Predicting ventricular arrhythmia inducibility in ajmaline-induced Brugada type I pattern: Validation of the dST-Tiso interval.

INTRODUCTION: The dST-Tiso is a newly proposed electrocardiographic (ECG) marker during Brugada (BrS) type I pattern, that predicts the likelihood of ventricular arrhythmia (VA) inducibility in patients with ajmaline-induced pattern. The objective of this study was to validate the effectiveness of this criterion using an independent data set. METHODS: Consecutive patients exhibiting a BrS type I ECG pattern following ajmaline administration underwent programmed ventricular stimulation (PVS). dST-Tiso interval was measured in all patients and tested as a predictor for positive VA inducibility. RESULTS: Among 128 patients (median age 43 years, 59% male) with drug-induced BrS type I ECG pattern who underwent PVS, 32 (25.0%) had VA inducibility that required defibrillation. Compared to noninducible subjects, those with positive PVS were more commonly male (81% vs. 51%, p = 0.003), had longer PQ (165 vs. 160 ms, p = 0.016) and dST-Tiso (310 vs. 230 ms, p < 0.001) intervals, and shorter QT interval (412 vs. 420 ms, p = 0.022). When treated as a continuous variable, dST-Tiso confirmed significant association with VA inducibility, with an adjusted odds ratio of 1.02 (95% confidence interval: 1.01-1.03, p < 0.001) for each 1 ms increase in duration. A dST-Tiso interval >300 ms yielded a sensitivity of 75%, a specificity of 86%, and positive and negative predictive values of 69% and 91%, respectively. CONCLUSION: The validation of the model based on the dST-Tiso interval >300 ms confirmed its high accuracy in predicting VA inducibility in drug-induced BrS type I pattern. This straightforward ECG marker might be linked to the extent of the electrical substrate of the disease.

Pulsed field ablation technology for pulmonary vein and left atrial posterior wall isolation in patients with persistent atrial fibrillation.

INTRODUCTION: Limited data exist on pulsed-field ablation (PFA) in patients with persistent atrial fibrillation (PeAF) undergoing left atrial posterior wall isolation (LAPWI). METHODS: The Advanced TecHnologies For SuccEssful AblatioN of AF in Clinical Practice (ATHENA) prospective registry included consecutive patients referred for PeAF catheter ablation at 9 Italian centers, treated with the FARAPULSETM-PFA system. The primary efficacy and safety study endpoints were the acute LAPWI rate, freedom from arrhythmic recurrences and the incidence of major periprocedural complications. Patients undergoing pulmonary vein isolation (PVI) alone, PWI + LAPWI and redo procedures were compared. RESULTS: Among 249 patients, 21.7% had long-standing PeAF, 79.5% were male; mean age was 63 ± 9 years. LAPWI was performed in 57.6% of cases, with 15.3% being redo procedures. Median skin-to-skin times (PVI-only 68 [60-90] vs. PVI + LAPWI 70 [59-88] mins) did not differ between groups. 45.8% LAPWI cases were approached with a 3D-mapping system, and 37.3% with intracardiac echocardiography. LAPWI was achieved in all patients by means of PFA alone, in 88.8% cases at first pass. LAPWI was validated either by an Ultrahigh-density mapping system or by recording electrical activity + pacing maneuvers. No major complications occurred, while 2.4% minor complications were detected. During a median follow-up of 273 [191-379] days, 41 patients (16.5%) experienced an arrhythmic recurrence after the 90-day blanking period, with a mean time to recurrence of 223 ± 100 days and no differences among ablation strategies. CONCLUSION: LAPWI with PFA demonstrates feasibility, rapidity, and safety in real-world practice, offering a viable alternative for PeAF patients. LAPWI is achievable even with a fluoroscopy-only method and does not significantly extend overall procedural times.

Temperature and ST-segment morphology remote monitoring: new perspectives for implantable cardiac monitors in Brugada syndrome.

INTRODUCTION: Patients with Brugada syndrome (BrS) face an increased risk of ventricular arrhythmias and sudden cardiac death. Implantable cardiac monitors (ICMs) have emerged as effective tools for detecting arrhythmias in BrS. Technological advancements, including temperature sensors and improved subcutaneous electrocardiogram (subECG) signal quality, hold promise for further enhancing their utility in this population. METHODS AND RESULTS: We present a case of a 40-year-old man exhibiting a BrS type 2 pattern on 12-lead ECG, who underwent ICM insertion (BIOMONITOR IIIm, BIOTRONIK) due to drug-induced BrS type 1 pattern and a history of syncope, with a negative response to programmed ventricular stimulation. The device contains an integrated temperature sensor and can transmit daily vital data, such as mean heart rate and physical activity. Several months later, remote alerts indicated a temperature increase, along with transmitted subECGs suggesting a fever-induced BrS type 1 pattern. The patient was promptly advised to commence antipyretic therapy. Over the following days, remotely monitored parameters showed decreases in mean temperature, physical activity, and mean heart rate, without further recurrence of abnormal subECGs. CONCLUSION: ICMs offer valuable insights beyond arrhythmia detection in BrS. Early detection of fever using embedded temperature sensors may improve patient management, while continuous subECG morphological analysis has the potential to enhance risk stratification in BrS patients.

Investigating Deep Sedation With Intravenous Ketamine in Spontaneous Respiration During Pulsed-Field Ablation.

OBJECTIVES: The authors report their experience of a protocol for deep sedation with ketamine in spontaneous respiration during the pulsed-field ablation (PFA) of atrial fibrillation (AF). DESIGN: Observational, prospective, nonrandomized fashion. SETTING: Single-center hospitalized patients. PARTICIPANTS: All consecutive patients undergoing PFA of AF. INTERVENTIONS: Patients undergoing deep sedation with intravenous ketamine. MEASUREMENTS AND MAIN RESULTS: The authors' sedation protocol involves the intravenous administration of fentanyl (1.5 µg/kg) and midazolam (2 mg) at low doses before local anesthesia with lidocaine. A ketamine adjunct (1 mg/kg) in 5-minute boluses was injected about 5 minutes before the first PFA delivery. The authors enrolled 117 patients (age = 59 ± 10 y, 74.4% males, body mass index = 27.6 ± 5 kg/m2, fluoroscopy time = 24 ± 14 minutes, skin-to-skin time = 80 ± 40 minutes and PFA LA dwell time = 24 ± 7 minutes). By the end of the procedure, pulmonary vein isolation had been achieved in all patients using PFA alone. The mean time under sedation was 54.9 ± 6 minutes, with 92 patients (79%) being sedated for <1 hour. A satisfactory Ramsay Sedation Scale level before ketamine administration was achieved in all patients, except one (80.3% of the patients with rank 3; 18.4% with rank 2). In all procedures, the satisfaction level was found acceptable by both the patient and the primary operator (satisfactory in 98.2% of cases). All patients achieved a Numeric Rating Scale for Pain ≤3 (none or mild). No major procedure or anesthesia-related complications were reported. CONCLUSION: The authors' standardized sedation protocol with the administration of drugs with rapid onset and pharmacologic offset at low doses was safe and effective, with an optimal degree of patient and operator satisfaction.

Do nomadic lactobacilli fit as potential vaginal probiotics? The answer lies in a successful selective multi-step and scoring approach.

BACKGROUND: The goal of this study was to create a multi-strain probiotic gel that would foster a lactobacilli-dominated vaginal microbiota in pregnant women and ensure appropriate eubiosis for the newborn. Nomadic lactobacilli (95 strains), mostly isolated from food sources, were preliminarily screened for functional traits before being characterized for their capability to inhibit the two vaginal pathogens Streptococcus agalactiae and Candida albicans, which may lead to adverse pregnancy-related outcomes. Eight best-performing strains were chosen and furtherly investigated for their ability to produce biofilm. Lastly, the two selected potential probiotic candidates were analyzed in vitro for their ability to reduce the inflammation caused by C. albicans infection on the reconstituted human vaginal epithelium (HVE). RESULTS: Lactiplantibacillus plantarum produced both isomers of lactic acid, while Lacticaseibacillus paracasei produced only L-isomer. The production of hydrogen peroxide was strain-dependent, with the highest concentrations found within Lact. paracasei strains. The auto-aggregation capacity and hydrophobicity traits were species-independent. S. agalactiae 88II3 was strongly inhibited both at pH 7.0 and 4.0, whereas the inhibition of C. albicans UNIBZ54 was less frequent. Overall, L. plantarum strains had the highest pathogen inhibition and functional scoring. L. plantarum C5 and POM1, which were selected as potential probiotic candidates also based on their ability to form biofilms, were able to counteract the inflammation process caused by C. albicans infection in the HVE model. CONCLUSIONS: Our multi-step and cumulative scoring-based approach was proven successful in mining and highlighting the probiotic potential of two nomadic lactobacilli strains (L. plantarum C5 and POM1), being applicable to preserve and improve human vaginal health.

Sedation strategies for pulsed-field ablation of atrial fibrillation: focus on deep sedation with intravenous ketamine in spontaneous respiration.

AIMS: A standardized sedation protocol for pulsed-field ablation (PFA) of atrial fibrillation (AF) through irreversible cellular electroporation has not been well established. We report our experience of a protocol for deep sedation with ketamine in spontaneous respiration during the PFA of AF. METHODS AND RESULTS: All consecutive patients undergoing PFA for AF at our center were included. Our sedation protocol involves the intravenous administration of fentanyl (1.5 mcg/kg) and midazolam (2 mg) at low doses before local anesthesia with lidocaine. A ketamine adjunct (1 mg/kg) was injected about 5 minutes before the first PFA delivery. We enrolled 66 patients (age = 59 ± 9 years, 78.8% males, body mass index = 28.8 ± 5 kg/m2, fluoroscopy time = 21[15-30] min, skin-to-skin time = 75[60-100] min and PFA LA dwell time = 25[22-28] min). By the end of the procedure, PVI had been achieved in all patients by means of PFA alone. The mean time under sedation was 56.4 ± 6 min, with 50 (76%) patients being sedated for less than 1 hour. A satisfactory Ramsey Sedation Scale level before ketamine infusion was achieved in all patients except one (78.8% of the patients with rank 3; 19.7% with rank 2). In all procedures, the satisfaction level was found to be acceptable by both the patient and the primary operator (Score = 0 in 98.5% of cases). All patients reported none or mild pain. No major procedure or anesthesia-related complications were reported. CONCLUSION: Our standardized sedation protocol with the administration of drugs with rapid onset and pharmacological offset at low doses was safe and effective, with an optimal degree of patient and operator satisfaction. CLINICAL TRIAL REGISTRATION: Advanced TecHnologies For SuccEssful AblatioN of AF in Clinical Practice (ATHENA). URL: http://clinicaltrials.gov/Identifier: NCT05617456.

Pulmonary vein isolation by means of a novel cryoballoon technology in paroxysmal atrial fibrillation patients: 1-year outcome from a large Italian multicenter clinical registry.

INTRODUCTION: Recently, a new cryoballoon (CB) technology (POLARx; Boston Scientific) has come onto the market. Preliminary data have shown that its acute safety and efficacy are similar to those of the first-generation CB. The aim of this study was to assess the medium-term outcome of pulmonary vein isolation (PVI) with the POLARxTM CB in a large multicenter registry. METHODS: We prospectively collected data on 125 consecutive patients with paroxysmal atrial fibrillation (AF) who underwent PVI by means of a novel CB system. Two cases of transient phrenic nerve palsy occurred, with full recovery in the 48h post procedure; no major procedure-related adverse events were reported. During the 90-day blanking period, 4 (3.2%) patients experienced an early recurrence. After the blanking period, over a mean follow-up of 411 ± 62 days, 19 patients (15.2%) suffered an AF/atrial tachycardia (AT) recurrence. The 1-year freedom from AF/AT recurrence was 86.4% (n = 17): 10 (8%) patients had an AF recurrence, 6 (4.8%) had an AT occurrence and 1 (0.8%) suffered both events. Patients with AF/AT recurrences had both a shorter deflation time and total deflation time. Moreover, CB ablations with measured TTI < 90 s and TTI < 60 s were more frequent in patients without AF/AT recurrence (88.5% and 77.4%, respectively) than in those who experienced at least one AF/AT recurrence (67.5% and 55.0%, p = .001 and p = .005, respectively). CONCLUSION: The novel POLARx cryo-balloon system is safe and effective for PV isolation, displaying a 1-year freedom from atrial arrhythmia recurrence of 86.4%, which is in line to that reported with AFA-Pro CB or RF ablation. CLINICAL TRIAL REGISTRATION: Catheter Ablation of Arrhythmias with a High-Density Mapping System in Real-World Practice (CHARISMA). URL: http://clinicaltrials.gov/ Identifier: NCT03793998. Registration date: January 4, 2019.

Genetic Determinants of Hydroxycinnamic Acid Metabolism in Heterofermentative Lactobacilli.

Phenolic acids are among the most abundant phenolic compounds in edible parts of plants. Lactic acid bacteria (LAB) metabolize phenolic acids, but the enzyme responsible for reducing hydroxycinnamic acids to phenylpropionic acids (HcrB) was only recently characterized in Lactobacillus plantarum In this study, heterofermentative LAB species were screened for their hydroxycinnamic acid metabolism. Data on strain-specific metabolism in combination with comparative genomic analyses identified homologs of HcrB as putative phenolic acid reductases. Par1 and HcrF both encode putative multidomain proteins with 25% and 63% amino acid identity to HcrB, respectively. Of these genes, par1 in L. rossiae and hcrF in L. fermentum were overexpressed in response to hydroxycinnamic acids. The deletion of par1 in L. rossiae led to the loss of phenolic acid metabolism. The strain-specific metabolism of phenolic acids was congruent with the genotype of lactobacilli; however, phenolic acid reductases were not identified in strains of Weissella cibaria that reduced hydroxycinnamic acids to phenylpropionic acids. Phylogenetic analysis of major genes involved in hydroxycinnamic acid metabolism in strains of the genus Lactobacillus revealed that Par1 was found to be the most widely distributed phenolic acid reductase, while HcrB was the least abundant, present in less than 9% of Lactobacillus spp. In conclusion, this study increased the knowledge on the genetic determinants of hydroxycinnamic acid metabolism, explaining the species- and strain-specific metabolic variations in lactobacilli and providing evidence of additional enzymes involved in hydroxycinnamic acid metabolism of lactobacilli.IMPORTANCE The metabolism of secondary plant metabolites, including phenolic compounds, by food-fermenting lactobacilli is a significant contributor to the safety, quality, and nutritional quality of fermented foods. The enzymes mediating hydrolysis, reduction, and decarboxylation of phenolic acid esters and phenolic acids in lactobacilli, however, are not fully characterized. The genomic analyses presented here provide evidence for three novel putative phenolic acid reductases. Matching comparative genomic analyses with phenotypic analysis and quantification of gene expression indicates that two of the three putative phenolic acid reductases, Par1 and HcrF, are involved in reduction of hydroxycinnamic acids to phenylpropionic acids; however, the activity of Par2 may be unrelated to phenolic acids and recognizes other secondary plant metabolites. These findings expand our knowledge on the metabolic potential of lactobacilli and facilitate future studies on activity and substrate specificity of enzymes involved in metabolism of phenolic compounds.

How fructophilic lactic acid bacteria may reduce the FODMAPs content in wheat-derived baked goods: a proof of concept.

BACKGROUND: FODMAPs (Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols) intake is associated with the onset of irritable bowel syndrome symptoms. FODMAPs in wheat-derived baked goods may be reduced via bioprocessing by endogenous enzymes and/or microbial fermentation. Because of the inherent enzyme activities, bread made by baker's yeast and sourdough may result in decreased levels of FODMAPs, whose values are, however, not enough low for people sensitive to FODMAPs. RESULTS: Our study investigated the complementary capability of targeted commercial enzymes and metabolically strictly fructophilic lactic acid bacteria (FLAB) to hydrolyze fructans and deplete fructose during wheat dough fermentation. FLAB strains displayed higher fructose consumption rate compared to conventional sourdough lactic acid bacteria. Fructose metabolism by FLAB was faster than glucose. The catabolism of mannitol with the goal of its reuse by FLAB was also investigated. Under sourdough conditions, higher fructans breakdown occurred in FLAB inoculated doughs compared to conventional sourdough bacteria. Preliminary trials allowed selecting Apilactobacillus kunkeei B23I and Fructobacillus fructosus MBIII5 as starter candidates, which were successfully applied in synergy with commercial invertase for low FODMAPs baking. CONCLUSIONS: Results of this study clearly demonstrated the potential of selected strictly FLAB to strongly reduce FODMAPs in wheat dough, especially under liquid-dough and high oxygenation conditions.

High-Value Compounds in Fruit, Vegetable and Cereal Byproducts: An Overview of Potential Sustainable Reuse and Exploitation.

Food waste (FW) represents a global and ever-growing issue that is attracting more attention due to its environmental, ethical, social and economic implications. Although a valuable quantity of bioactive components is still present in the residuals, nowadays most FW is destined for animal feeding, landfill disposal, composting and incineration. Aiming to valorize and recycle food byproducts, the development of novel and sustainable strategies to reduce the annual food loss appears an urgent need. In particular, plant byproducts are a plentiful source of high-value compounds that may be exploited as natural antioxidants, preservatives and supplements in the food industry, pharmaceuticals and cosmetics. In this review, a comprehensive overview of the main bioactive compounds in fruit, vegetable and cereal byproducts is provided. Additionally, the natural and suitable application of tailored enzymatic treatments and fermentation to recover high-value compounds from plant byproducts is discussed. Based on these promising strategies, a future expansion of green biotechnologies to revalorize the high quantity of byproducts is highly encouraging to reduce the food waste/losses and promote benefits on human health.

Fructose-rich niches traced the evolution of lactic acid bacteria toward fructophilic species.

Fructophilic lactic acid bacteria (FLAB) are found in fructose-rich habitats associated with flowers, fruits, fermented foods, and the gastrointestinal tract of several insects having a fructose-based diet. FLAB are heterofermentative lactobacilli that prefer fructose instead of glucose as carbon source, although additional electron acceptor substrates (e.g. oxygen) remarkably enhance their growth on glucose. As a newly discovered bacterial group, FLAB are gaining increasing interest. In this review, the ecological context in which these bacteria exist and evolve was resumed. The wide frequency of isolation of FLAB from fructose feeding insects has been deepened to reveal their ecological significance. Genomic, metabolic data, reductive evolution, and niche specialization of the main FLAB species have been discussed. Findings to date acquired are consistent with a metabolic model in which FLAB display a reliance on environmental niches and the degree of host specificity. In light of FLAB proximity to lactic acid bacteria generally considered to be safe, and due to their peculiar metabolic traits, FLAB may be successfully exploited in food and pharmaceutical applications.

Novel solid-state fermentation of bee-collected pollen emulating the natural fermentation process of bee bread.

Structure of lactic acid bacteria biota in ivy flowers, fresh bee-collected pollen (BCP), hive-stored bee bread, and honeybee gastrointestinal tract was investigated. Although a large microbial diversity characterized flowers and fresh BCP, most of lactic acid bacteria species disappeared throughout the bee bread maturation, giving way to Lactobacillus kunkeei and Fructobacillus fructosus to dominate long stored bee bread and honeybee crop. Adaptation of lactic acid bacteria was mainly related to species-specific, and, more in deep, to strain-specific features. Bee bread preservation seemed related to bacteria metabolites, produced especially by some L. kunkeei strains, which likely gave to lactic acid bacteria the capacity to outcompete other microbial groups. A protocol to ferment BCP was successfully set up, which included the mixed inoculum of selected L. kunkeei strains and Hanseniaspora uvarum AN8Y27B, almost emulating the spontaneous fermentation of bee bread. The strict relationship between lactic acid bacteria and yeasts during bee bread maturation was highlighted. The use of the selected starters increased the digestibility and bioavailability of nutrients and bioactive compounds naturally occurring in BCP. Our biotechnological protocol ensured a product microbiologically stable and safe. Conversely, raw BCP was more exposed to the uncontrolled growth of yeasts, moulds, and other bacterial groups.

How Lactobacillus plantarum shapes its transcriptome in response to contrasting habitats.

Triplets of Lactobacullus plantarum strains were isolated from nine contrasting habitats. Without any passage through other culture media, isolation and cultivation were on model media that strictly reproduced the chemical and physical conditions and stressors of the habitats of origin. Here, we demonstrated how L. plantarum regulates and shapes its transcriptome in response to contrasting habitats. Firstly, multivariate clustering analysis of transcriptional data (RNA-Seq), complemented with metabolomics and phenomics, grouped the strains according to the habitats of origin. Subsequently, selected strains from each habitat switched to repeated cultivation on MRS medium and transcriptomes homogenized into a unique cluster. Adaptation to this common medium mainly relied on activation of genes for phage- and prophage-related proteins and transposases. Finally, the comparison of growth across model media and with respect to MRS medium showed that 44% of the overall 3112 gene transcripts changed depending on the specific habitat. Regulation and shaping of transcriptomes mainly concerned carbohydrate acquisition, pyruvate catabolism, proteolytic system and amino acid, lipid and inorganic ion transport and metabolism, with contrasting responses for contrasting habitats. Pathways reconstruction demonstrated how the large genome size of L. plantarum imparts transcriptome and metabolic flexibility as the basic mechanism for a nomadic lifestyle.

Long-term outcome of pulmonary vein isolation in patients with paroxysmal atrial fibrillation and Brugada syndrome.

Aims: The therapeutical management of atrial fibrillation (AF) in the setting of Brugada syndrome (BS) might be challenging as many antiarrhythmic drugs (AADs) with sodium channel blocking properties might lead to to the development of ventricular arrhythmias. This study sought to evaluate the clinical outcome in a consecutive series of patients with BS having undergone pulmonary vein (PV) isolation by means of radiofrequency (RF) or cryoballoon (CB) ablation and the efficacy of catheter ablation for preventing inappropriate interventions delivered by implantable cardioverter defibrillators (ICD) on a 3-year follow up. Methods and results: Twenty-three consecutive patients with BS (13 males; mean age was 47 ± 18 years) having undergone PV isolation for drug-resistant paroxysmal AF were enrolled. Eleven patients (48%) had an ICD implanted of whom four had inappropriate shocks secondary to rapid AF. Over a mean follow-up period of 35.0 ± 25.4 months (median 36 months) the freedom from AF recurrence after the index PV isolation procedure was 74% without AADs. Patients with inappropriate ICD interventions for AF did not present futher ICD shocks after AF ablation. No major complications occurred. Conclusion: Catheter ablation is a valid therapeutic choice for patients with BS and paroxysmal AF considering the high success rates, the limitations of the AADs and the safety of the procedure, and it should be taken into consideration especially in those patients presenting inappropriate ICD shocks due to rapid AF.

Impact of the third generation cryoballoon on atrial fibrillation ablation: An useful tool?

BACKGROUND: Third-generation cryoballoon (CB3) is characterized by a 40% shorter distal tip designed to increase the rate of pulmonary veins real-time signal recording in order to measure time necessary to isolate veins, the "Time to effect" (TTE). Few data are currently available on clinical follow up of CB3 treated patients. METHODS: Sixtyeight consecutive patients (mean age 57.8 ±â€¯9.6 years, 48 male) with paroxysmal or persistent atrial fibrillation (AF) were enrolled. Thirthyfour (25 paroxysmal AF) underwent to a 28 mmCB3 pulmonary veins isolation and were compared to 34 treated (21 paroxysmal AF) with 28 mmCB2. RESULTS: CB3 use was correlated to significant increase of the possibility to measure TTE in every treated veins (left superior 82,35% vs 23,53%, left inferior 70,59% vs 38,24%, right superior 58,82% vs 14,71%, right inferior 52,94% vs 17,65%). When it is measured, TTE wasn't different between two groups. Higher nadir temperature was observed in CB3 patients (-39.4 ±â€¯5.2 °C vs -43.0 ±â€¯7.2 °C, p = 0.03). CB3 procedures were shorter (91.4 ±â€¯21.7 vs 110.9 ±â€¯31.8 min, p = 0.018), with a significant reduction in cryoenergy delivery time (24.2 ±â€¯8.5 vs 20.3 ±â€¯6.7 min, p < 0.05), and a significant reduction in left atrium dwell time (59.3 ±â€¯9.8 vs 69.3 ±â€¯10.8 min, p = 0.02, p < 0.05). At one year follow up period the Kaplan-Meier curve didn't show any significant difference in AF-free survival (Log p = 0,49). CONCLUSIONS: Novel CB3 is a useful tool in order to simplify AF cryoballoon ablation when compared to second generation cryoballoon, as observed in our experience. Follow up data seem confirm a clinical CB3 efficacy at least comparable CB2.

Metabolism of Fructophilic Lactic Acid Bacteria Isolated from the Apis mellifera L. Bee Gut: Phenolic Acids as External Electron Acceptors.

Fructophilic lactic acid bacteria (FLAB) are strongly associated with the gastrointestinal tracts (GITs) of Apis mellifera L. worker bees due to the consumption of fructose as a major carbohydrate. Seventy-seven presumptive lactic acid bacteria (LAB) were isolated from GITs of healthy A. mellifera L. adults, which were collected from 5 different geographical locations of the Apulia region of Italy. Almost all of the isolates showed fructophilic tendencies: these isolates were identified as Lactobacillus kunkeei (69%) or Fructobacillus fructosus (31%). A high-throughput phenotypic microarray targeting 190 carbon sources was used to determine that 83 compounds were differentially consumed. Phenotyping grouped the strains into two clusters, reflecting growth performance. The utilization of phenolic acids, such as p-coumaric, caffeic, syringic, or gallic acids, as electron acceptors was investigated in fructose-based medium. Almost all FLAB strains showed tolerance to high phenolic acid concentrations. p-Coumaric acid and caffeic acid were consumed by all FLAB strains through reductases or decarboxylases. Syringic and gallic acids were partially metabolized. The data collected suggest that FLAB require external electron acceptors to regenerate NADH. The use of phenolic acids as external electron acceptors by the 4 FLAB showing the highest phenolic acid reductase activity was investigated in glucose-based medium supplemented with p-coumaric acid. Metabolic responses observed through a phenotypic microarray suggested that FLAB may use p-coumaric acid as an external electron acceptor, enhancing glucose dissimilation but less efficiently than other external acceptors such as fructose or pyruvic acid.IMPORTANCE Fructophilic lactic acid bacteria (FLAB) remain to be fully explored. This study intends to link unique biochemical features of FLAB with their habitat. The quite unique FLAB phenome within the group lactic acid bacteria (LAB) may have practical relevance in food fermentations. The FLAB phenome may have implications for the levels of hexose metabolism products in fermented foods, as well as food probiotication. Due to the harsh conditions of honeybees' GITs, these bacteria had to develop specific physiological and biochemical characteristics, such as tolerance to phenolic acids. The screening of FLAB strains based on metabolic pathways involving phenolic acids may allow the selection of starter cultures with both technological and functional beneficial attributes. Bioconversion of phenolic compounds may contribute to the aroma attributes and biofunctionality of fermented foods. Thus, the selection of FLAB strains as starter cultures with specific enzymatic activities involving phenolic acids may have a promising role in food fermentations.

Exploitation of Leuconostoc mesenteroides strains to improve shelf life, rheological, sensory and functional features of prickly pear (Opuntia ficus-indica L.) fruit puree.

Strains of Leuconostoc mesenteroides were identified from raw prickly pear (Opuntia ficus-indica L.). Five autochthonous strains were selected based on the kinetics of growth and acidification on prickly pear fruit juice, and the capacity to synthesize exo-polysaccharides. All selected Leuc. mesenteroides strains showed an in vitro mucilage-degrading capability. A protocol for processing and storage of fermented prickly pear fruit puree (FP) was set up. Unstarted FP and chemically acidified FP were used as the controls. Starters grew and remained viable at elevated cell numbers during 21 days of storage at 4 °C. Contaminating Enterobacteriaceae and yeasts were found only in the controls. Viscosity and serum separation distinguished started FP compared to the controls. Colour parameters, browning index, sensory attributes, antimicrobial activity, vitamin C and betalains levels were positively affected by lactic acid fermentation. Increase of free radical scavenging activity in ethyl acetate soluble extract suggested an effect of selected strains on phenolic profiles. Started FP markedly inhibited the inflammatory status of Caco-2/TC7 cells, and also contributed to maintaining the integrity of tight junctions. Started FP scavenged the reactive oxygen species generated by H2O2 on Caco-2 cells. All selected strain variously affected the immunomodulatory activity towards anti- and pro-inflammatory cytokines.

Lactic acid fermentation as a tool to enhance the antioxidant properties of Myrtus communis berries.

BACKGROUND: Myrtle (Myrtus communis L.) is a medicinal and aromatic plant belonging to Myrtaceae family, which is largely diffused in the Mediterranean areas and mainly cultivated in Tunisia and Italy. To the best of our knowledge, no studies have already considered the use of the lactic acid fermentation to enhance the functional features of M. communis. This study aimed at using a selected lactic acid bacterium for increasing the antioxidant features of myrtle berries, with the perspective of producing a functional ingredient, dietary supplement or pharmaceutical preparation. The antioxidant activity was preliminarily evaluated through in vitro assays, further confirmed through ex vivo analysis on murine fibroblasts, and the profile of phenol compounds was characterized. RESULTS: Myrtle berries homogenate, containing yeast extract (0.4%, wt/vol), was fermented with Lactobacillus plantarum C2, previously selected from plant matrix. Chemically acidified homogenate, without bacterial inoculum and incubated under the same conditions, was used as the control. Compared to the control, fermented myrtle homogenate exhibited a marked antioxidant activity in vitro. The radical scavenging activity towards DPPH increased by 30%, and the inhibition of linoleic acid peroxidation was twice. The increased antioxidant activity was confirmed using Balb 3 T3 mouse fibroblasts, after inducing oxidative stress, and determining cell viability and radical scavenging activity through MTT and DCFH-DA assays, respectively. The lactic acid fermentation allowed increased concentrations of total phenols, flavonoids and anthocyanins, which were 5-10 times higher than those found for the non-fermented and chemically acidified control. As shown by HPLC analysis, the main increases were found for gallic and ellagic acids, and flavonols (myricetin and quercetin). The release of these antioxidant compounds would be strictly related to the esterase activities of L. plantarum. CONCLUSIONS: The lactic acid fermentation of myrtle berries is a suitable tool for novel applications as functional food dietary supplements or pharmaceutical preparations.

Metabolism of phenolic compounds by Lactobacillus spp. during fermentation of cherry juice and broccoli puree.

This study aimed to investigate the metabolism of phenolic acids and flavonoids during lactic acid fermentation of cherry juice and broccoli puree for potential food and pharmaceutical purposes. When fermenting cherry juice and broccoli puree, Lactobacillus spp. exhibited strain-specific metabolism of phenolic acid derivatives. The metabolism of protocatechuic, caffeic and p-coumaric acids through phenolic acid decarboxylases and reductases differed between mMRS and cherry juice and broccoli puree. The synthesis of reduced compounds was the highest during food fermentations and the substrate seemed to modulate the metabolism of phenolic compounds. The reduction of phenolic acids involves a hydrogen donor and the re-oxidation of the reduced co-factor NADH, which may provide a metabolic advantage through NAD(+) regeneration. Quinic acid reduction may replace fructose and pyruvate as hydrogen acceptors, and it may provide an energetic advantage to heterofermentative bacteria when growing in broccoli puree lacking of fructose. This study demonstrated that phenolics metabolism may confer a selective advantage for lactobacilli in vegetable and fruit fermentation, and the metabolic routes are strongly dependent on the intrinsic factors of substrate. Fermented cherry juice and broccoli puree, due to the selected bacterial bioconversion pathways, are enriched in phenolic derivative with high human bioavailability and biological activity.

Hydroxycinnamic acids used as external acceptors of electrons: an energetic advantage for strictly heterofermentative lactic acid bacteria.

The metabolism of hydroxycinnamic acids by strictly heterofermentative lactic acid bacteria (19 strains) was investigated as a potential alternative energy route. Lactobacillus curvatus PE5 was the most tolerant to hydroxycinnamic acids, followed by strains of Weissella spp., Lactobacillus brevis, Lactobacillus fermentum, and Leuconostoc mesenteroides, for which the MIC values were the same. The highest sensitivity was found for Lactobacillus rossiae strains. During growth in MRS broth, lactic acid bacteria reduced caffeic, p-coumaric, and ferulic acids into dihydrocaffeic, phloretic, and dihydroferulic acids, respectively, or decarboxylated hydroxycinnamic acids into the corresponding vinyl derivatives and then reduced the latter compounds to ethyl compounds. Reductase activities mainly emerged, and the activities of selected strains were further investigated in chemically defined basal medium (CDM) under anaerobic conditions. The end products of carbon metabolism were quantified, as were the levels of intracellular ATP and the NAD(+)/NADH ratio. Electron and carbon balances and theoretical ATP/glucose yields were also estimated. When CDM was supplemented with hydroxycinnamic acids, the synthesis of ethanol decreased and the concentration of acetic acid increased. The levels of these metabolites reflected on the alcohol dehydrogenase and acetate kinase activities. Overall, some biochemical traits distinguished the common metabolism of strictly heterofermentative strains: main reductase activity toward hydroxycinnamic acids, a shift from alcohol dehydrogenase to acetate kinase activities, an increase in the NAD(+)/NADH ratio, and the accumulation of supplementary intracellular ATP. Taken together, the above-described metabolic responses suggest that strictly heterofermentative lactic acid bacteria mainly use hydroxycinnamic acids as external acceptors of electrons.