Current challenges of microalgae applications: exploiting the potential of non-conventional microalgae species.

The intensified attention to health, the growth of an elderly population, the changing lifestyles, and the medical discoveries have increased demand for natural and nutrient-rich foods, shaping the popularity of microalgae products. Microalgae thanks to their metabolic versatility represent a promising solution for a 'green' economy, exploiting non-arable land, non-potable water, capturing carbon dioxide (CO2) and solar energy. The interest in microalgae is justified by their high content of bioactive molecules, such as amino acids, peptides, proteins, carbohydrates, polysaccharides, polyunsaturated fatty acids (as ω-3 fatty acids), pigments (as ß-carotene, astaxanthin, fucoxanthin, phycocyanin, zeaxanthin and lutein), or mineral elements. Such molecules are of interest for human and animal nutrition, cosmetic and biofuel production, for which microalgae are potential renewable sources. Microalgae, also, represent effective biological systems for treating a variety of wastewaters and can be used as a CO2 mitigation approach, helping to combat greenhouse gases and global warming emergencies. Recently a growing interest has focused on extremophilic microalgae species, which are easier to cultivate axenically and represent good candidates for open pond cultivation. In some cases, the cultivation and/or harvesting systems are still immature, but novel techniques appear as promising solutions to overcome such barriers. This review provides an overview on the actual microalgae cultivation systems and the current state of their biotechnological applications to obtain high value compounds or ingredients. Moreover, potential and future research opportunities for environment, human and animal benefits are pointed out. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Olive Mill Wastewater Fermented with Microbial Pools as a New Potential Functional Beverage.

Olive mill wastewater (OMWW) represents a by-product but also a source of biologically active compounds, and their recycling is a relevant strategy to recover income and to reduce environmental impact. The objective of the present study was to obtain a new functional beverage with a health-promoting effect starting from OMWW. Fresh OMWW were pre-treated through filtration and/or microfiltration and subjected to fermentation using strains belonging to Lactiplantibacillus plantarum, Candida boidinii and Wickerhamomyces anomalus. During fermentation, phenolic content and hydroxytyrosol were monitored. Moreover, the biological assay of microfiltered fermented OMWW was detected versus tumor cell lines and as anti-inflammatory activity. The results showed that in microfiltered OMWW, fermentation was successfully conducted, with the lowest pH values reached after 21 days. In addition, in all fermented samples, an increase in phenol and organic acid contents was detected. Particularly, in samples fermented with L. plantarum and C. boidinii in single and combined cultures, the concentration of hydroxytyrosol reached values of 925.6, 902.5 and 903.5 mg/L, respectively. Moreover, biological assays highlighted that fermentation determines an increase in the antioxidant and anti-inflammatory activity of OMWW. Lastly, an increment in the active permeability on Caco-2 cell line was also revealed. In conclusion, results of the present study confirmed that the process applied here represents an effective strategy to achieve a new functional beverage.

Metabolomic and Transcriptional Profiling of Oleuropein Bioconversion into Hydroxytyrosol during Table Olive Fermentation by Lactiplantibacillus plantarum.

This study aims to elucidate the mechanisms responsible for the bioconversion of oleuropein into low-molecular-weight phenolic compounds in two selected Lactiplantibacillus plantarum strains, namely, C11C8 and F3.5, under stress brine conditions and at two different temperatures (16°C and 30°C). For this purpose, we adopted an experimental strategy that combined high-resolution mass spectrometry, in silico functional analysis of glycoside hydrolase family 1 (GH1)-encoding candidate genes, and gene expression studies. The oleuropein hydrolysis products and the underlying enzymatic steps were identified, and a novel putative bgl gene was detected, using seven strains belonging to the same species as controls. According to metabolomic analysis, a new intermediate compound (decarboxymethyl dialdehydic form of oleuropein aglycone) was revealed. In addition, strain C11C8 showed a decrease in the oleuropein content greater than that of the F3.5 strain (30% versus 15%) at a temperature of 16°C. The highest increase in hydroxytyrosol was depicted by strain C11C8 at a temperature of 30°C. PCR assays and sequencing analyses revealed that both strains possess bglH1, bglH2, and bglH3 genes. Furthermore, a reverse transcription-PCR (RT-PCR) assay showed that bglH3 is the only gene transcribed under all tested conditions, while bglH2 is switched off in strain C11C8 grown at cold temperatures, and no transcription was detected for the bglH1 gene. The bglH3 gene encodes a 6-phospho-ß-glucosidase, suggesting how phospho-ß-glucosidase activity could belong to the overall metabolic strategy undertaken by L. plantarum to survive in an environment poor in free sugars, like table olives. IMPORTANCE In the present study, a new candidate gene, bglH3, responsible for the ß-glucosidase-positive phenotype in L. plantarum was detected, providing the basis for the future marker-assisted selection of L. plantarum starter strains with a ß-glucosidase-positive phenotype. Furthermore, the ability of selected strains to hydrolyze oleuropein at low temperatures is important for application as starter cultures on an industrial scale.

Polyphasic approach to study physico-chemical, microbiological and sensorial characteristics of artisanal Nicastrese goat's cheese.

Nicastrese goat's cheese is produced in the South of Italy under traditional procedures, from raw goat milk without any starter cultures addition. Samples from milk to ripened cheese provided by 4 different farms were subjected to a polyphasic approach to study their physico-chemical, microbiological and sensorial characteristics. In addition, volatile organic compounds formation in the final products was studied. Overall, gross composition and microbiological data revealed a significant variability among samples, which was confirmed by both the volatile organic compounds generated in the final products and by the sensorial data. Conventional technique allowed us to identify 720 isolates, mainly belonging to Lactococcus lactis, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus brevis, Leuconostoc mesenteroides, and Enterococcus faecalis. Culture-independent methods revealed shifts in the microbial community structure, with an increase in biodiversity of metabolically active bacterial species, from milk to cheese samples. Analysis of volatile organic compounds (VOCs) allowed the identification of 36 compounds; free fatty acids and ketones represented the main detected, followed by alcohols and esters. Moreover, statistical analysis was performed in order to correlate VOCs to bacterial species. Data showed that ester compounds as well as alcohol and aldehydes were positively correlated to NSLAB, indicating that the occurrence of L. casei, L. plantarum and L. brevis species is relevant for the VOCs formation in the final product.

Influence of PDO Ragusano cheese biofilm microbiota on flavour compounds formation.

The objectives of the present study were to characterize the biofilm microbiota of 11 different farms (from A to K), producing PDO Ragusano cheese, and to investigate on its ability to generate volatile organic compounds (VOCs) in milk samples inoculated with biofilm and incubated under Ragusano cheese making conditions. The biofilms were subjected to plate counting and PCR/T/DGGE analysis and the VOCs generated in incubated milk samples were evaluated through SmartNose, GC/O, and GC/MS. Streptococcus thermophilus was the dominant species both in biofilms and in incubated milks. Lactobacillus, Lactococcus, Enterococcus and Leuconostoc were also identified. Low levels of Pseudomonas spp. and yeasts counts were detected, whereas coliforms, Listeria monocytogenes and Salmonella spp., were never found. SmartNose and GC/O analyses were able to differentiate incubated milk samples on the basis of the odour compounds, highlighting that samples E and F overlapped and sample C was clearly separated from the others. These results complied with those acquired by GC/MS analysis, that detected in total 20 VOCs. Principal component analysis showed positive correlations (r > 0.6; P < 0.05) between some lactic acid bacteria (LAB) and VOCs: such as Enterococcus hirae with alcohols, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus casei and Lactobacillus delbrueckii with aldehydes, and Lactobacillus fermentum, Lactobacillus helveticus and Lactobacillus hilgardii with ketones. This work demonstrates that biofilm represents an excellent source of LAB biodiversity, which contribute to generate VOCs during the production of PDO Ragusano cheese.

Bacterial biota of women with bacterial vaginosis treated with lactoferrin: an open prospective randomized trial.

Background: Bacterial vaginosis is the most frequent condition associated to the vaginal microbiota imbalance, affecting about the 40-50% of women in the world. Even if antibiotics are effcetive for bacterial vaginosis treatment a long-term recurrence rates, higher than 70%, is recorded. Lactoferrin is an iron-binding glycoprotein with bacteriostatic and bactericidal properties. It owns the ability to protect the host against infection, by binding and regulating the iron needed for the bacterial proliferation. Objective: The present study was an open prospective randomized trial (registration no. SHI-EVE-2014.01) aimed at characterizing the bacterial biota of women affected by bacterial vaginosis (BV) and assessing the effects of two different lactoferrin concentrations (100 mg and 200 mg vaginal pessaries) on the composition and dynamics of the vaginal bacterial biota. Design: Sixty women with BV were recruited and randomized into two groups to receive lactoferrin pessaries for 10 days. Clinical evaluation was based on Amsel criteria and Nugent scores. Culture-dependent methods and Ion Torrent PGM sequencing of the 16S rRNA gene were applied to study in depth the overall structure of the vaginal bacterial biota and its dynamics during the treatment. Results: Vaginal lactoferrin administration modified the vaginal microbiota composition in patients with BV. During treatment, both 100 mg and 200 mg lactoferrin vaginal pessaries significantly decreased the occurrence of bacteria associated with BV, such as Gardnerella, Prevotella, and Lachnospira, and increased the occurrence of Lactobacillus species. The bacterial biota balance was maintained up to 2 weeks after treatment only in women treated with 200 mg lactoferrin pessaries. Conclusions: This study indicates that lactoferrin could be proposed as an alternative therapeutic approach for BV. Our data showed, for the first time, the dominance of Lactobacillus helveticus species during and after vaginal lactoferrin treatment.

A large factory-scale application of selected autochthonous lactic acid bacteria for PDO Pecorino Siciliano cheese production.

The main hypothesis of this study was that the autochthonous lactic acid bacteria (LAB) selected for their dairy traits are able to stabilize the production of PDO (Protected Denomination of Origin) Pecorino Siciliano cheese, preserving its typicality. The experimental plan included the application of a multi-strain lactic acid bacteria (LAB) culture, composed of starter (Lactococcus lactis subsp. lactis CAG4 and CAG37) and non starter (Enterococcus faecalis PSL71, Lactococcus garviae PSL67 and Streptococcus macedonicus PSL72) strains, during the traditional production of cheese at large scale level in six factories located in different areas of Sicily. The cheese making processes were followed from milk to ripened cheeses and the effects of the added LAB were evaluated on the microbiological, chemico-physical and sensorial characteristics of the final products. Results highlighted a high variability for all investigated parameters and the dominance of LAB cocci in bulk milk samples. The experimental curds showed a faster pH drop than control curds and the levels of LAB estimated in 5-month ripened experimental cheeses (7.59 and 7.27 Log CFU/g for rods and cocci, respectively) were higher than those of control cheeses (7.02 and 6.61 Log CFU/g for rods and cocci, respectively). The comparison of the bacterial isolates by randomly amplified polymorphic DNA (RAPD)-PCR evidenced the dominance of the added starter lactococci over native milk and vat LAB, while the added non starter LAB were found at almost the same levels of the indigenous strains. The sensory evaluation showed that the mixed LAB culture did not influence the majority of the sensory attributes of the cheeses and that each factory produced cheeses with unique characteristics. Finally, the multivariate statistical analysis based on all parameters evaluated on the ripened cheeses showed the dissimilarities and the relationships among cheeses. Thus, the main hypothesis of the work was accepted since the quality parameters of the final cheeses were stabilized, but all cheeses maintained their local typicality.

Comparative genomic and functional analysis of 100 Lactobacillus rhamnosus strains and their comparison with strain GG.

Lactobacillus rhamnosus is a lactic acid bacterium that is found in a large variety of ecological habitats, including artisanal and industrial dairy products, the oral cavity, intestinal tract or vagina. To gain insights into the genetic complexity and ecological versatility of the species L. rhamnosus, we examined the genomes and phenotypes of 100 L. rhamnosus strains isolated from diverse sources. The genomes of 100 L. rhamnosus strains were mapped onto the L. rhamnosus GG reference genome. These strains were phenotypically characterized for a wide range of metabolic, antagonistic, signalling and functional properties. Phylogenomic analysis showed multiple groupings of the species that could partly be associated with their ecological niches. We identified 17 highly variable regions that encode functions related to lifestyle, i.e. carbohydrate transport and metabolism, production of mucus-binding pili, bile salt resistance, prophages and CRISPR adaptive immunity. Integration of the phenotypic and genomic data revealed that some L. rhamnosus strains possibly resided in multiple niches, illustrating the dynamics of bacterial habitats. The present study showed two distinctive geno-phenotypes in the L. rhamnosus species. The geno-phenotype A suggests an adaptation to stable nutrient-rich niches, i.e. milk-derivative products, reflected by the alteration or loss of biological functions associated with antimicrobial activity spectrum, stress resistance, adaptability and fitness to a distinctive range of habitats. In contrast, the geno-phenotype B displays adequate traits to a variable environment, such as the intestinal tract, in terms of nutrient resources, bacterial population density and host effects.

Inside Current Winemaking Challenges: Exploiting the Potential of Conventional and Unconventional Yeasts.

Wine represents a complex matrix in which microbial interactions can strongly impact the quality of the final product. Numerous studies have focused on optimizing microbial approaches for addressing new challenges to enhance quality, typicity, and food safety. However, few studies have investigated yeasts of different genera as resources for obtaining wines with new, specific traits. Currently, based on the continuous changes in consumer demand, yeast selection within conventional Saccharomyces cerevisiae and unconventional non-Saccharomyces yeasts represents a suitable opportunity. Wine fermentation driven by indigenous yeasts, in the various stages, has achieved promising results in producing wines with desired characteristics, such as a reduced content of ethanol, SO2, and toxins, as well as an increased aromatic complexity. Therefore, the increasing interest in organic, biodynamic, natural, or clean wine represents a new challenge for the wine sector. This review aims at exploring the main features of different oenological yeasts to obtain wines reflecting the needs of current consumers in a sustainability context, providing an overview, and pointing out the role of microorganisms as valuable sources and biological approaches to explore potential and future research opportunities.

Protective Effect of Treated Olive Mill Wastewater on Target Bacteria and Mitochondrial Voltage-Dependent Anion-Selective Channel 1.

Olive mill wastewater, a by-product of the olive oil industry, represents an important resource, rich in bioactive compounds with antioxidant activity. In this study, two strategies to concentrate the bioactive components were used: the tangential membrane filtration (ultrafiltration and reverse osmosis) and the selective resin extraction. The concentrates were evaluated for physico-chemical characteristics and antioxidant activity. Furthermore, the antimicrobial activity and the effect on the mitochondrial voltage-dependent anion selective channel 1 were evaluated. The chemical results highlighted that the highest concentration of hydroxytyrosol (as 7204 mg/L) was revealed in the sample obtained by inverse osmosis while the highest concentration of oleuropein (10005 mg/L) was detected in the sample obtained by resin extraction. The latter sample exhibited the highest antimicrobial effects against Listeria monocytogenes, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. Both samples exhibited a high impact on the electrophysiological parameters of VDAC1 activity. These results showed that both valorization techniques, which can be reproduced at industrial scale, provided phenolic concentrates with antioxidant and antimicrobial activity useful for different future perspectives.

Effect of microbial fermentation on functional traits and volatiloma profile of pâté olive cake.

In this study, the pâté olive cake (POC), a by-product of the olive oil industry, was subjected to fermentation in a bioreactor using three microbial strains, Lactiplantibacillus plantarum, Wickerhamomyces anomalus and Candida boidinii, previously isolated from fermented table olive brines. Chemical, microbiological and molecular analyses were carried out at the beginning and at the end of fermentation. The lowest pH value (4.09) was reached after 10 days in sample inoculated with C. boidinii. Microbiological analyses exhibited the dominance of yeasts throughout the whole process (from 5.5 to 7.80 Log10 CFU/g), as confirmed by PCR-DGGE analysis. The microbial cultures affected both phenolic and volatile organic compound profiles. Moreover, the POC samples treated with different microbial strains were investigated for biological assays. The sample fermented with W. anomalus showed the greatest diffusion speed of transepithelial transport through Caco-2 cell, the highest inhibitory activity towards the tested cyclooxygenases and the highest antioxidant activity.

Effect of olive by-products feed supplementation on physicochemical and microbiological profile of Provola cheese.

Introduction: With the purpose to evaluate the effects of dietary olive cake, a source of bioactive phenolic compounds, as feed supplementation of lactating dairy cows on fatty acid composition, volatile organic compounds, and microbiological profiles of Provola cheese, we performed a two-arm study where control and experimental administered cows derived dairy have been compared. Methods: Our panel of analyses include metabolomics, physicochemical detected variables, culture dependent and independent analyses, and a stringent statistical approach aimful at disclosing only statistically significant results. Results and discussion: Looking at the physicochemical variable's profiles, a higher content of unsaturated fatty acids, polyunsaturated fatty acid, and conjugated linoleic acids as well of proteins were observed in experimental cheese samples, indicating the beneficial effect of dietary supplementation. Furthermore, based on volatilome composition, a clear cluster separation between control and experimental cheeses was obtained, mainly related to terpenes degradation, able of influencing their aroma and taste. Microbiological results showed a decrease of some spoilage related microbial groups in experimental cheeses, probably due to the inhibitory effect exerted by polyphenols compounds, that contrarily did not affect the core taxa of all cheese samples. This paper confirmed the promising utilization of olive by-product in farming practices to obtain more sustainable and safe dairy food products with lower environmental impact, mainly in Sicily and Mediterranean area, where waste disposal poses serious environmental and economic problems.

Diversity of bacterial population of table olives assessed by PCR-DGGE analysis.

Nocellara Etnea and Geracese table olives are produced according to traditional process, in which lactic acid bacteria (LAB) and yeasts are the dominant microorganisms. With the aim to evaluate the effect of selected starter cultures on dynamics of bacterial population during fermentation and on growth/survival of Listeria spp. artificially inoculated into the olive brine, a polyphasic approach based on the combination of culturing and PCR-DGGE analysis was applied. Plating results showed a different concentration of the major bacterial groups considered among cultivars and the beneficial effect of LAB starters, which clearly inhibited Enterobacteriaceae. Moreover, results indicated that the brine conditions applied did not support the growth/survival of Listeria monocytogenes strain, artificially inoculated, highlighting the importance of selecting right fermentation parameters for assuring microbiological safety of the final products. Comparison of DGGE profile of Nocellara Etnea and Geracese table olives, displayed a great difference among cultivars, revealing a wide biodiversity within Lactobacillus population during Geracese olives fermentation. Based on cloning and sequencing of the most dominant amplicons, the presence, among others, of Lactobacillus paracollinoides and Lactobacillus coryniformis in Geracese table olives was revealed in table olives for the first time.

Survival of Lactobacillus rhamnosus strains inoculated in cheese matrix during simulated human digestion.

Survival of probiotic bacteria during transit through the gastrointestinal (GI) tract is influenced by a number of environmental variables including stomach acidity, bile salts, digestive enzymes and food matrix. This study assessed survival of seven selected Lactobacillus rhamnosus strains delivered within a model cheese system to the human upper GI tract using a dynamic gastric model (DGM). Good survival rates for all tested strains were recorded during both simulated gastric and duodenal digestion. Strains H12, H25 and N24 demonstrated higher survival capacities during gastric digestion than L. rhamnosus GG strain used as control, with H12 and N24 continuing to grow during duodenal digestion. Strains L. rhamnosus F17, N24 and R61 showed adhesion properties to both HT-29 and Caco-2 cells. The ability to attach to the cheese matrix during digestion was confirmed by scanning electron microscopy, also indicating production of extracellular polysaccharides as a response to acid stress.

Olive Pomace and Pâté Olive Cake as Suitable Ingredients for Food and Feed.

Olive oil extraction generates several by-products that represent an environmental issue, mainly for Mediterranean countries where olive oil is mostly produced. These by-products represent an ecological issue for their phenolic components, such as oleuropein, hydroxytyrosol, and tyrosol. However, olive oil by-products can be treated and properly exploited in different fields for their health-promoting properties, and they represent great potential for the food and beverage, cosmetic, and pharmaceutical industries. Furthermore, recovery and treatment processes can contribute to efficient waste management, which can enhance the sustainability of the olive oil industry, and in turn, lead to relevant economic benefits. The solid waste, i.e., olive pomace, could be considered to be a suitable matrix or primary resource of molecules with high added value due to their high phenolic content. Olive pomace, at different moisture contents, is the main by-product obtained from two- or three-phase extraction systems. A commonly used centrifugal extraction system, i.e., a multiphase decanter (DMF), does not require the addition of water and can generate a new by-product called pâté or olive pomace cake, consisting of moist pulp that is rich in phenols, in particular, secoiridoids, without any trace of kernel. Although several reviews have been published on olive wastes, only a few reviews have specifically focused on the solid by-products. Therefore, the aim of the present review is to provide a comprehensive overview on the current valorization of the main solid olive oil by-products, in particular, olive pomace or pâté olive cake, highlighting their use in different fields, including human nutrition.

Insights on the bacterial composition of Parmigiano Reggiano Natural Whey Starter by a culture-dependent and 16S rRNA metabarcoding portrait.

Natural whey starters (NWS) are undefined bacterial communities produced daily from whey of the previous cheese-making round, by application of high temperature. As a result, in any dairy plant, NWS are continuously evolving, undefined mixtures of several strains and/or species of lactic acid bacteria, whose composition and performance strongly depend on the selective pressure acting during incubation. While NWS is critical to assure consistency to cheese-making process, little is known about the composition, functional features, and plant-to-plant fluctuations. Here, we integrated 16S rRNA metabarcoding and culture-dependent methods to profile bacterial communities of 10 NWS sampled in the production area of Parmigiano Reggiano cheese. 16S rRNA metabarcoding analysis revealed two main NWS community types, namely NWS type-H and NWS type-D. Lactobacillus helveticus was more abundant in NWS type-H, whilst Lactobacillus delbrueckii/St. thermophilus in NWS type-D, respectively. Based on the prediction of metagenome functions, NWS type-H samples were enriched in functional pathways related to galactose catabolism and purine metabolism, while NWS type-D in pathways related to aromatic and branched chain amino acid biosynthesis, which are flavor compound precursors. Culture-dependent approaches revealed low cultivability of individual colonies as axenic cultures and high genetic diversity in the pool of cultivable survivors. Co-culturing experiments showed that fermentative performance decreases by reducing the bacterial complexity of inoculum, suggesting that biotic interactions and cross-feeding relationships could take place in NWS communities, assuring phenotypic robustness. Even though our data cannot directly predict these ecological interactions, this study provides the basis for experiments targeted at understanding how selective regime affects composition, bacterial interaction, and fermentative performance in NWS.

Phenols recovered from olive mill wastewater as natural booster to fortify blood orange juice.

In the present study, a tangential membrane filtration system was applied to recover phenols from olive mill wastewater. The obtained concentrates were characterised for physico-chemical traits, antioxidant activity and antimicrobial effects. Results indicated that the highest concentration of hydroxytyrosol (7203.7 mg/L) was detected in the concentrate obtained by reverse osmosis, which also showed the highest antioxidant and antimicrobial activity. Moreover, the same concentrate was added, at different ratio, up to 4:250 v/v, into a commercial blood orange juice. The fortified juice with the addition of the concentrate, up to 2:250 v/v ratio, did not show off-flavour and off-odour compared to the control. Furthermore, after 60 days of refrigerated storage, the fortified juice exhibited a hydroxytyrosol content still complying with the daily intake recommended by EFSA health claim. The obtained results can be industrially useful in producing orange juice added with a natural antioxidant concentrate as a 'clean label' ingredient.

Antimicrobial activity of bovine lactoferrin against Gardnerella species clinical isolates.

Gardnerella species play a key role in the development and recurrence of Bacterial Vaginosis (BV), a common imbalance of the vaginal microbiota. Because of the high rates of BV recurrence reported after treatment with standard of care antibiotics, as well as the emergence of antibiotic-resistant BV, the development of alternative treatment approaches is needed. Bovine lactoferrin, a well studied iron-binding glycoprotein with selective antimicrobial activity, may ameliorate vaginal dysbiosis either alone or in combination with antibiotics. The present study evaluated the antimicrobial resistance/susceptibility profile of seventy-one presumptive G. vaginalis clinical isolates to metronidazole and clindamycin. In addition, the in vitro antimicrobial activity of Metrodora Therapeutics bovine Lactoferrin (MTbLF) against the tested clinical isolates, both alone and in combination with metronidazole and clindamycin, was in depth evaluated using defined-iron culture conditions. All 71 presumptive G. vaginalis clinical isolates exhibited resistance to metronidazole, with MIC values greater than 256 µg/ml. Different susceptibility profiles were detected for clindamycin. In detail, the vast majority of the tested strains (45%), exhibiting MIC lower than 2 µg/ml, were considered sensitive; 18 strains (25%) with MIC higher or equal to 8 µg/ml, were classified as resistant, whereas the remaining 21 (30%) were classified as intermediate. MTbLF was tested in culture medium at different concentrations (32, 16, 8, 4, 2, 1, and 0.5 mg/ml) showing ability to inhibit the growth of the tested presumptive G. vaginalis clinical isolates, including those metronidazole-resistant, in a dose-dependent and not in a strain-dependent manner. MTbLF, at concentrations ranging from 32 to 8 mg/ml, exerted a statistically different antimicrobial activity compared with lower concentrations (4, 2, 1, and 0.5 mg/ml). A synergistic effect between MTbLF (8 and 4 mg/ml) and clindamycin was revealed for all the tested strains. When tested in the absence of other sources of iron, MTbLF did not support the growth of the tested presumptive G. vaginalis clinical isolates. Bovine lactoferrin may be a potential candidate to treat Gardnerella species infection.

Characterization of Bifidobacterium asteroides Isolates.

Bifidobacteria have long been recognized as bacteria with probiotic and therapeutic features. The aim of this work is to characterize the Bifidobacterium asteroides BA15 and BA17 strains, isolated from honeybee gut, to evaluate its safety for human use. An in-depth assessment was carried out on safety properties (antibiotic resistance profiling, ß-hemolytic, DNase and gelatinase activities and virulence factor presence) and other properties (antimicrobial activity, auto-aggregation, co-aggregation and hydrophobicity). Based on phenotypic and genotypic characterization, both strains satisfied all the safety requirements. More specifically, genome analysis showed the absence of genes encoding for glycopeptide (vanA, vanB, vanC-1, vanC-2, vanD, vanE, vanG), resistance to tetracycline (tetM, tetL and tetO) and virulence genes (asa1, gelE, cylA, esp, hyl).

An alkaline ß-glucosidase isolated from an olive brine strain of Wickerhamomyces anomalus.

An efficient ß-glucosidase (ßG)-producing strain, Wickerhamomyces anomalus BS81, was isolated from naturally fermented olive brine and identified based on PCR/restriction fragment length polymorphism of the rDNA internal transcribed spacer and sequence analysis of the D1/D2 region of the 26S rRNA gene. The hydrolytic activity of the ßG had an optimum pH of 8.5 and an optimum temperature of 35 °C. The enzyme had high substrate specificity and high catalytic efficiency (K(m) 0.99 mM, V(max) 14 U g(-1) of cells) for p-nitrophenyl-ß-d-glucopyranoside. The enzyme was activated by increasing concentrations of NaCl, with maximum activity at 150 g L(-1) NaCl. Although ßGs have been purified and characterized from several other sources, the W. anomalusßG is unique among ßGs because its relative maximum activity occurs at alkaline pH and 35 °C. Moreover, the yeast strain has esterase activity that acts synergistically with ßG to degrade oleuropein to debitter table olives and olive oil.