CN117925414A - Aspergillus niger and application thereof, and method for preparing citric acid - Google Patents
Aspergillus niger and application thereof, and method for preparing citric acid Download PDFInfo
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- CN117925414A CN117925414A CN202211320532.4A CN202211320532A CN117925414A CN 117925414 A CN117925414 A CN 117925414A CN 202211320532 A CN202211320532 A CN 202211320532A CN 117925414 A CN117925414 A CN 117925414A
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to the field of bioengineering, and discloses Aspergillus niger, application thereof and a method for preparing citric acid. The preservation number of the aspergillus niger is CGMCC No.40040. The black yeast enzyme provided by the invention is used for fermenting to produce citric acid, so that the fermentation efficiency is obviously improved, and the yield and purity of the citric acid are obviously increased; moreover, the spore surface of the aspergillus niger is smooth, the density of mycelium pellets formed in the fermentation process is improved, more shorter branches are more prone to be formed instead of relatively fewer long branches, and therefore the aspergillus niger has higher stress resistance.
Description
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to aspergillus niger, application of aspergillus niger in citric acid production by fermentation and a method for preparing citric acid.
Background
Citric acid is widely used in the food industry, is an important edible organic acid, is an additive for foods such as beverages, candies, cans and the like, and can also be used as a feed additive, a nontoxic detergent, a mordant and the like. Meanwhile, the citric acid can also be used in the industrial fields of medicines, chemical industry, light industry, atomic energy, environmental protection treatment and the like, and is also an indispensable raw material for cosmetics, metal cleaning agents, resins, paperboard, tobacco, electroplating and the like. At present, the citric acid industry mainly adopts corn flour or uses corn flour combined with starchy raw materials such as wheat, sweet potatoes and the like as raw materials to compound an organic or inorganic nitrogen source to prepare a fermentation medium, and uses aspergillus niger as a fermentation strain to ferment and produce the citric acid.
During submerged fermentation of filamentous fungi, the morphological structure of the mycelium can significantly affect the rheological properties of the fermentation broth. When mycelium grows in a spherical shape, the viscosity of the culture solution is lower, the fermentation solution usually shows Newton-type fluid characteristics, the momentum, heat and mass transfer in deep culture are easier, when the mycelium pellet is gradually increased, a nutrition gradient is formed from outside to inside, the diffusion of nutrition components to the center of the pellet is limited, and the production of metabolites of the mycelium pellet inside is reduced; when hypha grows in a filiform way, the viscosity of the fermentation liquid is high, the fermentation liquid is in a non-Newtonian fluid characteristic, the liquid fluidity is poor, the momentum, the heat and the mass are difficult to transfer, the poor heat and mass transfer of a gas phase and a liquid phase are easily caused by insufficient mixing, and finally the production of metabolic products is also influenced. The micro-differences of mycelium pellet micro-morphology can possibly cause obvious differences of macroscopic fermentation behaviors, and the realization of ideal morphology control in the fermentation process is an important way of fermentation regulation.
The advantages and disadvantages of the citric acid strain play an important role in the yield and production cost of the citric acid, the high-yield citric acid fermentation is realized, the cost is reduced, and the domestication and the breeding of the citric acid strain are key. The strain breeding is generally carried out through separation, purification and screening, and finally a strain producing high acid is selected and applied to production. The citric acid strain is used as the basis of the fermentation industry, and the excellent breeding of the citric acid strain can effectively improve the yield and quality of products, and is also an important research and development direction in the field of citric acid fermentation.
Disclosure of Invention
The invention aims to provide a novel Aspergillus niger strain and application thereof, wherein the Aspergillus niger strain has higher density of mycelium pellets formed in the fermentation process, tends to form more short branches, has better stress resistance, and can obtain higher yield and purity of citric acid when being used for preparing the citric acid.
In order to achieve the above purpose, the invention provides an aspergillus niger (Aspergillus niger) with a preservation number of CGMCC No.40040.
In a second aspect, the invention provides the use of Aspergillus niger as described above in the fermentative production of citric acid.
In a third aspect, the present invention provides a method for producing citric acid, comprising inoculating Aspergillus niger having a preservation number of CGMCC No.40040 into a fermentation medium for fermentation.
Compared with the traditional Aspergillus niger strain, the spore surface of the Aspergillus niger disclosed by the invention is smoother, and the spore wall thickness is thinner, so that the novel mycelium is favorably generated, and mycelium pellets are easier to curl, so that the strain is easier to curl and wind into mycelium pellets in the germination process, and the compactness of the mycelium pellets in the later fermentation stage is improved. Meanwhile, as the winding effect of the mycelium is stronger, after the mycelium pellet is formed, the mycelium pellet is not suitable for generating long branches which grow independently but tends to generate shorter branches, so that the length of the branches can be reduced while the number of the branches of the mycelium pellet is increased, the mass transfer efficiency of the mycelium pellet is improved, and meanwhile, the single meristematic mycelium is not easily damaged by the shearing force of a strong flow field, so that the fermentation efficiency and the yield of the citric acid are improved.
The method for producing the citric acid by fermenting the aspergillus niger has the advantages that the fermentation efficiency is obviously improved, the yield of the citric acid is increased, and the impurity content in the fermentation acid production is low, and the citric acid is basically used; in addition, the aspergillus niger provided by the invention can rapidly produce the citric acid by using the starch raw material without using and decomposing the citric acid, so that the aspergillus niger has higher industrial application value.
Preservation of organisms
The strain provided by the invention is classified and named as Aspergillus niger Aspergillus niger, and is preserved in China general microbiological culture Collection center (CGMCC) for 01 and 10 days in 2022, wherein the preservation number is CGMCC No.40040, and the preservation address is North Star Xiyu No. 1 and No. 3 in the Chaiyang area of Beijing city.
Drawings
FIG. 1 shows a scanning electron microscope image of spores of a strain according to the invention.
FIG. 2 shows a microscopic image of mycelium pellets of the strain according to the present invention.
Fig. 3 shows an example graph for calculating the density of aspergillus niger mycelium pellets.
FIG. 4 shows an exemplary graph for calculating the number of branches of Aspergillus niger mycelium pellet.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
The first aspect of the invention provides an aspergillus niger (Aspergillus niger) with the preservation number of CGMCC No.40040.
The aspergillus niger of the invention is obtained by mutation and screening.
In a second aspect, the invention provides the use of Aspergillus niger as described above in the fermentative production of citric acid.
In a third aspect, the present invention provides a method for producing citric acid, comprising inoculating Aspergillus niger having a preservation number of CGMCC No.40040 into a fermentation medium for fermentation.
According to the actual production requirement, the aspergillus niger can be inoculated in the form of aspergillus niger spores and/or aspergillus niger mycelium pellets, and in general, the aspergillus niger spores are often used for culturing to obtain the aspergillus niger mycelium pellets in industrial production, and then the aspergillus niger mycelium pellets are used for fermentation to produce citric acid.
In a preferred embodiment of the present invention, the method for producing aspergillus niger mycelium pellet comprises: inoculating the aspergillus niger spores into a seed culture medium for culture to obtain aspergillus niger mycelium pellets.
According to the present invention, there is no particular requirement for the inoculum size of the spores of Aspergillus niger, but preferably the inoculum size of Aspergillus niger is 2X 10 5-5×105 spores/mL seed medium.
According to the present invention, the Aspergillus niger spores may be prepared by methods conventional in the art, such as activation and seed culture of Aspergillus niger species. The method of activation may comprise inoculating Aspergillus niger into PDA medium and culturing at 33-35 deg.C for 110-130h. The seed culture may comprise inoculating activated Aspergillus niger into bran medium, and culturing at 33-35deg.C for 220-250 hr to obtain Aspergillus niger spores. The PDA medium is typically formulated as: 180-210g/L of potato and 15-25g/L of glucose. The bran culture medium may be: mixtures of bran and rice hulls, bran, mixtures of bran and corncob, mixtures of bran and corn husks, and the like.
According to the present invention, the seed medium may be a conventional medium for culturing spores of Aspergillus niger, preferably the seed medium has a total sugar content of 80-150g/L, a total nitrogen content of 0.5-5g/L, and a pH of 5.5-6.5.
The method for testing the total sugar content is to test the reducing sugar and the non-reducing sugar in the grain by using GB/T5513-2019 grain and oil; the test method of the total nitrogen content is a Kjeldahl nitrogen determination method for measuring crude protein in GB/T6432-2018 feed.
The seed culture medium can contain starch sugar, wherein the starch sugar refers to sugar prepared from starch-containing grains, potatoes and the like by an acid method, an acid enzyme method or an enzyme method, and comprises maltose, glucose, fructose and the like. According to a specific embodiment, the starch sugar may be obtained by liquefying starch in combination with an amylase (such as an alpha-amylase and/or an isoamylase), and in particular, the method for preparing starch sugar may comprise: mixing starch slurry obtained from whole starch with amylase to obtain a mixture, and liquefying the mixture at pH5.2-6 and temperature of 90-105deg.C for 60-90 min to obtain starch liquefied solution, i.e. starch sugar-containing liquid.
In order to obtain a good culture effect, the seed culture medium may further contain a nitrogen source, wherein the nitrogen source may be an organic nitrogen source (such as corn steep liquor) and/or an inorganic nitrogen source (such as urea, ammonium nitrate, etc.), and the obtained seed culture medium may be used for culturing aspergillus niger spores after adjusting the pH to a pH suitable for culturing.
Preferably, the seed medium comprises starch liquefact and corn steep liquor.
Preferably, the starch liquefaction is used in an amount of 94-98 wt% and the corn steep liquor is used in an amount of 2-6 wt% based on the total weight of the seed medium.
According to the present invention, the conditions of the culture are not particularly limited, but preferably, the conditions of the culture include: the temperature is 32-36 ℃. Preferably, the culturing conditions further include: the time is 16-30h.
Mycelium pellets obtained after the aspergillus niger spores are cultured can be inoculated into a fermentation medium for fermentation to produce citric acid.
According to the present invention, the inoculum size of Aspergillus niger mycelium pellets is not particularly limited, but preferably, the inoculum size of Aspergillus niger mycelium pellets is 1X 10 4-1×105 mycelium pellets/mL fermentation medium.
The aspergillus niger of the present invention can be fermented to citric acid in the presence of a relatively high concentration of starch sugar, and thus, according to a preferred embodiment of the present invention, the fermentation medium contains starch sugar. In the present invention, the fermentation medium may be obtained by mixing a starch liquefied solution (carbon source) with a nitrogen source (e.g., corn steep liquor) and then adjusting the pH. Wherein, the starch liquefaction liquid is generally used in an amount of 95-98 wt% and the corn steep liquor is generally used in an amount of 2-5 wt% based on the total weight of the fermentation medium.
According to the present invention, the fermentation medium may be a conventional medium for fermentative production of citric acid, preferably the fermentation medium has a total sugar content of 150-240g/L, a total nitrogen content of 0.5-3g/L, and a pH of 4.5-5.5.
According to the present invention, the conditions of the fermentation are not particularly required, but preferably, the conditions of the fermentation include: the temperature is 35-38 ℃. Preferably, the conditions of the fermentation further comprise: the time is 50-80h.
According to the invention, in the culture and fermentation process, in order to ensure mass transfer and oxygen transfer, mixing and oxygen supply can be performed by adopting a ventilation and stirring mode, and a person skilled in the art can adjust the stirring rotation speed and ventilation amount according to actual conditions, and the description is omitted here.
According to the invention, the method further comprises the step of extracting citric acid from the fermented product. The extraction method can be calcium hydrogen citrate method, specifically comprises neutralizing fermented product with calcium carbonate to obtain calcium citrate precipitate, separating the precipitate calcium citrate, and reacting with sulfuric acid to obtain citric acid.
The present invention will be described in detail by examples.
In the following examples, the control strain Aspergillus niger was the strain used for production.
Sterilizing the culture medium at 121deg.C and 0.1MPa for 30 min; the testing method of the citric acid content (fermentation acid production) refers to GB 1886.235-2016 food safety national standard food additive citric acid; fermentation conversion = total acid yield/total sugar usage x 100%.
Preparing starch liquefaction mother liquor: the starch slurry obtained from whole starch was mixed with alpha-amylase (Novelin) to obtain a mixture, and the mixture was liquefied at pH5.5 and a temperature of 100℃for 80 minutes to obtain a starch liquefied mother liquor, and the total sugar content was 210g/L as measured with reference to GB/T5513-2019. And (5) diluting with water to obtain starch liquefied solutions with different total sugar contents.
Seed culture medium: 96 wt.% starch liquefier (120 g/L total sugar content), 4 wt.% corn steep liquor (75 g/L total nitrogen content), pH of about 6, sugar content of about 120g/L and nitrogen content of about 3g/L in the final seed medium.
Fermentation medium: the pH was adjusted to 5 with 97 wt% starch liquefier (total sugar content 210 g/L), 3wt% corn steep liquor (total nitrogen content 60 g/L), and the sugar content in the final fermentation medium was about 210g/L and the nitrogen content was about 1.8g/L.
Example 1
This example is intended to illustrate the spore morphology of the strain of the invention.
Spores of the strain Aspergillus niger CGMCC No.40040 are taken and the appearance is observed by a scanning electron microscope. As can be seen from the scanning electron microscope, the spore surface of the strain of the invention is smoother (figure 1), and the spore wall is thinner, basically about 600 nm.
Example 2
This example is intended to illustrate the mycelium pellet morphology of the strain of the present invention.
The fermentation broth in the fermentation process of the aspergillus niger CGMCC No.40040 is taken, the form of mycelium pellets is observed under a microscope (as shown in figure 2), the sizes of a projection area and a core area are measured, the number of branches is counted, the length of the branches is measured, and the density is calculated, and the specific results are shown in Table 1. The above results are all average values.
Wherein the morphology of the mycelium pellet of the strain of the present invention is shown in FIG. 2, it can be seen that the mycelium pellet of the strain of the present invention is denser, less branched and shorter than the conventional Aspergillus niger strain, and the shape is better toward regular spheres.
Wherein the density is the proportion of the projected area to the area of the core area (as shown in figure 3); as shown in fig. 4, there is a black marked branch of mycelium pellet.
The density of the strain of the invention is calculated to be 0.77, the average number of branches is 26, and the average branch length is 12.47 mu m. The strain provided by the invention has the advantages that the density and the branch number of the fungus ball are greatly improved, the length of branch hypha is obviously shortened, and the stress resistance of the fungus ball is greatly improved.
Compared with the traditional Aspergillus niger strain, the spore surface of the Aspergillus niger disclosed by the invention is smoother, and the spore wall thickness is thinner, so that the novel mycelium is favorably generated, and mycelium pellets are easier to curl, so that the strain is easier to curl and wind into mycelium pellets in the germination process, and the compactness of the mycelium pellets in the later fermentation stage is improved. Meanwhile, as the winding effect of the mycelium is stronger, after the mycelium pellet is formed, the mycelium pellet is not suitable for generating long branches which grow independently but tends to generate shorter branches, so that the length of the branches can be reduced while the number of the branches of the mycelium pellet is increased, the mass transfer efficiency of the mycelium pellet is improved, and meanwhile, the single meristematic mycelium is not easily damaged by the shearing force of a strong flow field, so that the fermentation efficiency and the yield of the citric acid are improved.
Example 3
This example is intended to illustrate the application of the strain of the invention to the production of citric acid.
Citric acid was produced by fermentation using the control strain and the strain according to the present invention, respectively, according to the following method.
Inoculating Aspergillus niger spores into a seed culture medium for culturing to obtain Aspergillus niger mycelium pellets, wherein the inoculum size of the Aspergillus niger is 3×10 5 spores relative to 1mL of the seed culture medium. The culture temperature was 34℃and the stirring speed during the culture was 100rpm, the aeration rate was 1:0.6vvm, and the culture time was 22 hours.
Inoculating the Aspergillus niger mycelium pellet obtained by culture into a fermentation medium for fermentation to produce citric acid, wherein the inoculating amount of the Aspergillus niger mycelium pellet is 5×10 4 Aspergillus niger mycelium pellets relative to 1mL fermentation medium. In the fermentation process, the fermentation temperature is controlled to be 37 ℃, the stirring speed is 80rpm, the ventilation amount is 1:0.4vvm, the fermentation time is 55h, and the volume of a fermentation tank for fermentation is 450L.
After the fermentation was completed, the content of citric acid in the fermentation acid production and the fermentation products thereof was measured, and the fermentation conversion rate and the purity of citric acid were calculated, and the results are shown in Table 1.
TABLE 1
| Strain | Fermentation acid production (g/L) | Fermentation conversion (%) | Purity of citric acid (%) |
| Control strain | 187 | 104 | 98.85 |
| The strain of the invention | 210 | 108 | 99.99 |
As can be seen from the data in Table 1, the strain of the present invention can produce more citric acid under the same fermentation conditions and fermentation period, has higher fermentation conversion rate, and has higher purity of citric acid in the fermentation product, which indicates that the strain of the present invention has better citric acid fermentation performance.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (10)
1. Aspergillus niger (Aspergillus niger) is characterized in that the collection number of the Aspergillus niger is CGMCC No.40040.
2. Use of aspergillus niger according to claim 1 for the fermentative production of citric acid.
3. A method for preparing citric acid, which is characterized in that the method comprises inoculating Aspergillus niger with the preservation number of CGMCC No.40040 into a fermentation medium for fermentation.
4. A method according to claim 3, wherein the aspergillus niger is inoculated in the form of aspergillus niger spores and/or aspergillus niger mycelium pellets.
5. The method of claim 4, wherein the method for producing the aspergillus niger mycelium pellet comprises the following steps: inoculating the aspergillus niger spores into a seed culture medium for culture to obtain aspergillus niger mycelium pellets.
6. The method of claim 5, wherein the inoculum size of the aspergillus niger spores is 2 x 10 5-5×105 spores/mL seed medium; and/or
The conditions of the culture include: the temperature is 32-36 ℃ and the time is 16-30h.
7. The method of claim 5, wherein the seed medium has a total sugar content of 80-150g/L, a total nitrogen content of 0.5-5g/L, and a pH of 5.5-6.5.
8. The process according to claim 2 or 4, wherein the fermentation medium has a total sugar content of 150-240g/L, a total nitrogen content of 0.5-3g/L and a pH of 4.5-5.5.
9. The method according to claim 4, wherein the inoculum size of the Aspergillus niger mycelium pellet is 1X 10 4-1×105 mycelium pellets/mL fermentation medium; and/or
The conditions of the fermentation include: the temperature is 35-38deg.C, and the time is 50-80h.
10. The method according to any one of claims 3-9, wherein the method further comprises the step of extracting citric acid from the fermented product.
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