Characterization of a Metal-Chelating Substance in Coffee

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1 Biosci. Biotechnol. Biochem., 69 (1), 26 30, 2005 Characterization of a Metal-Chelating Substance in Coffee Makiko TAKENAKA, y Naoko SATO, Hiromi ASAKAWA, XuWEN, Masatsune MURATA, and Seiichi HOMMA Department of Nutrition and Food Science, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo , Japan Received July 14, 2004; Accepted September 22, 2004 A metal-chelating substance in brewed coffee was separated and characterized by its chemical structure. This substance was a brown polymer. The contents of sugars, amino acids and phenolics in the substance were evaluated. This polymer contained small amounts of sugars and amino acids in its partial structure. After being decomposed by alkaline fusion, the decomposition products were identified by HPLC and GC MS. Several phenolics were detected in the decomposed products. To characterize this substance, various types of model compounds were prepared by roasting chlorogenic acid, sucrose, and (or) protein with cellulose powder. Among these model compounds, the polymer-forming ability was highest in the model prepared from all four of materials, but the metal-chelating ability was the highest in the model prepared from chlorogenic acid and cellulose. These results suggest that this metal-chelating substance was a melanoidin-like polymer formed by the decomposition and polymerization of sugars, amino acids and phenolics. Key words: coffee; metal-chelating; browning; melanoidin; phenolics Coffee beans, the seeds of Coffea canephora, are dried and roasted to give their characteristic flavor and brown color before the coffee is extracted with hot water. During roasting, various kinds of compounds are decomposed, condensed or polymerized to form heterogeneous and brownish polymers. The brown color is one of the most important characters of brewed coffee. Therefore, the browning reaction exerts an essential effect on the quality of the brewed coffee. The two major types of browning reaction in food processing and preservation are enzymatic browning and the Maillard reaction. 1) The former reaction involves phenolics which are oxidized by oxidase, the resulting oxidized products being polymerized to form brown pigments. This type of browning can often be observed during the storage of minimally processed vegetables or in the production of fruit juice and tea. The mechanism in the latter reaction of forming a Maillard reaction product (melanoidin) is assumed to consist of an early stage and advanced stage, although there is not yet sufficient knowledge about its chemical structure. 1) In the early stage, the aldehydic moiety of sugars and amino groups react to produce a component which has the partial structure of CHOH CO CH 2 NHR, while in the advanced stage, various aldehydes, ketones, and furfurals are formed from the early-stage products, and these react with amino compounds to form high-molecular-weight melanoidin. Coffee beans contain about 8% of phenolic compounds, 7% of reducing sugars and 12% of protein, 2) and coffee is one of the richest crops in chlorogenic acid and its derivatives. 3,4) The brown pigments in a coffee brew are formed by the reactions involving phenolic compounds, sugar and amino acids during roasting 2,5) because these compounds are easily decomposed and polymerized during this process. Brown products such as melanoidin and phenolic polymers have metal-chelating activity. 6 9) The coffee brew contains brown pigments and it has been reported that excessive consumption of brewed coffee inhibited iron absorption. 10) The brown pigments in the coffee brew or in roasted coffee beans should have a similar partial structure of melanoidin or a polymer of phenolic compounds. The zinc-chelating compounds in instant coffee were separated in this study into fractions by using the precipitation method and cellulose column chromatography. We report some chemical properties of the substance which had the strongest chelating ability. Materials and Methods Preparation of the zinc-chelating substance from instant coffee. Instant coffee of the lyophilized type (1 kg) was suspended in 3 liters of a 20 mm ZnCl 2 / 10 mm hexamethylenetetramine (hexamine) KCl buffer (ph 5.0) to form an insoluble Zn 2þ coffee complex. This was collected by centrifugation (1000 g, 13 min). The Zn 2þ coffee complex was dissolved in 1 liter of a 1% aqueous ammonia solution (sample A). Sample A was loaded into successive ion-exchange columns y To whom correspondence should be addressed. Present address: Food Processing Laboratory, Food Engineering Division, National Food Research Institute, Kannondai, Tsukuba, Ibaraki , Japan; Fax: ; tknk1221@nfri.affrc.go.jp

2 (5 20 cm) of Amberlite IRA-410 and Amberlite IR-120B. The non-adsorbed fractions were collected, lyophilized and applied to a cellulose column (5 40 cm, Cellulose Mikrokristallin, Merck Japan, Tokyo, Japan) which was eluted with 1% aqueous ammonia and 2-propanol mixtures (5:2, 1040 ml; 3:2, 165 ml; 1:1, 95 ml). The eluates were measured for their browning degree (absorbance at 470 nm) and Zn concentration. The Zn concentration was determined with an AA-670 atomic absorption spectrometer (Shimadzu, Kyoto, Japan). The fifth fraction (Ap-V) had the largest amount of Zn, i.e. the strongest metal-chelating ability, and was lyophilized for further analyses. Characterization of a Metal-Chelating Substance in Coffee 27 Amino acid analysis. Ap-V (10 mg) was hydrolyzed with 6 N HCl (1 ml) at 110 C for 24 hours. The hydrolysate was concentrated and dried in vacuo. The residue was dissolved in 0.01 N HCl (2 ml) and measured with a Hitachi 835 amino acid analyzer using the ninhydrin reaction. Determination of the phenolic content. The content of phenolics in the Ap-V sample was determined by the Folin Denis method. Ap-V was dissolved in water at a concentration of 0.1 mg/ml. A calibration curve was prepared by using chlorogenic acid (Nacalai Tesque, Japan) as the standard. Evaluation of the molecular weight. The molecular weight of Ap-V was evaluated by gel permeation chromatography (GPC) with molecular weight markers of polyethylene glycol. Ap-V was dissolved in a 20 mm phosphate buffer (ph 6.8) and chromatographed in a precolumn of TSK PWH (7:5 75 mm, Tosoh, Tokyo, Japan) and separation column of TSK-gel G-3000PW (7:5 600 mm, Tosoh, Tokyo, Japan) by HPLC (L- 6000; Hitachi, Tokyo, Japan). This was developed with a 200 mm NaCl/20 mm phosphate buffer (ph 6.8) at a flow rate of 1.0 ml/min with an RI-8000 detector (Showa Denko, Tokyo, Japan). Evaluation of the chelating activity. The tetramethyl murexide (TMM) method 11) was used. A TMM solution (10 mm), Ap-V solution (0.05%), and ZnCl 2 solution (from 20 to 100 mm) were prepared in a hexamine KCl buffer (ph 5.0). The sample solution (2.0 ml), TMM solution (0.2 ml) and ZnCl 2 solution (2.0 ml) were mixed together. After 30 min, the absorbance values at 460 nm and 530 nm were measured. The free zinc concentration was calibrated by using a standard curve. The total zinc concentration was determined by atomic absorption spectrometry, and the percentage of bound zinc was calculated. The amount of bound zinc was used to estimate the specific chelating activity, the total activity being defined as the specific activity multiplied by the sample weight. Sugar analysis. Ap-V (10 mg) was hydrolyzed with 2 N trifluoroacetic acid (2.4 ml) in a screw-capped test tube at 100 C for 3 hours, with the head-space gas displaced by N 2. The hydrolysate was concentrated and dried in vacuo. The residue was dissolved in water (400 ml) and measured with Hitachi sugar analyzer system, using post-column detection with the phosphate phenylhydrazin reaction and a Hitachi gel #3013- N column (4 150 mm). The detectors were set at 365 nm for ultraviolet, and at an excitation wavelength of 330 nm and an emission wavelength of 310 nm for fluorescence. The mobile phase was a sodium borate buffer, chromatography being performed at 70 C with the mobile phase flow rate of 0.4 ml/min. Chemical decomposition. Ap-V was decomposed by alkaline fusion. Ap-V (100 mg) and NaOH (500 mg) were heated in a crucible in a muffle furnace at 350 C for 20 min, before suspending the residue in water and extracting with diethyl ether to give the neutral and basic fraction. The aqueous phase was adjusted to ph 2 3 with 1 N HCl and extracted with diethyl ether to give the acidic fraction. Each fraction was then concentrated and used for the GC MS analysis. Spectra were recorded with a GCMS-QP5000 instrument (Shimadzu, Kyoto, Japan). The ion source temperature was kept at 220 C. A capillary column (DB-1, 0.25 mm, 0:25 mm 30 m) from J&W Scientific was used, the column oven temperature being programmed from 60 C to 220 C at a rate of 2 C/min and the injection port temperature being 200 C. The carrier gas (helium) was controlled at a flow rate of 1.0 ml/min. Mass spectra of the peaks were compared with those in the database of the GC MS instrument; and the compounds which mass spectra had high similarity (more than 80) to the standard compounds were collected. Those fractions were dissolved in methanol and applied to HPLC (Jasco, Tokyo, Japan), using a Mightysil RP-18 column (4:6 250 mm, Kanto Chemical, Tokyo, Japan). The detector was set at 280 nm. The mobile phase was a linear gradient of 0 50% acetonitrile in 4% acetonitrile/5% acetic acid over 30 min. Chromatography was performed at 40 C, the flow rate of the mobile phase being 1.0 ml/min. The major peaks were identified from their retention times and the UV spectral pattern of the standard compounds. Preparation of model heating products. Eight model heating products (models 1 8) were prepared with chlorogenic acid, sucrose, protein (bovine serum albumin), and cellulose microkristallin (Merck Japan, Tokyo, Japan) (the compositions are shown in Table 1). Cellulose is the major component of coffee beans. 2) The composition of model 1 is the closest to that of green coffee beans. These components were mixed and placed in test tubes and heated at 200 C for 30 min. After heating, the residue was suspended in water and dialyzed against sufficient water to remove the smaller molecules. Each dialysate was filtered and lyophilized. These water-soluble compounds are regarded as the model heating products.

3 28 M. TAKENAKA et al. Table 1. Compositions of the Model Heating Products Model No A: Chlorogenic acid (300 mg) þ þ þ þ B: Sucrose (300 mg) þ þ þ þ C: Protein (450 mg) þ þ þ þ D: Cellulose ([A + B + C] mg) þ þ þ þ þ þ þ þ þ, added;, not added. Evaluation of the metal-chelating activity of the model heating products. Each model reaction product was suspended overnight in 10 ml of a 20 mm ZnCl 2 / 10 mm hexamine buffer (ph 5.0) to chelate Zn 2þ and then dialyzed against the hexamine buffer solution. To the dialysate 50 ml of water was added, and the solution was measured for Zn concentration by atomic absorption spectrometry. Results and Discussion The yield of Ap-V was 360 mg from 1 kg of instant coffee. Ap-V is a brown amorphous powder, soluble in water, but insoluble in such organic solvents as methanol, ethanol, hexane, and ethyl acetate. The elemental composition was observed as C, 47.81%; N, 9.57%; and H, 5.27%. The calculated empirical formula of Ap-V was C 16 H 21 O 9 N 3 (C, 48.12%; O, 36.09%; N, 10.53%; H, 5.26%), which shows that Ap-V contained three molar equivalents of N in a skeleton of sixteen carbons. The molecular weight established by GPC was found to be about 35,000, suggesting that Ap-V was composed of about 88 units of C 16 H 21 O 9 N 3. The metal-chelating activity was evaluated by the same method as that Nakamura et al. used to determine the chelating activity of one of the zinc-chelating principals in instant coffee. 11) A Scatchard plot shows that Ap-V had two binding sites to zinc (Fig. 1), the dissociation constants being Kd ¼ 1: (M) and 8: (M). These results show that Ap-V had stronger affinity for zinc than citric acid, which has been thought of as main zinc chelator in instant coffee, having 13% of the total chelating activity of instant coffee. 11) Ap-V therefore contributes to the chelating activity of instant coffee as well as citric acid. The results of sugar and amino acid analyses are shown in Tables 2 and 3, respectively. These results suggest that most of the nitrogen in Ap-V did not exist as amino acids or proteins but in a melanoidin-like form. The contents of hexose and the amino acid residues calculated from the results of the analyses were about 3% and 4%, respectively. The content of phenolics calculated as chlorogenic acid, which was the major phenolic in green coffee beans, 3) was about 30%. Chlorogenic acids in green coffee beans may be incorporated into a melanoidin-like polymer during roasting. Merritt et al. have reported that, when green Fig. 1. Scatchard Plot of Zinc Binding by Ap-V. Kd ¼ 1: M and 8: M, n ¼ 1 and 2; r, mmol of bound zinc/mmol of Ap-V; n, number of binding sites; Kd, dissociation constant. Table 2. Hexose Composition of Ap-V Sugar Content (%, w/w) Rhamnose 0.11 Mannose 0.41 Arabinose 0.53 Galactose 1.25 Glucose 0.37 Total 2.67 Table 3. Amino Acid Composition of Ap-V Amino acid Content (%, w/w) Aspartic acid 0.26 Threonine 0.05 Serine 0.13 Glutamic acid 0.95 Glycine 0.70 Alanine 0.24 Cystein 0.14 Valine 0.15 Methionine 0.07 Leucine 0.18 Tyrosine 0.27 Phenylalanine 0.22 Lysine 0.03 Histidine 0.08 Arginine 0.05 Total 3.57 coffee was roasted, sucrose, protein and chlorogenic acid decreased by 7.0%, 8.6% and 4.0%, respectively, whereas the contents of cellulose, lignin, fat and ash did not change. 2) Sucrose may change partly to caramel and

4 mainly to melanoidin-like substance reacting with amino acid and protein. This notion is supported by the result that Ap-V had little sugar or amino acid residues in spite of the abundant content of sugars or proteins in green coffee beans. Ap-V was decomposed by alkaline fusion to provisionally examine its partial structure. In preliminary experiments, we attempted several methods to decompose Ap-V under weak conditions such as acid or alkaline hydrolysis and alkaline decomposition in glycerol, but Ap-V could not be effectively degraded by these methods. The yields of a neutral-basic fraction and an acidic fraction obtained after alkaline fusion were 5 mg and Characterization of a Metal-Chelating Substance in Coffee 29 6 mg, respectively from 360 mg of Ap-V. No definite peaks could be detected by HPLC from the neutral-basic fraction. Pyrogallol (0.02%, w/w Ap-V), protocatechuic acid (0.53%), catechol (0.41%), and p-hydroxybenzoic acid (0.14%) were detected by HPLC from the acidic fraction (Fig. 2). These fractions were subsequently analyzed by GC MS. Hardly any peaks were obtained from the neutral-basic fraction, while many peaks were detected from the acidic fraction (Fig. 3). Rational formulae estimated from the mass spectra were C 6 H 5 COOH (1), C 6 H 4 (OH)COOH (2 and 5), C 6 H 5 CH 2 CH(COOH) 2 (3), C 6 H 4 (OH)CH 2 COOH (4), and C 6 H 4 (OH)CH 3. These degradation products may have been derived from the chlorogenic acids moieties incorporated in Ap-V. Table 4 shows the yields of water-soluble substances prepared from the model heating products and the amounts of chelated Zn 2þ per 1 g of these model substances. The yield of model 1, which is closest to green coffee beans, was the highest. Models 6 8 hardly formed any water-soluble polymer. Considering in conjunction with the previous results regarding the partial structure of Ap-V, this result is consistent with Ap-V, a browned substance in roasted coffee beans, being a heating product of chlorogenic acids, sugar, and protein during roasting. In regard to the metal-chelating activity, this model tended to have higher activity as the ratio of chlorogenic acid in the model increased. Table 4. Yields and Zn-Chelating Abilities of the Model Substances Fig. 2. HPLC Chromatogram of the Acidic Fraction of Alkaline Fusion of Ap-V. 1, pyrogallol; 2, protocatechuic acid; 3, catechol; 4, p-hydroxybenzoic acid. Model No Yield (w/w, %) <0:01 <0:01 <0:01 Chelated Zn (mg/g of model) Fig. 3. GC MS Total Ion Chromatogram of the Acidic Fraction of Alkaline Fusion of Ap-V and Mass Spectra of Compounds 1 6.

5 30 M. TAKENAKA et al. Phenolics have metal-chelating activity, 9) and the activity of these models may have mainly arisen from their phenolic structure. In conclusion, the metal-chelating substance in coffee, Ap-V, was a brown melanoidin-like polymer derived from chlorogenic acids, sugars and proteins, and its metal-chelating activity may have been due to phenolic residues. Acknowledgments The authors are grateful to Nestle K.K. Japan for financial support. References 1) Friedman, M., Food browning and its preventation: an overview. J. Agric. Food Chem., 44, (1996). 2) Clifford, M. N., Volatile components. In Coffee, Vol. 1: Chemistry, eds. Clarke, R. J., and Macrae, R., Elsevier Applied Science Publishers, London, pp (1985). 3) Okada, H., Murata, M., and Homma, S., Hydroxycinnamic acid derivatives and p-coumaroyl-(l)-triptophan, a novel hydroxycinnamic acid derivative, from coffee beans. Biosci. Biotechnol. Biochem., 59, (1995). 4) Okada, H., Murata, M., Ohyazu, K., and Homma, S., Distinction between arabica and robusta coffee beans by hydroxycinnamic acid derivatives, especially by p- coumaroyl-(l)-tryptophan. Food Sci. Technol., 3, (1997). 5) Nakabayashi, T., and Watanabe, C., Chemical studies on the quality of coffee, part IV. Formation of brown pigments from chlorogenic acid by roasting. Nippon Shokuhin Kogyo Gakkaishi (in Japanese), 24, (1975). 6) Gomyo, T., and Horikoshi, M., On the interaction of melanoidin with metallic ions. Agric. Biol. Chem., 40, (1976). 7) Rendleman, Jr. J. A., Complexation of calcium by melanoidin and its role in determining bioavailability. J. Food Sci., 52, (1987). 8) Murray, L. W., and Connie, M. W., Maillard browning effects on in vitro availability of zinc. J. Food Sci., 53, (1988). 9) Fukushima, M., and Tanimura, A., Binding property of aluminium ion to dietary fiber, polyphenol compounds and organic acids. Nippon Shokuhin Kagaku Kogaku Kaishi (in Japanese), 42, (1995). 10) Mork, T. A., Lynch, S. R., and Cook, J. D., Inhibition of food iron absorption by coffee. Am. J. Clin. Nutr., 37, (1983). 11) Nakamura-Takada, Y., Shata, H., Minao, M., Ogawa, H., Sekiguchi, N., Murata, M., and Homma, S., Isolation of a zinc-chelating compound from instant coffee by the tetramethyl murexide method. Lebensm.-Wiss. u.-technol., 27, (1994).

Amino acids. Ing. Petrová Jaroslava. Workshop on Official Controls of Feed AGR 46230, , Ankara. Turkey ÚKZÚZ - NRL RO Praha 1

Amino acids. Ing. Petrová Jaroslava. Workshop on Official Controls of Feed AGR 46230, , Ankara. Turkey ÚKZÚZ - NRL RO Praha 1 Amino acids Ing. Petrová Jaroslava Workshop on Official Controls of Feed AGR 46230, 6. 7. 12. 2011, Ankara. Turkey 6.12.2011 ÚKZÚZ - NRL RO Praha 1 Content of this presentation 1. Function of amino acids