Sweet Wine Production from the Side-Stream of Industrial Corinthian Currant Processing: Product Quality, Antioxidant Capacity, and Volatilome.

In the frame of efforts to add value to the Mediterranean currant cultivation and processing sectors, which is essential for their sustainability, sweet wine production is proposed from the finishing side-stream (FSS) of premium quality Corinthian currants, involving complete fermentation using an alcohol-tolerant yeast followed by (i) the addition of FSS to extract sugars or (ii) syrup made from FSS to adjust sweetness. Wine was also made by (iii) ceasing fermentation at the desired sugar level by ethanol addition. The non-fortified wines had 15.2-15.5% ethanol, 115-145 g/L residual sugar, 7.2-7.6 g/L titratable acidity, low volatile acidity (VA; <0.33 g/L), 280-330 mg/L phenolic content (TPC) (as gallic acid), and 23.8-35.6 mg/L antioxidant capacity (AC) (as ascorbic acid). In total, 160 volatiles were identified by SPME GC-MS, including compounds derived from the grapes, the raisin drying, and the fermentation process. The non-fortified wines had better characteristics (mainly VA, AC, and TPC) than the fortified wine, while sweetness adjustment by FSS is the simplest and lowest cost method since it does not involve ethanol or syrup addition. The proposed methods can lead to good quality sweet wines with a characteristic fruity (grape/raisin) flavor that could be commercialized as specialty raisin beverages or liqueurs.

Antimicrobial activity of essential oils of cultivated oregano (Origanum vulgare), sage (Salvia officinalis), and thyme (Thymus vulgaris) against clinical isolates of Escherichia coli, Klebsiella oxytoca, and Klebsiella pneumoniae.

BACKGROUND: Oregano (Origanum vulgare), sage (Salvia officinalis), and thyme (Thymus vulgaris) are aromatic plants with ornamental, culinary, and phytotherapeutic use all over the world. In Europe, they are traditionally used in the southern countries, particularly in the Mediterranean region. The antimicrobial activities of the essential oils (EOs) derived from those plants have captured the attention of scientists as they could be used as alternatives to the increasing resistance of traditional antibiotics against pathogen infections. Therefore, significant interest in the cultivation of various aromatic and medicinal plants is recorded during the last years. However, to gain a proper and marketable chemotype various factors during the cultivation should be considered as the geographical morphology, climatic, and farming conditions. In this frame, we have studied the antimicrobial efficiency of the EOs from oregano, sage, and thyme cultivated under different conditions in a region of NE Greece in comparison to the data available in literature. METHODS: Plants were purchased from a certified supplier, planted, and cultivated in an experimental field under different conditions and harvested after 9 months. EOs were extracted by using a Clevenger apparatus and tested for their antibacterial properties (Minimum inhibitory concentration - MIC) against clinical isolates of multidrug resistant Escherichia coli (n=27), Klebsiella oxytoca (n=7), and Klebsiella pneumoniae (n=16) strains by using the broth microdilution assay. RESULTS: Our results showed that the most sensitive organism was K. oxytoca with a mean value of MIC of 0.9 µg/mL for oregano EOs and 8.1 µg/mL for thyme. The second most sensitive strain was K. pneumoniae with mean MIC values of 9.5 µg/mL for thyme and 73.5 µg/mL for oregano EOs. E. coli strains were among the most resistant to EOs antimicrobial action as the observed MICs were 24.8-28.6 µg/mL for thyme and above 125 µg/mL for thyme and sage. Most efficient were the EOs from thyme followed by those of oregano. CONCLUSIONS: With MIC values above 150 µg/mL, sage EOs did not show any antibacterial efficiency against the majority of the strains. However, no significant differences were observed concerning the antimicrobial action of all EOs originating from irrigated versus non-irrigated cultivated aromatic plants.

Chemical Profile Characterization of Fruit and Vegetable Juices after Fermentation with Probiotic Strains.

Lactic acid bacteria (LAB) are widely applied for fermentation purposes in dairy and non-dairy food matrices with beneficial technological and health-promoting properties. This study describes the effect of two lactic acid bacteria, namely, Lactiplantibacillus paracasei SP5 and Pediococcus pentosaceus SP2, on the phenolic profiles, antioxidant activities, total phenolic content (TPC), carotenoid content, and sensorial profile of two different mixed fruit juices. After 48 h of fermentation, both LABs retained viability over 9 Log CFU/mL in both juices. The TPC, zeaxanthin + lutein, ß-carotene content, and antioxidant activity (AA) were elevated for both LABs and mixed juices after 48 h of fermentation compared to control samples. Regarding the phenolic profile, both juices exhibited a significant decrease in chlorogenic acid levels, while quinic acid and tyrosol concentrations showed notable increases.

Assessment of the Physicochemical, Antioxidant, Microbial, and Sensory Attributes of Yogurt-Style Products Enriched with Probiotic-Fermented Aronia melanocarpa Berry Juice.

The aim() of this study was to create() various formulations of yogurt enriched with freeze()-dried adjuncts, namely() (i) probiotic Lactobacillus plantarum ATCC 14917 culture(), and (ii) L. plantarum ATCC 14917 fermented black chokeberry juice, along with a commercial() starter culture(). The goal was to enhance() functionality and optimize the nutritional() value() of the products. These new yogurt-style() formulations were subsequently() compared with commercially produced yogurt. All products demonstrated() favorable() physicochemical properties, and the probiotic strain() consistently() maintained viable() levels exceeding 7 log() cfu/g throughout() the entire() storage() period(). The fermented milk produced with the adjunct-free L. plantarum cells, as well as the yogurt produced with the proposed() lactobacilli-fermented chokeberry juice, exhibited the highest lactic acid() production() (1.44 g/100 g yogurt by the end of storage()). Levels of syneresis were observed at lower() values() in yogurt produced with freeze()-dried fermented chokeberry juice. Yogurts prepared() with the lactobacilli-fermented freeze()-dried chokeberry juice displayed elevated total() phenolic content() and antioxidant capacity() (25.74 µg GAE/g and 69.05 µmol TE/100 g, respectively()). Furthermore, sensory tests revealed a distinctive() fruity flavor() in samples incorporating fermented juice. The results demonstrate() that probiotic L. plantarum-fermented chokeberry juice enhances() both the antioxidant capacity() and the viability of beneficial() bacteria() in yogurt while it can be readily() applied and commercialized, especially in the form of a freeze()-dried formulation.

Natural Carotenoids: Recent Advances on Separation from Microbial Biomass and Methods of Analysis.

Biotechnologically produced carotenoids occupy an important place in the scientific research. Owing to their role as natural pigments and their high antioxidant properties, microbial carotenoids have been proposed as alternatives to their synthetic counterparts. To this end, many studies are focusing on their efficient and sustainable production from renewable substrates. Besides the development of an efficient upstream process, their separation and purification as well as their analysis from the microbial biomass confers another important aspect. Currently, the use of organic solvents constitutes the main extraction process; however, environmental concerns along with potential toxicity towards human health necessitate the employment of "greener" techniques. Hence, many research groups are focusing on applying emerging technologies such as ultrasounds, microwaves, ionic liquids or eutectic solvents for the separation of carotenoids from microbial cells. This review aims to summarize the progress on both the biotechnological production of carotenoids and the methods for their effective extraction. In the framework of circular economy and sustainability, the focus is given on green recovery methods targeting high-value applications such as novel functional foods and pharmaceuticals. Finally, methods for carotenoids identification and quantification are also discussed in order to create a roadmap for successful carotenoids analysis.

Genetic and phenotypic assessment of the antimicrobial activity of three potential probiotic lactobacilli against human enteropathogenic bacteria.

Introduction: Lactobacilli are avid producers of antimicrobial compounds responsible for their adaptation and survival in microbe-rich matrices. The bactericidal or bacteriostatic ability of lactic acid bacteria (LAB) can be exploited for the identification of novel antimicrobial compounds to be incorporated in functional foodstuffs or pharmaceutical supplements. In this study, the antimicrobial and antibiofilm properties of Lactiplantibacillus pentosus L33, Lactiplantibacillus plantarum L125 and Lacticaseibacillus paracasei SP5, previously isolated form fermented products, were examined, against clinical isolates of Staphylococcus aureus, Salmonella enterica subsp. enterica serovar Enteritidis and Escherichia coli. Methods: The ability of viable cells to inhibit pathogen colonization on HT-29 cell monolayers, as well as their co-aggregation capacity, were examined utilizing the competitive exclusion assay. The antimicrobial activity of cell-free culture supernatants (CFCS) was determined against planktonic cells and biofilms, using microbiological assays, confocal microscopy, and gene expression analysis of biofilm formation-related genes. Furthermore, in vitro analysis was supplemented with in silico prediction of bacteriocin clusters and of other loci involved in antimicrobial activity. Results: The three lactobacilli were able to limit the viability of planktonic cells of S. aureus and E. coli in suspension. Greater inhibition of biofilm formation was recorded after co-incubation of S. enterica with the CFCS of Lc. paracasei SP5. Predictions based on sequence revealed the ability of strains to produce single or two-peptide Class II bacteriocins, presenting sequence and structural conservation with functional bacteriocins. Discussion: The efficiency of the potentially probiotic bacteria to elicit antimicrobial effects presented a strain- and pathogen-specific pattern. Future studies, utilizing multi-omic approaches, will focus on the structural and functional characterization of molecules involved in the recorded phenotypes.

Nutritional Improvements of Sourdough Breads Made with Freeze-Dried Functional Adjuncts Based on Probiotic Lactiplantibacillus plantarum subsp. plantarum and Pomegranate Juice.

New types of sourdough breads are proposed, made with freeze-dried sourdough adjuncts based on: (i) Lactiplantibacillus plantarum subsp. plantarum ATCC 14917, a potential probiotic (LP) alone or (ii) with the addition of unfermented pomegranate juice (LPPO) and (iii) pomegranate juice fermented by the same strain (POLP). Physicochemical, microbiological, and nutritional characteristics (in vitro antioxidant capacity, AC, total phenolics, TPC, and phytate content) of the breads were evaluated and compared with commercial sourdough bread. All adjuncts performed well; the best results being those obtained by POLP. Specifically, the highest acidity (9.95 mL of 0.1 M NaOH) and organic acid content (3.02 and 0.95 g/kg, lactic and acetic acid, respectively) as well as better resistance to mold and rope spoilage (12 and 13 days, respectively) were observed for POLP3 bread (sourdough with 6% POLP). Significant nutritional improvements were observed by all adjuncts, in terms of TPC, AC, and phytate reduction (103 mg gallic acid/100 g, 232 mg Trolox/100 g, and 90.2%, respectively, for POLP3). In all cases, the higher the amount of adjunct, the better the results. Finally, the good sensory properties of the products indicate the suitability of the proposed adjuncts for sourdough breadmaking, while their application in freeze-dried, powdered form can facilitate commercial application.

Microbiological stability and sensorial valorization of thyme and oregano essential oils alone or combined with ethanolic pomegranate extracts in wine marinated pork meat.

In the present study, wine-based marinades containing ethanolic extract from pomegranate (Punica granatum L.), alone or in combination with two Essential Oils (Thyme & Oregano), were used for pork fillets marination and their antimicrobial activity, as well as their sensorial impact were evaluated. Likewise, the marinades exhibited promising results concerning their recorded antimicrobial activity versus Enterobacteriaceae, Total Mesophilic Bacteria, Yeasts/molds, Staphylococcus spp., Pseudomonas spp. & Lactic Acid Bacteria (LAB). The outcome demonstrated that pork fillets marinated with wine containing ethanolic extract of pomegranate and Oregano Essential Oil were more resistant to spoilage compared to all other samples; thus, their shelf-life was significantly extended (4 days in some cases). Triterpenes (maslinic, oleanolic and betulinic acid), monoterpenes (p-cymene, carvacrol, thymol, limonene), organic acids (citric & malic acid) and phenols, were the main constituents found in the plant extract, the wine and Essential Oils applied, as determined through LC-QTOF/MS and HPLC analysis. Additionally, the sensorial properties (color, tenderness, flavor and juiciness) of the marinated meat samples were not negatively influenced. Consequently, marinades of this type could be used as natural preservatives in meat products, with satisfying antimicrobial and organoleptic results.

Quality Characteristics of Novel Sourdough Breads Made with Functional Lacticaseibacillus paracasei SP5 and Prebiotic Food Matrices.

Lacticaseibacillus paracasei SP5, isolated from kefir, was assessed as a starter culture for sourdough bread making in freeze-dried form, both free (BSP5 bread) and immobilised on wheat bran (BIWB) and on a traditional flour/sour milk food, 'trahanas' (BITR). Physicochemical characteristics, shelf-life, volatilome, phytic acid, and sensory properties of the breads were evaluated. The BITR breads had higher acidity (9.05 ± 0.14 mL of 0.1 M NaOH/10 g) and organic acid content (g/Kg; 2.90 ± 0.05 lactic, 1.04 ± 0.02 acetic), which justifies the better resistance against mould and rope spoilage (>10 days). The highest number of volatiles (35) and at higher concentration (11.14 µg/g) were also found in BITR, which is in line with the sensory (consumer) evaluation regarding flavour. Finally, higher reduction of phytate (an antinutrient) was observed in all L. paracasei SP5 sourdoughs (83.3-90.7%) compared to the control samples (71.4%). The results support the use of the new strain for good quality sourdough bread.

Plant Extract and Essential Oil Application against Food-Borne Pathogens in Raw Pork Meat.

Herbal and plant extracts are being applied for a wide range of foods against different types of food-borne pathogens. In the present study, ethanolic and aqueous extracts (2% w/v) from cranberry (Vaccinium macrocarpon) and pomegranate (Punica granatum L.) plants were applied alone or in combination with two essential oils (thyme and oregano in a concentration of 0.150 µg/g) in pork meatballs and their antimicrobial activity was estimated. The extracts exhibited promising results (aqueous and ethanolic extracts of pomegranate and cranberry in a food-compatible concentration of 2% w/v) were applied to raw pork meatball production and their antimicrobial activity was recorded versus Enterobacteriaceae, total mesophilic bacteria, yeasts/molds, Staphylococcus spp., Pseudomonas spp. and lactic acid bacteria (LAB). The outcome demonstrated that meatballs containing aqueous extracts of pomegranate were more resistant to spoilage compared to all the other samples since they were preserved for more days. The chemical profiles of plant extracts were determined through LC-QTOF/MS and the chemical composition of the essential oils applied was determined with the use of GC/MS in order to identify the substances involved in the observed antimicrobial activity. Phenolic acids (quinic acid, chlorogenic acid), monoterpenes (p-cymene, carvacrol, thymol, limonene), organic acids (citric acid) and phenols were the main constituents found in the plant extracts and essential oils applied. These extracts of plant origin could be used as natural preservatives in meat products, even in low concentrations.

Genomic Insight Into Lacticaseibacillus paracasei SP5, Reveals Genes and Gene Clusters of Probiotic Interest and Biotechnological Potential.

The Lacticaseibacillus paracasei species is comprised by nomadic bacteria inhabiting a wide variety of ecological niches, from fermented foodstuffs to host-associated microenvironments. Lc. paracasei SP5 is a novel strain, originally isolated from kefir grains that presents desirable probiotic and biotechnological attributes. In this study, we applied genomic tools to further characterize the probiotic and biotechnological potential of the strain. Firstly, whole genome sequencing and assembly, were performed to construct the chromosome map of the strain and determine its genomic stability. Lc. paracasei SP5 carriers several insertion sequences, however, no plasmids or mobile elements were detected. Furthermore, phylogenomic and comparative genomic analyses were utilized to study the nomadic attributes of the strain, and more specifically, its metabolic capacity and ability to withstand environmental stresses imposed during food processing and passage through the gastrointestinal (GI) tract. More specifically, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Carbohydrate-active enzyme (CAZymes) analyses provided evidence for the ability of the stain to utilize an array of carbohydrates as growth substrates. Consequently, genes for heat, cold, osmotic shock, acidic pH, and bile salt tolerance were annotated. Importantly bioinformatic analysis showed that the novel strain does not harbor acquired antimicrobial resistance genes nor virulence factors, in agreement with previous experimental data. Putative bacteriocin biosynthesis clusters were identified using BAGEL4, suggesting its potential antimicrobial activity. Concerning microbe-host interactions, adhesins, moonlighting proteins, exopolysaccharide (EPS) biosynthesis genes and pilins mediating the adhesive phenotype were, also, pinpointed in the genome of Lc. paracasei SP5. Validation of this phenotype was performed by employing a microbiological method and confocal microscopy. Conclusively, Lc. paracasei SP5 harbors genes necessary for the manifestation of the probiotic character and application in the food industry. Upcoming studies will focus on the mechanisms of action of the novel strain at multiple levels.

Assessment of Antimicrobial Activity of Pomegranate, Cranberry, and Black Chokeberry Extracts against Foodborne Pathogens.

Herbal and plant extracts are being applied to a wide range of foods against different types of foodborne pathogens. In the present study, ethanolic and aqueous extracts of different concentrations (5% v/v, 10% v/v, and 20% v/v) from cranberry (Vaccinium macrocarpon), black chokeberry (Aronia melanocarpa), and pomegranate (Punica granatum L.) plants were applied in five concentrations (62.5 to 1000 mg/mL) against foodborne strains of Staphylococcus aureus,Escherichia coli, and Streptococcus pyogenes. The results revealed that a low concentration of solvents (5% v/v) did not exhibit decreased antimicrobial activity in comparison with higher solvent concentrations (10% and 20% v/v). Additionally, both aqueous and ethanolic extracts were highly effective against pathogens even in their low concentrations (62.5 mg/mL and 125 mg/mL). Likewise, the extracts exhibited promising results (aqueous extracts of pomegranate, cranberry, and black chokeberry in a food-compatible concentration of 2% w/v) were applied to raw pork meatball production, and their antimicrobial activity was recorded versus Enterobacteriaceae, total mesophilic bacteria (TMB), lactic acid bacteria (LAB), Staphylococcus spp., Pseudomonas spp., and yeasts/molds. The outcome demonstrated that meatballs that contained aqueous extracts of pomegranate were more resistant to spoilage compared to all of the other samples, as it was preserved for more days. Likewise, these extracts of a plant origin could be used as natural preservatives in meat products, even in their low concentrations.

Vinegar Production from Corinthian Currants Finishing Side-Stream: Development and Comparison of Methods Based on Immobilized Acetic Acid Bacteria.

Fruit wastes and side-streams can be used for vinegar production to create added value for the agri-food sector and enhance farmer incomes and local economies. In this study, methods for vinegar production by wild and selected acetic acid bacteria (the quick starter Acetobacter aceti and the acid-resistant Komagataeibacter europaeus), free (FC) and immobilized (IC) on a natural cellulosic carrier, are proposed using sweet wine made from the industrial finishing side-stream (FSS) of Corinthian currants as raw material. The results showed all cultures can produce vinegar with 46.65 ± 5.43 g/L acidity, from sweet FSS wine containing 5.08 ± 1.19% alcohol. The effect of immobilization was more obvious in the case of the selected culture, presenting better acetification efficiency, both fresh and after cold storage for 2 months. The vinegars had an antioxidant capacity of 263.5 ± 8.4 and 277.1 ± 6.7 mg/L (as ascorbic acid) and phenolic content 333.1 ± 12.0 and 222.2 ± 2.9 mg/L (as gallic acid) (for FC and IC, respectively). They also had a rich volatilome (140 compounds identified by SPME GC-MS), with higher percentages of esters identified in vinegars made by IC. The results are encouraging for vinegar production with IC of a mixed A. aceti and K. europaeus culture.

In Vitro Screening Potential Antibacterial Properties of the Greek Oregano Honey against Clinical Isolates of Helicobacter pylori.

Oregano honey is an exceedingly rare and distinct product, not commercially available, produced by bees bred in oregano fields of alpine altitudes at the mountainous area of Epirus, Greece. In ethnic popular medicine, this product is used as a therapeutic in various gastric diseases. To test this hypothesis, 14 strains of Helicobacter pylori (H. pylori), 6 isolated from gastric ulcers and 8 from cases of clinical gastritis, were employed in the present study. The above bacterial strains were exposed to various concentrations (75% v/v, 50% v/v, 25% v/v, 12.5% v/v, and 6% v/v) of 50 oregano honey samples by using the agar well method and the inhibition zones observed around each well were recorded. Although the inhibitory zones of the H. pylori isolated from the gastric ulcers were wide enough (0-34 mm), those strains, in general, appeared more resistant than the other eight (0-58 mm). The same result was observed when the same strains were tested against six antibiotics used in clinical practice. Extracts of oregano honey were prepared by extraction with four different organic solvents. N-hexane and chloroform extracts had the most potent antibacterial action. Finally, pure oregano honey and diethyl ether extracts of honey showed significant inhibitory activity against urease secreted by the pathogen. These results strongly indicate the susceptibility of H. pylori strains to the oregano honey by more than one mode of action. Consequently, this variety of honey seems to have potential therapeutic properties against gastric ulcers and gastritis, thus explaining the preference of the locals towards this traditional remedy.

Microbiological and Chemical Properties of Chokeberry Juice Fermented by Novel Lactic Acid Bacteria with Potential Probiotic Properties during Fermentation at 4 °C for 4 Weeks.

On the frame of this research survey, a novel potentially probiotic strain (Lactobacillus paracasei SP5) recently isolated from kefir grains was evaluated for chokeberry juice fermentation. Chokeberry juice was retrieved from the variety Aronia melanocarpa, a plant known to provide small, dark berries and to be one of the richest sources of antioxidants. The juice was subsequently fermented inoculating L. paracasei SP5 for 48 h at 30 °C. The fermented juices were left at 4 °C and tested regarding microbiological and physicochemical characteristics for 4 weeks. The potentially probiotic strain was proved capable of performing lactic acid fermentation at 30 °C. Cell viability of L. paracasei was detected in high levels during fermentation and the whole storage period, while the fermented juice showed higher levels of viability in juice with 40.3 g/L of initial sugar concentration. No ethanol was detected in the final fermented juice. Fermented chokeberry juice was characterized by aromatic desirable volatiles, which were retained in adequate levels for the whole storage period. Specifically, the occurrence of organic esters detected in fermented juices is considered as positive evidence of the provision of fruity and floral notes to the final product. During storage, total phenolics content and antioxidant activity were observed in higher levels in fermented chokeberry juice compared with non-fermented juice. Subsequently, fermentation of chokeberry juice by potentially probiotic lactic acid bacteria could provide high industrialization potential, providing the market with a nutritional beverage of good volatile quality with an enhanced shelf-life compared with an unfermented fresh juice.

Evaluation of Pediococcus pentosaceus SP2 as Starter Culture on Sourdough Bread Making.

In the present study, a novel Pediococcus pentosaceus SP2 strain, recently isolated from kefir grains, was evaluated as a starter culture in sourdough bread making. The novel starter was applied in fresh, freeze-dried, and freeze-dried immobilized (on wheat bran) form. The type of culture (fresh, freeze-dried, immobilized cells) influenced the bread characteristics. Specifically, the application of freeze-dried immobilized cells led to higher total titratable acidity (TTA) values (9.81 mL NaOH N/10), and the produced bread presented higher resistance to mold and rope spoilage. Moreover, the produced sourdough breads were significantly better in terms of pH, TTA, organic acids content, and resistance to mold and rope spoilage, compared to breads made with a commercial, wild microbiota, sourdough. The organic acids content was also significantly higher than the commercial sourdough sample (2.93 g/kg lactic acid; 1.01 g/kg acetic acid). Determination of volatile compounds through solid-phase microextraction (SPME) gas chromatography/mass spectrometry (GC/MS) analysis and sensorial assessments indicated no significant differences between the tested sourdough breads.

Functional pomegranate beverage production by fermentation with a novel synbiotic L. paracasei biocatalyst.

The recently isolated Lactobacillus paracasei K5 (a potential probiotic) was immobilized on delignified wheat bran (DWB) and was used to produce a functional pomegranate beverage. Fermentations were carried out for 24 h at different pH values (3.3, 3.6, and 3.9), and the fermented juices were stored for 4 weeks at 4 °C. In all cases, the immobilized biocatalyst was shown to be efficient for pomegranate juice fermentation, as indicated by analysis of sugars and organic acids, and ethanol production was maintained at low levels (0.5-1% v/v). The sugars consumed during fermentation were used mainly for cell mass production, while DWB exerted a prebiotic effect, protecting and enhancing the viability of L. paracasei. Optimum viability (9.8-11.7 log cfu/mL) was achieved at pH 3.9. The phenolic content and headspace volatiles were increased after fermentation and storage (112 ±â€¯17-213 ±â€¯30 mg GAE/100 mL and 27-46 compounds, respectively), indicating potential to produce good quality, synbiotic pomegranate beverages.

Isolation of a Lactobacillus paracasei Strain with Probiotic Attributes from Kefir Grains.

Κefir is a rich source of potentially probiotic bacteria. In the present study, firstly, in vitro screening for probiotic characteristics of ten lactic acid bacteria (LAB) isolated from kefir grains was performed. Strain AGR 4 was selected for further studies. Molecular characterization of strain AGR 4, confirmed that AGR 4 belongs to the Lactobacillus paracasei (reclassified to Lacticaseibacillus paracasei subsp. paracasei) species. Further testing revealed that L. paracasei AGR 4 displayed adhesion capacity on human adenocarcinoma cells, HT-29, similar to that of the reference strain, L. casei ATCC 393. In addition, the novel strain exerted significant time- and dose-dependent antiproliferative activity against HT-29 cells and human melanoma cell line, A375, as demonstrated by the sulforhodamine B cytotoxicity assay. Flow cytometry analysis was employed to investigate the mechanism of cellular death; however, it was found that AGR 4 did not act by inducing cell cycle arrest and/or apoptotic cell death. Taken together, these findings promote the probiotic character of the newly isolated strain L. paracasei AGR 4, while further studies are needed for the detailed description of its biological properties.

Assessment of the Immunomodulatory Properties of the Probiotic Strain Lactobacillus paracasei K5 in vitro and In Vivo.

Lactobacillus paracasei K5 is a lactic acid bacteria (LAB) strain that has been isolated from dairy products. Previous studies have established its probiotic potential in a series of in vitro tests, including molecular characterization, safety profiling, and tolerability of the gastrointestinal tract conditions. To characterize its beneficial actions on the host, we have shown previously that L. paracasei K5 adheres to Caco-2 cells and exerts anti-proliferative effects through the induction of apoptosis. In the present study, we focused on the immunomodulatory potential of this strain. We employed the dorsal-air-pouch mouse model of inflammation and recorded an eight-fold increase in the recruitment of immune cells in mice treated with the probiotic strain, compared to the control group. Analysis of the exudates revealed significant changes in the expression of pro-inflammatory mediators on site. Treatment of Caco-2 cells with L. paracasei K5 induced significant upregulation of cytokines interleukin-1α (IL-1α), ΙL-1ß, IL-6, tumor necrosis factor-alpha (TNF-α), the chemokine C-X-C motif ligand 2 (CXCL2), and the inflammation markers soluble intercellular adhesion molecule (sICAM) and metallopeptidase inhibitor-1 (TIMP-1). Transient induction of the Toll-like receptors (TLRs) 2, 4, 6, and 9 expression levels was recorded by real-time PCR analysis. These results highlight the immunomodulatory potential of this strain and further support its probiotic character.

Assessment of Ready-to-Use Freeze-dried Immobilized Biocatalysts as Innovative Starter Cultures in Sourdough Bread Making.

In the present study the effect of innovative biocatalysts as starter cultures in sourdough bread making was explored. The biocatalysts consisted of Lactobacillus paracasei K5 and Lactobacillus bulgaricus ATCC 11842 (in single and mixed form), immobilized on delignified wheat bran (DWB), and freeze dried without cryoprotectants. The parameters monitored were physicochemical characteristics, mold and rope spoilage appearance, volatile composition, and organoleptic characteristics. Results obtained showed that both biocatalysts exhibit good fermentative activity. However, the best results were achieved when freeze-dried immobilized L. paracasei K5 was applied as a single culture. In particular, the produced bread had a higher acidity (8.67 mL 0.1 N NaOH) and higher organic load (2.90 g/kg lactic acid and 1.11 g/kg acetic acid). This outcome was the main reason why this bread was preserved more regarding mold spoilage (14 days) and rope spoilage (12 days), respectively. In addition, the employment of freeze-dried immobilized L. paracasei K5 led to bread with better aromatic profile in terms of concentrations and number of volatile compounds produced as gas chromatography/mass spectrometry (GC/MS) analysis proved. Finally, no significant differences were observed through sensorial tests. Last but not least, it should be highlighted that the used microorganisms were cultured in cheese whey, minimizing the cost of the proposed biotechnological procedure.