Lactobacillus amylophilus isolate LAM1345 and its uses

Abstract

The present invention relates to the Lactobacillus amylophilus isolate LAM1345 and its uses. The present invention discloses a Lactobacillus amylophilus isolate LAM1345, which is deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) under the deposit number DSM 33510. The present invention also discloses that the Lactobacillus amylophilus isolate LAM1345 can be used to inhibit the conversion of choline to TMA and to treat diseases associated with elevated levels of TMA and / or TMAO.

Description

Technical Field

[0001] The present invention relates to an isolate of Lactobacillus amylovorus LAM1345, which is deposited under the accession number BCRC 910996 at the Biosource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI), a non-profit organization, and under the accession number DSM 33510 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). The Lactobacillus amylovorus LAM1345 isolate can be used to inhibit the conversion of choline to TMA and to treat diseases associated with elevated levels of TMA and / or TMAO. Background Art

[0002] Trimethylamine-N-oxide (TMAO) [chemical formula: (CH3)3NO] is an organic metabolite belonging to the amine oxide class. In recent years, reports have indicated that the accumulation of TMAO in the human body is one of the main factors triggering many diseases or accelerating their progression. When excessive choline is ingested, choline is metabolically converted to trimethylamine (TMA) in the gastrointestinal tract. After the generated TMA is transported to the liver via the blood, it is oxidized to TMAO by hepatic flavin monooxygenase 3 (FMO3), followed by a series of inflammatory reactions [e.g., macrophage accumulation in the blood vessel wall and promotion of platelet aggregation, etc.], which in turn lead to various diseases, including kidney diseases, diabetes mellitus, obesity, trimethylaminuria (also known as fish odor syndrome), and cardiovascular diseases, etc. These diseases have been collectively referred to as "diseases associated with elevated TMA and / or TMAO levels".

[0003] Previous studies have pointed out that in addition to the existing treatment methods for the above diseases, reducing the levels of TMA and TMAO in patients' blood can also be used as an effective alternative. Currently known drugs that can be used to reduce the levels of TMA and TMAO include 3,3-dimethyl-1-butanol (DMB), acetylsalicylic acid, FMO3 inhibitors, TMA lyase inhibitors, antibiotics, antimicrobials, antiplatelet agents, and sequestering agents for TMA and / or TMAO. However, the efficacy achieved by these drugs is still not ideal, and the main reasons include: individual differences in patients themselves, serious side effects and adverse effects of the drugs on patients. Therefore, how to effectively reduce the levels of TMA and / or TMAO without producing unwanted side effects has become the focus of attention and research in the current medical field.

[0004] Lactic acid bacteria (LAB) are a group of Gram-positive bacteria that can ferment sugars and produce lactic acid as the main metabolite. They are commonly found in dairy products, pickled products, and the intestinal mucosa of humans or animals. Lactic acid bacteria are generally recognized as safe (GRAS) and are familiar and widely used probiotics. They have been found to have the effects of inhibiting the growth of gastrointestinal pathogens, alleviating lactose intolerance, immunoregulation, anti-cancer, and reducing blood pressure. There are many types of lactic acid bacteria that can be used as probiotics, such as Lactobacillus, Lactococcus, Pediococcus, Streptococcus, and Enterococcus.

[0005] Previous studies have shown that some lactic acid bacteria strains have the effect of reducing the content of TMAO in the serum of mice. For example, in Qiu L. et al. (2018), Food Funct., 9:4299 - 4309, Qiu L. et al. administered Lactobacillus plantarum (also known as Lactobacillus plantarum) ZDY01 and ZDY04, Lactobacillus rhamnosus ZDY9, Lactobacillus casei ZDY8, and Lactobacillus bulgaricus ZDY5 to BALB / c mice respectively, and by measuring the content of TMAO in the serum of these mice, it was found that only Lactobacillus plantarum ZDY04 could effectively reduce the content of TMAO in the serum of mice. In addition, Qiu L. et al. used C57BL / 6J ApoE - / - mice as a mouse model with atherosclerosis and further administered Lactobacillus plantarum ZDY04 to it. Then, by measuring the content of TMAO in the serum of this mouse and observing the situation of atherosclerotic lesion formation, it was found that Lactobacillus plantarum ZDY04 could reduce the content of TMAO in the serum of mice with atherosclerosis and improve its atherosclerotic lesions, and thus was expected to be used to treat atherosclerosis and prevent atherosclerotic cardiovascular diseases.

[0006] Although there have been the above-mentioned literature reports, there is still a need in the art to screen out microorganisms that can reduce the levels of TMA and TMAO in the blood of patients for industrial use. Summary of the Invention

[0007] Thus, in a first aspect, the present invention provides Lactobacillus amylovorus LAM1345, which is deposited at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) under the deposit number BCRC 910996, and at the German Collection of Microorganisms and Cell Cultures (DSMZ) under the deposit number DSM 33510.

[0008] In a second aspect, the present invention provides the use of Lactobacillus amylovorus LAM1345 as described above for the preparation of a composition for treating diseases associated with an increase in the level of TMA and / or TMAO.

[0009] In a third aspect, the present invention provides a method for treating an individual having or suspected of having a disease associated with an elevated level of TMA and / or TMAO, which comprises administering to the individual Lactobacillus amylovorus LAM1345 as described above.

[0010] In a fourth aspect, the present invention provides the use of Lactobacillus amylovorus LAM1345 as described above for the preparation of a composition for inhibiting the conversion of choline to TMA.

[0011] In a fifth aspect, the present invention provides a method for inhibiting the conversion of choline to TMA in an individual, which comprises administering to the individual Lactobacillus amylovorus LAM1345 as described above.

[0012] For the use according to the present invention, the composition further comprises probiotics selected from the group consisting of Lactobacillus fermentum, Lactobacillus plantarum, and combinations thereof.

[0013] For the use according to the present invention, the composition is a pharmaceutical composition.

[0014] For the use according to the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0015] For the use according to the present invention, the pharmaceutical composition is in a dosage form for oral administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will be described in detail below in conjunction with the drawings and examples. Therefore, the above and other objects and features of the present invention will become more apparent by referring to the following description, the appended claims, and the accompanying drawings. In the drawings:

[0017] Figure 1 A phylogenetic tree drawn based on the alignment results of the lepA gene sequence of Lactobacillus isolate LAM1345 according to the present invention with the standard strains of Lactobacillus species in the NCBI gene database and the lepA gene sequences of existing Lactobacillus amylovorus isolates;

[0018] Figure 2 Showing the TMAO concentrations in the sera measured in each group of mice at the end of the 7th day after starting gavage with lactic acid bacteria, where the control group represents the mice gavaged with PBS; Comparative experimental group 1 represents the mice gavaged with the bacterial solution of Lactobacillus fermentum BCRC910896; Comparative experimental group 2 represents the mice gavaged with the bacterial solution of Lactobacillus plantarum BCRC 910897; and the experimental group represents the mice gavaged with the bacterial solution of Lactobacillus amylovorus LAM1345 BCRC 910996;

[0019] Figure 3 Shown is the TMA concentration in the serum measured in each group of mice at the end of day 7 after starting the gavage of lactic acid bacteria, where the control group represents mice gavaged with PBS; the single strain group represents mice gavaged with a bacterial solution of Lactobacillus amylovorus LAM1345 BCRC 910996; and the mixed strain group represents mice gavaged with a mixture containing 3 lactic acid bacteria strains; and

[0020] Figure 4 Shown is the TMAO concentration in the serum measured in each group of mice at the end of day 7 after starting the gavage of lactic acid bacteria, where the control group represents mice gavaged with PBS; the single strain group represents mice gavaged with a bacterial solution of Lactobacillus amylovorus LAM1345 BCRC 910996; the mixed strain group represents mice gavaged with a mixture containing 3 lactic acid bacteria strains; and "**" indicates p < 0.01 when compared with the control group. Detailed implementation mode

[0021] For the purposes of this specification, it will be clearly understood that the term "comprising" means "including but not limited to", and the term "comprises" has a corresponding meaning.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used to implement the present invention. Of course, the present invention is in no way limited to the methods and materials described.

[0023] The applicant isolated 1 lactic acid bacteria isolate from corn and was classified as Lactobacillus amylovorus through characterization. It was named "Lactobacillus amylovorus LAM1345" by the applicant and was deposited on April 23, 2020, with the deposit number BCRC 910996 at the Bioresource Collection and Research Center (BCRC of FIRDI) of the Food Industry Research and Development Institute, a corporation. This isolate was also deposited on April 27, 2020, with the deposit number DSM 33510 at the German Collection of Microorganisms and Cell Cultures (DSMZ) in accordance with the provisions of the Budapest Treaty.

[0024] The Lactobacillus amylophilus LAM1345 of the present invention has been proven through animal experiments to be able to effectively inhibit the conversion of choline into trimethylamine (TMA) and inhibit the formation of trimethylamine - N - oxide (TMAO). Based on the above - mentioned beneficial biological activities, the applicant believes that the Lactobacillus amylophilus LAM1345 of the present invention has a high potential for development into a treatment for diseases associated with elevated levels of TMA and / or TMAO.

[0025] Therefore, the present invention provides the use of the Lactobacillus amylophilus LAM1345 as described above for preparing a composition for treating diseases associated with elevated levels of TMA and / or TMAO.

[0026] As used herein, "treating" or "treatment" means preventing, reducing, alleviating, ameliorating, relieving, or controlling one or more clinical signs of a disease or disorder, and lowering, stopping, or reversing the progression of the severity of the condition or symptom being treated.

[0027] According to the present invention, the diseases associated with elevated levels of TMA and / or TMAO may include, but are not limited to: kidney diseases, diabetes mellitus, obesity, trimethylaminuria (also known as fish odor syndrome), cardiovascular diseases, and cancer.

[0028] The present invention also provides the use of the Lactobacillus amylophilus LAM1345 as described above for preparing a composition for inhibiting the conversion of choline into TMA.

[0029] According to the present invention, the inhibition of choline conversion to TMA may not require complete elimination of TMA production, and the degree of elimination only needs to be sufficient to achieve at least one of the following therapeutic effects on a disease associated with an increase in the level of TMA and / or TMAO: reducing the severity and / or duration of the disease, improving one or more symptoms of the disease, preventing the development, recurrence, onset, or progression of the disease, and enhancing or improving the efficacy of existing therapies for the disease, etc. For example, reducing TMA generated via choline metabolism by at least 1% to 100%. In a preferred embodiment of the present invention, TMA generated via choline metabolism is reduced by at least 30%.

[0030] According to the present invention, Lactobacillus amylovorus LAM1345 can be live or dead, concentrated or non-concentrated, liquid, paste, semi-solid, or solid [e.g., pellet, granule, or powder], and can be heat-inactivated, frozen, dried, or freeze-dried [e.g., in freeze-dried form or spray / fluid bed dried form]. In a preferred embodiment of the present invention, Lactobacillus amylovorus LAM1345 exists in liquid form.

[0031] According to the present invention, the composition may further comprise probiotics selected from the group consisting of Lactobacillus fermentum, Lactobacillus plantarum (also known as Lactobacillus plantarum), and combinations thereof. In a preferred embodiment of the present invention, the composition further comprises Lactobacillus fermentum BCRC 910896 and Lactobacillus plantarum BCRC 910897.

[0032] As used herein, the terms "probiotic" and "probiotic microbes" may be used interchangeably and refer to preparations of live microorganisms that, when ingested by humans or animals, can remain and survive in the gastrointestinal tract and can exert desired effects.

[0033] According to the present invention, the composition may be a pharmaceutical composition.

[0034] According to the present invention, the pharmaceutical composition can be manufactured into a dosage form suitable for oral administration by techniques well known to those skilled in the art, including, but not limited to: sterile powders, tablets, troches, lozenges, pellets, capsules, dispersible powders or granules, solutions, suspensions, emulsions, drops, syrups, elixirs, slurries, and the like.

[0035] According to the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier which is widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier may comprise one or more reagents selected from the following: solvent, buffer, emulsifier, suspending agent, decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome, and the like. The selection and amount of these reagents fall within the professional competence and routine techniques of those skilled in the art.

[0036] As used herein, the term "subject" means any mammalian animal of interest, such as humans, monkeys, cows, sheeps, horses, pigs, goats, dogs, cats, mice, and rats.

[0037] The present invention also provides a method for treating a subject having or suspected of having a disease associated with an elevated level of TMA and / or TMAO, which comprises administering Lactobacillus amylovorus LAM1345 as described above to the subject.

[0038] The present invention also provides a method for inhibiting the conversion of choline to TMA in a subject, which comprises administering Lactobacillus amylovorus LAM1345 as described above to the subject.

[0039] According to the present invention, the dosage and frequency of administration of Lactobacillus amylovorus LAM1345 will vary depending on the following factors: the severity of the disease to be treated, the route of administration, and the age, physical condition, and response of the subject to be treated. Generally, the composition may be administered orally or parenterally in a single dose or in divided doses.

[0040] The present invention will be further described with reference to the following embodiments. It should be understood, however, that the embodiments are for illustrative purposes only and should not be construed as limitations on the implementation of the present invention.

[0041] <Examples>

[0042] General experimental materials:

[0043] 1. Experimental animals:

[0044] The female C57BL / 6J mice (5 weeks old, weighing approximately 23 ± 2 g) used in the following examples were purchased from the Experimental Animal Center. All experimental animals were individually housed in an animal room with 12 hours of light and 12 hours of darkness, room temperature maintained at 23 ± 2 °C, and relative humidity maintained at 50 - 70%, and water and a choline-deficient diet (TD88052, Harlan Teklad, Madison, WI, USA) were provided ad libitum. All experimental procedures involving experimental animals complied with the regulations regarding animal protection in Taiwan, China, and were conducted in accordance with the guidelines of a certain institution in Taiwan, China.

[0045] Example 1. Screening and characterization of Lactobacillus isolate LAM1345

[0046] A. Source and isolation of Lactobacillus isolate LAM1345:

[0047] The applicant used corn (purchased from a traditional market in Taiwan, China) as the sample source and used Difco Lactobacilli MRS Agar to isolate and screen for Lactobacillus, and obtained 1 Lactobacillus isolate LAM1345.

[0048] To confirm the species to which Lactobacillus isolate LAM1345 belongs, Lactobacillus isolate LAM1345 was subjected to the following analyses in items B to D.

[0049] B. Preliminary tests:

[0050] Preliminary tests were conducted on the Lactobacillus isolate LAM1345. The test items included: Gram staining, morphological observation, motility, catalase reaction, growth under aerobic and anaerobic conditions, whether endospores were formed, and acid and bile salt tolerance assays, etc.

[0051] Based on the results of the preliminary tests, the Lactobacillus isolate LAM1345 was a Gram-positive bacillus, non-motile, without catalase activity, could grow under anaerobic conditions, did not form endospores, and had a survival rate of over 60% after being cultured in phosphate buffered saline (PBS) with a pH of 2 - 3 for 1 hour, and still had a survival rate as high as 93% after being cultured in MRS Broth (Difco, USA) containing 0.3% (v / v) bovine bile for 16 hours. Therefore, it was determined to have good acid and bile salt tolerance.

[0052] C. 16S rDNA sequence analysis:

[0053] First, the genomic DNA of the Lactobacillus isolate LAM1345 was extracted using a Genomic DNA Mini Kit (manufactured by Geneaid, product number GB100 / GB300). Then, using the obtained genomic DNA as a template, a pair of primers, forward primer F1 and reverse primer R1, designed for the 16S rDNA gene of bacteria and having the nucleotide sequences shown below, were used for polymerase chain reaction (PCR) to amplify the 16S rDNA fragment of the Lactobacillus isolate LAM1345. The reaction conditions for PCR are shown in Table 1 below.

[0054] Forward primer F1

[0055] 5’-agagtttgatcctggctcag-3’ (Sequence Identification Number: 1)

[0056] Reverse primer R1

[0057] 5’-ggttaccttgttacgact-3’ (SEQ ID NO: 2)

[0058] Table 1. Reaction conditions for PCR

[0059]

[0060] After completion of PCR, 2% agarose gel electrophoresis was used to confirm whether a PCR amplification product of approximately 1,500 bp in size was obtained, and the confirmed PCR product was recovered and purified from the gel.

[0061] After that, Genomics Biosci & Tech Co., Ltd was commissioned to perform sequencing analysis, and the 16S rDNA sequence of Lactobacillus isolate LAM1345 (SEQ ID NO: 3) was obtained. After comparison with the gene database on the NCBI website, it was found that the 16S rDNA sequence of Lactobacillus isolate LAM1345 had 99.9% similarity with a partial 16S rDNA sequence of Lactobacillus amylovorus DSM 20531 [Genbank accession number: AY944408.1].[[]]

[0062] D. Analysis of the leader peptidase A (lepA) gene sequence:

[0063] Using the genomic DNA obtained in item C above as a template, and using a pair of primers, forward primer F2 and reverse primer R2, which were designed for the bacterial lepA gene and had the nucleotide sequences shown below, to perform PCR. The reaction conditions for PCR are shown in Table 2 below.

[0064] Forward primer F2

[0065] 5’-ggdcacgtrgayttytcwtayga-3’ (SEQ ID NO: 4)

[0066] Reverse primer R2

[0067] 5’-gcatavccyttngtdrawgactt-3’ (SEQ ID NO: 5)

[0068] Table 2. Reaction conditions for PCR

[0069]

[0070] After completing PCR, 2% agarose gel electrophoresis was used to confirm whether a PCR amplification product of approximately 1,163 bp in size was obtained, and the confirmed PCR product was recovered and purified from the gel.

[0071] After that, Genomics BioSci & Tech Co., Ltd. was commissioned to perform sequencing analysis, and the lepA gene sequence of Lactobacillus isolate LAM1345 (Sequence Identification Number: 6) was obtained. After comparison with the gene database on the NCBI website, it was found that the lepA gene sequence of Lactobacillus isolate LAM1345 had 99.6% similarity with that of Lactobacillus amylovorus DSM 20531 T (at nucleotide residue positions 29946 to 30954 shown in the nucleotide sequence of Genbank accession number AZCM01000018.1).

[0072] E. Phylogenic analysis:

[0073] Regarding the phylogenic analysis, it was generally carried out with reference to Ramachandran P. et al. (2013), Appl. Environ. Microbiol., 79: 7220 - 7228. Briefly, the lepA gene sequence of this Lactobacillus isolate LAM1345 (Sequence Identification Number: 6) was compared with the reference strains of Lactobacillus spp. in the gene database on the NCBI website and the lepA gene sequences of existing Lactobacillus amylovorus isolates (including LAM1010 T , LAM0178, LAM1116, LAM1249, and LAM1250), and then a phylogenetic tree was constructed using the molecular evolutionary genetics analysis software version 7.0 (MEGA 7.0) (Pennsylvania State University, USA) by the Neighbor-joining method and the Kimura 2-parameter method. The results obtained are shown in Figure 1 Figure.

[0074] As can be seen from Figure 1 Figure, this Lactobacillus isolate LAM1345 has a relatively close genetic relationship with Lactobacillus amylovorus DSM 20531T and existing Lactobacillus amylovorus isolates.

[0075] Based on the experimental results of items B to E above, the Lactobacillus isolate LAM1345 of the present invention was preliminarily identified as Lactobacillus amylovorus. In order to confirm whether Lactobacillus amylovorus LAM1345 is a novel Lactobacillus amylovorus isolate, Lactobacillus amylovorus LAM1345 was further subjected to the following analyses in items F to H.

[0076] F. Repetitive extragenic palindromic-polymerase chain reaction (rep-PCR):

[0077] Regarding rep-PCR, it was generally carried out with reference to Rademaker J.L. and de bruijn F.J. (1997), DNA markers: protocols, applications and overviews, p. 151-171 and Rasschaert G. et al. (2005), J. Clin. Microbiol., 43: 3615-3623. Briefly, using the genomic DNA obtained in item C above as a template and primers ERIC1, ERIC2, BOXA, and GTG with the nucleotide sequences shown in Table 3 below, which are designed respectively for the enterobacterial repetitive intergenic consensus (ERIC) sequence, BOX sequence, and polytrinucleotide (GTG)5 sequence of bacteria, a PCR reaction was carried out using the reaction conditions shown in Table 4 below.

[0078] Table 3. Primer pairs used for rep-PCR

[0079]

[0080] Table 4. Reaction conditions for PCR

[0081]

[0082] After the PCR was completed, the obtained PCR amplification products were electrophoresed on a 2% agarose gel, then stained, and then observed and photographed under ultraviolet light, thereby obtaining the gene fingerprint map of the PCR products of Lactobacillus amylovorus LAM1345 of the present invention.

[0083] In addition, a genomic DNA Mini kit was used to extract the genomic DNA of the existing Lactobacillus amylovorus isolates LAM1010 T , LAM0178, LAM1116, LAM1249, and LAM1250, and PCR reactions and electrophoresis were performed according to the method described above to obtain the gene fingerprint maps of the PCR products of the 5 existing Lactobacillus amylovorus isolates.

[0084] After that, the gene fingerprint map of the PCR product of Lactobacillus amylovorus LAM1345 of the present invention was compared and analyzed with the gene fingerprint maps of the PCR products of the 5 existing Lactobacillus amylovorus isolates.

[0085] G. Random Amplified Polymorphic DNA (RAPD) analysis:

[0086] RAPD was generally carried out with reference to Akopyanz N. et al. (1992), Nucleic Acids Res., 20: 5137-5142. Briefly, using the genomic DNA obtained in item C above as a template and selecting 4 10-mer primers RAPD-A, RAPD-B, RAPD-C, and RAPD-1 with the nucleotide sequences shown in Table 5 below, PCR reactions were carried out under the reaction conditions shown in Table 6 below.

[0087] Table 5. Primer pairs used for RAPD

[0088]

[0089] Table 6. PCR reaction conditions

[0090]

[0091] After the completion of PCR, the obtained PCR amplification products were electrophoresed on a 2% agarose gel, then stained, and then observed and photographed under ultraviolet light to obtain the gene fingerprint map of the PCR product of Lactobacillus amylovorus LAM1345 of the present invention.

[0092] In addition, using the genomic DNA of the existing Lactobacillus amylovorus isolates LAM1010 T , LAM0178, LAM1116, LAM1249, and LAM1250 obtained in item F above as templates, and PCR reactions and electrophoresis were carried out according to the method described above to obtain the gene fingerprint maps of the PCR products of the 5 existing Lactobacillus amylovorus isolates.

[0093] Afterwards, the gene fingerprint patterns of the PCR products of Lactobacillus amylophilus LAM1345 of the present invention were compared and analyzed with the gene fingerprint patterns of the PCR products of 5 existing Lactobacillus amylophilus isolates.

[0094] H. Single nucleotide polymorphism (SNP) analysis:

[0095] Using the genomic DNAs of the existing Lactobacillus amylophilus isolates LAM1010 T , LAM0178, LAM1116, LAM1249, and LAM1250 obtained in item F above as templates, and according to the method described in item D above, the lepA gene sequences of these 5 Lactobacillus amylophilus isolates were analyzed, whereby the lepA gene sequences of these 5 Lactobacillus amylophilus isolates were obtained.

[0096] Then, the MEGA 7.0 software was used to perform multiple sequence alignment analysis on the lepA gene sequence of Lactobacillus amylophilus LAM1345 of the present invention obtained in item D above and the lepA gene sequences of the above 5 existing Lactobacillus amylophilus isolates to detect and identify the single nucleotide polymorphism (SNP) of the lepA gene. Afterwards, the genotyping of Lactobacillus amylophilus isolates was carried out by analyzing the genetic variation of SNPs located in the lepA gene among these 6 Lactobacillus amylophilus isolates.

[0097] The experimental results of items F to H above were summarized in Table 7 below. As can be seen from Table 7, combining these 3 genotyping methods, these 6 Lactobacillus amylophilus isolates were identified as 6 isolates with different genotypes.

[0098] Table 7. Genotypes identified by gene fingerprint pattern analysis of rep-PCR and RAPD and SNP analysis of the lepA gene

[0099]

[0100] Note 1: Different letters or symbols in each row represent different genotypes identified by the corresponding method.

[0101] Note 2: The lepA SNP analysis identified at least 7 SNP loci.

[0102] Based on the above results of characteristic identification, the applicant believes that: Lactobacillus amylovorus LAM1345 of the present invention is a novel Lactobacillus amylovorus isolate, which was deposited on April 23, 2020 under the deposit number BCRC 910996 at the Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute (FIRDI), No. 331, Shilin Road, Hsinchu City 300, Taiwan, China, and also deposited on April 27, 2020 under the deposit number DSM 33510 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ).

[0103] Example 2. Effect of Lactobacillus amylovorus LAM1345 BCRC 910996 on the production of trimethylamine-n-oxide (TMAO)

[0104] Experimental materials:

[0105] 1. The lactic acid bacteria strains used in this example have been deposited at the Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute (FIRDI), No. 331, Shilin Road, Hsinchu City 300, Taiwan, China and the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), respectively. For clarity, the relevant information of each lactic acid bacteria strain (including: deposit number and deposit date) has been integrated in Table 8 below.

[0106] Table 8. Deposit numbers and deposit dates of each lactic acid bacteria strain

[0107]

[0108]

[0109] 2. Preparation of the bacterial suspensions of each lactic acid bacterium:

[0110] The Lactobacillus amylovorus LAM1345 BCRC 910996 obtained in Example 1 above, the Lactobacillus fermentum BCRC 910896 of Item 1 in the above "Experimental Materials", and the Lactobacillus plantarum BCRC 910897 were respectively inoculated into MRS Broth (Difco, USA) and cultured at 37°C for 8 - 10 hours. After that, each lactic acid bacteria culture was centrifuged at 12,000 rpm at 4°C for 30 minutes, then the supernatant was discarded, and the pellets were washed with an appropriate amount of PBS, and then suspended with an appropriate amount of PBS and adjusted to a concentration of 10 8 -10 10 CFU / mL (the number of bacteria was counted using a plate count medium).

[0111] Experimental method:

[0112] A. Oral gavage of lactic acid bacteria strains:

[0113] First, the mice that had been fed a choline-deficient diet for 1 week in Item 1 of the above "General Experimental Materials" were randomly divided into 1 control group (n = 9), 2 comparative experimental groups (i.e., Comparative Experimental Groups 1 and 2), and 1 experimental group (n = 7 per group), and the supplied diet was changed to a choline diet containing 1% choline (TD14017, Harlan Teklad, Madison, WI, USA). At the same time, the mice in Comparative Experimental Groups 1 and 2 were respectively orally gavaged with the bacterial solutions of Lactobacillus fermentum BCRC 910896 and Lactobacillus plantarum BCRC 910897 (dose: 200 - 250 μL, 10 8 -10 10 CFU / mL), and the mice in the experimental group were orally gavaged with the bacterial solution of Lactobacillus amylovorus LAM1345 BCRC 910996 (dose: 200 - 250 μL, 10 8 -10 10 CFU / mL). As for the mice in the control group, they were orally gavaged with an equal volume of PBS. Each group of mice was orally gavaged once a day for a total of 7 days.

[0114] B. Preparation of serum samples:

[0115] On the 7th day after starting the gavage of lactic acid bacteria, before feeding, capillary tubes were used to collect blood from the orbital position of each group of mice. Subsequently, the obtained blood was left standing at room temperature for 2 hours to allow it to clot. Then, centrifugation was carried out at 2,000 rpm for 10 minutes at 4°C, and then filtered through a filter membrane with a pore size of 0.2 μm. The resulting serum samples were collected for the analysis in item C below.

[0116] Determination of C. TMAO concentration:

[0117] For each serum sample of each group of mice, 25 μL was taken and 10 μL of internal standard (1 ppm d9-TMAO, Cat No. T795792, Toronto Research Chemicals, Canada) was added. Subsequently, 30 μL of derivatization reagent [20 mg / mL ethyl bromoacetate] [prepared in acetonitrile] was added. Then, 1 μL of 26% ammonia solution was added and mixed evenly. Then, the derivatization reaction was carried out in a light-shielded environment at room temperature for 30 minutes. Then, 1 mL of 50% acetonitrile [prepared in 0.025% formic acid solution] was added to terminate the reaction. The TMAO concentration contained in the resulting mixtures of each group was determined by using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The UPLC analytical instrument used was the ACQUITY UPLC system (Waters). The operating parameters and conditions for UPLC analysis are shown in Table 9 below.

[0118] Table 9. Operating parameters and conditions for UPLC analysis

[0119]

[0120] Tandem mass spectrometry was carried out using Waters TQ-MS tandem quadrupole mass spectrometer (Waters TQ-MS tandem quadrupole mass spectrometer) (Milford, MA, USA).

[0121] In addition, for comparison, different concentrations of TMAO (1 - 6 ppm, dissolved in 50% acetonitrile) (purchased from Toronto Research Chemicals, Canada) were used as control standards and the same analysis was performed.

[0122] Afterwards, the experimental data of each group were expressed as "mean ± standard deviation (SD) of the mean". All data were analyzed by one - way analysis of variance (one - way ANOVA) and independent sample t - test to evaluate the differences between groups. If the obtained analysis result was p < 0.05, it represented statistical significance.

[0123] Results:

[0124] Figure 2 It shows the TMAO concentration in the serum measured in each group of mice at the end of the 7th day after starting gavage with lactic acid bacteria. From Figure 2 It can be seen that compared with the control group, the TMAO concentration in each comparison experimental group only showed a slightly decreased situation, while in the experimental group, it showed an obvious decrease, and the decrease amplitude could reach 28%. This experimental result shows that Lactobacillus amylophilus LAM1345 BCRC 910996 of the present invention can effectively and rapidly reduce the TMAO level.

[0125] Example 3. Effect of the combined use of Lactobacillus amylophilus LAM1345 BCRC 910996 and other lactic acid bacteria strains on the production of trimethylamine (TMA) and TMAO

[0126] Experimental materials:

[0127] 1. Preparation of a mixture containing 3 lactic acid bacteria strains:

[0128] The bacterial suspensions of each lactic acid bacterium obtained in item 2 of the "experimental materials" of Example 2 above, including the bacterial suspensions of Lactobacillus amylophilus LAM1345 BCRC 910996, Lactobacillus fermentum BCRC 910896, and Lactobacillus plantarum BCRC 910897, were mixed evenly at a ratio of 1:1:1 (v / v / v) to obtain a bacterial concentration of 10 8 -10 10A mixture containing 3 lactic acid bacteria strains at CFU / mL.

[0129] Experimental method:

[0130] A. Gavage of the mixture containing 3 lactic acid bacteria strains and preparation of serum samples:

[0131] First, the mice that had been fed a choline-deficient diet for 1 week in item 1 of the above "General experimental materials" were randomly divided into 1 control group, 1 single-strain group, and 1 mixed-strain group (n = 5 in each group), and the supplied diet was changed to a choline diet containing 1% choline. At the same time, the mice in the single-strain group were gavaged with a bacterial solution of Lactobacillus amylovorus LAM1345 BCRC910996 (dose: 200 - 250 μL, 10 8 -10 10 CFU / mL), while the mice in the mixed-strain group were gavaged with a mixture containing 3 lactic acid bacteria strains (dose: 200 - 250 μL, 10 8 -10 10 CFU / mL). As for the mice in the control group, they were gavaged with an equal volume of PBS. The mice in each group were gavaged once a day for a total of 7 days. Then, on the 7th day after starting the gavage of lactic acid bacteria, before feeding, the serum samples were prepared according to the method described in item B of the above Example 2, and thus the serum samples of the mice in each group were obtained and used for the analysis in item B below.

[0132] B. Determination of TMA and TMAO concentrations:

[0133] The determination of the TMAO concentration was carried out according to the method described in item C of the above Example 2. The determination of the TMA concentration was generally carried out in reference to the determination of the TMAO concentration, with the difference being that the internal standard in the sample to be measured was 1 ppm d9-TMA (Cat No. T795807, Toronto Research Chemicals, Canada), and the control standard was TMA (purchased from Toronto Research Chemicals, Canada).

[0134] Results:

[0135] Figure 3 and Figure 4 respectively show the TMA concentration and TMAO concentration in the serum measured in the mice in each group at the end of the 7th day after starting the gavage of lactic acid bacteria. From Figure 3 and Figure 4It can be seen that, compared with the control group, the TMA concentration and the TMAO concentration in the single-strain group both showed a significant decrease, and the mixed-strain group had a further decrease. The experimental results show that the use of Lactobacillus amylovorus LAM1345 BCRC 910996 of the present invention alone and its combined use with other lactic acid bacteria strains can effectively reduce the TMA level and the TMAO level.

[0136] Based on the above experimental results, the applicant believes that Lactobacillus amylovorus LAM1345 BCRC 910996 of the present invention can effectively inhibit the conversion of choline into TMA, and thus inhibit the formation of TMAO. Accordingly, Lactobacillus amylovorus LAM1345 BCRC 910996 of the present invention is expected to be used to treat diseases associated with elevated TMA and / or TMAO levels, and can be further used in combination with other probiotics with desired activities.

[0137] All patents and documents cited in this specification are incorporated herein by reference in their entirety. In case of any conflict, the detailed description of this application (including definitions) shall prevail.

[0138] Although the present invention has been described with reference to the above specific examples, it is obvious that many modifications and variations can be made without departing from the scope and spirit of the present invention. Therefore, it is intended that the present invention be limited only by what is shown in the appended claims.

[0139] Description of Biological Material Deposit Information

[0140] Deposit number: DSM 33510

[0141] Taxonomic name: Lactobacillus amylovorus LAM1345

[0142] Deposit date: April 27, 2020

[0143] Depositary institution: German Collection of Microorganisms and Cell Cultures

[0144] Address of the depositary institution: DSMZ - German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D - 38124 Braunschweig, Germany

[0145] Deposit number: DSM 33124

[0146] Taxonomic name: Lactobacillus fermentum LF1143

[0147] Date of deposit: May 16, 2019

[0148] Depositary institution: German Collection of Microorganisms and Cell Cultures GmbH (DSMZ)

[0149] Address of depositary institution: Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig, Germany

[0150] Accession number: DSM 33125

[0151] Taxonomic designation: Lactobacillus plantarum LP1145

[0152] Date of deposit: May 16, 2019

[0153] Depositary institution: German Collection of Microorganisms and Cell Cultures GmbH (DSMZ)

[0154] Address of depositary institution: Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig, Germany Sequence Listing <110> SYNBIO TECHNOLOGY CO., LTD. <120> Lactobacillus amylovorus LAM1345 Isolate and Uses Thereof <160> 14 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer F1 for amplifying 16S rDNA <400> 1 agagtttgat cctggctcag 20 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer R1 for amplifying 16S rDNA <400> 2 ggttaccttg ttacgact 18 <210> 3 <211> 1525 <212> DNA <213> Lactobacillus amylovorus <400> 3 tcaggacgaa cgctggcggc gtgcctaata catgcaagtc gagcgagcgg aaccaacaga 60 tttacttcgg taatgacgtt gggaaagcga gcggcggatg ggtgagtaac acgtggggaa 120 cctgccccta agtctgggat accatttgga aacaggtgct aataccggat aataaagcag 180 atcgcatgat cagcttttga aaggcggcgt aagctgtcgc taagggatgg ccccgcggtg 240 cattagctag ttggtaaggt aacggcttac caaggcgacg atgcatagcc gagttgagag 300 actgatcggc cacattggga ctgagacacg gcccaaactc ctacgggagg cagcagtagg 360 gaatcttcca caatggacgc aagtctgatg gagcaacgcc gcgtgagtga agaaggtttt 420 cggatcgtaa agctctgttg ttggtgaaga aggatagagg tagtaactgg cctttatttg 480 acggtaatca accagaaagt cacggctaac tacgtgccag cagccgcggt aatacgtagg 540 tggcaagcgt tgtccggatt tattgggcgt aaagcgagcg caggcggaaa aataagtcta 600 atgtgaaagc cctcggctta accgaggaac tgcatcggaa actgtttttc ttgagtgcag 660 aagaggagag tggaactcca tgtgtagcgg tggaatgcgt agatatatgg aagaacacca 720 gtggcgaagg cggctctctg gtctgcaact gacgctgagg ctcgaaagca tgggtagcga 780 acaggattag ataccctggt agtccatgcc gtaaacgatg agtgctaagt gttgggaggt 840 ttccgcctct cagtgctgca gctaacgcat taagcactcc gcctggggag tacgaccgca 900 aggttgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat 960 tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcta gtgcaatctg tagagatacg 1020 gagttccctt cggggacgct aagacaggtg gtgcatggct gtcgtcagct cgtgtcgtga 1080 gatgttgggt taagtcccgc aacgagcgca acccttgtta ttagttgcca gcattaagtt 1140 gggcactcta atgagactgc cggtgacaaa ccggaggaag gtggggatga cgtcaagtca 1200 tcatgcccct tatgacctgg gctacacacg tgctacaatg ggcagtacaa cgagaagcaa 1260 gcctgcgaag gcaagcgaat ctctgaaagc tgttctcagt tcggactgca gtctgcaact 1320 cgactgcacg aagctggaat cgctagtaat cgcggatcag cacgccgcgg tgaatacgtt 1380 cccgggcctt gtacacaccg cccgtcacac catgggagtc tgcaatgccc aaagccggtg 1440 gcctaacctt cgggaaggag ccgtctaagg cagggcagat gactggggtg aagtcgtaac 1500 aaggtagccg taggagaacc tgcgg 1525 <210> 4 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Forward primer F2 for amplifying lepA gene <220> <221> misc_feature <222> (3)..(3) <223> d represents a, g or t <220> <221> misc_feature <222> (9)..(9) <223> r represents g or a <220> <221> misc_feature <222> (12)..(12) <223> y represents t or c <220> <221> misc_feature <222> (15)..(15) <223> y represents t or c <220> <221> misc_feature <222> (18)..(18) <223> w represents a or t <220> <221> misc_feature <222> (21)..(21) <223> y represents t or c <400> 4 ggdcacgtrg ayttytcwta yga 23 <210> 5 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Reverse primer R2 for amplifying lepA gene <220> <221> misc_feature <222> (6)..(6) <223> v represents a, g or c <220> <221> misc_feature <222> (9)..(9) <223> y represents t or c <220> <221> misc_feature <222> (12)..(12) <223> n represents a, g, c or t <220> <221> misc_feature <222> (15)..(15) <223> d represents a, g or t <220> <221> misc_feature <222> (16)..(16) <223> r represents g or a <220> <221> misc_feature <222> (18)..(18) <223> w represents a or t <400> 5 gcatavccyt tngtdrawga ctt 23 <210> 6 <211> 1009 <212> DNA <213> Lactobacillus amylophilus <400> 6 cacaaggtgt gcaagctcag actttggcta acacttactt agcgatcgat gatgatttgg 60 caattttacc tgtaattaat aagatcgact tgccatctgc tgatattcct aagactaagg 120 aagaaattga agagatgctt ggtcttgatg cttctgaagc tgcggaagtt tctggtaaaa 180 ccggtcaagg catcaaggac atgctggaaa aagtagtaaa agacattcca gctccatctg 240 gtgatattac tgcgccactc aaggctttga tttttgactc aaaatatgat gactatcgtg 300 gtgtcgtaat gtcagtcaaa atcgaagacg gtacagttaa gcctggtgac cgagttcaaa 360 ttatgaatac tggtaaggaa tatgaagtta cagaagtagg tgtttcaagt ccacatccaa 420 ttaagaagga tatcttgatt gctggggatg taggttatat tactgccaac attaagtccg 480 tacgtgaaac tcgtgttggt gatactatca cccaagcaga taatcctact gccgaaccac 540 ttccaggtta ccgtcaaatt ccaccaatgg tttactctgg tatgtatcca gttgataacc 600 gtgattatga agacttaaag gaagctttgc aaaagttgca attaaacgat gcagctttag 660 aatttgaacc tgaaacttct actgctttag gcttcgggtt ccgttgtggt ttcttaggac 720 ttttgcatat ggatgttgtg caagaacgac tagaacaaga atttgatctt gatttaatta 780 tgactgcacc atccgttgac tatcatgcaa ttatgaatga tggctcaact aaggtaattg 840 ataacccatc agatttgcca gatgctggtg aatacaagga agtgcaagag ccttatgtta 900 aggcagaaat tatggtgcca aatgactttg ttggccctgt aatggaactt tgtcaaagaa 960 aacgtggcga atttgttacg atggactatc ttgataagta ccgcgttaa 1009 <210> 7 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer ERIC1 for rep-PCR <400> 7 atgtaagctc ctggggattc ac 22 <210> 8 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer ERIC2 for rep-PCR <400> 8 aagtaagtga ctggggtgag cg 22 <210> 9 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer BOXA for rep-PCR <400> 9 ctacggcaag gcgacgctga cg 22 <210> 10 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Primer GTG for rep-PCR <400> 10 gtggtggtgg tggtg 15 <210> 11 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Primer RAPD-A for RAPD <400> 11 ccgcagccaa 10 <210> 12 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Primer RAPD-B for RAPD <400> 12 aacgcgcaac 10 <210> 13 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Primer RAPD-C for RAPD <400> 13 gcggaaatag 10 <210> 14 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Primer RAPD-1 for RAPD <400> 14 ctcaggtcgc 10

Claims

1. A Lactobacillus amylovorus LAM1345, characterized in that: The Lactobacillus amylophilus LAM1345 was deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) under the accession number DSM 33510.

Images