Journal of Bioscience and Bioengineering

VOL. xx No. xx, 1e3, 2014

www.elsevier.com/locate/jbiosc

NOTE

Lactobacillus plantarum mediated fermentation of Psidium guajava L. fruit extract

Ravish Bhat, Lakshminarayana Chikkanayakanahalli Suryanarayana, Karunakara Alageri Chandrashekara,
Padma Krishnan, Anil Kush, and Puja Ravikumar*
Vittal Mallya Scientic Research Foundation, 94/3 & 94/5, 23rd Cross, 29th Main, BTM II Stage, Bangalore 560076, India
Received 18 February 2014; accepted 5 September 2014
Available online xxx

Sixteen hour fermentation of the white esh raw guava Lucknow 49 cultivar using Lactobacillus plantarum NCIM 2912
was taken up for enhancing the antioxidant potential. The fermented guava product with high antioxidant potential,
total phenolic content and short and medium chain fatty acids can be used as functional food.
2014, The Society for Biotechnology, Japan. All rights reserved.
[Key words: Psidium guajava L.; Phenolic content; Antioxidant activity; Fermented guava product; Fermentation; Functional foods]

Guava (Psidium guajava L.), family Myrtaceae, is a popular

commercial fruit from tropical and semitropical regions of world
with India accounting for 45% of total production followed by
Pakistan, Mexico and Brazil. Based on pigments produced in pulp,
guava fruits can be classied as white and pink cultivars. Nutritionally, guava fruit contains high amounts of fermentable sugars
such as fructose, glucose, sucrose, pectin, minerals (calcium and
phosphorus), antioxidants (vitamins A, B, C, E, niacin, lycopene,
carotinoids, polyphenols) and high dietary bers. Jimnez-Escrig
et al. (1) reported that guava pulp and peel are good sources of
antioxidant dietary ber with 7.79% total extractible phenols.
Vitamin C, E along with phenolics and carotinoids are potent free
radical scavengers. Raw guava fruits though rich in antioxidants;
are hard (due to high pectin content), less sweet and not preferred
for direct consumption. The antioxidant content in fruit declines as
they ripen (2) and the berries have a very limited shelf life. Guava
fruits are highly prone to chilling and mechanical injury followed
by fungal decay. Several post harvest techniques exist to preserve
guava fruits for retaining the nutritional properties and enhancing
the shelf life. Various products like guava pulp, concentrates, syrup,
paste, puree and yeast fermented wine are available. Sevda and
Rodrigues (3) reported that Saccharomyces cerevisiae NCIM 3095
strain was best suited for guava must fermentation for wine production. Increased consumer awareness about the benets of preventive health is a major contributor in increasing the demand of
functional foods. However, the perceptible off avors caused by
microbes pose a challenge to acceptability of these products. Flavor
masking technique helps address this problem by incorporating
attractive fruit avors (4,5). We took up fermentation of raw guava
fruit extract using Lactobacillus plantarum (NCIM 2912) and the
product was analyzed for antioxidant content, total phenolics and
production of short and medium chain fatty acids. To the best of our
knowledge this work is the rst attempt to improve nutritional

* Corresponding author. Tel.: 91 80 26 68 72 23; fax: 91 80 26 68 71 70.

E-mail address: puja@vmsrf.org (P. Ravikumar).

value of guava fruit extract by fermentation using lactic acid

bacteria.
Raw guava fruits (Lucknow49 variety) procured from Lalbagh
Horticulture Department, Bangalore were used in this study. Fruits
were round to oval in shape, green-pale yellow with white pulp
(each fruit weighing about 0.11 kg). L. plantarum NCIM 2912
(source: National Collection of Industrial Microorganisms, Pune,
India) was used for all the experiments. The cultures were revived
from glycerol stocks in de Man Rogosa Sharpe (MRS) broth and MRS


agar (Himedia, Mumbai, India) at 37 C for 48 h. A single isolated
colony was sub cultured in MRS broth and used for shake ask
fermentation. Methyl ester derivatives of butyric, caproic, caprylic,
capric and lauric acids, 2,20 -diphenyl-1-picryl hydrazyl (DPPH), 2,2azinobis 3-ethylbenzothiazolin 6-sulfonic acid (ABTS), butylated
hydroxy toluene (BHT); 3,5-dinitrosalicylic acid (DNS) were procured from Sigma Chemical Company (Mumbai, India). 2,4,6tripyridyl-s-triazine (TPTZ) was obtained from Alfa Aesar (Hyderabad, India). HPLC grade solvents and analytical reagent grade
chemicals were obtained from Merck (Mumbai, India).
The raw fruits were washed with double distilled water several
times followed by surface disinfection with 95% ethanol. Grated
guava was mixed with equal amount of sterile water (1:1 ratio
w/v) and pulverized in to slurry in an electric juicer blender
(Kenwood JE 720). To this pulp 0.25% (w/w) pectinase enzyme
(Tris enzymes, Mysore, India) was added and the mixture was


incubated at 38 C with shaking at 200 revolutions per min for 2 h
to obtain clear fruit extract. The slurry was ltered and extract was
diluted with sterile water (1:1 ratio) and supplemented with
following media components (Merck) (g/l): K2HPO4, 6.0; KH2PO4,
14.0; MgSO4. 7 H2O, 0.4; NaCl, 4.0; (NH4)2SO4, 2.0; Glycine, 3.0;
Sucrose, 50.0 and the pH was adjusted to 7.05  0.05 followed by
sterilization by autoclaving.
The fermentation experiments were carried out in triplicate as
separate asks and uninoculated media was maintained as unfer

mented control. Fermentation was carried out at 37 C for 16 h with
mild shaking at 120 rpm and sampling was done at an interval of
every 4 h starting from 0 h for measuring antioxidant activity and

1389-1723/$ e see front matter 2014, The Society for Biotechnology, Japan. All rights reserved.
http://dx.doi.org/10.1016/j.jbiosc.2014.09.007

Please cite this article in press as: Bhat, R., et al., Lactobacillus plantarum mediated fermentation of Psidium guajava L. fruit extract, J. Biosci.
Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.09.007

BHAT ET AL.

J. BIOSCI. BIOENG.,

FIG. 1. Microbial population, pH of the medium and reducing sugar utilization during fermentation of guava fruit extract.

In our study, guava extract fermentation using L. plantarum led

to increase in antioxidant activity, total phenolics, and short and
medium chain fatty acids. The increase in antioxidant potential is
observed because L. plantarum degrades some poly phenolic compounds into simple forms due to its wide portfolio of enzymes (i.e.,
b-glucosidase, p-coumaric acid decarboxylase, and decarboxylase).
The number of viable L. plantarum cells increased exponentially
during 4e8 h of fermentation and reached stationary growth phase
between 12 h and 16 h. The pH gradually dropped between 0 h and
8 h (pH 7.05e6.56); rapidly at 12 h (pH 5.65); reached 4.83 at 16 h.
Approximately 67% of available soluble sugars from fruit material
were utilized by L. plantarum by 12 h and consumption declined
thereafter as the microbial growth declined (Fig. 1). After 16 h of
fermentation the liquid supernatant of guava fermented broth was
brown colored aqueous solution with fruity odor and the lyophilized product was a hygroscopic, brown crystalline powder with
fruity fermented odor and freely miscible in water.
The DPPH free radical scavenging activity of fermented product
increased signicantly and exhibited highest value of 82.51% at 8 h.
Gradual decrease in antioxidant activity was observed in subsequent fermentation hours and it dropped to 77.29% at 16 h. ABTS
radical scavenging and FRAP activities were in consensus and
reached the highest levels by 16th h of fermentation (Table 1;
Fig. S1). The mechanism of antioxidant effect of LAB has been
studied and explained using intact cells and intracellular extracts of
LAB where these strains signicantly inhibited the Fe/H2O2
induced peroxidation of lipids and the intracellular extracts of LAB
possess metal chelating ability, reactive oxygen species (ROS)
scavenging and reduction activity properties. It has also been
illustrated that intracellular cell free extracts of different
L. plantarum strains show strong hydroxyl radical scavenging activity probably due to their ability to chelate Fe metal ions.

pH. The viability of L. plantarum during fermentation was checked

by plating on MRS agar plates.
The antioxidant potential of the fermented product was evaluated by determining the DPPH scavenging activity (6), ABTS radical
scavenging activity (7) and ferric reducing antioxidant power
(FRAP) assay (8). BHT was used to plot the standard graph and the
FRAP activity was expressed in terms of BHT equivalents (mg/mL).
The concentration of total phenolics in fermented product was
determined using colorimetric method as described by Singleton
et al. (9). The concentration of phenolics calculated from the standard graph using gallic acid and the phenolic content in fermented
product was expressed in terms of gallic acid equivalents (mg GAE/
mL). Reducing sugars were estimated in the fermentation medium
to quantify the amount of unutilized sugars and the estimation was
done by DNS method (10). D-Glucose (Himedia) was used to plot
standard graph (0.2e1.0 mg/mL). Detection and quantication of
short chain fatty acids (SCFAs) such as butyric acid (4:0) and medium chain fatty acids (MCFAs) like caproic acid (6:0), caprylic acid
(8:0), capric acid (10:0) and lauric acid (C12:0) was done by one
step liquideliquid extraction of fermented sample using chloroform:methanol (2:1) solvent system, conversion of free fatty acids
to fatty acid methyl esters followed by gas chromatographic analysis was done as described by Christie (11).
The mean values of percent free radical scavenging activity,
FRAP activity, total phenolics and short and medium chain fatty
acids in fermented product were obtained from triplicate asks of
two fermentation experiments and represented as mean  SE. The
correlation analysis was performed between total phenolic content
and antioxidant activities of fermented product using Pearson
correlation analysis in XLSTAT. The signicance of correlation was
done at P < 0.001 and the differences at P < 0.001 were considered
as statistically signicant.

TABLE 1. Antioxidant activity and total phenolic content in fermented guava product during fermentation. The uninoculated medium is used as control.
Fermentation
hours

Antioxidant activity
DPPH activity
Control

0
4
8
12
16

36.58
38.33
35.25
33.08
36.4







0.56
0.79
0.85
0.43
1.22

ABTS activity
FGP

37.01
60.49
82.51
80.81
77.29







Control
0.60
0.64
1.39
0.73
0.84

15.43
16.83
18.28
17.34
17.95







0.45
0.57
0.62
0.53
0.53

Total phenolic content (mg/mL

GAE)

FRAP activity
FGP

15.52
53.57
62.90
96.26
99.16







Control
0.28
2.08
2.55
0.49
0.21

0.81
0.80
0.75
0.73
0.71







0.04
0.02
0.02
0.02
0.01

FGP
0.80
0.93
1.20
1.60
2.20







0.02
0.06
0.03
0.06
0.08

Control
2.71
2.85
2.89
2.95
2.85







0.06
0.05
0.06
0.07
0.07

FGP
2.83
4.33
7.82
10.26
11.18







0.06
0.09
0.11
0.08
0.15

Each value in the table is the mean  standard error of two fermentations. DPPH and ABTS radical scavenging activity expressed in percentage. FRAP activity expressed in mg/
mL butylated hydroxy toluene (BHT) equivalents. GAE, gallic acid equivalent.

Please cite this article in press as: Bhat, R., et al., Lactobacillus plantarum mediated fermentation of Psidium guajava L. fruit extract, J. Biosci.
Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.09.007

VOL. xx, 2014

NOTE

TABLE 2. Short (SCFAs) and medium chain fatty acids (MCFAs) content in fermented
guava product (FGP).
Components (ng per 100 mL)
Butyrate (C-4)
Caproate (C-6)
Caprylate (C-8)
Caprate (C-10)
Laurate (C-12)

Unfermented control
1.30
1.00
2.40
1.58
9.67







0.39
0.34
0.31
0.27
0.36

Fermented guava product

17.85
62.03
34.93
6.97
17.97







0.68
0.55
0.62
0.52
0.51

The values are in ng/100 mL of fermented product; uninoculated medium is used as

control. Each value in the table is the mean  standard error of two fermentations.

L. plantarum mediated fermentation of guava increased the total

phenolics in fermented product to 66.48% (11.18 mg GAE/mL) as
compared to control that contains only 2.85 mg GAE/mL after 16 h
(Table 1). The total phenolic content in guava pulp and peel is
2.68 mg GAE/g and 5.87 mg GAE/g respectively as estimated and
reported in a previous study by Jimnez-Escrig et al. (1). We have
used whole fruit extract in our experiment and the total phenolic
content in the unfermented control is 2.85 mg GAE/mL of medium
is comparable to the values reported earlier. This increase in the
total phenolic content can be attributed to the enzymatic degradation of the polyphenol complexes by the fermenting microorganism. Duenas et al. (12) also studied the effect of L. plantarum
fermentation on total phenolic compounds in Vigna sinensis L. our
and reported that fermentation enhances the phenolic content
because it hydrolyzes complexes of polyphenols to other simpler
and biologically active ones.
A considerable increase in butyrate, caproate, caprylate, caprate
and laurate were observed in fermented product vis-a-vis unfermented control (Table 2; Fig. S2). The sugars in guava extract that
constitute the digestible carbohydrates of the fruit are converted to
SCFAs. Henningsson et al. (13) reported that a certain combination
of carbohydrate substrates may have an inuence on SCFA synthesis and these SCFAs and MCFAs (associated with gut health) are
produced by bacterial fermentation of indigestible carbohydrates in
colon. Vong and Stewart (14) reported that dietary fruit ber ferments to form SCFAs and butyric acid (SCFA) has been implicated in
preventing and treating colon diseases like ulcerative colitis (15,16).
The fermented guava product (FGP) with high antioxidant potential
and important fatty acids can be a ready to blend functional food
with multiple health benets. In our fermentation experiments we
observed that antioxidant potential is highest at 16th h of
fermentation as assessed by FRAP assay, DPPH assay and total
phenolic content. We optimized the process for harvesting at
16th h based on the antioxidant activity and fatty acid content of
product as well as the sugar utilization by microbes.
In conclusion, this study describes value addition to a common
tropical fruit guava using a probiotic strain L. plantarum. The fermented product is rich in antioxidant content, total phenolics, short
and medium chain fatty acids. The liquid concentrate of fermented
guava product is amenable for blending in a wide range of food
commodities like beverages, candies, bars, ice creams, etc. Further
optimization in the fermentation process using other LABs can help

in developing a ready to blend fermented guava product for fortication of a range of food items. Developing a value added fermented guava product will help circumvent the losses because of
highly perishable nature of fruits.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.jbiosc.2014.09.007.
Authors are thankful to the Department of Biotechnology, Government of India
(Grant Id: BT/PR-11099/FNS/20/388/2008) for nancial support. Authors declare no
conict of interest. This article does not contain any studies with human or animal
subjects.

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Please cite this article in press as: Bhat, R., et al., Lactobacillus plantarum mediated fermentation of Psidium guajava L. fruit extract, J. Biosci.
Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.09.007