Aspergillus niger seeds, methods for their production, and methods for the production of citric acid by aspergillus niger fermentation

Abstract

The present application relates to the field of citric acid fermentation, and discloses an aspergillus niger seed, a preparation method thereof and a method for fermenting citric acid by using aspergillus niger. The preparation method comprises the following steps: inoculating aspergillus niger spores into a seed culture medium, and culturing in the presence of a porous carrier to obtain the aspergillus niger seed; wherein the pore size of the porous carrier is 200-500 PPI. The aspergillus niger seed prepared by the method enables aspergillus niger to ferment in the form of mycelium during fermentation, which can reduce the inoculation amount, improve the acid production efficiency, and reduce energy consumption.

Description

Technical Field

[0001] This invention relates to the field of citric acid fermentation, specifically to a seed of Aspergillus niger, a method for preparing the seed, and a method for producing citric acid by fermenting Aspergillus niger. Background Technology

[0002] Currently, in the citric acid industry, the fermentation process for producing citric acid involves: Aspergillus niger spores are inoculated into the seed culture medium in a seed tank, where they germinate and grow into mycelial balls. These mycelial balls are then inoculated into the fermentation tank culture medium to synthesize and produce citric acid. However, if the mycelium is introduced into the fermentation tank in the form of mycelial balls, the mycelium is easily broken into fragments due to the shearing force from stirring, causing the fermentation broth to thicken and hindering acid production.

[0003] However, the small contact area between the mycelial balls and the nutrients and oxygen in the culture medium affects heat and mass transfer within the mycelial balls, often resulting in an anaerobic state and significantly reducing the efficiency of acid production by the mycelium. Since mass and heat transfer within the mycelial balls is restricted after formation, high ventilation and stirring speeds are required to enhance oxygen supply. However, excessively high stirring speeds can damage the mycelial balls, and high ventilation leads to high energy consumption. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems existing in the prior art and provide a method for preparing Aspergillus niger seeds and a method for fermenting Aspergillus niger to produce citric acid. This method enables Aspergillus niger to ferment in the form of mycelium during the fermentation process and can improve the acid production efficiency.

[0005] To achieve the above objectives, the present invention provides a method for preparing Aspergillus niger seeds, the method comprising: inoculating Aspergillus niger spores into a seed culture medium and culturing them in the presence of a porous carrier to obtain Aspergillus niger seeds;

[0006] The porous carrier has a pore size of 200-500 PPI.

[0007] Preferably, the amount of the porous carrier added is 5-10 g / L of seed culture medium.

[0008] A second aspect of the present invention provides Aspergillus niger seeds prepared by the method described above.

[0009] A third aspect of the present invention provides a method for producing citric acid by fermentation of Aspergillus niger, the method comprising inoculating Aspergillus niger seeds prepared by the method described above into a fermentation medium for fermentation to produce citric acid.

[0010] In this invention, a porous carrier is added to the seed culture medium, allowing the inoculated Aspergillus niger spores to be adsorbed onto the porous carrier for cultivation. The spores germinate and produce hyphae, at least part of which are contained within the pores of the porous carrier and protected from breakage, thus providing excellent seeds for subsequent fermentation.

[0011] The Aspergillus niger seeds prepared by the method of this invention and loaded onto a porous carrier are inoculated into a fermentation medium, which greatly avoids the problem of mycelia entangled into mycelial balls, thus reducing the mass and heat transfer efficiency. At the same time, the mycelia can fully contact the fermentation medium, which can greatly improve the acid production efficiency while reducing the inoculum amount, and reduce the stirring speed and ventilation volume, thus greatly reducing energy consumption. Detailed Implementation

[0012] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0013] The first aspect of the present invention provides a method for preparing Aspergillus niger seeds, the method comprising: inoculating Aspergillus niger spores into a seed culture medium and culturing them in the presence of a porous carrier to obtain Aspergillus niger seeds;

[0014] The porous carrier has a pore size of 200-500 PPI (for example, it can be 200, 250, 300, 350, 400, 450, 500 PPI or any range between any two values).

[0015] This invention involves inoculating Aspergillus niger spores into a seed culture medium containing a porous carrier, allowing the spores to adhere to the porous carrier and germinate into mycelia, thus avoiding the formation of mycelial balls. At the same time, it can also minimize the risk of the generated mycelia being sheared and broken into small segments during the culture process.

[0016] In this invention, PPI stands for Pore Density per Unit, which represents the average number of pores per unit inch of length. For example, 10 PPI means that there are an average of 10 pores per unit inch of length on the filter screen.

[0017] In this invention, preferably, the porous carrier has a pore size of 250-450 PPI. In this preferred embodiment, the effect of Aspergillus niger fermentation to produce citric acid can be further improved.

[0018] In this invention, the volume of a single porous carrier can be selected within a wide range; preferably, the volume of a single porous carrier is less than 4 cm³. 3 More preferably less than 2cm 3 .

[0019] The porous carrier can be any shape, such as a sphere or a polyhedron (preferably a regular polyhedron or other polyhedrons that are close to a regular polyhedron or a sphere).

[0020] In this invention, preferably, the diameter or side length of a single porous carrier is 1-2 cm.

[0021] The material used to prepare the porous carrier can be any material conventionally available in the art for preparing porous carriers. Preferably, the porous carrier material includes at least one of polyurethane, nano-silicon carbide, and propyl silica gel.

[0022] The amount of porous carrier added can be selected within a wide range. Preferably, the amount of porous carrier added is 5-10 g / L seed culture medium, more preferably 6-9 g / L seed culture medium, and even more preferably 6-8 g / L seed culture medium.

[0023] In this invention, the method for preparing Aspergillus niger spores can be a conventional method in the art, such as using wheat bran for the preparation of Aspergillus niger spores, as detailed in CN 112175837 A.

[0024] The inoculation amount of Aspergillus niger spores can be selected within a wide range. Preferably, the inoculation amount of Aspergillus niger spores is such that the content of Aspergillus niger spores in the seed culture medium after inoculation is 1×10⁻⁶. 5 -1×10 6 cfu / mL.

[0025] The method described in this invention can reduce the inoculum amount of Aspergillus niger spores by more than half.

[0026] The number of spores can be determined using methods known in the art. For example, the number of spores in Aspergillus niger bran koji, Aspergillus niger spore suspension, and Aspergillus niger seed liquid can all be determined using the hemocytocyte plate count method. Specifically, the method is as follows: 1g of Aspergillus niger bran koji is weighed into 5 groups, and 4% (v / v) sterile physiological saline is added to each group. The spores are then released into a free state using a magnetic stirrer and ultrasound. Spores are counted under a microscope, and the average value of the data obtained from the 5 groups is taken.

[0027] It should be understood that spores are often inoculated in the form of spore suspensions, and the amount of spores added and the spore content in the spore suspension can be controlled to determine the inoculation amount.

[0028] In this invention, the obtained spores are inoculated into a seed culture medium for cultivation. There are no particular limitations on the seed culture medium, which can be a conventional seed culture medium in the art, such as a starch raw material enzymatic hydrolysis liquefaction liquid diluted with water to a total sugar content of 5-20% by weight to obtain a seed culture solution.

[0029] In a preferred embodiment of the present invention, the seed culture medium comprises an enzymatic hydrolysis liquefaction solution of starchy raw material, ammonium sulfate, and dipotassium hydrogen phosphate. Preferably, the pH of the seed culture medium is 5-6.

[0030] In this invention, the content of each component in the seed culture medium can be selected within a wide range. Specifically, the total sugar content in the seed culture medium can be 5-20% by weight. Based on the dry weight of the total sugar, the content of ammonium sulfate can be 0.6-1.0% by weight, and the content of dipotassium hydrogen phosphate can be 0.2-0.5% by weight.

[0031] In this invention, the method for determining the total sugar content can be any method known in the art, preferably Fehling's method, referring to the national standard GB / T 50099-2008.

[0032] In this invention, the starchy raw material can be one or more of the starch-rich raw materials commonly used in the art, such as corn, wheat, sweet potato, potato, and rice, with corn being preferred.

[0033] In this invention, there are no particular limitations on the conditions for inoculating Aspergillus niger spores into the seed solution for cultivation. The conditions can be those commonly used in the art, such as: a cultivation temperature of 33-40°C and an initial pH of 5-6.

[0034] The culture time can be selected within a wide range, preferably 20-22 hours.

[0035] In this invention, the stirring speed is always based on a fermentation tank volume of 2m³. 3 For example, preferably, during the cultivation process, the stirring speed is controlled to be no higher than 90 rpm, and more preferably 70-80 rpm. Under these preferred conditions, the oxygen supply requirements can be met, while also promoting mycelial growth and maintaining a relatively loose state within the carrier, reducing the damage to the carrier and the mycelia attached to the carrier caused by the stirring shear force. It should be understood that when the volume of the fermenter is smaller, its stirring speed will be increased accordingly.

[0036] Preferably, during the cultivation process, the ventilation volume is controlled at 0.12-0.15 vvm. Under this preferred condition, the oxygen supply requirement can be met, energy consumption can be reduced, and at the same time, this ventilation condition is also conducive to the growth of Aspergillus niger and the formation of loose mycelia.

[0037] The term "ventilation rate" is usually expressed as the ratio of air volume passing through a unit volume of culture medium per minute (V / V·min or vvm). For example, an ventilation ratio of 1:0.1-1 is simply referred to as a ventilation rate of 0.1-1vvm.

[0038] In this invention, Aspergillus niger seeds can be obtained through cultivation, the Aspergillus niger seeds comprising Aspergillus niger hyphae loaded onto a porous carrier. It should be understood that the Aspergillus niger seeds also comprise Aspergillus niger hyphae free in the seed culture medium.

[0039] A second aspect of the present invention provides Aspergillus niger seeds prepared by the method described above.

[0040] It should be understood that the Aspergillus niger seeds comprise Aspergillus niger hyphae loaded onto a porous carrier.

[0041] A third aspect of the present invention provides a method for producing citric acid by fermentation of Aspergillus niger, the method comprising inoculating Aspergillus niger seeds prepared by the method described above into a fermentation medium for fermentation to produce citric acid.

[0042] Preferably, the inoculation amount of Aspergillus niger seeds is 7-10% of the volume of the fermentation medium.

[0043] The method for preparing citric acid by fermentation according to the present invention does not have special requirements for the composition of the fermentation medium, as long as it is a fermentation medium suitable for citric acid fermentation. Preferably, the fermentation medium contains enzymatic hydrolysate obtained from the enzymatic hydrolysis of starchy raw materials, and more preferably, the amount of enzymatic hydrolysate obtained from the enzymatic hydrolysis of starchy raw materials accounts for 80-100% by weight of the total amount of fermentation medium.

[0044] Generally, the product obtained from the enzymatic hydrolysis of starchy raw materials is called liquefied liquid. The liquefied liquid undergoes solid-liquid separation to obtain enzymatic hydrolysis residue and liquefied clear liquid. The liquefied clear liquid can typically be used to prepare fermentation media, or it can be mixed with the liquefied liquid for use in preparing fermentation media. Therefore, in this invention, the enzymatic hydrolysis product obtained from the enzymatic hydrolysis of starchy raw materials includes the liquefied clear liquid obtained through solid-liquid separation, as well as the liquefied liquid without solid-liquid separation, and a mixture of the two.

[0045] The fermentation medium is preferably obtained by mixing liquefied liquid and liquefied clear liquid with water or without mixing with water. More preferably, based on a total weight of 100 parts by weight of fermentation medium, the amount of liquefied clear liquid is 85-90 parts by weight and the amount of liquefied liquid is 10-15 parts by weight.

[0046] According to the method for preparing citric acid by fermentation of the present invention, the liquefied clear liquid can be prepared by various methods, for example, by the following method: pulverizing starchy raw materials, enzymatically hydrolyzing the pulverized product, and then separating the enzymatically hydrolyzed product into solid and liquid components to obtain the liquefied clear liquid and the enzymatic hydrolysis residue. The solid-liquid separation conditions are such that the solid content of the enzymatic hydrolysis residue is 45-55% by weight, preferably 49-51% by weight.

[0047] According to the method for preparing citric acid by fermentation of the present invention, the starchy raw material can be any starchy raw material known in the art that can be used for enzymatic hydrolysis and fermentation to prepare citric acid, such as one or more of corn, wheat, sweet potato, potato, and rice, preferably corn.

[0048] The enzymatic hydrolysis step can be performed using methods commonly used in the art, such as adding enzyme-producing microorganisms and / or enzymes to the pulverized product and incubating at the growth temperature of the enzyme-producing microorganisms and / or the temperature at which the enzymes are active. The enzyme-producing microorganisms are those capable of secreting amylase. The enzymes include amylase.

[0049] Since microbial growth produces byproducts, it is preferable to add the enzyme directly. The more enzyme used, the better. For cost considerations, it is preferable to use 15-50 enzyme activity units per gram of dry weight of the pulverized product.

[0050] In this invention, an enzyme activity unit is defined as the amount of enzyme required to convert 1 mg of starch into reducing sugar in 1 minute under conditions of pH 6.0 and temperature 70°C.

[0051] The temperature for enzymatic hydrolysis can vary within a wide range, preferably 70-105°C, and more preferably 90-95°C. Theoretically, the longer the hydrolysis time, the better. Considering equipment utilization, the preferred hydrolysis time is 90-150 minutes, and more preferably 100-120 minutes. The pH value for hydrolysis can vary within a wide range, preferably 5-7, and more preferably 5.4-6.2.

[0052] Amylase is a general term for a class of enzymes that can break down the glycosidic bonds of starch. Amylases generally include α-amylase, β-amylase, saccharifying enzymes, and isoamylase. These amylases can be obtained commercially.

[0053] In this invention, the methods and apparatus for solid-liquid separation are known to those skilled in the art, such as filter presses or centrifuges.

[0054] Preferably, during the fermentation process, the rotation speed is controlled to be no higher than 80 rpm, and more preferably 60-70 rpm.

[0055] Preferably, during the fermentation process, the ventilation volume is controlled to be 0.12-0.15 vvm.

[0056] Under the preferred rotation speed and ventilation rate, the oxygen supply requirement can be met, energy consumption can be reduced, and it is also beneficial for Aspergillus niger to ferment and produce citric acid.

[0057] The fermentation conditions can be selected within a wide range. Preferably, the fermentation conditions include a temperature of 33-40°C and an initial pH of 2-5.

[0058] In this invention, the Aspergillus niger seeds described herein are used for fermentation to produce citric acid. This avoids the thickening of the fermentation broth caused by the breakage of mycelia obtained by traditional cultivation methods when they are directly inoculated into the fermentation medium due to stirring and shearing forces. It also facilitates the separation of the mycelia and the culture medium after fermentation, reducing separation costs.

[0059] In addition, the inventors of this invention have also discovered that Aspergillus niger seeds loaded on a porous carrier can be reused, that is, they can be fermented multiple times. After 4-5 fermentations, the fermentation efficiency will decrease significantly.

[0060] In this invention, citric acid can be separated and refined according to the requirements of different industrial products, such as neutralization, acid hydrolysis, decolorization, concentration, crystallization, and packaging.

[0061] The present invention will be described in detail below through embodiments.

[0062] The Aspergillus niger was purchased from the National Standard Products Network, and the model number was Aspergillus niger 98003.

[0063] Acidity: The number of milligrams of potassium hydroxide (KOH) required to neutralize 1 gram of a chemical substance, determined by the method specified in GB1987-2007 Food Additives Citric Acid, in percentages.

[0064] Conversion rate: i.e., the conversion rate of citric acid, conversion rate (%) = (concentration of citric acid fermentation broth × volume of citric acid fermentation broth / weight of total sugar) × 100%, where the concentration of citric acid fermentation broth refers to acidity.

[0065] The mass concentration of NH3 in ammonia water is 4%, and the mass concentration of K2HPO4 in dipotassium hydrogen phosphate solution is 1%.

[0066] The total sugar content in seed culture media refers to the sugar content measured by Fehling's method as the reducing sugar content.

[0067] In the following examples and comparative examples, fermentation was carried out at 2m. 3 The fermentation is carried out in a fermentation tank.

[0068] In the following examples, each experiment was repeated three times, and the results were averaged.

[0069] Preparation Example 1

[0070] This preparation example illustrates the preparation of Aspergillus niger spores in this invention.

[0071] Mature wheat bran koji was rinsed with sterile water and filtered, then added to sterile water at 35℃ and pH 6.5. The mixture was then placed in a steel cylinder and incubated with shaking for 4 hours to obtain a uniformly dispersed spore suspension with a spore concentration of 3.0 × 10⁻⁶. 8 1 spore / ml suspension.

[0072] Preparation Example 2

[0073] This preparation example illustrates the preparation of the seed culture medium in this invention.

[0074] Corn was crushed to obtain the crushed product. The crushed product was mixed into a slurry at a concentration of 16% by weight. 30 units of amylase (Novozymes, α-amylase, this amylase was used in all embodiments of the present invention) were added per gram of crushed product. The mixture was fed into an injector and enzymatically hydrolyzed for 110 minutes at 95°C and pH 5.8 to obtain the enzymatic hydrolysate. The total sugar content of the enzymatic hydrolysate was determined using the Fehling's method.

[0075] Add the enzymatic hydrolysis liquefaction solution to a total sugar content of 4.5 t. Pour the liquefaction solution into a seed tank, then add 50 kg of ammonium sulfate and 25 kg of potassium dihydrogen phosphate to the seed tank. Adjust the pH to 5.8 and bring the volume to 40 ml. 3 Perform sterilization according to the normal sterilization procedure, and cool to 36°C after sterilization for later use.

[0076] Preparation Example 3

[0077] This preparation example illustrates the preparation of the fermentation medium in this invention.

[0078] The enzymatic hydrolysate was prepared according to the method in Preparation Example 2. A portion of the enzymatic hydrolysate was filtered using a hydraulic plate and frame filter press to separate the liquefied clear liquid and the enzymatic hydrolysis residue, wherein the solid content of the enzymatic hydrolysis residue was 50% by weight. The liquefied clear liquid and the enzymatic hydrolysate were mixed at a weight ratio of 10:1 and then sterilized to obtain the fermentation medium.

[0079] Example 1

[0080] This embodiment illustrates the preparation of Aspergillus niger seeds and the fermentation of citric acid according to the present invention.

[0081] (1) Preparation of Aspergillus niger seeds

[0082] Sterilized spherical porous carriers (made of polyurethane) with a diameter of 1 cm and a pore size of 350 PPI were added to the seed culture medium prepared in Preparation Example 2 for the cultivation of Aspergillus niger seeds. The amount of the porous carrier added was 8 g / L of seed culture medium.

[0083] The spore suspension obtained in Preparation Example 1 was inoculated into the seed culture medium prepared in Preparation Example 2, with an inoculation amount of 0.05% by volume, a culture temperature of 35±2℃, an aeration rate of 0.16 vvm, and a stirring speed of 80 rpm. After culturing for 26 hours, the culture was terminated, and Aspergillus niger seeds were obtained.

[0084] (2) Fermentation of citric acid

[0085] The Aspergillus niger seed liquid prepared in step (1) was added to the fermentation medium obtained in Preparation Example 3 to start fermentation. Based on the volume of the fermentation medium, the inoculum amount of the Aspergillus niger seed liquid was 10% by volume. The fermentation conditions included a temperature of 37°C, an initial pH of 4.5, an aeration rate of 0.15 vvm, a stirring speed of 70 rpm, and a reducing sugar content of 0 g / 100 mL. Fermentation was then stopped, and solid-liquid separation was performed to obtain the fermentation supernatant and the fermented cells.

[0086] The fermentation cycle was recorded, and the acidity of the supernatant after solid-liquid separation was measured. The conversion rate of citric acid was calculated. The specific results are shown in Table 1 (for Example 1-1).

[0087] In addition, the fermented cells were inoculated and fermented again in the manner of step (2) and repeated twice (that is, three consecutive fermentations). The acidity and citric acid conversion rate of each fermentation were measured, and the results are shown in Table 1 (Examples 1-2 and 1-3, respectively).

[0088] Example 2

[0089] This embodiment illustrates the preparation of Aspergillus niger seeds and the fermentation of citric acid according to the present invention.

[0090] (1) Preparation of Aspergillus niger seeds

[0091] Sterilized spherical porous carriers (made of polyurethane) with a diameter of 1.2 cm and a pore size of 250 PPI were added to the seed culture medium prepared in Preparation Example 2 for the cultivation of Aspergillus niger seeds. The amount of the porous carrier added was 9 g / L of seed culture medium.

[0092] The spore suspension obtained in Preparation Example 1 was inoculated into the seed culture medium prepared in Preparation Example 2, with an inoculation amount of 0.04% by volume, a culture temperature of 35±2℃, an aeration rate of 0.16 vvm, and a stirring speed of 80 rpm. After culturing for 25 hours, the culture was terminated, and Aspergillus niger seeds were obtained.

[0093] (2) Fermentation of citric acid

[0094] The Aspergillus niger seed liquid prepared in step (1) was added to the fermentation medium obtained in Preparation Example 3 to start fermentation. Based on the volume of the fermentation medium, the inoculum amount of Aspergillus niger was 10% by volume. The fermentation conditions included a temperature of 37°C, an initial pH of 4.5, an aeration rate of 0.16 vvm, and a reducing sugar content of 0 g / 100 mL. Fermentation was then stopped, and solid-liquid separation was performed to obtain the fermentation supernatant and the fermented cells.

[0095] The fermentation cycle was recorded, and the acidity of the supernatant after solid-liquid separation was measured. The conversion rate of citric acid was calculated. The specific results are shown in Table 1.

[0096] Example 3

[0097] This embodiment illustrates the preparation of Aspergillus niger seeds and the fermentation of citric acid according to the present invention.

[0098] (1) Preparation of Aspergillus niger seeds

[0099] Sterilized spherical porous carriers (made of polyurethane) with a diameter of 1.5 cm and a pore size of 450 PPI were added to the seed culture medium prepared in Preparation Example 2 for the cultivation of Aspergillus niger seeds. The amount of the porous carrier added was 10 g / L of seed culture medium.

[0100] The spore suspension obtained in Preparation Example 1 was inoculated into the seed culture medium prepared in Preparation Example 2, with an inoculation amount of 0.06% by volume, a culture temperature of 35±2℃, an aeration rate of 0.16 vvm, and a stirring speed of 80 rpm. After culturing for 25 hours, the culture was terminated, and Aspergillus niger seeds were obtained.

[0101] (2) Fermentation of citric acid

[0102] The Aspergillus niger seed liquid prepared in step (1) was added to the fermentation medium obtained in Preparation Example 3 to start fermentation. Based on the volume of the fermentation medium, the inoculum amount of Aspergillus niger was 10% by volume. The fermentation conditions included a temperature of 37°C, an initial pH of 4.5, an aeration rate of 0.16 vvm, and a reducing sugar content of 0 g / 100 mL. Fermentation was then stopped, and solid-liquid separation was performed to obtain the fermentation supernatant and the fermented cells.

[0103] The fermentation cycle was recorded, and the acidity of the supernatant after solid-liquid separation was measured. The conversion rate of citric acid was calculated. The specific results are shown in Table 1.

[0104] Example 4

[0105] This embodiment illustrates the preparation of Aspergillus niger seeds and the fermentation of citric acid according to the present invention.

[0106] The procedure is performed according to the method described in Example 1, except that the porous carrier is a spherical carrier with a diameter of 2 cm, a pore size of 500 PPI, and an addition amount of 10 g / L seed culture medium.

[0107] The fermentation cycle was recorded, and the acidity of the supernatant after solid-liquid separation was measured. The conversion rate of citric acid was calculated. The specific results are shown in Table 1.

[0108] Example 5

[0109] This embodiment illustrates the preparation of Aspergillus niger seeds and the fermentation of citric acid according to the present invention.

[0110] The procedure is performed according to the method described in Example 1, except that the porous carrier is a spherical carrier with a diameter of 3 cm, a pore size of 200 PPI, and an addition amount of 5 g / L seed culture medium.

[0111] The fermentation cycle was recorded, and the acidity of the supernatant after solid-liquid separation was measured. The conversion rate of citric acid was calculated. The specific results are shown in Table 1.

[0112] Comparative Example 1

[0113] This comparative example illustrates the conventional method for preparing Aspergillus niger seeds and the fermentation of citric acid.

[0114] The procedure is performed according to the method described in Example 1, except that a porous carrier is not used, that is, a porous carrier is not added to the seed culture medium.

[0115] The fermentation cycle was recorded, and the acidity of the supernatant after solid-liquid separation was measured. The conversion rate of citric acid was calculated. The specific results are shown in Table 1.

[0116] Furthermore, during the fermentation process, the low stirring speed and ventilation volume cause the mycelium to become loose and unable to effectively clump together into a spherical shape. The mycelium is not protected by the carrier, and under the shear force of stirring, the mycelium breaks continuously. The liquid becomes viscous, which is not conducive to oxygen transfer, resulting in low fermentation efficiency.

[0117] Comparative Example 2

[0118] This comparative example illustrates the conventional method for preparing Aspergillus niger seeds and the fermentation of citric acid.

[0119] The method described in Comparative Example 1 is followed, except that in step (1), the inoculation amount is 0.1% by volume, the stirring speed is adjusted to 100 rpm, and the ventilation volume is 0.2%; in step (2), the inoculation amount is 10% by volume, the stirring speed is adjusted to 100 rpm, and the ventilation volume is 0.2%.

[0120] The fermentation cycle was recorded, and the acidity of the supernatant after solid-liquid separation was measured. The conversion rate of citric acid was calculated. The specific results are shown in Table 1.

[0121] In addition, although the mycelium clumps together well under high rotation speed and large ventilation conditions, the fermentation efficiency is not high due to poor oxygen and mass transfer inside the mycelial balls. At the same time, since the mycelial balls are not adsorbed by the carrier, the individual cells are small, and they cannot be effectively separated during the final solid-liquid separation. Plate and frame filter presses are required for pressure filtration, and the mycelium cannot be reused.

[0122] Comparative Example 3

[0123] This comparative example illustrates the conventional method for preparing Aspergillus niger seeds and the fermentation of citric acid.

[0124] The operation is carried out according to the method described in Example 1, except that the pore size of the porous carrier is 10 PPI.

[0125] The fermentation cycle was recorded, and the acidity of the supernatant after solid-liquid separation was measured. The conversion rate of citric acid was calculated. The specific results are shown in Table 1.

[0126] During fermentation, the low stirring speed and low ventilation cause the mycelium to become loose and unable to effectively clump together into a ball. Although the carrier has a large pore size, it can provide some protection, but it still cannot effectively adsorb and protect the mycelium. Under the shear force of stirring, the mycelium breaks continuously, and the liquid becomes viscous, which is not conducive to oxygen transfer, resulting in low fermentation efficiency.

[0127] With larger pore sizes (e.g., 5 PPI), the porous carrier is basically unable to adsorb mycelia. Increasing the porous carrier actually makes the fermentation broth have higher dry matter content and is more viscous, and the fermentation effect is even worse than that of Comparative Example 1.

[0128] Table 1

[0129] Example number Fermentation cycle / h acidity% Lemon conversion rate % Example 1-1 48 17.5 99.45 Examples 1-2 48 17.5 99.46 Examples 1-3 49 17.4 99.32 Example 2 52 17.2 99.01 Example 3 53 17.2 98.95 Example 4 54 17.3 99.21 Example 5 57 17.2 99.03 Comparative Example 1 78 14.3 81.95 Comparative Example 2 64 16.7 94.91 Comparative Example 3 69 14.6 85.80

[0130] As can be seen from the results in Table 1, the fermentation technology of Aspergillus niger mycelium cultured in Examples 1-5 under the preferred conditions of low ventilation and low stirring speed, and immobilization and protection of the mycelium using the porous carrier described in this invention, has significant advantages in terms of cycle and conversion rate compared with Comparative Examples 1-3.

[0131] In Comparative Example 1, using the same culture medium and conditions but without any carrier, the mycelium was damaged into numerous broken strands during fermentation, the material became viscous, the fermentation cycle was significantly prolonged, and the fermentation conversion rate was extremely low, essentially resulting in fermentation failure. In Comparative Example 2, without adding a carrier, normal fermentation was achieved by increasing the stirring speed and aeration rate, but its fermentation cycle was longer than the previous example, and the fermentation effect was also poor. Although a porous carrier was added in Comparative Example 3, its large pore size prevented effective adsorption and protection of the mycelium, also leading to problems such as viscous liquid and low fermentation efficiency.

[0132] The technical solution described in this invention has significant advantages such as low spore inoculation amount in the seed tank, low stirring speed and low ventilation in the fermentation tank, and high mycelial fermentation efficiency.

[0133] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combining various technical features and carrier shapes in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for producing citric acid by fermentation of Aspergillus niger, characterized in that, This method involves inoculating Aspergillus niger seeds into a fermentation medium for fermentation to produce citric acid; The method for preparing Aspergillus niger seeds includes: inoculating Aspergillus niger spores into a seed culture medium and culturing them in the presence of a porous carrier to obtain Aspergillus niger seeds; wherein the Aspergillus niger is Aspergillus niger 98003; The porous carrier has a pore size of 300-400 PPI and is made of polyurethane; the diameter of a single porous carrier is 1-2 cm, and the shape is spherical; the amount of porous carrier added is 6-9 g / L of seed culture medium. The seed culture medium comprises enzymatic hydrolysis liquefaction of starch raw material, ammonium sulfate and dipotassium hydrogen phosphate, with a pH of 5-6; The inoculation amount of Aspergillus niger spores was such that the content of Aspergillus niger spores in the seed culture medium after inoculation was 0.5 × 10⁻⁶. 5 -2.5×10 5 cfu / mL; During the cultivation process, the rotation speed is controlled at 70-80 rpm, and the ventilation volume is controlled at 0.1-0.2 vvm; The culture conditions include: a temperature of 35-38℃, an initial pH of 5-7, and a time of 18-28h; The fermentation medium is an enzymatic hydrolysis product obtained by enzymatic hydrolysis of starch raw material, and the amount of the enzymatic hydrolysis product obtained by enzymatic hydrolysis of starch raw material accounts for 80-100% by weight of the total amount of fermentation medium. Based on the volume of the fermentation medium, the inoculation amount of Aspergillus niger seeds is 5-15% by volume. During the fermentation process, the rotation speed is controlled at 60-70 rpm; the ventilation rate is controlled at 0.12-0.15 vvm; the temperature is 33-40℃; and the initial pH is 2-5.