Food Chemistry 237 (2017) 957–965

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Starch digestibility and predicted glycemic index in the bread fortified

with pomelo (Citrus maxima) fruit segments
S.K. Reshmi a, M.L. Sudha b, M.N. Shashirekha a,⇑
a
Department of Fruit and Vegetable Technology, CSIR-CFTRI, Mysuru 570020, India
b
Flour Milling, Baking & Confectionery Technology Department, CSIR-CFTRI, Mysuru 570020, India

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this study was to evaluate the starch digestibility and predicted glycemic index in breads
Received 28 December 2016 incorporated with pomelo fruit (Citrus maxima) segments. Volume of the white and brown breads supple-
Received in revised form 8 April 2017 mented with pomelo fresh segments increased, while the crumb firmness decreased. Bread with 20%
Accepted 29 May 2017
fresh and 5% dry pomelo segments were sensorily acceptable. Bioactive components such as phenolics,
Available online 1 June 2017
flavonoids, naringin and carotenoids were retained to a greater extent in bread containing dry pomelo
segments. The pomelo incorporated bread had higher levels of resistant starch fractions (3.87–10.96%)
Keywords:
with low predicted glycemic index (62.97–53.13%), despite their higher total starch (69.87–75.47%) con-
Pomelo segments
Bread
tent compared to control bread. Thus pomelo segments in the product formulations lowered the glycemic
Naringin index probably by inhibiting carbohydrate hydrolyzing enzyme activity which could be attributed to nar-
Glycemic index ingin. Hence fortified bread prepared from pomelo fruit segment is recommended to gain nutritional
Biofunctional components value and to decrease the risk of diabetes.
Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction international trade and it has been recommended in herbal medi-

Recently, Innovative food products with health benefits are
increasingly becoming popular. Functional foods having a wide
range of phytochemical profiles exhibit therapeutic activity against
various health related disorders (Jenkins et al., 2008). The concept
of diet-based therapies is aimed at maximizing the physiological
benefits of various functional foods that require product develop-
ment (Siró, Kápolna, Kápolna, & Lugasi, 2008). Foods having high
protein and fiber content are now generally preferred by con-
sumers to maintain their health and to act against many diseases
like diabetes, obesity etc. So there is a new trend in the market
to develop a product that has health benefits with acceptable sen-
sory characteristics. Fruits are the basic components of human diet.
Apart from providing energy for metabolic pathways they also act
as precursor for protein synthesis and are a source of micronutri-
ents like vitamins and minerals. Citrus fruits have high economic
and medicinal value because of their multiple uses in the pharma-
ceutical, cosmetics, and food industries. These beneficial effects of
citrus fruits are attributed to their chemical constituents like
vitamins, dietary fiber, carotenoids, flavonoids, lipids, and essential
oils. Citrus fruits are the utmost value fruit crop in terms of
⇑ Corresponding author.
E-mail address: shashirekhaurs@gmail.com (M.N. Shashirekha).
cine as the source of diabetic medication (Andrade-Cetto, 1995).
Citrus maxima (Burm.) Merr., commonly known as pomelo is
one of the largest underutilized citrus fruits belonging to the family
Rutaceae. It has been reported to act as an appetizer, cardiac stim-
ulant, stomach tonic and also as a remedy for fever, insomnia, and
sore throat (Merina, Chandra, & Jibon, 2012). Further, it shows var-
ious pharmacological activities against oxidative stress (Mäkynen
et al., 2013), inflammation (Shivananda, Muralidhara, &
Jayaveera, 2013) and diabetes (Abdul, Shenoy, Hegde, Aamer, &
Shabaraya, 2014). Even though several reports are available on
the medicinal property of pomelo, there is a problem of its avail-
ability in large quantities. No commercial cultivation is undertaken
due to the bitterness and astringent nature of the fruits. Because of
this reason, it is less utilized by common people when compared to
other fruits like orange, lime and tangerines.
Baking is a process that has been adopted for centuries and bak-
ery products range from simple ingredients plain pastry to the cake
having numerous components. Bread is one of the bakery products
priced for its taste, aroma and texture. Bread making is a complex
process which includes mixing, proofing and baking (Dewettinck
et al., 2008). Bread is considered as a well-liked staple food
consumed as part of the daily diet worldwide. Annually
9 million
kg of bread are produced (Heenan, Dutour, Hamud, Harvey, &
Delahurry, 2008). The popularity of bakery products has

http://dx.doi.org/10.1016/j.foodchem.2017.05.138
0308-8146/Ó 2017 Elsevier Ltd. All rights reserved.
958 S.K. Reshmi et al. / Food Chemistry 237 (2017) 957–965
contributed to increased demand for ready to eat and convenient
food products such as bread, cakes, biscuits etc. So initiative has
been taken in this research work to use pomelo as a food fortificant
for increasing the consumption of this fruit for health benefits. This
study was conducted to develop value added white bread and
brown bread using pomelo fruit segments. The prepared products
were further analyzed for their glycemic index, retention of narin-
gin and other biofunctional components to ensure health promot-
ing properties of pomelo retained even after processing.
2. Materials and methods
Commercial wheat flour (10.2% moisture, 0.51% ash, 10.6% glu-
ten, 24 ml sedimentation value and 373 s falling number), com-
pressed yeast (Tower brand, Mumbai), sugar powder and
vegetable fat (Hindustan Unilever Ltd, Bangalore) were procured
from local market.
2.1. Pomelo fruit processing
The Citrus maxima (pomelo) fruits were obtained from the local
market of Mysuru, Karnataka, India during the month of February
2016. The fresh segments were separated from the fruit manually
and dried in hot air oven at 35 °C for overnight to obtain dry fruit
segments (residual moisture content of
5%).
2.2. Bread making characteristics
Effect of fresh pomelo fruit segments (0%, 10%, 20% and 30%)
and dried pomelo fruit segments (0%, 2.5%, 5% and 7.5%) on white
bread and brown bread making characteristics was studied (Sudha
& Leelavathi, 2008). The formulation used was flour: 100%, pomelo
fruit segments, compressed yeast: 2.0%, vegetable fat: 1%, salt:
1.0%; sugar: 2.5% and water. All the ingredients were mixed in a
Hobart mixer (Model N-50, Hobart, GmbH, Germany) with a flat
blade for 4 min at 61 rpm. The dough obtained was fermented in
a chamber maintained at 30 °C and 75% relative humidity (RH)
for 90 min. After 90 min, the dough was remixed and relaxed for
25 min, molded, proofed for 55 min at 30 °C, 85% RH and baked
for 25 min at 220 °C, cooled for physical and sensory evaluation.
Part of the bread samples were dried at 45 °C for 5 h, cooled,
homogenized and stored in poly propylene bags for various
estimations.
2.3. Evaluation of breads
Weight of the breads was taken and volume of the loaves was
measured by rapeseed displacement method (Sudha & Leelavathi,
2008). Bread crumb firmness, the objective measurement of tex-
ture was carried out in a texture analyzer (TAHDi, Stable Micro
Systems, Godalming, UK) by the standard AACC (2000) (74–09)
and 2.0 mm.s1 of pre-test speed and 1.67 mm.s1 of test speed
were used. Force required to compress 25% of the bread slice was
recorded using 35 mm diameter aluminium cylinder probe P-35.
Objective evaluation of color of bread crumb was measured using
the Hunter Lab Colour Measuring System (Colour Flex-EZ Hunter
Lab, USA) with a reflectance attachment of illuminant G against a
standard white board made of barium sulphate (100% whiteness).
Bread slice (3 cm  3 cm) was placed in the sample holder and the
reflectance from the surface measured.
2.4. Sensory evaluation
A scorecard containing the description for the desirable (cream-
ish white – color; crisp –texture; wholesome sweetish – taste) and
undesirable (dull or dark color; soft or hard – texture; unpleasant
taste) quality characteristics for various sensory attributes viz.
color of crust and crumb, texture, mouthfeel and overall quality
were given to the panelist consisting of men and women of 35–
50 age group. The panelist was then asked to assign scores for each
parameter as against the maximum scores given in the parenthesis
using a 7-point hedonic rating scale: excellent – 7, very good – 6,
good – 5, satisfactory – 4, fair – 3, poor – 2 and very poor – 1
(Rathi, Kawatra, & Sehgal, 2004).
2.5. Estimation of total sugars
The total sugars estimation in the samples was carried out using
the method described by Albalasmeh, Berhe, and Ghezzehei
(2013). An aliquot of 10 ll of sample was mixed with 300 ll of
5% aqueous solution of phenol. After 5 min of incubation 1.8 ml
of concentrated sulfuric acid was added rapidly to the mixture.
The test tubes were cooled and absorption read at 490 nm. Total
sugar content of the sample was expressed as equivalent to mg
glucose/g extract.
2.6. Estimation of reducing sugars
Reducing sugars were estimated based on the modified method
of Miller (1959). The sample (150 ll) was mixed with 1 ml of DNS
(3,5-Dinitrosalicylic acid) reagent in a test tube. The tubes were
placed in a boiling water bath for 10 min and cooled for ten to fif-
teen minutes at room temperature. Each solution was then diluted
with 2 ml of water, mixed thoroughly and absorbance was
recorded at 540 nm using spectrophotometer (Helios Alpha,
Thermo Electron Corporation, England, UK). Total reducing sugar
content of the samples was expressed as equivalent to mg glu-
cose/g extract.
2.7. Bioactive components
2.7.1. Estimation of total phenolic content
It was evaluated using a modified colorimetric method
described by Henríquez et al. (2010). The reaction mixture was
prepared by adding 100 ll of sample, 1.0 ml of Folin-Ciocalteau
reagent and 2.0 ml of 10% sodium carbonate solution. The mixture
was incubated for 60 min at room temperature, and the absor-
bance read at 765 nm using an UV–Vis spectrophotometer. The
measurement was compared with standard gallic acid solution.
The total phenolic content was expressed equivalent to mg gallic
acid/g extract.
2.7.2. Estimation of flavonoids
Flavonoids were estimated by a modified method of
Lallianrawna, Muthukumaran, Ralte, Gurusubramanian, and
Senthil Kumar (2013). To 0.9 ml of sample, 75 ll of 5% NaNO2 solu-
tion was added. After 5 min, 150 ll of 10% AlCl36H2O was added to
the mixture, which was kept at room temperature for 5 more min-
utes. This was followed by the addition of 0.5 ml of 1 M NaOH and
the total volume was made up to 2.5 ml with the addition of deio-
nised water. The resulting solution was mixed well and immedi-
ately, the absorbance was measured at 510 nm on a UV–VIS
spectrophotometer. For the blank, the extracts were replaced with
an equal volume of deionised water. Total flavonoid content of the
samples was expressed as the mg equivalent to catechin/g of
extract.
2.7.3. Estimation of carotenoids
The experiment was carried out by the modified procedure of
Carvalho et al. (2012). The sample (1 g) was homogenized in the
dark (to avoid photolysis of carotenoids) with 20 ml of acetone.
 S.K. Reshmi et al. / Food Chemistry 237 (2017) 957–965
The filtrate was further transferred to a separating funnel contain-
ing 10–15 ml of petroleum ether and mixed well. The lower aque-
ous layer was then transferred to another separating funnel and
the upper petroleum ether layer containing the carotenoids was
collected. The extraction was repeated until the aqueous layer
became colorless. A small amount of anhydrous sodium sulphate
was added to the petroleum ether extract to remove excess mois-
ture. The final volume of the petroleum ether extract was noted.
The absorbance of the yellow color was read in a spectrophotome-
ter at 450 nm using petroleum ether as blank.
A  VðmlÞ  104
Carotenoids content ðlg=gÞ ¼
A1cm
1%  PðgÞ
where A = Absorbance; V = Total extract volume; P = sample
weight; A1 = 2592 (b-carotene Extinction Coefficient in petroleum
ether).
2.7.4. Naringin content
The white and brown breads were analyzed for the presence of
naringin (bioactive compound) using HPLC (Shimadzu Class – VP
HPLC model used with SPD-10AVP (PDA detector)) by slightly
modifying the protocol of Pichaiyongvongdee & Haruenkit, 2009.
One gram of the sample was extracted in methanol and homoge-
nized for 30 min. The supernatant was passed through 0.45 mm
syringe filters and subjected to HPLC. A standard solution was pre-
pared by dissolving 1 mg of naringin in 1 ml with acetonitrile.
Supelco C18 (5 mm) column (15 cm  4.6 mm id), Supelco, USA
was used with chromatographic solvent mixture (mobile phase)
consisting of water: acetonitrile: (80:20 v/v) as mobile phase.
The solvents were degassed with vacuum before using in HPLC
analysis. The mobile phase was pumped with a LC-10 ATVP pump
at a flow rate of 1 ml/min. The injection volume was 20 ml and the
total run time was 15 min at 270 nm. Quantification of the com-
pound was evaluated by comparing the peak area with authentic
standards using peak processing post-run integration parameters
and external method: calibration type of Shimadzu class VP ver-
sion 6.14 SPI data acquisition software.
2.8. Total starch (TS)
The total starch in the bread samples was determined enzymat-
ically according to the method described by Goni, Garci-Alonso,
and Suara-Calirto (1997). The ground sample (50 mg) was dis-
persed in 6 ml of 2 M KOH and shaken at room temperature for
30 min. Three ml of 0.4 M Sodium acetate buffer pH 4.75 and
60 ml of amyloglucosidase (EC-3.2.1.3, Sigma-Aldrich Chemical
Company, St Louis, MO, USA) were added to this suspension and
incubated for 45 min at 60 °C in a controlled shaking water bath.
Starch was measured as glucose with glucose oxidase-peroxidase
(GODPOD) kit. Factor conversion from glucose to starch was 0.9.
2.9. Resistant starch (RS)
Resistant starch was estimated according to the method of Goni
et al. (1997). One hundred mg of each sample was incubated with
pepsin solution containing 20 mg of pepsin (EC-3.4.23.1, Sigma-
Aldrich Chemical Company, St Louis, USA) for 60 min at 40 °C for
protein removal. Then the starch was hydrolyzed by adding pan-
creatic a-amylase (EC-3.2.1.1, Sigma-Aldrich Chemical Company,
St Louis, USA) (10 mg/ml) solution containing amyloglucosidase
(AMG) for 16 h at 37 °C with constant shaking. After hydrolysis,
samples were washed thrice with ethanol (99% v/v and 50% etha-
nol). The separated pellet from supernatant was further digested
with 2 M KOH. Digested pellet and supernatant were separately
incubated with AMG. Glucose released was measured using a
959
glucose oxidase-peroxidase (GODPOD) reagent kit (K-GLOX, Mega-
zyme Bray, Co. Wicklow, Ireland) by absorbance at 510 nm against
the reagent blank. RS was calculated as glucose (mg)  0.9. Diges-
table starch (DS) has been calculated as difference between TS and
RS.
2.10. Predicted glycemic index (pGI)
A modified in vitro method based on the procedure of Goni et al.
(1997) was adopted. The ground sample (100 mg) was incubated
with 10 ml HCl–KCl buffer (pH 1.5) and 200 ll pepsin solution
(100 mg/ml HCl-KCl buffer) at 40 °C for 1 h with constant shaking.
The pH was raised by addition of 200 ll pancreatic a-amylase solu-
tion (1.5 mg/10 ml phosphate buffer; pH 7.8) and incubated at
37 °C for 45 min. Enzyme reaction was stopped with 70 ll Na2CO3
solution and samples diluted to 25 ml with tris-maleate buffer
(pH 6.9). Five ml of pancreatic a-amylase solution (3 U/5 ml
tris-maleate buffer) was thereafter added to the sample and incu-
bated at 37 °C with constant shaking. Aliquots of 1 ml were taken
at 30, 90 and 120 min from the samples and placed into boiling
water with vigorous shaking for 5 min to inactivate the enzyme
reaction. Samples were kept in refrigerator (4 °C) after each inacti-
vation until the end of incubation time (180 min).
All aliquots were treated with 3 ml of 0.4 M sodium acetate buf-
fer (pH 4.75) and 60 ll of amyloglucosidase (3300 U/ml) then incu-
bated at 60 °C for 45 min with constant shaking. After incubation,
volume was adjusted to 10 ml with distilled water, mixed properly
and centrifuged before transferring 0.1 ml aliquots of the solution
into glass test tubes for glucose measurement.
The glucose released was measured using a glucose oxidase-
peroxidase (GODPOD) kit (K-GLOX, Megazyme Bray, Co. Wicklow,
Ireland). Absorbance was measured at 510 nm against the reagent
blank using UV–vis spectrophotometer. The values were plotted on
a graph and the area under the concentration-over-time curve
(AUC) was determined using Sigmaplot 10.0 (Systat Software,
San Jose, CA, U.S.A.). The hydrolysis index (HI) was calculated as
the percentage of total glucose released from the samples as com-
pared to that released from standard glucose (0–180 min) (Barine
& Yorte, 2016). The predicted glycemic indices of the samples were
estimated according to the equation of Goni et al. (1997):
pGI = 39.71 + 0.549HI.
2.11. Statistical analysis
Data were statistically analyzed using Duncan’s new multiple
range tests using GraphPad Prism software version 4.03 for Win-
dows (San Diego, CA, USA) with different experimental groups
appropriate to the completely randomized design with four repli-
cates each as described by Sudha and Leelavathi (2008) at p  0.05.
3. Results and discussion
3.1. Quality characteristics of white and brown bread
The physical attributes of white and brown bread incorporated
with different levels of fresh and dry pomelo segments are pre-
sented in Table 1. The substitution of fresh pomelo segments
(0–30%) in white bread formulation increased the volume from
567 to 625 ml and decreased the specific volume from 4.05 to
3.98 ml/g whereas for brown bread the same increased from 425
to 485 ml and 2.77 to 2.97 ml/g respectively. However, incorpora-
tion of dry pomelo segments decreased the loaf volume and speci-
fic volume in both types of bread ranging from 575 to 534 ml and
3.96 to 3.34 ml/g for white bread and 480 to 410 ml and 2.94 to
2.31 ml/g for brown bread indicating that the volume of the brown
960 S.K. Reshmi et al. / Food Chemistry 237 (2017) 957–965

Table 1
Physical characteristics of white and brown bread supplemented with fresh/dry pomelo segments.

Pomelo segments (%) Volume (ml) Specific volume (ml/g) Crumb firmness (g force) Crumb color
L A b DE
White bread
0 567 ± 1.89c 4.05 ± 0.63a 397 ± 0.98e 67.57 ± 0.31a 0.13 ± 0.02 h 12.26 ± 0.03d 26.75 ± 0.28ef
Fresh
10 575 ± 0.87c 3.91 ± 0.89a 370 ± 1.07f 66.92 ± 0.35b 0.25 ± 0.02 h 12.09 ± 0.08d 26.36 ± 0.29ef
20 600 ± 1.44b 3.94 ± 1.06a 341 ± 1.16 g 65.33 ± 0.24b 0.56 ± 0.34 g 12.41 ± 0.10d 27.11 ± 0.24e
30 625 ± 1.86a 3.98 ± 1.76a 302 ± 1.22 g 65.91 ± 1.39b 1.01 ± 0.02f 12.48 ± 0.11 cd 27.21 ± 0.67e
Dry
2.5 575 ± 1.09c 3.96 ± 0.79a 308 ± 1.09 g 64.30 ± 0.01bc 0.83 ± 0.01f 12.45 ± 0.57c 29.17 ± 0.01d
5 540 ± 0.65d 3.46 ± 0.75b 401 ± 0.89e 62.31 ± 0.02c 1.12 ± 0.02f 12.78 ± 0.02c 27.01 ± 0.02e
7.5 535 ± 1.22d 3.34 ± 1.27b 429 ± 0.82e 61.08 ± 0.09c 1.26 ± 0.01e 12.92 ± 0.03c 27.30 ± 0.10e

Brown bread
0 425 ± 0.54 g 2.77 ± 1.45d 1056 ± 0.98a 51.95 ± 0.48d 3.66 ± 0.17d 15.02 ± 0.33a 42.19 ± 0.72bc
Fresh
10 450 ± 1.36f 2.84 ± 1.65c 777 ± 1.30c 50.61 ± 0.89d 3.92 ± 0.10c 14.94 ± 0.13b 42.16 ± 0.66bc
20 460 ± 1.11f 2.90 ± 1.39c 700 ± 0.92d 50.11 ± 1.13d 4.01 ± 0.22c 14.70 ± 0.20b 43.46 ± 1.11b
30 485 ± 1.90e 2.97 ± 0.85c 679 ± 0.77d 49.55 ± 0.56d 4.26 ± 0.07b 14.86 ± 0.16b 44.71 ± 0.57a
Dry
2.5 480 ± 0.48e 2.94 ± 0.47c 685 ± 1.09d 49.09 ± 0.19d 4.03 ± 0.07c 15.14 ± 0.11a 42.49 ± 0.68bc
5 455 ± 0.97f 2.67 ± 0.67d 743 ± 0.89c 47.59 ± 0.47e 4.73 ± 0.04b 15.56 ± 0.14a 43.85 ± 0.38b
7.5 410 ± 1.36 g 2.31 ± 1.08e 829 ± 1.11b 46.40 ± 0.41e 5.07 ± 0.06a 15.77 ± 0.13a 41.66 ± 0.42c
SEM (±) 1.02 0.11 5.3 1.01 0.92 0.45 2.01

Values are means ± standard deviation (n = 4); Values for a particular column followed by different letters differ significantly (p < 0.05); SEM – Standard error of mean at 32
degrees of freedom.

bread is comparatively lower than the white bread. Scores of soft-
ness attribute were in accordance with the results of texture anal-
ysis (Table 1). The bread supplemented with fresh segments in
both white (397–302 g/force) and brown (1056–679 g/force)
breads indicate that the softness of the bread increased with
increase in fresh segments. The crumb firmness increased in breads
supplemented with dried pomelo segments from 308 to
429 g/force (white bread) and 685 to 829 g/force (brown bread).
There was significant (p < 0.05) decrease in preference in all the
attributes evaluated as the percentage of pomelo segments (fresh/
dried) increased. It has been reported that the reduction in loaf vol-
ume could be due to the reduction in gluten content as a result of
supplementation (Sengev, Abu, & Gernah, 2013). Low gluten con-
tent of flour lowers the ability of the flour to extend (elasticity)
and retains the carbon dioxide produced during fermentation
thereby yielding a decreased loaf volume. According to Ragaee,
Guzar, Dhull, and Seetharaman (2011), partial substitution of
wheat flour with some grains such as barely, cellulose and oat
caused a reduction in volume of loaves of bread. This could be
explained by the fact that substitution of bread samples with
pomelo dry segments, caused gluten dilution and consequently
affected the optimal gluten matrix formation during the processing
of the breads (mixing, fermentation and baking).
the parameters a⁄. The bread samples partially substituted with
various concentration of pomelo had significantly (p < 0.05)
increased a⁄ value compared to control. The increasing amount of
pomelo segments in bread formula increased the redness gradually
with significant difference among all formulated breads. This could
be visually seen since the bread samples containing pomelo seg-
ments (fresh/dry) were pinkish compared to control which is of
white color. A very slight increase in the b⁄ (yellowness) value of
bread crumbs was observed (p < 0.05) with the addition of pomelo
segments by increasing the concentration. Color appeared to be a
very important criterion for the initial acceptability of the baked
product by the consumer. The color depends both on the physio-
chemical characteristics of the dough (i.e., water content, pH,
reducing sugars and amino acid content) and on the operating con-
ditions applied during baking which includes temperature, air
speed, relative humidity, and modes of heat transfer
(Schoenlechner, Szatmari, Bagdi, & Tömösközi, 2013). It was
observed that the color of the crumb sample of both white and
brown bread showed significant (p < 0.05) increase in redness
(a⁄ value) and yellowness (b⁄ value) but decrease in L⁄ value with
higher percentage of pomelo segments (Fig. 1a and b). This might
be attributed from the reddish color imparted by the fruit seg-
ments incorporated.

3.2. Color measurement 3.3. Sensory evaluation

The color of breads with pomelo fresh segments (10, 20 and
30%) and dry segments (2.5, 5 and 7.5%) differed statistically from
the control bread; formulated bread also significantly differed one
from the other (p < 0.05) by the addition of pomelo segments
(Table 1). The color value of brown breads is higher when com-
pared with that of white breads. However, the trend of L⁄, a⁄ and
b⁄ value in both white and brown bread were similar. They showed
a decrease (p < 0.05) in L value of pomelo segments (fresh/dry)
supplemented bread from lower to higher concentration which
indicates the increase in the development of darker color of the for-
mulated breads. But the breads differed significantly in relation to
The sensory evaluation of breads is presented in Table 2. The
evaluation was done on a seven-point hedonic scale. The crust
color, symmetry, texture, eating quality and overall quality of
white and brown breads were comparable to control bread. The
crust color of the white bread changed from dark brown to light
with increasing pomelo segments (fresh/dry). In brown bread,
the score was found to be similar in all formulations. The more
brownish bread appearance could be attributed to the high fiber
content in the bread (Hu, Yang, Ma, & Zhou, 2007). The brown color
of the bread is due to the caramelization and Maillard reaction, in
which protein and sugar of the flours react with each other during
 S.K. Reshmi et al. / Food Chemistry 237 (2017) 957–965
Fig. 1. Whole and sliced bread supplemented with fresh and dry fruit segments of pomelo.
baking process (Dhingra & Jood, 2001). The significant (p < 0.05)
decrease in likeness for crust and crumb as the level of supplemen-
tation increased could be ascribed to the bitter and sourness of the
bread which is related to the fruit. Generally, addition of increasing
concentration of pomelo segments had significant effects on sen-
sory attributes and overall acceptability of bread samples. Addition
of segments caused darker color and denser texture, in both forms
of breads at the level of 20% (fresh) and 5% (dry) which seems to be
acceptable for consumers, with citrus flavor and bitterness at
palatable levels. However, formulated breads beyond above men-
tioned concentration were very sour and bitter which seem to have
negative effect on consumer’s overall acceptability.
3.4. Bioactive components in breads
The bio functional properties in developed products were eval-
uated to determine their retention during processing. Since the
product is recommended for diabetic populations, the total sugar
and reducing sugar content of the product were also determined.
There was an increase in the content of total sugars and reducing
sugars with the increase in the concentration of fresh and dry
segments (Table 3). White bread showed lower range of sugar
content (4.47–6.97 mg/100 g) compared to brown breads which
ranged between 5.28 and 7.26 mg/100 g. Similar pattern of result
961
were observed in case of reducing sugar which ranged between
2.05–2.93 mg/100 g (white bread) and 2.49–3.47 mg/100 g
(brown bread). Kumar, Vijay, and Khan (2013) reported that the
total soluble sugar from pomelo juice is 4.87 mg/100 ml which
is in accordance with our results. Thus, addition of pomelo seg-
ments in bread formulation has shown a gradual increase in
sugar content.
The total phenolics and flavonoids in products were significantly
higher in breads supplemented with pomelo segments (Table 3).
The phenolic content varied from 60.12 to 90.19 mg GAE/100 g in
white breads and 65.11 to 95.66 mg GAE/100 g in brown breads.
The flavonoid content in white bread was 21.80–38.11 mg CE/100 g
and 23.32–42.0 mg CE/100 g in brown breads. The content
increased with the increase in pomelo segments. Breads supple-
mented with dry segments have shown prominent level of pheno-
lics and flavonoids compared to fresh segments. Phenolics are
secondary metabolites, which mainly include flavonoids, coumar-
ins, stilbenes and tannins. Recent interest in plant polyphenols
has focused on their potential benefits to human health. Several
previous reports have also suggested that the bioactive components
such as phenolics, flavonoids and their glycosides can act as an
effective inhibitors of a-glucosidases (Myung-Hee, Sung-Hoon,
Hae-Dong, Mee Sook, & Young-In, 2010), oxidative stress, microbial
growth and even as a remedy for other medical problems.
962 S.K. Reshmi et al. / Food Chemistry 237 (2017) 957–965

Table 2
Sensory evaluation of white and brown bread supplemented with fresh/dry pomelo segments.

Pomelo segments (%) Crust Crumb

Colour (7) Symmetry (7) Color (7) Texture (7) Eating quality (7) Overall quality (7)
White bread
0 6.0a 6.0a 6.0a 6.0a 5.5a 5.5a
Fresh
10 6.0a 6.0a 5.5b 6.0a 5.0b 5.0b
20 5.5b 6.0a 5.0c 6.0a 5.0b 5.0b
30 5.0c 6.0a 4.5d 5.5b 5.0b 4.0d
Dry
2.5 6.0a 6.0a 5.5b 5.5b 5.0b 5.5a
5 5.5b 6.0a 5.0c 5.0c 4.5c 5.0b
7.5 5.0c 5.5b 4.5d 4.5d 4.0d 4.5c

Brown bread
0 6.0a 6.0a 5.0a 4.5b 4.5a 5.0a
Fresh
10 6.0a 6.0a 5.0a 4.5b 4.5a 5.0a
20 5.5b 6.0a 4.5b 4.5b 4.0b 4.5b
30 5.5b 6.0a 4.5b 5.0a 3.5c 4.0c
Dry
2.5 5.5b 6.0a 4.5b 5.0a 4.5a 5.0a
5 5.5b 6.0a 4.0c 5.0a 4.0b 4.5b
7.5 5.5b 6.0a 4.0c 4.5b 3.0c 3.5d
SEM (±) 0.12 0.20 0.16 0.14 0.21 0.22

Values in the parenthesis indicate maximum score; Values for a particular column followed by different letters differ significantly (p < 0.05); SEM – Standard error of mean at
70 degrees of freedom.

Table 3
Bio-active components of white and brown bread supplemented with fresh/dry pomelo segments.

Pomelo segments (%) Sugars (g/100 g) Reducing sugars (g/100 g) Phenolics (mg GAE/100 g) Flavonoids (mg CE/100 g) Carotenoids (mg/100 g)
White bread
0 4.27 ± 0.09 1.66 ± 0.08 58.00 ± 1.09 20.13 ± 0.67 23.06 ± 0.98
Fresh
10 4.47 ± 0.16 2.06 ± 0.11 60.12 ± 1.06 21.80 ± 1.22 42.22 ± 0.89
20 4.88 ± 0.17 2.13 ± 0.11 63.5 ± 1.12 22.51 ± 0.78 70.05 ± 1.22
30 5.17 ± 0.11 2.45 ± 0.17 69.14 ± 0.74 25.92 ± 1.22 127.77 ± 1.08
Dry
2.5 5.07 ± 0.11 2.33 ± 0.08 66.85 ± 1.22 26.09 ± 1.34 100.35 ± 0.77
5 5.67 ± 0.15 2.56 ± 0.15 78.42 ± 1.19 32.56 ± 1.09 151.21 ± 1.05
7.5 6.97 ± 0.07 2.90 ± 0.13 90.19 ± 1.02 38.04 ± 1.35 231.78 ± 1.14

Brown bread
0 4.98 ± 0.18 2.32 ± 0.09 63.02 ± 1.22 21.80 ± 0.98 39.57 ± 0.65
Fresh
10 5.28 ± 0.08 2.49 ± 0.13 65.11 ± 1.35 23.32 ± 1.33 55.68 ± 1.06
20 5.47 ± 0.17 2.81 ± 0.11 68.98 ± 1.19 25.17 ± 1.22 84.34 ± 0.88
30 5.97 ± 0.11 3.29 ± 0.16 72.85 ± 1.27 26.94 ± 1.08 143.06 ± 0.74
Dry
2.5 5.80 ± 0.09 2.96 ± 0.08 73.36 ± 1.34 27.23 ± 1.45 111.25 ± 0.98
5 6.59 ± 1.18 3.36 ± 0.11 84.16 ± 1.09 35.72 ± 1.66 158.11 ± 0.85
7.5 7.26 ± 0.13 3.47 ± 0.16 95.66 ± 1.30 42.88 ± 1.54 244.53 ± 1.02

GAE-Gallic acid equivalent; CE- Catechin equivalent; Values are means ± standard deviation (n = 4).

The carotenoid content of the products ranged between 42.22–
231.78 mg/100 g (white bread) and 55.68–244.53 mg/100 g (brown
bread) (Table 3). In fact, the stability of carotenoids in foods is vari-
able. It depends not only on the extrinsic factors (heat treatment,
presence or absence of light) but also on the other characteristics
of the food matrices such as their chemical composition, oxygen
dissolved, size of the particles and the physical state of the carote-
noid in the food (Vásquez-Caicedo, Schilling, Carle, & Neidhart,
2007). Carotenoids also play a potential role in human health by
acting as biological antioxidants (Bendich, 1989), anti-cancer
(Nishino, 1998) and antimicrobial agents (Manimala &
Murugesan, 2014).
The nutritional factors (phenolics, flavonoid and carotenoid) are
high in brown bread. However, there is not much significant vari-
ation between both the bread types. According to Slavin (2004), the
product based on whole grain has components that are associated
with improved health status which include lignans, tocotrienols,
phenolic compounds, anti-nutrients and enzyme inhibitors. In the
grain-refining process, the bran is removed resulting in the loss
of components. Hence, refined grains product (white bread) has
 S.K. Reshmi et al. / Food Chemistry 237 (2017) 957–965
Table 4
In-vitro starch digestibility and predicted glycemic index of bread supplemented with pomelo fruit segments.
Pomelo segments (%) TS (%) RS (%)
White bread
0 69.27 ± 1.09 3.02 ± 1.40
Fresh* 69.87 ± 2.57 3.87 ± 1.64
Dry** 71.23 ± 1.77 4.96 ± 1.29
Brown bread
0 73.66 ± 1.56 4.98 ± 1.55
Fresh* 74.75 ± 2.38 5.17 ± 1.99
Dry** 75.47 ± 1.88 10.96 ± 1.30
*
-20%; **-5%; TS – Total starch; RS – Resistant starch; DS – Digestible starch; HI –Hydrolysis index; pGI – Predicted Glycemic index; Values are means ± standard deviation
(n = 4).
Fig. 2. Rate of starch hydrolysis of white and brown bread supplemented with fresh/dry pomelo segment.
shown lesser nutritional value compared whole wheat products
(brown bread).
3.5. Retention of naringin in supplemented breads
Naringin, a well-known flavanone glycoside of Citrus fruits,
possesses antioxidant, anti-inflammatory, anti-apoptotic, anti-
diabetic, anti-ulcer, anti-osteoporosis and anti-carcinogenic prop-
erties (Cui, Zhang, Sun, & Jia, 2012). Naringin is the bioactive
compound that dampens postprandial glycemic response and
offers a potential complementary approach in the management
of diabetes (Priscilla, Roy, Suresh, Kumar, & Thirumurugan,
2014). Hence, retention of naringin in processed products was
evaluated (Fig. S1). The retention of naringin in white bread was
60% in fresh segments and 70–80% in dry segments. In case of
brown bread, the retention were comparatively less. Fifty percent
and 65% retention were observed in brown bread formulation
incorporated with fresh and dry segments respectively. In both
types of breads, the formulated bread with dry segments has
showed better retention of naringin content with the minimum
loss. The percentage of retention in bread samples were calculated
based on the amount of naringin present in fresh and dry segments
taken for product development (Fig. S2). Thus, the naringin content
in the prepared bread could potentially act against carbohydrate
hydrolyzing enzymes which in turn prevents post-prandial
hyperglycemia.
3.6. Starch digestibility and glycemic index
Based on the sensory attributes of the products, 20% fresh and
5% dry segments incorporated white and brown breads were
963
DS (%) HI (%) pGI (%)
66.25 ± 0.56 48.00 ± 1.19 66.06 ± 1.53
66.00 ± 0.44 42.38 ± 2.11 62.97 ± 1.77
66.27 ± 0.32 35.42 ± 1.47 59.15 ± 1.69
68.68 ± 0.62 41.35 ± 1.27 62.41 ± 0.88
69.58 ± 0.84 33.69 ± 2.23 58.20 ± 1.94
64.51 ± 0.59 24.46 ± 1.68 53.13 ± 1.12
selected for further studies on in vitro starch digestibility. The level
(%) of TS, RS and DS of the developed bread products are presented
in Table 4. The TS content in the products ranged between 69.27
and 75.47%. Resistant starch (RS) content varied among products
with a range of 3.02–10.96% on dry basis. With reference to control
bread, the RS content was higher in brown bread compared to
white bread. High RS value of 10.96% was observed in 5% dry
pomelo segment supplemented brown bread which resulted in
the lower value of digestible starch (DS) of 64.51% that is signifi-
cantly different from the other formulated breads having high DS
value. In the present study, although the developed products had
similar starch content, they differed in the rate of starch digestion.
The variations may be due to differences in protein content, dietary
fiber, nature of starch and extent of starch gelatinization. Davis
(1994) reported that the wheat starch swells more slowly than
other starches, limiting the extent of starch gelatinization.
Chandrashekar and Kirleis (1988) reported that the presence of
protein bodies around starch granules may restrict granule swel-
ling and starch gelatinization and as a result reduces the suscepti-
bility to enzymatic attack. This may be partially responsible for the
low digestibility. In accordance to the above reports, the product
developed with the supplementation of pomelo segments (rich in
protein, dietary fiber and naringin content) has showed increased
level of resistant starch with the increase in concentration of fruit
segments.
Hydrolysis Indices (HI) calculated from the rate of hydrolysis
over time (Fig. 2) and the corresponding predicted Glycemic
Indices (pGI) are presented in Table 4. Among white and brown
breads; the white bread has shown higher hydrolysis index which
resulted in higher predicted glycemic index (pGI) ranging from
66.06 to 59.15%. The lowest HI and pGI was recorded in brown
964 S.K. Reshmi et al. / Food Chemistry 237 (2017) 957–965
breads that ranged between 62.41 and 53.13%. According to
Jenkins et al. (2008), foods with GI of 55, 56–69 and 70 are clas-
sified as low, medium and high GI, respectively. The observation of
low pGI in this study could be attributed to high fraction of RS
among samples. As stated previously, the bread with the pomelo
segments (rich in naringin) have shown lower and gradual release
of glucose than the control bread. These findings agree with the
fact that naringin and other bio-functional components act syner-
gistically to inhibit the enzymes involved in the post-prandial
hyperglycemia (Shen, Xu, & Lu, 2012). Brand-Miller (1994) has
demonstrated the therapeutic value of low GI diet in type 1 and
type 2 diabetic patients. Studies have also shown that diet with
low GI and high RS help in reducing insulin resistance, adjusts
blood glucose level, improves lipid metabolism and prevents car-
diovascular and cerebrovascular diseases (Chung, Sanguansri, &
Augustin, 2010). Thus, GI is related to nutritional quality of food
and a product with a low GI is preferable not only for individuals
with diabetes, but also for normal population (Björck & Asp, 1994).
4. Conclusion
Thus, the study indicates that supplementation of pomelo seg-
ments has a great potential in developing bakery products for
health benefits. Among the bread formulations, the physicochemi-
cal characteristics of bread supplemented with fresh fruit seg-
ments was better compared to dry fruit segments. The 20% fresh
and 5% dry supplemented bread (white and brown) were sensori-
ally acceptable. The bioactive components such as phenolics, flavo-
noids and carotenoids were found to be higher in supplemented
brown breads. The bioactive compound, naringin retained in bread
formulations had contributed to lower glycemic index by inhibit-
ing the digestive enzymes. Dry segment-incorporated bread
showed higher resistance towards starch digestion thereby lower-
ing the release of glucose. Inhibition of the carbohydrate hydrolyz-
ing enzymes involved in post-prandial hyperglycemia is an
important strategy for the management of type II diabetes. Thus,
the present finding suggests that the pomelo-fortified bread can
be recommended as suitable food for diabetic populations.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
We are grateful to Prof. Ram Rajasekharan, Director, CSIR-CFTRI,
Mysuru for constant encouragement throughout the course of
study and Department of Biotechnology (grant numbers: BT/
PR5994/FNS/20/563/2012), Govt. of India, New Delhi, India for
their financial support.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2017.
05.138.
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