Fatty Acid Composition of Lipids in Pot Marigold (Calendula Officinalis L.) Seed Genotypes Francisc V Dulf, Doru Pamfil, Adriana D Baciu* and Adela Pintea

Dulf et al. Chemistry Central Journal 2013, 7:8 http://journal.chemistrycentral.com/content/7/1/8

RESEARCH ARTICLE Open Access Fatty acid composition of lipids in pot marigold (Calendula officinalis L.) seed genotypes Francisc V Dulf, Doru Pamfil, Adriana D Baciu* and Adela Pintea

Abstract Background: Calendula officinalis L. (pot marigold) is an annual aromatic herb with yellow or golden-orange flowers, native to the Mediterranean climate areas. Their seeds contain significant amounts of oil (around 20%), of which about 60% is calendic acid. For these reasons, in Europe concentrated research efforts have been directed towards the development of pot marigold as an oilseed crop for industrial purposes. Results: The oil content and fatty acid composition of major lipid fractions in seeds from eleven genotypes of pot marigold (Calendula officinalis L.) were determined. The lipid content of seeds varied between 13.6 and 21.7 g oil/100 g seeds. The calendic and linoleic acids were the two dominant fatty acids in total lipid (51.4 to 57.6% and 28.5 to 31.9%) and triacylglycerol (45.7 to 54.7% and 22.6 to 29.2%) fractions. Polar lipids were also characterised by higher unsaturation ratios (with the PUFAs content between 60.4 and 66.4%), while saturates (consisted mainly of palmitic and very long-chain saturated fatty acids) were found in higher amounts in sterol esters (ranging between 49.3 and 55.7% of total fatty acids). Conclusions: All the pot marigold seed oils investigated contain high levels of calendic acid (more than 50% of total fatty acids), making them favorable for industrial use. The compositional differences between the genotypes should be considered when breeding and exploiting the pot marigold seeds for nutraceutical and pharmacological purposes. Keywords: Calendula officinalis L., Conjugated linolenic acids, Pot marigold, Seed oils, Fatty acids, Polar lipids, Triacylglycerols, Sterol esters, GC-MS

Background highly significant indicators of phylogenetic relationships Calendula officinalis L. (pot marigold), a member of the [11,12]. The seeds of pot marigold have a significant oil Asteraceae family, is an annual aromatic herb with yellow content (around 20%), of which about 60% is the un- or golden-orange flowers, native to the Mediterranean usual calendic acid (8 t, 10 t, 12c-18:3) [13-16]. Several climate areas, being also successfully cultivated in tem- studies demonstrated that calendic acid is synthesized in perate regions of the Earth for ornamental and medicinal Calendula seeds via desaturation of linoleic acid [17-21]. purposes [1]. The species have been reported to contain Due to its special structure – with three conjugated a variety of phytochemicals, including carbohydrates, double bonds – calendic acid and Calendula seeds oil lipids, phenolic compounds, steroids, terpenoids, toco- exhibit interesting chemical and physiological properties. pherols, carotenoids and quinones [2-5] with potential The seed oils such of Calendula officinalis L., health benefits [1,6-10]. Momordica charantia L. or Aleurites fordii Hemsl., Besides the usual fatty acids, a few plants are capable rich in conjugated linolenic acids (CLNAs) have a high rate to biosynthesize some unusual fatty acids, with special of oxidation and are used as raw materials in paints and chemical structure. Usually these fatty acids accumulate coatings industry, and have applications in the manufac- in storage tissues, while in green organs they are absent ture of cosmetics and some industrial polymers [19,22-24]. or present in very small amounts. The presence of un- For these reasons, in the last few years, a concentrated re- usual fatty acids is genetically determined and they are search effort in Europe has been directed towards the development of Calendula officinalis L. as an oilseed crop for * Correspondence: [email protected] industrial purposes [25] and for the engineering of University of Agricultural Sciences and Veterinary Medicine, Manastur 3-5, Cluj-Napoca 400372, Romania

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transgenic plants containing the metabolic route for the differences (p < 0.05) among genotypes, except for oil con- conjugated fatty acids biosynthesis [26,27]. tents of samples CO4 and CO6 versus CO9. The highest The increasing interest for plants producing conju- amounts of oils were found in the CO4 (21.7 g/100 g), gated fatty acids is also motivated by the recent find- CO6 (21.5 g/100 g) and CO11 (21.3 g/100 g), whereas the ings related to their biological effects. It has been genotypes CO1 (15.5 g/100 g), CO5 (15.3 g/100 g) and shown that CLNAs have an important body fat- CO9 (13.6 g/100 g), exhibited the lowest contents of the lowering effect [28] and possess anti-carcinogenic TLs. These values were similar to those reported by properties, exhibiting apoptotic activity against a wide Cromack and Smith [25] but much higher than those variety of tumor cells, such as the U-937 human observed by Ozgul- Yucel (5.9% oil in Turkish Calen- leukemic cancer cell line and the colon cancer cells dula seeds) [34] and Angelini et al. (5.4% oil in Italian (Caco-2) [24,29,30]. Bhaskar et al. [31] observed that CO seed crops from 1994) [35]. The TLs content of the the trans CLNAs exhibited stronger growth inhibition analyzed CO seeds in this study were also comparable and more DNA fragmentation in human colon cancer with those of some non-conventional vegetable oil sources cells than corresponding cis CLNA isomers. with unique phytochemical compositions, such as bitter To our knowledge, all the studies, excepting two short gourd (21%), cherry laurel (18.3%), pomegranate (18.1%), reports of Ul’chenko et al. [32] and Pintea et al. [33], re- blackthorn (16.5%), linseed dodder (15.5-20.7%), and cori- spectively, conducted on marigold seed oils determined the ander (12.7-18%) seeds [34,35]. fatty acid contents by analyzing only the total lipid matrix. Therefore, the aim of the present investigation was to Fatty acid composition compare the oil content and fatty acid compositions of The total lipid fatty acid composition as well as the fatty total lipids (TLs), triacylglycerols (TAGs), polar lipids acid composition of TAGs, PLs and SEs of the analyzed (PLs) and sterol esters (SEs) in seeds of eleven pot mari- pot marigold seed oils is presented in Tables 1 and 2. gold genotypes from six different locations in Europe, grown in the Transylvanian region (Romania). The infor- TL fatty acids mation obtained is helpful to identify suitable genotypes Nineteen fatty acids were identified in the studied pot for use in breeding programs of Calendula officinalis. marigold seed oils (Figure 2), including very low amounts of a hydroxy fatty acid, namely 9- hydroxy- trans-10, cis-12 Results and discussion octadecadienic-acid (9-HODE). Oil contents As expected, calendic acid [18:3 (8 t,10t,12c) (n-6)] The oil (total lipids) contents in eleven genotypes of pot was the predominant polyunsaturated fatty acid (PUFA) marigold (Calendula officinalis L.) (CO) seeds are pre- in all TL extracts, and its composition varied between sented in Figure 1.The values were found to vary between 51.47% (in CO8) and 57.63% of total fatty acids (in 13.6- 21.7 (g oil/100 g seeds). There were no significant CO4). The next most abundant fatty acid was linoleic acid [18:2 (n-6)] (28.50 to 31.86%), followed by oleic [18:1 (n-9)] (4.44 to 6.25%) and palmitic acids (16:0) 30 (3.86 to 4.55%). Small and very small (or trace) amounts (<2%) of stearic (18:0), β- calendic [18:3 (8 t,10t,12t) t CO4>CO9* CO6>CO9* (n-6)], elaidic [18:1 (9 ) (n-9)], arachidic (20:0), behenic α 20 (22:0), gondoic [20:1 (n-9)], - linolenic [18:3 (n-3)], linoelaidicic [18:2 (9 t,12 t) (n-6)], cis-7 hexadecenoic [16:1 (n-9)], palmitoleic [16:1 (n-7)], lauric (12:0), myristic (14:0), pentadecanoic (15:0), and margaric (17:0) 10 acids were also determined. Similar results for the calendic acid content (over 50%) were reported by Cromack and Smith [25] for two of nine hybrids of pot marigold Oil content (g/ 100g seeds) 100g (g/ content Oil 0 seeds grown in England, as well as by Cahoon et al. [26]. 0 Ozgul- Yucel concluded that Turkish calendula seed oil CO1 CO2 CO3 CO4 CO5 CO6 CO7 CO8 CO9 CO1 CO11 is characterized by high concentration of linoleic acid Calendula officinalis L. seeds (43.5%) and low content of CLNAs (calendic acid Figure 1 The oil content of Pot marigold (Calendula officinalis L.) (18.3%) + β- calendic (11.2%)) [34]. Moreover, the calen- seeds. CO1- CO11, pot marigold (Calendula officinalis L.) genotypes. dic acid levels reported here are considerably higher Results are given as mean ± SD (n = 3); *-significant difference, p <0.05 than those reported previously by Suzuki et al. [29] (using "Kruskal-Wallis non-parametric test" followed by "Dunn's Multiple (33.4%) and Angelini et al. [35] (16- 46%- in the Italian Comparison Test"). pot marigold seed oils, crops from 1993). Dulf et al. Chemistry Central Journal 2013, 7:8 Page 3 of 11 http://journal.chemistrycentral.com/content/7/1/8

Table 1 Fatty acid composition (%) in total lipids and individual lipid classes of different genotypes of pot marigold seed oils Species Fatty acids (%w/w of total fatty acids) 12:0 14:0 15:0 16:1 16:1 16:0 17:0 18:2 18:1 18:1 18:2 18:0 18:3 18:3 20:1 18:3 20:0 9-HODE 22:0 (n-9) (n-7) (n-6) (n-9) (9 t) (9 t,12 t) (n-3) (8 t,10 t, (n-9) (8 t,10 t, (n-9) (n-6) 12c) 12 t), (n-6) (n-6) CO1 TL 0.02 0.18 0.01 0.03 0.08 4.05 0.03 30.05 4.99 0.51 tr 1.87 0.09 55.93 0.14 0.85 0.36 0.63 0.18 TAG nd 0.15 0.08 0.03 0.08 5.62 tr 29.18 5.76 0.50 nd 2.62 0.15 54.67 0.10 0.60 0.46 nd nd PL 0.64 3.88 0.27 0.31 0.15 17.08 nd 60.78 6.49 0.70 tr 3.72 nd 4.29 tr 0.43 0.59 tr 0.67 SE 0.62 2.86 nd tr nd 20.02 nd 34.91 11.02 0.12 nd 2.53 nd 4.64 nd nd 2.45 nd 20.83 CO2 TL 0.03 0.30 0.01 0.03 0.07 4.33 0.04 28.50 4.49 0.53 0.02 1.84 0.10 57.22 0.16 0.77 0.42 0.89 0.25 TAG nd 0.13 0.02 0.04 0.18 7.98 0.04 25.10 9.10 1.10 nd 3.95 0.09 50.03 0.47 0.70 1.07 nd nd PL 0.41 4.44 0.15 0.16 0.08 20.61 0.17 55.02 7.04 1.01 1.06 3.94 nd 3.98 0.07 0.40 0.61 0.17 0.68 SE 0.91 3.05 nd 0.39 nd 20.23 nd 33.75 10.32 0.14 nd 2.97 nd 4.94 nd nd 2.53 nd 20.77 CO3 TL 0.01 0.15 0.01 0.03 0.07 3.93 0.02 31.79 5.06 0.54 0.06 1.73 0.10 54.07 0.15 0.79 0.38 0.95 0.17 TAG nd 0.14 0.02 0.04 0.18 10.21 0.07 24.30 10.02 0.73 nd 4.08 0.10 48.53 0.45 0.53 0.60 nd nd PL 0.17 2.45 0.12 0.13 0.12 17.66 0.15 61.33 6.88 1.13 0.76 3.43 nd 3.86 tr 0.46 0.50 0.13 0.72 SE 0.72 2.53 nd 0.33 nd 22.77 nd 32.34 12.00 0.20 nd 2.93 nd 2.69 nd nd 2.49 nd 21.00 CO4 TL 0.02 0.19 0.01 0.04 0.06 4.55 0.02 28.52 4.44 0.41 0.01 1.77 0.13 57.63 0.14 0.91 0.35 0.66 0.12 TAG nd 0.15 0.03 0.08 0.18 8.59 0.04 23.32 10.79 1.20 nd 4.20 0.08 49.02 0.53 0.51 1.28 nd nd PL 0.22 2.29 0.12 0.26 0.12 16.55 0.16 61.15 8.13 1.30 0.74 3.18 nd 4.19 0.10 0.34 0.46 0.16 0.53 SE 1.24 2.05 0.38 0.78 nd 21.78 nd 32.41 11.80 1.53 nd 3.51 nd 3.93 nd nd 2.68 nd 17.91 CO5 TL 0.02 0.22 0.01 0.03 0.07 4.22 0.03 31.47 6.19 0.55 0.07 1.88 0.11 52.52 0.15 0.83 0.40 0.98 0.25 TAG nd 0.15 0.02 0.05 0.25 8.15 0.04 24.75 13.27 1.15 nd 4.24 0.07 46.00 0.58 0.38 0.90 nd nd PL 0.26 3.23 0.16 0.17 0.12 20.31 0.19 57.96 7.69 1.20 0.77 3.68 nd 2.66 0.04 0.16 0.59 0.11 0.70 SE 1.01 2.11 nd tr nd 19.70 nd 33.52 10.31 0.37 nd 4.65 nd 4.60 nd nd 2.80 nd 20.93 CO6 TL 0.02 0.21 0.02 0.04 0.06 4.48 0.04 29.49 5.26 0.55 0.02 1.83 0.09 55.80 0.13 0.66 0.34 0.89 0.07 TAG nd 0.16 0.02 0.07 0.22 9.55 0.06 22.60 12.01 1.34 nd 4.42 0.06 47.85 0.48 0.33 0.83 nd nd PL 0.35 2.83 0.21 0.18 0.10 20.14 0.21 59.04 7.51 1.24 0.61 3.72 nd 2.61 tr 0.13 0.59 0.06 0.47 SE 1.59 2.90 nd tr nd 20.37 nd 32.33 12.29 0.17 nd 3.97 nd 3.75 nd nd 2.85 nd 19.78 CO7 TL 0.02 0.33 0.02 0.03 0.08 4.54 0.04 30.26 6.04 0.57 1.66 1.84 0.08 53.17 tr 0.46 0.38 0.38 0.11 TAG nd 0.16 0.02 0.10 0.22 8.62 0.08 24.62 12.65 1.13 nd 4.03 0.08 46.46 0.57 0.44 0.82 nd nd PL 0.29 2.74 0.13 0.16 0.14 19.90 0.22 60.46 7.20 1.13 tr 3.33 nd 2.77 0.11 0.13 0.53 0.11 0.65 SE 2.19 2.80 nd 0.71 nd 18.31 nd 33.89 10.83 0.22 nd 3.30 nd 3.77 nd nd 3.32 nd 20.66 CO8 TL 0.05 0.39 0.01 0.02 0.11 4.11 0.03 31.86 6.25 0.49 1.80 1.99 0.11 51.47 0.02 0.48 0.41 0.24 0.17 TAG nd 0.16 0.02 0.05 0.29 8.26 0.05 23.37 14.32 1.11 nd 4.77 0.06 45.73 0.53 0.43 0.85 nd nd PL 0.28 2.34 0.16 0.25 0.14 19.37 0.22 58.97 8.82 1.22 0.24 3.63 nd 2.91 tr 0.19 0.54 0.13 0.59 SE 1.75 3.14 nd 1.51 nd 23.77 nd 27.46 10.07 0.48 nd 3.03 nd 4.74 nd nd 3.76 nd 20.29 Dulf et al. Chemistry Central Journal 2013, 7:8 Page 4 of 11 http://journal.chemistrycentral.com/content/7/1/8

Table 1 Fatty acid composition (%) in total lipids and individual lipid classes of different genotypes of pot marigold seed oils (Continued) CO9 TL 0.03 0.19 0.02 0.02 0.10 3.98 0.05 30.81 4.98 0.53 1.34 1.87 0.12 54.21 tr 0.58 0.39 0.27 0.51 TAG nd 0.11 0.03 0.04 0.25 6.49 0.07 27.68 8.94 1.01 nd 3.59 0.10 50.06 0.47 0.53 0.63 nd nd PL 0.19 1.14 0.16 0.26 0.23 20.82 0.24 59.34 7.28 1.22 0.88 3.58 nd 2.95 0.05 0.17 0.62 0.10 0.77 SE 1.50 3.58 nd 1.05 nd 20.42 nd 32.38 9.68 0.24 nd 2.88 nd 3.69 nd nd 3.37 nd 21.21 CO10 TL 0.02 0.16 0.02 0.02 0.12 3.86 0.03 30.99 4.97 0.53 1.71 1.87 0.11 53.88 tr 0.65 0.36 0.29 0.41 TAG nd 0.08 0.01 0.05 0.31 6.99 0.05 27.93 9.09 1.06 nd 3.76 0.07 49.18 0.43 0.41 0.58 nd nd PL 0.12 0.95 0.15 0.25 0.25 20.28 0.27 61.51 6.98 1.16 0.35 3.51 nd 2.84 0.05 0.20 0.55 0.09 0.49 SE 1.86 3.31 nd tr nd 20.83 nd 32.16 10.11 0.23 nd 3.47 nd 3.43 tr nd 3.62 nd 20.98 CO11 TL 0.03 0.38 0.02 0.04 0.05 4.43 0.04 30.32 4.78 0.56 0.62 1.75 0.11 55.32 0.05 0.61 0.39 0.21 0.31 TAG nd 0.13 0.02 0.07 0.21 7.79 0.04 27.44 8.36 1.15 nd 3.43 0.09 49.85 0.39 0.48 0.55 nd nd PL 0.33 2.36 0.15 0.45 0.09 20.98 0.25 59.55 7.71 1.24 tr 3.31 nd 2.34 tr 0.13 0.51 0.04 0.56 SE 1.57 3.36 nd tr nd 20.93 nd 31.84 10.88 0.19 nd 3.26 nd 3.19 nd nd 3.61 nd 21.17 The values represent the means of three samples, analyzed individually in triplicate (n = 3x3). CO1- CO11, pot marigold (Calendula officinalis L.) genotypes. TL- total lipids, TAG- triacylglycerols, PL- polar lipids, SE- sterol esters, nd- not detected, tr- trace.

The available literature shows that the fatty acid com- fractions (TAGs, PLs and SEs) of each pot marigold position of oil seeds varies strongly according to their genotypes. origin/genotype, and geographical/climatic conditions of the growth areas [25,36]. It was also found that the ma- TAG fatty acids turity stage of the seeds is an important factor that influ- The fatty acid profiles of the TAGs were similar to that ences the accumulation of calendic acid in calendula of the profiles of the TL fractions, due to the dominance seeds oil. Pintea et al. [33] showed that during the mat- of the PUFAs (18:3 and 18:2 (n-6) fatty acids) in their uration period of the pot marigold seeds (0–2 weeks compositions (see Tables 1 and 2) and due to the fact after flower drops) the concentration of calendic acid that TAG are major components of the seeds oil. increased sharply and steadily (from 8.62% to 53%), ac- companied by a decrease in the amounts of linoleic and PL and SE fatty acids oleic acids. These observations are in agreement with The fatty acid composition of the PLs and SEs was dif- the presence of the specific conjugase which is able to ferent from that of the TL and TAG fractions in all the convert linoleic acid into calendic acid in Calendula pot marigold genotypes analyzed (Tables 1 and 2). seeds [18,26]. The stereospecific analysis of TAG proved The PL fractions were highly unsaturated, with the that calendic acid preferentially esterifies the sn-2 pos- linoleic acid content ranging from 55.02% (CO2) to ition of TAG [26,37]. 61.51% (CO10) of total fatty acids. Ul’chenko et al. The analysis of fatty acids classes showed statistically [32] studied the fatty acid compositions of the lipids significant differences (p < 0.05) with the exception of from seeds, leaves and flowers of Calendula officinalis PUFAs (Figure 3). The highest value of saturated fatty L. and reported lower value of linoleic acid (24.5%) in acid (SFAs) (p < 0.05) was registered in the TLs of Czech the phospholipids of seeds, than those determined in genotypes (CO11) (7.34%), whereas CO5, CO7 and CO8 the present work. were the richest sources of monounsaturated fatty acids With four exceptions (samples CO1-4), the calendic (MUFAs) (Figure 3A). On the other hand, small variations acid content in the PL fractions was lower than 3% (p < 0.05) were found in CLNAs contents (Figure 3B), with (Table 1). This conjugated fatty acid was found to be the highest proportions in CO4 (58.54%) and the lowest in below 1% in the phosphatidylcholine (PC) of Calendula CO8 (51.95%), respectively. As shown in Table 2, the levels officinalis seeds oil [26] or was not detected [32]. The of the PUFAs/SFAs (saturated fatty acids) ratios were differences between the reported data and our data can significantly higher (p <0.05)inTLs(duetothehigh be explained by the fact that we have investigated the values of 18:3 and 18:2 fatty acids) than in the lipid total polar lipids fraction which includes phospholipids Dulf et al. Chemistry Central Journal 2013, 7:8 Page 5 of 11 http://journal.chemistrycentral.com/content/7/1/8

Table 2 The composition (%) of fatty acid classes in total lipids and major lipid fractions from different genotypes of pot marigold seed oils Species Fatty acids (%w/w of total fatty acids) P P P P P SFAs MUFAs PUFAs VLCSFAs (≥20C) CLNAs PUFAs/ SFAs Mean SD Mean SD Mean SD Mean SD Mean SD CO1

TL 6.70d 0.25 5.75d 0.18 86.83a 2.25 0.54b 0.02 56.78a 1.76 12.96a

TAG 8.93c 0.35 6.47c 0.20 84.45a 2.30 0.46b 0.03 55.27a 1.55 9.46b

PL 26.85b 1.11 7.65b 0.22 65.50b 1.85 1.26b 0.04 4.72b 0.15 2.44c

SE 49.31a 1.68 11.14a 0.32 39.55c 1.68 23.28a 1.05 4.64b 0.12 0.80d CO2

TL 7.23d 0.22 5.27c 0.15 86.52a 2.30 0.67b 0.03 57.99a 1.65 11.97a

TAG 13.19c 0.30 10.89a 0.22 75.83b 2.27 1.07b 0.03 50.73b 1.38 5.75b

PL 31.01b 1.10 8.36b 0.18 60.46c 2.10 1.29b 0.05 4.38c 0.12 1.95c

SE 50.46a 1.80 10.85a 0.20 38.69d 1.50 23.30a 0.70 4.94c 0.15 0.77d CO3

TL 6.39d 0.19 5.85d 0.16 86.70a 2.38 0.54b 0.03 54.85a 1.32 13.56a

TAG 15.12c 0.35 11.42b 0.25 73.36b 1.95 0.60b 0.03 49.06b 1.75 4.85b

PL 25.20b 0.80 8.26c 0.18 66.41c 1.55 1.22b 0.04 4.32c 0.11 2.64c

SE 52.44a 1.60 12.53a 0.30 35.03d 1.15 23.49a 0.56 2.69c 0.10 0.67d CO4

TL 7.04d 0.16 5.09d 0.15 87.08a 2.42 0.47b 0.02 58.54a 1.58 12.37a

TAG 14.29c 0.31 12.78b 0.23 72.85b 2.10 1.28b 0.03 49.53b 1.65 5.10b

PL 23.51b 0.62 9.91c 0.22 66.42c 1.60 0.99b 0.03 4.53c 0.14 2.83c

SE 49.55a 1.55 14.11a 0.35 36.34d 1.11 20.59a 0.50 3.93c 0.11 0.73d CO5

TL 7.03d 0.17 6.99d 0.15 84.89a 2.30 0.66b 0.04 53.35a 1.25 12.08a

TAG 13.50c 0.28 15.30a 0.32 71.13b 2.00 0.90b 0.04 46.38b 1.60 5.27b

PL 29.12b 0.88 9.22c 0.20 61.55c 1.50 1.29b 0.03 2.82c 0.12 2.11c

SE 51.20a 1.65 10.68b 0.25 38.12d 1.20 23.73a 0.65 4.60c 0.14 0.74d CO6

TL 7.01d 0.20 6.03d 0.18 85.98a 2.35 0.41b 0.02 56.47a 1.35 12.26a

TAG 15.04c 0.32 14.12a 0.35 70.78b 2.10 0.83b 0.03 48.18b 1.62 4.71b

PL 28.52b 0.88 9.03c 0.25 62.39c 1.60 1.06b 0.02 2.74c 0.08 2.19c

SE 51.46a 1.70 12.46b 0.34 36.08d 1.15 22.63a 0.60 3.75c 0.09 0.70d CO7

TL 7.27d 0.22 6.72d 0.20 85.55a 2.40 0.48b 0.02 53.63a 1.30 11.77a

TAG 13.73c 0.30 14.67a 0.33 71.52b 2.15 0.82b 0.03 46.90b 1.25 5.21b

PL 27.79b 0.80 8.74c 0.24 63.36c 1.70 1.18b 0.03 2.90c 0.10 2.28c

SE 50.58a 1.48 11.76b 0.20 37.66d 1.10 23.98a 0.70 3.77c 0.15 0.74d CO8

TL 7.15d 0.17 6.90d 0.18 85.61a 2.29 0.57b 0.03 51.95a 1.38 11.98a

TAG 14.11c 0.40 16.30a 0.40 69.53b 1.98 0.85b 0.04 46.16b 1.30 4.93b

PL 27.13b 0.82 10.43c 0.28 62.31c 1.50 1.13b 0.03 3.10c 0.11 2.30c

SE 55.74a 1.85 12.06b 0.32 32.20d 0.88 24.05a 0.75 4.74c 0.14 0.58d Dulf et al. Chemistry Central Journal 2013, 7:8 Page 6 of 11 http://journal.chemistrycentral.com/content/7/1/8

Table 2 The composition (%) of fatty acid classes in total lipids and major lipid fractions from different genotypes of pot marigold seed oils (Continued) CO9

TL 7.04d 0.14 5.63c 0.16 86.94a 2.34 0.90b 0.03 54.80a 1.38 12.35a

TAG 10.92c 0.30 10.71a 0.31 78.27b 2.15 0.63b 0.04 50.59b 1.28 7.17b

PL 27.52b 0.72 9.04b 0.28 63.34c 1.52 1.39b 0.02 3.12c 0.06 2.30c

SE 52.96a 1.60 10.97a 0.30 36.07d 1.05 24.58a 0.65 3.69c 0.07 0.68d CO10

TL 6.74d 0.16 5.64c 0.14 87.22a 2.55 0.77b 0.03 54.52a 1.35 12.95a

TAG 11.47c 0.31 10.94a 0.28 77.52b 2.20 0.58b 0.03 49.59b 1.18 6.76b

PL 26.32b 0.72 8.69b 0.25 64.90c 1.55 1.04b 0.02 3.04c 0.08 2.47c

SE 54.07a 1.65 10.34a 0.26 35.59d 0.95 24.60a 0.68 3.43c 0.08 0.66d CO11

TL 7.34d 0.18 5.48d 0.12 86.87a 2.60 0.70b 0.03 55.93a 1.40 11.83a

TAG 11.96c 0.30 10.18b 0.25 77.77b 2.25 0.55b 0.04 50.33b 1.20 6.50b

PL 28.45b 0.70 9.49c 0.22 62.02c 1.52 1.07b 0.03 2.47c 0.08 2.18c

SE 53.90a 1.75 11.07a 0.27 35.03d 0.90 24.78a 0.80 3.19c 0.09 0.65d Values are given as mean ± SD of three samples, analyzed individually in triplicate (n = 3x3). Means in the same column followed by different subscript letters indicate significant differences (p < 0.05) among lipid classes of each genotype of pot marigold (ANOVA "Tukey's Multiple Comparison Test"). TL - total lipids, TAG- triacylglycerols, PL- polar lipids, SE- sterol esters.P SFAs- saturated fatty acids, MUFAs- monounsaturated fatty acids, PUFAs- polyunsaturated fatty acids, VLCSFAs- very long chain saturated fatty acids, CLNAs [18:3 (8trans,10trans,12cis) + 18:3 (8trans,10trans,12trans)] - conjugated linolenic acids.

100

9 6 / 8 \ 14 \

10 16 / / 18 15 2 17 \ 19 3 4 \ 11 13 / 1 \ \ 5 7 \ 0 Time 11.48 16.48 21.48 26.48 31.48 36.48 41.48 46.48 51.48 56.48 61.48 Figure 2 GC-MS chromatogram of FAMEs in the TLs of Calendula officinalis L. (CO2: cv. Prolifera nr. 214) seeds analysed with a BPx- 70 capillary column. Peaks: (1) lauric (12:0), (2) myristic (14:0), (3) pentadecanoic (15:0), (4) cis-7 hexadecenoic [16:1 (n-9)], (5) palmitoleic [16:1 (n-7)], (6) palmitic (16:0), (7) margaric (17:0), (8) linoleic [18:2 (n-6)], (9) oleic [18:1 (n-9)], (10) elaidic [18:1 (9 t) (n-9)], (11) linoelaidicic [18:2 (9 t,12 t) (n-6)], (12) stearic (18:0), (13) α- linolenic [18:3 (n-3)], (14) calendic [18:3 (8 t,10t,12c) (n-6)], (15) gondoic [20:1 (n-9)], (16) β- calendic [18:3 (8 t,10t,12t) (n-6)], (17) arachidic (20:0), (18) 9- hydroxy- trans-10, cis-12 octadecadienic (9- HODE), (19) behenic (22:0) acids. Dulf et al. Chemistry Central Journal 2013, 7:8 Page 7 of 11 http://journal.chemistrycentral.com/content/7/1/8

10 SFAs A MUFAs

abc abc ab abc a abc abc abc a cd bcd a a d bc b bc bcd bcd de cde e 5 % of fatty acids

CO1 CO2 CO3 CO4 CO5 CO6 CO7 CO8 CO9 CO1 CO2 CO3 CO4 CO5 CO6 CO7 CO8 CO9 CO10CO11 CO10CO11 Fatty acid classes

100 PUFAs B a a aa a a a a a aa CLNAs

a a ab ab abc 60 abc abc abc bc bc c

40 % of fatty acids

O7 O3 O6 CO1 CO2 CO3 CO4 CO5 CO6 C CO8 CO9 CO1 CO2 C CO4 CO5 C CO7 CO8 CO9 CO10CO11 CO10CO11 Fatty acid classes Figure 3 Comparative representation of fatty acid classes from total lipids of different genotypes of pot marigold (Calendula officinalis L.) seed oils. CO1- CO11, pot marigold (Calendula officinalis L.) genotypes. Values are mean ± SD of three samples, analyzed individually in triplicate (n = 3x3). Values with different letters (a-e) are significantly different (p < 0.05), using ANOVA "Tukey's Multiple Comparison Test". SFAs- saturated fatty acids, MUFAs- monounsaturated fatty acids, PUFAs- polyunsaturated fatty acids, CLNAs- conjugated linolenic acids. and glycolipids. Transgenic soybean and Arabidopsis mechanisms involving both desaturation and transacyla- seeds engineered to synthesize calendic acid (by cloning tion processes are involved in the biosynthesis of rich of the fatty acid conjugase from Calendula) accumulated CLNAs enriched TAG [26]. Same authors showed that moderate level of conjugated fatty acids. Calendic acid accumulation of conjugated fatty acids in PC of transgenic was found at comparable levels in PC and TAG fractions plants (soybean and Arabidopsis) negatively affected (85% in the sn-2 position of PC) proving that complex the appearance and the germination rate of seeds due Dulf et al. Chemistry Central Journal 2013, 7:8 Page 8 of 11 http://journal.chemistrycentral.com/content/7/1/8

to the special chemical and physical properties of optimal to reduce the risk of cardiovascular diseases CLNAs. In consequence, the selection of valuable [44,45]. Thus, the results of the present study show that genotypes of Calendula which are able to produce the Calendula officinalis oil, whatever the genotype ana- large amounts of oil enriched in CLNAs still has an lyzed in this paper, may reduce the risk of cardiovascular economical importance. diseases because both TLs and TAG presented PUFAs/ ThelevelsofSFAsinSEsweresignificantlyhigher SFAs ratios values are closed to the recommended PUFA/ (p < 0.05) than in the corresponding lipid fractions of SFA intake by nutrition scientists. eachgenotype(Table2).Theamountsofsaturated consisted mainly of palmitic (16:0) acid, very long- Conclusions chain saturated fatty acids (VLCSFAs) (more than 20 In the present paper, seeds of eleven genotypes of Calen- carbon atoms) and stearic (18:0) acid, respectively, and var- dula officinalis L. originating from six different locations ied between 49.31% (in CO1) and 55.74% (in CO8) of total in Europe, cultivated in Romania (Transylvanian) were fatty acids from SEs (Tables 1 and 2). These observations analyzed with respect to oil yields and fatty acid con- are in agreement with the data reported by Zlatanov [38], tents. To the best of our knowledge, data about detailed Kallio et al. [39] and Yang et al. [40] about the fatty acid fatty acid composition of main lipid fractions in pot composition of the phospholipids and the SE fractions of marigold seeds investigated in this study are not avail- other non-conventional seed oils. able in literature. In plant tissues, the very long-chain fatty acids (≥20 The oil content observed in most of the calendula seed carbon atoms) are precursors for the synthesis of lipids, samples studied was noted to range between 18 and 22 g such as cuticular waxes (on the aerial plant surfaces), su- oil/100 g seeds. The oil TAGs were similar in fatty acid berin (embedded in the cell walls of plant-environment composition to the TLs, containing substantial amounts interfaces), triacylglycerols (in seeds), and ceramides (in the of calendic and linoleic acids, making them excellent cell membranes) [41,42]. dietary sources of PUFAs, especially of CLNAs. The PL The TL, TAG and PL fractions of all analyzed pot fractions were highly unsaturated, due to the dominance marigold genotypes exhibited very low proportions of of the linoleic acid in their structures. A clear character- VLCSFAs (<1.50% of total fatty acids), whereas the SEs istic of the SEs from the pot marigold seed oils analyzed showed significantly higher (p < 0.05) amounts of this were the significantly high levels of SFAs, with consider- type of fatty acids (from 20.59% (CO4) to 24.78% able amounts of VLCSFAs. (CO11)) (Table 2). The compositional differences between the genotypes As shown in Table 2, in all extracts of pot marigold should be considered when breeding and exploiting the seeds, the PUFAs/SFAs ratios were significantly lower calendula seeds for industrial, nutraceutical or pharma- (p < 0.05) in SE and PL fractions than in the corresponding cological purposes. TLs or TAGs. A comprehensive study of the Diabetes and Nutrition Study Group of the Spanish Diabetes Association Materials and methods showed that a dietary PUFAs/SFAs ratio > 0.4 can greatly Seeds and chemicals reduce the risk of onset of diabetic complications [43]. Eleven genotypes of Calendula officinalis L. originating Moreover, in some earlier reports, the authors indicate that from six different locations in Europe (botanical gar- the values of this ratio comprised between 1.0 and 1.5, are dens and institutes) (Table 3) were cultivated on

Table 3 Genotypes of Calendula officinalis L. (CO) evaluated Samples Genotypes Sources CO1 C. officinalis L. D.a Humboldt-Universität zu Berlin, Institut für Biologie, Germany CO2 cv. Prolifera nr. 214 Botanische Garten der Universität Göttingen, Germany CO3 Bon-Bon Orange National Botanic Garden of Latvia, Salaspils, Latvia CO4 Bon-Bon Mix’ Hortus Botanicus Fominianus, Kiev, Ukraine CO5 cv. Radio Ökologisch-Botanischer Garten der Universität Bayreuth, Germany CO6 C. officinalis L. PL Hortus Farmacognosticus Academiae Medicinalis, Lublin, Poland CO7 C. officinalis L. I Instituto di Botanica e Orto Botanico Pierino Scaramella, Italy CO8 cv. Prycosnovjenie National Botanical Gardens Timirjazevska, Kiev, Ukraine CO9 cv. Pacific Beauty National Botanical Gardens Timirjazevska, Kiev, Ukraine CO10 cv. Zelenoye Serdtse National Botanical Gardens Timirjazevska, Kiev, Ukraine CO11 cv. Plamen Masarykova Univerzita Brno, Czech Republic Dulf et al. Chemistry Central Journal 2013, 7:8 Page 9 of 11 http://journal.chemistrycentral.com/content/7/1/8

experimental fields of the University of Agricultural Fatty acid analysis Sciences and Veterinary Medicine of Cluj- Napoca (Ro- The total lipid, PL, TAG and SE fractions were deriva- mania). The crops were established in the first half of tized by sodium methoxide catalysis [49]. The FAMEs May 2011, to a target population of 40 plants m-2. Plot were determined by gas chromatography–mass spec- area was prepared before (autumn of 2010) by trometry (GC-MS), using a PerkinElmer Clarus 600 T fertilization with animal manure. Nitrogen- based ferti- GC-MS (PerkinElmer, Inc., Shelton, U.S.A.) equipped lizers were applied during the vegetation period. The with a, BPx- 70 capillary column (60 m × 0.25 mm i.d., seeds were harvested manually at full maturity (end of 0.25 μm film; SGE, Ringwood, Australia). The initial September-beginning of October). oven temperature was 140°C, increased to 220°C with a All reagents (used for the oil extraction, fractionation rate of 2°C/min and then held at this temperature for and fatty acid methyl esters (FAMEs) preparation) and 25 min. Flow rate of the carrier gas He and the split lipid standards (used for identification of the lipid ratio were 0.8 ml/min and 1:24, respectively. The in- class) were of chromatographic grade (Sigma–Aldrich jector temperature was 210°C. The positive ion electron (St. Louis, MO, USA)). The thin layer chromatography impact (EI) mass spectra was recorded at an ionization (TLC) plates (silica gel 60 F254, 20 × 20 cm) were pur- energy of 70 eV and a trap current of 100 μA with a chased from Merck (Darmstadt, Germany).The FAMEs source temperature of 150°C. The mass scans were per- standard (37 component FAME Mix, SUPELCO, catalog formed within the range of m/z: 22–395 at a rate of 0.14 No: 47885-U) were purchased from Supelco (Bellefonte, scan/s with an intermediate time of 0.02 s between the PA, USA). scans. The injected volume was 0.5 μl. Identification of FAMEs was achieved by comparing their retention times with those of known standards (37component FAME Oil extraction and fractionation Mix, SUPELCO # 47885-U) and the resulting mass spec- The oils were extracted from 5 g of seeds, using a tra to those in our database (NIST MS Search 2.0). methanol/chloroform extraction procedure, according to Yang et al. [36] and Dulf et al. [46]. The sample was homo- Statistics genized in 50 mL methanol for 1 min using a homogeniser Three different samples of Calendula seeds for each (MICCRA D-9, Germany), 100 mL chloroform was added, genotype were assayed. The analytical results reported and homogenization was continued for further 2 min. The for the fatty acid compositions, are the average of tripli- mixture was filtered under vacuum through a Buchner cate measurements of three independent oils (n = 3x3). funnel and the solid residue was resuspended in 150 mL of The assumptions of equality of variances and normal chloroform: methanol (2:1, v/v) and homogenized for an- distribution of errors were checked for the tested re- other 3 min. The mixture was filtered again and washed sponse variables. Since the assumptions were satisfied, with 150 mL chloroform: methanol (2:1, v/v). The filtrates data were subjected to one-way ANOVA (repeated mea- were combined and cleaned with 0.88% potassium chloride sures ANOVA) and Tukey’s post hoc test. Statistical water solution and methanol: water (1:1, v/v) solution. The differences among oil samples were estimated using: bottom layer (with the purified lipids) was filtered before “Kruskal-Wallis non-parametric test” followed by “Dunn's the solvent was rotary evaporated. The total lipids recov- Multiple Comparison Test” (Graph Pad Prism Version 4.0, ered were transferred to vials with 4 mL chloroform (stock Graph Pad Software Inc., San Diego CA). A probability solution), and stored at −18°C for further analysis. value of p < 0.05 was considered to be statistical significant. Neutral and polar lipid fractions were separated by

TLC [47]. Lipid aliquots (0.2 ml of stock solution) were Abbreviations applied on the TLC plates and then developed in a mix- CLNAs: Conjugated linolenic acids; TLs: Total lipids; TAGs: Triacylglycerols; ture of petroleum ether: diethyl ether: acetic acid PLs: Polar lipids; SEs: Sterol esters; PUFAs: Polyunsaturated fatty acids; ’ ’ SFAs: Saturated fatty acids; MUFAs: Monounsaturated fatty acids; (85:15:1, v/v/v), sprayed with 2,7-dichlorofluoroscein/ VLCSFAs: Very long-chain saturated fatty acids; PC: Phosphatidylcholine; methanol (0.1% w/v) and viewed under UV light CO: Calendula officinalis; FAMEs: Fatty acid methyl esters; TLC: Thin layer (254 nm) [48]. The lipid classes were identified using chromatography; GC-MS: Gas chromatography–mass spectrometry. commercial standards and then scraped from the TLC plates. The first band (at the origin of the plates), corre- Competing interests The authors declare that they have no competing interests. sponding to the PLs was eluted from silica layer with methanol: chloroform (1:1, v/v), and the upper two Authors’ contributions major bands of TAGs and SEs respectively were eluted FVD and DP carried out the experimental design, interpretation of results with chloroform. The samples were filtered, the solvent and preparation of the paper. ADB contributed to the extraction of lipids. AP contributed to the separation, identification and quantification of the lipid was removed and the dry residue was subjected to trans- fractions and fatty acids from the samples. All authors read and approved esterification and gas chromatographic (GC) analysis. the final manuscript. Dulf et al. Chemistry Central Journal 2013, 7:8 Page 10 of 11 http://journal.chemistrycentral.com/content/7/1/8

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doi:10.1186/1752-153X-7-8 Cite this article as: Dulf et al.: Fatty acid composition of lipids in pot marigold (Calendula officinalis L.) seed genotypes. Chemistry Central Journal 2013 7:8.

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RESEARCH ARTICLE Open Access Fatty acids in berry lipids of six sea buckthorn (Hippophae rhamnoides L., subspecies carpatica) cultivars grown in Romania Francisc V Dulf

Abstract Background: A systematic mapping of the phytochemical composition of different sea buckthorn (Hippophae rhamnoides L.) fruit subspecies is still lacking. No data relating to the fatty acid composition of main lipid fractions from the berries of ssp. carpatica (Romania) have been previously reported. Results: The fatty acid composition of the total lipids (oils) and the major lipid fractions (PL, polar lipids; FFA, free fatty acids; TAG, triacylglycerols and SE, sterol esters) of the oils extracted from different parts of six sea buckthorn berry subspecies (ssp. carpatica) cultivated in Romania were investigated using the gas chromatography-mass spectrometry (GC-MS). The dominating fatty acids in pulp/peel and whole berry oils were palmitic (23-40%), oleic (20-53%) and palmitoleic (11-27%). In contrast to the pulp oils, seed oils had higher amount of polyunsaturated fatty acids (PUFAs) (65-72%). The fatty acid compositions of TAGs were very close to the compositions of corresponding seed and pulp oils. The major fatty acids in PLs of berry pulp/peel oils were oleic (20-40%), palmitic (17-27%), palmitoleic (10-22%) and linoleic (10%-20%) acids, whereas in seeds PLs, PUFAs prevailed. Comparing with the other lipid fractions the SEs had the highest contents of saturated fatty acids (SFAs). The fatty acid profiles of the FFA fractions were relatively similar to those of TAGs. Conclusions: All parts of the analyzed sea buckthorn berry cultivars (ssp. carpatica) exhibited higher oil content then the other European or Asiatic sea buckthorn subspecies. Moreover, the pulp/peel oils of ssp. carpatica were found to contain high levels of oleic acid and slightly lower amounts of linoleic and α-linolenic acids. The studied cultivars of sea buckthorn from Romania have proven to be potential sources of valuable oils. Keywords: Sea buckthorn, Hippophae rhamnoides L., Subspecies, Oil content, Fatty acids, Polar lipids, Free fatty acids, Triacylglycerols, Sterol esters, GC-MS

Background berries with an acidic and astringent taste and a high nu- Sea buckthorn (SB) (Hippophae rhamnoides L. Elaeag- tritional value. SB berries are rich in a variety of phyto- naceae) is a bush or a small tree, of the Elaeagnaceae chemicals with physiological properties such as vitamins family, naturally distributed in Eurasia. The classification (B, C, E and K), flavonoids, carotenoids, tocopherols and of genus Hippophae is still unclear. The most common many volatile compounds (i.e., aliphatic esters, alcohols species (sp.), H. rhamnoides, was classified in several and hydrocarbons [2-4]. Significant amounts of inositols subspecies (ssp.), including ssp. carpatica, which is na- and methylinositols were found in SB berries, which are tive in Romania [1]. Over the last decades the SB was supposed to contribute to health benefits of SB fruits domesticated in many countries from Asia, North and and derivatives [5]. SB fruit membranes are rich in caro- South America and Europe, not only for its soil and tenolipoprotein complexes with 61% phospholipids and water conservation ability but also for its yellow-orange 39% galactolipids, as structural components [6]. In vitro and clinical studies show that the SB fruits have positive effect in the treatment of type 1 diabetic patients im- Correspondence: [email protected] University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, proving the glucose and lipid metabolism [7], possess Manastur 3-5, 400372, Romania high anti-oxidant, hemostatic and anti-inflammatory

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effects [8,9] and help prevent cardiovascular disease and carpatica is the most cultivated sea buckthorn ssp. in cancer [10,11]. Romania. No data relating to the fatty acid composition In last years SB pulp and seed oils have become popu- of main lipid fractions from this berry ssp. have been lar food supplements playing important role in cancer previously reported. The purpose of the present study therapy [9]. Several studies have indicated that these was to characterize the fatty acid composition of the berry oils possess important immunostimulant, anti- total lipids (oils) and the major lipid fractions (PLs, ulcer and cholesterol-lowering effects, and may also be FFAs, TAGs and SEs) of the oils extracted from different used in treatment of various skin diseases [12-15]. fruit parts of six SB subspecies (ssp. carpatica) cultivated Both the seeds and soft parts (pulp/peel) of berries in Romania. show high amounts of oil. The contents of bioactive lipophilic compounds, (i.e., phytosterols (up to 23 g/kg Results and discussion in seed oil and up to 29 g/kg in pulp/peel oil), tocopher- Oil content of the SB materials ols and tocotrienols (up to 2.9 g/kg in seed oil and up to The oil content of seeds, soft parts and whole berries 1.8 g/kg in pulp oil) and carotenoids (up to 3.5 g/kg in (based on fresh weight, f.w.) of different SB cultivars pulp oil) are generally high in the extracted seed and (ssp. carpatica) are presented in Figure 1-A. The oil pulp/peel oils [2,16,17]. The existing studies reported amounts of the analyzed berry parts varied widely: 45– different chemical compositions for SB seed and pulp/ 84 g kg -1- in whole berries, 45- 88 g kg -1- in pulp/peel peel oils which vary widely depending on the subspecies, and 106–135 g kg -1- in seeds. The average oil content harvesting time of the fruits and the many other climatic in seeds of the studied SB ssp. (123 g kg -1) was signifi- and geographical conditions. Whereas the seed oil con- cantly higher (p < 0.05) than in soft parts (60 g kg -1) and tains high amounts of unsaturated fatty acids, with whole berries (62 g kg -1), respectively (Figure 1-B). linoleic (C18:2n-6) (30-40%) and α-linolenic (C18:3n-3) These results are similar with the oil contents of ssp. (20-35%) acid as the dominating fatty acids, the pulp/ mongolica, and higher than those reported for ssp. sinen- peel oil is rich in palmitoleic (C16:1n-7) (16-54%) and sis (97 g kg -1 seeds, f.w. and 41 g kg -1 berry, f.w.) [16]. palmitic acids (C16:0) (17-47%) being more saturated Yang et al. [17] determined the following amounts of oils [16,18,19]. The TAGs and PLs are the major lipid frac- for ssp. rhamnoides: 11% (f.w.) in seeds, 3% (f.w.) in soft tions in both of SB seed and pulp/peel oils [17]. parts and 3.5% (f.w.) in whole berries, respectively. A systematic mapping of the phytochemical compos- Gutierrez et al. [18] concluded that the drying methods ition of different SB fruits subspecies is still lacking. Ssp. of SB berry parts could affect the oil extraction yield.

Figure 1 Oil content (g kg -1 fresh weight) of sea buckthorn berries (ssp. carpatica): A- oil content of different parts of six cultivars; B- the average oil content in analyzed parts of berries (mean of six cultivars). Dulf Chemistry Central Journal 2012, 6:106 Page 3 of 12 http://journal.chemistrycentral.com/content/6/1/106

These authors reported significant differences between deviations were observed in the proportions of oleic (13- the total oil content of air-dried berry pulp (cultivar In- 21%) and linoleic (33-43%) acids. In contrast to the pulp dian-summer) and freeze-dried pulp (36% vs. 16% oils, seed oils had higher amounts of PUFAs (65-72%) (weight/weight, w/w)), whereas the total lipid recovery and lower proportions of MUFAs (16–21.5%) and SFAs from air-dried seeds and freeze-dried seeds were similar (11-16%), respectively (Table 2). These oils, character- (11% and 12% (w/w)). ized by high ratios of PUFAs/SFAs, with an extremely significant high value (p < 0.05) for cultivar C2 (6.25), Fatty acid composition in oil of pulp/peel, seeds and are susceptible to oxidative damage due to their high whole berries α-linolenic acid content (28-33%). Statistically signifi- The fatty acid compositions of pulp/peel, seeds and cant variations (p < 0.05) were observed between n-6/n-3 whole berries oils of six SB berry cultivars (ssp. carpa- ratios of analysed six seed oils, with all the values close to tica) are listed in Tables 1 and 2. Due to the dominance 1 (Table 2). This phenomenon could be explained by the of the pulp and peels in SB fruit, the composition of the ratio of linoleic to α-linolenic acid (close to 1:1), which is oil from the whole berry resembled that of the pulp/peel different from the main vegetable oils [20,21]. Generally oil. the proportions of unsaturated fatty acids from seed oils The fatty acid levels of the seed and berry pulp/peel obtained in this study were in accordance with those oil varied widely. reported by Yang and Kallio [17] and Yang et al. [22] for The dominating fatty acids in berry pulp/peel oils were ssp. sinensis and rhamnoides. The concentration of α- palmitic (16:0) (23-40%), oleic (18:1n-9) (20-53%) and linolenic was found slightly higher in air- and freeze- dried palmitoleic (16:1n-7) (11-27%). Small or trace amounts SB seed oils (~ 37% and ~ 39%, respectively) of cv. Indian- of vaccenic (18:1n-7), linoleic(18:2n-6), α-linolenic summer than in the corresponding oils from the present (18:3n-3), stearic (18:0), myristic (14:0), pentadecanoic work [18]. (15:0), cis-7 hexadecenoic (16:1n-9), margaric (17:0) and The high amount of palmitoleic acid, unusual for a two long chain fatty acids, arachidic (20:0) and eicose- vegetable oil, distinguishes the berry pulp/peel oils from noic (20:1n-9) acids were observed in all analyzed soft the seed oils of SB. This valuable fatty acid, which is an part oils. In two cultivars, C1 and C2, the proportions of important component of skin fat, has attracted an in- oleic acid (32.76% for C1 and 53.08% for C2) exceeded creasing interest due to its hypocholesterolemic and that of the palmitoleic acid (19.53% for C1 and 11.05% hypoglyceridemic activities [2]. for C2). From these results can be concluded that Comparing the average proportions (average of six cul- MUFAs were the dominant fatty acid classes (53-70%), tivars) of the fatty acid classes from the oils of different followed by SFAs (26-41%) and PUFAs (3-7%) (Table 2). parts of berries, the seed oil contained significantly lower The PUFA/SFA ratios were close to zero, with a signi- proportions of SFAs and MUFAs (p < 0.05), and signifi- ficantly high value (0.17) (p < 0.05) in pulp/peel oil of cantly higher amount of PUFAs (p < 0.05), than the C6. Statistically significant differences (p < 0.05) were whole berry and pulp/peel oils (Figure 2). observed between n-6/n-3 ratios of analyzed berry pulp/ peel oils, with the highest value in cultivar C4 (7.67) and Fatty acid composition in individual lipid fractions of oils the lowest in C6 (1.09), respectively (Table 2). from pulp/peel and seeds Similar amounts of palmitic (in cv. Indian-summer The fatty acid compositions of the main lipid classes and H. rhamnoides (India)), vaccenic (in cv. Indian-sum- (PLs, FFAs, TAGs and SEs) from pulp/peel and seed oils mer and ssp. sinensis)andα-linolenic (in cv. Indian- are presented in Tables 3, 4, 5 and 6. summer, H. rhamnoides (India) and H. salcifolia) acids were recently reported by different authors for berry Fatty acid composition of TAGs pulp oil. Higher proportions of palmitoleic acid and The fatty acid compositions of TAGs (Figure 3) were much lower levels of oleic acid were characteristic of the very close to the compositions of corresponding seed Finnish, Chinese and Canadian soft part SB oils, except- and pulp oils, with the same dominating fatty acid ing species H. tibetana which presented similar percen- classes (Table 1; Figure 4 (a), (b) and (c)). tages of (18:1n-9) with those of results from the present study [2,17,18]. Fatty acid composition of PLs Seed oils consisted mainly of linoleic, α-linolenic, oleic, The dominating fatty acids in descending order in berry palmitic and stearic acids, with minor or trace amounts pulp/peel oils were oleic (20-40%), palmitic (17-27%), of vaccenic, palmitoleic, arachidic, eicosenoic, myristic, palmitoleic (10-22%), linoleic (10%-20%) and α-linolenic pentadecanoic and margaric acids (Table 1). A notable (4-9%) acids (Table 3). In all PL fractions extremely sig- feature of the berry seed oils was the extremely low nificant differences (p < 0.05), were observed between level of palmitoleic acid (0.1-0.5%). The relatively high the proportions of fatty acid classes, with the MUFAs as Dulf Chemistry Central Journal 2012, 6:106 Page 4 of 12 http://journal.chemistrycentral.com/content/6/1/106

Table 1 Fatty acid composition (weight % of total fatty acids) of oils from whole berries, pulp/peel and seeds of different cultivars of H. rhamnoides L. (ssp. carpatica) fruits Sea buckthorn cultivars (ssp. carpatica) Fatty acid C1 C2 C3 C4 C5 C6 Whole berries C14:0 0.22 ± 0.05 0.61 ± 0.10 0.59 ± 0.10 0.25 ± 0.05 0.37 ± 0.03 0.33 ± 0.05 C15:0 tr 0.05 ± 0.02 0.04 ± 0.02 0.03 ± 0.01 0.03 ± 0.02 0.04 ± 0.01 C16:0 35.11 ± 0.80 20.80 ± 0.61 36.16 ± 0.84 37.33 ± 0.87 37.21 ± 0.89 33.32 ± 0.64 C16:1n-9 0.02 ± 0.01 0.14 ± 0.02 0.04 ± 0.02 0.02 ± 0.01 0.02 ± 0.01 0.03 ± 0.02 C16:1n-7 19.80 ± 0.55 9.63 ± 0.38 24.64 ± 0.46 23.70 ± 0.65 23.75 ± 0.75 19.65 ± 0.60 C17:0 0.03 ± 0.02 0.03 ± 0.02 0.02 ± 0.01 0.02 ± 0.01 0.02 ± 0.01 tr C18:0 1.41 ± 0.17 2.86 ± 0.14 0.94 ± 0.10 0.96 ± 0.12 0.82 ± 0.08 1.27 ± 0.10 C18:1n-9 30.47 ± 0.73 45.90 ± 0.80 22.29 ± 0.62 23.93 ± 0.73 24.85 ± 0.65 28.39 ± 0.91 C18:1n-7 6.78 ± 0.20 4.55 ± 0.30 6.23 ± 0.20 6.58 ± 0.22 5.76 ± 0.22 5.37 ± 0.17 C18:2n-6 3.05 ± 0.13 10.87 ± 0.38 6.24 ± 0.25 5.17 ± 0.20 4.57 ± 0.23 7.60 ± 0.25 C18:3n-3 2.90 ± 0.14 4.17 ± 0.15 2.67 ± 0.13 1.86 ± 0.14 2.41 ± 0.19 3.86 ± 0.16 C20:0 0.17 ± 0.05 0.23 ± 0.04 0.12 ± 0.02 0.12 ± 0.03 0.14 ± 0.03 0.13 ± 0.03 C20:1n-9 0.06 ± 0.02 0.15 ± 0.02 0.03 ± 0.01 0.02 ± 0.01 0.05 ± 0.02 tr Pulp/peel C14:0 0.23 ± 0.03 0.59 ± 0.06 0.46 ± 0.04 0.29 ± 0.05 0.42 ± 0.05 0.40 ± 0.04 C15:0 tr 0.04 ± 0.02 0.03 ± 0.02 0.03 ± 0.02 0.03 ± 0.01 0.04 ± 0.02 C16:0 34.62 ± 0.88 23.17 ± 0.63 39.11 ± 0.91 38.76 ± 1.11 39.22 ± 1.22 37.68 ± 1.12 C16:1n-9 0.04 ± 0.02 0.16 ± 0.04 0.02 ± 0.01 0.01 ± 0.01 0.02 ± 0.01 0.03 ± 0.02 C16:1n-7 19.53 ± 0.67 11.05 ± 0.44 26.70 ± 0.58 25.74 ± 0.96 26.19 ± 0.71 24.90 ± 0.90 C17:0 0.03 ± 0.02 0.02 ± 0.01 0.02 ± 0.01 0.02 ± 0.01 0.03 ± 0.02 0.02 ± 0.01 C18:0 1.25 ± 0.15 2.53 ± 0.07 0.84 ± 0.06 0.77 ± 0.08 0.61 ± 0.07 0.87 ± 0.08 C18:1n-9 32.76 ± 0.94 53.08 ± 1.12 20.81 ± 0.69 22.75 ± 0.75 24.41 ± 0.74 23.10 ± 0.82 C18:1n-7 6.41 ± 0.29 5.34 ± 0.16 6.41 ± 0.20 6.85 ± 0.25 5.72 ± 0.18 6.31 ± 0.19 C18:2n-6 4.06 ± 0.16 2.25 ± 0.10 4.57 ± 0.18 4.15 ± 0.16 2.57 ± 0.09 3.41 ± 0.10 C18:3n-3 0.84 ± 0.08 1.33 ± 0.07 0.90 ± 0.05 0.54 ± 0.04 0.63 ± 0.04 3.14 ± 0.11 C20:0 0.17 ± 0.03 0.24 ± 0.04 0.10 ± 0.02 0.07 ± 0.02 0.12 ± 0.03 0.10 ± 0.03 C20:1n-9 0.06 ± 0.03 0.20 ± 0.05 0.03 ± 0.01 tr 0.05 ± 0.02 tr Seeds C14:0 0.10 ± 0.02 0.09 ± 0.03 0.24 ± 0.03 0.15 ± 0.03 0.12 ± 0.02 0.09 ± 0.01 C15:0 0.11 ± 0.03 tr 0.30 ± 0.04 tr 0.12 ± 0.03 tr C16:0 9.12 ± 0.38 7.14 ± 0.26 12.44 ± 0.44 9.43 ± 0.33 10.29 ± 0.31 8.06 ± 0.28 C16:1n-9 nd nd nd nd nd nd C16:1n-7 0.53 ± 0.07 0.16 ± 0.03 0.36 ± 0.03 0.43 ± 0.06 0.33 ± 0.04 0.19 ± 0.03 C17:0 0.03 ± 0.01 0.03 ± 0.02 tr 0.05 ± 0.01 0.03 ± 0.01 tr C18:0 3.03 ± 0.07 3.84 ± 0.08 2.91 ± 0.09 3.68 ± 0.11 3.10 ± 0.10 2.98 ± 0.08 C18:1n-9 13.57 ± 0.53 14.89 ± 0.41 16.74 ± 0.66 15.49 ± 0.51 16.30 ± 0.60 20.09 ± 0.71 C18:1n-7 2.28 ± 0.11 1.38 ± 0.08 1.48 ± 0.10 2.22 ± 0.10 2.29 ± 0.11 1.27 ± 0.07 C18:2n-6 42.35 ± 0.95 42.12 ± 1.13 33.72 ± 0.98 36.98 ± 0.82 34.41 ± 1.04 38.93 ± 1.17 C18:3n-3 28.50 ± 0.55 29.78 ± 0.62 31.81 ± 0.72 30.98 ± 0.70 32.60 ± 0.80 28.13 ± 0.67 C20:0 0.37 ± 0.04 0.41 ± 0.04 0.21 ± 0.04 0.49 ± 0.03 0.35 ± 0.04 0.26 ± 0.04 C20:1n-9 tr 0.16 ± 0.03 tr 0.10 ± 0.02 0.06 ± 0.02 tr Values are mean ± SD of three samples of each fruit part, analyzed individually in triplicate; C1- C6, sea buckthorn (ssp. carpatica) cultivars. C14:0, myristic; C15:0, pentadecanoic; C16:0, palmitic; C16:1n-9, cis-7 hexadecenoic; C16:1n-7, palmitoleic; C17:0, margaric; C18:0, stearic; C18:1n-9, oleic; C18:1n-7, vaccenic; C18:2n-6, linoleic; C18:3n-3, α-linolenic; C20:0, arachidic; C20:1n-9, eicosenoic acids. Dulf Chemistry Central Journal 2012, 6:106 Page 5 of 12 http://journal.chemistrycentral.com/content/6/1/106

Table 2 Fatty acid composition (weight % of total fatty acids) of oils from different parts of sea buckthorn fruits (ssp. carpatica) Sea buckthorn cultivars (ssp. carpatica) Fatty acid classes C1 C2 C3 C4 C5 C6 Whole berries P SFAs 36.94 ± 1.09ab 24.58 ± 0.93c 37.87 ± 1.09ab 38.72 ± 1.09a 38.59 ± 1.06a 35.09 ± 0.83b P b b b b b b MUFAs 57.12 ± 1.51ab 60.37 ± 1.52 a 53.22 ± 1.31c 54.26 ± 1.62bc 54.43 ± 1.65bc 53.45 ± 1.70c P a a a a a a e a c d de b PUFAs 5.95 ± 0.27c 15.05 ± 0.53c 8.91 ± 0.38c 7.03 ± 0.34c 6.98 ± 0.42c 11.46 ± 0.41c PUFAs/SFAs 0.16d 0.61a 0.24c 0.18cd 0.18cd 0.33b n–6/n–3 1.05e 2.61b 2.34c 2.79a 1.90d 1.97d Pulp/peel P SFAs 36.30 ± 1.11b 26.59 ± 0.83c 40.56 ± 1.06a 39.95 ± 1.29a 40.41 ± 1.40a 39.11 ± 1.30ab P b b b b b b MUFAs 58.80 ± 1.95b 69.83 ± 1.81a 53.96 ± 1.49b 55.36 ± 1.97b 56.39 ± 1.66b 54.34 ± 1.93b P a a a a a a c d b c d a PUFAs 4.90 ± 0.24c 3.58 ± 0.17c 5.47 ± 0.23c 4.69 ± 0.20c 3.20 ± 0.13c 6.56 ± 0.21c PUFAs/SFAs 0.13ab 0.13ab 0.13ab 0.12bc 0.08c 0.17a n–6/n–3 4.83b 1.70d 5.05b 7.67a 4.11c 1.09e Seed P SFAs 12.77 ± 0.55bc 11.51 ± 0.43c 15.89 ± 0.64a 13.79 ± 0.51b 14.00 ± 0.51b 11.39 ± 0.41c P c c c c c c MUFAs 16.38 ± 0.71d 16.59 ± 0.55cd 18.58 ± 0.79b 18.24 ± 0.69bc 18.99 ± 0.77b 21.55 ± 0.81a P b b b b b b ab a c abc bc bc PUFAs 70.84 ± 1.50a 71.90 ± 1.75a 65.53 ± 1.70a 67.97 ± 1.52a 67.01 ± 1.84a 67.06 ± 1.84a PUFAs/SFAs 5.55c 6.25a 4.12e 4.93d 4.79d 5.89b n–6/n–3 1.49a 1.41b 1.06e 1.19d 1.06e 1.38c C1- C6, sea buckthorn (ssp. carpatica) cultivars. Values are mean ± SD of three samples of each fruit part, analyzed individually in triplicate. Means in the same row followed by different superscript letters indicate significant differences (p < 0.05) among cultivars (C1-C5); means in the same column followed by different subscript letters indicate significant differences (p < 0.05) between fatty acid classes of each cultivar; SFAs, saturated fatty acids; MUFAs, monounsaturated fatty acids; PUFAs, polyunsaturated fatty acids. the major fatty acids (Table 5). All the values of PUFA/ presented the highest values (p < 0.05) for PUFA/SFA SFA ratios were close to 1, varying between 0.67 (for ratios. The n-6/n-3 ratios varied between 1.4 (in C1) C4) and 1.36 (for C2), respectively. Comparing the and 4.1 (in C3) (Table 5). Recent studies have shown pulp/peel lipid fractions from the studied cultivars, PLs that a balanced intake of dietary PUFA and SFA (ranged between 1.0 and 1.5) can contribute to reduce cardiovascular diseases [23,24]. The glycerophospholipids from pulp/peel oils of subspecies sinensis, rhamnoides and mongolica presented greater amounts of the 18:2n-6 (25.7%, 24.2% and 32.1%, respectively) and 18:3n-3 (15.4%, 12.9% and 10%, respectively) fatty acids than those of corresponding PLs from the present study [16,17]. The phospholipid fractions from SB pulp oils of cv. Indian- summer exhibited much higher amounts of palmitoleic (22.7-25%) and lower amounts of oleic (1.4-2.4%) acids than coresponding samples of this work [18]. In seeds PLs, PUFAs were present in a significantly greater proportion (p < 0.05), than SFAs and MUFAs (Tables 4 and 6). The oleic and linoleic acid contents (Table 4) were comparable with the values reported for the seeds of Asian and European SB berries [16-18]. Small variations of n-6/n-3 ratios were observed for the seed oils PLs, the values (Table 6) being close to the recommended essential fatty acid balance, reported in Figure 2 Comparison of the fatty acid classes compositions (as literature [25]. As shown in Figure 4 (a) and (c) the aver- % of total fatty acids) from the oils of different parts of sea age value of MUFAs was significantly higher, in the berry buckthorn fruits (ssp. carpatica). pulp/peel oil PL than in the seed oil PL (53.5% vs 17.9%, http://journal.chemistrycentral.com/content/6/1/106 Dulf

Table 3 Fatty acid composition (weight % of total fatty acids) of individual lipid classes from pulp/peel oils of different cultivars (C1-C6) of sea buckthorn Journal Central Chemistry fruits (ssp. carpatica) Fatty acids (weight % of total fatty acids; mean ± SD, n = 3) Species C14:0 C15:0 C16:0 C16:1n-9 C16:1n-7 C17:0 C18:0 C18:1n-9 C18:1n-7 C18:2n-6 C18:3n-3 C20:0 C20:1n-9 C1 PL 0.36 ± 0.03 nd 24.48 ± 0.82 nd 14.57 ± 0.42 nd 1.34 ± 0.04 34.13 ± 0.85 6.21 ± 0.20 10.82 ± 0.45 7.54 ± 0.25 0.55 ± 0.04 nd FFA 1.25 ± 0.10 nd 32.09 ± 0.80 nd 17.70 ± 0.48 nd 18.20 ± 0.60 18.80 ± 0.57 3.94 ± 0.11 5.82 ± 0.22 2.20 ± 0.10 nd nd 2012, TAG 0.26 ± 0.04 0.03 ± 0.02 38.98 ± 1.10 0.12 ± 0.03 21.16 ± 0.52 0.06 ± 0.02 0.98 ± 0.10 28.98 ± 0.75 6.13 ± 0.25 2.69 ± 0.11 0.49 ± 0.04 0.11 ± 0.02 0.02 ± 0.01

SE 1.35 ± 0.12 nd 27.53 ± 0.90 0.90 ± 0.04 1.52 ± 0.09 0.52 ± 0.06 38.85 ± 1.11 20.24 ± 0.56 0.27 ± 0.05 6.19 ± 0.25 0.94 ± 0.04 1.70 ± 0.08 nd 6 :106 C2 PL 0.34 ± 0.04 nd 17.52 ± 0.58 nd 10.34 ± 0.38 nd 1.13 ± 0.04 39.57 ± 0.80 5.18 ± 0.19 17.09 ± 0.60 8.83 ± 0.28 tr nd FFA 1.50 ± 0.10 nd 33.98 ± 0.89 nd 14.83 ± 0.42 nd 17.26 ± 0.58 22.55 ± 0.40 3.60 ± 0.14 4.60 ± 0.18 1.68 ± 0.06 nd nd TAG 0.57 ± 0.04 0.02 ± 0.01 24.39 ± 0.78 0.32 ± 0.03 13.81 ± 0.46 tr 2.04 ± 0.12 48.83 ± 0.90 5.75 ± 0.18 2.52 ± 0.10 1.48 ± 0.12 0.13 ± 0.02 0.15 ± 0.02 SE 1.65 ± 0.09 nd 27.77 ± 0.63 0.60 ± 0.04 1.12 ± 0.11 0.42 ± 0.04 36.52 ± 0.84 22.82 ± 0.78 0.32 ± 0.04 6.60 ± 0.28 0.68 ± 0.10 1.50 ± 0.07 nd C3 PL 0.55 ± 0.05 nd 23.97 ± 0.48 nd 21.00 ± 0.58 nd 1.40 ± 0.15 20.55 ± 0.55 7.71 ± 0.30 19.45 ± 0.70 4.72 ± 0.18 0.64 ± 0.04 nd FFA 1.32 ± 0.08 nd 35.52 ± 0.95 nd 16.20 ± 0.62 nd 18.20 ± 0.53 18.84 ± 0.50 3.20 ± 0.12 4.82 ± 0.15 1.90 ± 0.06 nd nd TAG 0.38 ± 0.04 tr 40.16 ± 1.18 0.08 ± 0.02 26.31 ± 0.72 0.04 ± 0.02 0.83 ± 0.06 19.81 ± 0.40 6.70 ± 0.15 4.84 ± 0.14 0.63 ± 0.07 0.16 ± 0.02 0.08 ± 0.02 SE 1.42 ± 0.08 nd 26.53 ± 0.52 0.82 ± 0.03 1.42 ± 0.19 0.50 ± 0.05 39.89 ± 1.15 16.60 ± 0.42 0.82 ± 0.06 8.60 ± 0.28 1.20 ± 0.05 2.20 ± 0.10 nd C4 PL 0.72 ± 0.05 nd 27.22 ± 0.72 nd 19.90 ± 0.58 nd 0.88 ± 0.05 23.24 ± 0.62 8.08 ± 0.25 14.74 ± 0.48 4.75 ± 0.16 0.48 ± 0.04 nd FFA 1.88 ± 0.08 nd 36.42 ± 0.80 nd 16.72 ± 0.68 nd 16.85 ± 0.62 18.15 ± 0.45 3.12 ± 0.13 4.38 ± 0.16 2.48 ± 0.12 nd nd TAG 0.28 ± 0.04 tr 40.45 ± 1.12 0.03 ± 0.02 25.64 ± 0.72 0.02 ± 0.01 0.82 ± 0.04 21.17 ± 0.43 7.03 ± 0.28 3.96 ± 0.15 0.46 ± 0.04 0.14 ± 0.02 tr SE 0.88 ± 0.06 nd 29.22 ± 0.72 0.30 ± 0.04 0.90 ± 0.08 0.20 ± 0.04 33.18 ± 0.72 25.44 ± 0.70 1.20 ± 0.05 5.20 ± 0.20 1.60 ± 0.05 1.88 ± 0.09 nd C5 PL 0.26 ± 0.03 nd 20.27 ± 0.57 nd 22.12 ± 0.80 nd 3.48 ± 0.14 27.22 ± 0.60 7.31 ± 0.25 14.21 ± 0.32 4.60 ± 0.20 0.52 ± 0.03 nd FFA 1.72 ± 0.10 nd 30.54 ± 0.81 nd 14.65 ± 0.46 nd 16.90 ± 0.65 23.70 ± 0.82 4.20 ± 0.16 5.89 ± 0.19 2.40 ± 0.10 nd nd TAG 0.30 ± 0.04 tr 39.19 ± 0.91 0.06 ± 0.02 24.20 ± 0.52 0.02 ± 0.01 0.94 ± 0.10 24.94 ± 0.71 6.53 ± 0.22 2.93 ± 0.11 0.62 ± 0.06 0.20 ± 0.03 0.08 ± 0.02 SE 2.20 ± 0.09 nd 26.42 ± 0.52 0.70 ± 0.03 1.40 ± 0.11 0.70 ± 0.04 40.05 ± 0.92 19.60 ± 0.54 0.48 ± 0.06 5.80 ± 0.20 1.00 ± 0.05 1.65 ± 0.05 nd C6 PL 0.45 ± 0.05 nd 21.75 ± 0.57 nd 22.07 ± 0.60 nd 1.21 ± 0.09 25.83 ± 0.75 7.33 ± 0.30 15.10 ± 0.30 5.91 ± 0.19 0.36 ± 0.03 nd FFA 1.68 ± 0.08 nd 33.09 ± 0.61 nd 15.60 ± 0.42 nd 15.64 ± 0.45 21.78 ± 0.48 4.10 ± 0.15 5.95 ± 0.15 2.17 ± 0.10 nd nd

TAG 0.42 ± 0.04 0.05 ± 0.02 36.97 ± 1.13 0.08 ± 0.03 25.59 ± 0.92 0.03 ± 0.01 0.96 ± 0.09 24.82 ± 0.65 6.66 ± 0.28 3.53 ± 0.14 0.68 ± 0.05 0.13 ± 0.02 0.10 ± 0.03 12 of 6 Page SE 1.20 ± 0.06 nd 24.20 ± 0.61 1.10 ± 0.03 1.30 ± 0.12 0.88 ± 0.03 39.80 ± 0.88 22.34 ± 0.66 0.82 ± 0.05 5.70 ± 0.18 0.78 ± 0.07 1.88 ± 0.07 nd PL- polar lipids, FFA- free fatty acids, TAG- triacylglycerols, SE- sterol esters. http://journal.chemistrycentral.com/content/6/1/106 Dulf

Table 4 Fatty acid composition (weight % of total fatty acids) of individual lipid classes from seed oils of different cultivars (C1-C6) of sea buckthorn fruits Journal Central Chemistry (ssp. carpatica) Fatty acids (weight % of total fatty acids; mean ± SD, n = 3) Species C14:0 C15:0 C16:0 C16:1n-9 C16:1n-7 C17:0 C18:0 C18:1n-9 C18:1n-7 C18:2n-6 C18:3n-3 C20:0 C20:1n-9 C1 PL 0.16 ± 0.02 0.13 ± 0.03 17.21 ± 0.64 nd 0.26 ± 0.04 tr 6.30 ± 0.20 14.23 ± 0.57 4.32 ± 0.15 45.48 ± 1.22 11.09 ± 0.45 0.82 ± 0.04 tr FFA 0.46 ± 0.04 tr 25.33 ± 0.80 nd 0.41 ± 0.02 tr 9.13 ± 0.28 17.98 ± 0.62 4.56 ± 0.14 30.34 ± 0.90 11.79 ± 0.40 tr nd 2012, TAG 0.09 ± 0.02 0.14 ± 0.02 8.19 ± 0.25 nd 0.55 ± 0.02 0.04 ± 0.02 2.51 ± 0.16 17.94 ± 0.66 2.27 ± 0.09 43.65 ± 1.12 24.22 ± 0.82 0.29 ± 0.03 0.10 ± 0.02

SE 0.56 ± 0.05 0.05 ± 0.02 24.59 ± 0.62 nd 0.22 ± 0.03 0.39 ± 0.04 29.36 ± 0.77 13.37 ± 0.43 1.78 ± 0.08 18.05 ± 0.50 7.94 ± 0.22 3.68 ± 012 nd 6 :106 C2 PL 0.06 ± 0.02 0.06 ± 0.03 16.33 ± 0.42 nd 0.09 ± 0.02 tr 6.93 ± 0.28 14.56 ± 0.40 3.41 ± 0.15 46.98 ± 1.23 10.31 ± 0.38 0.96 ± 0.04 0.30 ± 0.03 FFA 1.20 ± 0.08 0.30 ± 0.02 26.32 ± 0.62 nd 0.20 ± 0.03 0.30 ± 0.04 11.20 ± 0.38 16.20 ± 0.48 3.10 ± 0.12 27.78 ± 0.85 12.20 ± 0.40 1.20 ± 0.05 nd TAG 0.06 ± 0.03 0.09 ± 0.02 5.63 ± 0.18 nd 0.16 ± 0.03 tr 2.32 ± 0.16 13.56 ± 0.54 1.25 ± 0.05 44.02 ± 1.10 32.68 ± 0.95 0.12 ± 0.03 0.12 ± 0.03 SE 0.25 ± 0.03 0.02 ± 0.01 26.20 ± 0.82 nd 0.30 ± 0.04 0.42 ± 0.05 28.40 ± 0.72 12.27 ± 0.50 1.65 ± 0.06 17.55 ± 0.68 8.74 ± 0.30 4.20 ± 0.18 nd C3 PL 0.15 ± 0.02 0.10 ± 0.03 18.69 ± 0.52 nd 0.15 ± 0.02 0.14 ± 0.03 8.64 ± 0.32 12.72 ± 0.52 4.05 ± 0.16 40.90 ± 0.95 13.33 ± 0.42 0.99 ± 0.06 0.15 ± 0.2 FFA 1.60 ± 0.08 0.20 ± 0.02 25.80 ± 0.76 nd 0.30 ± 0.03 0.60 ± 0.06 15.10 ± 0.44 15.20 ± 0.39 1.98 ± 0.05 25.80 ± 0.85 12.52 ± 0.52 0.90 ± 0.07 nd TAG tr tr 7.99 ± 0.28 nd 0.19 ± 0.02 tr 3.55 ± 0.20 17.72 ± 0.68 1.84 ± 0.06 36.05 ± 1.10 31.77 ± 0.88 0.60 ± 0.05 0.28 ± 0.04 SE 0.62 ± 0.03 0.04 ± 0.02 25.20 ± 0.78 nd 0.28 ± 0.03 0.26 ± 0.03 31.68 ± 0.88 10.82 ± 0.40 1.42 ± 0.06 16.80 ± 0.65 7.28 ± 0.28 5.60 ± 0.20 nd C4 PL 0.12 ± 0.03 0.06 ± 0.02 17.29 ± 0.50 nd 0.21 ± 0.03 0.11 ± 0.02 6.95 ± 0.25 12.61 ± 0.38 4.62 ± 0.17 43.08 ± 1.20 13.85 ± 0.52 1.10 ± 0.06 tr FFA 1.10 ± 0.06 0.15 ± 0.03 27.58 ± 0.60 nd 0.15 ± 0.02 0.25 ± 0.05 14.80 ± 0.38 12.85 ± 0.42 2.85 ± 0.10 27.10 ± 0.90 12.20 ± 0.40 0.97 ± 0.07 nd TAG 0.06 ± 0.02 0.12 ± 0.03 8.76 ± 0.30 nd 0.41 ± 0.04 0.04 ± 0.02 3.10 ± 0.10 15.55 ± 0.55 2.28 ± 0.12 36.84 ± 1.18 32.26 ± 0.80 0.47 ± 0.06 0.11 ± 0.03 SE 0.42 ± 0.03 0.02 ± 0.01 27.20 ± 0.72 nd 0.18 ± 0.02 0.35 ± 0.04 30.13 ± 1.00 11.25 ± 0.32 1.60 ± 0.07 16.15 ± 0.60 6.90 ± 0.20 5.80 ± 0.15 nd C5 PL 0.11 ± 0.02 tr 20.62 ± 0.80 nd 0.09 ± 0.03 0.10 ± 0.02 7.16 ± 0.22 12.33 ± 0.52 4.46 ± 0.20 40.86 ± 1.25 13.04 ± 0.38 1.22 ± 0.08 tr FFA 0.93 ± 0.04 0.23 ± 0.03 20.09 ± 0.78 nd tr tr 12.26 ± 0.40 13.37 ± 0.44 2.96 ± 0.12 29.53 ± 1.00 19.55 ± 0.62 1.09 ± 0.06 nd TAG 0.07 ± 0.03 tr 8.82 ± 0.32 nd 0.41 ± 0.05 0.02 ± 0.01 2.73 ± 0.09 15.75 ± 0.50 2.40 ± 0.10 35.28 ± 1.10 34.03 ± 1.12 0.41 ± 0.04 0.07 ± 0.02 SE 0.50 ± 0.03 tr 23.80 ± 0.84 nd 0.20 ± 0.04 0.30 ± 0.04 32.80 ± 0.98 12.30 ± 0.48 1.90 ± 0.06 15.85 ± 0.45 6.90 ± 0.25 5.45 ± 0.20 nd C6 PL 0.10 ± 0.02 0.08 ± 0.02 20.23 ± 0.54 nd 0.14 ± 0.03 0.23 ± 0.03 6.94 ± 0.18 15.30 ± 0.39 3.91 ± 0.16 41.54 ± 1.22 10.51 ± 0.42 1.02 ± 0.05 tr FFA 1.44 ± 0.06 0.28 ± 0.04 24.64 ± 0.78 nd 0.41 ± 0.06 0.50 ± 0.05 14.41 ± 0.38 14.01 ± 0.39 2.67 ± 0.12 26.12 ± 0.95 14.00 ± 0.60 1.53 ± 0.06 nd

TAG 0.05 ± 0.02 0.12 ± 0.03 7.24 ± 0.30 nd 0.29 ± 0.03 0.05 ± 0.01 2.86 ± 0.12 18.61 ± 0.52 1.77 ± 0.07 39.70 ± 1.12 28.85 ± 0.90 0.31 ± 0.03 0.16 ± 0.03 12 of 7 Page SE 0.30 ± 0.04 tr 25.20 ± 0.82 nd 0.28 ± 0.03 0.40 ± 0.06 31.80 ± 1.10 12.80 ± 0.42 1.30 ± 0.06 17.20 ± 0.50 5.80 ± 0.20 4.92 ± 0.18 nd PL- polar lipids, FFA- free fatty acids, TAG- triacylglycerols, SE- sterol esters. Dulf Chemistry Central Journal 2012, 6:106 Page 8 of 12 http://journal.chemistrycentral.com/content/6/1/106

Table 5 Fatty acid composition (weight % of total fatty Table 6 Fatty acid composition (weight % of total fatty acids) of individual lipid classes from pulp/peel oils of acids) of individual lipid classes from seed oils of different cultivars of sea buckthorn fruits (ssp. carpatica) different cultivars of sea buckthorn fruits (ssp. carpatica) Fatty acids (weight % of total fatty acids) Fatty acids (weight % of total fatty acids) P P P P P P Species SFA MUFA PUFA PUFA/ n-6/ Species SFA MUFA PUFA PUFA/ n-6/ SFA n-3 SFA n-3 C1 C1 b a c b c a PL 26.73 ± 0.93d 54.91 ± 1.47a 18.36 ± 0.70a 0.69a 1.44d PL 24.62 ± 0.93c 18.81 ± 0.76b 56.57 ± 1.67b 2.30b 4.10a a b c b c a FFA 51.54 ± 1.50b 40.44 ± 1.16b 8.02 ± 0.32b 0.16b 2.65c FFA 34.92 ± 1.12b 22.95 ± 0.78a 42.13 ± 1.30c 1.21c 2.57b b a c c b a TAG 40.41 ± 1.30c 56.41 ± 1.56a 3.18 ± 0.15c 0.08b 5.49b TAG 11.26 ± 0.50d 20.87 ± 0.79ab 67.87 ± 1.94a 6.03a 1.80d a b c a c b SE 69.94 ± 2.27a 22.93 ± 0.74c 7.13 ± 0.29b 0.10b 6.59a SE 58.63 ± 1.62a 15.37 ± 0.54c 25.99 ± 0.72d 0.44d 2.27c C2 C2 c a b b c a PL 18.99 ± 0.66d 55.09 ± 1.37b 25.92 ± 0.88a 1.36a 1.93c PL 24.34 ± 0.79c 18.36 ± 0.60a 57.29 ± 1.61b 2.35b 4.56a a b c a b a FFA 52.74 ± 1.57b 40.98 ± 0.96c 6.28 ± 0.24b 0.12b 2.74b FFA 40.52 ± 1.19b 19.50 ± 0.63a 39.98 ± 1.25c 0.99c 2.28b b a c c b a TAG 27.15 ± 0.97c 68.86 ± 1.59a 3.99 ± 0.22c 0.15b 1.71c TAG 8.22 ± 0.42d 15.08 ± 0.65b 76.70 ± 2.05a 9.33a 1.35d a b c a c b SE 67.86 ± 1.67a 24.86 ± 0.97d 7.28 ± 0.38b 0.11b 9.71a SE 59.49 ± 1.81a 14.22 ± 0.60b 26.29 ± 0.98d 0.44d 2.01c C3 C3 b a c b c a PL 26.56 ± 0.72d 49.26 ± 1.43b 24.18 ± 0.88a 0.91a 4.12c PL 28.70 ± 0.98c 17.07 ± 0.72b 54.23 ± 1.37b 1.89b 3.07a a b c a c b FFA 55.04 ± 1.56b 38.24 ± 1.24c 6.72 ± 0.21c 0.12b 2.54d FFA 44.20 ± 1.43b 17.48 ± 0.47b 38.32 ± 1.37c 0.87c 2.06c b a c c b a TAG 41.56 ± 1.32c 52.97 ± 1.31a 5.47 ± 0.21c 0.13b 7.67a TAG 12.14 ± 0.53d 20.03 ± 0.80a 67.83 ± 1.98a 5.59a 1.13d a b c a c b SE 70.54 ± 1.90a 19.66 ± 0.70d 9.80 ± 0.33b 0.14b 7.17b SE 63.40 ± 1.94a 12.52 ± 0.49c 24.08 ± 0.93d 0.38d 2.31b C4 C4 b a c b c a PL 29.29 ± 0.86d 51.21 ± 1.45a 19.49 ± 0.64a 0.67a 3.10b PL 25.63 ± 0.88c 17.44 ± 0.58ab 56.93 ± 1.72b 2.22b 3.11a a b c a c b FFA 55.15 ± 1.50b 37.99 ± 1.26b 6.86 ± 0.28b 0.12b 1.77c FFA 44.85 ± 1.19b 15.85 ± 0.54b 39.30 ± 1.30c 0.88c 2.22b b a c c b a TAG 41.70 ± 1.23c 53.87 ± 1.45a 4.43 ± 0.19c 0.11b 8.52a TAG 12.55 ± 0.53d 18.35 ± 0.74a 69.10 ± 1.98a 5.51a 1.14c a b c a c b SE 65.36 ± 1.63a 27.84 ± 0.87c 6.80 ± 0.25b 0.10b 3.25b SE 63.92 ± 1.95a 13.03 ± 0.41c 23.05 ± 0.80d 0.36d 2.34b C5 C5 b a c b c a PL 24.53 ± 0.77d 56.65 ± 1.65a 18.82 ± 0.52a 0.77a 3.09c PL 29.22 ± 1.14c 16.88 ± 0.75ab 53.90 ± 1.63b 1.84b 3.13a a b c b c a FFA 49.16 ± 1.56b 42.55 ± 1.44b 8.29 ± 0.29b 0.17b 2.45d FFA 34.60 ± 1.31b 16.32 ± 0.56b 49.08 ± 1.62c 1.42c 1.51c b a c c b a TAG 40.64 ± 1.09c 55.81 ± 1.42a 3.55 ± 0.17d 0.09c 4.77b TAG 12.05 ± 0.49d 18.63 ± 0.67a 69.32 ± 2.22a 5.75a 1.04d a b c a c b SE 71.02 ± 1.62a 22.18 ± 0.74c 6.80 ± 0.25c 0.10c 5.80a SE 62.85 ± 2.09a 14.40 ± 0.58c 22.75 ± 0.70d 0.36d 2.30b C6 C6 b a c b c a PL 23.77 ± 0.74d 55.22 ± 1.65a 21.01 ± 0.49a 0.88a 2.56c PL 28.60 ± 0.84c 19.35 ± 0.58a 52.05 ± 1.64b 1.82b 3.95a a b c a c b FFA 50.41 ± 1.14b 41.47 ± 1.05b 8.12 ± 0.25b 0.16b 2.75c FFA 42.80 ± 1.37b 17.08 ± 0.57b 40.12 ± 1.55c 0.94c 1.87c b a c c b a TAG 38.55 ± 1.31c 57.25 ± 1.91a 4.20 ± 0.19d 0.11c 5.22b TAG 10.62 ± 0.51d 20.83 ± 0.65a 68.55 ± 2.02a 6.46a 1.38d a b c a c b SE 67.96 ± 1.65a 25.56 ± 0.86c 6.48 ± 0.25c 0.10c 7.31a SE 62.62 ± 2.20a 14.38 ± 0.51c 23.00 ± 0.70d 0.37d 2.97b Values are mean ± SD of three samples, analyzed individually in triplicate Values are mean ± SD of three samples, analyzed individually in triplicate Means in the same row followed by different superscript letters indicate Means in the same row followed by different superscript letters indicate significant differences (p < 0.05) among fatty acid classes; means in the same significant differences (p < 0.05) among fatty acid classes; means in the same column followed by different subscript letters indicate significant differences column followed by different subscript letters indicate significant differences (p < 0.05) among lipid classes of each cultivar. (p < 0.05) among lipid classes of each cultivar. PL, polar lipids; FFA, free fatty acids; TAG, triacylglycerols; SE, steryl esters; SFA, PL, polar lipids; FFA, free fatty acids; TAG, triacylglycerols; SE, steryl esters; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. polyunsaturated fatty acids.

p < 0.001) and vice versa for PUFAs (21.3% vs 54.9%, of n-6/n-3 ratios of the berry pulp/peel oils SEs closely p < 0.001). resembled those of the berry pulp/peel oil TAGs, excepting cultivars C2 and C4 (see Table 5). Comparing with Fatty acid composition of SEs the other lipid fractions from these oils, the SEs had the The major fatty acids in ascending order in all berry soft highest content of SFAs (p < 0.05). This class of fatty acids part oils were linoleic (5-9%), oleic (16-26%), palmitic were also predominant in seed oil SEs due to their high (24-30%), and stearic (33-41%). The relatively high values content of palmitic and stearic acids (Tables 4 and 6). Dulf Chemistry Central Journal 2012, 6:106 Page 9 of 12 http://journal.chemistrycentral.com/content/6/1/106

Figure 3 GC-MS chromatogram of FAMEs from the TAGs of pulp/peel (a) and seeds (b) of sea buckthorn berries (ssp. carpatica).

It is interesting to note that the arachidic acid levels in seed oils FFAs, respectively (Tables 5 and 6). Low were around of 2% in pulp/peel oils SEs and between 3% levels of free fatty acids (2-4%) have been reported for and 6% in seed oils SEs. oils from air- and freeze- dried SB (cultivar Indian- Sum- The long chain saturated fatty acids, with more than mer) seeds and pulps by Gutierrez et al. [18], with the 20 carbons, are major structural components of plant similar fatty acid profiles to those of neutral lipids. The cuticular lipids [26]. quality of the vegetable oils depends on their lipid pro- Average proportions of MUFAs and SFAs were signifi- file. A high proportion of the free fatty acids offers an cantly higher in pulp/peel oils SEs than in seed oils unacceptable flavour to the oils [29]. Differences be- SEs (p < 0.01) and vice versa for PUFAs (p < 0.001) (see tween the fatty acid profiles of the studied lipid fractions Figure 4 (a), (b) and (c)). could be due to the different phases of biosynthesis and The levels of SFAs from studied SB oils SEs were com- accumulation of TAGs, SEs, PLs and fatty acids. In the parable to those reported for other berry SE fractions first stage PLs and SEs are synthesized with the SFAs as [27,28]. dominating fatty acid classes in their composition. The TAGs proportion, with high unsaturated fatty acid con- Fatty acid composition of FFA tent, increases in the second phase of biosynthesis The fatty acid profiles of the FFA fractions of pulp/peel [28,30,31]. and seed oils were relatively similar to those of TAGs excepting the proportions for stearic acid (in berry pulp/ Conclusions peel oils) and for palmitic, stearic and α-linolenic acids This study provides valuable information about the fatty (in seed oils), respectively (Tables 3 and 4). Generally, acid composition of the major lipid fractions (PLs, FFAs, the SFAs were the most representative in all analysed TAGs and SEs) in the oils extracted from different berry cultivars, followed by MUFAs in pulp/peel and PUFAs parts of six SB subspecies (ssp. carpatica). Dulf Chemistry Central Journal 2012, 6:106 Page 10 of 12 http://journal.chemistrycentral.com/content/6/1/106

Figure 4 The average proportions of fatty acid classes (3a- % of MUFAs, 3b- % of SFAs, 3c- % of PUFAs) in lipid fractions from pulp/ peel and seeds of sea buckthorn berries (ssp. carpatica).

Comparing with the other European or Asiatic SB sub- Seeds were isolated manually from the fruits just be- species, all berry parts of the analyzed cultivars exhibited fore analysis at the laboratory. higher oil content. Moreover, the pulp/peel oils of ssp. Standards of fatty acid methyl esters (37component carpatica were found to contain high levels of oleic acid FAME Mix, SUPELCO, catalog No: 47885-U) were pur- and slightly lower amounts of linoleic and α-linolenic acids. chased from Supelco (Bellefonte, PA, USA). All reagents, The PLs presented the highest PUFA/SFA ratios be- chemicals of analytical or HPLC purity and polar tween the analysed pulp/peel lipid fractions (from 0.67 lipid standards were purchased from Sigma–Aldrich to 1.36), values which were close to the recommended (St. Louis, MO, USA). The thin layer chromatography PUFA/SFA intake of nutrition scientists (1–1.5). (TLC) plates (silica gel 60 F254, 20 × 20 cm) were pur- The seed oils could be considered excellent sources of chased from Merck (Darmstadt, Germany). PUFAs due to their high contents of linoleic and α- linolenic acids which in human body are precursors of other long-chain n-3 and n-6 fatty acids. Lipid extraction The data obtained in the present work are useful to The oils of the whole berries, pulp/peel and seeds were identify suitable SB cultivars when organizing the berry extracted from 5 g of samples using a methanol/chloro- breeding programs and also provides important informa- form extraction procedure [17,32]. The sample was tion for food and pharmaceutical industry. homogenized in methanol (50 mL) for 1 min with a high-power homogeniser (MICCRA D-9, Germany), chloroform (100 mL) was added, and homogenization Methods was continued for a further 2 min. The mixture was fil- Samples and chemicals tered and the solid residue resuspended in chloroform: Berries of SB (Hippophae rhamnoides L., ssp. carpatica,cvs. methanol (2:1, v/v, 150 mL) and homogenized for an- Auras (C1), Serpenta (C2), Tiberiu (C3), Victoria (C4), other 3 min. The mixture was filtered again and washed Ovidiu (C5) and Silvia (C6)) were collected from the experi- with 150 mL chloroform: methanol (2:1, v/v). The fil- mental field of the Fruit Research Station- Bacau, Romania. trates were combined and cleaned with 0.88% potassium The fruits were collected during June to November of 2011 chloride water solution and methanol: water (1:1, v/v) at the stage of commercial maturity and were stored in poly- solution. The bottom layer containing the purified lipids ethylene bags at -20°C until analysis. was filtered before the solvent was removed on a rotary Dulf Chemistry Central Journal 2012, 6:106 Page 11 of 12 http://journal.chemistrycentral.com/content/6/1/106

evaporator. The lipid samples were transferred to vials ion electron impact (EI) mass spectra was recorded at an with 4 mL chloroform (stock solution), and stored at ionization energy of 70 eV and a trap current of 100 μA −18°C until they were analyzed. with a source temperature of 150°C. The mass scans were performed within the range of m/z: 22–395 at a rate of Fatty acid composition 0.14 scan/s with an intermediate time of 0.02 s between Fatty acid methyl esters (FAMEs) were obtained from the scans. The injection volume was 0.5 μl. Identification lipids using acid-catalysed transesterification procedure of FAMEs was done comparing their retention times with described by Christie [33]. those of known standards (37component FAME Mix, For total FAME analysis, 0.2 mL of each oil extract SUPELCO # 47885-U) and the resulting mass spectra to (stock solution) was dissolved in 1 ml toluene and then the ones from our database (NIST MS Search 2.0). methylated with 1% sulfuric acid in methanol (2 ml), using a 15 mL screw-cap Pyrex culture tube at 80°Cfor Statistical analyses 2 h. After cooling to room temperature, 5 ml of water All the extractions and GC-MS analysis were made in (with 5%NaCl) and 2 mL hexane were added. The hex- triplicate. Dates were expressed as mean ± S.D. Statis- ane layer was collected and concentrated before the tical differences among samples were estimated using FAMEs were applied to TLC plates. The loaded TLC Student’s t-test and ANOVA (Tukey’s Multiple Compari- plates were developed in a mixture of petroleum ether: son Test; GraphPad Prism Version 4.0, Graph Pad Soft- diethyl ether: acetic acid (85:15:1, v/v/v), sprayed with 2’, ware Inc., San Diego CA). P < 0.05 was accepted to be 7’-dichlorofluoroscein/methanol (0.1% w/v) and viewed statistical significant. under UV light (254 nm) [34]. The corresponding FAME band was scraped and eluted with chloroform. The Abbreviations eluent was removed with a gentle nitrogen stream. The Ssp: Subspecies; sp: Species; PLs: Polar lipids; FFAs: Free fatty acids; TAGs: Triacylglycerols; SEs: Sterol esters; PUFAs: Polyunsaturated fatty acids; FAMEs were dissolved in 1 mL hexane and placed into a SFAs: Saturated fatty acids; MUFAs: Monounsaturated fatty acids; SB: Sea gas chromatography (GC) vial. The vial was capped and buckthorn; f.w.: Fresh weight; w/w: Weight/weight; cv: Cultivars; FAMEs: Fatty placed at −18°C until GC analysis. acid methyl esters; TLC: Thin layer chromatography; GC-MS: Gas chromatography–mass spectrometry. The lipid classes (PLs, FFAs, TAGs and SEs) were separated also by TLC. For fractionation, 0.2 ml of each Competing interests oil (stock solution) was applied on the TLC plates, de- The author declares that he has no competing interests. veloped and viewed under UV light as above. The Authors’ contributions polar lipids remained at the origin of the plates (the FVD carried out all experiments and prepared the final manuscript. first band). The other major lipid class bands from TLC plates, were identified using commercial standards Acknowledgements (which were run in parallel with the samples) and then This work was financially supported by the Research Grant of University of Agricultural Sciences and Veterinary Medicine nr.1215/4, 2012, Cluj-Napoca, scraped from the plates. The bands for PLs and FFAs Romania. The author thanks dr. I.V. Rati and prof. dr Carmen Socaciu for were eluted with methanol: chloroform (1:1, v/v), and providing the sea buckthorn berries. the upper two major bands corresponding to TAGs and Received: 24 June 2012 Accepted: 17 September 2012 SE respectively, were eluted with chloroform. After the Published: 20 September 2012 chloroform was evaporated under a nitrogen stream, the lipid classes were methylated (20 min at reflux for PLs References and 2 h at reflux for the other lipid fractions). The ex- 1. 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Vodnar DC, Socaciu C: Green tea increases the survival yield of ipped with a SUPELCOWAX 10 column (60 m × Bifidobacteria in simulated gastrointestinal environment and during 0.25 mm i.d., 0.25 μm film thickness; Supelco Inc., refrigerated conditions. Chem Cent J 2012, 6:61. 5. Yang B, Zheng J, Kallio H: Influence of origin, harvesting time and Bellefonte, PA). The initial oven temperature was 140°C, weather conditions on content of inositols and methylinositols in sea increased to 220°C with a rate of 7°C/min and then held at buckthorn (Hippophaë rhamnoides) berries. Food Chem 2011, this temperature for 23 min. Flow rate of the carrier gas 125(2):388–396. 6. Pintea A, Marpeau A, Faye M, Socaciu C, Gleizes M: Polar lipid and fatty He and the split ratio were 0.8 ml/min and 1:24, respect- acid distribution in carotenolipoprotein complexes extracted from sea ively. The injector temperature was 210°C. The positive buckthorn fruits. Phytochem Analysis 2001, 12:293–298. 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Submit your manuscript here: http://www.chemistrycentral.com/manuscript/ Chemical Papers 66 (10) 925–934 (2012) DOI: 10.2478/s11696-012-0156-0

ORIGINAL PAPER

Fatty acid and phytosterol contents of some Romanian wild and cultivated berry pomaces‡

Francisc Vasile Dulf*, Sanda Andrei, Andrea Bunea, Carmen Socaciu

University of Agricultural Sciences and Veterinary Medicine, 400372, Cluj-Napoca, M˘an˘a¸stur 3–5, Romania

Received 26 October 2011; Revised 13 January 2012; Accepted 16 January 2012

The total oil content and composition of fatty acids and phytosterols of five Transylvanian (Roma- nia) pomaces of wild and cultivated blueberries (Vaccinium myrtillus), wild cowberry (Vaccinium vitis-idaea) and raspberry (Rubus idaeus), and cultivated black chokeberry (Aronia melanocarpa), were determined by capillary gas chromatography. Out of the five pomace oils, the percentages of polyunsaturated fatty acids (PUFAs) ranged from 37 % to 69 %. The lipid classes analysed (PLs – polar lipids, TGs – triacylglycerols, SEs – sterol esters) were separated and identified using thin-layer chromatography. TGs showed the highest PUFAs content (ranging from 41.9 % to 72.5 %) and PUFAs/SFAs (saturated fatty acids) ratios (in the range of 5.8–33.1 %). In the case of PL and SE fractions, the levels of SFA were significantly higher than in TGs. The total amount of sterols was in the range of 101.6–168.2 mg per 100 g of lipids of the pomaces analysed. The predominant phytosterols were β-sitosterol, stigmastanol + isofucosterol, and campesterol. The results indicated that the investigated pomace oils, due to their good balance between n–6 and n–3 fatty acids (except for chokeberry) and high β-sitosterol content, could be excellent sources of PUFAs and phytosterols, thus suggesting potential value-added utilisation of berry waste oils for preparing functional foods or food supplements. c 2012 Institute of Chemistry, Slovak Academy of Sciences

Keywords: berry pomace, vegetable oil, fatty acid, phytosterol

Introduction manian wild berries collected for both domestic consumption and for export, especially to countries in the The global food and agricultural industries pro- European Union. The fruits of all three species are rich duce large volumes of both liquid and solid wastes in anti-oxidants. Speciﬁc fractions from the berries of annually. Direct disposal of these agro-industrial by- V. myrtillus, V. vitis-idaea,andR. idaeus have been products in the environment creates serious environ- claimed to possess anti-inﬂammatory, anti-ulcer, anti- mental problems (Rodríguez Couto, 2008). Beverage carcinogenic properties (Zheng & Wang, 2003; Seeram and juice industry wastes, mainly consisting of seeds et al., 2006; Ogawa et al., 2008). and peels, have attracted considerable attention as po- The climate and geographical conditions of Tran- tential sources of natural anti-oxidants (e.g. polyphe- sylvania favour the cultivation of berries. Recently, nols) and other bioactive compounds such as fatty more and more farmers have begun to cultivate new acids and sterols (Yi et al., 2009). berry species which are not native to Transylvania, Blueberry (Vaccinium myrtillus), cowberries (Vac- like black chokeberries (Aronia melanocarpa Elliot), cinium vitis-idaea), and raspberry (Rubus idaeus)are whichbelongtotheRosaceae family (genus Aronia). distributed naturally throughout Transylvania (Ro- Wild and cultivated berries are commonly used mania). These constitute the largest proportion of Ro- in Romania to produce jam, juice, and tea. Seeds

*Corresponding author, e-mail: francisc [email protected] ‡ Presented at the 5th Meeting on Chemistry & Life 2011, Brno, 14–16 September 2011.

Unauthenticated Download Date | 11/24/15 5:32 AM 926 F. V. Dulf et al./Chemical Papers 66 (10) 925–934 (2012) and seed-containing press residues (pomaces), as by- Experimental products of the processing, are potential raw materials of valuable seed oils (Bushman et al., 2004; Adhikari Chemicals and samples preparation et al., 2008; Yang et al., 2011). The percentages of seeds in Rosaceae berries Standards of fatty acid methyl esters (37 compo- range from 4.4 % to 12.2 % of fresh fruits mass. nent FAME Mix, SUPELCO, catalogue No: 47885-U) The fat content of these seed species varies from were purchased from Supelco (Bellefonte, PA, USA). 6.9 % to 23.2 % of dry matter (Johansson et al., All reagents and chemicals of analytical or HPLC 1997). The fresh fruits of Ericanae berries con- purity, polar lipid and sterol standards were pur- tain 0.7–13.8 % of seeds by mass and the lipid chased from Sigma–Aldrich (St. Louis, MO, USA). amount of the seeds is in the range of 4.7–30.6 % Boron trifluoride/methanol complex (14 % BF3 solu- of dry matter (Johansson et al., 1997; Yang et al., tion in methanol), which was used for derivatisation of 2003). the fatty acids and thin-layer chromatography (TLC) Lipids are important structural components of an- plates (silica gel 60 F254) were purchased from Merck imal and plant cells. In addition to essential fatty (Darmstadt, Germany). acids, the phytosterols have also been found to play Wild blueberries (Vaccinium myrtillus), cowber- an important role in human nutrition. Several envi- ries (Vaccinium vitis-idaea), and raspberries (Rubus ronmental factors, such as climatic conditions and al- idaeus) (crops 2010) were collected in the north- titude, are known to influence the yield and chemical west of Transylvania (Romania). Cultivated blueber- composition of vegetable oils. Some previous reports ries and chokeberry (Aronia melanocarpa)werefrom have revealed that berry seeds oils contained signifi- the local berry farms (Zalau, Transylvania, Romania). cant amounts of essential fatty acids with a good bal- All the fruit samples were processed in our labora- ance between n–6 and n–3 fatty acids (Parry et al., tory within 24–48 h. After juice-pressing, the wet press 2005; Oh et al., 2007). Several studies have shown residues (pomaces) were analysed. that diets rich in n–3 fatty acids can prevent coro- The total lipids were extracted from 5 g of fresh wet nary heart disease and certain forms of cancer (Mail- berry pomaces (wbp) using a methanol/chloroform ex- lard et al., 2002; Schaefer, 2002; Kramer et al., 2004; traction procedure (Yang et al., 2001). The sample Parry et al., 2005; Ramadan & Moersel, 2006; Yang was homogenised in methanol (50 mL) for 1 min in et al., 2011). According to previous studies, the most a high-power homogeniser (MICCRA D-9, Germany), abundant fatty acids in berry seeds triglycerides are chloroform (100 mL) was added, and homogenisation oleic, linoleic, α-linolenic, palmitic, and stearic acids was continued for a further 2 min. The mixture was (Johansson et al., 1997; Zlatanov, 1999; Yang et al., filtered through a Büchner funnel and the solid residue 2011). re-suspended in chloroform/methanol (ϕr =2:1, Plant sterols are minor biologically active compo- 150 mL) and homogenised for 3 min. The mixture nents of foods of plant origin. The most common phy- was re-filtered and washed with 150 mL of chloro- tosterols in seed oils, β-sitosterol, campesterol, and form/methanol (ϕr = 2 : 1). The combined filtrates stigmasterol are structurally similar to cholesterol. were cleaned with 0.88 % aqueous potassium chloride Studies have shown the excellent ability of these plant- solution and methanol/water (ϕr = 1 : 1) solution. derived sterols to reduce cholesterol absorption in hu- The washing procedure was repeated, and the bottom man. For this reason they have become ingredients in layer containing the purified lipids was filtered before frequent use in the functional food industry (Toivo et the solvent was removed using rotary evaporator. The al., 2001; Lagarda et al., 2006). lipid samples were transferred to vials with chloroform The available studies on berries refer to the prop- (4 mL) and stored at –18 ◦C until they were analysed. erties of polyphenols, especially to their anti-oxidant Fatty acid methyl esters (FAMEs) were obtained activities (Heinonen, 2007; Seeram, 2008; Szajdek & from lipids using boron trifluoride/methanol com- Borowska, 2008; Nurmi et al., 2009; Paredes-López et plex according to the method of Morrison and Smith al., 2010), but there are only a few reports related to (1964). the lipid compositions of berry pomaces. For total FAMEs analysis, 0.3 mL (10–25 mg) of The present study attempts to characterise the each lipid extract was methylated with BF3/methanol composition of fatty acids in total lipids and indi- complex(1mL)ina15mLscrew-capPyrexculture vidual lipid classes (polar lipids, triacylglycerols, and tube at 80 ◦C for 1 h. After cooling to room temper- sterol esters) and the phytosterols content and com- ature, a few drops of water and hexane (2 mL) were position of the oils recovered from the five different added. The hexane layer was separated, solvent evap- Transylvanian (Romania) pomaces: wild and culti- orated and the FAMEs mixture was applied to TLC vated blueberries (Vaccinium myrtillus), wild cow- plates. The loaded TLC plates were developed using a berry (Vaccinium vitis-idaea) and raspberry (Rubus mixture of petroleum ether/diethyl ether/acetic acid idaeus), and cultivated black chokeberry (Aronia (ϕr = 85 : 15 : 1), sprayed at the edge of TLC plate melanocarpa). with 2,7-dichlorofluorescein/methanol (0.1 mass %)

Unauthenticated Download Date | 11/24/15 5:32 AM F. V. Dulf et al./Chemical Papers 66 (10) 925–934 (2012) 927 and visualised under UV light (254 nm) (Kramer et the bottle was washed with Milli-Q water (60 mL). al., 2004). The corresponding FAMEs band was re- The unsaponifiables (the total sterols) in the com- moved, eluted with chloroform, filtered and the chlo- bined solution were then extracted with diethyl ether roform was removed by a stream of nitrogen. The (3 × 50 mL). The ether layers were combined, washed FAMEs mixture was dissolved in hexane (1 mL) and with 5 % NaCl solution (3 × 50 mL), dried with transferred to a gas chromatographic (GC) vial. The sodium sulphate overnight, filtered and the solvent vial was capped and stored at –18 ◦C until GC analy- removed using rotary evaporator. The residues were sis. transferred into vials with petroleum ether and stored Neutral and polar lipids were also separated by until the derivatisation process. thin-layer chromatography. For fractionation of the The sterols were derivatised with a mixture of lipid classes, 0.4 mL (at least 15 mg) of each total N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) lipid extract was dried under a stream of nitrogen, re- and trimethylchlorosilane (TMCS) (ϕr =2:1),at solved in chloroform and chromatographed as above. 60 ◦Cfor2h. The polar lipids (PL, phospholipids, and glycolipids) GC analyses of sterol trimethylsilyl (TMS) ethers remained at the origin of the plates (the first band). were carried out using a Varian CP-Sil 5CB cap- The silica support containing the major lipid class illary column (100 % dimethyl polysiloxane, 25 m bands, identified with the help of commercial stan- × 0.25 mm i.d., 0.12 µm film thickness, Varian- dards, was scraped from the plates into tubes. The Chrompack, Middelburg, The Netherlands). A Shi- lowest band was eluted with methanol/chloroform (ϕr madzu GC-2010 gas-chromatograph equipped with a = 1 : 1) and the two higher major bands corresponding flame ionisation detector (FID) was used. The temper- to triacylglycerols (TGs) and sterol esters (SEs), were ature programme was: 3 min at 130 ◦C, 10 ◦Cmin−1 to eluted with chloroform. After removal of the chloro- 290 ◦C (for 5 min). The injector and FID temperatures form with nitrogen, the lipid classes were methylated were 260 ◦C and 290 ◦C, respectively. The injection vol- for 1 h, followed by the addition of water (1 mL) and ume was 0.5 µL. The carrier gas was helium (purity hexane (2 mL) to collect the FAMEs in hexane as 5.0; flow-rate of 1.1 mL min−1). above. Identification of sterols was based on a comparison of their relative retention times (RRt to β-sitosterol) FAMEs and sterol analyses with data from the literature (Kalo & Kuuranne, 2001; Phillips et al., 2005; Yang et al., 2001). A mixture of The FAMEs were analysed by gas chromatography- sterol standards (sitosterol 95 %, campesterol 98 %, mass spectrometry (GC-MS), using a Perkin–Elmer stigmasterol 95 %, and sitostanol 96.7 %) was stud- Clarus 600T GC-MS spectrometer (Perkin–Elmer, ied under the same conditions and the retention times Shelton, WA, USA) equipped with a highly polar BPx- (Rt) were used to assist the peak identification. The 70 capillary column (60 m × 0.25 mm i.d., 0.25 µm sterol concentrations were calculated using the area of film; SGE, Ringwood, Australia). The oven tempera- the internal standard peak (Li et al., 2007). ture was initially programmed at 140 ◦C, increased to 220 ◦Cat2◦Cmin−1 andthenheldatthistemper- Statistics ature for 25 min. The flow-rate of the carrier gas He and the split ratio were 0.8 mL min−1 and 1 : 24, re- All the extractions and GC analysis were made spectively. The injector temperature was 210 ◦C. The in triplicate. Data were expressed as mean ± S.D. positive ion electron impact(EI)massspectrawere Statistical differences between samples were tested recorded at the ionisation energy of 70 eV and a trap using ANOVA (Tukey’s Multiple Comparison Test; current of 100 µA with a source temperature of 150 ◦C. GraphPad Prism Version 4.0, GraphPad Software, San The mass scans were performed within the range of Diego, CA, USA). The level of significance was set at m/z 22–395 at a rate of 0.14 scan per s with an inter- p < 0.05. mediatetimeof0.02sbetweenthe scans. The injection volume was 1 µL. Results and discussion FAMEs were identified by comparison of the retention times with known standards and the result- Lipid contents ing mass spectra to those in our database (NIST MS Search 2.0). The data for the total lipids recovered from the Plant sterols were analysed using a modification of five Transylvanian (Romania) pomaces, from wild the procedure described by Yang et al. (2001). After and cultivated blueberries (Vaccinium myrtillus), the addition of 5α-cholestan-3β-ol (2 mg) as an in- wild cowberry (Vaccinium vitis-idaea) and raspberry ternal standard, the total crude lipids (100–350 mg) (Rubus idaeus), and cultivated chokeberry (Aronia were saponified by refluxing in 30 mL of a 1 M KOH melanocarpa) are summarised in Table 1. The berry ethanol/water (ϕr = 4 : 1) solution for 1 h. The mix- waste lipid yields (2.8–7.0 g per 100 g of wbp) var- ture was then transferred into a separatory funnel and ied significantly (p ≤ 0.05) within the fruits under

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Table 1. Lipid content in ﬁve Transylvanian (Romania) berry fresh berries (Johansson et al., 1997) were determined pomaces and reported by other authors.

Pomaces Lipid content per 100 g of wbp/g Fatty acids proﬁle

Blueberry (wild) 3.93 ± 0.30c Blueberry (cultivated) 2.80 ± 0.20d The proportions of fatty acids in total lipids (TLs), Cowberry (wild) 3.75 ± 0.25c,d polar lipids (PLs), triacylglycerols (TGs), and sterol Raspberry (wild) 7.00 ± 0.50a esters (SEs) are shown in Tables 2 and 3. Thirteen ± b Chokeberry (cultivated) 5.50 0.45 fatty acids were identified in the pomace extracts stud- Note: Data are expressed as mean ± SD of three samples of ied (Table 2), from which the most abundant were each berry pomace, analysed individually in triplicate; means linoleic [18:2 (n–6)], followed by oleic [18:1 (n–9)(Z)] followed by the same letter within the row are not significantly and palmitic (16:0) acid, together constituting more different (p > 0.05). than 70 % of the total FAMEs identified (Fig. 1). In TL fractions of the pomaces examined, the unsaturated fatty acids contents were responsible for analysis. The oil content in wild raspberry (7.0 g per large proportions (from 88.2 % in cultivated blue- 100 g of wbp), cultivated chokeberry (5.5 g per 100 g berry to 95.5 % in raspberry), of which the PUFAs of wbp), and wild blueberry pomaces (3.9 g per 100 g ranged between 37 % (cultivated blueberry) and 69 % of wbp) was significantly higher (p < 0.05) than in (raspberry) of the total FAMEs, and MUFAs varied the cultivated blueberry pomace (2.8 g per 100 g of between 24.9 % (chokeberry) and 51.2 % (cultivated wbp). The differences observed between the oil con- blueberry) of the total FAMEs (Table 3). tent of cowberry pomace and the blueberry pomaces The TGs fatty acid profiles were practically iden- (wild and cultivated) were not statistically significant tical to the profiles of the TLs (Table 2). Linoleic acid (p > 0.05) (Table 1). predominated in the TGs of raspberry (53.3 %) and The oil contents of the eight Rosaceae seed species chokeberry (69.2 %) pomaces. In TGs and TLs of blue- analysed by Johansson et al. (1997) ranged from 6.9 % berry and cowberry pomaces, the main component to 23.2 % on dry mass basis (% dm), whereas the was oleic acid followed by linoleic acid (Table 2). fresh berry oil contents varied from 8.1 g to 26.9 g The fatty acid composition of the SEs was dif- per kg of fresh berries. In chokeberry seeds, Zlatanov ferent from that of the TGs in all the berry po- (1999) reported an oil content of 19.3 g kg−1.The maces analysed. The amounts of saturated fatty acids total lipid content of whole chokeberries was found to (SFAs) in SEs were significantly higher (p < 0.05). be about 0.1 g per 100 g of fresh mass (fm) (Kulling & The percentages of saturated, mainly palmitic (16:0) Rawel, 2008). Oil contents of 15–22 % (fm) (Yang et and stearic (18:0), and very long-chain saturated fatty al., 2003), about 30 % (dm) (Johansson et al., 1997) acids (VLCSFAs) (more than 20 carbon atoms) were for Vaccinium berry seeds, and 4.1–8.5 g per kg of 74.6 % in chokeberry, 48.8 % in raspberry, 33.0–32.7 %

Fig. 1. GC-MS chromatogram of FAMEs from total lipids of black chokeberry (Aronia melanocarpa) pomace. Peaks of acids: (1) lauric (12:0), (2) myristic (14:0), (3) palmitoleic [16:1 (n–7)], (4) palmitic (16:0), (5) margaric (17:0), (6) α-linolenic [18:3 (n–3)], (7) linoleic [18:2 (n–6)], (8) oleic [18:1 (n–9)(Z)], (9) elaidic [18:1 (n–9)(E)], (10) stearic (18:0), (11) arachidic (20:0), (12) behenic (22:0).

Unauthenticated Download Date | 11/24/15 5:32 AM F. V. Dulf et al./Chemical Papers 66 (10) 925–934 (2012) 929 ef b d a d c c b d c b a 0.55 0.80 0.03 0.32 0.04 0.04 ± ± ± ± ± ± nd nd nd nd nd nd nd nd nd nd nd nd nd nd – pomace 0.14 9.82 0.40 0.53 17.11 18.21 ef b e b d a e c e b d a e b e a ef bc f b e c b a d bc f b d c g a 0.10 0.05 0.10 0.11 0.31 0.11 0.14 0.30 0.05 0.15 0.05 0.72 0.09 0.07 0.02 0.25 tr tr nd nd ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.43 0.15 f a e a s indicate significant differences –9) 20:0 22:0 0.02 0.01 tr tr tr ndnd 1.30 nd 1.66 nd 8.20 nd 0.49 nd 2.86 nd 8.27 nd 0.23 nd 4.14 ndnd 1.58 18.78 ndndnd 0.60 nd 0.87 0.22 5.34 ± ± 0.05 0.05 0.05) among lipid classes of each pomace < e c d a e d f b d c d a d d e b e c d a e c d b e c d a de c f b d c d b d c a a p 0.39 0.39 0.50 0.40 0.65 0.11 0.06 0.22 0.13 0.29 0.12 0.21 0.07 0.05 0.30 0.10 0.10 0.22 0.15 0.06 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.51 0.69 4.22 3.31 9.22 1.70 6.66 0.68 0.27 7.81 1.19 0.76 6.57 1.37 0.85 10.87 14.45 12.50 10.90 27.85 ) 18:0 20:1 ( n E fg bc e a e c h b e b e a e b g b e ab f a e b f ab ef c f a e c g b d a f b d b i b –9)( 0.06 0.11 0.02 0.10 0.05 0.09 0.09 0.07 0.08 0.06 0.05 0.10 0.06 0.02 0.02 0.11 0.07 0.07 0.05 0.08 n ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.37 1.44 0.15 0.41 0.38 2.18 0.33 0.40 0.43 0.51 0.20 0.33 0.23 0.83 0.30 0.68 0.93 0.36 0.48 0.55 ) 18:1 ( a b c d a a a c a a c c a a b b a b c d a a b c b a c b b a a a b a d b b a fg c Z 0.85 0.57 0.71 0.85 0.68 0.48 0.88 0.48 1.00 0.68 0.90 0.60 0.90 0.55 0.60 0.80 0.82 0.30 0.76 0.20 –9)( ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 5.80 47.93 20.08 52.23 30.80 50.74 17.77 50.60 25.18 50.44 18.58 52.30 27.28 26.22 21.32 24.40 25.95 23.47 10.52 22.99 b b a a b ab b c b c a a b b a c b ab a a b b a ab a a a c a a c d a b b c a a c d –6) 18:1 ( n 0.62 1.10 0.85 0.78 0.82 0.88 0.75 0.92 0.80 1.10 0.90 0.85 0.82 0.78 0.95 0.50 1.30 0.82 1.40 0.45 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 33.71 36.60 34.76 29.60 30.00 39.28 35.60 31.99 36.25 38.92 35.90 37.75 51.07 33.57 53.37 16.88 64.67 26.08 69.25 12.11 Fatty acid/mass % c a e c c a e b c b e d c c d a c b e c c a d b c a e c c a e b d b e a d b ef a –3) 18:2 ( n n 0.60 0.55 0.21 0.05 0.25 0.45 0.16 0.26 0.20 0.13 0.20 0.28 0.23 0.12 0.38 0.26 0.30 0.25 0.22 0.02 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 7.06 0.34 6.54 19.15 e e d b e a 0.03 0.01 0.02 0.28 tr 17.93 ndndnd 8.07 nd 2.22 9.00 6.45 ndndnd 2.72 6.31 nd 8.93 ndnd 8.22 nd 3.33 10.21 7.99 ndnd 4.22 7.64 ndnd 6.71 0.12 ± ± ± ± 0.12 0.02 0.12 7.53 d c b a d d c b c c b a c d c b d c b a d d c b d c b a d d d b c c a a c d d b 0.60 0.72 0.71 0.11 0.18 0.70 0.03 0.38 0.38 0.40 0.39 0.52 0.20 0.21 0.10 0.11 0.40 0.30 0.60 0.13 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 6.99 7.64 2.61 1.74 7.22 4.98 9.45 13.79 11.83 24.18 g e b g a f ef b f a e b d a d ab i b –7) 16:0 17:0 18:3 ( n 0.01 0.01 0.08 0.10 0.01 0.03 0.01 0.10 0.06 0.05 tr 3.28 tr 3.17 tr 5.46 nd 13.62 nd 10.75 ± ± ± ± ± ± ± ± ± ± 0.05 0.11 0.44 0.65 0.06 0.99 0.04 0.53 0.47 0.37 g b g a e b f a e b f a f c f b e c g a d b d b h a 0.02 0.02 0.07 0.01 0.04 0.03 0.12 0.10 0.09 0.02 0.15 0.03 0.09 nd nd 25.73 ± ± ± ± ± ± ± ± ± ± ± SD of three samples of each fruit pomace, analysed individually in triplicate; P1 – pomace 1 (wild blueberry), P2 – pomace 2 (cultivated blueberry), P3 2.92 1.86 1.29 0.04 1.43 0.17 3.01 ± e b fg a f f f b g a d b h a 0.10 0.10 0.13 0.18 0.10 0.02 0.03 0.17 ± ± ± 12:0 14:0 16:1 ( Fatty acid composition (mass % of total fatty acids) of total lipids and individual lipid classes from Transylvanian (Romania) berry pomaces SE 1.50 ± SE 0.92 ± SE 3.78 ± SE 2.48 ± SE 3.24 ± PL 1.39 PL ndPL nd nd nd nd 25.97 ± nd 24.22 ± PL nd 0.81 PL nd nd nd 27.46 ± TL tr 0.08 TL tr 0.15 TL tr 0.46 TL 0.03 TL 0.18 TG ndTG tr nd tr TG nd 0.02 ± TG nd 0.11 ± TG nd nd tr 1.12 0.05) within berry pomace lipid class; means in the same column followed by different subscript letters indicate significant differences ( < P1 P2 P3 P4 P5 Species p Table 2. Note: Values are mean 3 (wild cowberry), P4( – pomace 4 (wild raspberry), P5 – pomace 5 (cultivated chokeberry); means in the same row followed by different superscript letter sample (P1–P5); TL – total lipids, PL – polar lipids, TG – triacylglycerols, SE – steryl esters; tr – traces, nd – not detected.

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Table 3. Fatty acid composition (mass % of total fatty acids) of total lipids and individual lipid classes from ﬁve Transylvanian (Romania) berry pomaces

Fatty acid/mass % Species PUFAs/SFAs n–6/n–3 SFAs MUFAs PUFAs VLCSFAs (≥ 20C)

± c ± a ± b ± d TL c9.80 0.43c a48.35 0.92b c41.78 1.07ab a1.30 0.10b d4.26bb4.18bc ± a ± b ± a ± c PL 39.65 1.27a 21.52 0.68d 38.82 1.26bc 1.66 0.11b 0.98c 16.49a P1 ± c ± a ± b TG 3.86 0.17d 52.30 0.73a 43.76 1.11a – 11.33a 3.86c ± b ± b ± a ± c SE 32.74 1.16b 31.21 0.95c 36.05 0.98c 8.20 0.31a 1.10c 4.59b

± c ± a ± b ± d TL a11.70 0.50c a51.20 0.74a d37.00 1.03b d0.49 0.11c e3.16bb4.25c ± b ± c ± a ± d PL 38.00 1.15a 19.90 0.57c 42.00 1.01a 2.86 0.14b 1.10c 14.44a P2 ± c ± a ± b TG 7.16 0.30d 50.93 0.97a 41.91 0.95a –5.85a 5.64b ± b ± c ± a ± d SE 33.06 1.19b 26.02 0.63b 40.92 1.20a 8.27 0.30a 1.23c 3.58c

± c ± a ± b ± d TL d4.65 0.32c a50.87 1.08a c44.47 1.03ab e0.23 0.05b b9.56bb4.41bc ± b ± c ± a PL 38.67 1.10a 19.09 0.74c 42.25 1.22b –1.09c 11.69a P3 ± c ± a ± b TG 1.39 0.08d 52.50 0.95a 46.11 1.28a – 33.17a 3.52c ± c ± b ± a ± d SE 25.99 1.03b 28.26 0.80b 45.74 1.11a 4.14 0.15a 1.76c 4.72b

± c ± b ± a ± d TL d4.44 0.37c b26.56 0.99a a69.00 1.42a c0.62 0.15b a15.54bb2.85b ± a ± b ± a ± c PL 39.07 1.11b 23.14 0.60b 37.79 1.08b 1.58 0.05b 0.97c 7.95a P4 ± c ± b ± a ± d TG 2.69 0.28c 24.79 0.64ab 72.52 1.50a 0.20 0.06b 26.96a 2.79b ± a ± bc ± c ± b SE 48.85 1.89a 26.63 0.91a 24.52 0.75c 28.60 1.04a 0.50c 2.21b

± c ± b ± a ± d TL b10.06 0.76c b24.93 0.99a b65.01 1.35b b1.00 0.13c c6.46b a190.21b ± a ± d ± b ± c PL 56.34 1.72b 10.88 0.37b 32.79 1.04c 17.98 0.62b 0.58c 3.89c P5 ± c ± b ± a ± d TG 6.69 0.45d 23.94 0.87a 69.37 1.42a 0.75 0.06c 10.37a 577.08a ± a ± d ± c ± b SE 74.63 2.77a 6.72 0.33c 18.65 0.70d 23.55 1.05a 0.25c 1.85c

Note: Values are mean ± SD of three samples of each fruit pomace, analysed individually in triplicate; different subscript letters in front of the mean values in the same columns indicate significant differences (p < 0.05) between total lipids TLs of five analysed berry pomaces; means in the same row followed by different superscript letters indicate significant differences (p < 0.05) among fatty acid classes; means in the same column followed by different subscript letters indicate significant differences (p < 0.05) among lipid classes of each pomace sample (P1–P5); TLs – total lipids, PLs – polar lipids, TGs – triacylglycerols, SEs – steryl esters; SFAs – saturated fatty acids, MUFAs – monounsaturated fatty acids, PUFAs – polyunsaturated fatty acids, VLCSFAs – very long-chain saturated fatty acids. in blueberries, and 25.9 % in cowberry SEs. In the cor- TL fractions were found to contain very low propor- responding TGs, those quantities were 6.6 %, 2.6 %, tions of VLCSFAs, ranging from 0.2 % to 1.3 % of the 7.1–3.8 %, and 1.3 %, respectively. In polar lipid (PL) total FAMEs, whereas the ES classes exhibited much fractions, the content of SFAs was also significantly higher amounts of this type of fatty acids in all berry higher (p < 0.05) than in the corresponding TG and pomaces (Table 3). TL fractions (Table 3). These observations were in PUFAs, especially n–6 and n–3 essential fatty agreement with the data reported by Zlatanov (1999), acids, have many important biological effects (Simo- Yang et al. (2003), and Yi et al. (2009) on the fatty poulos, 2002). Numerous studies have shown that the acid composition of the phospholipids and SEs of other overall ratio of n–6 and n–3 fatty acids in the current seed oils. The differences observed between the fatty western diet is about 15–17:1 and for optimal health acid compositions of the lipid classes studied can be benefits the recommended essential fatty acid balance attributed to different phases of biosynthesis and ac- (n–6/n–3) varies between 2.5 and 5.0 (Maillard et al., cumulation of TGs, SEs, PLs, and fatty acids. PLs 2002; Schaefer, 2002; Simopoulos, 2002; Parry et al., and SEs are synthesised in the first stage and the 2005). The ratio of fatty acids, n–6 to n–3 PUFAs SFAs are accumulated in their composition. The sec- and PUFAs to SFAs varied depending on lipid frac- ond phase of biosynthesis is characterised by increased tion (Table 3). amounts of TGs with high unsaturated fatty acids Our results with regard to the ratios of n–6/n–3 in content (Sharma & Malik, 1994; Zlatanov, 1999; Zla- TLs of raspberry (2.8), wild blueberry (4.1), cultivated tanov et al., 2010). blueberry (4.2), and cowberry pomaces (4.4) were in The very long-chain saturated fatty acids (with agreement with the results of Oomah et al. (2000), more than 20 carbon atoms) (VLCSFAs) are impor- Parker et al. (2003), Parry and Yu (2004), and Parry tant structural components of the cuticular surface et al. (2005), reported for cold-pressed hemp seed and layers of plant origin tissues (Gurr et al., 2002). All different berry seed oils. The analysis of fatty acids in

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Table 4. Sterol content (% of total sterols) of ﬁve Transylvanian (Romania) berry pomaces

Pomaces Sterols RRt Blueberry Blueberry Cowberry Raspberry Chokeberry (wild) (cultivated) (wild) (wild) (cultivated)

± b ± a ± a Cholesterol 1.11 0.10e 2.15 0.20cde nd nd 1.84 0.10cd 0.890 ± a ± ab ± d ± b ± c Campesterol 7.12 0.38b 6.22 0.38b 2.82 0.20d 5.74 0.36bc 4.46 0.18bc 0.940 ± a ± b ± c ± b Stigmasterol 2.20 0.19de 1.28 0.10de 0.22 0.02e 1.46 0.11d nd 0.960 β ± ab ± b ± d ± c ± a -Sitosterol 73.10 3.15a 71.28 3.22a 55.18 2.22a 67.36 2.40a 74.42 3.12a 1.000 ± c ± ab ± bc ± a ± ab Stigmastanol + Isofucosterol 5.74 0.29bc 7.22 0.43b 6.58 0.36c 7.77 0.29bc 7.10 0.33b 1.010 1 β ± c ± b ± a ± b ± a Stigmasta-8,24(24 )-dien-3 -ol* 1.74 0.20de 2.46 0.21cde 5.26 0.23c 2.66 0.19d 5.76 0.25b 1.022 ± c ± b ± a Cycloartenol* 2.68 0.29de 4.24 0.30bcd tr 8.14 0.32b nd 1.027 1 β ± d ± c ± a ± e ± b Stigmasta-7,24(24 )-dien-3 -ol* 4.10 0.30cd 4.85 0.36bc 6.74 0.30c 1.24 0.10d 5.50 0.30b 1.030 ± b ± a 24-Methylenecycloartanol 1.79 0.20de tr 15.15 1.10b nd nd 1.050 ± d ± d ± a ± b ± c Citrostadienol 0.30 0.02e 0.10 0.01e 7.35 0.48c 5.63 0.22c 0.92 0.07d 1.070

Total sterol content** 142.90 ± 7.32b 101.68 ± 5.30c 168.21 ± 8.26a 150.22 ± 6.00b 148.22 ± 6.44b

Note: Values are mean ± SD of three samples of each berry pomace, analysed individually in triplicate; means in the same row followed by different superscript letters indicate significant differences (p < 0.05) among types of fruit pomaces; means in the same column followed by different subscript letters indicate significant differences (p < 0.05) within berry pomace sterols; RRt – retention times relative to β-sitosterol TMS ether (Rt = 24.904 min); RRt = RtS/RtSTMSE, where RtS is retention time (in min) of a sterol and RtSTMSE is retention time of β-sitosterol TMS ether (24.904 min); *tentative identification (Yang et al., 2003), **given in mg per 100 g of lipids; nd – not detected, tr – traces.

TLs and TGs of chokeberry pomace showed extremely berry (7.3 % of total sterols) and raspberry (5.6 % high levels of n–6/n–3 ratios (190.2 in TLs and 577.0 in of total sterols) pomaces. A higher proportion of TGs) due to the high values of 18:2 (n–6) and low val- cycloartenol was also found in raspberry pomace ues of 18:3 (n–3) (Table 2). In PL fractions of samples (8.1 % of total sterols) than in the other four samples of each pomace, the n–6/n–3 ratios were much higher (p < 0.05). Trace amounts of cholesterol were detected than those in the other three fractions (Table 3). only in blueberries and chokeberry pomace lipids (see As shown in Table 3, the TGs and TLs of wild cow- Table 4), data which were in agreement with those of berry (33.1 and 9.5) and raspberry (26.9 and 15.5) Zlatanov (1999). Phillips et al. (2002) also reported had the highest PUFAs/SFAs ratios, whereas in the very low concentrations of cholesterol (< 3mgper PL and SE fractions of five pomaces studied the low- 100 g of oil or fat) in 28 edible oils and fats. est values of these ratios were determined. Yi et al. β-Sitosterol was the most abundant phytosterol (2009) investigated the fatty acid composition of five in all samples. The highest concentration was de- lipid classes of two varieties of grape pomace powder termined in chokeberry pomace oil (74.4 % of total from the winemaking industry. They found the highest sterols), whereas the cowberry pomace oil had the values of PUFAs/SFAs ratios in TLs and TGs, rang- lowest amount of this sterol (55.1 % of total sterols) ing from 2.8 to 4.6 and the lowest in SE and PL frac- (p < 0.05). These high values of β-sitosterol reported tions, ranging from 0.7 to 1.9. According to Kang et al. in the present work are in agreement with data pub- (2005) and Guo et al. (2010), a dietary PUFAs/SFAs lished by Piironen et al. (2003), Yang et al. (2003), ratio varying between 1.0 and 1.5 is a desirable range and Zlatanov (1999). to reduce the risk of cardiovascular disease. In addition to the low-density lipoprotein (LDL)- cholesterol-lowering effect (Haydn Pritchard et al., Sterol composition 2003), recent epidemiological studies have shown that plant sterols may also possess an anti-cancer effect. The cowberry pomace oils had the highest to- Some previous reports have indicated that β-sitosterol tal phytosterol content (p < 0.05), followed by rasp- exhibits a significant anti-oxidant effect and anti- berry, chokeberry, wild blueberry, and cultivated genotoxic and immuno-stimulant potential (Kassen et blueberry pomaces (Table 4). The major compo- al. 2000; Park et al., 2003; Paniagua-Pérez et al., 2005, nents of the total sterol extracts analysed were 2008). Park et al. (2007) have shown that this phytos- β-sitosterol, stigmastanol + isofucosterol, campesterol exerts anti-proliferative and pro-apoptotic effects terol, stigmasta-7,24(241)-dien-3β-ol, and stigmasta- in human leukemic cells (U937) by the activation of a 8,24(241)-dien-3β-ol (Table 4). Low concentrations of cysteine-related protease (Caspase-3) and induction of stigmasterol were also determined. In cowberry po- pro-apoptotic protein Bax/apoptosis-suppressing Bcl- mace, 24-methylenecycloartanol was the second most 2 protein ratio. In another study, Awad et al. (2008) important sterol (15.1 % of total sterols). Relatively suggested that a β-sitosterol-rich diet could increase high levels of citrostadienol were detected only in cow- the anti-cancer activity of tamoxifen, which is the

Unauthenticated Download Date | 11/24/15 5:32 AM 932 F. V. Dulf et al./Chemical Papers 66 (10) 925–934 (2012) most common anti-oestrogen drug used to prevent or UEFISCSU, project number PNII – TE-168, code 109/2010. treat invasive breast cancer. The chokeberry and blueberry pomace oils showed References higher levels of β-sitosterol than those presented previously for other edible and non-traditional vegetable Adhikari, P., Hwang, K. T., Shin, M. K., Lee, B. K., Kim, oils (Phillips et al., 2002; Piironen et al., 2003). The S. K., Kim, S. Y., Lee, K. T., & Kim, S. Z. (2008). Tocols 111 amounts of total sterols reported in the literature for in caneberry seed oils. Food Chemistry, , 687–690. DOI: 10.1016/j.foodchem.2008.04.038. palm, olive, peanut, and soybean oils (Phillips et al., Awad, A. B., Barta, S. L., Fink, C. S., & Bradford, P. 2002), were similar to those determined in our study G. (2008). β-Sitosterol enhances tamoxifen effectiveness for berry pomace lipids. on breast cancer cells by affecting ceramide metabolism. Non-conventional vegetable oils have unique phy- Molecular Nutrition & Food Research, 52, 419–426. DOI: tochemical compositions with remarkable chemical 10.1002/mnfr.200700222. properties and anti-oxidant performance (Ramadan & Bushman, B. S., Phillips, B., Isbell, T., Ou, B., Crane, J. M., & Knapp, S. J. (2004). Chemical composition of caneberry Moersel, 2006). For this reason, they could replace sev- (Rubus spp.) seeds and oils and their antioxidant potential. eral traditional edible seed and fruit oils (e.g. olive oil). Journal of Agricultural and Food Chemistry, 52, 7982–7987. DOI: 10.1021/jf049149a. Conclusions Guo, Z., Miura, K., Turin, T. C., Hozawa, A., Okuda, N., Oka- mura, T., Saitoh, S., Sakata, K., Nakagawa, H., Okayama, The studies reporting data on the phytochemicals A., Yoshita, K., Kadowaki, T., Choudhury, S. R., Naka- mura, Y., Rodriguez, B. L., Curb, D. J., Elliott, P., Stamler, composition of agricultural-food wastes could be used J., & Ueshima, H. (2010). Relationship of the polyunsatu- as guidelines for more extensive industrial valorisa- rated to saturated fatty acid ratio to cardiovascular risk fac- tion of the fruit and vegetable by-products resulting tors and metabolic syndrome in Japanese: the INTERLIPID from different processing procedures. The berry fruits- study. Journal of Atherosclerosis and Thrombosis, 17, 777– processing industry supplies large amounts of peel and 784. DOI: 10.5551/jat.4135. seed residues which could be used as potential sources Gurr, M. I., Harwood, J. L., & Frayn, K. N. (2002). Lipid biochemistry: An introduction (5th ed.). London, UK: Blackwell of lipids with important applications in the cosmetics Science Ltd. and food industries. Haydn Pritchard, P., Li, M., Zamfir, C., Lukic, T., Novak, E., This study showed that wild raspberry pomace & Moghadasian, M. H. (2003). Comparison of cholesterol- contains a reasonably high quantity of oil which could lowering efficacy and anti-atherogenic properties of hydro- TM be explained by the naturally high seed content of genated versus non hydrogenated (Phytrol ) tall oil- the corresponding berries. Unfortunately, for advan- derived phytosterols in apo E-deficient mice. Cardiovascular Drugs and Therapy, 17, 443–449. DOI: 10.1023/b:card.00000 tageous edible oil production, it is necessary to have 15859.37581.f1. an appreciable berry processing industry in Romania. Heinonen, M. (2007). Antioxidant activity and antimicro- The TL and TG fractions of this berry pomace contain bial effect of berry phenolics – a Finnish perspective. about 17 % and 19 % linolenic acid, and about 51 % Molecular Nutrition & Food Research, 51, 684–691. DOI: to 53 % linoleic acid, making it an excellent dietary 10.1002/mnfr.200700006. source of 18:3 (n–3) and essential fatty acids. Johansson, A., Laakso, P., & Kallio, H. (1997). Characterization n n of seed oils of wild, edible Finnish berries. Zeitschrift für The TL fractions tested had –6 to –3 PUFAs ra- Lebensmittel-Untersuchung und -Forschung A, 204, 300–307. tios of 2.8 for raspberry, 4.1 for wild blueberry, 4.2 for DOI: 10.1007/s002170050081. cultivated blueberry, and 4.4 for cowberry pomaces, Kalo, P., & Kuuranne, T. (2001). Analysis of free and ester- values which are close to the n–6/n–3 PUFAs intake ified sterols in fats and oils by flash chromatography, gas recommended by nutritional scientists (< 5). The high chromatography and electrospray tandem mass spectrom- 935 ratios of n–6/n–3 PUFAs in TLs and TGs of choke- etry. Journal of Chromatography A, , 237–248. DOI: 10.1016/s0021-9673(01)01315-2. berry pomace suggest there should be restrictions on Kang, M. J., Shin, M. 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Unauthenticated The author has requested enhancement of the downloadedDownload file. Date All in-text | 11/24/15 references 5:32 AM underlined in blue are linked to publications on ResearchGate. Accepted Manuscript

Volatile profile, fatty acids composition and total phenolics content of brewers’ spent grain by-product with potential use in the development of new functional foods

Anca C. Fărcaş, Sonia A. Socaci, Francisc V. Dulf, Maria Tofană, Elena Mudura, Zoriţa Diaconeasa

PII: S0733-5210(15)00055-7 DOI: 10.1016/j.jcs.2015.04.003 Reference: YJCRS 1966

To appear in: Journal of Cereal Science

Received Date: 23 October 2014 Revised Date: 25 March 2015 Accepted Date: 2 April 2015

Please cite this article as: Fărcaş, A.C., Socaci, S.A., Dulf, F.V., Tofană, M., Mudura, E., Diaconeasa, Z., Volatile profile, fatty acids composition and total phenolics content of brewers’ spent grain by-product with potential use in the development of new functional foods, Journal of Cereal Science (2015), doi: 10.1016/j.jcs.2015.04.003.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT

1 Volatile profile, fatty acids composition and total phenolics content of brewers’ spent grain by-product

2 with potential use in the development of new functional foods

1 1* 2 1 1 1 4 Anca C. F ărca ș , Sonia A. Socaci , Francisc V. Dulf , Maria Tofan ă , Elena Mudura , Zorița Diaconeasa

6 1Faculty of Food Science and Technology, University of Agricultural Sciences and Veterinary Medicine Cluj-

7 Napoca, Cluj-Napoca 400372, Manastur 3-5, Romania; 2Faculty of Agriculture, University of Agricultural

8 Sciences and Veterinary Medicine Cluj-Napoca, Cluj-Napoca 400372, 3-5 Calea Mănăş tur Street, Romania

9 Corresponding author: Tel: +04 0264 596384; fax: +04 0264 593792; e-mail: [email protected]

10 (S.A. Socaci)

12 Abstract

13 Brewers’ spent grain (BSG) is the insoluble residue generated from the production of wort in the 14 brewing industry. This plant-derived by-product is known MANUSCRIPT to contain significant amounts of valuable 15 components, which remain unexploited in the brewing processes. Therefore, it is essential to develop a more

16 detailed characterization of BSG in order to highlight its potential in developing new value-added products

17 and simultaneously solve the environmental problems related to its discharge. The content of BSG in several

18 biologically active compounds (fatty acids, polyphenols, flavonoids, antioxidant capacity) as well as its

19 volatile fingerprint were assessed and compared with the composition of barley, malt and wheat flour samples.

20 The obtained results emphasized the importance and the opportunities of the re-use of this agro-industrial by-

21 product.

22 ACCEPTED

23 Keywords: brewers’ spent grain, volatile compounds, fatty acid, antioxidant compounds.

24 Chemical compounds studied in this article

ACCEPTED MANUSCRIPT

25 2-Methyl-propanal (PubChem CID: 6561); Hexanal (PubChem CID: 6184); Palmitic acid (PubChem CID:

26 985); Oleic acid (PubChem CID: 445639); Linoleic acid (PubChem CID: 5280450)

28 List of Abbreviations

29 BSG - brewers’ spent grain

30 BSGd - dried brewers’ spent grain

31 BSGl - lyophilized brewers’ spent grain

32 DPPH - 2,2-diphenyl-1-picrylhydrazyl

33 dw - dry weight

34 EI - electron ionisation

35 FAMEs - fatty acid methyl esters

36 fw - fresh weight

37 GAE - gallic acid equivalents 38 GC - gas chromatography MANUSCRIPT 39 GC-MS - gas chromatography – mass spectrometry

40 ITEX - in-tube extraction

41 MCf - Carafa malt

42 MCm - Caramunich malt

43 MP - Pilsner malt

44 MUFAs - monounsaturated fatty acids

45 PCA - principal component analysis

46 PUFAs - polyunsaturatedACCEPTED fatty acids

47 QE - quercetin equivalents

48 SB - spring barley

49 sd - standard deviation

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50 SFAs - saturated fatty acids

51 TIC - total ion chromatogram

52 TLs - total lipids

53 TPC - total phenolic content

54 tr - trace

55 VLCSFAs - very long chain saturated fatty acids

56 WF - wheat flour

57 WWF - wholemeal wheat flour

59 1. Introduction

60 Nowadays, increasing efforts are being directed towards the exploitation of agro-industrial by-

61 products, from both economic and environmental standpoints. Brewers’ spent grain (BSG) is an important by-

62 product from the brewing process, representing up to 30% (w/w) of the starting malted grain. BSG is therefore 63 a readily available, high volume and low cost by-product MANUSCRIPT within the brewing industry. It is estimated that 64 worldwide the annual output is around 30 million tons, about 200 tons of wet spent grain (70 to 80 % water

65 content) being produced per 10000 hl of produced beer (Kunze, 1996; Niemi et al., 2012).

66 The great interest shown in the last years in this by-product is due to its chemical composition that

67 permits its re-use in different areas (food ingredient, feed, raw material for microbiological or chemical

68 conversion, pharmaceutical, cosmetic or other industries) (Mussatto, 2013; Niemi et al., 2012; del Rio et al.,

69 2013). Basically, BSG is composed of the barley malt residual constituents and includes the barley grain husk,

70 in the greatest proportion, but also minor fractions of pericarp and fragments of endosperm (Mussatto et al.,

71 2006). Malt is one of ACCEPTED the key ingredients in brewing, providing the starch and the enzymes necessary to

72 produce the fermentable sugars that are turned by yeasts into alcohol in the fermentation process. Malt also

73 provides the colour and the flavour compounds, generated during the kilning step, which contribute to the final

74 character of the beer.

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75 As described in the literature (Santos et al., 2003) the composition of BSG is variable according to the

76 barley variety and harvest time, malting and mashing conditions, type and quality of secondary raw materials

77 added in the brewing process. Nevertheless, this plant-derived by-product is known to contain significant

78 amounts of valuable components, which remain unexploited in the brewing process.

79 BSG has high levels of dietary fibre, protein and particularly essential amino acids, as well as

80 appreciable levels of lipids, minerals, polyphenols and vitamins (Mussatto et al., 2006). These compounds,

81 when incorporated into human diets, may provide a number of benefits by lowering the risk of certain diseases

82 including cancer, gastrointestinal disorders, diabetes, obesity and coronary heart disease (Fastnaught, 2001).

83 BSG is still traditionally supplied to local farmers or, alternatively, it is composted, dried and

84 incinerated, dumped or anaerobically fermented (Fillaudeau et al. 2006). Therefore the development of

85 economically viable technologies for the exploitation of this agro-industrial by-product has been encouraged.

86 Although the by-products from the brewing and also from other fruit and vegetable processing industries are

87 considered agricultural wastes and an environmental problem, they are suitable to be used in the human diet 88 and efforts have been made to utilise them as functional ingredientsMANUSCRIPT (Rodriguez et al., 2006). Consequently, it 89 is essential to develop a detailed and comprehensive characterization of BSG in order to highlight its potential

90 as a valuable source of biologically active compounds in the development of new added-value food products.

91 Previous studies have been focused on determining the carbohydrate, protein and polyphenol

92 composition of BSG (Waters et al., 2012) and more recently on its lipid and lignan profile (Niemi et al., 2012;

93 del Rio et al., 2013). Besides the aforementioned classes of compounds, an important role in the acceptance of

94 a food product by the consumers is played by its aroma. For the exploitation of BSG as a food ingredient, its

95 volatile profile must also be taken into consideration. However there is limited information regarding the

96 volatile constituents of ACCEPTEDBSG (Ktenioudaki et al., 2013), the recent research on this area being directed to the

97 malt volatile profile characterization (Coghe et al., 2004; Dong et al., 2013). Thus, in order to complete the

98 existing information on BSG, the present study’s principal objective was to determine the BSG volatile profile

99 together with its composition in fatty acids, antioxidant activity, total polyphenolics and flavonoid content.

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100 2. Materials and methods

101 2.1 Materials

102 All the materials (malt, brewers’ spent grain) were supplied by the Microbrewery of the Faculty of Food

103 Science and Technology of University of Agricultural Sciences and Veterinary Medicine from Cluj-Napoca.

104 The BSG used in this work was obtained as a by-product from the mashing process of dark lager beer with

105 100% all grain malted barley (Weyermann Specialty Malting Company, Bamberg - Germany). Besides Pilsner

106 malt (MP) which imparts a malty-sweet and gentle note of honey to the beer, Caramunich (MCm) and Carafa

107 (MCf) malts were added in small amount (5-10%) to obtain a dark colour and to enhance the flavour

108 characteristics with notes of caramel, biscuit, coffee, cacao, dark chocolate and roastiness.

109 The high initial moisture content of fresh BSG (75-80%) and the presence of considerable levels of

110 polysaccharide and protein make it particularly susceptible to microbial degradation within a few days.

111 Therefore, the fresh BSG samples were preserved in two ways: by lyophilisation using laboratory freeze dryer

112 Alpha 1-2 Lyo Display Plus and by oven-drying at 78ºC for 12 hours to reach a moisture content of 6%. Then, 113 the dried samples were packed in sealed polyethylen eMANUSCRIPT bags and stored at room temperature while the 114 lyophilized samples were kept at -20 oC until further analyses.

115 A commercial wheat flour (WF) used for traditional bread making (type 650 according to ash content by

116 Romanian classification) with 14.5% moisture, 10.6% protein, 0.9% fat, 0.65% ash, 73.2% total starch and

117 0.6% fibre was purchased locally, Pambac, Bac ău - Romania. The wholemeal wheat flour (WWF) was also

118 purchased from a local producer (Panemar, Cluj-Napoca - Romania).

119 2.2 Chemicals

120 All reagents used for the lipid extraction and fatty acid methyl ester (FAMEs) preparation were of

121 chromatographic grade ACCEPTED(Sigma–Aldrich (St. Louis, MO, USA)). The FAMEs standard (37 component FAME

122 Mix, SUPELCO, catalog No: 47885-U) were purchased from Supelco (Bellefonte, PA, USA). The standard

123 compounds (gallic acid, quercetin) and reagents: 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocâlteu,

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124 methanol, aluminium chloride, sodium carbonate, sodium nitrite and sodium hydroxide were purchased from

125 Sigma-Aldrich or Merck (Darmstadt, Germany).

126 2.3. Extraction and analysis of volatile compounds

127 The extraction of volatile compounds was performed using the in-tube extraction technique (ITEX) as

128 described in our previous work (Socaci et al., 2014) using 3g of sample. The analysis of volatile compounds

129 was carried out on a GCMS QP-2010 (Shimadzu Scientific Instruments, Kyoto, Japan) model gas

130 chromatograph - mass spectrometer. The volatile compounds were separated on a Zebron ZB-5ms capillary

131 column of 50 m×0.32mm i.d and 0.25 m film thickness. The carrier gas was helium, 1ml/min and the split

132 ratio 1:20. The temperature program used for the column oven was: 40°C (held for 6 min) rising to 50°C at

133 2°C/min, heated to 250°C at 7°C/min and held for 2 min. The injector, ion-source and interface temperatures

134 were set at 250°C. The MS mode was electron impact (EI) at an ionization energy of 70 eV. The mass range

135 scanned was 40–400 m/z.

136 The volatile compounds were tentatively identified using the spectra of reference compounds from 137 NIST27 and NIST147 mass spectra libraries and verified MANUSCRIPT by comparison with retention indices drawn from 138 www.pherobase.com or www.flavornet.org (for columns with a similar stationary phase to the ZB-5ms

139 column). All peaks found in at least two of the three total ion chromatograms (TIC) were taken into account

140 when calculating the total area of peaks (100%) and the relative areas of the volatile compounds.

141 2.4. Determination of fatty acid composition

142 2.4.1 Extraction of lipids

143 The total lipids (TLs) of the samples were extracted using a chloroform: methanol mixture (Dulf et al.,

144 2013). The sample (10 g) was homogenized in methanol (20 mL) for 1 min with a high-power homogenizer

145 (MICCRA D-9, ART Prozess-undACCEPTED Labortechnik, Müllheim, Germany), then chloroform (40 mL) was added,

146 and homogenization continued for 2 min. The mixture was filtered and the solid residue was re-suspended in

147 chloroform: methanol mixture (2:1, v/v, 60 mL) and homogenized again for 3 min. The mixture was filtered,

148 and the residue was washed with chloroform: methanol (2:1, v/v, 60 mL). The filtrates and washings were

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149 combined and cleaned with 0.88% aqueous potassium chloride (45 mL) followed by methanol: aqueous

150 potassium chloride solution (1:1, v/v, 40 mL). The purified lipid layer was filtered and dried over anhydrous

151 sodium sulphate and the solvent was removed in a rotary evaporator (Laborata 4010 Digital, Heidolph,

152 Germany). The total lipids were weighed and the recovered oils were transferred into vials with 3 mL

153 chloroform, and stored at −18 °C for further analysis.

154 2.4.2 Transesterification procedure

155 Fatty acid methyl esters (FAMEs) of the total lipids were derivatized by acid–catalyzed

156 transesterification using 1% sulphuric acid in methanol (Christie, 1989). Lipids (1 mg) were re-suspended in 1

157 mL toluene in a Pyrex tube fitted with a condenser. Two millilitres of methanolic H 2SO 4 (1% v/v) were added,

158 and the mixture was refluxed for 2 hours at 80°C. Water (5 mL) containing potassium chloride (5%; w/v) was

159 added, and the transmethylated fatty acids extracted with hexane (2×5 mL) using Pasteur pipettes to separate

160 the layers. The hexane layer was washed with water (4 mL) containing 2 % potassium bicarbonate and dried

161 over anhydrous sodium sulphate. Finally, the solution was filtered and the solvent was removed under reduced 162 pressure in a rotary film evaporator. MANUSCRIPT 163 2.4.3 GC analysis of FAMEs

164 The FAMEs were determined by gas chromatography-mass spectrometry (GC-MS), using a

165 PerkinElmer Clarus 600 T GC-MS (PerkinElmer, Inc., Shelton, CT, USA) (Dulf et al., 2013). The initial oven

166 temperature was 140°C, programmed by 7°C/min to 220°C and kept 23 min at this temperature. The flow rate

167 of the carrier gas (helium) was 0.8 mL/min and the split ratio value was of 1:24. A sample of 0.5 L was

168 injected into a 60 m × 0.25 mm i.d., 0.25 m film thickness SUPELCOWAX 10 capillary columns (Supelco

169 Inc.) and the injector temperature was set at 210 °C. The positive ion electron impact (EI) mass spectra was

170 recorded at an ionizationACCEPTED energy of 70 eV and a trap current of 100 A with a source temperature of 150 °C.

171 The mass scans were performed within the range of m/z: 22–395 at a rate of 0.14 scan/s with an intermediate

172 time of 0.02 s between the scans.

173 The identification of FAMEs was accomplished by comparing their retention times with those of

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174 known standards and the resulting mass spectra to those in the database (NIST MS Search 2.0). The amount of

175 each fatty acid was expressed as percent of total fatty acid content.

176 2.5 Evaluation of the antioxidant activity of extracts and quantification of total phenolics and flavonoids

177 2.5.1 Extraction method

178 Phenolic compounds were extracted according to the method reported by Abdel-Aal et al. (2003) with

179 some minor modifications. All the analyzed samples (1 g) were homogenized in methanol containing HCl

180 (0.3%) using an ultraturax (Miccra D-9 KT Digitronic, Germany) and then were centrifuged at 4000 rpm for

181 10 min. Extracts were concentrated at 35 oC under reduced pressure (Rotavap Laborata 4010 Digital, Heidolph,

182 Germany) and stored at −18 °C for further analysis.

183 2.5.2 The total phenolic compounds assay

184 The Folin-Ciocalteu method estimates the total content of all phenolics present in the analyzed

185 samples, including flavonoids, anthocyanins and non-flavonoid phenolic compounds (Singleton et al., 1999).

186 Aliquots of 25 l sample were mixed with 1.8 ml distilled water in a 24 wells microplate. An aliquot of 120 l 187 of Folin-Ciocalteu reagent was added and mixed, followed, MANUSCRIPT after 5 minutes by the addition of 340 l Na 2CO 3 188 (7.5% in water) in order to create basic conditions (pH ~10) for the redox reaction between phenolic

189 compounds and Folin-Ciocalteu reagent. After incubation for 90 min at room temperature, the absorbance was

190 read at 750 nm using a microplate reader (BioTek Instruments, Winooski, VT), against the blank, in which the

191 standard or sample were replaced with methanol. Sample dilution was done when the recorded absorbance

192 value exceeded the linear range of the gallic acid calibration curve. The results (expressed as gallic acid

193 equivalents) were expressed as means ± standard deviation of triplicate analysis.

194 2.5.3 The total flavonoid assay

195 The total flavonoidACCEPTED content of the extracts was determined using the aluminium chloride colorimetric

196 method as described previously in other studies (Zhishen et al., 1999). The alcoholic extracts were diluted

197 with distilled water to a final volume of 5 ml and 300 l of 5% NaNO 2 were added. After 5 min, the mixture

198 was treated with 300 l AlCl 3 10% and, after 6 min, with 2 ml NaOH 1 N. The absorbance was measured

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199 against a prepared water blank at 510 nm, using a spectrophotometer (JASCO V-630 series, International Co.,

200 Ltd., Japan). Each determination was carried out in triplicate. The standard curve was performed using

201 different concentrations of quercetin solution (r=0.9941) and the flavonoid content was expressed as mg of

202 quercetin equivalents for 100g of fresh weight (fw).

203 2.5.4 Determination of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity

204 The DPPH scavenging activity assay was performed according to a method reported by Brand-

205 Williams et al. (1995). This method is based on the ability of stable free radicals of 2,2-diphenyl-1-

206 picrylhydrazyl to react with hydrogen donors. A DPPH solution (80 M) was freshly prepared in 95%

207 methanol. A volume of 250 l of this solution was allowed to react with 35 l of sample. The chemical

208 kinetics of the resulting solution was monitored at 515 nm for 30 minutes using a microplate reader BioTek

209 Synergy HT, BioTek Instruments, Winooski, VT. The antioxidant activity was calculated as follows:

210 % DPPH scavenging activity = (A0-A1/A 0) x 100, where

211 A0 was the absorbance of the control reaction and A 1 the absorbance in the presence of the sample. 212 2.6. Statistical analysis MANUSCRIPT 213 The discrimination between the barley, malt, BSG, wholemeal wheat flour and wheat flour samples

214 was achieved by subjecting the obtained chromatographic data (volatile and fatty acids profiles) together with

215 the results obtained for total phenolic content, total flavonoids content and antioxidant capacity to principal

216 component analysis (PCA) with cross validation (full model size and center data). All the statistical analyses

217 were performed using Unscrambler X software version 10.1 (CAMO Software AS Norway).

218 3. Results and discussion

219 3.1 ITEX/GC-MS profile of volatile compounds

220 The volatile fingerprintACCEPTED of BSG samples and those of spring barley (SB), MP, MCm, MCf, WF, WWF

221 samples were determined using the ITEX/GC-MS technique. This modern solventless technique was

222 previously used with success by our research group and also by others in the characterization of the volatile

223 profile of different vegetable matrices, including beers and wines (Laaks et al., 2014; Socaci et al., 2014

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224 Zapata et al., 2012). A total of 37 compounds were separated and tentatively identified in the analyzed

225 samples (Table 1). The identification of the volatiles was achieved by comparing the obtained mass spectra

226 with those from the NIST27 and NIST147 spectra libraries and also by the retention indices drawn from

227 www.pherobase.com or www.flavornet.org. Using the above mentioned database or other literature sources

228 (Dong et al., 2013; Ktenioudaki et al., 2013), the characteristic odour of each detected volatile compound is

229 also specified in Table 1. The volatile constituents found in the analyzed samples include alcohols, aldehydes,

230 ketones as well as furans and other classes of compounds. In the case of SB, the main volatile compounds

231 identified were hexanal (23.16%), 2-methyl-3-buten-2-ol (16.59%) and 2-pentyl-furan (16.72%). In relative

232 high percentage were also detected 3-methyl-butanal (8.46%), 1-pentanol (8.11%), 2-methyl-1-propanol

233 (4.29%), 1-penten-3-ol (3.98%) and toluene (4.15%). Aldehydes were the predominant chemical family in the

234 SB samples, accounting for 43.62% of total volatiles and imparting, based on their sensorial attributes, fresh

235 green and almond like notes (Dong et al., 2014). As shown in Table 1, four alcohols (2-methyl-3-buten-2-ol,

236 2-methyl-1-propanol, 1-penten-3-ol and 3-methyl-1-butanol) were identified in the SB samples, representing 237 32.97% of total volatiles. According to Dong et al. (2013), MANUSCRIPT these compounds are common lipid oxidation 238 products and have a green, sweet and fruity odour. Acetophenone, with a sweet, almond-like and floral aroma,

239 was the only compound from the ketone group found in all samples, but also the only ketone detected in SB,

240 WWF and WF samples. 2-Pentyl-furan was the only furan identified in the SB samples. This compound was

241 also the only furan found in barley flour by Cramer et al. (2005). In our barley sample it was detected in high

242 percentage (16.72%) while, in malt and BSG samples, it was present in smaller amounts (0.75-2.33%). It has a

243 pungent aroma at high concentration but a nutty odour when present at small levels (Dong et al., 2014). While

244 the volatile composition of malted and roasted barley has been widely investigated, the literature regarding the

245 volatile aroma compoundsACCEPTED from unprocessed brewing barley is scarce, our findings corroborating and at the

246 same time completing the recent research in the field (Cramer et al., 2005; Dong et al., 2014; Goncalves et al.,

247 2014).

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248 Even though aldehydes represented the main class of aroma compounds found in malt samples (MP,

249 MCm and MCf), their abundance was greater than in the SB sample and similar with that detected in BSG and

250 wheat flour samples. The predominant aldehydes found in all malt samples were 3-methyl-butanal, 2-methyl-

251 butanal, hexanal and 2-methyl-propanal. These compounds arise from the enzymatic oxidation of unsaturated

252 or polyunsaturated fatty acids of barley or from Maillard reactions that occur during the roasting process

253 (Dong et al., 2014; Ktenioudaki et al., 2013). The intense roasting process to which MCf is subjected, lead to

254 the formation of a larger variety of aldehydes in this type of malt. Furfural, heptanal, 2-heptanal were

255 identified only in MCf, while benzaldehyde and 2-octenal were found in both MCm and MCf. Among the

256 aldehydes detected only in MCf, furfural was present in high levels (7.39%), having an almond-like aroma.

257 Benzeneacetaldehyde, identified only in the MCm sample, together with benzaldehyde, contribute to the

258 green, cacao, almond and burnt sugar notes. Benzeneacetaldehyde was also identified in malt samples by

259 Dong et al. (2014) who proposed a possible pathway for its formation as a phenylalanine enzymolysis product.

260 Benzaldehyde and benzenacetaldehyde were also present in the analyzed BSG samples. The major ketone 261 found in all malt samples was 2-butanone, imparting aMANUSCRIPT butterscotch flavour, the larger percentage being 262 detected in MCf sample. As in the case of aldehydes, in the MCf sample was identified a greater number of

263 ketones as products of lipid oxidation or Maillard reaction (Ktenioudaki et al., 2013). The peroxidation of

264 lipids during the malting process leads to the formation of furan compounds. Besides 2-pentyl-furan, high

265 relative peak areas of 2-methyl-furan (9.97%) and 2,5-dimethyl-furan were also found in the MCf sample,

266 contributing to the sweet, chocolate like flavour. BSG samples (BSGd and BSGl) were characterized by

267 significant levels of aldehydes (95.02-98.45%), the representative ones being the same four as in the case of

268 malt samples (3-methyl-butanal, 2-methyl-propanal, 2-methyl-butanal and hexanal). The levels of 2-methyl-

269 propanal and 3-methyl-butanalACCEPTED were higher in BSG samples than in the malt sample, being responsible for the

270 malty, almond-like aroma. From the ketone group, only acetophenone was found in BSG samples, while the

271 furan compounds were represented by 2-pentyl-furan. Other compounds, among which toluene, propyl-

272 benzene and 1,3,5-trimethyl-benzene, were identified in the BSG samples, imparting a sweet, fruity, balsamic

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273 or green notes. Regarding the flour samples, both WF and WWF, were characterized by a small number of

274 detected aroma compounds, the major ones being 2-methyl-propanal and hexanal.

275 3.2 Fatty acid composition

276 Another important BSG macro-nutrient includes lipids and fatty acids. These compounds occur as

277 components of intercellular membranes, spherosomes (small oil droplets), starch, and protein bodies in all

278 parts of the cereal grain (Becker, 2007). According to recent studies, the predominant lipids identified in BSG

279 were triglyceride (55% to 67% of all identified lipid compounds), followed by a notable amount of free fatty

280 acids (from 18% up to 30%) with beneficial properties for health (Niemi et al., 2012; del Rio et al., 2013). The

281 total lipids (TLs) content varied from 0.89% (in WF sample) up to 6.61% (in the BSGd sample). The

282 concentration in lipids found in the BSG samples (5.94% and 6.61% respectively) was much higher than the

283 ones found in SB and malt samples.

284 The fatty acid composition of SB, malt, BSG and flour samples was determined by GC/MS-FID

285 analysis. A total of 26 fatty acids were identified in the analyzed samples, the most abundant being linoleic 286 (18:2(z,z)n-6), palmitic (16:0) and oleic (18:1n-9) acids (TableMANUSCRIPT 2). These data agree with previously published 287 work by Niemi et al., 2012, who also found these three fatty acids as the major ones in BSG. The α-linolenic

288 (18:3n-3) and stearic (18:0) acids were also present in all samples as well as small levels of myristic (14:0),

289 vaccenic (18:1n-7), arahidic (20:0), 11-eicosenoic (20:1n-9), behenic (22:0), lignoceric (24:0) acids. Only

290 minor changes in fatty acid composition occur during malting and mashing and therefore, the fatty acid

291 composition of BSG is similar to that of barley (Becker, 2007). Nevertheless, in barley, MP and MCm, the

292 linoleic acid relative peak areas are slightly higher than in BSG, while the palmitic acid is found in a greater

293 proportion in BSG compared with the barley and malt samples. In Figure 1 is presented the GC-MS

294 chromatogram of FAMEsACCEPTED of dried BSG sample. The fatty acid profiles of barley, malt and BSG samples were

295 qualitatively similar but different from those of wheat flour samples. Both WWF and WF samples contained a

296 smaller number of fatty acids, only 16 and 11 fatty acids being detected respectively. Lipids, as one of the

297 major constituents of BSG and with a wide range of industrial applications, make BSG an attractive and

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298 alternative source of bio-based lipids (del Rio et al., 2013), therefore the importance of an exhaustive

299 characterization of BSG composition.

300

301 3.3 Phenolic content and antioxidant activity

302 Cereals, especially barley and malt, were shown to contain more phytochemicals than previously

303 considered. The polyphenols of barley play an important role during the malting process as well as preventing

304 the significant enzymatic oxidation of polyunsaturated fatty acid (Guido et al., 2005). Because most of the

305 phenolic compounds of the barley grain are contained in the husk, BSG represents not only a rich source of

306 natural antioxidants but also an inexpensive alternative to synthetic antioxidants (Mussatto, 2013). Flavonoids,

307 as a class of phenolic compounds, are also present in BSG.

308 The content in total phenols, flavonoids and overall antioxidant capacity of the analyzed barley, malt,

309 BSG and flour samples is presented in Table 3. The concentration in total phenolics varied between 21.12 mg

310 GAE/100g fw (WF) and 335.88 mg GAE/100g fw (MCf). As expected, the lowest values for the total phenolic 311 content were obtained for the flour samples, while the MANUSCRIPT highest content was retrieved in the MCf sample, 312 followed by the BSGd sample (284.20 mg GAE/100g fw) and BSGl sample (291.47 mg GAE/100g fw). The

313 increase in phenolic concentration from barley to malt samples and also in BSG samples may be associated

314 with the enzymatic release of bound phenolic compounds during different stages of the malting process

315 (Dvo řáková et al., 2008). The determined flavonoid concentrations followed a similar pattern to the phenolic

316 compounds: MCm, MCf, BSGd and BSGl samples containing the larger amounts (8.97 – 13.16 mg QE/100g

317 fw), while SB and MP had smaller levels (5.28 – 6.17 mg QE/ 100g fw). Our data roughly agrees with

318 findings of other authors. Depending on the solvent used for extraction, Meneses et al. (2013) reported for

319 total phenols in BSG valuesACCEPTED ranging from 2.14 – 9.90 mg GAE/g and a flavonoid content varying between

320 0.02 and 4.61 mg QE/g. It is already known that the content in polyphenols is influenced not only by the

321 extraction technique but also by factors such as barley cultivar and the presence or absence of the hull.

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322 According to the results obtained for total phenols and flavonoid content, the greatest antioxidant

323 capacity was found for the MCm (57.87%) followed by BSGd and BSGl samples (55.95% and 53.78%

324 respectively). The increase of antioxidant activity of MCm and BSG samples compared to the barley sample is

325 attributed to the formation during the malting process of non-enzymatic browning compounds (e.g. Maillard

326 products) which may behave as antioxidants (Dvo řáková et al., 2008). Although MCf has recorded the highest

327 content of total polyphenols, the antioxidant activity is significantly reduced because it is obtained by roasting

328 at temperatures above 250ºC. Above this temperature, the insoluble compounds formed by polymerization

329 significantly reduce the antioxidant activity. Also, Coghe et al. (2006) demonstrated that the antioxidant

330 activity is directly proportional to the darkening of the malt colour, from 20% to 80%, if the roasting

331 temperature isn’t higher than 150°C.

332

333 3.4 Statistical analysis

334 Principal component analysis (PCA) was computed on the matrix containing as variables the 335 chromatographic data (volatile and fatty acids comp osition)MANUSCRIPT and the concentrations of total phenolic 336 compounds, flavonoids and antioxidant capacity of the studied samples. As shown in Figure 2, the first two

337 components explained 97% of the variance of data, leading to a very good discrimination of samples, even

338 between the two types of BSG samples (dried and lyophilized). The contribution of each variable to the

339 differentiation of samples can be assessed by computing the correlation loadings plot. The compounds found

340 in the outer ellipse have a greater influence on the specific pattern of each sample, indicating 100% of the

341 explained variance, while the ones with loading values near zero have similar concentrations in all samples.

342 Thus, it can be noticed that the antioxidant compounds, especially the polyphenol content significantly

343 contribute to the discriminationACCEPTED of samples, higher concentrations being specific for MCm, MCf, BSGd and

344 BSGl samples. The volatile profiles as well as the fatty acids composition of the samples also play an

345 important role in their characterization. Among the volatile compounds that may be considered as key

346 constituents, it can be mentioned 2-methyl-propanal, 3-methyl-butanal, hexanal, 2-methyl-3-buten-2-ol, 1-

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347 penten-3-ol, 1-pentanol, pentanal. From the fatty acid group, along with the total content in PUFAs, n-

348 6PUFAs and PUFAs/SFAs, linoleic acid, palmitic acid and also fatty acids found in smaller amounts (caproic,

349 caprylic, nervonic, Z-7-hexadecenoic or erucic acid) contribute to sample discrimination.

350 351 4. Conclusions

352 The content of BSG in several biologically active compounds (fatty acids, polyphenols, flavonoids,

353 antioxidant capacity) as well as its volatile fingerprint was assessed and compared with the composition of

354 barley, malt and wheat flour samples. The obtained results emphasized the importance and the opportunities of

355 the re-use of this agro-industrial by-product. The researches carried out in the last decade suggested a wide

356 range of possible industrial applications of BSG, including added-value food and pharmaceutical products.

357

358 Acknowledgements. The research was financially supported by a grant from the Romanian National

359 Authority for Scientific Research, CNCS – UEFISCDI, project number PN-II-ID-PCE-2011-3-0721 and a 360 grant of the Romanian Ministry of Education, CNCS – UEFISCDI,MANUSCRIPT project number PNII-RU-PD-2012-3-0245 361 (contract no. 92/25.06.2013).

362

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433

434

435 Figure captions:

436 Figure 1. GC-MS chromatogram of FAMEs in the TLs of dried BSG analyzed on a SUPELCOWAX 10

437 capillary column. Peaks: (1) Caproic, (6:0); (2) Caprylic, (8:0); (3) Capric, (10:0); (4) Lauric, (12:0); (5)

438 Myristic, (14:0); (6) Pentadecanoic, (15:0); (7) Azelaic, (AzA); (8) Palmitic, (16:0); (9) Z-7-Hexadecenoic, 439 [C16:1(n-9)]; (10) Palmitoleic, [16:1(n-7)]; (11) Margaric,MANUSCRIPT (C17:0); (12) Heptadecenoic, [17:1(n-9)]; (13) 440 Stearic, (18:0); (14) Oleic, [18:1(n-9)]; (15) Vacc enic, [18:1(n-7)]; (16) Linoleic, [18:2(Z,Z)(n-6)]; (17)

441 Linoelaidic, [18:2(E,E)(n-6)]; (18) α-Linolenic, [18:3(n-3)]; (19) Arachidic, (20:0); (20)11-Eicosenoic, [20:1(n-

442 9)]; (21) Eicosadienoic, [20:2 (n-6)]; (22) Heneicosanoic, (21:0); (23) Behenic, (22:0); (24) Erucic, [22:1(n-9)];

443 (25) Tricosanoic, (23:0); (26) Lignoceric, (24:0); (27) Nervonic, [24:1(n-9)] acids

444

445 Figure 2. Principal component analysis bi-plot (A) and correlation loading bi-plot (B) of volatile

446 compounds, fatty acids, total phenolic compounds, flavonoids and antioxidant capacity for the analyzed 447 spring barley, malt,ACCEPTED brewers’ spent grain and flour samples. For numbering see Table 1.

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Table 1. Mean relative peak areas (expressed as % from total peak areas) and standard deviations of volatile compounds from malt, BSG and wheat flour samples analyzed by HS-ITEX/GC-MS technique

No. Volatile compound Odour perception SB MP MCm MCf BSGd BSGl WWF WF Alcohols

4 2-Methyl-3-Buten-2-ol Green, earthy,oily 16.59±0.37 ------6 2-Methyl-1-Propanol Wine, solvent 4.29±0.24 ------9 1-Penten-3-ol Green, fruity, pungent, butter 3.98±0.35 - 0.50±0.08 0.82±0.02 - - - - Malty, alcoholic, fruity, 13 3-Methyl-1-Butanol - - 0.82±0.13 - - - - - whiskey, burnt 15 1-Pentanol Sweet, balsamic, fruity 8.11±0.28 - - 3.69±0.42 - - - - 29 3,5-Octadien-2-ol Fruity, fat, mushroom - - - 0.31±0.00 - - - - Total 32.97±1.24 - 1.32±0.22 4.82±0.45 - - - -

Aldehydes

2 2-Methyl-Propanal Wine, solvent, malty 1.97±0.19 19.04±0.11 13.88±0.47 3.63 ±0.33 21.62±0.04 30.34±0.00 65.71±4.06 49.20±0.83 Buttery, oily, dark chocolate, 7 3-Methyl-Butanal 8.46±0.09 38.48±0.15 36.64±1.00 13.31±0.24 48.34±0.21 40.68±0.04 - - cocoa, almod MANUSCRIPT 8 2-Methyl-Butanal Malty, buttery, oily, cocoa 3.36±0.32 26.40±0.03 18.58±0.03 13.79±0.28 13.10±0.47 11.55±0.25 - - 11 Pentanal Almond, malt, pungent - 0.42±0.04 1.39±0.23 5.30±0.09 2.38±0.53 1.64±0.14 - - 17 Hexanal Green, grass, fat 23.16±0.01 11.63±0.08 21.18±0.81 27.12±1.70 11.67±0.14 6.41±0.13 17.36±1.19 35.99±0.04 18 Furfural Bready, almond - - - 7.39±0.59 - - - - 20 Heptanal Fat, citrus, rancid - - - 0.42±0.02 - - - - 21 2-Heptenal Green, pungent - - - 0.35±0.01 - - - - 23 Benzaldehyde Almond, burnt sugar 3.09±0.03 - 0.68±0.17 1.83±0.14 0.74±0.04 3.16±0.11 - - 26 Octanal Fat, soap, lemon, green 1.26±0.06 0.35±0.07 0.12±0.04 0.37±0.01 - - - - 28 Benzeneacetaldehyde Harsh, green, honey, cocoa - - 0.32±0.01 - 0.21±0.02 0.35±0.03 - - 32 (E)-2-Octenal Green, nut, fat - - 0.08±0.02 0.56±0.01 - - - - 35 Nonanal Fat, citrus, green ACCEPTED 2.32±0.2 0.16±0.05 0.25±0.06 0.38±0.01 0.39±0.01 0.89±0.06 9.58±.27 6.80±0.72 36 (E)-2-Nonenal Fat, cucumber, paper - - 0.22±0.04 - - - - - 37 Decanal Soap, orange peel, tallow - - 0.10±0.02 - - - - - Total 43.62±0.25 96.48±0.13 93.44±0.12 74.45±0.10 98.45±0.05 95.02±0.64 92.65±0.80 91.99±0.14

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Ketones

3 2-Butanone Butterscotch - 2.65±0.14 1.34±0.37 3.40±0.45 - - - - 10 2-Pentanone Fruity, solvent - - - 1.54±0.03 - - - - 16 3-Hexanone Fruity, wax - - - 0.37±0.03 - - - - 19 2-heptanone Sulfur, pungent, green, fruity - - - 0.12±0.03 - - - - 33 Acetophenone Sweet, flower, almond 2.56±0.33 0.14±0.01 0.23±0.05 0.22±0.01 0.38±0.02 0.60±0.07 7.35±1.60 8.02±0.4 34 Fresh, sweat, woody, 3,5-Octadien-2-one mushroom - - - 0.14±0.03 - - - - Total 2.56±0.33 2.79±0.13 1.57±0.42 5.79±0.44 0.38±0.02 0.60±0.07 7.35±1.60 8.02±0.28

Furans

5 2-Methyl-Furan Chocolate, sweet, solvent, burnt - - - 9.97±0.47 - - - - 12 2,5-Dimethyl-Furan Ethereal, meaty - - - 1.69±0.10 - - - - 24 2-Pentyl-Furan Green beans, butter 16.72±0.06 0.75±0.12 1.09±0.01 1.86±0.13 1.21±0.06 2.33±0.56 - - Total 16.72±0.06 0.75±0.12 1.09±0.01 13.52±0.70 1.21±0.06 2.33±0.56 - -

Others

1 Methyl acetate Ethereal, solvent - - - 0.34±0.10 - - - - Pungent, caramel, fruity, MANUSCRIPT 14 Toluene 4.15±0.21 - 2.44±0.45 0.76±0.03 - 0.60±0.10 - - solvent 22 Propyl-Benzene Solvent, sweet - - - - - 0.23±0.04 - - 25 1,3,5-Trimethyl-Benzene Balsamic, green, ethereal - - - - - 0.67±0.07 - - 27 Limonene Citrus, mint - - - - - 0.58±0.05 - - 30 Benzene Fruity, sweet, paint thinner - - - 0.35±0.01 - - - - 3-Ethyl-2-methyl-1,3- 31 Nutty - - 0.18±0.01 - - - - - Hexadiene Total 4.15±0.21 - 2.62±0.45 1.45±0.24 - 2.08±0.01 - - SB – spring barley; MP – Pilsner malt; MCm – Caramunich malt; MCf – Carafa malt; BSGd – dried brewers’ spent grain; BSGl – lyophilized brewers’ spent grain ACCEPTED

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Table 2. Fatty acids composition (% of total fatty acids) of total lipid from malt, BSG and wheat flour samples analyzed by GC-MS

Fatty acids (% of total SB MP MCm MCf BSGd BSGl WWF WF fatty acids) 6:0 0.01±0.01 0.03±0.02 0.01±0.01 0.07±0.02 0.02±0.01 0.02±0.01 - - 8:0 0.01±0.01 tr 0.02±0.01 0.03±0.01 0.02±0.01 0.01±0.01 tr tr 10:0 0.01±0.01 0.01±0.01 0.01±0.01 - 0.01±0.01 0.01±0.01 - - 12:0 0.04±0.02 0.02±0.01 0.02±0.01 0.02±0.01 0.01±0.01 0.02±0.01 - - 14:0 0.41±0.04 0.21±0.02 0.17±0.02 0.17±0.02 0.30±0.02 0.28±0.02 0.09±0.01 0.32±0.02 15:0 0.11±0.02 0.09±0.03 0.10±0.02 0.07±0.02 0.14±0.01 0.14±0.02 0.07±0.02 - 16:0 21.23±1.05 22.26±1.10 21.04±0.90 23.19±1.00 25.81±1.15 25.32±1.20 17.53±0.70 19.02±0.80 16:1 (n-9) 0.03±0.02 0.04±0.02 0.06±0.01 0.04±0.01 0.07±0.02 0.07±0.02 0.06±0.03 - 16:1 (n-7) 0.10±0.01 0.12±0.03 0.12±0.02 0.09±0.02 0.13±0.02 0.14±0.01 0.12±0.02 - 17:0 0.07±0.02 0.07±0.01 0.09±0.01 0.07±0.02 0.10±0.01 0.08±0.01 0.08±0.02 - 17:1 (n-9) 0.03±0.02 0.03±0.01 0.03±0.02 0.03±0.01 0.03±0.01 0.03±0.01 - - 18:0 1.80±0.05 2.17±0.09 1.58±0.07 1.65±0.05 2.40±0.08 2.06±0.07 1.12±0.05 1.16±0.05 18:1 (n-9) 15.68±0.70 12.72±0.55 15.32±0.65 16.31±0.60 12.63±0.50 12.57±0.45 15.13±0.65 11.61±0.45 18:1 (n-7) 0.62±0.03 0.54±0.04 0.65±0.02 0.57±0.02MANUSCRIPT 0.70±0.02 0.62±0.02 0.79±0.03 0.61±0.03 18:2 (Z,Z) n-6 53.13±2.55 54.66±2.40 53.18±2.50 50.70±2.20 50.19±2.30 51.48±2.20 59.85±2.90 63.77±3.00 18:2 (E,E) n-6 0.04±0.01 - - 0.72±0.02 0.23±0.02 0.14±0.01 - - 18:3 (n-3) 4.82±0.22 5.11±0.20 5.78±0.25 4.37±0.20 5.18±0.20 5.09±0.22 4.19±0.20 2.59±0.10 20:00 0.30±0.02 0.34±0.03 0.22±0.02 0.21±0.02 0.34±0.02 0.30±0.02 0.12±0.02 0.15±0.01 20:1 (n-9) 0.71±0.04 0.68±0.03 0.88±0.03 0.95±0.03 0.78±0.02 0.70±0.03 0.49±0.04 0.28±0.02 20:2 (n-6) 0.08±0.03 0.09±0.02 0.11±0.02 0.08±0.02 0.09±0.01 0.11±0.02 - - 21:0 0.02±0.02 0.02±0.01 0.03±0.01 - 0.02±0.01 0.03±0.01 - - 22:0 0.33±0.03 0.41±0.02 0.19±0.01 0.18±0.02 0.32±0.02 0.29±0.02 0.20±0.02 0.23±0.03 22:1 (n-9) 0.10±0.01 0.08±0.01 0.11±0.02 0.13±0.02 0.12±0.01 0.15±0.01 0.06±0.01 - 23:00 0.05±0.02 0.06±0.02ACCEPTED 0.04±0.02 0.05±0.01 0.05±0.01 0.05±0.01 - - 24:0 0.19±0.02 0.18±0.02 0.16±0.03 0.18±0.02 0.23±0.02 0.20±0.02 0.09±0.01 0.26±0.02 24:1(n-9) 0.09±0.03 0.05±0.01 0.08±0.02 0.11±0.03 0.08±0.02 0.09±0.01 - - AzA 0.04±0.02 0.10±0.02 0.09±0.03 0.21±0.02 0.10±0.02 0.09±0.01 - - ∑SFAs 24.57±1.20 25.87±1.30 23.68±1.00 25.90±1.15 29.78±1.35 28.82±1.30 19.31±0.85 21.14±0.90 ACCEPTED MANUSCRIPT

∑MUFAs 17.36±0.75 14.26±0.65 17.25±0.70 18.23±0.75 14.53±0.60 14.36±0.65 16.65±0.70 12.50±0.50 ∑PUFAs 58.07±2.60 59.86±2.40 59.07±2.55 55.87±2.35 55.69±2.40 56.81±2.45 64.04±3.00 66.36±3.10 ∑n-3 PUFAs 4.82±0.22 5.11±0.20 5.78±0.23 4.37±0.20 5.18±0.18 5.09±0.20 4.19±0.15 2.59±0.10 ∑n-6 PUFAs 53.25±2.48 54.75±2.25 53.29±2.30 51.50±2.20 50.51±2.20 51.73±2.20 59.85±2.70 63.77±3.00 n-6/n-3 11.04 10.71 9.22 11.78 9.75 10.16 14.29 24.57 PUFAs / SFAs 2.36 2.31 2.49 2.16 1.87 1.97 3.32 3.14 ∑VLCSFA( ≥ 20C) 0.89±0.03 1.01±0.02 0.63±0.02 0.62±0.03 0.96±0.02 0.87±0.03 0.42±0.02 0.63±0.03 Total fat (% dw) 2.96±0.12 2.55±0.15 2.31±0.13 2.74±0.10 6.61±0.30 5.94±0.28 0.89±0.03 1.19±0.05 Values are mean ± SD of three samples, analyzed individually in triplicate (n=3x3). SB – spring barley; MP – Pilsner malt; MCm – Caramunich malt; MCf – Carafa malt; BSGd – dried brewers’ spent grain; BSGl – lyophilized brewers’ spent grain; SFAs-saturated fatty acids; MUFAs-monounsaturated fatty acids; PUFAs-polyunsaturated fatty acids; VLCSFAs-very long chain saturated fatty acids; tr-trace; Caproic, (6:0); Caprylic, (8:0); Capric, (10:0); Lauric, (12:0); Myristic, (14:0); Pentadecanoic, (15:0); Palmitic, (16:0); Z-7-Hexadecenoic, [C16:1(n-9)]; Palmitoleic, [16:1(n-7)]; Margaric, (C17:0); Heptadecenoic, [17:1(n-9)]; Stearic, (18:0); Oleic, [18:1(n-9)]; Vaccenic, [18:1(n-7)]; Linoleic, [18:2(Z,Z)(n-6)]; Linoelaidicic, [18:2(E,E)(n-6)]; α-Linolenic, [18:3(n- 3)]; Arachidic, (20:0); 11-Eicosenoic, [20:1(n-9)]; Eicosadienoic, [20:2 (n-6)]; Heneicosanoic, (21:0); Behenic, (22:0); Erucic, [22:1(n-9)]; Tricosanoic, (23:0); Lignoceric, (24:0); Nervonic, [24:1(n-9)]; Azelaic, AzA. MANUSCRIPT

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Table 3. Total phenols, flavonoids and radical scavenging activity values for spring barley,

Pilsner malt, Caramunich malt, Carafa malt, BSG, wheat flour and wholemeal flour.

Total phenols Flavonoids DPPH inhibition Sample (mg GAE/100 g fw) (mg QE/100 g fw) (%) Spring barley (SB) 133.93±2.45 6.17±0.11 43.17±0.07 Pilsner malt (MP) 148.42±0.51 5.28±0.13 46.36±0.1 Caramunich malt (MCm) 256.42±6.18 10.72±0.18 57.87±0.07 Carafa malt (MCf) 335.88±4.41 8.97±0.16 42.07±0.02 Dried BSG (BSGd) 284.20±3.07 13.16±0.27 55.95±0.28 Lyophilized BSG (BSGl) 291.47±2.89 10.35±0.16 53.78±0.07 Wheat flour (WF) 21.12±1.42 2.85±0.10 32.74±0.24 Wholemeal wheat flour (WWF) 64.68±3.48 3.18±0.15 37.54±0.36

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Highlights:

• Determination of volatile fingerprint of brewers’ spent grain by ITEX/GC-MS • Determination of fatty acids profile of brewers’ spent grain by GC-MS • Determination of antioxidant compounds and activity of brewers’ spent grain • Use of principal component analysis for sample discrimination

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ACCEPTED Vodnar et al. Microbial Cell Factories 2013, 12:92 http://www.microbialcellfactories.com/content/12/1/92

RESEARCH Open Access L (+)-lactic acid production by pellet-form Rhizopus oryzae NRRL 395 on biodiesel crude glycerol Dan C Vodnar1, Francisc V Dulf2, Oana L Pop1 and Carmen Socaciu1*

Abstract Background: Given its availability and low price, glycerol derived from biodiesel industry has become an ideal feedstock for the production of fuels and chemicals. A solution to reduce the negative environmental problems and the cost of biodiesel is to use crude glycerol as carbon source for microbial growth media in order to produce valuable organic chemicals. In the present paper, crude glycerol was used as carbon substrate for production of L (+)-lactic acid using pelletized fungus R. oryzae NRRL 395 on batch fermentation. More, the experiments were conducted on media supplemented with inorganic nutrients and lucerne green juice. Results: Crude and pure glycerols were first used to produce the highest biomass yield of R. oryzae NRRL 395. An enhanced lactic acid production then followed up using fed-batch fermentation with crude glycerol, inorganic nutrients and lucerne green juice. The optimal crude glycerol concentration for cultivating R. oryzae NRRL 395 was 75 g l-1, which resulted in a fungal biomass yield of 0.72 g g-1 in trial without lucerne green juice addition and 0.83 g g-1 in trial with lucerne green juice. The glycerol consumption rate was 1.04 g l-1 h-1 after 48 h in trial with crude glycerol 75 g l-1 whileintrialwithcrudeglycerol10gl-1 thelowestrateof0.12gl-1 h-1 was registered. The highest L (+)-lactic acid yield (3.72 g g-1) was obtained at the crude glycerol concentration of 75 g l-1 andLGJ25gl-1, and the concentration of lactic acid was approximately 48 g l-1. Conclusions: This work introduced sustainable opportunities for L (+)-lactic acid production via R. oryzae NRRL 395 fermentation on biodiesel crude glycerol media. The results showed good fungal growth on crude glycerol at 75 g l-1 concentration with lucerne green juice supplementation of 25 g l-1. Lucerne green juice provided a good source of nutrients for crude glycerol fermentation, without needs for supplementation with inorganic nutrients. Crude glycerol and lucerne green juice ratio influence the L (+)-lactic acid production, increasing the lactate productivity with the concentration of crude glycerol.

Background of sustainable alternatives to fossil fuels. Biodiesel, repre- Energy fuels for world consume are mainly derived for sent an alternative for the substitution of fossil fuels, finite and declining reserves of fossil hydrocarbons [1]. which is the most common biofuel in Europe [2]. Similar Now, there is a global dependency on fossil hydrocar- to the petroleum industry, biodiesel industry generate un- bons, which will not be environmentally and economic- wanted by-products [1]. Biodiesel production generate ally sustainable in the long term [1]. Nowadays, having huge amount of crude glycerol- one part of glycerol is the authority’s pessimistic prospects for the future, regard- produced at every ten parts of biodiesel [3] and has a ing the absolute dependency on fossil fuels, the political negative influence on the biodiesel price. The glycerine and economical policies are to stimulate the development phase from biodiesel industry causing the environmental problems regarding to the management of this by- product. A solution to reduce the negative environ- * Correspondence: [email protected] 1Food Science and Technology Department, Unit of Chemistry and mental problems and the cost of biodiesel is to use crude Biochemistry, University of Agricultural Sciences and Veterinary Medicine, 3-5 glycerol as carbon source for microbial growth media in Mănăştur str, Cluj-Napoca 400372, România order to produce valuable organic chemicals [4,5]. Full list of author information is available at the end of the article

© 2013 Vodnar et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Vodnar et al. Microbial Cell Factories 2013, 12:92 Page 2 of 9 http://www.microbialcellfactories.com/content/12/1/92

Lactic acid (lactate) and its derivates have many appli- fungal biomass yields on crude glycerol and pure glycerol cations in the food, pharmaceutical, and polymer indus- samples at 95% confidence (Figure 1). The highest specific tries [6]. Its most promising application is in being used biomass yield of 0.65 ± 0.02 g biomass increase/g initial bio- as a major raw material for the production of polylactic mass · g CODremoved) was obtained on crude glycerol sam- acid (PLA) [7]. In this context, lactic acid can be pro- ples. This demonstrated that R. oryzae NRRL 395 was able duced via biological route having the advantage of being to use crude glycerol as carbon source. These results were able to produce optically pure lactic acid, while chemical in agreement with those reported by Nitayavardhana et al. route produce racemic mixture with the requirements of [15], in which R. microsporus var. oligosporus showed a high temperature and pressure [8,9]. specific biomass yield of 0.64 ± 0.06 g on crude gly- Lactic acid from biological sources can be produced cerol. More, the work reported insignificant differences by both bacteria and fungi fermentation [10]. Production between fungal biomass yields on media with yeast ex- of L (+)-lactic acid via fermentative route has increased tract and crude glycerol. in the last decade [7]. For the industrial production of L As shown in Figure 2, the optimal crude glycerol con- (+)-lactic acid, it is necessary to provide cheap carbon centration for cultivating R. oryzae NRRL 395 was 75 g l-1, sources, and to obtain the optimal conditions of fermen- which resulted in a fungal biomass yield of 0.72 g g-1 in tation with higher yields and production rates [11]. Gen- trial without LGJ addition and 0.83 g g-1 in trial with LGJ. erally, bacterial fermentation has a higher yield [12]. With respect to the control (CG 100 g l-1, without nutri- Apart from the large group of bacteria, filamentous fungi, ent supplementation), at the optimal glycerol concentra- especially R. oryzae have been proving a good lactic acid tion (75 g l-1) and pH 5, an improvement in fungal producer. R. oryzae NRRL 395 can utilize crude glycerol biomass yield was observed with 0.2 g biomass when as carbon source, and unlike its competitors of lactic acid crude glycerol samples were supplemented with inorganic producing bacteria, tolerate high impurities, has lower nu- nutrients. These results were in agreement whit those trition requirements which reduce the fermentation cost reported by Nitayavardhana and Khanal, [16] which dem- and simplifies downstream product separation [13,14] it is onstrated over 200% increase in fungal biomass yield in more tolerant to a low pH environment and the fungal vinasse media supplemented with nitrogen and phos- biomass is easy to separate from broth [10]. phorus. Generally, the fungal biomass was lower in crude In the lactic acid production by R. oryzae NRRL 395,a glycerol media supplemented with nutrients than in considerable amount of chitin is produced [10], which vinasse. However, nutrient supplements had a lower solu- limited the mass transfer of oxygen and nutrients onto bility on crude glycerol, thus led to a slower fungal growth the fungus and the release of the organic acid media. and the use of low-cost nutrient-rich solution will over- Growing fungi in pellet form can reduce these problems. come this problem [16]. The aim of the current investigation was to further assess the potentialities of valorization of crude glycerol by pelletized R. oryzae NRRL 395,inorderto 0.8 produce L (+)-lactic acid in media supplemented with laboratory nutrients or with lucerne green juice. The pelletized fermentation hold great promise as potential removed bioconversion route of low-value glycerol streams to a 0.6 higher-value product like lactic acid.

Results and discussion 0.4 *** R. oryzae NRRL 395 biomass production R. oryzae NRRL 395 was successfully cultivated on various biofuel residues, but the extremely high organic content of the crude glycerol was a major concern prior to 0.2 start this work. Thus, the first study was conducted to evaluate the possibility of R. oryzae NRRL 395 to use the g biomass increase/g initial biomass·COD crude glycerol as sole carbon source. Nutrient supplemented 0.0 media containing different carbon sources 30 g l-1 including, Specific fungal biomass yield pure glycerol and crude glycerol were selected. The initial Crude Glycerol Pure Glycerol organic matters (COD) were different in the substrates. The Figure 1 Specific fungal biomass yield for samples with pure fungal biomass yield was used to evaluate the feasibility of R. glycerol and crude glycerol (30 g l-1) supplemented with oryzae NRRL 395 cultivation on these media. A statistical inorganic nutrients (IN). The bars are means of three determinations ± SD. *** Extremly significant according to T-test. analysis showed significant differences between the specific Vodnar et al. Microbial Cell Factories 2013, 12:92 Page 3 of 9 http://www.microbialcellfactories.com/content/12/1/92

Table 1 Characteristics of lucerne green juice 0.9 a b Parameters Values* 0.8 pH 5.98 ± 0.1 0.7 c Sugar [g/L] 16.8 ± 0.15 0.6 cd Ntot [g/L] 4.5 ± 0.11 0.5 d Ptot [g/L] 3.47 ± 0.11 0.4 DM [%] 6.12 ± 0.19

Biomass yield 0.3 e NO– [mg/L] 15.65 ± 1.62 0.2 2 e – 0.1 NO3 [mg/L] 7.50 ± 0.85 Cl– [mg/L] 1560 ± 64

(g biomassincrease/g initial biomass) 0.0 2– SO4 [mg/L] 596 ± 36 g/L g/L G 100 25g/L 3– C J PO4 [mg/L] 556.3 ± 24.16 +LGJ 60 + CG 10 g/L+ CGIN 40g/L+ INCG 75g/L+ IN L L +LG g/L +LGJ 90g/ g/ Na [mg/L] 81.7 ± 1.29

G 75 + CG 10 CG 40 C K [mg/L] 4968.5 ± 37.27 Figure 2 R. oryzae NRRL 395 biomass yield (g biomass increase/ Mg2+ [mg/L] 437.6 ± 16.2 g initial biomass) on media containing different ratio of crude Ca2+ [mg/L] 1289 ± 11.3 glycerol (CG) (10, 40, 75, 100 g l-1) supplemented with inorganic nutrients (IN) or with lucerne green juice (LGJ 90, 60, *All analysis are based on n (sample size) = 3. 25 g l-1). Means (n = 3) ± SD. Means with different letter are significantly different (p < 0.05). adapted itself to the media conditions without showing substantial changes in concentrations of either glucose or L (+)-lactic acid in the culture medium. In the next Another study shows that Lucerne green juice can be 2 days, R. oryzae NRRL 395 achieved a maximal yield of use as inexpensive nutrient in lactic acid fermentation L (+)-lactic acid after 72 hours of fermentation, indicated [17]. The extracted juice was successfully used as substi- by the major consumption of nutrients during this tute for expensive nutrients for cultivating Lactobacillus period. In the next 3 days, there was almost no add- paracasei 168. The study noticed that partial substitu- itional L (+)-lactic acid produced (the level remained tion of expensive media components was possible by constant around 59 g l-1). The glucose consumption de- using lucerne green juice. This finding suggested that creased sharply from 90.5 to 27 g l-1 after 48 h and all lucerne green juice constituted a nutrient supplement the glucose was largely used up after 72 h. For this ex- for crude glycerol fermentation, substituting the laboratory periment setup, 3 days of incubation were sufficient for expensive nutrients used for this kind of fermentations. the system to reach the stage highest concentration The lucerne green juice characteristics are summarized in (58.41 g l-1) of L (+)-lactic acid. These results were in Table 1. It is interesting to note that fungal growth in sam- agreement to those reported by Yao et al. [18], in which ples with crude glycerol 10 g l-1 and 90 g l-1 LGJ was not the final lactic acid concentration of 52 g l-1 was significantly different compared with crude glycerol 10 g l-1 achieved after 72 h of fermentation. supplemented with laboratory inorganic nutrients. As shown in Figure 2, a significant fungal biomass yield was 120 70 observed in crude glycerol supplemented with lucerne Glucose L-lactic acid -1 -1 60 green juice LGJ 60 g l (0.83 g biomass) and LGJ 25 g l 100 ) -1 )

(0.52 g biomass). This supports the potential of utilizing a -1 50

80 (g l low-cost lucerne green juice as a source of nutrients to en- 40 hance the growth of R. oryzae NRRL 395. 60 30 40

Kinetics study of lactic acid production in bioreactor l (g Glucose 20

Lactic acid production by R. oryzae NRRL 395 on glucose 20 10 L-Lactic acid substrate 0 0 First, the capacity of R. oryzae NRRL 395 for sugar 0 1 2 3 4 5 6 7 utilization was investigated on glucose as model media. The Time (days) observation of R. oryzae NRRL 395 in consuming glucose Figure 3 Substrate consumption and product yield curves of R. and producing L (+)-lactic acid was presented in Figure 3. oryzae NRRL 395 during the fermentation conditions on media -1 The initial concentration of glucose was 101 g l-1. Dur- with glucose (101 g l ). The bars are means of three determinations ± SD. ing the first day of growth, the R. oryzae NRRL 395 was Vodnar et al. Microbial Cell Factories 2013, 12:92 Page 4 of 9 http://www.microbialcellfactories.com/content/12/1/92

Lactic acid production by R. oryzae NRRL 395 on crude high pH [22]. Vially et al. [23] reported no lactate produc- glycerol supplemented with inorganic nutrients tion on glycerol or lactose when R. oryzae NRRL 395 Figure 4 demonstrated the trends of glycerol consump- UMIP 4.77 was used at a stirring rate of 200 rpm. In con- tion and the L (+)-lactic acid formulation in media with trast, Abe et al. [24] founded that R. oryzae NRRL 395 crude glycerol (CG), supplemented with inorganic nutri- grew vigorously on glycerol but poorly on sucrose and ents. R. oryzae NRRL 395 in the culture media with maltose. crude glycerol 10 g l-1 consumed 6.1 g l-1 glycerol and produced 1.75 g l-1 L (+)-lactic acid, showed a lactate Lactic acid production by R. oryzae NRRL 395 on crude yield of 0.34 g g-1 (Figure 5) and a productivity of glycerol supplemented with lucerne green juice 0.09 g l-1 h-1 (Figure 5). Increasing the crude glycerol Stirred –tank bioreactor experiments showed the R. oryzae concentration in the media was considered a possibility NRRL 395 capacities to grow and produce L (+)-lactic acid to reach higher lactic acid accumulation at the end of on crude glycerol media supplemented with lucerne green fermentations. The highest concentration of glycerol juice. As shown in Figure 7 and Figure 8, the highest L (61.5 g l-1) used by R. oryzae NRRL 395 was in fermenta- (+)-lactic acid yield (3.72 g g-1)wasobtainedatthecrude tion trial with 75 g l-1 addition of crude glycerol. In glycerol concentration of 75 g l-1 and LGJ 25 g l-1,andthe this case the lactate yield (Figure 5) and productivity concentration of lactic acid was approximately 48 g l-1. (Figure 6) was 1.64 g g-1 and 0.73 g l-1 h-1 respectively, When the LGJ 90 g l-1 was used, the consumption rate after 48 hours, but in a longer fermentation time (168 h) (Figure 8) of glycerol registered after 48 hours of fer- the final lactate yield was 2.31 g g-1. In trial with 40 g l-1 mentation was 0.12 g l-1 h-1while in trials with 60 g l-1 crude glycerol, the lactate yield was 1.59 g g-1 producing and 25 g l-1 LGJ the consumption rate was 0.51 g l-1 h-1 13.3 g l-1 lactic acid after 7 days of fermentation. The gly- and 1.2 g l-1 h-1, respectively. In comparison with glucose cerol consumption rate was 1.04 g l-1 h-1 after 48 h in trial (0.58 g g-1) (Figure 3) the lactic acid yield was higher in all with crude glycerol 75 g l-1 while in trial with crude gly- trials which used LGJ and less in trial with crude glycerol cerol 10 g l-1 the lowest rate of 0.12 g l-1 h-1 was regis- 10 g l-1 supplemented with inorganic nutrients (0.34 g g-1). tered. Based on this data, was apparent that crude glycerol Generally, the profile in bioreactor fermentation could be and inorganic nutrient supplements represented a good divided into two parts at the time point of 48 h (Figure 7). source for R. oryzae NRRL 395 growth and lactic acid pro- Prior to 48 h, 46.75 g l-1 L (+)-lactic acid was produced in duction. There are many factors that could influence the trial with 25 g l-1 LGJ addition, with a productivity of growth of R. oryzae NRRL 395 such as: medium nutrients, 0.93 g l-1 h-1 which was higher than the rest of the trials. pH, agitation, aeration, medium viscosity and inoculum From 48 h to 168 h, L (+)-lactic acid concentration reached size [19-21]. Some strains such as Rhizopus.sp.requires at a value higher than 47.1 g l-1. At the end of the fermenta- strong agitation to form pellets, while others strains needs tion 48.1 g l-1 L (+)-lactic acid was obtained. The pellet

10 CG 10g l-1 +IN 2.0 40 CG 40 g l-1 + IN 22 9 35 20 18

8 1.6 ) -1

) 30

) 16 ) 7 -1 -1 -1 14 l 6 1.2 25 g (g l 12 5 20 10 4 0.8 15 8

3 Glycerol (g l Glycerol (gl 10 6 2 0.4 4 L-Lactic acid 1 5 ( acid L-Lactic Glycerol L- lactic acid Glycerol L- lactic acid 2 0 0.0 0 0 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 Time (days) Time (days)

80 45 CG 75 g l-1+IN 70 40

35 ) ) 60 -1 -1 30 50 25 40 20 30 15 Glycerol (g l Glycerol 20 10 L-Lactic l acid (g 10 Glycerol L- lactic acid 5 0 0 0 1 2 3 4 5 6 7 Time (days) Figure 4 L (+)-lactic acid production and glycerol consumption obtained in trials with crude glycerol (CG) (10, 40, and 75 g l-1) supplemented with inorganic nutrients. The bars are means of three determinations ± SD. Vodnar et al. Microbial Cell Factories 2013, 12:92 Page 5 of 9 http://www.microbialcellfactories.com/content/12/1/92

40 30 10 4.0 CG 40 g l-1 +LGJ 60 g l-1 CG 10 g l-1 +LGJ 90g l-1 9 3.6 35 25 8 3.2 30

7 2.8 ) 20 ) ) ) -1

25 -1 -1 -1 6 2.4 l g 5 2.0 20 15 15 4 1.6 10 Glycerol (g l Glycerol

Glycerol (g l Glycerol 3 1.2 10 5 2 0.8 L-Lactic acid ( L-Lactic l acid (g 5 Glycerol L- lactic acid 1 L- lactic acid 0.4 Glycerol 0 0 0 0.0 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 Time (days) Time (days)

80 55 CG 75g l-1 +LGJ 25 g l-1 70 50 45 ) ) 60

40 -1 -1 50 35 30 40 25 30 20 Glycerol (g l 20 15 10 10 L-Lactic l acid (g Glycerol L- lactic acid 5 0 0 0 1 2 3 4 5 6 7 Time (days) Figure 5 L (+)-lactic acid yield (g L (+)-lactic acid/g glycerol) during R. oryzae NRRL 395 fermentation in media containing different ratio of crude glycerol (CG) (10, 40, and 75 g l-1) supplemented with inorganic nutrients (IN) or with lucerne green juice (LGJ) (90, 60, and 25 g l-1). The error bars in the figure indicate the standard deviations of three parallel replicates ± SD. formation observed in these trials might be attributed to the from low-cost and renewable biomass, the production of nitrogen and phosphorus content of lucerne green juice that lactic acid from various materials has gained attention re- increased the viscosity of culture medium and influenced cently. Fungal species and lactic acid bacteria (LAB) have the R. oryzae NRRL 395 growth. More, the potassium con- been investigated for production of lactic acid. Different tent and protein content of lucerne green juice might in- yields of L (+)-lactic acid had been reported during fermen- crease the growth rate of R oryzae in the culture conditions tation of crude glycerol by LAB strains (0.23-0.71 g g-1) [25] studied. Because of low price and availability, lucerne green or by fungi (0.25-0.86 g g-1) [23]. juice was a possible feedstock for lactate production. As the increasing interest in producing biotechnological products

CG 10g/L+ LGJ 90g/L CG 10g/L+ IN CG 40g/L+ LGJ 60g/L 4 CG 75g/L+ LGJ 25g/L 1.4 CG 40g/L+ IN

CG 10 g/L+ IN ) CG 75g/L+ IN -1 1.2 CG 40 g/L+ IN 1.0 CG 75 g/L+ IN 3 CG 10g/L+ LGJ 90g/L ] -1 0.8 CG 40g/L+ LGJ 60g/L h CG 75g/L+ LGJ 25g/L -1 0.6 2 [ g l 0.4 0.2 1 Consumption rate of glycerol of rate Consumption 0.0

0 1 2 3 4 5 6 7 L-Lactic acid Yield (g g Time (days) 0 Figure 6 L (+)-lactic acid productivity [concentration of L 0 1 2 3 4 5 6 7 (+)-lactic acid (in g l-1) / fermentation time (in h)] during R. oryzae NRRL 395 fermentation in media containing different Time (days) ration of crude glycerol (CG) (10, 40, and 75 g l-1) Figure 7 L (+)-lactic acid production and glycerol consumption supplemented with inorganic nutrients (IN) or with lucerne obtained in trials with crude glycerol (CG) (10, 40, and75 g l-1) green juice (LGJ) (90, 60, and 25 g l-1). The error bars in the figure supplemented with lucerne green juice (LGJ) (90, 60, and indicate the standard deviations of three parallel replicates ± SD. 25 g l-1). The bars are means of three determinations ± SD. Vodnar et al. Microbial Cell Factories 2013, 12:92 Page 6 of 9 http://www.microbialcellfactories.com/content/12/1/92 ) -1 1.0 h

-1 CG 10 g/L+ IN CG 40 g/L+ IN 0.8 CG 75 g/L+ IN CG 10g/L+ LGJ 90g/L 0.6 CG 40g/L+ LGJ 60g/L CG 75g/L+ LGJ 25g/L 0.4

0.2

0.0

L-Lactic acid Productivity (g l (g Productivity acid L-Lactic 0 1 2 3 4 5 6 7 Time (days) Figure 8 Consumption rate of glycerol [initial concentration of glycerol (g l-1) - residual concentration of glycerol (g l-1)]/fermentation time [in h], during R. oryzae NRRL 395 fermentation in media containing crude glycerol (CG) (10, 40, and 75 g l-1) supplemented with inorganic nutrients (IN) or with lucerne green juice (LGJ) (90, 60, and 25 g l-1). The error bars in the figure indicate the standard deviations of three parallel replicates ± SD.

FTIR characterization Methods FTIR spectroscopy was expected to be especially valu- Glycerol able in analyzing the phase structure and the product Crude glycerol was derived from the transesterification re- formulation during pelletized fungal fermentation. The action of soybean oil and ethanol catalyzed by NaOH. The infrared spectra of lactic acid, crude glycerol and the pH of glycerine phase was corrected with H2SO4 (1 N) to sample at the end of the fermentation process from trial eliminate the free alkalinity. It was then subject to heating with crude glycerol 75 g l-1 supplemented with inorganic (up to 120°C for approximatively 1 h) under agitation to nutrients were showed in Figure 9. The functional group eliminate ethanol. The sulphate resulting from the of glycerol, including O-H stretching at 3312 cm-1, C-H neutralization was separated by decantation during 24 h. stretching at 2983, 2935, 2883 cm-1, C-O stretching from 1100 cm-1 as primary alcohol to 1400 cm-1 represented Lucerne green juice the secondary alcohol, C-C and C-O stretching from From the pressing procedure, 2.13 kg green juice and 995 to 1050 cm-1 [26]. Specific peak for lactic acid was 2.87 kg press cake were obtained from 5 kg chopped located at 1112 cm-1 [27]. To study the lactic acid for- Lucerne. The juice was drawn off into plastic containers mulation, we followed the band corresponding to lactic stored until required, at temperature of −18°C. The import- acid, which showed changes in the spectra during fer- ant characteristics of lucerne green juice are summarised mentation processes. These signals validated the HPLC in Table 1. investigations regarding the concentration of lactic acid.

Conclusions R. oryzae NRRL 395 cultivation This work introduced sustainable opportunities for L The fungus R. oryzae NRRL 395 was obtained from (+)-lactic acid production via R. oryzae NRRL 395 fermenta- USAMV microorganism bank. The fungus was first grown tion on biodiesel crude glycerol media. The results showed on potato-dextrose agar (PDA) (Sifin) slants at 30°C for good fungal growth on crude glycerol at 75 g l-1 concentra- 7 days. For experiments, the fungal spores in the slant tion with lucerne green juice supplementation. Lucerne were resuspended in sterilized water maintaining at 3°C. green juice provided a good source of nutrients for crude In terms of achieving pellet form, the spore solution was glycerol fermentation, without required for supplementation inoculated in a 125 ml Erlenmeyer flask, containing 50 ml with inorganic nutrients. Crude glycerol and lucerne green of seed medium with a spore concentration of 1 x juice ratio influenced the L (+)-lactic acid production, in- 107spore/ml, and cultured at 27°C on shaker (Heidolph creased the lactate productivity once with the concentration Unimax 1100) set at 180 rot/min for 24 hours. The cul- of crude glycerol. Further research on scaling –up the tured temperature was set at 27°C. The pellet was process and monitorization by FTIR spectroscopy of L decanted by centrifugation at 15000 rpm for 15 min (+)-lactic acid production on bioreactor is required. and used for the following experiments. Vodnar et al. Microbial Cell Factories 2013, 12:92 Page 7 of 9 http://www.microbialcellfactories.com/content/12/1/92

0,5 1033 Glycerol 0.25 L-Lactic acid 1120 CG 75%+ W 25% 0.20 0,4 0.15

0.10 2935 1716 Absorbance [a.u] 0,3 0.05

0.00 2500 3000 3500 997 Wavenumber [cm-1] 820 0,2 1112 736 923

Absorbance [a.u] 850

1373 0,1 1210

0,0

1000 1500 Wavenumber [cm -1] Figure 9 Comparative FTIR fingerprint of glycerol, lactic acid and the sample at the end of the fermentation process from trial with crude glycerol (CG) (75 g l-1) supplemented with inorganic nutrients (IN).

Kinetic study of lactic acid production in bioreactor in a crucible for 2 h at 550°C in a preheated furnace. The kinetic study was performed on media containing After cooling, the crucible and ash were weighed. The -1 -1 -1 -1 10 g l ,40gl ,75gl and 100 g l crude glycerol with total nitrogen content (Ntot) was analyzed using the tap water and inorganic nutrient supplementation. Media standard method Kjeldahl. The colorimetric technique with inorganic nutrient supplementation contained 0.0092% was used to measure the total phosphorus with the mo- CaCl2 ·2H2O, 0.4% MgSO4 ·7H2O, 0.6% KH2PO4, and 1.6% lybdenum blue method. Anions and cations were deter- (NH4)2SO4. The second scenario consisted in the substitu- mined under the following conditions using the ion tion of inorganic nutrient supplements with lucerne green chromatograph Agilent 1200 Series HPLC: IonPac AS14 juice, thus the fermentation media containing 10 g l-1, (4 mm) column (anions) and IonPac CS12A (4 mm) column 40 g l-1,75gl-1 crude glycerol with lucerne green juice (cations). The eluent was 3 mM disodium carbonate, 1 mM 90 g l-1,60gl-1,and25gl-1. In all samples the pH was ad- sodium hydrogen carbonate (anions) and 23 mM sulfuric justed to 5.5 with calcium carbonate (35 g l-1). The bioreac- acid (cations) with a flow rate of 1 mL/min. The injection tor containing the medium was sterilized in autoclave at volume was 20 μL. Aliquots of the fermentation liquid were 121°C for 15 min. Inoculum was transferred to the fermen- taken every 24 h to determine L-lactic acid and gly- tation media at 1 x 107spore/ml. The bioreactor used for the cerol concentration. The content of lactic acid and gly- fermentative assays was the Electrolab FERMAC 360 with a cerol was determined by HPLC (Dionex) using a Eurokat vessel volume of 2.9 L, equipped with a digital control unit. H column (300 × 8 mm, 10 lm) and an differential refract- The fermentation was carried out at 27°C with 360 rpm agi- ometer RI-71 detector with a detection limit of 0.01 g/L. tation speed and 1 vvm aeration rate. The mobile phase was 0.01 NH2SO4 using isocratic elution with a flow rate of 0.9 mL/min. The chemical oxygen Analytical methods demand (COD) was determined by Standard Methods The lucerne green juice samples were dried to a con- #5220 [28]. stant weight at 102°C to dry matter (DM102). For total The yield of L (+)-lactic acid, productivity and the solids analysis, the dried residue was weighed and heated consumption rate of glycerol were calculated as: Vodnar et al. Microbial Cell Factories 2013, 12:92 Page 8 of 9 http://www.microbialcellfactories.com/content/12/1/92

Yield of lacticacid ¼ gLðÞþ ‐lacticacid=gglycerol

ÀÁ ‐ LðÞþ −lacticacid productivity ¼ concentrationof LðÞþ ‐lacticacid ing1 1 =fermentationtimeðÞ inh

ÀÁ ¼ −1 Consumption rate of glycerol initial concentration of glycerol g lÀÁ − −residual concentration of glycerol g l 1 =fermentation timeðÞ in h

Biomass yield and specific biomass yield Authors’ contributions CS, FVD, OLP carried out the chemical investigations regarding the Lucerne The biomass yield was determined following 96 hours of green juice and glycerol characterization. DV contributed to the fermentation fermentation. The inoculum for trials was fixed at 0.12 g experimental work, measuring the lactic acid and glycerol content and dry pellet biomass l-1. The collected samples after the recording the FTIR spectra, and wrote the manuscript. All authors read and approved the final manuscript. fermentation were centrifuged at 4000 rpm for 15 min.

The precipitated biomass was washed twice using 4 N Acknowledgements HCl to remove residual calcium carbonate and the bio- This work has supported by a research grant of University of Agricultural mass was determined by weighing the mass after drying Sciences and Veterinary Medicine, Cluj-Napoca. at 80°C overnight. Author details 1Food Science and Technology Department, Unit of Chemistry and Biomassyield ¼ gbiomassincrease=ginitialbiomass Biochemistry, University of Agricultural Sciences and Veterinary Medicine, 3-5 Mănăştur str, Cluj-Napoca 400372, România. 2Department of Environmental Specific biomass yield = grams biomass increase/gram and Plant Protection, University of Agricultural Sciences and Veterinary ă ăş initial biomass· COD removed, and was determined in Medicine, 3-5 M n tur str, Cluj-Napoca 400372, România. order to compare fungal growth in various samples with Received: 30 July 2013 Accepted: 7 October 2013 different initial organic content. Published: 10 October 2013

FTIR characterization of the fermentation processes References 1. Dobson R, Gray V, Rumbold K: Microbial utilization of crude glycerol for FTIR spectra using attenuated total reflectance (ATR) the production of value-added products. J Ind Microbiol Biotechnol 2012, and an internal reflection accessory made of composite 39:217–226. zinc selenide (ZnSe) and diamond crystals were obtained 2. Drozdzynska A, Leja K, Czaczyk K: Biotechnological production of 1,3- propanediol from crude glycerol. J Biotechnol Comp Bio Bionanotechnol on a Schimatzu IR Prestige- 21 spectrometer. Each spectrum 2011, 92:92–100. -1 was registered from 4000 to 500 cm .TheFTIRspectra 3. Rossi DM, da Costa JB, de Souza EA, Perelba MCR, Ayub MAZ: were recorded for all samples in parallel with controls. Three Bioconversion of residual glycerol from biodiesel synthesis into 1,3- propanediol and ethanol by isolated bacteria from environmental spectra were acquired for each trial variant at room consortia. Renew Energ 2012, 39:223–227. temperature. Each spectrum was composed of an aver- 4. Kosmider A, Drozdzynska A, Blaszka K, Leja K, Czaczyk K: Propionic acid age of 128 separate scans. The measuring time was ap- production by Propionibacterium freudenreichii ssp. shermanii using industrial wastes: crude glycerol and whey lactose. Pol J Environ Stud proximately 9 minutes per sample (n = 3), depending 2010, 19:1249–1253. on the number of scans per spectrum. Accordingly, as 5. Yang F, Hanna MA, Sun R: Value-added uses for crude glycerol-a the average number of scans increased, the measuring byproduct of biodiesel production. Biotechnol Biofuels 2012, 5:13. 6. Okano K, Tanaka T, Ogino C, Fukuda H, Kondo A: Biotechnological time increased. production of enantiomeric pure lactic acid from renewable resources: recent achievements, perspectives, and limits. Appl Microbiol Biotechnol Statistical analysis 2010, 85:413–423. 7. Zhao J, Xu L, Wang Y, Zhao X, Wang J, Garza E, Manow R, Zhou S: All experiments were conducted in triplicates and ANOVA Homofermentative production of optically pure L-lactic acid from xylose and t-tests were employed to determine if there was by genetically engineered Escherichia coli B. Microb Cell Fact 2013, 12:57. statistical difference between trials at significance level 8. Vaidya AN, Pandey RA, Mudliar S, Kumar MS, Chakrabarti T, Devotta S: Production and recovery of lactic acid for polylactide -an overview. Crit of p < 0.05 using Graph Prism 4.0 (Graph Pad Software Rev Environ Sci Technol 2005, 35:429–467. Inc., San Diego, CA, USA). 9. Oh H, Wee YJ, Yun JS, Han SH, Jung SW, Ryu HW: Lactic acid production from agricultural resources as cheap raw materials. Bioresour Technol Abbreviations 2005, 96:1492–1498. LGJ: Lucerne green juice; COD: Chemical oxygen demand; CG: Crude 10. Liu Y, Liao W, Chen S: Co-production of lactic acid and chitin using a glycerol; IN: Inorganic nutrients. pelletized filamentous fungus Rhizopus oryzae cultured on cull potatoes and glucose. J Appl Microbiol 2008, 105:1521–1528. Competing interests 11. Ilmén M, Koivuranta K, Ruohonen L, Rajgarhia V, Suominen P, Penttilä M: The authors declare that they have no competing interests. Production of l-lactic acid by the yeast Candida sonorensis expressing Vodnar et al. Microbial Cell Factories 2013, 12:92 Page 9 of 9 http://www.microbialcellfactories.com/content/12/1/92

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doi:10.1186/1475-2859-12-92 Cite this article as: Vodnar et al.: L (+)-lactic acid production by pellet- form Rhizopus oryzae NRRL 395 on biodiesel crude glycerol. Microbial Cell Submit your next manuscript to BioMed Central Factories 2013 12:92. and take full advantage of:

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Submit your manuscript at www.biomedcentral.com/submit Molecules 2013, 18, 11768-11782; doi:10.3390/molecules181011768 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article Lipid Classes and Fatty Acid Regiodistribution in Triacylglycerols of Seed Oils of Two Sambucus Species (S. nigra L. and S. ebulus L.)

Francisc Vasile Dulf 1, Ioan Oroian 1, Dan Cristian Vodnar 2,*, Carmen Socaciu 2 and Adela Pintea 3

1 Department of Environmental and Plant Protection, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Cluj-Napoca 400372, Manastur 3-5, Romania; E-Mails: [email protected] (F.V.D.); [email protected] (I.O.) 2 Faculty of Food Science and Technology, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Cluj-Napoca 400372, Manastur 3-5, Romania; E-Mail: [email protected] 3 Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Cluj-Napoca 400372, Manastur 3-5, Romania; E-Mail: [email protected]

* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +40-264-596-384 (ext.213); Fax: +40-264-593-792.

Received: 30 July 2013; in revised form: 2 September 2013; / Accepted: 4 September 2013 / Published: 25 September 2013

Abstract: The oil content and fatty acid composition of total lipids (TLs) and main lipid classes (NLs- neutral and PLs- polar lipids) in seeds of two wild Sambucus species (S. nigra and S. ebulus) from Transylvania (Romania) were determined by capillary gas chromatography (GC-MS). In addition, the positional distribution of fatty acids in seed triacylglycerols (TAGs) was determined by hydrolysis with pancreatic lipase. The seeds were found to be rich in fat (22.40–24.90 g/100g) with high amounts of polyunsaturated fatty acids (PUFAs) ranging from 68.96% (S. ebulus) to 75.15% (S. nigra). High ratios of PUFAs/SFAs (saturated fatty acids), ranging from 7.06 (S. nigra) to 7.64 (S. ebulus), and low ratios of n-6/n-3, ranging from 0.84 (S. nigra) to 1.51 (S. ebulus), were determined in both oils. The lipid classes/subclasses analyzed (PLs, MAGs—monoacylglycerols, DAGs—diacylglycerols, FFAs—free fatty acids, TAGs and SEs—sterol esters) were separated and identified using thin-layer chromatography. The fatty acid compositions of the TAG fractions were practically identical to the profiles of TLs, with the same dominating fatty acids in both analyzed species. SEs and FFAs, were characterized by high Molecules 2013, 18 11769

proportions of SFAs. The sn-2 position of TAGs was esterified predominantly with linoleic acid (43.56% for S. nigra and 50.41% for S. ebulus).

Keywords: Sambucus seed oils; fatty acid composition; lipid class; stereospecific analysis; GC-MS

1. Introduction

In recent years, non-traditional vegetable oils have been used more and more in the healthcare industry due to their therapeutic properties [1]. These oils have become attractive from a nutritional standpoint, due to their unique phytochemical composition and antioxidant properties [2–4]. Sambucus ebulus L. (also called dwarf elder, elderberry or danewort) and Sambucus nigra L. (also known as black or European elderberry) are native perennial herbs of the Adoxaceae family in the order of the Dipsacales. The genus Sambucus grows in temperate to subtropical regions of the World. The plants tolerate relatively poor soil conditions and prefer the sunlight-exposed locations, but they can also grow in semi-shade situations [5]. Sambucus nigra L., due to their dark blue/purple fruits which are desirable to birds, rapidly colonize the areas along roadways, forest edges, and fence lines [6]. Sambucus ebulus L. is known in Romanian folk medicine mainly for its bacteriostatic and diuretic action [7]. In many countries of the World, leaves, flowers and berries of these plants are traditionally used for several medicinal applications [5,8]. The small, fully ripened fruits of Sambucus species are rarely used for fresh consumption, and they are mainly processed into jams, jellies and juices. Several in vitro studies indicate that these berries, due to their high content of anthocyanins and other polyphenolics, possess important antioxidant activity and anticarcinogenic, immune-stimulating, antibacterial, antiallergic, antiviral and anti-inflammatory properties [9–13]. The berry seeds are byproducts of the beverage and juice processing industry and their direct disposal in the environment can create serious environmental problems. Recent studies have shown that berry seed residues could be used as raw materials for the production of non-conventional seed oils with unique chemical properties and wide applications in the healthcare industry [1]. The role of dietary fats and oils in human nutrition is determined by their composition [4]. Moreover, the positional distribution of fatty acids in triacylglycerols could affect the nutritional value of lipids [14]. The available scientific reports on Sambucus berries refer to the composition of phenolic compounds [6,11,15] and their anti-oxidant properties [10,12], but few studies relate to lipid compositions [12,16–19]. The study of Sambucus fruit seed oils for their main lipid constituents (neutral and polar), may lead to value-added utilization of these fats and enhance the profitability of the fruit processing industries. The objectives of this study were to compare the oil content and fatty acid composition of total lipids (TLs) and main lipid classes (polar lipids—PLs, monoacylglycerols—MAGs, diacylglycerols—DAGs, free fatty acids—FFAs, triacylglycerols—TAGs and sterol esters—SEs) in seeds of two wild Sambucus species (S. nigra and S. ebulus) growing on their natural sites in Transylvanian region (Romania). In addition, the positional distribution of fatty acids in seed TAGs was determined by enzymatic degradation with pancreatic lipase.

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2. Results and Discussion

2.1. Oil Content of the Seeds

The data for the total seed lipid contents (expressed on the basis of seed dry weight) from wild berries of the S. nigra and S. ebulus are summarized in Table 1. There was no significant difference (p < 0.05) in oil yield of the analyzed seed species (24.90 g/100 g for S. ebulus vs. 22.40 g/100 g for S. nigra seeds). Very little data are available in order to compare the lipid contents of the wild fruit seeds studied in the present paper. Fazio et al. [12] examined the seed oil of S. nigra and measured an oil content of 1.59 g oil/10 g of dry seed flour. Helbig et al. [20] studied the health-beneficial ingredients remaining in the waste of various berry seeds and reported 12% of recovered oil from elderberry seed press residues. Johansson et al. [19] found that the seeds of the berry species belonging to genera Vaccinium, Oxycoccus and Sambucus were similar, having oil contents in a narrow range, from 24% to 33% on dry weight basis.

2.2. Fatty Acids Profile

The fatty acid compositions of TLs and lipid classes/subclasses, polar (PLs) and neutral (MAGs, DAGs, FFAs, TAGs, SEs), from seeds of two Sambucus species are listed in Tables 1 and 2.

2.2.1. Fatty Acid Composition in TLs

According to the results shown in the Figure 1 and Table 1, twenty fatty acids were identified in both Sambucus seed oils. Comparing the TLs of two species, S. ebulus contained significantly higher proportion of oleic (C18:1n-9) and linoleic (C18:2n-6) acids (20.31% vs. 12.84%, p < 0.05 and 41.43% vs. 34.28%, p < 0.05, respectively) than S. nigra. The differences were greatest in the amounts of α-linolenic (C18:3n-3) (27.50% vs. 40.76%, p < 0.05) and palmitic (C16:0) (5.74% vs. 7.93%, p < 0.05) acids, but in the opposite direction. For S. nigra seed oils, Fazio et al. [12] reported a slightly lower α-linolenic acid content (approx. 32.10%) and similar values to those mentioned above for the other two major unsaturated fatty acids, linoleic (approx. 38.40%) and oleic (approx. 13.50%) acids, respectively. Small amounts of stearic (C18:0) (<3%) and very small (<1.30%) (or trace) percentages of vaccenic (C18:1n-7), cis-7 hexadecenoic (C16:1n-9), arachidic (C20:0), 11-eicosenoic (C20:1n-9), palmitoleic (C16:1n-7), myristic (C14:0), margaric (C17:0), 11,14-eicosadienoic (C20:2n-6), behenic (C22:0), erucic (C22:1n-9), pentadecanoic (C15:0), azelaic, eicosatrienoic (C20:3n-3), tricosanoic (C23:0) and lauric (C12:0) acids were also determined in both Sambucus species (Table 1). As shown in Table 2, statistically significant differences (p < 0.05) were found between fatty acid classes (excepting the very long chain saturated fatty acids fractions—VLCSFAs) of investigated berry seed TLs. The oil of both species contained high amounts of polyunsaturated fatty acids (PUFAs) ranging from 68.96% (S. ebulus) to 75.15% (S. nigra).

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Table 1. Fatty acid composition (% of total fatty acids) of total lipid and individual lipid class.

Sambucus nigra L. Sambucus ebulus L. Fatty acids TLs PLs MAGs DAGs FFAs TAGs SEs TLs PLs MAGs DAGs FFAs TAGs SEs 12:0 0.01 0.20 0.06 0.17 0.43 0.02 1.27 tr. 0.06 0.07 0.35 0.51 0.02 0.58 14:0 0.09 a 0.65 0.60 0.47 1.73 0.11 1.67 0.06 a 0.23 0.20 0.72 0.96 0.08 1.06 15:0 0.02 a 0.25 - - 0.51 0.03 0.73 0.01 a 0.09 0.07 0.28 0.43 0.02 0.46 AzA 0.02 a 0.42 - - - - 0.30 0.02 a 0.17 0.01 0.20 - - - 16:0 7.93 a 18.99 9.71 13.94 22.57 7.11 18.43 5.74 b 15.22 8.77 9.60 15.78 5.80 9.99 16:1,n-9 0.15 a 0.54 - 0.15 0.29 0.06 1.72 0.07 b 0.19 0.29 0.76 0.69 0.05 0.92 16:1,n-7 0.08 a 0.44 0.12 0.19 0.11 0.07 0.36 0.10 a 0.12 0.11 0.40 0.18 0.09 0.17 17:0 0.04 a 0.14 - 0.33 0.39 0.06 0.36 0.05 a 0.14 0.06 0.07 0.25 0.04 0.09 18:0 2.29 b 4.81 2.38 4.37 11.57 1.76 8.57 2.94 a 5.72 3.43 5.42 8.73 2.39 5.54 18:1, n-9 12.84 b 12.78 5.53 19.04 7.77 11.35 23.00 20.31 a 20.40 22.62 31.29 19.62 20.85 16.82 18:1, n-7 0.94 b 1.72 0.89 1.74 0.69 0.82 0.60 1.22 a 1.79 2.53 2.02 1.31 1.11 0.92 18:2, n-6 34.28 b 40.07 48.48 48.43 23.88 36.02 24.64 41.43 a 40.58 47.24 31.40 28.73 45.03 36.15 19:0 - 0.10 - - - - 0.33 - 0.05 - - 0.06 - - 18:3, n-3 40.76 a 11.34 27.50 9.44 29.50 42.31 10.39 27.50 b 13.16 13.19 12.82 19.00 24.21 19.02 20:0 0.15 a 0.66 - 0.39 0.32 0.06 1.99 0.14 a 0.66 0.14 0.38 0.73 0.06 0.32 2-OH-C16:0 - 0.63 ------0.20 - - - - - 20:1, n-9 0.14 b 0.36 - 0.39 - 0.10 0.38 0.28 a 0.34 0.39 0.74 0.39 0.17 0.49 20:2, n-6 0.07 a 0.07 - - - 0.02 - 0.03 b - 0.06 - - 0.01 - 20:3, n-3 0.04 - - - - 0.06 - tr. - - - - tr. - 22:0 0.08 a 3.35 - 0.69 - - 1.34 0.06 a 0.18 0.02 0.18 0.98 - 0.17 22:1, n-9 0.06 a 2.29 4.74 0.26 0.23 0.03 3.94 0.03 b 0.59 0.81 3.37 1.37 0.08 7.28 23:0 0.01 a 0.17 - - - - - 0.01 a 0.10 - - 0.27 - - Oil content (g/100 g seeds) 22.40 a 24.90 a The values represent the means of three samples, analyzed individually in triplicate (n = 3 × 3). TLs: total lipids, PLs: polar lipids, MAGs: monoacylglycerols, DAGs: diacylglycerols, FFAs: free fatty acids, TAGs: triacylglycerols, SEs: sterol esters, tr.: trace. Different superscript letters (a,b) in the same row mean significant differences between TLs of the two species (unpaired t-test). C12:0, lauric; C14:0, myristic; C15:0, pentadecanoic; AzA, azelaic; C16:0, palmitic; C16:1n-9, cis-7 hexadecenoic; C16:1n-7, palmitoleic; C17:0, margaric; C18:0, stearic; C18:1n-9, oleic; C18:1n-7,vaccenic; C18:2n-6, linoleic; C19:0, nonadecanoic; C18:3n-3, α-linolenic; C20:0, arachidic; 2-OH-C16:0, 2-hydroxy palmitic; C20:1n-9, 11-eicosenoic; C20:2n-6, 11,14-eicosadienoic; C20:3n-3, eicosatrienoic; C22:0, behenic; C22:1n-9, erucic; C23:0, tricosanoic acids. Molecules 2013, 18 11772

Table 2. The composition (%) of fatty acid classes in total lipids and major lipid fractions.

Fatty acids (% of total fatty acids) ∑ VLCSFAs PUFAs/ Species ∑ SFAs ∑ MUFAs ∑ PUFAs n-6/n-3 (≥20C) SFAs S.nigra c b a d TLs a10.64 ± 0.45 de b14.21 ± 0.55 d a75.15 ± 1.65 a a0.25 ± 0.05 de b0.84e a7.06b b c a d PLs 29.75 ± 1.15 b 18.14 ± 0.75 c 51.49 ± 1.25 c 4.18 ± 0.14 a 3.54b 1.73e b b a MAGs 12.75 ± 0.47 d 11.28 ± 0.38 e 75.97 ± 1.50 a - 1.76d 5.96c b b a c DAGs 20.36 ± 0.95 c 21.77 ± 0.95 b 57.87 ± 1.30 b 1.08 ± 0.12 c 5.13a 2.84d b c a d FFAs 37.53 ± 1.35 a 9.09 ± 0.35 f 53.38 ± 1.22 c 0.32 ± 0.08 d 0.81e 1.42f c b a d TAGs 9.15 ± 0.38 e 12.44 ± 0.50 de 78.42 ± 1.70 a 0.06 ± 0.02 e 0.85e 8.57a a b a c SEs 34.98 ± 1.20 a 30.00 ± 1.10 a 35.02 ± 1.30 d 3.33 ± 0.12 b 2.37c 1.00g S. ebulus c b a d TLs b9.02 ± 0.40 e a22.02 ± 0.80 c b68.96 ± 1.55 a a0.20 ± 0.05 d a1.51e a7.64b b b a c PLs 22.62 ± 0.78 b 23.44 ± 0.75 c 53.74 ± 1.30 c 0.94 ± 0.10 b 3.08b 2.38e c b a d MAGs 12.77 ± 0.35 d 26.75 ± 0.72 b 60.49 ± 1.60 b 0.16 ± 0.04 d 3.59a 4.74c c b a d DAGs 17.20 ± 0.72 c 38.57 ± 1.30 a 44.22 ± 1.22 d 0.57 ± 0.05 c 2.45c 2.57e b c a d FFAs 28.70 ± 1.10 a 23.57 ± 0.70 c 47.73 ± 1.26 d 1.97 ± 0.15 a 1.51e 1.66f c b a d TAGs 8.40 ± 0.35 e 22.35 ± 0.75 c 69.25 ± 1.60 a 0.06 ± 0.03 d 1.86d 8.24a c b a d SEs 18.22 ± 0.75 c 26.60 ± 0.70 b 55.17 ± 1.35 c 0.49 ± 0.04 c 1.90d 3.03d Values are mean ± SD of three samples, analyzed individually in triplicate (n = 3 × 3). Different subscript

letters (a, b) in front of the mean values in the same columns indicate significant differences (p < 0.05) between total lipids (TLs) of two analyzed seed species (unpaired t-test).; Means in the same row followed by different superscript letters (a, b, c, d) indicate significant differences (p < 0.05) among fatty acid classes; Means

in the same column followed by different subscript letters(a, b, c, d, e, f) indicate significant differences (p < 0.05) among lipid classes of each seed sample (ANOVA “Tukey’s Multiple Comparison Test”); SFAs—saturated fatty acids, MUFAs- monounsaturated fatty acids, PUFAs-polyunsaturated fatty acids, VLCSFAs—very long chain saturated fatty acids; TLs—total lipids, PLs—polar lipids, MAGs—monoacylglycerols, DAGs—diacylglycerols, FFAs—free fatty acids, TAGs—triacylglycerols, SEs—sterol esters.

Figure 1. GC-MS chromatogram of FAMEs in the TLs of Sambucus nigra L. seeds analyzed with a SUPELCOWAX 10 capillary column.

Peaks: (1) C12:0, lauric; (2) C14:0, myristic; (3) C15:0, pentadecanoic; (4) AzA, azelaic; (5) C16:0, palmitic; (6) C16:1n-9, cis-7 hexadecenoic; (7) C16:1n-7, palmitoleic; (8) C17:0, margaric; (9) C18:0, stearic; (10) C18:1n-9, oleic; (11) C18:1n-7,vaccenic; (12) C18:2n-6, linoleic; (13) C18:3n-3, α-linolenic; (14) C20:0, arachidic; (15) C20:1n-9, 11-eicosenoic; (16) C20:2n-6, 11,14-eicosadienoic; (17) C20:3n-3, eicosatrienoic; (18) C22:0, behenic; (19) C22:1n-9, erucic; (20) C23:0, tricosanoic acids. Molecules 2013, 18 11773

These levels are comparable to those of PUFA-rich vegetable oils, such as grape seed (65.40%), sunflower (66%), paprika seed (67.80%), perilla (69.90%), linseed (71.80%), blackcurrant seed (75.30%), safflower (77.30%) and hemp seed (79.10%) oils [21]. Moreover, the present analysis indicated that PUFAs profile of S. nigra seed oil resembles the seed oils of Ericaceae and sea buckthorn berries, whereas the proportion of oleic (C18:1n-9), linoleic (C18:2n-6) and α-linolenic (C18:3n-3) acids in TL fraction of S. ebulus seeds are similar to those of some seed oils of Rosaceae berries [19]. The oils of both species were characterized by high ratios of PUFAs/ saturated fatty acids (SFAs) (Table 2). These types of vegetable oils are susceptible to oxidative damage because of their high content in linoleic and α-linolenic acids [22]. The ratio of n-6 to n-3 fatty acids in TL of S. ebulus (1.51) was significantly higher (p < 0.05) than that in S. nigra TL (0.84). Epidemiological and clinical studies suggest that lowering the dietary n-6/n-3 fatty acid ratio may reduce the risk of coronary heart disease and cancer [23,24].

2.2.2. Fatty Acid Composition of TAGs

The data for the fatty acid compositions of TAGs in the seeds of two Sambucus species are shown in Tables 1 and 2. The fatty acid patterns of the TAG fractions (the major neutral lipid class of seed oils) were practically identical to the profiles of TLs, with the same dominating fatty acids in both analyzed species. Comparing data from the present study with those reported by Johansson et al. [19] for the main fatty acids from seed oil TAG of Sambucus racemosa, important differences in SFAs, MUFAs (monounsaturated fatty acids) and PUFAs contents were observed. However, the linoleic acid amount from S. ebulus seed oil TAG (45.03%) is similar to that determined in seed oil TAG of Sambucus racemosa (46.10%). The geographical and climatic conditions, as well as cultivating activities, genetic differences among species, maturity stages of the seeds and lipid extraction methods could explain these differences in fatty acid compositions [25,26].

2.2.3. Fatty Acid Composition of Minor Neutral Lipid (NL) Subclasses

The NL fraction of vegetable oils mainly consists of TAGs. However, small amounts of partial glycerides (MAGs and DAGs) and FFAs are always present, whose origin could be traced to biosynthetic and lipolytic (enzymatic or chemical) processes. All minor NL subclasses (MAGs, DAGs, FFAs and SEs) were highly unsaturated (Tables 1 and 2). The contents of unsaturated fatty acids (MUFAs+PUFAs), which mainly consisted of oleic, linoleic and α-linolenic acids (Table 1), ranged from 62.47% (in FFAs) to 87.25% (in MAGs) for S. nigra and 71.30% (in FFAs) to 87.24% (in MAGs) for S. ebulus (Table 2). The levels of SFAs in SEs (34.98%—S. nigra and 18.22%—S. ebulus) and FFAs (37.53%—S. nigra and 28.70%—S. ebulus) of both seed oil were significantly higher (p < 0.05) than in the other two NL fractions, due to the dominance of palmitic acid in their structures (Table 1). It is interesting to note that in SE fraction of S. nigra, the VLCSFAs, namely arachidic and behenic, were estimated in a relatively low but significant amount and comprised about 3.30% of total fatty acids. In higher plants, VLCSFAs (with more than 18 carbons) are essential structural components of plant cuticular lipids [27,28].

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As shown in Table 2, in SE and FFA fractions of S. nigra seed oil, the PUFAs/SFAs ratios were significantly lower (p < 0.05) (below 1.50) than in the other two corresponding NL subclasses (MAG and DAG ). Previous studies have shown that the values of this ratio comprised between 1.0 and 1.5, are optimal to reduce the risk of cardiovascular diseases [29,30].

2.2.4. Fatty Acid Composition of PLs

The major PUFAs in PLs of Sambucus seed oils were linoleic, and α-linolenic acids, together comprising more than 50% of the total fatty acids (Table 1). Comparing with the other lipid fractions, the PLs, similar to SEs and FFAs, were characterized by high percentages of SFAs (p < 0.05). The amounts of saturated (consisting mainly of palmitic, stearic and VLCS fatty acids) accounted for 29.75% and 22.62% of total fatty acids in S. nigra oil PL and S. ebulus oil PL, respectively (Table 2). These observations are in accordance with those of our previous studies [26,28] and with the data reported by Zlatanov [31], Kallio et al. [25], Yang et al. [32], Gutierrez et al. [22] and Ramadan et al. [1], regarding to the fatty acid composition of the PL, SE and FFA fractions of other non-conventional seed oils. The tested PLs had n-6 PUFAs to n-3 PUFAs ratios of 3.54 for S. nigra and 3.08 for S. ebulus. Very small amounts (<1%) of 2-hydroxy palmitic acid (2-OH-C16:0) were found in both PL fractions. These types of hydroxy fatty acids with important physicochemical and physiological properties in eukaryotic cells are synthesized by a sphingolipid fatty acid 2-hydroxylase, and are predominantly present in complex sphingolipids such as glucosylceramides and glycosylinositolphosphorylceramides [33]. In a recent study Herrero et al. [34] reported that 2-hydroxylated fatty acid-containing ceramides are involved in the mechanism of action of a novel synthetic antitumor drug (PM02734). Differences between the fatty acid compositions of the studied lipid fractions could be attributed to the different phases of biosynthesis and accumulation of TAGs, SEs, PLs and fatty acids [35,36]. Studies have shown that the membrane phospholipids are more labile to oxidation than emulsified triacylglycerols. However, when these polar lipids are in an oil phase, they are more stable to oxidation than the triacylglycerols or free fatty acids [37,38].

2.2.5. Positional Distribution of Fatty Acids in Seed TAGs

The positional distribution of fatty acids in Sambucus seed oils TAGs is shown in Table 3. The fatty acid composition of sn-1, 3 and sn-2 positions exhibited the similar patterns to the the total fatty acid composition of TAGs or TLs (Table 1). At all positions, the palmitic, oleic, linoleic and α-linolenic acids were the major fatty acids and together comprised more than 95% of total fatty acids (Table 3).The sn-2 position was esterified predominantly with linoleic acid: 43.56% in S. nigra seed oil vs. 50.41% in S. ebulus seed oil. Stearic, palmitic and α-linolenic acids were distributed primarily in the sn-1, -3 positions, while oleic acid was found in greater amount at the sn-2 position (Table 3). These are partially in accordance with the findings of Gunstone et al. [39]. Using an enzymatic method, these authors investigated the distribution of unsaturated fatty acids in vegetable TAGs and observed that linoleic acid was preferentially located in the secondary position, whereas oleic and α-linolenic acids were equally distributed in sn-1,-2, and -3 positions.

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Table 3. Positional distribution (% of total fatty acids) of fatty acids in TAGs of Sambucus seed oil samples analyzed.

Sambucus nigra L. Sambucus ebulus L. sn-position sn-position Fatty acids (%) sn-1,3 sn-2 sn-1,3 sn-2 (12:0) 0.03 0.01 0.04 0.01 (14:0) 0.11 0.05 0.11 0.06 (15:0) 0.03 - 0.03 0.02 (16:0) 8.57 0.97 7.78 1.20 (16:1,n-9) 0.06 0.05 0.09 0.10 (16:1,n-7) 0.06 0.05 0.09 0.10 (17:0) 0.04 - 0.06 - (18:0) 1.80 0.60 3.06 0.94 (18:1, n-9) 8.71 16.79 15.33 29.31 (18:1, n-7) 0.99 0.24 1.50 0.54 (18:2, n-6) 32.59 43.56 42.88 50.41 (18:3, n-3) 46.77 37.62 28.76 17.21 (20:0) 0.06 - 0.06 - (20:1, n-9) 0.08 - 0.17 - (20:2, n-6) 0.07 - 0.03 - (20:3, n-3) 0.04 - tr. - (22:1, n-9) - 0.06 - 0.10 Results are given as the average of triplicate determinations.C12:0, lauric; C14:0, myristic; C15:0, pentadecanoic; C16:0, palmitic; C16:1n-9, cis-7 hexadecenoic; C16:1n-7, palmitoleic; C17:0, margaric; C18:0, stearic; C18:1n-9, oleic; C18:1n-7, vaccenic; C18:2n-6, linoleic; C18:3n-3, α-linolenic; C20:0, arachidic; C20:1n-9, 11-eicosenoic; C20:2n-6, 11,14-eicosadienoic; C20:3n-3, eicosatrienoic; C22:1n-9, erucic.

The distribution patterns in sn-1,3 positions (Figure 2) were characterized by a higher percentage (p < 0.01) of PUFAs in S. nigra (79.46%) than in S. ebulus (71.67%). The difference was greatest in the proportions of MUFAs (9.89% vs. 17.19%, p < 0.001), but in the opposite direction. The ratio of n-6 to n-3 PUFAs was higher (p < 0.001) in S. ebulus (1.49) than in S. nigra (0.70). No significant difference (p > 0.05) was observed in the proportions of SFAs from the primary positions (10.64% in S. nigra and 11.14% in S. ebulus). In the case of sn-2 position of Sambucus seed oils TAGs (Figure 3), the levels of SFAs (2.23% vs. 1.63%, p < 0.001) and MUFAs (30.15% vs. 17.19%, p < 0.001) were significantly higher in S. ebulus than in S. nigra, and vice versa in PUFAs (67.62% vs. 81.18%, p < 0.001). It is generally known that unsaturated fatty acids are preferentially located in the sn-2 position and saturated fatty acids are distributed in sn-1 and sn-3 positions in TAGs of most vegetable oils [40,41]. The ratio of n-6 to n-3 PUFAs in the second position of S. ebulus (2.93) was significantly higher (p < 0.05) than that in S. nigra (1.16). These n-6/n-3 ratio values are close to the values recommended by Simopoulos [42] (n-6/n-3 = 1-5/1) as beneficial for good health.

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Figure 2. Positional distribution (%, means ± standard deviation, n = 3 × 3) of fatty acid classes in the TAGs sn-1, 3 positions of Sambucus seed oils.

100 S. nigra a** S. ebulus 80 b

40 -1,3 positions) -1,3 % of fatty acids sn

( a*** 20 a a b b a*** 0 3 As - FAs /n S MUFAs PUF n-6 Fatty acid classes Different letters (a,b) in the same group mean significant differences, ** p < 0.01, *** p < 0.001(unpaired t-test). SFAs—saturated fatty acids, MUFAs—monounsaturated fatty acids, PUFAs—polyunsaturated fatty acids, n-6/n-3—n-6 PUFAs to n-3 PUFAs ratios.

Figure 3. The percentage composition (means ± standard deviation, n = 3 × 3) of fatty acid classes in the TAGs sn-2 position of Sambucus seed, linseed and rapeseed oils.

100 S. nigra a*** S. ebulus 80 Linseed# b Rapeseed# 60 a** b a*** 40 -2 positions) a*** sn % of fatty acids ( 20 b b

a*** b a*** b 0 3 -6 -3 - n n /n SFAs -6 MUFAs PUFAs n Fatty acid classes # Reference [40]. Different letters (a,b) in the same group mean significant differences, ** p < 0.01, *** p < 0.001 (unpaired t-test). SFAs—saturated fatty acids, MUFAs—monounsaturated fatty acids, PUFAs—polyunsaturated fatty acids, n-6—(n-6 PUFAs), n-3—(n-3 PUFAs).

The n-3 PUFAs contents in sn-2 position of S. nigra (37.62%) and S. ebulus (17.21%) TAGs, due to their high content of α-linolenic acid, are much greater than that of most other plant oils, such as corn oil (0.7%), olive oil (0.8%) or soybean oil (7.1%), but comparable to that of rapeseed (20.3%) and linseed (59.8%) oils (Figure 3) [40]. For this reason, the Sambucus seed oils analyzed are optimal as food ingredients or food supplements to increase the intake of n-3 PUFAs. The α-linolenic acid is a precursor for the synthesis of longer chain n-3 PUFAs, such as eicosapentaenoic acid (20:5, n-3) and docosapentaenoic acid (22:5, n-3), which can promote visual, neural and vascular health [43]. Many

Molecules 2013, 18 11777 studies have reported that the fatty acids located in sn-1, 3 and sn-2 positions of TAG have different metabolic fates in the human body [14,44–46]. It was observed that fatty acids in the sn-2 position of TAG are directly absorbed by the intestine, whereas those of the primary positions are released before absorption.

3. Experimental

3.1. Samples and Chemicals

The ripe berries of S. nigra and S. ebulus were collected from different parts of wild bushes on slopes of the Carpathian Mountains (northwest of Transylvania, Romania). The fruits were collected during September to October of 2012 at the stage of commercial maturity and were identified with the help of experts from the Department of Environmental and Plant Protection, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Romania. Berries were stored in polyethylene bags at −20 °C until analysis. Seeds were isolated manually from frozen berries, water-washed, and dried at 40 °C to a moisture content of about 7% in an air-drifted oven. The lipid standards (used for identification of the lipid class) and chemicals [used for the total fat extraction, fractionation, enzymatic reaction and preparation of fatty acid methyl esters (FAMEs)] were of analytical grade (Sigma–Aldrich, St. Louis, MO, USA). Lipase from porcine pancreas (L-3126; Type II, 100–400 units/mg protein) was also purchased from Sigma-Aldrich. The thin layer chromatography (TLC) plates (silica gel 60 F254, 20 × 20 cm) were purchased from Merck (Darmstadt, Germany). The fatty acid methyl esters (FAMEs) standard (37 component FAME Mix, SUPELCO, catalog No: 47885-U) were obtained from Supelco (Bellefonte, PA, USA).

3.2. Extraction of Lipids

The TLs of the seeds were extracted using a chloroform/methanol mixture [26,47]. The sample (5g seeds) was homogenized in methanol (50 mL) for 1 min with a high-power homogeniser (MICCRA D-9, ART Prozess- und Labortechnik, Müllheim, Germany), then chloroform (100 mL) was added, and homogenization continued for 2 min. The mixture was filtered and the solid residue was resuspended in chloroform: methanol mixture (2:1, v/v, 150 mL) and homogenized again for 3 min. The mixture was filtered, and the residue was washed with chloroform: methanol (2:1, v/v, 150 mL). The filtrates and washings were combined and cleaned with 0.88% aqueous potassium chloride followed by methanol: water (1:1, v/v) solution. The purified lipid (bottom) layer was filtered and dried over anhydrous sodium sulfate and the solvent was removed in a rotary evaporator. The amount of lipids was noted. The recovered oils were transferred to vials with 4 mL chloroform (stock solution), and stored at −18 °C for further analysis.

3.3. Fractionation of TLs

Neutral (TAGs, MAGs, DAGs, FFAs and SEs) and PL fractions were separated by preparative TLC [28]. Aliquots of TL stock solutions (0.2 mL) were applied on the TLC plates, developed with petroleum ether: diethyl ether: acetic acid (80:20:2, v/v/v), sprayed with 2′,7′-dichlorofluoroscein/ methanol (0.1% w/v) and visualized under UV light (254 nm) [48]. The lipid classes/subclasses were

Molecules 2013, 18 11778 identified using commercial standards, which were run in parallel with the samples. The separated PLs, MAGs, DAGs and FFAs bands were scraped off, and extracted with a mixture of chloroform/methanol (2:1, v/v). The bands corresponding to TAGs and SEs were also scraped off and extracted with chloroform. After samples were filtered, the solvent was removed and the dry residue was subjected to transesterification and gas chromatographic (GC) analysis.

3.4. Positional Fatty Acid Composition

TAGs were purified from total lipid extracts by preparative TLC using the conditions described above for resolution of NLs and PLs. Pancreatic lipase was used to generate sn-2-MAGs from TAGs [46,49]. Five milligrams of purified TAGs were placed in a test tube (previously equilibrated to 40 °C) and mixed with Tris-HC1 buffer (1M; pH 7.6, 5 mL), 0.05% (w/v) bile salt solution (1.25 mL), aqueous calcium chloride solution (2.2%, w/v, 0.5 mL), and pancreatic lipase (5 mg). The mixture was vortexed for 1 min, and incubated in a water bath at 40 °C for 3 min. The enzymatic reaction was stopped by adding ethanol (1 mL) followed by HCl (6 M, 1 mL). The hydrolysis products were extracted from the reaction medium with diethyl ether (2 × 2 mL) and separated on TLC plates using a solvent system of petroleum ether: diethyl ether: acetic acid (70:30:1, v/v/v). Bands that co-migrated with MAGs and DAGs standards were scratched, extracted with chloroform: methanol (2:1, v/v) and subjected to GC analysis (after transesterification).

3.5. GC Analysis of FAMEs

Fatty acids of TL, NL, PL, MAG (sn-2) and DAG (sn-1, 3) fractions were derivatized by acid-catalyzed transesterification procedure [28,50]. The FAMEs were determined by gas chromatography-mass spectrometry (GC-MS), using a PerkinElmer Clarus 600 T GC-MS (PerkinElmer, Inc., Shelton, CT, USA) [28]. The initial oven temperature was 140 °C, programmed by 7 °C/min until 220 °C and kept 23 min at this temperature. The flow rate of the carrier gas (helium) was 0.8 mL/min and the split value was a ratio of 1:24. A sample of 0.5 μL was injected on a 60 m × 0.25 mm i.d., 0.25 μm film thickness SUPELCOWAX 10 (Supelco Inc.) capillary column. The injector temperature was set at 210 °C. The positive ion electron impact (EI) mass spectra was recorded at an ionization energy of 70 eV and a trap current of 100 μA with a source temperature of 150 °C. The mass scans were performed within the range of m/z: 22–395 at a rate of 0.14 scan/s with an intermediate time of 0.02 s between the scans. Identification of FAMEs was achieved by comparing their retention times with those of known standards (37component FAME Mix, SUPELCO # 47885-U) and the resulting mass spectra to those in our database (NIST MS Search 2.0). The amount of fatty acids was expressed as percent of total fatty acids.

3.6. Statistics

Three different samples of Sambucus seeds for each species were assayed. The analytical results reported for the fatty acid compositions, are the average of triplicate measurements of three independent oils (n = 3 × 3). Statistical differences among samples were estimated using Student’s t-test and ANOVA (one-way analysis of variance; Tukey’s Multiple Comparison Test; GraphPad

Molecules 2013, 18 11779

Prism Version 4.0, Graph Pad Software Inc., San Diego, CA, USA). A probability value of p < 0.05 was considered to be statistical significant.

4. Conclusions

In the present study the seeds of two wild grown Sambucus (nigra and ebulus) species from Romania (Transylvania) were analyzed with respect to oil yields and fatty acid contents of total lipids and corresponding fractions (neutral and polar). The positional distribution of fatty acids in seed TAGs were also determined to obtain detailed data to assess the chemical and nutritional properties of Sambucus seed oils and theirs potential for human consumption, as alternatives to the conventional vegetable oils. This work demonstrated that Sambucus seeds could be considered rich sources of oil (more than 22 g oil/100 g seeds). The oil TAGs were similar in fatty acid composition to the TLs, containing substantial amounts of α-linolenic, linoleic and oleic acids. The PL fractions and all the minor NL subclasses (MAGs, DAGs, FFAs and SEs) were also highly unsaturated. A clear characteristic of the SEs and FFAs were the significantly high levels of SFAs (over 18%), with considerable amounts of palmitic and stearic acids. Sambucus seed oils, with their high levels of α-linolenic acid together with a near 1:1 ratio of n-6 to n-3 PUFAs represent very balanced sources of essential PUFAs for human health. This conclusion is also supported by the results of the positional analysis of fatty acids in TAGs.

Acknowledgments

This work was supported by the Research Grant of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Romania (nr. 1346/ 08.02.2013), and a grant of the Romanian Ministry of Education, CNCS – UEFISCDI, project number PNII-RU-PD-2012-3-0245 (contract no. 92/25.06.2013).

Conflicts of Interest

The authors declare no conflict of interest.

References

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Sample Availability: Samples of Sambucus ebulus and Sambucus nigra seed oils, extracted by a chloroform/methanol mixture, are available from the authors.

© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

Article

pubs.acs.org/JAFC

Total Phenolic Contents, Antioxidant Activities, and Lipid Fractions from Berry Pomaces Obtained by Solid-State Fermentation of Two Sambucus Species with Aspergillus niger Francisc Vasile Dulf,†,‡ Dan Cristian Vodnar,*,§ Eva-Henrietta Dulf,∥ and Monica Ioana Tosa̧⊥

† Faculty of Agriculture, Department of Environmental and Plant Protection, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Manăs̆tuŗ 3-5, 400372 Cluj-Napoca, Romania ‡ SC Proplanta SRL, CCD-BIODIATECH, Trifoiului 12 G, 400372 Cluj-Napoca, Romania § Faculty of Food Science and Technology, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Manăs̆tuŗ 3-5, 400372 Cluj-Napoca, Romania ∥ Faculty of Automation and Computer Science, Department of Automation, Technical University of Cluj-Napoca, G. Baritiu 26-28, 400027 Cluj-Napoca, Romania ⊥ Faculty of Chemistry and Chemical Engineering, University Babes-Bolyai,̧ Biocatalysis Research Group, Arany Janoś 11, 400028 Cluj-Napoca, Romania

ABSTRACT: The aim of this study was to investigate the effect of solid-state fermentation (SSF) by Aspergillus niger on phenolic contents and antioxidant activity in Sambucus nigra L. and Sambucus ebulus L. berry pomaces. The effect of fermentation time on the total fats and major lipid classes (neutral and polar) was also investigated. During the SSF, the extractable phenolics increased with 18.82% for S. ebulus L. and 11.11% for S. nigra L. The levels of antioxidant activity of methanolic extracts were also significantly enhanced. The HPLC-MS analysis indicated that the cyanidin 3-sambubioside-5-glucoside is the major phenolic compound in both fermented Sambucus fruit residues. In the early stages of fungal growth, the extracted oils (with TAGs as major lipid fraction) increased with 12% for S. nigra L. and 10.50% for S. ebulus L. The GC-MS analysis showed that the SSF resulted in a slight increase of the linoleic and oleic acids level. KEYWORDS: solid- state fermentation, Aspergillus niger, Sambucus pomaces, polyphenols, antioxidant activity, lipids

■ INTRODUCTION consumed as fresh fruits, being processed mainly into juice, There is an increasing global trend toward the efficient, thereby generating large amounts of wastes. The available fi inexpensive, and environmentally rational utilization of scienti c reports on the Sambucus species focus mainly on their 3,8,9,12−15 agricultural and food byproducts. The direct disposal of these fruit’s composition and properties, and only a few wastes, especially those of berry pomaces, into soil or landfills studies relate to the berry pomaces, although they exhibit high can cause serious environmental and ecological problems anthocyanin (75−98% of total berry anthocyanins)4 and fat because of their low pH.1 Pomaces are the primary byproducts (22.40−24.90 g/100 g of seeds)16 contents. Even though the of the berry juice processing industry and consist mainly of the potential of Sambucus berry pomaces as sources of polyphenols processed skin, flesh, and seeds of the small fruits.2 The freshly and oil with high content of polyunsaturated fatty acids seems pressed berry pomaces are rich in fiber, containing small clear, there is less information on the potential of strategies for amounts of protein, minerals, and sugars. Due to their high the liberation and extraction of these biomolecules from the moisture content, these berry wastes are susceptible to rapid vegetable matrices. The economic value of the agro-industrial microbial growth. Among berries, fruits of the Sambucus species wastes could be easily increased by fungal pretreatment in the (herbs of the Adoxaceae family in the order of the Dipsacales) solid-state fermentation (SSF) system, prior to recovery of have the highest total phenolic and anthocyanin contents and, 3,4 valuable hydro- and lipophilic components. SSF consists of the therefore, the highest antioxidant potential. Moreover, the in microbial growth and product formation on solid matrix in the vitro studies indicate that the Sambucus berry extracts possess absence (or near absence) of free water. The substrate must important anticarcinogenic, immune-stimulating, antibacterial, ffi fl 5−9 contain su cient moisture to allow microbial growth and antiallergic, antiviral, and anti-in ammatory properties. The 17 elderberry (Sambucus nigra L.) was the 18th best selling herbal metabolism. The selection of a suitable microorganism is one dietary supplement in the Food, Drug, and Mass Market of the most important criteria in SSF. The fungus Aspergillus channel in the United States for 2011.10 In a 2010 report of the niger is a useful common species of the genus Aspergillus, which European Herb Growers Association (Europam), the Sambucus nigra L. flowers and berries were the most collected medicinal Received: January 29, 2015 plants in Romania for export trade, as well as for tea and phyto- Revised: March 18, 2015 pharmaceutical production.11 The small, fully ripened dark Accepted: March 18, 2015 blue/purple berries of the Sambucus species are rarely

© XXXX American Chemical Society A DOI: 10.1021/acs.jafc.5b00520 J. Agric. Food Chem. XXXX, XXX, XXX−XXX Journal of Agricultural and Food Chemistry Article is able to produce by SSF of different agro-industrial fermentation due to its potential for higher enzyme production. byproducts more than 19 types of enzymes (cellulase, A. niger was propagated according to the procedure developed pectinase, protease, etc.) and several added-value bioactive by Oberoi et al.23 The fungus was maintained on potato products (citric acid, alcohols, etc.).18 Most of the phenolic dextrose agar (PDA) plates at 4 °C. The culture of A. niger was compounds occur primarily in conjugated form with one or grown on PDA Petri dish and incubated at 30 °C. The fungal more sugar residues linked to hydroxyl groups. Association with spores were collected from the sporulation medium plates, organic acids, amines, and lipids is also common.19 These inoculated into sterile distilled water, and stored in the freezer. bounded forms of phenolics reduce their ability to function as Solid-State Fermentation. Fifty grams of solid substrates good antioxidants because the resonance stabilization of free (pH 5) in 500 mL Erlenmeyer flasks were thoroughly mixed radicals is highly dependent on the availability of free hydroxyl and autoclaved at 121 °C for 30 min. The substrates were groups on the phenolic rings.1 The free phenolics can be inoculated with spore suspension (2 × 107 spores/g of solid). released from the conjugated form via enzymatic hydrolysis After mixing, the flasks with inoculated substrates were with carbohydrate metabolizing enzymes (such as β-glucosi- incubated at 30 °C for 6 days. To carry out the chemical dase) produced by fungi during SSF of lignocellulosic wastes, characterization, samples were withdrawn at the beginning and improving the health functionality of these phytochemicals in every 24 h and stored at −20 °C. The experiment was this way.19 Enhancement of phenolic content and antioxidant performed in triplicate. Because the lipid accumulation process potential using SSF was reported in cranberry1 and apple in solid−state fermented medium is favored due to the low pomaces,19 pomegranate,20 and pineapple21 wastes and differ- content of nitrogen or phosphorus sources and an excess of ent cereals.22 To our knowledge, there is no data available in carbon source,23 in this study, the solid substrates were not the literature about the use of Sambucus berry pomaces as supplemented with any extra nutrient solution. support in SSF processes for the production of value-added Extraction of Phenolic Compounds. Extraction was compounds. The hydrolytic enzyme complex excreted by performed according to the method described by Veberic et Aspergillus niger in the SSF system, comprising mainly cellulase, al.7 with some minor modifications. The samples of berry pectinase, and protease, can also increase the lipid yields, pomaces (1 g) were extracted with 20 mL of extraction mixture hydrolyzing the polysaccharides from the seed cell walls and (hydrochloric acid/methanol/water in the ratio of 1:80:19) in releasing the extra oil from its bounded forms, such as an ultrasonic bath for 30 min at 40 °C. The mixtures were lipoproteins and lipopolysaccharides, which are otherwise centrifuged at 4000g for 10 min, and the supernatants were 23 nonextractable. Therefore, the objective of the present work filtered under vacuum through the glass frit. The filtrates were was to evaluate the influence of SSF by A. niger on phenolic evaporated to dryness under vacuum. The resulting dried contents and antioxidant activity in Sambucus nigra L. and extracts were dissolved in methanol and then used for phenolic Sambucus ebulus L. berry pomaces. Furthermore, the effect of content and antioxidant activity measurements. fermentation time on the total fats and major lipid classes Determination of Total Phenolic Content. The content (neutral and polar) in bioprocessed substrates was also of total polyphenols (TP) was determined by the Folin− investigated. This solid-state bioprocessing approach can Ciocalteu method24 using a Synergy HT Multi-Detection enhance the functional value of this berry byproduct. Microplate Reader with 96-well plates (BioTek Instruments, Inc., Winooski, VT, U.S.A.). A combination solution consisting ■ MATERIALS AND METHODS of 25 μL sample extract, 125 μL of Folin−Ciocalteu reagent μ Chemicals. Folin−Ciocalteu’s phenol reagent, gallic acid, (0.2 N), and 100 L of sodium carbonate (Na2CO3) solution sodium carbonate, acetic acid, acetonitrile, methanol, chloro- (7.5% w/v) was homogenized and incubated at room genic acid, rutin, cyanidin chloride, DPPH (1,1-diphenyl-2 temperature (25 °C) in the dark for 2 h. The absorbance was picrylhydrazyl), the lipid standards, and other chemicals (used measured at 760 nm against a methanol blank. Gallic acid for the total fat extraction, fractionation, and preparation of (0.01−1 mg/mL) was used as a standard, and the content of fatty acid methyl esters (FAMEs)) were purchased from Sigma- TP in the extract was expressed as gallic acid equivalents (GAE) Aldrich (Steinheim, Germany). The thin layer chromatography in mg/g dry weight (DW) of pomace. (TLC) plates (silica gel 60 F254, 20 × 20 cm) were purchased Phenolic Compound Analysis by High-Performance from Merck (Darmstadt, Germany). The FAMEs standard (37 Liquid Chromatography-Diode Array Detection-Electro- component FAME Mix, SUPELCO) was obtained from spray Ionization Mass Spectrometry (HPLC-DAD-ESI Supelco (Bellefonte, PA, U.S.A.). MS). An Agilent 1200 HPLC equipped with DAD detector, Raw Material. The freshly pressed berry pomaces were coupled with MS detector single quadrupole Agilent 6110 was obtained in our laboratory from the fully ripened berries of wild used to confirm and quantify each individual phenolic Sambucus nigra L. (black or European elderberry) and compound. Separations were carried out using an Eclipse Sambucus ebulus L. (dwarf elder, elderberry, or danewort) column, XDB C18 (4.6 × 150 mm, particle size 5 μm) (Agilent collected in September−October of 2013 in the northwest of Technologies, U.S.A.) at 25 °C. All samples were analyzed in Transylvania (Romania). triplicate on the basis of the method described by Pop et al.25 Culture Medium and Fermentation Conditions. The using as mobile phase 0.1% acetic acid/acetonitrile (99:1) in berry press residues of two Sambucus species were used as distilled water (v/v) (A) and 0.1% acetic acid in acetonitrile (v/ natural substrates for the SSF. The berry pomaces were v) (B) and the following gradient: 5% B (0−2 min), 5−40% B homogenized with a high-power homogenizer (MICCRA D-9, (2−18 min), 40−90% B (18−20 min), 90% B (20−24 min), Germany) and stored at −20 °C prior to use. The substrates 90−5% B (24−25 min), 5% B (25−30 min). The flow rate was were autoclaved for 30 min at 121 °C. The moisture content in 0.5 mL/min in all separations. For MS fragmentation, the the berry pomaces was 60% w/w. The fungal strain of ESI(+) module was applied, with a capillary voltage of 3000 V, Aspergillus niger (ATCC-6275; LGC Standards GmbH, Wesel at 350 °C and nitrogen flow of 8 L/min. Scan range was 100− Germany) was selected as a suitable fungus for solid-state 1000 m/z. The eluent was monitored by DAD, and the

B DOI: 10.1021/acs.jafc.5b00520 J. Agric. Food Chem. XXXX, XXX, XXX−XXX Journal of Agricultural and Food Chemistry Article

Figure 1. Total phenolic content (A), free radical scavenging activity of phenolics (B) and relationship between radical scavenging capacity (DPPH assay) and total phenolic content (C and D) of extracts from solid state fermented S. ebulus L. and S. nigra L. pomaces. Values are mean ± SD of three samples, analyzed individually in triplicate (n =3× 3). absorbance spectra (200−600 nm) were collected continuously Lipid Extraction. Total lipids (TLs) of berry pomaces were during the course of each run. The flavonols were detected at extracted from 5 g of samples, using a methanol/chloroform 340 nm and the anthocyanins at 520 nm.25 Data analysis was extraction method.27 carried out by Agilent ChemStation Software (Rev B.04.02 SP1, Isolation of Neutral (TAGs, MAGs, 1, 2- and 1, 3-DAGs, Palo Alto, California, U.S.A.). The identification of the FFAs, and SEs) and Polar lipid (PL) Fractions from TLs. phenolics was made according to UV−visible spectra, retention Using a previously described method,16,28,29 the TL extracts time, cochromatography with authentic standards (when were fractionated by TLC into seven fractions. The separated available), and mass spectra. The identified anthocyanins bands (PLs, MAGs, 1, 2- and 1, 3-DAGs and FFAs) were were quantified using cyanidin chloride as external standard scraped off, and extracted in a solvent mixture of chloroform/ and calculated as equivalents of cyanidin (mg cyanidin/100g methanol (2:1, v/v). The TAG and SE bands were also scraped ff fi dry weight (DW) of the substrate) (r2 = 0.9935). Using the o and extracted with chloroform. After ltration and solvent same quantification procedure, the chlorogenic acid was evaporation, the dry residues were subjected to transester- fi expressed in mg chlorogenic acid/100g DW of the substrate i cation and gas chromatographic (GC) analysis. μ (r2 = 0.9937) and quercetins were calculated as equivalents of Trinonadecanoylglycerol (70 g for TAGs) and nonadecanoic − μ μ rutin (mg rutin/100g DW of the substrate) (r2 = 0.9981).25 acid (10 50 g for PLs, MAGs, DAGs, SEs and 200 g for Determination of the Antioxidant Activity with the FFAs) were added as internal standards before the trans- fi DPPH Free Radical Scavenging Method. Amethod esteri cation process. described by Marghitas̆ ̧et al.26 with adaptation on the Analysis of Fatty Acids and Calculation of Lipid Fractions Content in TLs. The isolated lipid fractions were microplate reader was used to detect the 2,2-diphenyl-1- fi 16 picrylhydrazyl (DPPH) radical scavenging activity of the transesteri ed into FAMEs using the acid-catalyzed method. examined extracts. Methanol extract (40 μL) was mixed with The FAMEs were analyzed with a gas chromatograph (GC) 200 μL of DPPH solution (0.02 mg/mL) and left to stand 15 coupled to a mass spectrometer (MS): PerkinElmer Clarus 600 T GC-MS (PerkinElmer, Inc., Shelton, CT, U.S.A.)27 on a min at room temperature and then the absorbance was Supelcowax 10 (60 m × 0.25 mm i.d., 0.25 μm film thickness; measured at 517 nm. The percentage inhibition was calculated Supelco Inc., Bellefonte, PA, U.S.A.) capillary column. The using the equation: temperature program was set as follows: initial temperature, 140 °C, then to 220 °Cat7°C/min, and held at 220 °C for 23 Inhibitio n (%) min. The injector was set at 210 °C, with a 1:24 split ratio, and =−[1 (test sample absorbance/blank sample absorbance)] × 100 0.5 μL was injected. The column flow rate (He) was 0.8 mL/

C DOI: 10.1021/acs.jafc.5b00520 J. Agric. Food Chem. XXXX, XXX, XXX−XXX Journal of Agricultural and Food Chemistry Article

Table 1. Mean Polyphenolic Contents (mg/100g DW of the Substrate) of S. nigra L. and S. ebulus L. Pomaces During Solid- State Fermentation

anthocyanins cinnamic acid quercetins identified cyanidin 3- quercetin 3- quercetin 3- compound ([M + sambubioside-5- cyanidin 3- cyanidin 3,5- cyanidin 3- 5-caffeoylquinic acid rutinoside glucoside H] + ion, glucoside sambubioside diglucoside glucoside (chlorogenic acid) (rutin) (isoquercitrin) fragments) (743, 581, 449, 287) (581, 287) (611, 449, 287) (449, 287) (355, 181) (611, 303) (465, 303) peak 1 2 3 4 5 6 7 Sambucus nigra L. day of fermentation 0 44.94b ± 2.50 4.46b ± 0.15 18.70c ± 1.10 7.90a ± 0.35 4.16b ± 0.20 40.25c ± 2.10 25.45a ± 1.30 1 34.99e ± 1.65 2.76e ± 0.10 11.04d ± 0.48 3.48d ± 0.18 0.82f ± 0.06 30.92e ± 1.28 14.13d ± 0.65 2 38.58c ± 2.10 3.34d ± 0.16 21.44b ± 1.10 5.09c ± 0.30 3.17d ± 0.18 42.77b ± 1.85 18.83b ± 0.80 3 46.88a ± 2.20 4.62a ± 0.18 23.04a ± 1.18 5.93b ± 0.26 4.49a ± 0.22 45.50a ± 1.90 19.03b ± 0.85 4 46.45a ± 2.25 4.40b ± 0.20 18.80c ± 1.10 2.41e ± 0.15 3.52c ± 0.20 34.00d ± 1.65 16.18c ± 0.80 6 37.02d ± 1.72 4.16c ± 0.20 11.68d ± 0.60 1.80f ± 0.10 2.98e ± 0.21 31.07e ± 1.55 13.10e ± 0.65 Sambucus ebulus L. day of fermentation 0 28.90ab ± 1.40 3.56a ± 0.15 13.71a ± 0.72 2.59a ± 0.12 24.32ab ± 1.20 12.80a ± 0.65 9.85b ± 0.45 1 23.78e ± 1.20 2.43d ± 0.13 13.38ab ± 0.70 1.31e ± 0.07 18.16d ± 0.95 11.20b ± 0.60 7.85d ± 0.28 2 25.26d ± 1.18 2.84c ± 0.11 13.43ab ± 0.65 1.75c ± 0.09 23.85b ± 1.10 12.39a ± 0.70 9.92b ± 0.40 3 29.61a ± 1.62 2.94b ± 0.14 13.75 a ± 0.75 2.06b ± 0.10 25.04a ± 1.15 13.01a ± 0.65 10.69a ± 0.45 4 28.64b ± 1.45 2.46d ± 0.11 13.50ab ± 0.60 1.43d ± 0.07 21.20c ± 1.10 6.27cd ± 0.35 8.54c ± 0.40 6 27.52c ± 1.40 2.19e ± 0.11 13.19b ± 0.65 1.28e ± 0.08 20.50c ± 1.15 5.82d ± 0.30 7.34e ± 0.35 The values represent the means of three samples, analyzed individually in triplicate (n =3× 3). Totals with different superscript letters (a‑f) (within a column for the different Sambucus pomaces) were significantly different (repeated measures ANOVA “Tukey’s Multiple Comparison Test”, p < 0.05). min. The EI (positive ion electron impact) mass spectra were using the Rhizopus oligosporus. The rapid fall of the observed recorded at 70 eV and a trap current of 100 μA with a source measurable phenolics during the early stages of fermentation temperature of 150 °C. The mass scans were performed within suggests that the most phenolics were present in bound form the range of m/z:22−395 (0.14 scans/s with an intermediate and only a small amount was in free soluble- form. However, it time of 0.02 s between the scans). The FAMEs were identified should be noted that under the given fermentation conditions by comparing the retention times with those of known the phenolics increased only with 18.82% for S. ebulus L. and standards (37 components FAME Mix, Supelco no. 47885-U) 11.11% for S. nigra L. from initial values of 2.71 mg/g DW and and the resulting mass spectra to those in our database (NIST 4.05 mg/g DW for unfermented pomaces to 3.22 mg/g DW MS Search 2.0). Fatty acids were quantified by comparison to and 4.50 mg/g DW for fermented pomaces. Vattem et al.1 and peak areas of the methyl nonadecanoate without application of Ajila et al.19 reported that polyphenol content increases when any correction factor. The fatty acid compositions of lipid the β-glucosidase activity increases, showing that this fractions were expressed as molar percentages of the fatty acids. carbohydrate-cleaving enzyme plays an important role in the The neutral (TAGs, MAGs, 1,2- and 1,3-DAGs, FFAs, and SEs) bioconversion or liberation of free phenolics from different and polar lipid contents in TLs were calculated using an average pomaces during solid-state growth of Phanerochaete chrysospo- molecular weight of fatty acids of the fraction in question and rium, Lentinus edodes , and Rhizopus oligosporus. According to 1,21 were expressed as weight percentages (wt %) in oils. previous studies, the low nitrogen content of a media could Statistical Analysis. All values were expressed as the mean act as a barrier for free phenolic compounds production. It can ± standard deviation (SD) of three independent experiments be concluded that to improve the TP production, the SSF with samples in triplicate. Correlations between the antioxidant should be carried out with the solid substrate supplemented activity and total phenolic content were examined using with organic or inorganic nitrogen sources. Further inves- Pearson’s correlation. Statistical differences among samples tigation is needed. The measurable free phenolics for both were estimated using Student’s t test and ANOVA (repeated berry wastes showed a slight decrease after 3 days of measures ANOVA; Tukey’s Multiple Comparison Test; fermentation, probably due to the depletion of the nutrients GraphPad Prism Version 5.0, Graph Pad Software Inc., San and to the stress induced on the fungus. As response, it enabled Diego, CA). A probability value of p < 0.05 was considered to the lignifying and tannin forming peroxidase enzymes in the fi be statistically significant. fungus, causing the polymerization and ligni cation of the free phenolics and as an consequence, the intensity of the reaction 1 RESULTS AND DISCUSSION of the molecules with Folin-Ciocalteu reagent decreased. The ■ ranges for obtained phenolic contents are in accordance with Total Phenolics. During the first 3 days of growth, both literature data,4 indicating that significant amount of these Sambucus species showed a similar trend, the total phenolics biomolecules could be extracted from Sambucus berry pomaces, content decreased in the first 24 h and increased until the compared to whole berry or juice. Ozgen et al.30 reported in maximum yield (Day 3) (Figure 1A). Similar results were their study on 14 purple- black American elderberry accessions previously obtained by Correia et al.21 in the production of (Sambucus canadensis L.) TP content values between 3.30 and phenolics by the solid-state bioconversion of pineapple waste 3.90 mg/g fresh weight, whereas Bermudez-Sotó and Tomas-́

D DOI: 10.1021/acs.jafc.5b00520 J. Agric. Food Chem. XXXX, XXX, XXX−XXX Journal of Agricultural and Food Chemistry Article

Figure 2. HPLC chromatograms (detected wavelength at 280 nm) of A. niger fermented (A) S. nigra L. (fourth day of SSF) and (B) S. ebulus L. (second day of SSF) berry pomaces: (1) cyanidin 3-sambubioside-5- Figure 4. Lipid components in oils from the S. nigra L. (A) and S. glucoside; (2) cyanidin 3-sambubioside; (3) cyanidin 3,5-diglucoside; ebulus L. (B) berry press residues during solid-state fermentation. (4) cyanidin 3-glucoside; (5) 5-caﬀeoylquinic acid; (6) quercetin 3- Values are mean ± SD of three samples, analyzed individually in rutinoside; (7) quercetin 3-glucoside. triplicate (n =3× 3); * p < 0.05, ** p < 0.01, *** p < 0.001 (repeated measures ANOVA “Tukey’s Multiple Comparison Test”). TAGs − triacylglycerols, PL − polar lipids, MAG − monoacylglycerols, DAG − diacylglycerols, FFAs − free fatty acids, SEs − sterol esters.

Barberań3 determined 30.50 mg/mL of elderberry juice concentrate. The TP amounts for aqueous and methanol extracts of Sambucus ebulus L. fruits reported by Ebrahimzadeh et al.14 were 41.59 and 27.37 mg GAE/g of extract powder, respectively. In their study on anthocyanin and other polyphenolic profiles of different genotypes of S. canadensis and S. nigra L., Lee and Finn31 reported TP contents between 277 and 582 mg GAE/100 g berries, depending on the growing seasons and cultivators. It should be mentioned that the phenolic compounds from the Sambucus berries coexist with different toxic and allergenic lectins. However, Jimenez et al.9 showed that the negative effects of these lectins can be eliminated by a short-time heat treatment of berry-derived Figure 3. Time course of lipid production by strain Aspergillus niger products, without any significant reduction in the content of from the S. ebulus L. and S. nigra L. berry press residues during solid- ± ** the antioxidant compounds. state fermentation. Results are given as mean SD (n = 3); p < HPLC-DAD-MS Analysis of Phenolic Compounds. The 0.01, *** p < 0.001 (repeated measures ANOVA “Tukey’s Multiple Comparison Test”). contents of major phenolics in the berry press residues of two species of Sambucus were determined during fermentation (Table 1). The characteristic HPLC chromatograms are shown

E DOI: 10.1021/acs.jafc.5b00520 J. Agric. Food Chem. XXXX, XXX, XXX−XXX Journal of Agricultural and Food Chemistry Article

Figure 5. GC-MS chromatogram of FAMEs in the TLs of the S. ebulus L. berry pomaces after SSF with A. niger (the fourth day) analyzed with a Supelcowax 10 capillary column. Peak identification: (1) lauric (12:0); (2) myristic (14:0); (3) palmitic (16:0); (4) cis-7 hexadecenoic (16:1n-9); (5) palmitoleic (16:1n-7); (6) margaric (17:0); (7) stearic (18:0); (8) oleic (18:1n-9); (9) vaccenic (18:1n-7); (10) linoleic (18:2n-6); (11) nonadecanoic (19:0, internal standard); (12) α-linolenic (18:3n-3); (13) arachidic (20:0); (14) 11-eicosenoic (20:1n-9); (15) 11,14-eicosadienoic (20:2n-6); (16) eicosatrienoic (20:3n-3); (17) behenic (22:0); (18) erucic (22:1n-9); (19) tricosanoic (23:0); (20) lignoceric (24:0) acids. in Figure 2. Both species contained seven dominant level of cyanidin 3-sambubioside-5-glucoside increased signifi- polyphenolics; four anthocyanins (cyanidin 3-sambubioside-5- cantly (p < 0.05) with 2.46% by day 3 from initial value before glucoside, cyanidin 3-sambubioside, cyanidin 3,5-diglucoside gradually decreasing for the remaining period of fermentation. and cyanidin 3-glucoside), cinnamic acid (chlorogenic acid) The magnitude of this major anthocyanin change was higher and two quercetins (rutin and isoquercitrin). The fruits of for the S. nigra L. pomace, increased with 4.32% in the third Sambucus contain an appreciable amount of cyanidin as a before sharply decreasing in the sixth day (Table 1). The diglucoside (sambubioside), which is not common in most increase of the individual phenolic amounts during the first days berries.12 The proportion of the individual phenolics differed of fermentation may be explained by the enzymatic release of between species (Table 1). Cyanidin 3-sambubioside-5-gluco- bound phenolic compounds from the pomaces by means of side was the major phenolic compound in both residues of the lignin-degrading peroxidases and carbohydrate-cleaving en- Sambucus species. The next predominant polyphenolics in S. zymes expressed by the fungus during the initial stages of nigra L. were rutin, isoquercitrin and cyanidin 3, 5-diglucoside, growth, to allow penetration of the tissues and the use of whereas in S. ebulus L. the second major phenolic compound available nutrients from residues.19 The decline of individual was chlorogenic acid (Table 1 and Figure 2). The air-dried phenolic concentrations during the later course of growth could elderberries examined by Bridle and Garcia-Viguera32 also had be due to polymerization of the released phenolic compounds. cyanidin 3-sambubioside-5-glucoside as the major anthocyanin. Qin et al.,33 in their study on polyphenol and purine alkaloid Our findings concerning the identity and number of quercetins contents during solid-state fermentation of pu-erh tea leaves, in S. nigra L. press- residues match the literature data.8 reported that most of the flavone glycosides were degraded Quercetin-based glycosides were the major flavonol glycosides after bioconversion with Aspergillus fungi. in both processed Sambucus residues. The identified flavonols Antioxidant Activity. The ability of phenolic compounds were mostly rutinosides: from 61% (nonfermented) to 71% to inhibit DPPH (2,2′-diphenyl-1-picrylhydrazyl) free radical in (the third day of fermentation) of total flavonol glycosides of S. methanol extracts of two Sambucus berry press residues during nigra L. residues and from 56% (nonfermented) to 58% (the the course of solid-state bioprocessing was measured, and the first day of fermentation) of total flavonol glycosides of S. ebulus results are shown in Figure 1B. In the methanolic extracts of S. L. residues, while the remainders were conjugated to glucose nigra L. berry press residue the radical inhibition capacity (Table 1). In most cases berry juice or air-dried, freeze-dried or increased by 36.50% by day 4 compared with the initial value frozen fruits were analyzed.3,13,31 To our knowledge this is the (%I = 65) before sharply decreasing for the remaining period of first study investigating the variation of the amounts of phenolic the fungal growth. The increase in the radical inhibition activity compounds in wild-grown S. nigra L. and S. ebulus L. berry was higher for the S. ebulus L. pomace, which increased with pomaces in correlation to fermentation days in solid-state 66% in the second day versus initial value (%I = 51.20) before system by A. niger. As shown in Table 1, at the beginning stage gradually decreasing by day 4 (Figure 1B). The literature shows of fermentation (0−first day), the levels of all phenolic that the antioxidant activity of Sambucus berries is mainly compounds decreased significantly (p < 0.05) (except cyanidin connected with the presence of flavonols and anthocya- 3, 5-diglucoside from S. ebulus L. extract). After 24h, the nins.9,13,15 Moreover, Mandrone et al.15 have determined a contents of phenolics increased slightly during the course of strong correlation between the antioxidant activity of S. nigra L. SSF by day 3, after which the concentrations of individual fruits and their cyanidin-3-sambubioside-5-glucoside composi- polyphenols in both Sambucus berry residues decreased tion. Phenolic compounds in fruits and their processing wastes significantly (p < 0.05). In S. ebulus L. pomace extracts the have been ascribed important roles. Nontheless, there is

F DOI: 10.1021/acs.jafc.5b00520 J. Agric. Food Chem. XXXX, XXX, XXX−XXX ora fArclua n odChemistry Food and Agricultural of Journal Table 2. Fatty Acid Compositions (Molar %) of Total Lipids and Individual Lipid Classes Produced by Aspergillus niger in Solid-State Fermentation of S. ebulus L. Berry Press Residuesa

Sambucus ebulus L. fermentation time (day) 0 4 04040404040404 fatty acids TLs TAGs PLs MAGs 1,2-DAGs 1,3-DAGs FFAs SEs 12:0 0.01 tr 0.03 0.01 0.06 0.37 0.77 0.75 0.05 0.02 0.05 0.09 0.29 0.05 0.46 0.28 14:0 0.07 0.03 0.06 0.02 0.34 1.01 1.75 2.18 0.13 0.06 0.37 0.43 0.35 0.16 1.52 0.95 16:0 7.70 6.02 6.79 5.10 25.30 18.99 20.66 18.64 9.71 5.45 8.71 10.66 12.53 13.38 51.55 35.79 16:1n-9 0.05 0.02 0.05 0.02 0.28 0.34 tr 1.39 0.15 0.07 0.13 0.22 0.30 0.20 1.76 1.01 16:1n-7 0.10 0.04 0.09 0.07 0.15 0.43 - - 0.13 0.10 0.17 0.28 0.25 0.29 0.28 0.20 17:0 0.04 0.02 0.05 0.02 0.26 2.28 0.67 0.93 0.15 0.06 - - 0.19 0.17 0.30 0.20 18:0 2.78 1.08 2.63 0.99 9.26 12.57 28.03 13.07 3.90 4.31 4.76 13.59 5.00 4.93 8.89 6.82 18:1n-9 20.22 23.30 15.71 18.10 18.77 15.92 10.09 17.06 21.37 22.73 23.04 22.34 17.40 18.15 13.82 22.39 18:1n-7 1.08 0.47 1.02 0.42 1.83 1.14 1.15 2.47 1.88 0.86 1.70 1.13 0.88 3.11 0.49 0.86 18:2n-6 43.11 45.19 43.99 46.32 33.94 31.40 19.26 25.52 51.46 43.02 40.34 34.67 30.51 31.39 11.19 21.88 18:3n-3 23.78 23.34 28.62 28.67 7.53 10.47 7.48 10.06 9.97 22.87 18.02 13.13 15.38 17.16 3.41 6.03 20:0 0.16 0.07 0.09 0.05 1.09 1.70 1.63 tr 0.17 0.09 0.50 0.84 0.97 0.40 2.00 1.14 20:1n-9 0.18 0.07 0.15 0.06 0.27 - - - 0.30 0.14 0.35 0.34 0.13 0.46 0.23 0.25 20:2n-6 tr tr - - tr ------0.10 - -

G 20:3n-3 tr tr - - tr ------22:0 0.27 0.12 0.47 0.07 0.71 2.76 - - 0.14 0.06 0.56 0.70 5.47 0.87 2.77 1.76 22:1n-9 0.02 0.01 - - 0.23 0.61 8.51 7.92 0.50 0.17 1.31 1.57 0.10 - 1.35 0.46 23:0 0.02 0.01 ------0.07 tr - 24:0 0.42 0.21 0.24 0.08 ------10.26 9.11 - - *** ∑SFA 11.47a 7.56 b 10.36 6.34 37.01 39.68 53.50 35.58 14.24 10.04 14.95 26.32 35.05 29.14 67.48 46.93 ∑ VLCSFAs(≥20C) 0.87a* 0.41b 0.80 0.21 1.80 4.46 1.63 - 0.31 0.15 1.06 1.54 16.70 10.45 4.77 2.90 *** ∑MUFA 21.65b 23.92a 17.02 18.66 21.52 18.45 19.75 28.84 24.33 24.07 26.69 25.88 19.06 22.21 17.92 25.16 *** ∑PUFA 66.89b 68.53a 72.61 75.00 41.47 41.88 26.75 35.58 61.43 65.89 58.36 47.80 45.89 48.65 14.60 27.91 ∑n-3 PUFAs 23.78a* 23.34b 28.62 28.67 7.53 10.47 7.48 10.06 9.97 22.87 18.02 13.13 15.38 17.16 3.41 6.03 *** ∑n-6 PUFAs 43.11b 45.19a 43.99 46.32 33.94 31.40 19.26 25.52 51.46 43.02 40.34 34.67 30.51 31.49 11.19 21.88 ** n-6/n-3 1.81b 1.94a 1.54 1.62 4.50 3.00 2.57 2.54 5.16 1.88 2.24 2.64 1.98 1.84 3.28 3.63 *** PUFAs/SFAs 5.83b 9.07a 7.01 11.82 1.12 1.06 0.50 1.00 4.31 6.56 3.90 1.82 1.31 1.67 0.22 0.59 a The values represent the means of three samples, analyzed individually in triplicate (n =3× 3). TLs − total lipids, TAGs − triacylglycerols, PLs − polar lipids, MAGs − monoacylglycerols, DAGs − .Arc odChem. Food Agric. J. − − − − − − diacylglycerols, FFAs free fatty acids, SEs sterol esters; SFAs saturated fatty acids, MUFAs monounsaturated fatty acids, PUFAs polyunsaturated* fatty** acids, VLCSFAs*** very long chain saturated fatty acids; tr. − trace.Different superscript letters (a,b) in the same row mean significant differences between TLs of the two fermentation days. p < 0.05. p < 0.01. p < 0.001 (paired t-test). C12:0, lauric; C14:0, myristic; C16:0, palmitic; C16:1n-9, cis-7 hexadecenoic; C16:1n-7, palmitoleic; C17:0, margaric; C18:0, stearic; C18:1n-9, oleic; C18:1n-7,vaccenic; C18:2n-6, linoleic; C18:3n-3, α- O:10.1021/acs.jafc.5b00520 DOI: linolenic; C20:0, arachidic; C20:1n-9, 11-eicosenoic; C20:2n-6, 11,14-eicosadienoic; C20:3n-3, eicosatrienoic; C22:0, behenic; C22:1n-9, erucic; C23:0, tricosanoic; C24:0, lignoceric acids. XX X,XXX XXX, XXXX, Article − XXX ora fArclua n odChemistry Food and Agricultural of Journal Table 3. Fatty Acid Compositions (Molar %) of Total Lipids and Individual Lipid Classes Produced by Aspergillus niger in Solid-State Fermentation of S. nigra L. Berry Press Residuesa

Sambucus nigra L. fermentation time (day) 0 4 04040404040404 fatty acids TLs TAGs PLs MAGs 1,2-DAGs 1,3-DAGs FFAs SEs 12:0 0.01 0.01 - 0.01 0.21 0.75 0.60 0.41 0.06 0.05 0.21 0.54 0.30 0.02 0.71 1.57 14:0 0.09 0.08 0.06 0.06 0.68 1.01 2.06 1.81 0.31 0.28 0.71 1.59 0.79 0.13 3.42 2.57 16:0 8.46 6.88 7.46 5.82 28.22 19.74 27.08 9.38 12.61 7.33 13.77 11.95 19.79 15.30 25.38 21.19 16:1n-9 0.04 0.05 0.06 0.05 0.40 0.91 - - 0.31 0.41 tr 0.62 0.25 0.08 5.96 2.31 16:1n-7 0.06 0.06 0.06 0.06 0.29 0.69 tr 0.54 0.16 0.22 0.49 1.14 0.32 0.09 0.49 0.67 17:0 0.04 0.04 0.05 0.04 0.26 1.22 tr 0.86 0.07 - - - 0.34 0.09 0.91 0.44 18:0 2.40 1.02 1.99 1.10 8.85 13.81 20.12 6.93 4.51 5.23 6.31 10.85 6.90 4.29 18.99 6.80 18:1n-9 14.28 16.92 12.69 14.42 9.28 6.88 9.14 16.03 14.90 15.30 16.48 14.86 10.26 13.10 10.43 17.09 18:1n-7 0.85 0.64 0.77 0.79 1.24 1.14 4.09 0.52 1.37 0.90 1.64 0.92 0.74 1.07 0.32 0.77 18:2n-6 37.16 38.25 37.46 39.09 35.04 31.07 14.04 34.03 51.76 38.10 36.25 27.08 33.23 36.84 19.47 27.17 18:3n-3 36.05 35.59 39.10 38.26 10.20 16.57 7.43 25.21 12.48 31.83 20.07 26.95 25.28 28.23 9.23 13.68 20:0 0.17 0.10 0.15 0.10 1.17 1.47 - - 0.33 - 0.57 1.61 0.71 0.25 2.63 4.09 20:1n-9 0.13 0.09 0.11 0.09 0.18 0.00 - - 0.30 - 0.46 0.44 0.11 0.19 0.19 0.18 20:2n-6 tr 0.04 0.06 0.07 1.96 0.00 - - 0.19 - - - tr 0.09 - -

H 20:3n-3 tr 0.02 - 0.04 0.28 0.00 ------0.06 0.05 - - 22:0 0.15 0.14 - 0.02 1.73 4.73 1.86 4.27 0.22 0.36 1.69 1.46 0.44 0.14 0.98 1.47 22:1n-9 0.03 0.02 - - - 0.00 13.57 - 0.42 - 1.33 tr 0.10 - 0.74 - 23:0 tr - - - - 0.00 ------tr-0.17 - 24:0 0.08 0.06 - - - 0.00 ------0.37 0.05 - - *** ∑SFA 11.40a 8.33b 9.70 7.14 41.13 42.74 51.72 23.67 18.10 13.25 23.27 27.99 29.64 20.28 53.18 38.13 ∑ VLCSFAs(≥20C) 0.40a* 0.30b 0.15 0.12 2.91 6.20 1.86 4.27 0.55 0.36 2.26 3.07 1.52 0.44 3.78 5.56 *** ∑MUFA 15.39b 17.77a 13.68 15.40 11.40 9.61 26.80 17.09 17.47 16.82 20.41 17.98 11.78 14.51 18.12 21.02 ** ∑PUFA 73.21b 73.90a 76.61 77.46 47.47 47.64 21.48 59.24 64.43 69.93 56.32 54.03 58.57 65.21 28.70 40.85 ∑n-3 PUFAs 36.05a* 35.61b 39.10 38.30 10.48 16.57 7.43 25.21 12.48 31.83 20.07 26.95 25.35 28.29 9.23 13.68 *** ∑n-6 PUFAs 37.16b 38.29a 37.52 39.16 36.99 31.07 14.04 34.03 51.95 38.10 36.25 27.08 33.23 36.92 19.47 27.17 ** n-6/n-3 1.03b 1.08a 0.96 1.02 3.53 1.88 1.89 1.35 4.16 1.20 1.81 1.00 1.31 1.31 2.11 1.99 *** PU*FAs/SFAs 6.42b 8.87a 7.90 10.84 1.15 1.11 0.42 2.50 3.56 5.28 2.42 1.93 1.98 3.22 0.54 1.07 a The values represent the means of three samples, analyzed individually in triplicate (n =3× 3). TLs − total lipids, TAGs − triacylglycerols, PLs − polar lipids, MAGs − monoacylglycerols, DAGs − .Arc odChem. Food Agric. J. − − − − − − diacylglycerols, FFAs free fatty acids, SEs sterol esters; SFAs saturated fatty acids, MUFAs monounsaturated fatty acids, PUFAs polyunsaturated* fatty** acids, VLCSFAs*** very long chain saturated fatty acids; tr. − trace. Different superscript letters (a,b) in the same row mean significant differences between TLs of the two fermentation days. p < 0.05. p < 0.01. p < 0.001 (paired t- test). C12:0, lauric; C14:0, myristic; C16:0, palmitic; C16:1n-9, cis-7 hexadecenoic; C16:1n-7, palmitoleic; C17:0, margaric; C18:0, stearic; C18:1n-9, oleic; C18:1n-7,vaccenic; C18:2n-6, linoleic; C18:3n- O:10.1021/acs.jafc.5b00520 DOI: 3, α-linolenic; C20:0, arachidic; C20:1n-9, 11-eicosenoic; C20:2n-6, 11,14-eicosadienoic; C20:3n-3, eicosatrienoic; C22:0, behenic; C22:1n-9, erucic; C23:0, tricosanoic; C24:0, lignoceric acids. XX X,XXX XXX, XXXX, Article − XXX Journal of Agricultural and Food Chemistry Article confusing data about the correlation between antioxidant were pretreated with enzyme consortium obtained from the activity and TP contents. Several authors have reported that a individual culture of A. niger. high linear correlation exists between antioxidant potential and Neutral (TAGs, MAGs, 1,2- and 1,3-DAGs, FFAs, and TP contents in berries and different pomaces.12,19 However, on SEs) and Polar Lipid (PL) Contents of Oils from the the other hand, some scientific reports also claim that there is a Solid-State Fermented Pomaces. Profiles of the different bad correlation between these two parameters,12,34 which could lipid classes in the oils of the SSF S. ebulus L. and S. nigra L. be explained by the fact that different types of phenolic berry press residues are presented in Figure 4. The results compounds may contribute differently to total antioxidant showed that the major lipid fraction from SSF products (days 4 capacity of the analyzed plant matrix. The relationships and 6) was TAG, constituting about 90−94% of the fermented between the free radical scavenging activity (DPPH assay) berry-waste oils obtained in the early stages of fungal growth and total phenolic content of methanolic extracts from SSF S. (within 4 days) and about 57−62% of the oils in the sixth day ebulus L. and S. nigra L. berry press residues are shown in of fermentation. It should be noted that the proportions of Figure 1C,D. In both bioprocessed pomaces the radical FFAs increased significantly (p < 0.05) after the fourth day, inhibition capacity of the extracts showed no significant from average values of 1.50% (S. nigra L.) and 1.90% (S. ebulus correlations with the total phenolic content (R2 = 0.1887, p = L.) to averages of 32% (S. nigra L.) and 27.50% (S. ebulus L.) of 0.3301 for S. ebulus L. and R2 = 0.1657, p = 0.3648 for S. nigra the total extracted lipids in the sixth day of SSF. The quality of L., respectively). Our results are in agreement with some earlier the vegetable fat extracted from pomaces depends not only on reports1,21 which present weak correlations between these two the fatty acid composition but also on its lipid profile.16 Due to parameters among different bioprocessed fruit byproducts. Due the presence of lipase in the culture medium, TAGs are to the fact that these observations are common in studies that hydrolyzed to FFAs and DAGs or MAGs. A high amount of explore the ability of different fungi to produce enhanced levels FFAs indicates a low quality of the oil. Accumulation of this of phenolic antioxidants, it can be concluded that the SSF lipid fraction in fats leads to an unacceptable flavor, which can process had an effect on the correlation between total phenolic be prevented by inactivation of the enzymes involved in the content and the antioxidant potential of the fermented fruit lipid degradation.23 Among microorganisms, fungi are exten- byproducts. The possible explanation for this phenomenon sively recognized as the best lipase sources and are widely used could be that in certain stages of growth of fungi, the amount of in the food industry. It was reported that the lipase activity is the measurable phenolics decreases because of the polymer- induced by the presence of lipids in the medium.37 The same ization of the free phenolics, caused by the stress induced on authors had observed that in SSF system the lipase production the fungus. Vattem et al.1 and Di Majo et al.34 claim that the by Aspergillus niger was partially associated with the fungal inhibition of the free radicals tends to increase along with the growth, the synthesis of this enzyme starting after the second order of polymerization. day of fermentation. It is interesting to note that along Lipid Accumulation in SSF of the S. ebulus L. and S. fermentation processes, statistically significant (p < 0.05) nigra L. Berry Press Residues by A. niger. The lipid increases (3-fold for S. nigra L. and around 2-fold for S. ebulus recovery for both the Sambucus berry press residues showed a L.) in proportions of SEs were observed (Figure 5). These similar trend (Figure 3). After a significant increase (p < 0.05) positive changes can be attributed to the ability of the until day 4, there was a slight but statistically significant Aspergillus strains to synthesize SEs. According to the study decrease (p < 0.05) in the TL content for the remaining days of of Tőke et al.,38 the filamentous fungi (especially Aspergillus fermentation. The extracted lipids increased with 12% for S. spp.), in addition to their lipase production capacity, could nigra L., and 10.50% for S. ebulus L. compared with initial values produce significant amounts of sterol esterases under SSF of 21.41g/100 g DW and 22.20 g/100 g DW of unfermented conditions, enzymes able to efficiently catalyze the synthesis of pomaces to the maximum amounts of 24 g/100 g DW and sterol esters from phytosterols and free fatty acids. The free 24.53 g/100 g DW of fermented pomaces. The literature shows phytosterols and their esterified forms are known to reduce the that the enzyme consortia (cellulase, pectinase, protease, etc.) cholesterol level in blood.39 However, the sterol esters are secretion of the strains has a considerable impact on lipid yield, generally preferred in the food and nutraceutical industry and a strong correlation between these two parameters was because of their better fat-solubility and compatibility.38 Finally, found. The enzyme productivity by A. niger is a function of analysis of the various lipid fractions demonstrated that the culture conditions (temperature, pH, culture medium con- percentages of PLs also tends to increase (around 4-fold for S. ditions), amount of available carbon source (glucose, cellulose, nigra L. and more than 2-fold for S. ebulus L.) during hemicellulose, pectin, etc.), and C/N ratio.23,35 Previous fermentation processes (Figure 1 (A)). This observation is research conducted on hydrolytic enzyme production by A. consistent with experimental data provided by Oliveira et al.40 niger in SSF of different agro-residues have shown maximum showing that in oleaginous microorganisms, during lipid cellulase and protease activities after 72 or 96 h,23,36 when the turnover phase (which starts when the culture media is enzyme secretion had almost stabilized or decreased. These depleted of the carbon source), neutral lipid fraction (mainly observations are consistent with our results regarding the composed of TAGs) are preferentially degraded, while dynamics of the oil yields from Figure 3, with the maximum significant amounts of membranar polar lipids are synthesized. lipid amounts in day 4. At the early fermentation stage, fungi TLs Fatty Acid Compositions and Their Lipid Classes consume the readily available sugars (particularly glucose) and Obtained through Solid-State Fermented Products. The secrete hydrolytic enzymes; when the level of sugar monomer is GC-MS analysis (results presented inTables 2 and 3) showed too low, the strains start to use these enzymes to produce that the major fatty acid compositions of TLs from the solid- available sugars, resulting in a decrease of enzyme activity and state fermented pomaces (day 4) were linoleic acid (C18:2n-6, lipid yield.23 Oberoi et al.23 used different crude enzyme 45.19% in S. ebulus L. and 38.25% in S. nigra L.), α-linolenic extracts to improve the oil recovery from mustard seeds and acid (C18:3n-3, 23.34% in S. ebulus L. and 35.59% in S. nigra reported that about 7% more oil was recovered when seeds L.), oleic acid (C18:1n-9, 23.30% in S. ebulus L. and 16.92% in

I DOI: 10.1021/acs.jafc.5b00520 J. Agric. Food Chem. XXXX, XXX, XXX−XXX Journal of Agricultural and Food Chemistry Article

S. nigra L.) and palmitic acid (C16:0, 6.02% in S. ebulus L. and ingredients.49 The SEs exhibited significantly higher SFA levels 6.88% in S. nigra L.) (Figure 5). The fermentation resulted in a (38.13% in S. nigra L. and 46.93% in S. ebulus L.) than the other slight increase of the linoleic (C18:2n-6) and oleic (C18:1n-9) four NL fractions in fermented samples, because of the acids levels (43.11% and 20.22% in S. ebulus L. and 37.16% and dominance of palmitic acid (C16:0) in their structures. 14.28% in S. nigra L. unfermented pomaces, respectively). Furthermore, it was observed that by the SSF process, the SE These results are in line with earlier findings showing that fractions were enriched in n-3 and n-6 essential fatty acids. The strains of Aspergillus produce lipids with significant amounts of levels of n-3 PUFAs increased with 48% (in S. nigra L.) and oleic and linoleic acids.41,42 As shown in Tables 2 and 3, 77% (in S. ebulus L.), while the amounts of n-6 PUFAs statistically significant differences (p < 0.05) were found increased with 40% (in S. nigra L.) and 95% (in S. ebulus L.), between the proportions of fatty acid classes of investigated respectively. A comprehensive study of 91 patients tested the TLs, extracted from Sambucus wastes before and after benefit of omega-3 plant sterol esters (n-3-PSE) on lipid profile fermentation. Substantial decreases in the contents of saturated and other cardiovascular disease risk factors in subjects with fatty acids (SFAs) were observed for both fermented residues mixed hyperlipidemia.50 The study showed that n-3-PSE (from 11.47% to 7.56% in S. ebulus L., and from 11.40% to treatment decreased the serum triglyceride concentrations 8.33% in S. nigra L., respectively). The differences were also significantly (by 19%) without adversely affecting the plasma statistically significant (p < 0.05) for the content of mono- low-density lipoprotein cholesterol levels in mixed hyper- (MUFAs) and polyunsaturated fatty acids (PUFAs), the lipidemic patients, after a 12 week intervention period. average proportions in fermented substrates being slightly This global characterization may potentially provide the basis higher than those in the unfermented raw materials (Tables 2 for a sustainable integrated process of Sambucus berry pomaces and 3). The elevated levels of PUFAs from TLs of fermented exploitation as potential, inexpensive, and easily available berry press residues (68.53% in S. ebulus L. and 73.90% in S. sources of bioactive phytochemicals for the pharmaceutical nigra L.) determined in the present study are comparable to and food industries. those of PUFA-rich vegetable oils, such as grape seed, sunflower, linseed, blackcurrant seed, safflower, and hemp ■ AUTHOR INFORMATION seed oils.43 The results also showed that the TLs of both Corresponding Author bioprocessed wastes are characterized by high levels of PUFAs/ *E-mail: [email protected]. Tel.: +40-264-596 384 SFAs (8.87−9.07) and low levels of n-6/n-3-PUFAs (1.08− (ext. 213). Fax: +40-264-593-792. α 1.94) ratios. Due to their high linoleic and -linolenic acid Funding contents, the unsaturated fats are more susceptible to the 16 This work was supported by a grant of the Romanian Ministry oxidation process. The values of n-6/n-3 ratios reported in of Education, CNCS−UEFISCDI, project number PNII- this study are close to the optimal range for human health (2/ RUPD-2012-3-0245 (contract no. 92/25.06.2013). 1−4/1), suggested by the nutritional scientists.44 The profiles fi Notes of fatty acids in TAGs were practically identical to the pro les fi of the TLs, with the same dominating fatty acids in both solid- The authors declare no competing nancial interest. state fermented pomaces (Tables 2 and 3). The fatty acid REFERENCES composition of all minor lipid classes was somehow affected by ■ the fermentation period. The PL fractions of SSF berry wastes (1) Vattem, D. A.; Lin, Y. T.; Labbe, R. G.; Shetty, K. 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J DOI: 10.1021/acs.jafc.5b00520 J. Agric. Food Chem. XXXX, XXX, XXX−XXX Journal of Agricultural and Food Chemistry Article

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K DOI: 10.1021/acs.jafc.5b00520 J. Agric. Food Chem. XXXX, XXX, XXX−XXX Journal of Agricultural and Food Chemistry Article

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L DOI: 10.1021/acs.jafc.5b00520 J. Agric. Food Chem. XXXX, XXX, XXX−XXX Food Chemistry 209 (2016) 27–36

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Food Chemistry

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Effects of solid-state fermentation with two ﬁlamentous fungi on the total phenolic contents, ﬂavonoids, antioxidant activities and lipid fractions of plum fruit (Prunus domestica L.) by-products ⇑ Francisc Vasile Dulf a, , Dan Cristian Vodnar b, Carmen Socaciu b a Faculty of Agriculture, Department of Environmental and Plant Protection, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Ma˘na˘ßstur 3-5, 400372 Cluj-Napoca, Romania b Faculty of Food Science and Technology, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Ma˘na˘ßstur 3-5, 400372 Cluj-Napoca, Romania article info abstract

Article history: Evolutions of phenolic contents and antioxidant activities during solid-state fermentation (SSF) of plum Received 8 January 2016 pomaces (from the juice industry) and brandy distillery wastes with Aspergillus niger and Rhizopus oligos- Received in revised form 15 March 2016 porus were investigated. The effect of fermentation time on the oil content and major lipid classes in the Accepted 11 April 2016 plum kernels was also studied. Results showed that total phenolic (TP) amounts increased by over 30% for Available online 11 April 2016 SSF with Rhizopus oligosporus and by >21% for SSF with A. niger. The total ﬂavonoid contents presented similar tendencies to those of the TPs. The free radical scavenging activities of methanolic extracts were Keywords: also signiﬁcantly enhanced. The HPLC-MS analysis showed that quercetin-3-glucoside was the major Solid-state fermentation phenolic compound in both fermented plum by-products. The results also demonstrated that SSF not Aspergillus niger Rhizopus oligosporus only helped to achieve higher lipid recovery from plum kernels, but also resulted in oils with better qual- Plum by-products ity attributes (high sterol ester and n-3 PUFA-rich polar lipid contents). Polyphenols Ó 2016 Elsevier Ltd. All rights reserved. Antioxidant activity Lipids

1. Introduction mainly of flavonols, and caffeic acid derivatives (depending on the cultivar) with many health benefits, such as anticarcinogenic and In recent years, there has been an increasing trend towards the antimicrobial effects, and the ability to prevent heart diseases, more efficient utilization of agricultural and food by-products. The digestive system illnesses and osteoporosis, respectively (Kristl, insufficient collection and improper disposal of these wastes may Slekovec, Tojnko, & Unuk, 2011; Sójka et al., 2015). generate serious pollution problems and also a significant loss of Plums are widely distributed in most countries of the world. biomass which could potentially be used to produce various value- Romania is the 3rd highest world producer of these fruits, with added metabolites (Correia, McCue, Magalhães, Macêdo, & Shetty, 512,459 tons in 2013 (FAO, 2014). Using the plum fruits as raw 2004). Stone fruits, especially plums (Prunus domestica L.), are char- material, the Romanian food and beverage industry annually pro- acterized by high contents of dietary fibre, phenolics composed duces significant amounts of preserved fruits, juices and brandy called ‘‘Tßuica˘” or ‘‘Pa˘linca˘”. During industrial processing, tonnes of pomaces (press cake residues from juice industry), seeds Abbreviations: SSF, solid-state fermentation; TP, total phenolic; DPPH, 1,1- (stones) and brandy distillery wastes (spent fruit pulp/peels) are diphenyl-2 picrylhydrazyl; FAMEs, fatty acid methyl esters; TLC, thin layer produced and discarded. chromatography; PDA, potato dextrose agar; PP, plum pomace; WPBP, waste from plum brandy production; GAE, gallic acid equivalents; DW, dry weight; QE, The plum kernels contain a significant amount of oil quercetin equivalent; HPLC-DAD-ESIMS, high-performance liquid chromatography- (>45 g/100 g) with a remarkably high level of oleic acid, a moderate diode array detection-electro-spray ionization mass spectrometry; I%, percentage amount of linoleic acid and low contents of saturated fatty acids. inhibition of DPPH radical; RIC, radical inhibition capacity; TLs, total lipids; TAGs, Additionally, the oil also contains considerable amounts of other triacylglycerols; MAGs, monoacylglycerols; DAGs, diacylglycerols; FFAs, free fatty acids; SEs, sterol esters; PLs, polar lipids; GC-MS, gas chromatography mass compounds with beneficial effects such as phytosterols and toco- spectrometry; wt%, weight percentages; NL, neutral lipid; MUFAs, monounsatu- pherols (Matthaeus & Oezcan, 2009). rated fatty acids; PUFAs, polyunsaturated fatty acids; SFAs, saturated fatty acids. In the available literature there are few references on the ⇑ Corresponding author. polyphenol composition of plum by-products. As these wastes E-mail address: [email protected] (F.V. Dulf). http://dx.doi.org/10.1016/j.foodchem.2016.04.016 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved. 28 F.V. Dulf et al. / Food Chemistry 209 (2016) 27–36 are less recognized and studied raw materials, the only available the Transylvania region, Romania) collected in September of data are focused on unprocessed plum fruit composition and prop- 2014. The brandy distillery wastes (spent fruit pulp and peels, erties (Milala et al., 2013; Sójka et al., 2015). without stones) were obtained from a local distillery that uses Although the potential use of plums as sources of polyphenols the traditional methods for plum brandy production: alcoholic fer- and oil (from kernels) with a high content of monounsaturated mentation for 6–8 weeks in large barrels, followed by the distilla- fatty acids would seem advantageous, there is little information tion process (initially, the raw fermented material was heated to available concerning strategies for the liberation and extraction about 90–95 °C (2 h), after which the temperature was reduced of these bioactive compounds from the vegetable raw materials. to about 80 °C and maintained until the end of the distillation pro- The fungal pretreatment of the agro-industrial wastes using the cess). All of the by-products were dried in an oven (37 °C) until solid-state fermentation (SSF) system is a technology which could completely dry, ground to particle-sized fractions of 0.5–1 mm positively enhance the recovery of valuable hydro- and lipophilic and stored in a refrigerator before use. compounds. SSF is defined as a microbial culture that develops Folin-Ciocalteu’s phenol reagent, sodium carbonate (Na2CO3), on moist substrates in the absence of free water (Correia et al., sodium nitrite (NaNO2), ammonium nitrate (NH4NO3), hydrochlo- 2004). ric acid (HCl), aluminum chloride (AlCl3), sodium hydroxide Since plum pomaces are known to contain valuable nutrients (NaOH), sea salts, glucose, acetic acid, acetonitrile, methanol, gallic which can support the growth of microorganisms, exploitation of acid, quercetin, chlorogenic acid, rutin, cyaniding chloride, DPPH the nutritive potential of these wastes for the production of differ- (1,1-diphenyl-2 picrylhydrazyl), and the lipid standards and chem- ent high value compounds is a welcome technology. icals (used for oil extraction, fractionation, and preparation of fatty Most phenolic compounds occur primarily in conjugated form, acid methyl esters (FAMEs)) were purchased from Sigma–Aldrich linked to sugar moieties, but also to other compounds, such as (Steinheim, Germany). High performance TLC (thin layer chro- organic acids, amines and lipids. These conjugations reduce their matography) plates (silica gel 60 F254, 20 Â 20 cm) were obtained ability to function as good antioxidants, because the resonance sta- from Merck (Darmstadt, Germany) and the FAMEs standard (37 bilization of free radicals depends on the availability of free hydro- component FAME Mix, SUPELCO) was from Supelco (Bellefonte, xyl groups on the phenolic rings. The enzymatic hydrolysis of these PA, U.S.A.). phenolic conjugates with carbohydrate degrading enzymes produced by fungi during SSF appears to be an attractive means of 2.2. Microorganism maintenance and spore inoculum preparation increasing the concentration of free phenolics in agri-food by- products used as substrates in the fermentation processes (Ajila A. niger (ATCC-6275) and Rhizopus oligosporus (ATCC-22959) et al., 2012). Aspergillus niger, for example, is a microorganism (LGC Standards GmbH, Wesel Germany) were selected for SSF which has been used in many SSF studies due to its ability to syn- and were maintained on potato dextrose agar (PDA) slants and thesize >19 types of enzyme, such as cellulase, pectinase, protease, Petri plates at 4 °C(Oberoi et al., 2012). The fungal colonies were etc. This enzyme consortium, in addition to its capacity to liberate revived by transferring onto fresh PDA plates and cultured at room the phenolic compounds, also helps to hydrolyze the bonds temperature 10 days before use. between lipo-proteins and lipo-polysaccharides present in oilseeds, improving recovery of oil from the substrates (Dulf, 2.3. Solid-state fermentation (SSF) Vodnar, Dulf, & Tosßa, 2015; Falony, Armas, Mendoza, & Martínez Hernández, 2006). For SSF, 500 ml Erlenmeyer flasks containing 15 g solid sub- Rhizopus oligosporus is another important food-grade filamen- strates, 30 ml sea salt solution (30 gm/l), 1 g glucose and 1 g of tous fungus, which has been used in fermentation technologies NH4NO3 were used. The fermentation mediums were autoclaved for several centuries, especially in Asia, for the preparation of at 121 °C for 30 min and inoculated with spore suspension traditional fermented foods. It is able to produce high amounts of (2 Â 107 spores/g of solid) (Dulf et al., 2015). After being thor- b-glucosidase in SSF processes, that have a significant role in the oughly mixed, the fermentations (in triplicate) were conducted hydrolysis of phenolic glycosides (Lee, Hung, & Chou, 2008). for 14 days at 30 °C. During SSF, in order to carry out the chemical The potential of SSF for the improvement of phenolic contents characterization, 1 g of sample was removed after 2, 3 (for lipids), and antioxidant properties of different fruit pomaces has been 6, 9 and 14 days and stored at À20 °C. evaluated by employing several micro-organisms (Ajila et al., 2012; Correia et al., 2004; Vattem, Lin, Labbe, & Shetty, 2004). 2.4. Extraction and analysis of phenolic compounds To the best of our knowledge, there are no reported studies on the use of plum fruit by-products as supports in SSF systems for the The de-stoned plum residues (pomaces (PP) and brandy dis- production of value-added compounds. Therefore, the aim of this tillery wastes (WPBP)) (2 g) were individually extracted three study was to evaluate the changes in phenolic compositions and times with 20 ml of extraction mixture (hydrochloric acid/metha- antioxidant activity through the SSF of plum pomaces (from the nol/water in the ratio of 1:80:19) at 40 °C for 30 min in an ultra- juice industry) and brandy distillery wastes (spent fruit pulp and sonic bath (Dulf et al., 2015). After centrifugation (at 4000g for peels) with A. niger and Rhizopus oligosporus. Moreover, the influ- 10 min) and filtration of the supernatants, the filtrates were evap- ence of fermentation time on the oil content and major lipid orated to dryness under vacuum, dissolved in methanol and stored classes in solid state fermented plum kernels was also studied. (4 °C) until analysis (total and individual phenolics, total flavonoids and antioxidant activities).

2. Materials and methods 2.4.1. Total phenolic contents Determination of total phenolic (TP) level was performed by the 2.1. Materials and chemicals Folin-Ciocalteu method (Dulf et al., 2015; Singleton, Orthofer, & Lamuela-Raventos, 1999). An aliquot (25 lL) of each extract was The stones from fully ripened plums (Prunus domestica L.) were mixed with 125 ll of Folin-Ciocalteu reagent (0.2 N) and 100 ll removed and individually broken to obtain the intact kernels. The of 7.5% (w/v) Na2CO3 solution. The mixture was incubated for 2 h press cake residues (pomaces) were obtained in our laboratory in the dark at room temperature (25 °C). The absorbance against from de-stoned, dark blue plum fruits (cultivar Bistrita, grown in a methanol blank was recorded at 760 nm. A standard curve was F.V. Dulf et al. / Food Chemistry 209 (2016) 27–36 29 prepared using gallic acid (0.01–1 mg/ml), and the TP content in Pintea, 2013). The samples were analyzed with a gas chro- the extract was expressed as gallic acid equivalents (GAE) in matograph (GC) coupled to a mass spectrometer (MS): PerkinElmer mg/100 g dry weight (DW) of waste. Clarus 600 T GC–MS (PerkinElmer, Inc., Shelton, CT, U.S.A.) (Dulf et al., 2015). The GC column was a Supelcowax 10 2.4.2. Total flavonoid contents (60 m Â 0.25 mm i.d., 0.25 lm film thickness; Supelco Inc., Belle- Flavonoid levels were measured according to the aluminium fonte, PA, U.S.A.). The oven temperature was set at 140 °C, then chloride colorimetric method developed by Zhishen, Wengcheng, ramped to 220 °Cat7°C/min, and held at 220 °C for 23 min. The and Jianming (1999) using quercetin as the reference standard, injection volume was 0.5 ll (split ratio of 1: 24) and the injector as described by Ma˘rghitasß et al. (2009). The absorbance was mea- was set at 210 °C. Helium was used as the carrier gas with a sured immediately against a prepared reagent blank at 510 nm. constant flow rate of 0.8 ml/min. Mass spectra (E.I., positive ion Total flavonoid content was expressed as mg quercetin equivalent electron impact mode) were recorded at 70 eV and using a trap (mg QE/100 g DW). current of 100 lA with a source temperature of 150 °C. The MS was scanned from m/z 22–395 for all GC–MS experiments. 2.4.3. Analysis of phenolic compounds by high-performance liquid The FAME peak identification was based on comparison of both chromatography-diode array detection-electro-spray ionization mass retention time and MS of the unknown peak to those of known spectrometry (HPLC-DAD-ESIMS) standards and with data provided by an MS database (NIST MS Samples were analyzed with an Agilent 1200 HPLC with DAD Search 2.0). detector, coupled to an MS detector single quadrupole Agilent The quantification of each fatty acid was carried out by compar- 6110. Separations of phenolics were performed at 25 °Conan ison to peak area of the methyl nonadecanoate without application Eclipse column, XDB C18 (4.6 Â 150 mm, 5 lm) (Agilent Technolo- of any correction factor. The compositions of fatty acids in studied gies, U.S.A.). The binary gradient consisted of 0.1% acetic acid/ace- lipid fractions were expressed as molar percentages of the fatty tonitrile (99:1) in distilled water (v/v) (solvent A) and 0.1% acetic acids (molar% of total fatty acids). The neutral and polar lipid acid in acetonitrile (v/v) (solvent B) at a flow rate of 0.5 ml/min, fol- amounts in TLs were determined using an average molecular lowing the elution program used by Dulf et al. (2015): 0–2 min (5% weight of fatty acids of the fraction in question and were expressed B), 2–18 min (5–40% B), 18–20 min (40–90% B), 20–24 min (90% B), as weight percentages (wt%) in oils. 24–25 min (90–5% B), 25–30 min (5% B). Various phenolic compounds were identified by comparing the 2.6. Statistical analysis retention times, UV–visible and mass spectra of unknown peaks with the reference standards. For MS fragmentation, the E.I., posi- All tests were conducted in triplicate and the results were tive ion electron impact mode was applied, with scanning range expressed as mean ± standard deviation (SD). Correlations between 100 and 1000 m/z, capillary voltage 3000 V, at 350 °C between the antioxidant capacity and phenolics were determined and nitrogen flow of 8 L/min. The eluent was monitored by DAD, using Pearson’s correlation. Statistical differences among samples and the absorbance spectra (200–600 nm) were collected continu- were estimated using Student’s t test and ANOVA (repeated mea- ously in the course of each run. The flavonols were detected at sures ANOVA; Tukey’s Multiple Comparison Test; GraphPad Prism 340 nm and the anthocyanins at 520 nm (Dulf et al., 2015). Data Version 5.0, Graph Pad Software Inc., San Diego, CA). Differences analysis was performed using Agilent ChemStation Software (Rev between means at the 5% level values were considered statistically B.04.02 SP1, Palo Alto, California, U.S.A.). Anthocyanin levels were significant. determined using cyanidin chloride as the external standard and expressed as equivalents of cyanidin (mg cyanidin/100 g DW of substrate) (r2 = 0.9945). The chlorogenic and neochlorogenic acids 3. Results and discussion were expressed in mg chlorogenic acid/100 g DW of substrate (r2 = 0.9937) and flavonol glycosides were calculated as equiva- 3.1. Total phenolics and total flavonoids lents of rutin (mg rutin/100 g DW of substrate) (r2 = 0.9981). The results of the TP determination using the Folin-Ciocalteu 2.4.4. DPPH free radical scavenging assay method showed a similar trend during the first days of SSF for both Phenolic extracts of non- or fermented de-stoned plum residues strains of microorganism. The amounts of phenolics decreased in were subjected to DPPH radical scavenging activity assessment the first 48 h and increased significantly (p < 0.05) until the maxi- using the method described by Ma˘rghitasß et al. (2009). The per- mum yields (day 6 for WPBP and 9 for PP, respectively) (Fig. 1A). centage inhibition (I%) was calculated as [1 À (test sample absor- Similar tendencies of the measurable free phenolics in the early bance/blank sample absorbance)] Â 100. stages of fungal growth were also observed in our previous study on the effect of SSF by A. niger on phenolic contents in Sambucus 2.5. Plum kernels-lipid extraction, separation and methylation berry pomaces (Dulf et al., 2015), and in the work of Correia et al. (2004) regarding the production of phenolics by the SSF of pineap- Total lipids (TLs) of kernels from stones of fully ripened plum ple waste with Rhizopus oligosporus. Moreover, these authors sug- fruits were extracted from 1 g of sample with a solvent mixture gest that the low phenolic content observed during the first days of chloroform/methanol (2:1, v/v) (Dulf et al., 2015). of SSF could be explained by the fact that these compounds exist TLs were separated into neutral and polar lipid fractions (TAGs- predominantly in the membrane bound form and only a low triacylglycerols, MAGs-monoacylglycerols, DAGs-diacylglycerols, amount is in the free soluble form. Polyphenol content in the FFAs-free fatty acids, SEs-sterol esters and PLs-polar lipids) by extracts of plum by-products fermented with R. oligosporus TLC, using a previously described method (Dulf, Pamfil, Baciu, & increased showing values of 44.16% for PP (from 679.80 mg/g DW Pintea, 2013). to 980.00 mg/g DW) and 30.15% for WPBP (from 1295.31 mg/g Internal standards were added before methylation of the DW to 1685.96 mg/g DW) in relation to unfermented substrates, isolated fractions (80 lg trinonadecanoylglycerol for TAGs and while in plum wastes fermented by A. niger the phenolics increased 10–200 lg nonadecanoic acid for PLs, MAGs, DAGs, SEs and FFAs). to 35.32% (for PP) and 21.52% (for WPBP). These increases could be FAMEs were prepared by acid-catalyzed transesterification of attributed to the fungi b-glucosidases which are able to hydrolyze TLs and separated lipid fractions (Dulf, Oroian, Vodnar, Socaciu, & b-glucosidic linkages, mobilizing phenolics to react with the 30 F.V. Dulf et al. / Food Chemistry 209 (2016) 27–36

2000 900 WPBP- A.n. A B a*** WPBP- A.n. a*** WPBP- R.o. WPBP- R.o. 800 1600 b a*** 700 a*** b 1200 600 a*** a*** 1000 PP- A.n. 450 PP- A.n. PP- R.o. PP- R.o. 400 a*** a*** 800 b 350 b Total flavonoids (mg QE/100g DW) flavonoids QE/100g (mg Total Total phenolics (mg GAE/100g DW) 300 600

0 2 6 9 4 0 2 6 9 1 14 Day of fermentation Day of fermentation

100 a*** C

90 b a*** a**

80 a***

70 b WPBP- A.n. WPBP- R.o. 60 PP- A.n. % Inhibition of DPPH radical DPPH of Inhibition % PP- R.o. 50

0 2 6 9 14 Day of fermentation

Fig. 1. Total phenolic content (Folin–Ciocalteu method) (A), total ﬂavonoids (B) and free radical scavenging activity (DPPH assay) of phenolics (C) of extracts from solid state fermented plum by-products. Values are mean ± SD of triplicate determinations and different letters (a, b) indicate signiﬁcant differences (p < 0.05) (paired t-test). A.n. – Aspergillus niger, R.o. – Rhizopus oligosporus; PP – plum pomace; WPBP – waste from plum brandy production.

Folin–Ciocalteau reagent (Ajila et al., 2012). The measurable free In the current study, the values for the total phenolics and fla- phenolics for both plum by-products showed a slight decrease dur- vonoids obtained by SSF with R. oligosporus were only slightly ing the later stages of growth (after six days of fermentation for higher than those obtained by SSF with A. niger. Similar findings WPBP and nine days for PP) (Fig. 1A), which could be attributed were reported by Lee et al. (2008), who evaluated the total pheno- to the polymerization of the released phenolics by oxidative lic and anthocyanin contents of black beans during SSF with enzymes, activated as a response to the stress induced on the different species of GRAS (generally recognized as safe) filamen- fungus due to the nutrient depletion (Vattem et al., 2004). In the tous fungi (Aspergillus and Rhizopus). They concluded that the total literature, there are only a few studies on polyphenol composition extractable phenolic and anthocyanin content in black beans of plum pomaces. The available data shows the content and compo- increased after fermentation, depending on the starter microor- sition of phenolics in plum fruits. Recently, Sójka et al. (2015) eval- ganism. It is generally known that the previously mentioned uated the polyphenol composition of purified and freeze-dried microorganisms are capable of producing b-glucosidase. Moreover, water extracts from pomaces of three dark blue plum cultivars it was observed that the optimal conditions for the catalytic activ- and reported that the resulting pomace extracts were characterized ity of b-glucosidase vary with the source of enzyme (Lee et al., by high total phenolic contents comprising between 5 and 2008). 50 g/100 g of dry extract. According to Milala et al. (2013), the total As shown in Fig. 1(A, B) the WPBP samples (fermented and polyphenol content in plum pomaces (consisting mainly of peels) unfermented) exhibited markedly higher amounts of total pheno- depends on the cultivar and also on the drying parameters (air- lics and flavonoids than those of PP extracts. This feature might drying as opposed to freeze-drying). be explained by the origin of the starting material (WPBP), which The total flavonoid contents in solid-state processed plum by- can be considered a ‘‘double-processed” by-product (alcoholic fer- products showed a similar tendency with regards to the TPs mentation with Saccharomyces cerevisiae followed by the distilla- (Fig. 1B). After 2 days of SSF, both wastes showed steep increases tion process). Many studies have demonstrated that fermentation (p < 0.05) in flavonoid contents until the maximum yields were has a positive effect on the phenolic content and antioxidant activ- reached of 809.42 mg QE/100 g DW- by R. oligosporus, and 755.52 ity of different plant-based matrices, the degree of influence QE/100 g DW- by A. niger for WPBP (from the initial value of depending on the species of microorganism (Hur, Lee, Kim, Choi, 621.25 QE/100 g DW), and 435.60 mg QE/100 g DW- by R. oligos- & Kim, 2014). Strains of Saccharomyces cerevisiae have been shown porus and 421.87 QE/100 g DW- by A. niger for PP (from the initial to produce enzymes, including b-glucosidases, carboxylesterases, value of 306.21 QE/100 g DW). Similarly, a substantial increase and feruloyl esterases, which were found to be effective in releas- (over 50% of the initial value) in the levels of total flavonoids was ing insoluble-bound phenolics (Moore et al., 2007). Moreover, ther- also reported by Lin et al. (2014) in Aspergillus-fermented litchi mal processing (between 50 and 150 °C) was shown to release pericarp and its aqueous-organic extracted residues. more bound phenolic compounds due to the breakdown of cellular F.V. Dulf et al. / Food Chemistry 209 (2016) 27–36 31 constituents (Dewanto, Wu, Adom, & Liu, 2002). Although Overall, the SSF with both microorganisms had a significant disruption of cell walls can also trigger the release of oxidative (p < 0.05) effect on the evolution of phenolic proportions. In gen- and hydrolytic enzymes able to negatively affect the antioxidant eral, an increase in the individual phenolic amounts of all samples activity in fruits, the high processing temperature (P85 °C) deacti- (excepting isorhamnetin glycosides) was observed (Table 1). The vates these enzymes, thereby preventing the loss of phenolic quercetin-based glycosides were the major flavonol glycosides in compounds. both processed plum residues: from 57.30% (non-fermented) to The usefulness of wastes from plum brandy production is still 72.40% (fermented by A. niger) and 70.57% (fermented by R. oligos- underestimated. To the best of the authors’ knowledge, this is porus) (on the 9th day of SSF) of total flavonol glycosides of PP and the first study that attempts to determine the phenolic composi- from 86.50% (non-fermented) to 93.23% (A. niger – on the 9th day of tion of this by-product. SSF) and 94.63% (R. oligosporus – on the 14th day of SSF) of total flavonol glycosides of WPBP, while the remainders were isorhamnetin glycosides. The findings of this study regarding the decomposition 3.2. HPLC-DAD-MS analysis of phenolic compounds of isorhamnetin derivatives during SSF agree well with the findings of Moussa-Ayoub, El-Samahy, Kroh, and Rohn (2011), who reported The amounts of the main phenolics in the extracts of plum by- that all isorhamnetin glycosides in cactus pear peels were hydro- products were determined during fermentations with the strains of lyzed gradually to isorhamnetin by increasing the time of enzy- A. niger and R. oligosporus, respectively (Table 1). matic hydrolysis (using a mixture of pectinases and cellulases). The analyzed samples contained nine dominant polyphenolics; Previous studies on plum fruit skins and industrial plum two cinnamic acids (neochlorogenic and chlorogenic acids), five pomaces (Sójka et al., 2015) have shown that the predominant fla- flavonols (isoquercitrin, quercetin-3-galactoside, rutin and two vonol in the plum extracts was rutin, whereas neochlorogenic and isorhamnetin glycosides) and two anthocyanins (cyaniding-3- chlorogenic acids were the main hydroxycinnamic acids. Although glucoside and cyaniding-3-rutinoside) (Supplementary Fig. S1). the present study also found high amounts of rutin in the samples, These phenolic profiles were in general agreement with two previ- with maximum concentrations during the later stages of fermenta- ous reports on polyphenolic extracts obtained from plum pomaces tions (on the 9th or 14th day) (Table 1), however the isoquercitrin of various dark blue cultivars (Milala et al., 2013; Sójka et al., was the major flavonol in both processed plum residues. This result 2015). The proportion of the individual phenolics differed substan- could be explained by the ability of the filamentous fungi (espe- tially between PP and WPBP in both fermentation processes cially from the genus Aspergillus) to produce a-rhamnosidase (Lin (Table 1). Isoquercitrin was the major phenolic compound in both et al., 2014), an enzyme known for its capability of removing one processed plum residues. The next predominant phenolics in terminal a-rhamnose present in many natural glycosides. It is pos- fermented PP were cinnamic acids, whereas in WPBP the second sible that part of the rutin accumulated during SSF processes was major phenolic compound was rutin.

Table 1 Phenolics evolution (mg/100 g DW) as determined by HPLC-DAD-ESIMS during solid-state fermentation of plum by-products.

Cinnamic acids Flavonols Anthocyanins Phenolics ([M + H] + 3-CQA 5-CQA Q-3-gluc Q-3-gal⁄ Q-3-rut Isorha-3-gal⁄ Isorha-3-gluc⁄ Cya-3-gluc Cya-3-rut ion, fragments) (355, 181) (355,181) (465, 303) (465, 303) (611, 303) (479, 317) (479, 317) (449, 287) (595, 287) Peak 1 2 3 4567 89 Plum pomace FT (day) A. niger 0 23.45 ± 1.00c 22.35 ± 1.13c 22.95 ± 1.00e 11.05 ± 0.63c 20.50 ± 1.00e 25.23 ± 0.88a 15.38 ± 0.50a 4.70 ± 0.25b 6.50 ± 0.30b 2 23.58 ± 1.13c 22.58 ± 0.95c 24.10 ± 0.88d 10.55 ± 0.55d 22.08 ± 0.95d 23.53 ± 1.00b 11.00 ± 0.55b 5.50 ± 0.20a 6.95 ± 0.35a 6 26.00 ± 1.05b 25.80 ± 1.13b 30.05 ± 1.20c 11.93 ± 0.50b 23.45 ± 1.13c 23.08 ± 1.13b 11.38 ± 0.95b 3.50 ± 0.25c 4.00 ± 0.23c 9 27.95 ± 1.20a 33.00 ± 1.38a 36.08 ± 1.30a 13.63 ± 0.63a 26.13 ± 1.20a 19.40 ± 0.75c 9.50 ± 0.38c 2.75 ± 0.23d 3.20 ± 0.25d 14 19.85 ± 0.88d 24.23 ± 0.95bc 34.30 ± 1.05b 11.10 ± 0.50c 25.28 ± 1.13b 11.65 ± 0.55d 8.40 ± 0.45d 2.50 ± 0.23e 3.25 ± 0.25d R. oligosporus 0 23.45 ± 1.00c 22.35 ± 1.13e 22.95 ± 1.00d 11.50 ± 0.63c 20.50 ± 1.00b 25.23 ± 0.88a 15.38 ± 0.50a 4.70 ± 0.25b 6.50 ± 0.30b 2 23.53 ± 0.95c 24.83 ± 0.88c 24.53 ± 0.95c 12.20 ± 0.50b 17.93 ± 0.63d 21.08 ± 0.95b 11.35 ± 0.45b 4.13 ± 0.20c 7.25 ± 0.28a 6 24.60 ± 1.00b 25.80 ± 0.95b 26.23 ± 1.00b 12.70 ± 0.55a 19.45 ± 0.75c 16.35 ± 0.65c 10.85 ± 0.50c 5.63 ± 0.20a 5.25 ± 0.25d 9 25.15 ± 1.05a 26.18 ± 0.88a 26.68 ± 1.00a 12.80 ± 0.63a 21.10 ± 0.70a 15.53 ± 0.63d 9.78 ± 0.48d 3.25 ± 0.23d 5.75 ± 0.28c 14 21.95 ± 1.00d 24.23 ± 0.80d 24.50 ± 1.05c 10.35 ± 0.65d 15.40 ± 0.63e 13.58 ± 0.55e 8.65 ± 0.40e 3.25 ± 0.23d 4.25 ± 0.20e Waste from plum brandy production A. niger 0 33.00 ± 0.88e 19.95 ± 0.75c 91.70 ± 3.58e 25.78 ± 0.95e 41.65 ± 1.70c 18.93 ± 0.80a 5.93 ± 0.25b <1 <1 2 54.78 ± 1.78b 17.55 ± 0.80d 97.00 ± 3.80d 31.08 ± 1.00b 67.93 ± 2.75a 17.93 ± 0.88b 6.68 ± 0.28a <1 <1 6 48.98 ± 1.88d 19.95 ± 0.78c 99.43 ± 3.20c 28.55 ± 0.88d 65.33 ± 2.88b 8.88 ± 0.43c 5.00 ± 0.25d <1 <1 9 59.58 ± 2.05a 24.00 ± 0.88a 103.03 ± 4.10b 34.13 ± 1.13a 68.25 ± 3.00a 9.48 ± 0.38c 5.45 ± 0.23c <1 <1 14 52.80 ± 1.75c 21.28 ± 0.95b 119.75 ± 3.90a 29.78 ± 1.00c 63.80 ± 3.13b 9.48 ± 0.38c 5.06 ± 0.23d <1 <1 R. oligosporus 0 33.00 ± 0.88d 19.95 ± 0.75d 91.70 ± 3.58e 25.78 ± 0.95e 41.65 ± 1.70c 18.93 ± 0.80a 5.93 ± 0.25b <1 <1 2 45.05 ± 1.70ab 19.20 ± 0.70e 94.88 ± 3.85d 26.43 ± 0.88d 64.58 ± 2.75b 7.95 ± 0.35b 7.03 ± 0.30a <1 <1 6 40.90 ± 1.38c 20.83 ± 0.63c 98.25 ± 3.70c 28.20 ± 0.75c 72.93 ± 3.38a 6.95 ± 0.28cd 5.65 ± 0.23c <1 <1 9 44.40 ± 1.50b 24.55 ± 0.88a 101.18 ± 3.75b 34.05 ± 0.88b 71.90 ± 3.33a 6.58 ± 0.25d 4.80 ± 0.25e <1 <1 14 45.70 ± 1.63a 23.78 ± 0.88b 108.05 ± 4.00a 36.28 ± 1.00a 73.68 ± 3.13a 7.30 ± 0.28c 5.08 ± 0.28d <1 <1

Values (mean ± SD, n = 3) in the same column followed by different superscript letters (a–e) indicate significant differences (p < 0.05) among unfermented and fermented samples (separately for each fungus) (ANOVA ‘‘Tukey’s Multiple Comparison Test”). FT, fermentation time; 3-CQA, 3-caffeoylquinic acid (neochlorogenic acid); 5-CQA, 5- caffeoylquinic acid (chlorogenic acid); Q-3-gluc, quercetin-3-glucoside (isoquercitrin); Q-3-gal, quercetin-3-galactoside; Q-3-rut, quercetin-3-rutinoside (rutin); Isorha-3-gal, isorhamnetin-3-galactoside; Isorha-3-gluc, isorhamnetin-3-glucoside; Cya-3-gluc, cyaniding-3-glucoside; Cya-3-rut, cyaniding-3-rutinoside.⁄Tentative identification. 32 F.V. Dulf et al. / Food Chemistry 209 (2016) 27–36 hydrolyzed by the above-mentioned enzyme to isoquercitrin, thus equivalent value for PP (Fig. 1C). This behaviour of WPBP could causing an increase in the concentration of this flavonol in the be explained by its elevated content of Maillard reaction products samples. In addition, to explain the visibly higher concentrations with high antioxidant activities which probably accumulated dur- of quercetin derivatives from the WPBP (a heat-treated by- ing the alcoholic distillation process. product), the report of Oliveira, Coelho, Alexandre, Almeida, and The correlation coefficients between TPs, total flavonoid con- Pintado (2015) should also be taken into account, as they observed tents and the main individual phenolic compounds of solid-state that thermal processing may induce a significant increase in the processed plum by-products, as well as their antioxidant capacity rutin content of pasteurized strawberry fruits. Moreover, it was evaluated by the DPPH, are shown in Supplementary Table 1.In shown that chlorogenic acid, probably due to its esterified and more heat-labile structure, can protect rutin from decomposition by heating (Murakami, Yamaguchi, Takamura, & Atoba, 2004). A 100 A. niger According to You, Ahn, and Ji (2010), isoquercitrin is a more R. oligosporus bioavailable and more potent antiproliferative biomolecule than ** *** )* UF) 9 quercetin or its parent diglycoside (rutin). (3)>( )*** ,( ***,(9 ** 80 >(6) )* The extracts from fermented PP samples presented relatively (3) (6 )> low anthocyanin levels, while the WPBP presented only trace (3 amounts of these sensitive pigments (Table 1). Anthocyanins are highly unstable molecules and are sensitive to technological processing, especially when heat treatment is involved. During these 60 processes, the balance between the various chemical forms of anthocyanins moves rapidly toward the colourless pseudo-base form, which is destroyed by oxidation mechanisms (Welch, Wu,

& Simon, 2008). yield Lipid 40 The increases in phenolic levels in the early stages of SSF can be attributed to the release of cell-wall bound phenolics by the action (g/100g of kernel (DW)) of glycosidases and lignin degrading enzymes produced by the fungi. On the other hand, the decreases of the phenolic amounts 20 in the ﬁnal stages of fermentation may be explained by the initia- tion of polymerization processes of the free phenolics (Ajila et al., 2012). 0 3.3. Antioxidant activity UF 3 6 9 3 6 9 Day of fermentation The changes in the antioxidant activity of methanol extracts of two plum by-products during the SSF were measured using the DPPH radical inhibition capacity (RIC), and the results are B (UF)>(A.n.)**,(R.o.)** TAGs 100 presented in Fig. 1C. After each fermentation process, statistically PLs signiﬁcant increases (p < 0.05) in RIC of the examined extracts MAGs were registered. DAGs The maximum level of reduction power towards the DPPH rad- 50 ical occurred on the sixth day of fermentation for PP and after FFAs

* about nine days of SSF for WPBP. * SEs * ) When PP was used as a fermentation substrate, the RIC in the . o .

6 R methanolic extracts increased by 35.40% (for SSF with R. oligos- ( , * * * * porus) and 27.70% (for SSF with A. niger) by day 6 compared to * * * ) ) * . . ) . o n the initial value (I% = 65) before gradually decreasing for the . . o . R A ( ( R , remaining period of growth. The changes in the radical inhibition ( < * ) * , * * * ) F * . * activities were lower for the WPBP, which increased to 12.90% ) * 4 U . ) n ( . . o . n A (for SSF with R. oligosporus) and 10.60% (for SSF with A. niger)on . ( R < ( A ) , ( the ninth day versus the initial value (I% = 85) and remained high * F * <

Lipid components (wt%) Lipid ) * U ) F until the end of growth. Studies have shown that the antioxidant ( . * n U ) . ( . activity of plum fruits is strongly connected with the presence of A n ( . 2 < A ) ﬂavonoids (Kim, Jeong, & Lee, 2003), anthocyanins (Cevallos- ( F < ) U ( Casals, Byrne, Okie, & Cisneros-Zevallos, 2006) and caffeoylquinic F

U acid isomers (Kayano, Kikuzaki, Fukutsuka, Mitani, & Nakatani, ( 2002). Moreover, Kristl et al. (2011) have suggested that the antioxidant activity of plums may be underestimated in the litera- 0 ture. They found that the extractable antioxidants contributed less . . . F F UF .n. UF .n. UF .n. UF .n. U .n. U .n. .o. than 18% to the total antioxidant activity of this stone fruit, while A R.o A R.o A R.o A R.o. A R.o. A R Samples the hydrolysable tannins and non-extractable proanthocyanidins contributed significantly more. The few available studies that Fig. 2. The time course of oil production by Aspergillus niger (A.n.) and Rhizopus relate to plum by-products have also shown that the antioxidant oligosporus (R.o.) strains in plum kernels during SSF (A), and neutral and polar lipid capacity of these fruit wastes depends on the cultivar and the components of oils (the 3rd day of fermentation) (B). Results are given as mean ± SD (n = 3); ⁄ p<0.05, ⁄⁄ p < 0.01, ⁄⁄⁄ p<0.001 (repeated measures ANOVA ‘‘Tukey’s method of processing (Milala et al., 2013; Sójka et al., 2015). In Multiple Comparison Test”). TAGs – triacylglycerols, PLs – polar lipids, MAGs – the current work it was observed that the antioxidant activity of monoacylglycerols, DAGs – diacylglycerols, FFAs – free fatty acids, SEs – sterol unfermented WPBP was notably higher compared to the esters; UF – unfermented. F.V. Dulf et al. / Food Chemistry 209 (2016) 27–36 33 general, there were no significant correlations between the above- The results from the present study are in accordance with those mentioned compounds and RIC values. Total flavonoids of Correia et al. (2004) and Vattem et al. (2004), who also observed (R2 = 0.8826, p = 0.0177 for PP fermented with A. niger and poor correlations between the TP content of different solid-state R2 = 0.7859, p = 0.0451 for PP fermented with R. oligosporus), fermented fruit by-products and their antioxidant capacity. This quercetin-3-rutinoside (R2 = 0.9400, p = 0.0064 for WPBP phenomenon could be explained by the degree of polymerization fermented with A. niger and R2 = 0.9456, p = 0.0055 for WPBP and the ratio between monomeric and polymeric forms of the phe- fermented with R. oligosporus) and 3-caffeoylquinic acid nolic compounds in certain stages of fungal growth. Moreover, it is (R2 = 0.8861, p = 0.0169 for PP fermented with A. niger) were the generally recognized that the free radical scavenging activity of the only solid-state processed plum by-product constituents that corre- phenolic compounds tends to increase when the polymerization lated with the antioxidant capacity (Supplementary Table 1). order increases (Di Majo et al., 2008). Although the phenolic compounds in fruits have been reported to possess a wide range of biological activities, in the literature 3.4. Changes in lipid composition during SSF of the plum kernels by A. conflicting ideas and data can be found on the relationship niger and R. oligosporus between antioxidant capacity and individual or total phenolic concentrations. The data on total lipid composition of plum kernels fermented Some researchers have shown that a good or very good correla- with A. niger and R. oligosporus are shown in Fig. 2. tion exists between the antioxidant capacity evaluated by different Both filamentous fungi increased total lipids content until the assays and TP contents in stone fruits (Gil, Tomás-Barberán, Hess- third day of fermentation; this was especially higher on A. niger Pierce, & Kader, 2002) and their pomaces (Milala et al., 2013; Sójka (p < 0.05) than R. oligosporus despite both being considered non- et al., 2015). However, there are a few studies in which the authors oleaginous fungi (Oliveira et al., 2011). After day 3 the total lipids report a smaller linear correlation between these two parameters sharply decreased for the remaining days of fungal growth. Not fer- (Di Majo, La Guardia, Giammanco, La Neve, & Giammanco, 2008). mented plum kernels showed a lipid content of 53 g/100 g of ker- Moreover, Gil et al. (2002) reported that in nectarines and peaches, nel (DW), which is close to the values already reported by other both hydroxycinnamic acid derivatives and flavan-3-ols are signif- authors (Matthaeus & Oezcan, 2009). The extracted fats increased icantly correlated with antioxidant potential evaluated by the by 21.90% from the initial value for the fermented kernels with DPPH and ferric reducing ability plasma (FRAP) methods, whereas A. niger whereas for R. oligosporus the evolution of these com- flavonols and anthocyanins, are not. In the case of plums, the cor- pounds was practically insignificant (1.90%). It is obvious from relation was mainly with flavan-3-ols, whereas hydroxycinnamic the current results that A. niger has a better lipogenic capacity than derivatives were not correlated. R. oligosporus when grown on plum kernels.

Table 2 Fatty acid compositions (molar% of total fatty acids) (determined by GC–MS) of total lipids and individual lipid classes produced by Aspergillus niger and Rhizopus oligosporus in solid-state fermentation (day 3) of plum kernels.

Fatty acids (molar% of total fatty acids) Samples 12:0 14:0 16:0 16:1n-9 16:1n-7 17:0 18:0 18:1n-9 18:1n-7 18:2n-6 18:3n-3 20:0 20:1n-9 22:0 22:1 n-9 TLs UF tr. 0.04a 8.52a 0.03a 1.06a 0.06a 2.27a 62.92a 1.78b 22.93a 0.11a 0.13a 0.07a 0.07 tr. A. n. tr. 0.02a 5.34c 0.03a 0.82b 0.04b 1.52b 66.35a 1.79b 23.77a 0.09b 0.07b 0.06ab 0.07 0.04 R. o. 0.01 0.04a 6.10b 0.02a 0.83b 0.04b 1.58b 66.21a 1.91a 23.04a 0.07c 0.08b 0.05b –– PLs UF – 0.08 17.77 0.29 0.34 0.33 5.48 51.11 1.83 21.44 0.29 0.63 – 0.39 – A. n. 0.23 0.93 16.42 0.40 0.52 0.42 7.33 49.13 2.35 18.59 0.70 0.79 0.35 0.49 1.33 R. o. 2.24 1.57 13.86 0.25 0.57 0.37 6.40 49.67 3.22 18.02 0.44 0.72 0.66 0.51 1.50 MAGs UF 0.06 0.12 8.25 0.19 0.24 0.22 3.37 62.98 2.12 21.96 0.25 – – – 0.25 A. n. 1.67 5.17 38.31 4.24 – 0.79 30.43 7.25 – 4.85 – – – – 7.29 R. o. 1.14 4.86 35.92 6.09 – 0.71 26.85 10.15 – 4.09 – 0.27 – – 9.92 DAGs UF 0.37 0.57 11.02 0.37 0.68 – 5.75 59.54 2.69 19.01 – – – – – A. n. 0.31 0.53 14.03 0.44 0.29 0.24 4.49 53.67 1.93 23.02 0.20 0.12 0.18 – 0.55 R. o. 0.18 0.38 12.80 0.44 0.31 0.17 4.05 56.43 2.06 22.38 0.14 0.14 – – 0.53 FFAs UF 0.32 0.35 9.12 0.34 0.73 0.20 3.23 60.04 1.09 22.82 0.48 0.56 0.12 0.35 0.24 A. n. 0.58 2.26 24.40 2.20 – – 17.02 40.00 – 11.53 – – – – 2.01 R. o. 1.34 1.94 21.54 1.24 – – 11.67 44.97 – 14.90 – – – – – TAGs UF 0.01 0.03 7.55 0.04 1.04 0.06 2.06 64.97 1.66 22.31 0.09 0.12 0.06 – – A. n. 0.01 0.05 5.68 0.03 0.82 0.04 1.57 66.58 1.69 23.26 0.06 0.08 0.05 0.07 – R. o. 0.01 0.02 6.25 0.03 0.88 0.04 1.58 65.96 1.83 23.21 0.06 0.07 0.05 – – SEs UF 2.11 4.79 19.86 – – – tr. 34.08 – 26.51 10.95 – – – 1.71 A. n. 2.42 5.10 35.12 4.00 1.53 0.95 26.54 6.80 – 6.71 – 1.15 – – 9.69 R. o. 2.19 5.31 37.11 2.93 1.60 0.88 27.44 10.76 0.98 3.38 0.72 0.67 – – 6.03

Molar%Values are the means of three measurements (n = 3). Different superscript letters (a, b) in the same column indicate signiﬁcant differences (p < 0.05) among TLs of the unfermented and fermented substrates (ANOVA ‘‘Tukey’s Multiple Comparison Test”). TLs – total lipids, PLs – polar lipids, MAGs – monoacylglycerols, DAGs-diacylglycerols, FFAs – free fatty acids, TAGs – triacylglycerols, SEs – sterol esters, UF-unfermented, A.n.–Aspergillus niger, R.o. – Rhizopus oligosporus, tr. – trace. C12:0, lauric; C14:0, myristic; C16:0, palmitic; C16:1n-9, cis-7 hexadecenoic; C16:1n-7, palmitoleic; C17:0, margaric; C18:0, stearic; C18:1n-9, oleic; C18:1n-7, vaccenic; C18:2n-6, linoleic; C18:3n-3, a- linolenic; C20:0, arachidic; C20:1n-9, 11-eicosenoic; C22:0, behenic; C22:1n-9, erucic acids. 34 F.V. Dulf et al. / Food Chemistry 209 (2016) 27–36

The filamentous fungi are known to produce different enzymes SSF of plum kernels with A. niger and R. oligosporus were also mon- (cellulase, pectinase, protease, etc.) when grown on different sub- itored. The results showed that the major lipid fraction from SSF strates. Recent studies have shown that this enzyme consortia has products was TAG, constituting about 92.50% (A. niger) – 93.00% an essential role in the degradation of the oilseed cell wall, leading (R. oligosporus) of the fermented plum kernel oils obtained in the to release of most of the oil (generally bound to proteins as lipo- early stages of fungal growth (the 3rd day of SSF – with maximum proteins or to the polysaccharides as lipo-polysaccharides) lipid yields) (Fig. 2B) and about 65–75% of the oils for the second enmeshed in cellular structures (Oberoi et al., 2012). Several studies part of fermentations (results not shown). conducted on hydrolytic enzyme production using different agro- There were statistically significant (p < 0.05) changes in the industrial residues have reported maximum enzyme activity after studied lipid classes during SSF with both fungi (Fig. 2B), which 3 or 4 days of SSF, depending on culture conditions (Oberoi et al., might be explained by the presence of lipase in the fermentation 2012). These findings are in accordance with our results regarding medium. The FFAs, DAGs and MAGs increased after all fermenta- the dynamics of the oil yields shown in Fig. 2A, with the maximum tions (especially after the 3rd day of SSF, results not shown), which lipid contents detected on day 3. According to Botella, de Ory, was probably caused by the hydrolysis of TAGs. The presence of Webb, Cantero, and Blandino (2005) during the first phase of elevated FFAs level indicates a decline in quality or damage to an growth, the fungal strains use the readily available carbon sources oil, a phenomenon that can be stopped by inactivation of the (free sugars) and produce cell wall-degrading enzymes, but when lipid-degrading enzymes (myrosinase and lipase) (Oberoi et al., the concentration of free reducing sugars is too low, the microor- 2012). Filamentous fungi are broadly recognized as the best lipase ganisms begin to use these enzymes to obtain other carbon sources, producers. Moreover, it was showed that the lipase synthesis is leading to a decline in enzyme activity and lipid yield. fungal growth-associated (the enzyme production starting in the The available literature on fungal bioprocessing shows increases first stage of fungal growth) and its activity is positively influenced (slight or notable) or statistically significant reductions in total lipid by the presence of fats in the medium (Falony et al., 2006). content detected on different fermented substrates with Rhisopus Both fungal strains significantly improved (p < 0.05) the PLs fungi (Oduguwa, Edema, & Ayeni, 2008). On the other hand, Abu, (3-fold for A. niger and 4-fold for R. oligosporus) and SEs (8-fold Tewe, Losel, and Onifade (2000) investigated sweet potato SSF with for A. niger and 6-fold for R. oligosporus) contents during SSF pro- two Aspergillus species and observed a considerable increase in the cesses (Fig. 2B). These positive evolutions can be explained by lipid concentration during the fermentation period. A similar trend the ability of the filamentous fungi to secrete sterol esterases, was observed by Feng et al. (2014) during koji fermentation with enzymes able to mediate production of SEs from sterols and fatty Aspergillus oryzae and also in our previous study on SSF of berry acids available in the fermentation medium (Dulf et al., 2015). pomaces by A. niger (Dulf et al., 2015). Moreover, Oliveira et al. (2011) pointed out that microorganisms Changes in neutral lipid (triacylglycerols (TAGs), monoacylglyc- with lipogenic capacity could use excess lipids to produce erols (MAGs), diacylglycerols (DAGs), free fatty acids (FFAs), and membrane polar lipids, with important emulsifying properties that sterol esters (SEs)) and polar lipid (PL) compositions of oils during are useful in the food industry. It is important to note that the

Table 3 The composition (molar% of total fatty acids) of fatty acid classes in total lipids and major lipid fractions produced by Aspergillus niger and Rhizopus oligosporus in solid-state fermentation (day 3) of plum kernels.

Fatty acid classes (molar% of total fatty acids) Samples RSFAs RMUFAs RPUFAs Rn-3 PUFAs Rn-6 PUFAs Rn-9 UFAs n-6/n-3 PUFAs/SFAs TLs UF 11.09 ± 0.45a 65.86 ± 2.50a 23.04 ± 1.15a 0.11 ± 0.02a 22.93 ± 0.95a 63.02 ± 2.00a 208.45c 2.08c A. n. 7.06 ± 0.30b 69.09 ± 2.70a 23.86 ± 1.10a 0.09 ± 0.01b 23.77 ± 1.10a 66.48 ± 2.80a 264.11b 3.38a R. o. 7.86 ± 0.34b 69.03 ± 2.55a 23.12 ± 1.10a 0.07 ± 0.01c 23.04 ± 1.05a 66.29 ± 2.50a 329.14a 2.94b PLs UF 24.69 ± 1.10c 53.58 ± 2.50a 21.74 ± 0.95a 0.29 ± 0.02c 21.44 ± 0.90a 51.41 ± 2.25bc 73.93a 0.88a A. n. 26.62 ± 1.20a 54.09 ± 2.35a 19.29 ± 1.00ab 0.70 ± 0.03a 18.59 ± 0.95ab 51.22 ± 2.30c 26.55c 0.72b R. o. 25.68 ± 1.15b 55.87 ± 2.40a 18.45 ± 0.80b 0.44 ± 0.02b 18.02 ± 0.90b 52.08 ± 2.30a 40.95b 0.72b MAGs UF 12.01 ± 0.50b 65.77 ± 2.80a 22.21 ± 0.95a 0.25 ± 0.01 21.96 ± 0.85a 63.42 ± 2.80a 87.84 1.85a A. n. 76.37 ± 3.10a 18.79 ± 0.85c 4.85 ± 0.18b – 4.85 ± 0.18b 18.79 ± 0.85c – 0.06b R. o. 69.75 ± 2.90a 26.17 ± 1.10b 4.09 ± 0.20b – 4.09 ± 0.20b 26.17 ± 1.10b – 0.06b DAGs UF 17.71 ± 0.80a 63.28 ± 2.85a 19.01 ± 0.85b – 19.01 ± 0.85b 59.91 ± 2.65a – 1.07b A. n. 19.72 ± 0.95a 57.06 ± 2.40c 23.22 ± 1.00a 0.20 ± 0.02 23.02 ± 1.00a 54.85 ± 2.30a 115.10 1.18ab R. o. 17.72 ± 0.80a 59.77 ± 2.70b 22.52 ± 0.95a 0.14 ± 0.01 22.38 ± 0.95a 57.40 ± 2.45a 159.86 1.27a FFAs UF 14.14 ± 0.60c 62.56 ± 2.75a 23.30 ± 1.05a 0.48 ± 0.02 22.82 ± 1.10a 60.74 ± 2.80a 47.54 1.65a A. n. 44.26 ± 2.00a 44.21 ± 1.95c 11.53 ± 0.40c – 11.53 ± 0.40c 44.21 ± 1.95b – 0.26c R. o. 36.48 ± 1.50b 48.62 ± 2.15b 14.90 ± 0.50b – 14.90 ± 0.50b 48.62 ± 2.15b – 0.41b TAGs UF 9.83 ± 0.35a 67.77 ± 2.85a 22.39 ± 1.00a 0.09 ± 0.01a 22.31 ± 1.00a 65.07 ± 3.00a 247.89b 2.28c A. n. 7.51 ± 0.25c 69.17 ± 2.90a 23.32 ± 1.05a 0.06 ± 0.02a 23.26 ± 1.08a 66.65 ± 2.90a 387.66a 3.11a R. o. 7.97 ± 0.30b 68.75 ± 2.80a 23.28 ± 1.05a 0.06 ± 0.02a 23.21 ± 1.05a 66.04 ± 2.95a 386.83a 2.92b SEs UF 26.75 ± 1.15b 35.79 ± 1.55a 37.45 ± 1.60a 10.95 ± 0.40 26.51 ± 1.15a 35.79 ± 1.55a 2.42 1.40a A. n. 71.28 ± 3.10a 22.01 ± 0.90b 6.71 ± 0.25b – 6.71 ± 0.25b 20.48 ± 0.90b – 0.09b R. o. 73.61 ± 3.15a 22.29 ± 0.90b 4.10 ± 0.15c 0.72 ± 0.02 3.38 ± 0.10c 19.71 ± 0.85b 4.69 0.06b

Values (mean ± SD, n = 3) in the same column followed by different superscript letters (a–c) indicate significant differences (p < 0.05) among unfermented and fermented samples (separately for each lipid class) (ANOVA ‘‘Tukey’s Multiple Comparison Test”). UF – unfermented, A.n.–Aspergillus niger, R.o. – Rhizopus oligosporus. SFAs – saturated fatty acids, MUFAs – monounsaturated fatty acids, PUFAs – polyunsaturated fatty acids. F.V. Dulf et al. / Food Chemistry 209 (2016) 27–36 35 esterified form of phytosterols are frequently used in food formu- antioxidant potential determined by DPPH assay also increased lation due to their better fat-solubility. Furthermore, the plant ster- significantly over the course of growth. However, generally, in both ols (free and esterified) help lower the blood low-density bio-processed by-products, no significant correlations were lipoprotein (LDL) cholesterol concentration (Dulf, Unguresan, observed between phenolic compounds and antioxidant activities. Vodnar, & Socaciu, 2010). This study also demonstrated that SSF not only helped in higher oil recovery from plum kernels, but also resulted in lipids with better quality attributes (high SEs and n-3 PUFA-rich PLs contents). In 3.5. Changes in fatty acid compositions of TLs and their lipid classes the early stages of fungal growth, the extracted oils (with TAGs as during SSF of the plum kernels major lipid fraction) increased by 21.90% for the SSF with A. niger whereas for R. oligosporus the evolution was practically insignifi- Fatty acid compositions of the TLs, TAGs, PLs and minor neutral cant. The high oil content of plum kernels, comparable to rapeseed lipid (NL) fractions (MAGs, DAGs, FFAs and SEs) in the unprocessed or sunflower seeds, makes them highly suitable for commercial oil and solid-state fermented (day 3) plum kernels are shown in production. Tables 2 and 3. The utilization of plum by-products (including kernels) resulting The dominant fatty acids in the TLs were oleic (C18:1n-9) and from processing as waste could provide an extra source of income linoleic (C18:2n-6) acids, together comprising approximately 86% and at the same time help to reduce the solid waste disposal (in unfermented kernels) and 90% (in fermented kernels) of the problem. total fatty acids. The evolutions of these compounds during the SSF agree with previously reported results, which showed that strains of filamen- Conflict of interest tous fungi are able to synthesize fats with important levels of unsaturated fatty acids (Hui et al., 2010). The fermentation pro- The authors have no conflicts of interest to declare. cesses have caused slight but statistically significant (p < 0.05) decreases of the palmitic (C16:0) and stearic (C18:0) acids Acknowledgements (Table 2). The fatty acid compositions in TAGs were similar to those of TLs and showed a constant order during the different stages of This work was supported by a grant of the Romanian National SSF (mono-, MUFAs > polyunsaturated, PUFAs > saturated, SFAs Authority for Scientific Research and Innovation, CNCSÀUEFISCDI, fatty acids) (Table 3). Moreover, the studied oils are characterized project number PN-II-RU-TE-2014-4-1255. by low levels of PUFAs/SFAs and extremely high levels of n-6/ n-3-PUFAs ratios, because of the dominance (>63%) of n-9 PUFAs Appendix A. Supplementary data acids (especially oleic acid, C18:1) in their compositions. The elevated levels of MUFAs from the analyzed TLs are comparable Supplementary data associated with this article can be found, in to those of MUFA-rich (>60%) vegetable oils (rapeseed, apricot the online version, at http://dx.doi.org/10.1016/j.foodchem.2016. kernel, avocado, olive, etc.) (Dubois, Breton, Linder, Fanni, & 04.016. Parmentier, 2007). 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Predominant and Secondary Pollen Botanical Origins Inﬂuence the Carotenoid and Fatty Acid Proﬁle in Fresh Honeybee-Collected Pollen † † † ‡ ‡ Rodica Margă oan,̆ Liviu Al. Marghitas̆ ,̧Daniel S. Dezmirean, Francisc V. Dulf,*, Andrea Bunea, # † Sonia Ancuta̧ Socaci, and Otilia Bobiş † ‡ # Department of Technological Sciences, Animal Breeding, Department of Biochemistry, and Faculty of Food Science, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Manăs̆tuŗ 3-5, 400372 Cluj-Napoca, Romania

*S Supporting Information

ABSTRACT: Total and individual carotenoids, fatty acid composition of total lipids, and main lipid classes of 16 fresh bee- collected pollen samples from Romania were determined by high-performance liquid chromatography with photodiode array detection and capillary gas chromatography with mass detection. Analyzed samples were found rich in lutein, whereas β-criptoxanthin and β-carotene were present in a wide range of amounts correlated with predominant botanical origin of the samples. High amounts of lutein were correlated with the presence of Callendula oﬃcinalis, Taraxacum oﬃcinale and Anthylis sp. The highest amount of total lipids was found in samples where pollen from Brassica sp. was predominant. Lipid classes were dominated by polyunsaturated fatty acids. Saturated fatty acids were determined in variable amounts. Lipid and carotenoid contents present great variability, explained by the various botanical species present in the samples. KEYWORDS: bee-collected pollen, carotenoids, fatty acids, HPLC, GC, botanical origin

■ INTRODUCTION Lipids vary greatly in concentration in different plant species 18 As honey represents the energy source for bees, pollen is the pollen. Roulston and Cane show that ether-extractable material main source of other important nutrients such as proteins, lipids, from dry pollen of 62 plant species shows ranges of 0.8% in − minerals, and vitamins.1 3 The composition of bee pollen is Eucalyptus marginata (forrest eucalypt from Western Australia) fl to 18.9% for dandelion (Taraxacum officinale). Bee-collected variable and depends on the oral origin, being also related to ff seasonal environmental factors and processing methodologies.4 pollen originating from di erent botanical species will have a Honeybees obtain all of the proteins, lipids, minerals, and vita- lesser amount of lipids, due to the fact that plant pollen is mixed mins needed for their own consumption, brood rearing, and all with bee substances to form the pollen grain. The total lipid content of bee-collected pollen is generally physiological processes from pollen. The quantity and most of all 19 quality of collected pollen affects longevity, reproduction, and <10% dry weight, depending on the type of pollen. Fresh plant fi 5 pollen contains a higher level of fat than bee-collected or stored nally the productivity of the bee colony. 2 Bee pollen contains carbohydrates, lipids, proteins, vitamins pollen. The existing knowledge shows that the composition − A, B complex, C, D, and E, and polyphenols.3,6 8 For this equili- of pollen lipids varies greatly according to their botanical and 18,20,21 brate chemical composition, bee pollen is promoted as a healthy geographical origins. The fatty acids (especially essential food supplement, having a wide range of nutritional and ther- fatty acids) and other lipid classes play an important role in the − 21,22 apeutic properties.9 12 Depending on the processing steps and development, nutrition, and reproduction of bees. The total fl storage conditions, the content of different nutrients and vita- lipid fraction of honeybee-collected pollen (mono oral and mins in bee pollen can be different.13,14 multifloral) contains high levels of long-chain fatty acids (10−22 Carotenoids, a class of isoprenoids, are responsible for the carbon atoms in a molecule), the most abundant being linoleic 21 yellow, yellow-orange, red-orange, and red colors in fruits, (18:2n-6), α-linolenic (18:3n-3),and palmitic (16:0) acids. flowers, and other storage organs. Carotenoids have important Depending on the botanical origin, the fat composition in bee roles in human health; β-carotene, for example, constitutes the pollens is very different. With some exceptions (sunflower bee principal source of vitamin A, and dietary intake could lower the pollen), they contain higher amounts of unsaturated fatty 2,21 risk of different types of cancer or cardiovascular disease.15 acids. The composition of Romanian bee pollen has been shown to The color of pollen loads is determined by the presence of − pigments such as flavonoids and/or carotenoids.16 vary in terms of macronutrients and minerals.3,23 25 Lipids are important to honeybees first as a source of energy with some components of lipids involved in the synthesis of Received: December 11, 2013 17 reserve fat and glycogen and the membrane structure of cells. Revised: June 11, 2014 Fatty acids and sterols are also important in honeybee devel- Accepted: June 18, 2014 opment, nutrition, and reproduction. Published: June 18, 2014

Few studies on carotenoid content of bee pollen are published of extract was saponified with 30% methanolic KOH, at room tem- worldwide,1,13,16,26,27 and, to the best of our knowledge, no perature, in the dark, for 24 h. For the removal of soaps and alkalis, the studies related to carotenoid and also fatty acid composition of solution was washed three to four times with a sodium chloride saturated multifloral bee-collected pollen from Romanian (Transylvania) solution and distilled water. The organic layer containing carotenoids was dried over anhydrous sodium sulfate and evaporated to dryness.32 have been done, although pollen is often consumed as a food Samples were kept under nitrogen at −20 °C until further utilization. supplement. With respect to adopting the European Regulations ff Total carotenoids content was estimated spectrophotometrically regarding bee product labeling, the present work aims to o er from saponified samples, at 450 nm, using a Shimadzu UV-2102 PC new important information about the lipid classes present in scanning spectrophotometer. pollen samples and also about total and individual carotenoid com- HPLC Analysis. HPLC-DAD of all samples was performed on a position of multifloral pollen loads collected by bees in different system with a Waters 990 PDA detector, Kontron pumps and controller, regions from Transylvania (Romania). For the first time, a and a reversed phase C 18 column Zorbax ODS (250 × 4.6 mm), 5 μm. classification of pollen samples was achieved by subjecting the data The mobile phase consisted of mixtures of acetonitrile/water (9:1, v/v) obtained in chemical analysis to principal component analysis with 0.25% triethylamine (solvent A) and ethyl acetate with 0.25% (PCA) with cross-validation (full model size and center data). triethylamine (solvent B). The gradient started from 20% B to 50% B at 3 min. The percent of B increased to 75% at 12 min and continued isocratically up to 25 min. The flow rate was 1 mL/min. The chro- ■ MATERIALS AND METHODS matograms were monitored at 450 nm. fi Pollen Samples. Sixteen bee pollen samples (0.5 kg each) were The carotenoids identi cation was made by comparing the retention collected between May and August 2010 from three different counties in times of sample compounds with available standards and according to UV−vis spectra recorded by PDA detector. the Transylvania region (Romania) (Supporting Information Table S1). β β The areas were characterized by rich spontaneous and also cultivated Calibration curves for lutein, -cryptoxanthin, and -carotene were plants. Bee pollen was obtained with a bottom fitting pollen trap placed used for quantitative analysis. The results were expressed as micrograms in the front of the hives, daily harvested, cleaned of impurities, and per gram of pollen (fresh and dry weight). stored immediately at −18 °C until analysis. Fatty Acid Analysis. (a) Lipids Extraction. The total lipids (TLs) of the honeybee-collected pollen samples were extracted using a chloroform/ Physicochemical analyses of pollen samples were made according to 33,34 literature studies1 and existing pollen regulations, and results were pub- methanol mixture. The sample (5 g pollen) was homogenized in lished previously.24 methanol (20 mL) for 1 min with a high-power homogenizer (MICCRA Palynological Origin. The bases of palynological analysis were D-9, Germany), then chloroform (40 mL) was added, and homoge- fi the methods described by Almeida-Muradian et al.,1 Morais et al.,6 and nization was continued for 2 min. The mixture was ltered, and the solid Louvreaux et al.28 adapted for pollen load. residue was resuspended in chloroform/methanol mixture (2:1, v/v, A sample of 2 g, corresponding to approximately 200 pollen pellets, 60 mL) and homogenized again for 3 min. The mixture was again fi was considered to be representative for botanical origin determination. ltered, and the residue was washed with 60 mL chloroform/methanol fi The pellets were grouped into subsamples according to their color, and (2:1, v/v). The ltrates and washings were combined and cleaned with each subsample was weighed to calculate the percentage from the main an 0.88% potassium chloride water solution and a methanol/water fi fi bee-collected pollen samples.1,6 From each subsample, one microscopic solution (1:1, v/v). The puri ed lipid layer (bottom) was ltered and slide was prepared without acetolysis, by dissolving and washing the dried over anhydrous sodium sulfate, and the solvent was removed in a ‰ − rotary evaporator. The recovered total lipid fractions were transferred to pollen in diluted H2SO4 (5 ) and colored using a mixture of glycerin gelatin−fuxine for permanent preparation.28 vials with 4 mL of chloroform, forming the stock solution, and stored at − ° Slide examination was performed using a Nikon Eclipse 50i optic 18 C for further analysis. microscope at 1000× magnification (for identification) and 400× (b) GC Analysis of FAMEs. The fatty acid methyl esters (FAMEs) fi were prepared from TLs using the acid-catalyzed transesterification magni cation for counting. Five hundred pollen grains were counted 35 from every slide, and percentages of different botanical species were cal- procedure described by Christie. The FAMEs were determined by gas − 34 culated. For the identification of pollen type, reference pollen collection chromatography mass spectrometry (GC-MS). APerkinElmer slides from the Beekeeping Department, University of Agricultural Clarus 600 T GC-MS (PerkinElmer, Inc., Shelton, CT, USA) equipped Sciences and Veterinary Medicine from Cluj Napoca, were used, as well with a Supelcowax 10 (60 m × 0.25 mm i.d., 0.25 μm film thickness; as different pollen guides.29,30 Supelco Inc., Bellefonte, PA, USA) capillary column was used. The Chemicals and Reagents. All reagents (used for the lipid extrac- injector temperature was set at 210 °C. The temperature program for tion and fatty acid methyl esters (FAMEs) preparation) were of chro- the column was as follows: initial temperature, 140 °C; increase by 7 °C/ matographic grade (Sigma-Aldrich, St. Louis, MO, USA). The FAMEs min to 220 °C; and hold for 23 min. The flow rate of the carrier gas standard (37 component FAME Mix) were purchased from Supelco (helium) was 0.8 mL/min, and the split value was a ratio of 1:24. The (Bellefonte, PA, USA). injected volume was 0.5 μL. The positive ion electron impact (EI) mass Methanol, ethyl acetate, petroleum ether, diethyl ether, sodium spectra was recorded at an ionization energy of 70 eV and a trap current chloride, triethylamine, and anhydrous sodium sulfate were purchased of 100 μA with a source temperature of 150 °C. The mass scans were from Sigma Chemical Co. The purity of carotenoid standards (98% for performed within the range of m/z 22−395 at a rate of 0.14 scan/s with an β-cryptoxanthin, 96% for β-carotene, and 97.5% for lutein) was intermediate time of 0.02 s between the scans. Identification of FAMEs estimated by UV−vis spectra and by an individual HPLC analysis. was achieved by comparing their retention times with those of known Solvents used for carotenoid analysis (ethyl acetate, and acetonitrile) standards (37 component FAME Mix, Supelco no. 47885-U) and the were purchased from Merck. resulting mass spectra to those in our database (NIST MS Search 2.0). Analytical Methods. Carotenoids Identification. (a) Carotenoids The amount of fatty acids was expressed as percent of total fatty acids. Extraction. Total carotenoids were extracted from 5 g of fresh bee Statistical Analysis. Three different replicates of each honeybee- pollen with a mixture of methanol/ethyl acetate/petroleum ether (1:1:1, collected pollen were assayed. The analytical results reported for the v/v/v) containing BHT as antioxidant, following the procedure de- fatty acid compositions are the average of triplicate measurements of scribed by Breithaupt and Schwack.31 Extraction was carried out under three independent total lipid fractions (n =3× 3). Results reported for agitation and diminished light and was repeated until the bee pollen carotenoid determination represent the mean of three independent became colorless. The combined extracts were washed in a separation analyses. Statistical differences among samples were estimated using funnel with sodium chloride and diethyl ether, and the upper collected ANOVA (one-way analysis of variance; Tukey’s multiple-comparison phase was evaporated to drynes. test; GraphPad Prism version 4.0, Graph Pad Software Inc., San Diego, (b) Saponification. Every sample of obtained oleoresin was dissolved CA, USA). A probability value of p < 0.05 was considered to be in 20 mL of diethyl ether and divided for further analysis. Ten milliliters statistically significant.

6307 dx.doi.org/10.1021/jf5020318 | J. Agric. Food Chem. 2014, 62, 6306−6316 Journal of Agricultural and Food Chemistry Article Table 1. Plant Species Giving the Predominant, Secondary, and Minor Pollen in the Analyzed Samples

predominant pollen (>45%) secondary pollen (16−45%) important minor pollen (3−15%) minor pollen (<3%) sample family species family species family species family species P1 Rosaceae Crataegus Fagaceae Quercus sp. Brassicaceae Brassica sp. Asteraceae Taraxacum monogyna officinale Fabaceae Onobrychis viciifolia

P2 Rosaceae Filipendula Hypericaceae Hypericum sp. Lamiaceae Thymus sp. Fabaceae Trifolium pratense ulmaria Asteraceae Artemisia sp. Ericaceae Calluna vulgaris Onagraceae Epilobium sp.

P3 Rosaceae Filipendula Ericaceae Calluna sp. Asteraceae Artemisia sp. Onagraceae Epilobium sp. ulmaria Hypericaceae Hypericum sp. Asteraceae Taraxacum officinale Geraniaceae Geranium sp.

P4 Rosaceae Malus domestica Brassicaceae Brassica sp. Asteraceae Taraxacum officinale

P5 Fabaceae Trifolium repens Hypericaceae Hypericum sp. Fabaceae Anthillis sp. Asteraceae Taraxacum officinale Medicago sativa Asteraceae Artemisia sp. Fabaceae Onobrychis viciifolia Onagraceae Epilobium sp.

P6 Rosaceae Prunus sp. Brassicaceae Brassica sp. Asteraceae Taraxacum officinale

P7 Asteraceae Calendula Tiliaceae Tilia sp. Brassicaceae Brassica sp. Asteraceae Taraxacum officinalis officinale Fabaceae Trifolium pratense Fabaceae Vicia sativa Asteraceae Artemisia sp. Matricaria chamomilla Rosaceae Filipendula ulmaria

P8 Asteraceae Taraxacum Rosaceae Prunus sp. Asteraceae Cichorium sp. Fabaceae Anthillis sp. officinale Brassicaceae Brasica sp. Rosaceae Malus domestica

P9 Rosaceae Filipendula Hypericaceae Hypericum sp. Lamiaceae Thymus sp. Fabaceae Medicago sativa ulmaria Asteraceae Artemisia sp. Onobrychis viciifolia Onagraceae Epilobium sp. Rosaceae Rubus sp. Asteraceae Taraxacum officinale Ericaceae Calluna sp.

P10 Fabaceae Anthilis sp. Fabaceae Medicago sativa Asteraceae Calendula Fabaceae Onobrychis viciifolia officinalis Asteraceae Taraxacum Rosaceae Carduus sp. Lamiaceae Thymus sp. officinale Rubus sp. Geraniaceae Geranium sp. Filipendula ulmaria Salicaceae Salix sp.

P11 Rosaceae Rosa canina Hypericaceae Hypericum sp. Asteraceae Taraxacum Fabaceae Onobrychis viciifolia officinale Scrophulariaceae Verbascumsp. Matricaria Amorpha fruticosa chamomilla Rosaceae Filipendula ulmaria

P12 Fabaceae Anthillis sp. Rosaceae Rubus sp. Asteraceae Taraxacum officinale

6308 dx.doi.org/10.1021/jf5020318 | J. Agric. Food Chem. 2014, 62, 6306−6316 Journal of Agricultural and Food Chemistry Article Table 1. continued

predominant pollen (>45%) secondary pollen (16−45%) important minor pollen (3−15%) minor pollen (<3%) sample family species family species family species family species Medicago sp. Ericaceae Vaccinum sp. Asteraceae Carduus sp. Fabaceae Trifolium sp. Helianthus annuus Lamiaceae Thymus sp. Fabaceae Vicia sativa

P13 Brasicaceae Brassica sp. Rosaceae Rubus sp. Rosaceae Prunus sp. Fagaceae Quercus sp. Fabaceae Anthyllis sp. Fabaceae Medicago sativa

P14 Ericaceae Calluna vulgaris Rosaceae Filipendula Geraniaceae Geranium sp. Asteraceae Taraxacum ulmaria officinale Asteraceae Matricaria chamomilla Artemisia sp. Helianthus annuus

P15 Salicaceae Salix sp. Betulaceae Carpinus betulus Rosaceae Prunus sp. Asteraceae Taraxacum officinale

P16 Fabaceae Robinia Asteraceae Taraxacum Fabaceae Onobrychis viciifolia pseudoacacia officinale Anthyllis sp. Ericaceae Vaccinum sp. Fabaceae Trifolium pratense Vicia faba Salicaceae Salix sp.

Table 2. Individual Carotenoids Concentration in Bee Pollen Samples (HPLC Determination) and Total Carotenoid Content a (Spectrophotometric Determination), Expressed as Micrograms per Gram Sample

lutein β-cryptoxanthin β-carotene total carotenoid content sample fresh wt dry wt fresh wt dry wt fresh wt dry wt fresh wt dry wt P1 50.89 k 64.17 j,k 1.04 j 1.31 m 0.17 h 0.21 i 58.30 h,i 73.52 j,k P2 239.37 d 329.67 d 13.29 c 18.30 d 13.20 a 18.18 a 295.40 c 406.84 b P3 51.26 k 65.80 j,k 1.78 i,j 2.28 l,m tr tr 59.30 h,i 76.12 j,k P4 178.39 e,f 229.62 f 5.51 f 7.09 i tr tr 183.90 e,f 236.71 f P5 151.31 g 235.88 e,f 6.88 e 10.73 g 7.20 c 11.22 c 177.90 f 277.33 e P6 44.52 k 57.04 k 2.49 h,i 3.19 k,l 2.89 f 3.70 f,g 49.90 i 63.94 k P7 255.04 c,d 346.90 c,d 14.55 c 19.79 c,d nd nd 269.60 d 366.71 c P8 392.52 a 476.30 a 13.31 c 16.15 e 0.59 h 0.72 h,i 425.32 a 516.08 a P9 157.5 f,g 208.25 f 8.63 d 11.41 f,g 10.57 b 13.98 b 207.31 e 274.10 e P10 317.11 b 433.84 b 21.04 b 28.78 b 12.56 a 17.18 a 385.44 b 527.27 a P11 52.89 k 72.47 j,k 3.37 g,h 4.62 j 0.18 h 0.25 i 65.31 h,i 89.47 j,k P12 153.96 g 218.53 f 24.96 a 35.43 a tr tr 198.82 e,f 282.17 d,e P13 49.06 k 66.53 j,k 1.02 j 1.38 m tr tr 54.80 h,i 74.31 j,k P14 94.92 i 130.28 g,h 5.22 f 7.14 h,i 5.08 d 6.97 d 113.05 g 155.13 g,h P15 65.90 j,k 85.33 i,j 2.13 h,i,j 2.76 l,m 2.09 g 2.71 g 77.62 h 100.48 i,j P16 95.63 h,i 116.29 h 4.59 f,g 5.58 i,j 4.17 e 5.07 e 104.39 g 126.94 h,i aDiﬀerent letters in each column denote statistical diﬀerence at p < 0.05. The values represent the mean of three replicates, separately analyzed. nd, not detected; tr, traces.

The classification of pollen samples was achieved by subjecting the percentages as constituents. Six plant families were found in data obtained for palynological analysis, fatty acids, total lipids content, the 16 pollen samples as predominant pollen constituents (>45% β β lutein, -cryptoxanthin, -carotene, and total carotenoids to PCA with from the total content): Rosaceae (Crataegus monogyna, cross-validation (full model size and center data). All of the statistical analyses were performed using Unscrambler X software version 10.1 Filipendula ulmaria, Malus domestica, Prunus sp.,Rosa canina); (CAMO Software AS, Oslo, Norway). Fabaceae (Trifolium repens, Anthilis sp., Robinia pseudoacacia); Asteraceae (Calendula officinalis, Taraxacum officinale); Brassi- ■ RESULTS AND DISCUSSION caceae (Brassica sp.); Ericaceae (Calluna vulgaris); and Salicaceae Palynological Identification. Table 1 presents the (Salix sp.); another 10 botanical species were found to be identified plant families and species giving the predominant, secondary pollens for bee pollen samples (Table 1). secondary, and minor pollen in the analyzed samples. The Rosaceae family was dominant (>45%) in sevem pollen According to palynological analysis, all pollen samples were samples and was present as secondary pollen (16−45%) in found to be heterofloral, having different pollen types and another three samples, whereas the Fabaceae family was the

6309 dx.doi.org/10.1021/jf5020318 | J. Agric. Food Chem. 2014, 62, 6306−6316 Journal of Agricultural and Food Chemistry Article predominant pollen for three samples and the secondary pollen for another three samples. The Asteraceae family with Taraxacum officinale, Calendula officinalis and Carduus sp. was present in three different samples. Pollen botanical origin from the pollen pellets may vary according to the region of collection, vegetation available for bees at the collecting moment, and climatic conditions.36 Carotenoid Determination. The total carotenoid content in saponified honeybee-collected pollen samples was determined using UV−vis spectrophotometry. Quantitative analysis showed a wide range of concentrations for total carotenoid content: 49.90−425.32 μg/g in freshly collected bee pollen and 63.94−527.27 μg/g when reported to each sample’s dry weight (Table 2). The highest carotenoid concentration was found in sample P8, having Taraxacum officinale as predominant pollen, and the lowest in sample P6, Prunus spinosa predominant pollen. In Filipendula ulmaria bee pollen (samples P2, P3, and P9) total carotenoid content was fi fi quantified as 295.40, 59.30, and 207.31 μg/g fresh weight, Figure 1. Chromatogram of identi ed carotenoids from saponi ed extract of bee pollen P2. respectively. The results obtained for samples P2 and P9 are significantly different and also significantly different from P3 (lower catorenoid content), this fact being attributed to the secondary pollen found in this sample. In P2 and P9 Hypericum sp. is the secondary pollen (high content of total carotenoids quantified) and Calluna sp. the secondary pollen in P3, where low amounts of carotenoids were found. Taraxacum officinale is reported to be the richest natural vegetable source of β-carotene,37 above carrots, broccoli, and spinach.38 Filipendula ulmaria is also a rich source of β-carotene (36.32 μg/g plant).39 These data are additional proof of the results obtained in our study. No statistical differences on total carotenoid content were found between samples P1, P3, P11, and P13, reported as either fresh or dry weight. HPLC-PDA analysis of carotenoids from bee pollen allows the separation, identification, and quantification of individual carotenoids present in the samples. Obtained results are presented in Table 2. Lutein and β-cryptoxanthin were identified in all analyzed Figure 2. Total lipid content of honeybee-collected pollen samples from samples. ± Lutein was the major carotenoid found in pollen samples. The Romania (Transylvania). Results are given as the mean SD (n = 3). μ Values with different letters (a−g) are significantly different (p < 0.05), concentration varied between 44.52 and 392.52 g/g fresh ’ − μ using ANOVA Tukey s multiple-comparison test. P1 P16, samples of weight (57.04 and 476.30 g/g dry weight). The highest concen- multifloral pollen. tration was found in sample P8 (392.52 μg/g), followed by sample P10 (317.11 μg/g). The lowest lutein concentration was results regarding carotenoids are also reported on fresh and dry found in sample P6 (44.52 μg/g). Lutein concentration showed weight bases. no significant statistical difference for samples P1, P3, P6, P11, Almeida-Muradian et al.,1 analyzing 10 Brazilian bee pollen P13, and P15. samples, found a wide range of total carotenoid content: from The concentration of β-cryptoxanthin ranged between 1.02 “traces” (in 2 of the samples) to 451.50 μg/g fresh weight and 24.96 μg/g fresh weight; the highest concentration (found in (489.20 μg/g dry weight). Although pollen samples were sample P12) is significantly different from that of sample P10, in designated by the producers as monofloral pellets, botanical which the concentration was 21.04 μg/g. The lowest concen- origin analysis showed the presence of 17 plant taxa in the 10 tration of β-cryptoxanthin was found in samples P1 and P13 with samples. The authors concluded that botanical origin is directly no statistical differences between the mentioned samples (1.04 connected with carotenoid content; the highest value was and 1.02 μg/g fresh weight). obtained in pellets with high contents of Senecio and Vernonia With regard to β-carotene content, in some of the samples only pollen grains. The lack of β-carotene in all samples was attributed traces were detected, whereas the highest contents were found in to the drying process of the samples. samples P2 and P10 (13.20 and 12.56 μg/g fresh weight and Pereira de Melo and Almeida-Muradian,27 analyzing fresh and 18.18 and 17.18 μg/g dry weight). No statistical differences dehydrated pollen samples from Brazil, found total carotenoid between these two pollen samples was observed. contents in fresh bee pollen ranging from 27.08 to 344.6 μg/g. Most of the papers published on carotenoid determination β-Carotene content, quantified by HPLC by the same authors, from bee pollen (either total carotenoids or individual) report varied from 3.77 to 99.27 μg/g fresh bee pollen samples. With their results as micrograms per gram of fresh or dehydrated regard to total carotenoid content, the results obtained from sample. To have a better comparison with literature data, our Romanian samples are in agreement with amounts determined

6310 dx.doi.org/10.1021/jf5020318 | J. Agric. Food Chem. 2014, 62, 6306−6316 a Chemistry Food and Agricultural of Journal Table 3. Fatty Acid Composition (Percent of Total Fatty Acids) of Total Lipids in the Honeybee-Collected Pollen from Transylvania, Romania

pollen sampleb fatty acidc P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 mean 1. 6:0 0.05 0.03 0.08 0.05 0.05 0.04 0.11 0.15 0.03 0.10 0.02 0.08 0.12 0.07 0.04 0.06 2. 8:0 0.01 0.04 0.08 0.03 0.02 1.74 0.34 0.01 0.29 0.06 0.19 0.12 0.02 0.16 0.19 3. 10:0 0.05 0.97 1.68 0.17 0.98 0.11 2.23 0.16 0.87 0.79 0.29 0.20 0.20 0.34 0.04 0.08 0.57 4. 12:0 0.60 1.96 3.65 3.39 2.14 1.53 2.06 8.51 1.98 2.67 1.56 2.30 0.13 3.59 0.49 0.63 2.33 5. 14:0 1.10 2.26 3.62 1.21 2.46 0.77 1.13 0.18 2.08 1.69 0.62 0.83 1.86 0.89 0.69 0.35 1.36 6. 16:0 24.28 28.99 23.30 23.01 25.58 22.29 27.00 18.39 27.92 23.88 30.93 30.00 28.05 27.58 24.46 27.32 25.81 7. 18:0 3.25 1.99 2.21 4.32 2.07 3.24 2.51 8.99 2.17 3.32 1.63 2.67 3.09 2.01 3.50 1.62 3.04 8. 18:1n-9 14.41 4.18 8.06 8.51 3.86 15.34 8.78 8.60 4.61 9.52 5.23 6.81 5.15 6.54 14.28 5.55 8.09 9. 18:1(9t)n-9 0.99 0.52 0.34 0.42 0.54 0.36 0.56 0.46 0.67 0.33 1.02 0.42 0.60 0.42 0.36 0.88 0.56 10. 18:2n-6 33.21 20.86 24.68 26.99 25.31 30.75 13.50 22.06 19.85 20.28 7.62 14.79 11.44 23.63 30.65 29.10 22.17 11. 18:3n-3 20.28 35.87 28.44 29.25 33.77 22.48 35.15 29.58 37.04 34.76 49.37 38.43 46.93 29.40 24.26 32.33 32.96 12. 20:0 0.73 1.23 2.09 0.97 1.34 0.96 0.83 1.30 1.32 0.72 0.58 1.47 0.84 3.18 0.55 0.67 1.17 13. 20:1n-9 0.19 0.26 0.25 0.33 0.28 0.45 3.63 0.53 0.33 0.38 0.22 0.35 0.11 0.51 0.23 0.23 0.52 14. 22:0 0.87 0.88 1.56 1.30 1.58 1.66 0.77 0.74 1.13 1.26 0.85 1.46 1.37 1.83 0.49 1.02 1.17 ∑n-3 PUFAs 20.28 35.87 28.44 29.25 33.77 22.48 35.15 29.58 37.04 34.76 49.37 38.43 46.93 29.40 24.26 32.33 32.96 ∑n-6 PUFAs 33.21 20.86 24.68 26.99 25.31 30.75 13.50 22.06 19.85 20.28 7.62 14.79 11.44 23.63 30.65 29.10 22.17 ∑n-9 PUFAs 15.59 4.96 8.65 9.26 4.69 16.15 12.97 9.59 5.61 10.24 6.47 7.59 5.87 7.46 14.87 6.67 9.16

6311 n-6/n-3 1.64 0.58 0.87 0.92 0.75 1.37 0.38 0.75 0.54 0.58 0.15 0.38 0.24 0.80 1.26 0.90 0.76 a The values represent the means of three samples, analyzed individually in triplicate (n =3× 3). b P1−P16, samples of beepollen. c PUFAs, polyunsaturated fatty acids; caproic acid (6:0); caprylic acid (8:0); capric acid (10:0); lauric acid (12:0); myristic acid (14:0); palmitic acid (16:0); stearic acid (18:0); oleic acid (18:1n-9); elaidic acid [18:1(9t)n-9]; linoleic acid (18:2n-6); α-linolenic acid (18:3n-3); arachidic acid (20:0); 11-eicosenoic acid (20:1n-9); behenic acid (22:0). dx.doi.org/10.1021/jf5020318 | .Arc odChem. Food Agric. J. 04 2 6306 62, 2014, Article − 6316 Journal of Agricultural and Food Chemistry Article from Brazilian fresh samples, but β-carotene content was significantly lower in Romanian samples (from traces to 13.2 μg/g fresh bee pollen). This can be attributed to different flora and climatic conditions between the samples. Muniategui et al.26 found 50−150 μg/g of carotene and 140− 400 μg/g xanthophylls in four samples of fresh pollen. The same authors reported that total carotene content varied widely between samples. The small number of pollen samples analyzed in this case is not very conclusive, due to the fact that multifloral bee pollen is very different in composition because of the botanical origin of floral pollen. Also in this study, the high variability of total and individual carotenoids found could be attributed to climatic differences and the floral contribution to the pollen. Figure 3. GC-MS chromatogram of FAMEs in the TLs of the honeybee- collected multifloral pollen (P16) from Romania (Transylvania) The HPLC-PDA chromatogram for one pollen sample (P2) is analyzed with a Supelcowax 10 capillary column. Peak identification: presented in Figure 1. (1) caproic (6:0); (2) caprylic (8:0); (3) capric (10:0); (4) lauric Besides carotenoids, other classes of biologically active com- (12:0); (5) myristic (14:0); (6) palmitic (16:0); (7) stearic (18:0); (8) pounds make important contributions to the nutritional and oleic (18:1n-9); (9) elaidic [18:1(9t)n-9]; (10) linoleic (18:2n-6); (11) therapeutic properties of bee pollen. From previous research,3,25 α- linolenic (18:3 n-3); (12) arachidic (20:0); (13) 11-eicosenoic it is obvious that bee pollen can compete with other important (20:1(n-9); (14) behenic (22:0) acids. − sources of polyphenols,32,39 43 having also high amounts of 46 polyphenols and different classes of flavonoids. recommended by Simopoulos (n-6/n-3 = 1−5/1) as beneficial Lipid Contents. The TLs contents in 16 honeybee-collected for good health. pollen samples are presented in Figure 2. Generally, the yields Several studies suggest that lowering the dietary n-6/n-3 fatty varied significantly (p < 0.05) within the analyzed pollen samples, acid ratio may reduce the risk of coronary heart disease and 47,48 with the highest levels of fats in P13 (9.12 g/100 g bee-collected cancer. pollen) and the lowest in P6 (5.30 g/100 g bee-collected pollen). It is generally known that the dominant fatty acids present α 21 Correlation with botanical origin is again confirmed, the highest in pollens are palmitic, oleic, linoleic, and -linolenic acids. fi ff ff amount of lipids being found in the sample having Brassica sp. as Signi cant di erences were reported by di erent authors for compositions of myristic and arachidic acids.21,44 It is interesting predominant pollen. Bee pollen samples having high amounts of 49 total lipids (P7, P1, and P8) have in their botanical compositions to note that the study of Bastos et al., carried out on 14 pollen different amounts of Brassica sp. pollen. It is known that Brassica samples from Brazil, demonstrated that one of the predominat- sp. are rich sources of oleic, linoleic, and linolenic acids.44 Pollens ing fatty acids appeared to be arachidic acid (ranging from 8.90 to with high lipid concentrations are considered attractive to bees.45 42.7%). In contrast, the content of this long-chain saturated fatty Previous studies have shown that the cytoplasm of pollen can acid estimated in our study and those of other researchers did not exceed even 1.50%.21 contain twice as much lipids as found on the outermost layer 22 of the pollen wall (pollenkitt).2,18,22 The values obtained in this Manning mentioned that pollen lipids, dominated by long- chain unsaturated (linoleic and linolenic) and saturated (myristic work were similar to those observed in the samples of honeybee- and lauric) fatty acids, have bactericidal and antifungal properties, collected pollen originating from Poland (ranged from 6.74 to playing an important role in inhibiting the growth of the spore- 10.99% dry matter), but higher, on average, than those deter- forming bacteria Paenibacillus (American foulbrood), Melisso- mined for Korean (5.5% dw) and Chinese (6.2% dw) samples.21 ff coccus pluton (European larvae larvae foulbrood) and other Di erences in the lipid content of bee pollen may be attributed to microorganisms that inhabit the brood combs of beehives. For the geographical and climate conditions, apicultural practices, 21 larvae and young adult bees the fatty acids and other lipid classes and, last but not least, the genetic composition of plant species. are important sources of energy. The honeybees use the lipids for Fatty Acid Composition. The fatty acid compositions, total the synthesis of fat reserve and glycogen and for the production n-3, n-6, n-9 polyunsaturated fatty acids (PUFAs) and the n-6/ of royal jelly.2 n-3 ratios of TLs from the Romanian honeybee-collected pollen The statistical analysis of fatty acid classes showed significant are presented in Table 3. There were no qualitative differences in ff fi di erences (p < 0.05) between pollen samples (Figure 4). The the fatty acid pro les of the TLs in all samples, except for the highest values of saturated fatty acids (SFAs) (p < 0.05) were caproic acid (6:0) and caprylic acid (8:0), which are absent in the registered in the TLs of P12 and P14 (∼39%) (Figure 4A), TLs of P1 and P15, respectively. Fourteen fatty acids were iden- whereas P6 (16.15%), P1 (15.59%), and P15 (14.90%) were fi ti ed in the pollen extracts (Figure 3), from which the most the richest sources of monounsaturated fatty acids (MUFAs) α abundant was -linolenic (18:3n-3) acid, ranging between (Figure 4B). On the other hand, small variations (p < 0.05) were 20.28% (P1) and 49.37% (P11). The next most abundant fatty found in PUFAs contents (Figure 4C), with the highest pro- acids were linoleic acid (18:2n-6) (from 7.62%, P11, to 33.21% P1), portions in P16 (61.43%) and the lowest in P7 (48.65%). followed by palmitic (16:0) (from 18.39%, P8, to 30.93%, P11) Different ratios of saturated and unsaturated fatty acids as quality and oleic acids (18:1n-9) (from 3.86%, P5, to 15.34%, P6). Small index for pollen loads were determined. As shown in Figure 4D, and very small amounts (<3%) of stearic (18:0), myristic (14:0), the levels of the PUFAs/SFAs ratios were significantly higher (p lauric (12:0), arachidic (20:0), 11-eicosenoic (20:1n-9), behenic < 0.05) in TLs of P16 (due to the high values of 18:3 (32.33%) (22:0), elaidic [18:1(9 t)n-9), caproic (6:0), caprylic (8:0), and and 18:2 (29.10%) fatty acids) than in the TLs of the other 15 capric (10:0) acids were also determined. The ratios of n-6 to pollen samples. The UFAs (unsaturated fatty acids)/SFAs ratio, n-3 PUFAs ranged from 0.15 (P11) to 1.64 (P1) (Table 3). considered as a quality index of bee pollen, determined in the The average value of these ratios (0.76) is close to the values present study was similar to the values reported by Serra Bonvehi

6312 dx.doi.org/10.1021/jf5020318 | J. Agric. Food Chem. 2014, 62, 6306−6316 Journal of Agricultural and Food Chemistry Article

Figure 4. Comparative representation of fatty acid classes from total lipids of honeybee-collected pollen samples from Romania: (A) SFAs, saturated fatty acids; (B) MUFAs, monounsaturated fatty acids; (C) PUFAs, polyunsaturated fatty acids; ratio of polyunsaturated fatty acids to saturated fatty acids (D) and unsaturated fatty acids to saturated fatty acids (E) from multifloral bee collected pollen. Values are the mean ± SD of three samples, analyzed individually in triplicate (n =3× 3). Values with different letters (a−l) are significantly different (p < 0.05), using ANOVA Tukey’s multipl- comparison tes”.P1−P16, samples of multifloral pollen and Escora Jorda50 and Szczesną 21 and accounted for about 1.82 bee pollen, these pollen types being important providers of on average (Figure 4E). provitamin A. According to PCA, the botanical origin of pollen samples was The major carotenoids found in the samples were lutein, correlated with the amount of total lipids and fatty acid profile. β-cryptoxanthin, and β-carotene. Lutein was present in all High levels of total carotenoids were found in some of the samples as the major carotenoid. In contrast, β-carotene was samples, demonstrating the direct link of botanical species rich in identified and quantified only in some of the analyzed samples, carotenoids (Taraxacum, Calendula, and Filipendula sp.) and and there detected just in traces or very small amounts.

6313 dx.doi.org/10.1021/jf5020318 | J. Agric. Food Chem. 2014, 62, 6306−6316 Journal of Agricultural and Food Chemistry Article

The importance of individual variables can be assessed by computing the correlation loadings plot (Figure 6). The compounds from the inner ellipse indicate 50% of the explained variance, whereas those found in the outer ellipse indicate 100% of explained variance. Variables with loading values near zero have similar concentrations in all samples. Thus, it can be noted that total carotenoid content, lutein content, β-cryptoxanthin content, myristic acid (14:0) concentration, and Filipendula ulmaria predominant pollen significantly contribute to sample discrimination. According to the correlation loading plot, pollen samples 2, 3, and 9 are characterized by a high content in Filipendula ulmaria pollen and also in myristic acid compared to the other samples, whereas samples 7, 8, and 10 are richer in lutein, β-cryptoxanthin, and total carotenoids. With regard to carotenoid compounds, it has to be mentioned that a correlation loading plot Figure 5. Principal component analysis biplot for the analyzed pollen samples. Two dimensions together explained 88% of the data variance. showed that there is a strong positive correlation between total carotenoids and lutein. ■ ASSOCIATED CONTENT *S Supporting Information Table S1: Place of Origin and Harvesting Period for Bee- Collected Pollen Samples. This material is available free of charge via the Internet at http://pubs.acs.org. ■ AUTHOR INFORMATION Corresponding Author *(F.D.) Phone: 0040- 264-596384. Fax: 0040-264-593792. E-mail: [email protected]. Author Contributions All authors contributed equally to this paper Funding This work was supported by a grant of the Romanian Ministry of Figure 6. Correlation loading biplot for the analyzed pollen samples. Education, CNCS − UEFISCDI, Project PNII-RU-PD-2012-3- 0245 (Contract 92/25.06.2013), POS CCE project RoBeeTech, ’ Lipid and carotenoid amounts in bee-collected pollen pre- no. 206/2010 and is a part of R.M. s Ph.D. thesis. sented great variability between samples, which can be explained Notes by the various botanical origins of those samples. In general, the The authors declare no competing financial interest. investigated samples were characterized by high levels of α-linolenic (32.96% on average), palmitic (25.80, on average), ■ ABBREVIATION USED and linoleic (22.17% on average) acids. Well-known is the bene- HPLC-DAD, high-performance liquid chromatography with ficial effect of n-3 fatty acids in the prevention and management photodiode array detection; GC-MS, gas chromatography with of cardiovascular disease, hyperinsulinemia, and possibly type 2 mass spectrometry detection; PUFAs, polyunsaturated fatty 46 diabetes. Bee-collected pollen with high levels of α-linolenic acids; SFAs, saturated fatty acids; UFAs, unsaturated fatty acids; (n-3) acid together with a near 1:1 ratio of n-6 to n-3 PUFAs UV, ultraviolet; PCA, principal component analysis; FAMEs, represents also a very balanced source of essential PUFAs for fatty acid methyl esters; BHT, butylated hydroxytoluene; TLs, human health. total lipids; MUFAs, monounsaturated fatty acids The PCA is a chemometric method that can be used to establish interrelationships between different sample variables, ■ REFERENCES ff leading to the detection of similarities or di erences between the (1) Almeida-Muradian, L. B.; Pamplona, L. C.; Coimbra, S.; Barth, O. patterns of the analyzed samples. On the basis of the PCA of the M. Chemical composition and botanical evaluation of dried bee pollen palynological analysis data, the pollen samples studied were not pellets. J. Food Compos. 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Food Chemistry

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Identiﬁcation of the bioactive compounds and antioxidant, antimutagenic and antimicrobial activities of thermally processed agro-industrial waste ⇑ ⇑ Dan Cristian Vodnar a,b, , Lavinia Florina Ca˘linoiu a,b, Francisc Vasile Dulf c, , Bianca Eugenia Sßtefa˘nescu b,d, Gianina Crisßan d, Carmen Socaciu a,b a Faculty of Food Science and Technology, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Ma˘na˘ßstur 3-5, 400372 Cluj-Napoca, Romania b Institute of Life Sciences, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Ma˘na˘ßstur 3-5, 400372 Cluj-Napoca, Romania c Faculty of Agriculture, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Ma˘na˘ßstur 3-5, 400372 Cluj-Napoca, Romania d Department of Pharmaceutical Botany, Iuliu Hațieganu University of Medicine and Pharmacy, 12 I. Creanga˘ Street, Cluj-Napoca 400010, Romania article info abstract

Article history: The purpose of the research was to identify the bioactive compounds and to evaluate the antioxidant, Received 6 October 2016 antimutagenic and antimicrobial activities of the major Romanian agro-industrial wastes (apple peels, Received in revised form 21 March 2017 carrot pulp, white- and red-grape peels and red-beet peels and pulp) for the purpose of increasing the Accepted 22 March 2017 wastes’ value. Each type of waste material was analyzed without (fresh) and with thermal processing Available online 24 March 2017 (10 min, 80 °C). Based on the obtained results, the thermal process enhanced the total phenolic content. The highest antioxidant activity was exhibited by thermally processed red-grape waste followed by ther- Keywords: mally processed red-beet waste. Linoleic acid was the major fatty acid in all analyzed samples, but its Fresh content decreased signiﬁcantly during thermal processing. The carrot extracts have no antimicrobial Thermally processed Agro-food waste effects, while the thermally processed red-grape waste has the highest antimicrobial effect against the Phenolic content studied strains. The thermally processed red-grape sample has the highest antimutagenic activity toward S. typhimurium TA98 and TA100. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction cient collection and improper disposal of agro-industrial wastes can cause pollution problems and the loss of biomass, which can In recent years, there has been a worldwide concern regarding serve as a source of bioactive compounds (Shyamala & Jamuna, the use of industrial waste. The industrial waste is generated dur- 2010). By-products from the fruit and vegetable industry are par- ing processing of raw materials in the food industry. The insufﬁ- ticularly of interest because they are inexpensive and available in large quantities. Some of the agricultural by-products such as

Abbreviations: AF, apple waste fresh; CF, carrot waste fresh; WGF, white-grape apples and citrus fruits have indeed already been used in the pro- waste fresh; FAMEs, fatty acid methyl esters; RGF, red-grape waste fresh; BF, red- duction of dietary fiber (Figuerola, Hurtado, Estevez, Choffelle, & beet waste fresh; AT, apple waste thermally processed; CT, carrot waste thermally Asenjo, 2005). The compositions and physicochemical properties processed; WGT, white-grape waste thermally processed; RGT, red-grape waste of dietary fibers depend on both the characteristics of the raw thermally processed; BT, red-beet waste thermally processed; HPLC-DAD-ESI-MS, materials and the processing steps (Chau, Chen, & Lee, 2004). high-performance liquid chromatography-diode array detection-electrospray ionization mass spectrometry; I%, percentage inhibition of DPPH radical; RIC, radical The presence of bioactive molecules, such as fatty acids and inhibition capacity; TLs, total lipids; GC–MS, gas chromatograph mass spectrom- phenolic compounds, in agro-industrial waste makes fruit and veg- etry; wt%, weight percentages; MIC, minimum inhibitory concentration; MBC, etable leftovers more valuable for the food industry. Fruits and minimum bacterial concentration; SD, standard deviation; GAE, gallic acid equiv- vegetables contain bioactive compounds that impart health bene- alents; TF, total flavonoids; DW, dry weight; QE, quercetin equivalents; CFU, colony forming unit; TP, total phenolic/polyphenols. fits beyond basic nutrition (Oomah & Mazza, 2000). It has been ⇑ Corresponding authors at: University of Agricultural Sciences and Veterinary reported that the antioxidant capacity of fruits correlates to their Medicine Cluj-Napoca, Calea Ma˘na˘sßtur 3-5, 400372 Cluj-Napoca, Romania (D.C. total phenolic content and composition (Corral-Aguayo, Yahia, Vodnar). Carrilo-Lopez, & Gonzalez-Aguilar, 2008). Recent studies show that E-mail addresses: [email protected] (D.C. Vodnar), francisc_dulf@yahoo. com (F.V. Dulf). http://dx.doi.org/10.1016/j.foodchem.2017.03.131 0308-8146/Ó 2017 Elsevier Ltd. All rights reserved. 132 D.C. Vodnar et al. / Food Chemistry 231 (2017) 131–140 the antimicrobial potential of natural extracts is higher than that practical application of these wastes in the food industry. Through shown by synthetic antibiotics (Katalinic´ et al., 2010). the results reported in this study, it is expected that food- The Romanian fruit and vegetable juice and pulp industries processing industries may better direct their waste, thus avoiding stand out. Much of the waste, including apple peels, carrot pulp, a growing environmental problem. white and red grape peels and red beet peels and pulp, is discarded; however, this waste is rich in bioactive compounds and 2. Materials and methods can thus be improved and incorporated into food supplements (European Commission Final Report, 2010). 2.1. Materials and chemicals One of the five major Romanian agro-industrial wastes is apple peel. Studies report that apple peel, in addition to its ability to inhi- The apple peels (Ionatan of Voinesßti) (AW), carrot peels and bit lipid oxidation, has phytochemicals that exhibit cardioprotec- pulp (Nabuco variety) (CW), white (Feteasca˘ Regala˘) (WGW) and tive and anticancer properties (Knekt et al., 2002). These health red (Isabella variety) grape peels (RGW) and red beet (Beta vul- advantages of apple peels are correlated with the presence of flavo- garis) peels and pulp (RBW) were collected from a commercial nols, anthocyanins, flavan-3-ols, phenolic acids and dihydrochal- juice producer and transported in plastic containers at 20 °Cto cones (Boyer & Liu, 2004). Other previous results have indicated the University of Agricultural Sciences and Veterinary Medicine that approximately 80% of polyphenols are concentrated in apple Cluj-Napoca. The waste materials were immediately crumbled peel (Leccese, Bartolini, & Viti, 2009), whose total antioxidant and bioactive compounds were extracted as fresh samples (F). Each capacity is five-to-six-fold higher than that of apple flesh; the peel crumbled material was mixed with water (10 mL of distilled water also possesses unique flavonoids, such as quercetin glycosides, that for every 1 g of waste material) and thermally processed (10 min at are not found in the flesh (Rupasinghe & Kean, 2008). 80 °C), to make thermal samples (T). The results were calculated Another major Romanian agro-industrial waste is grape peel based on the dry matter. (white and red). Approximately 80% of the grape harvest is used Folin-Ciocalteu’s phenol reagent, sodium carbonate (Na2CO3), in the winemaking industry, resulting in huge amounts of waste sodium nitrate (NaNO2), ammonium nitrate (NH4NO3), hydrochlo- and a serious disposal problem. A high proportion of phenolic com- ric acid (HCl), aluminum chloride (AlCl3), sodium hydroxide pounds remains in the winemaking waste (Lafka, Sinanoglou, & (NaOH), sea salts, glucose, acetic acid, acetonitrile, methanol, gallic Lazos, 2007), and these compounds are effective as inhibitors of acid, quercetin, chlorogenic acid, rutin, cyanidin chloride, DPPH human low-density lipoprotein oxidation, in addition to other pos- (1,1-diphenyl-2-picrylhydrazyl), lipid standards and chemicals itive impacts that they have on human health (Folts, 2002). It is (used for oil extraction, fractionation and preparation of fatty acid known that there is a strong correlation between antioxidant methyl esters (FAMEs)) were purchased from Sigma-Aldrich capacity and the total phenolic compounds present (Corral- (Steinheim, Germany). For antimicrobial assays, Mueller-Hinton Aguayo et al., 2008). agar, thioglycollate broth with resazurin, and Mueller-Hinton Carrot pulp represents another important Romanian agro-food broth were purchased from BioMerieux (France) and Tween 80 waste. This particular waste has been shown to have high amounts and Broth Malt medium were purchased from Sigma-Aldrich of phenolic compounds and dietary fiber, which give some physical (Steinheim, Germany). characteristics to the carrot. For example, anthocyanins and carotenoids are responsible for the color, aroma and bitterness of carrots 2.2. Extraction and analysis of phenolic compounds (Gonçalves, Pinheiro, Abreu, Brandao, & Silva, 2010). Moreover, its phenolic acids have a strong antioxidant potential, and antho- The fresh (AF, CF, WGF, RGF, BF) and thermally processed sam- cyanins have been proven to reduce cardiovascular heart disease ples (AT, CT, WGT, RGT, BT) were individually extracted three times by decreasing the inflammation and lipid oxidation (Arscott & with 20 mL of extraction mixture (hydrochloric acid/methanol/ Tanumihardjo, 2010). water ratio of 1:80:19) at 40 °C for 30 min in an ultrasonic bath Red beet by-products are also found among major Romanian (Dulf, Vodnar, Dulf, & Tosa, 2015). After centrifugation (4000g for agro-industrial wastes. This type of waste, namely, the pomace/ 10 min), the supernatants were filtered; the filtrates were evapo- pulp from the juice industry, accounts for 15–30% of the raw mate- rated to dryness under vacuum, dissolved in methanol and stored rial and is usually discarded as feed or manure, even though it has a (4 °C) until analysis (total and individual phenolic compounds, high content of betalains. Betalains are water-soluble nitrogenous total flavonoids, antioxidant, antimutagenic and antimicrobial pigments, which consist of two main groups, the red betacyanins activities). and the yellow betaxanthins (Pedreno & Escribano, 2001). They actually give the color of the beet, and the phenolic portion of the peel has l-tryptophan, p-coumaric and ferulic acids and cyclo- 2.2.1. Total phenolic content dopa glucoside derivatives. Red beets are considered among the 10 Determination of total phenolic content (TP) was performed by most effective vegetables, in terms of antioxidant capacity, with using the Folin-Ciocalteu method (Dulf, Vodnar, & Socaciu, 2016; the largest amount of total phenolics being found in the peel Dulf et al., 2015). 25 ml of each extract was mixed with 125 mlof m (50%) (Kujala, Loponen, Kika, & Pihlaja, 2000). Thus, it is crucial Folin-Ciocalteu regent (0.2 N) and 100 l of 7.5% (w/v) Na2CO3 to explore red beet pulp and peel, as little is known about their solution. The mixture was incubated for 2 h in the dark at room in vivo absorption. temperature (25 °C). The absorbance against a methanol blank The utilization of Romanian agro-industrial waste could provide was recorded at 760 nm. A standard curve was prepared using gal- an extra source of income and, at the same time, help to reduce the lic acid (0.01–1 mg/mL), and the TP content in the extract was solid-waste disposal problem of the country. However, there is expressed as gallic acid equivalents (GAE) in mg/100 g dry weight limited information on the bioactive potential of these specific (DW) of waste. agro-food wastes after being treated by a thermal process. In this context, the present study is aimed at evaluating the phenol, flavo- 2.2.2. Total flavonoid content noid, and lipid (fatty acids) content, as well as the antioxidant, The total flavonoid content (TF) of the extracts was determined antimicrobial, and antimutagenic activities of these five major using methods described previously (Barakat & Rohn, 2014). A Romanian agro-industrial wastes, as both fresh and thermally pro- 100 ml aliquot of 2% aluminum chloride ethanol solution was added cessed matrices. The thermal treatment was used to test a future to 100 ml of the extracts and mixed. After incubating for 1 h at room D.C. Vodnar et al. / Food Chemistry 231 (2017) 131–140 133 temperature, the absorbance at 510 nm was measured against a chromatograph (GC) coupled to a mass spectrometer (MS); Perki- prepared regent blank. Total flavonoid content was expressed as nElmer Clarus 600T GC–MS (PerkinElmer, Inc., Shelton, CT, U.S.A) quercetin equivalents (QE) in mg/g dry weight (DW). (Dulf et al., 2015). The GC column was a Supelcowax 10 (60 m 0.25 mm i.d., 0.25 mm film thickness; Supelco INC., Belle- 2.2.3. Analysis of phenolic compounds by HPLC-DAD-ESI-MS fonte, PA, U.S.A). The oven temperature was set at 140 °C and then The characterization of the phenolic compounds in the samples increased to 220 °C. Helium was used as the carrier gas with a con- was carried out on an HPLC-DAD-ESI-MS system consisting of an stant flow rate of 0.8 mL/min. Mass spectra (E.I. positive-ion Agilent 1200 HPLC with DAD detector, coupled to an MS-detector electron-impact mode) were recorded at 70 eV using a trap current single-quadrupole Agilent 6110. Separations of phenolic com- of 100 mA with a source temperature of 150 °C. The MS was pounds were performed at 25 °C on an Eclipse column, XDB C18 scanned from m/z 22 to 395 for all GC–MS experiments. The FAME (4.6 150 mm, 5 mm) (Agilent Technologies, USA). The binary gra- peak identification was based on comparing both the retention dient consisted of 0.1% acetic acid/acetonitrile (99:1) in distilled time and MS of the unknown peak to those of known standards water (v/v) (solvent A) and 0.1% acetic acid in acetonitrile (v/v) and compounds listed in an MS database (NIST MS Search 2.0). (solvent B) at a flow rate of 0.5 mL/min, following the elution pro- The amount of each fatty acid was calculated as peak area percent- gram used by Dulf et al. (2015). age of total fatty acids. Various phenolic compounds were identified by comparing the retention times, UV–visible and mass spectra of unknown peaks 2.4. Antimicrobial and antifungal activity with reference standards. For MS fragmentation, the ESI (+) module was applied, with a scanning range between 100 and 1000 m/z, 2.4.1. Bacteria and culture conditions capillary voltage 3000 V, at 350 °C and with a nitrogen flow of 8 For this bioassay, six bacterial strains were used: four Gram- L/min. The eluent was monitored by DAD, and the absorbance positive bacteria (Staphylococcus aureus (ATCC 49444), Bacillus cer- spectra (200–600 nm) were collected continuously during the eus (ATCC 11778), Listeria monocytogenes (ATCC 19114), Enterococ- course of each run. The flavonols were detected at 340 nm and cus faecalis (ATCC 29212)), three Gram-negative bacteria the anthocyanins at 520 nm (Dulf et al., 2015). Data analysis was (Pseudomonas aeruginosa (ATCC 27853), Salmonella typhimurium performed using Agilent ChemStation Software (Rev B.04.02 SP1, (ATCC 14028) and Escherichia coli (ATCC 25922)) and two anaero- Palo Alto, California, U.S.A). The anthocyanin levels were deter- bic strains: Fusobacterium nucleatum (Gram-negative, ATCC mined using cyanidin chloride as the external standard and 25586) and Peptostreptococcus anaerobius (Gram-positive, ATCC expressed as equivalents of cyanidin (mg cyanidin/100 g DW of 27337). All tested microorganisms were obtained from Food substrate) (r2 = 0.9935). The chlorogenic and neochlorogenic acids Biotechnology Laboratory, UASVM CN, Romania. The aerobic were expressed in mg chlorogenic acid/100 g DW of substrate strains were cultured on Mueller-Hinton agar and cultures were (r2 = 0.9935) and flavonol glycosides were calculated as equiva- stored at 4 °C and subcultured once a month. Anaerobic bacteria lents of rutin (mg rutin/100 g DW of substrate) (r2 = 0.9987). were cultured overnight at 37 °C on thioglycollate broth with resazurin, stored at 4 °C and subcultured once a month. 2.2.4. DPPH free-radical-scavenging assay Phenolic extracts of fresh and thermally processed samples 2.4.2. Microdilution method were subject to DPPH radical-scavenging activity assessment, The modified microdilution technique was used to evaluate using the method described by Toma, Olah, Vlase, Mogosan, and antimicrobial activity. Briefly, fresh overnight cell suspensions Mocan (2015) and Dezsi et al. (2015). The percentage inhibition were adjusted with sterile saline solution to a concentration of (I%) was calculated as [1 (test sample absorbance/blank sample approximately 2 105 CFU/mL in a final volume of 100 lL per absorbance)] 100. well. The inoculum was stored at 4 °C for further use. Determina- tions of minimum inhibitory concentrations (MICs) were per- 2.3. Lipid extraction and GC–MS analysis of FAMEs formed by a serial dilution technique using 96-well plates. Different dilutions of the extracts were carried out with wells con- Total lipids (TLs) of fresh (apple peels, carrot peels and pulp, and taining 100 lL of Mueller-Hinton broth and, afterwards, 10 lLof white and red grape peels (AF, CF, WGF, RGF)) and thermally pro- inoculum was added to all the wells. The microplates were incu- cessed samples (AT, CT, WGT, RGT) were extracted using the bated for 24–48 h at 37 °C. The MIC of the samples was detected method described by Dulf et al. (2015). The red beet waste material after the addition of 20 lL (0.2 mg/mL) of resazurin solution to registered low content in lipids, and the results are not shown. Five each well, and the plates were incubated for 2 h at 37 °C. A change grams of each material was homogenized in 50 mL of methanol for from blue to pink indicates reduction of resazurin and, therefore, 1 min using a high-power homogenizer (MICCRA D-9, ART Prozess- bacterial growth. The MIC was defined as the lowest concentration und Labortechnik, Mullheim, Germany). One hundred mL of chlo- that prevented this color change. The minimum bactericidal con- roform was then added, and homogenization continued for centrations (MBCs) were determined by serial subcultivation of 2 min. The mixture was filtered, and the solid residue was resus- 2 lL into 96-well plates containing 100 lL of broth per well and pended in chloroform/methanol (2:1, v/v, 150 mL) and homoge- further incubation for 48 h at 37 °C. The lowest concentration with nized again for 3 min. After filtration of the mixture, the residue no visible growth was defined as the MBC, indicating death of was washed with 150 mL chloroform/methanol (2:1, v/v). The fil- 99.5% of the original inoculum. Streptomycin (Sigma P 7794, Santa trates and washes were combined and washed with 0.88% aqueous Clara, CA, USA) (0.05–3 mg/mL) was used as positive control for potassium chloride, followed by methanol/water (1:1, v/v) solu- bacterial growth. Water was used as negative control. tion. The purified lipid (bottom) layer was filtered and dried over anhydrous sodium sulfate, and the solvent was removed in a rotary 2.4.3. Antifungal activity evaporator. The amount of lipid was determined gravimetrically. To investigate the antifungal activities, the following fungi were The recovered oils were transferred to vials with 2 mL of chloro- used: Aspergillus flavus (ATCC 9643), Aspergillus niger (ATCC 6275), form (stock solution) and stored at 18 °C until further analysis. Candida albicans (ATCC 10231), Candida parapsilosis (ATCC 22019), FAMEs were prepared by acid-catalyzed transesterification of and Penicillium funiculosum (ATCC 56755), all bought from the TLs (Dulf, Oroian, Vodnar, Socaciu, and Pintea, 2013; Dulf, Pamfil, same source stated above. Cultures were maintained on malt agar Baciu, and Pintea, 2013). The samples were analyzed with a gas at 4 °C and subcultured every month. Spore suspensions 134 D.C. Vodnar et al. / Food Chemistry 231 (2017) 131–140

(1.5 105) were obtained by washing agar plates with sterile solu- the thermally processed samples of apple and white grapes had tion that contained 0.85% saline and 0.1% Tween 80 (v/v), which higher total phenolic content, red grape waste had the highest was then added to each well to a final volume of 100 lL. The min- (1990 ± 52.9 mg GAE/100 g dry weight), and carrot waste had the imum inhibitory (MIC) and minimum fungicidal (MFC) concentra- lowest (5.1 ± 0.25 mg GAE/100 g dry weight). Polyphenol content tion assays were performed using the microdilution method by in the extracts of carrot and red beet waste showed significantly preparing a serial of dilutions in 96-well plates. The extracts were (p < 0.05) higher values (37.5 mg GAE/100 g DW for CF and diluted in 0.85% saline (10 mg/mL), then added to microplates con- 14.1 mg GAE/100 g DW for BF) in fresh samples than in thermally taining Broth Malt medium with inoculum and incubated for 72 h processed samples (5.1 mg GAE/100 g dry weight for CT and 5.5 mg at 28 °C on a rotary shaker. The lowest concentrations without vis- GAE/100 g dry weight for BT). As an overall result, it can be stated ible growth (observed through a binocular microscope) were that fresh samples of carrot waste and red beet waste have the defined as minimal inhibitory concentrations (MICs). The fungici- lowest phenolic content and, during thermal processing, the phe- dal concentrations (MFCs) were determined by serial subcultiva- nolic content decreases significantly; in contrast, thermal process- tion of 2 lL of tested extracts that were dissolved in medium and ing increases the phenolic content in the rest of the samples. This inoculated for 72 h into microtiter plates containing 100 lLof can be attributed to the fact that extraction of intracellular con- broth per well and followed by further incubation 72 h at 28 °C. tents is enhanced by thermal treatment. Additionally, Wang, He, The lowest concentration with no visible growth was defined as and Chen (2014) reported that high temperature increased the the MFC, indicating death of 99.5% of the original inoculum. The content of phenolic compounds due to the hydrolysis of polysac- fungicide fluconazole (Sigma F 8929, Santa Clara, CA, USA) was charides. The TP content in fresh apple peels was (564 ± 39.5 mg used as positive control (1–3500 lg/mL). All the experiments were GAE/100 g DW); this value is 3.3-fold lower than those reported performed in duplicate and repeated thrice. Water was used as by Henríquez, Córdova, Almonacid, and Saavedra (2014) but agree negative control. with those reported by Drogoudi, Michailidis, and Pantelidis (2008). After thermal treatment (10 min at 80 °C), the polypheno- 2.5. Mutagenic and antimutagenic activity lics increased in AT to 20.56 ± 1.9 (mg GAE/100 g DW). The TP decrease (in CT and BT) can be explained by three possible mech- 2.5.1. Mutagenic and antimutagenicity test anisms: (1) the release of bound phenolic compounds; (2) the par- Mutagenic and antimutagenicity of fresh (AF, CF, WGF, RGF, BF) tial degradation of lignin, which can release phenolic acids; and (3) and thermally processed samples (AT, CT, WGT, RGT, BT) were the thermal degradation of the phenolic acids or catechins examined using the plate incorporation method (Maron & Ames, (Larrauri, Ruperez, & Saura-Calixto, 1997). Additionally, there is a 1983) described in detailed by Sarac and Sen (2014). 4-nitro-o- possibility that thermal treatment inactivates the enzymes responsible for the loss of phenolic compounds (like polyphenol oxidase) phenylenediamine (4-NPD, 3 mg/plate) and sodium azide (NaN3, 8 mg/plate) were used as positive controls for S. typhimurium or could also accelerate the activity of enzymes that participate in TA98 and S. typhimurium TA100. Ethanol: water (1:1, v/v) was used the biosynthesis of phenols (like phenylalanine ammonia-lyase) as negative control. The extracts were prepared at a concentration (Morales-de la Peña, Salvia-Trujillo, Rojas-Graü, & Martín-Belloso, of 5 mg/plate. The antimutagenicity of the reference mutagens in 2011). the absence of the extract was defined as 0% inhibition, and the The total flavonoids content was calculated and is displayed in antimutagenicity was calculated according to the formula given Fig. 1 (B); the obtained values range from 8.5 ± 1.1 (fresh carrot by Ong, Whong, Stewart, and Brockman (1986): %Inhibition = waste) to 1050 ± 62.1 mg (thermally processed red grape waste). [1 T/M] 100, where T is the number of revertants per plate in As observed for flavonoids, no significant differences were regis- the presence of mutagen and the extract, and M is the number of tered between fresh and thermally processed samples for apple revertants per plate in the positive control (without extract). The and white grape waste. The significant properties of flavonoids tests were performed in duplicate with three subsamples each, have produced a growing interest in their isolation and separation and the data is presented as the mean ± standard deviation (SD). from natural products and in the development of analytical data- Antimutagenicity was recorded as follows: strong: 40% or more bases of the flavonoid content of foods (Bajalan, Mohammadi, inhibition; moderate: 25–40% inhibition; low/none: 25% or less Alaei, & Pirbalouti, 2016). In the Medina-Meza and Barbosa- inhibition (Evandri et al., 2005). Cánovas (2015) study on grape peels processed by electric fields, the total flavonoid contents were 98 (mg/mL) quercetin eq in fresh samples, which increased by 96% when the sample was exposed to 2.6. Statistical analysis pulsed electric fields.

All tests were conducted in triplicate and the results were expressed as the mean ± standard deviation (SD). Correlations 3.2. Antioxidant activity between the antioxidant capacity and phenolic content were determined using Person’s correlation. Statistical differences The changes in antioxidant activity of the extracts were among samples were estimated using Student’s t-test (GraphPad assessed by measuring the DPPH radical-inhibition capacity (RIC), Prism Version 5.0, Graph Pad Software Inc., San Diego, CA). Differ- and the results are presented in Fig. 1(C). After thermal processing, ences between means at the 5% level were considered statistically statistically significant increases (58.37%) (p < 0.05) in RIC of red significant. grape waste was registered. Red-grape waste extract exhibits the highest scavenging activity, followed by red-beet waste extract. 3. Results and discussion The increase in antioxidant activities of thermally processed beet waste shows that the antioxidant activity depends not only on 3.1. Total phenolics and total flavonoids the presence of betacyanins but also on other polyphenols that could have increased during the treatments. These observations The total polyphenol content (TP) of extracts measured by the agree with the results of Dewanto, Wu, Adom, and Liu (2002), Folin-Ciocalteu method is shown in Fig. 1 (A). There were signifi- who found that thermal processing enhances the nutritional cant differences (p < 0.05) between the TPs content in the fresh value of tomatoes and corn by increasing the total antioxidant and thermally processed waste materials. As shown in Fig. 1 (A), activity. D.C. Vodnar et al. / Food Chemistry 231 (2017) 131–140 135

Fig. 1. Total phenolic content (Folin-Ciocalteu method) (A), total flavonoids content (B), antioxidant activity (C) and total lipid content (D) of the extracts, both fresh and thermally processed. Total phenolic content of the extract is expressed as gallic acid equivalents (GAE) in mg/100 g dry weight (DW) of waste. Total flavonoid content is expressed as quercetin equivalents (QE) in mg/g dry weight (DW). The percentage inhibition (I%) was calculated as [1 (test sample absorbance/blank sample absorbance)] 100. Values are reported as mean ± SD of triplicate determinations and different symbols (*, **, ***) indicate significant differences (p < 0.05) (paired t-test), while symbol (ns) indicate no significant difference. AF-apple waste fresh; AT-apple waste thermally processed; CF-carrot waste fresh; CT-carrot waste thermally processed; WGF-white-grape waste fresh; WGT-white-grape waste thermally processed; RGF-red-grape waste fresh; RGT-red-grape waste thermally processed; BF-red-beet waste fresh; BT-red-beet waste thermally processed.

3.3. Total lipid content two major phenols were detected: epicatechin and catechin-3-O- glucose. The flavonol group had three identified phenols: rutin, The total lipid yield of extracts was determined and the results quercitrin, and quercetin glucoside (Table 1). In almost all phenolic are presented in Fig. 1(D). The red-beet waste material registered cases, the quantities in the fresh and thermally processed samples low content in lipids and so the results are not shown. No signifi- substantially differed. The anthocyanin group was present only in cant differences between fresh and thermally processed samples of the red grape waste samples, and the malvidin-3-O-glucoside from apple, red and white grape waste were registered. The only signif- this group was the major phenolic compound identified in all the icant difference is for carrot waste samples, where, after the ther- extracts (13.958 [mg/%DW] in RGT and 13.015 [mg/%DW] in mal process, an increase of 2 g of lipids/100 g of waste material RGF). The next predominant phenolic is also within the same group was reported. In the study of Shyamala and Jamuna (2010), the car- identified in the red grape waste sample, and, depending on the rot pulp had a lipid content of 2.72%, a lower value than what we reference extract (fresh or thermally processed), could be either observed here. malvidin glucoside (8.365 [mg/%DW], when the reference is the thermally processed sample) or cyanidin-3-O-arabinoside (7.745 3.4. HPLC-DAD-ESI-MS analysis of phenolic compounds [mg/%DW], when the reference is the fresh sample). The flavonols are present in the apple peel tissues (apple samples), which agrees The analyzed samples contained sixteen phenolic compounds with a previous study by Rupasinghe et al. (2010). Additionally, the that originate from 5 phenolic groups: anthocyanins, cinnamic dihydrochalcone and flavonol groups were found only in the apple acids, dihydrochalcones, flavonols and flavan-3-ols. From the waste extracts, which agrees with a study that reported that apple anthocyanins group, four phenols were identified: petunidin glu- peels’ health advantages correlate with the presence of phenolic coside, malvidin glucoside, cyanidin-3-O-arabinoside and compounds, which include the flavonols and dihydrochalcones peonidin-3-O-glucoside. The five cinnamic acids identified were (Boyer & Liu, 2004). An interesting observation is the fact that, in caffeic acid, caffeic acid-4-O-glucoside, two caffeoylquinic acids some cases, the thermally processed samples show higher phenolic and a 3,4-dicaffeoylquinic acid. The dihydrochalcones identified content in comparison with the fresh samples. This agrees with a were phloridzin and phloretin glucoside. From the flavanol group, Morales-de la Peña et al. (2011) study, which reported that thermal 136 D.C. Vodnar et al. / Food Chemistry 231 (2017) 131–140

Table 1 Identiﬁcation of the phenolic compounds (mg/100 g DW) in the extracts via HPLC-DAD-ESI-MS method.

Phenolic [M+H]+ ion fragments Samples (mg/%DW) compounds AF AT CF CT WGF WGT RGF RGT BF BT Anthocyanins Petunidin 3-O-(60-p-coumaroyl)- 625, 317 2.454a 2.356b glucoside) Malvidin 3-O-(60-p-coumaroyl)- 639, 331 7.358b 8.365a glucoside Cyanidin 3-O-arabinoside 419, 287 7.745b 7.625a Peonidin 3-O-glucoside 463, 301 2.984a 1.892b Malvidin 3-O-glucoside 493, 331 13.015b 13.958a Cinnamic acid Caffeic acid 181, 163 2.138b 2.56a Caffeic acid-4-O-glucoside 343, 181,163 2.492b 3.165a 5-Caffeoylquinic acid 355, 181, 14.146a 4.265b 163 3-Caffeoylquinic acid 355, 181,163 1.27b 2.65a 3,4-Dicaffeoylquinic acid 515, 355 2.457a 0.263b Dihydrochalcones Phloridzin (Phloretin 20-O-glucoside) 437, 275 5.714a 4.652b Phloretin 20-O-xylosyl-glucoside 569, 437, 1.125a 0.958b 275 Flavan-3-ols Epicatechin 291 2.368a 2.261b Catechin 3-O-glucose 453, 291 2.262a 2.265a Flavonol Rutin (Quercetin 3-O-rutinoside) 611, 303 4.318 a 4.316a Quercitrin (Quercetin 3-O-rhamnoside) 449, 303 2.12b 2.46a Quercetin 3,40-O-diglucoside 627, 465,303 0.903b 1.362a Betacyanins Betanidin Betanidin 389,345 3.866b 3.952a Isobetanidin 389, 345 2.353b 2.456a Betanidin-5-O-b-glucoside 551, 389 7.006b 7.100a Isobetanidin-5-O-b-glucoside 551, 389 10.061a 10.074a

Values (mean ± SD, n = 3) in the same row followed by different superscript letters (a, b) indicate signiﬁcant differences (p < 0.05) between fresh and thermally processed samples of the same extract (Student’s t-test – GraphPad Prism Version 5.0, Graph Pad Software Inc., San Diego, CA).)

treatment can accelerate the activity of enzymes that participate in ated with a recovery of color (Stintzing & Carle, 2007). As beta- the biosynthesis of phenols, and with a Wang et al. (2014) study, cyanins undergo thermal treatment, it is known that pigments which reported that high temperature increases the content of will experience degradation and fluctuating chromatic stability phenolic compounds because of the hydrolysis of polysaccharides. (Herbach, Rohe, Stintzing, & Carle, 2006). Our findings agree with In accordance with this observation, in our study, a significant those reported by Harivaindaran, Rebecca, and Chandran (2008); increase in malvidin glucoside (13.68%) was registered after ther- specifically, as the temperature increases, the yield of betacyanin mal processing red grape waste; another major example was content increases (samples of Dragon fruit yielded the highest reported for caffeic acid 4-O-glucoside from apple waste, with a betacyanin content at 100 °C for 5 min). 27% (from 2.492 [mg%] to 3.165 [mg%]) increase after thermal treatment. On the other hand, samples of carrot waste contained 3.5. Fatty acid composition analyzed by GC–MS only the cinnamic acid group and this phenolic content decreased significantly with 70–90% (from 14.146 [mg/%DW] to 4.265 [mg/% The fatty acid profile of each sample is presented in Table 2.In DW]) during thermal processing. This agrees with Chandrasekara, almost all cases, the fatty acids were significantly different Naczk, and Shahidi (2012), who stated that thermal treatment between fresh and thermally processed samples, with six excep- can reduce the amount of phenolic compounds because the high tions whose differences were not significant. The exceptions are: temperature causes the polymerization or loss of thermolabile palmitic acid (16:0), in the case of CW; palmitoleic acid (16:1n- phenols. In both cases, a previous study (Rupasinghe, Laixin, 7), in the case of RGW and WGW; 20:1n-9 acid, in the case of Huber, & Pitts, 2008) demonstrated that processing technique AW and RGW and 16:1n-9 acid, in the case of RGW. The first fatty (baking) and temperatures influence phenolic content by increas- acids analyzed were lauric (12:0), myristic (14:0) and pentade- ing or decreasing different phenolic compounds. For instance, a canoic acid (15:0), all of which are more present in thermally pro- major, interesting finding of this study is that flavonols present cessed samples (e.g., in the case of WGW for all the above in apple skin powder are relatively resistant to thermal degrada- mentioned acids, the fresh samples have a value of 0 while the tion during baking, which is in agreement with our findings that TP samples have values ranging from 0.04 to 0.16% of TLs). A pos- flavonols are also not degraded during processing. Another exam- sible explanation for this observation is the fact that thermal treat- ple from the same literature study indicates that phloridzin values ment enhances the release of these fatty acids by breaking the cell decrease during baking (56% less), which also agrees with our walls. Apple waste that was thermally processed had the highest results when our thermal process (at just 80 °C) causes a loss of content of FAs. The dominant fatty acids in the TLs were palmitic approximately 10% of the phloridzin. acid (16:0), with a highest value of 19.98 in the CF sample; oleic The group of betacyanins contains four betanidin compounds acid (C18:1n-9), with a highest value of 19.32 in the RGT sample that were all identified in the red-beet waste extracts. With one and linoleic acid (c18:2n-6), with the highest value of 66.3 in exception (isobetanidin-5-O-b-glucoside), the amounts of all the WGF sample. reported betanidin compounds significantly increase during the With respect to palmitic acid (16:0), an interesting observation thermal process This could be explained by the fact that betanin occurred: in the cases of apple and carrot waste, the highest con- can regenerate by recondensation of hydrolysis products associ- tent was registered for fresh samples (a difference of 3.9% for apple D.C. Vodnar et al. / Food Chemistry 231 (2017) 131–140 137

and a difference of 21.75% for carrot), while in the cases of red and ** *** ** * white grape waste thermally processed samples actually had the

1.48 0 0.49 0.27 highest content. A similar result was observed for palmitoleic acid (16:1n-7), in which apple- and carrot-waste fresh samples regis-

*** *** *** *** tered the highest content. A possible explanation is that these

00.15 1.13 0.27 0.43 0.12 FAs are sensitive to thermal treatment, thereby decreasing the percentage that remains after thermal processing. In the case of C16:1n-9, thermally processed samples of apple, carrot and white *** *** * *** grape wastes registered the highest content. Stearic acid (18:0), as 00 0.18 0.03 0.58 0.02 well as oleic acid (18:1n-9) were consistently higher in content in all thermally processed samples. Linoleic acid (18:2n-6) was found *** *** *** ** in a higher proportion in the fresh samples of each type of extract 2.16 0.34 0.51.98 0.08 1.15 (an average of 60% in the fresh samples vs. 54% in thermally pro- ** *** *** ** cessed ones). A possible explanation for this is the fact that linoleic acid is very sensitive to heat; therefore, after a thermal treatment, 0.38 0.15 0.18 0.08 the concentration decreases. The distribution of high levels of linoleic acid in each sample (fresh and processed) agrees with s s ; C16:0, palmitic; C16:1n-9, cis-7 hexadecenoic; C16:1n-7, ** *** Kinsella’s results (1974) on grape seed oil (also containing 60%

0.210 0.290.22 2.77 00.23 n 0.88 linoleic acid). With respect to a-linolenic acid (18:3n-3), in apple and carrot waste samples (fresh and processed) the content of ** *** *** *** C18:3n-3 was high (6.83% in AF and 6.62% in CF), while in red

2.950.68 0.26 n 2.42 0.91 and white grape waste samples the level was relatively low (3.09% in WGT and 3.72% in RGT), the latter results being similar to Kinsella’s ﬁndings on grape seed oil (2.3%). An extremely inter- ** ** *** ** esting fact regarding the a-linolenic acid was that for apple and 6.83 6.62 2.972.15 1.77 0.49 0.14 0.22 0 0 1.24 0.63 0carrot 0 0 waste 0 0.63 the 0.22 highest content was registered in the case of fresh samples, while for red- and white-grape waste the highest values *** *** *** *** were reported for thermally processed samples. According to

49.46 63.89 60.956.53 3.09 3.72 Simopoulos (2002), nutrition societies recommend a precisely balanced ratio (<5:1) of these two types of FAs (18:2n-6 and 18:3n-3) to be necessary for optimal health. In Table 3, all the individual ** ** ** *** lipid classes are examined. All types of extracts were found to con- 0.741.02 43.980.8 54.61 5.90.87 5.87 3.69 0.46 0.09 tain very-long-chain saturated fatty acids (VLCSFA), with chain -linolenic; C20:0, arachidic; C20:1n-9, 11-eicosenoic; C21:0, heneicosylic; C22:0, behenic; C22:1n-9, erucic; C23:0,

a lengths exceeding 20 carbons. According to Gurr, Harwood, & *** *** *** *** Frayn (2002), these VLCSFA have a major role in the cuticular sur-

14.74 16.92 13.56 19.86 face layers of plant tissues; therefore, using only waste extracts (peels) in this study, our ﬁndings are in agreement with these ** *** *** ***

) in the same column indicate signiﬁcant differences (p < 0.05) among TLs of the fresh and thermally processed samples of the same works. Regardless of whether the heat treatment is applied or *** , 4.84 1.21 3.264.95 1.12 3.96 TLs not, apple samples, having the highest content in SFA, contained ** , * a much higher amount of VLCSFA than the other extracts. Accord- ** *** ** * ing to Simopoulos (2002), the two types of PUFAs (polyunsaturated

0.350.57 4.76 9.70.09 0.04 0.83 3.28 19.32 0.73 61.87 fatty acids), namely n-6 and n-3, are very important with respect to health and disease. A high ratio of n-6/n-3, ranging from 22.34 to

s s 28.82, was found in WGF and RGF. Additionally, these two types *** *** of extracts show the highest ratio of PUFAs/SFAs, ranging from Fatty acids (%) 0.280.32 0.27 0.18 n 0.240.15 n 1.45 3.92 to 4.13. The analysis of FAs show a high ratio of n-6/n-3 and much higher levels of n-6 PUFA in those speciﬁc cases in which a s

*** *** ** high value of 18:2n-6 was reported (Table 2). A low ratio of n-6/

0.20 0.39 0.13 0.8 0.07 0.14 0.09 n-3 was found in apple and carrot, precisely 7.24 in AF, increasing 2.9% after the thermal process, and a percentage of 9.66 in CF, *** *** *** *** decreasing 3.72% after the thermal process. Therefore, according to Simopoulos (2002), who stated that a lower ratio of omega-6/ 18.9316.41 0.64 10.46 0.46 11.75 omega-3 fatty acids is more desirable in reducing the risk of many

*** chronic diseases of high prevalence in Western societies and in s ** ** developing countries, apple and carrot wastes can be integrated

0.161 0.4 n 0.04 0.04 into a recommended healthy diet in different forms. PUFAs have a major effect in reducing the incidence of cardiovascular disease ** *** *** ** and cancer (Banni & Martin, 1998) and are therefore desirable in 0.56 0.21 0.16 0.13 the human diet. Simopoulos, in his study (2002), considers that the paper by Ahrens et al. from 1954 and subsequent work by Keys *** *** ** ** -test – GraphPad Prism Version 5.0, Graph Pad Software Inc., San Diego, CA). TLs – total lipids, C12:0, lauric; C14:0, myristic; C15:0, pentadecanoic t et al. ﬁrmly established the omega-6 fatty acids as the important 12:0 14:0 15:0 16:0 16:1 n-9 16:1 n-7 17:0 18:0 18:1 n-9 18:1 n-7 18:2 n-6 18:3 n-3fatty 20:0 20:1 n-9 acid 21:0 in 22:0 the 22:1 n-9 ﬁeld 23:0 of 24:0 cardiovascular disease. According to T 0.22 T 0.33 T 0.06 T 0.05 Simopoulos (2002), the optimal ratio of omega-6/omega-3 varies from 1/1 to 4/1, depending on the disease, but it is essential to decrease omega-6 intake while increasing the amount of omega- RG F 0 0.09 0 10.75 0.08 n Samples A F 0.17 0.42 0 19.67 CWG F F 0 0 0 0 0.4 0 19.98 10.16 0.07 0.16 0 3.89 12.07 0.59 66.3 Table 2 palmitoleic; C17:0, margaric; C18:0, stearic; C18:1n-9, oleic; C18:1n-7, vaccenic; C18:2n-6, linoleic; C18:3n-3, extract (Student’s tricosylic; C24:0,lignoceric. Molar % values are the mean of three measurements (n = 3). Different superscript symbols ( Fatty acid composition (molar% of total fatty acids) of total lipids for each sample, determined by GC–MS. 3 to prevent and manage of chronic disease. Additionally, the bal- 138 D.C. Vodnar et al. / Food Chemistry 231 (2017) 131–140

Table 3 Individual lipid classes and the GC–MS analyzes of FAME.

Fatty acid classes (%) RSFAs RMUFAs RPUFAs Rn-3 PUFAs Rn-6 PUFAs n-6/n-3 PUFAs/SFAs VLCSFA (>20c) Samples TLs A F 32 ± 0.9 11.72 ± 0.45 56.28 ± 2.75*** 6.83 ± 0.45*** 49.46 ± 1.9 *** 7.24 1.76*** 6.62 T 33.5 ± 0.88 ns 16.62 ± 0.68*** 49.88 ± 1.9 5.9 ± 0.3 43.98 ± 1.55 7.45 *** 1.49 8.41*** C F 24.32 ± 1.00** 5.18 ± 0.2 70.51 ± 2.55*** 6.62 ± 0.35*** 63.89 ± 2.4*** 9.66** 2.90 2.14*** T 20.63 ± 1.13 18.89 ± 0.6*** 60.48 ± 2.45 5.87 ± 0.6 54.61 ± 1.9 9.3 2.93** 1.69 WG F 17.69 ± 0.63 13.04 ± 0.3 69.27 ± 2.4*** 2.97 ± 0.1 66.3 ± 1.9*** 22.34*** 3.92** 3.64 T 21.09 ± 0.3*** 14.92 ± 0.6*** 63.99 ± 2.2 3.09 ± 0.1** 60.9 ± 2.00 19.73 3.03 5.34*** RG F 15.49 ± 0.65 20.49 ± 0.9 64.02 ± 2.1*** 2.15 ± 0.1 61.87 ± 1.6*** 28.82*** 4.13*** 1.34 T 18.55 ± 0.25*** 21.2 ± 0.5** 60.25 ± 1.5 3.72 ± 0.1*** 56.53 ± 1.5 15.2 3.25 2.53***

Values (mean ± SD, n = 3) in the same column followed by different superscript symbols (**, ***, ns) indicate signiﬁcant differences/no signiﬁcant differences (p < 0.05) among fresh and thermally processed samples of the same extract (separately for each lipid class) (Student’s t-test – GraphPad Prism Version 5.0, Graph Pad Software Inc., San Diego, CA). SFAs – saturated fatty acids, MUFAs – monounsaturated fatty acids, PUFAs – polyunsaturated fatty acids, VLCSFA – very long chain saturated fatty acids.

Table 4 Antimicrobial activity of waste extracts before and after thermal process.

Test items L. monocytogenes E. coli S. typhimurium P. aeruginosa S. aureus B. cereus E. faecalis F. nucleatum P. anaerobius mg/mL AF MIC 62.5 15.62 31.25 15.62 3.9 62.5 15.62 62.5 125 MBC 125 31.25 62.5 31.25 7.81 125 31.25 125 250 AT MIC 31.25 7.81 31.25 7.81 1.95 31.25 7.81 62.5 250 MBC 62.5 15.62 62.5 15.62 3.9 62.5 15.62 125 500 CF MIC 250 500 – 500 125 – 500 62.5 250 MBC 500 1000 – 1000 250 – 1000 125 500 CT MIC – – – – – – – 250 – MBC – – – – – – – 500 – WGF MIC 7.81 15.62 31.25 15.62 1.953 15.62 15.62 31.25 31.25 MBC 15.62 31.25 62.5 31.25 3.9 31.25 31.25 62.5 62.5 WGT MIC 3.9 3.9 15.62 7.81 1.953 7.81 7.81 15.62 15.62 MBC 7.81 7.81 31.25 15.62 3.9 15.62 15.62 31.25 31.25 RGF MIC 1.953 1.953 7.81 7.81 1.953 7.81 15.62 15.62 15.62 MBC 3.9 3.9 15.62 15.62 3.9 15.62 31.25 31.25 31.25 RGT MIC 1.953 1.953 3.9 3.9 1.953 3.9 7.81 7.81 7.81 MBC 3.9 3.9 7.81 7.81 3.9 7.81 15.62 15.62 15.62 BF MIC 1.953 1.953 7.81 7.81 1.953 15.62 31.25 31.25 31.25 MBC 3.9 3.9 15.62 15.62 3.9 31.25 62.5 62.5 62.5 BT MIC 15.62 7.81 15.62 3.9 31.25 31.25 15.62 15.62 15.62 MBC 31.25 15.62 31.25 7.81 62.5 62.5 31.25 31.25 31.25 Streptomycin mg/mL MIC 0.015 0.12 0.06 0.06 0.03 0.015 0.012 0.12 0.12 MBC 0.03 0.24 0.12 0.12 0.06 0.03 0.024 0.24 0.24

ance of omega-6 and omega-3 fatty acids is very important for wastes and betacyanins compounds in red beets. There are studies homeostasis and normal development. In general, in all analyzed that state that polyphenols have diverse antibacterial activity fractions, the thermal process significantly increased (p < 0.05) based on the fact that they attack a large number of bacteria. MUFAs and SFAs (carrot being an exception and, in the case of Because of the high antioxidant activity of thermally processed apple, no significant difference was reported), while unsaturated red-beet waste (activity increased by the presence of betacyanins), fatty acids decreased (Table 3). These results partially agree with it could be stated that the increased antibacterial activity that the previous findings of Dulf et al. (2016), who reported a decrease occurs in the presence of betacyanins directly correlates with their of PUFAs but also of MUFAs, after exposure to a fermentation pro- antioxidant activity. In all of the cases (except for the L. nucleatum cess and an increase of SFAs. A clear explanation of this partial sim- anaerobic strain), CT extract has shown zero antibacterial activity, ilarity is based on the different types of treatment (thermal and while CF had a very low inhibitory effect. By comparing the values, fermentation) within each study. PUFAs were the predominant it can be stated that carrot extracts have no antibacterial effects. fatty acids in the TLs because of the dominance of n-6 PUFAs, espe- The S. aureus strain is much more sensitive to almost all the cially linoleic acid C18:2n-6 (ranging from 43.98 – the lowest value extracts. Gram-positive bacterial strain, B. cereus, exhibits a higher in AT – to 66.3 – the highest value in WGF). resistance in comparison with S. aureus; the highest sensitivity of B. cereus was to RGT (MIC = 3.9 and MBC = 7.81), RGF and WGT 3.6. Evaluation of antibacterial effects (MIC = 7.81 and MBC = 15.62). In the case of E. faecalis, the minimum inhibitory concentration registered was 7.81 (mg/mL) for The investigated extracts exhibit antibacterial activity against AT and WGT extracts. A high inhibitory activity was registered bacteria strains. The results are summarized in Table 4. A great against the Gram-negative bacterium, E. coli, by the following range of bacteriostatic effects of the extracts were observed, extracts: RGF, RGT, BF with an MIC of 1.953 (mg/mL) and an depending on the strain studied. For L. monocytogenes, higher MBC of 3.9 (mg/mL), followed by WGT. Gram-negative bacteria, antibacterial activity was registered for RGF, RGT, and BF, with S. typhimurius and P. aeruginosa, were not as sensitive as E. coli an MIC of 1.953 (mg/mL). This result may be due to the increased toward extracts’ antibacterial effects, but red grape waste and content of phenolic compounds like anthocyanidins in red grape red-beet waste extracts, due to their high phenolic content and D.C. Vodnar et al. / Food Chemistry 231 (2017) 131–140 139 high antioxidant activity, exhibited the highest inhibitory results explained by the fact that the thermal process increases antioxi- against the studied strains (MIC = 3.9 and MBC = 7.81 for RGT). dant activity. RGT shows the highest antimutagenic potential, with Against P. aeruginosa, BT extract had the same high inhibitory an inhibition of 67.79% toward S. typhimurium. Some authors sta- effect as RGT extract. In the case of anaerobic strains, RGT had ted that plant compounds exhibiting antimutagenic properties the highest antibacterial effect. Against anaerobic bacteria, the have a series of possible applications in human health. With investigated extracts have relatively small antibacterial effects. In respect to TA100, apple, white- and red-grape wastes (in both almost all cases, the thermally processed samples have a better forms: fresh and thermally processed) exhibit a strong antimuta- inhibitory activity than fresh samples against studied strains. The genic activity, with higher inhibition in the case of thermally pro- highest inhibitory activity against almost all the strains is attribu- cessed samples. Carrot and red-beet waste samples register low ted to the thermally processed red-grape waste sample. A possible inhibition activity. The RGT sample shows again the highest explanation for this could be the release of phenolic compounds antimutagenic activity, with an inhibition of 71.81%, followed by during heat treatment, which directly correlates with the antibac- AF, AT, WGT and WGF. terial activity. When analyzing Table 4, it can also be concluded that the examined extracts had better inhibitory activity against 4. Conclusions Gram-positive bacterial strains than Gram-negative strains, with the least activity against anaerobic species. Overall, the results suggest that apple peel, carrot pulp, red and white grape peel and beetroot pulp/peel waste can be exploited for 3.7. Evaluation of antifungal activity their nutrients and antioxidant components and used to add value in food formulations. Hence, these results pave the way for utiliza- The antifungal activity of the investigated extracts against tion of food industry bio-waste that has bioactive potential after specific fungi is presented in Supplementary Table 1. The highest thermal treatment (10 min at 80 °C). antifungal effect was exhibited by RGT extract against Aspergillus The thermally processed samples, when compared with fresh flavus, with an MIC of 7.81 and an MFC of 15.62, followed by samples, have a stronger inhibitory activity against the studied RGF, WGT and AT. The Penicillium furniculosum and Candida parap- strains (bacteria and fungi). The carrot extracts have no antimicro- silosis strains were very resistant to the extracts’ antifungal effects; bial effects, while the best inhibitory activity against almost all the in the case of thermally processed white- and red-grape waste strains was attributed to thermally processed red-grape waste. extracts, no inhibitory activity is observed against Penicilinium. The increase in total antioxidant activities of thermally pro- Red-beet waste extracts exhibit a very small antifungal effect cessed samples shows that the antioxidant activity is enhanced against all studied strains. by thermal processing. Red-grape waste extract exhibited the highest scavenging activity, with a significant increase of 58.37% 3.8. Evaluation of mutagenic and antimutagenic activity (p < 0.05) in radical inhibition capacity after thermal processing. The fatty acid content is significantly different between fresh The extracts’ potential antimutagenicity toward S. typhimurium and thermally processed samples. Among all fatty acids measured, TA98 and TA100 was investigated. The results are summarized in linoleic acid (C18:2n-6) has the highest value in all analyzed sam- Table 5. With respect to S. typhimurium TA98, this assay indicates ples, with a peak of 66.3% of TLs in the WGF sample; however, its strong antimutagenic activity for AT, white- and red-grape waste content decreases during thermal processing. extracts (both forms) and BF. A moderate inhibition is exhibited The presence of bioactive compounds such as fatty acids and by AF and BT, with an inhibition of 37.95% and 34.35%, respec- phenolic compounds in agro-industrial waste enables fruits and tively. Regardless the type of extract, carrot had no antimutagenic vegetable leftovers to become more valuable for the food industry. activity toward S. typhimurium TA98, showing an inhibition of Use of these leftovers can provide an extra source of income, while 0.52%. In the case of white- and red-grape waste samples, an inter- in the same time reducing the waste disposal problem. esting observation is noticed: in both cases, the higher inhibition is exhibited by thermally processed samples. This aspect can be Conflict of interest

The authors have no conﬂicts of interest to declare.

Table 5 Antimutagenicity assay for S. typhimurium TA98 and TA100 bacterial strains. Acknowledgements

Test item Number of revertants This work was supported by the Partnership in Priority Areas TA 98 TA100 Programme-PNII, developed with the support of MEN-UEFISCDI Mean ± S.D. Inhibition% Mean ± S.D. Inhibition% (Project No.154 [PN-II-PT- PCCA-2013-4-0743]). Negative Control 9.45 ± 3.5a 9.45 ± 2.21a AF 121 ± 3.6 37.95 145 ± 6.5 58.8 AT 110 ± 2.1 43.58 136 ± 4.9 61.36 Appendix A. Supplementary data CF 194 ± 5.6 0.52 298 ± 7.2 15.34 CT 185 ± 4.7 5.12 295 ± 5.4 16.19 Supplementary data associated with this article can be found, in WGF 87 ± 3.76 55.38 189 ± 4.5 46.30 the online version, at http://dx.doi.org/10.1016/j.foodchem.2017. WGT 83 ± 2.54 57.43 174 ± 6.2 50.56 03.131. RGF 71 ± 3.16 63.58 115 ± 3.6 67.32 RGT 62.8 ± 3.0 67.79 99.2 ± 5.4 71.81 BF 113 ± 2.9 42.05 290 ± 6.8 17.61 References BT 128 ± 3.4 34.35 310 ± 6.4 11.93 b 4-NPD 195 ± 11.2 – – – Arscott, S. A., & Tanumihardjo, S. A. (2010). Carrots of many colors provide basic b NaN3 – – 352 ± 14.26 – nutrition and bioavailable phytochemicals acting as a functional food. Comprehensive Reviews in Food Science and Food Safety, 9(2), 223–239. a Values expressed are means ± S.D. of three replicates. b Bajalan, I., Mohammadi, M., Alaei, M., & Pirbalouti, A. G. (2016). Total phenolic and 4-NPD and NaN3 were used as positive controls for S. thyphimurium TA98 and ﬂavonoid contents and antioxidant activity of extracts from different TA100 strains, respectively. populations of lavandin. Industrial Crops and Products, 87, 255–260. 140 D.C. Vodnar et al. / Food Chemistry 231 (2017) 131–140

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