SK542015A3 - Strains Lactobacillus Plantarum LS/07 CCM 7766 and their use - Google Patents
Strains Lactobacillus Plantarum LS/07 CCM 7766 and their use Download PDFInfo
- Publication number
- SK542015A3 SK542015A3 SK54-2015A SK542015A SK542015A3 SK 542015 A3 SK542015 A3 SK 542015A3 SK 542015 A SK542015 A SK 542015A SK 542015 A3 SK542015 A3 SK 542015A3
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- Slovakia
- Prior art keywords
- lactobacillus plantarum
- ccm
- strain
- strains
- isolated
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Links
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
Je opísaný kmeň mikroorganizmov Lactobacillus Plantarum LS/07 CCM 7766 a spôsob jeho využitia.A strain of Lactobacillus Plantarum LS / 07 CCM 7766 and a method for its use is described.
Description
Oblasť technikyTechnical field
Vynález sa týka izolovaného kmeňa mikroorganizmov Lactobacillus Plantarum LS/07, zbierkové číslo CCM 7766, ktorý je zároveň prostriedkom pre prípravu preparátov a prídavkov do potravín pre účely preventívneho posilnenia črevnej bariéry. Kmeň Lactobacillus Plantarum LS/07 CCM 7766 bol vykultivovaný zo stolice zdravého človeka.The invention relates to an isolated strain of microorganisms Lactobacillus Plantarum LS / 07, collection number CCM 7766, which is also a means for the preparation of preparations and food additives for the purpose of preventive strengthening of the intestinal barrier. The Lactobacillus Plantarum strain LS / 07 CCM 7766 was cultivated from the faeces of a healthy human.
Doterajší stav technikyBACKGROUND OF THE INVENTION
Zdravie ľudskej populácie je jedným z hlavných atribútov fungovania ľudskej spoločnosti. Vysoké spoločenské nároky kladené na človeka prinášajú veľa negatívnych dopadov na jeho celkové zdravie. Stres, nutrične nevyvážená potrava a nezdravé návyky majú nepriaznivý dopad na črevnú mikroflóru človeka, čo sa môže neskôr negatívne prejaviť v celkovom zdraví ľudskej populácie. Civilizačné choroby vznikajú v dôsledku dlhodobého pôsobenia uvedených faktorov. Liečba civilizačných chorôb je dlhodobá a finančne vysoko nákladná, preto sa v súčasnej dobe čoraz viac presadzuje prevencia v boji proti civilizačným chorobám. Použitie vyselektovaných probiotických kmeňov môže vo veľkej miere zabezpečiť stabilitu zdravej črevnej mikroflóry s následným pozitívnym efektom na zdravie jedinca.The health of the human population is one of the main attributes of the functioning of human society. The high social demands placed on man have many negative impacts on his overall health. Stress, nutritionally unbalanced food and unhealthy habits have an adverse effect on the human intestinal microflora, which may later have a negative impact on the overall health of the human population. Civilization diseases are caused by the long-term effects of these factors. The treatment of civilization diseases is long-term and costly, therefore prevention in the fight against civilization diseases is increasingly being promoted. The use of selected probiotic strains can largely ensure the stability of a healthy intestinal microflora with a consequent positive effect on the health of the individual.
V súčasnosti sa izolované kmene laktobacilov vo veľkej miere využívajú ako súčasť funkčných potravín, alebo výživových doplnkov. Niektoré probiotické kmene využívané ako súčasť výživových doplnkov majú priaznivý vplyv na mikroflóru tráviaceho traktu človeka, takýmito kmeňmi sú Lactobacillus plantarum 299 a Lactobacillus plantarum 299v DSM 9843, uvedené úzko súvisiace kmene boli izolované z črevnej sliznice zdravého človeka (Molin a kol. 1993 a Johanson akol. 1993).Currently isolated strains of lactobacilli are extensively used as part of functional foods or nutritional supplements. Some probiotic strains used as part of nutritional supplements have a beneficial effect on the microflora of human digestive tract, such strains are Lactobacillus plantarum 299 and Lactobacillus plantarum 299 in DSM 9843, said closely related strains isolated from the intestinal mucosa of a healthy human (Molans et al. 1993).
Európsky patentový spis EP 1 904 074 opisuje použitie laktobacilov na zvýšenie absorpcie iónov železa, zinku a vápnika, týka sa použitia minimálne jedného špecifikovaného kmeňa Lactobacillus plantarum pre zvýšenie absorpcie ionov kovov u cicavcov a farmaceutickej kompozície, ktorá obsahuje aspoň jeden špecifikovaný kmeň Lactobacillus plantarum, pre použitie pri liečení anémie. V patente sa uvádza využitie úž známych patentovaných kmeňov Lactobacillus plantarum DSM 695, L. plantarum 299v, DSM 9843, L. Plantarum Heal 9, DSM 153121 L. plantarum, Heal 19, DSM 15 313, pre zvýšenie absorpcie iónov železa.EP 1 904 074 discloses the use of lactobacilli to increase the absorption of iron, zinc and calcium ions, relates to the use of at least one specified strain of Lactobacillus plantarum to increase the absorption of metal ions in mammals and a pharmaceutical composition comprising at least one specified strain of Lactobacillus plantarum for use in the treatment of anemia. The patent discloses the use of already known patented strains of Lactobacillus plantarum DSM 695, L. plantarum 299v, DSM 9843, L. Plantarum Heal 9, DSM 153121, L. plantarum, Heal 19, DSM 15 313, to increase the absorption of iron ions.
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Patentový spis W02Q15067947 opisuje vynález týkajúci sa kmeňa Lactobacillus plantarum a kompozície pre použitie pri modifikácii absorpcie cholesterolu u jedinca, alebo pri liečbe srdcových ochorení, cukrovky alebo obezity.WO02Q15067947 discloses an invention relating to a Lactobacillus plantarum strain and a composition for use in modifying cholesterol absorption in an individual, or in the treatment of heart disease, diabetes or obesity.
RU 2491079 Cl Komplexný probiotický preparát a spôsob jeho užívania. Preparát pozostáva zo zmesi laktobacilov (Lacobacillus plantarum, L. fermentum, L. acidophilus a bifidobaktérií (Bifidobacterium bifldum, B. longum). Tieto kmene boli získané z rastlín.RU 2491079 Cl Complex probiotic preparation and method of its use. The preparation consists of a mixture of lactobacilli (Lacobacillus plantarum, L. fermentum, L. acidophilus and bifidobacteria (Bifidobacterium bifldum, B. longum). These strains were obtained from plants.
ΕΡ230-9-870τ· Izolovaný kmeň Lactobacillus plantarum Tenšia DSM 21 380 ako antimikrobiálne a antihypertenzné probiotikum, potravinový produkt a kompozícia obsahujúca uvedený mikroorganizmus pre prípravu antihypertenzívneho lieku a spôsob potlačenia patogénov a neštartérových patogénov v potravinových produktoch. Tento kmeň bol izolovaný zo stolice zdravého dieťaťa v rámci porovnávacej štúdie vykonanej u estónskych a švédskych detí.ΡΡ230-9-870τ · Isolated Lactobacillus plantarum strain Thinner DSM 21 380 as an antimicrobial and antihypertensive probiotic, food product and composition containing said microorganism for the preparation of an antihypertensive drug and a method of controlling pathogens and non-starter pathogens in food products. This strain was isolated from the faeces of a healthy child as part of a comparative study conducted in Estonian and Swedish children.
Podstata vynálezuSUMMARY OF THE INVENTION
Vynález sa týka kmeňa mikroorganizmov Lactobacillus Plantarum LS/07 deponovaného v zbierke mikroorganizmov pod poradovým číslom CCM 7766 v Českej zbierke mikroorganizmov Brno dňa 2.7.2009 podľa podmienok Budapeštianskej zmluvy, ktorý metabolizuje zdraviu prospešné substancie, ktoré ho odlišujú od iných kmeňov mikroorganizmov rodu Lactobacillus izolovaných z človeka, zvierat a rastlín.The present invention relates to a strain of microorganisms Lactobacillus Plantarum LS / 07 deposited in the collection of microorganisms under sequence number CCM 7766 in the Czech Collection of Microorganisms Brno on 2 July 2009 under the terms of the Budapest Treaty that metabolizes health-promoting substances that distinguish it from other microorganisms isolated from Lactobacillus. humans, animals and plants.
Nový izolovaný kmeň, podľa vynálezu, v laboratórnych testoch preukázal vysokú schopnosť antibakteriálnej aktivity voči patogénnemu kmeňu Escherichia coli (CNTC Eck 63/59 uložený v Československej zbierke mikroorganizmov Praha). Lactobacillus plantarum LS/07 CCM 7766 patrí do skupiny baktérií mliečneho kvasenia produkujúci kyselinu mliečnu a ďalšie organické kyseliny. Vykonané enzymatické testy poukázali na to, že kmeň Lactobacillus plantarum LS/07 CCM 7766 neprodukujeenzým β-glukuronidázu, ktorý sa u človeka považuje za dôležitý marker vývoja karcinogenézy.The new isolated strain, according to the invention, has shown in laboratory tests a high ability of antibacterial activity against the pathogenic strain Escherichia coli (CNTC Eck 63/59 deposited in the Czechoslovak Collection of Microorganisms Prague). Lactobacillus plantarum LS / 07 CCM 7766 belongs to a group of lactic acid bacteria producing lactic acid and other organic acids. Enzyme tests showed that the Lactobacillus plantarum strain LS / 07 CCM 7766 did not produce β-glucuronidase, which is considered an important marker of carcinogenesis in humans.
Uvedený kmeň mikroorganizmov je aplikovaný ako samostatný výživový doplnok, výhodne vo forme toboliek alebo ako prídavok do výživových doplnkov pre účely preventívneho posilnenia čreynej bariéry a črevnej mikroflóry voči toxickým mutagénom a infekčným patogénom. Variantom vynálezu je, že Lactobacillus Plantarum LS/07 CCM 7766 izolovaný z človeka a nie zo zvierat je aplikovaný do funkčných potravín ako sú energetické cereálne tyčinky, mliečne a mäsové výrobky, výhodne pasterizovaná bryndza. V prípade využitia Lactobacillus Plantarum LS/07 CCM a » » * * · · tnSaid strain of microorganisms is applied as a separate nutritional supplement, preferably in the form of capsules or as an addition to nutritional supplements for the purpose of preventive strengthening of the clearing barrier and intestinal microflora against toxic mutagens and infectious pathogens. A variant of the invention is that Lactobacillus Plantarum LS / 07 CCM 7766 isolated from human and not from animals is applied to functional foods such as energy cereal bars, dairy and meat products, preferably pasteurized bryndza. If Lactobacillus Plantarum LS / 07 CCM is used, tn
7766 do pasterizovanej bryndze je navrátený požadovaný probiotický účinok funkčnej potraviny, ktorá je pasterizáciou zbavená živej mikroflóry lactobacilov pochádzajúcich zo zvierat.7766, the desired probiotic effect of the functional food is restored to the pasteurized bryndza, which is pasteurized and deprived of the living animal-derived lactobacilli microflora.
Výhodou nového kmeňa mikroorganizmov Lactobacillus Plantarum LS/07 CCM 7766, na ktorý sa vzťahuje podstata vynnálezu, je modulačný efekt použitého kmeňa sa prejavuje zvýšením počtu baktérií mliečneho kvasenia a znížením počtu koliformných mikroorganizmov v črevnom obsahu hrubého čreva. Aplikácia kmeňa má vplyv nielen na črevnú mikroflóru, ale aj na metabolické aktivity s priaznivým účinkom na lipidový profil.The advantage of the novel strain of Lactobacillus Plantarum LS / 07 CCM 7766, which is the subject of the present invention, is the modulating effect of the strain used is manifested by an increase in the number of lactic acid bacteria and a decrease in the number of coliform microorganisms in the intestinal colon. Application of the strain has an effect not only on the intestinal microflora, but also on metabolic activities with a beneficial effect on the lipid profile.
Laktobacilovýkmeň Lactobacillus plantarum LS/07 bol pôvodcami charakterizovaný v klinických štúdiách na potkanoch pričom boli zistené nasledujúce výhody kmeň podľa vynálezu:The lactobacillus strain Lactobacillus plantarum LS / 07 was characterized by the inventors in clinical studies in rats and the following advantages of the strain according to the invention were found:
Kmeň Lactobacillus plantarum LS/07 CCM 7766 nielen že neprodukuje enzým β-glukuronidáza, ktorý môže mať karcinogénne účinky, ale súčasne aj znižuje jeho aktivitu v črevnom obsahu pri experimentálne chemicky vyvolanom akútnom a chronickom zápale hrubého čreva u laboratórnych potkanov.The Lactobacillus plantarum strain LS / 07 CCM 7766 not only produces the β-glucuronidase enzyme, which may have carcinogenic effects, but also reduces its activity in the intestinal content of experimentally chemically induced acute and chronic colon inflammation in rats.
Histologické výsledky preukázali, že v skupinách zvierat, kde bol podávaný kmeň Lactobacillus plantarum LS/07, došlo k výraznému zníženiu a tlmeniu zápalu vo fixovanom tkanive kolónu potkanov v porovnaní s kontrolnou skupinou, kde uvedený kmeň nebol podávaný.Histological results showed that in the groups of animals receiving the Lactobacillus plantarum LS / 07 strain, there was a significant reduction and inhibition of inflammation in the fixed tissue of the rat colony compared to the control group where the strain was not administered.
Aplikovaním kmeňa Lactobacillus plantarum LS/07 CCM 7766 došlo k signifikantnej inhibícii (P<0,05) prozápalových cytokínov IL-2, IL-17 a TNF-α, a miernemu nárastu protizápalových regulačných cytokínov IL-10 a TGF-β oproti pozitívnej kontrolnej skupine zvierat.Application of Lactobacillus plantarum strain LS / 07 CCM 7766 significantly inhibited (P <0.05) the pro-inflammatory cytokines IL-2, IL-17 and TNF-α, and slightly increased the anti-inflammatory regulatory cytokines IL-10 and TGF-β compared to the positive control group of animals.
V skupinách zvierat, kde bol aplikovaný Lactobacillus plantarum LS/07 CCM 7766 bol zistený výrazne znížený počet populácie gramnegatívnych koliformných baktérií (4.96 ±0,32 CFU/g) v porovnaní s kontrolnou skupinou (5,45±0,l 1 CFU/g).In the groups of animals treated with Lactobacillus plantarum LS / 07 CCM 7766, a significantly reduced population of gram negative coliforms (4.96 ± 0.32 CFU / g) was found compared to the control group (5.45 ± 0.1 CFU / g). ).
Príklady uskutočnenia vynálezuDETAILED DESCRIPTION OF THE INVENTION
Príklad 1Example 1
V jednom príklade uskutočnenia vynálezu je kmeň vo forme biologicky čistej kultúry.In one embodiment, the strain is in the form of a biologically pure culture.
Príklad 2Example 2
Uvedený kmeň mikroorganizmov je aplikovaný ako samostatný výživový doplnok, výhodne vo forme toboliek alebo ako prídavok do výživových doplnkov pre účely preventívneho posilnenia črevnej bariéry a črevnej mikroflóry voči toxickým mutagénom a infekčným patogénom.Said strain of microorganisms is applied as a separate nutritional supplement, preferably in the form of capsules or as an addition to nutritional supplements for the purpose of preventively strengthening the intestinal barrier and intestinal microflora against toxic mutagens and infectious pathogens.
Príklad 3Example 3
Uvedený kmeň Lactobacillus Plantarum LS/07 CCM 7766 je aplikovaný do potravín ako sú energetické cereálne tyčinky, mliečne a mäsové výrobky alebo pasterizovaná bryndza.Said strain Lactobacillus Plantarum LS / 07 CCM 7766 is applied to foods such as energy cereal bars, dairy and meat products or pasteurized bryndza.
Pracovný postup prípravy kmeňa:Strain Preparation Procedure:
Kultivácia mirkoorganizmov rodu Lactobacillus Plantarum LS/07 CCM 6677 podľa nasledujúceho postupu:Cultivation of Lactobacillus Plantarum LS / 07 CCM 6677 microorganisms according to the following procedure:
Pomocou sterilnej odberovej súpravy je prevedený výter z konečníka človeka. Odberový tampón je ponorený do zásobnej skúmavky s fyziologickým roztokom o objeme 5 ml. Zo zásobnej skúmavky je odobraná vzorka s objemom 1 ml pomocou pipety a prenesená pipetou do skúmavky s fyziologickým roztokom o objeme 9 ml. Výsledkom riedenia je 10 násobné zriedenie vzorky. Nasleduje pokračovanie tohto postupu do zriedenia vzorky od 10'2 až do stupňa zriedenia 10’5. Zo skúmaviek s jednotlivými riedeniami sú odobrané vzorky 0,1 ml a aplikované na Petriho misky so selektívnou pôdou pre laktobacily pre rast baktérií mliečneho kvasenia. Naočkované platne sú uložené do špeciálnych inkubačných nádob, v ktorých je vyvíjač zabezpečujúci anaeróbne kultivačné prostredie. Vzduchotesne uzavreté nádoby sú uložené v biologickom termostate. Kultivácia laktobacilov je realizovaná pri teplote 37°C počas 48 hodín. Po 48 hodinovej inkubácii je posúdená veľkosť a tvar porastených kolónií, pričom porast je tvorený drobnými, okrúhlymi, nízko vypuklými kolóniami bielej farby do veľkosti až 2 mm.A sterile swab is made from a human's rectum using a sterile collection set. The collection swab is immersed in a 5 ml saline stock tube. A 1 ml sample is taken from the stock tube by pipette and pipetted into a 9 ml saline tube. The dilution results in a 10-fold dilution of the sample. Followed by the continuation of this process to the sample dilution from 10 2 to the degree of dilution 10 '5th Single dilution tubes are sampled 0.1 ml and applied to Lactobacilli selective soil petri dishes for lactic acid bacteria growth. The inoculated plates are placed in special incubation vessels in which the developer provides an anaerobic culture environment. The airtight containers are stored in a biological thermostat. The cultivation of lactobacilli is carried out at 37 ° C for 48 hours. After a 48-hour incubation, the size and shape of the overgrown colonies are assessed, the crop being formed by small, round, low-convex white colonies up to 2 mm in size.
Izolácia laktobacilovévo kmeňa je vykonaná podľa postupu:The isolation of the lactobacillus strain is carried out according to the procedure:
Sterilnou očkovacou kľučkou je opatrne (pod lupou) odobraná jedna kolónia baktérií z Pertiho misky a naočkovaná do skúmavky s laktobacilo-selektívnym LS bujónom v množstve 5 ml. uvedený postup je niekoľkokrát zopakovaný, pričom každá odobratá kolónia je naočkovaná do samostatnej skúmavky s bujónom. Preočkované kolónie sú inkubované v termostate pri teplote 37°C počas 18 hodín. Po inkubácii je z každej skúmavky odobraná porastená bujónová kultúra v množstve 0,1 ml a preočkovaná na Pertiho misky obsahujúce selektívny LS agar. Platne sú následne kultivované v termostate za anaeróbnych podmienok pri teplote 37°C počas 48 hodín. Po inkubácii je prevedené posudzovanie kolónií laktobacilov. Označené kolónie sú odobraté za účelom zistenia ich morfológie mikroskopicky farbiacou technikou Grama. Farbenie je vykonané nasledovne podľa schémy:A sterile seed loop is carefully removed (under a magnifying glass) from a single colony of bacteria from a Perti dish and inoculated into a 5 ml lactobacillus-selective LS broth tube. said procedure is repeated several times, each colony collected being inoculated into a separate broth tube. The inoculated colonies are incubated in a thermostate at 37 ° C for 18 hours. After incubation, 0.1 ml of overgrown broth culture is removed from each tube and inoculated onto Perti dishes containing selective LS agar. The plates are then cultured in a thermostate under anaerobic conditions at 37 ° C for 48 hours. After incubation, lactobacilli colonies are assessed. Marked colonies are picked to determine their morphology using the Gram-staining technique. The staining is carried out as follows:
Nasadenie kultúry na podložné sklíčko Vysušenie a fixácia nad plameňomPlace the culture on a slide Drying and fixing over a flame
AplikáciaApplication
Kryštálvioleť - 30 sekCrystalViolet - 30 sec
II
Opláchnutie destilovanou vodou AplikáciaRinsing with distilled water Application
Lugolov roztok - 30 sekúndLugol solution - 30 seconds
II
Opláchnutie destilovanou vodou AplikáciaRinsing with distilled water Application
Etanol 96% - 20 sekúndEthanol 96% - 20 seconds
ΨΨ
Opláchnutie destilovanou vodou AplikáciaRinsing with distilled water Application
Karbolfuchsín - 30 sekúndCarbolfuchsin - 30 seconds
II
Opláchnutie destilovanou vodou Vysušenie filtračným papieromRinse with distilled water Dry with filter paper
Očkovacie pôdy, spotrebný materiál a nástroje použité na izoláciu kmeňa sú sterilné. Všetky úkony týkajúce sa práce s mikrobiálnou kultúrou, sú vykonané v sterilnom očkovacom boxe.Vaccine soils, consumables and tools used to isolate the strain are sterile. All operations related to working with the microbial culture are performed in a sterile inoculation box.
Detekcia laktobacilov svetelným mikroskopom:Detection of lactobacilli by light microscopy:
Na posúdenie morfológie laktobacilov je použitý svetelný mikroskop Olympus s lOOOx násobným zväčšením. Laktobacily sú pozorované ako modré grampozitívne (G+) farbiace sa stredne silné a stredne dlhé nepohyblivé mikroorganizmy v tvare tyčinky.An Olympus light microscope with 100x magnification is used to assess the morphology of lactobacilli. Lactobacilli are observed as blue Gram positive (G +) staining medium-thick and medium-long immovable rod-like microorganisms.
Detekcia inhibičných zón kmeňa Lactobacillus plantarum LS/07 CCM 7766 proti patogénnemu Escherichia coli CNTC Eck 63/59 agar difúznym testom:Detection of inhibitory zones of Lactobacillus plantarum strain LS / 07 CCM 7766 against pathogenic Escherichia coli CNTC Eck 63/59 agar by diffusion tests:
Pripravená bujónová kultúra kmeňa Lactobacillus plantarum LS/07 je inkubovaná približne 18 hodín a zmeraná jej turbidimetrická hodnota na stupeň 0,5 McFarlanda, čo zodpovedá hodnote 1,5x108 mikroorganizmov. Z bujónovej kultúry je mikropipetou odobratá bujónová kultúra o objeme 0,050 ml a aplikovaná do jamky o veľkosti 4 mm (vyrezanej do stredu platne) selektívného LS agaru. Naočkované Petriho misky sú uložené do inkubačných nádob (GASPAK patent USA) a inkubované v biologickom termostate pri teplote 37 °C počas 48 hodín. Po 48 hodinách je na povrch agarových platní navrstvený agar v zložení, pepton 0,5g, kvasničný autolyzát lg, glukóza lg, agar 1,8 g, 100 ml destilovaná voda, pH 6,9, o objeme 3 ml, v ktorom je rozpustený bujón o objeme 0,3 ml obsahujúci nočnú kultúru porasteného kmeňa Escherichia coli serotyp O6:K2:H1 ATC 1938 CNTC Eck 63/59. Po aplikácií kmeňa, sú Petriho misky vložené do termostatu a inkubované pri teplote 37°C počas 18 hodín. Po inkubácií je zmeraná veľkosť inhibičných zón, pričom priemerný výsledok je získaný súčtom nameraných hodnôt jednotlivých meraní vydelených počtom testovaných platní. V príklade uskutočnenia sú použité odčítané hodnoty merania z troch platní.The prepared broth culture of Lactobacillus plantarum LS / 07 is incubated for approximately 18 hours and measured to a turbidimetric value of 0.5 McFarland, corresponding to 1.5x10 8 microorganisms. A broth culture of 0.050 ml volume is removed from the broth culture by a micropipette and applied to a 4 mm well (cut into the center of the plate) of selective LS agar. The inoculated Petri dishes are placed in incubation flasks (GASPAK US Patent) and incubated in a biological thermostate at 37 ° C for 48 hours. After 48 hours, the agar plates are superimposed on the surface of the agar plates: peptone 0.5g, yeast autolysate 1g, glucose 1g, agar 1.8g, 100ml distilled water, pH 6.9, in a volume of 3ml, in which it is dissolved 0.3 ml broth containing night culture of the overgrown Escherichia coli serotype O6: K2: H1 ATC 1938 CNTC Eck 63/59 strain. After application of the strain, Petri dishes are placed in a thermostat and incubated at 37 ° C for 18 hours. After incubation, the size of the inhibition zones is measured, the average result being obtained by summing the measured values of the individual measurements divided by the number of plates tested. In the exemplary embodiment, readings from three plates are used.
Molekulovo genetická detekcia kmeňa Lactobacillus plantarum LS/07 CCM 7766:Molecular genetic detection of Lactobacillus plantarum strain LS / 07 CCM 7766:
Na molekulovo genetickú detekciu izolovaného kmeňa je použitá vysoko citlivá a špecifická metóda techniky polymerázovej reťazovej reakcie (PCR). PCR zmes, použité špecifické primery, amplifikácia templátu a elektroforéza v agarózovom géli sú uskutočnené podľa literárnych zdrojov (Berthier a Ehrlich. 1998). Amplifikačné PCR produkty sú ofarbené etídium bromidom a detegované ultrafialovým svetlom, pričom je získaný špecifický PCR produkt o veľkosti 265 bázových párov.A highly sensitive and specific method of polymerase chain reaction (PCR) technique is used for molecular genetic detection of the isolated strain. The PCR mixture, the specific primers used, template amplification and agarose gel electrophoresis are performed according to literature sources (Berthier and Ehrlich. 1998). The amplification PCR products are stained with ethidium bromide and detected by ultraviolet light, yielding a specific 265 base pair PCR product.
Stanovenie organických kyselín:Determination of organic acids:
Koncentrácie organických kyselín (kyselina mliečna a octová) sú stanovené pomocou prístroja (CS ISOTACHOPHORETIC ANALYSER) technikou kapilárnej izotachoforézy. Izolovaný kmeň Lactobacillus plantarum LS/07 CCM 7766 najviac produkoval kyselinu mliečnu v porovnaní s kyselinou octovou. Hodnoty sú namerané po 24 hodinovej (kyselina mliečna - 78,66 mmol/1, kyselina octová - 7,91 mmol/1) a 48 hodinovej (kyselina mliečna - 226,31 mmol/1, kyselina octová 95,44 mmol/1) inkubácií v termostate pri teplote 37°C .The concentrations of organic acids (lactic and acetic acid) are determined using a CS ISOTACHOPHORETIC ANALYSER instrument using capillary isotachophoresis techniques. The isolated Lactobacillus plantarum strain LS / 07 CCM 7766 produced the most lactic acid compared to acetic acid. Values are measured after 24 hours (lactic acid - 78.66 mmol / l, acetic acid - 7.91 mmol / l) and 48 hours (lactic acid - 226.31 mmol / l, acetic acid 95.44 mmol / l) incubation in a thermostate at 37 ° C.
Stanovenie enzýmovej aktivityDetermination of enzyme activity
Na štúdium enzýmovej aktivity kmeňa Lactobacillus plantarum LS/07 CCM 7766 je použitý komerčný kit API ZYM (BIOMÉRIEUX®SA, Francúzko). Z dosiahnutých výsledkov je zistené, že testovaný kmeň neprodukuje enzým β-glukuronidázu, ktorý sa považuje za dôležitý marker v procese vývoja karcinogenézy kolorektálného karcinómu.A commercial API ZYM kit (BIOMERIEUX®SA, France) was used to study the enzyme activity of the Lactobacillus plantarum strain LS / 07 CCM 7766. From the results obtained, it is found that the test strain does not produce the enzyme β-glucuronidase, which is considered to be an important marker in the development of colorectal cancer carcinogenesis.
Experimenty na animálnych modelochExperiments on animal models
Izolovaný kmeň Lactobacillus plantarum LS/07 CCM 7766 je použitý v in vivo experimentálnych štúdiách na laboratórnych potkanoch. Experimentálne práce na laboratórnych zvieratách majú veľký význam pri štúdiu účinku izolovaných nových kmeňov laktobacilov v podmienkach in vivo. Nami izolovaný kmeň Lactobacillus plantarum LS/07 CCM 7766 zo stolice zdravého človeka sme použili v pokuse s laboratórnymi potkanmi. Potkany použité v pokuse (počet 51) pochádzali z (5 ff f. it iThe isolated strain Lactobacillus plantarum LS / 07 CCM 7766 is used in in vivo experimental studies in rats. Experimental work in laboratory animals is of great importance in studying the effect of isolated new strains of lactobacilli under in vivo conditions. The isolated strain of Lactobacillus plantarum LS / 07 CCM 7766 from the faeces of a healthy human was used in an experiment with rats. The rats used in the experiment (51) were from (5 ff f. It i
kmeňa Sprague Dawley boli vo veku štyroch mesiacov. Kmeň sme pripravovali denne čerstvý v rastovom bujóne pre laktobacily (priemerná koncentrácia 2,88 x 109 CFU/ml) a podávali sme ich denne potkanom 5 krát v týždni. Raz týždenne (tretí až siedmy týždeň) sme potkanom injekčné aplikovali chemický prokarcinogén 1,2 dymetylhidrazín-dyhidrochlorid (1,2 DMH). Počas celého experimentu (24 týždňov) sme nezaznamenali žiadne klinické zmeny (hnačky, úhyn) u pokusných skupín zvierat. Tým sa potvrdilo, že kmeň je bezpečný. Dosiahnuté výsledky pokusu potvrdzujú, že modulačný efekt použitého kmeňa sa prejavil zvýšením počtu baktérií mliečneho kvasenia a znížením počtu koliformných mikroorganizmov v črevnom obsahu hrubého čreva. Aplikácia kmeňa mala vplyv nielen na črevnú mikroflóru, ale aj na metabolické aktivity s priaznivým účinkom na lipidový profil. Sú zaznamenané pozitívne zmeny imunitných a histologických laboratórnych vyšetrení pokusných zvierat.Sprague Dawley were four months old. The strain was prepared daily in lactobacilli growth broth (average concentration 2.88 x 10 9 CFU / ml) and administered daily to rats 5 times a week. Once weekly (third to seven weeks), we injected rats with the chemical procarcinogen 1,2 dymethylhidrazine dyhidrochloride (1.2 DMH). During the whole experiment (24 weeks) no clinical changes (diarrhea, mortality) were observed in the experimental groups of animals. This confirmed that the strain is safe. The results of the experiment confirm that the modulating effect of the strain used was manifested by an increase in the number of lactic acid bacteria and a decrease in the number of coliform microorganisms in the intestinal contents of the colon. The application of the strain had an effect not only on the intestinal microflora, but also on metabolic activities with a beneficial effect on the lipid profile. Positive changes in immune and histological laboratory examinations of experimental animals are noted.
Priemyselná využiteľnosťIndustrial usability
Vyššie uvedené účinky kmeňa Lactobacillus Plantarum LS/07 CCM 7766 ho predurčujú na využitie pri modulácii črevnej mikroflóry ako aj pri prevencii zápalových chronických chorôb ľudí.The above-mentioned effects of the Lactobacillus Plantarum strain LS / 07 CCM 7766 predetermine it for use in the modulation of the intestinal microflora as well as in the prevention of inflammatory chronic human diseases.
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Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SK54-2015A SK542015A3 (en) | 2015-07-30 | 2015-07-30 | Strains Lactobacillus Plantarum LS/07 CCM 7766 and their use |
| CZ2018-101A CZ2018101A3 (en) | 2015-07-30 | 2016-07-30 | Microorganism strains Lactobacillus Plantarum LS/07 CCM 7766, and a product containing these strains |
| SK50011-2018A SK500112018A3 (en) | 2015-07-30 | 2016-07-30 | Microorganism strains Lactobacillus Plantarum LS/07 CCM 7766, product containing this strains |
| PCT/IB2016/054600 WO2017017662A1 (en) | 2015-07-30 | 2016-07-30 | Microorganism strain lactobacillus plantarum ls/07 ccm 7766, method of its use and food product |
| CZ2018-34733U CZ33363U1 (en) | 2015-07-30 | 2016-07-30 | A food product or nutritional supplement containing the Lactobacillus plantarum strain |
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| Application Number | Priority Date | Filing Date | Title |
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| SK54-2015A SK542015A3 (en) | 2015-07-30 | 2015-07-30 | Strains Lactobacillus Plantarum LS/07 CCM 7766 and their use |
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| SK54-2015A SK542015A3 (en) | 2015-07-30 | 2015-07-30 | Strains Lactobacillus Plantarum LS/07 CCM 7766 and their use |
| SK50011-2018A SK500112018A3 (en) | 2015-07-30 | 2016-07-30 | Microorganism strains Lactobacillus Plantarum LS/07 CCM 7766, product containing this strains |
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| CZ (2) | CZ2018101A3 (en) |
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| GB2369777B (en) * | 2000-10-05 | 2004-10-27 | St Ivel Ltd | Food products with antimicrobial lactic acid bacteria |
| EP1997499A1 (en) * | 2007-05-31 | 2008-12-03 | Puleva Biotech, S.A. | Mammalian milk microorganisms, compositions containing them and their use for the treatment of mastitis |
| KR101255050B1 (en) * | 2009-07-14 | 2013-04-16 | 씨제이제일제당 (주) | Novel lactobacillus plantarum and compositions comprising the same |
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| CZ33363U1 (en) | 2019-11-12 |
| CZ2018101A3 (en) | 2018-10-31 |
| WO2017017662A4 (en) | 2017-03-16 |
| WO2017017662A1 (en) | 2017-02-02 |
| SK500112018A3 (en) | 2018-12-03 |
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