Method for separating and purifying gulonic acid in fermentation liquor by coupling salting-out extraction with crystallization

Abstract

The invention relates to the technical field of separation and purification, and discloses a method for separating and purifying gulonic acid in fermentation liquor by coupling salting-out extraction with crystallization. According to the method, fermentation liquor is subjected to salting-out extraction treatment through a salting-out system composed of organic solvents including ethanol, isopropanol, tertiary butanol, n-butanol and acetone and inorganic salts including ammonium sulfate, sodium chloride, sodium carbonate and sodium bicarbonate, gulonic acid can be effectively enriched in a lower phase, thalli, most pigments, impure protein and other impurities are removed, and gulonic acid is easy to separate and purify. According to the method, the gulonic acid in the fermentation liquor is separated and purified through the salting-out extraction coupling crystallization technology, treatment such as centrifugation and membrane filtration does not need to be conducted on the gulonic acid fermentation liquor, the separation cost is reduced, meanwhile, the separation process is mild, operation is easy, and industrial production is facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of separation and purification, and in particular to a method for separating and purifying gulonic acid in a fermentation broth through salting-out extraction coupled with crystallization. Background Art

[0002] Vitamin C is a water-soluble vitamin that is widely found in plants, animals and some single-cell organisms. It is a trace element required by the human body and has multiple physiological effects, such as anti-oxidation and promoting collagen synthesis. Most plants and animals can synthesize vitamin C to meet their needs. However, due to the lack of L-gulono-1,4-lactone oxidase gene, a key enzyme in the vitamin C biosynthesis pathway, the human body cannot synthesize vitamin C independently and must ingest vitamin C exogenously to maintain normal human needs. When the human body lacks vitamin C for a long time, it will cause severe scurvy. Therefore, the market demand for vitamin C is still rising, and it has broad market prospects.

[0003] The industrial production of vitamin C mainly includes three methods: concentrated extraction, chemical synthesis, and biological fermentation. Among them, the two-step fermentation method is the most widely used, supplying more than 80% of the world's commercial vitamin C. This method uses D-sorbitol as the raw material and produces 2-keto-gulonic acid (2-KLG, referred to as gulonic acid) through two-step fermentation. 2-KLG needs to be separated from the fermentation broth before it can be further converted into vitamin C through enolization and lactonization.

[0004] The fermentation broth containing 2-KLG contains a lot of impurities such as mycelium, miscellaneous proteins and other suspended particles. Therefore, the separation and purification of 2-KLG prepared by the two-step fermentation method is difficult. In addition, the difficulty of the separation process is greatly increased due to the large amount of pigments and dark color contained in the fermentation broth. At present, the yield of 2-KLG is low, and the cost of separating gulonic acid downstream accounts for more than 70% of the total cost.

[0005] In the prior art, the methods for separating and purifying 2-KLG in fermentation broth mainly include heating precipitation method, chemical flocculation method and ultrafiltration method. However, the existing methods have the problems of low 2-KLG yield and insufficient purity of the separated product. For example, the patent with application publication number CN112028766A discloses a process for extracting gulonic acid, which filters the fermentation broth containing sodium gulonic acid through an ultrafiltration membrane system to remove macromolecular organic matter such as protein, mycelium, suspended matter and colloid in the fermentation broth, and then obtains the sodium gulonic acid product through ultrafiltration membrane filtration, acidification treatment, ion exchange conversion, electrolysis treatment, activated carbon adsorption, concentration treatment, activated carbon adsorption, and evaporation crystallization. The patented process has many steps, long operation time, and low yield. At the same time, the patented process needs to be processed by centrifugation, membrane filtration, etc., which will greatly increase the process cost due to the high price of ultrafiltration equipment, and centrifugation and membrane filtration will cause a large loss of product. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for separating and purifying gulonic acid in a fermentation broth by salting-out extraction coupled with crystallization.

[0007] The specific technical scheme of the present invention is: A method for separating and purifying gulonic acid in a fermentation broth by salting-out extraction coupled with crystallization comprises the following steps: Adding organic solvent and inorganic salt to the fermentation liquid of gulonic acid strain, stirring evenly, standing, layering, obtaining the lower phase enriched with gulonic acid, and separating the lower phase; wherein: The organic solvent is one or more of ethanol, isopropanol, tert-butanol, n-butanol and acetone; The inorganic salt is one or more of ammonium sulfate, sodium chloride, sodium carbonate and sodium bicarbonate.

[0008] The present invention uses a salting-out system composed of organic solvents such as ethanol, isopropanol, tert-butanol, n-butanol, and acetone, and inorganic salts such as ammonium sulfate, sodium chloride, sodium carbonate, and sodium bicarbonate to carry out salting-out extraction treatment on the fermentation broth, thereby effectively enriching gulonic acid in the lower phase, making it easy to separate and purify gulonic acid. The present invention separates and purifies gulonic acid in the fermentation broth through salting-out extraction coupled with crystallization technology, and does not require centrifugation, membrane filtration, and other treatments on the gulonic acid fermentation broth, thereby reducing the separation cost. At the same time, the separation process is mild, the operation is simple, and it is conducive to industrial production.

[0009] As a preferred embodiment of the above method, the organic solvent is isopropanol and / or tert-butanol, and the inorganic salt is ammonium sulfate.

[0010] As a preferred embodiment of the above method, in the mixed system of isopropanol, inorganic salt and fermentation broth of the gulonic acid strain, the mass proportion of isopropanol is 15% to 40%; in the mixed system of tert-butanol, inorganic salt and fermentation broth of the gulonic acid strain, the mass proportion of tert-butanol is 15% to 40%.

[0011] As a preferred embodiment of the above method, in the mixed system of organic solvent, inorganic salt and fermentation liquid of gulonic acid strain, the mass proportion of ammonium sulfate is 5% to 30%.

[0012] As a preferred embodiment of the above method, the pH of the fermentation broth is adjusted to 1-9.

[0013] As a preferred embodiment of the above method, the method further comprises the following steps: adding activated carbon to the separated lower phase and stirring or shaking the phase.

[0014] More preferably, the amount of activated carbon added is 0.5% to 3% of the total mass of the system.

[0015] As a preferred embodiment of the above method, the method further comprises the following steps: Add a crystallization agent to the separated lower phase, stir or shake at 20-40° C. for 0.5-4 hours, cool to 0-4° C. and place; the crystallization agent is one or more of isopropanol, tert-butyl alcohol and methanol.

[0016] Further preferably, in the method, the volume ratio of the crystallization agent to the lower phase is 1 to 3:1.

[0017] Compared with the prior art, the present invention has the following technical effects: (1) The present invention separates and purifies citric acid in the fermentation broth through salting-out extraction coupled with crystallization technology, and has a good effect. After salting-out extraction, the protein removal rate can reach 76.31%, the bacterial removal rate can reach 99.87%, and the citric acid recovery rate can reach 98.06%. The lower phase of the salting-out extraction is further crystallized, and the citric acid recovery rate can reach 68.92%, and the sodium sulfate removal rate is 89.37%.

[0018] (2) The present invention separates and purifies citric acid from the fermentation broth by using salting-out extraction coupled with crystallization technology. There is no need to subject the citric acid fermentation broth to treatments such as centrifugation and membrane filtration, thereby reducing the separation cost. At the same time, the separation process is gentle and easy to operate, which is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The effect of target substance concentration on gulonic acid standard solution in different salting-out extraction systems; Figure 2 The effect of the concentration change of ammonium sulfate on the separation effect in the isopropanol-ammonium sulfate system; Figure 3 The effect of isopropanol concentration change on the separation effect in the isopropanol-ammonium sulfate system; Figure 4 The effect of pH value of isopropanol-ammonium sulfate system on salting-out extraction of gulonic acid in fermentation broth; Figure 5 The effect of tert-butanol concentration on the separation effect in the tert-butanol-ammonium sulfate system; Figure 6 The effect of ammonium sulfate concentration on the separation effect in the tert-butyl alcohol-ammonium sulfate system; Figure 7 The effect of pH on the separation effect in the tert-butyl alcohol-ammonium sulfate system; Figure 8 It is the response surface optimization of tert-butyl alcohol-ammonium sulfate system; Fig. 9 The effect of activated carbon content on the lower phase solution of gulonic acid; Fig.10 The effect of activated carbon treatment time on the lower phase solution of gulonic acid; Fig.11 The effect of the volume ratio of the precipitating agent isopropanol to the lower phase solution on crystallization; Fig.12 The effect of the volume ratio of tert-butyl alcohol to the lower phase solution on crystallization; Fig.13 The effect of the volume ratio of the precipitating agent methanol to the lower phase solution on crystallization; Fig.14 The effect of termination temperature on crystallization. DETAILED DESCRIPTION

[0020] In the industrial production of gulonic acid fermentation, in order to prevent the produced gulonic acid from lowering the pH of the fermentation system and affecting the growth of bacteria, sodium hydroxide is added to maintain the pH between 7.00-7.25, so that gulonic acid is converted into sodium gulonic acid. Therefore, gulonic acid exists in the fermentation liquid in the form of sodium gulonic acid salt.

[0021] The present invention is to separate and purify gulonic acid from the fermentation broth of gulonic acid strains. The fermentation broth of gulonic acid strains can be understood as the fermentation broth obtained by fermenting engineering bacteria used for fermenting and producing gulonic acid in the field of bioengineering. The present invention is further described below in conjunction with the embodiments. A person of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only part of the embodiments of the present invention, rather than all of the embodiments. Therefore, based on the embodiments in the present invention, all other embodiments obtained by a person of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention.

[0022] Example 1 Screening of salting-out extraction The screening of the salting-out extraction system is carried out according to the following steps: (1) Using the standard solution of gulonic acid (gulonic acid dissolved in deionized water) as the object of investigation, different organic solvents (methanol, ethanol, isopropanol, tert-butanol, n-butanol and acetone) and inorganic salts ((NH 4 ) 2 SO 4 , NaCl, Na 2 CO 3 、NaHCO 3 ) were paired and mixed in pairs to form a salting-out extraction system, and the distribution behavior of citric acid in the salting-out extraction system composed of different salts and organic solvents was investigated.

[0023] Since the pH of gulonic acid fermentation broth is generally around 7.20, the pH of the primary screening system is set to 7.20, the organic solvent concentration is 25% (w / w), and the inorganic salt concentration is 20% (w / w). The operation steps are: in a 25mL stoppered test tube, add gulonic acid standard solution, then add inorganic salt, mix on a vortex mixer, add organic solvent after it is completely dissolved, vortex oscillate for about 3 minutes, mix thoroughly, let stand for phase separation, and after it is completely separated, read and record the volume of the upper and lower phases, and measure the concentration of each component in the upper and lower phases, and calculate the relative ratio (R), distribution coefficient (K) and recovery rate (Y) of the salting-out extraction system. The results are shown in Table 1.

[0024] The calculation formula of system relative efficiency (R) is as follows: Wherein, Vt and Vb are the volumes of the upper and lower phases (mL); The calculation formula of the system distribution coefficient (K) is as follows: Wherein, Ct and Cb are the concentrations of gulonic acid in the upper and lower phases (g / L); The recovery rate (Y) of the system is calculated as follows: Among them, Cb and Co are the concentration of gulonic acid in the lower phase and the concentration of gulonic acid standard solution (g / L), Vb, Vo are the volumes of the upper and lower phases and the standard solution (mL). Table 1 Distribution behavior of gulonic acid in different salting-out extraction systems As can be seen from Table 1, when the organic solvent is methanol, it is easy to cause the precipitation of the target substance of gluconic acid. When the salting-out extraction system is an organic solvent-sodium chloride / sodium bicarbonate, gluconic acid is also precipitated as a white precipitate. Although the ethanol-sodium carbonate system and the acetone-sodium carbonate system are phase-separated, the phase separation interface is not clear. The systems that were finally screened out with phase formation and clear interfaces are organic solvent-ammonium sulfate, isopropanol-sodium carbonate and n-butanol-sodium carbonate. In the salting-out extraction system formed by ammonium sulfate, the distribution coefficient of gluconic acid is ranked as follows: ethanol>acetone>isopropanol≈tert-butanol>n-butanol. The distribution coefficient is less than 1, indicating that gluconic acid is mainly distributed in the lower phase. It can also be seen from the recovery rate in Table 1 that the recovery rate of gluconic acid in the lower phase is above 96%, among which the highest recovery rate is 99.59% in the n-butanol-ammonium sulfate system, with a relative R of 0.54 and a distribution coefficient K of 0.007; the lowest is the ethanol-ammonium sulfate system, with a recovery rate of 96.02%, a relative R of 0.76, and a distribution coefficient K of 0.055.

[0025] (2) pH is one of the important factors affecting the distribution of salting-out extraction. Therefore, the distribution of salting-out extraction of citric acid standard solution under acidic pH conditions was investigated.

[0026] 1.75 is the pH of the unadjusted gulonic acid standard solution, and 7.20 is the pH of the fermentation broth in actual production. The systems with phase formation and clear interfaces in step (1) are organic solvent-ammonium sulfate, isopropanol-sodium carbonate and n-butanol-sodium carbonate. When the systems are isopropanol-sodium carbonate and n-butanol-sodium carbonate, since the pH of the system is under acidic conditions, adding Na 2 CO 3 A large number of bubbles will emerge and gush out of the reaction device. Taking the overall situation into consideration, the effect of the salting-out extraction system composed of organic solvents (ethanol, isopropanol, tert-butanol, n-butanol and acetone) and ammonium sulfate on the distribution behavior of gulonic acid under acidic conditions was investigated. The operation steps are: in a 25mL stoppered test tube, add gulonic acid standard solution, adjust the pH of the gulonic acid standard solution to 7.20 or 1.75, then add inorganic salts, mix well on a vortex mixer, and add organic solvent after it is completely dissolved, vortex oscillate for about 3 minutes, mix well, let stand for phase separation, and after it is completely separated, read and record the volume of the upper and lower phases, and measure the concentration of each component in the upper and lower phases, and calculate the relative ratio (R), distribution coefficient (K) and recovery rate (Y) of the salting-out extraction system. The results are shown in Table 1. The results are shown in Table 2.

[0027] Table 2 Effect of different salting-out extraction systems on the distribution behavior of gulonic acid under acidic conditions As can be seen from Table 2, after lowering the pH of the system, it can be seen that compared to R, there is no significant change, the distribution coefficient K increases, and at the same time, the recovery rate Y of gluconic acid in the lower phase decreases. When the pH (1.75) is lower than the pKa value of gluconic acid (2.10±0.54), gluconic acid is in a molecular state at this time, and molecular gluconic acid is easily extracted into the upper phase, the distribution coefficient increases, and the recovery rate of gluconic acid in the lower phase decreases. When the pH (1.75) is higher than the pKa value of gluconic acid (2.10±0.54), gluconic acid exists in a dissociated form and remains in the lower phase. This further proves that the pH of the extraction system is an important factor affecting the distribution of gluconic acid in the salting-out extraction system.

[0028] (3) The properties of the target substance itself will also affect the effect of salting-out extraction, such as the concentration of the target substance. Therefore, the effect of extract concentration on salting-out extraction of cloronal was investigated under different salting-out extraction systems (salting-out extraction systems composed of organic solvents (ethanol, isopropanol, tert-butanol, n-butanol and acetone) and ammonium sulfate, respectively).

[0029] The operation steps are as follows: add different concentrations of gulonic acid standard solution (30, 60, 120 mg / mL respectively) to a 25 mL stoppered test tube, then add ammonium sulfate respectively, mix well on a vortex mixer, add different organic solvents respectively after they are completely dissolved, vortex oscillate for about 3 minutes, mix thoroughly, let stand for phase separation, read and record the volume of the upper and lower phases after complete phase separation, and determine the concentration of each component in the upper and lower phases, and calculate the relative ratio (R), distribution coefficient (K) and recovery rate (Y) of the salting-out extraction system. The results are as follows Figure 1 shown.

[0030] from Figure 1 It can be seen that, with other conditions remaining unchanged, as the concentration of gulonic acid increases, R increases compared to the isopropanol-ammonium sulfate and tert-butanol-ammonium sulfate systems, while R increases compared to the other systems. The distribution coefficient K first increases and then decreases with the increase in the concentration of the gulonic acid standard solution, while the recovery rate of the gulonic acid lower phase is just the opposite (except for the n-butanol-ammonium sulfate system). The distribution coefficient K of the n-butanol-ammonium sulfate system decreases with the increase in the concentration of gulonic acid, while the recovery rate of the lower phase increases. This may be due to the different hydrophilicity and hydrophobicity of organic solvents.

[0031] Example 2 Salting-out extraction of gulonic acid in fermentation broth The content of gulonic acid prepared by the fermentation method is low, and the fermentation liquid also contains a large amount of impurities such as mycelium, miscellaneous proteins and other suspended particles, and contains more pigments, which makes the fermentation liquid darker in color. From the perspective of the salting-out extraction system obtained by screening gulonic acid standard products, gulonic acid must be retained in the lower phase to the maximum extent, and the ethanol-ammonium sulfate and acetone-ammonium sulfate systems must be excluded. This embodiment uses three systems of isopropanol-ammonium sulfate, tert-butanol-ammonium sulfate and n-butanol-ammonium sulfate, and takes gulonic acid fermentation liquid as the object of investigation. The gulonic acid recovery rate, protein removal rate, pigment removal rate and bacterial removal rate are used as indicators to investigate whether the salting-out extraction system can perform rough separation on the gulonic acid fermentation liquid and the effect of rough separation, and further screens to obtain a suitable gulonic acid fermentation liquid salting-out extraction system.

[0032] The operating method of salting-out extraction in this embodiment is as follows: adjust the pH of the gulonic acid fermentation broth to 7.2, add it to a 50mL centrifuge tube, add ammonium sulfate with a final concentration of 20% (w / w), dissolve and mix on a vortex oscillator, and after the inorganic salt is completely dissolved, add organic solvents such as isopropanol, tert-butanol, and n-butanol with a final concentration of 25% (w / w), shake and mix, balance, and centrifuge at 3000rpm for 5min. Read and record the upper and lower phase volumes, measure and calculate the pigment removal rate, protein removal rate, bacterial removal rate, and gulonic acid recovery rate. The results are shown in Table 3. The calculation formulas for pigment, protein, and bacterial removal rates are as follows: Among them, Ab and Ao refer to the absorbance of the lower phase and fermentation broth at wavelengths of 420, 595, and 650 nm, respectively. Vb and Vo refer to the volumes of the lower phase and fermentation broth, respectively (mL). Bacteria determination method: dilute the fermentation broth or the upper and lower phases after salting out extraction with distilled water by appropriate multiples, take 1mL and add 9mL 1mol / L HCl, mix well, and measure its absorbance at 650nm on a UV spectrophotometer using 1mol / L HCl as a blank control.

[0033] Pigment determination method: The fermentation liquid is centrifuged at 8000rpm / min for 10min, and the supernatant is diluted with distilled water by an appropriate multiple, and its absorbance is measured at 420nm on a UV spectrophotometer with distilled water as a blank control. The upper and lower phases after salting out extraction do not need to be centrifuged, and the samples are diluted and measured under the same conditions.

[0034] Soluble protein determination method: Coomassie brilliant blue method, the fermentation broth is centrifuged at 8000rpm / min for 10min, the supernatant is diluted with distilled water by an appropriate multiple, and the absorbance is measured at 595nm on a UV spectrophotometer with distilled water as a blank control. The upper and lower phases after salting out extraction do not need to be centrifuged, and the samples are diluted and measured under the same conditions.

[0035] Table 3 Effect of cloronate precipitation extraction in fermentation broth system Protein removal rate (%) Pigment removal rate (%) Bacteria removal rate (%) Recovery rate of gulonic acid (%) Isopropyl alcohol-ammonium sulfate 69.63±1.06 37.21±0.61 99.54±0.17 93.31±2.52 Tert-Butyl Alcohol-Ammonium Sulfate 65.68±1.81 31.07±0.50 99.73±0.23 91.82±1.28 n-Butanol-ammonium sulfate 65.16±1.25 16.33±0.56 99.00±0.10 92.11±1.08 The salting-out extraction effect is shown in Table 3. It can be seen that the isopropanol-ammonium sulfate and tert-butanol-ammonium sulfate systems have the best extraction effects. Among them, the protein removal rate of the isopropanol-ammonium sulfate system reaches 69.63%, the pigment removal rate reaches 37.21%, the bacterial removal rate reaches 99.54%, and the citric acid recovery rate reaches 93.31%; the protein removal rate of the tert-butanol-ammonium sulfate system reaches 65.68%, the pigment removal rate reaches 31.07%, the bacterial removal rate reaches 99.73%, and the citric acid recovery rate reaches 91.82%.

[0036] Example 3 Optimization of conditions for isopropanol-ammonium sulfate salting-out extraction of gulonic acid from fermentation broth In this embodiment, an isopropanol-ammonium sulfate salting-out extraction system is formed with different concentrations of isopropanol or different concentrations of ammonium sulfate to extract gulonic acid in fermentation broths of different pH values ​​by salting-out extraction. The isopropanol concentration (w / w) is set to 15%, 20%, 25%, 30%, 35%, and 40% respectively; the ammonium sulfate concentration (w / w) is set to 10%, 15%, 20%, 25%, and 30% respectively; and the pH value of the fermentation broth is adjusted to 1, 3, 5, 7.2, 9, and 11 respectively.

[0037] The operation method of salting out extraction in this example is the same as that in Example 2. Read and record the volumes of the upper and lower phases of the extraction phase separation, measure and calculate the pigment removal rate, protein removal rate, bacterial cell removal rate and gulonic acid recovery rate. Figure 2 , Figure 3 , Figure 4 .in, Figure 2 Figure A shows the effect of ammonium sulfate concentration on pigment removal rate, Figure B shows the effect of ammonium sulfate concentration on gulonic acid recovery, Figure C shows the effect of ammonium sulfate concentration on bacterial removal rate, and Figure D shows the effect of ammonium sulfate concentration on protein removal rate. Figure 3 A is the effect of isopropanol concentration on gulonic acid recovery, B is the effect of isopropanol concentration on protein removal rate, C is the effect of isopropanol concentration on pigment removal rate, and D is the effect of isopropanol concentration on protein removal rate; Figure 4 The results are as follows: the pigment removal rate, protein removal rate, bacterial removal rate and gulonic acid recovery rate of gulonic acid in the fermentation broth by salting out extraction at different pH values ​​in the isopropanol-ammonium sulfate system (40% (w / w) isopropanol-15% (w / w) ammonium sulfate).

[0038] from Figure 2 , Figure 3 , Figure 4It can be seen that the salting-out extraction system of 40% (w / w) isopropanol-15% (w / w) ammonium sulfate, pH 7.20 has the best effect. At this time, the pigment removal rate is 52.66%, the protein removal rate is 76.31%, the bacterial removal rate is 99.87%, and the citric acid recovery rate is 98.06%.

[0039] Example 4 Optimization of conditions for salting-out extraction of gulonic acid in fermentation broth by tert-butanol-ammonium sulfate This example uses response surface experiment to optimize the tert-butanol-ammonium sulfate salting-out extraction system, with pigment removal rate and protein removal rate as response values, tert-butanol concentration, ammonium sulfate concentration, and pH as response variables, and response surface related software Design-Expert 8.0 for experimental design to explore the optimal conditions. The operation method of salting-out extraction is the same as that of Example 2. The results are shown in Figure 2. Figure 8 shown.

[0040] Salting-out extraction experiments were conducted using salting-out extraction systems of 15% (w / w) isopropanol-15% (w / w) ammonium sulfate, 20% (w / w) isopropanol-15% (w / w) ammonium sulfate, 25% (w / w) isopropanol-15% (w / w) ammonium sulfate, 30% (w / w) isopropanol-15% (w / w) ammonium sulfate, 35% (w / w) isopropanol-15% (w / w) ammonium sulfate, and 40% (w / w) isopropanol-15% (w / w) ammonium sulfate. The pH of the fermentation broth was adjusted to 7.2, and the salting-out extraction operation was the same as in Example 2. Results are shown in Figure 5 .

[0041] Salting-out extraction experiments were conducted using salting-out extraction systems of 40% (w / w) isopropanol-5% (w / w) ammonium sulfate, 40% (w / w) isopropanol-10% (w / w) ammonium sulfate, 40% (w / w) isopropanol-15% (w / w) ammonium sulfate, 40% (w / w) isopropanol-20% (w / w) ammonium sulfate, 40% (w / w) isopropanol-25% (w / w) ammonium sulfate, and 40% (w / w) isopropanol-30% (w / w) ammonium sulfate. The pH of the fermentation broth was adjusted to 7.2, and the salting-out extraction operation was the same as in Example 2. Results are shown in Figure 6 .

[0042] The gulonic acid in the fermentation broth with different pH values ​​was extracted by salting out using a salting out extraction system of 40% (w / w) isopropanol-15% (w / w) ammonium sulfate. Figure 7 The pH of the fermentation liquid was adjusted to 1, 3, 5, 7.2, 9, and 11, respectively. The pH was adjusted using hydrochloric acid or sodium hydroxide.

[0043] like Figure 5-7As shown, it can be seen that with the increase of tert-butanol concentration, the pigment removal rate and protein removal rate show an overall upward trend. When the tert-butanol concentration reaches 40% (w / w), the pigment removal rate and protein removal rate are the highest. If the tert-butanol concentration continues to increase, the experiment will need to consume a large amount of tert-butanol, and it will also cause the inorganic salt ammonium sulfate and the target substance gulonic acid to precipitate in the system. Therefore, tert-butanol concentrations of 30%, 35%, and 40% (w / w) were selected for response surface experiments; with the increase of ammonium sulfate concentration, the pigment removal rate and protein removal rate showed an overall trend of first increasing and then decreasing. When the ammonium sulfate concentration reaches 15% (w / w), the protein removal rate is the highest; when the ammonium sulfate concentration reaches 20%, the pigment removal rate is the highest. This phenomenon shows that when the protein removal rate and pigment removal rate reach the maximum, continuing to increase the concentration of inorganic salt ammonium sulfate cannot further improve the protein removal rate and pigment removal rate, but will limit them. Therefore, ammonium sulfate concentrations of 10%, 15%, and 20% (w / w) were selected for response surface experiments; the pigment removal rate and protein removal rate showed an overall downward trend with the increase of pH. When the pH of the system is 1, the protein removal rate and pigment removal rate are the highest. Under acidic conditions, the product gulonic acid exists in molecular form. Under alkaline conditions, the product gulonic acid exists in the form of gulonic acid sodium salt. In actual production, the pH of the fermentation broth directly obtained is between 7.0-7.25. Therefore, in order to be close to actual production and reduce unnecessary solvent consumption, pH 3, 5, and 7 were selected for response surface experiments.

[0044] With tert-butyl alcohol concentration, ammonium sulfate concentration, and pH as response variables, and pigment removal rate and protein removal rate as response values, the experiment was carried out according to the response surface design scheme, and the response value data was filled in. The results are shown in Table 4 below.

[0045] Table 4 Design-Expert experimental design scheme and results The corresponding surface regression analysis was performed using Design-Expert 8.0 software, and the protein removal rate in the fermentation broth was used as the response value. The quadratic multinomial regression equation for the three factors of tert-butyl alcohol concentration, ammonium sulfate concentration, and pH was obtained: Y=70.68+1.92A+2.61B-5.72C+9.91AB+6.32AC+7.81BC+1.98A2-1.85B2+2.42C2 The results of the regression model validity and significance analysis and variance analysis are shown in Table 5. As shown in Table 5, the model is extremely significant (P<0.01), and the lack of fit term is not significant (P>0.05), indicating that the model has a good degree of fit and little interference from unknown factors. The model's coefficient of determination R2=0.9557, and the corrected coefficient of determination R2Adj=0.8986, indicating that 89.86% of the response value changes can be explained by the model, the experimental error is small, and the protein removal rate can be effectively analyzed and predicted. From the perspective of significance, the first-order term C and the second-order terms AB, AC, and BC in the model have extremely significant effects on the protein removal rate (P<0.01), and the first-order term B has a significant effect on the protein removal rate (P<0.05). According to the P value of each factor, the order of the influence of each parameter on the protein removal rate can be obtained: pH> ammonium sulfate concentration> tert-butyl alcohol concentration. The P values ​​of the second-order terms AB, AC, and BC are all less than 0.05, indicating that the three factors of tert-butyl alcohol concentration, ammonium sulfate concentration, and pH have interactive effects.

[0046] Table 5 Protein removal rate regression model and variance analysis Taking the pigment removal rate in the fermentation broth as the response value, the quadratic multinomial regression equation for the three factors of tert-butyl alcohol concentration, ammonium sulfate concentration, and pH was obtained: Y=46.95+4.78A+1.73B-9.86C+12.27AB+4.18AC-2.09BC-1.94A2-3.86B2+5.76C2 The results of variance analysis of the regression model validity and significance analysis are shown in Table 6. As shown in Table 6, the model is extremely significant (P<0.01), and the lack of fit term is not significant (P>0.05), indicating that the model has a good degree of fit and little interference from unknown factors. The model's coefficient of determination R2=0.9044, and the corrected coefficient of determination R2Adj=0.7814, indicating that 78.14% of the response value changes can be explained by the model, the experimental error is small, and the pigment removal rate can be effectively analyzed and predicted. From the perspective of significance, the first-order term C and the second-order term AB in the model have an extremely significant effect on the pigment removal rate (P<0.01), and the first-order term A has a significant effect on the pigment removal rate (P<0.05). According to the P value of each factor, the order of the influence of each parameter on the protein removal rate can be obtained: pH> tert-butyl alcohol concentration> ammonium sulfate concentration. The P<0.05 of the second-order term AB indicates that the two factors of tert-butyl alcohol concentration and ammonium sulfate concentration have an interactive effect.

[0047] Table 6 Pigment removal rate regression model and variance analysis After optimization using the response surface analysis software Design-Expert 8.0, it was found that the concentration of tert-butanol was 31.27% (w / w), the concentration of ammonium sulfate was 15.93% (w / w), and the pH was 3.57. The pigment removal and protein removal effects were the best, which were 53.28% and 77.06%, respectively. The bacterial removal rate and citric acid recovery rate were 99.58% and 95.72%, respectively. Since the result parameters are not integers, and the salting-out extraction system is small and difficult to operate, the parameters are integers, that is, the verification experiment was carried out under the conditions of 30% (w / w) tert-butanol, 15% (w / w) ammonium sulfate, and pH 3.57, and the average of the three experimental results was taken to ensure its accuracy. The verification results are shown in Table 7 below. It can be seen from Table 7 that under the salting-out extraction conditions of 30% (w / w) tert-butanol-15% (w / w) ammonium sulfate and pH 3.57, the standard deviations of the pigment removal rate and protein removal rate were 1.41% and 0.40%, respectively, which are slightly different from the model predicted values, indicating that this model can be used to predict the removal effect of protein and pigment in the tert-butanol-ammonium sulfate salting-out extraction of gulonic acid fermentation broth.

[0048] Table 7 Comparison of predicted values ​​and experimental values Example 5 Scale-up of the salting-out extraction system In this example, 40% (w / w) isopropanol-15% (w / w) ammonium sulfate was used as the salting-out extraction system to extract gulonic acid from the fermentation broth at pH 7.20. The mass of the salting-out extraction system in this example is 30g, 300g, 600g, 900g, and 1200g, respectively. The extraction and salting-out operation is referred to Example 2. After the fermentation broth is mixed with ammonium sulfate and isopropanol, it is stirred with an electric stirrer for 10 minutes. After the mixture is evenly mixed, it is allowed to stand at room temperature for 10 hours, and the upper and lower phases are taken for determination. The extraction results are shown in Table 8.

[0049] Table 8 Effect of system scale on the extraction effect of gulong acid precipitation It can be seen from Table 8 that after the system was scaled up, under the same operating conditions, the bacterial removal rate and citric acid recovery rate of the fermentation broth were not affected, and the pigment removal rate and protein removal rate were slightly reduced, indicating that the scale-up effect of the extraction system was small.

[0050] Example 5 Effect of Activated Carbon Treatment on the Lower Phase Solution of Salting-out Extraction (1) Take 7 portions of the lower phase solution after salting out extraction of the 30g system scale salting out extraction system in Example 4, of which 6 portions were added with 0.5, 1, 1.5, 2, 2.5, and 3% (w / w) activated carbon powder, respectively, and the other portion was not added with activated carbon as a control group. The 7 samples were placed in a shaking table at 25°C and 220rpm and shaken for 1 hour. After filtering each treated sample with filter paper, the supernatant was collected and the removal rate of pigment and protein and the recovery rate of citric acid were measured respectively. The results are as follows: Fig. 9 shown.

[0051] Depend on Fig. 9 It can be seen that adding 0.5-3% activated carbon to the lower phase solution can reduce pigments and impurities, and the removal effect is better as the amount of activated carbon added increases. Although the more activated carbon is added, the cleaner the pigments and impurities are removed, activated carbon can not only adsorb heat sources, pigments, and impurities, but also adsorb gulonic acid. As can be seen from the figure, as the amount of activated carbon increases, the content of gulonic acid also decreases significantly, so the content of activated carbon added is selected to be 2%.

[0052] (2) Investigate the effect of activated carbon action time.

[0053] Take 6 portions of the lower phase solution after salting out extraction of the 30g system scale salting out extraction system in Example 4, add 2% activated carbon powder to each, put each sample in a shaker at 25°C and 220rpm, and shake for 20, 40, 60, 80, 100, and 120 minutes respectively. After filtering each treated sample with filter paper, collect the supernatant, and measure the removal rate of pigment, protein and the recovery rate of citric acid respectively. The results are as follows: Fig.10 shown.

[0054] Depend on Fig.10 It can be seen that the amount of impurities adsorbed by activated carbon increases with the extension of the activation time within 20-120 minutes. However, as the activation time continues to increase, more and more citric acid is also adsorbed into the pores of activated carbon, so the activation time of activated carbon is selected to be 60 minutes.

[0055] Example 6 Crystallization of Gulonic Acid in the Lower Phase after Salting-out Extraction After the gulonic acid fermentation broth is extracted with a system of 40% (w / w) isopropanol-15% (w / w) ammonium sulfate, pH 7.20 and pretreated with activated carbon, the gulonic acid is distributed in the lower phase rich in ammonium sulfate. Sodium sulfate salt is generated in the lower phase after salting-out extraction, and the gulonic acid is refined by crystallization. Isopropanol, tert-butanol and methanol are selected as precipitants. The lower phase solution after salting-out extraction of the 300g system scale salting-out extraction system in Example 4 is used as the experimental raw material.

[0056] (1) The crystallization operation of this embodiment is as follows: the precipitation agent is mixed with the lower phase solvent, stirred in a 40°C water bath for 1 hour, then the temperature is lowered to 4°C, and the mixture is kept warm at 4°C until no more precipitation occurs. The secondary crystallization operation is as follows: the mixed system after the primary crystallization is filtered, the filtrate is added with the precipitation agent, the precipitation agent is added in the proportion of the primary crystallization, then the mixture is stirred in a 40°C water bath for 1 hour, then the temperature is lowered to 4°C, and the mixture is kept warm at 4°C until no more precipitation occurs. The tertiary crystallization operation is as follows: the mixed system after the secondary crystallization is filtered, the filtrate is added with the precipitation agent, the precipitation agent is added in the proportion of the primary crystallization, then the mixture is stirred in a 40°C water bath for 1 hour, then the temperature is lowered to 4°C, and the mixture is kept warm at 4°C until no more precipitation occurs.

[0057] When isopropanol is used as the precipitating agent, the results are as follows Fig.11 As shown, when the volume ratio of isopropanol to the lower phase solution is 1:1 or 2:1, the removal rate of sodium sulfate in the first crystallization is low, only 16.01% and 56.63%, while the recovery rate of gulonic acid is still high at this time, 96.02% and 74.90%, respectively. The second crystallization operation was carried out. Finally, when the volume ratio of isopropanol to the lower phase solution was 1:1, the removal rate of sodium sulfate was 38.76%, and the recovery rate of gulonic acid was 89.44%; when the volume ratio of isopropanol to the lower phase solution was 2:1, the removal rate of sodium sulfate was 78.49%, and the recovery rate of gulonic acid was 59.19%. When the volume ratio of isopropanol to the lower phase solution was 3:1, the recovery rate of gulonic acid in the first crystallization was only 66.83%, so the second operation was not carried out, and 55.68% of sodium sulfate could be removed at this time.

[0058] When tert-butyl alcohol is used as the precipitating agent, the results are as follows Fig.12 As shown, the effect of removing sodium sulfate by one crystallization is not good, so multiple crystallization operations are performed. When the volume ratio of isopropanol to the lower phase solution is 1:1, the operation is repeated three times (three crystallizations), and finally 48.49% of sodium sulfate is removed, while retaining 72.89% of gulonic acid; when the volume ratio of isopropanol to the lower phase solution is 2:1 or 3:1, the operation is repeated twice, and finally 53.15% and 54.08% of sodium sulfate are removed, respectively, while retaining 71.26% and 75.19% of gulonic acid.

[0059] When methanol is used as the precipitating agent, the results are as follows Fig.13 As shown, when the ratio of methanol solvent to lower phase solution is 1:1, most of the sodium sulfate is removed, with a removal rate of 92.75%, but a yellowish solid is obtained, which means that a large amount of sodium gulonic acid is precipitated. The recovery rate of sodium gulonic acid is only 22.92%, which also confirms this point, resulting in a reduction in product. When the ratio of methanol solvent to lower phase solution is reduced to 1:2 or 1:5, the recovery rate of gulonic acid increases, which are 77.32% and 85.16% respectively, while the corresponding removal rate of sodium sulfate decreases, which are 53.43% and 22.41% respectively.

[0060] It can be seen that the best effect is achieved when methanol is used as the precipitation agent.

[0061] (2) The volume ratio of methanol solvent to lower phase solution was fixed at 1:1, and the concentration of gluconic acid in the lower phase was taken as a single variable to investigate the effect of the concentration of gluconic acid in the lower phase on crystallization. The results are shown in Table 10. As can be seen from Table 10, when the ratio of methanol solvent to lower phase solution was 1:1, as the concentration of gluconic acid in the lower phase of salting-out extraction decreased, the recovery rate of gluconic acid showed a trend of first increasing and then decreasing, but had little effect on the removal of sodium sulfate, and remained at about 90% overall.

[0062] Table 10 Effect of gulonic acid concentration in the lower phase on crystallization (3) The volume ratio of methanol solvent to lower phase solution was fixed at 1:1, the concentration of gluconic acid in the lower phase was 57.09 mg / mL, and the termination temperature of the crystallization operation was used as a single variable to study the effect of the termination temperature on crystallization. The results are as follows: Fig.14 As shown. It can be seen that with the decrease of the termination temperature, the recovery of gulonic acid first increases and then decreases. When the termination temperature decreases from 10°C to 0°C, the recovery rate of gulonic acid increases from 64.74% to 69.34%. When the temperature continues to decrease to -5°C, the recovery rate decreases to 63.96%. The removal rate of sodium sulfate shows a slow increase trend with the decrease of the termination temperature. From 4°C to 0°C, the recovery rate of gulonic acid only increases by 0.42%, and the recovery rate of sodium sulfate only increases by 0.39%. Therefore, from the perspective of energy saving, the termination temperature of 4°C is the best. At this time, the recovery rate of gulonic acid can reach 68.92%, and the removal rate of sodium sulfate is 89.37%.

[0063] In summary, the operating conditions of cooling crystallization are: methanol is used as the precipitating agent, the volume ratio of methanol solvent to lower phase solution is 1:1, the termination temperature is 4°C, 40 mg / mL of gulonic acid, the gulonic acid recovery rate can reach 68.92%, and the sodium sulfate removal rate is 89.37%.

[0064] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0065] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for separating and purifying gulonic acid in a fermentation broth by salting-out extraction coupled with crystallization, characterized in that: The following steps are involved: Adding organic solvent and inorganic salt to the fermentation liquid of gulonic acid strain, stirring evenly, standing, layering, obtaining the lower phase enriched with gulonic acid, and separating the lower phase; wherein: The organic solvent is one or more of ethanol, isopropanol, tert-butanol, n-butanol and acetone; The inorganic salt is one or more of ammonium sulfate, sodium chloride, sodium carbonate and sodium bicarbonate.

2. The method for separating and purifying gulonic acid in a fermentation broth by salting-out extraction coupled with crystallization as claimed in claim 1, characterized in that: The organic solvent is isopropanol and / or tert-butanol, and the inorganic salt is ammonium sulfate.

3. A method for separating and purifying gulonic acid in a fermentation broth by salting-out extraction coupled with crystallization as claimed in claim 2, characterized in that: In the mixed system of isopropanol, inorganic salt and fermentation broth of gulonic acid strain, the mass proportion of isopropanol is 15% to 40%; in the mixed system of tert-butanol, inorganic salt and fermentation broth of gulonic acid strain, the mass proportion of tert-butanol is 15% to 40%.

4. A method for separating and purifying gulonic acid in a fermentation broth by salting-out extraction coupled with crystallization as claimed in claim 2, characterized in that: In the mixed system of organic solvent, inorganic salt and fermentation liquid of gulonic acid strain, the mass proportion of ammonium sulfate is 5% to 30%.

5. The method for separating and purifying gulonic acid in fermentation broth by salting-out extraction coupled with crystallization as claimed in claim 1, characterized in that: The pH of the fermentation broth is adjusted to 1-9.

6. The method for separating and purifying gulonic acid in a fermentation broth by salting-out extraction coupled with crystallization as claimed in claim 1, characterized in that: The following steps are also included: Add activated carbon to the separated lower phase and stir or shake it.

7. The method for separating and purifying gulonic acid in a fermentation broth by salting-out extraction coupled with crystallization as claimed in claim 1, characterized in that: The added amount of the activated carbon accounts for 0.5% to 3% of the total mass of the system.

8. The method for separating and purifying gulonic acid in fermentation broth by salting-out extraction coupled with crystallization as claimed in claim 1, characterized in that: The following steps are also included: Add a crystallization agent to the separated lower phase, stir or shake at 20-40° C. for 0.5-4 hours, cool to 0-4° C. and place; the crystallization agent is one or more of isopropanol, tert-butyl alcohol and methanol.

9. The method for separating and purifying gulonic acid in a fermentation broth by salting-out extraction coupled with crystallization as claimed in claim 1, characterized in that: The volume ratio of the crystallization agent to the lower phase is 1 to 3:1.

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