Study of the Resistance of Staphylococcus aureus Biofilm, Biofilm-Detached Cells, and Planktonic Cells to Microencapsulated Carvacrol Used Alone or Combined with Low-pH Treatment.

Microbial biofilms pose severe problems in the medical field and food industry, as they are the cause of many serious infections and food-borne diseases. The extreme biofilms' resistance to conventional anti-microbial treatments presents a major challenge to their elimination. In this study, the difference in resistance between Staphylococcus aureus DSMZ 12463 biofilms, biofilm-detached cells, and planktonic cells against microcapsules containing carvacrol was assessed. The antimicrobial/antibiofilm activity of low pH disinfection medium containing the microencapsulated carvacrol was also studied. In addition, the effect of low pH on the in vitro carvacrol release from microcapsules was investigated. The minimum inhibitory concentration of microencapsulated carvacrol was 0.625 mg mL-1. The results showed that biofilms exhibited greater resistance to microencapsulated carvacrol than the biofilm-detached cells and planktonic cells. Low pH treatment alone, by hydrochloric acid addition, showed no bactericidal effect on any of the three states of S. aureus strain. However, microencapsulated carvacrol was able to significantly reduce the planktonic cells and biofilm-detached cells below the detection limit (no bacterial counts), and the biofilm by approximatively 3 log CFU mL-1. In addition, results showed that microencapsulated carvacrol combined with low pH treatment reduced biofilm by more than 5 log CFU mL-1. Thus, the use of microencapsulated carvacrol in acidic environment could be a promising approach to combat biofilms from abiotic surfaces.

Two-Month Consumption of Orange Juice Enriched with Vitamin D3 and Probiotics Decreases Body Weight, Insulin Resistance, Blood Lipids, and Arterial Blood Pressure in High-Cardiometabolic-Risk Patients on a Westernized Type Diet: Results from a Randomized Clinical Trial.

This study examined the effects of orange juice (OJ) supplemented with vitamin D3 (2000 IU) and probiotics (Lacticaseibacillus casei Shirota and Lacticaseibacillus rhamnosus GG, 108 cfu/mL) on cardiometabolic risk factors in overweight and obese adults following a Westernized-type diet. Fifty-three high-risk individuals were randomly assigned to one of two groups. Over 8 weeks, one group consumed a vitamin D3 and probiotic-enriched OJ and the other regular OJ (control). Diets remained unchanged and were documented through food diaries. Measures of metabolic and inflammatory markers and blood pressure were measured at the start and end of the study. Post-intervention, the enriched OJ group showed the following significant metabolic improvements (without changes in triglycerides, inflammation, or central blood pressure): reduced fasting insulin, peripheral blood pressure, body weight (-1.4 kg 95% CI: -2.4, -0.4), energy (-270 kcal 95% CI: -553.2, -13.7), macronutrient (dietary fat -238 kcal 95% CI: -11.9, -1.0; carbohydrates -155 kcal 95% CI: -282.4, -27.3; sugars -16.1 g 95% CI: -11.9, -1.0) intake, and better lipid profiles (total cholesterol -10.3 mg/dL 95% CI: -21.4, 0.9; LDL-C -7 mg/dL 95% CI: -13.5, -0.5). The enriched OJ led to weight loss, less energy/macronutrient consumption, improved lipid profiles, and increased insulin sensitivity after 8 weeks in those following a Westernized diet, thus indicating potential benefits for cardiometabolic risk. This study was a part of FunJuice-T2EDK-01922, which was funded by the EU Regional Development Fund and Greek National Resources.

The impact of different acidic conditions and food substrates on Listeria monocytogenes biofilms development and removal using nanoencapsulated carvacrol.

Listeria monocytogenes biofilms present a significant challenge in the food industry. This study explores the impact of different acidic conditions of culture media and food matrices on the development and removal of biofilms developed on stainless steel surfaces by wild-type (WT) L. monocytogenes strains as well as in two mutant derivatives, ΔsigB and ΔagrA, that have defects in the general stress response and quorum sensing, respectively. Additionally, the study investigates the efficacy of nanoencapsulated carvacrol as an antimicrobial against L. monocytogenes biofilms developed in Tryptic Soy Broth (TSB) culture media acidified to different pH conditions (3.5, 4.5, 5.5, 6.5), and in food substrates (apple juice, strained yogurt, vegetable soup, semi-skimmed milk) having the same pH levels. No biofilm formation was observed for all L. monocytogenes strains at pH levels of 3.5 and 4.5 in both culture media and food substrates. However, at pH 5.5 and 6.5, increased biofilm levels were observed in both the culture media and food substrates, with the WT strain showing significantly higher biofilm formation (3.04-6.05 log CFU cm-2) than the mutant strains (2.30-5.48 log CFU cm-2). For both applications, the nanoencapsulated carvacrol demonstrated more potent antimicrobial activity against biofilms developed at pH 5.5 with 2.23 to 3.61 log reductions, compared to 1.58-2.95 log reductions at pH 6.5, with mutants being more vulnerable in acidic environments. In food substrates, nanoencapsulated carvacrol induced lower log reductions (1.58-2.90) than the ones in TSB (2.02-3.61). These findings provide valuable insights into the impact of different acidic conditions on the development of L. monocytogenes biofilms on stainless steel surfaces and the potential application of nanoencapsulated carvacrol as a biofilm control agent in food processing environments.

In vitro digestion and characterization of selenized Saccharomyces cerevisiae, Pichia fermentans and probiotic Saccharomyces boulardii.

BACKGROUND AND OBJECTIVE: Yeasts have the remarkable capability to transform and integrate inorganic selenium into their cellular structures, thereby enhancing its bioavailability and reducing its toxicity. In recent years, yeasts have attracted attention as potential alternative sources of protein. METHODS: This study explores the selenium accumulation potential of two less explored yeast strains, namely the probiotic Saccharomyces boulardii CCDM 2020 and Pichia fermentas CCDM 2012, in comparison to the extensively studied Saccharomyces cerevisiae CCDM 272. Our investigation encompassed diverse stress conditions. Subsequently, the selenized yeasts were subjected to an INFOGEST gastrointestinal model. The adherence and hydrophobicity were determined with undigested cells RESULTS: Stress conditions had an important role in influencing the quantity and size of selenium nanoparticles (SeNPs) generated by the tested yeasts. Remarkably, SeMet synthesis was limited to Pichia fermentas CCDM 2012 and S. boulardii CCDM 2020, with S. cerevisiae CCDM 272 not displaying SeMet production at all. Throughout the simulated gastrointestinal digestion, the most substantial release of SeCys2, SeMet, and SeNPs from the selenized yeasts occurred during the intestinal phase. Notably, exception was found in strain CCDM 272, where the majority of particles were released during the oral phase. CONCLUSION: The utilization of both traditional and non-traditional selenized yeast types, harnessed for their noted functional attributes, holds potential for expanding the range of products available while enhancing their nutritional value and health benefits.

Application of multi-functional lactic acid bacteria strains in a pilot scale feta cheese production.

Feta cheese is the most recognized Greek Protected Designation of Origin (PDO) product in the world. The addition of selected autochthonous lactic acid bacteria (LAB) strains to cheese milk as adjunct cultures is gaining more attention, since they can impact the nutritional, technological and sensory properties of cheeses, as well as improve the safety of the product. The aim of this study was to produce Feta cheese with enhanced quality and safety, and distinctive organoleptic characteristics by applying autochthonous lactic acid bacteria (LAB) with multi-functional properties as adjunct cultures. Feta cheeses were produced with the commercial lactococcal starter culture and the addition of 9 LAB strains (Lactococcus lactis SMX2 and SMX16, Levilactobacillus brevis SRX20, Lacticaseibacillus paracasei SRX10, Lactiplantibacillus plantarum FRX20 and FB1, Leuconostoc mesenteroides FMX3, FMX11, and FRX4, isolated from artisanal Greek cheeses) in different combinations to produce 13 cheese trials (12 Feta trials with the adjunct LAB isolates and the control trial). In addition, Feta cheese manufactured with FMX3 and SMX2 and control Feta cheese were artificially inoculated (4 log CFU/g) with Listeria monocytogenes (a cocktail of 4 acid or non-acid adapted strains). Cheese samples were monitored by microbiological and physicochemical analyses during ripening, and microbiological, physicochemical, molecular and sensory analyses during storage at 4°C. The results showed that after manufacture, the LAB population was ca. 9.0 log CFU/g at all samples, whereas during storage, their population declined to 6.5-7.0 log CFU/g. In the Listeria inoculated samples, Listeria was absent after 60 days (end of ripening) and after 90 days in the adjunct culture, and in the control trials, respectively. Moreover, the addition of selected strains, especially Lcb. paracasei SRX10, led to cheeses with desirable and distinctive organoleptic characteristics. Furthermore, randomly amplified polymorphic PCR (RAPD-PCR) molecular analysis confirmed that the multi-functional LAB strains were viable by the end of storage. Overall, the results of this study are promising for the use of autochthonous strains in various combinations with the commercial starter culture to satisfy industry requirements and consumer demands for traditional and high added value fermented products.

Depiction of the In Vitro and Genomic Basis of Resistance to Hop and High Hydrostatic Pressure of Lactiplantibacillus plantarum Isolated from Spoiled Beer.

Among the beer-spoiling microorganisms, the dominant ones belong to the genera Lactobacillus, Leuconostoc, Oenococcus, and Pediococcus. It is assumed that resistance to hop bitters correlates with resistance to other factors and can significantly impact the brewing industry. Beer preservation with high hydrostatic pressure eliminates the spoiling microorganisms while preserving all desired properties of the beer. Here, we present comprehensive in vitro and genomic analysis of the beer-spoiling Lactiplantibacillus plantarum KKP 3573 capacity to resist hop and high hydrostatic pressure. Lp. plantarum KKP 3573 is a strain isolated from spoiled beer. Our finding suggests that the growth rate of the strain depends on the medium variant, where a small concentration of beer (5 IBU) stimulates the growth, suggesting that the limited concentration has a positive effect on cell growth. At the same time, increased concentrations of 20 IBU, 30 IBU, and pure beer 43.6 IBU decreased the growth rate of the KKP 3573 strain. We observed that higher extract content in the pressurized beer increased microbial survivability. The wort and Vienna Lager beer can stimulate the baroprotective effect. The taxonomy of the novel strain was confirmed after whole genome sequencing (WGS) and comparative genomic analysis. More specifically, it contains a chromosome of 3.3 Mb with a GC content of 44.4%, indicative of the Lp. plantarum species. Accordingly, it possesses high genomic similarity (>98%) with other species members. Annotation algorithms revealed that the strain carries several genes involved in resistance to stress, including extreme temperature, hop bitters and high pressure, and adaptation to the brewing environment. Lastly, the strain does not code for toxins and virulence proteins and cannot produce biogenic amines.

Deciphering the growth responses and genotypic diversity of bioluminescent Photobacterium phosphoreum on chicken meat during aerobic refrigerated storage.

The advent of high-throughput sequencing technologies in recent years has revealed the unexpected presence of genus Photobacterium within the chicken meat spoilage ecosystem. This study was undertaken to decipher the occurrence, the growth patterns and the genotypic biodiversity of Photobacterium phosphoreum on chicken breast fillets stored aerobically at 4 °C through conventional microbiological methods and molecular techniques. Samples were periodically cultured on marine broth agar (MA; supplemented with meat extract and vancomycin) for the enumeration of presumptive bioluminescent Photobacterium spp. In total, 90 bioluminescent bacteria were recovered from the initial (time of first appearance), middle and end stages of storage. Concomitantly, 95 total psychrotrophic/psychrophilic bacteria were isolated from the same medium to assess the presence and diversity of non-luminous photobacteria. Genetic diversity between bioluminescent isolates was assessed with two PCR-based DNA fingerprinting methods, i.e. RAPD and rep-PCR. Moreover, the characterization of selected bacterial isolates at the genus and/or species level was performed by sequencing of the 16S rRNA and/or gyrB gene. Bioluminescent bacteria were scarcely encountered in fresh samples at population levels of ca. 2.0 log CFU/g, whilst total psychrotrophic/psychrophilic bacteria were found at levels of ca. 4.4 log CFU/g. As time proceeded and close to shelf-life end, bioluminescent bacteria were encountered at higher populations, and were found at levels of 5.3 and 7.0 log CFU/g in samples from the second and third batch, respectively. In the first batch their presence was occasional and at levels up to 3.9 log CFU/g. Accordingly, total psychrotrophic/psychrophilic bacteria exceeded 8.4 log CFU/g at the end of storage, suggesting the possible underestimation of bioluminescent populations following the specific cultivation conditions. Sequence analysis assigned bioluminescent isolates to Photobacterium phosphoreum, while genetic fingerprinting revealed high intra-species variability. Respectively, total psychrotrophs/psychrophiles were assigned to genera Pseudomonas, Shewanella, Psychrobacter, Acinetobacter, Vibrio and Photobacterium. Non-luminous photobacteria were not identified within the psychrotrophs/psychrophiles. Results of the present study reveal the intra- and inter-batch variability on the occurrence and growth responses of P. phosphoreum and highlight its potential role in the chicken meat spoilage consortium.

Short-Term Effects of Fruit Juice Enriched with Vitamin D3, n-3 PUFA, and Probiotics on Glycemic Responses: A Randomized Controlled Clinical Trial in Healthy Adults.

This study aimed to determine the glycemic index (GI) of a commercial mixed fruit juice (apple, orange, grape, and pomegranate; FJ) fortified with vitamin D3 or n-3 polyunsaturated fatty acids (PUFA) or probiotics, and their combination, and their effects on glycemic responses and salivary insulin concentrations. In a randomized controlled, double-blind, crossover study, 11 healthy participants (25 ± 2 years; five women; body mass index = 23 ± 1 kg/m2) were randomly assigned to receive five types of FJs [vitD (with vitamin D3); n-3 (with n-3 PUFA); probiotics (with Lacticaseibacillus casei Shirota and Lacticaseibacillus rhamnosus GG); vitD-n-3-probiotics FJ (combination of vitD3-n-3-probiotics), control (regular FJ)], all containing 50 g available carbohydrate, and glucose as reference drink. All FJs provided low GI values (control: 54; vitD3: 52; n-3: 51; probiotics: 50; and vitD-n-3-probiotics combination: 52, on glucose scale). Compared to the FJ control, the enriched FJs produced different postprandial glycemic and insulinemic responses and affected satiety scores. All FJ types, regardless of the added biofunctional ingredients, attenuated postprandial glycemic responses, which may offer advantages to glycemic control.

Role of Microbial Interactions across Food-Related Bacteria on Biofilm Population and Biofilm Decontamination by a TiO2-Nanoparticle-Based Surfactant.

Microbial interactions play an important role in initial cell adhesion and the endurance of biofilm toward disinfectant stresses. The present study aimed to evaluate the effect of microbial interactions on biofilm formation and the disinfecting activity of an innovative photocatalytic surfactant based on TiO2 nanoparticles. Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli, Leuconostoc spp., Latilactobacillus sakei, Serratia liquefaciens, Serratia proteomaculans, Citrobacter freundii, Hafnia alvei, Proteus vulgaris, Pseudomonas fragi, and Brochothrix thermosphacta left to form mono- or dual-species biofilms on stainless steel (SS) coupons. The effectiveness of the photocatalytic disinfectant after 2 h of exposure under UV light on biofilm decontamination was evaluated. The effect of one parameter i.e., exposure to UV or disinfectant, was also determined. According to the obtained results, the microbial load of a mature biofilm depended on the different species or dual species that had adhered to the surface, while the presence of other species could affect the biofilm population of a specific microbe (p < 0.05). The disinfectant strengthened the antimicrobial activity of UV, as, in most cases, the remaining biofilm population was below the detection limit of the method. Moreover, the presence of more than one species affected the resistance of the biofilm cells to UV and the disinfectant (p < 0.05). In conclusion, this study confirms that microbial interactions affected biofilm formation and decontamination, and it demonstrates the effectiveness of the surfactant with the photocatalytic TiO2 agent, suggesting that it could be an alternative agent with which to disinfect contaminated surfaces.

Genomic Analysis and In Vitro Investigation of the Hop Resistance Phenotype of Two Novel Loigolactobacillus backii Strains, Isolated from Spoiled Beer.

Loigolactobacillus backii is an important beer-spoiling species, exhibiting high hop tolerance. Here, we present the annotated whole genome sequence of two recently isolated strains, Lg. backii KKP 3565 and KKP 3566. Firstly, to study the genetic basis of the persistence of the two isolates in beer, a comprehensive bioinformatic analysis ensued. Their chromosome map was constructed, using whole-genome sequencing and assembly, revealing that the two strains carry genomes with a length of 2.79 Mb with a GC content of 40.68%. An average nucleotide identity (ANI) analysis demonstrated that the novel strains possess unique genomic sequences, also confirming their classification into the Lg. backii species. Their genome harbors numerous insertion sequences and plasmids, originating from other beer-spoiling species. Regarding their adaptation in brewery environment, homologous genes that confer resistance to hop were spotted, while the impact of hop bitters and pure beer on bacterial growth was investigated, in vitro. In brief, low hop concentrations were found to induce the proliferation of strains, while a higher concentration negatively affected their growth. Nonetheless, their ability to survive in pure beer indicated their tolerance to high hop concentrations. These results offer insight into the capacity of Lg. backii KKP 3566 and Lg. backii KKP 3566 to tolerate the extreme conditions prevalent in the brewery environment.

Corrigendum: Editorial: Biofilm formation and quorum sensing of foodborne microorganism.

[This corrects the article DOI: 10.3389/fmicb.2022.1107603.].

Mapping the Key Technological and Functional Characteristics of Indigenous Lactic Acid Bacteria Isolated from Greek Traditional Dairy Products.

The aim of the current study was to isolate indigenous lactic acid bacteria (LAB) from traditional Greek cheeses and assess their biochemical, technological, and functional characteristics, so as to develop novel cultures with multi-functional properties. Hence, 109 LAB isolates were recovered from traditional fresh cheeses and were evaluated in vitro for their gas production; proteolytic, lipolytic, and haemolytic activity; exopolysaccharide production (EPS); enzymatic potential; and ability to grow at 6.5% NaCl and at different pH, temperature, and anaerobic conditions. Consequently, 48 selected isolates were further evaluated for their survival under simulated gastrointestinal tract conditions, partial bile salt hydrolase activity, antibiotic resistance, and antimicrobial activity against pathogens. These isolates were also incorporated as co-cultures in yogurt production to examine their sensory characteristics and their survival in the product. Some prominent isolates that showed favorable technological and functional characteristics (good survival rates at low pH and bile salts, ability to produce ß-galactosidase, and EPS) and attributed desirable sensory characteristics to yogurt were Lactococcuslactis (SRX2, SRX3, SRX5, and SMX16), Lactobacillus paracasei SRX10, and Lactiplantibacillusplantarum (FRX7, FB1), while Leuconostoc mesenteroides FMX3 and L. lactis SMX2 showed an anti-listerial activity in vitro. The results of the present study are promising for the production of novel dairy functional products with an enhanced quality and safety.

Food Microbial Diversity.

The microbiological quality and safety of food could be assessed by mapping the microorganisms present in a particular type of food [...].

Evaluation of Plant Origin Essential Oils as Herbal Biocides for the Protection of Caves Belonging to Natural and Cultural Heritage Sites.

The present study concerns the serious issue of biodeterioration of the caves belonging to natural and cultural heritage sites due to the development of various microorganisms. Thus, a series of 18 essential oils (EOs) extracted from various Greek plants were evaluated in vitro (concentrations of 0.1, 0.2, 0.5, 1.0 and 5.0% v/v) against 35 bacterial and 31 fungi isolates (isolated from a Greek cave) and the antimicrobial activity was evident through the changes in optical density of microbial suspensions. In continuance, eight (8) representative bacterial and fungal isolates were further used to evaluate the minimum inhibitory concentration (MIC) and non-inhibitory concentration (NIC) values of the most effective EOs. According to the results, two EOs of Origanum vulgare were the most effective by inhibiting the growth of all the tested microorganisms at 0.1% (v/v), followed by that of Satureja thymbra which inhibited all bacterial isolates at 0.1% (v/v) and fungal isolates at 0.1, 0.2 and 0.5% (v/v) (depending on the isolate). The MIC ranged between 0.015-0.157 and 0.013-0.156 (v/v) for the bacterial and fungal isolates respectively, depending on the case. The current study demonstrated that conventional biocides may be replaced by herbal biocides with significant prospects for commercial exploitation.

Microbiota of Chicken Breast and Thigh Fillets Stored under Different Refrigeration Temperatures Assessed by Next-Generation Sequencing.

Chicken is one of the most widely consumed meats worldwide. The exploration of the bacterial diversity of chicken meat may provide new insights into the chicken-associated microbiome that will lead to moderation of food spoilage or safety. This study was undertaken to explore the bacterial communities of chicken breast and thigh fillets stored at refrigeration (0 °C and 5 °C) and slightly abuse (10 °C) temperatures for 5 days through conventional cultural methods along with next-generation sequencing (NGS) analysis. Total viable counts (TVC), Brochothrix thermosphacta, Pseudomonas spp., and lactic acid bacteria (LAB) were enumerated, while the bacterial communities were mapped through 16S rRNA gene amplicon sequencing. Chicken breast and thigh fillets possessed a complex bacterial structure that incorporated a total of >200 Operational Taxonomic Units (OTUs) at the genus level. The core microbiota of fresh samples consisted of Acinetobacter, Brochothrix, Flavobacterium, Pseudomonas, Psychrobacter, and Vibrionaceae (family). These genera persisted until the end of storage in >80% of samples, except Psychrobacter and Flavobacterium, while Photobacterium was also identified. Hierarchical clustering showed a distinction of samples based on storage time and chicken part. Conventional plate counting with growth media commonly used in spoilage studies did not always correspond to the microbial community profiles derived from NGS analysis, especially in Pseudomonas, Acinetobacter, Photobacterium, and Vibrionaceae. Results of the present study highlight Photobacterium and Vibrionaceae, in general, as potent chicken meat spoilers and suggest the necessity to combine classical microbiological methods along with NGS technologies to characterize chicken meat spoilage microbiota.

Exploring the Bacterial Communities of the Kaiafas Thermal Spring Anigrides Nymphes in Greece Prior to Rehabilitation Actions.

Anigrides Nymphes of Lake Kaiafas is a thermal spring that is well known for its therapeutical properties, as the hot water (32-34 °C) is rich in sulfur compounds and minerals. Nowadays, efforts are made from the Hellenic Republic to modernize the existing facilities and infrastructure networks of the area. To study the complex ecosystem of the thermal spring, we collected water from four sampling points (Lake, and Caves 1, 2, and 3). Filtration method was used for microbial enumeration. In parallel, total bacterial DNA was extracted and subjected to next-generation sequencing (NGS). A total of 166 different bacterial families were detected. Differences in families, genera, and species abundances were detected between the different sampling points. Specifically, Comamonadaceae was the most common family detected in Lake and Cave 3. Similarly, in Caves 1 and 2, Rhodobacteraceae was detected at a higher percentage compared to the rest of the families. Moreover, the detection of sequences assigned to waterborne or opportunistic pathogens, i.e., Enterobacteriaceae, Legionellaceae, Coxiellaceae, and Clostridiaceae, as well as Enterococcus and Vibrio, is of great importance. Although the presence of pathogens was not examined by quantitative PCR, the detection of their sequences strengthens the need of the planned rehabilitation actions of this natural environment in order to allow human swimming.

Microbial Diversity of Fermented Greek Table Olives of Halkidiki and Konservolia Varieties from Different Regions as Revealed by Metagenomic Analysis.

Current information from conventional microbiological methods on the microbial diversity of table olives is insufficient. Next-generation sequencing (NGS) technologies allow comprehensive analysis of their microbial community, providing microbial identity of table olive varieties and their designation of origin. The purpose of this study was to evaluate the bacterial and yeast diversity of fermented olives of two main Greek varieties collected from different regions-green olives, cv. Halkidiki, from Kavala and Halkidiki and black olives, cv. Konservolia, from Magnesia and Fthiotida-via conventional microbiological methods and NGS. Total viable counts (TVC), lactic acid bacteria (LAB), yeast and molds, and Enterobacteriaceae were enumerated. Microbial genomic DNA was directly extracted from the olives' surface and subjected to NGS for the identification of bacteria and yeast communities. Lactobacillaceae was the most abundant family in all samples. In relation to yeast diversity, Phaffomycetaceae was the most abundant yeast family in Konservolia olives from the Magnesia region, while Pichiaceae dominated the yeast microbiota in Konservolia olives from Fthiotida and in Halkidiki olives from both regions. Further analysis of the data employing multivariate analysis allowed for the first time the discrimination of cv. Konservolia and cv. Halkidiki table olives according to their geographical origin.