Chicken source lactobacillus craw mucus CC32 and application thereof

Abstract

The invention discloses a chicken source lactobacillus craw mucus CC32 and application thereof. The preservation number of the chicken source lactobacillus craw mucus CC32 is CCTCC No: M 20242859. The chicken source lactobacillus craw CC32 obtained through screening has the advantages that the feed utilization rate is increased, and digestion and absorption of nutrient substances in the feed are promoted; daily gain is increased, and feed conversion ratio is reduced; the feed additive has the characteristics of no pollution, no residue, biological environmental protection and the like, and has obvious effects in the aspects of regulating animal intestinal flora balance, promoting animal growth and increasing animal weight.

Description

Technical Field

[0001] The invention relates to the fields of microbiology and feed probiotics, and in particular to chicken-derived crop mucus Lactobacillus CC32 and applications thereof. Background Art

[0002] The intestine is the primary site for digestion and absorption of feed nutrients and defense against pathogens. Maintaining intestinal health is fundamental to overall host health and ensuring optimal feed utilization efficiency for animals. However, in modern poultry production, broilers often face various environmental stress challenges, such as rapid growth stress, heat stress, and high-density stocking stress. These stresses often cause broilers to be in a sub-healthy state, seriously threatening their intestinal health functions, causing inflammatory responses and triggering oxidative stress, thereby reducing feed utilization efficiency and even leading to death. The intestinal microbiome can affect host physiological functions, including digestion, absorption, and metabolism of feed nutrients, directly or through its metabolites, thereby affecting the efficient utilization of nutrients and promoting broiler growth.

[0003] At present, some studies have discovered some intestinal microorganisms that are closely related to the feed utilization efficiency of broilers. However, the research results to date are inconsistent and sometimes even contradictory. The low repeatability of microbial experiments may be due to the sensitivity of the intestinal flora to differences in diet, environment, management, age and breed. In addition, due to the complexity of the intestinal microbiome related to feed utilization efficiency, most studies only stay at the correlation level, without clearly identifying specific bacterial groups and analyzing the role and mechanism of their interaction with the host in affecting feed utilization efficiency. Therefore, how to discover key intestinal bacteria that can significantly improve feed utilization efficiency, and isolate and culture key chicken-derived probiotic strains in vitro, and further reveal their causal relationship with feed utilization efficiency through functional studies, is of great production significance for the development of products and nutritional regulation technologies that target key intestinal probiotics to improve broiler intestinal health and improve feed utilization efficiency. Summary of the Invention

[0004] The present invention aims to overcome the deficiencies of the prior art and provides a chicken-derived Lactobacillus mucosa CC32 and its application. The present invention screens and obtains chicken-derived Lactobacillus mucosa CC32 as a targeted key intestinal probiotic to improve the intestinal health of broiler chickens, thereby improving the feed utilization efficiency of broiler chickens.

[0005] To achieve the above purpose, the technical solution designed by the present invention is as follows: The present invention provides a chicken-derived crop mucus Lactobacillus ( Lactobacillusingluviei )CC32, its deposit number is CCTCC No: M 20242859.

[0006] The chicken-derived Lactobacillus ingluviei strain was isolated and screened from the cecum of Cobb broiler chickens and named Limosilactobacillus ingluviei CC32. 16S RNA gene sequence analysis confirmed that strain CC32 is Lactobacillus ingluviei. This strain was deposited with the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China, 430072) on December 18, 2024, under the accession number CCTCC M 20242859.

[0007] The microbiological characteristics of the chicken crop mucus Lactobacillus CC32 are as follows: When cultured anaerobically at 37°C for 48 hours on MRS solid medium, the colonies formed were light white with irregular edges and a diameter of approximately 2-3 mm. The strain CC32 entered the logarithmic growth phase after 6 hours and the plateau phase after 16 hours.

[0008] The present invention identified the probiotic effect of chicken-derived crop mucus Lactobacillus CC32 through an in vitro method, indicating that it is acid-resistant, bile-resistant, and resistant to the internal environment of the gastrointestinal tract, and has the potential to be a probiotic.

[0009] The present invention also provides a use of the chicken-derived crop mucus Lactobacillus CC32 in improving the intestinal health and feed utilization efficiency of broiler chickens.

[0010] The present invention also provides a use of the chicken-derived crop mucus Lactobacillus CC32 in the preparation of an additive for improving the intestinal health and feed utilization efficiency of broiler chickens.

[0011] The present invention also provides an additive for improving the intestinal health of broiler chickens, wherein the additive contains the above-mentioned chicken-derived crop mucus Lactobacillus CC32, wherein the concentration of chicken-derived crop mucus Lactobacillus is 10 9 ~10 10 CFU / mL.

[0012] The present invention also provides a broiler feed for improving the intestinal health of broilers, wherein the feed is prepared by adding the above-mentioned additive to the basic diet, wherein the effective viable bacteria count of chicken-derived crop mucus Lactobacillus in the feed is 10 9 CFU / kg.

[0013] Beneficial effects of the present invention: (1) The chicken-derived crop mucus Lactobacillus CC32 of the present invention has a certain acid resistance and can survive and grow in MRS culture medium with a pH of 3, indicating that it can tolerate the influence of gastric acid.

[0014] (2) The chicken-derived crop mucus Lactobacillus CC32 of the present invention has a strong bile salt tolerance and can grow and reproduce in MRS culture medium supplemented with 5 g / L bile salt.

[0015] (3) The chicken-derived crop mucus Lactobacillus CC32 of the present invention has the effect of improving the feed conversion rate of broiler chickens. This shows that crop mucus Lactobacillus CC32 can be used as a new type of probiotic additive and is widely used in broiler feed.

[0016] (4) The chicken-derived crop mucus Lactobacillus CC32 of the present invention is added to the basic diet, which has the effect of improving the intestinal health of broiler chickens and increasing feed utilization efficiency.

[0017] In summary, the chicken-derived crop mucus Lactobacillus CC32 screened by the present invention has the characteristics of improving feed utilization, promoting digestion and absorption of nutrients in feed, increasing daily weight gain, reducing feed-to-meat ratio, being pollution-free, residue-free, and being bio-friendly, and has significant effects in regulating the balance of animal intestinal flora, promoting animal growth, and increasing animal weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the colony morphology of chicken-derived crop mucus Lactobacillus CC32 cultured on MRS solid medium for 48 hours.

[0019] Figure 2 This is the Gram staining image of chicken-derived crop mucus Lactobacillus CC32.

[0020] Figure 3 This is the growth curve of chicken-derived crop mucus Lactobacillus CC32 under anaerobic conditions in MRS liquid culture medium.

[0021] Figure 4 This is a schematic diagram of the acid resistance test results of chicken-derived crop mucus Lactobacillus CC32.

[0022] Figure 5 This is a schematic diagram showing the bile salt tolerance test results of chicken-derived crop mucus Lactobacillus CC32.

[0023] Figure 6 This is the duodenal tissue morphology of broilers fed with broiler feed supplemented with chicken-derived crop mucus Lactobacillus CC32.

[0024] Figure 7 This is the Alcian blue staining image of the duodenal tissue of broilers fed with broiler feed supplemented with chicken-derived crop mucus Lactobacillus CC32. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below with reference to specific embodiments so that those skilled in the art can understand.

[0026] Example 1 Isolation and identification of chicken-derived crop mucus Lactobacillus CC32 1. Isolation, culture and morphological identification of strains Collect 1 g of cecal digesta from Cobb broiler chickens and place it in a test tube containing 9 mL of normal saline. Vortex and mix thoroughly to create a 1:10 dilution. This dilution is then diluted in 10-fold increments. Then, 1 mL of each of three appropriate dilutions is plated onto MRS medium. Incubate anaerobically at 37°C for 48 hours. Observe and record the colony morphology, and select well-growing single colonies for streak isolation and purification.

[0027] For strains in logarithmic growth phase with stable colony size, the single colony status is described, mainly including the size, color and colony edge status. The obtained single colony size is about 2-3mm, the color is light white, and the colony edge is irregular. See the colony morphology diagram for details. Figure 1 .

[0028] The MRS medium was prepared as follows: 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, and 0.05 g manganese sulfate. The medium was made up to 1 L with distilled water, the pH was adjusted to 7.2 ± 0.1, and sterilized at 121°C for 15 min.

[0029] 2. Gram staining of strains Place a drop of sterile distilled water on a glass slide, pick a single colony and dissolve it in water. After scraping, dry and fix it over an alcohol lamp. Add crystal violet stain for 2 minutes, rinse with water, and air dry. Add iodine solution for mordant staining for 2 minutes, rinse with water, and air dry. Add basic fuchsin ethanol solution for 50 seconds, rinse with water, and air dry. Observe under a standard optical microscope. The staining results show that the bacteria are Gram-positive rod-shaped rods (see Figure 2 ).

[0030] 3. Identification of strains Genomic DNA was extracted from the isolated strain using a bacterial genome pre-production kit (BL1044A, Biosharp). Universal primers were used to amplify the 16S rDNA sequence of the strain. The resulting 16S rDNA amplification product was subjected to 1% gel electrophoresis. Passing amplification sequences were sent to Wuhan Jinkairui Bioengineering Co., Ltd. for sequencing. A 1460-bp 16S rDNA sequence was obtained, as shown in SEQ ID NO: 1:

[0031] The above sequences were compared with the NCBI database to confirm that the isolated strain was Lactobacillus mucosa ( Lactobacillus ingluviei ), named CC32.

[0032] This strain was deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China, postal code 430072) on December 18, 2024, and was classified and named Limosilactobacillusingluviei CC32, with the deposit number CCTCC No: M 20242859.

[0033] Example 2: Stress resistance evaluation of chicken-derived crop mucus Lactobacillus CC32 1. Growth curve of chicken crop mucus Lactobacillus CC32 The strain CC32 was taken out of the -80°C freezer, a line was drawn on a plate, and the plate was placed in an anaerobic operating box at 37°C for 24 hours; a single colony was picked from the plate and inoculated into 10 mL of MRS medium, and anaerobically incubated at 37°C for 24 hours; three identical anaerobic tubes containing 10 mL of MRS medium (the formula is shown in Example 1) were prepared to constitute technical replicates, and the cultured bacterial solution was transferred to three 10 mL culture tubes at a 1% inoculum volume; all the culture solutions were placed in an anaerobic operating box at 37°C, and the OD was monitored every 2 hours using a microplate reader. 600 The absorbance value was monitored every 4 h after reaching the plateau phase, and the growth curve of strain CC32 was drawn.

[0034] The results showed that strain CC32 entered the logarithmic growth phase at 6 h and the plateau phase at 16 h (see Figure 3 ).

[0035] 2. Acid resistance test of chicken crop mucus Lactobacillus CC32 The activated strain CC32 was inoculated at 1% inoculation into MRS liquid culture medium with pH values ​​of 2.0, 3.0, 4.0 and 6.0, respectively. The culture was anaerobically cultured at 37°C for 12 h, with three replicates. The OD of each bacterial solution was measured using a microplate reader. 600 The absorbance value.

[0036] The results showed that chicken crop mucus Lactobacillus CC32 grew best in a medium with a pH of 6.0, and pH 4.0 had little effect on its growth. When the pH was 2, half of the bacteria were still able to survive and grow 12 hours after inoculation ( Figure 4 ), indicating that chicken-derived crop mucus Lactobacillus CC32 can tolerate the effects of gastric acid.

[0037] 3. Bile salt tolerance test of chicken crop mucus Lactobacillus CC32 The activated strain CC32 was inoculated at a 1% inoculum into MRS liquid culture medium with sodium taurocholate concentrations of 0 g / L, 1 g / L, 3 g / L, and 5 g / L, respectively. The culture was anaerobically cultured at 37°C for 12 h with three replicates. The OD600 absorbance of each group of bacterial liquid was measured using a microplate reader.

[0038] The results showed that the effects of 1 g / L and 3 g / L bile salt on chicken crop mucus Lactobacillus CC32 were relatively weak and almost negligible. After 12 hours of treatment with 5 g / L bile salt, the OD value of bacterial culture was still close to 1.0 (see Figure 5 ), indicating that the chicken crop mucus Lactobacillus CC32 has a strong bile salt tolerance.

[0039] Example 3 Preparation of an additive for improving intestinal health of broiler chickens The additive for improving the intestinal health of broiler chickens contains chicken-derived crop mucus Lactobacillus CC32, wherein the concentration of chicken-derived crop mucus Lactobacillus is 10 9 CFU / mL.

[0040] The preparation method of the additive for improving the intestinal health of broiler chickens comprises the following steps: a. Remove strain CC32 from the -80°C freezer, draw lines on the plate, and incubate the plate in an anaerobic chamber at 37°C for 24 hours. b. Pick a single colony from the plate and inoculate it into 10 mL of MRS medium and culture it anaerobically at 37°C for 24 h; obtain the fermentation broth, which is the additive. In the additive, the concentration of strain CC32 is 10 9 ~10 10 CFU / mL Among them, the preparation method of MRS culture medium is as follows: 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 6.0, and sterilize at 121℃ for 15min.

[0041] Example 4 Broiler feed to improve intestinal health of broiler chickens The broiler feed for improving the intestinal health of broilers was prepared by adding the additive prepared in Example 3 to the basic diet (Shandong New Hope Liuhe Group Co., Ltd.), wherein the effective viable count of chicken-derived crop mucus Lactobacillus CC32 in the feed was 10 9 CFU / kg.

[0042] Example 5 Application of Chicken Crop Lactobacillus CC32 in Improving Broiler Intestinal Health and Feed Utilization Efficiency The broiler model used in this example is as follows: 600 one-day-old male white-feathered broiler chickens (weighing 42±2g) were randomly divided into two groups, with 15 replicates in each group and 20 chickens in each replicate, as follows: Control group (BD): fed with basal diet, Treatment group (LI): fed with broiler feed; The broilers were fed with a basal diet starting from day 1 and continued for 21 days. Both groups were then fed with a basal diet until the broilers were marketed. During the experiment, the broilers had free access to feed and water.

[0043] The detection indicators are as follows: 1. Record the feed intake of broilers every day and calculate the average daily feed intake after the end of the experiment; record the body weight of broilers on the day of the start and end of the experiment and calculate the average daily weight gain; calculate the feed conversion rate based on feed intake and body weight gain, which is calculated as average daily feed intake / average daily weight gain.

[0044] 2. The duodenal tissue was carefully washed with pre-cooled saline, and small segments were cut and fixed in 4% paraformaldehyde. Paraffin sections were then made and the tissue morphology was analyzed by HE staining.

[0045] 3. Duodenal paraffin sections were stained with Alcian blue staining kit (Solarbio, G1560) for duodenal goblet cell staining analysis.

[0046] Table 1 Effects of adding chicken-derived crop mucus Lactobacillus CC32 to the diet on the production performance of broilers from week 1 to week 6 As can be seen from Table 1, the average daily weight gain of broilers in the treatment group (LI) was significantly ( P <0.05) was higher than that of the control group (BD), and the feed conversion rate was significantly lower than that of the control group ( P <0.05), indicating that the feed efficiency of broiler chickens supplemented with Lactobacillus CC32 from chicken crop mucus was better, and the production performance and feed utilization efficiency of broiler chickens were significantly improved.

[0047] The histomorphology of the duodenum was shown by HE staining (see Figure 6 ), the villi height of broilers in the treated group (LI) increased significantly compared with those in the control group (BD) ( P <0.05), indicating that broiler feed (diet supplemented with chicken-derived crop Lactobacillus CC32) increased the surface area of ​​broiler intestinal tract for absorption of dietary nutrients.

[0048] Alcian blue staining of the duodenum (see Figure 7), the broilers in the treatment group (LI) had more goblet cells that secreted mucus than the broilers in the control group (BD), indicating that the broiler feed (supplemented with chicken-derived crop mucus Lactobacillus CC32) improved the intestinal secretion, barrier and immune functions of broilers.

[0049] 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 inventiveness, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A chicken-derived crop mucus Lactobacillus ( Lactobacillus ingluviei )CC32, its deposit number is CCTCCNo: M 20242859.

2. A use of the chicken-derived crop mucus Lactobacillus CC32 according to claim 1 in improving the intestinal health and feed utilization efficiency of broiler chickens.

3. A use of the chicken-derived crop mucus Lactobacillus CC32 according to claim 1 in the preparation of an additive for improving the intestinal health and feed utilization efficiency of broiler chickens.

4. An additive for improving the intestinal health of broiler chickens, characterized by: The additive contains the chicken-derived crop mucus Lactobacillus CC32 according to claim 1, wherein the concentration of the chicken-derived crop mucus Lactobacillus is 10 9 CFU / mL.

5. A broiler feed for improving the intestinal health of broiler chickens, characterized by: The feed is a basic diet supplemented with the additive of claim 4, wherein the effective viable bacteria count of chicken-derived crop mucus Lactobacillus in the feed is 10 9 CFU / kg.

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