JP6239568B2 - Lactic acid bacteria and food additives, foods and drugs using the same - Google Patents

Description

  The present invention relates to lactic acid bacteria and food additives, foods, and drugs using the same, and particularly relates to lactic acid bacteria belonging to Lactobacillus plantarum and food additives, foods, and drugs using the same.

  Lactobacillus plantarum is known as a plant lactic acid bacterium (Patent Documents 1 and 2). Lactic acid bacteria such as Lactobacillus plantarum are known to exert physiological functions beneficial to humans that maintain the intestinal flora (intestinal flora) in a good state.

JP 2009-82127 A JP 2012-93 A

  In order for lactic acid bacteria to exert physiological functions to keep the intestinal flora in a good state, the lactic acid bacteria need to reach the intestine through the stomach without being killed by gastric acid. In other words, in order for lactic acid bacteria to function effectively in the intestine, the gastric acid resistance of the lactic acid bacteria is required to be high. Since the pH (hydrogen ion index) in the human stomach is about 1 to 2 when fasting and about 2 to 4 after meal, lactic acid bacteria with strong acid resistance at this level of acid concentration reach the intestine. Thus, the physiological function of keeping the intestinal flora in a good state is sufficiently exhibited, and it can be said that this is a useful lactic acid bacterium.

  The problem to be solved by the present invention is to provide a novel lactic acid bacterium that has a strong resistance to gastric acid and reaches the intestine with a high survival rate (survival rate against gastric acid environment), a food additive using the same, and a fermented seasoning for pickles It is to be.

  From one aspect, the lactic acid bacterium according to the present invention is a lactic acid bacterium PIC-NBN22 strain (accession number NITE P-1496) belonging to Lactobacillus plantarum.

  From another aspect, the lactic acid bacterium according to the present invention has a pH 3.5 survival rate of gastric acid environment of 260% or more, more preferably a pH 3.5 survival rate of gastric acid environment of 260% or more and pH 3.0. It is a lactic acid bacterium belonging to Lactobacillus plantarum having a gastric acid environment survival rate of 100% or more.

From another aspect, the lactic acid bacterium according to the present invention is a lactic acid bacterium belonging to at least the following Lactobacillus plantarum having a sugar utilization property.
Glycerol negative D-arabinose negative L-arabinose positive ribose positive D-xylose negative galactose positive glucose positive fructose positive mannose positive rhamnose negative inositol negative D-mannitol positive sorbitol positive N-acetylglucosamine positive amygdalin positive cervine sucrose positive Positive lactose positive melibiose negative sucrose positive trehalose positive raffinose negative gentiobiose positive

  The food additive according to the present invention contains the lactic acid bacteria according to the above-mentioned invention.

  The food according to the present invention contains the lactic acid bacteria according to the above-described invention.

  The method for producing pickles according to the present invention is a method for producing pickles having a fermentation-like fragrance, wherein the lactic acid bacteria according to the above-mentioned invention is added as a starter to the vegetables that are the raw materials of the pickles, and the vegetables are fermented by the lactic acid bacteria. .

  The lactic acid bacteria according to the present invention have high gastric acid resistance and reach the intestine with a high survival rate. Thereby, the lactic acid bacteria according to the present invention remarkably exert a beneficial physiological function to keep the intestinal flora in a good state.

The graph which shows the test result of the gastric acid tolerance measurement test of lactic acid bacteria PIC-NBN22 stock | strain (PIC-NBN lactic acid bacteria) and the lactic acid bacteria of a comparative example. ((A) is the survival rate of acid environment to gastric acid at pH 3.5, (B) is the survival rate of acid environment to gastric acid at pH 3.0, (C) is the survival rate of acid environment to gastric acid at pH 2.5) The graph which shows the measurement result of ATPase activity. (A) is a graph showing an analysis result by gas chromatography of a fermented seasoning liquid for pickles fermented using lactic acid bacteria PIC-NBN22 strain, (B) is a graph showing an equivalent analysis result of a comparative example without fermentation . The graph which shows the turbidity increase rate of the fermentation liquid of pH 5.5 and pH 4.5 after 42 hours of fermentation in the temperature environment at 20 degreeC. The graph which shows the measurement result of the ratio of the trans baxenoic acid in a microbial cell membrane fatty acid. The graph which shows the measurement result of the variation | change_quantity of IgA content. The photograph which shows the test result of the coculture of Helicobacter pylori.

  As a result of intensive studies, the present inventors have found a novel lactic acid bacterium PIC-NBN22 strain belonging to Lactobacillus plantarum. In the present invention, this lactic acid bacterium PIC-NBN22 strain is used.

  The lactic acid bacteria PIC-NBN22 strain was established in 2012 by the National Institute of Technology and Evaluation Microorganisms Deposited Center (2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan 292-0818). Deposited on the 25th of the month, the deposit number is NITE P-1496.

  The main bacteriological properties of lactic acid bacteria PIC-NBN22 strain are as follows. This mycological property and the 16S rDNA base sequence indicate taxonomically that the PIC-NBN22 strain belongs to Lactobacillus plantarum.

<Morphological properties>
Cell morphology: Aspergillus Size: 0.5 × 1.0-2.0 μm
Sporulation:-
Mobility:-
Catalase:-
Oxidase:-
Growth at 37 ° C: +
Growth at 45 ° C:-
Gram staining: Positive Colony color: Milky white Colony shape: Circular

<Sugar utilization (carbohydrate fermentation)>
Glycerol negative D-arabinose negative L-arabinose positive ribose positive D-xylose negative galactose positive glucose positive fructose positive mannose positive rhamnose negative inositol negative D-mannitol positive sorbitol positive N-acetylglucosamine positive amygdalin positive cervine sucrose positive Positive lactose positive melibiose negative sucrose positive trehalose positive raffinose negative gentiobiose positive

<Evaluation of gastric acid resistance>
In the present embodiment, lactic acid bacteria PIC-NBN22 strain and known Lactobacillus plantarum (comparative accession numbers NBRC3074, NBRC3075, NBRC12006, NBRC14712, NBRC14713, NBRC15891, NBRC101978, NBRC101978) are tested by the method and conditions described below. The gastric acid resistance of each was evaluated.

  Lactobacilli MRS Broth (manufactured by BD) is used as a basal medium, concentrated pepsin (manufactured by Mikuni Chemical Industry Co., Ltd.) is added to a final concentration of 0.3% (w / v), and each is combined with 2 ml of the test lactic acid bacteria culture solution. Three types of culture media were used which were adjusted to a total acid concentration of 10 ml using hydrochloric acid at pH 3.5, pH 3.0 and pH 2.5. This was incubated in a temperature environment of 37 ° C. for 3 hours, and the number of lactic acid bacteria after culturing relative to the number of lactic acid bacteria before the start of culture was calculated as the survival rate (%).

Table 1 and FIG. As shown in (C). FIG. 1 (A) shows the survival rate against acid environment at pH 3.5, FIG. 1 (B) shows the survival rate against acid environment at pH 3.0, and FIG. 1 (C) shows the survival rate against acid environment at pH 2.5. Each rate is shown.

As is apparent from the test results, the lactic acid bacterium PIC-NBN22 strain has a survival rate of acid environment to gastric acid at pH 3.5 of 260.00, viability of acid environment to gastric acid at pH 3.0 of 101.25, pH 3. All lactic acid bacteria reached the intestine without producing lactic acid bacteria that would die even at 0, and an increase in the number of bacteria was observed in the arrival process. Thus, the lactic acid bacteria PIC-NBN22 strain showed a markedly superior score compared with the comparative examples (Accession Nos. NBRC3074, NBRC3075, NBRC12006, NBRC14712, NBRC14713, NBRC15891, NBRC101977, NBRC101978), and the intestinal flora was good It can be expected that the beneficial physiological function (intestinal regulating action) for maintaining a stable state is remarkably exhibited.
<ATPase activity>

  In recent years, pickles and kimchi occupy most of the pickles market, and these pickles are manufactured with relatively low salt, so that harmful bacteria such as Escherichia coli are likely to propagate. In order to suppress the growth of harmful bacteria in a low salt environment, it is effective to control the inside of the factory to a low temperature environment of 20 ° C. or lower, and to adjust the pH of the seasoning liquid to 4.0 to 5.5. Yes.

  However, a low temperature environment of 20 ° C. or less and an environment of pH 4.0 to 5.5 are severe for growth even for lactic acid bacteria, which are useful bacteria, and often die even when added as a starter.

  On the other hand, lactic acid bacteria PIC-NBN22 strain is a lactic acid bacterium for pickles that shows excellent fermentability without dying even at 20 ° C. or less and at a pH of 4.0 to 5.5, and is a low temperature region that is often used in pickle production environments. Excellent fermentability at 20 ° C or lower.

The lactic acid bacteria PIC-NBN22 strain exhibits excellent fermentability even in an environment of pH 4.0 to 5.5. This is because of the characteristics of the ATPase activity of lactic acid bacteria PIC-NBN22 strain. ATPase is an enzyme that acts as a catalyst for ATP synthesis and decomposition reactions. At this time, proton H ⁺ is transported into and out of the membrane at the same time. When ATP is synthesized, ADP + Pi + 3H ⁺ out → ATP + 3H ⁺ in, and protons are taken into the membrane. This reaction is reversible, and when ATP is decomposed, ATP + 3H ⁺ in → ADP + Pi + 3H ⁺ out and protons are discharged out of the membrane.

  Bacteria that cannot tolerate low pH environments have low ATPase activity and allow protons to enter their membranes, lowering the pH in the body and making them unable to grow. Bacteria resistant to low pH environments have high ATPase activity and can prevent protons from entering the membrane. For this reason, bacteria resistant to low pH environments are characterized by high ATPase activity.

<Evaluation of ATPase activity>
According to the protocol described below, lactic acid bacteria PIC-NBN22 strain and the known Lactobacillus plantarum (Comparative Accession Nos. Activity was evaluated.

(1) Preparation of ATPase (a) Each Lactobacillus plantarum is cultured in an MRS liquid medium, and the cells are collected by centrifugation.
(B) 0.1 g of 25 mM Tris-HCl · 5 mM MgCl ₂ buffer (pH 8.0), 0.25 g of alumina and 4.5 μl of protease inhibitor are added to 0.1 g of microbial cells and ground in a mortar.
(C) Add 0.9 ml of 25 mM Tris-HCl · 5 mM MgCl ₂ buffer (pH 8.0), suspend the cells in the mortar well, and centrifuge at 1000 g × 10 minutes at 4 ° C.
(D) The supernatant is again centrifuged at 3000 g × 10 minutes at 4 ° C. The supernatant is crude ATPase.

(2) Measurement of ATPase activity 1000 μl of 50 mM Tris-HCl buffer (pH 7) containing 5 mM ATPNa / 5 mM MgCl ₂ and 200 μl of crude ATPase were reacted at 37 ° C. for 10 minutes, and then iced Chilled 0.1N · HCl: 600 μl was centrifuged at 10,000 g × 10 minutes, the supernatant: 1.6 ml and the coloring solution 4.4 ml were reacted at 18 ° C. for 10 minutes, and absorbance at 660 nm was measured. The coloring solution was an aqueous solution of 5N sulfuric acid: 10 ml, 2.5% ammonium molybdate: 10 ml, 3% hydrogen sulfate Na-1% paramethylaminophenol sulfuric acid: 10 ml, water: 40 ml.

  Since the crude ATPase and the substrate also contain free phosphoric acid, a blank test in which 0.1 N · HCl was added first was provided, and the amount of free phosphoric acid was determined from the subtracted value. The calibration curve was performed using potassium dihydrogen phosphate, and the Lowry method was used for protein quantification for calculating the ATPase specific activity. The measurement results are shown in Table 2 and FIG.

  Lactic acid bacteria PIC-NBN22 strain showed very high ATPase activity, and the ATPase activity under culture at pH 4.0 was as high as 5.7 nmol / mg / min or more.

<Evaluation of fermented seasoning liquid for pickles>
Lactic acid bacteria PIC-NBN22 strain can ferment what processed vegetables into a paste, and can give a complicated fragrance ingredient. Moreover, the pickle with a fermentation-like aroma can be manufactured by adding this to all the pickles (a shallow pickle, an old pickle, a bran pickle, kimchi).

(1) To a starter preparation (2% (w / v) glucose + 1% (w / v) yeast extract aqueous solution (sterilized)), a fish obtained from a glycerol stock of lactic acid bacteria PIC-NBN22 strain was added, A starter was prepared by culturing for 20 hours in a temperature environment of 30 ° C.

(2) Manufacture of fermented seasoning liquid for pickles Mixed with Chinese cabbage paste 10.0 g, upper white sugar 4.0 g, yeast extract 0.2 g, glucose 2.0 g, starter 2.0 g, water 181.8 ml, 30 ° C. In a temperature environment of 70 hours. Moreover, what was preserve | saved in the temperature environment of 0 degreeC and was not fermented was prepared for the comparative example.

(3) Analysis of aroma components by gas chromatography Gas chromatography (GC) was performed under the following conditions.
GC: HP6890 Series GC System manufactured by Hewlett-Packard Company
GC sampler: HP7694 Headspace Sampler manufactured by Hewlett-Packard Company
Integrator: HP6890 Series Integrator manufactured by Hewlett-Packard Company
Column: DB-WAX 125-7032 30 m × 0.53 mm 1.00 μm manufactured by Agilent
Column temperature: 40 ° C to 230 ° C
Temperature rising condition: held at 40 ° C. for 5 minutes, then heated at 10 ° C./min, and then kept at 230 ° C. for 5 minutes Carrier gas: He
Carrier gas flow rate: 50 cm / sec
Detector: Hydrogen flame ionization detector (FID)

  FIG. 3 (A) shows the GC analysis results of the fermented seasoning liquid for pickles fermented using the lactic acid bacteria PIC-NBN22 strain, and FIG. 3 (B) shows the GC analysis results of those not fermented. From this GC analysis result, in the fermented seasoning liquid for pickles fermented using lactic acid bacteria PIC-NBN22, peaks of various aroma components appeared compared to those not fermented, and the flavor of Chinese cabbage paste It turns out that it contributes to complexity.

  Thus, the lactic acid bacteria PIC-NBN22 strain can be provided as a fermented seasoning liquid for pickles having a beneficial physiological function of keeping the intestinal flora in a good state in a form that is delicious and easy to be ingested by humans. Also, when lactic acid bacteria PIC-NBN22 strain is added as a starter to vegetables such as Chinese cabbage that are raw materials for pickles, and vegetables are fermented by lactic acid bacteria, pickles with many flavors and complex flavors can be quickly produced. it can.

<Evaluation of fermentability for vegetables>
According to the protocol described below, the PIC-NBN22 strain and the known Lactobacillus plantarum (Comparative example accession numbers NBRC3074, NBRC3075, NBRC12006, NBRC14711, NBRC14712, NBRC14713, NBRC101973, NBRC101977, NBRC101977, NBRC101977, Fermentability was evaluated.

(1) Preparation of starter 1 g of glucose, 1 g of yeast extract and 48 ml of water are stirred, inoculated with colonies of each fungus after autoclaving, cultured at 30 ° C. for 20 hours, and then refrigerator at 5 ° C. until added to the pre-fermentation solution. Saved in.

(2) Preparation of pre-fermentation solution 5 g of Chinese cabbage paste, 0.5 g of glucose, 0.5 g of yeast extract, 0.1 g of soybean peptide, and 42.4 ml of water were stirred, stored in a refrigerator at 5 ° C. after autoclaving. The Chinese cabbage paste is prepared by pasting the Chinese cabbage without the core into a paste using a heat-resistant bag, heating at 95 ° C. for 30 minutes, cooling with running water for 20 minutes, and refrigerated.

(3) Culture of pre-fermentation solution 1.5 ml of each starter was added to the pre-fermentation solution (after autoclaving) and cultured at 30 ° C. The headspace was analyzed by GC about the sample of culture | cultivation 24 hours, and the composition of the aromatic component was investigated.

(4) Analysis of aroma components by gas chromatography Gas chromatography (GC) was performed under the following conditions.
GC: HP6890 Series GC System manufactured by Hewlett-Packard Company
GC sampler: HP7694 Headspace Sampler manufactured by Hewlett-Packard Company
Integrator: HP6890 Series Integrator manufactured by Hewlett-Packard Company
Column: DB-WAX 125-7032 30 m × 0.53 mm 1.00 μm manufactured by Agilent
Column temperature: 40 ° C to 230 ° C
Temperature rising condition: held at 40 ° C. for 5 minutes, then heated at 10 ° C./min, and then kept at 230 ° C. for 5 minutes Carrier gas: He
Carrier gas flow rate: 50 cm / sec
Detector: Hydrogen flame ionization detector (FID)

  Table 3 shows fermentation results of fermented edible fermented samples using lactic acid bacteria PIC-NBN22 strain, accession number, NBRC3074, NBRC3075, NBRC12006, NBRC14711, NBRC14712, NBRC14713, NBRC101973, NBRC101974, NBRC101977, NBRC101978, NBRC15891 The area ratio is shown.

  Thus, when the aroma component was analyzed, in pickles (Chinese cabbage) fermented using lactic acid bacteria PIC-NBN22 strain, the number of peaks indicating aroma was 21, which was the largest, that is, the aroma type was the most, Lactobacillus It was found that the plantarum contributes to the addition of a more complex flavor when fermenting Chinese cabbage.

<Evaluation of fermentation suitability in pickle production environment>
According to the protocol described below, PIC-NBN22 strain and known Lactobacillus plantarum (Comparative Examples Accession Numbers NBRC3074, NBRC3075, NBRC12006, NBRC14711, NBRC14712, NBRC14713, NBRC101973, NBRC101977, NBRC101977, NBRC101977, The fermentation suitability below was evaluated.

(1) Preparation of starter 1 g of glucose, 1 g of yeast extract and 48 ml of water are stirred, inoculated with colonies of each fungus after autoclaving, cultured at 30 ° C. for 20 hours, and then refrigerator at 5 ° C. until added to the pre-fermentation solution. Saved in. In addition, the turbidity of the culture solution was measured at an absorbance of 620 nm in order to calculate the added amount.

(2) Preparation of pre-fermentation solution 5 g of Chinese cabbage paste, 0.5 g of glucose, 0.5 g of yeast extract, 0.1 g of soybean peptide, and 42.4 ml of water were stirred, stored in a refrigerator at 5 ° C. after autoclaving. The Chinese cabbage paste is prepared by pasting the Chinese cabbage without the core into a paste using a heat-resistant bag, heating at 95 ° C. for 30 minutes, cooling with running water for 20 minutes, and refrigerated. Two pre-fermentation solutions were prepared per strain, one adjusted to pH 5.5 and the other adjusted to pH 4.5 with hydrochloric acid.

(3) Cultivation of pre-fermentation solution Calculate the other starter addition amounts with 1.5 ml of the starter addition amount with the lowest turbidity so that the number of added starter bacteria is constant, and add to the two types of pre-fermentation solution did. In order to match the total amount of the fermentation broth, distilled water was added to the pre-fermentation solution after the starter was added. And it sampled after culture | cultivating at 20 degreeC for 42 hours. Each fermentation broth was diluted 4 times, turbidity was measured at an absorbance of 620 nm with a spectrophotometer, and the growth rate of lactic acid bacteria was measured.

(4) Turbidity measurement The turbidity of pH 5.5 and pH 4.5 fermentation broth after 42 hours of fermentation in a temperature environment at 20 ° C. was measured, and the increase rate was calculated. The results are shown in FIG. 4 and Tables 4 (A) and (B). Table 4 (A) shows the turbidity increase rate of the fermentation broth at pH 4.5, and Table 4 (B) shows the turbidity increase rate of the fermentation broth at pH 5.5.

  As is clear from FIG. 4 and Tables 4 (A) and 4 (B), PIC-NBN22 lactic acid bacteria showed excellent fermentability in a severe environment of 20 ° C., pH 5.5 and pH 4.5. From this, it was found that PIC-NBN22 lactic acid bacteria are excellent in fermentability in a pickle production environment of low temperature and low pH and are suitable for fermentation in a pickle production environment.

<Evaluation of cell membrane fatty acid composition trans-vaccenic acid>
(1) Extraction procedure of fatty acid from lactic acid bacterial cells (a) The following Lactobacillus plantarum is cultured in a 4.0% NaCl-containing MRS liquid medium adjusted to pH 4.5, and the bacterial cells are collected by centrifugation.

  Lactic acid bacteria PIC-NBN22 strain and known Lactobacillus plantarum (comparative example accession number NBRC3074, NBRC3075, NBRC12006, NBRC14711, NBRC147712, NBRC14713, NBRC101973, NBRC10197, NBRC101978, NBRC101978, NBRC101978, NBRC101978)

(B) Add 5 ml of physiological saline, stir vigorously, and centrifuge again to recover the cells.
(C) Add 1g of aluminum oxide to 1g of cells and grind in a mortar.
(D) Add 1 ml of physiological saline and transfer to a Teflon (registered trademark) centrifuge tube.
(E) Add 5 ml methanol.
(F) Add 2.5 ml chloroform.
(G) Shake vigorously for 2 minutes and leave at room temperature for 10 minutes.
(H) Add 2.5 ml chloroform and shake vigorously for 30 seconds.
(I) Add 2.5 ml physiological saline and shake vigorously for 30 seconds.
(J) Centrifuge at 3000 rpm for 5 minutes.
(K) Collect the lower chloroform layer with a Pasteur pipette and transfer to a centrifuge tube made of Teflon (registered trademark).
(L) Centrifuge at 3000 rpm for 5 minutes.
(M) Carefully collect the lower chloroform layer with a Pasteur pipette and place it in an eggplant flask.
(N) Dry under reduced pressure in a hot water bath of 35 ° C or lower.
(O) Dissolve the dried product in 1 ml of hexane, transfer to a vial, add a small amount of magnesium sulfate and dehydrate overnight.
(P) After completion of dehydration, the hexane solution was used as the fatty acid extract.

(2) Methylation of fatty acid (a) To 0.5 ml of hexane solution, 1.0 ml of 0.5 mg / ml heptadecanoic acid / hexane solution and 0.5 ml of hexane are added and mixed.
(B) Add 0.2 ml of 2M potassium hydroxide / methanol solution and shake vigorously for 2 minutes.
(C) Collect 0.5 ml of the upper hexane layer, and inject a fixed volume of 5 ml with hexane into 1 μl gas chromatography.

(3) Analysis conditions by gas chromatography Gas chromatograph: GL Sciences GC-4000 Plus / FID
Column: TC-2560 0.25 mm I.D. D. × 100m df = 0.20μm
Column temperature: Incubate at 100 ° C. for 5 minutes and then increase to 230 ° C. at 10 ° C./min. Carrier gas: He
Split: 50ml / min

The results are shown in FIG.

  When exposed to harsh environments, lactic acid bacteria change the cell membrane fatty acid composition and create a membrane that matches the environment. Especially for low pH environments, transbacsenic acid is consumed to increase the acid resistance of the cell membrane. Lactic acid bacteria PIC-NBN22 strain has a lower proportion of transbaccenoic acid compared to other Lactobacillus plantarum when cultured in a low pH environment, and constructed the fatty acid composition of the cell membrane to obtain higher acid resistance I found out that

  Thus, since the lactic acid bacteria PIC-NBN22 strain has the acclimatization mechanism to the low pH environment, it can easily grow even in the pickle production process environment in which the seasoning at low pH is unavoidable.

  Lactic acid bacteria PIC-NBN22 strain has an immunostimulatory function by ingestion of intestinal IgA secretion, bile acid adsorption ability and cholesterol adsorption ability (blood pressure lowering function), and a function to suppress the growth of Helicobacter pylori by feeding. Use as a lactic acid bacterium. The lactic acid bacteria PIC-NBN22 strain is useful as a food additive having these health promotion functions. In addition, foods to which lactic acid bacteria PIC-NBN22 strain is added are useful as functional foods for promoting health. The above-described health promotion function was evaluated as described below.

<Evaluation of intestinal IgA secretion induction>
(1) Administration of PIC-NBN Lactic Acid Bacteria One group of 6-week-old BALB / c · Cr · Slc male mice grouped into 2 groups of 3 mice was given powdered PIC-NBN22 lactic acid bacteria with water for injection. The suspension used was administered, and the other group received a placebo (vehicle) as a control group. The administration period was 1 week in both groups, and oral gavage administration was performed, and the amount of administration liquid was 10 ml / kg.

The PIC-NBN22 lactic acid bacteria powder is a powder containing 4 × 10 ¹⁰ cells / g PIC-NBN22 lactic acid bacteria after culturing PIC-NBN22 lactic acid bacteria and recovering the cells, followed by drying with a vacuum freeze dryer.

(2) Measurement of IgA Feces were collected before administration and on the last day of administration, and the IgA content was measured as follows.

  The weight of feces was measured, 1 ml of the extract was added to the feces, and the mixture incubated at 4 ° C. for 1 hour was vigorously mixed for 30 minutes. It was centrifuged at 3000 g for 30 minutes at 4 ° C., and the supernatant was collected. The collected supernatant was diluted 2-fold, and the IgA concentration was measured using ELISA / KIT. Then, the IgA content in the feces was calculated from the measured IgA concentration and the weight of the feces used.

  The composition of the extract used was Phosphate Buffered Saline (Sigma) 49 ml, Protease Inhibitor Cocktail (Sigma) 500 μl, PMSF (Sigma) 500 μl, EDTA (Sigma) final concentration 50 mmol / L.

  The calculation result (change amount) of the IgA content is shown in FIG. Thus, when PIC-NBN22 lactic acid bacteria were given to mice for one week, the amount of IgA increase was significantly greater in the PIC-NBN22 lactic acid bacteria ingestion group than in the control ingestion group. From this, it was found that PIC-NBN22 lactic acid bacteria have an immunostimulatory effect.

<Evaluation of bile acid adsorption ability and cholesterol adsorption ability>
(1) Preparation of test substance PIC-NBN22 lactic acid bacteria were inoculated into an MRS liquid medium and cultured at 32 ° C. for 18 hours to prepare a preculture solution. A new MRS liquid medium was inoculated with 1% of the preculture and cultured at 32 ° C. for 18 hours. The culture solution was centrifuged to collect the cells, washed twice with sterilized water, and then freeze-dried.

(2) Measurement of bile acid adsorption capacity 12 mg of freeze-dried cells were suspended in 0.8 ml of a 1.25 mmol / L sodium taurocholate solution (10 mol / L phosphate buffer (pH 6.8)) and 2. Incubated for 5 hours. After centrifugation at 10,000 g and 4 ° C. for 5 minutes, the bile acid concentration in the supernatant was measured using Total Bile Acid Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.), and the ratio of bile acid adsorbed on the cells was determined. The bile acid adsorption rate (%) was 47.6 ± 0.9 (n = 3).

(3) Measurement of cholesterol adsorption ability 12 mg of freeze-dried cells were suspended in 0.4 ml of a cholesterol solution (100 μg / ml 60% ethanol) and incubated at 37 ° C. for 1 hour. Centrifugation was performed at 10,000 g and 4 ° C. for 5 minutes. To 0.1 ml of the supernatant, 0.3 ml of 33% KOH and 3 ml of 99.5% ethanol were added and incubated at 60 ° C. for 15 minutes. After cooling, 5 ml of hexane and 3 ml of pure water were added and stirred for 1 minute.

  After stirring, 3 ml of the hexane layer was collected and evaporated to dryness under a nitrogen stream, and then 2 ml of 0.05% phthalaldehyde-glacial acetic acid solution was added to dissolve the dried product. After standing at room temperature for 10 minutes, 1 ml of concentrated sulfuric acid was added, the absorbance (550 nm) was measured, the amount of cholesterol was determined, and the proportion of cholesterol adsorbed on the cells was calculated. The cholesterol adsorption rate (%) was 21.4 ± 1.4 (n = 3).

  Thus, it was found that PIC-NBN22 lactic acid bacteria adsorb cholesterol and bile acids. From this, it became clear that it has the effect of suppressing excessive cholesterol absorption into the body.

<Evaluation of growth inhibition ability of Helicobacter pylori>
(1) Culture of bacterial strain PIC-NBN22 lactic acid bacteria were anaerobically cultured at 30 ° C. using MRS medium. Helicobacter pylori was cultured in a microaerobic environment using BHI medium containing 10% inactivated fetal bovine serum.

(2) Preparation of culture solution A solution obtained by diluting the PIC-NBN22 lactic acid bacteria preculture solution 10-fold and culturing for 1 day under the above conditions was used as a lactic acid bacteria culture solution. Based on the absorbance at 600 nm, this solution was adjusted to 4 × 10 ⁸ cfu / ml and subjected to a co-culture test. A solution obtained by diluting the H. pylori pre-culture solution 250 times and culturing it for 1 day under the above conditions was used as the H. pylori culture solution. Based on the absorbance at 600 nm, this solution was adjusted to 7 × 10 ⁶ cfu / ml and subjected to a co-culture test.

(3) Co-culture test The above-mentioned lactic acid bacteria culture solution and H. pylori culture solution were added to 5 ml of FCS-containing BHI medium, and cultured at 37 ° C. for 22 hours under microaerobic conditions. The control group was prepared without the addition of lactic acid bacteria culture and cultured in the same manner. The number of Helicobacter pylori before and after the culture was calculated using a Helicobacter agar medium. The results are shown in Table 6.

  The left side of FIG. 7 is a photograph of the results of the co-culture test of the group to which PIC-NBN22 lactic acid bacteria were added, and the right side was photographed of the results of the co-culture test of the control group to which PIC-NBN22 lactic acid bacteria were not added. Is. In the control group, H. pylori grew to about 300 times, whereas in the group to which PIC-NBN22 lactic acid bacteria were added, there was almost no growth of H. pylori.

  As a summary, the characteristics of lactic acid bacteria PIC-NBN22 strain and other Lactobacillus plantarum are listed in Table 7. The evaluation criteria in this table are as follows.

ATPase activity: ≧ 5.0 nmol / mg / min
Less than 5.0 nmol / mg / min is x
Transbaxenoic acid ratio: 0.3% or less ○
Higher than 0.3%
Fermentation suitability of pickled vegetables in the environment of production: turbidity increase rate after culturing for 42 hours in an environment of 20 ° C., pH 5.5 and pH 4.5 is not less than 300%
The rate of increase in turbidity after culturing for 42 hours at 20 ° C., pH 5.5 and pH 4.5 is less than 300%.
Abundant variety of aroma components during vegetable fermentation: More than 20 types
Less than 20 types
Gastric acid tolerance: If the survival rate to acid environment at pH 3.5 is 260% or more and the survival rate to acid environment at pH 3.0 is 100% or more,
The survival rate against acid environment at pH 3.5 is less than 260% or the survival rate against acid environment at pH 3.0 is less than 100%.

  Thus, the lactic acid bacteria PIC-NBN22 strain is excellent in all of ATPase activity, the ratio of transbaccenoic acid, low temperature fermentability, abundant variety of aroma components at the time of vegetable fermentation, and gastric acid resistance.

  In addition to the fermented seasoning liquid for pickles, the lactic acid bacteria PIC-NBN22 strain is also used in breads, pasta, edible flour, rice, sushi, prepared dishes, sprinkles, soup, hot pot sauce, seasoning, spices It can be provided in the form of additives for foods such as tea, confectionery and beverages, and additives for lactic acid bacteria fermented foods such as yogurt.

  Lactic acid bacteria PIC-NBN22 strain is a powder, granule, tablet, liquid agent as a drug having an intestinal regulating action, an immunostimulatory action, a blood pressure lowering action, a bile acid adsorption ability, a cholesterol adsorption ability, and a Helicobacter pylori growth inhibitory action Can be provided in various forms such as an oral administration agent.

Claims (4)

  Lactic acid bacteria PIC-NBN22 strain (accession number NITE P-1496) belonging to Lactobacillus plantarum.   A food additive comprising the lactic acid bacterium according to claim 1.   A food comprising the lactic acid bacterium according to claim 1.   A drug comprising the lactic acid bacterium according to claim 1.