Yellow water oligosaccharide and its preparation method and application

Abstract

The present invention belongs to the field of food engineering technology and discloses a yellow water oligosaccharide and a preparation method and application thereof. The preparation method comprises the steps of raw material pretreatment, enzymatic hydrolysis, enzyme inactivation and impurity removal. The preparation method of the present invention is simple, the raw materials are readily available and inexpensive, and the prepared yellow water oligosaccharide can significantly increase the content of short-chain fatty acids in an in vitro fermentation liquid of feces, and has a significant effect on maintaining intestinal health and promoting fat decomposition and metabolism; and the yellow water oligosaccharide prepared by the method has a high yield and can meet production requirements.

Description

Technical Field

[0001] The present invention relates to the technical field of food engineering, in particular to yellow water oligosaccharide and a preparation method and application thereof. Background Art

[0002] Yellow water is a viscous, brownish-yellow liquid formed during the traditional solid-state liquor brewing process. The water produced by microbial metabolism and the water in the mash dissolve organic acids, tannins, pigments, soluble starch, proteins, reducing sugars, and other aromatic substances produced during fermentation, which then settle to the bottom of the cellar. Yellow water contains a large amount of residual soluble starch, which can be further degraded by enzymes and / or microorganisms to produce polysaccharides, oligosaccharides, and monosaccharides. Therefore, in-depth research on the sugars in yellow water, a byproduct of liquor production, will provide basic data for the rational development of yellow water resources, greatly improve its utilization rate, and provide important development strategies for liquor companies to achieve their "dual carbon" goals.

[0003] Oligosaccharides, also known as oligosaccharides, are indigestible sugars with special physiological functions, including improving intestinal flora and boosting immunity. They are essentially small, linear or branched polymers of 2-10 monosaccharide units connected by glycosidic bonds, placing them somewhere between monomeric monosaccharides and highly polymerized polysaccharides. Compared to polysaccharides, oligosaccharides offer significant advantages, including low viscosity, low degree of polymerization, low molecular weight, good water solubility, lack of antigenicity, ease of absorption, good bioavailability, and minimal accumulation in the host. Consequently, their development and application in various fields, including food, medicine, and health supplements, has become widespread. A significant market for oligosaccharide products has emerged both domestically and internationally.

[0004] At present, the methods for preparing oligosaccharides using polysaccharides as raw materials mainly include chemical degradation, physical degradation and enzymatic degradation. Traditional chemical degradation and physical degradation have problems such as violent reaction, high equipment maintenance cost, serious environmental pollution, low yield and difficulty in controlling product polymerization degree. The enzymatic hydrolysis method has the characteristics of mild reaction conditions, high extraction efficiency, easy process control and environmental friendliness. Therefore, the enzymatic hydrolysis method for preparing oligosaccharides has gradually become a research hotspot in recent years.

[0005] Meng Xiangyong, Shen Chi, Mao Jian, et al. studied the separation and purification steps of yellow rice wine polysaccharides (Separation, Purification and Physicochemical Properties of Yellow Rice Wine Polysaccharides [J]. Journal of Food and Biotechnology; 2017; 036(010): 1029-1035). Using Shaoxing yellow rice wine as raw material, ethanol precipitation and Sevag method were used to remove proteins to obtain crude yellow rice wine polysaccharides. The crude yellow rice wine polysaccharides were separated and purified by DEAE-Sepharose FF column and Sephadex G75 column, and the relative molecular mass and purity of the yellow rice wine polysaccharides were detected by high-performance gel permeation chromatography. The structural characteristics of the yellow rice wine polysaccharide component CRWP1 were further preliminarily analyzed by ultraviolet spectroscopy, infrared spectroscopy, and nuclear magnetic resonance spectroscopy. However, this separation and purification method does not meet the extraction rate requirements of yellow rice wine oligosaccharides and is not suitable for the extraction of yellow rice wine oligosaccharides.

[0006] The existing research on yellow water mainly focuses on the organic matter and microorganisms in yellow water. There are no reports on the preparation and extraction of oligosaccharides by enzymatic hydrolysis of yellow water polysaccharides and the study of the biological activity of oligosaccharides. Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides a yellow water oligosaccharide and its preparation method and application. Specifically, yellow water oligosaccharide is prepared by pre-treatment of yellow water, enzymatic hydrolysis of yellow water crude polysaccharides, enzyme inactivation and impurity removal. The oligosaccharide can significantly increase the content of short-chain fatty acids in the in vitro fermentation liquid of feces, and has a significant effect on maintaining intestinal health and promoting fat decomposition and metabolism.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] The present invention provides a method for preparing yellow water oligosaccharide, comprising the following steps:

[0010] S1. Pretreatment: Yellow water is precipitated with alcohol and then freeze-dried to obtain solid yellow water crude polysaccharide;

[0011] S2, enzymatic hydrolysis: solid yellow water crude polysaccharide is dissolved in PBS buffer to obtain yellow water crude polysaccharide solution, and complex enzyme is added for enzymatic hydrolysis to obtain enzymatic hydrolyzate;

[0012] S3, enzyme inactivation: heating the enzymatic hydrolyzate to inactivate the enzyme, cooling it to room temperature, centrifuging it, and filtering it to obtain the supernatant;

[0013] S4. Impurity removal: The supernatant is subjected to ultrafiltration and dialysis for impurity removal, and the yellow water oligosaccharide is obtained after freeze-drying.

[0014] Specifically, the pretreatment described in step S1 includes: centrifuging and filtering the yellow water to remove impurities such as rice husks and cellar mud, ethanol precipitation of the yellow water solution, then centrifuging to obtain a precipitate, and freeze-drying the precipitate to obtain solid yellow water crude polysaccharide.

[0015] Preferably, the flavor of the yellow water wine includes any one or more of light fragrance, sauce fragrance, strong fragrance, rich fragrance, sesame fragrance, rice fragrance, and phoenix fragrance.

[0016] Preferably, the complex enzyme in step S2 comprises α-amylase and pectinase; the addition amount of the complex enzyme is 5-15 μL / 10 mL yellow water crude polysaccharide solution.

[0017] Preferably, the volume ratio of the α-amylase to the pectinase is 1-5:1.

[0018] Further preferably, the volume ratio of the α-amylase to the pectinase is 1:1.

[0019] Preferably, the enzymatic hydrolysis conditions in step S2 are: pH 5-7, temperature 40° C.-55° C., and time 8 h-10 h.

[0020] Further preferably, the conditions for the enzymatic hydrolysis in step S2 are: pH 6, temperature 55° C., and time 9 h.

[0021] Preferably, the temperature for inactivating the enzyme in step S3 is 85°C-110°C.

[0022] More preferably, the temperature for inactivating the enzyme in step S3 is 100°C.

[0023] Preferably, the centrifugation conditions in step S3 are: temperature 20°C-25°C, rotation speed 8000rpm-12000rpm, and time 8min-12min.

[0024] Further preferably, the centrifugation conditions in step S3 are: temperature 20° C., rotation speed 10000 rpm, and time 10 min.

[0025] Preferably, the ultrafiltration membrane package in step S4 is 2000Da-4000Da.

[0026] Further preferably, the filter membrane package of the ultrafiltration in step S4 is 3000Da.

[0027] Preferably, the dialysis bag used in step S4 is 100Da-400Da; the dialysis conditions are: temperature 2°C-8°C, time 40h-52h.

[0028] Further preferably, the dialysis bag used in step S4 is 200Da; and the dialysis conditions are: temperature 2°C, time 40h.

[0029] The present invention also provides yellow water oligosaccharide, which is prepared by the above preparation method.

[0030] Preferably, the molecular weight of the yellow water oligosaccharide is 1828 Da; the yellow water oligosaccharide component includes glucose; and the yellow water oligosaccharide is α-configuration pyranose.

[0031] Specifically, the molecular weight of the yellow water oligosaccharide identified after purification is 1828 Da; the monosaccharide components in the yellow water oligosaccharide detected after purification include glucose; and the yellow water oligosaccharide identified after purification is pyranose with α-D configuration.

[0032] Preferably, the purification step comprises: purification via DE-52 ion exchange column and Sephadex G-15.

[0033] The present invention also provides a use of the yellow water oligosaccharide or the yellow water oligosaccharide prepared by the above preparation method in the preparation of weight loss functional foods, health products or medicines.

[0034] Preferably, the weight loss is achieved by increasing the content of short-chain fatty acids during intestinal fermentation.

[0035] Further preferably, the short-chain fatty acids include acetic acid, propionic acid and butyric acid.

[0036] The beneficial effects of the present invention are:

[0037] The preparation method of the present invention is simple, the raw materials are easily available and the price is low; the yellow water oligosaccharide prepared by the present invention can significantly increase the content of short-chain fatty acids in the in vitro fermentation liquid of feces, and has a significant effect on maintaining intestinal health and promoting fat decomposition and metabolism; the method of the present invention has a high yield and meets production requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a standard curve of molecular weight of narrow distribution polyethylene glycol;

[0039] Figure 2 This is a graph showing the molecular weight of the purified yellow water oligosaccharide from Example 1 of the present invention;

[0040] Figure 3 This is a diagram showing the monosaccharide composition of the purified yellow water oligosaccharide of Example 1 of the present invention;

[0041] Figure 4 This is an infrared analysis chart of the purified yellow water oligosaccharide of Example 1 of the present invention;

[0042] Figure 5 This is a graph showing changes in acetic acid content during in vitro fermentation;

[0043] Figure 6 This is a graph showing changes in propionic acid content during in vitro fermentation;

[0044] Figure 7 This is a graph showing the changes in butyric acid content during in vitro fermentation. DETAILED DESCRIPTION

[0045] The following examples are only intended to help understand the methods of the present invention and their core concepts. It should be noted that, for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications may be made to the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention. The following description of the disclosed embodiments enables professionals in this field to implement or use the present invention. Various modifications to these embodiments will be apparent to those 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 will not be limited to the embodiments shown herein, but may be applied to a wider range consistent with the principles and novel features disclosed herein. Although any methods and materials similar or equivalent to those described in the present invention may be used in the practice or testing of the present invention, preferred methods and materials are listed herein.

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0047] Experimental materials and instruments: Yellow water was obtained from Wuliangye Co., Ltd. in Yibin and stored at −20°C for future use; α-amylase (Ban-480L) and pectinase (Pectinex Ultra SP-L) were purchased from Novozymes (China) Biotechnology Co., Ltd.; monosaccharide standards (mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, N-acetylglucosamine, glucose, N-acetylglucosamine, galactose, xylose, arabinose, and fucose) were all chromatographic grade and purchased from Sigma; anhydrous ethanol (analytical grade) was purchased from Sinopharm Chemical Reagent Co., Ltd.; a high-speed refrigerated centrifuge (CR22N) was purchased from Hitachi, Ltd.; a high-performance liquid chromatograph (LC20) was purchased from Shimadzu Corporation; and a TSKgel GMPWXL aqueous gel chromatography columns were purchased from TOSOH (TSK), Japan; high-performance liquid chromatograph (U3000) was purchased from Thermo Fisher Scientific, USA; gas chromatograph (7890A) was purchased from Agilent Technologies, USA; infrared spectrometer (Nicolet™ iS™10) was purchased from Thermo Fisher Scientific, USA; ultrafiltration machine (ZJMP-16-078) and ultrafiltration membrane pack (PELLICON 2MINI) were purchased from MILLIPORE, USA; DE-52 ion exchange column was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; and Sephadex G-15 was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0048] Example 1 A method for preparing yellow water oligosaccharide

[0049] The following steps are involved:

[0050] S1. Pretreatment: After the yellow water is centrifuged and filtered to remove impurities such as rice husks and cellar mud, it is precipitated with anhydrous ethanol and freeze-dried at -50°C to obtain solid yellow water crude polysaccharide;

[0051] S2, enzymatic hydrolysis: solid yellow water crude polysaccharide was dissolved in PBS buffer with a pH of 6.0 to prepare a 1 mg / mL yellow water crude polysaccharide solution, and a complex enzyme (the volume ratio of α-amylase and pectinase was 1:1) was added for enzymatic hydrolysis, the amount of the complex enzyme added was 10 μL / 10 mL yellow water crude polysaccharide solution, the enzymatic hydrolysis pH was 6.0, the enzymatic hydrolysis temperature was 55° C., and the enzymatic hydrolysis time was 9 h to obtain an enzymatic hydrolyzate;

[0052] S3. Inactivation of enzyme: Heat the enzymatic solution to 100°C to inactivate the enzyme, cool to room temperature, and centrifuge (temperature 20°C, speed 10,000 rpm, time 10 min) to remove the precipitate. Filter the supernatant through a 0.45 μm filter membrane.

[0053] S4. Impurity Removal: The supernatant was ultrafiltered using a 3000 Da membrane to remove incompletely enzymatically hydrolyzed polysaccharide macromolecules. The permeate was then transferred to a 200 Da dialysis bag and dialyzed at 4°C for 48 hours to remove small molecule sugars, salts, and some pigments. After dialysis, the solution was freeze-dried at -50°C for 24 hours to obtain the yellow water oligosaccharide, with a yield of 28.24%.

[0054] Example 2 Preparation method of yellow water oligosaccharide

[0055] The following steps are involved:

[0056] S1. Pretreatment: After the yellow water is centrifuged and filtered to remove impurities such as rice husks and cellar mud, it is precipitated with anhydrous ethanol and freeze-dried at -50°C to obtain solid yellow water crude polysaccharide;

[0057] S2, enzymatic hydrolysis: solid yellow water crude polysaccharide was dissolved in PBS buffer with a pH of 6.0 to prepare a 1 mg / mL yellow water crude polysaccharide solution, and a complex enzyme (the volume ratio of α-amylase and pectinase was 5:1) was added for enzymatic hydrolysis, the amount of the complex enzyme added was 15 μL / 10 mL yellow water crude polysaccharide solution, the enzymatic hydrolysis pH was 7.0, the enzymatic hydrolysis temperature was 50° C., and the enzymatic hydrolysis time was 10 h to obtain an enzymatic hydrolyzate;

[0058] S3. Inactivation of enzyme: Heat the enzymatic solution at 110°C to inactivate the enzyme, cool to room temperature, and centrifuge (temperature 25°C, speed 12000 rpm, time 8 min) to remove the precipitate. Filter the supernatant through a 0.45 μm filter membrane.

[0059] S4. Impurity Removal: The supernatant was ultrafiltered using a 4000 Da membrane to remove incompletely enzymatically hydrolyzed polysaccharide macromolecules. The permeate was then placed into a 400 Da dialysis bag and dialyzed at 8°C for 52 hours to remove small molecule sugars, salts, and some pigments. After dialysis, the mixture was freeze-dried at -50°C for 24 hours to obtain the yellow water oligosaccharide, with a yield of 26.52%.

[0060] Example 3 Preparation method of yellow water oligosaccharide

[0061] The following steps are involved:

[0062] S1. Pretreatment: After the yellow water is centrifuged and filtered to remove impurities such as rice husks and cellar mud, it is precipitated with anhydrous ethanol and freeze-dried at -50°C to obtain solid yellow water crude polysaccharide;

[0063] S2, enzymatic hydrolysis: solid yellow water crude polysaccharide was dissolved in PBS buffer with a pH of 6.0 to prepare a 1 mg / mL yellow water crude polysaccharide solution, and a complex enzyme (the volume ratio of α-amylase and pectinase was 3:1) was added for enzymatic hydrolysis, the amount of the complex enzyme added was 5 μL / 10 mL yellow water crude polysaccharide solution, the enzymatic hydrolysis pH was 5.0, the enzymatic hydrolysis temperature was 40°C, and the enzymatic hydrolysis time was 8 h to obtain an enzymatic hydrolyzate;

[0064] S3. Inactivation of enzyme: Heat the enzymatic solution at 90°C to inactivate the enzyme, cool to room temperature, and centrifuge (temperature 22°C, speed 8000 rpm, time 12 min) to remove the precipitate. Filter the supernatant through a 0.45 μm filter membrane.

[0065] S4. Impurity Removal: The supernatant was ultrafiltered using a 2000 Da membrane to remove incompletely enzymatically hydrolyzed polysaccharide macromolecules. The permeate was then transferred to a 100 Da dialysis bag and dialyzed at 2°C for 40 hours to remove small molecule sugars, salts, and some pigments. After dialysis, the mixture was freeze-dried at -50°C for 24 hours to obtain the yellow water oligosaccharide, with a yield of 25.27%.

[0066] Comparative Example 1: Preparation method of yellow water oligosaccharide

[0067] Compared with Example 1, the difference is that the volume ratio of α-amylase to pectinase in step S2 is 1:3, and the other steps are the same as Example 1, with a yield of 18.77%.

[0068] Comparative Example 2: Preparation method of yellow water oligosaccharide

[0069] Compared with Example 1, the difference is that α-amylase is used instead of pectinase in step S2, and the other steps are the same as Example 1, with a yield of 19.93%.

[0070] Comparative Example 3: Preparation method of yellow water oligosaccharide

[0071] Compared with Example 1, the difference is that pectinase is used instead of α-amylase in step S2, and the other steps are the same as Example 1, with a yield of 11.24%.

[0072] Effect Example 1 Structural Analysis of Yellow Water Oligosaccharides

[0073] (1) The yellow water oligosaccharide obtained in Example 1 was purified by DE-52 ion exchange column and Sephadex G-15, and then the molecular weight was determined by high performance gel permeation chromatography. The conditions were: the chromatographic column was a TSKgel GMPWXL aqueous gel chromatographic column, the mobile phase was a 0.1M NaNO3 and 0.06% NaN3 aqueous solution, the flow rate was 0.6 mL / min, the column temperature was 35°C, and narrow distribution polyethylene glycols of different molecular weights (Mw 903000, 580000, 146000, 44200, 1000, 600) were used as standards to make a calibration curve, as shown in FIG. Figure 1 As shown; According to the calibration curve, the molecular weight of the sample is 1828Da, as shown Figure 2 shown.

[0074] (2) The yellow water oligosaccharide obtained in Example 1 was purified by DE-52 ion exchange column and Sephadex G-15, and the monosaccharide composition of the yellow water oligosaccharide was determined by high performance liquid chromatography: 3 mg of yellow water oligosaccharide was accurately weighed into a 10 mL ampoule, 3.0 mL of 2 mol / L trifluoroacetic acid (TFA) was added, nitrogen was filled, the tube was sealed, and acid hydrolysis was carried out at 120°C for 4 h. The sample was removed and methanol was added, TFA was evaporated by nitrogen, and 3.0 mL of water was added for re-dissolution. 250 μL of the sample solution was accurately pipetted into a 5 mL centrifuge tube, 250 μL of 0.6 mol / L NaOH and 500 μL of 0.4 mol / L PMP-methanol were added, and the reaction was carried out at 70°C for 1 h. Cooled in cold water for 10 min; 500 μL of 0.3 mol / L HCl was added for neutralization, and 1 mL of chloroform was added for 1 min, vortexed, and centrifuged at 3000 r / min for 10 min. The supernatant was carefully taken and extracted 3 times. Remove the supernatant and set aside. Accurately weigh appropriate amounts of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, N-acetylglucosamine, glucose, N-acetylglucosamine, galactose, xylose, arabinose, and fucose reference standards. Dissolve in water to dilute to a mixed reference solution containing 50 μg of each per 1 mL. Derivatize according to the above method. Determine monosaccharide composition using high-performance liquid chromatography (HPLC). HPLC conditions: Xtimate C18 4.6 x 200 mm 5 μm column, column temperature 30°C, flow rate 1.0 mL / min, mobile phase 0.05 M potassium dihydrogen phosphate (pH 6.70 with NaOH): acetonitrile = 83:17, detection wavelength 250 nm, injection volume 20 μL.

[0075] The results are as follows Figure 3 As shown, the results showed that the main component of yellow water oligosaccharides was glucose (95.41%), and it also contained small amounts of glucuronic acid (1.25%), mannose (1.04%), arabinose (0.87%), galactose (0.57%), xylose (0.52%) and ribose (0.34%).

[0076] (3) Infrared spectral analysis of yellow water oligosaccharides: The Fourier transform infrared spectra of yellow water oligosaccharides after purification by DE-52 ion exchange column and Sephadex G-15 were recorded by Nicolet iS10 FT-IR spectrometer with a scanning range of 4000 to 400 cm -1 , scanned 32 times. The spectrometer resolution is 4cm -1 The signal-to-noise ratio (S / N) was 50000: 1. Before measurement, 3 mg of the yellow oligosaccharide obtained in Example 1 was ground with KBr powder and pressed into thin sheets according to the KBr disk method before loading the sample.

[0077] The results are as follows Figure 4 As shown, 3337cm -1 The peaks near it are stretching vibration peaks of OH between or within polysaccharide molecules; at 2932 cm -1 The peak near it is the CH stretching vibration absorption peak of the alkyl group; at 1638cm -1 The three peaks are characteristic absorption peaks of sugar substances. -1 and 1417cm -1 It is the bending vibration peak of CH. At 1151cm -1 、1082cm -1 and 1024cm -1 The three absorption peaks near 848cm are the stretching vibration peaks of the pyranose ring, indicating that the yellow water oligosaccharide belongs to pyranose. -1 and 762cm -1 The absorption peak indicates the presence of α-glycosidic bonds in the sugar chain.

[0078] Effect Example 2 Effect of Yellow Water Oligosaccharide on the Content of Short-Chain Fatty Acids

[0079] (1) In vitro fermentation of fecal inoculum: Fecal samples from four adults (two males and two females, who had not taken antibiotics within 3 months and had no gastrointestinal diseases) were mixed and evenly dispersed in sterile PBS buffer (pH 7.0) to obtain a 10% (w / v) fecal suspension. The fecal suspension was centrifuged at 500×g for 5 min and placed in a clean bench for later use. The culture medium used was BHI medium, 6.0 g of tryptone, 0.6 g of anhydrous disodium hydrogen phosphate, 3 g of ox heart extract powder, 3 g of sodium chloride, and 1.2 g of glucose. Before the experiment, the prepared culture medium was added with different substrates and sterilized under high temperature and high pressure (121°C, 20 min). It was placed in a clean bench and cooled to room temperature. 29 mL of culture medium and 1 mL of fecal suspension were added to a sterilized 50 mL serum bottle. The negative control had no additional carbon source, and the positive control had an additional carbon source of 100 mg of inulin (purchased from Shanghai MacLean Biochemical Technology Co., Ltd., product number I811905). The composition of the culture medium for each experimental group is detailed in Table 1. A constant flow of N2 was purged through the serum bottles to exclude oxygen, and the caps were then rapidly screwed. All treatments were incubated with the fecal inoculum in a shaking incubator at 37°C, using anaerobic gas bags to maintain an anaerobic environment. Samples were removed and analyzed after 0, 6, 12, and 24 hours of fermentation.

[0080] Table 1 Fermentation broth ratios of each experimental group

[0081]

[0082] (2) Determination of short-chain fatty acid content in fermentation broth: Short-chain fatty acids were determined in the fermentation broth using gas chromatography. 1.5 mL of fermentation broth was taken at different times and centrifuged at 10,000 × g and 4 ° C for 5 min. 400 μL of fermentation broth was filtered through a 0.45 μm filter membrane, 20 μL of 2-ethylbutyric acid was added and mixed, 100 μL of 50% H2SO4 was added to acidify, vortexed (2000 rpm / min) and then 1000 μL of anhydrous ether was added. The mixture was vortexed (2000 rpm / min) and shaken for 10-15 seconds and then allowed to stand for 2 minutes for extraction. 3000 × g 4 ° C refrigerated centrifugation for 5 min, 700 μL of the upper organic phase was transferred to a 1.5 mL gas phase vial. The generated short-chain fatty acids were detected using a gas chromatograph equipped with a flame ionization detector (FID) using a DB-WAX detection column. Detection conditions: Initial column temperature 100°C for 1 minute, then increased at 5°C / min to 180°C, held for 2 minutes, then increased at 20°C / min to 230°C, held for 2 minutes. FID detector temperature 250°C, injector (syringe) temperature 250°C. Sample load 1 μL, split ratio 5:1, carrier gas N2 at a flow rate of 2.5 mL / min, makeup gas N2 at a purge flow rate of 30 mL / min, H2 flow rate 40 mL / min, and air flow rate 400 mL / min.

[0083] Short-chain fatty acids produced by in vitro fermentation Figure 5-7 As shown, Figure 5 The results showed that the acetic acid content of the yellow water oligosaccharide group at 6 h of fermentation was not significantly different from that of the other groups except inulin; after 12 h of fermentation, the acetic acid content of the Example 1, 2, and 3 groups was significantly higher than that of the Comparative Example 1, 2, and 3 groups and the negative control group; in particular, the acetic acid production after 24 h in Example 1 was 10.47 mM, which was not significantly different from that of the Example 2 and 3 groups, and was significantly higher than 6.36 mM of the negative control group, 9.70 mM of the inulin group, and the Comparative Example 1, 2, and 3 groups.

[0084] Figure 6 The changes in propionic acid content were shown. There was no significant difference in the propionic acid content of each group before 6 hours of fermentation. After 12 hours of fermentation, there was no significant difference in the propionic acid content produced by Examples 1, 2, 3 and fermented inulin, but they were all significantly higher than those of Comparative Examples 1, 2, 3 and the negative control group. It is worth noting that after 24 hours of fermentation, the propionic acid content of Examples 1, 2, 3 was significantly higher than that of Comparative Examples 1, 2, 3, the negative control group and the inulin group, among which the content of Example 1 was the highest, at 10.91 Mm.

[0085] like Figure 7 As shown, during fermentation for 0-6 h, there was no significant difference in the butyric acid content in each experimental group, all of which were lower than that in the inulin group; after 12 h, the butyric acid content in Example 1, 2, 3 groups and the inulin group began to increase significantly, and the butyric acid content in Example 1, 2, 3 groups was significantly higher than that in Comparative Example 1, 2, 3 groups and the negative control group; after fermentation for 24 h, the butyric acid content in Example 1 group was the highest, reaching 7.51 mM, which was not significantly different from that in Example 2 and 3 groups, but was significantly higher than that in the negative control group, inulin group and Comparative Example 1, 2, 3 groups.

[0086] Short-chain fatty acids are metabolites produced by the fermentation of oligosaccharides, non-starch polysaccharides, resistant starch or other dietary fibers by intestinal flora, mainly including acetic acid, propionic acid and butyric acid, etc., which can have an important impact on intestinal health. Studies have shown that short-chain fatty acids have a certain effect on maintaining intestinal health, regulating the immune system, promoting fat decomposition and metabolism, and regulating energy metabolism. The yellow water oligosaccharide prepared by the embodiment of the present invention can significantly increase the production of acetic acid, propionic acid and butyric acid short-chain fatty acids 24 hours after fermentation, and has excellent effects on maintaining intestinal health, promoting fat decomposition and metabolism, and preparing weight loss functional foods or health products or medicines.

[0087] The above further describes the present invention in conjunction with specific embodiments. However, these embodiments are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing yellow water oligosaccharide, characterized in that: The specific steps are as follows: S1. Pretreatment: The yellow water is centrifuged, filtered, and precipitated with alcohol, then centrifuged and freeze-dried to obtain solid yellow water crude polysaccharide; S2, enzymatic hydrolysis: solid yellow water crude polysaccharide is dissolved in PBS buffer to obtain yellow water crude polysaccharide solution, and complex enzyme is added for enzymatic hydrolysis to obtain enzymatic hydrolyzate; S3, enzyme inactivation: heating the enzymatic hydrolyzate to inactivate the enzyme, cooling it to room temperature, centrifuging it, and filtering it to obtain the supernatant; S4, impurity removal: ultrafiltration and dialysis are performed to remove impurities from the supernatant, and the yellow oligosaccharide is obtained after freeze-drying; The compound enzyme in step S2 includes α-amylase and pectinase; the addition amount of the compound enzyme is 5-15 μL / 10 mL yellow water crude polysaccharide solution; The volume ratio of the α-amylase to the pectinase is 1-5:1; wherein, the α-amylase Ban-480L and the pectinase PectinexUltra SP-L are both purchased from Novozymes (China) Biotechnology Co., Ltd.; The enzymatic hydrolysis conditions in step S2 are: pH 5-7, temperature 40°C-55°C, and time 8h-10h; The monosaccharide components in the yellow water oligosaccharide are composed of glucose, glucuronic acid, mannose, arabinose, galactose, xylose and ribose; and the yellow water oligosaccharide is pyranose of α-configuration.

2. The preparation method according to claim 1, characterized in that The temperature for inactivating the enzyme in step S3 is 85°C-110°C.

3. The preparation method according to claim 1, characterized in that The centrifugal conditions in step S3 are: temperature 20° C.-25° C., rotation speed 8000 rpm-12000 rpm, and time 8 min-12 min.

4. The preparation method according to claim 1, characterized in that The filter membrane bag of the ultrafiltration in step S4 is 2000Da-4000Da; the dialysis bag of the dialysis in step S4 is 100Da-400Da; the dialysis conditions are: temperature 2°C-8°C, time 40h-52h.

5. A yellow water oligosaccharide, characterized in that The invention is prepared by the preparation method according to any one of claims 1 to 4.

6. The yellow water oligosaccharide according to claim 5, characterized in that The molecular weight of the yellow water oligosaccharide is 1828 Da.

7. Use of the yellow water oligosaccharide according to any one of claims 5 to 6 or the yellow water oligosaccharide prepared by the preparation method according to any one of claims 1 to 4 in the preparation of weight loss functional foods, health products or medicines.

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