Functional properties, persistence, safety and efficacy of potential probiotic lactobacilli
Date
2012-05-21
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Abstract
Vähenenud sündimus ja pikem eluiga on suurendanud krooniliste haiguste levimust, mille patogeneesis on olulisel kohal oksüdatiivne stress. Samas on jätkuvalt olulisel kohal ka erinevad infektsioonid. Seetõttu vajab elanikkond haiguste ennetamiseks erinevaid meetmeid, sealhulgas tervislikumat toitumist. Mõned piimhappebakterid (sh laktobatsillid ja bifidobakterid) omavad antioksüdatiivset aktiivsust, kuid kahjuks pole selliste omadustega piimhappebaktereid kasutatud funktsionaalse toiduna, et vähendada oksüdatiivset stressi inimorganismis ja sellest tulenevalt mõjutada haiguste kulgu.
Probiootikum on inimeselt pärinev elus mittepatogeenne mikroob, mille manustamine on tervisele kasulik. Kuigi probiootikume peetakse üldiselt tervisele ohututeks, on iga potentsiaalset probiootikumi vaja põhjalikult testida. Vajalik on tagada probiootikumi tarbimise ohutus, püsivus seedetraktis ning probiootikumi funktsionaalsete omaduste avaldumine inimorganismis.
Käesoleva töö eesmärkideks olid: 1) määrata seitsme kommertsiaalsete probiootikumi entero- ja uropatogeenide vastast antagonistlikku toimet mikroaeroobses või anaeroobses keskkonnas, mis sarnaneb vastavale sooletrakti niššile ning määrata nende probiootikumide antioksüdatiivset aktiivsust in vitro katsetes; 2) hinnata sünbiootikumi, mis sisaldab kolme probiootikumi tüve (L. fermentum ME-3, L. paracasei 8700:2 ja B. longum 46) ja prebiootikumi raftiloos P95, tarbimise mõju vere oksüdatiivse stressi näitajale (oxLDL, BDC-LDL) tervetel inimestel; 3) hinnata TÜ mikrobioloogia instituudi Inimese Mikrobioota Biopanga laktobatsillide kollektsiooni tüvede koloniseerimisomadusi in vitro eksperimentidega, mis matkivad sooles olevaid tingimusi; 4) määrata L. plantarum TENSIA ja potentsiaalsete probiootiliste tüvede ohutust in vitro, loomkatsetes ja tervetel vabatahtlikel; 5) määrata eespool mainitud laktobatsilli tüvede elulemust ja püsivust tervete vabatahtlike seedetraktis.
Töö käigus selgus, et:
1. Seitsme kommertsiaalse probiootikumi antimikroobne aktiivsus oli erinevates keskkondades erinev ning sõltus konkreetsest probiootikumist ja patogeensest mikroobist. L. fermentum ME-3 omas kõrget antioksüdatiivset aktiivsust võrreldes teiste kommertsiaalsete probiootikumidega in vitro eksperimentides.
2. Sünbiootikumi tarbimine langetas süsteemset oksüdatiivset stressi, mis väljendus veres madala tihedusega lipoproteiinide dieenkonjugaatide (BDC-LDL) sisalduse vähenemises.
3. In vitro eksperimentidega, kus hinnatakse laktobatsillide koloniseerimisvõimet kirjeldavaid omadusi (autoagregatsioon, happe- ja sapitaluvus), on võimalik kindlaks määrata kõige sobivamaid potentsiaalseid probiootilisi laktobatsille inimuuringuteks.
4. Tuginedes in vitro ohutuse testidele (hemolüütilise aktiivsuse ja ülekantava antibiootikumresistentsuse puudumine) ja loomkatsetele olid viis potentsiaalset probiootikumi ja L. plantarum TENSIA ohutud.
5. Kapsuleeritud potentsiaalsete probiootiliste laktobatsillide tarbimine kõrgetes doosides ja probiootikumina patenteeritud L. plantarum TENSIA tarbimine ei põhjustanud raskeid seedetrakti vaevusi ega ka olulisi muutusi verenäitajates. Seega on uuritud tüvede tarbimine tervetel inimestel ohutu, mis on aluseks nende kasutamiseks funktsionaalse toiduna.
6. Potentsiaalsed probiootikumid ja L. plantarum TENSIA säilitasid eluvõime pärast sooletrakti läbimist, kuid tüvede püsivus sooletraktis erines tüveti ning kestis kolmest päevast kuni 5 nädalani pärast tüvede tarbimise lõpetamist.
The declined birth rates and longer life expectancy have led to increased prevalence of chronic disorders. Oxidative stress play important role in pathogenesis of these diseases. Moreover, several infections are still emerging. All this requires population based new preventive approaches, namely infection control and improved nutrition. Some lactic acid bacteria (including bifidobacteria and lactobacilli) possess antioxidative activity, but unfortunately, there are no lactobacilli with antioxidative properties used as functional food for reduction of oxidative stress in the human body, and consequently influence the disease course. Probiotics are live non-pathogenic microorganisms mostly of human origin which, when administered in adequate numbers confer a health benefit on the host. Although probiotics are generally considered safe, it is necessary to thoroughly test each potential probiotic. It is necessary to ensure the safety of consumption of probiotics and survival and persistence in gastrointestinal tract. However, studies focusing on expression of functional properties of a probiotic on healthy persons are lacking. The aims of the present study were: 1) to evaluate the in vitro antagonistic activity of some commercial probiotics against various gastric, entero- and urinary pathogens residing in distinct niches throughout the GI tract and to assess the antioxidative activity of the above mentioned probiotic strains; 2) to establish the efficacy of consumption of a synbiotic product containing selected commercial strains (L. fermentum ME-3, L. paracasei 8700:2, B. longum 46) and raftilose P95 in lowering the oxidative stress markers of blood (oxLDL, BDC-LDL) in healthy persons; 3) to screen the putative probiotic Lactobacillus strains of the intestinal tract for their colonizing potential using in vitro tests mimicking the GI tract conditions; 4) to assess the safety of the probiotic L. plantarum TENSIA (DSM 21380) and of previously selected six Lactobacillus strains using in vitro, animal experiments and in human studies; 5) to evaluate the survival and persistence of selected putative probiotic candidates and of the probiotic strain TENSIA in the GI tract of healthy volunteers after consumption. Current study revealed that: 1) Seven commercial probiotic strains expressed in vitro variable antimicrobial activity against different pathogens. L. fermentum ME-3 expressed the highest antioxidative activity among the tested 7 commercial strains in in vitro experiments. 2) Consumption of the synbiotic containing three commercial strains (L. paracasei 8700:2, B. longum 46 and L. fermentum ME-3) with prebiotic raftilose P95 decreased systemic oxidative stress manifested in the reduction of the circulating BDC-LDL values of the blood of volunteers, proving the antioxidative efficacy of the synbiotic. 3) Screening for the colonizing potential of Lactobacillus sp. (auto-aggregation, acid and bile tolerance) by in vitro experiments allowed selecting the most promising candidates for clinical trials with putative probiotic strains. 4) According to results of in vitro testing and animal experiments of L. plantarum TENSIA and of the putative 5 probiotic strains (absence of haemolytic activity, transferable antibiotic resistance and translocation) were considered safe for further human application. 5) The administration of the probiotic cheese comprising L. plantarum TENSIA and high doses of the capsulated Lactobacillus multistrains did not result in any serious adverse gastrointestinal complaints and/or abnormal values of blood indices. Thus, the safety of the capsulated Lactobacillus strains and the patented L. plantarum TENSIA strain, elaborated in probiotics was approved in clinical trials with healthy persons, as a basis for its application as functional food. 6) The putative probiotic strains tolerated well the passage through the GI tract. The persistence of a particular strain varied from 3 days up to 5 weeks after cessation of the consumption Lactobacillus strains.
The declined birth rates and longer life expectancy have led to increased prevalence of chronic disorders. Oxidative stress play important role in pathogenesis of these diseases. Moreover, several infections are still emerging. All this requires population based new preventive approaches, namely infection control and improved nutrition. Some lactic acid bacteria (including bifidobacteria and lactobacilli) possess antioxidative activity, but unfortunately, there are no lactobacilli with antioxidative properties used as functional food for reduction of oxidative stress in the human body, and consequently influence the disease course. Probiotics are live non-pathogenic microorganisms mostly of human origin which, when administered in adequate numbers confer a health benefit on the host. Although probiotics are generally considered safe, it is necessary to thoroughly test each potential probiotic. It is necessary to ensure the safety of consumption of probiotics and survival and persistence in gastrointestinal tract. However, studies focusing on expression of functional properties of a probiotic on healthy persons are lacking. The aims of the present study were: 1) to evaluate the in vitro antagonistic activity of some commercial probiotics against various gastric, entero- and urinary pathogens residing in distinct niches throughout the GI tract and to assess the antioxidative activity of the above mentioned probiotic strains; 2) to establish the efficacy of consumption of a synbiotic product containing selected commercial strains (L. fermentum ME-3, L. paracasei 8700:2, B. longum 46) and raftilose P95 in lowering the oxidative stress markers of blood (oxLDL, BDC-LDL) in healthy persons; 3) to screen the putative probiotic Lactobacillus strains of the intestinal tract for their colonizing potential using in vitro tests mimicking the GI tract conditions; 4) to assess the safety of the probiotic L. plantarum TENSIA (DSM 21380) and of previously selected six Lactobacillus strains using in vitro, animal experiments and in human studies; 5) to evaluate the survival and persistence of selected putative probiotic candidates and of the probiotic strain TENSIA in the GI tract of healthy volunteers after consumption. Current study revealed that: 1) Seven commercial probiotic strains expressed in vitro variable antimicrobial activity against different pathogens. L. fermentum ME-3 expressed the highest antioxidative activity among the tested 7 commercial strains in in vitro experiments. 2) Consumption of the synbiotic containing three commercial strains (L. paracasei 8700:2, B. longum 46 and L. fermentum ME-3) with prebiotic raftilose P95 decreased systemic oxidative stress manifested in the reduction of the circulating BDC-LDL values of the blood of volunteers, proving the antioxidative efficacy of the synbiotic. 3) Screening for the colonizing potential of Lactobacillus sp. (auto-aggregation, acid and bile tolerance) by in vitro experiments allowed selecting the most promising candidates for clinical trials with putative probiotic strains. 4) According to results of in vitro testing and animal experiments of L. plantarum TENSIA and of the putative 5 probiotic strains (absence of haemolytic activity, transferable antibiotic resistance and translocation) were considered safe for further human application. 5) The administration of the probiotic cheese comprising L. plantarum TENSIA and high doses of the capsulated Lactobacillus multistrains did not result in any serious adverse gastrointestinal complaints and/or abnormal values of blood indices. Thus, the safety of the capsulated Lactobacillus strains and the patented L. plantarum TENSIA strain, elaborated in probiotics was approved in clinical trials with healthy persons, as a basis for its application as functional food. 6) The putative probiotic strains tolerated well the passage through the GI tract. The persistence of a particular strain varied from 3 days up to 5 weeks after cessation of the consumption Lactobacillus strains.
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Keywords
probiootikumid, antioksüdantkaitse, oksüdatiivne stress, probiotics, antioxidant defence, oxidative stress