Lactobacillus hens C2-16-2 and application thereof

Abstract

The invention discloses a lactobacillus henryi C2-16-2 and application thereof. The preservation number of the lactobacillus henryi C2-16-2 is CCTCC (China Center for Type Culture Collection) NO: M 20241905. The lactobacillus henryi C2-16-2 disclosed by the invention has certain acid resistance, and can still grow in an MRS culture medium with the pH value of 3; the strain has certain salt tolerance, can grow in an MRS culture medium added with 4g / L cholate, and can grow in an MRS culture medium added with 8% of NaCl; after the hen lactobacillus C2-16-2 is added into the feed, the feed has the effect of promoting the development of intestinal tracts of chicks.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a Lactobacillus gallus C2-16-2 and an application thereof. Background Art

[0002] The intestinal health of poultry plays a vital role in its growth and development. As the main site for the digestion and absorption of nutrients, the health of the small intestine directly affects the growth rate and feed utilization efficiency of poultry. Especially in the breeding process of broilers, the development and functional improvement of the intestine is one of the key factors to improve production performance. In recent years, studies have shown that intestinal microbiota plays an important role in maintaining intestinal health, promoting nutrient absorption and enhancing immune function. Therefore, regulating the intestinal microbiota and promoting the colonization of beneficial bacteria have become important strategies to improve the intestinal health and overall growth performance of broilers.

[0003] In the process of poultry farming, how to effectively promote the healthy development of the small intestine of chickens and thus improve the overall growth performance is a key issue that needs to be solved. Current research mainly focuses on the impact of intestinal health on broiler growth, but research on the mechanism of optimizing intestinal morphology, structure and function by specific probiotic strains is still relatively weak. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a Lactobacillus gallinarum C2-16-2 and its application; Lactobacillus gallinarum is a lactic acid bacteria widely present in the intestinal tract of poultry, with good colonization ability and multiple potential benefits. The present invention studies the role and mechanism of Lactobacillus gallinarum C2-16-2 in the development of the small intestine of chickens, and by deeply exploring the effect of Lactobacillus gallinarum C2-16-2 on the development of the small intestine of chickens, a new scientific basis can be provided for the intestinal health management of broiler chickens.

[0005] To achieve the above purpose, the technical solution designed by the present invention is as follows:

[0006] The invention provides a Lactobacillus gallinarum C2-16-2, whose preservation number is CCTCC NO: M 20241905.

[0007] The above-mentioned C2-16-2 strain belongs to Lactobacillus gallinarum, and is named Lactobacillus gallinarum C2-16-2, abbreviated as Lactobacillus gallinarum C2-16-2; on September 2, 2024, the Lactobacillus gallinarum C2-16-2 was deposited in the China Center for Type Culture Collection, with the preservation address being Wuhan University, Wuhan City, Hubei Province, and its preservation number is: CCTCC NO: M 20241905.

[0008] The present invention also provides an application of the above-mentioned Lactobacillus gallus C2-16-2 in promoting the intestinal development of chicks.

[0009] The present invention also provides an application of the above-mentioned Lactobacillus gallus C2-16-2 in the preparation of an additive for promoting intestinal development in chicks.

[0010] The present invention also provides an additive for promoting intestinal development in chicks, wherein the additive contains Lactobacillus gallus C2-16-2, wherein the concentration of Lactobacillus gallus C2-16-2 is 10 9 -10 10 cfu / mL.

[0011] The present invention also provides a feed for promoting intestinal development in chicks, characterized in that: the feed is a basic diet supplemented with the above-mentioned additive or the above-mentioned Lactobacillus gallus C2-16-2, wherein the content of Lactobacillus gallus C2-16-2 is 1×10 11 -5x10 11 cfu / kg.

[0012] 6. The feed according to claim 5, characterized in that the content of the Lactobacillus gallus C2-16-2 is 10 11 cfu / kg.

[0013] Beneficial effects of the present invention:

[0014] (1) Lactobacillus gallinarum C2-16-2 has a certain acid resistance and can still grow in MRS medium with pH = 3;

[0015] (2) Lactobacillus gallinarum C2-16-2 has a certain salt tolerance and can grow in MRS medium supplemented with 4 g / L bile salt and in MRS medium supplemented with 8% NaCl;

[0016] (3) When Lactobacillus gallinarum C2-16-2 is added to feed, it has the effect of promoting intestinal development in chicks. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a comparison result of the genome amplified sequence of Lactobacillus gallinarum C2-16-2 in the NCBI database;

[0018] Figure 2 This is a picture of Lactobacillus gallinarum C2-16-2 cultured on MRS solid medium for 48 hours;

[0019] Figure 3 This is a Gram staining result of Lactobacillus gallinarum C2-16-2;

[0020] Figure 4 It is a growth curve of Lactobacillus gallinarum C2-16-2 in anaerobic conditions of MRS liquid medium;

[0021] Figure 5 This is a statistical result chart showing the improvement in the ratio of duodenal villus height to crypt depth in chicks after adding Lactobacillus gallinarum C2-16-2 to feed;

[0022] Figure 6 This is the result of immunohistochemical staining of ZO-1 protein in the ileum of chicks. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0024] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0025] The preparation method of the MRS solid culture medium in the embodiment of the present invention is as follows

[0026] 10g peptone, 5g beef powder, 4g yeast powder, 2g glucose, 1mL Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 15g agar powder, make up to 1L with distilled water, adjust the pH value to 7.2±0.1, and sterilize at 121℃ for 15min.

[0027] The preparation method of the MRS liquid culture medium in the embodiment of the present invention is as follows

[0028] 10g peptone, 5g beef powder, 4g yeast powder, 2g glucose, 1mL Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, make up to 1L with distilled water, adjust the pH value to 7.2±0.1, and sterilize at 121℃ for 15min.

[0029] The sources or configuration methods of the reagents in the embodiments of the present invention are as follows:

[0030] NaCl source: Sinopharm Chemical Reagent Co., Ltd.

[0031] Bile salt source: Beijing Solebow Technology Co., Ltd.

[0032] Preparation method of MRS culture medium containing different concentrations of NaCl: add different masses of NaCL to MRS culture medium and sterilize at 121℃ for 15 minutes.

[0033] Preparation method of MRS culture medium containing different concentrations of bile salts: add different masses of bile salts to MRS culture medium and sterilize at 121°C for 15 minutes.

[0034] The experiments in the embodiments of the present invention were all repeated at least three times, and the experimental data were statistically analyzed using SPSS24.0 software, using one-way analysis of variance, and P<0.05 was considered statistically significant.

[0035] Example 1

[0036] Isolation and Cultivation of Lactobacillus gallinarum C2-16-2

[0037] 1. Sample Collection

[0038] The samples were isolated from the small intestinal chyme of white-feathered broilers at the chicken farm of Huazhong Agricultural University. The organisms were separated and purified using MRS culture medium, and then anaerobically cultured for 48 hours in a constant temperature anaerobic operation box (Shanghai Longyue LAI-3) at 37°C. The anaerobic gas composition was N2:CO2:H2=80:10:10. Single colonies were picked for plate streak purification to obtain pure culture of each strain.

[0039] The genomic DNA of the isolated strain was extracted by referring to the bacterial genome advance kit (BL1044A, Biosharp), and detected by 1% agarose gel electrophoresis, without diffusion or tailing; and the quality and concentration of the extracted DNA were detected by ultraviolet spectrophotometer.

[0040] DNA samples with an A260 / 280nm ratio between 1.8 and 2.0 and not less than 20 ng / μL were considered qualified.

[0041] 2. Identification of Lactobacillus gallus C2-16-2

[0042] 2.1 16S rDNA PCR amplification

[0043] The genomic DNA of qualified strains was amplified by PCR using universal primers of 16S rDNA. The primers were synthesized by Wuhan Jinkairui Bioengineering Co., Ltd. The specific sequences are as follows:

[0044] Upstream primer 27F: 5′-AGAGTTTGATCATGGCTCAG-3′;

[0045] Downstream primer 1492R: 5′-TAGGGTTACCTTGTTACGACTT-3′.

[0046] PCR amplification system and program settings:

[0047] PCR reaction system: 20 μL system contains 1 μL of strain genomic DNA template, 1 μL of upstream and downstream primers, and 10 μL of Taq enzyme Mix (purchased from Nanjing Novozyme Biotechnology Co., Ltd.);

[0048] The PCR reaction procedure is as follows:

[0049] Stage 1: 95℃, 5min

[0050] Stage 2: 95℃, 10s

[0051] 48℃,15s

[0052] 72℃,90s

[0053] Stage 3: 72℃, 5min

[0054] Stage 4: 20℃, 10min

[0055] Among them, Stage 2 repeats 35 cycles

[0056] 2.2 Sequencing and identification of strain 16S rDNA:

[0057] The obtained 16S rDNA amplification product was subjected to 1% gel electrophoresis detection, and the qualified amplified sequence was sent to Wuhan Jinkairui Biotechnology Co., Ltd. for sequencing, and a 16S rDNA sequence of 1435 bp in length was obtained as shown in SEQ ID NO.1 in the sequence table:

[0058] 5’-GCTCCTTCCCGAAGGTTAGGCCACCGGCTTTGGGCATTGCAGACT TCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTGCAGACTGCAGTCCGAACTGAGAACAGCTTTCAGAGATTCGCTTGCCTTCGCAGGCTCGCTTCTCGTTGTACTGCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGCTGGCAACTAATAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTTAGCGTCCCCGAAGGGAACTCCTAATCTCTTAGGATGGCACTAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGAAAAACAGTTTCCGATGCAGTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTATTTTTCCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTGATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCCTTCTTCACCAACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATCGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATGCACCGCGGGGCCATCCCATAGCGACAGCTTACGCCGCCTTTTATAAGCTGATCATGCGATCTGCTTTCTTATCCGGTATTAGCACCTGTTTCCAAGTGGTATCCCAGACTATGGGGCAGGTTCCCCACGTGTTACTCACCCATCCGCCGCTCGCGTCCCCAGCGTCATTACCGAAGTAAATCTGCTGGTTCTGCTCGCTCGACTTGCATGTATT-3’

[0059] The 16S rRNA sequence of C2-16-2 was aligned on the NCBI database (https: / / blast.ncbi.nlm.nih.gov / ), and the bacterium with the highest homology to C2-16-2 was Lactobacillus gallinarum strain DPN164, with a similarity of 99.93%( Figure 1). It was confirmed that the isolated C2-16-2 strain belonged to Lactobacillus gallinarum, and was named Lactobacillus gallinarum C2-16-2, abbreviated as Lactobacillus gallinarum C2-16-2; on September 2, 2024, the Lactobacillus gallinarum C2-16-2 was deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan City, Hubei Province, and its deposit number being: CCTCC NO: M 20241905.

[0060] Example 2 Physical and chemical properties of Lactobacillus gallus C2-16-2 and its acid and salt resistance

[0061] 1. Physical and chemical properties of Lactobacillus gallus C2-16-2

[0062] 1.1 Colony morphology and characteristics of Lactobacillus gallus C2-16-2

[0063] The optimal growth conditions of Lactobacillus gallus C2-16-2 isolated in Example 1 were 37°C and anaerobic. The colonies cultured on MRS solid medium for 48 hours were light white, spherical, and about 1 mm in diameter. Figure 2 ).

[0064] Gram staining results showed that the bacteria were Gram-positive rod-shaped bacteria ( Figure 3 ).

[0065] 1.2 Plotting the growth curve of Lactobacillus gallus C2-16-2

[0066] From the 24-hour growth curve of Lactobacillus gallus C2-16-2 ( Figure 3 ) It can be seen that C2-16-2 enters the logarithmic growth phase at 6h and enters the plateau phase at 14h. The specific process is as follows:

[0067] 1) Take the C2-16-2 strain out of the -80°C freezer, draw a line on the plate, and place the plate in an anaerobic operating box at 37°C for 24 hours;

[0068] 2) Pick a single colony from the plate and inoculate it into 10 mL of MRS modified medium and culture it anaerobically at 37°C for 24 h;

[0069] 3) Prepare three identical anaerobic tubes containing 10 mL of MRS medium to form technical replicates, and transfer the cultured bacterial solution into three 10 mL culture tubes at a 1% inoculum volume;

[0070] All culture solutions were placed in an anaerobic operating box at 37°C and the OD600nm absorbance was monitored every 2 hours using an enzyme marker. After the plateau phase, the monitoring time was changed to every 6-8 hours to draw the growth curve of C2-16-2 ( Figure 4 )

[0071] 2. Acid and salt tolerance of Lactobacillus hen C2-16-2

[0072] 2.1 Detection of tolerance of Lactobacillus gallus C2-16-2 to different pH media

[0073] 1) Prepare MRS medium with pH=3.0, 4.0, 5.0, and 6.0 respectively, inoculate the fresh Lactobacillus gallus C2-16-2 seed culture liquid of the second generation into MRS medium with different pH values ​​at a ratio of 1:100, and culture anaerobically at 37°C for 14 h

[0074] 2) Use an ELISA reader to measure the OD600nm absorbance of the final culture solution.

[0075] 2.2 Tolerance of Lactobacillus gallus C2-16-2 to different salt concentrations

[0076] 1) Prepare MRS medium with NaCl concentration of 2%, 4%, 6%, and 8%, and prepare MRS medium with bile salt concentration of 1g / L, 2g / L, 3g / L, and 4g / L. Inoculate the fresh Lactobacillus gallus C2-16-2 seed culture liquid of the second generation into MRS medium with different salt concentrations at a ratio of 1:100, and culture anaerobically at 37°C for 14h

[0077] 2) Use an ELISA reader to measure the OD600nm absorbance of the final culture solution.

[0078] 2.3 The experimental results are as follows:

[0079] Lactobacillus gallinarum C2-16-2 has the ability to tolerate pH=3, 8% NacL and 4 g / L bile salts.

[0080] Table 1C2-16-2 Tolerance to acid and salt

[0081]

[0082] Example 3

[0083] The additive for promoting intestinal development of chicks contains Lactobacillus gallus C2-16-2, wherein the concentration of Lactobacillus gallus C2-16-2 is 10 9 -10 10 cfu / mL.

[0084] Example 4

[0085] A feed for promoting intestinal development in chicks is prepared by adding the additive of Example 3 to a basic diet, and the content of Lactobacillus gallus C2-16-2 in the feed is 10 11 cfu / kg.

[0086] Example 5 Application of Lactobacillus gallus C2-16-2 in promoting intestinal development in chicks

[0087] The selected broiler model is: the control group is fed with a basic diet, and the experimental group is fed with the feed of Example 4, and the feeding is continued for 14 days starting from 1 day of age; the specific test method is as follows:

[0088] 1. Grouping of broilers

[0089] A total of 160 one-day-old male white-feathered broiler chickens (weight 42±2g) were randomly divided into a control group (BD) and an experimental group (LG), with 8 replicates in each group and 10 chickens in each replicate. The broiler chickens were raised for 14 days.

[0090] 2. Analysis of the results of duodenum villus height and crypt depth in broiler chickens in different groups

[0091] After the two groups of broilers were treated for 14 days, they were slaughtered and sampled, and the villus height and crypt depth of the duodenal sections of broilers in different groups were observed using an optical microscope.

[0092] 3. The method for preparing duodenal tissue sections is as follows:

[0093] 1) Sampling and fixation

[0094] Take a 0.5 cm long segment of duodenum and fix it in 4% paraformaldehyde for 24 hours.

[0095] 2) Dehydration and transparency

[0096] Use alcohol from low to high concentration as dehydrating agent to gradually remove water from the tissue block. Then place the tissue block in xylene, a transparent agent that is soluble in both alcohol and paraffin, to make it transparent, and use xylene to replace the alcohol in the tissue block.

[0097] 3) Wax embedding

[0098] Place the transparent tissue block in the melted paraffin, put it in a wax melting box to keep it warm, and embed it after the paraffin is completely immersed in the tissue block. Pour the melted paraffin into a suitable container, quickly clamp the tissue block soaked in paraffin and put it into it, and cool it to solidify into a block.

[0099] 4) Slicing and patching

[0100] Fix the embedded wax block on the microtome and cut it into 5-8μm slices. Flatten it in heated water, stick it on a slide, and dry it in a 45℃ constant temperature box.

[0101] 5) Dewaxing and dyeing

[0102] After being soaked in distilled water, the slices were placed in a hematoxylin aqueous solution for staining for several minutes, and separated in acid water and ammonia water for several seconds each; rinsed in running water for 1 hour, dehydrated in 70% and 90% alcohol for 10 minutes each, and dyed in alcohol eosin staining solution for 2-3 minutes. The stained slices were dehydrated with pure alcohol and then made transparent with xylene.

[0103] 6) Seal the slide

[0104] Drop Canada balsam on the transparent sections and cover with a coverslip to seal.

[0105] 4. Results of immunohistochemical staining of ZO-1 protein in jejunum of broiler chickens in different groups

[0106] After the sections were dewaxed and rehydrated / antigen repaired / inactivated / blocked / primary antibody incubated / secondary antibody incubated / DAB color development / hematoxylin counterstaining / dehydrated / sealed, they were observed and photographed under an optical microscope.

[0107] Compared with the control group, the experimental group increased the abundance of ZO-1 protein in the jejunum ( Figures 5-6 ).

[0108] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A Lactobacillus gallinarum C2-16-2, whose deposit number is: CCTCC NO: M20241905.

2. An application of Lactobacillus gallus C2-16-2 according to claim 1 in promoting intestinal development in chicks.

3. Use of the Lactobacillus gallus C2-16-2 according to claim 1 in the preparation of an additive for promoting intestinal development in chicks.

4. An additive for promoting intestinal development in chicks, characterized in that: The additive contains Lactobacillus gallus C2-16-2, wherein the concentration of Lactobacillus gallus C2-16-2 is 10 9 -10 10 cfu / mL.

5. A feed for promoting intestinal development of chicks, characterized in that: The feed is a basic diet supplemented with the additive of claim 4 or the Lactobacillus gallus C2-16-2 of claim 1, wherein the content of Lactobacillus gallus C2-16-2 is 1×10 11 -5x10 11 cfu / kg.

6. The feed according to claim 5, characterized in that: The content of the Lactobacillus gallus C2-16-2 is 10 11 cfu / kg.

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