Impaginato 225 Adv. Hort. Sci., 2017 31(4): 225-233 DOI: 10.13128/ahs-20814 Detection of not allowed food-coloring additives (copper chlorophyllin, copper- sulphate) in green table olives sold on the Italian market C. Negro, L. De Bellis, E. Sabella, E. Nutricati, A. Luvisi, A. Miceli Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università di Salento, Via Monteroni, 165, 73100 Lecce, Italy. Key words: adulteration, Cu-chlorophyllin, E141ii, high performance liquid chro- matography, table green olives. Abstract: Table olives are a common and well-known food in the whole Mediterranean area, produced and consumed in great quantities. Many deep- green olives can be found on sale in the South of Italy. Sometimes a deep color could be the result of the fraudulent addition of a coloring agent (E141ii, cop- per chlorophyllins) during the pickling process, in spite of the European Union legislation that does not allow the addition of any colorant to fruits included table olives. The objectives of this study were to use a relatively simple method of detection of E141ii added to table olives, to verify the presence on the Italian market of artificially colored table olives, and to show that also CuSO4 can be employed for table olive re-greening. Compounds with chromatographic and spectral characteristics similar to the ones from the E141ii (Cu chlorin e6, Cu isochlorin e4, Cu pyropheophorbide a) were found in 8 samples out of 16. These results show that the fraudulent addition of colorant to table olives is a quite common practice. More pressing controls and analysis are required to ensure the complete food safety and the compliance with the current law. 1. Introduction Table olives are a common and well-known food in the whole Mediterranean area, produced and consumed in great quantities espe- cially in Southern Italy. Manufacturing techniques have been refined along the years, in order to optimize the quality and attractiveness of the final product and to cover different market niches. Table olives found on market’s shelves exhibit different colors and shapes, depending on culti- var type, ripening stage or processing method. There are green olives, spotted and fully ripened ones. In Southern Italy the Greek method and the Spanish method are commonly used to produce green table olives, while the Castelvetrano method covers a smaller but increasing market. With the Greek method, olives are washed with water and then stored in brine (5-8%). The addition of salt promotes the product preservation (*) Corresponding author: carmine.negro@unisalento.it Citation: NEGRO C., DE BELLIS L., SABELLA E., NUTRICATI E., LUVISI A., MICELI A., 2017 - Detection of not allowed food-coloring additives (copper chloro- phyllin, copper-sulphate) in green table olives sold on the italian market. - Adv. Hort. Sci., 31(4): 225-233 Copyright: © 2017 Negro C., De Bellis L., Sabella E., Nutricati E., Luvisi A., Miceli A. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 16 June 2017 Accepted for publication 22 September 2017 AHS Advances in Horticultural Science Adv. Hort. Sci., 2017 31(4): 225-233 226 and the development of fermentation-capable microorganisms. During this stage of treatment, olives slowly lose their bitterness (due to oleu- ropein’s enzymatic degradation) and acquires their final taste and properties. The development of lactic fermentation is favoured (olives average final pH of 5.2), in order to improve both the food safety and its organoleptic characteristics (Piga et al., 2001). The Spanish method, the most used worldwide, requires the olives to be treated with a solution of sodium-hydroxide and water in order to hydrolyze most of the oleuropeinic glucosides. Afterwards, olives are washed repeatedly with water and then stored in brine (5-6%) in which they naturally under- go a complete lactic fermentation, reaching an aver- age pH value of 4 (Garrido-Fernandez et al., 1997). Olives treated with the Castelvetrano method are plunged for 10 to 15 days in a sodium-hydroxide solution which concentration depends on olive cal- iber and their ripening stage. Later on, marine salt (NaCl) will be slowly added to the solution. After such treatment drupes will undergo several washings with water (Salvo et al., 1995) so that, in absence of a lac- tic fermentation, pH never goes below 6.5 (Fodale et al., 2007). Anyway, Castelvetrano type olives appears brilliant-green and with more compact pulp (Owen et al., 2003), so that they are preferred to green table olives prepared with the Greek or Spanish methods. As a matter of fact, treatment and storage in acid solutions are known to be the main responsables of chlorophylls degradation in table olives (Minguez Mosquera et al., 1989). The loss of the magnesium from the chlorophylls causes to turn into the corre- sponding pheophytins, shifting their color from green to brown (Scotter et al., 2005). At the end of the vari- ous pickling processes, therefore, olives had lost most part of their original chlorophylls (and of their original dye) becoming yellowish-green; in spite of that, many deep-green olives can be found on sale in the South of Italy, especially in the Bari Province. Their sometimes unnatural colour could be the result of the fraudulent addition of a colouring agent during the pickling process, in spite of the European Union legislation does not allow the addition of any colorant to fruit and table olives (EU, Reg CE 94/36/CE, 1994). Among the most used green food colorants, there are the chlorophyll-derived ones; Copper complexes of chlorophyillins, particularly, play a major role with- in this group. Copper chlorophyllins are manufac- tured by chlorophyll saponification which, in turn, is extracted by means of organic solvents from edible plant, such as alfalfa (Medicago sativa L.) and nettle (Urtica dioica L.) (Mortensen, 2006). The resulting chlorophyll-based salts are marketed as E141 col- orant and are available in two forms: E141i (liposolu- ble) and E141ii (hydrophilic). These salts are widely used as food colorants (e.g. ice creams, snacks, food decorations) but their addition to table olives and other fruits is expressly forbidden. In spite of that, E141ii (due to its hydrophilic properties and color stability) is sometimes fraudulently added to table olives during the pickling process as re-greening agent. The objectives of this study were to confirm the presence on the market of artificially colored table olives through the detect fraudulent color adulter- ation with E141ii, and to verify if the addition of Copper-sulphate during the pickling process could allow a re-greening of table olives as result of its interaction with Chlorophylls, leading to the forma- tion of Cu-chlorophyllins. Nowadays, Copper-sul- phate is not allowed and not even mentioned in food legislation, but it is known that it has been widely uti- lized by food manufacturers because of its re-green- ing properties on preserved vegetables (Cerutti, 2006) but its use is very dangerous because copper is toxic to the liver. Continuous consumption of olives treated with this compound could result in damage to the consumer due to its accumulation in the body (Stern, 2010). All this to generate a warning for con- sumers and public authorities. 2. Materials and Methods Chemicals All reagents were analytical or HPLC grade: Hexane and Water were supplied from Romil Ltd. (Cambrdge, UK); Methanol from Carlo Erba Reagents (Rodano, Italy); Acetic acid and tert-butyl metyl ether from J.T. Baker (Deventer, Netherlands). Chlorophyll a and b, pheophorbide a and copper sulfate were purchased from Sigma-Aldrich Co. (Saint Louis, USA). Sample of E-141ii colorant were supplied by Chimica D’agostino (Bari, Italy). Plant material Obviously deep-green table olives may have been fraudulently colored, while table olives appearing from pale-green to mustard-yellow are to be consid- ered not colored. Therefore, 16 table olives samples were purchased from local markets (Bari, Brindisi and Lecce): 9 olives were with a brilliant-green or deep- Negro et al. - Detection of not allowed food-coloring additives in green table olives in italian market 227 green color, while drupes from the remaining 7 sam- ples were pale-green or mustard-yellow. pH values of the brine were recorded with a PC 650 probe (Eutech instruments, Singapore). Due to the lack of informa- tion reported on the labels, we could not assess the cultivar and origin of each olive sample (Table 1). Pigment extraction All procedures were performed under dimmed green light to avoid any photo-oxidation of chloro- phylls. 40 g of olive pulp were collected and homoge- nized with 25 ml of Methanol-water solution (80/20, v/v) using an Ultra-Turrax T25 (Janke e Kunkle, IKA- Labortechnik, Germany); the resulting paste was fil- tered by means of a Buckner’s funnel with a paper fil- ter (Perfecte 2 extra rapida, Superfiltro Milano, Italy) and a suction flask connected to a vacuum pump. The solid residue was collected, added to 50 ml of Methanol and stirred for 1 hour; the resulting solu- tion underwent a second filtration step with the same procedure previously used. For same samples appearing still green, 50 ml of Methanol were added and a third stirring-filtering cycle has been per- formed. The filtrate was then mixed with an identical amount of hexane in a separating funnel in order to separate the lipophilic substances from the hydrophilic ones. The latter phase was recovered and evaporated under vacuum in a RE 111 rotavapor (Büchi, Flawil, Switzerland) at room temperature. The evaporation remnant was finally diluted up to 2 ml with Methanol and utilized for the HPLC/DAD analy- sis. This procedure was made up for extraction hydrophilic pigments preferably. CuSO4 addition tests In order to verify the possibility of re-greening using copper sulphate during the olive production process or the marketing, samples of olives were treated with CuSO4. It is known, in fact, that the addi- tion of copper stabilizes the tetrapyrrolic ring of pheophytin, resulting a re-greening of olive drupes (NIIR, 2004). A local food company provided us a sample of olives processed with the Castelvetrano method and stored in a Sodium-hydroxide/salt/water solution (pH 11.5). Four groups of 10 olives were taken from the sample: the first one was washed with water for 24 hours, stored in brine (6% NaCl, 0.6% Citric acid, 0.05% Ascorbic acid, pH: 2.3) for 48 hours (pH stabi- lized at 7.3) and then analyzed; the remaining 3 sub- samples were placed in beakers with 150 ml of their original packing solution, 1.5 g (1%), 7.5 g (5%) and 30 g (20%) of CuSO4 were then added. After 3 hours of stirring, the coloring solution was discarded and the samples were washed for 24 hours with water. The samples were then stored in brine solution for 48 hours until pH stabilization (pH 5.4). After the brine storage, all the sub-samples went through the before mentioned pigments extraction procedure. Another trial was run to test the effect of CuSO4 addition on olives treated with the classical Spanish method. 10 olives were selected from a sample which analysis already excluded any fraudulent colouration (S16). Drupes were first put in an alkaline solution (150 ml of 1% NaOH-water solution, pH: 12.8) until pH stabi- lization was reached (pH: 8.3), 30 g (20%) of CuSO4 were then added to this medium. The drupes were then immersed in brine solution; after 48 hours the pH reached a stable value of 4.0. The sample was then treated until pigment extraction following the already cited procedures. Analysis of chlorophylls compounds by HPLC/DAD The determination of pigment products were car- ried out by HPLC using a Agilent 1100 liquid chro- matograph fitted with a manual injector. A stainless steel column, Alltech Prontosil C30, 200 Å, 5 µ, 250× 4.6 mm I.D. was used. The column was protected by precolumn packed with the same material. Separation was performed using an elution gradient (flow rate 1 ml min-1) with the mobile phases (A) Methanol: distilled water: Acetic acid (90:10:0.5 v/v/v) and (B) tert-butyl metyl ether: Methanol: Acetic acid (100:10:0.5 v/v). The gradient scheme was 0-50% B in 30 minutes, 50-100% B in 10 minutes, 100% B for 5 minutes and 100-0% B in 5 minutes. Sample pH Colour Cultivar Origin S1 5.4 G n.r. n.r. S2 5.9 G n.r. Greece S3 7.1 G n.r. Greece S4 5.7 G Nocellara del Belice Italy (Sicily) S5 6.1 G n.r. n.r. S6 5.9 G n.r. Italy S7 5.1 G n.r. Italy S8 5.7 Y n.r. n.r. S9 6.4 G n.r. n.r. S10 4.2 Y Bella di Cerignola n.r. S11 3.8 Y n.r. n.r. S12 4.1 Y n.r. n.r. S13 5.0 Y n.r. n.r. S14 3.3 Y n.r. n.r. S15 4.0 Y n.r. n.r. S16 4.4 G n.r. n.r. Table 1 - Main characteristics of the olives samples analyzed G= green; Y= pale-green or mustard-yellow. n.r.= not reported. Adv. Hort. Sci., 2017 31(4): 225-233 228 Sequential detection was performed with a photodi- ode array detector (DAD) at 430 nm and 650 nm also the online UV-vis spectra were recorded from 250 to 800 nm. 100 µl of 1 mg/ml E141ii in methanol or 100 µl of extract was utilized for the analysis. To analyze the samples treated with CuSO4 the flow rate was lowered at 0.7 ml/min and the elution of B was set to 0-45% in 30 minutes, 45-100% in 10 minutes, 100% for 5 minutes and 100-0% in 5 minutes. Data were collected and processed with a LC Agilent ChemStation (revision software B.04.02). Pigments were identified by co-chromatography with authentic samples and/or by comparison their spectral charac- teristics with literature or compound standard when available. All analysis were made in duplicate. 3. Results and Discussion In order to identify and recognize the E141ii com- ponents likely to be present in olive samples, indus- trial copper chlorophyllin was analyzed by HPLC/DAD, injected and monitored at 650 nm. The resulting chromatogram shows 8 major peaks indi- cates with a to h in figure 1. The analysis of the elu- tion times and absorption spectra of the peaks (Fig. 2) compared with literature (Inoue et al., 1994; Chernomorsky et al., 1997; Mortensen and Geppel, 2007; Roca et al., 2010; Aparicio-Ruiz et al., 2011; Gandul-Rojas et al., 2012) allowed the tentative iden- tification of 7 of the 8 peaks: (a) Cu rhodin g7, (b) Cu chlorin e6, (c) Cu chlorin p6, (d) Cu isochlorin e4, (e) Cu 151-OH-lactone-pheophytin a (g) Cu rhodochlorin, (h) Cu pyropheophorbide a. We were unable to iden- tify without any doubt peak f. The main components of our E141ii standard eluted according to Montensen and Geppel (2007) procedure which uti- lize a chromatographic method similar, were: Cu Rhodin g7 < Cu Chlorin e6 < Cu Isochlorin e4 < Cu Pyropheophorbide a. Figures 3 and 4 show chromatograms resulting from the HPLC/DAD analysis of the studied samples; they have been recorded at 650 nm to facilitate the detection of chlorophylls and their derivatives, avoid- ing in the same time interferences from carotenoids. In 7 samples out of 16 (Fig. 3) we assessed the pres- ence of compounds with chromatographic character- istics similar to the ones from the industrial chloro- phyllin sample: Cu isochlorin e4 (peak d) and Cu pyropheophorbide a (peak h). This finding agrees with bibliographical data, since Cu isochlorin e4 is referred as one of the main component of commer- cial copper chlorophyllins (Inoue et al., 1994; Chernomorsky et al., 1997; Ferruzzi et al., 2002). Cu chlorin e6 (peak b) were found in 2 samples. Finally, the presence of Cu rhodin g7, Cu chlorin p6, Cu 151- OH-lactone-pheophytin a and Cu rhodochlorin was never observed. This result is supported by the work of Gandul-Rojas et al. (2012) which demonstrates Fig. 1 - HPLC/DAD analysis of food colorant E141ii, recorded at 650 nm, showing the peaks of the main identified com- ponents: a) Cu rhodin g7; b) Cu chlorin e6; c) Cu chlorin p6; d) Cu isochlorin e4; e) 151 OH lactone pheophytin a; f) unknown; g) Cu rhodochlorin; h) Cu pyropheophor- bide a. Fig. 2 - Spectra UV/Vis of the Copper chlorophyllins pigments found in the colorant E141ii and in some samples of green table olives. Negro et al. - Detection of not allowed food-coloring additives in green table olives in italian market 229 that the addition of E141ii to table olives led to the chromatographic identification of Cu-chlorin type compounds (mainly Cu chlorine e6 and Cu isochlorin e4) very different from chlorophyll derivatives usually found in green table olives. Moreover, Minguez- Mosquera et al. (1995) showed that under certain (still unexplained) circumstances, small amounts of Cu-chlorophyll compounds can be spontaneously synthesized within the olives, leading to localized pig- ment alteration on their surface (green staining alter- ation). The visual analysis of our samples led us to notice no traces of green staining on the drupes: Cu- compounds identified in our samples must therefore be the result of a colorant addition. In those samples we were also able to identify several other pigments, marked with numbers, such as chlorophyll b (peak 4; Fig. 3-S1); pheophitin a (peak 5; Fig. 3-S6 to 3-S8) and isochlorin e4 (peak 7; Fig. 3-S5 to 3-S8). Cu-compounds were never found in 8 of the ana- lyzed samples (Fig. 4). On the other hand, those sam- ples contained pigments identified as chlorophylls (e.g chlorophyll a; peak 6; Fig. 4-S9, 4S11, 4-S13, 4S16) and chlorophyll derivatives (e.g. pheophorbide a; peak 2; Fig. 4-S13, 4-S15, 4-S16, pheophytin a; peak 5; Fig. 4-S10 to 4-S16). Fig. 3 - HPLC/DAD chro- matograms recorded at 650 nm of the samples (S1-S8) con- taining Cu-compo- nents related to ones from E141ii col- orant. Peaks described in Table 2. Adv. Hort. Sci., 2017 31(4): 225-233 230 Gandul-Rojas et al . (2012) show that chro- matograms of olives treated with the Spanish method exhibit a series of major peaks belonging to Mg-free chlorophyll derivatives (mainly phaeo- phytins), while chromatograms of samples treated with the Castelvetrano method show peaks from chlorophylls or degraded chlorophylls with Mg (e.g. Glycoxylic acid chlorophylls; Formyl chlorophylls) in addition to the first ones. Table 2 indicates the chromatographic, spectral characteristics and origin of the chlorophyll derivates pigments identified in the commercial E141ii and in the olive samples. The results obtained are in agree- ment with the literature (Hynninen 1973; Inoue et al., 1994; Chernomorsky et al., 1997; Mortensen and Geppel, 2007; Roca et al., 2010; Aparicio-Ruiz et al., 2011; Gandul-Rojas et al., 2012). The pigments iden- tified are Cu chlorophyllins (peaks a-h), chlorophylls and derivatives (peaks 1-7). The analysis of the 9 samples initially suspected of have been fraudulently colored (indicated as G in Table 1), largely confirmed our hypothesys: 8 sam- ples contains Cu compounds related to the E141ii col- orant (Fig. 3). On the other hand, 6 out of the 7 sam- ples chosen accordingly to their pale-green or mus- tad-yellow dyes, do not contain compounds related to the food coloring agent, underlining that the addi- tion of E141ii during the pickling process could be the Fig. 4 - HPLC/DAD chro- matograms recorded at 650 nm of the sam- ples (S9-S16) do not contain Cu-compo- nents related to ones from E141ii colorant. Peaks described in Table 2. Negro et al. - Detection of not allowed food-coloring additives in green table olives in italian market 231 main responsible of the bright or deep-green dye of the analyzed olives. Concerning the addition of Copper-sulphate, the re-greening made at alkaline pH was more pro- nounced in samples treated with the addition of 5 and 20% CuSO4 in comparison respect to olives treat- ed with 1% CuSO4. In fact, untreated samples do not show peaks referable to Cu-chlorophyll compounds (Fig. 5-S17), while samples treated with 1, 5 and 20% of CuSO4 show well defined peaks in their chro- matograms: 2 of them were identified as Cu chlorin e6, (peak b) and Cu pyropheophorbide a (peak h) (Fig. 5-S18; 5-S19; 5-S20). The sample previously processed with Spanish method (therefore at acid pH) and treated with CuSO4, shows a lighter colour change with respect to the other three samples. This is supported by the very low adsorbance values of Cu chlorin e6 (peak b) pointed out in its chromatogram (Fig. 5-S21). The Copper of CuSO4 seems therefore to react only at alkaline pH (before the final acidification) with the degraded chlorophylls within the olives the same way it reacts with saponificated chlorophylls during industrial colorant production, leading to the synthesis of Cu-chlorophyll derivatives similar to the ones identified in the E141ii reference sample. Pigment Peak (z) Kc Soret Spectral data in HPLC eluent Sample (and/or E141ii dye) where the pig- ment is present ReferencesI II III IV V VI M R M R M R M R M R M R Chlorine e6 1 8.83 402 - - - - 500 18.02 528 25.66 (642) 9.45 662 5.47 S5 to S7, S10, S11, S13 to S16 [4] Cu rhodin g7 a 11.21 436 366 3.27 - - - - 577 6.85 - - 623 3.11 E141ii [3] Cu chlorin e6 b 12.42 408 (395) 1.21 - - - - 502 8.02 (588) 4.80 634 2.19 E141ii, S4, S8, S18 to S21 [2] [3] [4] [6] Cu chlorin p6 c 13.48 407 388 1.29 - - - - 500 19.52 (597) 9.76 640 2.22 E141ii [4] Pheophorbide a 2 17.77 408 (380) 1.53 (400) 1.10 507 9.33 537 9.34 609 9.76 666 2.01 S13, S15, S16 [2] [7] std Cu isochlorin e4 d 17.96 406 (394) 1.16 - - - - 501 18.71 (585) 11.65 627 2.20 E141ii, S1 to S7, S18 to S20 [2] [4] Cu 151-OH-lactone- pheophytin a e 19.07 412 392 1.27 - - 498 19.33 540 23.22 598 7.96 644 1.53 E141ii [5] Pheophytin b 3 20.75 436 - - 416 2.58 526 8.03 560 19.84 600 16.01 656 4.43 S6 [2] unknown f 21.93 417 (397) 1.86 - - 505 19.50 545 14.31 623 6.78 671 1.67 E141ii - Chlorophyll b 4 22.62 466 - - - - - - (550) 17.46 600 8.45 650 2.49 S1, S9 [2] std Pheophytin a 5 23.31 410 (380) 1.52 (400) 1.11 507 9.44 538 10.11 610 10.62 667 2.12 S5 to S7, S11, S13 to S16 [2] Cu rhodochlorin g 25.04 408 393 1.29 - - - - 498 21.50 (593) 9.67 636 1.83 E141ii [4] Chlorophyll a 6 26.16 430 (386) 1.70 (416) 1.12 - - (580) 9.80 618 5.10 665 1.13 S9, S11, S16 [2] std Cu pyropheophor- bide a h 26.31 424 (366) 1.78 403 1.15 510 19.60 554 606 5.44 652 1.13 E141ii, S1 to S4, S6, to S8, S18 to S20 [1] [4] [5] Isochlorin e4 7 28.55 400 - - 500 11.87 530 31.62 560 74.80 609 31.60 665 3.06 S5 to S8, S13, S16 [1] Table 2 - The chromatographic and spectroscopic characteristics of the chlorophyll derivates pigments present in the E141ii dye and in the analyzed green table olives Compounds derived from copper-free chlorophylls are indicated by numbers; the other, containing copper, present in the E141ii dye and in some samples, with letters. Retention factor. Kc = (tR - tM)/tM where tR is the retention time of the pigment peak and tM is the retention time of an unretained component. M = maximun absorbance (nm); R = quotient of absorbance at Soret band divided by absorbance at wavelength indicated. The values in parentheses indicate inflection points in the absorption spectrum. [1] Aparicio et al., 2011, [2] Gangul-Rojas et al., 2012; [3] Inoue et al., 1994; [4] Mortensen et al., 2007; [5] Roca et al., 2010, [6] Chernomorsky et al., 1997, [7] Hynninen, 1973, std = chemical standard. Adv. Hort. Sci., 2017 31(4): 225-233 232 4. Conclusions The use of a relatively simple method of detection of E141ii added to table olives shows that the fraudu- lent addition of colorant (or copper sulfate) to table olives is a quite common practice. In fact, the 50% of the analyzed samples possess copper chlorophyll pig- ments, most likely due to the addition of colorant E141ii, or, worse still, of copper sulphate during the production process. Our work has shown that in these samples there is often the presence of Cu chlo- rin e6 , Cu isochlorin e4 and Cu pyropheophorbide to which can therefore be considered markers for these adulterations. More controls and analysis are required to ensure the complete food safety and the compliance with the current law. The finding that even Copper-sulphate can be used to modify olive dye before marketing them, makes this need even more pressing, especially in relation to the severe liver damages it may cause. References APARICIO-RUIz R., RIEDL K.M., SCHWARTz S.J., 2011 - Identification and quantification of metallo-chlorophyll complexes in bright green table olives by High- Performance Liquid Chromatography-Mass spectrome- try Quadrupole/time-of-flight. - J. Agr. Food Chem., 59: 11100-11108. 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