Acetylated mannan oligosaccharide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of oligosaccharide modification, in particular to acetylated mannan oligosaccharide as well as a preparation method and application thereof. The method comprises the following steps: mixing mannan oligosaccharide, acetic anhydride and a catalyst, and reacting to obtain acetylated mannan oligosaccharide. Through acetylation, the water solubility of mannan oligosaccharide is reduced, the molecular weight is improved, and the surface hydrophobicity is changed, so that mannan oligosaccharide shows different microbial responses in the in-vitro fermentation process. Structurally-driven fermentation kinetics show that the release of short-chain fatty acids is delayed by the higher degree of acetylation substitution, and the enrichment of florae such as Bacteroides (Bacteroides), mucus eubacterium (Blauria), Parabacteroides (Parabacteroides), a Dorea strain (Dorea), Roseburia (Roseburia) and the like is promoted, so that the content of the short-chain fatty acids in the Saccharomyces cerevisiae is reduced, and the content of the short-chain fatty acids in the Saccharomyces cerevisiae in the Saccharomyces cerevisiae in the Saccharomyces cerevisiae in the Saccharomyces cerevisiae in the Saccharomyces cerevisiae in the Saccharomyces cerevisiae is reduced. Acetylated mannan oligosaccharide can be used as controllable prebiotics for realizing a fermentation effect in a specific region of an intestinal tract.

Description

Technical Field

[0001] This invention relates to the field of oligosaccharide modification technology, and in particular to an acetylated mannooligosaccharide, its preparation method, and its applications. Background Technology

[0002] Oligosaccharides represent a diverse class of indigestible carbohydrates that play a vital role in regulating gut microbiota and maintaining gut health. Among them, the most widely studied and applied are raffinose family oligosaccharides (raffinose, stachyose), fructooligosaccharides, and galactooligosaccharides, which possess prebiotic effects in promoting the growth of Bifidobacteria and Lactobacilli and improving host metabolism. However, all reported natural oligosaccharides limit their sustained efficacy in the distal gut due to their inherent hydrophilicity and rapid fermentation in the proximal colon due to their low molecular weight (MW). Rapidly fermented oligosaccharides are typically utilized by microbes in the proximal colon, leading to a surge in gas and SCFA production, which may fail to maintain colonic health throughout the distal region. In contrast, slowly fermented carbohydrates gradually and continuously release short-chain fatty acids (SCFAs) along the entire colon, helping to maintain microbial balance and promote distal gut health.

[0003] To address this issue, chemical modification of oligosaccharides has become a key option for adjusting their physicochemical properties and subsequent fermentation kinetics. However, the structural modification of small-molecule oligosaccharides faces multiple technical bottlenecks: on the one hand, the number of modifiable active sites on their molecular backbone is limited, and the reaction selectivity is poor, making them prone to over-modification or isomerization of modified sites, leading to a decrease in the purity of the target product; on the other hand, modified small-molecule oligosaccharides still retain strong hydrophilicity, and their physicochemical properties are only slightly different from those of unmodified oligosaccharides and reaction byproducts, making it difficult to efficiently separate and purify them from the aqueous phase using conventional chromatography, crystallization, and other methods during subsequent separation. This not only increases the complexity of the process and production costs but may also affect their prebiotic activity and application safety due to residual impurities; in addition, slight fluctuations in modification reaction conditions (such as temperature, pH, and catalyst) can easily lead to uneven molecular weight distribution of the product, further exacerbating the separation difficulty and limiting the large-scale preparation and practical application of modified oligosaccharides.

[0004] Therefore, how to provide a chemical modification method for small molecule oligosaccharides so that they can be used as controllable prebiotics to achieve fermentation in specific regions has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to provide an acetylated mannooligosaccharide, its preparation method, and its application, in order to solve the problems existing in the prior art.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: One of the technical solutions of this invention is a method for preparing acetylated mannooligosaccharides, comprising the following steps: Mann oligosaccharides, acetic anhydride, and a catalyst are mixed and reacted to obtain acetylated mann oligosaccharides.

[0007] The second technical solution of the present invention is an acetylated mannooligosaccharide prepared by the above-mentioned preparation method.

[0008] The third technical solution of the present invention is the application of the above-mentioned acetylated mannan oligosaccharide in the preparation of intestinal regulating drugs or functional foods.

[0009] Compared with the prior art, the present invention has the following beneficial effects: This invention reduces the water solubility of mannooligosaccharides (MOS) by acetylation, increases their molecular weight, and alters their surface hydrophobicity, leading to different microbial responses during in vitro fermentation. Structure-driven fermentation kinetics show that higher acetylation substitution delays the release of short-chain fatty acids (SCFAs) but promotes the release of Bacteroides (S.) Bacteroides ), via Myxobacterium spp. ( Blautia ), Parabacteroides ( Parabacteroides ), Dorea strain ( Dorea ) and Rochetomyces ( Roseburia Enrichment of microbial communities such as acetylated mannooligosaccharides (AMOS). These findings highlight the potential of AMOS as a controlled prebiotic for achieving regional fermentation effects and sustained metabolic benefits in the colon. Attached Figure Description

[0010] Figure 1 SEM microstructure images of MOS and AMOS; Figure 2 The graphs show the changes in total sugar and reducing sugar in MOS and AMOS (A), WSI and ESI (B), OAC (C), and WAC (D). Figure 3 The particle size distribution (A), particle size diagram (B), zeta potential diagram (C), DTG diagram (D), TG diagram (E), and antioxidant property diagram (F, G, H) are for MOS and AMOS. Figure 4 XRD patterns (A) and FT-IR patterns (B) of MOS and AMOS; Figure 5 For MOS and AMOS 1 H NMR spectrum; Figure 6 For MOS and AMOS 13 C NMR spectrum; Figure 7The graph shows the changes in pH (A), acetic acid (B), propionic acid (C), butyric acid (D), and total SCFAs yield (E) of MOS and AMOS after in vitro fecal microbial fermentation. Figure 8 Figure 1 shows the microbial diversity analysis of MOS and AMOS fecal bacteria after 24 h of fermentation; Note: CK0 group and CK group are samples fermented for 0 h and 24 h without sample addition, respectively; (A) Shannon dilution curve, α diversity index; (B) Shannon; (C) Simpson; (D) Chao1, β diversity; (E) PCA; Figure 9 Venn diagram (A), phylum-level bacterial community composition (B), and relative abundance diagrams of the top 5 bacteria at the phylum level (C, D, E, F, G) after 24 h of MOS and AMOS fecal microbiota fermentation. Figure 10 The bacterial community composition at the genus level for MOS and AMOS fecal bacteria after 24 h of fermentation (A), and the relative abundance of the top 6 bacteria at the genus level (B, C, D, E, F, G). Figure 11 This is a heatmap showing the correlation between environmental factors and the top 20 genera of bacteria in the bacterial community. Note: Color intensity is proportional to the relative abundance of each genera. Correlation coefficients are represented by color, with red indicating a positive correlation and blue indicating a negative correlation. Figure 12 The plot shows the LDA scores between environmental factors and the top 20 genera of bacterial communities. Note: Enriched bacterial communities with LDA scores greater than 4 are shown in the histogram. Figure 13 This is a LEfSe taxonomic phylogenetic tree showing the relationship between environmental factors and the top 20 genera of bacterial communities. Note: "p_" indicates phylum; "o_" indicates class; "c_" indicates order; "f_" indicates family. Figure 14 The diagram shows a functional pathway heatmap (A) and a schematic diagram of the effects of acetylation on the physicochemical properties of MOS and gut microbiota (B). Detailed Implementation

[0011] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0012] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0013] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0014] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0015] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0016] All raw materials used in this invention are commercially available or prepared using existing technologies. Mannooligosaccharides (MOS, purity ≥95%) were purchased from Zhengsheng Biotechnology Co., Ltd. (Shandong, China). Acetic anhydride was obtained from Tianjin Jiangtian Chemical Technology Co., Ltd. Dextran standards (T-10, 10 kDa; T-40, 40 kDa; T-70, 70 kDa; T-500, 500 kDa) were provided by Shanghai Yuanye Biotechnology Co., Ltd. All other reagents were analytical grade.

[0017] This invention provides a method for preparing acetylated mannooligosaccharides, comprising the following steps: Mann oligosaccharides, acetic anhydride, and a catalyst are mixed and reacted to obtain acetylated mann oligosaccharides.

[0018] In this invention, the ratio of mannan oligosaccharide to acetic anhydride is 5g:80mL.

[0019] In this invention, the catalyst comprises p-toluenesulfonic acid.

[0020] In this invention, the mass ratio of mannan oligosaccharide to catalyst is 25:1.

[0021] In this invention, the reaction temperature is 60°C and the time is 1 to 4 hours, for example, 1 hour, 2 hours, 3 hours or 4 hours.

[0022] In a specific embodiment of the present invention, after the reaction is completed, the mixture is cooled, an equal volume of ethanol is added to remove unreacted acetic anhydride, and the mixture is allowed to stand overnight. Then, it is concentrated by rotary evaporation, followed by the addition of water to precipitate acetylated mannooligosaccharides, and then centrifuged, washed, and dried to obtain acetylated mannooligosaccharides.

[0023] It is understandable that modifying the structure of small molecule oligosaccharides presents significant technical challenges, especially in the subsequent separation process where direct separation from the aqueous phase is difficult. However, the modification preparation method employed in this invention is an innovative reverse-phase technique that allows the modified oligosaccharides to be directly separated from the aqueous phase using their balanced hydrophobicity, thereby greatly improving production efficiency and reducing production costs.

[0024] The present invention also provides acetylated mannooligosaccharides prepared by the above-described preparation method.

[0025] This invention also provides the application of the above-mentioned acetylated mannan oligosaccharides in the preparation of intestinal regulating drugs or functional foods.

[0026] This invention prepared MOS with different degrees of substitution, analyzed their physicochemical properties (e.g., solubility, molecular weight, structural features by NMR, etc.) and gut microbiota fermentation characteristics, revealed the relationship between structural modification and changes in physicochemical properties, and established an interaction model between carbohydrate substrates and gut microbiota fermentation kinetics. Importantly, based on this invention, understanding the structural regulation of MOS behavior in the gut ecosystem may highlight new methods for precise prebiotic design, thereby enabling targeted regulation of the gut microbiota to combat diseases associated with dysbiosis.

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1 5 g of mannooligosaccharide (MOS) and 80 mL of acetic anhydride were mixed in a three-necked flask, followed by the addition of 0.2 g of p-toluenesulfonic acid. The mixture was then heated at 60 °C for 1 h. After the reaction, the mixture was cooled, and an equal volume of ethanol was added to remove unreacted acetic anhydride. The mixture was then allowed to stand overnight. The solution was then concentrated to 20 mL by rotary evaporation, followed by the addition of water to precipitate AMOS. The precipitate was centrifuged at 4,000 × g for 10 min, repeatedly washed with water, and then freeze-dried. The final product was acetylated mannooligosaccharide, denoted as A1MOS, with a degree of substitution of 2.42 ± 0.03.

[0029] Example 2 The only difference from Example 1 is that the heating reaction time is 2 hours; the acetylated mannooligosaccharide prepared is denoted as A2MOS, with a degree of substitution of 2.71 ± 0.02.

[0030] Example 3 The only difference from Example 1 is that the heating reaction time was 4 hours; the acetylated mannooligosaccharide prepared was designated A4MOS with a degree of substitution of 2.85 ± 0.01.

[0031] Test Example 1: Morphological characteristics of MOS before and after acetylation After bonding MOS powder onto a disc and spraying it with gold, the microstructure of the sample was scanned using a scanning electron microscope (SEM, SU1501, Hitachi, Japan) under low vacuum and accelerating voltage (25 kV).

[0032] SEM images (Figure 1) show that acetylation has a significant impact on the structure of MOS. MOS exhibits a smooth, spherical shape and well-defined particles. With increasing acetyl substitution, the AMOS samples gradually collapse, evolving from the initial rough and partially fragmented (A1MOS) to highly porous and irregular aggregates (A2MOS, A4MOS). This may be due to the dissociation of hydrogen bonds or the increased hydrophobicity resulting from the introduction of more acetyl groups on the MOS molecule.

[0033] Test Example 2: Solubility of the sample in water (WSI) and ethanol (ESI), and oil absorption capacity (OAC). Mix 0.2 g of sample with 20 mL of water or ethanol, shake for 1 h, and then centrifuge for 15 min (4,000 × g). Dry the supernatant at 60 °C to constant weight. WSI and ESI are calculated as follows: (1) (2) In the formula, M0 is the mass of MOS (g), and M1 and M2 are the masses of the dried supernatants of water and ethanol (g), respectively.

[0034] Mix MOS with oil (1:10, w:v), let stand overnight, then remove free oil. OAC is calculated as follows: (3) In the formula, the sample mass before and after adding oil is recorded as w0 and w1 (g), respectively.

[0035] Test Example 3: Determination of Surface Hydrophobicity (WCA) The surface hydrophobicity of the MOS samples was determined using the solid drop method (SDC-200S contact angle meter, Kunshan Xike Instrument Technology Co., Ltd.). All samples were measured at least three times.

[0036] Test Example 4: Changes in total sugar and reducing sugar in mannooligosaccharides after acetylation The content of reducing sugar and total sugar in MOS was determined by DNS colorimetry and phenol-sulfuric acid method. As shown in Figure 2A, the total sugar and reducing sugar content in AMOS decreases with increasing degree of substitution. This is because acetylation disrupts the sugar chain structure, leading to MOS degradation, and the introduction of a large number of acetyl groups spatially hinders the enzymatic or chemical hydrolysis of glycosidic bonds, thereby reducing the formation of free reducing ends. The solubility of AMOS in water decreases significantly, while its solubility in ethanol increases with increasing degree of substitution. Figure 2 (B) This indicates that the introduced acetyl group enhances the hydrophobicity of MOS, thereby reducing its hydrophilicity, while increasing its compatibility with organic solvents, which is consistent with the trend of surface hydrophobicity results. Figure 2 (D). AMOS has a higher OAC value than MOS, but A4MOS has the lowest ( Figure 2 (C). On the one hand, while acetylation increases surface hydrophobicity, excessive acetylation may reduce the interaction between MOS and oil due to excessive structural disruption, leading to the observed oil uptake trend. On the other hand, the higher polarity of A4MOS may alter the attraction-repulsion interaction between polar / nonpolar lipids and A4MOS, resulting in a decrease in OAC.

[0037] Test Example 5: Changes in particle size distribution and zeta potential of MOS after acetylation The particle size distribution and zeta potential of the MOS sample (1 mg / mL) were measured using a laser particle size analyzer (Bettersize 2600, China) and a zeta potential analyzer (Zetasizer Advance, UK).

[0038] Particle size distribution ( Figure 3Figure A) shows that after acetylation, the peak shifts significantly to the right, and the particle size of AMOS gradually decreases with the increase in the degree of substitution. This may be because acetylation promotes particle aggregation to form larger and more irregular particles, and as the degree of substitution increases, the greater the damage to the MOS structure, thus reducing its particle size, which can be observed in SEM ( Figure 1 ).

[0039] Zeta potential analysis ( Figure 3 Figure C) further reveals the surface charge changes caused by acetylation. MOS has the largest negative Zeta potential (-19.73 ± 1.72 mV), indicating strong electrostatic repulsion between particles, which helps to disperse them in the solution. After acetylation, the Zeta potential increases with the increase in the degree of substitution: A1MOS < A2MOS < A4MOS. The gradual increase in the Zeta potential indicates that acetylation reduces the number of exposed hydroxyl groups, which usually generate negative charges through deprotonation. When acetyl groups replace hydroxyl groups, the surface charge repulsion weakens, facilitating particle aggregation. In A4MOS, the Zeta potential even becomes positive, which means that excessive acetylation may introduce new surface interactions or change the charge distribution.

[0040] Test Example 6 Analysis of average molecular weight (M W ) The MOS samples were dissolved in water to <1 mg / mL and then filtered through a 0.22 - micron filter. The molecular weight of MOS was determined by high - performance liquid chromatography (HPLC, Agilent, USA), using a TSK gel G400PWXL chromatographic column, detected with a differential refractive index detector, a flow rate of 0.6 mL / min, an injection volume of 20 μL, and a standard curve was established using t - series dextrans.

[0041] The molecular weight (Mw) results (Table 1) show that the molecular structure of MOS changes significantly after acetylation. MOS has only one peak, corresponding to a molecular weight of 4.629 kDa, indicating a relatively uniform molecular size distribution. However, after acetylation, higher - molecular - weight components appear in all AMOS samples, indicating the formation of new molecular populations. The molecular weight of Peak1 decreases from 82.362 kDa (A1MOS) to 70.630 kDa (A4MOS), indicating that the higher the acetylation level, the more branched the structure may be and the lower the molecular weight.

[0042] Table 1 Molecular weights of MOS and AMOS

[0043] Test Example 7 Thermal property analysis of MOS before and after acetylation Weigh 5 mg of the sample in a crucible and then heat it from 30 °C to 600 °C at a rate of 10 °C / min using a thermogravimetric analyzer (TA Instrument, Q50, USA).

[0044] DTG and TG curves reveal the thermal stability of MOS and AMOS. The results of the DTG curve ( Figure 3 The results (D) show that A1MOS and A2MOS have higher thermal stability than MOS. However, with increasing substitution degree (from A1MOS to A4MOS), the maximum degradation rate temperature shifts to lower temperatures, indicating that overacetylation reduces the thermal stability of MOS. Moderate acetylation (A1MOS, A2MOS) is thought to enhance structural stability by introducing new crystalline regions. Figure 4 (A). In contrast, the decreased thermal stability observed in A4MOS is likely attributed to excessive disruption of the hydrogen bond network and sugar backbone caused by high-level acetylation, resulting in a structure with poorer thermal stability. TG curves indicate that the residual mass of AMOS is smaller than that of MOS ( Figure 3 (E). Acetylation can reduce ash mass to some extent, possibly because the acetyl group is an organic compound. The introduction of the acetyl group reduces the proportion of inorganic components, ultimately decreasing the residual ash mass.

[0045] Test Example 8: Antioxidant Activity Analysis of MOS Before and After Acetylation The antioxidant activity of MOS was represented by three methods: DPPH radical scavenging activity, ABTS radical scavenging activity, and FRAP.

[0046] The antioxidant capacity of MOS and AMOS was evaluated by measuring DPPH, ABTS, and FRAP, and the results reflected different free radical scavenging mechanisms. The free radical scavenging capacity of DPPH increased with increasing degree of substitution. Figure 3 The presence of acetyl groups (F) indicates that the introduction of acetyl groups enhances electron transfer capability or radical stability through electron delocalization along the carbohydrate backbone. The enhanced DPPH activity may also be due to increased hydrophobicity of the AMOS surface, which improves the accessibility of DPPH radicals in the organic phase. Conversely, both ABTS and FRAP values ​​decrease with increasing degree of substitution (F). Figure 3 The presence of GH (high-to-medium hydrogen) indicates a decrease in overall electron-donating ability. This reduction is likely due to the substitution of hydroxyl groups with acetylation, leading to a decrease in the number of hydrogen atoms available for radical quenching and metal ion reduction. Furthermore, the decrease in polarity and conformational changes caused by acetylation may limit the diffusion of hydrophilic groups and reduce metal-chelate interactions. In summary, these findings suggest that acetylation alters the antioxidant mechanism of MOS through a shift from hydrogen atom transfer dominance to single-electron transfer dominance, highlighting that the antioxidant behavior of AMOS is structurally regulated by and dependent on the degree of substitution.

[0047] Test Example 9: Crystallographic patterns of MOS before and after acetylation obtained by X-ray diffraction (XRD) Using an X-ray diffractometer (DMAX2500, Japan) at 10 min -1 The XRD spectra of the MOS samples were recorded using a scanning rate and a scanning range of 5-50°.

[0048] To investigate the crystallization pattern of acetylated MOS, XRD analysis was performed on the samples. Figure 4 As shown in Figure A, the MOS sample exhibits a broad diffraction peak at 2θ = 18°, indicating its amorphous nature. However, the X-ray diffraction of AMOS shifts to the right from 2θ = 18° to 2θ = 21°, revealing an amorphous structure with broad peaks, indicating that most of the hydroxyl groups in the MOS molecule are replaced by acetyl groups. Furthermore, AMOS displays a new diffraction peak at 2θ = 9°, indicating its V-shaped crystallinity. The introduction of acyl groups severely disrupts intramolecular and intermolecular hydrogen bonds, subsequently leading to the rearrangement of the AMOS molecule into a new ordered structure.

[0049] Test Example 10: Fourier Transform Infrared Spectroscopy (FTIR) The sample was ground into powder with KBr at a ratio of 1:150, compressed into tablets, and then analyzed using a Nicolet IS50 spectrometer (Thermo Nicolet, USA) at 400–4000 cm⁻¹. -1 The spectrum was obtained by scanning 16 times within the range.

[0050] Changes in the FTIR spectra provide direct evidence of functional group modification, confirming the introduction of acetyl groups and their impact on molecular structure. The spectra of MOS and its acetylated derivatives (AMOS) are shown below. Figure 4 As shown in Figure B. After acetylation, AMOS at 1740 cm⁻¹ -1 A strong absorption peak appeared at the C=O stretching vibration of the ester bond, confirming the successful introduction of the acetyl group. Furthermore, the peak intensity increased with increasing substitution degree, indicating a gradual increase in the degree of acetylation substitution. Compared to MOS, the peak intensity was higher in the 3200-3600 cm⁻¹ range. -1 The broad absorption band between these two groups (corresponding to OH stretching vibrations) exhibits a significantly reduced intensity in the AMOS sample and is negatively correlated with the degree of substitution. This indicates that the hydroxyl group is substituted with an acetyl group, reducing hydrogen bond interactions. The absorption band at 1000–1300 cm⁻¹ is significantly lower than that at 1000–1300 cm⁻¹. -1 The peaks within the range (corresponding to CO stretching vibrations) show significant changes and increased intensity in AMOS, further supporting the modification of glycosidic bonds through acetylation.

[0051] Test Example 11: Nuclear Magnetic Resonance (NMR) The sample was dissolved in DMSO (100 mg / mL). NMR spectroscopy was performed using a Bruker AVANCE III 600 NMR spectrometer (Germany) via 1D (1H and 1H) chromatography. 13 C) NMR spectroscopy analysis of MOS and AMOS.

[0052] 1 H and 13 Analysis of C10 NMR spectroscopy confirmed the structural modification of MOS after acetylation. 1 In the H NMR spectrum, the characteristic proton signal of MOS was mainly observed in the 3.0-5.5 ppm region. Figure 5 The α-hydroxyl group (-CH3) corresponds to the hydrogen atom in the sugar ring. After acetylation, additional peaks appear at approximately 2.0–2.3 ppm, which belong to the methyl proton (-CH3) of the acetyl group, indicating the successful introduction of the acetyl functional group. The signal intensity in this region increases with increasing acetylation degree, indicating a gradual modification of the MOS. 13 C10 NMR spectroscopy further confirmed these structural changes. Figure 6 MOS spectra show that the carbohydrate backbone signal is between 60-110 ppm, with anterior carbon peaks around 90-110 ppm. After acetylation, a new peak appears in the 170-175 ppm range, corresponding to the carbonyl group (C=O) of the acetyl moiety. Furthermore, the enhanced signal in the 2-30 ppm region represents the methyl group (-CH3) of the acetyl substituent. These changes in the two NMR spectra clearly demonstrate that acetylation alters the chemical environment of the MOS, successfully modifying its structural and functional properties.

[0053] Test Example 12: In vitro gut microbiota fermentation Written informed consent was obtained from all participants before recruitment. Fecal samples were collected from 7 healthy volunteers (3 men and 4 women, aged 22–28 years). Equal volumes of fecal samples from the 7 volunteers were first pooled together, and then the pooled samples were prepared into a slurry using PBS (0.1 M, pH 7.2) and culture medium. The culture medium consisted of: peptone (2 g / L), yeast extract (2 g / L), NaCl (0.1 g / L), K₂HPO₄ (0.04 g / L), KH₂PO₄ (0.04 g / L), NaHCO₃ (2 g / L), MgSO₄·7H₂O (0.01 g / L), CaCl₂·6H₂O (0.01 g / L), Tween 80 (2 mL / L), and heme chloride (0.05 g / L). Centrifuge tubes containing MOS and AMOS (10 mg / mL) were then incubated in an anaerobic incubator at 37°C for 24 hours, with untreated fecal liquid serving as a control. Samples were collected at 0 h, 6 h, 12 h, and 24 h and stored frozen at -80°C. For microbial community analysis, samples from the control groups at 0 h fermentation (CK0) and 24 h fermentation (CK) were used as control samples.

[0054] Statistical analysis Graphs were plotted using Origin 2018 64-bit software, and sample structure was analyzed using MestReNova software. Significance between samples was analyzed using IBM SPSS Statistics 26. p <0.05).

[0055] (1) pH measurement The pH of the sample was recorded at each time point using a pH meter (METTLER TOLEDO, Switzerland).

[0056] The pH value of the fermentation solution was used as an important indicator for evaluation during fermentation, and the results were as follows: Figure 7 As shown in Figure A, during fermentation, the pH value of the CK group (i.e., the control sample without added samples) was significantly higher than that of the other groups, while the pH value of MOS remained the lowest and decreased the fastest at 6 h, reflecting that MOS has a higher fermentation capacity and SCFA yield. After acetylation, the pH value of AMOS decreased significantly more slowly. The main reason for this trend is that the presence of acetyl groups restricts enzyme hydrolysis and slows down substrate degradation. Interestingly, the higher the degree of substitution, the lower the pH value, which seems to contradict the claim that acetyl groups can prevent enzyme hydrolysis. On the one hand, AMOS with a high degree of substitution has a lower molecular weight (Table 1) and is more easily utilized by gut microbiota. On the other hand, the introduced acetyl groups can be released during fermentation, which promotes the pH reduction of AMOS with a high degree of substitution.

[0057] (2) Determination of short-chain fatty acids after fermentation 20 µL of H₂SO₄ was added to 0.5 mL of the supernatant and vortexed for 30 s, followed by the addition of 0.8 mL of diethyl ether and vortexing again for 30 s. SCFAs were determined by gas chromatography (GC-2014, SHIMADZU) using a capillary column (Nukol™ 30 mm × 0.53 mm × 0.5 µm, SUPELCO, USA). The column temperature was increased from room temperature to 100 °C in split mode (split ratio 10:1) over 3 minutes; the vaporization chamber temperature was 230 °C; the flame ionization detector temperature was set to 275 °C; and nitrogen was used as the carrier gas (1 µL).

[0058] As shown in Figure 7B, it was observed that MOS produced the highest amount of acetic acid, while AMOS produced significantly less acetic acid compared to MOS. p <0.05%, but still higher than the CK group. This is because the steric hindrance and hydrophobicity of the acetyl groups in AMOS limit the accessibility of microbial enzymes (such as glycoside hydrolases) and slow down fermentation kinetics. In the AMOS group, the acetate content increases with increasing degree of substitution, which may be due to the conversion of acetyl groups to acetate and the decrease in molecular weight (Mw: A1MOS>A2MOS>A4MOS>MOS), consistent with the trend of pH. Figure 7 (A). It is worth noting that the content trends of propionic acid, butyric acid, and short-chain fatty acids are highly similar to those of acetic acid. Figure 7 The results (CE) indicate that the molecular weight of MOS samples is also an important factor affecting their fermentation properties. It has been reported that approximately 24% of acetic acid is converted to butyric acid and 3% to propionic acid. The relationship between the degree of substitution and SCFAs generation highlights the importance of structure customization in prebiotic design. Although MOS maximizes SCFAs yield, acetylated MOS (e.g., A4MOS) provides targeted delivery of sustained colonic benefits.

[0059] (3) Microbial community analysis Microbial DNA was extracted using the HiPure fecal DNA kit. Ribosomal RNA 16S rDNA target regions were then amplified by PCR. Bacterial 16S rDNA V3-V4 sequencing was performed by Novogene Corporation of Beijing on a NovaSeq sequencing system. Raw sequencing data were processed using the QIIME2 (version 2023.2) workflow. Quality filtering, noise reduction, chimera removal, and amplicon sequence variant (ASV) inference were all performed using the DADA2 plugin. Taxonomic classification was based on the SILVA138.1 reference database, using a 97% similarity threshold. All sequences were clustered into OTUs (operational taxonomic units), and species annotation and abundance analysis were performed. All samples were measured at least three times.

[0060] ① The effect of acetylation on microbial community diversity Shannon dilution curve ( Figure 8 As shown in Figure A, the curves for all samples approached saturation with increasing sequencing volume, ensuring data reliability. Overall, the Shannon and Simpson indices were primarily influenced by community diversity, while the Chao1 index was mainly affected by species abundance. The Shannon index (…) Figure 8 (B) indicates significant differences in microbial diversity among different groups, with the MOS group exhibiting the lowest microbial diversity, and the diversity of the AMOS group decreasing with increasing substitution degree. These results suggest that acetylation modification enhances the diversity of the MOS microbial community, while low-acylation AMOS (A1MOS) promotes a more balanced microbial community. Simpson index ( Figure 8 The results from the C group further validated the above findings. The Chao1 index (C) of the MOS group... Figure 8 The Chao1 index was lowest in the AMOS group (D), and gradually decreased with increasing substitution degree. This indicates that acetylation enhances microbial diversity, but excessive acetylation (A4MOS) slightly reduces species abundance. The diversity results suggest that MOS fermentation favors the dominant growth of specific microbial populations, while AMOS promotes a more heterogeneous microbial ecosystem, possibly due to structural modifications altering carbohydrate availability.

[0061] PCA chart ( Figure 8 Figure E shows the clustering of microbial communities among different treatment groups, with PC1 and PC2 variables accounting for 14.53% and 10.51% of the data differences, respectively. It can be observed that MOS is clearly separated from CK and CK0, indicating a significant change in the microbial composition of MOS. Although the AMOS group clusters closely together, they are also clearly separated, reflecting that different degrees of substitution in AMOS fermentation also lead to differences in gut microbiota composition.

[0062] ② Effects on gut microbiota composition at the phylum level The Venn diagram shows a total of 240 OTUs across all groups. Figure 9 The number of unique OTUs in the AMOS and AMOS groups were 275 (CK0), 188 (CK), 134 (MOS), 195 (A1MOS), 179 (A2MOS), and 118 (A4MOS), respectively. After 24 h of fermentation, the number of OTUs decreased in all groups. Among the MOS and AMOS groups, only the A1MOS group showed a slight increase in the number of OTUs compared to CK. The number of OTUs in AMOS decreased with increasing substitution degree, indicating that AMOS with a high substitution degree reduced the number of unique OTUs.

[0063] To elucidate the broad ecological effects of acetylation on the gut microbiota, we first analyzed the microbial community composition at the phylum level. Figure 9 (B) The dominant phylum in all AMOS groups was Firmicutes (Bacteria). Firmicutes Bacteroidetes ( Bacteroidota Proteobacteria ( Proteobacteria ) and Actinobacteria ( Actinobacteriota Firmicutes, comprising over 95% of the total microbial community, exhibited abundance variations strongly dependent on substitution degree, indicating that acetylation structurally regulates the interaction between AMOS and microorganisms. Firmicutes play a crucial role in the degradation of dietary polysaccharides and butyrate production (key metabolites for colonic cell energy and anti-inflammatory signaling). Their abundance in AMOS gradually increased with increasing substitution degree. Figure 9 (C), which may explain the elevated butyrate levels in the AMOS group, reflecting that acetylation promotes the proliferation of butyrate-producing strains within Firmicutes. Conversely, Bacteroidetes, which specialize in hydrolyzing complex carbohydrates via enzymatic sites on polysaccharides, showed increased abundance after MOS fermentation, but this was partially inhibited under high substitution conditions. Figure 9 (D). This likely reflects a shift in substrate availability with degree of substitution—high acetylation hinders glycosidic bond cleavage. Proteobacteria, which include various opportunistic pathogens, were significantly reduced in both the MOS and AMOS groups. Figure 9 The presence of E indicates that pathogenic bacteria were suppressed. After 24 hours of fermentation with MOS, the abundance of Actinobacteria significantly increased, while the addition of AMOS had no significant effect on the abundance of Actinobacteria. Figure 9 (F). Meanwhile, Desulfobacteria ( Desulfobacterota ) is a potentially harmful bacterium associated with intestinal inflammation. All experimental groups showed a reduction in the abundance of the phylum Dethiobacterium ( Figure 9Among the G groups, MOS contributed the most to reducing the abundance of Dethiobacterium. Overall, these findings suggest that acetylation not only alters the fermentation kinetics of MOS but also improves microbial selectivity, favoring the growth of butyrate-producing and anti-inflammatory bacteria, thereby enhancing gut ecological resilience in a substitution degree-dependent manner.

[0064] ③ The influence of gut microbiota composition at the genus level This study further explored the effects of MOS and AMOS on gut microbiota composition during in vitro fermentation at the genus level. Figure 10 (A). Compared with the CK group, MOS treatment reduced the number of Bifidobacterium spp. (A). Bifidobacterium Abundance increased nearly tenfold. Figure 10 (Medium B), which may stem from its ability to efficiently convert oligosaccharides into acetic acid and lactic acid, thereby promoting the proliferation of beneficial microorganisms. We found that AMOS has a significant advantage over MOS in promoting the growth of other beneficial bacteria. Specifically, Bacteroides spp., which have the ability to degrade complex polysaccharides and produce short-chain fatty acids ( Bacteroides The abundance of these MOS groups was significantly higher in the A1MOS and A2MOS groups than in the MOS group. p <0.05)( Figure 10 The C value indicates that moderate acetylation enhances the substrate availability of Bacteroides. *Myxobacterium* genus (known for producing short-chain fatty acids and maintaining the integrity of colonic mucus) Blautia In all AMOS groups, the abundance was higher than that in the MOS group. Figure 10 The presence of D in the middle layer suggests an enhanced supportive effect on intestinal barrier function. Similarly, *Parabacteroides* species (which participate in metabolic regulation through the metabolism of short-chain fatty acids and bile acids) Parabacteroides ), showing a significant substitution-dependent growth trend, with the A4MOS group reaching the highest abundance ( Figure 10 The *Dorea* genus (E) exhibits strong selectivity for highly acetylated substrates. Notably, potential butyrate producers... Dorea The abundance of was significantly increased in A1MOS, and remained higher in the A2MOS and A4MOS groups than in the MOS group. p <0.05)( Figure 10 The presence of F indicates that a lower degree of substitution is more conducive to its proliferation. Meanwhile, *Rhodotorula*, a key butyrate-producing bacterium crucial for anti-inflammatory effects and epithelial health, is also involved. Roseburia The AMOS treatment group also showed higher abundance than the MOS group, especially under high substitution conditions. Figure 10 The presence of butyrate (G) suggests that the immunomodulatory potential mediated by butyrate is enhanced. These results collectively confirm the importance of structural modification in regulating prebiotic activity. Acetylation enhances the prebiotic function of MOS in a substitution-dependent manner, providing an important basis for developing targeted prebiotic intervention strategies.

[0065] Linear discriminant analysis (LDA) combined with LEfSe was used to screen for bacterial communities with significant differences in each experimental group. Figure 12-13 LDA results showed that Faecalibacterium ( ) Faecalibacterium The bacteria were significantly enriched in the CK0 group (LDA>4), while the CK group was rich in Escherichia coli-Shigella spp. Escherichia-Shigella ), Sartella spp. Sutterella ) and bilirubin Bilophila () Figure 12 ). In the MOS group, Megamonas spp. ( Megamonas ) and Bifidobacterium spp. Bifidobacterium The main bacterial groups were genus *Parasartella*. After acylation treatment, the main bacterial groups in the AMOS group were *Parasartella* (…). Parasutterella (A1MOS group), Bacteroides ( Bacteroides ) and Prevotella spp. Prevotella (A2MOS group) and Parabacterium genus ( Parabacteroides (A4MOS group). Overall, these results indicate that acetylation treatment alters the key distribution of MOS in the gut microbiota and promotes the proliferation of specific bacteria.

[0066] ④ Correlation analysis between microbial communities and environmental factors The effects of different environmental factors (short-chain fatty acids and pH) on the gut bacterial community after fermentation were analyzed using correlation heatmaps. Results ( Figure 11 The results showed that Megamonas spp. ( Megamonas ), genus *Gastromycium* Megasphaera ), Parabacteroides ( Parabacteroides ) and Prevotella spp. Prevotella The levels of these bacteria were significantly positively correlated with major short-chain fatty acids (acetic acid, propionic acid, and butyric acid). These three bacteria may play important roles in short-chain fatty acid metabolism and maintaining intestinal homeostasis, and possess potential probiotic functions. Meanwhile, *Prevotella 9* (…) Prevotella_9 Bifidobacteria showed a positive correlation with acetic acid and butyric acid, while propionic acid was also positively correlated with Bifidobacteria. Prevotella 9 is a typical beneficial bacterium, possessing polysaccharide degradation genes or enzymes, and plays an important role in improving glucose metabolism and controlling blood sugar levels. Bifidobacteria (… Bifidobacterium Escherichia coli can produce acetic acid and lactic acid. The lactic acid produced is easily converted into propionic acid by other bacteria, which may explain the elevated propionic acid concentration. Conversely, *Shigella* spp. (…) Escherichia-Shigella ), Faecalibacterium genus ( Faecalibacterium ) and rare Micrococcus genus ( SubdoligranulumShort-chain fatty acid (SCFA) production showed a negative correlation with short-chain fatty acids and a positive correlation with pH. These bacteria may influence the intestinal environment by competing for nutrients or producing alkaline metabolites, potentially leading to gut microbiota imbalance. Furthermore, we noted a negative correlation between SCFA and pH, as SCFA lowers intestinal pH, and a low pH helps create an acidic intestinal environment, thereby preventing pathogen proliferation and maintaining gut microbial homeostasis. In conclusion, SCFA production is closely related to bacterial community composition. Figure 14 Figure B illustrates the mechanism by which acetylation affects the physicochemical properties of MOS and the gut microbiota.

[0067] ⑤ Functional prediction analysis of MOS and AMOS after fermentation Functional prediction data showed significant differences among the treatment groups in multiple metabolic pathways and biological functions. Figure 14 (A) The CK0 and CK groups showed relatively weak performance in various metabolic pathways, including nucleotide metabolism, translation, replication, and repair. The MOS group not only improved these functions but also showed positive correlations with energy metabolism and the immune system, while lipid metabolism and carbohydrate metabolism may have been reduced. In contrast, the AMOS group compensated for the deficiencies of MOS in biological metabolism and regulatory pathways, exhibiting a more balanced distribution and a high correlation with substitution degree. Furthermore, these groups also showed outstanding performance in metabolism, amino acid metabolism, cellular processes, and signal transduction, indicating that AMOS treatment promoted metabolic homeostasis and optimized biological function. In conclusion, MOS may have a significant impact on intestinal metabolism, while AMOS treatment plays a positive role in maintaining metabolic homeostasis, enhancing adaptability, and optimizing functional levels.

[0068] In summary, this invention elucidates how acetylation reshapes the chemical structure, physicochemical properties, and fermentation characteristics of mannooligosaccharides (MOS), thereby modulating their prebiotic potential. Acetylation reduces the water solubility of MOS, increases its molecular weight, and alters its surface hydrophobicity, leading to different microbial responses during in vitro fermentation. Structure-driven fermentation kinetics show that higher acetylation substitution delays the release of short-chain fatty acids (SCFAs) but promotes the release of Bacteroides (S. spp.). Bacteroides ), via Myxobacterium spp. ( Blautia ), Parabacteroides ( Parabacteroides ), Dorea strain ( Dorea ) and Rochetomyces ( Roseburia Enrichment of microbial communities such as [list of microorganisms]. These findings highlight the potential of acetylated mannooligosaccharides (AMOS) as a controlled prebiotic for achieving regional fermentation effects and sustained metabolic benefits in the colon. In vivo validation of AMOS function and its interactions with host metabolism could lead to the development of precision prebiotic formulations targeting specific microbiome regulation.

[0069] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A process for the preparation of acetylated manno-oligosaccharides, characterized in that, The method comprises the following steps: The acetylated mannan oligosaccharide is prepared by mixing mannan oligosaccharide, acetic anhydride and a catalyst and then reacting.

2. The production method according to claim 1, characterized by, The mass ratio of the mannan oligosaccharide to the acetic anhydride is 5g:80mL.

3. The production method according to claim 1, characterized by, The catalyst comprises p-toluenesulfonic acid.

4. The preparation method according to claim 1, characterized in that, The mass ratio of the mannan oligosaccharide to the catalyst is 25:

1.

5. The preparation method according to claim 1, characterized in that, The reaction is carried out at a temperature of 60℃ for 1-4 hours.

6. The acetylated mannan oligosaccharide prepared by the method of any one of claims 1-5.

7. The acetylated mannan oligosaccharide of claim 6 for use in the preparation of a drug for regulating the intestinal tract or a functional food.

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