«DETERMINATION OF CHEMICAL COMPOSITION OF ANATOLIAN CAROB POD (CERATONIA SILIQUA L.): SUGARS, AMINO AND ORGANIC ACIDS, MINERALS AND PHENOLIC COMPOUNDS ...»
DETERMINATION OF CHEMICAL COMPOSITION OF
ANATOLIAN CAROB POD (CERATONIA SILIQUA L.): SUGARS,
AMINO AND ORGANIC ACIDS, MINERALS AND
FAIK AHMET AYAZ1,5, HÜLYA TORUN1, SEMA AYAZ1,
PEDRO JOSÉ CORREIA2, MANUEL ALAIZ3, CARLOS SANZ3,
JIRI GRÚZ4 and MIROSLAV STRNAD4
Department of Biology Karadeniz Technical University 61080 Trabzon, Turkey Centro de Desenvolvimento de Ciências e Técnicas de Produçao Vegetal (CDCTPV) ˘ University of Algarve 8005-139 Faro, Portugal Department of Physiology and Technology of Plant Products Instituto de la Grasa Consejo Superior de Investigaciones Cientiﬁcas (CSIC) Padre García Tejero 4, 41012-Seville, Spain Laboratory of Groth Regulators Palacký University and Institute of Experimental Botany Academy of Sciences of the Czech Republic Šlechtitelu 11, 783 71 Olomouc, Czech Republic ˚ ABSTRACT Carob pod is the fruit of the carob tree (Ceratonia siliqua L. Fabaceae). The fruit and its products, sold both in large stores and local markets, contribute strongly to the diet of people living in the Mediterranean areas of Europe and Turkey. This study reports the composition of carob pods sampled in West and South Anatolia. Sucrose (437.3 mg/g dry weight), glucose (395.8 mg/g dry weight) and fructose (42.3 mg/g dry weight) were the major sugars identiﬁed and quantiﬁed in the fruit. Total phenolics (13.51 mg gallic acid equivalents [GAE]/g dry weight), proanthocyanidin (0.36 mg GAE/g dry weight), gallotannins (0.41 catechin equivalents [CE]/g dry weight) and Corresponding author. TEL: 90-462-377-3712; FAX: 90-462-325-3195; EMAIL: email@example.com Journal of Food Quality 30 (2007) 1040–1055. All Rights Reserved.
1040 © 2007, The Author(s) Journal compilation © 2007, Blackwell Publishing CHEMICAL COMPOSITION OF CAROB POD 1041 ﬂavanols (3.21 mg CE/g dry weight protein) content of the fruit were also determined. Gallic acid (3.27 mg/g dry weight) was the most abundant phenolic acid present in all three phenolic fractions (free, ester and glycoside) isolated from pods. Aspartic acid (18.25 mg/g dry weight protein) was the predominant amino acid in the pod protein fraction. Eight minerals were quantiﬁed in the fruit. Among the analyzed major minerals, K (9.70 mg/g dry weight) was the most abundant element present, and the pods were richer in Ca than in P and Mg. Levels of trace minerals were comparable to other plant species. The data are discussed in terms of the nutritional value of the carob pod.
The use of carob fruit and its food products in Turkey has been increasing in recent years. However, knowledge about the composition of carob fruit pod produced in Turkey as well as in Mediterranean countries is lacking. The present work describes a composition scale and the advantages to food technologists and consumers who use the fruit and its fruit products in their diets.
The results of the study can also aid in the assessment of adequate compositional information for further studies.
In many geographical regions, locally grown fruit and vegetables contribute substantially to local diet. The composition of such foods and their products is therefore a matter of considerable interest to nutritionists and food scientists.
The carob tree (Ceratonia siliqua L. Fabaceae) is a native evergreen plant of the Mediterranean area including West and South Anatolia (Turkey) (Chamberlain 1970; Batlle and Tous 1997). The nonﬂeshy and bean-like fruits, called “carob pods,” are a traditional part of the diet in the Mediterranean region, and the plant has been cultivated in the region for centuries for its edible fruits. The pod is light to dark brown, oblong, ﬂattened, straight or slightly curved, with a thickened margin, and ranges from 10 to 20 cm in length and 1.5–2 cm in width. The unripe pod is green, moist and very astringent, but the ripe pod is sweet. The broken pod has a characteristic odor caused by its 1.3% isobutyric acid content (Morton 1987). Current world production of carob pod has been estimated at about 310,000 tons per year, produced from about 200,000 hectares with very variable yields depending on the cultivar, region, and farming practices (Makris and Kefalas 2004).
1042 F.A. AYAZ ET AL.
Carob pod is widely used in the food industry to produce carob bean gum and locust bean gum, which are polysaccharides (galactomannans) (Morton 1987; Batlle and Tous 1997). Throughout the Mediterranean region including Turkey, gently milled carob pods are processed to a cocoa-like ﬂour which is sold as a “carob cocoa” in big stores and local markets. The milled ﬂour is often added to hot or cold milk for drinking (Morton 1987). The pod consists mainly of pulp (90%), which is rich in sugars (48–72%), but also may contain a large amount of condensed tannin (16–20%) (Würsch et al. 1984; Morton 1987; Saura-Calixto 1988; Bravo et al. 1994; Batlle and Tous 1997). Lower tannin values have been reported in some cases (Yousif and Alghzawi 2000).
Free sugars, organic acids and amino acids are natural constituents of many fruits and vegetables and play an important role in maintaining quality and determining nutrititive value (Ashoor and Knox 1982). The nature and the concentration of these constituents in fruits are also of interest because of their important organoleptic properties. Free sugars are one of the most important constituents of fruits and vegetables. Monosaccharides and disaccharides, such as fructose and glucose, are considered to be the major sugars in most fruits contributing to the ﬂavor of fruits (Shaw 1988). Amino acids and their derivatives are important for human nutrition and affect the quality of foods including taste, aroma and color (Belitz and Grosch 1999). Among the different substances that make up fruit and vegetables, amino acids are becoming increasingly important and, for various reasons, their analytical determination is becoming more necessary. First, the concentration of amino acids in fruit varies signiﬁcantly as a result of metabolic changes during growth, maturation and ripening (Gomis et al. 1990). Second, amino acid proﬁles vary from one species to another and among fruits of the same type but of different origin;
they can therefore be used to characterize fruit products (Gomis et al. 1990, 1992).
Phenolic compounds, nonnutrient but biologically active secondary plant metabolites which can act as antioxidants, are widely distributed in the Plant Kingdom and are present in many foods and beverages of plant origin. The acceptability of fruit and vegetables for human consumption may be affected by their content of phenolics (Shahidi and Naczk 1995). Interest in the role of phenolic antioxidants in human health has prompted research into the separation and characterization of active phenolic components in various plantderived foods (Häkkinen et al. 1999; Zuo et al. 2002; Ayaz et al. 2005).
Here, we report the results of chemical analyses carried out on carob tree fruit (carob pods) collected from various locations in West and South Anatolia (Turkey) where the plant is both cultivated and naturalized (Chamberlain 1970). To the best of our knowledge this is the ﬁrst such study undertaken on pods of carob trees grown in Turkey.
CHEMICAL COMPOSITION OF CAROB POD 1043
MATERIALS AND METHODS
Plant Material Carob pods (Ceratonia siliqua L.) were randomly harvested from various parts of several trees grown in different locations in western and southern parts of Anatolia in Turkey (250–300 m above sea level). The samples were collected in the morning from August to September in 2 consecutive years (2004, 2005). The carob pods were of the same physiological maturity (dark brown) and of uniform shape and size. Fruits (50 g per sample) collected from each natural habitat were combined to provide composite samples of 800 g.
Samples were sun dried, seeds were removed and the residue was ground in a mill (0.08 mm). From these stocks, three or four samples of gently milled pods were used for subsequent analyses. The grounded and milled samples were stored at -20C for further extraction.
Protein Determination Protein content was determined by elemental analysis using a LECO CHNS-932 analyzer (St. Joseph, MI), and was calculated as percentage nitrogen ¥ 6.25.
Amino Acids Samples containing 2 mg of protein were hydrolyzed using 6 mol/L HCl at 110C for 20 h under an inert nitrogen atmosphere and derivatized with diethyl ethoxymethylenemalonate. Amino acids were analyzed by reversedphase high-performance liquid chromatography (HPLC) using d,l-aaminobutyric acid as an internal standard following procedure of Alaiz et al.
(1992). Tryptophan was analyzed by HPLC after basic hydrolysis according to Yust et al. (2004).
Sugar and Organic Acid Extraction The ﬁnely grounded carob pods were defatted by repeated extraction (3 ¥ 1 h) with a mixture of petroleum ether and chloroform (1:1, v/v) at room temperature. The defatted samples were dried in vacuo then re-extracted twice with 20 mL 95% ethanol for 5 min as previously described (Pérez et al. 1997).
The homogenate was vacuum-ﬁltered through Whatman No. 1 ﬁlter paper (Whatman International Ltd., Maidstone, England) and the residue washed three times with 80% ethanol. The ﬁltrates were combined and evaporated to dryness using a rotary evaporator. The residue was redissolved in 80% ethanol and then centrifuged. The supernatant was collected, dried in vacuo and used for further analysis.
1044 F.A. AYAZ ET AL.
HPLC Analysis for Sugars and Organic Acids Sugars and organic acids were analyzed by a Hewlet-Packard (1090) liquid chromatograph equipped with a photodiode array detector (PDA) and a Waters 410 differential refractometer (Milipore Corp., Milford, MA) connected in series. Data were processed using a Hewlet-Packard 85-B computing system and a Beckman Analogue Interface Module 406 with Gold V.711 software. Isocratic separation of the compounds was carried out at a ﬂow rate of 0.4 mL/min on a stainless steel Ion-300 column (300 mm ¥ 7.8 mm, 10 mm) containing a cation-exchange polymer in the ionic hydrogen form, combined with an IonGuard GC801 precolumn (Interaction, San Jose, CA). Filtered (0.22 mm nylon) and degassed 0.0085 mol/L H2SO4 solution was used as the mobile phase. Both columns were maintained at 23C. Samples were dissolved in mobile phase, ﬁltered through a micro-ﬁlter (politetraﬁlvoretileno [PTFE] or Teﬂon, 4 mm, 0.22 mm) and 20 mL (50% of total sample volume before ﬁltration) was injected. The post column efﬂuent was introduced in sequence into the PDA detector (scanning range 210–300 nm; 1.2 nm resolution) and a refractive index detector (sensitivity setting 16¥, [Pérez et al. 1997]).
Determination of Total Phenolics, Flavanols and Tannins Total phenolics (Singleton and Rossi 1965) and ﬂavanols (Arnous et al.
2001) were determined using Folin-Ciocalteu reagent and DMACA (4dimethylamino]cinnamaldehyde) (Sigma Chemical Co., St. Louis, Mo) with calibration curves for gallic acid and (+) catechin, respectively. The proanthocyanidins (Price et al. 1978) and gallotannins (Inoue and Hagerman 1988) were determined by using vanillin-HCl and rhodanine assays, respectively.
Gallic acid was used to calibrate the quantiﬁcation of total phenolics and gallotannins, and ( ) catechin was used as a calibration standard to quantify total ﬂavanols and proanthocyanidins. Data were expressed as mg gallic acid equivalents (GAEs) or mg catechin equivalents (CEs)/g dry weight.
Extraction of Phenolic Acids from Carob Pod Phenolic acids, in particular phenolic fractions, were extracted and isolated according to Cvikrová et al. (1994). Triplicate 160-g fruit samples were treated with liquid N2 and ground in 80% methanol containing an antioxidant 2,6-ditercbutyl-b-cresol in an electrical high-speed blender. The homogenate was boiled under reﬂux for 20 min, ﬁltered and concentrated under vacuum in a rotary evaporator. The concentrate was acidiﬁed with 1 mol/L HCl to pH = 2 and then extracted four times with 100 mL diethyl ether. The organic phase was evaporated to dryness under vacuum at 40C, and the residue was dissolved in methanol, ﬁltered using a 0.45-mm microﬁlter (Whatman No. 1) and then used for the analysis of the free phenolic acids.
CHEMICAL COMPOSITION OF CAROB POD 1045 After extraction, the aqueous phase was divided into two parts. The ﬁrst half was hydrolyzed with 2-mol/L NaOH for 4.5 h under a nitrogen atmosphere at room temperature, then acidiﬁed with 6-mol/L HCl to pH = 2 and processed as described above for free acids (FAs). This fraction contained methanol-soluble phenolic esters (MSPEs).
One mole per liter HCl was added to the second half of the aqueous phase, and the concentrate (pH = 2) was placed under a nitrogen atmosphere and hydrolyzed for 1 h at 100C. The hydrolysate, containing methanol-soluble phenolic glycosides, was processed as described for the previous fractions.
Determination of Phenolic Acids Phenolic acids were analyzed by high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) as described earlier (Ayaz et al. 2005). Brieﬂy, internal standards of deuterium-labeled salicylic and phydroxybenzoic acids were added to all the extracted and ﬁltered sample solutions to a ﬁnal concentration of 10-5 mol/L. Ten microliters of the sample solutions were injected on a reversed phase column (Luna Phenyl-Hexyl, 5 mm, 250 ¥ 2 mm; Phenomenex, Torrance, CA). HPLC-MS analyses were performed on an Alliance 2690 Separations Module (Waters, Milford, MA) linked simultaneously to a PDA 996 (Waters) and a ZSpray Mass Detector (ZMD) mass (2000) single quadrupole mass spectrometer equipped with an electrospray interface (Micromass, Manchester, U.K.). Data were processed by MassLynx software (Data Handling System for Windows, version 4.0, Micromass, Altrincham, U.K.). The quantiﬁcation was based on the ratio of peak area of the analyte to the average peak area of the internal standards.
Deuterium labeled internal standards of (2,3,5,6-2H4) p-hydroxybenzoic and (3,4,5,6-2H4) salicylic acids were purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA).