Raw material and chemicals
The stones from fully ripened apricot (Prunus armeniaca L.) fruits were removed and individually broken to obtain the intact kernels. The press cake residues (pomaces—composed of fruit skins and pulp) were obtained in our laboratory from de-stoned of yellow apricot fruits collected in July 2016. The pomace and kernels were dried in oven (37 °C) until complete drying, ground and stored in refrigerator before use.
Folin-Ciocalteu’s phenol reagent, sodium carbonate (Na2CO3), sodium nitrite (NaNO2), ammonium nitrate (NH4NO3), hydrochloric acid (HCl), aluminum chloride (AlCl3), sodium hydroxide (NaOH), salts for nutrient solution, glucose, acetic acid, acetonitrile, methanol, phenolic standards, DPPH (1,1-diphenyl-2 picrylhydrazyl) were purchased from Sigma-Aldrich (Steinheim, Germany). The FAMEs (fatty acid methyl esters) standard (37 component FAME Mix, SUPELCO) was purchased from Supelco (Bellefonte, PA, USA). All chemicals and reagents used in this study were of analytical grade.
Culture medium and fermentation conditions
Culture medium
Aspergillus niger (ATCC-6275) and Rhizopus oligosporus (ATCC-22959) (LGC Standards GmbH, Wesel Germany) were selected as suitable fungi for SSF and were maintained on potato dextrose agar (PDA) slants and Petri plates at 4 °C [17]. The fungal spores were collected from the sporulation medium plates, inoculated into sterile distilled water, and stored in the freezer.
Solid-state fermentation
500 mL Erlenmeyer flasks containing 15 g solid substrates, 30 mL of a nutrient solution NaNO3 (4 g/L), K2HPO4 (2 g/L), MgSO4 (0.25 g/L), glucose (10 g/L) and NH4NO3 (1 g/L), were used for SSF. The fermentation mediums were autoclaved at 121 °C for 30 min and inoculated with spore suspension (2 × 107 spores/g of solid). After being thoroughly mixed, the fermentations were conducted for 14 days at 30 °C. The experiments were performed in triplicate. During SSF, 1 g of samples of the media were taken at different time points for analysis [16, 17].
Extraction and analysis of phenolic compounds
The apricot pomace samples (2 g) were individually extracted three times with 20 mL of extraction mixture (hydrochloric acid/methanol/water in the ratio of 1:80:19) at 40 °C for 30 min in an ultrasonic bath [16]. The resulting dried extracts were dissolved in methanol and stored (4 °C) until analysis (total and individual phenolics, total flavonoids and antioxidant activities).
Total phenolics
The total phenolic amounts were determined by the Folin–Ciocalteu method [26], using a Synergy HT Multi-Detection Microplate Reader with 96-well plates (BioTek Instruments, Inc., Winooski, VT, USA). An aliquot (25 μL) of each extract was mixed with 125 μL of Folin–Ciocalteu reagent (0.2 N) and 100 μL of 7.5% (w/v) Na2CO3 solution [16]. The absorbance against a methanol blank was recorded at 760 nm. A standard curve was prepared using gallic acid and the TP content in the extract was expressed as gallic acid equivalents (GAE) in mg/100 g fresh weight (FW) of waste.
Total flavonoids
The total flavonoid amounts were measured according to the aluminium chloride colorimetric method developed by Zhishen et al. [26] using quercetin as reference standard, as described by Dulf et al. [17]. The absorbance was measured at 510 nm. Total flavonoid content was expressed as mg quercetin equivalent (mg QE/100 g FW).
Analysis of individual phenolic compounds by HPLC–DAD-ESIMS (high-performance liquid chromatography-diode array detection-electro-spray ionization mass spectrometry)
The phenolic extracts were analyzed using an Agilent 1200 HPLC with DAD detector, coupled with MS detector single quadrupole Agilent 6110. The separations of phenolic compounds were performed at 25 °C on an Eclipse column, XDB C18 (4.6 × 150 mm, 5 μm) (Agilent Technologies, USA). The binary gradient consisted of 0.1% acetic acid/acetonitrile (99:1) in distilled water (v/v) (solvent A) and 0.1% acetic acid in acetonitrile (v/v) (solvent B) at a flow rate of 0.5 mL/min, following the elution program used by Dulf et al. [16]: 0–2 min (5% B), 2–18 min (5–40% B), 18–20 min (40–90% B), 20–24 min (90% B), 24–25 min (90–5% B), 25–30 min (5% B).
The phenolics were identified by comparing the retention times, UV- visible and mass spectra of unknown peaks with the reference standards. For MS fragmentation, the ESI(+) module was applied, with scanning range between 100 and 1000 m/z, capillary voltage 3000 V, at 350 °C and nitrogen flow of 8 L/min. The eluent was monitored by DAD, and the absorbance spectra (200–600 nm) were collected continuously in the course of each run. The flavonols were detected at 340 nm [17]. Data analysis was performed using Agilent ChemStation Software (Rev B.04.02 SP1, Palo Alto, California, USA). The chlorogenic and neochlorogenic acids were expressed in mg chlorogenic acid/100 g FW of substrate and flavonol glycosides were calculated as equivalents of rutin (mg rutin/100 g FW of substrate).
DPPH free radical scavenging assay
The antioxidant activity of the obtained phenolic extracts were determined by DPPH radical scavenging assay, using the method described by Dulf et al. [17]. The percentage inhibition (I%) was calculated as [1 − (test sample absorbance/blank sample absorbance)] × 100.
Oil extraction and fatty acid analysis
The non- and fermented (after 2, 6 and 9 days of SSF) apricot kernels (5 g) were extracted with 60 mL solution of chloroform: methanol (2:1, v/v) [17]. The oil contents were determined gravimetrically. An aliquot (10–15 mg) of each lipid extract was transesterified into FAMEs using the acid-catalyzed method [9] and analyzed by gas chromatography–mass spectrometry (GC–MS) using a previously described protocol [17]. A GC–MS (PerkinElmer Clarus 600 T GC–MS (PerkinElmer, Inc., Shelton, CT, USA)) equipped with a Supelcowax 10 capillary column was used (60 m × 0.25 mm i.d., 0.25 μm film thickness; Supelco Inc., Bellefonte, PA, USA). The column temperature was programmed from 140 to 220 °C at a rate of 7 °C/min and held for 23 min. Helium was used as carrier gas at a constant flow rate of 0.8 mL/min. The mass spectra were recorded in EI (positive ion electron impact) mode. The mass scans were performed from m/z 22 to 395. Identification of fatty acids was carried out by comparing their retention times with those of known standards and the generated mass spectral data with those of the NIST library (NIST MS Search 2.0).
Quantification of the fatty acids was achieved by the comparison of peak areas with internal standard (nonadecanoic acid, Sigma, Steinheim, Germany) which was added to the samples (200 μg) prior to methylation, without application of any correction factor. Fatty acid compositions of oils in apricot kernels were expressed as weight percentages of the total fatty acids.
Statistical analysis
All tests were conducted in triplicate and the results were presented as mean ± standard deviation (SD). Correlations among the antioxidant activity and phenolics were calculated using Pearson’s correlation. Statistical analyses were performed by Student’s t-test and ANOVA (repeated measures ANOVA; Tukey’s Multiple Comparison Test; GraphPad Prism Version 5.0, Graph Pad Software Inc., San Diego, CA). Differences between means at the 5% level were considered statistically significant.