Evaluation of the Enzymatic Production and Prebiotic Activity of Galactomannan Oligosaccharides Derived from Gleditsia microphylla

: Oligosaccharides have received considerable attention as prebiotics because they exhibit potential health beneﬁts related to their ability to modulate intestinal bacterial composition. This study evaluated the effects of galactomannan oligosaccharides (GMOS) derived from Gleditsia microphylla as a prebiotic on human intestinal bacteria. The β -mannanase used for the enzymatic hydrolysis of GMOS was produced by Trichoderma reesei Rut C-30. The enzymatic hydrolysis of GMOS was found to occur under optimal conditions at 50 ◦ C, pH 5, 20 U/g-GM, and 20 g/L, and resulted in a yield of 70.78% ± 1.34%. The purity of GMOS after puriﬁcation was 81.50%. Upon performing in vitro human fecal fermentation using GMOS as a carbon source, it was observed that GMOS effectively promoted the proliferation of intestinal bacteria, and the utilization efﬁciency of GMOS by intestinal bacteria was found to be at 98.40%. In addition, GMOS were found to have a stabilizing effect on intestinal pH. Additionally, 16S rRNA sequencing of GMOS revealed that GMOS signiﬁcantly affected the diversity of gut microbiota. Speciﬁcally, GMOS exhibited a signiﬁcant inhibitory effect on Fusobacteria at the phyla and genus level, and demonstrated a signiﬁcant inhibitory effect on Fusobacterium . Moreover, the results for the prediction of metabolic function analysis showed that GMOS had a signiﬁcant effect on the level two metabolism of carbohydrates, cofactors, and vitamins. Furthermore, during level three metabolism, the lipoic acid metabolism was signiﬁcantly affected by GMOS. These results provide a theoretical basis for the potential use of galactomannan oligosaccharides from Gleditsia microphylla as prebiotics for regulating human intestinal bacteria.


Introduction
The continual progress in technology and the ever-increasing drive towards enhanced productivity and contemporary dietary patterns have resulted in the prioritization of green and healthy foods that provide optimal nutrient composition. Consequently, food items rich in prebiotics, such as vegetables and fruits, have gained significant consumer attention. Gleditsia microphylla, a leguminous plant distributed widely throughout several provinces in China, contains galactomannan (GM) in its seed endosperm [1]. GM is a high-polymer polysaccharide with a mannose backbone and several branched galactose chains. Although the prebiotic activity associated with GM derived from other plants has been well documented [2,3], only a limited number of studies have explored the prebiotic activity of GM derived from Gleditsia microphylla.

Preparation of Gleditsia microphylla Galactomannans
In a 2 L beaker, 100 g of Gleditsia microphylla seed endosperms were immersed in 1 L of hot water (1:100, w/v) for 12 h. Subsequently, the mixture of endosperms with 1 L of water was centrifuged at 10,000 rpm for 5 min, and the supernatant obtained was subjected to ethanol precipitation using 65% ethanol. The sediment was preserved and washed three times before freeze drying.

Component Analysis of Gleditsia microphylla Galactomannans
Component analysis was performed as described by Tao et al. [17]. Galactomannan content (S GM content) was calculated as follows: S GM content (%) = C gal +C man ) × 0.9 × 0.087 L original dry sample(g) × 100% (1) C gal : Concentration of galactose. C man : Concentration of mannose.

Quantification of Gleditsia microphylla Galactomannan Oligosaccharides
According to the method described by Tao et al., anhydrous ethanol was added into the obtained enzymatic supernatant (50 • C, pH 5, 20 g/L substrate concentration, 20 U/g enzyme loading) until the ethanol concentration reached 65 wt.%. After centrifugation, the ethanol in the supernatant was evaporated. The supernatant was freeze dried and the molecular weight was determined.

Preparation of Gleditsia microphylla Galactomannan Oligosaccharides
During the enzymatic process, pretreated Gleditsia microphylla seeds were employed as a substrate with β-mannanase. At the end of the enzymatic process, the enzymatic solution in the conical flask was placed in boiling water for 7 min to inactivate the enzyme, and the suspension was centrifuged at 8000 rpm for 10 min to obtain the supernatant. Then, GMOS were obtained in the supernatant according to the method described in Section 2.4. After optimizing the substrate concentration, enzyme loading, reaction temperature, and pH in a 50 mL enzymatic hydrolysis system, the batch production process was performed. The equation for determining the yield of GMOS was as follows: Yield GM : yield of galactomannan. C Sgal : supernatant galactose concentration. C Sman : supernatant mannose concentration. C Sgal : substrate galactose concentration. C Sman : substrate galactose concentration.
2.6. Purification of Gleditsia microphylla Galactomannan Oligosaccharides 2.6.1. Removal of Pigment Pigment adsorption was performed using XAD16N resin (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), which was filled into a column and connected to a peristaltic pump. The supernatant was pumped into the resin column at a rate of 20 rpm, and the filtrate was collected. The absorbance of the filtrate was measured at 420 nm and compared to that of the unfiltered solution to determine the completion of pigment adsorption.

Monosaccharide Removal
Dialysis was conducted using a 200 Da dialysis bag (Hunan Yibo Biotechnology Co., Hunan, China) through a process that lasted for two days, with water changed three times per day, after which the fluid in the bag was collected. The fluid was filtered with a 0.22 µm nylon Acrodisc syringe filter, after which 20 µL was injected into a Dionex ICS-5000 system (Thermo Fisher, Waltham, MA, USA); the conditions used in the system were the same as those in Section 2.3. A galactose and mannose concentration of 0 indicated the complete removal of monosaccharides.
Using a 200 Da dialysis bag, the dialysis process was conducted for two days in doubledistilled water, which was changed three times per day, after which the fluid in the bag was collected. The fluid was filtered through a 0.22 µm nylon Acrodisc syringe filter, and then 20 µL of fluid was injected into a Dionex ICS-5000 system; the system conditions were the same as those described in Section 2.3. The completion of monosaccharide removal was performed to ensure the absence of galactose and mannose.

Collection of Human Fecal Matter and In Vitro Batch Fermentation of GMOS
Fecal samples were donated by three healthy volunteers (two males and one female, aged 22-27 years) who had not taken any antibiotic products in the past 3 months and had no intestinal diseases. The mixture of the freshly collected fecal samples and 0.1 mol/L PBS buffer (1:9, 30 mL total) was centrifuged at a low speed (500 rpm, 5 min) and the fecal suspension was extracted for the next step of the experiment. In vitro fermentation was carried out in a Dugbox anaerobic workstation (Ruskin Life Sciences, UK) at 37 • C for 0, 6, 12, 24, and 48 h. The original group was treated for 0 h, and all experiments were independently performed 5 times.

OD 600 of Intestinal Bacteria
Homogeneous fermentation broth (1 mL), fermented for 48 h, was placed in a 2 mL centrifuge tube for centrifugation (2000 rpm, 5 min), and the supernatant was removed. The precipitate was rinsed with physiological saline and centrifuged under the same conditions. This process was repeated thrice, and precipitates were collected. Finally, the precipitate was mixed with 1 mL of physiological saline and placed in a UV spectrophotometer (Shanghai Precision Scientific Instruments Co., Shanghai, China) for analysis at 600 nm.

Analysis of Short-Chain Fatty Acids
The supernatant, comprising 1 mL of centrifuged intestinal bacterial suspension (2000 rpm, 5 min), was filtered through a 0.25 µm membrane for the measurement of acetic acid, propionic acid, butyric acid, and lactic acid levels via HPLC 1260 (Agilent, Santa Clara, CA, USA). The measurement conditions for short-chain fatty acids were as follows: Bio-Rad Aminex HPX-87H (7.8 mm × 300 mm); column temperature: 55 • C; mobile phase: 5 mmol/L H 2 SO 4 ; flow rate: 0.6 mL/min; injection volume: 10 µL; detector: refractive index detector. Gut microbial DNA was extracted using a HiPure Soil DNA Kit (Magen, Guangzhou, China) according to the manufacturer's protocol. With the primers 341F: CCTACGGGNG-GCWGCAG and 806R: GGACTACHVGGGTATCTAAT, the 16S rDNA V3-V4 region of the ribosomal RNA gene was amplified by PCR (conditions of PCR: 94 • C for 2 min, followed by 30 cycles at 98 • C for 10 s, 62 • C for 30 s, and 68 • C for 30 s, and a final extension at 68 • C for 5 min). According to the manufacturer's instructions, the amplicons extracted from 2% agarose gels were purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using the ABI StepOnePlus Real-Time PCR System (Life Technologies, Foster City, CA, USA). Purified amplicons were pooled at equimolar concentrations and paired-end sequenced (2 × 250) on an Illumina platform according to standard protocols. Raw data from the Illumina platform for ineligible reads were filtered using FASTP software (version 0.18.0). Stitching was performed using FLASH (version 1.2.11), and ineligible sequences were filtered using QIIME (version 1.9.1). Finally, the sequences were clustered into OTUs (operational taxonomic units) by ≥97% similarity using UPARSE (version 9.2.64). The sequence with the highest abundance was selected as the representative sequence for each OTU. Intergroup-specific OTUs were analyzed using the R language VennDiagram package (version 1.6.16) and the UpSetR package (version 1.3.3) for Venn and upset graph analyses, respectively. Non-metric multi-dimensional scaling (NMDS) of (Un)-weighted UniFrac, Jaccard, and Bray-Curtis distances was generated in the R project Vegan package (version 2.5.3) and plotted in the R project ggplot2 package (version 2.2.1). To predict these functions, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the OTUs was performed using PICRUSt (version 2.1.4).

Utilization of GMOS by Intestinal Bacteria
The anaerobic fermentation solution (1 mL) was centrifuged (2000 rpm, 5 min), and an equal volume of 8% H 2 SO 4 was added to the supernatant. The acid digestion process was carried out in a sterilizer at 121 • C for 1 h. When the reaction was complete, the digested solution was moved out and cooled to room temperature. Then, 40 µL was added to 1 mL of the digested solution. The digested solution was analyzed using the Dionex ICS-5000 system under the same conditions as described in Section 2.3. The following equations were used: C OS : original sugar concentration. C FS : final sugar concentration.

Statistical Analysis
SPSS (2008) statistical software (Ver. 16.0 for Windows, SPSS Inc., Chicago, IL, USA) was used to analyze the data by ANOVA. Tukey's multiple range test was used to separate differences in means between treatment groups. p values below 0.05 were considered statistically significant.

Optimization of Enzymatic Hydrolysis Conditions for GMOS Production
To optimize the enzymatic hydrolysis of GMOS, Gleditsia microphylla seeds with a GM content of 28.9% were used as the substrate during the enzymatic hydrolysis of GMOS. Seeds were chosen instead of GM to avoid the extraction of GM and to maintain the low viscosity of the enzymatic system. The optimization of the enzymatic hydrolysis conditions was performed during the investigation of temperature, pH, enzyme loading, and substrate loading.
To investigate the effect of reaction temperature on the GMOS yield obtained from Gleditsia microphylla as well as the monosaccharide yield and enzyme selectivity toward the substrate, enzymatic hydrolysis reactions were conducted at a pH of 5 with an enzyme  Figure 1a. The results reveal that the temperature used during enzymatic hydrolysis had a significant impact on the GMOS yield. Specifically, the GMOS yield increased from 22.9% to 50.7% as the hydrolysis temperature increased from 30 • C to 50 • C, but then it decreased to 46.5% when the temperature was raised to 80 • C. Meanwhile, the enzyme selectivity decreased from 90.1% at 30 • C to 81.6% at 50 • C, and then increased to 93.2% at 70 • C, and finally dropped to 92.4%. Moreover, the highest monosaccharide yield of 11.4% was achieved at 50 • C. nificant impact on the GMOS yield. Specifically, the GMOS yield increased from 22.9 50.7% as the hydrolysis temperature increased from 30 °C to 50 °C, but then it decre to 46.5% when the temperature was raised to 80 °C. Meanwhile, the enzyme selec decreased from 90.1% at 30 °C to 81.6% at 50 °C, and then increased to 93.2% at 70 °C finally dropped to 92.4%. Moreover, the highest monosaccharide yield of 11.4% achieved at 50 °C.
To further investigate enzymatic hydrolysis optimization, an enzyme loading U/g-GM and substrate loading of 20 g/L were used with a temperature of 50 °C. The of pH on the GMOS yield was evaluated, and the results are shown in Figure 1b. A pH increased from 3 to 8, the GMOS yield increased from 39.5% (pH 3.0) to 51.4% (pH and then decreased to 42.6% (pH 8.0). Additionally, the enzyme selectivity for the strate increased from 79.7% to 83.9% as the pH increased from 3 to 6. However, whe pH was increased to 8, the enzyme selectivity for the substrate decreased to 81.6% monosaccharide yield ranged from 8.9% (pH 6) to 10.9% (pH 5). The impact of enzyme loading on GMOS yield was evaluated, as shown in Figu and the results indicated that as enzyme loading increased from 5 to 20 U/g-GM GMOS yield increased from 12.7% to 59.1%, respectively. However, when the en loading was further increased to 30 U/g GM, the GMOS yield increased only sligh 60.1%. Moreover, enzyme selectivity towards the substrate increased from 55.4% at 5 GM to 81.4% at 20 U/g-GM before declining to 77.5% at 30 U/g-GM. Therefore, 20 U/g was determined to be the optimal amount for enzyme loading.
Finally, the impact of substrate loading on GMOS yield was examined. As show Figure 1d, as the substrate loading increased from 10 to 40 g/L, the GMOS yield incre from 53.0% to 72.8%. However, at a substrate loading of 60 g/L, GMOS yield decreas To further investigate enzymatic hydrolysis optimization, an enzyme loading of 20 U/g-GM and substrate loading of 20 g/L were used with a temperature of 50 • C. The effect of pH on the GMOS yield was evaluated, and the results are shown in Figure 1b. As the pH increased from 3 to 8, the GMOS yield increased from 39.5% (pH 3.0) to 51.4% (pH 5.0) and then decreased to 42.6% (pH 8.0). Additionally, the enzyme selectivity for the substrate increased from 79.7% to 83.9% as the pH increased from 3 to 6. However, when the pH was increased to 8, the enzyme selectivity for the substrate decreased to 81.6%. The monosaccharide yield ranged from 8.9% (pH 6) to 10.9% (pH 5).
The impact of enzyme loading on GMOS yield was evaluated, as shown in Figure 1c, and the results indicated that as enzyme loading increased from 5 to 20 U/g-GM, the GMOS yield increased from 12.7% to 59.1%, respectively. However, when the enzyme loading was further increased to 30 U/g GM, the GMOS yield increased only slightly to 60.1%. Moreover, enzyme selectivity towards the substrate increased from 55.4% at 5 U/g-GM to 81.4% at 20 U/g-GM before declining to 77.5% at 30 U/g-GM. Therefore, 20 U/g GM was determined to be the optimal amount for enzyme loading.
Finally, the impact of substrate loading on GMOS yield was examined. As shown in Figure 1d, as the substrate loading increased from 10 to 40 g/L, the GMOS yield increased from 53.0% to 72.8%. However, at a substrate loading of 60 g/L, GMOS yield decreased to 19.9%. Moreover, the monosaccharide yield increased from 10.5% (10 g/L) to 22.3% (50 g/L) before declining to 20.8% at a substrate loading of 60 g/L, whereas the enzyme selectivity dropped from 83.4% (10 g/L) to 48.8% (60 g/L) because of changes in the monosaccharide yield. In summary, under these optimal conditions, i.e., 50 • C, pH 5, 20 U/g-GM, and 40 g/L, the GMOS yield was 72.8%, the enzyme selectivity to the substrate was 82.3%, and the number-average degree of polymerization of GMOS was 7.27.

Effect of GMOS on the Physiological Activity of Intestinal Bacteria
Ethanol precipitation of the enzymatic digests was performed to remove impurities, followed by centrifugation. Subsequently, the supernatant containing ethanol was evaporated at low temperatures until the ethanol was removed, and the supernatant was freeze dried to form solid GMOS. The obtained GMOS were purified until they were 85.0% pure, and they were used as a carbon source to evaluate in vitro intestinal bacterial fermentation for 48 h.
As shown in Figure 2a, a significant increase in gut microbial density was observed, with over a nine-fold increase, compared to the original density (p < 0.05). The superior absorption ability of GMOS was further demonstrated by the OD 600 value, which was 16.9% higher than that of GM at 48 h, compared to that observed with GM. Meanwhile, the utilization of GMOS by intestinal bacteria reached 98.4% (48 h), owing to the effective absorption of GMOS. The level of GMOS absorption was 24.8% higher than that of GM ( Figure 2b, p < 0.05). Intestinal bacteria produced SCFAs after consuming carbon sources, which altered the pH of the environment (Figure 2c, p < 0.05). However, in this study, the SCFA concentration in the control group was the highest among all groups during the entire fermentation process (Figure 2d, p < 0.05). Meanwhile, the pH of the control decreased to 6.8 (6 h) and then increased to 7.4 (48 h), and the pH of the GM and GMOS groups decreased to 5.9 (48 h) and 6.8 (48 h), respectively.
In summary, under these optimal conditions, i.e., 50 °C, pH 5, 20 U/g-GM, and 40 g/L, the GMOS yield was 72.8%, the enzyme selectivity to the substrate was 82.3%, and the number-average degree of polymerization of GMOS was 7.27.

Effect of GMOS on the Physiological Activity of Intestinal Bacteria
Ethanol precipitation of the enzymatic digests was performed to remove impurities, followed by centrifugation. Subsequently, the supernatant containing ethanol was evaporated at low temperatures until the ethanol was removed, and the supernatant was freeze dried to form solid GMOS. The obtained GMOS were purified until they were 85.0% pure, and they were used as a carbon source to evaluate in vitro intestinal bacterial fermentation for 48 h.
As shown in Figure 2a, a significant increase in gut microbial density was observed, with over a nine-fold increase, compared to the original density (p < 0.05). The superior absorption ability of GMOS was further demonstrated by the OD600 value, which was 16.9% higher than that of GM at 48 h, compared to that observed with GM. Meanwhile, the utilization of GMOS by intestinal bacteria reached 98.4% (48 h), owing to the effective absorption of GMOS. The level of GMOS absorption was 24.8% higher than that of GM ( Figure 2b, p < 0.05). Intestinal bacteria produced SCFAs after consuming carbon sources, which altered the pH of the environment (Figure 2c, p < 0.05). However, in this study, the SCFA concentration in the control group was the highest among all groups during the entire fermentation process (Figure 2d, p < 0.05). Meanwhile, the pH of the control decreased to 6.8 (6 h) and then increased to 7.4 (48 h), and the pH of the GM and GMOS groups decreased to 5.9 (48 h) and 6.8 (48 h), respectively. Following the completion of fermentation, the concentration of short-chain fatty acids (SCFAs) and lactic acid in the fermentation broth was quantified, and the findings are summarized in Table 1. Among the SCFAs, propionic acid emerged as the predominant component in the fermentation broth, exhibiting a higher concentration than acetic acid and butyric acid, with lactic acid demonstrating the lowest concentration. Specifically, the utilization of GMOS as the carbon source led to an elevated proportion of propionic acid, constituting 43.45%, as opposed to 38.13% when GM was utilized as the carbon source. However, it is noteworthy that the overall concentration of SCFAs was comparatively lower in both the GM and GMOS experimental groups in contrast to the control group.

Diversity Analysis of Human Intestinal Bacteria
To investigate the effects of GM and GMOS on the gut microbiota structure, 16s rRNA sequencing was performed on both the GM and GMOS groups. Figure 3 shows the rarefaction curve based on the Shannon index, which considers both the number of species and their evenness in the samples. It is generally assumed that an increase in the sequencing depth has no effect on species diversity when the curve tends to flatten. The results shown in Figure 2 indicate that the sequencing depth was adequate for analysis.
Following the completion of fermentation, the concentration of short-chain fatty acids (SCFAs) and lactic acid in the fermentation broth was quantified, and the findings are summarized in Table 1. Among the SCFAs, propionic acid emerged as the predominant component in the fermentation broth, exhibiting a higher concentration than acetic acid and butyric acid, with lactic acid demonstrating the lowest concentration. Specifically, the utilization of GMOS as the carbon source led to an elevated proportion of propionic acid, constituting 43.45%, as opposed to 38.13% when GM was utilized as the carbon source. However, it is noteworthy that the overall concentration of SCFAs was comparatively lower in both the GM and GMOS experimental groups in contrast to the control group.

Diversity Analysis of Human Intestinal Bacteria
To investigate the effects of GM and GMOS on the gut microbiota structure, 16s rRNA sequencing was performed on both the GM and GMOS groups. Figure 3 shows the rarefaction curve based on the Shannon index, which considers both the number of species and their evenness in the samples. It is generally assumed that an increase in the sequencing depth has no effect on species diversity when the curve tends to flatten. The results shown in Figure 2 indicate that the sequencing depth was adequate for analysis.

NMDS and Venn Analysis of 16S rRNA Sequencing Data
To investigate the effects of GMOS on the composition of intestinal bacteria during in vitro human fecal fermentation, we performed 16S rRNA sequencing. The resulting OTU data were evaluated by NMDS analysis (Figure 4a). Our results revealed a significant difference in the composition of intestinal bacteria between the control (without added sugars), GM, and GMOS groups, indicating that the addition of GMOS resulted in a distinct change in the composition of intestinal bacteria (stress < 0.05). In addition, the Venn diagram (Figure 4b) depicts the distribution of OTUs among the three groups and shows that 1848 OTUs in the GMOS group and 1088 OTUs in the GM group were different from

NMDS and Venn Analysis of 16S rRNA Sequencing Data
To investigate the effects of GMOS on the composition of intestinal bacteria during in vitro human fecal fermentation, we performed 16S rRNA sequencing. The resulting OTU data were evaluated by NMDS analysis (Figure 4a). Our results revealed a significant difference in the composition of intestinal bacteria between the control (without added sugars), GM, and GMOS groups, indicating that the addition of GMOS resulted in a distinct change in the composition of intestinal bacteria (stress < 0.05). In addition, the Venn diagram (Figure 4b) depicts the distribution of OTUs among the three groups and shows that 1848 OTUs in the GMOS group and 1088 OTUs in the GM group were different from those in the control group when GMOS or GM were used as the carbon source during fermentation, which is in accordance with the results of the NMDS analysis. those in the control group when GMOS or GM were used as the carbon source during fermentation, which is in accordance with the results of the NMDS analysis.

Alpha Diversity of Human Intestinal Bacteria
The Sobs, Shannon, and Simpson indices were calculated to analyze the alpha diversity of the samples ( Table 2). The Sobs index indicated that the GMOS group had significantly higher scores than the control and GM groups (p < 0.05). The Shannon index (p < 0.05) revealed that the diversity of the gut microbiota decreased with the addition of GM and GMOS. However, the Simpson index revealed only a slight difference between the GM and control groups, and no difference between the GMOS and control groups.

Changes in the Composition of Intestinal Bacteria
To investigate the effects of GM and GMOS on human intestinal bacteria, we analyzed differences at the phylum level (Figure 5a). Intestinal bacteria primarily belong to phyla such as Fusobacteria, Bacteroidetes, Proteobacteria, and Firmicutes. Compared with the control group, the relative abundance of the phylum Fusobacteria was significantly decreased in both the GM and GMOS groups (p < 0.05), from 45.82% to 13.12% and 19.13%, respectively. The relative abundance of the phylum Bacteroidetes varied between the GM and GMOS groups; it increased from 13.38% in the control group to 39.31% in the GM group (p < 0.05) and to 23.96% in the GMOS group. The relative abundance of Proteobacteria significantly increased in the GMOS group compared to the control group (p < 0.05), from 25.05% to 35.82%, and decreased from 25.05% to 11.98% in the GM group. However, GM and GMOS did not have a significant effect on the phyla Firmicutes.

Alpha Diversity of Human Intestinal Bacteria
The Sobs, Shannon, and Simpson indices were calculated to analyze the alpha diversity of the samples ( Table 2). The Sobs index indicated that the GMOS group had significantly higher scores than the control and GM groups (p < 0.05). The Shannon index (p < 0.05) revealed that the diversity of the gut microbiota decreased with the addition of GM and GMOS. However, the Simpson index revealed only a slight difference between the GM and control groups, and no difference between the GMOS and control groups.

Changes in the Composition of Intestinal Bacteria
To investigate the effects of GM and GMOS on human intestinal bacteria, we analyzed differences at the phylum level (Figure 5a). Intestinal bacteria primarily belong to phyla such as Fusobacteria, Bacteroidetes, Proteobacteria, and Firmicutes. Compared with the control group, the relative abundance of the phylum Fusobacteria was significantly decreased in both the GM and GMOS groups (p < 0.05), from 45.82% to 13.12% and 19.13%, respectively. The relative abundance of the phylum Bacteroidetes varied between the GM and GMOS groups; it increased from 13.38% in the control group to 39.31% in the GM group (p < 0.05) and to 23.96% in the GMOS group. The relative abundance of Proteobacteria significantly increased in the GMOS group compared to the control group (p < 0.05), from 25.05% to 35.82%, and decreased from 25.05% to 11.98% in the GM group. However, GM and GMOS did not have a significant effect on the phyla Firmicutes.
We analyzed the composition of intestinal bacteria at the genus level (Figure 5b). In the control group (p < 0.05), classifiable sequences were mainly composed of Parabacteroides (9.11%), Fusobacterium (45.82%), Escherichia-Shigella (15.12%), and Phascolarctobacterium (4.61%). These species composed 37.92%, 32.70%, 8.47%, and 7.07% of the sequences in the GM group (p < 0.05), respectively. Compared to the control group, the relative abundance levels of Parabacteroides, Phascolarctobacterium, and Escherichia-Shigella increased to 20.53%, 6.69%, and 17.25%, respectively, and the abundance level of Fusobacterium declined to 26.69% in the GMOS group (p < 0.05). Based on these results, GM was found to exhibit a significant effect on Parabacteroides, while GMOS had a significant impact on Fusobacterium. on 2023, 9, x FOR PEER REVIEW 10 of 15 We analyzed the composition of intestinal bacteria at the genus level (Figure 5b). In the control group (p < 0.05), classifiable sequences were mainly composed of Parabacteroides (9.11%), Fusobacterium (45.82%), Escherichia-Shigella (15.12%), and Phascolarctobacterium (4.61%). These species composed 37.92%, 32.70%, 8.47%, and 7.07% of the sequences in the GM group (p < 0.05), respectively. Compared to the control group, the relative abundance levels of Parabacteroides, Phascolarctobacterium, and Escherichia-Shigella increased to 20.53%, 6.69%, and 17.25%, respectively, and the abundance level of Fusobacterium declined to 26.69% in the GMOS group (p < 0.05). Based on these results, GM was found to exhibit a significant effect on Parabacteroides, while GMOS had a significant impact on Fusobacterium.

Functional Analysis of Intestinal Bacteria Affected by GMOS
Based on the 16S rRNA sequencing results, the PICRUSt2 method was used to predict the effects of intestinal bacteria on the metabolic function. As shown in Figure 6a, at level 2, the GMOS group was significantly affected by genetic information processing, replication and repair, carbohydrate metabolism, metabolism of cofactors and vitamins, metabolism of other amino acids, and energy metabolism. At level 3, 10 metabolic (as shown in Figure 6b) pathways were promoted, resulting in lipoic acid metabolism being affected the most.

Functional Analysis of Intestinal Bacteria Affected by GMOS
Based on the 16S rRNA sequencing results, the PICRUSt2 method was used to predict the effects of intestinal bacteria on the metabolic function. As shown in Figure 6a, at level 2, the GMOS group was significantly affected by genetic information processing, replication and repair, carbohydrate metabolism, metabolism of cofactors and vitamins, metabolism of other amino acids, and energy metabolism. At level 3, 10 metabolic (as shown in Figure 6b) pathways were promoted, resulting in lipoic acid metabolism being affected the most.

Discussion
An increase in the reaction temperature during enzymatic hydrolysis increases the activation energy of GM molecules [18] and improves the fluidity of the GM solution [19]. An increase in the temperature and pH within the optimal range for the reaction temperature and pH resulted in an increase in the activity of β-mannanase [20], which in turn increased the GMOS yield. However, when the temperature and pH exceeded the optimal range, β-mannanase activity decreased, leading to a decrease in the GMOS yield. In addition, the GMOS yield was related to the enzyme loading process. However, the GMOS yield improved by only 1.0% when enzyme loading was further increased to 30 U/g GM. This is attributable to the limited GM release rate of Gleditsia microphylla seed powder [21,22] and the limited number of binding sites between β-mannanase and GM in the enzymatic system [23,24]. Therefore, when the substrate loading increased to 40 g/L, the GMOS yield increased to 72.8%. However, the GMOS yield decreased when substrate loading was increased to 50 and 60 g/L, owing to the absorbent solution containing Gleditsia microphylla seed powder. The rapid decrease in the solution volume made it difficult to achieve the stirring of the enzyme system containing β-mannanase and GM. Eventually, the GMOS level and the enzyme selectivity to the substrate decreased [25].
GM and GMOS were used as carbon sources for the in vitro fermentation of human fecal matter. The level of proliferation of the gut microbes in the GMOS group was higher than that in the GM group (Figure 2a). This was attributable to the utilization of GMOS by intestinal bacteria being higher than that of GM [26]. It has been reported that a negative correlation exists between the pH and SCFA concentration [27]. During fermentation, the OD600 value of the control group was the lowest, and the intestinal bacteria consumed SCFAs, [28] causing the SCFA concentration in the GM and GMOS groups to be lower

Discussion
An increase in the reaction temperature during enzymatic hydrolysis increases the activation energy of GM molecules [18] and improves the fluidity of the GM solution [19]. An increase in the temperature and pH within the optimal range for the reaction temperature and pH resulted in an increase in the activity of β-mannanase [20], which in turn increased the GMOS yield. However, when the temperature and pH exceeded the optimal range, β-mannanase activity decreased, leading to a decrease in the GMOS yield. In addition, the GMOS yield was related to the enzyme loading process. However, the GMOS yield improved by only 1.0% when enzyme loading was further increased to 30 U/g GM. This is attributable to the limited GM release rate of Gleditsia microphylla seed powder [21,22] and the limited number of binding sites between β-mannanase and GM in the enzymatic system [23,24]. Therefore, when the substrate loading increased to 40 g/L, the GMOS yield increased to 72.8%. However, the GMOS yield decreased when substrate loading was increased to 50 and 60 g/L, owing to the absorbent solution containing Gleditsia microphylla seed powder. The rapid decrease in the solution volume made it difficult to achieve the stirring of the enzyme system containing β-mannanase and GM. Eventually, the GMOS level and the enzyme selectivity to the substrate decreased [25].
GM and GMOS were used as carbon sources for the in vitro fermentation of human fecal matter. The level of proliferation of the gut microbes in the GMOS group was higher than that in the GM group (Figure 2a). This was attributable to the utilization of GMOS by intestinal bacteria being higher than that of GM [26]. It has been reported that a negative correlation exists between the pH and SCFA concentration [27]. During fermentation, the OD 600 value of the control group was the lowest, and the intestinal bacteria consumed SCFAs, [28] causing the SCFA concentration in the GM and GMOS groups to be lower than that of the control group. Nevertheless, it was found that the GMOS was better in promoting the production of SCFAs, therefore, the reduction in polymerization did provide a beneficial effect on the prebiotic activity of GMOS. In addition, in this study, the GMOS added were oligosaccharide blends and the results showed that xylo-disaccharide and xylo-triose had better prebiotic activity [7]. Hence, we believe that further screening of GMOS with low polymerization will improve GMOS prebiotic activity.
At the phylum level, Fusobacteria were significantly inhibited in the GMOS group, and their effects were higher than those of the GM group. Polysaccharides with different molecular weights had varied effects on intestinal bacteria [29], and polysaccharides with lower molecular weights were utilized by intestinal bacteria more easily (Figure 2b). The inhibition of harmful bacteria by functional oligosaccharides occurs mainly through the proliferation of beneficial bacteria and improvement of the competitiveness between bene-ficial bacteria during the growth process, thus achieving the effect of inhibiting harmful bacteria [30]. The relative abundance of fusobacteria is positively associated with colon cancer [31]. The proliferation of Bacteroidetes was significantly higher in the GM group than in the GMOS group. The phylum Bacteroidetes can degrade polysaccharides [32] and contribute to the digestion of dietary fibers to produce propionic acid [33]. The relative abundance of Bacteroidetes in the GM group was higher than that in the GMOS group because GM must be degraded before it can be utilized by the gut microbiota [26]. GM increases the proliferation of Bacteroidetes.
At the genus level, there was a remarkable decrease in the relative abundance of Fusobacterium. It had been reported that the relative abundance of Fusobacterium was associated with the occurrence of colorectal cancer. The abundance of Fusobacterium was higher in the tumors of patients with colorectal carcinoma than in the healthy colon [34]. The effect of GM on Parabacteroides was greater than that on GMOS. Parabacteroides have the ability to metabolize carbohydrates and produce SCFAs [35], and have beneficial effects on metabolic disorders [36,37]. However, it is debatable whether Parabacteroides are beneficial or pathogenic. P. distasonis, a species belonging to the genus Parabacteroides, is thought to result in the fermentation and production of methane, which is associated with the pathogenesis of colon cancer. P. distasonis exhibits significant drug resistance [38]. Therefore, an assessment of the effects of GM and GMOS on Parabacteroides has not been conducted till date.
GMOS had maximal effects on lipoic acid metabolism. Lipoic acid, also known as alpha-lipoic acid (ALA), is a naturally occurring compound capable of reducing oxidative damage to deoxyribonucleic acids, proteins, and lipids in cell membranes [39], and directly scavenging reactive oxygen and nitrogen species [40]. In addition, ALA inhibited the proliferation of lung cancer cells in vitro and cancer growth in vivo by inducing mTORmediated inhibition of autophagy [41].

Conclusions
In this investigation, GMOS were produced through enzymatic degradation of GM using β-mannanase enzyme under controlled conditions of 50 • C, pH 5, 20 U/g-GM, and 40 g/L, thereby attaining a notable yield of 72.8%. Subsequent in vitro fermentation experiments employing GMOS as a carbon source demonstrated profound transformations in the composition of the human gut microbiota, and these alterations were statistically significant. Notably, GMOS exhibited a substantial proliferation of the phylum Bacteroidetes, alongside a discernible inhibitory effect on the phylum Fusobacteria. Furthermore, the impact of GMOS extended to the regulation of lipoic acid metabolism and other metabolic activities, which are intrinsically associated with mitochondrial health and result in compelling anti-inflammatory effects. Consequently, these functions are associated with mitochondrial health and result in anti-inflammatory effects. These findings suggest that GMOS can be used as a dietary supplement to improve gut health and to prevent or treat certain diseases.

Conflicts of Interest:
The authors declare that this study was conducted in the absence of any commercial or financial relationships that could be construed as potential conflict of interest.