Lactobacillus reuteri MRY6 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus reuteri MRY6 and application thereof, and the lactobacillus reuteri MRY6 is preserved in the China Center for Type Culture Collection on September 23, 2024; the address of the preservation unit is Wuhan University, Wuhan, China; the preservation number of the strain is CCTCC (China Center For Type Culture Collection) NO: The lactobacillus reuteri MRY6 is separated from faeces of healthy Hu sheep lambs of 0-7 days old, has the characteristic of rapid growth, and has the characteristics of improving the rumen dry matter digestibility, the mycoprotein content and the ammoniacal nitrogen content, improving the growth performance of newborn animals, reducing inflammation and improving intestinal flora composition.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to Lactobacillus reuteri MRY6 and applications thereof. Background Art

[0002] The gastrointestinal microbiota is crucial to key functional activities of livestock, such as digestive tract health, nutrient digestion and utilization, and immune response. The use of probiotics is an effective means of regulating the gastrointestinal microbiota. By introducing beneficial microorganisms (such as lactic acid bacteria, bifidobacteria, and yeast), it promotes the proliferation of beneficial bacteria in the intestine and inhibits the growth of harmful bacteria, thereby establishing a healthy, stable, and efficient gastrointestinal microbial ecosystem.

[0003] The gastrointestinal tract of newborn animals is almost sterile. After birth, microorganisms from the external environment begin to enter the animal's digestive tract and quickly colonize and develop. Therefore, this period is called the "critical window period" for the establishment of gastrointestinal microbial flora. At the same time, there is an obvious priority effect in the colonization of digestive tract microorganisms, that is, the state of the microbial community colonized in the digestive tract of newborn animals will significantly affect the subsequent microbial colonization and community formation. Therefore, by intervening in the "critical window period", it can have a long-term impact on the structure and function of the later microbial community, thereby continuously regulating the intestinal health, nutrient digestion and absorption, and growth performance of animals.

[0004] The newborn stage is a critical stage for the establishment of passive immunity and digestive tract microorganisms. At the same time, it also faces multiple problems of birth stress, digestive tract homeostasis and immune weakness. Therefore, the introduction of high-quality microorganisms in the newborn stage to improve their long-term health and growth performance is a technical problem that needs to be solved in the field of livestock breeding.

[0005] A large number of existing probiotic strains and flora have been tested in the prior art, but all of them have the following problems that are difficult to solve:

[0006] 1) The problem of digestive tract flora disturbance after the introduction of probiotics in the early stage of life;

[0007] 2) The problem of lowering passive immunity levels after the introduction of probiotics in the early stages of life;

[0008] 3) The problem of aggravated inflammatory response after the introduction of probiotics in the early stages of life;

[0009] 4) The introduction of probiotics in the early stages of life may cause excessive immune response in the digestive tract, leading to increased diarrhea rates;

[0010] 5) The problem of reduced growth performance after the introduction of probiotics in the initial stage.

[0011] Therefore, there is an urgent need to provide a probiotic strain that is suitable for the intestinal environment of newborn animals and can avoid the above problems. Summary of the invention

[0012] The object of the present invention is to provide a Limosilactobacillus reuteri MRY6 strain, which can improve the growth performance of neonatal animals, reduce inflammation, and affect the composition of the intestinal flora.

[0013] The object of the present invention is achieved by the following technical solutions:

[0014] The present invention provides a Limosilactobacillus reuteri MRY6, which was deposited at the China Center for Type Culture Collection on September 23, 2024; the address of the depositary institution: Wuhan University, Wuhan, China; the deposit number: CCTCC NO: M 20242030.

[0015] The present invention also provides a microbial inoculum, which comprises the Limosilactobacillus reuteri MRY6 described in claim 1.

[0016] Further, the microbial inoculum comprises a liquid inoculum or a solid inoculum.

[0017] Further, the concentration of Limosilactobacillus reuteri MRY6 in the liquid inoculum is 1×10 10 CFU / mL.

[0018] The present invention also provides an application of the Limosilactobacillus reuteri MRY6 or the microbial inoculum in the preparation of a product for promoting animal intestinal health.

[0019] Further, the promotion of animal intestinal health includes improving the growth performance of neonatal animals, reducing inflammation, and affecting the composition of the intestinal flora.

[0020] Further, the product comprises a feed additive.

[0021] Further, the promotion of animal intestinal health also includes reducing the diarrhea rate, regulating the level of animal inflammatory factors, enhancing the body immunity, regulating the health of the animal gastrointestinal tract, and regulating the function of the animal gastrointestinal barrier.

[0022] The present invention also provides a feed additive, which comprises the Limosilactobacillus reuteri MRY6 or the microbial inoculum.

[0023] Further, the feed additive also comprises excipients.

[0024] The beneficial effects of the present invention are as follows:

[0025] The present invention has conducted a wide range of screening from the feces and rumen of young cattle and sheep, and isolated a probiotic strain, Lactobacillus mucosae MRY6, suitable for the intestinal environment of neonatal animals, from the feces of healthy lambs. After neonatal lambs are orally perfused within 24 hours of birth, it can reduce the inflammatory response, improve the growth performance, improve the intestinal flora composition, and still maintain a long-term positive impact after discontinuation, while not significantly affecting the passive immunity level, providing a new available strain for the development of microbial preparations for young livestock. This Lactobacillus mucosae MRY6 is isolated from the feces of healthy Hu sheep lambs aged 0 to 7 days, has the characteristics of rapid growth, can improve the rumen dry matter digestibility, the content of microbial protein and ammonia nitrogen, improve the animal growth performance, reduce inflammation and improve the intestinal flora composition. The Lactobacillus mucosae MRY6 provided by the present invention is easy to culture in vitro, belongs to the feed additive permitted to be added in China, has good safety, has high heat resistance, has the potential to develop functional microbial additives, and has extremely high economic value when applied to the breeding of young livestock. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0027] Figure 1 It is the colony morphology diagram of Lactobacillus mucosae MRY6;

[0028] Figure 2 It is the phylogenetic tree diagram constructed for Lactobacillus mucosae MRY6;

[0029] Figure 3 It is the growth curve diagram of Lactobacillus mucosae MRY6 at different temperatures and pH conditions for 24 hours. Among them, A is for different temperatures and B is for different pH;

[0030] Figure 4 It is the Alpha diversity diagram of the intestinal flora of neonatal lambs by Lactobacillus mucosae MRY6. Among them, A is the richness index, B is the diversity index, and C is the evenness index;

[0031] Figure 5 It is the Beta diversity diagram of the intestinal flora of neonatal lambs by Lactobacillus mucosae MRY6. Among them, A is 3d (the 3rd day after birth), B is 7d (the 7th day after birth), C is 42d (the 42nd day after birth), and D is W7d (7 days after weaning);

[0032] Figure 6 The figure shows the intestinal flora composition of neonatal lambs by Limosilactobacillus reuteri. Among them, A - D show the effects of Limosilactobacillus reuteri on the intestinal flora of neonatal lambs at the phylum level. A is 3d (the 3rd day after birth), B is 7d (the 7th day after birth), C is 42d (the 42nd day after birth), and D is W7d (7 days after weaning); E - H show the effects of Limosilactobacillus reuteri on the intestinal flora of neonatal lambs at the genus level. E is 3d (the 3rd day after birth), F is 7d (the 7th day after birth), G is 42d (the 42nd day after birth), and H is W7d (7 days after weaning). Detailed implementation manners

[0033] The embodiments of the present invention are described in detail below. The embodiments are intended to explain the present invention and should not be construed as limiting the present invention. For those not specifying specific techniques or conditions in the embodiments, the techniques or conditions described in the literature in this field or according to the product specifications are followed. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0034] For a clearer understanding of the technical features, objectives, and effects of the present invention, the specific implementation manners of the present invention are now described.

[0035] In the present invention, the strain MRY6 was isolated from the feces of healthy Hu sheep lambs aged 0 - 7 days. Through morphological identification, the strain MRY6 was cultured on an MRS medium plate at 39°C for 24h, and the colony morphology was round, milky white, opaque, and the colony surface was moist and smooth( Figure 1 ). The results of molecular identification showed that the similarity of the 16S rRNA sequence of the strain MRY6 to Limosilactobacillus reuteri was 99.79%. At the same time, the construction results of the phylogenetic tree showed that the strain MRY6 clustered with Limosilactobacillus reuteri. Therefore, based on the comprehensive results of morphological and molecular biological identifications, the strain MRY6 is Limosilactobacillus reuteri (formerly known as Lactobacillus reuteri). This Limosilactobacillus reuteri MRY6 was deposited in the China Center for Type Culture Collection on September 23, 2024. The deposit address is Wuhan University, Wuhan, China, and the biological deposit number is CCTCC NO: M 20242030.

[0036] The present invention provides a microbial inoculant that can improve rumen dry matter digestibility (DMD), microbial crude protein (MCP), and ammonia nitrogen (NH3 - N), including the above - mentioned Limosilactobacillus reuteri MRY6 and excipients.

[0037] In the present invention, the microbial inoculum preferably includes a liquid inoculum or a solid inoculum. The concentration of the Lactobacillus mucosae strain in the liquid inoculum is 1×10 10 CFU / ml. The preparation method of the liquid inoculum includes the following steps:

[0038] The strain MRY6 is activated or amplified and cultured, washed with a buffer solution, and then made into a liquid inoculum.

[0039] In the present invention, the MRS medium is used for activation or culture. The activation or amplification culture temperature is preferably 37-39°C. The activation or amplification culture time is preferably 10-24 h, more preferably 12 h. The washing buffer solution is preferably physiological saline. The number of washing times is preferably 3-4 times. The method of making the liquid inoculum is preferably centrifugation and washing with physiological saline.

[0040] In the present invention, the Lactobacillus mucosae MRY6 includes improving animal growth performance, reducing inflammation, and affecting the intestinal flora composition. In the method for raising lambs, the Lactobacillus mucosae MRY6 is used as an additive and fed to the lambs through a syringe at 1 h, 12 h, 24 h, and 48 h after birth, 1*10 10 CFU / mL each time.

[0041] The present invention provides a feed additive for promoting the intestinal health of lambs, including the Lactobacillus mucosae MRY6 or the microbial inoculum and excipients.

[0042] The present invention has no special restrictions on the types of the excipients, and the excipients well-known in the art for feed additives can be used. The present invention has no special restrictions on the types of the feed additives, and the preparation methods well-known in the art for feed additives can be used.

[0043] The following examples are used to illustrate in detail a Lactobacillus mucosae strain MRY6, a microbial inoculum and their applications provided by the present invention, but they should not be construed as limiting the protection scope of the present invention.

[0044] The media and their preparation methods involved in the examples of the present invention:

[0045] Screening medium (LBS): peptone 10 g, beef extract powder 10 g, dipotassium hydrogen phosphate 2 g, sodium acetate 5 g, ammonium citrate 2 g, magnesium sulfate 0.2 g, manganese sulfate 0.05 g, glucose 20 g, calcium carbonate 5 g, Tween-80 1 ml, agar 25 g, distilled water 1000 mL, pH 6.0-6.5, autoclaved at 121°C for 30 minutes.

[0046] Isolation medium (MRS): 10 g of peptone, 8 g of beef extract powder, 4 g of yeast extract powder, 20 g of glucose, 2 g of dipotassium hydrogen phosphate, 2 g of disodium hydrogen citrate, 5 g of sodium acetate, 0.2 g of magnesium sulfate, 0.04 g of manganese sulfate, 20 g of calcium carbonate, 14 g of agar, 1 ml of Tween-80, 1000 mL of distilled water, pH 6.5 ± 0.2, sterilized at 121 °C for 30 min.

[0047] Method for measuring growth curves at different temperatures and different pH values in the examples:

[0048] After activating the MRY6 bacterial solution, inoculate it into the MRS liquid medium and anaerobically culture it under different temperature and different pH conditions. Measure the absorbance OD of the strains at each stage at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h respectively. 600 .

[0049] Method for measuring growth performance in the examples:

[0050] Weigh the lambs with an electronic scale before morning feeding at 0, 3, 7, 14, 21, 28, 35, 42 days after oral administration to lambs and 7 and 14 days after weaning respectively, and calculate their average daily gain (ADG).

[0051] Method for measuring immune indexes used in the examples:

[0052] Use the enzyme-linked immunosorbent assay (ELISA) method to measure the inflammatory factors (IL-1β, IL-6, TNF-α) in lamb serum.

[0053] Method for measuring intestinal microflora in the examples:

[0054] Send the fecal samples to Novogene Co., Ltd. in Beijing for 16S RNA gene sequencing using the Illumina NovaSeq 6000 high-throughput sequencing platform. Use the Soil Genomic DNA Extraction Kit (Catalog No.: DP336) to extract the sample DNA, and use the NanoDrop 2000 spectrophotometer to measure the concentration and purity of the obtained genomic DNA samples. Amplify the V3-V4 region of 16S rDNA by polymerase chain reaction (PCR). The library construction uses the The NEBNext UltraⅡ DNA Library Prep Kit (Cat.No.E7645B) kit. After clustering the high-quality sequences of OTUs with a sequence similarity threshold of 97% using the Vsearch software, align the representative sequences with the Silva138 database to obtain the taxonomic information of the species corresponding to each OTU. Use the QIME2 software for α and β diversity analysis.

[0055] Method for preparing rumen buffer solution used in the examples: The rumen buffer solution was prepared with reference to the method of Menke et al.

[0056] Method for measuring in vitro fermentation parameters in the examples:

[0057] At 2, 4, 6, 8, 10, 12, 24, 36, and 48 h of in vitro fermentation, a syringe was used to extract gas and the gas production was recorded. The gas production at 2, 4, 6, 8, 10, 12, 24, 36, and 48 h for each group was substituted into the gas production model to calculate the gas production parameters. The gas production model: GP = a + b(1 - e -ct ).

[0058] where t represents the fermentation time (h), GP represents the gas production at time t (mL), a represents the gas production of the rapidly degraded part (mL), b represents the gas production of the slowly degraded part (mL), c represents the gas production rate of b (% / h), and a + b represents the theoretical total gas production (mL).

[0059] The residue after 48 h of fermentation was filtered through a 300-mesh nylon bag and dried at 65 °C for 48 h. The dry matter (DM) in the diet and fermentation residue was determined with reference to the method of AOAC (2000). At 4, 8, 12, 24, 36, and 48 h of in vitro fermentation, 5 mL of the fermentation broth was extracted with a syringe and placed in a centrifuge tube. The MCP concentration was measured by the Coomassie brilliant blue method, and the NH3-N concentration was determined by the indophenol colorimetric method with reference to Chaney et al.

[0060] Isolation, purification, and identification of the strain in Example 1

[0061] Healthy Hu sheep lambs aged 0 - 7 days were selected, and fresh fecal samples were collected by the rectal fecal collection method and stored in 5 mL centrifuge tubes at -80 °C for later use. 1 g of the collected feces was serially diluted with sterile saline to make dilutions of 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 . 0.3 mL of each dilution was spread on MRS medium and incubated anaerobically at 37 °C for 24 - 48 h. Bacterial growth was observed after 24 h. Single colonies with a calcium dissolution zone on the LBS plate were transferred to MRS medium and isolated and purified by the four-zone streaking method. After morphological identification, when the strain MRY6 was cultured on the MRS medium plate at 37 °C for 24 h, the colony morphology was round, milky white, opaque, and the colony surface was moist and smooth ( Figure 1)。For the purified strain, colony DNA was extracted, and universal primers 1492R (SEQ ID NO: 1) (5’-GGTTACCTTGTTACGACTT-3’) and 27F (SEQ ID NO: 2) (5’-AGAGTTTGATCCTGGCTCAG-3’) were used. The PCR reaction volume was 25 μL: 2 μL of bacterial solution, 1 μL of upstream primer, 1 μL of downstream primer, 12.5 μL of Mix, and 8.5 μL of ddH2O. The PCR amplification program was pre-denaturation for 5 min (95 °C), denaturation for 1 min (94 °C), annealing for 1 min (47 °C), extension for 1.5 min (72 °C), with a total of 30 cycles, and finally extension for 10 min (72 °C). The PCR products were detected by 2% agarose gel electrophoresis, and the PCR products were sent to Shanghai Bioengineering Co., Ltd. for 16S rDNA sequencing. The obtained sequencing results were subjected to BLAST alignment on the NCBI website, and a phylogenetic tree of bacteria was constructed using MEGA7.0 based on the neighbor-joining method. The results are shown in Figure 2 . The results showed that strain MRY6 clustered with Lactobacillus mucosae reuteri, with a similarity of 99.79%.

[0062] Example 2 Determination of the growth curve of strain MRY6

[0063] Five temperature variables were set in the experiment, namely 28 °C, 35 °C, 37 °C, 39 °C, and 42 °C. The activated MRY6 bacterial solution was inoculated into anaerobic tubes containing an equal amount of MRS liquid medium at a ratio of 1%. Three replicates were set for each temperature. A small amount of bacterial solution was taken every 2 h to measure the OD of the bacterial solution 600 to determine the optimal in vitro culture temperature.

[0064] Bacteria were cultured at the optimal temperature, and 10 different pH values were set, namely 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5. The activated MRY6 bacterial solution was inoculated into anaerobic tubes containing MRS liquid medium with the above different pH values at an inoculation amount of 1%. Three replicates were set for each pH value. A small amount of bacterial solution was taken every 2 h to measure the OD of each group of bacterial solutions 600 to determine the optimal pH value. The results are shown in Figure 3 . The results showed that the optimal culture temperature of Lactobacillus mucosae reuteri MRY6 was 37 - 39 °C, and the optimal culture pH value was 6.5.

[0065] Example 3 Determination of the heat resistance of strain MRY6

[0066] The MRY6 bacterial solution was inoculated into MRS liquid medium at a ratio of 1%, anaerobically cultured at 37 °C for 14 h, and then plate counted after water bath at 70 °C, 75 °C, and 80 °C for 1, 3, and 5 min respectively.

[0067] Table 1 shows the tolerance of strain MRY6 to different temperatures. The results show that after 1 minute of water bath, the viable cell concentrations at 70 °C, 75 °C, and 80 °C are 7.0×10 7 , 6.33×10 7 , and 5.33×10 7 respectively. After 5 minutes of water bath, no viable cells can be detected at 75 °C and 80 °C by dilution plating.

[0068] Table 1 Tolerance of strain MRY6 to different temperatures

[0069]

[0070]

[0071] Example 4 Application of Limosilactobacillus reuteri MRY6 in animal feeding

[0072] The test site and test animals were provided by Baosen Animal Husbandry Co., Ltd. in Cangzhou City, Hebei Province. The test time was from November 2023 to February 2024. Twenty newborn Hu sheep lambs with the same parity (second parity), similar body weights (4.30 ± 0.50 kg), and consistent feeding management were selected and paired into two groups, namely the control group and the Limosilactobacillus reuteri MRY6 group, with 10 replicates in each group. At 1, 12, 24, and 48 hours after birth, 10 ml of normal saline and 10 ml of Limosilactobacillus reuteri MRY6 bacterial solution were fed via syringe respectively. The method for preparing the bacterial solution is described above.

[0073] Table 2 shows the effect of Limosilactobacillus reuteri MRY6 on the growth performance of lambs. The results show that the ADG7, ADG 14 , ADG 21 , and ADG 35 of the Limosilactobacillus reuteri MRY6 group are significantly higher than those of the control group, increasing by 38.10%, 23.81%, 21.05%, and 29.41% respectively compared with the control group.

[0074] Table 2 Effect of Limosilactobacillus reuteri MRY6 on the growth performance of lambs

[0075]

[0076] Table 3 shows the effect of Limosilactobacillus reuteri MRY6 on serum inflammatory factors in lambs. The results show that compared with the control group, the content of TNF-α in the Limosilactobacillus reuteri MRY6 group is significantly reduced, by 25.30%.

[0077] Table 3 Effect of Limosilactobacillus reuteri MRY6 on serum inflammatory factors in lambs

[0078]

[0079]

[0080] Figure 4 Effect of Lactobacillus reuteri MRY6 on the Alpha diversity of the intestinal flora of neonatal lambs. The results showed that the Richness index of the Lactobacillus reuteri MRY6 group was significantly higher than that of the control group at 3 days, and the Eveness index was significantly lower than that of the control group at 42 days.

[0081] Figure 5 Effect of Lactobacillus reuteri MRY6 on the Beta diversity of the intestinal flora of neonatal lambs. The results showed that there were significant differences in the intestinal flora of the two groups of lambs at 3 days and 42 days.

[0082] Figure 6 Effect of Lactobacillus reuteri on the composition of the intestinal flora of neonatal lambs. The results showed that at the phylum level, the Proteobacteria of the lambs in the Lactobacillus reuteri MRY6 group was significantly higher than that of the control group at 3 days, while the Firmicutes was significantly lower than that of the control group. Compared with the control group, the Verrucomicrobia of the lambs in the Lactobacillus reuteri MRY6 group was significantly increased at 3 and 42 days. At the genus level, Escherichia.Shigella, Eubacterium-coprostanoligenes-group, Muribaculaceae-uncultured-bacterium, Ruminococaceae-UCG.014, Alstipes, and Akkermansia of the lambs in the Lactobacillus reuteri MRY6 group were significantly higher than those in the CON group at 3 days after birth, and Butyricicoccus was significantly lower than that in the CON group; Hungatella, Caproiciproducene, and Flavonifractor of the lambs in the MRY6 group were significantly higher than those in the CON group at 7 days after birth, and Ruminococcus-gnavus-group was significantly lower than that in the CON group; Akkermansia of the lambs in the MRY6 group was significantly higher than that in the CON group at 42 days after birth, and Christensenellaceae-R.7-group, Rikenellaceae-RC9-gut-group, and Ruminiclostridium-6 were significantly lower than those in the CON group; Ruminococaceae-uncultured of the lambs in the MRY6 group was significantly higher than that in the CON group at 52 days after birth.

[0083] Example 5 Effects of Limosilactobacillus reuteri MRY6 on in vitro fermentation parameters

[0084] Using the addition level of the isolated Limosilactobacillus reuteri MRY6 (OD 600 = 1.621) as the experimental treatment, 1 control group and 4 experimental groups were designed, with 6 replicates in each group. Fresh rumen fluid was collected from the animal husbandry teaching base of Hebei Agricultural University, filtered through 4 layers of gauze, and mixed evenly with the rumen buffer solution described above in a ratio of 1:2, and then injected into a 500 ml fermentation bag. Using 6 g of the basal diet as the fermentation substrate, the addition amounts of Limosilactobacillus reuteri MRY6 were 0 μL, 40 μL, 80 μL, 120 μL, and 160 μL respectively. The fermentation bags were deoxygenated and sealed using a vacuum packaging machine, and then placed in a 39 °C constant temperature water bath for in vitro rumen fermentation at a rotation speed of 45 r / min for 48 h.

[0085] Table 4 shows the effects of the addition amount of Limosilactobacillus reuteri MRY6 on gas production and gas production parameters. The results showed that compared with the control group, the gas production in the bacterial liquid addition groups at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24, 26, and 48 h of fermentation was significantly higher than that in the control group (P < 0.05), and the gas production increased by 21.50 - 48.18%, and the gas production showed linear and quadratic curve changes with the addition dose (P < 0.05). The in vitro fermentation parameters showed that the gas production in the rapidly degraded part in the groups added with 120 μL and 160 μL of bacterial liquid was significantly lower than that in the control group (P < 0.05), and there was no significant effect in the groups added with 40 μL and 80 μL of bacterial liquid compared with the control group; the gas production in the slowly degraded part and the theoretical total gas production in the groups added with 40 μL, 80 μL, 120 μL, and 160 μL of bacterial liquid were higher than those in the control group (P < 0.05), and the gas production showed linear and quadratic curve changes with the addition dose (P < 0.01).

[0086] Table 4 Effects of the addition amount of Limosilactobacillus reuteri MRY6 on gas production and gas production parameters

[0087]

[0088] Note: Different lowercase letters in the same row of data of the addition amount of Limosilactobacillus reuteri MRY6 indicate significant differences (P < 0.05), and the same or no letters indicate no significant differences (P > 0.05); in the items, a is the gas production in the rapidly degraded part, b is the gas production in the slowly degraded part, c is the gas production rate of b, and a + b is the theoretical maximum gas production. - indicates gas production lag, and the gas production size of a is expressed in absolute value.

[0089] Table 5 shows the effects of Lactobacillus mucosae MRY6 strain on dry matter digestibility (DM), ammonium nitrogen (NH3-N), and microbial crude protein (MCP). The results show that the DM digestibility of the groups supplemented with 40 μL, 80 μL, 120 μL, and 160 μL of bacterial liquid was significantly higher than that of the control group (P < 0.05), increasing by 27.09%, 22.46%, 24.09%, and 24.54% respectively compared with the control group, and the DM digestibility showed linear and quadratic curve changes with the increase of the addition dose (P < 0.05); at 8 h of fermentation, the NH3-N concentration of the groups supplemented with 40 μL, 80 μL, 120 μL, and 160 μL of bacterial liquid was significantly higher than that of the control group (P < 0.05), increasing by 63.82%, 38.75%, 38.46%, and 31.34% respectively compared with the control group, and the NH3-N concentration showed quadratic curve changes with the increase of the addition dose (P < 0.05); at 48 h of fermentation, the NH3-N concentration of the groups supplemented with 120 μL and 160 μL of bacterial liquid was significantly higher than that of the control group, increasing by 34.63% and 57.20% respectively compared with the control group, and the NH3-N concentration showed linear and quadratic curve changes with the increase of the addition dose (P < 0.05); the MCP concentration of the bacterial liquid addition groups at 4, 8, 12, 24, and 48 h of fermentation was significantly higher than that of the control group (P < 0.05), and the MCP concentration showed linear and quadratic curve changes with the increase of the addition dose at 4, 12, 24, and 48 h of fermentation (P < 0.05).

[0090] Table 5 Effects of Lactobacillus mucosae MRY6 Strain on Dry Matter Digestibility (DM), Ammonium Nitrogen (NH3-N), and Microbial Protein (MCP)

[0091]

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A Lactobacillus reuteri MRY6, characterized in that: The Lactobacillus reuteri MRY6 was deposited in the China Center for Type Culture Collection on September 23, 2024; the address of the depository unit is Wuhan University, Wuhan, China; the deposit number is CCTCC NO: M 20242030.

2. A microbial agent, characterized in that: The microbial agent includes the Lactobacillus reuteri MRY6 according to claim 1.

3. The microbial agent according to claim 2, characterized in that: The microbial agent includes a liquid agent or a solid agent, and the concentration of Lactobacillus reuteri MRY6 in the liquid agent is 1×10 10 CFU / mL.

4. The microbial agent according to claim 3, characterized in that: The microbial agent can improve the digestibility of rumen dry matter, as well as the content of bacterial protein and ammonia nitrogen.

5. Use of the Lactobacillus reuteri MRY6 according to claim 1 or the microbial agent according to any one of claims 2 to 4 in the preparation of a product that promotes intestinal health in animals.

6. The use according to claim 5, characterized in that The promoting of intestinal health of newborn animals includes improving animal growth performance, reducing inflammation, and affecting the composition of intestinal flora.

7. The use according to claim 5, characterized in that The products include medicines, health products, foods, food additives, feeds and feed additives.

8. The use according to claim 5, characterized in that The promoting of intestinal health of animals also includes reducing diarrhea rate, regulating the level of inflammatory factors in animals, enhancing the body's immunity, regulating the gastrointestinal health of animals and regulating the gastrointestinal barrier function of animals.

9. A feed additive, characterized in that: The feed additive comprises the Lactobacillus reuteri MRY6 according to claim 1 or the microbial agent according to any one of claims 2-4.

10. The feed additive according to claim 9, characterized in that The feed additive also includes auxiliary materials.

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