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Ultrasonication is a non‐thermal method in food processing that has the advantage of preserving fruit juices without causing the common side‐effects associated with conventional heat treatments. Special attention was drawn to Alicyclobacillus acidoterrestris that can be found in concentrated fruit juices and which it is very difficult to inactivate by heat treatment proceedings. 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 made 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 μm), temperature (20, 40 and 60oC), 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). Contrary to expectations there was a growth activation of A. acidoterrestris DSM 3922. 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 minutes ranged from 4.580 to 5.869 log units, depending on the initial number of selected yeasts and moulds before treatment. The effect of ultrasound was mainly attributed to physical (cavitation, mechanical effects, micro‐mechanical shocks) and/or chemical effects due to formation of free radicals (H+ and OH‐ due to sonochemical reaction) formed by the decomposition of water inside the oscillating bubbles. Power ultrasound caused complete inactivation of the growth of yeasts and moulds at 60 °C and treatment times of 3, 6 and 9 minutes, regardless of the value of the amplitude. Number of vegetative cells of A. acidoterrestris were not reduced.

In recent years, due to a current focus on “healthy” food and a healthy lifestyle, the market for organic cereals has grown rapidly. Organic farming avoids the use of synthetically compounded fertilizers, pesticides or growth regulators, and prefers crop rotations, crop residues, animal and green manures, mechanical cultivation and biological pest control. Because of that, one can presume that the organic farming preserves biodiversity and that the organic products can be the source of different microbial species suitable for food industry. In this study the species diversity in organically grown wheat, spelt and rye was investigated. Identification was based on mass spectrometry using a Microflex LT ™ MALDI-TOF instrument. Also, fermentative and antimicrobial activities of identified species were determined in order to apply these microorganisms to sourdough fermentation. The fermentation products (lactic, acetic, formic and fenil-lactic acid) were determined by high pressure liquid chromatography, while the antimicrobial activity was determined using an overlay agar method. The results showed that the flour samples had a rich microflora. A total of 16 lactic acid bacteria and Candida crusei were isolated. Wheat flour showed less biodiversity than the rye and spelt flour. The most common genera in the tested flour were Lactobacillus and Enterococcus. Isolated Lactobacillus farciminis, Pediococcus pentosaceus, Leuconostoc citreum and Lactobacillus brevis showed the best fermentation activity. Lactobacillus brevis, Pediococcus pentosaceus, Weissella cibaria and Lactobacillus farciminis showed significant antimicrobial activity against A. niger and Penicillium sp.

Ochratoxin A (OTA) is a mycotoxin which contaminate a variety of foodstuffs worldwide and may be present in meat and offal of farm animals or in the products of animal origin made of contaminated raw materials. In process of food preservation, gamma (γ) irradiation shown to be an effective technique of decontamination as destroy pathogenic and spoilage microorganisms as well as reduces some toxins such as mycotoxins. The aim of this study was to investigate the efficiency of gamma irradiation on OTA reduction in meat products (n=24) using three types of homemade fermented sausages and cured meat products (ham and ribs) as frequently consumed meat product in Croatia. Final meat products produced from contaminated raw materials of muscle/adipose tissue were irradiated using gamma rays at doses of 7 and 10 kGy as doses applicable in food industry. Concentrations of OTA in pre- and post-processed products were quantified using a validated immunoassay (ELISA) method and expressed on the product dry matter. Before irradiation determined OTA concentrations were in range from 6.33 to 33.33 μg/kg. Following the 7 kGy-irradiation, the overall mean OTA reduction equalled to 14.1%, while the irradiation with a 10 kGy-dose achieved the overall mean toxin reduction of 23.7%, and maximall reached of 34.7%. The obtained results indicate that γ irradiation could reduce the OTA levels in meat product intended for human consumption, thus minimizing the human exposure to this carcinogenic mycotoxin, but also that due to the achieved reduction applied decontamination method is of limited value.

Maraština grapes, grown in Dalmatia, are used in the production of traditional dessert wine Prošek by using special grape drying techniques. The traditional sun-drying method requires particular climatic conditions, such as low humidity and hot temperatures. In recent years alternative methods, involving drying under controlled conditions, have been studied as safer and faster methods than the traditional sun-drying method. The objective of this work was to study how drying methods impact the volatile compounds and the quality of must from Maraština cv. grapes. Maraština grapes grown at Pelješac were harvested at 22 Brix and dried under two different conditions: three days at 50 C and twenty-two days under greenhouse conditions. Drying of grapes under greenhouse conditions and in the chamber was stopped when sugar content in the must reached 36.2 and 35.6 Brix, respectively. The volatile compounds of musts were determined using headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrophotometry (GC-MS). Drying at 50 C caused significant increase in the sum of relative concentrations of esters, aldehydes and terpenes and decrease in the sum of alcohols, while drying under the greenhouse conditions increased the sum of relative concentrations of alcohols, esters, aldehydes and terpenes. The major volatile compound in the must dried in the greenhouse was 1-hexanol, while 3-methyl-1-butanol was the predominat compound in the chamber-dried must. Relative concentration of -damascenone significantly decreased in the musts from dried grapes. The results of PCA analysis showed that the first two principal components are sufficient to describe 100% of the variation among the observed parameters measured for the samples (grapes and drying methods). The first principal component (PC1=77.51%) showed the domination of loadings for the content of ketones, aldehydes and esters pH, while in the second principal component (PC2=22.49%), alcohols and total acidity, as the observed parameters of quality, were dominant.

The aim of this research is functional characterisation of processed cereal/rice based product enriched with a lyophilized probiotic culture of Bifidobacterium animalis subsp. lactis BB-12, intended to be developed as probiotic baby food. The results show that the bacterial counts determined for B. animalis subsp. lactis BB-12, expressed as log CFU/g of product, were maintained above 106 CFU/g during storage period, what is the level requested for probiotic products. In order to properly identify B. animalis subsp. lactis BB-12 after implementation in processed cereal/rice based product, phenotypic and genotypic characterisations were performed. Analysis of sugar fermentation profiles and SDS-PAGE analysis of soluble cell proteins, together with PCR with specific primers for Bifidobacterium spp. and AFLP DNA fingerprints confirmed the presence of B. animalis subsp. lactis BB-12 in final product. Introduction of the probiotic strains underscores a need to exclude the presence of antibiotic resistance determinants. Analysis of B. animalis subsp. lactis BB-12 susceptibility to the representatives of all major antibiotic groups revealed that the strain was sensitive to the most antibiotic tested, but to gentamycin, neomycin and streptomycin what is related to aminoglycoside antibiotic instability in acid conditions. Further important functional characteristics, survival in simulated gastrointestinal tract conditions and competitive pathogen exclusion by B. animalis subsp. lactis BB-12 implemented in processed cereal/rice based product were evaluated. Comparison of the viable cells count determined for the B. animalis subsp. lactis BB-12 lyophilised culture and in processed cereal/rice based product, respectively revealed much higher viable cell counts when probiotic culture B. animalis subsp. lactis BB-12 was applied in combination with processed cereal/rice based product supporting the protective effect of the food matrix. In addition, antimicrobial activity of B. animalis subsp. lactis BB-12 against test microorganism Salmonella enterica serovar Typhimurium FP1 was assayed. According to obtained results B. animalis subsp. lactis BB-12 inhibits Salmonella enterica serovar Typhimurium FP1 growth. In conclusion, overall results of this research proved that processed cereal/rice based product positively influenced the functionality of B. animalis subsp. lactis BB-12.

Stevia rebaudiana (Bertoni) glycosides are extensively used as a high intensity sweetener with low calorie value. Glycosides are extracted from leaves to obtain steviosides and rebaudiosides as sweet components. According to EU and Croatian regulations, ground leaf powder is only to be used for extraction of glycosides. Besides solvents and extraction conditions, particle size and physical properties also influence the extraction process. In this research, dry Stevia rebaudiana (Bertoni) leaves were ground to a desired particle size and particle size fractions were separated by sieving. Obtained fractions were subjected to laser diffraction particle sizing in order to determine median diameter, Sauter diameter and particle size span. Fractions were also analysed by a powder rheometer to define powders cohesion and caking properties. A jolting volumeter was used for bulk density determination and reconstitution properties were determined by quick test methods. Experimental data was analysed by Spearman Correlation Coefficients. Sieving analysis resulted in five particle size fractions. Smaller particle size fractions exhibited typical bulk powder behaviour, while larger fractions contained a large amount of fractured leaves, resulting in a tea-like bulk rather than a powder. All the fractions were categorized as easy or free flowing based on cohesion index values. Cake strength values showed a significant increase (p

Isoflavones are group of phytochemicals associated with numerous benefits to the human health like prevention of cancer, cardiovascular diseases, osteoporosis and menopause symptoms. The major source of isoflavones in the diet is soybean, although many other legumes also contain these compounds. The aim of this study was to determine the content of individual isoflavones in different annual legume species collected in the Republic of Croatia and compare with soybean. Seven annual legume species: common bean (Phaseolus vulgaris L.), faba bean (Vicia faba L.), lentil (Lens culinaris Medic.), white lupin (Lupinus albus), chickpeas (Cicer arietinum L.), cowpea (Vigna unguiculata L.) and grass pea (Lathyrus sativus) were analysed and were compared with soybean (Glycine max (L.) Merr.).The ground samples of annual legume grains were extracted with ethanol and hydrolysed into their corresponding aglycones with hydrochloric acid. Isoflavones were identified by HPLC method, according to retention time compared with standards and quantified by the internal standard (β- naphthol) method. Total isoflavone content was expressed as sum of four isoflavones (aglycone): daidzein, genistein, glycitein and formononetin. The results showed wide variation in isoflavones content within and between species. The most dominant isoflavone, in all examined grain, was daidzein. The highest content of daidzein was found in grass pea accession S1 (123.61 mg/100g DM). In individual accessions of some examined legume species glycitein, genistein and formononetin was not detected. The accession Gradištanac (bean) had the highest content of glycitein and formononetin isoflavones (5.73 and 17.88 mg/100g DM), while the landrace faba bean from Pakovo selo had the highest content of genistein (67.96 mg/100g DM). The content of total isoflavones of 19 accessions of eight legume species ranged from 11.47 to 151.82 mg/100g DM, and the soybean species had the highest mean content with 120.9 mg/100g DM, while the greatest variation found within a grass pea species. The present study showed that examined legume species may be potential sources of isoflavones next to soybean.

Over 200 lactic acid bacteria (LAB) strains were isolated from autochthonous fresh cheeses and deposited in the Collection of Lactic Acid Bacteria (ZBMK) of Laboratory of Antibiotic, Enzyme, Probiotic and Starter Cultures Technologies, Faculty of Food Technology and Biotechnology, in order to selected Lactobacillus strains with important functional properties. After phenotypic characterisation, selected strains were assayed for their autoaggregation capacities and S-layer protein presence on bacterial cell surface by SDS-PAGE analysis, as these proteins are appointed to play important biological roles in Lactobacillus strains. Further research revealed that the autochthonous strain D6, which is identified as Lactobacillus brevis, is an S-layer protein carrying strain. This strain displayed the highest percentage of autoaggregation (70.37% ± 5.79 after 5h) among tested LAB strains, which has been affected after 5M guanidine hydrochloride removal of S-layer proteins (54.87±7.13 after 5h). Since S-layer proteins have been identified, between LAB, only among representatives of Lactobacillus species, it was of the special interest to study its functional role in Lb. brevis D6. Subsequently, the capacities of the Lb. brevis D6 to adhere to extracellular matrix proteins (ECM) collagen, fibronectin and lamin and to human intestinal epithelial Caco-2 cell lines in vitro were assessed. Removal of the intact S-layer proteins from the bacterial cell surface, reduced the adhesion of Lb. brevis D6 to fibronectin, collagen and laminin. Binding tests showed that in vitro adhesion to ECM proteins of Lb. brevis D6, is of a proteinase K-sensitive manner. Additionally, coaggregation abilities, pathogen exclusion and survival in simulated conditions of gastrointestinal tract (GIT) and during freeze drying of Lb. brevis D6, before and after guanidine hydrochloride extraction of S-layer proteins from cell surface, were examined. The results suggest that S-layer protein enhances certain probiotic activities, especially adhesion to different surfaces of Lb. brevis D6.

The aim of this study was to investigate the influence of isolated and characterized autochthonous starter cultures from the „Slavonski kulen“ in production technology and quality of „Slavonski kulen“. The raw mince production „Slavonski kulen“ (prepared according to traditional recipes) is added laboratory prepared lyophilized mixture of well defined autochthonous functional starter cultures composed by Lactobacillus and Staphylococcus strains. Prepared two groups of samples of „Slavonski kulen“ (with addition and without the addition of autochthonous starter culture bacteria (reference samples)) are in several stages of the manufacturing process carried out microbiological, physico-chemical and sensory analysis, continuous measurement of pH and losses on the weight of the“Slavonski kulen“. The results of the analysis showed that the addition of autochthonous starter culture bacteria in samples of „Slavonski kulen“ compared to reference samples, accelerates the ripening process, whereby the optimum sensory properties are achieved after 3 months, and that does not cause changes in specific smell, taste, colour and pH values which are typical of traditionally produced the sausages and reference samples. Also, the antimicrobial activity of autochthonous starter cultures reduced the risk of spoilage and contributed microbiological safety and longer shelf life of the product.

Paški cheese is the most famous Croatian cheese produced from raw ewe's milk. Complexity and varied sensory properties of such cheese manufactured from raw milk depends on ripening conditions, physico- chemical composition and microbial interactions. The objective of this study was to describe physico-chemical properties and microbial interactions of indigenous microflora (NSLAB) associated with the production of Paški cheese. Raw milk cheese samples were collected from three cheese dairy on the island Pag during ripening process. Cheese samples were collected in three batches of each cheese producer at day 0 and after 45 and 90 days of ripening. A total of 27 samples were subjected to the microbiological, biochemical and physico-chemical analyses. A total of 76 mesophilic lactobacilli isolates were investigated using culture-dependent approach combining phenotyping and genotyping. The combination of applied methods for identification has shown that L. brevis, L. plantarum and L. paracasei were predominant members of the NSALB population. The physico-chemical properties as pH, contents of dry matter, fat, protein and salt were analyzed. Results revealed that value of all measured parameters increases with ripening time, due to water loss during ripening. Additionally, the content of free amino acids (FAA), water-soluble oligopeptides, and LMWOA in cheese water extracts in ripened cheeses were analyzed. The results showed that dominant amino acids in ripened cheese were Ile, Lys, Glu, Val and Pro and total free amino acid content varied from 0.94 to 2.44 g FAA/100 g cheese dry matter. Dominant low-molecular weight organic acids were lactic acid, acetic acid and succinic acid. Obtained results can be considered as fundamental factors for the maintenance of the typical biochemical and sensorial features of Paški cheese which is base towards its selection, preservation and introduction for technological assessment and its PDO (Protected Designations of Origin) status.

In order to survive during the growth in a prolonged stationary phase in batch culture non-sporulating bacteria express wide variety of stress-response genes and alternative metabolic pathways. The lactic acid bacteria (LAB) are associated with various plant, animal and human niches and have ability to produce lactic acid during homo- or hetero-fermentative metabolism. Greater understanding of how LAB cells undergo change during prolonged starvation may facilitate new strategies for development of cell lines with advantageous traits. The purpose of this work was to examine the influence of long term incubation in nutrient wasted environment on Lactobacillus brevis L62 gene expression. The cells were grown for 75 days in spent MRS medium and 75-day-old isolates proteome was examined and compared with parent cells. Differential display estimated by 1-D and 2-D gel electrophoresis followed by MALDI-TOF/TOF protein identification revealed the key differences responsible for bacteria long-term survival in a hostile environment (deprivation of nutrition and detritus as growth media). Using two-dimensional electrophoresis, we quantitatively detected about 30 protein spots and identified 2 distinct proteins. Metabolic pathway changed on the proteome level could be successfully reconstructed by the data collected from the protein expression differences. A novel technique of de novo sequencing applied for the first time on presented model of Lactobacillus brevis revealed subtle differences in bacterial protein homology. Each variation is connected to different gene expression accomplished through the protein/peptide de novo sequencing.

The fluctuating stress conditions such as chemical and physical agents, low pH, low temperature and starvation increase the genomic mutation rate and influence on the lactic acid bacteria (LAB) evolution. On the base of common specific gene sets, species Lactobacillus brevis and Lactobacillus plantarum are classified as multi-niche LAB. We studied the survival response of L. brevis L62 and L. plantarum L73 during prolonged period of starvation, first cultured to the stationary phase in rich (MRS) and minimal (MML) medium and further kept in resultant spent medium, in terms of plasmid and chromosomal DNA status. The medium in which the cells were kept had a significant effect on the survival, plasmid status and chromosomal integrity of L. brevis L62 during 10 days starvation, as was shown in comparison between the MRS and MML medium. After 10 days starvation of L. brevis plasmid status has been investigated. The parent cells of L. brevis L62 and L. plantarum L73 contained three and two plasmids, respectively. The bacterium L. brevis L62 bears plasmids in size 5, 7 and 25 kb, while L. plantarum L73 harbors two plasmids approximately of 20 and 2.5 kb in size. These three plasmids were kept up in L. brevis during 10 days starvation in MML medium ; however after cultivation in rich MRS medium the smallest plasmid has been lost. It was noticed that curing of L. brevis L62 is connected with cell growth and low acidity media. Therefore, changes in gene expression of tested species were examined directly with observing protein status and indirectly controlling of appearance of antibiotic resistance, bacteriocins synthesis and sensitivity to hidrocloric acid and hydrogen peroxide. The antibiotic - resistance status of the both examined species was changed after prolonged pheriod of starvation. Better knowledge of L. brevis and L.plantarum genome evolution during prolonged starvation may facilitate new strategies for their development with advantageous characteristics.

The yeast cell wall consists of polysaccharides to which various proteins are attached. In order to investigate the role of non-covalently bound yeast cell wall proteins, Saccharomyces cerevisiae was transformed with BG1805 plasmids, each carrying gene for one protein of interest. Protein expression was under control of inducible GAL promoter. The aim was to determinate the mortality rate and detect possible morphological changes in wt+SCW4, wt+SCW10, wt+SCW11 and wt+BGL2. wt+SCW4 grown in media with no galactose added served as the negative control. Strains were monitored for two weeks after inoculation. The number of dead cells was estimated by methylene blue staining. All strains had increased mortality compared to the control. wt+SCW4 and wt+SCW10 show characteristic morphological changes which manifest as drastic cell volumen enlargement and presence of cells only with outline remains visible, so-called ghost cells. Characteristic volume enlargement could be the direct consequence of cell wall weakening that led to rupture of the cell and leakage of cellular contents. The fact that this features were not characteristic for wt+SCW11 and wt+BGL2 could indicate that this strains died for different reasons than wt+SCW4 and wt+SCW10.

Aflatoxin M1 (AFM1) is a very important toxin in milk and dairy products and it can be a potential threat to the health of consumers. Many microorganisms, including bacteria, yeasts, moulds, actinomycetes and algae are able to remove or degrade small amounts of aflatoxin in foods and feeds.Among the different potential decontaminating microorganisms, lactic acid bacteria represent unique groups, which are widely used in food fermentation and preservation. This study was carried out in order to determine the level of aflatoxin M1 contamination in raw milk, from randomly selected individual producers, from different regions of the continental Croatian, and to investigate the ability of dairy strains of lactic acid bacteria to degrade aflatoxin M1. Bacteria and AFM1 were incubated (48 h, 37 oC) and the amount of unbound AFM1 was determined by ELISA method. Lactobacillus rhamnosus strain GG (ATCC 53103) and Lactobacillus delbrueckii strain S1 can significantly (p