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Starch digestibility and predicted glycemic index in the bread fortified with pomelo (Citrus maxima) fruit segments
Accepted Manuscript Starch Digestibility and Predicted Glycemic Index in the Bread Fortified with Pomelo (Citrus maxima) Fruit Segments S.K. Reshmi, M.L. Sudha, M.N. Shashirekha PII: DOI: Reference:
S0308-8146(17)30949-4 http://dx.doi.org/10.1016/j.foodchem.2017.05.138 FOCH 21199
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
28 December 2016 8 April 2017 29 May 2017
Please cite this article as: Reshmi, S.K., Sudha, M.L., Shashirekha, M.N., Starch Digestibility and Predicted Glycemic Index in the Bread Fortified with Pomelo (Citrus maxima) Fruit Segments, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/j.foodchem.2017.05.138
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Starch Digestibility and Predicted Glycemic Index in the Bread Fortified with Pomelo
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(Citrus maxima) Fruit Segments
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Reshmi S. Ka, Sudha M. Lb, Shashirekha M. Na, *
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a
b
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Department of Fruit and Vegetable Technology
Flour Milling, Baking & Confectionery Technology Department
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CSIR-CFTRI, Mysuru-570020
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*
Corresponding author
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Tel.: 0821- 2515653
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E-mail: [email protected]
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Abstract
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The aim of this study was to evaluate the starch digestibility and predicted glycemic index in
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breads incorporated with pomelo fruit (Citrus maxima) segments. Volume of the white and
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brown breads supplemented with pomelo fresh segments increased, while the crumb firmness
20
decreased. Bread with 20% fresh and 5% dry pomelo segments were sensorily acceptable.
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Bioactive components such as phenolics, flavonoids, naringin and carotenoids were retained to a
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greater extent in bread containing dry pomelo segments. The pomelo - incorporated bread had
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higher levels of resistance starch fractions (3.87-10.96%) with low predicted glycemic index
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(62.97-53.13%), despite their higher total starch (69.87-75.47%) content compared to control
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bread. Thus pomelo segments in the product formulations lowered the glycemic index probably
26
by inhibiting carbohydrate hydrolyzing enzyme activity which could be attributed to naringin.
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Hence fortified bread prepared from pomelo fruit segment is recommended to gain nutritional
28
value and to decrease the risk of diabetes.
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Key words: Pomelo segments, bread, naringin, glycemic index, biofunctional components
2
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1. Introduction
33
Recently, Innovative food products with health benefits are increasingly becoming
34
popular. Functional foods having a wide range of phytochemical profiles exhibit therapeutic
35
activity against various health related disorders (Jenkins et al., 2008). The concept of diet-based
36
therapies is aimed at maximizing the physiological benefits of various functional foods that
37
require product development (Siró, Kápolna, Kápolna & Lugasi, 2008). Foods having high
38
protein and fiber content are now generally preferred by consumers to maintain their health and
39
to act against many diseases like diabetes, obesity etc. So there is a new trend in the market to
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develop a product that has health benefits with acceptable sensory characteristics. Fruits are the
41
basic components of human diet. Apart from providing energy for metabolic pathways they also
42
act as precursor for protein synthesis and are a source of micronutrients like vitamins and
43
minerals. Citrus fruits have high economic and medicinal value because of their multiple uses in
44
the pharmaceutical, cosmetics, and food industries. These beneficial effects of citrus fruits are
45
attributed to their chemical constituents like vitamins, dietary fiber, carotenoids, flavonoids,
46
lipids, and essential oils. Citrus fruits are the utmost value fruit crop in terms of international
47
trade and it has been recommended in herbal medicine as the source of diabetic medication
48
(Andrade-Cetto, 1995).
49
Citrus maxima (Burm.) Merr., commonly known as pomelo is one of the largest
50
underutilized citrus fruits belonging to the family Rutaceae. It has been reported to act as an
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appetizer, cardiac stimulant, stomach tonic and also as a remedy for fever, insomnia, and sore
52
throat (Merina, Chandra & Jibon, 2012). Further, it shows various pharmacological activities
53
against oxidative stress (Mäkynen et al., 2013), inflammation (Shivananda, Muralidhara & 3
54
Jayaveera, 2013) and diabetes (Abdul, Shenoy, Hegde, Aamer & Shabaraya, 2014). Even though
55
several reports are available on the medicinal property of pomelo, there is a problem of its
56
availability in large quantities. No commercial cultivation is undertaken due to the bitterness and
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astringent nature of the fruits. Because of this reason, it is less utilized by common people when
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compared to other fruits like orange, lime and tangerines.
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Baking is a process that has been adopted for centuries and bakery products range from simple
60
ingredients plain pastry to the cake having numerous components. Bread is one of the bakery
61
products priced for its taste, aroma and texture. Bread making is a complex process which
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includes mixing, proofing and baking (Dewettinck, Van, Kuhne, Walle, Courtens & Gellynck,
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2008). Bread is considered as a well-liked staple food consumed as part of the daily diet
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worldwide. Annually ~ 9 million kg of bread are produced (Heenan, Dutour, Hamud, Harvey &
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Delahurry, 2008). The popularity of bakery products has contributed to increased demand for
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ready to eat and convenient food products such as bread, cakes, biscuits etc. So initiative has
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been taken in this research work to use pomelo as a food fortificant for increasing the
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consumption of this fruit for health benefits. This study was conducted to develop value added
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white bread and brown bread using pomelo fruit segments. The prepared products were further
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analyzed for their glycemic index, retention of naringin and other biofunctional components to
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ensure health promoting properties of pomelo retained even after the processing.
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2. Materials and Methods
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Commercial wheat flour (10.2% moisture, 0.51% ash, 10.6% gluten, 24 ml sedimentation
74
value and 373 s falling number), compressed yeast (Tower brand, Mumbai), sugar powder and
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vegetable fat (Hindustan Unilever Ltd, Bangalore) were procured from local market. 4
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2.1. Pomelo fruit processing
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The Citrus maxima (pomelo) fruits were obtained from the local market of Mysuru,
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Karnataka, India during the month of February 2016. The fresh segments were separated from
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the fruit manually and dried in hot air oven at 35 ºC for overnight to obtain dry fruit segments
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(residual moisture content of ~5%).
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2.2. Bread making characteristics
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Effect of fresh pomelo fruit segments (0%, 10%, 20% and 30%) and dried pomelo fruit
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segments (0%, 2.5%, 5% and 7.5%) on white bread and brown bread making characteristics was
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studied (Sudha & Leelavathi, 2008). The formulation used was flour: 100%, pomelo fruit
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segments, compressed yeast: 2.0%, vegetable fat: 1%, salt: 1.0%; sugar: 2.5% and water. All the
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ingredients were mixed in a Hobart mixer (Model N-50, Hobart, GmbH, Germany) with a flat
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blade for 4 min at 61 rpm. The dough obtained was fermented in a chamber maintained at 300C
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and 75% relative humidity (RH) for 90 min. After 90 min, the dough was remixed and relaxed
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for 25 min, molded, proofed for 55 min at 300C, 85% RH and baked for 25 min at 2200C, cooled
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for physical and sensory evaluation. Part of the bread samples were dried at 450C for 5h, cooled,
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homogenized and stored in poly propylene bags for various estimations.
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2.3. Evaluation of breads
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Weight of the breads was taken and volume of the loaves was measured by rapeseed
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displacement method (Sudha & Leelavathi, 2008). Bread crumb firmness, the objective
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measurement of texture was carried out in a texture analyzer (TAHDi, Stable Micro Systems, 5
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Godalming, UK) by the standard AACC, (2000) (74-09) and 2.0 mm.s-1 of pre-test speed and
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1.67 mm.s-1 of test speed were used. Force required to compress 25% of the bread slice was
100
recorded using 35 mm diameter aluminium cylinder probe P-35. Objective evaluation of colour
101
of bread crumb was measured using the Hunter Lab Colour Measuring System (Colour Flex-EZ
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Hunter Lab, USA) with a reflectance attachment of illuminant G against a standard white board
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made of barium sulphate (100% whiteness). Bread slice (3cm x 3cm) was placed in the sample
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holder and the reflectance from the surface measured.
105 106
2.4. Sensory evaluation
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A scorecard containing the description for the desirable (creamish white – color; crisp –
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texture; wholesome sweetish – taste) and undesirable (dull or dark color; soft or hard – texture;
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unpleasant taste) quality characteristics for various sensory attributes viz. color of crust and
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crumb, texture, mouthfeel and overall quality were given to the panelist consisting of men and
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women of 35-50 age group. The panelist was then asked to assign scores for each parameter as
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against the maximum scores given in the parenthesis using a 7-point hedonic rating scale :
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excellent - 7, very good - 6, good - 5, satisfactory -4, fair - 3, poor - 2 and very poor -1 (Rathi,
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Kawatra, and Sehgal, 2004).
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2.5. Estimation of total sugars
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The total sugars estimation in the samples was carried out using the method described by
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Albalasmeh, Berhe, & Ghezzehei (2013). An aliquot of 10μl of sample was mixed with 300μl of
119
5% aqueous solution of phenol. After 5 min of incubation 1.8 mL of concentrated sulfuric acid 6
120
was added rapidly to the mixture. The test tubes were cooled and absorption read at 490 nm.
121
Total sugar content of the sample was expressed as equivalent to mg glucose/g extract.
122 123
2.6. Estimation of reducing sugars
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Reducing sugars were estimated based on the modified method of Miller (1959). The
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sample (150 μl) was mixed with 1ml of DNS (3,5-Dinitrosalicylic acid) reagent in a test tube.
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The tubes were placed in a boiling water bath for 10 min and cooled for ten to fifteen minutes at
127
room temperature. Each solution was then diluted with 2mL of water, mixed thoroughly and
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absorbance was recorded at 540 nm using spectrophotometer (Helios Alpha, Thermo Electron
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Corporation, England, UK). Total reducing sugar content of the samples was expressed as
130
equivalent to mg glucose/g extract.
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2.7. Bioactive Components
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2.7.1. Estimation of total phenolic content
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It was evaluated using a modified colorimetric method described by Henríquez et al.,
135
(2010). The reaction mixture was prepared by adding 100μl of sample, 1.0mL of Folin-
136
Ciocalteau reagent and 2.0 mL of 1% sodium carbonate solution. The mixture was incubated for
137
60 min at room temperature, and the absorbance read at 765nm using an UV-Vis
138
spectrophotometer. The measurement was compared with standard gallic acid solution. The total
139
phenolic content was expressed equivalent to mg gallic acid/g extract.
140 141 7
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2.7.2. Estimation of flavonoids
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Flavonoids were estimated by a modified method of Lallianrawna, Muthukumaran, Ralte,
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Gurusubramanian and Senthil Kumar (2013). To 0.9 ml of sample, 75μl of 5% NaNO2 solution
145
was added. After 5 min, 150μl of 10% AlCl3.6H2O was added to the mixture, which was kept at
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room temperature for 5 more minutes. This was followed by the addition of 0.5 ml of 1M NaOH
147
and the total volume was made up to 2.5 ml with the addition of deionised water. The resulting
148
solution was mixed well and immediately, the absorbance was measured at 510nm on a UV-VIS
149
spectrophotometer. For the blank, the extracts were replaced with an equal volume of deionised
150
water. Total flavonoid content of the samples was expressed as the mg equivalent to catchin /g of
151
extract.
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2.7.3. Estimation of carotenoids
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The experiment was carried out by the modified procedure of Carvalho et al., (2012). The
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sample (1g) was homogenized in the dark (to avoid photolysis of carotenoids) with 20ml of
155
acetone. The filtrate was further transferred to a separating funnel containing 10-15ml of
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petroleum ether and mixed well. The lower aqueous layer was then transferred to another
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separating funnel and the upper petroleum ether layer containing the carotenoids was collected.
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The extraction was repeated until the aqueous layer became colorless. A small amount of
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anhydrous sodium sulphate was added to the petroleum ether extract to remove excess moisture.
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The final volume of the petroleum ether extract was noted. The absorbance of the yellow color
161
was read in a spectrophotometer at 450nm using petroleum ether as blank.
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Carotenoids content (μg/g) = A × V (mL) × 104 A1cm × P (g) 8
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2.7.4. Naringin content
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The white and brown breads were analyzed for the presence of naringin (bioactive
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compound) using HPLC (Shimadzu Class – VP HPLC model used with SPD-10AVP (PDA
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detector)) by slightly modifying the protocol of Pichaiyongvongdee and Haruenkit, 2009. One
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gram of the sample was extracted in methanol and homogenized for 30 min. The supernatant was
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passed through 0.45 mm syringe filters and subjected to HPLC. A standard solution was
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prepared by dissolving 1mg of naringin in 1ml with acetonitrile. Supelco C18 (5µm) column (15
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cm x 4.6 mm id), Supelco, USA was used with chromatographic solvent mixture (mobile phase)
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consisting of water: acetonitrile: (80:20 v/v) as mobile phase. The solvents were degassed with
173
vacuum before using in HPLC analysis. The mobile phase was pumped with a LC-10 ATVP
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pump at a flow rate of 1 ml/min. The injection volume was 20µl and the total run time was 15
175
min at 270nm. Quantification of the compound was evaluated by comparing the peak area with
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authentic standards using peak processing post-run integration parameters and external method:
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calibration type of Shimadzu class VP version 6.14 SPI data acquisition software.
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2.8. Total Starch (TS)
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The total starch in the bread samples was determined enzymatically according to the method
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described by Goni, Garci-Alonso & Suara-Calirto (1997). The ground sample (50 mg) was
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dispersed in 6mL of 2M KOH and shaken at room temperature for 30 min. Three ml of 0.4M
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Sodium acetate buffer pH 4.75 and 60µL of amyloglucosidase (EC-3.2.1.3, Sigma- Aldrich
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Chemical Company, St Louis, MO, USA) were added to this suspension and incubated for 45
9
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min at 60°C in a controlled shaking water bath. Starch was measured as glucose with glucose
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oxidase-peroxidase (GODPOD) kit. Factor conversion from glucose to starch was 0.9.
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2.9. Resistant starch (RS)
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Resistant starch was estimated according to the method of Goni et al., (1997). One hundred
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mg of each sample was incubated with pepsin solution containing 20mg of pepsin (EC-3.4.23.1,
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Sigma- Aldrich Chemical Company, St Louis, USA) for 60 min at 40oC for protein removal.
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Then the starch was hydrolyzed by adding pancreatic α-amylase (EC-3.2.1.1, Sigma- Aldrich
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Chemical Company, St Louis, USA) (10 mg/ml) solution containing amyloglucosidase (AMG)
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for 16 h at 37⁰C with constant shaking. After hydrolysis, samples were washed thrice with
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ethanol (99% v/v and 50% ethanol). The separated pellet from supernatant was further digested
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with 2 M KOH. Digested pellet and supernatant were separately incubated with AMG. Glucose
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released was measured using a glucose oxidase-peroxidase (GODPOD) reagent kit (K-GLOX,
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Megazyme Bray, Co. Wicklow, Ireland) by absorbance at 510 nm against the reagent blank. RS
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was calculated as glucose (mg) x 0.9. Digestable starch (DS) has been calculated as difference
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between TS and RS.
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2.10. Predicted glycemic index (pGI)
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A modified in vitro method based on the procedure of Goni et al., (1997) was adopted.
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The ground sample (100mg) was incubated with 10 mL HCl–KCl buffer (pH 1.5) and 200 μL
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pepsin solution (100 mg/mL HCl-KCl buffer) at 40°C for 1 h with constant shaking. The pH was
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raised by addition of 200 μL pancreatic α-amylase solution (1.5 mg /10 mL phosphate buffer; pH
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7.8) and incubated at 37°C for 45 min. Enzyme reaction was stopped with 70 μL Na2CO3 10
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solution and samples diluted to 25 mL with tris-maleate buffer (pH 6.9). Five mL of pancreatic
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α-amylase solution (3 U /5 mL tris-maleate buffer) was thereafter added to the sample and
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incubated at 37°C with constant shaking. Aliquots of 1 mL were taken at 30, 90 and 120 min
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from the samples and placed into boiling water with vigorous shaking for 5 min to inactivate the
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enzyme reaction. Samples were kept in refrigerator (4°C) after each inactivation until the end of
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incubation time (180 min).
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All aliquots were treated with 3 ml of 0.4 M sodium acetate buffer (pH 4.75) and 60 μL
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of amyloglucosidase (3300 U/ml) then incubated at 60°C for 45 min with constant shaking. After
216
incubation, volume was adjusted to 10 mL with distilled water, mixed properly and centrifuged
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before transferring 0.1 mL aliquots of the solution into glass test tubes for glucose measurement.
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The glucose released was measured using a glucose oxidase-peroxidase (GODPOD) kit
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(K-GLOX, Megazyme Bray, Co. Wicklow, Ireland). Absorbance was measured at 510 nm
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against the reagent blank using UV-vis spectrophotometer. The values were plotted on a graph
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and the area under the concentration-over-time curve (AUC) was determined using Sigmaplot
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10.0 (Systat Software, San Jose, CA, U.S.A.). The hydrolysis index (HI) was calculated as the
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percentage of total glucose released from the samples as compared to that released from standard
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glucose (0–180 min) (Barine and Yorte, 2016). The predicted glycemic indices of the samples
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were estimated according to the equation of Goni et al. (1997): pGI = 39.71 + 0.549HI.
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2.11. Statistical analysis
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Data were statistically analyzed using Duncan’s new multiple range tests using GraphPad
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Prism software version 4.03 for Windows (San Diego, CA, USA) with different experimental 11
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groups appropriate to the completely randomized design with four replicates each as described
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by Sudha and Leelavathi (2008) at p < 0.05.
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3. Results and Discussion
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3.1. Quality characteristics of white and brown bread
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The physical attributes of white and brown bread incorporated with different levels of
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fresh and dry pomelo segments are presented in Table 1. The substitution of fresh pomelo
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segments (0 – 30%) in white bread formulation increased the volume from 567-625 ml and
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decreased the specific volume from 4.05-3.98 ml/g whereas for brown bread the same increased
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from 425-485 ml and 2.77-2.97 ml/g respectively. However, incorporation of dry pomelo
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segments decreased the loaf volume and specific volume in both types of bread ranging from
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575-534 ml and 3.96-3.34 ml/g for white bread and 480-410 ml and 2.94-2.31 ml/g for brown
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bread indicating that the volume of the brown bread is comparatively lower than the white bread.
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Scores of softness attribute were in accordance with the results of texture analysis (Table 1). The
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bread supplemented with fresh segments in both white (397-302 g/force) and brown (1056-679
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g/force) breads indicate that the softness of the bread increased with increase in fresh segments.
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The crumb firmness increased in breads supplemented with dried pomelo segments from 308-
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429 g/force (white bread) and 685-829 g/force (brown bread). There was significant (p < 0.05)
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decrease in preference in all the attributes evaluated as the percentage of pomelo segments
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(fresh/dried) increased. It has been reported that the reduction in loaf volume could be due to the
250
reduction in gluten content as a result of supplementation (Sengev, Abu & Gernah, 2013). Low
251
gluten content of flour lowers the ability of the flour to extend (elasticity) and retains the carbon
252
dioxide produced during fermentation thereby yielding a decreased loaf volume. According to 12
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Ragaee, Guzar, Dhull and Seetharaman (2011), partial substitution of wheat flour with some
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grains such as barely, cellulose and oat caused a reduction in volume of loaves of bread. This
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could be explained by the fact that substitution of bread samples with pomelo dry segments,
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caused gluten dilution and consequently affected the optimal gluten matrix formation during the
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processing of the breads (mixing, fermentation and baking).
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3.2. Color measurement
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The color of breads with pomelo fresh segments (10, 20 and 30%) and dry segments (2.5,
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5 and 7.5%) differed statistically from the control bread; formulated bread also significantly
261
differed one from the other (p < 0.05) by the addition of pomelo segments (Table 1). The color
262
value of brown breads is higher when compared with that of white breads. However, the trend of
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L*, a* and b* value in both white and brown bread were similar. They showed a decrease (p <
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0.05) in L value of pomelo segments (fresh/dry) supplemented bread from lower to higher
265
concentration which indicates the increase in the development of darker color of the formulated
266
breads. But the breads differed significantly in relation to the parameters a*. The bread samples
267
partially substituted with various concentration of pomelo had significantly (p<0.05) increased
268
a* value compared to control. The increasing amount of pomelo segments in bread formula
269
increased the redness gradually with significant difference among all formulated breads. This
270
could be visually seen since the bread samples containing pomelo segments (fresh/dry) were
271
pinkish compared to control which is of white color. A very slight increase in the b*
272
(yellowness) value of bread crumbs was observed (p<0.05) with the addition of pomelo segments
273
by increasing the concentration. Color appeared to be a very important criterion for the initial
274
acceptability of the baked product by the consumer. The color depends both on the physio13
275
chemical characteristics of the raw dough (i.e, water content, pH, reducing sugars and amino acid
276
content) and on the operating conditions applied during baking which includes temperature, air
277
speed, relative humidity, and modes of heat transfer (Schoenlechner, Szatmari, Bagdi &
278
Tömösközi, 2013). It was observed that the color of the crumb sample of both white and brown
279
bread showed significant (p < 0.05) increase in redness (a* value) and yellowness (b* value) but
280
decrease in L* value with higher percentage of pomelo segments (Fig. 1a and b). This might be
281
attributed from the reddish color imparted by the fruit segments incorporated.
282
3.3. Sensory evaluation
283
The sensory evaluation of breads is presented in Table 2. The evaluation was done on a
284
seven-point hedonic scale. The crust color, symmetry, texture, eating and overall qualities of
285
white and brown breads were comparable to control bread. The crust color of the white bread
286
changed from dark brown to light with increasing pomelo segments (fresh/dry). In brown bread,
287
the score was found to be similar in all formulations. The more brownish bread appearance could
288
be attributed to the high fiber content in the bread (Hu, Yang, Ma & Zhou, 2007). The brown
289
color of the bread is due to the caramelization and Maillard reaction, in which protein and sugar
290
of the flours react with each other during baking process (Dhingra & Jood, 2001). The significant
291
(p < 0.05) decrease in likeness for crust and crumb as the level of supplementation increased
292
could be ascribed to the bitter and sourness of the bread which is related to the fruit. Generally,
293
addition of increasing concentration of pomelo segments had significant effects on sensory
294
attributes and overall acceptability of bread samples. Addition of segments caused darker color
295
and denser texture, in both forms of breads at the level of 20% (fresh) and 5% (dry) which seems
296
to be acceptable for consumers, with citrus flavor and bitterness at palatable levels. However, 14
297
formulated breads beyond above mentioned concentration were very sour and bitter which seem
298
to have negative effect on consumer’s overall acceptability.
299
3.4. Bioactive components in breads
300
The bio functional properties in developed products were evaluated to determine their
301
retention during processing. Since the product is recommended for diabetic populations, the total
302
sugar and reducing sugar content of the product were also determined. There was an increase in
303
the content of total sugars and reducing sugars with the increase in the concentration of fresh and
304
dry segments (Table 3). White bread showed lower range of sugar content (4.47-6.97mg/100g)
305
compared to brown breads which ranged between 5.28-7.26mg/100g. Similar pattern of result
306
were observed in case of reducing sugar which ranged between 2.05-2.93mg/100g (white bread)
307
and 2.49-3.47mg/100g (brown bread). Kumar, Vijay and Khan (2013) reported that the total
308
soluble sugar from pomelo juice is 4.87mg/100ml which is in accordance with our results. Thus,
309
addition of pomelo segments in bread formulation has shown a gradual increase in sugar content.
310
The total phenolics and flavonoids in products were significantly higher in breads
311
supplemented with pomelo segments (Table 3). The phenolic content varied from 60.12-90.19mg
312
GAE/100g in white breads and 65.11-95.66mg GAE/100g in brown breads. The flavonoid
313
content in white bread was 21.80-38.11mg CE/100g and 23.32-42.0mg CE/100g in brown
314
breads. The content increased with the increase in pomelo segments. Breads supplemented with
315
dry segments have shown prominent level of phenolics and flavonoids compared to fresh
316
segments. Phenolics are secondary metabolites, which mainly include flavonoids, coumarins,
317
stilbenes and tannins. Recent interest in plant polyphenols has focused on their potential benefits
318
to human health. Several previous reports have also suggested that the bioactive components 15
319
such as phenolics, flavonoids and their glycosides can act as an effective inhibitors of α-
320
glucosidases (Myung-Hee, Sung-Hoon, Hae-Dong, Mee Sook & Young-In, 2010), oxidative
321
stress, microbial growth and even as a remedy for other medical problems.
322
The carotenoid content of the products ranged between 42.22-231.78µg/100g (white
323
bread) and 55.68-244.53 µg/100g (brown bread) (Table. 3). In fact, the stability of carotenoids in
324
foods is variable. It depends not only on the extrinsic factors (heat treatment, presence or absence
325
of light) but also on the other characteristics of the food matrices such as their chemical
326
composition, oxygen dissolved, size of the particles and the physical state of the carotenoid in
327
the food (Vásquez-Caicedo, Schilling, Carle & Neidhart, 2007). Carotenoids also play a potential
328
role in human health by acting as biological antioxidants (Bendich, 1989), anti-cancer (Nishino,
329
1998) and antimicrobial agents (Manimala & Murugesan, 2014).
330
The nutritional factors (phenolics, flavonoid and carotenoid) are high in brown bread.
331
However, there is not much significant variation between both the bread types. According to
332
Slavin (2004), the product based on whole grain has components that are associated with
333
improved health status which include lignans, tocotrienols, phenolic compounds, anti-nutrients
334
and enzyme inhibitors. In the grain-refining process, the bran is removed resulting in the loss of
335
components. Hence, refined grains product (white bread) has shown lesser nutritional value
336
compared whole wheat products (brown bread).
337
3.5. Retention of naringin in supplemented breads
338
Naringin, a well-known flavanone glycoside of Citrus fruits, possesses antioxidant, anti-
339
inflammatory, anti-apoptotic, anti-diabetic, anti-ulcer, anti-osteoporosis and anti-carcinogenic
340
properties (Cui, Zhang, Sun & Jia, 2012). Naringin is the bioactive compound that dampens 16
341
postprandial glycemic response and offers a potential complementary approach in the
342
management of diabetes (Priscilla, Roy, Suresh, Kumar, & Thirumurugan, 2014). Hence,
343
retention of naringin in processed products was evaluated (Fig. S1). The retention of naringin in
344
white bread was 60% in fresh segments and 70-80% in dry segments. In case of brown bread, the
345
retention were comparatively less. Fifty percent and 65% retention were observed in brown
346
bread formulation incorporated with fresh and dry segments respectively. In both types of
347
breads, the formulated bread with dry segments has showed better retention of naringin content
348
with the minimum loss. The percentage of retention in bread samples were calculated based on
349
the amount of naringin present in fresh and dry segments taken for product development (Fig.
350
S2). Thus, the naringin content in the prepared bread could potentially act against carbohydrate
351
hydrolyzing enzymes which in turn prevents post-prandial hyperglycemia.
352 353
3.6. Starch digestibility and glycemic index
354
Based on the sensory attributes of the products, 20% fresh and 5% dry segments
355
incorporated white and brown breads were selected for further studies on in-vitro starch
356
digestibility. The level (%) of TS, RS and DS of the developed bread products are presented in
357
Table 4. The TS content in the products ranged between 69.27 to 75.47 %. Resistant starch (RS)
358
content varied among products with a range of 3.02 – 10.96% on dry basis. With reference to
359
control bread, the RS content was higher in brown bread compared to white bread. High RS
360
value of 10.96% was observed in 5% dry pomelo segment supplemented brown bread which
361
resulted in the lower value of digestible starch (DS) of 64.51% that is significantly different from
362
the other formulated breads having high DS value. In the present study, although the developed 17
363
products had similar starch content, they differed in the rate of starch digestion. The variations
364
may be due to differences in protein content, dietary fiber, nature of starch and extent of starch
365
gelatinization. Davis (1994) reported that the wheat starch swells more slowly than other
366
starches, limiting the extent of starch gelatinization. Chandrashekar and Kirleis (1988) reported
367
that the presence of protein bodies around starch granules may restrict granule swelling and
368
starch gelatinization and as a result reduces the susceptibility to enzymatic attack. This may be
369
partially responsible for the low digestibility. In accordance to the above reports, the product
370
developed with the supplementation of pomelo segments (rich in protein, dietary fiber and
371
naringin content) has showed increased level of resistance starch with the increase in
372
concentration of fruit segments.
373
Hydrolysis Indices (HI) calculated from the rate of hydrolysis over time (Fig. 2) and the
374
corresponding predicted Glycemic Indices (pGI) are presented in Table 4. Among white and
375
brown breads; the white bread has shown higher hydrolysis index which resulted in higher
376
predicted glycemic index (pGI) ranging from 66.06-59.15%. The lowest HI and pGI was
377
recorded in brown breads that ranged between 62.41-53.13%. According to Jenkins et al.,
378
(2008), foods with GI of ≤ 55, 56-69 and ≥70 are classified as low, medium and high GI,
379
respectively. The observation of low pGI in this study could be attributed to high fraction of RS
380
among samples. As stated previously, the bread with the pomelo segments (rich in naringin) have
381
shown lower and gradual release of glucose than the control bread. These findings agree with the
382
fact that naringin and other bio-functional components act synergistically to inhibit the enzymes
383
involved in the post-prandial hyperglycemia (Shen, Xu & Lu, 2012). Brand-Miller (1994) has
384
demonstrated the therapeutic value of low GI diet in type 1 and type 2 diabetic patients. Studies 18
385
have also shown that diet with low GI and high RS help in reducing insulin resistance, adjusts
386
blood glucose level, improves lipid metabolism and prevents cardiovascular and cerebrovascular
387
diseases (Chung, Sanguansri, & Augustin, 2010). Thus, GI is related to nutritional quality of
388
food and a product with a low GI is preferable not only for individuals with diabetes, but also for
389
normal population (Björck & Asp, 1994).
390 391
4. Conclusion
392
Thus, the study indicates that supplementation of pomelo segments has a great potential in
393
developing bakery products for health benefits. Among the bread formulations, the
394
physicochemical characteristics of bread supplemented with fresh fruit segments was better
395
compared to dry fruit segments. The 20% fresh and 5% dry supplemented bread (white and
396
brown) were sensorially acceptable. The bioactive components such as phenolics, flavonoids and
397
carotenoids were found to be higher in supplemented brown breads. The bioactive compound,
398
naringin retained in bread formulations had contributed to lower glycemic index by inhibiting the
399
digestive enzymes. Dry segment-incorporated bread showed higher resistance towards starch
400
digestion thereby lowering the release of glucose. Inhibition of the carbohydrate hydrolyzing
401
enzymes involved in post-prandial hyperglycemia is an important strategy for the management of
402
type II diabetes. Thus, the present finding suggests that the pomelo-fortified bread can be
403
recommended as suitable food for diabetic populations.
404 405
19
406
Supplementary data: Naringin content in fruit segments (Figure S1); Naringin content in breads
407
(Figure S2).
408 409
Acknowledgments
410
We are grateful to Prof. Ram Rajasekharan, Director, CSIR-CFTRI, Mysuru for constant
411
encouragement throughout the course of study and Department of Biotechnology (grant
412
numbers: BT/PR5994/FNS/20/563/2012), Govt. of India, New Delhi, India for their financial
413
support.
414 415
Conflict of Interest
416
The authors declare no conflict of interest.
20
417
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Figure Captions
564
Figure 1: Whole and sliced bread supplemented with fresh and dry fruit segments of pomelo.
565
Figure 2: Rate of starch hydrolysis of white and brown bread supplemented with fresh/dry
566
pomelo
segment
28
567
Fig. 1. Whole and sliced bread supplemented with fresh and dry fruit segments of pomelo.
568
a. White breads
569
570 571 572
b. Brown breads
573
574 575 576 577 578 579 29
580 581 582 583 584
Fig. 2. Rate of starch hydrolysis of white and brown bread supplemented with fresh/dry
585
pomelo segments.
586 587 588 589
30
Pomelo segments (%)
Volume (ml)
Specific Volume (ml/g)
Crumb firmness (g force)
Crumb colour L
A
b
ΔE
White bread 0 Fresh 10 20 30 Dry 2.5
590 591
567±1.89c
4.05±0.63a
397±0.98
67.57±0.31a
0.13±0.02h
12.26±0.03d
26.75±0.28ef
575±0.87c 600±1.44b 625±1.86a
3.91±0.89a 3.94±1.06a 3.98±1.76a
370±1.07f 341±1.16g 302±1.22g
66.92±0.35b 65.33±0.24b 65.91±1.39b
0.25±0.02h 0.56±0.34g 1.01±0.02f
12.09±0.08d 12.41±0.10d 12.48±0.11cd
26.36±0.29ef 27.11±0.24e 27.21±0.67e
575±1.09c
3.96±0.79a
308±1.09g
64.30±0.01bc
0.83±0.01f
12.45±0.57c
29.17±0.01d
Table 1. Physical characteristics of white and brown bread supplemented with fresh/dry pomelo segments
31
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
425±0.54g
2.77±1.45d
Brown bread 1056±0.98a 51.95±0.48d
3.66±0.17d
15.02±0.33a
42.19±0.72bc
450±1.36f 460±1.11f 485±1.90e
2.84±1.65c 2.90±1.39c 2.97±0.85c
777 ±1.30c 700 ±0.92d 679 ±0.77d
50.61±0.89d 50.11±1.13d 49.55±0.56d
3.92±0.10c 4.01±0.22c 4.26±0.07b
14.94±0.13b 14.70±0.20b 14.86±0.16b
42.16±0.66bc 43.46±1.11b 44.71±0.57a
480±0.48e 455±0.97f 410±1.36g 1.02
2.94±0.47c 2.67±0.67d 2.31±1.08e 0.11
685±1.09d 743±0.89c 829±1.11b 5.3
49.09±0.19d 47.59±0.47e 46.40±0.41e 1.01
4.03±0.07c 4.73±0.04b 5.07±0.06a 0.92
15.14±0.11a 15.56±0.14a 15.77±0.13a 0.45
42.49±0.68bc 43.85±0.38b 41.66±0.42c 2.01
0 Fresh 10 20 30 Dry 2.5 5 7.5 SEM (+) 592 593 594 595 596 597 598 599 600 601 602 603 604
32
605 606 607 608 609 610
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
33
611 612
Table 2. Sensory evaluation of white and brown bread supplemented with fresh/dry pomelo segments Pomelo segments (%)
Crust Colour Symmetry
Color (7)
Crumb Texture Eating quality (7) (7)
Overall quality (7)
(7)
(7)
0
6.0a
White bread 6.0a 6.0a
6.0a
5.5a
5.5a
10 20 30
6.0a 5.5b 5.0c
6.0a 6.0a 6.0a
5.5b 5.0c 4.5d
6.0a 6.0a 5.5b
5.0b 5.0b 5.0b
5.0b 5.0b 4.0d
2.5 5 7.5
6.0a 5.5b 5.0c
5.5b 5.0c 4.5d
5.0b 4.5c 4.0d
5.5a 5.0b 4.5c
0
6.0a
6.0a 5.5b 6.0a 5.0c 5.5b 4.5d Brown bread 6.0a 5.0a
4.5b
4.5a
5.0a
10 20 30
6.0a 5.5b 5.5b
6.0a 6.0a 6.0a
5.0a 4.5b 4.5b
4.5b 4.5b 5.0a
4.5a 4.0b 3.5c
5.0a 4.5b 4.0c
2.5 5 7.5 SEM (+)
5.5b 5.5b 5.5b 0.12
6.0a 6.0a 6.0a 0.20
4.5b 4.0c 4.0c 0.16
5.0a 5.0a 4.5b 0.14
4.5a 4.0b 3.0c 0.21
5.0a 4.5b 3.5d 0.22
Fresh
Dry
Fresh
Dry
613 614 615 616 617 618
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
619 620 34
621 622 623 624 625
Table 3. Bio-active components of white and brown bread supplemented with fresh/dry pomelo segments Pomelo segments (%)
Sugars (g/100g)
Reducing sugars (g/100g)
Phenolics
Flavanoids
(mgGAE/100g) (mgCE/100g)
Carotenoids (µg/100g)
White bread 0
4.27±0.09
1.66±0.08
58.00±1.09
20.13±0.67
23.06±0.98
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
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
Fresh
Dry
Brown Bread 0
4.98±0.18
2.32±0.09
63.02±1.22
21.80±0.98
39.57±0.65
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
2.5
5.80±0.09
2.66±0.08
73.36±1.34
27.23±1.45
111.25±0.98
5
6.59±1.18
3.26±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
Fresh
Dry
626 627 628
GAE-Gallic acid equivalent; CE- Catechin equivalent ; Values are means ± standard deviation (n=4) 35
629 630 631 632 633
Table 4. In-vitro starch digestibility and predicted glycemic index of bread supplemented with pomelo fruit segments Pomelo Segments (%)
TS
RS
DS
HI
pGI
(%)
(%) (%) White bread
(%)
(%)
0
69.27±1.09
3.02±1.40
66.25±0.56
48.00±1.19
66.06±1.53
Fresh*
69.87±2.57
3.87±1.64
66.00±0.44
42.38±2.11
62.97±1.77
Dry**
71.23±1.77
4.96±1.29
66.27±0.32
35.42±1.47
59.15±1.69
Brown bread 0
73.66±1.56
4.98±1.55
68.68±0.62
41.35±1.27
62.41±0.88
Fresh*
74.75±2.38
5.17±1.99
69.58±0.84
33.69±2.23
58.20±1.94
Dry**
75.47±1.88
10.96±1.30
64.51±0.59
24.46±1.68
53.13±1.12
634 635 636 637
*-20%; **-5%; TS- Total starch; RS- Resistance starch; DS- Digestible starch; HI-Hydrolysis index; pGI- Predicted Glycemic index; Values are means ± standard deviation (n=4)
638 639
36
640
Research Highlights
641 642
Quality and Sensory of white and brown bread fortified with pomelo is studied
643
The presence of bioactive compounds were higher in bread supplemented with pomelo
644
The glycemic index of developed products were evaluated
645
This study offers a new approach for producing bread with a lower starch digestion rate.
646
37
S0308-8146(17)30949-4 http://dx.doi.org/10.1016/j.foodchem.2017.05.138 FOCH 21199
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
28 December 2016 8 April 2017 29 May 2017
Please cite this article as: Reshmi, S.K., Sudha, M.L., Shashirekha, M.N., Starch Digestibility and Predicted Glycemic Index in the Bread Fortified with Pomelo (Citrus maxima) Fruit Segments, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/j.foodchem.2017.05.138
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1
Starch Digestibility and Predicted Glycemic Index in the Bread Fortified with Pomelo
2
(Citrus maxima) Fruit Segments
3 4
Reshmi S. Ka, Sudha M. Lb, Shashirekha M. Na, *
5
a
b
6
Department of Fruit and Vegetable Technology
Flour Milling, Baking & Confectionery Technology Department
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CSIR-CFTRI, Mysuru-570020
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*
Corresponding author
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Tel.: 0821- 2515653
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E-mail: [email protected]
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Abstract
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The aim of this study was to evaluate the starch digestibility and predicted glycemic index in
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breads incorporated with pomelo fruit (Citrus maxima) segments. Volume of the white and
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brown breads supplemented with pomelo fresh segments increased, while the crumb firmness
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decreased. Bread with 20% fresh and 5% dry pomelo segments were sensorily acceptable.
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Bioactive components such as phenolics, flavonoids, naringin and carotenoids were retained to a
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greater extent in bread containing dry pomelo segments. The pomelo - incorporated bread had
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higher levels of resistance starch fractions (3.87-10.96%) with low predicted glycemic index
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(62.97-53.13%), despite their higher total starch (69.87-75.47%) content compared to control
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bread. Thus pomelo segments in the product formulations lowered the glycemic index probably
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by inhibiting carbohydrate hydrolyzing enzyme activity which could be attributed to naringin.
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Hence fortified bread prepared from pomelo fruit segment is recommended to gain nutritional
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value and to decrease the risk of diabetes.
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Key words: Pomelo segments, bread, naringin, glycemic index, biofunctional components
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1. Introduction
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Recently, Innovative food products with health benefits are increasingly becoming
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popular. Functional foods having a wide range of phytochemical profiles exhibit therapeutic
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activity against various health related disorders (Jenkins et al., 2008). The concept of diet-based
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therapies is aimed at maximizing the physiological benefits of various functional foods that
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require product development (Siró, Kápolna, Kápolna & Lugasi, 2008). Foods having high
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protein and fiber content are now generally preferred by consumers to maintain their health and
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to act against many diseases like diabetes, obesity etc. So there is a new trend in the market to
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develop a product that has health benefits with acceptable sensory characteristics. Fruits are the
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basic components of human diet. Apart from providing energy for metabolic pathways they also
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act as precursor for protein synthesis and are a source of micronutrients like vitamins and
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minerals. Citrus fruits have high economic and medicinal value because of their multiple uses in
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the pharmaceutical, cosmetics, and food industries. These beneficial effects of citrus fruits are
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attributed to their chemical constituents like vitamins, dietary fiber, carotenoids, flavonoids,
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lipids, and essential oils. Citrus fruits are the utmost value fruit crop in terms of international
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trade and it has been recommended in herbal medicine as the source of diabetic medication
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(Andrade-Cetto, 1995).
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Citrus maxima (Burm.) Merr., commonly known as pomelo is one of the largest
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underutilized citrus fruits belonging to the family Rutaceae. It has been reported to act as an
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appetizer, cardiac stimulant, stomach tonic and also as a remedy for fever, insomnia, and sore
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throat (Merina, Chandra & Jibon, 2012). Further, it shows various pharmacological activities
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against oxidative stress (Mäkynen et al., 2013), inflammation (Shivananda, Muralidhara & 3
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Jayaveera, 2013) and diabetes (Abdul, Shenoy, Hegde, Aamer & Shabaraya, 2014). Even though
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several reports are available on the medicinal property of pomelo, there is a problem of its
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availability in large quantities. No commercial cultivation is undertaken due to the bitterness and
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astringent nature of the fruits. Because of this reason, it is less utilized by common people when
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compared to other fruits like orange, lime and tangerines.
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Baking is a process that has been adopted for centuries and bakery products range from simple
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ingredients plain pastry to the cake having numerous components. Bread is one of the bakery
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products priced for its taste, aroma and texture. Bread making is a complex process which
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includes mixing, proofing and baking (Dewettinck, Van, Kuhne, Walle, Courtens & Gellynck,
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2008). Bread is considered as a well-liked staple food consumed as part of the daily diet
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worldwide. Annually ~ 9 million kg of bread are produced (Heenan, Dutour, Hamud, Harvey &
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Delahurry, 2008). The popularity of bakery products has contributed to increased demand for
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ready to eat and convenient food products such as bread, cakes, biscuits etc. So initiative has
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been taken in this research work to use pomelo as a food fortificant for increasing the
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consumption of this fruit for health benefits. This study was conducted to develop value added
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white bread and brown bread using pomelo fruit segments. The prepared products were further
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analyzed for their glycemic index, retention of naringin and other biofunctional components to
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ensure health promoting properties of pomelo retained even after the processing.
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2. Materials and Methods
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Commercial wheat flour (10.2% moisture, 0.51% ash, 10.6% gluten, 24 ml sedimentation
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value and 373 s falling number), compressed yeast (Tower brand, Mumbai), sugar powder and
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vegetable fat (Hindustan Unilever Ltd, Bangalore) were procured from local market. 4
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2.1. Pomelo fruit processing
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The Citrus maxima (pomelo) fruits were obtained from the local market of Mysuru,
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Karnataka, India during the month of February 2016. The fresh segments were separated from
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the fruit manually and dried in hot air oven at 35 ºC for overnight to obtain dry fruit segments
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(residual moisture content of ~5%).
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2.2. Bread making characteristics
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Effect of fresh pomelo fruit segments (0%, 10%, 20% and 30%) and dried pomelo fruit
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segments (0%, 2.5%, 5% and 7.5%) on white bread and brown bread making characteristics was
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studied (Sudha & Leelavathi, 2008). The formulation used was flour: 100%, pomelo fruit
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segments, compressed yeast: 2.0%, vegetable fat: 1%, salt: 1.0%; sugar: 2.5% and water. All the
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ingredients were mixed in a Hobart mixer (Model N-50, Hobart, GmbH, Germany) with a flat
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blade for 4 min at 61 rpm. The dough obtained was fermented in a chamber maintained at 300C
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and 75% relative humidity (RH) for 90 min. After 90 min, the dough was remixed and relaxed
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for 25 min, molded, proofed for 55 min at 300C, 85% RH and baked for 25 min at 2200C, cooled
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for physical and sensory evaluation. Part of the bread samples were dried at 450C for 5h, cooled,
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homogenized and stored in poly propylene bags for various estimations.
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2.3. Evaluation of breads
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Weight of the breads was taken and volume of the loaves was measured by rapeseed
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displacement method (Sudha & Leelavathi, 2008). Bread crumb firmness, the objective
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measurement of texture was carried out in a texture analyzer (TAHDi, Stable Micro Systems, 5
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Godalming, UK) by the standard AACC, (2000) (74-09) and 2.0 mm.s-1 of pre-test speed and
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1.67 mm.s-1 of test speed were used. Force required to compress 25% of the bread slice was
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recorded using 35 mm diameter aluminium cylinder probe P-35. Objective evaluation of colour
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of bread crumb was measured using the Hunter Lab Colour Measuring System (Colour Flex-EZ
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Hunter Lab, USA) with a reflectance attachment of illuminant G against a standard white board
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made of barium sulphate (100% whiteness). Bread slice (3cm x 3cm) was placed in the sample
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holder and the reflectance from the surface measured.
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2.4. Sensory evaluation
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A scorecard containing the description for the desirable (creamish white – color; crisp –
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texture; wholesome sweetish – taste) and undesirable (dull or dark color; soft or hard – texture;
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unpleasant taste) quality characteristics for various sensory attributes viz. color of crust and
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crumb, texture, mouthfeel and overall quality were given to the panelist consisting of men and
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women of 35-50 age group. The panelist was then asked to assign scores for each parameter as
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against the maximum scores given in the parenthesis using a 7-point hedonic rating scale :
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excellent - 7, very good - 6, good - 5, satisfactory -4, fair - 3, poor - 2 and very poor -1 (Rathi,
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Kawatra, and Sehgal, 2004).
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2.5. Estimation of total sugars
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The total sugars estimation in the samples was carried out using the method described by
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Albalasmeh, Berhe, & Ghezzehei (2013). An aliquot of 10μl of sample was mixed with 300μl of
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5% aqueous solution of phenol. After 5 min of incubation 1.8 mL of concentrated sulfuric acid 6
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was added rapidly to the mixture. The test tubes were cooled and absorption read at 490 nm.
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Total sugar content of the sample was expressed as equivalent to mg glucose/g extract.
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2.6. Estimation of reducing sugars
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Reducing sugars were estimated based on the modified method of Miller (1959). The
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sample (150 μl) was mixed with 1ml of DNS (3,5-Dinitrosalicylic acid) reagent in a test tube.
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The tubes were placed in a boiling water bath for 10 min and cooled for ten to fifteen minutes at
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room temperature. Each solution was then diluted with 2mL of water, mixed thoroughly and
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absorbance was recorded at 540 nm using spectrophotometer (Helios Alpha, Thermo Electron
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Corporation, England, UK). Total reducing sugar content of the samples was expressed as
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equivalent to mg glucose/g extract.
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2.7. Bioactive Components
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2.7.1. Estimation of total phenolic content
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It was evaluated using a modified colorimetric method described by Henríquez et al.,
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(2010). The reaction mixture was prepared by adding 100μl of sample, 1.0mL of Folin-
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Ciocalteau reagent and 2.0 mL of 1% sodium carbonate solution. The mixture was incubated for
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60 min at room temperature, and the absorbance read at 765nm using an UV-Vis
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spectrophotometer. The measurement was compared with standard gallic acid solution. The total
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phenolic content was expressed equivalent to mg gallic acid/g extract.
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2.7.2. Estimation of flavonoids
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Flavonoids were estimated by a modified method of Lallianrawna, Muthukumaran, Ralte,
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Gurusubramanian and Senthil Kumar (2013). To 0.9 ml of sample, 75μl of 5% NaNO2 solution
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was added. After 5 min, 150μl of 10% AlCl3.6H2O was added to the mixture, which was kept at
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room temperature for 5 more minutes. This was followed by the addition of 0.5 ml of 1M NaOH
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and the total volume was made up to 2.5 ml with the addition of deionised water. The resulting
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solution was mixed well and immediately, the absorbance was measured at 510nm on a UV-VIS
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spectrophotometer. For the blank, the extracts were replaced with an equal volume of deionised
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water. Total flavonoid content of the samples was expressed as the mg equivalent to catchin /g of
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extract.
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2.7.3. Estimation of carotenoids
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The experiment was carried out by the modified procedure of Carvalho et al., (2012). The
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sample (1g) was homogenized in the dark (to avoid photolysis of carotenoids) with 20ml of
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acetone. The filtrate was further transferred to a separating funnel containing 10-15ml of
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petroleum ether and mixed well. The lower aqueous layer was then transferred to another
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separating funnel and the upper petroleum ether layer containing the carotenoids was collected.
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The extraction was repeated until the aqueous layer became colorless. A small amount of
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anhydrous sodium sulphate was added to the petroleum ether extract to remove excess moisture.
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The final volume of the petroleum ether extract was noted. The absorbance of the yellow color
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was read in a spectrophotometer at 450nm using petroleum ether as blank.
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Carotenoids content (μg/g) = A × V (mL) × 104 A1cm × P (g) 8
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2.7.4. Naringin content
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The white and brown breads were analyzed for the presence of naringin (bioactive
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compound) using HPLC (Shimadzu Class – VP HPLC model used with SPD-10AVP (PDA
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detector)) by slightly modifying the protocol of Pichaiyongvongdee and Haruenkit, 2009. One
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gram of the sample was extracted in methanol and homogenized for 30 min. The supernatant was
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passed through 0.45 mm syringe filters and subjected to HPLC. A standard solution was
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prepared by dissolving 1mg of naringin in 1ml with acetonitrile. Supelco C18 (5µm) column (15
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cm x 4.6 mm id), Supelco, USA was used with chromatographic solvent mixture (mobile phase)
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consisting of water: acetonitrile: (80:20 v/v) as mobile phase. The solvents were degassed with
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vacuum before using in HPLC analysis. The mobile phase was pumped with a LC-10 ATVP
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pump at a flow rate of 1 ml/min. The injection volume was 20µl and the total run time was 15
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min at 270nm. Quantification of the compound was evaluated by comparing the peak area with
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authentic standards using peak processing post-run integration parameters and external method:
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calibration type of Shimadzu class VP version 6.14 SPI data acquisition software.
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2.8. Total Starch (TS)
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The total starch in the bread samples was determined enzymatically according to the method
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described by Goni, Garci-Alonso & Suara-Calirto (1997). The ground sample (50 mg) was
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dispersed in 6mL of 2M KOH and shaken at room temperature for 30 min. Three ml of 0.4M
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Sodium acetate buffer pH 4.75 and 60µL of amyloglucosidase (EC-3.2.1.3, Sigma- Aldrich
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Chemical Company, St Louis, MO, USA) were added to this suspension and incubated for 45
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min at 60°C in a controlled shaking water bath. Starch was measured as glucose with glucose
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oxidase-peroxidase (GODPOD) kit. Factor conversion from glucose to starch was 0.9.
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2.9. Resistant starch (RS)
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Resistant starch was estimated according to the method of Goni et al., (1997). One hundred
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mg of each sample was incubated with pepsin solution containing 20mg of pepsin (EC-3.4.23.1,
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Sigma- Aldrich Chemical Company, St Louis, USA) for 60 min at 40oC for protein removal.
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Then the starch was hydrolyzed by adding pancreatic α-amylase (EC-3.2.1.1, Sigma- Aldrich
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Chemical Company, St Louis, USA) (10 mg/ml) solution containing amyloglucosidase (AMG)
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for 16 h at 37⁰C with constant shaking. After hydrolysis, samples were washed thrice with
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ethanol (99% v/v and 50% ethanol). The separated pellet from supernatant was further digested
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with 2 M KOH. Digested pellet and supernatant were separately incubated with AMG. Glucose
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released was measured using a glucose oxidase-peroxidase (GODPOD) reagent kit (K-GLOX,
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Megazyme Bray, Co. Wicklow, Ireland) by absorbance at 510 nm against the reagent blank. RS
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was calculated as glucose (mg) x 0.9. Digestable starch (DS) has been calculated as difference
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between TS and RS.
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2.10. Predicted glycemic index (pGI)
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A modified in vitro method based on the procedure of Goni et al., (1997) was adopted.
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The ground sample (100mg) was incubated with 10 mL HCl–KCl buffer (pH 1.5) and 200 μL
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pepsin solution (100 mg/mL HCl-KCl buffer) at 40°C for 1 h with constant shaking. The pH was
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raised by addition of 200 μL pancreatic α-amylase solution (1.5 mg /10 mL phosphate buffer; pH
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7.8) and incubated at 37°C for 45 min. Enzyme reaction was stopped with 70 μL Na2CO3 10
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solution and samples diluted to 25 mL with tris-maleate buffer (pH 6.9). Five mL of pancreatic
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α-amylase solution (3 U /5 mL tris-maleate buffer) was thereafter added to the sample and
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incubated at 37°C with constant shaking. Aliquots of 1 mL were taken at 30, 90 and 120 min
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from the samples and placed into boiling water with vigorous shaking for 5 min to inactivate the
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enzyme reaction. Samples were kept in refrigerator (4°C) after each inactivation until the end of
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incubation time (180 min).
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All aliquots were treated with 3 ml of 0.4 M sodium acetate buffer (pH 4.75) and 60 μL
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of amyloglucosidase (3300 U/ml) then incubated at 60°C for 45 min with constant shaking. After
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incubation, volume was adjusted to 10 mL with distilled water, mixed properly and centrifuged
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before transferring 0.1 mL aliquots of the solution into glass test tubes for glucose measurement.
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The glucose released was measured using a glucose oxidase-peroxidase (GODPOD) kit
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(K-GLOX, Megazyme Bray, Co. Wicklow, Ireland). Absorbance was measured at 510 nm
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against the reagent blank using UV-vis spectrophotometer. The values were plotted on a graph
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and the area under the concentration-over-time curve (AUC) was determined using Sigmaplot
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10.0 (Systat Software, San Jose, CA, U.S.A.). The hydrolysis index (HI) was calculated as the
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percentage of total glucose released from the samples as compared to that released from standard
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glucose (0–180 min) (Barine and Yorte, 2016). The predicted glycemic indices of the samples
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were estimated according to the equation of Goni et al. (1997): pGI = 39.71 + 0.549HI.
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2.11. Statistical analysis
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Data were statistically analyzed using Duncan’s new multiple range tests using GraphPad
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Prism software version 4.03 for Windows (San Diego, CA, USA) with different experimental 11
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groups appropriate to the completely randomized design with four replicates each as described
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by Sudha and Leelavathi (2008) at p < 0.05.
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3. Results and Discussion
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3.1. Quality characteristics of white and brown bread
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The physical attributes of white and brown bread incorporated with different levels of
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fresh and dry pomelo segments are presented in Table 1. The substitution of fresh pomelo
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segments (0 – 30%) in white bread formulation increased the volume from 567-625 ml and
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decreased the specific volume from 4.05-3.98 ml/g whereas for brown bread the same increased
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from 425-485 ml and 2.77-2.97 ml/g respectively. However, incorporation of dry pomelo
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segments decreased the loaf volume and specific volume in both types of bread ranging from
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575-534 ml and 3.96-3.34 ml/g for white bread and 480-410 ml and 2.94-2.31 ml/g for brown
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bread indicating that the volume of the brown bread is comparatively lower than the white bread.
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Scores of softness attribute were in accordance with the results of texture analysis (Table 1). The
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bread supplemented with fresh segments in both white (397-302 g/force) and brown (1056-679
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g/force) breads indicate that the softness of the bread increased with increase in fresh segments.
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The crumb firmness increased in breads supplemented with dried pomelo segments from 308-
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429 g/force (white bread) and 685-829 g/force (brown bread). There was significant (p < 0.05)
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decrease in preference in all the attributes evaluated as the percentage of pomelo segments
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(fresh/dried) increased. It has been reported that the reduction in loaf volume could be due to the
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reduction in gluten content as a result of supplementation (Sengev, Abu & Gernah, 2013). Low
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gluten content of flour lowers the ability of the flour to extend (elasticity) and retains the carbon
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dioxide produced during fermentation thereby yielding a decreased loaf volume. According to 12
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Ragaee, Guzar, Dhull and Seetharaman (2011), partial substitution of wheat flour with some
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grains such as barely, cellulose and oat caused a reduction in volume of loaves of bread. This
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could be explained by the fact that substitution of bread samples with pomelo dry segments,
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caused gluten dilution and consequently affected the optimal gluten matrix formation during the
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processing of the breads (mixing, fermentation and baking).
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3.2. Color measurement
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The color of breads with pomelo fresh segments (10, 20 and 30%) and dry segments (2.5,
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5 and 7.5%) differed statistically from the control bread; formulated bread also significantly
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differed one from the other (p < 0.05) by the addition of pomelo segments (Table 1). The color
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value of brown breads is higher when compared with that of white breads. However, the trend of
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L*, a* and b* value in both white and brown bread were similar. They showed a decrease (p <
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0.05) in L value of pomelo segments (fresh/dry) supplemented bread from lower to higher
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concentration which indicates the increase in the development of darker color of the formulated
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breads. But the breads differed significantly in relation to the parameters a*. The bread samples
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partially substituted with various concentration of pomelo had significantly (p<0.05) increased
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a* value compared to control. The increasing amount of pomelo segments in bread formula
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increased the redness gradually with significant difference among all formulated breads. This
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could be visually seen since the bread samples containing pomelo segments (fresh/dry) were
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pinkish compared to control which is of white color. A very slight increase in the b*
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(yellowness) value of bread crumbs was observed (p<0.05) with the addition of pomelo segments
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by increasing the concentration. Color appeared to be a very important criterion for the initial
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acceptability of the baked product by the consumer. The color depends both on the physio13
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chemical characteristics of the raw dough (i.e, water content, pH, reducing sugars and amino acid
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content) and on the operating conditions applied during baking which includes temperature, air
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speed, relative humidity, and modes of heat transfer (Schoenlechner, Szatmari, Bagdi &
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Tömösközi, 2013). It was observed that the color of the crumb sample of both white and brown
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bread showed significant (p < 0.05) increase in redness (a* value) and yellowness (b* value) but
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decrease in L* value with higher percentage of pomelo segments (Fig. 1a and b). This might be
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attributed from the reddish color imparted by the fruit segments incorporated.
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3.3. Sensory evaluation
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The sensory evaluation of breads is presented in Table 2. The evaluation was done on a
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seven-point hedonic scale. The crust color, symmetry, texture, eating and overall qualities of
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white and brown breads were comparable to control bread. The crust color of the white bread
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changed from dark brown to light with increasing pomelo segments (fresh/dry). In brown bread,
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the score was found to be similar in all formulations. The more brownish bread appearance could
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be attributed to the high fiber content in the bread (Hu, Yang, Ma & Zhou, 2007). The brown
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color of the bread is due to the caramelization and Maillard reaction, in which protein and sugar
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of the flours react with each other during baking process (Dhingra & Jood, 2001). The significant
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(p < 0.05) decrease in likeness for crust and crumb as the level of supplementation increased
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could be ascribed to the bitter and sourness of the bread which is related to the fruit. Generally,
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addition of increasing concentration of pomelo segments had significant effects on sensory
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attributes and overall acceptability of bread samples. Addition of segments caused darker color
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and denser texture, in both forms of breads at the level of 20% (fresh) and 5% (dry) which seems
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to be acceptable for consumers, with citrus flavor and bitterness at palatable levels. However, 14
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formulated breads beyond above mentioned concentration were very sour and bitter which seem
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to have negative effect on consumer’s overall acceptability.
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3.4. Bioactive components in breads
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The bio functional properties in developed products were evaluated to determine their
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retention during processing. Since the product is recommended for diabetic populations, the total
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sugar and reducing sugar content of the product were also determined. There was an increase in
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the content of total sugars and reducing sugars with the increase in the concentration of fresh and
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dry segments (Table 3). White bread showed lower range of sugar content (4.47-6.97mg/100g)
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compared to brown breads which ranged between 5.28-7.26mg/100g. Similar pattern of result
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were observed in case of reducing sugar which ranged between 2.05-2.93mg/100g (white bread)
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and 2.49-3.47mg/100g (brown bread). Kumar, Vijay and Khan (2013) reported that the total
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soluble sugar from pomelo juice is 4.87mg/100ml which is in accordance with our results. Thus,
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addition of pomelo segments in bread formulation has shown a gradual increase in sugar content.
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The total phenolics and flavonoids in products were significantly higher in breads
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supplemented with pomelo segments (Table 3). The phenolic content varied from 60.12-90.19mg
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GAE/100g in white breads and 65.11-95.66mg GAE/100g in brown breads. The flavonoid
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content in white bread was 21.80-38.11mg CE/100g and 23.32-42.0mg CE/100g in brown
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breads. The content increased with the increase in pomelo segments. Breads supplemented with
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dry segments have shown prominent level of phenolics and flavonoids compared to fresh
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segments. Phenolics are secondary metabolites, which mainly include flavonoids, coumarins,
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stilbenes and tannins. Recent interest in plant polyphenols has focused on their potential benefits
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to human health. Several previous reports have also suggested that the bioactive components 15
319
such as phenolics, flavonoids and their glycosides can act as an effective inhibitors of α-
320
glucosidases (Myung-Hee, Sung-Hoon, Hae-Dong, Mee Sook & Young-In, 2010), oxidative
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stress, microbial growth and even as a remedy for other medical problems.
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The carotenoid content of the products ranged between 42.22-231.78µg/100g (white
323
bread) and 55.68-244.53 µg/100g (brown bread) (Table. 3). In fact, the stability of carotenoids in
324
foods is variable. It depends not only on the extrinsic factors (heat treatment, presence or absence
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of light) but also on the other characteristics of the food matrices such as their chemical
326
composition, oxygen dissolved, size of the particles and the physical state of the carotenoid in
327
the food (Vásquez-Caicedo, Schilling, Carle & Neidhart, 2007). Carotenoids also play a potential
328
role in human health by acting as biological antioxidants (Bendich, 1989), anti-cancer (Nishino,
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1998) and antimicrobial agents (Manimala & Murugesan, 2014).
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The nutritional factors (phenolics, flavonoid and carotenoid) are high in brown bread.
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However, there is not much significant variation between both the bread types. According to
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Slavin (2004), the product based on whole grain has components that are associated with
333
improved health status which include lignans, tocotrienols, phenolic compounds, anti-nutrients
334
and enzyme inhibitors. In the grain-refining process, the bran is removed resulting in the loss of
335
components. Hence, refined grains product (white bread) has shown lesser nutritional value
336
compared whole wheat products (brown bread).
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3.5. Retention of naringin in supplemented breads
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Naringin, a well-known flavanone glycoside of Citrus fruits, possesses antioxidant, anti-
339
inflammatory, anti-apoptotic, anti-diabetic, anti-ulcer, anti-osteoporosis and anti-carcinogenic
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properties (Cui, Zhang, Sun & Jia, 2012). Naringin is the bioactive compound that dampens 16
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postprandial glycemic response and offers a potential complementary approach in the
342
management of diabetes (Priscilla, Roy, Suresh, Kumar, & Thirumurugan, 2014). Hence,
343
retention of naringin in processed products was evaluated (Fig. S1). The retention of naringin in
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white bread was 60% in fresh segments and 70-80% in dry segments. In case of brown bread, the
345
retention were comparatively less. Fifty percent and 65% retention were observed in brown
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bread formulation incorporated with fresh and dry segments respectively. In both types of
347
breads, the formulated bread with dry segments has showed better retention of naringin content
348
with the minimum loss. The percentage of retention in bread samples were calculated based on
349
the amount of naringin present in fresh and dry segments taken for product development (Fig.
350
S2). Thus, the naringin content in the prepared bread could potentially act against carbohydrate
351
hydrolyzing enzymes which in turn prevents post-prandial hyperglycemia.
352 353
3.6. Starch digestibility and glycemic index
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Based on the sensory attributes of the products, 20% fresh and 5% dry segments
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incorporated white and brown breads were selected for further studies on in-vitro starch
356
digestibility. The level (%) of TS, RS and DS of the developed bread products are presented in
357
Table 4. The TS content in the products ranged between 69.27 to 75.47 %. Resistant starch (RS)
358
content varied among products with a range of 3.02 – 10.96% on dry basis. With reference to
359
control bread, the RS content was higher in brown bread compared to white bread. High RS
360
value of 10.96% was observed in 5% dry pomelo segment supplemented brown bread which
361
resulted in the lower value of digestible starch (DS) of 64.51% that is significantly different from
362
the other formulated breads having high DS value. In the present study, although the developed 17
363
products had similar starch content, they differed in the rate of starch digestion. The variations
364
may be due to differences in protein content, dietary fiber, nature of starch and extent of starch
365
gelatinization. Davis (1994) reported that the wheat starch swells more slowly than other
366
starches, limiting the extent of starch gelatinization. Chandrashekar and Kirleis (1988) reported
367
that the presence of protein bodies around starch granules may restrict granule swelling and
368
starch gelatinization and as a result reduces the susceptibility to enzymatic attack. This may be
369
partially responsible for the low digestibility. In accordance to the above reports, the product
370
developed with the supplementation of pomelo segments (rich in protein, dietary fiber and
371
naringin content) has showed increased level of resistance starch with the increase in
372
concentration of fruit segments.
373
Hydrolysis Indices (HI) calculated from the rate of hydrolysis over time (Fig. 2) and the
374
corresponding predicted Glycemic Indices (pGI) are presented in Table 4. Among white and
375
brown breads; the white bread has shown higher hydrolysis index which resulted in higher
376
predicted glycemic index (pGI) ranging from 66.06-59.15%. The lowest HI and pGI was
377
recorded in brown breads that ranged between 62.41-53.13%. According to Jenkins et al.,
378
(2008), foods with GI of ≤ 55, 56-69 and ≥70 are classified as low, medium and high GI,
379
respectively. The observation of low pGI in this study could be attributed to high fraction of RS
380
among samples. As stated previously, the bread with the pomelo segments (rich in naringin) have
381
shown lower and gradual release of glucose than the control bread. These findings agree with the
382
fact that naringin and other bio-functional components act synergistically to inhibit the enzymes
383
involved in the post-prandial hyperglycemia (Shen, Xu & Lu, 2012). Brand-Miller (1994) has
384
demonstrated the therapeutic value of low GI diet in type 1 and type 2 diabetic patients. Studies 18
385
have also shown that diet with low GI and high RS help in reducing insulin resistance, adjusts
386
blood glucose level, improves lipid metabolism and prevents cardiovascular and cerebrovascular
387
diseases (Chung, Sanguansri, & Augustin, 2010). Thus, GI is related to nutritional quality of
388
food and a product with a low GI is preferable not only for individuals with diabetes, but also for
389
normal population (Björck & Asp, 1994).
390 391
4. Conclusion
392
Thus, the study indicates that supplementation of pomelo segments has a great potential in
393
developing bakery products for health benefits. Among the bread formulations, the
394
physicochemical characteristics of bread supplemented with fresh fruit segments was better
395
compared to dry fruit segments. The 20% fresh and 5% dry supplemented bread (white and
396
brown) were sensorially acceptable. The bioactive components such as phenolics, flavonoids and
397
carotenoids were found to be higher in supplemented brown breads. The bioactive compound,
398
naringin retained in bread formulations had contributed to lower glycemic index by inhibiting the
399
digestive enzymes. Dry segment-incorporated bread showed higher resistance towards starch
400
digestion thereby lowering the release of glucose. Inhibition of the carbohydrate hydrolyzing
401
enzymes involved in post-prandial hyperglycemia is an important strategy for the management of
402
type II diabetes. Thus, the present finding suggests that the pomelo-fortified bread can be
403
recommended as suitable food for diabetic populations.
404 405
19
406
Supplementary data: Naringin content in fruit segments (Figure S1); Naringin content in breads
407
(Figure S2).
408 409
Acknowledgments
410
We are grateful to Prof. Ram Rajasekharan, Director, CSIR-CFTRI, Mysuru for constant
411
encouragement throughout the course of study and Department of Biotechnology (grant
412
numbers: BT/PR5994/FNS/20/563/2012), Govt. of India, New Delhi, India for their financial
413
support.
414 415
Conflict of Interest
416
The authors declare no conflict of interest.
20
417
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563
Figure Captions
564
Figure 1: Whole and sliced bread supplemented with fresh and dry fruit segments of pomelo.
565
Figure 2: Rate of starch hydrolysis of white and brown bread supplemented with fresh/dry
566
pomelo
segment
28
567
Fig. 1. Whole and sliced bread supplemented with fresh and dry fruit segments of pomelo.
568
a. White breads
569
570 571 572
b. Brown breads
573
574 575 576 577 578 579 29
580 581 582 583 584
Fig. 2. Rate of starch hydrolysis of white and brown bread supplemented with fresh/dry
585
pomelo segments.
586 587 588 589
30
Pomelo segments (%)
Volume (ml)
Specific Volume (ml/g)
Crumb firmness (g force)
Crumb colour L
A
b
ΔE
White bread 0 Fresh 10 20 30 Dry 2.5
590 591
567±1.89c
4.05±0.63a
397±0.98
67.57±0.31a
0.13±0.02h
12.26±0.03d
26.75±0.28ef
575±0.87c 600±1.44b 625±1.86a
3.91±0.89a 3.94±1.06a 3.98±1.76a
370±1.07f 341±1.16g 302±1.22g
66.92±0.35b 65.33±0.24b 65.91±1.39b
0.25±0.02h 0.56±0.34g 1.01±0.02f
12.09±0.08d 12.41±0.10d 12.48±0.11cd
26.36±0.29ef 27.11±0.24e 27.21±0.67e
575±1.09c
3.96±0.79a
308±1.09g
64.30±0.01bc
0.83±0.01f
12.45±0.57c
29.17±0.01d
Table 1. Physical characteristics of white and brown bread supplemented with fresh/dry pomelo segments
31
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
425±0.54g
2.77±1.45d
Brown bread 1056±0.98a 51.95±0.48d
3.66±0.17d
15.02±0.33a
42.19±0.72bc
450±1.36f 460±1.11f 485±1.90e
2.84±1.65c 2.90±1.39c 2.97±0.85c
777 ±1.30c 700 ±0.92d 679 ±0.77d
50.61±0.89d 50.11±1.13d 49.55±0.56d
3.92±0.10c 4.01±0.22c 4.26±0.07b
14.94±0.13b 14.70±0.20b 14.86±0.16b
42.16±0.66bc 43.46±1.11b 44.71±0.57a
480±0.48e 455±0.97f 410±1.36g 1.02
2.94±0.47c 2.67±0.67d 2.31±1.08e 0.11
685±1.09d 743±0.89c 829±1.11b 5.3
49.09±0.19d 47.59±0.47e 46.40±0.41e 1.01
4.03±0.07c 4.73±0.04b 5.07±0.06a 0.92
15.14±0.11a 15.56±0.14a 15.77±0.13a 0.45
42.49±0.68bc 43.85±0.38b 41.66±0.42c 2.01
0 Fresh 10 20 30 Dry 2.5 5 7.5 SEM (+) 592 593 594 595 596 597 598 599 600 601 602 603 604
32
605 606 607 608 609 610
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
33
611 612
Table 2. Sensory evaluation of white and brown bread supplemented with fresh/dry pomelo segments Pomelo segments (%)
Crust Colour Symmetry
Color (7)
Crumb Texture Eating quality (7) (7)
Overall quality (7)
(7)
(7)
0
6.0a
White bread 6.0a 6.0a
6.0a
5.5a
5.5a
10 20 30
6.0a 5.5b 5.0c
6.0a 6.0a 6.0a
5.5b 5.0c 4.5d
6.0a 6.0a 5.5b
5.0b 5.0b 5.0b
5.0b 5.0b 4.0d
2.5 5 7.5
6.0a 5.5b 5.0c
5.5b 5.0c 4.5d
5.0b 4.5c 4.0d
5.5a 5.0b 4.5c
0
6.0a
6.0a 5.5b 6.0a 5.0c 5.5b 4.5d Brown bread 6.0a 5.0a
4.5b
4.5a
5.0a
10 20 30
6.0a 5.5b 5.5b
6.0a 6.0a 6.0a
5.0a 4.5b 4.5b
4.5b 4.5b 5.0a
4.5a 4.0b 3.5c
5.0a 4.5b 4.0c
2.5 5 7.5 SEM (+)
5.5b 5.5b 5.5b 0.12
6.0a 6.0a 6.0a 0.20
4.5b 4.0c 4.0c 0.16
5.0a 5.0a 4.5b 0.14
4.5a 4.0b 3.0c 0.21
5.0a 4.5b 3.5d 0.22
Fresh
Dry
Fresh
Dry
613 614 615 616 617 618
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
619 620 34
621 622 623 624 625
Table 3. Bio-active components of white and brown bread supplemented with fresh/dry pomelo segments Pomelo segments (%)
Sugars (g/100g)
Reducing sugars (g/100g)
Phenolics
Flavanoids
(mgGAE/100g) (mgCE/100g)
Carotenoids (µg/100g)
White bread 0
4.27±0.09
1.66±0.08
58.00±1.09
20.13±0.67
23.06±0.98
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
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
Fresh
Dry
Brown Bread 0
4.98±0.18
2.32±0.09
63.02±1.22
21.80±0.98
39.57±0.65
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
2.5
5.80±0.09
2.66±0.08
73.36±1.34
27.23±1.45
111.25±0.98
5
6.59±1.18
3.26±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
Fresh
Dry
626 627 628
GAE-Gallic acid equivalent; CE- Catechin equivalent ; Values are means ± standard deviation (n=4) 35
629 630 631 632 633
Table 4. In-vitro starch digestibility and predicted glycemic index of bread supplemented with pomelo fruit segments Pomelo Segments (%)
TS
RS
DS
HI
pGI
(%)
(%) (%) White bread
(%)
(%)
0
69.27±1.09
3.02±1.40
66.25±0.56
48.00±1.19
66.06±1.53
Fresh*
69.87±2.57
3.87±1.64
66.00±0.44
42.38±2.11
62.97±1.77
Dry**
71.23±1.77
4.96±1.29
66.27±0.32
35.42±1.47
59.15±1.69
Brown bread 0
73.66±1.56
4.98±1.55
68.68±0.62
41.35±1.27
62.41±0.88
Fresh*
74.75±2.38
5.17±1.99
69.58±0.84
33.69±2.23
58.20±1.94
Dry**
75.47±1.88
10.96±1.30
64.51±0.59
24.46±1.68
53.13±1.12
634 635 636 637
*-20%; **-5%; TS- Total starch; RS- Resistance starch; DS- Digestible starch; HI-Hydrolysis index; pGI- Predicted Glycemic index; Values are means ± standard deviation (n=4)
638 639
36
640
Research Highlights
641 642
Quality and Sensory of white and brown bread fortified with pomelo is studied
643
The presence of bioactive compounds were higher in bread supplemented with pomelo
644
The glycemic index of developed products were evaluated
645
This study offers a new approach for producing bread with a lower starch digestion rate.
646
37