Center for Type Culture Collection (CCTCC) and has a preservation number of CCTCC NO: M 20241202. The strain is a bacteria with the ability of producing protease and cellulase, can promote the solid-state fermentation of yeast, and improve the true protein content of the yeast culture. The application provides a new strain resource and a fermentation method in the field of solid-state fermentation, and is helpful to improve the fermentation efficiency and product quality.

Description

Technical Field

[0001] This invention relates to the field of microorganisms, specifically to a... Mixta calida Strains, their preparation, and applications. Background Technology

[0002] Yeast starter originated in ancient China and is one of my country's unique national heritages. Made primarily from grains, it is a complex system possessing functions related to "materials, enzymes, and bacteria." Yeast starter contains a large number of culturable bacteria, among which... Mixta calida It has been reported to inhibit plant pathogens, degrade nicotine, nitrite, and ammonium nitrite, and has health protection applications in plant fermentation. It has been applied in plant biological control and cigar fermentation, but there are no related reports in the field of solid-state fermentation, which has a wider range of applications. Summary of the Invention

[0003] To address the above problems, the present invention provides a solution. Mixta calida The strain, its preparation and application: This strain has protease and cellulase activities and can increase the true protein content of yeast cultures in solid-state fermentation systems.

[0004] Specifically, the present invention proposes the following technical solution:

[0005] Technical Solution 1: A Mixta calida The bacteria, characterized in that, Mixta calida bacteria Mixta calida AMCC strain 10426 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO: M 20241202.

[0006] Technical Solution 2: As described in Technical Solution 1 Mixta calida The bacteria, characterized in that, Mixta calida The 16S rRNA gene sequence of AMCC strain 10426 is shown in SEQ ID NO.5.

[0007] Technical Solution 3: As described in Technical Solution 1 or 2 Mixta calida The bacteria, characterized in that, Mixta calida The gyrB gene sequence of AMCC strain 10426 is shown in SEQ ID NO.6.

[0008] Technical Solution 4: As described in any one of Technical Solutions 1-3 Mixta calida The bacteria, characterized in that, Mixta calida The AMCC 10426 strain produces a protease; preferably, the enzyme activity of the protease is 53-55 U / mL.

[0009] Technical Solution 5: As described in any one of Technical Solutions 1-4 Mixta calida The bacteria, characterized in that, Mixta calida The AMCC 10426 strain produces cellulase; preferably, the total enzyme activity of the cellulase is 0.1-0.2 U / mL;

[0010] Preferably, the cellulase includes β-glucosidase;

[0011] More preferably, the β-glucosidase has an enzyme activity of 0.2-0.3 U / L.

[0012] Technical Solution 6: A microbial agent, characterized in that the microbial agent contains any one of the following technical solutions 1-5 Mixta calida AMCC strain 10426.

[0013] Technical Solution 7: The microbial agent according to Technical Solution 6, characterized in that the microbial agent further includes excipients.

[0014] Technical Solution 8: A method for preparing the microbial agent described in Technical Solution 6 or 7, characterized in that the method includes the following steps:

[0015] (1) Any one of the technical solutions 1-5 Mixta calida Scale-up culture of AMCC 10426 strain;

[0016] (2) Add the product obtained in step (1) to a liquid culture medium and ferment it at 26-37℃.

[0017] Technical Solution 9: Any one of Technical Solutions 1-5 Mixta calida Application of AMCC 10426 strain or the microbial agent described in technical solution 6 or 7 in solid-state fermentation.

[0018] Technical Solution 10: The application according to Technical Solution 9, characterized in that, the Mixta calida strain AMCC10426 increases the true protein content in ferments;

[0019] Preferably, the Mixta calida The AMCC 10426 strain releases reducing sugars from the fermentation product, promoting solid-state fermentation of yeast.

[0020] Technical Solution 11: As described in any one of Technical Solutions 1-5 Mixta calida Application of AMCC 10426 strain or the microbial agent described in technical solution 6 or 7 in the fermentation preparation of fertilizer, feed, cigars and high-temperature koji.

[0021] Technical Solution 12: Any one of Technical Solutions 1-5Mixta calida Application of AMCC 10426 strain or the microbial agent described in technical solution 6 or 7 in plant protection.

[0022] Technical Solution 13: A high-temperature Daqu (a type of starter culture), wherein the high-temperature Daqu is produced by any one of technical solutions 1-5. Mixta calida It is prepared by fermentation of AMCC 10426 strain or the inoculum described in technical solution 6 or 7.

[0023] The beneficial effects of this invention include:

[0024] The invention obtained Mixta calida AMCC 10426 is a bacterium capable of producing proteases and cellulases, which can promote solid-state fermentation of yeast and increase the true protein content of yeast cultures.

[0025] This invention provides new strain resources and fermentation methods for the field of solid-state fermentation, helping to improve fermentation efficiency and product quality. Compared with existing technologies, this invention not only enriches the resources of solid-state fermentation strains but also expands the application scope. Mixta calida The strain has a wide range of applications and helps to improve fermentation efficiency and product quality, demonstrating significant advantages. Attached Figure Description

[0026] Figure 1 The image shown is from Example 1. Mixta calida Colony morphology of AMCC 10426 strain.

[0027] Figure 2 The image shown is from Example 1. Mixta calida Cell morphology of AMCC 10426 strain.

[0028] Figure 3 The image shown is from Example 1. Mixta calida Phylogenetic tree of AMCC 10426 strain.

[0029] Figure 4 The figure shows the true protein content of each fermentation group after fermentation in Example 3.

[0030] Information on strain preservation

[0031] The present invention provides Mixta calida Strain AMCC 10426 was deposited at the China Center for Type Culture Collection (CCTCC) on June 13, 2024, with accession number CCTCC NO: M 20241202. The deposit address is: Wuhan University, Wuhan, China, Postcode: 430072; Tel: 027-68754052.

[0032] The Max Kluyveromycin mentioned in the examples ( Kluyveromyces marxianusAMCC strain 31342 was deposited at the China Center for Type Culture Collection on December 30, 2022, with accession number CCTCC NO: M20222110. The depository address is: Wuhan University, Wuhan, China, Postcode: 430072; Telephone: 02768754052. It has been published in patent application with publication number CN118956623A. Detailed Implementation

[0033] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but...

[0034] This invention is not limited to the following technical solutions.

[0035] This invention provides Mixta calida AMCC strain 10426 is primarily used in solid-state fermentation. First, the strain was isolated and purified from the starter culture, and then identified as a specific strain through strain identification. Mixta calida This strain, named AMCC10426, exhibited the ability to produce protease and cellulase through enzyme activity testing. The protease hydrolyzes plant macromolecular proteins into polypeptides and free amino acids, while the cellulase hydrolyzes plant cellulose into fermentable sugars, indicating its potential for solid-state fermentation. Further verification through solid-state fermentation demonstrated that this strain possesses degradation capabilities in solid-state fermentation, promoting yeast solid-state fermentation and increasing the true protein content of yeast cultures. This strain can be used for fermentation preparation of fertilizers, feed, cigars, and high-temperature koji (a type of starter culture).

[0036] Solid-state fermentation, broadly speaking, refers to a class of processes that use insoluble solid substrates to cultivate microorganisms. This includes both submerged fermentation, where solids are suspended in a liquid, and processes that cultivate microorganisms on moist solid materials with little or no free water. In most cases, it refers to a biological reaction process in which one or more microorganisms ferment in a water-insoluble solid substrate with a certain level of moisture, in the absence of or with little to no free water. Examples include traditional industrial and agricultural production processes such as brewing, vinegar making, composting, and silage, as well as modern processes such as antibiotic production and β-glucosidase production. The processing of the traditional Chinese medicine *Zhongyao Shenqu* also falls under the category of solid-state fermentation.

[0037] High-temperature Daqu is a saccharification and fermentation agent used in brewing. Daqu is made from grains such as wheat, barley, and peas, which are crushed, shaped, and cultured to produce saccharification and fermentation agents for brewing. The highest fermentation temperature of high-temperature Daqu reaches above 60℃, usually between 60-65℃. High-temperature Daqu contains a rich variety and quantity of microorganisms, which can produce a variety of enzyme systems to promote saccharification and fermentation.

[0038] In some specific embodiments, the Mixta calidaAMCC strain 10426 produces protease;

[0039] The Mixta calida The AMCC 10426 strain was cultured in a protease-producing medium consisting of 45-55 g / L glucose, 8-12 g / L soybean peptone, 1.5-2.5 g / L KH2PO4, and 0.35-0.45 g / L MgSO4•7H2O.

[0040] The soybean peptone provides sufficient nitrogen to support the growth of the strain and the synthesis of protease. The soybean peptone can be any commercially available or homemade type. The inventors have found that as long as the total nitrogen content of the commercially available or homemade soybean peptone is ≥9% by weight, it can be used in this invention, preferably 10%-15%.

[0041] In some other embodiments, the enzyme activity of the protease is 53-55 U / mL.

[0042] In some other specific embodiments, the Mixta calida AMCC strain 10426 produces cellulase;

[0043] The Mixta calida The AMCC 10426 strain was cultured in a cellulase-producing medium containing 8-12 g / L sodium carboxymethyl cellulose, 1-3 g / L peptone, 0.4-0.6 g / L yeast extract, 1.3-1.7 g / L K₂HPO₄ and 2.3-2.7 g / L Na₂SO₄.

[0044] In this invention, peptone provides sufficient nitrogen for the strain. Any commercially available or self-made peptone can be used. The inventors have found that as long as the total nitrogen content of the peptone is ≥12.5%, amino nitrogen ≥2.5%, ash content ≤15%, and NaCl ≤5% by weight, it can be used in this invention. Preferably, the total nitrogen content of the peptone is 12.5-14%, amino nitrogen 2.5-4%, ash content 5-15%, and NaCl 3-5%.

[0045] The yeast paste can be any commercially available or homemade yeast paste. The inventors have found that as long as the total nitrogen content of the yeast paste is ≥7.2% by weight, it can be used in this invention.

[0046] In some other embodiments, the total enzyme activity of the cellulase is 0.1-0.2 U / mL.

[0047] In some other specific embodiments, the Mixta calidaAMCC strain 10426 was purified in LB solid medium, which consisted of 9-11 g tryptone, 4-6 g yeast extract, 9-11 g sodium chloride, 9-11 g agar and 1 L distilled water.

[0048] The tryptone can be any commercially available or self-made tryptone. The inventors have found that as long as the total nitrogen content of the tryptone is 12-16% by mass, the ash content is ≤6.0% and the NaCl content is ≤2.0%, it can be used in this invention.

[0049] The yeast extract can be any commercially available or homemade yeast extract. The inventors have found that as long as the total nitrogen of the yeast extract is ≥10%, amino nitrogen is ≥5%, ash content is ≤15%, NaCl content is ≤2%, and moisture content is ≤6% by weight, it can be used in this invention.

[0050] In some other specific embodiments, the Mixta calida Seed culture of AMCC 10426 strain was prepared in LB liquid medium, which consisted of 9-11 g tryptone, 4-6 g yeast extract, 9-11 g sodium chloride and 1 L distilled water.

[0051] The tryptone can be any commercially available or self-made tryptone. The inventors have found that as long as the total nitrogen content of the tryptone is 12-16% by mass, the ash content is ≤6.0% and the NaCl content is ≤2.0%, it can be used in this invention.

[0052] The yeast extract can be any commercially available or homemade yeast extract. The inventors have found that as long as the total nitrogen of the yeast extract is ≥10%, amino nitrogen is ≥5%, ash content is ≤15%, NaCl content is ≤2%, and moisture content is ≤6% by weight, it can be used in this invention.

[0053] The yeast used in this embodiment of the invention is Kluyveromyces martensii (Kluyveromyces). Kluyveromyces marxianus AMCC 31342 strain, which was obtained by mutagenesis, has been disclosed in patent application with publication number CN118956623A.

[0054] Max Kluyveromycin ( Kluyveromyces marxianus AMCC strain 31342 was purified in YPD solid medium, which consisted of 0.8-1.2 g yeast extract, 1.8-2.2 g glucose, 1.8-2.2 g peptone, 1.8-2.2 g agar and 100 mL distilled water.

[0055] The yeast extract can be any commercially available or homemade yeast extract. The inventors have found that as long as the total nitrogen of the yeast extract is ≥10%, amino nitrogen is ≥5%, ash content is ≤15%, NaCl content is ≤2%, and moisture content is ≤6% by weight, it can be used in this invention.

[0056] The peptone can be any commercially available or homemade peptone. The inventors have found that as long as the total nitrogen of the peptone is ≥12.5%, amino nitrogen is ≥2.5%, ash content is ≤15%, and NaCl is ≤5% by weight, it can be used in this invention. Preferably, the total nitrogen of the peptone is 12.5-14%, amino nitrogen is 2.5-4%, ash content is 5-15%, and NaCl is 3-5%.

[0057] Max Kluyveromycin ( Kluyveromyces marxianus Seed culture of AMCC 31342 strain was prepared in YPD liquid medium, which consisted of 0.8-1.2 g yeast extract, 1.8-2.2 g glucose, 1.8-2.2 g peptone and 100 mL distilled water.

[0058] The yeast extract can be any commercially available or homemade yeast extract. The inventors have found that as long as the total nitrogen of the yeast extract is ≥10%, amino nitrogen is ≥5%, ash content is ≤15%, NaCl content is ≤2%, and moisture content is ≤6% by weight, it can be used in this invention.

[0059] The peptone can be any commercially available or homemade peptone. The inventors have found that as long as the total nitrogen of the peptone is ≥12.5%, amino nitrogen is ≥2.5%, ash content is ≤15%, and NaCl is ≤5% by weight, it can be used in this invention. Preferably, the total nitrogen of the peptone is 12.5-14%, amino nitrogen is 2.5-4%, ash content is 5-15%, and NaCl is 3-5%.

[0060] Unless otherwise stated, all reagents and instruments used in the embodiments and comparative examples of this invention are conventional commercially available products. Information on the reagents and instruments used in this invention is provided in Tables 1 and 2 below.

[0061] Table 1 Reagent Information Table

[0062]

[0063]

[0064]

[0065]

[0066] Table 2 Instrument Information Table

[0067]

[0068] The composition of the culture media involved in the examples and comparative examples is as follows:

[0069] LB liquid medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, 1L distilled water, sterilized at 121℃ for 15min.

[0070] LB solid medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, 10g agar, 1L distilled water, sterilized at 121℃ for 15 min.

[0071] Protease-producing medium: 50g glucose, 10g soybean peptone, 2g KH2PO4, 0.4g MgSO4•7H2O, diluted to 1000 mL with sterile water, pH adjusted to 7, sterilized at 121℃ for 15 min.

[0072] Cellulase-producing culture medium: 10 g sodium carboxymethyl cellulose, 2 g peptone, 0.5 g yeast extract, 1.5 g K2HPO4, 2.5 g Na2SO4, bring the volume to 1000 mL with sterile water, adjust the pH to 7.0, and sterilize at 121℃ for 15 min.

[0073] YPD solid culture medium: 1 g yeast extract, 2 g glucose, 2 g peptone, 2 g agar, 100 mL distilled water, sterilized at 115℃ for 20 min.

[0074] YPD liquid culture medium: 1 g yeast extract, 2 g glucose, 2 g peptone, 100 mL distilled water, sterilized at 115℃ for 20 min.

[0075] Fermentation medium for distiller's grains: Take distiller's grains, add water at a material-to-water ratio of 1.5:1, mix evenly, and then dispense into sterilization bags and sterilize at 115℃ for 20 min as the medium for solid-state fermentation.

[0076] To better understand the technical solution of the present invention, the following describes the present invention in conjunction with specific embodiments.

[0077] The plan is explained in detail.

[0078] Example 1 Mixta calida Isolation and identification of AMCC 10426 strain

[0079] 1. Strains Isolation and Purification

[0080] Mixta calida Strain AMCC 10426 was isolated from brewing yeast. The specific isolation and purification methods are as follows:

[0081] Under aseptic conditions, dissolve 1g of Daqu powder in 9mL of sterile water, vortex to mix and prepare a bacterial suspension. Transfer 1mL of the bacterial suspension to 9mL of sterile water, and then dilute sequentially in 10-fold increments to a final volume of 10. -2 10 -3 10 -4 10 -5 10 -6 10 -7 Times, from 10 -5 10 -6 10 -7 Spread 200 μL of the diluted bacterial suspension onto LB agar plates and incubate at 37°C. Pile single colonies from the diluted plates and streak them 2-3 times to obtain a pure bacterial culture. Preserve the culture with glycerol and store at -80°C. Colonies should be pale yellow, irregularly shaped (circular), and have irregular edges (e.g., ...). Figure 1 As shown), the microscopic morphology is rod-shaped (e.g. Figure 2 (As shown).

[0082] 2. Strain identification

[0083] Identification of 16S rRNA and gyrB gene sequences of the strain: The preserved bacteria were inoculated into LB liquid medium and cultured overnight. Bacterial cells were collected by centrifugation, and the genome was extracted using a DNA extraction kit. PCR amplification was performed using universal primers for bacterial 16S rRNA (F: AGAGTTTGATCCGGCTCAG (SEQ ID NO.1); R: GGTTACCTTGTTACGACTT (SEQ ID NO.2)) and universal primers for gyrB (UP-1: GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA (SEQ ID NO.3); UP-2r: AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT (SEQ ID NO.4)). The amplification conditions were: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 45 s, 72℃ extension for 90 s, 30 cycles, and a final extension at 72℃ for 10 min. The PCR products were sequenced by Wuhan Qingke Biotechnology Co., Ltd. Mixta calida The 16S gene sequence (SEQ ID NO.5) of AMCC strain 10426 is as follows:

[0084]

[0085] Measured Mixta calida The gyrB gene sequence (SEQ ID NO.6) of AMCC strain 10426 is as follows:

[0086]

[0087] The sequencing sequences of the identified strains were compared using BLAST analysis in the GenBank database. Gene sequences with high consistency were downloaded, and a phylogenetic tree was constructed using MEGA-X software via neighbor-joining. The results are as follows: Figure 3 As shown. The screened strain AMCC 10426 and Mixta strains Mixta calida The strain belongs to the same branch and is most closely related in evolution. Furthermore, the BLAST alignment of the 16S rRNA gene sequence (SEQ ID NO. 5) and the gryB gene sequence (SEQ ID NO. 6) showed 100% homology. Therefore, the species of the obtained strain was identified as... Mixta calida and named Mixta calida AMCC 10426. It was deposited at the China Center for Type Culture Collection (CCTCC) on June 13, 2024, with accession number: CCTCC NO: M 20241202.

[0088] Example 2: Detection of enzyme production activity of strains

[0089] Will Mixta Calida AMCC strain 10426 was inoculated into LB liquid medium and cultured overnight at 37°C and 220 rpm on a shaker. Fresh bacterial culture was then inoculated at a rate of 2% (v / v) into cellulase-producing and protease-producing media, respectively. After 24 hours of culture, the fermentation supernatant was collected by centrifugation as crude enzyme solution. Protease activity was detected according to national standard GB1886.174—2024, and total cellulase activity was detected according to national standard GB / T 23881-2009 "Determination of Cellulase Activity in Feed - Filter Paper Method". The activity was further measured using p-nitrophenyl β-D-glucopyranoside (…). pUsing p-nitrophenyl-β-D-glucopyranoside (pNPG) as a substrate, β-glucosidase activity was detected spectrophotometrically. The method was as follows: 5 mmol / L p-nitrophenyl-β-D-glucopyranoside (pNPG) was prepared using sodium acetate solution at pH 5.0 as the substrate. 200 μL of the substrate was mixed thoroughly with 100 μL of crude enzyme solution and incubated at 50℃ for 60 min. After the reaction, 200 μL of 1 mol / L Na₂CO₃ was added to terminate the reaction. Three replicates were performed for each sample. A blank control of boiled and inactivated crude enzyme solution was used for instrument calibration to eliminate reagent interference. 300 μL of the supernatant was diluted in 1 mL of distilled water, and 200 μL of the diluted solution was transferred to the wells of an ELISA plate. The absorbance at 410 nm was measured using an ELISA reader. One unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol of p-nitrophenol per minute. Three replicates were performed. The measured protease activity (U / mL), total cellulase activity (U / mL), and β-glucosidase activity (U / L) are shown in Table 3.

[0090] Table 3

[0091]

[0092] As shown in Table 3, Mixta Calida Strain AMCC 10426 exhibits protease production capabilities, with an average protease activity of 53.4 U / mL. Furthermore, this strain also demonstrates cellulase production capabilities, with an average total cellulase (filter paper enzyme) activity of 0.12 U / mL and β-glucosidase activity of 0.27 U / L. This yeast strain, derived from the fermentation starter culture, possesses natural adaptability to distiller's grains and exhibits high protease activity, capable of breaking down plant proteins into small-molecule amino acids and peptides, thus demonstrating potential for promoting solid-state fermentation by yeast.

[0093] Example 3: Solid-state fermentation of strain

[0094] (1) Activation of strain

[0095] Preparation of bacterial culture: Take the glycerol tubes that have been preserved Mixta Calida AMCC 10426 strain was used in an aseptic environment. A small amount of bacterial suspension was taken with an inoculation loop and streaked onto an LB agar plate using the four-zone streak method. The plate was incubated upside down at 37°C for 48 h. Single colonies were picked and cultured overnight in LB liquid medium to prepare seed culture. 1 mL of seed culture was added to 100 mL of LB liquid medium at a 1% (v / v) inoculation rate and cultured at 37°C and 225 rpm for 24 h to obtain bacterial suspension.

[0096] Preparation of yeast culture: Take the Kluyveromyces martensii AMCC 31342 strain preserved in glycerol tubes, and under sterile conditions, use an inoculation loop to take a small amount of culture and streak it on a YPD plate using the four-zone streak method. Incubate at 30°C with the plate upside down for 48 hours. Pick a single colony and incubate it overnight in YPD liquid medium to prepare seed culture. Take 1 mL of seed culture at a 1% (v / v) inoculation rate and add it to 100 mL of YPD liquid medium. Incubate at 30°C with shaking at 180 rpm for 20 hours to obtain yeast culture.

[0097] (2) Solid-state fermentation

[0098] Solid-state fermentation of yeast: 10 mL of the above yeast culture was inoculated into 100 g of sterilized distiller's grains fermentation medium and fermented at 30°C for 7 days, turning the medium once a day. Samples were taken and dried on the first day of fermentation, the third day, and the seventh day. A total of 3 replicates were performed.

[0099] Bacterial solid-state fermentation group: Take 10 mL of the above bacteria Mixta Calida AMCC10426 bacterial culture was inoculated into 100 g of distiller's grains fermentation medium and fermented statically at 30℃ for 7 days, turning the medium once a day. Samples were taken and dried on the first day of fermentation, and on the third and seventh days of fermentation. A total of 3 replicates were performed.

[0100] Mixed fermentation group: 5 mL of the above yeast culture and 5 mL of bacterial culture were inoculated into 100 g of distiller's grains fermentation medium, and fermented statically at 30℃ for 7 days, turning the material once a day. Samples were taken and dried on the first day, the third day, and the seventh day of fermentation. A total of 3 replicates were performed.

[0101] (3) Evaluation of fermentation effect

[0102] The reducing sugars and true proteins in the yeast solid-state fermentation group, the bacterial solid-state fermentation group, and the mixed fermentation group were detected separately.

[0103] Reducing sugar detection: The 3,5-dinitrosalicylic acid (DNS) colorimetric method was used. The preparation method of the reducing sugar extract is as follows: On the day of fermentation and on the 1st, 3rd and 7th days of fermentation, weigh 1 g of the freshly fermented solid-state sample into a 250 mL Erlenmeyer flask, add a small amount of distilled water to make a paste, and then add 50 mL of distilled water to make up the volume. Boil at 100℃ for 5 min to leach the reducing sugar. Then transfer the extract (including precipitate) to a 50 mL centrifuge tube and centrifuge at 8000 r / min for 5 min. Wash the precipitate once with 20 mL of distilled water, centrifuge again, and collect the supernatant in a 100 mL volumetric flask. Dilute to the mark with distilled water and mix well. This is the 100-fold diluted reducing sugar test solution. The reduction sugar increase = (reducing sugar content in the solid-state fermented sample after fermentation - reducing sugar content in the solid-state fermented sample before fermentation) / (reducing sugar content in the solid-state fermented sample before fermentation) × 100%.

[0104] True protein detection: The fermentation samples obtained from solid-state fermentation (dried to constant weight at 65℃ on day 1, day 3, and day 7 of fermentation) were pulverized and sieved. 1 g was accurately weighed into a 200 mL beaker, 50 mL of water was added, and the mixture was heated to boiling. Then, 20 mL of copper sulfate solution and 20 mL of sodium hydroxide solution were added, and the mixture was stirred thoroughly with a glass rod. The mixture was allowed to stand for 1 hour, filtered through qualitative filter paper, and the precipitate was washed 5-6 times with hot water at 60-80℃. The filter paper was checked with 5 drops of barium chloride solution and 1 drop of hydrochloric acid solution until no white barium sulfate precipitate formed. The precipitate and filter paper were dried in a 65℃ oven for 2 hours. The precipitate was then transferred to a Kjeldahl flask, and nitrogen was determined using the semi-micro Kjeldahl method. The semi-micro Kjeldahl method is performed according to Q / YB.J19.134.

[0105] The method mentioned in DB13 / T 1098-2009, "Determination of Protein in Feed," uses the following formula to calculate true protein content: True protein content = Total nitrogen content precipitated after digestion × 6.25. True protein increase = (True protein content in the solid-state fermented sample after fermentation - True protein content in the solid-state fermented sample before fermentation) / (True protein content in the solid-state fermented sample before fermentation) × 100%.

[0106] The results of the reducing sugar content are shown in Table 4. Mixta Calida The bacterial fermentation group containing AMCC 10426 showed the highest reduction sugar release during fermentation, with the highest detected value being 2.79% on the first day of fermentation, a 191% increase compared to before fermentation (day 0). The reduction sugar contents on days 3 and 7 were 1.23% and 1.50%, respectively. The decrease in reduction sugar content in the later stages compared to day 1 may be related to microbial proliferation (bacterial proliferation consumes some of the reduction sugar). The reduction sugar content in the mixed fermentation group was higher than that in the yeast fermentation group on days 1 and 3, which is related to the release of reducing sugar by bacteria during fermentation. The lower reduction sugar content in the mixed fermentation group on day 7 compared to the yeast fermentation group may be related to the consumption of some reducing sugar by bacterial proliferation.

[0107] Table 4 Reducing sugar content in fermentation combinations

[0108]

[0109] True protein content is shown in Table 5 and Figure 4As shown, compared to before fermentation (day 0), the true protein content in the mixed fermentation group increased by 16.42% on day 7, the yeast fermentation group by 3.55%, and the bacterial fermentation group by 0.17%. The true protein content in each fermentation group represents the composition of the total protein in the system, which in solid-state fermentation mainly consists of plant and yeast proteins. The mixed fermentation group had the lowest residual reducing sugar content and the highest true protein content, indicating... Mixta Calida The nutrients, such as sugars, degraded by AMCC 10426 bacterial strain were consumed by yeast, and... Mixta Calida AMCC 10426 bacteria can produce proteases, which have the ability to break down large protein molecules into smaller polypeptides. This provides a more readily available nitrogen source for yeast, promoting yeast growth and the accumulation of yeast protein. (The above explanation is incomplete.) Mixta Calida The AMCC 10426 bacterial strain has the potential to promote solid-state fermentation of yeast.

[0110] Table 5. True protein content in fermentation combinations

[0111]

[0112] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A kind Mixta calida Fungus, characterized in that, The Mixta calida bacteria Mixta calida strain AMCC10426 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20241202.

2. As described in claim 1 Mixta calida Fungus, characterized in that, The Mixta calida The 16S rRNA gene sequence of strain AMCC10426 is shown in SEQ ID NO.

5.

3. As described in claim 1 Mixta calida Fungus, characterized in that, The Mixta calida The gyrB gene sequence of strain AMCC10426 is shown in SEQ ID NO.

6.

4. As described in claim 1 Mixta calida Fungus, characterized in that, The Mixta calida AMCC10426 strain produces protease.

5. The method according to claim 4 Mixta calida Fungus, characterized in that, The enzyme activity of the protease is 53-55 U / mL.

6. The method according to claim 1 Mixta calida Fungus, characterized in that, The Mixta calida AMCC10426 strain produces cellulase.

7. The method according to claim 6 Mixta calida Fungus, characterized in that, The total enzyme activity of the cellulase is 0.1-0.2 U / mL.

8. The method according to claim 6 Mixta calida Fungus, characterized in that, The cellulase includes β-glucosidase.

9. The method according to claim 8 Mixta calida Fungus, characterized in that, The β-glucosidase activity is 0.2-0.3 U / L.

10. A microbial agent, characterized in that, The microbial agent contains any one of the following as described in claims 1-9 Mixta calida AMCC strain 10426.

11. The microbial agent according to claim 10, characterized in that, The microbial agent also includes auxiliary materials.

12. A method for preparing the microbial agent according to claim 10, characterized in that, The method includes the following steps: (1) The embodiment of any one of claims 1-9 Mixta calida Scale-up culture of AMCC 10426 strain; (2) Add the product obtained in step (1) to a liquid culture medium and ferment it at 26-37℃.

13. The claim 1-9 Mixta calida Application of AMCC 10426 strain or the microbial agent as described in claim 10 or 11 in solid-state fermentation.

14. The application according to claim 13, characterized in that, The Mixta calida AMCC strain 10426 increases the true protein content in ferments.

15. The application according to claim 13, characterized in that, The Mixta calida The AMCC 10426 strain releases reducing sugars from the fermentation product, promoting solid-state fermentation of yeast.

16. The claim 1-9 Mixta calida Application of AMCC 10426 strain or the microbial agent as described in claim 10 or 11 in the fermentation preparation of fertilizers, feed, cigars and high-temperature koji.

17. The claim 1-9 Mixta calida Application of AMCC 10426 strain or the inoculant of claim 10 or 11 in plant protection.

18. A high-temperature Daqu (a type of Chinese liquor), characterized in that, The high-temperature Daqu liquor is as described in any one of claims 1-9. Mixta calida It is prepared by fermentation using AMCC 10426 strain or the inoculum described in claim 10 or 11.

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