CN110463824A - Ovshinsky lactobacillus strain BSLO 1801 and its application in Egg Production of Laying Hens - Google Patents
Ovshinsky lactobacillus strain BSLO 1801 and its application in Egg Production of Laying Hens Download PDFInfo
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Abstract
The present invention provides Ovshinsky lactobacillus strain BSLO 1801 and its applications in Egg Production of Laying Hens, belong to technical field of animal.The deposit number of Ovshinsky lactobacillus strain BSLO 1801 is CCTCC No.M 2019287.For improving performance in layers and improving the dedicated probiotics preparation of laying hen for quality of laying eggs, including the Ovshinsky lactobacillus strain BSLO 1801.Experiment shows after Ovshinsky lactobacillus strain BSLO 1801 is fed laying hen together with basal diet, can significantly improve performance in layers, the quality for improving egg, adjusts ileum intestinal flora and adjust hormone serum level.Therefore the present invention provides application of the egg feedstuff in layer breeding described in the Ovshinsky lactobacillus strain BSLO 1801.
Description
Technical field
The invention belongs to technical field of animal, and in particular to Ovshinsky lactobacillus strain BSLO 1801 and its in laying hen
Application in laying eggs.
Background technique
Egg is almost daily indispensable food, containing all nutriments needed by human, such as protein, fat, dimension life
Element and microelement etc., are often referred to as " ideal nutrition library " by people.The high laying rate of laying hen and preferable egg quality are almost
It is that raiser raises pursuing a goal for laying hen.
In the prior art, in order to improve the laying rate of laying hen and quality of laying eggs, often through adjustment chicken feed nutritional ingredient
The immunity of laying hen is improved simultaneously to realize.For example, the patent of publication number CN105432943A discloses a kind of ecological chicken feed
Production method, wherein formula in include complex enzyme formulation and probiotics, the feed of preparation not only has full of nutrition, in good taste
The characteristics of outside, there are also calmness compose oneself, bactericidal antiphlogistic, enhance disease resistance effect, increase laying hen egg production, improve egg wind
The effect of taste.In recent years, new probiotics strain is developed in the further investigation with people to probiotics, is improving laying hen
There are advantage outstanding, such as the patent disclosure of publication number CN105624070A in terms of Egg Quality and raising performance in layers
One plant of rhamnose lactic acid bacteria strain W27, the bacterial strain can promote host's Calcium and phosphorous absorption, can be improved eggshell strength, improve simultaneously
Performance in layers.
Summary of the invention
In view of this, the purpose of the present invention is to provide one plant of new lay eggs in raising quality and to improve laying rate of laying hen
Aspect shows the application of the Ovshinsky lactobacillus strain BSLO 1801 and the bacterial strain of excellent properties in Egg Production of Laying Hens.
The present invention provides Ovshinsky lactobacillus strain BSLO 1801 (Lactobacillus oris), the Ovshinsky cream bars
The deposit number of bacteria strain BSLO 1801 is CCTCC No.M 2019287.
The present invention provides a kind of dedicated probiotics systems of laying hen for improving performance in layers and improving quality of laying eggs
Agent, including the Ovshinsky lactobacillus strain BSLO 1801.
Preferably, the viable bacteria concentration of the Ovshinsky lactobacillus strain BSLO 1801 is 1 × 106CFU/kg~1 × 108CFU/
kg。
Preferably, the dedicated probiotics preparation of the laying hen further includes basal diet.
The present invention provides the Ovshinsky lactobacillus strain BSLO 1801 or the dedicated probiotics preparation of the laying hen in laying hen
Application in raising.
The present invention provides the Ovshinsky lactobacillus strain BSLO 1801 or the dedicated probiotics preparation of the laying hen to improve
Application in performance in layers.
The present invention provides the Ovshinsky lactobacillus strain BSLO 1801 or the dedicated probiotics preparation of the laying hen to improve
Application in egg quality.
The present invention provides the Ovshinsky lactobacillus strain BSLO 1801 or the dedicated probiotics preparation of the laying hen to adjust
Application in laying hen ileum intestinal flora.
The present invention provides the Ovshinsky lactobacillus strain BSLO 1801 or the dedicated probiotics preparation of the laying hen to adjust
Application in Laying-hen Serum hormone.
Preferably, the Laying-hen Serum hormone includes insulin and estradiol.
The present invention provides Ovshinsky lactobacillus strain BSLO 1801 (Lactobacillus oris), the Ovshinsky cream bars
The deposit number of bacteria strain BSLO 1801 is CCTCC No.M 2019287.Ovshinsky lactobacillus strain BSLO 1801 is blue by leather
Albert'stain Albert is positive, and microscopy is rod-shaped.Sequencing results through 16S rDNA show can by sequence homology analysis
Know, thus lactobacillus BSLO 1801 and 4864 homology similarity highest of Lactobacillus oris DSM determine separation
Bacterial strain BSLO1801 is Lactobacillus oris (Ovshinsky lactobacillus).
The present invention provides the Ovshinsky lactobacillus strain BSLO 1801 or the dedicated probiotics preparation of the laying hen in laying hen
Application in raising.Experiment shows after Ovshinsky lactobacillus strain BSLO 1801 is fed laying hen together with basal diet that energy is significant
It improves performance in layers (including reduced laying hen average weight and improve laying rate of laying hen), improve laying hen output egg
Quality (significantly improving Eggshell weight) adjusts ileum intestinal flora and (reduces Firmicutes to be averaged relative abundance；Belong in level
Significantly improve lactobacillus to be averaged relative abundance, reduce Romboutsia Pseudomonas and be averaged relative abundance) and adjust serum and swash
Plain horizontal (significantly reducing insulin level and estradiol content).
Biomaterial preservation information
Ovshinsky lactobacillus strain BSLO 1801 (Lactobacillus oris BSLO 1801), is preserved in Chinese Typical Representative
Culture collection, unit are referred to as CCTCC, and address is Wuhan, China, and Wuhan University, the preservation time is 04 month 2019 23
Day, deposit number is CCTCC No.M 2019287.
Detailed description of the invention
Fig. 1 is the species distribution histogram of two groups of samples Top20 in the case where door is horizontal, and wherein H group sample number is H1-H6；L
Group sample number is L1-L6；
Fig. 2 is the species distribution histogram of two groups of samples Top20 in the case where belonging to horizontal, and wherein H group sample number is H1-H6；L
Group sample number is L1-L6；
Fig. 3 is Specaccum species cumulative curve chart；
Fig. 4 is Beta diversity analysis as a result, wherein Fig. 4-A is PCA principal component analysis, Fig. 4-B be based on
The UPGMA clustering figure of WeightedUniFrac distance matrix；
Fig. 5 is KEGG the second ranking score Butut of PICRUSt prediction, and wherein A indicates H group, and B indicates L group；
Fig. 6 is the analysis of Spearman related network, and Green indicates negatively correlated, and red indicates to be positively correlated；
Fig. 7 is the species distribution histogram of two groups of Top20 in the case where door is horizontal；
Fig. 8 is the species distribution histogram of two groups of Top20 in the case where belonging to horizontal；
Fig. 9 is Specaccum species cumulative curve chart；
Figure 10 is PCA principal component analysis.
Specific embodiment
The present invention provides Ovshinsky lactobacillus strain BSLO 1801 (Lactobacillus oris), the Ovshinsky cream bars
The deposit number of bacteria strain BSLO 1801 is CCTCC No.M 2019287.Acid resistance the experimental results showed that, Ovshinsky lactobacillus bacterium
Strain survival rate under the conditions of pH value 4 of BSLO 1801 is up to 80%, and survival rate is up to 50% under conditions of pH value is 3, illustrates this Austria
1801 pairs of family name's lactobacillus BSLO sour well-tolerateds.Bile tolerance the experimental results showed that, Ovshinsky lactobacillus strain BSLO 1801 is in fowl
Survival rate has reached 50% under conditions of gallbladder salinity is 0.1%, and survival rate reaches under conditions of fowl gallbladder salinity is 0.2%
20%, it is relatively good to illustrate that the bacterial strain is resistant to fowl cholate.Decompose cholesterol the experimental results showed that, Ovshinsky lactobacillus strain
BSLO 1801 reaches 67.90% to cholesterol resolution ratio.
In the present invention, the cultural method of Ovshinsky lactobacillus strain BSLO 1801, preferably includes following steps: sterile item
Strain is inoculated in MRS fluid nutrient medium according to 2% volume ratio under part, Anaerobic culturel 16h under the conditions of 42 DEG C obtains Ovshinsky
The seed liquor of lactobacillus strain BSLO 1801.
The present invention provides a kind of dedicated probiotics systems of laying hen for improving performance in layers and improving quality of laying eggs
Agent, including the Ovshinsky lactobacillus strain BSLO 1801.
In the present invention, the viable bacteria concentration of the Ovshinsky lactobacillus strain BSLO 1801 is preferably 1 × 106CFU/kg~1
×108CFU/kg, more preferably 1 × 107CFU/kg.It is also preferable to include basal diets for the dedicated probiotics preparation of laying hen.This hair
It is bright that the type of the basal diet and source are not particularly limited, using egg feedstuff known in the art.It is described
Egg feedstuff is to be mixed to get 1801 seed liquor of Ovshinsky lactobacillus strain BSLO of culture with basal diet.
In the present invention, the same normal diet of feeding method of the dedicated probiotics preparation of the laying hen, laying hen are freely eaten, drink
Water, daily illumination 15h are fed 3 times daily.
The answering in layer breeding the present invention provides the Ovshinsky lactobacillus strain BSLO 1801 or the egg feedstuff
With.
The present invention provides the Ovshinsky lactobacillus strain BSLO 1801 or the egg feedstuff to improve laying hen productivity
Application in energy.Experiment shows that the experimental group for adding Ovshinsky lactobacillus BSLO 1801 all reduces laying hen average weight；With it is right
It is compared according to group, adds Ovshinsky lactobacillus A2 group (1 × 107CFU/kg it) significantly improves laying rate of laying hen (P < 0.05), A1 (1 ×
1067) and A3 group (1 × 10 CFU/kg8CFU/kg laying rate) is also improved, but difference is not significant (P > 0.05).
The present invention provides the Ovshinsky lactobacillus strain BSLO 1801 or the egg feedstuff in improving egg quality
Application.Experiment shows that the experimental group for adding Ovshinsky lactobacillus significantly improves Eggshell weight (P < 0.05), but albumen height,
Hough bit indicator is remarkably decreased (P < 0.05).
The present invention provides the Ovshinsky lactobacillus strain BSLO 1801 or the egg feedstuff to adjust laying hen ileum intestines
Application in road flora.Add Ovshinsky lactobacillus A2 group (1 × 107CFU/kg) compared with the control group, average opposite in door level
Abundance changed mainly Firmicutes (Firmicutes) and Actinobacteria (actinomyces door), A group firmicutes
The average relative abundance decline (P > 0.05) of door, the average relative abundance of actinomyces door rise (P > 0.05)；Belong to average opposite in level
Abundance changed mainly Romboutsia Pseudomonas and Lactobacillus (lactobacillus), A group significantly improve newborn bar
Pseudomonas is averaged relative abundance (P<0.05), reduces Romboutsia Pseudomonas and is averaged relative abundance (P>0.05).
The present invention provides the Ovshinsky lactobacillus strain BSLO 1801 or the egg feedstuff to swash in adjusting Laying-hen Serum
Application in element.The Laying-hen Serum hormone preferably includes insulin and estradiol.Compared with the control group, Ovshinsky lactobacillus is added
The laying hen that 1801 groups of BSLO, insulin (INS) is horizontal and estradiol (E2) is horizontal significantly reduces (P < 0.05).
Below with reference to embodiment to Ovshinsky lactobacillus BSLO 1801 provided by the invention and its application in Egg Production of Laying Hens
It is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Test material explanation
(1) experimental animal
First test laying hen: 42 same batches, the Roman egghen normally raised, by Sichuan Agricultural University's animal breeding
Center provides.
Second batch tests laying hen: the 350 age in days Luo Man eggs similar in 128 same batches, weight, in the later period of laying eggs
Chicken, by the note fresh egg cultivation of Lushan money, Co., Ltd is provided.
(2) animal experiment daily ration
Basal diet is formulated referring to NRC (1994) laying hen nutrition demand.Experimental basis daily ration composition and trophic level
It is shown in Table 1.
1 experimental basis daily ration of table composition
(3) culture medium prescription
LBS fluid nutrient medium: yeast extract 5.0g, pancreas casein peptone 10.0g, potassium dihydrogen phosphate 6.0g, ferrous sulfate
0.034g, magnesium sulfate 0.575g, glucose 20.0g, sodium acetate 25.0g, ammonium citrate 2.0g, manganese sulfate 0.12g, tween-
801.0mL, glacial acetic acid 1.3mL, distilled water 1000mL, pH value 4.0.
LBS solid medium: 17g agar, pH 4.0 are added in 1000mLLBS fluid nutrient medium.
MRS fluid nutrient medium: peptone 10g, beef extract 5.0g, yeast extract 4g, Tween-80 1.0mL, dipotassium hydrogen phosphate
2.0g, sodium acetate 2.0g, dibasic ammonium citrate 0.2g, magnesium sulfate (7H2O) 0.2g, manganese sulfate (4H2O) 0.05g, glucose
20.00g, distilled water 1000mL, pH value 6.2.
MRS solid medium: 17g agar, pH value 6.2 are added in 1000mLMRS fluid nutrient medium.
MRS-CHOL fluid nutrient medium: the MRS fluid nutrient medium of cholesterol containing 100mg/L and 0.3g/100mL fowl cholate.
(4) main agents
Dehydrated alcohol；Physiological saline；PBS buffer solution；Peptone, beef extract, yeast extract, Tween-80, phosphoric acid hydrogen dimethylamino,
Sodium acetate, dibasic ammonium citrate, magnesium sulfate (7H2O), manganese sulfate, glucose, agar etc. (being purchased from Wanke)；Total cholesterol (T-
CHO) testing cassete (Bioengineering Research Institute is built up in Nanjing)；Cholesterol, fowl cholate (it is limited to be purchased from Chengdu Kang Ruiyuan biotechnology
Company)；E.Z.N.A.TMFaeces DNA extracts kit (OmegaBio-Tek company, the U.S.)；Enzyme-linked immunosorbent assay kit (E2,
INS) (enzyme-linked biology) etc..
(5) key instrument
Spectrophotometer (Thermo scientific, the U.S.)；Optical microscopy (OLMPUS, Japan)；Gel imaging system
It unites in (Bio-Rad, the U.S.)；PCR instrument (Bio-Rad, the U.S.)；High-pressure sterilizing pot (Thermo Eletron 12Corpration,
The U.S.)；Microplate reader (Thermo scientific, the U.S.)；ND-2000 nucleic acid-protein analyzer
(Wilmington, the U.S.)；Ultra low temperature freezer (Thermo Eletron Corpration, the U.S.) etc..
Embodiment 1
The acquisition of 1 excrement sample
From first 42 Roman egghens, highest 6 laying hens of selection of egg production as H group, laying rate is 74.50 ±
4.28%, for 6 minimum laying hens of selection of egg production as L group, laying rate is 20.54 ± 7.12%.Its excrement sample of aseptic collection, ice
Box preservation is transported to Sichuan Agricultural University's Tiny ecosystem laboratory, and -30 DEG C save backup, and acquires complete in 10 minutes after laying hen defecation
At.
2 high-yield egg chickens and low yield laying hen analysis of intestinal microflora
Supreme Shanghai's style Sen Nuo Biotechnology Co., Ltd is sent to extract in the high-yield egg chicken excrement sample of acquisition and low yield layer hen manure sample
Faecal bacterial community total DNA examines total DNA quality, carries out both-end to community DNA segment using IlluminaMiSeq platform
(Paired-end) it is sequenced.
16S rDNA universal primer examining order is completed by upper Shanghai's style Sen Nuo Biotechnology Co., Ltd.Initial data is whole
It is analyzed after reason, filtering and quality evaluation, including OTU is divided and classification position identification, the microbe groups of each categorization levels
Number statistics, door belong to composition analysis in two categorization levels, and Metastats is analyzed, Alpha diversity index analysis (including
Tetra- species diversity index analysis of Simpson, Chao1, ACE and Shannon), (PCA is analyzed and is based on Beta diversity analysis
The UPGMA clustering of WeightedUniFrac distance matrix), the forecast analysis of PICRUSt metabolic function and dominant species interaction
The analysis of Spearman related network.
1.1, belong to two categorization levels on composition analysis
Under door is horizontal, two groups of single sample flora relative abundances are as shown in Figure 1, as can be seen from FIG. 1, Firmicutes is (thick
Wall bacterium door) it is dominant bacteria door absolute in H group, each sample content highest, main advantage bacterium is in L group sample
Proteobacteria (Proteobacteria) and Firmicutes (Firmicutes).
Under belonging to horizontal, two groups of single sample flora relative abundances are as shown in Fig. 2, as can be seen from FIG. 2, Lactobacillus
(lactobacillus) is the absolute Dominant genera of H group, the relative abundance highest in each sample, and Pseudomonas phase between L group sample
It is big to abundance difference, and Lactobacillus (lactobacillus) relative abundance in each sample of L group is lower.
Above data is subjected to comparison among groups:
In door level, first five dominant bacteria door of the relative abundance rankings of H group sample species is respectively as follows: Firmicutes (thickness
Wall bacterium door, average relative abundance be 86.30%), Actinobacteria (actinomyces door, average relative abundance be 6.10%),
Proteobacteria (Proteobacteria, average relative abundance be 3.84%), Fusobacteria (Fusobacterium door, it is average opposite
Abundance be 2.70%) and Bacteroidetes (Bacteroidetes, average relative abundance be 0.60%), L group sample species it is opposite
First five dominant bacteria door of abundance ranking be respectively as follows: Firmicutes (Firmicutes, average relative abundance be 51.28%),
Proteobacteria (Proteobacteria, average relative abundance be 37.04%), Actinobacteria (actinomyces door, average phase
To abundance be 5.83%), 2.42%) and [Thermi] (average phase (Bacteroidetes, average relative abundance are to Bacteroidetes
It is 2.29%) to abundance.There it can be seen that in door level, the ranking and relative abundance of dominant bacteria door between H group and L group
It is varied widely, and there is a dominant bacteria door type to be changed；It is analyzed by Metastats it is found that firmicutes
Door and the Proteobacteria relative abundance that is averaged are significantly changed (P < 0.05).
Belong in level, ten Pseudomonas is respectively as follows: Lactobacillus (newborn bar before the relative abundance ranking of H group sample species
Pseudomonas, average relative abundance be 67.57%), Unclassified-Peptostreptococcaceae (non-classified digestion chain
Coccus, average relative abundance be 3.82%), Enterococcus (enterococcus spp, average relative abundance be 3.25%),
Fusobacterium (Fusobacterium, average relative abundance be 2.70%), Unclassified-Ruminococcaceae (not
The wart germ category of classification, average relative abundance be 2.16%), Unclassified-Clostridiaceae (non-classified clostridium
Belong to, average relative abundance be 1.97%), Unclassified-Coriobacteriaceae (non-classified red stinkbug Pseudomonas, it is average
Relative abundance be 1.36%), Ruminococcus (Ruminococcus, average relative abundance be 1.23%), Unclassified-
1.15%) and Veillonella (Wei Yong Shi (non-classified xanthomonas, average relative abundance are to Xanthomonadaceae
1.12%) Coccus, average relative abundance are that ten Pseudomonas is respectively as follows: before the relative abundance ranking of L group sample species
(non-classified Peptostreptococcus, average relative abundance are Unclassified-Peptostreptococcaceae
14.44%), Turicibacter (average relative abundance be 9.59%), Unclassified-Xanthomonadaceae be not (
The yellow born of the same parents Pseudomonas of classification, average relative abundance be 8.20%), (Ochrobactrum, average relative abundance are Ochrobactrum
7.78%), Enterococcus (enterococcus, average relative abundance be 7.61%), Gallibacterium (put down by chicken Bacillus
Equal relative abundance be 6.56%), Lactobacillus (lactobacillus, average relative abundance be 4.99%),
Unclassified-Clostridiaceae (non-classified fusobacterium, average relative abundance be 3.54%), Burkholderia
(Burkholderia category, average relative abundance are that 2.89%) (deinococcus, average relative abundance are with Deinococcus
2.28%).Belong in level, before two groups of rankings in ten Dominant genera, not only superiority bacteria spp ranking is changed, kind classification
Also changed.Lactobacillus is the first Dominant genera in high-yield egg chicken intestinal flora in H group simultaneously, passes through Metastats
It is found that lactobacillus is averaged, relative abundance is significantly changed (P < 0.05) for analysis.
1.2Alpha diversity indices
By species accumulation curve judgement sample amount whether enough and estimate group's richness, show if curve steeply rises
Sample size is insufficient, needs to expand sampling scale；Conversely, then showing that sample size has been enough to reflect the richness of group.As a result such as Fig. 3
Shown, profile amplitude tends towards stability in figure, shows that this sequencing result can reflect laying hen intestinal flora feature.
For the diversity between comparing two groups, the first minimum sequencing to all samples in OTU abundance matrix 90%
Depth level, it is unified to carry out random double sampling (i.e. " sequence amount evens up processing "), to correct diversity caused by sequencing depth
Difference.Then, tetra- species diversity of Simpson, Chao1, ACE and Shannon is calculated to each sample respectively using QIIME software
Index.
The results are shown in Table 4, and H group Chao1 index and ACE index are lower than L group (P > 0.05).In Microbial diversity Journal of Sex Research
In, Chao1 index is used to estimate that the species number of physical presence in group, ACE richness estimation index are used to estimate real in group
Species number existing for border, it is however generally that, Chao1 or ACE index is bigger, shows that the richness of group is higher.As a result in, L group
Chao1 and ACE index is above H group, shows that the i.e. low yield laying hen group intestinal flora richness of L group is higher, and physical presence species
Number is higher than high yield group.
4 flora biodiversity index table * of table
* note: it is significant (P < 0.05) to infuse different lowercase letter indication differences with column data shoulder, identical lowercase or without word
Matrix shows that difference is not significant (P > 0.05)；All results are that ((X) ± standard deviation (SD) indicates average.
1.3Beta diversity analysis
As a result as shown in Fig. 4-A, as can be seen that two principal component analysis respectively illustrate total variable from PCA figure
The sample of 75.91% and 12.47%, H group and L group is all more dispersed, and individual difference is big, but a spacing is distributed between group and group
From illustrating that high-yield egg chicken and low yield laying hen intestinal microflora structure have different.It was found from UPGMA clustering
(see Fig. 4-B), H group sample clustering compare aggregation, and L group sample clustering is more dispersed, illustrate high-yield egg chicken intestinal microflora ratio
Low yield laying hen intestinal microflora is more like.
1.4PICRUSt metabolic function forecast analysis
As a result as shown in Fig 5, in 12 bacterial metabolism functions, with significant difference metabolic function have 7 (P <
0.05), it is respectively: nucleotide metabolism (Nucleotide Metabolism), other amino acid metabolisms (Metabolism
OfOtherAmino Acids), lipid-metabolism (Lipid Metabolism), enzyme be metabolized (Enzyme Families), carbon aquation
Close the anabolism (Biosynthesis of object metabolism (Carbohydrate Metabolism), other secondary metabolites
OfOther Secondary Metabolites) and amino acid metabolism (Amino Acid Metabolism).Wherein, laying hen intestines
Road flora function is mainly enriched in carbohydrate metabolism (Carbohydrate Metabolism) and amino acid metabolism (Amino
Acid Metabolism) on, in H group carbohydrate metabolism (Carbohydrate Metabolism) be significantly higher than L group (P <
0.05), amino acid metabolism (Amino AcidMetabolism) is substantially less than L group (P < 0.05).
The analysis of 1.5 dominant species interaction Spearman related networks
As a result as shown in fig. 6, in being located at preceding 46 Dominant generas, lactobacillus and Aeriscardovia belong in positive
It closes, with Burkholderia category (Burkholderia), bacillus faecalis category (Faeclibacterium), Bacteroides (Bacteroides)
It is directly negatively correlated Deng ten Pseudomonas, it is indirectly negatively correlated with remaining Pseudomonas.This shows in lactobacillus and laying hen intestinal flora
Most of Pseudomonas are competitive relations, and the difference of laying rate may be related with this Competition Characteristics of lactobacillus.
Embodiment 2
Promote the screening of laying rate of laying hen probiotics
This test plan analyzes high-yield egg chicken and low yield laying hen intestinal flora is poor by 16S rDNA high throughput sequencing technologies
It is different, it would be possible to carry out screening separation as probiotics, educable campylobacter bacteria.From high-yield egg chicken and low yield laying hen enterobacteriaceae
Cluster analysis test result it is found that lactobacillus relative abundance and Layer Production Performance in intestinal flora are closely related, therefore this
Test plan carries out separation screening to lactobacillus, it is desirable to filter out one plant of lactobacillus that can improve laying rate of laying hen.
(1) probiotics separation and Preliminary Identification
The all product 1.0g of first high-yield egg chicken excrement are weighed, are placed in the conical flask equipped with 99mL sterile saline, this
When test tube dilution be 10-2, sterile glass beads are added, shake up 30min on shaking table；It fullys shake, is uniformly mixed it, then
It draws in 0.5mL to next test tube, is diluted to 10 by 10 times of concentration gradients-6, then draw 10-6100 μ of sample of dilution
L is inoculated on LBS solid medium, and coating uniformly, is put into 42 DEG C of constant temperature incubation 48h of anaerobic culture box, chooses colonial morphology not
After same single colonie purifies culture 5 times respectively, thalli morphology is checked using Gram's stain, and picking single colonie is inoculated in
In MRS fluid nutrient medium, for 24 hours, bacterium solution and 50% concentration sterile glycerol are made by mixing in equal volume by 1:1 under aseptic condition for culture
Strain, -20 DEG C save backup.
(2) prepared by seed liquor
Strain is inoculated in MRS fluid nutrient medium according to 2% volume ratio, the Anaerobic culturel 16h under 42 DEG C of environment, as
The seed liquor of subsequent experimental.
(3) the acidproof detection of bacterial strain
By seed liquor according to 2% volume ratio be inoculated in pH value be respectively 3.0,4.0,6.0 MRS fluid nutrient medium in, in
42 DEG C of processing 2h.Concussion uniformly, takes bacteria suspension, detects viable count using colony counting method, is pair with the MRS culture solution of pH 6.0
According to calculating Strain survival rate.By comparing, it filters out the preferable bacterial strain of acid resistance and does experiment in next step.
Bacterial concentration × 100% of survival rate (%)=difference pH bacterial concentration/pH 6.0
(4) bacterial strain bile tolerance detects
It is respectively 0% that seed liquor, which is inoculated in fowl gallbladder salinity according to 2% volume ratio, 0.1%, 0.2% (w/v), pH
In 6.0 MRS fluid nutrient medium, after 42 DEG C of constant temperature incubation 2h, concussion uniformly, takes bacteria suspension, is deposited using colony counting method detection
Number (Formulas I) living calculates Strain survival rate so that the MRS culture solution of fowl cholate is not added as control.By comparing, bile tolerance is filtered out
The preferable bacterial strain of ability is done tests in next step.
Survival rate (%)=difference fowl gallbladder salinity bacterial concentration/0% fowl gallbladder salinity bacterial concentration × 100 Formulas I
(5) bacterial strain decomposes cholesterol ability detection
Seed liquor is inoculated in MRS-CHOL fluid nutrient medium by 2% (volume fraction), for 24 hours, concussion is equal for 42 DEG C of cultures
It is even, culture solution is drawn, 1000r/min is centrifuged 10min, and according to specification, (Nanjing is built using total cholesterol (T-CHO) testing cassete
At Bioengineering Research Institute) cholesterol level in culture solution is measured, detection lactobacillus strain decomposes cholesterol ability.
(6) bacterial strain is identified
By above-mentioned test, the preferable one plant of lactobacillus of comprehensive performance is chosen as target and promotees laying rate of laying hen probiotics,
And pass through measurement 16S rDNA Sequence Identification bacterial strain.
Step: the above-mentioned strain gene group total DNA filtered out is extracted, using 16S rDNA universal primer, forward primer
27F:5'-agagtttgatcctggctcag-3'(SEQ ID No.1), reverse primer 1492R:5'-
Cgttaccttgttacgactt-3'(SEQ ID No.2), it is expanded.PCR reaction system (25 μ L): 1 μ L of DNA profiling, upper,
Each 1 μ L (10nmol/L) of downstream primer, 12.5 μ 2 × PCRMasterMix of L, deionized water polishing.Pcr amplification reaction program:
95 DEG C of initial denaturation 5min；94 DEG C of 1min, 58 DEG C of 30s, 72 DEG C of 1min, totally 30 recycle；72 DEG C of extension 10min.With 1.5% agar
PCR product is sent to Huada gene company and is sequenced by sugared gel detection PCR product.Sequencing result is compared in NCBI, identification benefit
Raw strain category.
(2) promote the screening of laying rate of laying hen probiotics
The separation identification of 2.1 probiotics
Filter out 16 plants of bacterial strains, Gram's staining is all positive, microscopy be it is rod-shaped, be tentatively judged as lactobacillus.
2.2 bacterial strain acid resistance testing results
As can be seen from Table 5, lactobacillus strain S1, S5, S9, S13, S15 survival rate under the conditions of pH4 is up to 80%, pH3
Under conditions of survival rate be up to 50%, illustrate this five plants of lactobacillus to sour well-tolerated.
According to the above results, it is subsequent to choose relatively good lactobacillus strain S1, S5, S9, S13, S15 progress of acid resistance
Test.
5 bacterial strain acid resistance testing result (%) of table
2.3 bacterial strain bile tolerance testing results
As can be seen from Table 6, lactobacillus strain S5, S9 survival rate under conditions of fowl gallbladder salinity is 0.1% reaches
50%, survival rate has reached 20% under conditions of fowl gallbladder salinity is 0.2%, illustrates that this two plants of lactobacillus are resistant to fowl cholate
It is relatively good.
According to the above results, it is chosen in bile tolerance test and shows relatively good lactobacillus strain S5, S9.
6 bacterial strain bile tolerance test result (%) of table
2.4 bacterial strains decompose cholesterol result
Decomposing cholesterol, the results are shown in Table 7, and lactobacillus strain S9 cholesterol resolution ratio is 60.70%, lactobacillus strain
S5 cholesterol resolution ratio is 67.90%, and lactobacillus strain S5 cholesterol capacity of decomposition is higher than S9.According to the above results, choose each
The preferable lactobacillus S5 of aspect performance promotees laying rate of laying hen probiotics as potential, and carries out 16S rDNA sequencing.
7 bacterial strain of table decomposes cholesterol result (%)
2.516S the sequencing results of rDNA
By sequence homology analysis it is found that 4864 homology phase of lactobacillus S5 and Lactobacillus oris DSM
Like degree highest, thus determine that isolated strains S5 is Lactobacillus oris (Ovshinsky lactobacillus).It is named as BSLO
1801, it send to Wuhan Culture Collection and saves, fungi preservation number is CCTCC No.M 2019287.
Embodiment 3
Influence of the probiotics to later period laying hen of laying eggs
2.2.3 is promoted the probiotics behaved oneself best in the screening test of laying rate of laying hen probiotics application by this test plan
In laying hen, its influence to later period laying hen of laying eggs is studied.According to 2.2.3 promote laying rate of laying hen probiotics screening test as a result,
Show that bacterial strain Ovshinsky lactobacillus BSLO 1801 (Lactobacillus oris BSLO 1801) is showed most in 16 plants of lactobacillus
Good bacterial strain, therefore Ovshinsky lactobacillus BSLO1801 is applied to laying hen.
(1) laying hen test grouping and feeding management
128 350 age in days Roman egghens similar in same batch weight are chosen, are divided into 4 groups, every group of four repetition (every cages
For a repetition), 8 chickens of each repetition, feeding basal diet adapts to one week.By strain and according to the ratio of 2% (volume ratio)
It is inoculated in sterile MRS fluid nutrient medium, the Anaerobic culturel 16h under 42 DEG C of environment carries out count plate later, and viable count is about
1×109cfu/mL.- 20 DEG C of preservations after packing, the bacterium solution of preparation in three days.Ovshinsky lactobacillus BSLO1801 bacterium solution carries out later
It is grouped spice raising, is uniformly mixed, is fed 4 weeks.It is specifically grouped as follows: 1) control group: basal diet；2) A1 group: 1 ×
106Cfu/kg Ovshinsky lactobacillus+basal diet；3) A2 group: 1 × 107Cfu/kg Ovshinsky lactobacillus+basal diet；4) A3 group: 1 ×
108Cfu/kg Ovshinsky lactobacillus+basal diet.When raising, laying hen is freely eaten, drinks water, and daily illumination 15h feeds 3 daily
It is secondary, routinely carry out feeding management.Laundering period record egg number, and according to statistical result carry out appropriate adjustment, make control group with
Test group laying rate difference is not significant.
(2) animal sample acquires
One day 11:00 point empties produced egg before laying hen before the test, the diet that then feeding experiment prepares, then extremely
The time of second day 11:00 point is test 1d.Fasting is carried out before sampling but can't help drinking-water processing 12h.It is early in test 29d
Morning, every group of each repetition randomly select 2 chickens (every group of 8 chickens), carry out wing under venous blood collection 10mL in 15mL collecting pipe,
Institute's blood sampling is placed in after standing 30 minutes in 4 DEG C of environment, the isolated blood of 20min is centrifuged with 1500r/min revolving speed
Clearly, it saves backup for -20 DEG C；It then takes jugular vein depletion method to put to death chicken, takes the ileal contents of every chicken rapidly, rapidly
It is put in liquid nitrogen flash freezer, is subsequently transferred to save in -80 DEG C of refrigerators.
(3) influence of the probiotics to performance in layers
During test, clinical observation is carried out to each group laying hen daily, records the health status and death condition of daily laying hen.
11 points of 21d morning is tested to 11 points of morning for testing 28d, the laying hen of each processing group carried out as unit of repetition egg number,
Egg size, food consumption statistics, and make a record, weigh in 28d to laying hen, laying hen average weight, laying rate, egg counterpoise,
Feedstuff-egg ratio is based on counted numerical value and is calculated.
(4) influence of the probiotics to egg quality
According to the laying rate for testing last week, one group that lactobacillus group behaves oneself best is filtered out, in addition after control group is done
Continuous test (group echo to behave oneself best in three groups of lactobacillus is A group, and control group C flag is C group).At random choose respectively this two
12 eggs (three pieces of eggs of each repetition) that group 28d is produced, it is interior for 24 hours to send to Sichuan Agricultural University, school district, Yaan Animal nutrition institute
Egg product quality detection is carried out, index includes: eggshell strength, albumen height, yolk color, Hough unit, yolk specific gravity, eggshell ratio
Weight, average shell thickness.
(5) influence of the probiotics to ileum intestinal flora
According to operating instruction is used, E.Z.N.A. is usedTM(U.S. Omega Bio-Tek is public for faeces DNA extracts kit
Department) extract every laying hen 200mg ileal contents sample in total flora DNA.WithND-2000 nucleic acid egg
White analyzer detection nucleic acid concentration and purity, are subsequently placed in -80 DEG C of refrigerators and freeze.Total DNA sample is sent to Beijing source Nuo Hezhi
Co., Ltd examines, is sequenced.16S sequencing is based on IonS5TMXL microarray dataset, utilizes the side of single-ended sequencing (Single-End)
Method, building small fragment library carry out single-ended sequencing.After data processing, carry out door, belong to composition analysis in two categorization levels,
Metastats analysis, Alpha diversity indices and Beta diversity analysis.
(6) influence of the probiotics to serum hormone
According to operation instructions, using enzyme-linked immunosorbent assay kit (enzyme-linked biology) in serum estradiol (E2) and
Insulin (INS) content is detected.
The analysis of 2.3 data
Software is analyzed with SPSS 22 and carries out data statistics, analysis, is as a result expressed as average (X) ± standard deviation (SD) table
Show.Using R software, Specaccum species accumulation curve is drawn, using QIIME software development door and belongs to the abundance group in level
At table, PCA analysis is carried out by R software, carries out UPGMA cluster apart from square using UniFrac of the QIIME software to Weighted
Analysis.Use the Spearman hierarchical relationship network (rho>0.6 and value<0.01 P) between Mothur software building dominant genera.Two
Group or more with one-way analysis of variance method (one-wayANOVA) carry out multiple groups between significance test；Using Duncan multiple range
(Duncan ' smultiple-range) examines the multiple conspicuousness of progress to compare, and P < 0.05 has significant between being identified as two groups
Sex differernce.Significance test between two groups of groups is compared using independent sample T inspection between two groups, P < 0.05 is identified as two
There is significant difference between group
(3) influence result of the probiotics to later period laying hen of laying eggs
Influence of 3.1 probiotics to Layer Production Performance
In the test of entire laying hen, all groups do not occur laying hen death condition.The results are shown in Table 8 for remaining index, with
Control group is compared, addition Ovshinsky lactobacillus BSLO 1801 three processing groups all reduce laying hen average weight, wherein A2 group with
A3 group significantly reduces laying hen average weight (P < 0.05).In addition, addition Ovshinsky lactobacillus A2 group significantly improves Egg Production of Laying Hens
Rate (P<0.05), A1 and A3 group also improves laying rate, but difference is not significant (P>0.05).Compared with the control group, egg counterpoise and
Feedstuff-egg ratio is without significant difference (P > 0.05).
8 Layer Production Performance * of table
* note: it is significant (P < 0.05) to infuse different lowercase letter indication differences with column data shoulder, identical lowercase or without word
Matrix shows that difference is not significant (P > 0.05)；All results are that ((X) ± standard deviation (SD) indicates average.
Influence of 3.2 probiotics to Egg Quality
In three test groups, A2 group egg laying performance behaves oneself best, and is marked as A group, and control group C group echo is C group, will
12 eggs (three pieces of eggs of each repetition) that this two groups of 28d are produced, interior send to Sichuan Agricultural University Yaan school district animal is sought for 24 hours
Carried out egg product quality detection is supported, the results are shown in Table 9, and addition Ovshinsky lactobacillus group significantly improves Eggshell weight (P < 0.05),
But albumen height, Hough bit indicator are remarkably decreased (P < 0.05).Remaining index does not have significant changes.
9 Egg Quality Indexs measure result * of table
* note: it is significant (P < 0.05) to infuse different lowercase letter indication differences with column data shoulder, identical lowercase or without word
Matrix shows that difference is not significant (P > 0.05)；All results are that ((X) ± standard deviation (SD) indicates average.
3.3 ileum flora 16S rDNA high throughput analysis results
3.3.1 taxology composition analysis
The horizontal result of door is as shown in fig. 7, A group and C group Bacterial community are changed, and be averaged relative abundance ranking in A group
The bacterium door of first five be respectively as follows: Firmicutes (Firmicutes, average relative abundance be 81.90%), Actinobacteria (puts
Line bacterium door, average relative abundance be 16.48%), Bacteroidetes (Bacteroidetes, average relative abundance be 1.11%),
0.17%) and Deinococcus-Thermus (abnormal cocci-(Proteobacteria, average relative abundance are to Proteobacteria
0.16%) Thermus door, average relative abundance are.First five dominant bacteria door of average relative abundance ranking is respectively as follows: in C group
Firmicutes (Firmicutes, average relative abundance be 91.46%), Actinobacteria (actinomyces door, it is average relatively rich
Degree for 4.09%), unidentified-Bacteria (do not identify-Bacteriophyta, average relative abundance be 2.23%),
Bacteroidetes (Bacteroidetes, average relative abundance be 0.78%) and Tenericutes (no wall bacterium door, it is average relatively rich
0.77%) degree is.
The horizontal result of category is as shown in figure 8, significant changes, average relative abundance in A group has occurred in A group and C group Bacterial community
Ten Pseudomonas is respectively as follows: Romboutsia (average relative abundance is 35.45%), Lactobacillus (lactobacillus before ranking
Belong to, average relative abundance be 33.88%), Atopobium (unusual Pseudomonas, average relative abundance be 7.69%),
Enterococcus (enterococcus spp, average relative abundance be 7.64%), (average relative abundance is Aeriscardovia
4.33%), Rothia (Rothia, average relative abundance be 2.45%), Candidatus-Arthromitus (it is tentative-point
Save phycomycetes, average relative abundance be 0.65%), (streptococcus, average relative abundance are Streptococcus
0.59%) and Faecalibacterium (bacillus faecalis 0.63%), (Bacteroides, average relative abundance are to Bacteroides
Belong to, 0.58%) average relative abundance is.Ten superiority bacteria spp is respectively as follows: Romboutsia before average relative abundance ranking in C group
(average relative abundance be 68.02%), Lactobacillus (lactobacillus, average relative abundance be 8.68%),
Enterococcus (enterococcus spp, average relative abundance be 2.59%), (average relative abundance is Subdoligranulum
2.36%), unidentified-Bacteria (do not identify-bacterium category, average relative abundance be 2.14%),
Bifidobacterium (Bifidobacterium, average relative abundance be 1.86%), unidentified-Lachnospiraceae
(do not identify-Lachnospira, average relative abundance be 1.41%), Faecalitalea (average relative abundance is 1.22%),
Faecalicoccus (average relative abundance is 0.83%) and Blautia (average relative abundance is 0.81%).
Between two groups, in door level average relative abundance changed mainly Firmicutes (Firmicutes) and
Actinobacteria (actinomyces door), A group Firmicutes be averaged relative abundance decline (P > 0.05), and actinomyces are averaged opposite
Abundance rises (P > 0.05), and Proteobacteria (Proteobacteria) is averaged relative abundance without significant change；Belong to average in level
Relative abundance changed mainly Romboutsia Pseudomonas and Lactobacillus (lactobacillus), A group significantly improve
Lactobacillus is averaged relative abundance (P<0.05), reduces Romboutsia Pseudomonas and is averaged relative abundance (P>0.05).
3.3.2Alpha diversity indices calculates
As a result as shown in figure 9, species accumulation curve amplitude tends towards stability, show that this sequencing result can reflect laying hen ileum
Flora feature.
The results are shown in Table 10 for diversity indices, and A group Chao1 index and ACE index are lower than C group (P > 0.05), shows Austria
Family name's lactobacillus reduces laying hen ileum bacterial diversity, consistent with diversity indices result in 3.1.
10 flora biodiversity index table * of table
* note: it is significant (P < 0.05) to infuse different lowercase letter indication differences with column data shoulder, identical lowercase or without word
Matrix shows that difference is not significant (P > 0.05)；All results are that ((X) ± standard deviation (SD) indicates average.
3.3.3Beta diversity analysis
As a result as (abscissa indicates that first principal component, percentage then indicate tribute of the first principal component to sample difference to Figure 10
Offer value；Ordinate indicates that Second principal component, percentage indicate Second principal component, to the contribution margin of sample difference.Note: A group sample is compiled
Number be A1-A8；C group sample number is C1-C8) shown in, as can be seen that two principal component analysis respectively illustrate from PCA figure
The 28.57% and 18.5% of total variable；Sample A3 and other samples distance are dispersed in A group, in C group sample C4 and other samples away from
From more dispersed；But certain distance is distributed between A group and C group, both illustrate intestinal microflora structure be have it is certain poor
Different.Show after feeding laying hen Ovshinsky lactobacillus, laying hen ileum Bacterial community is changed.
Influence of 3.4 probiotics to serum hormone
As a result as shown in table 11, compared with the control group, insulin (INS) level and estradiol of Ovshinsky lactobacillus group laying hen
(E2) horizontal to significantly reduce (P < 0.05).
11 Laying-hen Serum hormonal readiness * of table
* note: it is significant (P < 0.05) to infuse different lowercase letter indication differences with column data shoulder, identical lowercase or without word
Matrix shows that difference is not significant (P > 0.05)；All results are that ((X) ± standard deviation (SD) indicates average.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Sichuan Agricultural University
<120>Ovshinsky lactobacillus strain BSLO 1801 and its application in Egg Production of Laying Hens
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
agagtttgat cctggctcag 20
<210> 2
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
cgttaccttg ttacgactt 19
Claims (10)
1. Ovshinsky lactobacillus strain BSLO 1801 (Lactobacillus oris), which is characterized in that the Ovshinsky lactobacillus bacterium
The deposit number of strain BSLO 1801 is CCTCC No.M 2019287.
2. a kind of dedicated probiotics preparation of laying hen for improving performance in layers and improving Egg Quality, which is characterized in that packet
Include Ovshinsky lactobacillus strain BSLO 1801 described in claim 1.
3. the dedicated probiotics preparation of laying hen according to claim 2, which is characterized in that the Ovshinsky lactobacillus strain BSLO
1801 viable bacteria concentration is 1 × 106CFU/kg~1 × 108CFU/kg。
4. the dedicated probiotics preparation of the laying hen according to Claims 2 or 3, which is characterized in that the dedicated probiotics system of laying hen
Agent further includes basal diet.
5. laying hen described in Ovshinsky lactobacillus strain BSLO 1801 or claim 2~4 any one described in claim 1 is dedicated
Application of the probiotics preparation in layer breeding.
6. laying hen described in Ovshinsky lactobacillus strain BSLO 1801 or claim 2~4 any one described in claim 1 is dedicated
Probiotics preparation is improving the application in performance in layers.
7. laying hen described in Ovshinsky lactobacillus strain BSLO 1801 or claim 2~4 any one described in claim 1 is dedicated
Probiotics preparation is improving the application in egg quality.
8. laying hen described in Ovshinsky lactobacillus strain BSLO 1801 or claim 2~4 any one described in claim 1 is dedicated
Probiotics preparation is adjusting the application in laying hen ileum intestinal flora.
9. laying hen described in Ovshinsky lactobacillus strain BSLO 1801 or claim 2~4 any one described in claim 1 is dedicated
Probiotics preparation is adjusting the application in Laying-hen Serum hormone.
10. applying according to claim 9, which is characterized in that the Laying-hen Serum hormone includes insulin and estradiol.
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