Assessment of the Antimicrobial, Antioxidant, and Antiproliferative Potential of Sideritis raeseri subps. raeseri Essential Oil.

The aim of the present study was to investigate the antimicrobial potential of Sideritis raeseri subps. raeseri essential oil (EO) against common food spoilage and pathogenic microorganisms and evaluate its antioxidant and antiproliferative activity. The EO was isolated by steam distillation and analyzed by GC/MS. The main constituents identified were geranyl-p-cymene (25.08%), geranyl-γ-terpinene (15.17%), and geranyl-linalool (14.04%). Initially, its activity against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Listeria monocytogenes, Salmonella Enteritidis, Salmonella Typhimurium, Pseudomonas fragi, Saccharomyces cerevisiae, and Aspergillus niger was screened by the disk diffusion method. Subsequently, minimum inhibitory concentration (MIC), non-inhibitory concentration (NIC), and minimum lethal concentration (MLC) values were determined. Growth inhibition of all microorganisms tested was documented, although it was significantly lower compared to gentamycin, ciproxin, and voriconazole, which were used as positive controls. In a next step, its direct antioxidant properties were examined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays, and the IC50 values were determined. The potential cytoprotective activity of the oil against H2O2-induced oxidative stress and DNA damage was studied in human immortalized keratinocyte (HaCaT) cells using the comet assay. Finally, the antiproliferative activity of the oil was evaluated against a panel of cancer cell lines including A375, Caco2, PC3, and DU145 and the non-cancerous HaCaT cell line using the sulforhodamine B (SRB) assay, and the EC50 values were determined. The oil demonstrated weak radical scavenging activity, noteworthy cytoprotective activity against H2O2-induced oxidative stress and DNA damage in HaCaT cells, and antiproliferative activity against all cell lines tested, being more sensitive against the in vitro model of skin melanoma.

Effect of Sugar Content on Quality Characteristics and Shelf-Life of Probiotic Dry-Fermented Sausages Produced by Free or Immobilized Lactobacillus casei ATCC 393.

The aim of the present study was to investigate the effect of sugar content (0, 0.30, and 0.60%) on quality attributes and shelf-life of dry-fermented sausages stored for 66 days containing free or immobilized Lactobacillus casei ATCC 393 on wheat. For comparison, dry-fermented sausages with no starter culture were also produced. Physicochemical characteristics ranged within the levels usually observed in fermented sausages, while a drastic decrease was recorded in numbers of enterobacteria, staphylococci, and pseudomonads during ripening in all cases. Noticeably, sugar addition and the probiotic culture resulted in significant increase of shelf-life, whereas levels of L. casei ATCC 393 after 66 days of ripening persisted above 6 log cfu/g. Sugar addition had a positive effect on sensory attributes; although all products were of high quality, the immobilized cells provided a distinctive characteristic aroma and a fine taste.

Effect of curing salts and probiotic cultures on the evolution of flavor compounds in dry-fermented sausages during ripening.

The effect of the concentration of curing salts, the nature of the probiotic culture (free or immobilized Lactobacillus casei ATCC 393 on wheat grains) and the ripening time on the generation of volatile compounds in probiotic dry-fermented sausages was investigated. Samples were collected after 1, 28 and 71 days of ripening and subjected to SPME GC/MS analysis. All factors affected significantly the concentration of all volatile compounds. Noticeably, the content of esters, organic acids and total volatiles was significantly increased after 28 and 71 days of ripening in almost all products. In most cases, reduction of curing salts content resulted in significant increase of esters and organic acids during ripening, whereas the opposite effect was observed in carbonyl compounds at days 28 and 71. Principal Component Analysis of the semi-quantitative data revealed that primarily the ripening process affected the volatile composition.

Effect of immobilized Lactobacillus casei on volatile compounds of heat treated probiotic dry-fermented sausages.

The effect of the amount of immobilized Lactobacillus casei ATCC 393 on wheat grains on the generation of volatile compounds during the production of heat treated probiotic dry-fermented sausages was investigated. For comparison reasons, sausages containing free L. casei cells or no starter culture as well as a similar commercial product were also included in the study. Samples ripened for 8 days and heat treated to 70-72°C for 8-10 min were subjected to Solid Phase Microextraction (SPME) Gas Chromatography/Mass Spectrometry (GC/MS) analysis. The starter culture affected significantly the production of volatile compounds. The highest content of esters and alcohols was observed in the sample containing 30 g of immobilized cells/kg of stuffing mixture, while the highest concentration of organic acids was observed in the sausages with no starter culture. In contrast, the commercial product contained the lowest concentration of volatiles. Principal component analysis of the semi-quantitative data revealed that the volatile composition was affected primarily by the nature and concentration of the starter culture.

In vitro study on the cell adhesion ability of immobilized lactobacilli on natural supports.

The aim of the present study was to investigate the effect of probiotic immobilization onto wheat grains, both wet and freeze dried, on the adhesion properties of the probiotic cells and make comparisons with wet and freeze dried free cells. Lactobacillus casei ATCC 393 and Lactobacillus plantarum NCIMB 8826 were used as model probiotic strains. The results showed satisfactory adhesion ability of free cells to a monolayer of Caco-2 cells (>1000CFU/100 Caco-2 cells for wet cells). Cell immobilization resulted in a significant decrease in adhesion, for both wet and freeze dried formulations, most likely because immobilized cells did not have direct access to the Caco-2 cells, but it still remained in adequate levels (>100CFU/100 Caco-2 cells for wet cells). No clear correlation could be observed between cell adhesion and the hydrophobicity of the bacterial cells, measured by the hexadecane adhesion assay. Most notably, immobilization enhanced the monolayer integrity of Caco-2 cells, demonstrated by a more than 2-fold increase in transepithelial electrical resistance (TEER) compared to free cells. SEM micrographs ascertained the adhesion of both immobilized and free cells to the brush border microvilli. Finally, the impact of the food matrix on the adhesion properties of probiotic bacteria and on the design of novel functional products is discussed.

Effect of immobilized Lactobacillus casei on the evolution of flavor compounds in probiotic dry-fermented sausages during ripening.

The effect of immobilized Lactobacillus casei ATCC 393 on wheat grains on the generation of volatile compounds in probiotic dry-fermented sausages during ripening was investigated. For comparison reasons, sausages containing free L. casei cells or no starter culture were also included in the study. Samples were collected after 1, 28 and 45days of ripening and subjected to SPME GC/MS analysis. Both the probiotic culture and the ripening process affected significantly the concentration of all volatile compounds. The significantly highest content of total volatiles, esters, alcohols and miscellaneous compounds was observed in sausages containing the highest amount of immobilized culture (300g/kg of stuffing mixture) ripened for 45days. Principal component analysis of the semi-quantitative data revealed that primarily the concentration of the immobilized probiotic culture affected the volatile composition.

Free and immobilized Lactobacillus casei ATCC 393 on whey protein as starter cultures for probiotic Feta-type cheese production.

The use of free and immobilized Lactobacillus casei ATCC 393 on whey protein as starter culture in probiotic Feta-type cheese production was evaluated. The probiotic cultures resulted in significantly higher acidity; lower pH; reduced counts of coliforms, enterobacteria, and staphylococci; and improved quality characteristics compared with cheese with no culture. Microbiological and strain-specific multiplex PCR analysis showed that both free and immobilized L. casei ATCC 393 were detected in the novel products at levels required for conferring a probiotic effect at the end of the ripening. The effect of starter culture on production of volatile compounds was investigated by the solid-phase microextraction gas chromatography-mass spectrometry analysis technique. The immobilized cells resulted in an improved profile of aroma-related compounds and the overall high quality of the novel products was ascertained by the preliminary sensory test. Finally, the high added value produced by exploitation of whey, which is an extremely polluting industrial waste, was highlighted and assessed.

Effective survival of immobilized Lactobacillus casei during ripening and heat treatment of probiotic dry-fermented sausages and investigation of the microbial dynamics.

The aim was the assessment of immobilized Lactobacillus casei ATCC 393 on wheat in the production of probiotic dry-fermented sausages and the investigation of the microbial dynamics. For comparison, sausages containing either free L. casei ATCC 393 or no starter culture were also prepared. During ripening, the numbers of lactobacilli exceeded 7 log cfu/g, while a drastic decrease was observed in enterobacteria, staphylococci and pseudomonas counts. Microbial diversity was further studied applying a PCR-DGGE protocol. Members of Lactobacillus, Leuconostoc, Lactococcus, Carnobacterium, Brochothrix, Bacillus and Debaryomyces were the main microbial populations detected. Microbiological and strain-specific multiplex PCR analysis confirmed that the levels of L. casei ATCC 393 in the samples after 66 days of ripening were above the minimum concentration for conferring a probiotic effect (≥ 6 log cfu/g). However, after heat treatment, this strain was detected at the above levels, only in sausages containing immobilized cells.

Monitoring survival of Lactobacillus casei ATCC 393 in probiotic yogurts using an efficient molecular tool.

The aim of the present study was to monitor the survival of the probiotic strain Lactobacillus casei ATCC 393 during refrigerated storage of natural regular yogurts compared with Lactobacillus delbrueckii ssp. bulgaricus. Both free and immobilized cells on supports of high industrial interest, such as fruits and oat pieces, were tested. Microbiological and strain-specific multiplex PCR analysis showed that both free and immobilized Lb. casei ATCC 393 were detected in the novel products at levels required to confer a probiotic effect (at least 6 log cfu/g) for longer periods than required by the dairy industry (≥ 30 d) during storage at 4°C. In contrast, the viable bacterial density of Lb. delbrueckii ssp. bulgaricus decreased to levels <6 log cfu/g after 14 d of cold storage. Of note, the final pH of all products was 4.2 to 4.3. Acid resistance or cold tolerance of Lb. casei ATCC 393 apparently allows for increased survival compared with Lb. delbrueckii ssp. bulgaricus in these yogurt formulations.

Distinct adhesion of probiotic strain Lactobacillus casei ATCC 393 to rat intestinal mucosa.

Adhesion to the intestine represents a critical parameter for probiotic action. In this study, the adhesion ability of Lactobacillus casei ATCC 393 to the gastrointestinal tract of Wistar rats was examined after single and daily administration of fermented milk containing either free or immobilized cells on apple pieces. The adhesion of the probiotic cells at the large intestine (cecum and colon) was recorded at levels ≥6 logCFU/g (suggested minimum levels for conferring a probiotic effect) following daily administration for 7 days by combining microbiological and strain-specific multiplex PCR analysis. Single dose administration resulted in slightly reduced counts (5 logCFU/g), while they were lower at the small intestine (duodenum, jejunum, ileum) (≤3 logCFU/g), indicating that adhesion was a targeted process. Of note, the levels of L. casei ATCC 393 were enhanced in the cecal and colon fluids both at single and daily administration of immobilized cells (6 and 7 logCFU/g, respectively). The adhesion of the GI tract was transient and thus daily consumption of probiotic products containing the specific strain is suggested as an important prerequisite for retaining its levels at an effective concentration.

Effect of probiotic-fermented milk administration on gastrointestinal survival of Lactobacillus casei ATCC 393 and modulation of intestinal microbial flora.

The aim of the present study was to assess the survival of free and immobilized Lactobacillus casei ATCC 393 on apple pieces, contained in probiotic-fermented milk, after gastrointestinal (GI) transit and to investigate the potential regulation of intestinal microbial flora in a rat model. In in vitro GI stress tolerance tests, immobilized L. casei ATCC 393 exhibited significantly higher survival rates compared to free cells. At a second stage, probiotic-fermented milk produced by either free or immobilized cells was administered orally at a single dose or daily for 9 days in Wistar rats. By 12 h after single-dose administration, both free and immobilized cells were detected by microbiological and molecular analysis at levels ≥6 logCFU/g of feces. Moreover, daily administration led to significant reduction of staphylococci, enterobacteria, coliforms and streptococci counts. In conclusion, L. casei ATCC 393 contained in fermented milk survived GI transit and modulated intestinal microbiota.