CN106906166A - One boar source Lactobacillus salivarius active bacteria formulation and application - Google Patents
One boar source Lactobacillus salivarius active bacteria formulation and application Download PDFInfo
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Abstract
The invention provides a boar source Lactobacillus salivarius active bacteria formulation and application, it is related to biological technical field, the active bacteria formulation of present invention production has prebiotic effect to pig, and can ensure that its field planting in enteron aisle, bacterial strain uses therefor is Gram-positive, it is identified as Lactobacillus salivarius, Main Biological is G+, thalline rod-short, amphimicrobian, non-refractory, there is certain depression effect to Escherichia coli and staphylococcus aureus, production technology includes slant strains, shake-flask seed liquid, fermentation tank, suspending agent and product, zoopery display use can reduce the diarrhea rate of pig, improve the speed of growth of pig, reduce feedstuff-meat ratio, reduce the antibiotic usage amount during pig is fed.
Description
Technical field
The present invention relates to biological technical field, and in particular to a boar source Lactobacillus salivarius active bacteria formulation and application.
Background technology
Probiotics is put forward first by Parker also known as probiotic in nineteen forty-seven, typically now refers to lead to after putting into
Cross the improvement host intestine flora ecological balance and play beneficial effect, reach the viable bacteria for improving host health level and health status
Preparation and its metabolite.
The mechanism of action of probiotics：1st, intestinal microecology balance is maintained：Probiotics is by transplanting the growth of pathogenic bacteria flora
Breeding, the balance of protection profitable strain, effectively prevent the generation of livestock and poultry, while promoting digesting and assimilating and reducing material for livestock and poultry
Meat ratio；2nd, Ant agonism：Probiotics is colonized in host intestine, forms fine and close Membranous flora, forms biological barrier, is prevented
Adhesion of the pathogen to enteron aisle；3rd, biology takes oxygen by force：Based on anaerobic environment, the field planting of probiotics can be consumed intact animal enteron aisle
A large amount of oxygen in enteron aisle, and then maintain anaerobic environment so that pathogen suppresses its growth and breeding because of anoxic；4th, body is strengthened
Immunity：Probiotics can stimulating animal produce interferon, the basic element of cell division, activating immune cell, improve immunoglobulin it is dense
Degree and macrophage activity, so as to strengthen body disease-resistant performance；5th, body digestion is promoted：Hydrolase, fermentation that probiotics produces
Enzyme and respiratory enzyme, are conducive to protein, fat and complex carbohydrates in degraded feed, so as to improve efficiency of feed utilization；6、
Suppress the generation of noxious material：Probiotics can produce H2O2 and natural antibiotics class material, reduce ammonia and amine etc. in enteron aisle poisonous
Material, suppresses to produce the growth of ammonia spoilage organisms, absorbs intestinal endotoxin, prevents diarrhoea, the few disease of solution；7th, environment purification, improves poultry
Product quality：The effect of probiotics declines the content of ammonia and amine in enteral and blood, reduces the cause excreted with excrement
The pernicious gas such as germ and ammonia, reduces the pollution to environment.
The selection of active bacteria formulation strain need to possess some and require：1st, strain is not pathogenic, nontoxicity；2nd, supported with stronger
The ability of anti-alimentary tract restraining factors, the tolerance to poor environment is stronger；3rd, growth and breeding speed is fast；4th, with stronger suction
Attached ability, can secrete various antibacterial substances and various digestive enzymeses；5th, the adaptability and repellence of environment are stronger to external world.Mesh
The preceding microorganism for microorganism formulation mainly includes lactic acid bacteria, Bifidobacterium, streptococcus fecalis, bacillus, saccharomycete, puts
Several major classes such as line bacterium, photosynthetic bacteria.
Lactic acid bacteria is that, using earliest, widest probiotics, it refers to that a class can ferment using carbohydrate generation greatly
The general name of the bacterium of lactic acid is measured, the metabolite of lactic acid bacteria mainly there are the antibacterial substances such as organic acid, hydrogen peroxide, bacteriocin, can
Pathogenic microorganism field planting in vivo is killed or prevented, suppresses the growth of pathogenic bacteria, in regulation host intestine microecological balance side
Face plays an important role.
What the China of table 1 ratified can be used for the lactobacillus list of food
Lactobacillus salivarius belongs to Lactobacillaceae, and lactobacillus widely exists in the enteron aisle of humans and animals, in recent years, saliva
Liquid lactobacillus is widely used in the production of humans and animals probiotics preparation as probiotics, is No. 65 bulletins of the Ministry of Public Health of China 2010 year
One of strain of food production processing that approval is used.Lactobacillus salivarius has certain suction-operated, can be good in intestines
Road surface is colonized and then plays its immunization, studies have found that Lactobacillus salivarius shows immunological regulation, anti-inflammatory and anti-infective
Characteristic, meanwhile, lactobacillus or its cell component can improve the immunologic function of animal, strengthen the immunity of body.Field planting exists
The growth of other bacteriums can be suppressed in the growth of lactobacillus metabolic process of enteron aisle by materials such as antibiotic, the bacteriocins of secretion,
And with pathogen competitive growth space and then play its antagonism.
Although the research report at present both at home and abroad to lactobacillus is a lot, passes through zoopery and then be applied to reality
Produce and also little for the report of feed addition.
The content of the invention
(1) technical problem for solving
In view of the shortcomings of the prior art, the invention provides a boar source Lactobacillus salivarius active bacteria formulation and application, use
This microbial inoculum can reduce the diarrhea rate of pig, improve the speed of growth of pig, reduce feedstuff-meat ratio, and reducing the antibiotic during pig is fed makes
Consumption, meets the requirement of green feed additive.
(2) technical scheme
To realize object above, the present invention is achieved by the following technical programs：
One boar source Lactobacillus salivarius active bacteria formulation, production technology is comprised the following steps：
(1) slant preservation strain is inoculated in liquid submerged culture base, concussion to logarithmic phase；
(2) above-mentioned cultured strain is accessed into 5L fermentation tanks by 5% inoculum concentration, is cultivated in culture medium to logarithmic growth
Phase, the formula of culture medium is：Peptone 0.8-1.2%, yeast extract 0.4-0.8%, beef extract 1-1.5%, glucose
1.3-2%, sodium acetate 0.1-0.6%, sodium citrate 0.15-0.25%, Tween 80 0.05-0.15%, MgSO4·7H2O
0.05-0.06%, MnSO4·4H2O 0.001-0.005%, K2HPO40.1-03% and CaCO30.5-1.5%, adjusts pH value
It is 5.5, it is 37 DEG C to control cultivation temperature, and whole process incubation time is 55-65 hours, and thalline quantity can reach 5 after fermentation ends
Hundred million/more than ml；
(3) after culture terminates, suspending agent is added to the bacterium solution in fermentation tank, is made active bacteria formulation.
Preferably, the strain in the step (1) is that separation screening is obtained from the excrement of pig.
Preferably, the separation screening of strain is comprised the following steps in the step (1)：Tool is separated from the rectum excrement of pig
There are the lactic acid bacteria strains of prebiotic effect；In vitro culture；Fermentation；Analyze influence of the isolated strains to pig intestine microenvironment；
Screening obtains the strain.
Preferably, the formula of culture medium is in the step (2)：Peptone 1%, yeast extract 0.6%, beef extract
1.2%th, glucose 1.6%, sodium acetate 0.5%, sodium citrate 0.215%, Tween 80 0.1%, MgSO4·7H2O
0.058%th, MnSO4·4H2O 0.005%, K2HPO40.2% and CaCO31%.
Preferably, the whole process incubation time is 60 hours, and thalline quantity reaches 500,000,000/more than ml after fermentation ends.
Preferably, application of the described pig source Lactobacillus salivarius active bacteria formulation in pig-breeding：This microbial inoculum can reduce pig
Diarrhea rate, improve pig the speed of growth, reduce feedstuff-meat ratio, reduce pig feed during antibiotic usage amount, meet green
The requirement of feed addictive.
(3) beneficial effect
The invention provides a boar source Lactobacillus salivarius active bacteria formulation and application, with advantages below：
1st, the present invention has low production cost, easy to use, the good advantage of using effect, it is adaptable to individuality cultivation and scale
The spread for changing pig farm is used.
2nd, the present invention can effectively reduce the generation of diarrhea of pigs rate, and diarrhea rate reduction by more than 60% had both been reduced to environment
Pollution, can reduce the antibiotic usage in pig growth course again, reduce pig cost, ensure meat quality.
3rd, the present invention can also improve the speed of growth of pig, reduce feedstuff-meat ratio, productivity effect be improve, by institute of the present invention
With strain isolation from the enteron aisle of pig, thalline is easier to be colonized in the enteron aisle of pig, and bacterial strain uses therefor belongs to probiotics, to pig without appointing
What side effect.
Specific embodiment
To make the purpose, technical scheme and advantage of the embodiment of the present invention clearer, below in conjunction with the embodiment of the present invention,
Technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is the present invention one
Divide embodiment, rather than whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art are not making
The every other embodiment obtained under the premise of creative work, belongs to the scope of protection of the invention.
Embodiment 1
The separation and identification of bacterial strain：
Excrement in pig rectum is taken with aseptic procedure, the fresh swine excrements of 1g are accurately weighed, is placed in 9mL physiological saline, shaken
15min is mixed, and carries out 10 times of gradient dilutions.Each dilution factor draws 100 μ L samples and adds RCM semisolid culturemediums, training
48h is supported, after bacterium colony is grown in culture medium the bacterial strain for selecting different colonial morphologies as much as possible is purified, to after purification
Bacterial strain carries out microscopy and determines its thalli morphology, while being measured to its biological characteristics.
Liquid tube inoculation is carried out to bacterial strain after purification, its genomic DNA is extracted, passed through by template of genomic DNA
PCR carries out 16S amplifications, and 16SrDNA amplifications use bacterial universal primers 27F：5 '-AGAGTTTGATCMTGGCTCAG-3 ' and
1492R：5 '-CGGYTACCTTGTTACGACTT-3 ' (primer is synthesized by Shanghai Invitrogen trade Co., Ltd), amplified fragments
Length is 1.5kb or so.
Picking single bacterium falls within 97 DEG C of high-temperature crushing cell membranes, PCR reaction systems in 10 μ l TE buffer solutions：The μ l of cumulative volume 25,
Including 2 × PCR Master Mix12.5 μ l；Concentration is each 1 μ l of 27F and 1492R primers of 10 μm of ol/L；The μ l of DNA profiling 1；dd
H2O 9.5μl。
PCR reaction conditions are：95 DEG C of predegeneration 5min, 94 DEG C of high-temperature denatured 45s, 53 DEG C of process annealing 45s, 72 DEG C of thermophilics
Extend 90s, cycle-index is 33, last 72 DEG C of insulations 10min.PCR reactions are needed to set and are replaced the moon of template DNA with ultra-pure water
Property control.
Verified through 1% agarose gel electrophoresis after reaction completion, sequence alignment is carried out after the completion of sequencing, determined bacterial strain
Classification position.Wherein, ZFB12 strain growths speed is fast, while having very strong tolerance to low acid, ensure that thalline is smooth
It is colonized into enteron aisle by the sour environment of stomach, and it has suppression well to Escherichia coli and staphylococcus aureus
System, the bacterium is identified as Lactobacillus salivarius, and Main Biological is G+, thalline rod-short is facultative to detest
Oxygen, non-refractory.
Embodiment 2
Bacterial strain Optimal Experimental:
(2.1) influences of the pH to Lactobacillus salivarius ZFB12
ZFB12 was cultivated to the logarithmic phase later stage in MRS culture mediums, and it is 2.0,3.0,4.0,5.0,6.0 to take bacterium solution in pH value
Physiological saline in, determine its viable count every 30min, as a result show, above-mentioned pH to Strain survival rate without considerable influence, when will
When pH is down to 1.0, thalline quantity has declined, therefore, ZFB12 can be with the sour environment that pH tolerant is 2.0, when pH is further
During reduction, bacterial strain declines to its tolerance.
(2.2) heat resistance of Lactobacillus salivarius ZFB12
ZFB12 was cultivated to the logarithmic phase later stage in MRS culture mediums, thalline is inoculated into and is pre-heated to 50 DEG C, 70 DEG C, 90 DEG C
Physiological saline in, determine viable count therein every 1min, as a result show, ZFB12 survival rates after 50 DEG C for the treatment of 2min are
93.7%, viable count is 41.2% after 70 DEG C for the treatment of 2min, and survival rate is 8.6% after 90 DEG C for the treatment of 2min.
(2.3) fungistatic effect of Lactobacillus salivarius ZFB12
Using Odontothrips loti, mixed after Escherichia coli and staphylococcus aureus are added into culture medium, be down flat plate, will
Zymotic fluid is accessed in Oxford cup, is as a result shown, ZFB12 is notable to the fungistatic effect of Escherichia coli and staphylococcus aureus.
(2.4) virulence experiment of Lactobacillus salivarius ZFB12
Lactobacillus salivarius ZFB12 bacterium solutions are configured to density for 1 × 106CFU/ml, respectively with every daily 1.0ml,
10.0ml bacterium solutions feed the healthy sucking pig of 10 ages in days, continuously take 5 days, observe all test pig active states, appetite, defecation,
And sucking pig whether any toxic action and the phenomena of mortality in 10 days.
As a result：Lactobacillus salivarius ZFB12 bacterium solutions feed the healthy sucking pig of 10 ages in days, continuously take 5 days, observe 10 days, can
See that sucking pig active state is good, body temperature, appetite are normal, discharge normal fecal stools, do not observe to the toxic effect of sucking pig and death,
Illustrate that the microbial inoculum is safe.
Embodiment 3
Practical application is tested：
(3.1) zoopery of Lactobacillus salivarius ZFB12 potus
The piglet for choosing 30 ages in days is tested, and is divided into four groups, and every group of three repetitions, each 6 piglet of repetition protect
The body weight and health for demonstrate,proving piglet are essentially identical, to be not added with the water of zymotic fluid as control, zymotic fluid are added the water fed
In, lactic acid bacteria final concentration respectively reaches 104,105 and 106CFU/ml, and cut off the water supply 2h before hello water, continuously feeds two weeks.
(3.2) the application dose experiment of Lactobacillus salivarius ZFB12 potus
Tested by two weeks and found, compared with the control, declining occurs in the experimental group grice diarrhoea rate for adding lactic acid bacteria,
Three experimental groups are compared, and during the final concentration of 105CFU/ml of lactic acid bacteria, grice diarrhoea rate is minimum, therefore, choose lactic acid bacteria final concentration
It is 105CFU/ml as optimal oral dose.
(3.3) Lactobacillus salivarius ZFB12 potus results of animal result compared with the control, as shown in table 2 and table 3：
Influence of the Lactobacillus salivarius potus of table 2 to weanling pig body weight
The diarrhoea situation observation result of table 3
To sum up, the embodiment of the present invention has the advantages that：The generation of diarrhea of pigs can be effectively reduced, piglet is effectively facilitated
Growth, improves feedstuff-meat ratio.
It should be noted that herein, such as first and second or the like relational terms are used merely to a reality
Body or operation make a distinction with another entity or operation, and not necessarily require or imply these entities or deposited between operating
In any this actual relation or order.And, term " including ", "comprising" or its any other variant be intended to
Nonexcludability is included, so that process, method, article or equipment including a series of key elements not only will including those
Element, but also other key elements including being not expressly set out, or also include being this process, method, article or equipment
Intrinsic key element.In the absence of more restrictions, the key element limited by sentence "including a ...", it is not excluded that
Also there is other identical element in process, method, article or equipment including the key element.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although with reference to the foregoing embodiments
The present invention has been described in detail, it will be understood by those within the art that：It still can be to foregoing each implementation
Technical scheme described in example is modified, or carries out equivalent to which part technical characteristic；And these modification or
Replace, do not make the spirit and scope of the essence disengaging various embodiments of the present invention technical scheme of appropriate technical solution.
Claims (6)
1. a boar source Lactobacillus salivarius active bacteria formulation, it is characterised in that production technology is comprised the following steps：
(1) slant preservation strain is inoculated in liquid submerged culture base, concussion to logarithmic phase；
(2) above-mentioned cultured strain is accessed into 5L fermentation tanks by 5% inoculum concentration, is cultivated to exponential phase in culture medium,
The formula of culture medium is：Peptone 0.8-1.2%, yeast extract 0.4-0.8%, beef extract 1-1.5%, glucose 1.3-
2%th, sodium acetate 0.1-0.6%, sodium citrate 0.15-0.25%, Tween 80 0.05-0.15%, MgSO4·7H2O 0.05-
0.06%th, MnSO4·4H2O 0.001-0.005%, K2HPO40.1-03% and CaCO30.5-1.5%, adjusting pH value is
5.5, it is 37 DEG C to control cultivation temperature, and whole process incubation time is 55-65 hours, and thalline quantity can reach 500,000,000 after fermentation ends
Individual/more than ml；
(3) after culture terminates, suspending agent is added to the bacterium solution in fermentation tank, is made active bacteria formulation.
2. pig source Lactobacillus salivarius active bacteria formulation as claimed in claim 1, it is characterised in that the strain in the step (1)
It is that separation screening is obtained from the excrement of pig.
3. pig source Lactobacillus salivarius active bacteria formulation as claimed in claim 2, it is characterised in that strain in the step (1)
Separation screening is comprised the following steps：The lactic acid bacteria strains with prebiotic effect are separated from the rectum excrement of pig；In vitro culture；Hair
Ferment；Analyze influence of the separated bacterial strain to pig intestine microenvironment；Screening obtains the strain.
4. pig source Lactobacillus salivarius active bacteria formulation as claimed in claim 1, it is characterised in that culture medium in the step (2)
Formula be：Peptone 1%, yeast extract 0.6%, beef extract 1.2%, glucose 1.6%, sodium acetate 0.5%, citric acid
Sodium 0.215%, Tween 80 0.1%, MgSO4·7H2O 0.058%, MnSO4·4H2O 0.005%, K2HPO40.2% He
CaCO31%.
5. pig source Lactobacillus salivarius active bacteria formulation as claimed in claim 1, it is characterised in that the whole process incubation time is
60 hours, thalline quantity reached 500,000,000/more than ml after fermentation ends.
6. application of the pig source Lactobacillus salivarius active bacteria formulation as claimed in claim 1 in pig-breeding.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107788240A (en) * | 2017-09-25 | 2018-03-13 | 江西农业大学 | A kind of piglet necrotic enteritis microecologic regulator and preparation method thereof, the feed comprising conditioning agent |
| CN108522873A (en) * | 2018-04-20 | 2018-09-14 | 安徽天邦生物技术有限公司 | A kind of lactic acid bacteria and combination of prebiotics product improving piglet growth performance |
| CN110051701A (en) * | 2019-05-29 | 2019-07-26 | 中国农业大学 | Lactobacillus salivarius is inhibiting the application in Porcine epidemic diarrhea virus proliferation |
| CN113388550A (en) * | 2021-07-16 | 2021-09-14 | 新希望六和股份有限公司 | Lactobacillus salivarius NHE-LsE33 and application thereof |
| CN114164157A (en) * | 2022-02-09 | 2022-03-11 | 中科宜康（北京）生物科技有限公司 | Lactobacillus salivarius strain ZK-88 for inhibiting inflammation, relieving swelling and aching of gum, and improving oral flora balance |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107788240A (en) * | 2017-09-25 | 2018-03-13 | 江西农业大学 | A kind of piglet necrotic enteritis microecologic regulator and preparation method thereof, the feed comprising conditioning agent |
| CN108522873A (en) * | 2018-04-20 | 2018-09-14 | 安徽天邦生物技术有限公司 | A kind of lactic acid bacteria and combination of prebiotics product improving piglet growth performance |
| CN110051701A (en) * | 2019-05-29 | 2019-07-26 | 中国农业大学 | Lactobacillus salivarius is inhibiting the application in Porcine epidemic diarrhea virus proliferation |
| CN113388550A (en) * | 2021-07-16 | 2021-09-14 | 新希望六和股份有限公司 | Lactobacillus salivarius NHE-LsE33 and application thereof |
| CN114164157A (en) * | 2022-02-09 | 2022-03-11 | 中科宜康（北京）生物科技有限公司 | Lactobacillus salivarius strain ZK-88 for inhibiting inflammation, relieving swelling and aching of gum, and improving oral flora balance |
| CN114164157B (en) * | 2022-02-09 | 2022-06-03 | 中科宜康（北京）生物科技有限公司 | Lactobacillus salivarius strain ZK-88 for inhibiting inflammation, relieving swelling and aching of gum, and improving oral flora balance |
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