Jadranka Frece

Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92

Aims: To investigate aggregation and adhesiveness of Lactobacillus acidophilus M92 to porcine ileal epithelial cells in vitro, and the influence of cell surface proteins on autoaggregation and adhesiveness of this strain.

Importance of S-layer proteins in probiotic activity of Lactobacillus acidophilus M92

Aims:  To investigate the functional role of surface layer proteins (S‐layer) in probiotic strain Lactobacillus acidophilus M92, especially its influence on adhesiveness to mouse ileal epithelial cells.

Functionality of the S-layer protein from the probiotic strain Lactobacillus helveticus M92

The objective of this study was the characterisation of the S-layer protein (SlpA) and its functional role in the probiotic activity of Lactobacillus helveticus M92. SlpA was isolated and identified by SDS-PAGE LC-MS/MS analysis. The slpA gene encoding the SlpA from L. helveticus M92 was sequenced and compared with other well characterised slpA genes. Sequence similarity searches revealed high homology with the SlpA of Lactobacillus strains. Purified SlpA showed significantly better immunomodulatory effects in orally immunised mice than L. helveticus M92 cells after SlpA removal. SlpA is involved in the autoaggregation of L. helveticus M92 cells and coaggregation of L. helveticus M92 with S. Typhimurium FP1 as these processes were negatively affected after SlpA removal from the cell surface. Therefore, the influence of oral treatment with L. helveticus M92 on an oral infection of mice by S. Typhimurium FP1 was investigated. Following the oral immunization of mice, with viable L. helveticus M92 and S. Typhimurium FP1 cells, the concentration in the luminal contents of total S-IgA and specific anti-Salmonella S-IgA antibodies, from all immunized mice was significantly higher compared to the control group or a group of mice infected only with S. Typhimurium FP1. These results demonstrate that the observed reduced infection by S. Typhimurium FP1 in mice with L. helveticus M92 is associated with competitive exclusion in the intestinal tract and enhanced immune protection conferred by the L. helveticus M92 and its SlpA.

Synbiotic effect of Lactobacillus helveticus M92 and prebiotics on the intestinal microflora and immune system of mice.

The synbiotic effect of the oral treatment of Swiss albino mice with milk-based diets supplemented with Lactobacillus helveticus M92 and various kinds of prebiotics was investigated. Survival, competition, adhesion and colonization, as well as, immunomodulating capability of Lb. helveticus M92, in synbiotic combination, in the gastrointestinal tract (GIT) of mice, were monitored. After the mice were fed with synbiotics, the lactic acid bacteria (LAB) counts in faeces were increased and reduction of enterobacteria and sulphite-reducing clostridia was observed. Similar results were obtained in homogenates of small and large intestine of mice on the 1st and 14th day, after feeding with synbiotics. After the mice were orally given viable Lb. helveticus M92 cells, alone or in combination with prebiotic, the concentration of faecal SIgA and total serum IgA antibodies from all immunized mice were higher compared with the control. The specific humoral immune response was not evoked after oral administration, therefore their synbiotic application is suitable. Among inulin, lactulose and raffinose, Lb. helveticus M92 in combination with inulin, has shown the best synbiotic effect on intestinal and faecal microflora and immune system of mice.

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.

Ochratoxin A reduction in meat sausages using processing methods practiced in households

The aim of this study was to investigate the possibilities of ochratoxin A (OTA) reduction in home-made meat products. Meat sausages (n = 50) produced from raw materials coming from pigs exposed to OTA-contaminated feed, were subject to common heat processes practiced in households (cooking, frying and baking). Concentrations of OTA in pre- and post-processed products were quantified using a validated immunoassay method, enzyme-linked immunosorbent assay, and confirmed using a high-performance liquid chromatography with fluorescence detection. In line with the differences in recipes used and the degree of OTA accumulation in raw materials, OTA concentrations established in Mediterranean and roast sausages were lower than those found in liver and blood sausages. Baking of contaminated sausages at the temperatures of 190–220°C (for 60 min) resulted in significant reduction of OTA levels (75.8%), while 30-min cooking (at 100°C) and frying (at 170°C) proved to be significantly less effective (e.g. yielding OTA reductions of 7.4% and 12.6%, respectively). The results pointed out that despite high OTA stability, heat processes are capable of reducing its concentration in home-made meat products, depending on the processing modality used.

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.

Influence of high power ultrasound on selected moulds, yeasts and Alicyclobacillus acidoterrestris in apple, cranberry and blueberry juice and nectar

HighlightsA. acidoterrestris DSM 3922 number were not significantly reduced by ultrasound.There was a growth activation of A. acidoterrestris DSM 3922.Complete inactivation of the of yeasts and moulds were achieved at treatments at 60 °C and times of 3, 6 and 9 min.Significant effect of temperature on the inactivation of yeasts and moulds were observed. ABSTRACT The purpose of this study was to investigate the effect of non‐thermal technology, high power ultrasound (HPU) on inactivation of Aspergillus ochraceus 318, Penicillium expansum 565, Rhodotorula sp. 74, Saccharomyces cerevisiae 5 and Alicyclobacillus acidoterrestris DSM 3922 in clear juices and nectars from apple, blueberry and cranberry juice concentrate. Inoculated juice and nectars were treated by high power ultrasound (20 kHz) according to procedure set by central composite design (CCD). Three operational parameters, amplitude (60, 90 and 120 &mgr;m), temperature (20, 40 and 60 °C), and treatment time (3, 6 or 9 min) were varied in order to observe the influence of ultrasound and combination of ultrasound and slight heating (thermosonication) on growth and inactivation of selected microorganisms. Number of vegetative cells of A. acidoterrestris DSM 3922 were not significantly reduced by high power ultrasound (p > 0.05), except in apple juice, where statistical significant (p < 0.05) influence of quadratic interaction of amplitude on bacteria reduction were observed. In all samples of fruit juices and nectars in terms of ultrasonic treatment at 60 °C and times of 3, 6 and 9 min, regardless of the value of the amplitude, complete inactivation of the growth of yeasts and moulds were achieved, while at 20 and 40 °C it is not observed. The value of reduction of cells of selected yeasts and moulds for ultrasound treatments at 60 °C and the duration of the 3, 6 and 9 min ranged from 3.556 to 5.934 log units, depending on the initial number of selected yeasts and moulds before treatment.