JP3766701B2 - Novel yeast, bread dough containing the yeast, and method for producing the dough - Google Patents

Description

まず、親株のスラントから、１白金耳、ＹＰＤ培地 ５ｍｌに植菌し、３０℃、１６時間振盪培養する。培養終了後、3000rpm、10分で遠心分離して集菌し、0.1M、pH7.0のリン酸バッファーで１回洗浄する。菌体を同じリン酸バッファー５ｍｌに懸濁し、ＥＭＳを０．１５ｍｌ添加し、３０℃、２時間、時々攪拌しながら処理し、３０００ｒｐｍ、１０分の遠心分離で集菌し、リン酸バッファーで洗浄後、20%Na₂S₂O₃を5ml加えて３０℃、１０分静置してＥＭＳを分解する。20%Na₂S₂O₃処理を２回行い、生理食塩水５ｍｌで３回洗浄し、生理食塩水５ｍｌに懸濁し、上記組成のＹＰＤ寒天プレートに適宜希釈して菌を塗布する。
【００５０】
（Ｂ）低温感受性酵母のスクリーニング
B-1.１次スクリーニング：マイクロプレート法
ブロムクレゾールパープル（BCP）を用い、高ｐＨでは青紫色、低ｐＨでは黄色になるBCPの性質を利用して発酵力をスクリーニングする方法である。
【００５１】
ＹＰＤ寒天プレートで生育した株を、１白金耳、ＹＰＤ培地200μlが入ったマイクロプレートに植菌し、３０℃、１６時間静置培養する。マイクロプレートの各ウエルにＢＣＰ含有ＹＰＤ培地（上記ＹＰＤ培地にブロムクレゾールパープル(BCP)30mg加えたもの）200μlを注入し、このマイクロプレート各2枚に、この培養ブロスを20μｌずつそれぞれ移植し、それぞれ１０℃、および３０℃で５時間静置培養する。３０℃培養区で生育が良く菌量が多く、よく発酵するためブロスのｐＨが下がり色が黄変し、かつ１０℃培養区で生育は良いが発酵が抑制されるためブロスのｐＨが下がらず色が黄変していない株を選択する。
【００５２】
B-2.２次スクリーニング：マイセル法：
２次スクリーニングはマイセル発酵の前後の重量差が大きい株を選択する方法である。使用する培地は以下の通りである。
【００５３】
糖蜜培地 マイセル液
糖蜜（糖として） ３０ｇ グルコース ８０ｇ
硫安 ５ｇ （ＮＨ₄ ）₂ ＨＰＯ₄ ５ｇ
燐安 ３ｇ ＫＨ₂ ＰＯ₄ ５ｇ
尿素 ２ｇ 水 １０００ｍｌ
水 １０００ｍｌ
１次スクリーニングで得られた株を糖蜜培地５ｍｌに１白金耳植菌して、３０℃、２４時間振盪培養する。この培養液５ｍｌを糖蜜培地５０ｍｌに移植し、さらに３０℃、２４時間振盪培養する。培養後、2本のフラスコから、培養液を併せて８０ｍｌとり、３０００ｒｐｍ、１０分遠心分離して集菌し、滅菌水で１回洗浄する。100mlのマイセル液に懸濁し、それぞれ５０ｍｌを、１０℃で１５時間、および３０℃で５時間培養した。マイセル発酵の前後の重量差をもってマイセル発酵力とし、３０℃で良く発酵しており、かつ１０℃で発酵力が低い株を選択する。
【００５４】
なお、本願発明の目的である、低温下で発酵力が抑制される酵母を取得する観点からは、変異処理した酵母を、増殖する酵母を死滅させる薬剤を含有するＹＰＤ培地に接種し、低温下で嫌気状態で培養する工程を加えることが、より効果的である。低温で増殖する菌体が死滅するので、スクリーニングの効率はさらに向上するからである。増殖する酵母を死滅させる薬剤としては、例えば、ナイスタチン、アンホテリシンＢ、シクロヘキシミドなどが挙げられるが、比較的低濃度で増殖細胞死滅効果のある薬剤であればいずれでもよい。薬剤の培地中の濃度は、ナイスタチンの場合１０μｇ／ｍｌ〜５０μｇ／ｍｌ、アンホテリシンＢの場合、１００μｇ／ｍｌ〜５００μｇ／ｍｌ、シクロヘキシミドの場合、１０００μｇ／ｍｌ〜５０００μｇ／ｍｌが適当である。
【００５５】
次に、インベルターゼ比活性の低い株のスクリーニング、育種について述べる。
【００５６】
インベルターゼ比活性の低い株の取得方法としては、一つは低温感受性株取得のための親株を選定する前により多くの菌株についてインベルターゼ活性を測定し、所望の範囲の比活性を持つ株を用いて低温感受性株の変異スクリーニング等を行うことが挙げられる。また、インベルターゼ比活性の低い胞子株を単離し、上記方法で得られた低温感受性株の胞子株と交雑株を形成させ、その胞子株を単離し、所望のインベルターゼ比活性の範囲内にある酵母を選択する等の方法を挙げることができるが、その方法については、スクリーニング、変異、交雑、細胞融合等いずれの方法を用いてもよく特に制限はない。
【００５７】
以上の方法で得られた酵母について、各種発酵力、冷蔵耐性、冷凍耐性、製パン性等の評価を行う。評価に使用する菌体は、以下の方法で調製し得る。
【００５８】

培地１００ｍｌを５００ｍｌ容坂口フラスコに分注、殺菌し、新鮮なスラントから１白金耳移植し、３０℃、２４時間振盪培養する。
【００５９】
第２種母：
第１種母と同じ培地１Ｌを５Ｌ容三角フラスコに分注、殺菌後、第１種母を全量植菌し、３０℃、２４時間振盪培養する。
【００６０】

１０Ｌ容ミニジャーファーメンターに培地１Ｌを加えて殺菌し、第２種母全量を加えて２Ｌとする。３０〜３５℃、ｐＨ４〜５（安水でコントロール）、３００ｒｐｍで攪拌しつつ、２ＮＬ／分通気して、１２時間培養する。
【００６１】
本培養：
培地組成：糖蜜（糖として） ３５０ ｇ 流加方式で添加する。
【００６２】
硫安 ４ ｇ
燐安 １ ｇ
尿素 ２．５ ｇ
１０Ｌ容ミニジャーファーメンターに培地１．３Ｌを加えて殺菌し、種培養液７００ｍｌを加えて２Ｌとする。３０〜３５℃、ｐＨ４〜５（安水でコントロール）、３００ｒｐｍで攪拌しつつ、２ＮＬ／分通気して、１０時間培養する。
【００６３】
上記培養ブロスを分離・水洗・濾過して、生菌体を得る。
【００６４】
上記に本発明の酵母のスクリーニング、育種法等について述べたが、低温感受性株の取得過程では１段階の変異処理だけでは、低温下での発酵力は抑制されても３０℃の発酵力も低下傾向にある。また、低インベルターゼ比活性株の取得では、低インベルターゼ比活性株との交雑株からの胞子株の単離という一回の操作では十分に所望の活性の株の取得ができない場合がある。このような場合には、次に挙げるような方法も実施し得る。
【００６５】
即ち、低温感受性株取得の場合には、変異処理を１回以上繰り返す方法、少なくとも１回の変異処理で得られた低温下で発酵が抑制される低温感受性変異株の胞子株と野生の２倍体である酵母の胞子株とを少なくとも１回戻し交雑させる方法、この低温感受性変異株と野生の２倍体である酵母とを少なくとも１回戻し細胞融合させる方法などである。
【００６６】
低インベルターゼ比活性株を取得する場合には、一回の交雑で得られた交雑株の胞子株と低インベルターゼ比活性株との交雑と、その胞子単離を１回以上行うなどの方法である。
【００６７】
なお、戻し交雑とは、変異株と野生株の胞子株を分離し、互いに異なる性をもった胞子株同志を交雑させて交雑株をとり、さらに交雑株から胞子株をとり、野生株の胞子株と交雑を行う操作を１回以上行うことである。これを行うことによって低温感受性（発酵に関する）の遺伝子と、野生株の優秀な発酵力の遺伝子を合わせ持つ菌株を取得することができる。
【００６８】
このように胞子株を取得できれば上述のような戻し交雑法が可能であるが、もし取得した変異株や野生株が胞子形成能が極端に低かったり、胞子出芽率が極端に低いため胞子株の取得が困難な場合は細胞融合法を用いればよい。常法に従ってマーカーをつけてもよいし、融合の頻度が高ければマーカーをつけなくとも融合は可能である。なお、マーカーについては例えば特開昭６３−２９４７７８に記載されているリジン要求株と呼吸欠損株との組み合わせが挙げられる。
【００６９】
【実施例】
次に実施例を挙げて本発明を更に詳細に説明するが、本発明は何等実施例により制限されるものではない。
【００７０】
（実施例１）
商品名カネカレッドイースト（鐘淵化学工業株式会社製）の菌株をFowellプレート（酢酸ナトリウム 0.4%、寒天2%、pH6.7）に塗布し、28℃、2日間培養して胞子を形成させ、得られた胞子株Ｒ−６Ｒを親株として、前記（Ａ）のＥＭＳ法による変異を行い、前記（Ｂ）のスクリーニングを行った結果、低温感受性のサッカロミセス・セレビシエＲ−６１８０−１を得た。
【００７１】
次に、この株に、商品名カネカレッドイーストから分離した多数の胞子株からスクリーニングされたインベルターゼ比活性の非常に低い株サッカロミセス・セレビシエＲ−１３Ｒを交雑させ、胞子を形成させ、生命工学工業技術研究所に寄託されている、インベルターゼ比活性が低く、低温感受性の胞子株サッカロミセス・セレビシエＲ−９１５２（ＦＥＲＭ Ｐ−１４６７５）、サッカロミセス・セレビシエＲ−９１５５（ＦＥＲＭ Ｐ−１４６７６）、およびサッカロミセス・セレビシエＲ−９１５６（ＦＥＲＭ Ｐ−１４６７７）を取得した。
【００７２】
これらの株のジャー培養を行い、インベルターゼ比活性、５℃高糖生地発酵力、３０℃高糖生地発酵力、３０℃復温高糖生地発酵力を測定すると共に、下記に示す方法で通常の加糖中種法菓子パンとストレート法菓子パンを通常の方法と冷蔵工程を経る方法の２つの方法で作製し、なたね置換法による比容積、パンの外観、内相の評価を行い、上記株の評価を行った。
【００７３】
なお、冷蔵貯蔵なしの通常のストレート法の製パンは、下記工程のうち冷蔵貯蔵工程なしとし、それ以外は同様の条件で製パンを行った。
【００７４】
加糖中種法菓子パンの配合及び工程：配合の数字は重量部を表す。
【００７５】

ストレート法による菓子パンの配合および工程：配合の数字は重量部を表す。

結果を表１、２、３及び４に示す。
【００７６】
【表１】

【００７７】
【表２】

【００７８】
【表３】

【００７９】
【表４】

【００８０】
表に示したように、本発明の酵母は、通常のイーストに比較して通常の中種法、ストレート法菓子パンでも良好なパンが得られ、また、冷蔵中の生地の膨張もなく冷蔵保存にともなう比容積の低下、外観、内相の劣化が少なく、菓子パン生地においても、冷蔵生地法において長期に良好なパンが得られることがわかる。
【００８１】
（実施例２）
実施例１と同様の方法で、インベルターゼ比活性が低く、低温感受性であり、かつ冷凍耐性であるサッカロミセス・セレビシエＲ−９１５６（ＦＥＲＭ Ｐ−１４６７７）、サッカロミセス・セレビシエＲ−９１６０（ＦＥＲＭ Ｐ−１４６７８）、およびサッカロミセス・セレビシエＲ−９１９４（ＦＥＲＭ Ｐ−１４６７９）を取得した。
【００８２】
これらの株を実施例１と同様にジャー培養を行い、インベルターゼ比活性、５℃高糖生地発酵力、３０℃高糖生地発酵力、３０℃復温高糖生地発酵力、−２０℃、１週間冷凍後の復温高糖生地発酵力を測定し、さらに、以下に示す方法で冷凍生地菓子パンを作製し、上記株の冷凍耐性を評価した。
【００８３】
配合：配合の数字は重量部を表す。
【００８４】
強力粉 ９０
薄力粉 １０
砂糖 ３０
食塩 １
油脂 ８
イースト ４
脱粉 ２
全卵 １０
イーストフード ０．１
水 ５０

なお、冷凍しない場合は、上記工程のうち、冷凍、解凍工程なしとし、それ以外は同様の条件で製パンを行った。
【００８５】
結果を表５、６および７に示す。
【００８６】
【表５】

【００８７】
【表６】

【００８８】
【表７】

【００８９】
表に示したように、本発明の酵母は、通常のイーストに比較して、冷凍保存にともなう比容積の低下、外観、内相の劣化が少なく、菓子パン生地においても、冷凍生地法において長期に良好なパンが得られることがわかる。
【００９０】
【発明の効果】
本発明の酵母を用いることにより、糖濃度の高い菓子パン等のリッチなパンにおいて、中種法、ストレート法、どちらにおいても従来の酵母に比べて良好なパンが得られる。さらに、この良好な性質を低温感受性株に導入することにより、冷蔵生地法、リタード法および冷凍生地法において、発酵が抑制され、生地膨張が少なく、冷蔵、冷凍後でも発酵力が低下せずに、良好なパンを得ることができる。特に、糖濃度が高いリッチなパン製法においても、従来不可能であった長期安定的に冷蔵、冷凍生地法において良好なパンが提供される。[0001]
[Industrial application fields]
The present invention relates to a novel yeast, bread dough containing the yeast, and a method for producing the dough. More specifically, it contains a novel yeast useful for pastry such as Danish and croissant including retard process, confectionery bread with high sugar concentration, and refrigerated or frozen dough in all types of bread, and this yeast. The present invention relates to bread dough and a method for producing the bread dough.
[0002]
[Prior art]
Conventionally, there has been a demand for baker's yeast that can produce good sweet bread with high fermentability even in sweet bread dough with a sugar concentration of 25% or more. About screening of yeast with high osmotic pressure resistance or invertase specific activity of yeast, etc. Consideration is being made. However, the relationship between the latter invertase specific activity and the fermenting power in confectionery bread dough has not been sufficiently studied, and invertase specific activity for fermenting power after refrigeration and freezing in high sugar doughs such as confectionery bread dough in particular. Currently, little research has been done on the involvement of. Therefore, the inventions focusing on the invertase specific activity are only those relating to baker's yeast having no or almost no invertase specific activity disclosed in JP-A-61-231993 and JP-A-3-80073. However, these inventions relate to a fructooligosaccharide non-assimilating yeast bred for use in fructooligosaccharide-containing bread. Therefore, these yeasts have the disadvantage that they are not sufficiently fermentable in high sugar dough such as confectionery bread to which a large amount of sugar is added, and are not practical enough as yeast for producing good confectionery bread. .
[0003]
From this situation, the invertase specific activity is more than enough to break down the sugar in the dough, but no yeast has been developed that has a very low specific activity and high fermentability of high sugar dough. Currently.
[0004]
Also, in the recent bakery industry, the frozen dough method is actively used to meet the needs of both rationalization of processes through labor saving and variety of varieties to meet various consumer needs. It has become like this. Furthermore, the so-called refrigerated dough method for aging the dough at the same time that the dough is stored in a refrigerated state without being frozen has been actively studied. However, in the case of conventional baker's yeast, the fermentation rate is slower than that at room temperature even if the dough is cooled to a low temperature, but the fermentation proceeds. Therefore, storage in the refrigerated dough is at most about one day. Although it has been put into practical use, it has not yet been widely applied.
[0005]
In the manufacture of pastries such as Danish and croissants, a so-called “retard method” is employed in which the dough is subjected to low-temperature treatment in order to fold the roll-in margarine into a beautiful layer. However, due to some dough fermentation in this retarding process, the roll-in margarine is not folded neatly, the resulting bread layer is not clean, and the refrigerated storage of the dough is practically impossible. This is not possible, leading to a decrease in the yield of the bread making process.
[0006]
In order to solve such problems, JP-A-5-76348, JP-A-5-24896, and JP-A-5-336872 describe yeasts that do not ferment during refrigeration but ferment above a certain temperature (cold-sensitive yeasts). However, the fermenting power after refrigerated storage (corresponding to the rewarming fermenting power in this specification) is about 60% of that before refrigerated storage. Therefore, when bread is produced using refrigerated dough, there is a disadvantage that high-quality bread with volume cannot be produced due to lack of fermentation power.
[0007]
In addition, the decrease in regenerative fermenting power of low-temperature sensitive yeast becomes more pronounced when the sugar concentration in the dough is increased, and in high sugar dough such as confectionery bread dough with an added amount of sugar per flour of 25% or more. The present condition is that the low temperature sensitive yeast in which the fermenting power after refrigeration has the fermenting power equivalent to that before refrigeration has not been developed yet.
[0008]
Even in the frozen dough method, after mixing the dough, some fermentation (so-called pre-freezing fermentation) occurs in the freezing process, and ethyl alcohol is produced in the dough, reducing the freezing tolerance of the yeast, opening and closing the freezer in the distribution process, etc. Due to the temperature rise due to poor storage conditions, there was a problem that even frozen doughs were partially thawed due to temperature rise and the quality deteriorated. And in order to suppress the fermentation before freezing, in the production of frozen dough, mixing is done at a low temperature like a refrigerator to keep the mixing temperature extremely low, and a huge amount of energy is used such as using a spiral quick freezer for quick freezing. What is needed is the actual situation.
[0009]
In order to solve such problems, Japanese Patent Application Laid-Open No. 4-234939 provides a baker's yeast having low-temperature sensitive and freezing-resistant properties, but the baker's yeast has a considerably higher fermenting power than ordinary baker's yeast. It is weak and problematic in terms of practicality.
[0010]
[Problems to be solved by the invention]
Therefore, there has been a demand for baker's yeast that can be sufficiently fermented with conventional medium seed method and straight method confectionery bread dough.
[0011]
In addition, the refrigerated dough method that can be distributed even in the refrigerated state without freezing the dough, and the dough expansion in the retarding process is less than conventional baker's yeast, has high commercial value, and can use the remaining dough and have a high product yield It has been desired to provide a novel yeast capable of producing a Lee product, a bread dough containing the yeast, and a method for producing the dough.
[0012]
Furthermore, it has been desired to provide a yeast having a high reheating fermentation power even when the fermenting power does not decrease even after refrigerated storage and the high sugar dough is stored refrigerated.
[0013]
In the frozen dough method, the ability to freeze is improved by quickly suppressing fermentation during the freezing process of the dough, and the production of frozen dough that is resistant to temperature rise due to storage conditions during the frozen distribution process. There has been a demand for a novel yeast that can be used and a frozen dough containing the yeast.
[0014]
[Means for Solving the Problems]
The present invention has been made to solve the above-mentioned problems, and the object of the invention is that the high sugar dough fermenting power is very high, and a good confectionery bread can be obtained by a normal medium seed method or straight method. Another object of the present invention is to provide a yeast that has refrigeration resistance and / or freezing resistance, has a small decrease in fermentation power after refrigeration and freezing storage, and is resistant to refrigeration storage with a high sugar dough.
[0015]
The present inventors conducted extensive yeast screening using strains obtained from nature or by screening from conserved strains or breeding operations such as mutation treatment, hybridization, and cell fusion. Yeast having a high sugar-dough fermentation was isolated, although the invertase specific activity was sufficient to decompose sugar in the dough, but the specific activity was quite low. As a result of further screening and breeding from these yeasts, the present inventors succeeded in obtaining the target strain and completed the present invention.
[0016]
The present invention belongs to the genus Saccharomyces and has the following properties:
(1) Invertase specific activity is 10 units or more, 150 units or less, and
(2) The fermentation power at 30 ° C. for 115 minutes in the high sugar dough is 350 ml or more,
It is related with the yeast which has.
[0017]
In a preferred embodiment, the yeast of the present invention further has the following properties:
(3) Fermentation power in high sugar dough at 5 ° C. for 24 hours is 10 ml or less, and
(4) The reheated high sugar dough fermentation power at 30 ° C. is 90% or more of the high sugar dough fermentation power at 115 ° C. for 115 minutes.
[0018]
In a preferred embodiment, the yeast of the present invention further has the following properties:
(5) It has the property that the reheated high sugar dough fermentation power at 30 ° C. after refrigerated storage at −20 ° C. for 1 week is 90% or more of the high sugar dough fermentation power at 115 ° C. for 115 minutes.
[0019]
As yeast having the above properties, the following yeasts:
Saccharomyces cerevisiae R-9152 (FERM P-14675),
Saccharomyces cerevisiae R-9155 (FERM P-14676),
Saccharomyces cerevisiae R-9156 (FERM P-14777),
Saccharomyces cerevisiae R-9160 (FERM P-14678), or
Saccharomyces cerevisiae R-9194 (FERM P-14679).
[0020]
The yeast can be suitably used for ordinary confectionery bread dough, refrigerated and / or frozen bread dough.
[0021]
The present invention further relates to a bread dough containing at least one yeast as described above. In a preferred embodiment, the bread dough is an ordinary confectionery bread dough, a refrigerated bread dough or a frozen bread dough.
[0022]
Moreover, this invention relates to the manufacturing method of the bread dough containing at least 1 sort (s) of the yeast as described above, In a suitable embodiment, it is a manufacturing method of normal confectionery bread dough, refrigerated bread dough, or frozen bread dough.
[0023]
By using the yeast of the present invention, which has a high sugar dough fermentation power and is highly fermented even after refrigeration and freezing, it is possible to produce good confectionery bread, which is the object of the present invention, and to have a good quality. This makes it possible to produce pastries by the retard method with a high yield, and further, frozen dough with high freezing resistance. In addition, it becomes possible to produce confectionery bread using high sugar dough by the refrigerated dough method. Therefore, the object of the present invention is achieved. The present invention will be specifically described below.
[0024]
The yeast of the present invention has at least the following properties:
(1) Invertase specific activity is 10 units or more, 150 units or less, and
(2) The fermentation power at 30 ° C. for 115 minutes in the high sugar dough is 350 ml or more,
Have
[0025]
If the invertase specific activity is lower than 10 units, the sugar in the dough cannot be decomposed sufficiently, so that sufficient fermenting power cannot be obtained. If the invertase specific activity is higher than 150 units, the decomposition rate of sugar in the high sugar dough increases. The osmotic pressure increases. As a result, fermentation is inhibited and sufficient high-sugar dough fermentation power cannot be obtained. On the other hand, if it is higher than 150 units, the sugar is decomposed more quickly during the refrigeration of the high sugar dough, so that the osmotic pressure rises and the regenerative fermentation power decreases. From such a point, the invertase specific activity is preferably 150 units or less.
[0026]
The yeast of the present invention further has the property that the fermentation power at 30 ° C. for 115 minutes in a high sugar dough is 350 ml or more. Since yeast having such properties is imparted with resistance at low temperatures and has sufficient fermentative power during bread production, the object of the present invention can be achieved.
[0027]
The yeast of the present invention further has the following properties:
(3) Fermentation power in high sugar dough at 5 ° C. for 24 hours is 10 ml or less, and
(4) The reheated high sugar dough fermentation power at 30 ° C. may be 90% or more of the high sugar dough fermentation power at 115 ° C. for 115 minutes.
[0028]
Because the fermenting power of high sugar dough at 5 ° C for 24 hours is 10ml or less, there is almost no expansion of the dough during refrigeration, making it easy to mold the dough by the retard method, etc., and the remaining dough can be reused become. In addition, the fermenting power of the reheated high-sugar dough at 30 ° C is 90% or more of the high-sugar dough fermentation power at 30 ° C for 115 minutes, so that sufficient fermenting power is maintained even after refrigeration. Good bread can be obtained.
[0029]
The yeast of the present invention further has the following properties:
(5) The reheated high sugar dough fermentation power at 30 ° C. after refrigerated storage for 1 week at −20 ° C. may be 90% or more of the high sugar dough fermentation power at 115 ° C. for 115 minutes. .
[0030]
When the property (5) is further added to the property (3), the freezing resistance is particularly enhanced, and a good bread with a large volume can be obtained even in the frozen dough method.
[0031]
Specific examples of yeast having the above properties include the following yeasts:
Saccharomyces cerevisiae R-9152 (FERM P-14675),
Saccharomyces cerevisiae R-9155 (FERM P-14676),
Saccharomyces cerevisiae R-9156 (FERM P-14777),
Saccharomyces cerevisiae R-9160 (FERM P-14678), or
Saccharomyces cerevisiae R-9194 (FERM P-14679).
[0032]
The property of the yeast of the present invention can be measured as follows.
[0033]
Invertase specific activity is defined as twice the amount (mg) of glucose produced from sucrose per minute by 1 g of cells as dry weight.
[0034]
Specifically, about 50 mg of yeast cells as a dry matter weight was suspended in 5 ml of 0.1 M acetate buffer (pH 5.0), and 0.2 ml thereof was added to 0.8 ml of 0.1 M acetate buffer, Further, 1 ml of 15 (W / V)% sucrose buffer solution is added and reacted at 30 ° C. for 10 minutes. The reaction was stopped by adding 1 ml of 1N NaOH, neutralized by adding 1 ml of 1N HCl, centrifuged (3000 rpm, 5 minutes) to remove the cells, and the glucose concentration of the supernatant was determined by the glucostat method (trade name: Glucose B-Test Wako: manufactured by Wako Pure Chemical Industries). The concentration of yeast to be measured is appropriately diluted depending on the level of invertase specific activity, and the measurement is performed in a range where the absorbance is linear in the measurement by the glucostat method.
[0035]
The dry weight of yeast is defined as the weight after drying yeast cells at 105 ° C. for 5 hours.
[0036]
Fermentation power in various high sugar doughs was measured as follows according to the high sugar dough fermenting power determined by the yeast industry association.
[0037]
(5 ℃ high sugar dough fermentation power)
The following ingredients:
100 g flour
Yeast 3 g
Sucrose 30 g
Salt 0.5 g
52 ml of water
Mix the dough with a temperature of 20 ± 1 ° C, put the dough into a cylinder, measure the amount of dough at the start in advance, and ferment the dough after 24 hours at 5 ° C. The difference from the amount of dough at the time was defined as 5 ° C. high sugar dough fermentation power.
(30 ℃ high sugar dough fermentation power)
Mixing the dough so that the kneading temperature is 30 ± 1 ° C by mixing the above ingredients, putting the dough into a cylinder, measuring the amount of dough at the start beforehand, and fermenting at 30 ° C for 115 minutes The difference between the amount and the amount of dough at the start is defined as 30 ° C. high sugar dough fermentation power.
[0038]
(30 ° C reheated high sugar dough fermentation power)
The reheating fermentation power is measured by fermenting a high sugar dough prepared in the same manner as the 5 ° C. high sugar dough fermentation power measurement at 5 ° C. for 24 hours and then reheating to 30 ° C. After rewarming, the fermentative power at 30 ° C. for 115 minutes is taken as 30 ° C. rewarmed high sugar dough fermenting power.
[0039]
(Fermentation power of reheated high sugar dough after 1 week of freezing)
Refreezing fermenting power after freezing is the same as the measurement of 30 ° C high sugar dough fermenting power. Fermented with high sugar dough at 30 ° C for 1 hour, degassed, frozen at -20 ° C for 1 week, and then reheated to 30 ° C. To measure. After the rewarming, the fermenting power at 30 ° C. for 115 minutes is defined as the rewarming high sugar dough fermenting power after one week of freezing.
[0040]
The bread dough of the present invention contains one or more yeasts having the above properties. The bread dough is preferably a high sugar dough such as a confectionery bread dough, a refrigerated dough, or a frozen dough. The bread dough of the present invention includes pastry or confectionery bread dough, such as Danish, croissant, bread, butter roll, Chinese bun, yeast donut, etc., all using baker's yeast as a swelling agent.
[0041]
The present invention further relates to a method for producing the above bread dough. The ingredients of the bread dough can be formulated according to each bread dough known to those skilled in the art.
[0042]
By using the yeast of the present invention, the high sugar dough fermenting power is increased and the high sugar dough fermenting power after refrigeration and freezing is increased because the invertase specific activity is sufficient to decompose sugar in the dough. However, it is considered that the effect of screening and breeding a considerably low yeast is great. In addition, although it is not clear why the fermentative power is suppressed at low temperatures, yeast fermentation uses extracellular sugars (glucose, fructose, sucrose, etc.) by the sugar permease (permease) present in the cell membrane. It is said that the transported saccharides are classified into ethyl alcohol and carbon dioxide gas by glycolytic enzymes, and the sugar permeation enzyme present in the cell membrane is especially in the case of the yeast. A protein specifically induced by temperature (heat shock protein) whose activity decreases at low temperature, and that part or all of the glycolytic enzyme present in the cytoplasm decreases in low temperature in the case of the yeast In the case of the yeast, the fermentation is inhibited, and in addition, the fermentation is delayed by suppressing the respiration ability particularly at a low temperature.
[0043]
In addition, it is generally known that a gene called LOW TEMPERATURE SENSITIVE (LTS) exists in the genus Saccharomyces, but this is related to a phenomenon in which "growth" is suppressed or killed at low or high temperatures. This is different from the phenomenon in which “fermentation” at low temperatures is suppressed.
[0044]
Hereinafter, a method for obtaining the yeast of the present invention will be described.
[0045]
First, a method for obtaining low-temperature sensitive yeast will be described.
[0046]
The yeast can be obtained by screening from nature, nitrosoguanidine, ethylmethanesulfonate, mutation treatment such as ultraviolet irradiation, breeding operations such as hybridization and cell fusion. Here, a method for obtaining a target yeast by mutation treatment is disclosed, but it goes without saying that the method for obtaining a yeast of the present invention is not limited to this method.
[0047]
In addition, when the single-stage mutation treatment is not sufficient, it is possible to use a method of repeating the mutation treatment, a method of obtaining a spore and backcrossing, a method of cell fusion, and the like. Details will be described later.
[0048]
(A) Yeast mutation method
Here, a mutation method using ethyl methanesulfonate (hereinafter referred to as EMS) will be described. The culture media used are as follows.
[0049]

First, 1 platinum ear and 5 ml of YPD medium are inoculated from the parent strain slant and cultured with shaking at 30 ° C. for 16 hours. After completion of the culture, the cells are collected by centrifugation at 3000 rpm for 10 minutes, and washed once with a phosphate buffer of 0.1 M, pH 7.0. Suspend the cells in 5 ml of the same phosphate buffer, add 0.15 ml of EMS, treat at 30 ° C. for 2 hours with occasional stirring, collect the cells by centrifugation at 3000 rpm for 10 minutes, and wash with phosphate buffer After, 20% Na ₂ S ₂ O _Three Add 5 ml of the solution, and leave it at 30 ° C. for 10 minutes to decompose EMS. 20% Na ₂ S ₂ O _Three The treatment is performed twice, washed 3 times with 5 ml of physiological saline, suspended in 5 ml of physiological saline, and appropriately diluted on a YPD agar plate having the above composition, and the bacteria are applied.
[0050]
(B) Screening for low temperature sensitive yeast
B-1. Primary screening: Microplate method
This is a method of screening fermentative power using bromcresol purple (BCP) by utilizing the property of BCP which becomes blue-violet at high pH and yellow at low pH.
[0051]
A strain grown on a YPD agar plate is inoculated into a microplate containing 1 platinum loop and 200 μl of YPD medium, followed by stationary culture at 30 ° C. for 16 hours. 200 μl of BCP-containing YPD medium (30 mg of bromocresol purple (BCP) added to the above YPD medium) was injected into each well of the microplate, and 20 μl of this culture broth was transplanted to each of the two microplates. Incubate at 10 ° C. and 30 ° C. for 5 hours. The broth grows well in the 30 ° C culture zone, has a large amount of bacteria, and ferments well, so the pH of the broth falls and the color turns yellow, and the growth in the 10 ° C culture zone is good but the fermentation is suppressed, so the pH of the broth does not drop Select a strain whose color is not yellowed.
[0052]
B-2. Secondary screening: Mycell method:
The secondary screening is a method for selecting a strain having a large weight difference before and after the mycelium fermentation. The culture media used are as follows.
[0053]
Molasses culture medium
Molasses (as sugar) 30g Glucose 80g
Ammonium sulfate 5g (NH _Four ) ₂ HPO _Four 5g
Phosphorus 3g KH ₂ PO _Four 5g
Urea 2g Water 1000ml
1000ml of water
One platinum loop of the strain obtained in the primary screening is inoculated into 5 ml of molasses medium and cultured with shaking at 30 ° C. for 24 hours. 5 ml of this culture solution is transplanted to 50 ml of molasses medium and further cultured with shaking at 30 ° C. for 24 hours. After culturing, 80 ml of the culture solution is taken from two flasks, collected by centrifugation at 3000 rpm for 10 minutes, and washed once with sterilized water. The suspension was suspended in 100 ml of mycelium, and 50 ml of each was cultured at 10 ° C for 15 hours and at 30 ° C for 5 hours. The difference in weight before and after the mycel fermentation is used as the fermentability of mycell, and a strain that is well fermented at 30 ° C and low in fermentability at 10 ° C is selected.
[0054]
In addition, from the viewpoint of obtaining yeast whose fermentability is suppressed at low temperatures, which is the object of the present invention, inoculated the mutant-treated yeast into a YPD medium containing a drug that kills the growing yeast, It is more effective to add a step of culturing in an anaerobic state. This is because the cells that grow at a low temperature are killed, and the screening efficiency is further improved. Examples of the agent for killing the growing yeast include nystatin, amphotericin B, cycloheximide and the like, and any agent can be used as long as it has a proliferation cell killing effect at a relatively low concentration. The concentration of the drug in the medium is suitably 10 μg / ml to 50 μg / ml for nystatin, 100 μg / ml to 500 μg / ml for amphotericin B, and 1000 μg / ml to 5000 μg / ml for cycloheximide.
[0055]
Next, screening and breeding of strains with low invertase specific activity will be described.
[0056]
One method for obtaining strains with low invertase specific activity is to measure the invertase activity for more strains before selecting a parent strain for cold-sensitive strain acquisition, and use strains with a specific activity in the desired range. Examples include performing mutation screening of low-temperature sensitive strains. Further, a spore strain having a low invertase specific activity is isolated, a spore strain of a low-temperature-sensitive strain obtained by the above method is formed and a hybrid strain is isolated, the spore strain is isolated, and a yeast within the range of the desired invertase specific activity There are no particular limitations on the method, and any method such as screening, mutation, hybridization, cell fusion, etc. may be used.
[0057]
About the yeast obtained by the above method, various fermentative power, refrigeration tolerance, freezing tolerance, bread-making property, etc. are evaluated. The microbial cells used for evaluation can be prepared by the following method.
[0058]

Dispense and sterilize 100 ml of medium in a 500 ml Sakaguchi flask, transfer 1 platinum loop from a fresh slant, and shake culture at 30 ° C. for 24 hours.
[0059]
Second kind mother:
Dispense 1 L of the same medium as the first seed mother into a 5 L Erlenmeyer flask, sterilize, inoculate the entire first seed mother, and culture by shaking at 30 ° C. for 24 hours.
[0060]

1 L of medium is added to a 10 L mini jar fermenter to sterilize, and the total amount of the second seed mother is added to 2 L. Incubate at 30 to 35 ° C., pH 4 to 5 (controlled with aqueous water), agitation at 300 rpm and aeration at 2 NL / min for 12 hours.
[0061]
Main culture:
Medium composition: molasses (as sugar) 350 g Add in fed-batch mode.
[0062]
Ammonium sulfate 4 g
Phosphorus 1 g
Urea 2.5 g
Sterilize by adding 1.3 L of medium to a 10 L mini jar fermenter, and add 700 ml of seed culture to 2 L. Incubate at 30 to 35 ° C., pH 4 to 5 (controlled with low water) and aeration at 2 rpm / min for 10 hours while stirring at 300 rpm.
[0063]
The culture broth is separated, washed and filtered to obtain viable cells.
[0064]
The yeast screening and breeding methods of the present invention have been described above. In the process of obtaining a low-temperature sensitive strain, the fermentation power at 30 ° C. tends to decrease even if the fermentation power at low temperature is suppressed by only one stage of mutation treatment. It is in. Moreover, in the acquisition of a low invertase specific activity strain, there may be a case where it is not possible to acquire a sufficiently active strain by a single operation of isolating a spore strain from a hybrid strain with a low invertase specific activity strain. In such a case, the following method can also be implemented.
[0065]
That is, in the case of obtaining a cold-sensitive strain, a method of repeating the mutation treatment one or more times, a spore strain of a cold-sensitive mutant strain that is inhibited by low-temperature fermentation obtained by at least one mutation treatment, and twice that of the wild For example, a method of backcrossing a yeast spore strain at least once, and a method of cell fusion of this cold-sensitive mutant strain and a wild diploid yeast at least once.
[0066]
In the case of obtaining a low invertase specific activity strain, it is a method of crossing a spore strain of a hybrid strain obtained by a single crossing with a low invertase specific activity strain and performing spore isolation at least once. .
[0067]
Backcrossing refers to isolating mutant and wild spore strains, crossing spore strains having different genders to each other to obtain a hybrid strain, further taking a spore strain from the hybrid strain, The operation of crossing with a stock is to be performed once or more. By doing this, it is possible to obtain a strain having both a low-temperature sensitive (fermentation-related) gene and a wild-type gene with excellent fermentative ability.
[0068]
If the spore strain can be obtained in this way, the backcross method as described above is possible, but if the obtained mutant strain or wild strain has extremely low spore-forming ability or the spore germination rate is extremely low, If acquisition is difficult, a cell fusion method may be used. A marker may be attached according to a conventional method, and if the frequency of fusion is high, fusion is possible without attaching a marker. Examples of the marker include combinations of lysine-requiring strains and respiratory deficient strains described in JP-A-63-294778.
[0069]
【Example】
EXAMPLES Next, although an Example is given and this invention is demonstrated still in detail, this invention is not restrict | limited by any Example.
[0070]
Example 1
The strain of Kaneka Red Yeast (manufactured by Kaneka Chemical Co., Ltd.) was applied to a Fowell plate (sodium acetate 0.4%, agar 2%, pH 6.7) and cultured at 28 ° C for 2 days to form spores. Using the obtained spore strain R-6R as a parent strain, mutation by the EMS method of the above (A) and screening of the above (B) were carried out, and as a result, low temperature sensitive Saccharomyces cerevisiae R-6180-1 was obtained.
[0071]
Next, this strain was crossed with Saccharomyces cerevisiae R-13R having a very low invertase specific activity screened from a large number of spore strains isolated from the trade name Kaneka Red Yeast to form spores. Low invertase specific activity, low temperature sensitive spore strains Saccharomyces cerevisiae R-9152 (FERM P-14675), Saccharomyces cerevisiae R-9155 (FERM P-14676), and Saccharomyces cerevisiae R deposited in the laboratory -9156 (FERM P-14777) was obtained.
[0072]
These strains are subjected to jar culture, and invertase specific activity, 5 ° C. high sugar dough fermentation power, 30 ° C. high sugar dough fermentation power, and 30 ° C. rewarmed high sugar dough fermentation power are measured. The sweetened middle-class confectionery bread and the straight confectionery bread are prepared by two methods, the normal method and the method of passing through the refrigeration process, and the specific volume, the appearance of the bread and the internal phase are evaluated by the rapeseed replacement method. Went.
[0073]
In addition, the bread making of the normal straight method without refrigerated storage did not carry out the refrigerated storage process among the following processes, and performed breadmaking on the same conditions other than that.
[0074]
Formulation and process of sweetened middle-class confectionery bread: the numbers in the formula represent parts by weight.
[0075]

Formulation and process of confectionery bread by the straight method: The numbers in the formula represent parts by weight.

The results are shown in Tables 1, 2, 3 and 4.
[0076]
[Table 1]

[0077]
[Table 2]

[0078]
[Table 3]

[0079]
[Table 4]

[0080]
As shown in the table, the yeast of the present invention can be obtained with a normal medium seed method or a straight method confectionery bread in comparison with ordinary yeast, and can be stored in a refrigerator without the expansion of the dough during the refrigeration. It can be seen that there is little reduction in specific volume, appearance, and deterioration of the internal phase, and that good bread can be obtained for confectionery bread dough for a long time by the refrigerated dough method.
[0081]
(Example 2)
In the same manner as in Example 1, Saccharomyces cerevisiae R-9156 (FERM P-14777), Saccharomyces cerevisiae R-9160 (FERM P-14678) are low invertase specific activity, low-temperature sensitive, and freeze-resistant , And Saccharomyces cerevisiae R-9194 (FERM P-14679).
[0082]
These strains were subjected to jar culture in the same manner as in Example 1, and were subjected to invertase specific activity, 5 ° C. high sugar dough fermentation power, 30 ° C. high sugar dough fermentation power, 30 ° C. rewarmed high sugar dough fermentation power, −20 ° C., 1 The reheated high-sugar dough fermenting power after weekly freezing was measured, and frozen dough cake bread was prepared by the method shown below, and the freezing tolerance of the strain was evaluated.
[0083]
Formulation: Formulation numbers represent parts by weight.
[0084]
Powerful powder 90
Soft flour 10
Sugar 30
Salt 1
Fats and oils 8
East 4
Powdering 2
Whole egg 10
East Food 0.1
Water 50

In addition, when not freezing, it was set as the absence of freezing and thawing | decompression process among the said processes, and bread-making was performed on the same conditions except it.
[0085]
The results are shown in Tables 5, 6 and 7.
[0086]
[Table 5]

[0087]
[Table 6]

[0088]
[Table 7]

[0089]
As shown in the table, the yeast of the present invention is less reduced in specific volume, appearance, and deterioration of the internal phase due to freezing compared to normal yeast, and in the pastry dough as well as in the frozen dough method for a long time. It can be seen that good bread is obtained.
[0090]
【The invention's effect】
By using the yeast of the present invention, in a rich bread such as a confectionery bread having a high sugar concentration, a good bread can be obtained in both the middle seed method and the straight method as compared with the conventional yeast. Furthermore, by introducing this good property into a low-temperature sensitive strain, fermentation is suppressed in the refrigerated dough method, retard method and frozen dough method, so that dough expansion is small, and fermenting power does not decrease even after refrigeration and freezing. Can get good bread. In particular, even in a rich bread manufacturing method with a high sugar concentration, a good bread can be provided in a long-term stable refrigerated and frozen dough method, which has been impossible in the past.

Claims (8)

It belongs to Saccharomyces cerevisiae, and has the following properties: (1) Invertase specific activity is 10 units or more and 150 units or less; (3) Fermentation power of high sugar dough at 5 ° C. for 24 hours is 10 ml or less, (4) Fermentation power of reheated high sugar dough at 30 ° C. is 90% or more of high sugar dough fermentation power at 115 ° C. for 115 minutes, 95 %, And (5) -20 ° C., the re-heated high sugar dough fermentation power at 30 ° C. after freezing storage for 1 week is 90% or more of the high sugar dough fermentation power at 30 ° C. for 115 minutes, 92.5 % Or less, and
Here, the high-sugar dough fermenting power at 5 ° C. for 24 hours has the following ingredients: 100 g flour, 3 g yeast, 30 g sucrose, 0.5 g salt, and 52 ml water. Mixing so that the dough is put in a cylinder, the amount of dough at the start is measured in advance, the difference between the amount of dough after fermentation at 5 ° C for 24 hours and the amount of dough at the start,
The high-sugar dough fermenting power at 30 ° C. for 115 minutes has the following ingredients: 100 g flour, 3 g yeast, 30 g sucrose, 0.5 g salt and 52 ml water so that the koji temperature is 30 ± 1 ° C. Mixing, putting the dough into a cylinder, measuring the amount of dough at the start in advance, and the difference between the amount of dough after fermentation at 30 ° C for 115 minutes and the amount of dough at the start,
The dough having 100 g of wheat flour, 3 g of yeast, 30 g of sucrose, 0.5 g of salt and 52 ml of water was fermented at 30 ° C. for 24 hours, and then reheated to 30 ° C. Later measured at 30 ° C, 115 minutes fermentation power,
The dough having the fermentative power of reheated high sugar dough at 30 ° C after freezing storage at -20 ° C for 1 week is fermented at 30 ° C for 1 hour with flour 100g, yeast 3g, sucrose 30g, salt 0.5g and water 52ml. After degassing, freezing at −20 ° C. for 1 week, and reheating to 30 ° C., fermenting power at 30 ° C. for 115 minutes,
Invertase activity is defined as twice the amount (mg) of glucose produced from sucrose per minute by 1 g of cells as dry weight.
yeast. The yeast, Sa Kkaromisesu cerevisiae R-9156 (FERMP-14677) , Saccharomyces cerevisiae R-9160 (FERM P-14678 ), or a Saccharomyces cerevisiae R-9194 (FERM P-14679 ), according to claim 1 Yeast as described in.   Bread dough containing at least one yeast according to any one of claims 1 to 2.   The bread dough according to claim 3, wherein the bread dough is refrigerated dough.   The bread dough according to claim 3, wherein the bread dough is a frozen dough.   A method for producing bread dough comprising at least one yeast according to any one of claims 1 to 2.   The method for producing bread dough according to claim 6, wherein the bread dough is refrigerated dough.   The method for producing bread dough according to claim 6, wherein the bread dough is frozen dough.