CN107164269A - A kind of lactobacillus paracasei, preparation and its application in pig feed - Google Patents
A kind of lactobacillus paracasei, preparation and its application in pig feed Download PDFInfo
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Abstract
The invention discloses a kind of lactobacillus paracasei (Lactobacillus paracasei) ZLP019, preserving number is：CGMCC No.11532.The bacterium has well acidproof, bile tolerance and antagonistic property.Lyophilized formulations are prepared into using the bacterium, Viable detection is high, convenient to be used in Feed Enterprise and plant.Said preparation is required in proportion to be directly appended in sow feed, it is possible to increase the reproductive performance of sow, improve suckling pig daily gain and survival rate, promote sow intestinal health, reduced harmful bacteria in excrement and discharge, be conducive to breeding environment friendly.
Description
Technical field
The present invention relates to biological feedstuff technical field, and in particular to a kind of lactobacillus paracasei, preparation and its in pig feed
In application.
Background technology
In Swine Production, sow carries the vital task of breeding piglet and suckling pig, and utilization periods are long, plays and makees
With big.But, usually there is constipation, loss of appetite, Farrowing Traits are poor, lactation amount is few, piglet survival ratio in the sow of large-scale pig farm
Low problem, has a strong impact on the Growth Results and economic benefit of plant.
Lactic acid bacteria has regulation and control animal intestinal micro-ecology balance, promotes nutrient digestion to absorb and promote body health
Effect, it is rationally feeding, it is expected to improve the integrated production capacity of sow.
During lactobacillus paracasei (Lactobacillus paracasei) is Lactobacillus (Lactobacillus)
One of population, is commonly applied to the production such as dairy products, health food, beverage, ice cream.In recent years, the Ministry of Agriculture《Feed addictive product
Plant catalogue (2013)》Allow lactobacillus paracasei to be applied to animal-breeding, promote the application in terms of feed cultivation.However,
About lactobacillus paracasei preparation, and preparation method thereof and feed cultivation in advanced technology it is fewer, Publication No. CN
103070287 A patent application " lactobacillus paracasei fermented feed and preparation method thereof, Zymolysis Equipment and purposes ", is disclosed
Lactobacillus paracasei liquid fermentation and lactobacillus paracasei solid fermentation step, fermented feed can reduce diarrhea of weaned piglets rate,
Improve growth speed of pigs, improve food conversion utilization ratio；The B of Publication No. CN 103039699 patent application " one kind mixing
Bacterium fermented feed and its methods for making and using same ", is disclosed using lactobacillus paracasei CICC20296, bacillus subtilis
ACCC10619 and saccharomyces cerevisiae CICC32236, carries out liquid fermentation and culture respectively, zymotic fluid by volume 1:1:After 1 mixing,
Solid fermentation culture is carried out again, is added to by a certain percentage in weanling pig feed, is improved Production Performance of Weaning Pigs.More than
The lactobacillus paracasei being related in patent is mainly used in the preparation of fermented feed, and its preparation process needs Zymolysis Equipment, fermentation
The time that process expends is longer, and production operation is relatively complicated, prepared by the lyophilized formulations for not yet relating to lactobacillus paracasei at present
Method, the lyophilized formulations for being also not involved with steady quality directly make an addition in feed and can improve sow reproductive performance in proportion
Method.
The content of the invention
It is a kind of new it is an object of the present invention to for the practical problem and demand in production practices, develop
Lactobacillus paracasei, the lactobacillus paracasei has stronger acidproof, bile tolerance and suppresses the characteristics such as harmful bacteria.
It is another object of the present invention to provide the lactobacillus paracasei preparation prepared by the lactobacillus paracasei
And preparation method thereof.
A further object of the present invention is that provide the lactobacillus paracasei is directly appended in pig feed in proportion
Using.
To realize that first purpose of the invention provides a kind of lactobacillus paracasei bacterial strain, one plant is filtered out from sodium selenite
The lactobacillus paracasei bacterial strain of function admirable, by the lactobacillus paracasei bacterial strain on October 22nd, 2015 in China Microbiological bacterium
Plant preservation administration committee common micro-organisms center (Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences microorganism
Research institute, postcode：100101) preservation is carried out, strain name is lactobacillus paracasei (Lactobacillus
Paracasei) ZLP019, deposit number is：CGMCC No.11532.
Lactobacillus paracasei (Lactobacillus paracasei) ZLP019 (CGMCC No.11532) is blue for leather
Family name's positive bacillus, single or paired arrangement, no gemma.Bacterium colony is that milky is opaque on MRS plating mediums, circular, grand
Rise, neat in edge.
Lactobacillus paracasei (Lactobacillus paracasei) ZLP019 (CGMCC that the present invention is provided
No.11532 it is specific as follows also with acidproof, bile tolerance and antagonistic property) in addition to above-mentioned growth characteristics:
1) survival rate that the lactobacillus paracasei acts in pH=2 environment 2 hours is 97.58%, is deposited within 4 hours
Motility rate is 76.22%, and the survival rate of effect 2 hours was up to the survival rate of more than 99%, 4 hour in pH is 3 and 4 sour environment
Up to more than 85%.
2) lactobacillus paracasei is in 0.1% and 0.2% cholate environment, 37 DEG C of incubation 3h, and survival rate is respectively
82.25% and 56.08%.
3) lactobacillus paracasei is to 3 boar common pathogens, such as Escherichia coli, salmonella, Staphylococcus aureus
Bacterium etc. has stronger inhibitory action, and antibacterial circle diameter is in 17~27mm.
Second object of the present invention provides a boar source lactobacillus paracasei preparation, and the preparation contains the newborn bar of secondary cheese
Bacterium (Lactobacillus paracasei) ZLP019, preserving number is：CGMCC No.11532.
Wherein it is preferred to, the viable count of lactobacillus paracasei (Lactobacillus paracasei) ZLP019 can
For 1 × 1011CFU/g and more than.
Pig source as described above lactobacillus paracasei preparation, can be used as a kind of feed addictive.
The present invention also provides the preparation method of the lactobacillus paracasei preparation, and it is to be fermented in liquid fermentation medium
Lactobacillus paracasei ZLP019, the zymotic fluid is centrifuged, emulsify it is lyophilized, the freeze-dried powder being prepared into be lactobacillus paracasei
Preparation.
Preferably, described preparation method may particularly include following steps：
(1) fermented and cultured：The lactobacillus paracasei ZLP019 is inoculated in by MRS fluid nutrient mediums with 1% inoculum concentration
In, 37 DEG C are cultivated 16~24 hours, so obtain inoculum after 2 generations of activation；
Inoculum is inoculated in the fermentation tank equipped with fluid nutrient medium with 1% inoculum concentration, 37 DEG C of design temperature,
Rotating speed 100rpm, maintains constant pH 5.5, starts feed supplement when fermentation was to 14 hours, adds feed supplement liquid, entered afterwards every 2 hours
Go once, altogether feed supplement 5 times；
When zymotic fluid pH is no longer reduced, fermentation is terminated, zymotic fluid is obtained；
(2) zymotic fluid is centrifuged：Zymotic fluid obtained above is centrifuged, bacterium mud is obtained；
(3) emulsify lyophilized：The bacterium mud that step (2) is obtained is mixed with the freeze drying protectant of fermentating liquid volume described in 1/3, high
After shear dispersion emulsifying machine emulsification, it is sub-packed in refrigerator tray, 1.5~2.5cm of thickness, pre-freeze 3 hours, vacuum freeze drying is set
- 50 DEG C of machine condensation temperature, vacuum 10Pa, after freezing 24 hours, produce the lyophilized formulations containing lactobacillus paracasei.
Preferably, the fluid nutrient medium by following concentration material composition：Glucose 30g/L, peptone 10g/L, ox
Meat extract 10g/L, sodium acetate 5g/L, yeast extract 10g/L, K2HPO4·3H2O 2g/L, diammonium hydrogen citrate 2g/L, MgSO4·
7H2O 0.5g/L, MnSO46.5,121 DEG C of sterilizing 15min of 0.2g/L, Tween-80 1ml/L, pH are standby.
The feed supplement liquid is glucose solution, and concentration is 300g/L.
Preferably, the freeze drying protectant is is respectively 14% skimmed milk, 8% marine alga containing mass percent concentration
Sugar, 5% dextrin, 1.5% Cys, 1% sodium glutamate, 0.5% sodium carboxymethylcellulose, remaining is water.
Application present invention also offers the pig source lactobacillus paracasei preparation of above-mentioned preparation as additive in feed.
Preferably, applied in pig feed, it is highly preferred that in latter half of gestation and the feed of nursery phase sow.
Further, the application is that lactobacillus paracasei ZLP019 preparations are added in basal diet, the secondary cheese breast
Viable counts of the bacillus ZLP019 in every kilogram of sow complete diet pellet is 2 × 109CFU。
Present invention also offers a kind of freeze drying protectant, its contain mass percent concentration be respectively 14% skimmed milk,
8% trehalose, 5% dextrin, 1.5% Cys, 1% sodium glutamate, 0.5% sodium carboxymethylcellulose,
70% water.
Beneficial effects of the present invention are：
Lactobacillus paracasei (Lactobacillus paracasei) ZLP019, CGMCC No.11532 of the present invention, tool
There are good acidproof, bile tolerance and antagonistic property.The lyophilized formulations being made up of the bacterium, can be added to pregnant directly as additive
It is pregnent in the sow feed of later stage and nursery phase, viable counts of the lactobacillus paracasei ZLP019 in complete feeds for milking sow is
2×109CFU/Kg, milking sow average daily gain improves 4.48%, and constipation rate reduces by 8.51 percentage points；Suckling pig is put down
Equal daily gain improves 8.90%, and number of weaned improves 8.65 percentage points, and diarrhea rate reduces by 31.92 percentage points；Sow excrement
Just middle harmful bacteria, such as coliform count, salmonella and the viable count of staphylococcus aureus are substantially reduced.As can be seen here,
Lactobacillus paracasei ZLP019 feeds the sow of latter half of gestation and nursery phase, it is possible to increase the reproductive performance of sow, improves and feeds
The daily gain and number of weaned of suckling piglet, can also reduce harmful bacteria in excrement and discharge, be conducive to breeding environment friendly.
Brief description of the drawings
Fig. 1 is lactobacillus paracasei ZLP019 colonial morphology.
Fig. 2 is lactobacillus paracasei ZLP019 16S rRNA phylogenetic trees collection of illustrative plates.
Fig. 3 is lactobacillus paracasei ZLP019 pheS phylogenetic trees collection of illustrative plates.
Fig. 4 is lactobacillus paracasei ZLP019 growth curve.
Embodiment
The present invention is discussed in detail with reference to embodiment, it should be appreciated that the scope of protection of present invention is not by described specific
The limitation of embodiment, the specific embodiment that the present invention is provided is only as the example for further illustrating the present invention, art technology
Personnel modify to the embodiment of the present invention with reference to the description of this case specification or some technical characteristics are carried out
Equivalent substitution, these should also fall into the protection domain of appended claims of the present invention without the improvement and replacement of creative work
Within.If not specified, the conventional technical means that technological means used is well known to those skilled in the art in embodiment.
The separation screening of the lactobacillus paracasei of embodiment 1 and identification
The healthy child care pig of selection is slaughtered, and enteron aisle, scraping intestinal mucosa are taken out in sterile working, are mixed with sterile saline, ten
10 are diluted to again-5、10-6、10-7, respectively take 0.1mL to be coated on MRS plating mediums, the 37 DEG C of cultures 24~48 of anaerobic jar method are small
When.Therefrom picking milky bacterium colony carries out preliminary screening, every Gram-positive, H2O2Enzyme test is negative, catalase is negative
Bacterial strain can primarily determine that as lactic acid bacteria the bacterial strain for obtaining primary dcreening operation carries out the specificity analysis such as acid and bile salt tolerance, antibacterial and tested, most
The lactic acid bacteria strains ZLP019 of one plant of function admirable is filtered out eventually, delivers to China General Microbiological DSMZ (CGMCC)
Identified, it is comprehensive through test datas such as cell morphology characteristic, physio-biochemical characteristics, 16S rRNA sequences and pheS gene orders
Analysis shows are closed, the dientification of bacteria is Lactobacillus paracasei.ZLP019 is Gram-positive bacillus, it is single or into
To arrangement, no gemma.Its colony characteristics on MRS plating mediums is that milky is opaque as shown in Figure 1, circular, grand
Rise, neat in edge.The qualification result of the bacterium physio-biochemical characteristics is shown in Table 1,16S rRNA gene orders such as SEQ ID No.1 institutes
Show, pheS gene orders are as shown in SEQ ID No.2.Phylogenetic analysis is carried out by the 16S rRNA genes to the bacterium, it enters
Change tree graph spectrum as shown in Figure 2；Phylogenetic analysis is carried out to the pheS genes of the bacterium, its chadogram collection of illustrative plates is as shown in Figure 3.Above-mentioned knot
Fruit shows that lactobacillus paracasei (Lactobacillus paracasei) ZLP019 that the present invention is provided is different from existing secondary dry
Lactobacillus paracasei, is new strains.
The lactobacillus paracasei ZLP019 of table 1 physiological and biochemical property
| Experimental project | As a result | Experimental project | As a result | Experimental project | As a result |
| Glycerine | - | Inositol | - | Synanthrin | + |
| Erythrite | - | Mannitol | + | Melezitose | + |
| D-R | - | Sorbierite | + | Gossypose | - |
| L-arabinose | - | Alpha-Methyl-D-MANNOSE glucoside | - | 2- keto-D-gluconate salt | - |
| D-ribose | + | Alpha-Methyl-D-Glucose glucoside | - | Beta-methyl-xyloside | - |
| D- xyloses | - | N- formyls-aminoglucose | + | Xylitol | - |
| L- xyloses | - | Amygdalin | + | Adonite | - |
| Glycogen | - | Arbutin | + | D- turanoses | + |
| D- galactolipins | + | Aesculin | + | D- lyxoses | - |
| D-Glucose | + | Cellobiose | + | D-Tag | + |
| D-Fructose | + | Maltose | + | D- fucoses | - |
| D-MANNOSE | + | Lactose | - | L-fucose | - |
| L- sorboses | - | Melibiose | - | D-R alcohol | - |
| L- rhamnoses | - | Sucrose | + | L-arabinose alcohol | - |
| Dulcitol | + | Trehalose | + | Starch | - |
Note：:"+", is positive；"-", is negative.
The lactobacillus paracasei bacterial strain is general in China Committee for Culture Collection of Microorganisms on October 22nd, 2015
Logical microorganism center (address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postal service is compiled
Code：100101) preservation is carried out, strain name is lactobacillus paracasei (Lactobacillus paracasei) ZLP019, preservation
Numbering is CGMCC No.11532.
Wherein, MRS solid cultures based formulas：Peptone 1g, beef extract 1g, yeast extract 0.5g, citric acid hydrogen diamine 0.2g,
Glucose 2g, K2HP040.2g, sodium acetate 0.2g, Tween-80 0.1ml, magnesium sulfate 0.058g, manganese sulfate 0.02g, agar 2g,
Water 100m1, pH 6-6.5,121 DEG C of autoclaving 15min.
The characteristic research of the lactobacillus paracasei of embodiment 2
1st, the growth curve of lactobacillus paracasei
The lactobacillus paracasei that the embodiment 1 of activation is obtained accesses MRS fluid nutrient mediums with 1% inoculum concentration, uses
Full automatic microorganism growth curve analyzer detects OD600Value, draws growth curve, its result as shown in Fig. 2 wherein abscissa
For incubation time, unit is hour, and ordinate is absorbance OD600Value.Test result indicates that, lactobacillus paracasei is cultivated in MRS
Grown rapidly in base, enter logarithmic phase in 4h or so, 11h or so enters stationary phase.
2nd, acid resistance is determined
The lactobacillus paracasei of activation is respectively connected to by 1% inoculum concentration in the MRS fluid nutrient mediums that pH is 2,3 and 4,
37 DEG C of 2,4 and 6h of insulation, using 0h as control, determine viable count, calculate survival rate, the results are shown in Table 2 respectively.
The lactobacillus paracasei acid resistance (%) of table 2
As a result illustrate, the survival rate that this lactobacillus paracasei acts on 2h in pH=2 sour environment is 97.58%, and 4 is small
When survival rate be 76.22%；The survival rate of effect 2 hours reaches more than 99%, work in pH is 3 and 4 sour environment
More than 85% is reached with the survival rate of 4 hours.Illustrate that the lactobacillus paracasei has very strong acid resistance, can be good at resistance to
By the gastric acid environment of animal.
2nd, bile tolerance performance measurement
The lactobacillus paracasei of activation is respectively connected to containing 0.1%, 0.2% and 0.3% Pig cholate by 1% inoculum concentration
MRS fluid nutrient mediums in, 37 DEG C insulation 3,6h, using 0h viable count for control, calculating survival rate, the results are shown in Table 3.
The lactobacillus paracasei bile tolerance performance measurement (%) of table 3
As a result illustrate, this bacterium acts on 3h in 0.1% cholate environment, its survival rate is 82.25%；In 0.2% cholate
3h is acted in environment, its survival rate is 56.08%；3h is acted under 0.3% gallbladder salinity, its survival rate is only 10.47%.
Illustrate that the lactobacillus paracasei has higher tolerance for 0.1% cholate.
3rd, bacteriostatic test
Using Odontothrips loti, the fungistatic effect of the lactobacillus paracasei zymotic fluid pig pathogenic bacteria common to 3 kinds is detected, as a result
It is shown in Table 4.
The lactobacillus paracasei bacteriostatic test of table 4
It can be seen from the results that the lactic acid bacteria is to this 3 kinds of harmful bacterias：(C838901 is purchased from Chinese beast to Escherichia coli
Pharmaceuticals supervise institute), cholera salmonella (C78-4 is purchased from China Veterinery Drug Inspection Office), staphylococcus aureus
(C56023 is purchased from China Veterinery Drug Inspection Office) has stronger inhibitory action, and antibacterial circle diameter reaches 17~27mm.
4th, drug sensitive test
The drug sensitive test of 26 kinds of antibiotic (OXOID drug sensitive test papers) has been carried out to lactobacillus paracasei, 5 are the results are shown in Table.
The lactobacillus paracasei drug sensitive test of table 5
Note：S is sensitivity；I is medium sensitivity；R is resistance.
As can be seen from Table 5, the bacterium is to ampicillin, tetracycline, Amoxicillin, azithromycin, polymyxin e, cephalo
Piperazine ketone, CTX, ceftriaxone, Enrofloxacin, amikacin, gentamicin, clindamycin, rifampin, Florfenicol, chlorine
The sensitivities such as mycin, it is husky to radonil, erythromycin, bacitracin, cefalexin, Ceftizoxime, ring third to Ofloxacin medium sensitivity
The antibiosis such as star, Norfloxacin, kanamycins, streptomysin, nystatin have drug resistance.
The preparation of the lactobacillus paracasei preparation of embodiment 3
1st, the fermented and cultured of lactobacillus paracasei
Lactobacillus paracasei (Lactobacillus paracasei) ZLP019 (preserving numbers that embodiment 1 is obtained：
CGMCC No.11532) it is inoculated in 1% inoculum concentration in MRS fluid nutrient mediums, 37 DEG C are cultivated 16~24 hours, so activation
Inoculum is obtained after 2 generations；Inoculum is inoculated in into the 50L equipped with 30L fluid nutrient mediums with 1% inoculum concentration to ferment
In tank, 37 DEG C of design temperature, rotating speed 100rpm maintain constant pH 5.5, start feed supplement when fermentation was to 14 hours, to fermentation tank
In once add 200mL feed supplement liquids, a feed supplement, altogether feed supplement 5 times were carried out every 2 hours afterwards；When zymotic fluid pH is no longer reduced
When, terminate fermentation.
Wherein, fluid nutrient medium composition is：Glucose 30g/L, peptone 10g/L, beef extract 10g/L, sodium acetate 5g/L,
Yeast extract 10g/L, K2HPO4·3H2O 2g/L, diammonium hydrogen citrate 2g/L, MgSO4·7H2O 0.5g/L, MnSO4 0.2g/
6.5,121 DEG C of sterilizing 15min of L, Tween-80 1ml/L, pH are standby.
The feed supplement liquid is glucose solution, and concentration is 300g/L, and 115 DEG C of sterilizing 15min are standby.
2nd, zymotic fluid is centrifuged
The zymotic fluid that the step 1 is obtained, with tube centrifuge separating thallus, obtains bacterium mud under the conditions of 19000rpm；
3rd, the preparation of lyophilized formulations
Bacterium mud is mixed with the freeze drying protectant of 1/3 fermentating liquid volume, using high-shearing dispersion emulsifying machine 3000r/min breasts
Freezed after changing 30min, produce lactobacillus paracasei lyophilized formulations.
Wherein, the bacteria suspension after emulsification is is sub-packed in refrigerator tray by lyophilisation condition, and 1.5~2.5cm of thickness, pre-freeze 3 is small
When, -50 DEG C of condensation temperature, vacuum 10Pa, freeze-drying time 24 hours are set.Wherein, freeze drying protectant is by following quality percentage
The material composition of specific concentration：Skimmed milk 14%, trehalose 8%, dextrin 5%, Cys 1.5%, sodium glutamate 1%, carboxylic
Sodium carboxymethylcellulose pyce 0.5%, remaining is water.It is formulated with this, the frozen-dried protective rate of lactobacillus paracasei is up to 88.20%.Wherein,
Frozen-dried protective rate calculation formula is：Frozen-dried protective rate (%)=(after lyophilized bacterium powder viable count × bacterium powder weight after lyophilized)/it is (lyophilized
Preceding bacteria suspension viable count × lyophilized preceding bacteria suspension volume) × 100.
The lactobacillus paracasei lyophilized formulations prepared are taken, are carried out with sterile saline after gradient dilution, it is flat using MRS
Plate medium culture surveys viable count, and the viable count for measuring lactobacillus paracasei in freeze-dried powder is 1.20 × 1011CFU/g。
4th, lactobacillus paracasei lyophilized formulations bin stability is determined
The lactobacillus paracasei lyophilized formulations of above-mentioned preparation are vacuum-packed with aluminium foil bag, -18 DEG C preserve 1 month, 6
Month, after 12 months, take bacterium powder to be carried out with sterile saline after gradient dilution, using MRS plating mediums survey viable count.By table
6 understand that lactobacillus paracasei lyophilized formulations Cord blood is after 12 months, and Viable detection still can reach 83.66%, the quality of the pharmaceutical preparations
It is stable.
The lactobacillus paracasei lyophilized formulations shelf characteric of table 6 is determined
Application of the lactobacillus paracasei preparation of embodiment 4 in sow produces
1st, experimental animal and packet
From health, parity be close, the binary sow 100 that grows up of pregnant 85 days, control group and test group 2 are randomly divided into
Individual group, every group of 50 repetitions each repeat 1 sow.Control group is normally fed, and test group adds on the basis of feeding basal diet
Plus the pig source lactobacillus paracasei lyophilized formulations (viable bacteria final concentration of 2 × 10 of complete diet pellet9CFU/Kg).Wherein, the basis of sow
Daily ration reference《Swine rearing standard》(2004) prepare, specific nutritional ingredient measured value is shown in Table 7.Sow is raised in same colony house,
Feeding management and immune programme for children etc. are performed by pig farm unified institutions.Test period is that sow gestation starts on the 85th day, young to lactation
Untill the age in days of pig 28 weans.
The basal diet nutritional ingredient measured value (%) of table 7
3rd, trial
During experiment, daily observation swinery growing state records food consumption and sows farrowing, litter weight, diarrhoea etc.；Experiment
After end, every group is taken the fresh excrement sample of 10 portions of sows, determines the work of lactic acid bacteria, Escherichia coli and staphylococcus aureus etc. in excrement
Bacterium number.Data statistics carries out one-way analysis of variance using the statistical softwares of SPSS 18.0, as a result with mean+SD table
Show.
4th, result and analysis
4.1 pig source lactobacillus paracasei preparations are shown in Table 8 to the result of the test of sow reproductive performance.
Influence of the pig source lactobacillus paracasei preparation of table 8 to sow reproductive performance
Note：Different with shoulder mark to represent significant difference, lowercase difference represents significant difference (P<0.05).
Compared with control group, test group piglet birth individual weight improves 7.01% (P<0.05), wean individual weight is improved
8.33% (P<0.05), number of weaned improves 8.65 percentage points of (P<0.05), suckling pig daily gain is improved
8.90% (P<0.05), milking sow daily ingestion amount improves 4.48% (P<0.05), suckling pig diarrhea rate reduces 31.92
Percentage point, milking sow constipation rate reduces 8.51 percentage points.As can be seen here, the feeding of pig source lactobacillus paracasei preparation is pregnant
It is pregnent the sow of later stage and nursery phase, it is possible to increase sow reproductive performance, promotes piglet health and survival rate to improve.
Influence result 9 of the 4.2 pig source lactobacillus paracasei preparations to milking sow fecal microorganism.
Influence of the pig source lactobacillus paracasei preparation of table 9 to milking sow fecal microorganism
Note：Different with shoulder mark to represent significant difference, lowercase difference represents significant difference (P<0.05).
As shown in Table 9, compared with control group, lactic acid bacteria number significantly improves (P in test group sow excrement<0.05), large intestine
Bacillus number and salmonella number substantially reduce (P<0.05).As can be seen here, pig source lactobacillus paracasei preparation feeding sow, can
The harmful bacteria in excrement is reduced, intestinal health and breeding environment, which are improved, positive role.
To sum up, this lactobacillus paracasei preparation feeding latter half of gestation and nursery phase sow, it is possible to increase sow it is numerous
Performance is grown, the daily gain and number of weaned of suckling pig is improved, promotes sow intestinal health, the harmful bacteria row in excrement is reduced
Put, be conducive to breeding environment friendly.
SEQUENCE LISTING
<110>Beijing City Agriculture and Forestry Institute
<120>A kind of lactobacillus paracasei, preparation and its application in pig feed
<130>
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1401
<212> DNA
<213>Lactobacillus paracasei（Lactobacillus paracasei）ZLP019 16S rRNA genetic fragments
<400> 1
ttgatgatcg gtgcttgcac cgagattcaa catggaacga gtggcggacg ggtgagtaac 60
acgtgggtaa cctgccctta agtgggggat aacatttgga aacagatgct aataccgcat 120
agatccaaga accgcatggt tcttggctga aagatggcgt aagctatcgc ttttggatgg 180
acccgcggcg tattagctag ttggtgaggt aatggctcac caaggcgatg atacgtagcc 240
gaactgagag gttgatcggc cacattggga ctgagacacg gcccaaactc ctacgggagg 300
cagcagtagg gaatcttcca caatggacgc aagtctgatg gagcaacgcc gcgtgagtga 360
agaaggcttt cgggtcgtaa aactctgttg ttggagaaga atggtcggca gagtaactgt 420
tgtcggcgtg acggtatcca accagaaagc cacggctaac tacgtgccag cagccgcggt 480
aatacgtagg tggcaagcgt tatccggatt tattgggcgt aaagcgagcg caggcggttt 540
tttaagtctg atgtgaaagc cctcggctta accgaggaag cgcatcggaa actgggaaac 600
ttgagtgcag aagaggacag tggaactcca tgtgtagcgg tgaaatgcgt agatatatgg 660
aagaacacca gtggcgaagg cggctgtctg gtctgtaact gacgctgagg ctcgaaagca 720
tgggtagcga acaggattag ataccctggt agtccatgcc gtaaacgatg aatgctaggt 780
gttggagggt ttccgccctt cagtgccgca gctaacgcat taagcattcc gcctggggag 840
tacgaccgca aggttgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat 900
gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatctt tttgatcacc 960
tgagagatca ggtttcccct tcgggggcaa aatgacaggt ggtgcatggt tgtcgtcagc 1020
tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttatg actagttgcc 1080
cagcatttag ttgggcactc tagtaagact gccggtgaca aaccggagga aggtggggat 1140
gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa tggatggtac 1200
aacgagttgc gagaccgcga ggtcaagcta atctcttaaa gccattctca gttcggactg 1260
taggctgcaa ctcgcctaca cgaagtcgga atcgctagta atcgcggatc agcacgccgc 1320
ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accatgagag tttgtaacac 1380
ccgaagccgg tggcgtaacc c 1401
<210> 2
<211> 394
<212> DNA
<213>PheS gene orders
<400> 2
caagtccaat gcaggcgcgg acaatggaaa agcacgactt taccaaagga ccgctgaaaa 60
tgattagccc tggggtggtt tatcgacgtg atgacgacga tgctactcat agccatcagt 120
ttcaccagat ggaaggactc gtcattgaca agcatataac catggctgat ctaaagggaa 180
ccttgttggc catgtgccaa cacgtgtttg gtaaagatcg gacaattcgc ttgcggccaa 240
gttattttcc atttacggag ccatccgttg aagttgatgt ttcctgtttt cgttgcggcg 300
gtaaaggttg cccggtttgc aaatataccg gttggattga agtgttaggt gccggcatgg 360
tgcatcccaa tgtgctacgg gcagcgaaca ttga 394
Claims (10)
1. a kind of lactobacillus paracasei (Lactobacillus paracasei) new strains ZLP019, its preserving number is CGMCC
No.11532。
2. a kind of lactobacillus paracasei preparation, it is characterised in that contain lactobacillus paracasei as claimed in claim 1
ZLP019。
3. a kind of feed addictive, it is characterised in that contain lactobacillus paracasei ZLP019 preparations as claimed in claim 2.
4. a kind of preparation method of lactobacillus paracasei preparation, it is characterised in that fermented and cultured is such as in liquid fermentation medium
Lactobacillus paracasei ZLP019 described in claim 1, obtains zymotic fluid, and the zymotic fluid is centrifuged, lyophilized, system is emulsified
It is standby into freeze-dried powder be lactobacillus paracasei preparation.
5. preparation method according to claim 4, it is characterised in that specifically include following steps：
(1) fermented and cultureds：The lactobacillus paracasei ZLP019 is inoculated in MRS fluid nutrient mediums with 1% inoculum concentration,
37 DEG C are cultivated 16~24 hours, so obtain inoculum after 2 generations of activation；
Inoculum is inoculated in the fermentation tank equipped with fluid nutrient medium with 1% inoculum concentration, 37 DEG C of design temperature, rotating speed
100rpm, maintains constant pH 5.5, starts to add feed supplement liquid when fermentation was to 14 hours, is carried out once every 2 hours afterwards, altogether
Feed supplement 5 times；
When zymotic fluid pH is no longer reduced, fermentation is terminated, zymotic fluid is obtained；
(2) zymotic fluids are centrifuged：The zymotic fluid centrifugation that the step (1) is obtained, obtains bacterium mud；
(3) emulsifications are lyophilized：The bacterium mud that the step (2) is obtained is mixed with the freeze drying protectant of fermentating liquid volume described in 1/3,
High-shearing dispersion emulsifying machine is emulsified, and is sub-packed in refrigerator tray, 1.5~2.5cm of thickness, pre-freeze 3 hours, sets vacuum freeze drying
- 50 DEG C of machine condensation temperature, vacuum 10Pa, after freezing 24 hours, produce lactobacillus paracasei preparation.
6. preparation method according to claim 5, it is characterised in that described fluid nutrient medium by following concentration material
Composition：Glucose 30g/L, peptone 10g/L, beef extract 10g/L, sodium acetate 5g/L, yeast extract 10g/L, K2HPO4·3H2O
2g/L, diammonium hydrogen citrate 2g/L, MgSO4·7H2O 0.5g/L, MnSO40.2g/L, Tween-80 1ml/L, pH 6.5；
The feed supplement liquid is glucose solution, and concentration is 300g/L；
The freeze drying protectant is is respectively 14% skimmed milk, 8% trehalose, 5% paste containing mass percent concentration
Essence, 1.5% Cys, 1% sodium glutamate, 0.5% sodium carboxymethylcellulose, remaining is water.
7. application of the lactobacillus paracasei preparation in feed described in claim 2.
8. application according to claim 7, it is characterised in that the feed is pig feed.
9. application according to claim 8, it is characterised in that the lactobacillus paracasei system is added in basal diet
Agent, it is 2 × 10 to make viable count of the lactobacillus paracasei in every kilogram of sow complete diet pellet9CFU。
10. a kind of freeze drying protectant, its contain mass percent concentration be respectively 14% skimmed milk, 8% trehalose, 5%
Dextrin, 1.5% Cys, 1% sodium glutamate, 0.5% sodium carboxymethylcellulose, remaining is water.
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| CN108315285A (en) * | 2018-05-08 | 2018-07-24 | 西藏高原之宝牦牛乳业股份有限公司 | A kind of Lactobacillus paracasei and preparation method thereof |
| CN108373984A (en) * | 2018-04-09 | 2018-08-07 | 北京博锦元生物科技有限公司 | A kind of Lactobacillus paracasei and its application |
| CN110804571A (en) * | 2019-12-02 | 2020-02-18 | 华中农业大学 | Compound lactobacillus preparation and application thereof in preparing feed additive |
| CN110810629A (en) * | 2018-08-07 | 2020-02-21 | 中国农业大学 | Probiotic preparation and application thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101768559A (en) * | 2008-12-31 | 2010-07-07 | 生展生物科技股份有限公司 | Lactobacillus, paracasei subsp. paracasei strain, bacteriostatic composition and applications thereof |
| CN106497828A (en) * | 2016-10-14 | 2017-03-15 | 北京市农林科学院 | A kind of application of lactobacillus casei and its lyophilized formulations in pig feed |
-
2017
- 2017-06-06 CN CN201710418582.9A patent/CN107164269B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101768559A (en) * | 2008-12-31 | 2010-07-07 | 生展生物科技股份有限公司 | Lactobacillus, paracasei subsp. paracasei strain, bacteriostatic composition and applications thereof |
| CN106497828A (en) * | 2016-10-14 | 2017-03-15 | 北京市农林科学院 | A kind of application of lactobacillus casei and its lyophilized formulations in pig feed |
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| CN108373984A (en) * | 2018-04-09 | 2018-08-07 | 北京博锦元生物科技有限公司 | A kind of Lactobacillus paracasei and its application |
| CN108315285A (en) * | 2018-05-08 | 2018-07-24 | 西藏高原之宝牦牛乳业股份有限公司 | A kind of Lactobacillus paracasei and preparation method thereof |
| CN110810629A (en) * | 2018-08-07 | 2020-02-21 | 中国农业大学 | Probiotic preparation and application thereof |
| CN110804571A (en) * | 2019-12-02 | 2020-02-18 | 华中农业大学 | Compound lactobacillus preparation and application thereof in preparing feed additive |
| CN110804571B (en) * | 2019-12-02 | 2021-03-05 | 华中农业大学 | Compound lactobacillus preparation and application thereof in preparing feed additive |
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| CN114292779A (en) * | 2021-12-22 | 2022-04-08 | 清远西周生物医药科技有限公司 | Lactobacillus paracasei freeze-dried powder, application and preparation method thereof |
| CN115141780A (en) * | 2022-07-19 | 2022-10-04 | 重庆市畜牧科学院 | Compound microbial agent and application thereof |
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| CN116121120B (en) * | 2022-11-30 | 2024-04-19 | 天津小薇生物科技有限公司 | Lactobacillus paracasei GF009 with antibacterial effect, preparation method of its progeny and application thereof |
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