CN116042413A - Microbial agent for fermenting aspergillus niger and preparing chitosan-containing oligosaccharide by taking chitosan as auxiliary carbon source and preparation method thereof - Google Patents

Microbial agent for fermenting aspergillus niger and preparing chitosan-containing oligosaccharide by taking chitosan as auxiliary carbon source and preparation method thereof Download PDF

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CN116042413A
CN116042413A CN202211687672.5A CN202211687672A CN116042413A CN 116042413 A CN116042413 A CN 116042413A CN 202211687672 A CN202211687672 A CN 202211687672A CN 116042413 A CN116042413 A CN 116042413A
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chitosan
aspergillus niger
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赵兰瑞
张俊庆
王春环
曹长旺
李宝健
徐振龙
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Shandong Zhizhidao Biotechnology Co ltd
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Abstract

The invention provides a microbial agent for fermenting Aspergillus niger and preparing chitosan-containing oligosaccharide by taking chitosan as an auxiliary carbon source and a preparation method thereof, wherein the effective viable count of the Aspergillus niger in the microbial agent is 2-20 hundred million/ml, and the content of the chitosan oligosaccharide is 1-1.6%; in the preparation process of the microbial agent, chitosan liquid is added into fermentation broth in a mode of adding chitosan, the chitosan is enzymatically hydrolyzed into chitosan oligosaccharide by using a metabolite of aspergillus niger, and the chitosan oligosaccharide is used as an auxiliary carbon source for fermentation, so that the enzymolysis cost of the chitosan is reduced, the addition of the carbon source in the culture process is reduced, and the fermentation cost is reduced. The microbial agent provided by the invention can produce a growth promoting effect on crops.

Description

Microbial agent for fermenting aspergillus niger and preparing chitosan-containing oligosaccharide by taking chitosan as auxiliary carbon source and preparation method thereof
Technical Field
The invention relates to the technical field of microbial agents, in particular to a microbial agent for fermenting aspergillus niger by taking chitosan as an auxiliary carbon source and preparing chitosan-containing oligosaccharide and a preparation method thereof.
Background
Chitosan is an alkaline polysaccharide, at C 2 Substituted in position by an acetamido group and an amino group, so that it contains a free amino group, has biodegradability, cell affinity and other properties; the activity of amino groups in chitosan molecules is large, so that the chitosan has excellent biological functions, and therefore, the research on chitosan as a functional biological material is increasing.
Chitosan exists in a large amount in nature, but is difficult to use due to poor solubility, so that the waste of chitosan is large. The chitosan is a good agricultural biological hormone after decomposition, and has good application prospect in agricultural planting, however, the decomposition process needs to be carried out under the conditions of acidity and enzyme addition, and the selling price of the chitosan enzyme in the market is as high as 5000-6000 yuan per kilogram, so that the cost of chitosan enzymolysis is high, and the chitosan enzymolysis is unfavorable for being applied to the agricultural field by the mode.
Chitosan is used as a substance similar to cellulose in structure, less research is conducted on the chitosan in fermentation, and how to use the chitosan as a carbon source in biological fermentation so as to improve the utilization rate and reduce the enzymolysis cost of the chitosan is of great significance to the industry.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a microbial agent for fermenting aspergillus niger by taking chitosan as an auxiliary carbon source and preparing chitosan oligosaccharide and a preparation method thereof, wherein the effective viable count of the aspergillus niger in the microbial agent is 2-20 hundred million/ml, and the content of the chitosan oligosaccharide is 1-1.6%; in the preparation process of the microbial agent, chitosan liquid is added into fermentation broth in a mode of adding chitosan, the chitosan is enzymatically hydrolyzed into chitosan oligosaccharide by using a metabolite of aspergillus niger, and the chitosan oligosaccharide is used as an auxiliary carbon source for fermentation, so that the enzymolysis cost of the chitosan is reduced, the addition of the carbon source in the culture process is reduced, and the fermentation cost is reduced. The microbial agent provided by the invention can produce a growth promoting effect on crops.
The technical scheme of the invention is as follows:
a microbial agent containing chitosan oligosaccharide is prepared by fermenting Aspergillus niger with chitosan as auxiliary carbon source, wherein the effective viable count of Aspergillus niger is 2-20 hundred million/ml, and the chitosan oligosaccharide content is 1-1.6%.
The preparation method for fermenting Aspergillus niger and preparing the microbial agent containing chitosan oligosaccharide by taking chitosan as an auxiliary carbon source comprises the following steps:
(1) Seed liquid:
200ml of PDA culture medium is filled in a 1L medicine bottle, and sterilization is carried out for 20min at 121 ℃ for standby;
inoculating Aspergillus niger flat plate stored in laboratory into shake flask under aseptic condition, culturing at 30deg.C and 180 rpm for 3-5 days, and performing microscopic examination without bacteria; in the seed solution, the thallus amount of Aspergillus niger is 7×10 8 -9×10 8 Individual spores/mL; the number of the aspergillus niger is CICC2215;
(2) Fermentation:
preparing a fermentation medium, inoculating the seed solution into the fermentation medium with an inoculum size of 2-5%, and culturing at 30 ℃ for 70-80 hours to obtain fermentation liquid, and performing microscopic examination;
(3) Bacterial agent:
when a large number of hyphae appear in the microscopic examination, chitosan solution is added into the fermentation liquor, the ratio of the weight of chitosan in the chitosan solution to the weight of the fermentation liquor is 0.5-2:100, the fermentation is continued for 120-155 hours at the temperature of 28-30 ℃, and the liquid microbial agent is obtained after centrifugation.
Preferably, in step (2), the fermentation medium comprises bran 45-55g, glucose 22-30g, ammonium sulfate 17-22g, K 2 HPO 4 0.7-1.5g、KCl 0.2-0.6g，MgSO 4 ·7H 2 O 0.4-0.7g，Na 2 SO 4 0.4-0.8g，FeSO 4 ·7H 2 O0.1-0.15 g, water is added to fix volume to 1L, and pH is natural.
Preferably, the fermentation medium is 50g of bran, 30g of glucose, 20g of ammonium sulfate and K 2 HPO 4 1g，KCl 0.5g，MgSO 4 ·7H 2 O 0.5g，Na 2 SO 4 0.5g，FeSO 4 ·7H 2 O0.1g, water is added to fix the volume to 1L, and the pH is natural.
Preferably, in step (3), the chitosan is used after deacetylation.
Preferably, in the step (3), the chitosan is pulverized to 100 mesh or more in advance so as to be easily dissolved in glacial acetic acid.
Preferably, in the step (3), the chitosan solution is prepared by the following steps: mixing chitosan and glacial acetic acid 2-3 hours before use, wherein the weight ratio of the chitosan to the glacial acetic acid is 1:5, dissolving chitosan in glacial acetic acid, and then feeding the solution into the fermentation liquor.
Preferably, in the step (3), since glacial acetic acid has great acidity and is easy to influence the fermentation process, the feeding time is controlled, so that adverse influence on the fermentation process is avoided; the feeding time is between 5 and 8 hours.
Preferably, in the step (3), the Aspergillus niger fermentation broth is centrifuged for 10min under the condition of 5000r/min, and the supernatant is respectively taken to obtain the microbial inoculum.
Aspergillus niger is a common microorganism and widely distributed in soil, grains and plant products, can generate various enzymes, gallic acid and other metabolites in the fermentation process, can provide nutrient elements for crop production, and has good application prospect; the fermentation process of the aspergillus niger is combined with the chitosan enzymolysis process, and the aspergillus niger metabolite is utilized to carry out enzymolysis on chitosan, so that the chitosan enzymolysis cost is effectively reduced, and the obtained microbial agent not only contains a large amount of metabolites, but also contains chitosan oligosaccharide after the enzymolysis of chitosan, so that the microbial agent can stimulate the crop to grow when being applied to crops, and the utilization rate of chitosan are improved.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, the fermentation of the aspergillus niger and the enzymolysis of the chitosan are synchronously carried out, the metabolic product of the aspergillus niger is utilized to carry out enzymolysis on the chitosan into chitosan oligosaccharide, and the chitosan oligosaccharide is used as an auxiliary carbon source for fermentation, so that the enzymolysis cost of the chitosan is reduced, the addition of the carbon source in the culture process is reduced, and the fermentation cost is reduced.
2. The microbial agent provided by the invention can stimulate the growth of crops and has a growth promoting effect on the crops.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the description of the embodiments or the prior art will be briefly described below, and it will be obvious to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.
FIG. 1 is a graph showing the change of the enzymolysis rate of chitosan added with different concentrations.
FIG. 2 is a graph showing the relationship between glucose change in the culture medium and the chitosan enzymolysis rate.
FIG. 3 shows the effect of chitosan on wheat growth promotion experiments 1.
FIG. 4 shows the effect of chitosan on wheat growth promotion experiments 2.
FIG. 5 shows the effect of chitosan on the experiment of promoting growth of cabbage.
Detailed Description
In order to better understand the technical solutions of the present invention, the following description will clearly and completely describe the technical solutions of the embodiments of the present invention in conjunction with the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
EXAMPLE 1 enzymatic hydrolysis of Chitosan at different concentrations in Aspergillus niger fermentation broth
(1) Seed liquid:
200ml of PDA culture medium is filled in a 1L medicine bottle, and sterilization is carried out for 20min at 121 ℃ for standby;
inoculating Aspergillus niger flat plate stored in laboratory into shake flask under aseptic condition, culturing at 30deg.C and 180 r/min for 4 days, and performing microscopic examination without bacteria; in the seed liquid, the thallus quantity of Aspergillus niger is 8 multiplied by 10 8 Individual spores/mL; the number of the aspergillus niger is CICC2215;
(2) Preparing a fermentation medium:
the fermentation medium comprises 50g of bran, 30g of glucose, 20g of ammonium sulfate and K 2 HPO 4 1g，KCl 0.5g，MgSO 4 ·7H 2 O 0.5g，Na 2 SO 4 0.5g，FeSO 4 ·7H 2 O0.1g, adding water to a constant volume to 1L, and naturally pH;
(3) Fermentation:
inoculating the seed solution into the fermentation culture medium in the step (2) with an inoculum size of 3%, and culturing at 30 ℃ for 72 hours to obtain a fermentation liquid, wherein a large number of mycelia are detected by microscopy; simultaneously carrying out 10 fermentations;
(4) Adding a chitosan solution:
preparing chitosan solution: mixing chitosan and glacial acetic acid in a weight ratio of 1:5 in advance for 2 hours, and dissolving chitosan in glacial acetic acid for later use;
respectively adding chitosan solution into 10 fermentation broths in the step (3), wherein the concentration of chitosan in the fermentation broths is respectively 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% and 5%; and (3) fermenting for 140 hours at 28 ℃, centrifuging for 10 minutes at 5000r/min, sampling, measuring the enzymolysis rate of chitosan, and drawing a graph, wherein the graph is shown in figure 1.
As can be seen from FIG. 1, when the concentration of chitosan added is between 0.5 and 2%, the hydrolysis state of chitosan is best, the decomposed sugar chains are mainly concentrated in 7 to 15 sugars, and the enzymolysis rate of the sugar chains of less than 30 molecules is more than 80%.
Example 2 determination of maximum glucose levels for chitosan substitution by progressively fewer glucose additions without affecting fermentation
(1) Preparation of the culture medium:
(1) the fermentation medium comprises 50g of bran, 30g of glucose, 20g of ammonium sulfate and K 2 HPO 4 1g，KCl 0.5g，MgSO 4 ·7H 2 O 0.5g，Na 2 SO 4 0.5g，FeSO 4 ·7H 2 O0.1g, adding water to a constant volume to 1L, and naturally pH;
(2) the fermentation medium comprises 50g of bran, 28g of glucose, 20g of ammonium sulfate and K 2 HPO 4 1g，KCl 0.5g，MgSO 4 ·7H 2 O 0.5g，Na 2 SO 4 0.5g，FeSO 4 ·7H 2 O0.1g, adding water to a constant volume to 1L, and naturally pH;
(3) the fermentation medium comprises 50g of bran, 26g of glucose, 20g of ammonium sulfate and K 2 HPO 4 1g，KCl 0.5g，MgSO 4 ·7H 2 O 0.5g，Na 2 SO 4 0.5g，FeSO 4 ·7H 2 O0.1g, adding water to a constant volume to 1L, and naturally pH;
(4) the fermentation medium comprises 50g of bran, 24g of glucose, 20g of ammonium sulfate and K 2 HPO 4 1g，KCl 0.5g，MgSO 4 ·7H 2 O 0.5g，Na 2 SO 4 0.5g，FeSO 4 ·7H 2 O0.1g, adding water to a constant volume to 1L, and naturally pH;
(5) the fermentation medium comprises 50g of bran, 22g of glucose, 20g of ammonium sulfate and K 2 HPO 4 1g，KCl0.5g，MgSO 4 ·7H 2 O 0.5g，Na 2 SO 4 0.5g，FeSO 4 ·7H 2 O0.1g, adding water to a constant volume to 1L, and naturally pH;
(6) the fermentation medium comprises 50g of bran, 20g of glucose, 20g of ammonium sulfate and K 2 HPO 4 1g，KCl 0.5g，MgSO 4 ·7H 2 O 0.5g，Na 2 SO 4 0.5g，FeSO 4 ·7H 2 O0.1g, adding water to a constant volume to 1L, and naturally pH;
(7) the fermentation medium comprises 50g of bran, 18g of glucose, 20g of ammonium sulfate and K 2 HPO 4 1g，KCl 0.5g，MgSO 4 ·7H 2 O 0.5g，Na 2 SO 4 0.5g，FeSO 4 ·7H 2 O0.1g, adding water to a constant volume to 1L, and naturally pH;
(8) the fermentation medium comprises 50g of bran, 16g of glucose, 20g of ammonium sulfate and K 2 HPO 4 1g，KCl 0.5g，MgSO 4 ·7H 2 O 0.5g，Na 2 SO 4 0.5g，FeSO 4 ·7H 2 O0.1g, adding water to a constant volume to 1L, and naturally pH;
(9) the fermentation medium comprises 50g of bran, 14g of glucose, 20g of ammonium sulfate and K 2 HPO 4 1g，KCl 0.5g，MgSO 4 ·7H 2 O 0.5g，Na 2 SO 4 0.5g，FeSO 4 ·7H 2 O0.1g, adding water to a constant volume to 1L, and naturally pH;
(2) Fermentation:
inoculating the seed solution of the embodiment 1 into the fermentation medium of the step (1) respectively at an inoculum size of 3%, and culturing at 30 ℃ for 72 hours to obtain a fermentation liquid, wherein a large number of mycelia are detected by microscopy;
(3) Adding chitosan:
and (3) adding 2% chitosan solution into the 9 fermentation liquids obtained in the step (3), continuously fermenting for 140 hours, centrifuging for 10 minutes under the condition of 5000r/min, sampling, and measuring the enzymolysis rate of chitosan (the enzymolysis rate obtained by detecting the molecular weight with the polymerization degree of less than 30 through liquid chromatography and the ratio of the molecular weight to the total addition amount) is shown in figure 2.
As can be seen from FIG. 2, when the glucose in the fermentation medium is reduced from 30g/L to 22g/L, the fermentation period and the mycelium spore amount are not changed significantly; when the glucose in the fermentation medium is less than 20g/L, the chitosan conversion rate is significantly reduced.
Example 3
A preparation method of a microbial agent for fermenting Aspergillus niger and preparing chitosan-containing oligosaccharide by taking chitosan as an auxiliary carbon source comprises the following steps:
200ml of PDA culture medium is filled in a 1L medicine bottle, and sterilization is carried out for 20min at 121 ℃ for standby;
inoculating Aspergillus niger flat plate stored in laboratory into shake flask under aseptic condition, culturing at 30deg.C and 180 r/min for 3 days, and performing microscopic examination without bacteria; in the seed solution, the thallus amount of Aspergillus niger is 7×10 8 Individual spores/mL; the number of the aspergillus niger is CICC2215;
(2) Fermentation:
preparing a fermentation medium, inoculating the seed solution into the fermentation medium with an inoculum size of 3%, and culturing at 30 ℃ for 72 hours to obtain a fermentation solution, and performing microscopic examination;
fermentation medium: 50g of bran, 30g of glucose, 20g of ammonium sulfate and K 2 HPO 4 1g，KCl 0.5g，MgSO 4 ·7H 2 O 0.5g，Na 2 SO 4 0.5g，FeSO 4 ·7H 2 O0.1g, adding water to a constant volume to 1L, and naturally pH;
(3) Bacterial agent:
when a large number of hyphae appear in the microscopic examination, chitosan solution is added into the fermentation liquor, the ratio of the weight of chitosan in the chitosan solution to the weight of the fermentation liquor is 0.5:100, the fermentation is continued for 140 hours at 29 ℃, and the liquid microbial agent is obtained after centrifugation.
Comparative example 1
The difference from example 3 is that no chitosan was added.
Example 4
A preparation method of a microbial agent for fermenting Aspergillus niger and preparing chitosan-containing oligosaccharide by taking chitosan as an auxiliary carbon source comprises the following steps:
200ml of PDA culture medium is filled in a 1L medicine bottle, and sterilization is carried out for 20min at 121 ℃ for standby;
inoculating Aspergillus niger flat plate stored in laboratory into shake flask under aseptic condition, culturing at 30deg.C and 180 r/min for 3 days, and performing microscopic examination without bacteria; in the seed solution, the thallus quantity of Aspergillus niger is 9×10 8 Individual spores/mL; the number of the aspergillus niger is CICC2215;
(2) Fermentation:
preparing a fermentation medium, inoculating the seed solution into the fermentation medium with an inoculum size of 2%, and culturing at 30 ℃ for 70 hours to obtain a fermentation solution, and performing microscopic examination;
fermentation medium: 45g of bran, 22g of glucose, 17g of ammonium sulfate and K 2 HPO 4 0.7g、KCl 0.2g，MgSO 4 ·7H 2 O 0.4g，Na 2 SO 4 0.4g，FeSO 4 ·7H 2 O0.1g, adding water to a constant volume to 1L, and naturally pH;
(3) Bacterial agent:
when a large number of hyphae appear in the microscopic examination, chitosan solution is added into the fermentation liquor, the ratio of the weight of chitosan in the chitosan solution to the weight of the fermentation liquor is 1:100, the fermentation is continued for 120 hours at 30 ℃, and the liquid microbial agent is obtained after centrifugation.
Example 5
A preparation method of a microbial agent for fermenting Aspergillus niger and preparing chitosan-containing oligosaccharide by taking chitosan as an auxiliary carbon source comprises the following steps:
(1) Seed liquid:
200ml of PDA culture medium is filled in a 1L medicine bottle, and sterilization is carried out for 20min at 121 ℃ for standby;
inoculating Aspergillus niger flat plate stored in laboratory into shake flask under aseptic condition, culturing at 30deg.C and 180 r/min for 5 days, and performing microscopic examination without bacteria; in the seed liquid, the thallus quantity of Aspergillus niger is 8 multiplied by 10 8 Individual spores/mL; the number of the aspergillus niger is CICC2215;
(2) Fermentation:
preparing a fermentation medium, inoculating the seed solution into the fermentation medium with an inoculum size of 2%, and culturing at 30 ℃ for 80 hours to obtain a fermentation solution, and performing microscopic examination;
fermentation medium: 55g of bran, 26g of glucose, 22g of ammonium sulfate and K 2 HPO 4 1.5g、KCl 0.6g，MgSO 4 ·7H 2 O 0.7g，Na 2 SO 4 0.8g，FeSO 4 ·7H 2 O0.15 g, adding water to a constant volume to 1L, and naturally pH;
(3) Bacterial agent:
when a large number of hyphae appear in the microscopic examination, chitosan solution is added into the fermentation liquor, the ratio of the weight of chitosan in the chitosan solution to the weight of the fermentation liquor is 1.5:100, the fermentation is continued for 155 hours at 30 ℃, and the liquid microbial agent is obtained after centrifugation.
Example 6 growth-promoting test
1. Material
The microbial agent of example 3;
fermentation broth of comparative example 1.
2. Test treatment
Seed selection: selecting a plurality of seeds of Chinese cabbage (New Beijing No. 3) and wheat (Dan Mai 26) with plump seeds and consistent sizes for standby.
Experiment group a: taking a sterilized plate, paving a piece of filter paper at the bottom, putting 15 grains of wheat seeds, diluting the microbial agent of the embodiment 3 by 5000 times to prepare a diluent, taking 15ml of the diluent, and putting the diluent into the plate;
control group a: the same amount of clear water as experimental group a was used;
experimental group B: taking a sterilized plate, paving a piece of filter paper at the bottom, putting 15 seeds of the Chinese cabbage into the plate, diluting the microbial agent of the embodiment 3 by 5000 times to prepare a diluent, taking 15ml of the diluent, and putting the diluent into the plate;
control group B: the same amount of clear water as experimental group B was used;
the experimental group A, the control group A, the experimental group B and the control group B are all cultivated in a 25-degree illumination incubator, and 5ml of water is supplemented every 2 days.
3. Observation of
After culturing for five days, starting to observe and record growth vigor every day; observing germination and rooting conditions of wheat and Chinese cabbage, wherein the growth conditions of wheat are shown in fig. 3-4, and the root growth results are shown in table 1; the growth condition of the cabbages is shown in fig. 5, and the root growth results are shown in table 2;
TABLE 1 wheat growth promotion test results
Length of upper part of leaf (cm) Root length (cm) Ground dry weight (g) Root system dry weight (g)
Control group 4.8 15.8 0.88 0.63
Experimental group 7.5 33.6 0.51 0.42
Growth rate (%) 66.7％ 112.6％ 72.5％ 51.2％
TABLE 2 results of cabbage growth promotion test
Germination percentage (%) Main root length (cm)
Control group 90.6 3.1
Test group 98.3 4.8
Increment ratio 7.7 54.8
In fig. 3, the left wheat is the experimental group and the right wheat is the control group; in fig. 4, the left wheat is the experimental group and the right wheat is the control group; in fig. 5, the left side chinese cabbage is the experimental group and the right side chinese cabbage is the control group.
As can be seen from fig. 3 to 5 and tables 1 and 2, the microbial agent of example 3 has a remarkable effect on promoting germination and growth as compared with the fermentation broth of comparative example 1.
Although the present invention has been described in detail by way of reference to preferred embodiments, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (8)

1. A microbial agent for fermenting Aspergillus niger by taking chitosan as an auxiliary carbon source and preparing chitosan oligosaccharide is characterized in that the effective viable count of the Aspergillus niger in the microbial agent is 2-20 hundred million/ml, and the chitosan oligosaccharide content is 1-1.6%.
2. The method for preparing the microbial agent containing chitosan oligosaccharide by fermenting aspergillus niger with chitosan as an auxiliary carbon source according to claim 1, which is characterized by comprising the following steps:
(1) Seed liquid:
200ml of PDA culture medium is filled in a 1L medicine bottle, and sterilization is carried out for 20min at 121 ℃ for standby;
inoculating Aspergillus niger flat plate stored in laboratory into shake flask under aseptic condition, culturing at 30deg.C and 180 rpm for 3-5 days, and performing microscopic examination without bacteria; in the seed solution, the thallus amount of Aspergillus niger is 7×10 8 -9×10 8 Individual spores/mL;
(2) Fermentation:
preparing a fermentation medium, inoculating the seed solution into the fermentation medium with an inoculum size of 2-5%, and culturing at 30 ℃ for 70-80 hours to obtain fermentation liquid, and performing microscopic examination;
(3) Bacterial agent:
when a large number of hyphae appear in the microscopic examination, chitosan solution is added into the fermentation liquor, and the ratio of the weight of chitosan in the chitosan solution to the weight of the fermentation liquor is 0.5-2:100, fermenting for 120-155 hours at 28-30 ℃, and centrifuging to obtain the liquid microbial agent.
3. The method for preparing a microbial agent for fermenting Aspergillus niger and preparing chitosan-containing oligosaccharides by using chitosan as an auxiliary carbon source as recited in claim 2, wherein in the step (2), the fermentation medium comprises bran 45-55g, glucose 25-35g, ammonium sulfate 17-22g, K 2 HPO 4 0.7-1.5g、KCl 0.2-0.6g，MgSO 4 ·7H 2 O 0.4-0.7g，Na 2 SO 4 0.4-0.8g，FeSO 4 ·7H 2 O0.1-0.15 g, water is added to fix volume to 1L, and pH is natural.
4. As claimed in claim 3 toThe preparation method of the microbial agent for fermenting Aspergillus niger and preparing chitosan oligosaccharide by taking chitosan as an auxiliary carbon source is characterized in that the fermentation medium comprises 50g of bran, 30g of glucose, 20g of ammonium sulfate and K 2 HPO 4 1g，KCl 0.5g，MgSO 4 ·7H 2 O 0.5g，Na 2 SO 4 0.5g，FeSO 4 ·7H 2 O0.1g, water is added to fix the volume to 1L, and the pH is natural.
5. The method for preparing a microbial agent for fermenting aspergillus niger and preparing chitosan-containing oligosaccharides by using chitosan as an auxiliary carbon source according to claim 2, wherein in the step (3), the chitosan is used after deacetylation.
6. The method for preparing a microbial agent for fermenting aspergillus niger and preparing chitosan-containing oligosaccharides by using chitosan as an auxiliary carbon source as claimed in claim 2, wherein in the step (3), the chitosan is crushed to more than 100 meshes in advance so as to be easily dissolved in glacial acetic acid.
7. The method for preparing the microbial agent for fermenting aspergillus niger and preparing chitosan-containing oligosaccharides by taking chitosan as an auxiliary carbon source as claimed in claim 2, wherein in the step (3), the preparation method of the chitosan solution is as follows: mixing chitosan and glacial acetic acid 2-3 hours before use, wherein the weight ratio of the chitosan to the glacial acetic acid is 0.5-3:100, chitosan is dissolved in glacial acetic acid and then fed into the fermentation broth.
8. The method for preparing a microbial agent for fermenting aspergillus niger and preparing chitosan-containing oligosaccharides by using chitosan as an auxiliary carbon source as claimed in claim 2, wherein in the step (3), the feeding time is between 5 and 8 hours.
CN202211687672.5A 2022-12-27 2022-12-27 Microbial agent for fermenting aspergillus niger and preparing chitosan-containing oligosaccharide by taking chitosan as auxiliary carbon source and preparation method thereof Pending CN116042413A (en)

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