Method for high-value utilization of sweet sorghum stem juice

Abstract

The present application belongs to the technical field of recycling of agricultural and forestry wastes, and particularly relates to a method for high-value utilization of sweet sorghum stem juice. The present application provides a method for high-value utilization of sweet sorghum stem juice. The method solves the bottlenecks in the actual industrial application of sweet sorghum stem juice (a natural biomass raw material with complex components), such as the inhibition of the catalytic system by pigments, impurities and unknown components, and the difficulty in identifying, retaining and cooperatively separating high-value components (such as D-mannitol) in the raw material. The high-value utilization of D-glucose and D-fructose in sweet sorghum stem juice is successfully realized.

Description

Technical Field

[0001] This invention belongs to the field of agricultural and forestry waste recycling technology, and specifically relates to a method for high-value utilization of sweet sorghum stem juice. Background Technology

[0002] Sweet sorghum stalks are a biomass resource with great development potential. Their stalks are rich in fermentable sugars (glucose, fructose, etc.). However, their current utilization mode is still mainly low-value. Most of them are produced by fermenting the juice through pressing to produce fuel ethanol or directly used as silage. Some are roughly processed into syrup containing impurities, which means that the high-value-added components have not been deeply explored, which seriously restricts the value-added space of sweet sorghum stalks.

[0003] D-Mannitol is a natural hexose alcohol with low-calorie, antioxidant, and anti-caries properties. It has been proven to be an important precursor for anti-tumor drugs and immunostimulants, and is widely used in the food, medical, and chemical industries. my country permits the use of D-mannitol as a food additive and has established corresponding national food safety standards. D-Allulose is a currently attracting attention as a natural functional low-calorie sweetener, with a sweetness approximately 70% that of sucrose and only 0.4 kcal / g. It is a good sucrose substitute with physiological functions such as low calorie, fat reduction, antioxidant, and neuroprotective effects, and is widely used in the food, pharmaceutical, and health care fields. In July 2025, the National Health Commission of China officially approved D-Allulose as a new food ingredient, marking its official entry into the Chinese consumer market.

[0004] This invention provides a method for the high-value utilization of sweet sorghum stem juice, specifically involving the high-value utilization of D-glucose and D-fructose in sweet sorghum stem juice. D-glucose and D-fructose are converted into D-allulose, a high-value-added functional low-calorie sweetener, through whole-cell catalysis. Furthermore, fermentation and chromatography are used to achieve efficient separation and purification of D-allulose and D-mannitol. The purity of the obtained D-allulose solution is 96.5%, and the purity of the D-mannitol solution is 96.2%.

[0005] Therefore, there is an urgent need to develop synergistic technologies that integrate biocatalytic transformation and precise separation in order to achieve the targeted transformation of sugar components into high-value sweeteners and the high-value utilization of all components. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for the high-value utilization of sweet sorghum stem juice. It solves the bottlenecks faced in the practical industrial application of sweet sorghum stem juice (a complex natural biomass raw material): such as the inhibition of the catalytic system by pigments, impurities, and unknown components, as well as the challenges of identifying, retaining, and synergistically separating existing high-value components (such as D-mannitol) in the raw material. The method successfully achieves the high-value utilization of D-glucose and D-fructose in sweet sorghum stem juice.

[0007] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0008] The first aspect of this invention discloses a method for co-producing D-allulose and D-mannitol from sweet sorghum stalk juice, comprising the following steps:

[0009] (1) Raw material pretreatment and decolorization: The juice obtained by washing, crushing and pressing sweet sorghum straw is concentrated to obtain sweet sorghum stem juice concentrate; in order to facilitate centrifugation, the concentrate is mixed with water at a mass ratio of 1:4, and after centrifugation to remove solids, powdered activated carbon is added for decolorization treatment. The decolorization conditions are: activated carbon addition amount 3.9%-4.1% (w / w), stirring at room temperature for 10-15 hours, and decolorization rate ≥98.9%; after decolorization, the solution is obtained by centrifugation and filtration.

[0010] (2) Whole-cell catalytic transformation: Using the Bacillus subtilis engineered strain Bs.WB600-pMA5-dpe-gi, which co-expresses glucose isomerase and D-allulose 3-epimerase, as a whole-cell biocatalyst, at pH 7.0, Under conditions of 39-41 and 69-71 °C, the D-glucose and D-fructose in the clarified sugar solution are catalyzed to convert into D-allulose; the clarified sugar solution contains D-glucose, D-fructose, and D-mannitol, which is naturally present and retained from sweet sorghum straw.

[0011] (3) Selective fermentation to remove residual sugar: The reaction solution obtained in step (2) was fermented using Saccharomyces cerevisiae S288C under the following conditions: =React at 14-16, 30 ℃, 210-230 rpm for 21-23 hours to selectively remove unreacted D-glucose and D-fructose while retaining D-allulose and D-mannitol;

[0012] (4) Chromatographic separation and purification: The mixed sugar solution obtained in step (3) after removing residual sugar was separated using a chromatographic column filled with Dowex® 50WX4 (200-400 mesh) resin. The column length was 1 m and the column diameter was 0.98 cm. The separation conditions were: injection volume 0.5-1 mL, eluent water, circulating water bath temperature 50 ℃, and flow rate 2 mL / min. The D-mannitol elution fraction and the D-alulose elution fraction were collected separately to obtain high-purity D-mannitol and D-alulose.

[0013] The culture conditions for the Bacillus subtilis engineered strain in step (2) are as follows: cultured in TB medium supplemented with 1 mM Fe³⁺ at 37 ℃ and 240-260 rpm for 23-25 ​​hours; the composition of the TB medium is: 12 g / L peptone, 24 g / L yeast extract, and 4 mL / L glycerol. 17 mM, 72 mM.

[0014] After the whole-cell catalytic conversion in step (2) is completed, the concentration ratio of D-mannitol to D-alokulose in the reaction system is 1.86:1.

[0015] The sweet sorghum stem juice concentrate described in step (1) is obtained by boiling.

[0016] An engineered Bacillus subtilis strain for implementing the above method, the strain being Bs.WB600-pMA5-dpe-gi, co-expresses glucose isomerase and D-allulose 3-epimerase; the amino acid sequence of the glucose isomerase is shown in SEQ ID NO: 1, and the amino acid sequence of the D-allulose 3-epimerase is shown in SEQ ID NO: 2.

[0017] GI amino acid sequence:

[0018] MKYFKDIPEVKYEGPQSDNPFAFKYYNPDEIIDGKPLKDHLRFAIAYWHTFCATGSDPFGQPTIVRPWDKFSNRMDNAKARVEAAFEFFELLDVPFFCFHDRDIAPEGENLK ESNKNLDEIVSLIKEYLKTSKTKVLWGTANLFSHPRYVHGAATSCNADVFAYAAAQVKKALEVTKELGGENYVFWGGREGYETLLNTDMGLELDNLARFLHMAVEYAKEIGFD GQFLIEPKPKEPTKHQYDFDSAHVYGFLKKYDLDKYFKLNIEVNHATLAGHDFHHELRFARINNMLGSIDANMGDLLLGWDTDQFPTDVRLTTLAMYEVIKAGGFDKGGLNF DAKVRRGSFELEDLVIGHIAGMDAFAKGFKIAYKLVKDGVFDKFIDERYKSYKEGIGAKIVSGEANFKMLEEYALSLDKIENKSGKQELLEMILNKYMFSEHHHHHH (SEQID NO: 1).

[0019] DPE amino acid sequence:

[0020] MKYGIYYAYWEKEWNGDYKYYIDKISKLGFDILEISCGAFSDYYTKDQELIDIGKYAKEKGVTLTAGYGPHFNESLSSSEPNTQKQAISFWKETLRKLKLMDIHIVGGALYGYWPVDYSKPFDKKRDLENSIKNMKIISQYAEEYDIMMGMEVLNR FEGYMLNTCDEALAYVEEVGSSNVGVMLDTFHMNIEEDNIAAAAIRKAGDRLYHFHIGEGNRKVPGKGMLPWNEIGQALRDINYQHAAVMEPFVMQGGTVGHDIKIWRDIIGNCSEVTLDDMDAQSALHFVKHVFEVDDEDEDEDEDEDEDHHHHHH (SEQ ID NO: 2).

[0021] This invention is the first to propose using sweet sorghum stem juice, an agricultural waste extract with complex components, as a unified raw material. Through a complete process chain, it simultaneously achieves the efficient conversion of D-glucose and D-fructose into D-allulose, as well as the full retention and high-purity recovery of naturally occurring D-mannitol in the raw material.

[0022] Compared with the prior art, the beneficial effects of this invention are as follows:

[0023] 1. This invention is the first to achieve high-value, targeted utilization of all components of sweet sorghum stem juice, a complex matrix: This invention not only converts the D-glucose and D-fructose, which account for a relatively high proportion, into D-allulose, but also innovatively identifies and retains D-mannitol, which is naturally present in the raw material and has independent high value, throughout the process. Finally, two high-purity functional sweeteners are produced from the same raw material, which greatly improves the comprehensive utilization value and economy of the raw material.

[0024] This invention develops a set of synergistic integrated processes adapted to complex raw materials: In response to the characteristics of sweet sorghum stem juice, such as complex composition, deep color and impurities, this invention adopts a complete technology chain including specific decolorization process, whole-cell catalyst resistant to complex matrices, selective fermentation strategy and targeted chromatographic separation method.

[0025] 3. Through optimization, the process of this invention can efficiently convert the target sugar while selectively removing impurities (such as residual monosaccharides) without losing the target product, and ultimately achieves efficient separation of D-allulose and D-mannitol (both with purities >96%), which have similar properties. The entire process route is clear, the conditions are well-defined, and the reproducibility is good, possessing the potential for industrial scale-up. Attached Figure Description

[0026] Figure 1 Comparison of activated carbon decolorization;

[0027] Figure 2 In the image, A is a high-performance liquid chromatogram, and B is a mass spectrum.

[0028] Figure 3 High-performance liquid chromatography (HPLC) chromatograms before and after whole-cell catalytic reaction;

[0029] Figure 4 High-performance liquid chromatography (HPLC) chromatograms of yeast before and after fermentation;

[0030] Figure 5 The diagram shows that when the injection volume is 0.5 mL, A is the separation effect of the chromatographic column; B is the purity determination of D-mannitol; and C is the purity determination of D-allulose.

[0031] Figure 6 The diagram shows that when the injection volume is 1 mL, A is the separation effect of the chromatographic column; B is the purity determination of D-mannitol; and C is the purity determination of D-allulose. Detailed Implementation

[0032] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but this does not limit the present invention to the scope of the described embodiments. Process parameters not specified in the embodiments of this application can be performed according to conventional methods, and all raw materials used can be obtained through commercial channels.

[0033] Example 1: Decolorization of sweet sorghum stem juice

[0034] Weigh 10 g of concentrated sweet sorghum stalk juice into a 100 mL centrifuge tube, then add 40 g of double-deionized water, resuspend, and centrifuge at 10000 rpm for 10 min to remove solids. Add 4% Fujian Zhixing powdered activated carbon to the supernatant and stir on a magnetic stirrer at room temperature for 15 h. Measure the OD before decolorization. 420 =29.6, OD after decolorization 420 =0.331, and the calculated decolorization rate is (29.6 - 0.331) / 29.6 × 100% = 98.9%. The before-and-after decolorization effects of activated carbon are shown in the following figures. Figure 1 As shown.

[0035] Example 2 Identification of Unknown Components in Sweet Sorghum Juice

[0036] First, the unknown components in sweet sorghum stem juice were separated and collected using high-performance liquid chromatography (HPLC), with the HPLC separation conditions shown in Table 1. Then, the collected solution of the unknown components was analyzed by mass spectrometry (MS) using a linear orbital trap electron transfer dissociation source high-resolution mass spectrometer (LOSMS) with negative ion ESI. The mobile phase was 80% acetonitrile. The molecular weight of the unknown component was 182.07, similar to that of the rare sugar alcohols D-mannitol and D-sorbitol. Subsequently, the elution times of D-mannitol, D-sorbitol, and the unknown components in the sweet sorghum stem juice were analyzed by HPLC, and the resulting chromatograms are shown in Table 1. Figure 2 As shown in the figure, the elution time of the unknown component is consistent with that of D-mannitol, suggesting that the unknown component is likely mannitol. However, the elution times of D-mannitol and L-mannitol in liquid chromatography are usually the same. Next, we determined the optical rotation of the unknown component, D-mannitol, and L-mannitol.

[0037] The automatic polarimeter used was from Shanghai Instrument & Electronics Physical Optics Instrument Co., Ltd., with a polarimeter tube length of 10 cm. The optical rotation measurement system was as follows: 1 mL of the reaction system contained 0.4-10 mg / mL mannitol, 21.28 g / L ammonium molybdate tetrahydrate, and 20 µL concentrated sulfuric acid. After standing at room temperature for 200 min, the optical rotation was measured. The measured optical rotations were: D-mannitol (5 mg / mL) 0.679, L-mannitol (4 mg / mL) -0.541, and the unknown component (5.221 mg / mL) 0.709. The optical rotation of the unknown component was positive and close to that of D-mannitol. Through the above identification, the unknown component was confirmed to be D-mannitol, and sweet sorghum stalk juice contains D-glucose, D-fructose, and D-mannitol.

[0038] Table 1. High Performance Liquid Chromatography (HPLC) Separation Conditions and Parameters

[0039] condition parameter chromatographic column Carbomix Pb-NP (10: 8%, 7.8×300 mm, 10 μm) Differential refractive index detector RID-20A mobile phase Double deionized water Flow rate 0.5 mL / min Injection volume 80 μL Column temperature 78 ℃

[0040] Example 3 Whole-cell biocatalysis of D-allulose production from sweet sorghum stem juice

[0041] The Bacillus subtilis engineered strain Bs.WB600-pMA5-dpe-gi (the strain prepared in CN117646046A), which co-expresses glucose isomerase GI (GI amino acid sequence as shown in SEQ.1) and D-allulose 3-epimerase DPE (DPE amino acid sequence as shown in SEQ.2), was used as a whole-cell biocatalyst. The culture conditions of the Bacillus subtilis engineered strain Bs.WB600-pMA5-dpe-gi are as follows:

[0042] Inoculate 1% of the culture medium into TB medium and add Fe. 3+ The final concentration was 1 mM. The culture was carried out at 37 ℃ and 250 rpm for 24 h. Then, the culture was collected by centrifugation at 10000 rpm for 5 min, washed, and the bacterial sludge was collected again as a whole-cell biocatalyst.

[0043] Conditions for whole-cell biocatalysis of D-allulose production from sweet sorghum stem juice:

[0044] The pH of sweet sorghum stalk juice (containing 27.8 g / L D-glucose, 24.7 g / L D-fructose, and 11.4 g / L D-mannitol) was adjusted to 7.0 using 10 M sodium hydroxide solution, and then used for reselection of mycelial sludge to OD. 600 =40, the resuspended solution was placed in a 70 ℃ constant temperature water bath and reacted for 12 h, as follows Figure 3 As shown, D-allulose is produced in the reaction system, and the concentration of D-allulose is 6.1 g / L.

[0045] Example 4: Removal of unreacted D-glucose and D-fructose using yeast fermentation

[0046] S. cerevisiae S288C (yeast) was inoculated into YPD medium at a 1% inoculum and cultured at 30°C and 200 rpm for 12 h. The culture was then collected by centrifugation at 10,000 rpm for 5 min, washed, and the mycelial sludge was collected again. The yeast sludge was resuspended in the solution after the whole-cell catalytic reaction to OD. 600 =15, 30 ℃, 220 rpm fermentation for 22 h, results are as follows Figure 4 As shown, all unreacted D-glucose and D-fructose were metabolized and removed by yeast, while D-mannitol and D-aloxose remained in the reaction system.

[0047] Example 5: Chromatographic separation of D-mannitol and D-allulose

[0048] A 0.5 mL mixture of D-mannitol and D-alokulose (30% sugar content) was injected into a chromatographic column. The column packing material was Dowex® 50WX4 resin (200-400 mesh). The column jacket was kept in a circulating water bath at 50 °C. Elution was performed using deionized water as the eluent at a flow rate of 2 mL / min. The separation results were as follows: Figure 5 As shown in Figure A, the D-mannitol eluent collected from 28 min to 41 min was analyzed by high performance liquid chromatography, and the purity of D-mannitol was determined to be 96.2%. Figure 5 (B); collect the D-allulose eluate from 42 min to 59 min, and determine the purity of D-allulose by high performance liquid chromatography (HPLC) to be 96.5%. Figure 5 (C)

[0049] Example 6: Chromatographic separation of D-mannitol and D-allulose

[0050] 1 mL of a 30% D-mannitol and D-alokulose mixed sugar solution was injected into a chromatographic column. The column packing material was Dowex® 50WX4 resin (200-400 mesh). The column jacket was kept in a circulating water bath at 50 ℃. Elution was performed using deionized water as the eluent at a flow rate of 2 mL / min. The separation results are as follows: Figure 6 As shown in Figure A, the D-mannitol eluent collected from 28 min to 41 min was analyzed by high performance liquid chromatography, and the purity of D-mannitol was determined to be 96.4%. Figure 6 (B); collect the D-allulose eluate from 42 min to 59 min, and determine the purity of D-allulose by high performance liquid chromatography (HPLC) to be 96.5%. Figure 6 (C)

[0051] This invention is not limited to the above-described embodiments. Any changes in shape or structure are within the scope of protection of this invention. The scope of protection of this invention is defined by the appended claims. Those skilled in the art can make various changes, modifications, substitutions, combinations, and simplifications to these embodiments without departing from the principles and essence of this invention. All such changes and simplifications should be considered equivalent substitutions and fall within the scope of protection of this invention.

Claims

1. A method for high-value utilization of sweet sorghum stem juice, characterized in that, Includes the following steps: (1) Raw material pretreatment: The sweet sorghum stalks are washed, crushed and pressed to obtain sweet sorghum stem juice, and the juice is concentrated to obtain concentrated liquid; (2) Decolorization and clarification: The concentrated liquid is mixed with water, and after removing solid impurities, it is decolorized with activated carbon. After centrifugation and filtration, a clear sugar solution is obtained. (3) Whole-cell catalytic transformation: Using Bacillus subtilis engineered strains that co-express glucose isomerase and D-alulose 3-epimerase as whole-cell biocatalysts, the strains react with the clarified sugar solution under catalytic conditions to convert D-glucose and D-fructose in the clarified sugar solution into D-alulose, resulting in a reaction solution containing D-alulose and D-mannitol. (4) Fermentation to remove residual monosaccharides: Yeast is inoculated into the reaction solution obtained in step (3) for fermentation to selectively remove unreacted D-glucose and D-fructose; (5) Chromatographic separation and purification: The liquid after fermentation in step (4) is separated by chromatography, and the eluting components are collected to obtain high-purity D-alokulose and D-mannitol products.

2. The method according to claim 1, characterized in that, In step (2), the mass ratio of the concentrate to water is 1:4; the activated carbon is powdered activated carbon, and its addition amount is 3.9% to 4.1% of the total mass of the mixture. The mixture is stirred and decolorized at room temperature for 10 to 15 hours.

3. The method according to claim 1, characterized in that, In step (3), the culture conditions for the engineered Bacillus subtilis strain are as follows: cultured in TB medium supplemented with 1 mM Fe³⁺ at 37 ℃ and 240-260 rpm for 23-25 ​​hours in a shaker; the TB medium contains 12 g / L peptone, 24 g / L yeast extract, 4 mL / L glycerol, and 17 mM Fe³⁺. and 72mM .

4. The method according to claim 1, characterized in that, In step (3), the catalytic conditions are: the pH of the reaction system is 7.0, and the amount of the whole-cell biocatalyst added is based on... The value is 39-41, and the reaction temperature is 69-71℃.

5. The method according to claim 1, characterized in that, In step (4), the yeast is Saccharomyces cerevisiae. S. cerevisiae S288C; The fermentation conditions are as follows: the amount of yeast added is based on OD 600 The sample was 14-16, and fermented at 30 ℃ and 210-230 rpm for 21-23 hours.

6. The method according to claim 1, characterized in that, In step (5), the chromatographic column parameters used for the chromatographic separation are: column length 1 m, column diameter 0.98 cm, packed resin is Dowex® 50WX4, and particle size is 200-400 mesh; the chromatographic separation conditions are: injection volume 0.5-1 mL, column temperature 50 ℃, deionized water as mobile phase, and flow rate 2 mL / min.

7. An engineered Bacillus subtilis strain for implementing the method according to any one of claims 1-6, characterized in that, The strain is Bs .WB600-pMA5- dpe - gi It co-expresses glucose isomerase (GI) and D-allulose 3-epimerase (DPE); the amino acid sequence of the glucose isomerase is shown in SEQ ID NO: 1, and the amino acid sequence of the D-allulose 3-epimerase is shown in SEQ ID NO: 2.

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