Method for preparing high-value cellobiose from a medicinal and edible aesculus chinensis residue and application thereof

Abstract

The application discloses a method for preparing high-value cellobiose from a medicine and food homologous Radix Millettiae and application of the high-value cellobiose. The method comprises the following steps: extracting cellulose from Radix Millettiae; optimizing a beta-glucanase enzymolysis process by using a response surface method; after the enzymolysis liquid is subjected to selective removal of glucose through yeast fermentation, the high-value cellobiose product is obtained through acetone precipitation and freeze-drying. The process condition is mild and environment-friendly, high-value utilization of agricultural waste is realized, the obtained cellobiose product has high purity, and an economic and effective approach is provided for large-scale production of cellobiose. Animal experiments prove that the cellobiose product has a significant relieving effect on colonitis induced by dextran sulfate sodium in mice, can effectively improve disease symptoms, reduce oxidative stress, repair intestinal barrier and regulate inflammatory response, and has a good application prospect in preparation of functional food or medicine for relieving colonitis and high-value utilization of Radix Millettiae.

Description

Technical Field

[0001] This invention belongs to the field of plant polysaccharides, and specifically relates to a method and application for preparing high-value cellobiose from the medicinal and edible residue of *Smilax glabra*. Background Technology

[0002] Cellobioses are crystalline disaccharides composed of two glucose molecules linked by β-1,4-glycosidic bonds. They are the basic repeating unit of cellulose polysaccharide chains and a key intermediate in the biodegradation of cellulose. As an important functional oligosaccharide, cellobioses are water-soluble, non-digestible, and selectively fermentable by gut microbiota, thus being considered a potential prebiotic. In recent years, their physiological activities in regulating gut microbiota, enhancing immunity, and improving intestinal barrier function have received increasing attention, demonstrating their potential application in functional foods, animal feed, and pharmaceutical excipients.

[0003] Currently, the production of cellobiose mainly relies on the hydrolysis of cellulose, with the main methods including chemical, enzymatic, and microbial methods. Chemical hydrolysis typically uses dilute acids (such as sulfuric acid and hydrochloric acid) to treat lignocellulose raw materials under heating conditions. However, this process involves numerous side reactions, easily producing byproducts such as glucose and hydroxymethylfurfural, resulting in low yields and purity of the target product, and high costs for subsequent separation and purification. Enzymatic hydrolysis is more promising due to its mild conditions and high specificity. For example, using a commercially available cellulase complex from *Trichoderma reesei* to treat pretreated microcrystalline cellulose at suitable temperature and pH can directionally generate cellobiose. However, commercially available cellulase preparations often contain highly active β-glucosidase, which further hydrolyzes cellobiose into glucose. Therefore, modifying the enzyme system through screening or genetic engineering (e.g., inhibiting or knocking out β-glucosidase activity), or using immobilized enzyme technology to control the reaction process, are key strategies for improving cellobiose yield. Microbial transformation utilizes certain strains that can directly secrete cellulase systems and accumulate cellobiose (such as some fungi and genetically engineered yeasts). However, this method currently suffers from low yields and long production cycles, and remains in the laboratory research stage. Overall, existing mainstream preparation technologies heavily rely on purified cellulose raw materials (such as microcrystalline cellulose, wood, and cotton), resulting in relatively high prices for commercially available cellobiose (currently, the market price can reach tens to hundreds of RMB per gram), and a limited range of sources. This severely restricts its large-scale application and in-depth functional research in various fields.

[0004] In view of the above bottlenecks, seeking new biomass raw materials that are cheap, readily available, and rich in cellulose, and developing efficient and low-cost green preparation processes that match them have become the key to breaking through the industrial application of cellobiose. In this context, a large amount of plant waste generated during the processing of food and Chinese medicinal materials has attracted the attention of researchers because it is rich in cellulose and is often discarded. Millettia speciosa Champ., also known as Millettia dielsiana Harms, is a well-known medicinal and edible homologous plant in the southern region of China. After its rhizomes are used to extract medicinal ingredients, make soups, or soak in wine, a large amount of solid waste will be generated. Research shows that the crude fiber content in the roots of Millettia speciosa Champ. can be as high as 32.03%, which means that there are rich cellulose resources in its processing waste. At present, these waste residues are usually discarded as solid waste, and the resource utilization has not been realized, resulting in great waste and environmental protection pressure. Therefore, using Millettia speciosa Champ. residue as a new type of cellulose raw material with distinct regional characteristics for the preparation of high-value-added product cellobiose not only conforms to the concepts of circular economy and green manufacturing but also provides innovative ideas for broadening the production sources of cellobiose. Millettia speciosa Champ.)

[0005] In terms of physiological activity, cellobiose is considered a potential prebiotic and can be fermented by microorganisms. In recent years, most research has started from animal feed and plant additives. Ren Bing et al. found in "Effects of Different Addition Levels of Oligosaccharides on Growth Performance and Serum Physiological and Biochemical Indexes of Broiler Chickens" (Feed Research. 2016, (24): 1-5.) that adding oligosaccharides containing cellobiose to broiler feed can effectively improve the activities of antioxidant enzymes such as serum T-SOD, CAT, GSH-Px, and GR, thereby improving the immune ability of broilers and their growth performance. Kong Meng found in "Research on the Immune Mechanism of Lettuce Induced by Cellobiose and Evaluation of Field Application" (Chinese Academy of Agricultural Sciences, 2025) that cellobiose can enhance the disease resistance of lettuce by activating immune-related transcriptional reprogramming and has broad-spectrum resistance to fungal diseases. Zheng Peng found in "Enzymatic Synthesis of Oligosaccharides and Their Promotion of the Growth Performance of Intestinal Probiotics" (Nanchang University, 2024) that cellobiose can significantly promote the proliferation of Lactobacillus casei, Lactobacillus acidophilus, and Lactobacillus paracasei, with better effects than fructooligosaccharide and sucrose, and can promote the production of short-chain fatty acids. The above experiments show that cellobiose has the activities of restoring intestinal health and immune activation, but the specific mechanism is still unclear. Colitis is a common chronic inflammatory bowel disease, characterized by impaired intestinal barrier function, elevated oxidative stress levels, and overexpression of pro-inflammatory factors. Currently, the clinical treatment drugs often have side effects, so finding safe and effective relievers from natural products has become a research hotspot. As a prebiotic, cellobiose has been proven to regulate the intestinal flora, but its direct relieving effect and mechanism on experimental colitis, especially cellobiose derived from Millettia speciosa Champ. residue, have not been systematically reported.

[0006] Based on the above research background and technical status quo, the purpose of this study is to use the processing waste residue of the traditional Chinese medicine Millettia speciosa Champ. in the Lingnan region as raw material, and establish a method for preparing high-value cellobiose (purity > 90%) from the residue of Millettia speciosa Champ. which is both medicine and food by optimizing the pretreatment and cellulase hydrolysis process. And the biological activity of cellobiose prepared from the regional waste residue of Millettia speciosa Champ. which is both medicine and food as raw material was studied and verified, realizing the leap from "waste residue" to "high-value functional product", and opening up a new and promising path for the comprehensive high-value utilization of by-products of traditional Chinese medicine. Summary of the Invention

[0007] In order to overcome the shortcomings of the unsystematic optimization of the cellobiose preparation process and the有待提高的产率 in the prior art, the primary object of the present invention is to provide a method for preparing high-value cellobiose from the residue of Millettia speciosa Champ. which is both medicine and food.

[0008] Another object of the present invention is to provide a method for efficiently preparing cellobiose by systematically optimizing the enzymatic hydrolysis process parameters by the response surface method.

[0009] Another object of the present invention is to provide the application of high-value cellobiose in the preparation of functional foods or drugs for preventing and / or alleviating colitis, and it is confirmed through systematic animal experiments that it can significantly improve the disease activity index, reduce oxidative stress, repair the intestinal barrier and regulate the balance of inflammatory factors.

[0010] The objects of the present invention are achieved by the following solutions: The present invention provides a method for preparing high-value cellobiose from the residue of Millettia speciosa Champ. which is both medicine and food, comprising the following steps: (1) Pretreat the residue of Millettia speciosa Champ. to remove starch, lignin and hemicellulose, and obtain cellulose of the residue of Millettia speciosa Champ.; (2) Use β-glucanase to hydrolyze the cellulose of the residue of Millettia speciosa Champ. obtained in step (1) to obtain a hydrolysis solution, wherein the enzymatic hydrolysis process parameters are optimized and determined by the response surface method; (3) Add yeast to the hydrolysis solution obtained in step (2) for fermentation to remove glucose; (4) Concentrate the liquid after fermentation in step (3), and use dialysis to remove small molecules and salt ions (5) Precipitate the liquid after dialysis in step (4) with acetone, collect the precipitate and dry it to obtain high-value cellobiose.

[0011] Further, in step (1), the residue of Millettia speciosa Champ. refers to the root residue of Millettia speciosa Champ. after a series of extraction and separation of functional active substances or processing such as brewing and decocting.

[0012] Note: The part "有待提高产率" in the original text seems to be an incomplete expression. I translated it as "有待提高的产率" according to the context. If there is a more accurate expression, please let me know and I will correct it.Further, in step (1), the pretreatment process includes: hot water treatment to remove dust and impurities from the *Smilax glabra* residue, using amylase to remove starch, using acidic sodium chlorite solution to remove lignin, using potassium hydroxide solution to remove hemicellulose, and then washing and drying to obtain *Smilax glabra* residue cellulose.

[0013] Further, in step (2), the enzymatic hydrolysis process parameters are: temperature 40-50℃, pH 4-6, reaction time 4-12h; the mass ratio of β-glucanase to cellulose from *Smilax glabra* is 0.5; and the enzymatic hydrolysis is carried out in a citrate-disodium hydrogen phosphate buffer system.

[0014] Furthermore, in step (2), the response surface methodology optimization is performed using enzymatic hydrolysis temperature (B), pH value (C), and reaction time (A) as independent variables, and cellobiose (COS-2) yield as the response value, employing a Box-Behnken design.

[0015] Furthermore, in step (2), the optimal process parameters for enzymatic hydrolysis were determined by the established quadratic multinomial regression model: temperature 45±2℃, pH 5.0±0.2, and reaction time 8±0.5 h. Under these optimal conditions, the yield of cellobiose can reach 17.81%.

[0016] Furthermore, in step (3), the yeast is Saccharomyces cerevisiae, and the amount added is 0.25% (w / v, g / L) of the volume of the enzymatic hydrolysate; the fermentation conditions are 28-32℃, 180-220rpm shaking culture for 22-26h. Under these conditions, most of the glucose can be effectively removed, while cellobiose is retained to the maximum extent.

[0017] Furthermore, in step (4), the concentration is carried out by rotary evaporation; the dialysis is carried out using a 100-500 Da dialysis bag, with stirring and dialysis at 4°C for 8 hours, and the water is changed every 2 hours.

[0018] Furthermore, in step (5), the volume ratio of the dialysis liquid to acetone is 1:8-1:10, the precipitation temperature is 2-8℃, and the precipitation time is not less than 12h.

[0019] Furthermore, in step (5), the volume ratio of the dialyzed liquid to acetone is 1:9, and the precipitation temperature is 4℃.

[0020] The present invention also provides a high-value cellobiose prepared by the above method.

[0021] Furthermore, high-value cellobiose is a high-purity cellobiose containing a small amount of glucose.

[0022] This invention also provides the application of the above-mentioned high-value cellobiose in the preparation of functional foods or medicines for the prevention and / or relief of colitis.

[0023] Furthermore, high-value fiber disaccharide plays a role in preventing and / or alleviating colitis through one or more of the following pathways: reducing the disease activity index (DAI), alleviating oxidative stress damage in colonic tissue, repairing the intestinal tight junction barrier, and regulating the balance of pro-inflammatory and anti-inflammatory factors.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) This invention uses waste residue from the processing of *Smilax glabra* as raw material, realizing the high-value utilization of waste at low cost and with sustainable sources. The β-glucanase enzymatic hydrolysis process was systematically optimized using response surface methodology, clarifying the interaction between temperature, pH, and time, significantly improving the directional yield of cellobiose, with good process repeatability, suitable for scale-up production. By selectively removing glucose through yeast fermentation and purifying with acetone precipitation, the purity of cellobiose in the final cellobiose product (high-value cellobiose) was significantly improved (the cellobiose content in the freeze-dried product can reach over 92.80%).

[0025] (2) The entire preparation process is mild and does not require strong acids or bases, making it environmentally friendly and in line with the concept of green chemistry.

[0026] (3) Animal experiments were conducted to verify that cellobiose has a significant ability to alleviate colitis in mice. It has the potential to be prepared into functional foods and drugs, and there is also a prospect for the high-value utilization of *Smilax glabra* residue. Attached Figure Description

[0027] Figure 1 This is an ion chromatogram of cellulose from *Smilax china* in Example 1.

[0028] Figure 2 The images show the 3D response surface plot and contour plot of the interaction between time A and temperature B in Example 2.

[0029] Figure 3 The images show the 3D response surface plot and contour plot of the interaction between time A and pH C in Example 2.

[0030] Figure 4 The images show the 3D response surface plot and contour plot of the interaction between temperature B and pH C in Example 2.

[0031] Figure 5 The image shows the ion chromatogram of the cellobiose product prepared under the optimized conditions in Example 2.

[0032] Figure 6 Ion chromatograms of glucose, cellobiose, cellotriose, cellotetraose, and cellopentaose standards.

[0033] Figure 7Graph of DAI scores of mice in each group in Example 4 within 10 days after DSS modeling treatment.

[0034] Figure 8 Graph of colon length of mice in each group after sample collection in Example 4.

[0035] Figure 9 Graph of spleen index of mice in each group after sample collection in Example 4.

[0036] Figure 10 Graph of MDA content in spleen tissues of mice in each group in Example 4.

[0037] Figure 11 Graph of immunohistochemical results of MPO index in colon tissues of mice in each group in Example 4.

[0038] Figure 12 Graph of HE staining of colon tissues of mice in each group in Example 4.

[0039] Figure 13 Graph of immunofluorescence of ZO-1 protein in colon tissues of mice in each group in Example 4.

[0040] Figure 14 Graph of immunofluorescence of Occludin protein in colon tissues of mice in each group in Example 4.

[0041] Figure 15 Graph of results of TNF-α, IL-1β, IL-6, and IL-10 indexes in sera of mice in each group in Example 4.

[0042] Figure 16 Graph of results of TNF-α, IL-1β, IL-6, and IL-10 indexes in colon tissues of mice in each group in Example 4. Detailed implementation manners

[0043] The present invention will be further described in detail below in conjunction with examples and drawings, but the implementation manners of the present invention are not limited thereto. For those conditions not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For those reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0044] The reagents used in the examples can be conventionally purchased from the market without special instructions.

[0045] In the present invention, "Millettia speciosa Champ." is a plant of the genus Millettia in the legume family and is a plant with both medicinal and edible uses. Millettia Speciosa Champ)" is a plant of the genus Millettia in the legume family and is a plant with both medicinal and edible uses.

[0046] Example 1: Isolation and extraction of Millettia speciosa Champ. residue cellulose 1. Preparation and characterization 180 g of dried *Smilax glabra* residue was weighed and added to distilled water at a material-to-liquid ratio of 1:20 (g / mL). 0.2% (w / w) of α-amylase was added, and the mixture was stirred in a water bath at 55℃ for 2 h. After inactivating the enzyme in a boiling water bath, the mixture was filtered. The residue was washed and dried to obtain crude *Smilax glabra* fiber. The crude fiber was then reacted with a 7.5% (v / v) sodium chlorite solution (pH 4.0) at 75℃ for 2 h. The precipitate was collected by centrifugation, washed until neutral, and dried to obtain lignin-free fiber. The lignin-free fiber was then reacted with a 10% (v / v) KOH solution (solid-to-liquid ratio 1:20 (g / mL)) at 25℃ for 12 h. The mixture was filtered, and the residue was washed until neutral and then washed with ethanol. After drying, the residue was pulverized and sieved to obtain *Smilax glabra* cellulose. The monosaccharide composition of the extracted cellulose was analyzed using ion chromatography to determine its purity.

[0047] 2. Results The cellulose obtained from *Smilax glabra* residue was weighed and measured, yielding a cellulose yield of 24.92%. The ion chromatogram of *Smilax glabra* residue cellulose is shown below. Figure 1 As shown, the purity of the cellulose was tested to be 94.69%.

[0048] Example 2: Response surface methodology for optimizing enzymatic hydrolysis to prepare cellobiose 1. Basic Enzymatic Hydrolysis Experiment: Weigh 50 mg of *Smilax glabra* cellulose into a centrifuge tube, add 5 mL of citrate-disodium hydrogen phosphate buffer solution (pH 4, pH 5, pH 6), preheat, add β-glucanase (β-glucanase to *Smilax glabra* cellulose mass ratio 0.5), and react with shaking at 40℃, 45℃, and 50℃ for 4 h, 8 h, and 12 h, respectively. Inactivate the enzyme in a boiling water bath, centrifuge, collect the supernatant, filter through a 0.22 μm filter membrane, and detect the cellobiose content using high-performance anion exchange chromatography.

[0049] 2. Response Surface Optimization Experimental Design: Based on the single-factor experiments, three key factors were selected: reaction time (A), temperature (B), and pH value (C). Three levels were set for each factor, and a Box-Behnken design was used for response surface analysis experiments. The factor and level design are shown in Table 1, and the experimental scheme and results are shown in Table 2.

[0050] Table 1 Response surface methodology and level design for cellobiose preparation

[0051] Table 2 Experimental scheme and results for the preparation of cellobiose

[0052] 3. Model Building and Analysis: Using Design-Expert 13 and Origin 2021 software, 3D response surface plots and contour maps of the interaction between A (time), B (temperature), and C (liquid-to-material ratio) were obtained. Figure 2 , 3 As shown in Figure 4, Figure 2 The 3D plot and contour plot of the response surface of the interaction between time A and temperature B are shown. Figure 3 The 3D response surface plot and contour plot of the interaction between time A and pH C are shown. Figure 4 The 3D response surface plot and contour plot of the interaction between temperature B and pH C are shown. Multiple regression fitting was performed on the data using Design-Expert software, yielding a quadratic polynomial regression equation with COS-2 yield (Y) as the response value: Y = -21.8038 + 0.0807A + 0.9235B + 0.7684C - 0.0005AB - 0.0018AC +0.0085BC - 0.0030A 2 - 0.0104B 2 - 0.1137C 2 The model has a p-value < 0.01, the lack-of-fit term is not significant, and R0 is... 2 =0.9558, the model is significant and fits well.

[0053] 4. Determination and Validation of Optimal Conditions: Model analysis determined the optimal enzymatic hydrolysis conditions to be: 8 hours, 45℃, and pH 5.0. Three parallel validation experiments were conducted under these conditions, with an average cellobiose yield of 17.81%, close to the model's prediction, demonstrating the model's reliability.

[0054] 5. Glucose removal by yeast fermentation: Take the enzymatic hydrolysate prepared in Example 2 under optimized conditions (time 8h, temperature 45℃, pH 5.0), add 0.25% (w / v, g / L) of Angel Yeast (purchased from Angel Yeast Co., Ltd.), and ferment in a shaker at 30℃ and 200rpm for 24h. After fermentation, centrifuge at 10000rpm for 5min and collect the supernatant.

[0055] 6. Dialysis: The fermented liquid is evaporated by rotary evaporation at 50℃. The concentrate is then dialyzed using a 100-500 Da dialysis bag at 4℃ for 8 hours. The water is changed every 2 hours. The liquid is collected after dialysis is completed.

[0056] 7. Acetone precipitation and drying: The dialyzed liquid was slowly added dropwise to 9 times its volume of anhydrous acetone pre-cooled to -20°C while stirring. The mixture was allowed to stand at 4°C for 12 hours to precipitate. Then, it was centrifuged at 5000 rpm for 10 minutes, and the precipitate was collected. The precipitate was then freeze-dried under vacuum for 48 hours to obtain a white, loose powdery cellobiose product, which is the high-value cellobiose.

[0057] Example 3: Product Analysis and Characterization The cellobiose product prepared under the optimized conditions in Example 2 was analyzed by high-performance anion exchange chromatography. Figure 5 The ion chromatograms of the cellobiose product prepared under the optimized conditions in Example 2 are shown. Figure 6 The ion chromatograms of glucose, cellobiose, cellotriose, cellotetraose, and cellopentaose standards are shown. The results indicate that the cellobiose content in the cellobiose product reaches 92.80%. Based on the initial mass of cellulose from *Smilax china* residue, the final yield of cellobiose is 3.71%.

[0058] Example 4: Evaluation of the alleviating effect of high-value cellobiose on DSS-induced colitis in mice 1. Experimental Materials and Instruments 1.1 Experimental animals: SPF grade C57BL / 6 male mice, weighing 18-22g.

[0059] 1.2 Main reagents: sodium dextran sulfate (DSS, molecular weight 36,000-50,000), 5-aminosalicylic acid (5-ASA), high-value cellobiose (COS-2, cellobiose product prepared under the optimal conditions in Example 2), hematoxylin-eosin (HE) staining kit, malondialdehyde (MDA) kit, myeloperoxidase (MPO) antibody, tight junction protein ZO-1 and Occludin antibody, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10 ELISA detection kit, paraformaldehyde, etc.

[0060] 1.3 Main instruments: electronic balance, vernier calipers, microplate reader, tissue homogenizer, paraffin microtome, fluorescence microscope, imaging system, etc.

[0061] 2. Experimental Methods 2.1 Animal grouping and administration: Fifty mice were randomly divided into 5 groups (n=10): (1) Control group: mice were allowed free access to distilled water and were given physiological saline by gavage. (2) Model group (DSS): mice were allowed free access to 3% (g / mL) DSS solution for modeling and were given physiological saline by gavage. (3) Positive control group (5-ASA): mice were given DSS for modeling and were given pentaminosalicylic acid (50 mg / kg / d) by gavage. (4) Low-dose COS-2 group (COS-2-L): mice were given DSS for modeling and were given high-value cellobiose (500 mg / kg / d) by gavage. (5) High-dose COS-2 group (COS-2-H): mice were given DSS for modeling and were given high-value cellobiose (2000 mg / kg / d) by gavage. The experiment lasted for 28 days: Days 1-7 were for acclimatization feeding; Days 8-14, except for the control group, all other groups were given the corresponding drugs by gavage for pretreatment; Days 15-21, except for the control group, all other groups were allowed to drink 3% (g / mL) DSS solution to establish an acute colitis model, and continued to receive the drugs by gavage; Days 22-28, all mice resumed drinking distilled water, and continued to receive the drugs by gavage until the end of the experiment.

[0062] 2.2 Disease Activity Index (DAI) score: During the experiment, the mice’s weight change, fecal characteristics and bloody stool were recorded daily, and the DAI score was calculated according to the standard (the scoring standard is shown in Table 3: the sum of the scores of weight loss percentage, fecal consistency and bloody stool).

[0063] Table 3. Detailed Scoring Rules for DAI Scoring

[0064] 2.3 Sample Collection: Mice were sacrificed on day 29, and serum was collected. The colon was completely dissected, and its length was measured. The spleen was removed and weighed, and the spleen index (spleen mass mg / body weight g) was calculated. Colon tissue from the same location was taken; a portion was fixed in 4% paraformaldehyde for pathological examination, and the other portion was frozen at -80℃ for biochemical index detection.

[0065] 2.4 Determination of Oxidative Stress Levels: A portion of colon tissue was homogenized and prepared. Following strict adherence to the kit instructions, the MDA content in the tissue homogenate was determined using ELISA. Paraffin sections of the fixed colon tissue were then subjected to MPO immunohistochemical staining, and the images were analyzed.

[0066] 2.5 Intestinal barrier function assessment: HE staining for colon pathology: Paraffin sections of colon tissue were routinely stained with HE, and the integrity of the colonic mucosa structure and the degree of inflammatory cell infiltration were observed under an optical microscope, and histopathological scoring was performed.

[0067] Immunofluorescence staining: Frozen sections of the colon were stained with immunofluorescence for ZO-1 and Occludin proteins. The expression and distribution of tight junction proteins were observed and semi-quantitatively analyzed under a fluorescence microscope.

[0068] 2.6 Detection of inflammatory factor levels: The levels of pro-inflammatory factors (TNF-α, IL-1β, IL-6) and anti-inflammatory factors (IL-10) in mouse serum and colon tissue homogenate were detected by ELISA.

[0069] 2.7 Statistical analysis: Data are expressed as mean ± standard deviation. One-way ANOVA was performed using SPSS software. LSD-t test was used for comparisons between groups. P < 0.05 was considered statistically significant.

[0070] 3. Results and Discussion 3.1 Effects of high-value cellobiose on the general condition and DAI score of colitis mice The DAI scores of mice in each group within 10 days from the start of DSS modeling are shown in the following figures. Figure 7 As shown, all five groups experienced significant weight loss, loose stools, bloody stools, and reduced activity within 7 days of DSS treatment. The DAI index rapidly increased significantly compared to the control group (P<0.01), indicating successful establishment of the colitis model. Three days after the end of DSS treatment, compared to the model group, the positive control drug pentamifil (SASP) and different doses of COS-2 interventions alleviated the above symptoms to varying degrees, manifested as reduced weight loss, improved stool consistency, and reduced bloody stools. Among them, different doses of COS-2 groups (COS-L, COS-H) showed a significant reduction in DAI scores (P<0.05), which was superior to the positive control group (5-ASA).

[0071] 3.2 Effects of high-value fiber disaccharide on colon length and spleen index Figure 8 The diagram shows the colon length of each group of mice after tissue sampling. Figure 9 The spleen index of each group of mice after tissue sampling is shown. Figure 8 and Figure 9 As shown, the colon length in the model group mice was significantly shorter than that in the control group, and the spleen index was significantly increased, indicating significant colonic shortening and systemic immune activation. All intervention groups (5-ASA, COS-2-L, COS-2-H) were able to reverse colonic shortening to varying degrees; different doses of COS-2 (COS-2-L, COS-2-H) significantly reduced splenomegaly caused by inflammation (decreased spleen index), showing statistically significant differences compared to the model group (P<0.05).

[0072] 3.3 Effects of high-value cellobiose on oxidative stress levels in colonic tissue Figure 10 The graph shows the MDA content in the spleen tissue of mice in each group. Figure 11 The immunohistochemical results of MPO index in the colon tissue of mice in each group are shown. Figure 10 and Figure 11 As shown, the MDA content in the colon tissue of the model group was significantly increased, and the MPO immunohistochemical results showed a significant increase in positive cell expression, indicating severe oxidative stress damage. After COS-2 intervention, especially the low-dose COS-2 group (COS-2-L), the MDA content in the colon tissue was significantly reduced, and the positive expression of MPO was decreased, suggesting that COS-2 has good antioxidant capacity.

[0073] 3.4 The protective effect of high-value cellobiose on the colonic mucosal barrier HE staining results: HE staining images of colon tissue from each group of mice are shown below. Figure 12 As shown, the colonic mucosa in the control group was intact, and the glands were neatly arranged. In the model group, extensive shedding of the mucosal epithelium, extensive inflammatory cell infiltration in the lamina propria, and destruction or even absence of glandular structure were observed, resulting in extremely high pathological scores. Different doses of COS-2 (COS-2-L, COS-2-H) significantly reduced mucosal damage, decreased inflammatory cell infiltration, and better protection of glandular structure; the pathological scores of the low-dose group were significantly lower than those of the model group.

[0074] Immunofluorescence results: Figure 13 Immunofluorescence images of ZO-1 protein in the colon tissue of mice in each group are shown. Figure 14 Immunofluorescence images of Occludin protein in the colon tissue of mice in each group are shown. Figure 13 and Figure 14 As shown, in the blank group, ZO-1 and Occludin proteins were continuously and clearly expressed along the intestinal epithelial cell membrane. In the model group, the expression of both proteins was significantly reduced, and their distribution was discontinuous or even absent. After COS-2 administration, especially in the low-dose COS-2 group (COS-2-L), the expression and proper distribution of ZO-1 and Occludin proteins were significantly promoted, approaching the levels of the blank group.

[0075] 3.5 The regulatory effect of high-value cellobiose on the level of inflammatory factors Figure 15 The results of TNF-α, IL-1β, IL-6, and IL-10 levels in the serum of mice in each group are shown in the figure. Figure 16 The results of TNF-α, IL-1β, IL-6, and IL-10 levels in the colon tissue of mice in each group are shown in the figure. Figure 15 and Figure 16As shown, the levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in the serum and colon tissue of mice in the model group were significantly increased, while the level of the anti-inflammatory factor IL-10 was decreased. COS-2 intervention selectively and significantly downregulated the levels of TNF-α, IL-1β, and IL-6 in the serum and colon, while upregulating the level of IL-10 (P<0.05 or P<0.01 vs. model group), indicating that COS-2 exerts its anti-inflammatory effect by regulating the balance of pro-inflammatory / anti-inflammatory factors.

[0076] 4. Conclusion This embodiment demonstrates, through a DSS-induced mouse model of acute colitis, that the high-value cellobiose prepared in this invention can effectively alleviate colitis symptoms. Its mechanism is closely related to improving oxidative stress, protecting the integrity of the intestinal mucosal barrier, and regulating the balance of systemic and local inflammatory factor networks. This study provides solid experimental evidence for the use of cellobiose as a functional food or pharmaceutical excipient in the prevention and adjuvant treatment of colitis and other inflammatory bowel diseases.

[0077] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing high-value cellobiose from the residue of *Smilax glabra* (a plant with medicinal and edible properties), characterized in that... Includes the following steps: (1) Pre-treat the residue of *Smilax glabra* to remove starch, lignin and hemicellulose, and obtain *Smilax glabra* cellulose; (2) The cellulose obtained from step (1) was enzymatically hydrolyzed using β-glucanase to obtain an enzymatic hydrolysate. The enzymatic hydrolysis process parameters were determined by response surface methodology. (3) Add yeast to the enzymatic hydrolysate obtained in step (2) for fermentation to remove glucose; (4) Concentrate the liquid after fermentation in step (3) and remove small molecules and salt ions by dialysis. (5) The liquid after dialysis in step (4) is precipitated with acetone, the precipitate is collected and dried to obtain the high-value cellobiose.

2. The method according to claim 1, characterized in that, In step (1), the "Niu Dali Zha" refers to the root residue of Niu Dali after a series of functional active substance extraction, separation, or treatment such as brewing and boiling. The pretreatment process includes: hot water treatment to remove dust and impurities from the *Smilax glabra* residue, using amylase to remove starch, using acidic sodium chlorite solution to remove lignin, using potassium hydroxide solution to remove hemicellulose, and then washing and drying to obtain *Smilax glabra* residue cellulose.

3. The method according to claim 1, characterized in that, In step (2), the enzymatic hydrolysis process parameters are: temperature 40-50℃, pH 4-6, reaction time 4-12h; the mass ratio of β-glucanase to cellulose from *Smilax glabra* is 0.

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4. The method according to claim 1, characterized in that, In step (2), the response surface methodology optimization is performed using enzymatic hydrolysis temperature, pH value and reaction time as independent variables and cellobiose yield as the response value, employing a Box-Behnken design.

5. The method according to claim 1, characterized in that, In step (2), the optimal process parameters for enzymatic hydrolysis are: temperature 45±2℃, pH 5.0±0.2, and reaction time 8±0.5h.

6. The method according to claim 1, characterized in that, In step (3), the yeast is Angel brewing yeast, and the amount of yeast added is 0.25% of the volume of the enzyme hydrolysate in g / L. The fermentation conditions are 28-32℃, 180-220rpm shaking culture for 22-26h.

7. The method according to claim 1, characterized in that, In step (4), the dialysis bag is a cellulose dialysis bag with a capacity of 100-500 Da; in step (5), the volume ratio of the dialyzed liquid to acetone is 1:8-1:10, the precipitation temperature is 2-8℃, and the precipitation time is not less than 12h.

8. A high-value cellobiose prepared by the method according to any one of claims 1-7.

9. The use of the high-value fiber disaccharide according to claim 8 in the preparation of functional foods or medicines for the prevention and / or relief of colitis.

10. The application according to claim 9, characterized in that, The high-value cellobiose exerts its preventive and / or alleviating effects on colitis through one or more of the following pathways: reducing the disease activity index (DAI), alleviating oxidative stress damage in colonic tissue, repairing the intestinal tight junction barrier, and regulating the balance of pro-inflammatory and anti-inflammatory factors.

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