CN104719671B - A kind of compound essential oil of prevention of diarrhea in piglets and its preparation method and application - Google Patents
A kind of compound essential oil of prevention of diarrhea in piglets and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of compound essential oil of prevention of diarrhea in piglets, and the compound essential oil is prepared by each component of following mass fraction：100 parts 0.25 2.5 parts of cinnamon oil, 0.1 1 parts of caryophyllus oil, 0.25 2.5 parts of Thymol, activation of attapulgite.The present invention also provides its preparation method and application.The composite essential oil of the present invention can significantly reduce diarrhea of weaned piglets rate, promote feed intake, improve daily gain, improve gut flora state, while suffer from diarrhoea prevention with promoting the effect of growth more preferable to early-weaned pigletses.The composite essential oil of the present invention effectively reduces the quantity of enteron aisle Escherichia coli, improve gut flora state, this is probably because attapulgite passes through the absorption to virus, pathogen, toxin in alimentary canal, inhibit the growth of harmful intestinal tract bacteria, the growth of beneficial bacterium is promoted, recovers, maintain the ecological balance of gut flora.
Description
Technical field
The invention belongs to field of feed additive technology, and in particular to the compound essential oil and its system of a kind of prevention of diarrhea in piglets
Preparation Method and application.
Background technology
In the Swine Production of modernization, feeding piglet is as one of link of most critical.Piglet carries out early weaning
It is to improve sow productivity, improves the effective way of Swine Production economic benefit, but early-weaned pigletses are easily suffered from diarrhoea,
Plus it is various stress, result in the high incidence and case fatality rate of piglet, brought about great losses to Swine Production.Substantial amounts of research table
Bright, there are pathogenicity and the aspect of avirulence two the reason for diarrhea of weaned piglets occurs, and avirulence reason mainly includes daily ration
Antigen allergy, ablactation stress and piglet physiological development do not build it is complete, wherein, daily ration antigen allergy causes intestinal mucosa damage to be
The reason for most basic, most direct, infection and the scabies secondary infection of pathogenic microorganism exacerbate the incidence of disease of early-weaned pigletses diarrhoea
And diarrhoea degree, after diarrhoea occurs, avirulence factor often acts on piglet simultaneously with causative factors, becomes diarrhoea
It is serious and complicated, due to serious harm of the early-weaned pigletses diarrhoea to Swine Production so that the prevention to it seems especially heavy
Will.At present, the additive for weanling pig has zinc oxide, antibiotic, enzyme preparation, acidulant and oligosaccharides, paddy ammonia phthalein amine etc.,
Use in production practices to antibiotic and high zinc is more universal, however, generating confrontation while huge effect is brought
The problems such as drug resistance and medicament residue of raw plain product；Moreover, of increasing concern in food sanitation safe, environmental pollution is prevented
Control and be increasingly subject to pay attention to, safety and environmental friendliness livestock products are increasingly subject under the social environment of high praise, and antibiotic, high zinc etc. are to poultry
The additive that product quality or environment adversely affect generally uses the undoubtedly misgivings in the minds of consumer, and this virtually has
Damage the sound development of farming industry.Therefore, it is that present feed adds to find and study high-quality, safety and efficient Substitutes For Antibiotic
Add a development trend of agent industry.Many scholars are devoted to the feed addictive research of novel antibacterial sterilization in recent years, especially
It is safe natural plant extracts feed addictive.
The content of the invention
In order to solve problems of the prior art, the invention provides a kind of pure natural non-stimulated prevention piglet abdomen
The compound essential oil rushed down, therefore, the present invention also provides its preparation method and application.
The present invention provides a kind of compound essential oil of prevention of diarrhea in piglets, the compound essential oil by following mass fraction each group
Divide and be prepared：Cinnamon oil 0.25-2.5 parts, caryophyllus oil 0.1-1 parts, Thymol 0.25-2.5 parts, activation attapulgite 100
Part.
Cinnamon oil is the volatile oil that the dry hide of camphor tree section plant Chinese cassia tree and bark obtain through steam distillation, the master of cinnamon oil
It is cinnamic acid to want composition, and the growth to bacterium, yeast and mould has stronger inhibitory action.
Caryophyllus oil is rare medicine, is by the dry of plant clove of myrtaceae Eugeniacaryophyllata Thumb.
Dry bud is through the volatile oil obtained by steam distillation.The effect of with warm stomach, warm kidney, control the swollen, hiccup of stomach cold pain, vomiting and diarrhoea, numbness pain,
Colic, toothache, halitosis etc..Eugenol is mainly contained in caryophyllus oil（Egenol）, its content is more than 80%.
Thymol（5- methyl -2- isopropyl-phenols）, also known as thymol, it is present in the essential oil of various plants.In hundred
Fragrant phenol is used for making spices, preservative, medical bactericide and ascarifuge agent, Recent study and shows that Thymol has mite killing
The multiple biological activities such as antibacterial.
Three of the above essential oil, plant protection product is approved as by European Union, there is antibacterial and oxidation resistant effect, can be used as essence and
Preservative etc..Because above Chemical Composition of The Essential Oil is unstable, water is also insoluble in, therefore is more easily damaged or loses in production process
Lose.Using cyclodextrin wrapper technology, essential oil clathrate is made into, but the stability and availability of essential oil are improved, although improving
The quality of essential oil formulation, but widely using in production practices, stability is inadequate, and effect is also undesirable.The compound of the present invention
Essential oil is summarized by produce reality for many years, and carries out scientific composition, is determined by the application of extensive market.It is recessed by activity
Convex rod stone is adsorbed, and obtains stable feed addition vegetable active additive.It is smaller to the MIC of 2 boar source pathogenic bacteria, and
Compared 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
Additive.
Attapulgite is that a kind of crystalloid is hydrated alumina magnesia silicate, has unique layer chain structure feature, in its knot
Crystal lattice in structure be present, help to contain variable amount of Na in crystal+、Ca2+、Fe3+、Al3+, crystal is in needle-like, threadiness or fiber
Gather shape.Attapulgite has good colloidal nature and the higher adsorption capacities such as unique scattered, high temperature resistant, salt resistance alkali,
It can adsorb essential oil, prepare new plant feed additive, have the characteristics of pollution-free, noresidue, attapulgite is to piglet
The prevention and treatment of enterogastric diseases have no relevant report, and the present invention prepares compound essential oil using attapulgite absorption essential oil
For preparing preventing piglet diarrhea medicine and feed addictive, widely use, and obtain well in husbandry sector at present
Effect, the research of prevention and antibiotic, the perfect substitution of high zinc or part substitute for early-weaned pigletses diarrhoea provide
A kind of effective product, also to be furtherd investigate to attapulgite and its exploitation of compound formulation provides more theoretical foundations.
Preferably, the compound essential oil is prepared by each component of following mass fraction：1.25 parts of cinnamon oil, caryophyllus oil
0.8 part, 1.25 parts of Thymol, activation 100 parts of attapulgite.
The present invention also provides the preparation method of above-mentioned compound essential oil, is by the cinnamon oil, caryophyllus oil, Thymol of formula ratio
Ethanol is added, is added at room temperature in the activation attapulgite of formula ratio, vibration, suction filtration is drying to obtain.
Third object of the present invention is to provide application of the above-mentioned compound essential oil in the medicine of prevention of diarrhea in piglets is prepared.
Fourth object of the present invention is to provide above-mentioned compound essential oil and is preparing suppression Escherichia coli and/or detection of Salmonella medicine
In application.
The 5th purpose of the present invention is to provide above-mentioned compound essential oil in the feed addictive of prevention of diarrhea in piglets is prepared
Application.
The 6th purpose of the present invention is to provide a kind of feed addictive of prevention of diarrhea in piglets, in the feed addictive
Contain the compound essential oil described in claim 1 or 2.
The 7th purpose of the present invention is to provide a kind of feed of prevention of diarrhea in piglets, and the feed is by 96-98wt% bases
Compound essential oil composition described in daily ration and 2-4wt% claims 1 or 2.
When the mass ratio of basal diet and compound essential oil is 97:When 3, the best results of prevention of diarrhea in piglets.
The 8th purpose of the present invention is to provide the detection method of active ingredient in above-mentioned composite essential oil, it is characterised in that：
It is to be detected with high performance liquid chromatography, testing conditions are：With methanol-water-glacial acetic acid (60: 4: 2, volume ratio) as flowing
Phase, with 1.0mL/min speed sample introduction, chromatogram value is detected under 274 nm wavelength.Chromatographic column: Diamonsil C18；
Sampling volume is 20 μ L；Column temperature: 30℃.
The present invention adds 2-4% in basal diet, and composite essential oil of the invention can significantly reduce diarrhea of weaned piglets rate,
Promote feed intake, improve daily gain, improve gut flora state, while to early-weaned pigletses diarrhoea prevention with promoting growth
Effect is more preferable.
The experiment of the present invention shows that composite essential oil of the invention effectively reduces the quantity of enteron aisle Escherichia coli, improves
Gut flora state, this is probably because attapulgite is by the absorption to virus, pathogen, toxin in alimentary canal, is inhibited
The growth of harmful intestinal tract bacteria, the growth of beneficial bacterium is promoted, recover, maintain the ecological balance of gut flora.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, it is conventional method unless otherwise specified.Test material used, is city unless otherwise specified in following embodiments
Sell.
Test material：
(Guangdong Yun Hua Industrial Co., Ltd.s produce, product batch number for cinnamon oil, caryophyllus oil, Thymol：2014051105,
2014081022,201401009) above-mentioned three kinds of material our company are in public offering；Activate attapulgite:The triumphant enlightening biology in Gansu
Technology company produces.
Embodiment 1
The formula of the 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：
Cinnamon oil 0.25g, caryophyllus oil 0.5g, Thymol 2.5g are added into 50ml ethanol, add 100g's at room temperature
Activate in attapulgite, vibrate 10h, filter, 40 DEG C are drying to obtain.
Embodiment 2
The formula of the 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：
Cinnamon oil 1.25g, caryophyllus oil 0.8g, Thymol 1.25g are added into 50ml ethanol, add 100g's at room temperature
Activate in attapulgite, vibrate 10h, filter, 40 DEG C are drying to obtain.
Embodiment 3
The formula of the 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：
Cinnamon oil 2.5g, caryophyllus oil 1g, Thymol 0.25g are added into 50ml ethanol, add 100g work at room temperature
Change in attapulgite, vibrate 10h, filter, 40 DEG C are drying to obtain.
Embodiment 4
The formula of the 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：
Cinnamon oil 0.25g, caryophyllus oil 0.1g, Thymol 2.5g are added into 50ml ethanol, add 100g's at room temperature
Activate in attapulgite, vibrate 10h, filter, 40 DEG C are drying to obtain.
Embodiment 5
The formula of the 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：
Cinnamon oil 1g, caryophyllus oil 0.3g, Thymol 1.5g are added into 50ml ethanol, add 100g activation at room temperature
In attapulgite, 10h is vibrated, is filtered, 40 DEG C are drying to obtain.
Comparative example 1
Essential oil：Cinnamon oil 4g, activation attapulgite 100g.Cinnamon oil 4g is added into 50ml ethanol, added at room temperature
In the activation attapulgite for entering 100g, 10h is vibrated, is filtered, 40 DEG C are drying to obtain.
Comparative example 2
Essential oil：Caryophyllus oil 4g, activation attapulgite 100g.Caryophyllus oil 4g is added into 50ml ethanol, added at room temperature
In the activation attapulgite for entering 100g, 10h is vibrated, is filtered, 40 DEG C are drying to obtain.
Comparative example 3
Essential oil：Thymol 4g, activation attapulgite 100g.Thymol 4g is added into 50ml ethanol, in room temperature
In lower addition 100g activation attapulgite, 10h is vibrated, is filtered, 40 DEG C are drying to obtain.
Embodiment 6
The compound essential oil of the present invention is tested as the feeding effect of feed addictive
1.1 experimental animal
Selection health, body weight is close, the CRP breed system B systems weanling pig 63 of age in days close (28 ± 2 days), formal to raise
Support 21 days phases.
1.2 experimental design and daily ration
Using single factor test completely randomized design, experimental animal is randomly divided into 7 processing, each processing sets 3 repetitions, often
Individual 3 pigs of repetition, the setting of test process and the quantity arrangement of animal refer to table 1.
The experimental design of table 1
Experimental basis daily ration designs with reference to pig farm actual conditions with reference to Chinese bacon hogs feeding standard, tests base
Plinth daily ration is powder, and basal diet formula and trophic level are shown in Table 2.
The basal diet formula of table 2 and trophic level
1.3 feeding management
Test swine rearing to feed in a circle, powder in nursery house, the feeding piglet of same repetition, free choice feeding and drinking-water,
Health in house is swept daily, rinses water fountain, by normal immunological program immunity, carries out observing and recording for experiment piglet daily.It is dynamic
Thing experiment is carried out on Guangdong Yangjiang breeding pig farm.
1.4 test index
1.4.1 daily gain, feed intake and feedstuff-meat ratio
Claim initial weight on an empty stomach after morning in on-test, then weigh 1 time within every two weeks, ramming material 1 time, united in units of repetition
Feed intake is counted, and calculates daily gain and feedstuff-meat ratio.As a result referring to table 3.
Influence of the compound essential oil of the present invention of table 3 to Production Performance of Weaning Pigs
1.4.1.1 average daily gain (ADFI)
From table 4:The average daily gain (ADFI) that 3 groups of embodiment is compared with control group height.
1.4.1.2 average daily gain (ADG)
As a result show:The daily gain highest of embodiment 2, test group is obviously higher than control group.
1.4.1.3 feedstuff-meat ratio (F/G)
As a result show:The feedstuff-meat ratio of embodiment 2 is minimum, and test group is less than control group.
1.4.2 Scours index and diarrhea rate
Observe grice diarrhoea situation, record diarrhoea piglet head number daily within experimental period, and given according to the situation of excrement
Different fractions, in units of repetition, Scours index is counted, diarrhea rate is calculated, referring specifically to table 4.By faecal scores be 4 points, 5
Person is divided to be defined as suffering from diarrhoea.
The standards of grading of the Scours index of table 4
The calculation formula of Scours index and diarrhea rate is as follows:
Scours index=stools scored sum/experiment pig's head number
Diarrhea rate=diarrhoea pig's head number of days/experiment pig's head number of days × %.Concrete outcome is referring to table 5.
Influence of the compound essential oil of the present invention of table 5 to diarrhea of weaned piglets
As a result show:1st week Scours index, 2 groups of embodiment are minimum.And test group is low compared with control group.
2nd week Scours index, 2 groups of embodiment are minimum.And test group is low compared with control group.
3rd week Scours index, 2 groups of embodiment are minimum.And test group is low compared with control group.
1.4.3 faecal bacterial community counts
Bacterial species:Escherichia coli, Bacillus acidi lactici
(l) preparation of selective medium:Corresponding selective medium is bought, is heated after being prepared according to operation instruction molten
Change, then autoclaving is standby.
(2) sample:Often repeat to randomly select 1 sodium selenite the 15th day morning in testing, with pretreated clean modeling
Pocket, sterile working take fresh excreta, send laboratory back to immediately.
(3) dilute:After sample transports laboratory back, in weighing about 1.0g excrement samples on superclean bench in sterile test tube, add
Enter dilution 9ml first prepare and sterilized, 3-5 minutes are shaken on magnetic force oscillator, this is 10-1, a period of time is stood,
Filled with the accurate ml of Aspirate supernatant 1 of micropipette rifle in the test tube of 9ml sterile diluents and carry out 10-2Dilution, after vibration, then
10 are carried out successively-3-10-7Dilute again.
(4) inoculation and culture:
Escherichia coli:After the culture medium of well in advance acts on 12h in 37 DEG C of incubators, it is placed in aseptic superclean bench.
By high dilution, excrement sample 10 is taken respectively with micro liquid sampler-4-10-6Dilution 0.1ml coatings are inoculated in Escherichia coli choosing
On selecting property culture medium (Yihong methylene blue culture medium) plate (each dilution factor sets 3 repetitions), aerobic culture 24 in 37 DEG C of incubators is put
Hour.
Bacillus acidi lactici:After the culture medium of well in advance acts on 12h in 37 DEG C of incubators, it is placed in aseptic superclean bench.
By high dilution, excrement sample 10 is taken respectively with micro liquid sampler-5-10-7Times dilution 0.1ml coating is inoculated in Bacillus acidi lactici
On selective medium (LBS) (each dilution factor sets 3 repetitions), aerobic culture 48 hours in 37 DEG C of incubators are put.
(5) Bacteria Identification is with counting:After incubation time arrives, bacterium is entered according to flora and cellular morphology, Gram's staining
Row identification.On the methylene blue flat board of Yihong, most E. coli clones are in dark purple black, circular, neat in edge, and surface is smooth wet
Profit, often with there is metallic luster；Gram's staining is feminine gender；It is in the rod-short of both ends blunt circle under microscope.Bacillus acidi lactici is trained in LBS
It is the petite of milky slightly projection on foster base, Gram's staining is the positive.
To E. coli plate, dark purple black or atropurpureus are chosen, surface is smooth, raised, neat in edge, and quality is soft, viscous,
Diameter 1.0-3.0mm bacterium colony is counted.To Bacillus acidi lactici, milky is chosen, slightly partially yellow, surface is smooth, raised, quality
Soft, diameter 0.6-2.7mm bacterium colony is counted.
Bacterium colony counts general requirement, has 30 or more to be counted by each plate, finally averages as bacterium
Experimental record number.
The calculation formula of total number of bacteria is in 1g samples:
Bacterial population=record number × extension rate × 1000/100
Unit is designated as:CFU/g.
Bacterium number contained by every gram of sample is obtained, finally with the logarithm (Log of bacterium number in every gram of fresh excrement sample10Cfu/g) represent.
Concrete outcome is referring to table 6.
Influence of the compound essential oil of the present invention of table 6 to microorganism species
Note:Microflora is represented with lgCFU/g excrement samples
As a result show:2 groups of coliform counts of embodiment are minimum.Test group is low compared with control group.And breast/big-and-middle, embodiment 2
Group highest, and test group is above control group.
Detection is completed in academy of agricultural sciences of Guangdong Province veterinary institute above.
The above results are described using average ± standard deviation mode, and test data uses spssl0.0 softwares progress side
Difference is analysed,
If significant difference carries out Multiple range test using DuncanShi methods.
2 discuss
This experiment shows that the composite essential oil of 3% present invention is added in basal diet can significantly reduce diarrhea of weaned piglets
Rate, promote feed intake, improve daily gain, improve gut flora state, while the prevention of early-weaned pigletses diarrhoea is given birth to promoting
Long effect is more preferable.
This experiment shows that composite essential oil of the invention effectively reduces the quantity of enteron aisle Escherichia coli, improves enteron aisle
Flora state, this may be because that attapulgite by the absorption to virus, pathogen, toxin in alimentary canal, inhibits enteron aisle to have
The growth of evil bacterium, promotes the growth of beneficial bacterium, recovers, maintains the ecological balance of gut flora.
Embodiment 7
The bacteriostatic test and effective substance method of the compound essential oil of the present invention 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, the master of cinnamon oil
It is cinnamic acid to want composition, and the growth to bacterium, yeast and mould has stronger inhibitory action.Caryophyllus oil main component is eugenol
With thymol etc., it can suppress and kill multiple-microorganism.Thyme linaloe oil is thymic extract, and main component is Thymol
And carvacrol, plant protection product is approved as by European Union, there is antibacterial and oxidation resistant effect, can be used as essence and preservative etc..Most
Small antibacterial mass concentration (MIC) can be usually used in determining MIC as the evaluation index of plants essential oil (EO) antibacterial action power
Method have disc diffusion method, agar dilution, broth dilution method and optical densitometric method etc..This experiment selection is accurately micro-
The micro broth dilution method of plate, measure cinnamon oil, caryophyllus oil, Thymol and their main component cinnamic acid, eugenol and hundred
In fragrant phenol to the MIC of 2 boar source pathogenic bacteria, and be natural compared with the antibacterial effect of colistin sulfate
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
Cinnamon oil, cinnamic acid, caryophyllus oil, eugenol, Thymol, the compound essential oil of embodiment 2（Cinnamon oil：Caryophyllus oil：
Thymol=1.25:0.8:1.25）；Nutrient broth fluid nutrient medium, the extensive and profound in meaning star biotechnology responsibility Co., Ltd in Beijing； MH
Agar (MHA)；Resazurin, Sigma companies.
1.1.2 strain
For trying strain pig source enterotoxigenic escherichia coli K88 and pig source cholera detection of Salmonella A72, by Hua Zhong Agriculture University beast
Medical college provides.
1.1.3 instrument
High performance liquid chromatograph (types of Waters 1525, with Waters2489 detectors).
1.2 method
1.2.1 essential oil Contents of Main Components detects
1mL cinnamic acids and eugenol, Thymol are accurately measured respectively, are weighed 1g and are respectively placed in a 100mL volumetric flask
In, it is diluted to cinnamic aldehyde content is 1%, eugenol content is 1% and Thymol mass concentration is respectively with chromatogram methanol
10 μ g/mL storing solution.Cinnamic acid is diluted to 0.0001,0.0005,0.001,0.005,0.01 and 0.02 μ g/ with methanol
ML standard items；By eugenol into the standard items for being diluted to 0.005,0.01,0.05,0.1,0.25 and 0.5 μ g/mL；By hundred
In fragrant phenol be diluted to 1.0,5.0,10.0,50.0,100.0 and 250.0 μ g/mL standard items to establish standard curve.Take 0.1mL
Sample is dissolved in 250mL volumetric flasks, through 30min ultrasounds, crosses 0.22 μm of filter membrane, testing sample is made.With methanol-water-glacial acetic acid
(60: 4: 2, volume ratio) are used as mobile phase, and with 1.0mL/min speed sample introduction, chromatogram value is detected under 274 nm wavelength.
Chromatographic column: Diamonsil C18；Sampling volume is 20 μ L；Column temperature: 30℃.
Using peak area as ordinate, the mass concentration of cinnamic acid, eugenol and Thymol draws standard song for abscissa
Line, and calculate regression equation:
Cinnamic acid:Y=13404X+25928, R2=0.999, it is good in 0.01-0.4 μ L/mL linear relationships.
Eugenol:Y=15123X+22670, R2=0.999, it is good in 0.01-0.4 μ L/mL linear relationships.
Thymol:Y=14825X-15883, R2=0.999, it is good in 5-500 μ g/mL linear relationships.
Cinnamon oil, caryophyllus oil and Thymol are diluted to suitable quality concentration, carried out according to above-mentioned condition and method
Detection, calculate the content of main component in each essential oil.Referring to table 7.
The Contents of Main Components of the cinnamon oil of table 7, caryophyllus oil and Thymol
1.2.2 the preparation of antibacterial decoction
By 2 kinds of essential oils through 0.22 μm of filtration sterilization（Cinnamon oil and caryophyllus oil）With 3 main components（Cinnamic acid, cloves
Phenol and Thymol）And the composite essential oil of the present invention is according to essential oil: Tween 80: dinitro sulfoxide=2: 1: 1 volume ratio mixes
Close, vortex 1min is made into the decoction that volume fraction is 1/2.The decoction that 102.4 μ L volume fractions are 1/2 is taken, with through HTHP
The nutrient broth fluid nutrient medium of sterilizing is diluted to 4mL, vortex 1min, and it is 12800 μ g/L that quality of liquid medicine concentration, which is made,.Use again
Quality of liquid medicine concentration is diluted to 12800,6400,3200,1600,800,400,200 and 100 μ g/L by 2 times of dilution methods respectively
It is stand-by etc. mass concentration (experiment proves that the mass concentration of Tween 80 and dinitro sulfoxide does not interfere with suppression less than 1% in decoction
The result of bacterium experiment).
1.2.3 the activation of strain and the preparation of bacterium solution
E.coli K88 and detection of Salmonella A72 are inoculated into nutrient broth fluid nutrient medium respectively, in 37 DEG C of shaking tables, with
150r/min speed concussion and cultivate 12h；Take 10 μ L to be added in 5mL nutrient broth fluid nutrient mediums from obtained bacterium solution with
Identical CMC model 12h；It is once rear stand-by to expand culture again with the same terms.
Using colony counting method, hanged bacterium is diluted by 10 times of dilution methods after the bacterium solution fully shaking vortex 1min activated
Liquid, draw 10-6、10-7With 10-8Each 100 μ L of bacteria suspension, are uniformly coated on sterile Letheen Broth culture medium, are put into 37 DEG C
12h is cultivated in constant incubator, each 2 repetitions of gradient, selects optimal plate count, and calculate in every milliliter of bacteria suspension
Clump count.Again by the clump count calculated, it is stand-by that bacterium solution is diluted to 500,000 CFU.
1.2.4 the preparation of indicator
0.025g resazurins accurately are weighed, are dissolved in 100mL ultra-pure waters, through autoclave sterilization, 0.25g/L mass is made
The sterile indicator of concentration is stand-by.
1.2.5 bacteriostatic test method
Using resazurin indicator micro-dilution method measure cinnamon oil, cinnamic acid, caryophyllus oil, eugenol, Thymol and
The Antibacterial Activity of compound essential oil prepared by embodiment 2.First in 96 porocyte culture plates of sterilizing each hole add 2 μ L,
0.25g/L resazurin indicator, according still further to the order from high concentration to low concentration, sequentially add 100 μ L diluted it is antibacterial
Decoction, each mass concentration do the repetition of 3 longitudinal directions side by side.Then take 3 holes only to add 100 μ L culture mediums to be used as to compare, 3
The bacterium solution that hole only adds 100 μ L to dilute does positive control, and 3 holes only add 100 μ L decoctions to do negative control.Again into each test hole
The bacterium solution for 500,000 CFU that 100 μ L have diluted is added, 100 μ L nutrient meats are added in control, positive control and each hole of negative control
Soup supplies volume, is consistent with test group.To reduce liquid evaporation in hole, cover after 96 orifice plate lids again with sterile fresh-keeping
Film seals plate.96 porocyte culture plates made are placed in 37 DEG C of constant incubators and cultivated, result is observed after 24h.
Result judgement:Resazurin solution is blueness, will be changed into red when there is bacterial growth.It is sterile after incubated
Long minimum drug quality concentration of being born is MIC.Concrete outcome is referring to table 8.
Several decoction minimum inhibitory concentrations of table 8（μg/L）
As seen from Table 7:3 kinds of essential oils and its main component are to the synthesis bacteriostasis of 2 boar source pathogenic bacteria:Cinnamic acid
＞ cinnamon oils ＞ Thymols >=eugenol and caryophyllus oil.The composite essential oil of the present embodiment 2 is 200 μ g/ to the MIC for trying bacterium
L.The MIC of cinnamon oil and cinnamic acid is the 1/4 and 1/2 of the composite essential oil of embodiment 2；The antibacterial MIC of Thymol is implementation
The 1/8 of the composite essential oil of example 2；The antibacterial MIC of eugenol and caryophyllus oil is the 1/8 and 1/16 of the composite essential oil of embodiment 2.
The fungistatic effect that cinnamon oil and cinnamic acid are shown to K88 and A72 is identical, to E.coli K88 and detection of Salmonella
A82 MIC is respectively 800 and 400 μ g/L, and show to play bacteriostasis in cinnamon oil is cinnamic acid.Eugenol is to 2 plants for examination
The MIC of bacterium is identical, is 1600 μ g/L, and caryophyllus oil is different to the MIC of 2 plants of bacterium, respectively 1600 and 3200 μ g/L.Hundred
In fragrant phenol be 1600 μ g/L to the bacteriostasis of 2 plants of bacterium.And the composite essential oil antibacterial activity of the embodiment of the present invention 2 is much high
In monomer oil, it may be possible to have certain coordinative role between Thymol or Multiple components, this also needs further to study.
The present invention is realized to being detected while 3 compositions by the high performance liquid chromatography of foundation in a system.Should
Method high sensitivity, reproducible, finding speed is fast and simple to operate, practical under experimental condition.Under detection method, volatilization
Each component obtains sufficiently effective separation in oil, has reached the requirement to wherein Major Components.This experiment has only been done to supplying
The quantitative detection of main composition in essential oil is tried, all the components are not detected, can if subsequently being required to essential oil component
Analyzed using the method for gas chromatography mass spectrometry.
Finally it should be noted that：The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention,
Although the present invention is described in detail with reference to the foregoing embodiments, for those skilled in the art, it still may be used
To be modified to the technical scheme described in foregoing embodiments, or equivalent substitution is carried out to which part technical characteristic.
Within the spirit and principles of the invention, any modification, equivalent substitution and improvements made etc., it should be included in the present invention's
Within protection domain.
Claims (8)
- A kind of 1. compound essential oil of prevention of diarrhea in piglets, it is characterised in that：The compound essential oil by following mass fraction each group Divide and be prepared：Cinnamon oil 0.25-2.5 parts, caryophyllus oil 0.1-1 parts, Thymol 0.25-2.5 parts, activation attapulgite 100 Part.
- 2. compound essential oil according to claim 1, it is characterised in that：The compound essential oil by following mass fraction each group Divide and be prepared：100 parts 1.25 parts of cinnamon oil, 0.8 part of caryophyllus oil, 1.25 parts of Thymol, activation of attapulgite.
- 3. the preparation method of the compound essential oil described in claim 1 or 2, it is characterised in that：It is by the cinnamon oil of formula ratio, cloves Oil, Thymol add ethanol, add at room temperature in the activation attapulgite of formula ratio, and vibration, suction filtration is drying to obtain.
- 4. application of the compound essential oil described in claim 1 or 2 in the medicine of prevention of diarrhea in piglets is prepared.
- 5. the compound essential oil described in claim 1 or 2 is preparing the application in suppressing Escherichia coli and/or detection of Salmonella medicine.
- 6. application of the compound essential oil described in claim 1 or 2 in the feed addictive of prevention of diarrhea in piglets is prepared.
- A kind of 7. feed addictive of prevention of diarrhea in piglets, it is characterised in that：In the feed addictive containing claim 1 or Compound essential oil described in 2.
- A kind of 8. feed of prevention of diarrhea in piglets, it is characterised in that：The feed is weighed by 96-98wt% basal diets and 2-4wt% Profit requires the compound essential oil composition described in 1 or 2.
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| 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 |
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