A method for preparing xylitol crystals from fermentation broth

Abstract

This invention discloses a method for preparing xylonic acid crystals from fermentation broth, belonging to the field of biochemical production technology. This invention utilizes *Gluconobacterium oxidans* to biocatalyze xylose fermentation to produce a high-concentration potassium xylose fermentation broth. The fermentation broth is centrifuged to obtain a clear potassium xylose solution, which is then concentrated and dried to obtain solid potassium xylose. This solid solution is thoroughly dissolved in anhydrous methanol to form a homogeneous potassium xylose solution. Concentrated sulfuric acid is then added dropwise and mixed to simultaneously carry out the acidification reaction and crystallization process, ultimately obtaining high-purity xylonic acid crystals. This application, through the combined use of biocatalysis and crystallization technologies, achieves high-purity xylonic acid crystals for the first time based on xylose raw materials, providing a new method and technology for the efficient conversion and utilization of xylose.

Description

Technical Field

[0001] This invention belongs to the field of biochemical production technology, and more specifically, relates to a method for preparing xyloic acid crystals from fermentation broth. Background Technology

[0002] Xylic acid, derived from the selective oxidation of xylose, the second most abundant sugar in nature, is a promising organic acid and has been identified by the U.S. Department of Energy as one of the 30 most valuable chemical substances. Xylic acid has broad application potential, including as a food and pharmaceutical ingredient, a precursor to 1,2,4-butanetriol, a highly efficient biocatalyst for organic conversion, the ability to blend hydroxy acids and viscose fibers to obtain cooling fibrous fabrics, the development of novel non-energy-based edible acidulants, and the creation of cooling fiber blends.

[0003] Xylan acid can be produced in the laboratory via electrochemical or chemical oxidation. However, these processes require high temperatures, high pressures, or the use of precious metals to achieve high yields and selectivity, and cause serious environmental pollution. Research on the production and application of xylan acid, both domestically and internationally, is limited and mainly focused on the 1970s and 1980s. Little is known about the metabolic and purification pathways of microbial production of xylan acid, which requires further investigation. Xylan acid is derived from the oxidation of xylose, which is the main product of hemicellulose hydrolysate. Utilizing xylose from hemicellulose to produce xylan acid will significantly reduce production costs, accelerate the industrial production of xylan acid, increase the added value of hemicellulose, and effectively utilize agricultural waste. This is of great significance for the utilization of lignocellulose in agricultural waste, as well as for environmental protection and sustainable development. The bioefficiency and yield of xylose to xylan acid conversion are high, showing broad development prospects. Green and efficient bioconversion technologies have attracted attention to the biopathological pathway of xylose to xylan acid produced by *Gluconobacterium*.

[0004] From the perspective of organic acid purification techniques, crystallization is a long-established separation technique and an important tool for preparing and separating solid products. However, due to the unique polyhydroxy structure of xylose, it is extremely hydrophilic and highly soluble, even deliquescing immediately in air. This characteristic has made the purification and preparation of xylose products through crystallization a truly unprecedented achievement. Since the crystal lattice is highly sensitive to minute changes in the molecular structure of compounds, crystallization is more conducive to obtaining high-purity substances. Therefore, the effective separation of high-purity xylose from fermentation broth by crystallization has significant industrial application value. Furthermore, currently, only analytical reports on xylose salt crystals exist, while research on the preparation and structural analysis of pure xylose crystals remains a gap in the scientific community. Summary of the Invention

[0005] In view of the above-mentioned problems in the prior art, the technical problem to be solved by the present invention is to provide a method for preparing xyloic acid crystals from fermentation broth.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing xylan acid crystals from fermentation broth: potassium xylanate is extracted from fermentation broth containing potassium xylanate, and then xylan acid crystals are prepared using the potassium xylanate as a raw material through a concentrated sulfuric acid-methanol solvent system.

[0008] Furthermore, the method for preparing xylan acid crystals from fermentation broth includes the following steps:

[0009] (1) After centrifuging the fermentation broth containing potassium xylose, the supernatant was obtained, concentrated, and freeze-dried to obtain potassium xylose;

[0010] (2) Dissolve the potassium xylose obtained in step (1) in methanol to obtain a potassium xylose methanol solution;

[0011] (3) Add 98% concentrated sulfuric acid dropwise to potassium xylitol methanol solution;

[0012] (4) After the addition is complete, the system is placed in a low-temperature environment to produce xylitol crystals.

[0013] Further, the method for preparing the fermentation broth containing potassium xylose in step (1) is as follows: inoculate glucosamine oxidase into a culture medium containing xylose for fermentation culture, and after the culture is completed, adjust the pH to 12-14 with potassium hydroxide.

[0014] Further, the specific method of concentration in step (1) is as follows: heat the supernatant to 40-60℃ and evaporate it to a water content of less than 20%.

[0015] Furthermore, in step (2), the mass ratio of potassium xylose to methanol in the potassium xylose methanol solution is 0.8 to 1.2:2, and the methanol is anhydrous methanol.

[0016] Further, in step (3), the mass ratio of potassium xylose in the potassium xylose solution to that of 98% concentrated sulfuric acid is <3.

[0017] Furthermore, the specific temperature of the low-temperature environment described in step (4) is 0-4℃.

[0018] Xyloic acid crystals prepared by any of the methods described.

[0019] The application of the xylosonic acid crystals in crystal analysis, drug research, or food production.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] (1) This invention establishes a xylan acid crystallization strategy: potassium xylanate homogeneous solution is formed by fully dissolving it in anhydrous methanol solvent, and then concentrated sulfuric acid is added dropwise and mixed to achieve simultaneous acidification and crystallization, ultimately obtaining high-purity xylan acid crystals. This method makes full use of methanol as an acidification buffer and crystallization solvent, and efficiently separates and prepares a large amount of high-purity xylan acid crystals from fermentation broth in an economical and simple manner. The obtained xylan acid crystals can be used for scientific analysis and research on the structure of xylan acid crystals, etc., solving a problem in the industry;

[0022] (2) The method of the present invention is simple in steps, reasonable in design, easy to implement, and can be used for the industrial production of xylitol crystals, and has extremely high economic value. Attached Figure Description

[0023] Figure 1 Image of the supernatant of potassium xylitol solution after adding 98% concentrated sulfuric acid;

[0024] Figure 2 The HPLC chromatograms of xylosonic acid crystallization process are shown (S1 and S2 are the sampling locations).

[0025] Figure 3 The image shows xylan acid crystals after a 4-hour ice-water bath (xylan acid crystals adhere to the bottom of the bottle).

[0026] Figure 4 This is a diagram illustrating the crystallization process of xylosonic acid.

[0027] Figure 5 The NMR and HPLC spectra of the product xylosonic acid crystals are shown.

[0028] Figure 6 A schematic diagram of the biocatalytic preparation of high-purity xyloic acid from xylose by *Gluconobacterium oxidans*. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments. The scope of protection of the present invention is not limited to the embodiments, and any modifications made by those skilled in the art within the scope defined by the claims also fall within the scope of protection of the present invention. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art. Unless otherwise specified, the experimental materials used in the following embodiments were all purchased.

[0030] The *Glucosamine oxytocinobacter* NL71 used in the following examples is a strain that has been domesticated and selected over a long period of time from the ATCC 621-H strain (the *Glucosamine oxytocinobacter* strain is ATCC 621-H, which comes from the American Type Culture Collection (ATCC)).

[0031] Seed culture medium: sorbitol 50g / L; yeast extract 5g / L.

[0032] Growth medium: sorbitol 100g / L; yeast extract 10g / L.

[0033] The catalytic culture medium for the fermenter system consists of xylose 100 g / L (feed in batches), glucose 2.5 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium sulfate 5 g / L, and yeast extract 15 g / L. The pH is adjusted using KOH, NaOH, or CaCO3.

[0034] Example 1:

[0035] The preparation of sodium xylorate fermentation broth includes the following steps:

[0036] Gluconobacter oxydans NL71 was cultured in seed culture and proliferation culture medium, and then inoculated into a 3-L fermenter containing 2.0L of catalytic medium for aeration culture. The specific culture conditions were: aeration rate of 3 vvm, temperature of 30℃, pressure of 0.02-0.0 MPa, and rotation speed of 300 rpm. After 72 hours of fermentation, the sodium xylorate content in the fermentation broth reached approximately 400 g / L. Excess NaOH powder was added for neutralization, and the pH was adjusted to 12-14.

[0037] The preparation of calcium xylanate fermentation broth includes the following steps:

[0038] Gluconobacter oxydans NL71 was cultured in seed culture and proliferation culture medium, and then inoculated into a 3-L fermenter containing 2.0L of catalytic medium for aeration culture. The specific culture conditions were: aeration rate of 3 vvm, temperature of 30℃, pressure of 0.02-0.0 MPa, and rotation speed of 300 rpm. After 72 hours of fermentation, the calcium xylose content in the fermentation broth reached approximately 100-200 g / L. Excess CaCO3 powder was added for neutralization, and the pH was adjusted to 6-7.

[0039] The preparation of potassium xylanate fermentation broth includes the following steps:

[0040] Gluconobacter oxydans NL71 was cultured in seed culture and proliferation culture medium, and then inoculated into a 3-L fermenter containing 2.0L of catalytic medium for aeration culture. The specific culture conditions were: aeration rate of 3 vvm, temperature of 30℃, pressure of 0.02-0.0 MPa, and rotation speed of 300 rpm. After 72 hours of fermentation, the potassium xylose content in the fermentation broth reached approximately 400 g / L. Excess KOH powder was added for neutralization, and the pH was adjusted to 12-14. The components and contents of the potassium xylose fermentation broth are shown in the table below.

[0041] Potassium xylocate 400 Bacterial cells 0.68 Magnesium sulfate 0.5 Monopotassium phosphate 1 Dipotassium phosphate 2 Ammonium sulfate 5 Potassium gluconate / 2-ketogluconate 2.5 Others 15

[0042] Example 2: Preparation of xylan crystals from sodium xylanate fermentation broth

[0043] The sodium xylanate fermentation broth obtained in Example 1 was centrifuged to remove precipitates such as *Gluconobacterium oxysporum* cells, and the supernatant was retained. The supernatant was then concentrated at approximately 55°C to a water content of 18-20%, resulting in a yellow, transparent, gel-like solution. This solution was then freeze-dried at -78°C for 12 hours to obtain freeze-dried sodium xylanate solid. The obtained sodium xylanate is sparingly soluble in methanol and cannot be crystallized by acidification.

[0044] Example 3: Preparation of xylan crystals from calcium xylanate fermentation broth

[0045] The calcium xylose fermentation broth obtained in Example 1 was centrifuged to remove precipitates such as *Gluconobacter oxidans* cells, and the supernatant was retained. The supernatant was then concentrated at approximately 55°C to a water content of 18-20%, resulting in a yellow, transparent, gel-like solution. This solution was then freeze-dried at -78°C for 12 hours to obtain freeze-dried calcium xylose solid. Although calcium xylose is soluble in methanol, the calcium ions in calcium xylose react directly with excess sulfate ions to form calcium sulfate precipitate, preventing the production of pure xylose crystals.

[0046] Example 4: Preparation of xylose crystals from potassium xylose fermentation broth

[0047] After centrifuging the high-concentration potassium xylose fermentation broth obtained in Example 1, the precipitate such as glucosamine oxidase cells was removed, and the supernatant was retained. The supernatant was then concentrated at about 55°C to a water content of 18-20%, which was a yellow transparent gel-like solution. The solution was then freeze-dried at -78°C for 12 hours to obtain potassium xylose freeze-dried solid.

[0048] The lyophilized potassium xylose solid was dissolved in pure methanol under heating conditions (the mass ratio of lyophilized potassium xylose solid to methanol was about 1:2), and then centrifuged and the potassium xylose-methanol solution was retained.

[0049] Adding 98% concentrated sulfuric acid dropwise to a centrifuged potassium xylose-methanol solution is problematic. Since excessive addition of 98% concentrated sulfuric acid would cause potassium xylose to carbonize, the amount of 98% concentrated sulfuric acid added should be such that the mass ratio of potassium xylose to 98% concentrated sulfuric acid in the solution is less than 3. Because concentrated sulfuric acid has a higher density than methanol, layering is observed after adding the sulfuric acid. The upper layer is mainly a yellow potassium xylose-methanol solution, and the lower layer is mainly concentrated sulfuric acid. Figures 1-2 Chromatographic analysis showed that after xylose ions entered the lower layer of concentrated sulfuric acid, they rapidly combined with hydrogen ions to form xylose crystals, while the xylose ions in the upper layer remained in a free state. Figure 2 As shown, the concentration of xylitol ions in the lower layer is only 1 / 21 of that in the upper layer of methanol.

[0050] After adding concentrated sulfuric acid, the potassium xylose-methanol mixed solution was placed in an ice-water bath at 4°C for 0-48 hours. Xylose in the lower layer of the solution first formed crystals. Figure 3 Subsequently, K2SO4 crystals gradually formed in the solution and adhered to the bottle wall. The content of relevant substances in the solution during the 0-48h process was detected and analyzed, which can be divided into three stages (…). Figure 4 ):

[0051] The first stage, from 0 to 4 hours, is when xylose crystals are formed. Xylose crystallizes in the lower layer of sulfuric acid solution at the bottom of the bottle. The high concentration of hydrogen ions inhibits the crystallization process of potassium sulfate. During this process, xylose ions in the lower layer combine rapidly with hydrogen ions to produce crystals. Therefore, the concentration of xylose ions in the lower layer is relatively low during this stage.

[0052] The second stage lasts from 4 to 16 hours: due to diffusion, the concentrations of various ions in the upper and lower layers of solution gradually reach a stable state.

[0053] The third stage is from 16 to 24 hours: due to the low solubility of potassium sulfate, K2SO4 granular crystals are produced that adhere to the bottle wall.

[0054] Acidification of potassium xylanate-methanol solution with 0.2 mL of 98% concentrated sulfuric acid for 48 h yielded a total of 1.34 g of mixed solid, including 0.5 g of potassium sulfate crystals and 0.84 g of xylanate crystals, with a xylanate crystal yield of 67.2%. If the supernatant solution is poured off at the end of the first stage (4 h), 0.84 g of pure xylanate flaky crystals can be obtained.

[0055] The pure xylanic acid crystals obtained were characterized by nuclear magnetic resonance (NMR). The carbon and hydrogen spectra of the NMR spectrum showed that the obtained xylanic acid crystals were similar to those of the xylanic acid standard. Figure 1 The xylan acid crystals were analyzed by HPLC, yielding a single-peak chromatogram indicating extremely high purity, which corroborates the NMR analysis results. Figure 5 ).

[0056] In summary, this invention develops a green biotechnology that can prepare large quantities of high-purity xylanic acid crystals in a short time. The obtained xylanic acid crystals can be used for scientific analysis and research on the structure of xylanic acid crystals or for drug research, etc. Figure 6 ).

Claims

1. A method for preparing xylan acid crystals from fermentation broth, characterized in that, Includes the following steps: (1) After centrifuging the fermentation broth containing potassium xylose, the supernatant is obtained, concentrated and freeze-dried to obtain potassium xylose. The preparation method of the fermentation broth containing potassium xylose is as follows: inoculate glucosamine oxidase into a culture medium containing xylose for fermentation culture. After the culture is completed, adjust the pH to 12-14 with potassium hydroxide to obtain the fermentation broth containing potassium xylose. (2) Dissolve the potassium xylose obtained in step (1) in methanol to obtain a potassium xylose methanol solution; (3) Add 98% concentrated sulfuric acid dropwise to potassium xylose methanol solution, wherein the mass ratio of potassium xylose to 98% concentrated sulfuric acid is <3; (4) After the addition is complete, the system is placed in a low temperature environment of 0-4℃. Only xylan acid crystals are formed within 0-4 hours. Xylan acid crystallizes in the lower sulfuric acid solution. The high concentration of hydrogen ions inhibits the crystallization process of potassium sulfate. Pour out the upper solution and separate pure xylan acid crystals.

2. The method for preparing xylan acid crystals from fermentation broth according to claim 1, characterized in that, The specific method for concentration in step (1) is as follows: heat the supernatant to 40-60℃ and evaporate it until the water content is below 20%.

3. The method for preparing xylan acid crystals from fermentation broth according to claim 2, characterized in that, In step (2), the mass ratio of potassium xylose to methanol in the potassium xylose methanol solution is 0.8 to 1.2:2, and the methanol is anhydrous methanol.

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