CN108277240B - Process for preparing citric acid by fermentation - Google Patents
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Abstract
The invention belongs to the technical field of microbial fermentation, and discloses a process for preparing citric acid by fermentation, which comprises the following steps: step 1) preparing a fermentation culture medium, step 2) activating an Aspergillus niger seed liquid, and step 3) fermenting to produce acid. The fermentation process of the invention has low cost and high citric acid yield.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a process for preparing citric acid by fermentation.
Background
Citric acid, also known as citric acid, is known by the scientific name 2-hydroxy-l, 2, 3-propanetricarboxylic acid (2-hydroxy-1, 2, 3-propane tricarboxylic acid) as a white crystalline powder, is odorless, often contains one molecule of crystal water or no crystal water, and is readily soluble in water. It is one of the main intermediates of the triacid cycle in organisms and therefore has important metabolic significance in almost all organisms. Citric acid has wide application in the fields of food, medicine, cosmetics, chemical industry and the like. Since citric acid has a refreshing sour taste, it is widely used as an acidic flavoring agent for various beverages and snacks, or an antioxidant for edible oils. In addition, citric acid has the effect of preventing or eliminating skin pigmentation, and thus citric acid can be used in the pharmaceutical and cosmetic industries. The citric acid solution and the salt solution thereof can also be used as a builder of textiles, can precipitate metal ions on the textiles, and simultaneously can be used as a formic acid-free crease-resistant finishing agent of pure cotton textiles. About 70% of the citric acid in the world market is used in the food industry, 10% in medicine and cosmetics, 15% in the chemical industry and 5% in others. At present, the worldwide consumption of citric acid reaches 120 ten thousand tons, the annual output reaches 160 ten thousand tons, the market trade volume is more than 90 ten thousand tons, and the annual growth rate is increased by 7 percent year by year. China is the biggest export country of citric acid in the world, production technology is in the forefront of the world, annual export amount exceeds half of the total world trade amount, and China is mainly sold to Europe, Japan and the like. Therefore, the production of citric acid is of great significance.
The production method of citric acid mainly comprises natural extraction method, chemical synthesis method and biological fermentation method. Biological fermentation is the most commonly used method. The citric acid fermentation mode comprises the following steps: solid state surface fermentation using penicillium and aspergillus fungi; carrying out submerged fermentation by using aspergillus fungi; solid state culture, continuous culture, and multi-stage fermentation. At present, citric acid is commercially produced mainly from starch as a substrate by fermentation of Aspergillus niger. The process of fermenting citric acid by aspergillus niger mainly comprises the following steps: utilizing a carbon source in the culture medium; accumulating citric acid after EMP pathway; citric acid is secreted from mitochondria to the cytoplasm; citric acid is secreted intracellularly into the culture medium.
The citric acid fermentation industry needs to solve the problems in industrial production mainly including strains, fermentation medium raw materials and extraction. In the fermentation of bacterial species, various microorganisms can accumulate citric acid, such as Aspergillus niger, Penicillium, Aspergillus nidulans, etc., and some yeasts can also utilize acetic acid, alkanes, etc. to produce citric acid. The strains used in the actual industrial production are all new strains which are screened and bred. The development of molecular biological engineering provides a wider space for the breeding of excellent strains, and reports of replacing the original citric acid producing strain with a gene recombinant strain are numerous, and the 70-90 th generation in the 20 th century is the high-yield period of new citric acid strains in China. Aspergillus niger is a food-grade safe filamentous fungus, has the advantages of rich enzyme system, high fermentation efficiency, few byproducts and the like, can well regulate and control the flux of glycolysis and the secretion of citric acid from mitochondria and cytoplasm, is still an important direction for the development of citric acid fermentation, and is also an important way for improving the productivity of citric acid. Up to now, aspergillus niger is still the most widely used citric acid producing bacterium in industry. The method has the advantages that the acid production mechanism of the existing high-yield strain is researched, the physiological processes of the whole growth and metabolism of the strain are analyzed, and the basis is provided for the explanation of the acid production mechanism and the improvement of the strain, so that the method is significant. "the review on the improvement of the strain of citric acid fermentation bacteria Aspergillus niger, biotechnology 2005" discloses the improvement mode of citric acid fermentation bacteria Aspergillus niger, including the modes of mutagenesis, transgenic technology and the like to the strain, which improves the acid yield of the strain to a certain extent; the preliminary research on citric acid fermentation of corn flour raw materials, industrial microorganisms 2000' researches the process parameters of citric acid fermentation by taking corn flour as the raw material, and optimizes an acid production system. How to further optimize an acid production system for microbial fermentation to improve the acid production and reduce the fermentation cost is a technical problem to be solved by the fermentation industry.
Disclosure of Invention
The invention aims to overcome the defects of high cost of a fermentation culture medium, low citric acid yield and the like in the prior art, and provides a process for preparing citric acid by fermentation.
The invention is realized by the following technical scheme:
a process for preparing citric acid by fermentation comprises the following steps: step 1) preparing a fermentation culture medium, step 2) activating an Aspergillus niger seed liquid, and step 3) fermenting to produce acid.
Specifically, the process comprises the following steps:
step 1) preparation of a fermentation medium: crushing corn straws, mixing the crushed corn straws with corn husks according to the mass ratio of 2:1, crushing the mixture to a particle size of more than 50 meshes, adding the crushed mixture into water with the weight of 5-7 times of that of the mixture, heating the mixture to 80 ℃ while stirring, preserving the heat for 30min, adding 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.01g/L of ferrous sulfate heptahydrate and 0.01g/L of manganese sulfate monohydrate, carrying out moist heat sterilization at 121 ℃ for 30min, and cooling the mixture to obtain a trichoderma culture solution; inoculating Trichoderma reesei into a Trichoderma culture solution according to the inoculation amount of 10%, culturing for 72-96h, controlling the stirring speed at 100rpm during culturing, controlling the culture temperature at 30-32 ℃, and controlling the pH value in the culture fermentation process to be 4-5 by feeding ammonia water; after the culture is finished, filtering to remove thalli, adding methanol and calcium hydroxide into the obtained liquid, and uniformly stirring to obtain a fermentation culture medium;
step 2) activating aspergillus niger seed liquid: culturing Aspergillus niger for 24h according to a conventional method to obtain an Aspergillus niger seed solution, and then carrying out ultrasonic treatment on the seed solution;
step 3), fermenting to produce acid: inoculating the Aspergillus niger seed liquid obtained in the step 2) into a fermentation tank containing the fermentation culture medium obtained in the step 1) according to the inoculation amount of 8-10% for fermentation culture, wherein the pH value is controlled to be 4-4.5 by feeding ammonia water in the fermentation process, and the fermentation time is 60 hours.
Preferably, the first and second electrodes are formed of a metal,
in the step 1), the adding amount of the methanol is 0.2 percent, the volume ratio is high, and the adding amount of the calcium hydroxide is 10wt percent.
Preferably, the first and second electrodes are formed of a metal,
the ultrasonic treatment parameters are as follows: the ultrasonic frequency is 30KHz, the power is 6W, the ultrasonic interval is 15s, the ultrasonic time is 1s, and the total ultrasonic time is 160 s.
Preferably, the first and second electrodes are formed of a metal,
in the step 3), fermentation parameters are as follows: the fermentation temperature is 34-36 ℃, the tank pressure is 0.03-0.04MPa, and the air quantity is 500-600L/h.
Compared with the prior art, the invention has the advantages that the following aspects are mainly included but not limited:
corn is a main raw material of a fermentation enterprise, corn flour is used for fermenting and preparing sugars, most of corn straws are discarded, most of corn husks are used as feed, the added value is low, and certain waste is caused; the corn stalks and the corn husks can be comprehensively treated, so that the waste is changed into valuable, and the additional value is improved;
aspergillus niger is directly adopted to ferment agricultural wastes such as straw powder, corn bran and the like, the yield of citric acid is only 10-20g/L, the sugar conversion rate is less than 60%, and the method cannot be industrially applied. The trichoderma reesei degrades corn straws and corn husks to obtain a solution containing reducing sugar, and is suitable for aspergillus niger to ferment; however, tests show that when trichoderma reesei and aspergillus niger are fermented together, although the two strains can be symbiotically better, the yield of citric acid is reduced, probably because trichoderma reesei utilizes more nitrogen sources, so that the nutrient substances of aspergillus niger are relatively insufficient, or trichoderma reesei generates extracellular secretion to inhibit the citric acid pathway of aspergillus niger, so that the yield of citric acid is reduced; therefore, after the culture of the trichoderma reesei is finished, the sterilization is carried out, and mycoprotein byproducts can be generated;
aspergillus niger can utilize reducing sugar or non-reducing sugar generated by decomposing cellulose by Trichoderma reesei, but in the fermentation process, along with the accumulation of citric acid, the fermentation pH is sharply reduced, particularly when the pH is reduced to below 2.00, the activity of saccharifying enzyme is damaged, the supply rate of glucose is reduced, the synthesis rate of products is reduced, and the content of total sugar in fermentation residue is higher; ammonia water is required to be fed back to control the pH value to be about 4 in the fermentation process, otherwise the conversion rate of the citric acid is reduced; the higher residual sugar problem significantly reduces the conversion ratio between substrate and product, and further aggravates the difficulty of product separation and purification steps;
according to the invention, the low-concentration methanol is added into the culture medium, so that the capacity of citric acid passing through cell membranes can be increased, and the secretion level of citric acid is improved; the low-intensity ultrasonic pretreatment of the seed liquid promotes the water permeation of the strain, can activate the strain, shortens the fermentation culture time of the strain, and improves the yield of citric acid, and compared with the method without ultrasonic treatment, the yield of citric acid can be improved by about 5 percent.
Detailed Description
Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products and methods described herein may be made and utilized without departing from the spirit, scope, and spirit of the invention. For a further understanding of the present invention, reference will now be made in detail to the following examples.
Example 1
A process for preparing citric acid by fermentation comprises the following steps:
preparing a fermentation medium: crushing corn straws, mixing the crushed corn straws with corn husks according to the mass ratio of 2:1, crushing the mixture into particles with the particle size of 50 meshes, adding the particles into water with the weight of 5 times of the particle size, heating the mixture to 80 ℃ while stirring, preserving the heat for 30min, adding 1g/L potassium dihydrogen phosphate, 1g/L dipotassium hydrogen phosphate, 0.2g/L magnesium sulfate heptahydrate, 0.01g/L ferrous sulfate heptahydrate and 0.01g/L manganese sulfate monohydrate, performing moist heat sterilization at 121 ℃ for 30min, and cooling the mixture to obtain a trichoderma culture solution; trichoderma reesei ATCC 13631 was inoculated at 10% (inoculation density 5X 10)6cfu/mL) is inoculated into a trichoderma culture solution, and the trichoderma culture solution is cultured for 72 hours, wherein the stirring speed is controlled to be 100rpm, the culture temperature is 30-32 ℃, and the pH value in the culture fermentation process is controlled to be 4-5 by feeding ammonia water; after the culture is finished, filtering to remove thalli, adding 0.2% (volume ratio) of methanol and 10wt% of calcium hydroxide into the obtained liquid, and uniformly stirring to obtain a fermentation medium (the content of reducing sugar is 68g/L, and the total content of protein and polypeptide is 8.1 g/L); the thallus can be used for preparing mycoprotein;
activating an Aspergillus niger seed solution: culturing Aspergillus niger Co827 for 24h according to a conventional method to obtain Aspergillus niger seed liquid, and performing ultrasonic treatment on the seed liquid, wherein the ultrasonic frequency is controlled to be 30KHz, the power is controlled to be 6W, the ultrasonic interval is controlled to be 15s, the ultrasonic time is controlled to be 1s, and the total ultrasonic time is controlled to be 160 s;
fermentation and acid production: inoculating the Aspergillus niger seed liquid obtained in the step 2) according to the inoculation amount of 8% (the inoculation density is 1 multiplied by 10)7cfu/mL) is inoculated into a 50L fermentation tank containing 30L of the fermentation medium obtained in the step 1) for fermentation culture, the fermentation temperature is 34 ℃, the tank pressure is 0.03MPa, the air volume is 600L/h, the pH is controlled at 4 by feeding ammonia water in the fermentation process, and the fermentation time is 60 h; the acid yield is 151g/L, the average conversion rate is 96 percent, and the residual sugar content is 0.18 percent.
Example 2
A process for preparing citric acid by fermentation comprises the following steps:
preparing a fermentation medium: crushing corn stalks, mixing with corn husks according to a mass ratio of 2:1, crushing to obtain particles with a particle size of 100 meshes, adding the particles into 6 times of water by weight, heating to 80 ℃ while stirring, and preserving heat for 30min, thenThen adding 1g/L potassium dihydrogen phosphate, 1g/L dipotassium hydrogen phosphate, 0.2g/L magnesium sulfate heptahydrate, 0.01g/L ferrous sulfate heptahydrate and 0.01g/L manganese sulfate monohydrate, carrying out moist heat sterilization at 121 ℃ for 30min, and cooling to obtain a trichoderma culture solution; the Trichoderma reesei was inoculated at a rate of 10% (inoculation density: 5X 10)6cfu/mL) is inoculated into a trichoderma culture solution, the culture is carried out for 96h, the stirring speed is controlled to be 100rpm during the culture, the culture temperature is 30-32 ℃, and the pH value in the culture fermentation process is controlled to be 4-5 by feeding ammonia water; after the culture is finished, filtering to remove thalli, adding 0.2% (volume ratio) of methanol and 10wt% of calcium hydroxide into the obtained liquid, and uniformly stirring to obtain a fermentation medium (the content of reducing sugar is 73g/L, and the total content of protein and polypeptide is 8.7 g/L); the thallus can be used for preparing mycoprotein;
activating an Aspergillus niger seed solution: culturing Aspergillus niger (ATCC 1015) for 24h according to a conventional method to obtain Aspergillus niger seed liquid, and performing ultrasonic treatment on the seed liquid, wherein the ultrasonic frequency is controlled to be 30KHz, the power is 6W, the ultrasonic interval is 15s, the ultrasonic time is 1s, and the total ultrasonic time is 160 s;
fermentation and acid production: inoculating the Aspergillus niger seed liquid obtained in the step 2) according to the inoculation amount of 10% (the inoculation density is 1 multiplied by 10)7cfu/mL) is inoculated into a 50L fermentation tank containing 30L of the fermentation medium obtained in the step 1) for fermentation culture, the fermentation temperature is 35 ℃, the tank pressure is 0.04MPa, the air volume is 500L/h, the pH is controlled at 4.3 by feeding ammonia water in the fermentation process, and the fermentation time is 60 h; the acid yield is 143g/L, the average conversion rate is 94%, and the residual sugar content is 0.27%.
Comparative example 1
Aspergillus niger fermentation tank culture medium: 3kg of corn flour, 2000U/g of alpha-amylase, 230 CaCl 230 g and the balance of water. The preparation method comprises the following steps: adding corn flour into water, adjusting pH to 6.0-6.5, adding high temperature resistant alpha-amylase, heating to 70 deg.C, maintaining for 1h, rapidly heating to 90 deg.C, and maintaining for 45-60 min; heating and boiling for 5-10min, adding 5% ammonium sulfate, 10% calcium hydroxide, and 10% defoaming agent. The fermentation process was the same as in example 1; the amount of acid and citric acid produced is 137g/L, and the fermentation period is 68 h.
Example 3
Taking example 1 as an example, the acid production conditions of mixed fermentation of trichoderma reesei and aspergillus niger are compared, and the specific comparison results are shown in table 1:
TABLE 1
| Fermentation mode | The acid yield is g/L, | the amount of residual sugar% | Fermentation period h |
| Mixed fermentation | 129 | 0.21 | 60 |
| Example 1 | 151 | 0.18 | 60 |
As shown in table 1 above, trichoderma reesei and aspergillus niger are fermented together, although the two strains can be symbiotically better, the yield of citric acid is reduced by 16%, probably because trichoderma reesei utilizes more nitrogen sources, so that the nutrient substances of aspergillus niger are relatively insufficient, or because trichoderma reesei generates extracellular secretion to inhibit the citric acid pathway of aspergillus niger, so that the yield of citric acid is reduced; therefore, in the present invention, it is necessary to remove the cells after the culture of Trichoderma reesei has been completed.
Example 4
The influence of the methanol addition in the fermentation medium on the fermentation acid production effect is set as 0, 0.1%, 0.2%, 0.4% and 0.8% and the rest of the fermentation system is the same as that in example 1, and is specifically shown in table 2:
TABLE 2
| The addition amount is v/v% | The acid yield is g/L, | fermentation period h |
| 0 | 140 | 60 |
| 0.1 | 147 | 60 |
| 0.2 | 151 | 60 |
| 0.4 | 145 | 60 |
| 0.8 | 137 | 60 |
As shown in table 2, the amount of acid produced increased with the increase in the amount of methanol, reached a maximum at 0.2% and decreased with the increase in the amount of methanol, so the amount of acid produced was selected to be most suitably 0.2%.
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (1)
1. A process for the fermentative preparation of citric acid, said process comprising the steps of:
step 1) preparation of a fermentation medium: crushing corn straws, mixing the crushed corn straws with corn husks according to the mass ratio of 2:1, crushing the mixture into particles with the particle size of 50-100 meshes, adding the particles into water with the weight of 5-6 times, heating the mixture to 80 ℃ while stirring, preserving the heat for 30min, adding 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.01g/L of ferrous sulfate heptahydrate and 0.01g/L of manganese sulfate monohydrate, performing moist heat sterilization at 121 ℃ for 30min, and cooling to obtain a trichoderma culture solution; inoculating Trichoderma reesei into a Trichoderma culture solution according to the inoculum size of 10%, culturing for 72-96h, controlling the stirring speed at 100rpm during culturing, controlling the culture temperature at 30-32 ℃, and controlling the pH value in the fermentation process to be 4-5 by feeding ammonia water; after the culture is finished, filtering to remove thalli, adding methanol and calcium hydroxide into the obtained liquid, and uniformly stirring to obtain a fermentation culture medium;
step 2) activating aspergillus niger seed liquid: culturing Aspergillus niger for 24h according to a conventional method to obtain an Aspergillus niger seed solution, and then carrying out ultrasonic treatment on the seed solution;
step 3), fermenting to produce acid: inoculating the Aspergillus niger seed liquid obtained in the step 2) into a fermentation tank containing the fermentation culture medium obtained in the step 1) according to the inoculation amount of 8-10% for fermentation culture, wherein the pH is controlled to be 4-4.3 by feeding ammonia water in the fermentation process, and the fermentation time is 60 hours;
in the step 1), the adding amount of the methanol is 0.2 percent by volume, and the adding amount of the calcium hydroxide is 10 percent by weight;
the ultrasonic treatment parameters are as follows: the ultrasonic frequency is 30KHz, the power is 6W, the ultrasonic interval is 15s, the ultrasonic time is 1s, and the total ultrasonic time is 160 s;
in the step 3), fermentation parameters are as follows: the fermentation temperature is 34-35 ℃, the tank pressure is 0.03-0.04MPa, and the air quantity is 500-600L/h.
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| CN113604365B (en) * | 2021-09-02 | 2023-09-15 | 江南大学 | Ultrasonic wave control mycelium pellet size and method for producing citric acid by using same |
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Inventor after: Dong Liqing Inventor after: Wang Zheng Inventor after: Tang Yongqiang Inventor after: Wang Feng Inventor after: Li Hang Inventor before: Wang Zheng Inventor before: Dong Liqing Inventor before: Tang Yongqiang Inventor before: Wang Feng Inventor before: Li Hang |
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