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.

Exploration of the shelf-life difference between chilled beef and pork with similar initial levels of bacterial contamination.

This study compared the shelf-life of beef and pork longissimus lumborum muscles (loins) that had the same initial bacterial loads and were held under the same chilled storage conditions. To identify the underlying pathways, comparisons were conducted from the perspective of the spoilage indicators; protease/lipase activity, and the volatile organic compounds (VOC) generated over 28 d of chilled storage. The initial total viable microbial count (TVC) on Day 0 for both type of meat was 4.3 log10 CFU/g. It was found that the TVC of beef and pork did not differ throughout the total chilled storage period and both ultimately exceeded 7 log10 CFU/g after 28 d. Based on total volatile basic nitrogen (TVB-N) guidelines, pork was spoilt after 21 d of chilled storage and therefore 7 d earlier than beef. Changes in the concentration of VOC spoilage biomarkers, including 1-octen-3-ol, 1-octanol, nonanal, and others, confirmed that pork had a shorter shelf-life than beef. An important reason for the difference in shelf-life between the two types of meat was that pork had a higher protease activity, although the beef had higher levels of total lipase activity. These findings help us understand the differences in the spoilage process of raw meat from different species and explore specific measures to control the spoilage of beef or pork.

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.

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.

Exploring yeast diversity of dry-salted naturally black olives from Greek retail outlets with culture dependent and independent molecular methods.

In the present study, the physicochemical (pH, water activity, moisture content, salt concentration) classical plate counts (total viable counts, yeasts, lactic acid bacteria, Staphylococcus aureus, Pseudomonas spp., Enterobacteriaceae) and amplicon sequencing of naturally black dry-salted olives obtained from different retail outlets of the Greek market were investigated. According to the results, the values of the physicochemical characteristics presented great variability among the samples. Specifically, pH and water activity (aw) values ranged between 4.0 and 5.0, as well as between 0.58 and 0.91, respectively. Moisture content varied between 17.3 and 56.7 % (g Η2Ο/100 g of olive pulp), whereas salt concentration ranged from 5.26 to 9.15 % (g NaCl/100 g of olive pulp). No lactic acid bacteria, S. aureus, Pseudomonas spp. and Enterobacteriaceae were detected. The mycobiota consisted of yeasts that were further characterized and identified by culture-dependent (rep-PCR, ITS-PCR, and RFLP) and amplicon target sequencing (ATS). Pichia membranifaciens, Candida sorbosivorans, Citeromyces nyonsensis, Candida etchelsii, Wickerhamomyces subpelliculosus, Candida apicola, Wickerhamomyces anomalus, Torulaspora delbrueckii and Candida versatilis were the dominant species according to ITS sequencing (culture-dependent), while ATS revealed the dominance of C. etchelsii, Pichia triangularis, P. membranifaciens, and C. versatilis among samples. The results of this study demonstrated considerable variability in quality attributes among the different commercial samples of dry-salted olives, reflecting a lack of standardization in the processing of this commercial style. However, the majority of the samples were characterized by satisfactory microbiological and hygienic quality and complied with the requirements of the trade standard for table olives of the International Olive Council (IOC) for this processing style in terms of salt concentration. In addition, the diversity of yeast species was elucidated for the first time in commercially available products, increasing our knowledge on the microbial ecology of this traditional food. Further investigation into the technological and multifunctional traits of the dominant yeast species may result in better control during dry-salting and enhance the quality and shelf-life of the final product.

Evaluating the Quality of Cheese Slices Packaged with Na-Alginate Edible Films Supplemented with Functional Lactic Acid Bacteria Cultures after High-Pressure Processing.

The aim of the current study was to assess the efficacy of Na-alginate edible films as vehicles for delivering lactic acid bacteria (LAB) with functional properties to sliced cheeses, with or without high-pressure processing (HPP). A three-strain LAB cocktail (Lactococcus lactis Τ4, Leuconostoc mesenteroides Τ25 and Lacticaseibacillus paracasei Τ26) was incorporated into Na-alginate solution in a final population of 9 log CFU/mL. The cheese slices (without or with HPP treatment at 500 MPa for 2 min) were packaged in contact with the LAB edible films (LEFs), and subsequently vacuum packed and stored at 4 °C. Cheese slices without the addition of films, with or without HPP treatment, were used as controls. In all cases, microbiological, pH and sensory analyses were performed, while the presence and the relative abundance of each strain during storage was evaluated using Random Amplified Polymorphic DNA-PCR (RAPD-PCR). In addition, organic acid determination and peptide analysis were performed using high-performance liquid chromatography. The results showed that in cheeses without HPP treatment, the microbiota consisted mostly of mesophilic LAB and lactococci (>7.0 log CFU/g), while HPP caused a reduction in the indigenous microbiota population of approximately 1−1.5 log CFU/g. In the LEF samples, the populations of mesophilic LAB and lactococci were maintained at levels of >6.35 log CFU/g during storage, regardless of the HPP treatment. Sensory evaluation revealed that the LEF samples without HPP had a slightly more acidic taste compared to the control, whereas the HPP-LEF samples exhibited the best organoleptic characteristics. RAPD-PCR confirmed that the recovered strains were attributed to the three strains that had been entrapped in the films, while the strain distribution during storage was random. Overall, the results of the study are promising since the functional LAB strains were successfully delivered to the products by the edible films until the end of storage.

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.

Use of Fourier Transform Infrared Spectroscopy for Monitoring the Shelf Life and Safety of Yogurts Supplemented With a Lactobacillus plantarum Strain With Probiotic Potential.

The current study aimed to explore the performance of a probiotic Lactobacillus strain as an adjunct culture in yogurt production and to assess Fourier transform infrared spectroscopy as a rapid, noninvasive analytical technique to evaluate the quality and the shelf life of yogurt during storage. In this respect, bovine milk (full-fat) was inoculated with the typical yogurt starter culture without (control case) or with the further addition of Lactobacillus plantarum T571 as an adjunct (probiotic case). The milk was also inoculated with a cocktail mixture of three strains of Listeria monocytogenes in two different initial levels of inoculum, and the fermentation process was followed. Accordingly, yogurt samples were stored at 4 and 12°C, and microbiological, physicochemical, molecular, and sensory analyses were performed during storage. Results showed that the lactic acid bacteria exceeded 7 log CFU/g during storage in all samples, where the probiotic samples displayed higher acidity, lower pH, and reduced counts of Lb. monocytogenes in a shorter period than the control ones at both temperatures. Pulsed-field gel electrophoresis verified the presence of the probiotic strain until the end of storage at both temperatures and in adequate amounts, whereas the survival and the distribution of Listeria strains depended on the case. The sensory evaluation showed that the probiotic samples had desirable organoleptic characteristics, similar to the control. Finally, the spectral data collected from the yogurt samples during storage were correlated with microbiological counts and sensory data. Partial least squares and support vector machine regression and classification models were developed to provide quantitative estimations of yogurt microbiological counts and qualitative estimations of their sensory status, respectively, based on Fourier transform infrared fingerprints. The developed models exhibited satisfactory performance, and the acquired results were promising for the rapid estimation of the microbiological counts and sensory status of yogurt.

Spoilage Potential of Pseudomonas (P. fragi, P. putida) and LAB (Leuconostoc mesenteroides, Lactobacillus sakei) Strains and Their Volatilome Profile During Storage of Sterile Pork Meat Using GC/MS and Data Analytics.

The aim of the present study was to investigate the evolution of the volatile compounds of aerobically stored sterile pork meat as a consequence of the metabolic activities of inoculated specific spoilage microorganisms. Thus, Pseudomonas fragi, Pseudomonas putida, Lactobacillus sakei and Leuconostoc mesenteroides were inoculated in monocultures, dual cultures and a cocktail culture of all strains on sterile pork meat stored aerobically at 4 and 10 °C. Microbiological and sensory analyses, as well as pH measurements, were performed, along with headspace solid-phase microextraction gas chromatography/mass spectroscopy (headspace SPME-GC/MS) analysis. Data analytics were used to correlate the volatile compounds with the spoilage potential of each stain using multivariate data analysis. The results for the sensory discrimination showed that the volatiles that dominated in spoiled samples consisted mostly of alcohols, ketones and two esters (butyl acetate and ethyl acetate), while at fresh samples, dimethyl sulfide, furans, acetoin and ethyl lactate were detected. On the other hand, 2-butanone, diacetyl and acetaldehyde were among the volatile compounds that were mainly correlated with the inoculated meat during storage. In addition, P. fragi was positively correlated with a higher number of volatiles compared to the other strains, strengthening the hypothesis that volatile compound production is strain-dependent.

Quality and Safety of Fresh Chicken Fillets after High Pressure Processing: Survival of Indigenous Brochothrix thermosphacta and Inoculated Listeria monocytogenes.

The effect of high-pressure processing (HPP) on Listeria monocytogenes, the indigenous microbiota and the shelf-life of chicken fillets was evaluated. Chicken fillets were inoculated with different inocula (2, 4, and 6 log CFU/g) of a 4-strain cocktail of L. monocytogenes, vacuum-packed, processed or not with HPP (500 MPa/10 min) and stored at 4 °C and 12 °C. Total viable counts (TVC), L. monocytogenes, Pseudomonas spp., Brochothrix thermosphacta, lactic acid bacteria (LAB), Enterobacteriaceae and yeasts/molds were determined along with the pH and sensory analysis. Pulsed-field gel electrophoresis (PFGE) was used to monitor the succession of indigenous Brochothrix isolates and inoculated Listeria strains. The main spoilage microorganism of HPP-treated samples was B. thermosphacta detected after 3 days of storage. HPP decreased the inoculated Listeria population. For the low and medium inoculum case it was detected throughout the shelf-life at both temperatures in populations near to the detection limit or after enrichment. In the high inoculum case, the pathogen decreased ≥5-log cycles after HPP, while increased subsequently to 1.6 and 4.5 log CFU/g at 4 °C and 12 °C, respectively, by the end of the shelf-life. PFGE showed that Brochothrix isolates exhibited a significant diversity among control samples, whereas this was limited for the HPP-treated samples. The survival and distribution of different Listeria strains depended on the initial inoculum and storage temperature. In conclusion, HPP increased the shelf-life (for 5 and 4 days, at 4 °C and 12 °C, respectively) and enhanced the safety of chicken meat.

Greek functional Feta cheese: Enhancing quality and safety using a Lactobacillus plantarum strain with probiotic potential.

The aim of the study was to investigate the performance of Lactobacillus plantarum T571 with probiotic potential as a co-starter culture in Feta cheese production and in its long-term storage. For this reason, Feta cheese was manufactured without (control) or with the probiotic T571 strain (probiotic) and then monitored during storage at 4 and 12 °C, respectively. The products were also inoculated with Listeria monocytogenes (3-strain cocktail). The results showed that lactic acid bacteria exceeded 6 log CFU/g during storage in all trials. The probiotic samples displayed higher acidity (≈1.5% lactic acid) and lower pH (≈4.2). Coliforms and L. monocytogenes, were inactivated in shorter time in probiotic samples, in comparison with the control ones. Pulsed field gel electrophoresis verified the presence of the probiotic strain in the cheese, until the end of storage at both temperatures, whilst the survival and distribution of the pathogenic strains depended on the trial. The sensory evaluation showed that the probiotic cheeses had desirable sensory characteristics similar to the control ones, with scores of ca. 8 on a 10 cm-scale. Finally, Fourier transform infrared spectroscopy was applied for the first time during Feta cheese storage with promising results for the rapid estimation of the microbiological counts and sensory status of cheese. Results showed that Lb. plantarum T571 highlighted desirable and robust technological properties and may be used in cheese making as an adjunct culture.

Effect of high pressure processing on the survival of Salmonella Enteritidis and shelf-life of chicken fillets.

High pressure processing (HPP) is a preservation technology alternative to heat treatment that is mild for food, but effectively inactivates the spoilage microbiota and foodborne pathogens of several foods. The purpose of the current study was to evaluate the effect of HPP on Salmonella ser. Enteritidis, indigenous microbiota and shelf-life of chicken fillets. Chicken fillets were inoculated with S. Enteritidis at three different initial inocula (3, 5, 7 log CFU/g), packed under vacuum, treated or not with HPP (500 MPa/10 min) and stored at 4 and 12 °C. Total viable counts, S. Enteritidis, pseudomonads, Brochothrix thermosphacta, lactic acid bacteria, Enterobacteriaceae and yeasts/molds populations were determined in parallel with sensory analysis of non-inoculated samples. The HPP resulted in the reduction of the pathogen population below the detection limit of the enumeration method (0.48 log CFU/g), irrespective of the inoculum. During the shelf life of the HPP samples, the pathogens population remained below or near the detection limit of the enumeration method at both temperatures, except from the high inoculum case that an increase was observed at 12 °C. At the low inoculum level, the pathogen could not be detected with the enrichment method after the first storage days (2nd day for 4 °C and 0 day for 12 °C). The survival of Salmonella strains was assessed by pulsed field gel electrophoresis and it was shown that the survival of the different strains depended on the inoculum and storage temperature. Regarding the indigenous microbiota, Br. thermosphacta was reported for the first time to be the main spoilage microorganism that survived and dominated after the HPP. From the results it was evident that, HPP may enhance the safety and increase the shelf life (6 at 4 °C and 2 days at 12 °C) of chicken meat.

Molecular Detection of Two Potential Probiotic Lactobacilli Strains and Evaluation of Their Performance as Starter Adjuncts in Yogurt Production.

A molecular method for efficient and accurate detection and identification of two potential probiotic lactobacilli strains isolated from fermented olives, namely Lactobacillus pentosus B281 and Lb. plantarum B282, was developed in the present study. Random Amplified Polymorphic DNA (RAPD) analysis was performed, and strain specific primers were designed and applied in a multiplex polymerase chain reaction (PCR) assay. The specificity of the assay was tested and successfully confirmed in 27 and 22 lactobacilli strains for Lb. pentosus B281 and Lb. plantarum B282, respectively. Moreover, the two strains were used as starter cultures in yogurt production. Cell enumeration followed by multiplex PCR analysis demonstrated that the two strains were present in yogurt samples at levels ≥6 log CFU/g even after 35 days of storage at 4 °C. Microbiological analysis showed that lactobacilli and streptococci were present within usual levels, whereas enterobacteriaceae and yeast/mold counts were not detected as expected. Although the pH values of the novel products were slightly lower than the control ones, the yogurt containing the probiotic cultures scored similar values compared to the control in a series of sensory tests. Overall, these results demonstrated the possible use of the two strains as starter adjuncts in the production of yogurt with potential probiotic properties.

Genotypic characterization of Brochothrix thermosphacta isolated during storage of minced pork under aerobic or modified atmosphere packaging conditions.

A total of 306 colonies were isolated from the selective medium for Brochothrix spp., during the spoilage of minced pork stored at 0, 5, 10 and 15 °C and packed aerobically and under modified atmosphere packaging conditions (MAP). Brochothrix biodiversity was assessed by Pulsed Field Gel Electrophoresis (PFGE), and representative strains were further analysed by Rep-PCR using primer (GTG)5 and Sau-PCR with primers SAG1 and SAG2. Although, different results were obtained from the different methods, a significant diversity among isolates recovered from aerobic conditions was observed. On the contrary, isolates from MAP showed a lower degree of heterogeneity. The storage conditions affected the Brochothrix diversity, the strains isolated in the initial stage being different from the ones present at the final stage of storage at chill temperatures. A representative number of isolates, based on the results of the clustering by molecular methods, were subjected to 16S rRNA gene sequencing, revealing that all belonged to Brochothrix thermosphacta.