A method for separating and purifying gallic acid in a fermentation broth by coupling salting-out extraction with crystallization

Abstract

The present application relates to the technical field of separation and purification, and discloses a method for separating and purifying gulonic acid in fermentation liquor by salting-out extraction and coupling crystallization, which comprises the following steps: preparing a salting-out system by using organic solvents (ethanol, isopropyl alcohol, tert-butyl alcohol, n-butyl alcohol and acetone) and inorganic salts (ammonium sulfate, sodium chloride, sodium carbonate and sodium bicarbonate), and then carrying out salting-out extraction on the fermentation liquor, so that the gulonic acid can be effectively enriched in the lower phase, and the impurities such as bacteria, most of the pigments and impure proteins can be removed, thus making the gulonic acid easy to separate and purify.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of separation and purification technology, and particularly relates to a method for separating and purifying gulonic acid in fermentation broth by salting-out extraction coupled with crystallization. BACKGROUND

[0002] Vitamin C is a water-soluble vitamin, which is widely present in plants, animals and some single-cell organisms, and is a trace element required by the human body. Vitamin C has various physiological functions, such as antioxidant effect and collagen synthesis promotion. Most animals and plants can synthesize vitamin C to meet the demand. 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 by itself, and thus must obtain vitamin C from external sources to maintain normal human needs. When the human body lacks vitamin C for a long time, it will cause serious scurvy. Therefore, the market demand for vitamin C is still rising, and vitamin C has a broad market prospect.

[0003] There are three ways for industrial production of vitamin C, including concentrated extraction, chemical synthesis and biological fermentation. The two-step fermentation method is the most widely used method, which supplies more than 80% of commercial vitamin C in the world. The method uses D-sorbitol as raw material to generate 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 to be further converted into vitamin C through enolization and lactonization.

[0004] The fermentation broth containing 2-KLG contains a large amount of mycelium, impurities and other suspended particles, and thus the separation and purification of 2-KLG prepared by the two-step fermentation method is difficult. In addition, the fermentation broth contains a large amount of pigment and has a dark color, which greatly increases the difficulty of the separation process. At present, the yield of 2-KLG is low, and the cost of downstream separation of gulonic acid accounts for more than 70% of the total cost.

[0005] In the prior art, the methods for separating and purifying 2-KLG in the fermentation broth mainly include three methods of heating precipitation, chemical flocculation and ultrafiltration, but the existing methods have the problems of low 2-KLG yield and insufficient purity of the separated product. For example, a patent with the application publication number CN112028766A discloses a gulonic acid refining process, which filters the fermentation broth containing sodium gulonic acid through an ultrafiltration membrane system to remove proteins, mycelia, suspended solids and colloidal macromolecules in the fermentation broth, and then obtains a 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 patent process has the problems of many steps, long operation time and low yield. At the same time, the patent process needs to be treated by centrifugation and membrane filtration. Since the ultrafiltration equipment is expensive, it will greatly increase the process cost, and centrifugation and membrane filtration will cause a large loss of product. SUMMARY

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

[0007] The specific technical scheme of the present application is as follows:

[0008] A method for separating and purifying gulonic acid in fermentation broth by salting-out extraction coupled with crystallization, comprising the following steps:

[0009] An organic solvent and an inorganic salt are added to the fermentation broth of gulonic acid strain, stirred uniformly, left to stand, separated into layers, and the lower phase enriched with gulonic acid is obtained, and the lower phase is separated; wherein:

[0010] The organic solvent is one or more of ethanol, isopropanol, tert-butanol, n-butanol and acetone;

[0011] The inorganic salt is one or more of ammonium sulfate, sodium chloride, sodium carbonate and sodium bicarbonate.

[0012] The present application can effectively enrich gulonic acid in the lower phase by using the salting-out extraction system composed of the organic solvents of ethanol, isopropanol, tert-butanol, n-butanol and acetone, and the inorganic salts of ammonium sulfate, sodium chloride, sodium carbonate and sodium bicarbonate, so that the gulonic acid is easy to separate and purify. The present application separates and purifies gulonic acid in the fermentation broth by the salting-out extraction coupled with crystallization technology, without the need for centrifugation, membrane filtration and other treatments of the gulonic acid fermentation broth, thereby reducing the separation cost, and at the same time, the separation process is mild and simple to operate, which is beneficial to industrial production.

[0013] As a preferred method of the above method, the organic solvent is isopropanol or / and tert-butanol, and the inorganic salt is ammonium sulfate.

[0014] As the above-mentioned method is preferred, in the mixed system of isopropyl alcohol, inorganic salt and the fermentation liquor of the strain of gulonic acid, the mass percentage of the isopropyl alcohol is 15% to 40%; in the mixed system of tertiary butyl alcohol, inorganic salt and the fermentation liquor of the strain of gulonic acid, the mass percentage of the tertiary butyl alcohol is 15% to 40%.

[0015] As the above-mentioned method is preferred, in the mixed system of organic solvent, inorganic salt and the fermentation liquor of the strain of gulonic acid, the mass percentage of the ammonium sulfate is 5% to 30%.

[0016] As the above-mentioned method is preferred, the pH of the fermentation liquor is adjusted to 1 to 9.

[0017] As the above-mentioned method is preferred, the method further comprises the following steps: adding activated carbon into the separated lower phase and stirring or oscillating treatment.

[0018] Further preferably, the added amount of the activated carbon accounts for 0.5% to 3% of the total mass of the system.

[0019] As the above-mentioned method is preferred, the method further comprises the following steps:

[0020] Adding a crystallization precipitant into the separated lower phase, stirring or oscillating treatment at 20 to 40℃ for 0.5 to 4 hours, and placing after cooling to 0 to 4℃; the crystallization precipitant is one or several of isopropyl alcohol, tertiary butyl alcohol and methanol.

[0021] Further preferably, the volume ratio of the crystallization precipitant to the lower phase in the method is 1 to 3:1.

[0022] Compared with the prior art, the present application has the following technical effects:

[0023] (1) The present application realizes separation and purification of gulonic acid in the fermentation liquor through salting-out extraction coupled with crystallization technology, which has good effects. After salting-out extraction, the protein removal rate can reach 76.31%, the bacteria removal rate can reach 99.87%, and the gulonic acid recovery rate can reach 98.06%. Further crystallization of the lower phase after salting-out extraction can recover gulonic acid with a recovery rate of 68.92% and remove sodium sulfate with a removal rate of 89.37%.

[0024] (2) The present application separates and purifies gulonic acid in the fermentation liquor through salting-out extraction coupled with crystallization technology, which does not need to centrifuge or filter the gulonic acid fermentation liquor, thereby reducing the separation cost. Meanwhile, the separation process is mild and simple to operate, which is conducive to industrialized production. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Effects of the concentration of target substances in different salting-out extraction systems on gulonic acid standard solution;

[0026] Figure 2Effect of the concentration of ammonium sulfate in isopropanol-ammonium sulfate system on the separation effect;

[0027] Figure 3 Effect of the concentration of isopropanol in isopropanol-ammonium sulfate system on the separation effect;

[0028] Figure 4 Effect of pH in isopropanol-ammonium sulfate system on the salting-out extraction of the fermentation liquor of gulonic acid;

[0029] Figure 5 Effect of the concentration of tert-butyl alcohol in tert-butyl alcohol-ammonium sulfate system on the separation effect;

[0030] Figure 6 Effect of the concentration of ammonium sulfate in tert-butyl alcohol-ammonium sulfate system on the separation effect;

[0031] Figure 7 Effect of pH in tert-butyl alcohol-ammonium sulfate system on the separation effect;

[0032] Figure 8 Response surface optimization of tert-butyl alcohol-ammonium sulfate system;

[0033] Figure 9 Effect of the content of activated carbon on the lower phase solution of gulonic acid;

[0034] Figure 10 Effect of the treatment time of activated carbon on the lower phase solution of gulonic acid;

[0035] Figure 11 Effect of the volume ratio of precipitant isopropanol to the lower phase solution on the crystallization;

[0036] Figure 12 Effect of the volume ratio of precipitant tert-butyl alcohol to the lower phase solution on the crystallization;

[0037] Figure 13 Effect of the volume ratio of precipitant methanol to the lower phase solution on the crystallization;

[0038] Figure 14 Effect of the termination temperature on the crystallization. DETAILED DESCRIPTION

[0039] In the industrial production of gulonic acid fermentation, in order to not make the produced gulonic acid reduce the pH of the fermentation system and affect the growth of the bacterial body, sodium hydroxide is added to keep the pH between 7.00-7.25, so that the gulonic acid is transformed into sodium gulonate, and therefore the gulonic acid exists in the form of sodium gulonate salt in the fermentation liquor.

[0040] The present application is to isolate and purify gulonic acid from the fermentation broth of gulonic acid strain. The fermentation broth of gulonic acid strain can be understood as the fermentation broth obtained by fermentation of the engineering bacteria used in the field of bioengineering for fermentation production of gulonic acid. The present application will be further described below in combination with examples. Those skilled in the art will be able to realize the present application based on the description. In addition, the examples of the present application involved in the following description are generally only a part of the examples of the present application, not all the examples. Therefore, all the other examples obtained by those skilled in the art based on the examples in the present application without making creative efforts should belong to the protection scope of the present application.

[0041] Example 1 Screening of salting-out extraction

[0042] The screening of salting-out extraction system is carried out according to the following steps:

[0043] (1) The gulonic acid standard solution (gulonic acid dissolved in deionized water to prepare) is taken as the investigation object, different organic solvents (methanol, ethanol, isopropanol, tert-butanol, n-butanol and acetone) and inorganic salts ((NH4)2SO4, NaCl, Na2CO3, NaHCO3) are paired and mixed to form a salting-out extraction system, and the distribution behavior of gulonic acid in the salting-out extraction system composed of different salts and organic solvents is investigated.

[0044] Since the pH of gulonic acid fermentation broth is generally about 7.20, the pH of the primary screening system is set to 7.20, the concentration of organic solvent is 25% (w / w), and the concentration of inorganic salt is 20% (w / w). The operation steps are as follows: in a 25 mL test tube with a plug, add the gulonic acid standard solution, then add the inorganic salt, mix on a vortex mixer, add the organic solvent after it is completely dissolved, vortex for about 3 min, mix thoroughly, stand to separate the phases, read and record the volume of the upper and lower phases, and measure the concentration of each component in the upper and lower phases, calculate the phase ratio (R), distribution coefficient (K) and recovery rate (Y) of the salting-out extraction system. The results are shown in Table 1.

[0045] The calculation formula of the phase ratio (R) of the system is as follows:

[0046]

[0047] Wherein, Vt and Vb are the volume of the upper and lower phases (mL);

[0048] The calculation formula of the distribution coefficient (K) of the system is as follows:

[0049]

[0050] Wherein, Ct and Cb are the concentration of gulonic acid in the upper and lower phases (g / L);

[0051] The formula for calculating the recovery rate (Y) of the system is as follows:

[0052]

[0053] wherein Cb and Co are the concentrations of the lower-phase gulonic acid and the gulonic acid standard solution (g / L), respectively, Vb and Vo are the volumes of the lower phase and the standard solution (mL), respectively.

[0054] Vo respectively the volumes of the upper and lower phases and the standard solution (mL).

[0055] Table 1 Partitioning behavior of gulonic acid in different salting-out extraction systems

[0056]

[0057] As can be seen from Table 1, when the organic solvent is methanol, it is easy to cause the precipitation of the target substance gulonic acid. When the salting-out extraction system is an organic solvent-sodium chloride / sodium bicarbonate, gulonic acid also precipitates as a white precipitate. Although the ethanol-sodium carbonate system and the acetone-sodium carbonate system are phase-separated, the phase interface is not clear. The finally selected phase-forming and interface-clearing systems are the organic solvent-ammonium sulfate, isopropanol-sodium carbonate and n-butanol-sodium carbonate. In the ammonium sulfate-formed salting-out extraction system, the partition coefficient of gulonic acid is ranked as follows: ethanol > acetone > isopropanol ≈ tert-butanol > n-butanol, and the partition coefficient is less than 1, indicating that gulonic acid is mainly distributed in the lower phase. As can be seen from Table 1, the recovery rate of gulonic 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 phase ratio R of 0.54 and a partition coefficient K of 0.007; the lowest recovery rate is 96.02% in the ethanol-ammonium sulfate system, with a phase ratio R of 0.76 and a partition coefficient K of 0.055.

[0058] (2) pH is one of the important factors affecting the salting-out extraction partition, therefore, the partition of the standard solution of gulonic acid in the phase-forming system under acidic conditions was investigated.

[0059] 1.75 is the pH of the standard solution of gulonic acid without pH adjustment, and 7.20 is the pH of the fermentation broth in actual production. The systems that phase-separate and have clear interfaces are organic solvent-ammonium sulfate, isopropanol-sodium carbonate, and n-butanol-sodium carbonate. When the system is isopropanol-sodium carbonate and n-butanol-sodium carbonate, a large amount of bubbles will be generated and the reaction device will be overflowed after the addition of Na2CO3 because the pH of the system is in an acidic condition. The influence of the salting-out extraction system composed of an organic solvent (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 as follows: in a 25 mL test tube with a stopper, a standard solution of gulonic acid was added, the pH of the standard solution of gulonic acid was adjusted to 7.20 or 1.75, an inorganic salt was added, and the mixture was mixed on a vortex mixer until it was completely dissolved. Then, an organic solvent was added, the mixture was vortexed for about 3 min to mix thoroughly, and the mixture was allowed to stand to separate into two phases. After the mixture was completely separated into two phases, the volumes of the upper and lower phases were read and recorded, and the concentrations of the components in the upper and lower phases were determined. The phase ratio (R), distribution coefficient (K), and recovery rate (Y) of the salting-out extraction system were calculated. The results are shown in Table 1.

[0060] Table 2 Influence of different salting-out extraction systems on the distribution behavior of gulonic acid under acidic conditions

[0061]

[0062] As can be seen from Table 2, when the pH of the system is reduced, the phase ratio R does not change significantly, the distribution coefficient K increases, and the recovery rate Y of gulonic acid in the lower phase decreases. When the pH (1.75) is lower than the pKa value (2.10 ± 0.54) of gulonic acid, the gulonic acid is in a molecular state, which is easily extracted into the upper phase, the distribution coefficient increases, and the recovery rate of gulonic acid in the lower phase decreases. When the pH (1.75) is higher than the pKa value (2.10 ± 0.54) of gulonic acid, the gulonic 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 gulonic acid in the salting-out extraction system.

[0063] (3) The properties of the target substance itself, such as the concentration of the target substance, can also affect the effect of salting-out extraction. Therefore, the influence of the concentration of the extract on the salting-out extraction of gulonic acid under different salting-out extraction systems (salting-out extraction systems composed of an organic solvent (ethanol, isopropanol, tert-butanol, n-butanol, and acetone) and ammonium sulfate) was investigated.

[0064] The operation steps are: in a 25 mL test tube with a plug, add different concentrations of gulonic acid standard solution (30, 60, 120 mg / mL respectively), then add ammonium sulfate, mix on a vortex mixer, dissolve completely, then add different organic solvents, vortex for about 3 min, mix thoroughly, separate the phases, read and record the volume of the upper and lower phases, and measure the concentration of each component in the upper and lower phases, calculate the phase ratio (R), distribution coefficient (K) and recovery rate (Y) of the salting-out extraction system. The results are shown in Table 1. Figure 1

[0065] As can be seen from Figure 1 , under other conditions, as the concentration of gulonic acid increases, the phase ratio R of the isopropyl alcohol-ammonium sulfate and tert-butyl alcohol-ammonium sulfate systems increases, and the phase ratio R of the remaining systems increases. The distribution coefficient K increases first and then decreases with the increase of the concentration of gulonic acid standard solution, and the recovery rate of gulonic acid in the lower phase is just the opposite (except for n-butanol-ammonium sulfate system). The distribution coefficient K of the n-butanol-ammonium sulfate system decreases with the increase of the concentration of gulonic acid, and the recovery rate of the lower phase increases. This may be due to the different hydrophilic and hydrophobic properties of the organic solvents.

[0066] Example 2 Salting-out extraction of gulonic acid in fermentation broth

[0067] The gulonic acid prepared by fermentation method has low content, and the fermentation broth also contains a large amount of mycelium, impurities and other suspended particles, and contains a lot of pigment, which causes the color of the fermentation broth to be dark. From the point of view of the salting-out extraction system selected by the gulonic acid standard, the maximum retention of gulonic acid in the lower phase is required, and the ethanol-ammonium sulfate and acetone-ammonium sulfate systems are excluded. In this embodiment, isopropyl alcohol-ammonium sulfate, tert-butyl alcohol-ammonium sulfate and n-butanol-ammonium sulfate systems are used, and gulonic acid fermentation broth is used as the research object, and gulonic acid recovery rate, protein removal rate, pigment removal rate and bacteria removal rate are used as indicators to investigate whether the salting-out extraction system can be used for crude separation of gulonic acid fermentation broth and the effect of crude separation, and further select the appropriate salting-out extraction system for gulonic acid fermentation broth.

[0068] The operation method of salting-out extraction in this embodiment is: adjust the pH of gulonic acid fermentation broth to 7.2, add 50 mL centrifuge tube, add ammonium sulfate with a final concentration of 20% (w / w), dissolve and mix on a vortex oscillator, after the inorganic salt is completely dissolved, add organic solvents isopropyl alcohol, tert-butyl alcohol, n-butanol with a final concentration of 25% (w / w) respectively, shake and mix, balance, centrifuge at 3000 rpm for 5 min. Read and record the volume of the upper and lower phases, measure and calculate the pigment removal rate, protein removal rate and bacteria removal rate, and gulonic acid recovery rate. The results are shown in Table 3. The calculation formula of pigment, protein and bacteria removal rate is as follows:

[0069] ​

[0070] Wherein, Ab, Ao respectively refer to the absorbance of the lower phase and the fermentation broth at wavelength 420, 595, 650 nm

[0071] ; Vb, Vo respectively refer to the volume (mL) of the lower phase and the fermentation broth.

[0072] Bacterial cell determination method: dilute the fermentation broth or the upper and lower phases after salting-out extraction with distilled water by appropriate times, take 1 mL and add 9 mL of 1 mol / L HCl, mix well, and take the absorbance at 650 nm in the ultraviolet spectrophotometer with 1 mol / L HCl as blank control.

[0073] Pigment determination method: centrifuge the fermentation broth at 8000 rpm / min for 10 min, take the supernatant and dilute with distilled water by appropriate times, take the absorbance at 420 nm in the ultraviolet spectrophotometer with distilled water as blank control. The upper and lower phases after salting-out extraction do not need to be centrifuged, and the samples are diluted and determined under the same conditions.

[0074] Soluble protein determination method: Coomassie brilliant blue method, centrifuge the fermentation broth at 8000 rpm / min for 10 min, take the supernatant and dilute with distilled water by appropriate times, take the absorbance at 595 nm in the ultraviolet spectrophotometer with distilled water as blank control. The upper and lower phases after salting-out extraction do not need to be centrifuged, and the samples are diluted and determined under the same conditions.

[0075] Table 3 Effect of salting-out extraction of gulonic acid in fermentation broth

[0076] System Protein removal rate (%) Color removal rate (%) Bacterial cell removal rate (%) Gulonic acid recovery rate (%) Isopropanol-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

[0077] The salting-out extraction effect is shown in Table 3, and it can be seen that the isopropanol-ammonium sulfate and tert-butyl alcohol-ammonium sulfate systems have the best extraction effect, wherein the protein removal rate of the isopropanol-ammonium sulfate system reaches 69.63%, the pigment removal rate reaches 37.21%, the bacterial cell removal rate reaches 99.54%, and the gulonic acid recovery rate reaches 93.31%; the protein removal rate of the tert-butyl alcohol-ammonium sulfate system reaches 65.68%, the pigment removal rate reaches 31.07%, the bacterial cell removal rate reaches 99.73%, and the gulonic acid recovery rate reaches 91.82%.

[0078] Example 3 Optimization of the conditions for salting-out extraction of gulonic acid in fermentation broth by isopropanol-ammonium sulfate

[0079] This example is to different concentrations of isopropanol or different concentrations of ammonium sulfate composed of isopropanol-ammonium sulfate salting-out extraction system, salting-out extraction of different pH of the fermentation broth in the gulonic acid. Among them, the concentration of isopropanol (w / w) is set as: 15%, 20%, 25%, 30%, 35%, 40%; the concentration of ammonium sulfate (w / w) is set as: 10%, 15%, 20%, 25%, 30%; the pH of the fermentation broth is adjusted to 1, 3, 5, 7.2, 9, 11.

[0080] The operation method of salting-out extraction in this example is the same as that in Example 2. The upper and lower phase volumes of the extraction phase separation are read and recorded, and the pigment removal rate, protein removal rate and cell removal rate, and gulonic acid recovery rate are measured and calculated. The results are shown in Figure 2 , Figure 3 , Figure 4 . Among them, Figure 2 , Figure A is the effect of ammonium sulfate concentration on the pigment removal rate, Figure B is the effect of ammonium sulfate concentration on the gulonic acid recovery, Figure C is the effect of ammonium sulfate concentration on the cell removal rate, and Figure D is the effect of ammonium sulfate concentration on the protein removal rate; Figure 3 , A is the effect of isopropanol concentration on the gulonic acid recovery, B is the effect of isopropanol concentration on the protein removal rate, C is the effect of isopropanol concentration on the pigment removal rate, and D is the effect of isopropanol concentration on the protein removal rate; Figure 4 is the results of the pigment removal rate, protein removal rate and cell removal rate, and gulonic acid recovery rate of the isopropanol-ammonium sulfate system (40% (w / w) isopropanol-15% (w / w) ammonium sulfate) in the salting-out extraction of gulonic acid in the fermentation broth at different pH.

[0081] From Figure 2 , Figure 3 , Figure 4 , it can be seen that the 40% (w / w) isopropanol-15% (w / w) ammonium sulfate, pH 7.20 salting-out extraction system has the best effect, at this time the pigment removal rate is 52.66%, the protein removal rate is 76.31%, the cell removal rate is 99.87%, and the gulonic acid recovery rate is 98.06%.

[0082] Example 4 Optimization of t-butyl alcohol-ammonium sulfate salting-out extraction of gulonic acid in fermentation broth This example uses response surface test to optimize the t-butyl alcohol-ammonium sulfate salting-out extraction system, with pigment removal rate and protein removal rate as response values, t-butyl alcohol concentration, ammonium sulfate concentration and pH as response variables, using response surface related software Design-Expert 8.0 for test design, to explore the optimal conditions. The operation method of salting-out extraction is the same as that in Example 2, and the results are shown in Figure 8 .

[0083] The salting-out extraction experiments were carried out with the salting-out extraction systems 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, 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. The results are shown in Table 2. Figure 5 .

[0084] The salting-out extraction experiments were carried out with the salting-out extraction systems 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, 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. The results are shown in Table 3. Figure 6 .

[0085] The salting-out extraction experiments were carried out with the salting-out extraction systems 40% (w / w) isopropanol-15% (w / w) ammonium sulfate, the pH of the fermentation broth was adjusted to 1, 3, 5, 7.2, 9, and 11, respectively. The pH adjustment was carried out using hydrochloric acid or sodium hydroxide. Figure 7 .

[0086] As Figures 5-7As shown in the figure, it can be seen that with the increase of the concentration of tert-butyl alcohol, the pigment removal rate and the protein removal rate show an overall upward trend. When the concentration of tert-butyl alcohol reaches 40% (w / w), the pigment removal rate and the protein removal rate are the highest. If the concentration of tert-butyl alcohol continues to increase, a large amount of tert-butyl alcohol will be consumed in the experiment, and the inorganic salt ammonium sulfate and the target substance gulonic acid in the system will also precipitate. Therefore, the concentration of tert-butyl alcohol is selected as 30%, 35%, and 40% (w / w) for the response surface experiment; with the increase of the concentration of ammonium sulfate, the pigment removal rate and the protein removal rate show an overall trend of first increasing and then decreasing. When the concentration of ammonium sulfate reaches 15% (w / w), the protein removal rate is the highest; when the concentration of ammonium sulfate reaches 20%, the pigment removal rate is the highest. This phenomenon shows that when the protein removal rate and the pigment removal rate reach the maximum, further increasing the concentration of inorganic salt ammonium sulfate cannot further improve the protein removal rate and the pigment removal rate, but rather limits them. Therefore, the concentration of ammonium sulfate is selected as 10%, 15%, and 20% (w / w) for the response surface experiment; the pigment removal rate and the protein removal rate show an overall downward trend with the increase of pH. When the pH of the system is 1, the protein removal rate and the pigment removal rate are the highest. Under acidic conditions, the product gulonic acid exists in the form of molecules. Under alkaline conditions, the product gulonic acid exists in the form of sodium gulonate. In actual production, the pH of the directly obtained fermentation broth is between 7.0 and 7.25. Therefore, in order to approach actual production and reduce unnecessary solvent consumption, the pH is selected as 3, 5, and 7 for the response surface experiment.

[0087] With the concentration of tert-butyl alcohol, the concentration of ammonium sulfate, and pH as response variables, and the pigment removal rate and the protein removal rate as response values, experiments were carried out according to the response surface design scheme, and the response value data were filled in, as shown in Table 4.

[0088] Table 4 Design-Expert experimental design scheme and results

[0089]

[0090] According to the response surface regression analysis by Design-Expert 8.0 software, with the protein removal rate in the fermentation broth as the response value, a quadratic multinomial regression equation about the three factors of the concentration of tert-butyl alcohol, the concentration of ammonium sulfate, and pH was obtained:

[0091] Y = 70.68 + 1.92A + 2.61B - 5.72C + 9.91AB + 6.32AC + 7.81BC + 1.98A2 - 1.85B2 + 2.42C2

[0092] The regression model effectiveness and significance analysis, variance analysis results are shown in Table 5. As shown in Table 5, the model is extremely significant (P < 0.01), and the misfit term is not significant (P > 0.05), indicating that the fitting degree of the model is good, and the unknown factor interference is small. The determination coefficient R2 of the model is 0.9557, and the adjusted determination coefficient R2Adj is 0.8986, indicating that 89.86% of the response value change can be explained by the model, and the test error is small, which can effectively analyze and predict the protein removal rate. From the significance, the linear term C and the quadratic term AB, AC, BC in the model have extremely significant influence on the protein removal rate (P < 0.01), and the linear term B has significant influence 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 is: pH > ammonium sulfate concentration > tert-butyl alcohol concentration. The P values of the quadratic terms AB, AC and BC are all less than 0.05, indicating that there is an interaction between tert-butyl alcohol concentration, ammonium sulfate concentration and pH.

[0093] Table 5 Regression model and variance analysis of protein removal rate

[0094]

[0095]

[0096] A quadratic multinomial regression equation about tert-butyl alcohol concentration, ammonium sulfate concentration and pH is obtained with the pigment removal rate in the fermentation broth as the response value:

[0097] Y = 46.95 + 4.78A + 1.73B - 9.86C + 12.27AB + 4.18AC - 2.09BC - 1.94A2 - 3.86B2 + 5.76C2

[0098] The regression model effectiveness and significance analysis, variance analysis results are shown in Table 6. As shown in Table 6, the model is extremely significant (P < 0.01), and the misfit term is not significant (P > 0.05), indicating that the fitting degree of the model is good, and the unknown factor interference is small. The determination coefficient R2 of the model is 0.9044, and the adjusted determination coefficient R2Adj is 0.7814, indicating that 78.14% of the response value change can be explained by the model, and the test error is small, which can effectively analyze and predict the pigment removal rate. From the significance, the linear term C and the quadratic term AB in the model have extremely significant influence on the pigment removal rate (P < 0.01), and the linear term A has significant influence 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 is: pH > tert-butyl alcohol concentration > ammonium sulfate concentration. The P value of the quadratic term AB is less than 0.05, indicating that there is an interaction between tert-butyl alcohol concentration and ammonium sulfate concentration.

[0099] Table 6 Regression model and variance analysis of pigment removal rate

[0100]

[0101]

[0102] After optimization by response surface analysis software Design-Expert 8.0, the tertiary butyl alcohol concentration is 31.27% (w / w), the ammonium sulfate concentration is 15.93% (w / w), and the pH is 3.57, and the pigment removal and protein removal effects are the best, which are 53.28% and 77.06% respectively, and the cell removal rate and gulonic acid recovery rate are 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 rounded to integers, that is, the verification experiment is carried out under the conditions of 30% (w / w) tertiary butyl alcohol, 15% (w / w) ammonium sulfate and pH 3.57, and the average value of three experimental results is taken to ensure its accuracy. The verification results are shown in Table 7. As can be seen from Table 7, under the salting-out extraction conditions of 30% (w / w) tertiary butyl alcohol-15% (w / w) ammonium sulfate and pH 3.57, the standard deviation of the pigment removal rate and the protein removal rate is 1.41% and 0.40% respectively, which is relatively small compared with the model predicted value, indicating that this model can be used for the prediction of the removal effect of proteins and pigments in the salting-out extraction of gulonic acid fermentation broth by tertiary butyl alcohol-ammonium sulfate.

[0103] Table 7 Comparison of predicted values and experimental values

[0104]

[0105] Example 5 Scaling-up of salting-out extraction system

[0106] In this example, 40% (w / w) isopropyl alcohol-15% (w / w) ammonium sulfate is used as the salting-out extraction system to extract gulonic acid from fermentation broth with pH 7.20. The mass of the salting-out extraction system in this example is 30g, 300g, 600g, 900g and 1200g respectively, and the extraction salting-out operation is referred to Example 2. After the fermentation broth is mixed with ammonium sulfate and isopropyl alcohol, it is stirred for 10 min by an electric stirrer, and then it is left to stand at room temperature for 10 h, and the upper and lower phases are taken for determination. The extraction conditions are shown in Table 8.

[0107] Table 8 Influence of system scale on gulonic acid salting-out extraction effect

[0108]

[0109]

[0110] 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.

[0111] Example 5 Effect of Activated Carbon Treatment on Salting-Out Extraction Lower Phase Solution

[0112] (1) Take 7 portions of the lower phase solution after salting out extraction of the 30g 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, protein and the recovery rate of citric acid were measured respectively. The results are shown in Figure 2. Figure 9 shown.

[0113] Depend on Figure 9 It can be seen that adding 0.5-3% activated carbon to the lower phase solution can help reduce pigments and impurities, and the removal effect is better as the amount of activated carbon added increases. Although more activated carbon is added, the pigments and impurities are removed more cleanly, activated carbon not only absorbs heat sources, pigments, and impurities, but also absorbs gulonic acid. As can be seen from the figure, the gulonic acid content decreases significantly with increasing activated carbon dosage, so the activated carbon content is selected to be 2%.

[0114] (2) Investigate the effect of activated carbon action time.

[0115] Take 6 portions of the lower phase solution after salting out extraction of the 30g system in Example 4, add 2% activated carbon powder to each, put each sample into 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 gulonic acid respectively. The results are as follows: Figure 10 shown.

[0116] Depend on Figure 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 extend, more and more gulonic acid is also adsorbed into the pores of the activated carbon, so the activation time of activated carbon is selected to be 60 minutes.

[0117] Example 6 Crystallization of Gulonic Acid in the Lower Phase after Salting-Out Extraction

[0118] The gulonic acid fermentation broth is extracted and pretreated with 40% (w / w) isopropyl alcohol-15% (w / w) ammonium sulfate, pH 7.20, and the gulonic acid is distributed in the lower phase rich in ammonium sulfate. After salting-out extraction, sodium sulfate salt is generated in the lower phase, and the gulonic acid is refined by crystallization. Isopropyl alcohol, t-butyl alcohol and methanol are selected as precipitants. The lower phase solution after salting-out extraction of the 300 g system scale salting-out extraction system in Example 4 is used as the experimental raw material.

[0119] (1) The crystallization operation in this example is as follows: the precipitant 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 kept at 4°C until no more precipitate is generated. The secondary crystallization operation is as follows: the mixed system after the first crystallization is filtered, the filtrate is taken and added with the precipitant, the precipitant is added according to the proportion of the first crystallization, then stirred in a 40°C water bath for 1 hour, then the temperature is lowered to 4°C, and kept at 4°C until no more precipitate is generated. The tertiary crystallization operation is as follows: the mixed system after the secondary crystallization is filtered, the filtrate is taken and added with the precipitant, the precipitant is added according to the proportion of the first crystallization, then stirred in a 40°C water bath for 1 hour, then the temperature is lowered to 4°C, and kept at 4°C until no more precipitate is generated.

[0120] When isopropyl alcohol is used as the precipitant, the results are shown in Table 1. Figure 11 As shown in Table 1, when the volume ratio of isopropyl alcohol 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, 96.02% and 74.90% respectively. After the second crystallization operation, when the volume ratio of isopropyl alcohol to the lower phase solution is 1:1, the removal rate of sodium sulfate is 38.76%, and the recovery rate of gulonic acid is 89.44%; when the volume ratio of isopropyl alcohol to the lower phase solution is 2:1, the removal rate of sodium sulfate is 78.49%, and the recovery rate of gulonic acid is 59.19%. When the volume ratio of isopropyl alcohol to the lower phase solution is 3:1, the recovery rate of gulonic acid in the first crystallization is only 66.83%, so the second operation is not performed, and 55.68% of sodium sulfate can be removed.

[0121] When t-butyl alcohol is used as the precipitant, the results are shown in Table 2. Figure 12 As shown in Table 2, the removal effect of sodium sulfate in the first crystallization is not good, so multiple crystallization operations are performed. When the volume ratio of isopropyl alcohol to the lower phase solution is 1:1, the operation is repeated three times (three times of crystallization), finally 48.49% of sodium sulfate is removed, and 72.89% of gulonic acid is retained; when the volume ratio of isopropyl alcohol to the lower phase solution is 2:1 or 3:1, the operation is repeated twice, finally 53.15% and 54.08% of sodium sulfate are removed, and 71.26% and 75.19% of gulonic acid are retained respectively.

[0122] When methanol is used as the precipitant, the results are shown in Table 3. Figure 13As shown in Table 9, when the volume ratio of methanol solvent to the lower phase solution is 1:1, the removal of sodium sulfate is mostly, with a removal rate of 92.75%, but a yellowish solid is obtained, indicating that a large amount of sodium coumarate is precipitated, and the recovery rate of sodium coumarate is only 22.92%, which also proves this point, resulting in a decrease in product. When the volume ratio of methanol solvent to the lower phase solution is reduced to 1:2 or 1:5, the recovery rate of coumaric acid increases to 77.32% and 85.16%, respectively, and the removal rate of sodium sulfate decreases to 53.43% and 22.41%, respectively.

[0123] As can be seen, methanol as a precipitant has the best effect.

[0124] (2) The volume ratio of methanol solvent to the lower phase solution is fixed at 1:1, and the concentration of coumaric acid in the lower phase is a single variable. The effect of the concentration of coumaric acid in the lower phase on crystallization is investigated, and the results are shown in Table 10. As shown in Table 10, when the volume ratio of methanol solvent to the lower phase solution is 1:1, as the concentration of coumaric acid in the salt extraction lower phase decreases, the recovery rate of coumaric acid first increases and then decreases, but the removal of sodium sulfate is not affected, and the overall remains at about 90%.

[0125] Table 10 Effect of coumaric acid concentration in the lower phase on crystallization

[0126]

[0127] (3) The volume ratio of methanol solvent to the lower phase solution is fixed at 1:1, the concentration of coumaric acid in the lower phase is 57.09 mg / mL, and the termination temperature of the crystallization operation is a single variable. The effect of the termination temperature on crystallization is studied, and the results are shown in Table 11. Figure 14 As can be seen, as the termination temperature decreases, the recovery rate of coumaric acid first increases and then decreases. When the termination temperature decreases from 10°C to 0°C, the recovery rate of coumaric acid increases from 64.74% to 69.34%, and 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 increasing trend as the termination temperature decreases. From 4°C to 0°C, the recovery rate of coumaric acid only increases by 0.42%, and the recovery rate of sodium sulfate also only increases by 0.39%. Therefore, from the perspective of energy saving, the termination temperature of 4°C is optimal, at which the recovery rate of coumaric acid can reach 68.92% and the removal rate of sodium sulfate is 89.37%.

[0128] In summary, the operating conditions of temperature reduction crystallization are as follows: methanol is used as a precipitant, the volume ratio of methanol solvent to the lower phase solution is 1:1, the termination temperature is 4°C, and the concentration of coumaric acid is 40 mg / mL. The recovery rate of coumaric acid can reach 68.92%, and the removal rate of sodium sulfate is 89.37%.

[0129] The raw materials and equipment used in the present application are common raw materials and equipment in the art unless otherwise specified; the methods used in the present application are conventional methods in the art unless otherwise specified.

[0130] The above is only a preferred embodiment of the present application, and does not limit the present application in any way. Any simple modification, change and equivalent transformation of the above embodiment based on the technical essence of the present application still falls within the protection scope of the technical solution of the present application.

Claims

1. A method for separating and purifying gulonic acid from a fermentation broth by salting-out extraction coupled with crystallization, characterized in that: The method comprises the following steps: The pH of the fermentation liquor is adjusted to 3-9, an organic solvent and an inorganic salt are added to the fermentation liquor of the strain of gulonic acid bacteria, the mixture is stirred uniformly, and then is left to stand, and the lower phase enriched with gulonic acid is separated from the upper phase. The organic solvent is isopropyl alcohol or / and tert-butyl alcohol, and the inorganic salt is ammonium sulfate. In the mixed system of isopropyl alcohol, inorganic salt and fermentation liquor of the strain of gulonic acid bacteria, the mass percentage of isopropyl alcohol is 15%-40%; in the mixed system of tert-butyl alcohol, inorganic salt and fermentation liquor of the strain of gulonic acid bacteria, the mass percentage of tert-butyl alcohol is 15%-40%. In the mixed system of organic solvent, ammonium sulfate and fermentation liquor of the strain of gulonic acid bacteria, the mass percentage of ammonium sulfate is 5%-30%. The gulonic acid is 2-keto-gulonic acid.

2. The method for separating and purifying the liquid of fermentation of the ancient dragon acid by coupling crystallization of salting extraction according to claim 1, characterized in that: The method further comprises the following steps: Active carbon is added to the separated lower phase, and the mixture is stirred or shaken.

3. The method for separating and purifying gulonic acid in fermentation liquor by coupling salting-out extraction and crystallization according to claim 2, characterized in that: The amount of active carbon added accounts for 0.5%-3% of the total mass of the system.

4. The method for separating and purifying the liquid of fermentation of the ancient dragon acid by coupling crystallization of salting extraction according to claim 1, characterized in that: The method further comprises the following steps: A crystallization precipitant is added to the separated lower phase, the mixture is stirred or shaken at 20-40℃ for 0.5-4 hours, and then is left to stand after being cooled to 0-4℃; the crystallization precipitant is one or more of isopropyl alcohol, tert-butyl alcohol and methanol.

5. The method for separating and purifying the liquid of fermentation of the ancient dragon acid by coupling crystallization of salting extraction according to claim 1, characterized in that: The volume ratio of the crystallization precipitant to the lower phase is 1-3:

1. The volume ratio of the crystallization precipitant to the lower phase is 1-3:1.

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