Promotion of lactose isomerization to fructose and lactulose in one batch by immobilized enzymes on bacterial cellulose membranes.

The production of the sugars fructose and lactulose from lactose using the enzymes ß-galactosidase and glucose isomerase immobilized on bacterial cellulose (BC) membranes has been investigated. Lactose is hydrolyzed by ß-galactosidase at 30 °C to glucose and galactose at a high conversion rate, while at the same temperature, glucose isomerase is not effective in converting the produced glucose to fructose. The rate of the isomerization reaction of glucose to fructose at 70 °C has been studied. Two types of enzyme immobilization were investigated: immobilization in one stage and immobilization in two stages. The results showed that BC membrane increased three-fold the yield and the reaction rate of fructose and lactulose production from lactose. The noteworthy enhancement of BC membranes' impact on the isomerization reaction by immobilized enzymes grants permission for a novel research avenue within the context of white biotechnology development. Additionally, this effect amplifies the role of BC in sustainability and the circular economy.

Antilisterial activity of raw sheep milk from two native Epirus breeds: Culture-dependent identification, bacteriocin gene detection and primary safety evaluation of the antagonistic LAB biota.

Raw milk from native small ruminant breeds in Epirus, Greece, is a valuable natural source of autochthonous lactic acid bacteria (LAB) strains with superior biotechnological properties. In this study, two bulk milks (RM1, RM2) from two local sheep yards, intended for traditional Kefalotyri cheese production, were preselected for bacteriocin-like antilisterial activity by in vitro tests. Their antagonistic LAB biota was quantified followed by polyphasic (16S rRNA gene sequencing; IGS for Enterococcus; a multiplex-PCR for Leuconostoc) identification of 42 LAB (RM1/18; RM2/24) isolates further evaluated for bacteriocin encoding genes and primary safety traits. Representative isolates of the numerically dominant mesophilic LAB were Leuconostoc mesenteroides (10) in both RMs, Streptococcus parauberis (7) in RM2, and Lactococcus lactis (1) in RM1; the subdominant thermophilic LAB isolates were Enterococcus durans (8), E. faecium (6), E. faecalis (3), E. hirae (1), E. hermanniensis (1), Streptococcus lutetiensis (2), S. equinus (1) and S. gallolyticus (1). Based on their rpoB, araA, dsr and sorA profiles, six Ln. mesenteroides strains (8 isolates) were atypical lying between the subspecies mesenteroides and dextranicum, whereas two strains profiled with Ln. mesenteroides subsp. jonggajibkimchi that is first-time reported in Greek dairy food. Two RM1 E. faecium strain biotypes (3 isolates) showed strong, enterocin-mediated antilisterial activity due to entA/entB/entP possession. One E. durans from RM1 possessed entA and entP, while additional nine RM2 isolates of the E. faecium/durans group processed entA or entP singly. All showed direct (cell-associated) antilisterial activity only, as also both S. lutetiensis strains from RM2 did strongly. Desirably, no LAB isolate was ß-hemolyrtic, or cytolysin-positive, or possessed vanA, vanB for vancomycin resistance, or agg, espA, hyl, and IS16 virulence genes. However, all three E. faecalis from RM2 possessed gelE and/or ace virulence genes. In conclusion, all Ln. mesenteroides strains, the two safe, enterocin A-B-P-producing E. faecium strains, and the two antilisterial S. lutetiensis strains should be validated further as potential costarter or adjunct cultures in Kefalotyri cheese. The prevalence of α-hemolytic pyogenic streptococci in raw milk, mainly S. parauberis in RM2, requires consideration in respect to subclinical mastitis in sheep and the farm hygiene overall.

Genomic and Phenotypic Characterization of Mastitis-Causing Staphylococci and Probiotic Lactic Acid Bacteria Isolated from Raw Sheep's Milk.

Dairy products play a crucial role in human nutrition as they provide essential nutrients. However, the presence of diverse microorganisms in these products can pose challenges to food safety and quality. Here, we provide a comprehensive molecular characterization of a diverse collection of lactic acid bacteria (LAB) and staphylococci isolated from raw sheep's milk. Whole-genome sequencing, phenotypic characterization, and bioinformatics were employed to gain insight into the genetic composition and functional attributes of these bacteria. Bioinformatics analysis revealed the presence of various genetic elements. Important toxin-related genes in staphylococci that contribute to their pathogenic potential were identified and confirmed using phenotypic assays, while adherence-related genes, which are essential for attachment to host tissues, surfaces in the dairy environment, and the creation of biofilms, were also present. Interestingly, the Staphylococcus aureus isolates belonged to sequence type 5, which largely consists of methicillin-susceptible isolates that have been involved in severe nosocomial infections. Although genes encoding methicillin resistance were not identified, multiple resistance genes (RGs) conferring resistance to aminoglycosides, macrolides, and fluroquinolones were found. In contrast, LAB had few inherently present RGs and no virulence genes, suggesting their likely safe status as food additives in dairy products. LAB were also richer in bacteriocins and carbohydrate-active enzymes, indicating their potential to suppress pathogens and effectively utilize carbohydrate substrates, respectively. Additionally, mobile genetic elements, present in both LAB and staphylococci, may facilitate the acquisition and dissemination of genetic traits, including RGs, virulence genes, and metabolic factors, with implications for food quality and public health. The molecular and phenotypic characterization presented herein contributes to the effort to mitigate risks and infections (e.g., mastitis) and enhance the safety and quality of milk and products thereof.

A Comparative Genomic and Safety Assessment of Six Lactiplantibacillus plantarum subsp. argentoratensis Strains Isolated from Spontaneously Fermented Greek Wheat Sourdoughs for Potential Biotechnological Application.

The comparative genome analysis of six Lactiplantibacillus plantarum subsp. argentoratensis strains previously isolated from spontaneously fermented Greek wheat sourdoughs is presented. Genomic attributes related to food safety have been studied according to the European Food Safety Authority (EFSA) suggestions for the use of lactic acid bacteria (LAB) in the production of foods. Bioinformatic analysis revealed a complete set of genes for maltose, sucrose, glucose, and fructose fermentation; conversion of fructose to mannitol; folate and riboflavin biosynthesis; acetoin production; conversion of citrate to oxaloacetate; and the ability to produce antimicrobial compounds (plantaricins). Pathogenic factors were absent but some antibiotic resistance genes were detected. CRISPR and cas genes were present as well as various mobile genetic elements (MGEs) such as plasmids, prophages, and insertion sequences. The production of biogenic amines by these strains was not possible due to the absence of key genes in their genome except lysine decarboxylase associated with cadaverine; however, potential degradation of these substances was identified due to the presence of a blue copper oxidase precursor and a multicopper oxidase protein family. Finally, comparative genomics and pan-genome analysis showed genetic differences between the strains (e.g., variable pln locus), and it facilitated the identification of various phenotypic and probiotic-related properties.

Fermented Foods: New Concepts and Technologies for the Development of New Products, Quality Control.

Fermentation has been of great interest for humans since antiquity and has been extensively used in all cultures worldwide [...].

Microbial Ecology of Sheep Milk, Artisanal Feta, and Kefalograviera Cheeses. Part II: Technological, Safety, and Probiotic Attributes of Lactic Acid Bacteria Isolates.

The aim of the present study was to examine 189 LAB strains belonging to the species Enterococcus faecium, E. faecalis, Lactococcus lactis, Pediococcus pentosaceus, Leuconostoc mesenteroides, Lactiplantibacillus pentosus, Latilactobacillus curvatus, Lp. plantarum, Levilactobacillus brevis, and Weissella paramesenteroides isolated form sheep milk, Feta and Kefalograviera cheeses at different ripening stages, for their technological compatibility with dairy products manufacturing, their activities that may compromise safety of the dairy products as well as their capacity to survive in the human gastrointestinal tract. For that purpose, milk acidification and coagulation capacity, caseinolytic, lipolytic, hemolytic, gelatinolytic, and bile salt hydrolase activity, production of exopolysaccharides, antimicrobial compounds, and biogenic amines, as well as acid and bile salt tolerance and antibiotic susceptibility were examined. The faster acidifying strains were Lc. lactis DRD 2658 and P. pentosaceus DRD 2657 that reduced the pH value of skim milk, within 6 h to 5.97 and 5.92, respectively. Strains able to perform weak caseinolysis were detected in all species assessed. On the contrary, lipolytic activity, production of exopolysaccharides, amino acid decarboxylation, hemolytic, gelatinase, and bile salt hydrolase activity were not detected. Variable susceptibility to the antibiotics examined was detected among LAB strains. However, in the majority of the cases, resistance was evident. None of the strains assessed, managed to survive to exposure at pH value 1. On the contrary, 25.9 and 88.9% of the strains survived after exposure at pH values 2 and 3, respectively; the reduction of the population was larger in the first case. The strains survived well after exposure to bile salts. The strain-dependent character of the properties examined was verified. Many strains, belonging to different species, have presented very interesting properties; however, further examination is needed before their potential use as starter or adjunct cultures.

Microbial Ecology of Artisanal Feta and Kefalograviera Cheeses, Part I: Bacterial Community and Its Functional Characteristics with Focus on Lactic Acid Bacteria as Determined by Culture-Dependent Methods and Phenotype Microarrays.

Artisanal cheesemaking is still performed using practices and conditions derived from tradition. Feta and Kefalograviera cheeses are very popular in Greece and have met worldwide commercial success. However, there is a lack of knowledge regarding their lactic acid microecosystem composition and species dynamics during ripening. Thus, the aim of the present study was to assess the microecosystem as well as the autochthonous lactic acid microbiota during the ripening of artisanal Feta and Kefalograviera cheeses. For that purpose, raw sheep's milk intended for cheesemaking, as well as Feta and Kefalograviera cheeses during early and late ripening were analyzed, and the lactic acid microbiota was identified using the classical phenotypic approach, clustering with PCR-RAPD and identification with sequencing of the 16S-rRNA gene, as well as with the Biolog GEN III microplates. In addition, the functional properties of the bacterial community were evaluated using the Biolog EcoPlates, which consists of 31 different carbon sources. In general, concordance between the techniques used was achieved. The most frequently isolated species from raw sheep's milk were Enteroroccus faecium, Lactiplantibacillus plantarum and Pediococcus pentosaceus. The microecosystem of Feta cheese in the early ripening stage was dominated by Lp. plantarum and E. faecium, whereas, in late ripening, the microecosystem was dominated by Weissella paramesenteroides. The microecosystem of Kefalograviera cheese in the early ripening stage was dominated by Levilactobacillus brevis and E. faecium, and in late ripening by W. paramesenteroides and E. faecium. Finally, Carbohydrates was the main carbon source category that metabolized by all microbial communities, but the extent of their utilization was varied. Kefalograviera samples, especially at early ripening, demonstrated higher metabolic activity compared to Feta cheese. However, dominating species within microbial communities of the cheese samples were not significantly different.

Consolidated bioprocessing of lactose into lactic acid and ethanol using non-engineered cell factories.

The aim of this study is the consolidated bioprocessing of lactose into lactic acid and ethanol using non-engineered Cell Factories (CFs). Therefore, two different types of composite biocatalysts (CF1-CF2) based on Saccharomyces cerevisiae with immobilized microorganism or enzyme on starch gel (SG) were prepared for 5% w/v lactose fermentation. In CF1, S. cerevisiae was covered with SG containing Lactobacillus casei, Lactobacillus bulgaricus, Kluyveromyces marxianus CF1a-c. S. cerevisiae/SG-ß-galactosidase (CF1d) was also used for simultaneous saccharification and fermentation (SSF) of lactose. In CF2, S. cerevisiae immobilized on tubular cellulose (TC) was covered with SG containing the aforementioned microorganisms (CF2a-c). The wet CF1d resulted in 96% of the theoretical ethanol yield while the wet CF1b and freeze-dried CF2b resulted in 89% of the theoretical lactic acid yield. The repeated batches using the CF2a-c exhibited better results than using CF1a-c. Subsequently, the freeze-dried CF2 as preservative and more manageable were verified for future exploitation of whey.

Assessment of the Spoilage Microbiota during Refrigerated (4 °C) Vacuum-Packed Storage of Fresh Greek Anthotyros Whey Cheese without or with a Crude Enterocin A-B-P-Containing Extract.

Although fresh whey cheeses are prone to rapid deterioration, mainly by psychrotrophic Gram-negative bacteria and lactic acid bacteria (LAB), data on the specific spoilage species in traditional Greek whey cheeses are scarce. Therefore, this study quantified growth and characterized the primary spoilage bacteria in fresh Anthotyros whey cheeses stored at 4 °C in a vacuum for 40 days, without or with an added 5% (v/w) of an enterocin A-B-P crude extract (CEntE). Psychrotrophic Pseudomonas spp., Aeromonas spp., Hafnia spp. and Serratia spp. grew faster than LAB during early storage. However, LAB outgrew the Gram-negative bacteria and prevailed by mid to late storage in all cheese batches, causing a strong or milder batch-dependent natural acidification. Two major non-slime-producing and two minor biotypes of Leuconostoc-like bacteria, all identified as Leuconostoc mesenteroides by 16S rRNA sequencing, dominated the LAB association (76.7%), which also included four subdominant Carnobacterium maltaromaticum biotypes (10.9%), one Leuconostoc lactis biotype (3.3%) and few Lactococcus (1.6%), mesophilic Lactobacillus (0.8%) and Enterococcus (0.8%). Growth and distribution of LAB and Gram-negative species were strongly batch-dependent and plant-dependent. The CEntE neither retarded growth nor altered the whey cheese spoilage association but enhanced LAB growth and the declines of Gram-negative bacteria by late storage.

Assessment of the spoilage microbiota in minced free-range chicken meat during storage at 4 C in retail modified atmosphere packages.

This study assessed the evolution of spoilage microbiota in association with the changes in pH and concentrations of lactic and acetic acids in retail oxygen-free modified atmosphere (30:70 CO2/N2) packages (MAP) of minced free-range chicken meat during storage at 4 °C for 10 days. MAP retarded growth of spoilage lactic acid bacteria (LAB) below 6.5 log cfu/g and fully suppressed growth of pseudomonads, enterobacteria, enterococci, staphylococci and yeasts. Two distinct Latilactobacillus sakei strain biotypes were predominant and Leuconostoc carnosum, Carnobacterium divergens, Latilactobacillus fuchuensis and Weissella koreensis were subdominant at spoilage. The chicken meat pH ranged from 5.8 to 6.1. l-lactate (832 mg/100 g on day-0) decreased slightly on day-7. d-lactate remained constantly below 20 mg/100 g, whereas acetate (0-59 mg/100 g) increased 5-fold on day-7. All MAP samples developed off-odors on day-7 and a strong 'blown-pack' sulfur-type of spoilage on day-10. However, neither the predominant Lb. sakei nor other LAB or gram-negative isolates formed H2S in vitro, except for C. divergens.

High-quality draft genome sequence data of six Lactiplantibacillus plantarum subsp. argentoratensis strains isolated from various Greek wheat sourdoughs.

Lactiplantibacillus plantarum is a species found in a wide range of foods and other commodities. It can be used as starter or adjunct culture in fermented foods. Herein the annotated high-quality draft genome (scaffolds) of six L. plantarum subsp. argentoratensis strains (LQC 2320, LQC 2422, LQC 2441, LQC 2485, LQC 2516 and LQC 2520) isolated from various Greek wheat sourdoughs is presented. Raw sequence reads were quality checked, assembled into larger contiguous sequences and scaffolds were annotated. The total size of the genomes ranged from 3.13 Mb to 3.49 Mb and the GC content from 45.02% to 45.13%. The total number of coding and non-coding genes were between 3268 and 3723 (3091 to 3492 protein-coding genes, 62 to 107 repeat-region, 54 to 59 tRNAs and 2 to 5 rRNAs, 20 to 30 crispr-repeats, 17 to 26 crispr-spacers and 2 to 4 crispr-arrays). The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession numbers JAEQMR000000000, JAEQMQ000000000, JAEQMP000000000, JAEQMO000000000, JAEQMN000000000 and JAEQMM000000000. The version described in this paper is version JAEQMR010000000, JAEQMQ010000000, JAEQMP010000000, JAEQMO010000000, JAEQMN010000000 and JAEQMM010000000. Raw sequence reads have been submitted in the Sequence Read Archive (SRA) under the BioProject accession number PRJNA689714 (BioSample accession numbers SAMN17215143, SAMN17215144, SAMN17215145, SAMN17215146, SAMN17215147 and SAMN17215148 and SRA accession numbers SRR13357463, SRR13357464, SRR13357465, SRR13357466, SRR13357467, SRR13357468).

Technological and Safety Attributes of Lactic Acid Bacteria and Yeasts Isolated from Spontaneously Fermented Greek Wheat Sourdoughs.

The aim of the present study was to assess the technological and safety potential of 207 lactic acid bacteria (LAB) and 195 yeast strains isolated from spontaneously fermented Greek wheat sourdoughs. More accurately, the amylolytic, proteolytic, lipolytic, phytase and amino acid decarboxylase activities, along with the production of exopolysaccharides and antimicrobial compounds by the LAB and yeast isolates, were assessed. A well diffusion assay revealed seven proteolytic LAB and eight yeast strains; hydrolysis of tributyrin was evident only in 11 LAB strains. A further Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) indicated partial hydrolysis of gluten. Lipolysis kinetics over 21 days was applied, exhibiting that lipolytic activity ranged from 6.25 to 65.50 AU/mL. Thirteen LAB inhibited Penicillium olsonii and Aspergillus niger growth and 12 yeast strains inhibited Pe. chrysogenum growth. Twenty-one Lactiplantibacillus plantarum strains exhibited inhibitory activity against Listeria monocytogenes, as well as several sourdough-associated isolates. The structural gene encoding plantaricin 423 was detected in 19 Lcb. plantarum strains, while the structural genes encoding plantaricins NC8, PlnE/F, PlnJ/K, and S were detected in two Lcb. plantarum strains. None of the microbial strains tested exhibited exopolysaccharide (EPS) production, amino acid decarboxylase, amylolytic or phytase activity. The technological and safety potential of the Lcb. plantarum and Wickerhamomyces anomalus strains was highlighted, since some of them exhibited proteolytic, lipolytic, antibacterial and antimould activities.

Safety Evaluation, Biogenic Amine Formation, and Enzymatic Activity Profiles of Autochthonous Enterocin-Producing Greek Cheese Isolates of the Enterococcus faecium/durans Group.

Autochthonous single (Ent+) or multiple (m-Ent+) enterocin-producing strains of dairy enterococci show promise for use as bioprotective adjunct cultures in traditional cheese technologies, provided they possess no pathogenic traits. This study evaluated safety, decarboxylase activity, and enzymatic (API ZYM) activity profiles of nine Ent+ or m-Ent+ Greek cheese isolates previously assigned to four distinct E. faecium (represented by the isolates KE64 (entA), GL31 (entA), KE82 (entA-entB-entP) and KE77 (entA-entB-entP-bac31)) and two E. durans (represented by the isolates KE100 (entP) and KE108 (entP-bac31-cyl)) strain genotypes. No strain was ß-hemolytic or harbored vanA and vanB or the virulence genes agg, ace, espA, IS16, hyl, or gelE. All strains were of moderate to high sensitivity to ampicillin, ciprofloxacin, chloramphenicol, erythromycin, gentamicin, penicillin, tetracycline, and vancomycin, except for the E. faecium KE64 and KE82 strains, which were resistant to erythromycin and penicillin. All cheese strains showed moderate to strong esterase-lipase and aminopeptidase activities and formed tyramine, but none formed histamine in vitro. In conclusion, all Ent+ or m-Ent+ strain genotypes of the E. faecium/durans group, except for the cyl-positive E. durans KE108, were safe for use as adjunct cultures in traditional Greek cheeses. Further in situ biotechnological evaluations of the strains in real cheese-making trials are required.

Cell factory models of non-engineered S. cerevisiae containing lactase in a second layer for lactose fermentation in one batch.

The objective of this project was to ferment lactose and whey to ethanol in one-step process. Models of cell factory of non-engineered S.cerevisiae have been proposed to ferment lactose. The cell factory of non-engineered S. cerevisiae/SG-lactase was prepared by the addition, of a starch gel solution containing lactase on non-engineered S. cerevisiae, and freeze drying of it. The 2-layer non engineered S.cerevisiae-TC/SG-lactase factory was prepared by immobilizing S. cerevisiae on the internal layer of tubular cellulose (TC), and the lactase enzyme was contained in the upper layer of starch gel (SG) covering cells of S. cerevisiae. Using such cell factory for the fermentation of lactose, alcohol yield of 23-32 mL/L at lactose conversion of 71-100%. The improvement in alcohol yield by cell factory versus co-immobilization of lactase enzyme and S. cerevisiae on alginates, was found in the range of 28-78%. Likewise, the cell factories are more effective than engineered S. cerevisiae. The fermentation of whey instead of lactose resulted in a significant reduction of the fermentation time. Freeze-dried cell factories led to improved results as compared with non-freeze dried. When lactase was substituted with L. casei, ethanol and lactic acid were produced simultaneously at high concentrations, but in a much longer fermentation time. The cell factories can be considered as models for white biotechnology using lactose containing raw materials. This suggested cell factory model can be applied for other bioconversions using the appropriate enzymes and cells, in the frame of White Biotechnology without genetic modification.

Microbial Ecology of Greek Wheat Sourdoughs, Identified by a Culture-Dependent and a Culture-Independent Approach.

The aim of the present study was to assess the microecosystem of 13 homemade spontaneously fermented wheat sourdoughs from different regions of Greece, through the combined use of culture-dependent (classical approach; clustering by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) and identification by PCR species-specific for Lactiplantibacillus plantarum, and sequencing of the 16S-rRNA and 26S-rRNA gene, for Lactic Acid Bacteria (LAB) and yeasts, respectively) and independent approaches [DNA- and RNA-based PCR-Denaturing Gradient Gel Electrophoresis (DGGE)]. The pH and Total Titratable Acidity (TTA) values ranged from 3.64-5.05 and from 0.50-1.59% lactic acid, respectively. Yeast and lactic acid bacteria populations ranged within 4.60-6.32 and 6.28-9.20 log CFU/g, respectively. The yeast: LAB ratio varied from 1:23-1:10,000. A total of 207 bacterial and 195 yeast isolates were obtained and a culture-dependent assessment of their taxonomic affiliation revealed dominance of Lb. plantarum in three sourdoughs, Levilactobacillus brevis in four sourdoughs and co-dominance of these species in two sourdoughs. In addition, Companilactobacillusparalimentarius dominated in two sourdoughs and Fructilactobacillussanfranciscensis and Latilactobacillus sakei in one sourdough each. Lactococcus lactis, Lb. curvatus, Leuconostoc citreum, Ln. mesenteroides and Lb. zymae were also recovered from some samples. Regarding the yeast microbiota, it was dominated by Saccharomyces cerevisiae in 11 sourdoughs and Pichia membranifaciens and P. fermentans in one sourdough each. Wickerhamomyces anomalus and Kazachstania humilis were also recovered from one sample. RNA-based PCR-DGGE provided with nearly identical results with DNA-based one; in only one sample the latter provided an additional band. In general, the limitations of this approach, namely co-migration of amplicons from different species to the same electrophoretic position and multiband profile of specific isolates, greatly reduced resolution capacity, which resulted in only partial verification of the microbial ecology detected by culture-dependent approach in the majority of sourdough samples. Our knowledge regarding the microecosystem of spontaneously fermented Greek wheat-based sourdoughs was expanded, through the study of sourdoughs originating from regions of Greece that were not previously assessed.

Semi-Industrial Production of Kashkaval of Pindos Cheese Using Sheep or a Mixture of Sheep-Goat Milk and the Utilization of the Whey for Manufacturing Urda Cheese.

Kashkaval of Pindos cheese was successfully produced using 100% sheep milk (KS) or with the addition of 10% goat milk (KG). Urda cheese was manufactured using 100% sheep milk (US) or 90% sheep milk-10% goat (UG) whey from the production of kashkaval of Pindos cheese. Both cheeses were made taking into account their traditional cheese-making methods. The cheeses were assessed for their chemical, microbiological and organoleptic characteristics. Generally, no significant differences were observed between KS and KG cheese and between US and UG cheese regarding their physicochemical, textural characteristics, soluble nitrogen fraction and total fatty acid content. The fat content of Urda cheese was low, in accordance with the demand of consumers for healthy products. KS cheeses showed higher total volatile compounds than KG cheeses at 60 and 90 days of ripening and storage as well as lower counts of thermophilic-mesophilic lactic acid bacteria. No differences were observed in the microbial counts between US and UG cheeses. Acetone, hexanal, 2 heptanone, ethanol and toluene were found in abundance in Urda cheeses. Both kashkaval of Pindos and Urda cheeses received high scores during the organoleptic evaluation. The obtained data may lead to the production of both cheeses with standard characteristic according to the traditional recipes and improve their recognition.

Enhanced Aromatic Profile and Functionality of Cheese Whey Beverages by Incorporation of Probiotic Cells Immobilized on Pistacia terebinthus Resin.

: In the present study, cheese whey was utilized for the development of a novel functional beverage, using Lactobacillus casei ATCC 393 probiotic cells immobilized on Pistacia terebinthus resin (pissa Paphos). Evaluation of shelf life of the produced beverages showed that spoilage microorganisms were not observed in beverages containing P. terebinthus resin. Terpenes' rich content might have contributed to the antimicrobial activity of the produced beverages; however, no significant effect on the viability of the immobilized probiotic cells was obtained. Whey beverages containing the immobilized biocatalyst retained a high viability (>1 × 106 CFU/g) of probiotic cells during a storage period of 30 days at 4 °C. The superiority of whey beverages containing the immobilized biocatalyst was also highlighted by GC-MS analysis, while the enhanced aromatic profile, which was mostly attributed to the higher concentration of terpenes, was also detected during the sensory evaluation performed. Conclusively, this study indicated the high commercialization potential of these novel functional whey beverages, within the frame of a sustainable dairy waste valorization approach. To the best of our knowledge, this is the first food-oriented approach within the guidelines of the circular economy reported in the literature, using the autochthonous Pistacia terebinthus resin for the production of functional whey beverages.

Probiotics in Food Systems: Significance and Emerging Strategies Towards Improved Viability and Delivery of Enhanced Beneficial Value.

Preserving the efficacy of probiotic bacteria exhibits paramount challenges that need to be addressed during the development of functional food products. Several factors have been claimed to be responsible for reducing the viability of probiotics including matrix acidity, level of oxygen in products, presence of other lactic acid bacteria, and sensitivity to metabolites produced by other competing bacteria. Several approaches are undertaken to improve and sustain microbial cell viability, like strain selection, immobilization technologies, synbiotics development etc. Among them, cell immobilization in various carriers, including composite carrier matrix systems has recently attracted interest targeting to protect probiotics from different types of environmental stress (e.g., pH and heat treatments). Likewise, to successfully deliver the probiotics in the large intestine, cells must survive food processing and storage, and withstand the stress conditions encountered in the upper gastrointestinal tract. Hence, the appropriate selection of probiotics and their effective delivery remains a technological challenge with special focus on sustaining the viability of the probiotic culture in the formulated product. Development of synbiotic combinations exhibits another approach of functional food to stimulate the growth of probiotics. The aim of the current review is to summarize the strategies and the novel techniques adopted to enhance the viability of probiotics.

Pistacia terebinthus Resin as Yeast Immobilization Support for Alcoholic Fermentation.

A natural resin retrieved from Pistacia terebinthus tree was evaluated as an immobilization carrier of Saccharomyces cerevisiae AXAZ-1 cells targeting successive fermentation batches of sugar synthetic mediums. Fermentation times below 54 h were recorded at temperatures 28-14 °C. In total, 147 compounds were detected using gas chromatography-mass spectrometry (GC-MS) analysis, including alcohols, esters, ketones, aldehydes, acids, and terpenes. Principal component analysis indicated that the state of cells (free/immobilized) and the fermentation temperature primarily affected terpenes' composition. Importantly, no spoilage of the fermented beverages was noted during 90 days of storage at room temperature, most likely due to the high content of extracted terpenoids and phenols (up to 579.01 mg L-1 and 171.8 mg gallic acid equivalent L-1, respectively). Likewise, the developed novel biocatalyst (yeast cells immobilized within Pistacia terebinthus resin) was suitable for the production of low alcohol beverages with an enhanced aromatic profile. The obtained results revealed that the proposed bioprocess shows great commercialization potential in the new fast-growing low-alcohol beverages sector.

Growth Capacity of a Novel Potential Probiotic Lactobacillus paracasei K5 Strain Incorporated in Industrial White Brined Cheese as an Adjunct Culture.

In this study, a novel potential probiotic strain Lactobacillus paracasei K5, previously isolated from traditional Greek Feta cheese and kefir grains, was evaluated as an adjunct culture for industrial white brined cheese production. Targeting industrial applications, apart from free cell cultures, a novel ready-to-use freeze-dried immobilized biocatalyst was prepared. The biocatalyst composed of L. paracasei K5 cells immobilized on delignified wheat bran prebiotic carrier and was freeze-dried without cryoprotectants. The adjunct free or immobilized culture was added separately without prior adaptation during white brined cheese manufacture and the produced cheeses were compared with commercial white brined cheeses. Several parameters that affect the acceptability and quality of the cheeses, including microbial populations, physicochemical parameters, volatile by-products and organoleptic characteristics, were analyzed through 70 days of storage. Results showed that the viability of the adjunct culture added either free or immobilized remained in high levels (7 to 8 log cfu/g) during maturation and storage. In addition, all white brined cheeses with the adjunct probiotic culture showed a sharp decrease in spoilage and pathogenic microorganisms such as enterobacteria, salmonella, staphylococci and coliforms during cheese maturation, especially when compared with the commercial white brined cheeses. Finally, after maturation time exceeded, all cheeses were characterized as safe for human consumption. Cheeses volatile compounds were significantly enhanced by the incorporated immobilized biocatalysts. These findings indicate the potential industrial use of freeze-dried ready-to-use immobilized lactobacilli as reinforcement adjunct cultures for the production of good-quality functional cheese products. PRACTICAL APPLICATION: The launch on market of novel foods developed by the incorporation of functional ingredients provides potential benefits to consumers' diet and new business opportunities for producers. Probiotic food products are one significant category of functional foods. Thus, this study focused on the development of a novel ready-to-use freeze-dried potential probiotic biocatalyst for functional white brined cheese production. The potential industrial application of such biocatalysts is highlighted by their incorporation as adjunct cultures that resulted in good-quality functional cheeses.