JP4526620B2 - New lactic acid strain - Google Patents
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- JP4526620B2 JP4526620B2 JP27933299A JP27933299A JP4526620B2 JP 4526620 B2 JP4526620 B2 JP 4526620B2 JP 27933299 A JP27933299 A JP 27933299A JP 27933299 A JP27933299 A JP 27933299A JP 4526620 B2 JP4526620 B2 JP 4526620B2
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Description
【０００１】
【発明の属する技術分野】
本発明は、細胞膜結合性アデノシントリホスファターゼ(ATPase)活性が低下しているラクトバチルス・デルブルッキー・サブスピーシズ・ブルガリクス(Lactobacillus delbrueckii subsp. bulgaricus) 変異株に関する。
【０００２】
【従来の技術】
ATPaseは、マグネシウムイオン又はカルシウムイオンの存在下、アデノシン三リン酸のγ位のリン酸を加水分解してアデノシン二リン酸と無機リン酸に分解する酵素であるが、この加水分解反応の際に生成するエネルギーが生体にとって重要であることが知られている。そして、乳酸菌のラクトバチルス・デルブルッキー・サブスピーシズ・ブルガリクス(Lactobacillus delbrueckii subsp. bulgaricus) には、大腸菌等と同様に細胞膜結合性ATPaseが存在することが知られている。しかし、この細胞膜結合性ATPaseがラクトバチルス・デルブルッキー・サブスピーシズ・ブルガリクス(Lactobacillus delbrueckii subsp. bulgaricus) の糖代謝やエネルギー生産に及ぼす影響については解明されていない。さらに、この細胞膜結合性ATPase活性が低下しているラクトバチルス・デルブルッキー・サブスピーシズ・ブルガリクス(Lactobacillus delbrueckii subsp. bulgaricus) の菌株は知られていない。
【０００３】
一方、大腸菌K-12株のピルビン酸生産菌より形質導入によって細胞膜結合性ATPase欠失変異株を誘導し、その発酵パターンを親株と比較したところ、細胞膜結合性ATPase欠失変異株では菌体のエネルギーレベルが低下するため解糖系が活性化し、菌体当たりの糖消費及びピルビン酸生産能が顕著に増大することが知られている。
【０００４】
また、細胞膜結合性ATPase活性が低下している乳酸桿菌ラクトバチルス・ヘルベチカス(Lactobacillus helveticus)菌株 (特許第 2824821号公報) 及び乳酸球菌ラクトコッカス・ラクチス・サブスピーシズ・ラクチス(Lactococcus lactis subsp.lactis)菌株 (特開平9-9954号公報) を選択して、それぞれの菌株を使用して乳酸酸度の上昇を抑制した発酵乳製品が提案されている。
【０００５】
【発明が解決しようとする課題】
本発明者らは、ラクトバチルス・デルブルッキー・サブスピーシズ・ブルガリクス(Lactobacillus delbrueckii subsp. bulgaricus) に関し、種々研究を進めていたところ、細胞膜結合性ATPase活性が低下したラクトバチルス・デルブルッキー・サブスピーシズ・ブルガリクス(Lactobacillus delbrueckii subsp. bulgaricus) を見出した。したがって、本発明は、細胞膜結合性ATPase活性が低下しているラクトバチルス・デルブルッキー・サブスピーシズ・ブルガリクス(Lactobacillus delbrueckii subsp. bulgaricus) 変異株を提供することを課題とする。
本発明の細胞膜結合性ATPase活性が低下しているラクトバチルス・デルブルッキー・サブスピーシズ・ブルガリクス(Lactobacillus delbrueckii subsp. bulgaricus) 変異株は、酸性側で急速に乳酸生成及び生育が低下するので、発酵乳やチーズ等の発酵乳製品を製造する際に有用である。
【０００６】
【課題を解決するするための手段】
本発明の細胞膜結合性ATPase活性が低下しているラクトバチルス・デルブルッキー・サブスピーシズ・ブルガリクス(Lactobacillus delbrueckii subsp. bulgaricus) 変異株は、以下のようにして得ることができる。
ラクトバチルス・デルブルッキー・サブスピーシズ・ブルガリクス(Lactobacillus delbrueckii subsp. bulgaricus) 親株からネオマイシン自然耐性株を取得し、このネオマイシン自然耐性株の中、親株よりも生育が低下した生育低下株を得る。そして、この生育低下株のATPase活性を測定し、親株のATPase活性と比較して10％以下にATPase活性が低下したATPase活性低下株を得る。
【０００７】
このようにして得られたラクトバチルス・デルブルッキー・サブスピーシズ・ブルガリクス(Lactobacillus delbrueckii subsp. bulgaricus) のATPase活性低下株は、発酵過程においてpHが低下すると本来、 H+ を排出する細胞膜結合性ATPaseが低下しているため、 H+ がうまく排出されずに菌体内のpHが低下する。
そのため、その生育が抑制され、乳酸の生成量が低下するので、発酵後に乳酸の生成を抑制する必要がある発酵乳製品等の製造に利用することが可能である。
親株とATPase活性低下株の発酵特性を比較すると、培養20時間まではその生育に差は認められないが、その後ATPase活性低下株の生育が低下した。培養36時間後には、ATPase活性低下株の酸生成能は極端に低下し、培養液のpHは親株4.06に対し4.52であった。
【０００８】
以下に実施例を示し、本発明を詳しく説明する。
【実施例１】
(1) ネオマイシン耐性株の取得
市販の発酵乳から分離したラクトバチルス・デルブルッキー・サブスピーシズ・ブルガリクス(Lactobacillus delbrueckii subsp. bulgaricus) SBT 0164を親株としてネオマイシン自然耐性株を取得した。すなわち、親株を市販のＭＲＳ液体培地を１／２希釈した培地（１／２ＭＲＳ液体培地）３ml中に接種し、37℃で一晩培養した後、得られた培養物を3,500rpm、10分、４℃で遠心集菌後、0.85％塩化ナトリウム溶液で２回洗浄し、同溶液３mlに懸濁した。その懸濁菌液を25〜50μg/ml濃度のネオマイシンを含む１／２ＭＲＳ液体培地寒天平板培地（１／２ＭＲＳ液体培地に寒天 15g/lを添加した培地）に 0.1mlを塗抹し、37℃で3 日間嫌気培養した。そして、生育したネオマイシン耐性株 108株を取得した。
【０００９】
(2) 生育低下株の取得
上記のネオマイシン耐性株を１／２ＭＲＳ液体培地中で振盪培養し、親株よりも生育が低下した生育低下株４株を取得した。
【００１０】
(3) ATPase活性の測定
上記の生育低下株のATPase活性を測定した。なお、ATPase活性の測定は次のような方法により行った。
まず、生育低下株を１／２ＭＲＳ液体培地が３ml入ったスクリューキャップ付き試験管で16時間培養したものを１／２ＭＲＳ液体培地３mlの入ったスクリューキャップ付き試験管に660 nmにおける吸光度が約0.03となるように接種し、37℃で静置培養した。18時間培養して得られた定常期の菌体を 8,000×g 、10分、4 ℃で遠心集菌後、同条件で 2.5mM塩化マグネシウムを含む 100mMトリス−塩酸緩衝液(pH 8)で２回洗浄した。その後、湿菌体1gを同緩衝液５mlに懸濁して超音波処理し、４℃で遠心分離 (20,000×ｇ、10分間) して上清を回収した。そして、この上清を４℃で超遠心分離(100,000×ｇ、１時間) して沈澱を回収し、同緩衝液に懸濁して粗酵素液とした。粗酵素液は氷冷して４℃で保存し、24時間以内に使用した。
一方、 5.0mM塩化マグネシウムを含む50mMトリス−塩酸緩衝液(pH 7)に基質として５mMアデノシントリリン酸ナトリウムを溶解して基質溶液とした。
【００１１】
上記の基質溶液 500μl と粗酵素液 100μl を混合し、37℃で10分間反応させた後、氷冷した0.1N塩酸 300μl を添加して反応を停止した。そして、表１に示した組成の発色液 2.1mlを添加して18℃で10分間発色させた後、3,000rpmで10分間遠心分離して沈澱を除去し、 660nmにおける吸光度を測定した。
標準物質をKH2PO4とした標準曲線より遊離された無機リン酸をその吸光度より算出した。酵素活性は１分間に遊離した無機リン酸量で示した。比活性は、ウシ血清アルブミン（BSA)を標準とし、Bio-Rad プロテイン・アッセイキットで測定した蛋白質1mg あたりの酵素活性で示した(H.Kobayashi and Anraku, J.Biochem, 71, 387-399(1972))。
【００１２】
【表１】
────────────────────────────────────
5N硫酸 10ml
2.5％モリブデン酸アンモニウム 10ml
３％硫酸水素ナトリウム−１％パラメチルアミノフェノール硫酸 10ml
水 40ml
────────────────────────────────────
【００１３】
(4) 低ATPase活性株の取得
表２に示した通り、親株のATPase活性の10％以下のATPase活性を示した４株を取得した。なお、ATPase活性の最も低い株は親株のATPase活性の７％であった。このATPase活性の最も低い株、ATPase活性低下株Ｂ株をラクトバチルス・デルブルッキー・サブスピーシズ・ブルガリクス(Lactobacillus delbrueckii subsp. bulgaricus) SBT 10757 として、工業技術院生命工学工業技術研究所に寄託した (受託番号:FERM P-17555)。
【００１４】
【表２】
なお、このラクトバチルス・デルブルッキー・サブスピーシズ・ブルガリクス(Lactobacillus delbrueckii subsp. bulgaricus) SBT 10757 は、親株のラクトバチルス・デルブルッキー・サブスピーシズ・ブルガリクス(Lactobacillus delbrueckii subsp. bulgaricus) SBT 0164と同様、以下に示す性質を有していたので、ラクトバチルス・デルブルッキー・サブスピーシズ・ブルガリクス(Lactobacillus delbrueckii subsp. bulgaricus) と同定し得る。
【００１６】
Ａ 形態的性状
(1)細胞の形： 桿菌
(2)運動性： なし
(3)胞子の有無： なし
(4)グラム染色性： 陽性
Ｂ 培地上の生育状態
(1)培養温度15℃ 生育しない
(2)培養温度45℃ 生育する
【００１７】
Ｃ 生理学的性質
(1)カタラーゼ： 陰性
(2)グルコースよりガスを産生しない。
(3)グルコン酸よりガスを産生しない。
(4)グルコースよりホモ乳酸発酵によりＤ（−）乳酸を産生する。
(5)各種炭水化物の分解性
1. グルコース： ＋
2. ラクトース： ＋
3. フラクトース： ＋
4. マンノース： −
5. ガラクトース： −
6. シュークロース： −
7. マルトース： −
8. セロビオース： −
9. トレハロース： −
10. メリビオース： −
11. ラフィノース： −
12. メレテトース： −
13. マンニトール： −
14. ソルビトール： −
15. ユースクリン： −
16. サリシン： −
17. アミグダリン： −
【００１８】
【試験例１】
表３に示した培地を充填した 300ml容三角フラスコに、親株であるラクトバチルス・デルブルッキー・サブスピーシズ・ブルガリクス(Lactobacillus delbrueckii subsp. bulgaricus) SBT 0164とATPase活性低下株としてラクトバチルス・デルブルッキー・サブスピーシズ・ブルガリクス(Lactobacillus delbrueckii subsp. bulgaricus) SBT 10757 をそれぞれ接種し、培養温度37℃で発酵特性を比較し、図１に示した。
【００１９】
【表３】
────────────────────
ペプトン 5 (g/l)
肉エキス 2.5
酵母エキス 2.5
グルコース 10
第二リン酸カリウム 1
ツイーン80 0.5
クエン酸二アンモニウム 1
酢酸ナトリウム 2.5
硫酸マグネシウム 0.05
硫酸マンガン 0.025
────────────────────
【００２０】
発酵開始から発酵20時間までは親株及びATPase活性低下株共に生育に差は認められないが、それ以降はATPase活性低下株の方が約20％ほど生育が低かった。発酵36時間後のpHは親株が4.06、ATPase活性低下株は4.52であり、ATPase活性低下株の酸生成能は親株に比べて顕著に低下していた。
【００２１】
【発明の効果】
本発明のラクトバチルス・デルブルッキー・サブスピーシズ・ブルガリクス(Lactobacillus delbrueckii subsp. bulgaricus) のATPase活性低下株は、発酵過程においてpHが低下するとその生育が抑制されるので、発酵後に乳酸の生成を抑制する必要がある発酵乳製品等を製造する際に有用である。
【図面の簡単な説明】
図１親株とATPase活性低下株との発酵特性を比較したものである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a Lactobacillus delbrueckii subsp. Bulgaricus mutant strain having reduced cell membrane-bound adenosine triphosphatase (ATPase) activity.
[0002]
[Prior art]
ATPase is an enzyme that hydrolyzes the phosphate at the γ position of adenosine triphosphate in the presence of magnesium ion or calcium ion to decompose it into adenosine diphosphate and inorganic phosphate. It is known that the energy generated is important for the living body. In addition, it is known that Lactobacillus delbrueckii subsp. Bulgaricus, which is a lactic acid bacterium, contains cell membrane-bound ATPase as in E. coli and the like. However, the effect of this cell membrane-bound ATPase on the sugar metabolism and energy production of Lactobacillus delbrueckii subsp. Bulgaricus has not been elucidated. Furthermore, a strain of Lactobacillus delbrueckii subsp. Bulgaricus having a reduced cell membrane-bound ATPase activity is not known.
[0003]
On the other hand, a cell membrane-bound ATPase deletion mutant was induced by transduction from a pyruvate-producing bacterium of E. coli K-12, and the fermentation pattern was compared with the parent strain. It is known that the glycolysis system is activated because the energy level is lowered, and the sugar consumption and the ability to produce pyruvic acid per cell are significantly increased.
[0004]
Further, Lactobacillus Lactobacillus helveticus that is a cell membrane-bound ATPase activity is decreased (Lactobacillus helveticus) strains (Japanese Patent No. 2824821) and Lactococcus lactis ssp lactis (Lactococcus lactis subsp. Lactis) strain (JP-A-9-9954) is selected, and fermented milk products that suppress the increase in the lactic acid acidity using each strain have been proposed.
[0005]
[Problems to be solved by the invention]
The present inventors have conducted various studies on Lactobacillus delbrueckii subsp. Bulgaricus, and as a result, Lactobacillus delbrueckii subsp. Cucumber (Lactobacillus delbrueckii subsp. Bulgaricus ) was found. Therefore, an object of the present invention is to provide a Lactobacillus delbrueckii subsp. Bulgaricus mutant strain having reduced cell membrane-bound ATPase activity.
The Lactobacillus delbrueckii subsp. Bulgaricus mutant strain having reduced cell membrane-bound ATPase activity according to the present invention has a rapid decrease in lactic acid production and growth on the acidic side. It is useful when producing fermented milk products such as cheese and cheese.
[0006]
[Means for Solving the Problems]
The Lactobacillus delbrueckii subsp. Bulgaricus mutant strain having reduced cell membrane-bound ATPase activity of the present invention can be obtained as follows.
Lactobacillus del Burukki ssp. Bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus) to get the neomycin natural resistant strains from the parent strain, the inside of neomycin natural resistant strains to obtain a growth decline strain growth was lower than the parent strain. Then, the ATPase activity of this growth-reduced strain is measured, and an ATPase activity-reduced strain having an ATPase activity reduced to 10% or less compared to the parent strain's ATPase activity is obtained.
[0007]
The Lactobacillus delbrueckii subsp. Bulgaricus ATPase activity-reduced strain obtained in this way has a membrane-bound ATPase that excretes H + when pH decreases during the fermentation process. Since it is lowered, H + is not excreted well, and the pH in the microbial cell is lowered.
Therefore, since the growth is suppressed and the production amount of lactic acid is reduced, it can be used for the production of fermented milk products and the like that need to suppress the production of lactic acid after fermentation.
When the fermentation characteristics of the parent strain and the ATPase activity-reduced strain were compared, no difference was observed in the growth until 20 hours of culture, but the growth of the ATPase activity-reduced strain subsequently decreased. After 36 hours of culturing, the acid-producing ability of the ATPase activity-reduced strain was extremely reduced, and the pH of the culture solution was 4.52 compared to 4.06 of the parent strain.
[0008]
The following examples illustrate the invention in detail.
[Example 1]
(1) Acquisition of neomycin resistant strain A neomycin natural resistant strain was acquired using Lactobacillus delbrueckii subsp. Bulgaricus SBT 0164 as a parent strain isolated from commercially available fermented milk. That is, the parent strain was inoculated into 3 ml of a commercially available MRS liquid medium diluted 1/2 (1/2 MRS liquid medium) and cultured overnight at 37 ° C., and then the resulting culture was 3,500 rpm, 10 minutes, After centrifugation at 4 ° C., the cells were washed twice with 0.85% sodium chloride solution and suspended in 3 ml of the same solution. 0.1 ml of the suspension was smeared on a 1/2 MRS liquid medium agar plate medium containing neomycin at a concentration of 25-50 μg / ml (medium obtained by adding 15 g / l of agar to 1/2 MRS liquid medium), and at 37 ° C. Anaerobic culture was performed for 3 days. Then, 108 grown neomycin resistant strains were obtained.
[0009]
(2) Acquisition of growth-reduced strains The above-mentioned neomycin-resistant strain was shake-cultured in a 1/2 MRS liquid medium to obtain 4 growth-reduced strains whose growth was lower than that of the parent strain.
[0010]
(3) Measurement of ATPase activity The ATPase activity of the above-mentioned growth-reduced strain was measured. The ATPase activity was measured by the following method.
First, a growth-reduced strain cultured for 16 hours in a test tube with a screw cap containing 3 ml of 1/2 MRS liquid medium was added to a test tube with a screw cap containing 3 ml of 1/2 MRS liquid medium, and the absorbance at 660 nm was about 0.03. And inoculated and incubated at 37 ° C. Steady-phase cells obtained by culturing for 18 hours were collected by centrifugation at 8,000 xg for 10 minutes at 4 ° C, then 2 times with 100 mM Tris-HCl buffer (pH 8) containing 2.5 mM magnesium chloride under the same conditions. Washed twice. Thereafter, 1 g of wet cells were suspended in 5 ml of the same buffer and sonicated, and centrifuged (20,000 × g, 10 minutes) at 4 ° C. to recover the supernatant. The supernatant was ultracentrifuged at 4 ° C. (100,000 × g, 1 hour) to recover the precipitate, and suspended in the same buffer to obtain a crude enzyme solution. The crude enzyme solution was ice-cooled and stored at 4 ° C. and used within 24 hours.
On the other hand, 5 mM sodium adenosine triphosphate as a substrate was dissolved in 50 mM Tris-HCl buffer (pH 7) containing 5.0 mM magnesium chloride to prepare a substrate solution.
[0011]
500 μl of the above substrate solution and 100 μl of the crude enzyme solution were mixed and reacted at 37 ° C. for 10 minutes, and then the reaction was stopped by adding 300 μl of ice-cooled 0.1N hydrochloric acid. Then, 2.1 ml of a coloring solution having the composition shown in Table 1 was added to cause color development at 18 ° C. for 10 minutes, followed by centrifugation at 3,000 rpm for 10 minutes to remove the precipitate, and the absorbance at 660 nm was measured.
Inorganic phosphoric acid liberated from a standard curve with KH 2 PO 4 as the standard substance was calculated from its absorbance. Enzyme activity was expressed as the amount of inorganic phosphate released per minute. Specific activity was expressed as enzyme activity per mg of protein measured with the Bio-Rad protein assay kit using bovine serum albumin (BSA) as a standard (H. Kobayashi and Anraku, J. Biochem, 71, 387-399 ( 1972)).
[0012]
[Table 1]
────────────────────────────────────
5N sulfuric acid 10ml
2.5% ammonium molybdate 10ml
3% sodium hydrogen sulfate-1% paramethylaminophenol sulfuric acid 10ml
40ml water
────────────────────────────────────
[0013]
(4) Acquisition of low ATPase activity strains As shown in Table 2, 4 strains having ATPase activity of 10% or less of the ATPase activity of the parent strain were obtained. The strain with the lowest ATPase activity was 7% of the ATPase activity of the parent strain. The strain with the lowest ATPase activity and the B strain with reduced ATPase activity was deposited at the National Institute of Advanced Industrial Science and Technology as Lactobacillus delbrueckii subsp. Bulgaricus SBT 10757 Number: FERM P-17555).
[0014]
[Table 2]
It should be noted that this Lactobacillus del Burukki ssp. Bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus) SBT 10757 , as well as the parent strain of Lactobacillus del Burukki ssp. Bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus) SBT 0164, to the following Since it had the property shown, it can be identified as Lactobacillus delbrueckii subsp. Bulgaricus .
[0016]
A Morphological properties
(1) Cell shape: Neisseria gonorrhoeae
(2) Mobility: None
(3) Presence or absence of spores: None
(4) Gram staining: Positive B Growth condition on medium
(1) Culture temperature 15 ° C
(2) Growing temperature 45 ℃
C Physiological properties
(1) Catalase: negative
(2) Does not produce gas from glucose.
(3) Does not produce gas from gluconic acid.
(4) D (-) lactic acid is produced from glucose by homolactic fermentation.
(5) Degradability of various carbohydrates
1. Glucose: +
2. Lactose: +
3. Fructose: +
4. Mannose: −
5. Galactose: −
6. Sucrose: −
7. Maltose: −
8. Cellobiose: −
9. Trehalose: −
10. Melibiose: −
11. Raffinose: −
12. Meletetose: −
13. Mannitol: −
14. Sorbitol: −
15. Youth Clean: −
16. Salicin: −
17. Amygdalin: −
[0018]
[Test Example 1]
In 300ml Erlenmeyer flask filled with medium shown in Table 3, Lactobacillus del Burukki ssp bulgaricus parent strain (Lactobacillus delbrueckii subsp. Bulgaricus) SBT 0164 and Lactobacillus del Burukki ssp as ATPase activity decreased strain -Bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus ) SBT 10757 was inoculated, and the fermentation characteristics were compared at a culture temperature of 37 ° C.
[0019]
[Table 3]
────────────────────
Peptone 5 (g / l)
Meat extract 2.5
Yeast extract 2.5
Tween 80 0.5
Sodium acetate 2.5
Magnesium sulfate 0.05
Manganese sulfate 0.025
────────────────────
[0020]
From the start of fermentation to 20 hours after fermentation, there was no difference in the growth of both the parent strain and the ATPase activity-reduced strain, but after that, the ATPase activity-reduced strain was about 20% less grown. The pH after fermentation for 36 hours was 4.06 for the parent strain and 4.52 for the ATPase activity-reduced strain, and the acid-producing ability of the ATPase activity-reduced strain was significantly lower than that of the parent strain.
[0021]
【The invention's effect】
ATPase activity decreased strain of Lactobacillus del Burukki ssp bulgaricus of the present invention (Lactobacillus delbrueckii subsp. Bulgaricus), because its growth the pH is reduced in the fermentation process is suppressed, inhibiting the production of lactic acid after fermentation It is useful when producing necessary fermented milk products.
[Brief description of the drawings]
FIG. 1 is a comparison of fermentation characteristics between a parent strain and a strain with reduced ATPase activity.
Claims (2)
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| WO2023038072A1 (en) | 2021-09-09 | 2023-03-16 | 株式会社明治 | Lactic acid bacterium, lactic acid bacterium starter, fermented milk, method for manufacturing fermented milk, and method for screening lactic acid bacterium |
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| JPH099954A (en) * | 1995-07-04 | 1997-01-14 | Snow Brand Milk Prod Co Ltd | New lactic acid strain |
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