Bagasse enzymatic hydrolysate detoxification process and application of bagasse enzymatic hydrolysate detoxification process in xanthan gum production

Abstract

The invention discloses a bagasse enzymatic hydrolysate detoxification process and application thereof in xanthan gum production, and relates to the technical field of bagasse enzymatic hydrolysate treatment and xanthan gum production. According to the bagasse enzymatic hydrolysate detoxification process, a quick lime detoxification method or a calcium phosphate detoxification method is adopted to treat bagasse enzymatic hydrolysate, and weak acid components, phenolic substances and furfural substances in the bagasse enzymatic hydrolysate are removed. The quick lime detoxification method comprises the following steps: adding quantitative calcium oxide solid into the bagasse enzymatic hydrolysate, continuously stirring, standing, primarily filtering to remove the solid, introducing carbon dioxide gas until the pH value of filtrate is less than or equal to 8, filtering again to remove the solid, and sterilizing the filtrate at high temperature. The detoxification process can efficiently remove inhibitors such as organic acids and / or inorganic acids, phenolic substances and furfural substances in the bagasse enzymatic hydrolysate, reduce inhibition on fermentation growth of xanthomonas campestris, significantly improve the yield of xanthan gum, provide a new application direction for the bagasse enzymatic hydrolysate, provide a new fermentation raw material for xanthan gum, and reduce the production cost. And the production cost is reduced.

Description

Technical Field

[0001] This invention relates to the field of sugarcane bagasse enzymatic hydrolysate treatment and xanthan gum production technology, and in particular to a sugarcane bagasse enzymatic hydrolysate detoxification process and its application in xanthan gum production. Background Technology

[0002] By 2025, my country's biomanufacturing industry had reached a scale of trillions of yuan. Globally, the biomanufacturing industry is projected to generate $30 trillion in economic value by 2050, accounting for one-third of global manufacturing. The carbon and nitrogen sources for biomanufacturing mainly come from deep-processed grain products. Currently, my country imports over 100 million tons of grain annually, while its fermentation industry demands 200 million tons of grain. Microbial polysaccharides, including xanthan gum, are primarily obtained through fermentation using grain as a biomass carbon source. In today's increasingly severe food crisis, achieving the substitution of non-grain biomass for "grain biomass" in biomanufacturing, including the microbial polysaccharide industry, is of great significance for developing a circular economy in biomanufacturing and ensuring my country's food and ecological security.

[0003] Sugarcane bagasse, a solid waste from the sugar industry, is a typical non-grain biomass carbon source. It is mainly composed of cellulose, hemicellulose, and lignin, with an annual output of approximately 11 million tons, and is rich in fermentable sugars. Through pretreatment and enzymatic hydrolysis, the cellulose and hemicellulose in sugarcane bagasse can be efficiently converted into fermentable sugars (mainly glucose and xylose), making it a highly promising non-grain biomass carbon source suitable for microbial fermentation. Although the enzymatic hydrolysate of sugarcane bagasse has a very broad potential as a biomass carbon source, its practical application faces significant challenges: after conversion, the hydrolysate still contains aliphatic acids, esters (acetic acid esters), phenolic compounds (different compounds obtained from lignin hydrolysis), and sugar dehydration products, including furfural and 5-hydroxymethylfurfural (5-HMF), which can severely affect microbial growth and product synthesis.

[0004] Currently, there are several mature pretreatment methods for fibrous and hemicellulose materials to hydrolyze and obtain sugars. The obtained sugars can be used for fermentation to produce biofuels. For example, CN104254613A discloses a method for detoxifying hydrolysates obtained from lignocellulosic biomass and a method for producing ethanol from the detoxified hydrolysates. The method includes the following steps: (a) mixing a starting solution of lignocellulosic hydrolysates obtained from lignocellulosic biomass with a first base or a mixture of first bases in an amount sufficient to raise the pH of the solution to between 3 and 8, the starting solution containing a mixture of fermentable sugars, furfural, and aliphatic acids; and (b) mixing the solution produced in step (a) with a second base or a mixture of second bases in an amount sufficient to raise the pH of the solution to between 7 and 10, the mixing duration being sufficient to eliminate at least 40% of the furfural in the lignocellulosic hydrolysates, thereby reducing the toxicity of the lignocellulosic hydrolysates. However, the above method is complex to operate, and it is uncertain whether the detoxification method can effectively remove the inhibitor of Xanthomonas oryzae, a xanthan gum-producing bacterium, from sugarcane bagasse enzymatic hydrolysate.

[0005] Therefore, optimizing the "detoxification" process of fermentable sugars in sugarcane bagasse enzymatic hydrolysate from non-grain biomass sources, and exploring the feasibility and efficiency of converting xanthan gum into sugarcane bagasse enzymatic hydrolysate through fermentation, is of great significance. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a new, efficient and safe detoxification process for sugarcane bagasse enzymatic hydrolysate in response to the shortcomings of the prior art. This invention also discloses the application of detoxified sugarcane bagasse fermentation broth as a substrate in the preparation of xanthan gum.

[0007] The technical solution adopted by this invention to solve its technical problem is:

[0008] A detoxification process for sugarcane bagasse enzymatic hydrolysate involves treating the sugarcane bagasse enzymatic hydrolysate with quicklime or calcium phosphate to remove weak acid components, phenolic substances, and furfural substances.

[0009] The specific operation of the quicklime detoxification method is as follows: a certain amount of calcium oxide solid is added to the sugarcane bagasse enzymatic hydrolysate, stirred continuously, and after standing, the solid is removed by initial filtration. Then, carbon dioxide gas is introduced to make the pH of the filtrate ≤ 8, and the solid is removed by filtration again. The filtrate is sterilized at high temperature to obtain the sugarcane bagasse enzymatic detoxification solution.

[0010] The sugarcane bagasse enzymatic hydrolysate detoxification process of this application utilizes CaO dissolved in water to generate Ca(OH)2. This firstly neutralizes the organic and / or inorganic acids in the hydrolysate, converting them into corresponding calcium salts. The organic acids are then removed by filtration. Secondly, under alkaline conditions, phenolic substances that disrupt cell membranes react with CaO.2+ The reaction produces an insoluble precipitate. Furfural compounds (HMF and FFR) decompose into less toxic products (such as furfuryl alcohol and furoic acid) under alkaline and heated conditions, thereby efficiently removing inhibitors of xanthan gum fermentation strains from the sugarcane bagasse enzymatic hydrolysate and reducing its inhibitory effect on Xanthomonas oryzae. In addition, compared with the existing saturated quicklime detoxification method, which uses concentrated acid (concentrated sulfuric acid H2SO4) to acidify the treatment solution with a pH of 10 to a pH of 5.5, the sugarcane bagasse enzymatic hydrolysate detoxification process of this invention adjusts the pH of the sugarcane bagasse enzymatic hydrolysate back to a weakly acidic state by introducing CO2. The introduction of CO2 is more conducive to slowing down the rate of pH adjustment of the sugarcane bagasse enzymatic hydrolysate. The flow of CO2 also facilitates solution mixing, improves the uniformity of solution pH, facilitates pH control, prevents excessive pH adjustment, and thus increases the yield of xanthan gum prepared by fermentation using sugarcane bagasse enzymatic detoxification solution as a substrate.

[0011] In one exemplary embodiment, a detoxification process for sugarcane bagasse enzymatic hydrolysate includes the following steps:

[0012] S1. Neutralization and detoxification: Add calcium oxide solid to bagasse enzymatic hydrolysate at a dosage of 2~3.0 g / L, stir continuously for more than 6 hours, let stand, and then filter to remove solids for the first time.

[0013] S2, fine-tuning pH by introducing CO2: After introducing carbon dioxide gas to bring the pH of the filtrate obtained from S1 filtration to 5.87~8, filter again to remove solids. The filtrate is then sterilized at 115℃ for 20 min to obtain sugarcane bagasse enzymatic detoxification solution.

[0014] The sugarcane bagasse enzymatic hydrolysate detoxification process quantifies the addition of calcium oxide solids, which is more direct than the existing saturated quicklime method that controls the amount of limestone emulsion added based on pH value. This avoids the problem of excessive calcium oxide addition due to pH value detection deviations caused by lag in solution mixing homogeneity. This improves the detoxification effect of sugarcane bagasse enzymatic hydrolysate on inhibiting the fermentation and growth of Xanthomonas campestris, while preventing excessive CaO from forming large amounts of fine gypsum precipitates that adsorb and encapsulate the sugar solution, leading to further loss of usable carbon sources. Furthermore, it avoids the excessive CaO... 2+ The phenomenon that a high osmotic pressure environment inhibits / affects the growth of Xanthomonas brassicae.

[0015] Preferably, in S1, the amount of calcium oxide solid added is 2.40 g / L.

[0016] In S2, the amount of CO2 introduced is controlled so that the pH of the filtrate obtained from S1 filtration is ≤7, and the pH is further optimized to reach 5.87-6.0.

[0017] The specific operation of the calcium phosphate detoxification method is as follows: the sugarcane bagasse enzymatic hydrolysate is adjusted to pH ≤ 3 with phosphoric acid, and then the pH is adjusted to 7 with solid CaO. After standing for more than 1 hour, the solid calcium phosphate is removed by filtration, and the filtrate is sterilized at high temperature to obtain the sugarcane bagasse enzymatic detoxification solution.

[0018] The aforementioned calcium phosphate detoxification method is based on calcium phosphate produced from phosphoric acid and CaO. Firstly, it can neutralize the weak acid components and complex phenolic substances in the bagasse enzymatic hydrolysate. Secondly, as a flocculated precipitate with a huge specific surface area, calcium phosphate can adsorb weak acids, furfural substances (mainly HMF and FFR), and phenolic substances under the action of surface tension, thereby achieving a detoxification effect and contributing to an increase in xanthan gum production.

[0019] Another technical solution adopted by the present invention to solve its technical problem is:

[0020] An application of a sugarcane bagasse enzymatic hydrolysate detoxification process in xanthan gum production, wherein the sugarcane bagasse enzymatic detoxification solution prepared by the above-mentioned sugarcane bagasse enzymatic hydrolysate detoxification process is used as a fermentation substrate to produce xanthan gum.

[0021] An application of a sugarcane bagasse enzymatic hydrolysate detoxification process in xanthan gum production involves adding Xanthomonas oryzae at 10% (V / V) to sugarcane bagasse enzymatic hydrolysate detoxification solution prepared using the above-mentioned sugarcane bagasse enzymatic hydrolysate detoxification process, fermenting and culturing for more than 96 hours, then centrifuging the fermentation broth to obtain the supernatant for alcohol precipitation, then filtering under reduced pressure to obtain the precipitate, washing and drying it to obtain xanthan gum.

[0022] The beneficial effects of the sugarcane bagasse enzymatic hydrolysate detoxification process of this invention are as follows:

[0023] The sugarcane bagasse enzymatic hydrolysate detoxification process of this invention improves upon the existing saturated quicklime method by quantitatively adding CaO to prevent excessive addition. CaO dissolves in water to form Ca(OH)₂, which first neutralizes organic and / or inorganic acids in the enzymatic hydrolysate, converting them into corresponding calcium salts. Organic acids are then removed by filtration. Secondly, under alkaline conditions, phenolic substances that disrupt cell membranes react with CaO. 2+The reaction produces an insoluble precipitate. Furfural compounds (HMF and FFR) decompose into less toxic products (such as furfuryl alcohol and furoic acid) under alkaline and heated conditions, thereby efficiently removing inhibitors of xanthan gum fermentation strains from sugarcane bagasse enzymatic hydrolysate and reducing their inhibitory effect on Xanthomonas spp. Compared to the existing saturated quicklime method that controls the amount of limestone emulsion added via pH, quantitative CaO addition is more direct and controllable. It effectively avoids the phenomenon of excessive calcium oxide addition due to pH detection deviation caused by lag in solution mixing homogeneity, thus improving the detoxification effect of sugarcane bagasse enzymatic hydrolysate on the fermentation and growth inhibition of Xanthomonas spp. It also avoids the formation of a large amount of fine gypsum precipitate by excessive CaO, which would adsorb and encapsulate the sugar solution, resulting in further loss of usable carbon sources. Furthermore, it avoids the phenomenon of excessive Ca2+ creating a high osmotic pressure environment that inhibits / affects the growth of Xanthomonas spp.

[0024] Simultaneously, CO2 hydrolysis forms a weak acid, which reacts with Ca... 2+ The excess Ca added during the detoxification process of sugarcane bagasse enzymatic hydrolysate is removed in the production of calcium carbonate via reaction. 2+ Furthermore, the pH of the sugarcane bagasse enzymatic hydrolysate was adjusted back to the slightly acidic level suitable for the growth of Xanthomonas oryzae. Compared with the existing saturated quicklime detoxification method, the introduction of CO2 is more conducive to slowing down the pH adjustment rate of the sugarcane bagasse enzymatic hydrolysate. The flow of CO2 also facilitates solution mixing, improves the uniformity of solution pH, makes pH control easier, prevents excessive pH adjustment, and thus increases the yield of xanthan gum prepared by fermentation using sugarcane bagasse enzymatic detoxification solution as a substrate.

[0025] The sugarcane bagasse enzymatic hydrolysate detoxification process of the present invention uses calcium phosphate detoxification based on the production of calcium phosphate from phosphoric acid and CaO. This calcium phosphate can neutralize the weak acid components and complex phenolic substances in the sugarcane bagasse enzymatic hydrolysate. At the same time, as a flocculation precipitate with a huge specific surface area, calcium phosphate can adsorb weak acids, furfural substances (mainly HMF and FFR) and phenolic substances under the action of surface tension, thereby achieving a detoxification effect and promoting an increase in xanthan gum production.

[0026] The application of the sugarcane bagasse enzymatic hydrolysate detoxification process of this invention in xanthan gum production provides a new application direction for sugarcane bagasse enzymatic hydrolysate, expands its application scope, and provides a new raw material for xanthan gum production. The sugarcane bagasse enzymatically detoxified hydrolysate obtained through the detoxification process, when used as a fermentation substrate to produce xanthan gum, achieves a xanthan gum yield as high as 22.93 g / L, compared to 1.69 g / L for direct fermentation of undetoxified sugarcane bagasse enzymatic hydrolysate. -1The yield increased by 1256.8%, approaching the xanthan gum yield of Xanthomonas oryzae on commonly used fermentation media. This indicates that the sugarcane bagasse enzymatic hydrolysate detoxified solution obtained by the sugarcane bagasse enzymatic hydrolysate detoxification process of this invention can replace existing fermentation media, further reducing the production cost of xanthan gum, and has a large application market and huge economic value. Attached Figure Description

[0027] Figure 1 —High-performance liquid chromatograms of sugarcane bagasse enzymatic hydrolysate samples, HMF standards, FFR standards, and mixed standards;

[0028] a: High-performance liquid chromatography (HPLC) chromatogram of HMF standard, b: HPLC chromatogram of FFR standard, c: HPLC chromatogram of mixed HMF and FFR standard, d: HPLC chromatogram of untreated sugarcane bagasse enzymatic hydrolysate.

[0029] Figure 2 —The FFR extraction rates of several organic reagents with different ratios in Comparative Example 1 of this invention;

[0030] Figure 3 —The extraction rate of HMF for several organic reagents with different ratios in Comparative Example 1 of this invention;

[0031] Figure 4 —The yield of xanthan gum after fermentation following extraction with organic reagents of different ratios in Comparative Example 1 of this invention;

[0032] Figure 5 —The xanthan gum yield of Comparative Example 2 of this invention was obtained by adding fermentation medium with equal concentrations of HMF and FFR before and after extraction.

[0033] Figure 6 —A comparative analysis of sugarcane bagasse enzymatic hydrolysate obtained by a sugarcane bagasse enzymatic hydrolysate detoxification process (calcium phosphate detoxification method) in Example 1 of this invention, untreated sugarcane bagasse enzymatic hydrolysate, and conventional fermentation culture medium used for xanthan gum fermentation.

[0034] Figure 7 —Comparative analysis of sugarcane bagasse enzymatic hydrolysate detoxification solution obtained by different CaO addition amounts in the sugarcane bagasse enzymatic hydrolysate detoxification process (quicklime detoxification method) in Examples 2-7 of this invention, used for xanthan gum fermentation.

[0035] Figure 8 —Infrared spectral analysis of xanthan gum produced by fermentation of sugarcane bagasse enzymatic detoxification solution obtained in Examples 1 and 5 of this invention.

[0036] Figure 9 —Comparative analysis of sugarcane bagasse enzymatic hydrolysate detoxification solution obtained by different CO2 injection rates in Examples 8-10 of this invention for xanthan gum fermentation.

[0037] Figure 10 - High performance ion chromatography (HPLC) results of xanthan gum produced by enzymatic detoxification fermentation of sugarcane bagasse obtained in Examples 1 and 5 of this invention and commercial xanthan gum.

[0038] 1: Fucose; 2: Galactose; 3: Rhamnose; 4: Arabinose; 5: Glucosamine; 6: Galactose; 7: Glucose; 8: Xylose; 9: Mannose; 10: Fructose; 11: Galacturonic acid; 12: Glucuronic acid.

[0039] Figure 11 - NMR spectra of xanthan gum produced by enzymatic detoxification fermentation of sugarcane bagasse obtained in Examples 1 and 5 of this invention and commercial xanthan gum;

[0040] Among them, (a) commercial grade xanthan gum; (b) polysaccharide obtained by fermentation of bagasse hydrolysate in Example 5 after treatment with the "CaO-CO2" method; and (c) polysaccharide obtained by fermentation of bagasse hydrolysate in Example 1 after treatment with calcium phosphate detoxification method. Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0042] The strain: Xanthomonas brasiliensis was preserved in the Key Laboratory of Sugar Chemistry and Biotechnology, Ministry of Education, College of Bioengineering, Jiangnan University;

[0043] Raw materials: Sugarcane bagasse enzymatic hydrolysate was provided by Guangxi Nongken Mingyang Biochemical Co., Ltd.;

[0044] Instrument: Shimadzu LC-2010HT high performance liquid chromatograph (UV detector);

[0045] Reagents: Furfural (FFR) was purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd.; 5-hydroxymethylfurfural (HMF) was purchased from Aladdin Biochemical Technology Co., Ltd.; n-butanol, n-hexane, ethyl acetate, dichloromethane, chloroform, sucrose, beef extract, yeast extract, agar, sodium nitrate, magnesium sulfate heptahydrate, dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, and ferrous sulfate were purchased from Sinopharm Chemical Reagent Co., Ltd.; tryptone was purchased from Beijing Bairddi Biotechnology Co., Ltd., and calcium oxide was purchased from Shanghai Titan Technology Co., Ltd.

[0046] 1. High-performance liquid chromatography conditions for determining FFR and HMF

[0047] High performance liquid chromatography conditions

[0048] The UV detector was 285 nm; the chromatographic column was an Ultimate XB-C18 column (250 mm × 4.6 mm, 5 μm), and the column temperature was 35 ℃. Under the HPLC detection elution gradient conditions: 0–2 min, methanol-0.5% formic acid (15:85, v / v); 2 min–24 min, methanol-0.5% formic acid (50:50, v / v); 24 min–27 min, methanol-0.5% formic acid (100:0, v / v); 27 min–29 min, methanol-0.5% formic acid (100:0, v / v); 29 min–45 min, methanol-0.5% formic acid (15:85, v / v). The sample was filtered through a 0.22 μm filter membrane, and 10 μl was injected after the instrument stabilized.

[0049] Preparation of standard solutions

[0050] Accurately weigh 500 mg of furfural (FFR) and 500 mg of 5-hydroxymethylfurfural (HMF) using an analytical balance, and place each into a 1000 mL volumetric flask. Dilute to volume with purified water to prepare a standard stock solution of 500 mg / L for subsequent experiments. When preparing the standard curve, the stock solution is gradually diluted to prepare standard solutions of different concentrations as shown in Table 1.

[0051] Table 1. Concentration Series of Liquid Standard Solutions

[0052]

[0053] See Figure 1 The elution positions of HMF and FFR were determined using HMF and FFR standards, as well as a mixed standard of HMF and FFR. The elution time of HMF was 12.256 min, and that of FFR was 13.880 min. The composition of sugarcane bagasse enzymatic hydrolysate is complex; the true elution positions of HMF and FFR were determined by comparing the standard and sample images. The elution time of HMF in the sugarcane bagasse enzymatic hydrolysate was 12.502 min, and that of FFR was 14.373 min.

[0054] 2. Culture medium components

[0055] Solid culture medium (g / L): sucrose 20, tryptone 5, beef extract 3, yeast extract 1, agar 20, pH = 7.0;

[0056] Seed culture medium (g / L): sucrose 20, tryptone 5, beef extract 3, yeast extract 1, pH = 7.0;

[0057] Fermentation medium (g / L): Glucose 40, Tryptone 2.0, Sodium nitrate 2.0, Magnesium sulfate heptahydrate 2.5, Dipotassium hydrogen phosphate trihydrate 3.5, Potassium dihydrogen phosphate 2.0, Ferrous sulfate 0.01, pH = 7.0.

[0058] 3. Xanthan gum production and extraction from Xanthan bacillus fermentation.

[0059] Xanthomonas colonies of rapeseed were passaged from petri dishes every two weeks. The preserved inoculum was streaked onto petri dishes and incubated at 30 °C for 48 h. Large, plump single colonies were selected and transferred to seed culture medium, which was then incubated at 30 °C and 200 r / min for 18 h. A 10% inoculum was then added to sugarcane bagasse enzymatic hydrolysate, which had undergone different detoxification processes to obtain the sugarcane enzymatically detoxified solution, and fermented for 96 h. After fermentation, the fermentation broth was centrifuged at 4 °C and 8000 r / min for 35 min. The supernatant was collected and three times its volume of 95% ethanol was added for alcohol precipitation. The precipitate was collected by vacuum filtration, washed with alcohol, and dried in a 60 °C oven to constant weight. The xanthan gum yield was then weighed and calculated.

[0060] 4. Quicklime detoxification method

[0061] A measured amount of solid calcium oxide was added to the bagasse enzymatic hydrolysate and stirred continuously for more than 6 hours. After standing, the solid was removed by filtration, and carbon dioxide gas was then introduced until the pH reached the specified values ​​of 5.87, 6, 7, and 8. The solid was then removed by filtration, and the filtrate was sterilized at 115℃ for 20 minutes. This filtrate was then used for Xanthomonas fermentation of wild rapeseed to obtain xanthan gum detoxified with quicklime.

[0062] Comparative Example 1 (Organic Reagent Extraction Detoxification Method)

[0063] Extraction was performed using different organic reagents (chloroform, dichloromethane, n-hexane, n-butanol, ethyl acetate) at different volume ratios (organic reagent: sugarcane bagasse hydrolysate = 1:2; 1:1; 2:1; 3:1). Sugarcane bagasse hydrolysate and organic solvent were added sequentially to a 250 mL separatory funnel according to the ratio. The mixture was shaken at room temperature for 30 min, then allowed to stand for 5 h. After complete separation of the two phases, the aqueous phase was collected by pouring it out from the top and bottom openings respectively. This aqueous phase was used for subsequent high-performance liquid chromatography analysis and as a fermentation substrate for xanthan gum fermentation.

[0064] The concentrations of HMF and FFR in the aqueous phase and sugarcane bagasse enzymatic hydrolysate obtained after extraction (the concentration of HMF before extraction was 18.49 mg / L and the concentration of FFR was 35.60 mg / L) were determined, and the extraction rate was calculated.

[0065] Extraction rate % = (c(initial concentration) - c(concentration after extraction)) / (c(initial concentration))

[0066] The results of FFR and HMF in the sample are as follows Figure 2 As shown. By Figure 2 It is evident that as the aqueous phase to organic phase ratio decreases, the extraction rates of several reagents for FFR and HMF gradually increase, while the extraction rates of FFR and HMF for several organic reagents tend to stabilize. n-Butanol exhibits higher extraction rates for both HMF and FFR in sugarcane bagasse enzymatic hydrolysate than the other extractants, especially for HMF. This may be because n-butanol, as a polar solvent, contains hydroxyl groups that can form hydrogen bonds with the carbonyl oxygen and furan epoxy in HMF and furfural, resulting in strong solubility. Dichloromethane and trichloromethane show similar extraction efficiencies for HMF and FFR, but both are higher than ethyl acetate. This is because these two chlorinated solvents are more polar than esters. n-Hexane, however, has extremely poor extraction effects for HMF and FFR because it is a non-polar solvent and difficult to mix with polar solvents. Nevertheless, it can extract non-polar, lipid-soluble impurities from the organic phase, thereby purifying FFR and HMF. Considering toxicity and reagent recovery, chloroform and dichloromethane are toxic and volatile, while n-hexane has a low extraction rate, and n-butanol is easily soluble in water. Therefore, ethyl acetate was subsequently selected as the organic extractant for FFR and HMF of sugarcane bagasse enzymatic hydrolysate.

[0067] See Figure 4 Using n-butanol as the extractant and the aqueous phase after extraction as the fermentation medium, the xanthan gum fermentation yield was higher than other groups. Compared to the control group that directly used untreated sugarcane bagasse enzymatic hydrolysate as the fermentation broth, the applicant's yield increased slightly from 1.68 g / L to 2.433 g / L, but without significant change. Figure 2 and Figure 3 This indicates that organic extraction can effectively remove HMF and FFR from sugarcane bagasse hydrolysate, but it does not have a significant detoxification effect. The treated sugarcane bagasse hydrolysate is difficult for Xanthomonas oryzae to utilize directly.

[0068] Comparative Example 2

[0069] Xanthomonas oryzae was fermented and cultured in the fermentation medium of this application (without HMF and FFR), and in fermentation medium containing equal concentrations of HMF and FFR before extraction (HMF = 18.9 mg / L, FFR = 35.6 mg / L) and after extraction (HMF = 6.58 mg / L, FFR = 7.55 mg / L) with the addition of sugarcane bagasse enzymatic hydrolysate, respectively, for the production of xanthan gum. The xanthan gum yield was as follows: Figure 5 As shown.

[0070] Depend on Figure 5The results showed that the xanthan gum yields of fermentation media with equal concentrations of HMF and FFR before extraction (HMF = 18.9 mg / L, FFR = 35.6 mg / L) and after extraction (HMF = 6.58 mg / L, FFR = 7.55 mg / L) were 22.04 g / L, 23.22 g / L, and 25.77 g / L, respectively, compared to the fermentation media without HMF and FFR. The yields after extraction with equal concentrations of HMF and FFR were slightly higher than those before extraction, but slightly lower than those in the control group (without HMF and FFR). This indicates that HMF and FFR affect xanthan gum production by *Xanthomonas campestris*, and the higher the concentration of HMF and FFR in the fermentation broth, the greater the impact on xanthan gum yield. However, the yields among the three groups were not significantly different, suggesting that the HMF and FFR content in the sugarcane bagasse hydrolysate has little effect on xanthan gum yield, and other substances may also play a role.

[0071] Example 1

[0072] This embodiment describes a detoxification process for sugarcane bagasse enzymatic hydrolysate. The process uses calcium phosphate to treat the hydrolysate, removing weak acid components, phenolic substances, and furfural compounds. Specifically, the hydrolysate is adjusted to pH 3 with phosphoric acid, and then further adjusted to pH 7 with solid CaO. After standing for 1 hour, it is filtered to remove the solid calcium phosphate. The filtrate is then sterilized at 115°C for 20 minutes to obtain the detoxified sugarcane bagasse enzymatic hydrolysate.

[0073] Streaking of preserved Xanthomonas campestris strains on petri dishes and incubating at 30 °C for 48 h was performed. Large, plump single colonies were selected and transferred to seed culture medium, where they were cultured at 30 °C and 200 r / min for 18 h. Then, a 10% inoculum was added to the sugarcane enzymatically detoxified solution obtained by the calcium phosphate detoxification method in this embodiment, and fermented for 96 h. After fermentation, the fermentation broth was centrifuged at 8000 r / min for 35 min at 4 °C. The supernatant was collected and three times its volume of 95% ethanol was added for alcohol precipitation. The precipitate was collected by vacuum filtration, washed with alcohol, and dried in a 60 °C oven to constant weight. The xanthan gum yield was then weighed and calculated.

[0074] Depend on Figure 6 It can be seen that the group treated with calcium phosphate detoxification had a significantly higher yield than the group that fermented sugarcane bagasse hydrolysate directly without treatment. The xanthan gum yield increased from 1.69 g / L to 17.5 g / L, but it was still lower than the xanthan gum yield produced by Xanthomonas oryzae fermentation in the fermentation medium (25.77 g / L).

[0075] Examples 2-10

[0076] A detoxification process for sugarcane bagasse enzymatic hydrolysate involves treating the sugarcane bagasse enzymatic hydrolysate with quicklime to remove weak acid components, phenolic substances, and furfural substances; the process includes the following steps:

[0077] S1. Neutralization and detoxification: Add a quantitative amount of calcium oxide solid to the sugarcane bagasse enzymatic hydrolysate, stir continuously for 6 hours, and remove the solid by initial filtration after standing.

[0078] S2, fine-tuning pH by introducing CO2: After introducing carbon dioxide gas to make the pH of the filtrate obtained from S1 filtration ≤ 8, filter again to remove solids. The filtrate is then sterilized at 115℃ for 20 min to obtain sugarcane bagasse enzymatic detoxification solution.

[0079] Table 1. Process parameters for sugarcane bagasse enzymatic hydrolysate detoxification process (quicklime detoxification method) in Examples 2-10

[0080]

[0081] Streaking of preserved Xanthomonas campestris strains on petri dishes and incubating at 30 °C for 48 h resulted in the selection of large, plump single colonies for seed culture at 30 °C and 200 r / min for 18 h. A 10% inoculum was then added to the sugarcane enzymatically detoxified liquid obtained using the quicklime detoxification method described in Examples 2-10, and fermented for 96 h. After fermentation, the fermentation broth was centrifuged at 8000 r / min for 35 min at 4 °C. The supernatant was then precipitated with three times its volume of 95% ethanol. The precipitate was collected by vacuum filtration, washed with alcohol, and dried in a 60 °C oven to constant weight. The xanthan gum yield was then weighed and calculated.

[0082] The sugarcane bagasse enzymatic hydrolysate detoxification solution obtained by the sugarcane bagasse enzymatic hydrolysate detoxification process (quicklime detoxification method) in Examples 2-7 was used as a fermentation substrate, and its xanthan gum yield was as follows: Figure 7 As shown. By Figure 7 It can be seen that the yield of sugarcane bagasse enzymatic hydrolysate gradually increases with the increase of CaO addition, reaching a maximum yield of 22.93 g / L when the CaO addition is 2.4 g / L. Compared with the xanthan gum yield of 17.5 g / L obtained from the fermentation of sugarcane bagasse enzymatic hydrolysate treated with calcium phosphate detoxification, the yield is increased by 31.02%. When the CaO addition exceeds 2.4 g / L and continues to be added in excess, the xanthan gum yield gradually decreases.

[0083] When the amount of CaO added is ≤2.4 g / L, CaO dissolves in water to form Ca(OH)2, which can neutralize the organic / inorganic acid components in the enzymatic hydrolysate, converting them into the corresponding calcium salts. Otherwise, excess undissociated acid molecules will freely diffuse into the neutral to slightly alkaline intracellular environment, dissociating into acid radicals and protons that cannot freely diffuse from the intracellular environment, further lowering the intracellular pH and affecting various intracellular enzymes that require a neutral pH to function properly. Simultaneously, to restore the normal pH, bacteria need to consume a large amount of ATP to actively transport protons back to the extracellular environment, which is detrimental to normal bacterial growth and metabolism. Secondly, under alkaline conditions, phenolic substances that damage the cell membrane will react with Ca... 2+ The reaction produces an insoluble precipitate. Finally, furfural compounds (HMF and FFR) decompose into less toxic products (such as furfuryl alcohol and furoic acid) under alkaline and heated conditions. Therefore, it promotes Xanthomonas oryzae to utilize sugarcane bagasse enzymes to decompose nutrients in the venom and ferment to produce xanthan gum, thereby increasing the yield of xanthan gum.

[0084] When the added CaO concentration exceeds 2.4 g / L, further addition of CaO leads to a decrease in xanthan gum yield. This is because excess CaO first reacts with reducing sugars under strongly alkaline conditions, easily causing aldose-enrichediol rearrangement. This not only reduces the total amount of fermentable sugars by bacteria but also generates enrichediol structures that further degrade into various organic acids. Secondly, excess CaO generates a large amount of fine gypsum precipitate, which adsorbs and encapsulates the sugar solution, resulting in further loss of usable carbon sources. Finally, excess CaO... 2+ This can lead to excessively high ion concentrations, creating a high osmotic pressure environment that is unfavorable for bacterial growth.

[0085] Xanthan gum obtained from the enzymatic detoxification fermentation of sugarcane bagasse treated in Examples 1 and 5 was compared with commercial xanthan gum using infrared spectroscopy. The results are as follows: Figure 8 As shown. In infrared scanning, 3200–3500 cm⁻¹ -1 The absorption peak is caused by the axial deformation of the OH group; 2900 cm⁻¹ -1 The absorption peak at 1600 cm⁻¹ is caused by axial deformation of -CH₂. -1 The absorption peak at this point is caused by the -C=O stretching vibration on pyruvate; the absorption peaks of ester, carboxyl, aldehyde, and ketone groups are in the range of 1700–1730 cm⁻¹. -1 ,and Figure 8 At 1700 cm -1 The absorption peak at 800–1500 cm⁻¹ should be due to the carboxyl groups in xanthan gum. -1 The region exhibits the most absorption peaks within the range. These peaks are caused by axial deformation of functional groups and stretching vibrations throughout the molecule, and are crucial for distinguishing between the two compounds. Figure 8The infrared scanning results of the polysaccharides obtained by the two treatment methods were consistent with those of commercial-grade xanthan gum. Therefore, it can be determined that the polysaccharide produced by Xanthomonas oryzae using detoxified sugarcane bagasse enzymatic hydrolysate is xanthan gum.

[0086] Based on the experimental results of Examples 2-7, in order to achieve the lowest production cost and the highest xanthan gum yield, the applicant selected the addition of 2.40 g / L CaO to the sugarcane bagasse enzymatic hydrolysate and further adjusted the final pH of the sugarcane bagasse enzymatic hydrolysate to determine the optimal CO2 injection rate. Specific conditions are as shown in Examples 8-10. The sugarcane bagasse enzymatic detoxification solutions obtained from the treatments in Examples 5 and 8-10 were used for xanthan gum production, and the corresponding xanthan gum yields are as follows: Figure 9 As shown.

[0087] Depend on Figure 9 It can be seen that the higher the CO2 flow rate, the greater the xanthan gum yield. The yield reaches its maximum at pH 6, at 21.88 g / L. With continued increases in CO2 flow rate, the solution pH gradually decreases, eventually reaching 5.87 and remaining stable, with a slight decrease in yield to 20.44 g / L. This indicates that maintaining a slightly acidic pH in the sugarcane bagasse enzymatic detoxification solution is more conducive to the growth of *Xanthomonas aeruginosa* and the synthesis of xanthan gum.

[0088] The polysaccharide colloids produced by calcium phosphate detoxification and quicklime detoxification methods were analyzed by high-performance ion chromatography after hydrolysis with trifluoroacetic acid, as shown in the following figures. Figure 10 As shown. Comparison Figure 10 The peak elution results of the monosaccharide standards showed that the monosaccharide composition of xanthan gum fermented from bagasse enzymatic hydrolysate treated with calcium phosphate detoxification was glucose:xylose:mannose:glucuronic acid = 6.4:2.6:1:0.38. The monosaccharide composition of xanthan gum fermented from bagasse enzymatic hydrolysate treated with quicklime detoxification was glucose:xylose:mannose:glucuronic acid = 11.8:5.8:1:0.34.

[0089] like Figure 11 As shown, the peak around 1.2 ppm is caused by the H in the pyruvate group on the xanthan gum side chain; the H absorption peak in the acetyl group on the xanthan gum side chain is at 1.8–1.9 ppm; the peak between 2.1–2.7 ppm is caused by the H in the -CH2 group on uronic acid or glucuronic acid, which corresponds to glucuronic acid on the side chain in xanthan gum; the H absorption peak on the hydroxyl group is at 3.4–3.9 ppm. Figure 11 It can be seen that the 1H NMR absorption peaks of polysaccharides produced from bagasse enzymatic hydrolysate after treatment with calcium phosphate and quicklime are basically consistent with those of commercial-grade xanthan gum. Figure 8 The infrared spectral results shown can basically confirm that the two newly produced polysaccharides are xanthan gum.

[0090] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A detoxification process for sugarcane bagasse enzymatic hydrolysate, characterized in that, Sugarcane bagasse enzymatic hydrolysate was treated using quicklime or calcium phosphate detoxification methods to remove weak acid components, phenolic substances, and furfural substances from the sugarcane bagasse enzymatic hydrolysate.

2. The sugarcane bagasse enzymatic hydrolysate detoxification process as described in claim 1, characterized in that, The specific operation of the quicklime detoxification method is as follows: a certain amount of calcium oxide solid is added to the sugarcane bagasse enzymatic hydrolysate, stirred continuously, and after standing, the solid is removed by initial filtration. Then, carbon dioxide gas is introduced to make the pH of the filtrate ≤ 8, and the solid is removed by filtration again. The filtrate is sterilized at high temperature to obtain the sugarcane bagasse enzymatic detoxification solution.

3. The sugarcane bagasse enzymatic hydrolysate detoxification process as described in claim 2, characterized in that, The quicklime detoxification method includes the following steps: S1. Neutralization and detoxification: Add calcium oxide solid to bagasse enzymatic hydrolysate at a dosage of 2~3.0 g / L, stir continuously for more than 6 hours, let stand, and then filter out the solid for the first time. S2, fine-tuning pH by introducing CO2: After introducing carbon dioxide gas to bring the pH of the filtrate obtained from S1 filtration to 5.87~8, filter again to remove solids. The filtrate is then sterilized at 115℃ for 20 min to obtain sugarcane bagasse enzymatic detoxification solution.

4. The sugarcane bagasse enzymatic hydrolysate detoxification process as described in claim 3, characterized in that, In S1, the amount of calcium oxide solid added is 2.40 g / L.

5. The sugarcane bagasse enzymatic hydrolysate detoxification process as described in claim 4, characterized in that, In S2, the amount of CO2 introduced is controlled so that the pH of the filtrate obtained from S1 filtration is ≤7.

6. The sugarcane bagasse enzymatic hydrolysate detoxification process as described in claim 5, characterized in that, In S2, the amount of CO2 introduced is controlled so that the pH of the filtrate obtained from S1 filtration reaches 5.87-6.

0.

7. The sugarcane bagasse enzymatic hydrolysate detoxification process as described in claim 1, characterized in that, The specific operation of the calcium phosphate detoxification method is as follows: the sugarcane bagasse enzymatic hydrolysate is adjusted to pH ≤ 3 with phosphoric acid, and then the pH is adjusted to 7 with solid CaO. After standing for more than 1 hour, the solid calcium phosphate is removed by filtration, and the filtrate is sterilized at high temperature to obtain the sugarcane bagasse enzymatic detoxification solution.

8. The application of a sugarcane bagasse enzymatic hydrolysis detoxification process in xanthan gum production, characterized in that, Sugarcane bagasse enzymatic detoxification solution prepared using the sugarcane bagasse enzymatic hydrolysis detoxification process as described in any one of claims 1 to 7 is used as a fermentation substrate to produce xanthan gum.

9. The application of the sugarcane bagasse enzymatic hydrolysis detoxification process as described in claim 8 in xanthan gum production, characterized in that, Xanthomonas aeruginosa was added to the sugarcane bagasse enzymatic detoxification solution prepared using the above-mentioned sugarcane bagasse enzymatic hydrolysis detoxification process. The mixture was fermented for more than 96 hours. The supernatant obtained by centrifugation of the fermentation broth was then subjected to alcohol precipitation. The precipitate obtained by vacuum filtration was washed and dried to obtain xanthan gum.

10. The application of the sugarcane bagasse enzymatic hydrolysis detoxification process as described in claim 9 in xanthan gum production, characterized in that, The amount of Xanthomonas brasiliensis added was 10% (V / V) of the sugarcane bagasse enzymatic detoxification solution.

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