International Food Research Journal 23(3): 1199-1206 (2016)

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Isolation and characterization of native and modified starch from adlay

(Coix lacryma jobi-L.)
1,2*
Capule, A.B. and 2Trinidad, T. P.
1
Department of Science and Technology, Food and Nutrition Research Institute, Bicutan, Taguig,
Metro Manila, Philippines
2
The Graduate School, University of Santo Tomas, España Blvd., Manila, Philippines

Article history Abstract

Received: 26 June 2015 Adlay (Coix lacryma jobi-L. var. Tapul) starch was isolated and subjected to heat-moisture
Received in revised form: treatment to produce a modified starch. Chemical, physical and functional properties were
14 September 2015 analysed using standard methods and compared with modified cornstarch and modified tapioca
Accepted: 17 October 2015
starch. Modification caused a decrease in moisture and increase in protein, dietary fibre and
ash content. Starch colour showed a decrease in the lightness of the modified Tapul starch.
Scanning electron microscopy (SEM) showed various cracks, fissures and holes on the starch
Keywords surface after modification. Water absorption capacity increased while swelling and solubility
decreased. Significant changes in pasting and gelatinization properties were also observed.
Adlay
Tapul
The findings provide evidence of altered starch properties after heat-moisture treatment which
Starch may have potential application in canned food products, sauces and bread.
Isolation
Heat-moisture treatment
Chemical
Physical
Functional properties © All Rights Reserved

Introduction typhoon by substituting this grain to rice. Tapul grains

differ in colour to the waxy adlay variety because it
In plants, starch is the main carbohydrate reserve comes in grey orange colour but similarly spherical
and an important ingredient in human nutrition (Kim in shape with a groove at one end. Its grain structure
et al., 2008). It is found in different botanical sources is similar to the waxy adlay and resembles that of
such as cereal, legume, root, tuber and unripe fruit the ordinary rice, only slightly bigger. Its proximate
(Ashogbon and Akintayo, 2014). Food starches are composition based on FNRI (2011) analysis on adlay
typically used as thickeners and stabilizers in foods grits indicates significant amount of energy (356
such as puddings, custards, soups, sauces, gravies, kcal) and protein (12.8 grams).
pie fillings and salad dressings, but still provide Adlay has long been used in traditional Oriental
many other uses. However, the industrial utilization and Chinese medicines, baked products, soups, tea,
of native starch is limited due to high rate of and distilled liquor as flour or whole grain (Arora,
retrogradation, insolubility in water and fluctuation 1977). It has also been reported that adlay grains
in viscosity during thermal processing (Ashogbon may have anti-tumor activity (Numata et al., 1994)
and Akintayo, 2014). A modified food starch is and be effective against viral infection (Hidaka et
produced when it undergoes one or more chemical al., 1992). Adlay varieties have long been used as
or physical modifications, allowing it to function a nourishing cereal, added in soups and broths in the
properly under high heat and/or shear which usually form of flour and whole grain. In Thailand, a non-
happened during processing. dairy drink from Job’s tears is available in the market
Adlay (Coix lacryma-jobi L.) or Job’s tears is an as an alternative health food. Animal and human
underutilized cereal crop in the Philippines which clinical trials demonstrated that the consumption of
shows potential as a source of starch. There are de-hulled flour and seed of Job’s tear can improve
four known adlay varieties in the Philippines which lipid metabolism, thereby decreasing the risk of
are differentiated by the colour of their seed coats. heart disease. In addition, it could reduce liver fat
Tapul is a non-waxy variety of adlay which has accumulation and protect from tumor stimulating
been discovered in the Philippines in 2010 when an compounds (Chang et al., 2003; Yu et al., 2005).
indigenous tribe in Zamboanga was able to survive a

*Corresponding author.
Email: alfeelynb@[Link]
1200 Capule et al./IFRJ 23(3): 1199-1206

Some bioactive compounds in Job’s tear, especially fat, ash, dietary fibre and amylose), physical (colour,
coixenolide, inhibited tumors, prevented cancer and starch granule structure and pH) and functional
provided protection against viral infection (Hung and properties (water absorption, swelling, solubility,
Chang, 2003; Chun et al., 2004). In a recent study pasting and gelatinization). The results may be
by Yang et al. (2013) adlay is also shown to prevent compared to that of commercially available modified
osteoporosis. Owing to its perceived nutritional starches to ascertain whether the former can be an
and health value, adlay is increasingly utilized in alternative modified starch source. Moreover, since
functional foods and drinks (Li and Corke, 1999). there is no sustainability issue, the use of this cereal
In the Philippines, adlay has been overlooked crop to produce modified starch may provide added
in the past years particularly in its suitability as a livelihood opportunities to Filipino farmers and
food product. Thus, despite perceived nutritional people in the rural areas.
and health-promoting benefits, promoting it in a
country where rice is considered as a staple food Materials and Methods
may be difficult. In order to optimize the utilization
of adlay, there is a need to conduct further study on Materials
its components to produce value-added products to Adlay (Coix lacryma jobi-L var. Tapul)
increase awareness and preference of the Filipinos was obtained from Southern Tagalog Integrated
to this underutilized crop. Transforming adlay into Agricultural Research Center (STIARC) in Lipa
either flour or starch can increase its utilization City, Batangas, Philippines. Modified corn starch
since in this form it can be used as a vehicle for and modified tapioca starch were purchased from
micronutrient fortification. However, utilization of Wills International, Bicutan, Taguig City, Philippines
adlay starch can be enhanced by modification. and used as standards. All chemical reagents used
Heat-moisture treatment or HMT is a physical were of analytical reagent grade.
method of starch modification. It is based on the
principle of heating the starch at a temperature Starch isolation
above its gelatinization point (70-130oC) for one to The Tapul starch was obtained following the
several hours under controlled moisture conditions modified alkaline steeping method of Sira and Amaiz
(15-35%) (Haghayageh and Schoenlechner, 2011). (2004). The grains were steeped in 0.25% sodium
This type of modification is simple because it only hydroxide solution overnight at room temperature,
requires heating, safe because no chemicals are used washed in excess water until neutral and ground for
and therefore relatively cheaper to produce (Jacobs 2 min at high speed in a Waring blender with enough
and Delcour, 1998; Chung, Moon and Chun, 2000; volumes of distilled water. The homogenate obtained
Jobling, 2004). Morever, HMT has been shown to was passed through an 80-mesh sieve and then
provide important properties for food application as further filtered in a 200-mesh sieve. The filtrate were
this method is shown to restrict swelling, increase pooled and re-suspended in water, manually stirred
starch paste stability and increase gelatinization for 5 min and allowed to settle for 2 hours. The
temperature which are all desirable for noodle supernatant is discarded and the starch is repeatedly
manufacture and canned foods (Collado et al., 2001; re-suspended in water, stirred and allowed to settle
Adebowale et al., 2005; Takahashi et al., 2005; and re-suspended in water again until the wash water
Julianti et al., 2013). has reached neutral pH. Finally, the starch is dried
Although adlay grains are high in starch, few in a drying oven at 45oC overnight until 12-14%
studies have been done on the physicochemical moisture content is reached. The dried starch was
properties of its starches (Li and Corke, 1999; ground, passed through an 80 micron sieve and stored
Ramirez, 1996a,b; Thongngam and Dechkunchon, in LDPE bags at ambient temperature till further use.
2007; Kim et al., 2008; Chaisiricharoenkul et al.,
2011) which may provide useful information for Removal of protein from starch
industry purpose and future product development. In The starch from Tapul was isolated using the
addition, its suitability as a raw material for HMT had modified method of Sira and Amaiz (2004). However,
not been studied. In order to develop value-added initial chemical analysis showed the starch to be
products from adlay, understanding and knowledge contaminated with protein (>1%), hence, the method
of its functional properties must be considered. was modified to remove the remaining adhering
Hence, the objective of this study is to characterize protein. The concentration of the alkaline steeping
adlay starch modified by heat-moisture treatment in medium was increased from 0.25% to 0.50%. To
terms of its chemical composition (moisture, protein, ensure removal of any remaining adhering protein,
 Capule et al./IFRJ 23(3): 1199-1206 1201

the starch isolated was re-suspended in 500 ml of meter standardized with buffer solutions pH 4 and
0.25% NaOH, stirred manually for 10 minutes and pH 7.
allowed to soak overnight. The soaked starch was
decanted, washed and re-suspended again in 0.10% Scanning electron microscopy (SEM)
NaOH overnight. The starch was allowed to settle, Dried and finely ground native and heat-moisture
decanted and washed several times with tap water treated adlay starches were placed on aluminum
until the washings reached neutral pH. The starch specimen holders with double-sided adhesive tapes.
was then dried in an oven at 45oC overnight. The The starches were coated with thin film of gold under
dried starch was then blended for 2 minutes, sieved vacuum using JEOL JFL-1200 FINE COATER.
at 80-mesh, packed and stored at room temperature. Coated samples were examined using JSM 5310
The stored Tapul starches were then subjected to (JEOL Ltd., Japan) scanning electron microscope
HMT (30% moisture, 110oC for 8 hours). operated at 20 kV. The images were viewed at 1000x
magnification.
Starch modification
Isolated native Tapul starch (NTS) was modified Water absorption capacity (WAC)
through heat-moisture treatment (HMT) following The water absorption capacity (WAC) of the
the modified method of Collado et al. (2001). Fifty starches was determined using the method of Sosulski
(50) grams of starch was adjusted to 30-35% moisture et al. (1976). One gram of the starch sample was
content by soaking in distilled water and equilibrated mixed with 10 ml distilled water and let stand at room
at 4oC overnight. The conditioned starch samples temperature for thirty (30) minutes, then centrifuged
were placed in petri dishes, covered with an aluminum at 3,000 rpm for 30 minutes. Water absorption was
foil and heated in a drying oven at 110oC for 8 hours. measured as percent water bound per gram starch.
The starch samples were mixed occasionally using a
mixer glass for even heat distribution. The starches Swelling power and solubility
were removed from heat and allowed to cool at Swelling power (SP) and solubility were
ambient temperature, packed in PE bags, sealed and determined according to Li and Yeh (2001) with
stored till further use. modifications. The sample was weighed and distilled
water was added into a centrifuge tube with a screw
Chemical analyses cap. The centrifuge tubes were heated at 55oC, 75oC
The chemical analysis of both native and heat- and 95oC for 30 min with occasional stirring. The tubes
moisture treated adlay starch were determined were cooled in an iced water bath and centrifuged at
according to AOAC (2000). The conversion factor 3,000 rpm for 15 min. The supernatant was carefully
of protein was 5.85. The amylose content was removed and starch sediment was weighed. The
determined using the Megazyme Rapid Test Kit clear supernatant was removed into a pre-weighed
(Megazyme International Ireland Ltd.) based on the aluminum dish and evaporated at 130oC for 2 hours,
principle of concanavalin-A binding method (Gibson cooled and then weighed. The swollen sediment and
et al., 1996). dried supernatant were weighed to determine the
swelling power and solubility as follows:
Physical analyses
The colour and colour difference were analyzed Swelling power (%) = [sediment weight / (initial
with a chromameter (CR-300; Minolta, Tokyo, sample weight x (100% - %solubility))] × 100;
Japan) using the Hunter system, which identifies Solubility (%) = [dry supernatant weight / initial
colour using three attributes: L (white = 100, black = sample weight] × 100
0), a (red = positive, green = negative) and b (yellow
= positive, blue = negative). The colour difference Pasting properties
(ΔE), a measure of the distance in colour space The pasting properties of the starch samples
between two colours, was determined by comparison were measured with a Rapid Visco-Analyzer (RVA,
to a white standard tile with colorimeter values of Newport Scientific, Warriwood, Australia), according
L = 94.5; a = -1.0 and b = 0.2, using the following to Li and Corke (1999). A 2.5 g of sample (dry basis)
relationship: was weighed into an RVA canister and deionized
water was added to obtain the total sample weight of
ΔE = √ ΔL2 + Δa2 + Δb2 28 grams. The sample was then mixed for 30 seconds
to obtain a lump-free sample. The time-temperature
The pH was determined using the Jenway pH profile was: holding for 2 minutes at 50oC, heating
1202 Capule et al./IFRJ 23(3): 1199-1206

to 95oC in 7.5 minutes, holding for 5 minutes at

95oC, cooling to 50oC in 7.5 minutes and holding
for 5 minutes at 50oC. The peak viscosity, setback,
breakdown and the final viscosity were recorded.

Gelatinization properties
The gelatinization temperature of native and
modified adlay starches were studied using a
differential scanning calorimeter (DSC-Q100, TA
Instruments Waters, USA) equipped with a TA
Universal Analysis software. Water (8 µl) was added
Figure 1. Scanning electron micrograph of Tapul starch a)
using a microsyringe to starch (4.0 mg, db) in the Native and b) HMT starch
DSC pans, which were then sealed and allowed to
equilibrate for at least 30 minutes. The sealed pans digestibility after HMT is attributed to changes that
were then heated (20-120˚C at 10˚C/min) to gelatinize occur in the starch granule particularly when starches
the starch. An empty pan was used as a reference. are heated at temperatures below its gelatinization
The onset of gelatinization (To), peak temperature point at high moisture content (>20˚C), thereby
(Tp), completion temperature (Tc) and gelatinization making the starch susceptible to enzymatic attack
enthalpy (∆H, J/g) were determined. (Jacobs and Delcour, 1998). In terms of amylose
content, the value did not change even after HMT
Statistical analysis and the obtained value is slightly lower compared
Analysis of Variance (ANOVA) was used to to the study of Li and Corke (1999) who reported a
analyze significant differences between the native and range of 15.9-25.8% for normal coix starches. The
heat-moisture treated Tapul starches, and between amylose content of starches is important as it affects
modified Tapul starches and standard modified swelling, gelatinization, pasting, retrogradation and
starches. enzymatic vulnerability of starches. Regardless of the
starch origin, HMT can cause a decrease in amylose
Results and Discussion leaching and formation of amylose-lipid complexes
which may cause an underestimation of the amylose
Chemical properties content.
Result of the chemical analyses of native and
heat-moisture treated Tapul starch and standard Physical properties
modified starches are presented in Table 1. Following Result of the physical analyses of NTS, HMTTS
modification, the moisture content of heat-moisture and standard modified starches is presented in Table
treated Tapul starch decreased in comparison to the 2. The colour of the finished starch is an important
standard modified starches. Moisture content of a characteristic with white as the ultimate objective
powdered product depends on the drying temperature, (Wang et al., 2000). A starch with an L value >90
length of drying time and humidity conditions to is considered to have a satisfactory lightness degree.
which the powdered product was exposed. It also After HMT, the L value decreased but is still within
played a significant role in the flow properties of a the acceptable range. In addition, low chroma value
food (Lawal, 2004). Re-suspension of the starch paste is desired for starches. The parameter a was observed
in alkali was able to significantly lower the protein to be negative (-) and reduced after heat-moisture
content of the starch as illustrated in Table 1. Protein treatment but b was observed to be positive (+) and
content in starch is related to starch purity and ideally found to increase after HMT which may affect the
should not be present in starch. However, increase whiteness of the modified adlay starch. In terms of
in protein content after HMT of Tapul starch may colour difference, ΔE increased after HMT which
be attributed to the residual proteins still embedded could be due to reaction of the amino group in the
in the starch granules which were released when proteins present in the starch during modification but
the starch granules were subjected to HMT. Ash ΔE was small (<10) to be detected visually (Kim et
content increased after HMT which may be due to the al., 2008; Yaseen and Shouk, 2011).
contaminating sodium from repeated alkali treatment. The pH is an indicator of the degree of acidity
This is also an indication of the process used to or alkalinity of a substance. It is also necessary that
isolate the starch from adlay. The total dietary fibre of the pH of starch is near neutrality so it may be used
Tapul starch after HMT increased. Increase in starch in industries where changes in pH of products are
 Capule et al./IFRJ 23(3): 1199-1206 1203

Table 1. Chemical properties of native and modified Tapul starch as compared to standards

*
NTS – native Tapul starch; HMTTS – heat-moisture treated Tapul starch; MCS – modified corn
starch; MTS – modified tapioca starch; n.d. = not detected
Data are reported on dry basis and values are means ± s.d.’s of duplicate determinations.
a ,b, c, d
Different letters within the same row indicate a significant difference p<0.05.

Table 2. Physical properties and WAC of native and modified Tapul starch as compared
to standards

*
NTS, HMTTS, MCS, MTS are as defined in Table 1.
Values are means ± s.d’s of triplicate determinations
a ,b, c, d
Different letters within the same row indicate a significant difference p<0.05.

not required. After HMT, the pH value obtained was products. It affects functional properties such as
lower and not significantly different from the standard viscosity, which is a very important indicator of
modified starches. Thus, heat-moisture treated Tapul bulking and consistency of products. Heat-moisture
starch may be used in food products where these treatment of Tapul starch resulted in an increase
standard modified starches are typically used. in the WAC from 209.50% to 265.60% with the
Result of scanning electron microscopy on increase significantly higher than the standard
the shape and surface characteristics of native and (Table 2). This could be attributed to the changes
treated starches are shown in Figure 1. Native in the available water binding sites when starch was
Tapul starch granules are shown to be round and modified. This finding is also in line with the studies
polygonal in shape that varies in size and with porous of Balasubramanian (2011) and Iheagwara (2013)
surfaces. The average granule sizes of the native where heat-moisture treated finger millet starch and
starch were 5.2 and 12.7 µm respectively, hence, sweet potato starch obtained the highest WAC value.
Tapul starch granules may be considered small. The swelling power of both native and modified
HMT did not alter the granule shape and size of the starches from Tapul increased with increasing
starch granules which was similar to the previous temperature as presented in Figure 2. Beginning from
study on HMT of finger millet starch (Adebowale the temperature of 75ºC, the swelling powers of both
et al., 2005). However, when viewed under SEM, native and modified starches from Tapul increased to
HMT adlay starch showed various cracks and holes a maximum. However, swelling power is restricted
on the surface. This may be attributed to the mode after HMT. In general, HMT causes restricted
of preparation and modification used that may have swelling and this may be attributed to increase in
affected the morphology of the starch granules. starch crystallinity causing restricted entry of water
within the starch matrices. This is in line with the
Functional properties report of Noomhorm and Hormdok (2007) on rice
Water absorption capacity or (WAC) is an starches.
important parameter that determines starch use in The solubility of both native and modified
1204 Capule et al./IFRJ 23(3): 1199-1206

Figure 2. Effect of swelling and solubility on NTS, HMTTS, MCS and MTS
starches from Tapul is also shown in Figure 2. With heating and shearing as compared to NTS and MTS
increasing temperatures, the solubility of starches which is in accordance with the study of Adebowale
was substantially increased. This might be due to the et al. (2005), Noomhorm and Hormdok (2007) and
fact that the starch contained a high amount of small Olayinka et al. (2008). The final viscosity increased
size starch molecules and some big and small holes after modification of the Tapul starch. The FV value
appeared on the surface of the starch granules. Upon is a measure of the ability of the starch to form paste
swelling, the surface holes may allow a number of or gel during cooling. This could mean that modified
the small starch molecules to leach out into the water. non-waxy adlay starch molecules re-associate easily
However, a reduction in solubility of NTS occurred during cooling to produce a stable gel or starch
after HMT at 95oC as compared to the standard paste. The setback value is a reflection of the degree
modified starches. This is in line with the results of retrogradation of starch pastes. After HMT, the
of Adebowale et al. (2002), who studied the effects setback value was reduced. This was probably related
of HMT on the solubility of Bambarra groundnut to the low amylose content, increasing swelling power
starch at 95oC where after HMT, the starch showed with a higher proportion of a short branch-chain of
maximum hydrophilicity with minimum amylose amylopectin. According to Chaisiricharoenkul et
leaching hence, becoming the least soluble. In al. (2011), Job’s tear starches are characterized by a
addition, chemically-modified starches have higher short-chain amylopectin. A low setback value could
solubility compared to physically modified starches. mean that during cooling, modified Tapul starch paste
Pasting of starch is a process following does not retrograde easily. Overall, higher viscosities
gelatinization in the dissolution of starch. Result and decreased retrogradation after HMT of Tapul
obtained from the RVA is presented in Table 3. It starch show potential application for food products
involves starch granule swelling and amylose that require thermal processing such as in canned
leaching from the granule until total disruption of products as well as a thickener in sauces and soups
starch granules occurred. Starch pasting is affected since it can extend viscosity longer.
by presence of amylose, lipids, protein, swelling After HMT, there was an increase in the
power and chain length distribution of amylopectin gelatinization properties (To, Tp, Tc and Tc-To)
(Li et al., 2008). An increase in pasting temperature for Tapul starch. Native Tapul starch showed a
suggests resistance of adlay starch to gelatinization gelatinization temperature ranging from 74-77˚C
after HMT. From Table 3, it was shown that Tapul corresponding to a narrow gelatinization range. After
starches had higher peak viscosities, lower trough and HMT, there was a widening of the gelatinization
final viscosities but with higher pasting temperatures temperature ranging from 73-81ºC and is also
compared with the standard starches. This may be different from the standards (61-74ºC). The enthalpy
attributed to increase in water absorption capacity or ∆H after HMT also increased (0.50-16.98 J/g)
and the interactions with traces of lipid and protein indicative of more heat stability and may be correlated
components with amylose in the starch may have to the relative crystallinities of the adlay starch as
affected pasting properties of starch. In terms of compared to ∆H values of the standard modified
breakdown, HMTTS showed a reduced breakdown starches (12-14 J/g). It may also be attributed to the
value compared to NTS and the standard. The characteristic of HMT starch to induce formation of
breakdown value is an estimate of the paste resistance new crystallites with different stabilities. A starch
to disintegration due to heat and shear. A decrease in with higher degree of crystallinity maintains its
breakdown value after HMT is an indication that the structural stability, consequently making granule
modified Tapul starch is more stable during continued resistant toward gelatinization since water molecules
 Capule et al./IFRJ 23(3): 1199-1206 1205

Table 3. Pasting properties of native and modified Tapul starch as compared to standards

*
NTS, HMTTS, MCS, MTS are as defined in Table 1.
Breakdown is P minus TV; Setback is FV minus PV; Peak time is time in seconds to reach the peak viscosity;
Pasting temperature is the temperature at the onset of rise in viscosity

cannot easily penetrate the crystallites requiring funding support of the Department of Science and
more energy and thus exhibiting a higher transition Technology-Science Education Institute (DOST-
temperature. Overall, HMT starches are shown to SEI) and free adlay samples from Southern Tagalog
have different gelatinization profile from chemically Integrated Agricultural Research Center (STIARC).
modified starches and this is important to ascertain
whether HMT Tapul starch can have functionalities References
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