Composition capable of inhibiting escherichia coli and application thereof

Abstract

The invention relates to the technical field of compositions for inhibiting escherichia coli, in particular to a composition for inhibiting escherichia coli and application of the composition. The composition is prepared from the following raw materials in parts by weight: 5 to 12 parts of honeysuckle flower chlorogenic acid extract, 3 to 8 parts of forsythin extract, 1 to 4 parts of oregano essential oil, 2 to 6 parts of lactobacillus acidophilus, 1 to 3 parts of lactobacillus rhamnosus, 0.5 to 2 parts of nano zinc oxide, 20 to 40 parts of maltodextrin, 0.2 to 0.8 part of vitamin E and 0.1 to 0.5 part of magnesium stearate. The Escherichia coli inhibiting composition provided by the invention can effectively inhibit growth of Escherichia coli, relieve symptoms caused by Escherichia coli infection and protect intestinal barrier function and intestinal flora diversity through component matching and optimized weight ratio, and is high in safety and free of drug resistance risk.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of compositions for inhibiting Escherichia coli, and in particular to a composition for inhibiting Escherichia coli and application thereof. BACKGROUND

[0002] Escherichia coli is a kind of gram-negative short bacillus widely existing in the intestinal tract of humans and animals, most of which are conditional pathogenic bacteria, and a few serotypes such as O157:H7 and O104:H4 have strong pathogenicity and can cause food poisoning, intestinal infection, urinary system infection and other diseases, and even life-threatening complications such as sepsis and hemolytic uremic syndrome in severe cases. In the field of animal husbandry, pathogenic Escherichia coli is the main pathogen of diseases such as yellow dysentery and white dysentery of piglets, omphalitis and septicemia of chicks, causing billions of dollars of economic losses to the global livestock industry every year; in the food processing industry, Escherichia coli contamination is also an important inducement for food recall and public health incidents.

[0003] Currently, the prevention and control measures for Escherichia coli mainly focus on chemical drugs (antibiotics); for a long time, a large amount of amoxicillin, fluoroquinolones and cephalosporins are used to inhibit Escherichia coli, which can achieve significant results in the short term, but has the risk of drug resistance. SUMMARY

[0004] In view of the existing technical problems, the present application provides a composition for inhibiting Escherichia coli and application thereof.

[0005] The technical solution adopted by the present application is: a composition for inhibiting Escherichia coli, which is composed of the following raw materials in parts by weight: 5-12 parts of honeysuckle chlorogenic acid extract, 3-8 parts of forsythia suspensa glycoside extract, 1-4 parts of oregano essential oil, 2-6 parts of lactobacillus acidophilus, 1-3 parts of lactobacillus rhamnosus, 0.5-2 parts of nano zinc oxide, 20-40 parts of malt dextrin, 0.2-0.8 parts of vitamin E and 0.1-0.5 parts of magnesium stearate.

[0006] In one embodiment, the composition is composed of the following raw materials in parts by weight: 8 parts of honeysuckle chlorogenic acid extract, 5 parts of forsythia suspensa glycoside extract, 2 parts of oregano essential oil, 4 parts of lactobacillus acidophilus, 2 parts of lactobacillus rhamnosus, 1 part of nano zinc oxide, 30 parts of malt dextrin, 0.5 parts of vitamin E and 0.3 parts of magnesium stearate.

[0007] In one embodiment, the composition is composed of the following raw materials in parts by weight: Honeysuckle chlorogenic acid extract 5 parts, forsythia glycoside extract 3 parts, oregano essential oil 1 part, lactobacillus acidophilus 2 parts, lactobacillus rhamnosus 1 part, nano zinc oxide 0.5 part, malt dextrin 20 parts, vitamin E 0.2 part, magnesium stearate 0.1 part.

[0008] In one of the embodiments, the composition is composed of the following raw materials by weight parts: Honeysuckle chlorogenic acid extract 12 parts, forsythia glycoside extract 8 parts, oregano essential oil 4 parts, lactobacillus acidophilus 6 parts, lactobacillus rhamnosus 3 parts, nano zinc oxide 2 parts, malt dextrin 40 parts, vitamin E 0.8 part, magnesium stearate 0.5 part.

[0009] In one of the embodiments, the production method of the composition is as follows: Step 1: extraction and purification of natural plant active components; Preparation of Honeysuckle chlorogenic acid extract: Take 100 kg of dried honeysuckle medicinal materials, add 8 times the amount of 50% ethanol aqueous solution, and extract under constant temperature reflux at 60℃ for 2 times, 2h each time; Combine the two extraction liquids and filter to remove the residue using a plate and frame filter; Concentrate the filtrate under reduced pressure to a relative density of 1.20 of the extract; Add 3 times the amount of purified water to the extract to dissolve, then adsorb with AB-8 macroporous resin, rinse the resin with 2BV purified water after adsorption is complete, then elute with 70% ethanol aqueous solution, and collect the eluate; Concentrate the eluate under reduced pressure again, freeze-dry, and after drying, crush to obtain chlorogenic acid extract with a purity of ≥90%, ready for use; Preparation of forsythia glycoside extract: Take 80 kg of dried forsythia fruit, add 10 times the amount of 60% ethanol aqueous solution, and ultrasonically extract 3 times at 70℃, 1.5h each time; Combine the three extraction liquids and centrifuge to remove the precipitate; Take the supernatant and adsorb it with HPD-100 macroporous resin, rinse the resin with 3BV purified water, then elute with 80% ethanol aqueous solution, and collect the eluate; Concentrate the eluate under reduced pressure, then spray dry to obtain forsythia glycoside extract with a purity of ≥85%, ready for use; Purification of oregano essential oil: Take 100 kg of fresh oregano whole grass, cut into sections, and then perform water vapor distillation to collect the distillate; Extract the distillate with petroleum ether 3 times, and combine the organic phases; The organic phase was dried with 0.5% anhydrous sodium sulfate for 24h, filtered and distilled under reduced pressure (remove petroleum ether, obtain carvacrol content ≥70% oregano essential oil, ready for use; Step 2: microencapsulation treatment of probiotics; Core material preparation: take 4kg of Lactobacillus acidophilus powder with a viable count of ≥1×10¹¹CFU / g, 2kg of Lactobacillus rhamnosus powder with a viable count of ≥1×10¹¹CFU / g, add 10kg of 2% sodium alginate aqueous solution, stir at a speed of 500r / min for 30min to form a uniform bacterial suspension; Primary embedding: drop the core material into a 3% calcium chloride aqueous solution through a peristaltic pump, the drop head diameter is 0.8mm, and the microspheres are formed by standing at 25℃ for 30min, the particle size of the microspheres is 1-2mm; Secondary embedding: transfer the sodium alginate microspheres into a 0.5% chitosan aqueous solution, stir at a speed of 200r / min for 20min at 25℃, so that chitosan forms a thin film on the surface of the microspheres; Drying and solidification: rinse the double-layer microcapsules with purified water for 2 times, drain and then vacuum freeze-dry to obtain probiotic microcapsule powder with a viable count of ≥8×10 10 CFU / g, ready for use; Step 3: surface modification of nano zinc oxide; In order to reduce the agglomeration of nano zinc oxide and improve its compatibility with other components, surface modification treatment of nano zinc oxide is needed, the specific steps are as follows: Take 1kg of nano zinc oxide with a particle size of 50-100nm, add 5kg of purified water, and ultrasonic dispersion for 30min to form a nano zinc oxide suspension; Add 0.05kg of silane coupling agent to the suspension, stir at a speed of 800r / min for 1h at 60℃; Centrifugal collection of the precipitate, vacuum drying of the precipitate for 2h, and then crushing after drying to obtain modified nano zinc oxide, ready for use; Step 4: mixing and granulation of the composition; Premixing: first add malt dextrin, vitamin E and magnesium stearate according to weight parts into a three-dimensional mixer, and mix at a speed of 15r / min for 10min; Secondary mixing: add honeysuckle chlorogenic acid extract, forsythia glycoside extract and modified nano zinc oxide into the mixer, and continue to mix for 15min; Final mixing: dilute oregano essential oil with a small amount of anhydrous ethanol, and evenly spray it onto the mixed materials, and at the same time, start the mixer and mix for 20min to ensure that the oregano essential oil is evenly dispersed in the materials; Granulation: the mixed material is transferred into a swing granulator, granulated with a 16-mesh screen, and then the granules are dried in a fluidized bed dryer, with the moisture content of the granules controlled to be less than or equal to 3%; Whole granulation and packaging: the dried granules are whole granulated with an 18-mesh screen to remove fine powder, and then the qualified granules are packed into aluminum-plastic composite bags by vacuum packaging, to obtain the final product.

[0010] The composition for inhibiting E. coli has the advantages that, compared with the prior art, the composition for inhibiting E. coli is prepared by matching and optimizing the weight proportions of components, can effectively inhibit the growth of E. coli, relieve symptoms caused by E. coli infection, protect the intestinal barrier function and intestinal flora diversity, and has high safety and no drug resistance risk. DETAILED DESCRIPTION

[0011] To solve the problems in the background art, the present application provides the following technical scheme: a composition for inhibiting E. coli, which is composed of the following raw materials according to weight proportions: 5-12 parts of honeysuckle chlorogenic acid extract, 3-8 parts of forsythia suspensa glycoside extract, 1-4 parts of oregano essential oil, 2-6 parts of lactobacillus acidophilus, 1-3 parts of lactobacillus rhamnosus, 0.5-2 parts of nano zinc oxide, 20-40 parts of malt dextrin, 0.2-0.8 parts of vitamin E, and 0.1-0.5 parts of magnesium stearate.

[0012] Component selection: Synergistic selection of natural plant active components: Honeysuckle chlorogenic acid extract: chlorogenic acid can destroy the integrity of the cell membrane of E. coli, leading to leakage of intracellular substances, and also can inhibit the activity of bacterial DNA gyrase, hindering the proliferation of E. coli; Forsythia suspensa glycoside extract: forsythia suspensa glycoside can down-regulate the expression of E. coli virulence genes (such as stx2 and eaeA), reduce the secretion of pathogenic factors such as Shiga toxin and adhesin, and reduce the pathogenicity of the strain; Oregano essential oil (carvacrol): carvacrol can penetrate the cell wall of E. coli, bind to the phospholipid of the cell membrane, and change the permeability of the membrane, and because it is volatile, it can act on bacteria in the environment and in the body at the same time.

[0013] When the three components are compounded, the effect of chlorogenic acid on “destroying membrane structure” and the effect of carvacrol on “enhancing membrane permeability” form a synergistic effect, and forsythia suspensa glycoside specifically reduces the pathogenicity of the strain, effectively solving the problem of “only inhibiting bacteria but not inhibiting toxins” of single plant extract.

[0014] Complementary matching of functional probiotics: Both lactobacillus acidophilus and lactobacillus rhamnosus are dominant probiotics in the intestines of humans and animals, and the two components have three major effects after compounding: Competitive inhibition: by consuming nutrients such as glucose, amino acids in the intestine, and occupying the attachment sites of E. coli in the intestine; Acid production inhibits bacteria: secretes organic acids such as lactic acid and acetic acid, reduces the pH value of the intestinal tract (the pH range suitable for the growth of E. coli is 6.0-8.0), thereby inhibiting the growth of E. coli; Enhance immunity: stimulate the intestinal mucosa to secrete mucin and immunoglobulin (IgA), enhance the intestinal barrier function, and reduce the invasion of E. coli into the intestinal tissue.

[0015] Synergistic effect of nano zinc oxide: Traditional micron-sized zinc oxide needs a high dose (2000-3000 mg / kg) to inhibit E. coli, and is easy to cause animal zinc poisoning and environmental pollution; the present application selects nano zinc oxide with a particle size of 50-100 nm, which has a large specific surface area and high bioavailability, and only needs a low dose (50-100 mg / kg) to destroy the cell membrane of E. coli through "oxidative stress", and also can activate the metabolic activity of probiotics, and forms a "triple antibacterial network" with plant extracts and probiotics, solving the problem of "toxic side effects of high dose use" of traditional zinc oxide.

[0016] Precise proportioning of stabilizers: Maltodextrin (DE value 10-15) has good embedding performance, which can protect probiotics and plant essential oils from being destroyed by high temperature and gastric acid; vitamin E can inhibit the oxidative degradation of plant essential oils, prolonging the shelf life of the composition; magnesium stearate as an anti-caking agent can prevent the composition from caking, ensuring product uniformity. The synergistic effect of the three ensures that the active ingredients in the composition are not lost during production, storage and use.

[0017] Example 1: The composition is composed of the following raw materials by weight: Honeysuckle chlorogenic acid extract 8 parts, forsythia glycoside extract 5 parts, oregano essential oil 2 parts, lactobacillus acidophilus 4 parts, lactobacillus rhamnosus 2 parts, nano zinc oxide 1 part, maltodextrin 30 parts, vitamin E 0.5 parts, and magnesium stearate 0.3 parts.

[0018] Example 2: The composition is composed of the following raw materials by weight: Honeysuckle chlorogenic acid extract 5 parts, forsythia glycoside extract 3 parts, oregano essential oil 1 part, lactobacillus acidophilus 2 parts, lactobacillus rhamnosus 1 part, nano zinc oxide 0.5 parts, maltodextrin 20 parts, vitamin E 0.2 parts, and magnesium stearate 0.1 parts.

[0019] Example 3: The composition is composed of the following raw materials by weight: Honeysuckle chlorogenic acid extract 12 parts, forsythia glycoside extract 8 parts, oregano essential oil 4 parts, lactobacillus acidophilus 6 parts, lactobacillus rhamnosus 3 parts, nano zinc oxide 2 parts, malt dextrin 40 parts, vitamin E 0.8 parts, magnesium stearate 0.5 parts.

[0020] The production method of the composition is as follows: Step 1: extraction and purification of natural plant active components; Preparation of Honeysuckle chlorogenic acid extract: Take 100 kg of dried honeysuckle medicinal materials (ground to 20 mesh) and add 8 times the amount of 50% ethanol aqueous solution. Reflux extraction at 60°C for 2 times, 2 hours each time; Combine the two extraction liquids and filter them using a plate and frame filter (filter cloth pore size 0.22 μm) to remove the residue; Concentrate the filtrate under reduced pressure (vacuum degree -0.08 MPa, temperature 55°C) to a relative density of 1.20 (measured at 60°C) extract; Dissolve the extract in 3 times the amount of purified water, then adsorb it with AB-8 macroporous resin (flow rate 2 BV / h). After adsorption, rinse the resin with 2 BV of purified water, then elute it with 70% ethanol aqueous solution (flow rate 1 BV / h) and collect the eluate; Concentrate the eluate under reduced pressure again, freeze-dry it (temperature -50°C, vacuum degree 10 Pa), and grind it after drying to obtain chlorogenic acid extract with a purity of ≥90%, which is ready for use.

[0021] Preparation of forsythia glycoside extract: Take 80 kg of dried forsythia fruit (ground to 20 mesh) and add 10 times the amount of 60% ethanol aqueous solution. Ultrasonic extraction at 70°C (power 300 W) for 3 times, 1.5 hours each time; Combine the three extraction liquids and centrifuge (3000 r / min, 15 min) to remove the precipitate; Take the supernatant and adsorb it with HPD-100 macroporous resin (flow rate 1.5 BV / h). After rinsing the resin with 3 BV of purified water, elute it with 80% ethanol aqueous solution (flow rate 1 BV / h) and collect the eluate; Concentrate the eluate under reduced pressure (vacuum degree -0.09 MPa, temperature 60°C), then spray dry it (inlet temperature 180°C, outlet temperature 80°C) to obtain forsythia glycoside extract with a purity of ≥85%, which is ready for use.

[0022] Purification of oregano essential oil: Take 100 kg of fresh oregano whole grass, cut it into sections and perform water vapor distillation (distillation temperature 100°C, distillation time 4 hours) to collect the distillate; Extract the distillate with petroleum ether (boiling range 60-90°C) 3 times and combine the organic phases; The organic phase was dried by adding 0.5% anhydrous sodium sulfate for 24 h, filtered, and then distilled under reduced pressure (vacuum degree -0.08 MPa, temperature 40°C) to remove petroleum ether to obtain oregano essential oil with a carvacrol content of ≥70%, which was used as needed.

[0023] Step 2: Microencapsulation of probiotics To improve the survival rate of probiotics during production and storage, the present application uses a "sodium alginate-chitosan" double-layer microencapsulation method, the specific steps are as follows: Core material preparation: Take 4 kg of Lactobacillus acidophilus powder with a viable count of ≥1×10¹¹ CFU / g and 2 kg of Lactobacillus rhamnosus powder with a viable count of ≥1×10¹¹ CFU / g, add 10 kg of 2% sodium alginate aqueous solution (40°C constant temperature), stir at a speed of 500 r / min for 30 min to form a uniform bacterial suspension (i.e. core material).

[0024] Primary embedding (sodium alginate layer): The core material is dropped into a 3% calcium chloride aqueous solution through a peristaltic pump (flow rate 5 mL / min), the drop head diameter is 0.8 mm, and it is placed at 25°C for 30 min to solidify, forming sodium alginate microspheres with a particle size of 1-2 mm.

[0025] Secondary embedding (chitosan layer): The sodium alginate microspheres are transferred into a 0.5% chitosan aqueous solution (pH 5.5), and stirred at a speed of 200 r / min for 20 min at 25°C to form a thin film of chitosan on the surface of the microspheres; Drying and solidification: The double-layer microcapsules are washed twice with purified water, drained, and then vacuum freeze-dried (temperature -45°C, vacuum degree 15 Pa) to obtain probiotic microcapsule powder with a viable count of ≥8×10 10 CFU / g, which is used as needed.

[0026] Step 3: Surface modification of nano zinc oxide To reduce the agglomeration of nano zinc oxide and improve its compatibility with other components, surface modification treatment of nano zinc oxide is required, the specific steps are as follows: Take 1 kg of nano zinc oxide with a particle size of 50-100 nm, add 5 kg of purified water, and ultrasonic disperse (power 500 W) for 30 min to form a nano zinc oxide suspension; Add 0.05 kg of silane coupling agent (KH-550) to the suspension, stir at a speed of 800 r / min for 1 h at 60°C; Centrifuge (5000 r / min, 20 min) to collect the precipitate, vacuum dry (temperature 80°C, vacuum degree -0.07 MPa) the precipitate for 2 h, and then crush it after drying to obtain modified nano zinc oxide, which is used as needed.

[0027] Step 4: Mixing and granulation of the composition Premixing: According to the preferred weight proportions (8kg of honeysuckle chlorogenic acid extract, 5kg of forsythoside extract, 2kg of oregano essential oil, 6kg of probiotic microcapsule powder, 1kg of modified nano zinc oxide, 30kg of maltodextrin, 0.5kg of vitamin E, and 0.3kg of magnesium stearate), first add the maltodextrin, vitamin E, and magnesium stearate to a three-dimensional mixer (model SYH-100) and mix at a speed of 15r / min for 10min; Secondary mixing: Add honeysuckle chlorogenic acid extract, forsythoside extract and modified nano zinc oxide to the mixer and continue mixing for 15 minutes; Final mixing: Dilute the oregano essential oil with a small amount of anhydrous ethanol (volume ratio 1:1), spray it evenly into the mixture, and turn on the mixer at the same time to mix for 20 minutes to ensure that the oregano essential oil is evenly dispersed in the material; Granulation: The mixed materials are transferred to a gyratory pellet mill (model YK-160) and granulated using a 16-mesh sieve. The pellets are then placed in a fluidized bed dryer (model FG-100) for drying (inlet air temperature 60℃, outlet air temperature 40℃), controlling the moisture content of the pellets to ≤3%. Granulation and Packaging: The dried granules are granulated using an 18-mesh sieve to remove fine powder. The qualified granules are then packed into aluminum-plastic composite bags (to protect against light and moisture) and vacuum-packed to obtain the final product.

[0028] Experimental examples and comparative examples: (a) Experimental materials; Experimental animals: SPF-grade female ICR mice, 6-8 weeks old, weighing 20±2g, totaling 180 mice. The housing environment was controlled at a temperature of 23±2℃ and a humidity of 50±5%, using a 12-hour light-dark cycle. The mice had free access to food and water.

[0029] Pathogenic bacteria: Escherichia coli O157:H7 standard strain (ATCC43895), provided by the China Institute of Veterinary Drug Control. The strain was inoculated into LB liquid medium and cultured at 37℃ and 180 rpm for 12 h with shaking. The bacterial concentration was then adjusted to 1×10⁻⁶. 9 CFU / mL, for later use.

[0030] Experimental drugs: The composition of the present invention (prepared according to Example 1, denoted as "Experimental Group A"); Compositions with different ratios (Example 2, Example 3) (referred to as "Experimental Group B" and "Experimental Group C"); Comparative drugs (referred to as "Comparative Examples 1-5").

[0031] Detection reagent: E. coli selective medium (MacConkey agar), mouse IL-6 (interleukin-6) ELISA kit, mouse TNF-α (tumor necrosis factor-α) ELISA kit.

[0032] (II) Experimental grouping and treatment; 180 mice were randomly divided into 10 groups, 20 mice in each group, and the specific grouping and treatment methods are as follows: Blank control group: normal feeding, not infected with E. coli, and not given any drug; Model control group: infected with E. coli O157:H7, and not given drug; Experimental group A: after infection with E. coli, the preferred composition of the present application was administered by gavage at a dose of 1 g / kg of body weight, once a day, for 7 consecutive days; Experimental group B: after infection with E. coli, the composition of "honeysuckle chlorogenic acid extract 5 parts + forsythia glycoside extract 3 parts + oregano essential oil 1 part + lactobacillus acidophilus 2 parts + lactobacillus rhamnosus 1 part + nano zinc oxide 0.5 part + malt dextrin 20 parts + vitamin E 0.2 part + magnesium stearate 0.1 part" was administered by gavage at a dose of 1 g / kg of body weight, once a day, for 7 consecutive days; Experimental group C: after infection with E. coli, the composition of "honeysuckle chlorogenic acid extract 12 parts + forsythia glycoside extract 8 parts + oregano essential oil 4 parts + lactobacillus acidophilus 6 parts + lactobacillus rhamnosus 3 parts + nano zinc oxide 2 parts + malt dextrin 40 parts + vitamin E 0.8 part + magnesium stearate 0.5 part" was administered by gavage at a dose of 1 g / kg of body weight, once a day, for 7 consecutive days; Comparative example 1: after infection with E. coli, "honeysuckle chlorogenic acid extract 8 parts + forsythia glycoside extract 5 parts + oregano essential oil 2 parts + malt dextrin 30 parts + vitamin E 0.5 part + magnesium stearate 0.3 part" (not containing probiotics and nano zinc oxide) was administered by gavage at a dose of 1 g / kg of body weight, once a day, for 7 consecutive days; Comparative example 2: after infection with E. coli, "lactobacillus acidophilus 4 parts + lactobacillus rhamnosus 2 parts + malt dextrin 30 parts + vitamin E 0.5 part + magnesium stearate 0.3 part" (not containing plant extracts and nano zinc oxide) was administered by gavage at a dose of 1 g / kg of body weight, once a day, for 7 consecutive days; Comparative example 3: after infection with E. coli, "nano zinc oxide 1 part + malt dextrin 30 parts + vitamin E 0.5 part + magnesium stearate 0.3 part" (not containing plant extracts and probiotics) was administered by gavage at a dose of 1 g / kg of body weight, once a day, for 7 consecutive days; Comparative example 4: after infection with E. coli, amoxicillin (commercial veterinary antibiotic) was administered by gavage at a dose of 50 mg / kg of body weight, once a day, for 7 consecutive days; Example 5: After infection of E. coli, the commercially available "Honeysuckle-Lactobacillus compound preparation" was administered orally at a dose of 1 g / kg of body weight, once a day, for 7 consecutive days.

[0033] Infection model establishment: On the first day of the experiment, all mice except the blank control group were fasted for 12 hours without water restriction, and then 0.2 mL of E. coli O157:H7 bacterial solution (concentration 1 x 10 9 CFU / mL) was administered orally to establish a mouse E. coli infection model. After 24 hours of bacterial administration, the corresponding drugs were administered to the mice in each experimental group.

[0034] (Three) detection index and method General state observation of mice: The mental state (lively / lethargic), appetite (food intake changes), and diarrhea (stool characteristics, diarrhea rate) of mice were observed daily, and the number of deaths in each group was recorded to calculate the mortality rate.

[0035] Intestinal E. coli number determination: On the 7th day of the experiment, 10 mice were randomly selected from each group and sacrificed by cervical dislocation. 0.1 g of cecal contents was collected aseptically, 0.9 mL of sterile normal saline was added, and it was diluted to 10⁻ 6 , 10⁻ 7 , 10⁻ 8 times, 100 μL of the diluted solution was taken and spread on MacConkey agar medium, and incubated at 37°C for 24 hours. The number of red colonies (typical E. coli colonies) was counted, and the CFU value of E. coli per gram of cecal contents was calculated (expressed as lgCFU / g).

[0036] Changes in mouse body weight: The body weight of mice was measured on the 1st day (before infection), the 4th day, and the 7th day of the experiment, and the body weight gain rate of mice in each group was calculated (body weight gain rate = (final body weight - initial body weight) / initial body weight x 100%).

[0037] Detection of intestinal inflammatory factor levels: On the 7th day of the experiment, after the mice were sacrificed, 0.5 g of small intestinal mucosa was collected aseptically, 4.5 mL of normal saline was added, and it was homogenized and centrifuged (3000 r / min, 10 min). The supernatant was taken, and the content of IL-6 and TNF-α was detected according to the operation steps of the ELISA kit instruction manual.

[0038] Intestinal flora diversity analysis: The cecal contents were collected, total DNA was extracted, and 16S rRNA gene sequencing technology (Illumina MiSeq platform) was used to analyze the intestinal flora alpha diversity (including Shannon index, Simpson index) to evaluate the effect of the composition on intestinal flora balance.

[0039] (Four) experimental results and analysis; General state observation results of mice (Table 1)

[0040] Analysis: After the model control group mice were infected with E. coli, obvious mental depression, decreased appetite, diarrhea and death were observed, indicating that the E. coli infection model was successfully established; the diarrhea rate (10%-15%) and mortality rate (0%) of the mice in the experimental groups A and C were significantly lower than those in the experimental group B (diarrhea rate 30%, mortality rate 5%) and each comparative example, and the mental state and appetite recovery of the mice were better, which indicated that the composition with the preferred ratio (experimental group A) had the best improvement effect on the clinical symptoms caused by E. coli infection in mice.

[0041] Intestinal E. coli quantity determination results (Table 2);

[0042] Analysis: The number of E. coli in the cecum of the model control group mice was significantly higher than that of the blank control group (P<0.01); the number of E. coli in the cecum of the mice in the experimental group A (6.58±0.28 lg CFU / g) was close to that of the blank control group, and was 3.29 lg CFU / g lower than that of the model control group, although it was slightly higher than that of comparative example 4 (antibiotic group), but there was no statistical difference (P>0.05); the inhibition effect of the experimental group A on E. coli was significantly better than that of the experimental groups B and C and comparative examples 1-3 and 5 (P<0.01), which indicated that the composition with the preferred ratio could effectively reduce the number of E. coli in the intestine, and the bacteriostatic effect was close to that of antibiotics, and there was no risk of drug resistance.

[0043] Mouse weight change results (Table 3)

[0044] Analysis: The model control group mice showed obvious weight loss (7-day weight gain rate was-10.97%) due to E. coli infection; the 7-day weight gain rate of the mice in the experimental group A (10.31%) was lower than that of the blank control group (14.71%), but was significantly higher than that of the experimental groups B and C and each comparative example (P<0.01), and there was no statistical difference with comparative example 4 (antibiotic group) (P>0.05), which indicated that the composition with the preferred ratio could effectively alleviate the weight loss caused by E. coli infection and promote the growth of mice.

[0045] Intestinal inflammatory factor level detection results (Table 4);

[0046] Analysis: The IL-6 and TNF-a levels in the intestinal mucosa of the model control group mice were significantly increased (P<0.01), indicating that the E. coli infection triggered a severe intestinal inflammatory response; the IL-6 (25.68±4.12 pg / mL) and TNF-a (22.53±3.57 pg / mL) levels in the intestinal mucosa of the mice in the experimental group A were close to those in the blank control group and were significantly lower than those in the experimental groups B and C and the respective comparative examples (P<0.01), and were not statistically different from those in comparative example 4 (antibiotic group) (P>0.05), which indicates that the composition with the preferred ratio can effectively inhibit the intestinal inflammatory response and protect the intestinal mucosal barrier.

[0047] Results of intestinal flora diversity analysis (Table 5)

[0048] Analysis: The higher the Shannon index and the closer the Simpson index to 1, the richer the intestinal flora diversity. The intestinal flora diversity of the model control group mice was significantly reduced (P<0.01); the Shannon index (3.58±0.18) and Simpson index (0.89±0.02) of the intestinal flora of the mice in the experimental group A were slightly lower than those in the blank control group, but were significantly higher than those in the experimental groups B and C and the respective comparative examples (P<0.01) and were much higher than those in comparative example 4 (antibiotic group), which indicates that the composition with the preferred ratio can effectively protect the intestinal flora diversity while inhibiting the growth of E. coli, avoiding the problem of intestinal flora imbalance caused by antibiotics.

[0049] Comparative example analysis and optimal ratio determination (I) Comparative examples 1-3: antibacterial effect of single category component The experimental results of comparative example 1 (containing only plant extracts), comparative example 2 (containing only probiotics), and comparative example 3 (containing only nano zinc oxide) are as follows: Comparative example 3 had the worst inhibitory effect on E. coli (only reducing the number of E. coli in the intestine by 0.66 lg CFU / g) and the mice had a significant decrease in body weight (body weight gain rate -12.23%), which indicates that the use of nano zinc oxide alone has limited antibacterial effect at a low dose and cannot alleviate the symptoms caused by E. coli infection. The antibacterial effects of comparative examples 1 and 2 were better than that of comparative example 3, but were still significantly lower than that of experimental group A (P<0.01), and the mice had a high diarrhea rate (45%-50%) and intestinal inflammatory factor level, which indicates that the use of plant extracts or probiotics alone has certain antibacterial effect, but cannot form a synergistic effect and cannot effectively control E. coli infection.

[0050] (II) Experimental groups B and C and experimental group A: differences in effects of different ratios The comparison results of experimental group B (low-dose component), experimental group C (high-dose component) and experimental group A (optimal ratio) show that: Experimental group B has lower dosage of each active component, and the inhibition effect on E. coli (the number of E. coli in the intestine decreases by 1.95 lg CFU / g), the diarrhea rate of mice (30%) and the weight gain rate (1.09%) are all significantly different from those of experimental group A (P<0.01), which indicates that insufficient dosage of components will weaken the synergistic effect among the components; Experimental group C has higher dosage of each active component, and the inhibition effect on E. coli (the number of E. coli in the intestine decreases by 3.02 lg CFU / g) is close to that of experimental group A, but the intestinal flora diversity (Shannon index 3.32) is slightly lower than that of experimental group A, and the production cost is higher (high-dose components increase the raw material cost by about 30%), which indicates that too high dosage of components will not only not significantly improve the inhibition effect, but also increase the production cost, and even may have a slight impact on intestinal flora.

[0051] (Three) Comparative Examples 4-5: Comparison with the effects of existing products; The experimental results of Comparative Example 4 (antibiotic) and Comparative Example 5 (commercially available composite preparation) show that: The inhibition effect of Comparative Example 4 (amoxicillin) on E. coli (the number of E. coli in the intestine decreases by 3.42 lg CFU / g) is close to that of experimental group A, but the intestinal flora diversity (Shannon index 2.85) is much lower than that of experimental group A, and there is a risk of drug resistance, which cannot be used for a long time; The inhibition effect of Comparative Example 5 (commercially available honeysuckle-lactic acid bacteria composite preparation) on E. coli (the number of E. coli in the intestine decreases by 2.14 lg CFU / g) and the diarrhea rate of mice (40%) are both significantly different from those of experimental group A (P<0.01), which indicates that the component matching and ratio of the existing commercially available composite preparation are unreasonable, the synergistic effect among the components is weak, and the overall effect is limited.

[0052] (Four) Optimal ratio determination; According to the above analysis, the preferred weight ratio (experimental group A) of the present application is: honeysuckle chlorogenic acid extract 8 parts, forsythia nucifera extract 5 parts, oregano essential oil 2 parts, lactobacillus acidophilus 4 parts, lactobacillus rhamnosus 2 parts, nano zinc oxide 1 part, malt dextrin 30 parts, vitamin E 0.5 parts, and magnesium stearate 0.3 parts, which has the following significant advantages: Significant inhibition effect: can effectively reduce the number of E. coli in the intestine (decrease by 3.29 lg CFU / g), and the effect is close to that of antibiotics; Obvious symptom improvement: can make the diarrhea rate of mice only 10%, the mortality rate 0%, and the weight gain rate 10.31%, and the intestinal inflammation factor level returns to normal; Intestinal protection is good: while inhibiting E. coli, it can protect the diversity of intestinal flora and avoid intestinal flora imbalance; Reasonable cost: the dosage of each component is moderate, the raw material cost is lower than that of the experimental group C, and there is no risk of antibiotic resistance.

[0053] The E. coli inhibiting composition designed in the application has the following significant beneficial effects compared with the prior art through the innovative compound mode of "natural plant extract + functional probiotics + synergistic minerals" and the optimized ratio: No drug resistance risk, high safety: all components of the composition are natural plant extracts, probiotics and food-grade minerals, and do not contain antibiotic ingredients, so long-term use will not cause E. coli to develop drug resistance. The LD 50 >5g / kg, which belongs to the actual non-toxic level and can be safely applied in the fields of aquaculture, food industry and clinical auxiliary treatment.

[0054] Stable bacteriostatic effect and strong synergy: the "triple bacteriostatic network" formed by plant extracts (destroying cell membrane structure, inhibiting strain toxicity), probiotics (competing for nutrients, secreting organic acids to inhibit bacteria) and nano zinc oxide (destroying cell membrane through oxidative stress, enhancing the activity of other components) has significant synergistic effect among components, and the inhibitory effect on E. coli is close to that of antibiotics, and can effectively reduce the pathogenicity of the strain, solving the problem of poor bacteriostatic effect of single component.

[0055] Protecting intestinal barrier and maintaining flora balance: while inhibiting the growth of E. coli, probiotics and plant extracts can stimulate the secretion of mucin and IgA in intestinal mucosa, enhance the function of intestinal barrier, and will not destroy the diversity of intestinal flora (Shannon index reaches 3.58), avoiding the problem of intestinal flora imbalance caused by antibiotics.

[0056] Wide application range and convenient application: the composition can be made into granules, powders, oral liquids and other dosage forms, which can be applied in aquaculture (preventing and controlling E. coli disease in livestock and poultry) through water drinking and feed adding, used as food preservative (inhibiting E. coli contamination in food), and used as dietary supplement for human intestinal health maintenance (auxiliary treatment of mild E. coli infection).

[0057] Mature production process, easy to industrialize: the extraction, microencapsulation, mixing and granulation processes used in the production process are mature food / drug processing methods, the required equipment is easy to obtain, and the operation is simple. Through microencapsulation and surface modification treatment, the stability of probiotics and nano zinc oxide is effectively improved, the shelf life of the product can reach more than 18 months, and industrial production is easy to realize.

[0058] The E. coli inhibiting composition provided by the application can effectively inhibit the growth of E. coli (make the number of E. coli in the intestinal tract decrease by 3.29 lg CFU / g), relieve the symptoms caused by E. coli infection (diarrhea rate 10%, mortality rate 0%), protect the intestinal barrier function and intestinal flora diversity, and is safe and has no drug resistance risk. The ratio of "8 parts of honeysuckle chlorogenic acid extract + 5 parts of forsythia glycoside extract + 2 parts of oregano essential oil + 4 parts of lactobacillus acidophilus + 2 parts of lactobacillus rhamnosus + 1 part of nano zinc oxide + 30 parts of malt dextrin + 0.5 part of vitamin E + 0.3 part of magnesium stearate" is the optimal ratio.

[0059] Although embodiments of the application have been shown and described, it is to be understood that the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A composition having inhibitory effect on Escherichia coli, characterized by comprising, The composition consists of the following raw materials by weight parts: Honeysuckle chlorogenic acid extract 5-12 parts, forsythia glycoside extract 3-8 parts, oregano essential oil 1-4 parts, lactobacillus acidophilus 2-6 parts, lactobacillus rhamnosus 1-3 parts, nano zinc oxide 0.5-2 parts, malt dextrin 20-40 parts, vitamin E 0.2-0.8 parts, and magnesium stearate 0.1-0.5 parts.

2. The composition for inhibiting Escherichia coli according to claim 1, wherein The composition consists of the following raw materials by weight parts: Honeysuckle chlorogenic acid extract 8 parts, forsythia glycoside extract 5 parts, oregano essential oil 2 parts, lactobacillus acidophilus 4 parts, lactobacillus rhamnosus 2 parts, nano zinc oxide 1 part, malt dextrin 30 parts, vitamin E 0.5 parts, and magnesium stearate 0.3 parts.

3. The composition for inhibiting E. coli according to claim 1, wherein The composition consists of the following raw materials by weight parts: Honeysuckle chlorogenic acid extract 5 parts, forsythia glycoside extract 3 parts, oregano essential oil 1 part, lactobacillus acidophilus 2 parts, lactobacillus rhamnosus 1 part, nano zinc oxide 0.5 part, malt dextrin 20 parts, vitamin E 0.2 parts, and magnesium stearate 0.1 parts.

4. The composition for inhibiting E. coli according to claim 1, wherein The composition consists of the following raw materials by weight parts: Honeysuckle chlorogenic acid extract 12 parts, forsythia glycoside extract 8 parts, oregano essential oil 4 parts, lactobacillus acidophilus 6 parts, lactobacillus rhamnosus 3 parts, nano zinc oxide 2 parts, malt dextrin 40 parts, vitamin E 0.8 parts, and magnesium stearate 0.5 parts.

5. The composition for inhibiting E. coli according to claim 1, wherein The production method of the composition is as follows: Step 1: extraction and purification of natural plant active components; Preparation of honeysuckle chlorogenic acid extract: Take 100 kg of dried honeysuckle medicinal materials, add 8 times the amount of 50% ethanol aqueous solution, and extract under constant temperature reflux at 60℃ for 2 times, each for 2 h; Combine the two extraction liquids and filter to remove the dregs using a plate and frame filter; Concentrate the filtrate under reduced pressure to a relative density of 1.20; Add 3 times the amount of purified water to the extract to dissolve, then adsorb with AB-8 macroporous resin, rinse the resin with 2 BV of purified water after adsorption is complete, then elute with 70% ethanol aqueous solution and collect the eluate; Concentrate the eluate under reduced pressure again, freeze-dry, and pulverize after drying to obtain chlorogenic acid extract with a purity of ≥90%, which is ready for use; Preparation of forsythia glycoside extract: Take 80 kg of dried forsythia fruit, add 10 times the amount of 60% ethanol aqueous solution, and ultrasonically extract 3 times at 70℃, each for 1.5 h; Combine the three extraction liquids and centrifuge to remove the precipitate; Take the supernatant and adsorb it with HPD-100 macroporous resin, rinse the resin with 3 BV of purified water, then elute with 80% ethanol aqueous solution and collect the eluate; Concentrate the eluate under reduced pressure, then spray dry to obtain forsythia glycoside extract with a purity of ≥85%, which is ready for use; Purification of oregano essential oil: Take 100 kg of fresh oregano whole grass, cut it into sections, and perform water vapor distillation to collect the distillate; Extract the distillate with petroleum ether 3 times and combine the organic phases; Add 0.5% anhydrous sodium sulfate to the organic phase and dry for 24 h, then filter and distill under reduced pressure (remove petroleum ether) to obtain oregano essential oil with a thymol content of ≥70%, which is ready for use; Step 2: microencapsulation treatment of probiotics; Core material preparation: 4 kg of Lactobacillus acidophilus powder with a viable count of ≥1×10¹¹ CFU / g, 2 kg of Lactobacillus rhamnosus powder with a viable count of ≥1×10¹¹ CFU / g, 10 kg of 2% sodium alginate aqueous solution, stirring at 500 r / min for 30 min to form a uniform bacterial suspension; Primary embedding: the core material is dropped into 3% calcium chloride aqueous solution through a peristaltic pump, the drop head diameter is 0.8 mm, and the sodium alginate microspheres with a particle size of 1-2 mm are formed by standing and solidifying at 25°C for 30 min; Secondary embedding: the sodium alginate microspheres are transferred into 0.5% chitosan aqueous solution, and the chitosan forms a thin film on the surface of the microspheres by stirring at 200 r / min for 20 min at 25°C; Dry solidification: the double-layer microcapsules were washed twice with purified water, and then vacuum freeze-dried after draining to obtain probiotic microcapsule powder with a viable count of ≥8×10 10 CFU / g, ready for use; Step 3: surface modification of nano zinc oxide; In order to reduce the agglomeration of nano zinc oxide and improve its compatibility with other components, surface modification treatment of nano zinc oxide is required, and the specific steps are as follows: Take 1 kg of nano zinc oxide with a particle size of 50-100 nm, add 5 kg of purified water, and ultrasonic dispersion for 30 min to form a nano zinc oxide suspension; Add 0.05 kg of silane coupling agent to the suspension, stir at 800 r / min at 60°C for 1 h; Centrifugal collection of the precipitate, vacuum drying of the precipitate for 2 h, and crushing after drying to obtain modified nano zinc oxide for standby; Step 4: mixing and granulation of the composition; Premixing: according to the weight parts, first add malt dextrin, vitamin E and magnesium stearate into a three-dimensional mixer, and mix at 15 r / min for 10 min; Secondary mixing: add honeysuckle chlorogenic acid extract, forsythia glycoside extract and modified nano zinc oxide into the mixer, and continue mixing for 15 min; Final mixing: dilute the oregano essential oil with a small amount of anhydrous ethanol, and uniformly spray it onto the mixed materials, while starting the mixer, and mixing for 20 min to ensure uniform dispersion of the oregano essential oil in the materials; Granulation: transfer the mixed materials into a swing granulator, and granulate with a 16 mesh screen, then dry the granules in a fluidized bed dryer, and control the moisture content of the granules to ≤3%; Whole granulation and packaging: use a 18 mesh screen to whole granulate the dried granules, remove the fine powder, then pack the qualified granules into aluminum-plastic composite bags, and vacuum package to obtain the final product.

6. A product for inhibiting E. coli, characterized in that, A composition for inhibiting E. coli according to any one of claims 2-4. A composition for inhibiting E. coli according to any one of claims 2-4.

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