JP5294659B2 - Fermentation and culture method, lotus root ferment extract and fermented extract blend - Google Patents

Fermentation and culture method, lotus root ferment extract and fermented extract blend Download PDF

Info

Publication number
JP5294659B2
JP5294659B2 JP2008061930A JP2008061930A JP5294659B2 JP 5294659 B2 JP5294659 B2 JP 5294659B2 JP 2008061930 A JP2008061930 A JP 2008061930A JP 2008061930 A JP2008061930 A JP 2008061930A JP 5294659 B2 JP5294659 B2 JP 5294659B2
Authority
JP
Japan
Prior art keywords
lotus root
extract
buckwheat
gram
bacteria
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
JP2008061930A
Other languages
Japanese (ja)
Other versions
JP2008255108A (en
Inventor
源一郎 杣
孝志 西澤
千恵 河内
裕之 稲川
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biomedical Research Group Inc
Original Assignee
Biomedical Research Group Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biomedical Research Group Inc filed Critical Biomedical Research Group Inc
Priority to JP2008061930A priority Critical patent/JP5294659B2/en
Publication of JP2008255108A publication Critical patent/JP2008255108A/en
Application granted granted Critical
Publication of JP5294659B2 publication Critical patent/JP5294659B2/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Landscapes

  • Medicines Containing Plant Substances (AREA)
  • Fodder In General (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Cosmetics (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Description

本発明は、ヒトを含む哺乳動物(具体的には家畜、愛玩動物など)、鳥類(具体的には養鶏、愛玩鳥類など)、両生類、は虫類、魚類(具体的には、愛玩魚類など)、無脊椎動物及び植物などの動植物に及ぶ医薬品、医薬部外品、化粧品、機能性食品、飼料、肥料及び浴用剤などに添加しても安全な免疫賦活物質含有食用グラム陰性菌によるソバ又はレンコン発酵方法、ソバ又はレンコン発酵によって得られる免疫賦活物質含有のエキスなどに関する。   The present invention includes mammals including humans (specifically, domestic animals, pets, etc.), birds (specifically, poultry farming, pets, etc.), amphibians, reptiles, fish (specifically, pets, etc.), Buckwheat or lotus root fermented by edible gram-negative bacteria containing immunostimulatory substances that are safe to add to pharmaceuticals, quasi-drugs, cosmetics, functional foods, feeds, fertilizers, bathing agents, etc. for animals and plants such as invertebrates and plants The present invention relates to a method, an extract containing an immunostimulatory substance obtained by buckwheat or lotus root fermentation.

ヒトを含む哺乳動物(具体的には家畜、愛玩動物など)、鳥類(具体的には養鶏、愛玩鳥類など)、両生類、は虫類、魚類(具体的には、愛玩魚類など)、無脊椎動物及び植物に関して、感染防除技術を含む疾病予防・治療法を確立することは喫緊の課題である。しかもこれを達成する上では、化学物質を用いず、環境汚染がなく、耐性菌を生ずることなく、人体に蓄積性がない方法が強く求められている。本発明者らは如上の課題に関して、日本では古くより食されているソバとレンコンに着目した。ソバやレンコンは単に主食のデンプン源としてだけでなく、健康食品的に取り扱われてきている。   Mammals including humans (specifically, domestic animals, pets, etc.), birds (specifically poultry farming, pets, etc.), amphibians, reptiles, fish (specifically, pets, etc.), invertebrates and It is an urgent task to establish disease prevention and treatment methods including infection control technology for plants. Moreover, in order to achieve this, there is a strong demand for a method that does not use chemical substances, does not cause environmental pollution, does not produce resistant bacteria, and does not accumulate in the human body. The present inventors paid attention to buckwheat and lotus root that have been eaten in Japan for a long time. Buckwheat and lotus root have been treated not only as a starch source for staple foods but also as health foods.

本発明者らは、グラム陰性菌の膜成分であるリポ多糖に免疫賦活作用があることを見出している。そこで、古来より食されているソバやレンコンに存在するグラム陰性菌に注目した。すなわち、食品の製造に用いられるグラム陰性菌にリムラス陽性糖脂質なかんずくリポ多糖が存在すれば、この事実はリムラス陽性糖脂質あるいはリポ多糖の食経験を立証することになる。すでに小麦粉常在菌であるパントエア・アグロメランスから得たリポ多糖を用いることができることを見出した(非特許文献1)。このことは、リムラス陽性糖脂質あるいはリポ多糖が経皮、経口投与で安心・安全であることを強力に示す知見であると共に、これら物質を用いた新たな化粧品・食品などのヘルスケア商品、医薬品などの開発を可能とするはずである。   The present inventors have found that lipopolysaccharide, which is a membrane component of Gram-negative bacteria, has an immunostimulatory effect. Therefore, we focused on Gram-negative bacteria present in buckwheat and lotus root that have been eaten since ancient times. That is, if Limulus-positive glycolipids, especially lipopolysaccharides, are present in Gram-negative bacteria used in the production of food, this fact demonstrates the dietary experience of Limulus-positive glycolipids or lipopolysaccharides. It has been found that lipopolysaccharide obtained from Pantoea agglomerans, which is already a flour-resident bacterium, can be used (Non-patent Document 1). This is a powerful finding that rimlas-positive glycolipids or lipopolysaccharides are safe and safe for transdermal and oral administration, as well as new health care products such as cosmetics and foods and pharmaceuticals using these substances. Should be possible.

本発明が提供するソバ又はレンコン発酵エキスとは発酵及び培養を行って得られる培養液そのもの、これを固液分離して得られる液体成分、及び固液分離して得られる固体成分に抽出過程を加え得られる液体成分などを総称する名称である。すなわち、ソバ又はレンコン発酵エキスは本発明に係る発酵及び培養方法によって得られる培養液そのもの、及び、その培養液の全部又は一部分を用いて調製することのできるエキスをすべて含んでいる。言うまでもないがソバ又はレンコン発酵エキスは乾燥してエキス末として利用することもできるし、エキス末を任意の濃度に適当な溶液例えば生理食塩水入りの燐酸緩衝液などに溶解して利用することもできる。
河内千恵ほか: 小麦発酵抽出物の自然免疫調節作用、NEW FOOD INDUSTRY 48, 19-26(2006). 若命浩二:ソバポリフェノールの利用開発,ソバの栄養,(社)日本ソバ協会,49-54(2000) 小砂憲一ほか:食品と開発,33,1143-1145(1998) 我妻千尋ほか:PMP(ソバポリフェノール)のTriton誘発高脂血症モデルに対する影響,第2回日本代替医療学会学術集会要旨,85(1999) 石黒綾ほか:化学発がんに対するAHCCとPMPの相乗効果,第2回日本代替医療学会学術集会要旨,85(1999) Kayashita, J. et al.: Hypocholesterolemiceffect of buckwheat protein extract in rats fed cholesterol enriched diets,Nutr. Res., 15, 691-698(1995) Kayashita, J. et al.: Consumption ofbuckwheat protein lowers plasma cholesteroland raises fecal neutral sterols incholesterol-fed rats because of its low digestability, J. Nutr., 127,1395-1400(1997) Kayashita, J. et al.: Feeding of buckwheatprotein extract reduces hepatic triglyceride concentration, adipose tissueweight, and hepatic lipogenesis in rats, Nutr. Biochem., 7, 555-559(1996) Powers EM. Efficacy of the Ryu nonstainingKOH technique for rapidly determining Gram reactions of food-bone and waterbonebacteria and yeasts. Appl. Environ. Microbiol. (1995) 61, 3756-3758.
The buckwheat or lotus root fermented extract provided by the present invention is a culture solution itself obtained by fermentation and culture, a liquid component obtained by solid-liquid separation of this, and a solid component obtained by solid-liquid separation. It is a name that collectively refers to liquid components that are obtained. That is, the buckwheat or lotus root fermented extract contains all of the culture solution itself obtained by the fermentation and culture method according to the present invention, and all the extracts that can be prepared using all or part of the culture solution. Needless to say, the buckwheat or lotus root fermented extract can be dried and used as an extract powder, or the extract powder can be used by dissolving it in an appropriate solution such as a phosphate buffer containing physiological saline. it can.
Chie Kawauchi et al .: Innate immune regulation of fermented wheat extract, NEW FOOD INDUSTRY 48, 19-26 (2006). Wakamei Koji: Utilization development of buckwheat polyphenol, buckwheat nutrition, Japan Buckwheat Association, 49-54 (2000) Kenichi Kosuna et al .: Food and Development, 33, 1143-1145 (1998) Chihiro Gazuma et al .: Effects of PMP (buckwheat polyphenol) on Triton-induced hyperlipidemia model, Abstracts of the 2nd Annual Meeting of the Japanese Society for Alternative Medicine, 85 (1999) Aya Ishiguro et al .: Synergistic effect of AHCC and PMP on chemical carcinogenesis, Abstracts of the 2nd Annual Meeting of the Japanese Society for Alternative Medicine, 85 (1999) Kayashita, J. et al .: Hypocholesterolemic effect of buckwheat protein extract in rats fed cholesterol enriched diets, Nutr. Res., 15, 691-698 (1995) Kayashita, J. et al .: Consumption of buckwheat protein lowers plasma cholesterol and raises fecal neutral sterols incholesterol-fed rats because of its low digestability, J. Nutr., 127, 1395-1400 (1997) Kayashita, J. et al .: Feeding of buckwheatprotein extract reduces hepatic triglyceride concentration, adipose tissueweight, and hepatic lipogenesis in rats, Nutr. Biochem., 7, 555-559 (1996) Powers EM. Efficacy of the Ryu nonstainingKOH technique for rapidly determining Gram reactions of food-bone and waterbonebacteria and yeasts.Appl.Environ.Microbiol. (1995) 61, 3756-3758.

ソバ(学名:Fagopyrum esculentum)はタデ科の植物で、米、麦の主要穀類より生産量は少ないものの、世界の広い地域で生産されている食用植物である。食用のほか、ソバは漢方で用いられており、『本朝食鑑』(1697)では「気味甘く、微寒にして毒なし、気を降ろし、腸胃の滓穢積滞を寛にす。水腫・白濁・泄利・腹痛・上気を治し、或は気盛んにして湿熱あるものによろし」とあり、ソバが、健康維持に有用な役割を持つことが伺える。   Buckwheat (scientific name: Fagopyrum esculentum) is an edible plant that is produced in a large area of the world, although it produces less than the main grains of rice and wheat. In addition to edible, buckwheat is used in traditional Chinese medicine, and in the book book Breakfast (1697), “It's so sweet, chilled, non-poisonous, distracted, and relaxed intestinal stomach stagnation. It is said that he cures white turbidity, excretion, abdominal pain, upper air, or has a vigorous and moist heat, ”indicating that buckwheat has a useful role in maintaining health.

ソバはカテキン類のほか,ソバ種実は穀類で唯一,フラボノイド化合物であるルチンを含んでいる。ルチンは毛細血管の強化作用を有し高血圧や糖尿病に有効であるといわれている。また、ソバ種実ポリフェノールは,細胞実験での抗酸化能の確認(非特許文献2)に加え,生体内では,マウス脳中の過酸化脂質の低下作用(非特許文献3),トライトン(Triton)誘発高脂血症モデルラットに対する血清中性脂肪の上昇抑制効果(非特許文献4),化学発がんモデル(皮膚がん)マウスに対する発がん抑制効果(非特許文献5)が明らかにされている.ポリフェノール以外では,タンパク抽出物(BWPE)がコレステロール低下作用(非特許文献6,7)をもつことが報告されている。加えて、ソバタンパクは肝臓トリグリセライド濃度の低下,精巣上体付着脂肪組織重量と腎付着脂肪組織重量の減少をもたらすことが明らかにされている(非特許文献8)。しかしながら、これらのソバの機能性成分は、ソバ自体の成分の解析であり、ソバに付着・共生している細菌由来の免疫賦活成分について解析されたことはこれまでになく、その機能性は不明であった。   In addition to catechins, buckwheat seeds are the only cereal and contain rutin, a flavonoid compound. Rutin is said to have an effect of strengthening capillaries and is effective for hypertension and diabetes. In addition, the buckwheat seed polyphenols are confirmed to have antioxidant ability in cell experiments (Non-patent Document 2), and in vivo, lipid peroxide lowering action in the mouse brain (Non-patent Document 3), Triton Inhibition of serum triglycerides in induced hyperlipidemia model rats (Non-patent Document 4) and inhibition of carcinogenesis in chemical carcinogenic model (skin cancer) mice have been clarified (Non-patent Document 5). In addition to polyphenols, it has been reported that protein extracts (BWPE) have a cholesterol-lowering effect (Non-patent Documents 6 and 7). In addition, buckwheat protein has been shown to cause a decrease in liver triglyceride concentration and a decrease in epididymal and adipose tissue weight (Non-patent Document 8). However, the functional component of these buckwheat is an analysis of the component of buckwheat itself, and it has never been analyzed for an immunostimulatory component derived from bacteria attached to and symbiotic to buckwheat, and its functionality is unknown Met.

レンコンは、ハスの地下茎が肥大した物で、食用に栽培され食物繊維やビタミンCなどの栄養素を豊富に含んでいる野菜であり、胃腸粘膜を保護するムチンや抗酸化作用を有するポリフェノールなど生理活性を示す物質を多く含んでいる。また、漢方の分野では咳や痰の沈静化、炎症の抑制といった目的でレンコンを使用しており、アレルギー症状の改善に対しても効果が期待される。地下茎の接合部分(普段は食用としない箇所)を生薬名「藕節(グウセツ)」と称し,吐血,胃潰瘍,十二指腸潰瘍,下血などに止血を目的として民間薬的に利用されている。止血作用については含有タンニンによるものと考えられている。しかしながら、これらレンコンについてもその機能性成分は、レンコン自体の成分の解析であり、レンコンに付着・共生している細菌由来の免疫賦活成分について解析されたことはこれまでになく、その機能性は不明であった。   Lotus root is an enlarged lotus lotus rhizome, cultivated for foods and rich in dietary fiber and vitamin C and other nutrients. It contains a lot of substances. In the field of Chinese medicine, lotus root is used for the purpose of calming cough and sputum and suppressing inflammation, and it is expected to be effective for improving allergic symptoms. The joint part of the rhizome (usually not edible) is called the crude drug name “Gutsutsu”, and is used as a folk medicine for hemostasis, gastric ulcer, duodenal ulcer, and melena. The hemostatic effect is considered to be due to the contained tannin. However, the functional components of these lotus roots are analysis of the components of the lotus root itself, and the immunostimulatory components derived from bacteria adhering to and living together with the lotus roots have not been analyzed so far, and their functionality is It was unknown.

本発明は、上記問題点に鑑み、食経験の長いレンコンに付着・共生するグラム陰性菌単独又は混合で発酵し該菌を培養する方法及び該方法で得られるエキス、エキス配合物を提供することを目的とする。 In view of the above problems, the present invention provides a method of culturing a gram-negative bacterium that adheres and symbiotic to a lotus root with a long diet alone or in a mixture and cultures the bacterium, and an extract and extract composition obtained by the method. With the goal.

本発明はレンコンの機能性を増強するために、免疫賦活機能を有するグラム陰性菌が有するリポ多糖に着目している。本発明は実施例に記載した微生物に限定されるわけではなく、免疫賦活機能を有する食経験のあるグラム陰性菌である、セラチア・フィカリア(Serratia ficaria)、セラチア・プリムチカ(Serratia plymuthica)、エンテロバクター・アムニゲナス(Enterobacter amnigenus)、エンテロバクター・クロアカ(Enterobacter cloacae)、ラーネラ・アクアティリス(Rahnella aquatilis)、レクレルシア・アデカルボキシラータ(Leclercia adecarboxylata)などの他の微生物にも適応できることは明らかである。さらに、本発明により提供されるレンコン発酵エキスは、経口及び経皮投与で安全に免疫を活性化することができるので、医薬品、並びに、栄養補助や特定機能性をもつ食品を含む食品、スキンケア製品、飼料及びペットフードなど動植物の健康維持を目的とした広範な用途に配合して用いることができる。 The present invention focuses on lipopolysaccharide possessed by gram-negative bacteria having an immunostimulatory function in order to enhance the functionality of lotus root . The present invention is not limited to the microorganisms described in the Examples, but is a gram-negative bacterium having an immunostimulatory function, such as Serratia ficaria, Serratia plymuthica, Enterobacter It is clear that it can also be applied to other microorganisms such as Enterobacter amnigenus, Enterobacter cloacae, Rahnella aquatilis, Leclercia adecarboxylata. Furthermore, since the lotus root fermented extract provided by the present invention can safely activate immunity by oral and transdermal administration, pharmaceuticals, foods including nutritional supplements and foods with specific functions, skin care products It can be used in a wide range of applications for the purpose of maintaining the health of animals and plants such as feed and pet food.

本発明の発酵及び培養方法は、レンコンに由来する素材を、前記レンコンに付着又は共生するグラム陰性菌によって発酵させて、同時に該菌を培養することを特徴とする。 METHOD fermentation and culture of the present invention, a material derived from lotus root, fermented by gram-negative bacteria to adhere or symbiotic to said lotus root, characterized in that simultaneously culturing fungus.

また、前記レンコンに由来する素材はレンコンに力学的操作を加えることによって得られることが望ましい。 Furthermore, material derived from the lotus root is preferably obtained by adding a mechanical operation on lotus root.

また、本発明のレンコン発酵エキスは、上記発酵及び培養方法で得られることを特徴とする。 Moreover, the lotus root fermented extract of the present invention is obtained by the above fermentation and culture method.

また、本発明のレンコン発酵エキス末は、上記植物発酵エキスから得られることを特徴とする。 Moreover, the lotus root fermented extract powder of the present invention is obtained from the above plant fermented extract.

また、本発明のレンコン発酵エキス配合物は、上記レンコン発酵エキス又は上記レンコン発酵エキス末が配合されていることを特徴とする。 Moreover, the lotus root fermented extract blend of the present invention is characterized in that the lotus root fermented extract or the lotus root fermented extract powder is blended.

また、上記レンコン発酵エキス配合物は、医薬品、動物用医薬品、医薬部外品、化粧品、食品、機能性食品、飼料、植物用肥料、植物用医薬品又は浴用剤であることを特徴とする。
Moreover, the lotus root fermented extract blend is a pharmaceutical, veterinary drug, quasi-drug, cosmetic, food, functional food, feed, plant fertilizer, plant drug, or bath preparation.

以下、本発明の好適な実施の形態について詳細に説明する。   Hereinafter, preferred embodiments of the present invention will be described in detail.

ソバ粉に含まれるリムラス陽性糖脂質含量
ソバ粉に含まれるリムラス陽性糖脂質量を測定した。ソバ粉(内層ソバ粉、中層ソバ粉、外層ソバ粉)、ソバの皮及びソバの実の粉砕物を各2.00g秤量し、それぞれに注射用水を加えて20mlとした。ボルテックスミキサーにて攪拌し懸濁液を調製した。懸濁液の各1mlを1.5mlチューブに移し、ブロックヒーターを用いて95℃で30分間加熱した。加熱終了後、遠心分離を行い、上清をリムラス陽性糖脂質測定用の原液とした。生化学工業のエンドスペシーのキットを用いて測定した。方法は、キット添付のプロトコールに従った。測定された糖脂質含量はIP-PA1の重量換算値として算出した。その結果を表1に示した。いずれのサンプルからも糖脂質が検出された。糖脂質含量は、ソバの実を破砕して抽出したものが最も高いものであった。

Figure 0005294659
Limulus-positive glycolipid content contained in buckwheat flour The amount of limulus-positive glycolipid contained in buckwheat flour was measured. Buckwheat flour (inner layer buckwheat flour, middle layer buckwheat flour, outer layer buckwheat flour), buckwheat peel and buckwheat crushed powder were weighed 2.00 g each, and water for injection was added to each to make 20 ml. The suspension was prepared by stirring with a vortex mixer. 1 ml of each suspension was transferred to a 1.5 ml tube and heated at 95 ° C. for 30 minutes using a block heater. After completion of the heating, centrifugation was performed, and the supernatant was used as a stock solution for measuring Limulus positive glycolipid. Measurements were made using Seikagaku's Endspecy kit. The method followed the protocol attached to the kit. The measured glycolipid content was calculated as a weight converted value of IP-PA1. The results are shown in Table 1. Glycolipids were detected from all samples. The highest glycolipid content was obtained by crushing and extracting buckwheat berries.
Figure 0005294659

ソバからの微生物の分離
実施例1の結果から、各種ソバサンプルにグラム陰性菌が存在することが推測された。そこで、次にソバからの微生物の単離を行った。ソバ粉、ソバの皮及びソバの実の粉砕物を各2.00g秤量し、それぞれに滅菌済みの蒸留水(大塚注射用水)を加えて20mlとした。ボルテックスミキサーにて攪拌し得られた懸濁液を普通寒天培地の平板プレートに塗抹し、30℃の恒温槽内で2日間分離培養を行った。培養後、各プレート上に認められたコロニー数(生菌数)を計測し、ソバ粉の単位重量当たりの生菌数を算出し表2に示した。菌数については、内層粉は菌数が最も少なく、ソバの皮が最も高かった。コロニーの形態によって分けられる菌の種類と割合は標品間で大きな違いは認められず、黄色のコロニーの菌がほぼ9割を占めた。黄色のコロニーについては、コロニーのサイズが大きいものと小さいものの少なくとも2種類が含まれ、ほぼ半々の割合で認められた。標品間での認められた菌の種類の違いについては、外層粉とソバの実の粉砕物では橙色のコロニーが認められたが、内層粉、中層粉及びソバの皮では認められなかった。各標品のプレートから菌種が異なる可能性が高いと判定したコロニー38種について純培養を行った。

Figure 0005294659
Separation of microorganisms from buckwheat From the results of Example 1, it was inferred that gram-negative bacteria were present in various buckwheat samples. Therefore, we next isolated microorganisms from buckwheat. 2.00 g each of buckwheat flour, buckwheat skin and buckwheat crushed powder were weighed, and sterilized distilled water (Otsuka water for injection) was added to each to make 20 ml. The suspension obtained by stirring with a vortex mixer was smeared on a flat plate of a normal agar medium, and separated and cultured in a constant temperature bath at 30 ° C. for 2 days. After culturing, the number of colonies (viable bacteria count) found on each plate was counted, and the viable bacteria count per unit weight of buckwheat flour was calculated and shown in Table 2. As for the number of bacteria, the inner layer powder had the smallest number of bacteria and the highest in buckwheat skin. There were no significant differences in the types and proportions of bacteria divided by colony morphology, and yellow colonies accounted for almost 90%. Regarding yellow colonies, there were at least two types of colonies with large and small colonies. Regarding the difference in the types of bacteria observed between the preparations, orange colonies were observed in the ground powder and buckwheat crushed powder, but not in the inner powder, middle powder and buckwheat skin. Pure culture was performed on 38 types of colonies that were determined to have a high possibility of different bacterial species from the plates of each sample.
Figure 0005294659

ソバ付着グラム陰性菌、グラム陽性菌の判定
Ryuの方法 (非特許文献9)を用いた。実施例2で得られたコロニーの形態より菌種が異なると推定されたコロニーを選択し、別の普通寒天培地プレートにまき、菌の単離を行った。各菌のコロニーより、菌体を掻き取りスライドグラス上に移し、3%KOH水溶液5μlを菌体にかけ、菌体をかき混ぜた。1分以内に粘り、ゲル化し糸を引くようになればKOHの反応に陽性と判定し、グラム陰性菌とした。菌体をかき混ぜても変化が認められず、糸を引かない場合は、KOHの反応に陰性と判定し、グラム陽性菌と判定した。分離した38種の微生物のうち、10菌株がグラム陽性菌と判定され、残りの27菌株がグラム陰性菌と判定された。黄色コロニーの菌株は全て、グラム陰性と判定された。ソバ粉サンプルに存在する生菌数の約9割がグラム陰性細菌であることがわかった。
Determination of buckwheat-attached gram-negative and gram-positive bacteria
Ryu's method (Non-Patent Document 9) was used. A colony presumed to have a different bacterial species from the form of the colony obtained in Example 2 was selected, spread on another normal agar plate, and the bacteria were isolated. From each colony of bacteria, the bacterial cells were scraped and transferred onto a slide glass, 5 μl of 3% KOH aqueous solution was applied to the bacterial cells, and the bacterial cells were mixed. If it became sticky and gelled within 1 minute, it was judged positive for KOH reaction, and it was regarded as a Gram-negative bacterium. When no change was observed even when the cells were stirred, and the thread was not pulled, it was determined to be negative for the KOH reaction and determined to be a Gram-positive bacterium. Of the 38 microorganisms isolated, 10 strains were determined as gram-positive bacteria, and the remaining 27 strains were determined as gram-negative bacteria. All strains of yellow colonies were determined to be gram negative. About 90% of the viable bacteria present in the buckwheat flour samples were found to be gram-negative bacteria.

ソバ付着菌種の同定
グラム陰性菌と判定された27菌株について判定した。ブドウ糖発酵性の有無、チトクローム・オキシダーゼの有無、ID-テスト EB20(日水製薬)を用いて菌種同定した。
(1).ブドウ糖発酵性
TSI寒天培地でブドウ糖発酵性を試験した。実施例3でグラム陰性菌と判定した菌株を用いた。TSI寒天培地(日水製薬株式会社)に被検菌を高層部にせん刺後、斜面部に塗布し、30℃の恒温槽にて18〜24時間培養した。ブドウ糖発酵性の菌は高層部が黄色に変化し、ブドウ糖非発酵性の菌は高層部の色は朱色で変化しない。ブドウ糖のみを分解する菌は高層部が黄変し、斜面部は無変化。乳糖、白糖共に、又はいずれか一方の分解菌は高層、斜面部共に黄変。ガス産生があれば高層部に気泡又はき裂を生じる。硫化水素の産生があれば高層部が濃く変化する。その結果、9株がブドウ糖非発酵性グラム陰性菌、18菌株がブドウ糖発酵性グラム陰性菌と判定された(表3)。
(2).チトクローム・オキシダーゼ
Ryuの反応とTSI寒天培地での培養から、ブドウ糖発酵性のグラム陰性菌と判定された18菌株を用いた。チトクローム・オキシダーゼ試験用ろ紙(チトクローム・オキシダーゼ試験用ろ紙、日水製薬株式会社)1片をシャーレに入れ、精製水数滴を滴下してろ紙全体を湿らせ、直ちにその上に固形培地上の培養菌をループで塗布した。1分以内に塗布部分が深青色を呈する菌をチトクローム・オキシダーゼ陽性菌と判定した。塗布部分に変化が認められない場合はその菌をチトクローム・オキシダーゼ陰性菌と判定した。その結果、18株ともにオキシダーゼ陰性のグラム陰性菌であった(表3)。
(3). IDテスト・EB-20
TSI寒天培地にて、ブドウ糖発酵性グラム陰性菌と判定された18菌株についてブドウ糖発酵性グラム陰性桿菌を同定することができるIDテスト・EB-20(日水製薬株式会社)で推定される菌種の同定を行った。普通寒天培地上で増殖培養した被検菌をループを用いて、IDテスト・EBブイヨン1本(2.5ml/tube)に懸濁した。接種菌液の菌液濃度は溶液1ml中に細菌3×108個相当とした。調製した菌液をIDテスト・EBプレートの各ホールの段差の部分に接種した。H2S、LDC、ADH、ODC、URE、INO、SOR及びARAの8項目には、菌液接種後、流動パラフィンを3滴重層して空気を遮断した。プレートにふたをして30℃の恒温槽の中で18から24時間培養した。生化学的性状の成績は、培養後添付の色調判定表と比較して陽性・陰性を判定した。PPA、IND、VPの3項目は培養後、IDテスト・EB(code 06628、日水製薬株式会社)試薬を加えてから判定した。判定結果に基づいてIDテスト・EB-20・解析プロファイル(5版)により、対応する菌種を調べた。グラム陰性菌と判定された各菌株の分類・同定結果を表3にまとめた。

Figure 0005294659
ID・テストEB-20で候補となった菌種を示した。表中では属名は省略してある。
S. ficaria:Serratia ficaria、S. plymuthica:Serratia plymuthica、
E. amnigenus:Enterobacter amnigenus、E. cloacae:Enterobacter cloacae
E. agglomerans:Enterobacter agglomerans、R.aquatilis:Rahnella aquatilis
L. adecarboxylata: Leclercia adecarboxylata
Identification of buckwheat adherent species 27 strains determined to be gram-negative bacteria were determined. The presence or absence of glucose fermentability, presence or absence of cytochrome oxidase, and ID-test EB20 (Nissui Pharmaceutical) were used to identify the bacterial species.
(1) Glucose fermentability
Glucose fermentability was tested on TSI agar medium. The strain determined to be a Gram-negative bacterium in Example 3 was used. The test bacteria were punctured in a high-layer part on a TSI agar medium (Nissui Pharmaceutical Co., Ltd.), then applied to the slope part, and cultured in a thermostatic bath at 30 ° C. for 18 to 24 hours. Glucose-fermenting fungi turn yellow at the upper part, and non-glucose fermenting fungi are vermilion in color. The fungus that decomposes only glucose turns yellow in the upper part and remains unchanged on the slope. Lactose, sucrose, or one of the degrading bacteria is yellowish in both the upper and sloped areas. If there is gas production, bubbles or cracks are generated in the upper part. If there is production of hydrogen sulfide, the upper part changes deeply. As a result, 9 strains were determined to be glucose non-fermentable gram-negative bacteria, and 18 strains were determined to be glucose-fermentable gram-negative bacteria (Table 3).
(2). Cytochrome oxidase
Eighteen strains determined to be glucose-fermenting gram-negative bacteria from Ryu reaction and culture on TSI agar medium were used. Place a piece of cytochrome oxidase test filter paper (cytochrome oxidase test filter paper, Nissui Pharmaceutical Co., Ltd.) in a petri dish, add a few drops of purified water to moisten the entire filter paper, and immediately culture on the solid medium. The fungus was applied in a loop. Bacteria whose application portion had a deep blue color within 1 minute were determined as cytochrome oxidase positive bacteria. When no change was observed in the applied part, the bacterium was determined to be a cytochrome oxidase-negative bacterium. As a result, all 18 strains were oxidase-negative gram-negative bacteria (Table 3).
(3). ID test EB-20
Bacterial species estimated by ID test EB-20 (Nissui Pharmaceutical Co., Ltd.) that can identify glucose-fermenting gram-negative bacilli for 18 strains determined to be glucose-fermenting gram-negative on TSI agar medium Was identified. The test bacteria grown and cultured on a normal agar medium were suspended in one ID test EB broth (2.5 ml / tube) using a loop. The bacterial solution concentration of the inoculum was equivalent to 3 × 10 8 bacteria in 1 ml of the solution. The prepared bacterial solution was inoculated into the step portion of each hole of the ID test / EB plate. In 8 items of H 2 S, LDC, ADH, ODC, URE, INO, SOR and ARA, three drops of liquid paraffin were layered after the bacterial solution was inoculated to block the air. The plate was covered and cultured for 18 to 24 hours in a constant temperature bath at 30 ° C. The results of biochemical properties were judged as positive or negative compared with the color judgment table attached after culture. Three items of PPA, IND, and VP were determined after adding an ID test / EB (code 06628, Nissui Pharmaceutical) reagent after culturing. Based on the determination results, the corresponding bacterial species were examined by ID test, EB-20, and analysis profile (5th edition). Table 3 summarizes the classification and identification results of each strain determined to be a Gram-negative bacterium.
Figure 0005294659
Species that were candidates for ID / test EB-20 were shown. The genus name is omitted in the table.
S. ficaria: Serratia ficaria, S. plymuthica: Serratia plymuthica,
E. amnigenus: Enterobacter amnigenus, E. cloacae: Enterobacter cloacae
E. agglomerans: Enterobacter agglomerans, R.aquatilis: Rahnella aquatilis
L. adecarboxylata: Leclercia adecarboxylata

パントエア・アグロメンランスの抗体を用いた同定
IDテストで同定を試みたグラム陰性菌の菌株の中にパントエア・アグロメランスの可能性が高いと判定された菌株が18種中に7種あった。いずれもコロニーは黄色でありパントエア・アグロメランスと類似していた。そこで、これらの7種について、小麦より分離したパントエア・アグロメランスより得られるリムラス陽性糖脂質のIP-PA1と同様の糖鎖構造を有する菌か調べる目的で、糖脂質 IP-PA1に特異的に反応するモノクローナル抗体を用いた免疫学的な同定を試みた。各被検菌について、プレートより菌を10mg程度掻き取り、予め重量を測定しておいた1.5mlチューブに移した。菌体の湿重量10mgに対して90μlの蒸留水を加え、100mg/mlの菌体の懸濁液を調製した。ブロックヒーターを用いて、95℃にて30分間加熱し糖脂質を抽出した。加熱後、遠心上清を糖脂質の加熱抽出液原液とした。原液10μlをPBS(-)990μlの入った1.5mlに加え、100倍希釈を調製した。各菌株から調製した希釈液を、50μlずつ、96穴イムノプレート(マキシソープ、NUNC)のAからHのウエルに添加した。陰性コントロールとしてのPBS(-)のみと陽性のコントロールとしてのIP-PA1の10μg/mlのPBS(-)溶液をそれぞれ同様に50μlずつ添加し、一晩、室温にて放置して固相化した。その後、3%牛血清アルブミン(BSA)を含むPBS(-)溶液を、抗体その他のタンパクの非特異的吸着を防止目的でウエル当たり250μl加え、室温で1時間放置した。その後、0.05%Tween 20を含むTBS(10mM Tris HCl pH7.5、150mM NaCl) にて3回洗浄後、列ごと(A列:細胞培養の培養液のみ、B列:4E11、C列:20A8、D列:32G3、E列:34G2、F列:49H5、G列:86F12)にIP-PA1に特異的モノクローナル抗体
(IP-PA1特異的モノクローナル抗体は以下の方法で作製した。パントエア・アグロメランス加熱死菌を完全フロイトアジュバントと混合し、BALB/cマウス腹腔内に一匹当り1×10個投与し、2週間間隔で3回腹腔内に投与した。最終投与3日後に、脾臓から細胞を取り出し、ミエローマ(P3U1)と50%ポリエチレングリコール(平均分子量1000)を用いて細胞融合した。融合細胞を10%FBS(牛胎児血清)を含むHAT培地に懸濁させ96穴平底プレートで37℃、5%CO下で静置しながら、7日間培養し、コロニー形成が見られた上清中を、IP-PA1を抗原とした公知慣用のELISA(Enzyme-Linked ImmunoSorbent Assay)法で測定した。上記で得られたモノクローナル抗体産生細胞を公知慣用の限界希釈法を用いて、クローニングした。その結果、抗IP-PA1マウスモノクローナルIgG抗体を得た。)
を50μlずつ加えた。室温で1時間反応させ、次いで、ウエルを0.05%Tween 20を含むTBSで3回洗浄し、1%BSA含有PBS(-)溶液で1000倍に希釈したアルカリフォスファターゼ結合抗マウスIgG、M、A免疫グロブリン抗体(シグマ社製)を50μlずつ、ウエルに入れ、室温で1時間放置した。その後、0.05%Tween 20を含むTBSで4回洗浄し、1mg/mlになるようにp-ニトロフェニルリン酸二ナトリウム(和光純薬工業製)を基質緩衝液に溶解した溶液を100μl/ウエルの割合で入れ、室温で1時間放置した後、2規定の水酸化ナトリウム水溶液50μl/ウエルを入れて反応を停止させ、マイクロプレートリーダー(モデル550、BIO-RAD)にて415nmの吸光度を測定した。その結果、7菌株中、2菌株が6種のIP-PA1特異的抗体に対して反応性を示した(表4)。

Figure 0005294659
陽性のコントロールであるIP-PA1での吸光度値を100、陰性のコントロールであるPBS(-)での吸光度値を0とした場合の、各菌の抽出液を添加したウエルの吸光度値の割合を%で示した。 Identification using pantoea agglomerence antibodies
Among 18 strains of Gram-negative bacteria that were identified by the ID test, 7 of 18 strains were determined to have a high possibility of Pantoea agglomerans. All colonies were yellow and similar to Pantoea agglomerans. Therefore, these 7 species specifically react with glycolipid IP-PA1 in order to investigate whether the rimlas-positive glycolipid obtained from Pantoea agglomerans isolated from wheat has the same sugar chain structure as IP-PA1. We attempted immunological identification using monoclonal antibodies. About each test bacterium, about 10 mg of the bacterium was scraped from the plate and transferred to a 1.5 ml tube in which the weight was measured in advance. 90 μl of distilled water was added to 10 mg of the wet weight of the microbial cells to prepare a 100 mg / ml microbial cell suspension. Glycolipids were extracted by heating at 95 ° C. for 30 minutes using a block heater. After the heating, the centrifugal supernatant was used as a heated extract solution of glycolipid. 10 μl of the stock solution was added to 1.5 ml containing 990 μl of PBS (−) to prepare a 100-fold dilution. 50 μl of the diluted solution prepared from each strain was added to wells A to H of a 96-well immunoplate (Maxisorp, NUNC). PBS (-) alone as a negative control and 10 μg / ml PBS (-) solution of IP-PA1 as a positive control were added in the same manner, 50 μl each, and allowed to stand overnight at room temperature to solidify. . Thereafter, 250 μl of a PBS (−) solution containing 3% bovine serum albumin (BSA) was added per well for the purpose of preventing non-specific adsorption of antibodies and other proteins, and left at room temperature for 1 hour. Then, after washing 3 times with TBS (10 mM Tris HCl pH 7.5, 150 mM NaCl) containing 0.05% Tween 20, every row (row A: cell culture medium only, row B: 4E11, row C: 20A8, D column: 32G3, E column: 34G2, F column: 49H5, G column: 86F12) IP-PA1-specific monoclonal antibody (IP-PA1-specific monoclonal antibody was prepared by the following method: Pantoea agglomerans heat death Bacteria were mixed with complete Freud's adjuvant, administered 1 x 10 8 per mouse intraperitoneally in BALB / c mice, and administered intraperitoneally 3 times at intervals of 2 weeks, 3 days after the final administration, cells were removed from the spleen The cells were fused with myeloma (P3U1) and 50% polyethylene glycol (average molecular weight 1000), and the fused cells were suspended in HAT medium containing 10% FBS (fetal calf serum) at 37 ° C. in a 96-well flat bottom plate, 5 ° C. % CO while standing at 2 under, and cultured for 7 days, colonies The resulting supernatant was measured by a well-known conventional ELISA (Enzyme-Linked ImmunoSorbent Assay) method using IP-PA1 as an antigen. As a result, an anti-IP-PA1 mouse monoclonal IgG antibody was obtained.)
Was added in 50 μl aliquots. The reaction was allowed to proceed for 1 hour at room temperature, and then the wells were washed 3 times with TBS containing 0.05% Tween 20, and diluted 1000-fold with a PBS (-) solution containing 1% BSA to immunize alkaline phosphatase-conjugated anti-mouse IgG, M, A 50 μl of globulin antibody (manufactured by Sigma) was added to each well and allowed to stand at room temperature for 1 hour. Thereafter, the plate was washed 4 times with TBS containing 0.05% Tween 20, and a solution of p-nitrophenyl phosphate disodium (manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in a substrate buffer to a concentration of 1 mg / ml was 100 μl / well. The mixture was allowed to stand at room temperature for 1 hour, and then the reaction was stopped by adding 50 μl / well of a 2N aqueous sodium hydroxide solution, and the absorbance at 415 nm was measured with a microplate reader (model 550, BIO-RAD). As a result, 2 out of 7 strains showed reactivity to 6 types of IP-PA1-specific antibodies (Table 4).
Figure 0005294659
When the absorbance value in IP-PA1 (positive control) is 100 and the absorbance value in PBS (-) (negative control) is 0, the ratio of the absorbance value of the wells to which each bacterial extract was added Indicated in%.

ソバ発酵エキス
ソバ粉0.5gと塩類(リン酸水素二ナトリウム七水和物1.28g、リン酸二水素カリウム0.3g、塩化ナトリウム50mg、塩化アンモニウム100mg、1M硫酸マグネシウム水溶液0.2ml、1M塩化カルシウム水溶液0.01ml)と水を加え全量を100mlとし1リットルの坂口フラスコに入れた。これをオートクレーブ(TOMY BS−325、120℃、20分)で滅菌した。実施例4の中層粉No.2のコロニー(パントエア・アグロメランス、表3においては旧称であるエンテロバクター・アグロメランスと表記)の分離菌株を種菌として、滅菌した溶液に加え、30℃にて一晩培養した。遠心分離器(日立、高速冷却遠心機 SCR−20B、5000rpm、20分間、4℃)により沈殿を回収した。沈殿にリン酸緩衝液を加えて懸濁し、全量100mlとして、沸騰水浴中で30分間加熱抽出した。加熱終了後、室温まで冷却し、本液を遠心分離(日立、高速冷却遠心機 SCR−20B、10000rpm、20分間、20℃)した。遠心後、淡黄色の上清を得た。この上清80mlあたりに8.9mlの5モル塩化ナトリウム溶液を加え、これに178mlのエタノールを加えた。これを、冷凍庫(−90℃)で一晩放置後、本液を遠心分離(日立、高速冷却遠心機SCR−20B、10000rpm、20分間、4℃)した。上清を除いて得た沈殿を風乾し、蒸留水に溶解し、10mlのソバ発酵エキス溶液を得た。本エキスの固形分は、0.3mlを予め秤量した1.5mlプラスチックチューブに移し、凍結後、凍結乾燥機にて、凍結乾燥を行ったところ11.5mgであった(3.8%)。
Buckwheat fermented extract buckwheat flour 0.5g and salts (disodium hydrogen phosphate heptahydrate 1.28g, potassium dihydrogen phosphate 0.3g, sodium chloride 50mg, ammonium chloride 100mg, 1M magnesium sulfate aqueous solution 0.2ml, 1M calcium chloride aqueous solution 0.01 ml) and water were added to make a total volume of 100 ml, and placed in a 1 liter Sakaguchi flask. This was sterilized with an autoclave (TOMY BS-325, 120 ° C., 20 minutes). The isolated strain of the middle layer powder No. 2 of Example 4 (Pantoeia agglomerans , referred to as “Enterobacter agglomerans” in Table 3 ) is used as an inoculum and added to a sterilized solution and cultured at 30 ° C. overnight. did. The precipitate was collected by a centrifuge (Hitachi, high-speed cooling centrifuge SCR-20B, 5000 rpm, 20 minutes, 4 ° C.). The precipitate was suspended by adding a phosphate buffer solution to a total volume of 100 ml, and extracted by heating in a boiling water bath for 30 minutes. After completion of the heating, the mixture was cooled to room temperature and centrifuged (Hitachi, high-speed cooling centrifuge SCR-20B, 10000 rpm, 20 minutes, 20 ° C.). After centrifugation, a pale yellow supernatant was obtained. To 80 ml of this supernatant, 8.9 ml of 5 molar sodium chloride solution was added, and 178 ml of ethanol was added thereto. This was left overnight in a freezer (−90 ° C.), and then the liquid was centrifuged (Hitachi, high-speed cooling centrifuge SCR-20B, 10000 rpm, 20 minutes, 4 ° C.). The precipitate obtained by removing the supernatant was air-dried and dissolved in distilled water to obtain 10 ml of a buckwheat fermented extract solution. The solid content of this extract was 11.5 mg (3.8%) when transferred to a 1.5 ml plastic tube weighed 0.3 ml in advance and freeze-dried with a freeze dryer after freezing.

ブラッドフォード法によるタンパク質定量を、BSAを標準タンパク質として、ソバ発酵エキスを測定した。糖含量の測定はフェノール硫酸法によりグルコースを標準糖として測定した。核酸含有量の測定は100倍希釈したサンプルの210〜340nmの吸光度測定を行った。260nmの吸光度から320nmの吸光度を引いた値と、DNAとしての吸光度1.0 ODあたり、50μgとしての最大含有量を算出した。リムラスアッセイによるリムラス活性物質含有量の測定はリムラス活性物質量は生化学工業のトキシカラーシステムを用い、標準リムラス活性物質として、生化学工業Et-1を用いた。測定結果を表5に示した。表5のソバ発酵エキスについての数値は乾燥重量の1gあたりの含有量をmgで表示した。

Figure 0005294659
Protein quantification by the Bradford method was performed, and buckwheat fermentation extract was measured using BSA as a standard protein. The sugar content was measured by the phenol-sulfuric acid method using glucose as a standard sugar. The nucleic acid content was measured by measuring absorbance at 210 to 340 nm of a sample diluted 100 times. The value obtained by subtracting the absorbance at 320 nm from the absorbance at 260 nm and the maximum content as 50 μg per absorbance of 1.0 OD as DNA were calculated. The Limulus active substance content was measured by the Limulus assay using the Toxicolor system of Seikagaku Corporation for the Limulus active substance content, and Seikagaku Corporation Et-1 as the standard Limulus active substance. The measurement results are shown in Table 5. The numerical values for the fermented buckwheat extract in Table 5 are expressed in mg of content per gram of dry weight.
Figure 0005294659

ソバ発酵エキスのアトピー性皮膚炎抑制効果
ソバ発酵エキスのアトピー性皮膚炎に対する効果を調べるために、I型アレルギーモデルを導入した。一群5匹の雄性のBALB/cマウスに抗ジニトロフェニル、マウスモノクローナル抗体(IgE)を1μg/マウスで静脈投与した。一時間後に実施例6で製造したソバ発酵エキス(10μg/マウス)を腹部皮内投与又は経口投与(1mg/マウス)し、さらに1時間後に、マウスの耳介の表裏に0.25%ジニトロフルオロベンゼン含有アセトン−オリーブオイル混合溶液(4対1)をアレルゲンとして20μl塗布した。塗布後、1、2、24及び48時間目の耳介の厚さをシクネスゲージで測定し、塗布直前の厚さとの差(△)を浮腫の程度とした。薬剤投与の効果は、アレルゲン投与1時間後に認められる早期反応と、24時間後に誘導される遅発反応、それぞれの抑制を以下の式によって求められる抑制率で評価した。{抑制率 = (1−薬剤投与後の△耳介の浮腫/対照の△耳介の浮腫)×100}結果を表6に示す。表から明らかなように、ソバ発酵エキスは皮内投与でも、経口投与でもアレルギー反応を抑制した。

Figure 0005294659
Inhibitory effect of buckwheat fermented extract on atopic dermatitis In order to examine the effect of buckwheat fermented extract on atopic dermatitis, a type I allergy model was introduced. Anti-dinitrophenyl and mouse monoclonal antibody (IgE) were intravenously administered at 1 μg / mouse to a group of 5 male BALB / c mice. One hour later, the fermented buckwheat extract (10 μg / mouse) produced in Example 6 was administered intraperitoneally or orally (1 mg / mouse), and after 1 hour, 0.25% dinitrofluoro was added to the front and back of the mouse auricle. 20 μl of benzene-containing acetone-olive oil mixed solution (4 to 1) was applied as an allergen. After application, the thickness of the pinna at 1, 2, 24, and 48 hours was measured with a thickness gauge, and the difference (Δ) from the thickness immediately before application was defined as the degree of edema. The effects of drug administration were evaluated based on the inhibition rate obtained by the following formula: early reaction observed 1 hour after allergen administration and delayed reaction induced 24 hours later. {Inhibition rate = (1-Auricular edema after drug administration / Control △ auricular edema) × 100} The results are shown in Table 6. As is clear from the table, the fermented buckwheat extract suppressed the allergic reaction both intradermally and orally.
Figure 0005294659

レンコンに含まれるリムラス陽性糖脂質含量
レンコンに含まれるリムラス陽性糖脂質量を測定した。レンコンに付いている土を水道水にて洗い落とした後、滅菌蒸留水にて3回洗浄した。その後、節部分と可食部に切り分け、それぞれについて、すりおろし、キムワイプにくるんだ後、圧搾して絞り汁を得た。絞り汁を遠心分離し、上清を新鮮標品からの抽出液として試験に供した。また、接続部と可食部に切り分けた後、薄く切り分け、40℃にて3日間乾燥剤を入れた乾燥機にて乾燥させた。乾燥した標品を乳鉢・乳棒を用いて細かく砕き粉末状とした。各粉末2.0gに蒸留水を加え20mlとした後、粉末を懸濁し、沸騰水浴中にて20分間加熱抽出を行った。加熱後、室温まで冷却したところ、各標品共に、ゲル化した(含まれる炭水化物によるものと推定される)為、一部をさらに蒸留水にて5倍希釈し、懸濁・攪拌し、遠心分離を行いその上清(以下、「レンコン希釈液」という。)を試験に供した。レンコンは3本用いて、各サンプルを調製した。生化学工業のエンドスペシーのキットを用いて測定した。方法は、キット添付のプロトコールに従った。測定された糖脂質含量はIP-PA1の重量換算値として算出した。その結果を表7に示した。いずれのサンプルからも糖脂質が検出された。

Figure 0005294659
Limulus-positive glycolipid content contained in lotus root The amount of limulus-positive glycolipid contained in lotus root was measured. The soil attached to the lotus root was washed away with tap water and then washed three times with sterilized distilled water. After that, it was cut into a knot portion and an edible portion, and each was grated, wrapped in a Kimwipe, and then squeezed to obtain juice. The juice was centrifuged and the supernatant was used as an extract from a fresh sample. Moreover, after dividing into a connection part and an edible part, it sliced thinly and dried with the dryer which put the desiccant for 3 days at 40 degreeC. The dried sample was finely pulverized with a mortar and pestle to form a powder. Distilled water was added to 2.0 g of each powder to make 20 ml, and the powder was suspended and extracted by heating in a boiling water bath for 20 minutes. After heating, when cooled to room temperature, each sample gelled (it is estimated to be due to the carbohydrates contained), so a part was further diluted 5 times with distilled water, suspended and stirred, and centrifuged. After separation, the supernatant (hereinafter referred to as “lotus diluted solution”) was subjected to the test. Each sample was prepared using 3 lotus roots. Measurements were made using Seikagaku's Endspecy kit. The method followed the protocol attached to the kit. The measured glycolipid content was calculated as a weight converted value of IP-PA1. The results are shown in Table 7. Glycolipids were detected from all samples.
Figure 0005294659

レンコン抽出液のマクロファージ活性化能(一酸化窒素産生能)
マウスのマクロファージ系細胞RAW264.7を用いたレンコン抽出物の免疫作用の測定はRAW264.7細胞を細胞濃度を8×105cells/mlとして、その100μlを96ウエルプレートの各ウエルに移し、6時間後に試験に用いた。RAW264.7細胞に各レンコン希釈液を50μlを添加し、1時間37℃、5%CO2インキュベータ内で培養した。その後、LPSp(パントエア・アグロメランスから得られる低分子量リポ多糖)400ng/mlを含む培養液50μl(終濃度100ng/ml:これを「LPSpの高純度標品」とする)又は、培養液のみを50μl添加し、さらに23時間培養を行った。培養終了後、上清50μlを回収し常法に従いGriess試薬を用いて培養液中の一酸化窒素の代謝物である亜硝酸イオン量を測定した。
Macrophage activation ability of lotus root extract (nitrogen monoxide production ability)
Measurement of immunity of lotus root extract using mouse macrophage cell line RAW264.7 was carried out with RAW264.7 cells at a cell concentration of 8 × 10 5 cells / ml, and 100 μl was transferred to each well of a 96-well plate. Used for testing after hours. 50 μl of each lotus root dilution was added to RAW264.7 cells and cultured in a 5% CO 2 incubator at 37 ° C. for 1 hour. After that, culture medium containing 400 ng / ml LPSp (low molecular weight lipopolysaccharide obtained from Pantoea agglomerans) 50 μl (final concentration 100 ng / ml : this is the “LPSp high-purity sample” ) or 50 μl of culture medium alone After addition, the cells were further cultured for 23 hours. After completion of the culture, 50 μl of the supernatant was collected, and the amount of nitrite ion that is a metabolite of nitric oxide in the culture solution was measured using Griess reagent according to a conventional method.

測定結果を表8に示した。節部分および可食部の乾燥標品の抽出液を添加した場合、各産地の標品共に添加量に依存してRAW264.7細胞からのNO産生の増加が認められた。節部分の新鮮標品からの抽出液を添加した場合、各産地の標品共に添加量に依存してRAW264.7細胞からのNO産生の増加が認められた。可食部の新鮮標品からの抽出液を添加した場合、各産地の標品共に終濃度10ng/ml添加量においてRAW264.7細胞からのNO産生が認められた。   The measurement results are shown in Table 8. When the extract of the dried preparation of the knot part and the edible part was added, the NO production from RAW264.7 cells was observed depending on the amount added for both preparations of each production area. When the extract from the fresh preparation of the knot portion was added, an increase in NO production from RAW264.7 cells was observed depending on the amount added for each preparation in each production area. When extracts from fresh edible samples were added, NO production from RAW264.7 cells was observed at the final concentration of 10 ng / ml for both samples.

以上結果から抽出液自体にRAW264.7細胞からのNO産生を誘導する活性のあることが明らかとなった。これらの活性は、LPSpの高純度標品に比較すると1/10程度であった。

Figure 0005294659
The above results revealed that the extract itself has an activity to induce NO production from RAW264.7 cells. These activities were about 1/10 compared with the high purity sample of LPSp.
Figure 0005294659

レンコン付着細菌の分離・単離
レンコンの各根茎接続部の一部を蒸留水に懸濁し、段階希釈後、100μlずつを普通寒天培地にまきこみ、30℃の恒温槽内でインキュベートした。4日間のインキュベートの後、プレート上に認められる各コロニーについて、別の普通寒天培地プレートにまき、菌の単離を行った。また、同時にプレート上の菌数、菌腫内訳から単位重量当たりの菌の存在数について算出した。
Separation and isolation of lotus-adherent bacteria A part of each rhizome connection part of lotus root was suspended in distilled water, and after serial dilution, 100 μl was spread on a normal agar medium and incubated in a 30 ° C. constant temperature bath. After 4 days of incubation, each colony found on the plate was seeded on a separate normal agar plate to isolate the bacteria. Simultaneously, the number of bacteria per unit weight was calculated from the number of bacteria on the plate and the breakdown of myoma.

レンコン付着細菌の同定
純培養で得られた各菌のコロニーを用い、グラム陰性か陽性をRyuの方法に基づき3%KOHに対する溶解性により決定した。グラム陰性菌と判別された菌株についてはID-test EB20(日水製薬)を用いて同定を行った。
Identification of lotus-adherent bacteria Using colonies of each bacterium obtained by pure culture, Gram negative or positive was determined by solubility in 3% KOH based on Ryu's method. The strains identified as gram-negative bacteria were identified using ID-test EB20 (Nissui Pharmaceutical).

その結果、レンコンの根から、1.4×108/gから2.0×108/gの細菌が認められた。各レンコンに共通して、わずかに黄色のコロニーの菌(グラム陰性菌と判定)が約全コロニー数の1割から2割程度を占めた。菌数の割合としては少ないが(1%以下)共通してコロニーの周囲が黄色で中心部が緑色のグラム陰性菌と判定された菌株が認められた。計59菌株単離したところ、グラム陰性菌と判定した51菌株の内、オキシダーゼ試験で陰性を示した(腸内細菌科の可能性が高い)15菌株についてID-test EB20で同定を試みたところ、セラチア・プリムチカ(Serratia plymuthica)、パントエア・アグロメランス(Pantoea agglomerans)と同定された菌株があった。残りの13菌株については該当する種が認められなかった。 As a result, 1.4 × 10 8 / g to 2.0 × 10 8 / g bacteria were observed from the roots of the lotus root. In common with each lotus root, slightly yellow colony bacteria (determined as Gram-negative bacteria) accounted for about 10 to 20% of the total number of colonies. Although the ratio of the number of bacteria was small (1% or less), a common bacterial strain that was judged to be a Gram-negative bacterium with a yellow color around the colony and a green center was observed. A total of 59 strains were isolated. Among 51 strains judged to be Gram-negative bacteria, 15 strains that were negative in the oxidase test (highly likely to be Enterobacteriaceae) were identified using ID-test EB20. , Serratia Purimuchika (Serratia plymuthica), there was a Pantoea agglomerans (Pantoea agglomerans) and identified strains. No corresponding species was found for the remaining 13 strains.

レンコン発酵エキス
各種グラム陰性菌のレンコン培養エキス
レンコンをスライスし、70℃の乾燥器に1週間乾燥させ、これを乳鉢ですりつぶしてレンコン粉末を得た。レンコン粉0.5gと塩類(リン酸水素二ナトリウム七水和物1.28g、リン酸二水素カリウム0.3g、塩化ナトリウム50mg、塩化アンモニウム100mg、1M硫酸マグネシウム水溶液0.2ml、1M塩化カルシウム水溶液0.01ml)と水を加え全量を100mlとした。これをオートクレーブで滅菌し、実施例11においてパントエア・アグロメランスと同定された一つのコロニー、又は、実施例11においてグラム陰性菌と同定された51菌株の内、セラチア・プリムチカとも、パントエア・アグロメランスとも同定されなかった菌体のコロニー(合計3サンプル)を加え、30℃にて一晩培養した。その後、培養液を95℃にて30分間加熱し糖脂質を抽出した。加熱後、遠心上清することでレンコン発酵エキスが得られた。
Lotus root fermentation extract Lotus root culture extract of various gram-negative bacteria Lotus root slices were sliced and dried in a dryer at 70 ° C. for 1 week and ground in a mortar to obtain lotus root powder. Lotus root powder 0.5g and salt (disodium hydrogen phosphate heptahydrate 1.28g, potassium dihydrogen phosphate 0.3g, sodium chloride 50mg, ammonium chloride 100mg, 1M magnesium sulfate aqueous solution 0.2ml, 1M calcium chloride aqueous solution 0.01ml) Water was added to make a total volume of 100 ml. This was sterilized in an autoclave, a colony was identified as Pantoea agglomerans in Example 11, or, among the 51 strains identified as Gram-negative bacteria in Example 11, with Serratia Purimuchika, identified with Pantoea agglomerans The bacterial cell colonies (three samples in total) that were not added were added and cultured overnight at 30 ° C. Thereafter, the culture solution was heated at 95 ° C. for 30 minutes to extract glycolipids. The lotus root fermented extract was obtained by centrifuging the supernatant after heating.

本エキス中の乾燥重量は0.3mlを予め秤量した1.5mlプラスチックチューブに移し、凍結後、凍結乾燥機にて、凍結乾燥を行ったところ4.5mgであった(固形分1.5%)。   The dry weight of this extract was transferred to a 1.5 ml plastic tube that weighed 0.3 ml in advance, and after freezing, it was lyophilized with a lyophilizer and found to be 4.5 mg (solid content 1.5% ).

ブラッドフォード法によるタンパク質定量を、BSAを標準タンパク質として、レンコン発酵エキスを測定した。糖含量の測定はフェノール硫酸法によりグルコースを標準糖として測定した。核酸含有量の測定は100倍希釈したサンプルの210〜340nmの吸光度測定を行った。260nmの吸光度から320nmの吸光度を引いた値と、DNAとしての吸光度1.0 ODあたり、50μgとしての最大含有量を算出した。リムラスアッセイによるリムラス活性物質含有量の測定はリムラス活性物質量は生化学工業のトキシカラーシステムを用い、標準リムラス活性物質として、生化学工業Et-1を用いた。測定結果を表9に示した。表9のレンコン発酵エキスについての数値は乾燥重量の1gあたりの含有量をmgで表示した。

Figure 0005294659
Protein quantification by Bradford method was carried out by measuring lotus root ferment extract using BSA as a standard protein. The sugar content was measured by the phenol-sulfuric acid method using glucose as a standard sugar. The nucleic acid content was measured by measuring absorbance at 210 to 340 nm of a sample diluted 100 times. The value obtained by subtracting the absorbance at 320 nm from the absorbance at 260 nm and the maximum content as 50 μg per absorbance of 1.0 OD as DNA were calculated. The Limulus active substance content was measured by the Limulus assay using the Toxicolor system of Seikagaku Corporation for the Limulus active substance content, and Seikagaku Corporation Et-1 as the standard Limulus active substance. The measurement results are shown in Table 9. The numerical value for the lotus root fermented extract in Table 9 indicates the content per 1 g of dry weight in mg.
Figure 0005294659

レンコン発酵エキス入りアメの製造
原材料としてグラニュー糖、水飴、水に実施例12で製造したレンコン発酵エキスを加えたものを5:5:5:1の割合で混合し、加熱して120℃〜160℃で煮詰めた。これを冷却用鉄板上で冷却し、棒状に引き伸ばして1g前後の粒状に成型し飴を得た。本アメ適量を水20mlに入れ、加熱することで溶解させた。この溶液中のレンコン発酵エキス有効成分としてリポ多糖量を測定したところ、8.9μg/gであった。このアメを、風邪をひいてのどの痛みのある男女6名に摂取させた。その後、直ちにのどの痛みに対するアンケート調査を行った。のどの痛みについては、6名とも痛みが軽減したと感じた(一標本符号検定:P<0.03)。
Manufacture of candy containing lotus root fermented extract Granulated sugar, starch syrup, and water obtained by adding lotus root fermented extract prepared in Example 12 at a ratio of 5: 5: 5: 1 and heated to 120 ° C. to 160 ° C. Boiled at ℃. This was cooled on a cooling iron plate, stretched into a rod shape, and formed into a granule of about 1 g to obtain a soot. An appropriate amount of this candy was put in 20 ml of water and dissolved by heating. When the amount of lipopolysaccharide as an active ingredient of the lotus root extract in this solution was measured, it was 8.9 μg / g. This candy was ingested by 6 men and women who had a cold and had a sore throat. After that, we immediately conducted a questionnaire survey for sore throat. Regarding the sore throat, all six felt that the pain was reduced (one-sample sign test: P <0.03).

Claims (6)

レンコンに由来する素材を、前記レンコンに付着又は共生するグラム陰性菌によって発酵させて、同時に該菌を培養することを特徴とする発酵及び培養方法。 A fermentation and culture method comprising fermenting a lotus root- derived material with a gram-negative bacterium adhering or symbiotic to the lotus root , and simultaneously culturing the bacterium. 前記レンコンに由来する素材はレンコンに力学的操作を加えることによって得られることを特徴とする請求項1記載の発酵及び培養方法。 The material is fermented and cultured method according to claim 1 characterized in that it is obtained by adding a mechanical operation lotus root derived from the lotus root. 請求項1又は2記載の発酵及び培養方法で得られることを特徴とするレンコン発酵エキス。 A lotus root fermented extract obtained by the fermentation and culture method according to claim 1 or 2. 請求項3記載の植物発酵エキスから得られることを特徴とするレンコン発酵エキス末。 A lotus root fermented extract powder obtained from the plant fermented extract according to claim 3. 請求項3記載のレンコン発酵エキス又は請求項4記載のレンコン発酵エキス末が配合されていることを特徴とするレンコン発酵エキス配合物。 Lotus root ferment extract formulation, characterized in that claim 3 lotus fermentation extract powder of lotus root ferment extract or claim 4, wherein according are blended. 前記レンコン発酵エキス配合物が医薬品、動物用医薬品、医薬部外品、化粧品、食品、機能性食品、飼料、植物用肥料、植物用医薬品又は浴用剤であることを特徴とする請求項5記載のレンコン発酵エキス配合物。 The said lotus root fermented extract blend is a pharmaceutical, veterinary drug, quasi-drug, cosmetic, food, functional food, feed, plant fertilizer, plant drug, or bath preparation. Lotus root fermented extract formulation.
JP2008061930A 2007-03-11 2008-03-11 Fermentation and culture method, lotus root ferment extract and fermented extract blend Active JP5294659B2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2008061930A JP5294659B2 (en) 2007-03-11 2008-03-11 Fermentation and culture method, lotus root ferment extract and fermented extract blend

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP2007061139 2007-03-11
JP2007061139 2007-03-11
JP2008061930A JP5294659B2 (en) 2007-03-11 2008-03-11 Fermentation and culture method, lotus root ferment extract and fermented extract blend

Publications (2)

Publication Number Publication Date
JP2008255108A JP2008255108A (en) 2008-10-23
JP5294659B2 true JP5294659B2 (en) 2013-09-18

Family

ID=39979033

Family Applications (1)

Application Number Title Priority Date Filing Date
JP2008061930A Active JP5294659B2 (en) 2007-03-11 2008-03-11 Fermentation and culture method, lotus root ferment extract and fermented extract blend

Country Status (1)

Country Link
JP (1) JP5294659B2 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102871047B (en) * 2012-09-11 2014-02-05 临泉县金禾面粉有限公司 Pigeon Chinese wolfberry soup dried noodles and preparation method thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101228993B1 (en) * 2003-09-26 2013-02-01 바이오메디칼 리서치 그룹 인코포레이티드 Fermentation and culture method, fermented plant extract, fermented plant extract powder and composition containing the fermented plant extract

Also Published As

Publication number Publication date
JP2008255108A (en) 2008-10-23

Similar Documents

Publication Publication Date Title
JP4704377B2 (en) Fermentation and culture method, plant fermented extract, plant fermented extract powder and blended plant fermented extract
KR101554960B1 (en) USE OF LACTOBACILLUS FOR INCREASING THE ABSORPTION OF A METAL CHOSEN FROM Fe, Zn, Ca AND IONS THEREOF
CN102271535B (en) Sialic acid producing bacteria
CN103649304B (en) Microbial bacteria lactobacillus plantarum (LACTOBACILLUS PLANTARUM) MCC1 DSM 23881 be separated and uses thereof
Afzaal et al. Nutritional health perspective of natto: A critical review
CN105229142B (en) immunostimulant
US9345730B2 (en) Methods of treating impaired glucose metabolism via administration of algal biomass
CN102210454B (en) Microbial compound preparation and preparation method thereof
Ngoufack et al. Viability and in vivo hypocholesterolemic effect of Lactobacillus plantarum 29V in local honey
JP5294659B2 (en) Fermentation and culture method, lotus root ferment extract and fermented extract blend
JP2009209132A (en) Antiallergic composition using new strain, lactobacillus crispatus kt-11, kt-23 and kt-25
Aleid Industrial biotechnology: date palm fruit applications
CN102604851B (en) Lactococcus lactis capable of lowering cholesterol and producing extracellular polysaccharide
US11730778B2 (en) Method of increasing the level and production of metabolized phytonutrients in a subject
KR102368626B1 (en) Composition for Type I Allergy
JP2011037822A (en) Enzyme-treated product and aspergillus fermentation treated product of dioscorea esculenta rhizome
Adedayo et al. Effect of nutriment from monoculture fermentation of Adasonia digitata seeds by Penicillium chrysogenum on haematology and functional endpoints of intra and extrahepatic tissues in rats
KR102197145B1 (en) Mixed culture strains (no. kctc13836bp or no. kctc13837bp) and synbiotics capable of enhancing beneficial microbes and supressing hazardous microbes in human gut
Ngongang et al. Isolation and identification of cholesterol lowering probiotic yeast from palm raffia (Raffia mambillensis) wine
TWI358266B (en)
Ebu et al. The effect of probiotic containing Lactobacillus fermentum, Lactobacillus plantenrun and Weissalla ciberia on some blood parameters of broiler chicken
TWI458438B (en) Fermentation and culture methods, plant fermentation extracts, plant fermentation extract powders, and plant fermentation extract complexes
JP2018050488A (en) Immunostimulatory composition
Yu et al. Radical Scavenging and Hypolipidemic Activity of Aqueous Extracts from Labadou (A Traditional Fermented Soybean Food of Southern China)
Carvalho In vitro impact of Tenebrio molitor insect flour on human gut microbiota

Legal Events

Date Code Title Description
A621 Written request for application examination

Free format text: JAPANESE INTERMEDIATE CODE: A621

Effective date: 20110117

A131 Notification of reasons for refusal

Free format text: JAPANESE INTERMEDIATE CODE: A131

Effective date: 20121212

A521 Request for written amendment filed

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20130212

A131 Notification of reasons for refusal

Free format text: JAPANESE INTERMEDIATE CODE: A131

Effective date: 20130313

A521 Request for written amendment filed

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20130513

TRDD Decision of grant or rejection written
A01 Written decision to grant a patent or to grant a registration (utility model)

Free format text: JAPANESE INTERMEDIATE CODE: A01

Effective date: 20130529

A61 First payment of annual fees (during grant procedure)

Free format text: JAPANESE INTERMEDIATE CODE: A61

Effective date: 20130611

R150 Certificate of patent or registration of utility model

Ref document number: 5294659

Country of ref document: JP

Free format text: JAPANESE INTERMEDIATE CODE: R150

Free format text: JAPANESE INTERMEDIATE CODE: R150

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250