Frane Delaš

Comparison of conventional and molecular methods for the routine confirmation of Listeria monocytogenes in milk products produced domestically in Croatia.

Conventional methods for the identification of Listeria in foodstuffs are generally cumbersome and time consuming. The use of primary enrichment in half strength Fraser broth and the use of PALCAM agar were assessed in comparison with API Listeria and polymerase chain reaction (PCR) for their ability to accurately detect and confirm the presence of List. monocytogenes in milk products. The aim of our work was to detect List. monocytogenes in domestic unpasteurised milk, fresh cheese and cream of raw milk taken from four different district of Zagreb-Croatia using conventional (microbiological and biochemical - API test) and PCR methods. Of the 180 milk products samples tested, 27.6% were presumptively positive for Listeria on PALCAM agar. Only 21.3% of samples were confirmed to be positive for Listeria by API Listeria test, and 17.3% were confirmed to be positive for List. monocytogenes by PCR amplification of the hly gene (64 bp). PCR was able to eliminate the false positive and detect all List. monocytogenes in the milk products, unlike the conventional methods used in the industry. These results indicate a low presence of this pathogen in this area (Zagreb) of Croatia. PCR proves to be a sensitive and rapid technique to be included in the procedure of detection of List. monocytogenes in food products and this method is considerably faster than current standard methods.

Antifungal effect of eugenol and carvacrol against foodborne pathogens Aspergillus carbonarius and Penicillium roqueforti in improving safety of fresh-cut watermelon

Background: Essential oil components eugenol and carvacrol (ranging between 100 and 200 ppm for carvacrol and between 250 and 750 ppm for eugenol) were tested for antifungal activity against foodborne pathogenic fungal species Aspergillus carbonarius A1102 and Penicillium roqueforti PTFKK29 in in vitro and in situ conditions. Materials and Methods: In vitro antifungal activity of eugenol and carvacrol was evaluated by macrobroth method, while watermelon Citrullus lanatus L. Sorento slices were used for antifungal assays in situ. Results: Selected components, eugenol and carvacrol showed significant inhibitory effect against tested fungi (A. carbonarius A1102 and P. roqueforti PTFKK29) in yeast extract sucrose broth, as well as in in situ conditions. The minimal inhibitory concentration (MIC) of eugenol against A. carbonarius A1102 determined by macrobroth method was 2000 ppm, while against P. roqueforti PTFKK29 determined MIC was 1000 ppm. Carvacrol inhibited growth of A. carbonarius A1102 at minimal concentration of 500 ppm, while against P. roqueforti PTFKK29, MIC was 250 ppm. The assays in real food system watermelon slices for eugenol and carvacrol show that the inhibitory effect against both selected fungal species was concentration dependent. Furthermore, our results showed that antifungal effect of carvacrol as well as eugenol applied on watermelon slices in all concentrations was a result of effective synergy between an active antifungal compound and lower incubation temperature (15°C) in inhibition of A. carbonarius A1102. Conclusion: The present study suggests that the use of eugenol and carvacrol is promising natural alternative to the use of food chemical preservatives, in order to improve safety and quality of fresh-cut and ready-to-eat fruits.

Growth inhibition of Aspergillus ochraceus ZMPBF 318 and Penicillium expansum ZMPBF 565 by four essential oils.

Fungi are recognized for their superior aptitudes to produce a large variety of extracellular proteins, organic acids and other metabolites, and for their capacities to adapt to severe environmental constraints and for the biotreatment of wastewater. Moulds of the genera Aspergillus and Penicillium are common contaminants in food and are especially dangerous because they have the capability to produce toxic metabolites mycotoxins. Essential oils are successful as a natural way to protect food from mould contamination. Essential oils of cinnamon, lavender, rosemary and sage at 1 % (v/v) concentration in Yeast media inoculated with spores (final concentration 106 mL-1 media) of Aspergillus ochraceus ZMPBF 318 and Penicillium expansum ZMPBF 565 were evaluated for their potential in the control of growth of fungal biomass as pure and mixed culture. Cinnamon showed the best inhibitory effect (100 %). Inhibitory effect of lavender was better on Aspergillus ochraceus (nearly 100 %) while on Penicillium expansum inhibitory effect was decreasing (to 57 %). Sage and rosemary showed the opposite effects. Inhibitory effect on Aspergillus ochraceus grows during the cultivation, while on Penicillium expansum decreases during the period of cultivation. The findings showed that fungi can be controlled by essential oils treatment, especially with cinnamon.Growth Inhibition of Aspergillus Ochraceus ZMPBF 318 and Penicillium Expansum ZMPBF 565 by Four Essential Oils Fungi produce a large variety of extracellular proteins, organic acids, and other metabolites and can adapt to several environmental conditions. Mycotoxin-producing moulds of the genera Aspergillus and Penicillium are common food contaminants. One of the natural ways to protect food from mould contamination is to use essential oils. In this study, we evaluated the effect of essential oils of cinnamon, lavender, rosemary, and sage at 1 % (v/v) concentration in yeast media inoculated with spores (final concentration 106 mL-1 media) of Aspergillus ochraceus ZMPBF 318 and Penicillium expansum ZMPBF 565, alone or in combination, on fungal biomass. Cinnamon showed the best inhibitory effect (100 %). Lavender oil best inhibited the growth of Aspergillus ochraceus (nearly 100 %), and was less successful with Penicillium expansum (having dropped to 57 % on day 28). With cultivation time the inhibitory effect of sage and rosemary oil grew for Aspergillus ochraceus and dropped for Penicillium expansum. These results suggest that fungi can be controlled with essential oils, especially with cinnamon oil. Inhibicija rasta plijesni Aspergillus ochraceus ZMPBF 318 i Penicillium expansum ZMPBF 565 djelovanjem četiriju eteričnih ulja Plijesni su poznate po svojoj visokoj sposobnosti proizvodnje različitih izvanstaničnih proteina, organskih kiselina i drugih metabolita i po svojoj mogućnosti prilagodbe na nepovoljne okolišne uvjete, a primjenjuju se i u obradi otpadnih voda. Plijesni iz rodova Aspergillus i Penicillium česti su kontaminanti u hrani i posebno opasne jer tvore toksične metabolite mikotoksine. Eterična ulja mogu se primijeniti kao prirodna sredstva za zaštitu hrane od kontaminacije plijesnima. U radu su prikazani rezultati istraživanja utjecaja eteričnih ulja cimeta, lavande, ružmarina i kadulje na kontrolu rasta biomase plijesni Aspergillus ochraceus ZMPBF 318 i Penicillium expansum ZMPBF 565, u obliku čistih i miješanih kultura. Ulja su dodavana u koncentraciji od 1 % (v/v), a podloga (kvaščev ekstrakt) bila je nacijepljena suspenzijama spora plijesni (106 mL-1 podloge). Eterično ulje cimeta pokazalo je najveći inhibitorni učinak (100 %). Inhibitorni učinak eteričnog ulja lavande bio je veći na rast Aspergillusa ochraceusa (skoro 100 %) nego Penicilliuma expansuma (57 %). Eterična ulja kadulje i ružmarina pokazala su suprotne učinke. Inhibitorni učinak na Aspergillus ochraceus tijekom perioda uzgoja je rastao, a na Penicillium expansum opadao. Rezultati pokazuju da se rast plijesni može kontrolirati primjenom eteričnih ulja, a posebno uljem cimeta. Također upućuju na ekonomsku vrijednost takvih tretmana.

Biodegradation of Olive Mill Wastewater by Trichosporon Cutaneum and Geotrichum Candidum

Biodegradation of Olive Mill Wastewater by Trichosporon Cutaneum and Geotrichum Candidum Olive oil production generates large volumes of wastewater. These wastewaters are characterised by high chemical oxygen demand (COD), high content of microbial growth-inhibiting compounds such as phenolic compounds and tannins, and dark colour. The aim of this study was to investigate biodegradation of olive mill wastewater (OMW) by yeasts Trichosporon cutaneum and Geotrichum candidum. The yeast Trichosporon cutaneum was used because it has a high potential to biodegrade phenolic compounds and a wide range of toxic compounds. The yeast Geotrichum candidum was used to see how successful it is in biodegrading compounds that give the dark colour to the wastewater. Under aerobic conditions, Trichosporon cutaneum removed 88 % of COD and 64 % of phenolic compounds, while the dark colour remained. Geotrichum candidum grown in static conditions reduced COD and colour further by 77 % and 47 %, respectively. This investigation has shown that Trichosporon cutaneum under aerobic conditions and Geotrichum candidum under facultative anaerobic conditions could be used successfully in a two-step biodegradation process. Further investigation of OMW treatment by selected yeasts should contribute to better understanding of biodegradation and decolourisation and should include ecotoxicological evaluation of the treated OMW. Biorazgradnja otpadne vode iz procesa proizvodnje maslinova ulja s pomoću Trichosporon cutaneum i Geotrichum Candidum Tijekom proizvodnje maslinova ulja nastaju velike količine otpadne vode. Karakterizira je visoka kemijska potrošnja kisika (KPK), visoka koncentracija sastojaka koji inhibiraju rast mikroorganizama, poput fenolnih spojeva i tanina i tamna boja. Cilj ovog rada je bio istražiti biorazgradnju otpadne vode iz procesa proizvodnje maslinova ulja s pomoću kvasaca Trichosporon cutaneum i Geotrichum candidum. Kvasac Trichosporon cutaneum u ovom je istraživanju uporabljen zbog svog visokog potencijala za biorazgradnju fenolnih i drugih toksičnih spojeva. Kvasac Geotrichum candidum uporabljen je kako bi se istražilo koliko je uspješan u biorazgradnji spojeva koji otpadnoj vodi daju tamnu boju. Pri aerobnim uvjetima Trichosporon cutaneum uklonio je 88 % KPK i 64 % fenolnih spojeva, dok tamna boja otpadne vode preostaje. Geotrichum candidum u statičkim je uvjetima smanjio KPK i boju za 77 %, odnosno 47 %. Ovo je istraživanje pokazalo da Trichosporon cutaneum pri aerobnim uvjetima i Geotrichum candidum pri fakultativno anaerobnim uvjetima mogu biti uspješno uporabljeni u dvostupanjskom procesu biorazgradnje. Daljnje istraživanje obrade otpadne vode iz procesa proizvodnje maslinova ulja s pomoću odabranih kvasaca trebalo bi pridonijeti boljem razumijevanju biorazgradnje i uklanjanju boje i trebalo bi biti provedeno zajedno sa ekotoksikološkim vrednovanjem obrađene otpadne vode.

The effects of wild thyme (Thymus serpyllum L.) essential oil components against ochratoxin-producing Aspergilli.

Abstract The aim of this study was to determine the effects of the essential oil of Thymus serpyllum L. and of its components thymol and total phenols (total phenolic content, TPC) extracted from the plant on the growth and mycotoxin production of Aspergillus ochraceus, A. carbonarius, and A. niger. Minimal inhibitory concentration (MIC) determined for the essential oil and thymol, and selected concentration of the TPC extract inhibited fungal growth and ochratoxin A biosynthesis by more than 60 %, depending on the conditions and duration of incubation with the fungi. Essential oil showed the strongest inhibitory effect which may have been related to the synergistic or cumulative effects of its components Cilj ovog rada bio je odrediti utjecaj eteričnog ulja majčine dušice (Thymus serpyllum), uljne komponente timola i ukupnih fenola ekstrahiranih iz biljke, na rast plijesni i sintezu mikotoksina. U odabranim uvjetima minimalna inhibitorna koncentracija (MIC) eteričnog ulja i timola, koja je određena u ovom radu, i koncentracija ukupnih fenola (TPC) inhibirale su više od 60 % rasta odabranih plijesni i biosinteze okratoksina A. Njihova inhibitorna učinkovitost ovisila je o uvjetima i trajanju inkubacije s plijesnima. U odnosu na timol i ekstrakt eterično ulje pokazalo je snažniji inhibitorni utjecaj, koji je uvjetovan sinergističkim ili kumulativnim djelovanjem uljnih komponenata.

A survey of the microbiological quality of Sharri, a hard mountain cheese from Kosovo

The microbiological quality of a hard mountain unpasteurised sheep cheese from three randomly selected manufacturing locations in Kosovo was investigated. Forty-eight samples of row milk, coagulum, 8–10 days ripening cheese and of ready to eat cheese (45-days in brine) were tested. Seventy-five per cent of raw milk samples failed to comply with EU regulation 853/2004. All of coagulum and ripened cheese failed to comply with EU regulation 2073/2005 on process hygiene criteria. Despite the high incidence of coagulase-positive staphylococci even in the final product [>105 colony-forming units (cfu)/g], Staphylococcal enterotoxin was detected in none of the samples and no samples were positive for Listeria monocytogenes and Salmonella enteritidis.

Antifungal and Antipatulin Activity of Gluconobacter oxydans Isolated from Apple Surface

Fungicides are the most common agents used in postharvest treatment of fruit and are the most effective against blue mould, primarily caused by Penicillium expansum. Alternatively, blue mould can be treated with antagonistic microorganisms naturally occurring on fruit, such as the bacterium Gluconobacter oxydans. The aim of this study was to establish the antifungal potential of the G. oxydans 1J strain isolated from apple surface against Penicillium expansum in culture and apple juice and to compare it with the efficiency of a reference strain G. oxydans ATCC 621H. The highest antifungal activity of G. oxydans 1J was observed between days 3 and 9 with no colony growth, while on day 12, P. expansum colony diameter was reduced to 42.3 % of the control diameter. Although G. oxydans 1J did not fully inhibit mould growth, it showed a high level of efficiency and completely prevented patulin accumulation in apple juice. Sažetak Tretiranje voća fungicidima, nakon berbe, uobičajeni je način suzbijanja plave plijesni. Međutim, propadanje voća može se spriječiti i upotrebom antagonističkih mikroorganizama, kao što je bakterija Gluconobacter oxydans. Svrha ovoga rada bila je izolirati prirodnu mikrobnu populaciju s površine jabuka i istražiti moguće inhibitorno djelovanje Gluconobacter oxydans 1J na plavu plijesan, Penicillium expansum, najvažnijeg uzročnika kvarenja jabuka u skladištu. Najveća antifungalna aktivnost bakterije primijećena je između 3. i 9. dana, kada nije zabilježen porast kolonija, a nakon 12. dana promjer kolonije plijesni bio je manji za 42,3 %. Iako istraživana bakterija Gluconobacter oxydans 1J nije u potpunosti inhibirala rast plijesni u jabučnom soku pokazala je visoku razinu učinkovitosti (od 86 % do 95 %). Gluconobacter oxydans 1J djelomično inhibira rast plijesni i u potpunosti biosintezu patulina, ovisno o vremenu i uvjetima uzgoja.

The Effects Of Wild Thyme (Thymus Serpyllum L.) Essential Oil Components Against Ochratoxin-Producing Aspergilli / Majčina Dušica (Thymus Serpyllum L.) I Njezine Komponente Protiv Okratoksikotvornih Vrsta Aspergillusa

Abstract The aim of this study was to determine the effects of the essential oil of Thymus serpyllum L. and of its components thymol and total phenols (total phenolic content, TPC) extracted from the plant on the growth and mycotoxin production of Aspergillus ochraceus, A. carbonarius, and A. niger. Minimal inhibitory concentration (MIC) determined for the essential oil and thymol, and selected concentration of the TPC extract inhibited fungal growth and ochratoxin A biosynthesis by more than 60 %, depending on the conditions and duration of incubation with the fungi. Essential oil showed the strongest inhibitory effect which may have been related to the synergistic or cumulative effects of its components Cilj ovog rada bio je odrediti utjecaj eteričnog ulja majčine dušice (Thymus serpyllum), uljne komponente timola i ukupnih fenola ekstrahiranih iz biljke, na rast plijesni i sintezu mikotoksina. U odabranim uvjetima minimalna inhibitorna koncentracija (MIC) eteričnog ulja i timola, koja je određena u ovom radu, i koncentracija ukupnih fenola (TPC) inhibirale su više od 60 % rasta odabranih plijesni i biosinteze okratoksina A. Njihova inhibitorna učinkovitost ovisila je o uvjetima i trajanju inkubacije s plijesnima. U odnosu na timol i ekstrakt eterično ulje pokazalo je snažniji inhibitorni utjecaj, koji je uvjetovan sinergističkim ili kumulativnim djelovanjem uljnih komponenata.

[Azo dyes, their environmental effects, and defining a strategy for their biodegradation and detoxification].

Azo Boje, Njihov Utjecaj Na Okoliš I Potencijal Biotehnološke Strategije Za Njihovu Biorazgradnju I Detoksifikaciju Intenzivan industrijski razvoj popraćen je sve većom kompleksnošću sastava otpadnih voda, što u smislu učinkovite zaštite okoliša i održivog razvoja nalaže potrebu pospješivanja kvalitete postojećih te uvođenjem novih postupaka obrade otpadnih voda, kao iznimno važnog čimbenika u interakciji čovjeka i okoliša. Posebnu znanstveno-tehnološku pozornost zahtijevaju novosintetizirani ksenobiotici, poput azo-boja, koji su u prirodi veoma teško razgradivi. Azo-boje podložne su bioakumulaciji, a zbog alergijskih, kancerogenih, mutagenih i teratogenih svojstava nerijetko su prijetnja zdravlju ljudi i očuvanju okoliša. Primjenu fizikalnokemijskih metoda za uklanjanje azo-boja iz otpadnih voda često ograničavaju visoke cijene, potrebe za odlaganjem nastalog štetnog mulja ili nastanak toksičnih sastojaka razgradnje. Biotehnološki postupci su, zbog mogućnosti ekonomične provedbe i postizanja potpune biorazgradnje, a time i detoksifikacije, sve zastupljeniji u obradi svih vrsta otpadnih voda, pa tako i onih koje sadržavaju azo-boje. Azo Dyes, Their Environmental Effects, and Defining a Strategy for Their Biodegradation and Detoxification Intense industrial development has been accompanied by the production of wastewaters of very complex content, which pose a serious hazard to the environment, put at risk sustainable development, and call for new treatment technologies that would more effectively address the issue. One particular challenge in terms of science and technology is how to biodegrade xenobiotics such as azo dyes, which practically do not degrade under natural environmental conditions. These compounds tend to bioaccumulate in the environment, and have allergenic, carcinogenic, mutagenic, and teratogenic properties for humans. Removal of azo dyes from effluents is mostly based on physical-chemical methods. These methods are often very costly and limited, as they accumulate concentrated sludge, which also poses a significant secondary disposal problem, or produce toxic end-products. Biotechnological approach may offer alternative, lowcost biological treatment systems that can completely biodegrade and detoxify even the hard-to-biodegrade azo dyes.

Majčina dušica (Thymus serpyllum L.) i njezine komponente protiv okratoksikotvornih vrsta Aspergillusa

Abstract The aim of this study was to determine the effects of the essential oil of Thymus serpyllum L. and of its components thymol and total phenols (total phenolic content, TPC) extracted from the plant on the growth and mycotoxin production of Aspergillus ochraceus, A. carbonarius, and A. niger. Minimal inhibitory concentration (MIC) determined for the essential oil and thymol, and selected concentration of the TPC extract inhibited fungal growth and ochratoxin A biosynthesis by more than 60 %, depending on the conditions and duration of incubation with the fungi. Essential oil showed the strongest inhibitory effect which may have been related to the synergistic or cumulative effects of its components Cilj ovog rada bio je odrediti utjecaj eteričnog ulja majčine dušice (Thymus serpyllum), uljne komponente timola i ukupnih fenola ekstrahiranih iz biljke, na rast plijesni i sintezu mikotoksina. U odabranim uvjetima minimalna inhibitorna koncentracija (MIC) eteričnog ulja i timola, koja je određena u ovom radu, i koncentracija ukupnih fenola (TPC) inhibirale su više od 60 % rasta odabranih plijesni i biosinteze okratoksina A. Njihova inhibitorna učinkovitost ovisila je o uvjetima i trajanju inkubacije s plijesnima. U odnosu na timol i ekstrakt eterično ulje pokazalo je snažniji inhibitorni utjecaj, koji je uvjetovan sinergističkim ili kumulativnim djelovanjem uljnih komponenata.