JP7121867B1 - Lactic acid bacteria - Google Patents
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 162
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 81
- 239000004310 lactic acid Substances 0.000 title claims abstract description 81
- 241000894006 Bacteria Species 0.000 title claims abstract description 77
- 241000604136 Pediococcus sp. Species 0.000 claims abstract description 9
- 235000013305 food Nutrition 0.000 claims abstract description 8
- 239000000843 powder Substances 0.000 claims abstract description 6
- 230000000694 effects Effects 0.000 abstract description 19
- 238000011156 evaluation Methods 0.000 abstract description 14
- 239000001257 hydrogen Substances 0.000 abstract description 5
- 229910052739 hydrogen Inorganic materials 0.000 abstract description 5
- 244000005700 microbiome Species 0.000 abstract description 4
- LFYJSSARVMHQJB-QIXNEVBVSA-N bakuchiol Chemical compound CC(C)=CCC[C@@](C)(C=C)\C=C\C1=CC=C(O)C=C1 LFYJSSARVMHQJB-QIXNEVBVSA-N 0.000 abstract description 2
- 125000004435 hydrogen atom Chemical class [H]* 0.000 abstract description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 34
- 238000000354 decomposition reaction Methods 0.000 description 12
- 210000004211 gastric acid Anatomy 0.000 description 12
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 description 11
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Abstract
【課題】過酸化水素の分解活性に優れた乳酸菌を提供する。【解決手段】本乳酸菌は、２０２１年１１月２６日付で、独立行政法人 製品評価技術基盤機構 特許微生物寄託センター（ＮＰＭＤ）（郵便番号２９２－０８１８ 日本国千葉県木更津市かずさ鎌足２－５－８ １２２号室）に受託されている。本乳酸菌は、受託番号がＮＩＴＥ Ｐ－０３５６３であるペディオコッカス ｓｐ．（Pediococcus sp.）の新規の乳酸菌である。本乳酸菌は、粉末であってもよい。本乳酸菌は飲食品であってもよい。【選択図】なし[Problem] To provide a lactic acid bacterium having excellent hydrogen peroxide-decomposing activity. [Solution] This lactic acid bacterium is dated November 26, 2021, National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (NPMD) (zip code 292-0818, 2-5 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan). 8 122 room). This lactic acid bacterium is Pediococcus sp., whose accession number is NITE P-03563. (Pediococcus sp.) novel lactic acid bacteria. The present lactic acid bacteria may be powder. The present lactic acid bacteria may be food and drink. [Selection figure] None
Description
本発明は、乳酸菌に関する。 The present invention relates to lactic acid bacteria.
生体内で発生する過酸化水素（Ｈ２Ｏ２）は、活性酸素の一種であり、生体内においては有害な物質であることが知られている。ヒトについても、多くの疾病と過酸化水素との関連が示唆されている。そこで、過酸化水素の分解活性（過酸化水素消去作用）をもつとして、例えば特許文献１には、ラクトバチルス・プランタラム（Lactobacillus plantarum） ＴＹ－１５７２株（ＮＩＴＥ Ｐ－９０）由来の過酸化物分解酵素が記載されている。この過酸化物分解酵素は、ＮＡＤ（Ｐ）Ｈの存在下、過酸化物に高い反応性を示し、過酸化水素にも反応する基質特異性と、ＳＤＳポリアクリルアミドゲル電気泳動による測定において４３ｋＤａの分子量とを有する。 Hydrogen peroxide (H 2 O 2 ) generated in vivo is a kind of active oxygen and is known to be a harmful substance in vivo. In humans, many diseases have also been suggested to be associated with hydrogen peroxide. Therefore, for example, Patent Document 1 describes a peroxide derived from Lactobacillus plantarum strain TY-1572 (NITE P-90) as having hydrogen peroxide decomposition activity (hydrogen peroxide scavenging action) Degradative enzymes are described. This peroxide-degrading enzyme exhibits high reactivity to peroxides in the presence of NAD(P)H, has substrate specificity that also reacts to hydrogen peroxide, and has a molecular weight of 43 kDa as measured by SDS polyacrylamide gel electrophoresis. and molecular weight.
乳酸菌については、ヒトの体内において種々の優れた働きをすることから、乳酸菌（乳酸菌の培養物、当該培養物から集菌された菌体を含む）を摂取することの意義は大きい。例えば、特許文献２には、ラクトバチルス・プランタラム（Lactobacillus plantarum）に属する乳酸菌またはペディオコッカス属（Pediococcus）に属する乳酸菌を培養して得られる培養物、この培養物から集菌された菌体を有効成分とする抗酸化剤が記載されている。 Since lactic acid bacteria perform various excellent functions in the human body, it is of great significance to ingest lactic acid bacteria (including cultures of lactic acid bacteria and cells collected from such cultures). For example, Patent Document 2 discloses a culture obtained by culturing lactic acid bacteria belonging to Lactobacillus plantarum or lactic acid bacteria belonging to the genus Pediococcus, and bacterial cells collected from this culture. are described as active ingredients.
乳酸菌は、周知の通り、ヒトをはじめとする哺乳類の生体内にも多種存在するが、摂取した場合に発現する働き（機能）及びその強弱は個体毎に異なる。すなわち、ある個体において良好な働きを十分に示す乳酸菌であっても、他の個体において同様な働きを示すとは限らない。したがって、過酸化水素の分解活性をもつ乳酸菌が新たに見いだされることは、摂取の選択肢が増えることになり望ましい。 As is well known, there are many types of lactic acid bacteria in mammals including humans, but the actions (functions) that are expressed when they are ingested and their strengths and weaknesses differ from individual to individual. In other words, even if a lactic acid bacterium shows sufficient action in a certain individual, it does not necessarily show the same action in another individual. Therefore, the discovery of new lactic acid bacteria with hydrogen peroxide-decomposing activity is desirable because it will increase options for ingestion.
そこで、本発明は、過酸化水素の分解活性に優れた乳酸菌を提供することを目的とする。 Accordingly, an object of the present invention is to provide a lactic acid bacterium having excellent hydrogen peroxide-decomposing activity.
本発明は、受託番号がＮＩＴＥ Ｐ－０３５６３であるペディオコッカス ｓｐ．（Pediococcus sp.）の乳酸菌である。 The present invention relates to Pediococcus sp. (Pediococcus sp.) lactic acid bacteria.
上記乳酸菌は、粉末であってもよい。乳酸菌は飲食品であってもよい。 Powder may be sufficient as the said lactic acid bacteria. Lactic acid bacteria may be food and drink.
本発明の乳酸菌は、過酸化水素の分解活性に優れる。 The lactic acid bacterium of the present invention has excellent hydrogen peroxide-decomposing activity.
本実施形態において、乳酸菌（以下、本乳酸菌と称する）は、ペディオコッカス ｓｐ．（Pediococcus sp.）の新規の乳酸菌株であり、２０２１年１１月２６日付で、独立行政法人 製品評価技術基盤機構 特許微生物寄託センター（ＮＰＭＤ）（郵便番号２９２－０８１８ 日本国千葉県木更津市かずさ鎌足２－５－８ １２２号室）に、受託番号ＮＩＴＥ Ｐ－０３５６３にて寄託されている。 In the present embodiment, the lactic acid bacterium (hereinafter referred to as the present lactic acid bacterium) is Pediococcus sp. (Pediococcus sp.), and on November 26, 2021, the National Institute of Technology and Evaluation Patent Microorganism Depositary Center (NPMD) (zip code 292-0818, Kisarazu City, Chiba Prefecture, Japan) 2-5-8 Room 122) under accession number NITE P-03563.
本乳酸菌は、糠から単離されたものである。 This lactic acid bacterium is isolated from rice bran.
本乳酸菌の菌学的性状は以下の通りである。
１．形態的性状
下記はいずれもＭＲＳ寒天培地での３０℃、４８時間、好気培養による。
（１）形態 ：球菌（大きさφは１．０μｍ以上１．２μｍ以下の範囲内）
（２）グラム染色性 ：＋（陽性）
（３）胞子形成（芽胞形成）：－（陰性）
（４）運動性 ：－（陰性）
（５）コロニー形態：白色、円形、光沢有り
The bacteriological properties of this lactic acid bacterium are as follows.
1. Morphological Properties All of the following are aerobic cultures on MRS agar medium at 30° C. for 48 hours.
(1) Morphology: coccus (size φ is in the range of 1.0 μm or more and 1.2 μm or less)
(2) Gram staining: + (positive)
(3) sporulation (spore formation): - (negative)
(4) Motility: - (negative)
(5) Colony morphology: white, circular, glossy
２．生理的性質
（１）生育能
１５℃での生育能：＋（陽性）
３７℃での生育能：＋（陽性）
４５℃での生育能：＋（陽性）
（２）カタラーゼ反応 ：＋（陽性）
（３）各種糖質の発酵性（糖資化性）：表１に示す。測定は、ＡＰＩ５０ＣＨ（ビオメリュー・ジャパン株式会社製）を用いて実施した。１５種の糖質について発酵性が認められている。表１において、「＋」は陽性、「－」は陰性を示す。
2. Physiological properties (1) Growth ability Growth ability at 15°C: + (positive)
Growth ability at 37°C: + (positive)
Growth ability at 45°C: + (positive)
(2) Catalase reaction: + (positive)
(3) Fermentability of various sugars (sugar utilization): shown in Table 1. The measurement was performed using API50CH (manufactured by bioMérieux Japan Co., Ltd.). Fermentability is recognized for 15 kinds of carbohydrates. In Table 1, "+" indicates positive and "-" indicates negative.
（４）各種培地での増殖性
本乳酸菌を前培養と本培養とにより以下のように培養し、本培養開始前における培養液のｐＨ（水素イオン濃度指数）及び培養液のＯＤ（光学密度、波長は６６０ｎｍ、以下ＯＤ６６０と記載する）と、本培養終了後における培養液のｐＨ及び培養液のＯＤ６６０とを測定した。本乳酸菌を含む凍結グリセロールストックをＭＲＳ寒天培地に接種し、３７℃、２４時間培養した。前培養は、このＭＲＳ寒天培地上に出現したコロニーの１白金耳分を、ねじ口試験管に収容された１０ｍｌのＭＲＳ液体培地に接種し、３７℃、２４時間静置することにより行った。本培養は、前培養で得られた前培養液を、ねじ口試験管に収容された１０ｍｌの培地に対して１％（ｖ/ｖ）接種し、３７℃、２４時間静置することにより行った。本培養で用いた上記１０ｍｌの培地は、培地Ａ～培地Ｆの６種である。培地ＡはＭＲＳ（乳酸菌用）、培地ＢはＧＡＭ（嫌気性菌用）、培地ＣはＹＭ（真菌用）、培地ＤはＭ１７（乳酸菌用）、培地ＥはＳｋｉｍ ｍｉｌｋ（乳酸菌用、動物性）、培地Ｆは肉汁培地（乳酸菌等用）であり、これらの各成分配合及び菌にとっての用途は表２に示す。なお、表２は、東京農業学菌株カタログ第三版２０２０による。ｐＨ及びＯＤ６６０の結果は表３に示す。表３において、「０ｈ」は本培養開始時、「２４ｈ」は本培養終了後であることを意味し、ｐＨの「低下度」とＯＤの「増加度」とは、本培養終了後の値から開始前の値を減算して求めた値である。
(4) Proliferation in various media The present lactic acid bacterium was cultured by pre-culture and main culture as follows, and the pH (hydrogen ion concentration index) of the culture solution and the OD (optical density, The wavelength was 660 nm, hereinafter referred to as OD 660 ), and the pH and OD 660 of the culture solution after completion of the main culture were measured. A frozen glycerol stock containing this lactic acid bacterium was inoculated onto an MRS agar medium and cultured at 37° C. for 24 hours. Preculture was carried out by inoculating 10 ml of MRS liquid medium contained in a screw cap test tube with one platinum loop of colonies that appeared on the MRS agar medium and allowing it to stand at 37° C. for 24 hours. The main culture was carried out by inoculating 1% (v/v) of the preculture solution obtained in the preculture to 10 ml of medium contained in a screw-capped test tube and allowing it to stand at 37°C for 24 hours. rice field. The above 10 ml of medium used in the main culture are 6 types of medium A to medium F. Medium A is MRS (for lactic acid bacteria), medium B is GAM (for anaerobes), medium C is YM (for fungi), medium D is M17 (for lactic acid bacteria), medium E is Skim milk (for lactic acid bacteria, animal) , Medium F is a gravy medium (for lactic acid bacteria, etc.). In addition, Table 2 is according to Tokyo Nogaku strain catalog 3rd edition 2020. The pH and OD 660 results are shown in Table 3. In Table 3, "0h" means at the start of the main culture, and "24h" means after the end of the main culture. is the value obtained by subtracting the value before the start from
表３の結果から、本乳酸菌は、培地Ｅにおいては増殖は認められず、その他の培地Ａ～Ｄ、Ｆにおいて増殖が認められた。このように、本乳酸菌は、入手しやすい多くの培地で培養することができる。また、培地Ａにおいて最も増殖し、その増殖性が非常に高いことから、培養が容易であることがわかる。 From the results in Table 3, the present lactic acid bacterium did not grow in medium E, but grew in other mediums A to D and F. Thus, the present lactic acid bacterium can be cultured in many readily available media. In addition, the growth rate is highest in medium A, and the growth rate is very high, indicating that culturing is easy.
（５）過酸化水素の分解活性
過酸化水素（Ｈ２Ｏ２）は、水素原子にヒドロペルオキシド基を有する過酸化物であり、本乳酸菌の過酸化水素の分解活性を評価した。本乳酸菌は、まず、ＭＲＳ液体培地で３０℃、２４時間静置培養した。この培養物から培養菌体を回収して、培養菌体を含有する菌体懸濁液を被検液とした。回収は、１００００ｒｐｍ、５分の遠心分離し、リン酸緩衝液（ｐＨ７．４）にて洗浄した後に、さらに、１００００ｒｐｍ、５分の遠心分離を行うことにより行った。被検液はＯＤ６６０＝１．６となるようにリン酸緩衝液（ｐＨ７．４）を加えて濃度調整し、その培養菌体を遠心分離により取得した。この菌体に終濃度３ｍＭとなるように過酸化水素を添加した。分解反応条件は、３７℃、１時間とし、反応終了後の反応液を遠心分離し、上清をリン酸緩衝液（ｐＨ７．４）を加えて３０倍希釈し、過酸化水素の残存量を、呈色反応（ＦＯＸ ａｓｓａｙ）にて測定した。この測定結果から過酸化水素の減少濃度（単位；μＭ（マイクロモル））を求めた。
(5) Hydrogen peroxide decomposition activity Hydrogen peroxide (H 2 O 2 ) is a peroxide having a hydroperoxide group on a hydrogen atom, and the hydrogen peroxide decomposition activity of the present lactic acid bacteria was evaluated. This lactic acid bacterium was first statically cultured in an MRS liquid medium at 30° C. for 24 hours. Cultured cells were recovered from this culture, and a cell suspension containing the cultured cells was used as a test solution. Recovery was performed by centrifuging at 10,000 rpm for 5 minutes, washing with a phosphate buffer (pH 7.4), and then centrifuging at 10,000 rpm for 5 minutes. Phosphate buffer (pH 7.4) was added to the test solution to adjust the concentration so that OD 660 =1.6, and the cultured cells were obtained by centrifugation. Hydrogen peroxide was added to the cells to a final concentration of 3 mM. The decomposition reaction conditions were 37° C. for 1 hour, the reaction solution after completion of the reaction was centrifuged, the supernatant was diluted 30-fold with phosphate buffer (pH 7.4), and the remaining amount of hydrogen peroxide was , color reaction (FOX assay). From this measurement result, the reduced concentration of hydrogen peroxide (unit: μM (micromoles)) was determined.
過酸化水素の残存量の測定方法は、以下である。試験管に分解反応終了後の上清を２００μｌ入れ、これに２５ｍＭ硫酸を０．８ｍｌ、Ｆｏｘ ａｓｓａｙ緩衝液を１．０ｍｌ添加して懸濁した溶液を、室温にて３０分反応させ、この反応溶液について分光光度計にて５４０ｎｍにおける吸光度を測定した。Ｆｏｘ ａｓｓａｙ緩衝液は、２５ｍＭ硫酸に硫酸アンモニウム第二鉄六水和物とキシレノールオレンジとをそれぞれ終濃度２００μＭとなるように溶解し調製した。過酸化水素の標準物質の吸光度より作成した検量線にて上清中の各成分濃度を算出した。なお、後述のクメンヒドロキシペルオキシドに対する分解性の評価においては、検量線をクメンヒドロキシペルオキシドの標準物質の吸光度により作成した。 The method for measuring the residual amount of hydrogen peroxide is as follows. 200 μl of the supernatant after completion of the decomposition reaction was placed in a test tube, and 0.8 ml of 25 mM sulfuric acid and 1.0 ml of Fox assay buffer were added to suspend the solution, which was then reacted at room temperature for 30 minutes. The absorbance at 540 nm of the solution was measured with a spectrophotometer. Fox assay buffer was prepared by dissolving ferric ammonium sulfate hexahydrate and xylenol orange in 25 mM sulfuric acid to a final concentration of 200 μM. The concentration of each component in the supernatant was calculated using a calibration curve prepared from the absorbance of a hydrogen peroxide standard substance. In the evaluation of degradability to cumene hydroperoxide, which will be described later, a calibration curve was prepared from the absorbance of a standard substance of cumene hydroperoxide.
また、比較として、下記の標準菌株ａ～ｉについても同様に評価した。標準菌株ａ～ｉは以下の通りである。標準菌株ａは、本乳酸菌に近縁な標準菌株であり、標準菌株ｂ～ｉは、ヒトの腸内に通常存在すると言われる標準的な菌株である。
標準菌株ａ；Pediococcus acidilactici NRIC0115
標準菌株ｂ；Lactobacillus rhamnosus NRIC1043
標準菌株ｃ；Enterococcus faecalis NRIC1142
標準菌株ｄ；Lactobacillus casei subsp. casei NRIC1042
標準菌株ｅ；Lactobacillus acidophilus NRIC1547
標準菌株ｆ；Lactococcus lactis subsp. lactis NRIC1149
標準菌株ｇ；Lactobacillus delbrueckii subsp. bulgaricus NRIC1688
標準菌株ｈ；Lactobacillus delbrueckii subsp.delbrueckii NRIC1053
標準菌株ｉ；Lactobacillus gasseri NRIC1546
For comparison, the following standard strains a to i were also evaluated in the same manner. Standard strains a to i are as follows. The standard strain a is a standard strain closely related to the present lactic acid bacterium, and the standard strains b to i are standard strains said to normally exist in the human intestine.
Standard strain a; Pediococcus acidilactici NRIC0115
Standard strain b; Lactobacillus rhamnosus NRIC1043
Type strain c; Enterococcus faecalis NRIC1142
Standard strain d; Lactobacillus casei subsp. casei NRIC1042
Standard strain e; Lactobacillus acidophilus NRIC1547
Standard strain f: Lactococcus lactis subsp. lactis NRIC1149
Standard strain g; Lactobacillus delbrueckii subsp. bulgaricus NRIC1688
Standard strain h; Lactobacillus delbrueckii subsp.delbrueckii NRIC1053
Standard strain i; Lactobacillus gasseri NRIC1546
本乳酸菌と各標準菌株ａ～ｉとの各結果は図１Ａに示す。なお、図１Ａ及び他の各図において、本乳酸菌は「ｘ」で示す。１００μＭを残存率１００％とした場合に、本乳酸菌は、過酸化水素の減少濃度が３２．９μＭであり、ヒトの腸内に通常存在すると言われる標準菌株ｂ～ｉと比べて、過酸化水素の減少濃度が約２倍であり、本乳酸菌株に近縁な標準菌株ａと比べても大きいことがわかる。このように、本乳酸菌は過酸化水素の分解活性に優れる。 The results for this lactic acid bacterium and each of the standard strains a to i are shown in FIG. 1A. In addition, in FIG. 1A and other figures, this lactic acid bacterium is indicated by "x". When 100 μM is taken as 100% residual rate, this lactic acid bacterium has a reduced concentration of hydrogen peroxide of 32.9 μM, and compared to standard strains b to i that are said to normally exist in the human intestine, hydrogen peroxide is about twice as large as that of the standard strain a, which is closely related to the present lactic acid bacteria strain. Thus, the present lactic acid bacterium is excellent in hydrogen peroxide-decomposing activity.
本乳酸菌の、クメンヒドロキシペルオキシドに対する分解活性も評価した。クメンヒドロキシペルオキシドは、ヒトの生体内には通常存在するものではないものの、過酸化物のひとつではある。評価方法及び条件は、分解反応条件を３７℃、３時間とした以外は、過酸化水素に対する分解活性の場合と同様である。また、標準菌株ａ～ｉについても同様にクメンヒドロキシペルオキシドの分解活性を評価した。 The decomposition activity of this lactic acid bacterium for cumene hydroperoxide was also evaluated. Cumene hydroperoxide is one of the peroxides, although it does not normally exist in the human body. The evaluation method and conditions were the same as those for the decomposition activity against hydrogen peroxide, except that the decomposition reaction conditions were 37° C. and 3 hours. In addition, the standard strains a to i were similarly evaluated for their cumene hydroperoxide-degrading activity.
本乳酸菌と各標準菌株ａ～ｉとの各結果は図１Ｂに示す。図１Ｂに示すように、本乳酸菌はクメンヒドロキシペルオキシドに対しても分解活性を示し、分解活性は腸内に通常存在すると言われる標準菌株ｂ、ｃ、ｆ、ｈと比べて低いものの、標準菌株ｄ、ｅ、ｇ、ｉに比べて高く、また、近縁な標準菌株ａと比べても高い。このように、本乳酸菌は、クメンヒドロキシペルオキシドの分解にも一定の効果を示し、過酸化水素以外の過酸化物の分解活性にも優れることがわかる。 The results for this lactic acid bacterium and each of the standard strains a to i are shown in FIG. 1B. As shown in FIG. 1B, this lactic acid bacterium also exhibits decomposition activity against cumene hydroperoxide, and although the decomposition activity is lower than that of the standard strains b, c, f, and h, which are said to normally exist in the intestine, the standard strain Higher than d, e, g and i, and higher than the closely related standard strain a. Thus, it can be seen that the present lactic acid bacterium exhibits a certain effect in decomposing cumene hydroperoxide and is also excellent in decomposing activity for peroxides other than hydrogen peroxide.
（６）胃酸及び胆汁酸に対する耐性
（６－１）胃酸耐性
本乳酸菌及び標準菌株ａ～ｉについて胃酸耐性について以下の方法で評価した。すなわち、ｐＨ２．５に調整したＭＲＳ液体培地に終濃度０．０４％となるようペプシンを添加した液体培地を人工胃液培地として調製し、この人工胃液培地を用いた評価を、胃酸耐性の評価とした。本乳酸菌及び標準菌株ａ～ｉは、前述の方法と同様に培養及び遠心分離して菌体を回収し、回収菌体をリン酸緩衝液にて洗浄した後、ＯＤ６６０＝１．０に菌液を調製し、人工胃液培地に１％接種して３時間処理した。
(6) Tolerance to gastric acid and bile acid (6-1) Gastric acid resistance This lactic acid bacterium and standard strains a to i were evaluated for gastric acid resistance by the following method. That is, an artificial gastric juice medium was prepared by adding pepsin to a final concentration of 0.04% in the MRS liquid medium adjusted to pH 2.5, and the evaluation using this artificial gastric juice medium was regarded as the evaluation of gastric acid resistance. did. This lactic acid bacterium and standard strains a to i were cultured and centrifuged in the same manner as described above to collect the cells, washed the collected cells with a phosphate buffer, and then reduced the OD 660 to 1.0. A liquid was prepared, inoculated 1% in an artificial gastric juice medium, and treated for 3 hours.
評価結果は、図２Ａに示す。この結果によると、ヒトの腸内に通常存在すると言われる標準菌株ｂ～ｈは生存率が０％に近い一方で、本乳酸菌は５０％を超える生存率を示しており、非常に高いことがわかる。このことから、本乳酸菌はヒトの腸内に通常存在すると言われる多くの乳酸菌よりも優れた胃酸耐性を示すことを期待することができる。 The evaluation results are shown in FIG. 2A. According to this result, the standard strains b to h, which are said to normally exist in the human intestine, have a survival rate close to 0%, while the present lactic acid bacterium has a survival rate of over 50%, which is very high. Recognize. From this, it can be expected that the present lactic acid bacterium exhibits gastric acid resistance superior to many lactic acid bacteria that are said to normally exist in the human intestine.
（６－２）胃酸及び胆汁酸耐性
本乳酸菌及び標準菌株ａ～ｉについて胆汁酸耐性について以下の方法で評価した。ＭＲＳ液体培地に終濃度０．３％ｏｘｙｇａｌｌとなるよう添加した培地を人工胆汁酸培地とした。上記の胃酸処理を経た処理液を人工胆汁酸培地に対して１％添加して２０時間処理し、この処理後の生菌数（単位；ｃｆｕ／ｍｌ）を、胆汁酸耐性として評価した。
(6-2) Gastric acid and bile acid resistance Bile acid resistance of the present lactic acid bacteria and standard strains a to i was evaluated by the following method. A medium added to the MRS liquid medium to a final concentration of 0.3% oxygall was used as an artificial bile acid medium. 1% of the treatment solution that had undergone the gastric acid treatment was added to the artificial bile acid medium and treated for 20 hours.
評価結果は、図２Ｂに示す。この結果によると、本乳酸菌は生菌数が多く、その生菌数はいずれの標準菌株ａ～ｉと比べても高いことがわかる。したがって、本乳酸菌は胃酸及び胆汁酸に対して優れた耐性を示すことが期待できる。このことから、ヒトの腸内に通常存在すると言われる多くの乳酸菌よりも胃酸耐性及び胆汁酸耐性を示すこと、すなわち、生きたまま腸に届くことが期待される。 The evaluation results are shown in FIG. 2B. According to the results, it can be seen that the present lactic acid bacterium has a large number of viable bacteria, and the viable cell count is higher than any of the standard strains a to i. Therefore, the present lactic acid bacterium can be expected to exhibit excellent resistance to gastric acid and bile acid. From this, it is expected that it will show more gastric acid resistance and bile acid resistance than many lactic acid bacteria that are said to normally exist in the human intestine, that is, it will reach the intestine alive.
（７）乳酸産生能
胃酸及び胆汁酸処理された場合の本乳酸菌の活性を評価するために、人工胃液培地（ｐＨ２．５）処理（処理時間は３ｈ）及びその後の人工胆汁酸培地（０．３％ｏｘｙｇａｌｌ）処理（処理時間は２０ｈ）を経た後の乳酸量（乳酸産生能）を測定した。なお、本乳酸菌との比較として、標準菌株ａ～ｉについても同様に乳酸量を測定した。
(7) Lactic acid-producing ability In order to evaluate the activity of this lactic acid bacterium when treated with gastric acid and bile acid, treatment with artificial gastric juice medium (pH 2.5) (treatment time: 3 h) and subsequent artificial bile acid medium (0. 3% oxygall) treatment (treatment time was 20 hours) and then the amount of lactic acid (lactic acid producing ability) was measured. For comparison with this lactic acid bacterium, the lactic acid content of the standard strains a to i was also measured.
乳酸量の測定は、イオン排除クロマトグラフィ、及びポストカラムｐＨ緩衝化電気伝導度検出法を用いて行った。分析条件は以下のとおりである。
・カラム ：Ｓｈｉｍ－ｐａｃｋ ＳＣＲ－１０２Ｈ ２本（８ｍｍ×３００ｍｍ）、ガードカラム付き
・ポンプ１ ：移動相：５ｍＭ ｐ－トルエンスルホン酸水溶液
流量：０．８ｍｌ／ｍｉｎ
・ポンプ２ ：反応液：５ｍＭ ｐ－トルエンスルホン酸、１００μＭ ＥＤＴＡ含有２０ｍＭ Ｂｉｓ－Ｔｒｉｓ水溶液
流量：０．８ｍｌ／ｍｉｎ
・カラム温度 ：４０℃
・検出器 ：ＰＯＬＡＲＩＴＹ：＋、ＤＩＳＰＬＡＹ：ＢＡＣＫ ＧＲＯＵＮＤ、ＲＥＳＰＯＮＳＥ：ＳＬＯＷ、ＧＡＩＮ：１
・サンプル注入量：１０μｌ
・１サイクル ：３５分
Measurement of lactate content was performed using ion exclusion chromatography and post-column pH-buffered conductivity detection. Analysis conditions are as follows.
・ Column: Shim-pack SCR-102H 2 (8 mm × 300 mm), with guard column ・ Pump 1: Mobile phase: 5 mM p-toluenesulfonic acid aqueous solution
Flow rate: 0.8ml/min
・Pump 2: Reaction solution: 20 mM Bis-Tris aqueous solution containing 5 mM p-toluenesulfonic acid and 100 μM EDTA
Flow rate: 0.8ml/min
・Column temperature: 40°C
・Detector: POLARITY: +, DISPLAY: BACK GROUND, RESPONSE: SLOW, GAIN: 1
・Sample injection volume: 10 μl
・1 cycle: 35 minutes
評価結果は、図３に示す。本乳酸菌は、ヒトの腸内に通常存在する標準菌株ｂ～ｉと比べて、産生した乳酸量が非常に多く、近縁の標準菌株ａと比べても、産生した乳酸量が多い。すなわち、本乳酸菌は、活性がより高い状態が保持され、活性が高い状態で腸に届くことが期待される。 Evaluation results are shown in FIG. The present lactic acid bacterium produces a very large amount of lactic acid compared to the standard strains b to i that normally exist in the human intestine, and also produces a large amount of lactic acid compared to the closely related standard strain a. That is, the present lactic acid bacterium is expected to maintain a higher activity state and reach the intestine in a highly active state.
３．１６Ｓ ｒＤＮＡ遺伝子解析結果
１６Ｓ ｒＤＮＡ部分塩基配列について解析を行った。解析条件は以下である。なお、解析は（株）テクノスルガ・ラボにて行い、以下の解析条件等は、（株）テクノスルガ・ラボの「細菌 Standard-Full報告書」による。
・ＤＮＡ抽出：アクロモペプチターゼ（FUJIFILM Wako Pure Chemical,Japan）
・ＰＣＲ増幅：PrimeSTAR HS DNA Polymerase（Takara Bio,Japan）
・サイクルシークエンス：BigDye Terminator v3.1 Cycle Sequencing Kit（Applied Biosystems,USA）
・使用プライマー：PCR増幅；９Ｆ，１５１０Ｒ
シークエンス；９Ｆ，５１５Ｆ，１０９９Ｆ，５３６Ｒ，９２６Ｒ，１５１０Ｒ
・シークエンス：ABI PRISM 3130 xl Genetic Analyzer System(Applied Biosystems)
・塩基配列決定：ChromasPro2.1（Technelysium ,AUS)
・ＢＬＡＳＴ相同性検索：解析ソフトウエア：ENKI(TechnoSuruga Laboratory)
データベース DB-BA15.0(TechnoSuruga Laboratory)
国際塩基配列データベース（DDBJ/ENA(EMBL)/GenBank）
検索日：２０２０年１月２３日
3. Results of 16S rDNA gene analysis The 16S rDNA partial base sequence was analyzed. Analysis conditions are as follows. The analysis was performed at Techno Suruga Lab Co., Ltd., and the following analysis conditions, etc. are based on Techno Suruga Lab Co., Ltd.'s "Bacteria Standard-Full Report".
・DNA extraction: Achromopeptidase (FUJIFILM Wako Pure Chemical, Japan)
・PCR amplification: PrimeSTAR HS DNA Polymerase (Takara Bio, Japan)
・Cycle sequencing: BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA)
・Primers used: PCR amplification; 9F, 1510R
Sequence; 9F, 515F, 1099F, 536R, 926R, 1510R
・Sequence: ABI PRISM 3130 xl Genetic Analyzer System (Applied Biosystems)
・Nucleotide sequencing: ChromasPro2.1 (Technelysium, AUS)
・BLAST homology search: Analysis software: ENKI (TechnoSuruga Laboratory)
Database DB-BA15.0 (Techno Suruga Laboratory)
International Nucleotide Sequence Database (DDBJ/ENA(EMBL)/GenBank)
Search date: January 23, 2020
微生物同定システム「ＥＮＫＩ（登録商標）」を用いたＤＢ-ＢＡ（微生物同定データベース、細菌用）に対するＢＬＡＳＴ（Basic Local Alignment Search Tool）相同性検索の結果、本乳酸菌の１６Ｓ ｒＤＮＡ部分塩基配列は、Pediococcus acidilactici ＤＳＭ ２０２８４Ｔ（ＡＪ３０５３２０）に対して相同率９９．５％を示した。上記Pediococcus acidilactici ＤＳＭ ２０２８４Ｔ株は、Pediococcus acidilactici ＮＲＩＣ０１１５と同じ菌株である。ＤＢ－ＢＡに対する相同性検索で得られた塩基配列を基に解析した簡易分子系統樹の推定の結果、本乳酸菌はPediococcus acidilactici ＤＳＭ ２０２８４Ｔ（ＡＪ３０５３２０）と１００％の高いブーストラップ値で支持されるクラスターを形成し、近縁であることが示された。本系統解析は近隣結合法にて行っており、その際の塩基置換モデルとしてはKimura-2-parameterを用い、樹形の信頼性評価はブートストラップ法（１０００反復）にて行っている。 As a result of BLAST (Basic Local Alignment Search Tool) homology search against DB-BA (microorganism identification database, for bacteria) using the microorganism identification system "ENKI (registered trademark)", the 16S rDNA partial nucleotide sequence of this lactic acid bacterium is Pediococcus acidilactici DSM 20284T (AJ305320) showed a homology rate of 99.5%. The Pediococcus acidilactici DSM 20284T strain is the same strain as Pediococcus acidilactici NRIC0115. As a result of estimation of a simple molecular phylogenetic tree analyzed based on the nucleotide sequence obtained by homology search against DB-BA, this lactic acid bacterium is a cluster supported by Pediococcus acidilactici DSM 20284T (AJ305320) and a high bootstrap value of 100%. and was shown to be closely related. This phylogenetic analysis was performed by the neighbor-joining method, Kimura-2-parameter was used as the base substitution model, and the bootstrap method (1000 iterations) was used to evaluate the reliability of the tree shape.
また、（株）テクノスルガ・ラボによる、本乳酸菌の国際塩基配列データベースに対するＢＬＡＳＴ検索結果を表４に示す。相同性スコアで上位３０に検索された１６Ｓ ｒＤＮＡ塩基配列データを示すものである。以上の結果より、本乳酸菌はPediococcus acidilacticiに近縁なPediococcus sp.であることが判った。 In addition, Table 4 shows the BLAST search results for the international base sequence database of this lactic acid bacterium by Technosuruga Labo Co., Ltd. 16S rDNA base sequence data retrieved in the top 30 by homology score are shown. From the above results, it was found that this lactic acid bacterium is Pediococcus sp., which is closely related to Pediococcus acidilactici.
本乳酸菌の１６Ｓ ｒＤＮＡ部分塩基配列は、配列表に示す。 The 16S rDNA partial base sequence of this lactic acid bacterium is shown in the sequence listing.
本乳酸菌は、粉末にすることができる。すなわち、粉末の集合体である粉体の態様で、保管、流通、使用することができる。本例における本乳酸菌の粉末は、粒径が概ね試験用ふるい（日本工業規格ＪＩＳ Ｚ８８０１－１）公称目開き３５５μｍパス（メッシュＮｏ．４５を通過）、６３μｍオン（メッシュＮｏ．２３０上に残留）の範囲内である。粉末化は、以下の方法で行うことができる。すなわち、前述のように培養して得られた培養液を、遠心分離することにより上清を除去して本乳酸菌の菌体を得て、得られた菌体を凍結した後、真空乾燥（減圧乾燥）し、粉砕する方法である。遠心分離の回転数は、概ね１００００ｒｐｍでよい。ただし、粉末化の方法はこの方法に限られず、乳酸菌の粉末化として知られる公知の手法を用いてもよい。 The present lactic acid bacteria can be powdered. That is, it can be stored, distributed, and used in the form of powder, which is an aggregate of powder. The powder of this lactic acid bacterium in this example has a particle size of approximately 355 μm pass (pass through mesh No. 45), 63 μm on (residue on mesh No. 230) using a test sieve (Japanese Industrial Standard JIS Z8801-1). is within the range of Powderization can be performed by the following method. That is, the culture solution obtained by culturing as described above is centrifuged to remove the supernatant to obtain the cells of the present lactic acid bacterium. drying) and pulverizing. The speed of centrifugation may be approximately 10000 rpm. However, the method of pulverization is not limited to this method, and a known technique known as pulverization of lactic acid bacteria may be used.
本乳酸菌は、喫食することができる飲食品（食品及び飲料）として利用することができる。したがって、本乳酸菌は、他の飲食品とともに喫食してもよいし、単独で喫食してもよい。他の飲食品と喫食する例としては、ヨーグルト、ジャム、ふりかけ等の食品とともに喫食することができる。単独で喫食する場合には、粉末とされた本乳酸菌を、例えば水等の飲料により経口摂取する方法が挙げられる。 The present lactic acid bacteria can be used as edible food and drink (food and beverage). Therefore, the present lactic acid bacterium may be eaten together with other food or drink, or may be eaten alone. As an example of eating with other foods and drinks, it can be eaten with foods such as yoghurt, jam, and furikake. When the lactic acid bacterium is eaten alone, a method of orally ingesting the powdered lactic acid bacterium in a drink such as water can be used.
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| JP2008104375A (en) * | 2006-10-24 | 2008-05-08 | Taiyo Corp | Peroxide degrading enzyme |
| JP2009191276A (en) * | 2009-05-27 | 2009-08-27 | Taiyo Corp | Lactic bacterium exhibiting peroxide decomposition characteristic |
| WO2022058798A2 (en) * | 2020-09-18 | 2022-03-24 | Biocc Oü | Microorganism strain pediococcus acidilactici tak 589 coccobest as an antimicrobial and antioxidant probiotic |
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| JP2008104375A (en) * | 2006-10-24 | 2008-05-08 | Taiyo Corp | Peroxide degrading enzyme |
| JP2009191276A (en) * | 2009-05-27 | 2009-08-27 | Taiyo Corp | Lactic bacterium exhibiting peroxide decomposition characteristic |
| WO2022058798A2 (en) * | 2020-09-18 | 2022-03-24 | Biocc Oü | Microorganism strain pediococcus acidilactici tak 589 coccobest as an antimicrobial and antioxidant probiotic |
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