Patent Application
20210171897
The present invention relates to a novel lactic acid bacteria strain and a composition comprising the same. In addition, the present invention relates to an antibacterial composition, a cosmetic composition, and a functional food comprising the lactic acid bacteria strain or culture supernatant thereof.
Lactic acid bacteria decompose carbohydrates, such as, glucose or lactose, and produce lactic acids using the carbohydrates decomposition products. Currently about 300 to 400 lactic acid bacteria are known. Various kinds of lactic acid bacteria are widely distributed in digestive tracts of humans or animals, fermented foods, for example, milk, yogurt, cheese, kimchi, etc., plants and soil, and the like. The representative lactic acid bacteria include Lactobacillus, Lactococcus, Streptococcus, Leuconostoc, Pediococcus, and Bifidobacterium.
Organic acids, such as, lactic acids and acetic acids produced by lactic acid bacteria exhibit excellent antibacterial activity against harmful bacteria grown in neutral and basic environments, and researches have been conducted to use lactic acid bacteria as preservatives for food, etc. using the antimicrobial properties. In addition, the bacteriocin produced by the lactic acid bacteria is a peptide or a protein-based antibacterial material, and the strains producing these bacteriocins exhibit strong antibacterial activity mainly against the strains which are phylogenetically similar to themselves. Accordingly, attempts have been made to isolate a peptide or a protein having antibacterial activities from lactic acid bacteria, identify characteristics thereof, and develop the same as an antibacterial material or an anti-inflammatory therapeutic agent. In particular, the bacteriocin has an advantage in developing the same as an antibacterial material or an anti-inflammatory therapeutic agent in that the bacteriocin is composed of a peptide or a protein and thus is easily decomposed in a body and thus there is no problem of human toxicity. For example, Nisin has been developed and widely used as a bacteriocin having a broad range of antimicrobial activity.
The tight junction of intestinal epithelial cells is important to maintain homeostasis of the intestinal immune functions. Claudin-4, Zo-1 and Occludin-4 are known as proteins constituting the tight junction, and, in particular, Claudin-1, Zo-1 and Occludin-4 are involved in the tight junctions of skin epithelial cells. It has been found that the function of tight junction is damaged in allergy caused by food, enteritis, autoimmune diseases, and Celiac disease and inflammatory bowel diseases, etc. (Fasano, A., Pathological and therapeutical implications of macromolecule passage through the tight junction. In Tight Junctions, CRC Press, Inc., Boca Raton, Fla. 697-722 (2001)). The tight junction acts as a barrier against the macromolecules entering into a human body. In a healthy state, only a small amount of immunologically active antigens can be introduced into the body through the barrier of the intestinal mucosa. If the tight junction system is damaged by radiation, chemotherapy or toxin or the like, a detrimental immune response leading to harmful autoimmune diseases and food allergy occurs. In the normal intestine, the intestinal immune response is adjusted to maintain the homeostasis of the intestine. The Celiac disease (CD) is a kind of chronic autoimmune disease, and gluten, which is a major protein fraction of wheat, is known as a factor leading to this disease. The digested gluten or derivatives thereof is deamidated by tissue transglutaminase (tTG) after passing through the small intestine epithelial cell wall and induces harmful T cell-mediated immune response while being involved in class II MHC molecules. When the tight junction system is damaged by such as the CD, the paracellular leak (leaky gut) and a response to the environmental antigens occur.
The inflammatory bowel disease (IBD) refers to an undesirable immune response developing in the intestine. Two types of IBD have been known: Crohn's disease and ulcerative colitis (UC). These two types of inflammatory bowel disease show an abnormal profile of T cell-mediated immunity. In patients with Crohn's disease, a strong Th1 response is induced, and in the colon of a UC patient, the Th2 response is raised. In the IBD, the barrier function of the intestine is destroyed. For example, the Crohn's disease is associated with increased permeability of the intestinal barrier (Oshitani, et al. Int. J Mol. Med. 15: 407-10; Ye Arch, et al, Am J Physiol.-Gastro Arch. And Cover Physiol. 290: 496-504, Wilmington, Del. et al. Clin. Exp. Immunol. 142: 275-284).
A tight junction is a barrier that is present in epithelial cells and that secures adjacent cell membrane sides to each other and prevents the flow of water and bodily fluids through the cell's side space. As the stratum corneum is damaged, the concentration of calcium ions is reduced together with the loss of a body fluid existing between the stratum corneum and the granular layer, so that the tight junctions of the upper portion of the granular layer is opened, and the dendrites of the Langerhans cells located just below the tight junctions are raised above the tight functions. The protein antigens with large molecular weight passing through the damaged stratum corneum are collected and recognized by the dendrite of Langerhans cell and transferred to local lymph nodes, and the serine-family proteases of the protein antigens activate PAR-2 present in the keratinocyte membrane. On the other hand, the bacteria that penetrate the stratum corneum activate the toll-like receptor (TLR). The keratinocytes secret various chemokines and cytokines, such as, TNF-α, IL-1 and thymic stromal lymphopoietin (TSLP), etc. to promote the onset of atopic dermatitis and exacerbate the disease. In addition, the expression of the tight junction proteins is significantly reduced in the skin of the atopic dermatitis patient comparing to the normal person. Recently, the Claudin-1 gene (CDN1) mutation has been reported among the atopic dermatitis patients and it is reported that the reduction of Claudin-1 in the tight junctions is one of the reasons for herpes virus infection (De Benedetto A, Rafaels N M, McGirt L Y, Ivanov A I, Georas S N, Cheadle C, Berger A E, Zhang K, Vidyasagar S, Yoshida T, Boguniewicz M, Hata T, Schneider L C, Hanifin J M, Gallo R L, Novak N, Weidinger S, Beaty T H, Leung D Y, Barnes K C, Beck L A. Tight junction defects in patients with atopic dermatitis. J Allergy Clin Immunol 2011; 127:773p 786e7.9).
We, inventors, have strived to isolate lactic acid bacteria strains having excellent antibacterial activity as well as antifungal activity, and as a result, confirmed that lactic acid bacteria strains isolated from Korean traditional foods, such as, fermented soybean paste or the like exhibit wide range of antibacterial activities and outstanding antifungal activities. We confirmed that the identified novel lactic acid bacteria belong to the Pediococcus pentosaceus and named it as Pediococcus pentosaceus AB160011.
The purpose of the present invention is to provide a novel lactic acid bacteria strain having antibacterial and antifungal activities.
In addition, the present invention provides a novel lactic acid bacteria strain which increases the expression of proteins constituting tight junctions.
In addition, the objective of the present invention is to provide a functional food for improving inflammatory bowel disease and immune diseases, comprising the lactic acid bacteria strains of the present invention.
In addition, the objective of the present invention is to provide an antibacterial and antifungal composition comprising the lactic acid bacteria strains above or cell-free culture supernatant of the lactic acid bacteria strains of the present invention.
In addition, the present invention provides a cosmetic composition comprising the lactic acid bacteria strains above or cell-free culture supernatant of the lactic acid bacteria strains of the present invention.
To achieve the purpose, the present invention provides a novel Pediococcus pentosaceus AB160011 strain.
The Pediococcus pentosaceus AB160011, according to the present invention, has excellent acid resistance and bile resistance, and exhibits antibacterial and antifungal activities. In addition, it has been found that the strain of the present invention and the culture supernatant thereof significantly increase the expression of the proteins constituting the tight junctions of the epithelial cells of the skin and the intestine, and has been shown to have an effect of enhancing skin moisturization according to clinical trials. In addition, the novel lactic acid bacteria strain culture supernatant has anti-inflammatory and antioxidant effects.
The present invention will now be described in more detail with reference to the following examples. The following examples are merely illustrative of the present invention, and it is to be understood that the scope of the invention is not limited to the following examples.
Isolation and Identification, and Examples of Manufacture
(1) Isolation of Lactic Acid Bacteria
Various kinds of lactic acid bacteria were isolated from fermented products including kimchi, soy sauce, and soybean paste prepared in Korea in order to screen lactic acid bacteria strains having antibacterial and antifungal activities, and cultured in a MRS (Difco 288110) solid medium containing 2% agar using a 10-fold dilution method to obtain the colonies. The resulting colonies were classified according to size, color, and characteristics, and then streaked onto another MRS medium to separate a total of 1,785 lactic acid bacteria of the target strains. Each colony of the isolated lactic acid bacteria was streaked again onto MRS agar plates and incubated at 37° C. for 24 hours. The isolated lactic acid bacteria were inoculated into the sterilized MRS broth and incubated at 37□ for 24 hours, followed by the addition of 20%(v/v) glycerol and stored in a ultra-low temperature freezer at −80° C.
(2) Strain Identification
Morphology Analysis
In order to identify the strains, the morphology of the colonies was observed under a microscope after being cultured in MRS plate media. The colonies' morphology is shown in Table 1, and
Pediococcus
pentosaceus
Identification by 16S rRNA
The novel strain of the present invention was found to have a 16S rRNA sequence as shown in SEQ ID NO: 1. Based on this, the isolated strain was found to have high similarity to Pediococcus pentosaceus AJ3053 and named Pediococcus pentosaceus AB160011, and deposited with deposition accession no. KCCM11954P at Korean Culture Center of Microorganisms (KCCM) on Dec. 7, 2016.
Identification by DNA Sequence Analysis
It was confirmed that the novel strain of the present invention has 98.97% homology with Pediococcus pentosaceus (ATCC25745 ATP), according to DNA sequence analysis (Gepard, Nucleotide sequences Dot plot) (see
Cell Culture Property
The novel lactic acid bacteria strains of the present invention were inoculated into a sterile MRS broth medium and incubated at 37° C. to observe changes in turbidity (Ultrospec2100pro, OD625 nm) and acidity (Orion star A211, pH).
Cultivation of Lactic Acid Bacteria Strains and Freeze Drying of the Cell-Free Culture Supernatant
The composition of the medium for culturing lactic acid bacteria is shown in Table 2, and the manufacturing process is largely classified into as follows: seed culture, main culture, separation of strains and cell-free culture supernatant, and freeze-drying. The seed culture refers to the process of culturing the strains to a volume of 10% of the final culture volume, depending on the size of the target final culture volume. After the seed culture is completed, the obtained seed culture solution including the strains is inoculated into another sterilized culture medium having larger volume and cultured, which is main culture. After the completion of the said main culture, the lactic acid bacteria and the culture supernatant are separated by centrifugation and/or using ceramic filter to obtain cell-free strain culture supernatant. The separated lactic acid bacteria and the cell-free culture supernatant are freeze-dried through a pre-freezing process and processed into a final powder form stored at −20□ for the following use.
In order to select lactic acid bacteria inhibiting the growth of pathogenic bacteria, the following bacteria were obtained from KCCM and cultured in the solid media presented in Table 3 below. Bacteria: Staphylococcus aureus (KCCM11335), Escherichia coli (KCCM11234), Bacillus cereus (KCCM40935), Staphylococcus epidermidis (KCCM40416), Pseudomonas (KCCM11321), Propionibacterium aeruginosa (KCCM41747), Salmonella enterica subsp. enterica (KCCM11806), Listeria monocytogenes (KCCM40307). Fungi (including fungi): Cryptococcus neoformans (KCCM50785), Candida albicans (KCCM11282), Aspergillus fumigatus (ATCC Mya-4609), Aspergillus niger (KCCM60332), Fusarium oxysporum (KCCM44187), Penicillium chrysogenum (KCCM60353) and Malassezia furfur (KCCM12679). Next, each colony of the pathogen was inoculated into a nutrient broth medium and incubated for 24 hours at an appropriate temperature (37° C. for E coli, Staphylococcus aureus and Bacillus; 30° C. for Cryptococcus and Candida), followed by the addition of 20% glycerol, and then stored at −80□ cryogenic freezer. In the case of Aspergillus, Fusarium and Penicillium, the spores were harvested with PBS (phosphate buffer saline) from YPD medium (Sigma-Aldrich) after 7 days incubation at 30° C., followed by the addition of 20% glycerol, and then stored at −80□, cryogenic freezer.
E.
coli
Staphylococcus
aureus
Bacillus
cereus
Cryptococcus
neoformans
Candida
albicans
Aspergillus
fumigatus
Aspergillus
niger
Fusarium
oxysporum
Penicillium
chrysogenum
For the isolated strains, their antimicrobial and antifungal activities were detected. First, each isolated strain stored at −80° C. was inoculated onto MRS solid medium and incubated at 37□ for 24 hours, and then the respective colonies were taken and suspended in 500 μl of
PBS to prepare a sample solution for subsequent antibacterial and antifungal activity experiments. Next, media for antimicrobial and antifungal activity tests of the isolation strains were prepared. To this end, the bacteria and fungi cultured and stored in Example 1 were inoculated into a nutrient media suitable for each growth and incubated for 24 hours. Next, the soft agar medium containing MRS (1%, w/w) was sterilized at 121□ for 15 minutes, cooled to 55□, and each culture medium including bacteria or fungi obtained above was inoculated with 1% (v/v) on the said MRS medium. The soft agar MRS medium inoculated with the bacteria or fungi was then poured into a disposable petri dish and placed at room temperature to prepare an overlay medium for antibacterial or antifungal activity experiments. A sterilized 8 mm paper disk (Adventec, 51020693) was placed on the obtained overlay medium, 50 μl of the isolated lactic acid bacteria or cell-free culture supernatant of the present invention was added, and incubated at the appropriate growth temperature of each bacteria or fungi for 24 hours. After the culture was completed, the diameter of the clear zone for the pathogens was measured to determine the antibacterial activity and antifungal activity of the isolated lactic acid bacteria. The antibacterial activity and antifungal activity above was indicated according to the diameter of the clear zone as follows: 8 mm or more: +, 13 mm or more: ++, 15 mm or more: +++, 17 mm or more: ++++.
The isolated lactic acid bacteria strains were cultured in Lactobacilli MRS broth for more than 16 hours and then the 100 μl of the bacterial culture was inoculated into 100 μl of MRS broth with different pH conditions (pH7.0, pH2.5) in a 96 well plate, followed by incubation at 37□ for 24 hours. The survival rate of the lactic acid bacteria was measured by comparing the absorbances of the pH7.0 culture medium and the pH2.5 culture medium at 620 nm using Microplate reader. The cell viability for the acid resistance test was measured in the following manner.
Viability (%)=OD24 h, pH 2.5/OD24 h, pH 7.0×100
To confirm bile resistance, the isolated lactic acid bacteria strain was cultured in Lactobacilli MRS broth for more than 16 hours and then 100 μl of the bacterial culture was inoculated into 100 μl of MRS broth with two conditions (MRS broth, MRS broth+0.3% oxgall) in a 96 well plate, followed by incubation at 37□ for 24 hours. The survival rate was measured by comparing the absorbance at 620 nm using Microplate reader. The cell viability for the bile resistance test was measured in the following manner.
Viability Measurement (%)=OD24 h, MRS+0.3% Oxgall/OD24 h, MRS×100
The acid resistance and bile resistance of the novel lactic acid bacteria of the present invention were 86.87% and 99.37%, respectively, and were found to have excellent properties exceeding the general standard of 70%.
The induction of expression of the intestinal TJ constituent proteins was confirmed by using Caco-2 cells (obtained from Korean Cell Line bank), intestinal epithelial cells. As shown in
The minimum inhibitory concentration (MIC) measurement was performed by a standard test method (Wiegand et al. Nat Protoc. 2008). First, E. coli, S. aureus or B. cereus was inoculated onto a sterile test solid nutrient medium (MHB, Muller Histone Broth, Difco) and cultured. The C. neoformans or C. albicans was inoculated on YM nutrient medium (Difco) and A. fumigatus was inoculated on YPD nutrient medium (Difco), and incubated at 37□ for 24 hours. The bacteria were then inoculated into sterile liquid medium (MHB) and the fungus was inoculated into RPMI1640 medium, respectively. Next, the cell-free bacterial culture supernatant of the present invention was added to MHB or RPMI1640 medium obtained above and incubated at 37° C. for 24 hours to measure the turbidity of the pathogenic microorganisms. The cell-free culture supernatant solution was prepared by dissolving the freeze-dried lactic acid bacteria culture supernatant in the triple distilled water. The serial 2×dilution of the supernatant solution said was used to confirm the lowest concentration of the culture supernatant inhibiting the visible growth of pathogens. To confirm the antibacterial and antifungal activities, the test above was repeated three times, and the OD (620 nm) value of the culture supernatant was detected using microplate spectrophotometer (Multiskan FC A28696). As shown in Table 4, the lactic acid bacteria culture supernatant of the present invention exhibited extensive inhibitory activity against Gram-positive and Gram-negative bacteria and fungi.
Antifungal activity and MIC test were performed according to the standard test method (CLSI document M38-A2, 2008). The culture supernatant prepared in the example above was serially 2×diluted for this experiment. Each well of each 96 well was inoculated with mold or fungi spores at a concentration of 2×103 spores/ml. The plate was incubated at 35° C. for 48 hours. The MIC was determined by microscopic observation to the lowest concentration of the cell-free culture supernatant inhibiting the visible growth of the fungi or mold. The experiment was repeated three times to confirm the antifungal activity. Table 4 shows the results.
Escherichia
coli
Staphylococcus
aureus
Staphylococcus
epidermidis
Propioni
bacterium
acnes
Bacillus
cereus
Pseudomonas
aeruginosa
Salmonella
Listeria
Cryptococus
neoformans
Candida
albicans
Aspergillus
niger
Fusarium
oxysporum
Penicillium
chrysogenum
Malassezia
furfur
Experiments were conducted to determine whether the antibacterial and antifungal activity against the pathogens by the cell-free culture supernatant of the lactic acid bacteria has thermal stability. The concentrated culture supernatant dissolved solution was prepared in the same manner as in the above example and was divided to two groups of non-heat treatment and heat treatment to determine the MIC showing antibacterial and antifungal activities. For the heat-treated group, the culture supernatant solution was heat-treated in 800 water bath (Biofree, BF60AC) for 30 minutes or in 121□ autoclave for 15 minutes. The results of the experiments are shown in Table 5.
E.
coli
S.
aureus
B.
cereus
C.
neoformans
C.
albicans
Experiments were conducted to determine whether the antibacterial and antifungal activity against pathogens of the lactic acid bacteria culture supernatant has pH stability. The concentrated culture supernatant solution prepared in the same manner as in the above example was treated with non-pH adjusted group or pH adjusted group to measure the MIC showing antibacterial and antifungal activities.
The pH was adjusted using lactic acids and NaHCO3. The results of the experiments are shown in Table 6 below.
E.
coli
S.
aureus
B.
cereus
C.
neoformans
C.
albicans
The Pediococcus pentosaceus AB160011 of the present invention, Lactobacillus brevis and E. coli were cultured until OD600 reached 0.8, respectively. Then, 0.5, 1 and 1.5 ml of Pediococcus pentosaceus AB160011, 1 ml of Lactobacillus brevis and 1 ml of E. coli were used to remove the supernatant using centrifuge, and the precipitated cells were pooled and washed with distilled water (DW). After performing centrifugation again, the supernatant was removed and Pediococcus pentosaceus AB160011 was pooled in 5 μl of DW, and each Lactobacillus brevis and E. coli were pooled in 1 ml of DW. Then, each Lactobacillus brevis and Escherichia coli, which were pooled in 1 ml of DW, were spread evenly in the MRS solid media. Then, Pediococcus pentosaceus AB1600115, which was pooled in 5 μl of DW, was spotted on the MRS medium above where Lactobacillus brevis or Escherichia coli, was spread, at a constant distance. The results were confirmed after 6˜20 hours of incubation and are shown in
The amount of Nitric Oxide, an inflammatory response indicator, was measured. First, RAW264.7 cells were seeded in 12 well plates with 1×105 cells per well and were incubated overnight. The 0.2 g of the lyophilized sample of the cell-free culture supernatant of Pediococcus pentosaceus AB160011 was dissolved in 1 ml of DMEM. The RAW264.7 cells were treated with 0.25, 0.3, 0.35, 0.4, 0.45 and 0.5% (wt/v) of the cell-free culture supernatant dissolved in DMEM above, and incubated for 24 hours. The amount of NO was determined by measuring the absorbance at 540 nm using Nitric Oxide Colorimetric Assay kit (BioVision). As a result, the said culture supernatant of the present invention did not induce an inflammatory response. The results are shown in
RAW R264.7 cells were seeded into 12 well plates at a concentration of 1×105 cells per well. One day after the seeding, each well was treated with Pediococcus pentosaceus AB160011 at each concentration of 5×107, 1×108 and 2×108 CFU/ml for 2 hours and then the amount of NO was measured at 540 nm using Nitric Oxide Colorimetric Assay kit (Biovision, Korea). As a result, the lactic acid bacteria of the present invention did not induce an inflammatory response. The results are shown in
One day after the seed inoculation of 1×105 cells per well in a 12 well plate of RAW R264.7 cells, 1×108 or 2×108 cfu/ml of Pediococcus pentosaceus AB160011 were added to each well, and each well was treated with 1 μg/ml of LPS for 24 hours to induce inflammation. The amount of NO was determined at 540 nm using Nitric Oxide Colorimetric Assay kit (Biovision, Korea). As a result, the lactic acid bacteria of the present invention have been shown to inhibit inflammation reactions. The results are shown in
The DPPH (1,1-diphenyl-2-picrylhydrazyl), which is a water-soluble material having a chemically stabilized free radical, has a maximum absorbance between 515 nm and 520 nm, and the radical (DPPH) is extinguished and discolored when the material encounters a material having an antioxidant activity. It is known that a chemically stable 1-diphenyl-2-picylhydryl radical (DPPH) can be used to analyze an antioxidant effects of the extracts containing several antioxidant components, a beverage, an oil, a pure phenol compound, and the like. The cell-free bacterial culture supernatant of the present invention was added to 500 ml of 0.2 mM DPPH, wherein the said culture supernatant was applied at each concentration of 0.1, 0.25, 0.3, 0.35, 0.4, 0.45 and 0.5 (wt/v)% (or 0.1, 0.25, 0.5, 0.75, 1, 1.5 and 2 (wt/v)%) while adjusting the final volume of the mixture to 1 ml. The absorbance was measured at 540 nm after the reaction in the dark room for 30 minutes. As a result, as shown in
A skin irritation test using a SKINETHIC human skin model (EPISKIN) was carried out at Korea Testing & Research Institute (KTR) according to the guidelines for testing toxicology for cosmetics and animal replacement tests (V)/skin irritation test method (Ministry of Food and Drug Safety, MFDS; Guideline-0752-01). The 16 μl of the test article of the cell-free bacterial culture supernatant was applied to the said human skin model with 1 minute intervals per tissue and exposed for 42 minutes (±1) in the clean bench. The test material was then removed and washed a total of 25 times with PBS (1 ml at one-time). The reversible damage to the said human skin model was evaluated by incubation the treated human skin at 37□ for 42 hours±1 hour in a 5% CO2 incubator. Each group of the three groups of the negative control, the positive control, and the test material group was set to have three repetitive samples. The cell viability of the test article was determined to be 86.3±2.9% over 50% of the cell viability baseline and determined to be non-irritating to the skin. The results are shown in
In order to evaluate the effects of improving skin moisturizing of the cosmetic essence product containing the cell-free bacterial supernatant of the present invention, a clinical test was performed at KTR (Korea). The skin moisturizing assay and abnormal response assay were performed for 22 females aged between 29 years and 59 years using Corneometer both before and after the use of the product. The results of skin moisturization have shown a statistically significant increase of the moisture content in the forehead, both cheeks and jaw areas two and four weeks after use compared to before the product was used (see
The present invention confirms that a novel lactic acid bacterial strain isolated from soybean paste, a Korean traditional fermented food, and the cell-free bacterial culture supernatant thereof have excellent antibacterial and antifungal activities, and induce the expression of proteins involved in TJ (tight junctions) of skin and intestinal epithelial cells. In addition, the bacterial strain and the supernatant of present invention have anti-inflammatory and antioxidant effects, and have effects of enhancing skin moisturization according to clinical trials. Therefore, the lactic acid bacterial strain of the present invention could be widely used in a wide range of industrial sectors such as health functional foods, cosmetics and preservatives.
Deposit institution name: Korean Culture Center of Microorganisms (overseas).
Deposit No.: KCCM11954P
Deposit Date: 20161207
<110>AmtixBioCo., Ltd.
<120>A novel Pediocococcus pentosaceus AB160011 and composition comprising thereof
<130>AMTIX17P01KR
<160>1
<170>KoPatent In 3.0
<210>1
<211>2849
<212>DNA
<213>Pediococcus pentosaceus
<400>1
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2017-0149048 | Nov 2017 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2018/007045 | 6/21/2018 | WO | 00 |
