Total phenolics and antioxidant potential of Syzygium malaccense leaf extract.
Table 1 presents the total phenolic content and antioxidant potential found in the red-Jambo leaf extract that was added to the vinegars produced in the present work.
Bioactivity parameters
|
Values
|
Total phenolics (mg Gallic Acid Equivalents/10g)
|
385.4 ±0.03
|
Antioxidant activity
|
ABTS (µmol Trolox equivalent/10g)
|
41.83 ±0.008
|
DPPH (µmol Trolox equivalent/10g)
|
11.82 ±0.019
|
FRAP (µmol FeSO4/10g)
|
54.15 ±0.051
|
Table 1. Total phenolics and antioxidant activity in red-Jambo leaf extract.
Extracts of red-Jambo leaves are still little used. Recent evidence reported that its leaves are rich in phenolic compounds and flavonoids, in addition to having an antioxidant activity 13. A high content of total phenolics (385.4 mg Gallic Acid Equivalents - GAE/10g) was found in the lyophilized leaf extract. However, values somewhat higher (537.70 mg GAE/10g) have been reported by Batista et al. 21 in the leaf extract obtained using methanol. Different parameters including genetic aspects of the plant, the time and period/season of harvesting, and agronomic conditions related to the crops of the plant, among others, can contribute to obtaining extracts with varying contents of total phenolics.
The antioxidant activity of polyphenols is attributed to their Redox properties, which allows them to act as reducing agents, hydrogen donors, singlet oxygen scavengers, and in metal-chelation 22. In this context, appreciable scavenging potential of DPPH (11.82 µmol Trolox equivalent - TE/10g) and ABTS (41.83 µmol TE/10g) radicals were found in the present work. Similarly, the extract showed a ferric ion reduction capacity of 54.15 µmol FeSO4/10g. The values found in the present study are much higher than those reported previously by Savi et al. 13 in a similar extract (ABTS: 0.853 µmol TE/kg, DPPH: 0.666 µmol TE/kg, FRAP: 1.267 µmol TE/kg).
Fermentation profile of must vinification.
The profiles of alcoholic fermentations of musts based on pineapple pulp and peel are shown in Figure 1a-b The fermentation time was set at 120 h when the release of CO2 ended, and consumption of at least 95% of the sugar content of the musts. The inoculum used in alcoholic fermentations exhibited high cellular activity concerning growth, substrate consumption, and fermentative activity.
The hydration and cultivation of commercial yeast in malt-extract broth and subsequent suspension in pineapple juice resulted in obtaining a metabolically-active inoculum, which was verified by the absence of the lag phase of yeast growth. In this sense, a linear increase in the concentration of yeast cells was observed in the first 48 h of cultivation, when the maximum cell concentrations (@ Log 16 CFU/mL) were obtained both in the musts of pineapple pulp (Figure 1a), and pineapple peel (Fibure 1b).
Cell growth was accompanied by effective substrate consumption and ethanol production, and especially in the pineapple-pulp cultivated cultures. In 72 h of the pulp fermentation, 94.73% of the substrate was consumed, and produced an ethanol concentration of 56.04 g/L. On the other hand, lower values of substrate assimilation (61.6%) and ethanol production (48.61 g/L) were observed in peel-based musts (72 h). At the end of the alcoholic fermentations, similar values of consumption of total reducing sugars (ART) were verified in musts obtained from the pulp (97.6%) and peel (96.7%). The yeast used in the present work also showed similar substrate assimilation values (96.5%) in must formulated with blackberry and honey as previously described by Fonseca et al. 12.
Regarding ethanol accumulation in the fermented broth, higher amounts were observed in musts formulated with pulp (66.2 g/L) than with peel (57.4 g/L). The substrate assimilation profile by the yeast observed in 72 h of fermentation suggests that the hydrolyzed pineapple peels present in the must hindered the assimilation of sugars. Such behavior may be related to the complexity of the chemical composition of the pineapple peels. In fact, in addition to the fermentable sugars (glucose, fructose, sucrose) present in the peels, are the high contents of structural polysaccharides (cellulose, hemicellulose, pectin) and lignin 23, as well as, phenolic compounds, alkaloids, flavonoids, tannins and saponins, and other secondary compounds, which reportedly can have antimicrobial potential 24,25.
The fermentative parameters of yeast cultivation in medium based on pineapple pulp and peel are described in Table 2. Determining such parameters is essential to evaluate the efficiency and yield of alcoholic fermentation, allowing a better understanding and comparison of the process.
Fermentation parameters
|
Wines - Alcoholic Fermentation
|
Pulp
|
Peel
|
Ethanol production (PF)
|
66.2 g/L ±2.11
|
57.4 g/L ±1.88
|
Ethanol yield (YP/S)
|
0.28 g/g ±0.01
|
0.27 g/g ±0.01
|
Volumetric productivity in ethanol (QP)
|
0.55 g/L.h ±0.03
|
0.48 g/L.h ±0.02
|
Overall substrate consumption rate (QS)
|
1.94 g/L.h ±0.07
|
1.75 g/L.h ±0.05
|
Efficiency of alcoholic fermentation (ŋ)
|
54.8% ±0.0
|
52.8% ±0.0
|
Overall percentage of substrate consumption (YC)
|
97.5% ±1.2
|
96.7% ±1.1
|
Maximum specific growth rate (µmáx)
|
0.37 h-1
|
0.34 h-1
|
Table 2. Fermentation parameters of must vinification.
As observed with the assimilation of fermentable sugars, the ethanol content accumulated at the end of the fermentation was 15.3% higher in the broth fermented with pulp compared to that of peel. Similarly, Alvarenga et al. 26 reported that the addition of pineapple peels in musts formulated with the pulp contributed to a reduction in ethanol production. Roda et al. 4 Roda et al. (2017), and Chalchisa and Dereje 27 also reported lower ethanol production values in wines produced from musts formulated with pineapple peels (47.34 g/L and 47.02 g/L, respectively). It is important to note that although greater ethanol production was obtained in pulp wines, the ethanol yields were similar in both the pulp (YP/S: 0.28 g/g) and peel (YP/S: 0.27 g/g) wines. The overall percentage of substrate consumption (YC) was also similar under both fermentation conditions (97.5% and 96.7%). Likewise, little difference was found concerning the process efficiency parameter values (h: 54.8% and 52.8%). Regarding the overall substrate consumption rate (QS), fermentation with pulp showed values 10.9% higher than those found in fermentation with peel. Corroborating the substrate assimilation profile in the exponential growth phase, the maximum specific rate of yeast growth was slightly higher in fermentation with the pulp (µmax: 0.37 h-1), suggesting that during this phase, a higher percentage of the substrate was directed to cell growth in fermentation of the pulp compared to the peel (µmax: 0.34 h-1). The fermentation results show that pineapple peels have potential as a raw material for formulating musts intended for alcoholic fermentation. Although the musts formulated with pure pulp stood out in fermentation, the peels showed good fermentative capacity.
Table 3 describes the physical-chemical and bioactivity parameters of pineapple pulp and peel wines.
Parameter analyzed
|
Wines - Alcoholic Fermentation
|
Pulp
|
Peel
|
pH
|
3.4a ±0.01
|
3.69b ±0.01
|
Titratable acidity (g/100mL)
|
7.6a ±0.01
|
4.6b ±0.057
|
Total reducing sugars (g/L)
|
5.86a ±0.006
|
7.24b ±0.004
|
Ethanol % (v/v)
|
8.39a ±0.020
|
7.28b ±0.006
|
Density (kg/m3)
|
987.0a
|
969.0b
|
Free sulfur dioxide – SO2 (mg/mL)
|
nd
|
nd
|
Total sulfur dioxide – SO2 (mg/mL)
|
nd
|
nd
|
Phenolic compounds
|
Total phenolics (mg GAE/L)
|
188.97a ±0.004
|
110.53b ±0.023
|
Catechin (mg/L)
|
31.63
|
27.88
|
Caffeic acid (mg/L)
|
< DL
|
1.73
|
p-Coumaric acid (mg/L)
|
0.35
|
4.05
|
Ferulic acid (mg/L)
|
< DL
|
1.48
|
Organic acids
|
Ascorbic acid (g/L)
|
2.7
|
1.9
|
Citric acid (g/L)
|
6.2
|
1.0
|
Malic acid (g/L)
|
1.9
|
0.7
|
Oxalic acid (g/L)
|
7.8
|
4.91
|
Succinic acid (g/L)
|
2.8
|
1.2
|
Antioxidant activity
|
ABTS (µmol TE/100 mL)
|
274.0a ±0.019
|
211.0b ±0.001
|
DPPH (µmol TE/100 mL)
|
129.0a ±0.013
|
139.0b ±0.017
|
FRAP (µmol FeSO4/100 mL)
|
562.6a ±0.075
|
258.1b ±0.089
|
Color
|
L*
|
42.73a ±0.1
|
52.75b ±0.01
|
a*
|
-0.78a ±0.15
|
-0.16 b ±0.01
|
b*
|
2.61a ±0.24
|
13.2b ±0.01
|
Hº
|
110.9a ±3.53
|
90.7b ±0.03
|
C*
|
2.42a ±0.22
|
13.2b ±0.01
|
DE
|
14.59
|
Table 3. Physicochemical and bioactive parameters of pineapple pulp and peel wines.
GAE: gallic acid equivalent, TE: trolox equivalents (Trolox-Equivalent Antioxidant Capacity), ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), DPPH: 2,2-Diphenyl-1-picrylhydrazyl, FRAP: ferric reducing antioxidant power, nd: not detected, <DL: values below detection limit, L*: luminosity, a*[(−) green to (+) red] and b* [(−) blue to + yellow] chromaticity coordinates, ho, Hue angle and C*, Chromaticity, DE: Total color difference
Different letters on the same line differ statistically at the 95% confidence interval (p < 0.05).
Pulp and peel wines had pH values of 3.94 (pulp) and 3.69 (peel) and titratable acidity expressed in acetic acid of 0.76 g/100 mL (pulp) and 0 .46 g/100 mL (peel). Final pH values between 3.5 and 4.0 were reported by Cunha et al. 28 in blackberry wines and Fonseca et al. 12 in mixed blackberry and honey wines produced in a vinification process where the same yeast used in the present study was also used. The acidity of a wine is basically due to the presence of organic acids from the fruit itself, such as malic acid, tartaric acid, citric acid, and the production of acids during fermentation, such as acetic acid. 29. Both pineapple wines obtained showed prominent acidity, especially the pulp wine, possibly because it contains higher amounts of organic acids derived from the fruit itself. Qi et al. (2017) obtained pineapple wines with lower acidity (0.229 g/100 mL) than was found in this work. This can be attributed both to the characteristics of the fruit and the metabolic properties of the yeast used in the winemaking process. Low pH values during alcoholic fermentation can prevent the growth of undesirable microbiota, and therefore, enhance the quality of the final products.
The alcohol contents present in the pulp and peel wines were 8.39% (v/v) and 7.28% (v/v), respectively. Such values are higher than those reported by Alvarenga et al. 26 in wine from the musts formulated with pineapple pulp (6.8% v/v) and pulp plus peel (100g/kg) (5.9% v/v). Lower ethanol values were also reported by Roda et al. 4 in wines of pineapple peel (6.0%, v/v). It is important to point out that the ethanol content present in wines destined for acetification is a parameter of importance that must be analyzed and adjusted where necessary. Very high ethanol content in wines can lead to vinegars with a high acetic acid content, resulting in a very acidic product that does not meet the legal standard. Associated with excess acidity, vinegars with ethanol contents outside the legislation standard can also be produced. Chalchisa and Dereje (2021) reported the concentration of ethanol in wine must be less than 7.5% to obtain good quality vinegar. However, it is important to consider the acetification process used, the acetic acid bacteria used in the ethanol-to-acetic acid bioconversion, as well as ethanol losses incurred by evaporation during the acetification process.
Pineapple pulp and peel wines showed density values of 987 and 969 kg/m3, respectively, that agreed with those reported by Akanni Ahoussi et al. 30 in pineapple wine (995.0 kg/m3). Similar values (998.2 kg/m3) were also found by Queiroz, Rabelo and Santos (2019) in alcoholic-fermented pineapple juice. The density of the wine varied according to the amount of sugars and ethanol present in the product.
Brazilian legislation 32 establishes a maximum limit to the addition of 300 mg/L of sodium metabisulfite in musts that are destined for comercial alcoholic fermentation. No sulfurous anhydride (SO2) residues were detected in the pineapple wines produced, which was to be expected as the musts were not sanitized with metabisulfite prior to the fermentation stage. Slow pasteurization was the method that we chose to sanitize the musts before alcoholic fermentation.
As shown in Table 3, appreciable total phenolic content was found in both the pulp wine (188.97 mg GAE/L) and peel wine (110.53 mg GAE/L). Different contents of total phenolics have been reported in the scientific literature. Pino and Queris 33, when evaluating the content of total phenolic compounds in pineapple wines, found lower values (108.0 mg GAE/L) than those found in the present work. On the other hand, Zhang et al. (2020) found higher values in pineapple peel wine (675.43 mg GAE/L). The different phenolic contents may be associated with the origin of the fruit, the degree of maturation, and the vinification process used 34.
The use of high-performance liquid chromatography with diode array detection allowed the identification and quantitation of phenolic acids: caffeic acid, coumaric acid, ferulic acid, and the flavonoid catechin in pineapple pulp and peel wines, as outlined in Table 3. Among the phenolic compounds, the flavonoid, catechin, was found in higher concentrations in both the pulp (31.63 mg/L) and peel (27.88 mg/L) wines. p-Coumaric acid was also found in relatively considerable amounts (0.35 mg/L and 4.05 mg/L) in both wines. On the other hand, caffeic acid (1.73 mg/L) and ferulic acid (1.48 mg/L) were identified only in the peel wine samples. Catechin (107.39 µmol/L) and ferulic acid (139.70 µmol/L) were the main compounds found in pineapple peel extracts by Li et al. 35. Ferulic acid has been reported in pineapple wine by Roda et al. 4 at concentrations lower (0.138 µg/L) than those obtained in the present work (1.48 mg/L), and no detectable levels were found in the pulp wine.
The pulp and peel wines, in addition to being rich in total phenolic content, showed high antioxidant potential estimated by the DPPH, ABTS, and FRAP techniques. The pulp wine had an ABTS scavenging capacity of 274.0 µmol TE/100 mL, a slightly higher value than the peel wine (211.0 µmol TE/100 mL). Similarly, appreciable DPPH scavenging potential was also observed in both wines (129.0 µmol TE/100 mL and 139 µmol TE/100 mL, respectively). Regarding the ferric ion reducing potential, pulp wine (562.6 µmol FeSO4/100 mL) stood out from peel wines (258.1 µmol TE/100 mL). The higher antioxidant capacity found in the pineapple pulp wines can be explained by the higher concentration of phenolic compounds and organic acids commonly found in the fruit pulp, which have antioxidant activity 36,37. There is a lack of reports regarding the antioxidant activity of pineapple wines in the scientific literature. Higher values were reported by Fonseca et al. 12 in blueberry wine and honey for all of the antioxidant methods evaluated. Similarly, Cunha et al. 28 also found higher values in blackberry wine for the DPPH (1395.2 µmol TE/100 mL) and ABTS (2124.0 µmol TE/100 mL) methods assessing antioxidant activity.
Organic acids such as ascorbic (vitamin C), citric, malic, oxalic, and succinic acids were found in pineapple pulp and peel used in alcoholic fermentation. Citric (6.2 g/L) and oxalic (7.8 g/L) acids were the predominant organic acids in pulp wine. Oxalic acid (4.91 g/L) and ascorbic acid (1.9 g/L) predominated in pineapple peel wine. Such results show that the pulp and the peels are rich in organic acids. It is essential to highlight that the composition and the amounts of organic acids present in the fruit can vary greatly depending on the variety, as well as the stage of fruit maturation, considering, for example, that the content of organic acids in the initial stages of fruit development is directly related to the supply of substrates for the respiratory processes 38.
Regarding the color of the samples, it is important to mention that in the CIELAB space, the luminosity coordinate (L*) varies from black (0) to white (100); the a* coordinate varies between green (-a) and red (+a), and the b* coordinate varies from blue (-b) to yellow (+b). The hue angle (hº) starts on the +a* axis (red) and is expressed in degrees: 0° corresponds to +a (red), 90° corresponds to +b (yellow), 180° corresponds to −a (green ), and 270 ° corresponds to −b (blue). C* chroma is 0 at the center of the color axis and increases with distance from it 39. Although this method does not provide a precise definition of color, it can effectively show differences in the color of the pulp and peel wine. Instrumental color characterization showed that samples of wine from pulp and peel showed statistically significant differences (p<0.05) between the color parameters L*, a*, b*, and chroma (C*). The L* values of both pulp and peel wines indicate a tendency towards a grayer color than white. The wine made from peel presented a higher luminosity (52.75) than the wine produced from the fruit’s pulp (42.73). Considering that luminosity is understood as the effectiveness of light in generating the sensation of brightness or clarity when perceived by the human eye, it can be said that the wine sample from peel tends to be lighter than the pulp sample. In fact, visually, the peel wine sample was visually clearer than the pulp wine.
Values of the a* coordinate (negative values) indicate a green direction, while the b* coordinate values show a yellow trend (positive values) in both samples. The color of the peel wine, in particular, tended more towards yellow (b*: 13.2) than the pulp wine (b*: 2.61), as indicated by the values of the green-yellow color coordinate. Another color aspect that differentiated the peel wine from the wine obtained from the pulp was its saturation (C*: 13.2), which was more noticeable than the pulp wine (C*: 2.24) that presented a more neutral color. Regarding tonality (hº), which is a qualitative attribute of color, there was a significant difference (p<0.05) between the two samples. Corroborating the results of the parameters L*a*b*, C*, and hº, a statistically significant total difference in color was verified between pulp and peel wine samples.
It is worth noting that there was a total difference in color between the pineapple-derived wine samples, with an DE of 14.58 being verified. Such behavior could be justified by the fact that both samples presented soft green-reddish tones, but with a tendency to yellow as observed by a* and b* coordinates, in addition to the peel wine presenting saturation values 5.5 times higher than the pulp.
Acetic fermentation and pineapple pulp and peel vinegar: physicochemical and bioactive properties.
The ethanol-to-acetic acid bioconversion profile of the pineapple pulp (Figure 2a) and peel (Figure 2b) wines reveal a good acetification efficiency of the acetic acid bacteria isolated from colonial vinegar and used as inoculum.
The acetification of the pulp wine occurred in 144 h, when a content of 7.06 g/100 mL of acetic acid, consumption of 88.3% of ethanol, 92.6% efficiency, and 0.49 g/L.h volumetric productivity in acetic acid were verified. On the other hand, although ethanol assimilation by the acetic acid bacteria was similar in the two acetification processes (88.3% and 86.6%), a longer acetification time was observed in the fermentation of wine formulated with pineapple peels. After 264 h of acetification, the acetic acid content was 5.66 g/100 mL, corresponding to an acetic fermentation efficiency of 87.3% and volumetric productivity of 0.21 g/L.h. Tanamool, Chantarangsee and Soemphol 40 reported a maximum acetic acid content in vinegar produced from pineapple peels in processes using co-inoculation of yeasts and thermotolerant-acetic acid bacteria of 7.2% (v/v) in 16 days of cultivation.
The higher performance observed in the acetification of the pulp wine compared to the peel wine could be explained in part by the greater nutritional richness of the fruit pulp than the peel. Another aspect that can be considered is the phenolic acid composition of the peel wine. In fact, extracts containing phenolic acids commonly have antimicrobial activity, and such activity can vary considerably depending on the amounts and types of phenolic acids present 41. Caffeic acid can interfere with the synthesis of bacterial cell wall macromolecules. Ferulic acid and catechins can modify the charge and hydrophobicity of the cell surface of gram-positive and gram-negative bacteria, leading to cell death by extravasation of the cytoplasmic material. p-Coumaric acid acts as an antimicrobial by disrupting the bacterial cell membrane and binding to bacterial DNA, inhibiting cellular functions 41.
Table 4 shows the physicochemical parameters of both vinegars. The pH ranged from 3.45 to 3.65, which is similar to that reported by Roda et al. 4 and Chalchisa and Dereje 27 in pineapple peel vinegar; pH values of 3.0 and 3.5, respectively.
Parameter analyzed
|
Vinegar
|
Pulp
|
Peel
|
Pulp + extract
|
Peel + extract
|
pH
|
3.64 ±0.01
|
3.65 ± 0.01
|
3.45 ±0.01
|
3.48 ±0.01
|
Total acidity (g/100 mL)
|
5.5b,c ±0.06
|
4.5b ± 0.04
|
5.58a,c ±0.06
|
4.73b ± 0.04
|
Ethanol % (v/v)
|
0.97a ±0.1
|
0.61b ±0.1
|
0.97a ±0.1
|
0.97a ±0.1
|
Mineral residue (g/L)
|
4.06a ±0.01
|
2.40b ±0.01
|
3.95a ±0.01
|
2.52b ±0.01
|
Total dry extract (g/L)
|
30.74a ±0.01
|
10.93b ±0.01
|
31.04a ±0.01
|
11.47b ±0.01
|
Reduced dry extract (g/L)
|
24.88a ±0.01
|
6.22b ±0.01
|
25.13a ±0.01
|
6.71b ±0.01
|
Density (g/mL)
|
1.024c ±0.00
|
1.026b ±0.00
|
1.066a ±0.00
|
1.011b,c ±0.,00
|
Sulfates (g/L)
|
nd*
|
nd*
|
nd*
|
nd*
|
|
Phenolic acids and Flavonoids
|
Total phenolics (mg GAE/L)
|
364.45b ±0.01
|
222.94c ±0.01
|
443.59a ±0,01
|
337.63b ±0.01
|
Catequina (mg/L)
|
23.38
|
12.13
|
27.88
|
12.88
|
Epicatequina (mg/L)
|
0.9
|
10.02
|
34.36
|
11.03
|
Ácido caféico (mg/L)
|
3.42
|
12.77
|
< DL
|
14.89
|
Ácido cumárico (mg/L)
|
< DL
|
11.6
|
7.61
|
12.02
|
Ácido ferúlico (mg/L)
|
4.85
|
8.46
|
11.56
|
5.88
|
Ácido gálico (mg/L)
|
18.09
|
3.22
|
19.23
|
3.79
|
|
Antioxidant activity
|
ABTS (µmol/100 mL)
|
410.5c ±0.01
|
266.6b ±0.01
|
547.1a ±0.01
|
337.5b,c ±0.01
|
DPPH (µmol/100 mL)
|
216.4a ±0.01
|
227.8a ±0.01
|
249.8a ±0.01
|
277.5a ±0.01
|
FRAP (µmol/100 mL)
|
402.8a ±0.04
|
277.8b ±0.02
|
675.8a ±0.01
|
542.3a ±0.01
|
|
Organic acids
|
Ascorbic acid (g/L)
|
1.0
|
0.90
|
1.3
|
0.96
|
Citric acid (g/L)
|
7.41
|
1.46
|
7.46
|
1.48
|
Malic acid (g/L)
|
1.8
|
0.20
|
2.0
|
0.27
|
Oxalic acid (g/L)
|
9.04
|
4.75
|
9.09
|
4.78
|
Succinic acid (g/L)
|
2.1
|
0.70
|
2.5
|
0.78
|
|
Color
|
L*
|
45.39a ±0.01
|
42.29b ±0.07
|
45.28a ±0.03
|
43.12c ±0.07
|
a*
|
-1.07a ±0.01
|
-0.74b ±0.01
|
-1.13a ±0.02
|
-0.71b ±0.01
|
b*
|
-0.53b ±0.01
|
0.76b ±0.06
|
-0.53b ±0.01
|
1.15a ±0.09
|
hº
|
206.54a ±0.73
|
135.01b ±0.4
|
205.63a ±0.7
|
122.64c ±0.2
|
C*
|
1.2a, b ±0.01
|
1.07b ±0.01
|
1.26a, b ±0.01
|
1.36a ±0.07
|
|
DE
|
|
3.20
|
2.29
|
Table 4. physicochemical and bioactive parameters of pineapple pulp and peel vinegar.
GAE: gallic acid equivalent, TE: trolox equivalents (Trolox-Equivalent Antioxidant Capacity), ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), DPPH: 2,2-Diphenyl-1-picrylhydrazyl, FRAP: ferric reducing antioxidant power, nd: not detected, <DL: values below detection limit
L*, luminosity; a*[(−) green to (+) red] and b* [(−) blue to + yellow] chromaticity coordinates, ho, Hue angle and C*, Chromaticity. DE: Total color difference. Different letters on the same line differ statistically at the 95% confidence interval (p < 0.05).
Peel vinegar had lower acidity (4.5%) than the pulp vinegar (5.5%), as judged by the lower content of acetic acid present in these vinegars (Figure 2a-b). Roda et al., (2017) and Raji et al. (2012) observed similar acidity values (5.0% and 4.77%, respectively) in the vinegars of pineapple peel.
Brazilian legislation establishes that the minimum volatile acidity of commercially-produced vinegars must be equivalent to 4.0 g of acetic acid in 100 mL of the product. Acidity in acetic acid is a physicochemical parameter of importance as it reflects the quality of vinegar, since it influences the flavor and acceptability of the product. Vinegar with acidity higher than 5.5% is commonly not acceptable by consumers. On the other hand, vinegar with low acidity produced by the traditional fermentation method is susceptible to contamination by the nematode, Anguillula aceti (vinegar eels) 28.
The content of residual ethanol present in pineapple pulp vinegar (0.97%, v/v), pulp vinegar enriched with red-Jambo extract (0.97%, v/v), peel vinegar (0.61%, v/v), and peel vinegar enriched with this extract (0.97%, v/v) are in accordance with Brazilian legislation, which establishes maximum values of 1% ethanol (v/v). The values found were similar to those reported by Roda et al. 4 in pineapple peel vinegar (0.50% v/v). It should be noted that small amounts of residual ethanol are necessary since acetic bacteria can promote the degradation of acetic acid due to a lack of ethanol 43.
Fixed mineral residue (ash) values in fruit vinegar are also established by Brazilian legislation, which must be at least 1 g/L, and in this sense, all commercial vinegar produced presents adequate values. The total dry extract parameter refers to the content of minerals and organic matter that persist after evaporating water and volatile substances from the vinegar (CUNHA et al., 2016). The values found in pulp vinegar are close to those described in the literature for fruit vinegars. The values of the total dry extract were lower in the vinegar samples obtained from the peels than compared to the pulp vinegar samples. This was probably due to the dilution of the peels in obtaining the must for vinification and wine production. Different contents of total dry extract in fruit peel vinegar have been reported in the literature. Prisacaru et al. 44 reported values between 2.11 g/100 mL and 26.43 g/100 mL, while values between 6.9 g/L and 10.59 g/L were mentioned by 45. The Brazilian legislation determines a minimum amount of 6 g/L of reduced dry extract in fruit vinegars, the values found in the present work are in accordance with the current legislation.
Regarding density, the vinegar of pulp (1.024 g/mL), peels (1.026 g/mL), pulp with plant extract (1.066 g/mL), and peels with plant extract (1.011 g/mL) are in agreement with what was reported by Raji et al. 42 in pineapple peel vinegar (1.08 g/mL). High amounts of phenolic compounds (pulp vinegar: 364.45 mg GAE/L, peel vinegar: 222.94 mg GAE/L, pulp+extrat- vinegar: 443.60 mg GAE/L and peel+extrat- vinegar: 337.63 mg GAE/L) were observed in all of the pineapple vinegar samples. It should be noted that pineapple peel vinegar, however, had lower total phenolic contents than those found in the pulp vinegar. In fact, the pulp wine used in acetic fermentation already had a higher content of phenolic substances from the pineapple fruit itself. Another interesting observed aspect is that the wine acetification process increased the phenolic content. This phenomenon occurred because acetic fermentation was conducted by the traditional vinegar system produced in wooden vats. Phenolic compounds migrate from the wooden walls of the acetification barrel and into the vinegar. The substances supplied by the wood will depend on the type of wood and the roasting of the barrel, the relationship between the contact surface and the volume of liquid, and the contact time 46.
The addition of red-Jambo extract in pineapple pulp and peel vinegars promoted the enrichment of the total phenolic content. Among the phenolic substances identified in the samples, the highest epicatechin concentrations were found in the pulp vinegar plus the extract (34.36 mg/L). Caffeic acid (14.89 mg/L) was peel vinegar's most prominent phenolic compound. Gallic (862.61 µg/mL) and caffeic (218.91 µg/mL) acids were reported by Mohamad et al. 47 as the major phenolic compounds in pineapple pulp vinegar.
In addition to having high contents of total phenolics, the vinegar produced also had an appreciable ability to scavenge DPPH and ABTS radicals and ferric reducing antioxidant power (FRAP). Regarding the capture capacity of the ABTS radical, values of 410.5 µmol/100 mL (pulp vinegar), 266.6 µmol/100 mL (peel vinegar), 547.1 µmol/100 mL (pulp+extract- vinegar) 337.5 µmol were obtained /100 mL (peel+extract- vinegar). The ABTS radical scavenging potential of the pulp vinegar was higher for the peel vinegar. Similarly, pulp vinegar showed greater ferric ion reducing potential (pulp vinegar: 402.8 µmol/100 mL and pulp+extract- vinegar: 675.8 8 µmol/100 mL) compared to peel vinegars (peel vinegar: 277.8 µmol/100 mL and peel+extract vinegar: 542.3 µmol/100 mL).On the other hand, peel vinegar was more efficient in capturing the DPPH radical (peel vinegar: 227.8 µmol/100 mL and peel+extract vinegar: 277.5 µmol/100 mL) than pulp vinegar (pulp vinegar: 216.4 µmol /100 mL and pulp+extract vinegar: 249.8 µmol/100 mL). The chemical nature of the bioactive compounds present in the vinegars, including chemical structure, polarity and hydrophobicity, strongly influence their free-radical scavenging capacity or reducing antioxidant potential. In this sense, more than one method is commonly used to evaluate the antioxidant potential of the same sample since the antioxidant evaluation methods are correlated with the mechanisms of antioxidant action 48. Several studies of antioxidant activity in vinegar samples have been described in the scientific literature. However, there is some difficulty in comparing results due to the diversity of methods and expression of results. Fonseca et al. 12 reported similar values for the scavenging of ABTS (368.39 to 402.15 μmol TE/100 mL) and DPPH (186.73 to 211.39 μmol TE/100 mL) radicals in blueberry and honey vinegar. Regarding the FRAP potential, these authors found much higher values (1881.45 to 1884.5 μmol FeSO4 / 100 mL) in relation to the vinegar obtained in the present study.
The same organic acids present in the pineapple pulp and peel wines were found in the vinegar samples. Like what was observed in pulp wines, citric (pulp vinegar: 7.41 g/L and pulp+extract vinegar: 7.46 g/L) and oxalic (pulp vinegar: 9.04 g/L and pulp+extract vinegar: 9.09 g/L) acids were the predominant organic acids in pulp vinegar. Oxalic acid (peel vinegar: 4.75 g/L and peel+extract vinegar: 4.78) and citric acid (peel vinegar: 1.46 g/L and peel+extract vinegar: 1.48 g/L) predominated in pineapple peel vinegar. The enrichment of vinegar with red-Jambo extract did not promote statistically significant changes in the composition of organic acids.
Pineapple pulp vinegar showed luminosity (L*: 45.39) close to the spectrum observed in wine (L*: 42.73). On the other hand, the peel vinegar (with and without the addition of extract) showed slightly lower luminosity values (peel vinegar: 42.29 and peel+extract vinegar: 43.12) than those found in the peel wine samples (L*: 52.75). The reduction in luminosity values indicates that the acetic fermentation process carried out in wooden barrels contributes to a certain decrease in the perception of brightness and clarity of the peel vinegar samples. This phenomenon may be associated with the probable extraction of compounds from the wood of the acetification barrel, associated with the chemical composition of these vinegars, which present differences in the concentrations of phenolic compounds compared to pulp vinegar. The color properties of different types of vinegar can change depending upon the color of the raw material and the technology used in the production 49.
Regarding the a* coordinate (red index), the acetic fermentation contributed to a green tendency, which was more pronounced in the peel vinegar samples. Similarly, the acetification of wines also reduced the values of the b* coordinate (yellow index: tendency from yellow to blue), especially in the vinegar of peels. The acetification of the wines led to an intensification of the hº coordinate (hue angle), increasing the tendency to green (from yellow to green) in the peel vinegar samples (peel vinegar:135.01 and peel+extract vinegar: 122.64). A more pronounced increase in the values of the hº coordinate was observed in the pulp vinegar samples in relation to the pulp wines, with a tendency to intensify the blue hue (peel vinegar: 206.54, peel+extract vinegar: 205.63). The evaluation of the chromaticity index (C*) of the samples indicates that the acetification led to the reduction of the saturation index of the vinegar samples concerning the wines, this phenomenon being more pronounced in the vinegar of pineapple peels. The enrichment of pulp vinegar with red-Jambo extract did not promote statistically significant changes in the color parameters of the pulp vinegar samples (L* a* b*, hº, and C*). On the other hand, adding the extract to the peel vinegar led to changes in the instrumentally-detectable color parameters (L* b*, hº, and C*) (peel vinegar versus peel+extract vinegar). It is essential to highlight that the evaluation of the total color difference (DE) indicates that adding extract to the samples did not promote visually-detectable changes when comparing samples with or without added extract. Some works described in the scientific literature and based on the study described by Stokes, Fairchild, and Berns (1992) mention that color differences less than 2.15 are not perceptible to the human eye 39. The results of DE found in the present work are close to this threshold, especially in relation to vinegar obtained from pineapple peels (Table 4).
Antimicrobial potential.
Vinegar has been recognized as an antimicrobial substance for a long time, and several studies have shown vinegars of different origins can act as antimicrobial agents against different pathogens 49. Table 5 shows the results of the susceptibility of different bacterial strains (gram-negative and gram-positive) and yeasts to the pulp and peel vinegar produced in the present work.
Diffusion disk tests and evaluation of the minimum inhibitory concentration (MIC) showed that all vinegars, with or without added red-Jumbo extract, presented inhibition potential against the microorganisms studied.
Microorganism
|
|
Vinegar samples
|
|
pulp
|
peel
|
Pulp+extract
|
Peel+extract
|
alcohol
|
PA
|
Staphylococcus aureus ATCC 25923
|
ID (mm)
|
14.67a, c ±0.8
|
7.33a ±1.,8
|
23.03a, c ±2.7
|
13.01a, c ±1.3
|
17.6c ±1.5
|
42.0b ±0.5
|
MIC (µL/mL)
|
16.0
|
16.0
|
16.0
|
16.0
|
*
|
*
|
MBC (µL/mL)
|
50.5
|
50.5
|
50.5
|
16.0
|
*
|
*
|
Escherichia coli ATCC 25922
|
ID (mm)
|
14.02b ±0.7
|
8.33b ±0.9
|
17.67b ±3.1
|
15.67 b ±2.9
|
13.7 b±1.8
|
50.0a±0.5
|
MIC (µL/mL)
|
5.0
|
16.0
|
5.0
|
16.0
|
*
|
*
|
MBC (µL/mL)
|
#
|
#
|
#
|
#
|
*
|
*
|
Salmonella enterica typhimurium ATCC 19659
|
ID (mm)
|
13.03a, b ±2.0
|
5.66b ±0.8
|
14.02a, c±1.3
|
12.33b, c ±1.5
|
12.0b, c ±1.1
|
44.0a ±0.8
|
MIC (µL/mL)
|
16.0
|
16.0
|
16.0
|
16.0
|
*
|
*
|
MBC (µL/mL)
|
151.5
|
50.5
|
151.5
|
50.5
|
*
|
*
|
Bacilus subtilis ATCC 0028
|
ID (mm)
|
12.66b ±0.9
|
9.01b ±1.1
|
18.7a, b ±3.7
|
12.50b ±1.3
|
10.0b ±0.6
|
36.0a ±0.9
|
MIC (µL/mL)
|
5.0
|
16.0
|
5.0
|
16.0
|
*
|
*
|
MBC (µL/mL)
|
16.0
|
50.5
|
16.0
|
50.5
|
*
|
*
|
Candida albicans ATCC 118804
|
ID (mm)
|
20.0b ±4.4
|
16.0b ±2.8
|
20.0b ±2.2
|
12.0b ±1.7
|
12.0b ±1.5
|
50.0a ±1.0
|
MIC (µL/mL)
|
16.0
|
16.0
|
16.0
|
16.0
|
*
|
*
|
MFC (µL/mL)
|
50.5
|
151.5
|
50.5
|
151.5
|
*
|
*
|
Candida tropicalis ATCC13803
|
ID (mm)
|
20.0b ±2.2
|
20.0b ±1.5
|
22.0b ±1.1
|
22.0b ±2.0
|
22.0b ±1.7
|
50.0a ±0.9
|
MIC (µL/mL)
|
16.0
|
16.0
|
16.0
|
16.0
|
*
|
*
|
MFC (µL/mL)
|
50.5
|
50.5
|
50.5
|
50.5
|
*
|
*
|
Table 5. Antimicrobial and antifungal potential of vinegar samples.
AS: standard antimicrobial (Tetracycline for bacteria, and Fluconazole for fungi), ID: Diameter of the inhibition zones (inhibition halo), MIC: minimum inhibitory concentration, MBC: minimum bactericidal concentration, MFC: Minimum fungicide concentration, *: no evaluated, #: no inhibition.
Pineapple pulp vinegar promoted greater inhibition halos against both bacteria and yeasts when compared to peel vinegar. Diffusion disk tests also showed that the enrichment of vinegar with red-Jambo extract potentiated the antimicrobial activity. Another aspect observed is the pulp, and peel vinegars promoted inhibition diameters similar to those observed using commercial alcohol vinegar (4.0 g acetic acid/100 mL). The minimum concentrations necessary to inhibit the microorganisms studied ranged from 5 µL/mL to 16 µL/mL. This range was wider regarding the bactericidal concentration, ranging from 15.5 µL/mL to 151.5 µL/mL. Similarly, the concentrations required for yeast inhibition ranged from 16 µL/mL to 151.5 µL/mL. These values indicate that the sensitivity of the microorganisms studied against the vinegar samples was relatively variable. In agreement with the results obtained, Ozturk et al. 51 reported a high variability in a study with twenty samples of traditional vinegar produced in Turkey (homemade vinegar) in relation to the sensitivity of the bacteria studied.
Bacillus subtilis (G+) was the microorganism most sensitive to pure pulp vinegar and enriched with the extract, with inhibition at a 5.0 µL/mL concentration and cell death at a concentration of 16.0 µL/mL. Escherichia coli (G-) was the most resistant strain, being inhibited at a concentration of 5.0 µL/mL in pulp vinegar (with or without the added plant extract) and 16.0 µL/mL in peel vinegar (with or without plant extract), but showed resistance to the biocidal activity of the vinegar samples. Similarly, Ousaaid et al. 52 reported Escherichia coli as the microbial strain most resistant to antimicrobial activity (MIC: 3.125 μL/mL; MBC: 6.25 μL/mL) in apple cider vinegar.
In evaluating the antimicrobial capacity against the yeasts, we observed that the pineapple pulp vinegar showed more significant antimicrobial potential against the Candida albicans strain than the peel vinegar. Pulp vinegar was able to inhibit this yeast at a 16.0 µL/mL concentration and promote its death at a 50.0 µL/mL concentration. However, there was no observable antimicrobial potentiation of the red-Jumbo extract against the two Candida tropicalis yeast strains evaluated, thus maintaining its minimum inhibitory concentration and fungicidal concentration.
The enrichment of peel vinegar with red-Jambo extract potentiated the antimicrobial activity against the Gram-positive bacterium S. aureus, with MBC values ranging from 50.5 μL/mL (peel vinegar) to 16 μL/mL (peel+extract vinegar). On the other hand, adding the red-Jumbo extract to the vinegar did not potentiate the antimicrobial activity against the other microorganisms. The antimicrobial potential of vinegar is associated with the presence of organic acids, which have antimicrobial activity, especially acetic acid, which can cross the bacterial membrane and promote a reduction in intracellular pH, consequently causing the death of the microorganism 52. Weak organic acids cross the cell membrane in the undissociated form and dissociate according to intracellular pH, releasing a proton into the cytoplasm 51.