WO2020056536A1 - Citric acid-producing microorganism strain and method for producing citric acid by fermenting starch sugar therefor - Google Patents

Abstract

Disclosed are a citric acid-producing microorganism strain and a method for producing citric acid by fermenting starch sugar therefor. The strain is Aspergillus niger 101-HAC11 with the he preservation number of CGMCC No.12480. Moreover, also disclosed are a culture medium formula and a fermentation process control process for producing citric acid by using starch sugar as a carbon source. The yield of citric acid produced by the strain is 166.8 g/L. Compared with the prior art, the citric acid yield of the recombinant Aspergillus niger strain is 220.34 g/L, and the yield is increased by 32.10%. The method has high speed, simple production process, high saccharic acid conversion rate, and high production efficiency, and is applicable to automated large-scale industrial production, and the products are easy to separate.

Description

A citric acid-producing microorganism strain and a method for producing citric acid by fermenting starch sugar Technical field

The invention belongs to the technical field of fermentation engineering, and relates to a method for genetically constructing a high fermentation intensity Aspergillus niger strain to produce citric acid by applying starch sugar fermentation. More specifically: a genetically engineered strain of Aspergillus niger with high fermentation strength, adding starch organic sugar as a fermentation carbon source, adding other organic and inorganic nitrogen sources, and a method for producing citric acid by fermentation with essential metal salt components.

Background technique

Citric acid is a common organic acid. It is widely used in food and beverage, pharmaceutical, chemical and cosmetics industries. It is currently the world's most demanded organic acid.

In 1784, C.W. Scheler used citrate precipitation method to prepare citric acid. At the end of the 19th century, the prototype of the biological fermentation method was formed. In 1916, most Aspergillus species such as Aspergillus oryzae, Aspergillus awamori, Aspergillus green and Aspergillus niger, and Aspergillus niger were tested by Tom and Curry to produce citric acid, and Aspergillus niger topped the list. Before the middle of the 20th century, shallow-plate fermentation was the mainstream. In 1923, the world's first factory to produce citric acid by shallow-plate fermentation with Aspergillus niger was in Fitzer Corporation. After 1950, a deep fermentation method was gradually established. Compared with shallow-plate fermentation, deep-layer fermentation has shorter cycles, higher yields, smaller floor space, and less labor. It is very advantageous for industrial production and has become the main method for citric acid production.

China's earliest report by Tang Tenghan and others in 1942 described the production of citric acid by fermentation. In 1952, citric acid was prepared by shallow-plate fermentation. In 1959, the 200L large-scale deep-fermentation citric acid test of the Ministry of Industry and Fermentation Industry Science Institute was initially successful. After 1966, the Tianjin Industrial Microbiology Research Institute and the Shanghai Industrial Microbiology Research Institute successively used dried potato powder as the raw material to prepare Citric acid, with successive successes, has identified a major process route for the production of citric acid in China. Since 2000, citric acid production in our country has used corn flour to spray liquefied sugar solution at high temperature, apply liquid submerged fermentation of Aspergillus niger strain to produce citric acid, and use calcium salt method and sulfuric acid hydrolysis to separate and extract citric acid. With the technological progress of China's fermentation industry and the increasing environmental protection requirements of the production process, the production of citric acid by this process has been unable to meet the requirements of high-intensity fermentation citric acid.

The main reasons are as follows:

(1) Application of corn flour liquefaction liquid with some corn dregs to co-ferment, resulting in viscous fermentation mash. At present, the total sugar of fermented mash is 17% -18%. If the total sugar of mash is increased, the liquid will be viscous. The degree of further increase, the fermentation process requires too much ventilation and stirring energy consumption to achieve dissolved oxygen, and industrialization cannot be achieved, so the acid production in a single tank is limited to about 18%, which cannot be improved.

(2) The corn liquefaction liquid contains various saccharides such as dextrin, oligosaccharides, oligosaccharides, maltose, and isomaltose, and contains different kinds of proteins, resulting in complicated fermentation products and various heteroacids. Fermented mash has various components, which makes it difficult to separate and extract citric acid. At present, the citric acid extraction uses the calcium salt method, which is most conducive to the removal of hetero acids, but at the same time, calcium sulfate waste residue can not be processed, and industrial waste residue is formed. Because it is difficult to separate and purify citric acid from fermented mash, the citric acid has been extracted by the calcium salt method, and calcium sulfate has become a hidden danger of solid waste for various citric acid production companies.

(3) At the end of citric acid fermentation, the mycelial ball of Aspergillus niger and the unusable corn dregs in the fermentation solid waste are mixed together and can only be dried to enter the feed market. High value-added products such as sterols.

(4) Because citric acid fermentation uses corn flour liquefied liquid as raw material, it is greatly affected by corn quality, such as corn kernel drying temperature, corn producing area, corn variety, corn storage time, etc. will affect the carbon and nitrogen ratio in corn liquefied liquid. , Leading to unstable fermentation process, affecting citric acid production.

(5) At present, citric acid fermentation uses corn flour liquefied liquid as a raw material, which causes difficulty in measuring and controlling dissolved oxygen and pH in the fermentation process, and automatic control and continuous production of the fermentation process cannot be achieved.

Based on the above reasons, it is imperative to develop citric acid fermentation based on starch sugar.

The citric acid process based on starch sugar fermentation has the following advantages:

(1) The powdery sugar is the solid material in the fermentation broth, the viscosity of the fermentation mash is low, and the initial sugar concentration can be increased to 25%, and the acid production in a single tank can be more than 25%, which improves the labor production efficiency.

(2) Starch sugar can be completely dissolved in water, and the viscosity of the fermentation mash is low, which is good for dissolved oxygen. The energy consumption for aeration and stirring in the fermentation process is greatly reduced, and the energy saving goal of the citric acid fermentation process is achieved.

(3) Starch sugar fermentation produces less citric acid and less citric acid. The purity of citric acid is high, and the concentration of other easy carbon compounds is low, which is conducive to the separation and extraction of citric acid. Method to eliminate calcium sulfate waste from the calcium salt method.

(4) Aspergillus niger mycelium ball produced by citric acid fermentation of starch sugar can be directly separated from the fermentation mash, and used for the extraction of ammonia sugar and sterol to realize high value-added products.

(5) Starch sugar fermentation citric acid fermentation broth has no solids except for Aspergillus niger mycelium bulbs, which is beneficial for the online observation of mycelium bulbs during the fermentation process, and the online inspection of the fermentation broth can be carried out to achieve automatic control and Accurate optimization of the fermentation process, and finally the realization of continuous fermentation processes such as starch sugar addition, hypha ball cutting and replication.

The key technical difficulties in achieving citric acid fermentation of starch sugar:

(1) The type and concentration of organic nitrogen source added during the citric acid fermentation of starch sugar is the key to achieve high conversion of citric acid fermentation.

(2) A good mycelium ball can be formed during the citric acid fermentation of starch sugar.

(3) High-conversion strain of citric acid for starch sugar fermentation.

Summary of the Invention

The purpose of the present invention is to provide a medium formula for fermentation production of citric acid by using Aspergillus niger 101-HAC11 (CGMCC No. 12480) using starch sugar as a carbon source and a fermentation process control process.

Another object of the present invention is to provide a strain of Aspergillus niger 101-HAC11, which is a strain microbially fermenting starch sugar with high citric acid production, and is classified as Aspergillus niger, and the deposit number is CGMCC No. 12480.

Another object of the present invention is to disclose a method for producing citric acid by fermenting starch sugars using Aspergillus niger 101-HAC11 (CGMCC No. 12480) strain.

To achieve the above object, the present invention discloses the following technical content:

The genetically engineered Aspergillus niger 101-HAC11 microbial fermentation strain was constructed and classified as Aspergillus niger with the accession number CGMCC No. 12480.

The strain CGMCC No. 12480 provided by the present invention is a new strain of Aspergillus niger capable of fermenting citric acid with starch sugars and being constructed by genetic engineering. The strain is used to produce citric acid by fermenting the saccharide raw materials. The classification belongs to Aspergillus niger ), The deposit number is CGMCC No. 12480, and the deposit date is June 12, 2016. Deposited place: General Microbial Center of China Microbial Strain Collection Management Committee. This strain is a citric acid-producing strain Aspergillus niger WLG CGMCC No. 10142, deposited on December 11, 2014, and deposited at the General Microbial Center of the China Microbial Species Collection Management Committee. Aspergillus niger WLG CGMCC No. 10142 was the starting strain. A high-fermentation-strength citric acid strain obtained by overexpressing the aox1 gene, the pyruvate carboxylase gene, and the pyruvate dehydrogenase gene was constructed by genetic engineering.

The physical and chemical properties of Aspergillus niger 101-HAC11 microbial fermentation strain, deposit number CGMCC No. 12480 are as follows: it can efficiently express the key enzymes pyruvate carboxylase, pyruvate dehydrogenase, and alternate oxidase in the citric acid synthesis process.

In the present invention, the inducible promoter Pgla is used to start the PC protein expression in Aspergillus niger, and the constitutive promoter PgpdA is used to start the pyruvate dehydrogenase PD gene, the oxidase Aox1 gene is alternated, and the pyruvate carboxylase PC gene expression is enhanced. As well as activity, increase pyruvate content. Further enhance the oxidative metabolic flux of acetyl CoA and NADH, thereby increasing the production of citric acid and the conversion rate of sugar and acid.

In the method of the present invention, the citric acid production strain Aspergillus niger WLG CGMCC No. 10142 is used as a starting strain for genetic engineering transformation. The citric acid yield of the starting strain is 166.8 g / L. Compared with the prior art, the recombinant Aspergillus niger The citric acid yield of the strain was 220.34 g / L, and the yield increased by 32.10%.

The present invention discloses a method for producing citric acid by fermenting starch sugar with CGMCC No. 12480 strain, which is characterized by:

(1) The fermentation strain used is Aspergillus niger 101-HAC11 deposit number CGMCC No. 12480, and the composition of the fermentation medium is (g / L): 200-260g of starch sugar material, 60-80g / L of corn pulp, (NH ₄ ) ₂ SO ₄ 9 ～ 11g / L, MgSO ₄ · 7H ₂ O 0.05 ～ 0.15g / L, K ₂ HPO, 4 ～ 6g / L, CuSO ₄ 0.8mg / L ZnSO ₄ 0.02g / L, CaCl ₂ 2.0g / L, KCl 0.1g / L, FeSO ₄ 0.34mg / L pH 5.0 ～ 5.5;

(2) Control conditions of fermentation culture: 10% to 15% (v / v) of the inoculation amount is controlled, the seed age is controlled to 27 hours, the cultivated seeds are connected to the fermentation medium, and the cultivation temperature is 30 to 40 ° C. The fermentation aeration is 0.1 ～ 0.15m ³ / m ³ .min, the tank pressure is 0.07 ～ 0.12MPa, the stirring speed is 80 ～ 100 rpm, the fermentation time is 50 ～ 60 hours, the sterile air is passed during the fermentation process, and the fermentation results are lemon. The acid yield is 220-260g / L, and the sugar-acid conversion rate is more than 102%. It has the characteristics of fast, simple production process, simple production process, high sugar-acid conversion, suitable for automated large-scale industrial production, high production efficiency, easy product separation, and so on.

The preparation method of the present invention, wherein the starch sugar material comprises: starch (including: corn starch, potato starch, cassava starch) after liquefaction, saccharification and filtering of a sugar-containing solution, wherein the sugar includes glucose, sucrose, and fructose , Maltose, no color dextrin.

The present invention is described in more detail as follows:

A fermenting strain of a citric acid-producing microorganism, the classification is Aspergillus niger 101-HAC11, and the deposit number is CGMCC No. 12480. The construction method of the strain is: the pyruvate carboxylase PC gene and pyruvate in Aspergillus niger Multiple copies of the dehydrogenase PD gene and the alternate oxidase Aox1 gene were integrated into the Aspergillus niger genome, and the recombinant Aspergillus niger strain was used to ferment to produce citric acid; the expression of the pyruvate carboxylase PC gene was regulated by the Pgla promoter, and acetone The acid dehydrogenase PD gene and the alternate oxidase Aox1 gene are regulated by the PgpdA promoter.

The nucleotide sequence of the pyruvate carboxylase PC gene is shown in SEQ ID NO. 1, the Pgla promoter is an inducible promoter, and the nucleotide sequence of the promoter is shown in SEQ ID NO. 2.

A pyruvate carboxylase PC gene expression frame, the expression frame comprising a Pgla promoter, a pyruvate carboxylase PC gene, a HYG resistance marker, and a trp terminator, arranged in the order of Pgla-PC-HYG-trp . The nucleotide sequence of the trp terminator is shown in SEQ ID NO.3. The nucleotide sequence of the pyruvate dehydrogenase PD gene is shown in SEQ ID No. 4; the nucleotide sequence of the alternating oxidase Aox1 gene is shown in SEQ ID NO. 5, and the PgpdA promoter is a constitutive promoter The nucleotide sequence of the promoter is shown in SEQ ID NO.6. The nucleotide sequence of the HYG gene is SEQ ID NO: 7.

The method for preparing the recombinant Aspergillus niger according to the present invention includes the following steps:

② Construction of the Pyruvate carboxylase PC gene expression frame Pgla-PC-HYG-trp;

② Construction of pyruvate dehydrogenase PD gene and alternate oxidase Aox1 gene expression frame PgpdA-PD-Aox1-HYG-trp;

③ The gene expression frames prepared in steps ① and ② are transformed into Aspergillus niger, and the recombinant Aspergillus niger is obtained through resistance screening and PCR identification.

The nucleotide sequence of the HYG gene in the resistance expression box in step ① is shown in SEQ ID NO.7.

(2) Liquid submerged fermentation of Aspergillus niger 101-HAC11 for citric acid production and fermentation process control:

a. The effect of different organic nitrogen sources on Aspergillus niger 101-HAC11 starch saccharide citric acid fermentation; different organic nitrogen sources have significant effects on Aspergillus niger 101-HAC11 citric acid fermentation, sugar acid conversion rate, etc. The results are shown in Figure 1.

As can be seen from Figure 1, the order of the effects of the four nitrogen sources on acid production is corn slurry> soy protein peptone> protein feed> yeast meal. When corn slurry is added, the acid production can reach up to 9.27%, and the sugar-acid conversion rate is higher than other nitrogen. Sources, and relatively few reducing sugars, followed by soybean protein, fermenting performance of adding corn pulp and soybean peptone is better than adding other organic nitrogen sources. Based on the above considerations of acid production, conversion rate, residual reducing sugar, and bacterial morphology, among the four organic nitrogen sources, corn pulp was finally selected as the most suitable organic nitrogen source for citrate corn serum fermentation.

b. Determination of the amount of corn slurry added

Corn pulp is determined as the best organic nitrogen source. The concentration of corn pulp is one of the important factors related to the efficient fermentation of citric acid. The optimal concentration of corn pulp is determined by measuring the final acid production and the calculation of sugar-acid conversion. The results are shown in Figure 2. It can be seen from FIG. 2 that when the corn pulp concentration is 0.7%, the acid yield and sugar acid conversion rate of Aspergillus niger are the highest, and the increase in corn pulp acid production will not increase. Comprehensive comparison of acid production and conversion index, 0.7% corn pulp was selected as the best organic nitrogen source for fermentation medium.

c.Effects of different concentrations of (NH ₄ ) ₂ SO ₄ on fermentation of citrate serum

After adding 0.7% corn slurry to the fermentation medium, different final concentrations of (NH ₄ ) ₂ SO _{4 were added} to determine the optimal concentration of (NH ₄ ) ₂ SO ₄ in the fermentation broth by determining the final acid production. The result is shown in Figure 3. As can be seen from FIG. 3, when the total concentration of (NH ₄ ) ₂ SO ₄ in the fermentation broth is 0.1%, both the acid production and the sugar-acid conversion rate are highest. When other concentrations of (NH ₄ ) ₂ SO ₄ are added, the acid production and sugar-acid conversion are not ideal, so 0.1% is selected as the optimum total concentration of (NH ₄ ) ₂ SO ₄ .

d.Effect of different concentrations of inositol on fermentation of citrate serum

Different concentrations of inositol were added to the fermentation medium. Finally, the final acid production was measured and the mycelial morphology was observed to determine the optimal amount of inositol in the fermentation broth of the supernatant. The results are shown in Figure 4. As can be seen in Figure 4, from the perspective of acid production and sugar-acid conversion, the addition of a certain amount of inositol has a significant effect on increasing acid production. The fermentation acid production with a certain amount of inositol is higher than the fermentation acid production without the addition of inositol. Among them, when the inositol was added at a total concentration of 0.007%, the rate of acid production and sugar acid conversion was the highest. When the concentration of inositol continued to increase, the acid production no longer increased and the consistency of mycelium decreased, and mycelium spread more. Considering the above factors, 0.007% inositol was selected as the optimal concentration.

e. Effect of metal ion concentration on fermentation

Various concentrations of MgSO ₄ , CuSO ₄ , ZnSO ₄ , CaCl ₂ , KCl, and FeSO ₄ were selected for orthogonal experiments. The results of the orthogonal experiments are shown in Table 1.

Table 1 Orthogonal test results

Table 2 Orthogonal test variance analysis

From Table 2, it can be seen that among the different levels of the six factors MgSO ₄ , CuSO ₄ , ZnSO ₄ , CaCl ₂ , KCl, and FeSO _4, it can be known from the range analysis that the major and minor order of the influencing factors is: CaCl ₂ ＞ MgSO ₄ ＞ KCl ＞ FeSO ₄ ＞ ZnSO ₄ ＞ CuSO ₄ , the optimal combination is A1B1C1D1E1F3, and the optimal concentration after optimization: MgSO ₄ 0.1g / L, CuSO ₄ 0.8mg / L ZnSO ₄ 0.02g / L, CaCl ₂ 2.0g / L , KCl 0.1g / L, FeSO ₄ 0.34mg / L.

Under the optimal concentration culture conditions, a verification test was performed on the optimization results. The volume of the 500 mL triangle bottle was 50 mL, 36.5 ° C, and 330 r / min for citric acid fermentation for 72 h. The result of the verification is 9.96%, which is higher than that of any group in the orthogonal experiment, and the advantage effect of the best combination is verified.

f. Optimum fermentation conditions for starch sugar serum

(1) Effect of different initial sugar concentrations on citric acid fermentation

After selecting different starch saccharin liquids for fermentation, the citric acid yield was measured. The residual sugar results are shown in Table 3.

Table 3 Citric acid production after fermentation

As can be seen from Table 3, when the initial sugar concentration was 20% -22%, the sugar-acid conversion rate reached 100% or more at the end of fermentation, and the residual sugar concentration was at a relatively low level.

(2) Effect of seed age on citric acid fermentation

Seed experiments with different seed ages were inoculated for fermentation experiments, the final citric acid content, residual reducing sugars were recorded, and the corresponding fermentation cycle was recorded. The results are shown in Table 4.

Table 4 Effect of different ages on citric acid fermentation

It can be known from Table 4 that with the increase of seed age, the citric acid production gradually increases, and the acid production is the highest at the age of 27h, so the age of 27h is selected as the best time for transplanting Aspergillus niger.

(3) Effect of different process control on fermentation of citric acid serum

Different process controls make the dissolved oxygen concentration in the fermentation broth significantly different, and the dissolved oxygen concentration has a great effect on citric acid fermentation. Samples were taken every 8 hours from the start of fermentation to determine the changes in citric acid concentration and pH in the fermentation broth. The results are shown in Figure 5. It can be seen from FIG. 5 that, from 0 to 16 hours, a large amount of dissolved oxygen is better than staged ventilation; from 16 to 64 hours, the solution A is higher than the solution B. With reference to FIG. 5, in the solution B, the dissolved oxygen is too high in the early fermentation stage, which may cause excessive proliferation of bacteria, which may cause the Aspergillus niger to enter the citric acid accumulation period prematurely, and the pH value may fall too quickly. Low pH will affect the saccharification enzyme activity, and then affect the utilization of raw materials, so that the residual sugar content in the fermentation broth is too high, so the solution B in the late fermentation stage is not as good as the solution A. Finally, choose the three-stage ventilation of scheme A.

It can be seen from FIG. 5 that in the early fermentation period (0-16h), the ventilation volume is controlled at 0.15m ³ / m ³ .min, the bacteria proliferate in large numbers, the metabolism is normal, and the pH of the fermentation broth is 3.5. This pH value is beneficial to maintaining the fermentation enzyme saccharification enzyme Vitality to increase the reducing sugar content. After 16h of fermentation, Aspergillus niger enters the rapid acid production period. The ventilation volume is adjusted to 0.225m ³ / m ³ .min and the rotation speed is 90 ～ 100r / min at this time. The metabolism is more vigorous, and more reducing sugar is converted into citric acid. At this time, the reducing sugar content begins to decrease sharply, and the acid production begins to increase greatly. After 48 hours, the fermentation is nearing the end, and the ventilation volume is adjusted to: 0.10 m ³ / m ³ .min, the speed is 85 ～ 95r / min, at this time the reducing sugar is at a low level, the dissolved oxygen is high, the pH value is relatively stable, and the ventilation and speed can be reduced. At the end of the 58h fermentation, the citric acid yield was 220.3 g / L, the sugar-acid conversion was 102.8%, and the amount of residual reducing sugar was 0.1%.

The citric acid-producing microorganism fermentation strain and the preparation method disclosed by the present invention have the following positive effects:

(1) Fermented mash with powdered sugar as raw material has low viscosity, and can increase the initial sugar concentration to 25%, achieve single-pot acid production above 25%, and improve labor production efficiency.

(2) Starch sugar can be completely dissolved in water, which is beneficial to dissolved oxygen. The energy consumption for aeration and stirring in the fermentation process is greatly reduced, and the energy saving goal of the citric acid fermentation process is achieved.

The nucleotide sequence of the Pgla promoter is SEQ ID NO: 2

Trp terminator nucleotide sequence SEQ ID NO: 3

The nucleotide sequence of PD gene SEQ ID NO4

Aox1 gene nucleotide sequence SEQ ID NO: 5

The nucleotide sequence of the PgpdA promoter is SEQ ID NO: 6

The nucleotide sequence of HYG gene SEQ ID NO: 7

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Effect of different organic nitrogen sources on citric acid fermentation;

Figure 2 Effect of different corn pulp contents on citric acid fermentation;

Figure 3 Effect of different (NH ₄ ) ₂ SO ₄ content on citric acid fermentation;

Figure 4 Effect of different inositol contents on citric acid fermentation;

Figure 5: The effect of different processes on the accumulation of 8-hour citric acid; A: three-stage ventilation of scheme A; B: acid production curve of large ventilation of scheme B.

detailed description

The present invention is described below through specific embodiments. Unless otherwise specified, the technical means used in the present invention are all methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative, rather than limiting the scope of the invention, which is only limited by the scope and spirit of the claims. For those skilled in the art, without departing from the spirit and scope of the present invention, various changes or modifications to the material composition and amount used in these embodiments also belong to the protection scope of the present invention. The raw materials and reagents used in the present invention are all commercially available.

Example 1

A method for constructing a fermenting strain of a citric acid-producing microorganism, which integrates multiple copies of a pyruvate carboxylase PC gene, a pyruvate dehydrogenase PD gene, and an alternate oxidase Aox1 gene from Aspergillus niger into the Aspergillus niger genome. The recombinant Aspergillus niger strain produces citric acid through fermentation; the expression of the pyruvate carboxylase PC gene is regulated by the Pgla promoter, and the pyruvate dehydrogenase PD gene and the alternating oxidase Aox1 gene are regulated by the PgpdA promoter.

Example 2

Method for preparing citric acid by using Aspergillus niger strain 101-HAC11 deposit number CGMCC No. 12480:

(1) The fermentation strain used is Aspergillus niger 101-HAC11 deposit number CGMCC No. 12480. The composition of the fermentation medium is (g / L): 200 g glucose, 60 g / L corn slurry, (NH ₄ ) ₂ SO ₄ 9g / L, MgSO ₄ · 7H ₂ O 0.05g / L, K ₂ HPO, 4g / L, CuSO ₄ 0.8mg / L ZnSO ₄ 0.02g / L, CaCl ₂ 2.0g / L, KCl 0.1g / L, FeSO ₄ 0.34mg / L pH 5.0 ～ 5.5;

(2) Fermentation control conditions: Controlled by 10% (v / v) inoculation amount, seed age is controlled at 27 hours, the cultivated seeds are connected to the fermentation medium, the cultivation temperature is 30 ° C, and the fermentation aeration is 0.1m ³ / m ³ .min, tank pressure 0.07 MPa, stirring speed 80 rpm, fermentation time 50 hours, sterile air is introduced during fermentation, the citric acid yield is 220 g / L, and the sugar-acid conversion rate is 102%. .

Example 3

Method for preparing citric acid by using Aspergillus niger strain 101-HAC11 deposit number CGMCC No. 12480:

(1) The fermentation strain used is Aspergillus niger 101-HAC11 deposit number CGMCC No. 12480, and the fermentation medium composition is (g / L): glucose 260g, corn slurry 80g / L, (NH ₄ ) ₂ SO ₄ 11g / L, MgSO ₄ · 7H ₂ O 0.15g / L, K ₂ HPO 6g / L, CuSO ₄ 0.8mg / L ZnSO ₄ 0.02g / L, CaCl ₂ 2.0g / L, KCl 0.1g / L, FeSO ₄ 0.34mg / L pH 5.0 ～ 5.5;

(2) Control conditions of fermentation culture: Controlled by inoculation amount of 15% (v / v), seed age is controlled at 27 hours, the cultivated seeds are connected to the fermentation medium, the cultivation temperature is 40 ° C, and the fermentation aeration is 0.15m ³ / m ³ .min, tank pressure of 0.12 MPa, stirring speed of 100 rpm, fermentation time of 60 hours, sterile air was introduced during fermentation, fermentation yield was 260 g / L, sugar acid conversion rate was 100% .

Example 4

Method for preparing citric acid by using Aspergillus niger strain 101-HAC11 deposit number CGMCC No. 12480:

(1) The fermentation strain used is Aspergillus niger 101-HAC11 deposit number CGMCC No. 12480, and the fermentation medium composition is (g / L): corn or potato starch saccharification solution is calculated based on 200g of glucose and 60g of corn pulp / L, (NH ₄ ) ₂ SO ₄ 9g / L, MgSO ₄ · 7H ₂ O 0.05g / L, K ₂ HPO 4g / L, CuSO ₄ 0.8mg / L ZnSO ₄ 0.02g / L, CaCl ₂ 2.0g / L, KCl 0.1g / L, FeSO ₄ 0.34mg / L pH 5.0 ～ 5.5;

(2) Fermentation control conditions: Controlled by 10% (v / v) inoculation amount, seed age is controlled at 27 hours, the cultivated seeds are connected to the fermentation medium, the cultivation temperature is 30 ° C, and the fermentation aeration is 0.1m ³ / m ³ .min, tank pressure 0.07 MPa, stirring speed 80 rpm, fermentation time 50 hours, sterile air is introduced during fermentation, the citric acid yield is 220 g / L, and the sugar-acid conversion rate is 102%. .

Example 5

Method for preparing citric acid by using Aspergillus niger strain 101-HAC11 deposit number CGMCC No. 12480:

(1) The fermentation strain used is Aspergillus niger 101-HAC11 deposit number CGMCC No. 12480, and the fermentation medium composition is (g / L): corn or potato starch saccharification solution is calculated based on 260g of glucose and 80g of corn pulp / L, (NH ₄ ) ₂ SO ₄ 11g / L, MgSO ₄ · 7H ₂ O 0.15g / L, K ₂ HPO 6g / L, CuSO ₄ 0.8mg / L ZnSO ₄ 0.02g / L, CaCl ₂ 2.0g / L, KCl 0.1g / L, FeSO ₄ 0.34mg / L pH 5.0 ～ 5.5;

(2) Control conditions of fermentation culture: Controlled by inoculation amount of 15% (v / v), seed age is controlled at 27 hours, the cultivated seeds are connected to the fermentation medium, the cultivation temperature is 40 ° C, and the fermentation aeration is 0.15m ³ / m ³ .min, tank pressure of 0.12 MPa, stirring speed of 100 rpm, fermentation time of 60 hours, sterile air was introduced during fermentation, fermentation yield was 260 g / L, sugar acid conversion rate was 100% .

Example 6

Method for preparing citric acid by using Aspergillus niger strain 101-HAC11 deposit number CGMCC No. 12480:

(1) The fermentation strain used is Aspergillus niger 101-HAC11 deposit number CGMCC No. 12480, and the fermentation medium composition is (g / L): sucrose or maltose 200g, corn pulp 60g / L, (NH ₄ ) ₂ SO ₄ 9g / L, MgSO ₄ · 7H ₂ O 0.05g / L, K ₂ HPO 4g / L, CuSO ₄ 0.8mg / L ZnSO ₄ 0.02g / L, CaCl ₂ 2.0g / L, KCl 0.1g / L, FeSO ₄ 0.34mg / L pH 5.0 ～ 5.5;

(2) Fermentation control conditions: Controlled by 10% (v / v) inoculation amount, seed age is controlled at 27 hours, the cultivated seeds are connected to the fermentation medium, the cultivation temperature is 30 ° C, and the fermentation aeration is 0.1m ³ / m ³ .min, tank pressure 0.07 MPa, stirring speed 80 rpm, fermentation time 50 hours, sterile air is introduced during fermentation, the citric acid yield is 220 g / L, and the sugar-acid conversion rate is 102%. .

Example 7

Method for preparing citric acid by using Aspergillus niger strain 101-HAC11 deposit number CGMCC No. 12480:

(1) The fermentation strain used is Aspergillus niger 101-HAC11 deposit number CGMCC No. 12480, and the fermentation medium composition is (g / L): sucrose or maltose 260g, corn pulp 80g / L, (NH ₄ ) ₂ SO ₄ 11g / L, MgSO ₄ · 7H ₂ O 0.15g / L, K ₂ HPO, 6g / L, CuSO ₄ 0.8mg / L ZnSO ₄ 0.02g / L, CaCl ₂ 2.0g / L, KCl 0.1g / L , FeSO ₄ 0.34mg / L pH 5.0 ～ 5.5;

(2) Control conditions of fermentation culture: Controlled by inoculation amount of 15% (v / v), seed age is controlled at 27 hours, the cultivated seeds are connected to the fermentation medium, the cultivation temperature is 40 ° C, and the fermentation aeration is 0.15m ³ / m ³ .min, tank pressure of 0.12 MPa, stirring speed of 100 rpm, fermentation time of 60 hours, sterile air was introduced during fermentation, fermentation yield was 260 g / L, sugar acid conversion rate was 100% .

Claims (4)

1. A fermenting strain of citric acid-producing microorganism, its classification is Aspergillus niger 101-HAC11, deposit number CGMCC No. 12480
2. The method for constructing a fermentation strain of a citric acid-producing microorganism according to claim 1, characterized in that the strain integrates a pyruvate carboxylase PC gene, a pyruvate dehydrogenase PD gene, and an alternate oxidase Aox1 gene in Aspergillus niger. On the Aspergillus niger genome, the recombinant Aspergillus niger strain is used to produce citric acid; the expression of the pyruvate carboxylase PC gene is regulated by the Pgla promoter, and the pyruvate dehydrogenase PD gene and the alternate oxidase Aox1 gene are activated by PgpdA Subregulation.
3. A method for preparing citric acid by using Aspergillus niger strain 101-HAC11 deposit number CGMCC No. 12480 according to claim 1:

   (1) The fermentation strain used is Aspergillus niger 101-HAC11 deposit number CGMCC No. 12480, and the composition of the fermentation medium is (g / L): 200-260g of starch sugar material, 60-80g / L of corn pulp, (NH ₄ ) ₂ SO ₄ 9 ～ 11g / L, MgSO ₄ · 7H ₂ O 0.05 ～ 0.15g / L, K ₂ HPO, 4 ～ 6g / L, CuSO ₄ 0.8mg / L ZnSO ₄ 0.02g / L, CaCl ₂ 2.0g / L, KCl 0.1g / L, FeSO ₄ 0.34mg / L pH 5.0 ～ 5.5;

   (2) Control conditions for fermentation culture: The inoculation amount is controlled to 10% to 15% (v / v), the seed age is controlled to 24 to 27 hours, the cultivated seeds are connected to the fermentation tank, the cultivation temperature is 30 to 40 ° C, and the fermentation is aerated The amount is 0.1 ～ 0.15m ³ / m ³ .min, the tank pressure is 0.07 ～ 0.12MPa, the stirring speed is 80 ～ 100 rpm, the fermentation time is 50 ～ 60 hours, the sterile air is passed during the fermentation process, and the citric acid yield is obtained as a result of fermentation. 220 ～ 260g / L, sugar-acid conversion rate is 100% ～ 102%
4. The preparation method according to claim 9, wherein the starch sugar material comprises starch, including corn starch, potato starch, cassava starch after liquefaction and saccharification, and a sugar-containing solution filtered, and the sugar includes glucose, sucrose, fructose, Maltose, no color dextrin.

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