A method for preparing oligogalactose prebiotics from cellulose

Abstract

The present application belongs to the technical field of biomass conversion and functional food ingredients, and discloses a method for preparing oligogalactose prebiotics from cellulose. The method comprises the following steps: mixing cellulose with a galactose aqueous solution and drying to obtain galactose pre-impregnated cellulose; impregnating an acid catalyst solution on the galactose pre-impregnated cellulose and drying to obtain cellulose impregnated with galactose and an acid catalyst; performing solvent-free ball milling treatment on the cellulose impregnated with galactose and an acid catalyst to make the cellulose simultaneously undergo depolymerization and grafting reaction with galactose, and obtain a crude product; and performing purification treatment on the crude product to remove residual acid catalyst and obtain oligogalactose prebiotics. The introduction of galactose branches in the oligogalactose prepared by the present application greatly improves the water solubility of oligosaccharides, and the final product not only has high yield, but also has prebiotic activity comparable to that of high-cost enzyme method commercial products or even better for specific strains.

Description

Technical Field

[0001] This invention relates to the technical field of biomass conversion and functional food ingredients, specifically to a method for preparing galactooligosaccharide prebiotics from cellulose. Background Technology

[0002] Cellulose is the most abundant renewable biopolymer on Earth, and its transformation into high-value-added functional products, such as prebiotics, has significant economic and environmental value. Prebiotics, especially galacto-oligosaccharides (GOS), can selectively promote the growth of beneficial gut bacteria and have become important functional food ingredients.

[0003] Currently, research on the preparation of prebiotic oligosaccharides from cellulose mainly faces the following technical bottlenecks: (1) Low conversion efficiency: The dense crystalline structure and high stubbornness of cellulose severely limit its accessibility to acids or enzymes, resulting in a generally low conversion rate to water-soluble oligosaccharides. (2) Insufficient water solubility and activity of the product: The water solubility of linear β-(1→4) cellulose oligosaccharides obtained by traditional hydrolysis decreases sharply with the increase of degree of polymerization, making it difficult for probiotics to utilize them effectively and limiting the activity of prebiotics. (3) Complex branching process: To improve water solubility, existing technologies usually adopt a two-step or multi-step method of "degradation followed by modification" to introduce branches into oligosaccharides. This method has a long process route, high energy consumption, low branching efficiency, and often requires the use of expensive sugar donors or organic solvents, which does not meet the requirements of green chemical industry. (4) Process control and cost issues: To dissolve cellulose, existing technologies mostly rely on special media such as ionic liquids and eutectic solvents, which have problems such as high cost, difficulty in recycling, and easy to cause excessive hydrolysis to generate a large amount of monosaccharides (such as glucose), and poor selectivity of target oligosaccharides.

[0004] Recently, mechanical ball milling has been used as a physical pretreatment method to break down the crystallinity of cellulose, but its function is limited to improving the efficiency of subsequent (enzymatic / acid) hydrolysis and does not directly lead to structurally well-defined branched oligosaccharide products.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The present invention aims to solve at least one of the above technical problems, and provides a method for preparing galactooligosaccharide prebiotics from cellulose.

[0007] Compared with the prior art, the present invention has the following beneficial effects:

[0008] This invention, through a stepwise design of first impregnating galactose and then impregnating an acid catalyst, not only achieves uniform dispersion of the acid catalyst, but more importantly, pre-loads the galactose substrate within the cellulose microstructure. This allows the newly formed chain ends to undergo in-situ grafting reactions with adjacent galactose molecules once the cellulose depolymerizes during subsequent ball milling. This overcomes the single function of traditional impregnation, achieving a synergistic effect of "pre-loading-catalysis-grafting".

[0009] In this invention, solvent-free ball milling is no longer a simple physical crushing process, but a core reaction step that integrates cellulose amorphization, acid-catalyzed depolymerization, mechanical activation of glycosidic bonds, and promotion of dehydration condensation grafting. The conversion from solid crystalline cellulose to water-soluble branched oligosaccharides can be completed in one step.

[0010] This invention abandons the traditional multi-step, complex process of degradation followed by modification. Through a clever coupling of stepwise impregnation and mechanochemistry, degradation and grafting are completed simultaneously in a single solvent-free system in one step. This process is simple, energy-efficient, requires no subsequent addition of glycosyl donors, and is both green and economical.

[0011] The introduction of galactose side chains into the oligogalactosides prepared by this invention greatly improves the water solubility of the oligosaccharides. The final product not only has a high yield, but its prebiotic activity is comparable to, or even superior to, high-cost enzymatic commercial products for certain strains. Attached Figure Description

[0012] Figure 1 The effect of ball milling time (0–5 h) on the synthesis of galactose-grafted branched-chain dextran oligosaccharide (BGO-Gal) from impregnated cellulose in Example 2: (a) BGO-Gal yield; (b) glucose yield; (c) water solubility of BGO-Gal; (d) XRD patterns of BGO-Gal at different ball milling times.

[0013] Figure 2 The effect of ball milling time (0-5h) on the synthesis of branched-chain glucan oligosaccharides (BGO-Gal) grafted with galactose from impregnated cellulose in Example 2 is as follows: (a) galactose conversion rate; (b) galactose grafting rate and grafting selectivity (%); (c) ¹H-NMR spectra of BGO-Gal with ball milling times of 0h and 4h.

[0014] Figure 3 The effect of the cellulose to galactose substrate ratio in Example 3 on the synthesis of galactose-grafted branched-chain dextran oligosaccharide (BGO-Gal) from impregnated cellulose by ball milling was investigated: (a) the yield of BGO-Gal to glucose; (b) the water solubility of BGO-Gal; (c) the galactose conversion rate; and (d) the branching rate and branching selectivity of galactose (%).

[0015] Figure 4The ¹H-NMR spectra of the branched-chain dextran oligomers (BGO-Gal) of grafted galactose synthesized by impregnation with different cellulose to galactose substrate ratios (4:1 to 11:1) in Example 3 are shown.

[0016] Figure 5 (a) and (b) are the MALDI-TOFMS spectra of galactose-grafted branched-chain dextran oligosaccharide BGO-Gal 4:1 and commercial enzymatically produced galacto-oligosaccharide, respectively, in Example 4; Note: The number x in parentheses represents the oligosaccharide (sodium adduct) with a degree of polymerization of x; Average degree of polymerization ( This is calculated based on the peak height.

[0017] Figure 6 MALDI-TOF MS degree of polymerization plot of branched-chain dextran oligosaccharides (BGO-Gal) with different dry weight ratios of cellulose to galactose (7:1, 9:1, 11:1) in Example 4.

[0018] Figure 7 (a) and (b) respectively show the galactose-grafted linear glucosamine (BGO-Gal) and commercially available enzymatically processed galacto-oligosaccharides in Example 4. 1 ¹H-NMR glycosidic bond results;

[0019] Figure 8 (a), (b), and (c) represent different carbon source pairs in Example 5. L. rhamnosus Final cell density of ATCC 53103 (in OD) 600 The effects of count, viable count (in Log(CFU / mL)), and metabolite concentration were investigated; data were based on three biological replicates and expressed as mean ± standard deviation, with different letters indicating statistical significance at the p<0.05 level;

[0020] Figure 9 In Example 5 L. rhamnosus Growth curves and maximum growth rates of ATCC 53103 in mMRS medium supplemented with different carbon sources (18 g / L); data are presented as mean ± standard deviation based on three biological replicates, with different letters indicating statistical significance at the p < 0.05 level.

[0021] Figure 10 This is the growth of specific Lactobacillus, Bifidobacterium and Pediococcus strains in Example 5 when using grafted galactose branched glucan oligomers and commercial enzymatically produced galacto-oligosaccharides as carbon sources, respectively.

[0022] Figure 11 (a) and (b) show the degree of polymerization analysis of the branched dextran oligomers in Comparative Example 1 using MALDI-TOF MS and glycosidic bond analysis, respectively.1 H-NMR results. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] The first embodiment of the present invention provides a method for preparing galactooligosaccharide prebiotics from cellulose, comprising the following steps:

[0025] S101, cellulose is mixed with galactose aqueous solution and dried to obtain galactose pre-impregnated cellulose; then acid catalyst solution is impregnated on the galactose pre-impregnated cellulose, and after drying, cellulose impregnated with galactose and acid catalyst is obtained.

[0026] The cellulose described in this invention refers to a cellulose raw material with a glucan backbone linked by β-(1→4) glycosidic bonds, and whose glucose units retain free hydroxyl groups (-OH) available for chemical reaction. Its core characteristic lies in retaining sufficient nucleophilic hydroxyl sites to participate in subsequent acid-catalyzed glycosylation grafting reactions. Therefore, the raw materials applicable to this invention include all forms of cellulose that have not undergone hydroxyl functional group derivatization (such as esterification or etherification). Examples include high-purity / refined cellulose: microcrystalline cellulose, α-cellulose, filter paper cellulose, etc.; natural plant fibers: cotton, hemp, bamboo, wood, etc.; agricultural and food processing residues: corn stalks, wheat stalks, rice straw, sugarcane bagasse, corn cobs, soybean residue, etc.

[0027] In this embodiment, the galactose aqueous solution is first mixed with cellulose. The purpose is to use water molecules to cause the cellulose to swell slightly, while loading and embedding galactose molecules into the surface and pore structure of the cellulose fibers. This allows the galactose to adhere tightly to the cellulose in solid form, preventing it from scattering during subsequent ball milling and ensuring that it is in close molecular-level contact with the cellulose depolymerization fragments that will be generated.

[0028] After galactose loading is completed, an ethanol solution of the acid catalyst is impregnated into the galactose-preimpregnated cellulose. This step aims to uniformly disperse and position the catalyst in the galactose-loaded cellulose matrix.

[0029] The stepwise impregnation sequence described above is crucial. If galactose and acid are mixed and impregnated together, the strong acid environment may cause partial hydrolysis or self-polymerization of galactose during the impregnation stage, consuming the glycosyl donor. This invention employs a stepwise method, first fixing the donor (galactose) and then introducing the catalyst (acid), ensuring that the catalyst's main function is to act on the depolymerization of cellulose and the subsequent controllable grafting reaction.

[0030] If only galactose impregnation is used without an acid catalyst, acid protons cannot catalyze the hydrolysis of cellulose into oligosaccharides, and the product is only a mixture of cellulose and galactose, without any water-soluble oligosaccharide products. If only an acid catalyst is used for impregnation without galactose impregnation, only the acid hydrolysis of cellulose occurs to produce linear cellulosic sugars and glucose. The glucose monosaccharide can undergo a grafting reaction with the cellulosic sugars, but the final product has poor water solubility.

[0031] In some preferred embodiments, the dry weight ratio of cellulose to galactose is 1:1 to 20:1. This ratio directly determines the amount of galactose available for grafting and is one of the core parameters for controlling the branching rate and water solubility of the final product. If the ratio is too low, excessive galactose may lead to self-polymerization or waste; if the ratio is too high, the branching will be insufficient and the improvement in the water solubility of the product will be limited.

[0032] In some preferred embodiments, the dry weight ratio of cellulose to galactose is 4:1 to 7:1, within which the grafting efficiency of galactose and the overall performance of the product are optimally balanced.

[0033] In this embodiment of the invention, the core function of the acid catalyst is to provide hydrogen protons (H⁺). It is an acidic substance that can provide hydrogen protons (H⁺) under the "stepwise impregnation-ball milling" process conditions, thereby catalyzing the depolymerization of cellulose and the galactose grafting reaction. Therefore, from the perspective of its working principle, any acidic substance that can effectively provide hydrogen protons in this system is applicable. This includes common liquid acids (such as sulfuric acid, hydrochloric acid, phosphoric acid, etc.) and solid acids that can provide surface protons (such as macromolecular organic sulfonic acids, etc.). Their common function is to reduce the activation energy of cellulose hydrolysis and subsequent glycosylation grafting reactions through protonation.

[0034] When the acid catalyst is sulfuric acid, the amount of sulfuric acid used is 0.1-10% of the mass of cellulose.

[0035] S102, the cellulose impregnated with galactose and acid catalyst is subjected to solvent-free ball milling to simultaneously depolymerize the cellulose and undergo a grafting reaction with galactose to obtain a crude product.

[0036] In this step, ball milling acts as a mechanochemical reactor, rather than a traditional pretreatment process. Its purpose is to achieve cellulose depolymerization and galactose grafting in a single step through the synergistic use of mechanical, thermal, and chemical energy, without the addition of external solvents.

[0037] The intense impact, shearing, and frictional mechanical forces generated by high-speed ball milling directly disrupt the crystalline regions of cellulose, transforming it into an amorphous state. This process significantly increases the accessibility of cellulose to acid catalysis. Simultaneously, the mechanical force directly acts on the glycosidic bonds of the cellulose chains, causing mechanochemical breakage and generating dextran oligosaccharide fragments with varying degrees of polymerization (i.e., depolymerization). While the mechanical force is applied, the pre-impregnated acid catalyst is uniformly activated. It catalyzes both the hydrolysis (depolymerization) of the aforementioned glycosidic bonds and the acid-catalyzed dehydration condensation reaction (i.e., glycosylation) between the hydroxyl groups at the reduced ends of the newly generated dextran oligosaccharide fragments and the anomeric hydroxyl groups of adjacent pre-loaded galactose molecules. This achieves simultaneous degradation and grafting.

[0038] Solvent-free systems avoid the dilution and isolation of reactants by solvent molecules, forcing cellulose, galactose, and acid catalysts into close, high-frequency solid-solid contact under mechanical forces, greatly improving reaction efficiency. At the same time, it eliminates solvent recovery, pollution, and cost issues, making the process greener.

[0039] Ball milling time is a key parameter for controlling the degree of reaction and product structure. If the time is too short, depolymerization and grafting will be insufficient; if the time is too long, excessive degradation of the product may occur. In this embodiment of the invention, the ball milling time is 1-10 hours, preferably 3-5 hours, and more preferably 4 hours. Experiments show that ball milling for 4 hours can achieve the highest galactose grafting selectivity (>95%) while obtaining a high oligosaccharide yield, and achieve optimal water solubility.

[0040] The rotational speed of the ball milling process can be selected within the conventional range for performing such mechanochemical reactions in the art, for example, 300-500 rpm.

[0041] S103, the crude product is purified to remove residual acid catalyst, yielding galactooligosaccharide prebiotic.

[0042] This step is a routine post-processing step, and unreacted acid catalyst remains in the crude product after the reaction. Separation is achieved by utilizing the significant difference in solubility between the target galactooligosaccharide product and the acid catalyst in alcohol solvents. Oligosaccharides have extremely low solubility in ethanol, while the acid catalyst and its salts are soluble.

[0043] The purification process includes washing the crude product with an alcohol solvent, centrifuging, and repeating the process to remove the acid catalyst.

[0044] The second embodiment of the present invention provides a galactooligosaccharide prebiotic, which is prepared by the method of the first embodiment.

[0045] This product is a direct chemical product resulting from the synergistic effect of stepwise impregnation and solvent-free mechanochemical treatment. It comprises a backbone linked by β-(1→4) glycosidic bonds, and galactose side chains linked on the backbone by α-(1→2), α-(1→4), and / or α-(1→6) glycosidic bonds. This oligogalactose prebiotic has a degree of polymerization of 2-15, with an average degree of polymerization of 5-9. The formation of the β-(1→4) backbone originates from the selective depolymerization of cellulose synergistically catalyzed by ball milling and acid, with well-controlled process control to avoid complete hydrolysis to glucose. The formation of the α-galactose side chains originates from the in-situ dehydration condensation of pre-loaded galactose with the ends of the nascent dextran chains under mechanical force and acid catalysis. The reaction tends to form thermodynamically more stable α-configuration bonds, especially the sterically less hindrance α-(1→6) bonds.

[0046] Linear β-(1→4)glucan oligosaccharides have poor water solubility. Introducing hydrophilic galactose side chains, particularly the steric hindrance created by α-type side chains, effectively disrupts the hydrogen bond self-aggregation between oligosaccharide molecules, significantly improving their water solubility. The α-(1→6) and other bond types they contain are similar to the key structural units of commercial prebiotic GOS, thus enabling them to be recognized and utilized by probiotic-related enzyme systems.

[0047] The preparation method and effects of galactooligosaccharide prebiotics are described in detail below through several specific examples.

[0048] The detection methods used in the following embodiments are as follows:

[0049] (1) High performance liquid chromatography analysis

[0050] The yield of soluble sugar products was quantitatively analyzed using high-performance liquid chromatography (HPLC). The simplified procedure is as follows: The analytical sample was prepared by dissolving 50 mg of the purified product in 10 mL of water and then filtering through a 0.22 μm PTFE membrane. HPLC analysis was performed using a 1260 / 1290 Infinity LC system equipped with a Bio-Rad Aminex HPX-87H analytical column and a differential refractive index detector (RID-10A). The column temperature was set at 55 °C. The eluent was 5 mmol / L H₂SO₄ solution, and the flow rate was set at 0.5 mL / min.

[0051] The yield of galactose-grafted branched-chain dextran oligosaccharide (BGO-Gal) was quantified by a post-hydrolysis process. The simplified procedure was as follows: 20 mg of the purified product was mixed with 5 mL of 4% (w / v) H₂SO₄ solution, followed by hydrolysis (130°C, 1 h). The residual glucose yield (Y) was then determined. G ), soluble oligomer yield (Y)O ), galactose conversion rate (C G ), galactose branch selectivity (S B ) and galactose branching rate (B G Use the following formula to calculate.

[0052] ;

[0053] ;

[0054] ;

[0055] ;

[0056] ;

[0057] In the formula, M GBM M represents the amount of glucose (mol) after synthesis. GC M represents the amount of glucose (mol) in a cellulose feedstock. GOH M represents the amount of glucose (mol) after hydrolysis. GA and M GB M represents the amount of galactose (mol) before and after synthesis; GHA and M GHB The values ​​represent the amount of galactose before and after hydrolysis (in moles), respectively.

[0058] (2) Water solubility test of BGO-Gal

[0059] The purified BGO-Gal product was redissolved in 100 mL of deionized water and stirred continuously at 25°C and 400 rpm for 2 hours. Undissolved solid residues were then separated by filtration and quantified gravimetrically. The formula for calculating the water solubility of BGO-Gal is:

[0060] In the formula, m0 and m i The numbers represent the initial mass (g) of BGO-Gal and the mass (g) of the undissolved solid residue of BGO-Gal, respectively.

[0061] (3) X-ray diffraction analysis

[0062] XRD analysis was performed using an X-ray diffractometer (Rigaku, MiniFlex-600) equipped with a Cu-Kα microfocusing X-ray source (wavelength 1.5418 Å) and a Vantec 500 surface detector. Samples were pressed into flat cellulose pads (approximately 1 mm thick) and analyzed in step scan mode with a 2θ angle range of 5° to 40° and a scan rate of 10° / min. The Segal crystallinity index (CrI) was calculated from the experimental diffraction patterns after background subtraction using the following formula.

[0063] Among them, I (c+a) The peak intensity of cellulose Iβ at 2θ = 22.7° or cellulose II at 2θ = 21.8°; a The peak intensity is represented by the peak intensity of disordered cellulose Iβ at 2θ=18° or cellulose II at 2θ=16°.

[0064] (4) Degree of aggregation (DP) was analyzed by MALDI-TOF MS.

[0065] The degree of polymerization (DP) of the BGO-Gal samples was analyzed by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) on a linear MALDI-TOF mass spectrometer (Ultraflextreme, Bruker). The simplified procedure was as follows: 1 μL of analyte solution (2 mg / mL, dissolved in a 2,5-dihydroxybenzoic acid matrix) was added dropwise onto an unpolished stainless steel MTP384 target plate to prepare the sample. Spectra were acquired in linear positive ion mode at 90% laser energy, with a detection range of 0–10000 m / z. The major mass peak (the mass-to-charge ratio m / z of the sodium adduct of the oligomer in the mass spectrum) was identified using the following formula:

[0066] m / z = 18 + 162.1427x + 23 (Na⁺); where x represents the oligomer with degree of polymerization x, and 162.1427 corresponds to the mass of the dehydrated glucose unit (AGU).

[0067] (5) Glycosidic bond analysis by ¹H-NMR

[0068] Glycosidic bonds in BGO-Gal were analyzed by ¹H NMR spectroscopy. Sample preparation was as follows: 5 wt.% BGO-Gal was dissolved in D₂O containing 10 mMDSS (internal standard). The solution was gently stirred (100 rpm, 5 min) and then sonicated (15 min) to ensure complete dissolution. After filtration, ¹H NMR spectra were acquired on a Bruker 400 MHz NMR spectrometer with 24 scans. Key glycosidic bond signals (chemical shifts δ = 5.5 to 4.1 ppm) were assigned, including anomeric protons (α- and β-reducing ends, α-RE and β-RE) and specific glycosidic bond linkage positions (α(1→3), α(1→6), and β(1→4)). The relative abundance of each glycosidic bond was determined by its integrated peak area (A0). Hi ) accounts for the peak area of ​​total glycosidic bonds (∑A Hi The relative abundance is quantified by the ratio of A to A, and the formula is: Relative Abundance = A / A Hi / (∑A Hi ).

[0069] (6) In vitro probiotic fermentation experiment

[0070] *Lactobacillus rhamnosus* ATCC 53103, *Lactobacillus reuteri* ATCC 6475, *Pediococcus pentosaceus* ATCC 33316, and *Pediococcus acidilactici* DSM 20284 were purchased from the Guangdong Provincial Microbial Culture Collection Center. *Lactobacillus plantarum* WCFS1, *Bifidobacterium lactis* BB-12, and *Bifidobacterium animalis* DSM 10140 were strains preserved in the applicant's laboratory. All strains were preserved in MRS broth containing 15% glycerol (with 0.05% L-cysteine ​​added for the two Bifidobacterium strains) at -80°C. The frozen strains were activated by streak plating onto MRS agar plates and anaerobic incubation at 37°C for 24 hours. Subsequently, single colonies were picked and transferred to MRS broth, and anaerobically cultured overnight at 37°C. The fresh bacterial culture was then inoculated at the initial OD value. 600The culture medium was transferred to modified MRS medium at a concentration of 0.1 g / L for subculturing. The modified MRS medium consisted of 1.0% peptone, 1.0% beef extract, 0.5% yeast extract, 0.2% dipotassium hydrogen phosphate, 0.2% ammonium citrate, 0.01% magnesium sulfate, 0.005% manganese sulfate (all percentages are by volume), 0.1% Tween 80, and 0.05% L-cysteine, with BGO-Gal sterilized through a 0.22 μm filter added as the sole carbon source. Other carbon sources tested included glucose, galactose, lactose, and commercial GOS. The concentration of all tested carbon sources in the final medium (on a dry matter basis) was set at 18 g / L. Given that yeast extract itself contains sugars (17.9% carbohydrates, including 4.3% glucose, 2.2% galactose, 1.4% xylose, 5.5% mannose and 3.5% trehalose), a modified MRS medium without added exogenous carbon sources was set up as a blank control.

[0071] After anaerobic culture at 37°C for 24 h in modified MRS media with different carbon sources, the final cell density was manually measured using a UV-Vis spectrophotometer. Bacterial viability was determined using plate counting. For growth curve analysis, 200 μL of inoculated medium was pipetted into 96-well plates, placed in a microplate reader, and anaerobically cultured at 37°C for 24 h, with OD readings automatically taken every 1 h. 600 Values. Growth curve data are not used for direct comparison of final cell density because the absolute OD values ​​measured by UV spectrophotometer and ELISA reader are different. 600 The values ​​are not directly comparable. The maximum growth rate was calculated by linear fitting of the exponential growth phase. After the growth experiment, the supernatant was collected by centrifugation and filtration through a 0.22 μm filter membrane, and residual sugars and metabolites (including lactic acid, formic acid, acetic acid, propionic acid, and butyric acid) were analyzed by HPLC.

[0072] All microbial growth experiments were performed in triplicate. Statistical comparisons were performed using Tukey's HSD test (JMP Pro 15 software). Statistical significance was determined by different letter labels, with p < 0.05 considered significant.

[0073] Example 1: Preparation of galacto-oligosaccharide prebiotics from microcrystalline cellulose under standard conditions

[0074] (1) Stepwise impregnation: Galactose aqueous solution (2.19 g galactose dissolved in 5 mL water) was mixed evenly with 8.75 g microcrystalline cellulose (Avicel); then, the mixture was dried at 70°C for 3 h to obtain cellulose pre-impregnated with galactose (at this time, the dry weight ratio of cellulose to galactose was 4:1, and sulfuric acid accounted for about 2.9% of the dry weight of cellulose); 140 μL of 98% H2SO4 was mixed evenly with 3 mL of anhydrous ethanol to prepare H2SO4-ethanol solution; the H2SO4-ethanol solution was added to the cellulose pre-impregnated with galactose and mixed evenly; the mixture was dried again at 70°C for 1 h to obtain cellulose impregnated with galactose and sulfuric acid;

[0075] (2) Solvent-free ball milling: The cellulose impregnated with galactose and sulfuric acid was ball milled using a QM-QX planetary ball mill at a speed of 400 rpm for a total time of 4 hours. To prevent overheating of the system, intermittent ball milling was used, i.e., after every 15 minutes of operation, the milling was paused for 5 minutes, and the crude product was obtained after the milling was completed.

[0076] (3) Purification: After mixing the crude product with excess anhydrous ethanol, centrifuge and discard the supernatant containing residual sulfuric acid. Collect the solid residue containing oligosaccharides. Repeat this washing process three times with anhydrous ethanol. Dry the final solid precipitate at 60°C for 2 hours to obtain the purified galactooligosaccharide prebiotic product, namely galactooligosaccharide grafted branched-chain glucan oligosaccharide (BGO-Gal).

[0077] Example 2: Effect of ball milling time on product synthesis

[0078] To investigate the effect of ball milling time, the dry weight ratio of cellulose to galactose was fixed at 4:1, the amount of sulfuric acid was 2.9%, and the rotation speed was 400 rpm. Ball milling experiments were conducted for 0, 1, 2, 3, 4, and 5 hours (0 hours represents impregnation without ball milling). Except for the ball milling time, the other steps were the same as in Example 1.

[0079] The BGO-Gal products prepared at different ball milling times were analyzed, and the results are as follows: Figure 1 As shown. From Figure 1 (a) It can be seen that when the ball milling time increased to 2 h, the yield of BGO-Gal rapidly increased from 9.5% to 33.0%. This is mainly attributed to the mechanical force generated during ball milling. The applied shear stress effectively disrupted the crystalline regions of cellulose, significantly improving the accessibility of acid depolymerization using sulfuric acid catalyst. From the ball milling time of 2 h onwards, the yield increase of BGO-Gal slowed down, reaching 41.9% after 5 h of ball milling. The yield of glucose remained at a low level (<0.12%). Figure 1(b) This may be because glucose can further participate in chemical grafting reactions to form oligomers (degree of polymerization ≥2), rather than accumulating as monomers. Figure 1 (c) It can be seen that the water solubility of BGO-Gal increased from 15.1% to 33.3% with ball milling time extended to 3 hours, and increased sharply to 53.1% when ball milling time reached 4 hours. The initial increase in water solubility of BGO-Gal may be due to the depolymerization of impregnated cellulose to form short-chain dextran oligomers, while the subsequent significant increase in water solubility may be attributed to the successful grafting of galactose or glucose onto the main chain of the dextran oligomers. The formation of galactose / glucose side chains in BGO-Gal may help improve its water solubility and prevent further degradation into monosaccharides.

[0080] Figure 1 (d) shows the XRD analysis results of BGO-Gal obtained at different ball milling times, where BM-0h to BM-5h represent BGO-Gal milled for 0h to 5h respectively. The study found that the unmilled sample (BM-0h) exhibited a distinct diffraction peak at 2θ = 22.5° (corresponding to the (200) crystal plane), a characteristic peak of cellulose type I, with a crystallinity index as high as 68.3% due to its dense hydrogen bond network. As the ball milling time increased to 3h, the half-width at half-maximum (FWHM) of the (200) diffraction peak increased from 1.2° to 3.5°, and the corresponding crystallite size decreased from 5.2 nm to 2.1 nm, indicating that mechanical force led to crystal breakage. When the ball milling time exceeded 3h, the peak intensity decreased significantly (relative crystallinity decreased to 21.4%), and at 5h, it completely transformed into a broad amorphous diffuse peak centered at 2θ = 20.5°. This transition from a crystalline to an amorphous state is attributed to the following synergistic mechanisms: (1) the intense shear force generated during ball milling directly disrupts the intramolecular and intermolecular hydrogen bond network of cellulose, particularly the van der Waals interactions along the (200) crystal plane; (2) sulfuric acid catalyzes the cleavage of β(1→4) glycosidic bonds in cellulose, producing short-chain oligomers, leading to a reduction in crystallite size; and (3) the introduction of galactose side chains creates steric hindrance, inhibiting intermolecular interactions between the formed oligomers. Figure 1 (c) shows that the transition from crystalline to amorphous state is closely related to the significant increase in water solubility of BGO-Gal (from 15.1% to 53.1%), which strongly demonstrates the close relationship between the molecular structure of BGO-Gal and its solubility.

[0081] In addition, the effects of ball milling time on the galactose conversion rate and branching structure in BGO-Gal were investigated, and the results are as follows: Figure 2 As shown. Figure 2(a) shows that as the ball milling time increased from 0 h to 3 h, the galactose conversion rate moderately increased from 51.2% to 58.7%, and further increased to 61.1% when the ball milling time reached 5 h. Figure 2 (b) It is evident that during the ball milling time of 0–4 h, the initial galactose grafting rate increased sharply (from 25.3% to 56.7%), demonstrating a significant enhancement in the chemical grafting reaction of galactose as ball milling progressed. Subsequently, when the ball milling time was extended to 5 h, the galactose grafting rate only slightly increased to 57.9%, possibly because the mechanical energy required for further chemical grafting reactions was insufficient. On the other hand, the selectivity of galactose branching showed a more pronounced dependence on ball milling time. In the initial stage of ball milling (0–1 h), the selectivity jumped sharply from 49.4% to 75.5%, indicating that the mechanical force generated by ball milling rapidly activated the reaction sites (hydroxyl groups) on galactose and dextran oligomers. Subsequently, the selectivity steadily increased with ball milling time, reaching and stabilizing at 95.3% at 4 h, while when the ball milling time was further extended to 5 h, the selectivity only slightly decreased to 94.7%. The above results demonstrate that, under the synergistic effect of mechanical force and sulfuric acid catalyst, galactose tends to undergo chemical grafting reactions with dextran oligomers rather than undergoing self-polymerization. Based on this, the optimal ball milling time was determined to be 4 hours, which ensures a high galactose grafting rate while avoiding excessive depolymerization of BGO-Gal.

[0082] The glycosidic bonds and reducing end structures in the BGO-Gal sample (BM-4h) prepared by ball milling for 4 h were characterized by ¹H-NMR and compared with those of the unmilled sample (BM-0h). The results are as follows: Figure 2As shown in (c). Based on our previous study (Liang, H.; Ye, S.; Liu, Q.; et al. Confined synthesis of glucan oligomers from glucose inzeolites. Green Chem. 2025, 27 (6), 1714-1722, DOI: 10.1039 / D4GC05126B.), peaks in the δ 5.4–4.2 ppm range were detected and classified into different types of glycosidic bonds. The relative content (%) of each glycosidic bond was quantified according to the corresponding peak area, and the results are listed in Table 1. Compared with the unmilled sample (BM-0 h), after 4 h of ball milling, the peak at δ 4.42 ppm corresponding to the β(1→4) bond decreased significantly from 70.4% to 57.1%, indicating that the cellulose backbone underwent significant depolymerization. In contrast, the peaks corresponding to α(1→6) bonds at δ 4.86 ppm and α(1→2) bonds at δ 5.13 ppm increased from 8.4% to 20.3% and from 4.2% to 7.5%, respectively. Furthermore, a small amount of α(1→4) bonds (2.2%) was observed at δ 5.38 ppm. ¹H-NMR analysis confirmed that in the unmilled oligomers (impregnated only with galactose and sulfuric acid), the α- and β-configurations accounted for 15.2% and 84.8%, respectively. After ball milling, the proportion of the α-configuration increased to 34.4%, while the proportion of the β-configuration decreased to 65.6%. The significant increase in α-glycosidic bonds demonstrates that galactose was successfully grafted onto the dextran oligomer backbone, forming a highly branched and water-soluble BGO-Gal.

[0083] Table 1. Effect of ball milling time (BM, 0–5 h) on the distribution of glycosidic bond types in branched-chain glucan oligosaccharides (BGO-Gal) synthesized from cellulose-impregnated branched-chain glucan oligosaccharides grafted with galactose.

[0084] .

[0085] The above results demonstrate the successful conversion of crystalline cellulose into water-soluble BGO-Gal via mechanochemical methods. Studies on ball milling time (0-5 h) show that ball milling for 5 h yields the highest BGO-Gal yield (41.9%) and the best water solubility (57.8%).

[0086] Example 3 Effect of the dry weight ratio of cellulose to galactose on product synthesis

[0087] To investigate the effect of substrate ratio, the ball milling time was fixed at 4 hours, the sulfuric acid dosage was 2.9%, and the rotation speed was 400 rpm. During the impregnation stage, the dry weight ratio of cellulose to galactose was adjusted to 11:1, 9:1, 7:1, 6:1, 5:1, and 4:1, respectively (the total amount of cellulose was kept constant at 8.75 g). The preparation method was the same as in Example 1.

[0088] The BGO-Gal products prepared at different mass ratios were analyzed, and the results are as follows: Figure 3 As shown. From Figure 3 (a) It can be seen that when the cellulose to galactose ratio decreased from 11:1 to 8:1, the BGO-Gal yield gradually increased from 33.8% to 39.8%. This suggests that the increased galactose loading may have promoted oligomer formation by enhancing the chemical grafting reaction. However, further increasing the galactose loading (reducing the cellulose to galactose ratio to 4:1) did not result in a higher BGO-Gal yield. This is likely because galactose impregnation had reached saturation, and the availability of sulfuric acid catalysts for acidic depolymerization and chemical grafting reactions was limited. Regardless of the change in galactose loading, the glucose yield remained below 0.5%. Figure 3 (b) It can be seen that galactose loading significantly affected the water solubility of BGO-Gal. When the cellulose to galactose ratio increased from 11:1 to 7:1, the water solubility increased from 32.1% to 53.1%. This is mainly attributed to the grafting of galactose side groups onto the linearly β(1→4) linked dextran backbone, resulting in an increase in the galactose grafting rate (from 39.7% to 66.3%). Figure 3 (d) also confirms this. This indicates that the initial galactose concentration favors the chemical grafting reaction between galactose and the dextran oligomers generated from cellulose depolymerization. However, when the galactose loading is further increased (cellulose to galactose ratio from 6:1 to 4:1), the galactose conversion reaches a plateau (71.2–75.2%), while the grafting selectivity decreases (from 97.5% to 82.7%). Figure 3 (c) and 3(d)). This trend is mainly due to the thermodynamic equilibrium of the chemical grafting reaction, where excess galactose cannot be further grafted into BGO-Gal.

[0089] The glycosidic bonds of BGO-Gal synthesized with different cellulose to galactose substrate ratios were characterized by ¹H-NMR. Figure 4The relative content of each type of glycosidic bond was quantified by peak area, and the results are summarized in Table 2. With the increase of galactose loading during cellulose impregnation (cellulose:galactose ratio decreased from 11:1 to 7:1), more newly formed α(1→6), α(1→4), and α(1→2) bonds were observed in BGO-Gal, while β(1→4) bonds decreased accordingly. This clearly indicates that a higher galactose loading mainly enhances the chemical grafting reaction between galactose and the dextran oligomers generated by cellulose depolymerization, resulting in a higher degree of branching in the formed BGO-Gal. This trend is also consistent with the improved water solubility of BGO-Gal synthesized under high galactose loading. Interestingly, compared to... Figure 4 (a) and Figure 4 (b) It can be found that BGO-Gal synthesized with a higher galactose loading contains more α-reducing ends, while the relative contents of its α(1→6), α(1→4) and α(1→2) bonds are relatively low.

[0090] Table 2. Distribution of glycosidic bonds in branched-chain dextran oligomers (BGO-Gal) of grafted galactose synthesized by impregnation with different cellulose and galactose ratios.

[0091] .

[0092] The results indicate that BGO-Gal synthesized using different cellulose to galactose substrate ratios (from 11:1 to 4:1) during the impregnation stage achieved the highest yield (39.8%) at a ratio of 8:1. However, better water solubility was obtained using smaller ratios (53.1% at 7:1 and 46.2% at 4:1), which may be due to the formation of diverse α-glycosidic bonds.

[0093] Example 4: Structural characterization of the product and its comparison with commercial products

[0094] The BGO-Gal 4:1 synthesized under preferred conditions (cellulose to galactose dry weight ratio 4:1, ball milling for 4 hours, preparation method the same as in Example 1) was characterized in detail and compared with commercial enzymatic galacto-oligosaccharides (GOS) (trade name: AlphaGOS®; manufacturer: Olygose, Venette, France).

[0095] (1) Degree of polymerization analysis: The degree of polymerization distribution of the synthesized BGO-Gal 4:1 and GOS was analyzed using MALDI-TOF MS positive ion mode. The results are as follows: Figure 5 As shown. From Figure 5(a) It can be seen that the degree of polymerization of BGO-Gal 4:1 synthesized in Example 1 is mainly concentrated between 2 and 10, with higher abundance of trimers and tetramers, while the proportion of components with DP of 11-16 is relatively low. In contrast, the degree of polymerization range of commercial GOS is relatively lower and narrower (DP 2-6), with the highest abundance at DP 4. Based on the peak height ratio, the relative percentage of each degree of polymerization and the average degree of polymerization ( The results were quantified and are shown in Table 3 below. The average degree of polymerization of BGO-Gal 4:1 was 5.7, slightly higher than that of commercial GOS (3.8). This difference stems from the difference in raw materials and preparation methods: commercial GOS is usually prepared by enzymatic hydrolysis of raffinose oligosaccharides present in legumes.

[0096] In addition, the degree of polymerization distribution of BGO-Gal synthesized under different substrate ratios in Example 3 was compared, and the results are as follows: Figure 6 As shown in Table 3, the average degree of polymerization of BGO-Gal was positively correlated with the cellulose content during impregnation, as expected: the average degrees of polymerization for BGO-Gal 7:1, 9:1, and 11:1 were 7.6, 7.7, and 7.8, respectively. This suggests that the lower the cellulose loading, the higher the degree of depolymerization may be. Consistent with this, BGO-Gal 4:1, with the highest galactose loading, showed the lowest average degree of polymerization (5.7), similar to commercial GOS, and may therefore exhibit prebiotic potential.

[0097] (2) Glycosidic bond analysis: The glycosidic bond composition of the synthesized BGO-Gal 4:1 and commercial GOS was characterized by ¹H-NMR, and the results are as follows: Figure 7 As shown. From Figure 7 (a) It can be seen that BGO-Gal 4:1 still contains 58% β(1→4) bonds, which are inherited from cellulose and constitute the backbone of the dextran oligomer. The newly formed α(1→6), α(1→2), and α(1→4) bonds account for 11.5%, 4.5%, and 0.6% respectively in BGO-Gal 4:1. They originate from the chemical grafting reaction between the dextran oligomers generated by the depolymerization of galactose and cellulose.

[0098] Chemical grafting is essentially an acid-catalyzed dehydration condensation reaction that occurs between the anomeric hydroxyl group (C1–OH) of galactose (glycosyl donor) and the nucleophilic hydroxyl group (e.g., C6–OH, C2–OH, C4–OH) of the dextran oligomer (glycosyl acceptor). α(1→6) bonds are more readily formed than other types of bonds because the C6–OH on the glycosyl acceptor is a primary alcohol, which has less steric hindrance compared to secondary hydroxyl groups (e.g., C2–OH, C4–OH), thus making the formed α(1→6) bonds thermodynamically more stable. Overall, the formation of α(1→6) and α(1→2) branches in BGO-Gal, despite its higher degree of polymerization, significantly improves its solubility in water. This contributes to the stability of the product and prevents further degradation into monosaccharides, consistent with the synthetic data from the aforementioned examples.

[0099] from Figure 7 (b) It can be seen that commercial GOS mainly contains α(1→6) bonds (70.9%), with very few α(1→4) bonds (0.1%). Based on its degree of polymerization analysis, it is inferred that the main oligosaccharide components in commercial GOS are mannotriose (DP 3, Galp-α(1→6)-Galp-α(1→6)-Glcp) and stachyotetraose (DP 4, Galp-α(1→6)-Galp-α(1→6)-Glcp). Overall, the BGO-Gal synthesized in this invention is similar to commercial GOS in glycosidic bond type, and therefore may be utilized by probiotics to exert prebiotic potential.

[0100] Example 5 Evaluation of the prebiotic activity of the product

[0101] (1) Experimental strains

[0102] Lactobacillus genus: Lactobacillus rhamnosus ( L. rhamnosus ATCC 53103 (LGG), Lactobacillus plantarum ( L. plantarum WCFS1, Lactobacillus reuteri ( L. reuteri ATCC 6475; Bifidobacterium: Bifidobacterium lactis ( B. lactis BB-12, Bifidobacterium animalis ( B. animalis DSM 10140; Pediococcus spp.: Pediococcus pentosaceus ( P. pentosaceus ATCC 33316, Pediococcus lactis ( P. acidilactici DSM 20284.

[0103] (2) Carbon source design and experimental methods

[0104] Carbon source setup (taking a detailed test of Lactobacillus rhamnosus ATCC 53103 as an example): A comprehensive control and experimental group were set up, with a carbon source concentration of 18 g / L, as detailed below:

[0105] Monosaccharide and disaccharide controls: Glucose, Galactose, Lactose, Cellobiose; Key commercial product and method controls: GOS (commercial enzymatic galactooligosaccharide from Example 4), BGO (branched dextran oligomers without galactose impregnation prepared in Comparative Example 1); Product series of this invention: BGO-Gal prepared using different cellulose to galactose mass ratios (11:1, 9:1, 7:1, 6:1, 4:1), denoted as BGO-Gal11:1 to BGO-Gal4:1, prepared using the same method as in Example 1; Blank control: Blank (mMRS medium without any carbon source).

[0106] Cultivation and Detection: Each probiotic strain was inoculated into mMRS medium containing different carbon sources and cultured anaerobically at 37°C for 24 h. After cultivation, samples were taken to determine: (a) final cell density (OD). 600 (a) Viable count (expressed as Log(CFU / mL) by plate count); (b) Concentration of major metabolites (lactic acid, acetic acid, and formic acid).

[0107] (3) Growth-promoting effect on Lactobacillus rhamnosus ATCC 53103

[0108] from Figure 8 It can be seen that for probiotics L. rhamnosus For ATCC 53103, glucose, galactose, and cellobiose are good carbon sources for its growth (OD). 600 The values ​​were 4.4, 3.8, and 4.2 respectively, while lactose resulted in extremely limited growth (OD). 600 0.5), with no statistically significant difference compared to the blank control group. Growth curve data ( Figure 9 This also indicates that L. rhamnosus ATCC 53103 grows very poorly on lactose (maximum specific growth rate μ). max 0.03), compared to better growth on glucose, galactose, and cellobiose (μ). max The values ​​were 0.17, 0.10, and 0.18 respectively. This is likely because... L. rhamnosusATCC 53103 lacks a gene encoding β-galactosidase for the hydrolysis of lactose, but it possesses a functional bgl operon that encodes an enzyme system (phosphotransferase system, phosphoβ-glucosidase) that can be used for the transport and metabolism of cellobiose.

[0109] BGO-Gal 4:1 resulted in the highest cell density (OD). 600 5.8), highest viable count (9.4 log(CFU / mL)) and fastest bacterial growth rate (μg / mL). max 0.15 (Figure S4(b)). This is likely due to the higher degree of branching, lower average degree of polymerization, and higher water solubility of BGO-Gal 4:1 among the tested BGO-Gal samples. Consistent with this, BGO-Gal 11:1 has a higher average degree of polymerization (7.8), a higher content of β(1→4) bonds in the main chain (62.6%), and lower water solubility, which may have led to poorer bacterial growth. Furthermore, LGG's growth performance on BGO and commercial GOS was significantly worse than that of BGO-Gal 4:1, possibly because the highly branched nature of BGO-Gal provides LGG with multiple types of glycosidic bonds for utilization. It is evident that the better growth-promoting ability is associated with BGO-Gal having a lower cellulose to galactose substrate ratio (i.e., a higher galactose grafting degree).

[0110] from Figure 8 (c) It can be seen that, L. rhamnosus ATCC 53103 fermentation of BGO-Gal primarily produces lactic acid (47–182 mM), as well as lower levels of acetic acid (6–13 mM) and formic acid (13–27 mM), both of which are beneficial short-chain fatty acids.

[0111] (4) Evaluation of the potential of broad-spectrum prebiotics and strain selectivity

[0112] To investigate the applicability of BGO-Gal to different probiotics, a 4:1 ratio of BGO-Gal with commercial GOS was used to test six other probiotic strains. The results are as follows: Figure 10 As shown.

[0113] from Figure 10 It can be seen that when BGO-Gal 4:1 or commercial GOS is used as the sole carbon source, the OD of Lactobacillus reuteri and Lactobacillus plantarum are significantly different. 600 The values ​​were similar, with no significant difference (p>0.05). For both Bifidobacterium strains, commercial GOS was significantly more effective than BGO-Gal 4:1 in promoting their growth, specifically manifested in higher OD values. 600Values ​​(Bifidobacterium lactis: 5.9 vs. 5.0; Bifidobacterium animalis: 2.8 vs. 0.8). In contrast, BGO-Gal 4:1 showed significantly better growth-promoting effects on Pediococcus pentosaceus and Pediococcus lactis than commercial GOS. This difference in growth-promoting effects between BGO-Gal and commercial GOS across different strains is determined by whether the strains possess genes encoding transport and hydrolytic enzymes for metabolizing short-chain oligosaccharides, and the expression of these genes is influenced by the size (DP range) of the oligosaccharide molecule and the type of glycosidic bonds it contains.

[0114] Comparative Example 1: Branched dextran oligomers without galactose impregnation

[0115] To demonstrate the necessity of the galactose impregnation step for introducing prebiotic active side chains, this comparative example was set up and compared with a portion of the BGO-Gal in Example 3.

[0116] Preparation method: Except for the absence of galactose impregnation, the other main process parameters are the same as in Example 3. Specifically, 8.75 g of microcrystalline cellulose was impregnated with an H2SO4-ethanol solution prepared in the same manner as in Example 3, dried, ball-milled, and purified using the same steps. The resulting product was designated as branched dextran oligomer BGO.

[0117] The structure of the prepared BGO was characterized, and the results are as follows: Figure 11 As shown in Table 3. Figure 11 (a) MALDI-TOF mass spectrometry analysis showed that the BGO mainly consisted of oligomers with a degree of polymerization of 7 to 11, with an average degree of polymerization of 8.8, which is relatively higher than the average degree of polymerization (5.7-7.8) of the galactose-impregnated BGO-Gal series. This may be because the galactose incorporated during ball milling promoted the dehydration condensation reaction between it and the depolymerized BGO, thereby further enhancing the depolymerization of cellulose. Figure 11 (b)¹H-NMR analysis showed that the glycosidic bonds in this BGO were mainly β(1→4) bonds (51.6%), with a low content of α(1→6) bonds (24.5%), and very low content of α(1→2) and α(1→4) bonds (1.9% and 2.1%, respectively). This indicates that ball milling of acidified cellulose successfully produced branched dextran oligomers with novel glycosidic bonds, which were formed through dehydration condensation, further validating the reaction pathway proposed in the glycosidic bond analysis of Example 4 (2).

[0118] Table 3. Relative percentage of degree of polymerization (DP) of grafted galactose branched dextran oligosaccharides (BGO-Gal, with different dry weight ratios of cellulose to galactose from 4:1 to 11:1), branched dextran oligosaccharides (BGO), and commercially available enzymatically produced galacto-oligosaccharides (GOS).

[0119] .

[0120] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing galactooligosaccharide prebiotics from cellulose, characterized in that, Includes the following steps: Cellulose was mixed with an aqueous galactose solution and dried to obtain galactose-preimpregnated cellulose; The acid catalyst solution is then impregnated onto the galactose-preimpregnated cellulose, and after drying, cellulose impregnated with galactose and acid catalyst is obtained. The cellulose impregnated with galactose and acid catalyst was subjected to solvent-free ball milling to simultaneously depolymerize the cellulose and undergo a grafting reaction with galactose, resulting in a crude product. The crude product was purified to remove residual acid catalysts, yielding galactooligosaccharide prebiotics.

2. The method as described in claim 1, characterized in that, The dry weight ratio of cellulose to galactose is 1:1 to 20:

1.

3. The method as described in claim 2, characterized in that, The dry weight ratio of cellulose to galactose is 4:1 to 7:

1.

4. The method according to any one of claims 1-3, characterized in that, The acid catalyst comprises one or more of sulfuric acid, phosphoric acid, hydrochloric acid, and organic sulfonic acid.

5. The method as described in claim 4, characterized in that, The acid catalyst is sulfuric acid, and the amount of sulfuric acid used is 0.1-10% of the mass of cellulose.

6. The method according to any one of claims 1-3, characterized in that, The ball milling process is carried out at a speed of 300-500 rpm for 1-10 hours.

7. The method as described in claim 6, characterized in that, The ball milling process takes 3-5 hours.

8. The method according to any one of claims 1-3, characterized in that, The purification process includes washing the crude product with an alcohol solvent, centrifuging, and repeating the operation to remove the acid catalyst.

9. A galacto-oligosaccharide prebiotic, characterized in that, Prepared by the method described in any one of claims 1-8.

10. The galactooligosaccharide prebiotic as described in claim 9, characterized in that, It comprises a main chain linked by β-(1→4) glycosidic bonds, and galactose side chains linked on the main chain by α-(1→2), α-(1→4) and / or α-(1→6) glycosidic bonds; the degree of polymerization of the oligogalactose prebiotic is 2-15, and the average degree of polymerization is 5-9.

Images