Investigation of the Microbiome of Industrial PDO Sfela Cheese and Its Artisanal Variants Using 16S rDNA Amplicon Sequencing and Shotgun Metagenomics.

Sfela is a white brined Greek cheese of protected designation of origin (PDO) produced in the Peloponnese region from ovine, caprine milk, or a mixture of the two. Despite the PDO status of Sfela, very few studies have addressed its properties, including its microbiology. For this reason, we decided to investigate the microbiome of two PDO industrial Sfela cheese samples along with two non-PDO variants, namely Sfela touloumotiri and Xerosfeli. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), 16S rDNA amplicon sequencing and shotgun metagenomics analysis were used to identify the microbiome of these traditional cheeses. Cultured-based analysis showed that the most frequent species that could be isolated from Sfela cheese were Enterococcus faecium, Lactiplantibacillus plantarum, Levilactobacillus brevis, Pediococcus pentosaceus and Streptococcus thermophilus. Shotgun analysis suggested that in industrial Sfela 1, Str. thermophilus dominated, while industrial Sfela 2 contained high levels of Lactococcus lactis. The two artisanal samples, Sfela touloumotiri and Xerosfeli, were dominated by Tetragenococcus halophilus and Str. thermophilus, respectively. Debaryomyces hansenii was the only yeast species with abundance > 1% present exclusively in the Sfela touloumotiri sample. Identifying additional yeast species in the shotgun data was challenging, possibly due to their low abundance. Sfela cheese appears to contain a rather complex microbial ecosystem and thus needs to be further studied and understood. This might be crucial for improving and standardizing both its production and safety measures.

The Rising Role of Omics and Meta-Omics in Table Olive Research.

Table olives are often the result of fermentation, a process where microorganisms transform raw materials into the final product. The microbial community can significantly impact the organoleptic characteristics and safety of table olives, and it is influenced by various factors, including the processing methods. Traditional culture-dependent techniques capture only a fraction of table olives' intricate microbiota, prompting a shift toward culture-independent methods to address this knowledge gap. This review explores recent advances in table olive research through omics and meta-omics approaches. Genomic analysis of microorganisms isolated from table olives has revealed multiple genes linked to technological and probiotic attributes. An increasing number of studies concern metagenomics and metabolomics analyses of table olives. The former offers comprehensive insights into microbial diversity and function, while the latter identifies aroma and flavor determinants. Although proteomics and transcriptomics studies remain limited in the field, they have the potential to reveal deeper layers of table olives' microbiome composition and functionality. Despite the challenges associated with implementing multi-omics approaches, such as the reliance on advanced bioinformatics tools and computational resources, they hold the promise of groundbreaking advances in table olive processing technology.

The Effects of Insect Infestation on Stored Agricultural Products and the Quality of Food.

In this review article, we focus on the effects of insect pests on the quality of stored cereals and legume grains. The changes in the amino-acid content, the quality of proteins, carbohydrates, and lipids, and the technological characteristics of the raw materials when infested by specific insects are presented. The differences reported concerning the rate and kind of infestation effects are related to the trophic habits of the infesting insect species, the variation of the component distribution in the different species of grains, and the length of the storage period. For example, wheat germ and brans feeders such as Trogoderma granarium may cause a higher reduction in proteins than endosperm feeders such as Rhyzopertha dominica, since the germ and brans contain higher concentrations of proteins. Trogoderma granarium may also cause higher reduction in lipids than R. dominica in wheat, maize and sorghum, in which most of the lipids exist in the germ. Furthermore, infestation with insects such as Tribolium castaneum may downgrade the overall quality of wheat flour, by increasing the moisture content, the number of insect fragments, the color change, the concentration of uric acid, the microbial growth, and the prevalence of aflatoxins. Whenever possible, the significance of the insect infestation and the concomitant compositional alterations on human health are presented. It should be highlighted that understanding the impact of insect infestation on stored agricultural products and the quality of food will be crucial for the required food security in the future.

Applying Image-Based Food-Recognition Systems on Dietary Assessment: A Systematic Review.

Dietary assessment can be crucial for the overall well-being of humans and, at least in some instances, for the prevention and management of chronic, life-threatening diseases. Recall and manual record-keeping methods for food-intake monitoring are available, but often inaccurate when applied for a long period of time. On the other hand, automatic record-keeping approaches that adopt mobile cameras and computer vision methods seem to simplify the process and can improve current human-centric diet-monitoring methods. Here we present an extended critical literature overview of image-based food-recognition systems (IBFRS) combining a camera of the user's mobile device with computer vision methods and publicly available food datasets (PAFDs). In brief, such systems consist of several phases, such as the segmentation of the food items on the plate, the classification of the food items in a specific food category, and the estimation phase of volume, calories, or nutrients of each food item. A total of 159 studies were screened in this systematic review of IBFRS. A detailed overview of the methods adopted in each of the 78 included studies of this systematic review of IBFRS is provided along with their performance on PAFDs. Studies that included IBFRS without presenting their performance in at least 1 of the above-mentioned phases were excluded. Among the included studies, 45 (58%) studies adopted deep learning methods and especially convolutional neural networks (CNNs) in at least 1 phase of the IBFRS with input PAFDs. Among the implemented techniques, CNNs outperform all other approaches on the PAFDs with a large volume of data, since the richness of these datasets provides adequate training resources for such algorithms. We also present evidence for the benefits of application of IBFRS in professional dietetic practice. Furthermore, challenges related to the IBFRS presented here are also thoroughly discussed along with future directions.

Evaluation of Plantaricin Genes Expression During Fermentation of Raphanus sativus Roots with a Plantaricin-Producing Lactobacillus plantarum Starter.

The aim of the present study was to assess the transcription of the plnE/F, plnN, plnG, plnD and plnI genes during lactic acid fermentation of radish (Raphanus sativus) roots by Lactobacillus plantarum strain LQC 740 at 20 and 30 °C. At both temperatures, this strain dominated the fermentation process, as indicated by (GTG)5 analysis. A total of five pln genes were detected in the genome of this strain, namely plnE/F, plnN, plnG, plnD and plnI. Regarding plantaricin genes expression, no regulation was observed in the majority of the samples at both temperatures, therefore, the transcription of the pln genes was not affected by the experimental conditions, i.e. radish fermentation vs. growth in MRS broth. Although transcription of the pln genes was similar between the two conditions, bacteriocin activity was different. The maximum plantaricin activity was 87.5 AU/mL during radish fermentation and 700 AU/mL during growth in MRS broth. Thus, no apparent correlation between bacteriocin activity and transcription level of the five pln genes could be established.

Probiotic Features of Lactic Acid Bacteria Isolated from a Diverse Pool of Traditional Greek Dairy Products Regarding Specific Strain-Host Interactions.

The increased consumers' interest on the positive role of food in wellbeing and health underscores the need to determine new probiotic microorganisms. Triggered by the fact that artisanal food products can be a valuable source of novel probiotic strains, 106 lactic acid bacteria, all isolated from traditional Greek dairy products, namely Feta, Kasseri, Xynotyri, Graviera, Formaela, Galotyri, and Kefalotyri cheeses as well as yogurt and milk, were studied for probiotic properties. Based on their survival at pH 2.5 and their stability in the presence of bile salts, 20 strains were selected for further analysis. These strains exhibited diverse susceptibility to commonly used antibiotics, while none was hemolytic. Seven out of the 20 strains produced functional bile salt hydrolases in vitro. The only antimicrobial activity detected of Streptococcus thermophilus ACA-DC 26 against the oral pathogen Streptococcus mutans LMG 14558T was attributed to compound(s) of proteinaceous nature. Two Lactobacillus plantarum strains, namely ACA-DC 2640 and ACA-DC 4039, displayed the highest adhesion according to a collagen-based microplate assay and by using ΗΤ-29 and Caco-2 cells. Co-cultivation of THP-1 cells with selected strains indicated a tendency for anti-inflammatory modulation by Lactobacillus plantarum ACA-DC 2640 as well as Streptococcus thermophilus ACA-DC 26 and ACA-DC 170, as shown by an increase in IL10 mRNA levels. Moreover, milk cell-free supernatants of Lactobacillus plantarum ACA-DC 2640 and ACA-DC 4039 exhibited strong angiotensin I-converting enzyme inhibition. To conclude, new isolates presenting interesting probiotic features were described and should be further investigated as health-promoting factors.

Lactic acid bacteria population dynamics during spontaneous fermentation of radish (Raphanus sativus L.) roots in brine.

The aim of the present study was to assess the microecosystem development and the dynamics of the lactic acid bacteria population during spontaneous fermentation of radish (Raphanus sativus L.) roots in brine at 20 and 30 °C. In both temperatures, lactic acid bacteria prevailed the fermentation; as a result, the pH value was reduced to ca. 3.6 and total titrable acidity increased to ca. 0.4% lactic acid. Enterococci population increased and formed a secondary microbiota while pseudomonads, Enterobacteriaceae and yeasts/molds populations were below enumeration limit already before the middle of fermentation. Pediococcus pentosaceus dominated during the first days, followed by Lactobacillus plantarum that prevailed the fermentation until the end. Lactobacillus brevis was also detected during the final days of fermentation. A succession at sub-species level was revealed by the combination of RAPD-PCR and rep-PCR analyses. Glucose and fructose were the main carbohydrates detected in brine and were metabolized into lactic acid, acetic acid and ethanol.

Evaluation of Two Lactic Acid Bacteria Starter Cultures for the Fermentation of Natural Black Table Olives (Olea europaea L cv Kalamon).

The production of Greek-style natural black table olives remains an empirical process relying on spontaneous fermentation despite its economic significance. For this reason producers often resort to increased NaCl concentration of the brine to secure quality of the product. In this study we employ two lactic acid bacteria Leuconostoc mesenteroides subsp. mesenteroides Lm139 and Lactobacillus pentosus DSM 16366 as starters in separate laboratory low salinity fermentations of "Kalamon" cultivar olives, processed according to the Greek-style method. L. mesenteroides subsp. mesenteroides Lm139 was previously isolated from Kalamon olives laboratory spontaneous fermentations, while L. pentosus DSM 16366 was isolated from fermenting green olives prepared according to the Spanish-style method. Spontaneous olives fermentation was also performed as a control. Microbiological and physicochemical analyses of the brines revealed that the use of the starters had a significant effect on the olives fermentation, leading to a faster acidification due to the more efficient consumption of soluble sugars in the brines. The final pH value reached by each starter culture used indicates a successful lactic fermentation. The production of lactic acid by the starters and the concomitant drop of the pH value proved to inhibit enterobacteria in a shorter period of time compared to the spontaneous fermentation. Concluding, the use of either of the two lactic acid bacteria as starters in Greek-style Kalamon olives fermentation could lead to a more controllable fermentation at lower salinities. The resulting product could be of higher quality with extended shelf-life while being at the same time safer for the consumer.

Milk protein fragments induce the biosynthesis of macedocin, the lantibiotic produced by Streptococcus macedonicus ACA-DC 198.

The aim of the present work was to study the mode of the induction of the biosynthesis of macedocin, the lantibiotic produced by Streptococcus macedonicus ACA-DC 198. Macedocin was produced when the strain was grown in milk but not in MRS or M17 broth. No autoinduction mechanism was observed. Production did not depend on the presence of lactose or galactose in the culture medium or on a coculture of the producer strain with macedocin-sensitive or macedocin-resistant strains. Induction seemed to depend on the presence of one or more heat-stable protein components produced when S. macedonicus ACA-DC 198 was grown in milk. The partial purification of the induction factor was performed by a combination of chromatography methods, and its activity was confirmed by a reverse transcription-PCR approach (RT-PCR). Mass spectrometric (MS) and tandem mass spectrometric (MS/MS) analyses of an induction-active fraction showed the presence of several peptides of low molecular mass corresponding to fragments of alpha(S1)- and beta-casein as well as beta-lactoglobulin. The chemically synthesized alpha(S1)-casein fragment 37-55 (2,253.65 Da) was proven to be able to induce macedocin biosynthesis. This is the first time that milk protein degradation fragments are reported to exhibit a bacteriocin induction activity.

In vitro and in vivo safety evaluation of the bacteriocin producer Streptococcus macedonicus ACA-DC 198.

Streptococcus macedonicus ACA-DC 198, a bacteriocin producer isolated from Greek Kasseri cheese, was used in a series of in vitro and in vivo experiments in order to evaluate its pathogenic potential. The strain was examined in vitro for haemolytic activity, antibiotic resistance and presence of pathogenicity genes encountered in Streptococcus pyogenes. Subsequently, the strain was orally administered to mice (8.9 log cfu daily), continuously over a period of 12 weeks, in order to ascertain the effects of its long term consumption on animal health and gastric inflammation. S. macedonicus ACA-DC 198 was found to be non-haemolytic and sensitive to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, streptomycin, tetracycline, and vancomycin, with the only resistance observed against kanamycin. PCR amplification and DNA-DNA hybridization did not reveal the presence of any of the S.pyogenes pathogenicity genes examined, namely emm, scpA, hasA, speB, smez2, speJ, sagAB, hylA, ska, speF, speG, slo, hylP2 and mga. In the mouse study, no detrimental effects were observed in the behaviour, general well being, weight gain and water consumption of the animals receiving S. macedonicus ACA-DC 198. Histologic analysis showed no evidence of inflammation in the stomach of the animals receiving S. macedonicus ACA-DC 198, while faecal microbiological analysis revealed that the strain retained its viability passing through the mouse gastrointestinal tract. Finally, no evidence of translocation to the liver, spleen and mesenteric lymph nodes was observed. In conclusion, none of the examined virulence determinants were detected in S. macedonicus ACA-DC 198 and its long term, high dosage oral administration did not appear to induce any pathogenic effect in mice.

Characterization of the gene cluster involved in the biosynthesis of macedocin, the lantibiotic produced by Streptococcus macedonicus.

Streptococcus macedonicus ACA-DC 198, a food-grade isolate from naturally fermented Greek Kasseri cheese, produces a lantibiotic named macedocin that has been previously purified and characterized. In the present study, a 15,171 bp region in the S. macedonicus ACA-DC 198 chromosome, containing the biosynthetic gene cluster of macedocin, has been sequenced. This region consists of 10 ORFs, which correspond to the genes (mcd genes) involved in macedocin biosynthesis, regulation and immunity. The mcd genes are organized in two operons and their role is predicted on the basis of similarities to genes of known lantibiotics. Compared with its closest match, the streptococcin A-FF22 gene cluster, the macedocin one contains an additional structural gene and an insertion sequence between the regulatory and the biosynthetic operons.

Rapid detection and identification of Streptococcus macedonicus by species-specific PCR and DNA hybridisation.

The aim of this study was to develop a simple and specific method for the rapid detection and identification of Streptococcus macedonicus. The method was based on polymerase chain reaction (PCR) using species-specific primers derived from the 16S rRNA gene. Specific identification was proven on seven S. macedonicus strains, while 16 strains belonging to different lactic acid bacteria species were tested negative. The PCR assay was capable of detecting 100 pg of S. macedonicus DNA, and it was also efficient on single colonies of the bacterium. Furthermore, the same bacterial strains were used for the specificity evaluation of a S. macedonicus species-specific probe. Neither species-specific PCR nor DNA hybridisation experiments could differentiate Streptococcus waius from S. macedonicus, due to the identity of the 16S rRNA gene of the two species, indicating high phylogenetical relatedness. This was further confirmed by the comparative sequence analysis of the 16S-23S rRNA intergenic regions. It was thus clearly demonstrated that S. waius, recently described as a novel Streptococcus species, is phylogenetically identical to S. macedonicus.