Methods of using symbiotic compositions to resist salmonella typhimurium infection

Abstract

The present invention provides a method for producing a recombinant bacterium, which is characterized in that the recombinant bacterium is used for producing a recombinant bacterium, and the recombinant bacterium is obtained by using a recombinant bacterium, which is preserved in the Deersche Sammlarg von Mikroorgani sung Zel lkulturen and has been preserved in the Deersche Sammlarg von Mikroorgani sung Zel lkulturen with a preservation number of DSM 32786. The present invention relates to a method for resisting Salmonella typhimurium infection by using a culture of Lactobacillus rhamnosus LRH10 having a preservation number of DSMZ GmbH, Lactobacillus paracasei LPC12 having a preservation number of DSMZ GmbH, Lactobacillus fermentum LF26 having a preservation number of DSMZ GmbH, Streptococcus thermophilus ST30 having a preservation number of DSMZ 32788, and Lactobacillus helveticus LH43 having a preservation number of DSMZ 32787, and more specifically, to a method for resisting Salmonella typhimurium infection by using a culture of Lactobacillus rhamnosus LRH10 having a preservation number of DSMZ GmbH.

Description

Technical Field

[0001] This invention relates to a method for treating Salmonella typhimurium infection using a composition containing cultures of five lactic acid bacteria strains. Background Technology

[0002] Salmonella typhimurium is a Gram-negative bacterium that colonizes the intestines of animals (such as pigs or humans) via Peyer's patches, particularly the ileum, cecum, and spiral colon. Salmonella typhimurium infection [also known as salmonellosis] can occur through ingestion of contaminated food sources or exposure to contaminated environments (including veterinary clinics). The main symptoms of salmonellosis include fever, loss of appetite, diarrhea, vomiting, and abdominal pain. Salmonella typhimurium most commonly causes gastroenteritis in humans.

[0003] Salmonella infection is typically treated with antibiotics such as apramycin, ceftiofur, trimethoprim-sulfamethoxazole (TMP-SMX), and gentamicin. However, these antibiotics can lead to antibiotic resistance in Salmonella typhimurium and may cause serious side effects and adverse reactions.

[0004] Probiotics are normal flora that reside in the gut and are believed to play an important role in regulating proper intestinal immunity and digestion by balancing the intestinal microflora. These beneficial microorganisms are widely used in dietary supplements containing live microorganisms to help restore the balance of the gut microbiota. Many species of lactic acid bacteria (LAB) are generally recognized as safe (GRAS) and are widely used as probiotics. Common LAB species include: Lactobacillus spp., Lactococcus spp., Pediococcus spp., Streptococcus spp., Enterococcus spp., Bifidobacterium spp., Bacillus spp., and Leuconostoc spp., etc.

[0005] Previous studies have shown that certain strains of LAB are effective against Salmonella typhimurium infection. For example, Streptococcus thermophilus St-21, Lactobacillus helveticus SP-27, and Lactobacillus rhamnosus GG have all been shown to inhibit the growth of Salmonella typhimurium, thereby achieving anti-Salmonella typhimurium infection efficacy (J. Piatek1 et al. (2019), Beneficial Microbes, 10(2):211-217 and BingjieDuan et al. (2021), Microb. Pathog., 156:104939).

[0006] Despite the above, there remains a need to develop a new strategy that can effectively combat Salmonella typhimurium infection. Summary of the Invention

[0007] Therefore, the object of the present invention is to provide a method for combating Salmonella typhimurium infection, which can reduce at least one disadvantage of the prior art.

[0008] The method involves administering to individuals in need of the following bacteria: *Lactobacillus rhamnosus* LRH10 (accession number DSM 32786, deposited at the German Microbiological Collection Center (DSMZ) GmbH), *Lactobacillus paracasei* LPC12 (accession number DSM 32785, deposited at DSMZ GmbH), *Lactobacillus fermentum* LF26 (accession number DSM32784, deposited at DSMZ GmbH), *Streptococcus thermophilus* ST30 (accession number DSM 32788, deposited at DSMZ GmbH), and *Streptococcus thermophilus* ST30 (accession number DSM 32787, deposited at DSMZ GmbH). A composition of cultures of Lactobacillus shelveticus LH43 from GmbH. Attached Figure Description

[0009] Other features and advantages of the present invention will become apparent from the following detailed description and specific examples and the accompanying drawings, wherein:

[0010] Figure 1 The diameters of the inhibition zones measured in each group in Example 1 below are shown, where the symbol “*” indicates p<0.05 (compared with single-cell-LAB supernatant CG1 to CG5 and BCG). Detailed Implementation

[0011] It should be understood that if any prior publication is cited herein, such prior publication does not constitute an admission that it forms part of the common general knowledge in the field in China or any other country.

[0012] For the purposes of this instruction manual, it will be clearly understood that the word "comprising" means "including but not limited to", and the word "comprises" has the corresponding meaning.

[0013] 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 similar or equivalent methods and materials that can be used to practice the invention. Of course, the invention is by no means limited to the methods and materials described.

[0014] This invention provides a method for treating Salmonella typhimurium infection, comprising administering to an individual in need a composition comprising a culture of Lactobacillus rhamnosus LRH10 deposited at the German Microbiological Collection Center (DSMZ) GmbH (accession number DSM 32786), Lactobacillus paracasei LPC12 deposited at DSMZ GmbH (accession number DSM 32785), Lactobacillus fermentum LF26 deposited at DSMZ GmbH (accession number DSM 32784), Streptococcus thermophilus ST30 deposited at DSMZ GmbH (accession number DSM 32788), and Lactobacillus helveticus LH43 deposited at DSMZ GmbH (accession number DSM 32787).

[0015] As used herein, the terms “against Salmonella typhimurium infection” or “anti-Salmonella typhimurium infection” mean prevention of infection with Salmonella typhimurium, inhibition of Salmonella typhimurium replication, and / or treatment and / or prevention of infectious diseases caused by Salmonella typhimurium.

[0016] As used herein, the terms “administering” and “administration” mean introducing, providing, or delivering to an individual, by any suitable means, the composition described above to perform its intended utility.

[0017] As used herein, the term "subject" refers to any mammal of interest, such as humans, monkeys, hamsters, cattle, sheep, horses, pigs, goats, dogs, cats, mice, and rats. In some specific instances, the subject is a human.

[0018] According to the present invention, cultures of various lactic acid bacteria strains can be prepared by culturing the lactic acid bacteria strains as described above in a liquid or solid culture medium suitable for growth and / or proliferation.

[0019] In some specific examples, cultures of individual lactic acid bacteria strains can be prepared by mixing the lactic acid bacteria strains as described above at 10... 5 Up to 10 9 The lactic acid bacteria strain is prepared by culturing in a suitable liquid or solid medium at a concentration of CFU / mL. In an illustrative example, the strain is prepared at 10... 6 Up to 10 8 The CFU / mL concentration is used to culture the organism in suitable liquid or solid culture media.

[0020] As used in this article, the term “culturing” may be used interchangeably with other terms such as “fermentation” and “cultivation”.

[0021] The procedures and conditions for culturing lactic acid bacteria strains can be adjusted according to actual requirements. In this regard, those skilled in the art may refer to journal articles, for example, Kimoto-Nira H. et al. (2012), J Dairy Sci., 95(4):2176-2185. doi:10.3168 / jds.2011-4824.

[0022] In some specific cases, a suitable liquid culture medium for culturing lactic acid bacteria strains is 10% skim milk.

[0023] In some specific cases, the culture of this lactic acid bacteria strain is a liquid culture.

[0024] In certain specific examples, the bacterial count ratio of *Lactobacillus rhamnosus* LRH10, *Lactobacillus paracasei* LPC12, *Lactobacillus fermentum* LF26, *Streptococcus thermophilus* ST30, and *Lactobacillus helveticus* LH43 in this composition falls between 5.1:29.7:1.9:47.8:1.2 and 10.9:41.3:3.4:53.2:

[0025] Within the range of 5.7.

[0026] In an exemplary specific example, the composition contains Lactobacillus rhamnosus LRH10, Lactobacillus paracasei LPC12, Lactobacillus fermentum LF26, Streptococcus thermophilus ST30, and Lactobacillus helveticus LH43 in a ratio of 7.8:35.5:2.6:50.4:3.3.

[0027] According to the present invention, the liquid culture may be substantially free of bacterial cells.

[0028] As used herein, the term "substantially free of" means that the liquid culture lacks a meaningful amount of a specifically identified component (e.g., lactic acid bacteria cells). In some specific examples, the amount of lactic acid bacteria cells has no measurable effect on the properties of the liquid culture. In other specific examples, the liquid culture contains no cells at all.

[0029] According to the present invention, the liquid culture that is substantially free of bacteria is obtained by performing separation treatment on the culture formed after culturing lactic acid bacteria strains to remove the bacteria therein.

[0030] According to the present invention, the separation process can be performed using techniques well known to those skilled in the art. Examples of the separation process may include, but are not limited to, filtration, centrifugation (such as multi-stage centrifugation), concentration, and combinations thereof.

[0031] In an illustrative specific example, the liquid culture, which is essentially devoid of bacteria, is obtained by centrifuging a culture formed after culturing a strain of lactic acid bacteria.

[0032] According to the present invention, the composition can be formulated into a food product using standard techniques known to those skilled in the art. For example, the composition can be added directly to edible materials or used to prepare intermediate compositions suitable for subsequent addition to edible materials [e.g., food additives or premixes].

[0033] As used herein, the term "food product" means any article or substance that may be ingested by an individual. Examples of food products may include, but are not limited to: milk powder, fermented milk, yogurt, cheese, beverages (e.g., tea, coffee, etc.), functional beverages, pasta products, baked goods, desserts, confectionery, fermented foods, animal feed, health foods, baby food, and dietary supplements.

[0034] According to the present invention, the composition can be prepared in the form of a pharmaceutical composition (e.g., a medicine). This pharmaceutical composition can be manufactured into a dosage form suitable for oral, parenteral, or topical administration using techniques well known to those skilled in the art.

[0035] According to the present invention, dosage forms suitable for oral administration include, but are not limited to: sterile powders, lozenges, tablets, oral tablets, pills, capsules, dispersible powders or fine particles, solutions, suspensions, emulsions, syrups, elixirs, concentrates, drops, and the like.

[0036] Regarding non-enteric administration, the pharmaceutical compositions according to the present invention can be formulated into injectable products, for example, sterile aqueous solutions or dispersions.

[0037] The pharmaceutical composition according to the present invention can be administered via one of the following non-enteric routes: intraperitoneal injection, intrapleural injection, intramuscular injection, intravenous injection, intraarterial injection, intraarticular injection, intrasynovial injection, intrathecal injection, intracranial injection, intraepidermal injection, subcutaneous injection, intradermal injection, intralesional injection, and sublingual administration. In some specific examples, the pharmaceutical composition can be administered via intravenous injection.

[0038] According to the present invention, the pharmaceutical composition can be manufactured into a topical preparation suitable for application to the skin using techniques known to those skilled in the art. The topical preparation includes, but is not limited to, emulsions, gels, ointments, creams, patches, liniments, powders, aerosols, sprays, lotions, emulsions, pastes, foams, drops, suspensions, ointments, and bandages.

[0039] The pharmaceutical compositions according to the present invention may further comprise a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing techniques. For example, such a pharmaceutically acceptable carrier may comprise one or more agents selected from the following: solvents, buffers, emulsifiers, suspending agents, disintegrants, disintegrants, dispersants, binders, excipients, stabilizers, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption delay agents, liposomes, and the like. The selection and quantity of these agents fall within the scope of expertise and practice of those skilled in the art.

[0040] The dosage and frequency of administration of the composition according to the present invention will vary depending on the following factors: the severity of the disease or condition to be improved, the route of administration, and the age, physical condition, and response of the individual to be improved. Generally, the composition may be administered in a single dose or in multiple doses.

[0041] Detailed Description of Preferred Embodiments

[0042] The present invention will be further described with reference to the following embodiments. However, it should be understood that the following embodiments are for illustrative purposes only and should not be construed as limiting the implementation of the present invention.

[0043] Example

[0044] General experimental materials:

[0045] 1. Lactic acid bacteria (LAB) strains:

[0046] Five strains of lactic acid bacteria, namely Lactobacillus rhamnosus LRH10, Lactobacillus paracasei LPC12, Lactobacillus fermentum LF26, Streptococcus thermophilus ST30, and Lactobacillus helveticus LH43 (all of which have been disclosed in US2020 / 0008437 A1 and are publicly available), have been deposited in accordance with the provisions of the Budapest Treaty at the German Microbiological Collection Center (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ) GmbH (Inhoffenstraβe 7B, 38124 Braunschweig, Germany).

[0047] The relevant information for each LAB strain (including accession number and accession date) has been compiled in Table 1 below.

[0048] Table 1

[0049] LAB strain Collection Number Preservation date Lactobacillus rhamnosus LRH10 DSM 32786 2018 / 04 / 03 Lactobacillus paracasei LPC12 DSM 32785 2018 / 04 / 03 Lactobacillus fermentum LF26 DSM 32784 2018 / 04 / 03 Streptococcus thermophilus ST30 DSM 32788 2018 / 04 / 03 Lactobacillus helveticus LH43 DSM 32787 2018 / 04 / 03

[0050] 2. The Salmonella typhimurium used in the following experiment was isolated from the intestines of chickens by the applicant.

[0051] General experimental methods:

[0052] 1. Statistical analysis:

[0053] All experiments described below were repeated three times. Data from all experimental groups are expressed as mean ± standard deviation (SD) and were analyzed using one-way analysis of variance (ANOVA) followed by the Tukey-Kramer test to assess differences between groups. Statistical significance was defined as p < 0.05.

[0054] Example 1. Evaluation of the efficacy of liquid culture of the LAB strain according to the present invention against Salmonella typhimurium:

[0055] Experimental methods:

[0056] Preparation of cell culture supernatant for strain A.LAB:

[0057] The *Lactobacillus rhamnosus* LRH10, *Lactobacillus paracasei* LPC12, *Lactobacillus fermentum* LF26, and *Lactobacillus helveticus* LH43, as described in item 1 of the "General Experimental Materials" section above, were each inoculated into Difco. TM Lactobacilli MRS (DeMan, Rogosa and Sharpe) broth (Cat. No. 288130, BD Difco), and Streptococcus thermophilus ST30 as described in item 1 of "General Experimental Materials" above, inoculated into M17 broth. The five LAB strains were cultured under aerobic conditions in an incubator (37°C) for 16 hours to obtain their respective inoculum.

[0058] Next, each inoculum source was inoculated into 10% skim milk at an inoculation rate of 2% (v / v) and then cultured aerobically in an incubator (37°C) for 16 hours to obtain single-cell-LAB liquid cultures of each LAB strain. In addition, *Lactobacillus rhamnosus* LRH10, *Lactobacillus paracasei* LPC12, *Lactobacillus fermentum* LF26, *Streptococcus thermophilus* ST30, and *Lactobacillus helveticus* LH43 were inoculated into 10% skim milk at a volume ratio of 1:1:1:1:1 [inoculation rate of each inoculum source: 0.4% (v / v)]. The cultures were then incubated under aerobic conditions in an incubator (37°C) for 16 hours to obtain a five-strain-LAB liquid culture. The bacterial counts of each LAB strain in the five-strain-LAB liquid culture were counted using techniques well known to those skilled in the art. The results showed that the bacterial count ratio of *Lactobacillus rhamnosus* LRH10, *Lactobacillus paracasei* LPC12, *Lactobacillus fermentum* LF26, *Streptococcus thermophilus* ST30, and *Lactobacillus helveticus* LH43 in the five-strain-LAB liquid culture was 7.8:35.5:2.6:50.4:3.3.

[0059] Subsequently, the five single-strain LAB liquid cultures and the five-strain LAB liquid cultures were centrifuged at 5,000 rpm for 10 minutes, and the resulting cell culture supernatants were collected for use in the following experiments.

[0060] B. Determination of antibacterial activity against Salmonella typhimurium:

[0061] Five cell culture supernatants obtained from five single-strain LAB liquid cultures as described in Section A above were designated as single-strain LAB supernatant groups, namely, single-strain LAB supernatant comparison groups 1 to 5 (abbreviated as single-strain LAB supernatants CG1 to CG5, as shown in Table 2 below). Furthermore, cell culture supernatants obtained from five-strain LAB liquid cultures as described in Section A above were designated as five-strain LAB supernatant groups, namely, five-strain LAB supernatant experimental group 1 (abbreviated as five-strain LAB supernatant EG1, as shown in Table 2 below). Additionally, 10% skim milk was used as a blank control group (abbreviated as BCG).

[0062] Table 2

[0063]

[0064] The *Salmonella typhimurium* described in item 2 of the "General Experimental Materials" section above was inoculated into tryptic soy broth (TSB) medium. The cells were then cultured in an incubator (37°C) under aerobic conditions for 16 hours.

[0065] After centrifugation at 5,000 rpm for 10 minutes at room temperature, the resulting cell pellet was collected. This cell pellet was then diluted with 5 mL of Dulbecco's phosphate-buffered saline (DPBS), pH 7.39 (Thermo Scientific). TM The bacteria were washed twice and then dispersed using DPBS to obtain a bacterial concentration of 10. 9 A bacterial suspension of CFU / mL was prepared. This suspension was then mixed with an appropriate amount of 0.85% sterile physiological saline to obtain a bacterial concentration of 10. 7 CFU / mL bacterial suspension (with dilution factor 10) 2 (To prepare).

[0066] Next, 100 μL of bacterial suspension was added to a TSB agar plate, followed by the addition of sterile glass beads. The TSB agar plate was then shaken to ensure even distribution of the bacterial suspension on the TSB agar medium. The glass beads were then poured out, and seven wells were created in the TSB agar medium using a sterile pipette tip (9.25 mm). Subsequently, 100 μL each of single-crystal LAB supernatant CG1 to CG5, five-crystal LAB supernatant EG1, and BCG were added to different wells of the TSB agar medium, and the medium was then incubated aerobically in an incubator (37°C) for 16 hours.

[0067] The diameter of the inhibition zone in each group was determined using techniques well known to those skilled in the art. The resulting data were analyzed according to the method described in section 1 of the "General Experimental Methods" above.

[0068] result:

[0069] Figure 1 Display the diameter of the inhibition zone measured for each group. For example... Figure 1 As shown, the diameter of the inhibition zone measured in the five-strain LAB supernatant EG1 is larger than that of the single-strain LAB supernatant CG1 to CG5.

[0070] The results of this experiment show that the cell culture supernatant obtained by co-cultivation of Lactobacillus rhamnosus LRH10, Lactobacillus paracasei LPC12, Lactobacillus fermentum LF26, Streptococcus thermophilus ST30 and Lactobacillus helveticus LH43 exhibits satisfactory efficacy in inhibiting the growth of Salmonella typhimurium.

[0071] While the invention has been described with reference to specific examples which are considered exemplary, it should be understood that this disclosure is not limited to the disclosed specific examples, but is intended to cover various different configurations included in the spirit and scope of the broadest interpretation, to include all such modifications and equivalent configurations.

Claims

1. A method for preventing infection with Salmonella typhimurium, comprising administering to an individual in need thereof a composition comprising a culture of Lactobacillus rhamnosus LRH10 deposited with the German Collection of Microorganisms (DSMZ) GmbH under the deposit number DSM 32786, Lactobacillus paracasei LPC 12 deposited with the DSMZ GmbH under the deposit number DSM 32785, Lactobacillus fermentum LF26 deposited with the DSMZ GmbH under the deposit number DSM 32784, Streptococcus thermophilus ST30 deposited with the DSMZ GmbH under the deposit number DSM 32788, and Lactobacillus helveticus LH43 deposited with the DSMZ GmbH under the deposit number DSM 32787.

2. The method of claim 1, wherein each of the cultures is a liquid culture.

3. The method according to claim 2, wherein in the composition, the bacterial count ratio of Lactobacillus rhamnosus LRH10, Lactobacillus paracasei LPC12, Lactobacillus fermentum LF26, Streptococcus thermophilus ST30 and Lactobacillus helveticus LH43 falls within the range of 5.1:29.7:1.9:47.8:1.2 to 10.9:41.3:3.4:53.2:5.

7.

4. The method according to claim 2, wherein the liquid culture contains substantially no bacterial cells.

5. The method of claim 1, wherein the composition is formulated as a food product.

6. The method of claim 1, wherein the composition is formulated as a pharmaceutical composition.

7. The method of claim 6, wherein the pharmaceutical composition is in a dosage form selected from the group consisting of an oral dosage form, a parenteral dosage form, and a topical dosage form.

8. A composition for use in preparing a medicament or food product for use in combating Salmonella typhimurium infection in an individual, wherein the composition comprises cultures of Lactobacillus rhamnosus LRH10 deposited at the German Collection of Microorganisms (DSMZ) GmbH under the accession number DSM 32786, Lactobacillus paracasei LPC12 deposited at DSMZ GmbH under the accession number DSM 32785, Lactobacillus fermentum LF26 deposited at DSMZ GmbH under the accession number DSM 32784, Streptococcus thermophilus ST30 deposited at DSMZ GmbH under the accession number DSM 32788, and Lactobacillus helveticus LH43 deposited at DSMZ GmbH under the accession number DSM 32787.

9. The use according to claim 8, wherein each of the cultures is a liquid culture.

10. The use according to claim 9, wherein in the composition, the bacterial count ratio of Lactobacillus rhamnosus LRH10, Lactobacillus paracasei LPC12, Lactobacillus fermentum LF26, Streptococcus thermophilus ST30 and Lactobacillus helveticus LH43 falls within the range of 5.1:29.7:1.9:47.8:1.2 to 10.9:41.3:3.4:53.2:5.

7.

11. The use according to claim 9, wherein the liquid culture contains substantially no bacterial cells.

12. The use according to claim 8, wherein the medicament is in a dosage form selected from the group consisting of an oral dosage form, a parenteral dosage form, and a topical dosage form.

13. A composition for resisting infection with Salmonella typhimurium, comprising cultures of Lactobacillus rhamnosus LRH10 deposited in the German Collection of Microorganisms (DSMZ) GmbH under the deposit number DSM 32786, Lactobacillus paracasei LPC 12 deposited in the DSMZ GmbH under the deposit number DSM 32785, Lactobacillus fermentum LF26 deposited in the DSMZ GmbH under the deposit number DSM 32784, Streptococcus thermophilus ST30 deposited in the DSMZ GmbH under the deposit number DSM 32788, and Lactobacillus helveticus LH43 deposited in the DSMZ GmbH under the deposit number DSM 32787.

14. The composition of claim 13, wherein each of the cultures is a liquid culture.

15. The composition according to claim 14, wherein in the composition, the bacterial count ratio of Lactobacillus rhamnosus LRH10, Lactobacillus paracasei LPC 12, Lactobacillus fermentum LF26, Streptococcus thermophilus ST30 and Lactobacillus helveticus LH43 falls within the range of 5.1:29.7:1.9:47.8:1.2 to 10.9:41.3:3.4:53.2:5.

7. The composition according to claim 14 , wherein the liquid culture contains substantially no bacterial cells.

17. The composition of claim 13 formulated as a food product.

18. The composition according to claim 13, which is formulated as a pharmaceutical composition.

19. The composition of claim 18, wherein the pharmaceutical composition is in a dosage form selected from the group consisting of an oral dosage form, a parenteral dosage form, and a topical dosage form.

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