A low-substituted mannose oligosaccharide, and a preparation method and application thereof

Abstract

This invention provides a low-substituted mannooligosaccharide, its preparation method, and its applications. The low-substituted mannooligosaccharide has a substitution degree of 2-15%. The preparation method includes the following steps: S1, pretreatment; S2, enzymatic hydrolysis; wherein the amount of α-galactosidase added is 0.1-10 mg / g substrate, and the amount of mannanase added is 0.1-5 mg / g substrate. The low-substituted mannooligosaccharide is used as a cellulase activity inhibitor to regulate the saccharification rate during biomass enzymatic hydrolysis, or as a prebiotic in functional foods or feed additives. This invention prepares a low-substituted mannooligosaccharide with a substitution degree of 2-15%. Compared with using mannanase alone, the method of this invention can reduce the amount of mannanase used by 30-70%, while increasing the yield of mannooligosaccharide by 5-30%, and can achieve gradient regulation of the galactose side chain substitution degree of mannooligosaccharide.

Description

Technical Field

[0001] This invention relates to the field of manno oligosaccharide preparation technology, specifically to a low-substitution-degree manno oligosaccharide, its preparation method, and its applications. Background Technology

[0002] Galactomannan is a class of functional polysaccharides widely found in the seeds of legumes (such as guar gum, locust bean gum, and konjac) and the cell walls of some microorganisms. Its structure consists of a mannose backbone linked by β-1,4-glycosidic bonds and galactose side chains linked by α-1,6-glycosidic bonds. The degree of galactose substitution on the backbone (i.e., degree of substitution, DS) determines the physicochemical properties and biological activity of galactomannan. The degree of substitution of naturally derived galactomannan is typically between 30% and 40%.

[0003] Mannooligosaccharides (MOS) are partially hydrolyzed products of galactomannan and possess prebiotic functions such as promoting the proliferation of beneficial bacteria, inhibiting the adhesion of pathogenic bacteria, and regulating immunity. They are widely used in functional foods and feed additives. Furthermore, recent studies have found that oligosaccharides with specific structures can also serve as regulators of cellulase activity, used to control the saccharification rate in biomass energy production processes.

[0004] Existing methods for preparing mannan oligosaccharides mainly include acid hydrolysis, enzymatic hydrolysis, and combinations thereof. Among these, enzymatic hydrolysis has become the mainstream method due to its mild conditions, high specificity, and environmental friendliness. Common enzymatic hydrolysis strategies involve using β-mannanase alone, or in combination with α-galactosidase and β-mannanase for synergistic degradation. However, the common goal of existing research is to completely remove the galactose side chain (i.e., degree of substitution ≈ 0%) to obtain structurally homogeneous mannan oligosaccharides (such as mannobiose and mannotriose), or to completely degrade them into monosaccharides for use in biofuel production. For example, patent application CN110747242A discloses a method for producing galactose, mannose, and mannan oligosaccharides by hydrolyzing guar gum with a combination of mannanase and galactosidase, utilizing the galactomannan substrate guar gum to prepare mannose.

[0005] However, existing technologies have the following shortcomings: The product structure is simple: existing methods focus on obtaining mannooligosaccharides with a degree of substitution ≈ 0%, and there is a lack of research and preparation methods for the intermediate region between the degree of substitution 0% and the natural high substitution. Pretreatment limitations: Conventional ammonia pretreatment is only used to remove impurities such as lignin and cannot actively participate in product structure design. The degree of substitution control depends entirely on the enzymatic hydrolysis conditions, making it difficult to achieve precise control. Limited Functional Applications: Mannooligosaccharides with a substitution degree of ≈0% have a homogeneous structure and relatively singular function, making it difficult to simultaneously meet the needs of multifunctional applications such as prebiotics and cellulase inhibitors; there are no reports in the existing technology of using mannooligosaccharides with a specific substitution degree range as cellulase activity inhibitors, and there is also a lack of technical solutions to achieve functional gradients by regulating the substitution degree.

[0006] Inefficient use of raw materials: For complex biomass such as waste coffee grounds, conventional pretreatment is difficult to effectively remove inhibitors and retain the complete structure of the target polysaccharide, which affects the efficiency of subsequent enzymatic hydrolysis and product quality.

[0007] Therefore, developing a method to prepare intermediate-region mannooligosaccharides with a degree of substitution of 2-15%, achieving gradient regulation of product structure, and expanding its application in multifunctional fields such as prebiotics and cellulase inhibitors, has important theoretical significance and practical value. Summary of the Invention

[0008] To address the aforementioned problems, this invention provides a low-substituted mannooligosaccharide, its preparation method, and its applications.

[0009] The technical solution of this invention is: A low-substituted mannooligosaccharide, wherein the degree of substitution of the low-substituted mannooligosaccharide is 2-15%, and the degree of substitution refers to the molar percentage of mannose residues in the mannooligosaccharide molecule that are linked to galactose side chains. The low-substituted mannooligosaccharide is obtained by adding α-galactosidase and mannanase to galactomannan-containing biomass for synergistic enzymatic hydrolysis and purification.

[0010] Furthermore, the galactomannan-containing biomass is one or more of waste coffee grounds, guar gum, konjac, and locust bean gum.

[0011] Note: By rationally selecting biomass types containing galactomannan, covering a variety of biomass containing galactomannan, especially waste coffee grounds, the resource utilization of waste can be achieved, which has environmental and cost advantages.

[0012] This invention also provides a method for preparing the above-mentioned low-substituted mannooligosaccharide, comprising the following steps: S1. Pretreatment: Take biomass containing galactomannan, pretreat it with ammonia solution, filter, wash with water, and dry it for later use. S2. Enzymatic hydrolysis: The pretreated biomass containing galactomannan was suspended in sodium citrate buffer as a substrate, the pH was adjusted to 3-7, an enzyme composition containing α-galactosidase and mannanase was added and the enzymatic hydrolysis reaction was carried out for 48±3h to obtain the enzymatic hydrolysis reaction solution. The enzymatic hydrolysis reaction solution was separated and purified to obtain the low-substituted mannooligosaccharide product. The amount of α-galactosidase added is 0.1~10 mg / g substrate, and the amount of mannanase added is 0.1~5 mg / g substrate.

[0013] Furthermore, in S1, the galactomannan-containing biomass is pulverized and passed through a 40-60 mesh sieve. The addition ratio of the galactomannan-containing biomass to the ammonia solution is 1g: 5-20mL, and the mass percentage of the ammonia solution is 5-20wt%. During pretreatment, it is pretreated at 40-80℃ for 4-6h and washed with water until neutral.

[0014] Note: By controlling parameters such as ammonia concentration and temperature, inhibitors such as lignin can be effectively removed while preserving the integrity of the substrate structure, creating conditions for subsequent precise enzymatic hydrolysis.

[0015] Preferably, in S1, a portion of pyruvate solution is added during pretreatment. The pyruvate solution is prepared by mixing pyruvate and deionized water at a volume ratio of 1:5. The amount of pyruvate solution added is 0.5-2% of the ammonia solution. Specifically, the ammonia solution is divided into two-thirds volume of a first ammonia solution and one-third volume of a second ammonia solution. The pyruvate solution is divided into three equal volumes. The galactomannan-containing biomass is mixed with the first ammonia solution and heated to the specified temperature. The mixture is stirred at 150-200 rpm for 30-45 minutes. The first portion of pyruvate solution is added dropwise, and the mixture is stirred for another 45-60 minutes. The second portion of pyruvate solution is added dropwise, and the mixture is stirred for another 45-60 minutes. The third portion of pyruvate solution is added dropwise, and the mixture is stirred for another 45-60 minutes. The second ammonia solution is added, and the mixture is stirred until the total reaction time is reached. The solid product is collected by centrifugation, washed with deionized water until neutral, and then dried at 40-50°C for later use.

[0016] Explanation: By adding pyruvate in stages during pretreatment, pyruvate can form reversible ester bonds with the galactose side chain, creating temporary protection in the amorphous region and partially preserving the side chain. This allows ammonia to preferentially swell the amorphous region, concentrating the protection in a specific area and forming a gradient structure. The protecting group is then slowly hydrolyzed during the enzymatic hydrolysis stage, achieving gradient pruning of the side chain and ultimately precisely controlling the degree of substitution between 2% and 15%.

[0017] Furthermore, in S2, the molar concentration of the sodium citrate buffer solution is 50±5mM, and the temperature is 30~70℃.

[0018] Note: The optimal pH for α-galactosidase and mannanase is achieved by adjusting the pH to ensure enzyme activity. At this pH, the hydrolysis rate of the pyruvate protecting group is moderate, enabling dynamic release.

[0019] Furthermore, in S2, the separation and purification steps include: sequentially subjecting the enzymatic hydrolysis reaction solution to enzyme inactivation, solid-liquid separation, desalting, monosaccharide removal, and drying; the enzyme inactivation is performed by heating in a boiling water bath for 5-15 minutes; the solid-liquid separation is performed by centrifugation at 10000g for 5-10 minutes; the desalting is performed by ion exchange resin desalting or membrane separation desalting; the monosaccharide removal is performed by yeast fermentation or activated carbon adsorption chromatography; and the drying is performed by spray drying, freeze drying, or vacuum drying.

[0020] Note: The enzyme is inactivated by heating in a boiling water bath to terminate the reaction. Denatured proteins and insoluble matter are removed by centrifugation to obtain a high-purity mannan oligosaccharide product.

[0021] The present invention also provides an application of the aforementioned low-substituted mannooligosaccharide, which is used as a cellulase activity inhibitor to regulate the saccharification rate during biomass enzymatic hydrolysis.

[0022] Explanation: By utilizing the steric hindrance of low-substituted mannan oligosaccharides to selectively inhibit cellulase activity, the saccharification rate of biomass can be precisely controlled, thereby improving the production efficiency of cellulosic ethanol.

[0023] The present invention also provides an application of the aforementioned low-substitution degree mannooligosaccharide, wherein the low-substitution degree mannooligosaccharide is used as a prebiotic in functional foods or feed additives.

[0024] Note: By utilizing the low-substituted regions on the surface of low-substituted mannan oligosaccharides, the proliferation of beneficial intestinal bacteria can be promoted. It can be used as a functional food or feed additive to replace antibiotics and improve intestinal health.

[0025] The beneficial effects of this invention are: (1) The present invention prepares low-substituted mannan oligosaccharides with a degree of substitution of 2-15%. Compared with the use of mannanase alone, the method of the present invention can reduce the amount of mannanase by 30-70% and increase the yield of mannan oligosaccharides by 5-30%. By adjusting the amount of α-galactosidase added and the enzymatic hydrolysis time, the gradient regulation of the galactose side chain substitution degree of mannan oligosaccharides can be achieved.

[0026] (2) This invention introduces the reversible protection mechanism of pyruvate into the ammonia pretreatment system, upgrading the pretreatment from cleaning the substrate to structural design. By adding pyruvate in stages, controlling the concentration of ammonia and the reaction time, the degree of substitution can be precisely controlled. At the same time, ammonia is low in cost and easy to remove, and pyruvate is a food-grade additive that meets the requirements for food / feed applications. The operation is simple, and only the addition of pyruvate in stages is added on the basis of the original ammonia treatment, making industrialization easy.

[0027] (3) The low-substitution degree mannan oligosaccharide of the present invention has dual functions: on the one hand, it can be used as a prebiotic. Its surface low-substitution region has a simple structure and is easily utilized by beneficial bacteria in the intestine, promoting the proliferation of beneficial bacteria; on the other hand, it can be used as a cellulase activity inhibitor. Its internal high-substitution region and overall gradient structure form a unique aggregation state, selectively inhibiting cellulase. The inhibition rate of cellobiase I is 15-35%, and the inhibition rate of endoglucanase II is 15-30%. Moreover, the inhibition intensity is negatively correlated with the degree of substitution of galactose side chain. Attached Figure Description

[0028] Figure 1 This is a graph showing the effect of the amount of α-galactosidase added on the degree of substitution of the galactose side chain of mannan oligosaccharide in Experiment Example 1. Figure 2 This is the dose-inhibition relationship of low-substituted mannan oligosaccharides on endoglucanase II and cellobiase I in Experiment Example 2; Figure 3 This is a scatter plot showing the correlation between the degree of substitution of the galactose side chain of mannan oligosaccharide and the cellulase inhibition rate in Experiment Example 2. Detailed Implementation

[0029] Example 1: A low-substitution degree mannan oligosaccharide with a substitution degree of 5.2%. The substitution degree refers to the molar percentage of mannose residues in the mannan oligosaccharide molecule that are linked to galactose side chains. The low-substitution degree mannan oligosaccharide is obtained by adding α-galactosidase and mannanase to galactomannan-containing biomass for synergistic enzymatic hydrolysis, followed by separation and purification. The galactomannan-containing biomass is waste coffee grounds.

[0030] Example 2: The difference between this example and Example 1 is that the galactomannan-containing biomass is made by mixing waste coffee grounds and konjac in a 1:1 mass ratio.

[0031] Example 3: The difference between this example and Example 1 is that the galactomannan-containing biomass is guar gum.

[0032] Example 4: The difference between this example and Example 1 is that the biomass containing galactomannan is locust bean gum.

[0033] Example 5: The difference between this example and Example 1 is that the galactomannan-containing biomass is a mixture of guar gum and locust bean gum in a 1:1 mass ratio.

[0034] Example 6: The difference between this example and Example 1 is that the biomass containing galactomannan is konjac.

[0035] Example 7: The difference between this example and Example 1 is that the degree of substitution of the low-substitution mannooligosaccharide is 2%.

[0036] Example 8: The difference between this example and Example 1 is that the degree of substitution of the low-substitution mannooligosaccharide is 4%.

[0037] Example 9: The difference between this example and Example 1 is that the degree of substitution of the low-substitution mannan oligosaccharide is 8%.

[0038] Example 10: The difference between this example and Example 1 is that the degree of substitution of the low-substitution mannooligosaccharide is 10%.

[0039] Example 11: The difference between this example and Example 1 is that the degree of substitution of the low-substitution mannooligosaccharide is 15%.

[0040] Example 12: This example is a method for preparing a low-substituted mannooligosaccharide as described in Example 1, including the following steps: S1. Pretreatment: Take the biomass containing galactomannan, crush it and pass it through a 40-mesh sieve. After pretreatment with ammonia solution, filter, wash and dry it for later use. The addition ratio of galactomannan biomass to ammonia solution is 1g:12mL, and the mass percentage of ammonia solution is 15wt%. Pretreatment is carried out at 50℃ for 5h, and then washed with water until neutral. S2. Enzymatic hydrolysis: Pretreated biomass containing galactomannan was suspended in sodium citrate buffer as a substrate. The pH was adjusted to 5. The concentration of the sodium citrate buffer was 50 mM. The pH was 5, and the temperature was 50°C. An enzyme composition containing α-galactosidase and mannanase was added, and the enzymatic hydrolysis reaction was carried out for 48 h to obtain the enzymatic hydrolysis solution. The enzymatic hydrolysis solution was separated and purified to obtain the low-substituted mannooligosaccharide product. In S2, the separation and purification steps included: the enzymatic hydrolysis solution was subjected to enzyme inactivation, solid-liquid separation, desalting, removal of monosaccharides, and drying in sequence. Enzyme inactivation was carried out by heating in a boiling water bath. 10 min; solid-liquid separation was performed by centrifugation at 10000g for 8 min; desalination was performed by ion exchange resin desalination, using cation exchange resin and anion exchange resin to adsorb cations and anions respectively to achieve desalination, with a cation exchange resin to anion exchange resin volume ratio of 1:1 and a flow rate of 2 BV / h; monosaccharide removal was performed by yeast fermentation, utilizing yeast microorganisms to preferentially utilize monosaccharides (glucose, galactose, mannose) for growth and metabolism, rather than oligosaccharides, and centrifugation was performed after fermentation to remove yeast cells, obtaining an oligosaccharide solution without monosaccharides, with a shaking speed of 200 rpm and a fermentation time of 24 h; drying was performed by spray drying, atomizing the concentrated liquid into fine droplets, and instantly evaporating the water in a hot air stream to obtain a dry powder, with an inlet air temperature of 200℃, an outlet air temperature of 80℃, and a centrifugation speed of 20000 rpm, to obtain a low-substituted manno-oligosaccharide product; The amount of α-galactosidase added was 0.1~10 mg / g substrate, and the amount of mannanase added was 0.1~5 mg / g substrate.

[0041] Example 13: The difference between this example and Example 12 is that in S1, the addition ratio of galactomannan biomass to ammonia solution is 1g:5mL, the mass percentage of ammonia solution is 20wt%, and the pretreatment is carried out at 40℃ for 6h, followed by washing with water until neutral.

[0042] Example 14: The difference between this example and Example 12 is that in S1, the addition ratio of galactomannan biomass to ammonia solution is 1g:20mL, the mass percentage of ammonia solution is 5wt%, and the pretreatment is carried out at 80℃ for 4h, followed by washing with water until neutral.

[0043] Example 15: This example differs from Example 12 in that: in S1, a portion of pyruvate solution is added during pretreatment. The pyruvate solution is prepared by mixing pyruvate and deionized water at a volume ratio of 1:5. The amount of pyruvate solution added is 1% of the ammonia solution. The specific method is as follows: the entire ammonia solution is divided into 2 / 3 volume of first ammonia solution and 1 / 3 volume of second ammonia solution. The entire pyruvate solution is divided into 3 equal volumes. The biomass containing galactomannan is mixed with the first ammonia solution and heated to the desired temperature. The mixture is stirred at 175 rpm for 40 minutes. The first portion of pyruvate solution is added dropwise, and the mixture is stirred for another 50 minutes. The second portion of pyruvate solution is added dropwise, and the mixture is stirred for another 60 minutes. The third portion of pyruvate solution is added dropwise, and the mixture is stirred for another 60 minutes. The second ammonia solution is added, and the mixture is stirred until the total reaction time is reached. The solid product is collected by centrifugation, washed with deionized water until neutral, and then dried at 48°C for later use.

[0044] Example 16: This example differs from Example 13 in that: in S1, a portion of pyruvate solution is added during pretreatment. The pyruvate solution is prepared by mixing pyruvate and deionized water at a volume ratio of 1:5. The amount of pyruvate solution added is 0.5% of the ammonia solution. The specific method is as follows: the entire ammonia solution is divided into 2 / 3 volume of first ammonia solution and 1 / 3 volume of second ammonia solution. The entire pyruvate solution is divided into 3 equal volumes. The biomass containing galactomannan is mixed with the first ammonia solution and heated to the desired temperature. The mixture is stirred at 150 rpm for 30 minutes. The first portion of pyruvate solution is added dropwise, and the mixture is stirred for another 60 minutes. The second portion of pyruvate solution is added dropwise, and the mixture is stirred for another 45 minutes. The third portion of pyruvate solution is added dropwise, and the mixture is stirred for another 45 minutes. The second ammonia solution is added, and the mixture is stirred until the total reaction time is reached. The solid product is collected by centrifugation, washed with deionized water until neutral, and then dried at 40°C for later use.

[0045] Example 17: This example differs from Example 14 in that: in S1, a portion of pyruvate solution is added during pretreatment. The pyruvate solution is prepared by mixing pyruvate and deionized water at a volume ratio of 1:5. The amount of pyruvate solution added is 2% of the ammonia solution. The specific method is as follows: the entire ammonia solution is divided into 2 / 3 volume of first ammonia solution and 1 / 3 volume of second ammonia solution. The entire pyruvate solution is divided into 3 equal volumes. The biomass containing galactomannan is mixed with the first ammonia solution and heated to the desired temperature. The mixture is stirred at 200 rpm for 45 minutes. The first portion of pyruvate solution is added dropwise, and the mixture is stirred for another 45 minutes. The second portion of pyruvate solution is added dropwise, and the mixture is stirred for another 50 minutes. The third portion of pyruvate solution is added dropwise, and the mixture is stirred for another 60 minutes. The second ammonia solution is added, and the mixture is stirred until the total reaction time is reached. The solid product is collected by centrifugation, washed with deionized water until neutral, and then dried at 50°C for later use.

[0046] Example 18: The difference between this example and Example 12 is that in S1, the biomass containing galactomannan is pulverized and passed through a 60-mesh sieve. Example 19: The difference between this example and Example 12 is that in S2, the pH is adjusted to 3, the molar concentration of sodium citrate buffer is 55mM, the temperature is 30°C, and an enzyme composition containing α-galactosidase and mannanase is added for enzymatic hydrolysis reaction for 45h.

[0047] Example 20: This example differs from Example 12 in that: in S2, the pH is adjusted to 7, the concentration of sodium citrate buffer is 45 mM, the temperature is 70°C, and an enzyme composition containing α-galactosidase and mannanase is added for enzymatic hydrolysis reaction for 51 h.

[0048] Example 21: This example differs from Example 12 in that: In S2, the separation and purification steps include: sequentially subjecting the enzymatic hydrolysis reaction solution to enzyme inactivation, solid-liquid separation, desalting, monosaccharide removal, and drying; enzyme inactivation is achieved by heating in a boiling water bath for 5 minutes; solid-liquid separation is achieved by centrifugation at 10000g for 5 minutes; desalting is achieved by membrane separation, utilizing the selective permeability of a nanofiltration membrane with a molecular weight cutoff of 250 Da, allowing water and salt ions to pass through while retaining oligosaccharides; monosaccharide removal is achieved by activated carbon adsorption chromatography, utilizing the adsorption-desorption difference of activated carbon on monosaccharides and oligosaccharides for separation. Monosaccharides are weakly adsorbed on activated carbon and are easily eluted by low-concentration ethanol, while oligosaccharides are strongly adsorbed and require high-concentration ethanol for desorption. The flow rate is 1 BV / h, and the activated carbon dosage is 0.2% (w / v); drying is achieved by freeze drying, pre-freezing the sugar solution into a solid state, and directly sublimating the water under vacuum conditions to obtain a loose and porous sponge-like dried product, suitable for small-batch products, which can retain biological activity to the greatest extent.

[0049] Example 22: This example differs from Example 12 in that: in S2, the separation and purification steps include: sequentially subjecting the enzymatic hydrolysis reaction solution to enzyme inactivation, solid-liquid separation, desalting, removal of monosaccharides, and drying; enzyme inactivation is performed by heating in a boiling water bath for 15 minutes; solid-liquid separation is performed by centrifugation at 10000g for 10 minutes; drying is performed by vacuum drying, heating under reduced pressure to lower the boiling point of water and reduce the damage to heat-sensitive substances, with a drying temperature of 60℃ and a vacuum degree of 0.09 MPa.

[0050] Example 23: This example is an application of the low-substituted mannooligosaccharide in Example 10. The low-substituted mannooligosaccharide is used as a cellulase activity inhibitor to regulate the saccharification rate during biomass enzymatic hydrolysis.

[0051] Example 24: This example is an application of the low-substitution degree mannan oligosaccharide in Example 1, in which the low-substitution degree mannan oligosaccharide is used as a prebiotic in functional foods or feed additives.

[0052] Experimental Example 1: To verify the effect of α-galactosidase on the degree of substitution of low-substituted mannan oligosaccharides in the method of the present invention, the following experiments were conducted. Comparative Example 1 was untreated; Comparative Example 2 was treated with only mannanase, and the remaining steps were the same as in Example 12; Comparative Example 3 was treated with only α-galactosidase, and the remaining steps were the same as in Example 12. The degree of substitution of low-substituted mannan oligosaccharides in each group of experiments was tested.

[0053] Specific steps for the degree of substitution experiment: Weigh 10.0 mg of mannan oligosaccharide sample, place it in a hydrolysis tube, add 1.0 mL of 2M trifluoroacetic acid solution, seal the tube with nitrogen, and hydrolyze at 100℃ for 4 hours. After cooling the hydrolysate, evaporate it to dryness at 50℃ by rotary evaporation. Add 1 mL of methanol and repeat the evaporation twice to remove residual trifluoroacetic acid. Redissolve the residue with 1.0 mL of ultrapure water to obtain a monosaccharide hydrolysate. Take 100 μL of the monosaccharide hydrolysate, add 100 μL of 0.5 M PMP methanol solution and 100 μL of 0.3 M NaOH solution, vortex to mix, and react at 70℃ for 60 minutes. After cooling the reaction solution, add 100 μL of 0.3 M HCl to neutralize, add 1 mL of chloroform for extraction, discard the lower organic phase, and repeat the extraction twice. Take the upper aqueous phase and filter it through a 0.45 μm filter membrane for HPLC analysis.

[0054] Chromatographic conditions: C18 column (4.6 × 250 mm, 5 μm), column temperature 30℃, mobile phase 0.1 M phosphate buffer (pH 7.0)-acetonitrile (83:17, v / v), flow rate 1.0 mL / min, UV detection wavelength 245 nm, injection volume 20 μL.

[0055] Standard curves were established by derivatizing mannose and galactose standards of a series of concentrations using the same method. The contents of mannose and galactose in the samples were calculated, and the degree of substitution of the galactose side chain was calculated using the following formula: Galactose substitution degree (%) = [moles of galactose / (moles of mannose + moles of galactose)] × 100%.

[0056] Testing showed that Comparative Example 1 did not generate mannan oligosaccharides, while Comparative Example 2 had a substitution degree of 18.4%. This demonstrates that by employing the specific amount of α-galactosidase added according to this invention, along with the pretreatment method, low-substitution-degree mannan oligosaccharides can be stably obtained. This is because the enzymatic hydrolysis time is 48 hours. α-galactosidase is an exoglycosidase that sequentially removes galactose residues from the non-reducing end of the substrate. α-galactosidase rapidly hydrolyzes easily accessible galactose side chains, causing a rapid decrease in the galactose substitution degree of the mannan oligosaccharides, and a simultaneous and rapid increase in their inhibition rate against cellulase. In the middle of the reaction (around 24 hours), the side chain hydrolysis rate slows down, the product structure becomes more linear, and the inhibition rate increases more slowly. After 48 hours of reaction, the product structure and inhibitory effect tend to stabilize.

[0057] like Figure 1 As shown, the degree of substitution of the galactose side chain in mannooligosaccharides decreased gradually with increasing α-galactosidase dosage. When the α-galactosidase dosage was 0.5-5.0 mg / g substrate, the degree of substitution could be controlled within the range of 2-15%, achieving gradient regulation of the product structure. Therefore, by controlling the enzymatic hydrolysis time within the range of 6-72 hours, gradient regulation of the galactose side chain substitution degree (2-15%) and cellulase inhibition intensity (inhibition rate 55-72%) of mannooligosaccharides can be achieved.

[0058] Experimental Example 2: To verify the inhibitory effects of the low-substituted mannooligosaccharide of the present invention on cellobiase I (CBHI), endoglucanase II (EGII) and β-glucosidase (GB), the following experiment was conducted: The required enzymes (8 mg / g DM) CBHI, (2 mg / g) DM EGII, and (0.2 mg / g DM) BG were prepared using PASC (0.5% w / v) as substrate, with the addition of MOS and aG. The total volume was 1 mL, and the incubation period was 50°C, 200 mL, for 24 hours. BG inhibition experiment: Substrate: Cellobiose (2% w / v) was used as substrate, and the required enzyme BG (0.2 mg / g DM) was added. MOS and aG were added. The reaction was carried out at 50℃ and 200 rpm for 3 hours. After the reaction, the enzyme was inactivated by heating in a boiling water bath for 10 minutes. The mixture was centrifuged (10,000 x g for 10 minutes). The glucose content was analyzed by HPLC, and the glucose yield and inhibition rate were calculated.

[0059] like Figure 2 As shown, the inhibition rate of low-substituted mannooligosaccharides against cellobiase I (CBHI) increased with increasing concentration, exhibiting a good dose-dependent relationship. This result indicates that low-substituted mannooligosaccharides have a significant inhibitory effect on cellobiase 1 and endoglucanase I.

[0060] like Figure 3 As shown, the inhibition rates of mannan oligosaccharides against cellobiase I and endoglucanase II were negatively correlated with the degree of substitution of the galactose side chain; the lower the degree of substitution, the stronger the inhibitory effect. When the degree of substitution decreased from 12.1% to 3.1%, the inhibition rate of CBHI increased from 24.6% to 32.8%, and the inhibition rate of EGII increased from 23.5% to 28.5%.

[0061] This is because galactomannan exhibits significant steric hindrance. High side-chain density creates strong steric hindrance, making it difficult for them to enter the active site of cellulase. α-Galactositase hydrolyzes α-1,6-galactosidic bonds, gradually removing the galactose side chains from galactomannan or mannooligosaccharides (MOS), increasing the accessibility of mannanase. After α-galactositase cleaves the side chains, the main chain is gradually exposed, increasing the number of mannanase binding sites. MOS with low substitution degrees has fewer side chains and a more linear structure, making it easier to enter the active site and compete with the substrate for binding, resulting in stronger inhibition of cellulase. This finding provides a theoretical basis for the application of mannooligosaccharides as regulators of cellulase activity.

Claims

1. A low-substitution-degree mannooligosaccharide, characterized in that, The degree of substitution of the low-substituted mannooligosaccharide is 2-15%. The low-substituted mannooligosaccharide is obtained by adding α-galactosidase and mannanase to galactomannan-containing biomass for synergistic enzymatic hydrolysis, followed by separation and purification.

2. The low-substitution-degree mannooligosaccharide according to claim 1, characterized in that, The galactomannan-containing biomass is one or more of waste coffee grounds, guar gum, konjac, and locust bean gum.

3. A method for preparing a low-substituted mannooligosaccharide according to any one of claims 1 to 2, characterized in that, Includes the following steps: S1. Pretreatment: Take biomass containing galactomannan, pretreat it with ammonia solution, filter, wash with water, and dry it for later use. S2. Enzymatic hydrolysis: The pretreated biomass containing galactomannan was suspended in sodium citrate buffer as a substrate, the pH was adjusted to 3-7, an enzyme composition containing α-galactosidase and mannanase was added and the enzymatic hydrolysis reaction was carried out for 48±3h to obtain the enzymatic hydrolysis reaction solution. The enzymatic hydrolysis reaction solution was separated and purified to obtain the low-substituted mannooligosaccharide product. The amount of α-galactosidase added is 0.1~10 mg / g substrate, and the amount of mannanase added is 0.1~5 mg / g substrate.

4. The method for preparing a low-substituted mannooligosaccharide according to claim 3, characterized in that, In S1, the galactomannan-containing biomass is pulverized and passed through a 40-60 mesh sieve. The addition ratio of the galactomannan-containing biomass to the ammonia solution is 1g: 5-20mL, and the mass percentage of the ammonia solution is 5-20wt%. During pretreatment, it is pretreated at 40-80℃ for 4-6h and washed with water until neutral.

5. The method for preparing a low-substituted mannooligosaccharide according to claim 4, characterized in that, In S1, a portion of pyruvate solution is added during pretreatment. The pyruvate solution is prepared by mixing pyruvate and deionized water at a volume ratio of 1:

5. The amount of pyruvate solution added is 0.5-2% of the ammonia solution. The specific method is as follows: the entire ammonia solution is divided into 2 / 3 volume of first ammonia solution and 1 / 3 volume of second ammonia solution. The entire pyruvate solution is divided into 3 equal volumes. The galactomannan-containing biomass is mixed with the first ammonia solution and heated to the specified temperature. The mixture is stirred at 150-200 rpm for 30-45 minutes. The first portion of pyruvate solution is added dropwise, and the mixture is stirred for another 45-60 minutes. The second portion of pyruvate solution is added dropwise, and the mixture is stirred for another 45-60 minutes. The third portion of pyruvate solution is added dropwise, and the mixture is stirred for another 45-60 minutes. The second ammonia solution is added, and the mixture is stirred until the total reaction time is reached. The solid product is collected by centrifugation. The solid product is washed with deionized water until neutral and then dried at 40-50°C for later use.

6. The method for preparing a low-substituted mannooligosaccharide according to claim 3, characterized in that, In S2, the molar concentration of the sodium citrate buffer solution is 50±5mM, and the temperature is 30~70℃.

7. The method for preparing a low-substituted mannooligosaccharide according to claim 3, characterized in that, In S2, the separation and purification steps include: sequentially subjecting the enzymatic hydrolysis reaction solution to enzyme inactivation, solid-liquid separation, desalting, monosaccharide removal, and drying; the enzyme inactivation is performed by heating in a boiling water bath for 5-15 minutes; the solid-liquid separation is performed by centrifugation at 10000g for 5-10 minutes; the desalting is performed by ion exchange resin desalting or membrane separation desalting; the monosaccharide removal is performed by yeast fermentation or activated carbon adsorption chromatography; and the drying is performed by spray drying, freeze drying, or vacuum drying.

8. The application of a low-substitution degree mannooligosaccharide according to any one of claims 1 to 2, characterized in that, The low-substituted mannooligosaccharide was used as a cellulase activity inhibitor to regulate the saccharification rate during biomass enzymatic hydrolysis.

9. The application of a low-substitution degree mannooligosaccharide according to any one of claims 1 to 2, characterized in that, The low-substituted mannooligosaccharide can be used as a prebiotic in functional foods or feed additives.

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