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.

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.

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.

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.

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.

Prebiotic effects of citrus pectic oligosaccharides.

Citrus peel wastes, one of the major by-products of the agri-food industry, are a source of value-added compounds. In this work, a commercial pectin (PEC1) and one obtained by direct extraction from citrus fibre (PEC2), were hydrolysed and ultrafiltered at different cut-off (100, 50 and 30 kDa) and then tested in broth with four different probiotic strains: Lacticaseibacillus paracasei, Limosilactobacillus fermentum, Lacticaseibacillus rhamnosus and Bifidobacterium animalis subsp. paracasei. In addition, the probiotic strains selected for their growth rate with the fractions of the two pectins were tested for tolerance to different pH values and bile salts. This study demonstrated that POS2 (product from the enzymatic hydrolysis by PEC2) showed greater prebiotic activity than POS1 (product from the enzymatic hydrolysis by PEC1). The results confirmed that citrus pectic oligosaccharides could be used as emerging prebiotics with improved properties due to their antimicrobial and modulating-microbiota ability.

Microbial Application to Improve Olive Mill Wastewater Phenolic Extracts.

Olive mill wastewater (OMW) contains valuable and interesting bioactive compounds, among which is hydroxytyrosol, which is characterized by a remarkable antioxidant activity. Due to the health claims related to olive polyphenols, the aim of this study was to obtain an extract from OMW with an increased level of hydroxytyrosol by means of microbial enzymatic activity. For this purpose, four commercial adsorbent resins were selected and tested. The beta-glucosidase and esterase activity of strains of Wickerhamomyces anomalus, Lactiplantibacillus plantarum, and Saccharomyces cerevisiae were also investigated and compared to those of a commercial enzyme and an Aspergillus niger strain. The W. anomalus strain showed the best enzymatic performances. The SP207 resin showed the best efficiency in selective recovery of hydroxytyrosol, tyrosol, oleuropein, and total phenols. The bioconversion test of the OMW extract was assessed by using both culture broths and pellets of the tested strains. The results demonstrated that the pellets of W. anomalus and L. plantarum were the most effective in hydroxytyrosol increasing in phenolic extract. The interesting results suggest the possibility to study new formulations of OMW phenolic extracts with multifunctional microorganisms.

Bioactive compounds and antioxidant activity of four rose hip species from spontaneous Sicilian flora.

The morphological characteristics and health-promoting components (anthocyanins, phenolic compounds, carotenoids, and ascorbic acid) together with the in-vitro antioxidant capacity of hips collected from four rose species (Rosa canina, R. corymbifera, R. micrantha, and R. sempervirens) growing in Sicily (South Italy) were evaluated. The results revealed significant variability among the rose species as the hips collected from R. canina and R. sempervirens exhibited the highest total polyphenol contents (6784.5 and 6241.2 mg GAE/100 g DW, respectively) and highest antioxidant activities, whereas R. canina and R. micrantha hips had the highest total anthocyanin contents (2.94 and 3.86 mg CGE/100 g DW, respectively). The data showed that the analysed Sicilian rose hips have good nutritional quality, making them suitable as functional foods according to their biochemical composition and as promising sources of natural antioxidants. Also, these findings confirm that rose pseudo-fruits contain significant amounts of secondary metabolites that may positively affect human health.

Transcriptomic Analysis of Orange Fruit Treated with Pomegranate Peel Extract (PGE).

A Pomegranate Peel Extract (PGE) has been proposed as a natural antifungal substance with a wide range of activity against plant diseases. Previous studies showed that the extract has a direct antimicrobial activity and can elicit resistance responses in plant host tissues. In the present study, the transcriptomic response of orange fruit toward PGE treatments was evaluated. RNA-seq analyses, conducted on wounded fruits 0, 6, and 24 h after PGE applications, showed a significantly different transcriptome in treated oranges as compared to control samples. The majority (273) of the deferentially expressed genes (DEGs) were highly up-regulated compared to only 8 genes that were down-regulated. Gene Ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis showed the involvement of 1233 gene ontology (GO) terms and 35 KEGG metabolic pathways. Among these, important defense pathways were induced and antibiotic biosynthesis was the most enriched one. These findings may explain the underlying preventive and curative activity of PGE against plant diseases.

Effect of Sequential Inoculum of Beta-Glucosidase Positive and Probiotic Strains on Brine Fermentation to Obtain Low Salt Sicilian Table Olives.

In the present study, the ß-glucosidase positive strain Lactobacillus plantarum F3. 3 was used as starter during the fermentation of Sicilian table olives (Nocellara Etnea cultivar) at two different salt concentrations (5 and 8%), in order to accelerate the debittering process. The latter was monitored through the increase of hydroxytyrosol compound. In addition, the potential probiotic Lactobacillus paracasei N24 strain was added after 60 days of fermentation. Un-inoculated brine samples at 5 and 8% of salt were used as control. The fermentation was monitored till 120 days through physico-chemical and microbiological analyses. In addition, volatile organic compounds and sensorial analyses were performed during the process and at the end of the fermentation, respectively. Lactic acid bacteria and yeasts were, in depth, studied by molecular methods and the occurrence of the potential probiotic N24 strain in the final products was determined. Results highlighted that inoculated brines exhibited a higher acidification and debittering rate than control ones. In addition, inoculated brines at 5% of salt exhibited higher polyphenols (hydoxytyrosol, tyrosol, and verbascoside) content compared to samples at 8% of NaCl, suggesting a stronger oleuropeinolytic activity of the starter at low salt concentration. Lactobacilli and yeasts dominated during the fermentation process, with the highest occurrence of L. plantarum and Wickerhamomyces anomalus, respectively. Moreover, the potential probiotic L. paracasei N24 strain was able to survive in the final product. Hence, the sequential inoculum of beta-glucosidase positive and potential probiotic strains could be proposed as a suitable technology to produce low salt Sicilian table olives.

Evaluation of a Pomegranate Peel Extract as an Alternative Means to Control Olive Anthracnose.

Olive anthracnose is caused by different species of Colletotrichum spp. and may be regarded as the most damaging disease of olive fruit worldwide, greatly affecting quality and quantity of the productions. A pomegranate peel extract (PGE) proved very effective in controlling the disease. The extract had a strong in vitro fungicidal activity against Colletotrichum acutatum sensu stricto, was very effective in both preventive and curative trials with artificially inoculated fruit, and induced resistance in treated olive tissues. In field trials, PGE was significantly more effective than copper, which is traditionally used to control the disease. The highest level of protection was achieved by applying the extract in the early ascending phase of the disease outbreaks because natural rots were completely inhibited with PGE at 12 g/liter and were reduced by 98.6 and by 93.0% on plants treated with PGE at 6 and 3 g/liter, respectively. Two treatments carried out 30 and 15 days before the expected epidemic outbreak reduced the incidence of the disease by 77.6, 57.0, and 51.8%, depending on the PGE concentration. The analysis of epiphytic populations showed a strong antimicrobial activity of PGE, which sharply reduced both fungal and bacterial populations. Because PGE was obtained from a natural matrix using safe chemicals and did not have any apparent phytotoxic effect on treated olive fruit, it may be regarded as a safe and effective natural antifungal preparation to control olive anthracnose and improve olive productions.

Screening of the anthocyanin profile and in vitro pancreatic lipase inhibition by anthocyanin-containing extracts of fruits, vegetables, legumes and cereals.

BACKGROUND: The phytotherapic treatment of overweight and/or moderate obesity is growing widely, thus there is a great interest towards the phenolic compounds of fruits and vegetables which may inhibit pancreatic lipase enzyme. In this study, we report the chemical composition and in vitro pancreatic lipase inhibitory activity of 13 freeze-dried anthocyanin-containing extracts of different Mediterranean plants: fruits (blood orange, pomegranate, blackberry, mulberry and sumac), citrus by-products (blood orange peel), citrus vegetative tissues (young lemon shoots); vegetables (red cabbage and violet cauliflower), legume seeds (black bean), cereals (black rice), and cereal processing by-products (black rice hull). Total phenols and anthocyanins were determined. Individual anthocyanins were identified by UHPLC-PDA-ESI/MSn . RESULTS: Results revealed a wide variation in the distribution of anthocyanin compounds. Blood orange and pomegranate juice extracts had the highest total anthocyanin content and exhibited the strongest inhibition of pancreatic lipase in vitro. CONCLUSION: Inhibitory activity was positively correlated with anthocyanin content. In appropriate formulations, anthocyanin-containing extracts could find a use as anti-obesity agents. © 2016 Society of Chemical Industry.

Larvicidal Effects of Four Citrus Peel Essential Oils Against the Arbovirus Vector Aedes albopictus (Diptera: Culicidae).

In this study, we evaluated the larvicidal activity of four citrus essential oils (EOs; sweet orange, mandarin, bergamot, and lemon) against the arbovirus vector Aedes albopictus (Skuse) (Diptera: Culicidae) under laboratory conditions. Through gas chromatography-mass spectrometry (GC-MS) analyses, we found that in sweet orange, mandarin, and lemon EOs, limonene was the most abundant compound, whereas linalyl acetate was the most abundant in the bergamot EO. All tested EOs showed a marked larvicidal activity, in particular sweet orange, lemon, and bergamot that killed all treated larvae. After 24 h of exposure, the LC50 values of the tested citrus EOs ranged from 145.27 (lemon EO) to 318.07 mg liter(-1) (mandarin EO), while LC(95) ranged from 295.13 to 832.44 mg liter(-1). After 48 h of exposure, the estimated LC(50) values decreased to values ranging from 117.29 to 209.38 mg liter(-1), while LC(95) ranged from 231.85 to 537.36 mg liter(-1). The results obtained from these evaluations, together with the large availability at reasonable costs of citrus EOs, are promising for the potential development of a new botanical mosquitocide.

Chemical Characterization of Different Sumac and Pomegranate Extracts Effective against Botrytis cinerea Rots.

Pomegranate (Punica granatum L.) peel and sumac (Rhus coriaria L.) fruit and leaf extracts were chemically characterized and their ability to inhibit table grape (cv. Italia) rots caused by Botrytis cinerea was evaluated on artificially inoculated berries. Different extraction methods were applied and extracts were characterized through Ultra Fast High Performance Liquid Chromatography coupled to Photodiode array detector and Electrospray ionization Mass spectrometer (UPLC-PDA-ESI/MSn) for their phenol and anthocyanin contents. The concentrated pomegranate peel extract (PGE-C) was the richest in phenols (66.97 g gallic acid equivalents/kg) while the concentrated sumac extract from fruits (SUF-C) showed the highest anthocyanin amount (171.96 mg cyanidin 3-glucoside equivalents/kg). Both phenolic and anthocyanin profile of pomegranate and sumac extracts were quite different: pomegranate extract was rich in cyanidin 3-glucoside, pelargonidin 3-glucoside and ellagic acid derivatives, while sumac extract was characterized by 7-methyl-cyanidin 3-galactoside and gallic acid derivatives. The concentrated extracts from both pomegranate peel and sumac leaves significantly reduced the development of Botrytis rots. In particular, the extract from pomegranate peel completely inhibited the pathogen at different intervals of time (0, 12, and 24 h) between treatment and pathogen inoculation on fruits maintained at 22-24 °C and high relative humidity (RH). This extract may represent a valuable alternative to control postharvest fungal rots in view of its high efficacy because of the low cost of pomegranate peel, which is a waste product of processing factories.

Phenolic trend and hygienic quality of green table olives fermented with Lactobacillus plantarum starter culture.

In this paper, four different olive cultivars from Southern Italy, Carolea, Cassanese, Nocellara del Belice, Nocellara Messinese table olives, produced according to traditional fermentation processes, were evaluated with the aim of assessing the effect of selected starter cultures on growth of bacterial population and on olive phenols during fermentation. Only Cassanese and Nocellara del Belice inoculated samples reached a safe pH value under 4.6 after 90 days while maintaining it until the end of storage. The most representative phenols in brine samples analysed by HPLC-MS/MS were hydroxytyrosol and verbascoside. Among the analysed phenols, only hydroxytyrosol, caffeic acid and ferulic acid always increased during fermentation, while the others increased up to 90-120 days and then decreased. The Principal Component Analysis (PCA) performed on pH and phenol values highlighted three clusters of olive cultivars. Throughout the brining period, lactic acid bacteria were always present while staphylococci and coliform bacteria disappeared after 30 and 90 days, respectively.

Qualitative and nutraceutical aspects of lemon fruits grown on the mountainsides of the Mount Etna: A first step for a protected designation of origin or protected geographical indication application of the brand name 'Limone dell'Etna'.

The cultivation of lemon (Citrus limon (L.) Burm f.) has occurred for centuries at the mountainsides of the Mount Etna the largest active volcano in Europe (Sicily, Italy). The peculiar geographical, soil and climatic conditions that characterize this area have recently prompted citrus growers to launch the brand name 'Limone dell'Etna' for the lemon fruits produced in that area. The present research focused on evaluating the quality and nutraceutical properties of the 'Limone dell'Etna' fruit juice ('Primofiore', 'Bianchetto', and 'Verdello' blooming) to enhance the value of a product that, due to its peculiar qualities, could be awarded with one of the European certified labels protecting the lemon geographical origin. Qualitative parameters, health-promoting components (such as ascorbic acid, flavonoids, and hydroxycinnamic acids) and the in-vitro total antioxidant capacity (ORAC, FCR) of fruit juice of two different varieties ('Femminello zagara bianca' and 'Monachello') were determined at fruit maturity. The results showed that the Bianchetto and Verdello lemon fruit exhibited higher levels of ascorbic acid than those recorded for the Primofiore production. The amount of flavonoids in Verdello fruits of both cultivars was the highest, as was reflected in the highest antioxidant activity. Thus, the 'Limone dell'Etna' production, with particular reference to the Verdello fruit, must be considered as a new, valuable and original source of natural antioxidants to be valorized and even exploited in the processing industry.

Cholesterol removal capability of lactic acid bacteria and related cell membrane fatty acid modifications.

Milk and dairy products play an important role in a healthy diet because of their high nutritional value, even if they represent a source of lipids and cholesterol. Nowadays, some commercially hypocholesterolemic products are available, which contain lactic acid bacteria (LAB). Therefore, the aims of this study were to test and compare the cholesterol removal abilities of different LAB species and to investigate the capacity of the cholesterol to change the cellular fatty acid composition of microorganisms. Fifty-eight strains of dairy LAB were studied for their ability to remove cholesterol during 24 h of growth. Two of them, L. plantarum 885 and L. acidophilus LA-5®, showed the higher reduction capability. For these strains, the cellular fatty acid composition was studied. They showed a different behaviour, which appeared related to the needs of the cells to maintain the characteristics of membrane fluidity, but was dependent upon their original fatty acid composition. Further studies are required to better characterise the LAB strains to be used to develop fermented dairy products with reduced cholesterol content or be able to induce hypocholesterolemic effects. It will also be interesting to investigate the possible modifications of the cell membrane caused by cholesterol and its possible involvement in cell metabolism.

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.

Effect of kaolin and copper based products and of starter cultures on green table olive fermentation.

In the present study table olives treated in field with kaolin and copper based products against "olive-fruit fly" were fermented using two selected strains of lactic acid bacteria (LAB). The fermentation process was monitored up to 260 days from brining through physico-chemical, microbiological and sensorial analyses. Results showed a dominance of LAB and yeasts and low level of Enterobacteriaceae counts throughout the whole process both in un-treated and treated samples. When investigating the effect of the single treatments on microbial dynamics, ANOVA results highlighted that copper based products affected significantly the control sample, while the sample inoculated with LAB starters maintained high level throughout the process, guaranteeing the fermentation process. Different behavior was revealed by yeasts population, which was partially influenced by copper treatment at the beginning of the fermentation. The polyphasic approach used in the present study, which combined sensory evaluation to microbial counts and physico-chemical characteristics, let to the conclusion on the importance of starter cultures in fermentation of table olives especially those treated with "non-conventional" pesticide, which could be used to prevent olive fly damage.