阅读说明：本技术 一种基于香菇柄中性多糖的果汁抑菌剂 (Fruit juice bacteriostatic agent based on neutral polysaccharide of lentinus edodes stems ) 是由 邓红 刘靓 孟永宏 梁佳蕊 田丹 郭玉蓉 于 2021-01-25 设计创作，主要内容包括：本发明公开了一种基于香菇柄中性多糖的果汁抑菌剂,其由香菇柄中性多糖与苹果幼果多酚按质量比为1:1～4混合而成。本发明利用香菇柄中性多糖复配苹果幼果多酚制备天然果汁抑菌剂,香菇柄中性多糖和苹果幼果多酚均为天然物质,不会对人体造成危害,其中香菇柄中性多糖具有抗菌、抗氧化、抗癌等作用,对大肠杆菌、金黄色葡萄球菌及枯草芽孢杆菌等具有明显的抑制作用,苹果幼果多酚能有效抑制微生物活动,两者复配在保留原果汁天然风味基础上能较彻底、更有效的抑制果汁中微生物生长,不仅可以有效抑制大肠杆菌、金黄色葡萄球菌和沙门氏菌生长,还可以高效抑制脂环酸芽孢杆菌生长,防止由微生物引起的腐败,延长果汁的保藏期。(The invention discloses a fruit juice bacteriostatic agent based on neutral lentinus edodes stem polysaccharide, which is prepared by mixing the neutral lentinus edodes stem polysaccharide and young apple polyphenol in a mass ratio of 1: 1-4. The natural fruit juice bacteriostatic agent is prepared by compounding the lentinus edodes stem neutral polysaccharide with the apple young fruit polyphenol, wherein the lentinus edodes stem neutral polysaccharide and the apple young fruit polyphenol are natural substances and do not cause harm to human bodies, the lentinus edodes stem neutral polysaccharide has the effects of resisting bacteria, oxidation, cancer and the like, and has an obvious inhibiting effect on escherichia coli, staphylococcus aureus, bacillus subtilis and the like, the apple young fruit polyphenol can effectively inhibit the activity of microorganisms, and the two are compounded on the basis of retaining the natural flavor of the original fruit juice, so that the microbial growth in the fruit juice can be completely and effectively inhibited, the growth of escherichia coli, staphylococcus aureus and salmonella can be effectively inhibited, the growth of alicyclobacillus can be effectively inhibited, the spoilage caused by microorganisms is prevented, and the preservation period of the fruit juice is prolonged.)

1. A fruit juice bacteriostatic agent based on mushroom stem neutral polysaccharide is characterized in that: the bacteriostatic agent is prepared by mixing neutral lentinus edodes stem polysaccharide and apple young fruit polyphenol in a mass ratio of 1: 1-4.

2. The mushroom stem neutral polysaccharide based fruit juice bacteriostatic agent according to claim 1, wherein: the bacteriostatic agent is prepared by mixing neutral lentinus edodes stem polysaccharide and apple young fruit polyphenol in a mass ratio of 1: 3.

3. The mushroom stem neutral polysaccharide based fruit juice bacteriostatic agent according to claim 1 or 2, wherein: the lentinus edodes stem neutral polysaccharide is extracted by the following method:

(1) pretreatment of

Crushing lentinus edodes stems, grinding the crushed lentinus edodes stems to 40-60 meshes, immersing the crushed lentinus edodes stems in petroleum ether for 10-12 hours to degrease, and performing vacuum filtration to obtain degreased lentinus edodes stem powder;

(2) extraction of lentinus edodes stem crude polysaccharide

Adding 15-25 times of mass of distilled water into degreased lentinus edodes stem powder, extracting for 2-4 hours in a constant-temperature water bath at 80-90 ℃, cooling, centrifuging, concentrating supernate under reduced pressure until the content of soluble solids is more than or equal to 4.50mg/mL, adding absolute ethyl alcohol with the volume being 8-10 times of that of the concentrated solution, precipitating for 2-4 hours at 70-80 ℃, centrifuging, recovering degreased crude polysaccharide, and fully dissolving with distilled water to obtain 0.8-1.0 mg/mL degreased crude polysaccharide solution;

(3) enzyme deproteinization

Dissolving papain in phosphate buffer solution with pH of 6.0, fixing volume, and preparing enzyme solution with concentration of 1 mg/mL; adding enzyme liquid with the mass of 8-12% of that of the defatted crude polysaccharide solution into the defatted crude polysaccharide solution, fully mixing the mixture, carrying out enzymolysis for 1-2 hours at 50-55 ℃, carrying out enzyme deactivation in a boiling water bath for 8-10 minutes, centrifuging to remove precipitates, carrying out reduced pressure concentration until the content of soluble solids is more than or equal to 4.50mg/mL, adding absolute ethyl alcohol with the volume of 8-10 times that of a concentrated solution, carrying out precipitation for 2-4 hours at 70-80 ℃, centrifugally recovering defatted and deproteinized polysaccharide, and fully dissolving the defatted and deproteinized polysaccharide with distilled water to obtain a defatted and deproteinized polysaccharide solution with the volume of 0.8-1.0 mg;

(4) separating and purifying cellulose column

Filling the treated DEAE-52 cellulose into a column by a wet method, and balancing for 20-24 hours by using distilled water; and adding the degreased deproteinized polysaccharide solution into a treated DEAE-52 cellulose column, eluting with distilled water, controlling the flow rate to be 8-12 mL/min, detecting the polysaccharide by adopting a phenol-sulfuric acid method, collecting the peak part of the positive reaction of the polysaccharide by the phenol-sulfuric acid method, concentrating under reduced pressure until the content of soluble solids is more than or equal to 4.50mg/mL, adding absolute ethyl alcohol with the volume being 8-10 times of that of the concentrated solution, precipitating for 2-4 hours at 70-80 ℃, centrifugally recovering the polysaccharide, and freeze-drying to obtain the purified lentinus edodes stem neutral polysaccharide.

4. The mushroom stem neutral polysaccharide based fruit juice bacteriostatic agent according to claim 3, wherein: in the step (4), the DEAE-52 cellulose treatment method comprises the following steps: soaking DEAE-52 cellulose filler in 0.5mol/L sodium hydroxide aqueous solution for 10-12 hours, fully washing with distilled water until the filler is neutral, performing suction filtration, then soaking in 0.5mol/L HCl aqueous solution for 10-12 hours, washing with distilled water until the filler is neutral, performing suction filtration, finally soaking in 0.5mol/L NaOH aqueous solution for 10-12 hours, and washing with distilled water until the filler is neutral.

5. The mushroom stem neutral polysaccharide based fruit juice bacteriostatic agent according to claim 1 or 2, wherein: the apple young fruit polyphenol is extracted by the following method:

crushing young apples, extracting for 2-4 hours by using ethanol water solution with volume concentration of 65-75%, wherein the feed-liquid ratio of the young apples to the ethanol water solution is 1g: 5-15 mL, evaporating and concentrating until the volume of concentrated solution is 0.10-0.15 times of the volume of the feed solution, adsorbing by using macroporous adsorption resin, wherein the adsorption rate is 1.0-2.0 BV/h, eluting by using ethanol water solution with volume concentration of 65-75%, wherein the elution rate is 1.0-2.0 BV/h, evaporating to remove ethanol, and drying to obtain young apple polyphenol.

Technical Field

The invention belongs to the technical field of fruit juice bacteriostats, and particularly relates to a fruit juice bacteriostat based on lentinus edodes stem neutral polysaccharide.

Background

In recent years, with the improvement of the awareness of food safety of consumers, quality problems become the most basic control of the fruit juice beverage industry. Spoilage of fruit juices is a very complex process. The microorganisms causing the fruit juice to decay are mainly escherichia coli, saccharomycetes, salmonella and bacillus, and are mostly derived from raw and auxiliary materials and production environment thereof.

The method for controlling the microorganisms in the fruit juice is various, and the physical method and the chemical method have good inhibition effect on the microorganisms in the fruit juice. The physical method mainly adopts pasteurization, ultraviolet radiation treatment, ultrasonic treatment, ultrahigh pressure sterilization and the like to control the pollution of harmful bacteria. The chemical bacteriostasis is mainly to add bacteriostats such as benzoic acid, Nisin, natamycin, ozone and the like. In recent years, with the increasing importance on concepts such as nature and health, many natural antibacterial ingredients enter the visual field of people. CN106262766A, CN106172730A, CN107996913A and other patents disclose that persimmon pectin, anthocyanin and polysaccharide composition, chufa peel extract and other natural substances can inhibit the growth of microorganisms in fruit juice. Many relevant documents report that natural substances such as rosemary extract, yeast extract, grape seed extract, pomegranate seed extract, citrus extract, pepper extract, and the like can also inhibit microbial growth in fruit juice. However, most of the natural substances found at present have relatively poor bacteriostatic effect, can not comprehensively inhibit some special but extremely harmful bacteria in the fruit juice, such as alicyclobacillus, and the flavor quality of the fruit juice is influenced by the fragrance of the natural substances. Therefore, the fruit juice industry is in need of developing natural bacteriostatic components with high efficiency and without affecting the flavor of the fruit juice.

Disclosure of Invention

The invention aims to overcome the defects of the existing bacteriostatic components, and provides a healthy, tasteless and good bacteriostatic effect fruit juice bacteriostatic agent based on lentinus edodes stem neutral polysaccharide, which can effectively inhibit microbial decay of fruit juice and prolong the shelf life of the fruit juice.

Aiming at the purposes, the fruit juice bacteriostatic agent based on the neutral lentinus edodes stem polysaccharide is prepared by mixing the neutral lentinus edodes stem polysaccharide and the young apple polyphenol in a mass ratio of 1: 1-4.

The fruit juice bacteriostatic agent is preferably prepared by mixing lentinus edodes stem neutral polysaccharide and apple young fruit polyphenol according to the mass ratio of 1: 3.

The lentinus edodes stem neutral polysaccharide is extracted by the following method:

(1) pretreatment of

Crushing the lentinus edodes stems, grinding the crushed lentinus edodes stems to 40-60 meshes, immersing the crushed lentinus edodes stems in petroleum ether for 10-12 hours to degrease, and performing vacuum filtration to obtain degreased lentinus edodes stem powder.

(2) Extraction of lentinus edodes stem crude polysaccharide

Adding 15-25 times of mass of distilled water into degreased lentinus edodes stem powder, extracting for 2-4 hours in a constant-temperature water bath at 80-90 ℃, cooling, centrifuging, concentrating supernate under reduced pressure until the content of soluble solids is more than or equal to 4.50mg/mL, adding absolute ethyl alcohol with the volume being 8-10 times of that of the concentrated solution, precipitating for 2-4 hours at 70-80 ℃, centrifuging, recovering degreased crude polysaccharide, and fully dissolving with distilled water to obtain 0.8-1.0 mg/mL degreased crude polysaccharide solution.

(3) Enzyme deproteinization

Dissolving papain in phosphate buffer solution with pH of 6.0, fixing volume, and preparing enzyme solution with concentration of 1 mg/mL; adding enzyme liquid with the mass of 8-12% of that of the defatted crude polysaccharide solution into the defatted crude polysaccharide solution, fully mixing the mixture, carrying out enzymolysis for 1-2 hours at 50-55 ℃, carrying out enzyme deactivation in boiling water bath for 8-10 minutes, centrifuging to remove precipitates, carrying out reduced pressure concentration until the content of soluble solids is more than or equal to 4.50mg/mL, adding absolute ethyl alcohol with the volume of 8-10 times of that of a concentrated solution, carrying out precipitation for 2-4 hours at 70-80 ℃, centrifugally recovering the defatted deproteinized polysaccharide, and fully dissolving the defatted deproteinized polysaccharide with distilled water to obtain a 0.8-1.0 mg/mL defatted deproteinized polysaccharide solution.

(4) Separating and purifying cellulose column

Filling the treated DEAE-52 cellulose into a column by a wet method, and balancing for 20-24 hours by using distilled water; and adding the degreased deproteinized polysaccharide solution into a treated DEAE-52 cellulose column, eluting with distilled water, controlling the flow rate to be 8-12 mL/min, detecting the polysaccharide by adopting a phenol-sulfuric acid method, collecting the peak part of the positive reaction of the polysaccharide by the phenol-sulfuric acid method, concentrating under reduced pressure until the content of soluble solids is more than or equal to 4.50mg/mL, adding absolute ethyl alcohol with the volume being 8-10 times of that of the concentrated solution, precipitating for 2-4 hours at 70-80 ℃, centrifugally recovering the polysaccharide, and freeze-drying to obtain the purified lentinus edodes stem neutral polysaccharide.

In the step (4), the method for treating the DEAE-52 cellulose comprises the following steps: soaking DEAE-52 cellulose filler in 0.5mol/L sodium hydroxide aqueous solution for 10-12 hours, fully washing with distilled water until the filler is neutral, performing suction filtration, then soaking in 0.5mol/L HCl aqueous solution for 10-12 hours, washing with distilled water until the filler is neutral, performing suction filtration, finally soaking in 0.5mol/L NaOH aqueous solution for 10-12 hours, and washing with distilled water until the filler is neutral.

The polyphenol of the young apple is extracted by the following method:

crushing young apples, extracting for 2-4 hours by using ethanol water solution with volume concentration of 65-75%, wherein the feed-liquid ratio of the young apples to the ethanol water solution is 1g: 5-15 mL, evaporating and concentrating until the volume of concentrated solution is 0.10-0.15 times of the volume of the feed solution, adsorbing by using macroporous adsorption resin, wherein the adsorption rate is 1.0-2.0 BV/h, eluting by using ethanol water solution with volume concentration of 65-75%, wherein the elution rate is 1.0-2.0 BV/h, evaporating to remove ethanol, and drying to obtain young apple polyphenol.

The use method of the fruit juice bacteriostatic agent comprises the following steps: adding the fruit juice bacteriostatic agent into NFC fruit juice according to the concentration of 300-500 mg/L, uniformly mixing, sealing, performing ultrahigh pressure sterilization, and storing at low temperature, wherein the storage period can be prolonged to about one year.

The invention has the following beneficial effects:

the mushroom stem neutral polysaccharide in the bacteriostatic agent is a colorless and tasteless natural substance with antibacterial, antioxidant and anticancer functions and has obvious inhibiting effect on escherichia coli, staphylococcus aureus, bacillus subtilis and the like; the apple young fruit polyphenol is a phenolic substance which is extracted from apple young fruits and can effectively inhibit microbial activities. According to the invention, the natural fruit juice bacteriostatic agent is prepared by compounding the neutral lentinus edodes stem polysaccharide with the young apple polyphenol, and the neutral lentinus edodes stem polysaccharide and the young apple polyphenol are natural substances and do not cause harm to human bodies; the fruit juice preservative has low cost and low energy consumption, can completely and more effectively inhibit the growth of microorganisms in the fruit juice on the basis of keeping the natural flavor of the original fruit juice, can effectively inhibit the growth of escherichia coli, staphylococcus aureus and salmonella, can also effectively inhibit the growth of alicyclobacillus, prevents the putrefaction caused by microorganisms, and prolongs the preservation period of the fruit juice.

Detailed description of the invention

The present invention will be described in further detail with reference to examples, but the scope of the present invention is not limited to these examples.

Example 1

1. Extraction of neutral polysaccharide from mushroom stem

(1) Pretreatment of

Crushing 100.00g of lentinus edodes stems, grinding to 40-60 meshes, then immersing in petroleum ether for pretreatment for 12 hours at room temperature to degrease, carrying out suction filtration under reduced pressure, recovering 97.2g of filter residues, and drying in a constant-temperature drying oven at 60 ℃ to obtain degreased lentinus edodes stem powder.

(2) Extraction of lentinus edodes stem crude polysaccharide

Weighing 50g of the degreased lentinus edodes stem powder, adding 1kg of distilled water, extracting for 3 hours in a constant-temperature water bath at 90 ℃, cooling, centrifuging to remove precipitates, concentrating the supernate under reduced pressure to 100mL (the content of soluble solid matters is approximately equal to 4.61mg/mL), adding 900mL of absolute ethyl alcohol, precipitating for 3 hours at 75 ℃, centrifuging to recover 456.3mg of degreased crude polysaccharide, and adding 500mL of distilled water to fully dissolve to obtain 0.91mg/mL of degreased crude polysaccharide solution.

(3) Enzyme deproteinization

100mg of papain was weighed, dissolved in phosphate buffer solution with pH 6.0 and transferred to a 100mL volumetric flask for constant volume to obtain an enzyme solution with a concentration of 1 mg/mL. Adding 50mL of enzyme solution into the degreased crude polysaccharide solution, fully and uniformly mixing, carrying out enzymolysis for 1 hour at 55 ℃, carrying out enzyme deactivation for 10 minutes in a boiling water bath, centrifuging to remove precipitates, concentrating the supernate under reduced pressure to 100mL (the content of soluble solids is approximately equal to 4.56mg/mL), adding 900mL of absolute ethyl alcohol, precipitating for 3 hours at 75 ℃, centrifugally recovering 441.2mg of degreased deproteinized polysaccharide, adding 500mL of distilled water, and fully dissolving to obtain 0.88mg/mL of degreased deproteinized polysaccharide solution.

(4) Separating and purifying cellulose column

Soaking DEAE-52 cellulose filler in 0.5mol/L sodium hydroxide aqueous solution for 12 hours, fully washing with distilled water to neutrality, filtering, then soaking in 0.5mol/L HCl aqueous solution for 12 hours, washing with distilled water to neutrality, filtering, finally soaking in 0.5mol/L NaOH aqueous solution for 12 hours, and washing with distilled water to neutrality. Filling the treated DEAE-52 cellulose into a column by a wet method, and balancing for 24 hours by using distilled water; then adding the degreased deproteinized polysaccharide solution into a treated DEAE-52 cellulose column, eluting with distilled water, controlling the flow rate at 10mL/min, collecting 20mL in each test tube, collecting 25 tubes, detecting the OD value at 490nm by using a phenol-sulfuric acid method, collecting the peak part of the positive reaction of the phenol-sulfuric acid method polysaccharide, concentrating under reduced pressure to 30mL (the content of soluble solid matters is approximately equal to 5.93mg/mL), adding 270mL of absolute ethyl alcohol, precipitating for 3 hours at 75 ℃, centrifuging, recovering the polysaccharide, and freeze-drying to obtain 176.5mg of shiitake stem neutral polysaccharide with the purity of 92.1%.

2. Extraction of apple young fruit polyphenol

Crushing 20g of young apple fruits, extracting the crushed young apple fruits for 3 hours at room temperature by using 200mL of 70% ethanol aqueous solution, then carrying out reduced pressure evaporation and concentration to 20mL, adsorbing the crushed young apple fruits by using X-5 macroporous adsorption resin at the adsorption rate of 1.5BV/h, eluting the crushed young apple fruits by using 40mL of 70% ethanol aqueous solution at the volume concentration of 1.5BV/h, evaporating to remove ethanol, and drying the crushed young apple fruits for 12 hours to obtain 400mg of young apple fruit polyphenol with the purity of 75.5%.

3. Preparing fruit juice bacteriostatic agent

And (3) uniformly mixing 100mg of lentinus edodes stem neutral polysaccharide and 300mg of apple young fruit polyphenol to obtain the fruit juice bacteriostatic agent.

Example 2

In this example, the mushroom stem neutral polysaccharide and the apple young fruit polyphenol are extracted according to the method of example 1, and then 100mg of the mushroom stem neutral polysaccharide and 100mg of the apple young fruit polyphenol are uniformly mixed to obtain the fruit juice bacteriostatic agent.

Example 3

In this example, the mushroom stem neutral polysaccharide and the apple young fruit polyphenol are extracted according to the method of example 1, and then 100mg of the mushroom stem neutral polysaccharide and 200mg of the apple young fruit polyphenol are uniformly mixed to obtain the fruit juice bacteriostatic agent.

Example 4

In this example, the mushroom stem neutral polysaccharide and the apple young fruit polyphenol are extracted according to the method of example 1, and then 100mg of the mushroom stem neutral polysaccharide and 400mg of the apple young fruit polyphenol are uniformly mixed to obtain the fruit juice bacteriostatic agent.

In order to prove the beneficial effects of the invention, the inventor selects a microdilution method which has higher sensitivity and can quantitatively detect the in-vitro antibacterial activity of the medicine, namely, the antibacterial activity of two components of lentinus edodes stem neutral polysaccharide and apple young fruit polyphenol in the fruit juice antibacterial agent is evaluated by using Minimum Inhibitory Concentration (MIC). The minimum inhibitory concentration of the two components is determined by adopting a resazurin color development method. Resazurin is a blue non-fluorescent dye that can be reduced to a pink fluorescent dye by a variety of reductases in living cells, while inactive cells do not have the metabolic capacity to reduce it. The method reflects the bacteriostatic ability of an object to be detected through the change of the color of a bacterial liquid: after the indicator, the object to be detected and the bacterial liquid are co-cultured for a period of time, if the bacterial liquid turns red, the object to be detected has no bacteriostatic activity, and if the bacterial liquid keeps blue, the object to be detected has bacteriostatic activity. The MIC of the neutral polysaccharide in the lentinus edodes stems and the young apple polyphenol is measured by adopting a 96-well plate dilution gradient method, and the specific test method is as follows:

inoculating bacteria such as Escherichia coli and Staphylococcus aureus into MRS broth, and culturing at 37 deg.C for 12 hr; alicyclobacillus was inoculated in K's medium and cultured at 55 ℃ for 24 hours. Diluting the above microorganism culture with culture medium to 10⁶～10⁷CFU/mL of bacterial suspension. 7.5mL of indicator solution (100. mu.g/mL of resazurin aqueous solution) was mixed with 5mL of the bacterial suspension, and 180. mu.L of the mixture was mixed in the first row of the 96-well plate, and 100. mu.L of the mixture was mixed in the other rows. Diluting a substance to be detected to a proper concentration by DMSO, adding 20 mu L of samples into a first row of a 96-well plate, performing three controls on each sample, uniformly mixing the samples in the first row, taking 100 mu L of the solution, transferring the solution to a second row, uniformly mixing the solutions, sequentially diluting the solutions by two times to form a certain concentration gradient in the other rows as above, culturing the solutions in a constant-temperature incubator at 37 ℃ for 12 hours, and observing the color change and the turbidity degree to determine the MIC value. The results are shown in Table 1.

TABLE 1 minimum inhibitory concentration (unit: μ g/mL) of two ingredients

	Neutral polysaccharide of Lentinus edodes stem	Apple young fruit polyphenol
			Escherichia coli	45	30
Staphylococcus aureus	35	45
			Salmonella	15	7.5
Alicyclobacillus	85	275

The experimental results in table 1 show that the lentinus edodes stem neutral polysaccharide and the apple fruitlet polyphenol have certain bacteriostatic effects on escherichia coli, salmonella, staphylococcus aureus, alicyclobacillus acidocaldarius and the like. Compared with the apple young fruit polyphenol, the inhibition capacity of the lentinus edodes stem neutral polysaccharide on alicyclobacillus is improved by 5-6 times.

Further in vitro study of lentinus edodes stems using the Bliss independent modelThe neutral polysaccharide and the apple fruitlet polyphenol have synergistic inhibition effect on escherichia coli, staphylococcus aureus, salmonella and alicyclobacillus. In the case of Alicyclobacillus, it is inoculated in K's medium, cultured at 55 ℃ for 24 hours, and its culture is diluted to 10 concentration with the medium⁶～10⁷CFU/mL of bacterial suspension. mu.L of the bacterial suspension is added into each hole of a 16-hole plate, mushroom stem neutral polysaccharide with sub-inhibitory concentration (0.5MIC is 42.5 mu g/mL) and apple young fruit polyphenol with sub-inhibitory concentration (0.5MIC is 137.5 mu g/mL) are respectively added into each hole, and the mushroom stem neutral polysaccharide and the apple young fruit polyphenol with sub-inhibitory concentration are simultaneously added for overnight culture for 12 hours to detect the OD value.

Synergy was calculated using the formula: s ═ S (OD)₁/OD₀)×(OD₂/OD₀)-(OD₁₂/OD₀) Wherein OD₀The optical density value of the bacteria without adding the test substance is shown; OD₁Represents the optical density value, OD, of the mushroom stem neutral polysaccharide alone₂Indicating the optical density, OD, of the apple young fruit with only polyphenol added₁₂The optical density value is shown when the neutral polysaccharide of the mushroom stem and the polyphenol of the apple young fruit are added simultaneously; the synergy value (S) corresponds to the following cutoff value: zero indicates neutrality, positive values indicate synergy, and negative values indicate antagonism. With higher positive values indicating higher synergy. The results are shown in Table 2.

TABLE 2 synergistic bacteriostatic action

Bacterial strain	Synergistic antibacterial action (S)
		Escherichia coli	0.02
Staphylococcus aureus	0.02
		Salmonella	0.03
Alicyclobacillus	0.09

Mixing the lentinus edodes stem neutral polysaccharide and the apple young fruit polyphenol according to the mass ratio of 1:1, 1:2, 1:3 and 1:4 respectively, and carrying out synergistic bacteriostatic ability detection on different compounding ratios. In the case of Alicyclobacillus, it is inoculated in K's medium, cultured at 55 ℃ for 24 hours, and its culture is diluted to 10 concentration with the medium⁶～10⁷CFU/mL of bacterial suspension. Adding 100 μ L of the above bacterial suspension into each well of a 16-well plate, adding 200 μ g/mL, 133 μ g/mL, 100 μ g/mL, 80 μ g/mL of lentinus edodes stem neutral polysaccharide, 200 μ g/mL, 266 μ g/mL, 300 μ g/mL, 320 μ g/mL of apple young fruit polyphenol into each well, and simultaneously adding 200 μ g/mL of lentinus edodes stem neutral polysaccharide and 200 μ g/mL of apple young fruit polyphenol, 133 μ g/mL of lentinus edodes stem neutral polysaccharide and 266 μ g/mL of apple young fruit polyphenol, 100 μ g/mL of lentinus edodes stem neutral polysaccharide and 300 μ g/mL of apple young fruit polyphenol, 80 μ g/mL of lentinus edodes stem neutral polysaccharide and 320 μ g/mL of apple young fruit polyphenol, culturing overnight for 12 hours, and detecting OD value. The synergy was calculated as above. The results are shown in Table 3.

TABLE 3 synergistic bacteriostatic ability in different proportions

	1:1	1:2	1:3	1:4
					Escherichia coli	0.04	0.06	0.09	0.03
Staphylococcus aureus	0.03	0.06	0.07	0.04
					Salmonella	0.07	0.05	0.05	0.03
Alicyclobacillus	0.04	0.03	0.07	0.03

The results in tables 2 and 3 show that the mushroom stem neutral polysaccharide and the apple fruitlet polyphenol have a certain synergistic effect, particularly the synergistic value (S) of the alicyclic acid bacillus is 0.09, the fruit juice has a good synergistic inhibition effect on common bacteria in the fruit juice, and when the two are compounded in a mass ratio of 1:3, the synergistic inhibition effect is optimal.

The inventor further applies the fruit juice bacteriostatic agent prepared in the embodiment 1 of the invention to apple juice and kiwi fruit juice, and the specific method comprises the following steps: 100mg of mushroom stem neutral polysaccharide and 300mg of apple polyphenol are dissolved in 10mL of distilled water, and the solution is concentrated to 1mL under reduced pressure to prepare a mixed solution of about 400 mg/mL. Adding 2 drops (about 0.1mL) of bacteriostatic agent into 100mL of NFC apple juice and 100mL of NFC kiwi fruit juice respectively, sealing, performing ultra-high pressure sterilization, and storing at low temperature. The experimental result shows that the storage period of the apple juice can be prolonged to 382 days, and the storage period of the kiwi fruit juice can be prolonged to 350 days. And the NFC apple juice and the NFC kiwi fruit juice which are not added with the bacteriostatic agent and are only subjected to ultra-high pressure sterilization are stored at low temperature, and the storage periods of the NFC apple juice and the NFC kiwi fruit juice are respectively about 180 days and about 150 days.