CN107828866A - A kind of application for the macleaya cordata conditioning agent for improving weanling pig immune performance - Google Patents
A kind of application for the macleaya cordata conditioning agent for improving weanling pig immune performance Download PDFInfo
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Abstract
The invention belongs to livestock-raising technical field, and in particular to a kind of application for the macleaya cordata conditioning agent for improving weanling pig immune performance；Comprise the following steps:A1, bacteriostatic test；A2, Animal experiment；A3, test cell line；Macleaya cordata conditioning agent has obvious fungistatic effect to pathogenic microorganisms：Macleaya cordata conditioning agent has fungistatic effect to gram-positive bacteria or Gram-negative bacteria, and macleaya cordata conditioning agent is to pathogenic microorganism antimicrobial spectrum testing result：Staphylococcus aureus ＞ bacillus subtilis ＞ Escherichia coli ＞ salmonellas, there is fungistatic effect to weanling pig enteropahtogenic microganism Escherichia coli, salmonella and staphylococcus aureus using macleaya cordata conditioning agent, especially to most notable to staphylococcus aureus fungistatic effect.
Description
Technical field
The invention belongs to livestock-raising technical field, and in particular to a kind of macleaya cordata for improving weanling pig immune performance is adjusted
Save the application of agent.
Background technology
It is long-term largely to cause bacterial drug resistance and medicament residue to cause social greatly concern using antibiotic, and forbid making
The adverse effect of pig-breeding industry is gradually embodied with antibiotic, many bacteriosises stage a comeback, and cause live pig
The general level of the health declines, efficiency of feed utilization reduces.There is presently no any additive can effectively improve pig as antibiotic
Production performance and improvement efficiency of feed utilization, if may result in food consumption increase and pig without using antibiotic in pig feed
The speed of growth declines, and therefore, stops using antibioticses feed addictive, must just have new Substitutes For Antibiotic, to protect
Card live pig health and production efficiency, currently used enzyme preparation, trace mineral supplement, organic acid can improve pig in various degree
Production performance and efficiency of feed utilization, but can not completely substitute antibiotics use.
At present, it is that special feed such as SDPP is directly applied in piglet diet to produce existing common method to piglet
To increase piglet blood IgG antibody level, and sp act material is used to can induce the research that IgG contents rise in animal body
It is relatively weak with application.
Therefore, seek safe and efficient Substitutes For Antibiotic has turned into improvement live pig health and has improved the important of production performance
Demand.
The content of the invention
The present invention proposes a kind of application for the macleaya cordata conditioning agent for improving weanling pig immune performance, can improve piglet machine
Body immune performance, improve piglet disease-resistant ability.
In order to achieve the above object, the present invention is achieved through the following technical solutions：
A kind of application for the macleaya cordata conditioning agent for improving weanling pig immune performance, comprises the following steps:
A1, bacteriostatic test
From Escherichia coli, salmonella, staphylococcus aureus, bacillus subtilis as subjects, measure is not
With the inhibitory effect and minimal inhibitory concentration of concentration macleaya cordata extracts, macleaya cordata extracts are to Escherichia coli, salmonella, gold
Staphylococcus aureus, the minimal inhibitory concentration of bacillus subtilis be respectively 1280 μ g/ml, 1280 μ g/ml, 2560 μ g/ml,
2560μg/ml；
A2, Animal experiment
It is experimental animal from 60 PIC, five yuan of hybridization piglets, the wean of 35 ages in days, tests and set using 1 × 4+1 randomizations
Meter, is randomly divided into 5 treatment groups, every group of 4 repetitions, each to repeat 3, it is each handle piglet feed respectively addition 0mg/kg,
The experiment daily ration of 2.5mg/kg, 5.0mg/kg, 7.5mg/kg macleaya cordata extracts, last group feeding addition 50mg/kg soil are mould
The experiment daily ration of element, free choice feeding and drinking-water, are routinely immunized, and determine each group production performance index, experiment piglet is in 60
Age in days, all piglet vena cava anteriors blood sampling 5mL, for determining blood parameters.Result of the test shows that macleaya cordata extracts are most
Broiler diets are 4.5mg/kg.
A3, test cell line
From pig back fibroblast, it is separately added into macleaya cordata after stimulating its immunological stress with 10 μ g/ml lipopolysaccharides and carries
Thing is taken to final concentration of 0ng/ml, 50ng/ml, 100ng/ml, 150ng/ml, positive control is adds terramycin to final concentration of
50 μ g/ml, after cultivating 24 hours, RNA is extracted, reverse transcription, finally carries out Real-time fluorescent quantitations, determines IgG and SOD
Gene mRNA relative expression quantity, macleaya cordata extracts can improve weanling pig Immunoglobulin in Serum G, nitric oxide, molten
The content of bacterium enzyme, superoxide dismutase, reduce mda content in serum.
Further, in step A1, the preparation process of macleaya cordata extracts is：Take the overground part after macleaya cordata drying
Point, it is crushed to after 40-50 mesh thicknesses, adds 80% ethanol that pH is 5, backflow is filtered after 2 hours, merging filtrate and dense
It is complete to be reduced to ethanol recovery, adds isometric water, stands, must precipitate, removes supernatant, is obtained after filter residue vacuum drying orange
Powdered macleaya cordata extracts.
Further, in step A1, inhibitory effect determine the step of be：0.1ml is pipetted respectively is in exponential phase
Escherichia coli, salmonella, staphylococcus aureus, the bacterium solution of bacillus subtilis, are spread evenly across broth medium
Surface, diameter 6mm sterilizing filter papers piece is taken to immerse in 1.5mg/ml, 3.0mg/ml, 4.5mg/ml macleaya cordata extracts solution
Taken out after 40min, treat that dry be put into is inoculated with flat board, flat board is placed in 37 DEG C of incubators after cultivating 24h and observed, every kind of bacterium takes 3
Scraps of paper antibacterial circle diameter average value records.
Further, in step A1, minimal inhibitory concentration determine the step of be：Win using micro-broth dilution method
Extract is returned to the minimal inhibitory concentration of different microorganisms, takes sterile test tube 13, in addition to the 1st pipe adds 1.6ml meat soups, remaining
Often pipe adds meat soup 1ml, and adding 2560 μ g/ml macleaya cordata extracts 0.4ml in the 1st pipe mixes, and then draws 1ml to the 2nd and manages,
The pipes of 1ml to the 3rd are drawn after mixing again, so continuous doubling dilution is drawn 1ml from the 11st pipe and discarded to the 11st pipe, and the 12nd
Manage as the growth control of not drug containing, above-mentioned each 1ml of the bacterium solution prepared is then added in every pipe, test tube is placed in 37 DEG C of temperature
After cultivating 24h in case, the minimal inhibitory concentration that not muddy test tube concentration is tested bacterium is visually observed.
Compared with prior art, beneficial effects of the present invention are：
1. macleaya cordata conditioning agent has obvious fungistatic effect to pathogenic microorganisms：Macleaya cordata conditioning agent is to gram-positive bacteria
Or Gram-negative bacteria has fungistatic effect, macleaya cordata conditioning agent is to pathogenic microorganism antimicrobial spectrum testing result：Golden yellow Portugal
Grape coccus ＞ bacillus subtilis ＞ Escherichia coli ＞ salmonellas, it is micro- to weanling pig enteropathogenic using macleaya cordata conditioning agent
Organism Escherichia coli, salmonella and staphylococcus aureus have fungistatic effect, especially to antibacterial to staphylococcus aureus
Most pronounced effects.
2. macleaya cordata conditioning agent is used in mixed way with piglet diet, daily gain can be significantly improved and improve feed profit
With rate.
3. macleaya cordata conditioning agent improves weanling pig immune performance：Macleaya cordata conditioning agent ratio is not added with macleaya cordata conditioning agent group
Piglet pole significantly improves piglet serum IgG content up to 5~6 times, and macleaya cordata conditioning agent carries than addition antibiotic positive group group piglet
High 3~4 times of serum IgG content.Macleaya cordata conditioning agent significantly improves weanling pig serum NO level, NOS and LSZ contents, using winning
Return conditioning agent and can obviously improve piglet body and exempt from performance, improve piglet disease-resistant ability.
Brief description of the drawings
Fig. 1 is Macleaya cordata alkaloids standard items chromatogram；
Fig. 2 is Macleaya cordata alkaloids extract chromatogram；
Fig. 3 is various concentrations macleaya cordata extracts to enteric microorganism inhibition schematic diagram；
In Fig. 3, A:Escherichia coli；B：Salmonella；C：Staphylococcus aureus；D bacillus subtilises.
Embodiment
In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, below in conjunction with embodiment, to this hair
Bright preferred embodiment is described in detail, to facilitate the technical staff to understand.
Embodiment 1
A kind of application for the macleaya cordata conditioning agent for improving weanling pig immune performance, comprises the following steps:
A1, bacteriostatic test
From Escherichia coli, salmonella, staphylococcus aureus, bacillus subtilis as subjects, measure is not
With the inhibitory effect and minimal inhibitory concentration of concentration macleaya cordata extracts, macleaya cordata extracts are to Escherichia coli, salmonella, gold
Staphylococcus aureus, the minimal inhibitory concentration of bacillus subtilis be respectively 1280 μ g/ml, 1280 μ g/ml, 2560 μ g/ml,
2560μg/ml；
A2, Animal experiment
It is experimental animal from 60 PIC, five yuan of hybridization piglets, the wean of 35 ages in days, tests and set using 1 × 4+1 randomizations
Meter, is randomly divided into 5 treatment groups, every group of 4 repetitions, each to repeat 3, it is each handle piglet feed respectively addition 0mg/kg,
The experiment daily ration of 2.5mg/kg, 5.0mg/kg, 7.5mg/kg macleaya cordata extracts, last group feeding addition 50mg/kg soil are mould
The experiment daily ration of element, free choice feeding and drinking-water, are routinely immunized, and determine each group production performance index, experiment piglet is in 60
Age in days, all piglet vena cava anteriors blood sampling 5mL, for determining blood parameters, macleaya cordata extraction optimum addition is
4.5mg/kg；
A3, test cell line
From pig back fibroblast, it is separately added into macleaya cordata after stimulating its immunological stress with 10 μ g/ml lipopolysaccharides and carries
Thing is taken to final concentration of 0ng/ml, 50ng/ml, 100ng/ml, 150ng/ml, positive control is adds terramycin to final concentration of
50 μ g/ml, after cultivating 24 hours, RNA is extracted, reverse transcription, finally carries out Real-time fluorescent quantitations, determines IgG and SOD
Gene mRNA relative expression quantity, macleaya cordata extracts can improve weanling pig Immunoglobulin in Serum G, nitric oxide, molten
The content of bacterium enzyme, superoxide dismutase, reduce mda content in serum.
Macleaya cordata conditioning agent has obvious fungistatic effect to pathogenic microorganisms：Macleaya cordata conditioning agent to gram-positive bacteria or
Gram-negative bacteria has fungistatic effect, and macleaya cordata conditioning agent is to pathogenic microorganism antimicrobial spectrum testing result：Golden yellow grape
Coccus ＞ bacillus subtilis ＞ Escherichia coli ＞ salmonellas, using macleaya cordata conditioning agent to the micro- life of weanling pig enteropathogenic
Thing Escherichia coli, salmonella and staphylococcus aureus have fungistatic effect, especially to the antibacterial effect of staphylococcus aureus
Fruit is most notable.
Embodiment 2
On the basis of embodiment 1, in step A1, the preparation process of macleaya cordata extracts is：Macleaya cordata is taken to dry it
Aerial part afterwards, it is crushed to after 40-50 mesh thicknesses, adds 80% ethanol that pH is 5, backflow is filtered after 2 hours, merged
Filtrate and to be concentrated into ethanol recovery complete, adds isometric water, stands, must precipitate, remove supernatant, filter residue is dried in vacuo it
After obtain orange powder shape macleaya cordata extracts,
It is higher to extract obtained macleaya cordata extracts purity, it is simple to operate.
Embodiment 3
On the basis of embodiment 1-2, in step A1, inhibitory effect determine the step of be：Pipette respectively at 0.1ml
In the Escherichia coli of exponential phase, salmonella, staphylococcus aureus, bacillus subtilis bacterium solution, be spread evenly across
Broth medium surface, take diameter 6mm sterilizing filter papers piece to immerse 1.5mg/ml, 3.0mg/ml, 4.5mg/ml macleaya cordata and carry
Take in thing solution and taken out after 40min, treat that dry be put into is inoculated with flat board, flat board is placed in 37 DEG C of incubators after cultivating 24h and observed, often
Kind bacterium takes 3 scraps of paper antibacterial circle diameter average values records, and macleaya cordata conditioning agent improves weanling pig immune performance：Macleaya cordata is adjusted
Agent ratio is not added with macleaya cordata conditioning agent group piglet pole and significantly improves piglet serum IgG content up to 5~6 times, macleaya cordata conditioning agent ratio
Add antibiotic positive group group piglet and improve 3~4 times of serum IgG content.Macleaya cordata conditioning agent significantly improves weanling pig serum
NO, NOS and LSZ content.Piglet body is can obviously improve using macleaya cordata conditioning agent and exempts from performance, improves piglet disease-resistant ability.
Embodiment 4
On the basis of embodiment 1-3, in step A1, minimal inhibitory concentration determine the step of be：Using micro meat soup
Dilution macleaya cordata extracts take sterile test tube 13, except the 1st pipe adds to the minimal inhibitory concentration of different microorganisms
Outside 1.6ml meat soups, often pipe adds meat soup 1ml for remaining, and adding 2560 μ g/ml macleaya cordata extracts 0.4ml in the 1st pipe mixes, so
The pipes of 1ml to the 2nd are drawn afterwards, draw the pipes of 1ml to the 3rd after mixing again, and so continuous doubling dilution is managed to the 11st pipe, and from the 11st
Middle absorption 1ml is discarded, and the 12nd pipe is the growth control of not drug containing, and it is each that the above-mentioned bacterium solution prepared is then added in every pipe
1ml, test tube is placed in 37 DEG C of incubators after cultivating 24h, it is the minimum antibacterial of tested bacterium to visually observe not muddy test tube concentration
Concentration, macleaya cordata conditioning agent have obvious fungistatic effect to pathogenic microorganisms：Macleaya cordata conditioning agent is to gram-positive bacteria or leather
Lan Shi negative bacteriums have fungistatic effect, and macleaya cordata conditioning agent is to pathogenic microorganism antimicrobial spectrum testing result：Staphylococcus aureus
Bacterium ＞ bacillus subtilis ＞ Escherichia coli ＞ salmonellas, using macleaya cordata conditioning agent to weanling pig enteropahtogenic microganism
Escherichia coli, salmonella and staphylococcus aureus have fungistatic effect, especially to staphylococcus aureus fungistatic effect
It is most notable.
Experimental analysis
1st, macleaya cordata extracts are to Antimicrobial effect
1.1 macleaya cordata extracts alkaloids detect
This experiment uses high performance liquid chromatography, using acetonitrile and phosphate aqueous solution as mobile phase under 270nm absorbing wavelengths
It is respectively 41.41% and 19.23% to detect sanguinarine and chelerythrine alkali content in macleaya cordata extracts.In macleaya cordata extracts
Sanguinarine and Chelerythrine chromatogram are as depicted in figs. 1 and 2：
From Fig. 1 and Fig. 2, the macleaya cordata extracts sanguinarine of the inventive method extraction and chelerythrine alkali content compared with
It is high.
The inhibitory effect measure of 1.2 macleaya cordata extracts
Increase with macleaya cordata extracts solution concentration, to Escherichia coli, salmonella, staphylococcus aureus, withered grass bud
The inhibition enhancing of spore bacillus, antibacterial circle diameter also gradually increase, as shown in Figure 3：Suppression of the macleaya cordata extracts to 4 kinds of bacteriums
Effect processed is respectively：Staphylococcus aureus ＞ bacillus subtilis ＞ Escherichia coli ＞ salmonellas.
The minimal inhibitory concentration measure of 1.3 macleaya cordata extracts
By macleaya cordata extracts solution by 2560 μ g/ml, 1280 μ g/ml, 640 μ g/ml, 320 μ g/ml, 160 μ g/ml,
80 μ g/ml, 40 μ g/ml concentration gradient measure minimal inhibitory concentration, the results are shown in Table 2.It has been observed that macleaya cordata extracts dilute
Degree is bigger, and meat soup is more muddy, illustrates that bacterial growth is more.Macleaya cordata extracts are to Escherichia coli, salmonella, golden yellow grape
Coccus, the minimal inhibitory concentration of bacillus subtilis are respectively 1280 μ g/ml, 1280 μ g/ml, 2560 μ g/ml, 2560 μ g/ml.
The minimal inhibitory concentration of the macleaya cordata extracts of table 1
Note：- no long bacterium is represented ,+a small amount of long bacterium is represented, ++ the long bacterium of appropriateness is represented, +++ represent a large amount of long bacterium.
Finally illustrate, preferred embodiment above is merely illustrative of the technical solution of the present invention and unrestricted, although logical
Cross above preferred embodiment the present invention is described in detail, it is to be understood by those skilled in the art that can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Claims (4)
- A kind of 1. application for the macleaya cordata conditioning agent for improving weanling pig immune performance, it is characterised in that:Comprise the following steps:A1, bacteriostatic testIt is different dense as subjects, measure from Escherichia coli, salmonella, staphylococcus aureus, bacillus subtilis The inhibitory effect and minimal inhibitory concentration of macleaya cordata extracts are spent, macleaya cordata extracts are to Escherichia coli, salmonella, golden yellow Staphylococcus, the minimal inhibitory concentration of bacillus subtilis are respectively 1280 μ g/ml, 1280 μ g/ml, 2560 μ g/ml, 2560 μ g/ml；A2, Animal experimentIt is experimental animal from 60 PIC, five yuan of hybridization piglets, the wean of 35 ages in days, tests and use 1 × 4+1 Randomized Designs, with Machine is divided into 5 treatment groups, and every group of 4 repetitions, each to repeat 3, each processing piglet feeds addition 0mg/kg, 2.5mg/ respectively The experiment daily ration of kg, 5.0mg/kg, 7.5mg/kg macleaya cordata extracts, the examination of last group feeding addition 50mg/kg terramycin Daily ration, free choice feeding and drinking-water are tested, is routinely immunized, determines each group production performance index, experiment piglet is in 60 ages in days, institute There is piglet vena cava anterior blood sampling 5mL, for determining blood parameters, macleaya cordata extraction optimum addition is 4.5mg/kg；A3, test cell lineFrom pig back fibroblast, macleaya cordata extracts are separately added into after stimulating its immunological stress with 10 μ g/ml lipopolysaccharides To final concentration of 0ng/ml, 50ng/ml, 100ng/ml, 150ng/ml, positive control is addition terramycin to final concentration of 50 μ G/ml, after cultivating 24 hours, RNA is extracted, reverse transcription, finally carries out Real-time fluorescent quantitations, determines IgG and sod gene MRNA relative expression quantities, macleaya cordata extracts can improve weanling pig Immunoglobulin in Serum G, nitric oxide, lysozyme, The content of superoxide dismutase, reduce mda content in serum.
- 2. a kind of application of macleaya cordata conditioning agent for improving weanling pig immune performance according to claim 1, its feature It is:In step A1, the preparation process of macleaya cordata extracts is：The aerial part after macleaya cordata drying is taken, is crushed to 40- After 50 mesh thicknesses, add pH be 5 80% ethanol, backflow 2 hours after filter, merging filtrate and be concentrated into ethanol recovery Completely, isometric water is added, stands, must precipitate, supernatant is removed, orange powder shape macleaya cordata is obtained after filter residue vacuum drying Extract.
- 3. a kind of application of macleaya cordata conditioning agent for improving weanling pig immune performance according to claim 1, its feature It is:In step A1, inhibitory effect determine the step of be：0.1ml is pipetted respectively is in the Escherichia coli of exponential phase, sand Door Salmonella, staphylococcus aureus, the bacterium solution of bacillus subtilis, are spread evenly across broth medium surface, take diameter 6mm sterilizing filter papers piece is immersed in 1.5mg/ml, 3.0mg/ml, 4.5mg/ml macleaya cordata extracts solution to be taken out after 40min, waits to do It is put into inoculation flat board, flat board is placed in 37 DEG C of incubators after cultivating 24h and observed, every kind of bacterium takes 3 scraps of paper antibacterial circle diameters to put down Average records.
- 4. a kind of application of macleaya cordata conditioning agent for improving weanling pig immune performance according to claim 1, its feature It is:In step A1, minimal inhibitory concentration determine the step of be：Using micro-broth dilution method macleaya cordata extracts pair The minimal inhibitory concentration of different microorganisms, sterile test tube 13 is taken, in addition to the 1st pipe adds 1.6ml meat soups, often pipe adds meat for remaining Soup 1ml, add 2560 μ g/ml macleaya cordata extracts 0.4ml in the 1st pipe and mix, then draw 1ml to the 2nd and manage, inhaled again after mixing 1ml to the 3rd is taken to manage, so continuous doubling dilution is drawn 1ml from the 11st pipe and discarded to the 11st pipe, and the 12nd pipe is not drug containing The growth control of thing, above-mentioned each 1ml of the bacterium solution prepared is then added in every pipe, test tube is placed in 37 DEG C of incubators and cultivated After 24h, the minimal inhibitory concentration that not muddy test tube concentration is tested bacterium is visually observed.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110218688A (en) * | 2019-07-05 | 2019-09-10 | 中国农业科学院特产研究所 | Hai Neishi bacillus LY-23, microbial inoculum and its application and the product for applying it |
| CN110812389A (en) * | 2019-11-29 | 2020-02-21 | 广东容大生物股份有限公司 | Soluble granule for improving pig immunity and preparation method thereof |
Citations (1)
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110218688A (en) * | 2019-07-05 | 2019-09-10 | 中国农业科学院特产研究所 | Hai Neishi bacillus LY-23, microbial inoculum and its application and the product for applying it |
| CN110218688B (en) * | 2019-07-05 | 2021-01-29 | 中国农业科学院特产研究所 | Bacillus marinus LY-23, microbial inoculum and application thereof and product using same |
| CN110812389A (en) * | 2019-11-29 | 2020-02-21 | 广东容大生物股份有限公司 | Soluble granule for improving pig immunity and preparation method thereof |
| CN110812389B (en) * | 2019-11-29 | 2021-12-21 | 广东容大生物股份有限公司 | Soluble granule for improving pig immunity and preparation method thereof |
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