Eur Food Res Technol (2005) 220:31–36 DOI 10.1007/s00217-004-1054-7
ORIGINAL PAPER
Rafael Llorach · Francisco A. Toms-Barbern · Federico Ferreres
Functionalisation of commercial chicken soup with enriched polyphenol extract from vegetable by-products Received: 1 March 2004 / Published online: 16 November 2004 Springer-Verlag 2004
Abstract Ready-to-eat foods such as soups are in great demand by consumers, owing to the changes in lifestyle over the last half-century. In this context, the addition of new health-promoting active ingredients such as polyphenols could represent an important way to increase the intake of these compounds. Three different by-products from artichoke, lettuce and cauliflower handling and commercialisation have been use to obtain enriched polyphenol extract using a water extraction protocol. The artichoke by-products extract was composed of caffeic acid derivatives while the lettuce and the cauliflower byproducts extract were composed of both caffeic acid derivatives and flavonols. The amounts of these compounds were evaluated with HPLC-DAD; it transpired that the artichoke by-products extract had the highest levels of polyphenols (100 mg of polyphenols/g of dry extract), followed by lettuce by-products extract (46 mg of polyphenols/g of dry extract) and cauliflower byproducts extract (34 mg of polyphenols/g of dry extract). A sensory panel with four trained judges evaluated the addition of different amounts of extracts. Both artichoke and lettuce by-products extract could be added to the soup to a maximum amount of 10 mg of extract/mL of soup and cauliflower with 5 mg of extract/mL of soup, while still improving the grade of acceptability of the soup with respect to the original soup. In addition, antioxidant capacity was evaluated as free radical scavenging activity (ABTS·+ assay) and the ability to reduce the 2,4,6-tripyridyl-s-triazine (TPTZ)-Fe(III) complex to TPTZ-Fe(II) (FRAP assay). The antioxidative capacity increased with addition of the extracts between 3.5 times and 13 times (ABTS·+ assay) as well as between 23 times and 85 times (FRAP assay). The results obtained indicate that these byproducts could provide the extracts with antioxidant R. Llorach · F. A. Toms-Barbern · F. Ferreres ()) Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC, P.O. Box 4195, 30080 Murcia, Spain e-mail:
[email protected] Fax: +34-968-396213
phenolics that could be used to functionalise foods. Obviously, before incorporating these by-product extracts as dietary complements, it is necessary to carry out further studies about their toxicity (i.e. possible residual presence of pesticides), in vivo activity, and bioavailability. Keywords Chicken soup · Polyphenol extract · Functionalisation · Sensor assessment · High-performance liquid chromatography–diode-array detection
Introduction Huge amounts of by-products are produced during the industrial processing of vegetables. These residues are responsible for important environmental problems in the industry [1], and diminishing their environmental impact has been the subject of increasing concern in recent years. In general, by-products from handling and commercialisation of vegetables have been traditionally valorised as animal feedstuff [2], in fibre production [3], and in fuel production [4]. In recent years, a number of studies have proposed some vegetable by-products being valorised as a source of natural antioxidants [5, 6, 7, 8]. In fact, artichoke, lettuce and cauliflower by-products have been proposed as cheap sources of enriched polyphenol extracts [9, 10]. The consumption of polyphenol-rich foods or beverages seems to be associated with the prevention of some types of diseases [11]. The role of flavonoids in the prevention of these diseases is mainly related to the prevention of the LDL oxidation [11, 12] through scavenging activity against peroxyl and hydroxyl radicals [12]. The modern life-style has produced an increase in the consumption of “ready-to-eat” foods (canned, refrigerated, etc) that generally show smaller amounts of healthpromoting compounds. Functional foods try to contribute to a good, balanced diet by providing foodstuffs with “added-value”: adding new ingredients that increase their health-promoting properties by increasing bioavailability of active compounds, etc. [13]. In this context, addition of
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phenolic-enriched extracts could be a feasible strategy to develop functional foods. In fact, Larrosa et al. [14] have reported that different vegetable by-product extracts could be applied to functionalise tomato juice. The aim of this work is to use artichoke, lettuce and cauliflower water extracts to functionalise commercial chicken soup, improving its phenolic profile as well as its antioxidative capacity. In addition, a trained panel was used to evaluate the different modified soups, selecting the better by-product concentration extracts for possible industrial use.
HPLC Analysis Of the original and functionalised soups, 20-mL samples were analysed using an HPLC system equipped with a pump Model L6200 (Merck Hitachi) and Shimadzu SPD-MSA photodiode array UV-VIS detector. Separations were achieved on a Lichrocart column (Merck) (RP-18, 250.4 cm; 5 mm particle size). The mobile phase was water with 5% formic acid (v/v) (solvent A) and HPLC grade methanol (solvent B) at a flow rate of 1 mL/min. The linear gradient started with 10% B in A to reach 15% B at 5 min, 20% B at 30, 50% B at 35 min followed by 50% isocratic for 5 min and 90% B at 45 min. Chromatograms were recorded at 335 nm. Phenolic compound identification and quantification
Materials and methods Plant material Three different kinds of by-products were used. Artichoke byproducts are produced by industrial canning processing of artichoke and are mainly blanched bracts, receptacles and stems. Lettuce byproducts are produced by fresh-cut salad production and are composed mainly of external leaves. Typical cauliflower by-products mainly consist of leaves and, in lesser amounts, stems. The artichoke by-products were supplied by Halcon (Campos del Rio, Murcia), the lettuce by-products were supplied by Kernel (Los Alcazares, Murcia), while the cauliflower by-products were supplied by Agrosol (Lorca, Murcia). By-product extracts A recently reported method to obtain the polyphenol-enriched extracts from by-products [10] was used to obtain the extracts. Each by-product (1 kg) was extracted by reflux with boiling water (1:2 w/v, 1:4 w/v and 1:3 w/v for artichoke, cauliflower and lettuce byproducts respectively) for 1 h. The extracts were cooled at room temperature and then filtered through Whatman No. 1 filter paper (Whatman, Maidstone, England). The extracts were freeze-dried at –50 C and stored. Functionalisation of chicken soup Tetra bricks of commercial chicken soup were acquired from a local supermarket. Commercial soup was functionalised and assayed immediately after opening. The by-product extracts were added to the chicken soup in different concentrations. Artichoke by-product and lettuce by-product extracts were assayed in 5, 10 and 20 mg extract/mL of soup and cauliflower by-products extracts in 2.5, 5 and 10 mg extract/mL soup. These samples of functionalised soup were evaluated by a sensory evaluation panel in order to select the optimal concentration as described below. The control was defined as soup without by-product extracts. Sensory evaluation panel A test panel composed of four trained judges evaluated the grade of acceptability (tolerance changes in both colour and flavour) in modified soup. Acceptability was scored on a 9-point scale (9=excellent; 5=moderate acceptance, marketable limit; and 1=poor, inedible). The panel of trained judges gave the score 6 to the original soup. The aim of this evaluation was obtain a functionalised soup equal to or better than the original soup.
The identification of phenolic compounds was carried out according to their UV spectra and retention times as previously reported by Llorach et al. [9, 10], and lettuce by-products extract flavonoids as described by Dupont et al. [15]. Caffeic acid derivatives were quantified by comparison with external standards as chlorogenic acid (5-O-caffeoylquinic acid), and flavonoids as rutin (quercetin3-rutinoside). Antioxidant activity The free radical scavenging activity (ABTS·+ assay) as well as ferric reducing ability [ferric reducing antioxidant power (FRAP) assay] were use to evaluate the antioxidant capacity of the functionalisated soups. ABTS·+ assay. The reaction was started by adding 5 mL of sample (original and functionalisated soups) to the cuvette containing 32 mM (water solution) (995 mL) of the free radical (ABTS·+). The radical was chemically generated with MnO2 as described by Espn and Wichers [16]. The experiments were always performed on freshly made up soups. The final volume of the assay was 1 mL. The disappearance of ABTS·+ was determined by measuring the decrease in absorbance at 414 nm for 1 h at 25 C in the spectrophotometer. The control was defined as soup without by-product extracts. FRAP assay. The FRAP assay was performed according to Benzie and Strain [17] with some modifications. The freshly made up FRAP solution contained 25 mL of 0.3 M acetate buffer (pH 3.6) plus 2.5 mL of 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) solution in 40 mM HCl (previously prepared) and 2.5 mL of 20 mM ferric chloride (FeCl3 6 H2O). This solution was used as blank. Warmed (37 C) FRAP solution (995 mL) was mixed with 5 mL of samples (original and functionalised soups). The ferric-reducing ability of samples soups was measured by monitoring the increase of absorbance at 593 nm for 45 min. The control was defined as soup without by-product extracts. All the antioxidant assays were repeated three times and the coefficient of variation was always less than 10%. In addition, calibration curves were made for each assay using Trolox as standard. The antioxidant activity (ABTS·+ and FRAP assays) was expressed as Trolox equivalent antioxidant activity (TEAC) following the nomenclature of Rice-Evans and Miller [18]. Statistical analysis The effect of type of by-product and concentration of extract on the acceptability of modified chicken soup was assessed using ANOVA and mean values compared using the least significant difference (LSD) test. Type of by-product, concentration of extract and their interactions were considered the main factors.
33 Table 1 Effect of type of byproduct and amount of extract on acceptability of modified chicken soups. LSD values are in brackets. Acceptability values are the mean(n=4 judges)
By-product sample extracts
Concentration of extract
Acceptability
(mg extract/mL soup) Original soup +ABa
0.0 5.0 10.0 20.0 5.0 10.0 20.0 2.5 5.0 10.0
+LBb +CLBc By-product sample extracts Concentration of extract By-product sample extractsconcentration
6.00 5.25 7.75 4.25 6.25 8.25 2.75 5.75 7.75 3.00 (0.4)** (0.3)*** (0.7)***
* P=0.05; ** P=0.01; *** P=0.001 a Soup with artichoke by-products extract added b Soup with lettuce by-product extract added c Soup with cauliflower by-products extract added
Generally, a high acceptability grade was observed after addition of by-product extracts. This acceptability was significantly affected by both the type of by-product and the concentration of the extract (Table 1). The artichoke and lettuce by-product extracts scored significantly better than the cauliflower by-product. Regarding the concentrations of the extracts of both artichoke and lettuce by-products, scores for more than 10 mg of extract/mL of soup were poor, especially those for 20 mg of lettuce by-products extract/mL of soup (Table 1). Nevertheless, concentrations lower than 10 mg extract/mL soup showed scores around the marketable limit. The soup with 2.5 mg of cauliflower by-product added scored higher than the marketable limit while for 10 mg the score decreased drastically (Table 1). The poor scores for higher concentrations of both lettuce and cauliflower by-product extracts were owing to changes in colour and smell. Finally, the most highly scored concentrations were 10 mg of extract/mL of soup for both artichoke and lettuce by-product extracts, and 5 mg/mL of soup for cauliflower by-product extract.
of dicaffeoylquinic acid (Fig. 1B, 6, 7). Lettuce byproduct extract was composed of both caffeic acid derivatives and flavonoids. Chicoric acid (dicaffeoyltartaric acid) (Fig. 1C, 4) was the main caffeic acid derivative. Regarding the flavonoids, both flavone (as traces) and flavonols were identified. Luteolin-glucuronide was identified as flavone (Fig. 1C, 5) and various quercentin glucoside derivatives were identified as flavonols (Fig. 1C, 6–8). Cauliflower by-product extract was also composed of both caffeic acid derivatives and flavonols (kaempferol acylglucoside derivatives). The main flavonoids identified were kaempferol-3-O-sophoroside-7-Oglucoside (Fig. 1D, 3) and its sinapoyl derivative (kaempferol-3-O-(sinapoylsophoroside)-7-O-glucoside) (Fig. 1D, 4). The phenolic contents of the different extracts varied depending on the fresh by-products. The content in artichoke by-products extract was the highest, with 100 mg of phenolic compound/g freeze-dried extract, followed by lettuce by-products extract and finally by cauliflower byproducts extract (Table 2). Taking into account the maximum concentration selected by the sensory evaluation panel, the soups with artichoke added showed the highest levels of total phenolics, followed by lettuce and finally by cauliflower (Table 2).
Phenolic composition and quantification
Antioxidant capacity
HPLC profiles of original and functionalised soup are shown in Fig. 1A-D. Phenolic compounds were not detected in original soup. The phenolic compositions of artichoke and cauliflower by-products have been previously reported by Llorach et al. [9, 10]. Artichoke by-product extract was composed mainly of caffeic acid derivatives, chlorogenic acid (Fig. 1B, 2) being the main phenolic compound. Others caffeic acid derivatives identified were cynarin (1, 5-O-dicaffeoylquinic acid) (Fig. 1B, 3) as well as different isomers
Two in vitro antioxidant assays were approached as a routine way to assess the potential antioxidant capacity of both original and modified soups. Further extrapolation to in vivo systems requires further research (bioavailability, structure-activity relationship, etc), far from the aim of the present study.
Results and discussion Sensory evaluation panel
ABTS·+ assay. Functionalised soup samples showed a good scavenging activity against ABTS·+ radicals (Table 2). Non-modified soup showed a residual activity that
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0,035€0,001 3,08€0,07 1,00€0,05 0,8€0,01 0.17€0,01 2.26€0,05 0.73€0,04 0.59€0,01 – 1€0.012 0.46€0.035 0.18€0,0265 – – 0.06€0.001 0.16€0.026 – 1€0.012 0.4€0.034 0.02€0.0005 10 10 5
c
b
a
Soup with artichoke by-products extract added Soup with lettuce by-product extract added Soup with cauliflower by-products extract added d Data were obtained from Table 1
– 100€1.2 46€3.6 33.8€0,25 – – 6€0.1 32€0.2
Flavonoids Caffeic acid derivatives
– 100€1.2 40€3.4 1.8€0.05 Original soup +ABa +LBb +CLBc
FRAP
(mg TEAC/mL soup)
ABTS Flavonoids
Total phenolics (mg/mL soup)
Caffeic acid derivatives
(mg extract/mL soup)d (mg/g dry extract)
Total phenolics
Concentration Phenolic compounds
Fig. 1A–D HPLC profiles of original soup and functionalisated soup. A Original soup. B Soup with artichoke by-product extract added (10 mg of extract/mL of soup). 1 Neochlorogenic acid; 2 chlorogenic acid; 3 cynarin; 4 and 5 other caffeic acid derivatives; 6 1, 5+3, 5 dicaffeoylquinic acids; 7 1, 4+4, 5 dicaffeoylquinic acids; 8 other caffeoylquinic derivatives. C Soup with lettuce by-product extract added (10 mg of extract/mL of soup). 1 Caffeoyltartaric acid 2 chlorogenic acid; 3 isochlorogenic acid; 4 chicoric acid; 5 luteolin 7-O-glucuronide; 6 quercetin-3-O-glucuronide; 7 quercetin-3-Oglucoside; 8 quercetin-3-O-(6-O-malonyl)-glucoside. D Soup with cauliflower by-product extract added (5 mg of extract/mL of soup). 1 Neochlorogenic acid; 2 chlorogenic acid; 3 kaempferol-3-Osophoroside-7-O-glucoside; 4 kaempferol-3-O-(sinapoylsophoroside)-7-O-glucoside; 5 kaempferol-3-O-sophoroside; 6 kaempferol3-O-(disinapoylsophotrioside)-7-O-glucoside
Table 2 Phenolic content and antioxidant capacities of original and modified soups. –Not detected
FRAP assay. The modified soups showed good antioxidant capacity, estimated from their ability to reduce TPTZ-Fe(III) complex to TPTZ-Fe(II) (Table 2). A significant increase was observed in all modified samples, the artichoke by-product producing the largest increment, followed by those of lettuce and cauliflower (Table 2). In this case, the control soup showed negligible activity. Both tea and red wine have shown a good capacity to reduce Fe(III) to Fe(II) [18]. In this context, soups with artichoke by-product added showed higher antioxidant capacity than both tea and red wine, while soups with both lettuce and cauliflower by-products added showed lower antioxidant capacity. A serving of soup is about 250 mL. From the values presented in Table 1, a serving of soup with artichoke byproducts added provides about 250 mg of phenolic compounds, the amount that would be provided by approximately 40 g of fresh artichoke [20]. The soup with lettuce by-products added provides 115 mg of phenolic compounds, the amount provided by approximately 350 g of fresh lettuce [21, 22]. The soup modified with cauliflower by-products provides 45 mg of phenolic compounds. It is difficult to extrapolate this to fresh cauliflower, since its edible parts are inflorescences and its by-products are leaves and stems, which have a different polyphenolic composition. However, from the quantitative point of view, about 250 g of fresh cauliflower would be necessary
Antioxidant capacity
could be attributable to any component not specified in the ingredient labels. A significant increase in antioxidant activity (around 13 times) was observed when the soup was functionalised with 10 mg of artichoke by-products extract. Regarding the soup modified with lettuce byproducts extract, this increment was 4 times, and when the cauliflower by-products extract was used, the increase was 3.5 times. Tea and red wine have been demonstrated to be important sources of antioxidants. In this context, red wine and tea showed an antioxidant activity of 1.25 mg of TEAC [19]. Soups with artichoke by-products extract added showed higher antioxidant activity than tea and red wine, while the soups with lettuce and cauliflower byproducts extracts added showed lower antioxidant activity (Table 2).
Phenolic compounds
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to obtain the same amount of polyphenols as the modified cauliflower soup provides [20]. The results obtained indicate that these by-products could provide extracts with antioxidant phenolics that could be used to functionalise foods. Obviously, before incorporating these by-products as dietary complements, it will be necessary to carry out further studies on their toxicity (i.e. possible residual presence of pesticides), in vivo activity and bioavailability.
References 1. Bonilla F, Mayen M, Merida J, Medina M (1999) Food Chem 66:209–215 2. Martnez Teruel A, Snchez J, Megas M D, Barrera JA, Yez A, Ruiperez F (1998) Arch Zootec 47:33–42 3. Femenia A, Robertson A, Waldron K, Selvendran R (1981) J Sci Food Agric 77:511–518 4. Hang YD (1987) Food Technol 41:115–117 5. Rodrguez de Sotillo D, Hadley M, Holm ET (1994) J Food Sci 59:1031–1033 6. Visioli F, Romani A, Mulinacci N, Zarini S, Conte D, Vincieri F, Galli C (1999) J Agric Food Chem 47:3397–3401 7. Lu Y, Foo L (2000) Food Chem 68:81–85 8. Moure A, Cruz M J, Franco D, Domnguez J M, Sineiro J, Domnguez H, Nfflez MJ, Paraj JC (2001) Food Chem 72:145–171
9. Llorach R, Espn JC, Toms-Barbern FA, Ferreres F (2002) J Agric Food Chem 50:3458–3464 10. Llorach R, Espn JC, Toms-Barbern FA, Ferreres F (2003) J Agric Food Chem 51:2181–2187 11. Scalbert A, Willansom G (2000) J Nutr 130 [Suppl]:S2073S2085 12. Rankin S, de Whaley C, Hoult J, Jessup W, Wilkins G, Gollard J, Leake D (1993) Biochem Pharmacol 45:67–75 13. Roberfroid, MB (2000) J Clinl Nutr 71:1660–1664 14. Larrosa M, Llorach R, Espn JC, Toms-Barbern FA (2002) Lebensm Wiss Technol 35:532–542 15. Dupont S, Mondi Z, Willamson G, Price K. (2000) J Agric Food Chem 48:3957–3964 16. Espn JC, Wichers HJ (2000) J Food Biochem 24:225–250 17. Benzie IFF, Strain JJ (1996) Anal Biochem 239:70–76 18. Rice-Evans CA, Miller NJ (1994) Methods Enzymol 234:279– 293 19. Gil MI, Toms-Barbern FA, Hess-Pierce B, Holcroft DM, Kader AA (2000) J Agric Food Chem 48:4581–4589 20. Gil-Izquierdo A, Gil MI, Conesa MA, Ferreres F (2001) Innov Food Sci Emerg Technol 2:199–202 21. Toms-Barbern FA, Ferreres F, Gil MI (2000) Antioxidant phenolic metabolites from fruit and vegetables and changes during postharvest storage and processing. In: Rahman A (ed) Bioactive natural products (part D). Elsevier, Amsterdam, pp 739–795 22. Hollman PCH, Arts ICW (2000) J Sci Food Agric 80:1081– 1093
