Culture method for increasing protein content of aspergillus niger mycelium by using corn steep liquor as culture medium

Abstract

The invention discloses a culture method for increasing the protein content of aspergillus niger mycelia by using corn steep liquor as a culture medium, and belongs to the technical field of biological fermentation. Aiming at insufficient resource utilization of corn steep liquor and substitute protein production requirements, a corn processing by-product corn steep liquor is creatively adopted to directly replace a traditional PDB culture medium, and the mycelium protein content is remarkably increased by optimizing a fermentation system. Results show that the corn steep liquor is used as a culture medium, aspergillus niger with the preservation number of CCTCC M 2013703 is inoculated, and aspergillus niger mycelium protein can be obtained through fermentation. The protein content of the mycelium obtained by the process disclosed by the invention reaches 50.62-53.56%, and the total amino acid content is increased by 64-76%. According to the method, high-value conversion of agricultural corn byproducts is realized, and an efficient and low-cost industrial solution is provided for alternative protein production.

Description

Technical Field

[0001] This invention relates to a cultivation method for increasing the protein content of Aspergillus niger mycelium using corn steep liquor as a culture medium, belonging to the field of biofermentation technology. Background Technology

[0002] Corn steep liquor is one of the main byproducts of the wet starch production process from corn, with an annual output of 800,000 to 900,000 tons. Rich in protein and carbohydrates, corn steep liquor is currently mainly used as a fermentation medium, but its usage is limited. Actively seeking new ways to utilize corn steep liquor resources is the primary solution to the problem of its difficult processing.

[0003] In ancient China, Aspergillus niger was already used in the traditional production of soy sauce, rice wine, and other foods. In the 1920s, Pfizer, an international company, screened out Aspergillus niger strains capable of efficiently producing citric acid, and in 1919, it was first applied to the industrial production of citric acid, thus beginning the era of Aspergillus niger as an industrial fermentation strain. For over a century, Aspergillus niger has been gradually domesticated and improved into a multi-purpose cell factory capable of converting various inexpensive substrates into a series of valuable metabolites.

[0004] In response to the growing human health crisis and animal ethics issues, the market demand for meat alternatives continues to increase. Aspergillus niger, as a filamentous microorganism, has emerged as a highly promising alternative to meat protein due to its natural fibrous structure, high protein content, and excellent amino acid composition. Filamentous microorganisms not only provide essential structural polysaccharides and high-quality proteins, but their unique fibrous properties also significantly simplify the processing technology for mimicking meat textures, offering an efficient solution for developing novel plant-based protein products.

[0005] However, in current technologies using corn steep liquor fermentation of Aspergillus niger for protein production, the corn steep liquor is always diluted and added as an additive to the Aspergillus niger culture medium for fermentation, or prepared as a dry powder and added in small amounts to promote mycelial protein fermentation. For example, Chinese patent CN119464092B, "Aspergillus niger, inoculum, application, and method for high-yield mycelial protein production," adds 0.1-1 g / L of corn steep liquor dry powder; Chinese patent CN120025914A, "A fermentation method for Aspergillus niger and its application in controlling cell morphology and producing phytase," uses a corn steep liquor concentration of 3-5% in the fermentation medium. It is evident that currently, in the fermentation process of Aspergillus niger, Aspergillus niger has low tolerance to high-concentration corn steep liquor, making it impossible to directly use high-concentration corn steep liquor for fermentation, resulting in reduced corn steep liquor utilization and increased energy consumption. Summary of the Invention

[0006] The purpose of this invention is to address existing problems by providing a cultivation method that uses corn steep liquor as a culture medium to increase the protein content of Aspergillus niger mycelium.

[0007] This invention is achieved through the following technical solution:

[0008] The first objective of this invention is to provide a method for cultivating Aspergillus niger mycelium using corn steep liquor as a culture medium to increase the protein content, comprising the following:

[0009] S1. Dilute the corn steep liquor to a solids content of 5-20%, and sterilize it to obtain corn steep liquor culture medium;

[0010] S2. Inoculate 5-20% Aspergillus niger seed liquid into corn steep liquor medium according to the volume ratio, and ferment at 20-40℃ for 1-5 days to obtain Aspergillus niger mycelial fermentation broth.

[0011] In one embodiment of the present invention, the Aspergillus niger is preserved with the accession number CCTCC NO:M2013703.

[0012] In one embodiment of the present invention, the Aspergillus niger seed solution is prepared by inoculating at a rate of 1% to 3% with a concentration of not less than 1×10⁻⁶. 6 Aspergillus niger spore suspension of CFU / mL was inoculated into PDB medium and cultured at 28–32℃ for 20–30 h to obtain the spores.

[0013] In one embodiment of the present invention, the Aspergillus niger spore suspension is obtained by eluting spores with physiological saline containing Tween-80 and then diluting.

[0014] In one embodiment of the present invention, in step S2, the rotation speed during fermentation is 400-600 r / min.

[0015] In one embodiment of the present invention, in step S2, the aeration ratio during fermentation is 0.4 to 0.6 vvm / L.

[0016] In one embodiment of the present invention, the Aspergillus niger mycelium fermentation broth further includes the step of collecting the mycelium by filtering with gauze.

[0017] In one embodiment of the present invention, the mycelium is obtained by vacuum freeze-drying to obtain dried mycelium.

[0018] A second objective of this invention is to provide Aspergillus niger mycelium prepared by the method described.

[0019] A third objective of this invention is to provide the application of the aforementioned Aspergillus niger mycelium in the preparation of mycelial proteins.

[0020] The beneficial effects of this invention are:

[0021] Compared with the prior art, the present invention has the following advantages: the mycelial protein is obtained by fermentation using corn steep liquor as a culture medium, which is easier to operate, lower in cost, and easier to plan and promote; the separate management of each stage of mycelial growth conditions is more in line with the growth pattern of mycelium at each stage, which helps to increase the yield of Aspergillus niger mycelium and the protein content; compared with the traditional PDB culture medium, the mycelial protein content is significantly improved and the amino acid composition of the mycelium is significantly improved, which can not only solve the problem of difficult processing of corn steep liquor, a by-product of corn processing, but also significantly improve its economic benefits. Detailed Implementation

[0022] The present invention will be further illustrated below with specific examples. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0023] Source of raw materials

[0024] Aspergillus niger (CCTCC NO: M 2013703) is disclosed in CN103937681A as a food-grade strain of Aspergillus niger and its application in the degradation of zearalenone in zearalenone.

[0025] PDA culture medium was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0026] The technical solution of the present invention will be described in detail below with reference to specific embodiments. In the following embodiments, unless otherwise specified, the reagents, materials and equipment used can be purchased commercially, prepared by conventional methods, or commonly used in the industry.

[0027] Example 1:

[0028] A method for increasing the protein content of Aspergillus niger mycelium using corn steep liquor as a culture medium, comprising the following:

[0029] (1) Preparation of culture medium: dilute corn steep liquor with a solid content of 40% to 10%;

[0030] (2) Seed culture preparation: Aspergillus niger was removed from -80℃, revived, inoculated into PDA medium, and cultured at 28℃ for 5 days. The spores were then washed off with sterile physiological saline containing Tween-80, and the spore concentration was adjusted to 1×10⁻⁶. 6 CFU / mL yields a suspension of Aspergillus niger spores. PDB medium is pumped into a 5L fermenter for sterilization. After sterilization and cooling, the temperature is raised to 30℃ and kept at that temperature. At a rotation speed of 500r / min, 2% Aspergillus niger spore suspension is added, and the culture time is 24h to obtain the seed culture medium.

[0031] (3) Culture medium sterilization: The corn steep liquor was loaded into a 10L liquid fermentation tank. The loading volume of the culture tank was 60% of the fermentation tank, which was 6L. The loaded liquid fermentation tank was placed in a high temperature and high pressure sterilizer at 121℃ for 20min for sterilization. After sterilization, the temperature was lowered to below 30℃ before inoculation.

[0032] (4) Fermentation management: control the fermentation time to 60h, control the fermentation temperature to 25℃, control the seed liquid inoculation amount to 10%, control the fermentation tank speed to 500r / min, and control the ventilation ratio to 0.5vvm / L.

[0033] (5) Cultivation: Cultivate under the above management conditions, and harvest the mycelium through gauze after fermentation.

[0034] (6) Mycelial drying: The mycelium obtained in (5) is placed in a stainless steel tray and vacuum freeze-dried. First, the mycelium is pre-cooled in a -80℃ refrigerator and then placed in a vacuum freeze-drying substrate for 48 hours to obtain dried mycelium.

[0035] Example 2

[0036] A method for increasing the protein content of Aspergillus niger mycelium using corn steep liquor as a culture medium, comprising the following:

[0037] (1) Preparation of culture medium: dilute corn steep liquor with a solid content of 40% to 10%;

[0038] (2) Seed culture preparation: Aspergillus niger was removed from -80℃, revived, inoculated into PDA medium, and cultured at 28℃ for 5 days. The spores were then washed off with sterile physiological saline containing Tween-80, and the spore concentration was adjusted to 1×10⁻⁶. 6 CFU / mL yields a suspension of Aspergillus niger spores. PDB medium is pumped into a 5L fermenter for sterilization. After sterilization and cooling, the temperature is raised to 30℃ and kept at that temperature. At a rotation speed of 500r / min, 2% Aspergillus niger spore suspension is added, and the culture time is 24h to obtain the seed culture medium.

[0039] (3) Culture medium sterilization: The corn steep liquor was loaded into a 10L liquid fermentation tank. The loading volume of the culture tank was 60% of the fermentation tank, which was 6L. The loaded liquid fermentation tank was placed in a high temperature and high pressure sterilizer at 121℃ for 20min for sterilization. After sterilization, the temperature was lowered to below 30℃ before inoculation.

[0040] (4) Fermentation management: control the fermentation time to 60h, control the fermentation temperature to 30℃, control the seed liquid inoculation amount to 10%, control the fermentation tank speed to 500r / min, and the ventilation ratio to 0.5vvm / L.

[0041] (5) Cultivation: Cultivate under the above management conditions, and harvest the mycelium through gauze after fermentation.

[0042] (6) Mycelial drying: The mycelium obtained in (5) is placed in a stainless steel tray and vacuum freeze-dried. First, the mycelium is pre-cooled in a -80℃ refrigerator and then placed in a vacuum freeze-drying substrate for 48 hours to obtain dried mycelium.

[0043] Example 3

[0044] A method for increasing the protein content of Aspergillus niger mycelium using corn steep liquor as a culture medium, comprising the following:

[0045] (1) Preparation of culture medium: Dilute corn steep liquor with a solid content of 40% to 5%;

[0046] (2) Seed culture preparation: Aspergillus niger was taken out from -80℃, revived, inoculated into PDA medium and cultured at 28℃ for 5 days. The spores were washed off with sterile physiological saline containing Tween-80 and the spore concentration was adjusted to 1×106 CFU / mL to obtain Aspergillus niger spore suspension. PDB medium was pumped into a 5L fermenter for sterilization. After sterilization and cooling, the temperature was raised to 30℃ and kept warm. 2% Aspergillus niger spore suspension was inoculated at a speed of 500r / min and cultured for 24h to obtain seed culture.

[0047] (3) Culture medium sterilization: The corn steep liquor was loaded into a 10L liquid fermentation tank. The loading volume of the culture tank was 60% of the fermentation tank, which was 6L. The loaded liquid fermentation tank was placed in a high temperature and high pressure sterilizer at 121℃ for 20min for sterilization. After sterilization, the temperature was lowered to below 30℃ before inoculation.

[0048] (4) Fermentation management: control the fermentation time to 60h, control the fermentation temperature to 30℃, control the seed liquid inoculation amount to 10%, control the fermentation tank speed to 500r / min, and the ventilation ratio to 0.5vvm / L.

[0049] (5) Cultivation: Cultivate under the above management conditions, and harvest the mycelium through gauze after fermentation.

[0050] (6) Mycelial drying: The mycelium obtained in (5) is placed in a stainless steel tray and vacuum freeze-dried. First, the mycelium is pre-cooled in a -80℃ refrigerator and then placed in a vacuum freeze-drying substrate for 48 hours to obtain dried mycelium.

[0051] Example 4

[0052] A method for increasing the protein content of Aspergillus niger mycelium using corn steep liquor as a culture medium, comprising the following:

[0053] (1) Preparation of culture medium: Dilute corn steep liquor with a solid content of 40% to 15%;

[0054] (2) Seed culture preparation: Aspergillus niger was removed from -80℃, revived, inoculated into PDA medium, and cultured at 28℃ for 5 days. The spores were then washed off with sterile physiological saline containing Tween-80, and the spore concentration was adjusted to 1×10⁻⁶. 6 CFU / mL yields a suspension of Aspergillus niger spores. PDB medium is pumped into a 5L fermenter for sterilization. After sterilization and cooling, the temperature is raised to 30℃ and kept at that temperature. At a rotation speed of 500r / min, 2% Aspergillus niger spore suspension is added, and the culture time is 24h to obtain the seed culture medium.

[0055] (3) Culture medium sterilization: The corn steep liquor was loaded into a 10L liquid fermentation tank. The loading volume of the culture tank was 60% of the fermentation tank, which was 6L. The loaded liquid fermentation tank was placed in a high temperature and high pressure sterilizer at 121℃ for 20min for sterilization. After sterilization, the temperature was lowered to below 30℃ before inoculation.

[0056] (4) Fermentation management: control the fermentation time to 60h, control the fermentation temperature to 30℃, control the seed liquid inoculation amount to 10%, control the fermentation tank speed to 500r / min, and the ventilation ratio to 0.5vvm / L.

[0057] (5) Cultivation: Cultivate under the above management conditions, and harvest the mycelium through gauze after fermentation.

[0058] (6) Mycelial drying: The mycelium obtained in (5) is placed in a stainless steel tray and vacuum freeze-dried. First, the mycelium is pre-cooled in a -80℃ refrigerator and then placed in a vacuum freeze-drying substrate for 48 hours to obtain dried mycelium.

[0059] Example 5

[0060] A method for increasing the protein content of Aspergillus niger mycelium using corn steep liquor as a culture medium, comprising the following:

[0061] (1) Preparation of culture medium: dilute corn steep liquor with a solid content of 40% to 10%;

[0062] (2) Seed culture preparation: Aspergillus niger was removed from -80℃, revived, inoculated into PDA medium, and cultured at 28℃ for 5 days. The spores were then washed off with sterile physiological saline containing Tween-80, and the spore concentration was adjusted to 1×10⁻⁶. 6 CFU / mL yields a suspension of Aspergillus niger spores. PDB medium is pumped into a 5L fermenter for sterilization. After sterilization and cooling, the temperature is raised to 30℃ and kept at that temperature. At a rotation speed of 500r / min, 2% Aspergillus niger spore suspension is added, and the culture time is 24h to obtain the seed culture medium.

[0063] (3) Culture medium sterilization: The corn steep liquor was loaded into a 10L liquid fermentation tank. The loading volume of the culture tank was 60% of the fermentation tank, which was 6L. The loaded liquid fermentation tank was placed in a high temperature and high pressure sterilizer at 121℃ for 20min for sterilization. After sterilization, the temperature was lowered to below 30℃ before inoculation.

[0064] (4) Fermentation management: control the fermentation time to 72h, control the fermentation temperature to 30℃, control the seed liquid inoculation amount to 10%, control the fermentation tank speed to 500r / min, and the ventilation ratio to 0.5vvm / L.

[0065] (5) Cultivation: Cultivate under the above management conditions, and harvest the mycelium through gauze after fermentation.

[0066] (6) Mycelial drying: The mycelium obtained in (5) is placed in a stainless steel tray and vacuum freeze-dried. First, the mycelium is pre-cooled in a -80℃ refrigerator and then placed in a vacuum freeze-drying substrate for 48 hours to obtain dried mycelium.

[0067] Example 6

[0068] A method for increasing the protein content of Aspergillus niger mycelium using corn steep liquor as a culture medium, comprising the following:

[0069] (1) Preparation of culture medium: dilute corn steep liquor with a solid content of 40% to 10%;

[0070] (2) Seed culture preparation: Aspergillus niger was removed from -80℃, revived, inoculated into PDA medium, and cultured at 28℃ for 5 days. The spores were then washed off with sterile physiological saline containing Tween-80, and the spore concentration was adjusted to 1×10⁻⁶. 6 CFU / mL yields a suspension of Aspergillus niger spores. PDB medium is pumped into a 5L fermenter for sterilization. After sterilization and cooling, the temperature is raised to 30℃ and kept at that temperature. At a rotation speed of 500r / min, 2% Aspergillus niger spore suspension is added, and the culture time is 24h to obtain the seed culture medium.

[0071] (3) Culture medium sterilization: The corn steep liquor was loaded into a 10L liquid fermentation tank. The loading volume of the culture tank was 60% of the fermentation tank, which was 6L. The loaded liquid fermentation tank was placed in a high temperature and high pressure sterilizer at 121℃ for 20min for sterilization. After sterilization, the temperature was lowered to below 30℃ before inoculation.

[0072] (4) Fermentation management: control the fermentation time to 48h, control the fermentation temperature to 30℃, control the seed liquid inoculation amount to 10%, control the fermentation tank speed to 500r / min, and the ventilation ratio to 0.5vvm / L.

[0073] (5) Cultivation: Cultivate under the above management conditions, and harvest the mycelium through gauze after fermentation.

[0074] (6) Mycelial drying: The mycelium obtained in (5) is placed in a stainless steel tray and vacuum freeze-dried. First, the mycelium is pre-cooled in a -80℃ refrigerator and then placed in a vacuum freeze-drying substrate for 48 hours to obtain dried mycelium.

[0075] Example 7

[0076] A method for increasing the protein content of Aspergillus niger mycelium using corn steep liquor as a culture medium, comprising the following:

[0077] (1) Preparation of culture medium: dilute corn steep liquor with a solid content of 40% to 10%;

[0078] (2) Seed culture preparation: Aspergillus niger was taken out from -80℃, revived, inoculated into PDA medium and cultured at 28℃ for 5 days. The spores were washed off with sterile physiological saline containing Tween-80 and the spore concentration was adjusted to 1×106 CFU / mL to obtain Aspergillus niger spore suspension. PDB medium was pumped into a 5L fermenter for sterilization. After sterilization and cooling, the temperature was raised to 30℃ and kept warm. 2% Aspergillus niger spore suspension was inoculated at a speed of 500r / min and cultured for 24h to obtain seed culture.

[0079] (3) Culture medium sterilization: The corn steep liquor was loaded into a 10L liquid fermentation tank. The loading volume of the culture tank was 60% of the fermentation tank, which was 6L. The loaded liquid fermentation tank was placed in a high temperature and high pressure sterilizer at 121℃ for 20min for sterilization. After sterilization, the temperature was lowered to below 30℃ before inoculation.

[0080] (4) Fermentation management: control the fermentation time to 60h, control the fermentation temperature to 30℃, control the seed liquid inoculation amount to 5%, control the fermentation tank speed to 500r / min, and the ventilation ratio to 0.5vvm / L.

[0081] (5) Cultivation: Cultivate under the above management conditions, and harvest the mycelium through gauze after fermentation.

[0082] (6) Mycelial drying: The mycelium obtained in (5) is placed in a stainless steel tray and vacuum freeze-dried. First, the mycelium is pre-cooled in a -80℃ refrigerator and then placed in a vacuum freeze-drying substrate for 48 hours to obtain dried mycelium.

[0083] Example 8

[0084] A method for increasing the protein content of Aspergillus niger mycelium using corn steep liquor as a culture medium, comprising the following:

[0085] (1) Preparation of culture medium: dilute corn steep liquor with a solid content of 40% to 10%;

[0086] (2) Seed culture preparation: Aspergillus niger was removed from -80℃, revived, inoculated into PDA medium, and cultured at 28℃ for 5 days. The spores were then washed off with sterile physiological saline containing Tween-80, and the spore concentration was adjusted to 1×10⁻⁶. 6 CFU / mL yields a suspension of Aspergillus niger spores. PDB medium is pumped into a 5L fermenter for sterilization. After sterilization and cooling, the temperature is raised to 30℃ and kept at that temperature. At a rotation speed of 500r / min, 2% Aspergillus niger spore suspension is added, and the culture time is 24h to obtain the seed culture medium.

[0087] (3) Culture medium sterilization: The corn steep liquor from step (2) is loaded into a 10L liquid fermentation tank. The loading volume of the culture is 60% of the fermentation tank, which is 6L. The loaded liquid fermentation tank is placed in a high temperature and high pressure sterilizer at 121℃ for 20min for sterilization. After sterilization, the temperature is lowered to below 30℃ before inoculation.

[0088] (4) Fermentation management: control the fermentation time to 60h, control the fermentation temperature to 30℃, control the seed liquid inoculation amount to 15%, control the fermentation tank speed to 500r / min, and the ventilation ratio to 0.5vvm / L.

[0089] (5) Cultivation: Cultivate under the above management conditions, and harvest the mycelium through gauze after fermentation.

[0090] (6) Mycelial drying: The mycelium obtained in (5) is placed in a stainless steel tray and vacuum freeze-dried. First, the mycelium is pre-cooled in a -80℃ refrigerator and then placed in a vacuum freeze-drying substrate for 48 hours to obtain dried mycelium.

[0091] The protein and amino acid contents of the mycelia prepared in Examples 1-8 were determined according to standards GB / T 6432-2018 and GB / T 18246-200, respectively; the results are shown in Table 1.

[0092] Table 1. Changes in bacterial cell yield, total protein, and total amino acids

[0093]

Claims

1. A method for cultivating Aspergillus niger mycelium using corn steep liquor as a culture medium to increase its protein content, characterized in that, Includes the following: S1. Dilute the corn steep liquor to a solids content of 5-20%, and sterilize it to obtain corn steep liquor culture medium; S2. Inoculate 5-20% Aspergillus niger seed liquid into corn steep liquor medium according to the volume ratio, and ferment at 20-40℃ for 1-5 days to obtain Aspergillus niger mycelial fermentation broth.

2. The cultivation method according to claim 1, characterized in that, The preservation number of the Aspergillus niger is CCTCC NO:M2013703.

3. The cultivation method according to claim 1, characterized in that, The Aspergillus niger seed solution is prepared by inoculating at a rate of 1% to 3% with at least 1×10⁻⁶ spores. 6 Aspergillus niger spore suspension of CFU / mL was inoculated into PDB medium and cultured at 28–32℃ for 20–30 h to obtain the spores.

4. The cultivation method according to claim 3, characterized in that, The Aspergillus niger spore suspension was obtained by eluting spores with physiological saline containing Tween-80 and then diluting.

5. The cultivation method according to claim 1, characterized in that, In step S2, the fermentation speed is 400–600 r / min.

6. The cultivation method according to claim 1, characterized in that, In step S2, the aeration ratio during fermentation is 0.4–0.6 vvm / L.

7. The cultivation method according to claim 1, characterized in that, The Aspergillus niger mycelium fermentation broth also includes a step of collecting the mycelium by filtering with gauze.

8. The cultivation method according to claim 1, characterized in that, The mycelium was obtained by vacuum freeze-drying.

9. Aspergillus niger mycelium prepared by the method according to any one of claims 1 to 8.

10. The use of the Aspergillus niger mycelium according to claim 9 in the preparation of Aspergillus niger mycelium protein.

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