KR102626750B1 - Lacticaseibacillus casei WiKim0135 strain with excellent chiroinositol producing ability and uses for lowering blood sugar thereof - Google Patents

Abstract

The present invention relates to a Lacticajay Bacillus casei WiKim0135 strain with excellent chiroinositol production ability and its use in lowering blood sugar. The Lacticajay Bacillus casei WiKim0135 strain according to the present invention has excellent heat resistance and is not affected by gastric acid and bile acid. It has an excellent ability to produce chiroinositol, which can help lower blood sugar levels, so it can be used as a probiotic strain.

Description

Lacticaseibacillus casei WiKim0135 strain with excellent chiroinositol producing ability and uses for lowering blood sugar thereof}

The present invention relates to Lacticajay Bacillus casei WiKim0135 strain with excellent chiroinositol production ability and its use in lowering blood sugar.

Worldwide, the number of people with diabetes is rapidly increasing due to population aging, increase in obesity, lack of exercise, changes in eating habits, and improvement in living standards.

Among these, diabetes refers to a metabolic disorder of multiple etiologies characterized by chronic hyperglycemia resulting from defects in insulin secretion or insulin action, and occurs when abnormally high blood glucose persists for a long period of time. Various complications occur due to chronic metabolic disorders and subsequent chronic vascular damage.

Diabetes is a representative adult metabolic disease that affects approximately 5% of the world's population, and the resulting human and economic losses are truly enormous. Most diabetic patients are taking oral treatments, but no safe treatments have been developed to date. Insulin resistance is known to be the most important mechanistic cause, but the exact mechanism is not yet clear, but it has been revealed that a combination of genetic predisposition and environment plays a role.

Diabetes is the third most serious disease worldwide, and the number of diabetes patients is expected to be approximately 250 million by 2010, and continued growth is expected in Korea as well. Non-insulin dependent diabetes (NIDDM), which is the 7th leading cause of death in Korea and accounts for more than 90% of all diabetic patients, usually develops after the age of 40 and is called adult-onset diabetes, which means it does not produce enough insulin. It is a metabolic disorder caused by the inability to use or not utilize it properly.

Although the cause of NIDDM is not clearly known, it is thought that both environmental factors, such as Westernized diet and lifestyle, and genetic factors, such as obesity and lack of exercise, play a role. NIDDM is usually treated first with diet and exercise therapy, and if the treatment effect of these methods is not sufficient, drugs are generally used, and in many cases, insulin is used.

Treatment methods for diabetes include diet therapy, exercise therapy, and drug therapy. Among these, drug therapy has the problem of causing various side effects (discomfort, rejection, hypoglycemia, etc.) because it uses chemicals. there is.

In addition, insulin is necessary for patients whose blood sugar cannot be controlled by diet and oral hypoglycemic agents, but since insulin is a protein, it is hydrolyzed and inactivated in the digestive tract, so it cannot be ingested orally and has the disadvantage of having to be injected intravenously or subcutaneously.

Meanwhile, the ice plant ( Mesembryanthemum crystallinum , common ice plant) is a halophyte belonging to the genus Mesembryanthemum of the order Mesembryanthemum. Its origin is the Namib Desert in South Africa. It normally performs C3 photosynthesis to adapt to the desert climate, but under extreme dry or salty conditions, it is a halophyte. It is a selective CAM (facultative CAM) plant that switches to CAM photosynthesis. Transparent crystals (bladder cells) on the surface contain inositol, beta-carotene, and abundant minerals, and are known to be high in pinitol, which is effective in treating diabetes.

Research on the efficacy of ice plant began in earnest in the early 2000s when it was known that it contains a large amount of pinitol among its ingredients. Additionally, ice plant is mainly being studied to have physiological effects such as lowering blood sugar, lowering blood pressure, and suppressing neutral fat accumulation.

In particular, pinitol, a substance contained in large quantities in ice plants, is converted into chiro-inositol during biological metabolism, and is known as a key molecule involved in moving blood glucose into cells, similar to insulin, and is a vegetable insulin. plays the role of

D-chiro-inositol (cis-1,2,4-trans-3,5,6-cyclohexol) is myo-inositol (cis-1,2,3,5- It is a stereoisomer of trans-4,6-cyclohexanehexol) in which the 3-hydroxyl group is epimerized. Chiroinositol is mainly found in eukaryotes and is biosynthesized by epimerization of myo-inositol. Chiroinositol is a major component of the insulin signaling system and is known to be a substance that significantly improves insulin action when administered by injection or oral administration. Although this is not currently registered as a medicine, it has been reported to be highly effective in lowering blood sugar levels and improving side effects in patients with non-insulin-dependent diabetes. Chiroinositol exists in extremely small quantities in nature, making its production difficult.

Currently, methods for synthesizing chiroinositol using chemical synthesis or microbial fermentation have been developed. Chiroinositol can be chemically synthesized mainly through hydrochloric acid hydrolysis of pinitol (D-pinitol) or kasugamycin. However, the raw materials pinitol or kasugamycin are expensive and the by-products are not easy to separate, so it is not economical. This is a low situation. An efficient way to produce chiroinositol is to bioconvert it from myo-inositol using microorganisms that produce the enzyme Myo-inositol dehydrogenase, but it is still in the early research stage.

Lacticaseibacillus casei is a strain that can be used as a probiotic approved in accordance with the standards and specifications of the Health Functional Food Code of the Ministry of Food and Drug Safety (2019) to balance and improve the intestinal microbial community. It is known to help proliferate beneficial bacteria in the intestines, suppress harmful bacteria, and help maintain smooth bowel movements. Our unique kimchi, which contains a mixture of various types and ingredients of seasoning such as garlic and red pepper powder, contains live plant-based lactic acid bacteria that are naturally high in salt and live in a strongly acidic environment and have strong survival even in acidic digestive juices in the stomach and small intestine, and Lactica J bacillus. L. casei, L. paracasei , and L. plantarum are probiotic lactic acid bacteria derived from kimchi with high survival and antibacterial activity in gastric acid and bile acid environments. It is known to have a better probiotic effect than dairy-derived lactic acid bacteria due to its effects such as decomposing bile acid, reducing blood cholesterol levels, protecting intestinal cells, suppressing colitis and irritable bowel syndrome, and increasing immunity. In addition, Bacillus casei lactic acid bacteria can convert myo-inositol to chiroinositol by producing the enzyme Myo-inositol dehydrogenase.

Therefore, there is an urgent need for the development of technology to convert ice plants with high myo-inositol content into high-concentration chiroinositol by fermenting them with the kimchi lactic acid bacteria Lactica J Bacillus casei WiKim0135 strain.

Republic of Korea Patent Publication No. 10-2014-0040509

Accordingly, the present inventors confirmed that chiroinositol can be produced in high yield using Lactica J Bacillus casei WiKim0135 strain, and that the produced chiroinositol is significantly superior in anti-diabetic effect.

Accordingly, the purpose of the present invention is to provide Lactica Jay Bacillus casei WiKim0135 strain with excellent chiroinositol production ability.

Another object of the present invention is to provide a composition for producing chiroinositol containing Lactica J Bacillus casei WiKim0135 strain, a culture of the strain, a concentrate of the culture, a dried material of the culture, or a culture supernatant of the strain.

Another object of the present invention is to provide a method for producing chiroinositol, which includes a culturing step of culturing the Bacillus casei WiKim0135 strain.

Another object of the present invention relates to a food composition containing chiroinositol produced by culturing Bacillus casei WiKim0135 strain.

Another object of the present invention relates to the use of Bacillus casei WiKim0135 strain for producing chiroinositol.

The present invention relates to Lacticajay Bacillus casei WiKim0135 strain with excellent chiroinositol production ability and its use in lowering blood sugar.

Hereinafter, the present invention will be described in more detail.

An example of the present invention relates to the Lacticaseibacillus casei WiKim0135 strain, which has excellent chiroinositol production ability.

As used herein, the term “chiroinositol” is a substance that can be produced by removing the methyl group through acid decomposition of Pinitol. It is a substance commonly found in the body and is known as one of the mediators involved in the insulin signaling system.

In the present invention, chiroinositol can exist in two forms: enantiomeric D-chiroinositol and L-chiroinositol, and has the following formula (I).

[Formula I]

In this specification, the term “Pinitol” is a type of water-soluble carbohydrate and is one of the ingredients contained in legumes, pine needles, etc. Pinitol has been found to be effective in controlling blood sugar levels in animal experiments in countries around the world since 1987. When consumed, pinitol is converted to chiroinositol in the body. In addition, it was confirmed that when pinitol was ingested in experimental animals with reduced blood sugar control function, blood sugar control function was significantly improved.

In the present invention, the Lactica J Bacillus casei WiKim0135 strain was deposited with KCTC (Korean Collection For Type Cultures), Republic of Korea, on December 13, 2021, with accession number KCTC 14820BP.

In the present invention, the 16s rRNA sequence of Bacillus casei WiKim0135 strain may include the base sequence of SEQ ID NO: 3, but is not limited thereto.

Another example of the present invention is a key containing at least one selected from the group consisting of Lactica J Bacillus casei WiKim0135 strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, and a culture supernatant of the strain. It relates to a composition for producing inositol.

In the present invention, the Lactica J Bacillus casei WiKim0135 strain may be deposited under the accession number KCTC 14820BP, but is not limited thereto.

In the present invention, the 16s rRNA sequence of Bacillus casei WiKim0135 strain may include the base sequence of SEQ ID NO: 3, but is not limited thereto.

In the present invention, the composition may include a compound of the following formula (I), but is not limited thereto.

[Formula I]

Another example of the present invention relates to a chiroinositol production method including a culturing step of culturing the Lacticaseibacillus casei WiKim0135 strain.

In the present invention, the Lactica J Bacillus casei WiKim0135 strain may be deposited under the accession number KCTC 14820BP, but is not limited thereto.

In the present invention, the 16s rRNA sequence of Bacillus casei WiKim0135 strain may include the nucleotide sequence of SEQ ID NO: 3, but is not limited thereto.

In the present invention, chiroinositol may be a compound of the following formula (I), but is not limited thereto.

[Formula I]

In the present invention, the culturing step is performed by cultivating the strain at pH 3 to 9, pH 3 to 8, pH 3 to 7, pH 3 to 6, pH 3 to 5, pH 4 to 9, pH 4 to 8, pH 4 to 7, pH It may include culturing at pH 4 to 6, pH 4 to 5, for example, pH 4.0, but is not limited thereto.

In the present invention, the culturing step is 0.1 to 1.0%, 0.1 to 0.9%, 0.1 to 0.8%, 0.1 to 0.7%, 0.1 to 0.6%, 0.1 to 0.5%, 0.2 to 1.0%, 0.2 to 1.0%, 0.2%. to 0.9%, 0.2 to 0.8%, 0.2 to 0.7%, 0.2 to 0.6%, 0.2 to 0.5%, 0.3 to 1.0%, 0.3 to 0.9%, 0.3 to 0.8%, 0.3 to 0.7%, 0.3 to 0.6%, 0.3 Culturing in a medium containing 0.5%, 0.4 to 1.0%, 0.4 to 0.9%, 0.4 to 0.8%, 0.4 to 0.7%, 0.4 to 0.6%, for example, 0.5% bile salt It may include, but is not limited to this.

In the present invention, the culturing step is performed by culturing the strain at 20 to 45°C, 20 to 40°C, 25 to 45°C, 25 to 40°C, 30 to 45°C, 30 to 40°C, 35 to 45°C, 35 to 40°C, e.g. For example, it may include culturing at 40°C, but is not limited thereto.

In the present invention, the culturing step is performed by cultivating Lactica J Bacillus casei WiKim0135 strain at 0.05 to 3.0% (v/v), 0.05 to 2.8% (v/v), 0.05 to 2.5% (v/v), 0.05 to 2.3%. (v/v), 0.05 to 2.0 % (v/v), 0.05 to 1.8 % (v/v), 0.05 to 1.5 % (v/v), 0.05 to 1.3 % (v/v), 0.05 to 1.0 % (v/v), 0.05 to 0.8% (v/v), 0.05 to 0.5% (v/v), 0.05 to 0.3% (v/v), for example, 0.1% (v/v). It may include, but is not limited to this.

In the present invention, the culturing step is performed on the Lactica J Bacillus casei WiKim0135 strain for 1 to 10 days, 1 to 7 days, 1 to 4 days, 2 to 10 days, 2 to 7 days, 2 to 4 days, for example, It may include culturing for 3 days, but is not limited thereto.

In the present invention, the culturing step is performed by adding 1.0 to 20.0% (w/w), 1.0 to 18.0% (v/v), 1.0 to 15.0% (v/v), 1.0 to 13.0% (v/) of ice plant powder in the medium. v), 1.0 to 11.0 % (v/v), 3.0 to 20.0 % (v/v), 3.0 to 18.0 % (v/v), 3.0 to 15.0 % (v/v), 3.0 to 13.0 % (v/ v), 3.0 to 11.0 % (v/v), 5.0 to 20.0 % (v/v), 5.0 to 18.0 % (v/v), 5.0 to 15.0 % (v/v), 5.0 to 13.0 % (v/ v), 5.0 to 11.0 % (v/v), 7.0 to 20.0 % (v/v), 7.0 to 18.0 % (v/v), 7.0 to 15.0 % (v/v), 7.0 to 13.0 % (v/ v), it may include adding 7.0 to 11.0% (v/v), for example, 10% (w/w), but is not limited thereto.

Another example of the present invention relates to a method for producing chiroinositol comprising the following steps:

A culture step of culturing the Bacillus casei WiKim0135 strain in a medium; and

Separation step to separate the culture from the medium.

In the present invention, the medium contains galactose, glucose, fructose, mannose, mannitol, N-acetylglucosamime, amygdalin, and arbutin ( arbutin, esculin, salicin, D-cellobiose, D-lactose, D-trehalose, D-melezitose (D- It may include, but is not limited to, one or more carbon sources selected from the group consisting of melezitose, gentiobiose, D-tagatose, and potassium gluconate.

The present invention relates to a Lacticajay Bacillus casei WiKim0135 strain with excellent chiroinositol production ability and its use in lowering blood sugar. The Lacticajay Bacillus casei WiKim0135 strain according to the present invention has excellent heat resistance and is not affected by gastric acid and bile acid. It has an excellent ability to produce chiroinositol, which can help lower blood sugar levels, so it can be used as a probiotic strain.

Figure 1 is a graph showing the results of comparing the myo-inositol decomposition activity of 17 different types of lactic acid bacteria derived from kimchi according to an embodiment of the present invention.
Figure 2 is a diagram showing the results of comparing sugar availability for 49 carbon sources of strains according to an embodiment of the present invention.
Figure 3 is a schematic diagram showing the base sequence analysis of the 16S rRNA gene of the Lactica Jay Bacillus casei WiKim0135 strain according to an embodiment of the present invention.
Figure 4 is a GC/FID chromatograph showing simultaneous analysis of three indicator components, pinitol, myo-inositol, and chiroinositol, according to an embodiment of the present invention.
Figure 5 is a graph showing the results of comparing the content of chiroinositol according to fermentation conditions according to an embodiment of the present invention.
Figure 6 is a graph showing the results of comparing the acid resistance, bile resistance, and heat resistance of Lactica Jay Bacillus casei WiKim0135 strain according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1. Selection of strains

The myo-inositol decomposition activity of various kimchi-derived lactic acid bacteria was compared. To isolate kimchi lactic acid bacteria, various types of kimchi (14 types) were collected, and the kimchi crushed liquid was spread and cultured on MRS solid medium to pure isolate microbial colonies. 200 types of kimchi lactic acid bacteria were isolated, and 17 types of kimchi lactic acid bacteria were initially selected through analysis of a myo-inositol assay kit (Megazyme, Ireland). After comparing the myo-inositol decomposition activity of each kimchi lactic acid bacteria, the The results are shown in Figure 1.

As can be seen in Figure 1, it was confirmed that among 17 types of kimchi-derived lactic acid bacteria, Lactica J Bacillus casei species was the most excellent in myo-inositol decomposition activity. As a result of searching the enzymes of each lactic acid bacteria species in an open database (Uniprot.org) where you can search proteins, it was found that Bacillus casei has an iol operon that can decompose myo-inositol and synthesize acetyl-coA. and Lactobacillus plantarum and Lactobacillus pentosus had only inositol transporter and inositol phosphate phosphatase.

Therefore, the Lactica J Bacillus casei strain was finally selected as a kimchi microorganism with excellent myo-inositol decomposition ability.

Example 2. Identification of selection strains

2-1. Sugar availability test of strains

The sugar availability of 49 carbon sources of the finally selected kimchi lactic acid bacteria Lactica J Bacillus casei strain was confirmed using the API CHL 50 kit, a biochemical analysis method. The carbon source fermented during the cultivation period produces acid and lowers the pH. API 50 CHL medium contains a pH indicator to read positive (yellow) and negative (purple) by color change, and the sugars for 49 carbon sources of the strain. Usability was confirmed, and the results are shown in Table 1 and Figure 2.

No. API CH50B Lacticaseibacillus casei WiKim0135 0 Control - One Glycerol - 2 Erythritol - 3 D-Arabinose - 4 L-Arabinose - 5 Ribose - 6 D-Xylose - 7 L-Xylose - 8 Adonitol - 9 β Methyl-xyloside - 10 Galactose + 11 D-Glucose + 12 D-Fructose + 13 D-Mannose + 14 L-Sorbose - 15 Rhamnose - 16 Dulcitol - 17 Inositol - 18 Mannitol + 19 Sorbitol - 20 α Methyl-D-mannoside - 21 α Methyl-D-glucosamine - 22 N-Acetyl glucosamine + 23 Amygdaline + 24 Arbutine + 25 Esculine + 26 Salicine + 27 Cellobiose + 28 Maltose - 29 Lactose + 30 Melibiose - 31 Saccharose - 32 Trehalose + 33 Inulin - 34 Melezitose + 35 D-Raffinose - 36 Amidon - 37 Glycogen - 38 Xylitol - 39 β Gentiobiose + 40 D-Turanose - 41 D-Lyxose - 42 D-Tagatose + 43 D-Fucose - 44 L-Fucose - 45 D-Arabitol - 46 L-Arabitol - 47 Gluconate + 48 2 ceto-gluconate - 49 5 ceto-gluconate -

As can be seen in Table 1 and Figure 2, galactose, glucose, fructose, mannose, mannitol, N-acetylglucosamime, amygdalin ( amygdalin, arbutin, esculin, salicin, D-cellobiose, D-lactose, D-trehalose, D-mel It was confirmed that D-melezitose, gentiobiose, D-tagatose, potassium gluconate, etc. can be used as carbon sources.

2-2. Molecular biological analysis and phylogenetic analysis of strains

The final selected strain of kimchi lactic acid bacteria, Bacillus casei, was identified molecularly through 16s rDNA sequence analysis. The strain was inoculated into MRS medium and then cultured at 30°C for 2 days. Next, the harvested strains were washed twice in sterilized saline solution, and then their DNA was extracted using the DNeasy tissue kit (Qiagen, Valecia, CA, USA). To amplify the 16S rDNA gene of the extracted DNA, the universal primers shown in Table 2 below were used.

sequence number designation Sequence (5'---> 3') bp One Forward primer GGATTAGATACCCTGGTA 18 2 Reverse primer CCGTCAATTCMTTTRAGTTT 20

For PCR reaction, 10 μl of Takara Perfect Premix (Takara, Japan) containing 0.4 mM dNTP, 0.5 units of Taq polymerase, and 4 mM Mg ²⁺ was added with 1 μl of DNA template (20 μg/mL) and 1.0 μM of DNA template. 1 μl each of the forward primer and 1.0 μM reverse primer were added, and the remainder was added with distilled water to make a total volume of 20 μl.

PCR amplification was performed using a Mastercycler gradient (Eppendorf, Hamburg, Germany), and the PCR reaction was at 95°C for 5 minutes (initial denaturation), 94°C for 45 seconds (denaturation), 52°C for 45 seconds (annealing), and 72°C. 30 cycles of 1 minute (extension) were performed, and a final extension was performed at 72°C for 5 minutes.

The total nucleotide sequence of 855 bp was determined as the 16s rRNA coding nucleotide sequence of the isolated strain, the Bacillus casei strain, and the results are shown in Table 3 below.

sequence number designation Sequence (5'---> 3') bp 3 16s rRNA GCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAAATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGCAAGCTATCGCTTTTGGATGGACCCGCGGGCGTATTAGCTAGTTGGGTGAGGTAACGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGC AGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCC TCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATT AAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGC 855

Based on the base sequence performed above, the homology test of the base sequence was performed using the Blast program (http://www.ncbi.nlm.nhi.gov) against registered information (Genebank database). As a result, it was confirmed that the Lacticaseibacillus casei strain was 99% identical to Lacticaseibacillus casei .

The partial nucleotide sequence of 16 rRNA (SEQ ID NO: 3) was registered in GenBank under accession number MN759454. Lactica J Bacillus casei strains showed genetic similarity as shown in Table 4 and Figure 3 below.

Genes of Bacillus casei strains for which base sequence analysis was performed Identification results through NCBI BlastN analysis accession number Similarity (%) 16S rRNA Lacticaseibacillus casei MN759454 99

Therefore, the strain was named Lactica J Bacillus casei WiKim0135, and was deposited with the Korean Collection For Type Cultures (KCTC), Republic of Korea, on December 13, 2021, and given accession number KCTC 14820BP.

Example 3. Chiroinositol conversion ability analysis

3-1. Observation of strain growth in medium containing ice plant

The medium (10% (w/v) ice plant powder and 2% (w/v) glucose) was sterilized at 121°C for 15 minutes. Next, after adding 1% Celluclast and 1% viscozyme, the enzyme reaction was performed at 50°C, and the enzyme reaction was inactivated by heat treatment at 121°C for 10 minutes to prepare a medium to inoculate the strain. Next, to maximize chiroinositol production in the medium, the primary fermentation was performed by inoculating 3.9x10 ⁸ CFU/mL of Bacillus subtilis WiKim0134 and shaking incubation at 37°C for 2 days. Next, in secondary fermentation, 0.1% (v/v) of Lactica J Bacillus casei WiKim0135 strain was inoculated and fermented for 3, 6, and 9 days, and then the number of viable bacteria was measured. After vaccination, the viable cell count was confirmed to be 6.1x10 ⁸ CFU/ml on the 3rd day, 1.7x10 ⁸ CFU/ml on the 6th day, and 4.0x10 ⁷ CFU/ml on the 9th day.

3-2. Gas chromatographic analysis method

GC-FID (Gas Chromatography-Flame Ionization Detector) was performed under the conditions in Table 5 below to simultaneously analyze the three indicator components, pinitol, chiroinositol, and myo-inositol, and the results are shown in Figures 4 and 5. .

device GC-FID column HP-5 capillary column
(0.32 mm idx30 m, 0.25 um) injection temperature
(Inject Temp.) 280℃ column temperature
(Column Temp.) Maintain at 150 ℃ for 5 minutes, then increase to 300 ℃ at a rate of 7.5 ℃/min and maintain for 10 minutes. Detection Temp. 300℃ flow rate He 1 ml/min Split ratio 10:1 Run time 35min

As can be seen in Figures 4 and 5, the sample fermented for 3 days with Lactica J Bacillus casei WiKim0135 strain showed the highest chiroinositol production of about 302.48 mg/kg, and the chiroinositol content of the untreated group was about 105.96. At mg/kg, compared to the untreated group, the chiroinositol production ability was shown to be about 2.85 times increased during ice plant fermentation using the Lactica J Bacillus casei WiKim0135 strain.

As the strain fermentation progressed to the 9th day, the chiroinositol content decreased. This can be confirmed to be due to a decrease in the active ingredient due to the decrease in the number of viable bacteria, as the number of viable bacteria of the Lactica J Bacillus casei WiKim0135 strain also decreased.

Therefore, it was confirmed that Lactica J Bacillus casei WiKim0135 strain is an excellent starter for ice plant fermentation in producing chiroinositol for lowering blood sugar.

Example 4. Investigation of acid resistance, bile resistance and heat resistance of strains

In order for probiotic lactic acid bacteria to reach the intestinal tract, it is important not only to have acid resistance to gastric juice, but also to have resistance to bile acids secreted from the pancreas to the duodenum. In addition, the probiotic activity of probiotic lactic acid bacteria is reduced when exposed to a high temperature environment during the formulation process, so the thermal stability of lactic acid bacteria is one of the important criteria for selecting probiotics. Accordingly, the probiotic characteristics of Lactica J Bacillus casei WiKim0135 strain, including acid resistance, bile resistance, and heat resistance, were confirmed.

As an acid resistance evaluation, the Lactica J Bacillus casei WiKim0135 strain was cultured for 2 hours at 30°C in MRS medium adjusted to pH 4.0, and it was confirmed that it had acid resistance with a survival rate of about 98% ( p<0.05 ).

In addition, as a result of evaluating bile resistance, the Lactica J Bacillus casei WiKim0135 strain was cultured in MRS liquid medium containing 0.5% bile salt for 24 hours, maintaining a survival rate of about 95% and having a significant effect on bile acids. It showed excellent stability as it was not subjected to any damage.

As a result of heat resistance evaluation, the Lactica J Bacillus casei WiKim0135 strain was treated at 40°C for 1 hour, showing a survival rate of 107% and strong heat resistance.

Through this, it was confirmed that Lactica J Bacillus casei WiKim0135 strain had a high survival rate even in acidic, biliary and high temperature environments and exhibited excellent probiotic effects.

Korea Research Institute of Bioscience and Biotechnology (KCTC) KCTC14820BP 20211213

<110> KOREA PRIME PHARM. CO., LTD. KOREA FOOD RESEARCH INSTITUTE <120> Lacticaseibacillus casei WiKim0135 strain with excellent chiroinositol producing ability and uses for lowering blood sugar of that <130> PN210482 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 1 ggattagata ccctggta 18 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 2 ccgtcaattc mtttragttt 20 <210> 3 <211> 855 <212> DNA <213> Artificial Sequence <220> <223> 16s rRNA <400> 3 gcggacgggt gagtaacacg tgggtaacct gcccttaagt gggggataac atttggaaac 60 agatgctaat accgcataaa tccaagaacc gcatggttct tggctgaaag atggcgcaag 120 ctatcgcttt tggatggacc cgcggcgtat tagctagttg gtgaggtaac ggctcaccaa 180 ggcgatgata cgtagccgaa ctgagaggtt gatcggccac attgggactg agaacacggcc 240 caaactccta cgggaggcag cagtagggaa tcttccacaa tggacgcaag tctgatggag 300 caacgccgcg tgagtgaaga aggctttcgg gtcgtaaaac tctgttgttg gagaagaatg 360 gtcggcagag taactgttgt cggcgtgacg gtatccaacc agaaagccac ggctaactac 420 gtgccagcag ccgcggtaat acgtaggtgg caagcgttat ccggattttat tgggcgtaaa 480 gcgagcgcag gcggtttttt aagtctgatg tgaaagccct cggcttaacc gaggaagcgc 540 atcggaaact gggaaacttg agtgcagaag aggacagtgg aactccatgt gtagcggtga 600 aatgcgtaga tatatggaag aacaccagtg gcgaaggcgg ctgtctggtc tgtaactgac 660 gctgaggctc gaaagcatgg gtagcgaaca ggattagata ccctggtagt ccatgccgta 720 aacgatgaat gctaggtgtt ggagggtttc cgcccttcag tgccgcagct aacgcattaa 780 gcattccgcc tggggagtac gaccgcaagg ttgaaactca aaggaattga cggggccccg 840 cacaagcggt ggagc 855

Claims (13)

Lacticaseibacillus casei WiKim0135 strain with excellent chiroinositol production ability, deposited with accession number KCTC 14820BP. delete The strain according to claim 1, wherein the 16s rRNA sequence of the Bacillus casei WiKim0135 strain includes the nucleotide sequence of SEQ ID NO: 3. Lacticaseibacillus casei WiKim0135 strain deposited with accession number KCTC 14820BP, culture of the strain, concentrate of the culture, dried material of the culture, and at least one selected from the group consisting of culture supernatant of the strain A composition for producing chiroinositol, comprising: delete The composition according to claim 4, wherein the 16s rRNA sequence of the Bacillus casei WiKim0135 strain includes the base sequence of SEQ ID NO: 3. 5. The composition of claim 4, wherein the composition comprises a compound of formula (I):
[Formula I]
A chiroinositol production method including a cultivation step of culturing the Lacticaseibacillus casei WiKim0135 strain deposited with accession number KCTC 14820BP. delete The method of producing chiroinositol according to claim 8, wherein the 16s rRNA sequence of the Bacillus casei WiKim0135 strain includes the nucleotide sequence of SEQ ID NO: 3. The method of producing chiroinositol according to claim 8, wherein the chiroinositol is a compound of formula (I) below.
[Formula I]
Method for producing chiroinositol comprising the following steps:
A culture step of cultivating the Lactica J Bacillus casei WiKim0135 strain deposited with accession number KCTC 14820BP in a medium; and
Separation step to separate the culture from the medium. The method of claim 12, wherein the medium contains galactose, glucose, fructose, mannose, mannitol, N-acetylglucosamime, and amygdalin. , arbutin, esculin, salicin, D-cellobiose, D-lactose, D-trehalose, D-melezitose A method for producing chiroinositol, comprising at least one carbon source selected from the group consisting of (D-melezitose), gentiobiose, D-tagatose, and potassium gluconate.