A modification method for specific pomelo pulp pomace dietary fiber

Abstract

The present invention discloses a method for modifying specific pomelo pomace dietary fiber, belonging to the technical field of dietary fiber modification. The method for modifying specific pomelo pomace dietary fiber disclosed by the present invention uses pomelo pomace as raw material, undergoes a boiling water bath, cellulase hydrolysis, papain hydrolysis, alcohol precipitation, and freeze-drying to obtain pomelo pomace soluble dietary fiber; inoculates activated Clostridium butyricum into RCM liquid culture medium supplemented with pomelo pomace soluble dietary fiber for fermentation, controlling the inoculation amount, fermentation time, fermentation temperature, and substrate concentration; and then freeze-spray-drying to obtain modified pomelo pomace dietary fiber. The method for modifying specific pomelo pomace dietary fiber disclosed by the present invention provides a modified pomelo pomace dietary fiber that has excellent α-amylase activity inhibition and excellent digestion resistance, and inhibits α-amylase activity by regulating intestinal flora.

Description

Technical Field

[0001] The invention relates to the technical field of dietary fiber modification, and more particularly to a method for modifying specific pomelo pulp and pomace dietary fiber. Background Art

[0002] Dietary fiber, known as the seventh nutrient, has physiological functions such as regulating intestinal flora, preventing intestinal diseases, lowering blood lipids and cholesterol, and reducing the risk of cardiovascular disease. It is widely used in functional or health foods. Currently, dietary fiber mainly comes from fruits, vegetables, nuts, grains and their by-products. As one of the most widely consumed fruits in the world, pomelo produces a large number of by-products during its processing, such as pulp residue. A large amount of by-products are directly discarded into landfills, causing serious environmental pollution, and inefficient treatment methods result in high costs. Pomelo pulp and pomace contain a large amount of soluble dietary fiber that is beneficial to the human body, making it an excellent source of dietary fiber raw materials. However, due to the excessively long molecular chains and high molecular weight of dietary fiber, most of it cannot be utilized by the intestinal flora after being digested in the human gastrointestinal tract and reaching the colon, affecting its biological activity.

[0003] Therefore, providing a method for modifying specific pomelo pulp and pomace dietary fiber is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0004] In view of this, the present invention provides a method for modifying specific pomelo pulp and pomace dietary fiber. The prepared modified pomelo pulp and pomace dietary fiber has good inhibitory effect on α-amylase activity and regulation of intestinal flora activity.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for modifying specific pomelo pulp pomace dietary fiber comprises the following steps:

[0007] (1) Using pomelo pulp pomace as raw material, the process is subjected to a boiling water bath, cellulase hydrolysis, papain hydrolysis, alcohol precipitation, and freeze-drying to obtain pomelo pulp pomace soluble dietary fiber;

[0008] (2) inoculating the activated Clostridium butyricum into RCM liquid medium (clostridium proliferation medium) added with soluble dietary fiber from pomelo pulp and pomace for fermentation, controlling the inoculation amount, fermentation time, fermentation temperature, and substrate concentration; and then freeze-spray drying to obtain modified pomelo pulp and pomace dietary fiber;

[0009] The modified pomelo pulp pomace dietary fiber consists of t-Araf, t-Glcp, 6-Glcp, 4-Glcp, 3,4-Glcp, 4,6-Glcp, 3,6-Galp, 2-Glcp, and 3-Galp, with a molar ratio of 3.60:4.10:2.80:9.30:1.0:3.20:2.60:1.50:1.40.

[0010] Furthermore, step (1) specifically includes the following steps:

[0011] (1-1) Dried pomelo pulp is ultrafinely ground at a working pressure of 0.5-0.7 MPa and a sorting frequency of 15-25 Hz to obtain pomelo pulp pomace; the pomelo pulp pomace is mixed with deionized water at a ratio of 1:10-20 (w / v), 10-15% (v / v) acetic acid-sodium acetate buffer is added, and the mixture is mixed thoroughly. The mixture is incubated at 80-100°C for 1-2 hours and cooled to 25°C to obtain a mixed solution A;

[0012] (1-2) Add 0.03-0.05% (w / v) cellulase to mixed solution A, react at 40-50°C for 1.0-2.0 h, terminate the enzyme reaction by treating at 90°C for 10 min, and cool to 25°C to obtain mixed solution B; the cellulase activity is 200 U / mL, and the pH is 4.5-5.5;

[0013] (1-3) Add 0.05-0.07% (w / v) papain to mixed solution B, react in a water bath at 50-70°C for 30-60 minutes, then terminate the reaction by treating at 90°C for 10 minutes, and cool to 25°C to obtain mixed solution C; the papain has an enzyme activity of 100 U / mL and a pH of 6.0-7.0;

[0014] (1-4) adding 85-95% (v / v) ethanol at a volume ratio of 3-4:1 to the mixed solution C, leaving the mixture overnight to obtain a precipitate, and performing centrifugal freeze-drying to obtain soluble dietary fiber from pomelo pulp and pomace.

[0015] The beneficial effects of the above technical solution are: if the amount of pomelo pulp and pomace added is too much, the yield and purity of dietary fiber from the pomace will be reduced; if the amount of pomelo pulp and pomace added is too little, the extraction efficiency will be reduced.

[0016] Adding too much cellulase and papain will increase the cost; adding too little will reduce the purity of dietary fiber in pomelo pulp and pomace.

[0017] Furthermore, step (2) specifically includes the following steps:

[0018] (2-1) Inoculate Clostridium butyricum into RCM liquid culture medium and culture it at 35-40℃ for 48-72h to obtain activated Clostridium butyricum. 600 =1.50-1.70;

[0019] (2-2) 0.4-0.6% (v / v) activated Clostridium butyricum is inoculated into RCM liquid culture medium in which glucose is replaced by 1.0-2.0% (w / v) soluble dietary fiber from pomelo pulp and pomace. The culture medium is reacted at 35-40° C. for 30-40 hours. The modified pomelo pulp and pomace dietary fiber is then prepared by freeze spray drying.

[0020] The beneficial effects of the above technical solution are: if the activation time is too short, the strain is in a hysteresis period, while if the time is too long, the strain is in a decline period, both of which affect the activity of the strain.

[0021] The inoculation amount, the amount of dietary fiber added from pomelo pulp and pomace, the reaction temperature and time all affected the structure of the modified dietary fiber, thereby affecting the inhibition of α-amylase activity.

[0022] Furthermore, the composition of the RCM liquid culture medium is as follows: yeast extract 3 mg / mL, beef extract 10 mg / mL, tryptone 10 mg / mL, glucose 5 mg / mL, soluble starch 1 mg / mL, sodium chloride 5 mg / mL, sodium acetate trihydrate 3 mg / mL, cysteine hydrochloride 0.5 mg / mL, 0.01% methylene blue, pH 7.0.

[0023] Furthermore, the modified pomelo pulp and pomace dietary fiber is prepared by the method.

[0024] Furthermore, the modified pomelo pulp pomace dietary fiber is used to inhibit the activity of α-amylase.

[0025] Furthermore, the modified pomelo pulp pomace dietary fiber is used in regulating intestinal flora.

[0026] Furthermore, the modified pomelo pulp pomace dietary fiber inhibits the proliferation of Shigella, Enterobacter cloacae, Acidaminococcus, Dorsalella, and Megamonas, and promotes the growth of Lactobacillus, Bifidobacterium, and Collinsella.

[0027] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses a method for modifying a specific pomelo pulp pomace dietary fiber. The modified pomelo pulp pomace dietary fiber prepared has a specific sugar chain structure. The pomelo pulp pomace dietary fiber with this structure has excellent α-amylase activity inhibition and excellent digestion resistance, and inhibits α-amylase activity by regulating intestinal flora. The preparation method of the modified pomelo pulp pomace dietary fiber of the present invention is simple, and the prepared specific pomelo pulp pomace can effectively regulate α-amylase activity. DETAILED DESCRIPTION

[0028] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0029] Example 1

[0030] A method for modifying specific pomelo pulp pomace dietary fiber comprises the following steps:

[0031] Dried pomelo pulp was ultrafinely ground (working pressure 0.6 MPa, sorting frequency 20 Hz) to obtain pomelo pomace. The pomelo pomace was mixed with deionized water at a ratio of 1:10 (w / v), and 10% (v / v) acetic acid-sodium acetate buffer (0.2 mol / L, pH 4.6) was added and mixed. The mixture was incubated at 100°C for 1 h and cooled to 25°C to obtain mixed solution A.

[0032] 0.03% (w / v) cellulase (200 U / mL, pH = 4.9) was added to the mixed solution A, reacted at 50°C for 1.5 hours, then treated at 90°C for 10 minutes to terminate the enzyme reaction, and cooled to 25°C to obtain mixed solution B;

[0033] 0.06% (w / v) papain (100 U / mL, pH 6.5) was added to mixed solution B, and the mixture was reacted in a water bath at 60°C for 30 min. The enzyme reaction was terminated by treating at 90°C for 10 min, and the mixture was cooled to 25°C to obtain mixed solution C.

[0034] 95% (v / v) ethanol was added to the mixed solution C at a volume ratio of 4:1, and the mixture was allowed to stand overnight to obtain a precipitate, which was then centrifuged and freeze-dried to obtain pomelo pulp and pomace dietary fiber.

[0035] The preserved Clostridium butyricum (CGMCC 1.5205) was inoculated into RCM liquid medium (3 mg / mL yeast extract, 10 mg / mL beef extract, 10 mg / mL tryptone, 5 mg / mL glucose, 1 mg / mL soluble starch, 5 mg / mL sodium chloride, 3 mg / mL sodium acetate trihydrate, 0.5 mg / mL cysteine hydrochloride, 0.01% methylene blue, pH 7.0) and cultured anaerobically at 37°C for 48 h to obtain activated Clostridium butyricum. Its OD 600 =1.70;

[0036] 0.5% (v / v) activated Clostridium butyricum was inoculated into RCM culture medium with 1.5% (w / v) grapefruit pomace dietary fiber replacing glucose, reacted at 37°C for 36 hours, and freeze-spray dried to prepare modified grapefruit pomace dietary fiber; its structure was identified as mainly composed of t-Araf, t-Glcp, 6-Glcp, 4-Glcp, 3,4-Glcp, 4,6-Glcp, 3,6-Galp, 2-Glcp, and 3-Galp, with a molar ratio of 3.60:4.10:2.80:9.30:1.0:3.20:2.60:1.50:1.40.

[0037] Example 2

[0038] A method for modifying specific pomelo pulp pomace dietary fiber comprises the following steps:

[0039] Dried pomelo pulp was ultrafinely ground (working pressure 0.5 MPa, sorting frequency 25 Hz) to obtain pomelo pulp pomace. The pomelo pulp pomace was mixed with deionized water at a ratio of 1:15 (w / v), and 15% (v / v) acetic acid-sodium acetate buffer (0.2 mol / L, pH 4.6) was added and mixed. The mixture was incubated at 80°C for 1.5 h and cooled to 25°C to obtain mixed solution A.

[0040] 0.04% (w / v) cellulase (200 U / mL, pH = 4.5) was added to the mixed solution A, reacted at 40°C for 1.0 h, then treated at 90°C for 10 min to terminate the enzyme reaction, and cooled to 25°C to obtain mixed solution B;

[0041] 0.05% (w / v) papain (100 U / mL, pH = 6.0) was added to mixed solution B, and the mixture was reacted in a water bath at 50°C for 30 minutes. The enzyme reaction was terminated by treating at 90°C for 10 minutes, and the mixture was cooled to 25°C to obtain mixed solution C.

[0042] 85% (v / v) ethanol was added to the mixed solution C at a volume ratio of 3:1, and the mixture was allowed to stand overnight to obtain a precipitate, which was then centrifuged and freeze-dried to obtain pomelo pulp and pomace dietary fiber.

[0043] The preserved Clostridium butyricum (CGMCC 1.5205) was inoculated into RCM liquid medium (3 mg / mL yeast extract, 10 mg / mL beef extract, 10 mg / mL tryptone, 5 mg / mL glucose, 1 mg / mL soluble starch, 5 mg / mL sodium chloride, 3 mg / mL sodium acetate trihydrate, 0.5 mg / mL cysteine hydrochloride, 0.01% methylene blue, pH 7.0) and cultured anaerobically at 35°C for 48 h to obtain activated Clostridium butyricum. Its OD 600 =1.60;

[0044] 0.4% (v / v) activated Clostridium butyricum was inoculated into RCM culture medium with 1.0% (w / v) grapefruit pomace dietary fiber replacing glucose, reacted at 35°C for 30 hours, and freeze-spray dried to prepare modified grapefruit pomace dietary fiber; its structure was identified to be mainly composed of t-Araf, t-Glcp, 6-Glcp, 4-Glcp, 3,4-Glcp, 4,6-Glcp, 3,6-Galp, 2-Glcp, and 3-Galp, with a molar ratio of 3.60:4.10:2.80:9.30:1.0:3.20:2.60:1.50:1.40.

[0045] Example 3

[0046] A method for modifying specific pomelo pulp pomace dietary fiber comprises the following steps:

[0047] Dried pomelo pulp was ultrafinely ground (working pressure 0.7 MPa, sorting frequency 15 Hz) to obtain pomelo pomace. The pomelo pomace was mixed with deionized water at a ratio of 1:20 (w / v), and 15% (v / v) acetic acid-sodium acetate buffer (0.2 mol / L, pH 4.6) was added and mixed. The mixture was incubated at 90°C for 2 h and cooled to 25°C to obtain mixed solution A.

[0048] 0.05% (w / v) cellulase (200 U / mL, pH = 5.5) was added to the mixed solution A, reacted at 50°C for 2.0 h, then treated at 90°C for 10 min to terminate the enzyme reaction, and cooled to 25°C to obtain mixed solution B;

[0049] 0.07% (w / v) papain (100 U / mL, pH = 7.0) was added to mixed solution B, and the mixture was reacted in a water bath at 70°C for 60 min. The enzyme reaction was terminated by treating at 90°C for 10 min, and the mixture was cooled to 25°C to obtain mixed solution C.

[0050] 90% (v / v) ethanol was added to the mixed solution C at a volume ratio of 3.5:1, and the mixture was allowed to stand overnight to obtain a precipitate, which was then centrifuged and freeze-dried to obtain pomelo pulp pomace dietary fiber.

[0051] The preserved Clostridium butyricum (CGMCC 1.5205) was inoculated into RCM liquid medium (3 mg / mL yeast extract, 10 mg / mL beef extract, 10 mg / mL tryptone, 5 mg / mL glucose, 1 mg / mL soluble starch, 5 mg / mL sodium chloride, 3 mg / mL sodium acetate trihydrate, 0.5 mg / mL cysteine hydrochloride, 0.01% methylene blue, pH 7.0) and cultured anaerobically at 40°C for 72 h to obtain activated Clostridium butyricum. Its OD 600 =1.50;

[0052] 0.6% (v / v) activated Clostridium butyricum was inoculated into RCM culture medium with 2.0% (w / v) grapefruit pomace dietary fiber replacing glucose, reacted at 40°C for 40 h, and freeze-spray dried to prepare modified grapefruit pomace dietary fiber; its structure was identified as mainly composed of t-Araf, t-Glcp, 6-Glcp, 4-Glcp, 3,4-Glcp, 4,6-Glcp, 3,6-Galp, 2-Glcp, and 3-Galp, with a molar ratio of 3.60:4.10:2.80:9.30:1.0:3.20:2.60:1.50:1.40.

[0053] Comparative Example 1

[0054] A method for modifying pomelo pulp pomace dietary fiber comprises the following steps:

[0055] Dried pomelo pulp was ultrafinely ground (working pressure 0.6 MPa, sorting frequency 20 Hz) to obtain pomelo pomace. The pomelo pomace was mixed with deionized water at a ratio of 1:10 (w / v), and 10% (v / v) acetic acid-sodium acetate buffer (0.2 mol / L, pH 4.6) was added and mixed. The mixture was incubated at 100°C for 1 h and cooled to 25°C to obtain mixed solution A.

[0056] 0.03% (w / v) cellulase (200 U / mL, pH = 4.9) was added to the mixed solution A, reacted at 50°C for 1.5 hours, then treated at 90°C for 10 minutes to terminate the enzyme reaction, and cooled to 25°C to obtain mixed solution B;

[0057] 0.06% (w / v) papain (100 U / mL, pH 6.5) was added to mixed solution B, and the mixture was reacted in a water bath at 60°C for 30 min. The enzyme reaction was terminated by treating at 90°C for 10 min, and the mixture was cooled to 25°C to obtain mixed solution C.

[0058] 95% (v / v) ethanol was added to the mixed solution C at a volume ratio of 4:1, and the mixture was allowed to stand overnight to obtain a precipitate, which was then centrifuged and freeze-dried to obtain pomelo pulp and pomace dietary fiber.

[0059] Comparative Example 2 (physical method modified dietary fiber)

[0060] A method for modifying pomelo pulp pomace dietary fiber comprises the following steps:

[0061] Dried pomelo pulp was ultrafinely ground (working pressure 0.6 MPa, sorting frequency 20 Hz) to obtain pomelo pomace. The pomelo pomace was mixed with deionized water at a ratio of 1:10 (w / v), and 10% (v / v) acetic acid-sodium acetate buffer (0.2 mol / L, pH 4.6) was added and mixed. The mixture was incubated at 100°C for 1 h and cooled to 25°C to obtain mixed solution A.

[0062] 0.03% (w / v) cellulase (200 U / mL, pH = 4.9) was added to the mixed solution A, reacted at 50°C for 1.5 hours, then treated at 90°C for 10 minutes to terminate the enzyme reaction, and cooled to 25°C to obtain mixed solution B;

[0063] 0.06% (w / v) papain (100 U / mL, pH 6.5) was added to mixed solution B, and the mixture was reacted in a water bath at 60°C for 30 min. The enzyme reaction was terminated by treating at 90°C for 10 min, and the mixture was cooled to 25°C to obtain mixed solution C.

[0064] 95% (v / v) ethanol was added to the mixed solution C at a volume ratio of 4:1, and the mixture was allowed to stand overnight to obtain a precipitate, which was then centrifuged and freeze-dried to obtain pomelo pulp and pomace dietary fiber.

[0065] A 1.5% (w / v) aqueous solution of pomelo pulp and pomace dietary fiber was placed in an ultrahigh pressure chamber and maintained at a pressure of 400 MPa for 60 minutes, and then freeze-spray-dried to obtain physically modified pomelo pulp and pomace dietary fiber.

[0066] Comparative Example 3 (Chemically Modified Dietary Fiber)

[0067] A method for modifying pomelo pulp pomace dietary fiber comprises the following steps:

[0068] Dried pomelo pulp was ultrafinely ground (working pressure 0.6 MPa, sorting frequency 20 Hz) to obtain pomelo pomace. The pomelo pomace was mixed with deionized water at a ratio of 1:10 (w / v), and 10% (v / v) acetic acid-sodium acetate buffer (0.2 mol / L, pH 4.6) was added and mixed. The mixture was incubated at 100°C for 1 h and cooled to 25°C to obtain mixed solution A.

[0069] 0.03% (w / v) cellulase (200 U / mL, pH = 4.9) was added to the mixed solution A, reacted at 50°C for 1.5 hours, then treated at 90°C for 10 minutes to terminate the enzyme reaction, and cooled to 25°C to obtain mixed solution B;

[0070] 0.06% (w / v) papain (100 U / mL, pH 6.5) was added to mixed solution B, and the mixture was reacted in a water bath at 60°C for 30 min. The enzyme reaction was terminated by treating at 90°C for 10 min, and the mixture was cooled to 25°C to obtain mixed solution C.

[0071] 95% (v / v) ethanol was added to the mixed solution C at a volume ratio of 4:1, and the mixture was allowed to stand overnight to obtain a precipitate, which was then centrifuged and freeze-dried to obtain pomelo pulp and pomace dietary fiber.

[0072] A 1.5% (w / v) aqueous solution of pomelo pulp and pomace dietary fiber was reacted with 5% (v / v) 1 mol / L NaOH for 6 h, and then freeze-spray-dried to prepare chemically modified pomelo pulp and pomace dietary fiber.

[0073] Test example

[0074] (1) Methylation analysis

[0075] The SDF samples (2 mg) prepared in Examples 1-3 and Comparative Examples 1-3 were mixed with dried NaOH powder (40 mg) and dissolved in 1 mL of anhydrous dimethyl sulfoxide (DMSO). The methylation reaction was then terminated with 1 mL of methyl iodide and 2 mL of distilled water. After reacting with 4 mol / L trifluoroacetic acid (TFA, 0.5 mL) for 1.5 h, the methylated SDF was mixed with 60 mg of NaBH₄ for 8 h, followed by the addition of 2 mL of dichloromethane. The samples were then analyzed using a gas chromatography-mass spectrometer. GC-MS conditions included: Column: RXI-5SIL MS (30 m × 0.25 mm × 0.25 μm); programmed heating conditions: starting temperature of 120°C, heating from 3°C / min to 250°C / min, and holding for 5 min; injection temperature of 250°C; detector temperature of 250°C / min; carrier gas: helium at a rate of 1 mL / min.

[0076] (2) Determination of α-amylase activity

[0077] Pancreatic α-amylase solution (150 U / mL, 0.1 mL) was mixed with 2 mL of 2% potato starch solution (w / v) and various SDF solutions (0.4 mL, 10 mg / mL). For a blank control, 0.4 mL of deionized water was used instead of the SDF solution. The mixture was incubated at pH 7.0 and 37°C for 1 hour. The reaction was terminated by the addition of 2.0 mL of 0.1 mol / L NaOH. Each mixture was centrifuged at 4800 × g for 10 minutes, and the supernatant was collected for glucose determination using the dinitrosalicylic acid method.

[0078] The structure and α-amylase inhibition activity of the modified pomelo pulp dietary fiber (SDF) prepared in Examples 1-3 and Comparative Examples 1-3 were analyzed and tested. The test results are shown in Table 1.

[0079] Table 1 Dietary fiber structure of modified pomelo pulp and inhibition of α-amylase activity

[0080]

[0081]

[0082] Note: NA means that the glycosidic bond does not exist in the modified pomelo pulp SDF.

[0083] As can be seen from Table 1, Examples 1-3 have the same monosaccharide composition and the molar ratio of each sugar residue is consistent; while Comparative Examples 1-3 have a different sugar chain structure from Examples 1-3, resulting in their α-amylase inhibitory activity being significantly lower than that of Examples 1-3, indicating that the modified pomelo pulp pomace dietary fiber with a specific structure prepared by the present invention has good application prospects in terms of α-amylase inhibitory activity.

[0084] In addition, the present invention also specifically tests and analyzes various performance indicators of the prepared modified pomelo pulp pomace dietary fiber. The test standards and analysis results are as follows:

[0085] (3) In vitro simulated digestion

[0086] First, simulated saliva fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) were prepared. The formulas are shown in Table 2.

[0087] Table 2 Preparation of simulated digestion stock solution

[0088]

[0089] To simulate salivary digestion, 1 g of SDF was dissolved in 4 mL of distilled water. This solution was then mixed with 3.5 mL of SSF, 0.5 mL of 75 U / mL α-amylase, 25 μL of 0.3 mol / L CaCl₂ solution, and 975 μL of distilled water. The mixture was then incubated in a shaking waterbath at 37°C for 2–5 min to obtain a salivary digestion solution. To simulate gastric digestion, 7.5 mL of SGF, 1.6 mL of 2000 U / mL porcine pepsin, 5 μL of 0.3 mol / L CaCl₂, and 695 μL of distilled water were added to the salivary digestion solution. The pH of the mixture was adjusted to 2.0 with 6 mol / L HCl, and the mixture was then incubated in a 37°C waterbath for 2 h. Intestinal digestion was then continued by adding 11 mL of SIF, 5.0 mL of 100 U / mL trypsin, 2.5 mL of 0.04 mg / mL bile, 40 μL of 0.3 mol / L CaCl₂, and 1.31 mL of distilled water. After adjusting the pH to 7.0 with 1 mol / L NaOH, the reaction was continued in a 37°C water bath for 2 h. The flasks were flushed with N2 to remove air during each reaction step. At the end of each step, aliquots were collected and centrifuged at 4°C (10,000 g, 5 min). The supernatant was lyophilized and stored at -20°C until further analysis. The results are shown in Table 3. The residue obtained after intestinal digestion was then lyophilized and used for colonic fermentation.

[0090] Table 3 Simulated digestion characteristics of dietary fiber from pomelo pulp and pomace

[0091]

[0092] As shown in Table 3, Examples 1-3 were resistant to degradation in saliva; only a small amount of degradation occurred in gastric juice, with a retention rate of over 94%; and after digestion in intestinal fluid, the retention rate was over 88%. In contrast, the unmodified Comparative Example 1 was significantly degraded after gastrointestinal digestion, with a retention rate of only 43%. Compared to Examples 1-3, Comparative Examples 2 and 3 were also significantly degraded after gastrointestinal digestion.

[0093] (4) In vitro fecal fermentation

[0094] Fecal samples were collected using the same criteria as for fresh stool samples. Fresh stool was collected from four healthy adult volunteers (two men and two women) on the same morning. The volunteers had no recent history of gastrointestinal illness and had not taken antibiotics or probiotics for at least three months. The experiment began within two hours of the initial stool collection to ensure fresh fecal microbiota. All subsequent manipulations were performed in an anaerobic chamber. After stool sample collection, 30 g of the middle portion of each sample was sampled, for a total of 120 g of stool sample.

[0095] Human fecal pre-culture medium consists of 10 g tryptone, 5 g yeast, 10 g sodium chloride, 5 g glucose, and 6 g maltose, dissolved in distilled water to a final volume of 1 L and sterilized at 121°C for 15 min. Under anaerobic conditions, 120 g of human fecal sample was added to the human fecal pre-culture medium to a final volume of 600 mL. The mixture was immediately stirred thoroughly with a sterilized glass rod and filtered through a double layer of medical gauze to prepare a 20% (w / v) fecal suspension. The suspension was then anaerobically pre-cultured in a TC-2212B thermostat at 37°C and 160 rpm for 48 h.

[0096] Modified pomelo pulp and pomace dietary fiber were subjected to in vitro colonic fermentation with human feces. The fermentation medium (1 L) contained 2.0 g peptone, 2.0 g yeast extract, 0.1 g sodium chloride, 2.0 g NaHCO₃, 0.01 g MgSO₄·7H₂O, 0.5 g L-cysteine hydrochloride, 0.04 g KH₂PO₄, 0.04 g K₂HPO₄, 0.5 g bile salts, 5.1 mg CaCl₂, 2 mL Tween 80, 0.02 g heme, 10 μL vitamin K₁, and 1 mg resazurin. The mixture was dissolved in distilled water to a volume of 1 L and sterilized at 121°C for 15 min.

[0097] 1 mL of 20% (w / v) fecal suspension and 4 mL of fermentation medium were added to each anaerobic sealed tube. The anaerobic sealed tubes containing 50 mg of simulated digested pomelo pulp pomace (the freeze-dried residue obtained after intestinal digestion in step (3) above) soluble dietary fiber were designed as the experimental group, and the anaerobic sealed tubes without pomelo pulp pomace soluble dietary fiber were designed as the control group. All anaerobic sealed tubes were placed in an anaerobic incubator for incubation. At 24 hours of fermentation, three samples were taken from each group and immediately placed in an ice water bath. After centrifugation at 10,000×g for 15 minutes at -4°C, the supernatant and precipitate were stored separately and immediately placed in dry ice, and then stored at -80°C for future use.

[0098] (5) Determination of short-chain fatty acid (SCFA) content during fermentation

[0099] The supernatant collected in step (4) was used for SCFA analysis. Standards of acetic acid, propionic acid, isobutyric acid, isovaleric acid, and valeric acid were diluted to a certain concentration gradient for subsequent gas chromatography analysis. Standard curves plotted against retention time and organic acid concentration were used to determine the content of the five organic acids in the sample. Organic acid standards and samples at different time points after fermentation were analyzed by gas chromatography using an Agilent 7890N GC and an Agilent 5975C QMS system. A 30 m × 250 μm I.S., 0.25 μm column (HP-INNOW AX, 190901N-213, J&W Scientific, Agilent Technologies Inc., USA) was used. Nitrogen was supplied at a flow rate of 1.2 mL / min with a split ratio of 1:50. The initial column temperature was maintained at 40°C for 1 min, then increased to 250°C at a rate of 5°C / min and maintained for 10 min. The temperature of the FID detector and injection port was maintained at 250°C. The injection volume was 0.5 μL and the run time was 50.5 min. The results are shown in Table 4.

[0100] (6) 16S rRNA gene sequencing and bioinformatics data analysis

[0101] The precipitate collected in step (4) was used for 16S rRNA analysis.

[0102] (6-1) DNA extraction and PCR amplification

[0103] according to Total DNA of the microbial community was extracted according to the instructions of the soil DNA kit (Omega Bio-tek, Norcross, GA, US). The quality of the extracted DNA was checked by 1% agarose gel electrophoresis, and the DNA concentration and purity were determined using NanoDrop2000. The V3-V4 variable region of the 16S rRNA gene was amplified by PCR using 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCT AAT-3'). The amplification procedure was as follows: pre-denaturation at 95°C for 3 min, 27 cycles (denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s), followed by stable extension at 72°C for 10 min, and finally storage at 10°C (PCR instrument: ABI). 9700). The PCR reaction system was as follows: 4 μL of 5× TransStart FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of upstream primer (5 μM), 0.8 μL of downstream primer (5 μM), 0.4 μL of TransStart FastPfu DNA polymerase, 10 ng of template DNA, and ddH2O to 20 μL. Each sample was replicated three times.

[0104] (6-2) Illumina Miseq sequencing

[0105] PCR products from the same sample were mixed and recovered using 2% agarose gel. The recovered products were purified using AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA), detected by 2% agarose gel electrophoresis, and analyzed using Quantus TM The recovered products were detected and quantified using a Fluorometer (Promega, USA). The NEXTflex™ Rapid DNA-Seq Kit (Bioo Scientific, USA) was used for library construction: (1) adapter ligation; (2) magnetic bead screening to remove adapter self-ligated fragments; (3) PCR amplification to enrich the library template; (4) magnetic bead recovery of the PCR product to obtain the final library. Sequencing was performed using the Illumina Miseq PE300 / NovaSeq PE250 platform (Shanghai Meiji Biopharmaceutical Technology Co., Ltd.). The raw data were uploaded to the NCBI SRA database.

[0106] (6-3) Data Processing

[0107] The raw sequencing sequences were quality controlled using fastp (https: / / github.com / OpenGene / fastp, version 0.20.0) software and spliced using FLASH (http: / / www.cbcb.umd.edu / software / flash, version 1.2.7) software:

[0108] 1) Filter the bases with a quality value below 20 at the end of the reads. Set a 50 bp window. If the average quality value within the window is lower than 20, cut off the bases at the end of the window. Filter reads below 50 bp after quality control and remove reads containing N bases.

[0109] 2) Based on the overlap relationship between PE reads, paired reads are spliced (merged) into a sequence with a minimum overlap length of 10 bp;

[0110] 3) The maximum mismatch ratio allowed in the overlap region of the spliced sequence is 0.2, and non-compliant sequences are screened;

[0111] 4) Samples are distinguished based on the barcodes and primers at both ends of the sequence, and the sequence direction is adjusted. The allowed number of barcode mismatches is 0, and the maximum number of primer mismatches is 2.

[0112] UPARSE (Edgar, 2013) software (http: / / drive5.com / uparse / , version 7.1) was used to cluster sequences into OTUs at a similarity of 97% (Edgar, 2013; Stackebrandt et al., 1994), and chimeras were removed. Each sequence was annotated to species taxonomy using the RDP classifier (http: / / rdp.cme.msu.edu / , version 2.2) and aligned to the Silva 16S rRNA database (version 138) with an alignment threshold of 70%. The results are shown in Table 4.

[0113] Table 4 Effects of pomelo pulp dietary fiber on SCFA production and composition of intestinal flora

[0114]

[0115]

[0116] As can be seen from Table 4, compared with Comparative Examples 1-3, Examples 1-3 significantly promoted the production of acetic acid, propionic acid, valeric acid, isobutyric acid and isovaleric acid by the intestinal flora, especially the production of propionic acid and isobutyric acid; in terms of the effect on the composition of the intestinal flora, compared with Comparative Examples 1-3, Examples 1-3 significantly inhibited the proliferation of Shigella, Enterobacter cloacae, Acidinococcus, Dorea, and Megamonas, and promoted the growth of Lactobacillus, Bifidobacterium, and Collinsella.

[0117] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for modifying specific pomelo pulp pomace dietary fiber, characterized in that: The following steps are involved: (1) Using pomelo pulp pomace as raw material, the process is subjected to boiling water bath, cellulase hydrolysis, papain hydrolysis, alcohol precipitation, and freeze-drying to obtain pomelo pulp pomace soluble dietary fiber; the process specifically comprises the following steps: (1-1) Dried pomelo pulp was ultrafinely ground at a working pressure of 0.5-0.7 MPa and a sorting frequency of 15-25 Hz to obtain pomelo pulp pomace. The pomelo pulp pomace was mixed with deionized water at a ratio of 1:10-20 (w / v), and 10-15% (v / v) acetic acid-sodium acetate buffer was added and mixed. The mixture was incubated at 80-100°C for 1-2 h and cooled to 25°C to obtain a mixture A. (1-2) Adding 0.03-0.05% (w / v) cellulase to mixed solution A, reacting at 40-50°C for 1.0-2.0 h, then terminating the enzyme reaction by treating at 90°C for 10 min, and cooling to 25°C to obtain mixed solution B; the cellulase has an enzyme activity of 200 U / mL and a pH of 4.5-5.5; (1-3) Adding 0.05-0.07% (w / v) papain to mixed solution B, reacting in a water bath at a temperature of 50-70°C for 30-60 minutes, then terminating the enzyme reaction by treating at 90°C for 10 minutes, and cooling to 25°C to obtain mixed solution C; the papain has an enzyme activity of 100 U / mL and a pH of 6.0-7.0; (1-4) adding 85-95% (v / v) ethanol at a volume ratio of 3-4:1 to the mixed solution C, leaving the mixture overnight to obtain a precipitate, which was then centrifuged and freeze-dried to obtain soluble dietary fiber from pomelo pulp; (2) The activated Clostridium butyricum is inoculated into the RCM liquid culture medium added with soluble dietary fiber from pomelo pulp and pomace for fermentation, and the inoculation amount, fermentation time, fermentation temperature, and substrate concentration are controlled; and then the modified pomelo pulp and pomace dietary fiber is obtained by freeze spray drying; the specific steps include: (2-1) Inoculate Clostridium butyricum into RCM liquid culture medium and culture it at 35-40℃ for 48-72 hours to obtain activated Clostridium butyricum. 600 =1.50-1.70; (2-2) 0.4-0.6% (v / v) activated Clostridium butyricum was inoculated into RCM liquid medium containing 1.0-2.0% (w / v) soluble dietary fiber from pomelo pulp and pomace instead of glucose, and the mixture was reacted at 35-40°C for 30-40 h; the mixture was then freeze-spray-dried to prepare modified pomelo pulp and pomace dietary fiber; The modified pomelo pulp pomace dietary fiber is composed of t-Ara f t-Glc p , 6-Glc p , 4-Glc p 、3,4-Glc p , 4,6-Glc p 、3,6-Gal p、 2-Glc p、 3-Gal p The composition has a molar ratio of 3.60:4.10:2.80:9.30:1.0:3.20:2.60:1.50:1.

40.

2. The method for modifying a specific pomelo pulp pomace dietary fiber according to claim 1, characterized in that: The composition of the RCM liquid medium is as follows: yeast extract 3 mg / mL, beef extract 10 mg / mL, tryptone 10 mg / mL, glucose 5 mg / mL, soluble starch 1 mg / mL, sodium chloride 5 mg / mL, sodium acetate trihydrate 3 mg / mL, cysteine hydrochloride 0.5 mg / mL, 0.01% methylene blue, pH 7.

0.

3. Modified pomelo pulp and pomace dietary fiber prepared by the method according to any one of claims 1 to 2.

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