CN114214370B - Method for improving organic acid production efficiency of aspergillus - Google Patents
Method for improving organic acid production efficiency of aspergillus Download PDFInfo
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Abstract
The invention discloses a method for improving the efficiency of aspergillus for producing organic acid, which comprises the steps of sterilizing an aspergillus fermentation culture medium, adding waste cordyceps fermentation liquor into the aspergillus fermentation culture medium to obtain an improved aspergillus fermentation culture medium, inoculating 10% of aspergillus seed liquid into the improved aspergillus fermentation culture medium, and carrying out shake culture on a shaking table after fully and uniformly mixing to obtain the organic acid produced by aspergillus. The method adopts a non-genetic engineering method, changes the nutrient composition of the aspergillus fermentation medium by adding the waste cordyceps sinensis fermentation liquid externally, strengthens the intracellular synthesis approach, improves the production intensity of the organic acid, shortens the fermentation period, improves the production efficiency of the organic acid and reduces the fermentation cost. The invention solves the problems of long period and low efficiency of the organic acid production by aspergillus fermentation in the prior art through simple exogenous addition.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for improving efficiency of aspergillus for producing organic acid.
Background
The organic acid refers to an acidic organic compound (excluding amino acids) containing carboxyl groups widely existing in living organisms, and includes malic acid, citric acid, fumaric acid, and the like. The organic acids are mostly involved in the life process of animal and plant metabolism, can be used as metabolic intermediate products or have remarkable biological activity, are important raw materials for organic synthesis and medical product development, have wide application in the fields of food, medicines, chemical industry and the like, and have great market application value.
Most of organic acids are intermediate products in microbial metabolic pathways, and the process for producing the organic acids by fermenting the microorganisms by taking renewable biomass resources as raw materials has great development prospect. The aspergillus is regarded as an ideal microorganism for producing organic acid because the nutrient source required by the fermentation process is simple, and the production cost can be obviously reduced, but the problems of long fermentation period and low production efficiency exist in the production of organic acid by utilizing the fermentation of the aspergillus. The existing method for solving the problem is mainly through strain breeding, but the breeding of organic acid production strains in the prior art is still focused on improving the acid production of the strains, and the breeding method for shortening the fermentation period and improving the production efficiency of the organic acid is provided.
Microbial fermentation is accompanied by the production of a large number of fermentation by-products, such as organic acids, nucleosides, amino acids, proteins, and the like. The common fermentation liquor treatment method is to use the thalli as an organic fertilizer after simple treatment. This reduces the added value of the product, which is a waste of resources. The fermentation liquor generated after fermentation is treated and regenerated by adopting a proper strategy, so that resource waste and environmental pollution can be greatly reduced. If the components of the culture medium can be changed by recycling the waste fermentation liquor, the fermentation period of the strain is shortened, the production efficiency is improved, the production cost of the organic acid fermented by the microorganisms is favorably reduced, and the method has a better application prospect in the industry of producing the organic acid by the microbial fermentation.
Through searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide a method for improving the efficiency of aspergillus for producing organic acid.
The technical scheme adopted by the invention for solving the technical problem is as follows:
a method for improving the efficiency of organic acid production by aspergillus comprises the steps of sterilizing an aspergillus fermentation culture medium, adding waste cordyceps fermentation liquor into the aspergillus fermentation culture medium to obtain an improved aspergillus fermentation culture medium, inoculating 10% of aspergillus seed liquid into the improved aspergillus fermentation culture medium, fully mixing uniformly, and performing shake culture on a shaking table to obtain the organic acid produced by aspergillus.
Further, the aspergillus is aspergillus oryzae or aspergillus niger.
Further, the Aspergillus oryzae is Aspergillus oryzae NRRL 3488 and the Aspergillus niger is Aspergillus niger ATCC 1015.
Further, the aspergillus fermentation medium is as follows: 8-11% of glucose, 0.02-0.04% of metal ions, 2-3% of nitrogen source, and sterilizing at 115 ℃ for 30 min to obtain the product;
wherein the percentage is the final concentration by mass;
the aspergillus seed solution is prepared as follows:
2 to 4 percent of glucose, 0.01 to 0.02 percent of metal ions, 1 to 2 percent of nitrogen source, sterilization at 115 ℃ for 30 min, inoculation of 1.5 multiplied by 10 aspergillus spores6Culturing at 30 deg.C and 200 rpm for 14-20 hr per mL to obtain the final product;
wherein the percentage is the final concentration by mass.
Further, the volume ratio of the waste cordyceps sinensis fermentation liquor to the fermentation medium is as follows: 1-3: 5.
further, the waste cordyceps sinensis fermentation liquor is a mixed liquor of one or more than two of cordyceps militaris fermentation liquor, Xinjiang cordyceps sinensis fermentation liquor and Liangshan cordyceps sinensis fermentation liquor.
Further, the volume ratio of the waste cordyceps sinensis fermentation liquor to the fermentation medium is as follows: 1-3: 5, wherein the waste cordyceps sinensis fermentation liquor is a single cordyceps sinensis fermentation liquor or a mixed liquor of more than two cordyceps sinensis fermentation liquors;
or when the two mixed liquids of the cordyceps militaris fermentation liquid, the Xinjiang cordyceps fermentation liquid and the Liangshan cordyceps fermentation liquid are added at the same time, the addition amount of each cordyceps fermentation liquid is 1/2 of the total volume of the mixed liquids, and when the cordyceps militaris fermentation liquid, the Xinjiang cordyceps fermentation liquid and the Liangshan cordyceps fermentation liquid are added at the same time, the addition amount of each cordyceps fermentation liquid is 1/3 of the total volume of the mixed liquids.
Further, the organic acid is malic acid or citric acid.
Further, the preparation method of the waste cordyceps sinensis fermentation liquor comprises the following steps:
(1) collecting waste liquid generated after cordyceps sinensis is subjected to liquid submerged fermentation and mycelium extraction, filtering by adopting two layers of gauze, and collecting filtrate a;
(2) filtering the filtrate a by adopting a WSW-6 type microbial film filter to obtain a filtrate b;
(3) concentrating the filtrate b to 1/3 of the original volume to obtain concentrated fermentation liquor c;
(4) and (4) filtering the concentrated fermentation liquor c through a filter membrane to obtain the used waste cordyceps sinensis fermentation liquor.
Further, the conditions of the aspergillus fermentation culture are as follows: culturing at 25-35 ℃ and 180-220 r/min for 48-96 h.
The beneficial effects obtained by the invention are as follows:
1. the method of the invention adopts a non-genetic engineering method, changes the nutrient composition of the aspergillus fermentation medium by adding the waste cordyceps sinensis fermentation liquid externally, improves the biosynthesis capability of organic acid, improves the production intensity of the organic acid, shortens the fermentation period, improves the production efficiency of the organic acid and reduces the fermentation cost. The invention solves the problems of long period and low efficiency of the organic acid produced by fermentation of aspergillus in the prior art by simple exogenous addition.
2. The cordyceps sinensis fermentation liquor used in the invention contains a small amount of saccharides, fat, growth factors and rich slow-acting nitrogen sources such as proteins and peptides existing in a macromolecular form, is further degraded into micromolecular peptides and amino acids, and then is absorbed and utilized by microorganisms, and the utilization speed is slow, thus being beneficial to the formation of metabolites. The waste cordyceps sinensis fermentation broth is added into the aspergillus fermentation medium, the proportion of the quick-acting nitrogen source and the slow-acting nitrogen source is regulated, the coordination of the growth period of the thallus and the formation period of the metabolite is controlled, and the aims of shortening the fermentation period and improving the yield are fulfilled.
3. The cordyceps sinensis fermentation liquor used in the invention has the function of inhibiting bacteria within the limit of the adding amount of the cordyceps sinensis fermentation liquor.
4. The method has simple steps, is convenient and feasible, only needs to add a certain proportion of waste cordyceps sinensis fermentation broth into the aspergillus fermentation medium, and is suitable for large-scale green and high-efficiency industrial production of organic acid.
5. The method recycles the waste fermentation liquor, improves the added value of the product, is not limited by seasons, and greatly reduces resource waste and environmental pollution.
Drawings
FIG. 1 is a graph showing the sugar consumption of different waste cordyceps sinensis fermentation liquids added to an Aspergillus oryzae fermentation medium according to the present invention;
FIG. 2 is a graph showing the production intensity of malic acid by adding different waste cordyceps fermentation liquids to an Aspergillus oryzae fermentation medium according to the present invention;
FIG. 3 is a graph showing sugar consumption of fermentation liquid obtained by adding different waste Cordyceps sinensis fermentation liquid into Aspergillus niger fermentation medium;
FIG. 4 is a graph showing the production intensity of citric acid by adding different waste cordyceps sinensis fermentation broth into an Aspergillus niger fermentation medium;
FIG. 5 is a graph showing sugar consumption of Aspergillus oryzae fermentation medium in combination with different waste Cordyceps fermentation broth;
FIG. 6 is a graph showing the intensity of malic acid production by the combined action of different waste cordyceps fermentation broth in an Aspergillus oryzae fermentation medium according to the present invention;
FIG. 7 is a graph showing sugar consumption of Aspergillus niger fermentation medium in combination with different fermentation liquids of waste Cordyceps sinensis;
FIG. 8 is a graph showing the production intensity of citric acid in the Aspergillus niger fermentation medium under the combined action of different waste Cordyceps fermentation broth.
Detailed Description
The present invention will be further described in detail with reference to examples for better understanding, but the scope of the present invention is not limited to the examples.
The raw materials used in the invention are all conventional commercial products if not specified, the method used in the invention is all conventional in the field if not specified, and the mass of each substance used in the invention is all conventional use mass.
A method for improving the efficiency of organic acid production by aspergillus comprises the steps of sterilizing an aspergillus fermentation culture medium, adding waste cordyceps fermentation liquor into the aspergillus fermentation culture medium to obtain an improved aspergillus fermentation culture medium, inoculating 10% of aspergillus seed liquid into the improved aspergillus fermentation culture medium, fully mixing uniformly, and performing shake culture on a shaking table to obtain the organic acid produced by aspergillus.
Preferably, the aspergillus is aspergillus oryzae or aspergillus niger.
Preferably, the Aspergillus oryzae is Aspergillus oryzae NRRL 3488 and the Aspergillus niger is Aspergillus niger ATCC 1015.
Preferably, the aspergillus fermentation medium is as follows: 8-11% of glucose, 0.02-0.04% of metal ions, 2-3% of nitrogen source, and sterilizing at 115 ℃ for 30 min to obtain the product;
wherein the percentage is the final concentration by mass;
the aspergillus seed solution is prepared as follows:
2 to 4 percent of glucose, 0.01 to 0.02 percent of metal ions, 1 to 2 percent of nitrogen source, sterilization at 115 ℃ for 30 min, inoculation of 1.5 multiplied by 10 aspergillus spores6Culturing at 30 deg.C and 200 rpm for 14-20 hr per mL to obtain the final product;
wherein the percentage is the final concentration by mass.
Preferably, the volume ratio of the waste cordyceps sinensis fermentation liquid to the fermentation culture medium is as follows: 1-3: 5.
preferably, the waste cordyceps sinensis fermentation liquid is one or a mixture of more than two of cordyceps militaris fermentation liquid, Xinjiang cordyceps sinensis fermentation liquid and Liangshan cordyceps sinensis fermentation liquid.
Preferably, the volume ratio of the waste cordyceps sinensis fermentation liquid to the fermentation culture medium is as follows: 1-3: 5, wherein the waste cordyceps sinensis fermentation liquor is a single cordyceps sinensis fermentation liquor or a mixed liquor of more than two cordyceps sinensis fermentation liquors;
or when the two mixed liquids of the cordyceps militaris fermentation liquid, the Xinjiang cordyceps fermentation liquid and the Liangshan cordyceps fermentation liquid are added at the same time, the addition amount of each cordyceps fermentation liquid is 1/2 of the total volume of the mixed liquids, and when the cordyceps militaris fermentation liquid, the Xinjiang cordyceps fermentation liquid and the Liangshan cordyceps fermentation liquid are added at the same time, the addition amount of each cordyceps fermentation liquid is 1/3 of the total volume of the mixed liquids.
Preferably, the organic acid is malic acid or citric acid.
Preferably, the preparation method of the waste cordyceps sinensis fermentation liquor comprises the following steps:
(1) collecting waste liquid generated after cordyceps sinensis is subjected to liquid submerged fermentation and mycelium extraction, filtering by adopting two layers of gauze, and collecting filtrate a;
(2) filtering the filtrate a by adopting a WSW-6 type microbial film filter to obtain a filtrate b;
(3) concentrating the filtrate b to 1/3 of the original volume to obtain concentrated fermentation liquor c;
(4) and (4) filtering the concentrated fermentation liquor c through a filter membrane to obtain the used waste cordyceps sinensis fermentation liquor.
Preferably, the conditions of the aspergillus fermentation culture are as follows: culturing at 25-35 ℃ and 180-220 r/min for 48-96 h.
Specifically, the preparation and detection are as follows:
the culture medium preparation and the method for measuring the residual sugar and organic acid content of the fermentation liquor can be as follows:
(1) preparation of a culture medium:
preparing a seed culture medium: 2-4% of glucose, 0.01-0.02% of metal ions, 1-2% of nitrogen source, sterilizing at 115 ℃ for 30 min, inoculating 1.5 multiplied by 10 spores6Culturing at 30 deg.C and 200 rpm for 14-20 hr per mL.
Fermentation medium: 8-11% of glucose, 0.02-0.04% of metal ions and 2-3% of nitrogen source, sterilizing at 115 ℃ for 30 min, adding a certain volume of waste cordyceps sinensis fermentation broth into the fermentation medium to obtain an improved fermentation medium, wherein the volume of the waste cordyceps sinensis fermentation broth is marked as P1 and P2, inoculating the improved fermentation medium into seed liquid 10%, and the fermentation condition is that the temperature is 30-35 ℃, the stirring speed is 200 rpm, so that the fermentation is stopped when the residual sugar content of the culture media P1 and P2 reaches single digit, and the organic acid fermentation broth is obtained.
(2) And (3) measuring the glucose concentration in the fermentation liquor: taking a proper amount of fermentation liquor, centrifuging for 5 min at 12000 Xg, diluting the supernatant by 100 times with deionized water, and measuring by using an SBA-40E biosensor analyzer.
(3) Measuring the content of organic acid in the fermentation liquor: fully and uniformly mixing the fermentation liquor, quickly taking 2 mL of the fermentation liquor in a 10 mL EP tube, and slowly adding HCl with the same volume and the concentration of 2M for full acidolysis; then centrifuging at 12000 Xg for 5 min, taking supernatant, diluting by 20-50 times, and filtering with 0.22 μm water system filter membrane. The High Performance Liquid Chromatography (HPLC) ultraviolet detection method is carried out by using a Berkele Aminex HPX-87H chromatographic column with a mobile phase of 5 mM dilute sulfuric acid, setting the column temperature at 65 ℃, the flow rate of the mobile phase at 0.8 mL/min and the wavelength at 210 nm.
Example 1 method for improving malic acid production efficiency by Aspergillus by adding waste Cordyceps fermentation broth
The operation steps of a plurality of experimental groups are basically the same, the difference is only that the types and the dosage of the waste cordyceps sinensis fermentation liquor are different, and the details are shown in a table 1:
recording the time of fermentation culture (time from residual sugar amount to single digit) in the experimental groups 1-3, detecting the change of glucose content in the fermentation culture process, and calculating the production efficiency of the organic acid.
No addition group a: the group is a control group of experiment groups 1-3, and organic acid is produced by fermentation by the same method as the experiment groups, except that waste cordyceps sinensis fermentation liquor is not added into a fermentation culture medium.
The production efficiency of the organic acid of the experimental groups 1-3 and the control group A is compared to verify the improvement effect of the exogenous addition of the waste cordyceps fermentation liquor on the production efficiency of the organic acid of the aspergillus. The results of the control group A and the experimental groups 1 to 3 are shown in FIG. 1 and FIG. 2.
As shown in fig. 1 and fig. 2, the fermentation time of the non-additive control group a is 96 hours, the fermentation time of the experimental groups 1-3 is 60 hours, 48 hours and 60 hours, respectively, after the waste cordyceps sinensis fermentation liquid is exogenously added to the fermentation medium, the fermentation time is remarkably shortened and the production efficiency is remarkably improved compared with the non-additive control group; wherein, the experiment group 1 is improved by 67.86%, the experiment group 2 is improved by 110.71%, and the experiment group 3 is improved by 64.29%.
In order to verify the superposition synergistic effect among the experimental groups 1-3, the effect of the superposition waste cordyceps sinensis fermentation liquor on the improvement of the organic acid production efficiency of aspergillus is verified according to the addition of 50% of the waste cordyceps sinensis fermentation liquor. Among them, the results of the control group C and the experimental groups 1+2, 1+3, 2+3, 1+2+3 are shown in fig. 5 and fig. 6.
As shown in fig. 5 and 6, the fermentation time of the control group C without addition is 96h, and the fermentation times of the experimental groups 1+2, 1+3, 2+3, 1+2+3 are 60 h, 48 h and 36h, respectively, after the fermentation culture medium is overlapped with the waste cordyceps sinensis fermentation liquid, compared with the control group without addition and the single addition group, the fermentation time is shortened, and the production efficiency is significantly improved; wherein, the experimental groups 1+2, 1+3, 2+3 and 1+2+3 are respectively improved by 78.57%, 103.57%, 96.43% and 132.14%.
Embodiment 2 method for improving citric acid production efficiency of aspergillus by adding waste cordyceps sinensis fermentation liquor externally
The operation steps of a plurality of experimental groups are basically the same, the difference is only that the types and the dosage of the waste cordyceps sinensis fermentation liquor are different, and the details are shown in a table 2:
recording the time of fermentation culture (the time from residual sugar amount to single digit) in the specific experimental groups 4-6, detecting the change of the glucose content in the fermentation culture process, and calculating the production efficiency of the organic acid.
No additive group B: the group is a control group of experiment groups 4-6, and organic acid is produced by fermentation by the same method as the experiment groups, except that waste cordyceps sinensis fermentation liquor is not added into a fermentation culture medium.
The production efficiency of the organic acid of the experimental groups 4 to 6 and the control group B is compared to verify the improvement effect of the exogenously added waste cordyceps sinensis fermentation liquor on the production efficiency of the organic acid of aspergillus. The results of control group B and examples 4 to 6 are shown in FIG. 3 and FIG. 4.
As shown in FIGS. 3 and 4, the fermentation time of the control group was 96 hours, and the fermentation times of examples 4 to 6 were 60 hours, 72 hours and 72 hours, respectively. After the fermentation culture medium is added with the waste cordyceps sinensis fermentation liquid externally, compared with a control group, the fermentation time is obviously shortened, and the production efficiency is obviously improved; wherein, the improvement of example 4 is 79.31%, the improvement of example 5 is 51.72%, and the improvement of example 6 is 62.07%.
In order to verify the synergistic effect of the experimental groups 4-6, the waste cordyceps sinensis fermentation liquor is added according to 50% of the addition amount of the waste cordyceps sinensis fermentation liquor, and the effect of the waste cordyceps sinensis fermentation liquor on improving the organic acid production efficiency of aspergillus is verified. Among them, the results of the control group D and the experimental groups 4+5, 4+6, 5+6, 4+5+6 are shown in fig. 7 and fig. 8.
As shown in fig. 7 and 8, the fermentation time of the non-additive control group D is 96 hours, and the fermentation times of the experimental groups 4+5, 4+6, 5+6, 4+5+6 are 60 hours, 48 hours and 36 hours, respectively, after the waste cordyceps sinensis fermentation liquid is added to the fermentation medium in a superposed manner, compared with the non-additive control group and the single additive group, the fermentation time is shortened, and the production efficiency is significantly improved; among them, the experimental groups 4+5, 4+6, 5+6 and 4+5+6 are improved by 96.4%, 89.29%, 85.71% and 107.14%. Therefore, the result of the superposition of the waste cordyceps sinensis fermentation liquid is obviously superior to the result of the single use of each combination, and the substance superposition has a synergistic effect.
The result shows that the efficiency of producing organic acid by aspergillus can be obviously improved by adding the waste cordyceps sinensis fermentation liquor through external sources, and the additive effect is better than that of single addition.
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, alterations and modifications are possible without departing from the spirit and scope of this disclosure and appended claims, and accordingly, the scope of this disclosure is not limited to the embodiments disclosed.
Claims (1)
1. A method for improving the efficiency of organic acid production by aspergillus is characterized in that: sterilizing an aspergillus fermentation culture medium, adding waste cordyceps sinensis fermentation liquid into the aspergillus fermentation culture medium to obtain an improved aspergillus fermentation culture medium, inoculating 10% of aspergillus seed liquid into the improved aspergillus fermentation culture medium, fully and uniformly mixing, and performing shake culture on a shaking table to obtain organic acid produced by aspergillus;
the aspergillus is aspergillus oryzae or aspergillus niger;
the Aspergillus oryzae is Aspergillus oryzae NRRL 3488, and the Aspergillus niger is Aspergillus niger ATCC 1015;
the aspergillus fermentation medium comprises: 8-11% of glucose, 0.02-0.04% of metal ions, 2-3% of nitrogen source, and sterilizing at 115 ℃ for 30 min to obtain the product;
wherein the percentage is the final concentration by mass;
the aspergillus seed solution is prepared as follows:
2-4% of glucose, 0.01-0.02% of metal ions, 1-2% of nitrogen source, sterilizing at 115 ℃ for 30 min, inoculating 1.5 × 10 Aspergillus spores6Culturing at 30 deg.C and 200 rpm for 14-20 hr per mL to obtain the final product;
wherein the percentage is the final concentration by mass;
the waste cordyceps fermentation liquor is one or more of cordyceps militaris fermentation liquor, Xinjiang cordyceps fermentation liquor and Liangshan cordyceps fermentation liquor;
the volume ratio of the waste cordyceps sinensis fermentation liquor to the fermentation culture medium is 1-3: 5;
when the two mixed liquids of the cordyceps militaris fermentation liquid, the Xinjiang cordyceps fermentation liquid and the Liangshan cordyceps fermentation liquid are added at the same time, the addition amount of each cordyceps fermentation liquid is 1/2 of the total volume of the mixed liquids, and when the cordyceps militaris fermentation liquid, the Xinjiang cordyceps fermentation liquid and the Liangshan cordyceps fermentation liquid are added at the same time, the addition amount of each cordyceps fermentation liquid is 1/3 of the total volume of the mixed liquids;
the organic acid is malic acid and citric acid;
the fermentation culture conditions of the aspergillus are as follows: culturing at 25-35 ℃ and 180-220 r/min for 36-96 h;
the preparation method of the waste cordyceps sinensis fermentation liquor comprises the following steps:
(1) collecting waste liquid generated after cordyceps sinensis is subjected to liquid submerged fermentation and hypha extraction, filtering by adopting two layers of gauze, and collecting filtrate a;
(2) filtering the filtrate a by adopting a WSW-6 type microbial film filter to obtain a filtrate b;
(3) concentrating the filtrate b to 1/3 to obtain concentrated fermentation liquor c;
(4) and (4) filtering the concentrated fermentation liquor c through a filter membrane to obtain the used waste cordyceps sinensis fermentation liquor.
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