A strain of Aspergillus niger and method and application thereof for producing sodium gluconate by fermentation

Abstract

The invention discloses a strain of Aspergillus niger ( Aspergillus niger ) OSYZTT‑02, when the initial sugar concentration is 30%‑32%, the high temperature fermentation is 40℃‑42℃, and the dissolved oxygen content is 30%‑32%, 10% of the seed liquid is inoculated into the fermentation tank together with the crude enzyme solution for fermentation, and 32% of sodium hydroxide is used to adjust the pH to 5.2 for fermentation. The fermentation cycle is 16h, and the glucose consumption rate reaches 17.68g / (L·h), and the conversion rate of sodium gluconate can reach 96.43%. It can reduce the impact of ambient temperature on fermentation production, and at the same time, the mycelium obtained by fermentation production can be reused to obtain crude enzyme solution, which can save the cost of purchasing enzymes while increasing the fermentation speed, shortening the fermentation cycle, shortening the profit recovery time of the enterprise, and improving the profit of the enterprise.

Description

Technical Field

[0001] The invention relates to the field of biochemical engineering, and in particular to an Aspergillus niger strain and a method and application for producing sodium gluconate by fermentation. Background Art

[0002] Sodium gluconate is a widely used polyhydroxy organic acid salt, which is widely used in food, medicine, light industry, chemical industry and other industries. In the food industry, it can be used as a nutritional supplement, food preservative, quality improver, buffer and low-salt (salt-free) food; in the pharmaceutical industry, it can be used as an acid-base balance agent; sodium gluconate has good chelating and corrosion and scale inhibition effects, and is widely used in surface cleaning agents and water quality stabilizers; sodium gluconate can increase the plasticity, strength and durability of concrete, and is widely used in the construction industry as a water reducer and retarder; in addition, sodium gluconate is also the basic raw material for the preparation of gluconolactone, gluconates (zinc, copper, ferrous salts), etc.

[0003] The main production methods of sodium gluconate are catalytic oxidation and microbial fermentation. The catalytic oxidation method uses glucose as raw material and precious metals as catalysts to catalytically oxidize glucose to produce gluconic acid, which is then neutralized with sodium hydroxide to produce sodium gluconate. This method has the disadvantages of high production cost, harsh reaction conditions, many side reactions, and serious environmental pollution. The microbial fermentation method uses sugar as raw material, which is converted into gluconic acid by microorganisms, and then sodium gluconate is obtained by adding sodium hydroxide solution during the fermentation process. This method has the advantages of mild conditions, simple and environmentally friendly process route, and low product cost, and has become the main production method of sodium gluconate.

[0004] Currently, the most common strain used for sodium gluconate fermentation production is Aspergillus niger, but the strain's poor high temperature resistance is a problem faced by related companies. The optimum temperature for Aspergillus niger growth is generally 33°C-37°C. When the fermentation tank temperature exceeds 40°C, the strain growth and enzyme production rate drop significantly. Especially in summer, the temperature in the production workshop can reach 40°C. In order to ensure normal production, a large amount of cooling water must be consumed to cool the fermentation tank, thereby consuming a lot of energy. In the patent number CN103667375A, entitled A method for preparing sodium gluconate using Aspergillus niger fermentation, the fermentation temperature of Aspergillus niger is 30±2°C, while in the patent number CN105385713A, entitled A method for sodium gluconate fermentation with bacterial cell recycling, the fermentation temperature of Aspergillus niger is 36°C-37°C;

[0005] Based on the fact that Aspergillus niger has some restrictions on the fermentation temperature, in recent years, glucose has been directly catalyzed and treated by enzymatic method, and sodium gluconate is obtained by acidification with sodium hydroxide, such as patent number CN118147243A, which is called a method for preparing sodium gluconate, in which glucose oxidase and catalase are added to starch saccharification liquid, and sodium hydroxide solution is used for acidification, and the enzymolysis cycle reaches 21 hours. Although the reaction conditions and enzymolysis cycle are more advantageous than fermentation with Aspergillus niger, glucose oxidase and catalase are added once and cannot be recovered during the enzymolysis catalysis process, which increases the cost of raw materials invisibly. Summary of the invention

[0006] The object of the present invention is to provide a strain of Aspergillus niger for producing sodium gluconate by fermentation, and the Aspergillus niger (OSYGN005) is obtained by ultraviolet mutagenesis and domestication using the laboratory preserved strain. Aspergillus niger ) OSYZTT-02, classified and named: Aspergillus niger ( Aspergillus niger ) OSYZTT-02, has been deposited in China Center for Type Culture Collection (CCTCC), Wuhan University, China, on June 17, 2024, with the deposit number CCTCC NO: M 20241233;

[0007] The second object of the present invention is to provide a fermentation method for producing sodium gluconate, wherein a crude enzyme liquid prepared from mycelium after the fermentation of sodium gluconate by Aspergillus niger is fermented at a high temperature of 42° C. together with Aspergillus niger seed liquid, so that the fermentation cycle is 16 hours, the conversion rate of sodium gluconate reaches 96.43%, and the profit recovery time of the enterprise is shortened;

[0008] The third object of the present invention is to provide a process for preparing a crude enzyme solution, wherein the mycelium after fermentation is collected and broken to obtain the crude enzyme solution, which is then recycled to improve the utilization rate of industrial intermediates.

[0009] The fourth object of the present invention is to provide the use of Aspergillus niger OSYZTT-02 in the preparation of sodium gluconate.

[0010] In order to realize the above technology, the present invention adopts the following technical measures:

[0011] Aspergillus niger Aspergillus niger )Obtaining OSYZTT-02:

[0012] The laboratory-preserved strain OSYGN005 was domesticated by UV mutagenesis to obtain Aspergillus niger ( Aspergillus niger )OSYZTT-02, classified and named: Aspergillus niger ( Aspergillus niger) OSYZTT-02, has been deposited in China Center for Type Culture Collection (CCTCC), Wuhan University, China, on June 17, 2024, with the deposit number CCTCCNO: M 20241233;

[0013] A method for producing sodium gluconate by fermentation comprises the following steps:

[0014] (1) Culture of bacteria: Aspergillus niger ( Aspergillus niger ) OSYZTT-02 is placed in a seed culture solution, cultured to obtain a seed solution for later use;

[0015] (2) Preparation of crude enzyme solution: The prepared seed solution is inoculated into the fermentation medium for fermentation. After the fermentation is completed, the fermentation solution is filtered to obtain mycelium. The mycelium is ruptured and inactivated to obtain crude enzyme solution of catalase and glucose oxidase. The filtered clear solution is concentrated and crystallized to obtain sodium gluconate.

[0016] (3) Fermentation culture: The prepared seed liquid and the crude enzyme liquid in the previous fermentation tank are inoculated into the fermentation medium for fermentation. After the fermentation is completed, the fermentation liquid is filtered to obtain the crude enzyme liquid from the mycelium, which is used for the fermentation in the next fermentation tank. The clear liquid is concentrated and crystallized to obtain sodium gluconate.

[0017] The Aspergillus niger ( Aspergillus niger ) OSYZTT-02 fermentation conditions are: 40℃-42℃ culture, rotation speed 150r / min, dissolved oxygen concentration 30%-32%, pH adjusted to 5.2 with 32% sodium hydroxide;

[0018] Seed culture medium: glucose 15-18%, ammonium sulfate 0.2-0.6g / L, dipotassium hydrogen phosphate 0.05-0.1g / L, ammonium nitrate 0.1-0.3g / L, magnesium sulfate 0.1-0.3g / L, sodium carbonate 2%, acetate buffer 60mmol / L, pH 5-6;

[0019] The fermentation medium is: glucose 32%-35%, ammonium sulfate 0.2-0.6g / L, dipotassium hydrogen phosphate 0.05-0.1g / L, ammonium nitrate 0.1-0.3g / L, magnesium sulfate 0.1-0.3g / L;

[0020] Preparation process of crude enzyme solution: the mycelium obtained by filtering the fermentation liquid through a candle filter is added to an acetic acid-sodium acetate buffer with a pH of 5-6, and the mycelium is broken by lysozyme, and then centrifuged and filtered through a centrifuge to obtain a supernatant, which is the crude enzyme solution.

[0021] In summary, the beneficial effects of the present application are as follows:

[0022] 1. Aspergillus niger screened by the present invention ( Aspergillus niger ) OSYZTT-02 achieves high temperature fermentation at 40℃-42℃, reducing the impact of ambient temperature on the fermentation of sodium gluconate by the strain and improving the fermentation efficiency of the strain;

[0023] 2. The present invention uses 10% seed liquid inoculation amount and crude enzyme liquid to inoculate into the fermentation tank for fermentation. When the initial sugar concentration is 30%-32%, the high temperature fermentation is 40°C-42°C, and the dissolved oxygen content is 30%-32%, 32% sodium hydroxide is used to adjust the pH to 5.2 for fermentation. The fermentation cycle is 16h, and the glucose consumption rate reaches 17.68g / (L·h), the conversion rate of sodium gluconate can reach 96.43%, and the residual sugar is less than 0.7g / L, achieving high output and low loss;

[0024] 3. The present invention utilizes fermentation to extract crude enzyme solution for the fermentation production of sodium gluconate, which can save enzyme purchase expenses and reduce enterprise expenditure costs;

[0025] 4. The inorganic salt culture medium used in the present invention for fermentation culture can greatly reduce the expenditure cost of raw materials, achieve low addition and high output, and improve the recovery benefits of the enterprise. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Other features, objects and advantages of the present application will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:

[0027] Figure 1 is the standard curve of sodium gluconate;

[0028] Figure 2 The influence of the number of passages of the strain of the present invention on its fermentation;

[0029] Figure 3 To study the effect of fermentation temperature on the production of sodium gluconate by the strain;

[0030] Figure 4 The effect of different inoculants on the fermentation production of sodium gluconate;

[0031] Figure 5 The effect of initial sugar concentration on the fermentation production of sodium gluconate;

[0032] Figure 6 The effect of the amount of seed solution added on the fermentation production of sodium gluconate;

[0033] Figure 7 The effect of dissolved oxygen on the fermentation production of sodium gluconate. DETAILED DESCRIPTION

[0034] The present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the relevant invention, rather than to limit the invention. It is also necessary to explain that, for ease of description, only the parts related to the invention are shown in the accompanying drawings.

[0035] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0036] Example 1

[0037] Aspergillus niger Aspergillus niger ) Acquisition of OSYZTT-02

[0038] The laboratory preserved strain OSYGN005 was activated and enriched using a seed solid culture medium at 37°C. After spores were produced, the spores were eluted with physiological saline to obtain a spore suspension, which was then placed under an ultraviolet lamp for 10-20 minutes and repeatedly irradiated to induce mutation. The induced spore suspension was then diluted and spread on a screening culture medium for plate screening at 42°C. Colonies with a larger diameter of a blue circle (larger than 0.5 cm in diameter) were selected. The selected colonies were cultured in a liquid shake flask using a seed liquid culture medium at 42°C and a rotation speed of 150 r / min. The content of gluconic acid in the shake flask was detected, and strains with a higher content of gluconic acid in the shake flask were selected. These strains were then subjected to high temperature acclimation culture, and the strains finally obtained were named Aspergillus niger ( Aspergillus niger )OSYZTT-02, the Aspergillus niger ( Aspergillus niger ) The OSYZTT-02 strain was deposited in the China Type Culture Collection of Wuhan University on June 17, 2024, with the deposit number CCTCC NO: M 20241233, and the classification name: Aspergillus niger ( Aspergillus niger )OSYZTT-02.

[0039] Seed liquid culture medium: glucose 15%, ammonium sulfate 0.6g / L, dipotassium hydrogen phosphate 0.1g / L, ammonium nitrate 0.3g / L, magnesium sulfate 0.3g / L, sodium carbonate 2%, acetate buffer 60mmol / L, pH 5-6;

[0040] The solid seed culture medium is a liquid seed culture medium with 2% agar added;

[0041] Screening culture medium: glucose 15%, ammonium sulfate 0.6g / L, dipotassium hydrogen phosphate 0.1g / L, ammonium nitrate 0.3g / L, magnesium sulfate 0.3g / L, calcium carbonate 0.35%, soluble starch 1%, potassium iodide 0.17%, sodium deoxycholate 0.02%, agar powder 1.5%, acetate buffer 60mmol / L, pH 5-6.

[0042] Method for detecting sodium gluconate by indicator colorimetric method:

[0043] Accurately weigh 2.18g (±0.001g) of sodium gluconate that has been dried to constant weight at 105°C, dissolve it with glacial acetic acid at a slight heat, cool it, and dilute it to 100ml with glacial acetic acid. Take 10ml, 20ml, 30ml, 40ml, 50ml, and 60ml of sodium gluconate acetic acid solution, dilute it to 100ml with glacial acetic acid, and prepare it to concentrations of 0.01mol / L, 0.02mol / L, 0.03mol / L, 0.04mol / L, 0.05mol / L, and 0.06mol / L, respectively. Then take the same volume (10mL) of sodium gluconate solution of different concentrations, add a few drops of quinaldine red solution, and titrate it with 0.10mol / LHClO4 standard solution until the colorless end point, and draw a standard curve. The standard curve is as shown below. Figure 1 shown.

[0044] Detection method of sodium gluconate in shake flask: Take 10mL of supernatant obtained after centrifugation of the shake flask liquid, add 3 drops of quinaldine red solution, and titrate with 0.10mol / LHClO4 standard solution until colorless, and record the volume V of 0.10mol / LHClO4 standard solution consumed. 耗 According to the standard curve formula, y=81.114x-0.0173, y is the consumption of 0.10mol / L HClO4 standard solution V 耗 , x is the sodium gluconate content in the supernatant in mol / L, calculate the sodium gluconate content in the shake flask.

[0045] Sodium gluconate content g / L=x×M1

[0046] M1: Molecular weight of sodium gluconate, 218.

[0047] Example 2

[0048] Aspergillus niger Aspergillus niger ) OSYZTT-02 genetic stability experiment

[0049] In order to verify the genetic stability of this strain, a flat screening medium was used, and ten generations were inoculated on the plate continuously. The colonies on each generation of plates were picked up with an inoculation loop and transferred to the seed liquid medium for shake flask culture. The culture conditions were: 42°C, rotation speed 150r / min, and culture time 8h. Then, each generation was fermented in a 5L fermenter containing 3L fermentation medium with an inoculation amount of 5%. The fermentation conditions were: 42°C, rotation speed 150r / min, fermentation dissolved oxygen content of 30%, and 32% sodium hydroxide solution was used to adjust the pH value to 5.2. When the residual sugar concentration in the fermentation liquid was lower than 3g / L, the fermentation was considered to be over. After the fermentation was completed, the fermentation liquid was centrifuged and the content of sodium gluconate in the fermentation liquid was detected by indicator colorimetric method. Each generation of Aspergillus niger ( Aspergillus niger ) After OSYZTT-02 fermentation is completed, the content of sodium gluconate in the fermentation liquid is as follows Figure 2 shown.

[0050] Seed liquid culture medium: glucose 15%, ammonium sulfate 0.6g / L, dipotassium hydrogen phosphate 0.1g / L, ammonium nitrate 0.3g / L, magnesium sulfate 0.3g / L, sodium carbonate 2%, acetate buffer 60mmol / L, pH 5-6;

[0051] Fermentation medium: glucose 25%, ammonium sulfate 0.4g / L, dipotassium hydrogen phosphate 0.1g / L, ammonium nitrate 0.2g / L, magnesium sulfate 0.2g / L;

[0052] like Figure 2 As shown, it can be seen that after ten generations of subculture and fermentation, it was found that the contents of sodium gluconate in the fermentation broth of each generation of strain fermentation were 261.58g / L, 259.77g / L, 262.19g / L, 260.57g / L, 260.17g / L, 258.95g / L, 258.80g / L, 260.97g / L, 260.37g / L and 258.92g / L respectively. It can be seen that after ten generations of subculture, the fluctuation of the content of sodium gluconate in the fermentation broth of each generation was less than 5%, indicating that the screened Aspergillus niger strain has good genetic stability and can be used for fermentation production.

[0053] Example 3

[0054] Effect of fermentation temperature on fermentation of strains

[0055] The activated Aspergillus niger ( Aspergillus niger ) OSYZTT-02 was cultured in a shake flask for 8h-12h, and then inoculated into a 5L fermentation tank containing 3L fermentation medium for fermentation culture. The pH value was adjusted to 5.2 with 32% sodium hydroxide solution. The fermentation culture temperature was 38℃, 40℃, 42℃, 44℃, and 46℃, the rotation speed was 150r / min, and the dissolved oxygen content was 30% until the fermentation was completed;

[0056] When the fermentation time is 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, and 36h, the fermentation liquid is taken and the sodium gluconate content in the fermentation liquid is detected by the indicator colorimetric method. The test results are as follows: Figure 3 shown.

[0057] Depend on Figure 3 It can be seen that when the fermentation temperature is 38°C, 40°C, and 42°C, the curves for producing sodium gluconate by fermentation are basically the same. When the fermentation temperature is 44°C and 46°C, after fermentation and cultivation, the content of sodium gluconate in the fermentation liquid does not increase substantially, and the growth of Aspergillus niger may be limited because of the high temperature. It is shown that the Aspergillus niger strain obtained by the present invention is suitable for growing in an environment where the temperature is 38°C-42°C. During summer fermentation, the temperature in the fermentation tank is higher than 40°C, and when the fermentation temperature is 37°C, bacterial contamination is likely to occur, so the optimum temperature of the Aspergillus niger strain obtained by the present invention is set to 40°C-42°C, which can reduce the probability of bacterial contamination on the one hand, and enable the Aspergillus niger strain to produce sodium gluconate by normal fermentation in a high temperature environment in summer on the other hand.

[0058] Example 4

[0059] Effects of crude enzyme solution, seed solution, and crude enzyme solution + seed solution on the fermentation production of sodium gluconate by strains

[0060] Preparation of seed solution: Activated Aspergillus niger ( Aspergillus niger ) After OSYZTT-02 was cultured in a shake flask for 8-12 hours, the seed solution was obtained and set aside;

[0061] Preparation of crude enzyme solution: Inoculate the seed solution at a 10% inoculation rate into a sterilized fermentation tank for fermentation. Fermentation conditions: fermentation temperature 42°C, rotation speed 150r / min, dissolved oxygen content 30%, use 32% sodium hydroxide solution to adjust the pH to 5.2, ferment for 28h-32h. After the fermentation is completed, pass the fermentation solution through a candle filter to obtain mycelium. The filtered mycelium is added to an acetic acid-sodium acetate buffer with a pH of 5-6. The wet weight of the mycelium: the volume of the buffer is 1:2. Lysozyme is added to the mixed mycelium buffer solution. The added volume of lysozyme: the volume of mycelium buffer solution is 1:100, that is, 10µl of lysozyme is added to 1ml of mycelium buffer solution, shake well and let stand for 1-2h, then centrifuge to obtain the supernatant. The obtained supernatant is the crude enzyme solution for standby use.

[0062] The fermentation clear liquid obtained by filtering through the candle filter enters the subsequent treatment process of sodium gluconate, adjusts the pH to neutral, and after heating to 60-70°C, it is filtered through a plate and frame to remove excess protein and mycelium, and then concentrated and crystallized to obtain sodium gluconate.

[0063] The prepared seed solution, crude enzyme solution, and seed solution + crude enzyme solution were inoculated into a 5L fermentation tank containing 3L fermentation medium for fermentation. The fermentation medium was: 25% glucose, 0.4g / L ammonium sulfate, 0.1g / L dipotassium hydrogen phosphate, 0.2g / L ammonium nitrate, and 0.2g / L magnesium sulfate. The fermentation conditions were: fermentation temperature 42°C, rotation speed 150r / min, dissolved oxygen content 30%, and pH was adjusted to 5.2 using 32% sodium hydroxide solution.

[0064] (1) The inoculation amount of seed solution is: 10%;

[0065] (2) The inoculation amount of crude enzyme solution is: the total amount of crude enzyme solution prepared by the crude enzyme solution preparation process;

[0066] (3) The inoculation amount of seed solution + crude enzyme solution is: all crude enzyme solution prepared by the crude enzyme solution preparation process + 2% seed solution;

[0067] When the fermentation time is 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, and 36h, the fermentation liquid is taken and the sodium gluconate content in the fermentation liquid is detected by the indicator colorimetric method. The test results are as follows: Figure 4 shown.

[0068] Depend on Figure 4 It can be seen that when the seed liquid, crude enzyme liquid, and seed liquid + crude enzyme liquid are added to the fermentation liquid for fermentation to produce sodium gluconate, the final concentration of sodium gluconate in the fermentation liquid hovers around 260 g / L, but the fermentation period of the seed liquid + crude enzyme liquid is significantly shorter than that of the seed liquid and crude enzyme liquid, that is, the fermentation period of the seed liquid + crude enzyme liquid can be shortened to 16 h-20 h, which not only indicates that the enzyme activity in the Aspergillus niger mycelium after the fermentation is completed can continue to be utilized to generate sodium gluconate from glucose, but also when it participates in the fermentation production of sodium gluconate together with the seed liquid, it can greatly improve the conversion rate of glucose, reduce its fermentation period, and shorten the profit recovery time of the enterprise;

[0069] Fermenting the seed liquid + crude enzyme liquid together facilitates the recycling preparation of the crude enzyme liquid, reduces the preparation steps of the crude enzyme liquid, can shorten the conversion time of the product, and reduce the investment in production costs.

[0070] Example 5

[0071] Effect of initial sugar concentration on the fermentation production of sodium gluconate

[0072] The seed solution + crude enzyme solution prepared in Example 4 were respectively introduced into 5L fermentation tanks containing 3L fermentation medium with different raw material ratios for fermentation.

[0073] The fermentation medium in the fermenter is:

[0074] Glucose 25%, ammonium sulfate 0.4g / L, dipotassium hydrogen phosphate 0.1g / L, ammonium nitrate 0.2g / L, magnesium sulfate 0.2g / L;

[0075] Glucose 28%, ammonium sulfate 0.4g / L, dipotassium hydrogen phosphate 0.1g / L, ammonium nitrate 0.2g / L, magnesium sulfate 0.2g / L;

[0076] Glucose 30%, ammonium sulfate 0.4g / L, dipotassium hydrogen phosphate 0.1g / L, ammonium nitrate 0.2g / L, magnesium sulfate 0.2g / L;

[0077] Glucose 32%, ammonium sulfate 0.4g / L, dipotassium hydrogen phosphate 0.1g / L, ammonium nitrate 0.2g / L, magnesium sulfate 0.2g / L;

[0078] Glucose 35%, ammonium sulfate 0.4g / L, dipotassium hydrogen phosphate 0.1g / L, ammonium nitrate 0.2g / L, magnesium sulfate 0.2g / L;

[0079] Glucose 37%, ammonium sulfate 0.4g / L, dipotassium hydrogen phosphate 0.1g / L, ammonium nitrate 0.2g / L, magnesium sulfate 0.2g / L;

[0080] The fermentation conditions were: fermentation temperature 42°C, rotation speed 150r / min, dissolved oxygen content 30%, and pH adjusted to 5.2 using 32% sodium hydroxide solution;

[0081] When the fermentation time is 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, and 36h, the fermentation liquid is taken and the sodium gluconate content in the fermentation liquid is detected by the indicator colorimetric method. The test results are as follows: Figure 5 shown.

[0082] Depend on Figure 5 It can be seen that when the inoculation amount of seed liquid + crude enzyme liquid is the same, when the concentration of the initial sugar liquid is between 25%-32%, the growth rate of sodium gluconate in the fermentation liquid is greater than the growth rate when the initial sugar concentration is 35%-37%, and when the initial sugar concentration is between 25%-32%, the fermentation cycle is 16h-20h, which is significantly less than the fermentation cycle of 28h-32h when the initial sugar concentration is 35%-37%. This may be because the initial sugar concentration is too high, which affects the osmotic pressure of the Aspergillus niger strain, thereby affecting the strain's use of glucose to produce sodium gluconate. And when the initial sugar concentration is 25%-28%, the sodium gluconate content is significantly lower than the sodium gluconate content produced when the initial sugar concentration is 30%-32%.

[0083] In summary, the initial sugar concentration is set between 30% and 32%, and the fermentation of seed liquid + crude enzyme solution is used with a fermentation cycle of 16h-20h, which can maximize the production capacity and the sodium gluconate content is 312.29g / L-331.72g / L.

[0084] Example 6

[0085] Effect of Seed Liquid Addition Amount on Fermentation Production of Sodium Gluconate

[0086] The prepared seed solution + crude enzyme solution was inoculated into a 5L fermentation tank filled with 3L fermentation medium for fermentation. The fermentation medium in the fermentation tank was: 32% glucose, 0.4g / L ammonium sulfate, 0.1g / L dipotassium hydrogen phosphate, 0.2g / L ammonium nitrate, and 0.2g / L magnesium sulfate;

[0087] The seed solution access amount was 2%, 4%, 6%, 8% and 10% respectively, and the fermentation conditions were: fermentation temperature 42℃, rotation speed 150r / min, dissolved oxygen content 30%, and pH was adjusted to 5.2 using 32% sodium hydroxide solution;

[0088] When the fermentation time is 4h, 8h, 12h, 16h, 20h, 24h, and 28h, the fermentation liquid is taken and the sodium gluconate content in the fermentation liquid is detected by the indicator colorimetric method. The test results are as follows: Figure 6 shown.

[0089] When the initial sugar concentration was 32% and the dissolved oxygen content in the fermentation liquid was 30%, the results of adding different seed liquid inoculation amounts to the crude enzyme liquid were as follows: Figure 6 As shown in the figure, when the amount of seed liquid added continues to increase, the content of sodium gluconate in the fermentation liquid increases in the same fermentation time period, which means that the more seed liquid is added, the faster the production efficiency of sodium gluconate will be. However, according to empirical values, the optimal inoculation amount is 10% of the seed liquid inoculation amount. Therefore, a 10% seed liquid inoculation amount + crude enzyme solution is used, the initial sugar concentration is 32%, the fermentation cycle is shortened to 16h, and the content of sodium gluconate in the fermentation liquid is 330.26g / L-331.54g / L.

[0090] Example 7

[0091] Effect of dissolved oxygen on the fermentation production of sodium gluconate

[0092] The 10% seed solution inoculation amount + crude enzyme solution prepared in Example 6 was inoculated into 3L fermentation medium and 5L fermentation tanks with different dissolved oxygen contents. The fermentation medium in the fermentation tanks was: 32% glucose, 0.4g / L ammonium sulfate, 0.1g / L dipotassium hydrogen phosphate, 0.2g / L ammonium nitrate, and 0.2g / L magnesium sulfate;

[0093] The dissolved oxygen content in different fermentation tanks was 25%, 28%, 30%, 32%, 35% and 37% respectively; the fermentation conditions were: fermentation temperature 42°C, rotation speed 150r / min, pH adjusted to 5.2 using 32% sodium hydroxide solution;

[0094] When the fermentation time is 4h, 8h, 12h, 16h, 20h, 24h, and 28h, the fermentation liquid is taken and the sodium gluconate content in the fermentation liquid is detected by the indicator colorimetric method. The test results are as follows: Figure 7 shown.

[0095] Depend on Figure 7 It can be seen that when the dissolved oxygen content in the fermentation broth is 30%-32%, the growth rate of sodium gluconate in the fermentation broth is the fastest, and when the dissolved oxygen content is 25-28%, the growth rate of sodium gluconate is lower than that of the fermentation system with a dissolved oxygen content of 30%-32%. This may be because the dissolved oxygen content is too low, which will affect the growth rate of the strain and thus affect the production of sodium gluconate; when the dissolved oxygen content is 35%-37%, the production rate of sodium gluconate is greatly reduced, which may be because the intense ventilation affects the growth of the strain.

[0096] Therefore, when the initial sugar concentration is 32%, 10% seed liquid inoculation + crude enzyme solution is used for fermentation, the dissolved oxygen content is 30%-32%, and the fermentation effect is best when 32% sodium hydroxide is used to adjust the pH to 5.2. The entire fermentation cycle is 16h, so that the sodium gluconate after fermentation can reach 330.35g / L, the glucose consumption rate reaches 17.68g / (L·h), the conversion rate of sodium gluconate can reach 96.43%, and the residual sugar is less than 0.7g / L.

[0097] Glucose consumption rate =

[0098] S1: glucose concentration in the fermentation broth at the initial stage of fermentation, g / L;

[0099] V1: volume of the fermentation broth after sterilization at the initial stage of fermentation, L;

[0100] S3: Residual sugar concentration at the end of fermentation, g / L

[0101] V2: the volume of the tank after fermentation, L;

[0102] H: fermentation time, h.

[0103] Glucose conversion rate =

[0104] S1: glucose concentration in the fermentation broth at the initial stage of fermentation, g / L;

[0105] V1: volume of the fermentation broth after sterilization at the initial stage of fermentation, L;

[0106] M1: molecular weight of glucose;

[0107] S2: sodium gluconate concentration after fermentation, g / L;

[0108] V2: the volume of the tank after fermentation, L;

[0109] M2: molecular weight of sodium gluconate;

[0110] Detection of glucose in fermentation broth at the initial stage of fermentation:

[0111] Take 1 mL of fermentation liquid and dilute it to 500 mL volumetric flask, shake well and set aside.

[0112] Pipette 5mL of Film A and B solution into a 250mL conical flask, add 5mL of diluted sample solution, add 0.1% glucose standard titration solution, titrate under boiling condition until the blue color disappears, and record the consumed volume. (At the same time, a blank titration should be performed, that is, 5mL of sample solution is replaced with 5mL of water)

[0113] Glucose% = (Vempty - Vconsumption) * 0.1% * n / Vtake

[0114] Vblank-the volume of glucose standard solution consumed in the blank

[0115] Vconsumption - Volume of glucose standard solution consumed by the sample

[0116] n-sample dilution factor

[0117] Vtake - sample volume.

[0118] The detection method of residual sugar in fermentation broth is the same as that of glucose in fermentation broth at the initial stage of fermentation.

[0119] The inoculation amount of the seed solution is the ratio of the volume of the seed solution to the volume of the fermentation solution.

[0120] Preparation method of acetic acid-sodium acetate buffer with a pH of 5-6: dissolve 120 g of anhydrous sodium acetate (or 199 g of CH3COONa·3H2O) in an appropriate amount of water, add 9 mL of glacial acetic acid, and dilute to 1 L with water.

[0121] The above description is only an explanation of the preferred embodiments of the present application and the technical principles and other schemes used. At the same time, the scope of the invention involved in the present application is not limited to the technical scheme formed by a specific combination of the above technical features, but also includes other technical schemes formed by any combination of the above technical features or their equivalent features without departing from the inventive concept. For example, the above features are replaced with the technical features with similar functions disclosed in the present application (but not limited to) to form a technical scheme.

Claims

1. A strain of Aspergillus niger, characterized in that: Aspergillus niger Aspergillus niger ) OSYZTT-02, the deposit number is: CCTCC NO:M 20241233.

2. A method for producing sodium gluconate by fermentation, characterized in that: Utilizing the Aspergillus niger described in claim 1 ( Aspergillus niger ) OSYZTT-02 A method for producing sodium gluconate by fermentation.

3. A method for producing sodium gluconate by fermentation according to claim 2, characterized in that: The following steps are involved: (1) Culture of bacteria: Aspergillus niger ( Aspergillus niger ) OSYZTT-02 is placed in a seed culture solution, cultured to obtain a seed solution for later use; (2) Preparation of crude enzyme solution: The prepared seed solution is inoculated into the fermentation medium for fermentation. After the fermentation is completed, the fermentation solution is filtered to obtain mycelium. The mycelium is ruptured and inactivated to obtain crude enzyme solution of catalase and glucose oxidase. The filtered clear solution is concentrated and crystallized to obtain sodium gluconate. (3) Fermentation culture: The prepared seed liquid and the crude enzyme liquid in the previous fermentation tank are inoculated into the fermentation medium for fermentation. After the fermentation is completed, the fermentation liquid is filtered to obtain the crude enzyme liquid from the mycelium, which is used for the fermentation in the next fermentation tank. The clear liquid is concentrated and crystallized to obtain sodium gluconate.

4. A method for producing sodium gluconate by fermentation according to claim 3, characterized in that: The seed culture medium is: glucose 15-18%, ammonium sulfate 0.2-0.6g / L, dipotassium hydrogen phosphate 0.05-0.1g / L, ammonium nitrate 0.1-0.3g / L, magnesium sulfate 0.1-0.3g / L, sodium carbonate 2%, acetate buffer 60mmol / L, pH5-6; seed culture control conditions: culture temperature 40-42℃, rotation speed 150r / min.

5. A method for producing sodium gluconate by fermentation according to claim 3, characterized in that: The fermentation medium is: glucose 32%-35%, ammonium sulfate 0.2-0.6g / L, dipotassium hydrogen phosphate 0.05-0.1g / L, ammonium nitrate 0.1-0.3g / L, magnesium sulfate 0.1-0.3g / L, culture control conditions: culture temperature 40-42°C, dissolved oxygen concentration 30%-32%, rotation speed 150r / min, pH 5.2 adjusted with 32% sodium hydroxide.

6. A method for producing sodium gluconate by fermentation according to claim 3, characterized in that: Preparation process of crude enzyme solution: the mycelium obtained by filtering the fermentation liquid through a candle filter is added to an acetic acid-sodium acetate buffer with a pH of 5-6, and the mycelium is broken by lysozyme, and then centrifuged and filtered through a centrifuge to obtain a supernatant, which is the crude enzyme solution.

7. Use of Aspergillus niger according to claim 1 in fermentation production of sodium gluconate.

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