Fermentation of Orange Peels by Lactic Acid Bacteria: Impact on Phenolic Composition and Antioxidant Activity.

Orange processing generates peel by-products rich in phenolic compounds, particularly flavanones like hesperidin and narirutin, offering potential health benefits. Utilizing these by-products is of significant interest in supporting Spain's circular bioeconomy. Therefore, the aim of this study was to investigate the fermentation of orange peels by different lactic acid bacteria (LAB) strains and its impact on phenolic composition and antioxidant activity. Three different LAB strains, two Lactiplantibacillus plantarum, and one Levilactobacillus brevis were utilized. The phenolic compounds were measured by HPLC-ESI-TOF-MS, and antioxidant activity was assessed using DPPH and ABTS methods. The growth of the LAB strains varied, showing initial increases followed by gradual declines, with strain-specific patterns observed. Medium acidification occurred during fermentation. A phenolic analysis revealed an 11% increase in phenolic acids in peels fermented by La. plantarum CECT 9567-C4 after 24 h, attributed to glycosylation by LAB enzymes. The flavonoid content exhibited diverse trends, with Le. brevis showing an 8% increase. The antioxidant assays demonstrated strain- and time-dependent variations. Positive correlations were found between antioxidant activity and total phenolic compounds. The results underscore the importance of bacterial selection and fermentation time for tailored phenolic composition and antioxidant activity in orange peel extracts. LAB fermentation, particularly with La. plantarum CECT 9567 and Le. brevis, holds promise for enhancing the recovery of phenolic compounds and augmenting antioxidant activity in orange peels, suggesting potential applications in food and beverage processing.

Detection of intrathecal IgG antibody for varicella and measles diagnosis by evaluation and comparison of a commercial IgG chemiluminescent immunoassay with two ELISAs.

OBJECTIVES: Analyse alternative methods of intrathecal antibody detection by comparing chemiluminescent immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA) techniques to determine if CLIA can replace ELISA in the diagnosis of CNS infections. METHODS: A panel of 280 paired samples-cerebrospinal fluid (CSF) and serum-with known antibody reactivities (Varicella, n = 60; Measles, n = 120) and negative samples (n = 100) were used to evaluate the performance of six serological test kits (Enzygnost, VirClia®, and Serion ELISA (Measles and Variella). RESULTS: For Measles virus IgG, the VirClia® IgG monotest revealed 97% and 94% positive and negative agreement to the Enzygnost as reference test, respectively. In contrast, Serion ELISA kits yielded values of 18% and 90%. For the Varicella Zoster virus (VZV) IgG, the VirClia® IgG monotest showed 97% and 90% positive and negative agreement compared to Enzygnost. The Serion ELISA kits showed values of 55% and 86%, respectively. ROC analysis revealed that the areas under the curve for Measles and VZV IgGs were 0.7 and 0.852, respectively, using the Serion kit, and 0.963 and 0.955, for Vircell S.L CLIA technique. VirClia® monotest values were calculated using an antibody index cut-off of 1.3. CONCLUSION: The findings indicate that CLIA testing can improve antibody detection in CSF samples, aiding the diagnosis of infectious neurological impairments.

Impact of Lactic Acid Bacteria Fermentation on Phenolic Compounds and Antioxidant Activity of Avocado Leaf Extracts.

The growing global consumption of avocados, associated with contents including bioactive compounds with numerous health-promoting properties, is producing a large amount of agro wastes around the world. Different management approaches are available for the recovery of bioactive compounds from wastes as potential ingredients for use in the production of functional foods and nutraceuticals. Lactic acid fermentation can be used to exploit nutritional potential and add value to agro wastes. In this study, fermentations with lactic acid bacteria were carried out in avocado leaves, and the total phenolic content and the antioxidant activity were determined by DPPH and FRAP assays from hydroalcoholic extracts obtained from fermented avocado leaves. Fifteen new phenolic compounds were identified for the first time in avocado leaves by HPLC-ESI-TOF-MS. L. plantarum CECT 748T and P. pentosaceus CECT 4695T showed the highest antioxidant activity. The sum of phenolic compounds was increased by 71, 62, 55 and 21% in fermentations with P. pentosaceus CECT 4695T, L. brevis CECT 5354, P. acidilactici CECT 5765T and L. plantarum CECT 9567, respectively, while it was reduced in the fermentation with L. plantarum 748T by 21% as demonstrated by HPLC-ESI-TOF-MS. Biotransformations induced by bacterial metabolism modified the phenolic compound profile of avocado leaves in a strain-specific-dependent manner. P. pentosaceus CECT 4695T significantly increased kaempferol, P. pentosaceus 4695T, L. brevis 5354 and L. plantarum 9567 increased rutin, and dihydro-p-coumaric acid was increased by the five selected lactic acid bacteria. Total flavonoids were highly increased after fermentations with the five selected lactic acid bacteria but flavonoid glucosides were decreased by L. plantarum 748T, which was related to its higher antioxidant activity. Our results suggest that lactic acid bacteria led the hydrolysis of compounds by enzymatic activity such as glycosidases or decarboxylase and the release of phenolics bound to the plant cell wall, thus improving their bioavailability.

Comparative Extraction of Phenolic Compounds from Olive Leaves Using a Sonotrode and an Ultrasonic Bath and the Evaluation of Both Antioxidant and Antimicrobial Activity.

A sonotrode ultrasound-assisted extraction of phenolic compounds from olive leaves has been developed using a Box-Behnken design to optimize the effects of solvent composition and ultrasound parameters. The determination of single phenolic compounds was performed by HPLC-MS and the highest recovery in total compounds, oleuropein and hydroxytyrosol was achieved using EtOH/H2O (55:45, v/v), 8 min and 100% of amplitude. The optimal conditions were applied on leaves from seven olive cultivars grown under the same conditions and the results were compared with those found by using a conventional ultrasonic bath, obtaining no statistical differences. Moreover, antioxidant activity by FRAP, DPPH and ABTS in these olive leaf extracts was evaluated and they exhibited a significant correlation with oleuropein and total phenolic content. All cultivars of olive leaf extracts were found to be active against S. aureus and methicillin-resistant S. aureus with minimum bactericidal concentration (MBC) values) that ranged from 5.5 to 22.5 mg mL-1. No extracts showed antimicrobial activity against C. albicans. The percentages of mycelium reduction in B. cinerea ranged from 2.2 and 18.1%. Therefore, sonotrode could be considered as an efficient and fast extraction technique that could be easily scaled-up at industrial level, thus allowing for olive leaves to be revalorized.

Essential Oils from Fruit and Vegetables, Aromatic Herbs, and Spices: Composition, Antioxidant, and Antimicrobial Activities.

In the field of food preservation, encapsulated Essential Oils (EOs) could be the best non-toxic and eco-friendly tool for food preservative applications substituting the chemicals ones that have several disadvantages for the environment and health. Thirteen commercial EOs from plants, fruits, and vegetables were characterized by GC-MS. The antioxidant activity was measured by DPPH and ABTS techniques. Antimicrobial activity was assessed by agar well-diffusion method and the Minimum Inhibitory Concentration (MIC) by agar dilution method against six bacteria, Candida albicans, and Botrytis cinerea. All the EOs tested have demonstrated antioxidant activity in the range of IC50 0.01-105.32 mg/mL. Between them, cinnamon EOs were the best, followed by oregano and thyme EOs. Fennel EO showed the lowest radical scavenging. MIC values ranged from 0.14 to 9 mg/mL. C. cassia, thyme, and oregano EOs were the most effective against the bacterial species tested, and the yeast C. albicans. On the contrary, citric fruit EOs showed low or no inhibition against most bacterial strains. The percentages of inhibition of mycelia growth of B. cinerea ranged from 3.4 to 98.5%. Thyme, oregano, mint, and fennel EOs showed the highest inhibition.

Draft genome sequence of Lactobacillus plantarum C4 (CECT 9567), a potential probiotic strain isolated from kefir.

Lactobacillus plantarum C4 (CECT 9567) was isolated from kefir and has been extensively studied because of its probiotic properties. Here we report the genome sequence of this strain. The genome consists of 3,221,350 bp, and contains 3058 CDSs with an average G + C content of 44.5%. The genome harbors genes encoding the AraC-family transcription regulator, the penicillin-binding protein Pbp2A, and the Na+/H+ antiporter NapA3, which have important roles in the survival of lactobacilli in the gastrointestinal tract. Also, the genome encodes the catalase KatE, NADH peroxidase and glutathione peroxidase, which enable anaerobic respiration, and a nitrate reductase complex, which enable anaerobic respiration. Additionally, genes encoding plantaricins and sactipeptides, and genes involved in the use of fructooligosaccharides and in the production of butyric acid were also identified. BLASTn analysis revealed that 91.4% of CDSs in C4 genome aligned with those of the reference strain L. plantarum WCFS1, with a mean identity of 98.96%. The genome information of L. plantarum C4 provides the basis for understanding the probiotic properties of C4 and to consider its use as a potential component of functional foods.

Adamdec1, Ednrb and Ptgs1/Cox1, inflammation genes upregulated in the intestinal mucosa of obese rats, are downregulated by three probiotic strains.

We have previously reported that administration of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 to obese Zucker-Lepr fa/fa rats attenuates liver steatosis and exerts anti-inflammatory effects. The goal of the present work was to investigate the modulation of gene expression in intestinal mucosa samples of obese Zucker-Lepr fa/fa rats fed the probiotic strains using a DNA microarray and postgenomic techniques. We also measured secretory IgA content in the gut and lipopolysaccharide (LPS)-binding protein (LBP) in serum. Expression of three genes (Adamdec1, Ednrb and Ptgs1/Cox1) was up-regulated in the intestinal mucosa of the obese rats compared with that in the rats when they were still lean. Probiotic administration down-regulated expression of Adamdec1 and Ednrb at the mRNA and protein levels and that of Ptgs1/Cox1 at the mRNA level, and this effect was in part mediated by a decrease in both macrophage and dendritic cell populations. Probiotic treatment also increased secretory IgA content and diminished the LBP concentration. Based on results reported in this work and else where, we propose a possible mechanism of action for these bacterial strains.

A Lactobacillus plantarum strain isolated from kefir protects against intestinal infection with Yersinia enterocolitica O9 and modulates immunity in mice.

Lactobacillus plantarum C4, previously isolated from kefir and characterized as a potential probiotic strain, was tested for its protective and immunomodulatory capacity in a murine model of yersiniosis. The inoculation of BALB/c mice with a low pathogenicity serotype O9 strain of Yersinia enterocolitica results in a prolonged intestinal infection with colonization of Peyer's patches. Pretreatment with C4 was without effect on fecal excretion of yersiniae, but shortened the colonization of Peyer's patches. This protective effect was associated with pro-inflammatory status in the intestinal mucosa (TNF-α production in infected mice was increased by C4) and an increase in total IgA secretion. At a systemic level, C4 did not promote a pro-inflammatory response, although production of the immunoregulatory cytokine IFN-γ was enhanced. These findings suggest that L. plantarum C4 can increase resistance to intestinal infections through its immunomodulatory activity.

Lack of correlation between in vitro antibiosis and in vivo protection against enteropathogenic bacteria by probiotic lactobacilli.

Increased resistance to infection is one of the beneficial effects attributed to probiotic microorganisms. This effect may be due to several mechanisms: production of inhibitory substances, blocking of adhesion sites on the intestinal surface, competition for nutrients and stimulation of mucosal and systemic immunity. The present study aimed to investigate the correlation between in vitro and in vivo antimicrobial activity of probiotic lactobacilli. The agar spot test was used to show that twenty Lactobacillus strains were able to inhibit the enteropathogenic bacterium Yersinia enterocolitica. This inhibition was mainly attributable to a decrease in pH resulting from dextrose fermentation by lactobacilli. The inhibition of Y. enterocolitica, Salmonella enterica serovar Typhimurium and Listeria monocytogenes by two probiotic strains, Lactobacillus casei C1 and Lactobacillus plantarum C4, was also associated with the pH decrease. However, both strains lacked protective effects in mouse experimental infection models, with the exception of long-lasting pre-treatment with L. plantarum C4, which exerted a partial protective effect against S. Typhimurium that was attributable to an immunostimulatory mechanism. Our results show that in vitro antibiosis tests do not provide useful information on the probiotic potential of Lactobacillus strains.

Clinic-epidemiologic study of human infection by Granada virus, a new phlebovirus within the sandfly fever Naples serocomplex.

Granada virus (GRV), a new phlebovirus within the Naples serocomplex, has been recently described in phlebotomine sandflies from Spain. The presence of anti-GRV immunoglobulin G (IgG) antibodies was investigated by indirect fluorescence assay (IFA) and neutralization test (NT) in 920 serum samples from the Granada population. By IFA, an overall GRV seroprevalence of 15.8% (N = 145) was observed, significantly increasing up to 65 years. NT was positive in 18% of anti-GRV IFA-positive samples. IgG antibodies against Toscana virus (TOSV), a hyperendemic phlebovirus within Granada province, were detected in 40% of anti-GRV-positive cases. Anti-GRV IgM antibodies were detected in 36 (6.6%) of 547 acute-phase serum samples from individuals with febrile illness, exanthema, and/or acute respiratory infection. All positives were anti-TOSV IgM-negative. GRV may infect humans, with most cases being asymptomatic. The codetection of anti-GRV and anti-TOSV IgG antibodies could be attributable to cross-reactivity or exposure to the same transmission vector.

[Evaluation of the LightCycler® SeptiFast test in newborns and infants with clinical suspicion of sepsis]. / Evaluación de la técnica LightCycler® SeptiFast en recién nacidos y lactantes con sospecha de sepsis.

INTRODUCTION: Neonatal sepsis is a significant cause of morbidity and mortality. Early diagnosis and prompt antimicrobial therapy are crucial for a favorable outcome of the newborn child. Blood culture, the current "gold standard" method for diagnosing bloodstream infections, has a low sensitivity in newborns. We evaluated the multiplex real-time PCR LightCycler(®) SeptiFast (LC-SF) for detection of bloodstream infections in newborns, compared with conventional blood culture. METHODS: A total of 42 blood samples were obtained from 35 subjects presenting with a febrile episode and hospitalized in neonatal intensive care unit at Hospital Universitario Virgen de las Nieves. Two samples were collected during each febrile episode in order to carry out LC-SF assay and blood culture, respectively. RESULTS: Sensitivity and specificity of 79% and 87%, respectively, compared with clinical diagnosis, were obtained for LC-SF. Contamination rate of blood cultures was 16.7%, mainly due to coagulase-negative staphylococci (CoNS) and viridans groups of streptococci. Contamination rate of LC-SF by CoNS was 2.4%. Concordance between LC-SF and blood culture was moderate (kappa index: 0.369). LC-SF demonstrated a higher concordance (kappa index: 0.729) with the final clinical diagnosis than blood culture (kappa index: 0.238). CONCLUSION: LC-SF assay could be a useful diagnostic tool, along with a conventional blood culture, in newborn, for confirming or ruling out those cases that blood culture could not determine, shortening the time to result to 7 hours.

Telithromycin inhibits the production of proinflammatory mediators and the activation of NF-kappaB in in vitro-stimulated murine cells.

Telithromycin is a ketolide antibiotic with anti-inflammatory properties. To investigate the mechanisms of these effects, we examined the in vitro immunomodulatory activity of telithromycin on murine splenocytes and the murine macrophage cell line RAW 264.7. Spleen cells from BALB/c-untreated mice and RAW 264.7 macrophages were cultured in the presence of telithromycin. Proliferation and apoptosis (colorimetric assay), and cytokine production (enzyme immunoassay) of spleen cells in response to lipopolysaccharide and concanavalin A (Con A), and nitric oxide (NO) (colorimetric assay) and cytokine production by lipopolysaccharide-stimulated RAW 264.7 cells were determined. Telithromycin moderately enhanced lymphocyte proliferation in response to lipopolysaccharide and Con A, and enhanced apoptosis induced by camptothecin in mitogen-stimulated splenocytes. Con A-induced IFN-gamma production was suppressed and lipopolysaccharide-induced IL-10 production was increased in spleen cell cultures with telithromycin. Lipopolysaccharide-induced secretion of NO and tumor necrosis factor-alpha (TNF-alpha) was suppressed by telithromycin in RAW 264.7 cultures. Lipopolysaccharide-induced activation of NF-kappaB transcription factor and TNF-alpha promoter in RAW 264.7 macrophages transitorily transfected with luciferase reporter constructs was also inhibited by the ketolide. The suppressive effect of telithromycin on NF-kappaB activation was confirmed by Western blot and enzyme immunoassay. These results suggest that telithromycin exerts anti-inflammatory activity mediated by the inhibition of activation of NF-kappaB.

Effects of telithromycin in in vitro and in vivo models of lipopolysaccharide-induced airway inflammation.

BACKGROUND: The ketolide antibiotic telithromycin (TEL) exerts immunomodulatory and antiinflammatory effects in vitro and in a mouse model of septic shock. We studied the antiinflammatory activity of TEL in in vitro and in vivo models of airway inflammation induced by lipopolysaccharide (LPS). METHODS: We measured the effects of TEL on the response of RAW 264.7 macrophages to LPS and of murine lung epithelial (MLE)-12 cells to supernatants of LPS-stimulated RAW 264.7 macrophages. Macrophage inflammatory protein (MIP)-2 and tumor necrosis factor (TNF)-alpha production, nuclear factor (NF)-kappaB activation, and apoptosis were determined. Acute airway inflammation was induced in untreated and TEL-treated BALB/c mice by nebulization with LPS. Total number of leukocytes, macrophages, and neutrophils, the protein concentration, and nitrite and cytokine levels were determined in the BAL fluid. RESULTS: TEL inhibited in a dose-dependent manner the production of MIP-2 and TNF-alpha by LPS-stimulated RAW 264.7 macrophages, and the production of MIP-2 by MLE-12 epithelial cells to supernatants of LPS-stimulated RAW 264.7 macrophages. NF-kappaB activation was inhibited and apoptosis was increased in both cell lines by TEL. The LPS-induced influx of neutrophils in BAL fluid was decreased by TEL pretreatment. TEL also reduced protein, nitrite, MIP-2, and TNF-alpha levels in the BAL fluid of LPS-nebulized animals. CONCLUSIONS: We have provided evidence that TEL exerts potent antiinflammatory effects in LPS-induced airways injury. We propose that TEL acts in the early phase of inflammation by reducing the release of inflammatory mediators through NF-kappaB inhibition, and in the later phase through enhancement of inflammatory cell apoptosis.

Administration of Lactobacillus casei and Lactobacillus plantarum affects the diversity of murine intestinal lactobacilli, but not the overall bacterial community structure.

Lactobacilli are normal inhabitants of the gastrointestinal tract (GIT) of many mammalian hosts. Their administration as probiotics in functional foods is currently a frequent practice, mainly because of their benefits to host health. It is therefore of interest to study the impact of administration of exogenous strains of Lactobacillus normally used as probiotics upon endogenous microbial populations. For this purpose, fecal and intestinal tissue samples were analyzed in a mouse model fed with a mixture of Lactobacillus plantarum and Lactobacillus casei isolated from commercially available dairy products. The murine intestinal microbiota was studied by means of cultivation-independent 16S rRNA gene-targeted techniques, namely denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis of clone libraries. Multivariate statistical analysis was used to integrate datasets obtained from the different techniques applied. Whereas no differences were detected in the composition of the overall fecal bacterial community, changes were observed for intestinal tissue samples. Moreover, an increase in the diversity of gut lactobacilli was observed in fecal as well as intestinal tissue samples when mice received the mixture of L. casei and L. plantarum.

A strain of Lactobacillus plantarum affects segmented filamentous bacteria in the intestine of immunosuppressed mice.

Segmented filamentous bacteria (SFB) are present in the gastrointestinal tract of mice from weaning until the maturation of the immune system. Probiotic bacteria also have an effect on host immunity. To study the relationships established between these bacteria, samples from a mouse model fed with Lactobacillus plantarum under different immunological conditions were analysed. SFB populations were measured by a newly designed group-specific quantitative PCR assay. The results confirmed the presence of the probiotic in the intestine and an expansion of SFB in the ileum of immunocompromised mice, which was abolished upon administration of L. plantarum, an effect not described to date.

[Enterovirus 75, a new pathogenic virus in Granada province (Spain)]. / Enterovirus 75, un nuevo virus patógeno en nuestro medio.

INTRODUCTION: Members of the genus Enterovirus are usually investigated for their etiological role in neurological syndromes. However, they are often associated with other syndromes such as febrile illness, acute respiratory infection and exanthema. In this study, clinical and epidemiological data from five subjects with infection by the recently described enterovirus 75 were analyzed in the province of Granada (Spain). METHODS: Diagnosis at the genus level was carried out by viral culture in MRC-5 and rhabdomyosarcoma cell lines. Isolate serotypes were determined by RT-PCR of a fragment of the VP1 region and subsequent sequencing of the PCR products. RESULTS: Among the five enterovirus 75 isolated, two were detected in children with aseptic meningitis (1 month and 12 years old) and three in subjects with non-neurological syndromes, i.e. acute respiratory infection, febrile illness and gastroenteritis (all were aged less than one year). The five cases were detected between December 2005 and May 2006. All patients recovered without sequelae. CONCLUSION: These data demonstrate that enterovirus 75 circulates in the south of Spain and indicate that this enterovirus serotype may be implicated in less severe non-neurological syndromes, particularly in younger children, and mainly during the cold months of the year.

A selective differential medium for Lactobacillus plantarum.

The quantification of exogenous lactobacilli in faecal samples is frequently required for the evaluation of the intestinal colonization by probiotic bacteria. In this study, a selective and differential medium, designated LPSM, was developed for the culture of exogenous Lactobacillus plantarum. In quantitative assays, LPSM showed a sensitivity similar to those of enriched and Lactobacillus-adapted media. The presence of ciprofloxacin made LPSM inhibitory to most intestinal bacteria, including endogenous acid lactic bacteria, whereas exogenous L. plantarum strains grew producing a yellow color caused by acid production from sorbitol in the presence of bromocresol purple. The results showed that LPSM is suitable for detection and enumeration of L. plantarum in faecal samples.

Immunomodulation by non-absorbable antibiotics given by the intragastric route.

To test the role of bacterial fractions released from intestinal flora during immunomodulation by antimicrobial agents, BALB/c mice were treated with the non-absorbable antibiotics polymyxin B or teicoplanin by the intragastric route. The composition of faecal microbiota and the capacity of spleen cells to proliferate in response to B-cell and T-cell mitogens were assessed at several times during the treatment. Both antibiotics lowered the count of some bacteria of the intestinal flora and induced significant modifications in spleen cell ability to proliferate in response to mitogens. Thus, the active fractions released from intestinal bacteria during antibiotic treatments may be able to induce immunomodulating effects.

Influenza-like illness criteria were poorly related to laboratory-confirmed influenza in a sentinel surveillance study.

OBJECTIVE: To analyze the most related clinical data for influenza and the utility of influenza-like illness criteria as the clinical threshold for sampling in an influenza sentinel surveillance over a 3-year period. METHODS: Sentinel physicians collected throat specimens and data from outpatients with acute respiratory infection (< or = 72 hours duration). Laboratory-confirmed influenza infection was compared with independent symptoms and the influenza-like illness criteria, as defined by the Classification Committee of the World Organization of Family Doctors. RESULTS: From 1934 patients, 359 (18.56%) yielded positive results for influenza viruses. Only 199 (55.4%) of laboratory-confirmed cases fulfilled clinical criteria of influenza-like illness: positive and negative predictive value (PPV and NPV) of 0.36 and 0.88, respectively. Fever, cough, and rhinorrhea individually correlated with influenza infections (PPV: 0.30, 0.20, and 0.20, respectively; NPV: 0.92, 0.87, and 0.85, respectively). Multivariate analysis demonstrated that the correlation of influenza infection with the presence of fever and cough was similar to the correlation between influenza infection and influenza-like illness criteria (odds ratio 2.24 vs. 2.71, respectively). CONCLUSION: Influenza-like illness criteria are poorly related to laboratory-confirmed influenza. For early detection of influenza viruses in surveillance systems, a less restrictive clinical criterion (specifically, acute respiratory infection) perhaps should be followed.