CN108186784B

CN108186784B - Dandelion composite enzyme and preparation method and application thereof

Info

Publication number: CN108186784B
Application number: CN201810086338.1A
Authority: CN
Inventors: 姜立春; 田徽; 阮期平
Original assignee: Mianyang Normal University
Current assignee: Mianyang Normal University
Priority date: 2018-01-30
Filing date: 2018-01-30
Publication date: 2021-04-13
Anticipated expiration: 2038-01-30
Also published as: CN108186784A

Abstract

The invention discloses a dandelion composite enzyme and a preparation method and application thereof. The preparation method comprises the following steps: (1) crushing raw material medicines including dandelion and houttuynia cordata, and mixing 40-50 parts by weight of dandelion and 50-60 parts by weight of houttuynia cordata with water respectively to obtain turbid liquid; (2) carrying out enzymolysis on the turbid solution by adopting pectinase, cellulase, xylanase and trypsin to obtain an enzymolysis solution; (3) adding nutrient substances into the enzymolysis liquid, and then sterilizing to obtain a culture medium; (4) inoculating saccharomycetes into the culture medium for anaerobic fermentation culture for 18-30 h, then inoculating lactic acid bacteria for standing fermentation culture for 30-120 h, and obtaining a culture product; (5) and carrying out solid-liquid separation on the culture product to obtain the composite enzyme. The method can reduce the fermentation time, and the obtained composite ferment has more remarkable bacteriostatic action.

Description

Dandelion composite enzyme and preparation method and application thereof

Technical Field

The invention relates to a compound enzyme and a preparation method and application thereof, in particular to a dandelion compound enzyme and a preparation method and application thereof.

Background

The bacterial infection is acute systemic infection caused by the invasion of pathogenic bacteria or conditional pathogenic bacteria into blood circulation to grow and reproduce and produce toxins and other metabolites; clinically, the medicine is characterized by chills, hyperpyrexia, rash, arthralgia and hepatosplenomegaly; some may have septic shock and migratory foci. Invasion of pathogenic microorganisms into the blood from a wound or infected lesion in the body causes acute systemic infection. Clinically, some patients also have dysphoria, cold limbs, cyanosis, thready pulse, rapid respiration, and blood pressure decrease.

At present, many traditional Chinese medicines and health-care foods with antibacterial function are available, most of the traditional Chinese medicines are common traditional Chinese medicine processed products, and the curative effect is common. The compound enzyme is a substance with special biological activity containing amino acid, is present in all living animals and plants, and is an essential substance for maintaining the normal functions of the organism, digesting food, repairing tissues and other life activities. The complex enzyme acts in the human body. Except that the physiological function of a person is abnormal due to lack of enzymes caused by congenital reasons, the body of the person is vigorous and insufficient from young to old, the ingested nutrients are not easy to digest and absorb, the capability of synthesizing the enzymes by means of ingesting the nutrients is reduced, vicious circle is caused, metabolism tends to be slow, fatigue is easy to occur, and the person is not easy to recover once suffering from the disease. In order to delay senility, prevent and treat diseases and strengthen physique, various enzymes need to be supplemented. As a unique health product with life vitality, the composite yeast has a huge health promotion prospect.

CN107373252A discloses a preparation method of dandelion fermentation liquor, which comprises the steps of mixing 7-8 parts of dandelion and 1-3 parts of water in a mass ratio, and crushing the mixture until the average particle size is 1-3 mm to obtain dandelion pulp; adding 0.05-0.2% of 1 ten thousand U/g cellulase and 0.05-0.2% of 2-5 ten thousand U/g pectinase according to the mass of the dandelion pulp, and carrying out enzymolysis at the enzymolysis temperature of 40-50 ℃ for 1-5 hours to obtain dandelion enzymolysis liquid; and performing combined fermentation on the dandelion enzymolysis liquid for 35-50 days by adopting a plurality of beneficial bacteria such as lactobacillus acidophilus, lactobacillus plantarum, bacillus subtilis and the like to obtain the dandelion fermentation liquid. On the other hand, the fermentation time of the above patent documents is too long to be suitable for industrial production; on the other hand, the above patent documents only suggest that the dandelion ferment beverage can promote the proliferation of beneficial bacteria in the intestinal tract, inhibit the propagation of harmful bacteria and the formation of putrefactive substances, and play roles in regulating the balance of intestinal flora, enhancing immunity, promoting sleep, delaying aging, etc., and the bacteriostatic action is not significant.

Therefore, there is still a need for a method for preparing dandelion complex enzyme, which can shorten the fermentation time, thereby facilitating the industrial production. In addition, there is still a need to improve the bacteriostatic effect of dandelion complex enzyme.

Disclosure of Invention

The invention aims to provide a preparation method of dandelion composite ferment, which can shorten the fermentation time. The invention also aims to provide the dandelion composite enzyme, which has improved bacteriostatic effect. The invention further aims to provide application of the dandelion composite ferment. The purpose of the invention is realized by the following technical scheme.

On one hand, the invention provides a preparation method of dandelion composite enzyme, which comprises the following steps:

(1) crushing raw material medicines including dandelion and houttuynia cordata, and mixing 40-50 parts by weight of dandelion and 50-60 parts by weight of houttuynia cordata with water respectively to obtain turbid liquid;

(2) carrying out enzymolysis on the turbid solution by adopting pectinase, cellulase, xylanase and trypsin to obtain an enzymolysis solution;

(3) adding nutrient substances into the enzymolysis liquid, and then sterilizing to obtain a culture medium;

(4) inoculating saccharomycetes into the culture medium for anaerobic fermentation culture for 18-30 h, then inoculating lactic acid bacteria for standing fermentation culture for 30-120 h, and obtaining a culture product;

(5) and carrying out solid-liquid separation on the culture product to obtain the composite enzyme.

According to the preparation method of the present invention, preferably, in the step (1), the amount of water is 5 to 15 times of the total weight of the dandelion and the houttuynia cordata.

According to the preparation method of the present invention, preferably, in the step (1), 45 to 50 parts by weight of the dandelion and 50 to 55 parts by weight of the houttuynia cordata are used.

According to the preparation method provided by the invention, preferably, in the step (2), the pH value of enzymolysis is 6-7, the enzymolysis temperature is 50-65 ℃, and the enzymolysis time is 3-8 hours.

According to the preparation method of the present invention, preferably, in the step (2), based on the total weight of the turbid solution, the pectinase is used in an amount of 0.05 to 0.5 wt%, the cellulase is used in an amount of 0.05 to 0.5 wt%, the xylanase is used in an amount of 0.05 to 0.5 wt%, and the trypsin is used in an amount of 0.03 to 0.2 wt%.

According to the preparation method of the present invention, preferably, in the step (3), the nutrients include 0.1 to 0.5 wt% of yeast extract, 1 to 5 wt% of ammonium salt, 0.02 to 0.2 wt% of zinc salt, 0.02 to 0.2 wt% of magnesium salt, and 0.3 to 0.8 wt% of phosphate; the above weight percentages are based on the total weight of the enzymatic hydrolysate.

According to the preparation method provided by the invention, preferably, in the anaerobic fermentation culture in the step (4), the inoculation amount of the yeast is 0.3-1.5 wt% of the culture medium, the fermentation temperature is 26-35 ℃, and the fermentation time is 20-28 h.

According to the preparation method of the invention, preferably, in the standing fermentation culture in the step (4), the inoculation amount of the lactic acid bacteria is 0.2-1.0 wt% of the culture medium, the fermentation culture is carried out for 28-38 h at 35-40 ℃, and then the fermentation culture is carried out for 25-60 h at 60-75 ℃.

In another aspect, the present invention provides a dandelion complex enzyme, which is obtained by any one of the above preparation methods.

In another aspect, the invention provides a use of the dandelion complex enzyme in preparation of a drug or a health product with bacteriostatic action.

The invention reduces the fermentation time by selecting proper enzyme and flora, thereby being more beneficial to industrial production. The invention utilizes an enzyme method to carry out hydrolysis, and then utilizes saccharomycetes and lactic acid bacteria to carry out fermentation treatment on dandelion and houttuynia cordata, so that the product is rich in functional active ingredients of the original traditional Chinese medicine, and the bacteriostatic action of the product is improved.

Detailed Description

The present invention will be further described with reference to the following specific examples, but the scope of the present invention is not limited thereto.

The complex ferment of the invention can also be called as a fermentation composition. It can be fermentation stock solution or fermentation powder. The raw materials for fermentation comprise dandelion and houttuynia cordata; preferably only dandelion and houttuynia cordata.

Taraxacum mongolicum hand-Mazz belonging to perennial herbaceous plants; is rich in vitamin A, vitamin C and potassium, and also contains ferrum, calcium, vitamin B2, vitamin B1, magnesium, vitamin B6, folic acid and copper. The herba Taraxaci herb contains taraxasterol, choline, inulin, pectin, etc. The dandelion root contains dandelion alcohol, dandelion sterol, beta-resinol, stigmasterol, beta-sitosterol, choline, organic acid, fructose, sucrose, glucose, glucoside, etc. The herba Taraxaci has effects of clearing away heat and toxic materials, promoting urination, resolving hard mass, resisting bacteria and resisting tumor; can be used for treating acute mastitis, lymphadenitis, lymphoid tuberculosis, furunculosis, acute conjunctivitis, common cold with fever, acute tonsillitis, acute bronchitis, gastritis, hepatitis, cholecystitis, urinary tract infection, etc.

Houttuynia cordata (Houttuynia cordita Thunb.) contains decanoyl acetaldehyde, lauraldehyde, alpha-pinene, linalool and the like; also contains methyl n-nonyl ketone, camphene, myrcene, limonene, bornyl acetate, caryophyllene and the like; in addition, it contains Adoforin, hyperin, rutin, chlorogenic acid, beta-sitosterol, stearic acid, oleic acid, linoleic acid, etc. The herba Houttuyniae leaf contains quercetin; the flowers and ears contain isoquercitrin. Herba Houttuyniae has effects of clearing away heat and toxic materials, inducing diuresis and relieving swelling, and can be used for treating pneumonia, lung abscess, dysentery, malaria, edema, gonorrhea, leucorrhea, carbuncle, hemorrhoid, rectocele, eczema, alopecia, and scabies.

The preparation method of the dandelion composite enzyme comprises the following steps: (1) a crushing and dispersing step; (2) enzymolysis; (3) preparing a culture medium; (4) a fermentation step: (5) and (5) a separation step. Optionally, a concentration and drying step (6) can also be included. As described in detail below.

In the crushing and dispersing step, raw material medicines including dandelion and houttuynia cordata are crushed, and 40-50 parts by weight of dandelion and 50-60 parts by weight of houttuynia cordata are respectively mixed with water to obtain turbid liquid. The raw material medicaments such as dandelion, houttuynia and the like can be respectively crushed, then weighed and mixed with water; or weighing herba Taraxaci and herba Houttuyniae, pulverizing, and mixing with water. The crude drug can be pulverized to 20 meshes or less, preferably 50 meshes or less, and more preferably 100 meshes or less. The fermentation time can be further reduced by controlling the particle size of the raw material medicines. In the present invention, the dandelion may be 40 to 50 parts by weight, preferably 45 to 50 parts by weight. The amount of houttuynia cordata may be 50 to 60 parts by weight, preferably 50 to 55 parts by weight. According to one embodiment of the present invention, 50 parts by weight of dandelion and 50 parts by weight of houttuynia cordata are used. According to another embodiment of the present invention, 50 parts by weight of dandelion and 45 parts by weight of houttuynia cordata are used. The combination makes the composite ferment bacteriostatic action more obvious.

In the crushing and dispersing step, the amount of water is 5-15 times of the total weight of the dandelion and the houttuynia cordata; preferably 8 to 10 times. The adoption of the proportion is beneficial to the enzymolysis and fermentation processes. The water can be distilled water or deionized water, and the like, as long as the enzymolysis and fermentation processes are not influenced.

In the enzymolysis step, the pH value of enzymolysis can be 6-7, and preferably 6.5-6.8; the enzymolysis temperature can be 50-65 ℃, preferably 55-60 ℃, and more preferably 52-58 ℃; the enzymolysis time can be 3-8 h, preferably 3-6 h, and more preferably 3.5-5 h. According to a specific embodiment of the invention, the pH value of enzymolysis is 6.5, the enzymolysis temperature is 55 ℃, and the enzymolysis time is 3.5 h. By adopting the enzymolysis condition of the invention, the three biological enzymes can carry out full enzymolysis on the raw material medicines, thereby reducing the fermentation time.

In the enzymolysis step, pectinase, cellulase, xylanase and trypsin are adopted to carry out enzymolysis on the turbid liquid to obtain an enzymolysis liquid. The amount of the pectinase is 0.05-0.5 wt%, preferably 0.08-0.2 wt%, based on the total weight of the turbid solution; the dosage of the cellulase is 0.05 to 0.5 wt%, preferably 0.08 to 0.2 wt%; the dosage of the xylanase is 0.05-0.5 wt%, preferably 0.08-0.2 wt%; the amount of trypsin is 0.03-0.2 wt%, preferably 0.05-0.15 wt%, and more preferably 0.1-0.15 wt%. In the invention, the enzyme activity of the pectinase can be 15-55 ten thousand U/g, preferably 25-50 ten thousand U/g, and more preferably 35-50 ten thousand U/g. The enzyme activity of the cellulase can be 20-55 ten thousand U/g, preferably 25-55 ten thousand U/g, and more preferably 35-50 ten thousand U/g. The enzyme activity of the xylanase can be 15-60 ten thousand U/g, preferably 25-50 ten thousand U/g, and more preferably 35-50 ten thousand U/g. The enzyme activity of the trypsin can be 15-55 ten thousand U/g, preferably 25-50 ten thousand U/g, and more preferably 35-50 ten thousand U/g. According to a specific embodiment of the invention, the enzyme activity of the pectinase is 50 ten thousand U/g, the enzyme activity of the cellulase is 50 ten thousand U/g, the enzyme activity of the xylanase is 50 ten thousand U/g, and the enzyme activity of the trypsin is 50 ten thousand U/g.

When the biological enzymes with the types and the dosage are adopted, the raw material medicines can be fully enzymolyzed, and raw materials such as amino acid, monosaccharide, oligosaccharide and the like are provided for fermentation culture, so that the fermentation time is shortened.

In the preparation step of the culture medium of the present invention, nutrients are added to the enzymatic hydrolysate to obtain the culture medium. Preferably, the medium may be sterilized. Sterilization may be performed using methods known in the art and will not be described further herein. Based on the total weight of the enzymolysis liquid, the nutrient substances comprise 0.1-0.5 wt% of yeast extract, 1-5 wt% of ammonium salt, 0.02-0.2 wt% of zinc salt, 0.02-0.2 wt% of magnesium salt and 0.3-0.8 wt% of phosphate; the above weight percentages are based on the total weight of the enzymatic hydrolysate. Commercially available yeast extract can be used. The amount of the yeast extract is preferably 0.2-0.35 wt%, and preferably 0.3-0.35 wt%. The ammonium salt may be at least one selected from ammonium sulfate, ammonium nitrate or ammonium carbonate, preferably ammonium sulfate. The amount of the ammonium salt is preferably 1 to 3 wt%, more preferably 1.5 to 2.5 wt%. The magnesium salt may be at least one selected from magnesium sulfate, magnesium chloride, magnesium nitrate and magnesium carbonate, and preferably magnesium sulfate. The amount of the magnesium salt is preferably 0.03 to 0.15 wt%, more preferably 0.05 to 0.1 wt%. The phosphate may be at least one selected from sodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, calcium phosphate, and magnesium phosphate, and is preferably potassium dihydrogen phosphate. The amount of the phosphate is preferably 0.3 to 0.65 wt%, more preferably 0.35 to 0.5 wt%. The zinc salt can be at least one selected from zinc sulfate, zinc chloride and zinc nitrate, and is preferably zinc chloride; the amount of the zinc salt is preferably 0.03 to 0.1 wt%, more preferably 0.05 to 0.08 wt%. The nutrient may also contain potassium salts. The potassium salt is preferably the same as the phosphate salt, for example potassium phosphate, dipotassium hydrogen phosphate or potassium dihydrogen phosphate, preferably potassium dihydrogen phosphate. The nutrient substances are added into the enzymolysis liquid, so that the nutrient element requirement of fermentation culture of yeast and lactic acid bacteria can be effectively met, and the fermentation time can be shortened.

In the fermentation step, saccharomycetes are inoculated into the culture medium for anaerobic fermentation culture for 18-30 h, and then lactobacillus is inoculated for standing fermentation culture for 30-120 h to obtain a culture product. Compared with the prior art, the yeast fermentation can produce metabolites with bacteriostatic activity. In addition, the fermentation time of the invention is greatly reduced. In the anaerobic fermentation culture, the inoculation amount of the yeast is 0.3-1.5 wt%, preferably 0.2-1.0 wt%, and more preferably 0.5-0.8 wt% of the culture medium. The fermentation temperature is 26-35 ℃, preferably 28-32 ℃, and more preferably 28 ℃. The fermentation time is 18-30 h, preferably 10-25 h, and more preferably 20 h. Under the conditions, the function of the yeast can be fully exerted and the time is saved. In the static fermentation culture, the inoculation amount of the lactic acid bacteria is 0.2-1.0 wt%, preferably 0.2-0.8 wt%, and more preferably 0.5-0.6 wt% of the culture medium. Fermenting and culturing for 28-38 h, preferably 30-35 h and more preferably 32h at 35-40 ℃, preferably 25-39 ℃ and more preferably 37 ℃; then fermenting and culturing for 25-60 h, preferably 28-50 h, more preferably 30h at 60-75 ℃, preferably 62-72 ℃, more preferably 65 ℃. Examples of yeasts of the present invention include, but are not limited to, high activity dry yeasts produced by Angel Yeast Ltd. The lactic acid bacteria of the present invention may be selected from one or more of lactobacillus bulgaricus and streptococcus thermophilus; preferably a mixture of Lactobacillus bulgaricus and Streptococcus thermophilus. The weight ratio of the lactobacillus bulgaricus to the streptococcus thermophilus can be 1: 10-10: 1, preferably 1: 5-5: 1, and more preferably 1: 1.

In the separation step of the invention, the culture product is subjected to solid-liquid separation to obtain the composite ferment. Separating to obtain the composite ferment (fermentation stock solution) and solid residue. The method of solid-liquid separation may be centrifugation or filtration, and is preferably centrifugation.

In the concentration and drying step of the invention, the fermentation stock solution is concentrated and dried to obtain the fermentation powder. The concentration and drying may be carried out by a method of reducing pressure, which will not be described in detail.

The composite ferment is obtained by adopting the preparation method. The composite ferment can be fermentation liquor or fermentation powder. Complex ferments are sometimes also referred to as fermentation compositions, and the like. According to one embodiment of the invention, the baking powder has a water content of less than 5 wt%.

The composite ferment has the bacteriostatic action, so that the composite ferment can be used for preparing medicines or health-care products with the bacteriostatic action. The health product can be food with health promoting effect, such as candy, beverage, bread, etc. The medicine can be various conventional solid dosage forms or liquid dosage forms, and is not described in detail.

The test methods of the following examples and comparative examples are illustrated below:

< Total Flavonoids Change before and after fermentation >

1. Preparation of sample liquid

Fermentation sample: taking 0.3g of the fermented powder prepared in the example 4 (equivalent to 2.0g of dandelion and 2.0g of houttuynia cordata), and adding distilled water to a constant volume of 50ml to obtain the traditional Chinese medicine.

Water extract sample: soaking herba Taraxaci 2.0g and herba Houttuyniae 2.0g in water for 30min, decocting and extracting for 2 times, adding 10 times of water each time, extracting for 2 hr each time, filtering, mixing filtrates, concentrating, and diluting with distilled water to 50ml to obtain the final product.

2. Preparation of control solutions

Precisely weighing 10mg of rutin control substance dried at constant temperature of 120 deg.C to constant weight, placing in a 50mL measuring flask, adding appropriate amount of ethanol, ultrasonic treating to dissolve completely, cooling, adding appropriate amount of ethanol to scale mark, and shaking to obtain the desired control solution (containing anhydrous rutin 0.2mg per 1 mL).

3. Absorbance measurement of control

Precisely measuring 6mL of reference substance solution, respectively placing the reference substance solution in 50mL measuring bottles, respectively adding water to 12mL, dropwise adding 2mL of 5 wt% sodium nitrite solution, shaking up, placing for 6min, then respectively dropwise adding 2mL of 10 wt% aluminum nitrate solution, shaking up, placing for 6min, dropwise adding 20mL of 4% sodium hydroxide test solution, finally adding water to a scale mark, shaking up, placing for 15min, using a corresponding reagent as a blank control group, immediately irradiating an ultraviolet-visible spectrophotometry, and setting the wavelength at 500nm to measure the absorbance.

4. Determination of content

Putting 10mL of sample solution into 50mL measuring bottles respectively, adding water to 12mL respectively, dripping 2mL of 5 wt% sodium nitrite solution respectively, shaking up, standing for 6min, dripping 2mL of 10 wt% aluminum nitrate solution respectively, shaking up, standing for 6min, dripping 20mL of 4% sodium hydroxide test solution, adding water to a scale mark, shaking up, standing for 15min, using a corresponding reagent as a blank control group, immediately measuring the absorbance of each group of sample solution at a set wavelength of 500nm (3 parallel samples), and recording the absorbance of each group obtained in the experiment. And calculating the total flavone content in the two sample solutions according to the curve.

TABLE 1 measurement results of total flavone content in fermentation product sample and water extract sample

Example 1 Complex ferment

1) Pulverizing herba Taraxaci, sieving with pharmacopeia No. 3 sieve (50 mesh), pulverizing herba Houttuyniae, and sieving with pharmacopeia No. 3 sieve; then, 50 parts by weight of dandelion and 50 parts by weight of houttuynia cordata are mixed, and purified water in an amount of 10 times the total weight of dandelion and houttuynia cordata is added to prepare a turbid liquid.

2) Mixing the turbid solution with 0.1 wt% of pectinase (the enzyme activity is 50 ten thousand U/g), 0.1 wt% of cellulase (the enzyme activity is 50 ten thousand U/g), 0.1 wt% of xylanase (the enzyme activity is 50 ten thousand U/g) and 0.1 wt% of trypsin (50 ten thousand U/g) based on the total weight of the turbid solution, and carrying out enzymolysis for 3.5h under the conditions that the pH value is 6.5 and the temperature is 55 ℃ to obtain an enzymolysis solution.

3) Adding 0.3 wt% of yeast extract, 1.5 wt% of ammonium sulfate, 0.05 wt% of zinc chloride, 0.05 wt% of magnesium sulfate and 0.4 wt% of monopotassium phosphate into the enzymolysis liquid based on the total weight of the enzymolysis liquid, and sterilizing to obtain the culture medium.

4) Inoculating 0.8 wt% yeast (high activity dry yeast produced by Angel Yeast Co., Ltd.) into the culture medium, and performing anaerobic fermentation at 28 deg.C for 20 hr; then inoculating 0.5 wt% lactobacillus (Lactobacillus bulgaricus and Streptococcus thermophilus at a weight ratio of 1: 1), standing and fermenting at 37 deg.C for 32 hr, and standing and fermenting at 65 deg.C for 30 hr.

5) And (4) after the fermentation is finished, performing centrifugal treatment to obtain enzyme stock solution (composite enzyme) and solid residue.

EXAMPLE 2 disinfectant liquid

The fermentation liquid of example 1 was mixed with water at a weight ratio of 1:10 to obtain a disinfectant liquid.

Example 3 Complex ferment

1) Pulverizing herba Taraxaci, sieving with pharmacopeia No. 3 sieve (50 mesh), pulverizing herba Houttuyniae, and sieving with pharmacopeia No. 3 sieve; then, 50 parts by weight of dandelion and 45 parts by weight of houttuynia cordata are mixed, and purified water in an amount of 10 times the total weight of dandelion and houttuynia cordata is added to prepare a turbid liquid.

Example 4 baking powder

The fermentation liquid obtained in example 3 was concentrated under reduced pressure, and dried under reduced pressure to obtain a fermented powder. The water content of the obtained fermentation powder is less than 5 wt%, the activity of superoxide dismutase (SOD) is more than (400 +/-5) U/g, and the total flavone is more than 4 wt%.

Experimental example 1 bacteriostatic test

< preparation of drug solution >

Set of baking powder (invention): weighing 4.5g of baking powder in example 4, adding distilled water to a constant volume of 120ml, and obtaining the fermented powder.

Enzymolysis liquid group (before fermentation): concentrating and drying the enzymolysis liquid of the embodiment 3 to obtain powder, taking 4.5g of the powder, and fixing the volume to 120ml by using distilled water to obtain the enzyme-linked immunosorbent assay solution.

Dandelion group (after fermentation): the houttuynia cordata thunb is omitted, the other conditions are the same as the examples 3 and 4, 4.5g of fermentation powder is weighed, and distilled water is added to the fermentation powder to reach the constant volume of 120ml, so that the houttuynia cordata thunb is obtained.

Dandelion group (before fermentation): the houttuynia cordata thunb is omitted, other conditions are the same as the conditions in the embodiment 3, the enzymolysis liquid is concentrated and dried to obtain powder, 4.5g of the powder is weighed, and distilled water is added to the powder to reach the constant volume of 120ml, so that the houttuynia cordata thunb powder is obtained.

Herba houttuyniae english group (after fermentation): omitting dandelion, weighing 4.5g of baking powder under the same conditions as in examples 3 and 4, and adding distilled water to a constant volume of 120ml to obtain the dandelion beverage.

Houttuynia cordata group (before fermentation): omitting dandelion, concentrating and drying the enzymatic hydrolysate to obtain powder, weighing 4.5g of powder, adding distilled water to constant volume of 120ml, and obtaining the dandelion tea.

< Experimental method >

The nutrient agar medium was sterilized at 121 ℃ for 20 min. Preparing a flat plate by a pouring method, adding 1mL of bacterial suspension into each sterilized culture dish on a clean bench, pouring 20mL of culture medium, fully and uniformly mixing, drying condensed water, and waiting for solidification. And (3) clamping the sterilized Oxford cup by using sterile forceps, putting the sterilized Oxford cup on the flame of an alcohol burner, quickly passing a fire, vertically placing the sterilized Oxford cup on the surface of a culture medium, and slightly pressing the sterilized Oxford cup to ensure that no gap exists between the bottom of the Oxford cup and the culture medium. 4 Oxford cups were placed per plate. The middle oxford cup was compared with normal saline, and 200 μ L of each of the other oxford cups was filled with the liquid. The liquid medicine can not overflow out. Each liquid medicine pouring method is repeated for 3 times; culturing at 35 deg.C for 48 h. Observing, measuring and recording.

Test liquid medicine for colibacillus and golden yellow grapeThe inhibition of cocci with pseudomonas aeruginosa. Firstly culturing Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa liquid to make its bacterial liquid concentration reach about 1 × 10⁶cfu/mL as standard solution of test bacteria. The liquid medicine is respectively subjected to a plate bacteriostasis test with escherichia coli, staphylococcus aureus and pseudomonas aeruginosa standard liquid, the inhibition degree is tested, and the results are shown in table 2. Each set of experiments was done in 3 replicates. The experimental result shows that the compound ferment liquid medicine has obvious inhibition effect on escherichia coli, staphylococcus aureus and pseudomonas aeruginosa.

TABLE 2 results of zone of inhibition test (mm) by Oxford cup method

The present invention is not limited to the above-described embodiments, and any variations, modifications, and substitutions which may occur to those skilled in the art may be made without departing from the spirit of the invention.

Claims

1. The preparation method of the dandelion composite enzyme is characterized by comprising the following steps:

(1) crushing raw material medicines consisting of dandelion and houttuynia cordata, and mixing 40-50 parts by weight of dandelion and 50-60 parts by weight of houttuynia cordata with water respectively to obtain turbid liquid;

(2) carrying out enzymolysis on the turbid solution by adopting pectinase, cellulase, xylanase and trypsin to obtain an enzymolysis solution; based on the total weight of the turbid solution, the dosage of the pectinase is 0.08-0.2 wt%, and the dosage of the cellulase is 0.08-0.2 wt%; the dosage of the xylanase is 0.08-0.2 wt%, and the dosage of the trypsin is 0.1-0.15 wt%;

(3) adding nutrient substances into the enzymolysis liquid to obtain a culture medium;

(4) inoculating saccharomycetes into the culture medium for anaerobic fermentation culture for 10-25 h, then inoculating lactobacillus, standing, fermenting and culturing for 30-35 h at 35-40 ℃, and then heating to 62-72 ℃ for standing, fermenting and culturing for 28-50 h to obtain a culture product; wherein the inoculation amount of the yeast is 0.5-0.8 wt% of the culture medium, the inoculation amount of the lactic acid bacteria is 0.2-1.0 wt% of the culture medium, and the lactic acid bacteria is a mixture of Bulgaria bacillus and Streptococcus thermophilus in a weight ratio of 1: 10-10: 1;

2. The preparation method according to claim 1, wherein in the step (1), the amount of water is 5 to 15 times of the total weight of the dandelion and the houttuynia cordata.

3. The method according to claim 1, wherein in the step (1), 45 to 50 parts by weight of the dandelion and 50 to 55 parts by weight of the houttuynia cordata are used.

4. The preparation method according to claim 1, wherein in the step (2), the pH value of enzymolysis is 6-7, the enzymolysis temperature is 50-65 ℃, and the enzymolysis time is 3-8 h.

5. The method according to claim 4, wherein in the step (3), the nutrients comprise 0.1 to 0.5 wt% of yeast extract, 1 to 5 wt% of ammonium salt, 0.02 to 0.2 wt% of zinc salt, 0.02 to 0.2 wt% of magnesium salt and 0.3 to 0.8 wt% of phosphate; the above weight percentages are based on the total weight of the enzymatic hydrolysate.

6. The method according to claim 5, wherein the fermentation temperature of the yeast in the anaerobic fermentation culture of step (4) is 26-35 ℃.

7. A dandelion complex enzyme obtained by the preparation method of any one of claims 1 to 6.

8. The use of the dandelion complex enzyme according to claim 7 in the preparation of a drug or health product with bacteriostatic action.