Antibacterial application of mulberry polysaccharide

Abstract

The invention discloses antibacterial application of mulberry polysaccharide, and the mulberry polysaccharide is prepared by the following method: drying fresh mulberry, crushing, and adding into absolute ethyl alcohol for extraction; then taking the precipitate and drying; adding into distilled water, performing water bath, centrifuging, taking supernate, performing vacuum concentration, centrifuging again, taking precipitate, adding distilled water into the precipitate, redissolving, and drying to obtain mulberry polysaccharide; the mulberry polysaccharide obtained by the method has a relatively good inhibition effect on gram-negative bacteria escherichia coli and salmonella and gram-positive bacteria staphylococcus aureus; the antibacterial effect is enhanced along with the rise of the temperature, so that the heat stability is achieved, and meanwhile, the good antibacterial effect is achieved in both acid and alkali environments; the mulberry polysaccharide can be further prepared into a novel natural bacteriostatic agent and has a wide application prospect in the fields of daily chemicals and medicines.

Description

Technical Field

[0001] The present invention belongs to the field of functional products, and in particular relates to mulberry polysaccharide and its antibacterial application. Background Art

[0002] Mulberry polysaccharide is one of the important active ingredients in mulberry (Fructus Mori) and is often extracted from mulberry by solvent methods (such as water extraction and acid-base assisted methods). For example, patent CN104987431B discloses a method for extracting active mulberry polysaccharides, which includes drying, crushing, defatting pretreatment, freeze-thaw wall breaking treatment, hot water extraction, vacuum concentration and protein removal, ethanol precipitation, freeze-drying treatment, etc., in order to retain the polysaccharide's high antioxidant and α-amylase and α-glucosidase inhibitory activities.

[0003] Existing reports suggest that mulberry polysaccharides have medical functions such as regulating blood sugar and blood lipids, enhancing immunity, and improving metabolism. For example, patent CN11481564B discloses the use of mulberry polysaccharide MFP-90-2 in the preparation of anti-tumor drugs. However, existing research has mostly focused on its component analysis and antioxidant activity, and no research on the antibacterial activity of mulberry polysaccharides has been reported. Summary of the Invention

[0004] In view of the above problems, the present invention aims to provide an antibacterial use of mulberry polysaccharide.

[0005] Specifically, this application provides the following technical solutions:

[0006] First, the present application provides an application of mulberry polysaccharide in inhibiting bacterial growth; the above-mentioned bacteria include inhibiting at least one of Escherichia coli, Salmonella, and Staphylococcus aureus.

[0007] Furthermore, the above-mentioned mulberry polysaccharide is prepared by the following method:

[0008] Step 1: Dry fresh mulberries (60°C, 24h), then grind them into 60-80 mesh in a grinder to obtain mulberry powder I;

[0009] Step 2: adding the mulberry powder I obtained in step 1 to anhydrous ethanol and soaking it for 48 hours, stirring once every 12 hours; after standing, the solution is separated into layers, and the precipitate is spread on filter paper and dried at 60° C. to obtain mulberry powder II; in this step, the mass ratio of the added mulberry powder I to anhydrous ethanol is preferably 1.5:7;

[0010] Step 3: The mulberry powder II obtained by drying in step 2 is fed into a grinder and crushed to 60-80 mesh to obtain mulberry powder III; the mulberry powder III is added to distilled water, and then heated in an 80°C water bath for 2 hours, then naturally cooled to room temperature, and then slowly centrifuged at 4000 rpm for 30 minutes, and the supernatant is collected to obtain the extract; in this step, the mass volume ratio of mulberry powder III to distilled water is preferably 1:10 (g / mL).

[0011] Step 4: The extract obtained in step 3 was concentrated using a rotary evaporator (speed 200 rpm, vacuum 150 mbar, water bath temperature 50-60 degrees Celsius, preferably 56 ° C), the concentrate was centrifuged at 8000 rpm for 15 minutes, and then the supernatant was discarded. The precipitate was re-dissolved with distilled water, and the mass ratio of the precipitate to distilled water was preferably 1:1. The precipitate was freeze-dried again (-55 ° C, 48 h) to obtain the above-mentioned mulberry polysaccharide.

[0012] The mulberry polysaccharide prepared by the above method has a total sugar content of about 13.8 g / 100 g; the mulberry polysaccharide is mainly pectin-type RGI, and its core structure is composed of repeating disaccharide units composed of 1,4-linked α-D-galacturonic acid and 1,2-linked α-L-rhamnose alternatingly, supplemented by branches such as arabinoxylan and galactomannan.

[0013] Secondly, the present application provides an antibacterial preparation containing mulberry polysaccharides. This antibacterial preparation has a significant inhibitory effect on the growth of Escherichia coli, Salmonella, and Staphylococcus aureus, with the inhibitory effect on Escherichia coli being the most significant. It has a good inhibitory effect on bacterial growth in different temperature and pH environments.

[0014] This application first discovered and utilized the inhibitory effect of mulberry polysaccharides on Escherichia coli and Salmonella, which are Gram-negative bacteria, and Staphylococcus aureus, which is Gram-positive bacteria. In the examples, mulberry polysaccharides were used to interfere with the growth of Escherichia coli at different concentrations, temperatures, and pH values, and the diameter of the inhibition zone and the minimum inhibitory concentration after the intervention were measured to evaluate its antibacterial efficacy. In one embodiment of the present application, the diameters of the inhibition zones of Escherichia coli at different concentrations of mulberry polysaccharides were 5.60-5.85 mm, 6.05-6.30 mm, 6.95-7.15 mm, 7.85-8.00 mm, and 8.20-8.30 mm, respectively.

[0015] In one embodiment of the present application, the effects of different concentrations, temperatures, and pH on the antibacterial effect of mulberry polysaccharide were studied, and the minimum inhibitory concentration of mulberry polysaccharide against Escherichia coli was determined to be 25 mg / mL.

[0016] The present invention designs and optimizes a polysaccharide extraction process, adopting a hot water extraction method and an alcohol precipitation method to produce mulberry polysaccharides. It is found for the first time that mulberry polysaccharides have a significant inhibitory effect on the growth of Escherichia coli, Salmonella, and Staphylococcus aureus. Furthermore, an antibacterial preparation containing mulberry polysaccharides is prepared, which can be widely used in the fields of daily chemicals and medicine, such as the development of natural antibacterial products such as antibacterial toothpaste, shampoo, and antibacterial sprays, and has broad market application development potential. Compared with the existing preparation and application of mulberry polysaccharides, the present application has the following beneficial effects:

[0017] 1. Efficient extraction and stable quality: This application uses ethanol soaking combined with freeze drying process, and the polysaccharide extraction rate reaches 59%, which is better than the traditional water extraction method (usually less than 50%), and the phenol-sulfuric acid method shows a good linear relationship (R 2 =0.994), ensuring quality control;

[0018] 2. Broad-spectrum antibacterial properties: Mulberry polysaccharides have significant inhibitory effects on common pathogens such as Escherichia coli and Staphylococcus aureus, and their antibacterial efficacy is better than that of similar natural polysaccharides;

[0019] 3. Strong tolerance to environmental influences: The antibacterial activity of mulberry polysaccharide is enhanced after heat treatment below 80°C, and it still maintains high antibacterial efficacy in an acidic environment (pH = 2), making it suitable for a variety of scenarios such as food preservation and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the standard glucose curve corresponding to the determination of mulberry polysaccharide content.

[0021] Figure 2 The antibacterial effect of mulberry polysaccharides on Escherichia coli at different temperatures.

[0022] Figure 3 It is the antibacterial effect of mulberry polysaccharides with different pH values on Escherichia coli. DETAILED DESCRIPTION

[0023] The preparation and bacterial growth inhibition of the present invention are described below through specific examples. The examples are only used to explain the present invention and are not used to limit the scope of protection of the present invention.

[0024] The mulberries used in the examples were commercially available fresh mulberries, and the reagents, raw materials, etc. used were all obtained from commercial sources unless otherwise specified.

[0025] Example 1 Preparation of mulberry polysaccharide

[0026] Step 1: First, spread commercially available fresh mulberries on a square plate and put them into an oven to dry (60°C, 24 hours). After drying, pour them into a grinder and grind them into 60-80 mesh to obtain mulberry powder I;

[0027] Step 2: Under room temperature, immerse the mulberry powder I obtained in step 1 in anhydrous ethanol (the mass ratio of the two is 1.5:7) for 48 hours, stirring once every 12 hours; after standing, the solution is separated into layers, the upper layer of anhydrous ethanol is removed, and the precipitate is spread on filter paper, and then placed in an oven (60°C) for drying to obtain mulberry powder II;

[0028] Step 3: feeding the mulberry powder II obtained by drying in step 2 into a grinder and grinding it into 60-80 mesh, i.e., mulberry powder III, for later use;

[0029] Take 20g of mulberry powder III, pour it into 200mL of distilled water, place it in a conical flask, and heat it in a water bath (80℃, 2h). After the solution cools to room temperature, centrifuge it at a slow speed (4000rpm, 30min) and collect the supernatant, which is the extract;

[0030] Step 4: The extract obtained in step 3 was concentrated using a rotary evaporator for 45 minutes (speed 200 rpm, vacuum 150 mbar, water bath temperature 56 ° C) to obtain a concentrate; the concentrate was centrifuged at high speed (8000 rpm, 15 min), the solution was separated after centrifugation, the supernatant was discarded, the precipitate was taken out and re-dissolved with distilled water at a mass ratio of 1:1, placed in a culture dish, and then freeze-dried (-55 ° C, 48 h) to finally obtain mulberry polysaccharide.

[0031] In this example, the polysaccharide content was determined using the phenol-sulfuric acid method (see "Tian Fengming et al. Optimization of Determination Conditions for Euphorbia pekinensis Polysaccharides by the Phenol-sulfuric Acid Method. Proceedings of the 2015 Annual Meeting of the Chinese Society of Agricultural Engineering, 2015"). Under the action of concentrated sulfuric acid, the polysaccharide hydrolyzes to form monosaccharides, rapidly dehydrates to form furfural derivatives, and then condenses with phenol to form an orange-yellow compound with no significant color change. At a wavelength of 490 nm and within a certain concentration range, the absorbance is linearly proportional to the polysaccharide content. Therefore, the absorbance can be measured spectrophotometrically, and a standard curve can be used to quantitatively determine the polysaccharide content of the sample. This was used to determine the polysaccharide content of mulberry. A standard curve was plotted using the concentration (X) of the glucose standard solution as the horizontal axis and the absorbance (Y) as the vertical axis. Take 7 mg / mL mulberry polysaccharide and measure the absorbance in the same way. Substitute it into the calculation to obtain the horizontal coordinate value X1. Calculate the polysaccharide content according to the following formula: Polysaccharide content % = X1 × 1000 / 7, and obtain the mulberry polysaccharide content.

[0032] The results are as follows Figure 1 As shown, the standard glucose curve was drawn using Excel, and a good linear relationship was found between X and Y. The regression equation was Y = 0.6464X-0.017, r 2= 0.994. Substituting the absorbance of mulberry polysaccharide Y1 into this equation, we obtain X = 0.413. The results show that the average polysaccharide content in mulberry is 59%.

[0033] Example 2 Determination of antibacterial ability of mulberry polysaccharides at different concentrations

[0034] Evaluation of the inhibitory ability of mulberry polysaccharides against Escherichia coli, Salmonella, and Staphylococcus aureus.

[0035] Step 1: Prepare a bacterial suspension using commercially available ATCC and CICC standard strains (including Escherichia coli, Salmonella, and Staphylococcus aureus). First, activate the strains on a slant medium at a constant temperature for 3 hours. Then, aseptically transfer the strains to LB solid medium and incubate at 37°C for 12 hours. Select representative colonies and inoculate them into a shake flask containing LB liquid medium. Maintain the same temperature and shake at 180 rpm for 8 hours to obtain the initial bacterial suspension.

[0036] Step 2: Dilute the initial bacterial solution prepared in step 1 to 2.0×10 5 CFU / mL.

[0037] Step 3: Determine the antibacterial efficacy of the samples using the filter paper diffusion method.

[0038] Place 5mm circular filter paper pieces in the polysaccharide solution and sterile water respectively and soak for 6 hours. Pipette 0.1ml of Escherichia coli, Staphylococcus aureus, and Salmonella suspension respectively and place them on the plate, spread them to make them evenly distributed. Use sterilized tweezers to pick up three soaked filter paper pieces and stick them on the surface of the plate with bacteria. At the same time, use filter paper pieces soaked in sterilized distilled water as a control. Place the plate upside down in a constant temperature incubator and incubate at 37°C for 24 hours. Measure the diameter of the inhibition zone. The specific measurement method is to use the cross-cross method to measure the diameter of the inhibition zone.

[0039] The results showed that for Gram-negative bacteria Escherichia coli and Salmonella, as well as Gram-positive bacteria Staphylococcus aureus, the diameter of the inhibition zone increased with increasing concentration of mulberry polysaccharide. Mulberry polysaccharide had a strong antibacterial effect on all tested bacteria. At the same concentration, mulberry polysaccharide had the best inhibitory effect on Escherichia coli. The experimental results also showed that the inhibitory effect of mulberry polysaccharide was significantly positively correlated with its concentration, and the higher the concentration of polysaccharide, the more significant its inhibitory effect on the colony.

[0040] Step 4: Determine the MIC of the sample against Escherichia coli using the microbroth method.

[0041] Under sterile conditions, 2×10 5CFU / mL: 90 μL MH liquid medium (purchased from Beijing Solebow Technology Co., Ltd.). 50 μL LB liquid medium was added to wells 2 through 11. 10 μL of 200.0 mg / mL sample solution was added to the first well and mixed thoroughly. 50 μL of the mixture was pipetted and added to the second well. This was continued to the 10th well, and 50 μL from the 10th well was discarded. 50 μL of bacterial liquid (drug-free) was added to well 11 as a blank control. After incubation of the drug and bacterial suspension for 6-8 hours, the MIC for the sample against the bacteria was determined when the absorbance value at 600 nm on a microplate reader was less than 50% of that of the control.

[0042] The test results are shown in Tables 1 and 2.

[0043] Table 1 Antibacterial effect of mulberry polysaccharides at different concentrations

[0044]

[0045] Note: The data are the average of 3 tests. “-” indicates the diameter of the non-inhibitory zone.

[0046] Table 2 Minimum inhibitory concentration of polysaccharides

[0047]

[0048] The above test results show that the inhibitory concentration of mulberry polysaccharide on Escherichia coli is the lowest, which is 25 mg / mL, which is consistent with the results of the inhibition zone.

[0049] Example 3 Determination of the antibacterial ability of mulberry polysaccharides at different temperatures

[0050] A 200 mg / mL solution of mulberry polysaccharide prepared using the method of Example 1 was placed in a constant temperature water bath at 37, 60, 80, and 100°C for 1 hour. After cooling to room temperature, a 5 mm diameter filter paper was placed in each of the mulberry polysaccharide solutions. Each experiment was repeated in triplicate. Sterile distilled water was used as a control.

[0051] Use a pipette to drop 0.1 mL of the E. coli suspension onto a plate-filled petri dish and spread it with a glass rod. Use sterile tweezers to pick up three pieces of soaked filter paper and place them on the plate containing the bacteria. Place the plate upside down in a constant-temperature incubator at 37°C for 24 hours. Measure the diameter of the inhibition zone using the cross-hatch method.

[0052] The results are as follows Figure 2 As shown in the figure, the inhibitory effect of mulberry polysaccharide on the bacterial activity at different temperatures shows an upward trend at 37-80℃, and the inhibitory effect of mulberry polysaccharide decreases significantly after 80℃, which indicates that temperature has a certain influence on the inhibitory effect of mulberry polysaccharide on the bacterial activity.

[0053] Example 4 Determination of the antibacterial ability of mulberry polysaccharides at different pH values

[0054] The 200mg / mL mulberry polysaccharide solution prepared by the method of Example 1 was treated with 0.5mol / L HCl solution and 0.5mol / L NaOH solution, and mulberry polysaccharide solutions of different pH values of 2.0, 4.0, 6.0, 7.0, 8.0, and 10.0 were prepared respectively. Using the filter paper diffusion method, 5mm filter papers were placed in different mulberry polysaccharide solutions of pH for 2h. 0.1mL of Escherichia coli suspension was dropped into a culture dish on which a flat plate had been poured, and dispersed by coating with a glass rod. Three pieces of soaked filter paper were clamped with sterilized tweezers and placed on the flat surface with the strain. The flat plate was placed upside down in a constant temperature incubator and cultured at 37°C for 24h. The diameter of the inhibition zone was then measured using the cross method.

[0055] The results are as follows Figure 3 As shown in the figure, under the condition of pH 2.0, mulberry polysaccharide has the highest inhibitory effect on Escherichia coli, and its inhibition zone diameter is 7.56 mm. When the pH value is greater than 2, the antibacterial activity of mulberry polysaccharide decreases with the increase of pH.

[0056] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. Application of mulberry polysaccharide in inhibiting bacterial growth.

2. The use according to claim 1, characterized in that The bacteria include at least one of Escherichia coli, Salmonella, and Staphylococcus aureus.

3. The use according to claim 1, characterized in that The mulberry polysaccharide is prepared by the following method: 1) The fresh mulberries were dried and crushed to 60-80 mesh to obtain mulberry powder I; 2) Soaking mulberry powder I in anhydrous ethanol for 48 hours, separating the layers, and drying and crushing the precipitate to 60-80 mesh to obtain mulberry powder III; 3) Add mulberry powder III to distilled water, heat in an 80°C water bath for 2 hours, and then centrifuge. The supernatant is the extract. 4) The extract obtained in step 3) is vacuum concentrated and then centrifuged, the precipitate is added into distilled water for redissolution, and then freeze-dried to obtain the mulberry polysaccharide.

4. The use according to claim 3, characterized in that The drying temperature in step 1) is 60°C.

5. The use according to claim 3, characterized in that In step 2), the mass ratio of the added mulberry powder I to anhydrous ethanol is 1.5:

7.

6. The use according to claim 3, characterized in that In step 3), the mass-to-volume ratio of the added mulberry powder III to distilled water is 1:10, and the unit of mass-to-volume ratio is g / mL.

7. The use according to claim 3, characterized in that In step 4), the mass ratio of the precipitate to distilled water is 1:

1.

8. The use according to claim 3, characterized in that In step 4), the temperature of the vacuum concentration is 50-60°C, and the freeze drying refers to drying at -55°C.

9. The use according to claim 3, characterized in that In step 3), the centrifugation refers to centrifugation at 4000 rpm for 30 min; in step 4), the centrifugation refers to centrifugation at 8000 rpm for 15 min.

10. An antibacterial preparation, characterized in that The antibacterial preparation contains mulberry polysaccharide, which is prepared by the following method: 1) The fresh mulberries were dried at 60 ° C and crushed to 60-80 mesh to obtain mulberry powder I; 2) Mulberry powder I was added to anhydrous ethanol and soaked for 48 hours. The solution was separated and the precipitate was dried at 60°C and crushed to 60-80 mesh size to obtain mulberry powder III. The mass ratio of mulberry powder I to anhydrous ethanol was 1.5:

7. 3) Add mulberry powder III to distilled water, heat in an 80°C water bath for 2 hours, and then centrifuge at 4000 rpm for 30 minutes. The resulting supernatant is the extract. The mass-to-volume ratio of mulberry powder III to distilled water is 1:

10. The unit of mass-to-volume ratio is g / mL. 4) The extract obtained in step 3) is concentrated in vacuo at 50-60° C., centrifuged at 8000 rpm for 15 min, the precipitate is added to an equal amount of distilled water for redissolution, and then freeze-dried at -55° C. to obtain the mulberry polysaccharide.

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