Probiotic Concept - Probiotics as Food Supplements and Probiotics as Biotherapeutics

Svjetska zdravstvena kriza, posebice u liječenju infektivnih bolesti zbog alarmantnog stanja s antibiotičkom rezistencijom te niz kroničnih bolesti koje ugrožavaju ljudsko zdravlje, proizvelo je situaciju za uspostavljanje koncepta funkcionalne hrane još 1980. godine, u Japanu. Dakle, hrana je funkcionalna ako sadrži sastojke koji pozitivno djeluju na jednu ili više ciljanih funkcija u tijelu. Ciljno mjesto djelovanja probiotika, prebiotika, odnosno sinbiotika, kao funkcionalnih dodataka hrani, je gastrointestinalni sustav. Taj sustav je najveći imuno organ u ljudskom organizmu s aktivnom površinom od oko 300 m2, a sadrži vrlo kompleksnu mikrobnu populaciju (crijevnu mikrofloru) ukupne mase od 1,2 kg. Ovako golema mikrobna biomasa i njezina metabolizamska aktivnost nisu bez utjecaja, mogu imati pozitivne i negativne učinke na ljudsko zdravlje. Ravnoteža ovog mikrobnog sustava je dinamična i može se poremetiti pod utjecajem starenja, liječenja, stresa, prehrane i drugih čimbenika iz okoliša. Probiotički, prebiotički, odnosno sinbiotički koncept podrazumijeva ponovno uspostavljanje ravnoteže crijevne mikroflore i usmjeravanje njenog metabolizma u proizvodnju metabolita korisnih za zdravlje. Obećavajući rezultati s prvom generacijom probiotika nude novu generaciju probiotika (živih lijekova) gdje će se kombinacija različitih probiotičkih sojeva s komplementarnim djelovanjem koristiti u liječenju različitih gastrointestinalnih i urogenitalnih bolesti ili će se koristiti kao vektori za vakcine, imunoglobuline i druge terapijske proteine. Rad se bazira na prikazu objavljenih rezultata znanstvenih istraživanja koja su provedena u Laboratoriju za tehnologiju antibiotika, enzima, probiotika i starter kultura na Prehrambeno-biotehnološkom fakultetu Sveučilišta u Zagrebu.World health crises, especially in a prevention of infective diseases and as result of the wide spread of antibiotic resistance and the number of chronic diseases that disturb human health, has lead, already in the 1980s, to the establishment of the functional food concept (FOSHU Food for specifi ed health use) in Japan. Hence, the food is defi ned as functional “ if it contains a food component (whether a nutrient or not) which affects one or a limited number of function(s) in the body in a targeted way so as to have positive effects”. Target place of action of probiotics, prebiotics or synbiotics, as functional food supplements, is gastrointestinal tract. Gastrointestinal tract represents the largest immune organ in our body with an active space of approximately 300 m2 and it contains a complex microbial ecosystem (gut microfl ora) with a total weight of 1.2 kg. Such a complex microbial biomass and it metabolic activity can have positive but also negative infl uence on human health. The balance of gut microfl ora is dynamic and could be disturbed by the infl uence of different factors like ageing processes, healing, stress, diet, and many other environmental factors. Probiotic, prebiotic and synbiotic concept imply the reestablishment of the disturbed gut microfl ora and direction of it metabolism to the production of bioactive metabolites benefi cial for health. The promising results of a first generation of probiotics offer new generation of probiotics (biotherapeutics) that will often contain mixture of probiotic strains with complementary characteristics, tailormade for different gastrointestinal and urogenital diseases, or as delivery systems for vaccines, immunoglobulins and other protein based therapies. In this paper the review of the published results of the investigations, performed in the Laboratory of Antibiotic, Enzyme, Probiotic and Starter Culture Technology at Faculty of Food Technology and Biotechnology University of Zagreb, are presented

Karakterizacija i primjena autohtonih starter-kultura u proizvodnji svježeg sira

The use of commercial starter cultures in fresh cheese production from pasteurized milk results in the loss of typical characteristics of artisan fresh cheese due to the replacement of complex native microbiota with a defined starter culture. Hence, the aim of this research is to isolate and characterize dominant lactic acid bacteria (LAB) in artisan fresh cheese and to evaluate their capacity as autochthonous starter cultures for fresh cheese production. Fifteen most prevalent Gram-positive, catalase-negative and asporogenous bacterial strains were selected for a more detailed characterization. Eleven lactic acid bacterial strains were determined to be homofermentative cocci and four heterofermentative lactobacilli. Further phenotypic and genotypic analyses revealed that those were two different LAB strains with high acidifying and proteolytic activity, identified as Lactobacillus fermentum A8 and Enterococcus faecium A7. These two autochthonous strains, alone or in combination with commercial starter, were used to produce different types of fresh cheese, which were evaluated by a panel. Conventional culturing, isolation, identification and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) procedures, applied to the total fresh cheese DNA extracts, were employed to define and monitor the viability of the introduced LAB strains and their effect on the final product characteristics. Production of fresh cheese using a combination of commercial starter culture and selected autochthonous strains resulted in improved sensorial properties, which were more similar to the ones of spontaneously fermented fresh cheese than to those of cheese produced with only starter culture or selected strains. After 10 days of storage, that cheese retained the best sensorial properties in comparison with all other types of cheese. The presence of inoculated autochthonous and starter cultures and their identification was demonstrated by DGGE analysis. The obtained results indicate that autochthonous strains have a strong potential to enrich the flavour of industrially produced fresh type cheese under controlled conditions.Primjenom komercijalnih starter-kultura u proizvodnji svježeg sira iz pasteriziranog mlijeka gube se specifična svojstva tradicionalno proizvedenog svježeg sira zbog zamjene prirodno prisutne kompleksne mikrobiote definiranim komercijalnim starter-kulturama. Stoga je svrha ovih istraživanja bila izolirati i okarakterizirati bakterije mliječne kiseline (BMK) koje prevladavaju u tradicionalnom svježem siru i ustanoviti mogućnost njihove primjene kao autohtonih starter-kultura za proizvodnju svježeg sira. Za detaljniju je karakterizaciju odabrano petnaest Gram-pozitivnih, katalaza-negativnih i nesporogenih bakterijskih sojeva koji su prevladavali u autohtonoj mikrobnoj populaciji izoliranoj iz svježeg sira. Jedanaest ih je okarakterizirano kao homofermentativni koki, a četiri kao heterofermentativni laktobacili. Na temelju fenotipske i genotipske analize ustanovljeno je da se radi o dva različita soja BMK koji brzo zakiseljavaju hranjivi medij i imaju proteolitičku aktivnost, a identificirani su kao Lactobacillus fermentum A8 i Enterococcus faecium A7. Ta su dva autohtona soja BMK, sama ili u kombinaciji s komercijalnom starter-kulturom, upotrijebljena za proizvodnju različitih uzoraka svježih sireva. Radi praćenja aktivnosti dodanih BMK i njihova utjecaja na svojstva konačnog proizvoda, provedene su kemijske i senzorske analize te konvencionalni uzgoj, izolacija i identifikacija bakterijskih sojeva iz uzoraka svježih sireva. Primjenom autohtonih sojeva u kombinaciji s komercijalnom starter-kulturom poboljšana su senzorska svojstva proizvedenih svježih sireva u usporedbi s onima u koje je dodana ili samo komercijalna starter-kultura ili samo autohtona kultura. Isti su sirevi zadržali najbolja senzorska svojstva i nakon deset dana čuvanja. PCR elektroforezom u denaturirajućem gradijentu (PCR-DGGE) ekstrahirane ukupne DNA iz svježeg sira potvrđena je prisutnost i provedena identifikacija inokulirane autohtone i starter-kulture. Prema dobivenim rezultatima može se zaključiti da odabrani autohtoni sojevi BMK mogu poboljšati aromu svježih sireva koji se industrijski proizvode u kontroliranim uvjetima

Karakterizacija i primjena autohtonih starter-kultura u proizvodnji svježeg sira

The use of commercial starter cultures in fresh cheese production from pasteurized milk results in the loss of typical characteristics of artisan fresh cheese due to the replacement of complex native microbiota with a defined starter culture. Hence, the aim of this research is to isolate and characterize dominant lactic acid bacteria (LAB) in artisan fresh cheese and to evaluate their capacity as autochthonous starter cultures for fresh cheese production. Fifteen most prevalent Gram-positive, catalase-negative and asporogenous bacterial strains were selected for a more detailed characterization. Eleven lactic acid bacterial strains were determined to be homofermentative cocci and four heterofermentative lactobacilli. Further phenotypic and genotypic analyses revealed that those were two different LAB strains with high acidifying and proteolytic activity, identified as Lactobacillus fermentum A8 and Enterococcus faecium A7. These two autochthonous strains, alone or in combination with commercial starter, were used to produce different types of fresh cheese, which were evaluated by a panel. Conventional culturing, isolation, identification and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) procedures, applied to the total fresh cheese DNA extracts, were employed to define and monitor the viability of the introduced LAB strains and their effect on the final product characteristics. Production of fresh cheese using a combination of commercial starter culture and selected autochthonous strains resulted in improved sensorial properties, which were more similar to the ones of spontaneously fermented fresh cheese than to those of cheese produced with only starter culture or selected strains. After 10 days of storage, that cheese retained the best sensorial properties in comparison with all other types of cheese. The presence of inoculated autochthonous and starter cultures and their identification was demonstrated by DGGE analysis. The obtained results indicate that autochthonous strains have a strong potential to enrich the flavour of industrially produced fresh type cheese under controlled conditions.Primjenom komercijalnih starter-kultura u proizvodnji svježeg sira iz pasteriziranog mlijeka gube se specifična svojstva tradicionalno proizvedenog svježeg sira zbog zamjene prirodno prisutne kompleksne mikrobiote definiranim komercijalnim starter-kulturama. Stoga je svrha ovih istraživanja bila izolirati i okarakterizirati bakterije mliječne kiseline (BMK) koje prevladavaju u tradicionalnom svježem siru i ustanoviti mogućnost njihove primjene kao autohtonih starter-kultura za proizvodnju svježeg sira. Za detaljniju je karakterizaciju odabrano petnaest Gram-pozitivnih, katalaza-negativnih i nesporogenih bakterijskih sojeva koji su prevladavali u autohtonoj mikrobnoj populaciji izoliranoj iz svježeg sira. Jedanaest ih je okarakterizirano kao homofermentativni koki, a četiri kao heterofermentativni laktobacili. Na temelju fenotipske i genotipske analize ustanovljeno je da se radi o dva različita soja BMK koji brzo zakiseljavaju hranjivi medij i imaju proteolitičku aktivnost, a identificirani su kao Lactobacillus fermentum A8 i Enterococcus faecium A7. Ta su dva autohtona soja BMK, sama ili u kombinaciji s komercijalnom starter-kulturom, upotrijebljena za proizvodnju različitih uzoraka svježih sireva. Radi praćenja aktivnosti dodanih BMK i njihova utjecaja na svojstva konačnog proizvoda, provedene su kemijske i senzorske analize te konvencionalni uzgoj, izolacija i identifikacija bakterijskih sojeva iz uzoraka svježih sireva. Primjenom autohtonih sojeva u kombinaciji s komercijalnom starter-kulturom poboljšana su senzorska svojstva proizvedenih svježih sireva u usporedbi s onima u koje je dodana ili samo komercijalna starter-kultura ili samo autohtona kultura. Isti su sirevi zadržali najbolja senzorska svojstva i nakon deset dana čuvanja. PCR elektroforezom u denaturirajućem gradijentu (PCR-DGGE) ekstrahirane ukupne DNA iz svježeg sira potvrđena je prisutnost i provedena identifikacija inokulirane autohtone i starter-kulture. Prema dobivenim rezultatima može se zaključiti da odabrani autohtoni sojevi BMK mogu poboljšati aromu svježih sireva koji se industrijski proizvode u kontroliranim uvjetima

Antimikrobna aktivnost – najvažnije svojstvo probiotičkih bakterija i starter kultura

The antimicrobial activity of industrially important lactic acid bacteria as starter cultures and probiotic bacteria is the main subject of this review. This activity has been attributed to the production of metabolites such as organic acids (lactic and acetic acid), hydrogen peroxide, ethanol, diacetyl, acetaldehyde, acetoine, carbon dioxide, reuterin, reutericyclin and bacteriocins. The potential of using bacteriocins of lactic acid bacteria, primarily used as biopreservatives, represents a perspective, alternative antimicrobial strategy for continuously increasing problem with antibiotic resistance. Another strategy in resolving this problem is an application of probiotics for different gastrointestinal and urogenital infection therapies.Glavna je tema ovoga rada antimikrobno djelovanje industrijski važnih bakterija mliječne kiseline što se koriste kao starter kulture i probiotičke bakterije. Njihovo antimikrobno djelovanje potječe od proizvedenih metabolita, kao što su organske kiseline (mliječna i octena kiselina), vodikov peroksid, etanol, diacetil, acetaldehid, acetoin, ugljikov dioksid, reuterin, reutericiklin i bakteriocini. Primjena bakteriocina bakterija mliječne kiseline, u prvom redu kao biokonzervansa, može biti alternativa rješavanju sve većeg problema antibiotičke rezistencije. Druga je mogućnost u rješavanju toga problema primjena probiotika u terapiji različitih gastrointestinalnih i urogenitalnih infekcija

Haemodynamic stability during anaesthesia induction with propofol – impact of phenylephrine. A double-blind, randomised clinical trial.

Background. We studied the efects of a parallel phenylephrine infusion during bispectral index guided anaesthesia induction with propofol on haemodynamic parameters. We hypothesised that mean arterial pressure and cardiac index would be better maintained in the group of patients receiving the phenylephrine infusion during induction. Methods. We studied ASA I-III patients scheduled for oncological abdominal surgery. Forty patients randomly received either a 0.9% NaCl or a phenylephrine (0.5 μg/kg/min) infusion during the induction of anaesthesia with propofol to a bispectral index value of 60. Mean arterial pressure, stroke volume index and systemic vascular resistance index were recorded, starting at one minute before induction for 20 minutes, at one-minute intervals. Results. Afer induction of anaesthesia before intubation mean arterial pressure and stroke volume index decreased signifcantly compared to baseline in both groups, while the systemic vascular resistance index increased slightly. At the end of measurements, mean arterial pressure (66 ± 11 vs. 94 ± 14 mmHg; 0.9% NaCl vs. phenylephrine group p<0.01) and stroke volume index (34.2 ± 9.1 vs. 44.0 ± 9.7 ml/ m2; 0.9% NaCl vs. phenylephrine group p<0.01) were lower in both groups in comparison to baseline values, but were better maintained in the phenylephrine group, whereas systemic vascular resistance index was higher than at baseline (2308 ± 656 vs. 3198 ± 825 dynes s/cm5/m2; 0.9% NaCl vs. phenylephrine group p<0.01) with signifcant diferences between groups. Conclusion. Our study shows that a continuous phenylephrine infusion can attenuate the drop in mean arterial pressure and stroke volume index during anaesthesia induction with propofol

Influence of probiotic strain Bifidobacterium animalis subsp. lactis lafti® b94, inulin and transglutaminase on the properties of set- style yoghurt

Cilj ovog rada bio je istražiti utjecaj probiotičkog soja Bifidobacterium animalis subsp. lactis LAFTI® B94, inulina i transglutaminaze na kvalitetu i senzorska svojstva čvrstog jogurta. Za proizvodnju jogurta korišteno je svježe homogenizirano mlijeko s 3,3% mliječne masti kojemu je, uz jogurtne starter kulture, dodana probiotička kultura Bifidobacterium animalis subsp. lactis LAFTI® B94, inulin i enzim transglutaminaza, aktiviran 1 h i 30 min. pri 55 °C. Inaktivacija enzima provedena je postupkom pasterizacije 15 min. pri 85 °C. Proizvedeni su i kontrolni uzorci čvrstog jogurta, bez dodatka probiotičke kulture, inulina i transglutaminaze. U radu su određivana fizikalnokemijska i senzorska svojstva proizvedenih čvrstih jogurta. Radi pouzdane identifikacije probiotičkog soja Bifidobacterium animalis subsp. lactis LAFTI® B94, izolira-nog iz proizvedenog jogurta, provedena je SDS-poliakrilamidna gel elektroforeza (SDS-PAGE) ukupnih staničnih proteina i lančana reakcija polimera-zom (PCR) s početnicama specifičnim za rod Bifidobacterium. Proizvedeni čvrsti jogurti s dodanom probiotičkom kulturom, inulinom i transglutami-nazom su bili čvršći i pokazali su manju sinerezu, te su dobili više ocjene senzorskih svojstava u odnosu na kontrolne uzorke jogurta. Broj živih stanica Bifidobacterium animalis subsp. lactis LAFTI® B94 nakon 28 dana čuvanja bio je viši u uzorcima jogurta koji su sadržavali inulin kao prebiotik. Fermentacija jogurta s inulinom i transglutaminazom je kraće trajala od fermentacije jogurta bez dodatka inulina i transglutaminaze. SDS poliakrilamidnom gel elektroforezom ukupnih staničnih proteina i lančanom reakcijom polimeraze, primjenom početnica specifičnih za rod Bifidobacterium, potvrđena je prisutnost probiotičke kulture u proizvedenom čvrstom jogurtu u visokom broju (više od 106 stanica/mL).The aim of this research was to examine the influence of probiotic strain Bifidobacterium animalis subsp. lactis LAFTI® B94, inulin and transglutaminase on quality and sensory properties of set-style yoghurt. Fresh, homogenized milk with 3,3% of milk fat was used for yoghurt production, with addition of Bifidobacterium animalis subsp. lactis LAFTI® B94, inulin and transglutaminase activated during 1h 30 min at 55 °C. Enzyme inactivation was carried out by pasteurization of milk during 15 minutes at 85 °C. Control samples were prepared without addition of probiotic culture, inulin and transglutaminase. Physico-chemical parameters and sensory properties of produced set-style yoghurt have been determined. For reliable identification of probiotic strain Bifidobacterium animalis subsp. lactis LAFTI® B94, isolated from the produced yoghurt, SDS-PAGE of whole cell proteins and PCR with species specific primers for Bifidobacterium were carried out. It has been shown that produced set-style yoghurt with probiotic strain Bifidobacterium animalis subsp. lactis LAFTI® B94, inulin and transglutaminase had higher firmness, less syneresis and better sensory properties than control yoghurt samples. After 28 days of storage the viable count of Bifidobacterium animalis subsp. lactis LAFTI® B94 was higher in samples containing inulin as prebiotic. Fermentation of yoghurt samples containing inulin and transglutaminase lasted shorter than fermentation of samples without inulin and transglutaminase. The presence of high number of probiotic culture (more than 106 cells/mL) in produced set yoghurts was confirmed by SDS-PAGE of whole cell proteins and PCR with species specific primers for Bifidobacterium

Osjetljivost na antibiotike i antimikrobna aktivnost autohtonih starter kultura kao parametri sigurnosti njihove primjene u proizvodnji svježeg sira

The antibiotic susceptibility and antimicrobial activity, as food safety parameters important for application of autochthonous lactic acid bacteria (LAB), that previously satisfied technological criteria for functional starter cultures in fresh cheese production were examined. Soluble whole cell protein patterns of autochthonous LAB strains from fresh cheese, obtained by SDS-PAGE, revealed the presence of two predominant strains, which were identified as Lactobacillus fermentum A8 and Enterococcus faecium A7. These strains were not resistant and shown susceptibility to antibiotics: ampicillin, bacitracin, penicillin G, azithromycin, chloramphenicol, clarithromycin, clindamycin, spiramycin, tetracycline, streptomycin, neomycin, gentamicin, erythromycin, rifampicin and novobiocin. Lb. fermentum A8 strain displayed phenotypic resistance to vancomycin, but this resistance is intrinsic, not transferable and it is acceptable from the safety aspect. The capacity of Lb. fermentum A8 and Ec. faecium A7 to inhibit growth of test-microorganisms Listeria monocytogenes ATCC 11911, Escherichia coli 3014, Salmonella enterica serovar Typhimurium FP1 and Staphylococcus aureus 3048, was also analysed. According to obtained results, Lb. fermentum A8 and Ec. faecium A7 are safe from the aspect of spreading antibiotic resistance and could be useful as bioprotective cultures that inhibit common bacterial food contaminants, including L. monocytogenes.U ovom radu istražena je osjetljivost na antibiotike i antimikrobna aktivnost odabranih autohtonih sojeva bakterija mliječne kiseline, koji su u prethodnim istraživanjima zadovoljili tehnološke kriterije za funkcionalne starter kulture namijenjene proizvodnji svježeg sira. Prema profilu topljivih staničnih proteina, dobivenom SDS-PAGE metodom, ustanovljeno je da u tradicionalno proizvedenom svježem siru prevladavaju dvije vrste bakterija mliječne kiseline, identificirane kao Lactobacillus fermentum A8 i Enterococcus faecium A7. Ovi bakterijski sojevi nisu bili rezistentni, nego su pokazali osjetljivost na testirane antibiotike: ampicilin, bacitracin, penicillin G, azitromicin, kloramfenikol, klaritromicin, klindamicin, spiramicin, tetraciklin, streptomicin, neomicin, gentamicin, eritromicin, rifampicin i novobiocin. Bakterijski soj Lb. fermentum A8 posjeduje fenotipsku rezistenciju na vankomicin, ali ta je rezistencija urođena, nije prenosiva na druge bakterije i stoga je prihvatljiva s aspekta sigurnosti njegove primjene kao starter kulture. Ispitano je i antimikrobno djelovanje kultura Lb. fermentum A8 and Ec. faecium A7 prema test-mikroorganizmima: Listeria monocytogenes ATCC 11911, Escherichia coli 3014, Salmonella enterica serovar Typhimurium FP1 i Staphylococcus aureus 3048. Prema dobivenim rezultatima, Lb. fermentum A8 i Ec. faecium A7 su sigurni s aspekta širenja antibiotičke rezistencije i mogu biti korisne kao zaštitne kulture koje inhibiraju bakterije, najčešće kontaminante hrane, uključujući i L. monocytogenes

Antimikrobna aktivnost – najvažnije svojstvo probiotičkih bakterija i starter kultura

The antimicrobial activity of industrially important lactic acid bacteria as starter cultures and probiotic bacteria is the main subject of this review. This activity has been attributed to the production of metabolites such as organic acids (lactic and acetic acid), hydrogen peroxide, ethanol, diacetyl, acetaldehyde, acetoine, carbon dioxide, reuterin, reutericyclin and bacteriocins. The potential of using bacteriocins of lactic acid bacteria, primarily used as biopreservatives, represents a perspective, alternative antimicrobial strategy for continuously increasing problem with antibiotic resistance. Another strategy in resolving this problem is an application of probiotics for different gastrointestinal and urogenital infection therapies.Glavna je tema ovoga rada antimikrobno djelovanje industrijski važnih bakterija mliječne kiseline što se koriste kao starter kulture i probiotičke bakterije. Njihovo antimikrobno djelovanje potječe od proizvedenih metabolita, kao što su organske kiseline (mliječna i octena kiselina), vodikov peroksid, etanol, diacetil, acetaldehid, acetoin, ugljikov dioksid, reuterin, reutericiklin i bakteriocini. Primjena bakteriocina bakterija mliječne kiseline, u prvom redu kao biokonzervansa, može biti alternativa rješavanju sve većeg problema antibiotičke rezistencije. Druga je mogućnost u rješavanju toga problema primjena probiotika u terapiji različitih gastrointestinalnih i urogenitalnih infekcija