JP5561642B2 - Yogurt - Google Patents
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- JP5561642B2 JP5561642B2 JP2010111527A JP2010111527A JP5561642B2 JP 5561642 B2 JP5561642 B2 JP 5561642B2 JP 2010111527 A JP2010111527 A JP 2010111527A JP 2010111527 A JP2010111527 A JP 2010111527A JP 5561642 B2 JP5561642 B2 JP 5561642B2
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- 235000013618 yogurt Nutrition 0.000 title claims description 57
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 144
- 241000894006 Bacteria Species 0.000 claims description 83
- 239000004310 lactic acid Substances 0.000 claims description 72
- 235000014655 lactic acid Nutrition 0.000 claims description 72
- 235000000346 sugar Nutrition 0.000 claims description 15
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- 239000002504 physiological saline solution Substances 0.000 description 4
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- 241000901050 Bifidobacterium animalis subsp. lactis Species 0.000 description 3
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- QZSODHGHTMLVCM-UHFFFAOYSA-N Cl.Cl.[K] Chemical compound Cl.Cl.[K] QZSODHGHTMLVCM-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 2
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- 235000013527 bean curd Nutrition 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
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- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
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- Dairy Products (AREA)
Description
本発明はヨーグルトに関する。 The present invention relates to yogurt.
プロバイオティクスとは、消化管内の善玉菌(乳酸菌、ビフィズス菌など)を増やし、腸内細菌のバランスを保ち、病気になりにくい体を作る予防医学のことを言う。
乳酸菌を含む各種乳製品は、プロバイオティクスの観点からも重要な食品に位置づけられ、特にヨーグルトは、手軽に食することのできる食品として広く普及している。しかるに、せっかくヨーグルトを食しても、酸性の強い胃酸や胆汁液に触れることから、大部分の乳酸菌は死滅してしまい、生き残って腸にまで到達する乳酸菌は非常に少ないと言われている。
そこで、最近では、乳酸菌を耐酸性のカプセルに封入し、このカプセルをヨーグルトに混入させる方法が知られている(特許文献1)。また、耐酸性のある所謂植物性乳酸菌を選抜することによって、乳酸菌を腸にまで達することができるように工夫した乳酸飲料等も知られている(特許文献2)。
Probiotics refers to preventive medicine that increases the number of good bacteria (lactic acid bacteria, bifidobacteria, etc.) in the gastrointestinal tract, maintains the balance of enteric bacteria, and makes the body less susceptible to disease.
Various dairy products containing lactic acid bacteria are positioned as important foods from the viewpoint of probiotics, and particularly yogurt is widely used as a food that can be easily eaten . However, even if you eat yogurt , it is said that most of the lactic acid bacteria are killed and reach the intestines because they touch the highly acidic stomach acid and bile fluid.
Therefore, recently, a method is known in which lactic acid bacteria are enclosed in acid-resistant capsules and the capsules are mixed into yogurt (Patent Document 1). Also known is a lactic acid beverage or the like devised so that lactic acid bacteria can reach the intestine by selecting so-called vegetable lactic acid bacteria having acid resistance (Patent Document 2).
しかし、乳酸菌をカプセルに封入するのは厄介であり、コストもかかるという課題がある。また、特許文献2のものでは、呈味が従来のヨーグルトとは異なるものとなってしまうという課題がある。
本発明は上記課題を解決すべくなされたものであり、その目的とするところは、呈味を損なうことなく、またカプセルを用いることなく乳酸菌を有効に生存させ、腸にまで達するようにすることができるヨーグルトを提供するにある。
However, encapsulating lactic acid bacteria in a capsule is awkward and expensive. Moreover, the thing of patent document 2 has the subject that taste will become a thing different from the conventional yogurt.
The present invention has been made to solve the above-mentioned problems, and the object of the present invention is to make lactic acid bacteria survive effectively and reach the intestine without impairing taste and using capsules. There is in providing yogurt that can.
本発明に係るヨーグルトは、乳酸菌を含有するヨーグルトにおいて、牛乳からなる乳原料と、ブドウ糖、砂糖、乳糖、メリビノースおよびラフィノースのうちのいずれかの糖類とに、微粉砕された多孔質の乾燥おからが添加された後、乳酸菌で発酵され、前記多孔質の乾燥おからを0.1〜20wt%含有すると共に、該多孔質の乾燥おからの空隙部の内壁に乳酸菌が繁殖、定着していることを特徴とする。ここでは生おからではなく乾燥おからであることが重要である。元来、おから自身には乳酸菌が直ちに利用できるような炭素源を少量しか含まない。そのため、おからを乳酸菌で発酵させるためには低分子の糖類(例えば、ブドウ糖、砂糖、乳糖、メリビノース、ラフィノースなど)をおからに含ませることが大切である。生おからの場合、その多孔性の空隙部には水が存在している。そのため乳酸菌の栄養物は自然拡散で届けられるが時間を要する。一方、乾燥おからの場合には多孔性の空隙部には空気しか存在しない。乾燥おからを糖類など栄養成分の含まれる溶液に添加する場合、乳酸菌の栄養となる物質が瞬時のうちにおからの繊維の多孔内部に持ち込まれることになる。発酵時間をあまり長くとることのできないヨーグルト発酵に適している。このようにしておからの多孔内部が乳酸菌にとって快適な成育環境になっている。
結果、ヨーグルトの作製方法は極めて単純で乳酸発酵時に乾燥おからを含んでさえいればよく、特別な設備も操作も必要としない。
また、前記乾燥おからとして、乾燥おからをあらかじめ乳酸発酵させたおからを用いることもできる。この予め発酵させるということは、乳酸菌の餌となる糖を含ませた乾燥おからを比較的水分の低い状態でやや長い時間発酵させるような選択をすることで、乳酸菌自身が作る保護膜、即ち、バイオフィルムの生成を助長させることができ、この保護膜による酸や胆汁液に対する保護効果もさらに期待できる。
The yogurt according to the present invention is a yogurt containing lactic acid bacteria, and is a porous dried okara that is finely pulverized into a milk raw material consisting of milk and any sugar of glucose, sugar, lactose, melvinose and raffinose. After being added, the lactic acid bacteria are fermented with lactic acid bacteria, contain 0.1 to 20 wt% of the porous dried okara, and the lactic acid bacteria have propagated and settled on the inner walls of the voids from the porous dried okara. Features. It is important here to be dried okara, not raw okara. Originally, okara itself contains only a small amount of carbon sources that are readily available to lactic acid bacteria. Therefore, in order to ferment okara with lactic acid bacteria, it is important to contain low molecular sugars (for example, glucose, sugar, lactose, melvinose, raffinose, etc.) in the okara. In the case of fresh okara, water exists in the porous voids. Therefore, the nutrients of lactic acid bacteria are delivered by natural diffusion, but it takes time. On the other hand, in the case of dried okara, only air exists in the porous void. When dry okara is added to a solution containing nutrients such as sugars, a substance that serves as a nutrient for lactic acid bacteria is instantaneously brought into the porous interior of the fiber. It is suitable for yogurt fermentation that cannot take a long fermentation time. In this way, the porous interior is a comfortable growth environment for lactic acid bacteria.
As a result, the production method of yogurt is very simple and only needs to contain dried okara during lactic acid fermentation, and does not require any special equipment or operation.
As the dried okara, okara obtained by lactic acid fermentation of dried okara can be used. This pre-fermentation means that a protective film made by the lactic acid bacterium itself is selected by selecting the fermented dried okara containing the sugar that is the bait of the lactic acid bacterium for a relatively long time with a relatively low moisture content. The production of biofilms can be promoted, and the protective effect against acid and bile fluid by this protective film can be further expected.
本発明によれば、糖類が乾燥おからの繊維の多孔内部に持ち込まれて発酵され、乳酸菌が乾燥おからの空隙部の内壁に繁殖、定着されたヨーグルトを提供できる。さらに、呈味を損なうことなく、またカプセルを用いることなく乳酸菌を有効に生存させ、腸にまで達するようにすることができるヨーグルトを提供できる。 According to the present invention, it is possible to provide a yogurt in which saccharides are brought into the pores of dried okara fibers and fermented, and lactic acid bacteria are propagated and fixed on the inner walls of the voids from the dried okara. Furthermore, it is possible to provide a yogurt that can effectively survive lactic acid bacteria and reach the intestine without impairing the taste and without using a capsule.
以下本発明の好適な実施の形態を添付図面に基づいて詳細に説明する。
乾燥おからの製造方法は種々のものが知られている(例えば特開平5−68503、特開2000−4817など)。
本実施の形態において用いる乾燥おからは、その製造方法が特に限定されるものではないが、いずれの製造方法においても、おからそのものは豆腐の製造過程で貯蔵たんぱく質が抽出された、主として細胞壁からなる残渣であるので、得られる乾燥おからは細胞壁により繋がった極めて空隙率の大きい多孔質構造をなしている。この空洞(空隙)部が乳酸菌の棲家となる(ただし、利用できる炭素源が必要)。
なお、乾燥おからは、菌数管理の上、急速乾燥し微粉砕されたものが望ましい。
図1はその乾燥おからの走査電子顕微鏡写真である。
Preferred embodiments of the present invention will be described below in detail with reference to the accompanying drawings.
Various methods for producing dried okara are known (for example, JP-A-5-68503, JP-A-2000-4817, etc.).
The production method of dried okara used in the present embodiment is not particularly limited, but in any production method, okara itself is extracted from the stored protein during the production of tofu, mainly from the cell wall. Therefore, the resulting dried okara has a porous structure with a very high porosity connected by cell walls. This hollow (void) part becomes a lactic acid bacteria house (however, an available carbon source is required).
The dried okara is preferably one that has been rapidly dried and finely pulverized under the control of the number of bacteria.
FIG. 1 is a scanning electron micrograph of the dried okara.
大豆細胞壁の構成糖のうちで最も多いのはガラクトースであり、その35wt%を占めるといわれている(「醸造物の成分」日本醸造協会編、1999)。このガラクトースは、乳酸菌の細胞壁を構成するレクチンと親和性が高い(Oxford Journals.Life Sciences.Glycobiology10(11)1193-1199、2000)。それ故、乳酸菌は大豆(おから)の細胞壁のガラクトースを足場として細胞壁上に繁殖することができる。具体的には、乾燥おからの空洞部内に多数の乳酸菌が繁殖、定着することが実際に確認された(図2の走査電子顕微鏡写真)。このように、乳酸菌が乾燥おからの空洞部内に定着することから、ヨーグルトとして食されたときに、胃酸や胆汁液に攻撃される度合いが減じ、生き残って腸にまで到達する乳酸菌が増えるのである。 It is said that galactose is the most abundant saccharide in the soy cell wall and occupies 35% by weight (“brewed ingredients” edited by Japan Brewing Association, 1999). This galactose has high affinity with the lectin constituting the cell wall of lactic acid bacteria (Oxford Journals. Life Sciences. Glycobiology 10 (11) 1193-1199, 2000). Therefore, lactic acid bacteria can propagate on the cell wall using galactose in the cell wall of soybean (okara) as a scaffold. Specifically, it was actually confirmed that a large number of lactic acid bacteria grew and settled in the cavity from dried okara (scanning electron micrograph in FIG. 2). In this way, lactic acid bacteria settle in the cavity of dried okara, so when eaten as yogurt, the degree of attack by gastric acid and bile fluid decreases, and lactic acid bacteria that survive and reach the intestine increase. .
しかも、大豆細胞壁の構成多糖では、ヘミセルロース(グルコース、キシロース)に次いでペクチン質が多い(約30wt%)。このペクチン質は、酸性では不溶(硬い)で、中性ないしはアルカリ性では可溶(膨潤する)である。したがって、胃や十二指腸を通過する際、胃酸や胆汁液によっては不溶でその形状を維持し、腸に達した際、アルカリ性の膵液などに触れて膨潤し、これにより乳酸菌が腸内に放出され易くなると考えられる。
乾燥おからの添加量は、0.1〜20wt%が好適である。0.1wt%未満であると、生きて腸にまで達する乳酸菌の量が少なく、20wt%よりも多いとヨーグルトの食感に影響を及ぼす。乾燥おからの添加量は、ヨーグルトとしての食感からは、0.1〜1 wt%程度が最適である。
Moreover, in the polysaccharide constituting the soybean cell wall, the amount of pectin is high next to hemicellulose (glucose, xylose) (about 30 wt%). This pectic substance is insoluble (hard) when acidic, and soluble (swells) when neutral or alkaline. Therefore, when passing through the stomach and duodenum, it is insoluble depending on stomach acid and bile fluid and maintains its shape. It is considered to be.
The addition amount from dried okara is preferably 0.1 to 20 wt%. If it is less than 0.1 wt%, the amount of lactic acid bacteria that live and reach the intestines is small, and if it exceeds 20 wt%, the texture of yogurt is affected. The amount added from dried okara is optimally about 0.1 to 1 wt% from the texture of yogurt.
<実施例1> 乾燥おからの乳酸発酵
まず、乾燥おからそのものをヨーグルト用の乳酸菌を用いて乳酸発酵させてみた。
1)発酵方法
乾燥おから(みすずコーポレーション製 ビーンフラワー#300) 100gに2.5%酢酸ナトリウムを含む20%糖蜜30gを加え、この130gを袋に入れ、オートクレーブ(121℃、20分間)して滅菌した。
発酵の種菌として、市販のヨーグルトから分離したヨーグルト分離菌(Lactobacillus delbuchii subsp. bulgaricus)の培養液8mLに水12mLを加えたもの、市販のサプリメントから分離した分離乳酸菌L.paracaseiの培養液8mLに水12mLを加えたもの、および市販乳酸菌種(協同乳業社製)(3乳酸菌:Lactobacillus delbuchii subsp. bulgaricus, Streptococcus thermophilus, Bifidobacterium lactis LKM512)の培養液8mLに水12mLを加えたものの3種類を使用した。
袋を密封し、35℃、14日間、発酵させた(水分39%、糖度2.9%)。
<Example 1> Lactic acid fermentation from dried okara First, the dried okara itself was lactic acid fermented using lactic acid bacteria for yogurt.
1) Fermentation method Dried okara (bean flour # 300, manufactured by Misuzu Corporation) Add 100 g of 30% 20% molasses containing 2.5% sodium acetate, place this 130 g in a bag, sterilize by autoclaving (121 ° C, 20 minutes) .
As an inoculum for fermentation, 12 mL of water was added to 8 mL of a culture solution of yogurt isolate (Lactobacillus delbuchii subsp. Bulgaricus) isolated from commercially available yogurt, and water was added to 8 mL of an isolated culture solution of L. paracasei isolated from a commercially available supplement. Three types were used: 12 mL added and commercially available lactic acid bacteria species (manufactured by Kyodo Daigyo Co., Ltd.) (3 lactic acid bacteria: Lactobacillus delbuchii subsp. Bulgaricus, Streptococcus thermophilus, Bifidobacterium lactis LKM512) culture solution 8 mL.
The bag was sealed and fermented at 35 ° C. for 14 days (water content 39%, sugar content 2.9%).
上記を表1に示す。
また、別にこれらの3菌をMRS培地で、35℃、24時間培養した培養液を生菌体対照とした。 Separately, a culture solution obtained by culturing these three bacteria in MRS medium at 35 ° C. for 24 hours was used as a living cell control.
2)仮想消化管での生存率調査
消化管での乳酸菌の生存をみるために胃液のpH(pH2-3、処理時間0.5-1h)と胆汁液(0.4%bile powder,0.5h)で処理(酸処理)し、生菌数を比較することでおからの効果を見た。
具体的な酸処理方法を次に示す。
酸処理方法:発酵おから1g、および生菌体対照区の培養液1gを4.2mL生理的食塩水に懸濁する。そこへ0.2M塩酸-塩酸カリウム緩衝液(pH1.18)5.0mLを加え、pHを2.0付近にする。37℃の水槽で40分間放置し、次いで、0. 4%濃度になるように10%胆汁液0.4mL加え、37℃の水槽で30分間処理する。次いで、室温で6M水酸化ナトリウム水溶液でpHを7.2-7.5付近の中性に戻し、これを界面活性剤(ツイーン80)0.5%含む生理的食塩水で希釈して、BCP添加プレート寒天培地、アネロパックジャー(三菱ガス化学株式会社製)を用いた嫌気培養を行い、生菌数を測定した。
酸未処理の群は、9mL生理食塩水で希釈し、BCP添加プレート寒天培地で生菌数を測定した。
2) Survival rate survey in the virtual digestive tract To examine the survival of lactic acid bacteria in the digestive tract, treatment with gastric pH (pH 2-3, treatment time 0.5-1h) and bile fluid (0.4% bile powder, 0.5h) ( Acid treatment), and the effect of okara was seen by comparing the number of viable bacteria.
A specific acid treatment method is shown below.
Acid treatment method: Suspend 1 g of fermented okara and 1 g of the culture solution of the viable cell control group in 4.2 mL of physiological saline. Thereto is added 5.0 mL of 0.2 M hydrochloric acid-potassium hydrochloride buffer (pH 1.18) to bring the pH to about 2.0. Leave in a 37 ° C water bath for 40 minutes, then add 0.4 mL of 10% bile solution to a concentration of 0.4% and treat in a 37 ° C water bath for 30 minutes. Next, the pH is returned to neutrality around 7.2-7.5 with 6M aqueous sodium hydroxide solution at room temperature, diluted with physiological saline containing 0.5% of a surfactant (Tween 80), and a BCP-added plate agar medium, Anero Anaerobic culture using a pack jar (Mitsubishi Gas Chemical Co., Ltd.) was performed, and the number of viable bacteria was measured.
The acid-untreated group was diluted with 9 mL of physiological saline, and the viable cell count was measured on a BCP-added plate agar medium.
結果を表2に示す。
表2から明らかなように、おからを含まない液体培養菌(対照)は、消化管対応の酸処理後の生菌数が、未処理と比較して1/10,000〜1/100,000に減少してしまう。一方、発酵おからの場合(試験)、酸処理後の乳酸菌の生存率は、未処理と比較して1/10〜1/100と極めて高いことがわかる。
なお、図2の走査電子顕微鏡写真は、市販乳酸菌種を用いた発酵おからの酸未処理における乳酸菌の繁殖状態を示す写真である。このように、乳酸菌は多孔質の乾燥おからの空洞部の内壁に繁殖、定着していることから、胃酸や胆汁液からの攻撃を受けにくく、生きて腸内にまで達することが多くなると考えられる。
The results are shown in Table 2.
As is clear from Table 2, the number of viable bacteria after acid treatment corresponding to the digestive tract was reduced to 1 / 10,000 to 1 / 100,000 in the liquid culture bacteria (control) that did not contain okara compared to untreated. End up. On the other hand, in the case of fermented okara (test), it can be seen that the survival rate of the lactic acid bacteria after the acid treatment is as extremely high as 1/10 to 1/100 compared with the untreated.
In addition, the scanning electron micrograph of FIG. 2 is a photograph which shows the reproduction state of the lactic acid bacteria in the acid untreated from fermentation okara using the commercially available lactic acid bacteria seed | species. In this way, lactic acid bacteria grow and settle on the inner wall of the cavity of porous dry okara, so it is difficult to be attacked by gastric acid and bile fluid, and it is likely to live and reach the intestines more often. It is done.
<実施例2>
1.ヨーグルトの作製
使用種
ヨーグルト作製の種菌として次の乳酸菌を用いた。
1)協同乳業:3乳酸菌(Lactobacillus delbuchii subsp. bulgaricus, Streptococcus thermophilus, Bifidobacterium lactis LKM512)
2)カフェファクトリー:カスピ海ヨーグルト(Lactococcus lactis subsp. cremoris)
3)プレーンヨーグルトA:小岩井(ビフィズス菌、アシドフィラス菌)
4)プレーンヨーグルトB:森永乳業(Bifidobacterium ロンガムBB536)
5)プレーンヨーグルトC:明治乳業(LG21)
6)プレーンヨーグルトD:明治乳業(Lactobacillus bulgaricus2038、Streptococcus thermophilus1131)
<Example 2>
1. As inoculum prepared <br/> used species yoghurt prepared yogurt using the following lactic acid bacteria.
1) Cooperative dairy industry: 3 lactic acid bacteria (Lactobacillus delbuchii subsp. Bulgaricus, Streptococcus thermophilus, Bifidobacterium lactis LKM512)
2) Cafe Factory: Caspian Sea yogurt (Lactococcus lactis subsp. Cremoris)
3) Plain yogurt A: Koiwai (Bifidobacterium, Acidophilus)
4) Plain yogurt B: Morinaga Milk Industry (Bifidobacterium longum BB536)
5) Plain Yogurt C: Meiji Dairy (LG21)
6) Plain yogurt D: Meiji Dairy (Lactobacillus bulgaricus2038, Streptococcus thermophilus1131)
種菌1)及び2)の場合
市販牛乳---500g
種菌 ----1g/包
砂糖 -----50g
乾燥おから(ビーンフラワー#300)--- 5wt%
Inoculum 1) and 2)
Commercial milk --- 500g
Inoculum ---- 1g / pack
Sugar ----- 50g
Dried Okara (Bean Flower # 300) --- 5wt%
ヨーグルト友種の場合
市販牛乳---500g
プレーンヨーグルト---50g
砂糖 -----50g
乾燥おから(ビーンフラワー#300)--- 5wt%
In the case of yogurt friend varieties Commercial milk --- 500g
Plain yogurt--50g
Sugar ----- 50g
Dried Okara (Bean Flower # 300) --- 5wt%
試験区(5wt%乾燥おからを含む)、対照区(おから無し)共に、35℃、16h発酵させた。上記のように、発酵を助けるため、砂糖を10wt%加えた。 Both the test group (including 5 wt% dried okara) and the control group (without okara) were fermented at 35 ° C. for 16 hours. As above, 10 wt% sugar was added to aid fermentation.
ヨーグルトの出来栄え=何れの菌も市販種菌や市販ヨーグルトをもとにした種菌であり、夫々表3のように美味しいヨーグルトが得られた。
<実施例3> おからによるヨーグルト発酵菌の生き残り調査
1.ヨーグルトの作製
協同乳業種菌:市販品(Lactobacillus delbuchii subsp. bulgaricus, Streptococcus thermophilus, Bifidobacterium lactis LKM512)を用いて乾燥おから(ビーンフラワー#300)8gと10%砂糖を含む牛乳160mLを46℃で発酵させた。発酵時間を標準的な12hと完熟させる40hとの2種類とした。Aとして乾燥おからを含んで乳酸発酵させたもの、Bとして乾燥おからを含まない従来のヨーグルト、CはBの12時間発酵させたヨーグルトに乾燥おからを単純に混合したものを調製した。
<Example 3> Survival survey of fermented yoghurt bacteria using okara Cooperative milk industry bacteria for yogurt production : Using commercially available products (Lactobacillus delbuchii subsp. Bulgaricus, Streptococcus thermophilus, Bifidobacterium lactis LKM512), fermented 8 g of dried okara (bean flour # 300) and 160 mL of milk containing 10% sugar at 46 ° C It was. There were two types of fermentation time: standard 12h and fully matured 40h. A was prepared by fermenting lactic acid with dried okara as A, B as a conventional yogurt without dried okara, and C was prepared by simply mixing dried okara with B for 12 hours fermented yogurt.
2.仮想消化管での生存率調査
胃から小腸上部の環境をin vitro で再現する目的でサンプル1gに対し、生理的食塩水6.1mL,0.2M塩酸-塩酸カリウム緩衝液(pH1.3)2.5mLを加えpHを2.0付近にし、37℃、40分間保持した。後、10wt%胆汁液0.4mLを加え、37℃、30分間処理した。処理後、6M水酸化ナトリウム水溶液(60-70μL)でpHを7.2-7.5付近に上げ、40分間放置した。このサンプルを前述同様希釈してBCP添加プレート寒天培地で生菌数を測定した。酸未処理の群も前述同様に生菌数を測定した。
2. Survival survey in the virtual digestive tract In order to reproduce the environment from the stomach to the upper small intestine in vitro, 6.1 mL of physiological saline and 2.5 mL of 0.2 M hydrochloric acid-potassium hydrochloride buffer (pH 1.3) are added to 1 g of the sample. In addition, the pH was adjusted to around 2.0 and maintained at 37 ° C. for 40 minutes. Thereafter, 0.4 mL of 10 wt% bile solution was added and treated at 37 ° C. for 30 minutes. After the treatment, the pH was raised to around 7.2-7.5 with 6M aqueous sodium hydroxide solution (60-70 μL) and left for 40 minutes. This sample was diluted in the same manner as described above, and the viable cell count was measured on a BCP-added plate agar medium. In the acid-untreated group, the number of viable bacteria was measured in the same manner as described above.
3.結果
結果を表4に示す。
4.12時間発酵
表4に示すように、乾燥おからと砂糖を含む牛乳を乳酸菌で46℃、12時間、発酵させてヨーグルトを作る時、従来のおからを含まない牛乳と遜色の無い菌数に至った。
これを前述の消化管を想定した酸処理すると、乾燥おからを含まない従来のヨーグルトと乾燥おからを発酵済みのヨーグルトへ単純に混合したものが、×108個から×103個まで5桁生菌数を減らしたのに対し、乾燥おからを含んで発酵させたものでは×108個から×105個での3桁の減少に留まった。以上の結果、乾燥おからを含んで発酵させたヨーグルトは無添加の普通ヨーグルトに比較して100倍の数の乳酸菌を保持していた。
4. Fermentation for 12 hours As shown in Table 4, when milk made with dried okara and sugar is fermented with lactic acid bacteria at 46 ° C for 12 hours to make yogurt, it does not look like milk that does not contain conventional okara. It reached the number.
When this is treated with the above-mentioned acid digestive tract, conventional yogurt that does not contain dried okara and dried okara are simply mixed with fermented yogurt from x10 8 to x10 3 5 While the number of sprouting bacteria was reduced, the one fermented with dried okara still had a three-digit decrease from × 10 8 to × 10 5 . As a result, yogurt fermented with dried okara retained 100 times the number of lactic acid bacteria as compared with normal yogurt without additives.
5.40時間発酵
ヨーグルトは12〜24時間で発酵を終了させるのが普通だが、ここでは乳酸菌の乾燥おからの空隙部へのさらなる進入を促進する目的で46℃、40時間、発酵させてヨーグルトを作った。
比較的温度に強い乳酸菌であったが、高温に加えて低pHの影響であろうか意図に反して生菌数の減少が認められた。
結果は12時間の場合と同様で、乾燥おからを発酵させたものでは2桁の菌数減少抑制効果であった。
5. Fermentation for 40 hours Yogurt usually finishes fermentation in 12-24 hours, but here it is fermented for 40 hours at 46 ° C for yogurt to promote further entry of the lactic acid bacteria into the voids made.
Although it was a lactic acid bacterium that was relatively resistant to temperature, a decrease in the number of viable bacteria was observed against the intention, whether it was an effect of low pH in addition to high temperature.
The result was the same as in the case of 12 hours, and fermented dried okara had a double-digit bacterial count reduction inhibitory effect.
6.保冷保存の影響
ヨーグルトは生産されて後、保冷されて流通し、顧客の手元に届くまで数日から2週間のタイムラグがある(一般的な賞味期限は10℃以下で2週間)。この間にもゆっくりと乳酸発酵は継続するとされている。
発酵終了後、保冷した場合のヨーグルトについておから添加効果が持続するか、あるいはさらに向上するか検討した。
6). Effect of cold storage Yogurt is produced, then cooled and distributed, and there is a time lag of several days to two weeks until it reaches the customer's hand (general shelf life is below 10 ° C for two weeks). During this time, lactic acid fermentation is said to continue slowly.
After the end of fermentation, it was investigated whether the effect of adding okara to yogurt when kept cold or to further improve it.
結果を表5に示す。
結果は表5に示すように、おからを含まない対照と比較すると、乾燥おからを加えて同時発酵させたものは、乳酸菌菌数が2桁多く酸処理等に耐えて生き残っている。
冷蔵保存によっておからの耐酸性などの効果が落ちることも上がることも無かった。
As shown in Table 5, as compared with the control not containing okara, the result of co-fermenting with dried okara survived by acid treatment and the like with the number of lactic acid bacteria increased by two orders of magnitude.
Refrigerated storage did not reduce or increase the effects of acid resistance from okara.
<実施例4> 人糞中の乳酸菌
おから入りヨーグルトを食べた場合、便の中に乳酸菌が認められるか調査した。
1.市販乳酸菌種の場合
8%砂糖と1%の乾燥おからを含んで発酵させたヨーグルトと乾燥おからを含まない通常のヨーグルトを市販乳酸菌で作製した。まず、通常のヨーグルト1日約100gを連続して4日間食べ、摂取最終日から48時間後、便を採取し凍結保存した。次いで、1%の乾燥おからを含んで発酵させたヨーグルトを同様に摂取し、便を採取し凍結保存した。
これら各々の便について、含まれる細菌の染色体DNAを抽出し、16SリボゾームDNA遺伝子の可変領域内で、乳酸菌に特異的なプライマーと一般的な細菌プライマーとを用いてGCクランプを付けたポリメラーゼ連鎖反応(PCR)を行った。増幅断片を変性剤濃度勾配ゲル電気泳動(DGGE)に掛けた。結果を図3に示す。
<Example 4> Lactic acid bacteria in human feces When eating oak yogurt, it was investigated whether lactic acid bacteria were observed in the stool.
1. For commercially available lactic acid bacteria species
Yogurt fermented with 8% sugar and 1% dried okara and normal yogurt without dried okara were made with commercially available lactic acid bacteria. First, about 100 g of normal yogurt was eaten continuously for 4 days, 48 hours after the last day of ingestion, stool was collected and stored frozen. Next, the fermented yogurt containing 1% dried okara was similarly taken, and the stool was collected and stored frozen.
For each of these stools, the chromosomal DNA of the contained bacteria was extracted, and the polymerase chain reaction with a GC clamp using a primer specific for lactic acid bacteria and a general bacterial primer within the variable region of the 16S ribosomal DNA gene (PCR) was performed. The amplified fragment was subjected to denaturing gradient gel electrophoresis (DGGE). The results are shown in FIG.
なお、プライマーとPCRの条件は次のとおりである。
16S リボゾームDNAの可変領域内で、乳酸菌類(Lactobacillus, Lueconostoc, Pediococcus, Wessellaの各属)に特異的なプライマーとしてフォワード側にS-G-Lab-0159-a-S-20(GGA AAC AG(A/G) TGC TAA TAC CG)をリバース側に一般的な細菌のS-*-Univ-0515-a-A-24(ATC GAT TTA CCG CGG CTG CTG GCA)を用いた(文献:Hans G.H.J. Heiligら、Appl.Environ.Microbiol.Jan.2002,p.114-123)。
PCRはTaqr-polymeraseを用い、染色体DNAとプライマーのアニーリング温度は63℃、35サイクルのDNA断片の増幅を行った。
Primer and PCR conditions are as follows.
SG-Lab-0159-aS-20 (GGA AAC AG (A / G)) on the forward side as a primer specific to lactic acid bacteria (genus Lactobacillus, Lueconostoc, Pediococcus, Wessella) within the variable region of 16S ribosomal DNA General bacterial S-*-Univ-0515-aA-24 (ATC GAT TTA CCG CGG CTG CTG GCA) was used on the reverse side of TGC TAA TAC CG (reference: Hans GHJ Heilig et al., Appl. Environ. Microbiol. Jan. 2002, p. 114-123).
For PCR, Taqr-polymerase was used, and DNA fragments were amplified for 35 cycles at an annealing temperature of chromosomal DNA and primers of 63 ° C.
図3は、上記市販乳酸菌を用いた場合のDGGEパターンを示す。
検出されたDNAのバンドの内、乳酸菌種のレーン1とおから入りヨーグルト摂取後の便のレーン3に共通するバンドa, b, cをゲルから切り出し、精製後、DNA配列を決定し、DNAデータベースに蓄積されている塩基配列情報から菌種を同定した。結果を表6にまとめた。
なお、DGGEの条件と染色条件は次のとおりである。
ゲルの変性剤の濃度勾配を35〜50%とした。ゲル温度60℃で定電圧220Vの条件で5時間電気泳動した。ガラス板からゲルを外し、ゲルスター核酸染色剤(Takara社製)を用いて室温、30分間DNAを染色し、紫外線イルミネーター上染色バンドを励起してDNAを検出した。
FIG. 3 shows a DGGE pattern when the above-mentioned commercially available lactic acid bacteria are used.
Of the detected DNA bands, bands a, b, and c common to lane 1 of lactic acid bacteria and lane 3 of stool after ingesting yogurt were cut out from the gel, purified, DNA sequence determined, DNA database Species were identified from the base sequence information accumulated in The results are summarized in Table 6.
The DGGE conditions and staining conditions are as follows.
The concentration gradient of the gel denaturant was 35-50%. Electrophoresis was performed at a gel temperature of 60 ° C. and a constant voltage of 220 V for 5 hours. The gel was removed from the glass plate, DNA was stained with a gel star nucleic acid stain (Takara) at room temperature for 30 minutes, and the staining band on an ultraviolet illuminator was excited to detect the DNA.
2.脱脂粉乳分離乳酸菌種の場合
1と同様に分離乳酸菌(Lactobacillus paracasei)を用いて1g乾燥おからを含むヨーグルトを作製し100gを1回のみ摂食した。この後、24時間、6日後、10日後の便を採取して、1と同様な方法で細菌のDGGEバンドを得た。
2. In the case of non-fat dry milk isolated lactic acid bacteria species As in 1, a yoghurt containing 1 g dried okara was prepared using isolated lactic acid bacteria (Lactobacillus paracasei), and 100 g was fed only once. Thereafter, stool samples were collected 24 hours, 6 days, and 10 days later, and bacterial DGGE bands were obtained in the same manner as in 1.
図4は、上記脱脂粉乳分離乳酸菌を用いた場合のDGGEパターンを示す。
レーン1の通常便になく、レーン2のおから入りヨーグルト摂取後の便にある、バンドを1と同様に遺伝子配列から菌を同定した。
結果、摂食後、24時間、6日後、及び10日を経過した人糞中に、ヨーグルト種に使用したLactobacillus paracaseiを発見した。
人糞中に乳酸菌の遺伝子断片が発見されたことが直ちに生きた乳酸菌の存在を意味するものではない。しかし、通常、ヒトが食べた食品は24時間で排泄されるとされている。摂食後、48時間以上経過してもなお乳酸菌DNA断片が存在していることはヨーグルトとして摂取した乳酸菌が生きたまま腸まで届き、増殖していることを示している。
FIG. 4 shows a DGGE pattern when the above skim milk powder isolated lactic acid bacterium is used.
Bacteria were identified from the gene sequence in the same manner as in band 1 in the stool after ingesting yogurt from lane 2 but not in normal lane in lane 1.
As a result, Lactobacillus paracasei used for yogurt was found in human feces after 24 hours, 6 days, and 10 days after feeding.
The discovery of a gene fragment of lactic acid bacteria in human feces does not imply the existence of live lactic acid bacteria. However, foods eaten by humans are usually excreted in 24 hours. The presence of the lactic acid bacteria DNA fragment after 48 hours after feeding indicates that the lactic acid bacteria ingested as yogurt reach the intestines alive and proliferate.
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