Z. Lebensm. Unters.-Forsch. 158, 87--92 (1975) © by J. F. Bergmann, ~[finehen 1975
Nonenzymic Browning. XII. Maillard Reactions in Green Coffee Beans on Storage J a n P o k o r n ~ , Nguy@fi-huy C6fl, E v a ~ m i d r k a l o v ~ , a n d G u s t a v g a n i 6 e k Department of Food Chemistry, Prague Institute of Chemical Technology, Prague (CSSR) Received September 13, 1974
N i c h t e n z y m a t i s c h e Br/~unung. X l I . M a i l l a r d - R e a k t i o n e n w g h r e n d d e r L a g e r u n g v o n grfinen K a f f e e b o h n e n
Zusammen]assun9. W/~hrend der Lagernng yon grfinen Kaffeebohnen bei erhShter Temperatur und konstanter Luftfeuchtigkeit roagieren die in den urspriingliehen Bohnen anwesenden reduzierenden Zucker mit freien Aminos~uren unter der Bildung yon farblosen unbestindigeu Produkten. Weitere reduzierende Zucker und freie Aminosiuren werden durch Hydrolyse yon Polysaeehariden und Proteinen gebildet. In der zweiten Reaktionsperiode ver~ndern sich die Gehalte an freien Aminos~uren mid Zuekern nur wenig, aber die Br/iunungsreaktionen werden intensiv. Diese Periode ist charakterisiert dutch Verminderung der sensorischen Qualit/it des Kaffeegetr~nks, besonders dessen Gernchs. Auch das in den Proteinen gebundene Lysin nimmt an Br~unungsreaktionen mittels eines farblosen intermedi~ren Produkts teil. Summary. During storage of green coffee beans at increased temperature and at constant humidity reducing sugars present in original beans react with free amino acids with formation of eolourless unstable products. Additional reducing sugars and free amino acids are produced by hydrolysis of polysaecharides and proteins, respectively. The second stage of storage is characterized by only slight changes in the content of free amino acids and sugars but by intensive browning reactions. This latter stage was characterized by deterioration of sensory quality of coffee beverage, especially of its odour. Lysine combined in protein was involved in browning reactions via colourless intermediary products. Introduction Green coffee beans are usually stored for a few years before roasting and consumption because the flavour of coffee beverage improves by storage of green beans for at least one year [1], however, coffee beans deteriorate by too long storage. The rate of deterioration processes depends on the water content depending in its turn on the relative humidity of atmosphere [2] and on other factors so that it is difficult to estimate the optimum storage time. I t was recently reported [3] that the quality of coffee depends on the content of various chemical constituents, so that the quality may be estimated by multiple linear regression analysis of the chemical composition of the sample. Products of Maillard reactions between free amino acids and reducing sugars are known to influence the flavour of foodstuffs. Changes of free amino acids indicate the advance of carbonyl-amino group reactions in food materials [4]. The content of free amino acids in green coffee is usually low, the maximum being 0.16% [5]. The maximum content of reducing sugars is 0.5% [6--8]. Ascorbic acid and other reductones, present in amounts of 24--180 ppm [9], may also contribute to Maillard reactions, possibly also via the Streeker degradation of free amino acids [10]. Therefore, we studied changes of free amino acids, reducing sugars and brown pigments in the course of storage of coffee beans at increased temperature to accelerate the browning process.
Experimental Material Colombia Coffee (1971 crop, stored for 12 months at ambient temperature) obtained 7.1% of water, 0.14% of total amino acids, 2.1% of ninhydrin-positive substances, expressed as glycine, extractable with 75% aqueous ethanol, and 0.52% of reducing sugars, (expressed as glucose). The amino acid composition of the total protein was as follows: lysine 9.1%, histidine 4.4%, arginine 6.9%, aspartic acid 9.6%, threonine 3.3%, serine 5.7%. glutamie acid 12.2%, proline 5.8%, glycine 7.7%, alanine 4.9%, cystine 0.9%, valine 5.3%, methionine 1.6%, isoleucine 2.2%, leueine 8.4%, tyrosine 2.5%, phenylalanine 3.2%, and ?-aminobutyric acid 2.0%. The sample was stored in a 200 ram-layer at 60° C in a closed container so that the water content remained constant during the experiment (51% relative humidity inside the container).
88 Analytical Methods Reducing sugars were determined by extracting 10.0 g of finely ground coffee three times, first with 100 ml, then with 50 ml of water for 30 rain. The filtrate was purified from amino acids by passing 5 ml of the extract through a 20 m m × 150 mm Dowex-50 WX-2 (200--~00 mesh) column and by washing with 100 ml of water [11 ]. Polyphenolic compounds were removed by diluting the eluate to 200 ml with water and by treating the solution with 1 g of polyamide powder Woelm [12]. The filtrate was analyzed for reducing sugars according to the Luff and Schoorl method [13]. Ninhydrin-active substances were extracted as above, the united filtrates were concentrated at a temperature < 35° C in vacuo. The residue was made up to 10 ml, and a mixture of 0.4 ml of the extract and 2 ml of 3.0% ninhydrin solution in 50% aqueous acetone was heated to 105° C for 15 rain [14]. The reaction mixture was diluted with methanol to 50 ml and the colour intensity measured at 570 nm (Spekol, C. Zeiss, Jena). The amino acid composition was determined with use of an automatic amino acid analyzer HD 1200 E (Z~vody Slovensk6ho n~rodn6ho povstania, n. p., 2iar n. H., ~SSR); the thiol group content according to MacDonnell et al. [15] ; the content of available lysine according to Carpenter [16], the content of free basic amino groups by means of Orange GG (Colour Index Acid Orange 10) according to Udy [17]. Soluble pigments were extracted with 75% aqueous ethanol (five extractions with a total of 100 ml of solvent per 10 g of sample) and measured using a Spekol (C. Zeiss, Jena) spectrophotometer. Total pigments were determined reflectemetrically with use of a Lovibond-Seholfield Tintometer 1A (Tintometer, Ltd., Salisbury, England) and evaluated by the trichromatic method. For a sensory evaluation of colour, a set of 9 finely ground samples was evaluated by rank test [18] and the results were tested by 20 judges; the probability level was 950/0. For sensory evaluation of odour and flavour, the sample was roasted in an electric laboratory roaster of 600 g capacity. The temperature of the roaster drum was 200--230° C, the roasting time was 20 rain, the average weight loss by roasting was 12.0°/o. Coffee beverage was prepared by infusing 7 g of finely ground coffee with 100 ml of boiling water and covering immediately with a glass cover. The extracts were tested 3--4 rain after preparation. Four samples were evaluated (the odour and the flavour on different series of test samples) at one time by ranking test [18] and results by 20 judges were evaluated after Kramer [19] on 95% probability level.
Results Changes of the c o n t e n t of reducing sugars were complicated (Fig. 1) as t h e i n i t i a l increase was followed b y a decrease a n d a n o t h e r increase. Changes of t o t a l n i n h y d r i n positive substances a n d of t o t a l a m i n o acids (as d e t e r m i n e d with a n a m i n o acid analyzer) were similar to one another. Storage periods when reducing sugars increased i n their c o n t e n t corresponded to periods of almost c o n s t a n t c o n t e n t of free a m i n o acids, while t h e r a p i d disappearance of free a m i n o acids took place a t t h e same time as a rapid d e s t r u c t i o n of reducing sugars.
120 P 100 80 60 40 20 0
0
o !
I
I
I
10
2O
3O
40
I
!
50 ~ 6 0
Fig. I. Changes of amino acids and reducing sugars during storage of green coffee. - - P -~ percentage of original amount; t ~ time of storage [days]; 0 reducing sugars; V total ninhydrin-posirive coumpounds as glycine; [] total amino acids determined with an amino acid analyzer
89
Changes in the composition of free amino acids on storage are shown in Table 1. Lysine and arginine were among the least stable amino acids, followed by y-aminobutyric acid and proline. Cystine and methionine were present only in traces, even in the original sample. The extract gave a positive xanthoprotein reaction [20, 21], but the three reactive subfraetions obtained by chromatography had R~-values different from that of tryptophan on a thin layer of silica gel G. Table 1. Changes of free amino acids during storage of coffee beans a t 60 ° C a n d a t a constant water content (7.3%) Amino acid
Content [izg/g] after storage time of original
Lysine 72 Histidine trace Arginine trace Aspartic acid 120 Threonine trace Serine 188 Glutamic 201 acid Proline 77 Glycine 25 Alanine 139 Cystine trace Valine 77 Methionine trace Isoleucine 35 Leucine 28 Tyrosine 37 Phenylalanine 51 7-Amino332 butyric acid
1 week
2 weeks
4 weeks
6 weeks
69 trace 20 117 trace 158 193
trace 0 0 75 0 115 142
trace 0 0 67 0 109 118
0 0 0 64 0 89 88
69 19 158 trace 66 trace 37 28 56 59 232
trace 20 123 0 40 0 23 20 37 51 130
trace 17 114 0 36 0 17 17 21 27 125
trace 12 103 0 27 0 14 15 trace trace 107
8 weeks 0 0 0 61 0 36 26 trace trace 63 0 21 0 trace trace trace trace 50
Changes of 7-aminobutyric acid and of total protein-constituing amino acids during short storage of green coffee beans are illustrated in Fig. 2. No free thiol groups were detected, even at the beginning of storage. Losses of lysine combined in
14100.,.
3.OF ~,51
~
430.,,7
120 P
!00
7.5
80
1.0
66
I ~ 0.51~
46 0
5
Io Fig. 2
15a
(3.00 2O
~
zf
o ~ ~°~
40
' 60
50On,.
'
80 t
Fig. 3
Fig. 2. Changes of free amino acids in the beginning of sf~rage of green coffee beans. - - P = percentage of the original a m o u n t ; d ~ time of storage [days]; l = total of protein-constituing amino acids; 2 -~ 7-aminobutyrie acid J~ig. 3. Course of browning during storage of green c o f f e e . - A ~ absorbance of extracts with 75% ethanol; t = time of storage [days]
90 protein did n o t exceed 5 % during storage (as determined b o t h according to Carpenter and to U d y ) and only after 100 days the content of basic groups decreased b y 25 %. The browning reaction, measured b y coloration of soluble pigments, was only slow during a period of 6 - - 7 weeks (Fig.3) b u t the intensity of coloration rapidly increased on further storage even when changes in free amino acids were slow in the last stage of storage. I n the case of total pigments, rapid changes of b o t h the trichromatic brightness and the saturation took place after 4 - - 6 weeks of storage (Table 2). Table 2. Changes of colour of green coffee on storage (evaluated after grinding) Time of storage days
Trichromatic determination dominant hue saturation nm %
brightness %
Sensory evaluation by rank test point total
0 1 4 14 28 42 56 60 80
580 578 575 578 578 582 581 582 584
41 40 40 38 39 28 23 21 20
42 26 52 81 99 120 112 181 156
31 30 26 26 42 41 44 44 48
Table 3. Effect of storage of green coffee on organoleptic properties of roasted coffee
Odour Flavour Colour
Rank total (points) after time of storage no storage 2 weeks 4 weeks
10 weeks
39 42 20
66 59 80
41 48 40
54 51 60
Note: Significant differences outside the range of 42--58 points (95°/o probability level) Results of the sensory evaluation show (Table 3) t h a t the sensory quality decreased during storage, especially in the last period when the browning reaction accelerated. Differences in the sensory quality were more perceptible in odour t h a n in flavour. Changes in coloration determined b y sensory test were significant except in the first few samples (Table 2).
Discussion The browning of coffee beans during storage can be caused: (i) by Maillard reactions of reducing sugars with free amino acids and with lysine combined in protein; (ii) by interaction of amino derivatives with quinones produced by oxidation of chlorogenie acids or other polyphenolle compounds; or (iii) by interaction of amino derivatives with oxidized coffee lipids, such as is observed in the case of mixtures of polyunsaturated fatty acid esters with protein [22]. In spite of a medium content of polyunsaturated fatty acids, i. e., 30--41% linoleie acid [23, 24] in coffee lipids, the role of oxidized lipids is not important, as the lipids are stabilized by natural inhibitors [25] and oleic and linoleie acid esters do not cause intensive browning [26]. Maillard reactions also contribute to the variability of contents of reducing sugars and of free amino acids in green coffee. For instance, glucose may be present only in traces [27], sometimes in larger amounts [6--8, 28--30]. The same is valid for free amino acids [5, 23, 31, 32]. Relatively low contents of total amino acids and trace contents of labile amino acids, such as eystine, methionine, and histidine (Table 1), indicate that some Maillard reactions have taken place even during the storage at ambient temperature before the experiment. A typical example is tryptophan, which is relatively stable in weakly acid medium, but which is rapidly destroyed by reaction with hexoses and other carbonylie derivatives [33]. Therefore, tryptophan was totally decomposed, even in the originM sample, and only several reaction products giving a positive xanthoprotein reaetion were detected in the extract.
91 Cysteine is very easily transformed by action of earbonyl compounds into semimercaptals [34, 35], which are further converted into thiazolidines [36]. Analogous compounds with ketones are less reactive [37]. However, these reactions do not lead to brown pigments on the contrary, cysteine has been reported [38, 39] as an inhibitor of browning. Because of this great reactivity, no free thiol groups were present, even in the original sample of green coffee, either in free or in combined amino acids. :Free cystine was decomposed as well, possibly via eysteine [40,41]. Reactions between free amino acids and reducing sugars at low water content are affected by the activity of water, pH value and temperature [42, 43]. In our experiments both the water content and the temperature were kept constant, but the pH value could change towards lower values, in spite of buffering capacity of the material. The course of changes of free amino acids and reducing sugars is complicated (Fig. 1) because additional amounts of the two reactants were produced during the storage by hydrolysis of proteins and of polysaccharides, respectively. The influence of hydrolysis is obvious, especially in the first reaction stage, before hydrolytic enzymes have become deactivated. I t may be seen in Fig. 2 that the content of ~-aminobutyric acid, i. e., an acid that is not bound in protein, decreased from the very beginning of storage, while the content of other amino acids combined in protein increased in the first stage of storage by hydrolytic reactions, and started to decrease only in later stages. Some amino acids and lower peptides also accompany browning reactions of protein [43]. The browning reaction (Fig. 3) consists of two phases: (1) first stage when amino acids are rapidly decomposed with formation of eolourless or light colonred compounds [A4s0~ 0.18 -[0.16 (1.5 --A_A)] where A.4. is the content of free amino acids [mg/g]; (2) second stage when the browning becomes more intensive [A,30 ~ 0.16 ~ 0.76 (1.0---AA)]. This behaviour is in agreement with the mechanism of N[aillard reactions. Brown products formed by Maillard reactions are partially soluble in 75% aqueous ethanol. The browning was a first order reaction (log A400~-- - - 0.38 ~ 0.98 t where t is the reaction time [d]) in agreement with the literature on nonenzymic browning reactions [44--47]. The ratio of absorbanees at 400 and 500 nm [A400:As00] decreased from 9.5 in the original coffee beans to 5.4 after 80 days of storage. This signifies that the dominant wavelength shifted to longer wavelengths on storage, probably by extension of conjugated double bond systems by dimerization of Schiff bases, aldolization and similar reactions [48--50]. Deducing sugars react not only with free amino acids but also with proteins, as the basic amino acids retain one reactive amino group, also when combined in protein [33, 51], e. g., when glucose was heated with casein, the contents of both available and total lysine, arginine and histidine decreased [52]. From the experimental data (Table 2) it is evident that some eolourless or light-coloured condensation products of lysine are formed as intermediary products. These products are then converted into brown pigments without appreciable change of available lysine.
References 1. Coste, R.: Les carriers et les eaf6s dans le monde, Vol. 3. Paris: Larose 1959 2. Streuli, H.: Handb. der Lebensmittelehemie, Bd. 6. Berlin, Heidelberg, New York: Springer 1970 3. Amorim, H.V., Malavolta, E., Tcixeira, A.A., Cruz, V.F., Melo,M., Guereio,M.A., Fossa, E., Breviglieri, 0., Ferrari, S.E., Silva, D.M. : VI e Colloque Intern. Chimie Caf6s, Bogota 1973 4. Holtermand, A.: St~rke 18, 319 (1966) 5. Underwood, G.E., Deatherage, F.E.: Food Res. 17, 419 (1952) 6. Slotta, K.H., bTeisser,K. : Ber. Deut. Chem. Ges. 72, 126 (1939) 7. Tcrrier, J.: Mitt. Geb. Lebensmittelunters. Hyg. 43, 307 (1952) 8. Hadorn, H., Suter, H. : Mitt. Gebiete Lebensm. Hyg. 47, 33 (1956) 9. Kuhn, A , Seh~fer, G. : Sfiddeut. Apotheker-Ztg. 79, 434 (1939) 10. SehSnberg, A., Moubaeher, R. : Chem. Rev. 50, 261 (1952) 11. Rohan, T.A., Stewart, T. S. : J. :Food Sei. 31, 206 (19661 12. Pokorn2,J., Karv&nek, M., Davldek, J.: Sb. Vysoke Skoly Chem.-Technol. Prazc E 10, 13 (1966) 13. Sehoorl, N.: Z. Untersueh. Lebensm. 57, 566 (1929) 14. Troll, V, Cannan, K. : J. Biol. Chem. 200, 803 (1953) 15. MaeDonnell, L.R., Silva, R.B., Feeney, R.E.: Arch. Bioehem. Biophys. 32, 288 (1951) 16. Milner, C. K., Westgarth, D. R. : J. Sci. Food Agr. 24, 873 (1973); Carpenter, K. J. : Bioehem. J. 77, 604 (1960) 17. Udy, D.C.: J. Am. Oil Chemists' Soc. 48, 29 A (1971) 18. Amerine,M.M., Pangborn, R.M., Roessler, E.B.: Principles of sensory evaluation of food. New York-London: Academic Press 1965 19. Kramer, A.: Food Teelmol. 17, 124 (1963) 20. Monnier, H., Besso, Z. : Helv. Chim. Aeta 35, 777 (1952) 21. Smirnov, B.P., Zheindov, V.L: Lab. Delo 12, 716 (1965) 22. Pokorn:~,J., E1-Zeany, B.A., Jan/~ek, G.: Z. Lebensm. Unters. -Forseh. 151, 31, 157 (1973)
92 23. Derbcly, M., Venot, C., Corte dos Santos, A., Benfrcd, J.A., Busson, F. : Qualitas Plant. Mater. Vegetabiles 18, 367 (1969) 24. Pokorn:~,J., Forman, L. : Nahrung 14, 631 (1970) 25. JaniSek, G., Pokorn~, J.: Z. Lebensm. Unters.-Forsch. 144, 189 {1970) 26. E1-Zeany, B.A., Pokorn~, J., JaniSek, G.: Z. Lebensm. Unters.-Forsch. In Press (Pt. XIII). 27. Wolfrom,M. L., Plunkett, R. A., Laver, M. L. : J. Agr. Food Chem. 8, 58 (1960) 28. Barbiroli, G.: Rass. Chim. 17, 261 (1965) 29. Pietet, C., Moreau, A.: Collog Intern. Chimie Cafes 4, 75 (1969) 30. Coassini-Lokar, L., Davanzo, S.: Univ. Studi Trieste, Fac. Econ. Commer., Ist. Merceol. Pubbl. 881 (1967) 31. Walter, W., Grigat, J. G., Heukeshoven, J.: Naturwissensehaften 57, 246 (1970) 32. Coassini-Lokar, L. :Fac. Econ. Univ. Studi Trieste 38, 17 (1967) 33. Heyns, K., lgoack, H.: Chem. Ber. 97, 415 (1964) 34. Armstrong, M.D., Vigneaud,V.du: J. Biol. Chem. 168, 373 (1947) 35. Armstrong, M.D., Vigneaud, V.du: J. Biol. Chem. 173, 749 (1948) 36. Bogner, R., Somogyi, L., Gyorgyde£k,Z. : J. Liebigs Ann. Chem. 738, 68 (1970) 37. Woodward, G.E., Sehroedcr, E.F.: J. Am. Chem. Soe. 59, 1690 (1937) 38. Arnold, R.G.: J. Dairy Sci. 52, 1857 (1969) 39. Okuhara, A., Saito, N.: Hakko Kogaku Zasshi 48, 177 (1970) 40. SchSberl, A. : Angew. Chem. 53, 227 (1940) 41. Routh, J.I. : J. Biol. Chem. 126, 147 (1938) 42. Lea, C.H., Hannan, R.S. : Bioehem. Biophys. Acta 8, 313 (1949) 43. Homma, S., Suzuki, N , Kato, H., Fujimaki, M.: Agr. Biol. Chem. (Japan) 84, 523 (1970) 44. Matsushima, Y. : Chem. Pharm. Bull. (Tokyo) 16, 2151 (1968) 45. Kozlova, N.Ya., Mel'nichenko, I.V., Yasnikov, h..A. : Ukr. Khim. Zh. 34, 263 (1968) 46. Koleheva, R.A., Kharin, S.E., Sapronov, A. R. : Izv. Vyssh. Ucheb. Zaved., Pishch. Tekhnol. 2, 206 (1970) 47. Kolka, S., Sokolowski, J.: Roczn. Chem. 44, 85 (1970) 48. Paquin, A.: Ber. Deut. Chem. Ges. 82, 316 (1949) 49. Emerson, W.S., Hess, S.U., Uhle, F.C.: J. Am. Chem. Soe. 68, 872 (1941) 50. Tiollais, R., Guillerm, H. : Compt. Rend. 286, 1798 (1953) 51. Iteyns, K., I~oaek, H.: Chem. Ber. 95, 720 (1962) 52. Patton, A.R., Hill, E.G., Foreman, E.I~.: Science (London) 167, 623 (1948) Doc. Dr. Jan Pokorn:~ Department of Food Chemistry Prague Institute of Chemical Technology Suehb~tarova 1905 CS-16628 Prague 6, Czechoslovakia
Nonenzymic Browning. XII. Maillard Reactions in Green Coffee Beans on Storage J a n P o k o r n ~ , Nguy@fi-huy C6fl, E v a ~ m i d r k a l o v ~ , a n d G u s t a v g a n i 6 e k Department of Food Chemistry, Prague Institute of Chemical Technology, Prague (CSSR) Received September 13, 1974
N i c h t e n z y m a t i s c h e Br/~unung. X l I . M a i l l a r d - R e a k t i o n e n w g h r e n d d e r L a g e r u n g v o n grfinen K a f f e e b o h n e n
Zusammen]assun9. W/~hrend der Lagernng yon grfinen Kaffeebohnen bei erhShter Temperatur und konstanter Luftfeuchtigkeit roagieren die in den urspriingliehen Bohnen anwesenden reduzierenden Zucker mit freien Aminos~uren unter der Bildung yon farblosen unbestindigeu Produkten. Weitere reduzierende Zucker und freie Aminosiuren werden durch Hydrolyse yon Polysaeehariden und Proteinen gebildet. In der zweiten Reaktionsperiode ver~ndern sich die Gehalte an freien Aminos~uren mid Zuekern nur wenig, aber die Br/iunungsreaktionen werden intensiv. Diese Periode ist charakterisiert dutch Verminderung der sensorischen Qualit/it des Kaffeegetr~nks, besonders dessen Gernchs. Auch das in den Proteinen gebundene Lysin nimmt an Br~unungsreaktionen mittels eines farblosen intermedi~ren Produkts teil. Summary. During storage of green coffee beans at increased temperature and at constant humidity reducing sugars present in original beans react with free amino acids with formation of eolourless unstable products. Additional reducing sugars and free amino acids are produced by hydrolysis of polysaecharides and proteins, respectively. The second stage of storage is characterized by only slight changes in the content of free amino acids and sugars but by intensive browning reactions. This latter stage was characterized by deterioration of sensory quality of coffee beverage, especially of its odour. Lysine combined in protein was involved in browning reactions via colourless intermediary products. Introduction Green coffee beans are usually stored for a few years before roasting and consumption because the flavour of coffee beverage improves by storage of green beans for at least one year [1], however, coffee beans deteriorate by too long storage. The rate of deterioration processes depends on the water content depending in its turn on the relative humidity of atmosphere [2] and on other factors so that it is difficult to estimate the optimum storage time. I t was recently reported [3] that the quality of coffee depends on the content of various chemical constituents, so that the quality may be estimated by multiple linear regression analysis of the chemical composition of the sample. Products of Maillard reactions between free amino acids and reducing sugars are known to influence the flavour of foodstuffs. Changes of free amino acids indicate the advance of carbonyl-amino group reactions in food materials [4]. The content of free amino acids in green coffee is usually low, the maximum being 0.16% [5]. The maximum content of reducing sugars is 0.5% [6--8]. Ascorbic acid and other reductones, present in amounts of 24--180 ppm [9], may also contribute to Maillard reactions, possibly also via the Streeker degradation of free amino acids [10]. Therefore, we studied changes of free amino acids, reducing sugars and brown pigments in the course of storage of coffee beans at increased temperature to accelerate the browning process.
Experimental Material Colombia Coffee (1971 crop, stored for 12 months at ambient temperature) obtained 7.1% of water, 0.14% of total amino acids, 2.1% of ninhydrin-positive substances, expressed as glycine, extractable with 75% aqueous ethanol, and 0.52% of reducing sugars, (expressed as glucose). The amino acid composition of the total protein was as follows: lysine 9.1%, histidine 4.4%, arginine 6.9%, aspartic acid 9.6%, threonine 3.3%, serine 5.7%. glutamie acid 12.2%, proline 5.8%, glycine 7.7%, alanine 4.9%, cystine 0.9%, valine 5.3%, methionine 1.6%, isoleucine 2.2%, leueine 8.4%, tyrosine 2.5%, phenylalanine 3.2%, and ?-aminobutyric acid 2.0%. The sample was stored in a 200 ram-layer at 60° C in a closed container so that the water content remained constant during the experiment (51% relative humidity inside the container).
88 Analytical Methods Reducing sugars were determined by extracting 10.0 g of finely ground coffee three times, first with 100 ml, then with 50 ml of water for 30 rain. The filtrate was purified from amino acids by passing 5 ml of the extract through a 20 m m × 150 mm Dowex-50 WX-2 (200--~00 mesh) column and by washing with 100 ml of water [11 ]. Polyphenolic compounds were removed by diluting the eluate to 200 ml with water and by treating the solution with 1 g of polyamide powder Woelm [12]. The filtrate was analyzed for reducing sugars according to the Luff and Schoorl method [13]. Ninhydrin-active substances were extracted as above, the united filtrates were concentrated at a temperature < 35° C in vacuo. The residue was made up to 10 ml, and a mixture of 0.4 ml of the extract and 2 ml of 3.0% ninhydrin solution in 50% aqueous acetone was heated to 105° C for 15 rain [14]. The reaction mixture was diluted with methanol to 50 ml and the colour intensity measured at 570 nm (Spekol, C. Zeiss, Jena). The amino acid composition was determined with use of an automatic amino acid analyzer HD 1200 E (Z~vody Slovensk6ho n~rodn6ho povstania, n. p., 2iar n. H., ~SSR); the thiol group content according to MacDonnell et al. [15] ; the content of available lysine according to Carpenter [16], the content of free basic amino groups by means of Orange GG (Colour Index Acid Orange 10) according to Udy [17]. Soluble pigments were extracted with 75% aqueous ethanol (five extractions with a total of 100 ml of solvent per 10 g of sample) and measured using a Spekol (C. Zeiss, Jena) spectrophotometer. Total pigments were determined reflectemetrically with use of a Lovibond-Seholfield Tintometer 1A (Tintometer, Ltd., Salisbury, England) and evaluated by the trichromatic method. For a sensory evaluation of colour, a set of 9 finely ground samples was evaluated by rank test [18] and the results were tested by 20 judges; the probability level was 950/0. For sensory evaluation of odour and flavour, the sample was roasted in an electric laboratory roaster of 600 g capacity. The temperature of the roaster drum was 200--230° C, the roasting time was 20 rain, the average weight loss by roasting was 12.0°/o. Coffee beverage was prepared by infusing 7 g of finely ground coffee with 100 ml of boiling water and covering immediately with a glass cover. The extracts were tested 3--4 rain after preparation. Four samples were evaluated (the odour and the flavour on different series of test samples) at one time by ranking test [18] and results by 20 judges were evaluated after Kramer [19] on 95% probability level.
Results Changes of the c o n t e n t of reducing sugars were complicated (Fig. 1) as t h e i n i t i a l increase was followed b y a decrease a n d a n o t h e r increase. Changes of t o t a l n i n h y d r i n positive substances a n d of t o t a l a m i n o acids (as d e t e r m i n e d with a n a m i n o acid analyzer) were similar to one another. Storage periods when reducing sugars increased i n their c o n t e n t corresponded to periods of almost c o n s t a n t c o n t e n t of free a m i n o acids, while t h e r a p i d disappearance of free a m i n o acids took place a t t h e same time as a rapid d e s t r u c t i o n of reducing sugars.
120 P 100 80 60 40 20 0
0
o !
I
I
I
10
2O
3O
40
I
!
50 ~ 6 0
Fig. I. Changes of amino acids and reducing sugars during storage of green coffee. - - P -~ percentage of original amount; t ~ time of storage [days]; 0 reducing sugars; V total ninhydrin-posirive coumpounds as glycine; [] total amino acids determined with an amino acid analyzer
89
Changes in the composition of free amino acids on storage are shown in Table 1. Lysine and arginine were among the least stable amino acids, followed by y-aminobutyric acid and proline. Cystine and methionine were present only in traces, even in the original sample. The extract gave a positive xanthoprotein reaction [20, 21], but the three reactive subfraetions obtained by chromatography had R~-values different from that of tryptophan on a thin layer of silica gel G. Table 1. Changes of free amino acids during storage of coffee beans a t 60 ° C a n d a t a constant water content (7.3%) Amino acid
Content [izg/g] after storage time of original
Lysine 72 Histidine trace Arginine trace Aspartic acid 120 Threonine trace Serine 188 Glutamic 201 acid Proline 77 Glycine 25 Alanine 139 Cystine trace Valine 77 Methionine trace Isoleucine 35 Leucine 28 Tyrosine 37 Phenylalanine 51 7-Amino332 butyric acid
1 week
2 weeks
4 weeks
6 weeks
69 trace 20 117 trace 158 193
trace 0 0 75 0 115 142
trace 0 0 67 0 109 118
0 0 0 64 0 89 88
69 19 158 trace 66 trace 37 28 56 59 232
trace 20 123 0 40 0 23 20 37 51 130
trace 17 114 0 36 0 17 17 21 27 125
trace 12 103 0 27 0 14 15 trace trace 107
8 weeks 0 0 0 61 0 36 26 trace trace 63 0 21 0 trace trace trace trace 50
Changes of 7-aminobutyric acid and of total protein-constituing amino acids during short storage of green coffee beans are illustrated in Fig. 2. No free thiol groups were detected, even at the beginning of storage. Losses of lysine combined in
14100.,.
3.OF ~,51
~
430.,,7
120 P
!00
7.5
80
1.0
66
I ~ 0.51~
46 0
5
Io Fig. 2
15a
(3.00 2O
~
zf
o ~ ~°~
40
' 60
50On,.
'
80 t
Fig. 3
Fig. 2. Changes of free amino acids in the beginning of sf~rage of green coffee beans. - - P = percentage of the original a m o u n t ; d ~ time of storage [days]; l = total of protein-constituing amino acids; 2 -~ 7-aminobutyrie acid J~ig. 3. Course of browning during storage of green c o f f e e . - A ~ absorbance of extracts with 75% ethanol; t = time of storage [days]
90 protein did n o t exceed 5 % during storage (as determined b o t h according to Carpenter and to U d y ) and only after 100 days the content of basic groups decreased b y 25 %. The browning reaction, measured b y coloration of soluble pigments, was only slow during a period of 6 - - 7 weeks (Fig.3) b u t the intensity of coloration rapidly increased on further storage even when changes in free amino acids were slow in the last stage of storage. I n the case of total pigments, rapid changes of b o t h the trichromatic brightness and the saturation took place after 4 - - 6 weeks of storage (Table 2). Table 2. Changes of colour of green coffee on storage (evaluated after grinding) Time of storage days
Trichromatic determination dominant hue saturation nm %
brightness %
Sensory evaluation by rank test point total
0 1 4 14 28 42 56 60 80
580 578 575 578 578 582 581 582 584
41 40 40 38 39 28 23 21 20
42 26 52 81 99 120 112 181 156
31 30 26 26 42 41 44 44 48
Table 3. Effect of storage of green coffee on organoleptic properties of roasted coffee
Odour Flavour Colour
Rank total (points) after time of storage no storage 2 weeks 4 weeks
10 weeks
39 42 20
66 59 80
41 48 40
54 51 60
Note: Significant differences outside the range of 42--58 points (95°/o probability level) Results of the sensory evaluation show (Table 3) t h a t the sensory quality decreased during storage, especially in the last period when the browning reaction accelerated. Differences in the sensory quality were more perceptible in odour t h a n in flavour. Changes in coloration determined b y sensory test were significant except in the first few samples (Table 2).
Discussion The browning of coffee beans during storage can be caused: (i) by Maillard reactions of reducing sugars with free amino acids and with lysine combined in protein; (ii) by interaction of amino derivatives with quinones produced by oxidation of chlorogenie acids or other polyphenolle compounds; or (iii) by interaction of amino derivatives with oxidized coffee lipids, such as is observed in the case of mixtures of polyunsaturated fatty acid esters with protein [22]. In spite of a medium content of polyunsaturated fatty acids, i. e., 30--41% linoleie acid [23, 24] in coffee lipids, the role of oxidized lipids is not important, as the lipids are stabilized by natural inhibitors [25] and oleic and linoleie acid esters do not cause intensive browning [26]. Maillard reactions also contribute to the variability of contents of reducing sugars and of free amino acids in green coffee. For instance, glucose may be present only in traces [27], sometimes in larger amounts [6--8, 28--30]. The same is valid for free amino acids [5, 23, 31, 32]. Relatively low contents of total amino acids and trace contents of labile amino acids, such as eystine, methionine, and histidine (Table 1), indicate that some Maillard reactions have taken place even during the storage at ambient temperature before the experiment. A typical example is tryptophan, which is relatively stable in weakly acid medium, but which is rapidly destroyed by reaction with hexoses and other carbonylie derivatives [33]. Therefore, tryptophan was totally decomposed, even in the originM sample, and only several reaction products giving a positive xanthoprotein reaetion were detected in the extract.
91 Cysteine is very easily transformed by action of earbonyl compounds into semimercaptals [34, 35], which are further converted into thiazolidines [36]. Analogous compounds with ketones are less reactive [37]. However, these reactions do not lead to brown pigments on the contrary, cysteine has been reported [38, 39] as an inhibitor of browning. Because of this great reactivity, no free thiol groups were present, even in the original sample of green coffee, either in free or in combined amino acids. :Free cystine was decomposed as well, possibly via eysteine [40,41]. Reactions between free amino acids and reducing sugars at low water content are affected by the activity of water, pH value and temperature [42, 43]. In our experiments both the water content and the temperature were kept constant, but the pH value could change towards lower values, in spite of buffering capacity of the material. The course of changes of free amino acids and reducing sugars is complicated (Fig. 1) because additional amounts of the two reactants were produced during the storage by hydrolysis of proteins and of polysaccharides, respectively. The influence of hydrolysis is obvious, especially in the first reaction stage, before hydrolytic enzymes have become deactivated. I t may be seen in Fig. 2 that the content of ~-aminobutyric acid, i. e., an acid that is not bound in protein, decreased from the very beginning of storage, while the content of other amino acids combined in protein increased in the first stage of storage by hydrolytic reactions, and started to decrease only in later stages. Some amino acids and lower peptides also accompany browning reactions of protein [43]. The browning reaction (Fig. 3) consists of two phases: (1) first stage when amino acids are rapidly decomposed with formation of eolourless or light colonred compounds [A4s0~ 0.18 -[0.16 (1.5 --A_A)] where A.4. is the content of free amino acids [mg/g]; (2) second stage when the browning becomes more intensive [A,30 ~ 0.16 ~ 0.76 (1.0---AA)]. This behaviour is in agreement with the mechanism of N[aillard reactions. Brown products formed by Maillard reactions are partially soluble in 75% aqueous ethanol. The browning was a first order reaction (log A400~-- - - 0.38 ~ 0.98 t where t is the reaction time [d]) in agreement with the literature on nonenzymic browning reactions [44--47]. The ratio of absorbanees at 400 and 500 nm [A400:As00] decreased from 9.5 in the original coffee beans to 5.4 after 80 days of storage. This signifies that the dominant wavelength shifted to longer wavelengths on storage, probably by extension of conjugated double bond systems by dimerization of Schiff bases, aldolization and similar reactions [48--50]. Deducing sugars react not only with free amino acids but also with proteins, as the basic amino acids retain one reactive amino group, also when combined in protein [33, 51], e. g., when glucose was heated with casein, the contents of both available and total lysine, arginine and histidine decreased [52]. From the experimental data (Table 2) it is evident that some eolourless or light-coloured condensation products of lysine are formed as intermediary products. These products are then converted into brown pigments without appreciable change of available lysine.
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