A strain of Lactobacillus delbrueckii and its application in promoting the development of animal digestive tract

Abstract

The present invention belongs to the field of microbial technology, and particularly relates to a strain of Lactobacillus delbrueckii and its application in promoting the development of the animal digestive tract. A strain of Lactobacillus delbrueckii, which is Lactobacillus delbrueckii subsp.bulgaricus strain RGY1, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on March 29, 2024, and its deposit number is: CGMCC No.30207. The culture supernatant of Lactobacillus delbrueckii RGY1 can significantly promote the proliferation of rumen epithelial cells, and is a postbiotic with high probiotic potential for promoting the development of the rumen of ruminants. Therefore, the strain is a lactic acid bacteria strain with high probiotic potential.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Lactobacillus delbrueckii and application thereof in promoting the development of animal digestive tract. Background Art

[0002] The development of the digestive tract of young animals affects the performance and health of the animals' later production performance, especially the rumen of ruminants. Promoting early rumen development can improve the animal's feed conversion efficiency, reduce the economic cost of breeding, and improve the production efficiency of ruminants. It is particularly important to find and develop feed additives that regulate the development of the digestive tract of young ruminants. Probiotics have attracted the attention of researchers and the industry for their advantages such as being green, residue-free, safe and growth-promoting. Among them, lactic acid bacteria, as a common probiotic, can regulate intestinal microecology, prevent and treat gastrointestinal inflammation, enhance the intestinal epithelial barrier, promote intestinal digestion and utilization of protein, and promote intestinal peristalsis to improve constipation in the digestive tract of humans and animals.

[0003] In addition to directly affecting the health and production status of livestock and poultry, livestock probiotics will also enter human life with livestock and poultry activities and animal products, and are closely related to human health and safety. Therefore, in the process of developing and applying livestock probiotics, it is crucial to conduct a comprehensive safety assessment on them. It is necessary to refer to the relevant regulations of various parties on human probiotics and apply strict safety and effectiveness screening standards. In addition to safety assessment, probiotics need to be colonized in the animal digestive tract to play a probiotic role by competing for intestinal adhesion sites and balancing intestinal flora. Due to the differences between individual animals and the complex acid, enzyme and microbial environment in the animal digestive tract, probiotics cannot be effectively colonized in the digestive tract of all animals, and their probiotic effects and scope of application are greatly reduced. However, some metabolites in the supernatant culture of the strain can directly play a probiotic role without being affected by the complex environment in the animal digestive tract. Therefore, finding probiotics whose supernatant can play a probiotic role will effectively guarantee the probiotic properties of the strain and expand the scope of application of probiotics. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Lactobacillus delbrueckii, which is sensitive to common antibiotics, non-hemolytic, and has no virulence genes. The strain supernatant of the strain can promote the proliferation of rumen epithelial cells and further promote the development of animal digestive tract.

[0005] The technical solution adopted by the present invention to solve its technical problem is:

[0006] A strain of Lactobacillus delbrueckii, which is strain RGY1 of Lactobacillus delbrueckii subspecies bulgaricus (Lactobacillusdelbrueckii), which was deposited on March 29, 2024 at the General Microbiology Center of China Culture Collection (CGMCC for short, address: No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and its deposit number is: CGMCC No.30207.

[0007] A bacterial agent containing the Lactobacillus delbrueckii of the present invention. The bacterial agent includes a culture solution containing the Lactobacillus delbrueckii strain of the present invention, a supernatant of the culture solution, and the like.

[0008] A Lactobacillus delbrueckii extract is a freeze-dried product of the supernatant of the bacterial solution produced by culturing the strain of the present invention.

[0009] An application of the Lactobacillus delbrueckii of the present invention, the bacterial agent of the present invention, or the Lactobacillus delbrueckii extract of the present invention in promoting the development of the digestive tract of animals, especially promoting the proliferation of rumen epithelial cells, and having the potential to promote rumen development.

[0010] A feed additive, comprising the Lactobacillus delbrueckii of the present invention or the bacterial agent of the present invention or the Lactobacillus delbrueckii extract of the present invention. Preferably, the bacterial agent or bacterial powder of the strain is 10 10 ~10 11 The added amount of CFU / head / day or the strain supernatant extract is mixed with animal diets such as conventional animal feed, milk replacer, drinking water, etc. and fed to animals, where the animals include pigs, poultry and ruminants.

[0011] An animal feed comprises the feed additive of the present invention. Further, the animals include pigs, poultry and ruminants.

[0012] An animal medicine prepared from the Lactobacillus delbrueckii of the present invention, the bacterial agent of the present invention, or the Lactobacillus delbrueckii extract of the present invention.

[0013] The complete 16S rDNA nucleotide sequence of the Lactobacillus delbrueckii strain is shown in SEQ ID No.1.

[0014] The Lactobacillus delbrueckii RGY1 provided by the present invention is separated from naturally fermented yak yogurt, is Gram-positive, sensitive to common antibiotics, non-hemolytic, and has no virulence genes. Yeast extract has been reported to promote rumen development. Compared with the MRS culture medium containing yeast extract, the culture supernatant of Lactobacillus delbrueckii RGY1 can significantly promote the proliferation of rumen epithelial cells, and is a postbiotic with high probiotic potential for promoting rumen development in ruminants. Therefore, the strain is a lactic acid bacteria strain with high probiotic potential. The strain can be used as an alternative strain for probiotic preparations for ruminants such as cattle and sheep, and the strain culture supernatant extract can be used as an alternative additive for probiotic preparations for ruminants such as cattle and sheep. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 These are photos of Lactobacillus delbrueckii RGY1 under an oil objective lens and a scanning electron microscope;

[0016] Figure 2 is the growth curve of Lactobacillus delbrueckii RGY1;

[0017] Figure 3 It is a schematic diagram of the antibiotic sensitivity results of Lactobacillus delbrueckii RGY1;

[0018] Figure 4 It is a schematic diagram of the hemolytic screening results of Lactobacillus delbrueckii RGY1;

[0019] Figure 5 This is a schematic diagram of the virulence gene screening results of Lactobacillus delbrueckii RGY1;

[0020] Figure 6 This is a graph showing the effect of Lactobacillus delbrueckii RGY1 on the viability of rumen epithelial cells. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described in detail below through specific embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any form of modification and / or change made to the present invention will fall within the protection scope of the present invention.

[0022] In the present invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0023] The reagents used in the following examples, unless otherwise specified, can be purchased from conventional biochemical reagent stores.

[0024] The formula of MRS liquid culture medium is as follows: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 20 g / L glucose, 2 g / L triammonium citrate, 5 g / L sodium acetate, 0.1 g / L MgSO4, 0.05 g / L MnSO4, 2 g / L K2HPO4, 1 g / L Tween 80, and 0.1 g / L 0.1% resazurin solution.

[0025] Example 1 Isolation and identification of Lactobacillus delbrueckii RGY1

[0026] Lactobacillus delbrueckii RGY1 was isolated according to the following method:

[0027] 1. Collect yogurt made from natural fermentation of yak milk, store it at 4°C and transport it back.

[0028] 2. Mix yogurt with sterile PBS buffer at a ratio of 1:9, take 0.2 mL and inoculate it into 10 mL MRS liquid culture medium, place it in an anaerobic glove box in a 37°C constant temperature culture room for enrichment culture for 24 hours, then take out 1 mL and dilute it step by step by 10 4 ~10 5 Times, take 100uL of the diluted bacterial solution and spread it on the MRS solid culture plate. Place it in an anaerobic incubator at 37℃ for incubation. After independent plaques grow on the plate, select a culture dish with 30 to 200 plaques, pick white, round and neatly edged plaques that meet the standard morphology of lactic acid bacteria with a sterile inoculation loop and dissolve them in 200μL sterile PBS solution. After mixing, streak the mixed bacterial solution on the surface of the MRS solid culture medium and place it in a 37℃ constant temperature incubator for 24 hours. After repeating the above picking and streaking process 3 times, select a single colony and dissolve it in 200uL sterile PBS solution, mix it, and use a disposable sterile syringe to inject it into 10mL MRS culture medium, and culture it overnight at 37℃, 180rpm horizontal constant temperature shaker. After the culture is completed, mix the bacterial solution with anaerobic glycerol in a volume ratio of 1:1 and transfer it to a -80℃ ultra-low temperature refrigerator for storage.

[0029] 3. The isolated bacterial liquid was used to extract total DNA, and the extracted DNA was amplified by PCR using bacterial universal primers 27F and 1492R (27F: 5′-AGAGTTTGATCCTGGCTCAG-3′, SEQ ID No.2, 1492R: 5′-GGTTACCTTGTTACGACTT-3′, SEQ ID No.3). The PCR product obtained in the experiment was sequenced, and the sequencing results were compared by BLAST comparison of the NCBI database. The strain was identified as Lactobacillus delbrueckii subsp.bulgaricus strain and named RGY1.

[0030] 4. Such as Figure 1 As shown, Gram staining was used to take pictures under an oil microscope, and the results showed that Lactobacillus delbrueckii RGY1 was a Gram-positive bacterium; the strain was morphologically characterized using a scanning electron microscope, and the results showed that Lactobacillus delbrueckii RGY1 was long rod-shaped, with a diameter of about 0.5 μm and a length of about 2 to 5 μm.

[0031] Example 2 Determination of the growth curve of Lactobacillus delbrueckii RGY1

[0032] Frozen Lactobacillus delbrueckii RGY1 was inoculated into MRS liquid medium for activation. After subculture for 2 times, the bacterial solution in the middle logarithmic growth period was inoculated into MRS liquid medium at an inoculation ratio of 2% (v / v), and cultured in an anaerobic glove box (85% N2, 10% CO2, 5% H2) at 37°C, and its OD value was measured in a visible light spectrophotometer at a wavelength of 600nm every 2 hours, and a growth curve was drawn with time (unit: hour) as the horizontal axis and OD600 value as the vertical axis, and three replicates were set.

[0033] The results are as follows Figure 2 As shown in the figure, the bacterial growth curve can generally be divided into four stages: hysteresis phase, logarithmic phase, stationary phase and decay phase. After subculture for 2 times, the hysteresis phase of Lactobacillus delbrueckii RGY1 is 0 to 2 hours, the logarithmic growth phase is 2 to 12 hours, and it enters the stationary phase after 12 hours.

[0034] Example 3 Antibiotic sensitivity test of Lactobacillus delbrueckii RGY1

[0035] 200 μL of the concentration of about 10 8 After the CFU / mL bacterial liquid of Lactobacillus delbrueckii RGY1 was dried, seven commonly used antibiotic test strips, including rifampicin, vancomycin, erythromycin, ampicillin, amoxicillin, tetracycline and streptomycin, were placed in the center of the culture dish and cultured under anaerobic conditions at 37°C for 18 hours for observation. Figure 3The figure is a schematic diagram of the antibiotic sensitivity results of Lactobacillus delbrueckii RGY1. The diameter of the drug sensitivity zone was measured with a vernier caliper, and the results are shown in Table 1. Lactobacillus delbrueckii RGY1 was sensitive to all seven antibiotics, indicating that the Lactobacillus delbrueckii RGY1 strain did not develop resistance to common antibiotics.

[0036] Table 1 Antibiotic sensitivity results of Lactobacillus delbrueckii RGY1

[0037]

[0038]

[0039] Note: R, MS and S represent insensitive, moderately sensitive and sensitive to the corresponding antibiotics, respectively.

[0040] Example 4 Hemolytic Screening of Lactobacillus delbrueckii RGY1

[0041] Prepare MRS agar as the blood agar matrix. After autoclaving, cool the matrix to 45-50°C, add 5% volume of sterile defibrinated sheep blood preheated to room temperature, rotate the flask to mix thoroughly while avoiding the formation of bubbles, and invert the plate to cool. Take an appropriate amount of the test bacterial solution and streak it on the blood agar plate. Incubate at 37°C for 24 hours to confirm its hemolytic property. Strains that produce green areas around the colonies (α-hemolysis) and have no effect on blood agar (γ-hemolysis) are identified as non-hemolytic strains, while strains that produce blood lysis areas around the colonies (β-hemolysis) are identified as hemolytic strains. Figure 4 As shown, Lactobacillus delbrueckii RGY1 had no effect on blood agar and was identified as a non-hemolytic strain, indicating that the Lactobacillus delbrueckii RGY1 strain could not adversely affect the host through hemolysis.

[0042] Example 5 Virulence gene screening of Lactobacillus delbrueckii RGY1

[0043] As shown in Table 2, RGY1 was amplified by PCR in bacterial solution according to the primer sequences of virulence genes gelE, cylA, esp, efaAfs, hyl, asa, hdc, ace, tdc, and odc. After amplification, the amplified product was detected by agarose gel electrophoresis to identify whether the strain genome contained virulence genes. Figure 5 As shown, the results showed that after PCR amplification of RGY1 using the above-mentioned primers of virulence genes, no bands of corresponding products were found, and the virulence genes gelE, cylA, esp, efaAfs, hyl, asa, hdc, ace, tdc and odc did not exist in RGY1, indicating that the Lactobacillus delbrueckii RGY1 strain was non-toxic and harmless to the host.

[0044] Table 2 Primers and PCR amplification conditions of virulence genes

[0045]

[0046]

[0047] Example 6 Evaluation of the effect of supernatant of Lactobacillus delbrueckii RGY1 strain on promoting proliferation of rumen epithelial cells in ruminants

[0048] Isolation of lamb rumen epithelial cells: Three 5-day-old Hu sheep lambs were selected, and the lambs were anesthetized by intramuscular injection of the anesthetic drug Lumianning at 0.001 mL / kg. After anesthesia, the lambs were slaughtered and the ventral rumen pouch tissue was collected. Wash the tissue 2 to 3 times with ice-cold buffer (D-Hank's balanced salt solution, containing 500 U / mL penicillin, 500 mg / mL streptomycin, 100 mg / mL gentamicin and 5 mg / mL amphotericin B) until the solution contains no visible impurities and color. Place the tissue in DMEM culture medium containing 1,000 U / mL penicillin, 1,000 mg / mL streptomycin, 200 mg / mL gentamicin and 10 mg / mL amphotericin B and transport it back to the laboratory. Cut the rumen epithelial tissue into 1 cm 2 5 pieces of rumen epithelial tissue from each sheep were taken and mixed to form a mixed sample of 3 sheep. After washing 5 times, the rumen epithelial tissue samples were pre-digested with 0.25% trypsin-EDTA solution, and the digestion solution was collected every 10 minutes, and fresh 0.25% trypsin-EDTA solution was added to continue digestion. The collected digestion solution was centrifuged at 300g for 5 minutes at 4°C to separate the digestion solution and cell pellet. After discarding the digestion solution, the cells were resuspended in DMEM and filtered with a 40μm nylon cell strainer (BD Falcon, San Jose, California, USA) to obtain rumen epithelial cells.

[0049] Culture of rumen epithelial cells: Rumen epithelial cells were cultured in a 5.0% CO2, 37°C incubator using a culture medium containing 90% DMEM (Gibco) + 10% fetal bovine serum (CellMax Cell Technology Co., Ltd., Beijing) + 10 ng / mL EGF (MCE) + 10 uM Y-27632 (MCE) + 1× insulin-transferrin-selenium (ITS-G, Gibco).

[0050] Preparation method of Lactobacillus delbrueckii RGY1 strain supernatant: RGY1 was inoculated into MRS liquid culture medium, cultured at 37°C in an anaerobic glove box for 48 hours, the strain culture medium was centrifuged at 8,000×g for 10 minutes, the supernatant was filtered through a 0.22 μm sterile filter to obtain a sterile strain supernatant, and stored at -80°C for use.

[0051] Rumen epithelial cells were treated with PBS buffer, MRS medium and supernatant of Lactobacillus delbrueckii RGY1 strain at a ratio of 5% of the volume of the cell culture medium. Cells were collected after 24 h and 48 h of treatment, and cell activity was evaluated using a cell counting kit (CCK-8, Beyotime, Shanghai) according to the instructions.

[0052] The results are as follows Figure 6 As shown, at 24h, compared with PBS, the supernatant of Lactobacillus delbrueckii RGY1 strain (P<0.01) and MRS medium (P<0.0001) significantly increased the relative activity of rumen epithelial cells; at 48h, there was no significant difference in the relative activity of rumen epithelial cells between the MRS medium treatment group and the PBS treatment group, while the supernatant of Lactobacillus delbrueckii RGY1 strain significantly increased the relative activity of rumen epithelial cells compared with PBS (P<0.01) and MRS medium (P<0.01). This result shows that the supernatant of Lactobacillus delbrueckii RGY1 strain can promote the proliferation of rumen epithelial cells and has the potential to promote rumen development.

[0053] Example 7 Preparation of supernatant extract of Lactobacillus delbrueckii RGY1 strain

[0054] Using fermentation technology, a large amount of high-concentration Lactobacillus delbrueckii RGY1 bacterial liquid was enriched in MRS liquid medium at 37°C and 200rpm. The bacterial liquid was divided into 250ml centrifuge tubes and filled with CO2, and the bacteria were precipitated by high-speed centrifugation for 20min at 8,000×g low temperature conditions. The strain supernatant was poured out and placed at -80°C for hardening for 2h. After freeze-drying using a vacuum freeze dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany), the supernatant extract of Lactobacillus delbrueckii RGY1 strain was obtained and stored at -20°C.

[0055] Example 8 Preparation of Lactobacillus delbrueckii RGY1 powder

[0056] Using fermentation technology, a large amount of high-concentration Lactobacillus delbrueckii RGY1 bacterial liquid was enriched in MRS liquid medium at 37°C and 200rpm. The bacterial liquid was divided into 250ml centrifuge tubes and filled with CO2, and the bacteria were precipitated by high-speed centrifugation at 8,000×g for 20min. After discarding the supernatant, 1000ml of freeze-drying protective agent was added to resuspend the precipitate and placed at -80°C for hardening for 2h. After freeze-drying using a vacuum freeze dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany), the bacterial powder of Lactobacillus delbrueckii RGY1 was obtained and stored at -20°C.

[0057] The preparation method of the lyophilized protective agent provided in this embodiment is as follows: 100 g of skim milk powder is dissolved in 1000 ml of boiling distilled water, and the dissolved oxygen is removed by bubbling in a microwave oven again, and CO2 is continuously introduced into the serum bottle (above the liquid surface) for cooling and standby use.

[0058] Example 9 Application of Lactobacillus delbrueckii RGY1

[0059] According to the summary of Announcement No. 2045 of the Ministry of Agriculture and Rural Affairs and subsequent revised announcements, as of July 2023, Lactobacillus delbrueckii subspecies Bulgariae is listed as a microbial category in the Chinese Feed Additive Variety Catalogue (2013), and its scope of application is farmed animals. The Lactobacillus delbrueckii RGY1 provided by the present invention can be applied to animal breeding as a feed additive, and the application method is: the bacterial powder of the strain is 10 10 ~10 11 The added amount of CFU / head / day or the strain supernatant extract is mixed with animal diets such as conventional animal feed, milk replacer, drinking water, etc. and fed to animals, where the animals include pigs, poultry and ruminants.

[0060] in conclusion:

[0061] The complex microecological environment in the animal digestive tract and the host's specific immune and metabolic regulation of microorganisms may cause the probiotics fed directly to be inactivated in the body, limiting the performance of the probiotic effect. The uniqueness of the Lactobacillus delbrueckii RGY1 of the present invention is that experiments have shown that the culture supernatant of the strain can be directly fed to animals and produce a probiotic effect. Directly feeding the supernatant of probiotics can avoid the problem of probiotics being inactivated in animals. However, since the supernatant is a liquid, it is relatively inconvenient during transportation, storage, and feeding. The present invention dries the supernatant to prepare a supernatant extract, which can be directly mixed and fed to animals as a feed additive after becoming a solid.

[0062] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0063] The above describes in detail a strain of Lactobacillus delbrueckii provided by the present invention and its application in promoting the development of the digestive tract of animals. Specific examples are used herein to illustrate the principle and implementation of the present invention, and the description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, the present invention can also be improved and modified in a number of ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A strain of Lactobacillus delbrueckii, which is Lactobacillus delbrueckii subspecies bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus ) strain RGY1, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on March 29, 2024, and its deposit number is: CGMCC No.30207.

2. A bacterial agent containing the Lactobacillus delbrueckii according to claim 1.

3. A Lactobacillus delbrueckii extract, characterized in that The Lactobacillus delbrueckii extract is a freeze-dried product of the supernatant of the bacterial solution produced by culturing the strain according to claim 1.

4. Use of the Lactobacillus delbrueckii described in claim 1, the bacterial agent described in claim 2, or the Lactobacillus delbrueckii extract described in claim 3 for improving the relative activity of rumen epithelial cells in vitro.

5. A feed additive, characterized in that: The feed additive comprises the Lactobacillus delbrueckii described in claim 1 or the bacterial agent described in claim 2 or the Lactobacillus delbrueckii extract described in claim 3.

6. A ruminant feed, characterized in that: The animal feed comprises the feed additive according to claim 5.

7. A ruminant medicine prepared from the Lactobacillus delbrueckii of claim 1, the bacterial agent of claim 2, or the Lactobacillus delbrueckii extract of claim 3.

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