KR102141569B1 - Novel Lactobacillus plantarum KCC-34 - Google Patents

Abstract

The present invention relates to a new lactic acid bacterium Lactobacillus plantarum KCC-34 strain, specifically, a new lactic acid bacterium Lactobacillus plantarum KCC-34 strain (microbial accession number KACC 92221P) isolated from alfalfa feed. .
The strain of the present invention is a novel lactic acid bacterium having very good probiotic properties such as antibacterial and antifungal activity, fermentation ability of various carbohydrates, various enzyme production ability, control using antibacterial agents, acid resistance, bile resistance, cohesiveness, hydrophobicity, antioxidant activity, etc. Therefore, it is very useful as a probiotic probiotic or in a food or feed or as a fermentation of fermented foods.

Description

New Lactobacillus plantarum KCC-34 strain {Novel Lactobacillus plantarum KCC-34}

The present invention relates to a new lactic acid bacterium Lactobacillus plantarum KCC-34 strain, specifically, a new lactic acid bacterium Lactobacillus plantarum KCC-34 strain (microbial accession number KACC 92221P) isolated from alfalfa feed. .

The human gastrointestinal tract has a complex and powerful microbial environment. This microbial environment changes with age, health and lifestyle, and plays an important role in inhibiting the action of pathogens and harmful food substances and promoting beneficial effects to the host. This effect is related to the tendency of microorganisms in the gastrointestinal tract, and the balance of these microorganisms in the gastrointestinal tract can lower the risk of gastrointestinal disease. Benefits of probiotics include improved lactose intolerance, improved bioavailability of nutrients and prevention of allergies, and anti-mutation, anti-cancer, anti-hypercholesterol, anti-hypertension, anti-osteoporosis and immunomodulatory effects have been reported. It has also been reported that probiotics can eliminate inflammatory bowel disease, irritable bowel syndrome, cirrhosis and constipation and reduce the risk of colon cancer, liver cancer and breast cancer.

Currently, the benefits of health-promoting probiotic foods are widely accepted worldwide. According to FAO/WHO, probiotics for health promotion are viable, active microorganisms that positively affect the health of the host when consumed in sufficient amounts (at least 10 ⁶ to 10 ⁷ cfu/g). Recent studies have reported the immunological properties of dead microorganisms. Most probiotic microorganisms produce lactic acid bacteria, of which Lactobacillus is one of the most basic fast-acting bacteria that improve human health. Therefore, the genomic sequence of several Lactobacillus species has been identified and used in a wide range of fermented foods. In addition, metabolic pathways related to Lactobacillus, gene regulation and function have been reported. Most researchers have thought that the best way to deliver probiotics is fermented milk, especially through fermented milk and yogurt.

Researchers today are more interested in producing new biotics, and believe that non-dairy probiotics could be a good basis for the development of functional probiotic foods for humans. Some of the well characterized probiotic microorganisms are commercially available worldwide. However, screening of new microorganisms is still receiving great attention from the fermented food industry.

Accordingly, the present inventor screened new microorganisms having excellent properties of probiotics from alfalfa feed, and as a result, the new lactic acid bacteria Lactobacillus plantarum KCC-34 strain has antibacterial and antifungal activity, fermentation ability of various carbohydrates, and various enzyme production capabilities, The present invention was completed after confirming that it exhibits excellent properties of probiotics such as control properties, acid resistance, bile resistance, cohesiveness, hydrophobicity, and antioxidant activity using an antibacterial agent.

J. Nutr. Health Aging 5, 80-91. Best Pract. Res. Clin. Gastroenterol. 18, 299-313. Cell 124, 837-848. Age. Res. Rev. 9, 107-116.

Therefore, the main object of the present invention is to provide a new strain having excellent properties as a probiotic.

Another object of the present invention is to provide a probiotic probiotic preparation, food and feed using the strain.

According to one aspect of the invention, the present invention provides a new lactic acid bacteria Lactobacillus plantarum ( Lactobacillus plantarum ) KCC-34 strain (microorganism accession number KACC 92221P).

According to another aspect of the present invention, the present invention provides a probiotic probiotic comprising the strain.

In the probiotic preparation of the present invention, it is preferable that it is a probiotic probiotic for human or livestock.

According to another aspect of the present invention, the present invention provides a food comprising the strain or the culture medium of the strain.

According to another aspect of the invention, the present invention provides a fermented food prepared by fermentation with the strain.

According to another aspect of the invention, the present invention provides a feed containing the strain or the culture medium of the strain.

According to another aspect of the invention, the present invention provides a fermented feed prepared by fermentation with the strain.

The strain of the present invention is a novel lactic acid bacterium having properties of very good probiotics such as antibacterial and antifungal activity, fermentation ability of various carbohydrates, various enzyme production ability, control using antibacterial agents, acid resistance, bile resistance, cohesiveness, hydrophobicity, antioxidant activity, etc. Therefore, it is very useful as a probiotic probiotic or in a food or feed or as a fermentation of fermented foods.

1 is a result of testing the antibacterial activity of the strain of the present invention.
2 is a result of experimenting the antifungal activity of the strain of the present invention.
Figure 3 is the result of experimenting the gastric juice environment resistance of the strain of the present invention.
4 is a result of testing the bile salt resistance of the strain of the present invention.
5 is a result of testing the auto-aggregation activity of the strain of the present invention.
6 is a result of testing the cell surface hydrophobicity of the strain of the present invention.
7 is a result of experimenting the DPPH free radical scavenging activity of the strain of the present invention.

The new lactic acid bacteria Lactobacillus plantarum KCC-34 strain of the present invention was isolated from Alfalfa, Medicago sativa feed, and microbial entrusted to the Korean Agricultural Culture Collection (KACC) It has been deposited under the number KACC 92221P.

The strain of the present invention can be cultured using a medium that is commonly used for culturing a strain of genus Lactobacillus, such as MRS. At this time, the culture temperature is preferably 35 ~ 40 ℃.

Some base sequences of the 16S rRNA gene of the strain of the present invention identified in the present invention are the same as SEQ ID NO: 1, and are described in NCBI Genbank as KP091750 (accession number). The strain of the present invention can be classified as a new strain belonging to Lactobacillus plantarum based on the result of the classification of the strain through the base sequence.

The strain of the present invention shows antibacterial activity against Escherichia coli , Enterococcus faecalis , Staphylococcus aureus and Pseudomonas aeruginosa , and may exhibit antifungal activity against Aspergillus clavatus , Penicillium chrysogenum and Aspergillus flavus .

The strain of the present invention can ferment carbohydrates such as L-arabinose, D-ribose, D-Adonitol, L-Rhamnose, Inositol (see Table 1). Therefore, it can be used to ferment materials containing these carbon sources.

The strain of the present invention exhibits susceptibility to antibiotics chloramphenicol, nitrofurantoin, tetracycline, streptomycine, colistin methane sulphonate, dicloxacillin, amikacin, gentamicin, cefoxitin, cefalexin, cefuroxime, and co-trimoxazole. , kanamycin, sulphafurazole and ampicillin.

The strain of the present invention can produce organic acids such as lactic acid and acetic acid.

The strain of the present invention can survive under acidic conditions of pH 2 to 3, and can also survive under conditions where a high concentration of bile salts such as oxgall is present, survive while passing through the digestive organs of the animal's stomach or small intestine to the intestine It is likely to reach alive.

Since the strain of the present invention exhibits auto-aggregation and hydrophobicity, it is advantageous to settle in the gut of animals.

The strain of the present invention may exhibit antioxidant activity such as DPPH free radical scavenging activity.

As described above, the strain of the present invention has very excellent properties as a probiotic, and thus can be used as a probiotic probiotic.

At this time, the probiotic may be made of the strain itself or a culture of the strain of the present invention, or may be in the form of additives such as excipients for ease of formulation.

In addition, the strain or culture of the present invention may be used as a food or feed material or additive. At this time, the culture can be used in a form that does not contain a cell, but in order to expect an effect as a probiotic, it is preferable to use a cell containing a cell.

In addition, the strain of the present invention can be used as a fermentation strain of fermented food or fermented feed. At this time, it is preferable to use a raw material containing a component capable of fermenting the strain of the present invention, as shown in Table 1 in the fermented food or fermented feed, the fermentation temperature is preferably 35 ~ 40 ℃.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

[Example]

1. Method

1-1. Isolation and identification of strains

Alfalfa ( Medicago sativa ) feed was collected at various locations located in Cheonan, South Korea, and stored at 4°C. 1 g of feed was suspended in 9 ml of 0.9% sterile physiological saline, and 100 μl of this suspension was poured into MRS agar (de Man-Rogosa-Shape). After solidifying the MRS agar medium at room temperature, the cells were cultured at 37°C for 48 hours. The resulting colonies were stained and antibacterial and antifungal activity was confirmed. The expected Lactobacillus strain was selected and stored in a 40% glycerol stock state at -80°C until further experiments were made. The isolated strains were identified by scanning electron microscopy (SEM).

1-2. Antibacterial activity

The antibacterial activity of the isolated strain was measured using the disk diffusion test method (Naghmouchi et al., 2006) (applied with a slight change).

The culture solution cultured for 24 hours was centrifuged at 9,000 g for 10 minutes at 4° C. to obtain a supernatant (CFS) that does not contain cells, and 50 μl of CFS was poured into each well on an MRS agar medium. The medium was maintained at room temperature for 30 minutes in aseptic conditions, and then cultured at 37°C for 2 days. After incubation, the anti-bacterial activity was measured by observing the inhibitory region around the well containing CFS.

1-3. Antifungal activity

The antifungal activity of the isolated strain was measured according to the method of Vijayakumar et al. (2015) (applied with a slight change).

25 ml of sterilized MRS agar medium was poured into a petri dish, and 50 µl of CFS cultured for 24 hours was poured into each well, followed by incubation at 37°C for 24 hours. Then, 10 ml of PDA (potato dextrose agar) containing 50 μl of the fungal suspension was poured onto MRS agar medium and incubated at 37° C. for 78 hours. After incubation, the anti-fungal activity was measured by observing the inhibition region.

1-4. Characteristics of the isolated Lactobacillus strain

Genomic DNA was extracted from the isolated strain and purified with QIAquick® PCR purification kit (Qiagen Ltd., Crawley, UK). For 16S ribosomal DNA gene sequencing, DNA was amplified using 5'-AGA GTT TGA TCG TGG CTC AG-3' forward primer and 5'-GCT TAC CTT GTT ACG ACT T-3' reverse primer (Sanger et. al., 1977). At this time, the PCR program conditions are as follows: 95°C/10 minutes, 95°C/40 seconds-58°C/1 minute 30 cycles, final 72°C/2 minutes. Based on the obtained 16S rRNA partial sequence, it was compared with the sequence of Lactobacillus using BLAST software. The identified 16S rRNA partial sequence of the isolated Lactobacillus strain was deposited in NCBI Genbank (Accession No. -KP091750).

1-5. Biochemical and physiological properties

The biochemical and physiological properties of the isolated Lactobacillus strains were investigated using the API 50 CHB system (bioMerieux, Inc, USA). To this end, the strain culture solution cultured for 24 hours was used for the strip, and the strip was prepared according to the manufacturer's instructions. Following the instructions, the strips were incubated at 37°C for 48 hours, and the results were observed.

1-6. Antimicrobial susceptibility

For the study of antimicrobial susceptibility, a standard single disc agar diffusion method (Bauer et al., 1996) was performed. 25 ml of sterilized MRS agar medium was poured into a petri dish, and the strain culture solution cultured for 24 hours was plated on MRS agar medium. Antibiotic discs were placed on the top of the MRS agar medium, placed at room temperature for 30 minutes, and incubated at 37°C for 48 hours. Subsequently, the susceptibility to the antibacterial agent was measured by observing the inhibition region.

1-7. Organic acid determination

Lactic acid and acetic acid were quantified using an HPLC spectrum system (Thermo, USA). The isolated Lactobacillus strains were incubated at 37°C for 48 hours, and then the culture samples were centrifuged at 4°C for 10 minutes at 9,000 g to collect CFS, which was used to quantify the organic acid. This experiment was repeated three times.

1-8. Probiotic properties of isolated Lactobacillus

1-8-1. Gastric juice of isolated strain Resistance to the environment

Analysis of resistance of the isolated Lactobacillus strain to the stimulated gastric juice was performed according to the method of Charteris et al. (1998). 20 ml of the culture solution cultured for 24 hours was centrifuged at 6,000 g for 20 minutes, the upper portion of the supernatant was discarded, and the cells were washed twice with 50 mM K ₂ HPO ₄ . 1 ml of the cell suspension was mixed with gastric juice (pepsin 3 mg/ml; sodium chloride 0.5% w/v; H ₂ O) stimulated at different pH (pH 2 and 3), and then 100 μl of the cell suspension was added to the MRS agar medium. Pour over time, and incubate at 37°C for 48 hours. Resistance was analyzed by counting the number of live cells.

1-8-2. Isolate strains Cholecystitis tolerance

The growth resistance of the isolated Lactobacillus strains against Oxgall and SDC was evaluated according to the method of Vinderola and Reinheimer (2003) (with a slight change). 1% of the culture solution cultured for 24 hours was inoculated into a MRS liquid medium containing 0.3% oxgall and 0.5% SDC, and cultured at 37°C for 48 hours. After cultivation, absorbance was measured at 600 nm and compared to a control group not treated with bile salts.

1-8-3. Auto aggregation

Auto-aggregation analysis was performed according to a modified protocol such as Del Re (2000). For the analysis, the isolated strain was cultured in MRS liquid medium at 37°C for 48 hours, and the culture solution was centrifuged at 8,000 g for 10 minutes. After discarding the upper portion of the supernatant, the cells were washed 3 times with PBS (phosphate buffered saline, pH 7.0) and resuspended in PBS to adjust the viable cell count to 10 ⁸ CFU/mL. 4 ml of the cell suspension was vortexed for 10 seconds, incubated at room temperature for different times (0, 1, 2, 3 hours), and then the OD600nm value was measured to calculate the autoaggregation percentage according to the following formula.

% Auto aggregation = [(OD _X -OD _y ) / OD _X ] x 100

OD _X : OD value according to culture time (1, 2, 3 hours)

OD _y : 0 hour OD value

1-8-4. Hydrophobicity

In vitro experiments were performed according to the method of Rosenberg et al. (1980) to investigate the bacterial adhesion properties to hydrocarbons. The culture solution cultured for 24 hours was centrifuged at 5,000 g for 15 minutes, and then the cell suspension was collected, washed with PBS, resuspended in PBS, and OD600nm was measured. Hydrocarbon xylene and 1 ml of chloroform were added to the same amount of cell suspension, vortexed for 2 minutes, and then left at 37°C for 10 minutes. The OD600nm value of the resulting aqueous phase was measured, and% hydrophobicity was determined according to the following equation.

% Hydrophobicity = [(OD _initial -OD _final ) / OD _initial )] x 100

1-8-5. Antioxidant activity (DPPH free radical scavenging activity)

The antioxidant properties of the isolated Lactobacillus strains were analyzed according to the modified method of Vijayakumar et al. (2013).

Cell suspensions of different concentrations (10, 20, 30, 40 and 50 μg/ml) were added to 1 ml of mixed solution of DPPH (0.05 mM), vortexed and incubated in the dark at room temperature. After incubation, antioxidant activity was measured at 517 nm. Antioxidant activity was measured according to the following formula by using only DPPH as a control.

DPPH free radical scavenging activity (%) = [(A _sample -A _blank ) / A _control ] x 100

1-9. Statistical analysis

All numerical data were obtained repeatedly and statistical analysis was performed using MS-Excel and SPSS 16 statistical analysis (SPSS Inc., Chicago, IL, USA). The results were expressed as mean ㅁ standard deviation, and when P <0.05, there was a significant difference.

2. Results

2-1. Isolation and identification of strains

25 strains were isolated from alfalfa feed collected from Cheonan. Although these strains showed similar properties in Gram positive, catalase negative, cocci, sporulation, and rod, among them, the KCC-34 strain has the best antifungal and antibacterial activity, one of the most important selection criteria in the potential for probiotics. Appeared. Through the 16S rRNA sequence analysis using BLAST, the KCC-34 strain was found to belong to Lactobacillus plantarum . This sequence is described in the NCBI Genbank as KP091750 (accession number).

2-2. Antibacterial and antifungal activity

As a result of measuring the antibacterial activity against various food-borne pathogens, KCC-34 has strong antibacterial activity against Escherichia coli (0.9mm), Enterococcus faecalis (0.6mm), Staphylococcus aureus (1.0mm) and Pseudomonas aeruginosa (0.8mm). It is shown (see FIG. 1).

In addition, as a result of specific antifungal activity against various fungal species, KCC-34 was Aspergillus clavatus (76.9±0.69), Penicillium It has been shown to inhibit the growth of pathogenic fungi such as chrysogenum (79.71±1.25) and Aspergillus flavus (77.10±1.58) (see Figure 2).

2-3. Physiological, biochemical and antibiotic susceptibility analysis

After incubation for 48 hours, KCC-34 has excellent ability to ferment carbohydrates such as L-arabinose, D-ribose, D-Adonitol, L-Rhamnose, Inositol, and erthritol, D-arabinose, etc. See Table 1). In addition, KCC-34 has been shown to produce various intracellular and extracellular enzymes (see Table 2). KCC-34 showed sensitivity to chloramphenicol, nitrofurantoin, tetracycline, streptomycine, colistin methane sulphonate, dicloxacillin, amikacin, gentamicin, cefoxitin, cefalexin, cefuroxime and co-trimoxazole, and showed resistance to kanamycin, sulphafurazole and ampicillin 3).

2-4. Organic acid determination

As a result of confirming and quantifying organic acids such as lactic acid and acetic acid of the separated KCC-34 strain by performing HPLC analysis, after 24 hours of incubation, KCC-34 produced lactic acid up to 305.47mg/ℓ and acetic acid up to 60.82mg/ℓ It turns out to produce.

2-5. Acid condition and bile salt resistance

According to FAO/WHO's guidelines, in order to evaluate probiotics in food, it must survive in the stomach and small intestine. Therefore, acidic pH of gastric juice and resistance to bile salts of the small intestine are the main selection criteria for good probiotic candidates. Thus, the KCC-34 of the present invention can survive (57-81%) at acidic pH (2 and 3) after 3 hours incubation (see FIG. 3), oxgall (50.28, 60.23%) and SDC (46.69, 53.99%) ) Was found to be resistant to bile salts (see Figure 4).

2-6. Auto-aggregation and hydrophobicity

The ability of the KCC-34 strain to interact with epithelial cells was investigated. 5, KCC-34 showed a high score (53-69%) for auto-aggregation after 3 hours of incubation. In addition, KCC-34 showed hydrophobicity for xylene (41.13%) and chloroform (57.28%) (see Fig. 6).

2-7. Antioxidant activity (DPPH free radical scavenging activity)

As a result of measuring the antioxidant activity of KCC-34 using a DPPH solution, KCC-34 showed concentration-dependent excellent DPPH free radical scavenging activity (see FIG. 7 ).

+: Positive,-: negative

+: Weak production, ++: normal production, +++: strong production

S: susceptibility (>10mm), R: resistance

Agricultural Biotechnology Research Institute KACC92221P 20180220

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Claims (7)

delete Lactobacillus plantarum ( Lactobacillus plantarum ) KCC-34 strain (microbial accession number KACC 92221P) probiotic probiotic comprising. According to claim 2,
The probiotic is a probiotic probiotic for human or livestock products. A food comprising the probiotic probiotic of claim 2. A fermented food prepared by fermenting the probiotic probiotic of claim 2. A feed comprising the probiotic probiotic of claim 2. A fermented feed prepared by fermenting the probiotic probiotic of claim 2.

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