Aspergillus niger, complex starter, fermentation bacteria and application thereof

Abstract

This invention relates to the field of fermentation technology, and specifically provides a *Aspergillus niger* strain with accession number CCTCC NO: M 20251780. This invention utilizes a selected multi-enzyme *Aspergillus niger* strain for solid-state fermentation to prepare a composite fermentation agent rich in various solid-state fermentation enzymes and mold spores. This agent serves as a functional adjuvant for molasses alcohol yeast fermentation, significantly improving the fermentation performance of the alcohol yeast. It substantially increases the fermentation speed and alcohol content of molasses alcohol, achieving an alcohol yield of 93.23%, effectively solving the problems of unstable fermentation performance and low alcohol yield in molasses alcohol yeast.

Description

Aspergillus niger, compound fermentation agent, fermentation inoculant and its application Technical Field

[0001] This invention relates to the field of fermentation technology, and in particular to Aspergillus niger, a compound fermentation agent, a fermentation inoculant, and their applications. Background Technology

[0002] Aspergillus niger is a common filamentous fungus widely used in traditional fermented food solid-state koji making, enzyme preparation production, bio-feed, and soil microbiology. Aspergillus niger grows vigorously and does not produce toxins. As a globally recognized safe and edible fungus, it holds an important position in industrial production and academic research. Studies have shown that Aspergillus niger can metabolize various enzymes, including cellulase, xylanase, amylase, protease, saccharifying enzyme, pectinase, lipase, glucose oxidase, tanninase, and phytase. Aspergillus niger can decompose various organic substances, including polysaccharides, fats, proteins, natural fibers, pectin, and insoluble compounds. The hydrolysis products are mostly monosaccharides, amino acids, and soluble carbohydrates that can be directly utilized by Saccharomyces cerevisiae.

[0003] Sugarcane molasses is one of the world's most important raw materials for fermentation industries, widely used in the production of alcohol, yeast, and organic acids. Sugarcane molasses contains approximately 40-45% fermentable sugars, 4-6% non-fermentable sugars, 3-6% crude protein, 10-15% colloids (mainly xylose gum, arabinose gum, and pectin), and 12-18% impurities (mainly cellulose, hemicellulose, and lignin). The cellulose impurities, non-fermentable sugars, and crude protein in molasses cannot be directly utilized by brewer's yeast.

[0004] The current molasses alcohol fermentation process requires the addition of a large amount of biological enzymes to decompose the substrate for absorption and utilization by alcohol fermentation yeast. However, the overall conversion efficiency is low. At the same time, sugarcane molasses contains a large amount of cellulose impurities, colloidal substances, and organic acids (lactic acid, butyric acid, and hexanoic acid) metabolized by a large number of acid-producing bacteria, which have a significant inhibitory effect on the growth and metabolic performance of alcohol yeast. This results in slow yeast reproduction and low alcohol content, which has a significant impact on the economic benefits of molasses alcohol enterprises.

[0005] Currently, there are three main problems hindering molasses alcohol fermentation: first, the fermentation speed is slow, resulting in a low alcohol yield of less than 90%; second, the fermentation process is unstable, with significant fluctuations in alcohol yield; and third, the large amount of enzymes required for molasses treatment leads to high costs. The main reasons for these problems are related to the fact that colloids, organic acids, and cellulose impurities in molasses can inhibit yeast growth, and existing enzyme treatment methods suffer from low efficiency and high costs.

[0006] Therefore, developing a fermentation agent that can relieve the growth inhibition of alcohol yeast by some organic acids, plant colloids, and cellulose impurities in molasses, effectively decompose nutrients in molasses for yeast absorption and utilization, reduce the amount of related enzyme preparations used, and significantly improve the fermentation performance of molasses alcohol yeast is crucial for improving the economic benefits of the molasses alcohol industry. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a *Aspergillus niger*, and the composite fermentation agent prepared from the above-mentioned *Aspergillus niger* is used as a functional auxiliary material for molasses alcohol yeast fermentation, thereby achieving a significant improvement in the fermentation performance of alcohol yeast.

[0008] This invention provides Aspergillus niger with accession number CCTCC NO: M 20251780.

[0009] The aforementioned Aspergillus niger metabolizes at least one of the following: cellulase, hemicellulase, acidic protease, glucosylamylase, α-amylase, pectinase, and glucose oxidase.

[0010] This Aspergillus niger was isolated from traditional baijiu cellar mud samples. The strain has the following characteristics: it has a strong ability to produce spores and can also metabolize a variety of enzyme systems that are beneficial for molasses alcohol fermentation, such as cellulase, hemicellulase, acidic protease, glucosylamylase, α-amylase, pectinase, and glucose oxidase.

[0011] Its ITS rDNA has the nucleic acid sequence shown in SEQ ID NO.1.

[0012] rDNA gene sequence of strain AM ITS:

[0013] .

[0014] The strain selection method described in this invention includes: isolating multiple strains of Aspergillus niger from traditional Sichuan Baijiu cellar mud samples, performing initial screening on casein plates, and secondary screening by shaking flask fermentation to produce enzyme systems such as hemicellulase, xylanase, pectinase, and glucosylamylase, to obtain a strain AM that can simultaneously and efficiently secrete related enzyme systems for molasses degradation treatment, which is identified as Aspergillus niger.

[0015] This invention provides a compound fermentation agent prepared from any of the above-mentioned technical solutions using Aspergillus niger.

[0016] The fermentation agent provided by this invention has the following specifications: mold spores > 500 million / g, hemicellulase > 15,000 u / g, xylanase > 2,000 u / g, acidic protease > 2,000 u / g, glucosylamylase > 1,000 u / g, and cellulase > 200 u / g.

[0017] This invention provides a method for preparing a compound fermentation agent, comprising the following steps:

[0018] The Aspergillus niger is cultured in liquid seed culture, then transferred to solid-state fermentation, dried, and pulverized to obtain the final product.

[0019] The inoculum amount for liquid-to-solid fermentation in this invention is 10-40%; specifically, it can be 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, or 40%.

[0020] According to the present invention, the liquid seed culture medium is formulated as follows: 12% molasses, 1% ammonium sulfate, 3% corn flour, with the remainder being water, pH 6.2, and sterilized at 121°C for 30 min.

[0021] In one embodiment, the liquid seed culture parameters are: temperature 33~35℃, specifically 33℃, 34℃, 35℃; rotation speed 150~180 r / min, specifically 150 r / min, 151 r / min, 152 r / min, 153 r / min, 154 r / min, 155 r / min, 156 r / min, 157 r / min, 158 r / min, 159 r / min, 160 r / min, 161 r / min. in, 162r / min, 163r / min, 164r / min, 165r / min, 166r / min, 167r / min, 168r / min, 169r / min, 170r / min, 171 r / min, 172r / min, 173r / min, 174r / min, 175r / min, 176r / min, 177r / min, 178r / min, 179r / min, 180r / min.

[0022] Aeration rate of 1.0~1.2 VVM, specifically 1 VVM, 1.1 VVM, or 1.2 VVM; fermentation time of 24~26 h, specifically 24 h, 25 h, or 26 h.

[0023] The solid fermentation culture medium formula of the present invention is: 65% wheat bran, 15% sugarcane bagasse, 8% corn flour and 12% soybean meal, sterilized at 121℃ for 50 min;

[0024] In one of the preferred embodiments, the solid-state fermentation adopts a variable temperature fermentation scheme as follows: the product temperature is controlled at 30-36℃ for 0-24h, 26-30℃ for 24-48h, and 22-26℃ for 48-64h.

[0025] The solid-state fermentation adopts an oxygen-limited culture scheme as follows: dissolved oxygen is controlled at 20% from 0 to 12 hours, 30% from 12 to 48 hours, and 20% from 48 to 64 hours; wherein the dissolved oxygen is 20% at 12 hours and 30% at 48 hours.

[0026] The preferred solid-state fermentation cultivation parameters are as follows: inoculum size 16-24%, specifically 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%; fermentation humidity 80-90%, specifically 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%; fermentation time 60-68h, specifically 60h, 61h, 62h, 63h, 64h, 65h, 66h, 67h, 68h.

[0027] This invention provides the application of Aspergillus niger described in the above-mentioned technical solution, the composite fermentation agent described in the above-mentioned technical solution, and the composite fermentation agent prepared by the preparation method described in the above-mentioned technical solution in the preparation of fermentation inoculants; the fermentation inoculant is a fermentation inoculant for plant-derived substrates.

[0028] The fermentation agent described in this invention has the function of degrading proteins, cellulose, hemicellulose, xylan, starch and / or pectin.

[0029] The plant-derived substrates described in this invention include: sugarcane bagasse, wheat bran, corn, soybean meal, rice straw, wheat straw, corn cob, cottonseed hulls, peanut shells, corn stalks, sorghum straw, millet straw, buckwheat straw, barley straw, highland barley straw, soybean straw, rapeseed straw, sesame cake, sunflower seed cake, rapeseed cake, tea seed cake, tung seed cake, palm kernel cake, coconut shell, corn stalks, sorghum straw, millet straw, buckwheat straw, barley straw, highland barley straw, and more. The raw materials include plant-derived materials such as hemp straw, jute straw, melon vine, pumpkin vine, winter melon vine, loofah vine, bitter melon vine, cucumber vine, zucchini vine, tomato vine, eggplant vine, chili pepper vine, green pepper vine, cowpea vine, pea vine, broad bean vine, edamame vine, hyacinth bean vine, sword bean vine, kidney bean vine, red bean vine, mung bean vine, black bean vine, and chickpea vine; sugarcane bagasse, wheat bran, corn flour, and soybean meal; preferably, the plant-derived substrate contains molasses.

[0030] The plant-derived substrates of this invention contain at least one of protein, cellulose, hemicellulose, xylan, starch and / or pectin.

[0031] The uses of microbial agents include, but are not limited to, brewing, soil improvement, or the production of bio-feed. The fermentation substrate can be solid or liquid; this invention does not limit the specific fermentation process.

[0032] For example, the microbial agent is used for brewing, and its substrates include sorghum, corn, rice, wheat, glutinous rice, barley, oats, highland barley, millet, buckwheat, beans (such as red beans and mung beans), tubers (such as sweet potatoes, potatoes, and cassava), fruits (such as grapes, apples, pears, cherries, strawberries, and blueberries), sugarcane, beets, malt, and various grain bran and tuber residues.

[0033] The microbial agent is used for soil improvement. Its substrates include straw (such as rice straw, wheat straw, corn straw, and soybean straw), livestock and poultry manure (such as chicken manure, cow manure, sheep manure, and pig manure), compost raw materials, humus, sawdust, bark, peat, mushroom residue (such as waste mushroom sticks after edible mushroom cultivation), green manure crops (such as alfalfa, milkvetch, and clover), and various plant residues and debris.

[0034] The microbial agent is used to prepare biological feed, and its substrates include soybean meal, rapeseed meal, cottonseed meal, peanut meal, sunflower seed meal, sesame meal, rapeseed cake, soybean cake, wheat bran, rice bran, corn flour, corn cob flour, wheat flour, wheat middlings, tuber flour (such as sweet potato flour, potato flour), straw flour (such as corn straw flour, wheat straw flour), silage raw materials (such as silage corn, silage alfalfa), distiller's grains, vinegar residues, fruit pomace (such as apple pomace, citrus pomace, grape pomace), beet pomace, sugarcane bagasse, and various plant-based feed raw materials.

[0035] This invention provides a fermentation agent, comprising alcoholic yeast and the compound fermentation agent described in the above-mentioned technical solutions or the compound fermentation agent prepared by any one of the preparation methods described in the above-mentioned technical solutions.

[0036] In a preferred embodiment of the present invention, the mass ratio of the compound fermenting agent to the alcoholic yeast is (5~15):100; particularly preferably 1:9.

[0037] This invention provides a method for molasses alcohol fermentation, which involves inoculating a substrate containing molasses into the fermentation agent described in the above technical solution, and then carrying out fermentation.

[0038] According to the present invention, the fermentation steps include: diluting sugarcane molasses, adding sucrose to adjust the molasses content, adding acid to the pH value, and then adding defoaming oil to the above-mentioned fermentation agent to obtain the product through fermentation.

[0039] In one embodiment of the present invention, the specific method for molasses alcohol fermentation is as follows: Raw sugarcane molasses is diluted to 27.5 Bx with 40°C warm water, sucrose is added to adjust the Bx to 33.5 Bx, the pH is adjusted to 4.8 with concentrated sulfuric acid, and a certain amount of defoaming oil is added. Molasses alcohol yeast is added at an inoculum rate of 0.1%, the fermentation temperature is 32°C, the fermentation speed is 100 r / min, and the fermentation time is 60 h.

[0040] This invention provides a *Aspergillus niger* strain with accession number CCTCC NO: M 20251780. This invention uses a selected multi-enzyme *Aspergillus niger* strain for solid-state fermentation to prepare a composite fermentation agent rich in biological enzymes and mold spores. This agent serves as a functional adjuvant for molasses alcohol yeast fermentation, significantly improving the fermentation performance of the alcohol yeast. It not only eliminates the inhibitory effects of some organic acids, plant colloids, and cellulose impurities in molasses on the growth of alcohol yeast, but also effectively decomposes nutrients in molasses for yeast absorption and utilization, reducing the amount of related enzyme preparations used. This significantly increases the fermentation speed and alcohol content of molasses alcohol, achieving an alcohol yield of 93.23%, effectively solving the problems of unstable fermentation performance and low alcohol yield in molasses alcohol yeast.

[0041] This patented compound fermentation agent is formulated into molasses alcohol yeast. When applied to molasses alcohol fermentation, the fermentation speed is significantly improved compared to the control, the alcohol content is increased by about 3% to 9%, and the alcohol yield reaches 93.23%.

[0042] This patented compound fermentation agent significantly improves the tolerance of alcohol yeast to various organic acids, and significantly enhances the yeast's reproductive and fermentation capabilities.

[0043] Biological Preservation Instructions

[0044] Biological material: Aspergillus niger AMCC 20191, deposited on August 5, 2025 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, accession number CCTCCNO: M 20251780. Detailed Implementation

[0045] This invention provides a bacterium, its application, and a modified soy protein isolate, as well as a method for its preparation. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of protection of this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0046] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0047] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

[0048] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0049] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and, unless otherwise stated, does not constitute a limitation on the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0050] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0051] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0052] The embodiments and comparative examples of this invention describe some examples, in which the embodiments illustrate certain implementations of the invention. However, this does not mean that the effects of the invention can only be achieved in these examples.

[0053] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a bacterium provided by the present invention, its application, modified soy protein isolate, and its preparation method.

[0054] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods.

[0055] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0056] Example 1: Screening of bacterial strains

[0057] Multiple Aspergillus niger strains (AM, AY-1, AY-2, RY-1, RY-2, BQ-1, MQ-1, HQ-1, DQ-1, and HT-1) screened in the laboratory were used as research subjects. Seed liquid was prepared according to the above process, followed by solid-state fermentation, drying, pulverization, and homogenization to obtain a compound fermentation agent. The enzyme activity of the compound fermentation agent was detected and analyzed, and the results are shown in Table 1.

[0058] The seed liquid formula is: 12% molasses, 1% ammonium sulfate, 3% corn flour, with the remainder being water, pH 6.2, sterilized at 121℃ for 30 minutes.

[0059] The solid-state fermentation formula is: 65% wheat bran, 15% sugarcane bagasse, 8% corn flour and 12% soybean meal, sterilized at 121℃ for 50 minutes.

[0060] The cultivation method was as follows: Different Aspergillus niger strains were scraped from slant agar and inoculated into shake flask seed culture medium. Fermentation was carried out at 33℃ and 180 rpm for 24 hours. Then, at a 4% inoculum size, the culture was transferred to a fermenter with seed culture medium at 34℃, 180 rpm, and an aeration rate of 1.0-1.2 VVM for 26 hours. Finally, at a 16% inoculum size, the mature seed fermentation broth was transferred to a solid-state fermentation medium. During the solid-state fermentation stage, variable temperature fermentation parameters were used: 33-36℃ for 0-24 hours, 28-33℃ for 24-48 hours, and 24-28℃ for 48-64 hours. Oxygen-limited culture parameters were used: 20% dissolved oxygen for 0-12 hours, 30% for 12-48 hours, and 20% for 48-64 hours. Humidity was controlled at 80-85% throughout the fermentation process for 64 hours. After fermentation, the culture was dried and pulverized to obtain the compound fermenting agent.

[0061] Table 1 Comparison of enzyme activity analysis of different strains of compound fermentation spawn

[0062] Example Cellulase (U / g) Hemicellulase (U / g) Xylanase (u / g) Acidic protease (U / g) Glucoamylase (U / g) Pectinase (u / g) Glucose oxidase (u / g) Example 1 (AM) 67221400485958032798376674 Comparative Example 1 (AY-1) 19817100357077503060161102 Comparative Example 2 (AY-2) 19015300247958362003231138 Comparative Example 3 (RY-1) 781380056334505890 5824 Comparative Example 4 (RY-2) 18017100899370052539857 Comparative Example 5 (BQ-1) 23018500108428993987224333 Comparative Example 6 (MQ-1) 1001200058018364312249245 Comparative Example 7 (HQ-1) 1058904459590288010241 Comparative Example 8 (DQ-1) 23776028910130300128142 Comparative Example 9 (HT-1) 2985303728909261016113 surface

[0063] The strain AM (AMCC20191) screened in Example 1 exhibits outstanding enzyme production performance during solid-state fermentation and contains a rich enzyme system. This strain AM was deposited on August 5, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251780.

[0064] Examples 2, 3, and Comparative Examples 1, 2, 3, 4, 5, and 6: Quality analysis of compound fermentation agents prepared by different processes.

[0065] Example 2: A compound fermentation agent was prepared using Aspergillus niger AM strain according to the method of Example 1. The difference from Example 1 is that the solid-state fermentation stage adopted variable temperature fermentation parameters: 0-24h temperature controlled at 35-36℃, 24-48h temperature controlled at 28-30℃, 48-64h temperature controlled at 24-26℃, humidity controlled at 85-90%, and fermentation time at 60h.

[0066] Example 3: A compound fermentation agent was prepared using Aspergillus niger AM strain according to the method of Example 1. The difference from Example 1 is that the inoculum amount for liquid-to-solid fermentation was 22%, the humidity was kept constant at 85±1% during the fermentation process, and the fermentation time was 68h.

[0067] Comparative Example 1: A compound fermentation agent was prepared using Aspergillus niger AM strain according to the method of Example 1. The difference from Example 3 is that the seed liquid formula is: 10% sucrose, 1% ammonium sulfate, 4% corn flour, and the balance is water, pH 6.2, sterilized at 121℃ for 30 min.

[0068] Comparative Example 2: A compound fermentation agent was prepared using Aspergillus niger AM strain according to the method of Example 1. The difference from Example 3 is that the solid-state fermentation formula is: 80% wheat bran, 8% corn flour and 12% soybean meal, sterilized at 121℃ for 50 min.

[0069] Comparative Example 3: A compound fermentation agent was prepared using Aspergillus niger AM strain according to the method of Example 1. The difference from Example 3 is that the solid-state fermentation formula is: 80% wheat bran, 20% corn flour, sterilized at 121℃ for 50 min.

[0070] Comparative Example 4: A compound fermentation agent was prepared using Aspergillus niger AM strain according to the method of Example 1. The difference from Example 3 was that the seed culture formulation consisted of 10% sucrose, 1% ammonium sulfate, 4% corn flour, with the remainder being water, pH 6.2, and sterilized at 121℃ for 30 min. The solid-state fermentation formulation consisted of 80% wheat bran, 8% corn flour, and 12% soybean meal, sterilized at 121℃ for 50 min.

[0071] Comparative Example 5: A compound fermentation agent was prepared using Aspergillus niger AM strain according to the method of Example 1, except that the seed liquid formulation was: 10% sucrose, 1% ammonium sulfate, 4% corn flour, with the balance being water, pH 6.2, and sterilized at 121℃ for 30 min. The solid-state fermentation formulation was: 80% wheat bran, 20% soybean meal, and sterilized at 121℃ for 50 min.

[0072] Comparative Example 6: A compound fermentation agent was prepared using Aspergillus niger AM strain according to the method of Example 1. The difference from Example 3 is that the solid-state fermentation formula is: 80% wheat bran, 20% soybean meal, sterilized at 121℃ for 50 min.

[0073] Seed culture of Aspergillus niger AM strain was prepared according to the above process, followed by solid-state fermentation, drying, pulverization, and homogenization to obtain a compound fermentation agent. The spore count and core enzyme activity of the compound fermentation agents prepared by different processes were detected and analyzed. The results are shown in Table 2.

[0074] Table 2 Comparison of enzyme activity in composite fermentation agents prepared by different processes

[0075] Examples of fungal spore count (100 million / g), hemicellulose enzyme (u / g), xylanase activity (u / g), acidic protease (u / g), glucoamylase (u / g), pectinase (u / g). Example 2: 31.25 20800 4930 5783 2991 406. Example 3: 37.25 22 100 5470 5083 3291 491. Comparative Example 1: 28.12 17990 3570 4664 273. 0220 Comparative Example 215.75 15670 3300 3458 139788 Comparative Example 313.98 15840 3070 3867 2949 142 Comparative Example 44.37 12700 1990 7380 1118 146 Comparative Example 54.86 9980 1670 7969 1096 189 Comparative Example 65.99 1321 0980 800 21367 321 surface

[0076] As can be seen from the comparison of data in Table 2, different processes have a significant impact on the core indicators of the compound fermentation agent: spore content, hemicellulase, xylanase and pectinase activity. The core indicators of the compound fermentation agent prepared under the process of Example 3 are the best: mold spores reach 3.725 billion / g, hemicellulase activity reaches 22,100 u / g, xylanase activity reaches 5,470 u / g and pectinase activity reaches 491 u / g.

[0077] Alcoholic yeast was mixed with compound fermentation agents prepared by different processes at a ratio of 9:1 to obtain compound molasses alcoholic yeast. The alcohol yield was tested according to the molasses alcohol fermentation method, and the results are shown in Table 3.

[0078] The specific method for molasses alcohol fermentation is as follows: Dilute raw sugarcane molasses with 40℃ warm water to 27.5 Bx (5.5 liters), add 550g of sucrose to adjust the Bx to 33.5 Bx, adjust the pH to 4.8 with concentrated sulfuric acid, and add a certain amount of defoaming oil. Dispense into 1000ml Erlenmeyer flasks, filling each flask with 800ml of liquid. Add molasses alcohol yeast containing a compound fermenting agent (10% compound fermenting agent) prepared in Examples 2-3 and Comparative Examples 1-6 of this invention at a 0.1% inoculation rate. Ferment at 32℃ and 100 r / min. After 60 hours of fermentation, detect the final alcohol content and the change in sugar content before and after fermentation, and calculate the alcohol yield.

[0079] Table 3 Comparison of alcohol yield from compound fermentation agents prepared using different processes and combined with molasses alcohol yeast

[0080] Example 1: Fermentation alcohol content (% vol), alcohol yield (%) Example 2: Alcohol yeast preparation 9.79%, 93.23% Example 3: Alcohol yeast preparation 9.68%, 92.19% Comparative Example 1: Alcohol yeast preparation 9.41%, 89.61% Comparative Example 2: Alcohol yeast preparation 9.32%, 88.76% Comparative Example 3: Alcohol yeast preparation 9.41%, 89.62% Comparative Example 4: Alcohol yeast preparation 8.97%, 85.42% Comparative Example 5: Alcohol yeast preparation 9.09%, 86.57% Comparative Example 6: Alcohol yeast preparation 8.89%, 84.67% surface

[0081] As shown in Table 3, the alcohol yield of the compound fermentation agent prepared according to the processes of Example 2 and Example 3, and the alcohol yeast prepared according to the process, reached more than 90%, which is 3-9% higher than that of the control group.

[0082] Examples 4, 5, and 6, and Comparative Examples 7, 8, 9, 10, and 11: Comparative Analysis of the Quality Advantages of Molasses Alcoholic Fermentation

[0083] The compound fermentation agent obtained in Example 2 was added to alcohol yeast at proportions of 5%, 10%, and 15% to obtain compound molasses alcohol yeast. Application tests were conducted according to the molasses alcohol fermentation method, and the results are shown in Tables 4 (Examples 4, 5, and 6).

[0084] To fully demonstrate the quality advantages of the compound molasses alcohol yeast obtained by this technology, comparative tests were conducted using pure alcohol yeast, alcohol yeast combined with enzyme preparations, and core competitor alcohol yeasts FK1, ML2, and LF2. The results are shown in Comparative Examples 7, 8, 9, 10, and 11 in Table 4.

[0085] The above method of combining alcohol yeast with enzyme preparation is as follows: 90% alcohol yeast, 4% acidic protease (200,000 / g), 3% hemicellulase (150,000 / g), and 3% pectinase (120,000 / g) are used to prepare compound molasses alcohol yeast.

[0086] Table 4. Analysis of the results of applying different proportions of compound molasses alcohol yeast to molasses alcohol fermentation.

[0087] Example 0h: Hundreds of millions of cells / ml; 8h: Hundreds of millions of cells / ml; 16h: Hundreds of millions of cells / ml; Final fermentation alcohol content (% VOL): %; Alcohol yield (%): Example 4: 0.17; 0.52; 1.43; 9.69; 9.29; Example 5: 0.15; 0.55; 1.47; 9.79; 9.24; Example 6: 0.13; 0.59; 1.49; 9.68; 9.19; Comparative Example 7 (Alcoholic yeast): 0.19; 0.50; 1.29; 9. 3388.5 Comparative Example 8 (Alcoholic yeast enzyme preparation) 0.19 0.59 1.31 9.47 90.19 Comparative Example 9 (Competitor yeast FK1) 0.16 0.53 1.30 9.33 88.85 Comparative Example 10 (Competitor yeast ML2) 0.18 0.50 1.33 9.28 88.38 Comparative Example 11 (Competitor yeast LF2) 0.12 0.52 1.32 9.44 89.90 surface

[0088] As shown in Table 4, the compound molasses alcohol yeast prepared in Examples 4, 5 and 6 of the present invention and applied to molasses alcohol fermentation have certain advantages over the control alcohol yeast and competing products in terms of comprehensive quality indicators such as yeast cell count, fermentation alcohol content and alcohol yield after 16 h. Among them, the application effect of the configuration scheme in Example 5 is the best, and the yeast cell count, fermentation alcohol content and alcohol yield are all at a high level in the industry.

[0089] In summary, the compound fermentation agent prepared using the Aspergillus niger AM strain and the matching fermentation process of this invention has good application potential in improving the alcohol yield in the field of molasses alcohol. It can significantly increase the alcohol yield of yeast in molasses alcohol fermentation while reducing the cost of enzyme preparations, thus creating more benefits for molasses alcohol fermentation enterprises.

[0090] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of Aspergillus niger, characterized in that, Its accession number is CCTCC NO: M20251780; it simultaneously metabolizes cellulase, hemicellulase, acidic protease, glucosylamylase, xylanase, pectinase and glucose oxidase.

2. The Aspergillus niger according to claim 1, characterized in that, Its ITS rDNA has the nucleic acid sequence shown in SEQ ID NO.

1.

3. A compound fermentation agent, prepared by solid-state fermentation of *Aspergillus niger* according to any one of claims 1-2; wherein the solid-state fermentation culture medium formula is: 65% wheat bran, 15% sugarcane bagasse, 8% corn flour and 12% soybean meal, sterilized at 121℃ for 50 min; the preparation method of the compound fermentation agent includes: The Aspergillus niger is cultured in liquid seed culture, then transferred to solid-state fermentation, dried, and pulverized to obtain the product. The culture medium formula for the liquid seed is: 12% molasses, 1% ammonium sulfate, 3% corn flour, with the balance being water, pH 6.2, sterilized at 121℃ for 30 min; the culture parameters for the liquid seed are: temperature 33~35℃, rotation speed 150~180 r / min, aeration rate 1.0~1.2 VVM, and fermentation time 24~26 h; the culture medium formula for the solid fermentation is: 65% wheat bran, 15% bagasse, 8% corn flour, and 12% soybean meal, sterilized at 121℃ for 50 min.

4. The compound fermentation agent according to claim 3, characterized in that, Among them, the number of mold spores is >500 million / g, hemicellulase is >15,000 u / g, xylanase is >2,000 u / g, acidic protease is >2,000 u / g, glucosylamylase is >1,000 u / g, and cellulase is >200 u / g.

5. The method for preparing the composite fermentation agent according to claim 3, characterized in that, The process includes the following steps: *Aspergillus niger* is cultured in a liquid seed culture, then transferred to solid-state fermentation, dried, and pulverized to obtain the final product. The liquid seed culture medium is formulated as follows: 12% molasses, 1% ammonium sulfate, 3% corn flour, with the remainder being water, pH 6.2, sterilized at 121℃ for 30 min. The liquid seed culture parameters are: temperature 33-35℃, rotation speed 150-180 r / min, aeration rate 1.0-1.2 VVM, and fermentation time 24-26 h. The solid-state fermentation culture medium is formulated as follows: 65% wheat bran, 15% bagasse, 8% corn flour, and 12% soybean meal, sterilized at 121℃ for 50 min.

6. The preparation method according to claim 5, characterized in that, The solid-state fermentation temperature-controlled fermentation scheme is as follows: temperature controlled at 30-36℃ for 0-24h, 26-30℃ for 24-48h, and 22-26℃ for 48-64h; the solid-state fermentation oxygen-limited culture scheme is as follows: dissolved oxygen controlled at 20% for 0-12h, 30% for 12-48h, and 20% for 48-64h; the solid-state fermentation culture parameters are as follows: inoculum size 16-24%, fermentation humidity 80-90%, and fermentation time 60-68h.

7. The application of the Aspergillus niger according to any one of claims 1 to 2, the compound fermentation agent according to claim 3, and the compound fermentation agent prepared by the preparation method according to any one of claims 5 to 6 in the preparation of fermentation agents; wherein the fermentation agent is a fermentation agent for plant-derived substrates.

8. The application according to claim 7, characterized in that, The fermentation agent has the function of degrading proteins, cellulose, hemicellulose, xylan, starch and / or pectin.

9. The application according to claim 7, characterized in that, The plant-derived substrate includes one or more of the following: sugarcane bagasse, wheat bran, corn, soybean cake, rice straw, wheat straw, corn cob, cottonseed hulls, peanut shells, corn stalks, sorghum straw, millet straw, buckwheat straw, barley straw, highland barley straw, soybean straw, rapeseed straw, sesame cake, sunflower seed cake, rapeseed cake, tea seed cake, tung seed cake, palm kernel cake, coconut shell, corn stalks, sorghum straw, millet straw, buckwheat straw, barley straw, highland barley straw, flax straw, hemp straw, jute straw, melon vine, pumpkin vine, winter melon vine, loofah vine, bitter melon vine, cucumber vine, zucchini vine, tomato vine, eggplant vine, chili pepper vine, green pepper vine, cowpea vine, pea vine, broad bean vine, edamame vine, hyacinth bean vine, sword bean vine, kidney bean vine, red bean vine, mung bean vine, black bean vine, or chickpea vine.

10. A fermentation inoculant, characterized in that, It includes alcohol yeast and the compound fermentation agent according to claim 3 or the compound fermentation agent prepared by the preparation method according to any one of claims 5 to 6.

11. The fermentation agent according to claim 10, characterized in that, The mass ratio of the compound fermentation agent to alcoholic yeast is (5~15):

100.

12. A method for molasses alcoholic fermentation, characterized in that, The substrate containing molasses is inoculated into the fermentation agent of claim 10, and then fermentation is carried out.

13. The method according to claim 12, characterized in that, The fermentation steps include: diluting sugarcane molasses, adding sucrose to adjust the molasses content, adjusting the pH value with acid, then adding defoaming oil and adding the fermentation agent described in claim 10, and fermenting to obtain the product.

14. The method according to claim 13, characterized in that, The dilution was carried out with 40°C warm water to a concentration of 27.5 Bx; the pH was adjusted to 4.8 with concentrated sulfuric acid.

15. The method according to claim 12, characterized in that, The fermentation temperature was 32℃, the rotation speed was 100 r / min, and the fermentation time was 60 h.