Phenolic compounds, flavonoids, lipids and antioxidant potential of apricot (Prunus armeniaca L.) pomace fermented by two filamentous fungal strains in solid state system

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

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.

Total phenolic and flavonoid contents. HPLC–MS analysis of individual phenolic compounds

The total phenolic amounts determined by Folin-Ciocalteu procedure showed a similar increasing trend over the first 6 days of solid-state fermentation for both filamentous fungal strains. This trend has continued only for fermentation with R. oligosporus until day 9, after that the total soluble phenolics sharply decreased for the remaining days of SSF (Fig. 1).

The increase in total phenolic content was higher when R. oligosporus was used for fermentation (78%-day 9), compared to A. niger (34%-day 6). These increases of measurable free phenolics contents could be attributed to the fungal-derived β-glucosidases which can hydrolyze β-glucosidic bonds, mobilizing the free phenolic compounds to react with the Folin–Ciocalteau reagent [14]. Similar tendencies in phenolic contents were also observed in our previous studies [16, 17]. The free phenolics amounts showed significant decrease in the second part of fermentations (Fig. 1) which could be due to the polymerization and lignification of the released free phenolics by lignifying and tannin forming peroxidases, activated in response to the stress induced on the microorganism due to the nutrient deficiencies [18].

The total flavonoid contents of solid-state processed apricot by-products showed similar trends as total phenolic amounts (Fig. 2).

In the first 6 days of fermentation with A. niger, and after 9 days of SSF by R. oligosporus, significant increases were observed in flavonoid contents until the maximum yields of 29 mg QE/100 g pomace, FW-by A. niger and 36 mg QE/100 g pomace, FW-by R. oligosporus, respectively (from the initial value of 26 mg QE/100 g FW). An important increase in the levels of total flavonoids was also observed by Lin et al. [19] in Aspergillus-fermented litchi pericarp. According to Ruiz et al. [4], the total polyphenol and flavonoid contents in apricot strongly depend on varieties.

The quantities of the main phenolics in the extracts of apricot pomaces were determined during solid-state fermentations (Table 1), using HPLC–DAD-MS. All samples contained four dominant phenolics: two cinnamic acids (3-caffeoylquinic and 5-caffeoylquinic acids) and two flavonols (quercetin-3-rutinoside and quercetin-3(6″acetyl-glucoside)) (Fig. 3).

Phenolics ([M + H] + ion, fragments	Cinnamic acids		Flavonols
	3-CQA (355, 181)	5-CQA (355, 181)	Q-3-rut (611, 303)	Q-3-ac-gluc* (517,303)
Aspergillus niger
FD
0	7.81 ± 0.31a	15.14 ± 0.61a	16.50 ± 0.66a	5.71 ± 0.23a
2	5.23 ± 0.21b	12.17 ± 0.49c	15.65 ± 0.63b	4.81 ± 0.19b
6	4.93 ± 0.20c	12.65 ± 0.51b	15.61 ± 0.62b	4.26 ± 0.17c
9	4.58 ± 0.18d	11.78 ± 0.47d	14.50 ± 0.58c	4.15 ± 0.17d
14	4.23 ± 0.17e	11.26 ± 0.45e	14.13 ± 0.57d	4.07 ± 0.16e
Rhizopus oligosporus
0	7.81 ± 0.31a	15.14 ± 0.61a	16.50 ± 0.66b	5.71 ± 0.23a
2	5.06 ± 0.20e	10.21 ± 0.41e	15.69 ± 0.63d	4.70 ± 0.19d
6	5.76 ± 0.23d	11.74 ± 0.47d	15.98 ± 0.64c	4.89 ± 0.20c
9	6.59 ± 0.26b	13.48 ± 0.54b	18.17 ± 0.73a	5.15 ± 0.21b
14	5.98 ± 0.24c	12.39 ± 0.50c	16.58 ± 0.66b	4.59 ± 0.18e

Values (mean ± SD, n = 3) in the same column with different letters (a–e) significantly differ (p < 0.05) (ANOVA “Tukey’s Multiple Comparison Test”). FD fermentation day, 3-CQA 3-caffeoylquinic acid (neochlorogenic acid), 5-CQA 5-caffeoylquinic acid (chlorogenic acid), Q-3-rut quercetin-3-rutinoside (rutin), Q-3-ac-gluc Quercetin-3(6″acetyl-glucoside) * Tentative identification

These phenolic profiles were in general agreement with the study of Ruiz et al. [4] on phenolic composition in peels of different apricot varieties. Chlorogenic acid and rutin were the major phenolics in both processed pomaces (Table 1). Overall, the SSF with both fungal strains had significant effect (p < 0.05) on evolution of phenolic amounts. In general, a decrease of the individual phenolic concentrations in all samples (excepting quercetin-3-rutinoside, as main flavonol in fermented samples with R. oligosporus) was observed (Table 1).

The usefulness of by-products from the beverage industry is still underestimated. To our knowledge this is the first study investigating the variation of the amounts of phenolic compounds in apricot pomaces from the beverage industry in correlation to fermentation days in solid-state system with these two filamentous fungi.

Antioxidant activity

The evolution of the antioxidant potential of methanol extracts from solid state fermented apricot by-products were measured using the DPPH radical scavenging assay and the results are presented in Fig. 4. The DPPH assay is widely used to determine antioxidant activity of phenolic compounds in natural plant extracts. This assay is based on the capacity of stable free radicals of DPPH to react with hydrogen donors.

After each SSF process, statistically significant increases in antioxidant activity levels (p < 0.05) of the analyzed extracts were registered. The antioxidant capacity increased by over 18% for both fungal fermentations by day 2 compared with the initial value (%I = 70) before gradually decreasing for the remaining period of growth.

In the case of SSF with A. niger, weak positive (0.1 < r < 0.3), but statistically not significant (p > 0.05) correlations were found between antioxidant capacity (determined by DPPH assay) and total phenolic and flavonoid contents (Fig. 5). It is also worth to mention that the values obtained for SSF with R. oligosporus correlated negatively (r < 0, p > 0.05) (Fig. 5). Moreover, the results presented in Fig. 6 also revealed a negative relationship between the concentrations of individual phenolic compounds and antioxidant activity of the methanolic extracts.

These correlation analyses suggested that the individual phenolic compounds could not be the key constituents responsible for the free radical scavenging activity of studied fermented samples.

These findings are mainly in agreement with our previous observations [16, 17] and with reported data from other authors [3, 18]. In all these reports (on different bio-processed agro-food wastes and cereals), weak correlations between polyphenolic contents and antioxidant capacities were found. This may be caused by the polymerization of phenolic monomers due to the stress induced on the fungus in certain phases of its growth. Many studies have shown that increasing degree of polymerization enhances the effectiveness of phenolics against a variety of free radical species due to the increment of the hydroxyl groups in addition to the extensive conjugations between double bonds and carbonyl groups from their structures [20, 21].

Changes in lipid and fatty acid compositions during SSF of the apricot kernels by A. niger and R. oligosporus

The data on oil content of apricot kernels processed with A. niger and R. oligosporus are shown in Fig. 7. Both fungal strains increased the total lipid content until the second day of fermentation.

The unfermented apricot kernels showed a fat content of 29.50 g/100 g of kernel (FW), which is close to the values already reported in the literature [22]. The extracted lipids increased significantly (p < 0.05) by 18.64% from the initial value for the solid state fermented kernels with A. niger whereas for R. oligosporus the evolution of these biomolecules was statistically insignificant (p > 0.05) (1.50%). It can be concluded that A. niger has a better lipogenic effect than R. oligosporus when grown on apricot kernels.

Recent studies have shown that the enzymes (cellulase, pectinase, protease, etc.) produced by the filamentous fungi in solid state system have a determining role in degradation of oil seeds cell wall, leading to the release of most of the lipids (generally bound to proteins or to the polyglucides) enmeshed in cellular structures [23]. The researchers have reported maximum enzyme activity until 48–72 h of SSF, depending on culture conditions (available fermentable sugars (carbon source), carbon to nitrogen (C:N) ratio of the fermentation medium, temperature, pH, etc.) after which the enzyme production had stabilized or decreased. These observations are in agreement with our findings regarding the dynamics of the lipid yields presented in Fig. 7, with the maximum oil amounts in the 2nd day of SSF.

The evolutions of the major fatty acids during the SSF are in agreement with the previously reported data, which demonstrated that the filamentous fungi are able to produce lipids with considerable proportions of unsaturated fatty acids [25].