A preparation method and application of low-polarity quinoa saponin monomer

Abstract

The present invention belongs to the technical field of quinoa extraction, and particularly relates to a preparation method and application of a low-polarity quinoa saponin monomer. The quinoa bran is directly subjected to heat treatment and air drying, and then the saponin components therein are enriched, separated and purified to obtain the obtained product. Through high-heat conversion, a low-polarity quinoa saponin monomer QPhy is obtained in the purified extract, and the obtained low-polarity quinoa saponin monomer QPhy has a purity of more than 90%. The low-polarity quinoa saponin monomer QPhy obtained by the present invention is verified by in vitro antibacterial experiments to be able to inhibit the growth of foodborne pathogens, and the protective effect of the low-polarity quinoa saponin QPhy on mice infected with lethal Listeria monocytogenes is verified by in vivo antibacterial experiments. The quinoa saponin monomer QPhy has a good antibacterial effect on foodborne pathogens and can be used to prepare drugs against foodborne pathogens.

Description

Technical Field

[0001] The invention belongs to the technical field of quinoa extraction, and particularly relates to a preparation method and application of a low-polar quinoa saponin monomer. Background Art

[0002] Quinoa is a nutritious grain native to South America and is known as a "superfood". Quinoa is rich in bioactive ingredients such as polyphenols, saponins, and flavonoids. A review of literature shows that quinoa saponins have antibacterial activity. Application number CN201610529182.0 is a quinoa saponin with enhanced antibacterial effect and a preparation method thereof. The quinoa saponin prepared by alkaline treatment and heating can inhibit oral bacteria. Since sodium hydroxide is required in the preparation process, it is easy to cause danger to personnel, and the prepared saponin components are a mixture, and the active substances are unclear.

[0003] Foodborne pathogens are pathogenic bacteria that can cause food poisoning or spread through food, and can directly or indirectly contaminate food and water sources. Oral infection in humans can lead to the occurrence of intestinal infectious diseases, food poisoning, and the spread of infectious diseases in livestock and poultry. With the overuse of antibiotics, bacterial resistance continues to increase. Therefore, it is necessary to develop new drugs to inhibit foodborne pathogens. Summary of the invention

[0004] The object of the present invention is to provide a preparation method and application of low-polarity quinoa saponin monomers to solve the problems raised in the above background technology.

[0005] In order to achieve the above technical purpose, the present invention provides a method for preparing a low-polarity quinoa saponin monomer, comprising the following steps:

[0006] S1. Weigh quinoa bran, heat with superheated steam at 110°C-120°C for 1-2h, dry to a moisture content of less than 10%, crush the dried quinoa bran and pass it through a 40-mesh sieve, extract with 70% ethanol at 60°C for 0.5h, and then concentrate it once in a vacuum decompression concentrator until the concentrate has no alcohol taste, use HP-20 macroporous resin with a volume of 15-30 times the concentrate to purify saponins from the concentrated concentrate to obtain an eluent, and use a vacuum decompression concentrator to concentrate the eluent for a second time, recover ethanol, and then spray-dry the concentrated solution obtained by the second concentration at 100-120°C to obtain a quinoa saponin mixture;

[0007] S2. The quinoa saponin mixture obtained in S1 is sequentially eluted with 20%, 40% and 80% methanol-water gradients through a reverse chromatographic column, the eluate after elution with 80% methanol-water is collected, the eluate is concentrated and dried using a rotary evaporator and methanol is recovered, and low-polarity quinoa saponin monomers are obtained after concentration and drying, i.e., phytolaccagenic acid with a purity of more than 90% is obtained.

[0008] As a further improvement, in step S1, the amount of 70% ethanol used is 10 to 15 times the mass of the crushed quinoa bran.

[0009] As a further improvement, in step S1, during the first concentration process, the pressure of the vacuum decompression concentration tank is 0.09 MPa and the temperature is 40-50°C; during the second concentration process, the pressure of the vacuum decompression concentration tank is 0.09 MPa and the temperature is 40-50°C.

[0010] As a further improvement, in step S1, the specific steps for saponin purification are: injecting the concentrated solution obtained by the first concentration into a chromatographic column filled with HP-20 macroporous resin, allowing it to stand for adsorption for 8 to 12 hours, then flushing the chromatographic column with 2BV of distilled water, and continuing to flush the chromatographic column with 2BV of 95% ethanol water, and collecting the eluate.

[0011] As a further improvement, in step S2, the reverse chromatographic column is filled with reverse silica gel filler, and the filling amount of the reverse silica gel filler is 15 to 30 times the mass of the quinoa saponin mixture.

[0012] As a further improvement, in step S2, the temperature of the rotary evaporator is 40-60°C and the pressure is 0.09 MPa.

[0013] The present invention also provides the use of low-polarity quinoa saponin monomers in the preparation of drugs for resisting foodborne pathogenic bacteria.

[0014] The present invention provides a method for preparing a low-polarity quinoa saponin monomer, which is obtained by directly heating quinoa bran and drying it, and then enriching, separating and purifying the saponin components therein. Through high-heat conversion, a low-polarity quinoa saponin monomer QPhy is obtained in a purified extract, and the obtained low-polarity quinoa saponin monomer QPhy has a purity of 90%.

[0015] The in vitro antibacterial experiment verified that the low-polarity quinoa saponin monomer QPhy obtained by the present invention can inhibit the growth of foodborne pathogenic bacteria. The in vivo antibacterial experiment verified the protective effect of the low-polarity quinoa saponin QPhy on mice infected with lethal Listeria monocytogenes. The quinoa saponin monomer QPhy has a good antibacterial effect on foodborne pathogenic bacteria, can be used as a drug against foodborne pathogens, and can also be used in the food industry as an additive, flavor regulator and preservative to achieve high-value use of quinoa. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 It is the HPLC chromatogram of quinoa saponin obtained by ordinary treatment and quinoa saponin monomer QPhy obtained by high heat treatment in Example 1, wherein A is the HPLC chromatogram of quinoa saponin obtained by ordinary treatment, and B is the HPLC chromatogram of low-polarity quinoa saponin monomer QPhy;

[0018] Figure 2 is the high-resolution mass spectrum of the low-polarity quinoa saponin monomer QPhy in Example 1;

[0019] Figure 3 It is a graph showing the safety evaluation results of the low-polarity quinoa saponin monomer QPhy in Example 4, wherein A is the cell viability results of the quinoa saponin monomer Qphy group and the quinoa saponin QPhy3 group, and B is the cell number results of the 125 μg / mL quinoa saponin monomer Qphy group, the 125 μg / mL quinoa saponin QPhy3 group and the blank group;

[0020] Figure 4 1 is a graph showing the antibacterial results of the in vitro antibacterial experiment on Bacillus cereus and Staphylococcus aureus in Example 5, wherein A is a graph showing the results of Bacillus cereus as the test strain, and B is a graph showing the results of Staphylococcus aureus as the test strain;

[0021] Figure 5 This is a graph showing the survival rate of infected mice in the in vivo antibacterial experiment in Example 6. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] Example 1 This example provides a method for preparing a low-polar quinoa saponin monomer QPhy, and the specific steps are as follows:

[0028] S1. Weigh quinoa bran, heat it with superheated steam at 120°C for 1h, dry it to a moisture content of less than 10%, crush the dried quinoa bran and pass it through a 40-mesh sieve, then extract it with 70% ethanol at 60°C for 0.5h, wherein the amount of ethanol used is 15 times the mass of the crushed quinoa bran, then concentrate it once at 0.09MPa and 40°C in a vacuum decompression concentrator until the concentrate has no alcohol taste, then use HP-20 macroporous resin with 15 times the volume of the concentrate to purify saponins from the concentrate obtained by the once concentration to obtain an eluent, and use a vacuum decompression concentrator to concentrate the eluent for a second time, and recover ethanol, the pressure of the second concentration is 0.09MPa, the temperature is 50°C, and then the concentrate obtained by the second concentration is spray-dried at 100°C to obtain a quinoa saponin mixture;

[0029] Specifically, the saponin purification is to inject the concentrate obtained by the primary concentration into a chromatographic column filled with HP-20 macroporous resin, let it stand for adsorption for 8 hours, then rinse the chromatographic column with 2BV of distilled water, continue to rinse the chromatographic column with 2BV of 95% ethanol water, and collect the eluate;

[0030] S2. The quinoa saponin mixture obtained in S1 is sequentially eluted with 20%, 40% and 80% methanol-water gradients through a reverse chromatographic column, and the eluate after elution with 80% methanol-water is collected, and the eluate is concentrated and dried by a rotary evaporator and methanol is recovered, and low-polarity quinoa saponin monomers are obtained after concentration and drying, that is, low-polarity phytolaccagenic acid (Phytolaccagenic acid) with a purity of more than 90% is obtained, which is named quinoa saponin monomer QPhy. The reverse chromatographic column is filled with reverse silica gel filler, and the filling amount of the reverse silica gel filler is 15 times the mass of the quinoa saponin mixture, and the temperature of the rotary evaporator is 40°C and the pressure is 0.09MPa.

[0031] In this example, the low-grade quinoa saponin monomer QPhy prepared above and the quinoa saponin treated in ordinary manner were analyzed by high performance liquid chromatography.

[0032] The common processing method of quinoa saponins is as follows: weigh 1 g of quinoa bran, then add 10 mL of 70% methanol aqueous solution, ultrasonically extract for 30 minutes under the condition of ultrasonic power of 700 w, take the supernatant and filter it through a 0.45 μm filter membrane for high performance liquid chromatography analysis.

[0033] The QPhy monomer treatment method is as follows: accurately weigh 2 mg of the low-grade quinoa saponin monomer QPhy prepared in this example, add 1 mL of 70% methanol aqueous solution, fully dissolve, and then filter through a 0.45 μm filter membrane for high performance liquid chromatography analysis.

[0034] The conditions for HPLC were:

[0035] Chromatographic column YMC ODS-Pack, 4.6 mm × 250 mm, film thickness 5 μm;

[0036] The column oven temperature was 25°C, the detection wavelength was 202 nm, and the flow rate was 1 mL / min;

[0037] Mobile phase A was ultrapure water, and mobile phase B was acetonitrile;

[0038] The elution conditions were as follows: 0-5 min, the volume percentage of mobile phase B changed from 0 to 10%, 5-10 min, the volume percentage of mobile phase B changed from 10% to 20%, 10-15 min, the volume percentage of mobile phase B changed from 20% to 28%, 15-35 min, the volume percentage of mobile phase B changed from 28% to 40%, 35-40 min, the volume percentage of mobile phase B changed from 40% to 60%, 40-50 min, the volume percentage of mobile phase B changed from 60% to 70%, 50-60 min, the volume percentage of mobile phase B was maintained at 70%, 60-65 min, the volume percentage of mobile phase B changed from 70% to 10%;

[0039] like Figure 1 Shown are HPLC chromatograms of quinoa saponins treated in the ordinary manner and the low polarity quinoa saponin monomer QPhy prepared in this embodiment, wherein A is the HPLC chromatogram of quinoa saponins treated in the ordinary manner, and B is the HPLC chromatogram of QPhy prepared in this embodiment.

[0040] from Figure 1 It can be seen that the polar monomer QPhy3 is the main component of the quinoa saponins in the conventional treatment. After pyrolysis, the low-polar quinoa saponin monomers prepared in this embodiment are mainly QPhy.

[0041] 1 mg of quinoa saponin monomer QPhy was accurately weighed, and 5 mL of mass spectrometry grade methanol was used to fully dissolve the weighed QPhy to obtain a QPhy methanol solution with a concentration of 200 μg / mL. The Qphy methanol solution with a concentration of 200 μg / mL was directly measured by a high-resolution mass spectrometer Q Exactive mass spectrometer (Thermo) to obtain a high-resolution mass spectrum, as shown in FIG. Figure 2 shown.

[0042] The mass spectrometry conditions were:

[0043] The capillary voltage was -1500 V in negative ion mode, the cone voltage was 30 V, the ion source temperature was 320 °C, the desolvation temperature was 500 °C, and the scanning range was 80~1200 m / z.

[0044] Depend on Figure 2 It can be seen that the molecular parent ion peak of this compound is 515.3347 [MH - ].

[0045] The quinoa saponin monomer QPhy obtained in this example has the following physical and chemical properties: white powder, purple-red in vanillin-perchloric acid solution, insoluble in water, soluble in alcohols and saponins. 13The CNMR chemical shift values ​​δ are C-28 (δ=179.80), C-30 (δ=177.36), C-13 (δ=147.37), C-12 (δ=122.74), C-3 (δ=72.46), C-23 (δ=65.93, no glycosidic bond. The compound was determined to be QPhy, and the specific structural formula is as follows:

[0046] .

[0047] Example 2 This example provides a method for preparing a low-polarity quinoa saponin monomer QPhy, and the specific steps are as follows:

[0048] S1. Weigh quinoa bran, heat it with superheated steam at 110°C for 2h, dry it to a moisture content of less than 10%, crush the dried quinoa bran and pass it through a 40-mesh sieve, then extract it with 70% ethanol at 60°C for 0.5h, wherein the amount of ethanol used is 10 times the mass of the crushed quinoa bran, then concentrate it once at 0.09MPa and 50°C in a vacuum decompression concentrator until the concentrate has no alcohol taste, then use 30 times the volume of the concentrate HP-20 macroporous resin to purify the concentrate for saponin to obtain an eluent, and use a vacuum decompression concentrator to concentrate the eluent for a second time, and recover ethanol, the pressure of the second concentration is 0.09MPa, the temperature is 40°C, and then the concentrate obtained by the second concentration is spray-dried at 120°C to obtain a quinoa saponin mixture;

[0049] Specifically, the saponin purification is to inject the concentrated solution obtained by the first concentration into a chromatographic column filled with HP-20 macroporous resin, let it stand for adsorption for 12 hours, then rinse the chromatographic column with 2BV distilled water, continue to rinse the chromatographic column with 2BV, 95% ethanol water, and collect the eluate;

[0050] S2. The quinoa saponin mixture obtained in S1 is sequentially eluted with 20%, 40% and 80% methanol-water gradients through a reverse chromatographic column, and the eluate after elution with 80% methanol-water is collected, and the eluate is concentrated and dried by a rotary evaporator and methanol is recovered, and low-polarity quinoa saponin monomers are obtained after concentration and drying, that is, phytolaccagenic acid with a purity of more than 90% is obtained, which is named quinoa saponin monomer QPhy. The reverse chromatographic column is filled with reverse silica gel filler, and the filling amount of the reverse silica gel filler is 30 times the mass of the quinoa saponin mixture, and the temperature of the rotary evaporator is 60° C. and the pressure is 0.09 MPa.

[0051] Example 3 In this example, Bacillus cereus was used as the test strain to determine the MIC and MBC values ​​of quinoa saponin monomer QPhy. The specific method is as follows:

[0052] The three test ingredients, quinoa saponin QPhy3, quinoa saponin monomer QPhy, and positive control cefradine (purchased from Shandong Lukang Pharmaceutical Co., Ltd.), were dissolved in DMSO to prepare a 100 mg / mL stock solution.

[0053] The mother solutions were diluted with physiological saline using the two-fold dilution method to form drug solutions with decreasing concentration gradients, specifically 100 mg / mL, 50 mg / mL, 25 mg / mL, 12.5 mg / mL, 6.25 mg / mL, 3.125 mg / mL, and 1.5625 mg / mL.

[0054] In a 96-well plate, the three test components prepared above were added to each well, with a concentration of 2 μL per well. Then, 198 μL of bacterial suspension was added to each well, wherein the content of Bacillus cereus in the bacterial suspension was 10 4 CFU / mL, so that the final concentrations of the three test components in the 96-well plate are 1000μg / mL, 500μg / mL, 250μg / mL, 125μg / mL, 62.5μg / mL, 31.25μg / mL, and 15.625μg / mL, and then 2μL DMSO and 198μL bacterial suspension are added to the negative control wells.

[0055] The prepared 96-well plate was placed in an incubator, incubated for 12 hours and observed. The bacterial growth in the negative control well was used as a reference. If there was no bacterial growth, the test was invalid. The lowest drug concentration at which no bacterial growth was observed by naked eye was the minimum inhibitory concentration (MIC) of the saponin monomer for the tested strains. The data was recorded. The drug concentration at which no bacterial growth was observed was dipped into the agar medium with an inoculation loop, and the lowest drug concentration at which no colonies grew was used as the minimum bactericidal concentration (MBC) of the saponin monomer for the tested strains. The experimental results are shown in Table 1.

[0056] As can be seen from Table 1, among quinoa saponins, the MIC value of quinoa saponin monomer QPhy is 125.000 μg / mL, and the MBC value is 1000.000 μg / mL, and quinoa saponin QPhy3 monomer has no obvious antibacterial activity.

[0057] Example 4 In this example, the safety of the quinoa saponin monomer QPhy obtained in Example 1 was evaluated. The specific method is as follows:

[0058] 1 mg of the quinoa saponin monomer QPhy obtained in Example 1 was accurately weighed, dissolved with 10 μL of DMSO, and added to 990 μL of complete culture medium to prepare a 1 mg / mL saponin mother solution, and then diluted with complete culture medium according to the MIC of the quinoa saponin monomer QPhy obtained in Example 3 to a quinoa saponin monomer QPhy concentration of 125 μg / mL.

[0059] Take GES-1 cells grown to 80% coverage of the cell flask and use 5×10 5 The cells were inoculated at a concentration of 100 / mL in a 96-well plate and incubated at 37°C and 5% CO 2 After culturing in a cell culture incubator for 24 hours, the original culture medium was discarded, the cells were washed once with PBS, and then a culture medium containing quinoa saponin monomer QPhy at a concentration of 125 μg / mL was added. The cells were incubated at 37°C and 5% CO 2 After culturing in the cell culture incubator for 22 hours, complete medium containing CCK-8 reagent was added and the cells were incubated at 37°C and 5% CO 2 The cells were cultured in the cell culture incubator for 2 hours as the drug-added group.

[0060] Take another GES-1 cell that has grown to 80% coverage of the cell bottle and use 5×10 5 The cells were inoculated at a concentration of 100 / mL in a 96-well plate and incubated at 37°C and 5% CO 2 After culturing in a cell culture incubator for 24 hours, the original culture medium was discarded, the cells were washed once with PBS, and then incubated at 37°C and 5% CO 2 After culturing in the cell culture incubator for 22 hours, complete medium containing CCK-8 reagent was added and the cells were incubated at 37°C and 5% CO 2 The cells were cultured in the cell culture incubator for 2 hours, which was designated as the drug-free group.

[0061] Take another 96-well plate that has not been inoculated with GES-1 cells and incubate it at 37°C and 5% CO 2 After culturing in a cell culture incubator for 24 hours, the original culture medium was discarded, the cells were washed once with PBS, and then incubated at 37°C and 5% CO 2 After culturing in the cell culture incubator for 22 hours, complete medium containing CCK-8 reagent was added and the cells were incubated at 37°C and 5% CO 2 The cells were cultured in the cell culture incubator for 2 hours as a blank group.

[0062] The above three groups of samples were set up in 4 parallel groups, and then their OD values ​​were detected by ELISA. 450 .

[0063] Calculate according to the formula: cell viability = [OD (drug added) - OD (blank)] / [OD (0 drug added) - OD (blank)] × 100%.

[0064] The quinoa saponin monomer Qphy with a concentration of 125 μg / mL was diluted to a quinoa saponin monomer Qphy with a concentration of 62.5 μg / mL.

[0065] Five groups of GES-1 cells were taken, and two of the groups of GES-1 cells were treated with 125 μg / mL and 62.5 μg / mL of quinoa saponin monomer Qphy for 24 h, respectively. Then, the other two groups of GES-1 cells were treated with 125 μg / mL and 62.5 μg / mL of quinoa saponin QPhy3 for 24 h, respectively. The last group was used as the control group, and no drugs were added to the control group. The survival rate of GES-1 cells in each group was then detected by CCK-8 method. The results are shown in the figure. Figure 3 As shown, A is the cell viability results of the quinoa saponin monomer Qphy group and the quinoa saponin QPhy3 group, and B is the cell number results of the 125 μg / mL quinoa saponin monomer Qphy group, the 125 μg / mL quinoa saponin QPhy3 group and the blank group.

[0066] from Figure 3 It can be seen that compared with the control group, quinoa saponin monomer QPhy has a slight proliferation-promoting effect on GES-1 cells and is non-toxic. After treating GES-1 cells with corresponding concentrations of quinoa saponin QPhy3 for 24 hours, the percentage of dead cells is much higher than that of quinoa saponin monomer QPhy.

[0067] Example 5 This example demonstrates that the low-polarity quinoa saponin monomer QPhy of the present invention can inhibit the growth of foodborne pathogenic bacteria through in vitro antibacterial experiments. The specific method is as follows:

[0068] Staphylococcus aureus (purchased from Guangdong Microbiological Culture Collection Center) and Bacillus cereus (purchased from Guangdong Microbiological Culture Collection Center) were used as test strains, and the antibacterial effect of saponins was studied by the paper strip method.

[0069] 0.1 mL, containing 1.5×10 8 CFU / mL Staphylococcus aureus suspension and a suspension containing 1.5×10 8 The bacterial suspension of CFU / mL Bacillus cereus was evenly spread on the entire nutrient agar solid culture medium plate.

[0070] Weigh quinoa bran, crush it and pass it through a 40-mesh sieve, then use 70% ethanol to extract it at 60°C for 0.5h, wherein the amount of ethanol used is 15 times the mass of the crushed quinoa bran, then use a vacuum decompression concentration tank to concentrate it once at 0.09MPa and 40°C until the concentrate has no alcohol taste, then use HP-20 macroporous resin with a volume of 15 times the concentrate to purify saponins from the concentrate to obtain an eluent, and use a vacuum decompression concentration tank to concentrate the eluent for a second time, and recover ethanol, the pressure of the second concentration is 0.09MPa, and the temperature is 50°C, and then the concentrate obtained by the second concentration is spray-dried at 100°C to obtain crude quinoa saponins before treatment;

[0071] Specifically, the saponin purification is to inject the concentrated solution obtained by the first concentration into a chromatographic column filled with HP-20 macroporous resin, let it stand for adsorption for 8 hours, then rinse the macroporous resin with 2BV distilled water, continue to rinse the chromatographic column with 2BV, 95% ethanol water, and collect the eluate.

[0072] The crude quinoa saponins before treatment obtained above were sequentially eluted with 20%, 40% and 80% methanol-water gradient through a reverse chromatographic column, the eluate after elution with 80% methanol-water was collected, and the eluate was concentrated and dried using a rotary evaporator to recover methanol. After concentration and drying, quinoa saponin QPhy3 was obtained, wherein the reverse chromatographic column was filled with reverse silica gel filler, the filling amount of the reverse silica gel filler was 15 times the mass of the quinoa saponin mixture, the temperature of the rotary evaporator was 40°C, and the pressure was 0.09MPa.

[0073] The quinoa saponin mixture obtained in Example 1 was used as the treated crude quinoa saponins, and the crude quinoa saponins before treatment, the crude quinoa saponins after treatment, the quinoa saponins QPhy3, the quinoa saponin monomer QPhy obtained in Example 1, penicillin (purchased from Shandong Lukang Pharmaceutical Co., Ltd.), and cephradine were dissolved in 75% ethanol solution to prepare 5 mg / mL sample solutions.

[0074] Take 7 paper pieces, No. 1-7, and then put paper piece No. 1 into 10 μL ethanol solution as a negative control group, then take it out and dry it, add paper pieces No. 2-5 to 10 μL of crude quinoa saponins before treatment, crude quinoa saponins after treatment, quinoa saponins monomer QPhy3, and quinoa saponins monomer QPhy sample solutions, respectively, take them out and dry them, and repeat the operation twice, add 10 μL of penicillin and cefradine sample solutions to paper pieces No. 6-7, then take them out and dry them, after all the paper pieces are dried, add the paper pieces to a solid culture medium plate containing Staphylococcus aureus, put them in a constant temperature incubator at 37°C for culture, and take out the plate after 6 hours of culture for observation.

[0075] The experimental method for Bacillus cereus as the test strain is the same as that for Staphylococcus aureus. Three parallel groups are set for each strain. The experimental results are shown in Figure 4 As shown, A is a solid culture medium for Bacillus cereus, and B is a solid culture medium for Staphylococcus aureus.

[0076] Depend on Figure 4 It can be seen that the crude quinoa saponin solution before treatment had no obvious inhibitory effect on Bacillus cereus and Staphylococcus aureus; after treatment, the crude quinoa saponin solution and quinoa saponin monomer QPhy both showed a certain inhibitory effect, and the main quinoa saponin monomer QPhy3 in the crude quinoa saponin solution before treatment had no obvious inhibitory effect on Bacillus cereus and Staphylococcus aureus.

[0077] Then the diameters of the inhibition zones of No. 1-7 were measured respectively, and the results are shown in Table 2.

[0078] Example 6 This example verifies the protective effect of low-polarity quinoa saponin QPhy on mice infected with lethal Listeria monocytogenes (purchased from Guangdong Microbiological Culture Collection Center) through an in vivo antibacterial experiment. The specific method is as follows:

[0079] Thirty female C57BL / 6J mice weighing 18-20 g, with stable weight gain and shiny hair after suitable feeding, were selected and divided into 5 groups according to the random principle, with 6 mice in each group, namely blank group (Cont group), negative group (Neg group), penicillin treatment group (Pos group), quinoa crude saponin treatment group before treatment (QS group), and quinoa crude saponin treatment group rich in quinoa saponin QPhy (QPhy group).

[0080] The QS group and the QPhy group were pretreated with 5 mg / mL aqueous solution of quinoa crude saponins before treatment and 5 mg / mL aqueous solution of quinoa crude saponins after treatment by intragastric gavage of equal volume for 14 days, and the gavage volume was 10 mL / kg / d. The other three groups did not receive any pretreatment. After 14 days, except for the blank group, the other groups were intraperitoneally injected with 0.1 mL and 10 6 CFU / mL monocytogenes bacterial suspension. After infection, the mice in the QS group and QPhy group continued to be gavaged with the same gavage dose as the pretreatment. The mice in the positive group were intraperitoneally injected with 0.1mL, 50,000 units / 0.1mL penicillin injection, while the negative group and the blank group were not treated. All groups of mice were provided with sufficient drinking water and feed, and their survival rates were observed. The experimental results are shown in Figure 5 shown.

[0081] Depend on Figure 5It can be seen that the survival rate of mice in the negative group within seven days was 33.3%, the survival rate of the blank group and the positive group within seven days was 100%, the survival rate of the QS group within seven days was 66.6%, and the survival rate of the QPhy group within seven days was 100%. Therefore, the low-polar quinoa saponin monomer QPhy has an obvious preventive and protective effect on mice infected with Listeria.

[0082] The present invention obtains quinoa low-polarity saponin substance QPhy through high-temperature conversion of water vapor. The quinoa saponin monomer QPhy obtained after treatment has a good antibacterial effect on foodborne pathogens and can be used as a drug against foodborne pathogens in the pharmaceutical and food industries.

[0083] The specific implementation of the present invention described above does not constitute a limitation on the protection scope of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing low-polarity quinoa saponin monomer, characterized in that: The following steps are involved: S1. Weigh quinoa bran, heat with superheated steam at 110°C-120°C for 1-2h, dry to a moisture content of less than 10%, crush the dried quinoa bran and pass it through a 40-mesh sieve, extract with 70% ethanol at 60°C for 0.5h, and then concentrate it once in a vacuum decompression concentrator until the concentrate has no alcohol taste, use HP-20 macroporous resin with a volume of 15-30 times the concentrate to purify saponins from the concentrated concentrate to obtain an eluent, and use a vacuum decompression concentrator to concentrate the eluent for a second time, recover ethanol, and then spray-dry the concentrated solution obtained by the second concentration at 100-120°C to obtain a quinoa saponin mixture; S2. The quinoa saponin mixture obtained in S1 is sequentially eluted with 20%, 40% and 80% methanol-water gradients through a reverse chromatographic column, the eluate after elution with 80% methanol-water is collected, the eluate is concentrated and dried using a rotary evaporator and methanol is recovered, and low-polarity quinoa saponin monomers are obtained after concentration and drying, i.e., phytolaccagenic acid with a purity of more than 90% is obtained.

2. The method for preparing low-polarity quinoa saponin monomer according to claim 1, characterized in that: In step S1, the amount of 70% ethanol used is 10 to 15 times the mass of the crushed quinoa bran.

3. The method for preparing low-polarity quinoa saponin monomer according to claim 1, characterized in that: In step S1, during the primary concentration process, the pressure of the vacuum decompression concentration tank is 0.09 MPa and the temperature is 40-50°C. During the secondary concentration process, the pressure of the vacuum decompression concentration tank is 0.09 MPa and the temperature is 40-50°C.

4. The method for preparing low-polarity quinoa saponin monomer according to claim 1, characterized in that: In step S1, the specific steps of saponin purification are: injecting the concentrated solution obtained by the first concentration into a chromatographic column filled with HP-20 macroporous resin, allowing it to stand for adsorption for 8 to 12 hours, then flushing the chromatographic column with 2BV of distilled water, and then flushing the chromatographic column with 2BV of 95% ethanol water, and collecting the eluate.

5. The method for preparing low-polarity quinoa saponin monomer according to claim 1, characterized in that: In step S2, the reverse chromatographic column is filled with reverse silica gel filler, and the filling amount of the reverse silica gel filler is 15 to 30 times the mass of the quinoa saponin mixture.

6. The method for preparing low-polarity quinoa saponin monomer according to claim 1, characterized in that: In step S2, the temperature of the rotary evaporator is 40-60°C and the pressure is 0.09 MPa.

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