The use of roasting kinetics data to characterize natural and artificial chocolate aroma precursors

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

535

T h e u s e of r o a s t i n g k i n e t i c s data t o c h a r a c t e r i z e n a t u r a l a n d artificial c h o c o l a t e aroma precursors G. P. Rizzi a n d P . R. Bunke Procter & Gamble Company, Miami Valley Laboratories Cincinnati, Ohio 45239-8707 (U.S.A.)

Abstract Chocolate aroma precursors were investigated in conjunction with a study aimed at delivering enhanced chocolate flavor in thin layer roasting of homogenized raw cocoa beans. Chocolate aroma precursors were isolated from fermented cacao beans and characterized by kinetic data obtained in controlled roasting studies. A rate-limiting first order loss of amino nitrogen was observed which is consistent with a bimolecular amine/carbonyl reaction mechanism. Rate constants and Arrhenius parameters served as a guide for design and development of simplified, artificial precursor systems. Fractionation of natural precursors with ion exchange extraction and activated carbon adsorption established the significance of hydrophobic amino acids Uke leu, ileu, val, tyr, trp and phe in chocolate aroma formation. A synthetic precursor mixture containing hydrophobic amino acids and reducing sugars generated authentic chocolate aroma and provided the basis for the development of a practical chocolate flavor enhancer. 1.

INTRODUCTION

A study of chocolate aroma precursors was undertaken as a part of related process work aimed at enhancing aroma production during continuous raw bean Hquor roasting. The objective of our study was to define a simplified, synthetic precursor mixture which could serve as an adjunct source of chocolate flavor in contemporary roasting processes. Traditionally, chocolate aroma is produced by roasting cocoa beans which have previously undergone fermentative and drying treatments. During fermentation, flavor precursors are formed which react at roast temperatures to form endproduct flavor compounds. In conventional processing roasted cocoa beans are macerated to form chocolate Hquor or "mass" which is further processed to produce cocoa butter, cocoa powder or confectionary chocolate [1]. In traditional batch processing, large variations in bean sizes and low thermal conductivity can result in uneven roasting of individual beans and less than optimum flavor production.

536

In recent years traditional batch processing of beans has partially been replaced by theoretically more uniform raw liquor roasting [2]. In continuous, raw liquor processing, raw (pre-fermented) beans are finely ground and uniformly roasted in thin-layer roasters. In thin layers, fast heat transfer is possible and optimum aroma is formed in a few seconds. Thin-layer roasters also provide the opportunity to add supplemental precursors to the raw liquor before roasting. For example, in the so-called LSCP process [3], raw Uquors are modified by reducing sugar addition and by enzyme pretreatment. Extensive research has led to the conclusion that chocolate aroma is a result of complex chemical reactions of amino acids, peptides, sugars and possibly flavonoid compounds during roasting [4, 5]. It was suggested [6,7] and later proven by l^C-tracer studies [8] that Strecker degradations of amino acids (probably fueled by Maillard reaction derived dicarbonyls) are the source of volatile aldehydes, a key ingredient of chocolate aroma. Also, model studies suggest that other important chocolate aroma components Hke aliphatic acids, a, P-unsaturated aldehydes, sulfur compounds and alkylpyrazines all originate as secondary products of the Strecker degradation [9]. Recently, it has also been shown in model studies that Amadori compound decomposition contributes directly to the formation of chocolate aroma and that the composition of aroma compounds formed in these reactions is a function of water activity [10]. Knowledge of roasting chemistry has already led to formulations of synthetic precursor mixtures that generate chocolate aroma under simulated roasting conditions [11]. Quantitative changes of amino acids during roasting correlate well with the formation of chocolate aroma volatiles. IQnetic studies on cacao roasting done with whole beans showed an apparently linear (zero order) loss of free amino acids with time and concomitant production of aldehydes and pyrazines [12, 13]. Chocolate aroma is also formed without the bean matrix when isolated natural precursors are heated under roasting conditions [7]. Changes in total amino acid level during bean extract roasting were shown to depend on time, temperature and initial moisture content; and subsequently, time/temperature data obtained in the model studies were used to obtain optimized chocolate flavor in factory scale, whole bean roasting. Based on related studies on synthetic amino acid/sugar mixtures [7], it was also proposed that chocolate aroma can be afi'ected by the way individual amino acids decompose at different rates at a given temperature. The goal of our experimentation was to gather roasting ]dnetic data in the early stages of bean extract roasting and to use this data to guide the development of a simplified, synthetic chocolate aroma precursor system. 2. MATERIALS AND METHODS Commercially available fermented/dried cocoa beans were used. Activated carbon was Merck U.S.P. activated charcoal.All analytical reagents, standard reference compounds and solvents were commercial materials of analytical grade

537

purity and used as received. 2.1. Cocoa bean extraction Beans, including shells were dry-pulverized to < 1 mm diameter particle size in a Waring blender and extracted with 80-20 v/v methanol-water for 18 hrs. at 22^C. Resulting slurries were filtered, methanol was evaporated under vacuum and the aqueous residues were fireeze-dried to obtain 7-12 % yields of dry,powdered extracts. Low fermented beans like Arriba and Sanchez gave deep purple extracts characteristic of intact anthocyanin pigments; however, extracts of well fermented beans Hke Ghana and Bahia were brown, apparently as a result of the action of pol5rphenoloxidase and oxygen. 2.2. Bean extract roasting Prior to roasting, freeze-dried extracts were stored in glass chambers provided with fixed relative humidity to obtain samples with definite, equilibrium water content (Table 2). For organoleptic evaluation, 0.1 gm. samples were oven heated at various temperatures and times in closed, screwcapped (2 oz.) glass bottles. After reaching room temperature, the roast aromas were evaluated by sniffing, and results are also summarized in Table 2. For kinetic measurements, 10 mL. glass ampules containing ca. 0.05 gm. of bean extracts or synthetic precursor mixtures were equilibrated to a ^ 0.13, flame-sealed and heated (totally submerged) in a thermostated oil bath. To obtain rate data, several identical samples were thermostated at t = 0 time. Then, samples were removed sequentially at time intervals and cooled rapidly to room temperature before analysis. On analysis, the ampules were opened and contents were dissolved/dispersed in water by sonication. Soluble products were isolated by filtration and aUquots of filtrates were analyzed for total amino nitrogen content. Optionally, solutes of aqueous solutions were prepared for roasting by freezedrying the samples directly in bottles or ampules prior to the equilibration step. 2.3. Fractionation of Ghana bean extract (GBE) GBE was washed with ethyl acetate to remove residual lipids, stirred in methanol and centrifuged to separate relatively high molecular weight (insoluble) materials. The methanoHc supernatent was evaporated and the residue was dissolved in 5:95 v/v acetic acid-water before passing it through a glass column containing a cation exchange resin (sulfonated polystyrene, H"*"form). Vacuum concentration of the eluant yielded "neutral plus acidic material", GBE/NA. Further elution of the column with 10:90 v/v pyridine-water followed by concentration and freeze-drying provided a "basic" fraction, GBE/B. 2.4. Activated carbon adsorption treatment of GBE basic fraction A 0.5% aqueous solution of GBE/B was treated with 1% activated carbon and stirred for 6 hrs. at 22^0 (ambient pH 3.98). Carbon was removed by filtration and the filtrate was evaporated to yield a white solid, GBE/B 1 (38.7% recovery based on GBE/B). The recovered carbon was sequentially extracted by stirring it

538 at 220C, first with (a) 20:80 v/v pyridine-water (pH 7.64) and then with (b) 100 % acetic acid. Each extract was isolated by filtration and vacuum concentrated to yield residues GBE/B2 and GBE/B3 representing 49.6% and 8.0% recovery (based on GBE/B) ficom (a) and (b) repectively. The proximate analysis of subficactions B, B l and B2 are presented in Table 3. 2.5. Analytical methods Individual amino acids were separated and quantified by high performance liquid chromatography (HPLC) analysis after reaction with o-phthalaldehyde to form fluorescent derivatives. Several unidentified amino compounds were observed which may have been Amadori rearrangement products [10]. A fluorescence emission detector was used to quantify the results. Total amino nitrogen was determined colorimetrically with a ninhydrin reagent [14] using a glycine standard on aliquots adjusted to pH 5.0 with a citrate buffer. Sugars and glycerol were analyzed directly by HPLC on an IBM amino column using 80:20 acetonitrile-water as eluant and a refractive index detector. HPLC was also used to quantify methyl xanthines (theobromine and caffeine) and epicatechin. Karl Fischer water and Kjeldahl nitrogen were assayed by automated procedures. Total phenols were determined by a standard procedure using Folin-Ciocalteu reagent [15] and results are expressed in terms of gallic acid. 3.

RESULTS AND DISCUSSION

3.1. Isolation of chocolate aroma precursors Four varieties of dried cocoa beans representing a wide range of prefermentation were extracted with aqueous methanol and freeze-dried to obtain apparently dry powders containing aroma precursors (Table 1). Extracts of the more fermented (Bahia and Ghana) beans contained more free amino acids, total amino N and less sucrose, than the lower fermented Arriba and Sanchez varieties. Both Bahia and Ghana contained less epicatechin, an indicator of fermentation-degree, probably due to degradation by polyphenoloxidase. All four extracts contained about the same levels of total reducing sugars, however the mole ratios of amino N/reducing sugars were clearly higher for Bahia and Ghana. Fructose was the dominant reducing sugar in all four bean extracts. Preliminary roasting experiments showed that the strongest, most authentic chocolate aromas were generated by heating Bahia or Ghana extracts to 120-130OC for 3060 min. at an initial moisture content adjusted to 4.4-4.7 % ( a ^ 0.13), Table 2. Based on organoleptic performance, we selected Ghana extract as our model for synthetic precursor design. 3.2. Roasting characteristics of cacao bean extracts Kinetic measurements of extract roasting were based on loss of amino N versus time since aroma formation is known to be tightly coupled to amino acid/peptide reactions in whole bean roasting. Amino N content initially dropped rapidly during roasting (0-60 min), but its rate of decrease slowed considerably at longer roast times. We found, in agreement with published data, that in

539 Table 1 Partial composition of cocoa bean extracts Component

Arriba

Bahia

Ghana

Sanchez

aspartic acid* glutamic acid serine histidine glutamine glycine arginine alanine tyrosine tryptophane methionine valine phenylalanine isoleucine leucine lysine

0.06% 0.33 0.07 tr 0 0.02 0.05 0.29 0.17 tr tr 0.22 0.29 0.14 0.41 tr

0.08% 0.40 0.14 tr 0 0.02 0.14 0.46 0.46 0.08 0.03 0.44 0.86 0.29 0.98 0.13

0.12% 0.66 0.16 tr 0 0.06 0.12 0.63 0.54 0.08 tr 0.61 0.95 0.37 1.09 0.10

0.05% 0.31 0.10 tr 0 0.04 0.07 0.31 0.27 tr tr 0.28 0.39 0.21 0.51 tr

glycerin fructose glucose sucrose

2.0 4.9 3.3 7.1

0.8 4.0 1.9 0.4

1.3 6.2 2.1 3.6

2.4 4.3 5.1 7.8

water Kjeldahl N amino N epicatechin

3.73 3.99 0.34 2.1

3.26 5.45 0.59 3.0

4.07 5.49 0.72 1.4

3.02 3.31 0.38 4.3

mol amino N/mol reducing sugars

0.53

1.28

1.12

0.52

* Amino acids listed in order of elution during HPLC analysis.

general, reducing sugars disappeared almost completely during roasting leaving some amino N unreacted at long roast times. During the first sixty minutes of roasting, amino N loss was well described by the integrated rate equation (1). ln[(Ct-C60)/Co-C60)] = - k i t

(r2 > 0.9)

(1)

540 Table 2 Effect of water activity (a^) on aromas generated during bean extract roasting

Extract

Water Activity, a ^

K. F. Water (%)

Aroma

Bahia

0.0 0.13 0.53 0.75

1.52 4.43 10.24 19.13

strong chocolate strong chocolate weak chocolate, winey weak chocolate, musty

Ghana

0.0

1.16

0.13 0.53 0.75

4.69 11.90 23.34

chocolate, hot chocolate milk chocolate, malty very weak chocolate very weak. mushroomy

Arriba

0.0 0.13 0.53 0.75

1.04 4.19 10.18 22.13

Sanchez

0.0 0.13

1.54 4.29

0.53 0.75

11.54 20.41

sweet chocolate chocolate hquor very weak chocolate very weak, si. burnt strong chocolate sharp, chocolate Uquor musty, fecal very weak, musty

Roasting conditions: 120^0 for 60 min.

where CQ, CQQ and C^ represent percent amino N in the roasted sample at times 0, 60 and t min. respectively, t is the roasting time in min. and k^ is a first order rate constant in m i n ' l . Rate constants were calculated from roasting kinetic data as the negative slope of the Hnear regression Unes obtained from plots of In [concentration ratio] versus roasting time. Roasting half-time (ti/2) in min. is equal to In 2/ki. The rate datajoer se tells us Uttle about reaction mechanism except to say that the rate of amino N loss with time is proportional to remaining amino N in the 0-60 min. roasting range. However, the kinetic order of amino N loss observed is consistent with the accepted mechanisms for the earliest stages of the Maillard reaction or the Strecker degradation. Kinetic roasting data for bean extracts spanning a 90-135^0 temperature range are summarized in Table 3. Predictably, the rates of amino N loss

541 Table 3 Roasting kinetic data for cocoa bean extracts at 90, 120 and 135^C with initial a ^ 0.13

ki (min-1), [ti/2 (min) ] Extract Bahia Ghana Arriba Sanchez

90OC 0.0070, 0.0076, 0.0077, 0.0062,

[99] [91] [90] [112]

120OC 0.023, 0.035, 0.045, 0.032,

[30] [20] [15] [22]

1350C 0.037, 0.039, 0.043, 0.040,

[19] [18] [16] [17]

Ea (KCalM-1) 11.1 11.5 12.4 12.8

increased markedly with roasting temperature, however more rate increase was observed between 90 and 120^0 than between 120 and 135^C. A less obvious variation was observed by comparing rate constants at the same temperature for a variety of extracts. At 90 and 135^0, k^ did not vary much across bean varieties. However, at 120^0, we observed as much as two-fold rate differences among the four bean extracts. To us this was an indication that an aroma precursor system based on amino acid/pep tide chemistry was highly operative in fermented extracts at 120^C. Since optimum chocolate aroma development also took place at 120^0, we also concluded that a k i of ca. 0.025 m i n ' l was a suitable design value for synthetic precursor development in this temperature range. The sensitivity of roasting chemistry to temperature change is also quantified by the activation energy parameter Ea defined in the Arrehnius equation (2). Inki = - Ea/RT + Inkg

(2)

In this equation, k l is a rate constant and Ea is an activation energy whose unit is defined by the units of R and T. A plot of Inkj versus 1/T, where T = roast temperature in ^C + 273 produced straight Unes in the 90-135^C roasting range (r2 > 0.9) whose slopes times -R (1.987 Cal-deg'l-mole"l) gave Ea in Cal-mole"l. For bean extract roasting (Table 3) extracts of highly fermented beans (Bahia and Ghana) showed less temperature effect on roasting rate (Ea, ca. 11 KCalmole'l) than lesser fermented bean extracts (Ea, ca. 12-13 KCal-mole"l). 3.3. Synthetic chocolate aroma precursor systems Ghana bean extract (GBE) was fractionated to identify components that are most responsible for producing chocolate aroma during roasting. Fractions were tested for aroma generating potential by roasting them both with and without added sugars (Table 5). Starting with a Uterature-suggested procedure [4] we isolated an aromagenic "basic fraction" (GBE/B) using cation-exchange

542

Table 4 Partial analysis of Ghana bean basic fraction (GBE/B) and results of partitioning by activated carbon.

Not adsorbed by carbon

Adsorbed by carbon

Component

Initial GBE/B

GBE/B 1

GBE/B2

unk-1* unk-2 glycine glutamine asparagine aspartic acid tryptophane unk-3 serine threonine isoleucine glutamic acid tyrosine unk-4 valine alanine phenylalanine leucine

0.0 relative % 0.0 0.5 0.5 0.5 1.4 1.4 2.4 2.4 2.4 6.3 7.7 7.7 9.6 9.6 11.5 14.9 21.2

0.0 relative % 0.0 0.9 0.4 0.4 1.5 0.0 2.8 3.1 3.7 8.3 8.7 0.7 10.8 12.2 14.4 2.6 29.6

0.8 relative % 0.8 0.0 0.0 0.0 0.8 3.1 0.8 0.8 0.8 3.1 2.3 23.7 3.8 2.3 2.3 45.0 9.2

theobromine caffeine total phenols** total amino N ash

16.0 absolute % 2.9 15.3 3.3 0.68

0.6 absolute % 0.2 1.6 6.4 1.21

29.0 absolute % 5.4 10.2 2.2 0.41

* Amino acids and related unknowns Usted in increasing amounts found in GBE/B. ** As gallic acid. chromatography. Fraction GBE/B contained free amino acids, methylxanthines and phenolic materials of unknown structure (Table 4). Because GBE/B contained viable precursors, it was fractionated further by adsorption onto activated carbon. Material not adsorbed to carbon, GBE/B 1 failed to produce chocolate aroma; however, GBE/B2 isolated from carbon by aqueous-pyridine extraction did produce chocolate aroma in conjunction with sugars.

543

Table 5 Identification of chocolate aroma precursors in Ghana bean fi-actions

ADDED REDUCING SUGAR NO YES 1 GHANA BEAN EXTRACT (GBE)

X

CHOC. AROMA FORMED YES NO X

1

cation exchange

1 1 1

BASIC FRACTION (GBE/B)

X

X X

X

1 activated carbon adsorption

1

1

NON-ADSORBED MATERIAL

1

(GBE/Bl)

X

X

X

X

1 1 ADSORBED MATERIAL 1 (GBE/B2)

X

X X

X

Freeze-dried mixtures roasted at 120^C for 30 min at initial a ^ 0.13. Sugars = mixture of fructose, glucose and sucrose in same relative proportions found in Ghana bean extract; mole ratio of amino N/reducing sugars 1:1.

Interestingly, GBE/B2 contained a predominance of hydrophobic amino acids, in particular the benzenoid derivatives tyrosine, phenylalanine and tryptophane. In addition to amino acids, the carbon adsorbant also retained most of the methylxanthines and some phenoUc materials. A sixteen component synthetic precursor mixture was formulated based on the ratio of amino acids and methylxanthines found in GBE/B2 and a mixture of sugars in the same relative proportions observed in a Ghana bean extract (Table 6). On roasting at 120^0 at initial a ^ 0.13, the mixture developed a strong aldehydic, chocolate-like aroma in 30 min. Roasting kinetics data closely resembled those of natural extract roasting; however, k j for the synthetic was somewhat more sensitive to temperature variation (Ea, 14.9 KCal-mole-1) than the naturals. Differences in aroma quahty were seen between synthetic and

544

Table 1 aoie 6b Sixteen-component synthetic precursor mix and related roasting kinetics Component phenylalanine tyrosine leucine tryptophane isoleucine alanine glutamic acid aspartic acid serine threonine lysine AMINO ACIDS (total) fructose glucose sucrose SUGARS (total) theobromine caffeine XANTHINES (total)

Relative % of classes 45.0 23.7 9.2 3.1 3.1 2.3 2.3 0.76 0.76 0.76 0.76 100

Absolute % in mixture 13.4 7.1 2.7 0.92 0.92 0.69 0.69 0.23 0.23 0.23 0.23 29.8

66.7 12.8 20.5 100

28.6 5.5 8.8 42.9

85.0 15.0 100

23.2 4.1 27.3

mM/gm of mixture 0.81 0.39 0.21 0.045 0.070 0.077 0.047 0.017 0.022 0.019 0.016 1.72 1.59 0.31 0.49 1.90 (reducing only)

Roasting Kinetics Data Roasting temp, "C 90 120 135 Ea (KCalM-1)

Rate constants, ki (min'^) 0.0051 0.029 0.039 14.9

natural systems which may have been due to the unidentified phenolic components in GBE/B2. In retrospect, it was found later that a stiU better chocolate aroma could be generated by heating the sixteen component mixture for very short times (5-15 min.) at ca. 180^0.

545 A further attempt toward simplification led to a nine-component synthetic mixture (Table 7) containing only four amino acids. During roasting, the kinetic data of this mixture closely paralleled those of natural extracts and reasonably good chocolate-like aroma was formed at 120-130^C.

Table 7 Nine-component, simplified precursor system and related roasting kinetics data

Relative % of classes

Component

Absolute % in mixture

mM/gm in mixture

tyrosine phenylalanine tryptophane leucine AMINO ACIDS(total) fructose glucose sucrose SUGARS (total)

42.0 34.0 17.0 7.0 100

13.6 11.0 5.5 2.3 32.4

0.75 0.67 0.27 0.18 1.87

67.0 13.0 20.0 100

27.7 5.4 8.3 41.4

1.54 0.30 0.24 1.84 (reducing)

caffeine theobromine XANTHINES (total)

15.0 85.0 100

3.9 22.3 26.2

Roasting Kinetics Data

Roasting temp, OQ

Rate constants, ki (min'l)

90 120 135

0.0056 0.026 0.041

Ea(KCalM-l)

13.1

546 4.

ACKNOWLEDGEMENTS The authors thank L. V. Haynes and L. Ngo for their analytical support.

5.

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