CN113265350B - Bifidobacterium W8118 and application thereof - Google Patents
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Abstract
The invention provides a bifidobacteriumBifidobacteriumsp.) W8118, the preservation number of which is CGMCC NO.21789 in China general microbiological culture Collection center, and the honey bee bifidobacterium is found to achieve the inhibition effect on the honey bee cocci of the honey bee by secreting antibacterial peptide through separating the supernatant of the bifidobacterium in the test; in an in-vitro bacteriostatic circle experiment, the bee bifidobacterium and a fermentation product thereof can directly inhibit the bee hive pneumococcus; in vivo experiments, the single-bacterium bees fed with the bifidobacteria have better inhibition effect on the melissococcus apis; the invention is suitable for industrial production and market popularization and application.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to bifidobacterium W8118 and application thereof.
Background
About 20 million animals with pollination function are known in the world, with 99.5% of the pollinators. According to statistics, the service value of the insect pollinating 1.8 million tons of agricultural products of 22 main crops in China in 2005 is up to 8860.5 million yuan, which accounts for 1.3% of GDP in the year. The bees are important pollination insects, and the value of the bee pollination insects is that the bee pollination insects create pollination service value for crops besides products such as honey, propolis, beeswax and royal jelly. Estimated by the food and agricultural organizations of the united nations, over 70 of the approximately hundreds of crops that provide 90% of the world's food are bee pollinated, producing an economic value of over 2000 billion dollars worldwide each year. The bee pollination contributes to the production of more than 30 crops in China, the economic value is about three billion yuan, which accounts for 36.25 percent of the total output value of the crops and is equivalent to 12.30 percent of the total output value of national agriculture. Obviously, the bees play an important role in guaranteeing the crop yield and maintaining the food and ecological safety in China.
In recent years, with the rapid development of economy, bees are threatened to various degrees. The main factors threatening the survival of bees include viruses, mites, bacteria, microsporidian, and the like. The combined action of these factors causes the bees to suddenly appear a 'swarm failure disorder' phenomenon (CCD) that the swarm worker bees cannot return to the nest, and the survival of the bees is seriously influenced. According to the American society of bee culture of America (AIA) in 2006-2007, 384 bee farmers are investigated and counted, and the average loss amount of bees caused by the bee colony failure disorder is up to 37.6%.
European foul brood is a malignant bacterial digestive tract infectious disease in bee larvae, and the pathogenic bacteria of the disease are Behcet bee cocci (Behcet's bee:)Melissococcus pluton) Mainly comprises the following steps. The bee-hive bee-coccus is a gram-positive bacterium, the shape of the bacterium is oblong, is in a needle shape, and is distributed singly or in pairs or is arranged in a chain shape. The melissococcus nidus mainly attacks bee larvae of 1-2 days old, has a latency period of 2-3 days, and has foul rot of European larvae, so that the larvae are not infected by bee larvae of 3-4 days oldThe cap was sealed and died a lot. The diseased larva collapses at the bottom of the honeycomb and is disorganized in position, the larva becomes soft and decays, the corpse residue has sour odor but no viscosity, the color of the corpse gradually changes from pale to yellow and finally to brown or even tan, the ring lines are fuzzy or disappear, and the phenomenon of 'flower arrangement and spleen' of the vacant house and the ovary are alternated appears. The disease mostly occurs in two seasons of spring and autumn, the disease condition is mild in autumn, and the disease condition is less severe in summer; the main characteristics of European foul brood are rapid spread and onset.
Research shows that European foul brood mainly occurs in the bee larva stage, and adult bees play the role of a propagator of the European foul brood, carry the melissococcus nidus and cause the European foul brood to be propagated in bee colonies and even among different bee colonies and bee yards, so compared with bee larvae, adult bees are more suitable for being used as subjects for epidemiological research and treatment of the European foul brood.
When the bee field is treated, because no effective treatment means is available and antibiotics are abused, pathogenic bacteria generate drug resistance, and the microecological balance of the intestinal tracts of bees is destroyed, so that the disease is further worsened; meanwhile, the application of antibiotics can cause residues in bee products, influence the quality of the products and bring food safety threats. At present, no report is available about the application of bifidobacterium in the control of European foul brood.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides bifidobacteriumBifidobacteriumsp.) W8118, which is preserved in China general microbiological culture Collection center (CGMCC) on 2021, 2 months and 1 days, with the preservation number of CGMCC No.21789 address: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.
Western honeybee (obtained from Lactobacillus plantarum W8118 Jilin of the invention)Apis mellifera Linnaeus) Extracting total DNA of the strain, amplifying target fragments by using a bacterial 16SrDNA universal primer, recovering, cloning and sequencing the fragments, wherein the 16srDNA sequence is shown as SEQ ID NO. 1; similarity alignment of 16SrDNA was performed using BLAST from NCBI on the sequence of the bacterial 16SrDNA gene fragment, and the results showed a match with doubleThe relation of the bifidobacterium is recent, the similarity reaches 99.85 percent, so the strain is identified as the bifidobacterium which is named as bifidobacterium W8118; the fermentation medium can be TPY medium or liquid medium prepared by adding distilled water into TRY medium powder;
another object of the present invention is to provide the above-mentioned BifidobacteriumBifidobacteriumsp.) W8118 is applied to inhibiting the melissococcus nidulans, wherein the bifidobacterium W8118 or the fermentation product thereof is used for preparing a microbial agent or feed for inhibiting the melissococcus nidulans; the bifidobacterium W8118 or a fermentation product thereof is used as an active ingredient for preparing a microbial agent or feed for preventing and treating European foul brood;
the component (or effective component) of the pathogenic bacteria microbial agent or feed for preventing and treating European larval foul brood is bifidobacterium W8118 or a fermentation product thereof, and one or more than one kind of the pathogenic bacteria microbial agent or auxiliary materials acceptable on the feed can be added to prepare a dosage form suitable for the microbial agent or the feed, and the dosage form is added according to the conventional addition, wherein the effective bacteria number is not less than 2 multiplied by 1011cfu/g；
The invention has the beneficial effects that:
the present invention provides BifidobacteriumBifidobacteriumsp.) W8118, in the test, through the separation of the supernatant of the bifidobacterium, the bifidobacterium W8118 is found to achieve the inhibition effect on the melissococcus meliae through secreting antibacterial peptide; in an in vitro bacteriostatic circle experiment, the bifidobacterium W8118 and a fermentation product thereof can directly inhibit the bee hive coccus; in vivo experiments, the single-fungus honeybees fed with the bifidobacterium W8118 have better inhibition effect on the melissococcus nidus; the bifidobacterium W8118 and the fermentation product thereof can effectively improve the European foul brood symptom of the bees, and the microbial inoculum or feed is environment-friendly, pollution-free, free from influencing the quality of bee products, and suitable for industrial production and market popularization and application.
Drawings
FIG. 1 is a schematic diagram of the experimental results of the in vitro inhibition zone of Bifidobacterium W8118 in example 3 of the present invention;
FIG. 2 is a schematic diagram showing the result of the inhibition effect of the bifidobacterium suspension solution on the bee horneri in the bee in vivo experiment;
FIG. 3 shows the results of the anti SMASH analysis;
FIG. 4 is a graph showing the results of purification of antibacterial substances in the supernatant of Bifidobacterium (upper panel) and the results of inhibition of Melissa apis (lower panel).
Detailed Description
The present invention is further illustrated by the following figures and examples, but the scope of the present invention is not limited thereto, and the methods in the examples are performed according to the conventional methods unless otherwise specified, the reagents used are commercially available reagents or prepared according to the conventional methods, and the media are commercially available products and used according to the instructions.
Example 1: isolation and identification of bifidobacteria
Capturing western bees from Jilin, removing the bee intestinal canal, manually grinding with a grinding rod in 100 μ L25% (v/v) glycerol to break the intestinal wall, and storing in an ultralow temperature medical refrigerator at-80 deg.C; streaking the intestinal tract sample in three regions of a TPY culture medium, and culturing in a 5% carbon dioxide incubator at 35 ℃ for 2-3 days; observing colony morphology, selecting single colony, streaking on TPY culture medium in eight regions, and purifying and culturing in 5% carbon dioxide incubator at 35 deg.C; selecting a plurality of single colonies on a culture medium, numbering the single colonies respectively, and selecting 20 single colonies for 16srDNA sequencing.
The total DNA of the bacteria is extracted by adopting a bacterial genome DNA extraction kit of Tiangen Biochemical technology limited company, adopting a 16S rDNA universal primer (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R:5'-GGTTACCTTGTTACGACTT-3') and adding into a 50 mu L PCR system (ddH)2O19 μ L, 2 × SanTaq PCR Mix 25 μ L, Primer 27F 2 μ L, Primer 1492R 2 μ L, template DNA 2 μ L) and PCR conditions were: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, renaturation at 60 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; finally, extension is carried out for 5min at 72 ℃.
The 16s rRNA amplification sequencing result of the embodiment shows that the 16s rRNA sequencing result of the strain with the serial number of W8118 is shown as SEQ ID NO. 1; the comparison in the database by using BLAST tool shows that the related strain W8118 bifidobacteria has the nearest relation and the similarity reaches 99.85 percent. Therefore, the strain was identified as bifidobacterium, named bifidobacterium W8118.
Example 2: preparation of bifidobacterium W8118 freeze-dried microbial inoculum
Activating bee bifidobacterium W8118 by using a TPY culture medium, wherein the inoculation amount is 3%, transferring the activated strain into a TPY liquid culture medium according to the inoculation amount of 4%, culturing for 25h at 37 ℃, and centrifuging (5000g, 15 min) to obtain the strain for later use. Adding protective agent (freeze-drying conventional protective agent) into the thallus at a volume ratio of 3: 1, and mixing well for later use. Before freeze-drying, the sample is pre-frozen at-80 ℃ in an ultra-low temperature refrigerator for 3h, then freeze-dried in three stages at 512Pa for 6h, then 256Pa for 5h, and then 103Pa until a powder product is obtained. The freeze-dried microbial inoculum is respectively stored at 4 ℃ and 25 ℃ for bacterial powder, the viable count of each bacterial powder is measured every 2 months, and 3 experiments are carried out in parallel; the results are shown in the following table; as can be seen from the table, the viable count of the freeze-dried microbial inoculum is still kept at a higher level after being stored for 10 months at 4 ℃ and 25 ℃; the stability at 25 ℃ is different from that at 4 ℃, but the survival level is still kept at 90%; the effective number of Bacillus bifidus W8118 powder is not less than 2 × 1011cfu/g；
Example 3: bifidobacterium W8118 in vitro bacteriostatic circle experiment
(1) Preparation of test product bifidobacterium sterile supernatant
Inoculating Bifidobacterium W8118 separated in the above experiment into TPY culture medium (TRY culture medium powder 36.65g, adding distilled water to 1L, sterilizing at 121 deg.C for 15 min), culturing for 5 days, centrifuging the culture solution at 8000g by low temperature high speed centrifuge for 10min, and collecting supernatant; finally filtering the supernatant through a filter membrane with the aperture of 0.22 mu m to obtain sterile bifidobacterium supernatant;
(2) culture and bacteriostatic circle experiment of bee hive micrococcus
In 1Adding 5mL of 1 XPBS buffer solution into a 0mL centrifuge tube, scraping all the honey bee hives in the optimal growth state on a flat plate into the centrifuge tube by using an inoculating ring, uniformly oscillating by using a vortex oscillator to prepare a bacterial suspension, placing 50 mu L of the bacterial suspension on a KSBHI flat plate, and uniformly coating; preparing 10mm holes on the coated agar medium; placing 100 mu L of sterile supernatant of bifidobacterium W8118 into the prepared holes; place the plate in 5% CO2Culturing in an incubator at 35 deg.C for 3-4 days; the formation of a zone of inhibition was observed around the hole.
The results are shown in fig. 1, from which it can be seen that the sterile supernatant of bifidobacterium W8118 has a better inhibitory effect on honey bee colonies.
Example 4: animal experiment of Bifidobacterium W8118 on Pediococcus meliae
1. Material
(1) Preparation of a test product bifidobacterium suspension solution: taking 1 g of lyophilized powder prepared in example 2 (wherein the effective viable count is not less than 10)11cfu/g), placing the mixture into a 1L beaker, adding a mixed solution of 450 mL of sucrose solution with the mass concentration of 50% and 450 mL of 1 XPBS buffer solution, stirring and suspending, and simultaneously adding 100 g of pollen to continue suspending to obtain 1L of bifidobacterium suspension solution;
(2) bee raising and testing
Bee breeding conditions are as follows: group breeding, wherein the breeding temperature and humidity are as follows: culturing at 35 deg.C and 40% -70% in dark place; the conditions of the incubator are always kept stable to ensure the reliability of test results, and the bees are divided into a sterile bee group and a bifidobacterium W8118 single-bacterium bee group;
constructing a sterile bee model: taking out the imagoes which are not feathered from the spleens of the bees, putting the imagoes into a sterile lunch box, putting the imagoes into the sterile incubator, culturing for 1-2 days at 35 ℃, adding a centrifugal tube containing 2mL of 50% sucrose solution into each lunch box, feathering the imagoes after about one day, wiping the perforated disposable cup with 75% alcohol or 84 disinfectant after the imagoes are feathered, and sterilizing for 15min under ultraviolet light; then picking out 25 bees from the lunch box, transferring the bees into a sterile disposable cup (3 groups of parallel tests), respectively putting centrifugal tubes filled with 50% sterile sucrose aqueous solution and sterile pollen into the cup for feeding the bees, and fixing the centrifugal tubes by using adhesive tapes; culturing in sterile environment for 7 days to obtain sterile bee.
Construction of Bifidobacterium W8118 Monomycosis bees: taking out the imagoes which are not eclosion from the spleens of the bees, putting the imagoes into a sterile lunch box, culturing the imagoes in the sterile incubator for 1 to 2 days at 35 ℃, adding a centrifugal tube containing 2mL of 50 percent sucrose solution into each lunch box, and eclosion after about one day. After the feathering, the perforated disposable cup is wiped by 75 percent alcohol or 84 disinfectant and sterilized for 15min under ultraviolet; then picking 25 bees from the lunch box and transferring the bees into a sterile disposable cup (3 groups of parallel tests), adding the bifidobacterium W8118 suspension solution into a 2mL centrifuge tube, feeding the sterile bees and sterile pollen at the same time, and placing the sterile centrifuge tube in a sterile incubator for culturing for 7 days at 35 ℃ to obtain a bifidobacterium W8118 single-bacterium bee model;
pathogen infection: respectively culturing the aseptic honeybees and the bifidobacterium W8118 single-bacterium group honeybees for seven days, and then respectively carrying out dip dyeing by using the meliococcus alvei;
collecting and processing samples: taking intestinal tracts of all bees seven days after the bee hive is infected with the bee cocci; and extracting bee intestinal genome, and extracting genome from each bee intestinal by CTAB method.
The dissected intestine was transferred to 728 μ L CTAB buffer and 20 μ L of 20mg/mL proteinase K solution, homogenized for 30s with a pestle, and transferred to a 2mL tube containing 500 μ L of 0.1 mm sterile zirconia beads; lysis with MO BIO Vortex Genie for 3min and centrifugation at 12000 g for 5 min; the supernatant was taken and transferred to a sterile 1.5 mL tube and incubated at 56 ℃ for 30 min, 5. mu.L RNase A was added, after incubation at 37 ℃ for 30 min, 400. mu.L phenol-chloroform-isoamyl alcohol (25: 24: 1) organic phase was added and centrifuged at 14000 rpm for 5min, the supernatant was transferred to a new 1.5 mL tube and 50. mu.L sodium acetate and 500. mu.L isopropyl alcohol were added, after centrifugation at 14000 rpm for 30 min, the precipitate was washed twice with 70% ethanol and finally suspended in 50. mu.L nuclease-removed water and stored at-20 ℃.
The method for detecting the bee hive cocci comprises the following steps: real-time fluorescent quantitative polymerase chain reaction using QuantStaudio 1 polymerase chain reaction produced by Thermo FisherThe formula reaction instrument is used for carrying out a test experiment on the quantity of the bee hive coccus; the total copy of the gene extracted from the bee intestinal tract is amplified by using designed primers specific to the meliococcus alvei, and the total copy is measured by the corresponding relation between the fluorescence CT value and the logarithmic value of the total copy number. Wherein the forward primer is MP-qpcr-69-f (5'-TGTTGTTAGAGAAGAATAGGGGAA-3'), the reverse primer is MP-qpcr-69-r (5'-CGTGGCTTTCTGGTTAGA-3'), and bee actin is used (bee actinactinAB 023025) Gene copy number normalization between samples was performed, forward primer actin-F (5'-TGCCAACACTGTCCTTTCTG-3'), reverse primer actin-R (5'-AGAATTGACCCACCAATCCA-3'); the qPCR test is carried out by adopting a dye method, and AceQ Universal SYBR qPCR Master Mix dye method kit produced by Nanjing Novozam is selected to prepare a qPCR system, wherein the total amount of the system is 20 mu L, and the qPCR system comprises 10 mu L of 2 XChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech company), 0.4 mu L of forward primer, 0.4 mu L of reverse primer, 1 mu L of sample to be detected and 8.2 mu L of double distilled water. Adding templates with different dilution concentrations into a prepared system to carry out qPCR test, and calculating to obtain a corresponding relation between a logarithmic value of absolute copy number and a cycle number (CT value) experienced when a fluorescence signal of a sample in each hole reaches a set threshold value; then, after diluting a sample to be detected in a proper amount, adding the diluted sample into a prepared system to perform a qPCR test, and substituting the obtained CT value into a drawn standard curve to obtain the absolute copy number corresponding to the sample; qPCR uses a three-step method, and the above cycle conditions are 95 ℃ for 30s, 3-10 s at 95 ℃ for 10-30 s at 60 ℃ (40 cycles), 15 s at 95 ℃ for 60 s and 15 s at 95 ℃.
The results are shown in fig. 2, and it can be seen from the results in fig. 2 that the bifidobacterium W8118 monosomy honeybee group bees have better resistance inhibition effect against the melissococcus apis than the aseptic honeybee group infected with the melissococcus apis.
Example 5: bioinformatic analysis of the type III lantipeptide biosynthetic Gene Cluster of Bifidobacterium
The CTAB method was used to extract the genome of a single bacterium, the total genomic DNA of W8118 was sequenced by Illumina Nova6000 platform and genome assembly was performed with SOAP denov 2 software, the integrity of the genome was detected with the CheckM method, finally the genome-wide average nucleotide homology (ANI) was calculated by FastANI and the genome was annotated with the Prokka software.
Analyzing and bioinformatics identifying a bifidobacterium W8118 gene cluster by using online software anti SMASH, and analyzing each gene in the gene cluster by using BAGEL 3; and performing comparison analysis on the nucleotide sequence and the protein sequence of each gene in the gene cluster by Blast software on an NCBI website.
As a result of the anti SMASH analysis, FIG. 3 shows that the Protein kinase, Lanthionine synthase C-like Protein, which is annotated by pHmm, is predicted to be a type III Lanthionine synthetic Protein: and (4) micKC.
Example 6: separation of supernatant of bifidobacterium and verification of bacteriostatic activity
Bifidobacterium W8118 was cultured in TPY liquid medium at 37 ℃ for 5 days and centrifuged in a high-speed refrigerated centrifuge (Thermo Scientific, Waltham, MA, USA) at 8000rpm at 4 ℃ for 15min to remove bacterial cells; subsequently, the remaining liquid was filtered through a membrane with a pore size of 0.22 μm to produce a cell-free supernatant.
The obtained supernatant was purified by Superdex 30 Increate 10/300 GL in Ä KTA pure protein isolation and purification platform (GE Healthcare, Marlborough, USA); the operating parameters are as follows: equilibrium volume is 2 Column Volumes (CV); eluting at pH 5.2; elution volume was 1.5 CV; ultraviolet light of 280 nm; the flow rate is 0.3 mL/min, and liquid is collected every 3 min; the antibacterial activity of each collected substance was determined using a liquid inhibition experiment (100 μ L of the isolate was mixed with TPY liquid 1:1 and inoculated with 10 μ L of honey bee hive coccus OD = 0.8).
The results are shown in fig. 4, and the substance under the first peak after liquid phase purification has obvious inhibitory effect on the melissococcus apis and is identified as the lantibiotide type III.
In a word, the experimental results show that the number of the bee colonies in the hive can be better controlled to be 10 by the experimental group fed with the bifidobacterium W81186One/intestine. The bifidobacterium W8118 plays a good role in inhibiting the melissococcus nidus by secreting III-type lanthionineThereby achieving the protection effect on the bees, and the animal experiment result is consistent with the in vitro bacteriostatic circle experiment result.
Sequence listing
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ctaatcctgt tcgctcccca cgctttcgct cctcagcgtc agtgacggcc cagagacctg 720
ccttcgccat cggtgttctt cccgatatct acacattcca ccgttacacc gggaattcca 780
gtctccccta ccgcactcca gtccgcccgt acccggcgcc gatccaccgt taagcgatgg 840
actttcacac cagacgcgac gaaccgccta cgagcccttt acgcccaata aatccggata 900
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Claims (2)
1. A BifidobacteriumBifidobacteriumsp.) W8118 with the preservation number of CGMCC NO.21789 in China Committee for culture Collection of microorganisms.
2. Use of Bifidobacterium W8118 as claimed in claim 1 or its fermentation supernatant for preparing medicine for inhibiting Apis mellifera: (Melissococcus pluton) The microbial inoculum or the feed.
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| CN114574396B (en) * | 2022-03-18 | 2024-02-06 | 中国农业大学 | Probiotic composition and application thereof |
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2889057A1 (en) * | 2005-08-01 | 2007-02-02 | Oreal | Topical cosmetic or dermatological composition, especially for treating or preventing sensitive or dry skin, comprises a microorganism and a polyunsaturated fatty acid |
| CN101679935A (en) * | 2007-05-03 | 2010-03-24 | 托比亚斯·奥洛夫松 | Novel bacteria isolated from fresh honey or the honey producing tract of honey bees |
| FR2937536A1 (en) * | 2008-10-28 | 2010-04-30 | Oreal | Cosmetic use of a lysate of Bifidobacterium species or one of its fractions to treat and/or prevent states of oily scalp |
| CN103260437A (en) * | 2010-10-14 | 2013-08-21 | 阿莎营养科学公司 | Optimized nutritional formulations, methods for selection of tailored diets therefrom, and methods of use thereof |
| WO2014097338A1 (en) * | 2012-12-11 | 2014-06-26 | Trovo' Stefano | Useful composition for the biological control of bee diseases |
| CN107616984A (en) * | 2017-01-13 | 2018-01-23 | 江苏西宏生物医药有限公司 | A kind of anti-diarrhea enteral nutrition composition |
| CN109182165A (en) * | 2018-08-21 | 2019-01-11 | 云南农业大学 | One plant of Lactobacillus helveticus and its application during bee raising |
| CN112458016A (en) * | 2020-11-27 | 2021-03-09 | 石家庄君乐宝乳业有限公司 | Bifidobacterium bifidum i771, and separation and purification method and application thereof |
| CN113265351A (en) * | 2021-05-11 | 2021-08-17 | 昆明理工大学 | Lactobacillus W8172 and application thereof |
| CN214178660U (en) * | 2020-12-28 | 2021-09-14 | 昆明理工大学 | Feeding box for bee toxicology and nutriology experiments |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2323450T3 (en) * | 2001-02-19 | 2009-07-16 | Societe Des Produits Nestle S.A. | CONSUMABLE PRODUCT THAT CONTAINS PROBIOTICS. |
| FR2985664B1 (en) * | 2012-01-18 | 2014-08-29 | Gilles Albert Chanforan | PROBIOTIC CONSISTS OF BACTERIA FROM THE BACILLI CLASS INTENDED FOR COLONIES OF BEES OF THE GENUS APIS FOR THE PREVENTION OF WINTER MORTALITIES AND COLONY COLLAPSE SYNDROME |
| US11529412B2 (en) * | 2015-11-30 | 2022-12-20 | Seed Health, Inc. | Method and system for protecting honey bees from pesticides |
| US10933128B2 (en) * | 2015-11-30 | 2021-03-02 | Joseph E. Kovarik | Method and system for protecting honey bees from pesticides |
| US10675347B2 (en) * | 2015-11-30 | 2020-06-09 | Joseph E. Kovarik | Method and system for protecting honey bees from fipronil pesticides |
| US10086024B2 (en) * | 2015-11-30 | 2018-10-02 | Joseph E. Kovarik | Method and system for protecting honey bees, bats and butterflies from neonicotinoid pesticides |
| US20200306325A1 (en) * | 2019-03-06 | 2020-10-01 | Oralta, Inc. | Probiotic consortia for oral health |
| CN113215039B (en) * | 2021-05-11 | 2022-04-15 | 中国农业大学 | Lactobacillus W8173 and application thereof |
-
2021
- 2021-05-11 CN CN202110509842.XA patent/CN113265350B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2889057A1 (en) * | 2005-08-01 | 2007-02-02 | Oreal | Topical cosmetic or dermatological composition, especially for treating or preventing sensitive or dry skin, comprises a microorganism and a polyunsaturated fatty acid |
| CN101679935A (en) * | 2007-05-03 | 2010-03-24 | 托比亚斯·奥洛夫松 | Novel bacteria isolated from fresh honey or the honey producing tract of honey bees |
| FR2937536A1 (en) * | 2008-10-28 | 2010-04-30 | Oreal | Cosmetic use of a lysate of Bifidobacterium species or one of its fractions to treat and/or prevent states of oily scalp |
| CN103260437A (en) * | 2010-10-14 | 2013-08-21 | 阿莎营养科学公司 | Optimized nutritional formulations, methods for selection of tailored diets therefrom, and methods of use thereof |
| WO2014097338A1 (en) * | 2012-12-11 | 2014-06-26 | Trovo' Stefano | Useful composition for the biological control of bee diseases |
| CN107616984A (en) * | 2017-01-13 | 2018-01-23 | 江苏西宏生物医药有限公司 | A kind of anti-diarrhea enteral nutrition composition |
| CN109182165A (en) * | 2018-08-21 | 2019-01-11 | 云南农业大学 | One plant of Lactobacillus helveticus and its application during bee raising |
| CN112458016A (en) * | 2020-11-27 | 2021-03-09 | 石家庄君乐宝乳业有限公司 | Bifidobacterium bifidum i771, and separation and purification method and application thereof |
| CN214178660U (en) * | 2020-12-28 | 2021-09-14 | 昆明理工大学 | Feeding box for bee toxicology and nutriology experiments |
| CN113265351A (en) * | 2021-05-11 | 2021-08-17 | 昆明理工大学 | Lactobacillus W8172 and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| "Characterization of bifidobacteria in the digestive tract of the Japanese honeybee,Apis cerana japonica";Meihua Wu等;《Journal of Invertebrate Pathology》;20121012;第112卷;第88-93页 * |
| "The Curious Case of Achromobacter eurydice,a Gram-Variable Pleomorphic Bacterium Associated with European Foulbrood Disease in Honeybees";Silvio Erler等;《Microb Ecol》;20170621;第1-6页 * |
| "肠道菌群在蜜蜂健康与疾病中的作用研究进展";陈奕霏等;《微生物学杂志》;20210415;第41卷(第2期);第92-100页 * |
| "蜜蜂肠道微生物功能及应用研究进展";黄祚骅等;《四川农业科技》;20190515(第05期);第47-49页 * |
| "西方蜜蜂工蜂肠道菌群定殖起始点和定殖稳态点的结构比较分析";董志祥等;《微生物学报》;20200518;第60卷(第7期);第1447-1457页 * |
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