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ISSN: 2320-5407 Int. J. Adv. Res. 6(5), 506-512
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Journal Homepage: -www.journalijar.com
Article DOI:10.21474/IJAR01/7058
DOI URL: http://dx.doi.org/10.21474/IJAR01/7058
RESEARCH ARTICLE
A PRELIMINARY STUDY ON ESTIMATED GLYCAEMIC INDEX AND MICROSTRUCTURE OF
STARCH, IN BOILED CUCURBITA MOSCHATA (SQUASH) FOUND IN SRI LANKA.
Akurange Sujeevi Dammadinna Wickramasinghe1, Alahendra Acharige Navinda Kalpitha 1, Poruthotage
Pradeep Rasika Perera2 and Udumalagala Gamage Chandrika2.
1. Department of Allied Health Sciences, Faculty of Medical Sciences, University of Sri Jayewardenepura,
Gangodawila, Sri Lanka.
2. Department of Biochemistry, Faculty of Medical Sciences, University of Sri Jayewardenepura, Gangodawila,
Sri Lanka.
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Manuscript Info Abstract
……………………. ………………………………………………………………
Manuscript History
Received: 07 March 2018
Final Accepted: 09 April 2018
Published: May 2018
Keywords:-
Bioaccessibility, In vitro digestion,
Starch, Brightfield microscopy, Squash
There is an increased tendency to use non-pharmacological strategies
such as dietary interventions in health related problems. Especially in
the case of chronic illnesses, dietary interventions are used along with
pharmacological treatment for proper management of patients. Dietary
interventions are helpful not only in the management but also in
prevention of most of the long term illnesses. In Sri Lanka, squash are
commonly consumed as a soup or with major meals usually after
traditional cooking. Starchy vegetables with high glycemic index lead
to rapidly elevated blood glucose levels, which is associated with risk
of obesity and diabetes mellitus. Therefore identification of thermal
processing methods that can be used as dietary interventions will help
to improve quality of life. The aim of this study was to evaluate the
estimated glycaemic index and microstructure of starch in boiled
preparation of squash found in Sri Lanka. Estimated glycaemic index
was determined using in vitro digestion procedures. Brightfield
florescence microscopy was used to observe the changes in
microstructure. The estimated glycaemic index of boiled squash was
13.1 ± 4.1 and microscopy showed a high degree of cell disruption and
release of starch out of cells in thermally processed preparation when
compared to raw sample. Foods with a low glycaemic index (<55), help
slow absorption of carbohydrates and prevent extreme blood glucose
fluctuations. Therefore the results of this study conclude that boiled
squash is safe for obese and diabetic population. Further studies with
other cooking methods will be needed to provide more knowledge.
Copy Right, IJAR, 2018,. All rights reserved.
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Introduction:-
Starch is the main carbohydrate in human nutrition and is a major component in plant foods. It is the most common
digestible polysaccharide found in plants. Common sources of starch include grains, potatoes, legumes and other
vegetables. Starch is found in all parts of plants (leaves, stems, roots, tubers, seeds). Starch is a complex
carbohydrate made up of two components, amylose and amylopectin. The digestion of starch begins in the mouth
with salivary α-amylase which hydrolyzes amylose and amylopectin, forming dextrins; short chain polysaccharides.
Corresponding Author:- Akurange Sujeevi Dammadinna Wickramasinghe.
Address:- Department of Allied Health Sciences, Faculty of Medical Sciences, University of Sri
Jayewardenepura, Gangodawila, Sri Lanka.
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Salivary amylase action continues in the stomach until gastric acid lowers the pH and inactivates the enzyme.
Dextrins are further digested in the small intestine when pancreatic α-amylase is secreted. Dextrins are broken down
by pancreatic α-amylase into disaccharides such as maltose, isomaltose. By the action of several disaccharidases
such as isomaltase, maltase, theses disaccharides are further breakdown into monosaccharides such as glucose which
will be absorbed into the body (Champe et al. 2008).
Carbohydrates provide a significant fraction of energy in the diet of most of the organisms including humans.
Glycemic Index (GI) ranks carbohydrate-containing foods in a 0 to 100 scale on how quickly and how much they
elevate blood glucose levels. Foods can be classified as having a low (<55), intermediate (55-70), or high (>70) GI.
GI can be estimated by in vitro rate and extent of starch digestibility, which is called Estimated Glycemic Index
(EGI) (Lemlioglu-austin 2012). Foods with a low GI produce slower and lower rise in blood glucose level compared
to foods with high GI (Nutrition Education Materials Online 2014). Foods with low GI also tend to create a sense of
satiety over a long period of time and may be helpful in limiting caloric intake (Champe et al. 2008). Therefore
foods with low GI are beneficial in controlling and reducing the risk of diabetes mellitus and obesity.
Many studies have revealed that processing methods of starchy vegetables can affect the microstructure and GI. An
in vivo study done on some commonly eaten West Indian carbohydrate-rich vegetables such as yams and potatoes
has shown that foods processed by roasting or baking may result in higher GI whereas boiling of foods may
contribute to a lower GI diet (Bahado-Singh et al. 2006).
Effect of thermal processing on microstructure has been investigated previously. Some nutrients found in plants are
protected in nature against degradation inside cells by attaching to membranes, occluding inside cell organelles, or
binding to cell walls, but this natural protection lowers bioavailability. Thermal and physical processing,
mastication, and to a limited extent digestion break down the cell walls, making the release of nutrients from the
food matrix easier and rendering them available for absorption in the intestine (Parada and Aguilera 2007).
According to degree of the heat treatment applied, tissue and cell disruption increase and there is more release of
nutrients from cells which increase bioaccessibility of those nutrients. Thermal properties of pumpkin and squash
starch has been studied in China and the results indicate that swelling and solubility of squash starch increases with
rise in temperature (Yin and Wang 2016).
Studies on GI have been done using both in vivo and in vitro methods on different types of natural and processed
foods. The in vitro methods have improved for reliable determination of bioavailability of genuine in vivo
metabolites, as they are rapid, cheap, and circumvent ethical issues related to the use of humans or animals (Parada
and Aguilera 2007). Concern for development and utilization of in vitro methods has been increased lately and
several studies also have been done to find EGI using in vitro methods. Methods to determine total starch content in
foods and in vitro digestion procedures to determine EGI have been developed and they gave similar results to in
vivo GI methods (McCleary et al. 1997) (Goñi et al. 1997). Using these methods, studies have been done for
different preparations of vegetables such as sweet potatoes (Allen et al. 2012). Studies have also been done to see
whether in vivo GI values can be predicted using EGI values obtained from in vitro procedures and it has been
concluded that in vivo and in vitro GI values are much identical (Vera et al. 2002, Monro and Mishra 2010). For
example Goni and Garcia-Alonso has proved in their study on processed potatoes that the Estimated Glycaemic
Index (EGI) obtained from starch digestibility procedure was in accordance with the reported GI values, for potatoes
processed in the same way (Garcia-Alonso and Goni 2000). Hence it is fair to apply in vitro GI results for living
beings.
Studies on GI of vegetables such as cassava (staple foods of African population) have been reported mostly from
African countries. An in vivo study shows that cassava paste gives relatively high GI (GI= 86) and another study
report from Tanzania tells that cassava flour has a relatively low GI (GI=49.84) and is good for treatment and
management of diabetes (Kouamé et al. 2014, Ruhembe et al. 2014). These studies and another in vivo study on
Jamaican sweet potatoes show that method of food processing has a significant impact on GI (Bahado-Singh et al.
2011).
There are limited studies reported on glycemic index of fruits of Cucurbitaceae family such as pumpkin and squash.
But studies on important nutrients such as carotenoids in those vegetables have been reported. One such in vitro
study done in Sri Lanka has revealed that squash curry contains 44.6 ± 12.3 µg/g (dry weight, n=6) β-carotene and
only 6.3 ± 1.4 µg/g (dry weight, n=6), (14%) is bioaccessible. In the same study, it has been found that the pumpkin
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curry had more bioaccessibility (32.3 %) when compared to boiled pumpkin (18.7 %) since the degree of heat
treatment is higher in the preparation of curry. Other than the degree of heat treatment, addition of coconut milk
(fat) has made an hydrophobic environment which increased bioaccessibility of carotenoids (Priyadarshani and
Chandrika 2007).
In Sri Lanka, traditional preparations such as curry (boiled with coconut milk) and boiled preparations of squash are
common. Squash in boiled form is a commonly consumed preparation. Hence, examining the effect of boiling on
EGI and microstructure of Squash is pertinent to encourage consumption of foods low in starch by obese and
diabetic subjects. There are varieties of some non-leafy vegetables in Sri Lanka where investigations for starch have
not yet been done. There are various kinds of cooking methods in Sri Lanka for these vegetables and the effect of
them on the microstructure of starch and GI are not known. Hence, there is a need for a more complete database on
above parameters of these vegetables in terms of food as eaten. This study will provide a platform and
encouragement for further studies.
Methodology:-
Sampling:-
Squash (Cucurbita moschata) were purchased at random from three different places in Colombo district. A sample
from each (100 g × 3) was taken for analysis.
Thermal processing:-
Thermal processing (boiling) was done in different houses at different days randomly. The 100 g samples were
boiled separately. Squash was washed thoroughly and cut into pieces of 1.5 × 1.5 × 1.5 cm3 in size and allowed to
boil in salty water for about 15 minutes in an uncovered pan.
Determination of total starch in boiled squash:-
The procedure was conducted according to method established by McCleary et al (McCleary et al. 1997).
From homogenously crushed samples of boiled squash 50.0 mg were accurately weighed and 3 mL of distilled water
was added carefully to moisten the sample. An amount of 3 mL of 4 M KOH was added to it. It was shaken at room
temperature for 30 minutes using a vortex. An amount of 3 mL of 0.4 M sodium acetate buffer (pH 4.75) was added
to it. pH was adjusted to 4.75 with 1 M and 2 M HCl. An amount of 80 µL of amyloglucosidase from Aspergillus
niger (Sigma-Aldrich A7095) was added to it. It was mixed well and incubated at 60 °C for 45 minutes in a shaking
incubator. The mixture was centrifuged at 3000 ×g for 15 min. The supernatant liquid was collected into a 500 mL
volumetric flask using a pipette. Volume was made up to 500 mL by adding distilled water. Glucose content for
each sample was determined using Glucose Oxidase - Peroxidase kit (BioSystems REF 11538). Procedure was done
in triplicate for each sample. Same procedure was carried out for three samples from fresh white bread as reference
samples.
Determination of EGI of boiled squash by in vitro digestion procedure:-
The procedure was conducted according to method established by Goni et al (Goñi et al. 1997).
From the homogenously crushed samples of boiled squash, 50.0 mg was accurately weighed and were homogenized
in 5 mL of HCl-KCl mixture (pH 1.5) for 1 minute using a homogenizer with controlled speed (level 4). The
samples were incubated at 40 oC for 60 minutes in a shaking incubator with 0.1 mL of pepsin (Himedia RM 084)
solution (0.2 mL of HCl-KCl mixture containing 1 mg of pepsin from porcine gastric mucosa). An amount of 7.5
mL of Tris-malate buffer was added to make pH to 6.9. An amount of 2.5 mL alpha amylase solution (5 mL of Tris-
Malate buffer containing 2.6 UI of alpha amylase from porcine pancreas) was added. The flasks were placed in a
shaking incubator at 37 oC.
Aliquots (0.1 mL) were taken every 30 minutes from 0 to 3 hour. Alpha amylase was inactivated by immediately
placing the tubes in a boiling water bath for 10 minutes with vigorous shaking for every 30 seconds. Then 1 mL of
0.4 M sodium acetate buffer (pH= 4.75) and 30 µL of amyloglucosidase were added. The samples were incubated at
60 oC for 45 minutes to hydrolyze the starch into glucose. Finally, the glucose concentration was measured using a
Glucose Oxidase-Peroxidase kit.
Amount of starch in mg was calculated as glucose in mg × 0.9. The digestion curves were prepared according to the
following non-linear equation established by Goni et al (Goñi et al. 1997).
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Where is the percentage of starch hydrolyzed at time t (min); is the equilibrium percentage of starch
hydrolyzed after 180 min; and k is the kinetic constant. The variables and k were estimated for each sample
using SPSS for windows 21.
The area under the hydrolysis curve (AUC) was calculated for each sample using the equation;
Where tf is the final time (180 min) and to is the initial time (0 min).
Hydrolysis Index (HI) was obtained by dividing the AUC of each sample by corresponding AUC of reference
sample (fresh white bread, GI=100). Finally EGI was predicted with the formula;
Procedure was done in triplicate for each boiled squash sample. Same procedure was carried out for three samples
from fresh white bread as reference samples.
Microscopy of starch granules:-
Brightfield microscopy was done using FSX 100 Olympus microscope equipped with FSX-BSW software.
Brightfield microscopy images at ×20 and ×40 magnification were obtained for raw and boiled samples of Squash.
For visualization of starch granules, slides were stained with iodine and they were examined and photographed.
Determination of Moisture Content:-
Moisture contents of raw and boiled squash were determined by drying amounts from each sample in triplicate in an
oven at 60 °C until a constant weight was obtained. The percentage moisture for each sample was calculated based
on the average weight of the three dried samples.
Results:-
EGI of boiled squash:-
The EGI of boiled squash was 13.05 ± 4.10 (n=3) with a moisture content of 92.68 %.
Effect of boiling on microstructure:-
Brightfield microscopy images of starch in YFM preparations are shown in Figure 1 below. Under microscopy
iodine stained starch granules were observed in blue-purple colour. Boiling of squash has caused cell separation,
disruption as well as starch gelatinization where starch granules swell and rupture and starch become solubilized
(Ratnayake and Jackson 2009).
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(A) Raw squash sample shows intact cells with cell walls which are closely bound to each other. Starch present as
globules in some cells in limited numbers. (B) Boiled squash sample shows two cells with less prominent cell walls
containing one large starch cluster per each cell. (Left column shows brightfield images in ×20 magnification and
right column shows same corresponding images in ×40 magnification)
Discussion:-
The principle aim of the present study was to investigate the effect of boiling on the EGI and microstructure of
starch in squash (Cucurbita moschata) found in Sri Lanka. Brightfield microscopy was carried out to visualize
starch. EGI were determined by in vitro digestion procedures that simulated the human gastrointestinal digestion.
Starch in plant foods is generally organized into granules inside amyloplasts and can be visualized using staining
techniques (Brackmann et al. 2011). Changes in microstructure such as starch gelatinization, cell separation,
plasmolysis etc. can be result due to thermal processing (Parada and Aguilera 2007).
This study shows that boiled squash has a GI of 13.05. According to GI indexing system of 0-100 boiled squash is a
low GI food. Changes in microstructure as described above were evident in boiled squash when compared to raw
squash.
Similar results have been reported from several studies which conducted in similar manner to this study but on
different kind of vegetables. A study of EGI values of boiled and fried forms of hausa potato (Solenostemon
rotundifolius poir) shows that they have intermediate GI and safe for patients with diabetes mellitus (Eleazu et al.
2017) In a comparative study of yellow fleshed manioc in boiled and curry form has revealed that boiled preparation
has lower EGI compared to curry form and boiled form is safe for people with diabetes mellitus (Wickramasinghe et
al. 2016). Another in vitro study on evaluating effect of processing on starch in potatoes shows that starch
digestibility is improved after processing and it is also affected by the processing methods. For example boiled and
B
A
Figure 1:-Brightfield microscopy images of starch granules in squash
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mashed potatoes showed the highest rate of digestion but raw potato was hardly digested (Garcia-Alonso and Goni
2000).
Findings on starch contents in plant foods provide solutions for some of the major nutrient related diseases such as
diabetes and obesity. Hence the main concern is to find ways to process foods in a way that give low amounts of
glucose to body for prevention and management of Diabetes and obesity. Further studies on other preparation
methods of squash and also on other Sri Lankan non leafy vegetables are needed to provide more knowledge on this
topic. Future studies can use this study as a platform and generate more data which will help improve health.
The EGI of boiled squash is lower than the EGI of vegetables such as boiled yellow fleshed manioc (24.6) in Sri
Lanka (Wickramasinghe et al. 2016).
Results of this study clearly show that boiling has an effect of disrupting cell membranes and solubilizing starch
thereby facilitating the release of starch from cells during digestion. According to the EGI value boiled squash is a
low GI food and safe for people with diabetes and obesity.
Acknowledgement:-
The chemical analyses were supported by Division Funds of the Department of Biochemistry of the Faculty of
Medical Sciences at University of Sri Jayewardenepura.
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