Cellulose oligosaccharide mixture as well as preparation method and application thereof
By extracting cellulose from *Smilax glabra* residue and preparing oligosaccharides using β-glucanase hydrolysis and yeast fermentation, the pollution and cost issues in oligosaccharide preparation have been solved. This method achieves efficient and environmentally friendly preparation of oligosaccharides with anti-inflammatory activity, and has broad application potential.
Patent Information
- Application Number
- CN202511761045.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-27
- Publication Date
- 2026-02-03
AI Technical Summary
Existing methods for preparing cellulosic sugars suffer from problems such as high pollution, high cost, expensive equipment, and difficulty in achieving large-scale production. In particular, the use of β-glucosidase in enzymatic hydrolysis results in low yields. Finding commercially available enzyme formulations at reasonable prices is a critical issue that urgently needs to be addressed.
Using *Smilax glabra* residue as raw material, water-soluble components, lignin, and hemicellulose were removed. Cellulose oligosaccharides were prepared by enzymatic hydrolysis with β-glucanase combined with yeast fermentation and acetone precipitation. The enzymatic hydrolysis conditions were optimized to improve purity and yield.
This study achieved efficient and environmentally friendly preparation of fiber oligosaccharides, which significantly inhibited the expression of macrophage inflammatory factors TNF-α, IL-1β, and IL-6, exhibiting anti-inflammatory activity and can be applied to the development of functional foods and drugs.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant polysaccharides, and specifically relates to a mixture of fiber oligosaccharides, its preparation method, and its application. Background Technology
[0002] Niu Dali is a legume belonging to the genus Millettia, mainly distributed in the Lingnan region of my country. Its scientific name is Millettia Speciosa Champ., and it is also known as Tian Niu Li, Dali Shu, and Xue Teng in folk terms.
[0003] *Smilax glabra* is a traditional Chinese medicinal plant, used for both food and medicine. Its root has a long history of medicinal use, dating back to the Qing Dynasty medical classic *Luchuan Materia Medica*. Records indicate that *Smilax glabra* root has effects such as tonifying deficiency and moistening the lungs, strengthening muscles and tendons, clearing heat and detoxifying. In daily life, it is often used in soups and to make medicinal wine. With the continuous development of biomedicine and pharmacology, the anti-inflammatory, liver-protective, immune-regulating, anti-fatigue, and anti-stress pharmacological effects of *Smilax glabra* have been increasingly confirmed and discovered, all related to the active substances in its root. As early as the 1990s, scholars both domestically and internationally studied its chemical composition. Currently, various chemical components have been isolated and identified from *Smilax glabra* root, mainly flavonoids, alkaloids, sterols, terpenes, coumarins and lignans, organic acids, and polysaccharides.
[0004] The root of *Smilax glabra* contains a large amount of cellulose. Studies have shown that the crude fiber content of *Smilax glabra* from Hainan is as high as 32.03%. Whether used in daily life for making medicinal wine or soup, or in industrial production, the extraction of active ingredients from *Smilax glabra* root produces a large amount of waste residue, the main component of which is cellulose. This indicates that the cellulose in *Smilax glabra* residue is a cheap and renewable biomass resource; however, this cellulose is often wasted and cannot be effectively utilized. The hydrolysis of cellulose usually produces many cello-oligosaccharides (COS) as intermediate products. Short-chain cello-oligosaccharides (degree of polymerization ≤ 6) are non-digestible oligosaccharides linked by β-1,4 glycosidic bonds of glucose. They are a promising water-soluble dietary fiber. COS can be fermented by microorganisms and is considered a potential prebiotic, which can be added to food and animal feed to enhance and improve gastrointestinal function.
[0005] Ren Bing et al., in their study "Effects of Different Addition Levels of Fiber Oligosaccharides on Growth Performance and Serum Physiological and Biochemical Indicators of Broilers" (Feed Research, 2016, (24): 1-5.), found that adding fiber oligosaccharides to broiler feed could effectively increase the activity of antioxidant enzymes such as serum T-SOD, CAT, GSH-Px, and GR, thereby improving the body's immunity, growth performance, and growth rate of broilers. Wang Jie et al., in their study "Effects of Compound Probiotics and Fiber Oligosaccharides on Growth Performance, Fecal Microorganisms, and Serum Indicators of Weaned Piglets" (Chinese Journal of Animal Nutrition, 2016, 28(3):881-890), used weaned piglets as the research subjects and found that fiber oligosaccharides effectively increased the serum TP, ALB, and GLB content and SOD and GSH-Px activities of piglets, which helped improve the body's immune function. At the same time, the intake of fiber oligosaccharides could inhibit the growth and reproduction of harmful bacteria - Escherichia coli - in piglets, further improving the growth performance of piglets. In their study, "Regulatory Effects of Oligosaccharides on Soil Microbial Diversity and Community Structure" (Chinese Journal of Agrometeorology, 2022, 43(6):464-473), Qian Yuanchao et al. applied a 50 mg / L oligosaccharide solution to soil and incubated it for 6 days before analyzing the composition and diversity distribution characteristics of the soil microbial community. The results showed that oligosaccharides significantly altered the community structure of bacteria and fungi, increasing the number of bacterial species observed. Specifically, the relative abundance of the beneficial genus *Pseudomonas* increased by 215% after treatment with oligosaccharides (COS), while significantly inhibiting harmful genus species. These experiments demonstrate that oligosaccharides also regulate bacterial and fungal communities in plants and soil, promoting plant growth and controlling soil microecology; however, the specific mechanisms remain unclear.
[0006] The preparation of cellulose oligosaccharides includes physicochemical methods and enzymatic methods. Among the physicochemical methods, acid hydrolysis is commonly used. Due to the stubborn nature of cellulose, strong acids are usually used for hydrolysis. This method is simple and fast, but it generates significant pollution during the preparation of cellulose oligosaccharides, and large-scale wastewater treatment costs are high in industrial production. In addition, hydrothermal treatment is also a common method for preparing oligosaccharides. The raw materials are treated under high temperature and pressure to produce oligosaccharides, but hydrothermal treatment requires high purity of raw materials and has high equipment costs, so it is not the optimal choice for preparing cellulose oligosaccharides. Enzymatic methods are a mild, efficient and environmentally friendly method for preparing cellulose oligosaccharides compared to physicochemical methods. Enzymatic methods are further divided into enzymatic hydrolysis and enzymatic synthesis. For example, Zheng Peng et al. used a three-enzyme cascade reaction of sucrose phosphorylase, cellobiose phosphorylase and cellobiose phosphorylase to prepare a mixed cellulose oligosaccharide with trisaccharides as the main component in their paper "Synthesis of Cellulose Oligosaccharides that Promote the Growth of Intestinal Probiotics by Multi-enzyme Cascade Reaction" (Chinese Journal of Biotechnology, 2023, 39 (08)). Enzymatic synthesis can regulate the degree of polymerization of products, but it is costly, complex, and difficult to scale up for mass production. In contrast, enzymatic hydrolysis is simpler and easier to operate. Enzymatic hydrolysis typically uses cellulase and β-glucanase; however, cellulase contains β-glucosidase, which further breaks down cellulosic oligosaccharides into glucose, significantly reducing the yield of cellulosic oligosaccharides. Furthermore, some high-efficiency, high-purity β-glucanases used in research are expensive. Therefore, finding commercially available enzyme formulations at a reasonable price for enzymatic hydrolysis is a pressing issue.
[0007] In summary, current research on the preparation of fiber oligosaccharides is insufficient. This invention aims to provide a mild, efficient, and environmentally friendly method for preparing fiber oligosaccharides, while also reducing industrial costs through the selection of enzyme preparations in the early screening process. Summary of the Invention
[0008] The purpose of this invention is to provide a fiber oligosaccharide mixture, its preparation method, and its application, in order to solve the problems in the prior art.
[0009] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, this application provides a method for preparing a mixture of fiber oligosaccharides, comprising the following steps: (1) Take the residue of *Smilax glabra*, remove water-soluble components, starch, lignin and hemicellulose to obtain *Smilax glabra* cellulose; (2) The cellulose obtained in step (1) is enzymatically hydrolyzed, and then the hydrolysate is collected; (3) Remove glucose from the enzymatic hydrolysate obtained in step (2) by yeast fermentation; (4) Concentrate the fermentation broth after step (3), precipitate it with acetone, and finally freeze-dry the precipitate to obtain cellulose oligosaccharide.
[0010] In step (1), "Niu Dali Zha" refers to the plant root residue remaining after the main active substances, including flavonoids and polysaccharides, are extracted from Niu Dali.
[0011] Furthermore, in step (1) to remove water-soluble and starch components, the ratio of *Euphorbia hirta* residue to water (W / V, g / mL) is 1:20. 0.2% (w / w) of α-amylase is added, and the mixture is stirred in a water bath at 55°C for 2 hours to obtain crude fiber from *Euphorbia hirta*.
[0012] Further, in step (1) the lignin removal step, the crude fiber of *Smilax glabra* is mixed with a 7.5% sodium chlorite solution with pH=4, the material-to-liquid ratio is 1:20, and the reaction is carried out in a water bath at 75°C for 2 hours to obtain lignin-free fiber.
[0013] Furthermore, in step (1) of removing hemicellulose, the lignin-removing fiber is uniformly mixed with 10% KOH solution, the mass ratio of lignin-removing fiber to KOH solution is 1:20, and the mixture is stirred in a water bath at 25°C for 12 hours to obtain Niu Dali cellulose.
[0014] Furthermore, in step (2), the enzymatic hydrolysis is carried out under the following conditions: 0.4 w / w enzyme-to-bottom ratio, pH 5.5, and 50°C for 8 hours.
[0015] 6. The method for preparing the fiber oligosaccharide mixture according to claims 1-4, characterized in that: In step (3), the amount of yeast added is 0.4% (w / v, g / mL).
[0016] Furthermore, in step (4), after rotary evaporation, the precipitate is added to acetone pre-cooled at 4°C. The ratio of acetone to concentrate is 9:1. The precipitate is carried out at 4°C for 12 hours. The parameters for centrifugation are 5000 rpm, 10 min, and 4°C.
[0017] Secondly, this application also provides a fiber oligosaccharide mixture, which is prepared by the aforementioned method for preparing fiber oligosaccharide mixtures.
[0018] Thirdly, this application also provides the application of fiber oligosaccharide mixtures in the preparation of anti-inflammatory functional foods, anti-inflammatory functional drugs, immune-enhancing functional foods, and immune-enhancing functional drugs.
[0019] Compared with existing processes and technologies, the present invention has the following advantages and beneficial effects: This invention uses *Smilax glabra* residue as raw material. The process involves first removing water-soluble components, lignin, and hemicellulose to extract cellulose, then enzymatically hydrolyzing with β-glucanase, purifying through yeast fermentation, and precipitating with acetone to obtain *Smilax glabra* residue cellulose oligosaccharide. Its composition, purity, and chemical structure were analyzed and identified. In vitro experiments showed that this polysaccharide possesses significant anti-inflammatory capabilities; it can exert anti-inflammatory activity by inhibiting the expression of macrophage inflammatory factors TNF-α, IL-1β, and IL-6. It has potential for application in the preparation of functional foods and pharmaceuticals, and also shows promise for the high-value utilization of *Smilax glabra* residue. Attached Figure Description
[0020] Figure 1 This is the ion chromatogram of *Smilax glabra* cellulose in Example 2.
[0021] Figure 2 This is the standard curve table for monosaccharides in Example 2.
[0022] Figure 3 This is a standard curve of β-glucanase activity in Example 2.
[0023] Figure 4 This is the ion chromatogram of the cellulose oligosaccharide standard in Example 2.
[0024] Figure 5 This is an ion chromatogram of the oligosaccharide from *Smilax glabra* fiber in Example 2.
[0025] Figure 6 The graph shows the single-factor curves for the preparation of *Smilax glabra* fiber oligosaccharide in Example 2, where a) temperature, b) pH value, c) enzyme-to-bottom ratio, and d) time.
[0026] Figure 7 This is a graph showing the glucose content in the enzymatic hydrolysate after yeast fermentation in Example 2.
[0027] Figure 8 This is the ion chromatogram of the final product of *Smilax glabra* fiber oligosaccharide.
[0028] Figure 9 This diagram illustrates the toxic effects of COS on RAW264.7 macrophages.
[0029] Figure 10 The figure shows the effect of low, medium, and high concentrations of COS on the release of the cytokine TNF-α produced by RAW264.7 macrophages.
[0030] Figure 11 The figure shows the effect of low, medium, and high concentrations of COS on the release of the cytokine IL-6 produced by RAW264.7 macrophages.
[0031] Figure 12The figure shows the effect of low, medium, and high concentrations of COS on the release of the cytokine IL-1β from RAW264.7 macrophages. Detailed Implementation
[0032] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.
[0033] Unless otherwise specified, all reagents used in the examples are commercially available.
[0034] In this invention, "Millettia Speciosa Champ" is a plant belonging to the genus Millettia in the legume family, and is a plant that is both medicinal and edible.
[0035] Example 1: Isolation and extraction of MSCC from *Smilax glabra* cellulose 1. Extraction of crude fiber from *Smilax glabra* Weigh 100g of dried *Smilax glabra* residue and mix it with distilled water in a beaker at a ratio of 1:20 (w / v, g / ml). Add 0.2% (w / w) α-amylase and incubate in a water bath at 55℃ for 2 h. Inactivate the enzyme by boiling in a water bath. Collect the residue and wash it three times with distilled water until the filtrate is clear. Finally, wash it with 95% ethanol and dry the residue in a forced-air drying oven at 60℃ for 16 h.
[0036] 2. Removal of lignin from crude fiber of *Smilax glabra* The crude fiber of *Smilax glabra* was uniformly mixed with a 7.5% sodium chlorite solution (the pH of which was adjusted to 3.8-4.0 beforehand with HCl solution), at a mass ratio of 1:20. The mixture was heated in a water bath at 75°C for 2 hours with constant stirring. The precipitate was then collected by centrifugation and washed repeatedly until the supernatant obtained by centrifugation was neutral in pH. The washing solution was then replaced with 95% ethanol, and the filter residue was washed once more. The residue was then dried at 60°C for 16 hours to obtain lignin-free fiber.
[0037] 3. Removal of coarse fiber and hemicellulose from *Smilax glabra* (a type of herb) The lignin-free fiber was uniformly mixed with a 10% KOH solution at a mass ratio of 1:20. The mixture was stirred continuously at 25°C for 12 hours. The residue was then collected by filtration and washed multiple times with distilled water until the filtrate was pH neutral. The residue was then washed again with 95% ethanol and finally dried at 60°C for 16 hours. The residue was then pulverized and sieved to obtain *Euphorbia lactea* residue cellulose.
[0038] 4. Purity determination of *Smilax glabra* cellulose The monosaccharide composition of the extracted *Smilax glabra* cellulose was analyzed using ion chromatography. First, the *Smilax glabra* cellulose sample was pretreated: 5.0 mg of the *Smilax glabra* cellulose sample was weighed and placed in a serum vial. 1 mL of 72% sulfuric acid was added to the vial, and after standing for 12 h, 4 mL of ultrapure water was added. The vial was gently shaken and then sealed. The *Smilax glabra* cellulose was hydrolyzed at 105℃ for 6 h. During this time, the vial was shaken periodically. After hydrolysis, the liquid was allowed to cool, the pH was adjusted to neutral, and the solution was brought to a final volume of 50 mL. The diluted solution was filtered through a 0.22 μm aqueous filter to remove insoluble matter. Standard solutions of eight monosaccharides were used as standards for quantification.
[0039] 5. Results 5.1 Cellulose yield Based on the final weight of the cellulose in *Smilax glabra*, the cellulose content in the residue is calculated to be 55.39%.
[0040] 5.2 Cellulose Purity Results according to Figure 1 The monosaccharide ion chromatogram and the monosaccharide standard curve in Table 1 were used to qualitatively and quantitatively analyze the cellulose of *Achyranthes bidentata*, and the purity of the cellulose was calculated to be 92.32%. After removing most of the lignin and hemicellulose, the probability of producing miscellaneous sugars, such as xylose, in the subsequent enzymatic hydrolysis of *Achyranthes bidentata* residue was reduced, effectively improving the enzymatic hydrolysis efficiency.
[0041] Example 2: Preparation of *Smilax glabra* fiber oligosaccharide Oligosaccharides of *Smilax glabra* fiber were prepared by enzymatic hydrolysis, fermentation, concentration and precipitation, and freeze-drying.
[0042] 1. Determination of β-glucanase activity The enzyme activity of the enzymes used was determined in accordance with the People's Republic of China industry standard NY / T 911-2004 to determine the specific dosage.
[0043] 2. Enzymatic hydrolysis of *Smilax glabra* cellulose and determination of optimal conditions Weigh 1g of dried *Smilax glabra* cellulose into a 200ml beaker, add 100ml of 0.05M citrate-disodium hydrogen phosphate buffer, and preheat at a certain temperature with shaking for 2 hours. After the temperature stabilizes, add a certain amount of β-glucanase, continue the reaction for a certain time, and then inactivate the enzyme in a boiling water bath for 10 minutes. Centrifuge and collect the supernatant, and store at 4℃. Filter the enzyme solution through a 0.22μm aqueous filter membrane and perform high-performance anion exchange chromatography for detection.
[0044] 3. Fermentation of *Achyranthes bidentata* enzymatic hydrolysate and determination of optimal conditions Add a certain amount of yeast to the enzymatic hydrolysate from the previous step for fermentation. Centrifuge the fermentation broth, collect the supernatant, filter it through a 0.22 aqueous filter membrane, and perform high-performance anion exchange chromatography for detection.
[0045] 4. Concentration and precipitation of enzymatic hydrolysate after fermentation of *Smilax glabra* The fermented enzymatic hydrolysate was concentrated by vacuum rotary evaporation at 50°C to remove ethanol and most of the liquid. The concentrate was added to a pre-cooled acetone solution at 4°C (concentrate:acetone = 1:9) and precipitated at 4°C for 24 hours. The precipitate was obtained by filtration and further freeze-dried under vacuum for 24 hours to obtain cellulosic acid.
[0046] 5. Ion chromatographic analysis of dried *Smilax glabra* fiber oligosaccharides Weigh 5 mg of *Smilax glabra* fiber oligosaccharide sample into a centrifuge tube, dissolve it in ultrapure water, and bring the volume to 100 ml. Filter the solution through a 0.22 aqueous membrane and perform high-performance anion exchange chromatography.
[0047] 6. Results 6.1 Results of β-glucanase activity Enzyme activity standard curve as follows Figure 3 As shown, the calculated enzyme activity of β-glucanase is 1.498 × 10⁻⁶. 6 U / g.
[0048] 6.2 Qualitative and quantitative analysis of *Smilax glabra* cellulose according to Figure 4 Ion chromatogram of fiber oligosaccharide standards and Figure 5 Ion chromatograms of the enzymatic hydrolysate are used for qualitative and quantitative analysis of the sample. Based on the elution time, it can be determined whether the enzymatic hydrolysate contains glucose, cellobiose, cellotriose, cellotetraose, and cellopentaose.
[0049] 6.3 Results of single-factor enzymatic hydrolysis of *Smilax glabra* cellulose like Figure 6 As shown, the optimal conditions for enzymatic hydrolysis of *Smilax glabra* cellulose are: temperature 50℃, pH 5.5, time 8h, and enzyme-to-base ratio 0.4. Under these conditions, the yield of cellobiose in the hydrolysate reaches 19.2%, which is far higher than the enzyme effect of Maclean's and the original leaf in the current experimental reagents (2%). At the same time, compared with *Smilax glabra* residue without removing lignin and hemicellulose, its enzymatic hydrolysis effect is also better.
[0050] 6.4 Results of Single-Factor Fermentation of *Smilax glabra* Cellulose like Figure 7 As shown, the optimal fermentation conditions for *Achyranthes bidentata* enzymatic hydrolysate are a yeast addition of 0.4%. Under these conditions, the glucose content in the hydrolysate decreases to 5% of its pre-fermentation level, while the cellobiose content remains almost unchanged.
[0051] 6.5 Quantitative Analysis of the Final Product of *Smilax glabra* Fiber Oligosaccharide according to Figure 8 Ion chromatograms show that the final product of *Smilax glabra* fiber oligosaccharide has a ratio of glucose: cellobiose: cellotriose: cellopentaose = 4.9:37.8:1:2.4. Example 3: Study on the anti-inflammatory activity of *Smilax glabra* fiber oligosaccharide Reagents: DMEM high glucose medium (manufactured by Gibco, USA), FBS (manufactured by Gibco, USA), and antibiotics (manufactured by Gibco, USA). Mix DMEM high glucose medium and fetal bovine serum at a ratio of 9:1 (w / v, (mg / mL)), add 1% (volume ratio) of antibiotics, and mix thoroughly to obtain DMEM complete medium.
[0052] Instruments: cell culture incubator, enzyme-linked immunosorbent assay (ELISA) reader, low-speed centrifuge, etc.
[0053] The anti-inflammatory activity of Cosminoides (COS) was evaluated by measuring its regulation of inflammatory factor levels in a LPS-stimulated RAW264.7 cell model.
[0054] 1. Cytotoxicity assay of COS RAW264.7 cells were cultured to the logarithmic growth phase, and cell counts were performed. Cells were then cultured at a density of 5 × 10⁶ cells / year. 4 Cell suspension at 100 μL / well was evenly seeded into 96-well plates, resulting in 5000 cells per well. After culturing for 24 h in a 5% CO2, 37°C cell culture incubator, the culture medium in the 96-well plates was discarded, and three groups were randomly assigned: a blank group, a control group, and an experimental group. The blank group contained no cells and used only complete culture medium; the control group received complete culture medium; the experimental group received 100 μL of complete culture medium containing 10, 50, 100, 200, 400, or 800 μg / mL COS-2 standard. After 24 h, 10 μL of CCK-8 reagent was added, and after incubation for 30 min, cell viability was measured at 450 nm. A relative proliferation rate of 80% or higher was considered acceptable for toxicity. The relative proliferation rate was calculated using the following formula: Relative proliferation rate (%) = (Sample A / Blank control A) x 100% Relative proliferation rate (%) = (Sample A / Blank control A) x 100% 2. Anti-inflammatory effect of COS on RAW264.7 cell model RAW264.7 cells were cultured to the logarithmic growth phase, and cell counts were performed. Cells were then cultured at a density of 5 × 10⁶ cells / year. 4Cell suspension of 100 μL / mL was evenly seeded into 96-well plates, resulting in 5000 cells per well. Four randomized groups were established: a blank control group, a model group, a positive control group, and a sample group. Except for the blank control group, all three groups (model, sample, and positive) were induced with 4 μg / mL LPS for 24 h to induce inflammation. Subsequently, the model group was treated with complete culture medium, and the sample group was treated with COS samples at three concentration gradients (100, 400, and 800 μg / mL). After 4 h of culture, the expression levels of inflammatory factors TNF-α, IL-6, and IL-1β were measured according to the inflammatory factor assay kit. The blank control group consisted of RAW264.7 cells cultured in DMEM complete medium; the model group consisted of cells induced with 4 μg / ml LPS for 24 h, followed by the addition of complete medium; and the sample group consisted of cells induced with 4 μg / ml LPS for 24 h, followed by treatment with COS samples for 4 h. Three concentration gradients were established: 100 μg / mL, 400 μg / mL, and 800 μg / mL.
[0055] 3. Results 3.1 Effect of COS on the viability of RAW264.7 cells Figure 9 The results showed that the survival rate of COS-treated RAW264.7 cells was above 100% within the range of 10-800 µg / mL, indicating that the sample was non-toxic to RAW264.7 cells. Therefore, three concentration gradients of 100, 400, and 800 µg / mL were ultimately selected for the study of anti-inflammatory activity. 3.2 Effect of COS on TNF-α levels in RAW264.7 cell model Figure 10 The results showed that low, medium and high concentrations of COS treatment reduced the expression of TNF-α in RAW264.7 cells of the inflammation model in a dose-dependent manner, with the release of TNF-α at 800 µg / mL being 80% of that in the control group.
[0056] 3.3 Effect of COS on IL-6 levels in RAW264.7 cell model Figure 11 The results showed that low, medium and high concentrations of COS treatment reduced the expression of IL-6 in the RAW264.7 inflammatory model cells in a dose-dependent manner, with the release of IL-6 at 800 µg / mL being 61% of that in the control group.
[0057] 3.4 Effect of COS on IL-1β levels in RAW264.7 cell model Figure 12The results showed that low, medium and high concentrations of COS treatment reduced the expression of IL-1β in the RAW264.7 inflammatory model cells in a dose-dependent manner, with the release of IL-1β at 800 µg / mL being 47% of that in the control group.
[0058] This invention uses *Smilax glabra* residue as raw material. The process involves first removing water-soluble components, lignin, and hemicellulose to extract cellulose, then enzymatically hydrolyzing with β-glucanase, purifying through yeast fermentation, and precipitating with acetone to obtain *Smilax glabra* residue cellulose oligosaccharide. Its composition, purity, and chemical structure were analyzed and identified. In vitro experiments showed that this polysaccharide possesses significant anti-inflammatory capabilities; it can exert anti-inflammatory activity by inhibiting the expression of macrophage inflammatory factors TNF-α, IL-1β, and IL-6. It has potential for application in the preparation of functional foods and pharmaceuticals, and also shows promise for the high-value utilization of *Smilax glabra* residue.
[0059] It should also be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.
Claims
1. A method for preparing a mixture of fiber oligosaccharides, characterized in that: Includes the following steps: (1) Take the residue of *Smilax glabra*, remove water-soluble components, starch, lignin and hemicellulose to obtain *Smilax glabra* cellulose; (2) The cellulose obtained in step (1) is enzymatically hydrolyzed, and then the hydrolysate is collected; (3) Remove glucose from the enzymatic hydrolysate obtained in step (2) by yeast fermentation; (4) Concentrate the fermentation broth after step (3), precipitate it with acetone, and finally freeze-dry the precipitate to obtain cellulose oligosaccharide.
2. The method for preparing the fiber oligosaccharide mixture according to claim 1, characterized in that: In step (1) Removal of water-soluble and starch components, the ratio of *Euphorbia hirta* residue to water (W / V, g / mL) is 1:
20. 0.2% (w / w) of α-amylase is added, and the mixture is stirred in a water bath at 55°C for 2 hours to obtain crude fiber from *Euphorbia hirta*.
3. The method for preparing the fiber oligosaccharide mixture according to claim 2, characterized in that: In step (1) lignin removal, the crude fiber of *Smilax glabra* is mixed with a 7.5% sodium chlorite solution with pH=4 at a ratio of 1:
20. The mixture is stirred in a water bath at 75°C for 2 hours to obtain lignin-free fiber.
4. The method for preparing the fiber oligosaccharide mixture according to claim 3, characterized in that: In step (1), the hemicellulose removal process involves uniformly mixing the lignin-removing fiber with a 10% KOH solution at a mass ratio of 1:
20. The mixture is stirred in a water bath at 25°C for 12 hours to obtain niudali cellulose.
5. The method for preparing the fiber oligosaccharide mixture according to any one of claims 1-4, characterized in that: In step (2), the enzymatic hydrolysis conditions are 0.4 w / w enzyme-to-bottom ratio, pH 5.5, and 50℃ for 8 hours.
6. The method for preparing the fiber oligosaccharide mixture according to claims 1-4, characterized in that: In step (3), the amount of yeast added is 0.4% (w / v, g / mL).
7. The method for preparing the fiber oligosaccharide mixture according to claims 1-4, characterized in that: In step (4), after rotary evaporation, the precipitate is added to acetone pre-cooled at 4°C. The ratio of acetone to concentrate is 9:
1. The precipitate is carried out at 4°C for 12 hours. The parameters for centrifugation are 5000 rpm, 10 min, and 4°C.
8. A mixture of fiber oligosaccharides, characterized in that, It is prepared by the method for preparing the fiber oligosaccharide mixture according to any one of claims 1-7.
9. The use of the fiber oligosaccharide mixture according to claim 8 in the preparation of anti-inflammatory functional foods, anti-inflammatory functional drugs, immune-enhancing functional foods, and immune-enhancing functional drugs.