High-activity chinese yam polysaccharide and preparation method therefor

Abstract

A high-activity Chinese yam polysaccharide and a preparation method therefor, relating to the technical field of nutritional food production. The method comprises: subjecting Chinese yam slurry to ultra-high pressure treatment to obtain ultra-high pressure-treated Chinese yam slurry; inoculating Lactobacillus plantarum subsp. plantarum into the ultra-high pressure-treated Chinese yam slurry, and fermenting to obtain Chinese yam fermentation broth; subjecting the Chinese yam fermentation broth to hot water extraction and centrifugation, collecting the supernatant, and concentrating and drying same to obtain a Chinese yam polysaccharide. The combination of ultra-high pressure and fermentation treatments can significantly increase the yield of Chinese yam polysaccharides, and the obtained Chinese yam polysaccharides have strong inhibitory effects on both lipase and amylase. The preparation method for the Chinese yam polysaccharide is simple, low-cost, energy-efficient, and suitable for large-scale production.

Description

Highly active yam polysaccharide and preparation method thereof Technical Field

[0001] The invention belongs to the technical field of nutritious food production, and particularly relates to a highly active yam polysaccharide and a preparation method thereof. Background Art

[0002] Chinese yam, also known as yam and yam, is rich in nutritional value and possesses numerous physiological and health benefits, making it an excellent medicinal food. Traditional Chinese medicine believes that Chinese yam has benefits such as strengthening the spleen and stopping diarrhea. Modern medical research indicates that polysaccharides are the main active functional component of Chinese yam, exhibiting immunomodulatory, diabetes-modulating, and antibacterial properties. Consequently, research on Chinese yam polysaccharides has attracted widespread attention. Chinese yam polysaccharides have been shown to alleviate colonic pathological damage in mice with DSS-induced colitis, inhibit activation of the colonic inflammatory signaling pathway NF-κB, and restore mRNA expression of the tight junction protein ZO-1, alleviating colitis. Chinese yam polysaccharides have also been shown to increase insulin secretion and improve the function of damaged pancreatic β cells in diabetic rats, exerting a hypoglycemic effect. As a natural active functional factor, Chinese yam polysaccharides hold great promise for development and utilization. However, the high starch content in Chinese yam significantly hinders its dissolution. Furthermore, the presence of Chinese yam polysaccharides in the cell walls, along with components such as cellulose and hemicellulose, also hinder their dissolution. Current extraction methods for Chinese yam polysaccharides include hot water extraction, ultrasound-assisted extraction, and enzyme-assisted extraction. However, hot water extraction is inefficient, and other methods are only suitable for small-scale laboratory-scale polysaccharide preparation and are energy-intensive, making them unsuitable for large-scale production. These drawbacks of yam polysaccharide extraction, such as low extraction yields and high energy consumption, have limited its development and utilization, necessitating the development of new technologies to address this issue.

[0003] Summary of the Invention

[0004] The present invention provides a highly active yam polysaccharide and a preparation method thereof. The yam polysaccharide obtained by the method of the present invention has a high yield and has significant inhibitory activity against lipase and amylase, and has high extraction efficiency, low energy consumption, and is environmentally friendly.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The invention provides a preparation method of highly active yam polysaccharide, comprising the following steps: subjecting yam slurry to ultrahigh pressure treatment to obtain ultrahigh pressure treated yam slurry; inoculating Lactobacillus plantarum subspecies into the ultrahigh pressure treated yam slurry, fermenting, and obtaining yam fermentation liquid; subjecting the yam fermentation liquid to hot water extraction, centrifugation, and concentrating and drying the supernatant to obtain yam polysaccharide.

[0007] Preferably, fresh yam and water are mixed and beaten in a mass ratio of 1:0.5 to 3 to obtain preliminary yam slurry.

[0008] Preferably, before the ultrahigh pressure treatment, the preliminary yam slurry is mixed with water to obtain yam slurry; the mass volume ratio of fresh yam to water in the yam slurry is 1:4 to 1:8.

[0009] Preferably, the pressure of the ultrahigh pressure treatment is 200-600 MPa, and the holding time is 5-11 minutes.

[0010] Preferably, the Lactobacillus plantarum subspecies plantarum needs to be cultured in MRS broth medium until a bacterial liquid is used.

[0011] Preferably, the bacterial concentration of Lactobacillus plantarum subspecies is 0.5×10 9 ~1.5×10 9 cfu / mL.

[0012] Preferably, the inoculation amount of the Lactobacillus plantarum subspecies plantarum is 2-6% based on the volume percentage of the fresh yam mass and the Lactobacillus plantarum subspecies plantarum bacterial liquid.

[0013] Preferably, the fermentation temperature is 35-40°C.

[0014] Preferably, the fermentation time is 1 to 4 days.

[0015] Preferably, the yam slurry after ultrahigh pressure treatment needs to be sterilized at 70-90° C. for 20-40 minutes and then inoculated with Lactobacillus plantarum subsp. plantarum for fermentation.

[0016] Preferably, water is added to the yam fermentation liquid so that 8 to 15 mL of water is contained in 1 gram of fresh yam, and hot water extraction is performed.

[0017] Preferably, the temperature of the hot water extraction is 80-90°C.

[0018] Preferably, the time is 2 to 5 hours.

[0019] Preferably, the concentration is to 1 / 6 to 1 / 3 of the original volume.

[0020] Preferably, the concentrate is deproteinized by the sevag method, and after the organic reagent is removed by rotary evaporation, it is placed in water for 36 to 60 hours using a dialysis bag with a molecular weight cutoff of 7000 to 9000 Da. The dialysate is collected and dried to obtain the yam polysaccharide extract.

[0021] The present invention also provides high-activity yam polysaccharide obtained by the preparation method.

[0022] The present invention also provides the use of the high-activity yam polysaccharide or the high-activity yam polysaccharide obtained by the preparation method in preparing products that inhibit the activity of lipase and / or amylase.

[0023] The present invention also provides the use of the highly active yam polysaccharide or the highly active yam polysaccharide obtained by the preparation method in preparing edible products for obese or hyperglycemic patients.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The preparation method of yam polysaccharide of the present invention is simple, low-cost, and easy to scale up.

[0026] (2) The extraction and preparation of yam polysaccharide of the present invention adopts a combined treatment of ultrahigh pressure and lactic acid bacteria fermentation. Compared with the existing extraction and preparation methods, the extraction rate of yam polysaccharide is significantly improved.

[0027] (3) The yam polysaccharide prepared by the present invention has a significant effect of inhibiting the activity of lipase and amylase compared with the conventionally extracted yam polysaccharide, and is suitable for consumption by obese patients, hyperglycemic patients or diabetic patients. It also has a better effect of promoting the proliferation and colonization of probiotics, and can be used for the development of yam functional foods, which is of great significance to the deep processing of yam. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Fig. 1. Selection results of fermentation strains.

[0029] Figure 2. Results of ultra-high pressure single-factor experiments.

[0030] Fig. 3 Results of single-factor fermentation experiment. DETAILED DESCRIPTION

[0031] The invention provides a preparation method of highly active yam polysaccharide, comprising the following steps: subjecting yam slurry to ultrahigh pressure treatment to obtain ultrahigh pressure treated yam slurry; inoculating Lactobacillus plantarum subspecies into the ultrahigh pressure treated yam slurry, fermenting, and obtaining yam fermentation liquid; subjecting the yam fermentation liquid to hot water extraction, centrifugation, and concentrating and drying the supernatant to obtain yam polysaccharide.

[0032] In the present invention, the mass volume ratio of fresh yam to water in the yam slurry is 1:4 to 1:8 (final material-liquid ratio). The present invention peels the fresh yam, then mixes and beats it in a ratio of fresh yam mass to water mass of 1:0.5 to 1.5, then adds water to the beaten initial yam slurry until the mass volume ratio of fresh yam to water in the system is 1:4 to 1:8, and mixes to obtain yam slurry. To ensure that the particle size of the yam is basically consistent, the present invention first beats the fresh yam in a certain ratio, and then adds a corresponding amount of water in the final ratio to obtain a yam slurry for subsequent experiments.

[0033] In the present invention, when ultra-high pressure is used to treat fresh yam pulp, the high pressure can not only destroy the cell wall structure, making the polysaccharides in the cells more easily dissolve, but also change the crystal structure of starch, change its gelatinization properties, increase solubility, and reduce viscosity. However, the ultra-high pressure pressure, treatment time, and material-liquid ratio will affect the yam polysaccharide extraction rate. When the pressure is too high, the yam cell wall will be severely damaged, causing impurities to dissolve, resulting in reduced polysaccharide extraction purity and extraction rate; when the pressure is too low, the degree of cell wall damage is limited, polysaccharide dissolution is hindered, and the yield is reduced; when the pressure holding time is short, polysaccharide dissolution is insufficient and the yield is not high; when the pressure holding time is too long, the polysaccharide is completely dissolved, and the yield does not increase with the extension of the treatment time, and energy consumption will increase. Therefore, the pressure of the ultra-high pressure treatment described in the present invention is 200-600MPa, and the pressure holding time is 5-11min; preferably, the pressure is 300-500MPa, and the pressure holding time is 6-10min.

[0034] In the present invention, the ultra-high pressure treated yam slurry needs to be sterilized. The purpose of the sterilization of the present invention is to kill various strains in the yam slurry and to achieve the gelatinization of yam starch. The sterilization method selected by the present invention is obtained after comparing conventional high-temperature sterilization and pasteurization. Conventional high-temperature sterilization (121°C, 15min) will change the structure of polysaccharides in yam, and excessively high temperature will cause degradation of yam polysaccharide chains, thereby reducing its biological activity. However, when pasteurized at 60-80°C (70°C is optimal) and treated for 20-40min (30min is optimal), the structure and biological activity of the active polysaccharides in yam will not be changed. At the same time, the miscellaneous bacteria in the yam slurry can be removed, and the starch in the yam can be gelatinized, thereby being better utilized by lactic acid bacteria.

[0035] In the present invention, the selected Lactobacillus plantarum subspecies was obtained by experimental screening. By comparing different strains, it was found that when the bacteria was used to ferment yam pulp, the yield of yam polysaccharides was the highest. The Lactobacillus plantarum subspecies described in the embodiment of the present invention was purchased from Guangdong Provincial Microbial Culture Collection Center, and the Lactobacillus plantarum subspecies was numbered GIM 1.380. The inoculation amount of the Lactobacillus plantarum subspecies described in the present invention is closely related to the growth state of the Lactobacillus plantarum subspecies, and will also affect the fermentation time. When the inoculation amount is too low, the fermentation time required is too long; when the inoculation amount is too much, the lactic acid bacteria grow excessively, and it will also secrete carbohydrate enzymes to degrade polysaccharides, resulting in a decrease in the polysaccharide yield. Therefore, the plant lactic acid bacteria inoculation amount selected by the present invention (according to the volume percentage of the fresh yam mass and the Lactobacillus plantarum subspecies bacterial liquid) is 2-6%, preferably 3-5%; the fermentation temperature is 35-40°C, preferably 37°C, and the fermentation time is 1-4d, preferably 1.5-2.5d. The bacterial concentration of the Lactobacillus plantarum subspecies described in the present invention is 0.5×10 9 ~1.5×10 9cfu / mL, preferably 1×10 9 cfu / mL. The bacterial liquid obtaining method of plantarum subspecies plantarum of the present invention comprises the steps: adopt the method for plate streaking to be coated in MRS agar medium and cultivate 48h after taking out the plantarum subspecies plantarum bacterial strain preserved in liquid nitrogen, select single colony inoculation to cultivate in 10mL MRS broth after 12h centrifugation (3000g, 10min), abandon supernatant, then add 10mL MRS broth and cultivate 12h. After cultivating, centrifugation (3000g, 10min) abandons supernatant, adds 5mL physiological saline (0.9%NaCl) and mixes and obtains plantarum subspecies plantarum bacterial liquid.

[0036] In the present invention, water is added to the fermentation broth at a final material-liquid ratio of 8 to 12 mL / g, and the broth is placed in 80 to 90°C hot water for extraction for 2 to 5 hours, centrifuged at 3000 to 5000 r / min, and the supernatant is taken. After concentrating to 1 / 6 to 1 / 3 of the original volume, the sevag method is used for deproteinization (1 / 4 volume of sevag reagent is added and shaken vigorously for 30 minutes, and the configuration of sevag reagent is chloroform: n-butanol = 4:1). After rotary evaporation to remove the organic reagent, a dialysis bag with a molecular weight cutoff of 7500 to 8500 Da is placed in pure water for dialyzation for 40 to 55 hours, and the dialysate is taken and dried to obtain the yam polysaccharide extract. The hot water extraction of the present invention is preferably extracted at 80°C for 3 hours. In the hot water extraction of the present invention, too high a heating temperature may cause the destruction of the polysaccharide components, and too low a temperature may cause insufficient extraction. The present invention is preferably concentrated to 1 / 5 of the original volume.

[0037] The present invention also provides high-activity yam polysaccharide obtained by the preparation method.

[0038] The present invention also provides the use of the highly active yam polysaccharide or the highly active yam polysaccharide obtained by the preparation method in the preparation of products that inhibit the activity of lipase and amylase. The present invention's inhibition of pancreatic lipase has been used as a target in the treatment of obesity; α-amylase inhibitors can slow the digestion of carbohydrates in patients with hyperglycemia or diabetes, resulting in a decrease in the rate of glucose absorption, thereby reducing the rise in postprandial blood sugar. Therefore, the highly active yam polysaccharide prepared by the present invention can be used as an inhibitor of lipase and amylase, and has a certain preventive and / or therapeutic effect on patients with obesity, hyperglycemia or diabetes. The yam polysaccharide of the present invention can also be used to promote the proliferation and colonization of probiotics.

[0039] In the present invention, unless otherwise specified, all components or reagents are commercially available products well known to those skilled in the art.

[0040] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0041] Example 1

[0042] This embodiment provides a method for preparing highly active yam polysaccharide, comprising the following steps:

[0043] 1) Ultrahigh pressure treatment: 100 g of fresh Chinese yam was slurried with water in a 1:1 mass ratio. Water was then added to the initial slurry until the system had a ratio of fresh Chinese yam to water of 1:5 (W / V, g / mL). The mixture was thoroughly mixed, sealed in a polyethylene bag, and then ultrahigh pressure treated at 500 MPa for 10 min.

[0044] 2) Lactobacillus fermentation: After taking out the plantarum Lactobacillus subsp. plantarum strain (GIM1.380) preserved in liquid nitrogen, the plate streaking method was adopted to be coated on MRS agar medium and cultured for 48h. After selecting a single colony and inoculating it into 10mL MRS broth and cultivating for 12h, the supernatant was discarded by centrifugation (3000g, 10min), and then 10mL MRS broth was added and cultivated for 12h. After the cultivation was completed, the supernatant was discarded by centrifugation (3000g, 10min), 5mL physiological saline (0.9% NaCl) was added and mixed to obtain the plantarum Lactobacillus subsp. plantarum bacterial liquid, and the bacterial concentration was 1×10 9 cfu / mL;

[0045] The ultrahigh pressure treated yam slurry was sterilized at 80° C. for 30 min, inoculated with 5% (5 ml of Lactobacillus plantarum subspecies plantarum bacterial solution was inoculated into 100 g of fresh yam) Lactobacillus plantarum subspecies plantarum bacterial solution, and fermented at 37° C. for 2 days to obtain yam fermentation liquid.

[0046] 3) Extraction: Add water to the yam fermentation broth at a final material-liquid ratio of 10 mL / g (1 g of fresh yam contains 10 mL of water), place it in 80°C hot water for extraction for 3 hours, centrifuge at 4000 r / min, take the supernatant, concentrate it to 1 / 5 of the original volume, and then use the sevag method to deproteinize (add 1 / 4 volume of sevag reagent and shake vigorously for 30 minutes, the configuration of sevag reagent is chloroform: n-butanol = 4:1), after rotary evaporation to remove the organic reagent, use a dialysis bag with a molecular weight cutoff of 8000 Da to dialyze in pure water for 48 hours, take the dialysate, and dry it to obtain the yam polysaccharide extract.

[0047] Example 2

[0048] This embodiment provides a method for preparing highly active yam polysaccharide, comprising the following steps:

[0049] 1) Ultrahigh pressure treatment: 100 g of fresh Chinese yam was slurried with water in a 1:1 mass ratio. Water was then added to the initial slurry until the system had a ratio of fresh Chinese yam to water of 1:7 (g / mL). The mixture was thoroughly mixed and sealed in a polyethylene bag before ultrahigh pressure treatment at 300 MPa for 8 min.

[0050] 2) Lactobacillus fermentation: The preparation of the Lactobacillus plantarum subsp. plantarum bacterial solution, the strain used, and the bacterial concentration were the same as those in Example 1;

[0051] The ultrahigh pressure treated yam slurry was sterilized at 80° C. for 30 min, inoculated with 5% (5 ml of Lactobacillus plantarum subspecies plantarum bacterial solution was inoculated into 100 g of fresh yam) Lactobacillus plantarum subspecies plantarum bacterial solution, and fermented at 37° C. for 3 days to obtain yam fermentation liquid.

[0052] 3) Extraction: Add water to the yam fermentation broth at a final material-liquid ratio of 10 mL / g (1 g of fresh yam contains 10 mL of water), place it in 85°C hot water for extraction for 3 hours, centrifuge at 4000 r / min, take the supernatant, concentrate it to 1 / 5 of the original volume, and then use the sevag method to deproteinize (add 1 / 4 volume of sevag reagent and shake vigorously for 30 minutes, the configuration of sevag reagent is chloroform: n-butanol = 4:1), after rotary evaporation to remove the organic reagent, use a dialysis bag with a molecular weight cutoff of 8000 Da to dialyze in pure water for 48 hours, take the dialysate, and dry it to obtain the yam polysaccharide extract.

[0053] Example 3

[0054] This embodiment provides a method for preparing highly active yam polysaccharide, comprising the following steps:

[0055] 1) Ultrahigh pressure treatment: 100 g of fresh Chinese yam was slurried with water in a mass ratio of 1:1. Water was then added to the initial slurry until the system had a ratio of fresh Chinese yam to water of 1:7 (g / mL). The mixture was thoroughly mixed and sealed in a polyethylene bag before ultrahigh pressure treatment at 500 MPa for 6 min.

[0056] 2) Lactobacillus fermentation: The preparation of the Lactobacillus plantarum subsp. plantarum bacterial solution, the strain used, and the bacterial concentration were the same as those in Example 1;

[0057] The ultrahigh pressure treated yam slurry was sterilized at 80° C. for 30 min, inoculated with 3% (3 ml of Lactobacillus plantarum subspecies plantarum bacterial solution was inoculated into 100 g of fresh yam) Lactobacillus plantarum subspecies plantarum bacterial solution, and fermented at 37° C. for 2 days to obtain yam fermentation liquid.

[0058] 3) Extraction: Add water to the yam fermentation broth at a final material-liquid ratio of 10 mL / g (1 g of fresh yam contains 10 mL of water), place it in 90°C hot water for extraction for 3 hours, centrifuge at 4000 r / min, take the supernatant, concentrate it to 1 / 5 of the original volume, and then use the sevag method to deproteinize (add 1 / 4 volume of sevag reagent and shake vigorously for 30 minutes, the configuration of sevag reagent is chloroform: n-butanol = 4:1), after rotary evaporation to remove the organic reagent, use a dialysis bag with a molecular weight cutoff of 8000 Da to dialyze in pure water for 48 hours, take the dialysate, and dry it to obtain the yam polysaccharide extract.

[0059] Example 4

[0060] This embodiment provides a method for preparing highly active yam polysaccharide, comprising the following steps:

[0061] 1) Ultrahigh pressure treatment: 100 g of fresh Chinese yam was slurried with water in a 1:1 mass ratio. Water was then added to the initial slurry until the system had a ratio of fresh Chinese yam to water of 1:7 (g / mL). The mixture was thoroughly mixed and sealed in a polyethylene bag. The mixture was then ultrahigh pressure treated at 400 MPa for 6 min.

[0062] 2) Lactobacillus fermentation: The preparation of the Lactobacillus plantarum subsp. plantarum bacterial solution, the strain used, and the bacterial concentration were the same as those in Example 1;

[0063] The ultrahigh pressure treated yam slurry was sterilized at 80° C. for 30 min, inoculated with 5% (5 ml of Lactobacillus plantarum subspecies plantarum bacterial solution was inoculated into 100 g of fresh yam) Lactobacillus plantarum subspecies plantarum bacterial solution, and fermented at 37° C. for 3 days to obtain yam fermentation liquid.

[0064] 3) Extraction: Add water to the yam fermentation broth at a final material-liquid ratio of 10 mL / g (1 g of fresh yam contains 10 mL of water), place it in 90°C hot water for extraction for 3 hours, centrifuge at 4000 r / min, take the supernatant, concentrate it to 1 / 5 of the original volume, and then use the sevag method to deproteinize (add 1 / 4 volume of sevag reagent and shake vigorously for 30 minutes, the configuration of sevag reagent is chloroform: n-butanol = 4:1), after rotary evaporation to remove the organic reagent, use a dialysis bag with a molecular weight cutoff of 8000 Da to dialyze in pure water for 48 hours, take the dialysate, and dry it to obtain the yam polysaccharide extract.

[0065] Test method 1: The polysaccharide content of the yam polysaccharide extracts prepared in Examples 1 to 4 was determined and the polysaccharide yield was calculated. The specific method is as follows:

[0066] Total sugar content determination: The phenol-sulfuric acid method was used to determine the total sugar content of the extract, using glucose as the standard. The principle of determination is that at high temperatures, sulfuric acid decomposes polysaccharides into monosaccharides, which are rapidly dehydrated to form aldehyde derivatives. These react with phenol to form an orange-yellow compound with a maximum absorbance at 490 nm, and a linear relationship is observed within a certain range. Glucose (dried to constant weight at 105°C before use) was prepared into standard solutions of varying concentrations (0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.08 mg / mL, and 0.1 mg / mL). The polysaccharide sample concentration was 0.1 mg / mL. To 1 mL of the sample to be tested, 1 mL of 5% phenol solution was added, followed by 5 mL of concentrated sulfuric acid. The mixture was allowed to stand for 30 minutes, and the absorbance was measured at 490 nm. A standard curve was constructed with glucose concentration as the horizontal axis and absorbance as the vertical axis, and the total sugar content of the polysaccharide sample was calculated based on this standard curve.

[0067] Reducing Sugar Content Determination: The reducing sugar content in the extract was calculated using the DNS method, using glucose as the standard. The assay principle is that dinitrosalicylic acid (DNS) undergoes an oxidation-reduction reaction with reducing sugars to produce 3-amino-5-nitrosalicylic acid, which has a maximum absorbance at 540 nm and exhibits a linear relationship within a certain range. DNS reagent preparation: Add 6.3 g of DNS and 262 ml of 2 mol / L sodium hydroxide to 500 ml of a hot aqueous solution containing 185 g of potassium sodium tartrate. Add 5 g of phenol and 5 g of sodium sulfite, stir to dissolve, and cool to a volume of 1000 ml with water. This prepares the DNS reagent and stores in a brown bottle at 4°C until use. Glucose (dried to constant weight at 105°C before use) was prepared into standard solutions of varying concentrations (0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1 mg / mL). The polysaccharide sample concentration was 5 mg / mL. Take 1 mL of the sample to be tested, add 0.75 mL of DNS reagent, shake well, heat in a boiling water bath for 5 minutes, quickly cool to room temperature in an ice-water bath, add 10.75 mL of distilled water, vortex to mix, and measure the absorbance at 540 nm. Develop a standard curve with glucose concentration as the x-axis and absorbance as the y-axis, and calculate the total sugar content in the polysaccharide sample based on this standard curve.

[0068] Polysaccharide content = total sugar - reducing sugar content;

[0069] Extraction yield = polysaccharide content / fresh yam mass × 100%.

[0070] Table 1 Extraction yield of yam polysaccharides prepared in Examples 1-4

[0071] Example 5 Process Optimization in the Preparation Method of Yam Polysaccharide

[0072] 1. Screening of strains: A total of 9 different strains and strain combinations were selected to ferment Chinese yam, namely Lactobacillus fermentum GIM1.1796, Lactobacillus acidophilus GIM 1.731, Lactobacillus plantarum subsp. plantarum GIM 1.380, Lactobacillus casei GIM 1.410, Bacillus subtilis GIM 1.372, Aspergillus niger GIM 3.576, Rhizopus oryzae GIM 3.127, Trichoderma viride GIM 3.443, Lactobacillus plantarum subsp. plantarum GIM 1.380+Bacillus subtilis GIM 1.372. The above strains were all purchased from Guangdong Provincial Microbial Culture Collection Center. The difference from the experimental steps of Example 1 is that in the mold-related experiments, the final material-liquid ratio of Chinese yam slurry to water in step (1) is 1:2, the inoculation amount is 1%, and the inoculation concentration is about 1×10 6 CFU / mL; in the experiment using Lactobacillus plantarum subspecies plantarum GIM 1.380 + Bacillus subtilis GIM 1.372, the volume ratio of Lactobacillus plantarum subspecies plantarum bacterial solution + Bacillus subtilis bacterial solution in the mixed bacterial solution was 1:1, and the other experimental steps were the same as in Example 1.

[0073] The preparation method of the above-mentioned Lactobacillus liquid is the same as that in Example 1.

[0074] Regarding the preparation of mold culture liquid: the preserved strains (Aspergillus niger GIM 3.576, Rhizopus oryzae GIM 3.127, Trichoderma viride GIM 3.443) were spread on PDA plate culture medium and cultured at 30°C for 5-7 days to grow mycelium. 10 mL of sterile water was added, and the mycelium on the agar surface was carefully scraped off. The mycelium was sucked into a sterilized centrifuge tube and shaken. After mixing evenly, it was transferred to a nucleic acid purification column filled with sterilized absorbent cotton for filtration. The filtrate was transferred to a 50 mL centrifuge tube to obtain a spore suspension for use.

[0075] Preparation of a Bacillus subtilis culture solution: A Bacillus subtilis strain (GIM 1.372) stored in liquid nitrogen was streaked onto LB agar and incubated for 48 hours. A single colony was selected and inoculated into 10 mL of LB broth, incubated for 12 hours, centrifuged (3000 g, 10 minutes), and the supernatant discarded. The culture was then added to 10 mL of LB broth and incubated for another 12 hours. After completion of the incubation, the culture was centrifuged (3000 g, 10 minutes), the supernatant discarded, and 5 mL of physiological saline (0.9% NaCl) was added and mixed to obtain a Bacillus subtilis culture solution.

[0076] The results are shown in FIG1 . Lactobacillus plantarum subsp. plantarum (GIM 1.380) can effectively increase the yield of yam polysaccharides compared with other strains.

[0077] 2. Ultra-high pressure single factor experiment

[0078] 1) Optimization of the material-liquid ratio (yam: water, W:V): The material-liquid ratio was changed to 1:2, 1:3, 1:4, 1:5, and 1:6, with a pressure of 400 MPa, a treatment time of 8 minutes, and 20 g of fresh yam. The remaining steps were the same as those in Example 1.

[0079] 2) Pressure optimization: The extraction pressure was changed to 200, 300, 400, 500, and 600 MPa, the holding time was 8 min, the molar material-liquid ratio was 1:4, and 20 g of fresh yam was used. The remaining steps were the same as the ultrahigh pressure treatment steps in Example 1.

[0080] 3) Optimization of pressure holding time: changing the holding time to 2, 4, 6, 8, and 10 min, the extraction pressure to 400 MPa, the material-liquid ratio to 1:4, and the fresh yam to 20 g. The remaining steps were the same as the ultrahigh pressure treatment steps in Example 1.

[0081] Polysaccharide Extraction: Take the ultrahigh pressure treated sample (containing 20g of fresh yam) and add the appropriate amount of distilled water at a material-to-liquid ratio of 1:10. Extract in an 80°C waterbath for 3 hours. Centrifuge at 4000 rpm, collect the supernatant, concentrate it, and dilute it to 20ml. Add four volumes of 95% ethanol and let it stand at 4°C overnight. Centrifuge the precipitate, redissolve it, and dilute it to 100ml. Determine the polysaccharide content using the phenol-sulfuric acid method and the DNS method, respectively. The difference between the two values ​​is approximately equal to the polysaccharide content. The ratio of the polysaccharide content to the mass of the fresh yam is the polysaccharide extraction rate. The results are shown in Figure 2.

[0082] As shown in Figure 2, in the ultrahigh pressure process, the extraction rate of polysaccharides increases within a certain range with the increase of the solid-liquid ratio, pressure and holding time. In each single-factor experiment, the polysaccharide extraction rate reaches the highest when the solid-liquid ratio is 1:5, the pressure is 400 MPa, and the holding time is 10 min.

[0083] 3. Fermentation single factor experiment

[0084] 1) Optimization of the material-liquid ratio (yam: water, W:V): the material-liquid ratio was changed to 1:2, 1:4, 1:6, 1:8, and 1:10, the inoculation amount was 2%, and the fermentation time was 2 days.

[0085] 2) Optimization of inoculation amount: Change the inoculation amount (according to the mass of yam, the bacterial concentration is 1×10 9 cfu / mL) was 1%, 2%, 3%, 4%, and 5%, the fermentation time was 2 days, and the material-liquid ratio was 1:4.

[0086] 3) Fermentation time optimization: the fermentation time was changed to 1d, 2d, 3d, 4d, and 5d, the inoculation amount was 2%, and the material-liquid ratio was 1:4.

[0087] The fermentation steps of yam according to the above-set process parameters include: beating the yam at a ratio of 1:1 (yam: water, W:V), weighing 20g and placing it in a 150ml conical flask, adding a certain amount of water according to different material-liquid ratios and sealing the flask, placing it in a sterilizer for pasteurization (80°C, 30 minutes), inoculating Lactobacillus plantarum subspecies plantarum (GIM 1.380) according to different inoculum sizes, and fermenting at 37°C for different fermentation times.

[0088] Extraction and determination of polysaccharides: After fermentation is complete, samples are taken out and the remaining determination steps are the same as step 2 of this example. The results are shown in Figure 3.

[0089] As shown in Figure 3, in the single-factor fermentation experiment, the polysaccharide extraction rate showed a trend of first increasing and then decreasing with the increase of the solid-liquid ratio, inoculation size and fermentation time. When the polysaccharide extraction rate reached the highest point, the various factor conditions were: solid-liquid ratio (1:6), inoculation size (4%), and fermentation time (2d).

[0090] 4. Orthogonal experiment

[0091] The extraction process of yam polysaccharide was optimized by a five-factor three-water orthogonal test. The experimental process settings and results are shown in Tables 2 and 3.

[0092] Table 2 Comparison of polysaccharides obtained by different extraction processes

[0093] Table 3 Test results of inter-subject effects in orthogonal experiment

[0094] The results in Tables 2 and 3 indicate that the optimal process is: inoculum concentration 5%, fermentation time 2 days, material-liquid ratio 1:5, pressure 500 MPa, and pressure holding time 10 minutes. Since the experimental metric is polysaccharide yield, and higher is better, the optimal solution should be determined by selecting the level corresponding to the maximum K value of each factor, where K1, K2, and K3 correspond to levels 1, 2, and 3, respectively. Table 3 shows that inoculum concentration, fermentation time, pressure, and pressure holding time significantly affect polysaccharide yield. Therefore, when determining the optimal level, the maximum level should be selected: inoculum concentration 5%, fermentation time 2 days, pressure 500 MPa, and pressure holding time 10 minutes. The material-liquid ratio has no significant effect, so a material-liquid ratio of 1:5 is selected to maximize processing efficiency.

[0095] The above-mentioned preferred optimal process was verified using the steps described in Example 1, and the polysaccharide extraction yield was measured.

[0096] Comparative Example 1

[0097] The hot water method for extracting yam polysaccharides includes the following steps:

[0098] Take 20g of yam, add water at a solid-liquid ratio of 10mL / g to pulp the yam, place the yam pulp in 80℃ hot water for extraction for 3h, centrifuge at 4000r / min, take the supernatant by centrifugation, concentrate the supernatant to 1 / 5 of the original volume, and then use the sevag method to deproteinize (add 1 / 4 volume of sevag reagent and shake vigorously for 30min, the configuration of sevag reagent is chloroform: n-butanol = 4:1), after rotary evaporation to remove the organic reagent, use a dialysis bag with a molecular weight cutoff of 8000Da to dialyze in pure water for 48h, take the dialyzate and dry it to obtain the extract.

[0099] Comparative Example 2

[0100] The ultrahigh pressure method for extracting yam polysaccharides includes the following steps: taking 20g of yam, adding water at a solid-liquid ratio of 6mL / g to pulp the yam, placing it in a polyethylene bag and sealing it for ultrahigh pressure extraction, the ultrahigh pressure pressure is 500MPa, and the pressure holding time is 10min. After the ultrahigh pressure treatment, a certain amount of water is added according to the final solid-liquid ratio of 10mL / g, and the yam is placed in 80℃ hot water for extraction for 3h, centrifuged to obtain the supernatant, concentrated to 1 / 5 of the original volume, and then deproteinized by the sevag method (adding 1 / 4 volume of sevag reagent and shaking vigorously for 30min, the configuration of sevag reagent is chloroform: n-butanol = 4:1), after rotary evaporation to remove organic reagents, a dialysis bag with a molecular weight cutoff of 8000Da is used for dialysis in pure water for 48h, and the dialyzate is dried to obtain the extract.

[0101] Comparative Example 3

[0102] The method for extracting yam polysaccharide by fermentation comprises the following steps: taking 20 g of yam, adding water at a solid-liquid ratio of 6 mL / g to beat the yam into a pulp, inoculating a Lactobacillus plantarum subspecies plantarum bacterial liquid at a 4% inoculum amount into the yam pulp (the Lactobacillus plantarum subspecies plantarum strain, bacterial liquid preparation, and inoculation method are the same as those in Example 1), fermenting at 37° C. for 2 days, adding water after fermentation so that the final water / fermentation liquid ratio is 10 mL / g, subjecting the mixture to 80° C. hot water extraction for 3 hours, centrifuging at 4000 rpm to obtain a supernatant, concentrating the mixture to 1 / 5 of the original volume, and then deproteinizing the mixture by a Sevag method (adding 1 / 4 volume of a Sevag reagent and vigorously shaking the mixture for 30 minutes, wherein the configuration of the Sevag reagent is chloroform: n-butanol=4:1), removing organic reagents by rotary evaporation, and then dialyzing the mixture in pure water for 48 hours using a dialysis bag with a molecular weight cut-off of 8000 Da, and drying the dialyzed liquid to obtain an extract.

[0103] The extracts obtained in Example 1 and Comparative Examples 1-3 were subjected to polysaccharide yield determination, lipase inhibitory activity determination, and amylase inhibitory activity determination.

[0104] Determination of polysaccharide extraction yield and total sugar content: The method is the same as test method 1.

[0105] Test Method 2: Amylase Inhibitory Activity Assay: 100 μL of yam polysaccharide (0.1-20 mg / mL) solution and 100 μL of α-amylase (5 U / mL) solution were mixed and incubated at 37°C for 15 minutes. Then, 100 μL of 2 mg / mL soluble starch solution was added and incubated for 10 minutes. The reaction was then terminated by adding 225 μL of DNS solution in a boiling water bath for 5 minutes. The mixture was then cooled to room temperature and diluted fivefold with PBS (75 mM, pH 7.4). The absorbance was measured at 540 nm using a UV-Vis spectrophotometer. Acarbose was used as a positive control.

[0106] A1: yam polysaccharide + α-amylase + starch, A2: yam polysaccharide + soluble starch, A3: soluble starch + α-amylase, A4: soluble starch.

[0107] Test method 3: Pancreatic lipase inhibitory activity assay: 120 μL of soluble dietary fiber (0.1-20 mg / mL) solution and 40 μL of pancreatic lipase solution (1 mg / mL) were mixed and incubated at 37°C for 15 min. Then, 40 μL of 2 mM PNPB (4-nitrophenylbutyrate) solution was added and incubated at 37°C for 15 min. The absorbance at 405 nm was measured, and orlistat was used as a positive control.

[0108] A1: yam polysaccharide + pancreatic lipase + PNPB, A2: yam polysaccharide + PNPB, A3: pancreatic lipase + PNPB, A4: PNPB.

[0109] Table 4 Effects of different extraction processes on the yield and activity of yam polysaccharides

[0110] As shown in Table 4, compared with Comparative Examples 1-3, the extraction rate of yam polysaccharides using the present invention method reached over 10%, a significant improvement compared to the 3.66-6.65% extraction rate of yam polysaccharides obtained in the prior art. Furthermore, the inhibitory activity of the polysaccharides obtained using the present invention against pancreatic lipase and amylase was significantly enhanced. This demonstrates that the yam polysaccharides extracted using the present invention method have significantly improved both extraction rate and activity.

[0111] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A preparation method of highly active yam polysaccharide, characterized in that, It includes the following steps: Perform ultra-high pressure treatment on the yam slurry to obtain the ultra-high pressure treated yam slurry; Inoculate Lactobacillus plantarum subsp. plantarum into the ultra-high pressure treated yam slurry and ferment to obtain the yam fermentation broth; Perform hot water extraction on the yam fermentation broth, centrifuge, take the supernatant, concentrate and dry it to obtain yam polysaccharide.

2. The preparation method according to claim 1, characterized in that, Mix fresh yam and water at a mass ratio of 1:0.5 - 3 to make a slurry to obtain the preliminary yam slurry.

3. The preparation method according to claim 1, characterized in that, Before performing ultra-high pressure treatment, mix the preliminary yam slurry with water to obtain the yam slurry; the mass-to-volume ratio of fresh yam to water in the yam slurry is 1:4 - 1:

8.

4. The preparation method according to claim 1, characterized in that, The pressure of the ultra-high pressure treatment is 200 - 600 MPa, and the pressure holding time is 5 - 11 min.

5. The preparation method according to claim 1, characterized in that, The Lactobacillus plantarum subsp. plantarum needs to be cultured in MRS broth medium until the bacterial liquid is used.

6. The preparation method according to claim 1, characterized in that, The bacterial concentration of the Lactobacillus plantarum subsp. plantarum is 0.5×10 9 ~1.5×10 9 cfu / mL.

7. The preparation method according to claim 1, wherein The Lactobacillus plantarum subsp. plantarum is inoculated according to the volume percentage of the mass of fresh yam to the volume of the Lactobacillus plantarum subsp. plantarum bacterial liquid at 2 - 6%.

8. The preparation method according to claim 1, characterized in that, The temperature of the fermentation is 35 - 40 °C.

9. The preparation method according to claim 1, wherein The time of the fermentation is 1 - 4 d.

10. The preparation method according to claim 1, characterized in that, After ultra-high pressure treatment, the yam slurry needs to be sterilized at 70 - 90 °C for 20 - 40 min, and then inoculated with Lactobacillus plantarum subsp. plantarum for fermentation.

11. The preparation method according to claim 1, characterized in that, Add water to the yam fermentation broth according to the amount of 8 - 15 mL of water per 1 g of fresh yam, and perform hot water extraction.

12. The preparation method according to claim 1, characterized in that, The temperature of the hot water extraction is 80 - 90 °C.

13. The preparation method according to claim 1, characterized in that, The time of the hot water extraction is 2 - 5 h.

14. The preparation method according to claim 1, wherein Concentrate to 1 / 6 - 1 / 3 of the original volume.

15. The preparation method according to claim 1, characterized in that, Use the sevag method to deproteinize the concentrated solution, remove the organic reagent by rotary evaporation, then place it in a dialysis bag with a molecular weight cut-off of 7000 - 9000 Da and dialyze in water for 36 - 60 h. Take the dialysate and dry it to obtain the yam polysaccharide extract.

16. The highly active yam polysaccharide obtained by the preparation method according to any one of claims 1 - 15.

17. The application of the highly active yam polysaccharide according to claim 16 or the highly active yam polysaccharide obtained by the preparation method according to any one of claims 1 - 15 in the preparation of products for inhibiting lipase and / or amylase activities.

18. The application of the highly active yam polysaccharide according to claim 16 or the highly active yam polysaccharide obtained by the preparation method according to any one of claims 1 - 15 in the preparation of food products for obese or hyperglycemic patients.

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