Microwave-assisted unfermented cocoa bean: improving flavor precursor after acetic acid submersion

Abstract

Objective was to enhance flavor precursors of unfermented cocoa beans by soaking beans in acetic acid and further heated by microwave. Acetic acid concentration, microwave power, and microwave exposure time were investigated and screened using a one-variable-at-a-time approach. Optimum condition for degree of hydrolysis (DH) was determined by Response Surface Methodology using Box-Behnken Design. Results showed that flavor precursors increased at a higher acetic acid concentration, microwave power, and microwave exposure time. Optimum condition was achieved at acetic acid concentration of 1.21 M, microwave power at 450 W, and microwave exposure time of 4 min. The microwave-assisted cocoa bean had a DH of 38.99% and a reducing sugar of 0.98%. Microwave-assisted heating increased amino acid content, especially hydrophobic amino acids as flavor precursors, and the main volatile compounds, especially aldehyde and pyrazine. Thus, microwave-assisted heating is a promising alternative to improve flavor precursors of unfermented cocoa beans.

Introduction

Most of Indonesia’s cocoa products are produced by smallholder plantations, and only a small portion is produced by private plantations and state-owned plantations. The increase in Indonesia’s cocoa production is inversely proportional to the quality of cocoa. The produced cocoa beans have low quality because they don’t undergo a fermentation process, which causes the lack of the desired flavor precursors.

The fermentation process in cocoa beans produced alcohols and organic acids, predominantly acetic acid (Apriyanto 2016; Hernani et al. 2019). The biochemical reaction in the beans automatically changed, so the astringent taste in beans was reduced (Tarigan and Iflah 2018). Additionally, the flavor precursors were produced during fermentation: amino acids, especially leucine, alanine, phenylalanine, and tyrosine. Sugar was also converted into alcohol and acids (Sukha et al. 2017). These compounds were converted into cocoa flavor during the cocoa bean roasting (Voigt and Lieberei 2014). Furthermore, the volatile compounds in chocolate are pyrazines and aldehydes (Liu et al. 2014).

On the other hand, unfermented beans lack flavor precursors since they were produced during fermentation. Alternatives to increase flavor precursors in unfermented cocoa beans were acid submersion, adding an enzyme, and re-fermentation with a starter such as S. cerevisiae; L. plantarum; A. aceti (Hernani et al. 2019; Kadow et al. 2015; Yuniar et al. 2018). Incubation on fresh cocoa beans using an acid solution produced high amounts of amino acids and reduced sugar (Kadow et al. 2015). Besides, unfermented cocoa beans treated with proteolytic enzymes produced better flavor after roasting, but enzyme is expensive and needs more cost to apply in industries (Yuniar et al. 2018). Re-fermentation of unfermented cocoa beans using a starter resulted in producing an acetic acid at the end of the fermentation process, generated kills the cocoa beans, and activating the enzyme (Hernani et al. 2019). The enzyme hydrolyzed the protein into amino acid and peptide, which were the flavor precursor of cocoa. It’s important to note that the starter must be developed before applying this treatment (Afoakwa 2016; Hernani et al. 2019).

Amino acids were flavor precursor of cocoa beans, and it was developed during fermentation. Microwave has been demonstrated as an acceptable alternative to traditional protein hydrolysis conditions. Microwave-assisted heating accelerated the hydrolysis of protein into amino acids and many short peptides in a short time (Chen et al. 2014; Wang et al. 2022). It offers significant time-saving due to a short time. Furthermore, microwave roasting of cocoa beans produced flavor precursors and distinctive aroma profiles (Lemarcq et al. 2022). Besides, incubating unfermented cocoa beans using an acid solution has been proven to improve the quality equivalent to fermented cocoa beans (Misnawi et al. 2003).

Thus, precursor flavor in cocoa was produced by acid submersion, adding an enzyme, and re-fermenting with the starter. The objective was to investigate the effect of a combination submersion of acetic acid medium and high temperature heating using a microwave to enhance the flavor precursor of unfermented cocoa beans. Factors such as acetic acid concentration, microwave power, and microwave exposure time were evaluated. Degree of hydrolysis, amino acid content (especially hydrophobic amino acids), and the main volatile compounds (especially aldehyde and pyrazine) were analyzed.

Materials and methods

Materials and methods

Cocoa beans of the forastero variety were collected from Patuk, Gunung Kidul, Yogyakarta, Indonesia. Acetic acid, trichloroacetic acid, NaOH, HCl, and H₂SO₄ were analytical-grade chemicals obtained from Merck KGaA (Darmstadt, Germany).

Bean submersion in acetic acid on total acid

Unfermented dry cocoa beans (5 g) were soaked in 30 mL acetic acid at various concentrations (0.75 M, 1 M, 1.25 M, 1.5 M, and 1.75 M) for 1 h. The cocoa beans were drained before the total acid was analyzed.

Acetic acid concentration on the degree of hydrolysis

The soaked cocoa beans (5 g) from Sect. “Bean submersion in acetic acid on total acid” were heated by microwave at a power of 600 W for 3 min. After that, the cocoa beans were ground in a blender. Then the degree of hydrolysis of sample was determined.

Microwave power on the degree of hydrolysis

The unfermented dry cocoa beans (5 g) were soaked in 30 mL acetic acid (best concentration Sect. “Result and discussion”) for 1 h. The cocoa beans were drained and heated by microwave (Electrolux type EMM2318X, Sweden) at a power of 150 W, 300 W, 450 W, 600 W, and 800 W for 3 min. Then the cocoa beans were ground in a blender, and the sample was analyzed to determine the degree of hydrolysis.

Microwave exposure time on degree of hydrolysis

Unfermented dry cocoa beans (5 g) were soaked in 30 mL acetic acid (selected concentration) for 1 h. The cocoa beans were drained and heated with a microwave with a selected power for 2 min, 3 min, 4 min, 5 min, and 6 min. The cocoa beans were ground in a blender and the sample was analyzed to determine the degree of hydrolysis.

Optimization of enhancing precursor flavor

The first step in the experimental design process was screening. The screening process was a one-variable-at-a-time (OVAT), five levels in each variable used to establish the range level in optimization. The acetic acid concentration (0.75–1.75 M), microwave power (150–800 W), and microwave exposure time (2–6 min) were all investigated for the maximum degree of hydrolysis under optimum conditions.

A Box-Behnken Design is used to optimize flavor precursor enhancement, followed by Response Surface Methodology. The next optimization step will use three levels for each factor, including acetic acid concentration (1 M; 1.25 M; 1.5 M), microwave power (300 W, 450 W, 600 W), and exposure time (3 min, 4 min, 5 min). There were fifteen combinations used in this study, including three center points. The main response with the highest important level (+ + +) will be the degree of hydrolysis. Furthermore, free amino acid, reducing sugar, and volatile compounds of sample from the optimum condition were evaluated.

Bean acidity analysis

The acidity was analyzed using the titration method. Five grams of sample was dissolved with 25 ml of warm distilled water and stirred for 15 min. Then it was filtered, and the residue was taken and dissolved with 20 ml of warm distilled water and stirred for 15 min. Ten mL of filtrate was titrated using 0.1 N NaOH with a phenolphthalein indicator. The formation of a stable pink color indicated the endpoint of the titration.

Degree of hydrolysis analysis

The degree of hydrolysis was analyzed with (Laohakunjit et al. 2014) method. Sample aliquots were mixed with 10 mL 20% trichloroacetic acid (TCA) and centrifuged at 5000 rpm for 15 min. Dissolved nitrogen in the supernatant and total nitrogen were determined using the Kjeldahl method. The degree of hydrolysis is calculated using the following equation:

$$DegreeofHydrolysis\left(\mathit{DH}\right)=\frac{20\%TCASo\text{lub}leN}{\mathit{TotalN}}\times 100$$

Total N represents the amount of nitrogen in the hydrolyzed protein solution, and 20% TCA soluble N represents the amount of protein soluble in TCA (Laohakunjit et al. 2014).

Amino acid analysis

The amino acid compositions were analyzed using high-performance liquid chromatography (HPLC). This analysis followed the method of Selamassakul et al. (2020) with some modifications. Five g cocoa beans were defatted using a Soxhlet apparatus for 8 h with 25 mL of n-hexane solvent. Then the sample was dried at room temperature to remove the hexane solvent. 60 mg sample was added with 4 mL of 6N HCl and heated for 1 h at 110 °C. The solution was neutralized (pH 7) with 6N NaOH, diluted to 10 mL, and filtered through 0.2 μm Whatman filter paper. Samples of 50 μm were added with 300 μm of OPA (Orthophalaaldehid) and stirred for 5 min, then 10 μL was injected into HPLC. Column LiChrospher 100 RP-18 (5 μm) was used with a Thermo Ultimate 3000 RS Fluorescence Detector. Mobile phase A = CH₃OH: 500 mM sodium acetate: THF (2:96:2) pH 6.8 and B = 65% CH₃OH. The flow rate was 1.5 mL/min.

Reducing sugar analysis

Reducing sugar was analyzed with the Nelson-Somogyi method. One g of cocoa powder was dissolved in 50 mL warm water and then filtered. Pb acetate was added to 25 mL of the solution, then filtered and diluted to 50 mL. One mL of the solution was added with Na-oxalate, then filtered and diluted to 50 mL. One mL of the solution was added to a test tube, and 1 mL of Nelson’s reagent was added. The solution was heated in boiling water for 29 min. After cooling, 1 ml of arsenomolydate was added, and 7 ml of distilled water was added and homogenized. The absorbance was measured at 540 nm using a UV–Vis spectrophotometer.

Volatile content analysis

A headspace solid-phase microextraction (HS-SPME) coupled to gas chromatography-mass spectrometry (GC–MS) (Agilent 7890A with Agilent 5975C) was used, following the method from Caprioli et al. (2016) with some modification. Five g samples in 22 mL SPME vial heated at 60 °C for 50 min with SPME fiber DVB/CAR/PDMS 2 cm.

Statistical analysis

All experiments were repeated three times, and results were shown as means ± standard deviations. Response Surface Methodology (RSM) was performed using Design Expert 11.0 to obtain the optimum enhancing precursor flavor of unfermented cocoa beans. RSM and Box Behnken Design (BBD) were used to determine the optimized condition. The initial step in BBD is screening the levels of each parameter for the optimization step. After screening, the chosen levels for all parameters were processed using Design Expert software to optimize the degree of hydrolysis and forming the precursor flavor.

Result and discussion

Effect of acetic acid submersion on total acid

Acid content is one of the factors that affect protein hydrolysis. As shown in Fig. 1a, the total acid of cocoa beans increased significantly (2.08 times) with increasing the acetic acid concentration from 0.75 to 1.75 M (P < 0.05). An increase in acetic acid concentration is due to concentration difference between the acetic acid solution and cocoa beans, and it is through diffusion during incubation at an increase in acetic acid concentration. However, the result was lower than the total acid in the fermentation of cocoa beans reported by Peláez et al. (2016) and Apriyanto et al. (2016), which was an acidity level of 2.37% and 4–5% for fermented cocoa, respectively.

Fig. 1.

a Total acid of unfermented cocoa beans after acetic acid submersion for 1 h; b Degree of hydrolysis of unfermented cocoa beans after 1 h submersion in various acetic acid concentrations followed by microwave-assisted heating at 600 W for 3 min; c Degree of hydrolysis of unfermented cocoa beans at various microwave powers (watt) for 4 min after 1 h submersion in 1.25 M acetic acid solution; d Degree of hydrolysis of unfermented cocoa beans at various microwave exposure time (min) and microwave power of 450 W after submersion in 1.25 M acetic acid solution for 1 h

Effect of acetic acid concentration during microwave-assisted heating on degree of hydrolysis

The acetic acid concentration significantly affected the degree of hydrolysis (P < 0.05) during microwave-assisted heating (Fig. 1b). The degree of hydrolysis increased by 1.6 times with increasing the acetic acid concentration from 0.75 to 1.25 M. It is suggested that an increase in degree of hydrolysis is due to an increase in acetic concentration in cocoa beans (Fig. 1b). Increase in acetic acid concentration leads to higher acidity, which promotes the hydrolysis of proteins during microwave-assisted heating (Chen et al. 2014). Further increase in acetic acid concentration to 1.75 M did not significantly affect degree of hydrolysis (P < 0.05). Thus, the highest degree of hydrolysis was obtained at a concentration of 1.25 M.

Effect of microwave power on degree of hydrolysis

Based on the ANOVA, microwave power affected the degree of hydrolysis (P < 0.05). It can be seen that the degree of hydrolysis did not have a significant increase at an increase in microwave power from 150 to 300 W (Fig. 1c). But it had a significant increase (1.43 times) with an increase in microwave power to 450 W (P < 0.05). It is suggested that an increase in microwave power resulted in an increase in product temperature. Consequently, it accelerates protein degradation (Chen et al. 2014).

Further increase in microwave power to 600 did not significantly affect the degree of hydrolysis (P < 0.05). But a decrease in the value of the degree of hydrolysis occurred at an increase in microwave power from 600 to 800 W, namely about 1.12 times. It is suggested that higher microwave power increases cocoa bean temperature so that it causes a side effect of protein reaction. Therefore, the degree of hydrolysis decreases. Thus, the highest degree of hydrolysis was obtained at a microwave power of 450 W.

Effect of microwave exposure time on degree of hydrolysis

Based on the ANOVA, the exposure time affected the degree of hydrolysis (P < 0.05) during microwave heating (Fig. 1d). The degree of hydrolysis increased significantly with an increase in microwave exposure time from 2 to 4 min (P < 0.05), which was 1.17 times. In general, microwave exposure time is correlated with microwave energy applied to the beans, and beans receive more microwave energy at an increase in microwave exposure time. Therefore, bean temperature increases. As a result, the reaction rate increases so that protein hydrolysis increases. According to Ball and Key (2014), the reaction rate increases as the temperature increases due to the molecules achieving the minimum activation energy required for the reaction. Therefore, the degree of hydrolysis increased.

Further increase in exposure time to 5 min resulted in a decrease in the degree of hydrolysis. It is suggested that an increase in cocoa bean temperature and exposure time causes the obtained peptides to undergo other reactions (Zheng et al. 2021). Therefore, the degree of hydrolysis decreases. The best microwave exposure time was 4 min.

Optimization condition using response surface methodology

The linear parameters (acetic acid concentration (A), microwave power (B), exposure time (C), all quadratic parameters (A2, B2, C2), and the interaction between concentration and microwave power (AB) significantly affected the degree of hydrolysis (P < 0.05). The response of the degree of hydrolysis (Y_DH) was given in Eq. (1):

$$\begin{array}{cc}\hfill {\text{Y}}_{\text{DH}}=& 37,64+1,\text{25A}+2,0\text{1B}+1,\text{28C}-2,0\text{9AB}+0,\text{34AC}+0,\text{345BC}\hfill \\ \hfill & -5,\text{96A2}-1,\text{57B2}-4,\text{4C2}\hfill \\ \hfill \end{array}$$ 1

Figure 2a–c shows a 3D graph of the response surface, with the best degree of hydrolysis illustrated in the red area. The optimum degree of hydrolysis was obtained at the acid concentration of 1.21 M, 600 W of microwave power, and an exposure time of 4 min, which was 38.09%. The higher the acetic acid concentration and the higher temperature applied to the hydrolysis process, the degree of hydrolysis will increase to a certain point. An increase in acid concentration resulted in an increase in a hydrolysis rate until an optimum is obtained.

Fig. 2.

3D response surface of the effect of acetic acid concentration (a), microwave power (b), and microwave exposure time on degree of hydrolysis (c). Degree of hydrolysis of unfermented beans, microwave-assisted heating, and fermented beans (d)

The optimum condition for enhancing flavor precursors on unfermented cocoa beans was an acetic acid concentration of 1.21 M, microwave power of 450 W, and exposure time of 4 min. the desirability of this condition was 1,000, indicating that it was similar to the desired target. The verification step of the optimum condition was the following step. Under optimum conditions, the degree of hydrolysis was predicted to be 38.09%, and the verification finding shows a 38.99% degree of hydrolysis. The result showed that the verification value was close to the prediction. If the prediction error value is < 5%, the verification results were acceptable and verified. Besides, the degree of hydrolysis for microwave-assisted heating was higher than for unfermented cocoa beans. But it is still lower than fermented cocoa beans (Fig. 2d).

Character of precursor flavor on microwave-assisted cocoa beans

Free amino acid

The most abundant amino acids in unfermented cocoa were glutamic acid, followed by aspartic acid, glycine, alanine, and arginine (Table 1). The highest amino acids in microwave-assisted cocoa were glutamic acid, aspartic acid, tyrosine, arginine, and leucine. The highest amino acids in fermented cocoa were glutamic acid, aspartic acid, arginine, tyrosine, and leucine. However, the microwave-assisted heating of cocoa beans had a higher glutamic acid concentration than unfermented and fermented cocoa, namely 2,42 and 2.14 times, respectively. Aspartic acid in microwave-assisted cocoa was about 1.7 1.8 times higher than in fermented and unfermented cocoa, respectively. The results are similar to Apriyanto (2017) that the highest amino acids in unfermented and fermented cocoa were glutamic acid and aspartic acid.

Table 1.

The free amino acid of unfermented cocoa beans, microwave-assisted heating, and fermented beans

Free Amino Acid	Treatment
	Unfermented (% area)	Microwave-assisted (% area)	Fermented % area)
Phenylalanine	16.22	28.61	22.39
Valine	12.65	31.49	15.52
Isoleucine	10.19	20.47	12.37
Leucine	19.29	35.18	25.38
Alanine	24.01	21.86	23.82
Tyrosine	22.49	40.80	27.06
Glycine	24.52	17.36	22.45
Lysine	21.98	23.15	24.21
Arginine	23.85	35.89	27.53
Threonine	14.28	16.31	16.31
Methionine	9.83	–	11.39
Serine	22.57	22.65	24.65
Aspartic acid	38.54	69.98	41.06
Glutamic acid	50.49	122.53	57.05
Histidine	–	9.39	–

Part of hydrophobic amino acids, such as leucine, alanine, phenylalanine, and tyrosine, are amino acid precursors that contribute to the aroma formation of cocoa and chocolate (Sukha et al. 2017). The composition of hydrophobic amino acids in cocoa beans can provide information on the potential for aroma formation, but it can’t predict the exact flavor compounds that will be formed during roasting. Based on hydrophobicity, microwave-assisted heating cocoa beans produced higher hydrophobic amino acids than fermented and unfermented cocoa, namely phenylalanine, valine, isoleucine, leucine, alanine, tyrosine, arginine, and threonine (Table 1). It is suggested that the submersion of beans in acetic acid results in an increase in acid. A combination of acid and microwave-assisted heating enhances protein degradation to amino acids (Chen et al. 2014) so that the amino acid level in microwave-assisted cocoa beans more than in unfermented and fermented cocoa. The principal for producing high flavor precursors are the controlled heat effect and acidity (Kadow et al. (2015). Thus, microwave-assisted heating is a better alternative for developing flavor precursors.

Reducing sugar

The lowest reducing sugar was obtained in microwave-assisted heating cocoa beans, followed by fermented and unfermented beans (Fig. 3). It is expected that microwave-assisted heating of cocoa beans had lower reducing sugar because it is partially released during acetic acid submersion. Besides, it is converted into other components, such as acetic acid and furfural, during microwave-assisted heating (Rahkadima et al. 2022). Sugar is converted to other compounds in fermented beans, such as alcohol and acids, during bean fermentation (Peláez et al. 2016). The amount of reducing sugars decreased throughout the cocoa bean fermentation process, and therefore, reducing sugar content was highest in unfermented beans.

Fig. 3.

Reducing sugar of unfermented cocoa beans with acetic acid submersion and microwave treatment

Volatile compound

Roasting is the most important process during chocolate processing to form the chocolate flavor. Volatile compounds were formed during the roasting. The results show that there were 213 compounds that were grouped into 16 groups (Table 2). The totals of acids, lactones, amines, and benzenes in microwave-assisted heating cocoa beans were higher than the unfermented and fermented cocoa beans.

Table 2.

Volatile contents of unfermented cocoa beans, microwave-assisted heating, and fermented beans after roasting

No	Compound*	Detectedinthesample (%area)			Odordescription	References
		Unfermented	Microwave-assisted	Fermented
	Acid
	Total	11,72	59,69	40,10
1	Aceticacid	10,98	55,07	28,78	Vinegar, sour	Rottiers et al. (2019)
2	isobutyricacid	–	–	3,48	Rancid, butter	Rodriguez-Campos et al. (2012)
3	isovalericacid	–	–	6,69	Sweat, rancid	Rodriguez-Campos et al. (2012)
4	Propanoicacid	–	–	0,10	Pungent, rancid	Rodriguez-Campos et al. (2012)
	Pyrazine
	Total	4,28	5,69	18,03
1	2-ethyl-6-methyl-pyrazine,	0,06	0,21	0,27	Candy, sweet	de Andrade et al. (2021)
2	2,3-dimethyl-pyrazine,	0,09	0,22	0,55	Caramel, cocoa	Rottiers et al. (2019)
3	trimethyl-pyrazine,	0,22	0,43	2,94	Cocoa, roastednuts, peanut	Rottiers et al. (2019)
4	tetramethyl-pyrazine,	0,10	–	9,58	Chocolate, cocoa, coffee
	Aldehydesandketones
	Total	11,39	10,02	11,35
1	2-Heptanone	0,61	0,38	0,37	Fruity, coconut, floral, cheesy	Rottiers et al. (2019)
2	2-Nonanone	0,08	0,10	0,40	Fruity, fresh, sweet	Rottiers et al. (2019)
3	2-Pentanone	0,78	–	0,24	Fruity, sweet, cheesy	de Andrade et al. (2021)
4	2-Phenyl-2-butenal	0,02	–	0,56	Sweet, chocolate	Rottiers et al. (2019)
5	Acetaldehydes	4,77	1,81	2,06	Pungent, bitter	Ramos et al. (2014)
6	Acetophenone	–	0,72	–	Flowery, sweet	Rodriguez-Campos et al. (2012)
7	Benzaldehyde	0,17	0,18	2,19	Sweet, bitteralmond, cherry, nutty	(Afoakwa 2016; Rottiers et al. (2019)
8	Acetoin	0,07	0,12	1,47	Butter, cream	Rodriguez-Campos et al. (2012)
	Furan,furanones,pyran,pyranones,pyrroles
	Total	6,31	0,52	8,07
1	2-Acetylfuran	–	0,13	0,10	Sweet, balsamic, slightlycoffee	Ramos et al. (2014)
2	2-Furfurylacetate	–	0,14	–	fruity, banana	Ramos et al. (2014)
3	2-Acetylpyrrole	0,36	0,35	1,03	Chocolate, hazelnut, nutty	(de Andrade et al. (2021)
4	2-Phenylethylacetate	–	–	1,01	Fruity, sweet, honey, floral, flowery, honey	Afoakwa 2016; de Andrade et al. (2021)
5	Ethylbutyrate	0,94	–	0,08	Pineapple	Ramos et al. (2014)
6	Ethylphenylacetate	0,26	–	0,47	Fruit, sweet, honey-like, flowery, rose	de Andrade et al. (2021)
7	Isoamylacetate	0,4	–	2,28	Banana, pear, fruit	Rottiers et al. (2019)
8	Isobutylacetate	0,1	–	0,07	Fruit, apple, banana	de Andrade et al. (2021)
	Alcohol
	Total	29,93	11,73	9,77
1	2-Heptanol	1,45	2,17	0,27	Citrus, fruity, lemongrass	Rottiers et al. (2019)
2	2-Hexanol	–	0,05	–	Fruity, green, herbal	Rottiers et al. (2019)
3	2-Nonanol	0,05	0,46	0,60	Citrus, orange, waxy	Rottiers et al. (2019)
4	2-Octanol	–	0,06	0,55	Spicy, green, woody, earthy	Rottiers et al. (2019)
5	2-Pentanol	17,14	0,04	0,26	Fermented, ripebanana	Rottiers et al. (2019)
6	2,3-Butanediol	–	0,12	0,20	Cocoabutter, sweet, flowery, creamy, buttery	de Andrade et al. (2021)
7	Isoamylalcohol	–	0,73	0,55	Banana, fruity, fermented, cognac	Rottiers et al. (2019)
8	Acetylcarbinol	0,44	–	0,18	Sweet, honey	Rodriguez-Campos et al. (2012)
9	Ethanol	4,82	5,48	0,28	Undesirable	Rottiers et al. (2019)
	Lactones
	Totallactones	0,83	1,48	0,77
1	Butyrolactone	0,64	1,35	0,43	Must, flowery, almond, sweet, aromaticcreamy	de Andrade et al. (2021); Rottiers et al. (2019)
	Terpenes
	Total	0,86	0,69	0,79
1	cis-Linalooloxide	0,83	–	0,36	Sweet, floral, earthy, woody	Rottiers et al. (2019)
2	Linalool	0,73	0,42	0,39	Floral, rose, sweet, green, citrus	Rottiers et al. (2019)
3	Trans-Linalooloxide	0,32	0,29	–	Floral, citrus, sweet, earthy	Rottiers et al. (2019)
4	β-Myrcene	0,81	–	0,23	Spicy	Rottiers et al. (2019)
	Pyridines
	Totalpyridines	0,31	0,00	0,00
1	Pyridine,2-methyl-	0,27	–	–	Caramel-like, sweet	Afoakwa (2016)
	Amines,amides,nitriles
	Total	0,32	0,89	0,13
1	Benzonitrile	–	–	0,13	Almond	Anonim (2023)
	Ethers
	Total	5,14	0,00	2,03
	Benzenes
	Total	0,17	0,26	0,12
	Azoles
	Total	4,87	0,13	0,07
	Others
	Total	3,26	1,40	0,43
1	Phenol,2-methoxy-	–	0,25	–	Smoke, sweet, phenol, spicy	de Andradeetal(2021)
2	Phenol	0,14	–	0,25	Smoky	Rodriguez-Campos et al. (2012)
3	Caffeine	0,15	–	–

*Compounds contributed to flavor

Some short-chain carboxylic acids, such as acetic acid and isovaleric, were dominant in the aroma of unroasted Criollo cocoa (Dulce et al. 2021). The result showed that acid concentrations were high in microwave-assisted heating beans, especially acetic acid. A similar result was also reported by Torres-Moreno et al. (2014). Acetic acid has been associated with sour and vinegar-like notes, and it is considered the highest odor-active compound in unroasted and roasted cocoa (Rodriguez-Campos et al. 2012; Rottiers et al. 2019). Furthermore, some acids in microwave-assisted heating cocoa beans didn’t produce, i.e., benzene acetic acid, caproic acid, isobutyric acid, isocaproic acid, isovaleric acid, and propanoic acid.

The group of pyrazines is one of the most important volatile compounds in roasted cocoa beans. Pyrazines are the major compounds formed during Maillard reaction of amino acids and sugars (Torres-Moreno et al. 2014). The pyrazines in microwave-assisted cocoa were higher than in unfermented cocoa, and the pyrazines in fermented cocoa were the highest. Trimethyl-pyrazine was detected; this compound was detected in cocoa, roasted nut, and peanut notes (Rottiers et al. 2019). Overall, pyrazine compound has candy, sweet, caramel, cocoa, chocolate, roasted nuts, and coffee notes (Afoakwa 2016; de Andrade et al. 2021; Rottiers et al. 2019).

The total aldehyde and ketones in microwave-assisted heating cocoa beans were lower than in the unfermented and fermented cocoa. These compounds produce malty, chocolate, sweet, bitter, almond, cream, and fruity flavors (de Andrade et al. 2021; Rodriguez-Campos et al. 2012; Rottiers et al. 2019). The highest aldehyde and ketone groups in microwave-assisted cocoa were acetaldehyde, which has pungent-bitter notes (Ramos et al. 2014).

The total alcohol of microwave-assisted heating beans was lower than the unfermented cocoa. However, the total alcohol of microwave-assisted heating beans was higher than that of fermented cocoa. High alcohol contents are desirable in cocoa products with flowery, fruity, and candy notes (de Andrade et al. 2021; Rodriguez-Campos et al. 2012; Rottiers et al. 2019). Alcohol is thought to be formed through sugar or amino acid catabolism, and it causes the alcohol content in every cocoa can be different. It depends on the sugar and amino acid contents (Liu et al. 2019).

Furthermore, as seen in Table 2, the total esters in microwave-assisted heating cocoa beans were lowest compared to unfermented and fermented cocoa beans. Esters were formed by acid and alcohol esterification and produced floral and sweet notes (Lee et al. 2018). After roasting, several esters were identified in the sample, which had basically fruity and flowery notes (Torres-Moreno et al. 2014). They were important flavor components of natural products and fermented foods.

The total lactone of microwave-assisted heating beans was highest compared to unfermented and fermented cocoa beans, and butyrolactone was the highest produced in microwave-assisted heating cocoa beans. This compound had flowery, almond, sweet, aromatic, and creamy notes (de Andrade et al. 2021; Rottiers et al. 2019).

The amine group in the microwave-assisted cocoa heating beans was highest compared to unfermented and fermented cocoa beans. However, most amines produced in microwave-assisted heating cocoa beans were not produced in fermented and unfermented cocoa beans. It was presumably due to the degradation of amine to other volatile compounds. According to Ziegleder (2009), the amino acid structure determines the resulting aldehydes, amines, and acids, which can be produced by the degradation of amino acids as well as the resulting volatile compounds such as alcohols and esters.

The alkenes were also present in low concentrations. Those alkenes come from the decarboxylation occurring during the roasting of the fatty acid (Torres-Moreno et al. 2021). Thiazoles were also present in low concentrations. Finally, other compounds also contribute to the formation of flavor in cocoa.

Conclusion

Submersion of unfermented cocoa beans increased bean acidity. However, it was lower than the total acid in the fermented cocoa beans. An increase in acetic acid concentration enhanced protein hydrolysis during microwave-assisted heating of cocoa beans so that the degree of hydrolysis increased. The highest degree of hydrolysis was obtained at the submersion of beans in an acetic acid concentration of 1.25 M. Microwave power significantly affected the degree of hydrolysis. The highest degree of hydrolysis was obtained at a microwave power of 450 W. Besides, an increase in microwave exposure time increased protein hydrolysis and, subsequently degree of hydrolysis. The best microwave exposure time was 4 min. The optimum condition for enhancing flavor precursors of unfermented cocoa beans was obtained at acetic acid concentration of 1.21 M, microwave power of 450 W, and microwave exposure time of 4 min. Under optimum conditions, the predicted degree of hydrolysis was 38.09%, and the verified degree of hydrolysis was 38.99%. Besides, hydrophobic amino acids were flavor precursors for the aroma formation of cocoa and chocolate. Microwave-assisted heating of cocoa beans produced higher hydrophobic amino acids than fermented and unfermented cocoa, namely phenylalanine, valine, isoleucine, leucine, alanine, tyrosine, arginine, and threonine. Volatile compounds, such as acetic acid and pyrazines, were higher in microwave-assisted heating of cocoa beans after roasting. But total aldehyde and ketones, total alcohol, and total esters were lower than the unfermented cocoa beans. Thus, microwave-assisted heating is a better alternative for developing flavor precursors.

Abbreviations

DH

Degree of hydrolysis

OVAT

One-variable-at-a-time

RSM

Response surface methodology

BBD

Box behnken design

TCA

Trichloroacetic acid

Authors’ contributions

ADM: Formal Analysis, Investigation, Writing Original Draft; RY: Writing, Review & Editing, Supervision; CH: Conceptualization, Methodology, Validation, Resources, Writing, Review & Editing, Supervision.

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Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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