JP4981278B2 - Artificial cultivation method of Hanabiratake - Google Patents
Artificial cultivation method of Hanabiratake Download PDFInfo
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Description
本発明は、ハナビラタケ(Sparassis crispa)の新菌株、その菌株を用いた栽培方法及びハナビラタケの系統識別方法に関するものである。 TECHNICAL FIELD The present invention relates to a new strain of Hanabiratake (Sparassis crispa), a cultivation method using the strain, and a strain identification method of Hanabiratake.
ハナビラタケは唐松などの針葉樹にまれに生えるキノコで、幻のキノコと呼ばれている。子実体は有柄、柄は繰り返して枝を分ける。各枝は平たくなり、波型にうねりくねった花弁状、全体はハボタン状になる。白からクリーム色、1株の径は10〜30cmに達する。花弁状の各片は厚さ1ミリほどで、柔軟だが上部で歯切れがよい肉質である。食用のキノコで日本のほかにヨーロッパなどでも発見されている。 Hanabiratake is a rare mushroom that grows on conifers such as Karamatsu and is called a phantom mushroom. The fruiting body has a handle, and the handle repeats to divide the branches. Each branch is flat, wavy and petal-like, and the whole is a habutton. White to cream, the diameter of one strain reaches 10-30cm. Each petal-like piece is about 1 mm thick and is flexible but fleshy at the top. It is an edible mushroom that has been found in Europe as well as in Japan.
ハナビラタケは、免疫を活性化させるといわれているβグルカンを多く含むことが知られており、今までに乾燥重量100gあたり43.6g含有する例が報告されている(特許文献1参照)。 Hanabiratake is known to contain a large amount of β-glucan, which is said to activate immunity, and an example containing 43.6 g per 100 g of dry weight has been reported so far (see Patent Document 1).
一方、キノコは一般に、同じ種に属する菌株でありながら、採集された場所の違いにより菌糸の生育速度および子実体形成能力が著しく異なることが知られている。ハナビラタケにおいても系統の異なるものにおいて、βグルカンの含量や、芽だしの違いがあることがわかっている。従来、菌類の識別方法として、菌糸、胞子、および繁殖器官等の形態的特徴に基づく方法が知られている。具体的に、胞子等の形態的特徴に基づく方法とは、分生子、胞子、子のうか、子のう、子のう胞子、接合胞子、担子胞子等の大きさを比較して、菌類の同定、分類を行う方法である。しかしながら上述の形態学的特徴に基づく方法では、同種内における菌糸、菌根レベルでの特定系統の識別が不可能であるという問題を有していた。 On the other hand, mushrooms are generally strains belonging to the same species, but it is known that the growth rate of mycelia and the ability to form fruit bodies differ significantly depending on the location where they are collected. It is known that the different species of Hanabiratake have different β-glucan content and buds. Conventionally, methods based on morphological features such as hyphae, spores, and reproductive organs are known as methods for identifying fungi. Specifically, the method based on morphological features such as spores is the identification of fungi by comparing the size of conidia, spores, offspring, offspring, offspring spores, zygospores, basidio spores, etc. This is a method of classification. However, the method based on the above morphological characteristics has a problem that it is impossible to identify a specific strain at the hyphal and mycorrhiza level in the same species.
きのこの系統の識別方法のひとつとしてこれまで対峙培養が用いられている。対峙培養は二つ以上の試菌株の二核菌糸を保存スラントよりブロックの形で切り出し、それぞれを同一寒天平板培地の中央部に対峙して接種し、培養後、両コロニー境界部に帯線が生じるか否かを判定する方法である。帯線を形成する菌株は対象の菌株と系統が異なることが示される。しかし、菌糸体の培養が長期にわたることや、帯線を形成しない菌株間の系統を解析することは困難である。 Up to now, counter culture has been used as one of the methods for identifying mushroom lines. In anti-cultivation, binuclear mycelia of two or more test strains are cut out in blocks from a preserved slant, and each is inoculated against the central part of the same agar plate medium. This is a method for determining whether or not it occurs. It is shown that the strain forming the band is different from the strain of interest. However, it is difficult to analyze the mycelium culture for a long time and to analyze the strains between strains that do not form a band.
最近、遺伝子レベルでの解析をきのこなどの菌類に適用した例が報告されている(特許文献2、非特許文献1参照)。しかし、ハナビラタケにおいて遺伝子レベルでの解析がなされた報告はない。
βグルカンを効率的に摂取するためにはβグルカンの含有量が多い食品が望まれ、ハナビラタケは他のキノコに比べβグルカン含有量が多いため、効率的な摂取が可能である。そのため、従来のハナビラタケよりもさらに高含量のβグルカンを含むハナビラタケが望まれていた。 In order to ingest β-glucan efficiently, foods with a high content of β-glucan are desired. Hanabiratake has a higher content of β-glucan than other mushrooms, so that it can be efficiently ingested. For this reason, there is a demand for a garlic bamboo containing a higher content of β-glucan than a conventional garlic bamboo.
また、従来の形態学的特徴や対峙培養に基づく方法では、ハナビラタケの系統を評価するには不十分であった。 In addition, conventional methods based on morphological characteristics and anti-cultivation were insufficient to evaluate Hanabiratake strains.
本発明の目的は、従来のハナビラタケよりβグルカンを多く含み、施設において工業的に、高品質かつ安価に、短期間に効率よく製造することが可能なハナビラタケの新菌株を提供することにある。もう一つの本発明の目的は、ハナビラタケ子実体、菌糸体レベルでの特定系統の識別を可能とするハナビラタケの系統識別方法を提供することにある。 An object of the present invention is to provide a new strain of Hanabiratake, which contains more β-glucan than conventional Hanabiratake, and can be efficiently produced in a short period of time at a facility at high quality and at low cost. Another object of the present invention is to provide a Hanabiratake strain identification method that enables identification of a specific strain at the fruit body and mycelium level.
本発明者等は、このような課題を解決するために鋭意検討した結果、ハナビラタケを広く自然界から純粋分離培養を行い、スクリーニングといった単離工程を経て得られた特定の菌株が、βグルカンの含有量の高く、良好な子実体を形成することを見出し、本発明を完成するに到った。 As a result of intensive investigations to solve such problems, the present inventors have conducted a pure isolation culture of Hanabiratake from nature, and a specific strain obtained through an isolation process such as screening contains β-glucan. The inventors have found that a good fruiting body can be formed with a high amount, and have completed the present invention.
また、ハナビラタケ菌糸体および子実体の遺伝子レベルでの系統識別としてRAPD(Random Amplified polymorphic DNA)解析が有用であり、特定のプライマー配列を見出し、本発明を完成するに到った。 In addition, RAPD (Random Amplified polymorphic DNA) analysis is useful as a system identification at the gene level for the mycelium and fruit bodies of the moss, and the inventors have found a specific primer sequence and completed the present invention.
すなわち、本発明は、βグルカンを子実体乾燥重量100gあたり48.0g以上含むことを特徴とするハナビラタケを要旨とするものであり、好ましくは、ハナビラタケが、ハナビラタケ(Sparassis crispa)UT−18(FERM P−19768)、ハナビラタケ(Sparassis crispa)UT−21(FERM P−19769)、ハナビラタケ(Sparassis crispa)UT−31(FERM P−19770)及びハナビラタケ(Sparassis crispa)UT−33(FERM P−19771)並びにこれらの変異株からなる群から選択される菌株である前記のハナビラタケであり、また、ハナビラタケが、ハナビラタケ(Sparassis crispa)UT−18(FERM P−19768)、ハナビラタケ(Sparassis crispa)UT−21(FERM P−19769)、ハナビラタケ(Sparassis crispa)UT−31(FERM P−19770)又はハナビラタケ(Sparassis crispa)UT−33(FERM P−19771)との対峙試験において帯線を形成しない菌株である前記のハナビラタケである。 That is, the gist of the present invention is that of ganoderma, which is characterized by containing β-glucan in an amount of 48.0 g or more per 100 g of fruit body dry weight, and preferably, ganoderma bamboo is Sparassis crispa UT-18 (FERM). P-19768), Spassis crispa UT-21 (FERM P-19769), Spassis crispa UT-31 (FERM P-19770) and Spassis crispa UT-33 (FERM P-19777) and The above-mentioned Hanabiratake, which is a strain selected from the group consisting of these mutant strains. P-19769), Hanabirata A (Sparassis crispa) UT-31 (FERM P-19770) or Sparassis crispa (Sparassis crispa) UT-33 said Sparassis crispa is a strain that does not form a ribbon conductor in confronting studies with (FERM P-19771).
また、本発明は、βグルカンを乾燥重量100gあたり48.0g以上含む子実体を形成しうるハナビラタケを要旨とするものである。 In addition, the gist of the present invention is a garlic bamboo that can form fruit bodies containing 48.0 g or more of β-glucan per 100 g of dry weight.
また、本発明は、ハナビラタケが、ハナビラタケ(Sparassis crispa)UT−18(FERM P−19768)、ハナビラタケ(Sparassiscrispa)UT−21(FERM P−19769)、ハナビラタケ(Sparassiscrispa)UT−31(FERM P−19770)及びハナビラタケ(Sparassis crispa)UT−33(FERM P−19771)並びにこれらの変異株からなる群から選択されるハナビラタケを要旨とするものであり、また、ハナビラタケが、ハナビラタケ(Sparassis crispa)UT−18(FERM P−19768)、ハナビラタケ(Sparassis crispa)UT−21(FERM P−19769)、ハナビラタケ(Sparassis crispa)UT−31(FERM P−19770)又はハナビラタケ(Sparassis crispa)UT−33(FERM P−19771)との対峙試験において帯線を形成しないハナビラタケを要旨とするものである。 Further, according to the present invention, Hanabiratake is made up of Spassis crispa UT-18 (FERM P-19768), Hanabiratake (Sparassiscrispa) UT-21 (FERM P-19769), and Parassiscrispa UT-31 (FERM P-19770). ) And Spassis crispa UT-33 (FERM P-19771), and Hanabiratake selected from the group consisting of these mutant strains. (FERM P-19768), Hanabiratake (Sparassis crispa) UT-21 (FERM P-19769), Hanabiratake (Sparassis crispa) UT-31 (FERM P-19770) or Hanabiratake (Sparassis crispa) UT-33 (FERM P-19777) Vs.) The gist of this is Hanabiratake, which does not form a band in the wrinkle test.
また本発明の第二は、前記したハナビラタケを、大鋸屑に栄養分を添加した培地に接種し、培養・生育工程を経て、βグルカンを子実体乾燥重量100gあたり48.0g以上含む子実体を形成させた後、該子実体を収穫することを特徴とするハナビラタケの人工栽培方法を要旨とするものである。 The second aspect of the present invention is to inoculate the above-mentioned Hanabiratake into a medium obtained by adding nutrients to large sawdust, and through a culture / growth process, form a fruiting body containing 48.0 g or more of β-glucan per 100 g of the fruiting body dry weight. Then, the gist of the method for artificial cultivation of the garlic bamboo, which is characterized by harvesting the fruit bodies.
さらに本発明の第三は、RAPD(Random Amplified polymorphic DNA)解析を用いてハナビラタケの系統を識別することを特徴とするハナビラタケの系統識別方法を要旨とするものであり、好ましくは、RAPD(Random Amplified polymorphic DNA)解析が、プライマー対として、配列agcagcgcctca、配列ggcatggccttt又は配列ccgcagttagatを用いてPCR法を行い、電気泳動によりDNAパターンを比較する方法であるハナビラタケの系統識別方法である。 Furthermore, a third aspect of the present invention is a genotype of Hanabiratake, characterized by identifying the line of Hanabiratake using RAPD (Random Amplified polymorphic DNA) analysis, preferably RAPD (Random Amplified). polymorphic DNA) analysis is a system identification method for Hanabiratake, which is a method in which a PCR method is performed using the sequence aggagcgcctca, the sequence ggcatggccttt or the sequence ccgcagttagat as a primer pair, and the DNA patterns are compared by electrophoresis.
本発明によれば、子実体に含まれるβグルカン量がより多いハナビラタケを得ることができるため、機能性の高いハナビラタケを高能率で生産することができる。 According to the present invention, it is possible to obtain a bamboo shoot with a larger amount of β-glucan contained in the fruiting body, so that a highly functional banana fly can be produced with high efficiency.
また、本発明によれば、高い確度での系統識別が可能となるため、優良菌株の育種などに有効に利用することができる。 In addition, according to the present invention, it is possible to identify a line with high accuracy, and therefore it can be effectively used for breeding excellent strains.
本発明のハナビラタケは、絶乾した子実体重量100gあたりβグルカンを48.0g以上含有するものである。また、本発明のハナビラタケは、βグルカンを乾燥重量100gあたり48.0g以上含む子実体を形成しうるハナビラタケである。 The flower of the present invention contains 48.0 g or more of β-glucan per 100 g of completely dried fruit body weight. In addition, the flower of the present invention is a flower that can form fruit bodies containing 48.0 g or more of β-glucan per 100 g of dry weight.
本発明におけるβグルカン量は、以下の方法により得られた値をいう。
〔前処理〕
収穫したハナビラタケの子実体の凍結乾燥品0.25gを100ml三角フラスコに入れ、次に0.08Mリン酸緩衝液(pH6.0)を加えて全量25mlにする。熱耐性αアミラーゼ(シグマ社製)を1000Unit添加し、沸騰水中で、30分間インキュベートする。水酸化ナトリウムを用いて中和し、pH7.5とした。さら50mg/mlプロテアーゼ(シグマ社製)溶液を50μl添加し、さらに60℃、30分間インキュベートする。つぎに塩酸でpH4.3に調整し、アミログルコシダーゼ(シグマ社製)溶液を50μl加え、再び60℃、30分間インキュベートする。次に95%エタノールを4倍量添加し、室温で1時間以上静置する。生成した沈殿をガラス繊維ろ紙(Advantec社製、品番GA-100)を用い、ろ過し、回収する。80%エタノールで沈殿を洗浄し、さらにアセトンで洗浄する。
The amount of β-glucan in the present invention refers to a value obtained by the following method.
〔Preprocessing〕
Add 0.25 g of the lyophilized fruit body of the harvested bamboo leaf to a 100 ml Erlenmeyer flask, and then add 0.08M phosphate buffer (pH 6.0) to make a total volume of 25 ml. Add 1000 Unit of heat resistant α-amylase (Sigma) and incubate in boiling water for 30 minutes. Neutralization with sodium hydroxide gave a pH of 7.5. Furthermore, 50 μl of 50 mg / ml protease (manufactured by Sigma) is added, and further incubated at 60 ° C. for 30 minutes. Next, adjust to pH 4.3 with hydrochloric acid, add 50 μl of amyloglucosidase (Sigma) solution, and incubate again at 60 ° C. for 30 minutes. Next, add 4 volumes of 95% ethanol and let stand at room temperature for 1 hour or more. The produced precipitate is filtered and collected using glass fiber filter paper (manufactured by Advantec, product number GA-100). Wash the precipitate with 80% ethanol and then with acetone.
〔硫酸分解〕
回収した沈殿を72%硫酸5mlで懸濁する。4時間静置する。水70ml添加し、沸騰水中で2時間、加水分解を行う。その後、氷水で冷却し中和する。ろ紙(Advantec社製、品番GA-100)によりろ過し、ろ液をサンプルとした。
[Sulfuric acid decomposition]
The collected precipitate is suspended in 5 ml of 72% sulfuric acid. Leave for 4 hours. Add 70 ml of water and hydrolyze in boiling water for 2 hours. Then, it cools with ice water and neutralizes. It filtered with the filter paper (Advantec company make, product number GA-100), and used the filtrate as the sample.
〔グルコース量の測定〕
以上の処理により得られたサンプルをグルコースCIIテストワコー(和光純薬工業社製)によりグルコース量を測定し、βグルカンが0.9gあたり、グルコース1gに変換されたものとしてハナビラタケのβグルカン量に換算した。
[Measurement of glucose level]
The sample obtained by the above treatment was measured for glucose level with Glucose CII Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) and converted to 1 g glucose per 0.9 g of β-glucan converted to β-glucan level of Hanabiratake did.
〔水分率の測定と補正〕
測定に供したハナビラタケを105℃で3時間絶乾して水分率を測定し、前記のグルコース量から求めたβグルカン量より絶乾での子実体100gあたりのβグルカン重量に補正して、本発明でいうβグルカン量とした。
[Measurement and correction of moisture content]
The dried bamboo shoots were subjected to absolute drying at 105 ° C. for 3 hours to measure the moisture content, and the amount of β-glucan obtained from the above-mentioned glucose amount was corrected to the amount of β-glucan per 100 g of fruit body in the absolute dry state. The amount of β-glucan referred to in the invention was used.
本発明における子実体絶乾重量100gあたりβグルカンを48.0g以上含有するハナビラタケは、従来の栽培ビンによる人工栽培方法によって得られる。すなわち、1)栽培ビンに大鋸屑に栄養分を添加した固形培地を作成する。固形培地には接種孔を開けておくことが望ましい。2)この固形培地に種菌を接種する。3)接種した培地を通常の条件で培養・生育させる。 Hanabiratake containing 48.0 g or more of β-glucan per 100 g of completely dry weight of fruit bodies in the present invention can be obtained by a conventional artificial cultivation method using cultivation bottles. That is, 1) A solid medium is prepared by adding nutrients to large sawdust in a cultivation bottle. It is desirable to have an inoculation hole in the solid medium. 2) Inoculate this solid medium with inoculum. 3) Culture and grow the inoculated medium under normal conditions.
本発明で用いられる大鋸屑としては、マツ、モミ、ツガ、ブナ、シイなどの大鋸屑が挙げられるが、好ましくは針葉樹の大鋸屑が好ましく、この中でもカラマツの大鋸屑が好ましい。広葉樹と針葉樹の大鋸屑の混合物を用いることもでき、大鋸屑にコーンコブなどを混合してもよい。添加する栄養分としては、必要に応じ適宜添加することができるが、小麦粉などが好ましく、小麦粉を1%〜30%、好ましくは3%〜20%、さらに好ましくは5%〜10%を混合する。これを通常850ccのポリプロピレン製栽培ビンに約520g充填する。この際、培地中央に接種孔を開けておくと、接種量を多くすることができ培地全体に菌糸が回りやすくなり、また通気がよくなるため好ましい。接種孔を開けることにより結果として収穫までの期間が短縮でき、収量が上がるなどの利点が生じる。接種孔の大きさは断面積がほぼ80mm2以上あればよい。固形培地は、高圧殺菌などの方法により殺菌をしておく。 Examples of the large sawdust used in the present invention include large sawdust such as pine, fir, Tsuga, beech, shii and the like, preferably coniferous large sawdust, among which larch large sawdust is preferable. A mixture of hardwood and coniferous large sawdust can also be used, and corncob or the like may be mixed with the large sawdust. As a nutrient to be added, it can be appropriately added as necessary, but wheat flour or the like is preferable, and 1% to 30%, preferably 3% to 20%, more preferably 5% to 10% of flour is mixed. About 520 g of this is normally filled into an 850 cc polypropylene cultivation bottle. At this time, it is preferable to make an inoculation hole in the center of the medium because the amount of inoculation can be increased, so that the mycelia can easily rotate around the entire medium and the ventilation is improved. Opening the inoculation hole results in advantages such as shortening the time to harvest and increasing the yield. The size of the inoculation hole is sufficient if the cross-sectional area is approximately 80 mm 2 or more. The solid medium is sterilized by a method such as high pressure sterilization.
このような培地に同様のオガクズ培地で培養した種菌を接種する。接種量は通常8g〜18gである。種菌を接種した後、一定の条件下、すなわち温度は15℃〜30℃、好ましくは18℃〜28℃、20〜25℃が最も好ましく、湿度は50%〜80%、好ましくは55%〜75%、60%〜70%が最も好ましい条件下で培養し、培地に菌糸を回らせる。その後、芽出し操作として、菌掻きと異種キノコの接種をおこなう(詳細は、特開2002−369621号公報を参照)。異種キノコは、同様のオガクズ培地でホウライタケを培養して作成した種菌2gを用いた。その後、ビンのふたを取って、(括弧内削除:温度約19℃、湿度約90%の)栽培室に移し、子実体を成育させる。栽培室の温度は10〜30℃、好ましくは15〜28℃、最も好ましくは17〜25℃、湿度は80%以上、好ましくは85%以上、最も好ましくは90%以上である。通常、芽出し操作後約56日間の生育工程により成長が終了するので、子実体の収穫を行う。 Such a medium is inoculated with an inoculum cultured in the same sawdust medium. The inoculum is usually 8 to 18 g. After inoculation with the inoculum, under certain conditions, i.e., the temperature is 15 ° C-30 ° C, preferably 18 ° C-28 ° C, 20-25 ° C, and the humidity is 50% -80%, preferably 55% -75. %, 60% to 70% is cultured under the most preferable conditions, and the mycelium is rotated in the medium. Then, as a sprouting operation, bacterial scraping and inoculation with different types of mushrooms are performed (for details, see JP-A-2002-369621). As the heterologous mushrooms, 2 g of inoculum prepared by cultivating spinach mushrooms in the same sawdust medium was used. Then, remove the bottle lid and move it to the cultivation room (deleted in parentheses: temperature of about 19 ° C, humidity of about 90%) to grow the fruit body. The temperature of the cultivation room is 10 to 30 ° C., preferably 15 to 28 ° C., most preferably 17 to 25 ° C., and the humidity is 80% or more, preferably 85% or more, and most preferably 90% or more. Usually, since the growth is completed by the growing process for about 56 days after the sprouting operation, the fruiting body is harvested.
本発明者らは、ハナビラタケの保存菌株及び新たに自然界から単離した菌株について人工栽培により得られた子実体のβグルカン量を調べたところ、本発明の子実体乾燥重量100gあたり48.0g以上のβグルカンを含有する菌株を見出した。さらに好ましくは、子実体乾燥重量100gあたり50.0g以上のβグルカンを含有する菌株である。したがって、本発明のハナビラタケは以上のようなスクリーニング操作を行うことにより取得することができる。 The inventors of the present invention examined the amount of β-glucan in fruit bodies obtained by artificial cultivation with respect to the preserved strain of Hanabiratake and a newly isolated strain from the natural world, and found to be 48.0 g or more per 100 g of the dry weight of the fruit body of the present invention. A strain containing β-glucan was found. More preferably, it is a strain containing 50.0 g or more of β-glucan per 100 g of fruit body dry weight. Therefore, the flower of the present invention can be obtained by performing the above screening operation.
本発明のハナビラタケに属する具体的な菌株として、ハナビラタケ(Sparassiscrispa)UT−18(FERM P−19768)、ハナビラタケ(Sparassis crispa)UT−21(FERM P−19769)、ハナビラタケ(Sparassis crispa)UT−31(FERM P−19770)及びハナビラタケ(Sparassis crispa)UT−33(FERM P−19771)を挙げることができる。これらの菌株は、以下に記載する培養的・形態的性質及び生理学的・化学分類学的性質においては公知の菌株と大きな相違はないが、本発明の特徴であるβグルカン量において差があり、さらに後述するRAPD解析において公知菌株にはない特徴的なDNAバンドを有していることが明らかであるため、新菌株として独立行政法人産業技術総合研究所特許生物寄託センターに寄託した。 As specific strains belonging to the flower of the present invention, the following species can be used: Spassiscrispa UT-18 (FERM P-19768), Spassis crispa UT-21 (FERM P-19769), Hanabiratake (Sparassis crispa) UT-31 ( FERM P-19770) and Spassis crispa UT-33 (FERM P-19771). These strains are not significantly different from known strains in the culture / morphological properties and physiological / chemical taxonomic properties described below, but there is a difference in the amount of β-glucan that is a feature of the present invention, Furthermore, since it is clear that RAPD analysis described later has a characteristic DNA band not found in known strains, it was deposited as a new strain at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary.
次に、前記した新菌株の菌学的諸性質をそれぞれ示す。
〔ハナビラタケUT−18株〕
1)バレイショ・ブドウ糖寒天培地における生育状態
14日目でコロニー径は8mm、白色で密な菌糸、気菌糸を多量に生じる。22日目でコロニー径は19mmとなり、菌糸は白色で密、直線状に伸びる。気菌糸が多い。裏面は一様で変色は無い。
2)フェノールオキシダーゼ検定用培地〔0.1%没食子酸添加ポテト・グルコース寒天培地〕における生育状態
14日目でコロニー径は14mm、白色で密な菌糸、気菌糸を多量に生じる。22日目でコロニー径は25mmとなり、菌糸は白色で密、直線状に伸びる。気菌糸が多い。裏面は一様で変色は無い。
3)麦芽エキス寒天培地における生育状態
14日目でコロニー径は16mm、白色で密な菌糸、気菌糸を多量に生じる。22日目でコロニー径は31mmとなり、菌糸は白色で密、直線状に伸びる。気菌糸が多い。裏面は一様で変色は無い。
4)最適育成温度
各温度でそれぞれ培養したところ、最適生育温度は25℃付近であった。また、5℃ではほとんど育成せず、30℃付近ではまったく生育しなかった。
5)最適育成pH
生育培地を各pHに調整し、生育を調べたところ、最適pHは4〜5の間であった。また、本菌株の生育範囲は、pH4〜pH8の間であった。
Next, the bacteriological properties of the new strain described above are shown.
[Hanabiratake UT-18 shares]
1) Growth state in potato and glucose agar medium
On the 14th day, the colony diameter is 8 mm, and white and dense mycelium and aerial hyphae are produced in large quantities. On the 22nd day, the colony diameter is 19 mm, and the mycelium is white and dense, and extends linearly. There are many aerial hyphae. The back side is uniform and there is no discoloration.
2) Growth condition in phenol oxidase assay medium (potato / glucose agar medium with 0.1% gallic acid)
On the 14th day, the colony diameter is 14 mm, and white and dense mycelium and aerial hyphae are produced in large quantities. On the 22nd day, the colony diameter becomes 25 mm, and the mycelium is white and dense, and extends linearly. There are many aerial hyphae. The back side is uniform and there is no discoloration.
3) Growth condition in malt extract agar medium
On the 14th day, the colony diameter is 16mm, and white and dense hyphae and aerial hyphae are produced in large quantities. On the 22nd day, the colony diameter is 31 mm, and the mycelium is white, dense, and straight. There are many aerial hyphae. The back side is uniform and there is no discoloration.
4) Optimal growth temperature When cultured at each temperature, the optimal growth temperature was around 25 ° C. Moreover, it hardly grew at 5 ° C and did not grow at all at around 30 ° C.
5) Optimal growth pH
When the growth medium was adjusted to each pH and the growth was examined, the optimum pH was between 4 and 5. The growth range of this strain was between pH 4 and pH 8.
〔ハナビラタケUT−21株〕
1)バレイショ・ブドウ糖寒天培地における生育状態
14日目でコロニー径は13mm、白色で密な菌糸、気菌糸を多量に生じる。22日目でコロニー径は24mmとなり、菌糸は白色で密、直線状に伸びる。気菌糸が多い。裏面は一様で変色は無い。
2)フェノールオキシダーゼ検定用培地〔0.1%没食子酸添加ポテト・グルコース寒天培地〕における生育状態
14日目でコロニー径は13mm、白色で密な菌糸、気菌糸を多量に生じる。22日目でコロニー径は22mmとなり、菌糸は白色で密、直線状に伸びる。気菌糸が多い。裏面は一様で変色は無い。
3)麦芽エキス寒天培地における生育状態
14日目でコロニー径は17mm、白色で密な菌糸、気菌糸を多量に生じる。22日目でコロニー径は32mmとなり、菌糸は白色で密、直線状に伸びる。気菌糸が多い。裏面は一様で変色は無い。
4)最適育成温度
各温度でそれぞれ培養したところ、最適生育温度は25℃付近であった。また、5℃ではほとんど育成せず、30℃付近ではまったく生育しなかった。
5)最適育成pH
生育培地を各pHに調整し、生育を調べたところ、最適pHは4〜5の間であった。また、本菌株の生育範囲は、pH4〜pH8の間であった。
[Hanabiratake UT-21 strain]
1) Growth state in potato and glucose agar medium
On the 14th day, the colony diameter is 13mm, and white and dense hyphae and aerial hyphae are produced in large quantities. On the 22nd day, the colony diameter becomes 24 mm, and the mycelium is white and dense, and extends linearly. There are many aerial hyphae. The back side is uniform and there is no discoloration.
2) Growth condition in phenol oxidase assay medium (potato / glucose agar medium with 0.1% gallic acid)
On the 14th day, the colony diameter is 13mm, and white and dense hyphae and aerial hyphae are produced in large quantities. On the 22nd day, the colony diameter becomes 22 mm, and the mycelium is white and dense, and extends linearly. There are many aerial hyphae. The back side is uniform and there is no discoloration.
3) Growth condition in malt extract agar medium
On the 14th day, the colony diameter is 17mm, and white and dense hyphae and aerial hyphae are produced in large quantities. On the 22nd day, the colony diameter becomes 32 mm, and the mycelium is white, dense, and straight. There are many aerial hyphae. The back side is uniform and there is no discoloration.
4) Optimal growth temperature When cultured at each temperature, the optimal growth temperature was around 25 ° C. Moreover, it hardly grew at 5 ° C and did not grow at all at around 30 ° C.
5) Optimal growth pH
When the growth medium was adjusted to each pH and the growth was examined, the optimum pH was between 4 and 5. The growth range of this strain was between pH 4 and pH 8.
〔ハナビラタケUT-31株〕
1)バレイショ・ブドウ糖寒天培地における生育状態
14日目でコロニー径は10mm、白色で密な菌糸、気菌糸を多量に生じる。22日目でコロニー径は21mmとなり、菌糸は白色で密、直線状に伸びる。気菌糸が多い。裏面は一様で変色は無い。
2)フェノールオキシダーゼ検定用培地〔0.1%没食子酸添加ポテト・グルコース寒天培地〕における生育状態
14日目でコロニー径は9mm、白色で密な菌糸、気菌糸を多量に生じる。22日目でコロニー径は18mmとなり、菌糸は白色で密、直線状に伸びる。気菌糸が多い。裏面は一様で変色は無い。
3)麦芽エキス寒天培地における生育状態
14日目でコロニー径は15mm、白色で密な菌糸、気菌糸を多量に生じる。22日目でコロニー径は29mmとなり、菌糸は白色で密、直線状に伸びる。気菌糸が多い。裏面は一様で変色は無い。
4)最適育成温度
各温度でそれぞれ培養したところ、最適生育温度は25℃付近であった。また、5℃ではほとんど育成せず、30℃付近ではまったく生育しなかった。
5)最適育成pH
生育培地を各pHに調整し、生育を調べたところ、最適pHは4〜5の間であった。また、本菌株の生育範囲は、pH4〜pH8の間であった。
[Hanabiratake UT-31]
1) Growth state in potato and glucose agar medium
On the 14th day, the colony diameter is 10 mm, and white and dense mycelia and aerial hyphae are produced in large quantities. On the 22nd day, the colony diameter becomes 21 mm, and the mycelium is white and dense, and extends linearly. There are many aerial hyphae. The back side is uniform and there is no discoloration.
2) Growth condition in phenol oxidase assay medium (potato / glucose agar medium with 0.1% gallic acid)
On the 14th day, the colony diameter is 9 mm, and white and dense mycelium and aerial mycelium are produced in large quantities. On the 22nd day, the colony diameter becomes 18 mm, and the mycelium is white and dense, and extends linearly. There are many aerial hyphae. The back side is uniform and there is no discoloration.
3) Growth condition in malt extract agar medium
On the 14th day, the colony diameter is 15 mm, and a large amount of white and dense hyphae and aerial hyphae are produced. On the 22nd day, the colony diameter is 29 mm, and the mycelium is white, dense, and straight. There are many aerial hyphae. The back side is uniform and there is no discoloration.
4) Optimal growth temperature When cultured at each temperature, the optimal growth temperature was around 25 ° C. Moreover, it hardly grew at 5 ° C and did not grow at all at around 30 ° C.
5) Optimal growth pH
When the growth medium was adjusted to each pH and the growth was examined, the optimum pH was between 4 and 5. The growth range of this strain was between pH 4 and pH 8.
〔ハナビラタケUT−33株〕
1)バレイショ・ブドウ糖寒天培地における生育状態
14日目でコロニー径は12mm、白色で密な菌糸、気菌糸を多量に生じる。22日目でコロニー径は24mmとなり、菌糸は白色で密、直線状に伸びる。気菌糸が多い。裏面は一様で変色は無い。
2)フェノールオキシダーゼ検定用培地〔0.1%没食子酸添加ポテト・グルコース寒天培地〕における生育状態
14日目でコロニー径は13mm、白色で密な菌糸、気菌糸を多量に生じる。22日目でコロニー径は23mmとなり、菌糸は白色で密、直線状に伸びる。気菌糸が多い。裏面は一様で変色は無い。
3)麦芽エキス寒天培地における生育状態
14日目でコロニー径は19mm、白色で密な菌糸、気菌糸を多量に生じる。22日目でコロニー径は35mmとなり、菌糸は白色で密、直線状に伸びる。気菌糸が多い。裏面は一様で変色は無い。
4)最適育成温度
各温度でそれぞれ培養したところ、最適生育温度は25℃付近であった。また、5℃ではほとんど育成せず、30℃付近ではまったく生育しなかった。
5)最適育成pH
生育培地を各pHに調整し、生育を調べたところ、最適pHは4〜5の間であった。また、本菌株の生育範囲は、pH4〜pH8の間であった。
[Hanabiratake UT-33]
1) Growth state in potato and glucose agar medium
On the 14th day, the colony diameter is 12mm, and white and dense hyphae and aerial hyphae are produced in large quantities. On the 22nd day, the colony diameter becomes 24 mm, and the mycelium is white and dense, and extends linearly. There are many aerial hyphae. The back side is uniform and there is no discoloration.
2) Growth condition in phenol oxidase assay medium (potato / glucose agar medium with 0.1% gallic acid)
On the 14th day, the colony diameter is 13mm, and white and dense hyphae and aerial hyphae are produced in large quantities. On the 22nd day, the colony diameter becomes 23 mm, and the mycelium is white and dense, and extends linearly. There are many aerial hyphae. The back side is uniform and there is no discoloration.
3) Growth condition in malt extract agar medium
On the 14th day, the colony diameter is 19 mm and a large amount of white and dense mycelia and aerial mycelia are produced. On the 22nd day, the colony diameter becomes 35 mm, and the mycelium is white, dense, and straight. There are many aerial hyphae. The back side is uniform and there is no discoloration.
4) Optimal growth temperature When cultured at each temperature, the optimal growth temperature was around 25 ° C. Moreover, it hardly grew at 5 ° C and did not grow at all at around 30 ° C.
5) Optimal growth pH
When the growth medium was adjusted to each pH and the growth was examined, the optimum pH was between 4 and 5. The growth range of this strain was between pH 4 and pH 8.
上記のハナビラタケの新菌株は、本発明の第三のハナビラタケの系統識別方法によれば、公知の菌株と異なるDNAパターンを有しており、さらに各々とも相違するDNAパターンであることから各新菌株はそれぞれ系統の異なるハナビラタケであると考えられた。 According to the third method of the present invention, the above-mentioned strains of Hanabiratake have different DNA patterns from those of known strains, and each has a different DNA pattern. Were considered to be different habitats of different strains.
RAPD解析とは、通常のPCRと異なり、PCR反応条件、合成プライマーの塩基配列や長さなどを任意に変えることによって、得られる複数の部位から様々なPCR増幅産物のDNA多型を利用する方法である。プライマーとしては、通常10塩基から12塩基のプライマーが用いられる。DNAの多型は、得られたPCR増幅産物を電気泳動にかけることによるバンドパターンにより把握することができる。この方法によりハナビラタケの系統識別を行うには、ハナビラタケのゲノムDNAをランダムに増幅させ、電気泳動をかけた場合に他のハナビラタケには無い安定したバンドを探すことにより行うことができる。安定したバンドとは複数回、同じサンプルでRAPD解析を行ったときに、毎回確認されるバンドのことを示す。菌株の系統識別に用いるバンドは安定したバンドで、他の菌株のRAPD解析で同じDNA断片長が確認されないものが好ましい。また、菌株の系統識別に用いるバンドは1種類であってもよいし、2種類以上であってもよい。 Unlike normal PCR, RAPD analysis uses DNA polymorphisms of various PCR amplification products from multiple obtained sites by arbitrarily changing the PCR reaction conditions, base sequence and length of synthetic primers, etc. It is. As the primer, a primer having 10 to 12 bases is usually used. DNA polymorphism can be grasped by a band pattern obtained by subjecting the obtained PCR amplification product to electrophoresis. In order to identify strains of the moss, this method can be performed by randomly amplifying the genome DNA of the moss and searching for stable bands that are not found in other moss. A stable band refers to a band that is confirmed each time when RAPD analysis is performed on the same sample multiple times. The band used for strain identification is preferably a stable band and the same DNA fragment length is not confirmed by RAPD analysis of other strains. Further, the band used for strain identification of the strain may be one type or two or more types.
本発明者らは、このRAPD解析において、プライマーとして塩基配列ggcatggccttt、塩基配列ccgcagttagat又は塩基配列agcagcgcctcaを用いることで上記ハナビラタケに特異的なバンドを探し出した。具体的な方法及び本発明の各新菌株に特有の安定したバンドは以下のとおりである。
〔染色体DNAの調製〕
ハナビラタケUT−18、同UT−21、同UT−31及び同UT−33についてのハナビラタケから以下のようにして染色体DNAを抽出、調製した。ハナビラタケ子実体および菌糸体100 mgを液体窒素を入れた乳鉢中で磨細したのち、組織重量と等量の溶液(100 mM Na2HPO4, 50 mMクエン酸, pH6.0, 10 mM EDTA, 2% N-ラウロイルサルコシンナトリウム)中に懸濁し、65℃で30分間放置した。次に冷却遠心分離機によって7000 rpmで10分間遠心し、上清を回収した。フェノール:クロロホルム:イソアミルアルコール(=75:24:1)溶液を等量加えて処理し(2回)、冷却遠心分離機によって13000 rpmで15分間遠心し、上層を回収した。1/10容量の3M NaOAcを加えた後、2.5倍量のエタノールを加えた後、−80℃で20分間放置した。13000 rpmで20分間遠心を行い、DNAを沈殿した。得られたDNAをTES-buffer(組成 30 mM Tris-HCl, pH8.0, 5 mM EDTA, 50 mM NaCl)に加えてよく溶かした後、RNaseを終濃度100 μg/mlとなるように加えてRNAを分解した。等量のフェノール:クロロホルム液を加えて攪拌した後、微量冷却遠心分離機によって13000 rpmで15分間遠心し、水層(DNAを含む)を回収した。1/10容量の3M NaOAcを加えた後、2.5倍量のエタノールを加えた後、−80℃で20分間放置した。13000 rpmで20分間遠心を行い、DNAを沈殿した。DNAをTE-buffer(組成 10 mM Tris-HCl, pH7.5, 1 mM EDTA)に加えてよく溶かした後、得られたDNA量を定量した。その結果ハナビラタケの染色体DNAが約30〜100 μg得られた。
In this RAPD analysis, the present inventors searched for a band specific to the above-mentioned Hanabiratake by using the base sequence ggcatggccttt, the base sequence ccgcagttagat or the base sequence aggagcgcctca as a primer. Specific methods and stable bands specific to each new strain of the present invention are as follows.
(Preparation of chromosomal DNA)
Chromosomal DNA was extracted and prepared from Hanabiratake for Hanabiratake UT-18, UT-21, UT-31 and UT-33 as follows. After grinding 100 mg of the fruit body and mycelium in a mortar containing liquid nitrogen, a solution equivalent to the tissue weight (100 mM Na 2 HPO 4 , 50 mM citric acid, pH 6.0, 10 mM EDTA, 2% N-lauroyl sarcosine sodium) and left at 65 ° C. for 30 minutes. Next, the mixture was centrifuged at 7000 rpm for 10 minutes with a cooling centrifuge, and the supernatant was collected. An equal amount of a phenol: chloroform: isoamyl alcohol (= 75: 24: 1) solution was added for treatment (twice), and the mixture was centrifuged at 13000 rpm for 15 minutes with a cooling centrifuge, and the upper layer was recovered. After adding 1/10 volume of 3M NaOAc, 2.5 times the amount of ethanol was added, and the mixture was allowed to stand at −80 ° C. for 20 minutes. Centrifugation was performed at 13000 rpm for 20 minutes to precipitate DNA. Add the obtained DNA to TES-buffer (composition 30 mM Tris-HCl, pH 8.0, 5 mM EDTA, 50 mM NaCl) and dissolve well, then add RNase to a final concentration of 100 μg / ml. RNA was degraded. An equal amount of phenol: chloroform solution was added and stirred, and then centrifuged at 13000 rpm for 15 minutes with a microcooled centrifuge to recover the aqueous layer (including DNA). After adding 1/10 volume of 3M NaOAc, 2.5 times the amount of ethanol was added, and the mixture was allowed to stand at −80 ° C. for 20 minutes. Centrifugation was performed at 13000 rpm for 20 minutes to precipitate DNA. DNA was added to TE-buffer (composition 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) and dissolved well, and then the amount of DNA obtained was quantified. As a result, about 30-100 μg of chromosomal DNA was obtained.
〔PCRによる増幅〕
上記のようにして得られたハナビラタケの染色体DNA 5 ngをテンプレートとしてPCRを行った。プライマーには12塩基のプライマーを各種用いた。PCRの条件は変性温度94℃を30秒、アニーリング温度42℃で2分、伸長反応温度72℃3分間を45サイクル行った。TaqポリメラーゼはTaKaRa EX Taq Hot Start Version(宝酒造社)を用いた。
[Amplification by PCR]
PCR was carried out using 5 ng of chromosomal DNA of Hanabiratake obtained as described above as a template. Various primers having 12 bases were used. PCR was performed at a denaturation temperature of 94 ° C. for 30 seconds, an annealing temperature of 42 ° C. for 2 minutes, and an extension reaction temperature of 72 ° C. for 3 minutes for 45 cycles. TaKaRa EX Taq Hot Start Version (Takara Shuzo) was used as Taq polymerase.
〔電気泳動〕
PCR増幅産物のバンドパターンの確認を、TAEバッファーに1%の濃度で作成したアガロースゲルにPCR産物を5μlをローディングし、100V、27分間電気泳動により行った。DNAマーカーは200bp ladder (宝酒造社)を用いた。電気泳動後、ゲルをエチジウムブロマイドで染色し、トランスイルミネータ上でバンドの確認を行った。
[Electrophoresis]
The band pattern of the PCR amplification product was confirmed by loading 5 μl of the PCR product onto an agarose gel prepared at a concentration of 1% in TAE buffer and performing electrophoresis at 100 V for 27 minutes. A 200 bp ladder (Takara Shuzo) was used as the DNA marker. After electrophoresis, the gel was stained with ethidium bromide and the band was confirmed on a transilluminator.
〔結果〕
まず、PCRのプライマーとして配列ggcatggcctttを用いた。その結果RAPD解析から得られたバンドパターンを図1に示す。図1よりハナビラタケUT−33はプライマーとして配列ggcatggcctttを用いた場合に他の菌株には現れない2300±200 bpのバンドを示すこと、ハナビラタケUT−21はプライマーとして配列ggcatggcctttを用いた場合に他の菌株には現れない2500±200 bpのバンドを示すことが明らかになった。
〔result〕
First, the sequence ggcatggccttt was used as a PCR primer. As a result, the band pattern obtained from RAPD analysis is shown in FIG. From FIG. 1, Hanabiratake UT-33 shows a band of 2300 ± 200 bp that does not appear in other strains when the sequence ggcatggccttt is used as a primer. It was revealed that a 2500 ± 200 bp band that does not appear in the strain was exhibited.
次にPCRのプライマーとして配列ccgcagttagatを用いてRAPD解析して得られたバンドパターンを図2に示す。図2よりハナビラタケUT−31はプライマーとして配列ccgcagttagatを用いた場合に他の菌株には現れない1300±200 bpのバンドを示すことが明らかになった。 Next, FIG. 2 shows a band pattern obtained by RAPD analysis using the sequence ccgcagttagat as a PCR primer. From FIG. 2, it was clarified that Hanabiratake UT-31 shows a band of 1300 ± 200 bp that does not appear in other strains when the sequence ccgcagttagat is used as a primer.
さらにPCRのプライマーとして配列agcagcgcctcaを用いた場合のバンドパターンを図3に示す。図3よりハナビラタケUT−18はプライマーとして配列agcagcgcctcaを用いた場合に他の菌株には現れない1600±200 bpにバンドを示すことが明らかとなった。 Furthermore, FIG. 3 shows a band pattern when the sequence aggagcgcctca is used as a primer for PCR. From FIG. 3, it was revealed that Hanabiratake UT-18 shows a band at 1600 ± 200 bp that does not appear in other strains when the sequence aggagcgcctca is used as a primer.
本発明のハナビラタケには、上述した新菌株であるハナビラタケUT−18、ハナビラタケUT−21、ハナビラタケUT−31及びハナビラタケUT−33のほかに、これらの各菌株の変異株も含むものである。ここで変異株とは、これらの菌株に限定されるものではなく、上記の性質を有する菌株のことを言う。 In addition to Hanabiratake UT-18, Hanabiratake UT-21, Hanabiratake UT-31 and Hanabiratake UT-33, which are the above-mentioned new strains, the mutants of these strains are included in the present invention. Here, the mutant strain is not limited to these strains, but refers to a strain having the above properties.
さらに、本発明のハナビラタケの新菌株は、上述した方法を更に改良して、菌株を同定することが可能である。以下にその方法を示す。上述のRAPD法により得られたバンドパターンの内、その菌株に特異的なDNA断片に着目し、その塩基配列を解析することによって、菌株に特有の塩基配列を得た。この塩基配列を用い、ハナビラタケ菌株識別用プライマーを作製し、PCR法を用いることによって、さらに正確な同定を行うことが可能となった。つぎに、その具体的な方法を示す。 In addition, the new strain of the flower of the present invention can be identified by further improving the above-described method. The method is shown below. Of the band patterns obtained by the above-mentioned RAPD method, focusing on DNA fragments specific to the strain, and analyzing the base sequence, a base sequence specific to the strain was obtained. Using this base sequence, a primer for discriminating Hanabiratake strains was prepared, and it became possible to perform more accurate identification by using the PCR method. Next, the specific method is shown.
ハナビラタケUT−18を識別するためにプライマー配列として5’-tagcagcgcctcagcactat-3’及び5’-attagcagcgcctcacaatg-3’を用いてPCRを行った。PCRの条件は変性温度94℃を30秒、アニーリング温度60℃で30秒、伸長反応温度72℃90秒間を30サイクル行った。PCR反応後、上記の方法と同じ条件でPCR反応液の電気泳動を行った。その結果を図4に示す。図4によりUT−18にのみ1200-1600bpの間にDNA断片がひとつ確認される。他の菌株にこのプライマーを用いても1250bpの1200-1600bpの間にDNA断片は確認できない。よって、この方法によりハナビラタケUT−18の同定が可能である。 PCR was performed using 5'-tagcagcgcctcagcactat-3 'and 5'-attagcagcgcctcacaatg-3' as primer sequences to identify Hanabiratake UT-18. The PCR was performed at a denaturation temperature of 94 ° C. for 30 seconds, an annealing temperature of 60 ° C. for 30 seconds, and an extension reaction temperature of 72 ° C. for 90 seconds for 30 cycles. After the PCR reaction, the PCR reaction solution was electrophoresed under the same conditions as described above. The result is shown in FIG. According to FIG. 4, one DNA fragment is confirmed between 1200-1600 bp only in UT-18. Even if this primer is used for other strains, a DNA fragment cannot be confirmed between 1250 bp and 1200-1600 bp. Therefore, the Hanabiratake UT-18 can be identified by this method.
さらに、ハナビラタケUT−21を識別するためにプライマー配列として5’-atagggcgggggaataaagt-3’及び5’-ggataggggaatagccattgt-3’を用いてPCRを行った結果を図5に示す。図5より、UT−21および、UT−33において1800-2000bpの間にDNA断片が確認される。さらにプライマー配列として5’-acgaatagagcgggctgat-3’および5’-tcggacgaatacgaaagtacg-3’を用いてPCRを行った結果を図6に示す。図6によりUT−21において1400-1800bpの間にDNA断片が確認される。一方、UT−33において1800-2000bpの間にDNA断片が確認される。よって、この方法によりハナビラタケUT−21の同定が可能である。ハナビラタケUT−31を識別するためにプライマー配列として5’-gcctttgctattggagctgt-3’及び5’-ggcatggcctttgagatgta-3’を用いてPCRを行った結果を図7に示す。図7よりUT−31において2000-3000bpの間にDNA断片が確認される。他の菌株にこのプライマーを用いても2000-3000bpの間にDNA断片は確認できない。よって、この方法によりハナビラタケUT−31の同定が可能である。ハナビラタケUT−33を識別するためにプライマー配列として5’-acgaatagagcgggctgat-3’及び5’-tcggacgaatacgaaagtacg-3’を用いてPCRを行った結果を図6に示す。図6よりUT−33において1800-2000bpの間にDNA断片が確認される。他の菌株にこのプライマーを用いても1800-2000bpの間にDNA断片は確認できない。よって、この方法によってハナビラタケUT−33の同定が可能である。 Furthermore, FIG. 5 shows the results of PCR using 5′-atagggcgggggaataaagt-3 ′ and 5′-ggataggggaatagccattgt-3 ′ as primer sequences to identify Hanabiratake UT-21. From FIG. 5, a DNA fragment is confirmed between 1800 and 2000 bp in UT-21 and UT-33. Further, FIG. 6 shows the results of PCR using 5′-acgaatagagcgggctgat-3 ′ and 5′-tcggacgaatacgaaagtacg-3 ′ as primer sequences. According to FIG. 6, a DNA fragment is confirmed between 1400-1800 bp in UT-21. On the other hand, a DNA fragment is confirmed between 1800 and 2000 bp in UT-33. Therefore, it is possible to identify Hanabiratake UT-21 by this method. FIG. 7 shows the results of PCR using 5′-gcctttgctattggagctgt-3 ′ and 5′-ggcatggcctttgagatgta-3 ′ as primer sequences to identify Hanabiratake UT-31. From FIG. 7, a DNA fragment is confirmed between 2000 and 3000 bp in UT-31. Even if this primer is used for other strains, DNA fragments cannot be confirmed between 2000 and 3000 bp. Therefore, it is possible to identify Hanabiratake UT-31 by this method. FIG. 6 shows the results of PCR using 5'-acgaatagagcgggctgat-3 'and 5'-tcggacgaatacgaaagtacg-3' as primer sequences to identify Hanabiratake UT-33. From FIG. 6, a DNA fragment is confirmed between 1800 and 2000 bp in UT-33. Even if this primer is used for other strains, a DNA fragment cannot be confirmed between 1800 and 2000 bp. Therefore, it is possible to identify Hanabiratake UT-33 by this method.
さらに、本発明のハナビラタケには、ハナビラタケUT−18、ハナビラタケUT−21、ハナビラタケUT−31又はハナビラタケUT−33との対峙試験において帯線を形成しない菌株を含むものである。ここで対峙試験とは、二つ以上の試菌株の二核菌糸を保存スラントより3mm×3mm×3mmブロックの形で切り出し、それぞれを同一寒天平板培地の中央部に約2cm間隔で対峙して接種し、培養後、両コロニー境界部に帯線が生じるか否かを判定する方法のことをいい、帯線とは異なる試菌株同士のコロニー境界部の菌糸が融合しないために生じる境界線のことをいう。 Furthermore, Hanabiratake of the present invention includes strains that do not form a band in the confrontation test with Hanabiratake UT-18, Hanabiratake UT-21, Hanabiratake UT-31 or Hanabiratake UT-33. Here, the confrontation test refers to inoculating binuclear mycelium of two or more test strains in a 3mm x 3mm x 3mm block form from a preserved slant, and inoculating them at the center of the same agar plate medium at intervals of about 2cm. And after culture, it means a method to determine whether or not a band line is generated at the boundary part of both colonies, and a boundary line that occurs because the mycelium at the colony boundary part between test strains different from the band line does not fuse Say.
次に本発明のハナビラタケの栽培方法について説明する。本発明のハナビラタケの栽培方法は、上述した子実体乾燥重量100gあたりβグルカンを48.0g以上含有するハナビラタケの菌株を、大鋸屑に栄養分を添加した培地に接種し、培養・生育工程を経て、βグルカンを子実体乾燥重量100gあたり48.0g以上含む子実体を形成させた後、該子実体を収穫することを特徴とするものである。ハナビラタケとして新菌株を用いる以外は、従来の栽培ビンによる人工栽培方法により行なわれる。すなわち、1)栽培ビンに大鋸屑に栄養分を添加した固形培地を作成する。固形培地には接種孔を開けておくことが望ましい。2)この固形培地に種菌を接種する。3)接種した培地を通常の条件で培養・生育させる。 Next, a method for cultivating the flower of the present invention will be described. According to the method for cultivating the bamboo shoot of the present invention, the above-mentioned strain of 4 liters of β-glucan per 100 g of the fruit body dry weight is inoculated into a medium in which nutrients are added to the sawdust, and after the culture and growth process, A fruit body containing 48.0 g or more of glucan per 100 g of the fruit body dry weight is formed, and then the fruit body is harvested. Except for the use of a new strain as Hanabiratake, it is carried out by a conventional artificial cultivation method using cultivation bottles. That is, 1) A solid medium is prepared by adding nutrients to large sawdust in a cultivation bottle. It is desirable to have an inoculation hole in the solid medium. 2) Inoculate this solid medium with inoculum. 3) Culture and grow the inoculated medium under normal conditions.
本発明で用いられる大鋸屑としては、マツ、モミ、ツガ、ブナ、シイなどの大鋸屑が挙げられるが、好ましくは針葉樹の大鋸屑が好ましく、この中でもカラマツの大鋸屑が好ましい。広葉樹と針葉樹の大鋸屑の混合物を用いることもでき、大鋸屑にコーンコブなどを混合してもよい。添加する栄養分としては、必要に応じ適宜添加することができるが、小麦粉などが好ましく、小麦粉を1%〜30%、好ましくは3%〜20%、さらに好ましくは5%〜10%を混合する。これを通常850ccのポリプロピレン製栽培ビンに約520g充填する。この際、培地中央に接種孔を開けておくと、接種量を多くすることができ培地全体に菌糸が回りやすくなり、また通気がよくなるため好ましい。接種孔を開けることにより結果として収穫までの期間が短縮でき、収量が上がるなどの利点が生じる。接種孔の大きさは断面積がほぼ80mm2以上あればよい。固形培地は、高圧殺菌などの方法により殺菌をしておく。 Examples of the large sawdust used in the present invention include large sawdust such as pine, fir, Tsuga, beech, shii and the like, preferably coniferous large sawdust, among which larch large sawdust is preferable. A mixture of hardwood and coniferous large sawdust can also be used, and corncob or the like may be mixed with the large sawdust. As a nutrient to be added, it can be appropriately added as necessary, but wheat flour or the like is preferable, and 1% to 30%, preferably 3% to 20%, more preferably 5% to 10% of flour is mixed. About 520 g of this is normally filled into an 850 cc polypropylene cultivation bottle. At this time, it is preferable to make an inoculation hole in the center of the medium because the amount of inoculation can be increased, so that the mycelia can easily rotate around the entire medium and the ventilation is improved. Opening the inoculation hole results in advantages such as shortening the time to harvest and increasing the yield. The size of the inoculation hole may be about 80 mm 2 or more in cross-sectional area. The solid medium is sterilized by a method such as high pressure sterilization.
このような培地に同様のオガクズ培地で培養した種菌を接種する。接種量は通常8g〜18gである。種菌を接種した後、一定の条件下、すなわち温度は15℃〜30℃、好ましくは18℃〜28℃、20〜25℃が最も好ましく、湿度は50%〜80%、好ましくは55%〜75%、60%〜70%が最も好ましい条件下で培養し、培地に菌糸を回らせる。その後、芽出し操作として、菌掻きと異種キノコの接種(詳細は、特開2002−369621号公報を参照)をおこなう。異種キノコは、同様のオガクズ培地でホウライタケを培養して作成した種菌2gを用いた。その後、ビンのふたを取って、(括弧内削除:温度約19℃、湿度約90%の)栽培室に移し、子実体を成育させる。栽培室の温度は10〜30℃、好ましくは15〜28℃、最も好ましくは17〜25℃、湿度は80%以上、好ましくは85%以上、最も好ましくは90%以上である。通常、芽出し操作後約56日間の生育工程により成長が終了するので、子実体の収穫を行う。 Such a medium is inoculated with an inoculum cultured in the same sawdust medium. The inoculum is usually 8 to 18 g. After inoculation with the inoculum, under certain conditions, i.e., the temperature is 15 ° C-30 ° C, preferably 18 ° C-28 ° C, 20-25 ° C, and the humidity is 50% -80%, preferably 55% -75. %, 60% to 70% is cultured under the most preferable conditions, and the mycelium is rotated in the medium. Thereafter, as a sprouting operation, bacterial scraping and inoculation with different types of mushrooms are performed (for details, see JP-A-2002-369621). As the heterologous mushrooms, 2 g of inoculum prepared by cultivating spinach mushrooms in the same sawdust medium was used. Then, remove the bottle lid and move it to the cultivation room (deleted in parentheses: temperature of about 19 ° C, humidity of about 90%) to grow the fruit body. The temperature of the cultivation room is 10 to 30 ° C., preferably 15 to 28 ° C., most preferably 17 to 25 ° C., and the humidity is 80% or more, preferably 85% or more, and most preferably 90% or more. Usually, since the growth is completed by the growing process for about 56 days after the sprouting operation, the fruiting body is harvested.
実施例1
ハナビラタケUT−18(FERM P−19768)の種菌を、栽培用のオガクズ培地に接種した。オガクズ培地の培地基材にはカラマツのオガクズを用い、これに小麦粉を7%混合し、水を加えて水分を60%に調製した。これを850ccのポリプロピレンの栽培ビンに520g充填し、直径20mmの接種孔を市販の穴あけ機を用いて栽培ビンの中央に1つ空けた後110℃で3時間高圧殺菌した。その後、培地温度が30℃以下まで下がるのを待って種菌を接種し、これを23℃、湿度65%で培養した。接種後、49日ほどでハナビラタケの菌糸が充満した。これに菌掻きを行い、栽培ビンのふたを取って、温度19℃、湿度90%の栽培室に移した。その後、ハナビラタケの子実体原基の形成が240本中240本見られ、その形成率は100%であった。菌掻きから収穫までの期間は約56日間であった。子実体形成の見られた栽培ビン240本つき1本のあたりの平均収量は約100gであり、全収穫量は24.0kgであった。
Example 1
An inoculum of Hanabiratake UT-18 (FERM P-19768) was inoculated on a sawdust medium for cultivation. As a medium substrate for the sawdust medium, larch sawdust was used, 7% of flour was mixed therein, and water was added to prepare a water content of 60%. This was filled with 520 g of an 850 cc polypropylene cultivation bottle, and an inoculation hole with a diameter of 20 mm was made in the center of the cultivation bottle using a commercially available drilling machine, and then pasteurized at 110 ° C. for 3 hours. Thereafter, the inoculum was inoculated after the temperature of the medium dropped to 30 ° C. or lower, and this was cultured at 23 ° C. and humidity 65%. About 49 days after the inoculation, the mycelium of Hanabiratake was full. The fungi were scraped, the lid of the cultivation bottle was removed, and the cultivation bottle was moved to a cultivation room with a temperature of 19 ° C. and a humidity of 90%. Thereafter, formation of fruit body primordia of Hanabiratake was observed in 240 out of 240, and the formation rate was 100%. The period from fungus scraping to harvesting was about 56 days. The average yield per 240 cultivated bottles with fruiting body formation was about 100 g, and the total yield was 24.0 kg.
任意に選んだ栽培ビン3本から収穫したハナビラタケUT−18の子実体について、上述した方法によりβグルカン量を測定したところ、平均として乾燥重量100gあたり51.2gであった。 When the amount of β-glucan was measured by the method described above for the fruit bodies of Hanabiratake UT-18 harvested from three arbitrarily selected cultivation bottles, the average was 51.2 g per 100 g of dry weight.
実施例2
ハナビラタケUT−21(FERM P−19769)を接種した以外は、実施例1と全く同様にして栽培した。子実体原基の形成率は99.6%であり、栽培ビン239本につき1本あたりの平均収量は約100gであり、全収穫量は23.9kgであった。
Example 2
Cultivation was carried out in exactly the same manner as in Example 1, except that inoculated with Hanabiratake UT-21 (FERM P-19769). The formation rate of fruit body primordium was 99.6%, the average yield per one 239 cultivation bottles was about 100 g, and the total yield was 23.9 kg.
また、ハナビラタケUT−21のβグルカン量は、任意に選んだ栽培ビン3本からの子実体の平均として乾燥重量100gあたり50.0gであった。 Moreover, the amount of β-glucan of Hanabiratake UT-21 was 50.0 g per 100 g of dry weight as an average of fruit bodies from three arbitrarily selected cultivation bottles.
実施例3
ハナビラタケUT−31(FERM P−19770)を接種した以外は、実施例1と全く同様にして栽培した。子実体原基の形成率は99.2%であり、栽培ビン238本につき1本あたりの平均収量は約96gであり、全収穫量は22.8kgであった。
Example 3
Cultivated in exactly the same manner as in Example 1 except that it was inoculated with Hanabiratake UT-31 (FERM P-19770). The formation rate of fruiting body primordium was 99.2%, the average yield per 238 cultivation bottles was about 96 g, and the total yield was 22.8 kg.
また、ハナビラタケUT−33のβグルカン量は、任意に選んだ栽培ビン3本からの子実体の平均として乾燥重量100gあたり49.6gであった。 In addition, the amount of β-glucan of Hanabiratake UT-33 was 49.6 g per 100 g of dry weight as an average of fruit bodies from three arbitrarily selected cultivation bottles.
実施例4
ハナビラタケUT−33(FERM P−19771)を接種した以外は、実施例1と全く同様にして栽培した。子実体原基の形成率は99.6%であり、栽培ビン239本につき1本あたりの平均収量は約90gであり、全収穫量は21.5kgであった。
Example 4
Cultivated in exactly the same manner as in Example 1 except that it was inoculated with Hanabiratake UT-33 (FERM P-19771). The formation rate of fruiting body primordium was 99.6%, the average yield per 239 cultivation bottles was about 90 g, and the total yield was 21.5 kg.
また、ハナビラタケUT−33のβグルカン量は、任意に選んだ栽培ビン3本からの子実体の平均として乾燥重量100gあたり48.1gであった。 Further, the amount of β-glucan of Hanabiratake UT-33 was 48.1 g per 100 g of dry weight as an average of fruit bodies from three arbitrarily selected cultivation bottles.
比較例1
実施例1〜4と同様にして、NS422、NS424(以上、財団法人日本きのこ研究所より入手)、UT-15 UT-19 UT-23 UT-35(以上、ユニチカ株式会社保有菌株)を栽培し、β-グルカン含有量を測定した。乾燥重量100gあたりのβ-グルカン含量は、それぞれ33.6g、37.7g、43.0g、43.4g、44.0g、41.1gであった。
Comparative Example 1
In the same manner as in Examples 1 to 4, NS422, NS424 (above, obtained from Japan Mushroom Research Institute), UT-15 UT-19 UT-23 UT-35 (above, strain owned by Unitika Ltd.) are cultivated. Β-glucan content was measured. The β-glucan content per 100 g of dry weight was 33.6 g, 37.7 g, 43.0 g, 43.4 g, 44.0 g, and 41.1 g, respectively.
表1は実施例1〜4と比較例1の結果をまとめたものである。実施例1〜4のβ-グルカン含有量は他の菌株に比べて明らかに高いことが分かる。 Table 1 summarizes the results of Examples 1 to 4 and Comparative Example 1. It can be seen that the β-glucan content of Examples 1 to 4 is clearly higher than that of other strains.
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