CN111349570A - Method for preparing aspergillus niger seeds and fermentation method of citric acid - Google Patents
Method for preparing aspergillus niger seeds and fermentation method of citric acid Download PDFInfo
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Abstract
The invention relates to the technical field of microbial fermentation, and discloses a method for preparing Aspergillus niger seeds and a fermentation method for citric acid, wherein the culture of the preparation method for the Aspergillus niger seeds comprises a first seed culture and a second seed culture, the second seed culture is a multi-stage seed culture, and the fermentation method for the citric acid comprises the step of inoculating the Aspergillus niger seeds obtained by the first seed culture and the second seed culture into a citric acid fermentation culture medium for fermentation culture, so that the aim of continuously culturing the Aspergillus niger seeds is fulfilled under the condition of not increasing the culture quantity of bran koji, the production quantity of Aspergillus niger bran koji can be effectively reduced, the culture time of a seed tank is shortened, the utilization rate of the seed tank and the quality of the Aspergillus niger seeds are improved, and in addition, the quality and the stability of the citric acid can be further improved in the fermentation process of the citric acid.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a method for preparing aspergillus niger seeds and a fermentation method of citric acid.
Background
In the existing citric acid production process, the fermentation culture process flow is shown in fig. 3, and as the seeding tank and the fermentation tank are in one-to-one correspondence, a large amount of aspergillus niger moldy bran (each bottle of aspergillus niger moldy bran is an independent culture unit) is required to be prepared in the fermentation production process of citric acid, and particularly, the existing citric acid production process has the following defects: (1) the aspergillus niger moldy bran has high labor intensity and long manufacturing time; (2) in the early stage of seeding tank culture, spores are in a water-absorbing expansion period for 6-8 hours, so that the utilization rate of equipment is greatly reduced; (3) each seeding tank belongs to single batch culture, and the quality of the seeding tank is unstable.
Therefore, how to reduce the manufacturing amount of aspergillus niger moldy bran, shorten the culture time of the seed tank, improve the utilization rate of the seed tank and the quality of aspergillus niger seeds is an important problem to be solved urgently in citric acid production.
Disclosure of Invention
The invention aims to solve the problems of large preparation amount of Aspergillus niger mouldy bran, long culture time of a seed tank and low utilization rate and quality of the seed tank in the prior art, and provides a method for preparing Aspergillus niger seeds and a fermentation method of citric acid.
In order to achieve the above objects, the present invention provides, in one aspect, a method for preparing aspergillus niger seeds, comprising the steps of:
(1) inoculating aspergillus niger mouldy bran to an aspergillus niger seed culture medium, performing first seed culture to obtain a first seed culture solution, and performing coccus and hypha breaking and separation on the aspergillus niger in the first seed culture solution to obtain a first hypha and a first mycelial pellet;
(2) inoculating the first hypha obtained in the step (1) into an Aspergillus niger seed culture medium, performing second seed culture to obtain a second seed culture solution, and performing rupture and separation of cocci and hyphae on the Aspergillus niger in the second seed culture solution to obtain a second hypha and a second bacterial ball;
wherein the Aspergillus niger seed comprises a first cocci and/or a second cocci.
In a second aspect, the present invention provides a method for fermenting citric acid, comprising:
(1) preparing Aspergillus niger seeds according to the method;
(2) inoculating the Aspergillus niger seeds prepared in the step (1) into a citric acid fermentation culture medium for fermentation culture.
Through the technical scheme, the continuous culture of the Aspergillus niger seeds is realized under the condition that the culture quantity of the mouldy bran is not increased, the preparation quantity of the Aspergillus niger mouldy bran can be effectively reduced, the culture time of the seed tank is shortened, the utilization rate of the seed tank and the quality of the Aspergillus niger seeds are improved, and in addition, the quality and the stability of the citric acid can be further improved in the fermentation process of the citric acid. For example, the number of Aspergillus niger moldy bran culture bottles in examples 1 to 6 is only 100 (without increasing the amount of moldy bran culture), and the first fermentation, the second fermentation, and the third fermentation can be performed (only the first fermentation can be performed in example 6).
Drawings
FIG. 1 is a schematic of the procedure for preparing Aspergillus niger seeds and fermentation of citric acid in examples 1-5;
FIG. 2 is a schematic of the procedure for preparing Aspergillus niger seeds and citric acid fermentation in example 6;
FIG. 3 is a schematic of the procedure for preparing Aspergillus niger seeds and citric acid fermentation in comparative example 1.
Description of the reference numerals
1 first hypha 2 first hypha 3 second hypha 4 third hypha
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
In a first aspect the present invention provides a method for preparing aspergillus niger seeds, the method comprising the steps of:
(1) inoculating aspergillus niger mouldy bran to an aspergillus niger seed culture medium, performing first seed culture to obtain a first seed culture solution, and performing coccus and hypha breaking and separation on the aspergillus niger in the first seed culture solution to obtain a first hypha and a first mycelial pellet;
(2) inoculating the first hypha obtained in the step (1) into an Aspergillus niger seed culture medium, performing second seed culture to obtain a second seed culture solution, and performing rupture and separation of cocci and hyphae on the Aspergillus niger in the second seed culture solution to obtain a second hypha and a second bacterial ball;
wherein the Aspergillus niger seed comprises a first cocci and/or a second cocci.
In the present invention, the method for preparing aspergillus niger moldy bran is not particularly limited, and may be conventional as long as it can be used for preparing aspergillus niger moldy bran, for example, the method for preparing aspergillus niger moldy bran may include sterilizing a moldy bran medium, inoculating aspergillus niger strain into the sterilized moldy bran medium, and performing moldy bran culture.
According to a particular embodiment of the inventionIn the application mode, the inoculation amount of the aspergillus niger strains is 1 × 10 on the basis of each gram of mouldy bran culture medium5-7×105Aspergillus niger spores, preferably Aspergillus niger strains of 2 × 10/g of bran culture medium5-5×105Aspergillus niger spores, in the present invention, the Aspergillus niger spores are inoculated into the bran koji culture medium in the form of Aspergillus niger bran koji, and the number of the Aspergillus niger spores can be counted by a blood counting chamber under a microscope, specifically, the method is as follows: weighing 5 groups of Aspergillus niger and mouldy bran 1g, adding sterile physiological saline containing 4% of anthracene into each group, enabling spores to be in a free state through a magnetic stirrer and ultrasonic waves, counting the spores under a microscope respectively, and taking the average value of data obtained by the 5 groups.
In the present invention, the composition of the koji medium is not particularly limited, and may be conventional as long as it can be used for the koji medium for aspergillus niger koji culture, and for example, the koji medium may be wheat fiber husk in which the content of wheat fiber is 20 to 35 wt%, the content of water is 45 to 65 wt%, the content of starch is 1.5 to 5 wt%, and the content of protein is 5 to 15 wt%.
In the invention, the culture conditions of aspergillus niger moldy bran are not particularly limited, and the aspergillus niger moldy bran can be used for the existing aspergillus niger moldy bran culture. For example, the conditions for cultivation of koji include: temperature 30-37 deg.C, humidity 45-55%, and ventilation 0.1-0.3 volume: (volume. min.) culture time is 7-15 days; more preferably, the temperature is 32-35 ℃, the humidity is 47-53%, and the ventilation is 0.1-0.2 volume: (volume. min.) and the incubation time was 8 to 10 days.
In the present invention, the term "aeration amount" is generally expressed in terms of an aeration ratio, usually in terms of an air volume ratio per minute (V/V · min) passing through a unit volume of culture solution, for example, an aeration ratio of 1: 0.1-1, short for ventilation volume of 0.01-1 volume: (vol. min.).
According to the invention, before the aspergillus niger moldy bran culture medium is inoculated with the aspergillus niger moldy bran culture medium, the aspergillus niger moldy bran culture medium is sterilized, wherein the conventional method which can be used for sterilizing the moldy bran culture medium can be adopted. For example, the moldy bran medium can be sterilized at a temperature of 121-125 ℃ and a pressure of 0.1-0.15MPa for 45-90 min.
In the invention, the method for preparing aspergillus niger seeds can further comprise the steps of sampling and inspecting aspergillus niger mouldy bran before non-inoculation, inoculating qualified aspergillus niger mouldy bran into a seed tank for seed culture, and sterilizing unqualified aspergillus niger mouldy bran, wherein the qualified aspergillus niger mouldy bran refers to: by shake flask culture, no other infectious microbes are present except for the normal Aspergillus niger. The unqualified Aspergillus niger moldy bran refers to the abnormal shape of the Aspergillus niger obtained by shake flask culture or the existence of other mixed bacteria such as bacillus, saccharomycetes and the like. In the present invention, the sterilization treatment of the rejected aspergillus niger moldy bran may be performed by various methods known to those skilled in the art. For example, sterilization can be carried out at a temperature of 121 ℃ and 125 ℃ and a pressure of 0.1 to 0.15MPa for 45 to 90 min.
In the present invention, the conditions of the first seed culture may include: culturing at 30-40 deg.C for 20-40h, pH of 4-6, pot pressure of 60-90KPa, stirring speed of 70-100rpm, and ventilation air volume of 0.1-0.6 volume: (vol. min.).
According to a preferred embodiment of the present invention, before the first seed culture for 8 to 15 hours, the aeration air volume is 0.1 to 0.2 volume: (v.min), the aeration air volume is 0.2-0.5 v/v after the first seed culture for 8-15 h: (vol. min.).
In the present invention, the conditions for the second seed culture may include: culturing at 30-40 deg.C for 15-30h, pH of 4-6, pot pressure of 60-90KPa, stirring speed of 70-100rpm, and ventilation air volume of 0.1-0.6 volume: (vol. min.).
According to a preferred embodiment of the present invention, the conditions of the second seed culture may include: culturing at 35-40 deg.C for 15-25 hr, pH of 5-6, stirring at 80-90rpm, and ventilating with air flow rate of 0.2-0.5 volume: (vol. min.).
According to a specific embodiment of the present invention, the aspergillus niger seed culture medium may include a starch sugar solution and corn steep liquor, wherein the starch sugar solution has a total sugar content of 10-15%, the starch sugar solution has a content of 90-98% by weight and the corn steep liquor has a content of 2-10% by weight, based on the total weight of the aspergillus niger seed culture medium.
In the present invention, the method for disrupting and separating the cocci and hyphae is not specifically limited, and may be the existing method which can be used for disrupting and separating the cocci and hyphae in the present invention, and the disruption of the cocci and hyphae can be performed on aspergillus niger in the first seed culture solution and/or the second seed culture solution by stirring at a speed of 110-; the first hyphae and the first mushroom ball after being broken can be separated and/or the second hyphae and the second mushroom ball can be separated by centrifugation or filtration.
Further, in the present invention, the stirring speed at the time of disrupting cocci and hyphae may be increased from 70-100rpm to 110-150rpm, and in the present invention, the stirrer for stirring may be at least one of a propeller stirrer, a turbine stirrer and a paddle stirrer, as long as the hyphae and the cocci can be disrupted.
In the present invention, the length of the first hypha and/or the second hypha obtained by the separation may be 10 to 100 micrometers, and particularly, when the length of the first hypha and/or the second hypha is 30 to 50 micrometers, the first hypha and/or the second hypha further contribute to the progress of the second seed culture, thereby contributing to the improvement of the conversion rate of the citric acid.
In the present invention, when the separation is performed by centrifugation, a low-speed desktop centrifuge of model DT5-4, manufactured by Beijing Shibeili centrifuge Co., Ltd, can be used.
According to a specific embodiment of the invention, the separation is performed by means of filtration, the filtration is performed by means of a screen, the screen comprises a first screen, a second screen and a third screen, the aperture of the first screen can be 100-200 microns, the aperture of the second screen can be 50-100 microns, and the aperture of the third screen can be 30-50 microns, the first seed culture solution containing the first hyphae and the first pellet after being fractured (or the second seed culture solution containing the second hyphae and the second pellet after being fractured) passes through the first screen, the second screen and the third screen in sequence, wherein the first hyphae (or the second pellet) is obtained through the first screen, the first hyphae (or the second hyphae) with the length of 50-100 microns is obtained through the second screen, the first hyphae (or the second hyphae) with the length of 30-50 microns is obtained through the third screen, and adding the first bacterium ball (or the second bacterium ball) into the filtered first seed culture solution (or the second seed culture solution) to obtain a first seed culture solution containing the first bacterium ball (or a second seed culture solution containing the second bacterium ball).
In the invention, the inoculation amount of the first hyphae is 2 × 10 on the basis of an Aspergillus niger seed culture medium per milliliter4-1.0×105Root, preferably 4 × 104-7×104In the present invention, the number of hyphae is determined by means of an optical microscope, for example, a computer-based biomicroscope of the type XSP-16C, manufactured by Shanghai-Tian instruments Ltd.
According to the invention, before the first hyphae are inoculated to the Aspergillus niger seed culture medium, the Aspergillus niger seed culture medium needs to be sterilized, wherein the existing method for sterilizing the Aspergillus niger seed culture medium can be adopted. For example, the Aspergillus niger seed culture medium can be sterilized at a temperature of 121 ℃ and 125 ℃ and a pressure of 0.1-0.15MPa for 20-30 min.
In the present invention, the second seed culture in step (2) may be a multi-stage seed culture.
In the present invention, the multistage seed culture may be: and (3) carrying out coccus and hypha breakage and separation on the aspergillus niger in the seed culture solution obtained by the previous-stage seed culture to obtain a previous-stage coccus and a previous-stage hypha, inoculating the previous-stage hypha into an aspergillus niger seed culture medium, and carrying out next-stage seed culture.
According to the invention, before the primary hyphae are inoculated to the Aspergillus niger seed culture medium, the Aspergillus niger seed culture medium needs to be sterilized, wherein the conventional method for sterilizing the Aspergillus niger seed culture medium can be adopted. For example, the Aspergillus niger seed culture medium can be sterilized at a temperature of 121 ℃ and 125 ℃ and a pressure of 0.1-0.15MPa for 20-30 min.
In the present invention, the number of stages of the multistage seed culture is not particularly limited, and for example, three-stage, four-stage, five-stage and above-mentioned stages of continuous culture may be used, and according to a specific embodiment of the present invention, the second seed culture is three-stage culture, and the specific steps may be as follows:
(a) inoculating the first hypha into an Aspergillus niger seed culture medium, performing first-stage seed culture to obtain a first-stage seed culture solution, and performing coccus and hypha breakage and separation on the Aspergillus niger in the first-stage seed culture solution to obtain first-stage hypha and first-stage mycelial pellets;
(b) inoculating the first-stage hyphae into an Aspergillus niger seed culture medium, performing second-stage seed culture to obtain a second-stage seed culture solution, and performing coccus and hyphae breakage and separation on the Aspergillus niger in the second-stage seed culture solution to obtain second-stage hyphae and second-stage hypha balls;
(c) inoculating the second-stage hyphae into an Aspergillus niger seed culture medium, performing third-stage seed culture to obtain a third-stage seed culture solution, and performing coccus and hyphae breakage and separation on the Aspergillus niger in the third-stage seed culture solution to obtain third-stage hyphae and third-stage hypha balls;
the second hypha comprises a first-level hypha, a second-level hypha and a third-level hypha, and the second mushroom ball comprises a first-level mushroom ball, a second-level mushroom ball and a third-level mushroom ball.
In a second aspect, the present invention provides a method for fermenting citric acid, comprising:
(1) preparing Aspergillus niger seeds according to the method;
(2) inoculating the Aspergillus niger seeds prepared in the step (1) into a citric acid fermentation culture medium for fermentation culture.
In the invention, in the step (2), Aspergillus niger seeds are inoculated into the citric acid fermentation culture medium by adding a first culture solution containing the first bacteria balls and a second culture solution containing the second bacteria balls into the citric acid fermentation culture medium, and the inoculation amount of Aspergillus niger spores is 1 × 10 on the basis of per milliliter of the citric acid fermentation culture medium4-5×104And (4) respectively.
In the present invention, the conditions of the fermentation culture may include: the fermentation temperature is 35-45 deg.C, the fermentation time is 50-70h, pH is 3-5, stirring speed is 70-100rpm, and the total volume of fermentation broth in the fermentation tank is used as reference, and ventilation air volume makes the dissolved oxygen value in the fermentation broth 35-60%.
In the invention, before fermentation, the citric acid fermentation medium is sterilized, and the sterilization conditions can comprise: sterilizing at 60-90 deg.C, and keeping the temperature for 20-40 min.
In the present invention, the fermentation medium is not particularly limited, and may be any medium that can be used for citric acid fermentation, for example, the fermentation medium may include a starch sugar solution and corn steep liquor, and the total sugar content of the starch sugar solution is 15 to 25%; based on the total weight of the culture medium of the fermentation tank, the content of the starch sugar solution is 94-99 wt%, and the content of the corn steep liquor is 1-6 wt%.
In the present invention, the fermentation culture can be performed in a plurality of fermentors (as shown in FIG. 1) or in the same fermentor (as shown in FIG. 2), and preferably, the fermentors of the fermentation culture correspond to the seed tanks of the second seed culture one by one.
According to a specific embodiment of the present invention, when the second seed culture is a tertiary culture, the fermentation may comprise the steps of:
(I) inoculating aspergillus niger mouldy bran to an aspergillus niger seed culture medium, performing first seed culture to obtain a first seed culture solution, and performing coccus and hypha breaking and separation on the aspergillus niger in the first seed culture solution to obtain a first hypha and a first mycelial pellet; adding a first seed culture solution containing first bacterium balls into a first fermentation tank for first fermentation;
(II) inoculating the first hypha into an Aspergillus niger seed culture medium, performing first-stage seed culture to obtain a first-stage seed culture solution, and performing coccus and hypha breakage and separation on the Aspergillus niger in the first-stage seed culture solution to obtain first-stage hypha and first-stage mycelial pellets; adding a first-stage seed culture solution containing first-stage bacterium balls into a first-stage fermentation tank for first-stage fermentation;
(III) inoculating the first-stage hyphae into an Aspergillus niger culture medium, performing second-stage seed culture to obtain a second-stage seed culture solution, and performing coccus and hyphae breakage and separation on the Aspergillus niger in the second-stage seed culture solution to obtain second-stage hyphae and second-stage hypha balls; adding a second-stage seed culture solution containing second-stage bacterium balls into a second-stage fermentation tank for second-stage fermentation;
(IV) inoculating the second-stage hyphae into an Aspergillus niger culture medium, performing third-stage seed culture to obtain a third-stage seed culture solution, and performing coccus and hyphae breakage and separation on the Aspergillus niger in the third-stage seed culture solution to obtain third-stage hyphae and third-stage mycelial pellets; and adding a third-stage seed culture solution containing third-stage bacteria balls into a third-stage fermentation tank for third-stage fermentation.
In the step (IV), the aspergillus niger in the third-stage seed culture solution may not be subjected to the rupture and separation of cocci and hyphae, but the third-stage seed culture solution is directly added into a third-stage fermentation tank for third-stage fermentation.
Wherein the second hyphae comprise first-level hyphae, second-level hyphae and third-level hyphae; the second fungus ball includes first order fungus ball, second level fungus ball and third level fungus ball, the fermentation includes first fermentation, first order fermentation, second level fermentation and third level fermentation.
The present invention will be described in detail below by way of examples. In the following examples of the present invention,
the acidity parameter is measured by the method of GB 1987-2007 food additive citric acid;
conversion of citric acid, conversion (%) (concentration of citric acid fermentation broth × volume of citric acid fermentation broth/weight of total sugar) × 100%, wherein the concentration of the obtained citric acid fermentation broth is abbreviated as acidity.
Aspergillus niger was purchased from national standards net and was type Aspergillus niger 98003.
Example 1
This example illustrates the method for preparing Aspergillus niger seeds and the fermentation method of citric acid according to the present invention
This example illustrates the preparation of Aspergillus niger seeds and the citric acid fermentation according to the procedure shown in FIG. 1
(1) Taking 100 bottles of Aspergillus niger moldy bran culture bottles, adding an equal amount of wheat fiber peel into each Aspergillus niger moldy bran culture bottle, and sterilizing the wheat fiber peel at the temperature of 121 ℃ and the pressure of 0.12MPa for 50min to obtain a moldy bran culture medium, wherein the wheat fiber content is 25 wt%, the water content is 55 wt%, the starch content is 4 wt%, the protein content is 10 wt% of the wheat fiber peel, and the balance is impurities;
(2) inoculating Aspergillus niger strain (slant culture) into bran koji culture medium, wherein the inoculating amount of Aspergillus niger strain is 2 × 10 per gram of culture medium5Culturing Aspergillus niger bran at 34 deg.C, humidity of 50%, ventilation rate of 0.15 volume (volume/min), culturing time of 10 days, and turning over every 12 hr to obtain Aspergillus niger bran with spore number of 1.2 × 10 per gram9Spores;
(3) adding starch sugar solution and corn steep liquor into a first seed tank to obtain Aspergillus niger seed culture medium (wherein the content of the starch sugar solution is 96 wt%, the content of the corn steep liquor is 4 wt%, and the total sugar of the starch sugar solution is 12 wt%), sterilizing at 121 ℃ and 0.12MPa for 50min, quickly cooling to 36 ℃, completely inoculating Aspergillus niger mouldy bran (100 bottles of Aspergillus niger mouldy bran are qualified) into the Aspergillus niger seed culture medium in the first seed tank, wherein the inoculation amount of the Aspergillus niger mouldy bran enables the inoculation amount of Aspergillus niger spores in each milliliter of Aspergillus niger seed culture medium to be 2.5 × 105Spore. Then, the first seed culture was carried out for 28 hours under conditions of a stirring rotation speed of 90rpm, a temperature of 36 ℃, a pot pressure of 80KPa, and a pH of 4.5, and the aeration rate for the first 10 hours was 0.1 vol: (v. min), aeration for 10-28h was 0.5 vol: (vol. min.) to obtain a first seed culture;
(4) adjusting the stirring speed to 120rpm, breaking cocci and hyphae in the first seed culture solution, sequentially filtering by using screens (the aperture of the first screen is 100 micrometers, the aperture of the second screen is 50 micrometers, and the aperture of the third screen is 30 micrometers), obtaining first bacterial balls by using the first screen, obtaining first hyphae with the length of 50-100 micrometers by using the second screen, obtaining first hyphae with the length of 30-50 micrometers by using the third screen, and obtaining the first seed culture solution containing the first bacterial balls by using the first bacterial balls and the filtered first seed culture solution;
(5) adding a first seed culture solution containing first bacterium balls into a sterilized first fermentation tank containing a first fermentation culture medium, and performing first fermentation;
(6) subjecting the first mycelium (5 × 10) with length of 30-50 μm obtained in step (4)4Root first hypha) is added into a first-stage seed culture tank, first-stage seed culture is carried out to obtain first-stage seed culture solution, the stirring speed is adjusted to 120rpm, cocci and hyphae in the first-stage seed culture solution are broken, then filtering is carried out in sequence through a screen (the aperture of a first screen is 100 micrometers, the aperture of a second screen is 40 micrometers, and the aperture of a third screen is 30 micrometers), first-stage bacterium balls are obtained through the first screen, first-stage hyphae with the length of 40-100 micrometers are obtained through the second screen, first-stage hyphae with the length of 30-40 micrometers are obtained through the third screen, and the first-stage bacterium balls and the filtered first-stage seed culture solution are mixed to obtain first-stage seed culture solution containing the first-stage bacterium balls;
(7) adding a first-stage seed culture solution containing first-stage bacterium balls into a sterilized first-stage fermentation tank containing a first-stage fermentation culture medium, and performing first-stage fermentation;
(8) adding the first-stage hyphae with the length of 30-40 micrometers obtained in the step (6) into a second-stage seed culture tank, carrying out second-stage seed culture to obtain a second-stage seed culture solution, adjusting the stirring speed to 125rpm, breaking cocci and hyphae in the second-stage seed culture solution, sequentially filtering through a screen (the aperture of a first screen is 100 micrometers, the aperture of a second screen is 45 micrometers, and the aperture of a third screen is 30 micrometers), obtaining second-stage hyphae through the first screen, obtaining second-stage hyphae with the length of 45-100 micrometers through the second screen, obtaining second-stage hyphae with the length of 30-45 micrometers through the third screen, and obtaining the second-stage seed culture solution containing the second-stage hyphae through the second-stage hyphae and the filtered second-stage seed culture solution;
(9) adding a second-stage seed culture solution containing second-stage bacterium balls into a sterilized second-stage fermentation tank containing a second-stage fermentation culture medium, and performing second-stage fermentation;
(10) adding the second-stage hyphae with the length of 30-45 micrometers obtained in the step (8) into a third-stage seed culture tank, carrying out third-stage seed culture to obtain a third-stage seed culture solution, adjusting the stirring speed to 120rpm, breaking cocci and hyphae in the third-stage seed culture solution, sequentially filtering through a screen (the aperture of a first screen is 100 micrometers, the aperture of a second screen is 45 micrometers, and the aperture of a third screen is 30 micrometers), obtaining third-stage hyphae through the first screen, obtaining third-stage hyphae with the length of 45-100 micrometers through the second screen, obtaining third-stage hyphae with the length of 30-45 micrometers through the third screen, and obtaining a third-stage seed culture solution containing the third-stage hyphae through the third screen and the filtered third-stage seed culture solution;
(9) adding a third-stage seed culture solution containing third-stage bacteria balls into a sterilized third-stage fermentation tank containing a third-stage fermentation culture medium, and performing third-stage fermentation;
wherein, the Aspergillus niger seed culture medium of the first-stage seed culture, the second-stage seed culture and the third-stage seed culture is the same as the Aspergillus niger culture medium in the first-stage seed culture, the sterilization condition of the Aspergillus niger culture medium is also the same as the sterilization condition of the Aspergillus niger culture medium in the first-stage seed culture, and the culture time is 20h (the rest culture conditions are the same as the first-stage seed culture conditions);
the first fermentation culture medium, the first-stage fermentation culture medium, the second-stage fermentation culture medium and the third-stage fermentation culture medium respectively comprise starch sugar liquid and corn steep liquor, the total sugar content of the starch sugar liquid is 18 percent (the content of the starch sugar liquid is 95 percent by weight, and the content of the corn steep liquor is 5 percent by weight), and the starch sugar liquid is sterilized at the temperature of 90 ℃ for 20 minutes and then is rapidly cooled to 36 ℃;
the conditions of the first fermentation, the first stage fermentation, the second stage fermentation and the third stage fermentation are as follows: the fermentation temperature is 36 ℃, the fermentation time is 70h, the pH is 4, the stirring speed is 90rpm, and the aeration air quantity enables the dissolved oxygen value in the fermentation broth to be 50% based on the total volume of the fermentation broth in the fermentation tank.
The acidity and the citric acid conversion rate in the first fermenter, the first stage fermenter, the second stage fermenter, and the third stage fermenter were measured, respectively, and the results are shown in table 1.
Example 2
This example illustrates the method for preparing Aspergillus niger seeds and the fermentation method of citric acid according to the present invention
This example illustrates the preparation of Aspergillus niger seeds and the citric acid fermentation according to the procedure shown in FIG. 1
(1) Taking 100 bottles of Aspergillus niger moldy bran culture bottles, adding equal amount of wheat fiber peel into each Aspergillus niger moldy bran culture bottle, and sterilizing the wheat fiber peel at the temperature of 123 ℃ and the pressure of 0.14MPa for 60min to obtain a moldy bran culture medium, wherein the wheat fiber content is 30 wt%, the water content is 55 wt%, the starch content is 2 wt%, the protein content is 7 wt% of the wheat fiber peel, and the balance is impurities;
(2) inoculating Aspergillus niger strain (slant culture) into bran koji culture medium, wherein the inoculating amount of Aspergillus niger strain is 2 × 10 per gram of culture medium5And (4) spores. The culture conditions of the bran koji comprise: temperature 33 ℃, humidity 48%, aeration 0.1 volume: (vol. min.) the incubation time was 8 days and the debranning was performed every 12 hours. Obtaining Aspergillus niger moldy bran per gramThe number of spores contained in the bran koji is 1.5 × 109；
(3) Adding starch sugar solution and corn steep liquor into a first seed tank to obtain an Aspergillus niger seed culture medium (wherein the content of the starch sugar solution is 98 wt%, the content of the corn steep liquor is 2 wt%, and the total sugar of the starch sugar solution is 14 wt%), sterilizing at 123 ℃ and 0.14MPa for 60min, quickly cooling to 38 ℃, completely inoculating Aspergillus niger mouldy bran (100 bottles of Aspergillus niger mouldy bran are qualified) into the Aspergillus niger seed culture medium in the first seed tank, wherein the inoculation amount of the Aspergillus niger mouldy bran enables the inoculation amount of Aspergillus niger spores in each milliliter of the Aspergillus niger seed culture medium to be 2.5 × 105And (4) spores. Then, the first seed culture was carried out for 32 hours under conditions of stirring rotation speed of 70rpm, temperature of 38 ℃, pot pressure of 70KPa, pH of 5, and aeration for the first 12 hours was 0.2 vol: (v.min), aeration for 12-32h was 0.4 vol: (vol. min.) to obtain a first seed culture;
(4) adjusting the stirring speed to 110rpm, breaking cocci and hyphae in the first seed culture solution, sequentially filtering by using screens (the aperture of the first screen is 100 micrometers, the aperture of the second screen is 50 micrometers, and the aperture of the third screen is 30 micrometers), obtaining first bacterial balls by using the first screen, obtaining first hyphae with the length of 50-100 micrometers by using the second screen, obtaining first hyphae with the length of 30-50 micrometers by using the third screen, and obtaining the first seed culture solution containing the first bacterial balls by using the first bacterial balls and the filtered first seed culture solution;
(5) adding a first seed culture solution containing first bacterium balls into a sterilized first fermentation tank containing a first fermentation culture medium, and performing first fermentation;
(6) subjecting the first mycelium (6 × 10) with length of 30-50 μm obtained in step (4)4Root first hypha) is added into a first-stage seed culture tank, first-stage seed culture is carried out to obtain a first-stage seed culture solution, the stirring speed is adjusted to 110rpm, cocci and hypha in the first-stage seed culture solution are broken, and then the first-stage seed culture solution passes through a screen (the aperture of a first screen is 100 microns, the aperture of a second screen is 40 microns, and the aperture of a third screen is 30 microns)Rice), sequentially filtering, obtaining first-stage bacterium balls through a first screen, obtaining first-stage hyphae with the length of 40-100 microns through a second screen, obtaining first-stage hyphae with the length of 30-40 microns through a third screen, and obtaining first-stage seed culture solution containing the first-stage bacterium balls from the first-stage bacterium balls and the filtered first-stage seed culture solution;
(7) adding a first-stage seed culture solution containing first-stage bacterium balls into a sterilized first-stage fermentation tank containing a first-stage fermentation culture medium, and performing first-stage fermentation;
(8) adding the first-stage hyphae with the length of 30-40 micrometers obtained in the step (6) into a second-stage seed culture tank, carrying out second-stage seed culture to obtain a second-stage seed culture solution, adjusting the stirring speed to 110rpm, breaking cocci and hyphae in the second-stage seed culture solution, sequentially filtering through a screen (the aperture of a first screen is 100 micrometers, the aperture of a second screen is 45 micrometers, and the aperture of a third screen is 30 micrometers), obtaining second-stage hyphae through the first screen, obtaining second-stage hyphae with the length of 45-100 micrometers through the second screen, obtaining second-stage hyphae with the length of 30-45 micrometers through the third screen, and obtaining the second-stage seed culture solution containing the second-stage hyphae from the second-stage hyphae balls and the filtered second-stage seed culture solution;
(9) adding a second-stage seed culture solution containing second-stage bacterium balls into a sterilized second-stage fermentation tank containing a second-stage fermentation culture medium, and performing second-stage fermentation;
(10) adding the second-stage hyphae with the length of 30-45 micrometers obtained in the step (8) into a third-stage seed culture tank, carrying out third-stage seed culture to obtain a third-stage seed culture solution, adjusting the stirring speed to 110rpm, breaking cocci and hyphae in the third-stage seed culture solution, sequentially filtering through a screen (the aperture of a first screen is 100 micrometers, the aperture of a second screen is 45 micrometers, and the aperture of a third screen is 30 micrometers), obtaining third-stage hyphae through the first screen, obtaining third-stage hyphae with the length of 45-100 micrometers through the second screen, obtaining third-stage hyphae with the length of 30-45 micrometers through the third screen, and obtaining a third-stage seed culture solution containing the third-stage hyphae through the third screen and the filtered third-stage seed culture solution;
(9) adding a third-stage seed culture solution containing third-stage bacteria balls into a sterilized third-stage fermentation tank containing a third-stage fermentation culture medium, and performing third-stage fermentation;
wherein, the Aspergillus niger seed culture medium of the first-stage seed culture, the second-stage seed culture and the third-stage seed culture is the same as the Aspergillus niger culture medium in the first seed, the sterilization condition of the Aspergillus niger culture medium is also the same as the sterilization condition of the Aspergillus niger culture medium in the first seed, and the culture time is 18h (the other culture conditions are the same as the culture conditions of the first seed);
the first fermentation culture medium, the first-stage fermentation culture medium, the second-stage fermentation culture medium and the third-stage fermentation culture medium respectively comprise starch sugar liquid and corn steep liquor, the total sugar content of the starch sugar liquid is 20 percent (the content of the starch sugar liquid is 97 percent by weight, and the content of the corn steep liquor is 3 percent by weight), and the starch sugar liquid is sterilized at the temperature of 80 ℃ for 25 minutes and then rapidly cooled to 38 ℃;
the conditions of the first fermentation, the first stage fermentation, the second stage fermentation and the third stage fermentation are as follows: the fermentation temperature is 38 ℃, the time is 65h, the pH is 4, the stirring speed is 70rpm, and the aeration air quantity enables the dissolved oxygen value in the fermentation liquid to be 60% based on the total volume of the fermentation liquid in the fermentation tank.
The acidity and the citric acid conversion rate in the first fermenter, the first stage fermenter, the second stage fermenter, and the third stage fermenter were measured, respectively, and the results are shown in table 1.
Example 3
This example illustrates the method for preparing Aspergillus niger seeds and the fermentation method of citric acid according to the present invention
This example illustrates the preparation of Aspergillus niger seeds and the citric acid fermentation according to the procedure shown in FIG. 1
(1) Taking 100 bottles of Aspergillus niger moldy bran culture bottles, adding equal amount of wheat fiber peel into each Aspergillus niger moldy bran culture bottle, and sterilizing the wheat fiber peel at the temperature of 125 ℃ and the pressure of 0.15MPa for 45min to obtain a moldy bran culture medium, wherein the moldy bran culture medium is the wheat fiber peel with the content of 29 wt%, the content of 53 wt% of water, the content of 3 wt% of starch, the content of 9 wt% of protein and the balance of impurities;
(2) inoculating Aspergillus niger strain (slant culture) into bran koji culture medium, wherein the inoculating amount of Aspergillus niger strain is 3.5 × 10 per gram of culture medium5Culturing Aspergillus niger bran at 35 deg.C, 55% humidity, 0.2 vol (v.min) ventilation, 9 days culture time, and turning over every 12 hr to obtain Aspergillus niger bran with spore number of 1.4 × 10 per gram9；
(3) Adding starch sugar solution and corn steep liquor into a first seed tank to obtain Aspergillus niger seed culture medium (wherein the content of the starch sugar solution is 95 wt%, the content of the corn steep liquor is 5 wt%, and the total sugar of the starch sugar solution is 10 wt%), sterilizing at 125 deg.C and 0.15MPa for 45min, rapidly cooling to 40 deg.C, inoculating Aspergillus niger mouldy bran (100 bottles of Aspergillus niger mouldy bran are qualified) into the Aspergillus niger seed culture medium in the first seed tank, wherein the inoculation amount of the Aspergillus niger mouldy bran is such that the inoculation amount of Aspergillus niger spores in each milliliter of Aspergillus niger seed culture medium is 3 × 105And (4) spores. Then, the first seed culture was carried out for 36 hours under conditions of a stirring rotation speed of 80rpm, a temperature of 40 ℃, a pot pressure of 65KPa, and a pH value of 5.5, and the aeration rate for the first 15 hours was 0.15 vol: (v.min), aeration for 15-36h was 0.3 vol: (vol. min.) to obtain a first seed culture;
(4) adjusting the stirring speed to 135rpm, breaking cocci and hyphae in the first seed culture solution, sequentially filtering by using screens (the aperture of the first screen is 100 micrometers, the aperture of the second screen is 50 micrometers, and the aperture of the third screen is 30 micrometers), obtaining first bacterial balls by using the first screen, obtaining first hyphae with the length of 50-100 micrometers by using the second screen, obtaining first hyphae with the length of 30-50 micrometers by using the third screen, and obtaining the first seed culture solution containing the first bacterial balls by using the first bacterial balls and the filtered first seed culture solution;
(5) adding a first seed culture solution containing first bacterium balls into a sterilized first fermentation tank containing a first fermentation culture medium, and performing first fermentation;
(6) subjecting the first mycelium (5.5 × 10) with length of 30-50 μm obtained in step (4)4Root first hypha) is added into a first-stage seed culture tank, first-stage seed culture is carried out to obtain first-stage seed culture solution, the stirring speed is adjusted to 135rpm, cocci and hyphae in the first-stage seed culture solution are broken, then filtering is carried out in sequence through a screen (the aperture of a first screen is 100 micrometers, the aperture of a second screen is 40 micrometers, and the aperture of a third screen is 30 micrometers), first-stage bacteria balls are obtained through the first screen, first-stage hyphae with the length of 40-100 micrometers are obtained through the second screen, first-stage hyphae with the length of 30-40 micrometers are obtained through the third screen, and the first-stage bacteria balls and the filtered first-stage seed culture solution are mixed to obtain first-stage seed culture solution containing the first-stage bacteria balls;
(7) adding a first-stage seed culture solution containing first-stage bacterium balls into a sterilized first-stage fermentation tank containing a first-stage fermentation culture medium, and performing first-stage fermentation;
(8) adding the first-stage hyphae with the length of 30-40 micrometers obtained in the step (6) into a second-stage seed culture tank, carrying out second-stage seed culture to obtain a second-stage seed culture solution, adjusting the stirring speed to 135rpm, breaking cocci and hyphae in the second-stage seed culture solution, sequentially filtering through a screen (the aperture of a first screen is 100 micrometers, the aperture of a second screen is 45 micrometers, and the aperture of a third screen is 30 micrometers), obtaining second-stage hyphae through the first screen, obtaining second-stage hyphae with the length of 45-100 micrometers through the second screen, obtaining second-stage hyphae with the length of 30-45 micrometers through the third screen, and obtaining a second-stage seed culture solution containing second-stage hyphae balls in the second-stage seed culture solution after filtering;
(9) adding a second-stage seed culture solution containing second-stage bacterium balls into a sterilized second-stage fermentation tank containing a second-stage fermentation culture medium, and performing second-stage fermentation;
(10) adding the second-stage hyphae with the length of 30-45 micrometers obtained in the step (8) into a third-stage seed culture tank, carrying out third-stage seed culture to obtain a third-stage seed culture solution, adjusting the stirring speed to 135rpm, breaking cocci and hyphae in the third-stage seed culture solution, sequentially filtering through a screen (the aperture of a first screen is 100 micrometers, the aperture of a second screen is 45 micrometers, and the aperture of a third screen is 30 micrometers), obtaining third-stage hyphae through the first screen, obtaining third-stage hyphae with the length of 45-100 micrometers through the second screen, obtaining third-stage hyphae with the length of 30-45 micrometers through the third screen, and obtaining a third-stage seed culture solution containing the third-stage hyphae through the third screen and the filtered third-stage seed culture solution;
(9) adding a third-stage seed culture solution containing third-stage bacteria balls into a sterilized third-stage fermentation tank containing a third-stage fermentation culture medium, and performing third-stage fermentation;
wherein, the Aspergillus niger seed culture medium of the first-stage seed culture, the second-stage seed culture and the third-stage seed culture is the same as the Aspergillus niger culture medium in the first seed, the sterilization condition of the Aspergillus niger culture medium is also the same as the sterilization condition of the Aspergillus niger culture medium in the first seed, and the culture time is 22h (the other culture conditions are the same as the first seed culture conditions);
the first fermentation culture medium, the first-stage fermentation culture medium, the second-stage fermentation culture medium and the third-stage fermentation culture medium respectively comprise starch sugar liquid and corn steep liquor, the total sugar content of the starch sugar liquid is 23 percent (the content of the starch sugar liquid is 96 percent by weight, and the content of the corn steep liquor is 4 percent by weight), and the starch sugar liquid is sterilized at the temperature of 70 ℃ for 30 minutes and then rapidly cooled to 40 ℃;
the conditions of the first fermentation, the first stage fermentation, the second stage fermentation and the third stage fermentation are as follows: the fermentation temperature is 40 ℃, the time is 60h, the pH is 4, the stirring speed is 80rpm, and the aeration air quantity enables the dissolved oxygen value in the fermentation broth to be 55% based on the total volume of the fermentation broth in the fermentation tank.
The acidity and the citric acid conversion rate in the first fermenter, the first stage fermenter, the second stage fermenter, and the third stage fermenter were measured, respectively, and the results are shown in table 1.
Example 4
This example illustrates the method for preparing Aspergillus niger seeds and the fermentation method of citric acid according to the present invention
This example was carried out to prepare Aspergillus niger seeds and to perform citric acid fermentation according to the procedure shown in FIG. 1. the method of example 1 was followed, except that in step (6), first hyphae having a length of 50-100 μm were added to the first-stage seed culture tank to perform the first-stage seed culture;
in step (8), the first grade hyphae (8 × 10) with the length of 40-100 microns4Root first hypha) is added into a second-stage seed culture tank for second-stage seed culture;
and (10) adding second-level hyphae with the length of 45-100 microns into a third-level seed culture tank for third-level seed culture.
The acidity and the citric acid conversion rate in the first fermenter, the first stage fermenter, the second stage fermenter, and the third stage fermenter were measured, respectively, and the results are shown in table 1.
Example 5
This example illustrates the method for preparing Aspergillus niger seeds and the fermentation method of citric acid according to the present invention
This example of Aspergillus niger seed preparation and citric acid fermentation according to the procedure shown in FIG. 1 the procedure of example 1 was followed, except that the first seed culture was carried out under the following conditions: stirring at 90rpm, 36 ℃, 80KPa pot pressure, 4.5 pH and 0.3 volume of aeration: the seed culture was carried out for 28 hours under the condition of (volume/minute) to obtain a first seed culture solution.
The acidity and the citric acid conversion rate in the first fermenter, the first stage fermenter, the second stage fermenter, and the third stage fermenter were measured, respectively, and the results are shown in table 1.
Example 6
This example illustrates the method for preparing Aspergillus niger seeds and the fermentation method of citric acid according to the present invention
According to the method of example 1, except that the Aspergillus niger seed preparation and citric acid fermentation are performed according to the procedure shown in FIG. 2, a first seed culture solution containing first fungal globules, a second seed culture solution containing second fungal globules and a third seed culture solution containing third fungal globules are added to a fermentation tank for fermentation.
The acidity and citric acid conversion in the fermentor were determined and the results are shown in table 1.
Comparative example 1
This comparative example is illustrative of the prior art method for preparing Aspergillus niger seeds and citric acid fermentation method
The method of example 1 was followed except that the preparation of Aspergillus niger seeds and the citric acid fermentation were carried out according to the scheme shown in FIG. 3, with the following specific steps:
(1) taking 100 bottles of Aspergillus niger moldy bran culture bottles, adding an equal amount of wheat fiber peel into each Aspergillus niger moldy bran culture bottle, and sterilizing the wheat fiber peel at the temperature of 121 ℃ and the pressure of 0.12MPa for 50min to obtain a moldy bran culture medium, wherein the wheat fiber content is 25 wt%, the water content is 55 wt%, the starch content is 4 wt%, the protein content is 10 wt% of the wheat fiber peel, and the balance is impurities;
(2) inoculating Aspergillus niger strain (slant culture) into bran koji culture medium, wherein the inoculating amount of Aspergillus niger strain is 2 × 10 per gram of culture medium5Culturing Aspergillus niger bran at 34 deg.C, humidity of 50%, ventilation rate of 0.15 volume (volume/min), culturing time of 10 days, and turning over every 12 hr to obtain Aspergillus niger bran with spore number of 1.2 × 10 per gram9Spores;
(3) adding starch sugar solution and corn steep liquor into a first seed tank to obtain Aspergillus niger seed culture medium (wherein the starch sugar solution content is 96 wt%, the corn steep liquor content is 4 wt%, and the total sugar content of the starch sugar solution is 12 wt%), and heating at 121 deg.CSterilizing at 0.12MPa for 50min, rapidly cooling to 36 deg.C, and inoculating Aspergillus niger moldy bran (100 bottles of qualified Aspergillus niger moldy bran) into Aspergillus niger seed culture medium in the first seed tank, wherein the inoculation amount of Aspergillus niger moldy bran is 2.5 × 10 Aspergillus niger spores per ml of Aspergillus niger seed culture medium5And (4) respectively. Then, the first seed culture was carried out for 28 hours under conditions of a stirring rotation speed of 90rpm, a temperature of 36 ℃, a pot pressure of 80KPa, and a pH of 4.5, and the aeration rate for the first 10 hours was 0.1 vol: (v. min), aeration for 10-28h was 0.5 vol: (vol. min.) to obtain a first seed culture;
(4) adding the first seed culture solution into a fermentation tank for fermentation culture, wherein the fermentation culture medium comprises starch sugar solution and corn steep liquor, the total sugar content of the starch sugar solution is 18% (the content of the starch sugar solution is 95 wt%, and the content of the corn steep liquor is 5 wt%), sterilizing at 90 deg.C for 30min, and rapidly cooling to 36 deg.C;
the fermentation conditions were: the fermentation temperature is 36 ℃, the fermentation time is 70h, the pH is 4, the stirring speed is 90rpm, and the aeration air quantity enables the dissolved oxygen value in the fermentation broth to be 50% based on the total volume of the fermentation broth in the fermentation tank.
TABLE 1
As can be seen from the data of examples 1-6, comparative example 1 and Table 1, the method for preparing Aspergillus niger seeds and the fermentation method of citric acid according to the present invention can be used to perform the first fermentation, the second fermentation and the third fermentation (only the first fermentation can be performed in example 6) with only 100 Aspergillus niger koji flasks in examples 1-6, while the Aspergillus niger koji flasks in comparative example 1 are also 100, but the first fermentation can be performed only in comparative example 1, the multi-stage fermentation (the first fermentation, the second fermentation and the third fermentation) cannot be performed, and if the fermentation is performed again in comparative example 1, the Aspergillus niger koji should be cultured again, i.e., the continuous culture of Aspergillus niger seeds can be achieved without increasing the amount of Aspergillus oryzae culture in examples 1-6, and the amount of Aspergillus niger koji produced can be effectively reduced, Greatly shortens the culture time of the seeding tank, improves the utilization rate of the seeding tank and the quality of the Aspergillus niger seeds, and can further improve the quality and the stability of the citric acid in the fermentation process of the citric acid (compared with the comparative example 1, the acidity in the first fermentation tank and the conversion rate of the citric acid are both higher).
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (10)
1. A method of preparing aspergillus niger seeds, comprising the steps of:
(1) inoculating aspergillus niger mouldy bran to an aspergillus niger seed culture medium, performing first seed culture to obtain a first seed culture solution, and performing coccus and hypha breaking and separation on the aspergillus niger in the first seed culture solution to obtain a first hypha and a first mycelial pellet;
(2) inoculating the first hypha obtained in the step (1) into an Aspergillus niger seed culture medium, performing second seed culture to obtain a second seed culture solution, and performing rupture and separation of cocci and hyphae on the Aspergillus niger in the second seed culture solution to obtain a second hypha and a second bacterial ball;
wherein the Aspergillus niger seed comprises a first cocci and/or a second cocci.
2. The method of claim 1, wherein the second seed culture in step (2) is a multi-stage seed culture.
3. The method of claim 2, wherein the multi-stage seed culture is: and (3) carrying out coccus and hypha breakage and separation on the aspergillus niger in the seed culture solution obtained by the previous-stage seed culture to obtain a previous-stage coccus and a previous-stage hypha, inoculating the previous-stage hypha into an aspergillus niger seed culture medium, and carrying out next-stage seed culture.
4. The method according to any one of claims 1 to 3, wherein the Aspergillus niger in the first seed culture solution and/or the second seed culture solution is subjected to coccus and hyphal disruption by means of stirring at a speed of 110-150 rpm;
and separating the broken first hypha and the first mushroom ball and/or separating the second hypha and the second mushroom ball in a centrifugal or filtering mode.
5. The method according to claim 4, wherein the length of the separated first hyphae and/or second hyphae is 10-100 microns, preferably 30-50 microns.
6. The method of claim 1, wherein the aspergillus niger moldy bran is inoculated in an amount such that the aspergillus niger spores are inoculated in an amount of 1 × 10 per ml of aspergillus niger seed medium5-5×105Spores, preferably 2 × 105-4×105And (4) spores.
7. The method according to claim 1, wherein the first hyphae are inoculated in an amount of 2 × 10 on a per milliliter basis in an Aspergillus niger seed medium4-1.0×105Root, preferably 4 × 104-7×104And (4) root.
8. The method of claim 1, wherein the first seed culture conditions comprise: culturing at 30-40 deg.C for 20-40h, pH of 4-6, pot pressure of 60-90KPa, stirring speed of 70-100rpm, and ventilation air volume of 0.1-0.6 volume: (volume. min);
the second seed culture conditions include: culturing at 30-40 deg.C for 15-30h, pH of 4-6, pot pressure of 60-90KPa, stirring speed of 70-100rpm, and ventilation air volume of 0.1-0.6 volume: (volume. min);
preferably, the conditions of the second seed culture comprise: culturing at 35-40 deg.C for 15-25h, pH of 5-6, pot pressure of 60-80KPa, stirring speed of 80-90rpm, and ventilation air volume of 0.2-0.5 vol: (vol. min.).
9. A method for fermenting citric acid, which is characterized by comprising the following steps:
(1) preparing aspergillus niger seeds according to the method of any one of claims 1 to 8;
(2) inoculating the Aspergillus niger seeds prepared in the step (1) into a citric acid fermentation culture medium for fermentation culture.
10. The method of claim 1, wherein the conditions of the fermentation culture comprise: the fermentation temperature is 35-45 deg.C, the fermentation time is 50-70h, pH is 3-5, stirring speed is 70-100rpm, and the total volume of fermentation broth in the fermentation tank is used as reference, and ventilation air volume makes the dissolved oxygen value in the fermentation broth 35-60%.
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