Preživljavanje Streptococcus thermophilus, Lactobacillus acidophilus i Bifidobacterium spp. tijekom skladištenja fermentiranog mlijeka

The viable counts of Lactobacillus acidophilus (L. acidophilus), Bifidobacterium ssp. and Streptococcus salivarius subsp. thermophilus (Str. thermophilus) and the survival of these bacteria in commercial fermented milks during the four weeks storage at 7 ± 1 °C were investigated. The titratable acidity and pH value of samples were measured as well. The fermented milk samples in original packaging were supplied by three dairies (coded A, B and C) one-day post production. First analysis of samples were done 24 hours after manufacture. During storage, samples were tested in seven days intervals. Str. thermophilus populations were in the range of 3 x 108 to 9 x 108 cfu/g and survived at levels greater than 108 cfu/g till the end of the storage for all fermented milk samples. The highest counts of L. acidophilus were found in samples A, but also in samples B while in C remained above the recommended limit of one million viable cells per gram throughout the storage period. The most variable was the count of Bifidobacterium spp. Mean values for the viable count one day post-production ranged from 4.6x102 to 2.3x106/g.Određivanje broj Lactobacillus acidophilus (L. acidophilus), Bifidobacterium ssp. i Streptococcus salivarius subsp. thermophilus (Str. thermophilus) u komercijalnim fermentiranim uzorcima mlijeka te preživljavanje navedenih bakterija tijekom skladištenja kroz 4 tjedna pri temperaturi od 7± 1 °C. Također je provedeno i određivanje titracijske kiselosti i pH vrijednosti uzoraka. Fermentirani uzorci mlijeka u originalnoj ambalaži dobavljeni su iz tri mljekare (označene sa A, B i C) nakon prvog dana proizvodnje. Prva analiza provedena je nakon 24 sata od završetka proizvodnje. Tijekom skladištenja uzorci su analizirani u vremenskom intervalu od 7 dana. Populacija Str. thermophilus iznosila je od 3 x 108 do 9 x 108 cfu/g, a nivo preživljavanja na kraju skladištenja bio je veći od 108 cfu/g, za sve fermentirane uzorke mlijeka. Najveći broj L. acidophilus, tijekom skladištenja, određen je u uzorcima A, zatim u B dok se u C zadržao iznad preporučene vrijednosti od jedan milijun živih stanica po gramu. Najveće promjene uočene su u broju Bifidobacterium spp. Tako je srednja vrijednost živih stanica, nakon prvog dana proizvodnje, iznosila od 4.6 x 102 do 2.3 x 106/g

Antimicrobial Resistance of Common Zoonotic Bacteria in the Food Chain: An Emerging Threat

Antimicrobial resistance in the food chain is currently a subject of a major interest. The excessive use or rather misuse of antimicrobials coupled with a poor hygiene in the food production chain has led to a rise of resistant zoonotic bacteria, commonly transmitted by food. They pose a serious threat to human health. Campylobacteriosis is the leading bacterial food-borne illness and most commonly reported zoonosis in humans in the European Union for more than a decade. Salmonellosis is most frequently diagnosed in food-borne outbreaks. Fluoroquinolones are considered as critically important for treatment of severe cases of both zoonoses in humans. Due to an extremely prevalent resistant isolates, especially from broilers and meat, also the treatment of human Campylobacter infections with fluoroquinolones has become compromised. Salmonella isolates from poultry and poultry meat tend to be highly resistant to fluoroquinolones as well. Beside the resistance to this group of antibiotics, the threat of multiple drug resistant (MDR) Campylobacter and Salmonella strains is discussed in the light of most recent reports of animal, food and human clinical surveillance systems

Utjecaj antibiotika na bakterije mliječne kiseline probavnog sustava

Lactic acid bacteria (LAB) are common inhabitants of the gastrointestinal (GI) tract and have important role in maintaining the equilibrium of GI flora, which can be influenced by various factors like diets, antimicrobials and stress. Minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) of 6 antibiotics, commonly used in human medicine for 8 selected lactobacilli strains were determined by macrodilution and microdilution methods in liquid media and by diffusion method on agar plates. The effects of Penicillin G and Ampicillin on intestinal LAB were tested in vivo on mice as well. Lactobacilli were sensitive to Penicillin G, (penicillines and their derivatives) and Erythromycin (macrolides) by in vitro testing. Clyndamycin (pyranosid) showed moderate inhibitory effect. All lactobacilli strains were resistant to Kanamycin and Neomycin (aminoglycosides), while L. salivarius IM 124 has shown extra resistance to Erythromycin and Clyndamycin. The influence of orally administered Ampicillin showed no significant influence on LAB count in mice faeces. The effect of Penicillin G on mice LAB total count was significant, while no effect of orally administered lactobacilli was determined.Bakterije mliječne kiseline (BMK) su uobičajeni sudionici crijevne mikroflore ljudi i životinja. One imaju važnu ulogu u održavanju ravnoteže gastrointestinalne (GI) flore, na koju mogu utjecati različiti faktori poput prehrane (hrane) antimikrobnih supstancija i stresa. Pomoću makrodilucijske i mikrodilucijske metode u tekućoj podlozi te metode difuzije na agar pločama, određene su minimalne inhibicijske koncentracije (MIC) i minimalne baktericidne koncentracije (MBC) šest (6) antibiotika koji se svakodnevno upotrebljavaju u humanoj medicini za 8 sojeva BMK. Učinak penicilina G i ampicilina na BMK probavnog sustava testiran je također in vivo na laboratorijskim miševima. Laktobacili su se pokazali osjetljivim na penicilin G, na ampicilin (derivati penicilina) i na eritromicin (makrozidni antibiotik) prilikom testiranja in vitro. Klindamicin (piranozidni antibiotik) je pokazao umjereni inhibicijski učinak. Svi sojevi BMK pokazali su se otpornim na kanamicin i neomicin (aminoglikozidni antibiotici). Od svih testiranih BMK posebno se otpornim na eritromicin i klindamicin pokazao L. salivarius IM 124. Učinak oralno primjenjenog ampicilina, na broj BMK u stolici miševa nije statistički dokazan. Utjecaj penicilina G na broj BMK kod miševa pokazao se signifikantnim, ali to nije bio slučaj s oralno primijenjenim BMK

Adhesion of Two Lactobacillus gasseri Probiotic Strains on Caco-2 Cells

Previous in vitro and in vivo studies showed that two human isolates of Lactobacillus gasseri, LF221 and K7 are able to survive the passage through the gastrointestinal tract and to colonise intestines of pigs at least temporarily. The aim of this study was to examine the adhesion ability of LF221 and K7 strains to Caco-2 cells. Adhesion of lactobacilli from early stationary growth phase was examined at two pH values of DMEM buffer (4.5 and 7). Lactobacillus rhamnosus GG, a widely used strain with clinical evidences of its efficiency, served as a positive control. The number of lactobacilli added to each well was found to be crucial in the adhesion assay. When added, lactobacilli were in range of 2.5 · 106 to 2.5 · 108 cfu/well, the linear correlation between the number of adhered cells (log cfu) and the number of added cells (log cfu) was found for all three strains (R2 > 0.99) at both pH values (4.5 and 7). At the highest concentration of added K7 and GG cells tested (app. 109 cfu/well), the efficiency of adhesion was reduced. pH value of the medium strongly affected the adhesion, which was promoted in acidic conditions (pH=4.5). The adhesion of K7 strain was slightly weaker compared to GG strain at both pH values, while at pH=4.5 the adhesion of LF221 strain was even better than GG adhesion, at least at lower concentration of lactobacilli. The direct comparison of these strains was possible by regression analysis. At lower concentration of lactobacilli (2.5 · 106), the best efficiency of adhesion (% of adhered bacteria) was observed for the strain LF221, reaching the values of 7.8 and 1.9 % at pH=4.5 and 7, respectively, while at higher lactobacilli concentration the ration of adhesion was higher for GG strain (3.3 % at pH=4.5). In conclusion, strains LF221 and K7 were demonstrated to be adhesive, especially in acidic conditions. The level of adhesion of K7 and GG strains positively correlates with the number of added lactobacilli only up to the certain point when the saturation of potential binding sites on Caco-2 cells probably occurs. As the adhesion to Caco-2 cell cultures alone does not guarantee the adhesion of examined strains in vivo, additional studies on experimental animals are in progress and human clinical studies are planned as well

Kompetitivna prednost bakteriocinogenih sojeva konzorcija bakterija mliječne kiseline izoliranih iz sira proizvedenog od sirovog mlijeka

The presence of gene determinants for different bacteriocins has been already demonstrated in traditional Slovenian types of raw milk cheeses ‘Tolminc’ and ‘Kraški’. These genes were present also in the cultivable microbiota. In this research the aim was to establish how the presence of gene determinants for bacteriocins in microbial consortia is reflected in its antimicrobial activity. In addition, one of the goals was to determine whether the strains that carry gene determinants for bacteriocins have any competitive growth advantage in microbial population. Microbial consortium of ‘Tolminc’ cheese was propagated in milk and examined at the end of propagation its antimicrobial activity and the presence of gene determinants for bacteriocins. Comparison of the results obtained before and after propagation leaded to the conclusion that most of the strains possessing gene determinants for bacteriocins were unable to persist during propagation. The strains which did persist during propagation carried gene determinants for enterocins P, L50B and cytolysin. Antimicrobial activity of consortium before and after propagation was not substantially different and cannot be attributed to any of detected bacteriocins.Prisutnost genskih determinanti za stvaranje različitih vrsta bakteriocina već je ranijim istraživanjima utvrđena u slovenskim ovčjim sirevima “Tolminac” i “Kraški sir” koji se proizvode na tradicionalan način. Isti geni za izoliranu mikrobnu populaciju (konzorcij) također su potvrđeni i u kultiviranim uvjetima. U ovom radu pokušalo se utvrditi kako se prisutnost tih genskih determinanti u mikrobnom konzorciju odražava na njegovu antimikrobnu aktivnost. Osim toga, utvrđivao se i utjecaj bakterijskih sojeva koji nose gene za stvaranje bakteriocina na kompetitivnost rasta u mješovitoj populaciji. Mikrobni konzorcij izoliran iz sira “Tolminac” propagiran je u mlijeku 10 dana, nakon čega je utvrđena antimikrobna aktivnost kulture i prisutnost genskih determinanti za stvaranje bakteriocina. Usporedbom rezultata za sposobnost stvaranja bakteriocina prije i nakon precjepljivanja u mlijeku, utvrđen je gubitak te sposobnosti za većinu izoliranih sojeva. Sojevi koji su izdržali ponovljena precjepljivanja u mlijeku nosili su genske determinante za bakteriocine: enterocin P, enterocin L50B i citolizin. Antimikrobna aktivnost konzorcija prije i nakon precjepljivanja nije se značajno razlikovala te se ne može pripisati niti jednom od u ovom pokusu potvrđenih bakteriocina

Preživljavanje Streptococcus thermophilus, Lactobacillus acidophilus i Bifidobacterium spp. tijekom skladištenja fermentiranog mlijeka

The viable counts of Lactobacillus acidophilus (L. acidophilus), Bifidobacterium ssp. and Streptococcus salivarius subsp. thermophilus (Str. thermophilus) and the survival of these bacteria in commercial fermented milks during the four weeks storage at 7 ± 1 °C were investigated. The titratable acidity and pH value of samples were measured as well. The fermented milk samples in original packaging were supplied by three dairies (coded A, B and C) one-day post production. First analysis of samples were done 24 hours after manufacture. During storage, samples were tested in seven days intervals. Str. thermophilus populations were in the range of 3 x 108 to 9 x 108 cfu/g and survived at levels greater than 108 cfu/g till the end of the storage for all fermented milk samples. The highest counts of L. acidophilus were found in samples A, but also in samples B while in C remained above the recommended limit of one million viable cells per gram throughout the storage period. The most variable was the count of Bifidobacterium spp. Mean values for the viable count one day post-production ranged from 4.6x102 to 2.3x106/g.Određivanje broj Lactobacillus acidophilus (L. acidophilus), Bifidobacterium ssp. i Streptococcus salivarius subsp. thermophilus (Str. thermophilus) u komercijalnim fermentiranim uzorcima mlijeka te preživljavanje navedenih bakterija tijekom skladištenja kroz 4 tjedna pri temperaturi od 7± 1 °C. Također je provedeno i određivanje titracijske kiselosti i pH vrijednosti uzoraka. Fermentirani uzorci mlijeka u originalnoj ambalaži dobavljeni su iz tri mljekare (označene sa A, B i C) nakon prvog dana proizvodnje. Prva analiza provedena je nakon 24 sata od završetka proizvodnje. Tijekom skladištenja uzorci su analizirani u vremenskom intervalu od 7 dana. Populacija Str. thermophilus iznosila je od 3 x 108 do 9 x 108 cfu/g, a nivo preživljavanja na kraju skladištenja bio je veći od 108 cfu/g, za sve fermentirane uzorke mlijeka. Najveći broj L. acidophilus, tijekom skladištenja, određen je u uzorcima A, zatim u B dok se u C zadržao iznad preporučene vrijednosti od jedan milijun živih stanica po gramu. Najveće promjene uočene su u broju Bifidobacterium spp. Tako je srednja vrijednost živih stanica, nakon prvog dana proizvodnje, iznosila od 4.6 x 102 do 2.3 x 106/g

In-situ inhibicija Staphylococcus aureus bakterijama mliječne kiseline iz dva tradicionalna slovenska sira proizvedena od sirovog mlijeka

Bacteriocin(s) producing lactic acid bacteria naturally present in traditional cheeses represent an inexhaustive pool of microbes with safeguarding potential. Some bacteriocins produced by cheese lactic acid bacteria were already described as successful anti-staphylococcal agents. The presence of genes for bacteriocins with potential anti-staphylococcal activity was also demonstrated in two Slovenian traditional raw milk cheeses, “Tolminc” and “Kraški ovčji sir”. Same bacteriocin genes were also detected in viable lactic acid bacteria consortia’s isolated from “bacteriocin positive cheeses” on Rogosa, M17 and CATC agar media. The aim of the research was to elucidate whether or not this particular cheese consortia, in which bacteriocin genes were detected, actually exhibit anti-staphylococcal activity in milk and/or cheese. For this purpose different cheese consortia were selected in relation to versatility of detected bacteriocin genes and used to perform challenge tests against Staphylococcus aureus in milk and cheese. In milk following the time/temperature regime of traditional cheese production all cheese consortia effectively inhibited growth of Staphylococcus aureus in the range of app. 2 to 3 log. In cheese the inhibition of staphylococci was less pronounced but still evident since inhibition of app. 1.5 log was detected. Sole inhibition by lactic acid production was ruled out while linking inhibition directly to bacteriocin production would take some additional work.Bakterije mliječne kiseline (BMK), prirodno prisutne u tradicionalnim sirevima, predstavljaju neograničen izvor mikroba sa zaštitnim svojstvima. Za neke bakteriocine, koje proizvode BMK iz sira, antistafilokokna svojstva već su poznata. Prisutnost gena za bakteriocine s antistafilokoknim svojstvima potvrđena je u dva tradicionalna slovenska sira iz sirovog mlijeka; to su sir “Tolminc” i “Kraški ovčji sir”. Prisutnost istih bakteriocina potvrđena je i kod bakterija mliječne kiseline, koji su izolirali iz tih sireva na agar podlogama Rogosa, M17 i CATC. Cilj je bio provjeriti da li izolirani sojevi bakterija mliječne kiseline, koji nose gene za bakteriocine, zaista pokazuju antistafilokokna svojstva u mlijeku i/ili siru. Na temelju prisutnih gena za bakteriocine ispitana je antimikrobna aktivnost združene kulture bakterija mliječne kiseline prema bakteriji Staphylococcus aureus u mlijeku i siru. U mlijeku kojeg smo podvrgnuli vremensko-temperaturnom režimu proizvodnje tradicionalnih sireva, združena kultura bakterija mliječne kiseline inhibirala je rast bakterije Staphylococcus aureus u rasponu od 2 do 3 logaritamske jedinice. U siru je inhibicija stafilokoka bila nešto manje izražena, ali još uvijek očita sa 1,5 logaritamskih jedinica slabijim rastom S. aureus nego u kontrolnom siru. Učinak inhibicije izazvane mliječnom kiselinom bio je isključen, ali bi bilo potrebno izvesti još dodatna ispitivanja, da se utvrdi izravno inhibicijsko djelovanje bakteriocina na stafilokoke

Kompetitivna prednost bakteriocinogenih sojeva konzorcija bakterija mliječne kiseline izoliranih iz sira proizvedenog od sirovog mlijeka

The presence of gene determinants for different bacteriocins has been already demonstrated in traditional Slovenian types of raw milk cheeses ‘Tolminc’ and ‘Kraški’. These genes were present also in the cultivable microbiota. In this research the aim was to establish how the presence of gene determinants for bacteriocins in microbial consortia is reflected in its antimicrobial activity. In addition, one of the goals was to determine whether the strains that carry gene determinants for bacteriocins have any competitive growth advantage in microbial population. Microbial consortium of ‘Tolminc’ cheese was propagated in milk and examined at the end of propagation its antimicrobial activity and the presence of gene determinants for bacteriocins. Comparison of the results obtained before and after propagation leaded to the conclusion that most of the strains possessing gene determinants for bacteriocins were unable to persist during propagation. The strains which did persist during propagation carried gene determinants for enterocins P, L50B and cytolysin. Antimicrobial activity of consortium before and after propagation was not substantially different and cannot be attributed to any of detected bacteriocins.Prisutnost genskih determinanti za stvaranje različitih vrsta bakteriocina već je ranijim istraživanjima utvrđena u slovenskim ovčjim sirevima “Tolminac” i “Kraški sir” koji se proizvode na tradicionalan način. Isti geni za izoliranu mikrobnu populaciju (konzorcij) također su potvrđeni i u kultiviranim uvjetima. U ovom radu pokušalo se utvrditi kako se prisutnost tih genskih determinanti u mikrobnom konzorciju odražava na njegovu antimikrobnu aktivnost. Osim toga, utvrđivao se i utjecaj bakterijskih sojeva koji nose gene za stvaranje bakteriocina na kompetitivnost rasta u mješovitoj populaciji. Mikrobni konzorcij izoliran iz sira “Tolminac” propagiran je u mlijeku 10 dana, nakon čega je utvrđena antimikrobna aktivnost kulture i prisutnost genskih determinanti za stvaranje bakteriocina. Usporedbom rezultata za sposobnost stvaranja bakteriocina prije i nakon precjepljivanja u mlijeku, utvrđen je gubitak te sposobnosti za većinu izoliranih sojeva. Sojevi koji su izdržali ponovljena precjepljivanja u mlijeku nosili su genske determinante za bakteriocine: enterocin P, enterocin L50B i citolizin. Antimikrobna aktivnost konzorcija prije i nakon precjepljivanja nije se značajno razlikovala te se ne može pripisati niti jednom od u ovom pokusu potvrđenih bakteriocina

Karakterizacija bakteriocinogenih sojeva bakterija mliječne kiseline iz tradicionalnog slovenskog sira ‘Tolminc’

The purpose of this research was to examine traditional Slovenian ‘Tolminc’ cheese for the presence of lactic acid bacteria that produce several bacteriocins. The presence of gene determinants for different bacteriocins in this type of cheese and in the cultivable population of ‘Tolminc’ microbiota, have already been demonstrated, as well as its antimicrobial activity. Due to the difficulties in connecting the presence of gene determinants for bacteriocins with the observed antimicrobial activity it was decided to examine in this study the same features on the level of individual bacteriocinogenic strains. Like in previous results, enterococci and their bacteriocins prevailed in cheese microbial consortia. None of isolated strains inhibited growth of Staphylococcus aureus, while the other indicator strains were inhibited in a strain specific manner. Most of isolated strains carried gene determinants for cytolysin. On the basis of gene determinants for bacteriocins, antimicrobial activity, phenotyping by PhP (PhenePlateTM) system and PCR identification, some similarities found were among Enterococcus isolates.Cilj ovog rada bio je provjeriti prisutnost sojeva bakterija mliječne kiseline koji proizvode različite bakteriocine u tradicionalnom slovenskom siru ‘Tolminc’. Prisutnost genskih determinanti za pojedine bakteriocine u ovoj vrsti sira i izoliranoj populaciji mikrobiote sira ‘Tolminc’ već je bila prikazana, a također i njihova antimikrobna aktivnost. Zbog poteškoća pri povezivanju detektiranih genskih determinanti za bakteriocine i antimikrobne aktivnosti, odlučeno je u ovom radu analizirati ista svojstva i kod pojedinačnih bakteriocinogenih sojeva. Slično prethodnim istraživanjima, enterokoki i njihovi bakteriocini najbolje su bili zastupljeni. Nijedan od izoliranih sojeva nije inhibirao bakteriju Staphylococcus aureus, dok su ostale indikatorske mikroorganizme inhibirali različito. Većina sojeva nosila je genske determinante za bakteriocin citolizin. Na temelju genskih determinanti za bakteriocine, antimikrobne aktivnosti, fenotipizacije s PhP (PhenePlateTM) sistemom i identifikacije roda i vrste sojeva, mogu se naći neke sličnosti između Enterococcus sojeva

In-situ inhibicija Staphylococcus aureus bakterijama mliječne kiseline iz dva tradicionalna slovenska sira proizvedena od sirovog mlijeka

Bacteriocin(s) producing lactic acid bacteria naturally present in traditional cheeses represent an inexhaustive pool of microbes with safeguarding potential. Some bacteriocins produced by cheese lactic acid bacteria were already described as successful anti-staphylococcal agents. The presence of genes for bacteriocins with potential anti-staphylococcal activity was also demonstrated in two Slovenian traditional raw milk cheeses, “Tolminc” and “Kraški ovčji sir”. Same bacteriocin genes were also detected in viable lactic acid bacteria consortia’s isolated from “bacteriocin positive cheeses” on Rogosa, M17 and CATC agar media. The aim of the research was to elucidate whether or not this particular cheese consortia, in which bacteriocin genes were detected, actually exhibit anti-staphylococcal activity in milk and/or cheese. For this purpose different cheese consortia were selected in relation to versatility of detected bacteriocin genes and used to perform challenge tests against Staphylococcus aureus in milk and cheese. In milk following the time/temperature regime of traditional cheese production all cheese consortia effectively inhibited growth of Staphylococcus aureus in the range of app. 2 to 3 log. In cheese the inhibition of staphylococci was less pronounced but still evident since inhibition of app. 1.5 log was detected. Sole inhibition by lactic acid production was ruled out while linking inhibition directly to bacteriocin production would take some additional work.Bakterije mliječne kiseline (BMK), prirodno prisutne u tradicionalnim sirevima, predstavljaju neograničen izvor mikroba sa zaštitnim svojstvima. Za neke bakteriocine, koje proizvode BMK iz sira, antistafilokokna svojstva već su poznata. Prisutnost gena za bakteriocine s antistafilokoknim svojstvima potvrđena je u dva tradicionalna slovenska sira iz sirovog mlijeka; to su sir “Tolminc” i “Kraški ovčji sir”. Prisutnost istih bakteriocina potvrđena je i kod bakterija mliječne kiseline, koji su izolirali iz tih sireva na agar podlogama Rogosa, M17 i CATC. Cilj je bio provjeriti da li izolirani sojevi bakterija mliječne kiseline, koji nose gene za bakteriocine, zaista pokazuju antistafilokokna svojstva u mlijeku i/ili siru. Na temelju prisutnih gena za bakteriocine ispitana je antimikrobna aktivnost združene kulture bakterija mliječne kiseline prema bakteriji Staphylococcus aureus u mlijeku i siru. U mlijeku kojeg smo podvrgnuli vremensko-temperaturnom režimu proizvodnje tradicionalnih sireva, združena kultura bakterija mliječne kiseline inhibirala je rast bakterije Staphylococcus aureus u rasponu od 2 do 3 logaritamske jedinice. U siru je inhibicija stafilokoka bila nešto manje izražena, ali još uvijek očita sa 1,5 logaritamskih jedinica slabijim rastom S. aureus nego u kontrolnom siru. Učinak inhibicije izazvane mliječnom kiselinom bio je isključen, ali bi bilo potrebno izvesti još dodatna ispitivanja, da se utvrdi izravno inhibicijsko djelovanje bakteriocina na stafilokoke