KR102674853B1 - Novel Lactobacillus rhamnosus FB019 strain and food composition comprising thereof - Google Patents

Abstract

The present disclosure relates to a novel Lactobacillus rhamnosus FB019 strain and a food composition containing the same. The strain can significantly increase beneficial bacteria (eg, Lactobacillus genus) in the subject's intestinal flora. These strains can maintain the balance of the subject's normal intestinal flora and improve intestinal health. Additionally, the strain can significantly increase beneficial bacteria in the intestinal flora of subjects who frequently consume coffee. Therefore, the strain can be usefully used as a probiotic composition or food composition (e.g., health functional food composition).

Description

Novel Lactobacillus rhamnosus FB019 strain and food composition comprising the same {Novel Lactobacillus rhamnosus FB019 strain and food composition comprising thereof}

The present disclosure relates to a novel Lactobacillus rhamnosus FB019 strain and a food composition containing the same.

The number of microorganisms existing in the human body is 38 trillion, which is more than the number of human cells (30 trillion), and most of these microorganisms exist in the large intestine. Microorganisms existing in the human body that do not cause disease and form colonies are called normal flora. The normal flora plays a role in preventing pathogenic microorganisms harmful to humans from settling in the body. In particular, in the case of the large intestine, hundreds of species of microorganisms live in a complex ecosystem, so maintaining the normal flora is essential for humans to maintain a healthy state. It is important. However, the human intestinal flora can become imbalanced due to various external factors, and if the ratio of normal flora and harmful pathogenic microorganisms deviates from the normal range due to changes, it can become dysbiosis. Recent studies have shown that imbalances occurring in the intestines are associated with various symptoms or diseases. In particular, the intestinal flora can be affected by the foods a subject consumes, and the state of the intestinal flora can vary depending on which foods are frequently consumed.

As a favorite food preferred by Koreans, coffee is a frequently consumed item. Coffee is a favorite beverage that is consumed by processing and roasting green beans harvested from coffee trees, then extracting a mixture of one or two types of coffee beans, and because it contains a large amount of caffeine, the effect of coffee consumption on intestinal microorganisms is expected to be significant. do. In addition, Korea's coffee market size has grown to about 5 trillion won in 2021, ranking third in the world, and is expected to grow to about 8 trillion won in 2023. There is a need for the development of probiotics to maintain the intestinal flora of modern people who frequently consume coffee within a normal range.

In order to solve the above problem, the present disclosure aims to provide customized lactic acid bacteria that stably settle in the intestinal flora of modern people who frequently consume coffee and help the proliferation of beneficial bacteria.

The purpose of the present disclosure is to provide a probiotic composition or food composition containing a novel Lactobacillus rhamnosus strain.

1. One embodiment of the present disclosure may relate to the novel Lactobacillus rhamnosus FB019 ( Lactobacillus rhamnosus FB019) strain deposited with accession number KCTC14784BP.

2. In one embodiment, the FB019 strain may include the 16S rRNA base sequence shown in SEQ ID NO: 1.

3. One embodiment of the present disclosure may relate to a probiotic composition containing the FB019 strain.

4. In one embodiment, the probiotic composition may include strain FB019 at a concentration of 1Х10 ⁸ to 1Х10 ¹¹ CFU/g.

5. One embodiment of the present disclosure may relate to a food composition containing a probiotic composition.

6. In one embodiment, the food composition may further include prebiotics.

7. In one embodiment, the prebiotics include fructooligosaccharides, chicory fiber, inulin, isomaltooligosaccharides, lactitol, lactosucrose, lactulose, pyrodextrins, soy oligosaccharides, and xylo oligosaccharides. It may be one or more selected from the group consisting of (Xylo-oligosaccharide).

8. In one embodiment, the prebiotic can be chicory dietary fiber.

9. In one embodiment, the food composition may be in the form of powder, granule, tablet, capsule, liquid, powder, or fermented milk.

10. In one embodiment, the food composition may further include one or more of additives, carriers, excipients, and flavoring agents that can be used in food.

11. In one embodiment, the food composition may be a health functional food composition.

12. In one embodiment, the food composition may be a freeze-dried product.

13. In one embodiment, the food composition may be intended to increase beneficial bacteria in the intestinal flora of a subject with high coffee consumption.

14. In one embodiment, the subject may be a subject who consumes coffee 3 to 4 or more times per week.

15. One embodiment of the present disclosure may relate to a composition for food addition containing the FB019 strain.

16. One embodiment of the present disclosure may relate to a composition for animal feed containing the FB019 strain.

17. One embodiment of the present disclosure includes probiotics, and the probiotics are a novel Lactobacillus rhamnosus FB019 strain deposited with accession number KCTC14784BP, and promote beneficial bacteria in the intestinal flora of subjects who consume a lot of coffee. It may be about a health functional food composition for increasing

The novel Lactobacillus rhamnosus FB019 strain according to an embodiment of the present disclosure can excellently increase beneficial bacteria (eg, Lactobacillus genus) in the intestinal flora of a subject. These strains can maintain the balance of the subject's normal intestinal flora and improve intestinal health.

The novel Lactobacillus rhamnosus FB019 strain according to an embodiment of the present disclosure can significantly increase beneficial bacteria in the intestinal flora of subjects who frequently consume coffee.

The novel Lactobacillus rhamnosus FB019 strain according to an embodiment of the present disclosure can be usefully used as a probiotic composition or food composition (e.g., health functional food composition).

Figure 1 shows the growth level of the novel Lactobacillus rhamnosus FB019 strain and other Lactobacillus comparison strains according to an embodiment of the present disclosure.
Figure 2 shows the growth level of the FB019 strain and other Lactobacillus comparison strains according to the concentration of fructooligosaccharide, a prebiotic.
Figure 3 shows the relative ratio of intestinal flora when the FB019 strain and other Lactobacillus comparison strains were treated in subjects with high coffee consumption.
Figure 4 shows the relative ratio change of the Lactobacillus genus in subjects with high coffee consumption when treated with the FB019 strain.
Figure 5 shows the relative ratio of intestinal flora in a group that frequently consumes coffee and a group that consumes less coffee, and the relative ratio of intestinal flora before and after treatment with the FB019 strain.
Figure 6 shows the change in the ratio of intestinal beneficial bacteria (Lactobacillus genus) in a group that frequently consumes coffee and a group that consumes coffee infrequently depending on whether or not FB019 was treated.

The various embodiments or examples described in this document are illustrative for the purpose of clearly explaining the technical idea of the present disclosure, and are not intended to limit them to specific embodiments. The technical idea of the present disclosure includes various modifications, equivalents, alternatives, and embodiments selectively combined from all or part of each embodiment or example described in this document. Or includes examples. Additionally, the technical idea of the present disclosure is not limited to the various embodiments or examples or specific descriptions thereof presented below.

Terms used in this disclosure, including technical or scientific terms, unless otherwise defined, may have meanings commonly understood by those skilled in the art to which this disclosure pertains. Terms such as those defined in general dictionaries should be interpreted as having meanings consistent with the meanings they have in the context of the related technology, and unless clearly defined in the present disclosure, should not be interpreted as having an ideal or excessively formal meaning.

Expressions such as “includes,” “may include,” “includes,” “may be provided,” “has,” “may have,” etc. used in the present disclosure refer to the target feature, e.g. function, operation or component, etc.) exists, and does not exclude the presence of other additional features. In other words, such expressions should be understood as open-ended terms that imply the possibility of including other embodiments.

The singular expressions used in this disclosure may include plural meanings unless the context dictates otherwise, and this also applies to the singular expressions described in the claims.

As used in this disclosure, “A, B, and C,” “A, B, or C,” “A, B, and/or C,” or “at least one of A, B, and C,” “at least one of A, B, or C. “one,” “at least one selected from A, B, and/or C,” “at least one selected from A, B, and C,” “at least one selected from A, B, or C,” “selected from A, B, and/or C. Expressions such as “at least one” may mean each listed item or all possible combinations of the listed items.

As used in this disclosure, the term “about” may indicate a typical error range for each value, as is well known to those skilled in the art. In the context of numerical values or ranges set forth in this disclosure, in one embodiment this means ±20%, ±15%, ±10%, ±9%, ±8%, ±7% of the stated or claimed numerical values or ranges. , ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%.

Unless otherwise specified in context, descriptions of dimensions, values, and ranges used in the present disclosure are not limited only to the dimensions, values, and ranges, but may mean an equivalent range including these.

Various embodiments of the present disclosure are described below.

One embodiment of the present disclosure may relate to a novel Lactobacillus plantarum strain.

In one embodiment, the strain is Lactobacillus rhamnosus FB019 strain and may be a strain deposited at the Korea Research Institute of Bioscience and Biotechnology under accession number KCTC14785BP.

In one embodiment, the strain may include the 16S rRNA base sequence shown in SEQ ID NO: 1.

One embodiment of the present disclosure may relate to a probiotic composition containing the Lactobacillus rhamnosus FB019 strain.

In one embodiment, probiotics are microorganisms or components thereof that exhibit beneficial effects on the health of hosts such as humans and animals, and may specifically refer to lactic acid bacteria. Probiotic compositions can be used for edible and medicinal purposes, and there are no particular restrictions on the dosage, including absorption into the body, body weight, patient's age, gender, health status, diet, administration time, administration method, excretion rate, severity of disease, etc. It may change accordingly. In general, the probiotic composition can be administered in an amount of about 10 to about 1000 mg per kg of body weight, and specifically, about 50 to about 500 mg per kg of body weight.

In one embodiment, the probiotic composition may be prepared according to a conventional method for preparing a probiotic composition, for example, in the form of powder, granule, tablet, capsule, liquid, powder, fermented milk, culture suspension, or dry powder. However, it is not limited to this. In addition, the composition can be prepared in a conventional formulation by selecting an effective amount of Lactobacillus rhamnosus FB019 strain, which is the main ingredient, and one or two or more excipients, carriers, or additives.

In one embodiment, the carrier may be one or more selected from the group consisting of a diluent, lubricant, binder, disintegrant, sweetener, stabilizer, and preservative, and the additive may include one or more selected from the group consisting of flavoring, vitamins, and antioxidants. You can use it.

In one embodiment, the type of excipient, carrier, or additive is not limited, but specifically, the diluent includes lactose monohydrate, trehalose, corn starch, soybean oil, and microcrystalline cellulose. (microcrystalline cellulose) or mannitol (D-mannitorl), magnesium stearate or talc as a lubricant, and polyvinylpyrrolidone (PVP: polyvinyipyrolidone) or hydroxypropylcellulose (HPC: hydroxypropylcellulose) as a binder. You can choose. In one embodiment, the disintegrant is carboxymethylcellulose calcium (Ca-CMC), sodium starchglycolate, polacrylin potassium or cross-linked polyvinylpyrrolidone, and a sweetener. The stabilizers are white sugar, fructose, sorbitol or aspartame, and the stabilizers are carboxymethylcellulosesodium (Na-CMC), beta-cyclodextrin, white bee's wax or xanthan gum. You can choose from (xanthan gum), and as a preservative, you can choose from methyl p-hydroxy benzoate (methlparaben), propyl p-hydroxybenzoate (propylparaben), or potassium sorbate. .

In one embodiment, the probiotic composition may include Lactobacillus rhamnosus FB019 strain at a concentration of about 1Х10 ⁸ to about 1Х10 ¹¹ CFU/g. Specifically, the Lactobacillus rhamnosus FB019 strain is about 1Х10 ⁸ CFU/g, about 5Х10 ⁸ CFU/g, about 1Х10 ⁹ CFU/g, about 5Х10 ⁹ CFU/g, about 1Х10 ¹⁰ CFU/g, about 5Х10 ¹⁰ CFU/g. , or may be included in the probiotic composition at a concentration of about 1Х10 ¹¹ CFU/g, or may be included in the probiotic composition at a concentration corresponding to any value between the above concentration values.

In one embodiment, the probiotic composition can be administered to the subject 1, 2, 3, 4, 5, or 6 or more times per day.

One embodiment of the present disclosure may relate to a food composition containing the Lactobacillus rhamnosus FB019 strain or a probiotic composition containing the same according to an embodiment of the present disclosure.

The food composition of the present invention includes all foods in the conventional sense, and includes functional foods, functional beverages, nutritional supplements, health foods, and food additives. Includes all forms. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

In one embodiment, the food composition may be manufactured in the form of various foods, such as beverages, gum, tea, vitamin complexes, powders, granules, tablets, capsules, snacks, rice cakes, bread, etc., but is not limited thereto. No. The food composition includes, for example, meat, grains, caffeinated beverages, general beverages, chocolate, bread, snacks, confectionery, candy, pizza, jelly, noodles, gum, dairy products, ice cream, alcoholic beverages, alcohol, vitamin complexes, and It may be any one selected from other health supplements, but is not limited thereto.

In one embodiment, when the strain or a probiotic composition containing the same is included in a food composition, the amount is about 0.001% by weight to about 90% by weight, about 0.1% by weight to about 50% by weight, based on the total weight of the food composition, It may be about 0.1% by weight to about 20% by weight, about 0.1% by weight to about 10% by weight, or about 0.1% by weight to about 5% by weight, and may be less than the above range for long-term intake, but the active ingredient is safe. If there are no problems in this regard, it can be used in amounts exceeding the above range, so it is not limited thereto.

In one embodiment, the food composition contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its It may contain salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., and the above ingredients can be used independently or in combination. In one embodiment, the food composition may include the Lactobacillus rhamnosus FB019 strain as an active ingredient, as well as ingredients commonly added during food production. In one embodiment, added ingredients include, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. In one embodiment, carbohydrates include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, oligosaccharides, etc.), and polysaccharides (e.g., dextrins, cyclodextrins, etc.). In one embodiment, the flavoring agent is a common sugar and sugar alcohol such as xylitol, sorbitol, erythritol, etc., natural flavoring agent (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)). Synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

In one embodiment, the food composition may further include prebiotics. In one embodiment, the prebiotics include fructooligosaccharides, chicory dietary fiber, inulin, isomaltooligosaccharides (IMO), lactitol, lactosucrose, lactulose, pyrodextrins, soy oligosaccharides, and xylosaccharides. It may be one or more selected from the group consisting of oligosaccharides (Xylo-oligosaccharides), but is not limited thereto. In one embodiment, prebiotics may be included in the food composition in an amount of about 0.1 to about 10% by weight based on the total weight of the food composition.

In one embodiment, the food composition may include Lactobacillus rhamnosus FB019 strain at a concentration of about 1Х10 ⁸ to about 1Х10 ¹¹ CFU/g.

In one embodiment, the food composition may be in the form of a powder, granule, tablet, capsule, liquid, powder, or fermented milk.

In one embodiment, the food composition may further include one or more of additives, carriers, excipients, and flavoring agents that can be used in foods.

In one embodiment, the excipients include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline. It may include cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.

In one embodiment, the food composition may be a health functional food composition. Health functional foods of the present disclosure refer to foods manufactured and processed in the form of tablets, capsules, powders, granules, liquids, beverages, and pills using raw materials or ingredients with functional properties useful to the human body. Here, ‘functionality’ means controlling nutrients for the structure and function of the human body or obtaining useful effects for health purposes, such as physiological effects. The health functional food of the present disclosure can be manufactured by a method commonly used in the industry, and can be manufactured by adding raw materials and ingredients commonly added in the industry.

In one embodiment, the formulation of a health functional food can also be manufactured without limitation as long as it is a formulation recognized as a health functional food. The food composition or health functional food composition of the present disclosure can be manufactured in various forms, and unlike general drugs, it is made from food as a raw material and has the advantage of not having side effects that may occur when taking the drug for a long period of time, and being portable. It is excellent and can be taken as a supplement.

In one embodiment, the health functional food may further include other additives. In one embodiment, the additive refers to a food supplement that can be added to food, and can be appropriately selected and used by a person skilled in the art as it is added to manufacture each type of health functional food. Examples of additives include flavoring agents (e.g., orange oil, peppermint oil, or apple flavor, etc.), preservatives (e.g., methyl paraoxybenzoate, or propyl paraoxybenzoate, etc.), and pH adjusters (e.g., , acetic acid, phosphoric acid, sodium bicarbonate, sodium hydroxide, etc.), buffering agents, colorants, anti-foaming agents (e.g., silicone oil, etc.) may be used, but are not limited thereto. These additive components may be used alone or in combination, and the additive may be included in an amount of from about 0.001% by weight to about 50% by weight based on the total weight of the composition, but is not limited thereto.

In one embodiment, beverages among health functional foods may contain various flavoring agents or natural carbohydrates as additional ingredients like regular beverages. These beverages may also contain pulp for the manufacture of fruit and vegetable beverages. Here, natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame can be used. The content of the natural carbohydrate may be about 0.01 to 0.04 g, or about 0.02 to 0.03 g per 100 mL of the beverage according to the present disclosure.

In one embodiment, the food composition may be a freeze-dried product. These freeze-dried products can be manufactured according to conventional manufacturing methods widely known in the industry.

In one embodiment, the food composition may be a food composition for increasing beneficial bacteria in the intestinal flora of a subject who consumes a lot of coffee. In one embodiment, a subject who consumes a lot of coffee refers to a subject who consumes coffee frequently and may refer to a subject who consumes coffee three to four times or more per week. In one embodiment, a subject with a high coffee intake consumes coffee 1 or more times, 2 or more times, 3 or more times, 4 or more times, 5 or more times, 6 or more times, 7 or more times, 8 or more times, or 9 times per week. It may refer to a subject who consumes more than, or more than 10 times. Here, the coffee can be based on an espresso shot.

In one embodiment, coffee may refer to a food that uses coffee beans as a raw material. Specifically, coffee may refer to a beverage of choice that is consumed by processing and roasting coffee beans, then extracting a mixture of one or two or more types of coffee beans, and includes coffee beans, instant coffee (e.g. solid coffee mix products, tea bag coffee bean products, It may include, but is not limited to, liquid products containing coffee bean extract, etc.), or coffee drinks.

One embodiment of the present disclosure may relate to a composition for food addition containing the Lactobacillus rhamnosus FB019 strain. In one embodiment, the strain or culture thereof can be used as a food additive for foods such as kimchi, beverages, baby food, fermented milk, and bread. In one embodiment, the strain or culture thereof can be used as a starter for manufacturing fermented products. The fermented products include cheese, kimchi, fermented raw food products, etc. In one embodiment, fermented products using the strain or culture thereof can be produced according to conventional methods known in the art.

One embodiment of the present disclosure may relate to a composition for animal feed containing the Lactobacillus rhamnosus FB019 strain.

When used as a composition for animal feed, this composition may be a highly concentrated solution of 20 to 90% or may be prepared in powder or granule form. In one embodiment, the composition for animal feed contains organic acids such as citric acid, humalic acid, adipic acid, lactic acid, and malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, and polyphosphate (polymerized phosphate), polyphenol, and catechin. , alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid, etc., may additionally contain one or more of natural antioxidants. When used as feed, the composition for animal feed may be formulated in the form of a normal feed and may include common feed ingredients.

In one embodiment, the composition for animal feed or feed comprising the same may include grains such as ground or ground wheat, oats, barley, corn and rice; vegetable protein feeds, such as those based on rapeseed, soybean, and sunflower; Animal protein feeds such as blood meal, meat meal, bone meal and fish meal; It may further include dry ingredients such as sugar and dairy products, such as various powdered milk and whey powder, and may further include nutritional supplements, digestion and absorption enhancers, growth promoters, etc.

In one embodiment, the composition for animal feed or feed containing the same may be administered to animals alone or in combination with other feed additives in an edible carrier. Additionally, the composition for animal feed and feed containing the same can be easily administered to animals as a top dressing, by mixing them directly into animal feed, or in an oral dosage form separate from the feed. When the composition for animal feed or the feed containing the same is administered separately from animal feed, it can be prepared into an immediate-release or sustained-release formulation by combining it with a pharmaceutically acceptable edible carrier, as is well known in the art. These edible carriers may be solid or liquid, such as corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the feed additive may be a tablet, capsule, powder, troche or sugar-containing tablet, or a top dressing in microdisperse form. When a liquid carrier is used, the animal feed composition or feed containing the same may be in the form of a gelatin soft capsule, syrup, suspension, emulsion, or solution.

In one embodiment, the composition for animal feed or the feed containing the same may contain auxiliaries, such as preservatives, stabilizers, wetting or emulsifying agents, solution accelerators, etc. The feed additive can be used by adding it to animal feed by injecting, spraying, or mixing it.

In one embodiment, the composition for animal feed or feed containing the same can be applied to the diet of a number of animals, including mammals, poultry, and fish.

In one embodiment, the animals include mammals and can be used for pigs, cattle, sheep, goats, laboratory rodents, and laboratory rodents, as well as pets such as dogs, cats, etc., and poultry can be used for chickens. , it can be used for turkey, duck, goose, pheasant, and quail, and can be used for fish such as trout, but is not limited to this.

One embodiment of the present disclosure includes probiotics, and the probiotic is a novel Lactobacillus rhamnosus FB019 strain deposited under accession number KCTC14785BP, and is used to increase beneficial bacteria in the intestinal flora of subjects who consume a lot of coffee. It may be about a health functional food composition.

One embodiment of the present disclosure may relate to a method for increasing beneficial bacteria in the intestinal flora of a subject, comprising administering Lactobacillus rhamnosus FB019 strain or a composition containing the same to the subject.

One embodiment of the present disclosure may relate to the use of the Lactobacillus rhamnosus FB019 strain or a composition containing the same for increasing beneficial bacteria in the intestinal flora of a subject.

One embodiment of the present disclosure may relate to Lactobacillus rhamnosus FB019 strain or a composition containing the same for use in a method for increasing beneficial bacteria in the intestinal flora of a subject.

In one embodiment, the composition may be a probiotic composition, a food composition, a food additive composition, an animal feed composition, or a health functional food composition. In one embodiment, the strain or a composition containing it may be orally administered or ingested by a subject. In one embodiment, the subject may be a subject who consumes a lot of coffee, and may be a subject who consumes coffee three to four times or more per week.

The present disclosure will become clearer through the above-described embodiments and the following examples or experimental examples. Hereinafter, the present disclosure will be described in detail through examples or experimental examples so that those skilled in the art can easily understand and implement the present disclosure. However, these examples or experimental examples are for illustrative purposes only and the scope of the present disclosure is not limited to these examples or experimental examples.

[Example 1] Isolation and identification of Lactobacillus rhamnosus FB019 strain derived from newborn feces

As a result of isolating and identifying the strain from the feces of a Korean newborn, a new strain was discovered, which was named Lactobacillus rhamnosus FB019 ( Lactobacillus rhamnosus FB019). The FB019 strain was deposited at the Korea Research Institute of Bioscience and Biotechnology's Biological Resources Center (KCTC) on November 18, 2021 (Accession Number: KCTC14784BP).

The 16S rRNA sequence of strain FB019 is the same as SEQ ID NO: 1, and the characteristics of the strain are as follows.

1) Morphological characteristics (morphology when cultured on MRS solid medium at 37°C for 2 days)

① Cell type: Bacillus

② Mobility: None

③ Spore forming ability: None

④ Shape of fungi: circular, convex and smooth shape

2) Physiological characteristics

① Gram staining: Gram positive

② Optimum growth temperature: 33~37 ℃

③ Optimum growth pH: pH 5.0~5.5

④ Oxygen requirement: facultative anaerobic

3) Catalase: -

4) Gas formation: -

5) Indole production: -

6)Lactic acid production: +

[Experimental Example 1] Establishment of optimal intestinal flora culture system (In vitro Incubation model) conditions

(1) IRB and securing fecal samples for subject recruitment

To conduct an experiment to confirm the efficacy of the new FB019 strain, subjects were recruited and their fecal samples were obtained.

To recruit subjects and secure fecal samples, bioethics review was conducted and a review number was obtained through the Sejong University Bioethics Review Committee (IRB no.: SJU-BR-E-2020-025). Feces were collected from each subject using a one-time self-collection method using a sterilized cotton swab, and in order to minimize exposure to oxygen, they were immediately put into an in vitro intestinal flora culture system and homogenized together with the basic medium.

To minimize other factors that could affect the study results other than diet control, subjects who continuously consumed antibiotics and probiotic products and subjects with digestive tract-related diseases were excluded.

(2) Establishment of an in vitro intestinal flora culture system

To ensure stable cultivation of obligate anaerobes living in the human intestine and to minimize changes in flora, feces were collected and immediately transferred to an anaerobic chamber to conduct experiments under obligate anaerobic conditions. To prevent sample evaporation and cross-contamination, culture was performed after leaving the outermost part of the 96 well empty. Based on previous research, a medium composition and preparation method similar to the human intestinal condition was selected, and the optimal mixing ratio of the basic medium and fecal inoculum for culturing intestinal flora was selected. As a result, it was confirmed that 1/3 of the strains in the feces were stably cultured in the constructed basic medium composition. The pH of the final medium was adjusted to 7.0 ± 0.1, and the preparation method and composition of the medium are as follows.

(i) The medium components in Table 1, excluding heat-sensitive vitamin stock, trace mineral solution, and hemin solution, were added to 1 L of distilled water.

(ii) After stirring and mixing with a magnetic stirrer for 5 minutes, the pH was adjusted to 7.0 ± 0.1 using a pH meter. pH control was carried out by stirring on a stirrer, and titration was performed using 1M HCl and 1M NaOH.

(iii) After autoclaving, transfer to an anaerobic chamber, cool, continuously flow nitrogen gas into the basic medium to remove all dissolved oxygen in the basic medium, and add 1 ml of vitamin solution, 1 ml of trace element solution, and 1 ml of hemin solution to the basic medium. It was added and stirred.

Components of basic medium ingredient manufacturing company product no content Distilled water - - 1L peptone Difco 211677 2.0g enzyme extract Difco 21275 2.0g sodium bicarbonate Sigma Aldrich S5761 4.0 g Tween 80 Sigma Aldrich P8074-500 ml 2ml Pig stomach mucin Sigma Aldrich M1778-100g 4.0 g Blemishes (sodium cholate and sodium chenodeoxycholate) Sigma Aldrich C1254-25g;
C8261-1g 0.5 g (0.25 g sodium cholate and 0.25 g sodium chenodeoxycholate) sodium chloride Sigma Aldrich S5886-500g 1.0 g Potassium phosphate dibasic anhydrous Sigma Aldrich 60353 0.4g Potassium dihydrogen phosphate Sigma Aldrich P9791-100g 0.4g magnesium sulfate Sigma Aldrich M7506 0.1g Calcium chloride dehydrate Sigma Aldrich C8106 0.1g L-cysteine hydrochloride Sigma Aldrich C1276 0.5g Hemin Sigma Aldrich H9039-1g 5mg Vitamin K1 Sigma Aldrich 95271-1g 10ul

[Experimental Example 2] Growth test of Lactobacillus rhamnosus FB019 strain

(1) Lactobacillus rhamnosus FB019 strain and Lactobacillus comparison strains (Lac02, Lac03, Lac08, Lac09) were inoculated into MRS broth (BD Difco, Sparks, MD, USA) and cultured at 37°C for 24 hours. In the following experimental examples, the comparative strains are as follows.

Lac02: Lactobacillus rhamnosus YF3055

Lac03: Lactobacillus rhamnosus YF3048

Lac08: Lactobacillus plantarum FB091 (Accession number: KCTC14785BP)

Lac09: Lactobacillus casei FB005

After culturing, the growth level of each strain was confirmed by measuring the absorbance using spectrophotometry. As a result, as shown in Figure 1, it was confirmed that the FB019 strain according to an embodiment of the present disclosure grew smoothly at a similar level to other Lactobacillus comparison strains.

(2) To confirm the effect of prebiotics on the growth of strains, chicory dietary fiber powder (COSUCRA Group Warcoing S.A., Belgium) was added to MRS broth at different concentrations (0.5, 1, 1.5, 2, 5, 10%). was added and incubated at 25°C for 24 hours.

As a result, as shown in FIG. 2, it was confirmed that the growth of the Lactobacillus rhamnosus FB019 strain according to an embodiment of the present disclosure increased depending on the concentration of chicory dietary fiber powder. It was confirmed that chicory dietary fiber powder is effective as a prebiotic against Lactobacillus rhamnosus FB019 strain.

[Experimental Example 3] Effect on intestinal flora in subjects with high coffee intake

Fecal samples obtained from a total of 19 subjects who frequently consume coffee (subjects who consume coffee more than 3 to 4 times a week based on espresso shots; consume 1 shot of espresso per intake) were injected into the intestine according to Experimental Example 1. A flora culture system was applied. Afterwards, Lactobacillus rhamnosus FB019 strain and Lactobacillus comparison strain were treated in the culture system at a concentration of 2.00E+08 CFU/ml and cultured at 37°C for 24 hours. After culturing, the relative abundance of intestinal flora present in the samples was analyzed. Specifically, taxonomic classification was performed based on the QIIME2 pipeline, and alignment was performed using the SILVA database. Then, by relatively comparing the intestinal flora at the phylum, class, order, family, and genus levels, changes in the flora according to strain intake were confirmed and the results were reported. It is shown in 3.

As a result, as shown in Figure 3, compared to the control group (Control) in which only feces were cultured, the Lactobacillus genus was confirmed to be at the 1% level, in the group treated with the Lactobacillus rhamnosus FB019 strain, the Lactobacillus genus was improved by 7%. , which corresponds to a relatively high level compared to other comparative strain groups.

As a result of analyzing the effect of treatment with the Lactobacillus rhamnosus FB019 strain for each subject, it was confirmed that 16 out of 19 subjects (84%) showed an effect of increasing the Lactobacillus genus in the bacterial flora of the feces, as shown in Figure 4.

Therefore, these results show that the Lactobacillus rhamnosus FB019 strain is superior to other Lactobacillus comparison strains in increasing beneficial bacteria (Lactobacillus) in the intestinal flora of people who consume a lot of coffee, and maintains the balance of normal intestinal flora. It was confirmed that intestinal health can be significantly improved while maintaining it.

[Experimental Example 4] Changes in intestinal flora in subjects depending on the frequency of coffee consumption

An experiment was conducted to determine differences in the subject's intestinal flora depending on the frequency of coffee consumption and changes in the flora when lactic acid bacteria were consumed.

As in Example 3, there were a total of 19 subjects with frequent coffee consumption (subjects who consumed coffee more than 3 to 4 times a week) and those with low coffee consumption (subjects who did not consume coffee or consumed coffee less than 1 to 4 times a month). The intestinal flora culture system according to Experimental Example 1 was applied to fecal samples obtained from a total of 5 subjects. Afterwards, Lactobacillus rhamnosus FB019 strain was treated in the culture system at a concentration of 2.00E+08 CFU/ml and cultured at 37°C for 24 hours. After culturing, the relative abundance of intestinal flora present in the samples was analyzed. Specifically, taxonomic classification was performed based on the QIIME2 pipeline, and alignment was performed using the SILVA database. Then, by relatively comparing the intestinal flora at the phylum, class, order, family, and genus levels, changes in the flora according to strain intake were confirmed and the results were reported. It is shown in 5. In addition, based on the analysis results of the relative ratio of intestinal flora, the LDA score of beneficial bacteria was calculated using a Python-based LEfSe (Linear discriminant analysis Effect Size) analysis program, and the results are shown in Figure 6(A). The LDA score is a number derived from LDA effect size (LEfSe) analysis, and is calculated by reflecting statistical significance information, biological consistency, and effect size estimation. The results of confirming the relative proportion of intestinal beneficial bacteria (Lactobacillus genus) depending on whether or not FB019 was treated are shown in Figure 6(B).

According to Figure 5, as a result of confirming changes in the composition of intestinal microorganisms according to the frequency of coffee consumption, the relative content of the genus Prevotella was found to be higher in the group with frequent coffee consumption. On the other hand, in the group that consumed coffee less frequently, the genus Bacteroides was present at a high rate. From these results, it can be seen that changes may occur in the subject's intestinal flora depending on the frequency of coffee consumption. Additionally, in the group with frequent coffee consumption, the genera Prevotella and Lachnospiraceae were present at high rates when treated with the FB019 strain. In the group with low coffee consumption, the proportions of the genera Bacteroides and Lachnospiraeceae increased, and the proportions of the genera Prevotella and Escherichia decreased.

According to Figure 6, as a result of treating the FB019 strain in groups divided according to the frequency of coffee consumption, the relative content of the Lactobacillus genus increased significantly and significantly in the group that frequently consumed coffee. In particular, it was confirmed that the Lactobacillus genus increased statistically significantly in the group that frequently consumed coffee.

Specifically, according to the LDA score results in Figure 6(A), when the FB019 strain was treated, the relative content of the Lactobacillus genus significantly increased in the group that frequently consumed coffee, and even in the group that consumed coffee less frequently, the relative content of the Lactobacillus genus increased significantly. It can be seen that the content has increased. In addition, according to the relative abundance results for the Lactobacillus genus in Figure 6(B), in the case of the group that frequently consumed coffee, the relative abundance of the Lactobacillus genus present in the subject's intestinal flora was approximately as the FB019 strain was treated. It was confirmed that it increased from 0.01 to 0.06~0.07, a significant increase of 6 to 7 times, and the relative content of Lactobacillus was confirmed to increase even in the group that consumed coffee less frequently. In addition, when the FB019 strain was treated, it was confirmed that the relative content of the Lactobacillus genus in the group with frequent coffee consumption increased significantly compared to the group with low coffee consumption.

In summary, the Lactobacillus rhamnosus FB019 strain according to an embodiment of the present disclosure has the effect of increasing the content of the Lactobacillus genus in both groups with high coffee consumption (i.e., subjects with high coffee consumption) and groups with low coffee consumption. Accordingly, it was confirmed that the effect of increasing beneficial bacteria (Lactobacillus) present in the intestinal flora was excellent in all groups and that intestinal health could be significantly improved while maintaining the balance of normal intestinal flora. . In addition, the FB019 strain showed the effect of significantly increasing the relative content of the Lactobacillus genus in the group with frequent coffee consumption compared to the group with low coffee consumption, and accordingly, the FB019 strain increased the presence of beneficial bacteria in the intestinal flora of subjects with high coffee consumption. It may be useful as a food (or health functional food) to increase .

From the above description, a person skilled in the art to which the present disclosure pertains will be able to understand that the present disclosure can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the examples or experimental examples described above should be understood as illustrative in all respects and not restrictive. The scope of the present disclosure should be construed as including the meaning and scope of the claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present disclosure.

Korea Research Institute of Bioscience and Biotechnology KCTC14784BP 20211118 Korea Research Institute of Bioscience and Biotechnology KCTC14785BP 20211118

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Claims (17)

A novel Lactobacillus rhamnosus FB019 strain deposited under the accession number KCTC14784BP containing the 16S rRNA base sequence shown in SEQ ID NO: 1. A probiotic composition comprising the strain of claim 1. The probiotic composition according to claim 2, comprising the strain at a concentration of 1Х10 ⁸ to 1Х10 ¹¹ CFU/g. A food composition comprising the probiotic composition of claim 2. The food composition according to claim 4, further comprising prebiotics. The method of claim 5, wherein the prebiotics include fructooligosaccharides, chicory dietary fiber, inulin, isomaltooligosaccharides, lactitol, lactosucrose, lactulose, pyrodextrin, soy oligosaccharides, and xylo oligosaccharides. (Xylo-oligosaccharide). One or more food compositions selected from the group consisting of. The food composition according to claim 5, wherein the prebiotic is chicory dietary fiber. The food composition according to claim 4, which is in the form of powder, granule, tablet, capsule, liquid, powder or fermented milk. The food composition according to claim 4, further comprising one or more of additives, carriers, excipients, and flavoring agents that can be used in food. The food composition according to claim 4, which is a health functional food composition. The food composition according to claim 4, which is a freeze-dried product. The food composition according to claim 4, which is used to increase beneficial bacteria in the intestinal flora of subjects who consume a lot of coffee. The food composition according to claim 12, wherein the subject consumes coffee 3 to 4 times or more per week. A composition for food addition containing the strain of claim 1. A composition for animal feed containing the strain of claim 1. Contains probiotics,
The probiotic is a novel Lactobacillus rhamnosus FB019 strain deposited under the accession number KCTC14784BP containing the 16S rRNA base sequence shown in SEQ ID NO: 1,
A health functional food composition for increasing beneficial bacteria in the intestinal flora of subjects who consume a lot of coffee. delete

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