KR20240115996A - Bifidobacterium longum subsp. infantis DS2770 and Use Thereof - Google Patents

Abstract

The present invention relates to a novel Bifidobacterium longum subsp. Infantis DS2770 strain (KCTC 14811BP) and its use, which strain contains 3'-sialyllactose, a major component of breast milk. ) and 6'-sialyllactose (6'-sialyllactose) as the only carbon source, as it has the property of being able to grow by metabolizing them, so it is used to improve intestinal function in infants, improve immune function, and promote cognitive development. It can be usefully used.

Description

New Bifidobacterium longum subspecies infantis strain and use thereof {Bifidobacterium longum subsp. infantis DS2770 and Use Thereof}

The present invention relates to novel Bifidobacterium longum subsp. Infantis strains and uses thereof.

Human milk oligosaccharides (HMO) are very important components of breast milk and are the most abundant solid component after lactose and lipids. It provides healthy development and ideal nutritional balance for infants and contains numerous bioactive components such as immunoglobulins, hormones, oligosaccharides, etc.

Sialated oligosaccharides are one of the oligosaccharides that make up the second most abundant component in HMOs, promote the growth of bifidobacteria, and have characteristics of resistance to infectious diseases from pathogens (especially intestinal infectious diseases), immune function, and newborn brain development. . It may also have a positive effect on intestinal microflora, intestinal maturation, and inflammation. Sialated oligosaccharides have significant health benefits for newborns due to their role in supporting pathogen resistance, intestinal maturation, immune function, and cognitive development.

Microbial colonization of the neonatal gut is important for normal maturation and development. This initial microbiota establishment process, which is known to be influenced by multiple factors, constitutes an important determinant for immune health. If microbial colonization is lacking, you are easily exposed to diseases such as inflammatory diseases, obesity, and immune diseases.

There is a significant difference in the types of microorganisms that dominate the microbial colonization of newborns when they consume breast milk or formula. When breast milk is consumed, Bifidobacterium species dominate and beneficial species such as Lactobacillus and Staphylococcus increase, while when formula is consumed, in addition to Bifidobacterium species, Bifidobacterium species and Clostylium species increase. It has been reported that species such as In particular, Bifidobacterium infantis is a representative species known to have a group of enzymes that decompose breast milk oligosaccharides, and is known to play an important role in the intestinal health of newborns and the formation of early microbial communities.

Recently, the development of foods containing breast milk oligosaccharides has been actively conducted, but research on developing beneficial bacteria that effectively decompose breast milk oligosaccharides is relatively lacking. Recently, a patent has been developed on a strain that effectively utilizes fucosyllactose (FL). has been registered. Accordingly, in the present invention, 3'-Sialyllactose (hereinafter referred to as 3'-SL) and 6'-Sialyllactose (hereinafter referred to as 6'-SL), which are other main components of breast milk oligosaccharides, ) A new Bifidobacterium subspecies Infantis strain with excellent usability was isolated from newborn feces and its characteristics of using 3'-SL and 6'-SL as the sole carbon source were investigated to complete the present invention.

The purpose of the present invention is to provide a novel Bifidobacterium longum subsp. Infantis strain DS2770.

In addition, the present invention aims to provide a composition containing as an active ingredient at least one selected from the group consisting of the strain, the culture medium of the strain, the concentrate of the culture medium, the dried product of the culture medium, and the extract of the culture medium.

In addition, the present invention aims to provide a composition that is a pharmaceutical composition, health functional food composition, food composition, or feed composition for improving the intestinal health of infants.

Additionally, the present invention aims to provide a composition that is a pharmaceutical composition, health functional food composition, food composition, or feed composition for improving the immune function of infants.

Additionally, the present invention aims to provide a composition that is a pharmaceutical composition, health functional food composition, food composition, or feed composition for promoting cognitive development in infants.

In order to achieve the above object, one aspect of the present invention provides a strain of Bifidobacterium longum subsp. infantis DS2770 ( Bifidobacterium longum subsp. infantis DS2770) deposited with accession number KCTC 14811BP.

Another aspect of the present invention provides a Bifidobacterium longum subspecies Infantis DS2770 strain that has the activity of decomposing sialyllactose.

Another aspect of the present invention provides a Bifidobacterium longum subspecies Infantis DS2770 strain capable of growing even in an environment where sialyllactose is the only carbon source.

Meanwhile, in the present invention, the strain may preferably have one or more properties selected from acid resistance, bile resistance, antibiotic resistance, and intestinal adhesion ability.

Another aspect of the present invention includes as an active ingredient at least one selected from the group consisting of the Bifidobacterium longum subsp. Infantis DS2770 strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, and an extract of the culture. Provides a composition that

Another aspect of the present invention provides a composition that is a pharmaceutical composition, health functional food composition, food composition, or feed composition for improving the intestinal health of infants.

Another aspect of the present invention provides a composition that is a pharmaceutical composition, health functional food composition, food composition, or feed composition for improving the immune function of infants.

Another aspect of the present invention provides a composition that is a pharmaceutical composition, health functional food composition, food composition, or feed composition for promoting cognitive development in infants.

The Bifidobacterium longum subspecies Infantis DS2770 strain (KCTC 14811BP) of the present invention contains only 3'-sialyllactose and 6'-sialyllactose, which are major components of breast milk. They can grow by metabolizing them in an environment where they exist as a carbon source.

Therefore, the strain shows an excellent growth rate in an environment where 3'-sialyllactose and 6'-sialyllactose exist as the only carbon source.

In addition, the strain has excellent acid resistance, bile resistance, antibiotic resistance, and intestinal cell adhesion, making it suitable for use as probiotics.

In addition, the composition of the present invention containing the strain or culture medium of the strain has the effect of improving the intestinal function of infants, inhibiting the growth of pathogenic microorganisms, improving immune function, or promoting the development of cognitive function. There is.

However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

Figure 1 shows the growth curve of the Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention using 3'-sialyllactose (hereinafter, 3'-SL) as a carbon source.
Figure 2 shows the growth curve of the Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention using 6'-sialyllactose (hereinafter referred to as 6'-SL) as a carbon source and the amount of residual sialyllactose in the culture medium.
Figure 3 shows the acid resistance and bile resistance of the Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention.
Figure 4 shows the intestinal attachment rate of the Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention according to carbon source feeding.
Figure 5 shows a comparison of the measured antibiotic resistance values of the Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention and the EFSA guide strain.
Figure 6 shows the intestinal adhesion inhibition effect of the Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention against harmful bacteria.

Hereinafter, the present invention will be described in detail.

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

One. Novel Bifidobacterium longum subspecies infantis strain

One aspect of the present invention provides a novel Bifidobacterium longum subsp. infantis DS2770 ( Bifidobacterium longum subsp. infantis DS2770) strain.

The new Bifidobacterium longum subspecies Infantis strain of the present invention may be one in which 16S rRNA is encoded by the base sequence of SEQ ID NO: 1, and may in particular be the Bifidobacterium longum subspecies Infantis DS2770 strain, specifically in 2022. It may be a Bifidobacterium longum subspecies Infantis strain deposited at the Korea Research Institute of Bioscience and Biotechnology Microbial Resource Center (Korean Collection for Type Culture, KCTC) on December 7 with accession number KCTC 14811BP.

The term " Bifidobacterium longum subsp. Infantis strain" of the present invention refers to an Infantis strain of the subspecies taxon Bifidobacterium longum, wherein Bifidobacterium longum is a human It is a Gram-positive lactic acid bacterium present in the gastrointestinal tract, and is known to be a catalase-negative, rod-shaped bacterium and a member of the genus Bifidobacterium .

Probiotics refer to live microorganisms that provide health benefits and play an important role in suppressing harmful bacteria and increasing beneficial bacteria in the intestinal flora. In order to be recognized as a probiotic, it must survive gastric acid and bile acid, reach the small and large intestines, proliferate and settle in the intestine, exhibit useful effects in the intestine, and be non-toxic and non-pathogenic.

The strain of the present invention may have the ability to utilize one or more sugars selected from the group consisting of glucose, lactose, sucrose, maltose, mannose, and raffinose.

In terms of sugar utilization, the strain of the present invention may utilize glucose, lactose, sucrose, maltose, mannose, and raffinose as carbon sources.

In a specific embodiment of the present invention, the culture medium of the strain of the present invention and the KTCT 3249 standard strain was mixed with glucose (GLU), mannitol (MAN), lactose (LAC), sucrose (SAC), maltose (MAL), and selicin (SAL), respectively. , xylose (XYL), arabinose (ARA), gelatin (GEL), esculin (ESC), glycerol (GLY), cellobiose (CEL), mannose (MNE), melegitose (MLZ), raffinose. After inoculation into tubes containing ose (RAF), sorbitol (SOR), rhamnose (RHA), or trehalose (TRE), the sugar utilization ability of the strain was measured. As a result, the strain of the present invention can utilize glucose, lactose, sucrose, maltose, mannose, and raffinose, and mannitol, selicin, xylose, arabinose, gelatin, esculin, glycerol, cellobiose, and melegito. It was confirmed that ose, sorbitol, rhamnose and trehalose were not available. In addition, it was confirmed that the strain of the present invention, unlike the standard strain, cannot utilize mannitol, selicin, xylose, esculin, cellobiose, melegitose, and trehalose.

Therefore, the strain of the present invention not only differs from the standard strain in 16S rRNA base sequence, but also differs in terms of sugar utilization ability of the strain depending on the type of sugar, and was confirmed to be a new strain different from the conventional strain.

The strain of the present invention may have physiological characteristics that allow it to be used as a probiotic.

Specifically, the strain may have acid resistance. The acid resistance refers to the property of the strain to survive well in an acidic environment, in an environment of pH 5 or less, such as pH 4.5 or less, pH 4 or less, pH 3.5 or less, especially in an acidic environment of pH 3 or less. Even after 1 hour or more, such as 1.5 hours or more, 2 hours or more, 2.5 hours or more, especially 3 hours or more, the survival rate does not decrease, or rather, the survival rate increases by 220% or more, such as 225% or more, 230% or more, or 235%. This may mean an increase of more than 240%, especially more than 245%.

In a specific example of the present invention, the Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention showed a survival rate of about 247% even after being cultured for 3 hours at pH 3, confirming that it had excellent acid resistance.

Additionally, the strain may have bile resistance. The bile resistance refers to the ability of the strain to survive well in a biliary environment in which bile distributed from the gallbladder exists, where bile such as oxgall, bile salt, etc. is 5% or less, for example, 4.5% or less, In the biliary environment, present in concentrations of 4% or less, 3.5% or less, especially 3% or less, for more than 5 hours, such as more than 6 hours, more than 7 hours, more than 8 hours, more than 9 hours, more than 10 hours, more than 11 hours. , In particular, even after 12 hours or more, the survival rate does not decrease, or rather, it may mean that the survival rate increases by more than 200%, for example, more than 300%, and especially more than 400%.

In a specific example of the present invention, the Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention showed a survival rate of about 405% when cultured for 12 hours in a medium supplemented with 3% bile salts, showing excellent bile resistance. It was confirmed that

The Bifidobacterium longum subspecies Infantis DS2770 strain has excellent acid resistance and bile resistance, so it has the effect of being able to reach the intestines in a surviving state without dying in the stomach.

In addition, the strain may have intestinal adhesion ability. The intestinal adhesion ability refers to the property of the strain to attach to intestinal cells and not detach. If the intestinal adhesion ability is excellent, the time the strain can stay in the intestines increases, improving intestinal flora as a probiotic, improving bowel movements and intestinal health. This means that the effect of improving can be maintained for a long time. Specifically, the intestinal adhesion ability refers to the initial viable cell count after the strain reaches the intestine and attaches to intestinal cells and remains in the intestinal environment for more than 12 hours, such as more than 15 hours, more than 18 hours, more than 21 hours, especially more than 24 hours. This may mean that live bacteria of 1.0% or more, such as 1.1% or more, 1.2% or more, especially 1.3% or more, are attached to intestinal cells.

In a specific example of the present invention, the strain of the present invention was treated with the Caco-2 cell line and cultured with glucose as a carbon source, and after washing with a PBS solution, the intestinal attachment rate (%) of the strain was confirmed. As a result, the strain showed an intestinal adhesion rate of 1.3%, confirming that it had excellent intestinal adhesion ability.

Therefore, the novel Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention has excellent acid resistance and bile resistance, so it can reach the intestines in a surviving state without dying in the stomach, and even the strain that reaches the intestines adheres to the intestines. Because it has excellent properties, the time it can stay in the intestines increases, so the effects of improving intestinal flora, bowel movements, and intestinal health as probiotics can be maintained for a long time. Therefore, since the above strain satisfies all the advantageous conditions that must be met as a probiotic, the novel Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention is useful as a probiotic that improves intestinal flora, promotes bowel activity and intestinal health. can be used

On the other hand, the strain may have better intestinal attachment ability in an environment in which 3'-SL or 6'-SL exists as a carbon source compared to an environment in which glucose exists as a carbon source. Specifically, the intestinal adhesion ability is achieved by attaching to intestinal cells after the strain reaches the intestine and maintaining it for more than 12 hours, such as more than 15 hours, more than 18 hours, more than 21 hours, especially in the intestinal environment where 3'-SL exists as a carbon source. After 24 hours or more, it means that more than 2.0% of the initial viable bacteria count, for example, more than 2.5%, more than 3%, more than 3.5%, especially more than 4.0% of the viable bacteria are attached to the intestinal cells, or the strain is After reaching the intestine, it attaches to intestinal cells and 6'-SL exists as a carbon source in the intestinal environment. After 12 hours or more, such as 15 hours or more, 18 hours or more, 21 hours or more, especially 24 hours or more, the initial viable cell count This may mean that live bacteria of 2.0% or more, such as 2.3% or more, 2.6% or more, especially 2.9% or more, are attached to intestinal cells.

In addition, the strain has an intestinal attachment rate in an environment in which 3'-SL exists as a carbon source compared to an environment in which glucose exists as a carbon source, such as 2.5 times more, 3 times more, 3.5 times more, especially 3 times more. This may indicate a high attachment rate, and the strain is 1.4 times more, for example, 1.6 times more, 1.8 times more, 2 times more, especially 2.2 times more than that in an environment where 6'-SL exists as a carbon source compared to an environment where glucose exists as a carbon source. It may be that the intestinal attachment rate is more than twice as high.

In a specific experimental example of the present invention, the strain of the present invention was treated with the Caco-2 cell line, cultured with glucose, 3'-SL, or 6'-SL as a carbon source, respectively, and washed with a PBS solution, and then inoculated into the intestine of the strain. The attachment rate (%) was confirmed. As a result, the strain showed an intestinal attachment rate that was about 3 times and about 2.2 times higher when 3'-SL or 6'-SL was used as a carbon source, respectively, compared to when glucose was used as a carbon source, and the strain was 3 It was confirmed that the intestinal adhesion ability was better in an environment where '-SL or 6'-SL existed as a carbon source compared to an environment where glucose existed as a carbon source.

Additionally, the strain may not be resistant to antibiotics or may have low resistance to antibiotics. The resistance may be extrinsic resistance, and the extrinsic resistance refers to resistance that can be induced by introducing a resistance gene to an antibiotic into another external bacterium through a mobile means such as a plasmid or transposon. Therefore, the Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention, which does not exhibit extrinsic resistance to antibiotics, does not horizontally transfer resistance genes to harmful bacteria existing in the intestines, so the harmful bacteria in the intestines acquire extrinsic resistance to antibiotics. There is no fear of causing resistance problems.

Antibiotics to which the Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention does not show resistance are not particularly limited as long as they are known in the technical field to which the present invention pertains, and include, for example, penicillin-based antibiotics, tetracyclines-based antibiotics, These may be macrolide antibiotics, sulfonamide antibiotics, amphenicol antibiotics, aminoglycoside antibiotics, etc., specifically vancomycin and streptomycin. , erythromycin, clindamycin, tetracycline, and chloramphenicol.

In a specific example of the present invention, the DS2770 strain was tested for antibiotic resistance using MTS ^TM (MIC Test Strip) (Liofilchem). As a result, the strain was resistant to vancomycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol according to the EFSA guide (European Food Safety Authority Antibiotic Safety Standard) ) was confirmed to be lower than that stated. In particular, in Europe, cases where lactic acid bacteria can horizontally transfer antibiotic resistance genes to other bacteria cannot be commercialized for a long time, and in Korea, the Ministry of Food and Drug Safety recently added a provision regarding transfer of antibiotic resistance to the criteria for judging microorganisms as food raw materials. , the Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention has the advantage of being commercialized as its safety is recognized as a probiotic.

In addition, the Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention may inhibit the growth of harmful bacteria, and the strain may be used in an environment where 3'-SL or 6'-SL, especially 6'-SL, is present as a carbon source. This may inhibit the growth of the harmful bacteria by about three times or more compared to an environment where glucose exists as a carbon source.

In addition, the Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention may inhibit intestinal colonization of harmful bacteria, and the strain has 3'-SL or 6'-SL, especially 6'-SL, as a carbon source. Compared to an environment in which glucose exists as a carbon source, the intestinal colonization of the harmful bacteria may be suppressed by about two times or more.

In a specific example of the present invention, the DS2770 strain of the present invention is cultured in a medium containing glucose and 6'-SL as carbon sources, respectively, and then a competitive analysis and a defensive analysis are performed to determine the growth of beneficial and harmful bacteria according to the treatment of the DS2770 strain. and settlement characteristics were analyzed. As a result, it was confirmed that the growth and intestinal adhesion of harmful bacteria were further inhibited when 6'-SL was used compared to when glucose was used.

Therefore, the Bifidobacterium longum subsp. Infantis DS2770 strain of the present invention has excellent efficacy in suppressing the growth of harmful bacteria and inhibiting intestinal adhesion, especially when consumed using 6'-SL as a prebiotic.

According to another aspect of the present invention, the Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention may have the activity of decomposing sialyllactose.

The sialyllactose is one of the Human Milk Oligosaccharides (HMOs) most abundant in breast milk's colostrum, and can improve intestinal function, improve brain development and cognition, and increase immune function to prevent infections. , for example, 3' sialyllactose (3'-SL) or 6' sialyllactose (6'-SL), but is not limited thereto.

The human milk oligosaccharides (HMO) refer to multifunctional glycans that naturally exist in human breast milk.

Specifically, the sialyllactose is one of the oligosaccharides that accounts for the second most abundant component in HMO, can promote the growth of bifidobacteria, has resistance to infectious diseases caused by pathogens, and develops immune and cognitive functions in infants. Since it can help, it can act as a useful ingredient in the overall development of infants.

In addition, the Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention may be capable of growing even in an environment where sialyllactose is the only carbon source.

The sialyllactose can be understood in the same way as the sialyllactose described above.

In a specific example of the present invention, the availability of 3'-SL or 6'-SL of the Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention was measured, and as a result, the strain of the present invention was 3'-SL or It showed a high growth rate even in a medium environment where 6'-SL was present at a high concentration as the only carbon source, and it was confirmed that growth efficiency was excellent even in an environment where 3'-SL or 6'-SL was present as the only carbon source.

Therefore, the Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention has the activity of decomposing sialyllactose and can grow even in an environment where sialyllactose is the only carbon source, so it has excellent usability for the sialyllactose. Therefore, it can be useful as an active ingredient in compositions that help with the development of intestinal function, brain development, or immune function in infants who consume breast milk.

2. Composition containing the novel strain of the present invention

In another aspect of the present invention, the Bifidobacterium longum subsp. Infantis DS2770 strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, and an extract of the culture contain at least one selected from the group consisting of an active ingredient. It provides a composition comprising:

The culture medium is obtained by culturing the Bifidobacterium longum subspecies Infantis DS2770 strain, and may be the culture medium itself containing cells of the strain, or a culture supernatant obtained by removing cells therefrom, and may also be a culture supernatant obtained by removing cells therefrom. It may be filtrate, concentrate or dried. The culture medium from which the cells have been removed contains components produced and secreted by the Bifidobacterium longum subspecies Infantis DS2770 strain, such as metabolites, and has excellent acid resistance and bile resistance, so it survives in the stomach without dying. It may have probiotic activity that promotes the growth of beneficial bacteria.

The concentrate increases the solid concentration of the culture medium, and may be a concentrate of the culture medium containing the lactic acid bacteria cells or a concentrate of the culture supernatant from which the lactic acid bacteria cells have been removed. The concentrate may be concentrated by vacuum concentration, plate concentration, thin film concentration, etc., but is not limited thereto, and may be performed at a temperature of 40°C to 60°C using, for example, a known concentrator. Depending on the concentration of the concentrate, the content of the culture medium included in the composition of the present invention can be appropriately adjusted.

The dried product includes, but is not limited to, those dried through methods such as freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, spray drying, foam drying, high frequency drying, and infrared drying.

The extract means extracted from the culture medium or its concentrate, and may include an extract, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, or a crude or purified product thereof, or a fraction thereof.

In addition, the composition is suitable for ingestion together with the lactic acid bacteria of the present invention, inhibits the growth of harmful microorganisms upon ingestion, and may further include other types of known microorganisms that have the activity of improving the balance of intestinal flora. .

The composition containing the Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention is prepared by adding carriers, excipients and/or additives according to a method that can be easily performed by a person skilled in the art to which the present invention pertains. It can be manufactured in unit dose form by formulating it or by placing it in a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, capsule, gel (e.g., hydrogel), or freeze-dried agent, and the additives include a dispersant, A stabilizer or cryoprotectant may additionally be included.

Specifically, in the case of the freeze-drying agent, it involves freeze-drying the strain with a cryoprotectant and using it in the form of a powder, wherein the freeze-protectant includes skim milk powder, maltodextrin, dextrin, trehalose, maltose, lactose, mannitol, and cyclo. It may be dextrin, glycerol and/or honey. In addition, it includes mixing with a preservation carrier, adsorbing it, drying it, and solidifying it for use. The preservation carrier may be diatomaceous earth, activated carbon, and/or degreased steel.

The composition containing the Bifidobacterium longum subsp. Infantis DS2770 strain of the present invention includes at least one selected from the group consisting of a strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, and an extract of the culture. It can be manufactured through mixing with any one of carriers, excipients, or additives.

Descriptions of the strain, carrier, excipients and additives are as described above. When using a cryoprotectant as the additive, the composition containing the Bifidobacterium longum subspecies Infantis DS2770 strain is mixed with the strain and the cryoprotectant, and the mixture is frozen at -45 ° C to -30 ° C. Afterwards, it may be dried at 30°C to 40°C, ground in a blender, and manufactured in the form of freeze-dried powder. Specifically, the freezing process may be a process of vacuum freezing for 65 to 75 hours under temperature conditions of -45°C to -30°C and pressure conditions of 5 to 50 mTorr.

When the composition of the present invention further contains a cryoprotectant, damage or death of the strain can be suppressed during the process of freeze-drying the composition, and the freeze-dried composition has advantages in the storage, distribution, and storage process. . Additionally, the freeze-dried composition can be ingested in powder form, and in this case, the strain in the composition can grow or metabolize in the body and exhibit its activity.

3. Use of the new strain of the present invention or a composition containing the same

Another aspect of the present invention is that the composition containing the Bifidobacterium longum subsp. Infantis DS2770 strain may be a pharmaceutical composition, a health functional food composition, a food composition, or a feed composition.

Specifically, in another aspect of the present invention, the composition containing the Bifidobacterium longum subsp. Infantis DS2770 strain may be a pharmaceutical composition, a health functional food composition, a food composition, or a feed composition for improving the intestinal health of infants.

The improvement in intestinal health may be helpful for beneficial proliferation and suppression of harmful bacteria in the intestines, helpful for intestinal health by regulating immunity, or helpful for smooth bowel movements.

In the present invention, the term "improvement" refers to any action that includes improvement, suppression, or delay of symptoms, and may be used interchangeably with the above prevention or treatment.

If the composition is a pharmaceutical composition, it may be a pharmaceutical composition for improving intestinal health, and if the composition is a food or health functional food composition, it may be a food composition or a health functional food composition for improving intestinal health, and the composition This feed additive composition may be a feed additive composition for improving the intestinal health of animals other than humans.

In addition, the health functional food refers to a food using raw materials or ingredients with functionality useful to the human body, and has the functionality to maintain and improve health through maintaining the normal function of the human body or activating physiological functions. The health functional food of the present invention corresponds to a health functional food under the Health Functional Food Act. Since the Bifidobacterium longum subspecies Infantis DS2770 strain has probiotic activity when ingested, the strain, the culture fluid of the strain, the concentrate of the culture fluid, the dried product of the culture fluid, and the extract of the culture fluid have the function of improving intestinal health. As a functional raw material, it can be included as an active ingredient in health functional foods.

In addition, another aspect of the present invention is that the composition containing the Bifidobacterium longum subsp. Infantis DS2770 strain may be a pharmaceutical composition, a health functional food composition, a food composition, or a feed composition for improving the immune function of infants.

The term immunity refers to a mechanism for physiologically recognizing and excluding exogenous and endogenous foreign substances in the body of humans and animals, and maintaining homeostasis. Here, foreign substances are called antigens, and numerous types of white blood cells and proteins are involved in the process of removing them. The improvement of immune function may improve the function of white blood cells and proteins related to immune function or improve diseases or adverse health reactions caused by decreased immunity.

Diseases caused by decreased immune function may specifically include, but are not limited to, infectious diseases, inflammatory diseases, allergic diseases, or chronic fatigue.

The meaning of the improvement can be understood in the same way as the meaning of the improvement described above.

If the composition is a pharmaceutical composition, it may be a pharmaceutical composition for improving immune function, and if the composition is a food or health functional food composition, it may be a food composition or health functional food composition for improving immune function, and the composition In the case of a feed additive composition, it may be a feed additive composition for improving the immune function of animals other than humans.

In addition, the health functional food refers to a food using raw materials or ingredients with functionality useful to the human body, and has the functionality to maintain and improve health through maintaining the normal function of the human body or activating physiological functions. The health functional food of the present invention corresponds to a health functional food under the Health Functional Food Act. Since the Bifidobacterium longum subspecies Infantis DS2770 strain has probiotic activity when ingested, the strain, the culture fluid of the strain, the concentrate of the culture fluid, the dried product of the culture fluid, and the extract of the culture fluid have the function of improving immune function. As a functional raw material, it can be included as an active ingredient in health functional foods.

In addition, another aspect of the present invention is that the composition containing the Bifidobacterium longum subsp. Infantis DS2770 strain may be a pharmaceutical composition, health functional food composition, food composition, or feed composition for promoting cognitive development in infants.

The promotion of cognitive function development may be promoting the development of mental activities or processes that obtain knowledge and understanding through thinking, experience, and sensation. This may include processes such as knowledge, attention, memory and working memory, judgment and evaluation, reasoning and calculation, problem solving and decision making, and understanding and production of language.

If the composition is a pharmaceutical composition, it may be a pharmaceutical composition for promoting cognitive function development, and if the composition is a food or health functional food composition, it may be a food composition or health functional food composition for promoting cognitive function development, If the composition is a feed additive composition, it may be a feed additive composition for promoting cognitive function development in animals other than humans.

In addition, the health functional food refers to a food using raw materials or ingredients with functionality useful to the human body, and has the functionality to maintain and improve health through maintaining the normal function of the human body or activating physiological functions. The health functional food of the present invention corresponds to a health functional food under the Health Functional Food Act. Since the Bifidobacterium longum subspecies Infantis DS2770 strain has probiotic activity when ingested, the strain, the culture fluid of the strain, the concentrate of the culture fluid, the dried product of the culture fluid, and the extract of the culture fluid are used to promote cognitive function development. As a functional raw material, it can be included as an active ingredient in health functional foods.

The composition containing the strain is 0.00001% by weight to 80% by weight, for example, 0.00001% by weight to 60% by weight, 0.00001% by weight to 40% by weight, 0.00001% by weight to 30% by weight, 0.00001% by weight, based on the total weight of the composition. % to 20% by weight, 0.00001% to 10% by weight, 0.00001% to 5% by weight, 0.05% to 60% by weight, 0.05% to 40% by weight, 0.05% to 30% by weight, 0.05% to 0.05% by weight. 20% by weight, 0.05% to 10% by weight, 0.05% to 5% by weight, 0.1% to 60% by weight, 0.1% to 40% by weight, 0.1% to 30% by weight, 0.1% to 20% by weight %, 0.1 wt% to 10 wt%, or 0.1 wt% to 5 wt% of at least one selected from the group consisting of the strain, the culture medium of the strain, the concentrate of the culture medium, the dried product of the culture medium, and the extract of the culture medium. It can be included as an active ingredient. Also, at this time, the Bifidobacterium longum subspecies Infantis DS2770 strain may be included in an amount of 10 ⁵ CFU/g or more, 10 ⁶ CFU/g or more, for example, 10 ⁷ CFU/g or more.

The composition containing the Bifidobacterium longum subsp. Infantis DS2770 strain may be a pharmaceutical composition, food composition, health functional food composition, or feed additive composition.

The pharmaceutical composition is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art to which the present invention pertains. It may be manufactured by placing it in a multi-capacity container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet, capsule, or gel (e.g., hydrogel), and may additionally contain a dispersant or stabilizer. You can.

Additionally, the strain or culture medium thereof contained in the pharmaceutical composition may be transported in a pharmaceutically acceptable carrier such as colloidal suspension, powder, saline solution, lipid, liposome, microsphere, or nano spherical particle. there is. They may form complexes or associate with delivery vehicles and may be used in the art as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancers or fatty acids. It can be transported in vivo using known delivery systems.

In addition, pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyester, and other commonly used carriers. It may include, but is not limited to, vinyl pyrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, in addition to the above ingredients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. may be additionally included. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition can be administered orally or parenterally during clinical administration and can be used in the form of a general pharmaceutical preparation. That is, the pharmaceutical composition of the present invention can be administered in various oral and parenteral dosage forms during actual clinical administration. When formulated, diluents such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants are used. Or it is prepared using excipients. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain herbal extracts or herbal medicinal ferments with at least one excipient, such as starch, calcium carbonate, sucrose, or lactose. It is prepared by mixing gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, wethepsol, macrogol, Tween 61, cacao, laurel, glycerol, gelatin, etc. can be used.

The concentration of the active ingredient included in the pharmaceutical composition can be determined considering the purpose of treatment, patient's condition, required period, etc., and is not limited to a specific concentration range. The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type and severity of the patient's disease. It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition may be administered as an individual treatment, or in combination with other enterological treatments, and may be administered simultaneously, separately, or sequentially with conventional treatments, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, gender, condition, weight, absorption of the active ingredient in the body, inactivation rate, excretion rate, type of disease, and concomitant drug, route of administration, It can be increased or decreased depending on the severity of obesity, gender, weight, age, etc., and for example, about 0.0001 ㎍ to 500 mg, for example, 0.01 ㎍ to 100 mg per 1 kg of patient's body weight can be administered per day. Additionally, depending on the judgment of a doctor or pharmacist, it may be administered in divided doses several times a day at regular time intervals, for example, 2 to 3 times a day.

In addition, the food includes both edible raw materials and processed substances, meaning that it contains various digestible nutrients.

When using the food composition as a food additive, the food composition can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods.

There are no particular restrictions on the type of food. Foods to which the food composition can be added may be probiotic preparations, such as meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, It includes beverages, tea drinks, alcoholic beverages, vitamin complexes, and fermented foods, and includes all health foods in the conventional sense. In particular, the fermented food may be fermented milk, cheese or butter such as yogurt (hard type, soft type, drink type), lactic acid bacteria beverage, but is not limited thereto, and is not limited to any food manufactured by fermentation performed by fermentation microorganisms or lactic acid bacteria. Food and products may also be included.

Additionally, the food composition can be manufactured into food, especially functional food. The functional food includes ingredients commonly added during food production, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when manufactured as a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrins, cyclodextrins, etc.), or sugar alcohols (e.g., , xylitol, sorbitol, erythritol, etc.) are preferred. The flavoring agent may be a natural flavoring agent (e.g., thaumatin, stevia extract, etc.) or a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).

In general, the health functional food composition of the present invention may appropriately contain the above functional raw materials as active ingredients depending on the purpose of use (prevention or improvement). For example, it can be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less. However, in case of long-term intake for health purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

The health functional food composition can be manufactured in dosage forms such as tablets, granules, soft capsules, hard capsules, syrups, beverages, pills, soft extracts, and water baths. More specifically, the health food composition of the present invention can be manufactured in dosage forms such as tablets, granules, soft capsules, hard capsules, syrups, beverages, pills, soft extracts, dried extracts, and extraction baths. In addition, the health food composition of the present invention contains ingredients commonly added in the production of health functional foods, such as excipients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, and alginic acid, in addition to the ingredients described above in each formulation. It may further include salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, flavorings, pigments, preservatives, etc. The ratio of the added ingredients is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition.

In addition, the feed additive composition is an auxiliary substance mixed in small amounts in feed for the purpose of improving the productivity of livestock, etc., and the feed additive composition of the present invention corresponds to an auxiliary feed under the Feed Management Act.

In the present invention, the term "feed" means any natural or artificial diet, meal, etc., or a component of the meal, for or suitable for eating, ingestion, and digestion by an animal, and the animal is an animal other than a human. . The type of feed is not particularly limited, and feed commonly used in the art can be used. Non-limiting examples of the feed include plant feeds such as grains, roots and fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, cucurbits or grain by-products; Examples include animal feeds such as proteins, inorganic substances, fats and oils, minerals, fats and oils, single-cell proteins, zooplanktons or food. These may be used alone or in combination of two or more types.

Hereinafter, the present invention will be described in detail through examples.

However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[Example 1]

Bifidobacterium longum subsp of the present invention. Isolation and identification of Infantis DS2770 strain

Bifidobacterium longum subsp of the present invention was obtained from feces and breast milk of infants up to 3 years of age. Infantis DS2770 strain was isolated. To identify the above strain, the universal primers 27F (5'-AGA GTT TGA TCM TGG CTC A-3') and 1492R (5'-TAC GGY TAC CTT GTT ACG ACT T-3') were used to identify the DS2770 strain. The base sequence of 16S rRNA was analyzed (SEQ ID NO: 1).

As a result, the base sequence of the 16S rRNA showed 99.85% homology with the conventional strain, Bifidobacterium infantis ATCC 15697(T) strain, confirming that the strain of the invention was a novel strain.

In addition, as a result of homology analysis of the base sequence of the 16S rRNA, it was confirmed that the new strain corresponds to Bifidobacterium longum subspecies Infantis in phylogenetic classification, and the new strain of the present invention was classified as Bifidobacterium longum subspecies Infantis. It was named tis DS2770 ( Bifidobacterium longum subsp. infantis DS2770).

The strain was deposited at the Korean Collection for Type Culture (KCTC) of the Korea Research Institute of Bioscience and Biotechnology on December 7, 2022, and was assigned a deposit number of KCTC 14811BP.

[Example 2]

Confirmation of growth rate of the DS2770 strain of the present invention in 3'-sialyllactose (hereinafter, 3'-SL) and 6'-sialyllactose (hereinafter, 6'-SL) media

Bifidobacterium longum subsp of the present invention. Infantis strain DS2770, the same species strain DS2952, and the standard strain ( Bifidobacterium infantis , KCTC 3249) were cultured for 48 hours in 30 ml of liquid medium with carbon sources of 1% 3'-SL and 0.5% 6'-SL, respectively. Growth rates were compared.

In addition, in order to check the decomposition efficiency for 6'-SL, 2% (v/v) of the pre-cultured culture was inoculated into CDM medium with 0.5% of 6'-SL added as a carbon source, and then inoculated every 8 hours. The growth of the strain was measured by OD ₆₀₀ value. At this time, after centrifuging the collected samples, each supernatant was separated on an Decomposition products were examined. As a result, as shown in Figure 1, the DS2770 strain of the present invention is better than the standard strain (KCTC 3249) as well as the DS2952 strain of the same species in a medium containing 3'-SL or 6'-SL. It was confirmed that the growth rate was excellent. Specifically, when cultured for 48 hours, the DS2770 strain showed a superior growth rate than the DS2952 strain and the standard strain, and was confirmed to use and consume 6'-SL more efficiently than 3'-SL as a carbon source.

Additionally, as shown in Figure 2, Bifidobacterium longum subsp. The growth of the Infantis strain was confirmed to be superior to the standard strain or the DS2952 strain, which showed the highest OD ₆₀₀ value of 1.45 in 6'-SL medium at 24-48 hours after inoculation of the culture medium, and the DS2952 strain showed 1.10. When examining the amount of 6'-SL remaining in the medium at each culture and sample collection time, when 6'-SL was used as a carbon source, the DS2770 strain consumed most of the 6'-SL in about 40 hours of culture. In the KCTC3249 strain and the DS2952 strain, most of the 6'-SL appears to remain in the medium, and it can be confirmed that only the DS2770 strain specifically uses 6'-SL effectively.

[Example 3]

Characterization of probiotics of Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention

In order to confirm whether the Bifidobacterium longum subspecies Infantis DS2770 strain identified in Example 1 has characteristics that can be used as a probiotic, tests were conducted on sugar availability, acid resistance, bile resistance, intestinal adhesion ability, and antibiotic resistance in MRS medium. did.

3-1. Confirmation of sugar availability

In order to confirm the sugar utilization ability of the strain of the present invention, the DS2770 strain and the KTCT 3249 standard strain (control group) were prepared by plating on MRS agar medium. Using the API 20A kit (Bio-Merieux, France), the experiment was conducted by suspending strain colonies in API 20A Medium to a turbidity of 3 McFarland or higher and inoculating them into each well according to the provided instructions.

The types of sugars confirmed in this experiment were glucose (GLU), mannitol (MAN), lactose (LAC), sucrose (SAC), maltose (MAL), selicin (SAL), xylose (XYL), arabinose (ARA), Gelatin (GEL), esculin (ESC), glycerol (GLY), cellobiose (CEL), mannose (MNE), melegitose (MLZ), raffinose (RAF), sorbitol (SOR), rhamnose ( RHA) and trehalose (TRE).

After inoculating the prepared bacterial solution into a sugar tube and culturing for 24 hours, a drop of BCP reagent was added to all sugar tubes (except URE, GEL, and ESC), and the reaction results generated were read according to the reading table. For each reaction, the results are shown in Table 4 with + if positive and - if negative.

Tests GLU MAN LAC SAC MAL SAL XYL ARA GEL KCTC3249 ^T + + + + + + + - - DS2770 + - + + + - - - - Tests ESC GLY CEL MNE MLZ RAF SOR RHA TRE KCTC3249 ^T + - + + + + - - + DS2770 - - - + - + - - -

As a result, as shown in Table 1, the DS2770 strain of the present invention can utilize glucose, lactose, sucrose, maltose, mannose, and raffinose, as well as mannitol, selicin, xylose, arabinose, gelatin, esculin, It was confirmed that glycerol, cellobiose, melegitose, sorbitol, rhamnose and trehalose were not available.

In addition, it was confirmed that the strain of the present invention, unlike the standard strain, cannot utilize mannitol, selicin, xylose, esculin, cellobiose, melegitose, and trehalose.

3-2. Confirmation of acid resistance and bile resistance

The DS2770 strain of the present invention was tested to see if it had acid-resistant growth characteristics for use as a cosmetic composition, fermented food, or food additive.

First, the strain was pre-cultured at 37°C with MRS medium. To confirm the acid resistance of the strain, the strain concentration was adjusted to OD ₆₀₀ = 1, and the strain was inoculated at a concentration of 1% (v/v) in 10 ml of MRS medium adjusted to pH 3 and cultured for 3 hours. .

The survival rates of the strains were compared by comparing the total number of bacteria after 0 hours and 3 hours, and the survival rates after 3 hours were measured.

As a result, as shown in Table 2, the DS2770 strain of the present invention was confirmed to have excellent survival rate with the number of viable cells increasing by about 247% in acidic medium.

Acid resistance (pH 3) 0h 3h Viable count (CFU) Viable count (CFU) Survival rate (%) DS2770 ^{2.1 5.2} >200%

In addition, to confirm the bile resistance of the strain, the strain was adjusted to a concentration of 10 ⁵ CFU/ml and inoculated into 10 ml of fresh MRS medium containing 3% (w/v) (OXOID ^TM ) of bile salts. The survival rate of the strains was compared by comparing the total number of bacteria after 0 hours and 12 hours, and the survival rate after 12 hours was confirmed.

As a result, as shown in Table 3, the DS2770 strain of the present invention was confirmed to have an excellent survival rate with the number of viable cells increasing by about 405% in bile salt medium.

Bile tolerance (bile salts 3%) 0h 12h Viable count (CFU) Viable count (CFU) Survival rate (%) DS2770 ^{4 1.62} >200%

In summary, as shown in Figure 3, the DS2770 strain of the present invention showed excellent survival rates in both acid and bile salt media, and had superior acid resistance and bile resistance compared to the D2952 strain, which is a strain of the same species. When administered as a probiotic, it was judged that the survival rate would be high during passage through the intestinal tract.

3-3. Confirmation of intestinal adhesion ability

To evaluate the intestinal adhesion ability of the DS2770 strain of the present invention, the Caco-2 cell line was used. Specifically, the Caco-2 cell line was cultured in a 5% CO2 incubator (PHC, MCO-230AIC, Indonesia) at a temperature of 37°C using MEM medium containing 10% FBS and 1% penicillin-streptomycin, and 1x105 The cells were dispensed into 24-well plates at a cell/well concentration and cultured for 14 days. Then, the cultured cells were suspended in MEM medium to adjust the final concentration to 1x10 ⁸ CFU/ml, and 1ml was inoculated into each well and cultured for 2 hours. At this time, glucose, 3'-SL, and 6'-SL were used as carbon sources for the strain, respectively. Afterwards, the cultured strain was washed five times with PBS (Phosphate Buffered Saline) solution, treated with 0.5% trypsin-EDTA to remove unattached Caco-2 cells from the plate, and the removed cells were diluted with PBS and plated on MRS plate. After plating on the medium, the number of viable cells was measured after 24 hours.

As a result, as shown in Figure 4, the DS2770 strain of the present invention was confirmed to have excellent intestinal attachment ability by showing an intestinal attachment rate of 1.3% when glucose was used as a carbon source.

Additionally, as shown in Figure 4, when glucose was used as the carbon source, the intestinal adhesion ability was 2.3%, whereas when 3'-SL and 6'-SL were used as the carbon source, respectively, the intestinal adhesion ability was 4.0% and 2.9%, respectively. Since it exhibits excellent intestinal adhesion ability, it was confirmed that the DS2770 strain of the present invention has the effect of improving intestinal adhesion ability in an environment where 3'-SL or 6'-SL is present as a carbon source compared to an environment where glucose is present as a carbon source.

3-4. Confirmation of antibiotic resistance

To confirm the antibiotic resistance of the strain of the present invention, the DS2770 strain was prepared by plating on MRS agar medium. Antibiotic resistance tests were conducted on antibiotics commonly used in the EFSA (European Food Safety Authority) guide using MTS ^TM (MIC Test Strip) (Liofilchem) according to the manufacturer's instructions.

The antibiotics used in this experiment were vancomycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol, and the concentration at the point where the antibiotic strip and the inhibition zone meet was set as the minimum inhibitory concentration.

After the KGC1201 strain was spread on the prepared medium, an antibiotic strip was placed on it, and the culture was cultured at 37°C for 48 hours to measure the minimum inhibitory concentration (MIC) at which growth was inhibited. The results are shown in Table 4.

Strain name control Vancomycin streptomycin Erythromycin Clindamycin tetracycline Chloramphenicol EFSA guide
(μg/ml) 2 128 One One 8 4 DS2770
(μg/ml) Growth 1.6 15.6 0.4 0 1.6 3.1

As a result, as shown in Figure 5, the DS2770 strain of the present invention was confirmed to have lower antibiotic resistance to vancomycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol than the standard values of the EFSA guide. Therefore, since the Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention does not horizontally transfer resistance genes to harmful bacteria existing in the intestines, there is no concern that harmful bacteria in the intestines will acquire extrinsic resistance and cause resistance problems to antibiotics. It has advantages in being commercialized as a probiotic.

[Example 4]

Growth and settlement characteristics of harmful bacteria by new Bifidobacterium longum subspecies Infantis DS2770 strain

In order to confirm the growth of harmful bacteria and the intestinal colonization characteristics of the DS2770 strain of the present invention, competition assay, protection assay, and displacement assay were performed on Clostridium difficile. assay) was performed. Specifically, when the DS2770 strain of the present invention was cultured in a medium using glucose as a carbon source and a medium using 6'-SL as a carbon source, the degree of inhibition of Clostridium difficile growth was compared using a competitive analysis method, and the degree of inhibition of the growth of Clostridium difficile was compared. Using an analysis method, we compared the degree to which the attachment of Clostridium difficile bacteria was inhibited by the attachment of beneficial bacteria, and then used a desorption analysis method to compare the degree to which already attached Clostridium difficile bacteria were removed.

Specifically, first, lactic acid bacteria were cultured in 10 ml of liquid medium containing glucose and 6'-SL, and Clostridium difficile was cultured in 10 ml of RCM (Reinforced Clostridial Broth) liquid medium. Afterwards, the existing medium was removed using PBS, DS2770 and Clostridium difficile were adjusted to about 1Х10 ⁸ CFU/mL, and then suspended in MEM (Minimum Essential Medium), a medium for HT-29 cells.

(1) Competition assay was performed by simultaneously inoculating the cells with DS2770 and harmful bacteria prepared above and reacting them in a CO ₂ incubator at 37°C for 2 hours. Then, the plate was washed five times with PBS to remove non-attached strains. (2) In the Protectiom assay, DS2770 prepared in the same manner as above was first attached to cells for 1 hour, and non-attached strains were removed by washing with PBS 5 times. Then, harmful bacteria were inoculated and reacted for about 1 hour, and non-attached strains were incubated for about 1 hour. It was removed by washing five times with PBS. (3) In the displacement assay, harmful bacteria are first attached to the cells for 1 hour, then non-attached strains are removed by washing them 5 times with PBS, and then the DS2770 strain of the present invention is attached for 1 hour, and non-attached strains are washed with PBS for 5 times. It was removed by washing twice. The strains attached by each method were treated with 0.25% Trypsin-EDTA at 37°C for 10 minutes, the attached strains were removed from the plate, each DNA was extracted, quantitative PCR was performed using the primers shown in Table 5 below, and amplification was performed. The value was calculated using the delta delta ct method to confirm the ability to inhibit adhesion to Clostridium difficile of the 6'-SL-cultured strain compared to the glucose-cultured strain.

B. infantis specific gene Primer Sequence forward 5'-CTGAGATACGGCCCAGACTC-3' reverse 5'-CGCTTTGCTCCCCGATAAAAG-3' C. difficile specific gene forward 5'-AGGCCTTCGGGTCGTAAAAC-3' reverse 5'-CGGTTGAGCCGTAGCCTTTC-3'

As a result, as shown in Figure 6, it was confirmed that the strain grown using 6'-SL as a carbon source inhibited harmful bacteria three times more significantly in competition assay compared to the case where glucose was used as a carbon source. In addition, in the Protectiom assay, which inhibits the attachment of harmful bacteria following the attachment of beneficial bacteria, strains grown using 6'-SL as a carbon source were shown to inhibit the attachment of harmful bacteria about two times more, so 6'-SL was used as a prebiotic. Therefore, it was confirmed that when DS2770 was ingested, it had an inhibitory effect on the attachment of harmful bacteria to the intestines. Meanwhile, in the displacement assay to remove attached Clostridium difficile bacteria, there was no statistically significant difference between the case of using glucose as the carbon source and the case of using 6'-SL as the carbon source.

[Manufacturing example]

Manufacture of health functional food powder from new Bifidobacterium longum subspecies Infantis DS2770 strain

A health functional food powder containing the Bifidobacterium longum subspecies Infantis DS2770 strain of the present invention as an active ingredient was prepared. The Bifidobacterium longum subspecies Infantis DS2770 strain was cultured using optimized enrichment medium and culture conditions. After recovering the bacterial cells, add 5-50% (volume/weight) of maltodextrin or 5-50% (volume/weight) of trehalose or 5-50% (volume/weight) of cellulose, which are cryoprotectants, compared to the concentrate, freeze-dry, and grind to obtain lactic acid bacteria. The raw powder was manufactured.

In the above, representative embodiments of the present invention have been described by way of example, but the scope of the present invention is not limited to the specific embodiments described above, and those skilled in the art will understand that the scope of the present invention is within the scope stated in the claims of the present application. It will be possible to change it appropriately.

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC14811BP 20221207

Sequence list electronic file attached

Claims (10)

Bifidobacterium longum subsp. infantis DS2770 ( Bifidobacterium longum subsp. infantis DS2770) strain deposited with accession number KCTC 14811BP. In claim 1,
The strain is a strain that has the activity of decomposing sialyllactose. In claim 1,
The strain is capable of growing even in an environment where sialyllactose exists as the only carbon source. In claim 2 or claim 3,
A strain in which the sialyllactose is 3'-sialyllactose (3'-SL) or 6'-sialyllactose (6'-SL). In claim 1,
The strain is a strain that has the ability to utilize any one or more sugars selected from the group consisting of glucose, lactose, sucrose, maltose, mannose, and raffinose. In claim 1,
The strain contains one or more sugars selected from the group consisting of mannitol, selicin, xylose, arabinose, gelatin, esculin, glycerol, cellobiose, melegitose, sorbitol, rhamnose and trehalose. A strain that has no utility. Bifidobacterium longum subspecies Infantis DS2770 strain deposited with accession number KCTC 14811BP, containing as an active ingredient at least one selected from the group consisting of a culture of the strain, a concentrate of the culture, a dried product of the culture, and an extract of the culture. A composition that does. In claim 7,
The composition is a pharmaceutical composition, a health functional food composition, a food composition, or a feed composition for improving the intestinal health of infants. In claim 7,
The composition is a pharmaceutical composition, health functional food composition, food composition, or feed composition for improving the immune function of infants. In claim 7,
The composition is a pharmaceutical composition, health functional food composition, food composition, or feed composition for promoting cognitive development in infants.