Journal of Food Health and Bioenvironmental Science (January - April 2019), 12(1): 21-27 21

Journal of Food Health and Bioenvironmental Science

Journal homepage : http://jfhb.dusit.ac.th/

Development of Fermented Banana Vinegar: Chemical Characterization

and Antioxidant Activity

Wilawan Boonsupa1* Witaya Pimda2 Kanyarat Sreeninta1 Chooreeporn Yodon1

Nawarat Samorthong1 Benjarut Bou-on1 & Phanumat Hemwiphat1

1
Department of Biology, Faculty of Science and Technology, Rajabhat Maha Sarakham University, Mahasarakham
44000, Thailand
2
Faculty of Science and Liberal Arts, Rajamangala University of Technology Isan, Nakhon Ratchasima
30000, Thailand

Articleinfo Abstract

Article history:
Received : 17 December 2018 This study was carried out to examine the chemical properties, antioxidant
Revised : 23 February 2019
activities and sensory scores of banana vinegar produced from four banana cultivars,
Accepted : 25 March 2019
namely ‘ Khai Pra Tabong’, ‘Nak’, ‘Hin’, and ‘Phama Heak Kuk’. The initial
Keywords: soluble solid contents in the banana juice were adjusted to 25 ºBrix before taking
Chemical Properties, to fermentation. Alcoholic fermentation was conducted using Saccharomyces
Antioxidant Activity, cerevisiae as the inoculant while Acetobacter pasteurianus was used for acetous
Fruit Vinegar, Banana, Sensory fermentation. As observed, all samples during the alcoholic fermentation the levels
Evaluation
of soluble solids decreased continuously and the level of alcohol were found to
increase at the end of fermentation process. Notably, the wine produced from
‘Phama Heak Kuk’ cultivar exhibited the highest level of alcohol (9.54 %) and
exhibited the highest levels of antioxidant activity (87.04 %). Similar results were
observed for all samples during the acetous fermentation, in which the level of
alcohol dropped continuously and the levels of acetic acid were noted to elevate at
the end of the fermentation process. The highest levels of acetic acid (3.49 %) was
detected in the vinegars produced from ‘Phama Heak Kuk’ cultivar while those
produced form ‘Khai Pra Tabong’ cultivar exhibited the highest levels of antioxidant
activity (80.59 %). Sensory evaluation based on the 9-point hedonic scales showed
that the vinegars produced from ‘Khai Pra Tabong’ cultivar showed the highest
overall acceptability with an average score of 8.13, equivalent to the hedonic scale
of 9, which indicated a high pleasant level of the vinegar preference of the consumers.

Introduction
widely used seasonings in the world (Jo et al., 2013). In
Due to its availability in several different varieties addition to being primarily used as food seasoning, vin-
in every country, vinegar represents one of the most egar plays an important role in the production of food

* Corresponding Author Development of Fermented Banana Vinegar:

e-mail: wilawanboonsupa@ymail.com Chemical Characterization and Antioxidant Activity
22 Journal of Food Health and Bioenvironmental Science (January - April 2019), 12(1): 21-27

products since it is applied in a wide variety of products, compare the chemical properties, antioxidant activities
including sauces, ketchups and mayonnaise (Ho et al., and sensory scores of the banana vinegars produced via
2017). Moreover, vinegar has long been used in the a two-stage fermentation process from four cultivars,
treatment of many common ailments with claims of namely Khai Pra Tabong’, ‘Nak’, ‘Hin’, and ‘Phama
anti-infective, antitumor, and hyperglycemic properties Heak Kuk’. In this context, chemical properties were
(Johnston & Gaas, 2006; Wongsudarak & Nunium, 2013). assessed in terms of alcohol contents, glucose and fruc-
The production of vinegar is in general low in costs due tose contents, and acetic acid contents. Antioxidant ac-
to the fact that inexpensive raw materials like by-products tivities were determined by DPPH radical assays and
from food processing, fruit waste, substandard fruit and total phenolic contents. Sensory evaluation was per-
agricultural surpluses are utilized (Solieri & Giudici, formed based on the 9-point hedonic scale in order to
2009). The beneficial effects of vinegar might be due to determine the consumers preference.
bioactive substances such as amino acids, organic acids
orphenolic compounds derived from its raw materials Materials and methods
(Budak et al., 2014; Ghosh et al., 2016). Moreover, the
bioactive compounds in vinegars can be produced and/ 1. Chemicals and reagents
or increased through the overall vinegar fermentation 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH)
process (Solieri & Giudici, 2009), where phenolic com- was purchased from Sigma–Aldrich (Steinheim, Germa-
pounds are transformed into new antioxidative molecules ny). Iron (II) sulfate, iron (III) chloride 6-hydrate and
(Shahidi et al., 2008). Additionally, the aroma and flavor gallic acid standard were supplied by Fluka (Buchs,
of vinegars impacting on consumer acceptance is influ- Switzerland). 2,4,6-tri-2-pyridyl-2-triazine (TPTZ) and
enced by the raw materials used, the compounds formed sodium acetate 3-hydrate were obtained from HiMedia
during the fermentation process, and the fermentation Laboratories (Mumbai, India). Folin-ciocalteau reagent
type used (Callejon et al., 2008; Liang et al., 2016; Mo- was from Merck (Darmstadt, Germany) and sodium
rales et al., 2002; Ubeda et al., 2012). Recently, the de- carbonate (anhydrous) from Univar (Downers Grove, IL,
mand for fruit vinegars has increased due to their repu- USA). All other chemicals and solvents were purchased
tation as health food products, which help to promote from local manufacturers. Deionized water was prepared
different kinds of beneficial effects to consumers, such by a Milli-Q Water Purification system (Millipore, MA,
as having antidiabetic effects and lowering cholesterol USA).
levels in blood by inhibiting the oxidation of low densi- 2. Raw materials and fermentation
ty lipoproteins (LDLs), among other benefits (Chen et Banana fruits of four cultivars, namely
al., 2017). Khai Pra Tabong’ (The short stem is curved, thick peel.
Musa spp. Comprising dessert bananas and plan- When ripe had yellow color, orange flesh and sweet
tains, were among the world leading fruit crops as source flavor), ‘Nak’ (The raw banana is bright red, the ripe
of energy in the diet of people living in humid tropical banana is an orange-red color), ‘Hin’(Yellow thick peel,
regions (Sudhanyaratana creamy white pulp with a sweet flavor), and ‘Phama Heak
et al., 2016). Banana has firm pulp when the fruit Kuk’(The raw banana is a dark green color, the ripe fruit
is not ripe and soft pulp during maturation. It is known is an orange-red color), was used for the production of
that dessert banana pulp and peel contains some second- banana vinegars via a two-stage (alcoholic and acetous)
ary metabolites in their composition, e.g. catecholamines, fermentation process. Banana fruits of each cultivar were
phenolics, and carotenoid compounds as well as pyri- crushed and mixed with water at a ratio of 1:1 to prepare
doxine. Many of banana’s volatile compounds such as banana juice. After adjustment of the pH to 4.5 using 5%
ester and alcohols play an important role in the aromat- acetic acid and sugar content up to 25º Brix.
ic properties of banana. (Pereira & Maraschin, 2015). 3. Banana vinegar production
According to Coelho et al. (2017), who studied the Amylase enzyme from Mc-Zyme S.P. Corpo-
chemical composition and antioxidant activity of banana ration was applied in order to degrade the starch still
vinegar produced from fruit concentrates, the results present in the banana puree for 24 hours at 30ºC. The
revealed the total acidity of banana vinegar (5.4 %) and banana juice was pasteurized for 30 min at 60ºC. Alco-
the antioxidant activity by Frap (3.7 mmol Fe2SO4/L) holic fermentation was conducted for 5 days at room
For this purpose, this study was carried out to temperature under static conditions in plastic vessels

Development of Fermented Banana Vinegar: Boonsupa et al.

Chemical Characterization and Antioxidant Activity
 Journal of Food Health and Bioenvironmental Science (January - April 2019), 12(1): 21-27 23

containing 2 L of the banana juice inoculated with wine prepared by dissolving 0.004 g of DPPH in 95% ethanol,
yeast, Saccharomyces cerevisiae, (Wine & Scientific followed by adjustment of the solutions to a final volume
Equipment Ltd., Part) at a ratio of 0.75% (v/v). Prepara- of 100 mL. DPPH radical scavenging capacity (RSC)
tion of yeast inoculum was carried out by mixing 5 g of was calculated using the equation %RSC = (AC – AS/AC)
yeast powder with 60 mL of warm water. At the end of × 100, where AC and AS denote the absorbance of control
the fermentation process, the obtained wine was sepa- and sample, respectively.
rated from the sediment by allowing it to settle in glass 6. Total phenolic content analysis
bottles, followed by pasteurization for 30 min at 60ºC Total phenolic contents of the banana vinegars
and clarification for 45 days at 10ºC. Prior to acetous were determined using Folin-Ciocalteu reagent as de-
fermentation, modified from the method of Coelho et al scribed by Singleton et al (1999). Briefly, 1 mL of each
(2017), the alcohol content of the obtained wine was sample was diluted with 9.5 mL of distilled water and
adjusted to 6 %. Acetous fermentation was performed was then mixed with 0.5 mL of Folin-Ciocalteu reagent
for 15 days under the aforementioned conditions in glass and 2 mL of 10% Na2CO3 solution. After 30-min incu-
vessels containing 135 mL of the banana wine inoculat- bation at room temperature, absorbance was measured
ed with Acetobacter pasteurianus TISTR 521 at a ratio at 765 nm using a Shimadzu UV-1700 spectrophotome-
of 10% (v/v). Sampling was performed at given time- ter (Shimadzu, Japan). Results were expressed as mg
points to collect the two-stage fermented banana vinegars gallic acid equivalents in 1 mL of sample (mg GAE/mL).
by allowing them to settle in microtube and stored at 4ºC 7. Sensory evaluation
before the analyses. 200 g of the banana vinegars were mixed with
4. Chemical analysis 150 g of honey and 150 g of water to make drinking
Analysis of alcohol, acetic acid, glucose and vinegars and the obtained drinking vinegars were sub-
fructose contents modified from the method of Aguiar et jected to the sensory evaluation based on the 9-point
al. (2005). The analysis was performed on a Shimadzu hedonic scale by using 30 untrained panelists for 5 at-
HPLC-RID system (Shimadzu, Japan) consisting of tributes (sweet, color, odor, taste and overall acceptance)
Shimadzu LC-20AD pumps and RID-10A refractive with the scale 9 representing like extremely, 5 represent-
index detector. The analytical column was Aminex HPX- ing neither like nor dislike and 1 representing dislike
87H column (300 mm × 7.8 mm i.d., 9 µm, Bio-Rad extremely.
Laboratories, Inc., USA) coupled to a cationic exchange 8. Statistical analysis
precolumn (Bio-Rad Laboratories, Inc., USA). H2SO4 A randomized block design, with three repli-
(5 mM) was used as the mobile phase. The injection cates and four samples per replicate, was used to compare
volume was 20 mL with a flow rate of 0.6 mL/min. The the chemical properties, antioxidant activities and sen-
column temperature was set at 45ºC. sory evaluation of the banana vinegars produced from
Total soluble solids values of the wine were four banana cultivars. The results are expressed as the
measured using an AllA France refractometer (AllA mean ± standard deviation (SD) and data were analyzed
France, France) calibrated with distilled water. The using one-way analysis of variance (ANOVA) with
values were expressed as oBrix. Duncan multiple range test (DMRT) to determine the
5. Antioxidant activity significance between samples. In all cases, p < 0.05 was
Antioxidant activities of the vinegars were considered significant.
evaluated by DPPH radical assay (Brand-Williams et al.,
1995) in which 2,2-diphenyl-1-picrylhydrazyl hydrate Results and discussion
(DPPH) radical was used as a stable radical. In brief, 1.5
mL of each sample was added to 1.5 mL of 0.1 mM DPPH 1. Chemical properties of the banana wines
radical solution prepared in ethanol, and the mixture was and vinegars
incubated for 20 min at room temperature in the dark. The banana wines produced from four banana
After incubation, absorbance was measured at 517 nm cultivars via a 5-day alcoholic fermentation process
using a Shimadzu UV-1700 spectrophotometer (Shimad- using Saccharomyces cerevisiae as an inoculant were
zu, Japan), and the DPPH radical scavenging activities analyzed for their chemical compositions, and the results
were expressed as the percentage of the DPPH radical are presented in Fig. 1A. It was observed that at the end
elimination effect of vitamin C. Control solutions were of the fermentation, high alcohol content was detected

Boonsupa et al. Development of Fermented Banana Vinegar:

Chemical Characterization and Antioxidant Activity
 24 Journal of Food Health and Bioenvironmental Science (January - April 2019), 12(1): 21-27

in all the banana wines, indicating that sugars in the fructose was completely depleted while glucose remained
banana juice were rapidly converted to alcohol. The at 0.046 g/100 mL. In Fig.1D, total soluble solid of 4
banana wine produced from ‘Phama Heak Kuk’ cultivar wine were adjust to 25 oBrix after fermenting in 5 days,
contained the highest alcohol content of 9.54 %, which the results showed that Nak wine (15.47 °Brix) had
was higher than that (9.27%) detected in the lychee wines greater TSS than Khai Pra Tabong (12 °Brix). From the
produced in an earlier study (Chen & Liu, 2016). As experiment, it was found that the alcohol content in Khai
given in Fig. 1B, Glucose was rapidly utilized during Pra Tabong wine had greater values than Nak wine
the production of the banana wine as observed for all because yeast had the ability to consume TSS in the
samples, with the most rapidly utilized glucose observed Khai Pra Tabong effectively. During the 15-day acetous
after 1 day of the fermentation in ‘Nak’ cultivar. Notably, fermentation process, Oxidative fermentation is a
glucose was completely depleted in all wine samples fermentation process caused by bacteria that requires
after 4 days of the fermentation. Fructose was likely oxygen to respirate at the cellular level (Pongdam, 2017).
utilized more slowly as compared to glucose (Fig. 1C). The banana vinegars produced from the four banana
The most rapidly utilized fructose were observed in the wines using A. pasteurianus were analyzed for their
banana wine produced from ‘Phama Heak Kuk’, Khai chemical compositions, and the results are given in Fig.
Pra Tabong and Hin cultivar which was decreased more 2. All the banana vinegars showed a significant decrease
than Hin in 3 days of the fermentation. The rapid in the alcohol content as it was converted to acetic acid
utilization of glucose and fructose and the consequent by acetic acid bacteria, which was consistent with the
increase in the levels of alcohol confirmed that the yeast increased acetic acid content. However, the alcohols
dominated the fermentation, which was supported by an were not completely depleted, at the end of acetous
earlier study (Taniasuri et al., 2016) which elucidated the fermentation the vinegar produced from ‘Nak’ cultivar
rapid utilization of glucose and fructose in the production contained the highest alcohol content of 0.81 % while
of durian wine, in which at the end of the fermentation that produced from ‘Khai Pra Tabong’ cultivar had the 10

Fig. 1 Physicochemical properties of banana wine during a 5-days fermentation process alcohol (A) glucose (B) fructose (C) and total soluble solid (D)

Fig. 1 Physicochemical properties of banana wine during a 5-days fermentation process alcohol (A) glucose (B) fructose (C) and total
soluble solid (D)
Development of Fermented Banana Vinegar: Boonsupa et al.
Chemical Characterization and Antioxidant Activity
 11

Journal of Food Health and Bioenvironmental Science (January - April 2019), 12(1): 21-27 25

phenolic contents detected in the banana vinegars

produced from different banana cultivars via a two-stage
fermentation process are given in Table 2. It was noted
that the banana wine derived from ‘Nak’ cultivar
contained the highest levels (229.30 ± 0.59 mg/L) of
total phenolics. Similar results were observed for the
banana wine produced from the same cultivar, in which
the vinegar measured at the end of acetous fermentation
exhibited the highest total phenolic content of 243.98 ±
3.35 mg/L, which was much greater than that detected
in the purple sweet potato makgeolli vinegar (24.73 ±
0.04 mg/L) (Chun et al., 2014). The antioxidant activity
and total phenolic content were decreased in vinegar,
the results were in agreement with an earlier study of
Fig. 2Fig.
Physicochemical properties
2 Physicochemical of banana
properties vinegar
of banana fromfrom
vinegar banana 4 cultivarsTowantakavanit et al. (2011) which demonstrated that
4 cultivars
banana
duringa a15-day
during 15-dayfermentation
fermentationprocess
process the drop in total phenol level could be due to fermentation
process condensation and polymerization reactions as
lowest alcohol content of 0.47 %. In an earlier study (Li well as the formation of oxidative products and
et al., 2014) which elucidated that the alcohol content in precipitations occur.
Article Informationerinaceus vinegar was 0% after 9 days of
the Hericium
Received 17 December 2018
acetic fermentation. Regarding the acetous fermentation, Table 1 Antioxidant activities of the four banana vinegars produced via a
Revised 23 February 2019 two-stage fermentation process
at the end
Accepted 25 Marchof 2019
a 15-day acetous fermentation process,
acetic acid content was found to range from 2.27% to
DPPH (% inhibition)
3.49%, etwith
Boonsupa al. the highest value of 3.49% observed in the Cultivars
banana vinegar produced from ‘Phama Heak Kuk’ Wine Vinegar

cultivar and the lowest of 2.27 % produced from ‘Nak’ Nak 52.48 ± 1.87 c
47.44 ± 5.03c
Phama Heak Kuk 87.04 ± 0.00a 68.04 ± 1.49b
cultivar which was much greater than that obtained in a
Hin 72. 08 ± 0.05b 69.30 ± 8.09b
previous study (Li et al., 2014), in which an acetic acid Khai Pra Tabong 86.94 ± 0.05a 80.59 ± 2.30a
content of 21.56 mg/mL was detected in the H. erinaceus
Remark: Values with different letters in the same column are significantly
vinegar after 9 days of acetous fermentation. The acetic different according to Duncan’s multiple range test (p < 0.05).
acid content in vinegar was not up to the standard of
fermented vinegar (least 4% acetic acid), this could be Table 2 Total phenolic contents of the four banana vinegars produced via a
caused by the same condition in fermentation had the two-stage fermentation process

problem. Ex. The ability of survival of acetic acid

Total phenolic content (mg/L)
bacteria or the deficiency of aeration in fermentation. Cultivars
2. Total phenolic contents and antioxidant Wine Vinegar

activities Nak 229.30 ± 0.59 a

243.98 ± 3.35a
The levels of antioxidant activities of the Phama Heak Kuk 126.28 ± 0.21 d
102.23 ± 0.69d
Hin 214.57 ± 0.23b 198.26 ± 1.61b
banana vinegars are presented in Table 1. The results Khai Pra Tabong 166.77 ± 0.47c 115.92 ± 9.40c
showed that the banana wine derived from ‘Phama Heak
Remark: Values with different letters in the same column are significantly
Kuk’ and ‘Khai Pra Tabong’ cultivar exhibited the different according to Duncan’s multiple range test (p < 0.05).
highest antioxidant activity of 87.04% and 86.94%, which
was greater than that produced from citrus fruit (36.8 ±
0.09%) (Chen et al., 2017). On the other hand, the 3. Sensory evaluation
vinegar produced from ‘Khai Pra Tabong’ cultivar was The levels of consumers’ preference based on
observed to exhibit the highest antioxidant activity of the 9-point hedonic scale of the vinegar drinks; a blend
80.59 ± 2.30%, which was much greater than that of the vinegars made from different banana cultivars and
detected in the purple sweet potato makgeolli vinegar honey, are depicted in Table 3. The results showed that
(67.63 ± 0.17%) (Chun et al., 2014). The levels of total significant (p < 0.05) differences in sweet odor sour and

Boonsupa et al. Development of Fermented Banana Vinegar:

Chemical Characterization and Antioxidant Activity
26 Journal of Food Health and Bioenvironmental Science (January - April 2019), 12(1): 21-27

Table 3 Sensory scores of the drinking vinegars blended from the four References
fermented banana vinegars

Overall Aguiar, A., Nascimenta, R.A.D.A., Ferretti, L.P., & Goncalves,

Curtivars Sweet Color Odor Sour
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Nak 7.17 ± 1.90ab 6.57 ± 1.68ns 5.97 ± 1.52ab 6.63 ± 1.59b 7.57 ± 1.55ab
ethanol in commercial vinegars. Brazilian Journal of
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Phama 6.63 ± 1.47b 6.77 ± 1.38ns 5.80 ± 1.69b 6.60 ± 1.38b 6.93 ± 1.31bc Brand-Williams, W., Cuvelier, M.E., & Berset, C. (1995). Use
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Hin 7.87 ± 0.97a 7.30 ± 1.34ns 6.87 ± 1.89a 7.63 ± 1.13a 8.13 ± 1.14a activity. LWT - Food Science and Technology, 28,
Khai Pra 6.37 ± 1.90b 6.50 ± 1.63ns 5.70 ± 1.74b 6.73 ± 1.91b 6.70 ± 1.66c 25-30.
Tabong Budak, N.H., Aykin, E., Seydim, A.C., Greene, A.K., &
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vinegar. Journal of Food Science 79, 757-764.
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Boonsupa et al. Development of Fermented Banana Vinegar:

Chemical Characterization and Antioxidant Activity