JP2568537B2 - Method for enhancing diacetyl production by lactic acid bacteria - Google Patents
Method for enhancing diacetyl production by lactic acid bacteriaInfo
- Publication number
- JP2568537B2 JP2568537B2 JP3337887A JP3337887A JP2568537B2 JP 2568537 B2 JP2568537 B2 JP 2568537B2 JP 3337887 A JP3337887 A JP 3337887A JP 3337887 A JP3337887 A JP 3337887A JP 2568537 B2 JP2568537 B2 JP 2568537B2
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- Prior art keywords
- diacetyl
- lactic acid
- enhancing
- citric acid
- amount
- Prior art date
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Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は、乳酸菌の産生する主要な香気成分であるジ
アセチルの生成量の増強法に関するものである。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for enhancing the production amount of diacetyl, which is a main flavor component produced by lactic acid bacteria.
ジアセチルは醗酵バター，サワークリーム，チーズな
どの醗酵乳製品の主要な香気成分であり、マーガリン，
サマーソーセージ，はち蜜，コーヒー，パンなどでは芳
香として喜ばれている。乳酸菌によるジアセチル生成能
あるいは乳製品中のジアセチル生成量を高めるためにジ
アセチルの生成前駆体であるクエン酸を添加して培養す
る方法が古くから報告されており、またLeuconostoc cr
emorisにおいてはMn2+の添加が本菌の増殖ならびにクエ
ン酸の利用性を高めることが報告されている（T.M.Coga
n,J.Dairy Research,42,139（1975））。しかしStrepto
coccus lactis subsp.diacetylactisにおいては、培地
中へのクエン酸の添加を除いてジアセチルの生成を促進
する有効な成分は認められていない。Diacetyl is a major flavor component of fermented dairy products such as fermented butter, sour cream, cheese, etc.
In summer sausage, honey, coffee, bread, etc., it is delighted as an aroma. A method of culturing by adding citric acid, a precursor of diacetyl production, in order to increase the ability of lactic acid bacteria to produce diacetyl or the amount of diacetyl produced in dairy products has been reported for a long time.
In emoris, it has been reported that the addition of Mn 2+ enhances the growth of this bacterium and the availability of citric acid (TMCoga
n, J. Dairy Research, 42 , 139 (1975)). But Strepto
In coccus lactis subsp. diacetylactis, there is no effective component that promotes the production of diacetyl except for the addition of citric acid to the medium.
また、乳酸菌によるジアセチル生成機構については、
これまでR.A.Speckmanら（J.Bact.,95,174（1968））,
J.Stadholders（Milchwissenschaft,29,329（1674））
等の報告がある。いずれもクエン酸を代謝してジアセチ
ルを生成する経路につき記載している。Regarding the mechanism of diacetyl production by lactic acid bacteria,
RASpeckman et al. (J. Bact., 95 , 174 (1968)),
J. Stadholders (Milchwissenschaft, 29 , 329 (1674))
Etc. are reported. All of them describe the pathway of metabolizing citric acid to produce diacetyl.
さらにStreptococcus lactis subsp.diacetyl−actis
によるジアセチルの生成量を高めるために紫外線照射に
よる変異株の使用（R.K.Kuila,J.Dairy Sci.,61379（19
78））、固定化菌体の使用（J.Rossi,Milchwissenschaf
t,39,336（1984））などが報告されている。Furthermore, Streptococcus lactis subsp.diacetyl-actis
Of Mutants by UV Irradiation to Increase the Amount of Diacetyl Generated by UV (RKKuila, J. Dairy Sci., 61 379 (19
78)), use of immobilized cells (J. Rossi, Milchwissenschaf
t, 39 , 336 (1984)).
乳酸菌によるジアセチル生成量を高めるための上記の
従来技術につぎのようの改良を加えた。すなわち培地中
のクエン酸を効率よくジアセチルに変換させるための方
法につき種々検討を重ねたものである。The following improvements were added to the above-mentioned conventional technology for increasing the amount of diacetyl produced by lactic acid bacteria. That is, various studies have been made on a method for efficiently converting citric acid in the medium to diacetyl.
本発明は、ジアセチル生成促進物質を培養基質に添加
するだけの簡単な手段により、ジアセチル生成量を高め
ることを目的とするものである。An object of the present invention is to increase the amount of diacetyl production by a simple means of simply adding a diacetyl production promoting substance to a culture substrate.
本発明者らはStreptococcus lactis subsp.diacetyla
ctis,Leuconostoc cremoris,Leuconostoc lacits等のジ
アセチル生成に関する諸酵素活性の活性化因子につき鋭
意研究を重ねた結果、ジアセチル生成促進のためにはク
エン酸とともにCu2+,Fe2,Mo6+,Fe3+の１種または２種以
上を含有するようにこれらの塩類をそれぞれ強化した培
地で培養するとジアセチルの生成量を増大させるために
極めて有効であるとの知見を得て、本発明を完成した。The present inventors have proposed Streptococcus lactis subsp. Diacetyla
As a result of intensive studies on the activators of various enzyme activities related to diacetyl production such as ctis, Leuconostoc cremoris, and Leuconostoc lacits, Cu 2+ , Fe 2 , Mo 6+ , Fe 3 were added together with citric acid to promote diacetyl production. The present inventors have found that culturing these salts in a medium reinforced with each of them so as to contain one or more kinds of + is extremely effective for increasing the amount of diacetyl produced, and completed the present invention.
すなわち、本発明はクエン酸醗酵能を有す乳酸菌によ
りクエン酸からジアセチルを醗酵生成せしめるに際し
て、前記乳酸菌のクエン酸を含有する培養基質に鉄，銅
およびモリブデンの１種または２種以上を無機塩類また
は有機塩類の形で添加することを特徴とするものであ
る。That is, according to the present invention, when diacetyl is fermented from citric acid by a lactic acid bacterium capable of fermenting citric acid, one or more of iron, copper and molybdenum are added to a culture substrate containing citric acid of the lactic acid bacterium as an inorganic salt. Alternatively, it is characterized by being added in the form of organic salts.
本発明におけるクエン酸醗酵能を有する乳酸菌として
は、クエン酸醗酵能を有する乳酸菌であればいずれでも
使用できるが、Streptococcus lactis subsp.diacetyla
ctis及びLauconostoc cremorisがジアセチル生成能が優
れているので好ましい。As the lactic acid bacterium having a citric acid fermenting ability in the present invention, any lactic acid bacterium having a citric acid fermenting ability can be used, but Streptococcus lactis subsp. Diacetyla
ctis and Lauconostoc cremoris are preferred because of their excellent diacetyl producing ability.
培養基質として脱脂乳あるいは脱脂粉乳溶液を主に使
用する場合は、両者ともクエン酸を固形分中に約１％を
含有しているので、クエン酸を培養基質に添加しなくて
差支えないが、培養基質にクエン酸が含有ていないが、
含有量の少ない場合あるいはジアセチルを多量に生成さ
せる必要のある場合は、所望のジアセチル生成量に応じ
た量のクエン酸を培養基質に添加含有せしめる。なお添
加するクエン酸はクエン酸塩として添加するとよい。When skim milk or skim milk solution is mainly used as a culture substrate, both contain citric acid in a solid content of about 1%, so that citric acid may not be added to the culture substrate. The culture substrate does not contain citric acid,
When the content is small or when it is necessary to produce a large amount of diacetyl, an amount of citric acid corresponding to the desired amount of diacetyl produced is added to the culture substrate. The citric acid to be added may be added as a citrate.
本発明において培養基質に添加する鉄，銅およびモリ
ブデンの無機塩類または有機塩類は単独でもよいし、組
合せて添加してもよい。その添加量は鉄イオン，銅イオ
ン，モリブデンイオンの合計量が0.01mM〜10mM程度とな
るように添加することが好ましい。0.01mMより少ないと
ジアセチル生成の増強の効果が認められない。10mMより
多いと培養基質の着色，凝固などの現象を生ずるおそれ
がある。In the present invention, the inorganic or organic salts of iron, copper and molybdenum to be added to the culture substrate may be used alone or in combination. It is preferable to add the iron ion, the copper ion, and the molybdenum ion so that the total amount thereof is about 0.01 mM to 10 mM. If it is less than 0.01 mM, no effect of enhancing diacetyl production is observed. If it is more than 10 mM, phenomena such as coloring and coagulation of the culture substrate may occur.
なお、醗酵乳の培養基質となる乳中には、銅，鉄，モ
リブデンがそれぞれ３〜17μg/100ml,16〜62μg/100ml,
2〜15μg/100ml含まれていることが知られている（牛乳
の化学,P64,津郷友吉ら著，地球出版）。しかしこのよ
うな培養基質の金属イオン含量では乳酸菌のクエン酸代
謝に関与する酵素系を活性化するには不十分である。In addition, copper, iron, and molybdenum are 3 to 17 μg / 100 ml, 16 to 62 μg / 100 ml, respectively, in the milk serving as a culture substrate of the fermented milk.
It is known to contain 2-15 μg / 100 ml (Chemicals of milk, P64, Yugo Tsugo et al., Earth Publishing). However, the metal ion content of such a culture substrate is insufficient to activate an enzyme system involved in citrate metabolism of lactic acid bacteria.
つぎに、本発明の試験例を示す。 Next, test examples of the present invention will be described.
試験例１ Streptococcus lactis subsp.diacetylactis（ATCC 1
1007）のクエン酸代謝に関与する諸活性のうち、クエン
酸取込み活性、クエン酸リアーゼ活性ならびにジアセチ
ル生成活性につき検討し、第１表の結果を得た。Test Example 1 Streptococcus lactis subsp. Diacetylactis (ATCC 1
Among the various activities related to citrate metabolism of 1007), citrate uptake activity, citrate lyase activity and diacetyl generation activity were examined, and the results shown in Table 1 were obtained.
（１） クエン酸取込み活性測定法 Strepococcus lactis subsp.diacetylactis（ATCC 11
007）をMRS培地で培養し、滅菌水にて菌体洗浄後、0.1M
リン酸緩衝液（pH6.0）に懸濁する。体液9mlに0.2％ク
エン酸ナトリウムを1ml加え、30℃にて30分間反応す
る。反応後0.45μｍ（ポアサイズ）のフィルターにて除
菌した濾液中のクエン酸含量を測定する。反応前後にお
けるクエン酸量の変化より乾菌重量ｇ当りの30分間反応
させた際のクエン酸の菌体への取込み量を求める。 (1) Citrate uptake activity measurement method Strepococcus lactis subsp. Diacetylactis (ATCC 11
007) in an MRS medium, washed with sterile water, washed with 0.1M
Suspend in phosphate buffer (pH 6.0). 1 ml of 0.2% sodium citrate is added to 9 ml of body fluid, and reacted at 30 ° C. for 30 minutes. After the reaction, the content of citric acid in the filtrate that has been sterilized with a 0.45 μm (pore size) filter is measured. From the change in the amount of citric acid before and after the reaction, the amount of citric acid incorporated into the cells when the reaction is carried out for 30 minutes per g of dry bacteria is determined.
（２） クエン酸リアーゼ活性測定法 500mlのMRS培地にてStreptococcus lactis subsp,dia
cetylactis（ATCC 11007）を30℃にて20時間培養後、滅
菌水にて２回菌体洗浄を繰返し、0.1Mリン酸緩衝液（pH
6.0）で30mlの菌体懸濁液を調製する。本液を超音波処
理にて菌体破砕し、25,000r.p.m.にて遠心分離し、その
上澄液を一夜透析処理し、粗酵素液とする。5mMクエン
酸ナトリムウ,1mM塩化マグネシウムを含む0.1Mリン酸緩
衝液3mlに粗酵素液0.5mlを加え、30℃にて60分間反応さ
せ、沸騰水浴中で反応を停止する。本液を3000r.p.m.で
５分間遠心分離後、上澄液中の生成酢酸量を測定する。
クエン酸リアーゼ１単位は１μmolの酢酸を30℃１分間
で生成させるのに必要とする酵素量とし、比活性は酵素
タンパク1mgあたりの単位数とする。(2) Citrate lyase activity measurement method Streptococcus lactis subsp, dia in 500 ml of MRS medium
After culturing cetylactis (ATCC 11007) at 30 ° C. for 20 hours, the cells were washed twice with sterile water, and the cells were repeatedly washed with 0.1 M phosphate buffer (pH
Prepare 30 ml cell suspension in 6.0). This solution is sonicated to disrupt the cells, centrifuged at 25,000 rpm, and the supernatant is dialyzed overnight to obtain a crude enzyme solution. 0.5 ml of the crude enzyme solution is added to 3 ml of 0.1 M phosphate buffer containing 5 mM sodium citrate and 1 mM magnesium chloride, and the mixture is reacted at 30 ° C. for 60 minutes, and the reaction is stopped in a boiling water bath. After centrifuging this solution at 3000 rpm for 5 minutes, the amount of acetic acid produced in the supernatant is measured.
One unit of citrate lyase is the amount of enzyme required to produce 1 μmol of acetic acid at 30 ° C. for one minute, and the specific activity is the number of units per mg of enzyme protein.
（３） ジアセチル生成活性測定法 Kanekoら（Agric.Biol.Chem.,502639（1986））の方
法に従って測定した。(3) Method for measuring diacetyl-forming activity Measured according to the method of Kaneko et al. (Agric. Biol. Chem., 50 2639 (1986)).
第１表の結果からCu2+がクエン酸リアーゼ活性を、Cu
2+,Mo6+,Fe2+,Fe3+がジアセチル生成活性を高めること
がわかる。From the results in Table 1, Cu 2+ shows citrate lyase activity,
It can be seen that 2+ , Mo 6+ , Fe 2+ , and Fe 3+ enhance diacetyl generation activity.
試験例２ Cu2+,Mo6+,Fe2+,Fe3+をそれぞれ1.0mMとなるように添
加した10％脱脂粉乳培地にStreptococcus lactis subs
p.diacetylactis（ATCC 11007）またはLeuconostoc cre
moris（ATCC 19254）をそれぞれ接種し、30℃で20時間
培養後のジアセチル生成量を測定した。結果を第２表に
示す。Test Example 2 Streptococcus lactis subs. Was added to a 10% skim milk medium supplemented with 1.0 mM of Cu 2+ , Mo 6+ , Fe 2+ , and Fe 3+ respectively.
p.diacetylactis (ATCC 11007) or Leuconostoc cre
moris (ATCC 19254) were inoculated, and the amount of diacetyl produced after culturing at 30 ° C. for 20 hours was measured. The results are shown in Table 2.
ジアセチル測定法 Kanekoら（Agric.Biol.Chem.,50,2639（1986））の方
法に従って測定した。 Diacetyl measurement method The measurement was performed according to the method of Kaneko et al. (Agric. Biol. Chem., 50 , 2639 (1986)).
第２表の結果から、Cu2+,Mo6+,Fe2+,Fe3+の添加がジ
アセチル生成量を高めるために極めて有効であることが
わかる。この現象は、第１表が示すように、培地中より
菌体内にとり込んだクエン酸よりジアセチルにいたる代
謝において、金属イオンが有効に作用したことによりも
のと考えられる。The results in Table 2 show that the addition of Cu 2+ , Mo 6+ , Fe 2+ , and Fe 3+ is extremely effective in increasing the amount of diacetyl produced. This phenomenon is considered to be due to the effective action of metal ions on metabolism from citric acid taken up into the cells to diacetyl, as shown in Table 1.
実施例１ 30％脱脂粉乳溶液（0.05％酵母エキスを含む）100kg
に塩化第２鉄5g及びクエン酸ナトリウム10gを加え、95
℃にて15分間殺菌後、30℃に冷却し、Streptococcus la
ctis subsp.diacetylactis （ATCC 11007）を含むスタ
ーターを1.5kg接種し、これを30℃で16時間醗酵させて
得られた醗酵乳中のジアセチル生成量は30mg/kgであっ
た。Example 1 100 kg of 30% skim milk solution (including 0.05% yeast extract)
5 g of ferric chloride and 10 g of sodium citrate were added to
After sterilization at 15 ° C for 15 minutes, the mixture was cooled to 30 ° C and Streptococcus la
1.5 kg of a starter containing ctis subsp. diacetylactis (ATCC 11007) was inoculated and fermented at 30 ° C. for 16 hours, and the amount of diacetyl produced in the fermented milk obtained was 30 mg / kg.
上記したように本発明によれば、銅，鉄，モリブデン
を添加するという簡単な手段により、ジアセチルを主要
な香気成分としている醗酵乳製品のジアセチル生成量を
高めることにより、より芳香のすぐれた醗酵乳製品を得
ることができる。As described above, according to the present invention, the fermentation dairy product containing diacetyl as a main flavor component is increased in the amount of diacetyl produced by a simple means of adding copper, iron and molybdenum, thereby producing a fermentation product having a more aromatic flavor. Dairy products can be obtained.
また、高濃度にジアセチルを生成させることにより、
得られた醗酵製品はヨーグルト，バター，マーガリン，
パンなどのフレーバー付与物質として使用できる。Also, by producing diacetyl at a high concentration,
The resulting fermented products are yogurt, butter, margarine,
It can be used as a flavoring substance such as bread.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｃ１２Ｒ 1:46） ──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 6 Identification code Agency reference number FI Technical indication C12R 1:46)
Claims (4)
ン酸からジアセチルを醗酵生成せしめるに際して、前記
乳酸菌のクエン酸を含有する培養基質に鉄，銅およびモ
リブデンの１種または２種以上を無機塩類または有機塩
類の形で添加することを特徴とする乳酸菌によるジアセ
チル生成量の増強法。When a lactic acid bacterium capable of fermenting citric acid is used to ferment and produce diacetyl from citric acid, one or more of iron, copper and molybdenum are added to a citrate-containing culture substrate of the lactic acid bacterium as an inorganic salt or a salt thereof. A method for enhancing the amount of diacetyl produced by lactic acid bacteria, which is added in the form of organic salts.
銅イオン及びモリブデンイオンの合計量が0.01〜10mMと
なるように添加することを特徴とする特許請求の範囲第
１項記載の乳酸菌によるジアセチル生成量の増強法。2. The method according to claim 1, wherein the inorganic or organic salt is an iron ion,
2. The method for enhancing the amount of diacetyl produced by a lactic acid bacterium according to claim 1, wherein the copper ion and the molybdenum ion are added in a total amount of 0.01 to 10 mM.
ocus lactis subsp.diacetylactisであることを特徴と
する特許請求の範囲第１項および第２項のいずれかに記
載の乳酸菌によるジアセチル生成量の増強法。3. A lactic acid bacterium having a citric acid fermentation ability is Streptoc.
3. The method for enhancing diacetyl production by a lactic acid bacterium according to any one of claims 1 and 2, wherein the method is ocus lactis subsp. diacetylactis.
toc cremorisであることを特徴とする特許請求の範囲第
１項及び第２項のいずれかに記載の乳酸菌によるジアセ
チル生成量の増強法。4. A lactic acid bacterium having a citric acid fermentation ability is Leuconos.
3. The method for enhancing the amount of diacetyl produced by a lactic acid bacterium according to any one of claims 1 and 2, wherein the method is toc cremoris.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3337887A JP2568537B2 (en) | 1987-02-18 | 1987-02-18 | Method for enhancing diacetyl production by lactic acid bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3337887A JP2568537B2 (en) | 1987-02-18 | 1987-02-18 | Method for enhancing diacetyl production by lactic acid bacteria |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63202390A JPS63202390A (en) | 1988-08-22 |
| JP2568537B2 true JP2568537B2 (en) | 1997-01-08 |
Family
ID=12384931
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3337887A Expired - Lifetime JP2568537B2 (en) | 1987-02-18 | 1987-02-18 | Method for enhancing diacetyl production by lactic acid bacteria |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2568537B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2901008B2 (en) * | 1989-11-28 | 1999-06-02 | 明治乳業株式会社 | Diacetyl and acetoin fermentation by lactic acid bacteria |
-
1987
- 1987-02-18 JP JP3337887A patent/JP2568537B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPS63202390A (en) | 1988-08-22 |
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