1 H NMR, a Rapid Method to Monitor Organic Acids during Cupuassu (Theobroma grandiflorum Spreng) Processing ISIS M. FIGUEIREDO, ² NADIA R. PEREIRA, ‡ PRISCILLA EFRAIM, ‡ NELSON H. P. GARCı ÄA, ‡ NADIA R. RODRIGUES, § ANTO ˆ NIO MARSAIOLI,JR., ‡ AND ANITA J. MARSAIOLI* ,² Departamento de Quı ´mica Orga ˆnica, Instituto de Quı ´mica, Universidade Estadual de Campinas, P.O. Box 6154, 13084-971 Campinas, SP, Brazil, Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas, P.O. Box 6121, 13084-971 Campinas, SP, Brazil, and CPQBA/UNICAMP, P.O. Box 6171, 13084-971 Campinas, SP, Brazil The development of an analytical method using 1 H nuclear magnetic resonance ( 1 H NMR) spectrometry to monitor cupuassu (Theobroma grandiflorum Spreng) bean fermentation, drying, and roasting processes is reported. The analysis of organic acids and alcohols of crude water extracts of cupuassu ground kernels were monitored by HPLC and 1 H NMR spectroscopy. The residual protein signals caused deleterious effects on acid and alcohol quantifications. Therefore, the analytical procedures were optimized by sample cleanup and water suppression pulse sequences in order to obtain compatible data using HPLC and 1 H NMR. The quantification of lactic acid, acetic acid, and 2,3-butanediol by NMR is 5- to 10-fold faster than by HPLC, with the advantage of providing the identification of several chemical species in a single experiment. Application of these analytical conditions to some cupuassu samples revealed that this methodology can be applied to the quality profiles of fermentation and roasting processes. KEYWORDS: 1 H NMR spectroscopy; cupuassu; WET-CPMG; PRESAT; fermentation; roasting processes, microwave INTRODUCTION Cupuassu is a commercially important tree, naturally occur- ring in eastern Amazon (Brazil). The fruits have a white-yellow pulp with a pleasant acidic taste and a strong fragrance. The production of “cupulate” from cupuassu seeds (a product similar to chocolate) is a multistep process: after harvesting, the seeds are separated from the fruits and fermented. The dry fermented seeds are roasted, sieved, and finely ground. The resulting solid product (“liquor”) is the raw material for the “cupulate” industries (1-3). Flavor attributes such as flavor intensity, bitterness, astrin- gency, and acidity are assigned to kernel fermentation, drying, and roasting and caused by several reactions occurring during post-harvest processing (4). Roasting leads to development of specific aromas and formation of volatile organic compounds with desirable flavor properties (5, 6), such as esters (ethyl and butyl butyrate, ethyl and butyl 2-methylbutyrate), terpenoids (- linalool), and heterocyclic compounds (pyrazines, piperazine, 2,5-dihydro-2,5-dimethoxyfuran). Moreover, some undesirable volatile fermentation compounds are eliminated, such as organic acids, resulting in flavor enhancement (7, 8). The fermentation process is characterized by the production of organic acids (acetic acid and lactic acid) (9, 10), which are particularly impor- tant in defining cupuassu quality in the manufacture of “cupu- late”. It was reported that beans with high levels of acetic and lactic acid provided cocoa beans with low chocolate flavor (11). The bacterial production and degradation of acetic acid during fermentation are important to cupuassu curing and flavor precur- sor development. However, an excess of acetic acid causes an adverse effect on cupuassu flavor. Additionally, under anaerobic glucose fermentation there is formation of large amounts of lactate and traces of acetate, ethanol, formate, and 2,3- butanediol, which is markedly different from the composition of end products under aerobic fermentation when a mixture of lactate, acetate, and acetoin-diacetyl is produced (Figure 1)(12). Thus rapid methodologies to monitor fermentation and roasting are important in optimizing cupuassu manufacture, and 1 H nuclear magnetic resonance is one of the most adequate nondestructive, multinuclear, and noninvasive techniques, suc- cessfully employed for the simultaneous detection of several substances present in one sample. Additionally the spectrum acquisition time, requiring minimal sample preparation, enables the examination of many samples in a reduced time interval, as required for most food composition, authenticity, and quality control applications (13, 14). Notwithstanding these properties, * Corresponding author. E-mail: anita@iqm.unicamp.br. Phone: + 55 19 3788 3067. Fax: + 55 19 3788 3023. ² Departamento de Quı ´mica Orga ˆnica, Universidade Estadual de Campi- nas. ‡ Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas. § CPQBA/UNICAMP. 4102 J. Agric. Food Chem. 2006, 54, 4102-4106 10.1021/jf0525176 CCC: $33.50 © 2006 American Chemical Society Published on Web 05/17/2006