Polygonatum polysaccharide as well as extraction method and application thereof

Abstract

The invention belongs to the technical field of polysaccharide extraction, and discloses rhizoma polygonati polysaccharide as well as an extraction method and application thereof. The invention aims to solve the technical problems that the yield of the polygonatum polysaccharide is low and the antioxidant activity of the product polygonatum polysaccharide is not high in the existing extraction method of the polygonatum polysaccharide. According to the extraction method of the polygonatum sibiricum polysaccharide, the fermentation process is simple and convenient, the polygonatum sibiricum is fermented through saccharomycetes / lactobacillus paracasei, the yield of the polygonatum sibiricum polysaccharide can be further increased, and the antioxidant activity is remarkably enhanced. The polysaccharide yield of the polygonatum polysaccharide extracting solution can reach up to 83.28%, the scavenging rate of the product polygonatum polysaccharide on DPPH free radicals reaches up to 86.54%, the scavenging rate of ABTS free radicals reaches up to 99.77%, and the scavenging rate of hydroxyl free radicals reaches up to 99.69%. And compared with the rhizoma polygonati extracting solution extracted by a water immersion method and a yeast fermentation method, the polysaccharide yield of the rhizoma polygonati extracting solution extracted by lactobacillus paracasei is obviously improved, and is respectively increased by 53.82% and 36.12%. Compared with a yeast fermentation method and a water immersion method, the hydroxyl free radical scavenging rate is increased by 2.39% and 23.33% respectively. The yield of the polygonatum polysaccharide extracted by a lactobacillus paracasei fermentation method is greatly increased.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide extraction, and particularly relates to polygonatum polysaccharide and an extraction method and application thereof. Background Art

[0002] Polygonatum sibiricum ( Polygonatumsibiricum Polygonatum sibiricum belongs to the genus Polygonatum in the Liliaceae family. It prefers warm and humid climates and has strong adaptability. Polygonatum sibiricum is a dual-purpose medicinal and edible plant, widely used in food development. The Compendium of Materia Medica records that Polygonatum sibiricum has the effects of tonifying deficiency, relieving cold and heat, and replenishing essence. Recent studies have shown that the main active ingredients in Polygonatum sibiricum are polysaccharides, steroidal saponins, flavonoids, polyphenols, lignin, and amino acids. Polygonatum sibiricum polysaccharides have been shown to have antioxidant, anti-tumor, antiviral, and blood sugar and lipid-lowering properties.

[0003] Eating raw Polygonatum sibiricum can cause a numbness in the tongue and irritation to the throat and esophagus. This led to the development of a "nine-steaming and nine-drying" processing technique, which promotes the release of active ingredients, eliminates irritation, and ensures the stability of its medicinal properties. However, the Polygonatum sibiricum prepared in this process is dark in color and has a poor taste when brewed. Extracting Polygonatum sibiricum polysaccharides from Polygonatum sibiricum and producing a corresponding Polygonatum sibiricum polysaccharide product can overcome these shortcomings.

[0004] Currently, methods for extracting Polygonatum sibiricum polysaccharides include water extraction, acid-base extraction, ultrasound-assisted extraction, and microbial fermentation. Water extraction yields relatively low levels of Polygonatum sibiricum polysaccharides; acid-base extraction often hydrolyzes the polysaccharides, resulting in loss of activity; and ultrasound-assisted extraction, while it can improve extraction efficiency, is difficult to apply in actual production.

[0005] Microbial fermentation extraction is a biotransformation method that uses enzymes such as cellulase and pectinase produced by microorganisms to break down components of plant cell walls, such as cellulose and pectin. Patent publication number CN117604055A discloses a method for extracting polygonatum polysaccharide, which produces it by fermenting it four times with yeast. This method involves complex fermentation raw materials and numerous steps. Summary of the Invention

[0006] To overcome the low yield and low antioxidant activity of Polygonatum sibiricum polysaccharide in existing extraction methods, the present invention proposes a method for preparing Polygonatum sibiricum polysaccharide using a simple fermentation process. By fermenting Polygonatum sibiricum with edible fungi, the yield of Polygonatum sibiricum polysaccharide is further increased, and the antioxidant activity of the resulting Polygonatum sibiricum polysaccharide is higher than that obtained by water immersion.

[0007] In one aspect, the present invention relates to a method for extracting polygonatum polysaccharide, comprising: The pretreated polygonatum is placed in a container at a material-liquid ratio of 1:20-100; Inoculate edible fungi at an inoculum rate of 1% to 13%; After fermentation at 30°C for 12 to 72 hours, the supernatant was collected to obtain polygonatum polysaccharide.

[0008] Furthermore, in the method for extracting polygonatum polysaccharide provided by the present invention, the pretreatment comprises slicing polygonatum, drying in an oven at 50-60° C. to constant weight, then crushing and passing through a 60-mesh sieve to obtain polygonatum powder.

[0009] When yeast is used for edible fungi: Further, the material-liquid ratio is 1:76; Furthermore, the yeast is pretreated before inoculation, and the pretreatment includes: activating the yeast strain, placing the activated yeast single colony in a liquid culture medium for shaking culture to prepare a liquid strain.

[0010] Furthermore, the yeast was Saccharomyces cerevisiae ATCC9080 strain, the inoculation amount was 4.7%, and the fermentation time was 23 h.

[0011] When Lactobacillus paracasei is used as edible fungi: Furthermore, the material-liquid ratio is 1:80.

[0012] Furthermore, the Lactobacillus paracasei is pretreated before inoculation, and the pretreatment includes: activating the Lactobacillus paracasei strain, placing the activated Lactobacillus paracasei single colony in a liquid culture medium for shaking culture to prepare a liquid strain.

[0013] Furthermore, the inoculation rate is 3% to 11%.

[0014] Furthermore, the Lactobacillus paracasei is ATCC-334 strain, the inoculation amount is 6.6%, and the fermentation time is 34 hours.

[0015] Furthermore, in the method for extracting polygonatum polysaccharide provided by the present invention, the polygonatum is polygonatum yunnanensis, polygonatum sibiricum, Polygonatum sibiricum Red. Or Polygonatum multiflorum.

[0016] Polygonatum sibiricum Polygonatum kingianum Coll.et Hemsl. , Polygonatum Polygonatum sibiricum Red. Polygonatum multiflorum Polygonatum cyrtonema Hua The dried rhizome of the Chinese herb. Depending on its shape, it is commonly known as "big rhubarb," "chicken-headed rhubarb," or "ginger-shaped rhubarb." Harvested in spring and autumn, the fibrous roots are removed, washed, briefly blanched in boiling water or steamed until thoroughly cooked, and then dried (Chinese Pharmacopoeia, 2020 edition).

[0017] On the other hand, the present invention also relates to a polygonatum polysaccharide, which is extracted using the above-mentioned method for extracting polygonatum polysaccharide.

[0018] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects or advantages: This application used yeast / Lactobacillus paracasei to ferment Polygonatum sibiricum in a liquid-phase fermentation. Fermentation conditions were optimized using single-factor experiments and a Box-Behnken response surface methodology, and the antioxidant activity of Polygonatum sibiricum polysaccharides extracted by fermentation was determined. The results showed that the order of influence on the yield of Polygonatum sibiricum polysaccharides by yeast fermentation is solid-liquid ratio > inoculum size > fermentation time. The optimal conditions for optimizing the yield of Polygonatum sibiricum polysaccharides were an inoculum size of 4.7%, a solid-liquid ratio of 1:76, a fermentation time of 23 hours, and a fermentation temperature of 30°C. The Polygonatum sibiricum fermentation broth had scavenging rates of 74.34% for DPPH radicals, 99.77% for ABTS radicals, and 97.36% for hydroxyl radicals, representing increases of 59.43%, 31.00%, and 20.45%, respectively, compared to the water-extraction method. The yeast fermentation method significantly increased the yield of Polygonatum sibiricum polysaccharides, and the antioxidant activity of Polygonatum sibiricum polysaccharides was higher than that of the water-extraction method.

[0019] The influence of Lactobacillus paracasei fermentation on the yield of Polygonatum sibiricum polysaccharide is fermentation time > inoculation size > solid-liquid ratio, and the optimal conditions for optimizing the process of Polygonatum sibiricum polysaccharide yield are inoculation size 6.6%, solid-liquid ratio 1:80, fermentation time 34h, fermentation temperature 37℃, the polysaccharide yield of Polygonatum sibiricum extract is 83.28%, the scavenging rate of DPPH free radical is 86.54%, the scavenging rate of ABTS free radical is 99.54%, and the scavenging rate of hydroxyl free radical is 99.69%. Compared with the water immersion method and yeast fermentation method, this method significantly increased the polysaccharide yield of Polygonatum sibiricum extract, increasing by 53.82% and 36.12%, respectively. Its DPPH free radical scavenging rate was significantly higher than that of the water immersion method and yeast fermentation method, increasing by 85.59% and 16.41%, respectively. Its ABTS free radical scavenging rate was significantly higher than that of the water immersion method, but slightly lower than that of the yeast fermentation method, increasing by 30.70% and -0.23%, respectively. Its hydroxyl free radical scavenging rate was higher than that of the yeast fermentation method and water immersion methods, increasing by 2.39% and 23.33%, respectively. The Lactobacillus paracasei fermentation method significantly increased the yield of Polygonatum sibiricum polysaccharides, and the fermentation broth exhibited strong antioxidant activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. It is obvious that the drawings described below only illustrate some embodiments of the present invention. Those skilled in the art can derive other drawings based on these drawings without inventive effort.

[0021] Figure 1 This is the result of bacterial activation in Example 1. Figure 1 A in the figure is the uninoculated sample. Figure 1 B in the figure is the activated inoculated sample.

[0022] Figure 2 This is a comparison diagram of the liquid culture medium of Example 1. Figure 2 A and C in the experiment are MRS broth culture medium of Lactobacillus paracasei, and milk 1 and milk 2 are parallel experiments. Figure 2 Middle B is uninoculated MRS broth liquid culture medium.

[0023] Figure 3 The glucose standard curves of Examples 1 and 2 are shown.

[0024] Figure 4 This is a graph showing the effect of the inoculation amount on the yield of Polygonatum sibiricum polysaccharide in Example 1.

[0025] Figure 5 This is a diagram showing the effect of the material-liquid ratio on the yield of Polygonatum sibiricum polysaccharide in Example 1.

[0026] Figure 6 This is a graph showing the effect of fermentation time on the yield of Polygonatum sibiricum polysaccharide in Example 1.

[0027] Figure 7 is the response surface diagram of the interaction between factors in Example 1. Figure 7 (a) in the equation is the interaction between A (inoculation amount) and B (material-liquid ratio). Figure 7 (b) in the figure is the interaction between A (inoculation amount) and C (fermentation time). Figure 7 (c) in the figure is the interaction between B (material-liquid ratio) and C (fermentation time).

[0028] Figure 8 This is the result of bacterial activation in Example 2. Figure 8 A in the figure is the uninoculated sample. Figure 8 B in the figure is the activated inoculated sample.

[0029] Figure 9 This is a comparison diagram of the liquid culture medium of Example 2. Figure 9 A in the figure is uninoculated YPD liquid medium. Figure 9 B in the figure is YPD liquid culture medium for yeast.

[0030] Figure 10 This is a graph showing the effect of the inoculation amount on the yield of Polygonatum sibiricum polysaccharide provided in Example 2.

[0031] Figure 11 This is a diagram showing the effect of the material-liquid ratio on the yield of Polygonatum sibiricum polysaccharide provided in Example 2.

[0032] Figure 12 This is a graph showing the effect of fermentation time on the yield of Polygonatum sibiricum polysaccharide provided in Example 2.

[0033] Figure 13 The response surface diagram for the interaction between factors provided in Example 2. Figure 13(a) in the equation is the interaction between A (inoculation amount) and B (material-liquid ratio). Figure 13 (b) in the figure is the interaction between A (inoculation amount) and C (fermentation time). Figure 13 (c) in the figure is the interaction between B (material-liquid ratio) and C (fermentation time). DETAILED DESCRIPTION

[0034] The technical solutions of the present invention are described below with reference to the following examples. However, the present invention is not limited to the following examples. The experimental methods and detection methods in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0035] The experimental materials involved in the following examples are as follows: Lactobacillus paracasei ATCC-334 strain was stored in a -80°C refrigerator (Hefei Ai'erpu Laboratory Supplies Co., Ltd.), Saccharomyces cerevisiae ATCC9080 strain was stored in a -80°C refrigerator (Hefei Ai'erpu Laboratory Supplies Co., Ltd.); Polygonatum sibiricum medicinal material (Bozhou Sanyitang Pharmaceutical Co., Ltd.); Potato dextrose agar medium (PDA) (Qingdao Haibo); YPD liquid medium (Qingdao Haibo); MRS agar medium (Qingdao Haibo Biotechnology Co., Ltd.); MRS broth medium (Qingdao Haibo Biotechnology Co., Ltd.); Anhydrous glucose standard CAS: 50-99-7; Anthrone analysis Pure (Sinopharm Chemical Reagent Co., Ltd.); concentrated sulfuric acid, analytical grade (Xilong Scientific Co., Ltd.); 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) standard, CAS: 1898-66-4 (Tanmo Quality Inspection - Standard Material Center); 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) standard, CAS: 30931-67-0 (Shanghai MacLean Biochemical Technology Co., Ltd.); potassium persulfate (Sinopharm Chemical Reagent Co., Ltd.); ferrous sulfate (Sinopharm Chemical Reagent Co., Ltd.); hydrogen peroxide (Tianjin Damao Chemical Reagent Factory); salicylic acid (Sinopharm Chemical Reagent Co., Ltd.).

[0036] Experimental instruments: UV-1800 ultraviolet-visible spectrophotometer (Shimadzu Enterprise Management (China) Co., Ltd.); Shimadzu AUW220D analytical balance (Shanghai Xiangfan Instrument Co., Ltd.); HH digital display constant temperature water bath (Changzhou Guoyu Instrument Manufacturing Co., Ltd.); single electric furnace (Beijing Kewei Yongxing Instrument Co., Ltd.); LR10M large-capacity refrigerated centrifuge (Hunan Hexi Instrument Equipment Co., Ltd.); UPT-ll-10T Youpu series ultrapure water device (Sichuan Youpu Ultrapure Technology Co., Ltd.); DHG-9202-3SA electric constant temperature drying oven (Shanghai Xiyuan Scientific Instrument Co., Ltd.); DFY-300C 300g swing high-speed pulverizer (Wenling Linda Machinery Co., Ltd.); GI54DP vertical automatic pressure steam sterilizer (Zhiwei Instrument Co., Ltd.); ZWYC-2932 constant temperature incubation shaker (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.); OptiClean1300 clean bench (Likang Precision Technology Co., Ltd.).

[0037] Example 1: In this example, Lactobacillus paracasei was used to extract Polygonatum sibiricum polysaccharide.

[0038] 1. Test material processing 1.1 Polygonatum The root tuber of Polygonatum sibiricum was sliced, dried in an oven at 50-60°C to constant weight, and then crushed. The Polygonatum sibiricum powder was passed through a 60-mesh sieve and placed in a desiccator for later use.

[0039] 1.2 Reagent preparation To prepare MRS agar medium: Weigh 17.54 g of solid powder and completely dissolve it in 250 ml of distilled water. Heat to boiling to achieve dissolution. Once dissolved, divide the medium into Erlenmeyer flasks and autoclave at 121°C for 15 minutes for subsequent use.

[0040] To prepare MRS broth: Weigh 31.34 g of the solid powder and dissolve it in 600 mL of distilled water. Once dissolved, divide the medium into Erlenmeyer flasks and autoclave at 118°C for 15 minutes for later use.

[0041] 0.2% Anthrone-Sulfuric Acid Solution: Weigh 0.2g of anthrone into a beaker and slowly add 90% sulfuric acid solution. Stir with a glass rod to dissolve. After dissolution, a yellow transparent solution will be formed. Transfer it to a 100mL brown volumetric flask and set aside. (Prepare and use immediately) 1.3 Bacteria activation and cultivation 1.3.1 Activation of bacteria Take out the Lactobacillus paracasei ATCC-334 strain stored in a -80℃ refrigerator and place it in a clean workbench. After disinfecting your hands with alcohol cotton balls, wipe the table and light the alcohol lamp. Next to the alcohol lamp, crush the glass beads on the tube containing the strain to allow the buffer solution on the tube to slowly flow down and mix with the strain. Use a cotton swab stained with bacteria beads to draw a circle with a diameter of 1 cm in the MRS solid culture medium. Use the plate streaking method to streak the strain. After completion, turn the plate upside down and place it in a constant temperature incubation shaker at 37℃ for activation culture for 48 hours. The results are as follows: Figure 1 shown.

[0042] Depend on Figure 1 As can be seen, the blank plate (A) shows sterile growth, while the inoculated plate (B) shows milky white colonies with a sticky, creamy texture and neat edges, consistent with the colony morphology of Lactobacillus paracasei. This indicates that the operating environment is sterile and the first-generation seeds from the activated culture are contaminant-free.

[0043] 1.3.2 Liquid culture of bacterial strains In the clean bench, after disinfecting your hands with alcohol cotton balls, wipe the table, light the alcohol lamp, burn the inoculation loop next to the alcohol lamp and cool it, use the inoculation loop to pick a single colony on the activated plate and inoculate it into MRS broth liquid culture medium, and do a blank test at the same time, place it in a constant temperature culture shaker, shake and culture at 37℃ and 180r / min for 48h to make liquid bacteria, and put it in a 4℃ refrigerator for use. The results are as follows Figure 2 shown.

[0044] Depend on Figure 2 It can be seen that the blank group (A) appears as a yellow translucent liquid, and there is no bacterial growth in the liquid culture medium. The bottom of the Lactobacillus paracasei liquid culture medium (B) shows a milky white precipitate and has obvious turbidity, indicating that the bacteria have grown and multiplied in large quantities after liquid culture, the culture conditions are suitable, and there is no contamination from other bacteria.

[0045] 2 Extraction of Polygonatum sibiricum polysaccharide Place 2g of Polygonatum sibiricum powder in a 1000mL Erlenmeyer flask and add deionized water at a specific material-liquid ratio (1:20-100). Autoclave the flask at 121°C for 15 minutes. After cooling, inoculate the flask at a specific inoculum size (3%-11%) in a clean bench. Ferment the flask in a constant-temperature shaker at 37°C for a specific fermentation time (12-72 hours). After fermentation, aliquot the fermentation broth into centrifuge tubes and centrifuge at 4000 rpm for 20 minutes. The supernatant is the Polygonatum sibiricum extract.

[0046] 3. Determination of Yield of Polygonatum sibiricum Polysaccharide Glucose standard curve drawing: According to the 2020 edition of the Chinese Pharmacopoeia (take 32.94 mg of anhydrous glucose reference substance dried to constant weight at 105 ° C, accurately weighed, placed in a 100 mL volumetric flask, added water to dissolve and dilute to the scale, shake well, and the reference solution is obtained. Accurately measure 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, and 0.5 mL of the reference solution, respectively, and place them in 10 mL stoppered graduated test tubes, add water to 2.0 mL each, shake well, and Slowly add 0.2% anthrone-sulfuric acid solution dropwise to the mark in an ice-water bath, mix thoroughly, let cool, and then place in a water bath for 10 minutes. Remove from heat and immediately place in an ice-water bath to cool for 10 minutes. Remove from heat and use the corresponding reagent as a blank. Measure absorbance at 582 nm according to UV-Vis spectrophotometry (General Method 0401). Plot a standard curve with absorbance as the ordinate and concentration as the abscissa. The regression equation for the standard curve is: Y = 0.03922X + 0.1368, R 2 =0.9989. The standard curve is as follows Figure 3 shown.

[0047] Determination of the yield of Polygonatum sibiricum polysaccharide in the fermentation supernatant: 10 μL of fermentation broth was placed in a 10 mL stoppered test tube, and the OD582 absorbance was determined according to the preparation of glucose standard curve. Figure 1 : The polysaccharide content was calculated using a glucose standard curve, and the experiment was repeated three times.

[0048] Calculation formula: Polysaccharide yield (%) = amount of Polygonatum sibiricum polysaccharide / sample mass × 100% The following provides a parameter determination experiment for the Polygonatum sibiricum polysaccharide extraction process.

[0049] The fermentation broth under different test parameters was extracted according to the extraction steps of Polygonatum sibiricum polysaccharide in Example 1.

[0050] 1. Single-factor experiment 1. Determination of inoculum size The yield of Polygonatum sibiricum polysaccharide with different inoculation amounts was determined by UV-visible spectrophotometer. Figure 4 shown.

[0051] The appropriate inoculation amount has an important influence on the fermentation of Lactobacillus paracasei. If the inoculation amount is too low, it will lead to slow growth of Lactobacillus paracasei, and it will take a long time to form a dominant bacterial population, and it is easy to cause contamination by other bacteria in the process; if the inoculation amount is too high, the nutrients may be consumed too quickly by the bacteria, which is not conducive to the fermentation process. Figure 4 It can be seen that when the inoculum size is 7%, the PSP yield is the highest. When the inoculum size is greater than 7%, the PSP yield gradually decreases. It is more appropriate to choose an inoculum size of 7%.

[0052] 2 Determination of material-liquid ratio The yield of Polygonatum sibiricum polysaccharide after fermentation with different material-liquid ratios was determined by UV-visible spectrophotometer. Figure 5 shown.

[0053] The material-liquid ratio plays an important role in the fermentation process. If the material-liquid ratio is too low, the raw materials may not be enough to meet the nutritional needs of the strain in the later stage of fermentation, resulting in a decrease in growth rate and a decrease in the production of metabolites. On the contrary, if the material-liquid ratio is too high, it may cause a waste of raw materials and may have an adverse effect on the fermentation process, such as inhibiting the growth of the strain or affecting the metabolic pathway. Figure 5 The effect of the material-liquid ratio on the PSP content shows that as the material-liquid ratio increases, the PSP yield gradually increases. When the material-liquid ratio is 1:80, the PSP yield reaches its maximum. When the material-liquid ratio is greater than 1:80, the PSP yield gradually decreases. Therefore, a material-liquid ratio of 1:80 is more appropriate.

[0054] 3. Determination of fermentation time The yield of Polygonatum sibiricum polysaccharide after fermentation at different times was determined by UV-visible spectrophotometer. Figure 6 shown.

[0055] Depend on Figure 6 As can be seen, the PSP yield gradually increased with fermentation time, reaching its peak at 36 hours. Thereafter, the PSP content gradually decreased with fermentation time. This may be because the prolonged fermentation time led to insufficient nutrient supply in the later stages. To maintain their own growth, the microorganisms consumed sugars and other nutrients in the fermentation broth, thus affecting polysaccharide production.

[0056] 4 Response surface experiment The single-factor experiment showed that the three factors of inoculation amount, liquid-to-solid ratio and fermentation time had a significant effect on the indicators of the fermentation broth. Three levels of the three factors, inoculation amount (A), liquid-to-solid ratio (B) and fermentation time (C), were selected respectively, and the yield of Polygonatum sibiricum polysaccharide was used as an indicator to carry out the Box-Benhnken response surface design optimization experiment (Table 1).

[0057]

[0058] The test results are shown in Table 2. The quadratic regression equation with the polysaccharide yield of the fermentation broth as the response value is: Polysaccharide yield Y=+83.08-1.27*A+0.21*B-3.08*C+0.86*A*B-0.015*A*C+0.24*B*C-3.38*A 2 -6.04* B 2 -9.78* C 2

[0059] The variance analysis of the model is shown in Table 3. It is found that the model has reached an extremely significant level (P < 0.01), the P value of the lack of fit item is greater than 0.05, and R 2 =0.9993, R 2 adj =0.9984 indicates the model calibration relationship, indicating that the model has a good fit, that is, the above linear regression equation can predict the change of Polygonatum polysaccharide yield with each factor to a certain extent. The CV value is equal to 0.37%, indicating that the reliability and accuracy of the test are good. Secondly, the results of variance analysis show that factor A, factor C, AB interaction, A 2 、B 2 and C 2 It has a very significant effect on the yield of Polygonatum sibiricum polysaccharide; from the size of the F value of each factor, it can be seen that the influence of each factor on the yield of polysaccharide in the fermentation broth is ranked as: C>A>B.

[0060]

[0061] Note: P <0.01 is extremely significant, indicated by “**”; P <0.05 is significant, indicated by “*”; P >0.05, not significant.

[0062] The results of the interaction between the three factors of inoculation amount, liquid-to-solid ratio and fermentation time on the yield of polysaccharide in fermentation broth are shown in Figure 7 The interaction between inoculum size and liquid-to-liquid ratio was the most significant. Based on the above analysis and predictions from the Design Expert software, the optimal conditions for extracting Polygonatum sibiricum by fermentation with Lactobacillus paracasei were: inoculum size 6.63%, liquid-to-liquid ratio 1:80.03 g / mL, and fermentation time 34.12 h. The predicted polysaccharide yield in the fermentation broth was 83.44%. Based on practical considerations, the optimal conditions for extracting Polygonatum sibiricum were determined to be 6.6% inoculum size, 1:80 g / mL liquid-to-liquid ratio, 34 h fermentation time, and 37°C fermentation temperature. Three confirmatory tests were conducted under these conditions, resulting in a polysaccharide yield of 83.28%, which is consistent with the theoretical prediction. This demonstrates that the model fits well and that the regression equation is effective in analyzing and predicting Polygonatum sibiricum extraction conditions. This indicates that this process is the optimal process for extracting Polygonatum sibiricum polysaccharides by fermentation with Lactobacillus paracasei.

[0063] The antioxidant activity of the extracted polygonatum polysaccharide was determined as follows: Polygonatum polysaccharide was prepared according to the extraction method of polygonatum polysaccharide in Example 1, wherein the inoculation amount was 6.6%, the material-liquid ratio was determined to be 1:80 g / mL, the fermentation time was determined to be 34 h, and the fermentation temperature was 37°C.

[0064] A sample solution of Polygonatum sibiricum polysaccharide was prepared.

[0065] 1. Determination of DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) free radical scavenging ability Take 0.75 mL of the sample solution, add 1 mL of a 0.1 mg / mL DPPH solution, and then add anhydrous ethanol to 2 mL, mix thoroughly, and incubate in the dark for 30 minutes. Measure the absorbance (A1) at 517 nm. Also measure the absorbance (A0) of a mixture of 2 mL of the 0.1 mg / mL DPPH solution and 2 mL of anhydrous ethanol. Also measure the absorbance (A2) of a mixture of 2 mL of the sample solution and 2 mL of anhydrous ethanol. The results are shown in Table 4. The clearance rate of the Polygonatum sibiricum extract was calculated using the following formula.

[0066] DPPH clearance rate (%) = [ A 0 -( A 1 - A 2 ) / A 0 ]×100% Where A0 is the absorbance of the blank control group; A1 is the absorbance of the sample solution; A2 is the absorbance of anhydrous ethanol and the sample extract.

[0067] 2. Determination of the free radical scavenging ability of ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) Mix equal volumes of potassium persulfate (4.9 mmol / L) and ABTS solution (14 mmol / L), place in the dark at 20-25°C for 12-16 hours, and dilute with anhydrous ethanol before use to maintain the absorbance of the ABTS dilution at 734 nm within the range of 0.700 ± 0.020. Take 13 mL of the sample solution, add 3.9 mL of the ABTS dilution, mix the sample, and react for 30 minutes in the dark. The absorbance measured at 734 nm is A x ; Then take 0.5mL of ethanol and add 3.9mL of ABTS diluent, mix in the same way and the absorbance is measured as A0; take 13mL of sample solution, add 3.9mL of anhydrous ethanol, mix in the same way and the absorbance is measured as A j The results are shown in Table 4. The clearance rate of the Polygonatum sibiricum extract was calculated using the following formula.

[0068] ABTS free radical scavenging rate (%) = [( A 0 + A j - Ax ) / A 0 ]×100% 3. Determination of hydroxyl radical scavenging ability Prepare a 9 mmol / L FeSO₄ solution, a 9 mmol / L ethanol-salicylic acid solution, and an 8.8 mmol / L hydrogen peroxide (H₂O₂) solution in advance. Mix 12 mL of the sample solution with equal volumes of salicylic acid solution, FeSO₄ solution, and H₂O₂ solution, respectively. Incubate in a 37°C water bath for 15 minutes, and measure the OD at 510 nm (denoted as A1). Mix 1 mL of the sample solution with equal volumes of distilled water, salicylic acid solution, and FeSO₄ solution, and measure the OD at 510 nm (denoted as A2). Replace the sample with distilled water, keeping all other parameters unchanged, and measure the OD at 510 nm (denoted as A0). The results are shown in Table 4. The clearance rate of the Polygonatum sibiricum extract was calculated using the following formula.

[0069] Hydroxyl radical scavenging rate (%) = [( A 1 - A 2 ) / A 0 ]×100% Water immersion extraction steps: Take approximately 2g of Polygonatum sibiricum powder, dried to constant weight at 60°C, accurately weigh it, place it in a conical flask, add 160mL of purified water, and shake it in a constant-temperature incubator at 37°C for 36 hours to extract the Polygonatum sibiricum polysaccharide. After extraction, transfer the extract to a centrifuge tube and centrifuge at 4000 rpm for 20 minutes. Remove the supernatant, which is the Polygonatum sibiricum water extract.

[0070] Yeast fermentation extraction steps: take about 2g of polygonatum powder dried to constant weight at 60℃, accurately weigh it, place it in a 1000mL conical flask, add deionized water at a solid-liquid ratio of 1:76, sterilize it with high-pressure steam at 121℃ for 15 minutes, cool it down, inoculate it in an ultra-clean workbench at a 4.7% inoculation rate, and culture it in a constant temperature incubator at 30℃ and 180r / min for 23 hours. After fermentation, the fermentation liquid is divided into centrifuge tubes, centrifuged at 4000rpm for 20 minutes, and the supernatant is the polygonatum fermentation liquid.

[0071] The yield and antioxidant activity of polygonatum polysaccharide prepared by the fermentation method provided by the present invention were compared with those prepared by the water immersion method and yeast fermentation method. The comparison results are shown in Table 4.

[0072]

[0073] Table 4 shows that the yield and antioxidant activity of Polygonatum sibiricum polysaccharides extracted by fermentation were significantly higher than those by water extraction. For Polygonatum sibiricum polysaccharide yield, the results were as follows: Lactobacillus paracasei fermentation method > yeast fermentation method > water extraction method; for DPPH radical scavenging rate, the results were as follows: Lactobacillus paracasei fermentation method > yeast fermentation method > water extraction method; for ABTS radical scavenging rate, the results were as follows: yeast fermentation method > Lactobacillus paracasei fermentation method > water extraction method; and for hydroxyl radical scavenging rate, the results were as follows: Lactobacillus paracasei fermentation method > yeast fermentation method > water extraction method. This may be due to the metabolic activities of microorganisms, which decompose components of plant cell walls, such as cellulose, hemicellulose, and pectin. These components often surround polysaccharides, making them difficult to extract by water extraction. Microbial enzymes, such as cellulase and pectinase, help break down these structures, releasing more polysaccharides. The strong antioxidant activity may be related to the increased polysaccharide content and the production of antioxidant compounds, such as organic acids, polyphenols, and flavonoids, through microbial metabolism. Different microorganisms produce different enzymes with different activities, resulting in varying antioxidant activity.

[0074] Example 2: This example provides a method for extracting Polygonatum sibiricum polysaccharide.

[0075] 1. Test material processing 1.1 Polygonatum The root tuber of Polygonatum sibiricum was sliced, dried in an oven at 50-60°C to constant weight, and then crushed. The Polygonatum sibiricum powder was passed through a 60-mesh sieve and placed in a desiccator for later use.

[0076] 1.2 Reagent preparation Potato Dextrose Agar (PDA): Weigh 11.5 g of this product into a 250 mL beaker, add 200 mL of deionized water to dissolve, transfer to a 1000 mL Erlenmeyer flask, and autoclave at 115°C for 20 minutes. The sterilized medium may be reheated and melted only once.

[0077] Yeast extract peptone dextrose medium (YPD liquid medium): Weigh 10.0 g of this product into a 250 mL beaker, dissolve it in 200 mL of deionized water by heating, and place it into a 1000 mL Erlenmeyer flask. Autoclave at 121°C for 15 minutes and set aside.

[0078] 0.2% Anthrone-Sulfuric Acid Solution: Weigh 0.2g of anthrone into a beaker and slowly add 90% sulfuric acid solution. Stir with a glass rod to dissolve. After dissolution, a yellow transparent solution will be formed. Transfer it to a 100mL brown volumetric flask and set aside. (Prepare and use immediately) 1.3 Bacteria activation and cultivation 1.3.1 Activation of bacteria Take out the ATCC9080 strain of Saccharomyces cerevisiae stored in a -80°C refrigerator. In the clean bench, disinfect your hands with alcohol cotton balls, wipe the table, light the alcohol lamp, and crush the glass beads above the tube containing the strain next to the alcohol lamp to allow the buffer solution above the tube to slowly flow down and mix with the strain. Use a cotton swab dipped in bacteria beads to draw a circle with a diameter of 1 cm on the PDA solid culture medium. Use the plate streak method to streak the strain. After completion, turn the plate upside down and place it in a constant temperature incubation shaker at 30°C for activation culture for 48 hours. The results are as follows: Figure 8 shown.

[0079] Depend on Figure 8 As can be seen, the blank plate (A) shows sterile growth, while the inoculated plate (B) shows milky white, large, round or oval colonies. The surface is moist, smooth, and sticky, possibly glossy. The edges are neat, sometimes appearing raised, consistent with the colony morphology of yeast. This indicates that the operating environment is sterile and the first-generation seeds from the activated culture are free of contamination.

[0080] 1.3.2 Liquid culture of bacterial strains In the clean bench, after disinfecting your hands with alcohol cotton balls, wipe the table, light the alcohol lamp, burn the inoculation loop next to the alcohol lamp and cool it, pick a single colony from the activated plate with the inoculation loop and inoculate it into YPD liquid culture medium, and do a blank test at the same time, place it in a constant temperature culture shaker, shake and culture at 30℃ and 180r / min for 48h to make liquid bacteria, and put it in a 4℃ refrigerator for use. The results are as follows Figure 9 shown.

[0081] Depend on Figure 9 It can be seen that the blank group (A) appears as a yellow translucent liquid, and there is no bacterial growth in the liquid culture medium. The yeast liquid culture medium (B) shows a milky white precipitate at the bottom, with obvious turbidity, indicating that the bacteria grow and reproduce in large quantities after liquid culture, the culture conditions are suitable, and there is no contamination from other bacteria.

[0082] 2 Extraction of Polygonatum sibiricum polysaccharide 2g of Polygonatum sibiricum powder was placed in a 1000mL Erlenmeyer flask. Deionized water was added at a specific material-liquid ratio (1:20, 1:40, 1:60, 1:80, 1:100). The mixture was sterilized by high-pressure steam sterilization at 121°C for 15 minutes. After cooling, the mixture was inoculated in a clean bench at a specific inoculum size (1%, 3%, 5%, 7%, 9%, 11%, and 13%). Fermentation was carried out in a constant temperature shaker for a specific fermentation time (12 hours, 24 hours, 36 hours, 48 hours, and 72 hours) at 30°C. After fermentation, the fermentation broth was aliquoted into centrifuge tubes and centrifuged at 4000 rpm for 20 minutes. The supernatant was the fermentation broth.

[0083] 3. Determination of Yield of Polygonatum sibiricum Polysaccharide The yield of Polygonatum sibiricum polysaccharide can be determined by referring to the operation of Example 1.

[0084] The following is an experiment to determine the parameters of the Polygonatum sibiricum polysaccharide extraction process.

[0085] The fermentation broth under different test parameters was extracted according to the extraction steps of Polygonatum sibiricum polysaccharide in Example 2.

[0086] 1. Single-factor experiment 1. Determination of inoculum size The yield of Polygonatum sibiricum polysaccharide with different inoculation amounts was determined by UV-visible spectrophotometer. Figure 10 shown.

[0087] The appropriate inoculation amount has an important influence on yeast fermentation. If the inoculation amount is too low, the yeast will grow slowly, and it will take a long time to form a dominant bacterial group, and it is easy to cause contamination by foreign bacteria in the process. If the inoculation amount is too high, the nutrients may be consumed too quickly by the bacteria, which is not conducive to the fermentation process. Figure 4 It can be seen that when the inoculum size is 5%, the PSP yield is the highest. When the inoculum size is greater than 5%, the PSP yield gradually decreases. It is more appropriate to choose an inoculum size of 5%.

[0088] 2 Determination of material-liquid ratio The yield of Polygonatum sibiricum polysaccharide after fermentation with different material-liquid ratios was determined by UV-visible spectrophotometer. Figure 11 shown.

[0089] The material-liquid ratio plays an important role in the fermentation process. Whether the raw materials are sufficient determines whether the bacteria have enough nutrients for their growth and metabolism in the later stage of fermentation. Figure 11 The effect of the material-liquid ratio on the PSP content shows that as the material-liquid ratio increases, the PSP yield gradually increases. When the material-liquid ratio is 1:80, the PSP yield reaches its maximum. When the material-liquid ratio is greater than 1:80, the PSP yield gradually decreases. Therefore, a material-liquid ratio of 1:80 is more appropriate.

[0090] 3. Determination of fermentation time The yield of Polygonatum sibiricum polysaccharide after fermentation at different times was determined by UV-visible spectrophotometer. Figure 12 shown.

[0091] Depend on Figure 12As can be seen, the PSP yield gradually increased with fermentation time, reaching its peak at 24 hours. After that, the PSP content gradually decreased with fermentation time. This may be because the fermentation time was too long, resulting in insufficient nutrient supply in the later stages. To maintain their growth, the microorganisms consumed sugars and other nutrients in the fermentation broth.

[0092] 4 Response surface experiment Depend on Figure 13 It can be seen that the single-factor experiment showed that the three factors of inoculation amount, liquid-to-solid ratio and fermentation time had a significant effect on the indicators of the fermentation broth. Three levels of the three factors of inoculation amount (A), liquid-to-solid ratio (B) and fermentation time (C) were selected respectively, and the yield of Polygonatum sibiricum polysaccharide was used as an indicator to carry out the Box-Benhnken response surface design optimization experiment (Table 5).

[0093]

[0094] The test results are shown in Table 6. The quadratic regression equation with the polysaccharide yield of the fermentation broth as the response value is: Y=60.58-4.03*A-7.51*B-3.77*C-0.3750*AB+0.1275*AC+2.37*BC-15.89*A 2 -15.63*B 2 -17.03*C 2

[0095] The variance analysis of the model is shown in Table 7, and it is found that the model has reached an extremely significant level ( P <0.01), the lack-of-fit term is greater than 0.05, R 2 =0.9966, R 2 adj =0.9923 indicates the model calibration relationship, indicating that the model has a good fit, that is, the above linear regression equation can predict the change of the yield of Polygonatum sibiricum polysaccharide with each factor to a certain extent. The CV value is equal to 3.86%, indicating that the reliability and accuracy of the test are good. Secondly, the results of variance analysis show that factor A, factor B, factor C, factor A 2 、B 2 and C 2 It has a very significant effect on the yield of Polygonatum sibiricum polysaccharide, and the interaction between B and C has a significant effect on the polysaccharide yield; from the size of the P value of each factor, it can be seen that the influence of each factor on the polysaccharide yield in the fermentation broth is ranked as: B>A>C.

[0096]

[0097] Note: P≤0.01, extremely significant, indicated by “**”; P ≤0.05, significant, indicated by “*”; P >0.05, not significant.

[0098] The results of the interaction between the three factors of inoculation amount, liquid-to-solid ratio and fermentation time on the yield of polysaccharide in fermentation broth are shown in Figure 13 The interaction between the liquid-to-liquid ratio and fermentation time was the most significant. Based on the above analysis and predictions made by the Design Expert software, the optimal conditions for yeast fermentation of Polygonatum sibiricum are: inoculum size 4.767%, liquid-to-liquid ratio 1:75.399 g / mL, and fermentation time 23.259 h. The predicted polysaccharide yield in the fermentation broth is 61.929%. Based on practical considerations, the optimal fermentation conditions for Polygonatum sibiricum fermentation broth were determined to be an inoculum size of 4.7%, liquid-to-liquid ratio 1:76, fermentation time 23 h, and fermentation temperature 30°C. Three confirmatory experiments were conducted under these optimal culture conditions, resulting in a polysaccharide yield of 61.18%, which is consistent with theoretical predictions. This demonstrates that the model fits well and that the regression equation is effective in analyzing and predicting fermentation conditions. This indicates that this process is the optimal process for extracting Polygonatum sibiricum polysaccharides using yeast fermentation.

[0099] Next, the antioxidant activity of the extracted polygonatum polysaccharide was determined. Polygonatum polysaccharide was prepared according to the extraction method of polygonatum polysaccharide in Example 1, wherein the inoculation amount was 4.7%, the material-liquid ratio was 1:76, the fermentation time was 23 h, and the fermentation temperature was 30°C.

[0100] A sample solution of Polygonatum sibiricum polysaccharide was prepared.

[0101] 1. Determination of DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) free radical scavenging ability Refer to Example 1 for the determination of the DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) free radical scavenging ability.

[0102] 2. Determination of the free radical scavenging ability of ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) Refer to Example 1 for the determination of the free radical scavenging ability of ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid).

[0103] 3. Determination of hydroxyl radical scavenging ability Refer to the measurement procedure of the hydroxyl radical scavenging ability in Example 1.

[0104] The yield and antioxidant activity of Polygonatum sibiricum polysaccharide prepared by the fermentation method provided by the present invention and the water immersion method are compared, and the comparison results are shown in Table 8.

[0105]

[0106] As shown in Table 4, the yield and antioxidant activity of Polygonatum sibiricum polysaccharides extracted by fermentation were significantly higher than those extracted by water extraction. This is likely due to the fact that microorganisms, through their metabolic activities, decompose components of the plant cell wall, such as cellulose, hemicellulose, and pectin. These components often surround the polysaccharides, making them difficult to extract by water extraction. Microbial enzymes, such as cellulase and pectinase, help break down these structures and release more polysaccharides. The stronger antioxidant activity may be due to the fact that microorganisms, through their metabolic pathways, can transform compounds in the raw material, producing more antioxidant substances, such as organic acids, polyphenols, and flavonoids.

[0107] Comparative Example 1 This comparative example provides a comparison of the antioxidant activity / product purity of Polygonatum sibiricum polysaccharide prepared by publication number CN117604055A and the antioxidant activity / product purity of Polygonatum sibiricum polysaccharide prepared using the optimal extraction process parameters of the present application. This demonstrates that the present application not only has a higher yield, but also exhibits higher antioxidant activity when the product concentration is consistent.

[0108] As described above, the basic principles, main features and advantages of the present invention are well described. The above embodiments and descriptions are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the scope of protection determined by the present invention.

Claims

1. A method for extracting polygonatum polysaccharide, characterized in that: include: The pretreated polygonatum is placed in a container at a material-liquid ratio of 1:20 to 100; Inoculate the edible fungi at an inoculum rate of 1% to 13%; After fermentation at 30°C for 12 to 72 hours, the supernatant was collected to obtain polygonatum polysaccharide.

2. The extraction method according to claim 1, wherein The pretreatment comprises slicing the polygonatum, drying the polygonatum in an oven at 50-60° C. to a constant weight, then crushing the polygonatum, and passing the mixture through a 60-mesh sieve to obtain polygonatum powder.

3. The extraction method according to claim 2, characterized in that The material-liquid ratio is 1:

76.

4. The extraction method according to claim 3, characterized in that The edible fungus is yeast, which is pretreated before inoculation. The pretreatment includes: activating the yeast strain, placing the activated yeast single colony in a liquid culture medium for shaking culture to prepare a liquid strain.

5. The extraction method according to claim 4, characterized in that The yeast was Saccharomyces cerevisiae ATCC9080 strain, the inoculation amount was 4.7%, and the fermentation time was 23 h.

6. The extraction method according to claim 2, characterized in that The material-liquid ratio is: 1:

80.

7. The extraction method according to claim 6, characterized in that The edible fungus is Lactobacillus paracasei, and the inoculation amount is 3% to 11%.

8. The extraction method according to claim 7, characterized in that The Lactobacillus paracasei is pretreated before inoculation. The pretreatment includes: activating the Lactobacillus paracasei strain, placing the activated Lactobacillus paracasei single colony in a liquid culture medium for shaking culture to prepare a liquid strain.

9. The extraction method according to claim 8, characterized in that The Lactobacillus paracasei was ATCC-334 strain, the inoculation amount was 6.6%, and the fermentation time was 34 h.

10. A Polygonatum sibiricum polysaccharide, characterized in that: The method is extracted using any one of claims 1 to 9.

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