Preparation method and application of shatian pomelo peel pectin and bio-enzyme modified pectin

Abstract

The application discloses a shatian pomelo peel pectin and a preparation method and application of a biological enzyme modified pectin. The shatian pomelo peel is defatted, subcritical extraction is assisted by using citric acid, and after alcohol precipitation and dialysis, the shatian pomelo peel pectin is obtained, and the shatian pomelo peel pectin is further treated by using a biological enzyme-complex pectinase to obtain the complex pectinase modified pectin. The complex pectinase modified pectin enhances the fermentation capacity of the shatian pomelo peel pectin and changes the carbohydrate utilization, and meanwhile, the regulation effect on the intestinal health is improved. Meanwhile, the complex pectinase modified pectin can also significantly change the microbial composition of the fermentation liquor, promote the growth of potential probiotics and inhibit the proliferation of pathogens, so that the intestinal microbial flora forms a healthy mode. The method has the environmental protection and sustainability, can provide more kinds of modified pomelo peel pectins, and has wide application potential.

Description

Technical Field

[0001] This invention relates to the field of food technology, specifically to a method for preparing pectin from Shatin pomelo peel and its bio-enzyme-modified pectin, and its application. Background Technology

[0002] Grapefruit (Citrus grandis) is widely cultivated in southwestern China, Southeast Asia, and other Asian countries due to its high yield, ease of storage, and cultivation. This fruit contains not only pectin but also other bioactive substances such as polysaccharides, phenolic compounds, flavonoids, and vitamins. However, grapefruit peel contains a large amount of insoluble dietary fiber, making it difficult to chew and eat. Furthermore, the bitterness of naringin in grapefruit peel also affects its edible value. Therefore, exploring a new, efficient, and green method for extracting readily bioavailable pectin from grapefruit peel is of great significance.

[0003] Pectin is a soluble fiber found in plant cell walls, particularly abundant in fruits like grapefruit. The structure, extraction methods, applications, and potential health benefits of grapefruit pectin have been a subject of considerable interest. Homogalacturonan (HG) is the main component of the pectin backbone, accounting for approximately 65% ​​of the total pectin molecule. Another important component is rhamnolacturonic acid I (RG-I), comprising 20–35% of the total. RG-I is structured by a series of repeating disaccharide units containing rhamnoose residues and galacturonic acid (GalA) units. These GalA units alternate with rhamnoose residues, forming the unique structure of RG-I. Due to its complex molecular structure, pectin exhibits several important properties. By forming a network structure, pectin provides support and stability in plant cell walls, playing a crucial role in cell structure and mechanical properties. Simultaneously, pectin also plays an important role in food. It imparts unique texture, viscosity, and mouthfeel to plant-based ingredients such as fruits and vegetables, thereby affecting the overall taste and texture of the food.

[0004] Pectin is poorly digestible but can be utilized by gut microbes to generate metabolism and regulate the composition and / or activity of the gut microbiota, thereby producing beneficial physiological effects on the host. Furthermore, pectin is highly valued as a polysaccharide with health benefits. Pectins with different structural and / or compositional characteristics have been found to have potential in protecting and preventing gastrointestinal problems, lowering cholesterol, and regulating immunity. The health-supporting characteristics of pectin can be found in its fermentability and the corresponding changes in gut microbiota composition. In the gut, pectin is utilized by gut microbiota and fermented into microbial metabolites, such as short-chain fatty acids (SCFAs). Pectin can support proper regulation of the gut microbiota. For example, pectin derived from the pericarp of *Cucumis meteliferus* has been found to increase the abundance of beneficial bacteria such as *Bifidobacterium* in the gut microbiota. Soybean and citrus pectin increased the relative abundance of *Prevotella* in porcine colonic digests. The relative abundance of *Lactobacillus* was increased by beet pectin during rat cecal fermentation.

[0005] Pectin is popular as a dietary supplement due to its potential health benefits, such as promoting weight loss, improving digestion, regulating immunity, preventing colon cancer, and lowering cholesterol levels. Gut fermentability refers to the extent to which specific types of food are broken down and fermented by bacteria naturally present in the colon, and is further used to study its regulatory effects on microbial composition and metabolite production. Various complex polysaccharides, including pectin, have been used for in vitro fermentation to mimic the microbial fermentation processes that occur in the human gut. However, pectin obtained from different sources and extraction methods leads to variations in efficacy and activity range. Pectin extracted from sunflower and artichoke, as well as its enzyme-modified forms, have been observed to have similar abilities to promote the growth of beneficial gut bacteria during in vitro fermentation. It has been found that low-methoxyl pectin ferments faster than high-methoxyl pectin under in vitro and in vivo conditions in rats. Pectin with a highly branched regional structure can improve physiological properties and is beneficial to colonic health compared to commercial pectin. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned shortcomings of the prior art and to provide a method for preparing pectin from Shatin pomelo peel and its bio-enzyme-modified pectin, as well as its application.

[0007] The first objective of this invention is to provide a method for preparing pectin from Shatin pomelo peel.

[0008] The second objective of this invention is to provide a pectin extracted from pomelo peel using the preparation method described above.

[0009] The third objective of this invention is to provide a method for preparing bio-enzyme-modified pectin.

[0010] A fourth objective of this invention is to provide a bio-enzyme-modified pectin prepared by the aforementioned preparation method.

[0011] A fifth objective of this invention is to provide the application of the aforementioned pectin from Shatin pomelo peel and / or bio-enzyme-modified pectin in regulating intestinal flora and / or promoting the growth of probiotics.

[0012] To achieve the above objectives, the present invention is implemented through the following solution:

[0013] A method for preparing pectin from Shatin pomelo peel, the method comprising the following steps:

[0014] S11. Degrease the Shatin pomelo peel powder to obtain defatted Shatin pomelo peel powder;

[0015] S12. Mix the defatted pomelo peel powder with the chelating agent at a ratio of 1g:(18-22)mL, and perform subcritical extraction on the resulting mixed solution.

[0016] S13. Centrifuge the extract and adjust the pH to neutral, then perform alcohol precipitation and solid-liquid separation to obtain the crude precipitate;

[0017] S14. Dissolve the crude precipitate fully in water, dialyze it using an 8-14 kDa dialysis bag, and then freeze-dry it to obtain the final product.

[0018] Preferably, the method for defatting the pomelo peel powder in step S11 is to fully extract it with ether at 60-70°C, and then remove the ether.

[0019] More preferably, a Soxhlet extractor is used for reflux defatting.

[0020] More preferably, the mass-to-volume ratio of the pomelo peel powder to the ether is 1:(9-11).

[0021] More preferably, the mass-to-volume ratio of the pomelo peel powder to the ether is 1:10.

[0022] Preferably, the chelating agent in step S12 is citric acid.

[0023] Preferably, the ratio of the defatted pomelo peel powder to the chelating agent in step S12 is 1:20.

[0024] Preferably, the subcritical extraction step S12 is as follows: the pressure is increased from room temperature to 127°C and 0.15 MPa within 35 min and maintained for 25 min, and then the pressure is reduced to atmospheric pressure within 35 min.

[0025] Pectin from pomelo peel obtained by the above preparation method.

[0026] A method for preparing bio-enzyme-modified pectin, the method comprising the following steps:

[0027] S21. Dissolve the above-mentioned pectin from Shatin pomelo peel in a buffer solution and adjust the pH to 4-5;

[0028] S22. The pectin solution of Shatin pomelo peel is subjected to enzymatic hydrolysis, followed by heating inactivation and solid-liquid separation. The liquid is then dialyzed and freeze-dried to obtain the final product.

[0029] Preferably, the buffer solution in step S21 is an acetate-sodium acetate buffer solution.

[0030] Preferably, the pH in step S21 is 4.5.

[0031] Preferably, the bioenzyme mentioned in step S22 is pectin esterase, polygalacturonase, and pectin lyase.

[0032] More preferably, the pectin esterase, polygalacturonase and pectin lyase are formulated into a biological enzyme in a ratio of 1:3:1 with enzyme activity.

[0033] Preferably, the enzymatic hydrolysis time in step S22 is 0.5 to 2.5 hours.

[0034] A bio-enzyme-modified pectin prepared by the above-described preparation method.

[0035] The above-mentioned applications of pectin from Shatin pomelo peel and / or bio-enzyme-modified pectin in regulating intestinal flora and / or promoting the growth of probiotics.

[0036] Preferably, the gut microbiota is one or more of Fusobacteriota, Bacteroides, Faecalibacterium, Fusobacterium, Escherichia-Shigella, Sutterella, Prevotella, Lachnoclostridium, Klebsiella, Lachnospira, Dorea, and Tyzzerella.

[0037] Preferably, the promotion of probiotic growth is to increase the relative abundance of one or more of Prevotella, Lachnospira, Dorea, Tyzzerella, and Faecalibacterium.

[0038] The above-mentioned pectin from Shatin pomelo peel and / or bio-enzyme-modified pectin are used in the preparation of drugs, foods or health products that regulate the structure of intestinal flora.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] This invention discloses a method for preparing pectin from Shatin pomelo peel and its bio-enzyme-modified pectin, as well as its applications. The invention involves defatting Shatin pomelo peel, performing citric acid-assisted subcritical extraction, and then dialysis with alcohol precipitation to obtain Shatin pomelo peel pectin (CA-SWP). This is further processed by a bio-enzyme-complex pectinase to obtain enzyme-modified pectin with specific properties. The complex pectinase treatment significantly reduces the molecular weight of the Shatin pomelo peel pectin and produces pectin fragments of different molecular weights, generating pectin fractions with different monosaccharide molar ratios and compositions. Simultaneously, it significantly reduces the degree of methyl esterification of the pectin, affecting its physical properties and structure.

[0041] Infrared spectroscopy analysis showed that the pectin from Shatin pomelo peel, after modification with a compound pectinase, exhibited different intensities in its characteristic absorption peaks. Furthermore, the compound pectinase-modified pectin enhanced the fermentation capacity of Shatin pomelo peel pectin and altered carbohydrate utilization during fermentation. The low molecular weight Shatin pomelo peel pectin obtained through compound pectinase modification could increase the yield and rate of short-chain fatty acids during in vitro fermentation. If the molecular weight of natural Shatin pomelo peel pectin is relatively low, enzymatic hydrolysis with compound pectinase into even lower molecular weight pectin would only promote the yield and rate of short-chain fatty acids in the early fermentation stage. The low molecular weight Shatin pomelo peel pectin obtained through compound pectinase modification (especially from high molecular weight natural pectin) can significantly alter the microbial composition of the fermentation broth, promoting the growth of probiotics while inhibiting the proliferation of pathogens, thus fostering a healthy gut microbiota. Enzymatic hydrolysis of Shatin pomelo peel pectin using compound pectinase to obtain low molecular weight pectin with low methyl esterification can effectively enhance the regulatory effect of Shatin pomelo peel pectin on gut health. Attached Figure Description

[0042] Figure 1 This describes the extraction of pectin from Shatin pomelo peel and the enzymatic hydrolysis process using compound pectinase.

[0043] Figure 2Molecular weight distribution and Fourier transform infrared spectra of raw pectin and pectin modified with compound pectinase; A: pectin extracted by citric acid-assisted subcritical extraction (CA-SWP), pectin extracted by citric acid-assisted subcritical extraction after 0.5 h of enzymatic hydrolysis with compound pectinase (E1-CA-SWP), pectin extracted by citric acid-assisted subcritical extraction after 2.5 h of enzymatic hydrolysis with compound pectinase (E2-CA-SWP), and pectin extracted by EDTA-2Na-assisted hot water extraction (…). Molecular weight distribution of pomelo peel pectin extracted with EDTA-2Na-assisted hot water after hydrolysis with compound pectinase for 0.5 h (E1-E-HWP) and pomelo peel pectin extracted with EDTA-2Na-assisted hot water after hydrolysis with compound pectinase for 2.5 h (E2-E-HWP), and Fourier transform infrared spectra of CA-SWP, E1-CA-SWP, E2-CA-SWP, E-HWP, E1-E-HWP and E2-E-HWP.

[0044] Figure 3 For the preliminary assessment of pectin fermentation capacity during in vitro fermentation: A: Total uronic acid (UA) content of each group at different time points during in vitro fermentation; B: Total carbohydrate content of each group at different time points during in vitro fermentation; C: Dynamic changes in pH value of each group at different time points during in vitro fermentation; D: UA utilization rate of each group at different time points during in vitro fermentation; E: Total carbohydrate utilization rate of each group at different time points during in vitro fermentation; F: Total gas production of each group at different time points during in vitro fermentation.

[0045] Figure 4 The following are examples of molecular weight degradation of pectin during in vitro fermentation: A: Dynamic changes in the high performance size exclusion chromatography (HPSEC) chromatograms of CA-SWP molecular weight degradation during in vitro fermentation; B: Dynamic changes in the HPSEC chromatograms of E1-CA-SWP molecular weight degradation during in vitro fermentation; C: Dynamic changes in the HPSEC chromatograms of E2-CA-SWP molecular weight degradation during in vitro fermentation; D: Dynamic changes in the HPSEC chromatograms of E-HWP molecular weight degradation during in vitro fermentation; E: Dynamic changes in the HPSEC chromatograms of E1-E-HWP molecular weight degradation during in vitro fermentation; F: Dynamic changes in the HPSEC chromatograms of E2-E-HWP molecular weight degradation during in vitro fermentation.

[0046] Figure 5The following figures represent the dynamic changes in the monosaccharide composition and structure of pectin during in vitro fermentation. A: Dynamic changes in monosaccharide content of CA-SWP at different time points; B: Dynamic changes in monosaccharide content of E1-CA-SWP at different time points; C: Dynamic changes in monosaccharide content of E2-CA-SWP at different time points; D: Dynamic changes in monosaccharide content of E-HWP at different time points; E: Dynamic changes in monosaccharide content of E1-E-HWP at different time points; F: Dynamic changes in monosaccharide content of E2-E-HWP at different time points; G: Contribution of RG-I structure to the overall pectin (Rha / UA); H: Branching degree of RG-I ((Ara+Gal) / Rha); I: Linearity of pectin (UA / (Fuc+Gal+Ara+Rha+Xyl)).

[0047] Figure 6 The following data represent the dynamic changes in the production of short-chain fatty acids (SCFAs) in pectin during in vitro fermentation: A: Dynamic changes in acetic acid content (production or consumption) at different time points for each group; B: Dynamic changes in propionic acid content (production or consumption) at different time points for each group; C: Dynamic changes in butyric acid content (production or consumption) at different time points for each group; D: Dynamic changes in isobutyric acid content (production or consumption) at different time points for each group; E: Dynamic changes in valerate content (production or consumption) at different time points for each group; F: Dynamic changes in isovaleric acid content (production or consumption) at different time points for each group; G: Dynamic changes in total SCFAs content (production or consumption) at different time points for each group; H: Dynamic changes in total BCFAs content (production or consumption) at different time points for each group; I: Dynamic changes in the ratio of total SCFAs to total BCFAs at different time points for each group.

[0048] Figure 7 Analysis of the gut microbiota at the phylum and genus levels: A: Chao index of gut microbiota in all groups 24 h after in vitro fermentation and in the initial state (0 h); B: Shannon index of gut microbiota in all groups 24 h after in vitro fermentation and in the initial state (0 h); C: Principal coordinate analysis of gut microbiota in all groups 24 h after in vitro fermentation and in the initial state (0 h); D: Bacterial taxonomic distribution of gut microbiota at the phylum level in all groups 24 h after in vitro fermentation and in the initial state (0 h); E: Bacterial taxonomic distribution of gut microbiota at the genus level in all groups 24 h after in vitro fermentation and in the initial state (0 h); F: Heatmap analysis of the relative abundance of gut microbiota at the genus level in all groups 24 h after in vitro fermentation and in the initial state (0 h) (F).

[0049] Figure 8Correlation analysis was performed on gut microbiota, pectin characteristics, and metabolites. A: Correlation between bacterial genus, carbohydrates in the substrate, molecular weight of pectin, and degree of methylation (DM); B: Correlation between bacterial genus and SCFA production; C: Correlation between SCFA production, carbohydrates in the substrate, molecular weight of pectin, and degree of methylation (DM). Positive correlations are indicated in red, and negative correlations in blue. The intensity of the color is proportional to the correlation coefficient (Spearman correlation coefficient). Detailed Implementation

[0050] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0051] Example 1: Extraction of pectin from Shatin pomelo peel and preparation of pectin modified with compound pectinase

[0052] I. Experimental Methods

[0053] 1. Preparation of pectin from Shatin pomelo peel and pectin modified by compound pectinase

[0054] (1) Degreasing of Shatin pomelo peel

[0055] After washing and peeling the Shatin pomelo, the fresh pomelo peel is divided into 2cm×2cm pieces and dried in a constant temperature oven at 45℃ for 24 hours until the pieces become dry and hard. Then, it is crushed into powder and passed through a 60-mesh sieve to obtain pomelo peel powder (particle diameter <0.25mm).

[0056] To remove fat from the powder using a Soxhlet extractor: Pour anhydrous ether into a dry extraction tube, filling it to approximately 1 / 3 to 1 / 2 of its volume. Place the pomelo peel powder on filter paper and wrap it securely in a filter paper packet. Then, place the filter paper packet into the anhydrous ether in the extraction tube. Ensure the sample is completely submerged in the ether to fully dissolve the fat (the mass-to-volume ratio of pomelo peel powder to anhydrous ether is 1:10). Place the extraction tube containing the sample in a constant temperature water bath at 65°C. During extraction, maintain a reflux rate of ether drops at least 3 times per hour to ensure thorough extraction. Continue extraction until no oil residue is visible when the ether is dripped through the filter paper, indicating that the fat has been completely dissolved and extracted from the sample. Remove the extracted filter paper packet and place it in a fume hood to allow the ether to evaporate. Wait in the fume hood for a period of time until the ether has completely evaporated. Place the filter paper packet in a 50°C oven for 12 hours to dry. During this process, the ether residue further volatilizes, resulting in defatted Shatin pomelo peel powder. The Shatin pomelo peel pectin and pectinase-modified pectin treatment groups are as follows: Figure 1 As shown.

[0057] (2) Preparation of chelating agent extract

[0058] Citric acid extract: At room temperature, dissolve 5g of citric acid in 100mL of deionized water (50g / L). EDTA extract: At room temperature, dissolve 1g of EDTA-2Na in 100mL of deionized water (10g / L).

[0059] (3) Extraction of pectin from Shatin pomelo peel

[0060] ① Weigh the grapefruit peel powder and pass it through an 80-mesh sieve (particle diameter <0.18mm). Divide the sieved peel powder into two parts and perform EDTA-2Na-assisted hot water extraction and citric acid-assisted subcritical extraction on the other part, respectively. The specific methods are as follows:

[0061] EDTA-2Na-assisted hot water extraction: The screened fruit peel powder was dissolved in EDTA-2Na solution (10 g / L) at a mass-to-volume ratio of 1:20, and then subjected to heat treatment at 85℃ for 1.5 h.

[0062] Citric acid-assisted subcritical extraction: The screened fruit peel powder was dissolved in citric acid solution (50 g / L) at a mass-to-volume ratio of 1:20, and then subcritical extraction was carried out at 127℃ and 0.15 MPa for 35 min. Pectin was extracted using the subcritical extraction (G154TW) process (program settings: increase from room temperature to 127℃ and 0.15 MPa within 35 min, maintain for 25 min, and then decrease to atmospheric pressure within 35 min).

[0063] ② The extracts obtained by the above two methods were centrifuged at 3000g for 15 min, and then the pH was adjusted to 7.0 at 25℃. Subsequently, 4 times the volume of anhydrous ethanol was added to the supernatant, and the mixture was allowed to stand at 4℃ for 12 h before the precipitate was collected.

[0064] ③ Dissolve the precipitate collected in step ② completely with 4 times its weight of ultrapure water. Dialyze the precipitate through an 8-14 kDa dialysis bag at 4°C to remove small molecules, and then dry it in a freeze vacuum to obtain citric acid-assisted subcritical extraction pectin (CA-SWP) and EDTA-2Na-assisted hot water extraction pectin (E-HWP). Weigh the extracted pectins.

[0065] ④ Calculate the pectin yield of Shatin pomelo peel. The calculation formula is:

[0066] Yield (%) of pectin from Shatin pomelo peel = mass of extracted pectin from Shatin pomelo peel / dry weight of Shatin pomelo peel powder × 100%.

[0067] (4) Preparation of compound pectinase modified pectin

[0068] ① CA-SWP and E-HWP were dissolved in an acetate-sodium acetate buffer solution (0.05 mol / L) to make the concentration of the mixed solution reach 5 mg / mL, and the pH of the mixed solution was adjusted to 4.5 at 25℃.

[0069] ② A compound pectinase was prepared by mixing pectin esterase, polygalacturonase, and pectin lyase in a ratio of 1:3:1, resulting in a total enzyme activity of 3000 U / g. The compound pectinase was then added to the corresponding mixed solution to achieve an enzyme activity of 2.5 U / mL, and enzymatic hydrolysis was performed to obtain the enzymatically hydrolyzed pectin solution. Among them, CA-SWP and E-HWP were enzymatically hydrolyzed for 0.5 h and 2.5 h, respectively, to obtain different enzymatically hydrolyzed pectin solutions, specifically: pectin of Shatin grapefruit peel hydrolyzed for 0.5 h with citric acid-assisted subcritical extraction (E1-CA-SWP), pectin of Shatin grapefruit peel hydrolyzed for 2.5 h with citric acid-assisted subcritical extraction (E2-CA-SWP), pectin of Shatin grapefruit peel hydrolyzed for 0.5 h with EDTA-2Na-assisted hot water extraction (E1-E-HWP), and pectin of Shatin grapefruit peel hydrolyzed for 2.5 h with EDTA-2Na-assisted hot water extraction (E2-E-HWP).

[0070] ③ The above-mentioned enzymatically hydrolyzed pectin solution was heated at 90℃ for 15 min to denature and inactivate the compound pectinase. The inactivated enzymatically hydrolyzed pectin solution was filtered and dialyzed at 4℃ for 48 h through an 8-14 kDa dialysis bag to further remove small-molecule pectin fragments. Then, it was freeze-dried under vacuum to obtain the corresponding compound pectinase-modified pectin, which was then weighed.

[0071] ④ Calculate the yield of pectin modified by compound pectinase. The calculation formula is as follows:

[0072] Yield (%) of pectin modified with compound pectinase = (Mass of pectin modified with compound pectinase / Mass of pectin from Shatin grapefruit peel) × 100%

[0073] 2. Changes in the molecular weight of pectin

[0074] Take 5 mg each of the original pectin and the compound pectinase-modified pectin prepared by the above method, dissolve them in 1 mL of ultrapure water, and remove impurities through a 0.22 μm hydrophilic filter membrane to obtain 6 kinds of pectin samples.

[0075] The molecular weight (Mw) of six pectin samples was determined by high performance size exclusion chromatography (HPSEC): a TSK-GEL column and a TSK-GEL guard column were connected in series (TSKgel Guardcolumn SuperSW, 6 mm × 40 mm), and 4000, 3000, and 2500 Super AW (6 mm × 150 mm) columns were connected to an RI detector (RefractoMax 520). A 0.2 M NaNO3 solution was used as the mobile phase, and dextran solutions with Mw values ​​of 10, 40, 70, 500, and 2000 kDa were used as standards. At a column temperature of 55 °C, 20 μL of pectin sample and dextran were injected separately into the HPSEC system at a flow rate of 0.6 mL / min for analysis.

[0076] II. Experimental Results

[0077] As shown in Table 1, the pectin yield of both CA-SWP and E-HWP after 2.5 hours of enzymatic hydrolysis was approximately 8% lower than that after 0.5 hours of enzymatic hydrolysis. Furthermore, compared to the original pectin CA-SWP and E-HWP, the molecular weight of the pectin modified by the compound pectinase was significantly reduced (p<0.05) (Table 1 and...). Figure 2 In A), but the molecular weight of E2-CA-SWP (15.42kDa) is similar to that of E2-E-HWP (14.57kDa).

[0078] Therefore, the molecular weight of pectin modified with compound pectinase decreases significantly with increasing enzymatic hydrolysis time. Prolonged exposure to compound pectinase may lead to the formation of more low-molecular-weight pectin fragments or oligosaccharides, which are subsequently removed during dialysis, resulting in a relatively low pectin yield. This also indicates that under certain conditions, enzymatic hydrolysis of compound pectin can yield pectin of different molecular weights, and in some cases, these treated pectins may have similar molecular weights.

[0079] Table 1. Yields and molecular weights of raw pectin and enzyme-modified pectin.

[0080]

[0081] Example 2: Properties of pectin from Shatin pomelo peel and its modified pectin with compound pectinase

[0082] I. Monosaccharide Composition and Basic Characteristics of Pectin

[0083] 1. Experimental Methods

[0084] (1) Calculation of degree of methylation (DM)

[0085] The DM of a sample is determined by establishing a standard curve using the percentage of ester peak area (I) and the DM value. I = A 1745 / (A 1745 +A 1605 )×100%, A 1745 and A 1605 These represent the absorption peak areas of the methyl ester group and the carboxylate group, respectively. Standard curves were established using commercial pectins with different DM values ​​(DM: 3, 18, 33, 44, 55, 62.5, and 70.5%) to calculate the sample DM values.

[0086] (2) Monosaccharide composition analysis

[0087] ① Take 10 mg each of the original pectin and the compound pectinase modified pectin prepared in Example 1, mix them with an inositol solution (internal standard) containing 1 mg of inositol in a K-max tube, shake and mix well, and freeze dry to obtain lyophilized powder.

[0088] ② Perform pre-hydrolysis: Add 0.45 mL of 72% H2SO4 solution (v / v) to the above lyophilized powder, incubate in a constant temperature water bath at 30℃ for 1 hour, shaking every 20 minutes to ensure complete reaction between the lyophilized powder and concentrated sulfuric acid, to obtain the pre-hydrolysis product. Add 0.55 mL of 1M H2SO4 solution to the pre-hydrolysis product and hydrolyze in a metal bath at 100℃ for 3 hours to obtain the hydrolysis product.

[0089] ③ Carry out the reduction reaction. Add 25% NH3 solution (v / v) to the above hydrolysis product and check the pH value. If the pH value is less than 7, continue to add NH3 solution. Then add 0.45 mL of 3M NH3 solution (containing 150 mg / mL NaBH4), heat in a metal bath at 30°C for 1 h, and then cool in an ice bath to obtain the reduction product.

[0090] ④ Perform the acetylation step: Take 1 mL of the above reduction product, add 0.3 mL of N-methylimidazole and 2 mL of acetic anhydride, mix well, heat in a metal bath at 30 °C for 30 min, and then cool in an ice bath to obtain the acetylated product.

[0091] ⑤ Perform liquid-liquid extraction: Take 1.3 mL of the above acetylated product, add 3.9 mL of distilled water and 2.6 mL of dichloromethane, vortex mix, centrifuge at 1000 rpm for 10 min and discard the upper layer. Repeat this step 3 times.

[0092] ⑥ Remove residual dichloromethane in the lower layer solution by evaporation in a 40℃ metal bath;

[0093] ⑦ Add 2 mL of acetone to the lower layer solution, evaporate and dry in a metal bath at 40 °C, and repeat this step twice.

[0094] ⑧ Dissolve the dried product in 0.25 mL of acetone, mix thoroughly, transfer to a vial and store at -20 °C for subsequent gas chromatography (GC) analysis.

[0095] ⑨ Inject 2 μL of sample into a gas chromatograph (GC) (G2010Plus) equipped with a DB-255 column (15 m × 0.53 mm × 1 μm id) and a flame ionization detector (FID). The detector and injection temperatures were set to 300 °C and 250 °C, respectively. The GC temperature and gas program was as follows: hold at 200 °C for 1 min, then gradually increase to 210 °C at a rate of 10 °C / min and hold for 20 min to separate all monosaccharide peaks, using nitrogen as the carrier gas (3 mL / min).

[0096] (3) Determination of uronic acid content

[0097] Divide the 18 test tubes into 3 groups of 6. Add 100, 200, 300, 400, 500, and 600 μL of 100 μg / mL galacturonic acid standard solution to each group of test tubes, respectively. Then add 500, 400, 300, 200, 100, and 0 μL of distilled water (distilled water serves as a blank control instead of galacturonic acid solution) to each group of test tubes, respectively, and shake to mix.

[0098] Place all three sets of test tubes in an ice bath, then add 3.6 mL of 0.0125 M sodium tetraborate-concentrated sulfuric acid solution to each set of test tubes and shake to mix. Next, place all three sets of test tubes in a boiling water bath and remove them precisely after 5 minutes. Finally, place all three sets of test tubes in cold water to allow them to cool to room temperature.

[0099] Add 65 μL of 0.15% m-phenylphenol solution (v / v) to the first two groups of test tubes, and add 65 μL of 0.5% sodium hydroxide solution (w / v) to the third group of standard solutions as a sample blank control. Measure the absorbance at 520 nm. Plot a standard curve with absorbance on the ordinate and concentration on the abscissa.

[0100] The products of hydrolysis of uronic acid and sodium tetraborate under concentrated sulfuric acid at high temperature further react with m-phenylphenol to generate a pink derivative. This derivative exhibits maximum UV absorption at 520 nm, and its absorbance shows a linear relationship with the concentration of galacturonic acid within a certain concentration range. Therefore, using D-galacturonic acid as a standard, the uronic acid (UA) content of the hydrolyzed sample was quantitatively determined at 520 nm using the p-hydroxydiphenyl colorimetric method.

[0101] The total carbohydrate content is the sum of all monosaccharides measured, including rhamnose (Rha), fucose (Fuc), arabinose (Ara), xylose (Xyl), mannose (Man), galactose (Gal), glucose (Glc), and uronic acid (UA).

[0102] 2. Experimental Results

[0103] The results are shown in Table 2. Compared with the original pectin CA-SWP and E-HWP, the DM value of pectin modified by the compound pectinase decreased significantly with the extension of enzymatic hydrolysis time. The compound pectinase used in the experiment included pectin esterase, polygalacturonase, and pectin lyase. Among them, pectin esterase reduces the DM of pectin by removing methoxy groups, while polygalacturonase and pectin lyase reduce the Mw of pectin through hydrolysis. This resulted in the decrease of both the DM value and Mw of the compound pectinase-modified pectin with increasing enzyme treatment time (Table 2).

[0104] Compared with the original pectin, the total carbohydrate content of pectin modified with compound pectinase was reduced. However, with the extension of enzymatic hydrolysis time, the molar ratios of monosaccharides Rha, Fuc, Ara, Man, Gal, and Glc increased during the enzymatic hydrolysis of E-HWP (E2-E-HWP>E1-E-HWP>E-HWP). Interestingly, compared with CA-SWP (90.02%), the molar ratio of UA in pectin modified with compound pectinase was slightly increased, with E1-CA-SWP having a UA molar ratio of 91.41%, while E2-CA-SWP reached the highest value (91.63%), but the difference was not significant. However, a significant decrease in the molar ratio of UA was observed in both E-HWP and its compound pectinase-modified pectin (p<0.05), with E-HWP having a UA molar ratio of 89.34% and E2-E-HWP having a UA molar ratio of 84.85%.

[0105] Compared with the original pectin CA-SWP, the Rha / UA value of the composite pectinase-modified pectin thereof is decreased. Compared with the original pectin E-HWP, the value of (Ara+Gal) / Rha increases with the extension of enzymolysis time (E-HWP<E1-E-HWP<E2-E-HWP). Compared with the original pectin CA-SWP, the UA / (Fuc+Xyl+Rha+Ara+Gal) of the composite pectinase-modified pectin thereof increases with the extension of enzymolysis treatment time (CA-SWP<E1-CA-SWP<E2-CA-SWP), while the situation of E-HWP is opposite (E-HWP>E1-E-HWP>E2-E-HWP). In conclusion, the enzymatic action of composite pectinase on natural pectins with different structural characteristics such as CA-SWP and E-HWP produces enzymolyzed pectins with different monosaccharide molar ratios and structures.

[0106] Table 2 Physicochemical properties, monosaccharide molar ratios and structural patterns of original pectin and composite pectinase-modified pectin

[0107]

[0108]

[0109] II. Infrared spectra of original pectin and composite pectinase-modified pectin

[0110] 1. Experimental method

[0111] The lyophilized powder of original pectin and composite pectinase-modified pectin prepared in Example 1 was taken, and the Fourier transform infrared (FT-IR) spectrum of KBr compressed tablet of pectin lyophilized powder was recorded using a Nicolet iS50+iN10 spectrometer (Thermo Fisher Scientific), with a resolution of 4 cm -1 , the cumulative number of scans is 32, and the wavelength range is 4000-500 cm -1 . KBr disks were prepared using a salt:sample ratio of 90:10.

[0112] 2. Experimental results

[0113] The infrared spectra and characteristic absorption peaks of original pectins CA-SWP and E-HWP and their corresponding composite pectinase-modified pectins are shown as Figure 2 B in , wherein the absorption peak near 2940 cm -1 is attributed to the stretching vibration of C-H in alkyl or methyl groups, the absorption peak at 1745 cm -1 is caused by the stretching vibration of C=O in ester bonds, the peak at 1630 cm -1 is caused by the stretching vibration of C=O of carboxyl groups in pectin, while the peak at 1100 cm -1The nearby peaks are caused by the tensile vibrations of COC. This is consistent with previous studies on pectin. Furthermore, pristine pectins such as CA-SWP and E-HWP show peak values ​​at 1745 cm⁻¹. -1 The peak intensity at 1745 cm⁻¹ is higher than that of the corresponding compound pectinase-modified pectin. -1 The peak intensity at 1745 cm⁻¹. Lower esterification means fewer methyl groups; therefore, pectin with higher esterification has a higher peak intensity at 1745 cm⁻¹ compared to pectin with lower esterification. -1 The height is greater than 1635cm. -1 The higher absorption peak is consistent with the DM measurement results. In summary, the infrared spectroscopy analysis results further demonstrate that there are significant differences in chemical structure between natural pectin and pectinase-modified pectin.

[0114] Example 3: Effects of pectin from Shatin pomelo peel and its compound pectinase-modified pectin on simulated in vitro colonic fermentation.

[0115] I. Establishment of an in vitro fermentation experimental model

[0116] The in vitro simulated fermentation experiment was conducted based on previously published methods, with some modifications and adjustments made as needed. Fresh fecal samples were collected from eight healthy donors, including five men and three women, aged 20–24 years, who had not received any antibiotic treatment or had any digestive-related illnesses in the past three months.

[0117] Collected fecal samples were placed in 50 mL sterile centrifuge tubes containing 30 mL of PBS buffer (0.1 mol / L, pH 7.0) and 40 glass beads, with strict avoidance of contamination during the process. Weights before and after sampling were recorded, and concentrations were calculated. After vortexing homogenization, the samples were centrifuged at 500 g for 10 min. The supernatant bacterial solution from each tube was collected in a biosafety cabinet (pre-exposed to UV light for 20 min) and diluted to 0.1 g / mL for later use (stored at 4°C, used within 3 hours). The diluted fecal solutions (0.1 g / mL) from different donors were mixed in a 1:1 ratio to prepare a mixed diluted fecal bacterial solution (0.1 g / mL) for subsequent sample inoculation. Unused portions were mixed with 30% biological grade glycerol (glycerol and bacterial solution were prepared at a volume ratio of 3:7) and stored at -80°C.

[0118] The basic nutrient medium for in vitro fermentation was prepared according to the following formula: NaCl 4.5 g / L, K2HPO4 2.5 g / L, CaCl2·2H2O 0.45 g / L, MgSO4·7H2O 0.5 g / L, FeSO4·7H2O 0.005 g / L, taurine bile salt 0.05 g / L, cysteine ​​0.4 g / L, bacterial peptone 3.0 g / L, casein 3.0 g / L, 1% resazurin indicator 1 mL / L, heme 0.01 g / L, para-aminobenzoic acid 0.05 g / L, D-biotin 0.002 g / L, vitamin B-12 0.0005 g / L, vitamin K3 0.001 g / L, pantothenic acid 0.01 g / L, nicotinamide 0.005 g / L, and thiamine 0.004 g / L. In vitro fermentation was performed using 5 mL anaerobic fermentation tubes. The original pectin and pectin modified with compound pectinase prepared in Example 1 were dissolved in sterile culture medium to a concentration of 10 mg / mL. A basal nutrient growth medium without any other carbon source was used as a blank control (CON), and fructooligosaccharides (FOS) were used as a positive control. Strict anaerobic operation was performed in an anaerobic fermentation chamber, and diluted fecal supernatant was inoculated into the basal nutrient growth medium for in vitro fermentation (inoculation volume 10% of the total fermentation system volume). The anaerobic fermentation tubes were sealed and cultured at 37°C in a vibrating incubator (100 rpm) for 0, 3, 6, 9, 12, and 24 h to obtain the in vitro fermentation broths of original pectin and pectin modified with compound pectinase.

[0119] II. Changes in total carbohydrate content of in vitro fermentation broth

[0120] 1. Experimental Methods

[0121] The determination of total carbohydrate and uronic acid (UA) content was the same as in Example 2, except that the original pectin and compound pectinase modified pectin were replaced with the in vitro fermentation broth of original pectin and compound pectinase modified pectin.

[0122] 2. Experimental Results

[0123] After 24 hours of in vitro fermentation, there was no significant difference in the residual carbohydrate content of CA-SWP and its compound pectinase-modified pectin. Compared with E-HWP, the carbohydrate content of its compound pectinase-modified pectin was significantly reduced after 24 hours of fermentation, but there was no significant difference between E1-E-HWP and E2-E-HWP. Figure 3 (B in the text). The consumption of total UA was similar to that of total carbohydrates, except that at 24 h, the remaining UA in CA-SWP was significantly higher than that in E2-CA-SWP, but there was no significant difference between E1-CA-SWP and E2-CA-SWP. Figure 3 A in the middle.

[0124] The utilization rates of total carbohydrates and UA were calculated by dividing the consumption in one stage by the remaining amount in the previous stage. In CA-SWP and its compound pectinase-modified pectin, a trend was observed where the utilization of total carbohydrates and UA initially increased and then decreased. Figure 3 (D and E in the original text). The utilization rate of CA-SWP reached its maximum at 9–12 h and then declined, while E1-CA-SWP and E2-CA-SWP reached their maximum at 6–9 h and then began to decline. Interestingly, the carbohydrate and UA utilization rates of E-HWP and its compound pectinase-modified pectin showed an increasing trend over time, and the compound pectinase-modified pectin was higher than E-HWP at each stage. Notably, compared with the original pectin, the compound pectinase-modified pectin had higher carbohydrate and UA utilization rates within 3–9 h.

[0125] In each stage of in vitro fermentation, enzymatic treatment of pectin with higher Mw (e.g., E-HWP) improved the utilization of total carbohydrates and UA, while similar effects were only observed in the early fermentation stages with CA-SWP and its compound pectinase-modified pectin. In conclusion, improvements in pectin fermentation capacity and changes in carbohydrate utilization during fermentation can be achieved through enzymatic hydrolysis.

[0126] III. Changes in gas production and pH value of in vitro fermentation broth

[0127] 1. Experimental Methods

[0128] At the end of fermentation, gas production (mL) was recorded at a specific scale value by inserting a sterile piston-type needle through a rubber stopper into the tube without contacting the culture medium. The pH values ​​of fermentation broth samples at different time points were determined using a micro pH meter 962244 detector (PHS-3C).

[0129] 2. Experimental Results

[0130] The pH value of the CON group fluctuated slightly around 6.0 within 24 hours, without a significant downward trend. Among the six pectin fermentation samples, E-HWP showed the slowest pH decrease. Compared with E-HWP, the pH of the pectin modified by its compound pectinase decreased significantly after 24 hours of in vitro fermentation (p<0.05), with E2-E-HWP being significantly lower than E1-E-HWP (p<0.05). Figure 3 Interestingly, compared to CA-SWP, the pH of the pectin modified by its compound pectinase was significantly higher after 24 h (p<0.05), but there was no significant difference in pH between E1-CA-SWP and E2-CA-SWP. The pH of the fermentation substrate is affected by the content of microbial metabolites such as SCFAs; the higher the total SCFAs content, the lower the pH.

[0131] In summary, the gas production of E-HWP was lower than that of E1-E-HWP and E2-E-HWP at all time points, and there was a significant difference in gas production among E-HWP and its compound pectinase-modified pectin after 24 hours of in vitro fermentation (p<0.05). Figure 3 (F in the text). Furthermore, compared to E1-E-HWP, E2-E-HWP, with its smaller molecular weight, exhibited higher gas production at all time points, and was significantly higher than E1-E-HWP at 24 h. Similar gas production patterns were observed in E1-CA-SWP and E2-CA-SWP, but the gas production of CA-SWP was significantly higher than that of E1-CA-SWP and E2-CA-SWP after 24 h of fermentation. In summary, different pectin samples exhibited significant differences in their fermentation characteristics. Notably, pectinase treatment significantly affected the fermentation characteristics of pectin, particularly pH and gas production.

[0132] IV. Degradation of molecular weight during in vitro fermentation

[0133] 1. Experimental Methods

[0134] The molecular weight determination was the same as in Example 1, except that the original pectin and the compound pectinase-modified pectin were replaced with the in vitro fermentation broth of the original pectin and the compound pectinase-modified pectin.

[0135] 2. Experimental Results

[0136] The results are as follows Figure 4 As shown in Figures A through F, the RI response values ​​of CA-SWP and E1-CA-SWP decreased after 3 hours of in vitro fermentation, while Mw remained unchanged. Over time, the RI response value and Mw of E2-CA-SWP gradually decreased. Compared to CA-SWP, its compound pectinase-modified pectin produced smaller pectin fragments (Mw < 10 kDa) after 6 hours of fermentation and exhibited a higher RI response value.

[0137] In the E-HWP group, the Mw and RI response values ​​of pectin decreased with increasing fermentation time, while some residual pectin fragments with Mw greater than 70 kDa remained after 24 h. The Mw of E1-E-HWP and E2-E-HWP decreased within 3–9 h, then rapidly degraded within 9–12 h, and completely decomposed into smaller fragments (Mw < 10 kDa) within the following 12 h.

[0138] In summary, different pectin samples exhibited unique degradation characteristics during in vitro fermentation. Samples treated with a compound pectinase showed smaller pectin fragments and higher RI response values ​​in the early stages of in vitro fermentation, highlighting the modulating effect of the compound pectinase on the molecular structure and properties of pectin. This further indicates that the use of compound pectinase can affect in vitro degradation efficiency, thereby altering the pectin degradation process.

[0139] V. Utilization and Dynamic Changes in the Structural Pattern of Pectin Monosaccharides during In Vitro Fermentation

[0140] 1. Experimental Methods

[0141] The determination of monosaccharide content was the same as in Example 2, except that the original pectin and compound pectinase modified pectin were replaced with the in vitro fermentation broth of original pectin and compound pectinase modified pectin.

[0142] 2. Experimental Results

[0143] In the fermentation broth of CA-SWP and its compound pectinase-modified pectin, the contents of Gal, Glc, and Man were higher than those of other monosaccharides, and their levels decreased significantly within 24 hours of in vitro fermentation. Figure 5 (A to C in the original text). Similarly, in the fermentation broth of E-HWP and its compound pectinase-modified pectin, the top three monosaccharides were Ara, Gal, and Glc, which significantly decreased within 24 hours of in vitro fermentation. Figure 5 (D~F in the formula). However, the content and utilization rate of other monosaccharides in E-HWP, CA-SWP, and their compound pectinase-modified pectin are relatively low.

[0144] In E1-CA-SWP and E2-CA-SWP, the Rha / UA ratio increased with fermentation time, while in CA-SWP it decreased at 12 h. Figure 5 In the early fermentation stage (0–9 h), the (Ara+Gal) / Rha value in all pectin fermentation broth samples decreased over time. Figure 5 (H in the text). For example Figure 5 As shown in Figure I, the UA / (Fuc+Gal+Ara+Rha+Xyl) value in the E-HWP fermentation broth gradually increased with increasing fermentation time, while E1-E-HWP and E2-E-HWP showed a turning point and decreased at 12 h. In CA-SWP and its compound pectinase-modified pectin, the UA / (Fuc+Gal+Ara+Rha+Xyl) value increased in the early fermentation stage (0–6 h) and then showed a decreasing trend.

[0145] During in vitro fermentation, the monosaccharide content of pectin decreased to varying degrees, indicating that it was effectively utilized by the gut microbiota and reflecting structural changes in pectin during fermentation. Notably, compared to UA, pectin had a lower neutral sugar content, leading to a greater impact of UA degradation on total carbohydrate degradation, and consequently, similar degradation curves for UA and total carbohydrates. Although there were differences in the initial UA or total carbohydrate content in the fermentation broth of different pectin samples, there was no significant correlation between the fluctuating concentration range and the total SCFAs yield at the fermentation endpoint. This phenomenon can be attributed to the influence of pectin's Mw and DM on the final total SCFAs yield, rather than being caused by differences in the total carbohydrate or UA content in the fermentation substrate.

[0146] VI. Short-chain fatty acid formation

[0147] 1. Experimental Methods

[0148] Take 300 μL of the original pectin and the in vitro fermentation broth of pectin modified with compound pectinase, add 300 μL of diethylacetic acid (0.15 mg / mL, dissolved in 0.2 M HCl) and 75 μL of oxalic acid (0.15 M), and mix thoroughly. Centrifuge at 12000 g for 10 min, and transfer the supernatant to a vial for GC analysis.

[0149] Acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid were used as standards, and their processing methods were the same as those for fermentation samples. 2 μL of the supernatant was injected into a gas chromatograph (G2010Plus) equipped with a DB-FFAP column (30 m × 0.53 mm × 1.00 μm) and FID for SCFA determination. Standard curves were constructed using acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid. GC parameters were as follows: nitrogen as carrier gas, initial temperature 100 °C, increased to 160 °C at a rate of 5 °C / min, and held for 4 min.

[0150] 2. Experimental Results

[0151] During the in vitro fermentation of CA-SWP, E-HWP, and their compound pectinase-modified pectin, SCFAs mainly consisted of acetic acid, propionic acid, and butyric acid, with small amounts of valeric acid and isovaleric acid, while the isobutyric acid content was even lower. Compared with CA-SWP, no significant difference was observed in the production of total SCFAs and BCFAs in the compound pectinase-modified pectin within 3 hours of in vitro fermentation. Figure 6 (A-F in the text). However, at 6h and 9h of in vitro fermentation, the total SCFAs generated by E2-CA-SWP were significantly higher than those of CA-SWP or E1-CA-SWP (A-F). Figure 6(G in the text). Furthermore, there was no significant difference in the total SCFAs content between E1-CA-SWP and CA-SWP at these two time points. Subsequently, the total SCFAs production of CA-SWP approached that of E2-CA-SWP at 12 h, and then surpassed it at 24 h. The production trends of acetic acid, propionic acid, and butyric acid were similar to the trends in total SCFAs production.

[0152] Overall, E1-E-HWP and E2-E-HWP produced more SCFAs than E-HWP at all time points during in vitro fermentation, while E2-E-HWP produced more SCFAs than E1-E-HWP at different time points. Similar trends were observed in the production of acetic acid, butyric acid, propionic acid, and isobutyric acid in E-HWP and its compound pectinase-modified pectin. The SCFAs / BCFAs ratio can be used to assess the fermentation patterns of in vitro fermentation sample groups at different time points. The total SCFAs / BCFAs value increased over time in all pectin groups (…). Figure 6 The I in the figure indicates that microorganisms mainly utilize pectin as a carbohydrate energy source during fermentation and metabolize it to produce related metabolites.

[0153] SCFAs play a vital role in maintaining human health, and some bacterial genera can efficiently utilize carbohydrates to produce them. Acetic acid, produced by beneficial bacteria such as *Blautia*, has been shown to enhance gut health and inhibit *Escherichia coli*. Butyrate, a metabolite believed to modulate inflammation and enhance barrier function, is produced by *Lachnospiraceae* UCG-004. *Parabacteroides* possesses the physiological characteristics to metabolize carbohydrates and secrete SCFAs. Generally, increased relative abundance of these bacterial genera favors SCFA production, particularly acetic acid and butyrate. Propionate has been shown to have positive effects on suppressing appetite and combating obesity, while valerate has been shown to promote intestinal epithelial growth and protect the intestinal barrier; their increases may be beneficial for maintaining gut health. The exact mechanisms of the relationship between *Escherichia-Shigella*, *Bilophila*, *Klebsiella*, and SCFAs are not fully understood, but they may compete with other gut bacteria for the nutrients and resources required for SCFA production, thereby inhibiting the overall level of SCFA production.

[0154] Branched-chain fatty acids such as isobutyric acid and isovaleric acid originate from protein breakdown rather than carbohydrates, indicating that the increase in these fatty acids is due to the higher utilization of protein by microorganisms. The SCFAs / BCFAs levels increased with fermentation time in all pectin groups, suggesting that microorganisms in all pectin substrates preferentially utilize pectin as an energy source throughout the fermentation process.

[0155] Therefore, it can be inferred that pectin substrates provide a sufficient carbon source for microbial growth and reproduction throughout the fermentation process, which is crucial for further improving carbohydrate utilization and the production of beneficial metabolites such as SCFAs. Compared with pectin with lower Mw and DM characteristics, higher Mw and DM may hinder the microbial enzymatic degradation of pectin, resulting in lower carbohydrate utilization and lower short-chain fatty acid production. Overall, the DM and Mw values ​​of pectin are negatively correlated with the total SCFA levels, indicating that low Mw and low DM pectin are beneficial for the production of total SCFAs during in vitro fermentation, while high Mw pectin promotes the formation of BCFAs.

[0156] VII. Analysis of the composition of intestinal microbiota during in vitro fermentation

[0157] 1. Experimental Methods

[0158] Microbial DNA was extracted from fermentation broth samples using a fecal genomic DNA extraction kit (Solarbio, CHN) according to the manufacturer's instructions. DNA concentration was determined by agarose gel electrophoresis and NanoDrop2000 (Thermo Fisher, USA). The V3-V4 region of the bacterial 16S rRNA gene was amplified using universal primers 343F: 5'-TACGGRAGGCAGCAG-3' and 798R: 5'-AGGGTATCTAATCCT-3'. Primer sequences were removed from the raw data using cutadapt software. Qualified paired-end raw data underwent quality control analysis, including quality filtering, noise reduction, merging, and chimera removal, processed using the DADA2 method in the default parameters of QIIME 2. Representative sequences and amplicon sequence variant (ASV) abundance tables were obtained. Furthermore, representative sequences for each ASV were selected using QIME2 and annotated by comparison with the Silva database (version 138).

[0159] 2. Experimental Results

[0160] (1) Alpha diversity analysis

[0161] Compared with CON, a significant decrease in the Chao index was observed in CA-SWP, E-HWP, and their corresponding compound pectinase-modified pectins. Figure 7(A) There was no significant difference in the Chao index among E1-CA-SWP, E2-CA-SWP, and CA-SWP, while the Chao index of the pectin modified by the compound pectinase E-HWP was significantly lower than that of E-HWP, but the Chao index of E2-E-HWP was higher than that of E1-E-HWP. Interestingly, after 24 hours of in vitro fermentation, the Simpson index of all pectins was significantly higher than that of CON( Figure 7 (B) There was no significant difference in the Simpson index between CA-SWP and its compound pectinase-modified pectin. The changes in microbial Alpha diversity of CA-SWP and its compound pectinase-modified pectin were smaller than those of E-HWP and its compound pectinase-modified pectin, indicating that the enzymatic modification of CA-SWP had a smaller impact on microbial community stability. In addition, the Simpson index of E1-E-HWP was significantly lower than that of E-HWP or E2-E-HWP. The PCoA plot captured about 80% of the changes in microbial communities among ASVs in all substrates, of which PCoA1, PCoA2, and PCoA3 explained 37.71%, 22.68%, and 19.78%, respectively. Figure 7 (C in the middle).

[0162] Diversity analysis showed that the combined pectinase hydrolysis had a greater impact on the α-diversity of E-HWP compared to the inter-group differences in CA-SWP. PCoA analysis indicated that the inter-group differences in CA-SWP and its combined pectinase-modified pectin were smaller than those in E-HWP and its combined pectinase-modified pectin. In conclusion, the species richness and diversity of microorganisms in fermentation broth can be significantly altered by obtaining low-Mw pectin from the hydrolysis of high-Mw pectin (such as E-HWP).

[0163] (2) Analysis of gut microbiota composition during in vitro fermentation

[0164] Microbial composition analysis after in vitro fermentation showed that, at the phylum level, after 24 hours of in vitro fermentation, the microbial communities in the culture media of each pectin and CON group mainly consisted of Bacteroidota, Firmicutes, Proteobacteria, and Fusobacteriota. Figure 7 (D) The relative abundance of microorganisms in CA-SWP and its compound pectinase-modified pectin, except for Fusobacteriota which decreased with increasing enzyme treatment time, did not show significant changes in the relative abundance of the other three phyla. Compared with E-HWP, E1-E-HWP and E2-E-HWP had higher relative abundance of Bacteroidota, with E2-E-HWP having a higher abundance than E1-E-HWP. Overall, there were significant differences in the microbial community at the phylum level between E-HWP and its compound pectinase-modified pectin.

[0165] At the genus level, compared with the CON-24h group, the addition of different pectins significantly altered the microbial composition in the fermentation broth. Figure 7 (E in the table). The relative abundance changes of some microorganisms are further presented in Table 3. Compared with the CON-24h group, CA-SWP and E-HWP significantly increased the relative abundance of Bacteroides, and different degrees of inhibition or promotion of Bacteroides were observed in their corresponding compound pectinase modified pectin.

[0166] Overall, compared with the CON-24h group, CA-SWP, E-HWP, and their compound pectinase-modified pectin all promoted the relative abundance of Faecalibacterium in the fermentation broth to varying degrees, while inhibiting the relative abundance of Fusobacterium, Escherichia-Shigella, and Sutterella to varying degrees. Furthermore, as shown in Table 3, the original pectin and its compound pectinase-modified pectin had different effects on the relative abundance of Prevotella, Lachnoclostridium, and Klebsiella.

[0167] Table 3. Effects of pectin on the relative abundance (%) of microbial taxonomy at the genus level 24 h after in vitro fermentation

[0168]

[0169] Faecalibacterium is considered a next-generation probiotic that improves gut health by producing butyrate, reducing inflammation, and enhancing intestinal barrier function. Compared to the CON-24h group, the relative abundance of Faecalibacterium in the fermentation broths of raw pectin and its compound pectinase-modified pectin was significantly increased. Bacteroides maintain a complex and generally beneficial relationship with the human body in the gut, possessing a range of carbohydrate-utilizing enzymes that enable them to better utilize polysaccharides and produce large amounts of short-chain fatty acids through fermentation, providing energy to the host. The addition of pectin significantly increased its relative abundance in the fermentation broth. Compared to E-HWP, its compound pectinase-modified pectin had a significant effect on increasing the relative abundance of Bacteroides, and the effect was more pronounced with decreasing Mw. Lachnoclostridium is a potentially pathogenic bacterium whose relative abundance can be regulated by proteins of animal or plant origin. Notably, except for the high-Mw E-HWP, the relative abundance of Lachnoclostridium was decreased in all pectin groups compared to CON-24h. Klebsiella is considered a population-controlled gut commensal bacterium, but some of its species, such as Klebsiella pneumoniae, are often opportunistic pathogens. Compared to the CON-24h group, low-Mw CA-SWP pectin and its compound pectinase-modified pectin significantly reduced the relative abundance of Klebsiella.

[0170] The relative abundance of the top 30 microorganisms at the genus level in all pectin fermentation groups is shown in the heatmap. Figure 7 The results are shown in F). Overall, the microbial composition at the genus level changed significantly between the original pectin and the pectin modified with compound pectinase, especially in E-HWP and its compound pectinase-modified pectin. Compared with CA-SWP, the relative abundance of Lachnospira, Dorea, and Tyzzerella increased in E2-CA-SWP. Compared with E-HWP, the relative abundance of Subdoligranulum, Lachnoclostridium, and Coridextribacter decreased in E1-E-HWP and E2-E-HWP, while the relative abundance of Enterobacter and Bacteroides increased. Although E2-E-HWP and E2-CA-SWP have similar molecular weights, heatmap analysis showed significant differences in their microbial composition at the genus level. Overall, compared to E-HWP and its compound pectinase-modified pectin, CA-SWP and its compound pectinase-modified pectin can significantly increase the relative abundance of probiotics such as Faecalibacterium and inhibit the growth of pathogenic bacteria such as Escherichia-Shigella, which is beneficial to the gut microbiota.

[0171] In summary, in vitro fermentation of different types of pectin and pectin modified with compound pectinase significantly impacts the composition of the gut microbiota, particularly regulating the abundance of specific bacterial genera. These genera include probiotics, potentially pathogenic bacteria, and symbiotic bacteria in controlled quantities. Notably, the chemical properties of different pectins and the methods of compound pectinase treatment lead to variations in these effects on the microbial community. Therefore, strategies for promoting gut health and microbial balance require comprehensive consideration of factors such as pectin type and degree of enzymatic hydrolysis to achieve optimal results.

[0172] VIII. Correlation Analysis of Pectin Properties, Metabolites, and Gut Microbiota

[0173] 1. Experimental Methods

[0174] Correlation analysis was employed to investigate the relationships between different variables. Data related to the top 20 bacterial genera, carbohydrate content of pectin, Mw, and DM were collected, ensuring data completeness and accuracy. To accurately assess the associations between these variables, classic Pearson correlation analysis was chosen. In the actual analysis, Pearson correlation coefficients were calculated for each pair of variables, providing quantitative information about the relationships between different variables. A positive correlation coefficient indicates that as one variable increases, the other also increases; while a negative correlation coefficient indicates that an increase in one variable is accompanied by a decrease in the other. To determine whether the correlation was significant, p-values ​​of the correlation coefficients were calculated. Generally, when p < 0.05, the observed correlation can be considered significant, meaning the association between the variables is not accidental.

[0175] 2. Experimental Results

[0176] Figure 8 Figure A shows the correlations between the top 20 bacterial genera, carbohydrates in the fermentation broth substrate, and the corresponding Mw and DM of pectin. In summary, the effects of different monosaccharides on the relative abundance of the microbial community differed significantly at the genus level. Specifically, Gal, UA, and Escherichia-Shigella were negatively correlated, while Blautia and Parasturtella were positively correlated. Furthermore, the Mw and DM of pectin were significantly positively correlated with Colidextribacter and Sutterella, but significantly negatively correlated with Prevotella.

[0177] Figure 8Figure B shows the correlation between the top 20 bacterial genera and SCFA yields in the fermentation substrate after 24 hours of in vitro fermentation. On the one hand, *Blautia*, *Parabacteroides*, and *Lachnospiraceae_UCG-004* showed significant positive correlations with acetic acid, butyric acid, and total SCFAs. On the other hand, *Escherichia-Shigella*, *Bilophila*, and *Klebsiella* showed significant negative correlations with acetic acid, butyric acid, and total SCFAs.

[0178] Figure 8 The C-cell analysis examined the correlation between carbohydrate content, short-chain fatty acid (SCFA) yield, and pectin DM and Mw in various pectin fermentation substrates. The results showed that pectin Mw and DM were positively correlated with isovaleric acid and total branched-chain fatty acids (BCFAs), but negatively correlated with acetic acid, propionic acid, and butyric acid. Notably, there was no significant correlation between total carbohydrate and UA content and total SCFAs in the fermentation substrates. Different monosaccharides had different effects on SCFA yield; for example, Gal was significantly positively correlated with acetic acid and butyric acid, while Ara and Glc were significantly negatively correlated.

[0179] Sutterella is a widespread commensal bacterium in the human gastrointestinal tract with mild pro-inflammatory capabilities. Notably, Sutterella showed a significant positive correlation with the Mw and DM of pectin. This indicates that pectin modified with compound pectinase, characterized by low Mw and low DM, can effectively reduce the relative abundance of Sutterella compared to undiluted pectin. Escherichia-Shigella is generally considered pro-inflammatory and is a major pathogen associated with infectious diarrhea. Significance analysis showed that Gal and UA inhibited the proliferation of Escherichia-Shigella and promoted the growth of Blautia and Parasturtella. Fusobacterium is a genus of bacteria that includes many commensal species, but also some pathogens, such as Fusobacterium nucleatum. Compared to the CON-24h group, the relative abundance of these potential pathogens was significantly suppressed after the addition of pectin. Prevotella is a commensal bacterium in the gut that participates in the breakdown of polysaccharides, some of which have beneficial effects on glucose metabolism, while others have pathogenic properties that promote inflammatory diseases. The relative abundance of Prevotella was negatively correlated with the Mw of pectin, indicating that low-Mw pectin obtained through enzymatic hydrolysis had an inhibitory effect on it.

[0180] Correlation analysis showed a negative correlation between the molecular weight (Mw) of pectin and the total SCFA content. Throughout the in vitro fermentation process, pectin modified with low Mw by the compound pectinase from E-HWP produced higher total SCFAs compared to E-HWP. This was because the Mw of the compound pectinase-modified pectin gradually decreased with increasing hydrolysis time. In contrast, pectin modified with low Mw by CA-SWP and its compound pectinase significantly increased the total SCFA production in the early stages of in vitro fermentation, which was related to the rapid decrease in Mw within the first 6 hours of fermentation. Therefore, CA-SWP compound pectinase-modified pectin can significantly increase the total short-chain fatty acid yield in the early stages of in vitro fermentation and has a faster promoting effect on intestinal fermentation; while E-HWP compound pectinase-modified pectin only promotes the later stages of fermentation. Overall, compared to E-HWP, pectin with low Mw produced by compound pectinase hydrolysis can increase the yield and rate of SCFAs during in vitro fermentation. However, if the original pectin has a relatively low Mw, enzymatic hydrolysis into pectin with an even lower Mw will only promote the total SCFAs production and rate in the early fermentation stages.

[0181] Overall, the regulatory effect of pectin on the gut microbiota is closely related to changes in pectin DM, Mw, structural properties, and sugar composition caused by enzymatic hydrolysis with compound pectinase. Although E2-CA-SWP and E2-E-HWP have similar Mw and DM values, differences in their structure and composition lead to different microbial compositions. Compared with the original pectin sample, pectin modified with compound pectinase, which has lower Mw and DM values, significantly promotes the growth of potential probiotics and inhibits the proliferation of potential pathogens, resulting in a healthier gut microbiota. Furthermore, enzymatic modification of the original pectin E-HWP, which has a higher Mw, has a greater impact on the microbiota than CA-SWP. CA-SWP and its compound pectinase-modified pectin have less impact on gut microbiota stability than E-HWP and its compound pectinase-modified pectin, and can more effectively promote the growth of probiotics, inhibit the proliferation of pathogens, and accelerate and increase the production of short-chain fatty acids.

[0182] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of a bio-enzyme-modified pectin in the preparation of a drug that promotes the growth of probiotics, characterized in that, The probiotics are Tyzzerella , The preparation method of the bio-enzyme modified pectin includes the following steps: S1. Degrease the Shatin pomelo peel powder to obtain defatted Shatin pomelo peel powder; S2. The defatted pomelo peel powder and citric acid were thoroughly mixed at a ratio of 1 g: (18-22) mL, and the resulting mixture was subjected to subcritical extraction. S3. Centrifuge the extract and adjust the pH to neutral, then perform alcohol precipitation and solid-liquid separation to obtain the crude precipitate; S4. Dissolve the crude extract precipitate fully in water, dialyze it using an 8-14 kDa dialysis bag, and then freeze-dry it to obtain pectin from Shatin pomelo peel. S5. Dissolve the pectin from the peel of Shatin pomelo completely in a buffer solution and adjust the pH to 4-5; S6. The pectin solution of Shatin pomelo peel is subjected to enzymatic hydrolysis, followed by heating inactivation and solid-liquid separation. The liquid is then dialyzed and freeze-dried to obtain the enzyme-modified pectin. The bioenzymes are pectin esterase, polygalacturonase, and pectin lyase. The enzymatic hydrolysis time is 0.5–2.5 h; The enzymatic hydrolysis time was 0.5 h, and the monosaccharide molar ratio of the obtained bio-enzyme modified pectin was: rhamnose: fucose: arabinose: xylose: mannose: galactose: glucose: uronic acid = (0.92±0.0): (0.10±0.0): (0.97±0.2): (0.54±0.1): (0.55±0.2): (1.94±0.1): (1.94±0.2): (91.41±3.1); The enzymatic hydrolysis time was 2.5 h, and the monosaccharide molar ratio of the obtained bio-enzyme modified pectin was: rhamnose: fucose: arabinose: xylose: mannose: galactose: glucose: uronic acid = (1.00±0.0): (0.11±0.0): (1.06±0.3): (0.49±0.2): (0.59±0.1): (1.47±0.1): (1.44±0.1): (91.63±2.0).

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