High-Power Ultrasound (HPU) and Pulsed Electric Field (PEF) in the Hurdle Concept for the Preservation of Antioxidant Bioactive Compounds in Strawberry Juice-A Chemometric Evaluation-Part II.

In this work, the influence of high-power ultrasound (HPU) followed by pulsed electric field (PEF) in the hurdle concept (HPU + PEF) on the content of biologically active compounds (BACs) and antioxidant activity in strawberry juices stored at 4 °C/7 days was investigated. The HPU was performed with an amplitude of 25% and pulse of 50% during 2.5, 5.0 and 7.5 min, while the PEF was performed with an electric field strength of 30 kV cm-1 and frequency of 100 Hz during 1.5, 3 and 4.5 min. The results obtained indicate that the synergy of the mechanisms of action for technologies in the hurdle concept plays a critical role in the stability of BACs and antioxidant activity. Juices treated with HPU + PEF hurdle technology and kept at 4 °C for 7 days showed a statistically significant decrease in all BACs, antioxidant capacity and pH. Shorter HPU + PEF treatment times favored the preservation of BACs in juices. Regarding total phenolic compounds, flavonols, condensed tannins and antioxidant capacity, optimization of hurdle parameters showed that a shorter HPU treatment time of 2.5 min provided the best yield of these compounds. In summary, by optimizing and adjusting the parameters of the HPU/PEF technology, it is possible to produce functional strawberry juice.

Prevalence, Genetic Diversity, and Quantification of the RNA Genome of the Hepatitis E Virus in Slaughtered Pigs in Serbia.

The goal of this study conducted in Serbia was to detect HEV in pig liver samples from slaughterhouses, retail outlets, and environmental swabs over the course of a year. All positive HEV samples were measured and expressed as HEV gene copy numbers per gram of sample, and a representative number of samples were sequenced using the Sanger approach. A total of 45 HEV-positive samples were re-amplified using nested RT-PCR employing CODEHOP primers targeting ORF2 (493 nucleotides). The average prevalence of the HEV genotype 3 in all pig liver samples from the slaughterhouses was 29%, while HEV prevalence was 44% in liver samples from animals younger than 3 months. HEV RNA was found in thirteen out of sixty (22%) environmental swab samples that were taken from different surfaces along the slaughter line. Our findings confirmed seasonal patterns in HEV prevalence, with two picks (summer and winter periods) during the one-year examination. Among HEV-positive samples, the average viral particles for all positive liver samples was 4.41 ± 1.69 log10 genome copies per gram. Phylogenetic analysis revealed the majority of HEV strains (43/45) from Serbia were grouped in the HEV-3a subtype, while two strains were classified into the HEV-3c subtype, and one strain could not be classified into any of the HEV-3 subtypes.

Determination of antilisterial effect of some microbial isolates from traditional zlatar cheese during the fermentation of soft white cheese.

The aim of this study was to select autochthonous lactic acid bacteria (LAB) isolates with antilisterial activity from Zlatar cheese and to evaluate the ability of selected LAB to control Listeria monocytogenes growth during soft white cheese production. The genotype characterization of isolated LAB (n = 93) was done using PCR method by 16S rRNA sequencing. In this way, the following isolates were detected: Lactococcus lactis ssp. lactis (40 isolates), Enterococcus faecalis (30), Lactobacillus plantarum (12), Leuconostoc mesenteroides ssp. mesenteroides (3) Lactobacillus garvieae (3), Lactobacillus curvatus (2), Lactobacillus casei (1), Enterococcus faecium (1) and Staphylococcus hominis (1). Each isolated LAB was tested for bacteriocin-producing ability. It was determined that two LAB isolates had bactericidal properties: Lactococcus lactis ssp. lactis SRB/ZS/094 and Enterococcus faecalis SRB/ZS/090. Semi-purified of enterococcal bacteriocin (enterocin) was isolated using precipitation procedures with ammonium sulphate. Its properties were determined (strength and range of activities). Isolated enterocin and bacteriocin-producing Lactococcus strain showed significant antimicribial activity against Listeria monocytogenes, but still the inhibition activity of Staphylococcus aurues and Escherichia coli was not detected. Based on the obtained laboratory results, in the second phase of the research, the antilisterial effect of bacteriocin isolated from Enterococcus faecalis SRB/ZS/090 and cells Lactococcus lactis ssp. lactis SRB/ZS/094 were determined, that are added as additives in the production of soft white cheese through five variants. Cheese supplemented with enterocin (E2) had the lowest aerobic mesophilic bacteria count, indicating that enterocin (E2) play an important role for bio-preservation.

Sustainable Removal of Tolperisone from Waters by Application of Photocatalysis, Nanotechnology, and Chemometrics: Quantification, Environmental Toxicity, and Degradation Optimization.

Environmental pollution is an emerging global issue. Heterogenous photocatalytic degradation, which belongs to the advanced oxidation processes, is a promising sustainable technique for the removal of harmful pollutants (e.g., pharmaceuticals) from natural resources (surface and underground waters), as well as wastewaters. In our study, we examined the efficiency of photocatalytic degradation (with TiO2 and ZnO as photocatalysts) of tolperisone hydrochloride (TLP) and the effect of TLP and its degradation intermediates on germination, photosynthetic capacity, and biomass production of wheat. According to the UFLC-DAD and LC-ESI-MS results, we found that the complete degradation of TLP can be reached after 60.83 min of UV irradiation using TiO2 as a photocatalyst. Furthermore, we determined that germination, biomass production, and chlorophyll b (Chl b) were not related to the percentage of TLP after irradiation. Chlorophyll a (Chl a) (r = -0.61, p ≤ 0.05), Chl a+b (r = -0.56, p ≤ 0.05), and carotenoid (car) (r = -0.57, p ≤ 0.05) were strongly inversely (negatively) correlated with TLP, while Chl a+b/car (r = 0.36, p ≤ 0.05) was moderately (positively) related.

Prevalence, characterization, and antibiotic susceptibility of Cronobacter spp. in a milk powder processing environment: The first reported case in Serbia.

Cronobacter spp. are opportunistic foodborne pathogens that most often infect neonates and infants through contaminated powdered infant formula. No reports have been published in Serbia on the prevalence of Cronobacter spp. in powdered milk production environments. Consequently, this study aimed to determine the prevalence, molecular characterization, antimicrobial susceptibility, and biofilm-forming ability of Cronobacter spp. isolated from a milk powder plant. Hundred samples were collected from the production facility. Fifteen Cronobacter sakazakii strains were isolated and identified, giving a contamination rate of 15%. Using multi-locus sequence typing, the isolates were divided into five sequence types (STs). Cronobacter sakazakii ST4 (50%), ST1 (16.67%), and ST83 (16.67%) were the dominant STs isolated. A novel sequence type (ST759) was identified and registered in the Cronobacter MLST database. The results of the antibiotic susceptibility testing indicated that C. sakazakii strains were susceptible to piperacillin/tazobactam, ampicillin/sulbactam, and amoxicillin/clavulanate, especially to meropenem and cefotaxime. Most of the ST4 showed moderate-to-strong biofilm-forming ability. The presence of clinically relevant isolates (ST4, ST1, ST83, and ST8) revealed that the production plant is likely a potential concern for public health. Finally, finding new sequence types like the one detected in this study (ST759) underlines evolving genetic changes in C. sakazakii.

Screening and Molecular Characterization of Hepatitis E Virus in Slaughter Pigs in Serbia.

Hepatitis E virus (HEV) is a zoonotic virus that can cause acute hepatitis in humans. Besides the fecal-oral route, transmission can occur by consumption of undercooked pig liver. Genotype 3 is the most frequent genotype found in Europe. Studies on HEV in slaughter-age pigs have not been conducted in Serbia so far. Pork meat production and consumption in Serbia is on average, higher than in the rest of Europe. With the aim to identify the circulating HEV genotypes, pig livers and swab samples from three pig slaughterhouses located in three different sub-regions of Serbia were collected. A nested RT-PCR was used to amplify the hypervariable HEV ORF-1 region (334 bp). The amplicons yielded in this study were sequenced, and a molecular phylogeny analysis based on the maximum likelihood method, including HEV sequences reported in several other countries, was performed. The average prevalence of HEV genotype 3 in 3-month-old pigs was 34%. Phylogenetic analysis revealed the majority of HEV amplification fragments from Serbia were grouped in four clades within sub-genotype 3a and were also genetically related to German, Italian, Slovenian, and American HEV sequences. Sub-genotypes 3b and 3j were also found in a single pig each. This study provides the first analysis of the genetic diversity and circulation dynamics of HEV in pigs at slaughterhouses in Serbia.

Effects of Temperature Abuse on the Growth and Staphylococcal Enterotoxin A Gene (sea) Expression of Staphylococcus aureus in Milk.

The aim of this study was to determine the effects of different temperatures and storage time on Staphylococcus aureus growth, sea gene expression, and synthesis of staphylococcal enterotoxin A (SEA) in the pasteurized and UHT-pasteurized milk. Pasteurized and UHT-pasteurized milk were inoculated with 3.98 log10 CFU/mL of S. aureus (ATCC 13565). Inoculated milk samples were stored at 8°C, 15°C, and 22°C for 24, 48, and 72 h, respectively. SEA synthesis was detected with a fully automated miniVIDAS instrument using the Enzyme-Linked Fluorescent Assay (ELFA) technology. The patterns of gene regulation were detected by quantitative reverse transcriptase PCR. The 2-ΔΔCT method has been used as a relative quantification strategy for gene expression responses data analysis. The results indicated that growth rate, sea gene expression, and SEA synthesis were influenced by type of milk, storage time, and temperature. Incubation of milk at different temperatures (15°C and 22°C) and times was used to simulate inadequate transport and storage conditions. Storage of pasteurized milk at 22°C for 24 h significantly upregulated the expression of sea gene compared with milk stored at 8°C, which coincides with the achieved S. aureus number of 105 CFU/mL and detected amount of SEA. In addition, storage of UHT-pasteurized milk at 22°C for 24 h and at 15°C for 48 h significantly upregulated the sea gene expression compared with milk stored at 8°C, which coincides with the detected amount of SEA and the dynamics of S. aureus number change. It can, therefore, be concluded that implementing good hygiene practices to avoid pre- and post-heat treatment milk contamination and maintaining the cold chain at temperature <8°C throughout the entire dairy production chain are of paramount importance to decrease the risk of staphylococcal food poisoning.

Presence of the esp gene in Enterococcus faecium derived from oropharyngeal microbiota of haematology patients.

OBJECTIVES: Antibiotic use and immunocompromised status in haematology patients have been shown to determine the constituents of commensal microbiota with highly increased resistance, including vancomycin resistant enterococci. We compared the carriage of virulence factor genes and the capacity for biofilm formation in vancomycin resistant enterococci (VRE) originating from the oropharyngeal and stool cultures of haematology patients. DESIGN: PCR tests were used to investigate the presence of genes encoding pathogenicity factors (esp and hyl) in VRE isolates. The genotype of vancomycin resistance was investigated by multiplex PCR tests for vanA and vanB genes. PFGE typing was conducted to exclude the duplicate isolates. RESULTS: Of 3310 pharyngeal swabs taken from inpatients at a clinic for haematology, Enterococcus species were recovered in 6.46%. All VRE investigated were identified as Enterococcus faecium and were highly vancomycin resistant. VanA genotype was confirmed in all. In the group of oropharyngeal carriers (n = 8 patients), 15 strains were recovered from oropharyngeal specimens and PFGE typing revealed 5 types and 3 subtypes. Identical types of VRE in the oropharynx and stool cultures were found in three patients from this group. In the group of stool carriers (n = 24 patients) VRE were obtained from stools only and placed in 21 macro-restriction patterns. The esp gene was more common in VRE isolated from the oropharynx than in isolates from stools (p = 0.014). Results were not significant when we compared the presence of hyl genes in oropharyngeal isolates with those from stool cultures (p = 0.66) or when we investigated the association between esp and hyl gene carriage and capability of biofilm formation in non-repeated VRE. CONCLUSIONS: In the present study, isolation of VRE from the oropharynx in haematology patients was associated with esp gene carriage. Further research is needed to investigate the clinical and long-term effects of this finding.

A review of the current situation of aflatoxin M1 in cow's milk in Serbia: risk assessment and regulatory aspects.

The aim of this systematic review is to provide information regarding the incidence and levels of aflatoxin M1 (AFM1) in raw and heat processed cow's milk in Serbia during 2015-16 and to compare these with collected data on the occurrence of AFM1 in raw milk and dairy products during the last decade in our region. Estimation of dietary exposure (EDI) and hazard index (HI) calculations for different age groups of the population were also carried out, based on the AFM1 content of milk samples and on available food consumption data in Serbia. AFM1 was detected in 69.9% (984/1408) of raw milk samples in 2015 versus 84.9% (3094/3646) in 2016, while in heat-processed milk, AFM1 was detected in 77.8% (364/468) in 2015 versus 98.5% (753/765) in 2016. On the basis of the obtained results, 450 (9%) of raw and 14 (1.1%) of heat-processed milk samples were contaminated with AFM1 levels above the maximum permitted level in Serbia (0.25 µg kg-1). However, a large percentage of raw and heat processed milk in Serbia (30.1% and 17.3%, respectively) was contaminated with AFM1 levels above the maximum permitted level regulated in the European Union (0.05 µg kg-1). Therefore, in order to protect consumer health, it is extremely important to further control the level of aflatoxins in milk, and this should be considered as a high priority for risk management actions.

Tracking of Listeria monocytogenes in meat establishment using Whole Genome Sequencing as a food safety management tool: A proof of concept.

Repeated Listeria outbreaks particularly associated with Ready-To-Eat (RTE) delicatessen meat products have been reported annually at global level. The most frequent scenario that led to foodborne outbreaks was the post-thermal treatment cross-contamination of deli meat products during slicing and modified atmosphere packaging (MAP). The precondition for such cross contamination is the previous introduction of Listeria into meat processing facilities and subsequent colonization of the production environment, associated with formation of biofilms resilient to common sanitation procedures regularly applied in meat establishments. The use of Whole Genome Sequencing (WGS) can facilitate the understanding of contamination and colonization routes of pathogens within the food production environment and enable efficient pathogen tracking among different departments. This study aimed to: a) provide a proof of concept on practical use of WGS in a meat establishment to define the entry routes and spread pattern of L. monocytogenes, and b) to consider the regular use of WGS in meat processing establishments as a strong support of food safety management system. The results revealed that Listeria spp. was present in slaughter line, chilling chambers, deboning, slicing, MAP, as well as in corridors and dispatch (53 positive samples, out of 240). Eight L. monocytogenes isolates (out of 53) were identified from the slaughterhouse, chilling chambers, deboning, MAP and dispatch. L. monocytogenes isolates were of three different serotypes (1/2a, 1/2c, 4b) and correspondingly of three MLST sequence types. Overall, two pairs of L. monocytogenes isolates were genetically identical, i.e. two serotype 4b isolates (ST1), isolated from water drain at dispatch unit and two isolates obtained from slaughterhouse (floorwall junction at the carcass wash point) and MAP (water drain). These findings indicated that L. monocytogenes isolates identified in meat processing units (MAP, chilling chamber and dispatch) originated from the slaughter line. Further, all eight L. monocytogenes isolates were confirmed to be biofilm producers on glass and stainless steel surfaces. The identification of the main entry routes of L. monocytogenes into meat establishments and tracking the routes for spread of the pathogen are of essential importance to define appropriate risk mitigation strategies for L. monocytogenes in meat production environment. The routine use of WGS for bacterial characterization, as a strong support of food safety management system in meat establishments, will require the cost-effective approach. It may encompass in-house sequencing when sequencing equipment is used for multiple applications (e.g. WGS of pathogens, starter cultures and spoilage organisms).

Influence of Environmental Conditions on Norovirus Presence in Mussels Harvested in Montenegro.

This study comprises the first systematic survey of the occurrence of Norovirus in Mediterranean mussels from harvesting areas in Montenegro coast of Adriatic Sea. Mussels may accumulate contaminants of public health concern, including pathogenic bacteria and viruses. Microbiological monitoring of harvesting areas is based on count of Escherichia coli in bivalve molluscs in the European Union. It is assumed that E. coli does not reflect contamination with enteric viruses. A structured field study was undertaken at six locations in Bay of Kotor, Montenegro, in order to investigate plausible influence of environmental factors on the variability of E. coli and norovirus (NoV). From July 2015 to July 2016, a total of 72 samples of mussels were collected in coastal harvesting areas of the Montenegro. The samples were screened for NoV of genogroups GI and GII using reverse transcription-qPCR (RT-qPCR). There were 43% NoV positive samples with higher presence of genogroup GII (74.2%). With regard to influence of environmental conditions on Norovirus presence, we have proved seasonal pattern of virus occurrence i.e., the largest number of positive samples was noticed during winter, while other physico-chemical factors were not of great significance. It was found that count of E. coli did not correlate with Norovirus prevalence. From the aspect of food safety, an upgrade of monitoring plans could lead to obtaining safer products.

Molecular typing, pathogenicity factor genes and antimicrobial susceptibility of vancomycin resistant enterococci in Belgrade, Serbia.

In this study the distribution of species and antimicrobial resistance among vancomycin resistant enterococci (VRE) recovered from clinical specimens obtained from five hospitals in Belgrade was analyzed. Strains were further characterized by pulsed-field gel electrophoresis (PFGE). Polymerase chain reaction (PCR) was used to investigate the presence of vanA and vanB genes and pathogenicity factor genes. Identification of 194 VRE isolates revealed 154 Enterococcus faecium, 21 Enterococcus faecalis, 10 Enterococcus raffinosus and 9 Enterococcus gallinarum. This study revealed existence of 8 major clones of VRE. PCR determined vanA gene to be present in all of the VRE studied. Esp and hyl genes were present in 29.22% and 27.92% of E. faecium, respectively, and in 76.19% and 0 of E. faecalis, respectively. Esp and hyl genes were not found more frequently in members of predominant clones of E. faecium than in single isolates; nor was their presence connected to invasiveness.