Bioprocessing technology to exploit organic palm date (Phoenix dactylifera L. cultivar Siwi) fruit as a functional dietary supplement

Journal of Functional Foods 31 (2017) 9–19

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Bioprocessing technology to exploit organic palm date (Phoenix dactylifera L. cultivar Siwi) fruit as a functional dietary supplement Raffaella Di Cagno a, Pasquale Filannino b,⇑, Ivana Cavoski c, Alessia Lanera b, Bahaaaldin Mohamed Mamdouh c, Marco Gobbetti a a b c

Faculty of Sciences and Technology, Free University of Bolzano, 39100 Bolzano, Italy Department of Soil, Plant and Food Science, University of Bari Aldo Moro, 70126 Bari, Italy CIHEAM-MAIB, Mediterranean Agronomic Institute of Bari, 70010 Valenzano, Bari, Italy

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Article history: Received 29 August 2016 Received in revised form 13 December 2016 Accepted 15 January 2017

Keywords: Lactic acid bacteria Dietary fibers Phenolics Conjugated fatty acids Gut microbiota Short chain fatty acids

a b s t r a c t Date fruit (Phoenix dactylifera L.) is a suitable raw matrix for manufacturing of functional ingredients. The aptitude of selected autochthonous lactic acid bacteria for manufacturing of freeze-dried powder from fermented date fruits puree as dietary supplement with health-promoting features was investigated. Fermentation of date fruits puree with selected Lactobacillus plantarum strains allowed the highest concentration of c-amino butyric acid, conjugated fatty acids, and insoluble dietary fibers. The highest Trolox equivalent antioxidant activity was also induced. Lactic acid fermentation affected the profile of phenolic acids and flavonoids, enriching date fruits puree in phenolic derivatives with high human bioavailability. Fermented date fruits puree was also capable to modulate positively in vitro the growths of several cultivable gut microbial taxa, and enhanced the synthesis of short chain fatty acids. In conclusion, the lactic acid fermentation of date fruits is a suitable tool to achieve a standardized and marketable functional dietary supplement. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Date palm (Phoenix dactylifera L.) is a significant crop in arid and semi-arid regions of the world. The total world date fruit production for 2012 was 7,548,918 metric tons (FAOSTAT, 2012). Nowadays, worldwide production, utilization and industrialization of dates are continuously increasing (Chandrasekaran & Bahkali, 2013). The largest producers over the last years have been Egypt, with an average of 1.3 million metric tons per year, followed by Iran (over 1 million metric tons), Saudi Arabia (979,017 metric tons), United Arab Emirates (754,400 metric tons), Pakistan and Algeria (540,000 metric tons) and Sudan (333,500 metric tons) (El Hadrami & Al-Khayri, 2012). Date palm fruit has always been an important economic and nutritional source for traditional producer countries (El Hadrami & Al-Khayri, 2012). However, only recently dates gained interest due to their several health benefits. Several studies were aimed at identify and quantify various classes of phytochemicals and many in vitro animal studies have been performed (Baliga, Baliga, Kandathil, Bhat, & Vayalil, 2011; Rahmani, Aly, Ali,

⇑ Corresponding author. E-mail address: [email protected] (P. Filannino). http://dx.doi.org/10.1016/j.jff.2017.01.033 1756-4646/Ó 2017 Elsevier Ltd. All rights reserved.

Babiker, & Srikar, 2014). Nutritional value of dates is due to their high content of essential nutrients, which include carbohydrates, vitamins, minerals, dietary fibre, fatty acids, amino acids and proteins. B-complex vitamins, vitamin A and C are essential for metabolism of carbohydrates, fat and protein, synthesis of DNA, and as antioxidants (Baliga et al., 2011). Dates are rich in minerals (e.g., selenium, zinc, manganese, boron, calcium, cobalt, copper, and potassium), many of which act as cofactors for antioxidant enzymes. Selenium exerts its antioxidant function mainly as selenocysteine residue, which is an integral constituent of reactive oxygen species–scavenging seleno–enzymes. Zinc competes with copper for binding to the cell wall, thus decreasing the synthesis of hydroxyl radicals. Manganese is an important cofactor of mitochondrial superoxide dismutase (Vayalil, 2012). Date fruit is also a good source of dietary fibre. Literature data showed the importance of insoluble fibers of dates (e.g., cellulose, hemicelluloses, pectin, and lignin), capable of accelerating the activity of the gastro-intestinal (GI) tract and reducing the risk of constipation, preventing bowel cancer or diverticular disease, and improving the cardiac vitality (Baliga et al., 2011). Profiles of the fatty acids in the flesh and seeds of dates revealed the presence of saturated fatty acids, (e.g., capric, lauric, myristic, palmitic, stearic, margaric, arachidic, heneicosanoic, behenic and

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tricosanoic acids) and unsaturated fatty acids (e.g., palmitoleic, oleic, linoleic and linolenic acids) (El Hadrami & Al-Khayri, 2012). Besides, date fruit is also a good source of important phytochemicals, including phenolics (phenolic acids, anthocyanins, procyanidins and flavonoids), sterols, and carotenoids, known to positively affect the human health (Baliga et al., 2011). Preclinical studies have confirmed that dates may exert several beneficial functions in humans, such as antioxidant, immunostimulant, antimutagenic, antimicrobial, anti-inflammatory, anti-cancer and gastro-, hepato- and nephro-protective properties. The beneficial impact of date fruits on human gut microbiota and their potential prebiotic activity has been also recognized (Eid et al., 2014). The abundance and the relative low cost of dates fruits, as well as their nutritional value and emerging beneficial health effects offer new opportunities to turn the surplus production into value-added products such as paste, syrup, jam, date dip, date– honey, and fermented date–based products (Chandrasekaran & Bahkali, 2013). In this context, date fruit have been considered as potential raw matrices for manufacturing of value added products employing fermentation. Bioprocessing of plant matrices through microbial fermentation may have better capacity to exploit inherent bioactivities compared to industrial enzymatic processes. To the best of our knowledge, no studies have considered the use of autochthonous and selected lactic acid bacteria for fermentation of date fruit puree as an alternative biotechnology (Chandrasekaran & Bahkali, 2013). The enhancement of the biogenic activity of dates, which may rely into standardized and marketable products or novel applications, meets the increasing demand for functional foods and dietary supplements. An abundant literature showed the potential of lactic acid fermentation for maintaining and/or improving the nutritional and functional properties of fruits and vegetables (Di Cagno, Coda, De Angelis, & Gobbetti, 2013). Selection of starters within the autochthonous microbiota of fruits, especially for those that have inherent hostile composition, was also preferred since autochthonous cultures may ensure better performance compared to commercial strains (Di Cagno et al., 2013). Dates show a number of drawbacks (e.g., osmotic stress, high level of phenolic compounds) and require the development of an unrealized biotechnological protocol. This study aims at the isolation and identification of autochthonous lactic acid bacteria from organic date fruits collected from Bahariya Oasis (Egypt); the selection of pro-technological starters; and the setting up of the protocol for processing date fruit puree that aimed at guarantying improved health promoting features. In depth physical and chemical characterization and in vitro assays were combined to study the bioactive molecules responsible for the functional features, with a particular focus on antioxidant activity and phenolics. The impact of the fermented whole date fruit and date phenolic extracts on gut microbial taxa and short chain fatty acids synthesis was also assayed using pH-controlled mixed faecal batch cultures.

2. Materials and methods 2.1. Date fruits sampling, handling and storage Date palm (Phoenix dactylifera L. cultivar Siwi) fruits were collected at the beginning of September 2015 at Rutab stage (semi ripening stage) from certified organic fields located in Bahariya oasis (Egypt). Date fruits were collected from 4 different fields. For each sampling site, dates were randomly collected from at least 10 healthy plants using sterile gloves and bags in order to avoid cross contamination. Samples were packed in refrigerated box for the shipment, and stored at 4 °C prior the use.

2.2. Preliminary characterization of date fruits Fruits dimension and weight, percentage of pulp, and seeds weight were recorded. Ten measurements were averaged for each morphological parameter. Total titratable acidity (TTA) was determined on 10 g of date fruits homogenized with 90 mL of distilled water (Classic Blender, PBI International, Milan, Italy), and expressed as the amount (mL) of 0.1 M NaOH to achieve a pH of 8.3. Value of pH was measured by a Crison pH-meter (model 507; Crison, Milan, Italy). Soluble solids were determined using the digital refractometer ATAGO (Chemifarm srl, Parma, Italy). Refractive index was recorded and converted to °Brix. Measurements were performed at 25 ± 0.5 °C. The refractometer prism was cleaned with distilled water after each analysis. Proximate analysis of the date fruits included ash, protein, fat, water and carbohydrate contents, which were carried out according to the methods of the Association of Official Analytical Chemists (AOAC, 2000). All analyses were performed in triplicate. 2.3. Microbiological analyses of date fruits Ten grams of fruits were suspended into sterile 0.1% (w/v) peptone-water solution and homogenized with a Classic Blender 400 (PBI International) for 2 min at room temperature. Mesophilic lactic acid bacteria and yeasts were determined on MRS agar (Oxoid Ltd, Basingstoke, Hampshire, England) containing 0.1% of cycloheximide (Sigma Cemical Co.) at 30 °C for 48 and 72 h under anaerobic conditions, and on Malt Extract Agar (MEA, Oxoid), added of 150 ppm chloramphenicol, at 30 °C for 72 h, respectively. Total mesophilic bacteria were numbered on PCA agar (Oxoid) at 30 °C for 48 h. Total enterobacteria were also enumerated on Violet Red Bile Glucose Agar (VRBGA, Oxoid) at 37 °C for 24 h. 2.4. Isolation, typing and identification of lactic acid bacteria MRS broth containing 0.1% of cycloheximide was used as the enrichment medium. Fifty grams of fruits were suspended in 50 mL of MRS broth, and incubated at 30 °C for 72 h under stirring conditions (200 rpm). Aiming to isolate presumptive mesophilic lactic acid bacteria, serial dilutions were made and plated on MRS agar (Oxoid) at 30 °C for 48–72 h under anaerobic condition. At least ten colonies, with different morphology, were isolated from the highest dilutions of the MRS plates. Gram-positive, catalase-negative, non-motile rod and cocci isolates were cultivated in MRS broth at 30 °C for 24 h, and re-streaked into MRS agar. Genomic DNA was extracted as described by De Los ReyesGavilán, Limsowtin, Tailliez, Séchaud, and Accolas (1992) from 2 mL of MRS culture broth of each isolates. Two primer pairs (Sigma Chemical Co. Milan, Italy), LacbF/LacbR and LpCoF/LpCoR, were used to amplify 16S rRNA gene fragment of lactic acid bacteria (De Angelis et al., 2006). The expected amplicons of ca. 1400 and 1000 bp were eluted from the gel and purified by the GFXTM PCR DNA Gel Band Purification Kit (GE Healthcare, Buckinghmshire, UK). PCR products were separated by electrophoresis, purified as described above, and subjected to sequencing. Taxonomic strain identification was performed by comparing the sequences of each isolate with those reported in the Basic BLAST database (Altschul et al., 1997). Strains showing homology of at least 97% were considered to belong to the same species (Goebel & Stackebrandt, 1994). Randomly amplified polymorphic DNApolymerase chain reaction (RAPD-PCR) analysis was carried as described by Di Cagno et al. (2011), using primers M13, P7 and P4 (Invitrogen Life Technologies, Milan, Italy). Cultures were maintained as stocks in 15% (v/v) glycerol at 80 °C and routinely propagated at 30 °C for 24 h in MRS broth (Oxoid).

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2.5. Starter selection

2.8. Analysis of carbohydrates, organic acids, and free amino acids

Date fruits juice was chosen as growth model system to investigate the kinetics of growth and acidification of lactic acid bacteria strains. Fruits juice medium was prepared as described by Filannino, Cardinali, et al. (2014). Fruits, deprived of the seeds, were homogenized with a Classic Blender 400 (PBI International), centrifuged (10,000g, 20 min, 4 °C), heat treated (121 °C for 10 min), and stored at 20 °C before use. The sterility of the juice was confirmed through plate counts. Aiming to optimize the culture conditions, kinetics of growth and acidification were also determined on fruit juice diluted with sterile water to a final concentration of 75, 50, and 30% (v/v) of fruit juice. Strains of lactic acid bacteria were propagated on MRS broth at 30 °C for 24 h under stirring conditions (200 rpm). Twenty-fourhour-old cells were harvested by centrifugation (10,000g, 10 min at 4 °C), washed twice in 50 mM sterile potassium phosphate buffer (pH 7.0), re-suspended in sterile distilled water to a final optical density at 620 nm (OD620) of 2.5 (final cell number corresponding to ca. 9.0 log cfu mL 1) and used to inoculate the fruit juice to a final cell density corresponding to ca. 8.0 log cfu mL 1. Fruit juices were incubated at 30 °C for 60 h under stirring conditions (200 rpm). Growth was monitored by measuring the optical density at 620 nm or plating on MRS agar. The pH was measured by a Crison pH-meter (model 507; Crison). Kinetics of growth and acidification were determined and modelled according to the Gompertz equation as modified by Zwietering, Jongeberger, Roumbouts, and Van’t Riet (1990): y = k + A exp{ exp[(lmax or Vmax e/A)(k t) + 1]}, where k is the initial level of the dependent variable to be modelled (OD620 or pH units), A is the difference in OD620 or pH units (DpH) between inoculation and the stationary phase, lmax and Vmax are the maximum growth rate (expressed as OD620 units h 1) and the maximum acidification rate (expressed as pH h 1), respectively, k is the length of the lag phase (expressed in hours), and t is the time.

Equal volumes of perchloric acid (5%, v/v) were added to fermented DP, raw DP, and CA-DP aliquots as precipitating agent. The suspension was kept at 4 °C overnight, centrifuged (10,000g, 10 min), and filtered through a Millex-HA 0.22-mm pore size filter (Millipore Co., Bedford, MA, USA). Concentration of glucose and fructose was determined through HPLC analysis, using an ÄKTA Purifier system (GE Healthcare) equipped with a Spherisorb column (Waters, Millford, USA) and a Perkin Elmer 200a refractive index detector (Rizzello, Nionelli, Coda, De Angelis, & Gobbetti, 2010). Organic acids were determined by HPLC, using an ÄKTA Purifier system (GE Healthcare) equipped with an Aminex HPX-87H column (ion exclusion, Biorad) and a UV detector operating at 210 nm (Zeppa, Conterno, & Gerbi, 2001). Organic acids and sugars used as the standards were purchased from Sigma-Aldrich (Steinheim, Germany). Total and individual free amino acids (FAA) were analyzed by a Biochrom 30 series Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, England), with a Na-cationexchange column (20 by 0.46 cm inner diameter) as described by Rizzello, Coda, Mazzacane, Minervini, and Gobbetti (2012).

2.6. Fermentation of date fruits puree Dates fruits, deprived of the seeds, were blended by vertical food processor (mod R8 Robot Coupe, Bologna, Italy), and diluted with sterile water to a final concentration of 30% (v/v) of fruit puree (optimized condition chosen during starter selection). Resulting date fruit puree (DP) was heated at 90 °C for 20 min and cooled at 25 °C before the inoculum. Selected lactic acid bacteria strains were used as single starter or as mixed starter. Twentyfour-hour-old cells cultivated in MRS broth at 30 °C were prepared as previously described and used to inoculate DP to a final cell density corresponding to ca. 8.0 log cfu g 1. DP was fermented at 30 °C for 60 h. Samples were taken before and after fermentation. Raw DP (Raw–DP) without heat treatment, and DP chemically acidified with lactic acid (final pH of ca. 4.0) (CA–DP) incubated at 30 °C for 60 h were used as the controls. Microbiological analysis of fermented DP, Raw–DP, and CA–DP were carried out as described in Section 2.3. Raw–DP, CA–DP and fermented DP were freezedried through a LIO 5P 4 k freeze dryer (Cinquepascal s.r.l., Milan, Italy).

2.7. Physical-chemical analysis Soluble solids, total tritatable acidity (TTA), and pH were measured as described above. Soluble and insoluble dietary fibers were determined according to the AOAC enzymatic-gravimetric method 993.19 for soluble dietary fibre (SDF), and method 991.42 for insoluble dietary fibre (IDF) (AOAC, 2005).

2.9. Bioactive compounds extraction Antioxidant in vitro assay and bioactive compounds analysis were carried out using water-soluble (WSE), methanol-soluble (MSE), or ethyl acetate-soluble (ESE) extracts from raw DP, CA-DP and fermented DP (Proteggente et al., 2002). For WSE preparation, 1 g of freeze-dried DP was re-suspended in 10 mL of distilled water and sonicated for 15 min at room temperature. The supernatant was collected by centrifugation (3500g for 5 min at 15 °C), the extraction was repeated with 5 mL of distilled water, and the supernatants were combined. MSE were obtained by re-suspending 1g of freeze-dried DP in 10 mL aqueous methanol solution [20:80 (v:v)]. After sonication for 15 min at room temperature, the supernatant was collected by centrifugation (3500g for 5 min at 15 °C). The extraction was repeated with 5 mL of aqueous methanol solution, and the supernatants were combined. For ESE preparation, MSE was obtained as described above, and was evaporated under vacuum at 30 °C. Solids were redissolved with 5 mL of Milli-Q water and acidified to pH 1.5 with hydrochloric acid. The liquid-liquid extraction was carried out with 25 mL of ethyl acetate. The mixture was shaken every 10 min for 30 min. The liquid- extraction was repeated, and the extract was evaporated under vacuum at 30 °C. Solids were redissolved in 15 mL of methanol. 2.10. Antioxidant in vitro assays Free radical scavenging capacity was determined through DPPH and ABTS radical scavenging activity assays, and ferric reducing ability of plasma (FRAP) assay. All analyses were carried out using WSE, MSE or ESE extracts from fermented DP, Raw-DP or CA-DP. DPPH radical scavenging activity was measured by using the stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) as reported by Brand-Williams, Cuvelier, and Berset (1995). Ten lL of extract were added to 3 mL of 40 lM DPPH solution and mixed. Samples were kept under dark condition for 60 min at room temperature and the reaction was monitored by reading the absorbance at 517 nm. ABTS assay is based on the oxidation of the 2,20-azino bis-(3-ethylbenzothiazoline-6-sulphonic acid) di-ammonium salt (ABTS) by potassium persulphate to form a radical cation (ABTS) (Re et al., 1999). The reaction mixture was prepared by mixing 7.0 mM ABTS solution and 4.95 mM potassium persulfate solution in a ratio of 1:2. The mixture was allowed to react for 12 h at room temperature under dark condition. One mL of mixture was diluted with 60 mL of methanol to obtain an absorbance of 0.70 ± 0.02 at

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734 nm. Extract (20 lL) was allowed to react with 2 mL of the reaction mixture for 1 min under dark condition. The reaction was monitored by reading the absorbance at 734 nm. FRAP assay was performed according to (Benzie & Strain, 1996) with some modifications. The reaction mixture was prepared by mixing 200 mL of 300 mM acetate buffer (3.1 g sodium acetate and 16 mL glacial acetic acid, pH 3.6), 20 mL of 10 mM 2,4,6-tripyridyl-s-triazine solution in 40 mM HCl, and 20 mL of 20 mM ferric chloride solution. The mixture was heated in a water bath at 50 °C, and the extract (20 lL) was allowed to react with 2 mL of the reaction mixture for 4 min under dark condition. The reaction was monitored by reading the absorbance at 593 nm. All results are shown as Trolox equivalents.

acquired by full range acquisition covering 100–1000 m/z. For fragmentation study, a data dependent scan was performed by deploying the collision-induced dissociation (CID). Standard were infused into the electrospray ion source at 5 lg mL 1 in MeOH using a syringe pump at a flow rate of 250 lL min 1 to determine the collision energy. External standards were analyzed under the same conditions and used for identification by comparison of elution volume, mass spectrum, and UV absorbance. All chemicals were purchased from Sigma-Aldrich. Tentative identification of compounds was simultaneously performed with an UV and an HESI-triple quadrupole mass spectrometer detector when external standards were not available, and literature data for mass spectra and UV absorbance were used as reference. The detection limit ranged from 0.001 mg L 1 for phenolic acids to 0.005 mg L 1 for quercetin.

2.11. Total phenolic compounds and total flavonoids 2.13. In vitro digestion of date fruit puree (DP) Total phenolic compounds were assayed according to FolinCiocalteu method (Singleton & Rossi, 1965). Analyses were carried out using ESE from raw DP, CA-DP or fermented DP. The reaction mixture contained 200 lL of extract, 200 lL of Folin-Ciocalteu reagent, and 1 mL of distilled water. After 2 min, 1 mL of sodium carbonate solution 10% (w/v) was added. The mixture was shaken at 25 °C for 15 min. The absorbance was measured at 740 nm. Data were expressed as mg gallic acid equivalent (GAE) per 100 g dry weight. Total flavonoids content was measured according to (Zhishen, Mengcheng, & Jianming, 1999). The reaction mixture contained 1 mL aliquot of extract, 4 mL of distilled water, and 300 lL of 5% NaNO2. After 5 min, 0.3 mL of 10% AlCl3 was added. At 6 min, 2 mL of 1 M NaOH was added to the mixture. Immediately, the solution was diluted to a final volume of 10 mL with water and mixed thoroughly. Absorbance of the mixture was determined at 510 nm. Total flavonoids content was expressed as mg quercetin equivalents (QE) per 100 g dry weight. 2.12. Identification and quantification of bioactive compounds in ESE Separation, determination and quantification of bioactive compounds in ESE were performed by using a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Bremen, Germany) equipped with a photodiode array detector (PAD 3000) and a TSQ Quantum Access Max triple quadrupole mass spectrometer (Thermo Fisher Scientific, Basel, Switzerland) with a heated electrospray ionization (HESI) source. A Hypersil gold C18 (100  2.1 mm, 3 lm) column (Thermo Fisher Scientific) was used. Mobile phase consisted of (A) water + 0.1% formic acid and (B) methanol + 0.1% formic acid. A linear gradient program at a flow rate of 0.3 mL min 1 was used: 0.0–5.0 min from 5% to 10% (B), 5.0–15 min from 10% to 40% (B), 15-25 min from 40% to 95% (B), then 95% (B) for 5 min and 30-35 min from 95% to 5% (B). The injection volume was 5 lL and column oven was set at 35 °C. The method described here separated and quantified the compounds in 35 min. PAD analyses of compounds was performed at 280, 310, 320 and 360 nm wavelengths and a scan mode ranging from 220 to 500 nm was used. Mass spectrometer operated in negative ionization mode. HESI-source parameters were set as follows: spray voltage was 4000 V; vaporizer temperature 250 °C; sheath gas pressure 32 arbitrary units (AU), and auxiliary gas pressure was 10 AU; capillary temperature at 320 °C; and skimmer offset was 0 V. Argon was the collision gas used at a pressure of 1.5 mTorr. Collision-induced fragmentation experiments were carried out using argon, and collision energy was set at 30 V. Selected reaction monitoring (SRM) experiment for quantitative analysis was performed using two MS2 fragments for each compound. The Xcalibur software (version 2.1) was used for instrument control, data acquisition and data analysis. The MS spectra were also

The impact of the digested DP (DDP) on human gut microbiota was assayed using pH-controlled mixed faecal batch cultures (Eid et al., 2014). DDP was obtained through an enzymatic in vitro model that simulates the conditions of the physiological digestion (Eid et al., 2014). Freeze-dried raw DP, CA-DP or fermented DP (60 g) were added to 150 mL of distilled water and mixed in a stomacher for 2 min. Then the solution was mixed with aamylase (20 mg) in CaCl2 (1 mM, 6.25 mL) and incubated at 37 °C for 30 min under stirring condition. Pepsin (2.7 g) was dissolved in 25 mL 0.1 M HCl, and the mixed sample was added. The pH was adjusted to 2 using 6 M HCl and incubated at 37 °C for 2 h under stirring condition. Pancreatin (560 mg) and bile (3.5 g) were dissolved in 125 mL of 0.1 M NaHCO3 and the sample was added. pH was adjusted to 7.0 by using 6 M NaOH and incubated at 37 °C for 3 h under stirring condition (50 rpm). Samples were transferred to cellulose dialysis membranes (1 kDa molecular weight) to be dialysed against 0.01 M NaCl at 5 °C to remove low-molecular mass digestion products. After 15 h, the dialysis fluid was changed and dialysis continued for additional 2 h. DDP was freeze-dried and used in in vitro fermentation. All chemicals were purchased from Sigma-Aldrich. 2.14. Human faecal batch-culture fermentation Faecal samples were obtained from three separate individuals. All volunteers were in good health, had not been prescribed antibiotics for at least 6 months before the study and had no history of gastrointestinal disease. Samples were collected on site, on the day of the experiment and used immediately. A 1:10 (w/v) dilution of the samples in anaerobic phosphate buffer (0.1 M; pH 7.4) was prepared and homogenized in a stomacher for 2 min. Resulting faecal slurry was used to inoculate the batch-culture vessels. Batch-culture fermentation vessels (300 mL volume; one vessel per treatment) were sterilized and filled with 135 mL of sterile basal nutrient medium composed by peptone water (2 g L 1), yeast extract (2 g L 1), NaCl (0.1 g L 1), K2HPO4 (0.04 g L 1), KH2PO4 (0.04 g L 1), NaHCO3 (2 g L 1), MgSO4 7H2O (0.01 g L 1), CaCl2
6H2O (0.01 g L 1), Tween 80 (2 mL L 1), haemin (50 mg L 1), vitamin K1 (10 mL L 1), L-cysteine (0.5 g L 1), bile salts (0.5 g L 1), resazurin (1 mg L 1) and distilled water. The medium was gassed overnight with O2-free N2. Prior to inoculation of the faecal slurry, the temperature of the basal nutrient medium was maintained at 37 °C by using a circulating water-bath. The pH of the medium was maintained at 6.8, using both basic (1 M NaOH) and acidic (1 M HCl) solutions, through a pH controller. The batch fermenters were inoculated with 15 mL of faecal slurry (1:10, w/v). Four different experiment were carried out with: (i) ESE obtained from 60 g of freeze-dried raw DP, CA-DP or fermented DP; (ii) freeze-dried DDP obtained from 60 g of freeze-

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dried raw DP, CA-DP or fermented DP; (iii) or fructooligosaccharide (FOS, 1%, w/v raftilose); and (iv) a control vessel without a substrate was also used. These amounts were estimated to reflect levels of intake of about seven to ten pieces of date fruit (Eid et al., 2014). FOS were used as positive control for their wellknown bifidogenic effects (Bouhnik et al., 1999; Eid et al., 2014; Vernazza, Gibson, & Rastall, 2005). The batch fermenters were incubated under anaerobic conditions for 48 h. Batch cultures were carried out in triplicates. Samples (7 mL) were collected at five time points (0, 5, 10, 24 and 48 h) for enumeration of cultivable bacteria and short chain fatty acid analysis. 2.15. Enumeration of cultivable bacteria in faecal batch-culture Faecal batch-culture samples (10 g) were mixed with 90 mL of sterile physiological solution and homogenized. Counts of viable bacterial cell were carried out as described by Macfarlane, Quingley, Hopkins, Newton, and Macfarlane (1998) The following selective media were used: plate count agar (total facultative aerobes and anaerobes); Bifidobacterium agar modified (bifidobacteria) (Becton Dickinson France SA, Le Pont de Claix, France); M17 (lactococci and streptococci); Baird Parker (Staphylococcus); Wilkins-Chalgren anaerobe agar (total anaerobes); WilkinsChalgren anaerobe agar plus GN selective supplements and sheep blood defibrinated (Bacteroides, Porphyromonas and Prevotella genera); ChromoCult (total coliforms) (Merck KGaA, Germany); MacConkey agar No2 (enterobacteria); GSP agar (Merck KGaA) plus penicillin-G (60 g L 1) (Pseudomonas and Aeromonas genera); Slanetz and Bartley (Enterococci); Hoyle medium (Corynebacterium); and MRS agar incubated at 25 °C (lactobacilli and enterococci psychrotrophic) or 42 °C (lactobacilli and enterococci thermophilic). Except for Bifidobacterium agar modified, ChromoCult, and GSP agar, all media were purchased by Oxoid Ltd (Hampshire, England). 2.16. Short chain fatty acid (SCFA) analysis Undiluted aliquots (1.0 mL) were recovered from faecal batch culture and centrifuged (13,000g, 10 min). The supernatant was acidified with 6 M HCl (3:1, v/v) and incubated at room temperature for 10 min. The mixture was re-centrifuged at 13,000g for 5 min, filtered using a 0.2 lm PVDF filter (Millipore, Cork, Ireland), and used to determine SCFA by HPLC (Vernazza et al., 2005). An Äkta purifier system (GE Healthcare) was equipped with an Aminex HPX-87H column (ion exclusion; Bio-Rad), a UV detector operating at 210 nm. Elution was at 35 °C with a flow rate of 0.6 mL min 1, and 10 mM H2SO4 was used as the mobile phase. Calibration was performed using standard solutions of lactic, acetic, propionic, butyric, and succinic acids. 2.17. Statistical analysis Data were subjected to one-way ANOVA; pair-comparison of treatment means was achieved by Tukey’s procedure at P < 0.05, using the statistical software, Statistica for Windows (Statistica7.0 per Windows). Cell densities of cultivable bacteria in faecal batch culture were subjected to permutation analysis using PermutMatrix. 3. Results 3.1. Preliminary morphological, physical-chemical and microbiological characterization of date fruits The main morphological traits and physical-chemical features of date fruits (Phoenix dactylifera L. cultivar Siwi) are shown in

Table 1. Dates were characterized by low total titratable acidity (TTA), non-acidic pH, and high level of soluble solids. Cell density of presumptive mesophilic lactic acid bacteria, yeast, and total mesophilic bacteria was 2.3 ± 0.3, 3.4 ± 0.1, and 2.5 ± 0.2 Log cfu mL 1, respectively. 3.2. Isolation, typing and identification of lactic acid bacteria Forty Gram-positive, catalase-negative, non-motile cocci and rods, mesophilic, and able to acidify MRS broth were identified by partial sequencing of the 16S rRNA. The following species were identified: Leuconostoc mesenteroides (19 isolates), Lactobacillus plantarum (14), and Lactobacillus kunkeei (7). All forty isolates of presumptive lactic acid bacteria were subjected to RAPD-PCR analysis. The reproducibility of the RAPD fingerprints was assessed by comparing the PCR products obtained from three separate cultures of the same strain. At the similarity level of 85%, isolates were grouped into fifteen clusters (data not shown). Fifteen strains were chosen as representative of each cluster and further screened. 3.3. Starter selection Fifteen representative strains were screened based on the kinetics of growth and acidification in date fruit juice. Aiming to optimize the culture conditions, kinetics of growth and acidification were also determined on fruits juice diluted with sterile water to a final concentration of 75, 50, and 30% (v/v) of fruit juice. The latter condition (30%, v/v) allowed the best performances of growth and acidification for all the strains (Table 2). The initial cell density was ca. 8.0 log cfu mL 1. The increase of cell density (A) during 60 h of incubation ranged from 1.10 ± 0.06 to 2.88 ± 0.05 OD620 units. L. plantarum strains reached the highest A (2.48–2.88 OD620 units). The stationary phase of growth was reached after ca. 50 h by L. plantarum strains and after ca. 20–30 h by L. kunkeei and Leuc. mesenteroides strains. Lag phase ranged from 0.67 ± 0.03 (L. plantarum T1.15) to 8.91 ± 0.03 h (Leuc. mesenteroides O2.17). No significant (P > 0.05) growth was found when fruits juice 100% (v/v) was used as model medium, whereas a slight growth was found with fruits juice 75% (0.3 ± 0.04–0.8 ± 0.05 OD620 units) or 50% (v/v) (0.7 ± 0.04– 1.5 ± 0.06 OD620 units). The initial value of pH was 6.21 ± 0.02. After 60 h of fermentation of fruit juice 30%, v/v, the decrease of pH (DpH) ranged from 2.31 ± 0.06 to 2.51 ± 0.04 pH units. Based on the above results, L. plantarum T1.3, T1.15, O1.8, T2.8, and T1.18 were selected as the autochthonous starters to ferment DP 30% (v/v). 3.4. Fermentation of date fruits puree (DP) A protocol for processing of DP was set up, which included: (i) heat treatment (90 °C for 20 min); (ii) fermentation at 30 °C for Table 1 Morphological and physical-chemical parameters of date fruits (Phoenix dactylifera L. cultivar Siwi). Parameters Fruit length (mm) Fruit width (mm) Fruit weight (g) Seed weight (g) % pulp (w/w) pH TTA (mL 0.1 M NaOH/10 g) Soluble solids (°Brix) Water (g/100 g fresh weight) Ash (g/100 g fresh weight) Protein (g/100 g fresh weight) Fat (g/100 g fresh weight) Carbohydrate (g/100 g fresh weight)

33 ± 1 23 ± 0.5 14.5 ± 0.3 1.4 ± 0.1 90.3 ± 0.9 6.21 ± 0.03 0.3 ± 0.1 57.8 ± 4.2 39.9 ± 1.7 1.3 ± 0.1 1.8 ± 0.1 0.2 ± 0.08 52.6 ± 0.8

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Table 2 ParametersA of the growth and acidification kinetics of fifteen representative strains during fermentation of date fruit juice 30% (v/v) incubated aerobically under stirring conditions (200 rpm) at 30 °C for 60 h. Growth

Lactobacillus kunkeei O1.4 L. kunkeei T2.10 L. kunkeei O2.8 Lactobacillus plantarum T1.3 L. plantarum T1.15 L. plantarum O1.8 L. plantarum T2.8 L. plantarum T1.18 Leuconostoc mesenteroides T2.14 Leuc. mesenteroides O2.17 Leuc. mesenteroides O2.19 Leuc. mesenteroides O1.20 Leuc. mesenteroides T2.6 Leuc. mesenteroides T2.20 Leuc. mesenteroides T2.1

Acidification

A

lmax

k

DpH

Vmax

k

1.60 ± 0.05d 1.11 ± 0.04e 1.80 ± 0.03c 2.48 ± 0.07b 2.74 ± 0.08a 2.50 ± 0.04b 2.88 ± 0.09a 2.57 ± 0.04b 1.08 ± 0.06e 1.17 ± 0.09e 1.15 ± 0.07e 1.20 ± 0.08e 1.14 ± 0.07e 1.16 ± 0.07e 1.10 ± 0.06e

0.15 ± 0.01b 0.11 ± 0.03b 0.09 ± 0.01c 0.07 ± 0.01c 0.07 ± 0.02c 0.07 ± 0.03c 0.07 ± 0.02c 0.07 ± 0.01c 0.16 ± 0.02b 0.21 ± 0.01a 0.20 ± 0.02a 0.22 ± 0.02a 0.14 ± 0.03b 0.17 ± 0.03ab 0.14 ± 0.02b

6.64 ± 0.08e 6.45 ± 0.07f 2.98 ± 0.03j 4.07 ± 0.04i 0.67 ± 0.03l 4.39 ± 0.08g 0.79 ± 0.03k 4.16 ± 0.02h 8.56 ± 0.12b 8.91 ± 0.13a 8.81 ± 0.11a 8.95 ± 0.15a 7.92 ± 0.08d 8.34 ± 0.08c 7.95 ± 0.09d

2.35 ± 0.04d 2.42 ± 0.08abcd 2.48 ± 0.05ab 2.36 ± 0.09bcd 2.44 ± 0.04abc 2.51 ± 0.04a 2.46 ± 0.06abc 2.42 ± 0.07abcd 2.42 ± 0.08abcd 2.31 ± 0.06e 2.34 ± 0.07cd 2.36 ± 0.04cd 2.36 ± 0.03d 2.36 ± 0.05cd 2.36 ± 0.05cd

0.33 ± 0.02a 0.34 ± 0.02a 0.33 ± 0.03a 0.35 ± 0.01a 0.35 ± 0.02a 0.35 ± 0.01a 0.35 ± 0.03a 0.36 ± 0.02a 0.32 ± 0.02a 0.32 ± 0.03a 0.32 ± 0.02a 0.32 ± 0.03a 0.32 ± 0.04a 0.32 ± 0.02a 0.32 ± 0.02a

2.47 ± 0.05d 2.43 ± 0.07de 2.80 ± 0.05a 1.82 ± 0.08h 2.25 ± 0.09f 2.62 ± 0.07bc 2.34 ± 0.06ef 2.04 ± 0.04g 2.73 ± 0.05ab 2.55 ± 0.06cd 2.65 ± 0.07bc 2.67 ± 0.08abc 2.64 ± 0.04bc 2.68 ± 0.10abc 2.61 ± 0.07bc

a–l

Means within the column with different letters are significantly different (P < 0.05). Growth kinetics were modelled according to the Gompertz equation as modified by Zwietering et al. (1990): y = k + A exp{ exp[(lmax or Vmax e/A)(k t) + 1]}, where k is the initial level of the dependent variable to be modelled (OD620 or pH units), A is the difference in OD620 or pH units (DpH) between inoculation and the stationary phase, lmax and Vmax are the maximum growth rate (expressed as OD620 units h 1) and the maximum acidification rate (expressed as pH h 1), respectively, k is the length of the lag phase (expressed in hours), and t is the time. A

60 h by selected L. plantarum starters singly inoculated; and (iii) freeze-drying of fermented DP. The selected heat treatment conditions were aimed to reduce the cell density of the endogenous microbiota, as well to preserve the nutrients. Raw DP (Raw–DP) without heat treatment, and DP chemically acidified with lactic acid (CA–DP) incubated at 30 °C for 60 h were used as the controls. After 60 h of fermentation, cell numbers of presumptive lactic acid bacteria of started DP increased from ca. 8.0 Log CFU g 1 to 9.10–9.51 Log cfu g 1. Starters were monitored by RAPD-PCR analysis throughout the process. All strains were detectable after 60 h of fermentation at 30 °C (data not shown). Presumptive lactic acid bacteria of CA–DP reached cell densities of 1.78 ± 0.21 Log cfu g 1. Yeasts were found only in incubated CA–DP (1.50 ± 0.12 Log cfu g 1). Enterobacteria were absent in 10 g of all DP samples. 3.5. Physical-chemical analysis Raw DP had initial values of pH and TTA of 6.22 ± 0.03 and 0.2 ± 0.8 mL 0.1 M NaOH 10 g 1, respectively. After fermentation, the value of pH of started DP decreased to 3.7 ± 0.2–3.8 ± 0.1. TTA significantly (P < 0.05) increased for started DP to 3.8 ± 0.3– 4.1 ± 0.4 mL 0.1 M NaOH 10 g 1. CA–DP had pH and TTA of 3.62 ± 0.01 and 4.9 ± 0.4 mL 0.1 M NaOH 10 g 1, respectively. Values of soluble solids slightly (P > 0.05) decreased during processing of started DP (data not shown). Compared to Raw-DP, the level of soluble dietary fibers only slightly decreased in fermented DP, whereas completely disappeared in CA–DP (Fig. 1). The level of insoluble dietary fibers significantly (P < 0.05) increased (ca. 16%) in all fermented DP, whereas no significant (P > 0.05) change was found in CA–DP (Fig. 1). 3.6. Analysis of carbohydrates, organic acids, and free amino acids Glucose and fructose were the predominant carbohydrates of Raw-DP (112 ± 7 and 115 ± 6 g kg 1, dry weight). During fermentation, started DP showed a slight (P > 0.05) decrease of glucose and fructose (ca. 9%). Main microbial metabolite was lactic acid (19.1 ± 0.2–21.7 ± 0.2 g kg 1 dry weight). Before processing, the concentration of total free amino acids (FAA) of Raw-DP was 2.52 ± 0.01 g kg 1 dry weight, where Asp (508 ± 16 mg kg 1 dry weight), Glu (137 ± 12 mg kg 1 dry weight), Tyr (287 ± 3 mg kg 1

Fig. 1. Level (% dry weight) of soluble (blue bars) and insoluble (green bars) dietary fibers in freeze-dried Raw-DP, CA-DP or DP fermented at 30 °C for 60 h by the selected autochthonous Lactobacillus plantarum T1.3, T1.15, O1.8, T2.8, and T1.18 strains. Bars with different superscript letters differ significantly (P < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

dry weight), c-amino butyric acid (GABA) (848 ± 4 mg kg 1 dry weight), and Pro (428 ± 11 mg kg 1 dry weight) were the FAA found at the highest concentration. Total FAA almost remained unchanged in fermented DP (2.52 ± 0.01–2.55 ± 0.02 g kg 1 dry weight), but a significant (P < 0.05) increase of GABA was found (ca. 7%), consistently with a decrease of Glu (ca. 27–42%). In CA– DP, the concentration of total FAA increased to 3.2 ± 0.02 g kg 1 dry weight, but only a slight increase of c-amino butyric acid was found (ca. 2%). 3.7. Antioxidant activity Antioxidant capacities of plant extracts not only depend on extract composition but also on the test used (Dudonné, Vitrac, Coutiere, Woillez, & Mérillon, 2009; Roginsky & Lissi, 2005). More than one type of antioxidant capacity measurement needs to be performed to take into account the various modes of action of antioxidants and to obtain a good reliability. In this study, we

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determined the free radical scavenging capacities of DP extracts using DPPH, ABTS, and FRAP assay. The ABTS radical scavenging capacity assay is particularly suitable for plant extracts because the wavelength absorption at 734 nm remove the color interference (Li, Wong, Cheng, & Chen, 2008). Type of solvent markedly affects the extraction of bioactive compounds. Therefore, the analysis was carried out using WSE, MSE, or ESE extracts from freezedried Raw-DP, CA-DP or fermented DP (Table 3). Apart from the sample, the highest Trolox equivalent antioxidant capacity was found for WSE and MSE, where DPPH and FRAP assays revealed the highest values. Regardless of the assay and the extract, fermented DP and CA–DP induced the highest (P < 0.05) antioxidant activity compared to Raw-DP. WSE from fermented DP showed the highest (P < 0.05) values (3.02 ± 0.02–3.23 ± 0.01 mmol Trolox eq/100 g dry weight) with respect to CA–DP and Raw-DP. A remarkable increase (P < 0.05) of the antioxidant activity was also found in ESE with the ABTS assay compared to CA–DP and Raw-DP. The increase was particularly evident with DP fermented by L. plantarum T1.3 (0.35 ± 0.01) and T2.8 (0.35 ± 0.02) (Table 3).

3.8. Bioactive compounds in DP Total phenolic compounds and total flavonoids analysis was carried out using ESE. Raw-DP had initial levels of total phenolics and flavonoids of 11 ± 3 mg gallic acid eq. 100 g 1 dry weight and 19 ± 3 mg quercetin eq. 100 g 1 dry weight, respectively. Compared to Raw-DP, fermented DP and CA–DP showed higher (P < 0.05) amount of total phenolics (24 ± 3–31 ± 5 mg gallic acid eq. 100 g 1 dry weight) and flavonoids (40 ± 7–53 ± 6 mg quercetin eq. 100 g 1 dry weight). Only slight differences (P > 0.05) between fermented DP and CA–DP were found. Separation of bioactive compounds in ESE was carried out through UHPLC-PDA-HESI-MS/MS. The highest peaks were identified. External standards were analyzed under the same conditions and used for identification by comparison of retention time, mass spectrum, and UV absorbance. Literature data for mass spectra and UV absorbance were used when external standards were not available. Free phenolic acids were detected at 280 nm. p-Coumaric (m/z 163) and ferulic (m/z 193) acids were the most abundant phenolic acids (2.0 ± 0.1 and 5.1 ± 0.2 mg 100 g 1 dry weight, respectively) found in Raw-DP. Low amounts of protocatechuic (m/z 153), vanillic (m/z 167), and isoferulic (m/z 193) acids were also found (0.10 ± 0.02, 0.56 ± 0.08, and 0.71 ± 0.05 mg 100 g 1 dry weight, respectively) (Fig. 2 and Table S1). Only in fermented DP, p-coumaric and ferulic acids were almost completely metabolized, whereas a strong increase (P < 0.05) was found for protocatechuic acid (2.5 ± 0.1–4.1 ± 0.2 mg 100 g 1 dry weight), and to a lesser extent (P < 0.05) for p-hydroxy benzoic acid (m/z 137) (0.50 ± 0.01–0.70 ± 0.02 mg 100 g 1 dry weight) (Fig. 2). Free flavonoids were detected at 320 nm. In raw DP, naringenin methyl

ether (m/z 285) and proanthocyanidins-1 (m/z 577) were the most abundant (1.8 ± 0.1 and 1.8 ± 0.2 mg 100 g 1 dry weight, respectively), and low amounts of naringenin (m/z 579), isoquercetin (m/z 463), and proanthocyanidins-2 (m/z 577) were also found (0.75 ± 0.08, 0.35 ± 0.03, and 0.14 ± 0.02 mg 100 g 1 dry weight, respectively). In fermented DP, naringenin, naringenin methyl ether, proanthocyanidins-1, and proanthocyanidins-2 decreased (ca. 50%) (P < 0.05), whereas rutin (m/z 609) significantly (P < 0.05) increased (0.10 ± 0.02–0.09 ± 0.01 mg 100 g 1 dry weight). In CA–DP rutin was also detected (0.05 ± 0.01 mg 100 g 1 dry weight), and a slight decrease of naringenin, naringenin methyl ether, and proanthocyanidins-1 (ca. 25%) was observed. Fatty acid profiles were also investigated. Only in fermented DP, a remarkable synthesis (P < 0.05) of conjugated linolenic (m/z 277) and linoleic (m/z 279) acids was found (Fig. 2 and Table S1). 3.9. Enumeration of cultivable bacteria in faecal batch-culture Aiming to investigate the effects of ESE and digested extracts from DP (DDP) on human faecal microbiota, relatively selective media were used to enumerate cultivable cells of the main microbial groups. ESE and DDP were obtained from raw-DP, CA-DP, or DP fermented at 30 °C for 60 h by L. plantarum T1.3 and T2.8. These latter two strains were chosen for binary fermentation of DP as they showed the highest antioxidant activity in ESE. Enumeration of cultivable bacteria was carried out at 0, 5, 10, 24 and 48 h of incubation. Highest cell densities were found after 48 h of incubation (Fig. 3). Results showed that both extracts (ESE and DDP) from DP were capable to modulate significantly (P < 0.05) and in a different way the growth of specific bacterial taxa. ESE from Raw-DP significantly (P < 0.05) enhanced the growth of presumptive total coliform, enterobacteria, Pseudomonas, Aeromonas, and Corynebacterium genera. A weaker impact (P < 0.05) on presumptive bifidobacteria, lactobacilli, enterococci, and Bacteroides counts was found. On the contrary, ESE from fermented DP and DP–CA has been shown to reduce bacterial counts. Though digested extracts from Raw-DP exhibited a bifidogenic effect, it was significantly (P < 0.05) smaller than FOS used as positive control. Furthermore, it showed the highest (P < 0.05) cell density of presumptive total coliform. Compared to Raw–DP and CA–DP, the effect on bacterial composition change of the digested extracts from fermented DP was much closer to FOS. The highest (P < 0.05) cell density of lactobacilli and enterococci was found. 3.10. Short chain fatty acid (SCFA) analysis HPLC analysis was used to identify and quantify the main SCFA in faecal batch culture. Analysis was carried out at 0, 5, 10, 24 and 48 h of incubation. The highest amounts were found after 48 h of

Table 3 DPPH and ABTS radical scavenging activities, and ferric reducing ability of plasma (FRAP) of water-soluble (WSE), methanol-soluble (MSE), or ethyl acetate-soluble (ESE) extracts from freeze-dried raw dates puree (raw DP), chemically acidified DP (CA-DP) or DP fermented at 30 °C for 60 h by the selected autochthonous Lactobacillus plantarum T1.3, T1.15, O1.8, T2.8, and T1.18 strains. Results are shown as Trolox equivalents. Sample

Raw-DP CA-PD Lactobacillus L. plantarum L. plantarum L. plantarum L. plantarum a–f

plantarum T1.3 T1.15 O1.8 T2.8 T1.18

DPPH radical scavenging activity (mmol Trolox eq/100 g dry weight)

ABTS radical scavenging activity (mmol Trolox eq/100 g dry weight)

WSE

MSE

ESE

WSE

MSE

ESE

WSE

MSE

ESE

1.82 ± 0.01e 2.13 ± 0.02d 3.05 ± 0.01c 3.16 ± 0.03b 3.14 ± 0.02b 3.02 ± 0.02c 3.23 ± 0.01a

0.87 ± 0.01f 3.18 ± 0.02c 3.11 ± 0.04d 3.11 ± 0.03d 2.68 ± 0.02e 3.40 ± 0.04b 3.56 ± 0.01a

0.37 ± 0.02f 0.95 ± 0.01d 1.52 ± 0.02a 0.97 ± 0.03 cd 0.92 ± 0.01e 1.30 ± 0.03b 0.98 ± 0.01c

0.40 ± 0.02c 0.42 ± 0.03c 0.51 ± 0.01a 0.50 ± 0.03ab 0.51 ± 0.01a 0.47 ± 0.01b 0.50 ± 0.02ab

0.34 ± 0.01c 0.66 ± 0.02a 0.57 ± 0.01b 0.54 ± 0.02b 0.55 ± 0.02b 0.65 ± 0.01a 0.67 ± 0.01a

0.09 ± 0.01d 0.20 ± 0.01c 0.35 ± 0.01a 0.25 ± 0.03b 0.24 ± 0.01b 0.35 ± 0.01a 0.20 ± 0.01c

0.75 ± 0.02e 0.82 ± 0.02d 1.05 ± 0.03c 1.11 ± 0.01b 1.14 ± 0.01a 1.08 ± 0.01c 1.14 ± 0.04ab

0.69 ± 0.01d 1.25 ± 0.01b 1.18 ± 0.04c 1.31 ± 0.02a 1.23 ± 0.04b 1.24 ± 0.03b 1.31 ± 0.02a

0.22 ± 0.03d 0.64 ± 0.02bc 0.73 ± 0.04a 0.61 ± 0.01c 0.66 ± 0.04ab 0.70 ± 0.01a 0.69 ± 0.02a

Means within the column with different letters are significantly different (P < 0.05).

Ferric reducing ability of plasma (mmol Trolox eq/100 g dry weight)

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Fig. 2. Separation by UHPLC-PDA-HESI-MS/MS (280 nm) of bioactive compounds in ethyl acetate-soluble extracts (ESE) from freeze-dried raw dates puree (Raw-DP) (red profile), chemically acidified DP (CA-DP) (blue profile), or DP fermented at 30 °C for 60 h by Lactobacillus plantarum T1.3 (green profile). L. plantarum T1.3 was chosen as representative of selected autochthonous strains. 1. Protocatechuic acid; 2. p-hydroxy benzoic acid; 3. vanillic acid; 4. p-coumaric acid; 5. ferulic acid; 6. isoferulic acid; 7. conjugated linolenic acid; 8. conjugated linoleic acid. Data are representative of three biological replicates analyzed in triplicate. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. Pseudo-heatmaps showing the cell density (Log cfu mL 1) of cultivable bacteria after 48 h of incubation in batch culture supplemented with ethyl acetate-soluble extracts (ESE) (A), and digested extract (DDP) (B) from freeze-dried raw dates puree (Raw-DP), chemically acidified DP (CA-DP), or DP fermented at 30 °C for 60 h by Lactobacillus plantarum T1.3 and T2.8 (f-DP). A batch culture supplemented with fructo-oligosaccharide (FOS) was used as positive control.

incubation (Fig. 4). ESE, and especially digested extract from DP (DDP) were capable to modulate significantly (P < 0.05) SCFA levels. With ESE, only acetic and butyric acids were detected, whereas increases in lactic, succinic, acetic, propionic, and butyric acids were found after 48 h of incubation with DDP. The highest levels (P < 0.05) of lactic (8.1 ± 1.3 mM), succinic (7.9 ± 1.3 mM), acetic (5.7 ± 0.9 mM), and propionic (4.1 ± 0.7 mM) acids were found with DDP from fermented DP. FOS induced only the synthesis of succinic (2.2 ± 0.4 mM) and butyric (2.4 ± 0.2 mM) acids. 4. Discussion The high concentration of biogenic compounds and the intrinsic nutritional features of date fruit (Phoenix dactylifera L.) make it a

suitable raw matrix for manufacturing of functional ingredient, dietary supplement or pharmaceutical preparation. To our knowledge, this is the first study reporting the use of autochthonous lactic acid bacteria for manufacturing of freeze-dried powder from fermented date fruits puree as a dietary supplement with healthpromoting features. Lactic acid bacteria, intended as microbial cell factories, have been shown to increase the functionality of many plant matrices through their enzyme portfolio, which in turn promotes the synthesis of various metabolites and/or the release of biogenic compounds, which are mainly cryptic in the raw matrix (Di Cagno et al., 2013). First, autochthonous lactic acid bacteria were isolated from dates. A limited biodiversity of lactic acid bacteria was found. Lactobacillus plantarum, Leuconostoc mesenteroides, and Lactobacillus

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Fig. 4. Concentrations (mM) of main organic acids after 48 h of incubation in faecal batch culture supplemented with ethyl acetate-soluble extracts (ESE) (A) and digested extract (DDP) (B) from freeze-dried raw dates puree (Raw-DP), chemically acidified DP (CA-DP), or DP fermented at 30 °C for 60 h by Lactobacillus plantarum T1.3 and T2.8 (f-DP). A batch culture supplemented with fructo-oligosaccharide (FOS) was used as positive control. Bars with different superscript letters differ significantly (P < 0.05).

kunkeei were the three species identified. L. plantarum and Leuc. mesenteroides are usually found in the most common fruits (Di Cagno et al., 2013), whereas L. kunkeei are commonly isolated from fructose-rich fruits and flowers (Endo, Futagawa-Endo, & Dicks, 2009). Leuc. mesenteroides has been previously isolated from palm date sap (Manel, Nedia, Moktar, & Ali, 2011). In this study, five autochthonous L. plantarum strains were selected to ferment date puree (DP) since they were the best performing strains in terms of growth and acidification. Current data have been largely shown that L. plantarum well adapts to plant matrices fermentation (Filannino, Cardinali, et al., 2014; Filannino, Di Cagno, et al., 2016). An innovative biotechnological protocol was developed, which included a heat treatment (90 °C for 20 min), and the fermentation of DP (30%, v/v) at 30 °C for 60 h by selected L. plantarum starters. DP was diluted to a final concentration of 30% (v/v) based on preliminary screening; the dilution rate did not induce osmotic stress to the bacterial cells, and at the same time ensured a sufficient level of fermentable carbohydrates to allow the bacterial growth (Filannino, Cardinali, et al., 2014). Fermented DP was freezedried, and its functional features were compared to those of the raw DP (Raw–DP) and the chemically acidified DP (CA–DP), both used as controls. Date fruits puree is a good source of amino acids (2.52 ± 0.01 g kg 1 dry weight). Lactic acid fermentation of DP with selected strains allowed the highest concentration of c-amino butyric acid (GABA) (900–910 mg kg 1 dry weight), a non-protein amino acid with several physiological functions (e.g., induction of hypotension, diuretic and tranquilizer effect) (Oh & Oh, 2003; Wong, Bottiglieri, & Snead, 2003). The daily intake of 15 g of freeze-dried fermented DP guarantees an amount of GABA above the physiological threshold (ca. 10 mg/day), hypothesizing an in vivo human health benefit (Inoue et al., 2003). Compared to the two controls, fermented DP showed a significant increase (ca. 16%) of the level of insoluble dietary fibers. The increase ofi nsoluble dietary fibers was previously found during cooking and dehydration processes of legume flours (Aguilera, Benítez, Mollá, Esteban, & Martín-Cabrejas, 2011; Chang & Morris, 1990; VidalValverde & Frias, 1991). Probably, the process conditions of our protocol affected the dietary fibers level, although a role of lactic acid bacteria should not be neglected. Dietary fibre is frequently included in the formulation of functional foods/ingredients, due to its beneficial human health effects, which may profile microbial diversity and drive a cascade of dominating specialized micro-

biome pathways. Date dietary fibers, especially the insoluble fraction (e.g., cellulose, hemicelluloses, pectin, and lignin), are capable of accelerating the gastro-intestinal (GI) tract activity reducing the risk of constipation, as well may have also a preventive effect against some diseases (e.g., the digestive system cancers) (Kritchevsky, 1986; Nicklas, Farris, Myers, & Berenson, 1995; Souli et al., 2014). As usual in herbal medicine, the solvent extraction of bioactive compounds from permeable solid plant materials is a key step to obtain phytochemical-rich products. Antioxidant activity of WSE, MSE, and ESE extracts from fermented DP was investigated through DPPH and ABTS radical scavenging activity assays, and ferric reducing ability of plasma (FRAP) assay. Antioxidant activity of DP was positively affected by lactic acid fermentation. The highest Trolox equivalent antioxidant capacity was found for WSE from fermented DP compared to CA–DP and Raw-DP. The preservative effect of fermentation on vitamin C and water-soluble pigments levels certainly positively contributed to the activity of WSE (Di Cagno et al., 2016; Filannino, Cavoski, et al., 2016). Furthermore, a positive contribution by water-soluble peptides cannot be excluded (Filannino, Cavoski, et al., 2016; Rizzello et al., 2013). A remarkable increase of the antioxidant activity was also found in ESE from fermented DP, suggesting a role of ethyl acetate extractable compounds, like phenolics. However, phenolics show variable solubility in water and organic solvents, and might positively affect also the activity of WSE. Since the phenolic compounds in foods have attracted interest because of their health benefits, the level of various phenolics in ESE was investigated (Valdés et al., 2015). Fermentation affected the content and the profile of DP phenolic acids and flavonoids. During fermentation of DP, p-coumaric and ferulic acids were almost completely metabolized, whereas protocatechuic, p-hydroxy benzoic acid, and rutin increased. L. plantarum has been shown to metabolize p-coumaric and ferulic acids to the corresponding reduced or vinyl derivatives, which may exert higher biological activities than their precursors (Filannino, Bai, Di Cagno, Gobbetti, & Gänzle, 2015; Filannino, Gobbetti, De Angelis, & Di Cagno, 2014; Huang, de Paulis, & May, 2004; Nenadis, Zhang, & Tsimidou, 2003; Silva et al., 2000; Sánchez-Maldonado, Schieber, & Gänzle, 2011). Furthermore, esterase and glucosidase activities of selected L. plantarum strains lead to the release of phenolic acids bound to insoluble cell wall material, especially protocatechuic and p-hydroxybenzoic acids.

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Higher concentration of rutin may arise from the increased accessibility of non extractable phenolics (Bordenave, Hamaker, & Ferruzzi, 2014; Pérez-Jiménez, Díaz-Rubio, & Saura-Calixto, 2013), since the fermentation leads to the degradation of the molecules to which they are associated (carbohydrates or proteins) (Pérez-Jiménez et al., 2013). The slight changes found in CA-DP suggested an acid hydrolysis or the action of plant esterases (Svensson, Sekwati-Monang, Lutz, Schieber, & Gänzle, 2010). Protocatechuic acid has been demonstrated to exert strong antioxidant and antitumor effects, and induced apoptosis in HL-60 human leukemia cells (Tseng, Wang, Kao, & Chu, 1996; Tseng et al., 1998, 2000). Overall, food phenolics are not necessarily the most active within the human body, either because they have a low intrinsic activity or because they are poorly absorbed from the intestine, since are present in the unavailable form (e.g., esters, glycosides, or polymers). When fermentation occurs, the efficiency of absorption may be increased (Hole et al., 2012; Manach, Scalbert, Morand, Rémésy, & Jiménez, 2004; Pérez-Jiménez et al., 2013). Consequently, under the experimental conditions of this study, due to the bacterial bioconversion, fermented DP was enriched in phenolic derivatives with potentially high human bioavailability. Fermented DP resulted also enriched in conjugated linolenic and linoleic acids. Conjugated fatty acids have potential health or nutritional effects, including anticarcenogenic and antiatherogenic properties (Ha, Storkson, & Pariza, 1990; Lee, Kritchevsky, & Pariza, 1994; Pariza, Park, Cook, Albrigh, & Liu, 1996). The ability to reduce the catabolic effects of immune stimulation and to reduce body fat was also experimentally proved (Cook, Miller, Park, & Pariza, 1993; Pariza et al., 1996). The impact of ethyl acetate-soluble extracts (ESE) and digested extracts from DP (DDP) on gut microbiota and short chain fatty acids synthesis was also assayed using pH-controlled mixed faecal batch cultures. ESE mainly contained ethyl acetate-soluble phenolics. Phenolic compounds are subject to extensive biodegradation by the gut microbiota, although the effect of this metabolism on the growth of the microbiota is not completely understood (Del Rio et al., 2013; Manach et al., 2004). DDP contained all the components of the whole fruit subjected to enzymatic in vitro digestion that simulates the conditions of the physiological digestion. Bacterial growth was inhibited under treatment with ESE, whereas it was enhanced when exposed to DDP from fermented DP. The decreased bacterial growth with ESE was probably due to the use of pure phenolic extracts. For instance, increased level of rutin in fermented DP may enhance the antibacterial activities of other flavonoids (Arima, Ashida, & Danno, 2002). The presence of fibers in the DDP from fermented DP probably interfered with the selective bacterial growth, promoting lactobacilli and enterococci growth and decreasing total coliforms. Thus fermented DP was capable to modulate positively the growth of specific bacterial taxa, similarly to the effect of prebiotics such as FOS. Such changes may play a beneficial role in enhancing colon health and affecting the short chain fatty acids synthesis. Instead, CA-DP only inhibited bacterial growth, and Raw-DP only enhanced the growth of potentially pathogens bacterial group. HPLC analysis was used to identify and quantify the main short chain fatty acids produced in faecal batch cultures. Fermented DP positively affected the levels of lactic, succinic, acetic, and propionic acids. Synthesis of SCFA in the human colon is a highly dynamic process that follows complex enzymatic pathways and involves an extensive number of bacterial species belonging to different phyla (e.g., Actinobacteria, Firmicutes, Bacteroidetes) (Fernández et al., 2016). Short chain fatty acids exert energetic effects for eukaryotic cells, contribute to the normal homeostasis of colonocytes, act as strong antitumour compound for tumour colonocytes, and exert anti-inflammatory activity (Fernández et al., 2016).

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