Fermented rhizoma polygonati polysaccharide with weight-losing effect as well as preparation method and application of fermented rhizoma polygonati polysaccharide

Abstract

The invention provides a preparation method of probiotic fermented polygonatum sibiricum polysaccharide, and belongs to the field of traditional Chinese medicinal material processing. The preparation method comprises the following steps: (1) adding cellulase and pectinase into polygonatum kingianum, carrying out water extraction, carrying out enzyme deactivation after water extraction, and carrying out suction filtration to obtain filtrate; (2) preparing bacillus DU-106 and lactobacillus plantarum nbk-MA2 into bacterial powder, and adding water to obtain bacterial liquid; mixing the bacterial liquid with the filtrate, fermenting, performing suction filtration, and centrifuging to obtain supernate; performing alcohol precipitation to remove protein; carrying out ethanol precipitation by using absolute ethyl alcohol again, collecting the precipitate after centrifuging, and carrying out redissolution, dialysis and freeze-drying to obtain fermentation modified crude polysaccharide; and (3) purifying the fermented and modified crude polysaccharide to obtain a purified polysaccharide component. The obtained probiotic fermented polygonatum sibiricum polysaccharide has the effect of losing weight, and can obviously improve the obesity characteristics of mice, specifically, reducing body weight, reducing fat, reducing serum liver lipid level, relieving serum liver inflammation of the mice, weakening blood glucose insulin sensitivity, repairing liver injury and the like.

Description

Technical Field

[0001] The invention belongs to the field of Chinese medicinal material processing, and in particular relates to a fermented polygonatum polysaccharide with weight loss effect, and a preparation method and application thereof. Background Art

[0002] Obesity is a common metabolic disease in humans, and unhealthy diet and lifestyle are important causes of obesity. With the global obesity rate rising, it is crucial to effectively prevent and treat obesity and hyperlipidemia caused by poor diet. The pathophysiological basis of obesity includes energy metabolism disorders, increased adipose tissue, and oxidative stress imbalance. At present, the focus of obesity management is on lifestyle changes, dietary adjustments, and the combined use of drugs. However, the potential risks and side effects caused by long-term use of these drugs cannot be ignored, so it is necessary to explore effective and cost-effective strategies to alleviate obesity. Polygonatum sibiricum is a plant of the genus Polygonatum in the family Liliaceae. It is rich in a variety of bioactive ingredients, such as polysaccharides, polyphenols, sterols, flavonoids, alkaloids and trace elements. Polygonatum sibiricum polysaccharide, as an important functional active ingredient of Polygonatum sibiricum, is mainly composed of various monosaccharides such as glucose, mannose, arabinose and galactose, with a specific molecular weight, glycosidic bond type and sugar chain structure. Polygonatum sibiricum polysaccharide has good anti-aging, immunity enhancement, anti-cancer cell proliferation, liver protection and diabetes treatment effects. The traditional processing of Polygonatum sibiricum is mainly based on nine steaming and nine processing. Not only is the processing steps cumbersome, but with the increase of steaming times, harmful substances increase, and bioactive ingredients degrade, resulting in reduced functional activity.

[0003] Probiotic fermentation of polysaccharides is a common green biotechnology in modern polysaccharide modification technology. During the fermentation process, polysaccharides come into contact with probiotics, which usually leads to changes in the polysaccharide structure and a decrease in the molecular weight of the polysaccharide. These changes will directly affect the strength of its functional activity. Therefore, it is of certain application value to explore the fermented Polygonatum sibiricum polysaccharide with anti-obesity effect. Summary of the invention

[0004] The invention provides a novel method for extracting and modifying polygonatum polysaccharide. Polygonatum polysaccharide with significant weight loss effect is obtained by extracting polygonatum polysaccharide with the aid of enzymolysis and then fermenting and modifying it.

[0005] The present invention provides a method for preparing polygonatum polysaccharide by probiotic fermentation, comprising the following steps: (1) adding cellulase and pectinase to polygonatum cyrtonema to perform water extraction, inactivating the enzymes after water extraction, and filtering to obtain a filtrate; (2) preparing Bacillus DU-106 and Lactobacillus plantarum nbk-MA2 into bacterial powder, dissolving the bacterial powder in sterile water, and activating the bacterial solution; mixing the bacterial solution with the above filtrate, fermenting, filtering and centrifuging to obtain a supernatant; adding anhydrous ethanol to precipitate, collecting the precipitate by centrifugation, and re-dissolving with ultrapure water; adding trichloroacetic acid to precipitate protein, standing and centrifuging to obtain a supernatant; again using anhydrous ethanol to precipitate, collecting the precipitate after centrifugation, re-dissolving, dialysis, and freeze-drying to obtain a fermentation-modified crude polysaccharide; (3) separating and purifying the solution of the fermented and modified crude polysaccharide by medium pressure chromatography, and eluting with ultrapure water and sodium chloride solutions of different concentrations, respectively; collecting the polysaccharide components after elution with ultrapure water, concentrating by rotary evaporation, dialyzing, and freeze-drying to obtain purified polysaccharide components.

[0006] Furthermore, in step (1), the water extraction is specifically performed by adding deionized water by stirring and heating; wherein the stirring and heating time is 2 to 4 hours, and the stirring and heating temperature is 60 to 70°C; In step (1), during the water extraction, the solid-liquid ratio of Polygonatum cyrtonema to deionized water is 1:2-5.

[0007] Furthermore, in step (1), 2 to 5 μL of cellulase and pectinase are added to every 5 g of Polygonatum cyrtonema raw material, respectively; wherein the cellulase and pectinase are added in a volume ratio of 1:1.

[0008] Furthermore, in step (1), the enzyme inactivation treatment is specifically performed by heating with 100°C hot water for 5 to 10 min.

[0009] Furthermore, in step (2), the Bacillus DU-106 and Lactobacillus plantarum nbk-MA2 are mixed evenly into bacterial powder in a 1:1 viable bacterial count ratio; In step (2), during activation, the mass ratio of bacterial powder to sterilized water is 1-5:25, and the activation time is 30-60 min.

[0010] Furthermore, in step (2), the rotation speed used during fermentation is 150-180 rpm, the fermentation temperature is 37° C., and the fermentation time is 6 to 9 days.

[0011] Furthermore, in step (2), the volume ratio of the supernatant obtained by standing and centrifuging to anhydrous ethanol is 1:3-6, and the alcohol precipitation is carried out at 4°C overnight; In step (2), the mass concentration of the trichloroacetic acid is 3% to 5%; and the volume ratio of the trichloroacetic acid to the reconstituted solution is 1:1. Furthermore, in step (3), the crude polysaccharide obtained in step (2) after probiotic fermentation and modification is dissolved in water to prepare a solution of the fermented and modified crude polysaccharide with a concentration of 20-50 mg / ml. The present invention also provides probiotic fermented polygonatum polysaccharide prepared by any of the above-mentioned preparation methods.

[0012] The present invention also proposes the use of any of the above-mentioned probiotic fermented polygonatum polysaccharides in the preparation of foods, health products or medicines with weight loss effects. The present invention has the following advantages: The preparation method of probiotic fermented polygonatum polysaccharide provided by the present invention adopts the mode of enzymatic assisted extraction followed by probiotic fermentation treatment. Specifically, cellulase and pectinase are added to treat the polygonatum water extract during the water extraction process, so that macromolecular cellulose and pectin are enzymatically hydrolyzed into oligosaccharides and other small molecule sugars, thereby promoting the extraction of polygonatum polysaccharide. Lactobacillus DU-106 and Lactobacillus plantarum nbk-MA2 are inoculated into the polygonatum polysaccharide extract obtained after the water extraction for fermentation modification. The synergistic effect of the two strains promotes the modification of polygonatum polysaccharide. Subsequently, probiotic fermented polygonatum polysaccharide is obtained after purification, and the purity of the polysaccharide is high, and the residual impurities such as protein are greatly reduced.

[0013] The present invention studies the weight loss effect of the probiotic fermented polygonatum polysaccharide obtained by the preparation method through animal experiments. The results show that the probiotic fermented polygonatum polysaccharide has a weight loss effect and can significantly improve the obesity characteristics of mice, specifically including reducing body weight, reducing fat, reducing serum liver lipid levels, alleviating serum liver inflammation in mice, weakening blood sugar insulin sensitivity, repairing liver damage, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0015] Figure 1 This is a graph showing the separation, purification and elution curves of PKP and FPKP in Example 1 of the present invention; Figure 2 The molecular weight of PKP0 and FPKP0 in Test Example 1 of the present invention is determined; Figure 3 Determination of the monosaccharide composition of PKP0 and FPKP0 in Test Example 1 of the present invention; Figure 4 It is the Fourier infrared determination of PKP0 and FPKP0 in Test Example 1 of the present invention; Figure 5 The scanning electron microscopy results of PKP0 and FPKP0 in Test Example 1 of the present invention; Figure 6is the methylation result of PKP0 in Test Example 1 of the present invention; Figure 7 is the FPKP0 methylation result in Test Example 1 of the present invention; Figure 8 The effects of PKP0 and FPKP0 on body weight, food intake, diet and liver weight of C57BL / 6J mice in Experimental Example 1 of the present invention; Fig. 9 The effects of PKP0 and FPKP0 on the weight, coefficient, area and size of major adipose tissues, and adipocytes of C57BL / 6J mice in Experimental Example 1 of the present invention; Fig.10 The effects of PKP0 and FPKP0 on blood lipids and blood sugar in C57BL / 6J mice in Experimental Example 1 of the present invention; Fig.11 This is the effect of PKP0 and FPKP0 on liver inflammation in C57BL / 6J mice in Experimental Example 1 of the present invention. DETAILED DESCRIPTION

[0016] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. In the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0017] On the one hand, the embodiment of the present invention provides a method for preparing Polygonatum cyrtonema polysaccharide by probiotic fermentation, comprising the following steps: (1) adding cellulase and pectinase to polygonatum cyrtonema to perform water extraction, inactivating the enzymes after water extraction, and filtering to obtain a filtrate; (2) preparing Bacillus DU-106 and Lactobacillus plantarum nbk-MA2 into bacterial powder, dissolving the bacterial powder in sterile water, and activating the bacterial solution; mixing the bacterial solution with the above filtrate, fermenting, filtering and centrifuging to obtain a supernatant; adding anhydrous ethanol to precipitate, collecting the precipitate by centrifugation, and re-dissolving with ultrapure water; adding trichloroacetic acid to precipitate protein, standing and centrifuging to obtain a supernatant; again using anhydrous ethanol to precipitate, collecting the precipitate after centrifugation, re-dissolving, dialysis, and freeze-drying to obtain a fermentation-modified crude polysaccharide; (3) Separating and purifying the fermented modified crude polysaccharide solution by medium pressure chromatography, eluting with ultrapure water and sodium chloride solutions of different concentrations, respectively; collecting the polysaccharide components after elution with ultrapure water, concentrating by rotary evaporation, dialyzing, and freeze-drying to obtain purified polysaccharide components.

[0018] The preparation method of probiotic fermented polygonatum polysaccharide proposed in the embodiment of the present invention comprises adding cellulase and pectinase during water extraction, adding bacterial liquid (Bacillus DU-106, Lactobacillus plantarum nbk-MA2) for fermentation after water extraction, and the extraction method can bio-modify the polygonatum polysaccharide, and separate and purify the modified polysaccharide to obtain probiotic fermented polygonatum polysaccharide with high purity, which has significant anti-obesity function. In one embodiment of the present invention, in step (1), the Polygonatum cyrtonema is crushed Polygonatum cyrtonema.

[0019] In one embodiment of the present invention, in step (1), the water extraction is specifically performed by adding deionized water by stirring and heating, wherein the stirring and heating time is 2 to 4 hours and the stirring and heating temperature is 60 to 70°C.

[0020] In one embodiment of the present invention, in step (1), during the water extraction, the solid-liquid ratio of Polygonatum cyrtonema to deionized water is 1:2-5.

[0021] In one embodiment of the present invention, in step (1), 2 to 5 μL of cellulase and pectinase are added to every 5 g of Polygonatum cyrtonema raw material, respectively; wherein the cellulase and pectinase are added in a volume ratio of 1:1.

[0022] In one embodiment of the present invention, in step (1), the enzyme inactivation treatment is specifically performed by heating with 100° C. hot water for 5-10 minutes. In one embodiment of the present invention, in step (2), the Bacillus DU-106 and Lactobacillus plantarum nbk-MA2 are mixed evenly into bacterial powder in a 1:1 live bacterial count ratio.

[0023] In one embodiment of the present invention, in step (2), during activation, the mass ratio of bacterial powder to sterile water is 1-5:25, and the activation time is 30-60 min.

[0024] In one embodiment of the present invention, in step (2), the rotation speed used during fermentation is 150-180 rpm, and the fermentation temperature is 37°C.

[0025] In one embodiment of the present invention, in step (2), the fermentation time is 6 to 9 days.

[0026] In one embodiment of the present invention, in step (2), the centrifugal speed is 4000 rpm and the centrifugal time is 5 to 15 minutes.

[0027] In one embodiment of the present invention, in step (2), the volume ratio of the supernatant obtained by standing and centrifuging to anhydrous ethanol is 1:3-6, and the alcohol precipitation is carried out at 4°C overnight.

[0028] In one embodiment of the present invention, in step (2), the mass concentration of the trichloroacetic acid is 3% to 5%; and the volume ratio of the trichloroacetic acid to the reconstituted solution is 1:1.

[0029] In one embodiment of the present invention, in step (3), the fermented and modified crude polysaccharide obtained in step (2) is dissolved in water to prepare a fermented and modified crude polysaccharide solution with a concentration of 20-50 mg / ml. In one embodiment of the present invention, in step (3), the concentrations of the sodium chloride solutions of different concentrations are 0.1 M / ml, 0.2 M / ml, 0.3 M / ml, 0.4 M / ml, and 0.5 M / ml, respectively.

[0030] In one embodiment of the present invention, in step (3), before separation and purification, the solution of the fermented modified crude polysaccharide is filtered through a 0.22 μm microporous filter membrane to initially remove impurities.

[0031] In one embodiment of the present invention, in step (3), DEAE-52 cellulose filler is used for purification.

[0032] On the other hand, the present invention also provides a probiotic fermented polygonatum polysaccharide prepared by any of the above-mentioned preparation methods. The probiotic fermented polygonatum polysaccharide obtained by the present invention has an Mw of 2883, and its main monosaccharide components are glucose (Glc) and galactose (Gal), with a ratio of 95.3:4.7. The probiotic fermented polygonatum polysaccharide is a glucan with a main chain of →4)-Glcp-(1→ On the other hand, the embodiments of the present invention further provide the use of any of the above-mentioned probiotic fermented polygonatum polysaccharides in the preparation of foods, health products or medicines with weight loss effects.

[0033] The probiotic fermented Polygonatum sibiricum polysaccharide proposed in the embodiment of the present invention shows an outstanding effect in the treatment of obesity induced by a high-fat diet, providing a theoretical basis for the application of the probiotic in the food, health care products and pharmaceutical industries, and has broad development prospects. The present invention studies the efficacy of the probiotic fermented Polygonatum sibiricum polysaccharide obtained by the preparation method in weight loss through animal experiments. The results show that the obtained probiotic fermented Polygonatum sibiricum polysaccharide can significantly improve the obesity characteristics of mice, including reducing body weight, reducing adipose tissue, reducing serum liver lipid levels, relieving serum liver inflammation in mice, weakening blood sugar insulin sensitivity, and repairing liver damage.

[0034] The present invention will be described in detail below with reference to embodiments.

[0035] Some of the culture media involved in the present invention and embodiments are as follows: The Lactobacillus plantarum nbk-MA2 involved in this application was purchased from Nobike (Wuhan) Biotechnology Co., Ltd. Bacillus involved in this application Bacillus sp DU-106 was deposited in Guangdong Microbiological Culture Collection Center (GDMCC) on March 22, 2019, with the deposit number GDMCC No: 60621. Address: Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, zip code 510075.

[0036] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the art can be used. The raw materials or instruments used are all conventional products that can be obtained commercially, including but not limited to the raw materials or instruments used in the examples of this application.

[0037] Comparative Example 1 A method for preparing polygonatum polysaccharide comprises the following steps: (1) Extraction of Polygonatum sibiricum Polysaccharide 500 g of fresh rhizome of Polygonatum cyrtonema was washed to remove the sludge and peeled, then crushed and added with deionized water at a solid-liquid ratio of 1: 2 (w / v), and stirred continuously at 65 ℃ for 3 h for extraction; the filtrate was concentrated under vacuum and anhydrous ethanol was added at 1:4 (v / v), and allowed to stand at 4 ℃ overnight; the next day, centrifuged at 4000 rpm for 15 min, the precipitate was collected, dissolved in deionized water (100 mL), and 3% trichloroacetic acid was added at a volume ratio of 1:1, and allowed to stand for 2 h. The resulting solution was concentrated by rotary evaporation, and then precipitated with ethanol (1:4, v / v), precipitated at 4 ℃ overnight, collected by centrifugation, and re-dissolved in deionized water, and dialyzed (molecular weight cutoff 3.5k Da) to remove trichloroacetic acid and some small molecular impurities. Subsequently, the crude polysaccharide powder (PKP) of Polygonatum cyrtonema was obtained by freeze drying for 72 h.

[0038] (2) Purification of Polygonatum cyrtonema polysaccharide The PKP powder was dissolved in ultrapure water to prepare a 50 mg / ml crude polysaccharide solution. It was then filtered through a 0.22 μm pore size membrane, separated and purified using a medium pressure chromatography column, and purified using DEAE-52 cellulose filler. PKP was eluted with ultrapure water, 0.1 M / ml, 0.2 M / ml, 0.3 M / ml, 0.4 M / ml, and 0.5 M / ml sodium chloride solutions, respectively. At the same time, an automatic collector was used to collect the sample solution eluted from one tube of sample every 5 minutes. The elution curve of Polygonatum sibiricum polysaccharide was drawn with reference to the phenol-sulfuric acid method. The eluted solution was collected and concentrated by rotary evaporation, dialyzed with a 1000 Da dialysis bag until the conductivity tended to be stable, and freeze-dried to obtain a purified polysaccharide component, among which the purified polysaccharide component PKP0 was obtained by eluting with ultrapure water.

[0039] The elution curve is Figure 1As shown in Figure 1 (A), there are two main peaks, namely PKP0 (eluted with ultrapure water) and PKP3 (eluted with 0.1 mol / L NaCl), with peak area ratios of 51.8% and 48.2% respectively. The peak area of ​​the small peak in the figure is too small to be ignored. The PKP0 obtained after measurement and purification is the fraction with the highest PKP purity, with a purity of 89.32±0.71%.

[0040] Example 1 A method for preparing polygonatum polysaccharide fermented by probiotics comprises the following steps: (1) Preparation of probiotic fermented Polygonatum odoratum crude polysaccharide Take 500 g of fresh rhizomes of Polygonatum cyrtonema, wash and remove the sludge, peel, crush, add deionized water at a solid-liquid ratio of 1:2 (w / v), add cellulase and pectinase (200 μL each), and stir continuously for 3 h at 65 ℃ for extraction; after water extraction, inactivate the enzyme at 100 ℃ for 5 min, cool and transfer to a 1 L conical flask for use; (2) Bacillus DU-106 and Lactobacillus plantarum nbk-MA2 were mixed evenly at a 1:1 activity ratio, the mixed bacterial powder was weighed and activated in 37°C sterile water for 30 min, the mass ratio of the mixed bacterial powder to sterile water was 2:25, 5 mL of the activated bacterial suspension was added to the above 1 L conical flask under a sterile environment, and fermented at 37°C with shaking (150 rpm) for 7 days; After the fermentation, the filtrate of the fermented Polygonatum cyrtonema mixture was centrifuged (4000 rpm, 10 min, 25 ℃) to separate the supernatant; anhydrous ethanol was added to the supernatant at 1:4 (v / v) and allowed to stand at 4 ℃ overnight; the next day, the mixture was centrifuged at 4000 rpm for 15 min, the precipitate was collected, dissolved in deionized water (100 mL), and 3% trichloroacetic acid was added at a volume ratio of 1:1, and allowed to stand for 2 h. The resulting solution was concentrated by rotary evaporation, and then precipitated with ethanol (1:4, v / v), precipitated at 4 ℃ overnight, and the precipitate was collected by centrifugation and re-dissolved in deionized water, and dialyzed (molecular weight cutoff value 3.5 kDa) to remove trichloroacetic acid and some small molecular impurities. Then, the mixture was freeze-dried for 72 h to obtain the fermentation-modified crude polysaccharide (FPKP); (3) Purification of Polygonatum cyrtonema polysaccharide fermented by probiotics The FPKP powder was dissolved in ultrapure water to prepare a 50 mg / ml crude polysaccharide solution. It was then filtered through a 0.22 μm pore size membrane, separated and purified using a medium pressure chromatography column, and purified using DEAE-52 cellulose filler. FPKP was eluted with ultrapure water, 0.1 M / ml, 0.2 M / ml, 0.3 M / ml, 0.4 M / ml, and 0.5 M / ml sodium chloride solutions, respectively. At the same time, an automatic collector was used to collect the sample solution eluted from one tube of sample every 5 min. The elution curve of Polygonatum sibiricum polysaccharide was drawn with reference to the phenol-sulfuric acid method. The eluted solution was collected and concentrated by rotary evaporation, dialyzed with a 1000 Da dialysis bag until the conductivity tended to be stable, and freeze-dried to obtain a purified polysaccharide component, among which the purified polysaccharide component FPKP0 was obtained by eluting with ultrapure water.

[0041] The elution curve is Figure 1 (B) As shown. Using ultrapure water and NaCl solution (0.1 and 0.3 mol / L) to separate FPKP produced three peaks: FPKP0 (68.92%), FPKP1 (14.26%) and FPKP3 (16.82%). The polysaccharide components of FPKP were not detected under 0.2, 0.4 or 0.5 mol / L NaCl. This change in peak distribution may be due to the degradation of polysaccharides by microorganisms during probiotic fermentation, resulting in a decrease in the polysaccharide content of some components or complete disappearance.

[0042] The FPKP0 obtained after purification was measured to be the fraction with the highest FPKP purity, with a purity of 90.35±1.12%.

[0043] Test Example 1 Structural Identification of Polygonatum cyrtonema Polysaccharide Fermented by Probiotics The PKP0 obtained in Comparative Example 1 and the FPKP0 obtained in Example 1 were used as test samples, and their molecular weight and monosaccharide composition were determined, Fourier transform infrared spectrum scanning, scanning electron microscopy (SEM) and methylation analysis were performed to explore the structural differences between FPKP0 and PKP0 obtained after probiotic fermentation.

[0044] 1. Molecular weight determination Prepare a 5 mg / mL pullulan standard solution. Take 5 mg of PKP0 and FPKP0 samples, dissolve them in 1 mL of mobile phase solution, shake evenly, centrifuge at 12000 rpm for 10 min, and transfer to a sample injection bottle for later use.

[0045] The specific chromatographic detection conditions are as follows; mobile phase: 0.05M NaCl solution; chromatographic column: BRT105-103-101 (8×300 mm); flow rate: 1.0ml / min; column temperature: 40℃; injection volume: 25μl; detector: differential detector RID-20A; inject after preparation, integrate and analyze the data.

[0046] Place the standard sample in the sample tray and select the above chromatographic method for analysis to obtain the retention time, lgMp-RT (Mp peak molecular weight), lgMw-RT (Mw weight average molecular weight), lgMn-RT (Mn number average molecular weight). The standard curve equations are: y = -0.2702x + 11.508R² = 0.9991, R 2 = 0.9991; y = -0.2733x + 11.581, R² = 0.9989; y = -0.272x + 11.544, R² = 0.9988; Place the sample in the sample tray, select the above chromatographic method for analysis, obtain the chromatogram and retention time, substitute the retention time into the formula to obtain the molecular weight (Mp, Mw, Mn). The molecular weight determination results of the standard are shown in Table 1.

[0047] Table 1 Specific data of molecular weight (Mp, Mw, Mn) of standard products

[0048] like Figure 2 As shown in the figure, the gel permeation chromatography (GPC) results showed that both PKP0 and FPKP0 were symmetrical single peaks, indicating that the molecular weight distribution of the two sugars was uniform and had fewer impurities. The results in Table 2 showed that the molecular weight of FPKP0 was significantly reduced compared with PKP0, which may be related to the enzymatic hydrolysis of the uptake and utilization of polysaccharides and their secretion by lactic acid Bacillus DU-106 and plant lactobacillus nbk-MA2 during the fermentation process. The reduction in molecular weight may improve the solubility of FPKP0, which is more conducive to its absorption and bioavailability.

[0049] Table 2 Specific data of molecular weight (Mp, Mw, Mn) of PKP0 and FPKP0

[0050] 2. Determination of monosaccharide composition Sixteen kinds of monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid, mannuronic acid) were accurately weighed, and 2 mL of 3M trifluoroacetic acid solution (TFA) was added to the standard respectively. After hydrolysis at 120 °C for 3 h, nitrogen was blown until dry, and deionized water was added to mix evenly to prepare a standard mother solution. The mother solution was mixed with standard solutions of different concentrations for the determination of the molar ratio of monosaccharides in polysaccharides.

[0051] Similar to the pretreatment steps of the above 16 sugars, 5 mg of sample was accurately weighed, 2 mL of 3M TFA was added, and hydrolysis was carried out at 120℃ for 3h. The hydrolyzed product was dried, dissolved in ultrapure water, and 5 mL of deionized water was added to mix evenly. After centrifugation, the supernatant was analyzed by ion chromatography. It was passed through a Dionex Carbopac™ PA20 column (3×150 mm) with a mixed solution of water, 15 mM NaOH and 15 mM NaOH and 100 mM NaAc, with a flow rate of 0.3 mL / min and a column temperature of 30℃. The setting of the corresponding conditions changed according to time for detection and analysis. The sequence of the monosaccharide composition standard is shown in Table 3.

[0052] Table 3 Monosaccharide standard sequences of monosaccharide composition in polysaccharides

[0053] Figure 3 The monosaccharide composition of PKP0 and FPKP0 is shown. The main monosaccharide components of PKP0 are fructose (Fru) and glucose (Glc), with a ratio of Fru:Glc = 80.03:19.97. The main monosaccharide components of FPKP0 become glucose (Glc) and galactose (Gal), with a ratio of Glc:Gal = 95.3:4.7.

[0054] 3. Fourier transform infrared spectrum scanning 1.5 mg of PKP0, FPKP0 and 100 mg of sodium bromide were weighed respectively, ground thoroughly until no obvious particles were found, and then pressed into tablets. The Fourier transform infrared spectrometer was used for 4000-100 cm -1 Scan within range.

[0055] Figure 4AB are the FT-IR infrared analysis results of PKP0 and FPKP0. The absorption peak at the 3400 cm-1 band corresponds to the stretching vibration of -OH. The absorption peak at 2931 cm-1 is the stretching vibration of CH, which is the characteristic absorption peak of polysaccharides. The absorption peak at 1641 cm-1 is attributed to the vibration of C=O, and the absorption peak at 1415 cm-1 is the bending vibration of CH. The absorption peak at 1025.86 cm-1 is the stretching vibration of CO, and the asymmetric absorption peak at 930 cm-1 is the characteristic performance of pyranose (such as glucose, fructose and galactose). The FT-IR results of the two sugars are similar, which is consistent with the analysis of the monosaccharide composition results. Both are rich in the above chemical groups.

[0056] 4. Scanning electron microscopy (SEM) analysis After the sample was treated by ultrasonic dispersion, 5 μL was dropped on the surface of the silicon wafer, freeze-dried at -80 °C for 12 h using a freeze dryer, and then iridium sprayed for 60 s using an ion sputtering instrument. The prepared sample was placed in a scanning electron microscope, and the morphology was observed under the conditions of an accelerating voltage of 5.0 kV and a working distance of 12 mm. The image was gradually magnified from low magnification (1000x) to high magnification (50000x), and the InLens detector was used to obtain the best resolution and surface details.

[0057] PKP0 and FPKP0 were observed at 250, 500, 1000 and 2000X magnifications (Figure 5). Under 1000X magnification, FPKP0 was found to have a sparser structure and fewer bands than PKP0, and a smoother surface.

[0058] 5. Methylation Analysis Polysaccharide methylation: Weigh 5 mg of PKP0 and FPKP0 samples into a reaction bottle, add 1 mL of anhydrous DMSO and methylation reagent A solution, add solution B after ultrasonic dissolution, and react in a 30°C water bath with magnetic stirring for 1 h. Add 2 mL of ultrapure water to terminate, dialyze at 1000Da for 24 h, and freeze-dry. Infrared analysis confirms that methylation is complete.

[0059] Hydrolysis reduction: 1 mL 2M TFA was used to hydrolyze the methylated polysaccharide for 90 min, and then the mixture was dried by rotary evaporation. The residue was added with 2 ml double distilled water and reduced with 60 mg sodium borohydride for 8 h, neutralized with glacial acetic acid, and the sample was concentrated by rotary evaporation and dried at 101°C. 1 mL acetic anhydride was added to react at 100°C for 1 h, and then 10 mL pure water was added to terminate the reaction after cooling.

[0060] Extraction analysis: The product was washed with 3 mL CH 2 Cl 2Dissolve, transfer to a separatory funnel, add ultrapure water to shake, remove the upper aqueous phase, and repeat 4 times. 2 SO 4 Dry, evaporation concentrate to 1 mL, transfer to a light-proof injection bottle. Analyze using ThermoScientific 1300-7000 GC-MS, HP-INNOWAX column (30 m × 0.32 mm × 0.25 um), starting temperature 140 ° C, 1 ° C / min to 230 ° C, injection port and detector temperature conditions of 250 ° C, helium carrier gas, flow rate conditions of 1 mL / min.

[0061] Methylation analysis of PKP0 revealed eight major peaks ( Figure 6 ), with retention times of 23.254, 23.907, 27.652, 37.678, 38.386, 38.539, 41.909 and 54.048, respectively. They are Man-ol-(2→, Glc-ol-(2→, Glcp-(1→, →6)Man-ol / Glc-ol-(2→, →1)-Man-ol-(2→, →1)-Glc-ol-(2→, →6-Glcp-(1→ and →1,6)Man-ol / Glc-ol-(2→. Since the monosaccharide composition results show that the main monosaccharide components of the polysaccharide are fructose and glucose. Fructose is a ketose that is isomerized into mannose and glucose during the reduction process, and the resulting methyl glycosides are isomerized into mannitol and glucitol. Therefore, the two sugar residues of glucitol and mannitol are integrated, such as Figure 6As shown, the final sugar residues after integration are Fruf-(2→, Glcp-(1→, →6)Fruf-(2→, →1)-Fruf-(2→, →6-Glcp-(1→ and →1,6)Fruf-(2→. FPKP0 obtained 12 main peaks, with peak values ​​of 27.594, 38.042, 38.658, 41.100, 41.896, 49.358, 50.769, 53.711, 53.976, 54.432, 62.350 and 63.911, respectively. These residues are Glcp-(1→, →2)-Glcp-(1→, →3)-Glcp-(1→, →4)-Galp-(1→, →4)-Glcp-(1→, →3,4)-Glcp-(1→, →2,4)-Glcp-(1→, →2,6)-Glcp-(1→, →3,6)-Glcp-(1→, →4,6)-Glcp-(1→, →3,4,6)-Glcp-(1→ and →2,4,6)-Glcp-(1→). However, since the sugar residues of →3,4)-Glcp-(1→, →2,4)-Glcp-(1→, →2,6)-Glcp-(1→, →3,6)-Glcp-(1→, →3,4,6)-Glcp-(1→ and →2,4,6)-Glcp-(1→) account for less than 3%, they can be ignored. Therefore, we speculate that PKP0 is a fructan with →1)-Fruf-(2→ as the main chain, while FPKP0 is a glucan with →4)-Glcp-(1→ as the main chain.

[0062] Test Example 1 Verification of the anti-obesity activity of Polygonatum cyrtonema polysaccharide fermented by probiotics In this experimental example, the anti-obesity activity of the probiotic fermented Polygonatum cyrtonema polysaccharide obtained in Example 1 and Comparative Example 1 was verified by animal experiments.

[0063] 1. Experimental Animals C57BL / 6J male SPF mice (weight 19 g-22 g) were obtained from the Experimental Animal Center of Southern Medical University (certificate number: SCXK (Guangdong) 2022-0002) and were housed in the specific pathogen-free laboratory of South China Agricultural University. Mice were allowed to acclimate to animal feed and distilled water freely for 10 days. This animal experiment was reviewed and approved by the Experimental Ethics Review Committee of the Experimental Animal Center of South China Agricultural University [No.: 2024b034]. The experimental protocol followed the ethical principles of animal experiments and met the requirements of ethical experimental specifications.

[0064] 2. Experimental Grouping and Treatment After 10 days of adaptive feeding, the mice were randomly divided into 6 groups, each with 10 mice (n=10), including blank control group (NC), high-dose probiotic fermented Polygonatum sibiricum polysaccharide blank control group (NC+200FPKP0), long-term high-fat diet obesity group (HFD), long-term high-fat diet obesity probiotic fermented Polygonatum sibiricum polysaccharide low-dose group (HFD+50FPKP0), long-term high-fat diet obesity probiotic fermented Polygonatum sibiricum polysaccharide high-dose group (HFD+200FPKP0), and long-term high-fat diet obesity Polygonatum sibiricum polysaccharide high-dose group (HFD+200PKP0). The NC and HFD groups were gavaged with 0.1 mL of sterile saline per 10 g of mouse body weight per day. The HFD+50FPKP0 group was gavaged with 50 mg / kg d -1 The mice were gavaged with 200 mg / kg d of probiotic fermented Polygonatum cyrtonema polysaccharide, NC+200FPKP0, HFD+200FPKP0, and HFD+200PKP0, respectively. -1 The probiotics fermented or unfermented Polygonatum cyrtonema polysaccharide of the mice body weight. The experiment lasted for 10 weeks, the first 6 weeks were the modeling period, and the last 4 weeks were the intervention period.

[0065] The weight of mice was recorded once a week. After the adaptation period, 30 g of feed was given to each cage of mice every three days, and the feed was weighed every three days. The water intake was measured by giving each cage of mice 50 mL of sterile water each time, and the water intake was recorded every three days.

[0066] Two days before the mice were dissected, oral glucose tolerance test (OGTT) and insulin measurement (ITT) were performed after 8 h of water and food deprivation. The mice were gavaged with sterile glucose solution (dose of 2 g / kg mouse body weight) or intraperitoneally injected with sterile insulin injection (injection dose of 0.4U / kg mouse body weight). A small part of the mouse tail was cut with sterile scissors to allow a little blood to flow out. The start of gavage was recorded as 0 minutes, and blood was collected from the wound of the mouse every 30 minutes, tested with a blood glucose meter, and the changes in OGTT and ITT curves were drawn.

[0067] The mice were dissected at the 10th week of the experiment. The mice were anesthetized with ether, and blood samples were collected from the mice by cardiac bleeding. The samples were placed at 4°C for 12 hours to precipitate serum, and serum samples were obtained after refrigerated centrifugation (4°C, 4000 rpm / min, 15 min). In addition, the mice were dissected and the liver, inguinal fat, perirenal fat, shoulder fat, epididymal fat and other organs and tissues were collected and immediately placed at -80°C for use.

[0068] Phosphate buffer was added to the liver tissue, ground in a cryogenic grinder, centrifuged at 10,000 rpm / min for 10 min, and the supernatant was taken for testing. The mouse serum or liver homogenate supernatant was placed at room temperature for 15 min. Serum samples were used to determine fasting blood glucose and fasting insulin, among which TC (total cholesterol), TG (triglycerides), HDL-C (high-density lipoprotein cholesterol), LDL-C (low-density lipoprotein cholesterol), AST (aspartate aminotransferase), and ALT (alanine aminotransferase) were tested according to the instructions of the kit. Liver homogenate samples were tested according to the instructions of the TC, TG, IL-6, IL-10, IL-1β, and TNF-α kits.

[0069] 3. Statistical methods Data analysis and drawing were performed using statistical analysis software such as Rstudio 4.4.3, GrapPad Primsm 10.3.0, Flowjo, ImageJ, Python 4.43, ITOL, GLYCAM, Chiplot, Autoduck tools, Autoduck vina, Pymol 2.9, Discovery studio, Schrödinger, Github desktop, and Adobe illustrator. All results are expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) and Tukey post hoc test were used for comparison between groups. In the significant level annotation, different letters indicate significant differences between different groups (p < 0.05).

[0070] 4. Experimental Results 4.1 Effects of probiotic fermented Polygonatum sibiricum polysaccharide on body weight, food intake, water intake and liver weight of C57BL / 6J mice like Figure 8 As shown, on the last day, the body weight of mice in the HFD+200FPKP0 group and the HFD+200PKP0 group was significantly lower than that in the HFD group (P<0.05), and the body weight of the HFD+200FPKP0 group was even lower.

[0071] There were certain differences in the Lee's index of mice. Compared with the HFD group, the indexes of the HFD+200FPKP0 group and the HFD+200PKP0 group were significantly decreased (P<0.05), among which the effect of the HFD+200FPKP0 group was better than that of the HFD+200PKP0 group.

[0072] There was no significant difference in liver coefficient among the groups, but the liver weight of each group was significantly lower than that of the HFD group (P<0.05). In summary, most obesity-related indicators of HFD mice changed to a certain extent after being treated with PKP0 and FPKP0.

[0073] 4.2 Effects of probiotic fermented Polygonatum sibiricum polysaccharide on various adipose tissues of C57BL / 6J mice The main adipose tissues of mice include shoulder fat, inguinal fat, subcutaneous fat and epididymal white fat.

[0074] Depend on Fig. 9 It can be seen that compared with the HFD group, the weight of each adipose tissue of mice after intervention with each dose of probiotic fermented polysaccharide showed a significant decrease (P<0.05). Among them, supplementation of FPKP0 and PKP0 can increase the mass of peri-shoulder fat. Since peri-shoulder fat belongs to brown fat, its proper increase is beneficial to the heat production and energy consumption of adipose tissue. iWAT, SAT and eWAT are all white fat. Under the intervention of FPKP0 and PKP0, the mass of these tissues decreased significantly (P<0.05). In terms of reducing the weight of iWAT, the FPKP0 group was better than the PKP0 group. However, for the coefficients of various adipose tissues of mice, only the iWAT coefficient showed significant differences, and FPKP0 and PKP0 had a significant reduction effect after intervention (P<0.05), and there was no significant difference in the other adipose tissues. This proves that under the intervention of FPKP0 and PKP0, the weight of each adipose tissue of mice decreased to a certain extent, reducing the fat in the mice.

[0075] Depend on Fig. 9 It can be seen that the HFD group can see obvious large areas of overall fat, while after high-dose FPKP0 and PKP0 treatment, the fat area of ​​obese mice is smaller. After intervention with high-dose FPKP0 and PKP0 groups, the area distribution of fat cells tends to be concentrated, and the area is significantly reduced. Among them, the area of ​​fat cells in the high-dose FPKP0 group is significantly smaller than that in the PKP0 group. This proves that under the intervention of FPKP0 and PKP0, the volume of fat cells in mice is reduced and the ability to decompose fat is enhanced.

[0076] 4.3 Effects of probiotic fermented Polygonatum sibiricum polysaccharide on blood glucose and blood lipids in C57BL / 6J mice The blood glucose and blood lipids of mice are closely related to their health. Studies have shown that obese mice tend to have high levels of TC, TG, LDL-c and low levels of HDL-c.

[0077] Depend on Fig.10It can be seen that the HFD mice had significantly higher levels of TC, TG and LDL-c than the NC group (P<0.05), and under the intervention of FPKP0 and PKP0, the first three indicators decreased significantly (P<0.05), and HDL-c increased significantly (P<0.05). The serum transaminase content is proportional to the degree of liver damage, and excessive accumulation of liver lipids can cause liver damage, leading to increased transaminase. The transaminase level in the HFD group was higher. After treatment with PKP0 and FPKP0, ALT and AST, except for the low-dose FPKP0 group, showed no significant changes (P>0.05), while the levels of the other groups decreased significantly (P<0.05). This shows that FPKP0 and PKP0 both have a certain ability to regulate the blood lipid level of HFD mice, and the effect is significantly better than PKP0 in some indicators.

[0078] Studies have shown that obesity is often accompanied by symptoms such as increased blood sugar and insulin sensitivity. Fig.10 As shown, mice in the HFD group had higher blood glucose and insulin levels. After intervention with FPKP0 and PKP0, the blood glucose and insulin levels of obese mice decreased significantly (P<0.05), and the blood glucose of obese mice after FPKP0 intervention was significantly lower than that of obese mice after PKP0 intervention (P<0.05).

[0079] In addition, the area under the curve of OGTT and ITT experiments of HFD mice was significantly lower than that of FPKP0 and PKP0 intervention groups (P<0.05). In particular, the effect of FPKP0 intervention on obese mice in OGTT experiment was significantly better than that of PKP0 (P<0.05). In summary, FPKP0 and PKP0 can play a certain role in regulating blood sugar level and insulin sensitivity.

[0080] 4.4 Effects of probiotic fermented Polygonatum sibiricum polysaccharide on liver inflammation in C57BL / 6J mice The accumulation of lipids in the liver can be reflected by measuring TC and TG in liver homogenate. Excessive accumulation of fat can cause liver damage and induce inflammation.

[0081] like Fig.11 As shown in the figure, the TC and TG levels in the HFD group mice were higher, and their levels decreased significantly after FPKP0 and PKP0 intervention (P<0.05). Compared with the HFD group mice, the levels of inflammatory factors (IL-6, IL-1β and TNF-α) decreased significantly after FPKP0 and PKP0 intervention (P<0.05), while the level of IL-10 increased significantly (P<0.05). Therefore, after PKP0 and FPKP0 intervention, the liver damage of HFD mice was repaired and chronic liver inflammation was alleviated.

[0082] In summary, the study on the high-fat diet-induced obesity model in C57BL / 6J mice showed that both FPKP0 and PKP0 could significantly improve obesity characteristics, reduce serum lipid levels, improve insulin sensitivity, and relieve liver inflammation, among which the therapeutic effect of FPKP0 was more prominent.

[0083] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing polygonatum polysaccharide fermented by probiotics, characterized in that: The steps include: (1) adding cellulase and pectinase to polygonatum cyrtonema to perform water extraction, inactivating the enzymes after water extraction, and filtering to obtain a filtrate; (2) preparing Bacillus DU-106 and Lactobacillus plantarum nbk-MA2 into bacterial powder, dissolving the bacterial powder in sterile water, and activating the bacterial solution; mixing the bacterial solution with the above filtrate, fermenting, filtering and centrifuging to obtain a supernatant; adding anhydrous ethanol to precipitate, collecting the precipitate by centrifugation, and re-dissolving with ultrapure water; adding trichloroacetic acid to precipitate protein, standing and centrifuging to obtain a supernatant; again using anhydrous ethanol to precipitate, collecting the precipitate after centrifugation, re-dissolving, dialysis, and freeze-drying to obtain a fermentation-modified crude polysaccharide; (3) separating and purifying the solution of the fermented and modified crude polysaccharide by medium pressure chromatography, and eluting with ultrapure water and sodium chloride solutions of different concentrations, respectively; collecting the polysaccharide components after elution with ultrapure water, concentrating by rotary evaporation, dialyzing, and freeze-drying to obtain purified polysaccharide components.

2. The preparation method according to claim 1, characterized in that: In step (1), the water extraction is specifically performed by adding deionized water by stirring and heating; wherein the stirring and heating time is 2 to 4 hours and the stirring and heating temperature is 60 to 70°C; In step (1), during the water extraction, the solid-liquid ratio of Polygonatum cyrtonema to deionized water is 1:2-5.

3. The preparation method according to claim 1, characterized in that: In step (1), 2 to 5 μL of cellulase and pectinase are added to every 5 g of Polygonatum cyrtonema raw material, respectively; wherein the cellulase and pectinase are added in a volume ratio of 1:

1.

4. The preparation method according to claim 1, characterized in that: In step (1), the enzyme inactivation treatment is specifically performed by heating with 100°C hot water for 5 to 10 min.

5. The preparation method according to claim 1, characterized in that: In step (2), the Bacillus DU-106 and Lactobacillus plantarum nbk-MA2 are mixed evenly into bacterial powder in a ratio of 1:1 in terms of viable bacteria count; In step (2), during activation, the mass ratio of bacterial powder to sterilized water is 1-5:25, and the activation time is 30-60 min.

6. The preparation method according to claim 1, characterized in that: In step (2), the rotation speed used during fermentation is 150-180 rpm, the fermentation temperature is 37° C., and the fermentation time is 6 to 9 days.

7. The preparation method according to claim 1, characterized in that: In step (2), the volume ratio of the supernatant obtained by standing and centrifuging to anhydrous ethanol is 1:3-6, and the alcohol precipitation is carried out at 4°C overnight; In step (2), the mass concentration of the trichloroacetic acid is 3% to 5%; and the volume ratio of the trichloroacetic acid to the reconstituted solution is 1:

1.

8. The preparation method according to claim 1, characterized in that: In step (3), the crude polysaccharide obtained in step (2) after probiotic fermentation and modification is dissolved in water to prepare a solution of the fermented and modified crude polysaccharide with a concentration of 20-50 mg / ml.

9. Polygonatum sibiricum polysaccharide fermented by the preparation method according to any one of claims 1 to 8.

10. Use of the probiotic fermented polygonatum polysaccharide according to claim 7 in the preparation of food, health care products or medicines with weight loss effects.

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