Fermented beverages based on Hylocereus lemairei (Hook.) fruits: Chemical characterization and antioxidant capacity evaluation

Hylocereus lemairei (Hook.) is a plant popularly known as pitaya-purple, whose fruits are known as dragon fruit. This paper aimed to carry out the physical-chemical and chemical characterization of fermented beverages based on H. lemairei fruits. Fermented beverages were elaborated using two different commercial yeasts of Saccharomyces cerevisiae [Biolievito Bayanus (BB) and Arom Cuvée (AC)] and were characterized by NMR. In addition, the antioxidant capacity (DPPH and ABTS) and Total Phenolic Content (TPC) based on the Folin Ciocalteu method were determined. According to the legislation, the fermented beverages from BB and AC showed alcoholic levels of 12.9 and 12.5% (v/v) and pH of 3.9 and 3.8, respectively. The chemical compounds of both beverages were similar, whose major organic compounds are glycerol, myo-inositol, tyrosol, and citric and succinic acids. According to the DPPH and TPC evaluation, BB beverage (248.3 μM TE and 117.6 mg GAE L) presented higher antioxidant capacity when compared to the BC beverage (219.8 μM TE and 108.4 mg GAE L). In the ABTS assay, both beverages were not statistically different (p < 0.05). Tyrosol may be responsible for increasing the antioxidant capacity and phenolic compounds content when compared to the control juice used to prepare fermented beverages.


Introduction
Fermented fruit is defined as a beverage with an alcoholic degree from 4 to 14% (v/v, 20 °C). Alcoholic fermentation can be obtained from fresh and ripe fruits of a single species, as well as from the respective integral juice, concentrate or pulp (Brasil, 2009). The process of obtaining fermented fruits is very similar to the wine production process with different operations according to the fruit type. Alcoholic fermentation is a relatively efficient preservation process because it increases the shelf life of a food product and reduces refrigeration or other forms of preservation technology. This technique is suitable in remote areas, where other preservation technologies are not available or inaccessible (Battcock & Azam-Ali, 1998).
The species from the family Cactaceae was possibly originated in North, Central and South America. The genus Hylocereus is native from the tropical deciduous forests in Mexico, Western India, Central America and northern South America. The species are known as pitaya, whose fruits are called dragon fruit in Asia. Pitaya enhanced the interest of the consumer because it is exotic and a source of vitamins and minerals. Interesting for fruit growers is mainly based on their high commercial value (Bastos et al., 2006;Fernandes et al., 2017;Nunes et al., 2014).
In order to add value to the fruit and elaborate a new beverage with attractive colors, the species Hylocereus lemairei (Hook.) Britton & Rose from the Cactaceae family was selected. Its fruits are popularly known as pitaya-purple, pitaya-red, pitaia, pitaya, pitahya roja or dragon fruit. It is a succulent epiphyte species, and have been cultivated in different regions of Brazil, including the Amazon region. Its fruits are globose, red, berry-like and without thorns with purple fleshy pulp and black seeds. It has the same water-soluble pigment found in beet, known as betalain. These N-heterocyclic compounds are categorized as betacyanins (red-purple color) and betaxanthines (yellow-orange color), which are natural antioxidants This work aims to determine the physical-chemical parameters, as well as the chemical profiles of fermented beverages from H. lemairei fruits. These alcoholic beverages were prepared using two different commercial yeasts and the antioxidant capacity and total phenol content were determined.

Preparation of fermented beverages
Hylocereus lemairei fruits were purchased at the Adolpho Lisboa Market (Manaus, Amazonas). Fruits were washed and sanitized with peracetic acid (0.5 g L -1 ), cut and processed in a blender to obtain the juice. The yeastt was prepared in a 1.0:1.4 ratio (fruit/water, m/v), chaptalized to 21 °Brix and the pH (3.5) was corrected using tartaric acid. Fermentation process were carried out in glass bottles equipped with an airlock valve and conducted at 20 °C by the commercial yeasts Biolievito Bayanus (BB) and Arom Cuvée (AC) (Biotecsul, Caxias do Sul).
The fermentation evolution was monitored at regular intervals by the Total Soluble Solids -TSS (°Brix). Racking was performed after fermentation at 20 °C, followed by vacuum filtration with diatomaceous earth. The fermentation was stored in glass bottles with synthetic stoppers . The following physicochemical analyzes were carried out (density at 20 ºC, pH, alcohol content, total sugars, total acidity, volatile acidity, ash, and reduced dry extract) (Jacobson, 2006;Rizzon, 2010).

1 H NMR analysis of Hylocereus lemairei fermented beverages
A juice aliquot (550 µL) was mixed with 50 µL of D2O (0.6 mM TMSP-d4). After ethanol removal in the absence of light, the fermented beverages were solubilized in 600 µL of D2O (0.6 mM TMSP-d4). Samples were subjected to a Bruker ® Avance IIIHD Nuclear Magnetic Resonance Spectrometer (11.74 T, BBFO Plus SmartProbe ™) at 298 K. The ZGPR sequence was used for analyzing the samples and suppression of the residual water signal (4.8 ppm). The obtained spectra were processed by TopSpin™ 4.1.1 software. Chemical identification of the compounds was appropriately marked based on the chemical shift (H and C), coupling constant (J in Hertz), and comparison with literature data.

Scavenging capacity of DPPH . and ABTS .+ radicals
Juice and fermented beverage were subjected to the scavenging capacity of DPPH . and ABTS .+ radicals and the determination of total phenols was based on methodologies with marginal modifications on a microplate reader (Molyneux, 2004;Re et al., 1999;Souza et al., 2020). For the DPPH assay, 10 µL of the sample was added to 190 µL of the DPPH solution (60 µM) and incubated in a dark environment for 30 min. Subsequently, the absorbances were read on a Microplate Reader at 515 nm (Epoch 2, Biotek). A standard Trolox curve from 100 to 500 µM was obtained (y = -0.0007x + 0.5322, R 2 = 0.9999).
For the ABTS assay, the sample was mixed with the ABTS solution (1:100) and incubated in a dark environment for 6 min.
Measurements were performed in triplicate.

Statistical analysis
ANOVA (One-Way, Tukey's test, 95% significance) and Test-T (p < 0.05) to evaluate the assays. Results were expressed as mean ± standard deviation. The Minitab ® 18.1 software was employed in these analyses.

Physicochemical Analysis
The fermentation evolution was monitored by the content of TSS (ºBrix) over time regular intervals. The end of fermentation was determined when the TSS content stabilized on two consecutive measurements in 17 days with a TSS of 6 for both fermented beverages (Figure 1). Then, fermented beverages from H. lemairei fruits were subjected to analysis of physical-chemical parameters (Table 1). The fermented products obtained from different yeasts presented an alcohol content within the range established by the current legislation from 4 to 14% v/v, with 12.9 and 12.5% (v/v) for the yeasts BB and AC, respectively. These densities were found to be similar among the fermented samples, 0.988 g mL -1 . The pH of the beverage was 3.8 to 3.9. The values of total acidity in the beverage fermented by the yeasts BB and AC were 63.6 mEq L -1 and 62.1 mEq L -1 , respectively. The acidity observed by current legislation for fermented fruit ranges from 50 mEq L -1 to 130 mEq L -1 , so both fermented are in accordance with the reference values. The volatile acidity values were 9.6 mEq L -1 for BB and 10.0 mEq L -1 for the AC beverage, which are in agreement to the standards established by the legislation for fruit fermented (maximum 20.0 mEq L -1 ).
The alcoholic beverages produced by the yeasts BB and AC showed dry extract content of 21.2 g L -1 and 20.7 g L -1 , respectively. The ash concentration found in the AC beverage (3.1 g L -1 ) was slightly higher than that of BB 2.6 g L -1 . The average ashes content for fermented beverages was found from 1 to 3 g L -1 (Brasil, 2012).

1 H NMR analysis of juices and fermented beverages
The major signs characteristic of carbohydrates and organic acids were observed from the 1 H NMR spectrum ( Figure   2, Table 2). The signals at H 5.23 (d, J = 3.8 Hz) and at  The analysis of chemical profiles by 1 H NMR of H. lemairei fermented beverage obtained with the BB and AC yeasts were similar (Figure 3, Table 2). The correlation maps HSQC and HMBC contributed to the confirmation of the compounds.
The data were compared with the scientific literature (Al-Mekhlafi et al., 2021;Souza et al., 2020). After the fermentation process, no signs of sugars were observed, as there was a conversion to ethanol. After removed of ethanol, the analyzed extracts of both fermenters showed major signals of organic acids.
The signal at H 3.54 (dd, J = 11.7 and 6.5 Hz) and at H 3.64 (dd, J = 11.7 and 4.3 Hz) were directly linked to carbon at C 65.5. The multiplet at H 3.77 ppm linked to carbon at C 75.0 ppm was attributed to glycerol. The signal at H 4.05 ppm (t, J = 2.9 Hz) attached to carbon at C 75.1, H 3.61 (t, J = 9.5 Hz), H 3.52 (dd, J = 9.5 and 2.9 Hz) and H 3.26 (t, J = 9.5 Hz) was assigned to myo-inositol. The compounds glycerol, citric acid, and succinic acid were also found in the fermented beverage from dragon fruit (H. costaricensis). Higher glycerol content was observed in the fermentation with Saccharomyces cerevisiae when compared to different yeasts (Jiang et al., 2020). Glycerol is produced as a by-product of S. cerevisiae ethanolic fermentation process and is responsible for the smoothness and viscosity of wine (Scanes et al., 1998). Inositol has previously been identified in dragon fruit varieties (Al-Mekhlafi et al., 2021), which was not observed in the juice but was identified as one of the major coumpounds in fermented beverages.
Tyrosol is produced by Saccharomyces cerevisiae from L-tyrosine, anaerobically in the essential presence of glucose (Sentheshanmuganathan & Elsden, 1958). Beer and wine are beverages containing tyrosol. When these beverages are consumed, this simple phenolic compound is converted endogenously to hydroxytyrosol. This phenolic compound has been considered a powerful antioxidant in diets (Soldevila-Domenech et al., 2019). Temperature, alcoholic degree, sugar and amino acid concentrations, oxygen dissolved and pH have been influenced the glycerol biosynthesis, as well as in the production of aromatic compounds (Bordiga et al., 2016;Zhao;Procopio;Becker, 2015).

Antioxidant capacity and quantification of total phenols
The results from the antioxidant capacity based on the DPPH and ABTS assays, as well as the quantification of the total phenols are shown in the The scavenging capacity of the radicals observed from the DPPH and ABTS assays, as well as the presence of phenolic compounds in the fermented beverages can be attributed to the presence of the phenolic compound tyrosol (Souza et al., 2020). The content of phenolic compounds found in the fermented beverages was higher when compared to the fermented beverage with dragon fruit of 28.0 mg GAE L -1 elaborated by Souza and contributors (2018), and lower when compared to the fermented beverage from dragon fruit (H. costaricensis) of 280.6 mg GAE L -1 (Jiang et al., 2020). Resulted are expressed as means ± standard deviation (n=3). a-c Different letters in same column are significant (p-value < 0.05). μM TE = micromolar of Trolox equivalent, mg L -1 GAE = milligram of Gallic acid equivalent per liter of sample. Source: Authors.

Conclusion
The physical-chemical parameters analyzed in the Hylocereus lemairei fermented beverages prepared with the yeasts BB and AC are in accordance with the current legislation. The chemical profiles of the beverage extracts prepared with yeasts were similar, presenting as major compounds alcohols and organic acids, and phenolic compounds in lower concentration.
Tyrosol, a phenolic compound, is probably responsible for the antioxidant capacity of the fermented beverages, when compared to the control juice. There was a significant increase in the antioxidant activity after the fermentation process. Future research must be carried out to evaluate the influence of different parameters in the preparation of H. lemairei fermented beverages. The production of new fermented beverage from fruits encourages the cultivation by communities in the capital and interior of Amazonas State, as well as contributes to the formation of local productive arrangements.