Lactobacillus reuteri as well as application and feed thereof

Abstract

The invention relates to the technical field of biology, and discloses a strain of lactobacillus reuteri, the preservation number of the lactobacillus reuteri is CCTCC NO: M20231546, the preservation date is August 31, 2023, the preservation unit is China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the classification name is Limobacillus reuteri XY227, the preservation number is CCTCC NO: M20231546, the preservation number is CCTCC NO: M20231546, the preservation number is CCTCC NO: M20231546, the preservation number is CCTCC NO: M20231546, and the preservation number is CCTCC NO: M20231546. The bacterial strain can increase the content of amino acid in serum of rats, and further experiments surprisingly find that the bacterial strain can also increase the content of amino acid in serum and ileum of pigs, and the capacity of increasing branched chain amino acid is particularly outstanding; the invention further discloses application of the lactobacillus reuteri and a feed.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a strain of Lactobacillus reuteri and its use and feed. Background Art

[0002] With the upgrading of people's consumption demand, the goal of pig production has changed from pursuing quantity and production efficiency to improving product quality. my country is a major pig breeding country and a major pork consumer. The proportion of foreign pig breeds introduced from abroad is as high as 80%, mainly Yorkshire pigs, large white pigs and Duroc pigs. The introduced pig breeds have the advantages of fast growth, strong reproductive capacity and high feed utilization rate, but have shortcomings such as poor meat taste, low environmental resistance and poor meat color. Compared with lean meat-type three-way hybrid pigs, my country's local pig breed resources are diverse and rich in genetic diversity, have good environmental adaptability, excellent disease resistance and stress resistance, meat quality and tolerance to roughage. Ningxiang pigs, Rongchang pigs, Taihu pigs and Jinhua pigs are collectively known as my country's four famous pigs, among which Ningxiang pigs have excellent meat quality traits.

[0003] Meat is the main source of high-quality protein, and the composition of amino acids directly affects the nutritional value of meat products. Essential amino acids are vital to human health, and a lack of certain essential amino acids may lead to malnutrition. In addition, amino acids not only affect the nutrition of meat products, but also directly participate in the formation of flavor. For example, glutamic acid can increase the umami taste of meat, while other amino acids can affect the aroma and overall taste of meat products. Therefore, the amino acid content has an important impact on the nutrition and flavor of meat products.

[0004] Chinese patent application 200810247335.8 discloses a Lactobacillus pentosus, an active fresh-keeping flavoring agent and its application. The scheme provides a Lactobacillus pentosus (Lactobacillus pentosus) with a preservation number of CGMCC No. 2313 after separation and screening. It can be used with Pediococcus acidilactici (CGMCC No. 2312) to prepare an active fresh-keeping flavoring agent for Chinese and Western meat product processing. The active fresh-keeping flavoring agent of the scheme can remove or reduce the unpleasant smells such as raw fishy smell and mutton smell of meat through a simple process of constant temperature ripening, and competitively inhibit the growth of harmful microorganisms. At the same time, the meat is tenderized, the pH of the meat product is appropriately low, the growth of spoilage bacteria and pathogenic bacteria and the production of toxins are inhibited, the shelf life of the meat product is greatly extended, it is conducive to storage and circulation, it is safe to eat, has a high amino acid content, and the beneficial components are not destroyed, it is nutritious and easy to absorb.

[0005] Further observation of paragraph 69 of the description of the scheme shows that the principle of increasing the amino acid content in meat products is specifically as follows: "After the beneficial microorganisms in the preservative and flavoring agent ferment the meat, the protein is decomposed into amino acids, which greatly improves its digestibility and absorbability, and at the same time increases the essential amino acids and vitamins for the human body, so that its nutritional and health benefits are further enhanced." It can be seen that the increase in the amino acid content of meat in the scheme lies in the decomposition of the protein in the meat by the amino acids;

[0006] However, in life, fresh meat is also one of the common meat products consumed by consumers. It is equally important to increase the amino acid content in fresh meat. Providing the amino acid level in the body of the animal that serves as the source of fresh meat is also one of the means to increase the amino acid content of fresh meat.

[0007] In this regard, Chinese patent application 202210646957.8 discloses a Lactobacillus reuteri strain LN0214 and its derivative products and applications for reducing serum branched-chain amino acid levels. The scheme increases the metabolic level of branched-chain amino acids in serum by regulating the expression of one or more genes among BCAT1, B CAT2, BCKDHA, BCKDHB and PPM1K, thereby reducing serum branched-chain amino acid levels;

[0008] It can be seen that the above scheme clearly shows that Lactobacillus reuteri strain LN0214 not only does not increase the level of branched-chain amino acids in serum, but instead reduces the level of branched-chain amino acids in serum.

[0009] The problem that this scheme needs to solve: How to provide a strain that can increase the level of amino acids. Summary of the invention

[0010] The purpose of the present application is to provide a strain of Lactobacillus reuteri, which can increase the amino acid content in an organism by increasing the amino acid levels in the serum and ileum of the organism.

[0011] To achieve the above-mentioned purpose, the present application provides the following technical solution: a strain of Lactobacillus reuteri, the preservation number of the Lactobacillus reuteri is CCTCC NO: M20231546, the preservation date is August 31, 2023, the preservation unit is the China Center for Type Culture Collection, the preservation address is: Wuhan University, Wuhan, China, and the classification name is Limosilactobacillus reuteri XY227.

[0012] Preferably, the 16SrRNA sequence of Lactobacillus reuteri is as shown in SEQ ID NO:1.

[0013] Preferably, the Lactobacillus reuteri is isolated from Ningxiang pig feces.

[0014] In addition, the present application also discloses the use of the above-mentioned Lactobacillus reuteri for increasing the amino acid level in serum or ileum.

[0015] Preferably, the amino acid is at least one of leucine, isoleucine and valine.

[0016] In addition, the present application also discloses the use of the above-mentioned Lactobacillus reuteri for increasing the intramuscular fat content of pigs.

[0017] In addition, the present application also discloses the use of the above-mentioned Lactobacillus reuteri in preparing feed.

[0018] In addition, the application also discloses a feed containing the above-mentioned Lactobacillus reuteri.

[0019] Preferably, the live bacteria concentration of Lactobacillus reuteri in the feed is 0.8×10 11 ~1.2×10 11 CFU / kg.

[0020] The beneficial effects of this application are:

[0021] The Lactobacillus reuteri of the present application is isolated from the feces of Ningxiang pigs and can increase the amino acid content in rat serum. After further experiments, we surprisingly found that it can also increase the amino acid content in pig serum and ileum, and its ability to increase branched-chain amino acids is particularly outstanding. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the morphological characteristics of strain 1 (LM);

[0023] Figure 2 is a schematic diagram of the morphological characteristics of strain 2 (LR);

[0024] Figure 3 is a schematic diagram of the morphological characteristics of strain 3 (LS);

[0025] Figure 4 is the growth curve of strain 1 (LM);

[0026] Figure 5 is the growth curve of strain 2 (LR);

[0027] Figure 6 is the growth curve of strain 3 (LS);

[0028] Figure 7 The antibiotic sensitivity test results of strain 1 (LM) are shown in Figure 1. From left to right, the MRS solid culture medium with rifamycin, penicillin, erythromycin, and clindamycin susceptibility tablets added is shown.

[0029] Figure 8The antibiotic sensitivity test results of strain 2 (LR) are shown in Figure 1. From left to right, the MRS solid culture medium is supplemented with clindamycin, erythromycin, tetracycline, and ampicillin susceptibility tablets;

[0030] Fig. 9 The antibiotic sensitivity test results of strain 3 (LS) are shown in Figure 1. From left to right, the MRS solid culture medium is supplemented with penicillin, clindamycin, ampicillin, and rifamycin susceptibility tablets.

[0031] Fig.10 This is the acid resistance test result of strain 1 (LM);

[0032] Fig.11 This is the acid resistance test result of strain 2 (LR);

[0033] Fig.12 This is the acid resistance test result of strain 3 (LS);

[0034] Fig.13 This is a graph showing the bile salt tolerance test results of strain 1 (LM);

[0035] Fig.14 This is a graph showing the bile salt tolerance test results of strain 2 (LR);

[0036] Fig.15 This is a graph showing the bile salt tolerance test results of strain 3 (LS);

[0037] Fig.16 This is a comparison chart of the initial body weight of rats in the LM group, LR group, LS group and control group;

[0038] Fig.17 This is a comparison of the final body weight of rats in the LM group, LR group, LS group and control group;

[0039] Fig.18 This is a comparison chart of the food intake of rats in the LM group, LR group, LS group and control group;

[0040] Fig.19 The figure is a comparison of the levels of leucine, isoleucine, valine and total branched-chain amino acids in the ileum contents of rats in the LR group and those in the control group;

[0041] Fig. 20 The figure is a comparison of the serum leucine, isoleucine, valine and total branched-chain amino acid levels between the LR group and the control group;

[0042] Fig.21 This is a comparison chart of BCAA levels in the serum of fattening pigs in the Lactobacillus reuteri XY227 group and the control group;

[0043] Fig. 22This is a comparison chart of the amino acid levels in the ileum contents of fattening pigs in the Lactobacillus reuteri XY227 group and the control group;

[0044] Fig.23 This is a comparison chart of the amino acid levels in the colon contents of fattening pigs in the Lactobacillus reuteri XY227 group and the control group;

[0045] Fig.24 This is a comparison chart of the effect of Lactobacillus reuteri XY227 on the intramuscular fat content of the back muscle of fattening pigs and the control group;

[0046] Fig.25 This is a comparison of the oil red staining of the back muscle of fattening pigs by Lactobacillus reuteri XY227 and the control group. DETAILED DESCRIPTION

[0047] In the description of this application, it should be noted that if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0048] The present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0049] Example 1

[0050] 1.1 Isolation and identification of strains

[0051] (1) Isolation of strains

[0052] Fecal samples were collected from healthy Ningxiang fattening pigs for the isolation of Lactobacillus. Fecal samples were suspended in phosphate buffered saline (PBS) and then serially diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8The dilutions of different gradients were spread on MRS medium and cultured for two days in an anaerobic workstation at 37°C. The mixed gas in the workstation included 80% N2 by volume, 10% CO2 by volume, and 10% H2 by volume. Single white or milky colonies were picked from the plate, and the colonies were purified by streaking them multiple times on MRS medium. The purified single colonies were picked into liquid culture medium and cultured anaerobically. The bacterial solution was mixed with a 20% glycerol aqueous solution and stored in a refrigerator for later use.

[0053] (2) 16S rRNA gene sequence identification

[0054] The 16S rRNA gene of a single strain was amplified using universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-TACGGCTACCTTGTTA CGACTT-3') and then sequenced by the Sanger method. The measured 16S rRNA gene sequence was then compared with the NCBI nucleotide sequence database. Finally, three lactic acid bacteria were isolated, namely strain 1 (LM), strain 2 (LR) and strain 3 (LS).

[0055] The 16SrRNA gene sequence identification results of strain 1 (LM) are as follows:

[0056] ACGCCGGCGGTGTGCCTAATACATGCAAGTCGAACGCGTTGGCCCAAC

[0057] TGATTGAACGTGCTTGCACGGACTTGACGTTGGTTTACCAGCGAGTGGCGG

[0058] ACGGTGAGTAACACGTAGGTAACCTGCCCCAAAGCGGGGGATAACATTT

[0059] GGAAACAGATGCTAATACCGCATAACAATTTGAATCGCATGATTCAAATTTA

[0060] AAAGATGGCTTCGGCTATCACTTTGGGATGGACCTGCGGCGCATTAGCTTG

[0061] TTGGTAGGGTAACGGCCTACCAAGGCTGTGATGCGTAGCCGAGTTGAGAG

[0062] ACTGATCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGG

[0063] CAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACC

[0064] GCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAGAGAAGA

[0065] ACGTGCGTGAGAGCAACTGTTCACGCAGTGACGGTATCTAACCAGAAAGT

[0066] CACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTT

[0067] ATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTGATAAGTCTGAT

[0068] GTGAAAGCCTTTGGCTTAACCAAAGAAGTGCATCGGAAACTGTCAGACTT

[0069] GAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAG

[0070] ATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGA

[0071] CGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGT

[0072] CCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGT

[0073] GCCGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGT

[0074] TGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGG

[0075] TTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCC

[0076] AACCCTAGAGATAGGGCGTTTCCTTCGGGAACGCAATGACAGGTGGTGCAT

[0077] GGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGC

[0078] GCAACCCTTGTTACTAGTTGCCAGCATTCAGTTGGGCACTCTAGTGAGACT

[0079] GCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCC

[0080] CTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCG

[0081] AACTCGCGAGGGCAAGCTAATCTCTTAAAACCGTTCTCAGTTCGGACTGCA

[0082] GGCTGCAACTCGCCTGCACGAAGTCGGAATCGCTAGTAATCGCGGATCAGC

[0083] ATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCA

[0084] TGAGAGTTTGCAACACCCAAAGTCGGTGGGGTAACCCTTCGGGGAGCTAG

[0085] CCGCCTAAGGTGGGGCAGATGATTAGGGTG

[0086] The 16SrRNA sequence of the strain had a 100% similarity with the 16SrRNA sequence of Limosilactobacillus mu cosae in the NCBI GenBank database, indicating that the strain was Limosilactobacillus muc osae, named LM in this article.

[0087] The identification results of the 16SrRNA gene sequence (SEQ ID NO: 1) of strain 2 (LR) are as follows:

[0088]

[0089] The similarity between the 16S rRNA sequence of the strain and the 16S rRNA sequence of Limosilactobacillus reute ri in the NCBI GenBank database is 99.80%, indicating that the strain is Limosilactobacillus reute ri, named (LR) in this article.

[0090] The 16SrRNA gene sequence identification results of strain 3 (LS) are as follows:

[0091] CCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGACATGCTGATTCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTAAACCTCGCGGTCTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCGCCAGAGTGCCCAACTTAATGCTGGCAACTGACAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCACTTTGTCCCCGAAGGGAAAGCCTAATCTCTTAGGTGGTCAAAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTATTGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTACCCACGCTTTCGAACCTCAGCGTCAGTTACAGACCAGAGAGCCGCTTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGTCTTCCAGTTTCCAATGCACTACTCCGGTTAAGCCGAAGGCTTTCACATCAGACTTAAAAGACCGCCTGCGTTCCCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTGCTGGTTAGATACCGTCATCGAATGAACAGTTACTCTCACTCGTGTTCTTCTCTAACAACAGAGTTTTACGATCCGAAGACCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGT AGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAACCTCTCAGTTCGGCTACGTATCATCACCTTGGTAGGCCGTTACCCCACCAACTAGTTAATACGCCGCGGGTCCATCTAAAAGCGATAGCAGAACCATCTTTCATCTAAGGATCATGCGATCCTTAGAGATATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTTCTTTTAGGCAGGTTACCCACGTGT

[0092] The similarity between the 16S rRNA sequence of the strain and the 16S rRNA sequence of Ligilactobacillus salivarius in the NCBI GenBank database is 99.92%, indicating that the strain is Ligilactobacillus salivarius, named (LS) in this article.

[0093] 1.2 Identification of strains

[0094] The morphological characteristics of the above-mentioned preserved strains were identified with reference to Bergey's Manual of Systematic Bacteriology.

[0095] (1) Morphological characteristics of strains

[0096] Observe the strain morphology on MRS solid medium, such as Figure 1-3 As shown, strain 1 (LM) is irregularly serrated, milky white, and flat, about 2.5 to 3 mm in size;

[0097] Strain 2 (LR) is round, about 1 mm in size, milky white, and flat;

[0098] Strain 3 (LS) was round with a diameter of about 2 mm, milky white, and plump in shape.

[0099] All three strains are Gram-positive bacteria. Strain 1 (LM) has a long straight rod shape with rounded ends; strain 2 (LR) has a short rod shape with blunt ends; and strain 3 (LS) has a short rod shape with blunt ends.

[0100] 1.3 Growth curve of strains

[0101] (1) Test method: Inoculate the three activated bacterial cultures into multiple tubes of MRS liquid culture medium at a 1% inoculation rate, place in an incubator, and culture at 37°C for anaerobic incubation. Repeat 3 times at each time point. Use a microplate reader to measure the OD value of the culture medium every 2 hours. 600 The absorbance.

[0102] (2) Test results: Figure 4-6 As shown, the three lactobacilli had different growth rates and plateau phases, with LM entering the logarithmic growth phase at 6 h and reaching the plateau phase after 24 h;

[0103] LR entered the logarithmic growth phase at 6 h and reached the plateau phase after 14 h;

[0104] LS entered the logarithmic growth phase at 4 h and reached the plateau phase after 8 h.

[0105] 1.4 Antibiotic sensitivity of strains

[0106] (1) Test method: A certain amount of bacterial liquid is drawn from the seed liquid at a 1% inoculum volume and evenly spread on the MRS solid culture medium. Drug-sensitive tablets of streptomycin, gentamicin, tetracycline, erythromycin, penicillin, clindamycin, ciprofloxacin, kanamycin, chloramphenicol, ampicillin, and rifamycin are placed on the MRS agar culture medium. After 24 to 48 hours, the bacterial culture medium is removed and the diameter of the inhibition zone is measured with a vernier caliper.

[0107] (2) Test results: Reference Figure 7-9 As shown in Table 1 , LM is relatively sensitive to rifamycin, penicillin, erythromycin, and clindamycin;

[0108] LR is relatively sensitive to clindamycin, erythromycin, tetracycline, ampicillin, and rifamycin;

[0109] LS is sensitive to penicillin, clindamycin, ampicillin and rifamycin.

[0110] Table 1

[0111]

[0112]

[0113] 1.5 Test of acid resistance of strains

[0114] (1) Test method: A certain amount of bacterial solution was taken from the seed solution at a 1% inoculum volume and inoculated into MRS liquid culture medium with pH values ​​of 1.0, 2.0 and 3.0, and the OD was measured at 0h, 1h, 2h and 3h respectively. 600 The experiment was repeated four times and the survival rate of the strains was calculated.

[0115] (2) Test results: Reference Fig.10 , the survival rate of LM treated in pH = 1 medium for 1 h was 85%, the survival rate after 2 h was 80%, and the survival rate after 3 h was 77%;

[0116] The survival rate of LM treated in pH = 2 medium was 93% after 1 h, 91% after 2 h, and 87% after 3 h;

[0117] The survival rate of LM treated in pH = 3 medium was 105% after 1 h, 109% after 2 h, and 111% after 3 h;

[0118] refer to Fig.11 , the survival rate of LR treated in pH = 1 medium was 84% ​​for 1 h, 76% after 2 h, and 71% after 3 h;

[0119] The survival rate of LR treated in pH = 2 medium was 90% after 1 h, 89% after 2 h, and 88% after 3 h;

[0120] The survival rate of LR treated in pH=3 medium was 105% after 1 hour, 108% after 2 hours, and 109% after 3 hours.

[0121] refer to Fig.12 , the survival rate of LS treated in pH = 1 medium was 84% ​​for 1 h, 77% after 2 h, and 74% after 3 h;

[0122] The survival rate of LS treated in pH = 2 medium was 93% after 1 h, 94% after 2 h, and 97% after 3 h;

[0123] The survival rate of LS treated in pH=3 medium was 102% after 1 hour, 105% after 2 hours, and 110% after 3 hours.

[0124] 1.6 Bile salt tolerance test of strains

[0125] (1) Test method: A certain amount of bacterial liquid was taken from the seed liquid at a 1% inoculum volume and inoculated into MRS liquid culture medium with a pig bile salt concentration of 0.1%, 0.2%, and 0.3%, respectively. The culture medium was placed in an incubator and the OD was measured at 0h, 1h, 2h, and 3h. 600The experiment was repeated four times, and the survival rate of the strains was calculated.

[0126] (2) Test results: Reference Fig.13 , the survival rate of LM treated in 0.1% bile salt medium for 1 h was 99%, the survival rate after 2 h was 91%, and the survival rate after 3 h was 85%;

[0127] The survival rate of LM treated in 0.2% bile salt medium was 82% after 1 h, 70% after 2 h, and 62% after 3 h;

[0128] The survival rate of LM treated in 0.3% bile salt medium was 77% after 1 hour, 58% after 2 hours, and 48% after 3 hours.

[0129] refer to Fig.14 , the survival rate of LR treated in 0.1% bile salt medium for 1 h was 95%, the survival rate after 2 h was 84%, and the survival rate after 3 h was 75%;

[0130] The survival rate of LR treated in 0.2% bile salt medium was 77% after 1 h, 62% after 2 h, and 52% after 3 h;

[0131] The survival rate of LR treated in 0.3% bile salt medium was 71% after 1 hour, 53% after 2 hours, and 44% after 3 hours.

[0132] refer to Fig.15 , the survival rate of LS treated in 0.1% bile salt medium for 1 h was 90%, the survival rate after 2 h was 78%, and the survival rate after 3 h was 70%;

[0133] The survival rate of LS treated in 0.2% bile salt medium for 1 h was 76%, after 2 h the survival rate was 62%, and after 3 h the survival rate was 52%;

[0134] The survival rate of LS treated in 0.3% bile salt medium was 72% after 1 hour, 54% after 2 hours, and 46% after 3 hours.

[0135] Example 2

[0136] 2.1 Effects of strains on rat body weight and branched-chain amino acid metabolism

[0137] (1) Experimental materials and instruments

[0138] Lactobacillus mucosa (LM);

[0139] Lactobacillus reuteri (LR) (isolated from Ningxiang pig feces by our research group in the early stage, the deposit number is CCTCC NO: M20231546, the deposit date is August 31, 2023, the depository is China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, and the classification name is Limosilactobacillus reuteri XY227);

[0140] Lactobacillus salivarius (LS).

[0141] (2) Main biochemical reagents

[0142] Hydrochloric acid, n-hexane, sulfosalicylic acid

[0143] (3) Instruments and equipment

[0144] Amino acid analyzer, centrifuge

[0145] (4) Experimental design

[0146] Forty 7-week-old male SD rats (180-210 g) were purchased from Bo Ruixin Biotechnology Co., Ltd. (Hunan, China). After one week of adaptation, the rats were randomly assigned to four groups, with 10 replicates in each group. The control group was gavaged with sterile saline (NC), the mucosal Lactobacillus gavage group (LM, 10 9 CFU / mL), Lactobacillus reuteri gavage group (LR, 10 9 CFU / mL) and Lactobacillus salivarius gavage group (LS, 10 9 CFU / mL). Rats were housed in individually ventilated cages (IVCs) with a temperature controlled at 23-25°C and a light-dark cycle of 12 h. Rats were given free access to food and water. All rats received intragastric administration once a day with a dose of 1 mL each time for 8 consecutive weeks. All procedures were performed in accordance with the guidelines of the Animal Ethics Committee of Hunan Agricultural University (CACAHU 2022-1011).

[0147] 2.2 Effects of strains on body weight changes in rats

[0148] (1) Test method: The body weights of the rats in the above four groups were weighed and recorded every week until the end of the experiment.

[0149] (2) Test results: Reference Figure 16-18 There was no significant difference in the initial body weight of the four groups of rats; compared with the control group, the final body weight and body weight gain of the rats in the LR treatment group increased significantly (P < 0.05), while there was no significant difference in the LS or LM treatment groups. There was no significant difference in the feed intake among the four groups of rats during the whole experimental period.

[0150] It can be seen that LR can further improve the weight gain ability of rats under the condition of the same feed intake. Therefore, LR undoubtedly has a better application prospect in subsequent feed additives.

[0151] 2.3 Effect of strain (LR) on branched-chain amino acid content in rats

[0152] (1) Test method:

[0153] Serum: Pipette about 1 mL of serum into a centrifuge tube, centrifuge at 3000 rpm for 5 min, then pipette 500 μL of the supernatant into another centrifuge tube, add 500 μL of an aqueous solution of sulfosalicylic acid with a mass fraction of 8% (equal volume), mix well, and let stand for 15 min; centrifuge at 10000 rpm for 10 min, take the supernatant, filter through a 0.22 μM filter membrane, and place it on a high-speed amino acid analyzer (LA8080, HITACHI, Japan) for detection.

[0154] Ileal contents: Weigh about 100 mg of the crushed lyophilized sample of ileal contents, use 5 mL of 0.02N hydrochloric acid to homogenize, and ultrasonically extract for 30 minutes; transfer the supernatant to a 25 mL colorimetric tube by centrifugation, repeat the homogenization and extraction 2 to 3 times, combine the supernatant, and make up to volume. After shaking well, accurately pipette 2 mL of the filtrate into a centrifuge test tube, add 2 mL of n-hexane to defat (shake well and let stand, discard the upper layer after stratification, and take the lower layer); take 1 mL of the filtrate, add 1 mL of 8% sulfosalicylic acid aqueous solution, mix well, let stand overnight, at least 15 minutes; centrifuge at 10000 rpm for 10 minutes; take the supernatant, pass through a 0.22 μm filter membrane, and place it on a high-speed amino acid analyzer (LA8080, HITACHI, Japan) for detection.

[0155] (2) Test results: Reference Fig.19 , compared with the control group, the levels of leucine, isoleucine, valine and total branched-chain amino acids in the ileum contents of rats in the LR treatment group were significantly increased;

[0156] refer to Fig. 20 The levels of serum leucine, isoleucine, valine and total branched-chain amino acids were also significantly higher than those in the control group;

[0157] Further, in the scheme of Chinese patent application 202210646957.8 recorded above, Lactobacillus reuteri strain LN0214 has the effect of reducing serum branched-chain amino acid levels, however, experiments on rats in the present application found that Lactobacillus reuteri XY227 (LR) in the present application can increase serum leucine, isoleucine, valine and total branched-chain amino acid levels;

[0158] It can be seen that not all Lactobacillus reuteri have the same ability to increase branched-chain amino acids in serum.

[0159] Example 3 Effect of Lactobacillus reuteri on branched-chain amino acids in the digestive system of Duchang-Fattening Pigs

[0160] Construction method: 16 Duroc castrated boars weighing 60-65 kg in good health were randomly divided into two groups: 8 pigs in each group (single pen), and the Lactobacillus reuteri group was fed with a live Lactobacillus reuteri concentration of 1×10 11 CFU / kg of feed (Hunan Gold Medal Haotian Feed), and the control group was fed feed without any bacteria (Hunan Gold Medal Haotian Feed). The experimental pigs were free to eat and drink, and the pig house was regularly disinfected and ventilated.

[0161] (2) Test method: Serum: Pipette about 1 mL of serum into a centrifuge tube, centrifuge at 3000 rpm for 5 min, then pipette 500 μL of supernatant into another centrifuge tube, add 500 μL of 8% sulfosalicylic acid (equal volume), mix well, and let stand for 15 min; centrifuge at 10000 rpm for 10 min, take the supernatant, filter through a 0.22 μM filter membrane, and place on a high-speed amino acid analyzer (LA8080, HITACHI, Japan) for detection.

[0162] Ileal contents: Weigh about 100 mg of the crushed lyophilized sample of ileal contents, use 5 mL of 0.02N hydrochloric acid to homogenize, and ultrasonically extract for 30 minutes; transfer the supernatant to a 25 mL colorimetric tube by centrifugation, repeat the homogenization and extraction 2 to 3 times, combine the supernatant, and make up to volume. After shaking well, accurately pipette 2 mL of the filtrate into a centrifuge test tube, add 2 mL of n-hexane to defat (shake well and let stand, discard the upper layer after stratification, and take the lower layer); take 1 mL of the filtrate, add 1 mL of 8% sulfosalicylic acid, mix well, let stand overnight, at least 15 minutes; centrifuge at 10000 rpm for 10 minutes; take the supernatant, pass through a 0.22 μm filter membrane, and place it on a high-speed amino acid analyzer (LA8080, HITACHI, Japan) for detection.

[0163] Colon content: Weigh about 100 mg of pulverized colon content freeze-dried sample, use 5 mL of 0.02N hydrochloric acid to homogenize, ultrasonically extract for 30 minutes; transfer the supernatant to a 25 mL colorimetric tube by centrifugation, repeat the homogenization and extraction 2 to 3 times, combine the supernatant, and make up to volume. After shaking, accurately pipette 2 mL of the filtrate into a centrifuge test tube, add 2 mL of n-hexane to defat (shake well and let stand, discard the upper layer after stratification, and take the lower layer); take 1 mL of the filtrate, add 1 mL of 8% sulfosalicylic acid, mix well, let stand overnight, at least 15 minutes; centrifuge at 10000 rpm for 10 minutes; take the supernatant, pass through a 0.22 μm filter membrane, and place it on a high-speed amino acid analyzer (LA8080, HITACHI, Japan) for detection.

[0164] (3) Test results: Reference Figure 21-22 Compared with the control group, the levels of amino acids (BCAA), leucine (Leu), isoleucine (lle), and valine (Val) in the serum and ileum contents of fattening pigs in the Lactobacillus reuteri XY227 group were significantly increased. Fig.23 It can be seen that there was no significant difference in the levels of branched-chain amino acids (leucine, isoleucine, and valine) in colon contents.

[0165] It can be seen that the Lactobacillus reuteri XY227 in the present application can not only increase the amino acid level in pig serum, but also increase the amino acid level in pig ileum. The ileum is an important organ for absorbing amino acids in the digestive system. Undoubtedly, the amino acid content in pork will also increase accordingly.

[0166] Example 4 Effect of Lactobacillus reuteri on fat content and fatty acid composition in muscle of Duchang-Fattening Pigs

[0167] 4.1 Effect of Lactobacillus reuteri on fatty acid composition in muscle

[0168] (1) Test method: Use the freeze-dried muscle samples of two groups of Duroc fattening pigs obtained in Example 3, weigh about 0.5 g and place them in a 10 mL centrifuge tube, add 4 mL of a mixed solvent of benzene-petroleum ether (the volume ratio of benzene to petroleum ether is 1:1) to the tube, and then seal and extract for 24 hours. After the extraction, add 4 mL of potassium hydroxide-methanol solution (the molar concentration of gallium hydroxide is 0.4 mol / L) to the tube, shake on a vortex mixer for 3 minutes, and let it stand for 30 minutes. Add ultrapure water to the tube, centrifuge at 8000g for 5 minutes, take the upper solution, add an appropriate amount of anhydrous sodium sulfate to remove the water in the sample. Take 1 mL of the sample (if the content is too high, dilute it with n-hexane), pass it through a 0.22 μM organic filter membrane and then load it. Use a gas chromatograph (8890, Agilent, USA) to determine the fatty acid composition.

[0169] (2) Test results: Referring to Table 3, compared with the control group, supplementation with Lactobacillus reuteri XY227 can significantly increase the content of polyunsaturated fatty acids (PUFA), unsaturated fatty acids (UFA), n-3PUFA and n-6PUFA in the longissimus dorsi muscle of fattening pigs (P < 0.05), among which the contents of C10:0, C14:0, C18:2n6c, C22:0, C20:3n3 and C22:6n3 are all higher than those in the control group (P < 0.05). There is no significant difference in the content of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) between the two groups (P > 0.05).

[0170] Table 3

[0171]

[0172]

[0173] 4.2 Effect of Lactobacillus reuteri on intramuscular fat content in muscle

[0174] (1) Test method: Weigh 0.5 g (accurate to 0.0002 g) of the freeze-dried and crushed sample of the longissimus dorsi muscle on filter paper, fold it into a filter paper bag, mark the sample number with a pencil, place the fat bag in a 105°C oven to dry for 2 hours, put it in a desiccator to cool for 30 minutes, and then weigh it. Dry it again for 30 minutes, weigh it after cooling, and the difference between the two weighings must be less than 0.0008 g to ensure constant weighing. Place the fat bag after constant weight on a Soxhlet extractor for measurement. After the extraction is completed, place the fat bag in a fume hood to allow the residual petroleum ether to evaporate, then place it in a 105°C oven to dry for 2 hours, cool it in a desiccator for 30 minutes, and weigh it. Dry it again for 30 minutes, cool it and weigh it again, and the difference between the two weighings must be less than 0.001 g to ensure constant weighing.

[0175] (2) Test results: Reference Figure 24-25 , Lactobacillus reuteri XY227 significantly increased the intramuscular fat content of the longissimus dorsi muscle of fattening pigs;

[0176] It can be seen that Lactobacillus reuteri XY227 in the present application can not only increase the level of branched-chain amino acids in rat serum, but also significantly increase the amino acid levels in pig serum and ileum, and this effect is exactly the opposite of the effect of Lactobacillus reuteri strain LN0214 recorded in Chinese patent application 202210646957.8, which is to reduce the level of branched-chain amino acids in serum;

[0177] In this regard, Lactobacillus reuteri XY227 in this application exceeded our expectations;

[0178] At the same time, Lactobacillus reuteri XY227 not only has the above-mentioned effect of improving the amino acid level, but also, referring to the experimental content of Example 2, it can also improve the weight gain ability of rats that ingest this strain; and referring to the experimental content of Example 4, it can also increase the fat content in the muscles of pigs. It can be seen that Lactobacillus reuteri XY227 undoubtedly has huge application potential in animal husbandry.

Claims

1. A strain of Lactobacillus reuteri, characterized in that The preservation number of the Lactobacillus reuteri is CCTC C NO: M20231546, the preservation date is August 31, 2023, the preservation unit is China Center for Type Culture Collection, the preservation address is: Wuhan University, Wuhan, China, and the classification name is Limosilactobacillus reuteri XY227.

2. The Lactobacillus reuteri according to claim 1, characterized in that The 16SrRNA sequence of Lactobacillus reuteri is shown in SEQ ID NO:

1.

3. The Lactobacillus reuteri according to claim 1, characterized in that The Lactobacillus reuteri is isolated from Ningxiang pig feces.

4. Use of Lactobacillus reuteri as claimed in claim 1 for increasing the amino acid level in serum or ileum.

5. The use according to claim 4, characterized in that The amino acid is at least one of leucine, isoleucine and valine.

6. Use of Lactobacillus reuteri as claimed in claim 1 for increasing the intramuscular fat content of pigs.

7. Use of the Lactobacillus reuteri according to claim 1 in preparing feed.

8. A feed, characterized in that: Contains the Lactobacillus reuteri according to claim 1.

9. The feed according to claim 5, characterized in that The live bacterial concentration of Lactobacillus reuteri in feed is 0.8×10 11 ~1.2×10 11 CFU / kg.

Images