Lactobacillus khakensiensis H09 with intestinal motility and immunity improving function and application of lactobacillus khakensiensis H09
By screening Lactobacillus quinquefolius H09 and preparing freeze-dried bacterial agents, the problem of damage to the intestinal flora of bees was solved, and the bees' food intake, intestinal development and immunity were improved.
Patent Information
- Application Number
- CN202411053054.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-01
- Publication Date
- 2026-02-06
AI Technical Summary
In existing technologies, the use of antibiotics in commercial beekeeping leads to damage to the gut microbiota, and the lack of high-quality bee probiotic strains affects bee health and gut microbial diversity.
Lactobacillus quinquefolius H09 was screened and isolated, and prepared into a freeze-dried bacterial agent for use in the intestines of bees. It improved bee health by regulating food intake, intestinal development and immunity.
It significantly increases the amount of food consumed by bees, increases the length of intestinal development, promotes intestinal motility, and enhances the bees' immunity.
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Abstract
Description
Technical Field
[0001] This application belongs to the field of microbial technology, specifically relating to a strain of Lactobacillus quercetin H09 that can increase food intake, increase intestinal length, promote intestinal motility, and enhance immunity, and its application. Background Technology
[0002] Honeybees (Apis) belong to the order Hymenoptera, superfamily Apidae, and genus Apis. They are polyphagous, social insects. Commercially kept honeybees have high economic value as pollinators. However, some husbandry techniques used in commercial honey production can adversely affect the gut microbiota. For example, in the United States, antibiotics are frequently used to prevent bedbug and mite infections in commercially kept honeybees, but this can also damage the bees' gut flora and reduce gut microbial diversity.
[0003] Lactic acid bacteria are recognized as probiotics in the gut microbiota of humans and animals, benefiting the host's health. Currently, strains of Lactobacillus and Bifidobacterium are widely used in probiotic preparations for humans and other animals. For example, in mammal and fish farms, the use of probiotic strains can partially replace antibiotics, thereby improving animal growth and reducing disease incidence. Studies have shown that the application of Lactobacillus in bee colonies can inhibit Paenibacillus larvae, which causes American bedbug disease, and reduce the mortality rate of infected larvae. Furthermore, the application of lactobacillus preparations such as Lactobacillus kunkeei to bees can reduce larval mortality, increase hive weight, increase the size of bee fat bodies and related glands, and increase the amount of nutrient membranes around glands, thus providing bees with better digestive function. In addition, Lactobacillus can also regulate the bee gut microbiota, thereby promoting bee gut health.
[0004] Currently, the screening and research of lactic acid bacteria in the bee gut has made little progress, and there is a lack of high-quality probiotic strains for bees on the market. Therefore, there is a significant research and market demand for developing probiotic strains with beneficial functions. Summary of the Invention
[0005] To address the problems existing in the prior art, the purpose of this application is to provide a *Lactobacillus quercifolius* H09 that promotes bee health and its applications. This *Lactobacillus quercifolius* H09 was deposited on July 17, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, 100101, with accession number CGMCC NO. 31250.
[0006] Specifically, this application relates to the following aspects:
[0007] 1. A strain of Lactobacillus quercetin H09, with the accession number CGMCC NO.31250 from the China General Microbiological Culture Collection Center.
[0008] 2. The application of Lactobacillus quinquefolius H09 described in 1 above in regulating food intake, intestinal development, intestinal motility, and immunity.
[0009] 3. A microbial agent comprising Lactobacillus quinquefolius H09 as described in 1 above.
[0010] 4. The microbial agent according to 3 above, wherein the viable count of *Lactobacillus quercetin* H09 in the microbial agent is 1 × 10⁻⁶. 5 -1×10 13 CFU / g, preferably 1×10⁻⁶ 8 -1×10 11 CFU / g.
[0011] 5. The microbial agent according to claim 3 above, wherein the microbial agent further includes excipients permitted in the field of microbial preparations.
[0012] 6. A probiotic composition comprising Lactobacillus quinquefolius H09 as described in 1 above, or the probiotic agent described in any one of 3-5 above.
[0013] 7. The use of Lactobacillus quinquefolius H09 described in 1 above, the bacterial agent described in any one of 3-5 above, or the probiotic composition described in 6 above in the preparation of products for regulating food intake, regulating intestinal development, regulating intestinal motility, and regulating immunity.
[0014] 8. The use described in 7 above, wherein the article is food, feed, medicine, or health product.
[0015] The beneficial effects of this application are as follows:
[0016] This application aims to establish a system for screening and isolating probiotic strains from the intestines of bees, thereby laying the foundation for the commercial development and industrial application of bee probiotics.
[0017] This application provides a Lactobacillus quercetin H09 strain that can significantly increase food intake, increase intestinal length, promote intestinal motility, and enhance immunity. Attached Figure Description
[0018] The accompanying drawings are provided to better understand this application and do not constitute an undue limitation thereof. Wherein:
[0019] Figure 1 Results of the effect of Lactobacillus quinquefolius H09 on bee diet;
[0020] Figure 2 Results of the effect of Lactobacillus quinquefolius H09 on the intestinal development length of honeybees;
[0021] Figure 3 Results of the effect of Lactobacillus quinquefolius H09 on bee intestinal motility;
[0022] Figure 4 Results of the effect of Lactobacillus quinquefolius H09 on the expression of immune factors in bees; Detailed Implementation
[0023] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.
[0024] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.
[0025] Example
[0026] Example 1: Isolation and purification method of Lactobacillus quercetin H09
[0027] The honeybees were sourced from Mentougou District, Beijing. A complete gut sample from a healthy honeybee was placed in a 1.5ml sterile centrifuge tube, and 100ul of 25% (v / v) glycerol PBS was added. The sample was then ground on ice. The gut sample was streaked three times on lactic acid bacteria medium (MRS) containing 2% (w / v) fructose and 0.1% (w / v) cysteine, and incubated at 35℃ with 5% CO2 for 48-72 hours. Colony morphology was observed. Single colonies were picked and re-streaked eight times on MRS medium containing 2% (w / v) fructose and 0.1% (w / v) cysteine, and purified in a 35℃, 5% CO2 incubator. Several single colonies were selected from the medium, numbered, and 20 single colonies were selected for 16S rDNA sequencing.
[0028] The specific 16S rDNA sequence of this bacterium is shown below (SEQ ID NO:1):
[0029] CGAACGAGCTCTCCCAAATTGATTTTATGCTTGCATAAATGATTTTTGGATTCGGAGCGAGTGGCGAACTGGTGAGTAACACGTGGGTAACCTGCCCCGAAGCGGGGGATAACATTTGGAAACAAGTGCTAATACCGCATAATTAGTTGGAACCGCATGGTTCCAACTTGAAAGATGGCT CTGCTATCACTTTGGGATGGACCCGCGCCGTATTAGTTAGTTGGTGAGATAAAAGCCCACCAAGACGATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATG GAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAAAGAAGAACAAGTGTTAGAGTAACTGTTAACACTTTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGT AAAGCGAGCGCAGGCGGTTTTGTAAGTCTGCTGTGAAAGCCCTCAGCTCAACTGAGGAAGTGCAGTGGAAACTACAAAACTTGAGTACAGAAGAGGAAAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTTTCTGGTCTGTTACTG
[0030] Example 2: Preparation of freeze-dried bacterial agent
[0031] Lactobacillus quinquefolius H09 was activated with MRS medium containing 2% (w / v) fructose and 0.1% (w / v) cysteine at an inoculation rate of 2%. The activated strain was then transferred to 1 L of MRS medium at an inoculation rate of 5% and cultured at 35°C for 25 h. The cells were collected by centrifugation (6000 g, 15 min) for later use.
[0032] The harvested mycelium was added to the protectant at a volume ratio of 5:1 and mixed evenly for later use. The protectant was an aqueous solution with the following formula: trehalose 1% (w / v), sodium glutamate 0.5% (w / v), skim milk 1% (w / v), ascorbic acid 0.25% (w / v), and dextran 0.25% (w / v).
[0033] Before freeze-drying, the sample needs to be pre-frozen in an ultra-low temperature freezer at -80℃ for 8 hours. Then, a 5-stage freeze-drying process is adopted. The first stage is freeze-drying at 0 Pa pressure and -40℃ for 2 hours, followed by freeze-drying at 0 Pa pressure and -30℃ for 3 hours, then freeze-drying at 0 Pa pressure and -10℃ for 2 hours, and finally freeze-drying at 0 Pa pressure and 0℃ for 4 hours. Finally, freeze-drying is carried out at 0 Pa pressure and 25℃ for 24 hours until the powder product is obtained.
[0034] The freeze-dried bacterial agents were stored at 4℃ and 25℃ respectively. The viable cell count of each bacterial powder was measured every 2 months. The experiment was repeated in triplicate, and the results are shown in Table 1. As can be seen from the table, after 10 months of storage at 4℃ and 25℃, the viable cell count of the freeze-dried bacterial agents remained at a high level. Although the stability at 25℃ was somewhat different from that at 4℃, it still maintained a survival rate of over 90%. Among them, the effective bacterial count of Lactobacillus quinquefolius H09 bacterial powder was not less than 2×10¹³ cfu / g.
[0035] Table 1: Statistical count of viable Lactobacillus quinquefolius H09
[0036]
[0037]
[0038] Example 3: Effect of Lactobacillus quercetin H09 on bee food intake
[0039] 1. Preparatory work for the experiment
[0040] Weigh 500g of sucrose and dilute to 1L with ultrapure water to obtain a 50% (w / v) sucrose solution. Filter the solution through a 0.22μm filter membrane for sterilization to obtain the required 50% (w / v) sterile sucrose solution for the experiment. Dispense the purchased pollen (rapeseed pollen from Qinghai Province) into resealable bags, ensuring strict sealing, and sterilize with an electron beam at 30kGy for 4 hours to obtain sterile pollen.
[0041] 2. Selection and Cultivation of Experimental Bees
[0042] Select suitable honeycomb frames. Using a sterilized awl or tweezers, gently pry open the pupa caps at random points on the honeycomb frame to observe the maturity of the pupae. Continue prying open the caps around pupae that are darker in color and have not yet grown hair. When prying open the pupa caps, be careful to clean the surrounding area as much as possible to avoid bringing honeycomb debris with the pupae. Hold the sterilized tweezers firmly together and insert them from above the pupae, then open them to the sides. Gently pinch the thorax of the pupae to remove them, avoiding physical damage as much as possible. Note that pupae that have already developed wings should not be used.
[0043] Place the selected bee pupae into plastic square boxes that have been disinfected with 84 disinfectant and alcohol beforehand. Insert 2mL centrifuge tubes (with holes punched in the tube walls) containing sterile sucrose solution into the side or top of the box. Each square box can hold 100-150 bee pupae. Place the square boxes containing bee pupae in an incubator at 35℃ and 60-80% humidity overnight.
[0044] On the second day, observe the emergence of bee pupae (if they haven't emerged, culture them for another night). Gently remove the emerged bees and aliquot them into disposable transparent culture cups. Take a new sterile 2mL centrifuge tube, add 990μL of sterile PBS, then add 10μL of normal worker bee gut fluid, and mix well. Finally, fill the remaining space in the centrifuge tube with 50% sterile sucrose solution, and gently shake the centrifuge tube in your hand to mix thoroughly. Feed each cup of newly emerged bees with the above mixture and continue to incubate in a constant temperature incubator for two days.
[0045] 3. Animal grouping and experimental design
[0046] Two hundred bees were randomly divided into two groups of 100 bees each, with 25 bees per cage. The groups were a control group and an experimental group. The control group was fed a liquid diet of 50% (w / v) sugar water and a solid diet of rapeseed pollen powder. The experimental group was fed a liquid diet of 50% (w / v) sugar water and a solid diet of rapeseed pollen powder containing *Lactobacillus quinquefolius* H09 at a concentration of 10⁹ cfu / g. The added *Lactobacillus quinquefolius* H09 was the freeze-dried bacterial agent prepared as described in Example 2. The diet was changed daily for 7 consecutive days.
[0047] 4. Food intake testing
[0048] From day 1 to day 7 of feeding and rearing, weigh the bee food before and after each daily change for food quantity calculation. The bee food quantity is as follows: Figure 1 As shown, ** indicates a significance level less than 0.01. The results show that the *Lactobacillus quinquefolius* H09 described in this application significantly increases the food intake of bees.
[0049] Example 4: Effect of Lactobacillus quercetin H09 on intestinal development length in bees
[0050] 1. The experimental bees used to detect intestinal development length were bees treated with Lactobacillus quinquefolius H09 for 7 days as described in Example 3 above.
[0051] 2. Methods for detecting intestinal development length
[0052] Hold the bee by its thorax with tweezers, tail facing upwards. Use another pair of tweezers to grasp the last segment of the tail and slowly pull out the complete gut. Arrange the removed gut neatly on measuring paper, label with group information and a ruler, take a photograph, and measure the length using ImageJ software. The length of the bee's gut development is shown below. Figure 2 As shown, * indicates a significance level less than 0.05. The results show that the *Lactobacillus quinquefolius* H09 described in this application significantly increases the intestinal length of bees.
[0053] Example 5: Effects of Lactobacillus quercetin H09 on intestinal motility in bees
[0054] 1. The intestinal motility test bees were bees treated with Lactobacillus quinquefolius H09 for 7 days as described in Example 3 above.
[0055] 2. Methods for detecting intestinal motility
[0056] Take a 1.5ml centrifuge tube and punch a hole in the cap. Place the bees to be tested into the centrifuge tube with the hole, starve them in the incubator for 1-2 hours, and then transfer them to the test table. Use a pipette to take 5ul of 50% (w / v) sugar water containing 0.2% (w / v) Coomassie Brilliant Blue and feed the bees through the hole in the centrifuge tube or by gently opening the cap.
[0057] After feeding, place the bees in individual feeding cups and allow them to move freely. Precisely time the process; 30 minutes after feeding each bee, immediately extract the complete gut and place it on measuring paper. Gently stretch the gut into a straight line using tweezers. Lightly mark the start and end points of the gut, as well as the staining length, on the measuring paper with a marker. Take a photograph with a ruler. Measure the staining length and total gut length using ImageJ. The staining length / total gut length is the gut motility value.
[0058] Bee intestinal motility Figure 3 As shown, ** indicates a significance level less than 0.01. The results show that the *Lactobacillus quinquefolius* H09 described in this invention significantly promotes intestinal motility in bees.
[0059] Example 6: Effects of Lactobacillus quercetin H09 on bee immunity
[0060] 1. Immunity evaluation experimental bees were bees treated with Lactobacillus quinquefolius H09 as described in Example 3 above for 7 days.
[0061] 2. Total RNA was extracted from the heads of bees in the control group and the Lactobacillus quinquefolius H09-added group using the BTN81220 column-type insect RNA extraction kit. The three immune genes, Defensin, Abaecin, and Apidaecins, were quantitatively analyzed using an Applied Biosystems real-time quantitative PCR instrument.
[0062] The three primers for amplifying immune genes are shown in Table 2:
[0063] Table 2: Primer sequences for immune gene amplification
[0064]
[0065] Bee immunity such as Figure 4 As shown, * represents a significance level less than 0.05, ** represents a significance level less than 0.01, and **** represents a significance level less than 0.0001. The results show that the *Lactobacillus quinquefolius* H09 described in this invention significantly promotes the expression of immune factors in bees.
Claims
1. A strain of Lactobacillus quercetin H09, with the accession number CGMCC NO.31250 from the China General Microbiological Culture Collection Center.
2. The application of Lactobacillus quinquefolius H09 as described in claim 1 in regulating food intake, intestinal development, intestinal motility, and immunity.
3. A microbial agent comprising Lactobacillus quinquefolius H09 as described in claim 1.
4. The microbial agent according to claim 3, wherein the viable count of *Lactobacillus kunzhai* H09 in the microbial agent is 1 × 10⁻⁶. 5 -1×10 13 CFU / g, preferably 1×10⁻⁶ 8 -1×10 11 CFU / g.
5. The microbial agent according to claim 3, wherein the microbial agent further comprises excipients permitted in the field of microbial preparations.
6. A probiotic composition comprising Lactobacillus quinquefolius H09 as described in claim 1, or the probiotic agent as described in any one of claims 3-5.
7. The use of Lactobacillus quinquefolius H09 as described in claim 1, the bacterial agent as described in any one of claims 3-5, or the probiotic composition as described in claim 6 in the preparation of products for regulating food intake, regulating intestinal development, regulating intestinal motility, and regulating immunity.
8. The use according to claim 7, wherein the article is food, feed, medicine, or health product.