Influence of Aerobic and Anaerobic Moist Incubation on Selected Nonvolatile Constituents─Comparison to Traditionally Fermented Cocoa Beans

Abstract

Recently, moist incubation has been proposed as an alternative postharvest processing method for cocoa beans. During this treatment, unfermented and dried cocoa nibs are rehydrated with a lactic acid solution containing ethanol and subsequently incubated for 72 h at 45 °C before drying. Previous studies focused on the aroma formation during this treatment and the further processing of chocolate. The current study focused on the influence of aerobic and anaerobic moist incubation on selected nonvolatile components in comparison with the unfermented raw material and traditionally fermented cocoa. Total phenolic content and total flavan-3-ol content, contents of (+)-catechin, (−)-epicatechin, procyanidins B2 and C1, cinnamtannin A2, methylxanthines (theobromine and caffeine), contents of sugars (sucrose, D-glucose, and D-fructose) and free amino acids (17 proteinogenic amino acids) were determined. The fermentation index was also evaluated. The aerobically incubated and fermented cocoa showed low levels of phenolic compounds in comparison to the unfermented cocoa and the anaerobically incubated cocoa. The level of methylxanthines was unaffected by all treatments. The contents of reducing sugars were more than 2-fold higher after both incubation treatments compared to fermentation. The level of free amino acids liberated was highest after anaerobic incubation followed by fermentation and aerobic incubation. The aerobically incubated cocoa showed the highest FI, while the anaerobically incubated cocoa may be considered under-fermented (FI <1.0). Statistical analysis (ANOVA) showed significant differences between all treatments, which was verified by principal component analysis.

Amino acid profile behavior during the fermentation of Criollo cocoa beans

“…According to the correlation circle ( Fig. 8 c), the first component contained Phe, Leu, Arg, Lys, Iso, Val , His, Ala, and Cys (highlighted amino acids were those with the highest correlation, >94%), similar to that reported by Schlüter et al (2022) ; whereas, in the second component the amino acids with the highest contribution were Glu, Tyr, Ser, Pro, Gly and Asp (highlighted amino acid with the highest correlation, >89%); behavior similar to SF. In addition, multivariate analysis has shown that amino acids (primarily hydrophobic) prevailed at the end of the fermentation, similar to that reported by Deus et al (2021) , due to the ability of the inoculum to modulate the change in the concentration of aromatic precursors ( Sande et al, 2020 ), and influenced by the cacao origin, soil characteristics, climatic conditions, among others ( Balcázar-Zumaeta, Pajuelo-Muñoz, et al, 2023 ; Marseglia et al, 2014 ; Servent et al, 2018 ; Spizzirri et al, 2019 ), however further studies are warranted.…”

“…Table 1 , Table 2 , Table 3 show the concentration of 17 amino acids in the cocoa bean. It can be observed that the fermentation time affected the amino acids content ( p < 0.05), similar to that reported by Rottiers et al (2019) , evidencing that the fermentation process degrades the proteins due to the presence of microorganisms and enzymes ( Domínguez-Pérez, Beltrán-Barrientos, González-Córdova, Hernández-Mendoza, & Vallejo-Cordoba, 2020 ; Febrianto et al, 2022 ), which contribute to the formation of aromas during the cocoa bean processing ( Schlüter et al, 2022 ; Tamimi, Hidayat, Utami, & Witasari, 2023 ). An atypical behavior was observed in the identification of amino acids in SF in cocoa beans (day 3, Guadalupe), which could be related to the microbial behavior (lactic acid bacteria and nitrogenous yeasts) and carbon metabolism in a spontaneous (uncontrolled) fermentation ( Balcázar-Zumaeta, Castro-Alayo, et al, 2023 ; Deus et al, 2021 ) however, further studies are needed to explain this behavior.…”

“…In the first component, PCA showed that amino acids such as Tyr, Phe, Leu, Iso, Val, Lys , His, Cys, and Gly were the variables with the highest contribution to the first component ( Fig. 6 c, the highlighted amino acids were those with the highest correlation, > 93%), similar to that obtained by Schlüter et al (2022) where Gly, Tyr, and Phe contributed the highest and were correlated in the PCA correlation circle; while Ala, Arg , Asp, Glu, His and Ser were the amino acids with the highest contribution ( Fig. 7 c, the highlighted amino acids were those with the highest correlation, > 75%).…”

“…2 a, the cocoa from Copallín registered a low content during almost the entire fermentation process. The FAA content was below the value mentioned by Tchouatcheu et al (2019) , where a fermented bean contained around 800 to 1400 mg/100 g; this may be because the study was performed in an aerobic process (with turnovers) ( Balcázar-Zumaeta, Pajuelo-Muñoz, et al, 2023 ), producing the oxidation of flavan-3-ols, which reacted with nucleophilic groups of amino acids to form insoluble complexes ( Schlüter et al, 2022 ), and the presence of microorganisms which limited the production of FAA content ( Rahardjo et al, 2022 ).

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“…Given this, the use of inoculums during fermentation allows the synthesis of flavor compounds and derivatives ( Balcázar-Zumaeta, Pajuelo-Muñoz, et al, 2023 ); inoculums reported were yeasts ( Kluyveromyces marxianus, Pichia kluyveri , and Saccharomyces cerevisiae ), acetic acid bacteria ( Acetobacter pasteurianus, A. aceti, and A. tropicalis ), and lactic acid bacteria ( Lactobacillus fermentum and L. plantarum ) ( Balcázar-Zumaeta, Pajuelo-Muñoz, et al, 2023 ; Ooi, Ting, & Siow, 2020 ). In addition, the use of starter cultures is a method that improves the chemical composition of the fermented cocoa bean (with emphasis on amino acids) ( Febrianto, Wang, & Zhu, 2022 ), can modulate the fermentation time and influence the amino acids concentration ( Balcázar-Zumaeta, Castro-Alayo, et al, 2023 ; Castro-Alayo et al, 2019 ; Korcari et al, 2023 ; Sande et al, 2020 ; Schlüter, André, Hühn, Rohn, & Chetschik, 2022 ).…”