KR102647158B1 - Novel Pediococcus acidilatici strain and various uses of fermented soybean products using the strain - Google Patents

Abstract

The present invention is for the prevention, improvement or treatment of blood pressure, blood lipids, or blood lipids, including a novel strain capable of improving intestinal flora and a soybean fermented product fermented using the strain. It relates to a composition for improving lipids or intestinal flora, and can provide excellent food compositions and pharmaceutical compositions with lower side effects compared to synthetic drugs, while preventing, improving or treating blood pressure, improving blood lipids or improving intestinal flora.

Description

Novel Pediococcus acidilatici strain and various uses of fermented soybean products using the strain {Novel Pediococcus acidilatici strain and various uses of fermented soybean products using the strain}

The present invention relates to a novel strain and a composition for preventing, improving or treating high blood pressure, improving blood lipids or improving intestinal flora, and specifically, for preventing, improving or treating high blood pressure, improving blood lipids or improving intestinal flora. It relates to a composition for preventing, improving or treating high blood pressure, improving blood lipids or improving intestinal flora, including a novel strain and a fermented soybean product using the strain.

Hypertension is a circulatory disease in which blood pressure remains consistently higher than normal blood pressure. It is a very important cardiovascular disease that occurs in 15% of the total population worldwide, especially in 50% of the elderly population over 65 years of age. High blood pressure is one of the important causes of death that causes damage to tissues and organs such as the cardiovascular system, kidneys, and nervous system. The causes of high blood pressure are very diverse. Essential hypertension, the cause of which is not yet known, accounts for 90 to 95% of all high blood pressure patients, and the remaining high blood pressure occurs secondary to causes such as diabetes, kidney disease, and drug use. It accounts for 5%. Treatment methods also vary greatly depending on the cause, degree of hypertension, patient's age, and acceptance of treatment. Blood pressure is determined by the heart's blood output and peripheral vascular resistance. Therefore, hypertension has been traditionally treated using drugs that can lower these two factors. For example, diuretics (thiazides) or sympathetic nervous system inhibitors (α, β-Adrenergic antagonists; Clonidine, Prazocin) have been used. Recently, angiotensin receptor blockers, such as losartan (DuP753), SKF108566, or inactive angiotensin I, which inhibit the physiological action of angiotensin II, a powerful vasoconstrictor, have been used as treatments for hypertension. Drugs that inhibit the action of angiotensin converting enzyme, which converts angiotensin II into its form, such as capdopril, enalapril, ramipril, etc., are increasingly being used as antihypertensive agents. However, the treatment effect is often minimal or non-existent depending on the etiology of hypertension, such as geriatric hypertension.

Meanwhile, fat is an important nutrient along with protein and carbohydrates, and is especially useful as a high energy source. However, fat is high in calories, which promotes obesity and causes problems such as lifestyle diseases. In fact, when it is determined that the patient has hyperlipidemia (hypercholesterolemia, hypertriglyceridemia), diet therapy is used as the best treatment, and when appropriate diet therapy and exercise therapy are combined, symptoms often improve and normalize. However, because fat increases appetite and modern people often eat foods containing large amounts of fat, excessive fat intake has become a problem in developed countries where eating conditions are good. Many cases of hypertriglyceridemia are the result of overeating, lack of exercise, obesity, and excessive alcohol drinking, so they are often combined with high blood pressure or diabetes. Therefore, there are many cases where risk factors overlap or it is difficult to improve daily lifestyle habits, and the number of cases where active drug therapy is administered to prevent the onset of ischemic heart disease is increasing.

It is known that human intestinal health has a close impact on human health. When the balance of interactions between intestinal microorganisms and intestinal cells is disrupted due to various factors, various side effects such as an imbalance in the immune response are caused, which is known to cause intestinal diseases, metabolic diseases, immune diseases, etc. In order to improve the intestinal flora, research is continuously being conducted on probiotics using beneficial intestinal bacteria and prebiotics, which are compounds that serve as food for beneficial bacteria. Recently, natural products with few side effects have been used to increase beneficial intestinal bacteria and reduce harmful intestinal bacteria. Research to improve health is actively underway.

Public Patent Publication No. 10-2015-0088243 (2015.07.31) Public Patent Publication No. 10-2020-0039607 (2020.04.16) Public Patent Publication No. 10-2017-0135520 (2017.12.08)

In order to solve the above problems, the present invention seeks to provide lactic acid bacteria strains that can prevent, improve or treat high blood pressure, improve blood lipids, and improve intestinal flora.

In addition, it is intended to provide a composition for preventing, improving or treating high blood pressure, improving blood lipids, and improving intestinal flora, including fermented soybeans using the lactic acid bacteria.

In order to achieve the above object, the present invention provides lactic acid bacteria strains for preventing, improving or treating high blood pressure, improving blood lipids, or improving intestinal flora.

In addition, a composition containing the above strain for preventing, improving or treating high blood pressure, improving blood lipids or improving intestinal flora is provided.

In addition, it provides a composition for preventing, improving or treating high blood pressure, improving blood lipids or improving intestinal flora, comprising fermented soybeans using the above strain.

The lactic acid bacteria strain is not limited, but is preferably Pediococcus acidilactici .

In the present invention, the Pediococcus axidilactici may be a strain having the 16S rRNA sequence of SEQ ID NO: 1.

The composition may be a pharmaceutical composition for preventing, improving or treating high blood pressure, improving blood lipids, or improving intestinal flora.

Alternatively, the composition may be a food composition for preventing or improving high blood pressure, improving blood lipids, or improving intestinal flora.

The pharmaceutical composition may further include pharmaceutically acceptable excipients, carriers, disintegrants, binders, lubricants, binders, fragrances, etc.

In addition, the pharmaceutical composition can be manufactured into dosage forms such as tablets, capsules, sustained-release preparations, elixirs, suspensions, syrups, wafers, unit dose injection ampoules, and suspensions using methods commonly used in formulation in the art.

The administration methods of the pharmaceutical composition include oral administration, intravenous administration, intramuscular administration, intraarterial administration, intramedullary administration, intrathecal administration, intracardiac administration, transdermal administration, subcutaneous administration, intraperitoneal administration, intranasal administration, and enteral administration. , can be administered by methods such as topical administration, rectal administration, topical administration, dermal administration, mucosal administration, intraspinal administration, including oral and sublingual administration, and is preferably oral administration, but is not limited thereto. In addition, during the above administration, it can be formulated and administered in a formulation appropriate for each administration method, and the formulation can be formulated by a method widely known in the art.

The pharmaceutical composition may be appropriately selected depending on several factors, including the age, weight, health status, gender, administration time, administration route, excretion rate, drug formulation, and severity of the specific disease to be prevented or treated, and the number of administrations. It can be administered once or twice a day, every other day, every other week, or every other month.

The food composition may additionally include flavoring agents, flavoring agents, stabilizers, thickeners, preservatives, etc. commonly used in the art to produce food, such as lactose, dextrose, sucrose, Sorbitol, mannitol, xylitol, erythritol, malditol, starch, alginate, calcium phosphate, calcium silicate, maltodextrin, silicon dioxide, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydride. It may be oxybenzoate, talc, magnesium stearate, etc., but is not limited thereto.

Additionally, the food composition may be manufactured in the form of beverages, gum, tea, vitamin complexes, powders, granules, tablets, capsules, snacks, etc.

The food composition according to the present invention can be appropriately selected depending on the age, weight, health status, gender, administration time, content of active ingredients, severity of disease, etc. of the ingestion target, and the number of intakes is once or twice per day. You can take it every other day, every other week, or every other month.

In addition, the present invention provides a method for producing fermented soybean water for preventing, improving or treating high blood pressure, improving blood lipids or improving intestinal flora, comprising the following steps:

(a) treating a soybean powder solution with a proteolytic enzyme;

(b) fermenting the enzyme-treated soybean powder solution by adding a fermentation strain;

(c) Stirring and culturing the soybean powder solution of step (b).

The soybean powder in step (a) is not limited, but may preferably be a powder obtained by drying and pulverizing soybeans. Additionally, the soy powder solution can be prepared at 10 to 30% w/v, preferably 20% w/v, in a solvent such as water. If it is outside the above range, fermentation may not proceed properly in the subsequent steps.

The enzyme may be one or more selected from the product names Food Pro Alkaline protease, Prozyme 1000L, Prozyme 2000P, Alcalase, Flavourzyme, and Neutrase, and preferably Prozyme 2000p.

Additionally, the enzyme treatment may be performed at a temperature of 40 to 60°C, preferably 50°C, at 50 to 300 rpm, preferably 100 to 200 rpm, for 2 to 4 hours, preferably 3 hours.

If the temperature is below the temperature, the enzyme may not work because the temperature is low, and if it exceeds the temperature, there is a risk that the enzyme may be deformed. In addition, if the rpm is less than the above range, stirring may not be performed properly, and if it exceeds the above range, the stirring speed may be too fast and the enzyme function may not be exerted.

If the enzyme treatment time is less than the above range, the enzyme function may not function properly and the protein may not be properly decomposed, and if it exceeds the above range, there is a risk of the protein being overly decomposed.

The fermentation strain in step (b) is not limited, but is preferably Pediococcus acidilactici , more preferably a Pediococcus acidilactici strain having the 16S rRNA sequence of SEQ ID NO: 1. It can be.

In addition, the inoculum amount of the strain is 1×10 ⁷ to 1×10 ⁹ cfu/ml, preferably 1×10 ⁸ to 5×10 ⁸ cfu/ml, preferably 1×10 ⁸ to 3×10 ⁸ cfu/ ml, more preferably 2×10 ⁸ cfu/ml.

Additionally, the fermentation temperature may be 30 to 45°C and the fermentation time may be 24 to 72 hours, preferably 24 to 48 hours. If the temperature and time are below the above-mentioned temperature and time, fermentation may not proceed properly, and if it exceeds the above-mentioned range, there is a risk of over-fermentation.

The stirring and culturing in step (c) can be performed at a temperature of 36 to 38°C, preferably 37°C for 24 to 72 hours.

The present invention is for the prevention, improvement or treatment of blood pressure, blood lipids, or blood lipids, including a novel strain capable of improving intestinal flora and a soybean fermented product fermented using the strain. It relates to a composition for improving lipids or intestinal flora, and can provide excellent food compositions and pharmaceutical compositions with lower side effects compared to synthetic drugs, while preventing, improving or treating blood pressure, improving blood lipids or improving intestinal flora.

Figure 1 shows the results of evaluating the ACE inhibitory activity of fermented grains fermented with different strains.
Figure 2 shows the results of evaluating the ACE inhibitory activity of fermented grain products according to fermentation time.
Figure 3 shows the results of evaluating the ACE inhibitory activity of fermented grain products according to fermentation temperature.
Figure 4 shows the manufacturing process of fermented grain product according to the present invention.
Figure 5a shows the results of 72-hour soybean fermentation (cell growth and pH) LOT 1, Figure 5b shows the results of 72-hour soybean fermentation (cell growth and pH) LOT 2, and Figure 5c shows the results of 72-hour soybean fermentation (cell growth and pH) LOT 3. will be.
Figure 6 shows the results of evaluating the ACE inhibitory activity of Experimental Example 5.
Figure 7 shows the stability evaluation results for the gastrointestinal enzyme of Experimental Example 6.
Figure 8 shows the cytotoxicity results of Experimental Example 7.
Figure 9 shows the results of evaluating the effect of soy protein bioconversion material on NO production in HUVEC.
Figure 10 shows the results of evaluating the effect of soy protein bioconversion material on HO-1 activity in UVEC.
Figure 11 shows the results of evaluating the effect of soy protein bioconversion material on ET-1 production in HUVEC.
Figure 12 shows the results of evaluating the effect of soy protein bioconversion material on PGE2 production in HUVEC.
Figure 13 shows the results of oral administration of BSB on blood pressure in spontaneously hypertensive rats: (A) 50 mg/kg BSB, 250 mg/kg BSB, or 500 mg/kg compared to wistar rats administered 500 mg/kg BSB or DW. Systolic blood pressure in spontaneously hypertensive rats before and after treatment with BSB or 50 mg/kg Captopril. (B) Diastolic blood pressure of spontaneously hypertensive rats before and after treatment with 50 mg/kg BSB, 250 mg/kg BSB, or 500 mg/kg BSB or 50 mg/kg Captopril and wistar rats or DW administered 500 mg/kg BSB. Data were expressed as mean ± SD. Data points sharing the same letter were not significantly different (p > 0.05).
Figure 14 shows the time course of body weight change after oral administration before and after treatment in wistar rats administered 50 mg/kg BSB, 250 mg/kg BSB or 500 mg/kg BSB or 50 mg/kg Captopril and 500 mg/kg BSB or DW. . Data were expressed as mean (n = 5) ± SD. Data points sharing the same symbol (* or **) were not significantly different (p > 0.05).
Figure 15 shows the effect of BS consumption on body weight of experimental animals: each bar represents the mean ± SEM of five measurements, * indicates no significant difference (p > 0.05) using one-way analysis of variance and Tukey test. represents.
Figure 16 shows the effect of BSB consumption on serum triglycerides: each bar represents the mean ± SEM of five measurements, data points with * are significantly different (p < 0.05) using one-way ANOVA and Duncan's test. NOR: Wistar rat, HIG: BSB at 500 mg/kg BW, MEDIUM: BSB at 250 mg/kg BW and LOW: BSB at 50 mg/kg BW.
Figure 17 shows the effect of BSB consumption on serum cholesterol levels: each bar represents the mean ± SEM of five measurements, data points with * were not significantly different using one-way ANOVA and Duncan's test (p > 0.05) . NOR: Wistar rat, HIG: BSB at 500 mg/kg BW, MEDIUM: BSB at 250 mg/kg BW and LOW: BSB at 50 mg/kg BW.
Figure 18 shows the results of a comparison of gut microbial alpha diversity between each group, including species richness (expressed as Chao1) and evenness (expressed as Shannon and inverse Simpson exponents): boxplots with (*) were not significantly different (p > 0.05).
Figure 19: Distinct features of the intestinal microbiota composition in groups of mice. A and B) PCoA analysis results of 16S rRNA sequencing shown in 2D view. Each dot represents data from one rat and each eclipse represents the rat group they belong to.
Figure 20 shows the effects of BSB and Captopril administration on the main phyla of rat intestinal bacteria: spontaneously hypertensive rats (n = 5), BSB group (n = 5), Captopril group (n = 5) and Wistar Kyoto rats. Fecal samples were collected.
Figure 21 shows the 15 most abundant bacteria in the gut microbiome of experimental animals: HIGH: BSB at 500 mg/kg BW, MID: BSB at 250 mg/kg BW, and LOW: BSB at 50 mg/kg BW.
Figure 22 shows the effect of BSB and Captopril administration on the main phyla of rat intestinal bacteria: spontaneously hypertensive rats (n = 5), BSB group (n = 5), Captopril group (n = 5), and Fecal samples were collected from Wistar Kyoto rats.
Figure 23 shows the effect of BSB administration on serum branched chain amino acid levels: Normotensive = Wistar Kyoto rats. Each bar represents the peak area of an amino acid in a serum sample.
Figure 24: Effect of BSB administration on fecal levels. Each bar represents the peak area of an amino acid in a serum sample: WKY = Wistar Kyoto rat.
Figure 25: Effect of BSB administration on fecal levels. Each bar represents the peak area of an amino acid in a serum sample.

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

However, the following examples and experimental examples only illustrate the present invention, and the content of the present invention is not limited to the following examples and experimental examples.

<Experiment method>

1. Fermentation conditions and strain culture

In the present invention, rice protein and soya bean were provided by Erom Co., Ltd. and used as raw materials. The bacterial growth medium for lactic acid bacteria consisted of defatted rice flour treated with 10% (w/v) Prozyme 2000p in distilled water. The growth medium was sterilized by autoclaving at 121°C for 15 minutes before inoculation with lactic acid bacteria. Pediococcus acidilactici OHER4 (2x10 ⁸ cfu/mL) was transferred from overnight culture to 200mL of autoclaved growth medium (pH6). The medium was incubated at 37°C with 150 rpm agitation for 48 hours. The medium was then centrifuged at 10,000 The final fermentation product was designated as fermentation DP. The medium consisted of soybean powder treated with 20% (w/v) Prozyme 2000p in distilled water. The medium (200 mL, pH 6) was autoclaved at 121°C for 15 minutes, inoculated with Pediococcus acidilactici OHER4 (2 x ¹⁰⁸ cfu/mL), and cultured at 37°C at a shaking speed of 140 rpm for 48 hours. The medium was centrifuged at 11,000 xg for 10 minutes and the supernatant of the medium was recovered. The supernatant was stored at -80°C overnight, frozen, and dried using a TFD5505 tabletop freeze dryer (Gyeonggi-do Ilshin Biobase Co., Ltd.). Dried samples were stored at -18°C for further analysis. The dried fermented product was called fermented PSB.

2. Evaluation of ACE inhibitory activity

The ACE inhibitory activity of the substrate was tested using the Angiotensin I Converting Enzyme Activity (ACEI) kit, with some modifications to the manufacturer's protocol. After mixing 95 μL of ACEI dilution buffer and 5 μL of ACEi positive control vial, 10 μL of diluted ACEi solution was dispensed into a 96 well plate. After dispensing 40 μl of ACEi assay buffer to adjust the volume of the well to 50 μl, 20 μl of inhibitor was dispensed.

Captopril (positive control), ACEi assay buffer (negative control), and sample were dispensed in 20 μl each. ACE 1 substrate (3 μl) was mixed with 47 μl ACEi assay buffer and then transferred to each well and mixed. To draw an Abz-standard standard curve, the concentration of 50 μl of 1mM Abz-standard was adjusted to 100 μM using 450 μl of ACEi assay buffer. After transferring 100 μM of Abz-standard to a 96 well plate, it was serially diluted to 0, 200, 400, 600, 800, and 1000 pmol/well using ACE 1 assay buffer, and the absorbance was measured. After measuring the absorbance of each well, the ACE inhibitory activity was evaluated using the following equation.

Sample (sample ACE1 activity) = BXD/ (ΔT x V)

where B = Abz in Sample based on Std. curve slope (pmol)

ΔT = reaction time (min)

V = volume of sample added to the reaction well (mL)

D = sample dilution factorAngiotensin 1-Converting enzyme inhibition activity evaluation

3. Antihypertensive efficacy analysis (in vivo assay animal test)

All animal experiments were conducted in accordance with the ethical procedures and guidelines of Kangwon National University Animal Care and Management Committee approval number 90 KW-151127-1). A total of 25 male SHR (12 weeks old) and 10 Wistar rats weighing 270-360 g were obtained from Charles River Laboratories in Barcelona, Spain and used in the experiment. SHRs were separated into five groups by simple randomization and housed in a temperature-controlled room (23°C) under a 12-h light/dark cycle and provided with tap water and standard 5001 rodent laboratory chow (Purina, Saint Hubert, Quebec, Canada). Wistar rats were also divided into two groups (n=5). Indirect blood pressure in sleep-restricted rats was measured using a computer-assisted non-invasive blood pressure device (NIBP 76-0173 unit with LE5160R cuff & transducer, Sang Chung Commercial Co., Ltd, Kangnam-Ku, South Korea) using the non-invasive tail cuff method. was measured. Rats were kept at 37°C for 15 minutes to detect tail artery pulsations. To study the effect of bioconverted soybean (BS) intake on blood pressure, each group of hypertensive rats was administered 50 mg/kg of BS per kg of body weight, 250 mg/kg of BS per kg of body weight, 50 mg/kg of Captopril (Sigma-Rich), and 750 μL distilled water. It was administered once by gastric intubation. One group of Wistar rats was provided with normal diet and the other Wistar group was administered 500 mg/kg of BS once a day for 4 weeks.

Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured once daily for 4 weeks before (time 0) and after BS intake. To avoid stress during blood pressure measurement, the tail cuff was deflated after each measurement. Each value of SBP and DBP was obtained by averaging five successful measurements without signal interference. After 4 weeks of treatment, rats were anesthetized using pentobarbital at an intraperitoneal dose of 60 mg kg ^-1 body weight, and blood was extracted by cardiac puncture (5 mL per rat) with a 5 mL 23G syringe. Serum was recovered from the blood and stored at -80°C for further studies.

To assess the amount of food consumed by the rats, the amount of food fed to the rats was recorded each morning and the food remaining in the feeder was weighed the next morning. Differences in mass were recorded as the amount of food consumed by the rats. To evaluate body weight gain, the body weight of each rat in each group was recorded once a week (weeks 0-4), and body weight changes were recorded as weight gain or loss.

4. Scale-up test

In order to develop a mass production process for fermented soybean food materials using soybean (domestic soybean) raw material, production stability was confirmed by increasing the unit process using selected strains and fermentation conditions under laboratory-scale bioconversion conditions. The raw material used in the test was soybean (domestic soybean) powder. The enzyme for pretreatment was Prozyme 2000p commercial enzyme, which had both optimal activity and economic efficiency in previous studies. The fermentation strain had optimal activity. Pediococcus pentosaceus OHER4, which showed , was used. In each scale-up step, small-scale cultivation was first performed using a 5L jar fermenter, and then the volume was increased to a 70L fermenter to confirm production stability.

5. Selection of optimal conditions for the bioconversion process

Analysis of protein hydrolysis ability of finally selected excellent lactic acid bacteria strains and enzymes

- Growth promotion: Extracts for Pediococcus acidilactici SDL1405, Pediococcus acidilactici SDL1406, Pediococcus acidilactici SDL1402, Streptococcus thermophilus SCML300, Streptococcus thermophilus SCML337, Weissell acibaria SCCB2306, Enterococcus faecium SC54, Lactobacillus rhamnosus JDFM6 Growth promotion efficacy was evaluated. While culturing the strain in a 12-well plate, absorbance was measured at 600 nm using a spectrophotometer for 12 hours. Rice bran extract and general nutrient fortified medium (control) were mixed and used at a concentration of 10-1 in the culture medium.

- Degree of protein hydrolysis: O -phthaldialdehyde (OPA) was used to determine the degree of protein hydrolysis according to the method of Church et al. (Church et al. 1983). Briefly, the OPA solution is made by mixing 25 mL of 100 mM sodium tetraborate, 2.5 mL of 20% (w/w) sodium dodecyl sulfate, and 40 mg/1 mL of OPA with methanol with 100 mL of mercaptoethanol to a final volume of 50 mL with distilled water. . The sample protein fermentation product-low molecular weight peptide (5 mg/ml) is reacted in OPA solution (1.0 mL) at room temperature for 2 minutes, and then the absorbance (wavelength 340 nm) is measured. The amount of peptide released by water decomposition from the sample protein is determined from the standard curve of dipeptide Phe-Gly.

6. Metagenomic analysis

- Microbial DNA extraction: flora isolated from feces is measured using 16S rRNA bacterial genotype. Bacterial DNA was extracted from animal specimens using the PowerLyzer® Powersoil DNA® Isolation Kit (MoBio) according to the manufacturer's instructions. Subsamples of feces were taken only from the outer area of the specimen and weighed in PowerLyzer® glass bead tubes (MoBio) to reduce variability. FastPrep-24TM 5 G (MP Biomedical) was used to homogenize the samples at a speed of 5.5 m/s for four 90 s cycles with 60 screaks between each cycle.

- 1.1.2 16S rRNA bacterial gene sequence analysis: The extracted bacterial DNA was used as a template for initial PCR amplification of the V3-V4 hypervariable region of the 16S rRNA bacterial gene using a barcode fusion primer; 16SR_V4 (5'-CAAGCAGAAGACGGCATACGAGAT-barcode-AGTCAGTCAG CCGGACTACHVGGGTWTCTAAT-3') and 16SF_V3(5'-AATGATACGGCGACCACCGAGATCT `ACAC-barcode-TATGGTAATTGGCCTACGGGAGGGCAGCAG-3'). It also includes adapters for downstream Illumina MiSeq sequencing. Each sample is amplified with a pair of barcoding primers, and the PCR reagents used are Invitrogen AccuPrimeTMPfx SuperMix (part number 12344-040), 10 μM 16SR_V4 primer (1 μl), 10 μM 16SF_V3 primer (1 μl), and Ambion Nucleisprimer ( Generalized by catalog number AM9932). PCR conditions were as follows: 95°C for 2 min, followed by 30 cycles at 95°C for 20 s (rejection), 55°C for 15 s, and 5 min, ending with 72°C for 10 min (extension). Library clean-up was performed using the Invitrogen Sequal-Prep Normalization Plate Kit (Thermo Fisher). A large amount of PCR product was used to clean the library with a runoff volume of 12 μl. The Qubit DNA high-sensitivity assay was used to measure library concentration, and the Bioanalyzer DNA HS assay was used to measure library opening. Libraries were filled to equal volumes and sequencing was performed on an Illumina MiSeq machine using a read length of 2 × 250 bp at the Massey Genome Service (Massey University). Data obtained from Illumina MiSeq sequencing were analyzed using Quantitative Insights to Microbial Ecology (QIIME). A paired-end assembler for DNA sequencing was used to assemble forward and backward reads in a contiguous sequence ensuring a minimum overlap of 50 bp, with a minimum of 350 bp and a maximum of 500 bp. Chimeric filtered sequences and reads were clustered into operational taxonomic units based on an ID threshold of 97% using USEARCH 6.1 and UCLUST. Sequencing alignment using the Green genes core reference database (version 13.5) was performed using PyNAST. RDP Na ve Bayesian classifiers will be used to provide taxonomic tasks. There will be changes in library size (33,906 to 196,843), and all samples will be sparse to 33,000 due to the possibility of different sequencing depths, which may bias the diversity measure calculations.

- 2.1 Fecal short chain fatty acids (SCFA) analysis: SCFA will be measured by GC using a modified known method (Richardson et al. 1989). In a frozen state, 0.5 to 1.0 g of the animal specimen was weighed using a 15 ml eppendorf tube. Using 0.01 MPBS containing 2-ethylbutyric acid (5·56 mM) as an internal standard, an aqueous solution containing 5 mM 2-ethylbutyric acid (dilution factor 10) was prepared to be added to animal samples. Afterwards, the samples were kept on ice to disperse the animal material. The aqueous animal solution was centrifuged at 3000g at 4°C for 10 minutes, and 500 μl of the supernatant was transferred to a 2 ml eppendorf tube, acidified with 250 μl of concentrated hydrochloric acid, and 1000 μl of diethyl ether was added. To transfer the acid to the diethyl ether phase, the mixture was mixed using VORTEX for 10 seconds and centrifuged at 10,000g at 4°C for 5 minutes. In a vial, 100 μl of diethyl ether was induced with 1% butyldimethylchlorosilane (MTBSTFA + TBDMSCI, 99:1; Sigma-Aldrich) and 20 μl of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide in a water bath at 80°C for 20 minutes. Once cooled, the derivatized sample was transferred into a 200 μl vial. To ensure complete derivatization, the samples were left at room temperature for 48 hours and then analyzed using GC. A standard containing 2-ethylbutyric acid (5 mM) was prepared as an internal standard for derivatization along with the sample. Analysis was performed on a GC system (GC-2010 Plus) equipped with a flame ionization detector and Restek column (SH-Rtx-1, 30 m × 0·25 mm ID × 0·25 μm) (Shimadzu). The transport gas had a total flow rate of 21.2 ml/min and a pressure of 131.2 kPa, and nitrogen was used as supplementary gas. The temperature program started at 70°C and increased to 115°C at 6°C/min, with the final increase held at 60°C/min to 300°C for 3 minutes. The flow control mode was set to a linear speed of 37.5 cm/s, the injector temperature was 260°C, and the detector temperature was 310°C. Samples were injected in split injections (split ratio 10:1). The GC equipment was controlled and data processed using Shimadzu GC Work Station Lab Solutions version 5.3. The data collected provided a final sample result of μmol SCFA/g wet animal population.

<Experimental Example 1> Screening of lactic acid bacteria with high ACE inhibitory activity

After producing fermented products using various types of lactic acid bacteria for soybeans, the ACE inhibitory activity was evaluated to select strains with excellent activity. Domestic soybeans were ground into powder, treated with Porzyme 2000, and then fermented with several types of lactic acid bacteria.

The fermentation strains used in the experiment were Pediococcus acidilatici SDL1402, Pediococcus acidilatici SL1406, Pediococcus acidilatici SCL1420, Pediococcus acidilatici OHER4, Pediococcus acidilatici DM9, Pediococcs pentosaceus SDL141, Lactobacillus plantarum SDL1416, and Lactobacillus, which were identified as lactic acid bacteria among strains isolated from soybean paste. acillus plantarum JDFM44, Lactobacillus pentosus SC48, Lactobacillus arizonensis SC25, Lactobacillus curvatus JBNU38 and Lactobacillus bravis SDL1411 were used.

The basic growth medium for lactic acid bacteria was composed of 20% (w/v) soybean powder in distilled water. The medium was prepared by mixing and stirring after treatment with Prozyme 2000P prior to fermentation, and sterilized under high pressure at 121°C for 15 minutes before culturing with lactic acid bacteria. did.

Each strain was inoculated at 2x10 ⁸ cfu/ml in a 500 ml Erlenmeyer flask containing 200 ml of soybean powder medium (pH 6), and culture was performed at 37°C with agitation at 150 rpm. The prepared sample was heated at 90°C for 20 minutes to stop strain activity and sterilized, then concentrated, freeze-dried, and stored at -80°C for further analysis.

The Angiotensin I Converting Enzyme Activity (ACEI) kit was used to determine ACE inhibitory activity, and the manufacturer's protocol was partially modified.

After mixing 95 μL of ACEI dilution buffer and 5 μL of ACEi positive control vial, 10 μL of diluted ACEi solution was dispensed into a 96 well plate. After dispensing 40 μl of ACEi assay buffer to adjust the volume of the well to 50 μl, 20 μl of inhibitor was dispensed. Captopril (positive control), ACEi assay buffer (negative control), and sample were dispensed in 20 μl each. ACE 1 substrate (3 μl) was mixed with 47 μl ACEi assay buffer and then transferred to each well and mixed. To draw an Abz-standard standard curve, the concentration of 50 μl of 1mM Abz-standard was adjusted to 100 μM using 450 μl of ACEi assay buffer. After transferring 100 μM of Abz-standard to a 96 well plate, it was serially diluted to 0, 200, 400, 600, 800, and 1000 pmol/well using ACE 1 assay buffer, and the absorbance was measured. After measuring the absorbance of each well, the ACE inhibitory activity was evaluated using the following equation.

Sample (sample ACE1 activity) = BXD/ (ΔT x V)

where B = Abz in Sample based on Std. curve slope (pmol)

ΔT = reaction time (min)

V = volume of sample added to the reaction well (mL)

D = sample dilution factorAngiotensin 1-Converting enzyme inhibition activity evaluation

After fermentation, the sample fermented with Pediococcus acidilactici OHER4 showed the strongest ACE inhibitory activity (Figure 1), and as a result of analyzing the 16S rRNA sequencing (Macrogen Co., Korea) of the strain, the sequence as shown in Table 1 was confirmed, The sequence is shown in SEQ ID NO: 1.

Genetic information of the strain according to the present invention Strain name 16S rRNA sequence Pediococcus acidilatici OHER4 1 ggagggtttc cgcccttcag tgctgcagct aacgcattaa gtaatccgcc tggggagtac
61 gaccgcaagg ttgaaactca aaagaattga cgggggcccg cacaagcggt ggagcatgtg
121 gtttaattcg aagctacgcg aagaacctta ccaggtcttg acatcttctg ccaacctaag
181 agattaggcg ttcccttcgg ggacagaatg acaggtggtg catggttgtc gtcagctcgt
241 gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttattacta gttgccagca
301 ttcagttggg cactctagtg agactgccgg tgacaaaccg gaggaaggtg gggacgacgt
361 caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggat ggtacaacga
421 gtcgcgaaac cgcgaggttt agctaatctc ttaaaaccat tctcagttcg gactgtaggc
481 tgcaactcgc ctacacgaag tcggaatcgc tagtaatcgc ggatcagcat gccgcggtga
541 atacgttccc gggccttgta cacaccgccc gtcacaccat gagagtttgt aacacccaaaa
601 gccggtgggg

<Experimental Example 2> Evaluation of ACE inhibitory activity according to fermentation time

According to the results of Experiment 1 above, the following experiment was conducted using the strain of the present invention to evaluate whether the ACE inhibitory activity increases depending on the fermentation time of grain. The sample used in Experimental Example 1 was fermented for 0 hours (unfermented sample), 12 hours, 24 hours, 48 hours, and 72 hours, and the results are shown in Figure 2.

As a result of the experiment, it was observed that the ACE inhibitory activity of the fermented samples increased as the fermentation time increased, but samples fermented for 24 hours and 48 hours did not show a significant difference. Considering the economic feasibility of mass production, 24 hours and 48 hours were observed. The fermentation time was confirmed to be appropriate.

<Experimental Example 3> Evaluation of ACE inhibitory activity according to fermentation temperature

Using the sample of Experimental Example 1, the following experiment was conducted to evaluate whether the ACE inhibitory activity increases depending on the fermentation temperature. The samples were fermented with the strain according to the present invention (P ediococcus acidilactici OHER4) and cultured at 30, 37 and 45°C for 48 hours and then compared for ACE activity (Figure 3). As a result of the experiment, the inhibition of ACE activity significantly increased as the fermentation temperature increased, but there was no significant difference, so 37°C was set as the optimal process considering the economic efficiency of the process.

<Experimental Example 4> Fermentation process scale-up test Ⅰ: 5L fermenter fermentation trial production test

Soybeans were obtained domestically, dried and selected, then made into powder and used in the process. In the bioconversion process of soybean powder, Przyme 2000P was used as an enzyme according to laboratory-scale production conditions, and Pediococcus acidilactic i OHER4 was used as a fermentation strain. To select optimal conditions, a 20% w/v soybean powder solution was prepared and then treated with the selected enzyme, Prozyme 2000p (50°C, 150rpm, 3 hours) to prepare a soybean powder enzyme treatment solution. Pediococcus acidilatici OHER4 was centrifuged to separate the pellet and washed twice in distilled water. The recovered cell pellet was resuspended in sterilized distilled water at a concentration of 2 x 10 ⁸ CFU/ml and then inoculated into 10 mL of grain medium. and cultured with agitation at 37°C for 72 hours. To stop fermentation, the samples were heated at 95°C for 15 minutes and then freeze-dried. The process of each step was performed a total of 3 times (3 #Lots) to compare and evaluate the stability of the process, and each unit process and conditions are shown in Figure 4.

During the fermentation of soybean powder during the above process, the pH decreased from 5.8 to 3.9 in the first 24 hours and did not change further. Bacterial growth increased from 0 to 50 hours and began to decrease until 72 hours (Figures 5A-5C).

<Experimental Example 5> Evaluation of ACE inhibitory activity of fermented soybean food material

The ACE inhibitory activity was evaluated using LOTs 1 to 3 prepared in Experimental Example 4. As a comparative example, captopril, an angiotensin converting enzyme inhibitor known as an antihypertensive drug, was selected. Summarizing the experimental results, the ACE inhibitory activity was evaluated for samples fermented with the strain according to the present invention after enzymatic pretreatment of soybean powder. As a result, all samples produced in #3 Lot. were active against the raw soybean before fermentation. This was confirmed to be very excellent, confirming the suitability and stability of the production conditions (Figure 6).

<Experimental Example 6> Stability evaluation of gastrointestinal enzymes

To evaluate the stability of the fermented product of the present invention to gastrointestinal enzymes, an experiment was conducted as follows. We attempted to evaluate the stability of small-scale produced fermented soybean food materials by treating them with pepsin and pancreatin, digestive enzymes in the gastrointestinal tract, and then measuring their activity (Figure 7).

As a result of the experiment, the ACE inhibitory activity of the fermented soybean food material did not significantly decrease after treatment with pepsin and pancreatin, and the produced raw material was confirmed to maintain stability even in the digestive tract, confirming its high value as a food material.

<Experimental Example 7> Evaluation of antihypertensive efficacy (in vitro)

To evaluate the antihypertensive efficacy of the present invention, an experiment was conducted as follows. We sought to confirm the antihypertensive activity of soy protein material produced through a bioconversion process by evaluating its effect on indicators related to antihypertension using vascular endothelial cells (HUVEC).

Cytotoxicity evaluation : MTS assay was performed to confirm the cytotoxicity of fermented soybean extract in HUVEC. 1×10 ⁵ HUVEC cells were seeded in a 96 well plate and cultured for 24 hours. Afterwards, the fermented soybean extract was treated at a concentration of 2 to 20 μg/ml, cultured for 24 hours, and MTS (3-(4,5-dimethylthiazol-2-yl)5-(3-carboxy-methoxyphenyl)-2-( 20 μl of 4-sulfophenyl)-2H-tetrazolium) solution (Promega, USA) was added to each well, reacted for 4 hours in a 5% CO ₂ incubator at 37°C, and the absorbance was measured at 580 nm (Figure 8). . As a result of the experiment, it was found that after treating HUVEC cells with soy protein bioconversion material at concentrations of 2, 5, 10, and 20 μg/ml, it did not cause cell death depending on the treatment concentration.

Measurement of nitric oxide (NO) production activity : HUVEC were distributed at 1x10 ⁵ cells/well in a 96 well plate and cultured for 24 hours, then treated with fermented soybean extract at a concentration of 2~20μg/mL and TNF-α 20μg/mL for 24 hours. It was cultured for some time. The nitric oxide (NO) production activity of the culture medium obtained after culturing was measured by absorbance at 540 nm using a Nitric Oxide (NO) detection kit (iNtRON) (FIG. 9).

As a result of the experiment, the NO production rate was measured after treating HUVEC cells with soy protein bioconversion material at concentrations of 2, 5, 10, and 20 μg/ml, and it was confirmed that the production of NO significantly increased depending on the treatment concentration of the sample. I was able to. Therefore, it was evaluated that soy protein bioconversion material can be expected to have a blood pressure control function through the confirmation of blood vessels in hypertension by increasing NO production.

Hemoxygenase-1 (HO-1) activity measurement : HUVECs were distributed at 1x10 ⁵ cells/well in a 96 well plate and cultured for 24 hours, then treated with fermented soybean extract at a concentration of 2~20μg/mL and TNF-α 20μg/mL. and cultured for 24 hours. After culture, RNA from HUVEC cells was extracted and RT-PCR was performed using HO-1 primers to evaluate the expression rate of the HO-1 gene (FIG. 10).

As a result of the experiment, it was confirmed that the expression of HO-1 significantly increased with the treatment of soy protein bioconversion material. HO-1 plays an important role in antioxidant mechanisms, and especially in hypertension, it improves oxidative damage to vascular endothelial cells and promotes relaxation of blood vessels by promoting the production of carbon oxide. Therefore, it was evaluated that treatment with soy protein bioconversion material can prevent and improve blood vessel damage along with controlling blood pressure through vascular relaxation in hypertension.

Measurement of endothelin-1 (ET-1) production inhibitory activity : HUVEC cells were distributed in a 96-well plate at a concentration of 1.5 2, 5, 10, and 20 μg/mL) and cultured for 18 hours. A certain amount of supernatant was taken and the amount of ET-1 produced was measured using the Endothelin-1 Quantikine kit (R&D systems, USA) (FIG. 11).

As a result of the experiment, it was confirmed that the expression of ET-1 significantly increased with the treatment of soy protein bioconversion material. ET-1 is a protein factor that plays an important role in the constriction of blood vessels and particularly affects the increase in blood pressure in hypertension. Therefore, the soy protein bioconversion material was evaluated to have the function of regulating blood pressure through vasodilation in hypertension by reducing the production of ET-1.

Measurement of rostaglandin E2 production inhibitory activity : HUVEC cells were distributed in a 96-well plate at a concentration of 1.5 20 μg/mL) and cultured for 18 hours. A certain amount of supernatant was taken and PGE2 was measured using a PGE2 kit (R&D, Minneapolis, MN, USA) (FIG. 12).

As a result of the experiment, it was evaluated that the production of PGE-2 was significantly reduced by treatment with the soy protein bioconversion material, so it is expected that the treatment with the material will have the function of suppressing inflammation in the vascular endothelial cells and tissues caused by high blood pressure. do. Meanwhile, the activity was highest when the soy protein bioconversion material was treated at a concentration of 10 μg/mL, and it tended to increase again at 20 μg/mL.

<Experimental Example 8> Evaluation of antihypertensive efficacy (in vivo)

To evaluate the antihypertensive efficacy of the present invention, an experiment was conducted as follows. When BS was administered to SHR, blood pressure was significantly reduced, and captopril reduced blood pressure in SHR to a greater extent than BS. The experimental results showed that BS lowered blood pressure depending on the dose, and in the third week, there was no significant difference in systolic blood pressure between SHRs consuming 500 mg/kg body weight of BS and SHRs consuming 50 mg/kg captopril. Additionally, BS intake had no effect on blood pressure in normal (non-hypertensive) rats, and in general, BS regulated diastolic blood pressure better than captopril (Figure 13).

Additionally, consumption of BS did not significantly affect the amount of food consumed by the experimental rats (Figure 14), and consumption of BS did not affect the body weight of the experimental animals (Figure 15).

In addition, consumption of BS did not affect blood triglycerides in the experimental animals, but neutral fats significantly increased in the captopril-consuming group, which was a positive control (FIG. 16). Consumption of BS significantly increased blood HDL cholesterol and significantly decreased the LDL/VLDL cholesterol ratio in experimental animals (Figure 17).

<Experimental Example 9> Evaluation of the effect of improving intestinal flora

This experiment was conducted to evaluate the effect of the present invention on improving intestinal flora.

1. Alpha diversity

As a result of testing the richness of microbial species in each group using Chao1 analysis, the hypertensive rat negative group (SHR) was found to be high, but the evenness using Shannon analysis and inverse Simpson's index analysis was confirmed to be the lowest (Figure 18).

2. Beta diversity

16S rDNA sequencing was used to analyze the intestinal microbial communities of experimental animals, and differences between communities were analyzed as shown in a 2D chart. Intergroup differences in the intestinal microbial community through PCoA analysis showed significant differences between the hypertensive rat negative control group (SHR), Wistar normal rat (WKYR), and hypertensive rat test group fed fermented soybean product (BSB), as shown in rmflaa. , Meanwhile, there was no significant difference between the test groups fed with Wistar normal rats (WKYR) and fermented soybeans (BSB). It was evaluated that consumption of fermented soybeans made the distribution of the intestinal microbial community in hypertensive rats similar to that in normal rats (Figure 19).

3. Effect of BSB intake on SHR intestinal microbial flora

In experimental animals that consumed fermented soybeans (BSB), the level of Bacteriodetes among intestinal microorganisms increased significantly from 30% to 42%, and the level of Actinobacteria decreased from 22% to 2%. Verrucomicrobia was not seen in hypertensive rats (SHR), but the amount of Verrucomicrobia was significantly increased in hypertensive rats that consumed fermented soybeans (Figure 20).

4. Significant bacteria-boosting effects of BSB consumption

The levels of Akkermansia muciniphila and Rumminococus albus were significantly reduced in hypertensive rats (SHR), but consumption of fermented soybean extract (BSB) significantly increased the levels of Akkermansia muciniphila and Rumminocus albus in the intestines of hypertensive rats. Akkermansia muciniphila is known to play an important role in metabolic gastrointestinal disorders, and Rumminocus albus is known to produce major short-chain fatty acids (FIG. 21).

5. Unique gut microbial network-inducing effect of BSB intake

Based on the top 20 bacteria identified in the fecal samples of experimental animals, correlation analysis was performed using JMP software (AS institute Inc.). As a result, the network was displayed in cytoscape as shown in Figure 22, and the network was displayed in hypertensive rats (SHR). As a result of consuming fermented soybeans, negative interactions were found to decrease.

6. Effect of reducing branched chain amino acid levels in serum

Many studies have shown that an increase in branched chain amino acids in the blood is strongly correlated with high blood pressure. In this experiment, consumption of fermented soybeans (BSB) increased the valine content in the serum of hypertensive rats (SHR). was observed to decrease (Figure 23).

7. Effect of reducing the level of microbial short chain acids in feces

Consumption of fermented soybeans (BSB) was shown to improve the level of microbial short chain acids in the feces of hypertensive rats (SHR) (FIG. 24).

8. Effect of increasing levels of microbial aryl hydrocarbon receptor ligands in feces

It can be seen that the intake of fermented soybeans (BSB) increased the production of 2-oxindole-3-acetate by microorganisms, as shown in Figure 25. 2-oxindole-3-acetate is an aryl hydrocarbon receptor ligand and is known to alleviate metabolic syndrome.

<110> Erom Co., Ltd. KNU-Industry Cooperation Foundation <120> Novel Pediococcus acidilatici strain and various uses of fermented soybean products using the strain <130> 21-11927 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 610 <212> RNA <213> Artificial Sequence <220> <223> 16S rRNA sequence of Pediococcus acidilatici <400> 1 ggagggtttc cgcccttcag tgctgcagct aacgcattaa gtaatccgcc tggggagtac 60 gaccgcaagg ttgaaactca aaagaattga cgggggcccg cacaagcggt ggagcatgtg 120 gtttaattcg aagctacgcg aagaacctta ccaggtcttg acatcttctg ccaacctaag 180 agattaggcg ttcccttcgg ggacagaatg acaggtggtg catggttgtc gtcagctcgt 240 gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttattacta gttgccagca 300 ttcagttggg cactctagtg agactgccgg tgacaaaccg gaggaaggtg gggacgacgt 360 caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggat ggtacaacga 420 gtcgcgaaac cgcgaggttt agctaatctc ttaaaaccat tctcagttcg gactgtaggc 480 tgcaactcgc ctacacgaag tcggaatcgc tagtaatcgc ggatcagcat gccgcggtga 540 atacgttccc gggccttgta cacaccgccc gtcacaccat gagagtttgt aacacccaaa 600 gccggtgggg 610

Claims (10)

(a) treating a soybean powder solution with a proteolytic enzyme;
(b) fermenting the enzyme-treated soybean powder solution by adding a fermentation strain;
(c) stirring and culturing the soybean powder solution of step (b),
In step (a), the proteolytic enzyme is Prozyme 2000P,
A method for producing a fermented soybean product, characterized in that the fermentation strain in step (b) is Pediococcus axidyllactici having the 16S rRNA sequence of SEQ ID NO: 1. The method of producing a fermented soybean product according to claim 1, wherein in step (a), the soybean powder solution is 10 to 30% w/v. delete The method of claim 1, wherein the enzyme treatment in step (a) is performed at 40 to 60°C and 100 to 200 rpm for 2 to 4 hours. delete The method of claim 1, wherein the inoculum amount of the fermentation strain in step (b) is 1×10 ⁷ to 1×10 ⁹ cfu/ml. The method of claim 1, wherein the stirring and culturing conditions in step (c) are 24 to 72 hours at 36 to 38°C. A fermented soybean product produced by the method of any one of claims 1, 2, 4, 6, and 7. A food composition for preventing or improving high blood pressure, improving blood lipids, or improving intestinal flora, comprising the fermented soybean product of claim 8. A pharmaceutical composition for preventing, improving or treating high blood pressure, improving blood lipids or improving intestinal flora, comprising the fermented soybean product of claim 8.