Aspergillus niger and application thereof, and preparation method of chitosan

Abstract

The invention discloses aspergillus niger and application thereof and a preparation method of chitosan. The Aspergillus niger is Aspergillus niger WL08, the preservation number of the Aspergillus niger is CCTCC (China Center for Type Culture Collection) NO: M2025459, and the Aspergillus niger is preserved in the China Center for Type Culture Collection on March 13, 2025. The preparation method comprises the following steps: fermenting the aspergillus niger, extracting the chitin, and deacetylating the obtained chitin to obtain the chitosan. The aspergillus niger provided by the invention has the characteristics of fast thallus growth and high biomass, and further has the characteristics of fast growth speed, low fermentation cost and high chitin content in cell walls in combination with the culture medium and the culture mode provided by the invention. Meanwhile, the chitosan is produced by further combining with a fermentation mode disclosed by the invention, the extraction of the chitosan is greatly facilitated by optimizing a culture medium and fermentation conditions, and the chitosan is high in extraction rate and stable in quality, so that low-cost and efficient chitosan production is realized.

Description

Technical Field

[0001] The present invention relates to the technical fields of microbial fermentation technology and chitosan extraction technology. Specifically, it relates to Aspergillus niger, its applications, and a method for preparing chitosan. Background Art

[0002] Chitosan is a natural polymer derived from chitin, with good biocompatibility, biodegradability, and antibacterial properties, and is widely used in fields such as food, medicine, agriculture, and environmental protection. Currently, chitosan is mainly produced by chemical methods and fermentation methods.

[0003] Traditional chemical methods use chitin as a raw material and remove the acetyl group through treatment with strong alkali solutions and high temperatures to prepare chitosan. This method has the advantages of simple process and short production cycle, but it has obvious defects:

[0004] Environmental pollution: Chemical methods require the use of large amounts of strong alkalis (such as sodium hydroxide) and high-temperature heating, causing serious pollution to the environment and incurring high costs for treating wastewater and waste.

[0005] Unstable product quality: Due to the harsh reaction conditions, it is difficult to precisely control the degree of deacetylation and molecular weight of chitosan, resulting in inconsistent product performance.

[0006] Limited raw material sources: Chitin is mainly derived from the shells of crustacean aquatic products. The raw material supply is easily restricted by seasonality and regionality, and the processes of deproteinization and decalcification of the raw materials are complex.

[0007] Fermentation methods utilize microorganisms (such as fungi and bacteria) to metabolize and produce chitosan or chitosan analogs. However, the current fermentation methods have the following deficiencies:

[0008] Low production efficiency: The chitosan yields of existing fermentation strains are limited, and the fermentation cycle is long, making it difficult to meet the requirements of large-scale industrial production.

[0009] Insufficient process optimization: The regulation of fermentation conditions (such as carbon-nitrogen ratio, pH, temperature) has a significant impact on chitosan yield and quality, but relevant research is scarce and a standardized process has not yet been formed.

[0010] Poor strain stability: Many fermentation strains are prone to decline during long-term cultivation or industrial fermentation, affecting production stability and economy.

[0011] Aspergillus niger is a common filamentous fungus, widely used in the industrial production of organic acids (such as citric acid) and enzymes. In recent years, research has shown that Aspergillus niger has the potential to synthesize chitosan, but its research and application in this field are still in the initial stage. The current research and application still have the following problems:

[0012] Low chitosan yield: The existing Aspergillus niger strains have limited ability to produce chitosan under fermentation conditions, making it difficult to meet industrial demands.

[0013] Insufficient process optimization: The growth behavior and metabolic characteristics of Aspergillus niger under different nutritional conditions have not been fully clarified, and the existing fermentation processes still lack systematic optimization.

[0014] Insufficient anti-pollution ability: In industrial production, the fermentation system is prone to contamination by other microorganisms, and the existing Aspergillus niger strains have limited resistance to contamination.

[0015] Based on this, providing an Aspergillus niger with low culture cost, fast production speed, and capable of greatly improving chitosan yield and its application in the preparation of chitosan is an urgent problem to be solved in the present invention. Summary of the Invention

[0016] In view of the above-mentioned prior art, the purpose of the present invention is to provide a fermentation system for efficiently and quickly preparing chitosan with Aspergillus niger as the core, so as to solve the problems of high raw material cost, slow cell growth rate, and low chitosan content existing in the preparation of chitosan by the prior art.

[0017] To achieve the above purpose, the present invention provides an Aspergillus niger, which is Aspergillus niger WL08, with a preservation number of CCTCC NO: M2025459, and was preserved at the China Center for Type Culture Collection on March 13, 2025.

[0018] The present invention also provides an application of the above-mentioned Aspergillus niger in the preparation of chitosan.

[0019] The present invention also provides a method for preparing chitosan, which includes: fermenting the above-mentioned Aspergillus niger and then extracting chitin, and deacetylating the obtained chitin to obtain chitosan.

[0020] Preferably, the process of extracting chitin specifically includes:

[0021] S100. Cultivation of Aspergillus niger: Cultivate Aspergillus niger on a PDA medium to form spores, wash the obtained spores to prepare a spore solution; inoculate the prepared spore solution into a liquid seed medium for cultivation to obtain a seed solution;

[0022] S200. Fermentation of Aspergillus niger: Inoculate the seed solution obtained in step S100 into a fermentation medium for fermentation cultivation to obtain a fermentation broth;

[0023] S300. Extraction of chitin: Filter, wash, and dry the fermentation broth obtained in step S200 to obtain chitin.

[0024] Preferably, in step S200, the inoculation amount of the seed liquid in the fermentation medium is 5-20% by volume.

[0025] Preferably, in step S200, the fermentation medium comprises: 30-50 g / L of molasses, 5-10 g / L of corn steep liquor; 0.1-0.2 g / L of KH2PO4, 0.1-0.2 g / L of K2HPO4, 0.1-0.2 g / L of MgSO4, 0.1-0.2 g / L of CaCl2.

[0026] Preferably, in step S100, the culture conditions in the liquid seed medium are: the culture temperature is 25-30 °C, the rotation speed is 180-220 r / min, and the culture time is 18-24 h;

[0027] And / or, in step S200, the culture conditions in the fermentation medium are: the culture temperature is 25-30 °C, the pH value is 4.5-5.5, and the culture time is 32-40 h.

[0028] Preferably, the deacetylation treatment of chitin specifically comprises:

[0029] S400. After adding an aqueous NaOH solution with a concentration of 40-60 wt% to the obtained chitin, reacting at 80-120 °C for 24-48 h to obtain a chitosan crude product by deacetylation; or, adding an aqueous deacetylase solution with a concentration of 2-4 wt% to the obtained chitin, reacting at 40-50 °C for 18-30 h to obtain a chitosan crude product by deacetylation;

[0030] S500. Adding an acid solution to the deacetylated chitosan crude product until the pH value is 2-4, dissolving the chitosan crude product to obtain a chitosan solution; adding an alkali solution to the obtained chitosan solution to adjust the pH value to 6.5-7.5, precipitating chitosan, filtering, washing, and drying to obtain chitosan.

[0031] Preferably, the acid solution in step S500 is selected from hydrochloric acid and / or acetic acid.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention provides an excellent Aspergillus niger strain with high chitosan yield, which is characterized by fast mycelial growth and high biomass. Combined with the culture medium and culture method of the present invention, it further has the characteristics of fast growth rate, low fermentation cost, and high chitin content in the cell wall. At the same time, further combined with the fermentation method of the present invention to produce chitosan, by optimizing the culture medium and fermentation conditions, the extraction of chitosan is greatly facilitated, and the chitosan extraction rate is high and the quality is stable, thereby realizing low-cost and high-efficiency chitosan production. Brief Description of the Drawings

[0034] The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention, but do not constitute a limitation to the present invention. In the accompanying drawings:

[0035] Figure 1 is a flow chart of the preparation method of chitosan in the present invention;

[0036] Figure 2 is a picture of the fermentation broth obtained in Example 1 of the present invention;

[0037] Figure 3 is a picture of the chitosan obtained in Example 1 of the present invention. Detailed Description of the Invention

[0038] The following provides a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0039] In the present invention, the culture media used are all prepared with raw materials and preparation methods commonly used by those skilled in the art. The rotten water bamboo samples used in the present invention are from rotten water bamboo samples in Huangyan District, Taizhou City, Zhejiang Province, China. Without special instructions, the raw materials used in the present invention are all commercially available products commonly used by those skilled in the art, and will not be elaborated here. In the present invention, in order to further reduce production costs, the molasses used here is specifically the waste generated in the sugar industry, and it is used as a carbon source in the present invention.

[0040] Specifically, in the following specific embodiments of the present invention, the raw material components of some of the culture media used are as follows:

[0041] PDA culture medium: 200 g / L of potato (boiled for 30 min, filtered to obtain the juice), 20 g / L of glucose, and 20 g / L of agar.

[0042] Liquid seed culture medium: 30 g / L of glucose, 20 g / L of corn steep liquor, 2 g / L of KH2PO4, and 0.5 g / L of MgSO4·7H2O.

[0043] Preparation Example

[0044] 1. Sample pretreatment: Mix 10 g of rotten water bamboo samples with 90 mL of sterile normal saline, shake at 28 °C and 180 rpm for 30 min, and take the supernatant as the original bacterial suspension after standing.

[0045] 2. Selective pre-culture: The obtained original bacterial suspension was cultured in an acidic PDA medium containing 0.1% pectin (i.e., on the basis of the aforementioned PDA medium, acid solution was further added to adjust the pH to 4.0, and pectin was added until the overall proportion of pectin in it was 0.1% by weight, and 50 μg / mL chloramphenicol was added to inhibit bacteria) for 48 h at 28 °C with static culture to enrich acid-tolerant filamentous fungi and obtain a pre-culture solution.

[0046] 3. Gradient dilution and single colony isolation: The pre-culture solution obtained after the above selective pre-culture was serially diluted 10 -3 ~10 -6 times, and spread on a selective medium plate containing 0.05% chitosan, and cultured at 30 °C for 5 - 7 days. The target strains were preliminarily screened by observing colony morphology (typical Aspergillus niger characteristics with neat edges, well-developed aerial hyphae, and dark brown spores), and single colonies were obtained after three rounds of plate streaking purification. That is, the Aspergillus niger WL08 of the present invention was obtained.

[0047] Example 1

[0048] 1. Culture Aspergillus niger WL08 on PDA medium to form spores.

[0049] 2. The obtained spores were washed to prepare a spore solution, which was inoculated into a liquid seed medium and cultured at 28 °C and 200 rpm for 18 hours to obtain a seed solution.

[0050] 3. The obtained seed solution was inoculated into a fermentation medium (inoculation amount 10%), and cultured at 28 °C and pH 5.0 for 32 hours to obtain a fermentation broth.

[0051] 4. The fermentation broth was filtered to collect the thalli, and after washing the obtained thalli, they were dried at low temperature to obtain dry thalli containing chitin (i.e., Aspergillus niger thalli with chitin in the cell wall).

[0052] 5. The dry thalli were crushed and 50% NaOH solution was added, and the reaction was carried out at 100 °C for 24 h for deacetylation;

[0053] 6. Subsequently, hydrochloric acid was added to it until the pH value was 3 to dissolve it, and a solution containing chitosan was obtained; NaOH solution was added to the solution containing chitosan for neutralization, and then it was filtered, washed, and dried to obtain chitosan.

[0054] Among them, the components of the fermentation medium in step 3 were: molasses 30 g / L, corn steep liquor dry powder 5 g / L, KH2PO4 0.1 g / L, K2HPO4 0.1 g / L, MgSO4 0.1 g / L, CaCl2 0.1 g / L.

[0055] Weigh the dried bacterial cells (i.e., Aspergillus niger cells) directly in step 4 to detect their biomass, and weigh the chitosan obtained in step 5.

[0056] After detection, the biomass of Aspergillus niger is 36 g / L, the chitosan content prepared accounts for 12% of the dry weight of its biomass, and the chitosan yield is 4.32 g / L.

[0057] Example 2

[0058] Operate according to the method of Example 1, except that: the culture time in step 2 is 20 h; the culture time in step 3 is 36 h; step 5 is to add 3% deacetylase solution after crushing the dried bacterial cells and react at 45 °C for 24 h for deacetylation.

[0059] Among them, the components of the fermentation medium in step 3 are: molasses is 40 g / L, corn steep liquor dry powder is 8 g / L, KH2PO4 is 0.15 g / L, K2HPO4 is 0.15 g / L, MgSO4 is 0.15 g / L, and CaCl2 is 0.15 g / L.

[0060] After detection, the biomass of Aspergillus niger is 40 g / L, the chitosan content prepared accounts for 11% of the dry weight of its biomass, and the chitosan yield is 4.40 g / L.

[0061] Example 3

[0062] Operate according to the method of Example 1, except that: the culture time in step 2 is 22 h; the culture time in step 3 is 38 h; the concentration of NaOH solution in step 5 is 40%, and the reaction time is 48 h.

[0063] Among them, the components of the fermentation medium in step 3 are: molasses is 45 g / L, corn steep liquor dry powder is 9 g / L, KH2PO4 is 0.2 g / L, K2HPO4 is 0.2 g / L, MgSO4 is 0.1 g / L, and CaCl2 is 0.15 g / L.

[0064] After detection, the biomass of Aspergillus niger is 43 g / L, the chitosan content prepared accounts for 11.5% of the dry weight of its biomass, and the chitosan yield is 4.95 g / L.

[0065] Example 4

[0066] Operate according to the method of Example 1, except that: the culture time in step 2 is 24 h; the culture time in step 3 is 40 h; step 5 is to add 3% deacetylase solution after crushing the dried bacterial cells and react at 45 °C for 24 h for deacetylation.

[0067] Among them, the components of the fermentation medium in Step 3 are: molasses is 50 g / L, corn steep liquor dry powder is 10 g / L, KH2PO4 is 0.2 g / L, K2HPO4 is 0.2 g / L, MgSO4 is 0.2 g / L, and CaCl2 is 0.2 g / L.

[0068] After detection, the biomass of Aspergillus niger is 38 g / L, the chitosan content prepared accounts for 12% of the dry weight of its biomass, and the chitosan yield is 4.56 g / L.

[0069] Example 5

[0070] Operate according to the method of Example 4, the difference is: Step 5 is to add 50% NaOH solution after crushing the dried thallus, and react at 100 °C for 24 h for deacetylation.

[0071] After detection, the biomass of Aspergillus niger is 43 g / L, the chitosan content prepared accounts for 10% of the dry weight of its biomass, and the chitosan yield is 4.3 g / L.

[0072] Control Example

[0073] An article "Study on the Fermentation Production of Chitosan by Aspergillus niger" published in the journal "Grain Distribution Technology" obtained a dry cell weight of 21.38 g / L using a potato medium. Through electrolysis and alternating acid-base treatment, the chitosan yield was 13.7% and the degree of deacetylation was 86.1%. That is, the chitosan yield was 2.93 g / L.

[0074] The Aspergillus niger provided by the present invention can use a fermentation medium with cheap carbon and nitrogen sources (molasses as a carbon source and corn steep liquor as a nitrogen source) to achieve fermentation and extract chitosan, greatly reducing the production cost.

[0075] In the present invention, the molasses used is a by-product of the sugar industry, with a wide source and low price. Compared with some commonly used pure sugar carbon sources (such as glucose, etc.), its price has obvious advantages. Using a large amount of molasses as the carbon source of the fermentation medium can provide sufficient carbon skeletons and energy for the growth and metabolism of Aspergillus niger at a lower cost, meet the demand of Aspergillus niger for carbon sources during the fermentation process, and effectively reduce the raw material procurement cost on the premise of ensuring the normal growth of Aspergillus niger and the synthesis of chitosan precursor substances. At the same time, in the present invention, corn steep liquor, a by-product in the production process of corn starch, can be used as a nitrogen source. On the premise of ensuring the large-scale reproduction of Aspergillus niger and the active progress of chitosan-related metabolic pathways, compared with other high-quality organic nitrogen sources (such as peptone, etc.), the nitrogen source input cost is greatly reduced.

[0076] Under the premise that Aspergillus niger provided by the present invention uses inexpensive carbon and nitrogen sources such as 30 - 50 g / L molasses and 5 - 10 g / L corn steep liquor as components of the fermentation medium, under similar fermentation scale and time conditions (fermentation time 72 - 96 h, fermentation temperature 28 - 30 °C, etc.), the chitosan yield can reach 4 - 5 g / L, and the degree of deacetylation is over 90%. In the case of using inexpensive carbon and nitrogen sources, the yield is equivalent to or even higher than that of the traditional high-cost carbon and nitrogen source fermentation method, highlighting the advantages of this method in terms of the comprehensive cost-yield benefit.

[0077] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0078] In addition, it should be noted that, among the various specific technical features described in the above specific embodiments, without conflict, they can be combined in any appropriate way. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0079] Furthermore, any combination can be made between various different embodiments of the present invention as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. An Aspergillus niger, characterized in that, The Aspergillus niger is Aspergillus niger WL08, with a preservation number of CCTCC NO: M2025459, and it was preserved in the China Center for Type Culture Collection on March 13, 2025.

2. Use of the Aspergillus niger as described in claim 1 in the preparation of chitosan.

3. A preparation method of chitosan, characterized in that, The preparation method includes: fermenting the Aspergillus niger as described in claim 1 and then extracting chitin, and deacetylating the obtained chitin to obtain chitosan.

4. The preparation method according to claim 3, characterized in that, The process of extracting chitin specifically includes: S100. Cultivation of Aspergillus niger: Cultivate Aspergillus niger on a PDA medium to form spores, wash the obtained spores to prepare a spore solution; inoculate the prepared spore solution into a liquid seed medium for cultivation to obtain a seed solution; S200. Fermentation of Aspergillus niger: Inoculate the seed solution obtained in step S100 into a fermentation medium for fermentation culture to obtain a fermentation broth; S300. Extraction of chitin: Filter, wash, and dry the fermentation broth obtained in step S200 to obtain chitin.

5. The preparation method according to claim 4, characterized in that, In step S200, the inoculation amount of the seed solution in the fermentation medium is 5 - 20% (by volume).

6. The preparation method according to claim 4, characterized in that, In step S200, the fermentation medium includes: 30 - 50 g / L molasses, 5 - 10 g / L corn steep liquor; 0.1 - 0.2 g / L KH2PO4, 0.1 - 0.2 g / L K2HPO4, 0.1 - 0.2 g / L MgSO4, 0.1 - 0.2 g / L CaCl2.

7. The preparation method according to any one of claims 4 to 6, characterized in that, In step S100, the cultivation conditions in the liquid seed medium are: the cultivation temperature is 25 - 30 °C, the rotation speed is 180 - 220 r / min, and the cultivation time is 18 - 24 h; And / or, in step S200, the cultivation conditions in the fermentation medium are: the cultivation temperature is 25 - 30 °C, the pH value is 4.5 - 5.5, and the cultivation time is 32 - 40 h.

8. The preparation method according to claim 3, characterized in that, The deacetylation treatment of chitin specifically includes: S400. After adding an aqueous NaOH solution with a concentration of 40 - 60 wt% to the obtained chitin, react under the condition of 80 - 120 °C for 24 - 48 h to obtain a chitosan crude product by deacetylation; or, add an aqueous deacetylase solution with a concentration of 2 - 4 wt% to the obtained chitin, and react under the condition of 40 - 50 °C for 18 - 30 h to obtain a chitosan crude product by deacetylation; S500. Add an acid solution to the deacetylated chitosan crude product until the pH value is 2 - 4, dissolve the chitosan crude product to obtain a chitosan solution; add an alkali solution to the obtained chitosan solution to adjust the pH value to 6.5 - 7.5, precipitate chitosan, filter, wash, and dry to obtain chitosan.

9. The preparation method according to claim 8, characterized in that, The acid solution in step S500 is selected from hydrochloric acid and / or acetic acid.

Images