JPWO2010061575A1 - Yeast low-temperature fermentation promoter, yeast growth medium using the same, and method for producing fermented food and drink - Google Patents
Yeast low-temperature fermentation promoter, yeast growth medium using the same, and method for producing fermented food and drink Download PDFInfo
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Abstract
本発明の目的は、発泡性アルコール飲料などのように、製造時に低温での酵母発酵が行われる場合に、酵母の増殖活性の鈍化によって発酵効率が低下する問題を解決するものである。大豆蛋白質からグロブリンの一種であるβ−コングリシニンに富む画分を分画し、この画分を酵素分解したペプチドが酵母の低温増殖能を顕著に強化する作用を示すことを見出した。すなわち、本発明は、大豆β−コングリシニンの分解物を含むことを特徴とする酵母の低温発酵促進剤である。The object of the present invention is to solve the problem that fermentation efficiency decreases due to slowing of the growth activity of yeast when yeast fermentation is performed at a low temperature during production, such as effervescent alcoholic beverages. A fraction rich in β-conglycinin, a kind of globulin, was fractionated from soy protein, and the peptide obtained by enzymatic degradation of this fraction was found to have an effect of remarkably enhancing the low-temperature growth ability of yeast. That is, the present invention is a yeast low-temperature fermentation promoter characterized by containing a degradation product of soybean β-conglycinin.
Description
本発明は、酵母により低温域で発酵させて得られる食品の製造において、低温における酵母の発酵促進剤に関する。 The present invention relates to a yeast fermentation promoter at low temperatures in the production of foods obtained by fermentation at low temperatures with yeast.
酵母は、醤油・味噌・パン・酒といった多くの発酵飲食品を製造する過程で使われている。また、一部の酵母は分子生物学のモデル生物としても利用されている。このように酵母は産業界や学術界で広く利用されている微生物であり、温度や培地組成などの培養条件に関しては活発な研究が展開されている。酵母が効率良く増殖する上で、重要な環境因子の一つが温度である。酵母の至適温度は酵母の種類によって異なるが、30℃付近の温度でよく生育するものが多く、15℃以下という低温においては、酵母の増殖能力は大きく低下する。これは酵母の生育が環境ストレスの1つである低温ストレスによって阻害されるためと考えられる。 Yeast is used in the process of producing many fermented foods and drinks such as soy sauce, miso, bread, and sake. Some yeasts are also used as model organisms for molecular biology. Thus, yeast is a microorganism that is widely used in industry and academia, and active research is being conducted on culture conditions such as temperature and medium composition. One of the important environmental factors for the efficient growth of yeast is temperature. The optimum temperature of yeast varies depending on the type of yeast, but many of them grow well at temperatures around 30 ° C., and at low temperatures of 15 ° C. or less, the ability of yeast to grow is greatly reduced. This is thought to be because the growth of yeast is inhibited by low-temperature stress, which is one of environmental stresses.
また、酵母の増殖を左右するものに菌体外の栄養源の存在がある。酵母が増殖するには、糖などの炭素源、アミノ酸や蛋白質などの窒素源の他に、ビタミンなどの微量の栄養源が必要である。窒素源としては、通常硫酸アンモニウムのような無機物や、アミノ酸・タンパク質のような有機物が使われており、大豆を加水分解した大豆ペプチドも窒素源として利用されつつある。例えばビールや麦芽使用率が低いか又は麦芽を使用しないビール様飲料などの発泡性アルコール飲料の製造における酵母発酵において、大豆ペプチドが使用されている。しかしながら、発泡性アルコール飲料では、15℃以下という低温で発酵がしばしば行われているが、上述のように低温ストレスによって酵母の増殖活性が鈍くなり、これによって発酵効率が低下すると、発酵時間を長くする必要がある。この場合、風味の点では利点があるが、より短時間で低温発酵を終了することができれば製造効率上の利点はより大きい。 Moreover, the presence of nutrients outside the fungus is one that affects the growth of yeast. In order for yeast to grow, in addition to carbon sources such as sugar and nitrogen sources such as amino acids and proteins, trace nutrient sources such as vitamins are required. As the nitrogen source, inorganic substances such as ammonium sulfate and organic substances such as amino acids and proteins are usually used, and soybean peptides obtained by hydrolyzing soybean are also being used as nitrogen sources. For example, soy peptide is used in yeast fermentation in the production of effervescent alcoholic beverages such as beer-like beverages that have low beer and malt usage or do not use malt. However, in effervescent alcoholic beverages, fermentation is often performed at a low temperature of 15 ° C. or lower. However, as described above, yeast growth activity becomes dull due to low-temperature stress. There is a need to. In this case, there is an advantage in terms of flavor, but if the low-temperature fermentation can be completed in a shorter time, the advantage in production efficiency is greater.
このような課題に関連する知見として、アラビアガムとトラガントガムを添加して酵母を発酵させると、5℃でアルコールの産生が活発になることが分かっている(特許文献1)。また、大豆ペプチドを含む培地で培養した酵母は、冷凍に対して耐性が上昇することが記載されている(特許文献2)。しかしながら、特許文献1では、菌の増殖ではなくアルコールの生成量を見ており、菌の増殖活性に関しては言及されていない。また、15℃における効果も示されていない。また特許文献2の知見は非凍結域であって15℃以下という低温域における酵母の増殖について何ら教示するものではない。
As a knowledge related to such a problem, it has been found that when gum arabic and gum tragacanth are added to ferment yeast, alcohol production becomes active at 5 ° C. (Patent Document 1). Moreover, it is described that the yeast culture | cultivated by the culture medium containing a soybean peptide raises tolerance with respect to freezing (patent document 2). However, Patent Document 1 looks at the amount of alcohol produced rather than the growth of bacteria, and does not mention the growth activity of the bacteria. Also, the effect at 15 ° C. is not shown. Further, the knowledge of
本発明の目的は、発泡性アルコール飲料などのように、製造時に低温での酵母発酵が行われる場合に、酵母の増殖活性の鈍化によって発酵効率が低下する問題を解決するものである。 The object of the present invention is to solve the problem that fermentation efficiency decreases due to slowing of the growth activity of yeast when yeast fermentation is performed at a low temperature during production, such as effervescent alcoholic beverages.
上記課題に鑑み、本発明者は鋭意研究を行った結果、大豆蛋白質からグロブリンの一種であるβ−コングリシニンに富む画分を分画し、この画分を酵素分解したペプチドが酵母の低温増殖能を顕著に強化する作用を示すことを見出した。そして該画分の低温増殖能は、酵母の細胞内酵素の活性が低温によって低下し、酵母の活動が抑制される、いわゆる低温ストレスから保護する作用に基づくものであることを見出した。本発明はかかる知見に基づき完成するに到ったものである。 In view of the above problems, as a result of intensive studies, the present inventors have fractionated a fraction rich in β-conglycinin, which is a kind of globulin, from soybean protein, and the peptide obtained by enzymatic degradation of this fraction is capable of low-temperature growth of yeast. It has been found that it has an effect of remarkably strengthening. It was found that the low-temperature growth ability of the fraction was based on the action of protecting against so-called low-temperature stress, in which the activity of yeast intracellular enzymes was reduced by low temperature and the yeast activity was suppressed. The present invention has been completed based on this finding.
すなわち本発明は、
(1)大豆β−コングリシニンの分解物を含むことを特徴とする酵母の低温発酵促進剤、
(2)大豆β−コングリシニンの分解物中における分子量120〜500の画分の割合が60%以上である前記(1)記載の酵母の低温発酵促進剤、
(3)前記(1)記載の低温発酵促進剤を添加した酵母生育用培地、
(4)前記(1)記載の低温発酵促進剤を含む培地に酵母を接種し、15℃以下で発酵する発酵飲食品の製造法、
(5)大豆β−コングリシニンの分解物を含むことを特徴とする酵母の低温ストレス保護剤、である。That is, the present invention
(1) A low-temperature fermentation promoter for yeast, comprising a degradation product of soybean β-conglycinin,
(2) The low-temperature fermentation promoter for yeast according to (1), wherein the fraction of a molecular weight of 120 to 500 in the degradation product of soybean β-conglycinin is 60% or more,
(3) A yeast growth medium to which the low-temperature fermentation promoter according to (1) is added,
(4) A method for producing a fermented food or beverage that inoculates a medium containing the low-temperature fermentation promoter according to (1) and ferments at 15 ° C. or lower,
(5) A low-temperature stress protective agent for yeast, comprising a degradation product of soybean β-conglycinin.
本発明によれば、大豆β−コングリシニン分解物を培地に添加することにより、酵母を低温ストレスから保護し、その結果低温における酵母の増殖を促進することができる。発酵を早く進めるためには、低温下で酵母を増殖させることが必須であるが、本発明ではその増殖時間を短縮できることにより、従来よりも短時間で発酵食品の製造が可能となる。 According to the present invention, by adding soybean β-conglycinin degradation product to a culture medium, yeast can be protected from low-temperature stress, and as a result, yeast growth at low temperatures can be promoted. In order to advance the fermentation quickly, it is essential to grow the yeast at a low temperature, but in the present invention, the growth time can be shortened, so that the fermented food can be produced in a shorter time than before.
本発明の酵母の低温発酵促進剤は、大豆β−コングリシニンの分解物を含むことを特徴とするものである。以下、本発明の実施形態について詳細に説明する。 The yeast low-temperature fermentation promoter of the present invention is characterized by containing a degradation product of soybean β-conglycinin. Hereinafter, embodiments of the present invention will be described in detail.
(大豆β−コングリシニン)
大豆蛋白質を構成する蛋白質は、超遠心分析による沈降係数から、2S,7S,11S,15Sの各グロブリンに分類される。このうち、7Sグロブリン と11Sグロブリンはグロブリン画分の主要な構成蛋白成分である。このうち、免疫学的命名法にいうβ−コングリシニンは7Sグロブリンに、グリシニンは11Sグロブリンに実質的に相当するものである。また、大豆のグロブリン画分には、これら以外のマイナーな酸沈殿性大豆蛋白質群が存在し、これらはレシチンや糖脂質などの極性脂質を多く随伴するため、包括的に「脂質親和性蛋白質」(以下、「LP」と略記することがある。)と呼ばれている(国際公開WO2006/129647号公報参照)。(Soybean β-conglycinin)
Proteins constituting soy protein are classified into 2S, 7S, 11S, and 15S globulins based on sedimentation coefficients obtained by ultracentrifugation analysis. Of these, 7S globulin and 11S globulin are the main constituent protein components of the globulin fraction. Among these, β-conglycinin in the immunological nomenclature substantially corresponds to 7S globulin, and glycinin substantially corresponds to 11S globulin. In addition, the soybean globulin fraction contains a group of minor acid-precipitating soy proteins other than these, and these are accompanied by a large amount of polar lipids such as lecithin and glycolipids. (Hereinafter sometimes abbreviated as “LP”) (refer to International Publication WO2006 / 129647).
このように、大豆蛋白質はβ−コングリシニン(7Sグロブリン)、グリシニン(11Sグロブリン)、LPの3つに分画でき(上記公報参照)、本発明においてはこれらのうち、β−コングリシニンを原料として選択したものである。β−コングリシニンはα、α´、βという3つのサブユニットから構成される3量体であり、これには血中の中性脂肪を低減させる効果があることが知られている。 In this way, soybean protein can be fractionated into β-conglycinin (7S globulin), glycinin (11S globulin) and LP (see the above publication), and in the present invention, among these, β-conglycinin is selected as a raw material. It is a thing. β-conglycinin is a trimer composed of three subunits α, α ′, and β, and it is known that this has an effect of reducing neutral fat in blood.
大豆蛋白質からβ−コングリシニンに富む画分を分画する手段は公知の手段を用いることができる。特にβ−コングリシニンが粗蛋白質中40重量%以上含まれるのが好ましく、50重量%以上がより好ましい。好ましい製造法としては、国際公開WO2006/129647号公報やWO2002/028198号公報に記載される方法が挙げられる。β−コングリシニンに富む画分のβ−コングリシニンの純度は蛋白質中60%以上であるのが好ましく、80%以上がより好ましい。 A well-known means can be used as a means for fractionating a fraction rich in β-conglycinin from soybean protein. In particular, β-conglycinin is preferably contained in the crude protein in an amount of 40% by weight or more, and more preferably 50% by weight or more. Preferable production methods include those described in International Publications WO2006 / 129647 and WO2002 / 028198. The purity of β-conglycinin in the fraction rich in β-conglycinin is preferably 60% or more, more preferably 80% or more in the protein.
(大豆β−コングリシニンの分解物)
本発明のβ−コングリシニン分解物は、β−コングリシニンをプロテアーゼで分解することにより得られるペプチド混合物である。β−コングリシニン分解物は、分解度が高い方が好ましく、分解物中の分子量120〜500の画分の割合が60%以上であるのが好ましく、64%以上であるのがより好ましい。また非特許文献2によると、アミノ酸は遊離の形で存在するよりペプチド態である方が望ましいと考えられるため、分子量120未満の画分の割合は10%以下であるのが好ましく、5%以下であるのがより好ましい。さらに、ペプチド態はより低分子であることが望ましいことから、分子量500以上の画分の割合は40%以下が好ましく、35%以下がより好ましい。(Soybean β-conglycinin degradation product)
The β-conglycinin degradation product of the present invention is a peptide mixture obtained by degrading β-conglycinin with a protease. The β-conglycinin degradation product preferably has a high degree of degradation, and the proportion of the fraction having a molecular weight of 120 to 500 in the degradation product is preferably 60% or more, and more preferably 64% or more. Further, according to Non-Patent
酵素分解は上記大豆たん白質スラリーまたは水溶液を基質とし、プロテアーゼ処理を行う。ここで用いるプロテアーゼは動物起源、植物起源あるいは微生物起源は問わず、プロテアーゼの分類において「金属プロテアーゼ」,「酸性プロテアーゼ」,「チオールプロテアーゼ」,「セリンプロテアーゼ」に分類されるプロテアーゼ、好ましくは「金属プロテアーゼ」,「チオールプロテアーゼ」,「セリンプロテアーゼ」に分類されるプロテアーゼの中から適宜選択することができる。特に2種類以上、あるいは3種類以上の異なった分類に属する酵素を、順次もしくは同時に作用させる分解方法がジペプチドやトリペプチド等の比較的分子量の低いペプチドの割合を増加させることができ好ましい。 In the enzymatic degradation, the soybean protein slurry or the aqueous solution is used as a substrate to perform protease treatment. Proteases used here are of animal origin, plant origin or microbial origin, and are classified as “metal protease”, “acid protease”, “thiol protease”, “serine protease”, preferably “metal” The protease can be appropriately selected from proteases classified as “protease”, “thiol protease”, and “serine protease”. In particular, a degradation method in which enzymes belonging to two or more, or three or more different categories are sequentially or simultaneously acted is preferable because the ratio of peptides having a relatively low molecular weight such as dipeptides and tripeptides can be increased.
このプロテアーゼの分類は、酵素化学の分野において通常行われている活性中心のアミノ酸の種類による分類方法である。各々の代表として「金属プロテアーゼ」にはBacillus中性プロテイナーゼ, Streptomyces中性プロテイナーゼ, Aspergillus中性プロテイナーゼ,サモアーゼ等、「酸性プロテアーゼ」にはペプシン, Aspergillus酸性プロテイナーゼ,スミアームAP等、「チオールプロテアーゼ」にはブロメライン,パパイン等、「セリンプロテアーゼ」にはトリプシン,キモトリプシン,ズブチリシン, Streptomycesアルカリプロテアーゼ, アルカラーゼ, ビオプラーゼ等が挙げられる。これら以外の酵素でも作用pHや阻害剤との反応性により、その分類を確認することができる。活性中心が異なる酵素間では、基質への作用部位が大きく異なるために、「切れ残り」を減らし、効率良く酵素分解物を得ることができる。或いは異なった起源(起源生物)の酵素を併用することで、更に効率よく酵素分解物を製造することが出来る。同分類でも起源が異なれば、基質であるたんぱく質への作用部位も異なり、結果としてジペプチドやトリペプチドの割合を増やすことができる。これらプロテアーゼはエキソ活性が少ないものが好ましい。 This classification of protease is a classification method based on the type of amino acid at the active center, which is usually performed in the field of enzyme chemistry. As representatives of each, “metal protease” includes Bacillus neutral proteinase, Streptomyces neutral proteinase, Aspergillus neutral proteinase, samoyase, etc. “acid protease” includes pepsin, Aspergillus acid proteinase, Sumiarm AP, etc. Examples of “serine protease” such as bromelain, papain and the like include trypsin, chymotrypsin, subtilisin, Streptomyces alkaline protease, alcalase, biopase and the like. The classification of other enzymes can also be confirmed by the working pH and reactivity with inhibitors. Since the site of action on the substrate is greatly different between enzymes having different active centers, the “uncut residue” can be reduced, and an enzyme degradation product can be obtained efficiently. Alternatively, enzymatic degradation products can be produced more efficiently by using enzymes of different origins (origin organisms) in combination. Even in the same classification, if the origin is different, the site of action of the substrate protein is also different, and as a result, the proportion of dipeptides and tripeptides can be increased. These proteases are preferably those with low exo activity.
反応pHや反応温度は、用いるプロテアーゼの特性に合わせて設定すればよく、通常反応pHは至適pH付近で行い、反応温度は至適温度付近で行えばよい。概ね反応温度は20〜80℃、好ましくは40〜60℃で反応を行うことができる。反応後は酵素の失活するに十分な温度(60〜100℃程度)で加熱し、残存酵素活性を失活させる。
得られた反応液は、そのまま或いは濃縮して用いることもできるが、通常、殺菌して粉霧乾燥、凍結乾燥等して乾燥粉末の状態で利用することができる。また反応液を任意のpHに調整しておくこともでき、またpH調整時に発生する沈殿物や懸濁物を遠心分離や濾過等により除去することもできる。またさらに活性炭や吸着樹脂により精製を行うこともできる。The reaction pH and reaction temperature may be set in accordance with the characteristics of the protease to be used, and the reaction pH is usually carried out near the optimum pH, and the reaction temperature may be carried out near the optimum temperature. In general, the reaction can be carried out at a reaction temperature of 20 to 80 ° C, preferably 40 to 60 ° C. After the reaction, the enzyme is heated at a temperature sufficient to deactivate the enzyme (about 60 to 100 ° C.) to deactivate the remaining enzyme activity.
The obtained reaction liquid can be used as it is or after being concentrated, but it can usually be sterilized and used in the form of a dry powder after being powder-dried or freeze-dried. Further, the reaction solution can be adjusted to an arbitrary pH, and precipitates and suspensions generated during pH adjustment can be removed by centrifugation, filtration, or the like. Further, purification can be performed with activated carbon or an adsorption resin.
(分子量分布測定法)
得られたβ−コングリシニン分解物の分子量分布を測定する場合、以下のゲルろ過カラムを用いたHPLC法により測定することとする。カラム1本を使用してペプチド用ゲル濾過システムを組み、分子量マーカーとなる既知ペプチドをチャージし、分子量と保持時間の関係において検量線を求める(表1)。酵素分解した分解物(1%)を10,000×g、10分で遠心分離した上清を、ゲル濾過用溶媒で2倍希釈し、その5μlをアプライした。各分子量画分の含有量比率%については、全体の吸光度のチャート面積に対する、特定の分子量範囲(時間範囲)の面積の割合によって求めた。(カラム:Superdex Peptide 7.5/300GL、溶媒:1%SDS/10mMリン酸緩衝液, pH8.0、25℃、流速:0.25ml/min、検出:OD220nm)。(Molecular weight distribution measurement method)
When measuring the molecular weight distribution of the obtained β-conglycinin degradation product, it is determined by the HPLC method using the following gel filtration column. A gel filtration system for peptides is assembled using one column, a known peptide serving as a molecular weight marker is charged, and a calibration curve is obtained in the relationship between molecular weight and retention time (Table 1). The supernatant obtained by centrifuging the enzymatically degraded product (1%) at 10,000 × g for 10 minutes was diluted 2-fold with a solvent for gel filtration, and 5 μl thereof was applied. About the content ratio% of each molecular weight fraction, it calculated | required by the ratio of the area of the specific molecular weight range (time range) with respect to the chart area of the whole light absorbency. (Column: Superdex Peptide 7.5 / 300GL, solvent: 1% SDS / 10 mM phosphate buffer, pH 8.0, 25 ° C., flow rate: 0.25 ml / min, detection: OD220 nm).
(表1)分子量標準物質
(Table 1) Molecular weight reference materials
(酵母の低温発酵促進剤)
本発明は、上記のようにして得られたβ−コングリシニン分解物を酵母の低温発酵促進剤として用いることに特徴がある。通常、食品製造に利用される酵母の至適温度は20〜30℃程度であり、15℃以下の低温域においては酵母の増殖速度が著しく低下する。発酵食品の製造過程において、酵母の生育状態は重要な鍵となっており、発酵に費やされる日数も比較的長い。しかし、本発明の低温発酵促進剤により、低温における酵母の増殖速度を早めることができ、製造日数を短縮することが可能となる。
ここで本発明において「低温」とは室内温(20℃前後)よりも低い程度の温度をいい、具体的には15℃以下の温度を示し、より限定的には12℃以下である。ただし培養液が凍結してしまう凍結域の温度ではそもそも酵母が増殖できず、さらに酵母の活性の低下にも繋がるので、少なくとも培養液の凍結域の温度以上であることを示す。具体的には0℃以上、より限定的には3℃以上である。(Yeast low temperature fermentation promoter)
The present invention is characterized in that the β-conglycinin degradation product obtained as described above is used as a low-temperature fermentation promoter for yeast. Usually, the optimum temperature of yeast used for food production is about 20 to 30 ° C., and the growth rate of yeast is remarkably reduced in a low temperature range of 15 ° C. or lower. In the process of producing fermented food, the growth state of yeast is an important key, and the days spent for fermentation are relatively long. However, the low-temperature fermentation promoter of the present invention can increase the growth rate of yeast at low temperatures and shorten the number of production days.
Here, in the present invention, “low temperature” refers to a temperature lower than the room temperature (around 20 ° C.), specifically a temperature of 15 ° C. or less, and more specifically 12 ° C. or less. However, since the yeast cannot proliferate in the first place at the temperature of the freezing region where the culture broth freezes, and further leads to a decrease in the activity of the yeast, this indicates at least the temperature of the freezing region of the culture broth. Specifically, it is 0 ° C. or higher, more specifically 3 ° C. or higher.
(酵母培養用培地)
本発明の低温発酵促進剤を酵母の主要な窒素源の一つとして培地に配合し、酵母生育用培地とすることができる。例えば酵母の増殖培地中に0.1〜10%添加することにより得られる。酵母生育用培地の種類は、天然培地、合成培地、半合成培地のいずれでもよく、液体培地、固体培地のいずれでもよい。他の窒素源としてペプトン、酵母抽出物、酵母窒素ベース、硫化アンモニウム、アミノ酸、ヌクレオチド等も添加することができ、炭素源としてはグルコース、ガラクトース、ラフィノース、デキストロース等を添加することができる。
この酵母生育用培地で酵母を15℃以下の低温で培養すると、低温であっても増殖が活発となる。これは酵母の受ける低温ストレスからβ−コングリシニン分解物が酵母を保護する低温ストレス保護剤としての作用による。(Yeast culture medium)
The low-temperature fermentation promoter of the present invention can be blended in a medium as one of the main nitrogen sources of yeast to obtain a yeast growth medium. For example, it is obtained by adding 0.1 to 10% in a yeast growth medium. The type of the yeast growth medium may be any of a natural medium, a synthetic medium, and a semi-synthetic medium, and may be a liquid medium or a solid medium. Peptone, yeast extract, yeast nitrogen base, ammonium sulfide, amino acids, nucleotides and the like can be added as other nitrogen sources, and glucose, galactose, raffinose, dextrose and the like can be added as carbon sources.
When yeast is cultured in this yeast growth medium at a low temperature of 15 ° C. or lower, the growth becomes active even at low temperatures. This is due to the action of a β-conglycinin degradation product as a low-temperature stress protective agent that protects yeast from low-temperature stress experienced by yeast.
(発酵飲食品の製造)
本発明の低温発酵促進剤は上記のように市販の酵母生育用培地の培地成分として添加できるほか、低温での酵母発酵により製造される発酵飲食品の製造において、発酵前原料として混合し、これに酵母を接種して発酵させることも有効である。
低温で酵母発酵により製造されうる発酵飲食品の例としては、酒類としてビールや麦芽の使用率が低いもしくは0%であるビール様飲料等の発泡性アルコール飲料、ワイン、ウイスキー、清酒、焼酎などが挙げられ、調味料として醤油、みそ、食酢などが挙げられ、一般食品としてパン、発酵乳、発酵バター、ケフィール、漬物などが挙げられるが、これらに限定されるものではなく、低温で酵母発酵を行うあらゆる発酵飲食品に対して適用可能である。
発酵飲食品中への低温発酵促進剤の添加量は通常0.1〜10%とすることができる。(Manufacture of fermented food and drink)
The low-temperature fermentation promoter of the present invention can be added as a medium component of a commercially available yeast growth medium as described above. In addition, in the production of fermented foods and beverages produced by yeast fermentation at low temperatures, it is mixed as a raw material before fermentation. It is also effective to inoculate and ferment yeast.
Examples of fermented foods and beverages that can be produced by yeast fermentation at low temperatures include effervescent alcoholic beverages such as beer-like beverages with low or 0% use of beer and malt as liquors, wine, whiskey, sake, shochu, etc. Examples of seasonings include soy sauce, miso, and vinegar. Examples of general foods include bread, fermented milk, fermented butter, kefir, and pickles, but are not limited to these. Applicable to all fermented foods and beverages to be performed.
The amount of the low-temperature fermentation accelerator added to the fermented food or drink can be usually 0.1 to 10%.
以下、実施例により本発明のより具体的な実施態様について説明する。 Hereinafter, more specific embodiments of the present invention will be described by way of examples.
(製造例1)各種大豆蛋白質画分の調製
佐本らの方法(M Samoto etc, Food Chemistry, 102, 317-322, 2007.)に準じて以下の通り、β−コングリシニン、グリシニン、LPの各種大豆蛋白質画分を調製した。
1)低変性脱脂大豆に加熱処理を施してNSI(水溶性窒素指数、AOCS公式分析法BA-11-65 NSIによる)を低下させた脱脂大豆(NSI70%)の温水抽出スラリーを遠心分離機にてオカラ画分を除き脱脂豆乳とした。
2)次に脱脂豆乳のpHを5.8に調整して遠心分離機にて沈殿カード画分を回収した。この画分が「グリシニン画分」である。
3)次に、残りの上清のpHを5.0に調整し、55℃で10分間放置後、次いでpH5.5に調整後、遠心分離機にて沈殿カード画分を回収した。この画分が脂質親和性蛋白を多く含む「LP画分」である。
4)次に、残りの上清のpHを4.5に調整し、遠心分離機にて沈殿カード画分を回収した。この画分が「β−コングリシニン画分」である。
5)得られた各画分を中和して140℃×10秒の加熱処理後、噴霧乾燥した。
各画分の純度はグリシニン画分が96%、β−コングリシニン画分が91%であった。(Production Example 1) Preparation of various soybean protein fractions According to the method of Samoto et al. (M Samoto etc, Food Chemistry, 102, 317-322, 2007.) A soy protein fraction was prepared.
1) A hot water extraction slurry of defatted soybean (NSI 70%) with low NSI (water-soluble nitrogen index, according to AOCS official analysis method BA-11-65 NSI) by heat treatment of low-denatured defatted soybean in a centrifuge The okara fraction was removed to obtain defatted soy milk.
2) Next, the pH of the defatted soymilk was adjusted to 5.8, and the precipitated curd fraction was collected with a centrifuge. This fraction is the “glycinin fraction”.
3) Next, the pH of the remaining supernatant was adjusted to 5.0, allowed to stand at 55 ° C. for 10 minutes, then adjusted to pH 5.5, and then the precipitated card fraction was collected with a centrifuge. This fraction is an “LP fraction” containing a large amount of lipophilic protein.
4) Next, the pH of the remaining supernatant was adjusted to 4.5, and the precipitated card fraction was collected with a centrifuge. This fraction is the “β-conglycinin fraction”.
5) Each obtained fraction was neutralized, heat-treated at 140 ° C. for 10 seconds, and then spray-dried.
The purity of each fraction was 96% for the glycinin fraction and 91% for the β-conglycinin fraction.
(製造例2)分離大豆蛋白の調製
別途、低変性脱脂大豆から以下のように分離大豆蛋白を調製した。
1)低変性脱脂大豆の温水抽出スラリーを遠心分離機にてオカラ画分を除き脱脂豆乳とした。
2)得られた脱脂豆乳をpHを4.5に調整して等電点沈殿せしめ、遠心分離機にて酸沈殿カードを得て中和した。
3)次にこの中和液を噴霧乾燥し、分離大豆蛋白(SPI)とした。(Manufacture example 2) Preparation of isolation | separation soybean protein Separately, isolation | separation soybean protein was prepared from the low modified | denatured defatted soybean as follows.
1) Warm water extraction slurry of low-denatured defatted soybeans was defatted soymilk by removing the okara fraction with a centrifuge.
2) The obtained defatted soymilk was adjusted to pH 4.5 and subjected to isoelectric precipitation, and an acid precipitation card was obtained and neutralized with a centrifuge.
3) Next, this neutralized solution was spray-dried to obtain separated soybean protein (SPI).
(製造例3)各種大豆蛋白分解物の製造
製造例1,2で得られたβ−コングリシニン、グリシニン、LP及び分離大豆蛋白から国際公開WO2006/134752号公報を参考に、以下のようにプロテアーゼによる酵素分解物を調製した。
3%大豆たんぱく質溶液に対して、サモアーゼ(起源;Bacillus thermoproteolyticus, 金属プロテアーゼ, 大和化成)を対たん白質あたり2%加え、pH9.0, 58℃で60分間作用させた。次にビオプラーゼ(起源;Bacillus sp., セリンプロテアーゼ, ナガセケムテック)を対たんぱく質あたり1%加え、pH7.5, 58℃で60分間作用させた。スミチームFP(起源;Aspergillus sp., 金属プロテアーゼ, 新日本化学工業)を対たんぱく質あたり1%加え、pH7.5, 58℃で60分間作用させた。以上の処理の後、90℃, 20分で反応を停止した後、試料とした。
得られた4種類の酵素分解物の分子量を非特許文献2に記載の方法に従って分子量を測定した。測定結果を表2に示す。(Production Example 3) Production of various soybean protein digests From the β-conglycinin, glycinin, LP and isolated soybean protein obtained in Production Examples 1 and 2, with reference to International Publication WO2006 / 134752, the following protease Enzymatic degradation products were prepared.
2% Samoaase (source: Bacillus thermoproteolyticus, metalloprotease, Yamato Kasei) was added to 3% soy protein solution per protein and allowed to act at pH 9.0, 58 ° C. for 60 minutes. Next, 1% of biopase (source: Bacillus sp., Serine protease, Nagase Chemtech) per protein was added and allowed to act at pH 7.5, 58 ° C. for 60 minutes. Sumiteam FP (source: Aspergillus sp., Metalloprotease, Shin Nippon Chemical Industry) was added at 1% per protein and allowed to act at pH 7.5, 58 ° C for 60 minutes. After the above treatment, the reaction was stopped at 90 ° C. for 20 minutes, and then used as a sample.
The molecular weights of the obtained four types of enzyme degradation products were measured according to the method described in
(表2)β−コングリシニン分解物の分子量分布
(Table 2) Molecular weight distribution of β-conglycinin degradation product
β−コングリシニン分解物において、ジペプチドやトリペプチドが多く含まれる分子量120〜500の画分の割合が66.9%であり、60%以上の割合を占めていた。一方、遊離アミノ酸が多く含まれる分子量120未満の画分の割合は3.1%であり、10%に満たなかった。また分子量500以上の比較的高分子のペプチド画分の割合は30%であり、40%に満たなかった(表2)。他の分解物における画分割合もβ−コングリシニン分解物と概ね同等であり、分子量分布には大きな差はなく、いずれも同様の酵素分解が行われていることが示された。 In the β-conglycinin degradation product, the proportion of the fraction having a molecular weight of 120 to 500 containing a large amount of dipeptides and tripeptides was 66.9%, accounting for 60% or more. On the other hand, the ratio of the fraction containing a large amount of free amino acids and having a molecular weight of less than 120 was 3.1%, which was less than 10%. The proportion of the relatively high-molecular peptide fraction having a molecular weight of 500 or more was 30%, which was less than 40% (Table 2). The fraction ratio in other degradation products was almost the same as that of the β-conglycinin degradation product, and there was no significant difference in the molecular weight distribution, indicating that the same enzymatic degradation was performed in all cases.
(実施例1)
製造例3で得られたβ−コングリシニン分解物による低温増殖促進効果を見るために、酵母の低温培養実験を行った。比較対象として、同じく製造例3で得られたグリシニン分解物、LP分解物、分離大豆蛋白分解物を用いた。
培地は酵母生育用であるSD培地に窒素源として各種大豆蛋白分解物を添加した。具体的には、Yeast Nitrogen Base w/o amino acids and anmoniuum sulfate0.17%、グルコース2%、アデニン14.6mg/L、ウラシル20mg/L、リジン塩酸塩30mg/L、ロイシン90mg/L、ヒスチジン20mg/Lの混合物に、各種大豆蛋白分解物を乾物重量で1%となるように混合して培地を作成した。この培地に出芽酵母「YPH499株」を1mLあたりの細胞数が106個になるように植菌し、10℃での細胞数を経時的(0時間、24時間、72時間)に見た。Example 1
In order to see the low temperature growth promoting effect of the β-conglycinin degradation product obtained in Production Example 3, a low temperature culture experiment of yeast was performed. As comparative objects, the glycinin degradation product, LP degradation product, and separated soybean protein degradation product obtained in Production Example 3 were used.
As a medium, various soybean protein degradation products were added as a nitrogen source to an SD medium for yeast growth. Specifically, Yeast Nitrogen Base w / o amino acids and anmoniuum sulfate 0.17%,
図1の結果に示すとおり、β−コングリシニン分解物を添加した場合、他の大豆蛋白分解物を添加した場合と比較すると、72時間の発酵時間において酵母数に顕著な差がみられ、10℃という低温域において酵母の増殖が顕著に促進されていた。一方、他のグリシニン分解物やLP分解物は分離大豆蛋白分解物における酵母の増殖速度と同等であり、これらにはβ−コングリシニン分解物のような顕著な酵母の低温発酵促進効果はみられなかった。
以上の通り、同じ大豆蛋白質由来のペプチドでありながら、特に大豆蛋白質の画分の一つであるβ−コングリシニンのペプチドが顕著な酵母の低温発酵促進効果を有することが明らかとなった。
またβ−コングリシニン分解物を添加することで、低温域でも酵母が増殖可能になることが示されたが、これは低温によって細胞内酵素の活性が低下し、酵母の活動が抑制されるという現象がβ−コングリシニンによって緩和されているためと考えられる。その意味でβ−コングリシニン分解物は低温ストレス保護剤としての効果を有していると言える。As shown in the results of FIG. 1, when the β-conglycinin degradation product was added, a significant difference was observed in the number of yeasts at the fermentation time of 72 hours, compared with the case where other soybean protein degradation products were added. Yeast growth was significantly promoted in the low temperature range. On the other hand, other glycinin degradation products and LP degradation products are equivalent to the growth rate of yeast in the isolated soybean protein degradation product, and they do not have a significant yeast low-temperature fermentation promoting effect like β-conglycinin degradation products. It was.
As described above, it has been clarified that β-conglycinin peptide, which is one of the fractions of soybean protein, has a remarkable effect of promoting low-temperature fermentation of yeast while being peptides derived from the same soybean protein.
In addition, it was shown that by adding β-conglycinin degradation product, yeast can grow even in a low temperature range, but this is a phenomenon in which the activity of intracellular enzymes decreases and the activity of yeast is suppressed by low temperature. Is considered to be relaxed by β-conglycinin. In that sense, it can be said that the β-conglycinin degradation product has an effect as a low-temperature stress protective agent.
▲Total :分離大豆蛋白分解物
○7S :β−コングリシニン分解物
□11S:グリシニン分解物
*LP :LP(脂質親和性蛋白質)分解物▲ Total: Isolated soy protein degradation product ○ 7S: β-conglycinin degradation product □ 11S: Glycinin degradation product * LP: LP (lipophilic protein) degradation product
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PCT/JP2009/006317 WO2010061575A1 (en) | 2008-11-25 | 2009-11-24 | Promoter for low temperature fermentation of yeast, and culture medium for growing yeast and method for producing fermented food or beverage each of which utilizes the promoter |
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