Physicochemical and aromatic characterization of carob macerates produced by different maceration conditions

Abstract Carob liqueur is an alcoholic drink (minimum 15% v/v of ethanol and 100 g/L of sugar) typical for the Mediterranean countries. In the current work, carob macerate produced by maceration of carob pods in hydroalcoholic base at different maceration conditions was characterized for the first time based on its aroma compounds/profile, physicochemical parameters, and chromatic characteristics. The results confirm the migration process of bioactive compounds, aroma compounds, and sugars flowing from the carob pod to the hydroalcoholic base. Changes in ethanol concentration modify the physical properties of the solvent and influence the phenolic and aroma compounds extraction, color, and acidity of the obtained samples. The higher content of phenolic compounds was determinate in the samples obtained in the darkness. The amounts of phenols were in the range of some red fruit liqueurs or walnut liqueurs, and sugars (mostly sucrose) ranging between 96 and 107 g/L. Twenty‐six (out of total 94) aroma compounds were detected in all samples, of which 17 esters, 3 alcohols, 4 ketones, and 2 acids. Low molecular weight ethyl esters, ethyl hexanoate, ethyl 2‐methyl propanoate, ethyl octanoate, ethyl benzoate, ethyl butanoate, and ethyl cinnamate, were the most abundant. Carob pod maceration in 50% v/v hydroalcoholic base (1:5 solid to liquid ratio) in darkness at room temperature during 8 weeks can be recommended as optimal maceration conditions for production of the aromatic carob macerate with functional properties.

sugar) made of carob pods. Carob liqueur is produced by maceration of partially crushed carob pods in the hydroalcoholic base, with the addition of sugar. Maceration is a solid-liquid extraction technique employed to obtain alcoholic extracts of varied type of vegetable matrices (citrus peels, flowers, leaves, medicinal/aromatic herbs).
During the maceration process, various compounds that give a characteristic flavor and color as well as the biologically active compounds are extracted from the plant material into the hydroalcoholic base (Petrović, Vukosavljević, Đurović, Ntić, & Gorjanović, 2019;Rodríguez-Solana, Vázquez-Araújo, Salgado, Domínguez, & Cortés-Diéguez, 2016;Veljović et al., 2019). Macerate, the solution obtained by the maceration process, could be used as a base for liqueurs or further distilled to give a distillate of macerate. This distillate contains volatile substances transferred from the plant material into the macerate (colored) and then into the distillate (colorless). The distillate can be added to the macerate for further flavor enrichment (Buglass & Caven-Quantrill, 2012).
For example, at higher alcohol percentage essential oils, lipids, and resins are dissolved, while at lower alcohol percentage substances soluble in water (organic acids, bitter substances, and carbohydrates) are dissolved. There are only a few scientific researches on carob-based alcoholic drinks and on the nutritional characteristics of the carob liqueur (Rodríguez-Solana, Carlier, Costa, & Romano, 2018;Rodríguez-Solana, Salgado, et al., 2019). Rodríguez-Solana, Salgado et al. (2019) investigated the process of maceration of carob in order to extract as many of the bioactive compounds as possible.
The results showed that gallic acid is the most abundant bioactive compound in the carob liqueur, and the amount of both gallic acid and total phenolic compounds significantly dependent on the carob cultivar characteristics. In addition to phenolic compounds during maceration, other components of the carob pod are extracted into the macerate. Research by Rodríguez-Solana et al. (2018) showed that carob liqueur is rich in minerals: Na, K, Ca, Mg, Cu, and Fe were detected in all liqueur samples. The most common ones were potassium, sodium, and calcium.
The available scientific literature does not define the optimal parameters of the maceration process for the production of carob liqueur. The aim of this study was to investigate the influence of the maceration process parameters on physicochemical characteristics of carob macerates and to determine the aroma compounds that are characteristic for this type of alcoholic drink. The maceration time (1-12 weeks), the ratio of the plant:hydroalcoholic base/solid:liquid (1:5 and 1:10), alcoholic strength (30%, 50% and 70%), and exposure to sunlight or darkness were the studied parameters. In addition, total phenolic content, antioxidant activity, total sugar content, pH value, and chromatic parameters of the carob macerates were determined.
Sodium carbonate, sodium chloride, sodium acetate, and zinc sulfate heptahydrate were of analytical grade, obtained from Grammol. Acetic acid (glacial) was obtained from Carlo Erba Reagents (Barcelona, Spain) and hydrochloric acid (37%) from Fisher Scientific.

| Preparation of carob macerates
Carob macerates were prepared by the maceration of chopped carob pods (4 cm size pieces, unroasted) in hydroalcoholic base (mix of 96% synthetic ethanol and water). Carob pods were macerated in 500 ml of hydroalcoholic base (30, 50, and 70% v/v of ethanol) in different solid to liquid ratio (1:5 and 1:10) at room temperature exposed to sunlight as well to darkness. During the maceration, the samples were daily manual shaken. Samples were obtained after every week during 12 weeks of maceration. Carob pods were obtained from local producer from the island of Vis, Dalmatia, Croatia.

| HPLC analysis of extracted sugars
Concentrations of extracted sugars (sucrose, glucose, and fructose) in macerates at the end of maceration were determinate by HPLC. Prior the analysis, Carrez reagents were added to the sample and the precipitated proteins were removed by filtration (Lefebvre, Gabriel, Vayssier, & Fontagne-Faucher, 2002 Separation and elution were performed using phosphoric acid (0.1% w/w) as the mobile phase at a flow rate of 0.5 ml/min (Trontel, Baršić, Slavica, Šantek, & Novak, 2010). The column and the refractive index detector were maintained at 30°C. The sample injection volume was 10 μl. Qualitative and quantitative determination was based on the injection of pure standards (sucrose, fructose, and glucose). Quantification was done by external calibration preparing calibration curves of six points with concentrations of standards (sucrose, fructose, and glucose) 0.1-5 g/L.

| Determination of total phenolic content (TPC)
The total phenolic content (TPC) of each sample was determined by applying the Folin-Ciocalteu method (Singleton & Rossi Junior, 1965).
Briefly, 0.3 ml of diluted samples or standard solutions (gallic acid) were added to 6 ml of distilled water and 0.5 ml of the Folin-Ciocalteu reagent, mixed thoroughly and allowed to stand for 5 min. Then, 1.5 ml of saturated sodium carbonate solution was added, mixed well, and filled to a total volume of 10 ml with distilled water. The samples were left at room temperature for 2 hr in the darkness. The absorbance of the samples was measured at 760 nm with an UV/Vis spectrophotometer (Heλios β, Unicam). The calibration curve used for the quantification of the samples was prepared with different concentrations of gallic acid standard solution (50-300 mg/L).
The total phenolic content was expressed as mg of gallic acid equivalents (GAE) per 100 ml of carob macerate.

| Antioxidant activity
For the determination of antioxidative activity of carob macerates, ferric ion reducing antioxidant power (FRAP) was used. The FRAP assay was performed as previously described by Benzie and Strain (1999) with some modifications. FRAP reagent solution was made of the mixture of acetate buffering agent (300 mM, pH = 3.6), TPTZ (10 mM solution TPTZ in 40 mM HCl), and FeCl 3 * 6H 2 O (20 mM) in volume ratio 10:1:1, respectively). The working FRAP reagent was prepared fresh on the day of the analysis. All samples, standards, and reagents were preincubated at 37°C. The examined sample (80 μl) was mixed with FRAP reagent (2080 μl) and distilled water (240 μl). After the reaction at 37°C for 5 min, the absorbance at 593 nm was measured. The standard curve was constructed by using serial dilution (0.1-2.0 mM) of Trolox stock solution. The final results were expressed as mM Trolox equivalents.

| pH assay
The measurements of pH of all samples were done with Oakton pH 5 plus Meter pH meter (Oakton Instruments). The system was calibrated by placing pH-probe in buffer pH 4.

| Chromatic characteristics
The chromatic characteristics were determined on a Specord 50 Plus (Analytik Jena) and a 10-mm glass cell, by measuring the transmittance of the sample every 10 nm from 380 to 770 nm, with a D65 illuminant. Based on the transmittance values, some parameters were calculated: luminosity (L*); saturation (C*); chromaticity coordinates (a* and b*), and hue (h*) (OIV -Compendium of International

Methods of Analysis of Spirituous Beverages of Vitivinicultural
Origin, 2014).

| HS-SPME and GC/MS conditions and analysis
Headspace (HS)-solid-phase microextraction (SPME) and gas chromatography (GC) were applied for analysis of aroma compounds.
Aliquot of 8 ml of diluted sample (to the final concentration of 5% (v/v) ethanol) was pipetted into 20-mL glass vial, spiked with 2 g NaCl, and capped with silicone septa. Manual sampling was done using 50/30 μm DVB/CAR/PDMS (divinylbenzene/carboxen/ polydimethylsiloxane) 1 cm StableFlex fiber (Supelco), which is recommended for aroma compounds (volatiles and semivolatiles) (Câmara et al., 2007). Before use, fiber was conditioned according to manufacturer's instructions. After 10 min stabilization of the sample, fiber was exposed to the sample headspace for 40 min at 60°C with continuous magnetic stirring (Du, He, Li, Wanga, & Xiao, 2015). The SPME fiber was thermally desorbed in the programmed temperature vaporizer injector at 250°C during 5 min using splitless mode.

| Statistical analysis
Statistical analysis was carried out using the MS Excel tool XLStat (Addinsoft) and Statistica 12 (Statsoft Inc.) programs. A basic descriptive statistical analysis was followed by an analysis of variance (ANOVA) test for mean comparisons. Principal component analysis (PCA) was used to visualize the differences between carob macerates obtained after 12 weeks of carob pod maceration, based on physicochemical characteristics and aroma compounds.

| Total phenolic content (TPC), antioxidant activity (FRAP), total sugar content, pH, and color parameters of carob macerates
Starting from the idea of using carob pods for preparing new addedvalue liqueur, the present study was focused to determine and propose the optimal maceration process parameters for production of macerate with acceptable sensory and nutritional characteristics.
The sample description as well as the total phenolic content (TPC), antioxidant activities, total sugar content, and pH value of carob macerates obtained after 12 weeks of maceration are shown in Table 1.  TPC ranging from about 20 mg GAE/100 ml after the first week of maceration up to about 200 mg GAE/100 ml after 12 weeks of maceration, depending also on the tested parameters ( Figure 3).
In addition to phenolic compounds, during maceration, other carob compounds are extracted into the macerate. Sugar analysis showed that macerates containing, in average, values ranging between 96 and 107 g/L of total sugars in the case of higher carob quantity, while for the lower quantity the concentration is expectedly, half less. In the total sugar content, the most abundant were sucrose (≈75%), glucose (≈15%), and fructose (≈10%), (Figure 4). The exposure to sunlight did not have a significant impact on the amount of extracted sugars as well as the strength of the alcohol for the higher quantity of carob ( Figure 1). In macerates with the lower carob quantity, the lowest sugar concentration was measured at high TA B L E 1 Physicochemical characteristics of carob macerate obtained after 12 weeks of carob pod maceration in various strength of alcohol base (30, 50 and 70% v/v) and various solid/liquid ratio (1:5 = H and 1:10 = L) at room temperature exposed to sunlight (S) and darkness (D)

| Aroma compounds characterization of carob macerates
A total of 95 aroma compounds have been identified in carob macerates. Twenty-seven components were detected in all samples, of which 17 esters, 3 alcohols, 4 ketones, and 2 acids (  . These are exactly the conditions in which liqueurs are traditionally produced. Namely, many of the liqueurs are home-made fruit liqueurs produced with available agricultural raw materials, whose production are in accordance with some traditional recipes. These recipes commonly suggest fruit maceration in the sunlight, most likely due to faster extraction at higher temperatures (Paz, Fernández, Matías, & Pinto, 2014). In alignment to our research, it should be highlighted that such conditions should be avoided and these habits need to be changed due the sensitivity and degradation of phenolic compounds exposed to sunlight and in order to preserve liqueur antioxidant potential. Also, good manufacturing practice should be overwritten from wineries. Wine is filled in colored bottles which provide some protection from UV and visible light radiation, while liqueurs are usually filled in clear glass bottles, often in attractive shapes.
The strength of alcohol proved to be the most important parameter in phenolic and aroma compounds extraction, color, and acidity F I G U R E 6 Principal component analysis (PCA) score plot for carob macerates obtained after 12 weeks of carob pod maceration in various strength of alcohol base (30, 50, and 70% v/v) and various solid/liquid ratio (1:5 = H and 1:10 = L) at room temperature exposed to sunlight (S) and darkness (D), based on chromatic characteristics of macerates. The macerates prepared in 30% hydroalcoholic base had higher total acidity than the macerates prepared with higher alcoholic strength (50% and 70%). Acidity is also affected by the carob pod quantity. The macerates prepared with higher quantity of carob had higher total acidity than those prepared with lower quantity of carob ( Figure 1). It should be noted that although there are some common guidelines, optimal parameters for the production of liqueurs from different fruits may be different. Nour, Trandafir, and Central (2015) optimized the hydroalcoholic extraction conditions to maximize the anthocyanin content, total phenolic content, and antioxidant activity of bilberry extracts in order to obtain bilberry liqueur. Based on TPC, color parameters, and consumer preference, they selected the optimal maceration conditions: 70% v/v of ethanol, 40 g/L plant concentration, and 3 weeks of maceration process, significantly different from our optimum conditions for the carob.
Therefore, these results indicate the importance of another maceration parameters: the plant/fruit particle size and its structure because they have important influence on compounds extraction efficiency and consequently duration of the maceration. Using dry, leathery, and larger sized chopped carob pods (4 cm) in our research, longer extraction time is needed to obtain macerate with the highest TPC.
Detection of aroma compounds is one of the most important steps in the evaluation of spirits, liqueurs, and other types of alcoholic beverages quality (Chen, Capone, & Jeffery, 2019). The aroma is influenced by many factors, including the quality of the starting raw material together with variables within the production process (Sliwinska, Wisniewska, Dymerski, Wardencki, & Namiesnik, 2015).
The volatile aromatic compounds are mostly esters, higher alcohols, and aldehydes. Identification of these compounds is important to establish the flavor characteristics of a given spirit drink (Hanousek Čiča et al., 2019). This is the first scientific report of the volatile aroma compounds in carob macerate. The volatile organic compounds of carob fruit and flour were previously reported by Krokou, Stylianou, and Agapiou (2019 and heptanone were detected in carob fruit by Krokou et al. (2019).
In our experiment, heptanone was detected in only 4 of 12 macerates (in macerates with higher alcohol strength and higher quantity of macerated carob). Nonan-2-one is a plant metabolite, present in many fruits and spices (National Center for Biotechnology Information). It is a clear slightly yellow liquid. It is most represented in the sample 50_DH and significantly contributes to the yellow color of this macerate ( Figure 6). The esters generally have a pleasant aroma and a very intense odor, and they are important beverage aroma components (Lukić et al., 2011;Sliwinska et al., 2015). These compounds make a positive contribution to the general quality of the spirit, being responsible for their "fruity" and "floral" sensory properties (Câmara et al., 2007). Ethyl hexanoate presents a tropical fruit aroma and ethyl octanoate is associated with banana, pineapple, and brandy-like aromas (Genovese, Ugliano, Pessina, Gambuti, & Piombino, 2004;Rogerson & De Freitas, 2002). As many volatile esters, ethyl benzoate has a pleasant odor described as sweet, wintergreen, fruity, medicinal, cherry, and grape. Ethyl butanoate, present in many fruits, has a fruity odor, similar to pineapple and ethyl 2-methylpropanoate fruity, aromatic odor. Krokou et al. (2019) detected only 45 aroma compounds in carob fruit, significantly less than in our experiment, probably due to the application of SPME directly, without any solvent.
TA B L E 2 Average peak areas (%) of volatile aroma compounds that occur in all samples of carob macerate obtained after 12 weeks of carob pod maceration in various strength of alcohol base (30, 50 and 70% v/v) and various solid/liquid ratio (1:5-High and 1:10-Low) at room temperature exposed to sunlight and darkness

| CON CLUS IONS
In this study, optimal maceration process parameters for production of the aromatic and bioactive rich carob macerate are determined. During the maceration process, macerates changed in aroma properties, color, sugar content, phenols composition, and antioxidant activity depending on studied parameters. Changes in ethanol concentration modify the physical properties of the solvent and affect the macerates composition. Carob pod maceration in 50% v/v hydroalcoholic base in darkness, in solid to liquid ratio 1:5 at room temperature, can be recommended as maceration conditions for obtaining macerate of desired functional properties, sweetness, and desirable aroma compounds. Optimal maceration time is 6-8 weeks.
The total phenolic content was in the range of some red fruit liqueurs or walnut liqueurs, and sugars (mostly sucrose) ranging between 96 and 107 g/L. Ethyl esters, ethyl hexanoate, ethyl 2-methylpropanoate, ethyl octanoate, ethyl benzoate, ethyl butanoate, and ethyl cinnamate, are the compounds found in greater proportion in the carob macerates flavor.

ACK N OWLED G M ENTS
The research is funded by the Croatian Science Foundation, grant number: IP-11-2013_3304-TEUCLIC and paper was produced as part of the "Atrium of Knowledge" project co-financed by the European Union from the European Regional Development Fund and the Operational Programme Competitiveness and Cohesion 2014-2020.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

Jasna Mrvčić
https://orcid.org/0000-0001-8066-5851 F I G U R E 7 A biplot representation of the aroma compounds of carob macerates according to the principal component analysis