JP2017012163A - Lactobacillus plantarum llp5193 with high acid- and bile-resistance and high cell attaching ability and products containing it as the effective ingredient - Google Patents
Lactobacillus plantarum llp5193 with high acid- and bile-resistance and high cell attaching ability and products containing it as the effective ingredient Download PDFInfo
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Abstract
Description
本発明はキムチから分離された新規の乳酸菌であるラクトバシラス・プランタラムＬＬＰ５１９３及びこれを有効成分として含む製品に関するものである。ラクトバシラス・プランタラムＬＬＰ５１９３は植物性乳酸菌で、韓国人の腸内環境に適し、耐酸性及び耐胆汁性に優れており腸内に無事に到逹することができ、腸管細胞付着能に優れており腸内で安定に定着することができる。また、ラクトバシラス・プランタラムＬＬＰ５１９３に賦形剤を一定濃度で添加して製造した食品は食感及び耐摩損性が良くて剤形化に有利であり、粉末が多く発生せず、食べやすい。 The present invention relates to Lactobacillus plantarum LLP5193, which is a novel lactic acid bacterium isolated from kimchi, and a product containing this as an active ingredient. Lactobacillus plantarum LLP5193 is a plant lactic acid bacterium that is suitable for the Korean intestinal environment, has excellent acid resistance and bile resistance, can safely reach the intestine, and has excellent intestinal cell adhesion. It can settle stably in the intestine. In addition, a food produced by adding an excipient to Lactobacillus plantarum LLP5193 at a constant concentration has good texture and wear resistance, is advantageous for formulation, does not generate much powder, and is easy to eat.
人間は紀元前から乳酸菌を食品として用いた。乳酸菌は栄養が豊かで多様な生理活性物質を含んでいる。乳酸菌は整腸作用を有しており下痢や便秘を予防し、有害細菌の増殖を抑制して腸癌や老化を防止し、ビタミンを生成して発育を促進し、コレステロールを調節して成人病を予防し、免疫力を増強する効果がある。 Humans have used lactic acid bacteria as food since BC. Lactic acid bacteria are rich in nutrients and contain a variety of physiologically active substances. Lactic acid bacteria have an intestinal action that prevents diarrhea and constipation, suppresses the growth of harmful bacteria, prevents intestinal cancer and aging, generates vitamins to promote growth, regulates cholesterol, and controls adult diseases It has the effect of preventing and enhancing immunity.
また、乳酸菌から作られた食品は乳酸を含んでいるので、保存期間が長く、特有のさわやかな酸味があり、有害な微生物の増殖を抑制し、乳蛋白質の消化率を高め、カルシウム、鉄、リンの吸収を促進するなどの多くの利点がある。 In addition, foods made from lactic acid bacteria contain lactic acid, so it has a long shelf life, has a unique refreshing acidity, suppresses the growth of harmful microorganisms, increases the digestibility of milk proteins, calcium, iron, There are many advantages such as promoting absorption of phosphorus.
現在、韓国内で使用する乳酸菌は大部分が外国から持ち込んだもので、動物の腸内、原乳などから分離した動物性乳酸菌である。韓国人は西洋人とは違い、植物性食品を主食とするため、腸内環境が違い、腸の長さが長い。したがって、動物性乳酸菌よりは野菜、果物などの植物、キムチなどの果菜類を発酵した食品から分離した植物性乳酸菌を摂取することが効果面でより良い。 Currently, most of the lactic acid bacteria used in Korea are imported from abroad, and are animal lactic acid bacteria isolated from the intestines of animals and raw milk. Unlike Westerners, Koreans use vegetable foods as their staple food, so the intestinal environment is different and the length of the intestines is long. Therefore, it is better in terms of effectiveness to ingest plant lactic acid bacteria separated from foods obtained by fermenting vegetables, fruits and other plants, and fruit vegetables such as kimchi, rather than animal lactic acid bacteria.
大韓民国公開特許第１０−２００１−００７５５７７号は、キムチから分離したラクトバシラス・プランタラム菌株に関するものである。この従来技術によるラクトバシラス・プランタラム（Ｌａｃｔｏｂａｃｉｌｌｕｓ Ｐｌａｎｔａｒｕｍ）は植物性乳酸菌で、韓国人にうまく適用でき、耐酸性及び耐胆汁性に優れており腸内に無事に到逹することができるという利点を持つ。しかし、乳酸菌の腸管細胞付着能に対する知見がないという問題があった。また、乳酸菌の生存率を高めるためには、耐酸性及び耐胆汁性を一層高める必要がある。 Korean Patent No. 10-2001-0075577 relates to a Lactobacillus plantarum strain isolated from kimchi. Lactobacillus plantarum (Lactobacillus Plantarum) according to this prior art is a plant lactic acid bacterium, which can be successfully applied to Koreans, has excellent acid resistance and bile resistance, and can safely reach the intestines. . However, there is a problem that there is no knowledge about the ability of lactic acid bacteria to adhere to the intestinal cells. Moreover, in order to increase the survival rate of lactic acid bacteria, it is necessary to further improve acid resistance and bile resistance.
本発明は、キムチから分離され、韓国人の腸内環境に対する適応性の高い植物性乳酸菌であるラクトバシラス・プランタラムを提供することにその目的がある。 An object of the present invention is to provide Lactobacillus plantarum, which is a plant lactic acid bacterium isolated from kimchi and highly adaptable to the Korean intestinal environment.
また、本発明は、優れた耐酸性及び耐胆汁性を持ち、腸内に確実に到逹することができるラクトバシラス・プランタラムを提供することにその目的がある。 Another object of the present invention is to provide a Lactobacillus plantarum that has excellent acid resistance and bile resistance and can reliably reach the intestines.
また、本発明は、腸管細胞付着能に優れて乳酸菌の効果を十分に発揮することができるラクトバシラス・プランタラムを提供することにその目的がある。 Another object of the present invention is to provide Lactobacillus plantarum that is excellent in intestinal cell adhesion ability and can sufficiently exert the effect of lactic acid bacteria.
また、本発明は、ラクトバシラス・プランタラムに賦形剤を添加して食感及び耐摩損性に優れた食品を提供することにその目的がある。 Another object of the present invention is to provide a food having excellent texture and wear resistance by adding an excipient to Lactobacillus plantarum.
本発明の目的は以上に言及した目的に制限されない。本発明の目的は以下の説明からより明らかになり、特許請求の範囲に記載された手段及びその組合せから実現可能であろう。 The object of the present invention is not limited to the object mentioned above. Objects of the present invention will become more apparent from the following description, and may be realized by the means described in the claims and combinations thereof.
本発明は前記目的を達成するために下記のような構成を含む。 In order to achieve the above object, the present invention includes the following configurations.
本発明は、キムチから分離された耐酸性、耐胆汁性及び腸管細胞付着能に優れた乳酸菌であるラクトバシラス・プランタラムＬＬＰ５１９３（ＫＣＣＭ１１５９８Ｐ）を提供する。 The present invention provides Lactobacillus plantarum LLP5193 (KCCM11598P), which is a lactic acid bacterium isolated from kimchi and excellent in acid resistance, bile resistance and intestinal cell adhesion.
また、本発明は、前記ラクトバシラス・プランタラムＬＬＰ５１９３を有効成分として含む発酵乳及び凍結乾燥物を提供する。
本発明の凍結乾燥物は、デキストリン、米粉及びタピオカ澱粉からなる群から選択された一種以上の賦形剤をさらに含むこともできる。
この賦形剤として、タピオカ澱粉を３〜５重量％添加したものを用いることもできる。
Moreover, this invention provides the fermented milk and lyophilizate which contain the said Lactobacillus plantarum LLP5193 as an active ingredient.
The lyophilized product of the present invention may further contain one or more excipients selected from the group consisting of dextrin, rice flour and tapioca starch.
As this excipient, one added with 3 to 5% by weight of tapioca starch can also be used.
本発明によるラクトバシラス・プランタラムはキムチから分離した植物性乳酸菌なので、韓国人の腸内環境に適する。 Since the Lactobacillus plantarum according to the present invention is a plant lactic acid bacterium isolated from kimchi, it is suitable for the Korean intestinal environment.
また、本発明によるラクトバシラス・プランタラムは耐酸性及び耐胆汁性に優れて腸内に確実に到逹することができる。 In addition, the Lactobacillus plantarum according to the present invention is excellent in acid resistance and bile resistance and can reach the intestine with certainty.
また、本発明によるラクトバシラス・プランタラムは腸管細胞付着能に優れており腸内で安定的に定着することができるので、乳酸菌としての効果を十分に発揮することができる。 In addition, Lactobacillus plantarum according to the present invention is excellent in intestinal cell adhesion ability and can be stably settled in the intestine, so that the effect as a lactic acid bacterium can be sufficiently exerted.
また、本発明によるラクトバシラス・プランタラムから製造した製品は食感及び耐摩損性が良くて剤形化に有利で、食べやすい。 In addition, the product manufactured from Lactobacillus plantarum according to the present invention has good texture and wear resistance, is advantageous for formulation, and is easy to eat.
以下、実施例に基づいて本発明を詳細に説明する。本発明の実施例は、発明の要旨が変更されない限り、多様な形態に変形可能である。しかし、本発明の権利範囲が以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described in detail based on examples. The embodiments of the present invention can be modified into various forms as long as the gist of the invention is not changed. However, the scope of rights of the present invention is not limited to the following examples.
本発明の要旨をあいまいにすることができると判断されれば、公知の構成及び機能についての説明は省略する。この明細書において、“含む”とは、特に他の記載がない限り、他の構成要素をさらに含むことができることを意味する。 If it is determined that the gist of the present invention can be obscured, descriptions of known configurations and functions are omitted. In this specification, “including” means that other components can be further included unless otherwise specified.
本発明はキムチから分離した新規の植物性乳酸菌であるラクトバシラス・プランタラムＬＬＰ５１９３（寄託番号ＫＣＣＭ１１５９８Ｐ）を提供する。 The present invention provides Lactobacillus plantarum LLP5193 (deposit number KCCM11598P) which is a novel plant lactic acid bacterium isolated from kimchi.
近年、野菜や果物などの植物、キムチなどの果菜類を発酵した食品から分離した植物性乳酸菌が動物性乳酸菌に比べて優れた効果を持つという研究結果が相次いで発表されている。 In recent years, research results have been published that plant lactic acid bacteria isolated from fermented foods such as vegetables and fruits and fruit vegetables such as kimchi have superior effects compared to animal lactic acid bacteria.
動物性乳酸菌（牛乳発酵乳酸菌）は培地栄養分として炭水化物とともに蛋白質、脂肪などを使う。したがって、長期間の発酵過程で蛋白質と脂肪によって腐敗する菌株の数が増加する欠点がある。一方、植物性乳酸菌は培地栄養分として炭水化物のみを使うので、発酵時に腐敗しない。 Animal lactic acid bacteria (fermented milk lactic acid bacteria) use protein, fat, etc. along with carbohydrates as nutrients in the medium. Therefore, there is a drawback that the number of strains that are spoiled by protein and fat increases during a long-term fermentation process. On the other hand, plant lactic acid bacteria do not rot during fermentation because they use only carbohydrates as nutrients in the medium.
また、植物性乳酸菌は動物性乳酸菌に比べて栄養分が足りない所で生存するので、低ｐＨ、高塩濃度などの苛酷な環境でも強い生存力を持ち、培養が容易であり、腸内に安定的に到逹することができるという利点がある。さらに、韓国人は植物性食品を主食とするので、植物性乳酸菌に適した腸内環境を持つ。 In addition, plant lactic acid bacteria survive in places with less nutrients than animal lactic acid bacteria, so they have strong viability in harsh environments such as low pH and high salt concentration, are easy to culture, and are stable in the intestines. There is an advantage that it can be arrived at. Furthermore, since Koreans eat vegetable foods as a staple food, they have an intestinal environment suitable for plant lactic acid bacteria.
本発明によるラクトバシラス・プランタラムＬＬＰ５１９３は優れた耐酸性、耐胆汁性を持つ。 Lactobacillus plantarum LLP5193 according to the present invention has excellent acid resistance and bile resistance.
乳酸菌が整腸作用及び静菌作用に役立つためには、腸内に確実に到逹しなければならない。したがって、胃酸によって死滅せずに胃を通過するために、優れた耐酸性を持たなければならなく、十二指腸の上部から分泌される胆汁酸に対しても耐性を持たなければならない。 In order for lactic acid bacteria to be useful for intestinal action and bacteriostatic action, it must reach the intestine reliably. Therefore, in order to pass through the stomach without being killed by gastric acid, it must have excellent acid resistance and must be resistant to bile acids secreted from the upper part of the duodenum.
本発明によるラクトバシラス・プランタラムＬＬＰ５１９３は優れた細胞付着能を持つ。乳酸菌が腸内で効果を発揮するためには腸管細胞（Ｉｎｔｅｓｔｉｎａｌ ｃｅｌｌ）に付着しなければならないので、細胞付着能が重要である。 Lactobacillus plantarum LLP5193 according to the present invention has excellent cell adhesion ability. In order for lactic acid bacteria to exert their effects in the intestine, they must adhere to intestinal cells (Intestinal cells), and thus the cell adhesion ability is important.
本発明はラクトバシラス・プランタラムＬＬＰ５１９３に賦形剤を一定含量で添加することで、食感が良くて食べるのに適し、摩損率が低くて剤形化に有利な凍結乾燥物を提供する。 The present invention provides a freeze-dried product that has a good texture and is suitable for eating by adding an excipient to Lactobacillus plantarum LLP5193 at a constant content and has a low friability and is advantageous for formulation.
特に、嬰児や幼児は粉末がたくさん飛ぶような食品や硬い食品を食べにくいので、本発明による凍結乾燥物を使えば嬰児や幼児のための乳酸菌含有製品を製造するのに大いに役に立つことができる。 In particular, since infants and infants are hard to eat foods that are powdery and hard foods, the freeze-dried product according to the present invention can be very useful for producing lactic acid bacteria-containing products for infants and infants.
本発明は、キムチ試料を採取し、これから耐酸性、耐胆汁性及び細胞付着能に優れた菌株を選別する過程によって選択された乳酸菌であるラクトバシラス・プランタラムＬＬＰ５１９３を提供する。 The present invention provides Lactobacillus plantarum LLP5193, which is a lactic acid bacterium selected by a process of collecting a kimchi sample and selecting a strain excellent in acid resistance, bile resistance and cell adhesion ability from the sample.
また、本発明は、ラクトバシラス・プランタラムＬＬＰ５１９３を特定の条件で培養した後、特定の条件で発酵して発酵乳を製造する。また、前記発酵乳に一定含量の賦形剤を添加し、特定の条件で凍結乾燥して凍結乾燥物を製造する。 In the present invention, Lactobacillus plantarum LLP5193 is cultured under specific conditions, and then fermented under specific conditions to produce fermented milk. In addition, a certain amount of excipient is added to the fermented milk and freeze-dried under specific conditions to produce a freeze-dried product.
前記ラクトバシラス・プランタラムＬＬＰ５１９３は、ＭＲＳ培地または脱脂粉乳が２〜１０重量％（Ｓｋｉｍ ｍｉｌｋ ２０〜１００ｇ／Ｌ）含む商用牛乳培地で２５〜４２℃で１８〜３６時間培養できる。 The Lactobacillus plantarum LLP5193 can be cultured at 25-42 ° C. for 18-36 hours in a commercial milk medium containing 2-10% by weight of MRS medium or skim milk powder (Skim milk 20-100 g / L).
前記ラクトバシラス・プランタラムＬＬＰ５１９３の培養液０．５〜２重量％を脱脂粉乳が２〜１０重量％含まれた商用牛乳培地に添加した後、２５〜４２℃で１２〜２４時間発酵して発酵乳を製造することができる。 Fermented milk obtained by adding 0.5-2% by weight of the culture solution of Lactobacillus plantarum LLP5193 to a commercial milk medium containing 2-10% by weight of skim milk and fermenting at 25-42 ° C. for 12-24 hours Can be manufactured.
前記発酵乳にタピオカ澱粉１〜５重量％を添加し、零下７０℃で２〜６時間凍結させた後、５０ｍｔｏｒｒ及び零下８０℃を維持する凍結乾燥器で１８〜３６時間凍結乾燥して凍結乾燥物を製造することができる。 Tapioca starch was added to the fermented milk in an amount of 1 to 5% by weight, frozen at 70 ° C. for 2 to 6 hours, and then freeze-dried for 18 to 36 hours in a freeze dryer maintained at 50 mtorr and 80 ° C. Can be manufactured.
以下、本発明を実施例に基づいてより詳細に説明する。しかし、これらの実施例は本発明を例示するためのもので、本発明の範囲がこれらの実施例によって限定されるものではない。 Hereinafter, the present invention will be described in more detail based on examples. However, these examples are for the purpose of illustrating the present invention, and the scope of the present invention is not limited by these examples.
実施例１−ラクトバシラス・プランタラムＬＬＰ５１９３の選別 Example 1-Selection of Lactobacillus plantarum LLP5193
（１）試料の採取 (1) Sample collection
以下の［表１］のように全国各地の伝統在来市場で約１５０種のキムチ試料を採取した。 As shown in [Table 1] below, about 150 kinds of kimchi samples were collected at traditional traditional markets throughout the country.
採取した試料に滅菌蒸溜水を添加して希釈液を作った後、粉砕均質器（Ｓｔｏｍａｃｈｅｒ、商品名Ｐｒｏ−Ｍｅｄｉａ ＳＨ−００１、ＥＬＭＥＸ社製）で試料の内部を均質化した。 Sterile distilled water was added to the collected sample to prepare a diluted solution, and then the inside of the sample was homogenized with a pulverization homogenizer (Storcher, trade name: Pro-Media SH-001, manufactured by ELMEX).
前記希釈液を滅菌食塩水で段階別に希釈した後、０．１ｍｌを取った。これを０．００２重量％のＢＣＰ（Ｂｒｏｍｏｃｒｅｓｏｌ ｐｕｒｐｌｅ）と１．５重量％の寒天（Ａｇａｒ）が添加されたＭＲＳ固体培地に塗抹し、３７℃で維持される培養器で４８時間培養した。 The diluted solution was diluted stepwise with sterile saline, and 0.1 ml was taken. This was smeared on an MRS solid medium supplemented with 0.002% by weight of BCP (Bromocresol purple) and 1.5% by weight of agar (Agar), and cultured in an incubator maintained at 37 ° C. for 48 hours.
培養後、黄色の環を現すコロニー（Ｃｏｌｏｎｙ）を選別し、個体別に同一培地に２〜３回継代培養することによって各菌株を純粋分離した。 After the culture, colonies showing a yellow ring were selected, and each strain was purely isolated by subculturing 2-3 times in the same medium for each individual.
前記菌株を個体別にＭＲＳ液体培地に接種し、３７℃で４８時間再培養した後、４℃で冷蔵保管しながら使用した。 The above strains were individually inoculated into an MRS liquid medium, re-cultured at 37 ° C. for 48 hours, and then used while refrigerated at 4 ° C.
（２）菌株の選別 (2) Selection of strains
カコ−２（Ｃａｃｏ−２）細胞を用いて前記菌株の中で腸管細胞付着能に優れた菌株を選別した。 A strain having excellent intestinal cell adhesion ability was selected from the strains using Caco-2 cells.
Ｃａｃｏ−２細胞は６−ウェルプレート（６−ｗｅｌｌ ｐｌａｔｅ）に２×１０５ｃｅｌｌｓ／ｍｌ濃度で接種した後、３７℃、５％ＣＯ２の条件を維持する培養器で約１５日間培養し、単層（ｍｏｎｏｌａｙｅｒ）を形成したものを使った。 Caco-2 cells are inoculated in a 6-well plate at a concentration of 2 × 10 5 cells / ml, and then cultured in an incubator that maintains conditions of 37 ° C. and 5% CO 2 for about 15 days. What formed the monolayer (monolayer) was used.
前記菌株をＭＲＳ液状培地に接種し、３７℃で２４時間培養した後、遠心分離（８，０００ｒｐｍ、１０分）して菌体を回収した後、リン酸緩衝液（Ｐｏｔａｓｓｉｕｍ ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒ）を添加して菌株懸濁液を用意した。 The strain is inoculated into an MRS liquid medium, cultured at 37 ° C. for 24 hours, centrifuged (8,000 rpm, 10 minutes), and the cells are collected, and then a phosphate buffer (Potassium phosphate buffer) is added. A strain suspension was prepared.
前記菌株懸濁液を、Ｃａｃｏ−２細胞が入っている６−ウェルプレートに１ｍｌ添加し、３７℃、５％ＣＯ２の条件で２時間反応させた後、リン酸緩衝液で３回繰り返し洗浄した。 1 ml of the strain suspension is added to a 6-well plate containing Caco-2 cells, reacted for 2 hours at 37 ° C. and 5% CO 2 , and then washed three times with a phosphate buffer. did.
洗浄の終了した６−ウェルプレートにＭＲＳ液状培地２ｍｌを添加した後、滅菌されたスクレーパー（Ｓｃｒａｐｅｒ）でＣａｃｏ−２細胞及び菌株を掻き出した。これを１０ｍｌチューブに移した後、滅菌食塩水で多段希釈した。適正倍数の希釈液０．１ｍｌをＭＲＳ平板培地に接種した後、３７℃で４８時間培養した。ＭＲＳ平板培地上で培養されたコロニー数を確認して菌数を測定した。 After adding 2 ml of MRS liquid medium to the washed 6-well plate, Caco-2 cells and strains were scraped with a sterilized scraper. This was transferred to a 10 ml tube and then diluted in multiple stages with sterile saline. After inoculating an MRS plate medium with 0.1 ml of an appropriate multiple dilution, the cells were cultured at 37 ° C. for 48 hours. The number of colonies cultured on the MRS plate medium was confirmed and the number of bacteria was measured.
菌株の最初に添加した菌体の濃度と洗浄後の菌体濃度をそれぞれ測定して付着率（洗浄後菌体濃度／最初菌体濃度）を確認した。一番優秀な付着率を示した菌株を最終的に選別した。 The density | concentration of the microbial cell added at the beginning of the strain and the microbial cell concentration after washing | cleaning were measured, respectively, and the adhesion rate (microbe concentration after washing | cleaning / initial microbial cell concentration) was confirmed. The strain that showed the best adherence rate was finally selected.
最終的に選別された菌株をＭＲＳ液体培地に接種し、３７℃で４８時間培養して増殖させ、遠心分離（１０，０００ｇ、１０分）して菌体を獲得した。前記菌体をＭＲＳ液体培地：グリセロール（Ｇｌｙｃｅｒｏｌ）が４：１の割合で含まれた凍結保存溶液（Ｆｒｅｅｚｉｎｇ ｓｏｌｕｔｉｏｎ）に１ｍｌ添加した後、クリオチューブ（Ｃｒｙｏ−ｔｕｂｅ）に入れ、−７０℃で凍結保存した。凍結保存された菌株を後続のテストのためのスターターとして使った。 The finally selected strain was inoculated into MRS liquid medium, cultured at 37 ° C. for 48 hours to grow, and centrifuged (10,000 g, 10 minutes) to obtain bacterial cells. 1 ml of the above microbial cells was added to a freezing solution containing MRS liquid medium: glycerol (Glycerol) in a ratio of 4: 1, and then added to a cryotube (freezing at −70 ° C.). saved. The cryopreserved strain was used as a starter for subsequent testing.
実施例２−菌株の特徴分析及び同定 Example 2-Strain characterization and identification
（１）特徴分析 (1) Feature analysis
選別された菌株の形態学的及び生化学的特徴を分析した。以下の［表２］を参照して、前記菌株はグラム陽性、桿菌、胞子形成能及びカタラーゼ（Ｃａｔａｌａｓｅ）は陰性と確認された。 The morphological and biochemical characteristics of the selected strains were analyzed. With reference to the following [Table 2], it was confirmed that the strain was Gram positive, Neisseria gonorrhoeae, spore-forming ability and catalase were negative.
（２）菌株同定 (2) Strain identification
前記菌株の糖利用性をＡＰＩ ５０ ＣＨＬキット（Ｂｉｏｍｅｒｉｅｕｘ社製、Ｆｒａｎｃｅ）で分析した。以下の［表３］及び図１を参照して、ラクトバシラス・プランタラム標準菌株（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐｌａｎｔａｒｕｍ １）と９９．７％一致するが、トレハロース（Ｔｒｅｈａｌｏｓｅ）に対する利用性は９６％違うことが確認された。 The sugar utilization of the strain was analyzed with API 50 CHL kit (Biomerieux, France). Referring to [Table 3] and FIG. 1 below, it was confirmed that the utilization against trehalose was 96% different from the standard strain of Lactobacillus plantarum (Lactobacillus plantarum 1), which was 99.7%. .
前記菌株の１６Ｓ ｒＲＮＡ遺伝子塩基配列は、［配列表］に示す配列番号１の通りである。塩基配列の分析結果、前記菌株はラクトバシラス属菌株と最大９９％の相同性を持つ。これは図２から確認することができる。 The 16S rRNA gene base sequence of the strain is as shown in SEQ ID NO: 1 shown in [Sequence Listing]. As a result of nucleotide sequence analysis, the strain has a homology of up to 99% with the Lactobacillus strain. This can be confirmed from FIG.
よって、本発明者は前記菌株を新規のラクトバシラス・プランタラム菌株に同定し、ラクトバシラス・プランタラムＬＬＰ５１９３と名付け、韓国微生物保存センターに２０１４年１１月３日付けで寄託した（寄託番号ＫＣＣＭ１１５９８Ｐ）。 Therefore, the present inventor identified the above strain as a novel Lactobacillus plantarum strain, named it Lactobacillus plantarum LLP5193, and deposited it with the Korean Preservation Center on November 3, 2014 (deposit number KCCM11598P).
実施例３−ラクトバシラス・プランタラムＬＬＰ５１９３の耐酸性及び耐胆汁性測定 Example 3 Measurement of Acid Resistance and Bile Resistance of Lactobacillus plantarum LLP5193
口腔から乳酸菌を摂取すると、胃酸及び胆汁によって相当数が死滅する。したがって、小腸及び大腸まで到達する乳酸菌の数を増やすためには優れた耐酸性及び耐胆汁性が必要である。 When lactic acid bacteria are ingested from the oral cavity, a considerable number are killed by gastric acid and bile. Therefore, in order to increase the number of lactic acid bacteria reaching the small and large intestines, excellent acid resistance and bile resistance are required.
ラクトバシラス・プランタラムＬＬＰ５１９３をＭＲＳ平板培地に接種して３７℃で２４時間培養した。培養されたコロニーを採取し、ＭＲＳ液状培地で３７℃で２４時間静置培養して種菌培養液を製造した。 Lactobacillus plantarum LLP5193 was inoculated into MRS plate medium and cultured at 37 ° C. for 24 hours. The cultured colonies were collected and allowed to stand at 37 ° C. for 24 hours in an MRS liquid medium to produce an inoculum culture solution.
（１）耐酸性試験 (1) Acid resistance test
種菌培養液１％をＭＲＳ液体培地に接種し、３７℃で２４時間培養した。取得した培養液に１０％Ｈ２ＳＯ４を添加してｐＨ３．０の強酸性条件にし、２時間放置した。その後、試料を多段希釈した希釈液をＭＲＳ平板培地に塗抹し、３７℃で２４時間培養し、初期生菌数とｐＨ３．０処理後の生菌数を測定した。本発明において、耐酸性は（初期生菌数／ｐＨ処理後の生菌数）の割合で示す。 1% of the inoculum culture solution was inoculated into MRS liquid medium and cultured at 37 ° C. for 24 hours. To the obtained culture broth, 10% H 2 SO 4 was added to make it a strongly acidic condition of pH 3.0 and left for 2 hours. Thereafter, the diluted solution obtained by multi-stage dilution of the sample was smeared on the MRS plate medium and cultured at 37 ° C. for 24 hours, and the initial viable cell count and the viable cell count after pH 3.0 treatment were measured. In the present invention, acid resistance is expressed as a ratio of (initial viable cell count / viable cell count after pH treatment).
（２）耐胆汁性試験 (2) Bile resistance test
胆汁（ｂｉｌｅ）を０．１重量％、０．３重量％、０．５重量％で添加して製造したＭＲＳ平板培地を用意した。それぞれのＭＲＳ平板培地に種菌培養液を接種した後、３７℃で２４時間培養し、増殖状態を確認した。 An MRS plate medium produced by adding bile at 0.1 wt%, 0.3 wt%, and 0.5 wt% was prepared. Each MRS plate medium was inoculated with an inoculum culture solution and then cultured at 37 ° C. for 24 hours to confirm the growth state.
（３）試験結果 (3) Test results
それぞれの試験の比較群としてラクトバシラス・プランタラム標準菌株（ＫＣＴＣ２１００４、ＡＴＣＣ１４９１７）を用いた。耐酸性試験及び耐胆汁性試験の結果は以下の［表４］の通りである。 A Lactobacillus plantarum standard strain (KTCTC21004, ATCC14917) was used as a comparative group for each test. The results of acid resistance test and bile resistance test are as shown in [Table 4] below.
ラクトバシラス・プランタラムＬＬＰ５１９３はｐＨ３．０の強酸性条件下で、約２時間処理しても９９．６％の生存率を示した。また、０．５重量％の胆汁が添加された時にも生育が可能なことを確認した。 Lactobacillus plantarum LLP5193 showed a survival rate of 99.6% even after treatment for about 2 hours under strongly acidic conditions at pH 3.0. It was also confirmed that growth was possible when 0.5% by weight of bile was added.
したがって、ラクトバシラス・プランタラムＬＬＰ５１９３は優れた耐酸性及び耐胆汁性を持って腸まで到逹する確率が高いため、食品または健康機能食品として摂取する場合、乳酸菌の多様な効能を効果的に発揮することができる。 Therefore, since Lactobacillus plantarum LLP5193 has excellent acid resistance and bile resistance and has a high probability of reaching the intestine, when it is ingested as a food or health functional food, it effectively exhibits various effects of lactic acid bacteria. be able to.
実施例４−ラクトバシラス・プランタラムＬＬＰ５１９３の腸管細胞付着能測定 Example 4-Measurement of intestinal cell adhesion ability of Lactobacillus plantarum LLP5193
以上の実施例３からラクトバシラス・プランタラムＬＬＰ５１９３は耐酸性及び耐胆汁性に優れており腸まで到達する割合が他の乳酸菌に比べて高いことを確認した。 From the above Example 3, Lactobacillus plantarum LLP5193 was excellent in acid resistance and bile resistance, and it was confirmed that the rate of reaching the intestine was higher than other lactic acid bacteria.
しかし、乳酸菌が腸まで到達しても腸管細胞に付着しなければ遺失してしまうため、効果を正常に発揮することができない。したがって、細胞付着能が耐酸性及び耐胆汁性よりもっと重要な特性であると言える。 However, even if the lactic acid bacteria reach the intestines, they will be lost if they do not adhere to the intestinal cells, so that the effect cannot be exhibited normally. Therefore, it can be said that cell adhesion ability is a more important characteristic than acid resistance and bile resistance.
ラクトバシラス・プランタラムＬＬＰ５１９３の腸管細胞付着能を定量的に測定するとともに、付着形態を顕微鏡で観察した。 The intestinal cell adhesion ability of Lactobacillus plantarum LLP5193 was quantitatively measured, and the adhesion form was observed with a microscope.
（１）定量的測定 (1) Quantitative measurement
前記実施例１においてＣａｃｏ−２細胞を用いた腸管細胞付着能試験と同様の方法でラクトバシラス・プランタラムＬＬＰ５１９３の腸管細胞付着能を測定した。その結果は以下の［表５］の通りである。 Intestinal cell adhesion ability of Lactobacillus plantarum LLP5193 was measured in the same manner as in the intestinal cell adhesion ability test using Caco-2 cells in Example 1. The results are as shown in [Table 5] below.
総６回の試験を繰り返し遂行した。平均値で調べると、ラクトバシラス・プランタラムＬＬＰ５１９３が約４．５×１０９ＣＦＵ／ｍｌ投与されたとき、付着反応後、約５．１×１０８ＣＦＵ／ｍｌが付着していた。これから、一般的に微生物の濃度を示すログ値を取って付着率を計算したところ、約９０．２％［（８．７１ｌｏｇ／９．６５ｌｏｇ）×１００］と非常に高かった。 A total of 6 tests were repeated. Examining the average value, when Lactobacillus plantarum LLP5193 was administered at about 4.5 × 10 9 CFU / ml, about 5.1 × 10 8 CFU / ml adhered after the adhesion reaction. From this, a log value indicating the concentration of microorganisms was generally taken and the adhesion rate was calculated. As a result, it was very high, about 90.2% [(8.71 log / 9.65 log) × 100].
一般的なプロバイオティックス乳酸菌製品の摂取基準が１×１０８ＣＦＵ／ｍｌ（８．０ｌｏｇ）であることを考慮すれば、ラクトバシラス・プランタラムＬＬＰ５１９３の腸管細胞付着能が非常に優れることが分かる。 Considering that the intake standard of a general probiotic lactic acid bacteria product is 1 × 10 8 CFU / ml (8.0 log), it can be seen that the ability of Lactobacillus plantarum LLP5193 to adhere to intestinal cells is very excellent.
（２）顕微鏡観察 (2) Microscopic observation
Ｃａｃｏ−２細胞を用いた付着試験において、付着反応の終了後、培養液を一部採取した。前記培養液をスライドガラスに適量移した後、グラム染色（Ｇｒａｍ ｓｔａｉｎｉｎｇ）でラクトバシラス・プランタラムＬＬＰ５１９３を染色した。その結果は図３の通りである。 In the adhesion test using Caco-2 cells, a part of the culture solution was collected after completion of the adhesion reaction. An appropriate amount of the culture solution was transferred to a slide glass, and then Lactobacillus plantarum LLP5193 was stained with Gram staining. The result is as shown in FIG.
染色されたラクトバシラス・プランタラムＬＬＰ５１９３がＣａｃｏ−２細胞の周りに非常に多く付着されたことを確認することができる。 It can be confirmed that a large amount of stained Lactobacillus plantarum LLP5193 was attached around Caco-2 cells.
ラクトバシラス・プランタラムＬＬＰ５１９３は耐酸性及び耐胆汁性に優れるので、腸まで安定的に到達することができ、優れた細胞付着能を持つので、腸管細胞に多数が付着することができる。したがって、ラクトバシラス・プランタラムＬＬＰ５１９３は乳酸菌としての効果をとてもよく発揮することができる。 Since Lactobacillus plantarum LLP5193 is excellent in acid resistance and bile resistance, it can stably reach the intestine and has excellent cell adhesion ability, so that a large number can adhere to intestinal cells. Therefore, Lactobacillus plantarum LLP5193 can exhibit the effect as a lactic acid bacterium very well.
実施例５−ラクトバシラス・プランタラムＬＬＰ５１９３を有効成分として含む製品の製造 Example 5-Production of a product containing Lactobacillus plantarum LLP5193 as an active ingredient
（１）発酵乳の製造
ラクトバシラス・プランタラムＬＬＰ５１９３をＭＳ固体（ａｇａｒ）培地に接種した後、３７℃で２４時間培養して活性化した。活性化したラクトバシラス・プランタラムＬＬＰ５１９３のコロニーを、牛乳１００ｍｌに脱脂粉乳を５重量％添加した培地に接種し、カード（ｃｕｒｄ）が作られるまで３７℃で約１２時間培養した。種菌培養物１重量％を脱脂粉乳が５重量％添加された牛乳１Ｌと混合した後、カード（ｃｕｒｄ）が形成されるまで３７℃で約１２時間培養して発酵乳を製造した。
(1) Production of fermented milk Lactobacillus plantarum LLP5193 was inoculated into MS solid (agar) medium and then activated by culturing at 37 ° C for 24 hours. Activated Lactobacillus plantarum LLP5193 colonies were inoculated into a medium in which 5% by weight of nonfat dry milk was added to 100 ml of milk, and cultured at 37 ° C. for about 12 hours until a curd was produced. After mixing 1% by weight of the inoculum culture with 1 L of milk to which 5% by weight of skim milk powder was added, fermented milk was produced by culturing at 37 ° C. for about 12 hours until a curd was formed.
（２）凍結乾燥物の製造 (2) Production of freeze-dried product
前記発酵乳に一定濃度の賦形剤を添加して作った凍結乾燥物の生菌数、食感及び摩損率を測定した。 The viable cell count, texture and erosion rate of the freeze-dried product prepared by adding a certain concentration of excipient to the fermented milk were measured.
前記発酵乳にデキストリン、米粉、タピオカ澱粉を１重量％、３重量％、５重量％で添加した。その混合物を成形のためのモールドに投入し、極低温冷凍機（零下７０℃維持）で２時間急速冷凍させた。これを凍結乾燥器（イルシンバイオベース、ＭＣＦＤ Ｓｅｒｉｅｓ、大韓民国）に移した後、５０ｍｔｏｒｒ、零下８０℃で１８時間凍結乾燥して凍結乾燥物を製造した。 Dextrin, rice flour and tapioca starch were added to the fermented milk at 1 wt%, 3 wt% and 5 wt%. The mixture was put into a mold for molding and rapidly frozen in a cryogenic refrigerator (maintained at 70 ° C. below zero) for 2 hours. This was transferred to a lyophilizer (Ilsin Biobase, MCFD Series, South Korea), and then lyophilized at 50 mtorr and 80 ° C. for 18 hours to produce a lyophilized product.
凍結乾燥物の生菌数を測定した。凍結乾燥物を滅菌食塩水で希釈した後、適度な希釈液を取った。これをＭＲＳ固体培地に塗抹し、３７℃で４８時間培養した後、コロニーを計数した。 The viable count of the lyophilized product was measured. After the lyophilized product was diluted with sterile saline, an appropriate dilution was taken. This was smeared on an MRS solid medium, cultured at 37 ° C. for 48 hours, and then colonies were counted.
凍結乾燥物の食感を測定した。任意に５人を選別し、凍結乾燥物を摂取した後、硬さを評価した。 The texture of the lyophilized product was measured. After arbitrarily selecting 5 persons and taking a lyophilized product, the hardness was evaluated.
凍結乾燥物の摩損率を測定した。凍結乾燥物をローリング機に投入した後、５０回転させた。回転前の凍結乾燥物の重量と回転後の重量を測定した。摩損率は（回転前重量−回転後重量）／回転前重量×１００で示した。 The friability of the lyophilizate was measured. The lyophilized product was put into a rolling machine and then rotated 50 times. The weight of the lyophilized product before rotation and the weight after rotation were measured. The wear rate was expressed as (weight before rotation−weight after rotation) / weight before rotation × 100.
賦形剤を添加した凍結乾燥物の生菌数、食感及び摩損率の結果は以下の表６の通りである。 The results of the viable cell count, texture, and abrasion rate of the lyophilized product to which the excipient was added are shown in Table 6 below.
＊ １：硬い→５：柔らかい * 1: Hard → 5: Soft
賦形剤を添加することで、無添加の場合に比べて生菌数が高くなることが分かった。ただ、賦形剤の種類による違いは大きくないことが確認された。 It was found that the number of viable cells was increased by adding the excipient compared to the case of no addition. However, it was confirmed that the difference depending on the type of excipient was not significant.
賦形剤の添加による摩損率及び食感を調べたところ、賦形剤無添加の場合、食感は軟らかかったが、摩損率が６６．７％と高く、凍結乾燥した形態を保存することがほぼできなかった。 As a result of examining the wear rate and the texture when the excipient was added, the texture was soft when no excipient was added, but the wear rate was as high as 66.7% and the lyophilized form should be preserved. Was almost impossible.
デキストリンと米粉を使用した場合、同一濃度のタピオカ澱粉を使用した場合に比べて食感はほぼ同じ程度であったが摩損率が高かった。一番優れた耐摩損性と食感が見られたものはタピオカ澱粉を３重量％添加した凍結乾燥物であった。 When dextrin and rice flour were used, the texture was almost the same as when tapioca starch of the same concentration was used, but the wear rate was high. The lyophilized product to which 3% by weight of tapioca starch was added showed the most excellent abrasion resistance and texture.
したがって、タピオカ澱粉３重量％を添加したとき、耐摩損性が良くて粉末が多く発生せず、食感が良いので、嬰児や幼児のための食品を製造することができるという利点がある。 Therefore, when 3% by weight of tapioca starch is added, there is an advantage that foods for infants and infants can be manufactured because the wear resistance is good, a large amount of powder is not generated, and the texture is good.
凍結乾燥物にタピオカ澱粉を３重量％添加した実製品を図４に示した。 The actual product obtained by adding 3% by weight of tapioca starch to the lyophilized product is shown in FIG.
本発明によるラクトバシラス・プランタラムはキムチから分離した植物性乳酸菌として韓国人の腸内環境に適する。 The Lactobacillus plantarum according to the present invention is suitable for the Korean intestinal environment as a plant lactic acid bacterium isolated from kimchi.
また、本発明によるラクトバシラス・プランタラムは耐酸性及び耐胆汁性に優れており腸内に確実に到逹することができる。 In addition, Lactobacillus plantarum according to the present invention is excellent in acid resistance and bile resistance and can reach the intestine with certainty.
また、本発明によるラクトバシラス・プランタラムは腸管細胞付着能に優れており腸内に安定的に定着することができるので、乳酸菌としての効果を十分に発揮することができる。 In addition, the Lactobacillus plantarum according to the present invention is excellent in intestinal cell adhesion ability and can be stably settled in the intestine, so that the effect as a lactic acid bacterium can be sufficiently exerted.
また、本発明によるラクトバシラス・プランタラムから製造した製品は食感及び耐摩損性が良いので、剤形化に有利で、粉末が多く発生せず、食べやすい。 In addition, since the product produced from Lactobacillus plantarum according to the present invention has good texture and abrasion resistance, it is advantageous for formulation, does not generate much powder, and is easy to eat.
以上、本発明について詳細に説明した。ただ、本発明の権利範囲はこれに限定されなく、以下の特許請求の範囲によって決定される。 The present invention has been described in detail above. However, the scope of rights of the present invention is not limited to this, and is determined by the following claims.
ＫＣＣＭ１１５９８Ｐ KCCM11598P
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