An Integrated Comprehensive Peptidomics and In Silico Analysis of Bioactive Peptide-Rich Milk Fermented by Three Autochthonous Cocci Strains.

Bioactive peptides (BPs) are molecules of paramount importance with great potential for the development of functional foods, nutraceuticals or therapeutics for the prevention or treatment of various diseases. A functional BP-rich dairy product was produced by lyophilisation of bovine milk fermented by the autochthonous strains Lactococcus lactis subsp. lactis ZGBP5-51, Enterococcus faecium ZGBP5-52 and Enterococcus faecalis ZGBP5-53 isolated from the same artisanal fresh cheese. The efficiency of the proteolytic system of the implemented strains in the production of BPs was confirmed by a combined high-throughput mass spectrometry (MS)-based peptidome profiling and an in silico approach. First, peptides released by microbial fermentation were identified via a non-targeted peptide analysis (NTA) comprising reversed-phase nano-liquid chromatography (RP nano-LC) coupled with matrix-assisted laser desorption/ionisation-time-of-flight/time-of-flight (MALDI-TOF/TOF) MS, and then quantified by targeted peptide analysis (TA) involving RP ultrahigh-performance LC (RP-UHPLC) coupled with triple-quadrupole MS (QQQ-MS). A combined database and literature search revealed that 10 of the 25 peptides identified in this work have bioactive properties described in the literature. Finally, by combining the output of MS-based peptidome profiling with in silico bioactivity prediction tools, three peptides (75QFLPYPYYAKPA86, 40VAPFPEVFGK49, 117ARHPHPHLSF126), whose bioactive properties have not been previously reported in the literature, were identified as potential BP candidates.

Modulation of the Gut Microbiota by the Plantaricin-Producing Lactiplantibacillus plantarum D13, Analysed in the DSS-Induced Colitis Mouse Model.

Lactiplantibacillus plantarum D13 shows antistaphylococcal and antilisterial activity, probably due to the synthesis of a presumptive bacteriocin with antibiofilm capacity released in the cell-free supernatant (CFS), whose inhibitory effect is enhanced by cocultivation with susceptible strains. An in silico analysis of the genome of strain D13 confirmed the pln gene cluster. Genes associated with plantaricin biosynthesis, structure, transport, antimicrobial activity, and immunity of strain D13 were identified. Furthermore, the predicted homology-based 3D structures of the cyclic conformation of PlnE, PlnF, PlnJ, and PlnK revealed that PlnE and PlnK contain two helices, while PlnF and PlnJ contain one and two helices, respectively. The potential of the strain to modulate the intestinal microbiota in healthy or dextran sulphate sodium (DSS)-induced colitis mouse models was also investigated. Strain D13 decreased the disease activity index (DAI) and altered the gut microbiota of mice with DSS-induced colitis by increasing the ratio of beneficial microbial species (Allobaculum, Barnesiella) and decreasing those associated with inflammatory bowel disease (Candidatus Saccharimonas). This suggests that strain D13 helps to restore the gut microbiota after DSS-induced colitis, indicating its potential for further investigation as a probiotic strain for the prevention and treatment of colitis.

Evaluation of the Probiotic Properties of Lacticaseibacillus casei 431® Isolated from Food for Special Medical Purposes§.

Research background: Increasing awareness of the importance of nutrition in health promotion and disease prevention has driven to the development of foods for special medical purposes (FSMPs). In this study, the probiotic strain Lacticaseibacillus paracasei ssp. paracasei (Lacticaseibacillus casei 431®) was incorporated into FSMPs to develop an innovative product. The aim was to investigate the influence of the FSMP matrix on the specific probiotic properties of L. casei 431® in vitro. Experimental approach: A series of in vitro experiments were performed as part of the probiotic approach. After evaluation of antibiotic susceptibility profiles, functional properties such as survival under simulated gastrointestinal tract (GIT) conditions, bile salt deconjugation activities, cholesterol assimilation, antagonistic activity against spoilage bacteria and adhesion to Caco-2 cell line monolayers and extracellular matrix proteins were investigated. Results and conclusions: The L. casei 431® strain, both the lyophilised strain and the strain isolated from the FSMP matrix, effectively survived the simulated adverse gastrointestinal conditions without significant effects of the food matrix. The effect of the FSMP matrix on the deconjugation activity of the bile salts of L. casei 431® was minimal; however, cholesterol assimilation was increased by 16.4 %. L. casei 431® had antibacterial activity against related lactic acid bacteria regardless of whether it was used in FSMPs or not. Conversely, the probiotic strain isolated from FSMP matrix had significantly higher inhibitory activity against six potential pathogens than the lyophilised culture. The autoaggregation ability of the L. casei 431® cells was not affected by the FSMP matrix. The adhesion of L. casei 431® bacterial cells to the extracellular matrix proteins was reduced after treatment with proteinase K, with the highest adhesion observed to laminin. The adhesion of L. casei 431® reduced the binding of E. coli 3014 by 1.81 log units and the binding of S. Typhimurium FP1 to Caco-2 cell lines by 1.85 log units, suggesting the potential for competitive exclusion of these pathogens. Novelty and scientific contribution: The results support the positive effect of the FSMP matrix on the specific probiotic properties of L. casei 431®, such as antibacterial activity, bile salt deconjugation and cholesterol assimilation, while the incorporation of this probiotic strain adds functional value to the FSMPs. The synergistic effect achieved by the joint application of L. casei 431® and innovative FSMP matrix contributed to the development of the novel formulation of an improved functional food product with added value.

The Human Milk Microbiota Produces Potential Therapeutic Biomolecules and Shapes the Intestinal Microbiota of Infants.

Human milk not only provides a perfect balance of nutrients to meet all the needs of the infant in the first months of life but also contains a variety of bacteria that play a key role in tailoring the neonatal faecal microbiome. Microbiome analysis of human milk and infant faeces from mother-breastfed infant pairs was performed by sequencing the V1-V3 region of the 16S rRNA gene using the Illumina MiSeq platform. According to the results, there is a connection in the composition of the microbiome in each mother-breastfed infant pair, supporting the hypothesis that the infant's gut is colonised with bacteria from human milk. MiSeq sequencing also revealed high biodiversity of the human milk microbiome and the infant faecal microbiome, whose composition changes during lactation and infant development, respectively. A total of 28 genetically distinct strains were selected by hierarchical cluster analysis of RAPD-PCR (Random Amplified Polymorphic DNA-Polymerase Chain Reaction) electrophoresis profiles of 100 strains isolated from human milk and identified by 16S RNA sequencing. Since certain cellular molecules may support their use as probiotics, the next focus was to detect (S)-layer proteins, bacteriocins and exopolysaccharides (EPSs) that have potential as therapeutic biomolecules. SDS-PAGE (Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis) coupled with LC-MS (liquid chromatography-mass spectrometry) analysis revealed that four Levilactobacillus brevis strains expressed S-layer proteins, which were identified for the first time in strains isolated from human milk. The potential biosynthesis of plantaricin was detected in six Lactiplantibacillus plantarum strains by PCR analysis and in vitro antibacterial studies. 1H NMR (Proton Nuclear Magnetic Resonance) analysis confirmed EPS production in only one strain, Limosilactobacillus fermentum MC1. The overall microbiome analysis suggests that human milk contributes to the establishment of the intestinal microbiota of infants. In addition, it is a promising source of novel Lactobacillus strains expressing specific functional biomolecules.

Lyophilized alginate-based microspheres containing Lactobacillus fermentum D12, an exopolysaccharides producer, contribute to the strain's functionality in vitro.

Lactobacillus (Limosilactobacillus) fermentum D12 is an exopolysaccharide (EPS) producing strain whose genome contains a putative eps operon. Whole-genome analysis of D12 was performed to disclose the essential genes correlated with activation of precursor molecules, elongation and export of the polysaccharide chain, and regulation of EPS synthesis. These included the genes required for EPS biosynthesis such as epsA, B, C, D and E, also gt, wzx, and wzy and those involved in the activation of the precursor molecules galE, galT and galU. Both the biosynthesis and export mechanism of EPS were proposed based on functional annotation. When grown on MRS broth with an additional 2% w/v glucose, L. fermentum D12 secreted up to 200 mg/L of a mixture of EPSs, whose porous structure was visualized by scanning electron microscopy (SEM). Structural information obtained by 1HNMR spectroscopy together with composition and linkage analyses, suggested the presence of at least two different EPSs, a branched heteropolysaccharide containing t-Glcp and 2,6-linked Galf, and glycogen. Since recent reports showed that polysaccharides facilitate the probiotic-host interactions, we at first sought to evaluate the functional potential of L. fermentum D12. Strain D12 survived simulated gastrointestinal tract (GIT) conditions, exhibited antibacterial activity against enteropathogenic bacteria, adhered to Caco-2 cells in vitro, and as such showed potential for in vivo functionality. The EPS crude extract positively influenced D12 strain capacity to survive during freeze-drying and to adhere to extracellular matrix (ECM) proteins but did not interfere Caco-2 and mucin adherence when added at concentrations of 0.2, 0.5, and 1.0 mg/mL. Since the viable bacterial count of free D12 cells was 3 logarithmic units lower after the exposure to simulated GIT conditions than the initial count, the bacterial cells had been loaded into alginate for viability improvement. Microspheres of D12 cells, which were previously analyzed at SEM, significantly influenced their survival during freeze-drying and in simulated GIT conditions. Furthermore, the addition of the prebiotic substrates mannitol and lactulose improved the viability of L. fermentum D12 in freeze-dried alginate microspheres during 1-year storage at 4 °C compared to the control.

A Lactic Acid Bacteria Consortium Impacted the Content of Casein-Derived Biopeptides in Dried Fresh Cheese.

This study aimed to define a consortium of lactic acid bacteria (LAB) that will bring added value to dried fresh cheese through specific probiotic properties and the synthesis of bioactive peptides (biopeptides). The designed LAB consortium consisted of three Lactobacillus strains: S-layer carrying Levilactobacillus brevis D6, exopolysaccharides producing Limosilactobacillus fermentum D12 and plantaricin expressing Lactiplantibacillus plantarum D13, and one Enterococcus strain, Enterococcus faecium ZGZA7-10. Chosen autochthonous LAB strains exhibited efficient adherence to the Caco-2 cell line and impacted faecal microbiota biodiversity. The cheese produced by the LAB consortium showed better physicochemical, textural and sensory properties than the cheese produced by a commercial starter culture. Liquid chromatography coupled with matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (LC-MALDI-TOF/TOF) showed the presence of 18 specific biopeptides in dried fresh cheeses. Their identification and relative quantification was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using multiple reaction monitoring (MRM). The results also showed that their synthesis resulted mainly from ß-casein and also α-S1 casein degradation by proteolytic activities of the LAB consortium. The designed LAB consortium enhanced the functional value of the final product through impact on biopeptide concentrations and specific probiotic properties.

The functional capacity of plantaricin-producing Lactobacillus plantarum SF9C and S-layer-carrying Lactobacillus brevis SF9B to withstand gastrointestinal transit.

BACKGROUND: We evaluated the functional capacity of plantaricin-producing Lactobacillus plantarum SF9C and S-layer-carrying Lactobacillus brevis SF9B to withstand gastrointestinal transit and to compete among the gut microbiota in vivo. Considering the probiotic potential of Lb. brevis SF9B, this study aims to investigate the antibacterial activity of Lb. plantarum SF9C and their potential for in vivo colonisation in rats, which could be the basis for the investigation of their synergistic functionality. RESULTS: A plantaricin-encoding cluster was identified in Lb. plantarum SF9C, a strain which efficiently inhibited the growth of Listeria monocytogenes ATCC® 19111™ and Staphylococcus aureus 3048. Homology-based three-dimensional (3D) structures of SF9C plantaricins PlnJK and PlnEF were predicted using SWISS-MODEL workspace and the helical wheel representations of the plantaricin peptide helices were generated by HELIQUEST. Contrary to the plantaricin-producing SF9C strain, the S-layer-carrying SF9B strain excluded Escherichia coli 3014 and Salmonella enterica serovar Typhimurium FP1 from the adhesion to Caco-2 cells. Finally, PCR-DGGE analysis of the V2-V3 regions of the 16S rRNA gene confirmed the transit of the two selected lactobacilli through the gastrointestinal tract (GIT). Microbiome profiling via the Illumina MiSeq platform revealed the prevalence of Lactobacillus spp. in the gut microbiota of the Lactobacillus-treated rats, even on the 10th day after the Lactobacillus application, compared to the microbiota of the healthy and AlCl3-exposed rats before Lactobacillus treatment. CONCLUSION: The combined application of Lb. plantarum SF9C and Lb. brevis SF9B was able to influence the intestinal microbiota composition in rats, which was reflected in the increased abundance of Lactobacillus genus, but also in the altered abundances of other bacterial genera, either in the model of healthy or aberrant gut microbiota of rats. The antibacterial activity and capacity to withstand in GIT conditions contributed to the functional aspects of SF9C and SF9B strains that could be incorporated in the probiotic-containing functional foods with a possibility to positively modulate the gut microbiota composition.

Influence of Dehydrated Wheat/Rice Cereal Matrices on Probiotic Activity of Bifidobacterium animalis ssp. lactis BB-12®§.

Three novel dehydrated wheat/rice cereal functional products with an addition of well documented probiotic Bifidobacterium animalis ssp. lactis BB-12® (BB-12®) were developed in Podravka factory for the infants older than 4 months: instant rice cereal, instant rice cereal with fruits and instant wheat cereal with vanilla. Notably, the number of viable BB-12® cells in each of the novel products was higher than the required minimal number of probiotic cells per gram of product (106 CFU/g) during the storage period of 106 weeks. Therefore, BB-12® strain recovery and genome stability were evaluated by strain-specific polimerase chain reaction and amplified fragment length polymorphism fingerprinting analysis. Further aim was to evaluate the influence of these three different cereal food matrices on specific probiotic properties of BB-12® strain in vitro. Applied food matrices positively influenced the survival in the simulated conditions of the gastrointestinal tract and antagonistic activity against undesirable microorganisms, while no influence on auto- and coaggregation ability of B. animalis ssp. lactis BB-12® was observed. Adhesion to extracellular matrix proteins and intestinal epithelial Caco-2 cells together with antibacterial activity emphasized competitive pathogen exclusion from Caco-2 cells by probiotic strain BB-12®.

Biosynthesis of Oxytetracycline by Streptomyces rimosus:Past, Present and Future Directions in the Developmentof Tetracycline Antibiotics.

Natural tetracycline (TC) antibiotics were the first major class of therapeutics to earn the distinction of 'broad-spectrum antibiotics' and they have been used since the 1940s against a wide range of both Gram-positive and Gram-negative pathogens, mycoplasmas, intracellular chlamydiae, rickettsiae and protozoan parasites. The second generation of semisynthetic tetracyclines, such as minocycline and doxycycline, with improved antimicrobial potency, were introduced during the 1960s. Despite emerging resistance to TCs erupting during the 1980s, it was not until 2006, more than four decades later, that a third--generation TC, named tigecycline, was launched. In addition, two TC analogues, omadacycline and eravacycline, developed via (semi)synthetic and fully synthetic routes, respectively, are at present under clinical evaluation. Interestingly, despite very productive early work on the isolation of a Streptomyces aureofaciens mutant strain that produced 6-demethyl-7-chlortetracycline, the key intermediate in the production of second- and third-generation TCs, biosynthetic approaches in TC development have not been productive for more than 50 years. Relatively slow and tedious molecular biology approaches for the genetic manipulation of TC-producing actinobacteria, as well as an insufficient understanding of the enzymatic mechanisms involved in TC biosynthesis have significantly contributed to the low success of such biosynthetic engineering efforts. However, new opportunities in TC drug development have arisen thanks to a significant progress in the development of affordable and versatile biosynthetic engineering and synthetic biology approaches, and, importantly, to a much deeper understanding of TC biosynthesis, mostly gained over the last two decades.

Degradation of Oxytetracycline, Streptomycin, Sulphathiazole and Chloramphenicol Residues in Different Types of Honey.

Some of the most frequently used antibiotics in apiculture for the treatment of bacterial brood diseases are oxytetracycline, chloramphenicol, sulphathiazole and streptomycin. Therefore, the aim of this research is to determine the degradation kinetics of the residua these antibiotics leave behind in different types of honey stored in dark at 25 °C. The examined honey samples (N=74) originating from the Croatian market had satisfactory physicochemical properties and pollen characteristics. Quantitative analyses of antibiotic residues were done using HPLC and ELISA methods. No antibiotic residues were found in any of the tested acacia, chestnut, floral, meadow and honeydew honey samples (N=74). In the next step, each of the tested honey samples was supplemented with one of the following antibiotics (in mg/kg): oxytetracycline 10, chloramphenicol 200, sulphathiazole 200 or streptomycin 0.5. Relatively high mass fractions of antibiotics have been added to the honey because the stability of each antibiotic is highly dependent on the spiked antibiotic mass fraction and also on its chemical structure. During a 6-month storage in the dark at 25 °C, the dynamics of degradation of the studied antibiotics was proven to differ dependent on the type of honey into which the antibiotic was added. The half-life of oxytetracycline in the acacia, floral, meadow and honeydew honey stored in the dark at 25 °C was 15, 16, 17 and 19 days, respectively, while in the chestnut honey the decomposition failed to be seen even after 60 days of storage. In all examined honey samples, the half-life of chloramphenicol and sulphathiazole was proven to be longer than 6 months. The fastest decomposition was seen of oxytetracycline added into the acacia honey, followed by streptomycin and sulphathiazole added into the same, while the longest decomposition delay was seen when chloramphenicol was added into the floral honey. According to the results obtained using a linear model of degradation kinetics, the longest oxytetracycline degradation was expected to occur in chestnut honey (116 days). Chloramphenicol and sulphathiazole are preserved the longest in floral (for 661 and 581 days, respectively) and streptomycin in meadow honey (for 321 days).

Limosilactobacillus fermentum strains MC1 and D12: Functional properties and exopolysaccharides characterization.

Lactic acid bacteria (LAB) produce a broad spectrum of exopolysaccharides (EPSs), commonly used as texturizers in food products. Due to their potential contribution to LAB probiotic properties, like adhesion to human epithelial cells and competitive exclusion of pathogens from human intestinal epithelial cells, this study was focussed on the structural and functional characterization of the EPSs produced by two Limosilactobacillus fermentum strains - MC1, originating from mother's milk, and D12, autochthonous from Croatian smoked fresh cheese. Whole-genome sequencing and functional annotation of both L. fermentum strains by RAST server revealed the genes involved in EPS production and transport, with some differences in functionally related genes. EPSs were extracted from the cell surface of both bacterial strains and purified by size-exclusion chromatography. Structural characterization of the EPSs, achieved by chemical analyses and 1D and 2D NMR spectroscopy, showed that both strains produce an identical mixture of three different EPSs containing galactofuranose and glucopyranose residues. However, a comparison of the functional properties showed that the MC1 strain adhered better to the Caco-2 cell line and exhibited stronger antimicrobial effect against Salmonella enterica serovar Typhimurium FP1 than the D12 strain, which may be attributed to the potential bacteriocin activity of the MC1 strain.

Proteolytic activity of probiotic strain Lactobacillus helveticus M92.

The aim of this research was to investigate the potential of previously defined probiotic strain Lactobacillus helveticus M92 as functional starter culture for fermented dairy products. Therefore, proteolytic activity of L. helveticus M92 was investigated and compared with those of different representatives of probiotic and starter culture strains. Cluster analysis of AFLP fingerprints showed a difference of L. helveticus M92 compared to five other L. helveticus strains, but the percentage of similarity confirmed the identification on species level. Casein hydrolysis by L. helveticus M92 was monitored by agar-well diffusion test, SDS-PAGE and Anson's method. L. helveticus M92 exhibited the highest proteolytic activity among tested probiotic and starter cultures strains with the fastest acidification rate and the highest pH decrease after overnight incubation in skim milk. The presence of prtH2 gene was confirmed by PCR amplification with specific primers, while PCR product was not obtained after amplification with primers specific to prtH. Furthermore, SDS-PAGE LC-MS/MS analysis of insoluble proteome of L. helveticus M92 enabled identification of several proteins involved in proteolytic system of L. helveticus such as protease PrtM as well as proteins involved in Opp peptide transport system and the intracellular peptidases PepE, PepN, and PepQ.

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.

Estimation of the Withdrawal Time of Levamisole in Eggs after Oral Administration to Laying Hens.

This study was conducted to evaluate withdrawal time of levamisole in eggs after oral administration in laying hens at different doses. Sampling of eggs was conducted for 37 days after the end of treatment, and levamisole concentrations were measured by liquid chromatography-tandem mass spectrometry validated according to the Commission Decision 2002/657/EC. Estimated validation parameters were as follows: decision limit, 0.54 µg/kg; detection capability, 0.56 µg/kg; limit of detection, 0.04 µg/kg; limit of quantification, 0.15 µg/kg; accuracy (recovery), between 92.9 and 102.3%; precision (relative standard deviation), ≤4.62%; and within-laboratory precision (relative standard deviation), ≤5.19%. Levamisole residue levels were significantly higher in egg yolks than in egg whites. The highest levels of levamisole were detected on day 2 posttreatment in groups receiving 50 mg/kg of body weight (556.2 µg/kg in egg yolks and 166.5 µg/kg in egg whites). Significant elimination occurred within 5 days after the cessation of treatment in all groups, with an elimination half-life of 1.3 days. Levamisole was still detectable on day 30 after the end of treatment in egg whites (0.06 µg/kg) and on day 37 in egg yolks (0.06 µg/kg). The longest withdrawal time for levamisole in eggs (14.9 days) was determined in a group treated with 25 mg of levamisole per kg of body weight for two consecutive days. According to the results, oral treatment of laying hens with levamisole may result in noncompliant egg samples even 14 days after treatment.

Generation of Lactobacillus plantarum strains with improved potential to target gastrointestinal disorders related to sugar malabsorption.

Malabsorption of dietary sugars is a common cause of gastrointestinal discomfort, affecting up to one in three people with debilitating symptoms, such as abdominal pain, osmotic diarrhoea, bloating and flatulence. Besides dietary interventions, it has been suggested that ingestion of lactobacilli may alleviate these symptoms. The objectives of this study were to generate strains with improved potential to ameliorate sugar malabsorption related gastrointestinal disorders. Initial selection was made from 183 natural isolates of lactic acid bacteria, on the basis of broad sugar fermentation ability, absence of gas production, gastrointestinal survival and susceptibility to important medical antimicrobials. Two strains of L. plantarum (KR6 and M5) exhibited favourable characteristics for all criteria, and were further optimised through random mutagenesis and selection approaches. Ultraviolet light (UV) exposure resulted in mutants characterized by better survival (for 1.9 log and 1.4 log) in gastrointestinal conditions. Subsequent exposure to ethyl methanesulfonate (EMS) provided mutants with greater tolerance to glucose induced catabolic repression. UV and UV-EMS mutants of L. plantarum M5 showed improved adhesion ability. As a result of this optimisation, L. plantarum MP2026 and L. plantarum MP2420 have been identified as promising candidates for probiotics, intended for alleviation of gastrointestinal discomfort originating from sugar malabsorption.

Genome Sequence of Lactobacillus brevis Strain D6, Isolated from Smoked Fresh Cheese.

The autochthonousLactobacillus brevisstrain D6, isolated from smoked fresh cheese, carries a 45-kDa S-layer protein. Strain D6 has shown adhesion to extracellular matrix proteins and to Caco-2 intestinal epithelial cells, as well as immunomodulatory potential and beneficial milk technological properties. Hence, it could be used as a potential probiotic starter culture for cheese production.

Characterisation of the yeast and mould biota in traditional white pickled cheeses by culture-dependent and independent molecular techniques.

Artisanal white pickled cheese of Western Serbia is a product of complex microbial community which detection by culture-dependent method only is hampered by its limitations. Thus, in the present study, we used a culture-independent, semi-quantitative technique based on construction of an internal transcribed spacer (ITS)-clone library from metagenomic DNA. This approach, based on direct DNA extraction followed by amplification of fungal internal transcribed regions (ITS) cloned into plasmid and restricted by endonucleases, revealed greater species richness in analysed cheeses and their by-products (17 species in total) compared to the more commonly used techniques of the culture-dependent method (8 species) and LSU-DGGE (10 species). The most frequently occurring yeast species which are commonly associated with cheeses production were Debaryomyces hansenii, Kluyveromyces lactis and Candida zeylanoides. On the other hand, Yarrowia lipolytica and Galactomyces geotrichum were detected only in one cheese sample. Moreover, some species, mainly moulds (Filobasidium globisporum, Cladosporium sp., Aspergillus sp. or Alternaria sp.) were identified only by culture-independent methods. The discrepancies between the techniques were confirmed by low correlation factor and by different indices of general biodiversity and dominance of species. The ITS-clone library approach provides the opportunity to analyse complex fungal communities associated with food products.

Antimicrobial activity of ibuprofen: new perspectives on an "Old" non-antibiotic drug.

Pharmaceutical industry has been encountering antimicrobial activity of non-antibiotics during suitability tests carried out prior to routine pharmacopoeial microbiological purity analysis of finished dosage forms. These properties are usually ignored or perceived as a nuisance during pharmaceutical analysis. The aim of this study was: (i) to compare the available data to our method suitability test results carried out on products containing ibuprofen, i.e. to demonstrate that method suitability can be a valuable tool in identifying new antimicrobials, (ii) to demonstrate the antimicrobial activity of ibuprofen and ibuprofen lysine. Microbiological purity method suitability testing was carried out according to European Pharmacopoeia (EP), chapters 2.6.12. and 2.6.13. Antimicrobial activity of ibuprofen and ibuprofen lysine was demonstrated by a disk diffusion method, a modification of the European Committee for Antimicrobial Susceptibility Testing method (EUCAST), against test microorganisms recommended in the EP. It was confirmed that ibuprofen may be responsible for the broad spectrum of antimicrobial activity of the tested products, and that method suitability tests according to the EP can indeed be exploited by the scientific community in setting guidelines towards future research of new antimicrobials. In the disk diffusion assay, inhibition zones were obtained with more than 62.5 µg and 250 µg for Staphylococcus aureus, 125 µg and 250 µg for Bacillus subtilis, 31.3 µg and 125 µg for Candidaalbicans, 31.3 µg and 62.5 µg for Aspergillusbrasiliensis, of ibuprofen/disk, and ibuprofen lysine/disk, respectively. For Escherichiacoli, Pseudomonasaeruginosa and Salmonellatyphimurium inhibition zones were not obtained. Antimicrobial activity of ibuprofen is considered merely as a side effect, and it is not mentioned in the patient information leaflets of ibuprofen drugs. As such, for the patient, it could represent an advantage, but, it could also introduce additional risks during usage. Further microbiological, pharmacological and clinical trials are of great importance.

Evaluation of probiotic potential of yeasts isolated from traditional cheeses manufactured in Serbia and Croatia.

AIM: The aim of this study was to investigate the in vitro probiotic potential of dairy yeast isolates from artisanal cheeses manufactured in Serbia and Croatia. MATERIALS AND METHODS: Twelve yeast strains isolated from artisanal fresh soft and white brined cheeses manufactured in Serbia and Croatia were used in the study. Survival in chemically-simulated gastrointestinal conditions, adherence to epithelial intestinal cells and proliferation of gut-associated lymphoid tissue (GALT) cells were evaluated. RESULTS: The results revealed that two strains of Kluyvereomyces lactis ZIM 2408 and ZIM 2453 grew above one log unit (Δ log CFU/ml) in the complex colonic medium during 24 h of cultivation, while Torulaspora delbrueckii ZIM 2460 was the most resistant isolate in chemically-simulated conditions of gastric juice and upper intestinal tract. It was demonstrated that the strains K. lactis ZIM 2408 and ZIM2441 and Saccharomyces cerevisiae ZIM 2415 were highly adhesive to Caco-2 cells, while strains K. lactis ZIM 2408 and Debaryomyces hansenii ZIM 2415 exhibit the highest adhesion percentage to HT29-MTX cells. All strains significantly (P < 0.0001) decreased the proliferation of GALT cells, suggesting the possible strain-specific immunomodulatory potential of the isolates. CONCLUSION: The dairy yeast isolates exhibit strain-specific probiotic properties, particularly the strain K. lactis ZIM 2408, which appears to be the best probiotic candidate in terms of all three criteria. Taking into account their immunomodulatory potential, the yeast isolates could be further tested for specific probiotic applications and eventually included in functional food formulated for patients suffering from diseases associated with an increased inflammatory status.

Traditionally produced sauerkraut as source of autochthonous functional starter cultures.

Spontaneous sauerkraut fermentation was performed at industrial scale in "Prehrana Inc.", Varazdin, in order to select autochthonous lactic acid bacteria (LAB) which were evaluated according probiotic criteria and tested for their capacity as probiotic starter cultures. At the end of the spontaneous sauerkraut fermentation, total LAB counts reached 9.0×10(5) CFU/ml. This underlines that the need for addition of the well characterised probiotic cultures, in appropriate viable cell counts, would be valuable in probiotic sauerkraut production. Phenotypic characterisation through API 50 CHL and SDS-PAGE of cell protein patterns revealed that Lactobacillus plantarum is predominant LAB strain in homofermentative phase of fermentation. Autochthonous LAB isolates SF1, SF2, SF4, SF9 and SF15 were selected based on the survival in in vitro gastrointestinal tract conditions. RAPD fingerprints indicated that the selected autochthonous LAB were distinct from one another. All of the strains efficiently inhibited the growth of indicator strains and satisfied technological properties such as acidification rate, tolerance to NaCl and viability during freeze-drying. Strains Lb. paraplantarum SF9 and Lb. brevis SF15, identified by AFLP DNA fingerprints, have shown the best properties to be applied as probiotic starter cultures, because of their highest adhesion to Caco-2 cells and expression of specific, protective S-layer proteins of 45 kDa in size. With addition of these strains, probiotic attribute of the sauerkraut will be achieved, including health promoting, nutritional, technological and economic advantages in large scale industrial sauerkraut production.