Process Biochemistry xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Fermentation-based biotransformation of biaoctive phenolics and volatile

compounds from cashew apple juice by select lactic acid bacteria
⁎
Ratchadaporn Kaprasoba, Orapin Kerdchoechuena, , Natta Laohakunjita, Dipayan Sarkarb,
Kalidas Shettyb
a
School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi,49 Teintalay 25 Rd., Thakam, Bangkhuntein, Bangkok 10150, Thailand
b
Department of Plant Sciences, North Dakota State University, Fargo, ND 58102, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Select lactic acid bacteria (LAB); Lactobacillus plantarum, L. casei and L. acidophilus were targeted for enhancing
Antioxidant activity bioactives and flavor volatiles of cashew apple juice (CAJ) that is an underutilized byproduct from cashew nut
Lactic acid bacteria processing in Tropical countries. Results indicated the vitamin C and phenolic metabolites such as condensed
Phenolics tannin can be increased at certain stages such as at 12 h over the 48 h fermentation period. Whereas antioxidant
Vitamin C
activity based on DPPH and ABTS radical scavenging activity generally decreased from initial unfermented stage
Fruit juice fermentation
Flavor compounds
range of (75%–95%) to consistently in the 50% range by 48 h of fermentation and this follows the decrease in
viable counts. The fermentation process increased the condensed tannin contents in CAJ whereas hydrolysable
tannins decreased. In this study the changes in flavor volatile types were also analyzed over the course of CAJ
fermentation. The results indicated that LAB changed the flavor profiles of fermented CAJ and overall the fruity
odor decreased, but the whiskey and acid odor increased. These results provide the foundation to further target
the functional benefits of LAB-induced fermented CAJ for further human, animal, and plant health applications.

1. Introduction
Cashew (Anacardium occidentale) is very resilient and hardy tropical
plant with origins in tropical Brazil whose modified fruit called cashew
apple is a rich source of bioactive phytochemicals with potential for
diverse biological applications. Cashew belongs to genus Anacardium of
the family Anacardiaceae. Cashew apple is commonly an agricultural
by-product from cashew nut production and close to 90% of this fruit is
lost or underutilized [1]. Cashew apple can be consumed fresh or
processed into value-added juice, jams, jelly, syrups, various beverages
and other food products. Further it can be used as substrate for
fermentation or other biotransformations including enzyme-based
synthesis. Cashew apple is an excellent source of bioactive compounds,
minerals, vitamins and unique flavors [1,2]. Cashew apple juice (CAJ)
contains ascorbic acid that is 4X to 10X higher than orange and
pineapple juice [3]. Additionally it contains phenolic compounds such
as leucodelphinidin, anarcardic acid, cardol, tannins, and terpenoid
such as carotenoids that is an antioxidant with potential health benefits
[4,5]. Ascorbic acid and total phenolic compounds in particular are the
two classes of bioactives that can be targeted for enhancing functional
health benefits by novel microbial biotransformation. It is known that
the strong radical scavenging activity of proanthocyanidins (condensed
tannins) are also responsible for the typical astringent and acid taste of
some fruits [6] and could also have other functional health benefits.
Combining these tannin astringency cashew apples can be processed
into a beverage due to its fleshy pulp, soft peel, lack of seeds and high
sugar concentration. Further it has strong exotic flavor [7,8] from
volatile constituents such as, esters (38–80% of total volatiles), alcohols
(30% of total volatiles), acids (15% of total volatiles), aldehydes,
ketones, and ethers [2]. These flavor bioactives can be targeted for
further value addition through microbial bioatransformation, including
both aerobic growth and fermentation using food grade bacteria [9].
Fermentation is a well-established traditional technology across the
globe used to enhance the shelf-life, nutritional and organoleptic
qualities and remove undesirable compounds of primary food sub-
strates [10]. Further fermentation processes have now been targeted
and developed to increase the synthesis of biologically active microbial
metabolites for diverse functional benefits using suitable substrates or
appropriate nutrients [1] and with the right microbial target. Among
these targets lactic acid bacteria (LAB)-based fermentation clearly
contributes to improvement of nutritional value and digestibility of
various foods, decreasing lactose intolerance, controlling potential
infections and additionally has relevance to add further functional
bioactives [11]. Functional benefits of lactic acid bacteria due to their

⁎
Corresponding author.
E-mail address: orapin.ker@kmutt.ac.th (O. Kerdchoechuen).

http://dx.doi.org/10.1016/j.procbio.2017.05.019
Received 9 February 2017; Received in revised form 14 April 2017; Accepted 16 May 2017
1359-5113/ © 2017 Elsevier Ltd. All rights reserved.

Please cite this article as: Kaprasob, R., Process Biochemistry (2017), http://dx.doi.org/10.1016/j.procbio.2017.05.019
R. Kaprasob et al.
complex enzymes could also lead to the novel modification of flavor
attributes [12,13].
Therefore the genus Lactobacillus is an excellent target for value
addition of CAJ as it can grow in the pH range from mild acidic to
neutral values and temperatures from 2 to 53 °C, with optimum
temperature range of 30–40 °C and optimum pH range of 5.5–6.2
[14]. Among various species targets, Lactobacillus plantarum is the
species most frequently used to ferment food products of plant origin
[15]. In terms of antioxidant enrichment lactic acid bacteria with β-
glucosidase activity (including Lactobacillus acidophilus, Lactobacillus
casei, Lactobacillus plantarum, Lactobacillus fermentum, and Bifidobacter-
ium longum) have potential based on the ability to increase isoflavone
aglycone [16]. Lactobacillus acidophilus and Lactobacillus casei could
produce active enzymes such as amylase, lactate dehydrogenases,
peptidases and proteinase that can transform primary food matrix into
functional moieties. Specifically L. plantarum is known to produce
active enzymes such as amylase, β-glucosidase, decarboxylase, lactate
dehydrogenases, peptidase, phenolic acid decarboxylases, phenol re-
ductase, proteinase and tannase [17] and these have relevance for
diverse food substrate biotransformations. Based on this rationale
several fruit juice substrates such as pomegranate juice [18], blueberry,
strawberry, noni juice [19], and cashew apple juice [5] were fermented
using lactic acid bacteria.
Since cashew apple juice possess excellent bioactive potential to
produce a healthy functional food with excellent flavor attributes
[5,12,13,20] and further cashew tree can be grown in dry arid regions
of Thailand we have targeted cashew apple juice as a substrate for LAB-
based bioactive enrichment for diverse biological applications from
food-derived human health to animal and plant health applications.
Further we have targeted the analysis of volatile aroma constituent
through this targeted LAB-based biotransformation of CAJ. Based on
the above overall rationale the focused objective of this present study
was to evaluate the primary functional transformations of major
bioactives such as vitamin C, phenolics and aroma compounds and
derived overall antioxidant potential of fermented CAJ by select and
targeted Lactobacillus spp. (L. acidophilus, L. casei, and L. plantarum).
This initial study is essential as the key foundation to further advance
specific functional analysis and benefits targeted for human, animal and
crop food applications of LAB-biotransformed CAJ.
2. Materials and Methods
2.1. Materials and cashew apple juice (CAJ)
Other than mentioned specifically, all chemicals were purchased as
analytical grade from Sigma-Aldrich Chemical Co., Ltd. (Singapore).
Fresh cashew apples from the Heritage Grower Corporation Ltd.,
located in the South of Thailand were used in this study. The cashews
were harvest in the 80% mature stage at the crop season
(December–March 2014). Cashew apples were cleaned with filtered
water and then chopped into small pieces. The edible pieces were
homogenized and blended using a Waring blender for 5 min and stored
in freezer. After that stored CAJ were pasteurized for 15 min at 70 °C
and 20 g of CAJ was used for each fermentation study. The initial
cashew apple substrate contained 86.12 g total carbohydrate, 1.41 g of
protein, 2.07 g of fat and initial pH of 4.0.
2.2. Lactic acid bacteria strains and inoculum preparation
Lactobacillus acidophilus TISTR 1338, Lactobacillus casei TISTR 390,
and Lactobacillus plantarum TISTR 543 were obtained from the Thailand
Institute of Scientific and Technological Research (TISTR), Thailand.
The culture were activated at 30 °C for 24 h in 125 mL Erlenmeyer
flasks containing 25 mL of Difco lactobacilli deMan, Rogosa and Sharpe
(MRS) broth (BD, Sparks, MD, USA). Cell cultivation was carried out
statically in an incubator until the cell density reached 1.000 which
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Process Biochemistry xxx (xxxx) xxx–xxx
corresponds to 9.18 Log colony forming units (CFU) per milliliter, using
the McFarland scale [7]. Cell density was spectrophotometrically
determined at 600 nm and this culture was used as inoculum in the
fermentation process.
2.3. Cashew apple juice (CAJ) fermentation
Fermentation experiments were conducted in sealed Erlenmeyer
flasks (covered with aluminum foil), each containing 20 g of pasteur-
ized CAJ without supplementary nutrients or water. Two milliliters of
inoculum, containing 9.18 Log CFU/mL of various LAB strains, were
added to 125 mL Erlenmeyer’s flasks containing pasteurized CAJ. The
initial cell count in the juice was 8.18 Log CFU/mL. The fermentation
process was performed at 30 °C for 48 h. Samples were taken at 0, 12,
24, and 48 h for viable cell count and analysis of targeted phytochem-
icals, bioactivity and volatile compounds as described below.
2.3.1. Viable cell counts
Cell viable counts of fermented CAJ were obtained by serial dilution
with 0.7% NaCl solution until 10−6 dilutions. Aliquots of 0.1 mL of
dilution were plated, in triplicate in plates containing MRS Agar (spread
plate method). The plates were incubated for 36–48 h at 37 °C, and
plates containing 20–350 colonies were measured or recorded as Log
CFU/mL.
2.3.2. pH and total titratable acidity (%TTA)
The pH of fermented CAJ was measured during the fermentation
periods using a pH meter (Mettler Toledo, Fisher Scientific, UK)
calibrated with buffer solutions at pH 4.0 and 7.0 (Laboratory
Chemical, New Zealand). Total titratable acidity, expressed as percen-
tage of lactic acid, was determined by titration with 0.1 M NaOH using
the method of [21].
2.3.3. Total sugar and reducing sugar
Reducing sugar contents were analyzed as glucose equivalents by
Nelson-Somogyi method [22]. Total sugar contents were analyzed as
glucose equivalents by the phenol sulfuric acid method of Dubois et al.
[23]. Absorbance was determined on UV-Spectrophotometer (Biomate
3, Genesys 10, Thermo Electron Corporation, Germany).
2.3.4. Vitamin C contents
Vitamin C contents were determined following the AOAC method
for vitamin C (ascorbic acid) in vitamin preparations and juice [21]. A
total of 200 μL sample was added to 50 mL Erlenmeyer’s flasks and
mixed with 1.8 mL distilled water and 5 mL metaphosphoric acid-acetic
acid solution. The mixture (7 mL) was titrated with 2,6-dichloroindo-
phenol standard solution, its titration volume was recorded and used to
quantify vitamin C content of the samples (mg of ascorbic acid
equivalent (AAE)/L of sample). The indophenol solution was standar-
dized by titrating 1 mg/mL ascorbic acid standard solution (2 mL
ascorbic acid standard solution with 5 mL metaphosphoric acid-acetic
acid solution) and sample blanks.
2.3.5. Total phenolic contents
Total phenolics of samples were determined by the Folin–Ciocalteu
colorimetric method according to Huang et al. [24]. Briefly, sample
(10 μL) was added to the test tube and mixed with 90 μL distilled water
and 2 mL of 2% (w/v) Na2CO3 solution. After incubation for 3 min,
100 μL of Folin-Ciocalteu reagent was added. After standing for 30 min
at room temperature, the absorbance was measured at 750 nm, using
distilled water as a blank. Results were expressed as mg gallic acid
equivalents (GAE)/L of sample by using gallic acid standard curve
calibration (0.025–04 mg/mL).
2.3.6. Condensed tannin contents
Total tannins or condensed tannins were measured using the
R. Kaprasob et al.
modified vanillin assay described by Sun et al. [25]. Three milliliters of
4% methanol vanillin solution and 1.5 mL concentrated HCl were
added to 100 μL of sample. Reaction mixture was kept for 15 min,
and the absorbance was measured at 500 nm against methanol as a
blank. The amount of total condensed tannins was expressed as mg
(+)-catechin equivalent (CE) per L of sample through the calibration
curve with catechin (0.025–04 mg/mL). Triplicate measurements were
taken for all samples.
2.3.7. Hydrolysable tannin content
Hydrolysable tannins were determined according to the method of
Saad et al. [26]. A volume of 2.5 mL KIO3 aqueous solution (2.5% w/v)
was added to the test tube and heated for 7 min at 30 °C, and then
0.5 mL of sample was added. After an additional 2 min of tempering at
30 °C, the absorbance was measured at 550 nm, using distilled water as
a blank. Results were expressed as mg tannic acid equivalent (TAE) per
L of sample through the calibration curve using tannic acid solution
(5000 mg/mL) prepared by solubilization of 0.125 g of tannic acid in
25 mL of methanol (80%). Triplicate measurements were taken for all
samples.
2.3.8. Determination of antioxidant activity
2.3.8.1. ABTS radical cation decolorization (ABTS%+) scavenging
antioxidant activity assay. The ABTS%+ radical scavenging effect of
fermented samples using ABTS method was followed based on the
method of Shan et al. [27] with slight modifications. ABTS%+ radical
cation was generated by reacting 7 mM ABTS solution and 2.45 mM
potassium persulfate in the dark for 16 h at 20 °C. Before analysis, the
ABTS•+ solution was diluted with ethanol to obtain an absorbance of
0.700 ± 0.020 (starting absorbance) at 734 nm. Then, the absorbance
of diluted ABTS%+ solution was recorded. An aliquot of the sample
(10 μL) was added to the test tube and mixed with 70 μL distilled water.
After adding 4 mL diluted ABTS•+ solutions, which was prepared daily
to the sample, the absorbance was measured immediately at 734 nm
after exactly 6 min of initial mixing. Control was carried out with
distilled water instead of sample. Percentage of ABTS radical
scavenging antioxidant activity (%) was expressed as percentage
disappearance of ABTS•+.
2.3.8.2. DPPH radical scavenging antioxidant activity assay. The DPPH
radical scavenging effects of fermented samples were measured
according to the method of Bersuder et al. [28] with a slight
modification. A 10 μL aliquot of the sample was added to the test
tube and mixed with 30 μL of distilled water. Then, 2 mL of 0.1 mM
DPPH% dissolved in 95% ethanol was added to the sample. The mixture
was shaken and left for 30 min in the dark at room temperature.
Absorbance of the resulting solution was recorded at 517 nm. The
results were expressed as the percentage of DPPH% scavenging
antioxidant activity (%) [28].
2.3.9. Determination of volatile compounds
Analysis of volatile compounds in fermented CAJ was performed on
a headspace solid-phase microextraction (HS-SPME) gas chromatogha-
phy mass spectrometry (GC–MS) (Model 7890A MS, Agilent
Technologies, Santa Clara, CA, USA) operating with an ionization
voltage of 70 eV. The headspace sampling was carried out using a
Combi-Pal auto sampler (CTC Analytics AG, Switzerland). Each sample
(3 mL) was placed in a glass vial with a capacity of 20 mL and heated at
40 °C for 1 min in a GC–MS heating block for headspace analysis.
Volatile compounds were absorbed onto a SPME fibre (50/30 μm DVB/
Carboxen™; Supelco, Bellefonte, PA) for 7 min. After equilibrium, the
SPME fibre was desorbed into the injector port at 240 °C for 13 min,
and the injector was operated in splitless mode. Helium was used as the
carrier gas at 1.0 mL/min. Separation of volatiles was attained through
a HP-5 capillary column (30 m × 0.25 mm, 0.25 μm film thickness;
J & W Scientific Inc., Folsom, CA, USA). Temperature programming was
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Process Biochemistry xxx (xxxx) xxx–xxx
set at 80 °C initially followed by an increase to 230 °C at 5 °C/min, and
holding this temperature for 5 min, giving a total analysis time of
approximately 35 min. Identification of volatile compounds was based
on comparison of their retention time and mass spectrum with data in
the Wiley 275 and NIST libraries at a percentage of quality match over
85%, and the data was compared to previously published literatures.
Retention index (RI) values of samples were obtained from the
comparison with alkane standards (C8eC20). Results were reported as
relative abundance expressed as (compound peakarea/total compounds
peakarea) × 100.
2.4. Statistical analysis
All experiments were carried out in triplicate, and each sample was
analyzed in duplicate. The results are expressed as mean ± S.D.
(standard deviation). The SAS Statistical Analysis System (SAS
Institute, USA) was used to analyze the experimental data. Significant
differences (p < 0.05) between means were separated by Duncan’s
Multiple Range Test (DMRT).
3. Results and discussion
3.1. Viable cell counts
Cell viability changes in CAJ during fermentation at 37 °C for 48 h
with L. acidophilus, L. casei and L. plantarum are shown in Fig. 1a. All of
the lactic acid bacteria strains showed different growth characteristics
but growth declined significantly within 24 h. However no significant
decrease in LAB growth was observed after 24 h with L. casei and L.
plantarum. Population of L. acidophilus in CAJ decreased within 12 h
(from initial cell counts of 8.18 Log CFU/mL) lesser than L. casei and L.
plantarum, reaching 7.58 log CFU/mL after 12 h, however after 48 h the
viable cell counts of L. acidophilus was lower than other two LAB. No
significant decrease in cell viability of L. plantarum was observed
between 12 h up to 24 h of fermentation and reached 6.56 log CFU/
mL by 48 h. Meanwhile, cell viability of L. casei decreased overall and
was stable between 24 h and 48 h and reached 6.15 log CFU/mL by
48 h. For L. casei and L. plantarum the cell viability did not further
decline between 24 h and 48 h compared to the decline that was
observed between 12 h and 24 h. The decline in the viable cell counts
might be due to pH differences between pretreatment (MRS broth, pH
5.4) and fermentation medium, where the initial pH of cashew apple
juice was 4.0. The initial pH and nutrient differences potentially
induced stress on the microorganisms and thereby decreased microbial
growth at the early stage of fermentation (lag phase) [18]. At the end of
fermentation process, population of lactic acid bacteria in CAJ
remained at levels exceeding 6.00 log CFU/mL except in case of L.
acidophilus that was less than 6 log CFU/mL. Similar to the finding by
[13], where L. plantarum and L. acidophilus could survive in the high
acidity and low pH of fermented cereal. On the contrary L. casei was
unable to survive at the low pH and high acidity conditions in
fermented cabbage juice [29]. In another previous study, it was found
that L. acidophilus, L. casei, L. plantarum, and L. delbrueckii were capable
of rapid utilization of tomato juice for cell synthesis without nutrient
supplementation. The maximum growth was obtained after 48 h
fermentation at 30 °C. In addition, the primary metabolites formed
during the first fermentation, which were associated with growth and
the secondary metabolites were suggested to be formed in the declining
phase [13]. Overall cell viability is the key factor to design functional
food products [14] and our results indicated that the CAJ is able to
sustain a viable level of LAB even after 48 h fermentation. However
further studies are required to optimize viable LAB level to develop
functional food products with health benefits from LAB fermented
cashew apple juice.
R. Kaprasob et al.
Fig. 1. Change in population of Lactobacilli (a), pH value (—) and total titratable acidity (%TTA) (——) (b), reducing sugar (c) and total sugar (d) in fermented cashew apple juice (CAJ)
at 0, 12, 24, and 48 h of fermentation at 37 °C. Data is expressed as the average of three determinations ± SD.
3.2. pH and total titratable acidity (%TTA)
Changes in pH and total titratable acidity (TTA) in CAJ fermented
with L. acidophilus, L. casei and L. plantarum at 37 °C for 48 h are shown
in Fig. 1b. The pH slightly decreased for the first 12 h of fermentation
from initial pH of 4.0 and then sharply decreased during the declining
viability phase of lactic acid bacteria until 48 h. There was slightly
decreased pH in both CAJ fermented with L. casei and L. plantarum,
reaching 3.86–3.93 after 24 h from initial 4.0. Meanwhile, pH of the
CAJ fermented with L. acidophilus decreased rapidly from 4.00 to 3.90
after 12 h fermentation (p < 0.05). Thereafter, the pH slightly de-
creased until 48 h of fermentation. At the end of fermentation, CAJ
fermented with L. acidophilus showed the highest decrease of pH (3.70)
with significantly lower values (p < 0.05) than the pH in CAJ
fermented with L. casei (3.77) and L. plantarum (3.85). The decrease
in pH is generally associated with organic acids produced by lactic acid
bacteria as part of energy metabolism and growth (Raimbault & Tewe
[36]). These findings are consistent with research reported previously
for probiotic enriched roselle juice with L. plantarum and L. casei [30],
fruit juices during fermentation with L. plantarum [31], probiotic
enriched litchi juice with L. casei [32], and fermented drinks of oat,
barley, and malt substrates inoculated with L. plantarum and L.
acidophilus [13]. The most obvious reason was the secretion of organic
acids such as citric, acetic or lactic acids, causing decrease in pH.
According to the pH changes, the profile of total titratable acidity
slightly increased throughout fermentation period. A rapid production
of total titratable acidity at initial stage of 12 h fermentation was found
in CAJ fermented with L. acidophilus. Overall L. acidophilus produced
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Process Biochemistry xxx (xxxx) xxx–xxx
significantly more total titratable acidity than that of L. casei and L.
plantarum. At the end of fermentation, %TTA of CAJ fermented with L.
acidophilus increased from 0.66% to 1.36%, whereas%TTA of CAJ
fermented with L. casei and L. plantarum increased from 0.64% to 1.28%
and 0.65% to 1.24%, respectively (Fig. 1b). The greater amounts of
%TTA of CAJ fermented with L. acidophilus is potentially due to its
homofermentative nature. This group of lactic acid bacteria efficiently
metabolizes carbohydrates generating ATP which is subsequently used
for the biosynthesis of lactic acid and other end-products. Meanwhile, L.
plantarum and L. casei are facultatively heterofermentative, producing
lactic acid, acetic acid, CO2 and ethanol as main end-products [33].
Low pH is an important factor for controlling pathogenic and spoilage
bacteria [14]. The low pH and high acidity were in agreement with the
findings by Ibanoglu et al. [34] for tarhana fermentation and by
Salmerón et al. [13] for cereal fermentation. Sucrose hydrolysis to
lactic acid production at low pH values is likely the overriding
mechanism [14,35].
3.3. Total sugar and reducing sugar
Sugar content of the cashew apple has usually been assessed in
terms of total sugar (TS) or reducing sugar (RS). Changes in TS and RS
contents of fermented CAJ are shown in Fig. 1c and 1d. In all fermented
CAJ, the values of TS and RS decreased through fermentation
(p ≥ 0.05). The decrease of TS of CAJ fermented with L. plantarum
was greater than those of L. acidophilus and L. casei. Meanwhile, the
decrease of RS of CAJ fermented with L. acidophilus and L. casei was
greater than those of L. plantarum. Results indicated that CAJ fermented
R. Kaprasob et al.
Fig. 2. Ascorbic acid (a), total phenolic (b), condensed tannin (c), and hydrolysable tannin (d) content change in fermented cashew apple juice (CAJ) at 0, 12, 24, and 48 h of
fermentation at 37 °C. Data is expressed as the average of three determinations ± SD. Data that does not share the same capital case letter on top of the vertical bar are significantly
different at 5% according to Duncan’s multiple range test.
with L. plantarum consumed sugar at a much faster rate than of L.
acidophilus and L. casei but overall was not different at the end of the
fermentation. The decrease in sugar concentrations during fermentation
was largely due to not only bioconversion into lactic acid, but also the
utilization for growth and metabolism of lactic acid bacteria [36,37].
During fermentation, homo-fermentative lactic acid bacteria could
utilize glucose and sucrose via the Embden-Meyerhof pathway, yielding
lactic acid. Whereas, hetero-fermentative lactic acid bacteria could
utilize glucose and sucrose via the pentose phosphate pathway (PPP),
yielding lactic-acid, ethanol, and CO2, potentially resulting in lower pH
and high acidity [38]. Overall the results are in agreement with
Salmerón et al. [13] who stated that reducing sugar decreased during
fermentation of non-dairy substrates with L. plantarum.
3.4. The impact of fermentation on antioxidant compounds (Ascorbic acid,
total phenolic compound, condensed tannin and hydrolysable tannin)
The major contribution to the antioxidant capacity of fresh fruits
and vegetables is related to their content of vitamin C and phenolic
metabolites (total phenolic compound, condensed tannin and hydro-
lysable tannin) (Fig. 2) [6,38]. Phenolic metabolites are known to have
many nutritional and human health benefits, especially acting as strong
antioxidants [38]. Phenolic acids are associated with color, sensory
qualities, nutritional and antioxidant properties of foods [39,40].
Moreover, tannins (proanthocyanidin and hydrolysable) are the major
phenolics of the cashew apple [41]. Therefore, it is important to
consider the effect of fermentation with lactic acid bacteria on the
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Process Biochemistry xxx (xxxx) xxx–xxx
antioxidant compounds of CAJ. Initially, the CAJ (0 h) contained high
ascorbic acid contents, reaching 2471.40–2474.88 mg AAE/L (Fig. 2a)
and the total phenolic contents of CAJ (0 h) was in range of
122.99–124.92 mg GAE/L (Fig. 2b). The condensed and hydrolysable
tannin of CAJ (0 h) was in the range of 3.06–3.25 mg CE/L and
25.09–25.82 mg TAE/L, respectively (Fig. 2c and d). Fermentation
resulted in a positive effect on vitamin C and phenolic metabolites
contents (total phenolic compounds and condensed tannin) during
0–24 h fermentation, and then a decreasing trend that was not
significantly different (p ≥ 0.05), while fermentation resulted in de-
crease in hydrolysable tannin that was significant (p < 0.05). After
12 h of fermentation, a 11% increase in ascorbic acid was observed in
CAJ fermented with L. casei and L. plantarum and only 7% increase in
ascorbic acid was found in CAJ fermented with L. acidophilus (Fig. 2a),
which might be due to the fact that ascorbic acid could be additionally
synthesized by microorganisms [42]. L. acidophilus and L. casei showed
an 8% loss of ascorbic acid at the end of 48 h of fermentation, whereas
L. plantarum retained the ascorbic acid compared with the beginning of
the fermentation. These results were in agreement with Rakin et al.
[37], who reported an increase in ascorbic acid during fermentation of
beetroot juice with lactic acid bacteria. Zheng et al. [32] also reported
an increase in ascorbic acid of litchi juice when fermented for 18 h by L.
casei. In addition, the decrease in ascorbic acid during fermentation was
potentially due to degradation because of residual oxygen and ascorbic
acid oxidase and when the oxygen was totally depleted and there was
switch to low micro-aerobic or anaerobic phase [32,42].
Total phenolic content of CAJ fermented with L. acidophilus, L. casei,
R. Kaprasob et al. Process Biochemistry xxx (xxxx) xxx–xxx

and L. plantarum did not significantly increase and ranged from 118.43
to 128.20 mg GAE/L (p ≥ 0.05). Results showed that both homo-
fermentative and heterofermentative lactic acid bacteria maintained
total phenolics during fermentation. The L. plantarum in CAJ at 24 h
fermentation had the highest total phenolic content (128.20 mg GAE/
L). The result was in contrast with Filannino et al. [43] who reported
that the polyphenolic compounds of fermented pomegranate juice with
L. plantarum decreased during fermentation. Several studies have
reported that the levels of many compounds, such as beta-carotene,
polyphenols and flavonoids, had increased through lactic acid bacteria
fermentation [39,44]. Phenolics of cashew apple juice were gallic acid,
caffeic acid, and flavonoids which might combine or bind with sugar or
amino acid [45] and therefore were potentially more stable. Moreover,
cashew apple phenolics are commonly present in form of complex
structures and bound such as flavonoids and tannins (hydrolysable
tannins and condensed tannins) where degradation to simple soluble
mono-phenolics could be a possible detoxification mechanism for
bacteria and yeast [46]. During fermentation, total soluble phenolics
present in the fermented fruit substrate might also be mobilized and
degraded to small molecules, resulting in lower phenolic contents.
Lactic acid bacteria has a range of enzymes which might help in
degrading certain phenolic compounds [47].
Tannin content of CAJ fermented with L. acidophilus, L. casei, and L.
plantarum has usually been assessed in terms of hydrolysable tannin and
condensed tannin [48]. Condensed tannin contents in CAJ fermented
with lactic acid bacteria increased and ranged from 3.06 to 3.68 mg CE/
L, however the increased concentration is not statistically significant at
p ≥ 0.05. After 48 h of fermentation, L. plantarum associated CAJ had
the highest condensed tannin content (3.68 mg CE/L). An increase in
condensed tannin was approximately 6.69%, 10.22% and 16.85%
increment that were recorded for L. casei, L. acidophilus, and L.
plantarum after 48 h of fermentation, respectively. Although fermenta-
tion process of CAJ with lactic acid bacteria increased the condensed
tannin contents, hydrolysable tannins in fermented CAJ tended to
decrease (p < 0.05). Hydrolysable tannin contents in fermented CAJ Fig. 3. DPPH (a) and ABTS (b) free radical scavenging of fermented cashew apple juice
(CAJ) at 0, 12, 24, and 48 h of fermentation at 37 °C. Data is expressed as the average of
decreased throughout the fermentation process (p < 0.05) and it was
three determinations ± SD. Hundred percent inhibition (free radical scavenging) was
lowest when fermented with L. plantarum for 48 h (21.96 mg TAE/L) considered as change in absorbance from 0.700 to 0 at 734 nm for ABTS and 2.0–0 at
(Fig. 2d). Results indicated that hydrolysable tannins decreased while 517 nm for DPPH. Data that does not share the same capital case letter on top of the
condensed tannin increased, which along with the concentration of vertical bar are significantly different at 5% according to Duncan’s multiple range test.
other soluble phenolic acids may have contributed to the constant
concentration of total phenolic content in fermented cashew apple that CAJ has a good potential for free radical inhibitory activity and
juice. A significant decrease in hydrolysable tannin was approximately therefore reflecting good antioxidant activity potential. After 12 h of
4.78%, 9.45% and 14.15% reduction that were recorded for L. casei, L. fermentation, an additional increase in DPPH radical scavenging
acidophilus, and L. plantarum after 48 h of fermentation, respectively. activity of CAJ fermented with L. casei, L. plantarum, and L. acidophilus
The lowest hydrolysable tannin found in CAJ fermented with L. of approximately 9.13%, 6.53% and 4.14%, respectively was observed.
plantarum might be due to the enzyme tannase after 12 h growth on In the case of L. casei and L. plantarum, after 48 h of fermentation there
CAJ containing hydrolysable tannins. Tannase could catalyze the was approximately 4% reduction in DPPH radical scavenging activity,
hydrolysis reaction of ester bonds in hydrolysable tannins and gallic whereas L. acidophilus showed a 10.22% decrease in DPPH radical
acid ester resulting in a decrease in hydrolysable tannins [15]. It is scavenging activity. The ABTS radical scavenging activities of CAJ
known that high astringent taste of CAJ is potentially due to tannins fermented with L. plantarum, L. casei, and L. acidophilus was approxi-
and therefore decreasing hydrolysable tannin contents could contribute mately 9.15%, 14.80%, and 16.06% reduction, respectively, after 48 h
to reduced astringent taste in fermented CAJ. This will be confirmed in of fermentation (Fig. 3b). In the case of L. plantarum, the ABTS
future studies in the product development phase. scavenging value was higher than in the case of L. casei, and L.
acidophilus after 24 h of fermentation. Overall the three Lactobacillus
3.5. The impact of fermentation on antioxidant activity spp. had similar trends during fermentation period. However, no
significant difference (p ≥ 0.05) was observed between the DPPH and
Two systems were chosen to evaluate the antioxidant activity (using ABTS radical scavenging activity in CAJ fermented with three Lactoba-
ABTS•+ and DPPH% radical scavenging methods) of CAJ during cillus spp.
fermentation where ABTS•+ is based on hydrogen atom transfer A decrease in antioxidant activity can be associated with oxidation
mechanism (HAT), whereas DPPH% is based on electron transfer or degradation of antioxidant compounds [50]. The decrease of ABTS
mechanism (ET) [49]. These are widely used methods to evaluate the radical scavenging activity was in agreement with similar decrease in
antioxidant activity in a short time interval [8]. DPPH radical scavenging activity. The differences in the reduction
Fig. 3a and b indicated the changes in antioxidant activity (DPPH trends might be due to the fact that antioxidant activity of the food
and ABTS values) of fermented CAJ produced under LAB (L. acidophilus, depends on the synergistic and redox interactions among the different
L. casei, and L. plantarum) fermentation. All samples at 0 h had 75–77% compounds present in fruit. However, values obtained by the ABTS
DPPH and 97–99% ABTS radical scavenging activity which indicated

6
R. Kaprasob et al. Process Biochemistry xxx (xxxx) xxx–xxx

Table 1
Volatile compounds identified in cashew apple juice (CAJ) fermented with Lactobacillus acidophilus, Lactobacillus casei, and Lactobacillus plantarum during fermentation at 37 °C.

RIc Volatile compoundsa Peak area (×107) Odor descriptionb

(% Relative peak aread)

Lactobacillus acidophilus Lactobacillus casei Lactobacillus plantarum

0h 12 h 24 h 48 h 0h 12 h 24 h 48 h 0h 12 h 24 h 48 h

718 3-hydroxy-2-butanone 4.70 4.94 3.53 3.49 4.04 3.52 3.35 5.23 3.30 4.77 5.87 4.88 butter, cream
(acetoin) (6.70) (8.14) (7.63) (6.70) (6.71) (5.60) (5.14) (7.38) (4.95) (5.43) (7.68) (6.54)
736 3-methyl-1-butanol 11.40 11.27 6.72 6.46 10.14 8.27 8.36 11.41 9.23 13.30 14.80 14.20 whiskey, malt,
(18.43) (18.58) (14.51) (12.41) (16.84) (13.15) (12.80) (16.11) (13.85) (15.15) (19.38) (19.02) burnt
851 3-methyl butanoic acid – – – – – – – 1.96 – 1.93 2.90 2.48 sweat, acid,
(isovaleric acid) (2.77) (2.20) (3.80) (3.32) rancid
854 2-methyl butanoic acid 4.86 4.67 3.04 5.34 3.20 4.76 4.47 5.69 3.13 6.33 6.51 7.37 cheese, sweat
(7.85) (7.70) (6.56) (10.25) (5.31) (7.57) (6.85) (8.03) (4.70) (7.21) (8.53) (9.87)
873 ethyl-3-methyl-butanoate 12.04 11.32 6.06 7.41 12.24 10.47 13.50 10.53 16.68 17.96 12.71 12.01 fruit
(ethyl isovalerate) (19.45) (18.66) (13.09) (14.23) (20.33) (16.65) (20.68) (14.87) (25.01) (20.47) (16.64) (16.07)
877 1-hexanol 1.68 1.96 1.23 1.41 1.58 1.76 1.93 1.79 1.84 2.79 2.60 2.47 resin, flower,
(2.71) (3.23) (2.67) (2.70) (2.62) (2.80) (2.96) (2.53) (2.76) (3.18) (3.40) (3.31) green
940 ethyl-(2E)-2-methyl-2-butenoate 2.41 2.20 1.56 1.62 2.61 1.95 2.94 1.96 4.15 4.31 2.50 2.25 fruity type odor
((E)-ethyl tiglate) (3.90) (3.62) (3.36) (3.11) (4.33) (3.10) (4.51) (2.77) (6.23) (4.91) (3.27) (3.01)
962 benzaldehyde 3.07 3.26 2.91 2.52 2.41 3.58 2.87 3.15 3.63 4.48 4.13 3.94 almond, burnt
(4.96) (5.37) (6.29) (4.83) (4.00) (5.69) (4.39) (4.44) (5.44) (5.11) (5.41) (5.28) sugar
1002 ethyl hexanoate 2.05 2.22 1.61 1.65 2.02 1.98 3.03 2.23 2.31 2.93 1.46 1.43 apple peel, fruit
(3.32) (3.65) (3.47) (3.18) (3.36) (3.15) (4.64) (3.14) (3.47) (3.34) (1.91) (1.92)
1047 1-methyl-2-phenyl-1H-Indole 1.28 1.02 0.91 1.36 – – 0.99 1.51 1.15 1.43 1.16 1.20 –
(2.07) (1.69) (1.97) (2.61) (1.52) (2.13) (1.73) (1.63) (1.51) (1.61)
1059 2,6-dimethyl-4-heptanol 12.27 11.36 12.68 13.85 13.79 16.79 14.60 15.32 14.13 18.57 14.73 14.76 fruity and sweet
(19.84) (18.72) (27.40) (26.61) (22.90) (26.69) (22.36) (21.64) (21.19) (21.16) (19.28) (19.76) odor
1068 4-methyl-4-heptanol 5.26 4.91 5.20 5.64 6.05 6.93 6.40 6.45 5.94 7.95 6.40 6.49 piney, Citrus
(8.51) (8.09) (11.23) (10.83) (10.05) (11.02) (9.80) (9.10) (8.91) (9.06) (8.38) (8.69)
1108 3-methylbutyl-3-methyl- 0.84 1.35 0.84 1.13 0.53 1.19 1.33 1.84 0.98 0.83 – 0.66 fruity odor
butanoate (1.36) (2.23) (1.82) (2.17) (0.88) (1.89) (2.03) (2.60) (1.47) (0.94) (0.89)
(Isoamyl Isovalerate)
1124 2-phenylethanol – – – – 0.92 0.96 0.82 0.89 – – – – floral type odor
(1.54) (1.53) (1.26) (1.26)
1196 ethyl octanoate – 0.19 – 0.18 – 0.16 0.15 0.35 0.20 0.19 – – fruit, fat
(ethyl caprylate) (0.32) (0.35) (0.25) (0.23) (0.49) (0.30) (0.22)
1201 benzenepropanol – – – – – – – – – – 0.63 0.54 balsamic type
(3-phenyl-1-propanol) (0.82) (0.72) odor
1292 3,6-dimethoxy-9-(2- – – – – 0.68 0.57 0.55 0.52 – – – –
phenylethynyl)-fluoren-9-ol (1.12) (0.91) (0.84) (0.74)

−, Not detectable.
a
All compounds were identified by comparison with mass spectra and retention index database.
b
Odor descriptions were cited from www.flavornet.org and recent reports.
c
RI (retention index) based on HP-5 capillary column using a series of alkanes between C8 and C20.
d
% Relative peak area of volatile compounds is expressed as (compound peakarea/total compounds peakarea) × 100.

method were slightly higher than those found by the DPPH method.
This could be due to the fact that the ABTS method was more sensitive
to determine the antioxidant activity of samples as it could determine
antioxidant activity at lower inhibitor concentrations [49]. Increases in
antioxidant activities during the beginning of fermentation (12 h)
might be associated with the production of vitamin C and phenolic
compounds having antioxidant capacity. The decrease or increase in
antioxidant activity of phenolic compounds is affected by their chemi-
cal structure depending upon the group attached to a basic aglycone
[32]. Other studies vary, taking into account the impact of the
fermentation on antioxidant content, the type of fermentation and the
fermentation substrate. It is interesting to note that fermented CAJ
remained at over 50% radical scavenging activity after 48 h fermenta-
tion, indicating an overall good potential for free radical scavenging of
CAJ.
3.6. Identification of volatile compounds during fermentation
One of the most important characteristics of food products for
consumer palatability is the flavor volatile compounds. Table 1 show
these volatile compounds of fermented CAJ with three Lactobacillus spp.
which was identified by comparison of their mass spectra by SPME-
GC–MS on HP-5 column with libraries and published data. Results
indicate that alcohols, esters and acids were quantitatively the major
group of flavor volatile compounds produced throughout fermentation
of CAJ. In total, 12–13 flavor volatile compounds were identified in
fermented CAJ with three Lactobacillus spp. at 0 h incubation. Major
compounds were 2,6-dimethyl-4-heptanol (fruity and sweet aroma),
ethyl-3-methyl-butanoate (fruity aroma), and 3-methyl-1-butanol
(whiskey, malt, burnt aroma). As the fermentation time increased, acid
volatile compounds (2-methyl butanoic acid) in CAJ fermented with the
three Lactobacillus spp. increased, whereas ethyl-3-methyl butanoate
and ethyl-(2E)-2-methyl-butenoate decreased (Table 1). In all cases,
results indicated that the fruity odor decreased, but the whiskey and
acid odor increased. Moreover, 3-methyl butanoic acid is produced in
CAJ fermented with L. casei (at 48 h) and L. plantarum (at 12–48 h) and
was not detected in CAJ fermented with L. acidophilus. The increase of
unpleasant odor of fermented CAJ was due to 2-methyl butanoic acid
(sweaty and cheese) and 3-methyl butanoic acid (pungent/sour odor).
The three LAB strains were able to produce volatile compounds at
various levels when inoculated in CAJ potentially due to their distinct
protein and carbohydrate metabolic pathways. At 48 h, 2,6-dimethyl-4-
heptanol was detected in high quantities in CAJ fermented with three
LAB (13.85–15.32 × 107). 2-Phenylethanol and 3,6-dimethoxy-9-(2-
phenylethynyl)-fluoren-9-ol were only detected in CAJ fermented with
L. casei. Benzenepropanol was only detected at 24 and 48 h in CAJ

7
R. Kaprasob et al.
Fig. 4. Change in the concentration of ethanol (a), acetic acid (b), and ethyl acetate (c) in
fermented cashew apple juice (CAJ) at 0, 12, 24, and 48 h of fermentation at 37 °C. Data
is expressed as the average of three determinations ± SD.
fermented with L. plantarum. 3-Methyl-1-butanol was detected in high
quantities in fermented CAJ with L. casei and L. plantarum, whereas low
quantities were detected in fermented CAJ with L. acidophilus.
Moreover, 3-methyl-1-butanol contributing to the overripe cashew
apple odor [2] is derived from leucine and is considered the most
quantitatively important alcohol in Chinese ciders [51]. One important
identified volatile is 1-hexanol which is considered to be a character-
istic volatile compound of fruits and is produced during the enzymatic
oxidation process of linoleic acid [51]. Moreover, volatile compounds
of fermented CAJ were quite comparable with those reported for
fermented apple juice [20]. Overall it was found that inoculation with
LAB generated a change in flavor profiles of fermented CAJ.
The low quantities of volatile esters in fermented CAJ were ethyl-3-
methyl-butanoate, ethyl-(2E)-2-methyl-butenoate, ethyl hexanoate, 3-
methylbutyl-3-methyl-butanoate and ethyl octanoate (Table 1) which
were reported as the odor-active compounds in CAJ [2]. Volatile ester
compounds are responsible for sweet, fruity and cashew-like aroma
odors. According to [2,12], among the detected compounds, the
presence of ethyl-3-methyl butanoate, ethyl hexanoate, and ethyl-2-
methyl-2-butenoate contributed to the fruity and cashew-like aroma of
cashew apple-based alcoholic beverage.
The 3-hydroxy-2-butanone compound (acetoin) was detected in the
beginning of fermentation and was considered to be the most quantita-
tively important ketone in fermented CAJ. The acetoin potentially
resulted from LAB strains acting on the citric acid and the odor is
assigned to butter cream [52].
8
Process Biochemistry xxx (xxxx) xxx–xxx
The major other volatile compounds identified in fermented CAJ
with L. plantarum were ethanol (alcohol, winey, and sweet), whereas
ethyl acetate and acetic acid were detected in lower amounts (Fig. 4a).
In this study, ethanol was not detected in CAJ fermented with L.
acidophilus and L. casei. L. plantarum might have the ability to synthesize
ethanol during fermentation, due to the alcohol dehydrogenase activity,
an enzyme that is able to convert acetaldehyde to ethanol. Higher
concentrations of acetic acid in CAJ fermented with L. acidophilus were
detected between 24 and 48 h (Fig. 4b) which formed during fermenta-
tion due also its homofermentative nature. This group of lactic acid
bacteria efficiently metabolizes carbohydrates generating ATP which is
subsequently used for the biosynthesis of lactic acid and other end-
products. Meanwhile, L. plantarum and L. casei are facultatively
heterofermentative, producing lactic acid, acetic acid, CO2 and ethanol
as main end-products [33]. Acetic acid was found to be the most
abundant among short chain free fatty acids in fermented CAJ. Ethyl
acetate (odor note of pineapple) was detected in CAJ fermented with L.
plantarum which was higher than CAJ fermented with L. acidophilus and
L. casei over all fermentation period (Fig. 4c). These findings are
consistent with research reported previously for cereal-based probiotic
beverages with L. plantarum [13]. Moreover, the volatile production
depends on the substrate and lactobacilli strains in the fermentation
process.
4. Conclusions
Cashew apple juice is an excellent fruit substrate for fermentation-
based biotransformation into more value added bioactive and beneficial
volatile compounds using the 3 selected LAB; L. plantarum. L. casei and
L. acidophilus. The study indicated that bioactive moieties such as
vitamin C and phenolic compounds and associated antioxidants can be
beneficially modulated to higher levels in most cases but even when
reduced in the case of antioxidant activity and viable counts by end of
48 h the overall added benefits from LAB-induced fermentation remain.
Specifically the modulation of vitamin C and phenolic compounds hold
the best potential to further utilize the be benefits of CAJ fermentation
from this study for further functional applications in human, animal
and plant health where host beneficial responses could be targeted for
potential improvement from this CAJ byproduct. Another beneficial
aspect of LAB-induced biotransformation is the generation of diverse
array of flavor volatiles which when combined with other health
benefits can have relevance in range of food beverage design as
functional foods in future studies. Overall this study provides a sound
foundation to further add value to CAJ substrate through LAB-induced
fermentation and provides the rationale for further functional studies
for human, animal and plant health benefits.
Acknowledgements
This work was supported by the Royal Golden Jubilee PhD Program
(PHD/0186/2553) of Thailand, and National Research University
Project of Thailand. The cashew apple was kindly provided by
Heritage Grower Corporation Ltd.
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