JP2020018294A - Method for producing gallic acid and method for producing fermented tea, as well as lactic acid bacteria, lactic acid bacteria compositions, tea fermented products, and foods and drinks - Google Patents
Method for producing gallic acid and method for producing fermented tea, as well as lactic acid bacteria, lactic acid bacteria compositions, tea fermented products, and foods and drinks Download PDFInfo
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- JP2020018294A JP2020018294A JP2019134290A JP2019134290A JP2020018294A JP 2020018294 A JP2020018294 A JP 2020018294A JP 2019134290 A JP2019134290 A JP 2019134290A JP 2019134290 A JP2019134290 A JP 2019134290A JP 2020018294 A JP2020018294 A JP 2020018294A
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- lactic acid
- gallic acid
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- fermented
- tea
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- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 title claims abstract description 351
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 304
- 229940074391 gallic acid Drugs 0.000 title claims abstract description 174
- 235000004515 gallic acid Nutrition 0.000 title claims abstract description 174
- 241000894006 Bacteria Species 0.000 title claims abstract description 162
- 239000004310 lactic acid Substances 0.000 title claims abstract description 152
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 152
- 239000000203 mixture Substances 0.000 title claims abstract description 92
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 53
- 241001122767 Theaceae Species 0.000 title claims abstract 7
- 235000013616 tea Nutrition 0.000 title claims description 62
- 235000019225 fermented tea Nutrition 0.000 title claims description 54
- 235000013305 food Nutrition 0.000 title claims description 22
- 230000000694 effects Effects 0.000 claims abstract description 140
- 238000000855 fermentation Methods 0.000 claims abstract description 73
- 230000004151 fermentation Effects 0.000 claims abstract description 73
- 102000004031 Carboxy-Lyases Human genes 0.000 claims abstract description 67
- 108090000489 Carboxy-Lyases Proteins 0.000 claims abstract description 67
- 108010038851 tannase Proteins 0.000 claims abstract description 58
- 239000000284 extract Substances 0.000 claims abstract description 45
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims abstract description 34
- ADRVNXBAWSRFAJ-UHFFFAOYSA-N catechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3ccc(O)c(O)c3 ADRVNXBAWSRFAJ-UHFFFAOYSA-N 0.000 claims abstract description 29
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- PFTAWBLQPZVEMU-DZGCQCFKSA-N (+)-catechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-DZGCQCFKSA-N 0.000 claims abstract description 28
- WQGWDDDVZFFDIG-UHFFFAOYSA-N pyrogallol Chemical compound OC1=CC=CC(O)=C1O WQGWDDDVZFFDIG-UHFFFAOYSA-N 0.000 claims description 68
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- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 235000014058 juice drink Nutrition 0.000 description 1
- 229940004208 lactobacillus bulgaricus Drugs 0.000 description 1
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Tea And Coffee (AREA)
- General Preparation And Processing Of Foods (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
本発明は、没食子酸の製造方法及び茶発酵物の製造方法、並びに、乳酸菌、乳酸菌組成物、茶発酵物、及び飲食品に関する。 The present invention relates to a method for producing gallic acid and a method for producing a fermented tea product, as well as lactic acid bacteria, a lactic acid bacteria composition, a fermented tea product, and food and drink.
没食子酸は３，４，５−トリヒドロキシ安息香酸（3,4,5-Trihydroxybenzoic Acid）ともいい、抗酸化作用を有することが知られている。また、没食子酸を主要成分とするターミナリアベリリカの抽出物については、ヒトにおいて食後血中中性脂肪の上昇抑制作用（非特許文献１）を、及びマウスにおいて血糖値の上昇抑制作用（非特許文献２）を、それぞれ奏することが報告されており、これらの作用には該没食子酸が関与すると考えられている。そのため、没食子酸はメタボリックシンドローム改善への寄与が期待される成分として注目される。 Gallic acid is also called 3,4,5-trihydroxybenzoic acid, and is known to have an antioxidant effect. Further, with respect to the extract of Terminaria bellirica containing gallic acid as a main component, an effect of suppressing an increase in postprandial blood triglyceride in humans (Non-Patent Document 1) and an effect of suppressing an increase in blood glucose level in mice (Non-patent Document 1) Literature 2) has been reported, and it is thought that the gallic acid is involved in these actions. Therefore, gallic acid is attracting attention as a component expected to contribute to improvement of metabolic syndrome.
没食子酸の製造方法としては、化学合成による方法、天然物からの抽出による方法の他、酵素や発酵による方法が挙げられ、例えば、ガレート型カテキンにタンナーゼ処理を施し、その加水分解反応生成物として没食子酸を得る方法が知られている。また、前記ガレート型カテキンは茶や茶抽出物に多く含まれることが知られている。 Examples of the method for producing gallic acid include a method by chemical synthesis, a method by extraction from natural products, and a method by enzyme or fermentation.For example, a galenic catechin is subjected to tannase treatment, and as a hydrolysis reaction product thereof. Methods for obtaining gallic acid are known. In addition, it is known that the gallate-type catechin is contained in tea or tea extract in a large amount.
このような発酵に用いることが可能なタンナーゼ活性を有する菌としては、例えば、特表２００６−５２１８１７号公報（特許文献１）に、ヒト腸粘膜に付着する能力を有するタンナーゼ産生株としてLactobacillus plantarum種に属する乳酸菌が記載されている。しかしながら、かかる乳酸菌は前記タンナーゼ活性を有すると共に、没食子酸を脱炭酸する没食子酸脱炭酸酵素活性も有しているため、該タンナーゼ活性によって没食子酸が得られても前記没食子酸脱炭酸酵素活性によって脱炭酸されてピロガロールが生成してしまい、目的の没食子酸を効率的に得ることが困難であるという問題を有していた。また、例えば、特開２００９−１２４９４３号公報（特許文献２）には、タンナーゼ活性を有し、かつ、没食子酸脱炭酸酵素活性を有さない乳酸菌が記載されており、同文献には、前記乳酸菌を用いて没食子酸を含有する植物エキスを製造する方法も記載されている。 Examples of a bacterium having a tannase activity that can be used for such fermentation include, for example, Japanese Patent Application Laid-Open No. 2006-521817 (Patent Document 1) as a tannase-producing strain having the ability to adhere to human intestinal mucosa, Lactobacillus plantarum Lactic acid bacteria belonging to the group are described. However, such a lactic acid bacterium has the tannase activity and also has a gallic acid decarboxylase activity for decarboxylating gallic acid. Pyrogallol was generated by decarboxylation, and it was difficult to efficiently obtain the desired gallic acid. For example, Japanese Patent Application Laid-Open No. 2009-124943 (Patent Document 2) describes lactic acid bacteria having tannase activity and no gallic acid decarboxylase activity. A method for producing a plant extract containing gallic acid using lactic acid bacteria is also described.
しかしながら、特許文献２に記載の乳酸菌は、必ずしもタンナーゼ活性が強いものではないため、目的の没食子酸を十分に得ることが困難な場合がある。また、特許文献２に記載の方法に使用できる乳酸菌はイヌリン資化性を有する等、一部の菌株に限定されるために汎用性が低く、好ましい風味を奏する菌株を用いることができるとは限らない。 However, since the lactic acid bacteria described in Patent Document 2 do not always have strong tannase activity, it may be difficult to obtain the desired gallic acid sufficiently. In addition, lactic acid bacteria that can be used in the method described in Patent Literature 2 have inulin assimilation properties, and are limited to some strains. Therefore, versatility is low, and it is not always possible to use strains that exhibit favorable flavor. Absent.
本発明は、上記従来技術の有する課題に鑑みてなされたものであり、タンナーゼ活性及び没食子酸脱炭酸酵素活性を有する乳酸菌による発酵によって没食子酸を効率的に得ることができる没食子酸の製造方法を提供することを目的とする。 The present invention has been made in view of the above-mentioned problems of the related art, and provides a method for producing gallic acid capable of efficiently obtaining gallic acid by fermentation with lactic acid bacteria having tannase activity and gallic acid decarboxylase activity. The purpose is to provide.
本発明者らは、上記目的を達成すべく鋭意研究を行った結果、炭酸塩存在下において、タンナーゼ活性及び没食子酸脱炭酸酵素活性を有する乳酸菌をガレート型カテキンを含有する茶抽出物に接触させて発酵させることにより、没食子酸脱炭酸酵素活性が抑制されて、目的の没食子酸を効率よく容易に得られることを見い出した。また、かかる方法においては、タンナーゼ活性及び没食子酸脱炭酸酵素活性を有する乳酸菌であれば菌株に限定されず用いることができるため、汎用性及び応用性にも優れることを見い出した。 The present inventors have conducted intensive studies to achieve the above object, and as a result, in the presence of carbonate, contacting a lactic acid bacterium having tannase activity and gallic acid decarboxylase activity with a tea extract containing a gallate-type catechin. It has been found that the gallic acid decarboxylase activity is suppressed by fermentation, and the desired gallic acid can be obtained efficiently and easily. In addition, it has been found that in this method, any lactic acid bacterium having tannase activity and gallic acid decarboxylase activity can be used without being limited to strains, and therefore has excellent versatility and applicability.
すなわち、本発明は、
［１］没食子酸の製造方法であって、
炭酸塩と、ガレート型カテキンを含有する茶抽出物と、タンナーゼ活性及び没食子酸脱炭酸酵素活性を有する乳酸菌と、を含有する発酵ミックスを調製する工程と、
前記発酵ミックス中で前記茶抽出物を前記乳酸菌により発酵させて没食子酸を得る工程と、
を含む没食子酸の製造方法。
［２］前記乳酸菌がラクトバチルス属に属する乳酸菌である［１］に記載の没食子酸の製造方法。
［３］前記炭酸塩の濃度が前記発酵ミックス全量に対して０．１５ｗ／ｖ％以上である［１］又は［２］に記載の没食子酸の製造方法。
［４］茶発酵物の製造方法であって、
炭酸塩と、ガレート型カテキンを含有する茶抽出物と、タンナーゼ活性及び没食子酸脱炭酸酵素活性を有する乳酸菌と、を含有する発酵ミックスを調製する工程と、
前記発酵ミックス中で前記茶抽出物を前記乳酸菌により発酵させて没食子酸を含有する茶発酵物を得る工程と、
を含む茶発酵物の製造方法。
［５］前記茶発酵物におけるピロガロールの含有量が没食子酸の含有量１００質量部に対して５０質量部以下である［４］に記載の茶発酵物の製造方法。
［５１］前記乳酸菌がラクトバチルス属に属する乳酸菌である［４］又は［５］に記載の茶発酵物の製造方法。
［５２］前記炭酸塩の濃度が前記発酵ミックスの全量に対して０．１５ｗ／ｖ％以上である［４］、［５］又は［５１］に記載の茶発酵物の製造方法。
［６］［１］〜［３］のうちのいずれかに記載の没食子酸の製造方法、又は［４］〜［５］及び［５１］〜［５２］のうちのいずれかに記載の茶発酵物の製造方法に用いるための乳酸菌であり、タンナーゼ活性及び没食子酸脱炭酸酵素活性を有する乳酸菌。
［７］［１］〜［３］のうちのいずれかに記載の没食子酸の製造方法、又は［４］〜［５］及び［５１］〜［５２］のうちのいずれかに記載の茶発酵物の製造方法に用いるための乳酸菌組成物であり、［６］に記載の乳酸菌を含有する乳酸菌組成物。
［８］炭酸塩と、［６］に記載の乳酸菌、［７］に記載の乳酸菌組成物、及びこれらの処理物からなる群から選択される少なくとも１種と、没食子酸とを含有することを特徴とする茶発酵物。
［９］［８］に記載の茶発酵物を含有する飲食品。
［１０］ガレート型カテキンを含有する茶抽出物のタンナーゼ活性及び没食子酸脱炭酸酵素活性を有する乳酸菌による発酵において、炭酸塩を添加する、没食子酸脱炭酸酵素活性の抑制方法。
［１１］ガレート型カテキンを含有する茶抽出物のタンナーゼ活性及び没食子酸脱炭酸酵素活性を有する乳酸菌による発酵において、前記没食子酸脱炭酸酵素活性を抑制するための、炭酸塩の使用。
［１２］ガレート型カテキンを含有する茶抽出物のタンナーゼ活性及び没食子酸脱炭酸酵素活性を有する乳酸菌による発酵において、前記没食子酸脱炭酸酵素活性の抑制に使用される、炭酸塩。
を提供する。
That is, the present invention
[1] A method for producing gallic acid,
Carbonate, and a tea extract containing gallate-type catechin, and a lactic acid bacterium having tannase activity and gallic acid decarboxylase activity, and a step of preparing a fermentation mix containing
Fermenting the tea extract in the fermentation mix with the lactic acid bacteria to obtain gallic acid,
A method for producing gallic acid comprising:
[2] The method for producing gallic acid according to [1], wherein the lactic acid bacterium is a lactic acid bacterium belonging to the genus Lactobacillus.
[3] The method for producing gallic acid according to [1] or [2], wherein the concentration of the carbonate is 0.15 w / v% or more based on the total amount of the fermentation mix.
[4] A method for producing a fermented tea product,
Carbonate, and a tea extract containing gallate-type catechin, and a lactic acid bacterium having tannase activity and gallic acid decarboxylase activity, and a step of preparing a fermentation mix containing
A step of fermenting the tea extract in the fermentation mixture with the lactic acid bacteria to obtain a fermented tea product containing gallic acid,
A method for producing a fermented tea product comprising:
[5] The method for producing a fermented tea according to [4], wherein the content of pyrogallol in the fermented tea is 50 parts by mass or less based on 100 parts by mass of gallic acid.
[51] The method for producing a fermented tea product according to [4] or [5], wherein the lactic acid bacteria are lactic acid bacteria belonging to the genus Lactobacillus.
[52] The method for producing a fermented tea product according to [4], [5] or [51], wherein the concentration of the carbonate is 0.15 w / v% or more based on the total amount of the fermentation mix.
[6] The method for producing gallic acid according to any one of [1] to [3], or the tea fermentation according to any one of [4] to [5] and [51] to [52] A lactic acid bacterium for use in a method for producing a product, the lactic acid bacterium having tannase activity and gallic acid decarboxylase activity.
[7] The method for producing gallic acid according to any one of [1] to [3], or the tea fermentation according to any one of [4] to [5] and [51] to [52] A lactic acid bacterium composition for use in a method for producing a product, comprising the lactic acid bacterium according to [6].
[8] A composition comprising a carbonate, at least one selected from the group consisting of the lactic acid bacterium according to [6], the lactic acid bacterium composition according to [7], and a processed product thereof, and gallic acid. Characterized tea fermented product.
[9] A food or drink containing the fermented tea product according to [8].
[10] A method for suppressing gallic acid decarboxylase activity, comprising adding a carbonate in fermentation of a tea extract containing gallate-type catechin with lactic acid bacteria having tannase activity and gallic acid decarboxylase activity.
[11] Use of a carbonate to suppress the gallic acid decarboxylase activity in fermentation of a tea extract containing gallate-type catechin with lactic acid bacteria having tannase activity and gallic acid decarboxylase activity.
[12] A carbonate used for suppressing the gallic acid decarboxylase activity in fermentation of a tea extract containing gallate-type catechin with lactic acid bacteria having tannase activity and gallic acid decarboxylase activity.
I will provide a.
本発明によれば、タンナーゼ活性及び没食子酸脱炭酸酵素活性を有する乳酸菌による発酵によって没食子酸を効率的に得ることができる没食子酸の製造方法を提供することが可能となる。また、没食子酸を含有する茶発酵物の製造方法、これらの方法に用いる乳酸菌及び乳酸菌組成物、並びに、それによって得られる茶発酵物及び飲食品を提供することも可能となる。 ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to provide the manufacturing method of the gallic acid which can obtain gallic acid efficiently by fermentation with the lactic acid bacteria which have tannase activity and gallic acid decarboxylase activity. Further, it is also possible to provide a method for producing a fermented tea product containing gallic acid, lactic acid bacteria and a lactic acid bacteria composition used in these methods, and a fermented tea product and food and drink obtained by the method.
以下、本発明をその好適な実施形態に即して詳細に説明する。 Hereinafter, the present invention will be described in detail with reference to preferred embodiments.
＜没食子酸の製造方法、茶発酵物の製造方法＞
本発明の没食子酸の製造方法は、
炭酸塩と、ガレート型カテキンを含有する茶抽出物と、タンナーゼ活性及び没食子酸脱炭酸酵素活性を有する乳酸菌と、を含有する発酵ミックスを調製する工程と、
前記発酵ミックス中で前記茶抽出物を前記乳酸菌により発酵させて没食子酸を得る工程と、
を含む。また、本発明の茶発酵物の製造方法は、
炭酸塩と、ガレート型カテキンを含有する茶抽出物と、タンナーゼ活性及び没食子酸脱炭酸酵素活性を有する乳酸菌と、を含有する発酵ミックスを調製する工程と、
前記発酵ミックス中で前記茶抽出物を前記乳酸菌により発酵させて没食子酸を含有する茶発酵物を得る工程と、
を含む。
<Production method of gallic acid, production method of fermented tea>
The method for producing gallic acid of the present invention,
Carbonate, and a tea extract containing gallate-type catechin, and a lactic acid bacterium having tannase activity and gallic acid decarboxylase activity, and a step of preparing a fermentation mix containing
Fermenting the tea extract in the fermentation mix with the lactic acid bacteria to obtain gallic acid,
including. Further, the method for producing a fermented tea product of the present invention,
Carbonate, and a tea extract containing gallate-type catechin, and a lactic acid bacterium having tannase activity and gallic acid decarboxylase activity, and a step of preparing a fermentation mix containing
A step of fermenting the tea extract in the fermentation mixture with the lactic acid bacteria to obtain a fermented tea product containing gallic acid,
including.
（炭酸塩）
本発明においては、炭酸塩存在下において下記の茶抽出物を下記の乳酸菌で発酵させることにより、該乳酸菌の没食子酸脱炭酸酵素活性のみが抑制されて、目的の没食子酸を効率よく製造することが可能となる。前記炭酸塩によって前記没食子酸脱炭酸酵素活性のみが抑制される理由は必ずしも定かではないが、該炭酸塩を添加することで反応場が炭酸リッチ条件となって、二酸化炭素を遊離する脱炭酸反応が特異的に阻害されるためであると本発明者らは推察する。
(Carbonate)
In the present invention, by fermenting the following tea extract with the following lactic acid bacteria in the presence of carbonate, only the gallic acid decarboxylase activity of the lactic acid bacteria is suppressed, and the desired gallic acid is efficiently produced. Becomes possible. The reason why only the gallic acid decarboxylase activity is suppressed by the carbonate is not always clear, but by adding the carbonate, the reaction field becomes a carbonic acid-rich condition, and the decarboxylation reaction releasing carbon dioxide. The present inventors speculate that this is due to specific inhibition.
本発明において、炭酸塩とは、炭酸イオン（ＣＯ３ ２−）を含む化合物を示し、例えば、炭酸水素ナトリウム（重曹）、炭酸ナトリウム、炭酸水素カルシウム、炭酸カルシウム、炭酸マグネシウムが挙げられ、これらのうちの１種を単独であっても２種以上の組み合わせであってもよい。これらの中でも、本発明に係る炭酸塩としては、没食子酸脱炭酸酵素活性の抑制効果により優れる傾向にある観点から、炭酸水素ナトリウムであることが好ましい。 In the present invention, the carbonate, showed a compound containing a carbonate ion (CO 3 2-), for example, sodium bicarbonate (baking soda), sodium carbonate, calcium hydrogen carbonate, calcium carbonate, magnesium carbonate and the like, these One of them may be used alone or a combination of two or more may be used. Among them, the carbonate according to the present invention is preferably sodium hydrogencarbonate from the viewpoint that it tends to be more excellent in the effect of suppressing gallic acid decarboxylase activity.
（茶抽出物）
本発明において、茶抽出物とは、茶から抽出された混合物を示し、より具体的には、茶（例えば、Camellia siensis var. sinensis、Camellia siensis var.assamica、やぶきた種等のCamellia属）の葉及び／又は茎等から、抽出溶媒で抽出して得られる混合物を示す。前記茶の葉及び茎には、生の葉及び茎の他、煎茶、番茶、玉露、てん茶等の緑茶；鳥龍茶と呼ばれる半醗酵茶；紅茶と呼ばれる醗酵茶等の製茶も含まれ、前記茶の葉及び茎としてはこれらのうちの１種を単独であっても２種以上の組み合わせであってもよい。また、前記抽出溶媒としては、水（温水及び熱水を含む）、有機溶媒（例えば、エタノール等の水性有機溶媒）等が挙げられ、これらのうちの１種を単独であっても２種以上の組み合わせであってもよい。前記抽出の方法及び条件は、使用する茶の種類や前記抽出溶媒の種類等に応じて適宜決定できる。
(Tea extract)
In the present invention, the tea extract refers to a mixture extracted from tea, and more specifically, tea (for example, Camellia genus such as Camellia siensis var. Sinensis, Camellia siensis var. Fig. 3 shows a mixture obtained by extracting a leaf and / or stem from an extraction solvent. The tea leaves and stems include, in addition to raw leaves and stems, green tea such as sencha, bancha, gyokuro and tencha; semi-fermented tea called bird dragon tea; and fermented tea such as black tea and fermented tea. One of these tea leaves and stems may be used alone or in combination of two or more. Examples of the extraction solvent include water (including hot water and hot water), and organic solvents (for example, aqueous organic solvents such as ethanol). May be combined. The extraction method and conditions can be appropriately determined according to the type of tea used, the type of the extraction solvent, and the like.
本発明に係る茶抽出物としては、前記混合物を適宜公知の方法又はそれに準じた方法で濃縮・精製したものであってもよく、また、「サンフェノンＢＧ−５（太陽化学株式会社製）」、「サンフェノンＢＧ−３（太陽化学株式会社製）」、「カメリアエキス３０Ｓ（太陽化学株式会社製）」等の市販のものを適宜用いてもよい。 The tea extract according to the present invention may be a mixture obtained by concentrating and purifying the mixture by a known method or a method analogous thereto as appropriate, or “Sanphenone BG-5 (manufactured by Taiyo Chemical Co., Ltd.)” Commercially available products such as "Sanphenon BG-3 (manufactured by Taiyo Kagaku Co., Ltd.)" and "Camelia Extract 30S (manufactured by Taiyo Kagaku Co., Ltd.)" may be used as appropriate.
本発明に係る茶抽出物は、ガレート型カテキンを含有する。ガレート型カテキンとは、ガロイル基を有するカテキン類であり、例えば、エピガロカテキンガレート（ＥＧＣｇ）、エピカテキンガレート（ＥＣｇ）、カテキンガレート（Ｃｇ）、ガロカテキンガレート（ＧＣｇ）等が挙げられ、これらのうちの１種を単独であっても２種以上の組み合わせであってもよい。前記茶抽出物における前記ガレート型カテキンの含有量としては、特に制限されないが、乾燥固体質量換算で、３．０〜８．０質量％であることが好ましく、４．０〜７．０質量％であることがより好ましい。 The tea extract according to the present invention contains gallate-type catechin. The gallate-type catechin is a catechin having a galloyl group, for example, epigallocatechin gallate (EGCg), epicatechin gallate (ECg), catechin gallate (Cg), gallocatechin gallate (GCg), and the like. May be used alone or in combination of two or more. The content of the gallate-type catechin in the tea extract is not particularly limited, but is preferably 3.0 to 8.0% by mass, and preferably 4.0 to 7.0% by mass in terms of dry solid mass. Is more preferable.
本発明に係る茶抽出物としては、本発明の効果を阻害しない範囲内において、前記ガレート型カテキンの他に、さらに茶由来のその他成分を含有していてもよく、例えば、糖類、糖アルコール類、ミネラル類、ビタミン類、タンパク質、ペプチド、アミノ酸類、有機酸のうちの１種を単独で又は２種以上を組み合わせて含有していてよい。 As long as the tea extract according to the present invention does not impair the effects of the present invention, in addition to the gallate-type catechin, it may further contain other components derived from tea, for example, sugars, sugar alcohols , Minerals, vitamins, proteins, peptides, amino acids, and organic acids may be used alone or in combination of two or more.
（乳酸菌）
本発明の乳酸菌は、本発明の没食子酸の製造方法又は茶発酵物の製造方法に用いるための乳酸菌であり、タンナーゼ活性及び没食子酸脱炭酸酵素活性を有する。前記タンナーゼ活性及び前記没食子酸脱炭酸酵素活性をいずれも有することにより、タンナーゼ活性による没食子酸生成作用及び前記炭酸塩による没食子酸脱炭酸酵素活性抑制作用がいずれも十分に機能して特に効率よく没食子酸を製造することができる。
(Lactic acid bacteria)
The lactic acid bacterium of the present invention is a lactic acid bacterium used for the method of producing gallic acid or the method of producing a fermented tea product of the present invention, and has tannase activity and gallic acid decarboxylase activity. By having both the tannase activity and the gallic acid decarboxylase activity, both the gallic acid generating action by the tannase activity and the gallic acid decarboxylase activity inhibitory action by the carbonate function sufficiently and the gallic acid particularly efficiently. Acids can be produced.
本発明の乳酸菌はタンナーゼ活性及び没食子酸脱炭酸酵素活性をいずれも有するため、該乳酸菌によれば、通常は、下記の式（１）で示されるタンナーゼ活性及び式（２）で示される没食子酸脱炭酸酵素活性による反応が進行する。なお、下記式（１）中、Ｒは水素原子、水酸基等を示す。 Since the lactic acid bacterium of the present invention has both a tannase activity and a gallic acid decarboxylase activity, according to the lactic acid bacterium, usually, a tannase activity represented by the following formula (1) and a gallic acid represented by the following formula (2): The reaction by the decarboxylase activity proceeds. In the following formula (1), R represents a hydrogen atom, a hydroxyl group, or the like.
〔タンナーゼ活性〕
本発明において、タンナーゼ活性とは、上記式（１）に示される、ガレート型カテキンのエステル結合を加水分解する反応を触媒する活性である。前記ガレート型カテキンのエステル結合が加水分解されると、通常、ガレート型カテキンが非ガレート型カテキンに変換されるとともに、没食子酸が生成する。
(Tannase activity)
In the present invention, the tannase activity is an activity that catalyzes a reaction of hydrolyzing an ester bond of gallate-type catechin represented by the above formula (1). When the ester bond of the gallate-type catechin is hydrolyzed, gallate-type catechin is usually converted to non-gallate-type catechin, and gallic acid is generated.
本発明において、前記乳酸菌が前記タンナーゼ活性を有することは、例えば、Ｎｉｓｈｉｔａｎｉ Ｙ．ら、２００３年、Ｊｏｕｒｎａｌ ｏｆ Ｍｉｃｒｏｂｉｏｌｏｇｉｃａｌ Ｍｅｔｈｏｄｓ ５４、ｐ．２８１−２８４に記載の方法に準じた方法によって確認することができる。より具体的には、５ｍＭ没食子酸メチル（基質）を含むリン酸水素二ナトリウム（好ましくは、３３ｍＭ）水溶液（好ましくは、ｐＨ５．０）に対象の乳酸菌又はそれを含有する乳酸菌組成物を添加し、３０℃において好気条件で２４時間反応させた後の反応液中の没食子酸（反応生成物）を検出する方法が挙げられる。また、前記タンナーゼ活性の強さは、例えば、上記のＮｉｓｈｉｔａｎｉ Ｙ．らに記載の方法に準じた方法によって定量することができ、より具体的には、前記反応後の反応液の上清について、アルカリ条件下（好ましくは、ｐＨ８．６）における没食子酸に由来する呈色（緑色〜茶色）の吸光度を、分光光度計を用いて波長４５０ｎｍで測定し、例えば、Aspergillus ficuumによる検量線を用いて算出することによって定量することができる。 In the present invention, the fact that the lactic acid bacterium has the tannase activity is described in, for example, Nishitani Y. et al. Et al., 2003, Journal of Microbiological Methods 54, p. It can be confirmed by a method according to the method described in 281 to 284. More specifically, a target lactic acid bacterium or a lactic acid bacterium composition containing the same is added to an aqueous solution (preferably, pH 5.0) of disodium hydrogen phosphate (preferably 33 mM) containing 5 mM methyl gallate (substrate). And a method of detecting gallic acid (reaction product) in a reaction solution after reaction at 30 ° C. under aerobic conditions for 24 hours. Further, the strength of the tannase activity can be determined, for example, by the method described in Nishitani Y. Can be quantified by a method according to the method described in the above, and more specifically, the supernatant of the reaction solution after the reaction is derived from gallic acid under alkaline conditions (preferably, pH 8.6). The absorbance of the color (green to brown) can be quantified by measuring the absorbance at a wavelength of 450 nm using a spectrophotometer and calculating using, for example, a calibration curve by Aspergillus ficuum.
本発明の乳酸菌が有するタンナーゼ活性の強さとしては、上記の方法で定量したときの強さで、３０ｍＵ／ｍＬ以上であることが好ましく、５０ｍＵ／ｍＬ以上であることがより好ましい。前記タンナーゼ活性の強さの上限としては特に限定されないが、例えば、１５０ｍＵ／ｍＬ以下であることが好ましい。また、前記タンナーゼ活性の強さとしては、生菌数１×１０７ｃｆｕあたりで、４ｍＵ以上であることが好ましく、５ｍＵ以上であることがより好ましく、６ｍＵ以上であることがさらに好ましく、８ｍＵ以上であることがさらにより好ましい。前記生菌数１×１０７ｃｆｕあたりのタンナーゼ活性の強さの上限としても特に限定されないが、例えば、１５ｍＵ以下であることが好ましい。前記タンナーゼ活性の強さが前記下限未満であると、十分な量の没食子酸を得ることが困難となる傾向にある。 The strength of the tannase activity possessed by the lactic acid bacterium of the present invention is preferably 30 mU / mL or more, more preferably 50 mU / mL or more, as determined by the above method. The upper limit of the intensity of the tannase activity is not particularly limited, but is preferably, for example, 150 mU / mL or less. In addition, the intensity of the tannase activity is preferably 4 mU or more, more preferably 5 mU or more, still more preferably 6 mU or more, and more preferably 8 mU or more per 1 × 10 7 cfu of viable cells. Is even more preferred. The upper limit of the intensity of tannase activity per 1 × 10 7 cfu of viable cells is not particularly limited, but is preferably, for example, 15 mU or less. When the intensity of the tannase activity is less than the lower limit, it tends to be difficult to obtain a sufficient amount of gallic acid.
〔没食子酸脱炭酸酵素活性〕
本発明において、没食子酸脱炭酸酵素活性とは、上記式（２）に示される、没食子酸を脱炭酸してピロガロールを生成する活性である。ピロガロールは、１，２，３−トリヒドロキシベンゼン（1,2,3-Trihydroxybenzene）ともいう。
(Gallic acid decarboxylase activity)
In the present invention, the gallic acid decarboxylase activity is the activity of decarboxylating gallic acid to form pyrogallol, as shown in the above formula (2). Pyrogallol is also referred to as 1,2,3-trihydroxybenzene.
本発明において、前記乳酸菌が前記没食子酸脱炭酸酵素活性を有することは、例えば、特開平１１−５６３８号公報に記載の方法に準じた方法によって確認することができる。より具体的には、８０ｍＭ没食子酸（基質）を含むリン酸カリウム（好ましくは、０．１Ｍ）緩衝液（好ましくは、ｐＨ６．０）に対象の乳酸菌又はそれを含有する乳酸菌組成物を添加し、３０℃において好気条件で３０分間反応させた後の反応液中のピロガロール（反応生成物）を検出する方法が挙げられる。また、前記没食子酸脱炭酸酵素活性の強さは、例えば、前記反応後の反応液の上清について、高速液体クロマトグラフ（ＨＰＬＣ）を用いて生成したピロガロール量を測定し、１分間に１μｍｏｌのピロガロールを生成させることができる酵素量を１Ｕとして、定量することができる。 In the present invention, the fact that the lactic acid bacterium has the gallic acid decarboxylase activity can be confirmed, for example, by a method according to the method described in JP-A-11-5638. More specifically, a lactic acid bacterium of interest or a lactic acid bacterium composition containing the same is added to a potassium phosphate (preferably 0.1 M) buffer (preferably pH 6.0) containing 80 mM gallic acid (substrate). And a method of detecting pyrogallol (reaction product) in a reaction solution after reaction at 30 ° C. under aerobic conditions for 30 minutes. Further, the strength of the gallic acid decarboxylase activity can be determined, for example, by measuring the amount of pyrogallol generated on the supernatant of the reaction solution after the reaction using high performance liquid chromatography (HPLC), and measuring 1 μmol / min. Assuming that the amount of enzyme capable of producing pyrogallol is 1 U, it can be quantified.
本発明の乳酸菌が有する没食子酸脱炭酸酵素活性の強さとしては、上記の方法で定量したときの強さで、１５０ｍＵ／ｍＬ以上であることが好ましく、３００ｍＵ／ｍＬ以上であることがより好ましく、３５０ｍＵ／ｍＬ以上であることがさらに好ましく、４００ｍＵ／ｍＬ以上であることが特に好ましい。前記没食子酸脱炭酸酵素活性の強さの上限としては特に限定されないが、例えば、６００ｍＵ／ｍＬ以下であることが好ましい。また、前記没食子酸脱炭酸酵素活性の強さとしては、生菌数１×１０７ｃｆｕあたりで、０．５ｍＵ以上であることが好ましく、１．０ｍＵ以上であることがより好ましく、１．５ｍＵ以上であることがさらに好ましく、２．０ｍＵ以上であることがさらにより好ましい。前記生菌数１×１０７ｃｆｕあたりの没食子酸脱炭酸酵素活性の強さの上限としても特に限定されないが、３．５ｍＵ以下であることが好ましい。前記没食子酸脱炭酸酵素活性の強さが前記下限未満であると、タンナーゼ活性も低下したり、前記炭酸塩による没食子酸脱炭酸酵素活性の抑制作用が十分に機能しなかったりして得られる没食子酸の収量が逆に少なくなる場合がある。 The strength of the gallic acid decarboxylase activity possessed by the lactic acid bacterium of the present invention is preferably at least 150 mU / mL, more preferably at least 300 mU / mL, as determined by the above method. , 350 mU / mL or more, and particularly preferably 400 mU / mL or more. The upper limit of the gallic acid decarboxylase activity is not particularly limited, but is preferably, for example, 600 mU / mL or less. Further, the intensity of the gallic acid decarboxylase activity is preferably 0.5 mU or more, more preferably 1.0 mU or more, and more preferably 1.5 mU per 1 × 10 7 cfu of viable bacteria. More preferably, it is still more preferably 2.0 mU or more. The upper limit of the gallic acid decarboxylase activity per 1 × 10 7 cfu of the viable bacteria is not particularly limited, but it is preferably 3.5 mU or less. When the intensity of the gallic acid decarboxylase activity is less than the lower limit, tannase activity is reduced, and the gallic acid obtained due to insufficient inhibition of the gallic acid decarboxylase activity by the carbonate does not function. Conversely, the acid yield may be reduced.
本発明の没食子酸の製造方法及び茶発酵物の製造方法においては、前記タンナーゼ活性及び前記没食子酸脱炭酸酵素活性を有する乳酸菌であれば特に制限なく用いることができる。本発明の乳酸菌には、ラクトバチルス属（Lactobacillus）、ワイセラ属（Weissella）等の乳酸桿菌；ペディオコッカス属（Pediococcus）、ロイコノストック属（Leuconostoc）、ラクトコッカス属（Lactococcus）、ストレプトコッカス属（Streptococcus）、エンテロコッカス属（Enterococcus）等の乳酸球菌；ビフィドバクテリウム属（Bifidobacterium）等が含まれる。本発明の没食子酸の製造方法及び茶発酵物の製造方法においては、本発明の乳酸菌として、これらのうちの１種を単独で用いても２種以上の組み合わせて用いてもよいが、これらの中でも、本発明の乳酸菌としては、風味がより良好な生成物（発酵物、茶発酵物、飲食品）が得られやすい観点からは、ラクトバチルス属に属する乳酸菌であることが好ましい。 In the method for producing gallic acid and the method for producing a fermented tea product of the present invention, any lactic acid bacteria having the tannase activity and the gallic acid decarboxylase activity can be used without particular limitation. The lactic acid bacteria of the present invention include lactobacilli such as Lactobacillus and Weissella; Pediococcus, Leuconostoc, Lactococcus, and Streptococcus ( Lactococci such as Streptococcus and Enterococcus; Bifidobacterium and the like. In the method for producing gallic acid and the method for producing a fermented tea product of the present invention, the lactic acid bacteria of the present invention may be used alone or in combination of two or more. Among them, the lactic acid bacterium of the present invention is preferably a lactic acid bacterium belonging to the genus Lactobacillus from the viewpoint of easily obtaining a product having a better flavor (fermented product, fermented tea product, food or drink).
前記ラクトバチルス属に属する乳酸菌としては、例えば、ラクトバチルス・ブルガリカス（Lactobacillus bulgaricus）、ラクトバチルス・ジョンソニ（L.johnsonii）、ラクトバチルス・カゼイ（L.casei）、ラクトバチルス・パラカゼイ（L.paracasei）、ラクトバチルス・ガセリ（L.gasseri）、ラクトバチルス・ヘルベティカス（L.helveticus）、ラクトバチルス・ラムノーサス（L.rhamnosus）、ラクトバチルス・アシドフィラス（L.acidophilus）、ラクトバチルス・サリバリウス（L.salivarius）、ラクトバチルス・ペントーサス（L.pentosus）、ラクトバチルス・プランタラム（L.plantarum）、ラクトバチルス・ブレビス（L.brevis）、ラクトバチルス・ロイテリ（L.reuteri）、ラクトバチルス・スポロゲネス（L.sporogenes）が挙げられる。これらの中でも、本発明の乳酸菌としては、風味がより良好な生成物（発酵物、茶発酵物、飲食品）が得られやすい観点からは、ラクトバチルス・ペントーサス及びラクトバチルス・プランタラムのうちの少なくとも１種であることが好ましく、ラクトバチルス・ペントーサスであることがより好ましい。 Examples of the lactic acid bacteria belonging to the genus Lactobacillus include, for example, Lactobacillus bulgaricus, Lactobacillus johnsonii (L. johnsonii), Lactobacillus casei (L. casei), and Lactobacillus paracasei (L. paracasei). ), L. gasseri, L. helveticus, L. rhamnosus, L. acidophilus, L. salivarius ), Lactobacillus pentosus (L. pentosus), Lactobacillus plantarum (L. plantarum), Lactobacillus brevis (L. brevis), Lactobacillus reuteri (L. reuteri), Lactobacillus sporogenes (L. sporogenes). Among them, as the lactic acid bacteria of the present invention, from the viewpoint of easily obtaining a product having a better flavor (fermented product, fermented tea product, food and drink), the lactic acid bacterium of the Lactobacillus pentosus and Lactobacillus plantarum is preferred. It is preferably at least one kind, more preferably Lactobacillus pentosus.
前記ラクトバチルス・ペントーサスとしては、例えばラクトバチルス・ペントーサス ＯＬＬ２０３９６９株（受託番号：ＮＩＴＥ ＢＰ−０１９８６）、ラクトバチルス・ペントーサス ＯＬＬ２０３９８２株（受託番号：ＮＩＴＥ ＢＰ−０１９８７）、ラクトバチルス・ペントーサス ＯＬＬ２０３９８４株（受託番号：ＮＩＴＥ ＢＰ−０１９８８）が挙げられ［前記受託番号とは、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における各菌株の受託番号を示す。以下同様］、これらの中でも、ラクトバチルス・ペントーサス ＯＬＬ２０３９８４株（以下、場合により「ＯＬＬ２０３９８４株」という）及びラクトバチルス・ペントーサス ＯＬＬ２０３９８２株（以下、場合により「ＯＬＬ２０３９８２株」という）のうちの少なくとも１種であることが好ましく、ＯＬＬ２０３９８４株が特に好ましい。 As the Lactobacillus pentosus, for example, Lactobacillus pentosus OLL203969 strain (Accession number: NITE BP-01986), Lactobacillus pentosus OLL203982 strain (Accession number: NITE BP-01987), and Lactobacillus pentosus OLL20398 strain (Accession number) : NITE BP-01988) [The accession number indicates the accession number of each strain at the Patent Organism Depositary of the National Institute of Technology and Evaluation, National Institute of Technology and Evaluation. The same applies hereinafter), and among these, at least one of Lactobacillus pentosus OLL203984 strain (hereinafter, sometimes referred to as “OLL203984 strain”) and Lactobacillus pentosus OLL203982 strain (hereinafter, sometimes referred to as “OLL203982 strain”) is used. Is preferred, and the OLL203984 strain is particularly preferred.
なお、ＯＬＬ２０３９８４株において、上記の方法で定量される前記タンナーゼ活性の強さは７７．９８ｍＵ／ｍＬ、生菌数１×１０７ｃｆｕあたりで９．２８ｍＵであり、前記没食子酸脱炭酸酵素活性の強さは４６６ｍＵ／ｍＬ、生菌数１×１０７ｃｆｕあたりで２．７４ｍＵであった。また、ＯＬＬ２０３９８２株において、上記の方法で定量される前記タンナーゼ活性の強さは８５．２９ｍＵ／ｍＬ、生菌数１×１０７ｃｆｕあたりで５．８４ｍＵであり、前記没食子酸脱炭酸酵素活性の強さは３８６ｍＵ／ｍＬ、生菌数１×１０７ｃｆｕあたりで１．３６ｍＵであった。 In the OLL203984 strain, the intensity of the tannase activity determined by the above method is 77.98 mU / mL, 9.28 mU per 1 × 10 7 cfu of viable bacteria, and the gallic acid decarboxylase activity is The strength was 466 mU / mL, and 2.74 mU per 1 × 10 7 cfu of viable cells. In the OLL203982 strain, the intensity of the tannase activity determined by the above method was 85.29 mU / mL, 5.84 mU per 1 × 10 7 cfu of viable cells, and the gallic acid decarboxylase activity was The strength was 386 mU / mL, and 1.36 mU per 1 × 10 7 cfu of viable cells.
さらに、本発明の乳酸菌には、前記タンナーゼ活性及び前記没食子酸脱炭酸酵素活性を有しており、かつ、ＯＬＬ２０３９８４株又はＯＬＬ２０３９８２株の、継代株、人工変異株、自然変異株、又は遺伝子組み換え株である株も含まれ、本発明の没食子酸の製造方法及び茶発酵物の製造方法に好適に用いることができる。 Further, the lactic acid bacterium of the present invention has the tannase activity and the gallic acid decarboxylase activity, and is a subcultured strain, an artificial mutant strain, a natural mutant strain, or a genetically modified strain of the OLL203984 strain or the OLL203982 strain. A strain which is a strain is also included, and can be suitably used in the method for producing gallic acid and the method for producing a fermented tea product of the present invention.
本発明の乳酸菌の培養方法としては、特に制限されず、前記乳酸菌の種類に応じて、適宜公知の方法又はそれに準じた方法で培養して所望の菌数に調整することができる。 The method of culturing the lactic acid bacterium of the present invention is not particularly limited, and the lactic acid bacterium can be cultivated by a known method or a method equivalent thereto according to the type of the lactic acid bacterium to adjust the number of the desired bacterium.
（発酵）
本発明の没食子酸の製造方法及び茶発酵物の製造方法においては、前記炭酸塩存在下において、前記茶抽出物を前記乳酸菌により発酵させることにより、前記没食子酸脱炭酸酵素活性のみが抑制されて、目的の没食子酸を効率的に製造できる。本発明においては、先ず、前記炭酸塩、前記茶抽出物、及び前記乳酸菌を含有する発酵ミックスを調製する。
(fermentation)
In the method for producing gallic acid and the method for producing a fermented tea product of the present invention, in the presence of the carbonate, by fermenting the tea extract with the lactic acid bacterium, only the gallic acid decarboxylase activity is suppressed. The desired gallic acid can be produced efficiently. In the present invention, first, a fermentation mix containing the carbonate, the tea extract, and the lactic acid bacterium is prepared.
前記発酵ミックスとしては、水溶液であることが好ましい。前記水溶液の溶媒としては、水、リン酸緩衝液、生理食塩水、液体培地、一般に抽出した茶等が挙げられ、これらのうちの１種を単独であっても２種以上の組み合わせであってもよい。これらの中でも、発酵後の発酵ミックス（発酵物）をそのまま茶発酵物として利用可能であり、かつ、没食子酸の収率がより高くなる傾向にあるという観点からは、水であることが好ましい。 The fermentation mix is preferably an aqueous solution. Examples of the solvent for the aqueous solution include water, phosphate buffer, physiological saline, liquid culture media, and generally extracted tea. One of these alone or a combination of two or more thereof may be used. Is also good. Among these, water is preferred from the viewpoint that the fermented mix (fermented product) after fermentation can be used as it is as a tea fermented product, and the yield of gallic acid tends to be higher.
前記発酵ミックスにおける前記炭酸塩の濃度としては、発酵ミックス全量の体積に対して０．１５ｗ／ｖ％以上であることが好ましく、０．２ｗ／ｖ％以上であることがより好ましく、０．３ｗ／ｖ％以上であることがさらに好ましく、０．４ｗ／ｖ％以上であることがさらにより好ましい。前記炭酸塩の濃度の上限としては、０．８０ｗ／ｖ％以下であることが好ましく、０．７ｗ／ｖ％以下であることがより好ましく、０．６ｗ／ｖ％以下であることがさらに好ましい。このような炭酸塩の濃度の好ましい範囲としては、例えば、０．１５〜０．８ｗ／ｖ％、０．２〜０．７ｗ／ｖ％、０．３〜０．７ｗ／ｖ％、０．４〜０．６ｗ／ｖ％である。前記炭酸塩の濃度が前記下限未満であると、没食子酸脱炭酸酵素活性の抑制効果が低下して没食子酸を効率的に得ることが困難となる傾向にあり、他方、前記上限を超えると、得られる生成物（発酵物、茶発酵物、飲食品）の風味が著しく悪化する傾向にある。なお、本発明において、「ｗ／ｖ％」とは、全体積１００ｍＬに対する質量（ｇ）の割合を示す。 The concentration of the carbonate in the fermentation mix is preferably at least 0.15 w / v%, more preferably at least 0.2 w / v%, and more preferably at least 0.3 w / v%, based on the total volume of the fermentation mix. / V% or more, and even more preferably 0.4 w / v% or more. The upper limit of the concentration of the carbonate is preferably 0.80 w / v% or less, more preferably 0.7 w / v% or less, and even more preferably 0.6 w / v% or less. . The preferred range of the concentration of such a carbonate is, for example, 0.15 to 0.8 w / v%, 0.2 to 0.7 w / v%, 0.3 to 0.7 w / v%, 0.1 to 0.7 w / v%. 4 to 0.6 w / v%. When the concentration of the carbonate is less than the lower limit, the effect of suppressing gallic acid decarboxylase activity tends to decrease and it becomes difficult to efficiently obtain gallic acid.On the other hand, when the concentration exceeds the upper limit, The flavor of the resulting product (fermented product, fermented tea product, food or drink) tends to be significantly deteriorated. In addition, in this invention, "w / v%" shows the ratio of the mass (g) with respect to 100 mL of total volume.
前記発酵ミックスに含有される前記茶抽出物の形態としては、乾燥粉末等の固体；濃縮物、希釈物等の水溶液；スラリー状のいずれも形態であってもよい。かかる茶抽出物の濃度としては、特に制限されないが、発酵ミックス全量の体積に対して、ガレート型カテキンの含有量で、０．００５〜１．０ｗ／ｖ％となる濃度であることが好ましく、０．０１〜０．７０ｗ／ｖ％となる濃度であることがより好ましく、０．０２〜０．５０ｗ／ｖ％となる濃度であることがさらに好ましい。前記茶抽出物の濃度が前記下限未満であると、基質量が少なくなって没食子酸を十分に得ることが困難となる傾向にあり、他方、前記上限を超えると、没食子酸脱炭酸酵素活性の抑制効果が低下して没食子酸を効率的に得ることが困難となる傾向にある。前記茶抽出物の濃度としては、前記ガレート型カテキンの含有量で、得られる生成物（発酵物、茶発酵物、飲食品）の風味の観点からは０．０２〜０．２０ｗ／ｖ％となる濃度であることが特に好ましく、より没食子酸を効率的に得る観点からは０．１０〜０．５０ｗ／ｖ％となる濃度であることが特に好ましい。なお、本発明において、前記発酵ミックスの溶媒に上記の一般に抽出した茶が含まれる場合、前記茶抽出物の濃度（ガレート型カテキンの含有量）には、かかる茶に由来するガレート型カテキンの含有量も含む。 The form of the tea extract contained in the fermentation mix may be a solid such as a dry powder; an aqueous solution such as a concentrate or a diluent; or a slurry. The concentration of the tea extract is not particularly limited, but is preferably a concentration of 0.005 to 1.0 w / v% in terms of the content of gallate-type catechin with respect to the volume of the entire fermentation mix, The concentration is more preferably 0.01 to 0.70 w / v%, and still more preferably 0.02 to 0.50 w / v%. When the concentration of the tea extract is less than the lower limit, it tends to be difficult to sufficiently obtain gallic acid with a reduced base mass, while, when the concentration exceeds the upper limit, the gallic acid decarboxylase activity is reduced. There is a tendency that it is difficult to obtain gallic acid efficiently because the inhibitory effect is reduced. As the concentration of the tea extract, the content of the gallate-type catechin is 0.02 to 0.20 w / v% from the viewpoint of the flavor of the obtained product (fermented product, fermented tea product, food and drink). Is particularly preferable, and from the viewpoint of obtaining gallic acid more efficiently, the concentration is particularly preferably 0.10 to 0.50 w / v%. In the present invention, when the above-mentioned commonly extracted tea is contained in the solvent of the fermentation mix, the concentration of the tea extract (the content of gallate-type catechin) includes the content of gallate-type catechin derived from such tea. Including quantity.
前記発酵ミックスに含有される前記乳酸菌としては、該乳酸菌を含有する乳酸菌組成物の形態であってもよい。本発明において、前記乳酸菌組成物には、乳酸菌の培養終了後の培養上清、培地成分等である培養物；前記培養物の濃縮物、希釈物、乾燥物、凍結物等が含まれ、これらのうちの１種を単独であっても２種以上の組み合わせであってもよい。これらの中でも、本発明の乳酸菌としては、前記乳酸菌組成物の形態、より好ましくは、培養物又は培養物の濃縮物の形態で前記発酵ミックスに含有されることが好ましい。また、前記発酵ミックスに含有される前記乳酸菌としては、適宜公知の方法で賦活培養した乳酸菌又は乳酸菌組成物を用いてもよい。 The lactic acid bacteria contained in the fermentation mix may be in the form of a lactic acid bacteria composition containing the lactic acid bacteria. In the present invention, the lactic acid bacteria composition includes a culture supernatant, a culture medium component, and the like after completion of the culture of the lactic acid bacteria; a concentrate, a dilution, a dried product, a frozen product, and the like of the culture. May be used alone or in combination of two or more. Among these, the lactic acid bacteria of the present invention are preferably contained in the fermentation mix in the form of the lactic acid bacteria composition, more preferably in the form of a culture or a concentrate of the culture. In addition, as the lactic acid bacteria contained in the fermentation mix, lactic acid bacteria or a lactic acid bacteria composition that has been appropriately activated and cultured by a known method may be used.
前記発酵ミックスにおける前記乳酸菌の濃度としては、特に制限されないが、発酵ミックス全量の体積に対して、乳酸菌数換算で、１．０×１０８〜１．０×１０１０ｃｆｕ／ｍＬであることが好ましく、５．０×１０８〜１．０×１０１０ｃｆｕ／ｍＬであることがより好ましい。前記乳酸菌の濃度が前記下限未満であると、反応量が少なくなって没食子酸を十分に得ることが困難となる傾向にあり、他方、前記上限を超えると、炭酸塩による没食子酸脱炭酸酵素活性の抑制効果が低下して没食子酸を効率的に得ることが困難となる傾向にある。また、前記乳酸菌の濃度としては、得られる生成物（発酵物、茶発酵物、飲食品）の風味の観点からは５．０×１０８〜１．０×１０９ｃｆｕ／ｍＬであることが特に好ましく、より没食子酸を効率的に得る観点からは１．０×１０９〜５．０×１０９ｃｆｕ／ｍＬであることが特に好ましい。 The concentration of the lactic acid bacteria in the fermentation mix is not particularly limited, but may be 1.0 × 10 8 to 1.0 × 10 10 cfu / mL, in terms of the number of lactic acid bacteria, based on the total volume of the fermentation mix. More preferably, it is 5.0 × 10 8 to 1.0 × 10 10 cfu / mL. When the concentration of the lactic acid bacterium is less than the lower limit, the amount of reaction tends to be small and it is difficult to sufficiently obtain gallic acid. This tends to reduce the effect of suppressing galvanic acid, making it difficult to obtain gallic acid efficiently. In addition, the concentration of the lactic acid bacterium may be from 5.0 × 10 8 to 1.0 × 10 9 cfu / mL from the viewpoint of the flavor of the obtained product (fermented product, fermented tea product, food or drink). From the viewpoint of obtaining gallic acid more efficiently, it is particularly preferably from 1.0 × 10 9 to 5.0 × 10 9 cfu / mL.
また、前記発酵ミックスのｐＨとしては、前記乳酸菌の生育条件、前記タンナーゼ活性及び前記没食子酸脱炭酸酵素活性の至適条件、前記発酵ミックスの量等に応じて適宜選択することができるが、例えば、ｐＨ４．８〜８．３であることが好ましく、ｐＨ５．３〜７．８であることがより好ましい。前記ｐＨの範囲が前記範囲を外れると、タンナーゼ活性が低下して得られる没食子酸量が減少する傾向にある。 The pH of the fermentation mix can be appropriately selected according to the growth conditions of the lactic acid bacteria, the optimal conditions for the tannase activity and the gallic acid decarboxylase activity, and the amount of the fermentation mix. , PH 4.8 to 8.3, more preferably pH 5.3 to 7.8. If the pH range is outside the above range, the amount of gallic acid obtained due to a decrease in tannase activity tends to decrease.
前記発酵ミックスとしては、本発明の効果を阻害しない範囲内において、前記炭酸塩、前記茶抽出物、前記乳酸菌、及び前記溶媒の他に、さらにその他成分を含有していてもよく、例えば、糖類、糖アルコール類、ミネラル類、ビタミン類、タンパク質、ペプチド、アミノ酸類、有機酸、ｐＨ調整剤のうちの１種を単独で又は２種以上を組み合わせて含有していてよい。 As long as the effect of the present invention is not impaired, the fermentation mix may further contain other components in addition to the carbonate, the tea extract, the lactic acid bacterium, and the solvent. , Sugar alcohols, minerals, vitamins, proteins, peptides, amino acids, organic acids, and pH adjusters may be used alone or in combination of two or more.
本発明の没食子酸の製造方法及び茶発酵物の製造方法においては、次いで、前記発酵ミックス中で前記茶抽出物を前記乳酸菌により発酵させて、該発酵ミックス中に没食子酸を生成、蓄積させる。前記発酵の方法としては、前記乳酸菌の生育条件、前記タンナーゼ活性及び前記没食子酸脱炭酸酵素活性の至適条件、前記発酵ミックスの量等に応じて適宜選択することができ、特に制限されないが、例えば、温度２５〜４０℃、より好ましくは３０〜３７℃において、好気条件下で、１２〜７２時間、より好ましくは１８〜４８時間、前記発酵ミックスを静置又は撹拌（好ましくは、静置）することが好ましい。また、好気条件の代わりに、窒素通気条件下での発酵を採用することもできる。 In the method for producing gallic acid and the method for producing a fermented tea product of the present invention, the tea extract is then fermented in the fermentation mix with the lactic acid bacteria to produce and accumulate gallic acid in the fermentation mix. The fermentation method can be appropriately selected according to the growth conditions of the lactic acid bacteria, the optimal conditions for the tannase activity and the gallic acid decarboxylase activity, and the amount of the fermentation mix, but is not particularly limited. For example, the fermentation mix is allowed to stand or agitated (preferably, standing) at a temperature of 25 to 40 ° C, more preferably 30 to 37 ° C, under aerobic conditions for 12 to 72 hours, more preferably 18 to 48 hours. ) Is preferred. In addition, instead of aerobic conditions, fermentation under nitrogen aerated conditions can be employed.
前記発酵により、前記発酵後の発酵ミックス（発酵物）中に没食子酸を得ることができる。得られた没食子酸は、必要に応じて、前記発酵物の上清からこれを精製して採取することができる。前記採取の方法としては、適宜公知の方法を採用することができ、特に制限されず、例えば、酢酸エチル等の有機溶媒によって単離する方法、イオン交換樹脂を用いる方法、結晶化法、沈殿法等の方法を単独で又は組み合わせた方法が挙げられる。 By the fermentation, gallic acid can be obtained in the fermented mix (fermented product) after the fermentation. The resulting gallic acid can be purified and collected from the supernatant of the fermented product, if necessary. A known method can be appropriately adopted as the method for the collection, and is not particularly limited. For example, a method of isolating with an organic solvent such as ethyl acetate, a method of using an ion exchange resin, a crystallization method, a precipitation method And the like may be used alone or in combination.
（茶発酵物）
また、前記発酵後の発酵ミックス（発酵物）は、そのまま、又は必要に応じて濃縮、希釈、乾燥、若しくは凍結等することにより、本発明の茶発酵物とすることができる。また、前記発酵物における乳酸菌を破砕若しくは加熱処理等して、又は必要に応じてこれを濃縮、希釈、乾燥、若しくは凍結等することにより、本発明の茶発酵物とすることができる。したがって、本発明の茶発酵物は、生成された没食子酸を含有する。また、本発明の茶発酵物としては、前記炭酸塩と、前記乳酸菌、前記乳酸菌組成物、及びこれらの処理物からなる群から選択される少なくとも１種と、没食子酸とを含有することが好ましい。本発明において、前記乳酸菌及び乳酸菌組成物の処理物には、上記の乳酸菌又は乳酸菌組成物の破砕物及び加熱処理物、これらの濃縮物、希釈物、乾燥物、凍結物等が含まれ、これらのうちの１種を単独であっても２種以上の組み合わせであってもよい。また、本発明の茶発酵物としては、前記茶抽出物及び／又は前記発酵ミックスに必要に応じて含有される上記のその他成分のうちの１種又は２種以上をさらに含有していてもよい。
(Fermented tea)
Further, the fermented mix (fermented product) after the fermentation can be used as the fermented tea product of the present invention as it is or, if necessary, by concentration, dilution, drying, or freezing. Further, the lactic acid bacteria in the fermented product can be crushed or heat-treated, or, if necessary, concentrated, diluted, dried, or frozen to obtain the fermented tea product of the present invention. Therefore, the tea fermented product of the present invention contains the produced gallic acid. Further, the fermented tea product of the present invention preferably contains the carbonate, the lactic acid bacterium, the lactic acid bacterium composition, and at least one selected from the group consisting of processed products thereof, and gallic acid. . In the present invention, the processed product of the lactic acid bacterium and the lactic acid bacterium composition includes a crushed product and a heat-treated product of the lactic acid bacterium or the lactic acid bacterium composition, a concentrate thereof, a diluted product, a dried product, a frozen product, May be used alone or in combination of two or more. Further, the fermented tea product of the present invention may further contain one or more of the above-mentioned other components contained as necessary in the tea extract and / or the fermentation mix. .
本発明の茶発酵物において、前記炭酸塩の濃度は、前記発酵ミックスにおける濃度に相当する。また、本発明の茶発酵物において、前記乳酸菌、前記乳酸菌組成物、及びこれらの処理物の濃度は、前記発酵ミックスにおける乳酸菌濃度及び発酵の条件に対応する。また、本発明の茶発酵物において、没食子酸の濃度は、特に限定されないが、ピロガロールの含有量が、該没食子酸の含有量１００質量部に対して、５０質量部以下となる量（没食子酸の含有量／ピロガロールの含有量が２以上）であることが好ましく、４５質量部以下となる量（没食子酸の含有量／ピロガロールの含有量が２．２以上）であることがより好ましく、３０質量部以下となる量（没食子酸の含有量／ピロガロールの含有量が３．３以上）であることがさらに好ましい。本発明によれば、前記没食子酸脱炭酸酵素活性のみが抑制されるため、このようにピロガロールの生成が十分に抑制され、かつ、没食子酸を十分に含有量する茶発酵物を効率的に製造することができる。 In the fermented tea product of the present invention, the concentration of the carbonate corresponds to the concentration in the fermentation mix. In the fermented tea product of the present invention, the concentrations of the lactic acid bacteria, the lactic acid bacteria composition, and the processed product thereof correspond to the lactic acid bacteria concentration in the fermentation mix and the fermentation conditions. In the tea fermented product of the present invention, the concentration of gallic acid is not particularly limited, but the amount of pyrogallol is 50 parts by mass or less based on 100 parts by mass of the gallic acid (gallic acid). Content / pyrogallol content of 2 or more), more preferably 45 parts by mass or less (gallic acid content / pyrogallol content of 2.2 or more), more preferably 30 parts by mass. More preferably, the amount is not more than part by mass (the content of gallic acid / the content of pyrogallol is 3.3 or more). According to the present invention, since only the gallic acid decarboxylase activity is suppressed, the production of pyrogallol is thus sufficiently suppressed, and a tea fermented product containing a sufficient amount of gallic acid is efficiently produced. can do.
（飲食品等）
本発明の茶発酵物には上記のように没食子酸が含有されるため、飲食品の成分として好適に利用することができる。また、医薬品、化成品、化粧品等の有効成分としても利用することができる。本発明の茶発酵物を含有させることができる飲食品としては、特に制限されず、例えば、一般食品、機能性表示食品、栄養機能食品、特定保健用食品等が挙げられ、より具体的には、飲料（茶類、炭酸飲料、ココア、コーヒー、乳飲料、乳酸菌飲料、豆乳飲料、果汁・野菜汁飲料等）、加工食品（チョコレート、ガム、グミ、ゼリー等）、乳製品、サプリメント等が挙げられる。
(Food etc.)
Since the fermented tea product of the present invention contains gallic acid as described above, it can be suitably used as a component of food and drink. It can also be used as an active ingredient in pharmaceuticals, chemicals, cosmetics, and the like. Foods and beverages that can contain the tea fermented product of the present invention are not particularly limited, and include, for example, general foods, functionally labeled foods, nutritionally functional foods, foods for specified health uses, and more specifically. , Beverages (teas, carbonated drinks, cocoa, coffee, milk drinks, lactic acid drinks, soy milk drinks, fruit and vegetable juice drinks, etc.), processed foods (chocolate, gum, gummy, jelly, etc.), dairy products, supplements, etc. Can be
本発明の茶発酵物を含有する飲食品において、前記茶発酵物の濃度は特に制限されず、前記飲食品の目的及び形態に応じて適宜調整することができる。 In the food or drink containing the fermented tea product of the present invention, the concentration of the fermented tea product is not particularly limited, and can be appropriately adjusted depending on the purpose and form of the food or drink.
本発明の茶発酵物を含有する飲食品としては、他に、飲食品に含有させることが可能な各種成分をさらに含有してもよい。このような成分としては、特に制限されず、例えば、水、糖類、糖アルコール類、ミネラル類、ビタミン類、タンパク質、ペプチド、アミノ酸類、有機酸、ｐＨ調整剤、澱粉及び加工澱粉、食物繊維、果実・野菜及びその加工品、動物及び植物生薬エキス、天然由来高分子（コラーゲン、ヒアルロン酸、コンドロイチン等）、油脂、増粘剤、乳化剤、溶剤、界面活性剤、ゲル化剤、安定剤、緩衝剤、懸濁化剤、粘稠剤、賦形剤、崩壊剤、結合剤、流動化剤、保存料、着色料、香料、矯味剤、甘味剤等が挙げられ、これらのうちの１種を単独で含有していても２種以上を組み合わせて含有していてもよい。また、前記飲食品等においては、その製品において没食子酸によりもたらされる様々な作用・効能（例えば、抗酸化作用、食後血中中性脂肪の上昇抑制作用、血糖値の上昇抑制作用等）が表示されていてもよい。 The food or beverage containing the fermented tea product of the present invention may further contain various components that can be contained in the food or beverage. Such components are not particularly limited, and include, for example, water, sugars, sugar alcohols, minerals, vitamins, proteins, peptides, amino acids, organic acids, pH adjusters, starch and processed starch, dietary fiber, Fruits and vegetables and their processed products, animal and plant herbal extracts, naturally derived polymers (collagen, hyaluronic acid, chondroitin, etc.), oils and fats, thickeners, emulsifiers, solvents, surfactants, gelling agents, stabilizers, buffers Agents, suspending agents, thickeners, excipients, disintegrants, binders, fluidizers, preservatives, coloring agents, flavors, flavoring agents, sweeteners, and the like. They may be contained alone or in combination of two or more. In addition, in the foods and drinks, various actions and effects (eg, antioxidant action, action of suppressing increase in postprandial blood triglyceride, action of suppressing increase in blood glucose level, etc.) provided by gallic acid in the product are indicated. It may be.
以下、実施例及び比較例に基づいて本発明をより具体的に説明するが、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on Examples and Comparative Examples, but the present invention is not limited to the following Examples.
＜乳酸菌＞
以下の活性測定及び各調製例においては、乳酸菌１〜５として、それぞれ下記の乳酸菌株：
乳酸菌１：ラクトバチルス・ペントーサス（L.pentosus）ＯＬＬ２０３９８４株（受託番号：ＮＩＴＥ ＢＰ−０１９８８）、
乳酸菌２：ラクトバチルス・ペントーサス（L.pentosus）ＯＬＬ２０３９８２株（受託番号：ＮＩＴＥ ＢＰ−０１９８７）、
乳酸菌３：ラクトバチルス・プランタラム（L.plantarum；ＩＦＯ３０７４）、
乳酸菌４：ラクトバチルス・プランタラム（L.plantarum；ＮＣＩＭＢ１１９７４Ｔ）、及び
乳酸菌５：ラクトバチルス・ファーメンタム（L.fermentum；ＮＲＩＣ１７５２Ｔ）
を用いた。乳酸菌１〜２は本発明の出願人が分離した菌株であり、乳酸菌３〜５は各菌株ライブラリーから入手した菌株である。
<Lactic acid bacteria>
In the following activity measurements and preparation examples, the following lactic acid bacteria strains were used as lactic acid bacteria 1 to 5, respectively:
Lactic acid bacteria 1: Lactobacillus pentosus OLL203984 strain (Accession number: NITE BP-01988),
Lactic acid bacteria 2: Lactobacillus pentosus OLL203982 strain (Accession number: NITE BP-01987),
Lactic acid bacteria 3: Lactobacillus plantarum (L. plantarum; IFO3074),
Lactic acid bacteria 4: Lactobacillus plantarum (L. plantarum; NCIMB11974T), and lactic acid bacteria 5: Lactobacillus fermentum (L. fermentum; NRIC1752T)
Was used. Lactic acid bacteria 1 and 2 are strains isolated by the applicant of the present invention, and lactic acid bacteria 3 to 5 are strains obtained from each strain library.
＜活性測定＞
〔タンナーゼ活性試験〕
乳酸菌１〜５のタンナーゼ活性の強さを、下記のＮｉｓｈｉｔａｎｉ Ｙ．ら、２００３年、Ｊｏｕｒｎａｌ ｏｆ Ｍｉｃｒｏｂｉｏｌｏｇｉｃａｌ Ｍｅｔｈｏｄｓ ５４、ｐ．２８１−２８４に記載の方法に準じた方法によって定量した。
<Activity measurement>
(Tannase activity test)
The strength of the tannase activity of lactic acid bacteria 1 to 5 was determined according to Nishitani Y. Et al., 2003, Journal of Microbiological Methods 54, p. Quantification was performed by a method according to the method described in 281 to 284.
すなわち、先ず、Aspergillus ficuum（シグマ・アルドリッチ社製）を３３ｍＭリン酸二水素ナトリウム（ｐＨ５．０）に溶解し、検量線作成用に０．７８〜１２８００ｍＵの酵素溶液を調製した。調製した各酵素溶液から５０μＬずつ採取し、それぞれ予め用意しておいた基質溶液（５ｍＭ没食子酸メチル（シグマ・アルドリッチ社製）を含有する３３ｍＭリン酸水素二ナトリウム水溶液（ｐＨ５．０））５ｍＬに加えて混合した。これを３０℃で２４時間静置して反応させた後、各１００μＬを採取してエッペンドルフチューブに移し、飽和炭酸水素ナトリウム水溶液（ｐＨ８．６）を等量加えて、３７℃で２時間静置した。静置後、よく撹拌し、８，０００×ｇで２０秒間遠心分離した。遠心分離後の上清を１００μＬ採取して９６穴マイクロプレートに移し、波長４５０ｎｍにおける吸光度を測定し、得られた吸光度から検量線（タンナーゼ活性強さ−吸光度）を作成した。 That is, first, Aspergillus ficuum (manufactured by Sigma-Aldrich) was dissolved in 33 mM sodium dihydrogen phosphate (pH 5.0) to prepare an enzyme solution of 0.78 to 12800 mU for preparing a calibration curve. 50 μL of each prepared enzyme solution was collected and placed in 5 mL of a substrate solution (a 33 mM disodium hydrogen phosphate aqueous solution (pH 5.0) containing 5 mM methyl gallate (manufactured by Sigma-Aldrich)) prepared in advance. In addition, they were mixed. After allowing this to stand at 30 ° C. for 24 hours to react, 100 μL of each was collected, transferred to an Eppendorf tube, an equal amount of a saturated sodium hydrogen carbonate aqueous solution (pH 8.6) was added, and the mixture was allowed to stand at 37 ° C. for 2 hours. did. After standing, the mixture was stirred well and centrifuged at 8,000 × g for 20 seconds. 100 μL of the supernatant after centrifugation was collected, transferred to a 96-well microplate, the absorbance at a wavelength of 450 nm was measured, and a calibration curve (tannase activity intensity-absorbance) was prepared from the obtained absorbance.
次いで、乳酸菌１〜５を、それぞれ、ＭＲＳ寒天培地において、３７℃、嫌気条件下で２４時間培養した後、滅菌コンラージで掻き取り、０．４５μｍフィルターでろ過滅菌した上記基質溶液に懸濁させ、Ｏ．Ｄ．６６０ｎｍでの濁度が０．４になるように調製し、各試験溶液とした。次いで、前記試験溶液をそれぞれ１ｍＬ採取し、生理食塩水で段階希釈後、ＭＲＳ寒天培地に塗沫した。３７℃において、嫌気条件で４８時間培養後、出現したコロニー数を計測し、生菌数を測定した。また、各試験溶液の残りについては、それぞれ、３０℃において、好気条件で２４時間静置して反応させた後、１ｍＬを採取してエッペンドルフチューブに移し、８，０００×ｇで５分間遠心分離した。遠心分離後の上清を１００μＬ採取してエッペンドルフチューブに移し、飽和炭酸水素ナトリウム水溶液（ｐＨ８．６）を等量加えて、３７℃で２時間静置した。静置後、よく撹拌し、８，０００×ｇで２０秒間遠心分離した後の上清を１００μＬ採取して９６穴マイクロプレートに移し、波長４５０ｎｍにおける吸光度を測定した。得られた吸光度から前記検量線に基づいてタンナーゼ活性の強さ（ｍＵ／ｍＬ）を求めた。 Then, after culturing the lactic acid bacteria 1 to 5 on an MRS agar medium under anaerobic conditions at 37 ° C. for 24 hours, scraped with a sterile confluence, suspended in the above substrate solution sterilized by filtration with a 0.45 μm filter, O. D. Each test solution was prepared so that the turbidity at 660 nm was 0.4. Next, 1 mL of each of the test solutions was collected, serially diluted with physiological saline, and then spread on an MRS agar medium. After culturing at 37 ° C. under anaerobic conditions for 48 hours, the number of colonies that appeared was counted, and the number of viable bacteria was measured. The rest of each test solution was allowed to stand at 30 ° C. under aerobic conditions for 24 hours, and then 1 mL was collected, transferred to an Eppendorf tube, and centrifuged at 8,000 × g for 5 minutes. separated. 100 μL of the supernatant after centrifugation was collected, transferred to an Eppendorf tube, an equal amount of a saturated aqueous sodium hydrogen carbonate solution (pH 8.6) was added, and the mixture was allowed to stand at 37 ° C. for 2 hours. After standing still, the mixture was thoroughly stirred, centrifuged at 8,000 × g for 20 seconds, 100 μL of the supernatant was collected, transferred to a 96-well microplate, and the absorbance at a wavelength of 450 nm was measured. From the obtained absorbance, the intensity (mU / mL) of tannase activity was determined based on the calibration curve.
上記タンナーゼ活性試験の結果、乳酸菌１のタンナーゼ活性の強さは、７７．９８ｍＵ／ｍＬ、生菌数は８．４×１０７ｃｆｕ／ｍＬであり、生菌数１×１０７ｃｆｕあたりのタンナーゼ活性の強さは９．２８ｍＵであった。乳酸菌１〜５のタンナーゼ活性の強さ（活性（ｍＵ／ｍＬ））、生菌数（ｃｆｕ／ｍＬ）、及び生菌数あたりのタンナーゼ活性の強さ（活性／生菌数（ｍＵ／１０７ｃｆｕ））を、それぞれ、下記の表１に示す。 As a result of the above tannase activity test, the intensity of tannase activity of lactic acid bacterium 1 was 77.98 mU / mL, the number of viable bacteria was 8.4 × 10 7 cfu / mL, and tannase per 1 × 10 7 cfu of viable bacteria was measured. The activity intensity was 9.28 mU. The intensity of tannase activity (activity (mU / mL)), the viable cell count (cfu / mL), and the tannase activity per viable cell count (activity / viable cell count (mU / 10 7 ) of lactic acid bacteria 1 to 5 cfu)) are shown in Table 1 below.
〔没食子酸脱炭酸酵素活性試験〕
乳酸菌１〜５の没食子酸脱炭酸酵素活性の強さを、下記の特開平１１−５６３５８８号公報に記載の方法に準じた方法によって測定した。
(Gallic acid decarboxylase activity test)
The strength of gallic acid decarboxylase activity of the lactic acid bacteria 1 to 5 was measured by a method according to the method described in JP-A-11-563588 described below.
すなわち、先ず、乳酸菌１〜５を、それぞれ、没食子酸を０．３ｇ／Ｌ含有するＭＲＳ液体培地２０ｍＬにおいて、３７℃、嫌気条件下で２４時間培養した後、３０００×ｇで１０分間遠心分離して上清を除去した。遠心分離後のペレットに０．１Ｍリン酸カリウム緩衝液（ｐＨ６．０）８ｍＬを加えて良く懸濁し、各試験溶液とした。次いで、前記試験溶液をそれぞれ１ｍＬ採取し、生理食塩水で段階希釈後、ＭＲＳ寒天培地に塗沫した。３７℃において、嫌気条件で４８時間培養後、出現したコロニー数を計測し、生菌数を測定した。また、各試験溶液の残りについては、それぞれ、各０．５ｍＬ採取して小試験管に移した後、０．４５μｍフィルターでろ過滅菌した０．１Ｍアスコルビン酸水溶液１ｍＬ、及び０．４５μｍフィルターでろ過滅菌した基質溶液（８０ｍＭ没食子酸を含有する０．１Ｍリン酸カリウム緩衝液（ｐＨ６．０））０．５ｍＬを加えて混合した。これを３０℃において、好気条件で３０分間静置して反応させた後、氷冷し、３，０００×ｇで１０分間遠心分離した。遠心分離後の上清について、ＨＰＬＣ（ＨＰＬＣ（型番）：ＬＣ−２０ＡＴ（株式会社島津製作所製）、カラム：ＣＯＳＭＯＳＩＬ ｃｈｏｌｅｓｔｅｒ（４．６ｍｍ Ｉ．Ｄ．×１５０ｍｍ）（ナカライテスク株式会社製）、移動相：移動相Ａ；アセトニトリル：２０ｍｍｏｌ／Ｌリン酸緩衝液（ｐＨ２．５）＝１：９、移動相Ｂ；アセトニトリル：２０ｍｍｏｌ／Ｌリン酸緩衝液（ｐＨ２．５）＝３：７、グラジエント分析条件：０→２０分で、移動相Ａ；１００％→０％、移動相Ｂ；０％→１００％、流速：１．０ｍＬ／ｍｉｎ、検出波長：２００ｎｍ、試料注入量：１０μＬ）により分析を行い、測定されたピロガロールの生成量から、１分間に１μｍｏｌのピロガロールを生成するのに用いられる酵素量を１Ｕとして、没食子酸脱炭酸酵素活性の強さ（ｍＵ／ｍＬ）を求めた。 That is, first, each of the lactic acid bacteria 1 to 5 was cultured in 20 mL of an MRS liquid medium containing 0.3 g / L of gallic acid under anaerobic conditions at 37 ° C. for 24 hours, and then centrifuged at 3000 × g for 10 minutes. To remove the supernatant. To the pellet after centrifugation, 8 mL of 0.1 M potassium phosphate buffer (pH 6.0) was added and suspended well to prepare each test solution. Next, 1 mL of each of the test solutions was collected, serially diluted with physiological saline, and then spread on an MRS agar medium. After culturing at 37 ° C. under anaerobic conditions for 48 hours, the number of colonies that appeared was counted, and the number of viable bacteria was measured. For the rest of each test solution, each 0.5 mL was collected and transferred to a small test tube, and then filtered with a 0.45 μm filter, sterilized with a 0.1 M ascorbic acid aqueous solution 1 mL, and filtered with a 0.45 μm filter. 0.5 mL of a sterilized substrate solution (0.1 M potassium phosphate buffer (pH 6.0) containing 80 mM gallic acid) was added and mixed. This was allowed to stand at 30 ° C. under aerobic conditions for 30 minutes to react, then cooled on ice, and centrifuged at 3,000 × g for 10 minutes. HPLC (HPLC (model number): LC-20AT (manufactured by Shimadzu Corporation), column: COSMOSIL cholester (4.6 mm ID × 150 mm) (manufactured by Nacalai Tesque, Inc.) Phase: mobile phase A; acetonitrile: 20 mmol / L phosphate buffer (pH 2.5) = 1: 9, mobile phase B; acetonitrile: 20 mmol / L phosphate buffer (pH 2.5) = 3: 7, gradient analysis Conditions: 0 to 20 minutes, mobile phase A: 100% → 0%, mobile phase B: 0% → 100%, flow rate: 1.0 mL / min, detection wavelength: 200 nm, sample injection volume: 10 μL) Based on the amount of pyrogallol produced, the amount of enzyme used to produce 1 μmol of pyrogallol per minute was defined as 1 U, and gallic acid decarboxylation was performed. The intensity of the enzyme activity (mU / mL) was determined.
上記没食子酸脱炭酸酵素活性試験の結果、乳酸菌１の没食子酸脱炭酸酵素活性の強さは、４６６ｍＵ／ｍＬ、生菌数は１．７×１０９ｃｆｕ／ｍＬであり、生菌数１×１０７ｃｆｕあたりの没食子酸脱炭酸酵素活性の強さは２．７４ｍＵであった。乳酸菌１〜５の没食子酸脱炭酸酵素活性の強さ（活性（ｍＵ／ｍＬ））、生菌数（ｃｆｕ／ｍＬ）、及び生菌数あたりの没食子酸脱炭酸酵素活性の強さ（活性／生菌数（ｍＵ／１０７ｃｆｕ））を、それぞれ、下記の表２に示す。 As a result of the gallic acid decarboxylase activity test, the strength of gallic acid decarboxylase activity of lactic acid bacterium 1 was 466 mU / mL, the number of viable bacteria was 1.7 × 10 9 cfu / mL, and the number of viable bacteria 1 × The intensity of gallic acid decarboxylase activity per 10 7 cfu was 2.74 mU. The intensity of gallic acid decarboxylase activity of lactic acid bacteria 1 to 5 (activity (mU / mL)), viable cell count (cfu / mL), and the intensity of gallic acid decarboxylase activity per viable cell count (activity / The viable cell count (mU / 10 7 cfu) is shown in Table 2 below.
＜調製例１＞
（実施例１）
〔乳酸菌組成物〕
乳酸菌１を、ホエイペプチド、酵母エキス、グルコース、及び硫酸アンモニウムを含有する液体培地（ｐＨ４〜６）を用いて、３７℃で中和培養した。培養後の培地を遠心分離により濃縮し、凍結したものを以下の乳酸菌組成物として用いた。
<Preparation Example 1>
(Example 1)
(Lactic acid bacteria composition)
Lactic acid bacterium 1 was neutralized and cultured at 37 ° C. using a liquid medium (pH 4 to 6) containing whey peptide, yeast extract, glucose, and ammonium sulfate. The culture medium after the culture was concentrated by centrifugation, and the frozen medium was used as the following lactic acid bacteria composition.
〔発酵物〕
炭酸水素ナトリウム（重曹）、茶抽出物（緑茶葉熱水抽出物）、上記の乳酸菌組成物、及び水を混合し、炭酸水素ナトリウムの含有量が０．２ｗ／ｖ％、前記茶抽出物の含有量がガレート型カテキンの含有量で０．１８ｗ／ｖ％、前記乳酸菌組成物の含有量が乳酸菌数換算で１．２×１０９ｃｆｕ／ｍＬである発酵ミックスを６５ｍＬ調製した。次いで、前記発酵ミックスを３０℃、好気条件において３０時間静置して発酵させた。
(Fermented product)
A mixture of sodium hydrogencarbonate (baking soda), a tea extract (green tea leaf hot water extract), the lactic acid bacteria composition described above, and water is mixed with a sodium hydrogencarbonate content of 0.2 w / v%, 65 mL of a fermentation mix having a content of 0.18 w / v% in terms of gallate-type catechin content and 1.2 × 10 9 cfu / mL in terms of the number of lactic acid bacteria was prepared. Next, the fermentation mix was left to stand at 30 ° C. under aerobic conditions for 30 hours for fermentation.
〔没食子酸濃度測定〕
上記発酵後の発酵物（茶発酵物）を３，０００×ｇで１０分間遠心分離して得られた上清について、上記没食子酸脱炭酸酵素活性試験におけるピロガロールのＨＰＬＣによる測定と同様のＨＰＬＣ条件で、前記発酵物における没食子酸の濃度（μｇ／ｍＬ）を求めた。ただし、ＨＰＬＣ検出波長は２８０ｎｍとした。
(Gallic acid concentration measurement)
The supernatant obtained by centrifuging the fermented product (fermented tea product) after fermentation at 3,000 × g for 10 minutes was subjected to the same HPLC conditions as those for the pyrogallol HPLC measurement in the gallic acid decarboxylase activity test. The concentration of gallic acid (μg / mL) in the fermented product was determined. However, the HPLC detection wavelength was 280 nm.
〔ピロガロール濃度測定〕
上記発酵後の発酵物を０．５ｍＬ採取して小試験管に移した後、０．４５μｍフィルターでろ過滅菌した０．１Ｍアスコルビン酸水溶液１ｍＬ、及び０．４５μｍフィルターでろ過滅菌した基質溶液（８０ｍＭ没食子酸を含有する０．１Ｍリン酸カリウム緩衝液（ｐＨ６．０））０．５ｍＬを加えて混合した。これを３０℃において、好気条件で３０分間静置して反応させた後、氷冷し、３，０００×ｇで１０分間遠心分離した。遠心分離後の上清について、上記没食子酸脱炭酸酵素活性試験と同様にしてＨＰＬＣにより分析を行い、前記発酵物におけるピロガロール濃度（μｇ／ｍＬ）を求めた。
(Pyrogallol concentration measurement)
After collecting 0.5 mL of the fermented product after the above fermentation and transferring it to a small test tube, 1 mL of an aqueous 0.1 M ascorbic acid solution sterilized by filtration with a 0.45 μm filter and a substrate solution (80 mM) sterilized by filtration with a 0.45 μm filter 0.5 mL of a 0.1 M potassium phosphate buffer (pH 6.0) containing gallic acid was added and mixed. This was allowed to stand at 30 ° C. under aerobic conditions for 30 minutes to react, then cooled on ice, and centrifuged at 3,000 × g for 10 minutes. The supernatant after centrifugation was analyzed by HPLC in the same manner as in the above gallic acid decarboxylase activity test, and the pyrogallol concentration (μg / mL) in the fermented product was determined.
（実施例２）
前記発酵ミックスにおける炭酸水素ナトリウムの含有量を０．４ｗ／ｖ％としたこと以外は実施例１と同様にして発酵させ、発酵後の発酵物について、実施例１と同様にして没食子酸濃度（μｇ／ｍＬ）及びピロガロール濃度（μｇ／ｍＬ）を求めた。
(Example 2)
Fermentation was performed in the same manner as in Example 1 except that the content of sodium bicarbonate in the fermentation mix was set to 0.4 w / v%. μg / mL) and pyrogallol concentration (μg / mL).
（比較例１）
前記発酵ミックスに炭酸水素ナトリウムを添加しなかった（炭酸水素ナトリウムの含有量：０ｗ／ｖ％）こと以外は実施例１と同様にして発酵させ、発酵後の発酵物について、実施例１と同様にして没食子酸濃度（μｇ／ｍＬ）及びピロガロール濃度（μｇ／ｍＬ）を求めた。
(Comparative Example 1)
Fermentation was performed in the same manner as in Example 1 except that sodium bicarbonate was not added to the fermentation mix (content of sodium bicarbonate: 0 w / v%), and the fermented product after fermentation was the same as in Example 1. The gallic acid concentration (μg / mL) and pyrogallol concentration (μg / mL) were determined.
実施例１〜２及び比較例１で得られた各発酵物における没食子酸濃度（μｇ／ｍＬ）及びピロガロール濃度（μｇ／ｍＬ）を図１に示す。上記に示したように、乳酸菌１は、タンナーゼ活性及び没食子酸脱炭酸酵素活性をいずれも十分に有していることが確認されたが、図１に示したように、炭酸塩（炭酸水素ナトリウム）存在下においてかかる乳酸菌により茶抽出物を発酵させると、没食子酸脱炭酸酵素活性、すなわちピロガロールの生成のみが十分に抑制されて、没食子酸を効率的に得られることが確認された。 FIG. 1 shows the concentrations of gallic acid (μg / mL) and pyrogallol (μg / mL) in each of the fermented products obtained in Examples 1 and 2 and Comparative Example 1. As described above, lactic acid bacterium 1 was confirmed to have sufficient tannase activity and gallic acid decarboxylase activity, but as shown in FIG. ) It was confirmed that when the tea extract was fermented with such a lactic acid bacterium in the presence, only the gallic acid decarboxylase activity, that is, the production of pyrogallol alone was sufficiently suppressed, and gallic acid could be obtained efficiently.
＜調製例２＞
〔乳酸菌組成物〕
乳酸菌１〜４について、それぞれ、ＭＲＳ培地において、３７℃、嫌気条件下で１８時間の賦活培養を２回実施した後、培養液を遠心分離により濃縮し、０．８５ｗ／ｖ％生理食塩水で１回洗浄後、滅菌水で１×１０１０ｃｆｕ／ｍＬに調製した組成物を、乳酸菌組成物１〜４とした。また、乳酸菌１〜４に代えて乳酸菌５を用いたこと以外は同様にして調製した組成物を、乳酸菌組成物５とした。
<Preparation Example 2>
(Lactic acid bacteria composition)
For each of the lactic acid bacteria 1 to 4, the activation culture was performed twice in an MRS medium at 37 ° C. under anaerobic conditions for 18 hours, and the culture was concentrated by centrifugation, and then diluted with 0.85 w / v% physiological saline. After washing once, compositions prepared at 1 × 10 10 cfu / mL with sterile water were designated as lactic acid bacteria compositions 1 to 4. A composition prepared in the same manner except that lactic acid bacteria 5 were used instead of lactic acid bacteria 1 to 4 was designated as lactic acid bacteria composition 5.
〔発酵物〕
炭酸水素ナトリウム（重曹）、茶抽出物（緑茶葉熱水抽出物）、上記の乳酸菌組成物１〜４、及び水を混合し、炭酸水素ナトリウムの含有量が０．２、０．４、又は０．６ｗ／ｖ％、前記茶抽出物の含有量がガレート型カテキンの含有量で０．０９ｗ／ｖ％、前記各乳酸菌組成物の含有量が乳酸菌数換算で６．７×１０８ｃｆｕ／ｍＬである発酵ミックスを、それぞれ、５ｍＬ調製した。次いで、前記発酵ミックスを３０℃、好気条件において３０時間静置して発酵させた後、１１０℃で１分間加熱して殺菌し、発酵物（茶発酵物）（実施例３〜１４）をそれぞれ得た。
(Fermented product)
A mixture of sodium hydrogen carbonate (baking soda), a tea extract (green tea leaf hot water extract), the above lactic acid bacteria compositions 1 to 4, and water, wherein the content of sodium hydrogen carbonate is 0.2, 0.4, or 0.6 w / v%, the content of the tea extract is 0.09 w / v% in terms of the content of gallate-type catechin, and the content of each of the lactic acid bacteria compositions is 6.7 × 10 8 cfu / in terms of the number of lactic acid bacteria. 5 mL of each fermentation mix was prepared. Next, the fermented mix was left to stand at 30 ° C. under aerobic conditions for 30 hours for fermentation, and then sterilized by heating at 110 ° C. for 1 minute to obtain a fermented product (tea fermented product) (Examples 3 to 14). Got each.
また、乳酸菌組成物１〜４に代えて上記の乳酸菌組成物５を用いたこと以外は同様にして、発酵物（茶発酵物）（比較例７〜９）をそれぞれ得た。さらに、前記発酵ミックスに炭酸水素ナトリウムを添加しなかった（炭酸水素ナトリウムの含有量：０ｗ／ｖ％）こと以外は上記と同様にして、発酵物（茶発酵物）（比較例２〜６）をそれぞれ得た。 Fermented products (fermented tea products) (Comparative Examples 7 to 9) were obtained in the same manner except that the above-mentioned lactic acid bacteria composition 5 was used instead of the lactic acid bacteria compositions 1 to 4. Furthermore, a fermented product (fermented tea product) was prepared in the same manner as described above except that sodium hydrogencarbonate was not added to the fermentation mix (content of sodium bicarbonate: 0 w / v%) (Comparative Examples 2 to 6). Was obtained respectively.
〔没食子酸濃度測定〕
上記で得られた各発酵物を３，０００×ｇで１０分間遠心分離して得られた上清について、上記没食子酸脱炭酸酵素活性試験におけるピロガロールのＨＰＬＣによる測定と同様のＨＰＬＣ条件で、前記発酵物における没食子酸の濃度（μｇ／ｍＬ）を求めた。ただし、ＨＰＬＣ検出波長は２８０ｎｍとした。
(Gallic acid concentration measurement)
The supernatant obtained by centrifuging each of the fermented products obtained above at 3,000 × g for 10 minutes was analyzed under the same HPLC conditions as in the pyrogallol HPLC measurement in the gallic acid decarboxylase activity test. The concentration (μg / mL) of gallic acid in the fermented product was determined. However, the HPLC detection wavelength was 280 nm.
〔ピロガロール濃度測定〕
上記で得られた各発酵物を０．５ｍＬ採取して小試験管に移した後、０．４５μｍフィルターでろ過滅菌した０．１Ｍアスコルビン酸水溶液１ｍＬ、及び０．４５μｍフィルターでろ過滅菌した基質溶液（８０ｍＭ没食子酸を含有する０．１Ｍリン酸カリウム緩衝液（ｐＨ６．０））０．５ｍＬを加えて混合した。これを３０℃において、好気条件で３０分間静置して反応させた後、氷冷し、３，０００×ｇで１０分間遠心分離した。遠心分離後の上清について、上記没食子酸脱炭酸酵素活性試験と同様にしてＨＰＬＣにより分析を行い、前記発酵物におけるピロガロール濃度（μｇ／ｍＬ）を求めた。
(Pyrogallol concentration measurement)
After collecting 0.5 mL of each fermentation product obtained above and transferring it to a small test tube, 1 mL of a 0.1 M aqueous ascorbic acid solution sterilized by filtration with a 0.45 μm filter, and a substrate solution sterilized by filtration with a 0.45 μm filter 0.5 mL (0.1 M potassium phosphate buffer (pH 6.0) containing 80 mM gallic acid) was added and mixed. This was allowed to stand at 30 ° C. under aerobic conditions for 30 minutes to react, then cooled on ice, and centrifuged at 3,000 × g for 10 minutes. The supernatant after centrifugation was analyzed by HPLC in the same manner as in the above gallic acid decarboxylase activity test, and the pyrogallol concentration (μg / mL) in the fermented product was determined.
各実施例及び比較例における発酵ミックス中の炭酸水素ナトリウム濃度（重曹（ｗ／ｖ％））、得られた各発酵物における没食子酸濃度（没食子酸（ＧＡ）（μｇ／ｍＬ））、ピロガロール濃度（ピロガロール（ＰＧ）（μｇ／ｍＬ））、及びこれらの比（ＧＡ／ＰＧ）を、それぞれ、下記の表３に示す。 Sodium bicarbonate concentration (baking soda (w / v%)) in the fermented mix in each Example and Comparative Example, gallic acid concentration (gallic acid (GA) (μg / mL)), pyrogallol concentration in each obtained fermented product (Pyrogallol (PG) (μg / mL)) and their ratio (GA / PG) are shown in Table 3 below, respectively.
なお、発酵物における没食子酸濃度とピロガロール濃度との比（ＧＡ／ＰＧ）は、０．５以上でピロガロールの生成が十分に抑制されていると評価でき、２．０以上でピロガロールの生成のみが特に抑制されていると評価できる。 The ratio (GA / PG) between the gallic acid concentration and the pyrogallol concentration in the fermented product can be evaluated to be sufficient to suppress the formation of pyrogallol when the ratio is 0.5 or more, and only when the ratio is 2.0 or more, only the formation of pyrogallol is suppressed. In particular, it can be evaluated as being suppressed.
上記に示したように、乳酸菌１〜４は、タンナーゼ活性及び没食子酸脱炭酸酵素活性をいずれも十分に有していることが確認されたが、表３に示したように、炭酸塩（炭酸水素ナトリウム）存在下において茶抽出物を発酵させると、没食子酸脱炭酸酵素活性、すなわちピロガロールの生成のみが十分に抑制されて、没食子酸を効率的に得られることが確認された。また、前記炭酸塩をより高い濃度（例えば、０．２ｗ／ｖ％を超える濃度）とすると、ピロガロールの生成がより抑制され、没食子酸をさらに効率的に得られることが確認された。なお、表３において、タンナーゼ活性を有さない乳酸菌５を用いた発酵物においても若干の没食子酸の生成が認められたが、これは、主に発酵物の殺菌時にガレート型カテキンが炭酸水素ナトリウムと反応して加水分解されたことによって若干量が生じたものである。 As described above, it was confirmed that the lactic acid bacteria 1 to 4 had sufficient tannase activity and gallic acid decarboxylase activity. It was confirmed that when the tea extract was fermented in the presence of (sodium hydrogen), only the gallic acid decarboxylase activity, that is, the formation of pyrogallol, was sufficiently suppressed, and gallic acid could be obtained efficiently. Further, it was confirmed that when the concentration of the carbonate was higher (for example, a concentration exceeding 0.2 w / v%), the production of pyrogallol was further suppressed, and gallic acid could be obtained more efficiently. In Table 3, in the fermented product using the lactic acid bacterium 5 having no tannase activity, generation of some gallic acid was recognized. This is mainly because gallate-type catechin was converted to sodium bicarbonate during sterilization of the fermented product. And a small amount was produced by hydrolysis.
以上説明したように、本発明によれば、タンナーゼ活性及び没食子酸脱炭酸酵素活性を有する乳酸菌による発酵によって没食子酸を効率的に得ることができる没食子酸の製造方法を提供することが可能となる。また、没食子酸を含有する茶発酵物の製造方法、これらの方法に用いる乳酸菌及び乳酸菌組成物、並びに、それによって得られる茶発酵物及び飲食品を提供することも可能となる。 As described above, according to the present invention, it is possible to provide a method for producing gallic acid capable of efficiently obtaining gallic acid by fermentation with lactic acid bacteria having tannase activity and gallic acid decarboxylase activity. . Further, it is also possible to provide a method for producing a fermented tea product containing gallic acid, lactic acid bacteria and a lactic acid bacteria composition used in these methods, and a fermented tea product and food and drink obtained by the method.
１．
（１）識別の表示：Lactobacillus pentosus ＯＬＬ２０３９８４
（２）受託番号：ＮＩＴＥ ＢＰ−０１９８８
（３）原受託日：２０１５年１月５日
（４）寄託機関：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
２．
（１）識別の表示：Lactobacillus pentosus ＯＬＬ２０３９８２
（２）受託番号：ＮＩＴＥ ＢＰ−０１９８７
（３）原受託日：２０１５年１月５日
（４）寄託機関：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
1.
(1) Identification indication: Lactobacillus pentosus OLL203984
(2) Accession number: NITE BP-01988
(3) Original deposit date: January 5, 2015 (4) Deposited organization: Patented Microorganisms Depositary Center, National Institute of Technology and Evaluation 2.
(1) Indication of identification: Lactobacillus pentosus OLL203982
(2) Accession number: NITE BP-01987
(3) Original contract date: January 5, 2015 (4) Deposited organization: Patented Microorganisms Depositary Center, National Institute of Technology and Evaluation
Claims (9)
炭酸塩と、ガレート型カテキンを含有する茶抽出物と、タンナーゼ活性及び没食子酸脱炭酸酵素活性を有する乳酸菌と、を含有する発酵ミックスを調製する工程と、
前記発酵ミックス中で前記茶抽出物を前記乳酸菌により発酵させて没食子酸を得る工程と、
を含むことを特徴とする没食子酸の製造方法。 A method for producing gallic acid,
Carbonate, and a tea extract containing gallate-type catechin, and a lactic acid bacterium having tannase activity and gallic acid decarboxylase activity, and a step of preparing a fermentation mix containing
Fermenting the tea extract in the fermentation mix with the lactic acid bacteria to obtain gallic acid,
A method for producing gallic acid, comprising:
炭酸塩と、ガレート型カテキンを含有する茶抽出物と、タンナーゼ活性及び没食子酸脱炭酸酵素活性を有する乳酸菌と、を含有する発酵ミックスを調製する工程と、
前記発酵ミックス中で前記茶抽出物を前記乳酸菌により発酵させて没食子酸を含有する茶発酵物を得る工程と、
を含むことを特徴とする茶発酵物の製造方法。 A method for producing a fermented tea product,
Carbonate, and a tea extract containing gallate-type catechin, and a lactic acid bacterium having tannase activity and gallic acid decarboxylase activity, and a step of preparing a fermentation mix containing
A step of fermenting the tea extract in the fermentation mixture with the lactic acid bacteria to obtain a fermented tea product containing gallic acid,
A method for producing a fermented tea product, comprising:
A food or drink comprising the fermented tea product according to claim 8.
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| JP2022082467A (en) * | 2020-11-20 | 2022-06-01 | レッド・ブル・ゲゼルシャフト・ミット・ベシュレンクテル・ハフツング | Method for preparing extract containing gallic acid, aqueous concentrate containing gallic acid, and food product and food supplement containing that aqueous concentrate |
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| JP2022082467A (en) * | 2020-11-20 | 2022-06-01 | レッド・ブル・ゲゼルシャフト・ミット・ベシュレンクテル・ハフツング | Method for preparing extract containing gallic acid, aqueous concentrate containing gallic acid, and food product and food supplement containing that aqueous concentrate |
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