Bacterial strains, methods of using the same, foods or beverages containing the strains, and methods of selecting the strains

Abstract

To achieve a strain having both of antibacterial action and adhesion to the gastrointestinal tract.SOLUTION: A strain according to the present invention has antibacterial activity and mucin adhesion, the strain belonging to any species of Latilactobacillus sakei, Lactiplantibacillus paraplantarum, and Latilactobacillus curvatus.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to bacterial strains, methods for their use, foods or beverages containing the strains, and methods for selecting the strains. [Background technology]

[0002] Lactic acid bacteria play an important role in the production of dairy products such as yogurt and pickles, and lactic acid bacteria that produce bacteriocins (peptides with antibacterial properties) are also used in biopreservation for food preservation. Furthermore, lactic acid bacteria have recently become known as representative probiotics (live microorganisms that, when ingested in appropriate amounts, have beneficial effects on the host). One of the mechanisms by which probiotics regulate the intestinal health is the inhibition of the growth of pathogenic bacteria in the intestines by the antibacterial substances produced by probiotics. Numerous reports have been published on lactic acid bacteria strains with antibacterial properties; for example, Patent Document 1 discloses a lactic acid bacteria strain that produces antibacterial substances.

[0003] Furthermore, the ability to adhere to the digestive tract is also considered an important property for the use of lactic acid bacteria as probiotics, and is one of the indicators for selecting probiotics. For example, adhesion to the digestive mucosa improves the ability of lactic acid bacteria to colonize the digestive tract. Furthermore, infections with Helicobacter pylori and Salmonella are major problems, and the adhesion of these pathogenic bacteria to digestive epithelial cells may be inhibited by competition with lactic acid bacteria that have the ability to adhere to the digestive tract. Many reports have been published on the ability of lactic acid bacteria to adhere to digestive mucin (viscous glycoprotein); for example, Patent Document 2 discloses lactic acid bacteria that have the ability to adhere to mucin.

[0004] However, most previous reports have focused on the selection of lactic acid bacteria with either antibacterial or gastrointestinal adhesive properties. Therefore, when using them as probiotics, it has sometimes been necessary to mix multiple strains to achieve effectiveness. When using multiple lactic acid bacteria in combination, the growth rate of each strain and the influence of metabolic products may inhibit the growth of one strain, necessitating consideration of the mixed culture conditions. Under these conditions, there is a need for lactic acid bacteria strains that combine both functions to reduce the labor required for mixed culture and to utilize lactic acid bacteria efficiently. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2005-192553 [Patent Document 2] International Publication No. 2008 / 001676 Summary of the Invention [Problem to be solved by the invention]

[0006] One aspect of the present invention aims to achieve a strain that has both antibacterial activity and gut-adherence. [Means for solving the problem]

[0007] In order to solve the above problems, one embodiment of the present invention provides a bacterial strain belonging to any of the species Latilactobacillus sakei, Lactiplantibacillus paraplantarum, and Latilactobacillus curvatus, which has antibacterial activity and mucin adhesion at temperatures between 3°C and 40°C.

[0008] In order to solve the above-mentioned problems, one embodiment of the present invention provides a method for selecting a bacterial strain, which is a method for selecting, from bacterial strains derived from food, a bacterial strain belonging to any one of the species Latilactobacillus sakei, Lactiplantibacillus paraplantarum, and Latilactobacillus curvatus, which has antibacterial activity, mucin adhesion, salt tolerance, and gastrointestinal stress resistance. [Effects of the Invention]

[0009] According to one aspect of the present invention, a bacterial strain can be realized that has both antibacterial activity and the ability to adhere to the gastrointestinal tract. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows the effect of salt concentration on the mucin adhesion of selected lactic acid bacteria when cultured at 30°C. [Figure 2] FIG. 2 shows the results of measuring the mucin adhesion of selected lactic acid bacteria when cultured at 37°C. [Figure 3] FIG. 3 shows the results of measuring the mucin adhesion of selected lactic acid bacteria when cultured at 25°C. [Figure 4] FIG. 4 shows the results of measuring the mucin adhesion of selected lactic acid bacteria when cultured at 5°C. DETAILED DESCRIPTION OF THE INVENTION

[0011] [Strain] One embodiment of the present invention will be described in detail below. The bacterial strain according to this embodiment is a bacterial strain having antibacterial activity and mucin adhesion ability. Furthermore, the bacterial strain according to this embodiment is a strain belonging to any of the species Latilactobacillus sakei (hereinafter referred to as L. sakei), Lactiplantibacillus paraplantarum (hereinafter referred to as L. paraplantarum), and Latilactobacillus curvatus (hereinafter referred to as L. curvatus).

[0012] As a result of extensive research, the present inventors have discovered that strains belonging to the species L. sakei, L. paraplantarum, and L. curvatus possess antibacterial activity and mucin adhesion. These bacterial species have rarely been used as probiotics.

[0013] To date, most research and development of probiotics has focused on lactic acid bacteria derived from the human intestinal tract, such as Lactobacillus gasseri, or on starter bacteria (starters) used in the production of yogurt and cheese, which are the mainstream probiotic-containing foods. Therefore, strains such as L. sakei, which are rarely isolated from the human intestinal tract and have poor fermentation properties in milk, making them unsuitable as starters for dairy products, have not been evaluated as probiotics. Furthermore, the main sources of isolation of L. sakei and other bacterial species are fermented sausages and pickles, but these foods are often produced by spontaneous fermentation without the use of a starter, and development of starter strains for these foods has been slow. For this reason, there has been little knowledge about the potential use of L. sakei and other bacterial species as probiotics.

[0014] For example, JP-A-2007-502858 discloses lactic acid bacteria species that have antibacterial activity and the ability to adhere to mucin, but does not disclose any of L. sakei, L. paraplantarum, or L. curvatus.

[0015] Furthermore, Martin et al. (2009) J Dairy Res. 76:418-425 investigated the antibacterial spectrum of L. paraplantarum CRB7, but did not investigate its antibacterial activity against Bacillus species. Furthermore, CRB7 is an animal-derived lactic acid bacterium, and its antibacterial activity and mucin adhesion were investigated only at its optimal incubation temperature of 37°C. Therefore, it is unclear whether it retains its antibacterial activity at different incubation temperatures. Furthermore, no investigations have been conducted into the optimal incubation conditions for these functionalities, taking into account the conditions or environments in which lactic acid bacteria are used (e.g., the production and storage of pickles and fermented meat products; low-temperature environments, high-salt environments, etc.). Therefore, CRB7 has been a strain that exhibits antibacterial activity in food production, but presents challenges for its use as a probiotic in animals.

[0016] The bacterial strain according to this embodiment exhibits antibacterial activity over a wide temperature range, from 3°C to 40°C, and therefore the culture temperature is not limited. Furthermore, as will be described later, the bacterial strain according to this embodiment has antibacterial activity against the genus Bacillus and the like, and therefore can be suitably used as a useful microorganism in food production and storage, and as a probiotic that is effective in the intestines of a subject. In this specification, unless otherwise specified, the term "subject" refers to a subject that has been administered or ingested a bacterial strain or an extract thereof.

[0017] In this embodiment, "antibacterial activity" refers to the growth inhibitory effect on harmful bacteria that cause food poisoning (hereinafter referred to as "food poisoning bacteria"), food spoilage bacteria, and / or pathogenic bacteria. Therefore, when a bacterial strain has antibacterial activity, it is possible to improve the intestinal environment of a subject and prevent food spoilage and food poisoning.

[0018] Food poisoning bacteria and / or food spoilage bacteria are not particularly limited, but examples include Bacillus coagulans, Bacillus subtilis, Bacillus circulans, Listeria monocytogenesis, and Staphylococcus aureus.

[0019] Pathogens include, but are not limited to, Salmonella and Enterococcus faecalis, which are associated with opportunistic infections.

[0020] The antibacterial activity may be evaluated by a conventional method. For example, the culture solution of the bacterial strain is serially diluted in two-fold increments, and the maximum dilution concentration 2 at which an inhibition circle is formed in the culture of the food poisoning bacteria is determined. n The antibacterial activity value per mL of supernatant may be evaluated by defining it as (n+1)*100 / mL. In this case, the antibacterial activity value is preferably 100 or higher, more preferably 600 or higher, and even more preferably 1200 or higher against at least one food poisoning bacteria. When the antibacterial activity value of a bacterial strain falls within this range, the strain is expected to exert antibacterial activity when used in food production, thereby preventing food poisoning or food spoilage. Furthermore, when a subject orally ingests the bacterial strain, it is expected to exert antibacterial activity in the intestines, inhibiting the growth of pathogenic bacteria and thereby regulating the intestinal function.

[0021] The bacterial strain according to this embodiment has mucin adhesion properties over a wide temperature range from 3°C to 40°C. This allows the bacterial strain according to this embodiment to exhibit its mucin adhesion properties at various stages after being ingested by a subject. Specifically, since the bacterial strain according to this embodiment has mucin adhesion properties at temperatures of 30°C or less, it is expected that the bacterial strain cultured at a food manufacturing facility will exhibit mucin adhesion properties in the digestive tract of the subject immediately after oral ingestion by the subject. Furthermore, since the bacterial strain has mucin adhesion properties at temperatures of 37°C or more, it is possible for the bacterial strain that has grown in the intestinal tract of the subject to adhere to the intestinal tract.

[0022] In this embodiment, "mucin adhesiveness" refers to the property of a bacterial strain to adhere to mucin. If a bacterial strain has mucin adhesiveness, it can be expected that the bacterial strain will colonize the digestive tract and that pathogenic bacteria will be inhibited from colonizing the digestive tract. Mucin adhesiveness may be evaluated according to a conventionally known method.

[0023] For example, mucin adhesion can be quantitatively evaluated by staining the mucin-adhering bacterial strain with any staining solution. For example, the bacterial strain adhering to porcine gastric mucin can be stained with a crystal violet solution and quantified by absorbance at a wavelength of 595 nm. If a strain is found to have mucin adhesion equal to or greater than that of an arbitrary positive control strain, it can be determined to have mucin adhesion. If the bacterial strain falls within this range, it can be expected that the strain will colonize the digestive tract even when orally ingested by a subject.

[0024] Here, in this embodiment, "equivalent" refers to the absence of a significant difference between any value relating to the function of the strain of this embodiment and any reference value. A value relating to any function of the strain of this embodiment and the positive control strain can be statistically tested to determine that there is no significant difference, and the strains may be evaluated as "equivalent." For example, when determining whether there is a significant difference using a t-test, if p≧0.05, there is no significant difference and the strains can be determined to be "equivalent."

[0025] Even when a single strain of bacteria having antibacterial activity and mucin adhesion is used for probiotic purposes, it can exert an effect of preventing food poisoning and food spoilage, as well as an intestinal regulating effect by suppressing the growth of enteric pathogens through antibacterial activity and competition in the intestines of the subject. Furthermore, even when mixed culture is performed, by using the strains having antibacterial activity and mucin adhesion ability of this embodiment, the number of additional strains used during mixed culture can be reduced, making it easier to consider the conditions.

[0026] [Strain resistance to various culture conditions] Traditionally, Lactobacillus and Bifidobacteria, isolated from animals, have been widely used as effective probiotics. Because these bacteria generally require strict culture conditions, they are typically cultivated under uniform culture conditions (e.g., a culture temperature of 37°C and the use of MRS medium for lactic acid bacteria culture). On the other hand, when oral probiotics are taken, dairy products such as yogurt and fermented foods such as pickles and fermented meat products are also used. These production conditions may involve room temperature or low temperatures and / or high salt concentrations. Therefore, when using bacterial strains in fermented foods, it is necessary to investigate the effects of culture conditions similar to those used during production on the functionality of the bacterial strain and to verify the functionality before proceeding. Specifically, lactic acid bacteria that possess the aforementioned two functionalities of antibacterial activity and mucin adhesion, as well as a wide growth temperature range and high salt tolerance, are particularly preferred.

[0027] (growth temperature range) In one embodiment of the present invention, the bacterial strain may be one that exhibits antibacterial activity even when cultured at a temperature between 3°C and 30°C. The present inventors have discovered that the bacterial strain of this embodiment grows and exhibits antibacterial activity even at temperatures lower than the general culture temperature of 37°C. A bacterial strain that has a wide growth temperature range and possesses the dual functionalities of antibacterial activity and mucin adhesion can prevent food poisoning or food spoilage by exerting antibacterial activity during food production and / or storage, and can also exert mucin adhesion and antibacterial activity in the body when orally ingested, thereby regulating the intestines.

[0028] Furthermore, the inventors have found that the optimum temperature for the growth of the strain according to this embodiment is around 30° C., and the optimum temperature for antibacterial activity is around 25° C., and that the optimum temperatures for the growth of the strain and the antibacterial activity are different. Therefore, when an antibacterial substance derived from the strain is extracted and used, the strain according to this embodiment may be cultured at a temperature of 20° C. or higher and 30° C. or lower in order to improve the extraction efficiency of the antibacterial substance.

[0029] Furthermore, it has become clear that the optimum temperature for growth and the optimum temperature for producing antibacterial activity of the strain of this embodiment are different, making it possible to examine conditions based on temperature conditions in accordance with requirements of the production process, such as efficient fermentation or prevention of microbial contamination. In one embodiment of the present invention, the bacterial strain may be one that exhibits antibacterial activity even when cultured at a temperature of 3° C. or higher and 10° C. or lower. Lactic acid bacteria strains that exhibit antibacterial activity even at such temperatures also exhibit antibacterial activity during refrigerated storage, thereby achieving the effect of suppressing the growth of food poisoning bacteria or food spoilage bacteria even during refrigerated storage of food.

[0030] In one embodiment of the present invention, the bacterial strain may have antibacterial activity and the ability to adhere to mucin when cultured at a temperature of 30° C. or higher and 40° C. or lower. A bacterial strain that has antibacterial activity and the ability to adhere to mucin at such temperatures is expected to suppress the growth of pathogenic bacteria by producing antibacterial substances in the intestine and inhibiting adhesion of pathogenic bacteria to the intestinal epithelium through competition.

[0031] (Salt tolerance of the strain) In one embodiment of the present invention, the bacterial strain may be a strain having salt tolerance. In this embodiment, "salt tolerance" refers to the ability to grow in a medium containing salt. The salt-containing medium is not particularly limited as long as it contains salt, and may be, for example, a medium containing 3% (w / v) or more of salt.

[0032] The salt tolerance of a strain may be evaluated based on the salt concentration of the medium that does not affect the growth of the strain. For example, the strain may be tolerant to a salt concentration of 3% (w / v) or more, and preferably to a salt concentration of 6% (w / v) or more. Here, "having" tolerance means that it is capable of growth. Salt-tolerant strains can also be used favorably in foods with high salt content. In particular, the ability of a strain to grow in an environment with a salt concentration of 6% (w / v) is an essential property for a starter for fermented foods such as fermented sausages.

[0033] Furthermore, it is preferable that the mucin-adhering ability of the bacterial strain of this embodiment is not impaired even when cultured in a medium with a high salt concentration. The present inventors have found that some bacterial strains have higher mucin-adhering ability when cultured in a medium with a high salt concentration. Such bacterial strains are particularly suitable for use as starters for fermented foods.

[0034] The salt-tolerant strains can be used in foods with high salt content, such as fermented foods such as sausages and pickles, as well as miso and fish sauce.

[0035] [Gastrointestinal stress tolerance of bacterial strains]

[0036] In one embodiment of the present invention, the bacterial strain may have resistance to gastrointestinal stress. Resistance to gastrointestinal stress refers to the characteristic of having resistance to digestive fluids secreted from the gastrointestinal tract. In particular, resistance to gastric acid and bile acid is preferred.

[0037] In one embodiment of the present invention, the bacterial strain may have low pH resistance. In this embodiment, a low pH solution refers to a solution that mimics gastric acid, typically a solution with a pH of 1 to 3. Furthermore, in this embodiment, low pH resistance refers to the property of a bacterial strain that allows the strain to survive even when exposed to a low pH solution. For example, when a bacterial strain is incubated at 37°C for 1.5 hours in a low pH solution of pH 2.5 and then cultured on agar for 1 to 2 days, the viable cell count is preferably 7.0 (Log cfu / mL) or more, but may be 7.0 (Log cfu / mL) or less as long as the viable cell count is within a range that does not cause the bacterial strain to die. Furthermore, compared to the viable cell count of a bacterial strain cultured under normal conditions, it is preferable that the decrease in the viable cell count of a bacterial strain cultured under conditions exposed to a low pH solution is small, but the extent of this decrease is not particularly limited. A bacterial strain with low pH resistance can survive in the stomach even when orally ingested, and is therefore expected to reach the intestine alive.

[0038] Furthermore, in order to improve low pH tolerance, a protective agent such as milk may be used, enteric encapsulation may be performed, etc. By doing so, the strain can be used even when higher low pH tolerance is desired.

[0039] In one embodiment of the present invention, the strain may have bile acid tolerance. Here, in this embodiment, bile acid tolerance refers to tolerance to bile, and the method for evaluating it is not particularly limited. For example, bile acid tolerance may be evaluated as bile acid tolerance (%) according to the following formula, based on the growth of the strain when cultured in a culture medium containing no bile acid (bile acid-free sample) and a culture medium containing 0.3% bile acid (bile acid-added sample). Note that the OD 620 is the value obtained by evaluating the number of bacteria using optical density (OD) at a wavelength of 620 nm.

[0040] Bile acid tolerance (%)=OD 620 (bile acid added sample) / OD 620 (Bile acid-free sample) x 100

[0041] The bile acid tolerance of the bacterial strain of this embodiment is preferably equal to or higher than that of widely available probiotics.

[0042] A bacterial strain with low pH tolerance and bile acid tolerance, i.e., a bacterial strain with gastrointestinal stress resistance, can reach the intestinal tract as a live bacterium and survive in the intestine even when orally ingested by a subject, and is therefore expected to exert its function in the intestine. Furthermore, since the bacterial strain of this embodiment has mucin adhesion properties, live bacteria can settle in the intestine. Therefore, the bacterial strain of this embodiment can preferably obtain an intestinal regulating effect.

[0043] (Origin of the strain) In one embodiment of the present invention, the bacterial strain may be derived from food. Strains derived from food have a history of being consumed and are therefore highly safe.

[0044] The strain may be, for example, a strain derived from yogurt, pickles, miso, fish sauce, and the like.

[0045] Furthermore, it is preferable that the bacterial strain is derived from a plant-based food. Generally, animal-derived bacterial strains often have an optimum growth temperature of around 37°C. By deriving the bacterial strain from a plant-based food, it is possible to increase the possibility of selecting a strain with a lower optimum temperature.

[0046] (Specific examples of strains) The bacterial strains of this embodiment may be Latilactobacillus sakei strain A60 (deposit number: NITE P-03490) or strain 4-44 (deposit number: NITE P-03494), Lactiplantibacillus paraplantarum strain D02 (deposit number: NITE P-03491), or Latilactobacillus curvatus strain 4-36 (deposit number: NITE P-03492) or strain 4-43 (deposit number: NITE P-03493), or may be plasmid-free strains or mutants based on these. All of these strains were selected by the present inventors based on their antibacterial activity and mucin adhesion ability. Furthermore, these strains are tolerant to low and high temperatures, salts, low pH, and bile acids. Hereinafter, the five selected strains will be referred to as the "selected strains."

[0047] All selected strains were isolated from food and have a history of consumption, making them suitable for use in the food industry. The selected strains can be cultured at temperatures ranging from 5°C to 40°C, and some strains exhibit antibacterial activity even at a culture temperature of 5°C, making them highly useful under conditions where food is stored at low temperatures. Furthermore, the salt tolerance of the selected strains, particularly their ability to grow sufficiently in an environment with a salt concentration of 6%, is an essential property for starters of fermented foods such as fermented sausages. Some selected strains exhibit enhanced mucin adhesion when cultured in a medium containing salt, making them particularly suitable for use in this field. Because the strains of this embodiment are resistant to gastrointestinal stress, they are expected to reach the intestinal tract as live bacteria. This allows for colonization within the intestine. To further improve gastrointestinal stress resistance, protective agents such as milk and enterosoluble encapsulation may be used.

[0048] The bacterial strain of this embodiment exhibits antibacterial activity and adherence to mucin even at 37°C, and is therefore expected to exert antibacterial effects by producing antibacterial substances in the intestine and inhibiting adhesion of pathogenic bacteria to the intestinal epithelium.

[0049] As described above, the bacterial strain of this embodiment is derived from food and has salt tolerance and digestive tract stress resistance. Therefore, it is a bacterial strain that can be used in a wide range of applications. Furthermore, because the bacterial strain is multifunctional, it can also be preferably used in mixed culture.

[0050] A new strain may be prepared based on the selected strain. For example, when an antibiotic resistance gene is encoded in a plasmid of the selected strain, a plasmid-free strain of the selected strain may be prepared by any method and used.

[0051] Alternatively, mutants based on the selected strains may be produced. The mutants may be produced based on any of the selected strains. The method for producing the mutants is not particularly limited, and they may be produced based on conventionally known methods. For example, mutants may be produced by breeding, genome editing, etc.

[0052] The selected strains of this embodiment each have different characteristics, and their use may be considered based on their different characteristics.

[0053] [Method of using the strain] (Probiotic use) One embodiment of the present invention may be a method for using the strain of any of the above-described embodiments for probiotic purposes. The method for probiotic use is not particularly limited, but typically involves ingesting the strain as a probiotic and allowing it to colonize the intestine to obtain its effects.

[0054] For example, the strain may be cultured at 20° C. to 30° C. for probiotic use. At this culture temperature, the strain produces antibacterial substances, and therefore is preferably used when antibacterial activity is particularly required.

[0055] (food or drink) In one embodiment of the present invention, the strain may be a food or beverage containing the strain of any of the above-mentioned embodiments. The food or beverage may be, but is not limited to, a fermented food, a dairy product, a pickled vegetable, or the like.

[0056] [Strain selection method] In one embodiment of the present invention, the method for selecting a bacterial strain may be a method for selecting, from bacterial strains derived from food, a bacterial strain belonging to any one of the species Latilactobacillus sakei, Lactiplantibacillus paraplantarum, and Latilactobacillus curvatus, which has antibacterial activity, mucin adhesion, salt tolerance, and gastrointestinal stress resistance. The conditions and tests in the selection process may be appropriately changed for selection.

[0057] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0058] (summary) The present invention can also be expressed as follows.

[0059] The bacterial strain according to this embodiment 1 is a strain belonging to any of the species Latilactobacillus sakei, Lactiplantibacillus paraplantarum, and Latilactobacillus curvatus, which has antibacterial activity and mucin adhesion at temperatures between 3° C. and 40° C. Such a bacterial strain colonizes the intestinal tract when ingested and is capable of inhibiting the growth of food poisoning bacteria, food spoilage bacteria, or pathogenic bacteria at any stage from food production to colonization in the intestine.

[0060] The bacterial strain according to this aspect 2 is a bacterial strain in aspect 1 that exhibits antibacterial activity at temperatures between 3° C. and 30° C. Such a bacterial strain can suppress the growth of food poisoning bacteria and food spoilage bacteria during food production at low or normal temperatures.

[0061] The bacterial strain according to this aspect 3 is a bacterial strain that exhibits antibacterial activity at a temperature of 3° C. or higher and 10° C. or lower in aspect 1 or 2. Such a bacterial strain can suppress the growth of food poisoning bacteria and food spoilage bacteria even when stored at low temperatures.

[0062] The strain according to this aspect 4 is a strain having salt tolerance in any one of aspects 1 to 3. Such a strain can be suitably used when the strain is applied to foods containing salt.

[0063] The bacterial strain according to this aspect 5 is a bacterial strain having bile acid tolerance in any one of aspects 1 to 4. Such a bacterial strain has increased resistance to gastrointestinal stress.

[0064] The bacterial strain according to this aspect 6 is a bacterial strain having low pH resistance in any one of aspects 1 to 5. Such a bacterial strain can reach the intestines as live bacteria even when orally ingested.

[0065] The bacterial strain according to this aspect 7 is a bacterial strain derived from food in any of aspects 1 to 6. Such bacterial strains have a history of being eaten, and are therefore highly safe.

[0066] Aspect 8 of the present invention relates to a bacterial strain in any one of Aspects 1 to 7, wherein the bacterial strain is Latilactobacillus sakei strain A60 (deposit number: NITE P-03490) or strain 4-44 (deposit number: NITE P-03494), Lactiplantibacillus paraplantarum strain D02 (deposit number: NITE P-03491), or Latilactobacillus curvatus strain 4-36 (deposit number: NITE P-03492) or strain 4-43 (deposit number: NITE P-03493), or a plasmid-free strain or mutant based thereon. Such a bacterial strain has antibacterial activity, mucin adhesion, salt tolerance, and gastrointestinal stress resistance, and is a novel bacterial strain derived from food.

[0067] A bacterial strain according to this embodiment 9 is a method for using the bacterial strain according to any one of embodiments 1 to 8 for probiotic purposes. This configuration makes it possible to obtain the effect of improving the intestinal environment of the subject using the bacterial strain.

[0068] The bacterial strain according to this aspect 10 is a food or drink containing the bacterial strain according to any one of aspects 1 to 8. This configuration allows the subject to conveniently ingest the bacterial strain.

[0069] The method according to this embodiment 11 is a method for selecting a bacterial strain, in which a bacterial strain belonging to any one of the species Latilactobacillus sakei, Lactiplantibacillus paraplantarum, and Latilactobacillus curvatus having antibacterial activity, mucin adhesion, salt tolerance, and gastrointestinal stress resistance is selected from bacterial strains derived from food.

[0070] [Example] Example 1: Selection of strains Twenty-two strains from the lactic acid bacteria library held by the National Agriculture and Food Research Organization were subjected to the antibacterial activity test described in detail in Example 2, and 16 strains of lactic acid bacteria exhibiting excellent antibacterial activity were selected. Here, excellent antibacterial activity was evaluated based on the breadth of the antibacterial activity spectrum of the strain, the bacterial species inhibited by the antibacterial activity of the strain, and the like.

[0071] Of the 16 strains obtained, food-derived strains were further selected, resulting in the selection of five strains, A60, D02, 4-36, 4-43, and 4-44, belonging to the species Latilactobacillus sakei, Lactiplantibacillus paraplantarum, and Latilactobacillus curvatus. The species, strain names, and isolation sources of the selected lactic acid bacteria are shown in Table 1 below. Hereinafter, the five selected strains will be referred to as the "selected lactic acid bacteria strains." [Table 1]

[0072] From the results of the salt tolerance test in Example 3 described below, it was clear that A60, D02, and 4-44 were new strains different from the standard strain. Furthermore, from the results of inoculating L. curvatus into MRS liquid medium at 1% (v / v) and culturing at 40°C for 24 hours, it was clear that both 4-36 and 4-43 were new strains different from the standard strain. When L. curvatus was cultured at 40°C, the optical density at a wavelength of 620 nm (OD ) measured as the turbidity of the resulting culture solution was 0.01. 620 ) are shown in Table 2 below. The turbidity of the culture medium was measured under the same conditions as in Example 2 described below. T is the type strain of L. curvatus. [Table 2]

[0073] <Example 2: Antibacterial activity test> In the antibacterial activity test, the antibacterial activity of the selected lactic acid bacteria against indicator bacteria was tested.

[0074] (Test strain) The selected lactic acid bacteria strains were cultured overnight at 30°C in MRS liquid medium. Then, they were inoculated into fresh MRS liquid medium at 1% (v / v) and cultured before the test under each culture temperature condition shown in Table 2. When the culture temperature condition for the pre-test culture was 25°C, 30°C, or 37°C, the culture was carried out for 24 hours, and when it was 5°C, the culture was carried out for 2 weeks. After the culture, the culture solution of the selected lactic acid bacteria strain was used as the stock solution of the test strain and was subjected to measurement of the antibacterial activity value. The turbidity of the stock solution of the test strain was measured as the optical density (OD ) at a wavelength of 620 nm using a Spectronic 20 spectrophotometer (Bausch & Lomb, Rochester, NY, USA). 620 ) was evaluated.

[0075] Test strains for the antibacterial activity test were Pediococcus pentosaceus JCM 5885 (obtained from RIKEN, hereinafter also referred to as "JCM 5885") and Leuconostoc mesenteroides subsp. mesenteroides JCM 6124. T (Obtained from RIKEN, hereafter referred to as "JCM 6124 T "), Lactobacillus dextrinicus JCM 5887 T (Obtained from RIKEN, hereafter referred to as "JCM 5887 T "), Latilactobacillus sakei subsp sakei JCM 1157 T (Obtained from RIKEN, hereafter referred to as "JCM 1157 T "), Bacillus coagulans JCM 2257 T (Obtained from RIKEN, hereafter referred to as "JCM 2257 T "), Bacillus subtilis JCM 1465 T (Obtained from RIKEN, hereafter referred to as "JCM 1465 T "), Bacillus circulans JCM 2504 T(Obtained from RIKEN, hereafter referred to as "JCM 2504 T "), Listeria innocua JCM 32814 T (Obtained from RIKEN, hereafter referred to as "JCM 32814 T Each tester was cultured overnight under the liquid medium conditions and at the temperature shown in Table 2, and then inoculated into a new liquid medium of the same type at 1% (v / v). The culture was then cultured for 20 hours at the temperature shown in Table 2 and used on plates for antibacterial activity testing. The culture temperature for each tester was the optimum temperature for the culture of that tester.

[0076] To prepare plates for antibacterial activity testing, 7 mL of Lactobacilli Agar AOAC (LAA) medium or TSB-YE agar medium (1.5% (w / v) agar) was inoculated with 1% (v / v) of the culture solution of each test bacterium into 7 mL of sterilized and dissolved Lactobacilli Agar AOAC (LAA) medium or TSB-YE agar medium (1.5% (w / v) agar) that had been kept at 55°C, and the mixture was layered on a 10 mL MRS agar plate (1.5% (w / v) agar) and then dried at 40°C for 45 minutes.

[0077] The culture conditions for each selected lactic acid bacteria strain and test strain, as well as the test medium used for each test strain, are shown in Table 3 below.

[0078] [Table 3]

[0079] (Antibacterial activity test) The culture solution of the selected lactic acid bacteria (original test solution) was sterilized using a 0.25 μm filter, and then serially diluted in 2-fold increments with 0.1% (v / v) Tween-20 solution. 0 (Undiluted) to 2 11Dilutions up to 100x dilution were prepared. After dropping 10 μL of the test solution onto an antibacterial activity test plate, the test was incubated overnight at the optimal incubation temperature for each test bacterium, and the presence or absence of growth inhibition of the test bacterium was observed. As shown in Table 3, tests were conducted on all combinations of the selected lactic acid bacteria strains and test bacterium strains shown in Table 2. The formation of an inhibition zone was evaluated as indicating growth inhibition, and the absence of an inhibition zone was evaluated as indicating no growth inhibition. The antibacterial activity value was defined as (n+1)*100 / mL as the antibacterial activity value per mL of supernatant, and the antibacterial activity value of each selected lactic acid bacteria strain against each test bacterium strain was calculated. Here, n is the maximum dilution factor at which growth inhibition of the test bacterium was observed, multiplied by 2. n This is the value of n when expressed as

[0080] The antibacterial spectrum in the antibacterial activity test is shown in Tables 4 and 5. In Table 4, "Cultivation temperature" refers to the temperature conditions under which the selected lactic acid bacteria strains were cultured after inoculating them into MRS liquid medium at 1% (v / v) in preparation of the test strains, and indicates the OD 620 represents the turbidity of the original test solution. [Table 4] [Table 5]

[0081] (Antibacterial activity test results) As shown in Table 4, all of the selected lactic acid bacteria strains were found to have antibacterial activity against at least one type of test strain. An antibacterial activity value of 600 or higher is considered to be excellent, and an antibacterial activity value of 1200 or higher is considered to be particularly excellent.

[0082] Furthermore, in the culture before the test, the culture temperatures were 25°C, 30°C, and 37°C, and the OD at 30°C 620 The values were generally high, which was the optimal condition for the growth of the selected lactic acid bacteria strains. On the other hand, the antibacterial activity values of the selected lactic acid bacteria strains tended to be high at 25°C.

[0083] As shown in Table 5, in the culture before the test, the A60 strain, the 4-36 strain, and the 4-43 strain showed antibacterial activity against multiple test strains even under culture conditions at 5°C.

[0084] <Example 3: Salt tolerance test> (Test strain) The selected lactic acid bacteria strains and the standard strains of each selected lactic acid bacteria strain were used as test strains for salt tolerance tests. JCM 1157 was used as the standard strain of Latilactobacillus sakei. T , JCM 12533 as the standard strain of Lactiplantibacillus paraplantarum T , as the standard strain of Latilactobacillus curvatus, JCM 1096 T All of the standard strains were obtained from the Institute of Physical and Chemical Research.

[0085] (Salt tolerance test) MRS liquid medium containing 3, 6, 9, or 12% (w / v) sodium chloride was inoculated with 1% (v / v) MRS culture solution containing the selected lactic acid bacteria and each standard strain, which had been cultured overnight, and cultured at 30°C for 24 hours. After culture was completed, the growth of the bacterial cells was visually determined. The results are shown in Table 6. In Table 6, + indicates bacterial growth, ± indicates slight bacterial growth, and - indicates no bacterial growth at all. [Table 6]

[0086] (Salt tolerance test results) JCM 1157 T However, strain A60 and strain 4-44 could grow even when the salt concentration in the medium was 6%, and strain 4-44 in particular could grow even when the salt concentration was 9%. JCM 12533 T The strain 4-36 and the strain 4-43 were both JCM 1096. TThese results clearly show that the selected lactic acid bacteria strains were able to grow in media with a salt concentration of 6% or higher.

[0087] <Example 4: Mucin adhesion test> (Preparation of measurement wells) Porcine stomach mucin (partially purified type III porcin stomach, Sigma-Aldorich) was suspended in 50 mM carbonate / bicarbonate buffer (pH 9.6) at a concentration of 0.5–1 mg / mL, dispensed at 100 μL / well into a 96-well immunomicroplate (Maxisorp Nunc, Roskilde, Denmark), and left overnight at 4°C to immobilize the mucin to the wells. The wells were washed three times with PBS and blocked with 1% Tween-20 in PBS at 37°C for 1 hour.

[0088] (Preparation of measurement samples) The selected lactic acid bacteria strains were the same as those used in Examples 1 and 2, and Lacticaseibacillus rhamnosus GG strain (hereinafter referred to as "GG strain") was used as a positive control strain capable of adhering to porcine gastric mucin. The GG strain was purchased from the American Type Culture Collection (ATCC) (ATCC 53103, Manassas, VA). The selected lactic acid bacteria strains were cultured overnight in MRS liquid medium at 30°C, and the GG strain cells were cultured overnight at 37°C. They were washed twice with 0.85% sodium chloride solution and adjusted to an optical density of 1.0 at 620 nm with the same solution to prepare test strain solutions. 100 μL of the test strain solution was added to each well and incubated at 37°C for 2 hours to allow the cells to adhere to the mucin. The plate was washed three times with 200 μL of PBS containing 0.05% Tween-20 to remove any cells not adhering to the mucin. The plate was then dried at 55°C. 20 μL of 1% crystal violet solution (33% acetic acid) was added to the wells and left at room temperature for 45 minutes. After washing twice with PBS, 100 μL of 50 mM citric acid (pH 4.0) was added to the wells and left at room temperature for 45 minutes to obtain a measurement sample.

[0089] (Measurement of mucin adhesion) The absorbance of the measurement samples was measured at a wavelength of 595 nm using an absorbance meter (iMark, BioRad, CA, USA). A blank was performed using a well without mucin immobilized. Measurements were performed using multiple bacterial cells, and the average absorbance was evaluated as the absorbance.

[0090] (Mucin adhesion test results) The selected lactic acid bacteria strains had mucin adhesion properties equal to or greater than those of the GG strain, a positive control strain for adhesion to porcine gastric mucin. Since the GG strain is a common lactic acid bacterium that can be used for probiotics, the fact that the selected lactic acid bacteria strains exhibited mucin adhesion properties equal to or greater than that of the GG strain indicates that they are lactic acid bacteria strains with high potential for probiotic applications.

[0091] Example 5: Effect of culture conditions on mucin adhesion (Test strain) The selected lactic acid bacteria strains were subjected to a test to evaluate the effect of culture conditions on mucin adhesion.

[0092] (Evaluation of the effect of salt concentration on mucin adhesion) As in Example 3, selected lactic acid bacteria were inoculated into MRS liquid media with salt concentrations of 0% and 6%, and the strains cultured at 30°C were used in a mucin adhesion test. The mucin adhesion test was performed in the same manner as in Example 4. The absorbance at a wavelength of 595 nm for the bacterial cells cultured at each salt concentration for each strain is shown in Figure 1.

[0093] (result) As shown in Figure 1, all selected lactic acid bacteria strains exhibited mucin adhesion even when cultured in a medium with a 6% salt concentration. In particular, the A60 and 4-36 strains exhibited significantly higher mucin adhesion (P<0.001) than when the salt concentration was 0%. The D02 strain showed a tendency toward higher mucin adhesion (P<0.1).

[0094] (Evaluation of the effect of culture temperature on mucin adhesion) The mucin adhesion test was carried out in the same manner as in Example 5, except that the test strains were cultured at 5°C, 25°C, and 37°C before the test.

[0095] (result) As shown in Figure 2, the selected lactic acid bacteria strains exhibited adhesion to mucin even under culture conditions at 37°C, and their adhesion ability was equivalent to or significantly (P<0.05) higher than that of the GG strain.

[0096] As shown in Figure 3, the selected lactic acid bacteria strains exhibited adhesion to mucin even under culture conditions at 25°C, and their adhesion strength was either equivalent to or significantly (P<0.05) higher than that of the GG strain.

[0097] As shown in Figure 4, the D02, 4-36, 4-43, and 4-44 strains adhered to mucin even when cultured at 5°C, and their adhesion was significantly (P<0.05) higher than that of the GG strain.

[0098] <Example 6: Gastrointestinal stress tolerance test> (Test strain) The selected lactic acid bacteria strains and the GG strain were subjected to low pH tolerance tests and bile acid tolerance tests.

[0099] (Measurement of low pH tolerance) The selected lactic acid bacteria strains and the GG strain were cultured overnight at 30°C, and the GG strain was cultured at 37°C.

[0100] The test strains were centrifuged at 13,000 g for 10 minutes to harvest the cells and then washed twice with 0.85% sodium chloride solution. The test cells were then suspended in a 0.85% sodium chloride solution, which had been adjusted to pH 2.5 with 1N hydrochloric acid, and incubated at 37°C for 1.5 hours. Before and after exposure to the low-pH solution, aliquots of the cell suspension were collected and appropriately diluted with 0.85% sodium chloride solution. The diluted aliquots were then smeared on MRS agar medium (1.6% agar) and cultured for 1–2 days at the optimal incubation temperature for each test strain. The number of colonies grown on the agar was counted as the viable cell count. The logarithmic transformation of the viable cell counts is shown in Table 7. [Table 7]

[0101] (result) Since the selected lactic acid bacteria continued to grow even after exposure to a low pH solution, it was determined that the bacteria were sufficient for probiotic use.

[0102] (Measurement of bile acid tolerance) Bile acid-added samples were prepared by inoculating 1% (v / v) of MRS liquid medium containing 0.3% (w / v) dehydrated bile (Oxgall, BD) with MRS culture medium containing overnight cultures of each selected lactic acid bacterium and the GG strain, followed by incubation at 37°C for 24 hours. Bile acid-free samples were prepared in the same manner as the bile acid-added samples, except that dehydrated bile was not added.

[0103] After the incubation, the optical density (OD) at 620 nm was measured using the bile acid-added sample and the bile acid-free sample. 620 ) was measured, and bile acid tolerance was calculated using the following formula.

[0104] Bile acid tolerance (%)=OD 620 (bile acid added sample) / OD 620 (Bile acid-free sample) x 100

[0105] OD of each bile acid sample 620 The values and bile acid resistance (%) are shown in Table 8. [Table 8]

[0106] (result) As shown in Table 8, each selected lactic acid bacteria strain survived even when cultured in a bile acid solution containing 0.3% bile acid, demonstrating its resistance to 0.3% bile acid. Therefore, each selected lactic acid bacteria strain is expected to be able to survive in the intestine. In particular, A60 and 4-43 had better bile acid resistance than the GG strain, making them particularly suitable as probiotics. [Industrial Applicability]

[0107] The present invention can be used in the food industry. [Accession number]

[0108] Latilactobacillus sakei strain A60 (deposit number: NITE P-03490), Lactiplantibacillus paraplantarum strain D02 (deposit number: NITE P-03491), Latilactobacillus curvatus strain 4-36 (deposit number: NITE P-03492), 4-43 (deposit number: NITE P-03493), strain 4-44 (deposit number: NITE P-03494).

Claims

1. A bacterial strain which is Latilactobacillus sakei A60 strain (deposit number: NITE P-03490), or 4-44 strain (deposit number: NITE P-03494), Lactiplantibacillus paraplantarum D02 strain (deposit number: NITE P-03491), or Latilactobacillus curvatus 4-36 strain (deposit number: NITE P-03492), or 4-43 (deposit number: NITE P-03493).

2. The strain described in claim 1, which has antibacterial activity and mucin adhesion at temperatures between 3°C and 40°C.

3. The strain according to claim 1 or 2, which has antibacterial activity at a temperature of 3°C or higher and 30°C or lower.

4. The strain according to any one of claims 1 to 3, which has antibacterial activity at a temperature of 3°C or higher and 10°C or lower.

5. The strain according to any one of claims 1 to 4, which is salt-tolerant.

6. The strain according to any one of claims 1 to 5, which has bile tolerance.

7. The strain according to any one of claims 1 to 6, which has low pH tolerance.

8. The strain according to any one of claims 1 to 7, which is derived from food.

9. Use of the strain according to any one of claims 1 to 8 for probiotic purposes.

10. A food or beverage comprising the strain according to any one of claims 1 to 8.

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