JP7455554B2 - Method for producing fermented milk and method for enhancing flavor of fermented milk - Google Patents
Method for producing fermented milk and method for enhancing flavor of fermented milk Download PDFInfo
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
特許法第３０条第２項適用 １．２０１９年９月１２日 ｗｗｗ．ｉｔｏｅｎ．ｃｏ．ｊｐ／ｎｅｗｓ／ｄｅｔａｉｌ／ｉｄ＝２５３９７、ｗｗｗ．ｉｔｏｅｎ．ｃｏ．ｊｐ／ｐｒｏｄｕｃｔｓ／ｄｅｔａｉｌ．ｐｈｐ？ｉｄ＝２６１０、ｗｗｗ．ｉｔｏｅｎ．ｃｏ．ｊｐ／ｐｒｏｄｕｃｔｓ／ｄｅｔａｉｌ．ｐｈｐ？ｉｄ＝２６１１ にて発表 ２．２０１９年９月１２日 ニュースリリースにて発表 ３．２０１９年９月２３日 全国で販売Application of Article 30, Paragraph 2 of the Patent Act 1. September 12, 2019 www. itoen. co. jp/news/detail/id=25397, www. itoen. co. jp/products/detail. php? id=2610, www. itoen. co. jp/products/detail. php? Announced at id=2611 2. Announced in news release on September 12, 2019 3. Sold nationwide on September 23, 2019
本発明は、発酵乳の製造方法、発酵乳、発酵乳製品、および発酵乳の香味の増強方法に関する。 The present invention relates to a method for producing fermented milk, fermented milk, fermented milk products, and a method for enhancing the flavor of fermented milk.
発酵乳を使用した発酵乳製品、特にヨーグルト製品は、日本においても健康食品として広く認知されている。現在では嗜好性や飲食シーンの多様化により、ヨーグルト製品に求められる香味は多種多様となっている。 Fermented milk products, especially yogurt products, made with fermented milk are widely recognized as health foods in Japan. Nowadays, due to diversification of tastes and eating and drinking situations, the flavors required for yogurt products are becoming more and more diverse.
発酵乳製品に用いられる中温性乳酸菌としてラクトコッカス・クレモリス（Lactococcus lactis subsp. cremoris），ラクトコッカス・ラクティス（Lactococcus lactis subsp. lactis），ロイコノストック・クレモリス(Leuconostoc mesenteroides subsp. cremoris）等が知られている。例えばロイコノストック・クレモリスをスターターとして用いて発酵した発酵乳製品として、一般にフィンランドのヴィリ、スウェーデンのラングフィル、ノルウェーのテッテ、コーカサスのケフィール等が知られている。これらは独特の強いチーズ風味があり、また粘性が強いことによって滑らかな食感があるものとされる。また我国でもラクトコッカス・クレモリスを主発酵菌としてこれに酢酸菌を併用することによって、同様にチーズ風味と強い粘性を有する所謂カスピ海ヨーグルトが知られている。この場合、チーズ風味と粘性による食感が上記諸外国のものより比較的日本人の嗜好に適合し易いものとされて人気がある。 Lactococcus cremoris (Lactococcus lactis subsp. cremoris), Lactococcus lactis (Lactococcus lactis subsp. lactis), Leuconostoc mesenteroides subsp. cremoris, etc. are known as mesophilic lactic acid bacteria used in fermented dairy products. ing. For example, fermented dairy products fermented using Leuconostoc cremoris as a starter are generally known as Villi from Finland, Langfil from Sweden, Tette from Norway, and Kefir from the Caucasus. These have a unique strong cheese flavor and are said to have a smooth texture due to their strong viscosity. Also in Japan, so-called Caspian Sea yogurt, which similarly has a cheese flavor and strong viscosity, is known by using Lactococcus cremoris as the main fermenting bacteria in combination with acetic acid bacteria. In this case, it is popular because its cheese flavor and texture due to its viscosity are said to be more compatible with Japanese tastes than those from other countries.
特許文献１では、乳酸菌として上述したラクトコッカス・クレモリス、ラクトコッカス・ラクティス及びロイコノストック・クレモリスから選択される中温性乳酸菌のみをスターターとして用いたものは、発酵乳製品の保存中にチーズ臭が次第に強くなり、チーズ風味の変化が激しい傾向が顕著に見られることなどを問題点として指摘している（段落［０００４］等）。同文献ではこの問題点を解決し、保存中も安定した穏やかなチーズ香を呈し、日本人の嗜好に適合したチーズ風味の発酵乳製品を提供するために（段落［０００５］等）、上述した３種の中温性乳酸菌から選択される乳酸菌を、高温性乳酸菌であるストレプトコッカス・サーモフィラス（Streptococcus thermophilus）と併用することを提案している（特許請求の範囲、等）。 In Patent Document 1, a starter using only mesophilic lactic acid bacteria selected from the above-mentioned Lactococcus cremoris, Lactococcus lactis, and Leuconostoc cremoris as a lactic acid bacterium causes a cheese odor during storage of fermented dairy products. It points out as a problem that the cheese flavor tends to gradually become stronger and the cheese flavor changes drastically (paragraph [0004] etc.). In the same document, in order to solve this problem and provide a cheese-flavored fermented dairy product that exhibits a stable and mild cheese aroma even during storage and that suits the tastes of Japanese people (paragraph [0005] etc.), the above-mentioned It is proposed to use lactic acid bacteria selected from three types of mesophilic lactic acid bacteria together with Streptococcus thermophilus, which is a thermophilic lactic acid bacterium (claims, etc.).
また、ラクトコッカス属菌と他の乳酸菌とを併用した発酵乳製品として、特許文献２では、ビフィズス菌の生残性が高く、ホエイの栄養価とビフィズス菌の機能を十分に有する多機能のホエイ発酵飲料を提供するために、ビフィドバクテリウム（Bifidobacterium）属菌とラクトコッカス属菌とを併用することを提案している（特許請求の範囲、等）。 In addition, Patent Document 2 describes a fermented dairy product that uses Lactococcus bacteria and other lactic acid bacteria in combination, and describes a multifunctional whey product that has high survival of bifidobacteria and has the nutritional value of whey and the functions of bifidobacteria. In order to provide a fermented beverage, it is proposed to use bacteria of the genus Bifidobacterium and bacteria of the genus Lactococcus in combination (claims, etc.).
上述した通り、現在ではユーザーにおける嗜好性や飲食シーンの多様化により、ヨーグルト製品などの発酵乳製品に求められる香味は多種多様となっている。本発明では、市販の発酵乳製品のライトな味わいや、単調で複雑さのない味わいに飽きや物足りなさを感じている発酵乳製品のヘビーユーザーに着目した。特許文献１に開示の技術により、一般的な日本人の嗜好に適合した発酵乳製品が得られたとしても、また、特許文献２に開示の技術により、多機能性の発酵乳製品が得られたとしても、濃厚さと味の複雑さを兼ね揃えた発酵乳製品を求めるヘビーユーザーを必ずしも満足させるものではない。一方、濃厚な味わいを出すために、乳酸菌による発酵を促進させることが考えられるが、この場合は酸味が過剰になるという新たな問題が生じ得る。 As mentioned above, due to the diversification of user preferences and eating and drinking scenes, the flavors required for fermented dairy products such as yogurt products are now diverse. The present invention focuses on heavy users of fermented dairy products who feel bored or unsatisfied with the light taste or monotonous uncomplex taste of commercially available fermented dairy products. Even if the technology disclosed in Patent Document 1 yields fermented dairy products that meet the tastes of ordinary Japanese people, the technology disclosed in Patent Literature 2 does not yield multifunctional fermented dairy products. Even so, it does not necessarily satisfy heavy users who seek fermented dairy products with both richness and complexity of flavor. On the other hand, in order to produce a rich taste, it is possible to promote fermentation by lactic acid bacteria, but this may cause a new problem of excessive sourness.
本発明は、上記実情に鑑み開発されたものであり、酸味を抑制しつつ、ヘビーユーザー向けの濃厚で複雑な香味を有する発酵乳の製造方法、発酵乳、及び発酵乳製品を提供することを目的とする。また、本発明は、酸味を抑制しつつ、ヘビーユーザー向けの複雑で濃厚な香味を付与するための発酵乳の香味の増強方法を提供することを目的とする。 The present invention was developed in view of the above circumstances, and aims to provide a method for producing fermented milk, fermented milk, and fermented milk products that have a rich and complex flavor for heavy users while suppressing sourness. purpose. Another object of the present invention is to provide a method for enhancing the flavor of fermented milk for imparting a complex and rich flavor for heavy users while suppressing sourness.
本発明の実施形態に係る発明（以下において、「本実施形態」という。）は、以下の通りである。
［１］ 乳原料を含む原料混合物を調製すること、
上記原料混合物に、乳酸菌として少なくとも、ラクトコッカス・クレモリス及びラクトコッカス・ラクティスからなる２種のラクトコッカス属菌を添加すること、および
上記乳酸菌を２９℃～３５℃で７．０時間～１７．０時間培養し、上記乳原料を発酵させること
を含む、発酵乳の製造方法。
The invention according to the embodiment of the present invention (hereinafter referred to as "this embodiment") is as follows.
[1] Preparing a raw material mixture containing dairy raw materials,
Adding at least two types of Lactococcus bacteria consisting of Lactococcus cremoris and Lactococcus lactis as lactic acid bacteria to the raw material mixture; and A method for producing fermented milk, which comprises culturing for a period of time and fermenting the milk raw material.
［２］ 上記発酵乳に含まれる培養後の上記ラクトコッカス・クレモリスの菌数が、上記発酵乳１ｍｌ当たり、５．０×１０７～７．０×１０８であり、培養後の上記ラクトコッカス・ラクティスの菌数が、上記発酵乳１ｍｌ当たり、１．０×１０７～５．０×１０８である、［１］に記載の発酵乳の製造方法。 [2] The number of bacteria of the Lactococcus cremoris after the culture contained in the fermented milk is 5.0×10 7 to 7.0×10 8 per ml of the fermented milk, and the Lactococcus cremoris after the culture - The method for producing fermented milk according to [1], wherein the number of Lactis bacteria is 1.0 x 10 7 to 5.0 x 10 8 per ml of the fermented milk.
［３］ 上記原料混合物に、乳酸菌として、ラクトバチルス属菌、ストレプトコッカス属菌及びビフィドバクテリウム属菌からなる群から選ばれる１種又は２種以上をさらに添加する、［１］又は［２］に記載の発酵乳の製造方法。 [3] Further adding one or more lactic acid bacteria selected from the group consisting of Lactobacillus, Streptococcus, and Bifidobacterium to the raw material mixture, [1] or [2] The method for producing fermented milk described in .
［４］ 上記ラクトバチルス属菌が、ラクトバチルス・ヘルベティカス、ラクトバチルス・ブルガリクス、ラクトバチルス・カゼイ及びラクトバチルス・アシドフィルスからなる群から選択される乳酸菌であり、上記ストレプトコッカス属菌が、ストレプトコッカス・サーモフィルスであり、上記ビフィドバクテリウム属菌が、ラクティス、ビフィダム、ロンガム、インファンティスからなる群から選択される乳酸菌である、［３］に記載の発酵乳の製造方法。 [4] The Lactobacillus bacteria are lactic acid bacteria selected from the group consisting of Lactobacillus helveticus, Lactobacillus bulgaricus, Lactobacillus casei, and Lactobacillus acidophilus, and the Streptococcus bacteria are Streptococcus thermophilus. The method for producing fermented milk according to [3], wherein the Bifidobacterium genus is a lactic acid bacterium selected from the group consisting of Lactis, Bifidum, Longum, and Infantis.
［５］ 上記ラクトバチルス属菌を添加する場合、上記発酵乳に含まれる培養後の上記ラクトバチルス属菌の菌数が、上記発酵乳１ｍｌ当たり、１．０×１０８～９．０×１０８であり、上記ストレプトコッカス属菌を添加する場合、上記発酵乳に含まれる培養後の上記ストレプトコッカス属菌の菌数が、上記発酵乳１ｍｌ当たり、８．０×１０８～１．０×１０９であり、上記ビフィドバクテリウム属菌を添加する場合、上記発酵乳に含まれる培養後の上記ビフィドバクテリウム属菌の菌数が、上記発酵乳１ｍｌ当たり、２．０×１０６～７．０×１０６である、［３］又は［４］に記載の発酵乳の製造方法。 [5] When adding the Lactobacillus bacteria, the number of Lactobacillus bacteria contained in the fermented milk after culturing is 1.0×10 8 to 9.0×10 per ml of the fermented milk. 8 , and when the Streptococcus bacteria are added, the number of Streptococcus bacteria contained in the fermented milk after culture is 8.0×10 8 to 1.0×10 9 per ml of the fermented milk. When the Bifidobacterium genus bacteria are added, the number of Bifidobacterium bacteria contained in the fermented milk after culture is 2.0×10 6 to 7 per ml of the fermented milk. The method for producing fermented milk according to [3] or [4], which is .0×10 6 .
［６］ 上記ラクトバチルス属菌を添加する場合、上記発酵乳に含まれる培養後の上記ラクトバチルス属菌の菌数に対する培養後の上記２種のラクトコッカス属菌の合計菌数の比率（ラクトコッカス属菌／ラクトバチルス属菌）が、０．１～１０．０である、［３］～［５］の何れか１項に記載の発酵乳の製造方法。 [6] When adding the Lactobacillus bacteria, the ratio of the total number of the two types of Lactococcus bacteria after culture to the number of Lactobacillus bacteria after culture contained in the fermented milk The method for producing fermented milk according to any one of [3] to [5], wherein the number of bacteria of the genus Coccus/bacteria of the genus Lactobacillus is 0.1 to 10.0.
［７］ 上記ビフィドバクテリウム属菌を添加する場合、上記発酵乳に含まれる培養後の上記ビフィドバクテリウム属菌の菌数に対する培養後の上記２種のラクトコッカス属菌の合計菌数の比率（ラクトコッカス属菌／ビフィドバクテリウム属菌）が０．８～４０．０である、［３］～［６］の何れか１項に記載の発酵乳の製造方法。 [7] When adding the above Bifidobacterium bacteria, the total number of the two types of Lactococcus bacteria after culture relative to the number of Bifidobacterium bacteria after culture contained in the fermented milk. The method for producing fermented milk according to any one of [3] to [6], wherein the ratio (Lactococcus/Bifidobacterium) is 0.8 to 40.0.
［８］ 上記発酵乳に含まれる培養後の上記乳酸菌の総菌数が、上記発酵乳１ｍｌ当たり、１．０×１０８～１．０×１０１０である、［１］～［７］の何れか１項に記載の発酵乳の製造方法。 [8] The total number of lactic acid bacteria contained in the fermented milk after culture is 1.0 × 10 8 to 1.0 × 10 10 per ml of fermented milk, [1] to [7]. The method for producing fermented milk according to any one of items 1 to 1.
［９］ 上記原料混合物に含まれる少なくとも一部の乳糖を、乳糖分解酵素を用いて分解することを更に含む、［１］～［８］の何れか１項に記載の発酵乳の製造方法。 [9] The method for producing fermented milk according to any one of [1] to [8], further comprising decomposing at least a portion of lactose contained in the raw material mixture using a lactose degrading enzyme.
［１０］ 上記原料混合物が酵母抽出物を更に含有する、［１］～［９］の何れか１項に記載の発酵乳の製造方法。 [10] The method for producing fermented milk according to any one of [1] to [9], wherein the raw material mixture further contains a yeast extract.
［１１］ 上記発酵乳に含まれる無脂乳固形分が１．０～１５．０質量％である、［１］～［１０］の何れか１項に記載の発酵乳の製造方法。 [11] The method for producing fermented milk according to any one of [1] to [10], wherein the non-fat milk solid content contained in the fermented milk is 1.0 to 15.0% by mass.
［１２］ 上記発酵乳のｐＨが３．９０～５．００である、［１］～［１１］の何れか１項に記載の発酵乳の製造方法。 [12] The method for producing fermented milk according to any one of [1] to [11], wherein the fermented milk has a pH of 3.90 to 5.00.
［１３］ ［１］～［１２］の何れか１項に記載の発酵乳の製造方法により得られる発酵乳。 [13] Fermented milk obtained by the method for producing fermented milk according to any one of [1] to [12].
［１４］ ［１３］に記載の発酵乳を含む発酵乳製品。 [14] A fermented dairy product containing the fermented milk according to [13].
［１５］ 酸味を抑制しつつ複雑で濃厚な香味を発酵乳に付与する発酵乳の香味の増強方法であって、
乳原料の発酵に使用する乳酸菌として、少なくとも、ラクトコッカス・クレモリス及びラクトコッカス・ラクティスからなる２種のラクトコッカス属菌を選択すること、および
乳原料を、上記２種のラクトコッカス属菌を用いて、２９℃～３５℃で７．０時間～１７．０時間発酵させること
を含む、発酵乳の香味の増強方法。
[15] A method for enhancing the flavor of fermented milk, which imparts a complex and rich flavor to fermented milk while suppressing sourness,
At least two types of Lactococcus bacteria consisting of Lactococcus cremoris and Lactococcus lactis are selected as lactic acid bacteria used for fermentation of the milk raw material, and the above two types of Lactococcus bacteria are used for the milk raw material. A method for enhancing the flavor of fermented milk, comprising fermenting at 29° C. to 35° C. for 7.0 hours to 17.0 hours.
本発明により、酸味を抑制しつつ、ヘビーユーザー向けの濃厚で複雑な香味を有する発酵乳の製造方法、発酵乳、及び発酵乳製品を提供することが可能となった。また、本発明により、酸味を抑制しつつ、ヘビーユーザー向けの複雑で濃厚な香味を付与するための発酵乳の香味の増強方法を提供することが可能となった。 According to the present invention, it has become possible to provide a method for producing fermented milk, fermented milk, and fermented milk products that have a rich and complex flavor for heavy users while suppressing sourness. Furthermore, the present invention has made it possible to provide a method for enhancing the flavor of fermented milk for imparting a complex and rich flavor for heavy users while suppressing sourness.
以下、本実施形態について説明する。
特許文献１に開示された発酵乳の製造方法は、特定の中温性の乳酸菌と特定の高温性の乳酸菌とを併用することを要旨とするものであり、具体的に開示された乳酸菌の組み合わせは、中温性のラクトコッカス・クレモリスと高温性のストレプトコッカス・サーモフィラスの組み合わせのみである（実施例１及び実施例２）。また、特許文献２に開示された発酵乳の組み合わせは、ビフィドバクテリウム属菌とラクトコッカス属菌であり、具体的に開示された乳酸菌の組み合わせは、ビフィドバクテリウム・ロンガム（Bifidobacterium longum）とラクトコッカス・ラクティス・サブスピーシーズ・ラクティス（Lactococcus lactis subsp. lactis）のみである。本発明者等による鋭意研究により、特許文献１及び２に開示された上記乳酸菌の併用によっては、所望とする濃厚で複雑な香味を有する発酵乳は得らないことが明らかとなった。
This embodiment will be described below.
The gist of the method for producing fermented milk disclosed in Patent Document 1 is to use a specific mesophilic lactic acid bacterium and a specific thermophilic lactic acid bacterium in combination, and the specifically disclosed combination of lactic acid bacteria is , a combination of mesophilic Lactococcus cremoris and thermophilic Streptococcus thermophilus (Example 1 and Example 2). Further, the combination of fermented milk disclosed in Patent Document 2 is Bifidobacterium and Lactococcus, and the specifically disclosed combination of lactic acid bacteria is Bifidobacterium longum. and Lactococcus lactis subsp. lactis. Through intensive research by the present inventors, it has become clear that fermented milk having the desired rich and complex flavor cannot be obtained by combining the lactic acid bacteria disclosed in Patent Documents 1 and 2.
そして、本発明者等による更なる鋭意研究により、スターターとして、ラクトコッカス・クレモリス及びラクトコッカス・ラクティスという特定の２種のラクトコッカス属菌の存在下、乳原料を、特定温度で特定時間発酵させることが、酸味を抑制しつつ、ヘビーユーザー向けの複雑で濃厚な香味を有する発酵乳を得るために、極めて重要な技術的意義を有することが見出され、本発明を完成するに至ったものである。 Further intensive research by the present inventors has revealed that dairy raw materials are fermented at a specific temperature for a specific period of time in the presence of two specific Lactococcus bacteria, Lactococcus cremoris and Lactococcus lactis, as starters. It was discovered that this has extremely important technical significance in order to obtain fermented milk with a complex and rich flavor suitable for heavy users while suppressing sourness, and this led to the completion of the present invention. It is.
すなわち、本実施形態に係る発酵乳の製造方法は、乳原料を含む原料混合物を調製する原料混合物の調製工程、上記原料混合物に、乳酸菌として少なくとも、ラクトコッカス・クレモリス（Lactococcus lactis subsp. cremoris）及びラクトコッカス・ラクティス（Lactococcus lactis subsp. lactis）を添加する乳酸菌添加工程、および上記乳酸菌を、２９℃～３５℃で７．０時間～１７．０時間培養し、上記乳原料を発酵させる発酵工程を含む。本実施形態に係る発酵乳の製造方法は、加熱殺菌工程、乳糖分解工程、及び冷却工程の一つ以上を更に含んでいてよい。 That is, the method for producing fermented milk according to the present embodiment includes a raw material mixture preparation step of preparing a raw material mixture containing milk raw materials, and at least Lactococcus cremoris (Lactococcus lactis subsp. cremoris) and A lactic acid bacteria addition step in which Lactococcus lactis subsp. lactis is added, and a fermentation step in which the above-mentioned lactic acid bacteria are cultured at 29° C. to 35° C. for 7.0 hours to 17.0 hours to ferment the above milk raw material. include. The method for producing fermented milk according to the present embodiment may further include one or more of a heat sterilization step, a lactose decomposition step, and a cooling step.
本明細書中、発酵乳とは、乳原料を含む混合物中で乳酸菌を培養した発酵物をいい、乳等省令により定められている発酵乳、乳製品乳酸菌飲料、乳酸菌飲料などのいずれであってもよい。発酵乳の一例としてヨーグルトが挙げられ、ヨーグルトは、セットタイプヨーグルトやソフトタイプヨーグルトであってもよいし、ドリンクタイプタイプヨーグルトであってもよい。また、本発明によって製造された発酵乳を、フローズンヨーグルトやチーズの材料として用いることも可能である。また、発酵乳製品とは、上記発酵乳をそのまま、あるいはそれらに希釈またはシロップ添加等の加工処理を施した後、容器に充填・包装したもののことである。 In this specification, fermented milk refers to a fermented product obtained by culturing lactic acid bacteria in a mixture containing milk raw materials, and is any of the fermented milk, dairy products lactic acid bacteria drinks, lactic acid bacteria drinks, etc. specified by the Milk Ministerial Ordinance. Good too. Yogurt is an example of fermented milk, and the yogurt may be a set type yogurt, a soft type yogurt, or a drink type yogurt. Furthermore, the fermented milk produced according to the present invention can also be used as a material for frozen yogurt and cheese. In addition, fermented milk products refer to the fermented milk as it is, or those processed by processing such as dilution or addition of syrup, and then filled and packaged in containers.
本実施形態に係る発酵乳の製造方法に含まれる各工程について以下に説明する。
＜原料混合物の調製工程＞
本実施形態において調製される原料混合物に含まれる原料は、発酵乳を製造するために必要な原料であり、少なくとも乳原料を含む。乳原料としては、生乳、全脂乳、脱脂乳、ホエイ、クリーム等が挙げられ、１種を単独で用いてもよいし、２種以上を用いてもよい。原料混合物には、必要に応じて、酵母抽出物等が更に含まれていてもよい。原料混合物は、公知の方法に従って調製することができ、一形態において、水に脱脂乳等の少なくとも一種の乳原料、および必要に応じて添加される酵母抽出物等を溶解することにより調製される。原料混合物は、一形態において、最終的に得られる発酵乳の無脂乳固形分率（Ｓｏｌｉｄｓ Ｎｏｔ Ｆａｔ；ＳＮＦ）が１．０～１５．０質量％となるよう調整されることが好ましく、ＳＮＦが８．０～１４．０質量％となるよう調整されることがより好ましい。
Each step included in the method for producing fermented milk according to this embodiment will be described below.
<Preparation process of raw material mixture>
The raw materials contained in the raw material mixture prepared in this embodiment are raw materials necessary for producing fermented milk, and include at least milk raw materials. Examples of milk raw materials include raw milk, whole fat milk, skim milk, whey, cream, etc., and one type may be used alone or two or more types may be used. The raw material mixture may further contain yeast extract or the like, if necessary. The raw material mixture can be prepared according to a known method, and in one form, it is prepared by dissolving at least one dairy raw material such as skim milk in water, and yeast extract, etc. added as necessary. . In one form, the raw material mixture is preferably adjusted so that the solids not fat (SNF) of the finally obtained fermented milk is 1.0 to 15.0% by mass, and SNF It is more preferable that the amount is adjusted to 8.0 to 14.0% by mass.
＜加熱殺菌工程＞
本実施形態に係る発酵乳の製造方法は、一形態において、原料混合物の調製工程後に加熱殺菌工程を更に含んでもよい。加熱殺菌工程は、原料混合物を加熱して殺菌する工程である。加熱温度及び加熱時間は、例えば、原料混合物の雑菌を殺菌できる程度に調整して決定されることができ、一形態において、加熱温度は、８５℃～９５℃であってよく、加熱時間は、２０分～３０分であってよい。本実施形態において、加熱殺菌工程には、公知の方法を用いることができる。例えば、加熱殺菌工程では、プレート式熱交換器、チューブ式熱交換器、スチームインジェクション式加熱装置、スチームインフュージョン式加熱装置、通電式加熱装置などによって加熱処理を行えばよく、ジャケット付のタンクによって加熱処理を行ってもよい。
<Heating sterilization process>
In one form, the method for producing fermented milk according to the present embodiment may further include a heat sterilization step after the raw material mixture preparation step. The heat sterilization process is a process of heating and sterilizing the raw material mixture. The heating temperature and heating time can be determined, for example, by adjusting to the extent that germs in the raw material mixture can be sterilized, and in one embodiment, the heating temperature may be 85°C to 95°C, and the heating time is It may be 20 minutes to 30 minutes. In this embodiment, a known method can be used for the heat sterilization step. For example, in the heat sterilization process, heat treatment can be performed using a plate heat exchanger, a tube heat exchanger, a steam injection heating device, a steam infusion heating device, an electric current heating device, etc. Heat treatment may also be performed.
＜乳糖分解工程＞
本実施形態に係る発酵乳の製造方法は、他の形態において、原料混合物に含まれる少なくとも一部の乳糖を、乳糖分解酵素を用いて分解する工程を更に含んでもよい。
<Lactose decomposition process>
In another embodiment, the method for producing fermented milk according to this embodiment may further include a step of decomposing at least a portion of the lactose contained in the raw material mixture using lactase.
乳糖分解工程では、調製された原料混合物に乳糖分解酵素を添加することにより、原料混合物に含まれる少なくとも一部の乳糖を分解する。乳糖は、乳原料に由来して原料混合物に含まれている。乳糖分解酵素としては、例えば、ラクターゼを挙げることができる。ラクターゼは、乳糖を分解して、グルコースとガラクトースとを生成する。添加されるラクターゼの至適ｐＨが中性領域又は酸性領域であれば、添加されるラクターゼの種類は特に限定されない。例えば、市販されているラクターゼを原料混合物に添加することができる。 In the lactose decomposition step, at least part of the lactose contained in the raw material mixture is decomposed by adding a lactose degrading enzyme to the prepared raw material mixture. Lactose is derived from milk raw materials and is contained in the raw material mixture. Examples of lactases include lactase. Lactase breaks down lactose to produce glucose and galactose. The type of lactase to be added is not particularly limited as long as the optimum pH of the lactase to be added is in the neutral range or acidic range. For example, commercially available lactase can be added to the raw material mixture.
本実施形態に係る乳酸菌の製造方法が、加熱殺菌工程と乳糖分解工程とを含む場合、加熱殺菌工程は、乳糖分解工程の前に行ってもよいし、乳糖分解工程の後に行ってもよい。加熱殺菌工程が乳糖分解工程の前に行われる場合、後述する発酵工程において、乳糖分解酵素による乳糖の分解が発酵工程中にも継続されるため、発酵乳における乳糖濃度をさらに低減することができる。また、乳糖分解酵素の原料混合物への添加は、２種のラクトコッカス属菌が原料混合物に添加されるタイミングまでに行われることが好ましく、一形態において、乳糖分解酵素と２種のラクトコッカス属菌とを同時に原料混合物に添加してもよい。 When the method for producing lactic acid bacteria according to the present embodiment includes a heat sterilization step and a lactose decomposition step, the heat sterilization step may be performed before or after the lactose decomposition step. When the heat sterilization step is performed before the lactose decomposition step, the lactose decomposition by the lactose degrading enzyme continues during the fermentation step, which will be described later, so the lactose concentration in the fermented milk can be further reduced. . Further, it is preferable that the lactose-degrading enzyme is added to the raw material mixture before the two types of Lactococcus bacteria are added to the raw material mixture. The bacteria may be added to the raw material mixture at the same time.
ラクターゼが添加された原料混合物を、例えば５℃以上５０℃以下の温度範囲で保持することにより、ラクターゼによる乳糖の分解を促進させることができる。上述した通り、加熱殺菌工程後に乳糖分解工程が行われる場合、ラクターゼによる乳糖の分解は、乳酸菌スターターを原料混合物に添加した後においても継続する。 The decomposition of lactose by lactase can be promoted by maintaining the raw material mixture to which lactase has been added at a temperature range of, for example, 5° C. or higher and 50° C. or lower. As described above, when the lactose decomposition step is performed after the heat sterilization step, the decomposition of lactose by lactase continues even after the lactic acid bacteria starter is added to the raw material mixture.
乳酸菌分解酵素による乳糖の分解は、発酵終了の時点で、発酵乳における乳糖濃度が０．８６質量％以下となるまで行われることが好ましく、０．４３質量％以下となるまで行われることがより好ましく、０質量％となるまで行われることが更に好ましい。 The decomposition of lactose by lactic acid bacteria degrading enzyme is preferably carried out until the lactose concentration in the fermented milk becomes 0.86% by mass or less, more preferably 0.43% by mass or less, at the end of fermentation. Preferably, it is more preferably carried out until it reaches 0% by mass.
＜乳酸菌添加及び発酵工程＞
本実施形態に係る発酵乳の製造方法では、上述した原料混合物に、ラクトコッカス・クレモリス（Lactococcus lactis subsp. cremoris）及びラクトコッカス・ラクティス（Lactococcus lactis subsp. lactis）からなる２種のラクトコッカス属菌を添加する。ラクトコッカス・クレモリス及びラクトコッカス・ラクティスの菌株は市販されており、一般に入手可能である。
<Lactic acid bacteria addition and fermentation process>
In the method for producing fermented milk according to the present embodiment, two types of Lactococcus bacteria, consisting of Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis, are added to the above-mentioned raw material mixture. Add. Strains of Lactococcus cremoris and Lactococcus lactis are commercially available and generally available.
発酵工程において、乳原料の発酵は、２９．０℃～３５．０℃の発酵促進温度域に保持しながら７．０時間～１７．０時間行われる。この発酵促進温度域及び発酵時間は、酸味を抑制しつつ、濃厚で複雑な香味を有する発酵乳を得る観点から、上記２種のラクトコッカス属菌を併用することと技術的に密接な関連性を有するものである。上記発酵促進温度域は、一形態において、３０℃～３２℃であることが好ましい。また、上記発酵時間は、一形態において、１０時間～１２時間であることが好ましい。 In the fermentation process, fermentation of the milk raw material is carried out for 7.0 hours to 17.0 hours while maintaining the fermentation promotion temperature range of 29.0°C to 35.0°C. This fermentation acceleration temperature range and fermentation time are technically closely related to the combination of the above two types of Lactococcus bacteria, from the viewpoint of suppressing sourness and obtaining fermented milk with a rich and complex flavor. It has the following. In one embodiment, the fermentation promoting temperature range is preferably 30°C to 32°C. Further, in one embodiment, the fermentation time is preferably 10 to 12 hours.
ラクトコッカス・クレモリス及びラクトコッカス・ラクティスの原料混合物への添加量の比率は、菌数比（コロニー形成単位（ＦＣＵ）の比）で、ラクトコッカス・クレモリス：ラクトコッカス・ラクティス＝３：４～５：１であってよく、１：１～４：１であってよく、２：１～３：１であってよい。酸味を抑制しつつ、濃厚で複雑な香味を有する発酵乳を得る観点からこのような比率が好ましい。 The ratio of the amounts of Lactococcus cremoris and Lactococcus lactis added to the raw material mixture is the bacterial count ratio (colony forming unit (FCU) ratio): Lactococcus cremoris: Lactococcus lactis = 3:4-5 :1, 1:1 to 4:1, and 2:1 to 3:1. Such a ratio is preferable from the viewpoint of obtaining fermented milk having a rich and complex flavor while suppressing sourness.
本実施形態では、スターターとして上記２種のラクトコッカス属菌に加え、他の乳酸菌を更に添加してもよい。上記２種のラクトコッカス属菌と併用することが好ましい他の乳酸菌として、ラクトバチルス属菌、ストレプトコッカス属菌及びビフィドバクテリウム属菌からなる群から選ばれる乳酸菌が挙げられる。 In this embodiment, in addition to the above two types of Lactococcus bacteria, other lactic acid bacteria may be further added as a starter. Other lactic acid bacteria that are preferably used in combination with the above two species of Lactococcus include lactic acid bacteria selected from the group consisting of Lactobacillus, Streptococcus, and Bifidobacterium.
上記ラクトバチルス属菌としては、ラクトバチルス・ヘルベティカス（Lactobacillus helveticus）、ラクトバチルス・ブルガリクス（Lactobacillus bulgaricus）、ラクトバチルス・カゼイ（Lactobacillus casei）、及びラクトバチルス・アシドフィルス（Lactobacillus acidophilus）からなる群から選択される乳酸菌が挙げられ、上記ストレプトコッカス属菌としては、ストレプトコッカス・サーモフィルス（Streptococcus thermophilus）が挙げられ、上記ビフィドバクテリウム属菌としては、ビフィドバクテリウム・ラクティス（Bifidobacterium lactis）、ビフィドバクテリウム・ビフィダム（Bifidobacterium bifidum）、ビフィドバクテリウム・ロンガム（Bifidobacterium longum）、及びビフィドバクテリウム・インファンティス（Bifidobacterium infantis）からなる群から選択される乳酸菌が挙げられ、これらから選択される１種又は２種以上の乳酸菌を更に添加することができる。 The Lactobacillus bacteria are selected from the group consisting of Lactobacillus helveticus, Lactobacillus bulgaricus, Lactobacillus casei, and Lactobacillus acidophilus. Examples of the Streptococcus bacteria include Streptococcus thermophilus, and examples of the Bifidobacterium bacteria include Bifidobacterium lactis and Bifidobacterium lactis. Examples include lactic acid bacteria selected from the group consisting of Bifidobacterium bifidum, Bifidobacterium longum, and Bifidobacterium infantis; A species or two or more types of lactic acid bacteria can be further added.
原料混合物への乳酸菌の添加方法は特に制限されず、菌末の状態で添加することも、カルチャー（培養物）の状態で添加することもできる。菌末とは、菌を適当な培地で生育させ、遠心分離により分離後、凍結乾燥保護剤と混合してから凍結乾燥し、乾燥品を粉砕後、必要に応じて倍散剤と混合して得られる粉末状の製品である。カルチャーとは、菌を適当な培地で生育させて得られる液状組成物である。 The method of adding lactic acid bacteria to the raw material mixture is not particularly limited, and the lactic acid bacteria can be added in the form of bacterial powder or in the form of a culture. Bacterial powder is obtained by growing bacteria in an appropriate medium, separating it by centrifugation, mixing it with a lyophilization protectant, lyophilizing it, pulverizing the dried product, and mixing it with a dispersing agent if necessary. It is a powdered product. A culture is a liquid composition obtained by growing bacteria in an appropriate medium.
一形態において、ラクトコッカス・クレモリスの原料混合物への添加量は、発酵乳に含まれる培養後のラクトコッカス・クレモリスの菌数が、発酵乳１ｍｌ当たり、好ましくは５．０×１０７～７．０×１０８、より好ましくは５．０×１０７～５．０×１０８、さらに好ましくは５．２×１０７～２．０×１０８となるよう適宜調整される。 In one form, the amount of Lactococcus cremoris added to the raw material mixture is such that the number of Lactococcus cremoris bacteria after culture contained in the fermented milk is preferably 5.0 x 10 7 to 7.0 per ml of fermented milk. It is appropriately adjusted to be 0×10 8 , more preferably 5.0×10 7 to 5.0×10 8 , even more preferably 5.2×10 7 to 2.0×10 8 .
一形態において、ラクトコッカス・ラクティスの原料混合物への添加量は、発酵乳に含まれる培養後のラクトコッカス・ラクティスの菌数が、発酵乳１ｍｌ当たり、好ましくは１．０×１０７～５．０×１０８、より好ましくは１．５×１０７～２．０×１０８、さらに好ましくは２．０×１０７～１．０×１０８となるよう適宜調整される。 In one form, the amount of Lactococcus lactis added to the raw material mixture is such that the number of Lactococcus lactis bacteria after culture contained in the fermented milk is preferably 1.0 x 10 7 to 5.0 per ml of fermented milk. It is appropriately adjusted to be 0×10 8 , more preferably 1.5×10 7 to 2.0×10 8 , even more preferably 2.0×10 7 to 1.0×10 8 .
上記２種のラクトコッカス属菌に加え、上記ラクトバチルス属菌を添加する場合、一形態において、ラクトバチルス属菌の原料混合物への添加量は、発酵乳に含まれる培養後のラクトバチルス属菌の菌数が、発酵乳１ｍｌ当たり、好ましくは１．０×１０８～９．０×１０８、より好ましくは１．５×１０８～７．０×１０８となるよう適宜調整される。また、他の形態において、ラクトバチルス属菌の原料混合物への添加量は、発酵乳に含まれる培養後のラクトバチルス属菌の菌数に対する、培養後の上記２種のラクトコッカス属菌の合計菌数の比率（ラクトコッカス属菌／ラクトバチルス属菌）が、好ましくは０．１～１０．０、より好ましくは０．５～３．０となるよう適宜調整される。後掲の表２において、ラクトコッカス属菌／ラクトバチルス属菌＝Ｌｃ／Ｌｂと表記する。 When adding the above-mentioned Lactobacillus bacteria in addition to the above two types of Lactococcus bacteria, in one form, the amount of Lactobacillus bacteria added to the raw material mixture is the same as the Lactobacillus bacteria contained in the fermented milk after cultivation. The number of bacteria per ml of fermented milk is adjusted as appropriate so that it is preferably 1.0×10 8 to 9.0×10 8 , more preferably 1.5×10 8 to 7.0×10 8 . In another form, the amount of Lactobacillus bacteria added to the raw material mixture is the sum of the two types of Lactococcus bacteria after cultivation, relative to the number of Lactobacillus bacteria contained in the fermented milk after cultivation. The ratio of the number of bacteria (Lactococcus/Lactobacillus) is adjusted as appropriate so that it is preferably 0.1 to 10.0, more preferably 0.5 to 3.0. In Table 2 below, Lactococcus/Lactobacillus = Lc/Lb.
また、上記２種のラクトコッカス属菌に加え、上記ストレプトコッカス属菌を添加する場合、一形態において、ストレプトコッカス属菌の原料混合物への添加量は、発酵乳に含まれる培養後のストレプトコッカス属菌の菌数が、発酵乳１ｍｌ当たり、好ましくは８．０×１０８～１．０×１０9、より好ましくは９．０×１０８～９．５×１０8となるよう適宜調整される。 Furthermore, when adding the above-mentioned Streptococcus bacteria in addition to the above two types of Lactococcus bacteria, in one embodiment, the amount of Streptococcus bacteria added to the raw material mixture is the same as that of the Streptococcus bacteria after culture contained in the fermented milk. The number of bacteria is adjusted as appropriate so that it is preferably 8.0×10 8 to 1.0×10 9 , more preferably 9.0×10 8 to 9.5×10 8 per ml of fermented milk.
また、上記２種のラクトコッカス属菌に加え、上記ビフィドバクテリウム属菌を添加する場合、一形態において、ビフィドバクテリウム属菌の原料混合物への添加量は、発酵乳に含まれる培養後のビフィドバクテリウム属菌の菌数が、発酵乳１ｍｌ当たり、好ましくは２．０×１０６～７．０×１０6、より好ましくは２．５×１０６～６．５×１０６となるよう適宜調整される。また、他の形態において、ビフィドバクテリウム属菌の原料混合物への添加量は、発酵乳に含まれる培養後のビフィドバクテリウム属菌の菌数に対する上記２種のラクトコッカス属菌の合計菌数の比率（ラクトコッカス属菌／ビフィドバクテリウム属菌）が、好ましくは０．８～４０．０、より好ましくは１．０～３５．０となるよう適宜調整される。後掲の表２において、ラクトコッカス属菌／ビフィドバクテリウム属菌＝Ｌｃ／Ｂｂと表記する。 In addition, in addition to the above two types of Lactococcus bacteria, when adding the above Bifidobacterium bacteria, in one form, the amount of Bifidobacterium bacteria added to the raw material mixture is determined by the amount of culture contained in the fermented milk. The number of bacteria of the genus Bifidobacterium is preferably 2.0×10 6 to 7.0×10 6 , more preferably 2.5×10 6 to 6.5×10 6 per ml of fermented milk. It will be adjusted accordingly. In another form, the amount of Bifidobacterium added to the raw material mixture is the sum of the two types of Lactococcus to the number of Bifidobacterium after cultured in the fermented milk. The ratio of the number of bacteria (Lactococcus/Bifidobacterium) is adjusted as appropriate so that it is preferably 0.8 to 40.0, more preferably 1.0 to 35.0. In Table 2 below, Lactococcus/Bifidobacterium is expressed as Lc/Bb.
一形態において、上記２種のラクトコッカス属菌を含む乳酸菌の原料混合物への添加量の合計は、発酵乳に含まれる培養後の乳酸菌の総菌数が、発酵乳１ｍｌ当たり、好ましくは１．０×１０８～１．０×１０１０、より好ましくは１．０×１０9～４．０×１０9となるよう適宜調整される。 In one form, the total amount of lactic acid bacteria containing the two types of Lactococcus bacteria added to the raw material mixture is such that the total number of lactic acid bacteria contained in the fermented milk after culture is preferably 1.0 per ml of fermented milk. It is appropriately adjusted to be 0×10 8 to 1.0×10 10 , more preferably 1.0×10 9 to 4.0×10 9 .
また、本実施形態に係る発酵乳の製造方法により得られる発酵乳は、一形態において、１０℃におけるｐＨが３．９０～５．００となるよう調整されることが好ましい。 Further, in one form, the fermented milk obtained by the fermented milk production method according to the present embodiment is preferably adjusted to have a pH of 3.90 to 5.00 at 10°C.
以下、本実施形態をさらに詳細に説明する。
＜発酵乳の製造例＞
実施例１
水に、脱脂粉乳および酵母エキスを添加して溶解し、以下の組成の原料混合物を得た。次いで、得られた原料混合物を９０℃で２０分間殺菌した。加熱殺菌後の原料混合物に対し、０．０３質量％のラクターゼ（ＧＯＤＯ－ＹＮＬ；合同酒精社製）と、表１に記載の乳酸菌（何れもクリスチャンハンセン社製）を同表に記載の配合量で添加し、３０℃で１０時間培養した。その後、１０℃以下に冷却することにより、発酵乳を得た。尚、表１に記載の乳酸菌の菌数は、乳酸菌１ｍｌ当たりの菌数を示す。
This embodiment will be described in further detail below.
<Production example of fermented milk>
Example 1
Skim milk powder and yeast extract were added and dissolved in water to obtain a raw material mixture having the following composition. Then, the obtained raw material mixture was sterilized at 90° C. for 20 minutes. To the raw material mixture after heat sterilization, add 0.03% by mass of lactase (GODO-YNL; manufactured by Godo Shusei Co., Ltd.) and the lactic acid bacteria listed in Table 1 (all manufactured by Christian Hansen) in the amounts listed in the same table. and cultured at 30°C for 10 hours. Thereafter, fermented milk was obtained by cooling to 10° C. or lower. Note that the number of lactic acid bacteria listed in Table 1 indicates the number of bacteria per ml of lactic acid bacteria.
［原料混合物組成（原料混合物１００ｇ当たり）］
脱脂粉乳（明治脱脂粉乳；明治乳業社製） １３ｇ
ＳＫ酵母エキスＨｉ－ＫＣ（Ｔ）(日本製紙社製) ０．２ｇ
水 ８６．８ｇ
[Raw material mixture composition (per 100g of raw material mixture)]
Skimmed milk powder (Meiji skimmed milk powder; manufactured by Meiji Dairies Co., Ltd.) 13g
SK yeast extract Hi-KC (T) (manufactured by Nippon Paper Industries) 0.2g
Water 86.8g
実施例２～実施例９、比較例１～比較例９
実施例１の発酵乳の製造方法に対し、添加する乳酸菌の種類又は配合量、もしくは発酵温度又は発酵時間を表１に示す条件に変更した以外は、実施例１と同様の条件及び手段により、実施例２～実施例９、比較例１～比較例９の各発酵乳を得た。
Examples 2 to 9, Comparative Examples 1 to 9
Using the same conditions and means as in Example 1, except that the type or amount of lactic acid bacteria to be added, or the fermentation temperature or fermentation time was changed to the conditions shown in Table 1 in the method for producing fermented milk in Example 1. Fermented milks of Examples 2 to 9 and Comparative Examples 1 to 9 were obtained.
表１に示す発酵前後（培養前後）における乳酸菌の菌数の測定方法、得られた発酵乳の無脂乳固形分率（ＳＮＦ）およびｐＨの測定方法は、以下の通りである。 The method for measuring the number of lactic acid bacteria before and after fermentation (before and after culture) shown in Table 1, and the method for measuring the non-fat milk solids fraction (SNF) and pH of the obtained fermented milk are as follows.
＜乳酸菌の菌数の測定方法＞
本発明における各種乳酸菌の菌数の測定方法は、リアルタイムＰＣＲによって計測して得られた値である。
まず、各単一菌種を含む培養液を２等分し、一方を寒天培地に播種し、生菌数を測定した。もう一方の培養液を下記に記載の方法によってＤＮＡを抽出することで、菌数既知のＤＮＡサンプルを得た（スタンダード）。また、各実施例及び比較例の発酵前後それぞれのサンプルからも同様の方法によってＤＮＡを抽出した。
まず、培養液から２００μｌまたは各サンプルから５０μｌ回収し、遠心分離(１２０００ｒｐｍ、５分、４℃)を行った後、上清を除去した。その後、ＴＥＮバッファー(２０ｍＭ Ｔｒｉｓ－ＨＣｌ ｐＨ８．０、５ｍＭ ＥＤＴＡ ｐＨ８．０、１４０ｍＭ ＮａＣｌ)を１．０ｍｌ添加して懸濁した。遠心分離(１２０００ｒｐｍ、５分)を行い、上清を除去した後、ＴＥＮバッファーを１４０μｌ、１００ｍｇ/ｍｌリゾチーム溶液を６０μｌ添加して懸濁し、恒温槽にて３７℃で９０分インキュベートした。その後、１０％ ＳＤＳ溶液を２０μｌ添加し、軽く攪拌してから１００μｌのＴＥ飽和フェノールおよび１００μｌのクロロホルムを添加して激しく攪拌した。遠心分離(１２０００ｒｐｍ、１０分、４℃)を行い、水層２００μｌを新しいチューブに移した後、再び１００μｌのＴＥ飽和フェノールおよび１００μｌのクロロホルムを加え、激しく攪拌した。遠心分離(１２０００ｒｐｍ、１０分、４℃)を行い、水層１８０μｌを新しいチューブに移した後、３Ｍ酢酸ナトリウム (ｐＨ５．２)を２０μｌおよび５００μｌのエタノールを加え、激しく攪拌した。遠心分離（１２０００ｒｐｍ、１０分、４℃）を行い、上清を除去した後、３００μｌの７０％エタノールを添加して洗浄した。遠心分離（１２０００ｒｐｍ、１０分、４℃）を行い、上清を除去した後、５０μｌのＴＥバッファー(１０ｍＭ Ｔｒｉｓ－ＨＣｌ；ｐＨ８．０、１ｍＭ ＥＤＴＡ；ｐＨ８．０)に溶解させた。
<Method for measuring the number of lactic acid bacteria>
The method for measuring the number of various lactic acid bacteria in the present invention is a value obtained by measuring by real-time PCR.
First, a culture solution containing each single bacterial species was divided into two equal parts, one half was inoculated onto an agar medium, and the number of viable bacteria was measured. DNA was extracted from the other culture solution by the method described below to obtain a DNA sample with a known number of bacteria (standard). In addition, DNA was extracted by the same method from the samples before and after fermentation of each Example and Comparative Example.
First, 200 μl from the culture solution or 50 μl from each sample was collected, centrifuged (12000 rpm, 5 minutes, 4° C.), and then the supernatant was removed. Then, 1.0 ml of TEN buffer (20mM Tris-HCl pH8.0, 5mM EDTA pH8.0, 140mM NaCl) was added and suspended. After centrifugation (12,000 rpm, 5 minutes) and removal of the supernatant, 140 μl of TEN buffer and 60 μl of 100 mg/ml lysozyme solution were added and suspended, followed by incubation at 37° C. for 90 minutes in a constant temperature bath. Thereafter, 20 μl of 10% SDS solution was added and stirred gently, and then 100 μl of TE-saturated phenol and 100 μl of chloroform were added and stirred vigorously. After centrifugation (12,000 rpm, 10 minutes, 4°C) and transferring 200 μl of the aqueous layer to a new tube, 100 μl of TE-saturated phenol and 100 μl of chloroform were added again, followed by vigorous stirring. After centrifugation (12000 rpm, 10 minutes, 4°C) and transferring 180 μl of the aqueous layer to a new tube, 20 μl of 3M sodium acetate (pH 5.2) and 500 μl of ethanol were added, followed by vigorous stirring. After centrifugation (12000 rpm, 10 minutes, 4°C) and removal of the supernatant, 300 μl of 70% ethanol was added for washing. After centrifugation (12,000 rpm, 10 minutes, 4°C) and removal of the supernatant, it was dissolved in 50 μl of TE buffer (10 mM Tris-HCl; pH 8.0, 1 mM EDTA; pH 8.0).
段階的に希釈したスタンダード及び各サンプルのＤＮＡを、各乳酸菌種に対し特異的なプライマーを用いてＰＣＲに供し、得られたＣＴ値からスタンダードを元にサンプルの菌数を算出した。
なお、リアルタイムＰＣＲの反応は、下記表１に記載の各プライマーを用いて、９５℃、１５秒間→（９４℃、３０秒間→各プライマーのアニーリング温度、３０秒間→７２℃、３０秒間）×３５～４０サイクルにて、Bifidobacterium lactis、Lactobacillus acidophilus、Lactobacillus bulgaricus、Lactobacillus helveticus、Streptococcus thermophilus、Lactobacillus casei、Lactococcus lactis subsp. cremoris及びLactococcus lactis subsp. lactisの菌数及び総菌数をQuantStudio（登録商標）5 Food Safety Real-Time PCR System（Thermo Fisher Scientific社）により定量した。なお、菌数はサンプル１ｍｌ当りの菌数の対数値で表記した。
The serially diluted standard and DNA of each sample were subjected to PCR using primers specific to each lactic acid bacteria species, and the number of bacteria in the sample was calculated from the obtained CT value based on the standard.
The real-time PCR reaction was performed using each primer listed in Table 1 below at 95°C for 15 seconds → (94°C, 30 seconds → annealing temperature of each primer, 30 seconds → 72°C, 30 seconds) x 35 In ~40 cycles, the bacterial counts and total bacterial counts of Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus helveticus, Streptococcus thermophilus, Lactobacillus casei, Lactococcus lactis subsp. cremoris, and Lactococcus lactis subsp. lactis were measured using QuantStudio® 5 Food. Quantification was performed using the Safety Real-Time PCR System (Thermo Fisher Scientific). The number of bacteria was expressed as a logarithm of the number of bacteria per ml of sample.
＜ＳＮＦの測定方法＞
ＳＮＦ（無脂乳固形分）は、「乳等省令（乳及び乳製品の成分規格等に関する省令）」の「乳等の成分規格の試験法」に記載の定量方法によって測定した。
<Method of measuring SNF>
SNF (non-fat milk solids) was measured by the quantitative method described in the "Test Methods for Ingredient Standards for Milk, etc." of the "Ministerial Ordinance on Milk, etc. (Ministerial Ordinance on Ingredient Standards, etc. for Milk and Dairy Products)".
＜ｐＨの測定方法＞
ｐＨは、ガラス電極法により測定した。ポータブル型ｐＨメーター「Ｄ－７２（株式会社堀場製作所製）」について、ｐＨ標準溶液による校正を実施した。実施例及び比較例で得られたサンプルをビーカーに採取し、「Ｄ－７２」のガラス電極をサンプル中に挿入し、測定(機器の表示値を記録)した。
<Method for measuring pH>
pH was measured by a glass electrode method. A portable pH meter "D-72 (manufactured by Horiba, Ltd.)" was calibrated using a pH standard solution. The samples obtained in Examples and Comparative Examples were collected in beakers, a "D-72" glass electrode was inserted into the samples, and measurements were taken (the values displayed on the device were recorded).
＜試験例：官能評価＞
各発酵乳について、官能評価試験を行った。この官能評価試験は、飲料の開発を担当する訓練された５人のパネラーに委託して行った。培養後、１０℃以下に冷却された各試料について、＜酸味の抑制＞、＜味の複雑さ＞および＜濃厚さ＞の３項目について、以下の評価基準に基づき５人のパネラーの協議により評価点が決定された。官能評価は、陰性対照との対比により行われた。陰性対照として、上記で調製した原料混合物にビフィズスのみを添加し、４０℃で５時間発酵させた発酵乳を用いた。評価結果を表２に示す。
<Test example: Sensory evaluation>
A sensory evaluation test was conducted for each fermented milk. This sensory evaluation test was outsourced to five trained panelists in charge of beverage development. After culturing, each sample was cooled to below 10°C and evaluated in three categories: <suppression of sour taste>, <complexity of taste>, and <richness> based on the following evaluation criteria through discussion among five panelists. The point has been determined. Sensory evaluation was performed by comparison with a negative control. As a negative control, fermented milk in which only bifidus was added to the raw material mixture prepared above and fermented at 40° C. for 5 hours was used. The evaluation results are shown in Table 2.
＜酸味の抑制＞
３：酸味を感じないか、ほとんど感じない。非常に良好。
２：酸味を感じるが、陰性対照よりも弱く、良好。
１：酸味を強く感じる。陰性対照と同程度であり、問題あり。
<Suppression of acidity>
3: No or almost no sourness felt. Very good.
2: A sour taste is felt, but it is weaker and better than the negative control.
1: I feel a strong sour taste. It is at the same level as the negative control, so there is a problem.
＜味の複雑さ＞
３：舌に残る複雑な味わいに持続性があり、味の厚みを非常に感じる。非常に良好。
２：陰性対照のような味の単調さは感じず、複雑な味わいが舌に残り、味の厚みを感じる。良好。
１：陰性対照と同様に味が単調であり、問題あり。
<Complexity of taste>
3: The complex taste that remains on the tongue is persistent, and the taste is very thick. Very good.
2: The taste did not feel monotonous like the negative control, but the complex taste remained on the tongue and the taste was felt to be thick. Good.
1: Similar to the negative control, the taste is monotonous and problematic.
＜濃厚さ＞
３：濃厚さが強く、非常に良好。
２：陰性対照よりも濃厚さを感じ、良好。
１：陰性対照と同程度の濃厚さしかなく、物足りなさを感じる。問題あり。
<Thickness>
3: Strong richness and very good quality.
2: Felt richer than the negative control, good.
1: It is only as rich as the negative control and feels unsatisfactory. There is a problem.
＜総合評価＞
Ａ’：合計点数が９であり、非常に良好。
Ａ：合計点数が８であり、非常に良好。
Ｂ：合計点数が６～７であり、且つ「１」の評価が無く、良好。
Ｃ：合計点数が５以下であるか、評価に「１」があり、問題あり。
<Comprehensive evaluation>
A': Total score is 9, very good.
A: The total score is 8, which is very good.
B: The total score is 6 to 7, and there is no evaluation of "1", which is good.
C: The total score is 5 or less, or there is a "1" in the evaluation, indicating a problem.
なお、本発明は、上記実施形態に限定されるものではなく、実施段階ではその要旨を逸脱しない範囲で種々に変形することが可能である。また、各実施形態は適宜組み合わせて実施してもよく、その場合組み合わせた効果が得られる。更に、上記実施形態には種々の発明が含まれており、開示される複数の構成要件から選択された組み合わせにより種々の発明が抽出され得る。例えば、実施形態に示される全構成要件からいくつかの構成要件が削除されても、課題が解決でき、効果が得られる場合には、この構成要件が削除された構成が発明として抽出され得る。 Note that the present invention is not limited to the above-described embodiments, and can be variously modified at the implementation stage without departing from the gist thereof. Moreover, each embodiment may be implemented in combination as appropriate, and in that case, the combined effect can be obtained. Furthermore, the embodiments described above include various inventions, and various inventions can be extracted by combinations selected from the plurality of constituent features disclosed. For example, if a problem can be solved and an effect can be obtained even if some constituent features are deleted from all the constituent features shown in the embodiment, the configuration from which these constituent features are deleted can be extracted as an invention.
Claims (15)
前記原料混合物に、乳酸菌として少なくとも、ラクトコッカス・クレモリス及びラクトコッカス・ラクティスからなる２種のラクトコッカス属菌と、ラクトバチルス属菌およびストレプトコッカス属菌からなる群から選ばれる１種又は２種以上とを添加すること、および
前記乳酸菌を２９℃～３５℃で７．０時間～１７．０時間培養し、前記乳原料を発酵させること
を含む、発酵乳の製造方法。 preparing a raw material mixture containing a dairy raw material;
In the raw material mixture, at least two types of Lactococcus bacteria consisting of Lactococcus cremoris and Lactococcus lactis , and one or more types selected from the group consisting of Lactobacillus and Streptococcus bacteria are added to the raw material mixture. A method for producing fermented milk, the method comprising: adding, and culturing the lactic acid bacteria at 29° C. to 35° C. for 7.0 hours to 17.0 hours to ferment the milk raw material.
乳原料の発酵に使用する乳酸菌として、少なくとも、ラクトコッカス・クレモリス及びラクトコッカス・ラクティスからなる２種のラクトコッカス属菌と、ラクトバチルス属菌およびストレプトコッカス属菌からなる群から選ばれる１種又は２種以上とを選択すること、および
乳原料を、前記２種のラクトコッカス属菌を用いて、２９℃～３５℃で７．０時間～１７．０時間発酵させること
を含む、発酵乳の香味の増強方法。 A method for enhancing the flavor of fermented milk, which imparts a complex and rich flavor to fermented milk while suppressing sourness,
As lactic acid bacteria used for fermentation of milk raw materials, at least two types of Lactococcus bacteria consisting of Lactococcus cremoris and Lactococcus lactis , and one or two types selected from the group consisting of Lactobacillus and Streptococcus bacteria. and fermenting the milk raw material using the two types of Lactococcus bacteria at 29°C to 35°C for 7.0 hours to 17.0 hours. How to enhance.
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| JP2001120178A (en) | 1999-10-28 | 2001-05-08 | Yakult Honsha Co Ltd | Milk fermented food containing bifidobacterium |
| WO2016186151A1 (en) | 2015-05-18 | 2016-11-24 | 合同酒精株式会社 | Method for producing fermented milk |
| US20190000101A1 (en) | 2015-12-24 | 2019-01-03 | Danone S.A. | Use of Starch for Improving the Preparation of a Strained Fermented Dairy Product |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2001120178A (en) | 1999-10-28 | 2001-05-08 | Yakult Honsha Co Ltd | Milk fermented food containing bifidobacterium |
| WO2016186151A1 (en) | 2015-05-18 | 2016-11-24 | 合同酒精株式会社 | Method for producing fermented milk |
| US20190000101A1 (en) | 2015-12-24 | 2019-01-03 | Danone S.A. | Use of Starch for Improving the Preparation of a Strained Fermented Dairy Product |
Non-Patent Citations (1)
| Title |
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| Schleifer, K H., et al.，Transfer of Streptococcus lactis and Related Streptococci to the Genus Lactococcus gen. nov.，System. Appl. Microbiol.，1985年，Vol.6，p.183-195 |
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