CN104719671A - Compound essential oil for preventing diarrhea of piglets as well as preparation method and application of compound essential oil - Google Patents
Compound essential oil for preventing diarrhea of piglets as well as preparation method and application of compound essential oil Download PDFInfo
- Publication number
- CN104719671A CN104719671A CN201510132914.8A CN201510132914A CN104719671A CN 104719671 A CN104719671 A CN 104719671A CN 201510132914 A CN201510132914 A CN 201510132914A CN 104719671 A CN104719671 A CN 104719671A
- Authority
- CN
- China
- Prior art keywords
- essential oil
- compound essential
- oil
- piglets
- diarrhea
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The invention provides compound essential oil for preventing diarrhea of piglets as well as a preparation method and an application of the compound essential oil. The compound essential oil is prepared from the following components in parts by mass: 0.25-2.5 parts of cinnamon oil, 0.1-1 part of clove oil, 0.25-2.5 parts of thymol and 100 parts of activated attapulgite. The invention also provides a preparation method and an application of the compound essential oil. The compound essential oil can obviously reduce the diarrhea rate of weaned piglets, improve the food intake, improve the daily gain, improve the intestinal flora state, and meanwhile can prevent diarrhea of weaned piglets at the early stage and enhance the growth. The compound essential oil effectively reduces the amount of escherichia coli of intestinal tracts, improves the intestinal flora state, attapulgite can adsorb viruses, pathogenic bacteria and toxin in digestive tracts, inhibit the growth of harmful bacteria of the intestinal tracts, enhance the growth of beneficial bacteria, and recover and maintain the ecological balance of intestinal flora.
Description
Technical field
The invention belongs to field of feed additive technology, be specifically related to compound essential oil of a kind of prevention of diarrhea in piglets and its preparation method and application.
Background technology
In modern Swine Production, feeding piglet is one of link becoming most critical.It is improve sow productivity that piglet carries out early weaning, improve the effective way of Swine Production economic benefit, but early-weaned piglets is very easily suffered from diarrhoea, add various stress, result in high incidence and the case fatality rate of piglet, bring about great losses to Swine Production.Large quantifier elimination shows, the reason that diarrhea of weaned piglets occurs has pathogenicity and avirulence two aspects, it is irritated that avirulence reason mainly comprises daily ration antigen, not building of ablactation stress and piglet physiological development is complete, wherein, it is the most basic that daily ration antigen allergy causes intestinal mucosa to damage, the most direct reason, the infection of pathogenic microorganism and scabies secondary infection exacerbate the incidence of disease and diarrhoea degree that early-weaned piglets suffers from diarrhoea, after diarrhoea occurs, avirulence factor and causative factors often act on piglet simultaneously, diarrhoea is made to become serious and complicated, due to the serious harm of early-weaned piglets diarrhoea to Swine Production, make to seem especially important to its prevention.At present, additive for weanling pig has zinc oxide, antibiotic, enzyme preparation, acidulant and oligosaccharides, paddy ammonia phthalein amine etc., comparatively general to the use of antibiotic and high zinc in production practices, but, while bringing huge effect, create the problem such as drug resistance and medicament residue to antibiotic product; And, day by day receive publicity at food sanitation safe, the control of environmental pollution comes into one's own day by day, under the social environment that safety, green, environmental protection livestock products are praised highly day by day, antibiotic, Gao Xin etc. have generally using of the additive of adverse effect to be undoubtedly consumer's misgivings in the heart to livestock products quality or environment, this can damage the sound development of farming industry virtually.Therefore, find and research high-quality, safety and efficiently Substitutes For Antibiotic be a development trend of present feed supplement industry.Many scholars are devoted to the feed addictive research of novel antibacterial sterilization in recent years, especially safe natural plant extracts feed addictive.
Summary of the invention
In order to solve problems of the prior art, the invention provides the compound essential oil of the non-stimulated prevention of diarrhea in piglets of a kind of pure natural, for this reason, the present invention also provides its preparation method and application.
The invention provides a kind of compound essential oil of prevention of diarrhea in piglets, described compound essential oil is prepared from by each component of following mass fraction: cinnamon oil 0.25-2.5 part, caryophyllus oil 0.1-1 part, Thymol 0.25-2.5 part, activation attapulgite 100 parts.
Cinnamon oil is the volatile oil that the dry hide of camphor tree section plant Chinese cassia tree and bark obtain through steam distillation, and Chinese cassia tree main body of oil is cinnamic acid, has stronger inhibitory action to the growth of bacterium, yeast and mould.
Caryophyllus oil is famous and precious medicine, is by the volatile oil of the dry flower of plant clove of myrtaceae Eugeniacaryophyllata Thumb. through steam distillation gained.There is effect of warm stomach, warm kidney, control swollen, the hiccup of stomach cold pain, vomiting and diarrhoea, numbness pain, colic, toothache, halitosis etc.Main containing eugenol (Egenol) in caryophyllus oil, its content is more than 80%.
Thymol (5-methyl-2-isopropyl-phenol), has another name called thymol, is present in the essential oil of various plants.Thymol is used for making spices, anticorrisive agent, medical bactericide and ascarifuge agent, and Recent study shows that Thymol has and kills the multiple biologically actives such as mite is antibacterial.
Above three kinds of essential oils, are approved as plant protection product by European Union, have antibacterial and oxidation resistant effect, can be used as essence and anticorrisive agent etc.Because above Chemical Composition of The Essential Oil is unstable, also water insoluble, be therefore easily destroyed in production process or lose.Adopt cyclodextrin wrapper technology, be made into essential oil clathrate, can improve stability and the availability of essential oil, although improve the quality of essential oil formulation, but widely using in production practices, stability is inadequate, and effect is also undesirable.Compound essential oil of the present invention is by producing actual summary for many years, and carries out scientific composition, determined by market application widely.Adsorbed by active attapulgite stone, obtain stable feed and add vegetable active additive.Less to the MIC of 2 boar source pathogenic bacteria, and compare with the antibacterial effect of folk prescription essential oil, its antibacterial effect is better than folk prescription essential oil, is a kind of excellent natural plant feed additives.
Attapulgite is a kind of crystalloid hydrous magnesium aluminium silicate mineral, has unique layer chain structure feature, in its structure, there is crystal lattice, the Na containing non-quantitative in side crystal
+, Ca
2+, Fe
3+, Al
3+, crystal is needle-like, threadiness or fiber collection shape.Attapulgite has unique dispersion, high temperature resistant, the colloidal nature that anti-saline and alkaline grade is good and higher adsorption capacity, it can adsorb essential oil, prepare new plant feed additive, have pollution-free, the feature of noresidue, the prevention and treatment of attapulgite to piglet enterogastric diseases have no relevant report, the present invention's application attapulgite absorption essential oil prepares compound essential oil for the preparation of anti-grice diarrhoea medicine and feed addictive, widely use in husbandry sector at present, and obtain good effect, for prevention and the antibiotic of early-weaned piglets diarrhoea, the perfect substitution of high zinc or the research of Some substitute product provide a kind of effectively product, also for further investigate attapulgite and the exploitation of compound formulation provides more theoretical foundations.
Preferably, described compound essential oil is prepared from by each component of following mass fraction: cinnamon oil 1.25 parts, caryophyllus oil 0.8 part, Thymol 1.25 parts, activation attapulgite 100 parts.
The present invention also provides the preparation method of above-mentioned compound essential oil, is the cinnamon oil of formula ratio, caryophyllus oil, Thymol are added ethanol, at room temperature adds in the activation attapulgite of formula ratio, and vibration, suction filtration is drying to obtain.
3rd object of the present invention is to provide the application of above-mentioned compound essential oil in the medicine preparing prevention of diarrhea in piglets.
4th object of the present invention is to provide the application of above-mentioned compound essential oil in preparation suppression Escherichia coli and/or detection of Salmonella medicine.
5th object of the present invention is to provide the application of above-mentioned compound essential oil in the feed addictive preparing prevention of diarrhea in piglets.
6th object of the present invention is to provide a kind of feed addictive of prevention of diarrhea in piglets, containing the compound essential oil described in claim 1 or 2 in described feed addictive.
7th object of the present invention is to provide a kind of feed of prevention of diarrhea in piglets, and described feed is made up of the compound essential oil described in 96-98wt% basal diet and 2-4wt% claim 1 or 2.
When the mass ratio of basal diet and compound essential oil is 97:3, the best results of prevention of diarrhea in piglets.
8th object of the present invention is to provide the detection method of active ingredient in above-mentioned composite essential oil, it is characterized in that: be detect by high performance liquid chromatography, testing conditions is: with methanol-water-glacial acetic acid (60: 4: 2, volume ratio) as mobile phase, with the speed sample introduction of 1.0mL/min, under the wavelength of 274 nm, detect chromatogram value.Chromatographic column: Diamonsil C18; Sampling volume is 20 μ L; Column temperature: 30 DEG C.
The present invention adds 2-4% in basal diet, and composite essential oil of the present invention can significantly reduce diarrhea of weaned piglets rate, promotes feed intake, improves daily gain, improves gut flora state, the better effects if of simultaneously suffer from diarrhoea to early-weaned piglets prevention and growth promoting effects.
Test of the present invention shows, composite essential oil of the present invention effectively reduces the colibacillary quantity of enteron aisle, improve gut flora state, this may be because attapulgite is by the absorption to virus, pathogen, toxin in alimentary canal, inhibit the growth of harmful intestinal tract bacteria, facilitate the growth of beneficial bacterium, recover, maintain the ecological balance of gut flora.
Detailed description of the invention
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is commercially available.
Test material:
Cinnamon oil, caryophyllus oil, Thymol (Guangdong Yun Hua Industrial Co., Ltd. produces, product batch number: 2014051105,2014081022,201401009) above-mentioned three kinds of material our company are at public offering; Activation attapulgite: Gansu Kai Di biotech company produces.
embodiment 1
The formula of compound essential oil of the present invention is as follows:
Cinnamon oil 0.25g, caryophyllus oil 0.5g, Thymol 2.5g, activation attapulgite 100g.
The preparation method of compound essential oil of the present invention is:
By cinnamon oil 0.25g, caryophyllus oil 0.5g, Thymol 2.5g adds 50ml ethanol, at room temperature adds in the activation attapulgite of 100g, and vibration 10h, suction filtration, 40 DEG C are drying to obtain.
embodiment 2
The formula of compound essential oil of the present invention is as follows:
Cinnamon oil 1.25g, caryophyllus oil 0.8g, Thymol 1.25g, activation attapulgite 100g.
The preparation method of compound essential oil of the present invention is:
By cinnamon oil 1.25g, caryophyllus oil 0.8g, Thymol 1.25g adds 50ml ethanol, at room temperature adds in the activation attapulgite of 100g, and vibration 10h, suction filtration, 40 DEG C are drying to obtain.
embodiment 3
The formula of compound essential oil of the present invention is as follows:
Cinnamon oil 2.5g, caryophyllus oil 1g, Thymol 0.25g, activation attapulgite 100g.
The preparation method of compound essential oil of the present invention is:
By cinnamon oil 2.5g, caryophyllus oil 1g, Thymol 0.25g adds 50ml ethanol, at room temperature adds in the activation attapulgite of 100g, and vibration 10h, suction filtration, 40 DEG C are drying to obtain.
embodiment 4
The formula of compound essential oil of the present invention is as follows:
Cinnamon oil 0.25g, caryophyllus oil 0.1g, Thymol 2.5g, activation attapulgite 100g.
The preparation method of compound essential oil of the present invention is:
By cinnamon oil 0.25g, caryophyllus oil 0.1g, Thymol 2.5g adds 50ml ethanol, at room temperature adds in the activation attapulgite of 100g, and vibration 10h, suction filtration, 40 DEG C are drying to obtain.
embodiment 5
The formula of compound essential oil of the present invention is as follows:
Cinnamon oil 1g, caryophyllus oil 0.3g, Thymol 1.5g, activation attapulgite 100g.
The preparation method of compound essential oil of the present invention is:
By cinnamon oil 1g, caryophyllus oil 0.3g, Thymol 1.5g adds 50ml ethanol, at room temperature adds in the activation attapulgite of 100g, and vibration 10h, suction filtration, 40 DEG C are drying to obtain.
comparative example 1
Essential oil: cinnamon oil 4g, activation attapulgite 100g.Cinnamon oil 4g is added 50ml ethanol, at room temperature adds in the activation attapulgite of 100g, vibration 10h, suction filtration, 40 DEG C are drying to obtain.
comparative example 2
Essential oil: caryophyllus oil 4g, activation attapulgite 100g.Caryophyllus oil 4g is added 50ml ethanol, at room temperature adds in the activation attapulgite of 100g, vibration 10h, suction filtration, 40 DEG C are drying to obtain.
comparative example 3
Essential oil: Thymol 4g, activation attapulgite 100g.Thymol 4g is added 50ml ethanol, at room temperature adds in the activation attapulgite of 100g, vibration 10h, suction filtration, 40 DEG C are drying to obtain.
embodiment 6
Compound essential oil of the present invention is tested as the feeding effect of feed addictive
1.1 experimental animal
Select healthy, body weight is close, the CRP breed system B system weanling pig 63 of age in days close (28 ± 2 days), formal feeding period 21 days.
1.2 experimental design and daily ration
Adopt single factor test completely randomized design, experimental animal is divided at random 7 process, 3 repetitions are established in each process, each repetition 3 pigs, and the setting of test process and the quantity arrangement of animal refer to table 1.
Table 1 experimental design
Experimental basis daily ration, with reference to Chinese bacon hogs feeding standard, designs in conjunction with pig farm actual conditions, and experimental basis daily ration is powder, and basal diet formula and trophic level are in table 2.
Table 2 basal diet formula and trophic level
1.3 feeding and management
Test pig is raised in nursery house, and the feeding piglet of same repetition is in a circle, and powder is fed, free choice feeding and drinking-water, sweeps health in house every day, rinses water fountain, by normal immunological program immunity, carries out the observed and recorded of test piglet every day.Animal experiment carries out on breeding pig farm, Yangjiang, Guangdong.
1.4 test index
1.4.1 daily gain, feed intake and feedstuff-meat ratio
After on-test, claim initial weight on an empty stomach morning, then within every two weeks, weigh 1 time, ramming material 1 time, adds up feed intake in units of repeating, and calculates daily gain and feedstuff-meat ratio.Result is see table 3.
Table 3 compound essential oil of the present invention is on the impact of Production Performance of Weaning Pigs
1.4.1.1 average daily ingestion amount (ADFI)
From table 4: comparatively control group is high for the average daily ingestion amount (ADFI) of embodiment 3 groups.
1.4.1.2 average daily gain (ADG)
Result shows: the daily gain of embodiment 2 is the highest, and test group is all apparently higher than control group.
1.4.1.3 feedstuff-meat ratio (F/G)
Result shows: the feedstuff-meat ratio of embodiment 2 is minimum, and test group is lower than control group.
1.4.2 Scours index and diarrhea rate
Within experimental period, every day observes grice diarrhoea situation, record diarrhoea piglet head number, and gives different marks according to the situation of ight soil, and in units of repeating, statistics Scours index, calculates diarrhea rate, specifically see table 4.Be that 4 points, 5 points persons are defined as diarrhoea by faecal scores.
The standards of grading of table 4 Scours index
The computing formula of Scours index and diarrhea rate is as follows:
Scours index=stools scored sum/test pig's head number
Diarrhea rate=diarrhoea pig's head number of days/test pig's head number of days × %.Concrete outcome is see table 5.
Table 5 compound essential oil of the present invention is on the impact of diarrhea of weaned piglets
Result shows: the 1st week Scours index, embodiment 2 groups is minimum.And test group comparatively control group is low.
2nd week Scours index, embodiment 2 groups is minimum.And test group comparatively control group is low.
3rd week Scours index, embodiment 2 groups is minimum.And test group comparatively control group is low.
1.4.3 faecal bacterial community counting
Bacterial species: Escherichia coli, Bacillus acidi lactici
L the preparation of () selective medium: buy corresponding selective medium, prepare rear heating according to operation instruction and dissolve, then autoclaving is for subsequent use.
(2) sample: often repeat to randomly draw 1 sodium selenite the 15th day morning in test, with pretreated clean polybag, fresh excreta is got in sterile working, sends laboratory back to immediately.
(3) dilute: after laboratory transported back by sample, on superclean bench, take about 1.0g excrement sample in sterile test tube, add first prepare and sterilized dilution 9ml, magnetic force oscillator shakes 3-5 minute, and this is 10
-1, leave standstill a period of time, fill in the test tube of 9ml sterile diluent with micropipette rifle accurate Aspirate supernatant 1 ml and carry out 10
-2dilution, after vibration, then carries out 10 successively
-3-10
-7doubly dilution.
(4) inoculation and cultivation:
Escherichia coli: after the culture medium of well in advance acts on 12h in 37 DEG C of incubators, be placed in aseptic superclean bench.By high dilution, get excrement sample 10 respectively with micro liquid sampler
-4-10
-6dilution 0.1ml coating is inoculated in (each dilution factor establishes 3 repetitions) on E. coli selectable culture medium (Yihong methylene blue culture medium) plate, puts aerobic in 37 DEG C of incubators and cultivates 24 hours.
Bacillus acidi lactici: after the culture medium of well in advance acts on 12h in 37 DEG C of incubators, be placed in aseptic superclean bench.By high dilution, get excrement sample 10 respectively with micro liquid sampler
-5-10
-7times dilution 0.1ml coating is inoculated in Bacillus acidi lactici selective medium (LBS) upper (each dilution factor establishes 3 repetitions), puts aerobic in 37 DEG C of incubators and cultivates 48 hours.
(5) Bacteria Identification and counting: after incubation time arrives, identifies bacterium according to flora and cellular morphology, Gram's staining.On the methylene blue flat board of Yihong, most E. coli clones is dark purple black, circular, neat in edge, and smooth surface is moistening, often has metallic luster; Gram's staining is negative; Rod-short in the blunt circle in two ends under microscope.Bacillus acidi lactici is the petite of milky slightly projection on LBS culture medium, and Gram's staining is positive.
To E. coli plate, choose dark purple black or atropurpureus, smooth surface, projection, neat in edge, quality is soft, sticky, and the bacterium colony of diameter 1.0-3.0mm counts.To Bacillus acidi lactici, choose milky, slightly partially yellow, smooth surface, projection, quality is soft, and the bacterium colony of diameter 0.6-2.7mm counts.
Colony counting General Requirements, has 30 or more to count by each plate, finally averages as the experimental record number of bacterium.
In 1g sample, the computing formula of total number of bacteria is:
Bacterial population=record number × extension rate × 1000/100
Unit is designated as: CFU/g.
Obtain bacterium number contained by every gram of sample, finally use the logarithm (Log of bacterium number in every gram of fresh excrement sample
10cfu/g) represent.Concrete outcome is see table 6.
Table 6 compound essential oil of the present invention is on the impact of microorganism species
Note: Microflora lgCFU/g excrement sample represents
Result shows: embodiment 2 groups of coliform counts are minimum.Test group comparatively control group is low.And breast/big-and-middle, embodiment 2 groups is the highest, and test group is all higher than control group.
More than detect and complete at academy of agricultural sciences of Guangdong Province veterinary institute.
The above results adopts average ± standard deviation mode to be described, and test data adopts spssl0.0 software to carry out variance analysis,
If significant difference adopts DuncanShi method to carry out Multiple range test.
2 discuss
This test shows, adds 3% composite essential oil of the present invention and can significantly reduce diarrhea of weaned piglets rate, promote feed intake, improve daily gain, improve gut flora state in basal diet, the better effects if of simultaneously suffer from diarrhoea to early-weaned piglets prevention and growth promoting effects.
This test shows, composite essential oil of the present invention effectively reduces the colibacillary quantity of enteron aisle, improve gut flora state, this may because attapulgite be by the absorption to virus, pathogen, toxin in alimentary canal, inhibit the growth of harmful intestinal tract bacteria, promote the growth of beneficial bacterium, recover, maintain the ecological balance of gut flora.
embodiment 7
The bacteriostatic test of compound essential oil of the present invention and effective substance method use
Cinnamon oil is the volatile oil that the dry hide of camphor tree section plant Chinese cassia tree and bark obtain through steam distillation, and Chinese cassia tree main body of oil is cinnamic acid, has stronger inhibitory action to the growth of bacterium, yeast and mould.Caryophyllus oil main component is eugenol and thymol etc., can suppress and kill multiple-microorganism.Thyme linaloe oil is thymic extract, and main component is Thymol and carvacrol, is approved as plant protection product by European Union, has antibacterial and oxidation resistant effect, can be used as essence and anticorrisive agent etc.Minimum-bacteriostat mass concentration (MIC) can be used as the evaluation index of plants essential oil (EO) antibacterial action power, and the method being usually used in measuring MIC has disc diffusion method, agar dilution, broth dilution method and optical densitometric method etc.Microplate trace broth dilution method comparatively is accurately selected in this test, measure cinnamon oil, caryophyllus oil, Thymol and their main component cinnamic acid, eugenol and Thymol to the MIC of 2 boar source pathogenic bacteria, and compare with the antibacterial effect of colistin sulfate, for natural
The theoretical foundation that the development and utilization of plant feed additive provides.
1 materials and methods
1.1 material
1.1.1 essential oil and reagent
The compound essential oil (cinnamon oil: caryophyllus oil: Thymol=1.25:0.8:1.25) of cinnamon oil, cinnamic acid, caryophyllus oil, eugenol, Thymol, embodiment 2; Nutrient broth fluid nutrient medium, Beijing extensive and profound in meaning star biotechnology responsibility Co., Ltd; MH agar (MHA); Resazurin, Sigma company.
1.1.2 bacterial classification
For examination bacterial classification pig source enterotoxigenic escherichia coli K88 and pig source cholera detection of Salmonella A72, provided by Hua Zhong Agriculture University's veterinary college.
1.1.3 instrument
High performance liquid chromatograph (Waters 1525 type, joins Waters2489 detector).
1.2 method
1.2.1 essential oil Contents of Main Components detects
Accurately measure 1mL cinnamic acid and eugenol, Thymol respectively, take 1g and be respectively placed in a 100mL volumetric flask, with chromatogram methyl alcohol it is diluted to respectively cinnamic aldehyde content is 1%, eugenol content be 1% and Thymol mass concentration be the storing solution of 10 μ g/mL.With methyl alcohol, cinnamic acid is diluted to the standard items of 0.0001,0.0005,0.001,0.005,0.01 and 0.02 μ g/mL; Eugenol is become to be diluted to the standard items of 0.005,0.01,0.05,0.1,0.25 and 0.5 μ g/mL; Standard items Thymol being diluted to 1.0,5.0,10.0,50.0,100.0 and 250.0 μ g/mL carry out Criterion curve.Getting 0.1mL sample is dissolved in 250mL volumetric flask, ultrasonic through 30min, crosses 0.22 μm of filter membrane, makes testing sample.With methanol-water-glacial acetic acid (60: 4: 2, volume ratio) as mobile phase, with the speed sample introduction of 1.0mL/min, under the wavelength of 274 nm, detect chromatogram value.Chromatographic column: Diamonsil C18; Sampling volume is 20 μ L; Column temperature: 30 DEG C.
Take peak area as ordinate, the mass concentration of cinnamic acid, eugenol and Thymol is abscissa drawing standard curve, and calculates regression equation:
Cinnamic acid: Y=13404X+25928, R
2=0.999, good in 0.01-0.4 μ L/mL linear relationship.
Eugenol: Y=15123X+22670, R
2=0.999, good in 0.01-0.4 μ L/mL linear relationship.
Thymol: Y=14825X-15883, R
2=0.999, good in 5-500 μ g/mL linear relationship.
Cinnamon oil, caryophyllus oil and Thymol are diluted to suitable quality concentration, detect according to above-mentioned condition and method, calculate the content of main component in each essential oil.See table 7.
The Contents of Main Components of table 7 cinnamon oil, caryophyllus oil and Thymol
1.2.2 the preparation of antibacterial liquid
By through 2 kinds of essential oils (cinnamon oil and caryophyllus oil) of 0.22 μm of filtration sterilization and 3 main components (cinnamic acid, eugenol and Thymol) and composite essential oil of the present invention according to essential oil: Tween 80: dinitro sulfoxide=2: the volume ratio mixing of 1: 1, vortex 1min is made into the liquid that volume fraction is 1/2.Getting 102.4 μ L volume fractions is the liquid of 1/2, is diluted to 4mL, vortex 1min with the nutrient broth fluid nutrient medium through autoclave sterilization, and making quality of liquid medicine concentration is 12800 μ g/L.Adopting 2 times of dilution methods quality of liquid medicine concentration to be diluted respectively is again the mass concentrations stand-by (through verification experimental verification, in liquid, the mass concentration of Tween 80 and dinitro sulfoxide is less than the result that 1% can not affect bacteriostatic test) such as 12800,6400,3200,1600,800,400,200 and 100 μ g/L.
1.2.3 the activation of bacterial classification and the preparation of bacterium liquid
Respectively E.coli K88 and detection of Salmonella A72 are inoculated into nutrient broth fluid nutrient medium, in 37 DEG C of shaking tables, cultivate 12h with the concussion of the speed of 150r/min; From the bacterium liquid obtained, get 10 μ L join in 5mL nutrient broth fluid nutrient medium with identical CMC model 12h; Stand-by after again expanding cultivation once with the same terms.
Adopt colony counting method, dilute bacteria suspensions by 10 times of dilution methods after the bacterium liquid obtaining activating fully being shaken vortex 1min, draw 10
-6, 10
-7with 10
-8the each 100 μ L of bacteria suspension, are coated on aseptic Letheen Broth culture medium uniformly, put into 37 DEG C of constant incubators and cultivate 12h, and each gradient 2 repetition, selects best plate count, and calculate the clump count in every milliliter of bacteria suspension.Again by the clump count calculated, bacterium liquid is diluted to 500,000 CFU stand-by.
1.2.4 the preparation of indicator
Accurately take 0.025g resazurin, be dissolved in 100mL ultra-pure water, through autoclave sterilization, the aseptic indicator making 0.25g/L mass concentration is stand-by.
1.2.5 bacteriostatic test method
Resazurin indicator micro-dilution method is adopted to measure the Antibacterial Activity of compound essential oil prepared by cinnamon oil, cinnamic acid, caryophyllus oil, eugenol, Thymol and embodiment 2.First in 96 porocyte culture plates of sterilizing, each hole adds the resazurin indicator of 2 μ L, 0.25g/L, again according to the order from high concentration to low concentration, add the antibacterial liquid that 100 μ L have diluted successively, each mass concentration does 3 longitudinal direction repetitions side by side.Then get 3 holes and only add 100 μ L culture mediums in contrast, 3 holes only add the bacterium liquid that 100 μ L have diluted and do positive control, and 3 holes only add 100 μ L liquids and do negative control.In each test hole, add the bacterium liquid of 500,000 CFU that 100 μ L have diluted again, add 100 μ L nutrient broths in contrast, positive control and each hole of negative control and supply volume, be consistent with test group.For liquid evaporation in reduce orifice, with aseptic preservative film, plate is sealed again after covering 96 orifice plate lids.The 96 porocyte culture plates made are placed in 37 DEG C of constant incubators cultivate, observed result after 24h.
Result judges: resazurin solution, as blue, will become redness when there being bacterial growth.After constant temperature culture, the minimum drug quality concentration without colony growth is MIC.Concrete outcome is see table 8.
Several liquid minimum inhibitory concentration of table 8 (μ g/L)
As seen from Table 7: 3 kinds of essential oils and the comprehensive bacteriostasis of main component to 2 boar source pathogenic bacteria thereof are: cinnamic acid > cinnamon oil > Thymol >=eugenol and caryophyllus oil.The composite essential oil of the present embodiment 2 is 200 μ g/L to the MIC for examination bacterium.The MIC of cinnamon oil and cinnamic acid is 1/4 and 1/2 of the composite essential oil of embodiment 2; The antibacterial MIC of Thymol is 1/8 of the composite essential oil of embodiment 2; The antibacterial MIC of eugenol and caryophyllus oil is 1/8 and 1/16 of the composite essential oil of embodiment 2.
Cinnamon oil is identical with the fungistatic effect that cinnamic acid shows K88 with A72, is respectively 800 and 400 μ g/L to the MIC of E.coli K88 and detection of Salmonella A82, and what show to play bacteriostasis in cinnamon oil is cinnamic acid.Eugenol is identical for the MIC of examination bacterium to 2 strains, is 1600 μ g/L, and caryophyllus oil is different to the MIC of 2 strain bacterium, is respectively 1600 and 3200 μ g/L.The bacteriostasis of Thymol to 2 strain bacterium is 1600 μ g/L.And the composite essential oil antibacterial activity of the embodiment of the present invention 2 is far away higher than monomer whose oil, may be have certain coordinative role between Thymol or Multiple components, this also needs further research.
The high performance liquid chromatography of the present invention by setting up, realizes detecting while 3 compositions in a system.The method is highly sensitive, reproducible, finding speed is fast and simple to operate, practical under experimental condition.Under detection method, in volatile oil, each component obtains fully effective separation, reaches the requirement to wherein Major Components.This test has only been done the quantitative detection for main composition in examination essential oil, does not detect all the components, has requirement if follow-up to essential oil component, the method for gas chromatography mass spectrometry can be utilized to analyze.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. a compound essential oil for prevention of diarrhea in piglets, is characterized in that: described compound essential oil is prepared from by each component of following mass fraction: cinnamon oil 0.25-2.5 part, caryophyllus oil 0.1-1 part, Thymol 0.25-2.5 part, activation attapulgite 100 parts.
2. compound essential oil according to claim 1, is characterized in that: described compound essential oil is prepared from by each component of following mass fraction: cinnamon oil 1.25 parts, caryophyllus oil 0.8 part, Thymol 1.25 parts, activation attapulgite 100 parts.
3. the preparation method of the compound essential oil described in claim 1 or 2, is characterized in that: be that the cinnamon oil of formula ratio, caryophyllus oil, Thymol are added ethanol, at room temperature add in the activation attapulgite of formula ratio, and vibration, suction filtration is drying to obtain.
4. the application of the compound essential oil described in claim 1 or 2 in the medicine preparing prevention of diarrhea in piglets.
5. the compound essential oil described in claim 1 or 2 suppresses the application in Escherichia coli and/or detection of Salmonella medicine in preparation.
6. the application of the compound essential oil described in claim 1 or 2 in the feed addictive preparing prevention of diarrhea in piglets.
7. a feed addictive for prevention of diarrhea in piglets, is characterized in that: containing the compound essential oil described in claim 1 or 2 in described feed addictive.
8. a feed for prevention of diarrhea in piglets, is characterized in that: described feed is made up of the compound essential oil described in 96-98wt% basal diet and 2-4wt% claim 1 or 2.
9. the detection method of active ingredient in the composite essential oil described in claim 1 or 2, it is characterized in that: be detect by high performance liquid chromatography, testing conditions is: with methanol-water-glacial acetic acid (60: 4: 2, volume ratio) as mobile phase, with the speed sample introduction of 1.0mL/min, under the wavelength of 274 nm, detect chromatogram value;
Chromatographic column: Diamonsil C18; Sampling volume is 20 μ L; Column temperature: 30 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510132914.8A CN104719671B (en) | 2015-03-25 | 2015-03-25 | A kind of compound essential oil of prevention of diarrhea in piglets and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510132914.8A CN104719671B (en) | 2015-03-25 | 2015-03-25 | A kind of compound essential oil of prevention of diarrhea in piglets and its preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104719671A true CN104719671A (en) | 2015-06-24 |
| CN104719671B CN104719671B (en) | 2018-03-06 |
Family
ID=53444534
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510132914.8A Active CN104719671B (en) | 2015-03-25 | 2015-03-25 | A kind of compound essential oil of prevention of diarrhea in piglets and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104719671B (en) |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105146676A (en) * | 2015-08-07 | 2015-12-16 | 北京工商大学 | Escherichia coli inhibitor |
| CN107927349A (en) * | 2017-12-01 | 2018-04-20 | 四川黑虎零抗农业科技有限公司 | Nonreactive composite essential oil preparation |
| CN108077591A (en) * | 2018-01-16 | 2018-05-29 | 浙江大学 | A kind of livestock and poultry disease-resistant growth-promoting feed addictive and preparation method |
| CN108077600A (en) * | 2018-01-16 | 2018-05-29 | 浙江大学 | A kind of promotion animal and bird intestines health feed addictive and preparation method |
| CN109393183A (en) * | 2018-11-23 | 2019-03-01 | 湖南晶天科技实业有限公司 | A kind of feed addictive, its application and a kind of feed |
| CN109549001A (en) * | 2018-12-26 | 2019-04-02 | 无锡正大生物股份有限公司 | A kind of suckling piglet nanometer coating plants essential oil and preparation method thereof |
| CN110574831A (en) * | 2018-06-08 | 2019-12-17 | 湖南晶天科技实业有限公司 | Application of cinnamic aldehyde and/or allantoin in replacement of high zinc in piglet feed, feed additive or additive premix and feed |
| CN110574837A (en) * | 2018-06-08 | 2019-12-17 | 湖南晶天科技实业有限公司 | Application of paeonol in replacing high zinc in piglet feed, feed additive or additive premix and feed |
| CN111328918A (en) * | 2020-04-10 | 2020-06-26 | 中国科学院兰州化学物理研究所盱眙凹土应用技术研发中心 | Preparation method of attapulgite-based pH response antibacterial material |
| CN111617137A (en) * | 2020-06-30 | 2020-09-04 | 山东康裕生物科技有限公司 | Preparation method of eucalyptus oil and peppermint oil compound spray |
| CN112471347A (en) * | 2020-12-02 | 2021-03-12 | 安徽省农业科学院畜牧兽医研究所 | Composite plant extract additive for improving pig production performance and oxidation resistance |
| CN115136963A (en) * | 2022-07-13 | 2022-10-04 | 武汉天楚生物科技有限公司 | Acidic dry powder disinfectant and preparation method and application thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108378217A (en) * | 2018-01-19 | 2018-08-10 | 广东科贸职业学院 | A kind of natural plant essential oils compound formulation and its preparation method and application |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102038842A (en) * | 2010-12-01 | 2011-05-04 | 北京资源亚太动物药品有限公司 | Medicament for improving production performance of pigs and preparation method thereof |
| CN102987093A (en) * | 2012-10-29 | 2013-03-27 | 安佑（中国）动物营养研发有限公司 | Feeding plant essential oil additive as well as preparation and application of additive |
| CN101331915B (en) * | 2007-06-29 | 2013-04-03 | 帝斯曼知识产权资产管理有限公司 | Animal feed composition |
| CN103392922A (en) * | 2013-07-19 | 2013-11-20 | 安佑生物科技集团有限公司 | Compound feed additive capable of improving health level of animal |
-
2015
- 2015-03-25 CN CN201510132914.8A patent/CN104719671B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101331915B (en) * | 2007-06-29 | 2013-04-03 | 帝斯曼知识产权资产管理有限公司 | Animal feed composition |
| CN102038842A (en) * | 2010-12-01 | 2011-05-04 | 北京资源亚太动物药品有限公司 | Medicament for improving production performance of pigs and preparation method thereof |
| CN102987093A (en) * | 2012-10-29 | 2013-03-27 | 安佑（中国）动物营养研发有限公司 | Feeding plant essential oil additive as well as preparation and application of additive |
| CN103392922A (en) * | 2013-07-19 | 2013-11-20 | 安佑生物科技集团有限公司 | Compound feed additive capable of improving health level of animal |
Non-Patent Citations (1)
| Title |
|---|
| 窦茂鑫: "饲料添加剂肉桂油中肉桂醛含量的测定", 《饲料研究》 * |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105146676A (en) * | 2015-08-07 | 2015-12-16 | 北京工商大学 | Escherichia coli inhibitor |
| CN107927349A (en) * | 2017-12-01 | 2018-04-20 | 四川黑虎零抗农业科技有限公司 | Nonreactive composite essential oil preparation |
| CN108077591A (en) * | 2018-01-16 | 2018-05-29 | 浙江大学 | A kind of livestock and poultry disease-resistant growth-promoting feed addictive and preparation method |
| CN108077600A (en) * | 2018-01-16 | 2018-05-29 | 浙江大学 | A kind of promotion animal and bird intestines health feed addictive and preparation method |
| CN110574831A (en) * | 2018-06-08 | 2019-12-17 | 湖南晶天科技实业有限公司 | Application of cinnamic aldehyde and/or allantoin in replacement of high zinc in piglet feed, feed additive or additive premix and feed |
| CN110574837A (en) * | 2018-06-08 | 2019-12-17 | 湖南晶天科技实业有限公司 | Application of paeonol in replacing high zinc in piglet feed, feed additive or additive premix and feed |
| EP3804529A4 (en) * | 2018-06-08 | 2022-03-23 | Hunan Jingtian Technology Industry Co., Ltd. | Application of cinnamaldehyde and/or allantoin to replace high zinc amounts in suckling piglet feed, feed additive or additive premix, and feed |
| CN109393183A (en) * | 2018-11-23 | 2019-03-01 | 湖南晶天科技实业有限公司 | A kind of feed addictive, its application and a kind of feed |
| CN109549001A (en) * | 2018-12-26 | 2019-04-02 | 无锡正大生物股份有限公司 | A kind of suckling piglet nanometer coating plants essential oil and preparation method thereof |
| CN111328918A (en) * | 2020-04-10 | 2020-06-26 | 中国科学院兰州化学物理研究所盱眙凹土应用技术研发中心 | Preparation method of attapulgite-based pH response antibacterial material |
| CN111328918B (en) * | 2020-04-10 | 2022-04-08 | 中国科学院兰州化学物理研究所 | Preparation method of attapulgite-based pH response antibacterial material |
| US11700849B2 (en) | 2020-04-10 | 2023-07-18 | Lanzhou Institute Of Chemical Physics, Chinese Academy Of Sciences | Method for preparation of attapulgite-based pH-responsive antibacterial material |
| CN111617137A (en) * | 2020-06-30 | 2020-09-04 | 山东康裕生物科技有限公司 | Preparation method of eucalyptus oil and peppermint oil compound spray |
| CN112471347A (en) * | 2020-12-02 | 2021-03-12 | 安徽省农业科学院畜牧兽医研究所 | Composite plant extract additive for improving pig production performance and oxidation resistance |
| CN115136963A (en) * | 2022-07-13 | 2022-10-04 | 武汉天楚生物科技有限公司 | Acidic dry powder disinfectant and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104719671B (en) | 2018-03-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104719671B (en) | A kind of compound essential oil of prevention of diarrhea in piglets and its preparation method and application | |
| CN1883275B (en) | A botanical antibiotic and application thereof | |
| CN103168965B (en) | Anti-stress synbiotics traditional Chinese medicine composition applicable to chicken and preparation method of anti-stress synbiotics traditional Chinese medicine composition | |
| CN103875916B (en) | Chinese herbal medicine type feed mould inhibitor | |
| CN107744062A (en) | A kind of tea tree oil microemulsion Animal nutrition agent and preparation method thereof | |
| CN110314178B (en) | Pure natural compound essential oil with broad-spectrum bactericidal activity and preparation and application thereof | |
| CN110100965A (en) | A kind of derma type essential oil organic acid composite feed additive and its preparation method and application | |
| CN105287790B (en) | A kind of fevervine extract and its application | |
| CN107836466A (en) | A kind of tea tree ethereal oil natural disinfectant agent for animals and preparation method thereof | |
| CN106386853A (en) | Multi-effect active iodine disinfectant as well as preparation method and application thereof | |
| CN103783089B (en) | Compound Chinese herbal medicine disinfectant for tableware and kitchenware disinfection | |
| CN101411394A (en) | Use method of Schisandra chinensis as feedstuff additive | |
| JP2018509161A (en) | Archaea in bioactive animal feed, method for producing the composition, and method using the composition | |
| CN103039723A (en) | Broiler feed additive and preparation method thereof | |
| CN103190544B (en) | Duckling anti-stress synbiotics and traditional Chinese medicine composition and preparation method of composition | |
| CN103655684A (en) | Traditional Chinese herbal medicine microecological preparation capable of killing and inhibiting foot and mouth disease virus in vitro and in vivo | |
| CN1903254A (en) | Compound medicine for treating wind-heat type cold and preparing method thereof | |
| CN102939966A (en) | Compound disinfectant, preparation method and application thereof | |
| CN109843067A (en) | Composition comprising Duddingtonia flagrans | |
| CN107115549B (en) | A kind of application of livestock and poultry cultivation air modifying agent in the drug for preventing and treating porcine respiratory disease is prepared | |
| CN103190545B (en) | Gosling anti-stress synbiotics and traditional Chinese medicine composition and preparation method of composition | |
| CN105713855A (en) | Strains, application of strains, vaccine and preparation method of vaccine | |
| CN103975957A (en) | Preparation method of mixed formula preparation for controlling brown planthopper | |
| CN115804807B (en) | Traditional Chinese medicine compound composition for preventing and treating mycoplasma ovipneumoniae and preparation method and application thereof | |
| CN113456655B (en) | New application of propyl gallate synergistic anti-streptococcus suis antibiotic |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |