【発明の詳細な説明】[Detailed description of the invention]
〈産業上の利用分野〉
本発明は酵母の培養法、特に醤油、味噌等の醸
造食品の製造に好適な生菌数が多く、発酵能の強
い酵母を効率良く、連続的に得る方法に関する。
〈従来の技術〉
従来、醤油、味噌等の醸造において、別途人為
的に分離、培養された酵母を、該醸造工程で添加
する方法が一般に実施されている。
酵母の培養方法は、一般には回分培養法、流加
培養法、連続培養法の三方法があるが、醤油、味
噌等の醸造において用いられる酵母は、回分培養
法によつて培養されている。
また酵母の培養条件、特に溶存酸素に関する研
究には、例えば亜硫酸パルプ排液中の糖類を主な
炭素源とする培地でトルラ酵母を連続培養するに
際し、酸素ガス単独もしくは酸素と空気との混合
ガスを通気して培地中の溶存酸素濃度を３〜20p.
p.m.の範囲に保つもの（特開昭57−58881号公
報）、パン酵母の通気培養において、高濃度酸素
含有空気を培養槽に導入して、培養液中の溶存酸
素濃度を酸素分圧で340mmHg（30℃、16p.p.m.）
を超えないように制御するもの（特公昭51−9833
号公報）、サツカロミセス（Saccharomyces）属
の糖類を主炭素源とする通気培養法において菌濃
度が25％（酵母含水率68％）を超える場合に、培
養液中のアルコール濃度及び溶存酸素濃度を一定
範囲内に抑えるように栄養源を添加して培養する
もの（特開昭53−124677号公報）等がある。
〈発明が解決しようとする問題点〉
以上の如く、醤油、味噌等の醸造においては、
初発よりすべての栄養源を加えて行なう回分培養
法が簡単であり、よく利用されているのである
が、この方法では収率が悪く、また生産性も悪い
という欠点があつた。
さらに、得られた酵母を、醤油、味噌等の醸造
工程、例えば仕込初期の諸味に充分量の菌体を添
加してもすぐにはアルコールを生成せず、酵母が
発酵してアルコールを生成する迄に数日間を要
し、この間に野生酵母が繁殖する等の欠点があつ
た。
また酵母の培養液に糖等の栄養物を培養時間の
経過とともに少量ずつ添加する流加培養法も行な
われているのであるが、この流加培養法は、添加
する栄養物の濃度を任意に調節できる点に特徴が
あるのであるが、さらに生菌数の多い酵母を収率
よく、効率的に得るためには、培地中に添加され
る栄養物の濃度を常に一定範囲に制御することが
必要とされ、この制御を如何に簡単かつ確実に行
なうかが課題とされている。
いずれにしても、従来の醤油、味噌等の醸造に
おいて用いられている酵母の培養は、回分培養
法、流加培養法ともに、酵母の増殖末期で培養を
終了するものであり、それ以降における培養液を
制御して培養するものではない。
一方、前記した特開昭57−58881号公報等酵母
の連続培養法においては、酵母の増殖末期以降に
おける培養液の溶存酸素量に着目して培養液を制
御して酵母を培養するものではない。
本発明は、上述した課題を解決すべく成したも
のであつて、その目的とするところは、酵母を培
養するに際し、培養液中の溶存酸素量を常に最適
範囲内に簡単かつ確実に制御し、もつて醤油、味
噌等の醸造食品の製造に好適な生菌数が多く、発
酵能の強い酵母を効率良く、連続的に得る酵母の
培養法を提供することにある。
〈問題点を解決するための手段〉
本発明者等は、酵母を培養するに際し、増殖末
期以降における培養液中の溶存酸素を1p.p.m.以
下となるようにして培養することにより、上記課
題を解決できることを知り、本発明を完成した。
即ち、本発明は、酵母を培養するに際し、増殖
末期以降における培養液中の溶存酸素を1p.p.m.
以下となるようにして培養することを特徴とする
酵母の連続培養法である。
以下、本発明を詳細に説明する。
先ず、本発明に用いられる酵母としては、チゴ
サツカロミセス（Zygosaccharomyces）属、カ
ンデイダ（Candida）属、ピヒア（Pichia）属、
ハンゼヌラ（Hansenula）属等酵母菌であれば特
に制限はないが、チゴサツカロミセス・ルキシ
ー、カンデイダ・エツケルシー及びカンデイダ・
ベルサチルスに属する、例えばチゴサツカロミセ
ス・ルキシー（Zygosaccharomyces rouxii）
ATCC13356、ATCC14679、IAM4114、カンデ
イダ・エツケルシー（Candida etchellsii）
IFO1229、カンデイダ・ベルサチルス（Candida
versatilis）IFO10038、IFO10056等の醤油、味噌
等の醸造に通常用いられる酵母が、耐塩性を有す
るための雑菌汚染を防止する意味で好適に用いら
れる。
そして酵母培養培地としては、酵母が利用し得
る炭素源、窒素源、無機塩類、その他酵母の生育
に必要な成分を、適宜配合した合成培地、天然培
地が用いられる。
なお醤油、味噌等の如く、高食塩濃度の諸味へ
の添加を意図する場合には、該培地の食塩濃度を
５〜18％（Ｗ／Ｖ）程度に調整することが望まし
い。
次に酵母の培養は、振盪培養、通気培養、攪拌
培養、静置培養等の好気的、嫌気的培養等の適宜
な方法が採用されるが、特に好気的培養が好適で
ある。そして、例えば、通気培養を行なう場合、
その通気量は、培養液10当たり２〜20／
min.程度通気するのが望ましい。またその他の
培養温度、培地のPH等の培養条件は、通常酵母の
培養に用いられる条件が適用でき、さらに必要に
より、種培養、本培養等適宜拡大して培養するこ
ともできる。
このようにして酵母を培養すると、培養初期の
誘導期を経て、増殖期に移行し、菌体は著しく増
加する。通常、培養開始後、18〜24時間程度で増
殖は止まり、増殖末期以降は定常期に移行する。
回分培養法においては、ほぼこの時期に培養を
終了するのであるが、本発明においては、この増
殖末期以降における培養液中の溶存酸素を1p.p.
m.以下、好ましくは0.1〜1p.p.m.、より好ましく
は、0.1〜0.2p.p.m.となるように維持して培養す
る。
維持法としては、例えば、培養液の攪拌回数を
制御する、通気ガスの酸素分圧を制御する等の方
法を単独であるいは組み合わせて用いることがで
きる。
この培養液中の溶存酸素を1p.p.m.以下とする
のは、1p.p.m.を超えた溶存酸素量で培養して得
られた酵母は、菌体量は充分あつてもすぐには発
酵せず、アルコールの生成が遅れるからである。
一方、培養液中の溶存酸素があまり少なくなり
過ぎると、酵母の菌体量が低下する。
従つて培養液中の溶存酸素を上記した範囲、即
ち1p.p.m.以下、好ましくは0.1〜1p.p.m.、より好
ましくは0.1〜0.2p.p.m.の範囲となるように維持
して培養する。
このようにして増殖末期以降の培養液中の溶存
酸素を特定の範囲内に維持して培養することによ
り、生菌数が多く、発酵能の強い酵母を効率よく
得ることができる。
〈実施例〉
以下、実施例を示して本発明をさらに詳細に説
明する。
実施例 １
生醤油：15％（Ｖ／Ｖ）、グルコース：７％
（Ｗ／Ｖ）、食塩：11％（最終、Ｗ／Ｖ）をそれぞ
れ含有する液体培地（PH5.5）２を基礎培地と
し、これを５容ジヤーフアーメンターに投入
し、120℃、15分間オートクレーブで殺菌した。
該培地にチゴサツカロミセス・ルキシー
（Zygosaccharomyces rouxii）ATCC13356の培
養液を107個／mlとなるように接種し、温度30℃、
通気量1V／Ｖ・m.、攪拌数400r.p.m.で培養を開
始した。そして培養開始後18時間経過した時点
（増殖末期）により該培養液に添加用培地〔生醤
油：15％（Ｖ／Ｖ）、グルコース：７％（Ｗ／
Ｖ）、食塩：11％（最終、Ｗ／Ｖ）を含有する液
体培地（PH5.0）を120℃、15分間オートクレーブ
で殺菌したもの〕をシリコンチユーブを通じ、80
ml／hr.（希釈率0.04V／Ｖ・hr.）の速度で供給し
つつ、温度30℃、培養液のPH5.0、通気量1V／
Ｖ・m.、第１表に記載した溶存酸素量で、それ
ぞれ定常状態まで連続培養を行なつた。得られた
酵母培養液の生菌数を測定した。そしてこの酵母
のエタノール発酵能を判定するため、醤油諸味培
地（NaCl：14.8％、T.N.：1.2％、PH：5.60）100
ｇに上記培養液を５ml接種後、30℃で７日間静置
培養し、エタノール生成量を測定した。その結果
を第１表及び第１図に示した。
<Industrial Application Field> The present invention relates to a method for culturing yeast, and in particular to a method for efficiently and continuously obtaining yeast with a large number of viable bacteria and strong fermentation ability suitable for the production of brewed foods such as soy sauce and miso. <Prior Art> Conventionally, in the brewing of soy sauce, miso, etc., a method has generally been practiced in which yeast that has been separately artificially isolated and cultured is added during the brewing process. There are generally three methods for culturing yeast: a batch culture method, a fed-batch culture method, and a continuous culture method. Yeast used in brewing soy sauce, miso, etc. is cultured by the batch culture method. In addition, for research on yeast culture conditions, especially dissolved oxygen, for example, when continuously culturing Torula yeast in a medium whose main carbon source is sugars in sulfite pulp wastewater, oxygen gas alone or a mixture of oxygen and air is required. Aerate the medium to reduce the dissolved oxygen concentration in the medium to 3 to 20p.
In the aerated culture of baker's yeast, high-concentration oxygen-containing air is introduced into the culture tank, and the dissolved oxygen concentration in the culture solution is maintained at 340 mmHg (oxygen partial pressure). (30℃, 16p.pm)
(Tokukoku Sho 51-9833)
When the bacterial concentration exceeds 25% (yeast moisture content: 68%) in the aeration culture method using sugars of the genus Saccharomyces as the main carbon source, the alcohol concentration and dissolved oxygen concentration in the culture solution are kept constant. There is a method in which a nutrient source is added to the culture so as to keep it within a certain range (Japanese Patent Application Laid-open No. 124677/1983). <Problems to be solved by the invention> As mentioned above, in the brewing of soy sauce, miso, etc.
The batch culture method, in which all nutritional sources are added from the beginning, is simple and often used, but this method has the drawbacks of low yield and poor productivity. Furthermore, even if the obtained yeast is added to a sufficient amount of bacteria during the brewing process of soy sauce, miso, etc., for example to moromi at the initial stage of preparation, alcohol will not be produced immediately, but the yeast will ferment and produce alcohol. It took several days to complete the process, and there were drawbacks such as the proliferation of wild yeast during this time. There is also a fed-batch culture method in which nutrients such as sugar are added little by little to the yeast culture solution over the course of culture time. It is unique in that it can be adjusted, but in order to obtain yeast with a large number of viable bacteria in a high yield and efficiently, it is necessary to always control the concentration of nutrients added to the medium within a certain range. The problem is how to easily and reliably perform this control. In any case, in the conventional cultivation of yeast used in the brewing of soy sauce, miso, etc., in both the batch culture method and the fed-batch culture method, the culture is terminated at the final stage of yeast growth, and the subsequent culture is It is not a method for culturing by controlling the liquid. On the other hand, in the continuous cultivation method of yeast such as the above-mentioned Japanese Unexamined Patent Publication No. 57-58881, yeast is not cultured by controlling the culture solution by focusing on the amount of dissolved oxygen in the culture solution after the final stage of yeast growth. . The present invention has been made to solve the above-mentioned problems, and its purpose is to easily and reliably control the amount of dissolved oxygen in the culture medium always within the optimum range when culturing yeast. To provide a yeast culture method for efficiently and continuously obtaining yeast with a large number of viable bacteria and strong fermentation ability suitable for producing brewed foods such as soy sauce and miso. <Means for solving the problem> The present inventors have solved the above problem by culturing yeast in such a way that the dissolved oxygen in the culture solution after the final stage of growth is 1 p.pm or less. Knowing that this problem could be solved, the present invention was completed. That is, when culturing yeast, the present invention reduces dissolved oxygen in the culture solution to 1 p.pm after the final stage of growth.
This is a continuous yeast culture method characterized by culturing in the following manner. The present invention will be explained in detail below. First, yeasts used in the present invention include the genus Zygosaccharomyces, the genus Candida, the genus Pichia,
There is no particular restriction as long as it is a yeast of the genus Hansenula, etc., but yeasts such as Chigosatsucharomyces ruxii, Candida etskelsii, and Candida
Belonging to Versacillus, for example Zygosaccharomyces rouxii
ATCC13356, ATCC14679, IAM4114, Candida etchellsii
IFO1229, Candida versacillus
Yeasts commonly used for brewing soy sauce, miso, etc., such as IFO10038 and IFO10056, are preferably used to prevent bacterial contamination due to their salt tolerance. As the yeast culture medium, a synthetic medium or a natural medium containing a carbon source, a nitrogen source, inorganic salts, and other necessary components for the growth of yeast that can be used by yeast are appropriately blended. Note that when it is intended to be added to moromi with a high salt concentration such as soy sauce, miso, etc., it is desirable to adjust the salt concentration of the medium to about 5 to 18% (W/V). Next, for culturing the yeast, appropriate methods such as aerobic or anaerobic culture such as shaking culture, aeration culture, stirring culture, static culture, etc. are employed, and aerobic culture is particularly preferred. For example, when performing aerated culture,
The aeration rate is 2 to 20 per 10 of the culture solution.
It is desirable to ventilate the area to a minimum of 1. In addition, other culture conditions such as culture temperature and pH of the medium can be those normally used for yeast culture, and if necessary, culture can be expanded as appropriate such as seed culture and main culture. When yeast is cultured in this manner, it passes through a lag phase at the initial stage of culture and then enters a growth phase, where the number of bacterial cells increases significantly. Normally, proliferation stops approximately 18 to 24 hours after the start of culture, and after the final stage of proliferation, it shifts to the stationary phase. In the batch culture method, the culture ends at approximately this stage, but in the present invention, dissolved oxygen in the culture medium after this end stage of growth is reduced to 1 p.p.
m.m. or less, preferably 0.1 to 1 p.pm, more preferably 0.1 to 0.2 ppm, and cultured. As a maintenance method, for example, methods such as controlling the number of times the culture solution is stirred or controlling the oxygen partial pressure of the aeration gas can be used alone or in combination. The reason why the dissolved oxygen in this culture solution is set to 1 p.pm or less is because yeast obtained by culturing with a dissolved oxygen amount exceeding 1 p.pm will not ferment immediately even if there is a sufficient amount of bacterial cells. This is because the production of alcohol is delayed. On the other hand, if the dissolved oxygen in the culture solution becomes too low, the amount of yeast cells will decrease. Therefore, the culture is carried out while maintaining the dissolved oxygen in the culture solution within the above-mentioned range, that is, 1 p.pm or less, preferably 0.1 to 1 p.pm, more preferably 0.1 to 0.2 ppm. By culturing while maintaining the dissolved oxygen in the culture solution within a specific range after the final stage of growth, yeast with a large number of viable bacteria and strong fermentation ability can be efficiently obtained. <Examples> Hereinafter, the present invention will be explained in more detail by showing examples. Example 1 Raw soy sauce: 15% (V/V), glucose: 7%
(W/V), salt: 11% (final, W/V) 2 liquid medium (PH5.5) was used as the basal medium, and this was put into a 5-volume jar fermenter, and heated at 120℃ for 15 minutes. Sterilize by autoclaving for minutes.
A culture solution of Zygosaccharomyces rouxii ATCC13356 was inoculated into the medium at a concentration of 10 7 cells/ml, and the temperature was 30°C.
Culture was started at an aeration rate of 1 V/V·m. and a stirring rate of 400 rpm. Then, 18 hours after the start of culture (end of growth), the culture solution was added with a medium [raw soy sauce: 15% (V/V), glucose: 7% (W/V)].
V), salt: A liquid medium (PH5.0) containing 11% (final, W/V) sterilized in an autoclave at 120°C for 15 minutes] was passed through a silicone tube at 80°C.
While supplying at a rate of ml/hr. (dilution rate 0.04V/V・hr.), the temperature was 30℃, the pH of the culture solution was 5.0, and the aeration rate was 1V/hr.
Continuous cultivation was carried out at the dissolved oxygen levels listed in Table 1 until a steady state was reached. The number of viable bacteria in the obtained yeast culture solution was measured. In order to determine the ethanol fermentation ability of this yeast, soy sauce moromi medium (NaCl: 14.8%, TN: 1.2%, PH: 5.60) was used at 100%
After inoculating 5 ml of the above culture solution into G. g, the culture solution was statically cultured at 30° C. for 7 days, and the amount of ethanol produced was measured. The results are shown in Table 1 and Figure 1.
【表】
第１表及び第１図に示した如く、増殖末期以降
における培養液中の溶存酸素を1p.p.m.を超える
量にすると、生菌数は高くなるが、諸味中でのエ
タノール生成に遅れが生じ、逆に溶存酸素を
0.05p.p.m.未満とするとエタノール生成の遅れは
ほとんどないが、生菌数が低いレベルとなる。
しかしながら、本発明の範囲内である溶存酸素
を0.6〜0.7p.p.m.に維持した場合、生菌数も高く、
かつ諸味中に添加してもエタノール生成の遅れを
短縮できる発酵能の強い酵母を得ることができ
る。
実施例 ２
希釈率を0.06V／Ｖ・hr.とし、溶存酸素を0.1
〜0.2p.p.m.及び1.1〜1.4p.p.m.とする以外は実施
例１と全く同様にして酵母の連続培養を行ない、
培養液を得た。そして実施例１と同様に酵母生菌
数及び醤油諸味培地中でのエタノール生成量を測
定し、その結果を第２表及び第２図に示した。[Table] As shown in Table 1 and Figure 1, if the amount of dissolved oxygen in the culture solution exceeds 1 p.pm after the end of growth, the number of viable bacteria will increase, but the ethanol production in moromi will decrease. A delay occurs, and on the contrary, dissolved oxygen
If it is less than 0.05 ppm, there will be almost no delay in ethanol production, but the number of viable bacteria will be at a low level. However, when dissolved oxygen is maintained at 0.6 to 0.7 ppm, which is within the scope of the present invention, the number of viable bacteria is also high;
Moreover, it is possible to obtain a yeast with strong fermentation ability that can shorten the delay in ethanol production even when added to moromi. Example 2 The dilution rate was 0.06V/V・hr., and the dissolved oxygen was 0.1
Yeast was continuously cultured in exactly the same manner as in Example 1 except that the concentrations were ~0.2 ppm and 1.1 to 1.4 ppm,
A culture solution was obtained. Then, in the same manner as in Example 1, the number of viable yeast cells and the amount of ethanol produced in the soy sauce moromi medium were measured, and the results are shown in Table 2 and Figure 2.
【表】
第２表及び第２図に示した如く、溶存酸素を
0.1〜0.2p.p.m.に維持した場合、1p.p.m.を超えて
維持した場合に比べ、生菌数はほとんど変わらな
いにもかかわらず、諸味中でのエタノール生成の
遅れは認められなかつた。
実施例 ３
希釈率を0.08V／Ｖ・hr.とし、溶存酸素を
0.05p.p.m.未満、0.1〜0.2p.p.m.及び1.1〜1.5p.p.
m.とする以外は、実施例１と全く同様にして酵
母の連続培養を行ない、培養液を得た。そして実
施例１と同様に酵母生菌数及び醤油諸味培地中で
のエタノール生成量を測定し、その結果を第３表
及び第３図に示した。[Table] As shown in Table 2 and Figure 2, dissolved oxygen
When the concentration was maintained at 0.1 to 0.2 ppm, no delay in ethanol production in the moromi was observed, although the number of viable bacteria was almost unchanged compared to when the concentration was maintained at over 1 p.pm. Example 3 The dilution rate was 0.08V/V・hr., and the dissolved oxygen was
Less than 0.05ppm, 0.1~0.2ppm and 1.1~1.5ppm
Continuous culture of yeast was carried out in exactly the same manner as in Example 1 except that the yeast was cultured to obtain a culture solution. Then, as in Example 1, the number of viable yeast cells and the amount of ethanol produced in the soy sauce moromi medium were measured, and the results are shown in Table 3 and Figure 3.
【表】
第３表及び第３図に示した如く、溶存酸素を
1p.p.m.を超える量にすると、生菌数は高くなる
が諸味中でのエタノール生成に遅れが生じ、逆に
溶存酸素を0.05p.p.m.未満とするとエタノール生
成の遅れはほとんどないが、生菌数が低くなる。
しかしながら、溶存酸素を0.1〜0.2ppmとする
と、生菌数も高く、かつ醤油諸味中でのエタノー
ル生成の遅れは認められなかつた。
〈発明の効果〉
本発明によれば、著しく生菌数が多く、諸味に
添加してもエタノール生成の遅れが短縮でき、従
つてすぐにエタノールを生成するため、野生酵母
等の繁殖を抑制することができる酵母を効率良
く、連続的に得ることができ、特に醤油、、味噌
等の醸造食品の製造に好適に用いることができ、
本発明は産業上極めて有意義である。[Table] As shown in Table 3 and Figure 3, dissolved oxygen
If the amount exceeds 1p.pm, the number of viable bacteria will increase, but there will be a delay in ethanol production in the moromi.On the other hand, if the amount of dissolved oxygen is less than 0.05ppm, there will be almost no delay in ethanol production, but the number of viable bacteria will decrease. It gets lower.
However, when dissolved oxygen was set to 0.1 to 0.2 ppm, the number of viable bacteria was high, and no delay in ethanol production in soy sauce moromi was observed. <Effects of the Invention> According to the present invention, the number of viable bacteria is significantly large, and even when added to moromi, the delay in ethanol production can be shortened, and therefore ethanol is produced immediately, suppressing the proliferation of wild yeast, etc. It is possible to efficiently and continuously obtain yeast that can produce yeast, and it can be particularly suitably used in the production of brewed foods such as soy sauce and miso.
The present invention is extremely significant industrially.
【図面の簡単な説明】[Brief explanation of the drawing]
第１〜３図は、醤油諸味培地に酵母培養液を接
種し、30℃、７日間静置培養した時のエタノール
の経日的な生成量を示す図である。
Figures 1 to 3 are diagrams showing the amount of ethanol produced over time when a yeast culture solution was inoculated into a soy sauce moromi medium and cultured stationary at 30°C for 7 days.