3.1. Optimization of the Product Development by Traditional Maceration
The experiments were started by performing traditional macerations with and without agitation, using different pineapple presentations (Table 1
). As can be seen, in the case of freeze-dried pineapple, the concentration of pineapple used was considerably lower than that used in the other experiments, although the lowest amount of fresh pineapple used was 200 g/L. This strategy was based on the water loss that takes place in lyophilization processes. Since the freeze-drying process was performed in the laboratory, the initial amount of pineapple employed was known, and an amount of freeze-dried material corresponding to the initial 200 g/L of fresh pineapple was employed. In the case of dehydrated pineapple, the dehydration process was not carried out, but the product was acquired already prepared from the market. Therefore, the initial amount of fresh pineapple employed to obtain the dehydrated material was unknown. Therefore, the same amount as if it was fresh was employed, but taking into account that, actually, the initial amount of pineapple employed to obtain this dose was higher than 200 g/L. In all cases, a maceration time of 72 h was used [14
After separating the solid parts from the pineapple, different yields of macerated vinegar (product) were obtained depending on the pineapple presentation used. In the case of fresh pineapple, 100% of the product volume was recovered. For dehydrated pineapple, the yield was 45% and for freeze-dried pineapple, it was 95%. The dehydrated pineapple absorbed 55% of the product, which after the separation of the solid parts was discarded together with the residue.
Samples from all trials were tasted to try and discriminate between the types of traditional maceration. The samples that received the highest score with respect to pineapple aroma intensity on a 1 to 5 scale were those elaborated with 200 g/L dehydrated pineapple (4.8 points out of 5), followed by 400 g/L fresh pineapple without magnetic agitation (4.1 points out of 5). This was a logical result, taking into account that, due to the dehydration process, the amount of dehydrated pineapple employed actually came from a higher amount of fresh pineapple. Of these two, the one with the highest pineapple aroma score was the dehydrated pineapple sample; however, it should be taken into account that the price of the dehydrated pineapple was 4 times that of the fresh pineapple and that the losses in the macerated product implied a drop of 55% in the final yield. The price of the raw material is a very important aspect to be considered when dealing with the obtention of new products and should not be underestimated.
When analyzing the pros and cons of the two best trials, it was concluded that the samples corresponding to the traditional maceration with fresh pineapple were the best option compared to that of dehydrated pineapple vinegar. The price of the raw material was not the only variable considered, but taking into account that other cheaper options also presented a high value of pineapple character, the decision was made by taking all these aspects into account, together with the yield of the process. Therefore, the optimal conditions for traditional maceration were established as 400 g of fresh pineapple/L of vinegar, two daily stirrings, and a maceration time of 72 h.
3.2. Optimization of Microwave Maceration
Once the target product had been developed by traditional maceration, efforts were made to optimize the process when microwave energies were applied with the objective of elaborating a product that was similar or even better than the one obtained by traditional maceration, but in a shorter maceration time. For this purpose, a central composite design of experiments consisting of a response surface (22
+ 1 central point, 10 experiments, in duplicate) was established. The dose of fresh pineapple in all cases was 400 g/L, and the microwave power was 350 W. The parameters to be optimized were time (values limits: 3–10 min) and temperature (values limits: 30–60 °C). Total polyphenol index (TPI) and Folin–Ciocalteu index (FCI) were set as the response variables to be optimized because the objective was to obtain a product with the highest healthy character, related to the phenolic content. Table 2
presents the obtained parameters from the model.
Based on the evaluation of the statistic results, it was determined that temperature was the variable with the greatest influence on the concentration of polyphenols (Figure 1
), with a direct correlation. It was also observed, that the samples that had been obtained at higher temperatures presented higher TPI and FCI values. Therefore, high temperature values (60 °C) were used.
Subsequently, in order to corroborate the effect of the time variable, several additional experiments were carried out (in duplicate) while keeping the temperature steady at a high level (60 °C) and varying the extraction time as follows: 10, 12.5, 15 and 20 min. The TPI and FCI values obtained are displayed in Table 3
. For each one of these responses, significant differences were presented with different letters in the same column.
As can be seen, the highest TPI and FCI values were obtained when the extraction times were longer. Therefore, although initially the time variable appeared as non-significant in the experimental design for the interval 3–10 min, it seems that longer extraction times favor the increment of the polyphenolic content in the macerated vinegar samples.
On the other hand, we should note that the sensory analysis of the samples would focus on pineapple aroma intensity (data not shown). The results from such analyses revealed that longer maceration times did not significantly modify the vinegars obtained in relation to pineapple aroma, even when they presented greater polyphenolic contents.
Therefore, based on the results obtained for PTI, FCI, pineapple aroma and extraction time (all of them, important variables to be taken into account for the final decision), a pineapple concentration of 400 g/L, a power of 350 W, a temperature of 60 °C and maceration times of 10 and 20 min were adopted as the conditions to be employed in the subsequent microwave studies.
3.3. Comparison of the Extraction Methods
A comparison of the different optimized extraction methods (traditional maceration, microwave maceration) against ultrasound maceration carried out according to the conditions optimized by other authors was conducted [14
]. The FCI obtained by the different methodologies was measured and presented in Figure 2
As can be observed, all the extraction techniques significantly increased the FCI with respect to the initial unmacerated sample, where the traditional maceration together with the microwave extraction for 20 min was the highest, followed by the extraction with microwaves for 10 min and the extraction with ultrasound. Other authors have corroborated the fact that extraction techniques favor the transfer of polyphenolic compounds to oenological samples [24
On the other hand, the samples studied were subjected to sensory evaluation and arranged in order from lowest to highest pineapple aroma intensity and overall quality. The ranking results are shown in Table 4
As can be seen, all of the samples subjected to maceration with pineapple were ranked higher than the sample that had not been macerated. In addition, the traditional maceration sample was at the top, followed by the 10-min microwave maceration sample. However, in relation to the overall quality, it is interesting to mention that the sample to which ultrasound was applied presented defects in its aromatic profile, which caused the judges to rank it below the rest of the samples, including the initial unmacerated sample. In a previous study [15
], the use of dynamic ultrasonication for the maceration of citrus fruits with vinegar also provided the product with olfactory flaws, but the employment of static ultrasonication was successfully employed for the maceration of orange and lemon peels with vinegar [14
]. However, static ultrasound was ranked after traditional and microwave maceration when these three techniques were compared [15
]. Our results with pineapple corroborate this preference, because in this case, the judges positioned the vinegar sample that had been microwaved for 10 min in first place, ahead of the traditionally macerated sample.
These tests demonstrated that the microwave extraction samples had similar organoleptic characteristics to those obtained through traditional maceration and could therefore be a valid alternative for the successful elaboration of a quality product.
3.4. Analysis of Volatile Compounds
The pineapple macerated vinegar samples that had been obtained using the different extraction methods were subjected to analysis of volatile compounds using the Stir Bar Sorptive Extraction technique coupled to Gas Chromatography and Mass Spectrometry detection (SBSE-GC-MS). A total of 32 volatile compounds were identified as shown in Table 5
. The approximate number of chromatographic peaks present in the chromatograms was around 100, but due to the lack of commercial standards, only 32 were selected for the study. This selection was based on the previous experience of the researchers and it was focused on those compounds with a high percentage of matching against the library data (>85%).
As can be seen, a greater number of compounds were identified in the macerated samples (32 different compounds) compared to their presence in the initial vinegar that had not been macerated and where only 23 compounds were identified. The increase in the number of volatile compounds detected in Sherry vinegar after maceration with fruits had been previously reported by other authors [6
]. Some of the compounds found in the samples macerated with pineapple are directly related to the volatile compounds profile of pineapple. Among these, we should mention methyl octanoate, decanoic acid, ethyl hexanoate, 3-methyl-1-butanol, and ethyl acetate [3
]. Some compounds, such as methyl octanoate, ethyl heptanoate, 2-butyl acetate, linalool oxide, pentanoic acid, 2,6-dimethyl-4-heptanone, hexanoic acid, ethyl 2-methylbutyrate, and 2-methyl-1-propanol only appeared in the samples that had been macerated with pineapple, which seems to indicate that these compounds were exclusively contributed by the added pineapple. Previous studies [26
] have mentioned that esters, lactones, furanoids and sulfur compounds act as very significant components in pineapple aroma. Esters such as ethyl 2-methylbutanoate, methyl hexanoate and ethyl hexanoate provide fruity notes from fresh pineapple as well as from other fruits [27
]. Using odor threshold values and concentration data, other authors have concluded that some of the most important contributors to the aroma of fresh pineapple are: methyl 2-methylbutanoate, ethyl 2-methylbutanoate, ethyl acetate, ethyl hexanoate, ethyl butanoate, ethyl 2-methylpropanoate, methyl hexanoate, and methyl butanoate [28
], compounds that have been detected in our study.
The data obtained from the study of the volatile profile of the samples were subjected to ANOVA in order to identify any relevant differences between the different extraction techniques (Table 5
). The results from the ANOVA confirmed that most of the compounds had been influenced by the treatment, with the exception of 2,6-dimethyl-4-heptanone, 3-hexen-1-ol acetate, 2,3-butanediol diacetate, iso-butyric acid, methylbenzeneacetic acid and hexanoic acid. Similar results were obtained by other authors, who reported significant differences in the majority of the volatile compounds of vinegars macerated with fruits, taking into account the maceration procedure [15
]. As expected, the characteristic pineapple aroma compounds increased with maceration. The samples subjected to microwave maceration presented the largest amounts of compounds such as ethyl 2-methylbutyrate, ethyl hexanoate or ethyl heptanoate, among others. These compounds were also found in almost the same quantities in the traditionally macerated vinegar and, to a lesser extent, in the ultrasound macerated samples. In the traditionally macerated vinegar, methyl hexanoate or methyl octanoate, among others, were the most prominent compounds. On the other hand, 2-methyl-1-propanol and pentanoic acid were the most abundant compounds found in the ultrasonic extraction samples, followed by the traditional and microwave ones. Taking into account the influence of the maceration technique on the volatile profile, in general terms, it seems that microwaves and traditional macerations provoked a significant increase of a higher number of compounds, compared to ultrasound extraction (Table 4
), which is in agreement with previous research [15
]. This fact could be also related to the sensory results, which ranked the traditional and microwave macerated vinegars in the first positions, regarding pineapple aroma and overall quality.
In some cases, the content level of some particular volatile compounds was lower in the samples that had been macerated with pineapple. This is the case for compounds such as ethyl acetate, isoamyl acetate, benzaldehyde, isobutyric acid, 3-methylbutanoic acid, octanoic acid, and others. This phenomenon was more noticeable in the ultrasonic extraction samples, followed by the microwave ones. In this regard, some authors have reported a possible degradation of volatile compounds in white wine samples when subjected to ultrasound treatment [29
Finally, a multivariate principal component analysis was performed. This analysis included all the traditional maceration samples used for the optimization (T), all the microwave samples with extraction times equal to or longer than 10 min (M), the samples obtained by ultrasound extraction (U) and the unmacerated (initial) vinegar (Figure 3
Five of the principal components obtained had an eigenvalue >1 and explained 99.94% of the variation between samples. Component 1 (PC1) explained 43.77% while component 2 (PC2) explained 16.80% of the variability. PC1 was able to separate the samples that were subjected to maceration with pineapple from the initial sample, that had not been macerated. As can be seen in Table 6
, the 5 compounds that were most relevant to this component were: methyl salicylate, ethyl phenyl acetate, 2-phenethyl acetate, benzenemethanol, and 4-ethylphenol, of which 2-phenethyl acetate is closely related to pineapple aroma [30
]. Therefore, as expected, it was confirmed that maceration with pineapple greatly modifies the volatile profile of the resulting vinegar, with some pineapple-derived compounds as clear markers of the macerated samples.