CN116179617B - Method for preparing citric acid by fermentation - Google Patents
Method for preparing citric acid by fermentation Download PDFInfo
- Publication number
- CN116179617B CN116179617B CN202310436154.4A CN202310436154A CN116179617B CN 116179617 B CN116179617 B CN 116179617B CN 202310436154 A CN202310436154 A CN 202310436154A CN 116179617 B CN116179617 B CN 116179617B
- Authority
- CN
- China
- Prior art keywords
- citric acid
- fermentation
- liquid
- acid fermentation
- parts
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/48—Tricarboxylic acids, e.g. citric acid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
- C12R2001/685—Aspergillus niger
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
- C12R2001/69—Aspergillus oryzae
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention discloses a method for preparing citric acid by fermentation, and relates to the technical field of citric acid preparation. The method for preparing the citric acid by fermentation comprises the following steps: inoculating Neurospora crassa, aspergillus oryzae and Aspergillus niger in a citric acid fermentation medium, fermenting and culturing to obtain citric acid fermentation broth, and subjecting the citric acid fermentation broth to membrane sterilization, electrodialysis, decolorization, ion exchange, concentration and crystallization to obtain citric acid. The invention adopts the Neurospora crassa, the Aspergillus oryzae and the Aspergillus niger to carry out mixed fermentation, thereby shortening the fermentation period of the citric acid and improving the yield and the conversion rate of the citric acid.
Description
Technical Field
The invention relates to the technical field of citric acid preparation, in particular to a method for preparing citric acid by fermentation.
Background
Citric acid (C) 6 H 8 O 7 ) Citric acid, also known as 2-hydroxy propane-1, 2, 3-tricarboxylic acid, is a colorless transparent or translucent powder with a strong sour taste. As one of the main products of organism metabolism, as acidulant, preservative and plasticizer, the product is applied in food industry, medicine industry, textile industry, feeding industry and the like.
The existing production method of the citric acid comprises the following steps: natural purification, chemical synthesis and biological fermentation. Wherein, the natural purification method is to add lime milk into fruits and vegetables rich in citric acid to form calcium salt, and then to process with sulfuric acid and concentrate to obtain citric acid, but the natural purification method has low output and is not suitable for large-scale industrial production; when a chemical synthesis method is adopted, toxic substances are often generated, the production process is complex, and more expensive reagents than citric acid are required to be adopted, so that the method is not suitable for mass production; the biological fermentation principle is the most common method. In biological fermentation, more than 80% of the citric acid product is obtained by submerged fermentation of Aspergillus niger species in waste materials containing glucose or sugar processing by-products.
The key technology of the citric acid fermentation preparation process is as follows: fermentation raw materials, strain selection, fermentation process control and purification refining process, wherein in the aspect of strain selection, although aspergillus niger is food-grade safe filamentous fungi, has the advantages of rich enzyme system, less byproducts and the like, in order to further shorten the fermentation period and improve the yield and purity of citric acid, mutagenesis and transgenic treatment are often carried out on the aspergillus niger, but the use and research of genetic engineering need a great deal of capital and time support, are unfavorable for industrial large-scale popularization and application, and meanwhile, the mutagenesis or transgenic treatment is used as an emerging biotechnology means, but has the problems of immaturity and uncertainty, so that transgenic foods are accompanied with biosafety.
In the prior art, CN1673385A discloses a method for producing citric acid and saponin from yam plants by using biotechnology, which comprises the steps of preparing citric acid by co-fermenting bacillus subtilis, lactobacillus and aspergillus niger, wherein the conversion rate of the prepared citric acid is 35%, the purification yield is 85% (containing crystal water), the fermentation period is 65-70h, and the method for producing the citric acid by fermenting crop straws by mixed bacteria is disclosed by using trichoderma reesei, aspergillus niger and Phanerochaete chrysosporium for mixed fermentation, the fermentation period is 112h, and the yield of the citric acid is 108.6 g/L.
Disclosure of Invention
In view of the above prior art, the present invention aims to provide a method for preparing citric acid by fermentation. The method adopts the common fermentation of Neurospora crassa, aspergillus oryzae and Aspergillus niger to prepare the citric acid, so that the fermentation period of the citric acid is short, and the yield and the conversion rate are high.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides a method for preparing citric acid by fermentation, which comprises the following steps:
(1) Inoculating Neurospora crassa, aspergillus oryzae and Aspergillus niger in a citric acid fermentation medium, and fermenting and culturing to obtain citric acid fermentation broth;
(2) And purifying the citric acid fermentation liquor to obtain the citric acid.
Preferably, in the step (1), the Neurospora crassa seed solution, the Aspergillus oryzae seed solution and the Aspergillus niger seed solution are mixed according to the volume ratio of 1 (3-5) (4-6), so as to obtain mixed seed solution, and the mixed seed solution is inoculated into a citric acid fermentation medium for fermentation culture.
Further preferably, the inoculation amount of the mixed seed liquid is 15% -20% of the volume of the citric acid fermentation medium.
Further preferably, in the mixed seed solution, the viable count of Neurospora gossypii is not less than 1.8X10 6 cfu/mL, the viable count of Aspergillus oryzae is more than or equal to 1.2X10 6 cfu/mL, the viable count of Aspergillus niger is more than or equal to 2.6X10 5 cfu/mL。
Further preferably, the Neurospora crassa has a strain number of CICC 40204, the Aspergillus oryzae has a strain number of CICC 2397, and the Aspergillus niger has a strain number of CICC 40048.
Preferably, the fermentation conditions are: the fermentation temperature is 32-40 ℃, the initial ventilation is 0.1-0.2 v/(v.min), the initial stirring rotation speed is 180-220r/min, the initial tank pressure is 0.1-0.2MPa, the initial dissolved oxygen amount is 100%, the ventilation and the stirring rotation speed are regulated when the dissolved oxygen amount is less than or equal to 15% along with the fermentation, the dissolved oxygen amount is controlled to be 15-25%, and the fermentation is stopped when the reducing sugar in the citric acid fermentation medium is less than or equal to 4 g/L.
Preferably, in the step (1), the citric acid fermentation medium comprises the following raw materials in parts by weight: 100-120 parts of starch saccharification liquid, 4.5-5 parts of (NH) 4 ) 2 SO 4 3-4 parts of NH 4 Cl, 3-4 parts KH 2 PO 4 0.4-0.8 part VB 1 5-6 parts of Tween 80 and 110-130 parts of trace element mixed solution.
Further preferably, the trace element mixed solution comprises: 0.5g/L glycine, 3.0g/L MgSO 4 ·7H 2 O、3.0g/L FeSO 4 ·7H 2 O、0.1g/L CoSO 4 、0.1g/L CaCl 2 ·2H 2 O、0.1g/L ZnSO 4 ·7H 2 O、0.2g/L CuSO 4 ·5H 2 O、0.01g/L KAl(SO 4 ) 2 ·12H 2 O、0.01g/L H 3 BO 4 、0.01g/L Na 2 MoO 4 ·2H 2 O、0.01g/L MnSO 4 ·H 2 O。
Further preferably, the preparation method of the starch saccharification liquid comprises the following steps: pulverizing starchiness raw material, pulping, adding amylase, liquefying at 110-120deg.C for 60min to obtain starchiness liquefied solution, cooling to 50-60deg.C, adding saccharifying enzyme, and saccharifying at constant temperature for 30min to obtain starchiness saccharified solution.
Preferably, the starchy material is one or more of corn, potato, wheat and sorghum.
Preferably, the particle size of the crushed starchy material is 60-80 mesh.
Preferably, the concentration of the starchy raw material after size mixing is 20-30wt%.
Preferably, the amylase is added in an amount of 1-3% by mass of the starchy material.
Preferably, the saccharifying enzyme is added in an amount of 2-4% by mass of the starchy material.
Preferably, in the step (2), the purifying operation is as follows: and (3) carrying out membrane sterilization, electrodialysis, decolorization, ion exchange, concentration and crystallization on the citric acid fermentation broth to obtain the citric acid.
The invention has the beneficial effects that:
the invention adopts aerobic fermentation of Neurospora crassa, aspergillus oryzae and Aspergillus niger, and has the same requirement for nutrients, so that the bacteria have no antagonism. In addition, the invention combines a plurality of fermentation strains for use, and the invention has the synergistic effect of the Neurospora, the Aspergillus oryzae and the Aspergillus niger, is used for the fermentation preparation of the citric acid, can shorten the fermentation period of the citric acid, and improves the yield and the conversion rate of the citric acid.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background art, in the prior art, aspergillus niger is mostly used as a fermentation strain to prepare the citric acid, so that the yield and the conversion rate of the citric acid are improved, the fermentation period of the citric acid is shortened, mutagenesis and transgenic treatment are often carried out on the aspergillus niger, but the research and the use of genetic engineering need a large amount of capital and time support, the genetic engineering is not suitable for popularization and application of industrial development scale, and the gene subjected to the mutagenesis and the transgenic treatment has the biosafety problem. Meanwhile, in the prior art, although different strains are combined for preparing the citric acid by fermentation, the problems of long fermentation period, low citric acid conversion rate and low yield still exist.
Based on this, the invention provides a method for preparing citric acid by fermentation, comprising the following steps:
(1) Inoculating Neurospora crassa, aspergillus oryzae and Aspergillus niger in a citric acid fermentation medium, and fermenting and culturing to obtain citric acid fermentation broth;
(2) And (3) carrying out membrane sterilization, electrodialysis, decolorization, ion exchange, concentration and crystallization on the citric acid fermentation broth to obtain the citric acid.
In the step (1), the Neurospora crassa seed solution, the Aspergillus oryzae seed solution and the Aspergillus niger seed solution are mixed according to the volume ratio of 1 (3-5) to 4-6 to obtain mixed seed solution, and the mixed seed solution is inoculated into a citric acid fermentation culture medium for fermentation culture.
In the mixed seed liquid, the viable count of the Neurospora gossypii is more than or equal to 1.8X10 6 cfu/mL, the viable count of Aspergillus oryzae is more than or equal to 1.2X10 6 cfu/mL, the viable count of Aspergillus niger is more than or equal to 2.6X10 5 cfu/mL。
In the present invention, the specific mode and conditions of seed culture are not particularly limited as long as a seed solution having a good cell production state can be obtained.
Citric acid fermentation mediumComprises the following components in parts by weight: 100-120 parts of starch saccharification liquid, 4.5-5 parts of (NH) 4 ) 2 SO 4 3-4 parts of NH 4 Cl, 3-4 parts KH 2 PO 4 0.4-0.8 part VB 1 5-6 parts of Tween 80 and 110-130 parts of trace element mixed solution.
Wherein, the microelement mixture liquid comprises: 0.5g/L glycine, 3.0g/L MgSO 4 ·7H 2 O、3.0g/L FeSO 4 ·7H 2 O、0.1g/L CoSO 4 、0.1g/L CaCl 2 ·2H 2 O、0.1g/L ZnSO 4 ·7H 2 O、0.2g/L CuSO 4 ·5H 2 O、0.01g/L KAl(SO 4 ) 2 ·12H 2 O、0.01g/L H 3 BO 4 、0.01g/L Na 2 MoO 4 ·2H 2 O、0.01g/L MnSO 4 ·H 2 O。
The preparation method of the starch saccharification liquid comprises the following steps: crushing the starchiness raw material to the grain size of 50-60 meshes, regulating the slurry to ensure that the concentration of the starchiness raw material after the slurry is regulated is 20-30wt%, adding amylase, liquefying at 110-120 ℃ for 60min to obtain starchiness liquefied liquid, cooling the starchiness liquefied liquid to 50-60 ℃, adding saccharifying enzyme, and carrying out heat preservation saccharification for 30min to obtain the starchiness saccharified liquid.
The starchy material is one or more of corn, potato, wheat and sorghum.
The addition amount of amylase is 1-3% of the mass of the starchy raw material, and the addition amount of saccharifying enzyme is 2-4% of the mass of the starchy raw material.
The fermentation conditions are as follows: the fermentation temperature is 32-40 ℃, the initial ventilation is 0.1-0.2 v/(v.min), the initial stirring rotating speed is 180-220r/min, the initial tank pressure is 0.1-0.2MPa, the initial dissolved oxygen amount is 100%, the ventilation is regulated when the dissolved oxygen amount is less than or equal to 15% along with the fermentation, the dissolved oxygen amount is controlled to be 15-25%, and the fermentation is stopped when the reducing sugar in the citric acid fermentation medium is less than or equal to 4 g/L.
In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and are commercially available.
The Neurospora crassa (Neurospora sitophila) adopted in the following examples and comparative examples is purchased from China center for type culture Collection of microorganisms, and the strain number is CICC 40204; aspergillus oryzae (Aspergillus oryzae) is purchased from China center for type culture collection of Industrial microorganisms, and the strain number is CICC 2397; aspergillus nigerAspergillus niger) Purchased from China center for type culture Collection of microorganisms, and the strain number is CICC 40048; amylase was purchased from Jiangsu Miao biosciences, inc.; the saccharifying enzyme was purchased from Shandong Fengtai Biotech Co.
Example 1: preparation of citric acid
(1) Crushing corn to 70 meshes, adding water for pulping to ensure that the concentration of the corn after pulping is 25wt%, adding amylase, liquefying at 110 ℃ for 60min to obtain a starch liquefied liquid, cooling the starch liquefied liquid to 55 ℃, adding saccharifying enzyme, and carrying out heat preservation and saccharification for 30min to obtain a starch saccharified liquid, wherein the adding amount of the amylase is 2% of the mass of the corn, and the adding amount of the saccharifying enzyme is 3% of the mass of the corn;
110g of the starch saccharification liquid and 4.8g (NH) 4 ) 2 SO 4 、3.5gNH 4 Cl、3.5gKH 2 PO 4 、0.6gVB 1 Uniformly mixing 5.5g of Tween 80 and 120g of microelement mixture, adding water to 1000mL of constant volume to obtain a citric acid fermentation medium, wherein the microelement mixture comprises: 0.5g/L glycine, 3.0g/L MgSO 4 ·7H 2 O、3.0g/L FeSO 4 ·7H 2 O、0.1g/L CoSO 4 、0.1g/L CaCl 2 ·2H 2 O、0.1g/L ZnSO 4 ·7H 2 O、0.2g/L CuSO 4 ·5H 2 O、0.01g/L KAl(SO 4 ) 2 ·12H 2 O、0.01g/L H 3 BO 4 、0.01g/L Na 2 MoO 4 ·2H 2 O、0.01g/L MnSO 4 ·H 2 O；
Uniformly mixing the Neurospora crassa seed solution, the Aspergillus oryzae seed solution and the Aspergillus niger seed solution according to the volume ratio of 1:4:5 to obtain mixed seed solution, inoculating the mixed seed solution into a citric acid fermentation culture medium, fermenting at 37 ℃ with the inoculation amount of the mixed seed solution being 18% of the volume of the citric acid fermentation culture medium, wherein the initial ventilation amount of fermentation is 0.15 v/(v.min), the initial stirring rotating speed is 200r/min, the initial tank pressure is 0.15MPa, the initial dissolved oxygen amount is 100%, the ventilation amount and the stirring rotating speed are regulated when the dissolved oxygen amount is less than or equal to 15%, the dissolved oxygen amount is controlled to 15% -25%, and the fermentation is stopped when the reducing sugar in the citric acid fermentation culture medium is 4g/L, so as to obtain the citric acid fermentation liquid;
(2) And (3) performing membrane sterilization, electrodialysis, decolorization, ion exchange, concentration and crystallization on the citric acid fermentation broth prepared in the step (1) to obtain the citric acid.
Example 2: preparation of citric acid
(1) Crushing sweet potatoes to 60 meshes, adding water and pulping to ensure that the concentration of the sweet potatoes after pulping is 20wt%, adding amylase, liquefying at 100 ℃ for 60min to obtain a starch liquefied liquid, cooling the starch liquefied liquid to 50 ℃, adding saccharifying enzyme, and carrying out heat preservation and saccharification for 30min to obtain a starch saccharified liquid, wherein the adding amount of the amylase is 1% of the mass of the sweet potatoes, and the adding amount of the saccharifying enzyme is 3% of the mass of the sweet potatoes;
100g of the starch saccharification liquid and 4.5g (NH) 4 ) 2 SO 4 、3gNH 4 Cl、3gKH 2 PO 4 、0.6gVB 1 Uniformly mixing 5g of Tween 80 and 110g of microelement mixture, adding water to 1000mL of constant volume to obtain a citric acid fermentation medium, wherein the microelement mixture comprises: 0.5g/L glycine, 3.0g/L MgSO 4 ·7H 2 O、3.0g/L FeSO 4 ·7H 2 O、0.1g/L CoSO 4 、0.1g/L CaCl 2 ·2H 2 O、0.1g/L ZnSO 4 ·7H 2 O、0.2g/L CuSO 4 ·5H 2 O、0.01g/L KAl(SO 4 ) 2 ·12H 2 O、0.01g/L H 3 BO 4 、0.01g/L Na 2 MoO 4 ·2H 2 O、0.01g/L MnSO 4 ·H 2 O；
Uniformly mixing a Neurospora crassa seed solution, an Aspergillus oryzae seed solution and an Aspergillus niger seed solution according to a volume ratio of 1:3:4 to obtain a mixed seed solution, inoculating the mixed seed solution into a citric acid fermentation medium, fermenting and culturing at 32 ℃ with an inoculum size of 15% of the volume of the citric acid fermentation medium, wherein the initial ventilation amount of fermentation is 0.1 v/(v.min), the initial stirring rotation speed is 180 r/min, the initial tank pressure is 0.1MPa, the initial dissolved oxygen amount is 100%, the ventilation amount and the stirring rotation speed are regulated when the dissolved oxygen amount is less than or equal to 15%, and the fermentation is stopped when the reducing sugar in the citric acid fermentation medium is 4g/L, so as to obtain the citric acid fermentation liquid;
(2) And (3) performing membrane sterilization, electrodialysis, decolorization, ion exchange, concentration and crystallization on the citric acid fermentation broth prepared in the step (1) to obtain the citric acid.
Example 3: preparation of citric acid
(1) Crushing wheat to 80 meshes, adding water for pulping to ensure that the concentration of the wheat after pulping is 30wt%, adding amylase, liquefying at 120 ℃ for 60min to obtain a starch liquefied liquid, cooling the starch liquefied liquid to 60 ℃, adding saccharifying enzyme, and carrying out heat preservation and saccharification for 30min to obtain a starch saccharified liquid, wherein the adding amount of the amylase is 3% of the mass of the wheat, and the adding amount of the saccharifying enzyme is 4% of the mass of the wheat;
120g of the starchy saccharification liquid and 5g (NH) 4 ) 2 SO 4 、4gNH 4 Cl、4gKH 2 PO 4 、0.8gVB 1 Mixing evenly 6g of Tween 80 and 130g of microelement mixture, adding water to 1000mL of constant volume to obtain the citric acid fermentation medium, wherein the microelement mixture comprises: 0.5g/L glycine, 3.0g/L MgSO 4 ·7H 2 O、3.0g/L FeSO 4 ·7H 2 O、0.1g/L CoSO 4 、0.1g/L CaCl 2 ·2H 2 O、0.1g/L ZnSO 4 ·7H 2 O、0.2g/L CuSO 4 ·5H 2 O、0.01g/L KAl(SO 4 ) 2 ·12H 2 O、0.01g/L H 3 BO 4 、0.01g/L Na 2 MoO 4 ·2H 2 O、0.01g/L MnSO 4 ·H 2 O；
Uniformly mixing the Neurospora crassa seed solution, the Aspergillus oryzae seed solution and the Aspergillus niger seed solution according to the volume ratio of 1:5:6 to obtain mixed seed solution, inoculating the mixed seed solution into a citric acid fermentation culture medium, fermenting at 40 ℃ with the inoculation amount of the mixed seed solution being 20% of the volume of the citric acid fermentation culture medium, wherein the initial ventilation amount of fermentation is 0.2 v/(v.min), the initial stirring rotating speed is 220r/min, the initial tank pressure is 0.2MPa, the initial dissolved oxygen amount is 100%, the ventilation amount and the stirring rotating speed are regulated when the dissolved oxygen amount is less than or equal to 15%, the dissolved oxygen amount is controlled to 15% -25%, and the fermentation is stopped when the reducing sugar in the citric acid fermentation culture medium is 4g/L, so as to obtain the citric acid fermentation liquid;
(2) And (3) performing membrane sterilization, electrodialysis, decolorization, ion exchange, concentration and crystallization on the citric acid fermentation broth prepared in the step (1) to obtain the citric acid.
Comparative example 1: citric acid prepared by fermenting Aspergillus niger
(1) Inoculating Aspergillus niger seed liquid into a citric acid fermentation culture medium, wherein the citric acid fermentation culture medium is the same as the citric acid fermentation culture medium in the embodiment 1, fermenting at 37 ℃ with the inoculum size of 18% of the volume of the citric acid fermentation culture medium, wherein the initial ventilation amount of fermentation is 0.15 v/(v.min), the initial stirring rotation speed is 200r/min, the initial tank pressure is 0.15MPa, the initial dissolved oxygen amount is 100%, when the dissolved oxygen amount is less than or equal to 15%, the ventilation amount and the stirring rotation speed are regulated, the dissolved oxygen amount is controlled to 15% -25%, and when the reducing sugar in the citric acid fermentation culture medium is 4g/L, the fermentation is stopped, so that the citric acid fermentation liquid is obtained;
(2) And (3) performing membrane sterilization, electrodialysis, decolorization, ion exchange, concentration and crystallization on the citric acid fermentation broth prepared in the step (1) to obtain the citric acid.
Comparative example 2: citric acid prepared by Aspergillus oryzae fermentation
(1) Inoculating Aspergillus oryzae seed liquid into a citric acid fermentation culture medium, wherein the citric acid fermentation culture medium is the same as the citric acid fermentation culture medium in the embodiment 1, fermenting at 37 ℃ with the inoculum size of the Aspergillus oryzae seed liquid being 18% of the volume of the citric acid fermentation culture medium, wherein the initial ventilation amount of fermentation is 0.15 v/(v.min), the initial stirring rotation speed is 200r/min, the initial tank pressure is 0.15MPa, the initial dissolved oxygen amount is 100%, when the dissolved oxygen amount is less than or equal to 15%, the ventilation amount and the stirring rotation speed are regulated, the dissolved oxygen amount is controlled to be 15% -25%, and when the reducing sugar in the citric acid fermentation culture medium is 4g/L, the fermentation is stopped, so that the citric acid fermentation liquid is obtained;
(2) And (3) performing membrane sterilization, electrodialysis, decolorization, ion exchange, concentration and crystallization on the citric acid fermentation broth prepared in the step (1) to obtain the citric acid.
Comparative example 3: citric acid prepared by fermentation of Neurospora crassa
(1) Inoculating the Neurospora crassa seed liquid into a citric acid fermentation culture medium, wherein the citric acid fermentation culture medium is the same as the citric acid fermentation culture medium in the embodiment 1, fermenting at 37 ℃ with the inoculation amount of the Neurospora crassa seed liquid being 18% of the volume of the citric acid fermentation culture medium, wherein the initial ventilation amount of fermentation is 0.15 v/(v.min), the initial stirring rotating speed is 200r/min, the initial tank pressure is 0.15MPa, the initial dissolved oxygen amount is 100%, when the dissolved oxygen amount is less than or equal to 15%, the ventilation amount and the stirring rotating speed are regulated, the dissolved oxygen amount is controlled to be 15% -25%, and when the reducing sugar in the citric acid fermentation culture medium is 4g/L, stopping fermentation, so as to obtain the citric acid fermentation liquid;
(2) And (3) performing membrane sterilization, electrodialysis, decolorization, ion exchange, concentration and crystallization on the citric acid fermentation broth prepared in the step (1) to obtain the citric acid.
Comparative example 4: citric acid prepared by fermenting Aspergillus niger and Neurospora crassa
(1) Uniformly mixing a Neurospora crassa seed solution and an Aspergillus niger seed solution according to a volume ratio of 1:5 to obtain a mixed seed solution, inoculating the mixed seed solution into a citric acid fermentation medium, wherein the citric acid fermentation medium is the same as the citric acid fermentation medium in the embodiment 1, fermenting at 37 ℃ with an inoculum size of 18% of the volume of the citric acid fermentation medium, wherein the initial ventilation rate of fermentation is 0.15 v/(v.min), the initial stirring rotating speed is 200r/min, the initial tank pressure is 0.15MPa, the initial dissolved oxygen amount is 100%, the ventilation rate and the stirring rotating speed are regulated when the dissolved oxygen amount is less than or equal to 15%, the dissolved oxygen amount is controlled to be 15% -25% when the reducing sugar in the citric acid fermentation medium is 4g/L, and the citric acid fermentation liquid is obtained;
(2) And (3) performing membrane sterilization, electrodialysis, decolorization, ion exchange, concentration and crystallization on the citric acid fermentation broth prepared in the step (1) to obtain the citric acid.
Comparative example 5: citric acid prepared by fermenting Aspergillus niger and Aspergillus oryzae
(1) Uniformly mixing Aspergillus oryzae seed liquid and Aspergillus niger seed liquid according to a volume ratio of 4:5 to obtain mixed seed liquid, inoculating the mixed seed liquid into a citric acid fermentation medium, wherein the citric acid fermentation medium is the same as the citric acid fermentation medium in the embodiment 1, fermenting at 37 ℃ with an inoculation amount of 18% of the volume of the citric acid fermentation medium, wherein the initial ventilation amount of fermentation is 0.15 v/(v.min), the initial stirring rotation speed is 200r/min, the initial tank pressure is 0.15MPa, the initial dissolved oxygen amount is 100%, and the ventilation amount and the stirring rotation speed are regulated when the dissolved oxygen amount is less than or equal to 15%, and the fermentation is stopped when the reducing sugar in the citric acid fermentation medium is 4g/L, so as to obtain the citric acid fermentation liquid;
(2) And (3) performing membrane sterilization, electrodialysis, decolorization, ion exchange, concentration and crystallization on the citric acid fermentation broth prepared in the step (1) to obtain the citric acid.
Test example 1
The concentrations (i.e., acidity) of the citric acid fermentation broths in examples 1-3 and comparative examples 1-5 were examined according to GB/T1987-2007 standard, and the conversion of citric acid was calculated, and the fermentation periods and yields of citric acid in examples 1-3 and comparative examples 1-5 were counted, and the results are shown in Table 1.
Wherein conversion (%) = [ (concentration of citric acid broth×volume of broth)/weight of total sugar in citric acid broth ] ×100%;
the fermentation period of the citric acid is the time from the start of fermentation to the stop of fermentation;
the yield of citric acid is the ratio of the mass of citric acid after concentration and crystallization to the volume of citric acid fermentation broth.
Examples 1-3 are identical to the citric acid purification conditions employed in comparative examples 1-5.
TABLE 1 acidity, conversion, fermentation cycle, yield values of citric acid
As can be seen from Table 1, comparative examples 1 to 3 are citric acid prepared by fermentation using single Aspergillus niger, aspergillus oryzae and Neurospora crassa, respectively, the conversion rate of citric acid is 15.8 to 95.2%, the fermentation period is 61 to 142 hours, and the yield is 12 to 168g/L, and thus, when fermentation is performed using a single strain, the yield, conversion rate and fermentation period of citric acid are not ideal.
In the prior art, aspergillus niger is mainly used as a fermentation strain to prepare citric acid, and two strains are used for fermentation in comparison examples 4-5, wherein one of the strains is aspergillus niger, and as can be seen from comparison examples 4-5, aspergillus oryzae or Neurospora crassa are respectively added on the basis of the aspergillus niger, and compared with single strain fermentation, the fermentation period can be shortened, and the conversion rate, the yield and the acidity of the citric acid are improved, but the effect of the citric acid is a certain difference compared with the mixed fermentation of three strains of aspergillus niger, aspergillus oryzae and Neurospora crassa. Therefore, the invention adopts Aspergillus niger, aspergillus oryzae and Neurospora good to ferment cooperatively, can effectively improve the yield and conversion rate of citric acid and shorten the fermentation period.
The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.
Claims (1)
1. A method for the fermentative preparation of citric acid, comprising the steps of:
(1) Mixing the Neurospora crassa seed solution, the Aspergillus oryzae seed solution and the Aspergillus niger seed solution according to the volume ratio of 1 (3-5) (4-6) to obtain mixed seed solution, inoculating the mixed seed solution into a citric acid fermentation medium, and fermenting to obtain a citric acid fermentation liquid;
(2) Performing membrane sterilization, electrodialysis, decolorization, ion exchange, concentration and crystallization on the citric acid fermentation broth to obtain citric acid;
the citric acid fermentation medium comprises the following components in parts by weight: 100-120 parts of starch saccharification liquid, 4.5-5 parts of (NH) 4 ) 2 SO 4 3-4 parts of NH 4 Cl, 3-4 parts KH 2 PO 4 0.4-0.8 part VB 1 5-6 parts of Tween 80 and 110-130 parts of trace element mixed solution;
the preparation method of the starch saccharification liquid comprises the following steps: crushing a starchiness raw material, mixing pulp, adding amylase, liquefying at 110-120 ℃ for 60min to obtain starchiness liquefied liquid, cooling the starchiness liquefied liquid to 50-60 ℃, adding saccharifying enzyme, and carrying out heat preservation saccharification for 30min to obtain starchiness saccharified liquid; the starchy raw material is one or more of corn, potato, wheat and sorghum;
the microelement mixture liquid comprises: 0.5g/L glycine, 3.0g/L MgSO 4 ·7H 2 O、3.0g/L FeSO 4 ·7H 2 O、0.1g/L CoSO 4 、0.1g/L CaCl 2 ·2H 2 O、0.1g/L ZnSO 4 ·7H 2 O、0.2g/L CuSO 4 ·5H 2 O、0.01g/L KAl(SO 4 ) 2 ·12H 2 O、0.01g/L H 3 BO 4 、0.01g/L Na 2 MoO 4 ·2H 2 O、0.01g/L MnSO 4 ·H 2 O；
In the mixed seed liquid, the viable count of the Neurospora gossypii is more than or equal to 1.8X10 6 cfu/mL, the viable count of Aspergillus oryzae is more than or equal to 1.2X10 6 cfu/mL, the viable count of Aspergillus niger is more than or equal to 2.6X10 5 cfu/mL；
The inoculation amount of the mixed seed liquid is 15% -20% of the volume of the citric acid fermentation medium;
the fermentation conditions are as follows: the fermentation temperature is 32-40 ℃, the dissolved oxygen is 15-25%, and when the reducing sugar in the citric acid fermentation medium is less than or equal to 4g/L, the fermentation is stopped.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310436154.4A CN116179617B (en) | 2023-04-23 | 2023-04-23 | Method for preparing citric acid by fermentation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310436154.4A CN116179617B (en) | 2023-04-23 | 2023-04-23 | Method for preparing citric acid by fermentation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116179617A CN116179617A (en) | 2023-05-30 |
| CN116179617B true CN116179617B (en) | 2023-07-18 |
Family
ID=86449208
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310436154.4A Active CN116179617B (en) | 2023-04-23 | 2023-04-23 | Method for preparing citric acid by fermentation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116179617B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115094096A (en) * | 2022-07-11 | 2022-09-23 | 吉林中粮生化有限公司 | Method for improving citric acid fermentation intensity by using precise fermentation key technology |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1673385A (en) * | 2004-03-25 | 2005-09-28 | 刘华祥 | Method for producing citrin and saponin from tuber crops by biotechnology |
| CN102533877B (en) * | 2012-01-13 | 2014-02-26 | 中粮生物化学（安徽）股份有限公司 | Method for preparing citric acid by fermentation |
| CN105886556B (en) * | 2016-06-14 | 2019-03-01 | 徐州生物工程职业技术学院 | A kind of method of mixed fungus fermentation crop straws for producing citric acid |
| WO2018217588A1 (en) * | 2017-05-21 | 2018-11-29 | Innovanutra Inc. | Methods and compositions of incorporating a bioactive agent and use thereof |
| CN109852640B (en) * | 2017-11-30 | 2020-12-15 | 中粮生物科技股份有限公司 | Seed culture medium for preparing fermented citric acid from full starch, culture medium for fermenting citric acid and method for preparing citric acid from full starch |
-
2023
- 2023-04-23 CN CN202310436154.4A patent/CN116179617B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115094096A (en) * | 2022-07-11 | 2022-09-23 | 吉林中粮生化有限公司 | Method for improving citric acid fermentation intensity by using precise fermentation key technology |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116179617A (en) | 2023-05-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sawant et al. | Fungal citric acid production using waste materials: a mini-review | |
| Soccol et al. | New perspectives for citric acid production and application | |
| Singh Dhillon et al. | Recent advances in citric acid bio-production and recovery | |
| EP3640337B1 (en) | Method for continuously culturing aspergillus niger seeds and producing citric acid using same | |
| CN109207373B (en) | Microbial strain for high yield of citric acid and method for producing citric acid by fermenting starch sugar through microbial strain | |
| CN101912051A (en) | Fermentation process of sea cucumber compound feed | |
| CN111517860A (en) | Plant nutrient rich in seaweed active oligosaccharide and preparation method thereof | |
| CN101962664A (en) | Fermentation process for producing L-valine efficiently | |
| CN102206684A (en) | Fermentation technology for producing calcium lactate with sweet potatoes as raw material | |
| CN114621836A (en) | Yellow wine yeast and preparation method thereof | |
| CN106086093B (en) | Lactic acid fermentation bacteria residue pretreatment method and method for producing lactic acid by circular fermentation | |
| CN116179617B (en) | Method for preparing citric acid by fermentation | |
| CN106244638B (en) | Comprehensive utilization process for producing lactic acid by biomass circulating fermentation | |
| CN106047954B (en) | Method for producing lactic acid and co-producing protein feed through circulating fermentation | |
| CN114369627B (en) | Method for producing lactic acid by promoting co-fermentation of kitchen waste and mushroom residue by aspergillus niger | |
| CN105586367A (en) | Method for conducting fermentative production of citric acid by adding saccharifying enzyme stage by stage based on pH responses | |
| FI85501B (en) | FOERFARANDE FOER FRAMSTAELLNING AV POLYOLER GENOM PAO INDUSTRIELL SKALA BASERAD FERMENTATION AV SOCKER. | |
| CN102212484B (en) | Method for controlling growthform of filamentous fungi during fermentation process | |
| CN105087519B (en) | Gene engineering inulinase and its method that crystal diabetin is prepared as raw material using jerusalem artichoke | |
| CN1074242A (en) | The method that using solid distillers ' grains, yellow water by fermentation are produced lactic acid | |
| CN107849592A (en) | Use heat resistance bacillus fermentation next life lactic acid producing or the method for its salt | |
| CN114214370B (en) | Method for improving organic acid production efficiency of aspergillus | |
| CN101153247A (en) | Novel method of wine brewing with sweet potato raw meal yeast wine separation continuous fermentation | |
| CN109182407A (en) | A kind of tryptophan preparation method and its fermentation medium and tryptophan that use fermentation special nutritional member | |
| CN112176004B (en) | Production method of citric acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |