Use of lactobacillus reuteri 202212 in promoting growth and development of lambs

Abstract

The application discloses application of lactobacillus reuteri 202212 in promoting growth and development of lambs, and belongs to the technical field of microorganism application, and particularly relates to application of lactobacillus reuteri 202212 in promoting growth and development of lambs. Lactobacillus reuteri The lactobacillus reuteri 202212 is preserved in the China General Microbiological Culture Collection Center on April 26, 2022, and the preservation address is No. 1, Xibaixili, Chaoyang District, Beijing, and the preservation number is CGMCC No. 24773. The lactobacillus reuteri 202212 disclosed by the application can promote growth and development of lambs, regulate intestinal microorganism flora of lambs, and enhance immune function of lambs.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology, and in particular relates to the application of Lactobacillus reuteri 202212 in promoting the growth and development of lambs. Background Technology

[0002] During lamb growth and development, numerous factors, such as bacterial, viral, and parasitic diseases, hinder their progress. To combat the invasion of pathogenic microorganisms during development, antibiotics are frequently used in veterinary clinical practice. Overuse of antibiotics can disrupt the normal intestinal flora, leading to drug resistance and affecting subsequent disease treatment. Traditional farming methods rely on antibiotics to promote animal growth and control pathogens; however, with the rising risk of antibiotic resistance and related regulations, finding safe and effective alternatives has become an urgent need in the industry. Therefore, antibiotic alternatives have attracted considerable attention from researchers in related fields.

[0003] Probiotics, especially lactic acid bacteria, can colonize the intestines, secrete antibacterial substances, and enhance the intestinal immune barrier and overall immunity. Studies have shown that adding specific probiotics to feed can improve gut health, enhance immune function, and improve nutrient absorption efficiency in livestock, thereby promoting overall growth performance. For example, using probiotic preparations in lambs can improve growth performance and rumen microbiota composition in lambs with stunted growth; other literature indicates that probiotics can enhance intestinal barrier function and immune cell activity in lambs.

[0004] Lactobacillus reuteri ( Lactobacillus reuteri Lactobacillus reuteri is a very important class of probiotics. In the lamb population, the role of Lactobacillus reuteri in growth and development is still unclear, especially since different strains have different effects. The specific strains, dosages, administration methods, safety, and effects on gastrointestinal development, nutritional metabolism, and daily weight gain have not yet been fully revealed.

[0005] Against this backdrop, developing a strain of *Lactobacillus reuteri* that promotes lamb growth and development, and clarifying its mechanism of action (such as intestinal colonization, immune regulation, and enhanced nutrient absorption), would have significant scientific and industrial value. This would not only improve breeding efficiency and reduce disease incidence but also decrease antibiotic use, thus contributing to the sustainable development of animal husbandry and the improvement of animal product quality. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes the application of Lactobacillus reuteri 202212 in promoting lamb growth and development. The Lactobacillus reuteri 202212 described in this invention can promote lamb growth and development, regulate the intestinal microbiota of lambs, and enhance lamb immune function.

[0007] To achieve the above objectives, this invention provides the application of Lactobacillus reuteri 202212 in promoting lamb growth and development, wherein Lactobacillus reuteri ( Lactobacillus reuteri 202212 was deposited on April 26, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 24773.

[0008] Preferably, the Lactobacillus reuteri 202212 can increase the weight gain of lambs, thereby enabling lambs to achieve faster weight accumulation and compensatory growth during the weaning period or growth retardation period.

[0009] The present invention also provides the application of Lactobacillus reuteri 202212 in regulating the intestinal microbiota of lambs. Lactobacillus reuteri 202212 can increase the proportion of beneficial bacteria in the lamb's intestine, reduce the number of harmful bacteria, enhance intestinal barrier function, and reduce diarrhea and inflammatory response.

[0010] The present invention also provides the application of Lactobacillus reuteri 202212 in enhancing the immune function of lambs. Lactobacillus reuteri 202212 can increase the level of immunoglobulins in lambs, thereby enhancing the lambs' resistance to pathogenic microorganisms.

[0011] The present invention also provides a microbial agent for promoting the growth and development of lambs, including the Lactobacillus reuteri 202212.

[0012] Preferably, the effective viable count of Lactobacillus reuteri 202212 in the microbial agent is ≥10. 8 CFU / mL.

[0013] The present invention also provides a microbial agent for regulating the intestinal microbiota of lambs and enhancing lamb immunity, including the Lactobacillus reuteri 202212.

[0014] Preferably, the effective viable count of Lactobacillus reuteri 202212 in the microbial agent is ≥10. 8 CFU / mL.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: This invention provides the application of *Lactobacillus reuteri* 202212 in promoting lamb growth and development. The *Lactobacillus reuteri* 202212 described in this invention can promote lamb growth and development, regulate the intestinal microbiota of lambs, and enhance lamb immune function. Specifically, *Lactobacillus reuteri* 202212 has significant advantages in promoting lamb growth and development. Adding this strain can increase lamb weight gain, enabling lambs to achieve faster weight accumulation and compensatory growth during weaning or growth retardation periods. Intestinal health is improved, feed conversion efficiency is significantly increased, feed utilization is increased, and breeding costs are correspondingly reduced. Furthermore, *Lactobacillus reuteri* 202212 also has significant effects in regulating intestinal microecology, inhibiting pathogens, and enhancing immune function. This strain can increase the proportion of beneficial bacteria in the intestine, reduce the number of harmful bacteria, enhance intestinal barrier function, and reduce diarrhea and inflammatory responses. Simultaneously, its immunomodulatory effect is manifested in increasing immunoglobulin levels, thereby enhancing the lamb's resistance to pathogenic microorganisms. This strain is beneficial for improving lamb production performance, feeding safety and economic benefits, and has the potential to replace antibiotics and promote sustainable farming. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The results show the in vitro antibacterial activity of Lactobacillus reuteri 202212 cell-free culture supernatant. In the figure, A represents Staphylococcus aureus, B represents Escherichia coli, and C represents Salmonella typhimurium. MRS represents the MRS culture medium group, and L. reuteri represents the Lactobacillus reuteri 202212 cell-free culture supernatant group. The figures show… "and" "Representative difference significance analysis; Figure 2 This is a comparison chart of lamb weights between the experimental and control groups. In the chart, L. reuteri represents the experimental group, Con represents the control group, and "ns" and "" are used to indicate the weights of lambs. "Representative difference significance analysis; Figure 3 This is the standard curve for serum immunoglobulin IgG. Figure 4 The figure shows the serum immunoglobulin IgG levels in the experimental group of lambs. "Representative difference significance analysis; Figure 5 This is the standard curve for serum immunoglobulin IgA; Figure 6 The figure shows the serum immunoglobulin IgA level of lambs in the experimental group. "Representative difference significance analysis; Figure 7 This is a graph showing the β-diversity analysis of the gut microbiota in the experimental group of lambs; Figure 8 A graph showing the abundance of gut microbiota at the genus level in the experimental group of lambs; Figure 9 The figures show the D-lactic acid and DAO content of lambs in the experimental group, where A represents D-lactic acid content and B represents DAO content. "Representative difference significance analysis; Figure 10 This is a comparison chart of the diarrhea rate in lambs in the experimental group and the control group. In the chart, L. reuteri represents the experimental group and Con represents the control group. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The materials used in this invention were sourced as follows: MRS liquid culture medium was purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.; sheep immunoglobulin IgG detection kit and sheep immunoglobulin IgA detection kit were purchased from Quanzhou Ruixin Biotechnology Co., Ltd.; disposable vacuum blood collection tubes were purchased from Hebei Kangweishi Medical Technology Co., Ltd.; and DAO detection kit and D-lactic acid detection kit were purchased from Nanjing Jiancheng Bioengineering Institute.

[0023] Example 1 I. Lactobacillus reuteri 202212 ( L.reuteri Evaluation of the antibacterial effect of cell-free culture supernatant in vitro: Lactobacillus reuteri 202212 was inoculated into MRS liquid medium and cultured at 37°C for 24 h. Then, it was transferred to fresh MRS liquid medium at a 1% (v / v) inoculation rate and cultured for another 24 h. After culture, the fermentation broth was centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was collected and filtered through a 0.22 μm sterile filter to obtain cell-free supernatant (CFS) of L. reuteri 202212.

[0024] A mixed culture medium was prepared by mixing Lactobacillus reuteri 202212 cell-free culture supernatant with LB broth (Qingdao Haibo Biotechnology Co., Ltd.) at a volume ratio of 1:9. Staphylococcus aureus was then added at a volume ratio of 1:100. ‌ S.aureus ), Escherichia coli ( E. coli ) and Salmonella typhimurium ( S.typhimurium After incubation at 37℃ in a shaker for 12 hours, OD was measured. 600 The absorbance of bacteria at that time.

[0025] like Figure 1 China A~ Figure 1 As shown in Figure C, the cell-free culture supernatant of Lactobacillus reuteri 202212 significantly inhibited Staphylococcus aureus in vitro. S.aureus ), Escherichia coli ( E. coli ) and Salmonella typhimurium ( ‌ S.typhimurium Growth.

[0026] II. Supplementation with Lactobacillus reuteri 202212 ( L.reuteri Effects on lamb weight gain: Preparation of bacterial culture: Lactobacillus reuteri 202212 was inoculated into MRS liquid medium and cultured at 37°C for 24 h. Then, it was transferred to fresh MRS liquid medium at an inoculation rate of 1% (v / v) and cultured for another 24 h. The effective viable count in the bacterial culture was ≥10. 8 cfu / mL.

[0027] Forty healthy lambs were selected, including the experimental group ( L.reuteri Twenty lambs were divided into a bacterial culture group and a control group (MRS liquid culture medium group). Both groups were fed normal forage at 06:00 daily; at 13:00, they were supplemented with concentrate feed, with bacterial culture medium and MRS liquid culture medium added at a volume ratio of 1:200 to their original diet. Water was provided freely. The feeding period lasted 4 weeks. Lamb weights were measured before the start of the experiment, on the last day of the second week of feeding, and on the last day of the fourth week of feeding, and the average daily weight gain of the lambs was calculated.

[0028] like Figure 2 As shown, the average daily weight gain rate of lambs was calculated using the formula: (measured weight - initial weight) / initial weight. The experimental group received supplemental feeding. L.reuteri The bacterial solution promoted the daily weight gain of lambs throughout the trial period. L.reuteri The lambs in the experimental group fed with the bacterial solution showed no significant difference in weight between the experimental and control groups at week 2, but their weight increased significantly compared to the control group at week 4, indicating that supplemental feeding was effective. L.reuteri Bacterial solution can promote lamb fattening.

[0029] III. Supplementing with Lactobacillus reuteri 202212 ( L.reuteri Effects on lamb serum immunoglobulin IgG levels: IgG levels were detected using a goat immunoglobulin IgG assay kit. First, the concentration of the standard was plotted on the x-axis, and the corresponding absorbance (OD value) on the y-axis. Using computer software, a four-parameter logistic curve was fitted to create a standard curve equation. The sample concentration was then calculated using the sample absorbance (OD value) from the equation. The resulting standard curve R... 2 =0.997, indicating a good fit (e.g. Figure 3 ).

[0030] IgG is the main antibody component of serum, accounting for approximately 75% of the total serum immunoglobulins. It has antibacterial and antiviral properties and can effectively prevent corresponding infectious diseases. Blood was collected from lambs in the experimental group before the start of the experiment, on the last day of the second week of feeding, and on the last day of the fourth week of feeding. Serum was separated for IgG level detection. Blood was collected from the jugular vein and separated into disposable vacuum blood collection tubes for later use.

[0031] The results are as follows Figure 4 As shown, supplementing with Lactobacillus reuteri 202212 can significantly increase serum IgG levels and improve the immunity of sheep.

[0032] IV. Supplementing with Lactobacillus reuteri 202212 ( L.reuteri Effects on lamb serum immunoglobulin IgA levels: IgA levels were detected using a goat immunoglobulin IgA assay kit. First, the concentration of the standard was plotted on the x-axis, and the corresponding absorbance (OD value) on the y-axis. Using computer software, a four-parameter logistic curve (4-p1) was fitted to create a standard curve equation. The sample concentration was then calculated using the sample absorbance (OD value) from the equation. The resulting standard curve R0... 2 =0.999, indicating a good fit (e.g. Figure 5 (As shown).

[0033] IgA is the second most abundant immunoglobulin in the serum of normal animals, after IgG, accounting for 10% to 20% of the serum immunoglobulin content. It is the main antibody for mucosal immunity. Blood was collected from lambs in the experimental group before the start of the experiment, on the last day of the second week of feeding, and on the last day of the fourth week of feeding. Serum was separated for IgG level detection. Blood was collected from the jugular vein and separated into disposable vacuum blood collection tubes for later use.

[0034] The results are as follows Figure 6 As shown, in the experimental group of lambs supplemented with Lactobacillus reuteri 202212, the serum IgA content increased on the last day of the second week of feeding and the last day of the fourth week of feeding compared with the level before the start of the experiment, and the increase on the last day of the fourth week of feeding was significant compared with the level before the start of the experiment.

[0035] V. Supplement with Lactobacillus reuteri 202212 ( L.reuteri Effects on the gut microbiota of lambs: Feces from lambs in the experimental group were collected on the last day of the fourth week of feeding for amplicon sequencing.

[0036] On the last day of week 4 of the feeding trial, fresh fecal samples were collected from lambs in the experimental group, placed in sterile sampling tubes, quickly placed on dry ice, and then stored at -80°C. Subsequently, total microbial DNA was extracted from the fecal samples. After determining the DNA concentration and purity, specific variable regions of the bacterial 16S rRNA gene were selected for PCR amplification. After the amplification products passed agarose gel electrophoresis, they were purified, quantified, and library constructed. Amplicon sequencing was then performed using a high-throughput sequencing platform. This part was outsourced to Beijing Novogene Biotechnology Co., Ltd.

[0037] Changes in gut microbiota such as Figure 7 and Figure 8 As shown, Figure 7 The PCoA analysis results shown indicate a certain degree of separation in community structure between the 0d and 28d groups, suggesting that the β-diversity of lamb feces microorganisms changed after 28 days of feeding, and the overall structure of the gut microbiota shifted. Figure 8Species composition analysis at the genus level showed differences in fecal microbial community composition between the 0-day and 28-day groups. Compared to the 0-day group, the 28-day group... Escherichia Shigella The relative abundance decreased, while Lactobacillus , Akkermansia , Bacteroides , Rikenellaceae The increased relative abundance of bacteria such as _RC9_gut_group and UCG-005 indicates that the composition of the lamb's intestinal flora was remodeled after 28 days of feeding. The proportion of potentially beneficial bacteria and bacteria related to short-chain fatty acid production, intestinal barrier function and nutrient metabolism increased, suggesting that this treatment may have a certain regulatory effect on the lamb's intestinal microecology.

[0038] VI. Supplement with Lactobacillus reuteri 202212 ( L.reuteri Effects on the intestinal barrier of lambs: Blood was collected from the jugular vein of lambs, and serum was obtained by centrifugation. DAO and D-lactic acid were used as markers of intestinal barrier permeability, and D-lactic acid and DAO detection kits were used for detection.

[0039] The results are as follows Figure 9 China A and Figure 9 As shown in Figure B, the levels of D-lactic acid and DAO decreased on the last day of week 4 compared to before the start of the experiment.

[0040] VII. Effect of supplemental feeding with Lactobacillus reuteri 202212 (L. reuteri) on the diarrhea rate in lambs: From one day before the start of the experiment to the last day of the fourth week of feeding, the diarrhea rate of lambs in the control group and the experimental group was observed and statistically analyzed every two days.

[0041] The results are as follows Figure 10 As shown, supplementing with Lactobacillus reuteri 202212 can significantly reduce the rate of diarrhea in lambs.

[0042] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of Lactobacillus reuteri 202212 in promoting lamb growth and development, characterized in that, The Lactobacillus reuteri ( Lactobacillus reuteri 202212 was deposited on April 26, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 24773.

2. The application according to claim 1, characterized in that, The Lactobacillus reuteri 202212 can increase the weight gain of lambs, thereby enabling lambs to achieve faster weight accumulation and compensatory growth during weaning or growth retardation periods.

3. The application of Lactobacillus reuteri 202212 as described in claim 1 in regulating the intestinal microbiota of lambs, characterized in that, The Lactobacillus reuteri 202212 can increase the proportion of beneficial bacteria in the lamb's intestines, reduce the number of harmful bacteria, enhance intestinal barrier function, and reduce diarrhea and inflammatory response.

4. The application of Lactobacillus reuteri 202212 as described in claim 1 in enhancing the immune function of lambs, characterized in that, The Lactobacillus reuteri 202212 can increase the immunoglobulin level in lambs, thereby enhancing their resistance to pathogenic microorganisms.

5. A microbial agent for promoting the growth and development of lambs, characterized in that, Includes Lactobacillus reuteri 202212 as described in claim 1.

6. The microbial agent according to claim 5, characterized in that, The effective viable count of Lactobacillus reuteri 202212 in the microbial agent is ≥10. 8 CFU / mL.

7. A microbial agent for regulating the intestinal microbiota of lambs and enhancing their immunity, characterized in that, Includes Lactobacillus reuteri 202212 as described in claim 1.

8. The microbial agent according to claim 7, characterized in that, The effective viable count of Lactobacillus reuteri 202212 in the microbial agent is ≥10. 8 CFU / mL.

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