CN113215039B - Lactobacillus W8173 and application thereof - Google Patents

Abstract

The invention discloses a lactobacillus (lactobacillus sp.) W8173, the preservation number of which in China general microbiological culture Collection center is CGMCC NO.21787, the strain can improve the European foul brood symptom of bees, and can directly inhibit the bee hives in vitro bacteriostatic experiments; in vivo experiments, the single-bacterium honeybee fed with lactobacillus has a good inhibition effect on the bee horneri; through the separation of the supernatant of the lactobacillus W8173 culture solution, the secretion product of the lactobacillus W8173 has an inhibiting effect on the bee hive cocci.

Description

Lactobacillus W8173 and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus (A)lactobacillus sp.) W8173 and its uses.

Background

Bee (A)Apis mellifera) Is an important agricultural pollination insect, plays an important role in constructing ecological agriculture and bee product supply, and plays an important role in maintaining biological diversity and ecological balance for pollination of different vegetations. At present, about 36 crops such as apples, cotton, rape, soybeans and the like in China depend on the pollination effect of bees in large quantity, and the annual pollination value reaches 3042 million yuan. However, in recent years, the number of honeybee populations worldwide has declined year by year due to the reduction in honey plant diversity and habitat, interspecific competition, infection by pathogenic microorganisms and parasites, and the likeAnd (4) potential. Pathogenic microorganism and parasite infection are main inducement for sharp reduction of bee population quantity, and have the characteristics of strong destructiveness, obvious seasonality of disease occurrence, large difference of disease occurrence conditions in different regions and the like, so that bee populations are difficult to predict in the early disease occurrence stage and cannot be controlled pertinently, the health state of the bee populations is further deteriorated due to excessive application of abuse chemical drugs, the problem of drug residues of products is caused, and great influence is caused on the feeding enthusiasm of beekeepers and the pollination industry.

The European foul brood is second only to the American foul brood in the western bees, and the pathogenic bacteria of the European foul brood is the Behcet bee cocci (Melissococcus pluton) Gram-positive bacteria, are obligate pathogens of bees. The melissococcus nidus mainly attacks 1-2 days old bee larvae, so that 3-4 days old larvae die in large amount when not covered. The sick larvae collapse to the bottom of the nidus and are disorderly in position, and the bodies of the larvae become soft and rotten, so that the phenomenon of 'flower arrangement and seed spleens' between empty houses and ovaries can be caused. The occurrence of European foul brood is obvious seasonal, bee colonies are easy to occur in early spring and autumn, although the bee brood stage is mainly infected by the bee brood coccus, adult worker bees carry the bee brood coccus, so that the European foul brood is spread in the bee colonies and even among different bee colonies and bee yards, and the pathogenic mechanism of the bee brood coccus is not clear at present.

Generally, the abuse of antibiotics in bee farms due to the lack of effective treatment means not only causes drug resistance of pathogenic bacteria, but also destroys the microecological balance of the intestinal tracts of bees, and further worsens the disease. 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 relevant reports on the application of lactobacillus for controlling European foul brood are found.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a lactobacillus (A)lactobacillus sp.) W8173, which is preserved in China general microbiological culture Collection center (CGMCC) on 2 months and 1 day 2021, with the preservation number of CGMCC No.21787,address: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.

The Lactobacillus W8173 of the present invention is a Western bee (E.mellifera) obtained from JilinApis mellifera Linnaeus) Extracting total DNA of the strain, amplifying target fragments by using a bacterial 16S rRNA universal primer, recovering, cloning and sequencing the fragments, wherein the 16S rRNA sequence is shown as SEQ ID No. 1; similarity comparison of 16S rRNA is carried out on the sequence of the 16S rRNA gene fragment of the bacterium by using BLAST of NCBI, and the result shows that the similarity reaches 99.72 percent in a recent relation with lactobacillus Firm5, so that the strain is identified as lactobacillus and is named as lactobacillus W8173.

The invention also aims to apply the lactobacillus W8173 or the fermentation product thereof in preparing microbial inoculum or feed for treating European foul brood.

The application of the invention is that the lactobacillus W8173 or the fermentation product thereof is used for preparing microbial inoculum or feed for preventing and treating European foul brood, namely the lactobacillus W8173 or the fermentation product thereof is used as an active ingredient for preparing the microbial inoculum 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 lactobacillus W8173 or fermentation product thereof, and one or more than one kind of the pathogenic bacteria preventing and treating microbial agent or auxiliary material acceptable on the feed can be added to prepare a dosage form suitable for the microbial agent or feed, the dosage form is added according to the conventional addition amount, and the number of effective viable bacteria in the microbial agent or feed is not less than 10¹¹cfu/g。

The invention has the beneficial effects that:

the invention aims to lay a foundation for disease control of bees and market development and industrial application of novel probiotics; the lactobacillus W8173 provided by the invention can inhibit the action of the melissococcus nidus by secreting antibacterial peptide, and has great potential in treating European foul brood.

The lactobacillus W8173 provided by the invention can improve the European foul brood symptom of bees, and can directly inhibit the bee hives in an in vitro bacteriostatic circle experiment; in vivo experiments, the single-bacterium honeybee fed with the lactobacillus W8173 has a good inhibition effect on the melissococcus nidus; through the separation of the supernatant of the lactobacillus W8173, the lactobacillus achieves the inhibition effect on the meliococcus alvei through secreting the product; the microbial inoculum or the feed is environment-friendly, pollution-free, free of influence on quality of bee products, and suitable for industrial production and market popularization and application.

Drawings

FIG. 1 shows the results of in vitro bacteriostatic experiments on melissococcus nidus with the supernatant of Lactobacillus W8173;

FIG. 2 shows the results of in vitro bacteriostatic experiments on melissococcus nidus with concentrated supernatant of Lactobacillus W8173;

FIG. 3 shows the results of in vivo experiments of Lactobacillus W8173 on the bee hives;

FIG. 4 shows the purification results of the antibacterial substances in the culture supernatant of Lactobacillus strain W8173 (upper panel) and the results of the bacteriostatic experiment (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, 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 culture media are commercially available products and used according to the instructions;

wherein the MRS culture medium is: 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 2 g of dipotassium phosphate, 2 g of diammonium citrate, 5 g of sodium acetate, 20 g of glucose, 801 mL of tween-801, 0.5 g of magnesium sulfate, 0.25 g of manganese sulfate and 15 g of agar powder, wherein the volume is fixed to 1L by using distilled water, and the pH value is adjusted to 6.2-6.4.

Example 1: isolation and identification of Lactobacillus W8173

Capturing western bees from Jilin, dissecting and taking out complete intestinal tracts of the bees, manually grinding the intestinal tracts in 100 mu L of 25% (v/v) glycerol by using a grinding rod to grind and break the intestinal walls, and storing the intestinal tracts in an ultralow-temperature medical refrigerator at the temperature of-80 ℃; the intestinal tract sample is streaked on the upper three regions of a lactic acid bacteria culture Medium (MRS), and cultured in a 5% carbon dioxide incubator at 35 ℃ for 2-3 days. Observing colony morphology, selecting single colony, streaking on MRS culture medium in eight regions, and performing purification culture 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 16S rRNA sequencing.

The total DNA of the bacteria was extracted by using a bacterial genome DNA extraction kit of Tiangen Biochemical technology Ltd, using a 16S rRNA universal primer (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3') in a 50. mu.L PCR system (ddH)₂O19. mu.L, 2 XSantaQ PCR Mix 25. mu.L, Primer F2. mu.L, Primer R2. mu.L, template DNA 2. mu.L); the PCR conditions were: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, renaturation at 60 ℃ for 30s, extension at 72 ℃ for 1 min, and 30 cycles; finally, extension is carried out for 5min at 72 ℃.

The 16srDNA amplification sequencing result of the embodiment shows that the 16s rRNA sequencing result of the strain with the serial number of W8173 is shown as SEQ ID NO. 1; the comparison in the database by using BLAST tool shows that the strain W8173 related to the invention has the closest relationship with lactobacillus Firm-5, and the similarity reaches 100%. Therefore, the strain was identified as lactobacillus, named lactobacillus W8173.

Example 2: preparation of freeze-dried microbial inoculum

Activating lactobacillus Apis W8173 with MRS culture medium, inoculating 3% of the strain, transferring the activated strain into MRS liquid culture medium according to the inoculum size of 4%, culturing at 37 deg.C for 25h, centrifuging (5000 g, 15 min), and harvesting thallus for use. Adding protectant (trehalose 1%, sodium glutamate 0.5%, skimmed milk 1%, ascorbic acid 0.25%, dextran 0.25%, g/100mL distilled water) into the above thallus at volume ratio of 3: 1, and mixing well for 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 512 Pa for 6 h, then 256 Pa for 5h, and then 103 Pa until a powder product is obtained. The freeze-dried microbial inoculum is stored at 4 ℃ and 25 ℃ respectively, the viable count of each microbial inoculum 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 ℃; stable at 25 deg.CThe qualitative difference is certain when the product is stored at 4 ℃, but the survival level is still kept at 90%; the effective bacteria number of the lactobacillus W8173 powder is not less than 2 multiplied by 10¹¹ cfu/g；

。

Example 3: in vitro bacteriostatic experiment of lactobacillus W8173

(1) Preparation of a test product lactobacillus W8173 sterile supernatant: inoculating the lactobacillus W8173 freeze-dried microbial inoculum in the experiment into a lactobacillus culture medium (MRS broth culture medium powder 52 g, distilled water added to 1L, and sterilization at 121 ℃ for 15 min), culturing for 5 days, centrifuging the culture solution for 10 min by a low-temperature high-speed centrifuge 8000 g, taking supernatant, and finally filtering the supernatant through a filter membrane with the aperture of 0.22 mu m to obtain lactobacillus W8173 sterile supernatant;

(2) preparation of a test product lactobacillus W8173 concentrated sterile supernatant: concentrating 1L of the sterile supernatant of the lactobacillus W8173 into 100mL by a rotary evaporator (55 ℃), thus obtaining the concentrated sterile supernatant of the lactobacillus W8173;

(3) culture and bacteriostatic zone experiment of pathogenic bacterium meliococcus alvei

Adding 5 mL of 1 XPBS buffer solution into a 10 mL centrifuge tube, scraping all the honey bee nidus 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 10 mm holes on the coated agar medium; placing 100 mu L of lactobacillus W8173 sterile supernatant and concentrated sterile supernatant into 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 and fig. 2, and it can be seen that the sterile supernatant and the concentrate of lactobacillus W8173 have better inhibitory effect on the bee hives, and the comparison of fig. 1 and fig. 2 shows that the 10 times of the concentrate has better inhibitory effect than the sterile supernatant.

Example 4: animal experiment of Lactobacillus plantarum W8173 on Petasites apis mellifera

1. Material

Preparation of a test lactobacillus W8173 suspension solution: taking 1 g of lyophilized bacteria agent (wherein the effective viable count is not less than 10)¹¹ cfu/g), placing the mixture into a 1L beaker, adding a mixed solution of 450mL of 50% sucrose solution with mass concentration and 450mL of 1 XPBS buffer solution, stirring and suspending, and simultaneously adding 100 g of pollen to continue suspending to obtain the lactobacillus W8173 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 lactobacillus W8173 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 2 mL 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 15 min 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 Lactobacillus W8173 Single-bacterium honeybee: 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 2 mL 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 15 min under ultraviolet; and then picking 25 bees from a lunch box, transferring the bees into a sterile disposable cup (3 groups of parallel tests), adding the lactobacillus W8173 suspension solution into a 2 mL centrifuge tube, feeding the sterile bees and sterile pollen at the same time, and culturing the mixture in a sterile incubator at 35 ℃ for 7 days to obtain the lactobacillus W8173 single-bacterium bee model.

Pathogen infection: respectively culturing the aseptic honeybees in the aseptic honeybee group and the single-bacterium honeybees in the lactobacillus W8173 for seven days, and then respectively carrying out a dip dyeing test by using the honey bee dorsalis;

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 2 mL tube containing 500 μ L of 0.1mm sterile zirconia beads; lysis with MO BIO Vortex Genie for 3 min 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: testing the quantity of the bee hive coccus by using a QuantStaudio 1 real-time fluorescent quantitative polymerase chain reaction instrument produced by Thermo Fisher company; 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'); qPCR test is carried out by adopting a dye method, and AceQ Universal SYBR qPCR Master Mix dye method reagent produced by Nanjing Novozam is selectedThe qPCR system was prepared in a cassette format, wherein the total volume of the system was 20. mu.L, and included 10. mu.L of 2 XCAMQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd.), 0.4. mu.L of the forward primer, 0.4. mu.L of the reverse primer, 1. mu.L of the sample to be tested, 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. 3, and it can be seen from the results of fig. 3 that the lactobacillus W8173 suspension has a better inhibitory effect on the honey bee cocci than the aseptic bees infected with the honey bee cocci.

Example 5: separation and antibacterial activity verification of lactobacillus W8173 culture supernatant

Lactobacillus W8173 was cultured in MRS liquid medium at 37 ℃ for 5 days and centrifuged in a high-speed refrigerated centrifuge (Thermo Scientific, Waltham, MA, USA) at 8000 rpm at 4 ℃ for 15 min 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; collecting liquid 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 1:1 MRS liquid and inoculated with 10 μ L of honey bee hive coccus OD = 0.8).

The experimental result is shown in figure 4, and the purified Helveticin-J (peak 3) has obvious inhibitory effect on the bee hives;

the experimental result of the invention shows that the number of the bee-brood cocci in the intestinal tract of the bee of the lactobacillus W8173 is less than that of the control group, so that the number of the bee-brood cocci can be better controlled to be 10⁶ A plurality of; the lactobacillus W8173 has a good inhibition effect on the bee horneri by secreting products, thereby achieving a certain protection effect on bees; the animal experiment result is consistent with the in vitro bacteriostatic circle experiment result.

Sequence listing

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Claims (3)

1. A Lactobacillus (B)lactobacillus sp.) W8173 with the preservation number of CGMCC NO.21787 in China Committee for culture Collection of microorganisms.

2. Use of lactobacillus W8173 according to claim 1 for the preparation of a bacterial agent for the treatment of european foul brood.

3. Use of lactobacillus W8173 according to claim 1 for the preparation of a feed for the treatment of european foul brood.

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