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Enhancement of nutritionally significant constituents of black currant seeds by chemical elicitor application
Accepted Manuscript Enhancement of nutritionally significant constituents of black currant seeds by chemical elicitor application Gema Flores, María Luisa Ruiz del Castillo PII: DOI: Reference:
S0308-8146(15)01349-7 http://dx.doi.org/10.1016/j.foodchem.2015.09.006 FOCH 18088
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
7 May 2015 11 August 2015 2 September 2015
Please cite this article as: Flores, G., del Castillo, a.L.R., Enhancement of nutritionally significant constituents of black currant seeds by chemical elicitor application, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/ j.foodchem.2015.09.006
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Running-title header: Development of added-value blackcurrant seeds
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Enhancement of nutritionally significant constituents of black
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currant seeds by chemical elicitor application
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Gema Flores and María Luisa Ruiz del Castillo*
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Instituto de Ciencia y Tecnología de Alimentos y Nutrición. Consejo Superior de
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Investigaciones Científicas (ICTAN-CSIC), Juan de la Cierva 3, 28006 Madrid
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* Corresponding author: Dr. M.L. Ruiz del Castillo Instituto de Ciencia y Tecnología de Alimentos y Nutrición (ICTAN-CSIC) c/ Juan de la Cierva 3 28006 Madrid, SPAIN Telephone: 91- 5622900 Fax: 91- 564 48 53
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ABSTRACT
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seed oil contains high amounts of nutritionally desirable constituents such as ɣ-linolenic
41
acid (GLA), α-linolenic acid (ALA) and stearidonic acid (SA), as well as certain
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phenolic acids, which act as natural antioxidants. Fatty acids and phenolic acids of seeds
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from black currant cultivars after elicitation with methyl jasmonate (MJ) were
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examined. GLA contents around 25% with respect to total fatty acid content were
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measured in seeds after pre-harvest treatment of black currants with 0.02 mM MJ in
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0.05% Tween-20. High GLA samples also exhibited high SA content (higher than 10%
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with respect to total fatty acid content); however, ALA dropped (from 16 to 10%). High
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GLA content seeds also showed increased contents of gallic, caffeic, p-coumaric and
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ferulic acids. In particular, seeds from 0.02 mM MJ treated Ben Hope black currants
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exerted contents of gallic, caffeic, p-coumaric and ferulic acids of 201.4, 125.9, 201.3
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and 112.5 µg g-1 vs 124.3, 58.6, 165.4 and 95.8 µg g-1 measured in seeds from
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untreated Ben Hope black currants. Comparable results were obtained for Ben Alder
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and Ben Gairn berries. Chemical elicitation with 0.02 MJ is proposed as an industrial
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practice in such a way that, after consideration of quality issues, it would be obtained
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high added value black currant seeds.
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Keywords: black currant; by-products; fatty acids; methyl jasmonate; phenolic acids;
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seeds
Black currant seeds are obtained as a residue during juice production. Black currant
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2
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1. Introduction
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consumed as fresh fruits. Black currant berries have been largely demonstrated to have
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considerable medicinal properties attributable to their exceptional antioxidant activity
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compared with other fruits (Lister, Wilson, Sutton, & Morrison, 2002).
73
During black currant juice production, seeds are obtained in large quantities as a
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residue. Black currant seed oil contains high amounts of nutritionally desirable
75
compounds including fatty acids and phenolic acids (Ruiz del Castillo, Dobson,
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Brennan, & Gordon, 2002; Lu & Foo, 2003). Among the fatty acids occurring in black
77
currant seeds, ɣ-linolenic [GLA, 18:3 (n-6)] has attracted particular interest because of
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its positive effects on the attenuation of hypertension (Mills, Huang, & Poisson, 1996),
79
diabetes (Poisson, Narce, Huang, & Mills, 1996), and cancer (Das, 1996). Other fatty
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acids of nutritional significance in black currant seeds are α-linolenic acid [ALA, 18:3
81
(n-3)] and stearidonic acids [SA, 18:4 (n-3)]. In humans, ALA is the immediate
82
precursor to SA, which gives rise to eicosapentaenoic acid [EPA, 20:5 (n-3)], the
83
precursor of eicosanoids that posses anti-inflammatory and antithrombotic activities
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(Simopoulos, 1994).
85
GLA, ALA and SA, as well as all polyunsaturated fatty acids (PUFA), are susceptible to
86
oxidation. However, they are reasonably stable in the intact seeds due to the co-
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existence of phenolic compounds which act as potent natural antioxidants. Reports in
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the literature have described a black currant seed phenolic composition similar to that
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found in the berries, made up of phenolic acids, anthocyanins and certain flavonols (Lu
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& Foo, 2003). Phenolic acids are known as a kind of multipurpose bioactive agents for
91
their therapeutic and health protection effects (He, 2000). Bibliographic evidence has
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proved that phenolic acids can be particularly effective as potential protectors against
Black currants are mainly used to produce juices and jams although they are also
3
93
cancer and heart diseases (Jacob & Burri, 1996; Morton, Caccetta, Puddey, & Croft,
94
2000).
95
In recent years, we have studied the accumulation of various secondary metabolites in
96
plant foods through pre-harvest and post-harvest treatments with chemical elicitors. In
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particular, we have reported the promoting effect of methyl jasmonate (MJ), alone or in
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conjunction with ethanol, on the bioformation of aroma compounds in soft fruits
99
(Blanch, Flores, & Ruiz del Castillo, 2011; Blanch, & Ruiz del Castillo, 2012),
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anthocyanins in grapes (Flores, Blanch, & Ruiz del Castillo, 2015), flavonols in berries
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(de la Peña Moreno, Blanch, Flores, & Ruiz del Castillo, 2010) and lipid-derived
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compounds in potato (Ruiz del Castillo, Flores, & Blanch, 2010). Besides, increase of
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raspberry metabolomic enzyme activity (Flores & Ruiz del Castillo, 2014) as well as
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improvement of antioxidant and anti-inflammatory properties of MJ treated strawberry
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extracts have also been proved (Flores, Pérez, Gil, Blanch, & Ruiz del Castillo, 2013).
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Other researchers have also reported the positive effects of MJ treatments on disease
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resistance (Jin, Zheng, Tang, Rui & Wang, 2008) and as on phenolics (Yang et al.,
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2011) including phenolic acids in vegetables and fruits (Wang & Zheng, 2005; Wang,
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Bowman, & Ding, 2008).
110
However, concerning MJ elicitation on fatty acid formation, occasional studies have
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been accomplished to date (Czapski, Horbowicz, & Saniewski, 1992; Goldhaber-
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Pasillas, Mustafa, & Verpoorte, 2014). From these studies controversial results have
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been published in such a way that no clear conclusion about MJ impact on fatty acids
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has been reached. On the other hand reports about MJ effects on seeds are also scarce in
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the literature. In addition these reports deal with seed development rather than seed
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composition (Norastehnia, Sajedi, & Nojavan-Asghari, 2007). In fact, to the best of our
4
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knowledge, no study about MJ influence on black currant seed composition has been
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carried out so far.
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The goal of this research work was to determine for the first time the contents of fatty
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acids (GLA in particular but also others such as ALA and SA) and phenolic acids in
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pre-harvest MJ treated black currant seeds. Since seeds are obtained as a residue during
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black currant juice production, the final purpose of this study was to propose a
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procedure to obtain high-value products. After consideration of quality issues always
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required previous commercialisation, these products could be equally applied as a
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dietary supplement and as a cosmetic preparation.
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2. Materials and methods
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HPLC-grade acetonitrile was obtained from Lab Scan (Dublin, Ireland). HPLC-grade
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MeOH was supplied by VWR Inc. (Bridgeport, PA, USA). Ultrapure water was
133
collected from a purification system (Millipore Milford, MA, USA). MJ, tween 20,
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gallic, p-coumaric, caffeic and ferulic acid standards were supplied by Sigma
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(Steinheim, Germany). The methyl esters of myristic (C14:0), palmitic (C16:0), stearic
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(C18:0), arachidic (C20:0), oleic (C18:1), linoleic (C18:2), linolenic (C18:3) and
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stearidonic (C18:4) acids, sodium methoxide and 2,6-di-tert-butyl-4-methylphenol
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(BHT) were all purchased from Sigma-Aldrich (Milan, Italy). Ben Alder, Ben Hope and
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Gairn cultivars of black currants (Ribes nigrum L.) were used in this study. They were
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grown and treated in the field at the Scottish Crop Research Institute (Dundee, UK).
2.1. Samples and chemicals
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2.2. Preharvest MJ treatment
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Two-year-old black currant plants were employed for the treatments. Four plants from
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different rows were used per replicate. Black currant plants from each cultivar (Ben
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Hope, Ben Alder and Ben Gairn) were randomly divided into three different groups:
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those untreated and those subject to treatments with MJ, which was applied at two
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different concentrations. Group 1 (untreated-control plants) was not treated, group 2
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was sprayed with MJ 0.002 mM in 0.05% Tween-20, and group 3 was exposed to MJ
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0.02 mM in 0.05% Tween-20. The amounts of MJ used were 0.045 mg 100 -1 mL 0.05%
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Tween-20 and 0.45 mg 100-1 mL 0.05% Tween-20, respectively. The two treatments
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were carried out by applying a foliage-berry spray at a constant speed of 500 mL h-1 to
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runoff at the time that berries were still in green stage. Spraying was applied two more
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times at two-week intervals, in early black and later black stages, respectively. Black
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currant berries at black stages were picked up for seed extraction. Black currant seeds
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were carefully separated from pulp and skin. Approximately 1 g of intact seeds from
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each cultivar and each group were used for the analysis of both fatty acids and phenolic
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acids. Seeds were immediately dried until a moisture level of 9-10% and then stored at
161
4°C until analysis. Three replicates of the whole analytical procedure for both fatty
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acids and phenolic acids were performed for each sample. For fatty acids the overall
163
procedure included oil extraction, fatty acid methyl ester derivatization and gas
164
chromatographic (GC) analysis and for phenolic acids the overall procedure consisted
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of extraction, hydrolysis and high performance liquid chromatographic (HPLC)
166
analysis. That involves three replicates for each variety and each treatment.
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2.3. Oil Extraction
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Prior to the actual oil extraction, the dried seeds (1 g) were first cleaned by removing
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any remains of fruit, dirt and spoiled seeds. After that, 15 mL of hexane was added to
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the dried and cleaned seeds and the mixture was transferred to a test tube. The mixture
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was then homogenized for 4 min using an Ultra-Turrax blender, the crushed seeds in 15
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mL of hexane were extracted twice at 90°C for 2 h. The extracts were collected and the
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solvent was evaporated in a rotary evaporator at 35°C to dryness. Solventless lipids
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were immediately analysed in the dry extract as detailed in section 2.4. Defatted black
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currant seed residue was further extracted for phenolic acids as explained below (section
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2.5.).
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2.4. Fatty acid composition
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2.4.1. Fatty acid derivatization
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Transesterification of extracted fatty acids from black currant seeds to form fatty acid
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methyl esters (FAMEs) was performed based on the method previously proposed by
185
Ruiz del Castillo et al. (2002). 100 mg of sample was weighed and 2.0 mL of C21:0
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methyl ester internal standard solution (1.25 mg/mL) was added. Then, 2.0 mL of
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MeOH and 2.0 mL of 0.5 N sodium methoxide were also added to the mixture. The
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resulting combination was heated at 50 °C for 10 min. Subsequently, 100 µL of glacial
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acetic acid, 5 mL of saturated sodium chloride, and 3 mL of MeOH containing
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butylated hydroxytoluene (BHT; 50 ppm) were added. After shaking the tube and
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centrifuging the contents (5 min, 300 rpm), the upper layer was removed and put
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through an anhydrous sodium sulfate column. The sample was then ready for GC
193
analysis.
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2.4.2. Analysis of FAMEs by GC
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Fatty acids were analyzed in a gas chromatograph (Shimadzu model AOC-20i),
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equipped with a split/splitless injector system and a flame ionization detector (FID). To
198
accomplish the GC separations a 25 m x 0.25 mm i.d. capillary column coated with a
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0.25 µm layer of polyethylene glycol (007 Carbowax 20M, Quadrex). Helium was used
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as the carrier gas at a constant flow of 1 mL/min. Injector and detector temperatures
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were 250 °C and 320 °C, respectively. The injector was operated in the splitless mode.
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The GC column was programmed at 4 ºC/min from 70 ºC to 230 ºC (5 min). Data
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acquisition was accomplished with the Shimadzu MDGC solution system. The
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identification of the chromatographic peaks was made by comparing the retention time
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of the sample peaks with those of fatty acid methyl ester standards run under the same
206
conditions. In addition, retention time data from real-life samples were used to confirm
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the identities (Pérez, Ruiz del Castillo, Gil, Blanch, & Flores, 2015). GC analysis of
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each sample was carried out in triplicate. Total oil contents (% wt) were measured
209
gravimetrically (difference in weight before and after oil extraction) whereas total fatty
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acid contents as weight percents of seeds, were measured using the equation: AXA × CFx) × WIS
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% FAME = Σ
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where AXA = area accounts of individual FAME, AIS = area account of C21:0 methyl
215
ester internal standard, WIS = mass of internal standard added to the sample, WA = mass
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of seeds used, and CFX = theoretical correction factor relative to C21:0 methyl ester (IS)
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(Christie, 1989). Fatty acid composition was expressed as weight percents of total fatty
218
acids.
× 100 (AIS × WA)
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2.5. Phenolic acid composition
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2.5.1. Extraction and hydrolysis of phenolic acids
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Defatted black currant seed residues (approximately 0.6 g) were extracted twice with
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acidified methanol (4 mL) containing 1% (v/v) 1.2 M HCl. Tert-butylhydroquinone
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(TBHQ, 3.0 × 10-3 M) was added to the mixture which was then stirred at 90°C under
226
reflux for 2 h to hydrolyse the phenolic acid derivatives to the corresponding free forms.
227
Between extractions, the sample was centrifuged for 10 min at 2000 rpm. The combined
228
supernatants were collected, filtered through Whatman No. 1 filter paper and evaporated
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to dryness. The residual crude methanolic seed extract was weighed and immediately
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analysed. Extractions were accomplished in duplicate.
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2.5.2. Determination of phenolic acids
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A Konik-Tech model 560 (Barcelona, Spain) liquid chromatograph fitted with a manual
234
injection valve (model 7725i, Konik-Tech, Barcelona, Spain) and having a 20-µl sample
235
loop was used for the analyses. The separation was accomplished on a ODS reverse
236
phase (C18) column (250 nm × 4.6 mm i.d., 5-µm particle size, ACE, Madrid, Spain).
237
The elution was performed by using a solvent A (water containing 0.1% TFA) and B
238
(MeOH/CN, 80/20) at a constant flow rate of 1.0 ml/min. A linear gradient was applied
239
from the initial eluent composition (A:B, 70:30, v/v) up to A:B, 55:45 (v/v) for the first
240
5 min and then up to A:B, 45:55 (v/v) within 5 min. Subsequently, the composition was
241
modified up to A:B, 20:80 (v/v) within 10 min and finally to 100% B within another
242
more 10 min. The ultraviolet (UV) detector was programmed at 280 nm for the first 10
243
min, to detect gallic acid, and then at 320 nm until the end of the analysis for caffeic, p-
244
coumaric and ferulic acids. Blanks between consecutive runs were performed to assure
245
the washing of the equipment. Stock solutions of the standard compounds were
9
246
prepared in 70% (v/v) methanol to final concentration of 1 mg/mL. Each stock solution
247
was further diluted to obtain six concentrations of the standard. Calibration curves of
248
the standards were established on six data points, and each standard dilution was
249
injected in triplicate. Peak areas for the extracts and standards were integrated by use of
250
Konikrom Plus (KNK-725-240).
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2.6. Statistical analyses
253
Analysis of variance (ANOVA) of data on the influence of pre-harvest MJ treatment of
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black currant cultivars on the content of free fatty acids and phenolic acids in the seeds
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was performed using JMP Statistics software package version 8 (purchased from SAS
256
Institute Inc., North Carolina). The effect of the treatments was assessed by the Fisher
257
test. Differences between data were compared by least significant differences (LSD).
258
The values used were always the mean of the three replicates performed. Differences at
259
p ≤ 0.05 were considered to be significant.
260 261
3. Results and discussion
262
Table 1 represents the oil and total fatty acid (TFA) contents (weight percent in seeds)
263
from control and MJ treated black currants of different cultivars (ie, Ben Hope, Ben
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Alder and Ben Gairn). To estimate TFA, only the major FA (ie, myristic palmitic,
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stearic, arachidic, oleic, linoleic, linolenic, and stearidonic acids) were taken into
266
account. Although some other minor peaks were also detected, their identification was
267
uncertain and they only represented 8% of the total area. For this reason they were
268
considered negligible. For each sample, both the oil and TFA contents were determined
269
for three replicates. As seen in the table, the oil content of control black currant seeds
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was on the range of 25-30 w%. This oil content was considered to be approximately
10
271
equal to TFA content, but with the addition of small amounts of glycerol and
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nonsaponifiable matter. The values estimated in the present study are slightly higher
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than those previously reported in black currant seeds from different genotypes, in which
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total oil content values varied between 12 and 21% (Ruiz del Castillo, Dobson,
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Brennan, & Gordon, 2004; Dobson et al., 2012; Bada, León-Camacho, Copovi, &
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Alonso, 2014). There are several factors that can affect blackcurrant seed oil content,
277
such as breeding conditions, temperature and/or rainfall. As also seen in Table 1, MJ
278
treatment did not have significant effect on either oil or TFA contents whatever the
279
cultivar. It is probable that MJ affects differently individual fatty acids in such a way
280
that they are balanced in the total content.
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The fatty acid compositions (weight percent of total fatty acid) in seeds from control-
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untreated and MJ treated black currant cultivars are represented in Table 2. To get an
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insight of repeatability of the analytical method used, the Relative Standard Deviation
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(RSD) values calculated from three replicates for each sample (same variety and same
285
treatment) were estimated. The values obtained for fatty acid composition were in all
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cases < 6.2%. To evaluate differences between varieties and treatments, statistical
287
analysis was carried out. As a result, fatty acid composition in untreated black currant
288
seeds was statistically similar between cultivars. Besides, this composition agreed well
289
with those reported elsewhere for black currant seed oil (Gunstone, 1992; Ruiz del
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Castillo et al., 2002). Linoleic acid (LA) was the major component representing around
291
40-45% of TFA. We focused this study on MJ effect on the levels of ALA, SA and,
292
particularly, GLA because of their nutritional relevance. The ALA, SA and GLA
293
contents in untreated black currant seeds varied from 12.9 to 16.2%, from 3.2 to 4.5%
294
and from 16.2 to 18.8%, respectively depending on the cultivar. These values were in
295
the same range as that earlier described for seeds of black currant genotypes (Ruiz del
11
296
Castillo et al., 2002). As observed in Table 2, MJ only brought about variations in fatty
297
acid contents when using 0.02 MJ concentrations. Specifically, significant increase of
298
GLA and SA contents were measured whereas LA and ALA declined significantly after
299
elicitation with 0.02 MJ. However, this did not happen when lower MJ concentrations
300
(ie, 0.002) were applied. This general trend was cultivar independent, although the
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specific variations were differently marked according to the cultivar. For instance, Ben
302
Hope was the only cultivar which exhibited significant changes in the levels of the four
303
most relevant fatty acids (ie, LA, ALA, GLA and SA) after 0.02 mM MJ treatment.
304
Statistical comparisons between cultivars indicate that no significant differences could
305
be established between their behaviour to MJ treatments. The three of them exerted
306
from a statistical standpoint similar response to chemical elicitation. From Table 2, it is
307
also seen that MJ did not affect myristic (14:0), palmitic (16:0), stearic (18:0), arachidic
308
(20:0) and oleic acids (18:1) whatever MJ concentration. Little information about the
309
effects of chemical elicitation on fatty acid content is available. The results on MJ effect
310
herein shown are in accordance with those documented by Czapski et al. (1992) in
311
tomato fruit. They have also described decrease of LA content and no MJ effect on
312
myristic, palmitic, stearic and oleic acids. No data have been however reported about
313
MJ effect on ALA, SA and GLA. GLA is the intermediate in the bioconversion of
314
dietary LA to eicosanoids and ALA is the immediate precursor of SA (Gunstone, 1992).
315
Both the transformations of LA to GLA and of ALA to SA by the ∆6-desaturase
316
enzyme are considered to be the rate-limiting step in the pathways (Brenner, 1977). It is
317
therefore presumed that MJ activates ∆6-desaturase enzyme and thus promotes the
318
chemical pathway conducting the bioformation of these acids. However it is also
319
interesting to realise that the drop of LA described by Czapski et al (1992) in tomato
320
and in blackcurrant seeds in the present work after MJ elicitation contrasts with
12
321
previous results on inhibitory MJ effect on lipid oxidation previously found in potato
322
found, which would result in an increase of LA content (Ruiz del Castillo et al., 2010).
323
It is believed that MJ promoting effect on ∆6-desaturase enzyme is higher than its
324
inhibitory effect on enzymes responsible for lipid oxidation, such as lipoxygenase.
325
Therefore, the overall effect observed is a decrease of LA and ALA contents. From
326
these results it is deduced that MJ treatment effect cannot be easily predicted. In fact, it
327
largely depends on the particular number of pathways and enzymes in which the
328
compounds studied are involved.
329
There are only a few significant natural sources of GLA (Clough, 2001). The highest
330
GLA content has been described in borage (Borago officinalis L.), whose content varied
331
from 17 to 25%, and black currant seed oil, whose content is lower than 20% (Clough,
332
2001). In previous studies on black currant seeds, certain genotypes exhibited GLA
333
content > 20%, which is unusual for black currants (Ruiz del Castillo et al., 2004).
334
However, these genotypes did not show high SA content, which is also interesting
335
because of its biological properties. In the present work, we obtained seeds with GLA
336
content > 25% and SA content > 10% after chemical elicitation of black currants with
337
0.02 MJ. These values are higher than those reported for black currant seeds so far. It is
338
also worth mentioning that high GLA and SA content samples showed however ALA
339
content < 13%. Even so, black currant seeds treated with 0.02 MJ are recommendable
340
particularly for patients with certain pathologic conditions in which ∆6-desaturase
341
enzyme activity is impaired (Leventhal, Boyce, & Zurier, 1994; Wu & Meydani, 1996).
342
Table 3 depicts contents of phenolic acids in seeds of untreated and MJ treated black
343
currant cultivars. RSD values, calculated from three replicates for each sample, for
344
phenolic acid composition were in all cases < 10.1%. By comparing untreated-control
345
samples between cultivars, contents of phenolic acids were cultivar-independent;
13
346
although p-coumaric acid was always the most abundant followed by gallic, ferulic and
347
caffeic acids. Based on the extraction method used, the phenolic acid contents measured
348
were considered to be hydrolysis products of the corresponding glycosides (Stoehr &
349
Herrmann, 1975). Although the contents of phenolic acids here found in black currant
350
seeds are higher than those described in the berries (Jakobek,
351
Medvidovic-Kosanovic, 2007), similar composition between berries and seeds were
352
established being p-coumaric acid prevalent in both cases. In addition, the
353
concentrations here measured were comparable to those reported for phenolic acids in
354
seeds from fruits other than black currant (Pasko, Sajewicz, Gorinstein, & Zachwieja,
355
2008). The presence of p-coumaric acid as the major phenolic acid in black currant
356
seeds agrees with data earlier reported (Lu & Foo, 2003). These authors have also
357
described the occurrence of gallic, ferulic and caffeic acids in black currant seeds.
358
However, protocatechuic acid and p-hyroxybenzoic acid, also reported as black currant
359
seed constituents, were not detected in the present study. Compositional differences can
360
be attributed to the different clean-up method used and/or to the distinct cultivar.
361
As far as MJ effect is concerned, elicitation with 0.002 MJ concentration did not affect
362
significantly phenolic acid contents, except for p-coumaric acid in Ben Hope cultivar.
363
However, when higher MJ concentrations were sprayed to the plants (ie, 0.02 mM MJ)
364
significant increases were in general observed. In particular, gallic acid content
365
increased from 124.3 to 201.4 µg g-1, from 102.5 to 192.3 µg g-1 and from 115.9 to
366
165.7 µg g-1 in Ben Hope, Ben Alder and Ben Gairn, respectively. Caffeic acid
367
increased from 58.6 to 125.9 µg g-1, from 75.9 to 135.8 µg g-1 and from 65.4 to 150.4
368
µg g-1 in Ben Hope, Ben Alder and Ben Gairn, respectively. p-Coumaric acid increased
369
from 165.4 to 201.3 µg g-1, from 150.7 to 193.4 µg g-1 and from 165.2 to 221.3 µg g-1 in
370
Ben Hope, Ben Alder and Ben Gairn, respectively. An exception was ferulic acid,
14
Seruga, Novak, &
371
which was not significantly affected by the preharvest MJ treatments in Ben Hope black
372
currants, even when 0.02 mM MJ concentration was applied (apparent increase 95.8 to
373
112.5 µg g-1 was not significant). Increases from 88.6 to 164.9 µg g-1 and from 94.3 to
374
146.3 µg g-1 were however estimated for ferulic acid in Ben Alder and Ben Gairn,
375
respectively. It is assumed that MJ is capable of promoting phenylalanine ammonia-
376
lyase (PAL) enzyme activity resulting thus in higher contents of phenolic acids. PAL
377
enzyme regulates directly the bioformation of p-coumaric, caffeic and ferulic acids and
378
influences indirectly the pathway involved in the biosynthesis of gallic acid. Recently,
379
we have reported the impact of preharvest and postharvest MJ on PAL activity in red
380
raspberry (Flores & Ruiz del Castillo, 2014). The results reported by other authors in
381
MJ treated radish sprout also support this theory (Kim, Chen, Wang, & Choi, 2006).
382
The co-existence of high content of phenolic acids with fatty acids in black currant
383
seeds not only contributes to the improvement in the health-promoting properties of the
384
seeds but also helps stabilize fatty acids (GLA, LA, SA and ALA). Our data provide
385
evidence that it is possible to obtain black currant seeds with high contents of
386
nutritionally important compounds, such as GLA and SA, and naturally occurring
387
antioxidants by the application of chemical elicitors.
388 389
4. Conclusions
390
Pre-harvest treatment of black currants with 0.02 mM MJ in 0.05% Tween-20 enables
391
high GLA content seeds to be obtained. High GLA content black currant seeds also
392
possess high level of other nutritionally desirable compounds, such as SA and certain
393
phenolic acids (gallic, caffeic, p-coumaric and ferulic acids). These phenolic acids
394
contribute to the health-promoting properties of the seeds and, in turn, act as natural
395
antioxidants avoiding oxidation of the fatty acids. The improved positive attributes of
15
396
these seeds make them interesting for food, pharmacologic and cosmetic industries.
397
Since seeds are by-products in black currant juice production, their industrial
398
application proves the potential of chemical elicitation in obtaining high added value
399
products.
400
401
Acknowledgement
402
Authors thank the Comunidad Autónoma of Madrid (Spain) and European funding from
403
FEDER program (research project S2013/ABI-3028, AVANSECAL-CM) for financial
404
support. We thank Itziar Rodríguez for her help in performing part of the experimental
405
work. Dra. Gema Flores acknowledges CSIC for her JAE-Doc.
406 407
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Preservation, 35, 417-422.
21
Table 1. Oil and total fatty acid (TFA) contents (weight %) in seeds of black currant cultivars. Data obtained from samples untreated and treated with two different MJ concentrations are included.
Black Currant Cultivars Seed content
Ben Hope Control
OIL
25.5±0.3a
0.002 MJ 23.0±0.4a
TFA
21.2±0.2a
19.0±0.5b
Ben Alder 0.02 MJ 25.7±0.2a 22.9±0.3a
Control
Ben Gairn
29.2±0.3a
0.002 MJ 24.6±0.3a
0.02 MJ 20.8±0.4a
27.2±0.5a
22.8±0.1a
18.1±0.3b
Control 28.1±0.3a
0.002 MJ 29.5±0.5a
0.02 MJ 22.6±0.1a
25.2±0.4a
29.2±0.3a
20.1±0.2a
Different letters in the same row indicate significant (p < 0.05) differences between control and treated samples
22
Table 2. Fatty acid composition (weight %) in seeds from untreated and MJ treated black currant cultivars. Data are presented as means ± SD of triplicated samples.
BEN HOPE
FATTY ACIDS
UNTREATED
BEN ALDER
0.002 MJ
0.02 MJ
UNTREATED
BEN GAIRN
0.002 MJ
0.02 MJ
UNTREATED
0.002 MJ
0.02 MJ
n-14:0
0.5±0.1a
0.7±0.2a
0.4±0.1a
0.3±0.1a
0.5±0.1a
0.4±0.1a
0.7±0.1a
0.5±0.1a
0.3±0.1a
n-16:0
5.5±0.3a
5.8±0.4a
5.3±0.3a
4.1±0.3a
4.2±0.2a
4.8±0.2a
6.5±0.2a
5.2±0.2a
6.1±0.2a
n-18:0
1.2±0.2a
1.3±0.3a
1.1±0.4a
2.3±0.4a
1.8±0.2a
1.9±0.1a
2.2±0.1a
2.3±0.1a
2.8±0.1a
18:1 (n-9)
11.1±0.5a
10.9±0.5a
10.3±0.5a
12.2±0.4a
12.9±0.4a
12.5±0.2a
11.3±0.2a
13.8±0.3a
14.0±0.4a
18:2 (n-6) (LA)
40.6±0.2a
39.9±0.3a
30.3±0.4b
45.3±0.5a
45.4±0.5a
32.2±0.3a
43.3±0.5a
43.5±0.5a
27.2±0.4a
18:3 (n-6) (GLA)
18.8±0.4a
18.3±0.3a
25.5±0.3b
16.8±0.2a
17.1±0.3a
25.3±0.2a
16.2±0.3a
18.2±0.3a
26.7±0.4b
18:3 (n-3) (ALA)
16.2±0.3a
16.9±0.2a
12.4±0.2b
12.9±0.4a
11.7±0.4a
7.2±0.1b
14.5±0.4a
11.8±0.4a
8.0±0.1b
18:4 (n-3) (SA)
4.5±0.1a
4.4±0.4a
13.2±0.3b
3.2±0.4a
3.9±0.1a
13.1±0.4b
3.8±0.2a
3.5±0.1a
13.7±0.3 b
n-20:0
1.6±0.3a
1.8±0.2a
1.5±0.2a
2.9±0.2a
2.5±0.1a
2.6±0.1a
1.5±0.1a
1.2±0.1a
1.2±0.1b
Different letter in the same row between untreated and treated samples within the same cultivar indicates significant changes at p < 0.05 level
23
Table 3. Phenolic acid contents (expressed as µg per g dry weight ± standard deviation) in seeds from untreated and MJ treated black currant cultivars. Data are presented as means ± SD of triplicated samples. BEN HOPE
PHENOLIC ACIDS
UNTREATED
0.002 MJ
BEN ALDER 0.02 MJ
UNTREATED
0.002 MJ
BEN GAIRN 0.02 MJ
UNTREATED
0.002 MJ
0.02 MJ
Gallic
124.3 ± 0.2a
130.5± 0.5a
201.4± 0.4b
102.5 ± 0.6a 110.4± 0.4a
192.3± 0.3b
115.9± 0.5a
95.4± 0.3a
165.7± 0.5b
Caffeic
58.6 ± 0.3a
65.3± 0.2a
125.9± 0.2b
75.9 ± 0.3a
69.3± 0.1a
135.8± 0.4b
65.4± 0.5a
71.3± 0.3a
150.4± 0.1b
p-Coumaric
165.4 ± 0.3a
161.3± 0.1a
201.3± 0.3b
150.7± 0.4a 183.1± 0.5b
193.4± 0.3b
165.2± 0.4a
170.6± 0.2a
221.3± 0.4b
Ferulic
95.8 ± 0.4a
101.0± 0.1a
112.5± 0.3a
164.9± 0.3b
94.3± 0.3a
92.8± 0.6a
146.3± 0.3b
88.6± 0.2a
100.3± 0.2a
Within each row, values followed by different letters are significantly at p < 0.05 level
24
HIGHLIGHTS 1. Methyl jasmonate increase GLA and SA contents in black currant seeds. 2. Methyl jasmonate increases phenolic acid content in black currant seeds. 3. Chemical elicitation enables nutritional value of blackcurrant seeds to be increased. 4. Chemical elicitation is useful to obtain value-added products.
25
S0308-8146(15)01349-7 http://dx.doi.org/10.1016/j.foodchem.2015.09.006 FOCH 18088
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
7 May 2015 11 August 2015 2 September 2015
Please cite this article as: Flores, G., del Castillo, a.L.R., Enhancement of nutritionally significant constituents of black currant seeds by chemical elicitor application, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/ j.foodchem.2015.09.006
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Running-title header: Development of added-value blackcurrant seeds
10 11
Enhancement of nutritionally significant constituents of black
12
currant seeds by chemical elicitor application
13
Gema Flores and María Luisa Ruiz del Castillo*
14
Instituto de Ciencia y Tecnología de Alimentos y Nutrición. Consejo Superior de
15
Investigaciones Científicas (ICTAN-CSIC), Juan de la Cierva 3, 28006 Madrid
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
* Corresponding author: Dr. M.L. Ruiz del Castillo Instituto de Ciencia y Tecnología de Alimentos y Nutrición (ICTAN-CSIC) c/ Juan de la Cierva 3 28006 Madrid, SPAIN Telephone: 91- 5622900 Fax: 91- 564 48 53
1
37 38 39
ABSTRACT
40
seed oil contains high amounts of nutritionally desirable constituents such as ɣ-linolenic
41
acid (GLA), α-linolenic acid (ALA) and stearidonic acid (SA), as well as certain
42
phenolic acids, which act as natural antioxidants. Fatty acids and phenolic acids of seeds
43
from black currant cultivars after elicitation with methyl jasmonate (MJ) were
44
examined. GLA contents around 25% with respect to total fatty acid content were
45
measured in seeds after pre-harvest treatment of black currants with 0.02 mM MJ in
46
0.05% Tween-20. High GLA samples also exhibited high SA content (higher than 10%
47
with respect to total fatty acid content); however, ALA dropped (from 16 to 10%). High
48
GLA content seeds also showed increased contents of gallic, caffeic, p-coumaric and
49
ferulic acids. In particular, seeds from 0.02 mM MJ treated Ben Hope black currants
50
exerted contents of gallic, caffeic, p-coumaric and ferulic acids of 201.4, 125.9, 201.3
51
and 112.5 µg g-1 vs 124.3, 58.6, 165.4 and 95.8 µg g-1 measured in seeds from
52
untreated Ben Hope black currants. Comparable results were obtained for Ben Alder
53
and Ben Gairn berries. Chemical elicitation with 0.02 MJ is proposed as an industrial
54
practice in such a way that, after consideration of quality issues, it would be obtained
55
high added value black currant seeds.
56 57
Keywords: black currant; by-products; fatty acids; methyl jasmonate; phenolic acids;
58
seeds
Black currant seeds are obtained as a residue during juice production. Black currant
59 60 61 62 63 64 65 66
2
67 68 69
1. Introduction
70
consumed as fresh fruits. Black currant berries have been largely demonstrated to have
71
considerable medicinal properties attributable to their exceptional antioxidant activity
72
compared with other fruits (Lister, Wilson, Sutton, & Morrison, 2002).
73
During black currant juice production, seeds are obtained in large quantities as a
74
residue. Black currant seed oil contains high amounts of nutritionally desirable
75
compounds including fatty acids and phenolic acids (Ruiz del Castillo, Dobson,
76
Brennan, & Gordon, 2002; Lu & Foo, 2003). Among the fatty acids occurring in black
77
currant seeds, ɣ-linolenic [GLA, 18:3 (n-6)] has attracted particular interest because of
78
its positive effects on the attenuation of hypertension (Mills, Huang, & Poisson, 1996),
79
diabetes (Poisson, Narce, Huang, & Mills, 1996), and cancer (Das, 1996). Other fatty
80
acids of nutritional significance in black currant seeds are α-linolenic acid [ALA, 18:3
81
(n-3)] and stearidonic acids [SA, 18:4 (n-3)]. In humans, ALA is the immediate
82
precursor to SA, which gives rise to eicosapentaenoic acid [EPA, 20:5 (n-3)], the
83
precursor of eicosanoids that posses anti-inflammatory and antithrombotic activities
84
(Simopoulos, 1994).
85
GLA, ALA and SA, as well as all polyunsaturated fatty acids (PUFA), are susceptible to
86
oxidation. However, they are reasonably stable in the intact seeds due to the co-
87
existence of phenolic compounds which act as potent natural antioxidants. Reports in
88
the literature have described a black currant seed phenolic composition similar to that
89
found in the berries, made up of phenolic acids, anthocyanins and certain flavonols (Lu
90
& Foo, 2003). Phenolic acids are known as a kind of multipurpose bioactive agents for
91
their therapeutic and health protection effects (He, 2000). Bibliographic evidence has
92
proved that phenolic acids can be particularly effective as potential protectors against
Black currants are mainly used to produce juices and jams although they are also
3
93
cancer and heart diseases (Jacob & Burri, 1996; Morton, Caccetta, Puddey, & Croft,
94
2000).
95
In recent years, we have studied the accumulation of various secondary metabolites in
96
plant foods through pre-harvest and post-harvest treatments with chemical elicitors. In
97
particular, we have reported the promoting effect of methyl jasmonate (MJ), alone or in
98
conjunction with ethanol, on the bioformation of aroma compounds in soft fruits
99
(Blanch, Flores, & Ruiz del Castillo, 2011; Blanch, & Ruiz del Castillo, 2012),
100
anthocyanins in grapes (Flores, Blanch, & Ruiz del Castillo, 2015), flavonols in berries
101
(de la Peña Moreno, Blanch, Flores, & Ruiz del Castillo, 2010) and lipid-derived
102
compounds in potato (Ruiz del Castillo, Flores, & Blanch, 2010). Besides, increase of
103
raspberry metabolomic enzyme activity (Flores & Ruiz del Castillo, 2014) as well as
104
improvement of antioxidant and anti-inflammatory properties of MJ treated strawberry
105
extracts have also been proved (Flores, Pérez, Gil, Blanch, & Ruiz del Castillo, 2013).
106
Other researchers have also reported the positive effects of MJ treatments on disease
107
resistance (Jin, Zheng, Tang, Rui & Wang, 2008) and as on phenolics (Yang et al.,
108
2011) including phenolic acids in vegetables and fruits (Wang & Zheng, 2005; Wang,
109
Bowman, & Ding, 2008).
110
However, concerning MJ elicitation on fatty acid formation, occasional studies have
111
been accomplished to date (Czapski, Horbowicz, & Saniewski, 1992; Goldhaber-
112
Pasillas, Mustafa, & Verpoorte, 2014). From these studies controversial results have
113
been published in such a way that no clear conclusion about MJ impact on fatty acids
114
has been reached. On the other hand reports about MJ effects on seeds are also scarce in
115
the literature. In addition these reports deal with seed development rather than seed
116
composition (Norastehnia, Sajedi, & Nojavan-Asghari, 2007). In fact, to the best of our
4
117
knowledge, no study about MJ influence on black currant seed composition has been
118
carried out so far.
119
The goal of this research work was to determine for the first time the contents of fatty
120
acids (GLA in particular but also others such as ALA and SA) and phenolic acids in
121
pre-harvest MJ treated black currant seeds. Since seeds are obtained as a residue during
122
black currant juice production, the final purpose of this study was to propose a
123
procedure to obtain high-value products. After consideration of quality issues always
124
required previous commercialisation, these products could be equally applied as a
125
dietary supplement and as a cosmetic preparation.
126 127 128 129 130
2. Materials and methods
131
HPLC-grade acetonitrile was obtained from Lab Scan (Dublin, Ireland). HPLC-grade
132
MeOH was supplied by VWR Inc. (Bridgeport, PA, USA). Ultrapure water was
133
collected from a purification system (Millipore Milford, MA, USA). MJ, tween 20,
134
gallic, p-coumaric, caffeic and ferulic acid standards were supplied by Sigma
135
(Steinheim, Germany). The methyl esters of myristic (C14:0), palmitic (C16:0), stearic
136
(C18:0), arachidic (C20:0), oleic (C18:1), linoleic (C18:2), linolenic (C18:3) and
137
stearidonic (C18:4) acids, sodium methoxide and 2,6-di-tert-butyl-4-methylphenol
138
(BHT) were all purchased from Sigma-Aldrich (Milan, Italy). Ben Alder, Ben Hope and
139
Gairn cultivars of black currants (Ribes nigrum L.) were used in this study. They were
140
grown and treated in the field at the Scottish Crop Research Institute (Dundee, UK).
2.1. Samples and chemicals
141 142 143 144
5
145
2.2. Preharvest MJ treatment
146
Two-year-old black currant plants were employed for the treatments. Four plants from
147
different rows were used per replicate. Black currant plants from each cultivar (Ben
148
Hope, Ben Alder and Ben Gairn) were randomly divided into three different groups:
149
those untreated and those subject to treatments with MJ, which was applied at two
150
different concentrations. Group 1 (untreated-control plants) was not treated, group 2
151
was sprayed with MJ 0.002 mM in 0.05% Tween-20, and group 3 was exposed to MJ
152
0.02 mM in 0.05% Tween-20. The amounts of MJ used were 0.045 mg 100 -1 mL 0.05%
153
Tween-20 and 0.45 mg 100-1 mL 0.05% Tween-20, respectively. The two treatments
154
were carried out by applying a foliage-berry spray at a constant speed of 500 mL h-1 to
155
runoff at the time that berries were still in green stage. Spraying was applied two more
156
times at two-week intervals, in early black and later black stages, respectively. Black
157
currant berries at black stages were picked up for seed extraction. Black currant seeds
158
were carefully separated from pulp and skin. Approximately 1 g of intact seeds from
159
each cultivar and each group were used for the analysis of both fatty acids and phenolic
160
acids. Seeds were immediately dried until a moisture level of 9-10% and then stored at
161
4°C until analysis. Three replicates of the whole analytical procedure for both fatty
162
acids and phenolic acids were performed for each sample. For fatty acids the overall
163
procedure included oil extraction, fatty acid methyl ester derivatization and gas
164
chromatographic (GC) analysis and for phenolic acids the overall procedure consisted
165
of extraction, hydrolysis and high performance liquid chromatographic (HPLC)
166
analysis. That involves three replicates for each variety and each treatment.
167 168 169
6
170
2.3. Oil Extraction
171
Prior to the actual oil extraction, the dried seeds (1 g) were first cleaned by removing
172
any remains of fruit, dirt and spoiled seeds. After that, 15 mL of hexane was added to
173
the dried and cleaned seeds and the mixture was transferred to a test tube. The mixture
174
was then homogenized for 4 min using an Ultra-Turrax blender, the crushed seeds in 15
175
mL of hexane were extracted twice at 90°C for 2 h. The extracts were collected and the
176
solvent was evaporated in a rotary evaporator at 35°C to dryness. Solventless lipids
177
were immediately analysed in the dry extract as detailed in section 2.4. Defatted black
178
currant seed residue was further extracted for phenolic acids as explained below (section
179
2.5.).
180 181
2.4. Fatty acid composition
182
2.4.1. Fatty acid derivatization
183
Transesterification of extracted fatty acids from black currant seeds to form fatty acid
184
methyl esters (FAMEs) was performed based on the method previously proposed by
185
Ruiz del Castillo et al. (2002). 100 mg of sample was weighed and 2.0 mL of C21:0
186
methyl ester internal standard solution (1.25 mg/mL) was added. Then, 2.0 mL of
187
MeOH and 2.0 mL of 0.5 N sodium methoxide were also added to the mixture. The
188
resulting combination was heated at 50 °C for 10 min. Subsequently, 100 µL of glacial
189
acetic acid, 5 mL of saturated sodium chloride, and 3 mL of MeOH containing
190
butylated hydroxytoluene (BHT; 50 ppm) were added. After shaking the tube and
191
centrifuging the contents (5 min, 300 rpm), the upper layer was removed and put
192
through an anhydrous sodium sulfate column. The sample was then ready for GC
193
analysis.
194
7
195
2.4.2. Analysis of FAMEs by GC
196
Fatty acids were analyzed in a gas chromatograph (Shimadzu model AOC-20i),
197
equipped with a split/splitless injector system and a flame ionization detector (FID). To
198
accomplish the GC separations a 25 m x 0.25 mm i.d. capillary column coated with a
199
0.25 µm layer of polyethylene glycol (007 Carbowax 20M, Quadrex). Helium was used
200
as the carrier gas at a constant flow of 1 mL/min. Injector and detector temperatures
201
were 250 °C and 320 °C, respectively. The injector was operated in the splitless mode.
202
The GC column was programmed at 4 ºC/min from 70 ºC to 230 ºC (5 min). Data
203
acquisition was accomplished with the Shimadzu MDGC solution system. The
204
identification of the chromatographic peaks was made by comparing the retention time
205
of the sample peaks with those of fatty acid methyl ester standards run under the same
206
conditions. In addition, retention time data from real-life samples were used to confirm
207
the identities (Pérez, Ruiz del Castillo, Gil, Blanch, & Flores, 2015). GC analysis of
208
each sample was carried out in triplicate. Total oil contents (% wt) were measured
209
gravimetrically (difference in weight before and after oil extraction) whereas total fatty
210
acid contents as weight percents of seeds, were measured using the equation: AXA × CFx) × WIS
211 212 213
% FAME = Σ
214
where AXA = area accounts of individual FAME, AIS = area account of C21:0 methyl
215
ester internal standard, WIS = mass of internal standard added to the sample, WA = mass
216
of seeds used, and CFX = theoretical correction factor relative to C21:0 methyl ester (IS)
217
(Christie, 1989). Fatty acid composition was expressed as weight percents of total fatty
218
acids.
× 100 (AIS × WA)
219 220
8
221
2.5. Phenolic acid composition
222
2.5.1. Extraction and hydrolysis of phenolic acids
223
Defatted black currant seed residues (approximately 0.6 g) were extracted twice with
224
acidified methanol (4 mL) containing 1% (v/v) 1.2 M HCl. Tert-butylhydroquinone
225
(TBHQ, 3.0 × 10-3 M) was added to the mixture which was then stirred at 90°C under
226
reflux for 2 h to hydrolyse the phenolic acid derivatives to the corresponding free forms.
227
Between extractions, the sample was centrifuged for 10 min at 2000 rpm. The combined
228
supernatants were collected, filtered through Whatman No. 1 filter paper and evaporated
229
to dryness. The residual crude methanolic seed extract was weighed and immediately
230
analysed. Extractions were accomplished in duplicate.
231 232
2.5.2. Determination of phenolic acids
233
A Konik-Tech model 560 (Barcelona, Spain) liquid chromatograph fitted with a manual
234
injection valve (model 7725i, Konik-Tech, Barcelona, Spain) and having a 20-µl sample
235
loop was used for the analyses. The separation was accomplished on a ODS reverse
236
phase (C18) column (250 nm × 4.6 mm i.d., 5-µm particle size, ACE, Madrid, Spain).
237
The elution was performed by using a solvent A (water containing 0.1% TFA) and B
238
(MeOH/CN, 80/20) at a constant flow rate of 1.0 ml/min. A linear gradient was applied
239
from the initial eluent composition (A:B, 70:30, v/v) up to A:B, 55:45 (v/v) for the first
240
5 min and then up to A:B, 45:55 (v/v) within 5 min. Subsequently, the composition was
241
modified up to A:B, 20:80 (v/v) within 10 min and finally to 100% B within another
242
more 10 min. The ultraviolet (UV) detector was programmed at 280 nm for the first 10
243
min, to detect gallic acid, and then at 320 nm until the end of the analysis for caffeic, p-
244
coumaric and ferulic acids. Blanks between consecutive runs were performed to assure
245
the washing of the equipment. Stock solutions of the standard compounds were
9
246
prepared in 70% (v/v) methanol to final concentration of 1 mg/mL. Each stock solution
247
was further diluted to obtain six concentrations of the standard. Calibration curves of
248
the standards were established on six data points, and each standard dilution was
249
injected in triplicate. Peak areas for the extracts and standards were integrated by use of
250
Konikrom Plus (KNK-725-240).
251 252
2.6. Statistical analyses
253
Analysis of variance (ANOVA) of data on the influence of pre-harvest MJ treatment of
254
black currant cultivars on the content of free fatty acids and phenolic acids in the seeds
255
was performed using JMP Statistics software package version 8 (purchased from SAS
256
Institute Inc., North Carolina). The effect of the treatments was assessed by the Fisher
257
test. Differences between data were compared by least significant differences (LSD).
258
The values used were always the mean of the three replicates performed. Differences at
259
p ≤ 0.05 were considered to be significant.
260 261
3. Results and discussion
262
Table 1 represents the oil and total fatty acid (TFA) contents (weight percent in seeds)
263
from control and MJ treated black currants of different cultivars (ie, Ben Hope, Ben
264
Alder and Ben Gairn). To estimate TFA, only the major FA (ie, myristic palmitic,
265
stearic, arachidic, oleic, linoleic, linolenic, and stearidonic acids) were taken into
266
account. Although some other minor peaks were also detected, their identification was
267
uncertain and they only represented 8% of the total area. For this reason they were
268
considered negligible. For each sample, both the oil and TFA contents were determined
269
for three replicates. As seen in the table, the oil content of control black currant seeds
270
was on the range of 25-30 w%. This oil content was considered to be approximately
10
271
equal to TFA content, but with the addition of small amounts of glycerol and
272
nonsaponifiable matter. The values estimated in the present study are slightly higher
273
than those previously reported in black currant seeds from different genotypes, in which
274
total oil content values varied between 12 and 21% (Ruiz del Castillo, Dobson,
275
Brennan, & Gordon, 2004; Dobson et al., 2012; Bada, León-Camacho, Copovi, &
276
Alonso, 2014). There are several factors that can affect blackcurrant seed oil content,
277
such as breeding conditions, temperature and/or rainfall. As also seen in Table 1, MJ
278
treatment did not have significant effect on either oil or TFA contents whatever the
279
cultivar. It is probable that MJ affects differently individual fatty acids in such a way
280
that they are balanced in the total content.
281
The fatty acid compositions (weight percent of total fatty acid) in seeds from control-
282
untreated and MJ treated black currant cultivars are represented in Table 2. To get an
283
insight of repeatability of the analytical method used, the Relative Standard Deviation
284
(RSD) values calculated from three replicates for each sample (same variety and same
285
treatment) were estimated. The values obtained for fatty acid composition were in all
286
cases < 6.2%. To evaluate differences between varieties and treatments, statistical
287
analysis was carried out. As a result, fatty acid composition in untreated black currant
288
seeds was statistically similar between cultivars. Besides, this composition agreed well
289
with those reported elsewhere for black currant seed oil (Gunstone, 1992; Ruiz del
290
Castillo et al., 2002). Linoleic acid (LA) was the major component representing around
291
40-45% of TFA. We focused this study on MJ effect on the levels of ALA, SA and,
292
particularly, GLA because of their nutritional relevance. The ALA, SA and GLA
293
contents in untreated black currant seeds varied from 12.9 to 16.2%, from 3.2 to 4.5%
294
and from 16.2 to 18.8%, respectively depending on the cultivar. These values were in
295
the same range as that earlier described for seeds of black currant genotypes (Ruiz del
11
296
Castillo et al., 2002). As observed in Table 2, MJ only brought about variations in fatty
297
acid contents when using 0.02 MJ concentrations. Specifically, significant increase of
298
GLA and SA contents were measured whereas LA and ALA declined significantly after
299
elicitation with 0.02 MJ. However, this did not happen when lower MJ concentrations
300
(ie, 0.002) were applied. This general trend was cultivar independent, although the
301
specific variations were differently marked according to the cultivar. For instance, Ben
302
Hope was the only cultivar which exhibited significant changes in the levels of the four
303
most relevant fatty acids (ie, LA, ALA, GLA and SA) after 0.02 mM MJ treatment.
304
Statistical comparisons between cultivars indicate that no significant differences could
305
be established between their behaviour to MJ treatments. The three of them exerted
306
from a statistical standpoint similar response to chemical elicitation. From Table 2, it is
307
also seen that MJ did not affect myristic (14:0), palmitic (16:0), stearic (18:0), arachidic
308
(20:0) and oleic acids (18:1) whatever MJ concentration. Little information about the
309
effects of chemical elicitation on fatty acid content is available. The results on MJ effect
310
herein shown are in accordance with those documented by Czapski et al. (1992) in
311
tomato fruit. They have also described decrease of LA content and no MJ effect on
312
myristic, palmitic, stearic and oleic acids. No data have been however reported about
313
MJ effect on ALA, SA and GLA. GLA is the intermediate in the bioconversion of
314
dietary LA to eicosanoids and ALA is the immediate precursor of SA (Gunstone, 1992).
315
Both the transformations of LA to GLA and of ALA to SA by the ∆6-desaturase
316
enzyme are considered to be the rate-limiting step in the pathways (Brenner, 1977). It is
317
therefore presumed that MJ activates ∆6-desaturase enzyme and thus promotes the
318
chemical pathway conducting the bioformation of these acids. However it is also
319
interesting to realise that the drop of LA described by Czapski et al (1992) in tomato
320
and in blackcurrant seeds in the present work after MJ elicitation contrasts with
12
321
previous results on inhibitory MJ effect on lipid oxidation previously found in potato
322
found, which would result in an increase of LA content (Ruiz del Castillo et al., 2010).
323
It is believed that MJ promoting effect on ∆6-desaturase enzyme is higher than its
324
inhibitory effect on enzymes responsible for lipid oxidation, such as lipoxygenase.
325
Therefore, the overall effect observed is a decrease of LA and ALA contents. From
326
these results it is deduced that MJ treatment effect cannot be easily predicted. In fact, it
327
largely depends on the particular number of pathways and enzymes in which the
328
compounds studied are involved.
329
There are only a few significant natural sources of GLA (Clough, 2001). The highest
330
GLA content has been described in borage (Borago officinalis L.), whose content varied
331
from 17 to 25%, and black currant seed oil, whose content is lower than 20% (Clough,
332
2001). In previous studies on black currant seeds, certain genotypes exhibited GLA
333
content > 20%, which is unusual for black currants (Ruiz del Castillo et al., 2004).
334
However, these genotypes did not show high SA content, which is also interesting
335
because of its biological properties. In the present work, we obtained seeds with GLA
336
content > 25% and SA content > 10% after chemical elicitation of black currants with
337
0.02 MJ. These values are higher than those reported for black currant seeds so far. It is
338
also worth mentioning that high GLA and SA content samples showed however ALA
339
content < 13%. Even so, black currant seeds treated with 0.02 MJ are recommendable
340
particularly for patients with certain pathologic conditions in which ∆6-desaturase
341
enzyme activity is impaired (Leventhal, Boyce, & Zurier, 1994; Wu & Meydani, 1996).
342
Table 3 depicts contents of phenolic acids in seeds of untreated and MJ treated black
343
currant cultivars. RSD values, calculated from three replicates for each sample, for
344
phenolic acid composition were in all cases < 10.1%. By comparing untreated-control
345
samples between cultivars, contents of phenolic acids were cultivar-independent;
13
346
although p-coumaric acid was always the most abundant followed by gallic, ferulic and
347
caffeic acids. Based on the extraction method used, the phenolic acid contents measured
348
were considered to be hydrolysis products of the corresponding glycosides (Stoehr &
349
Herrmann, 1975). Although the contents of phenolic acids here found in black currant
350
seeds are higher than those described in the berries (Jakobek,
351
Medvidovic-Kosanovic, 2007), similar composition between berries and seeds were
352
established being p-coumaric acid prevalent in both cases. In addition, the
353
concentrations here measured were comparable to those reported for phenolic acids in
354
seeds from fruits other than black currant (Pasko, Sajewicz, Gorinstein, & Zachwieja,
355
2008). The presence of p-coumaric acid as the major phenolic acid in black currant
356
seeds agrees with data earlier reported (Lu & Foo, 2003). These authors have also
357
described the occurrence of gallic, ferulic and caffeic acids in black currant seeds.
358
However, protocatechuic acid and p-hyroxybenzoic acid, also reported as black currant
359
seed constituents, were not detected in the present study. Compositional differences can
360
be attributed to the different clean-up method used and/or to the distinct cultivar.
361
As far as MJ effect is concerned, elicitation with 0.002 MJ concentration did not affect
362
significantly phenolic acid contents, except for p-coumaric acid in Ben Hope cultivar.
363
However, when higher MJ concentrations were sprayed to the plants (ie, 0.02 mM MJ)
364
significant increases were in general observed. In particular, gallic acid content
365
increased from 124.3 to 201.4 µg g-1, from 102.5 to 192.3 µg g-1 and from 115.9 to
366
165.7 µg g-1 in Ben Hope, Ben Alder and Ben Gairn, respectively. Caffeic acid
367
increased from 58.6 to 125.9 µg g-1, from 75.9 to 135.8 µg g-1 and from 65.4 to 150.4
368
µg g-1 in Ben Hope, Ben Alder and Ben Gairn, respectively. p-Coumaric acid increased
369
from 165.4 to 201.3 µg g-1, from 150.7 to 193.4 µg g-1 and from 165.2 to 221.3 µg g-1 in
370
Ben Hope, Ben Alder and Ben Gairn, respectively. An exception was ferulic acid,
14
Seruga, Novak, &
371
which was not significantly affected by the preharvest MJ treatments in Ben Hope black
372
currants, even when 0.02 mM MJ concentration was applied (apparent increase 95.8 to
373
112.5 µg g-1 was not significant). Increases from 88.6 to 164.9 µg g-1 and from 94.3 to
374
146.3 µg g-1 were however estimated for ferulic acid in Ben Alder and Ben Gairn,
375
respectively. It is assumed that MJ is capable of promoting phenylalanine ammonia-
376
lyase (PAL) enzyme activity resulting thus in higher contents of phenolic acids. PAL
377
enzyme regulates directly the bioformation of p-coumaric, caffeic and ferulic acids and
378
influences indirectly the pathway involved in the biosynthesis of gallic acid. Recently,
379
we have reported the impact of preharvest and postharvest MJ on PAL activity in red
380
raspberry (Flores & Ruiz del Castillo, 2014). The results reported by other authors in
381
MJ treated radish sprout also support this theory (Kim, Chen, Wang, & Choi, 2006).
382
The co-existence of high content of phenolic acids with fatty acids in black currant
383
seeds not only contributes to the improvement in the health-promoting properties of the
384
seeds but also helps stabilize fatty acids (GLA, LA, SA and ALA). Our data provide
385
evidence that it is possible to obtain black currant seeds with high contents of
386
nutritionally important compounds, such as GLA and SA, and naturally occurring
387
antioxidants by the application of chemical elicitors.
388 389
4. Conclusions
390
Pre-harvest treatment of black currants with 0.02 mM MJ in 0.05% Tween-20 enables
391
high GLA content seeds to be obtained. High GLA content black currant seeds also
392
possess high level of other nutritionally desirable compounds, such as SA and certain
393
phenolic acids (gallic, caffeic, p-coumaric and ferulic acids). These phenolic acids
394
contribute to the health-promoting properties of the seeds and, in turn, act as natural
395
antioxidants avoiding oxidation of the fatty acids. The improved positive attributes of
15
396
these seeds make them interesting for food, pharmacologic and cosmetic industries.
397
Since seeds are by-products in black currant juice production, their industrial
398
application proves the potential of chemical elicitation in obtaining high added value
399
products.
400
401
Acknowledgement
402
Authors thank the Comunidad Autónoma of Madrid (Spain) and European funding from
403
FEDER program (research project S2013/ABI-3028, AVANSECAL-CM) for financial
404
support. We thank Itziar Rodríguez for her help in performing part of the experimental
405
work. Dra. Gema Flores acknowledges CSIC for her JAE-Doc.
406 407
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Table 1. Oil and total fatty acid (TFA) contents (weight %) in seeds of black currant cultivars. Data obtained from samples untreated and treated with two different MJ concentrations are included.
Black Currant Cultivars Seed content
Ben Hope Control
OIL
25.5±0.3a
0.002 MJ 23.0±0.4a
TFA
21.2±0.2a
19.0±0.5b
Ben Alder 0.02 MJ 25.7±0.2a 22.9±0.3a
Control
Ben Gairn
29.2±0.3a
0.002 MJ 24.6±0.3a
0.02 MJ 20.8±0.4a
27.2±0.5a
22.8±0.1a
18.1±0.3b
Control 28.1±0.3a
0.002 MJ 29.5±0.5a
0.02 MJ 22.6±0.1a
25.2±0.4a
29.2±0.3a
20.1±0.2a
Different letters in the same row indicate significant (p < 0.05) differences between control and treated samples
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Table 2. Fatty acid composition (weight %) in seeds from untreated and MJ treated black currant cultivars. Data are presented as means ± SD of triplicated samples.
BEN HOPE
FATTY ACIDS
UNTREATED
BEN ALDER
0.002 MJ
0.02 MJ
UNTREATED
BEN GAIRN
0.002 MJ
0.02 MJ
UNTREATED
0.002 MJ
0.02 MJ
n-14:0
0.5±0.1a
0.7±0.2a
0.4±0.1a
0.3±0.1a
0.5±0.1a
0.4±0.1a
0.7±0.1a
0.5±0.1a
0.3±0.1a
n-16:0
5.5±0.3a
5.8±0.4a
5.3±0.3a
4.1±0.3a
4.2±0.2a
4.8±0.2a
6.5±0.2a
5.2±0.2a
6.1±0.2a
n-18:0
1.2±0.2a
1.3±0.3a
1.1±0.4a
2.3±0.4a
1.8±0.2a
1.9±0.1a
2.2±0.1a
2.3±0.1a
2.8±0.1a
18:1 (n-9)
11.1±0.5a
10.9±0.5a
10.3±0.5a
12.2±0.4a
12.9±0.4a
12.5±0.2a
11.3±0.2a
13.8±0.3a
14.0±0.4a
18:2 (n-6) (LA)
40.6±0.2a
39.9±0.3a
30.3±0.4b
45.3±0.5a
45.4±0.5a
32.2±0.3a
43.3±0.5a
43.5±0.5a
27.2±0.4a
18:3 (n-6) (GLA)
18.8±0.4a
18.3±0.3a
25.5±0.3b
16.8±0.2a
17.1±0.3a
25.3±0.2a
16.2±0.3a
18.2±0.3a
26.7±0.4b
18:3 (n-3) (ALA)
16.2±0.3a
16.9±0.2a
12.4±0.2b
12.9±0.4a
11.7±0.4a
7.2±0.1b
14.5±0.4a
11.8±0.4a
8.0±0.1b
18:4 (n-3) (SA)
4.5±0.1a
4.4±0.4a
13.2±0.3b
3.2±0.4a
3.9±0.1a
13.1±0.4b
3.8±0.2a
3.5±0.1a
13.7±0.3 b
n-20:0
1.6±0.3a
1.8±0.2a
1.5±0.2a
2.9±0.2a
2.5±0.1a
2.6±0.1a
1.5±0.1a
1.2±0.1a
1.2±0.1b
Different letter in the same row between untreated and treated samples within the same cultivar indicates significant changes at p < 0.05 level
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Table 3. Phenolic acid contents (expressed as µg per g dry weight ± standard deviation) in seeds from untreated and MJ treated black currant cultivars. Data are presented as means ± SD of triplicated samples. BEN HOPE
PHENOLIC ACIDS
UNTREATED
0.002 MJ
BEN ALDER 0.02 MJ
UNTREATED
0.002 MJ
BEN GAIRN 0.02 MJ
UNTREATED
0.002 MJ
0.02 MJ
Gallic
124.3 ± 0.2a
130.5± 0.5a
201.4± 0.4b
102.5 ± 0.6a 110.4± 0.4a
192.3± 0.3b
115.9± 0.5a
95.4± 0.3a
165.7± 0.5b
Caffeic
58.6 ± 0.3a
65.3± 0.2a
125.9± 0.2b
75.9 ± 0.3a
69.3± 0.1a
135.8± 0.4b
65.4± 0.5a
71.3± 0.3a
150.4± 0.1b
p-Coumaric
165.4 ± 0.3a
161.3± 0.1a
201.3± 0.3b
150.7± 0.4a 183.1± 0.5b
193.4± 0.3b
165.2± 0.4a
170.6± 0.2a
221.3± 0.4b
Ferulic
95.8 ± 0.4a
101.0± 0.1a
112.5± 0.3a
164.9± 0.3b
94.3± 0.3a
92.8± 0.6a
146.3± 0.3b
88.6± 0.2a
100.3± 0.2a
Within each row, values followed by different letters are significantly at p < 0.05 level
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HIGHLIGHTS 1. Methyl jasmonate increase GLA and SA contents in black currant seeds. 2. Methyl jasmonate increases phenolic acid content in black currant seeds. 3. Chemical elicitation enables nutritional value of blackcurrant seeds to be increased. 4. Chemical elicitation is useful to obtain value-added products.
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