3.2. Successive Fermentations Batches by Various Sugars at Different Temperatures
The immobilized biocatalysts were used in successive batch fermentations using a synthetic medium that contained ~120 g L−1
of glucose, sucrose, and maltose, respectively (Table 1
). Batch fermentations were also tested at different temperatures (28, 21, 14, and 7 °C). In all cases, batch fermentations were conducted by the use of free cells for comparison reasons under the same conditions.
Fermentation kinetics proved to be more efficient in the case of immobilized cells providing lower fermentation times (p
< 0.05) compared with free cells (Table 1
). Regarding the fermentation temperature, the immobilized biocatalysts proved to achieve better kinetics in temperatures of 28, 21, and 14 °C, while increased fermentation values were observed at 7 °C. In all cases, fermentation kinetics remained in levels accepted by the industry. Similar increase in fermentation times at low temperatures has also been reported before by the psychrophilic strain AXAZ-1 [34
] or by adaptation of the Saccharomyces cerevisiae
]. In the present study, the fermentation times at low temperature (7 °C) proved to be slightly increased in the case of the immobilized yeast cells on P. terebinthus
resin when compared to immobilized cells of the same strain on other natural supports [26
]. A possible explanation to this outcome could be the hardening of the resin at a low temperature of 7 °C, which might result in diffusion difficulties between fermentation substrates and products. According to other studies, Pistacia resins are reported to provide antimicrobial properties [2
] and as a result, yeasts lower populations and increased fermentation times of the present study could be a consequence of the resins antimicrobial characteristics. Nevertheless, fermentations accomplished by the use of the novel immobilized biocatalyst proved to be successful despite the possible antimicrobial effects of the resin.
Ethanol production remained at similar levels in fermented beverages produced by immobilized or free cells. However, ethanol productivity in fermentations carried out by immobilized cells was higher (p
< 0.05) than free cells at all temperatures (Table 1
), as fermentation time was significantly lower in the case of the immobilized biocatalyst.
3.3. Formation of Major Volatile By-Products
Since the fermentation products are designated for potable alcohol production, which is usually added in various kinds of alcoholic drinks, such as liqueurs, sweet wines, and distillates, a study on the formation of major volatiles was conducted (Table 2
). Acetaldehyde, ethyl acetate, 1-propanol, isobutanol, and amyl alcohols are the major volatile by-products produced during fermentation and accounted for more than half of the total volatiles [40
]. In order to assess the contribution of the volatiles produced during fermentation and those derived from the immobilization support (P. terebinthus
resin) on the aroma and taste of the produced alcoholic liquids, an examination of fermented beverages for volatile by-products was carried out by both immobilized and free cells (Table 2
Aldehydes are the oxidation products of alcohols and are usually detected at very low levels in alcoholic beverages [41
]. Acetaldehyde specifically, can provide a pleasant fruity aroma when it is detected at low concentration. On the other hand, acetaldehyde can give a pungent, undesirable, irritating odor to the product when it is present at higher levels. Hence, it is crucial, especially in the case of potable alcohol, that sugar fermentation is performed by yeast strains that produce low amounts of acetaldehyde [40
]. In the present study, the detected acetaldehyde concentration ranged within acceptable levels [42
]. In most cases, acetaldehyde ranged in higher concentrations in glucose and sucrose fermented mediums compared to maltose while no effect was reported by the use of the starter culture (free or immobilized) (p
> 0.05) in the case where glucose and maltose-based media were used.
Ethyl acetate, the most important and abundant ester in wines, ranged in concentrations from 3.2 to 10.3 mg L−1
. Saccharomyces cerevisiae
strains usually produce intermediate levels of ethyl acetate while its concentration is desirable in levels between 120–150 mg L−1
, providing fruity notes, while at higher concentrations is considered unpleasant and may result in a spoiled, pungent tang aroma bouquet [43
Higher alcohols are considered the largest group of flavor compounds in wines and fermented liquids [41
]. Among them, 1-propanol, isobutanol, and amyl alcohols are considered crucial for aroma quality [43
]. In the current study, concentrations of 1-propanol, isobutanol, and amyl alcohols were up to 3 mg L−1
, 4.2 mg L−1
, and 19.7 mg L−1
, respectively. The low concentration of higher alcohols is an indication of high quality in wines and in fermented liquids, since high concentrations are related to off flavors. The methanol content ranged 12.9–50.4 mg L−1
, indicating the quality of the fermented products. Due to its toxic properties, concentrations >0.1–0.2 g L−1
are undesirable [43
In addition, it has already been established that phenolic compounds contribute to the sensory characteristics of alcoholic beverages [45
]. In the present study, the polyphenol content was significantly affected (p
< 0.05) by the fermentation temperature. Likewise, fermentations carried out by the immobilized biocatalyst resulted in significantly (p
< 0.05) higher content of polyphenols (Table 2
), while increased amounts were recorded between 21–28 °C. These results agree with previous studies reporting that the extracted quantity of polyphenols can be affected by the fermentation temperature and the solvent type [46
]. In addition, the presence of polyphenols can provide an antioxidant effect to the product. Hence, their present is desirable in alcoholic beverages which are produced without the addition of preservatives [47
]. The stability and content of polyphenols can also be affected by pH, metal ions, exposure to light, storage time, oxygen, temperature, and enzymatic activities [49
Propanol and isobutanol were also detected in limited concentrations and the results showed that their presence within the fermented liquid was significantly affected by the fermentation temperature (p < 0.05).
Overall, based on the above results, all products were considered of acceptable quality.
3.4. SPME GC-MS Analysis
Even though the chemical composition of the P. terebinthus
resin essential oils has been studied [50
], the effect of P. terebinthus
resin on the volatile composition of fermented liquids has never been reported before, as only triterpenes of the galls of the resin have been elucidated [51
]. In the present study, HS-SPME GC-MS technique was used to determine the volatile compounds in fermentations carried out by immobilized cells of S. cerevisiae
AXAZ-1 on P. terebinthus
resin, in comparison to fermentations by free cells, at 7, 14, 21, and 28 °C.
Qualitative results of the volatile compounds are presented in Table 3
. In total, 147 compounds were detected and identified in the alcoholic liquids fermented by immobilized or free cells. The compounds included mainly esters, organic acids, alcohols, carbonyl compounds, and terpenes.
Since the main aim of the study was to assess the potential extraction of terpenoides from Pistacia terebintus
resin into the new products, analysis of the volatile compounds was focused on the identified terpenes. Monoterpenes, oxygenated monoterpenes, and sesquiterpenes were mainly detected. Several terpenes, such as α
-pinene, verbenyl ethyl ether, linalool, dehydro-p
-cymene, bornyl acetate, 4-terpineol, L-trans
-phellandren-8-ol, borneol, verbenol, exo-hydroxycineol, verbenone, p
-methyl-acetophenone, myrtenol, and trans
-carveol were only detected in fermentations carried by the immobilized cells at all temperatures. However, the number of terpenes found in alcoholic liquids fermented by the immobilized cells decreased with the fermentation temperature (Table 3
). Thus, several terpenes, such as carvomenthol, trans
-pinane, 1,8-cineole, p
-terpineol, neral, geraniol, geranyl acetate, p
-myrtanol, piperitenone, β
-cresol, cuminyl alcohol, pseudoionone, longipinanol, and farnesol were only identified in the alcoholic liquids produced by the immobilized cells at temperatures ≥21 °C. On the other hand, camphene and thuja-2,4(10)-diene were only present in fermented products at 7 °C, but not at 28, 21, and 14 °C, probably due to vaporization at room temperature.
From a quantitative point of view, significantly (p
< 0.05) higher amounts of terpenes were detected in fermentations with immobilized cells in all cases, as expected (Table 4
). The content of terpenes also seemed to depend on the fermented sugar. Thus, the highest (p
< 0.05) concentration of terpenes was observed in sucrose fermentation at 7 °C (579.01 mg L−1
). On the contrary, a decrease was noticed at temperatures <28 °C in glucose and maltose fermentations.
Principal component analysis is used in exploratory analysis, as it gives graphical representations of inter-sample and inter-variable relationships and provides a way to reduce the complexity of the data.
The application of the PCA algorithm to data concerning terpenes content showed four distinctive groups (Figure 2
). The first group was composed by liquids fermented by immobilized cells at 28 °C, while the second group by samples fermented by immobilized cells at 21 and 14 °C. A third group consisting of liquids fermented by immobilized cells at 7 °C was evident. Finally, the fourth group contained all samples fermented with free cells projected at almost the same point, as the content of terpenes ranged in similar (low) levels.
Hence, the results indicated that primarily the nature of the cells (immobilized or free) and the fermentation temperature affected the terpenes’ composition.
3.6. Phenolic Content
Fermentations carried out by immobilized cells resulted in a significantly (p
< 0.05) higher content of polyphenols. More specifically, their average content in the case of free cells was 24.3–70.2 mg GAE L−1
in glucose synthetic medium fermentations, 34.3–49.3 mg GAE L−1
in sucrose fermentations, and 30.2–61.8 mg GAE L−1
in maltose fermentations, while in the case of immobilized cells the average content was 74.3–171.8 mg GAE L−1
, 66.0-109.3 mg GAE L−1
, and 63.6–113.5 mg GAE L−1
, respectively (Table 2
The extracted polyphenols content was significantly affected (p
< 0.05) by the fermentation temperature, while the highest polyphenol content was observed in fermentations carried out at 21 °C, in the case of immobilized cells (Figure 3
). The results agree with a previous study reporting that the extracted quantity of polyphenols can be affected by the fermentation temperature and the solvent type [46
3.7. Preliminary Sensory Evaluation and Resistance to Spoilage
The use of immobilized cells on P. terebinthus
resin provided a distinctive aromatic character to the fermented beverages, which was also detected throughout the whole storage period (90 days). The capacity of P. terebinthus
resin to provide unique aromatic characteristics to fermented products has also been reported by previous studies [2
]. Regarding sensory evaluation of the current study, the panel showed a high preference (p
< 0.05) on the fermented beverages produced by the immobilized cells (scores 7.42 ± 0.96) compared to the beverages produced by free cells (scores 5.21 ± 1.09).
It is noteworthy that the fermented alcoholic beverages remained for 90 days at room temperature without any treatment (e.g., addition of preservatives) and no contamination or spoilage was macroscopically observed. On the other hand, the fermented alcoholic beverages produced by free cells showed spoilage affects mainly attributed to overgrowth of white and green molds on the surface of the fermented liquids. Similar results were also reported by Schoina, Terpou, Angelika-Ioanna, Koutinas, Kanellaki, and Bosnea [13
] showing an improved resistance against spoilage microorganisms in yoghurts produced with incorporated P. terebintus
resin. This result may be attributed to the high antimicrobial and antioxidant effect of the resin deriving mainly from the high terpene content. Specifically, many of the terpenes detected in the fermented beverages produced by the immobilized biocatalyst have shown considerable antimicrobial and antioxidant activities. Kotan et al. reported a variable degree of antibacterial activities of α
-terpineol, borneol, carvone, linalool, 4-terpineol, bornyl acetate, 1,8-cineole, and geranyl acetate [52
], while Duru et al. observed antifungal activity of total and neutral fractions of essential oil obtained from the Pistacia lentiscus
resin against R. solani
]. Similarly, farnesol displayed antimicrobial action against Staphylococcus aureus
] and Staphylococcus epidermidis