Sains Malaysiana 38(3)(2009): 341345

Effect of Red Seaweed Polysaccharides Agar (Gracilaria changii) on Thermal Properties and Microstructure of Wheat Starch
(Kesan Polisakarida Agar daripada Rumpai Laut Merah (Gracilaria changii) ke atas Sifat Terma dan Mikrostruktur Kanji Gandum)
P. KHAIRUL FAIZAL, H. ROHASMIZAH, B. ABDUL SALAM & A.S. NORRAKIAH

This study has been carried out on the mixture of Gracilaria changii agar (0.1%, 0.2%, 0.4% and 0.8%) with wheat starch. Scanning electron microscopy (SEM) was performed for morphology observation, and starch thermal analysis were carried out to determine the properties of gelatinization and retrogradation. Proximate analysis has been determined for isolated wheat starch and agar. Through SEM, interaction was first observed at 64C for 0.4% agar but at 0.8% of agar, a more extensive bridging was formed which enveloped the starch granules. Differential scanning calorimetric (DSC) result shows that the addition of agar decreased the onset temperature (To) of gelatinization significantly (p<0.05) but increased the gelatinized enthalpy (Hgel), gelatinized temperature range (Rg) and Peak Height Index (PHI) significantly (p<0.05). Agar lowered the retrogradation enthalpy (Hret), retrogradation range (Rret) and retrogradation percentage (%R) of wheat starch significantly (p<0.05). Keywords: Agar; gelatinization; Gracilaria changii; retrogradation; starch granule Kajian telah dijalankan terhadap campuran agar Gracilaria changii (0.1%, 0.2%, 0.4% dan 0.8%) dengan kanji tepung gandum. Kajian ini meliputi mikroskopi pengimbas elektron (SEM) untuk pemerhatian morfologi. Analisis terma kanji juga dikaji bagi melihat sifat pengelatinan dan retrogradasi. Melalui SEM pada suhu 64C, interaksi mula dilihat pada aras 0.4% agar tetapi pada aras 0.8%, agar membentuk jejambat yang lebih ekstensif dan bertindak menyaluti granul kanji. Melalui kalorimetrik pengimbas perbezaan (DSC), penambahan agar merendahkan suhu permulaan penggelatinan (To) secara signifikan (p<0.05) sementara meningkatkan nilai entalpi penggelatinan (Hgel), julat penggelatinan (R) dan Indeks Ketinggian Puncak (PHI) dengan signifikan (p<0.05). Agar juga merendahkan entalpi retrogradasi (Hret), julat retrogradasi (R) dan peratusan retrogradasi (%R) kanji gandum secara bererti (p<0.05). Kata kunci: Agar; penggelatinan, Gracilaria changii; retrogradasi; granul kanji INTRODUCTION A pilot plant study was successfully ran on the Bay of Bengal Program (BOBP) with the involvement of Food and Agriculture Organization (FAO), Malaysia Department of Fisheries and other agencies on the cultivation of Gracilaria in Penang. However, during the 1980s, the Malaysian government redirected the fund on Gracilaria cultivation into shrimp farming (Anon 2007). As a result, less investment on the development on Gracilaria cultivation was recorded ever since. A few market studies for agar have been carried out by universities in Malaysia. More recently interest has been revived by the Fisheries Department and Universiti Sains Malaysia on both cultivation and improvement of agar extraction method. The genus of Glacilaria is one of the valuable economic assets which can be utilized as hydrocolloid and agar. According to Phang et al. (1996), Gracilaria changii was recorded in Malaysia and Thailand as one of the abundant agarophytic seaweeds. The use of hydrocolloid such as alginate and carageenan is very popular in food application since it has been proven to be able to increase several quality parameters. Xanthan and guar gum are sufficiently functional at very low levels to be cost effective. Christianson et al. (1981) found that guar gum, xanthan and carboxymethylcellulose (CMC) elevated wheat starch viscosity during gelatinization. Increased viscosities at the end of heat peak were caused by interaction of gums with starches. Gums will function successfully in some applications and have no effect on others. However, explanation for this variability is lacking (Heflich 1996). The objective of this research is to monitor the effect of Gracilaria changii agar addition at different levels on the gelatinization and retrogradation properties of wheat starch.
ABSTRAK

ABSTRACT

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MATERIALS & METHODS

ISOLATION OF WHEAT STARCH THROUGH ALKALINE EXTRACTION

procedure stated in MALAYSIAN STANDARD (1994) provided by SIRIM and AOAC (1990).
PREPARATION OF SAMPLE FOR DEFFERENTIAL SCANNING CALORIMETRY (DSC)

100g of wheat flour was suspended in 350 mL distilled water and 350 mL of 0.5% NaOH solution for 60 minutes and then centrifuged. Centrifugation was done at 3000 g for 10 minutes at 20C. Sediments were washed with 350 mL of distilled water for 30 minutes and again centrifuged and washed with 60 mL of distilled water for 15 minutes. After centrifugation, sediments were suspended in 80 mL of water, stirred for 10 minutes, neutralized using 1.0 M HCl and centrifuged. The upper greyish layer was removed and starch which is white in colour was suspended in 100 mL distilled water and sieved through 38 mm with additional of 300 mL distilled water. Starch sample was centrifuged and dried overnight, sieved through 250 mm using mortar and pestle (Schierbaum et al. 1991).
AGAR EXTRACTION

Agar from Gracilaria changii was extracted using alkaline treatment based on Hurtado-Ponce & Umezaki (1988) with slight modification. 25 g of red seaweed was soaked in a 1 L of 5% NaOH for 1 hour. After that, the sample was neutralized with 750 mL of 0.5% acetic acid for 1 hour. Extraction started when the sample was boiling in distilled water for an hour. The sample was then vacuum filtered to separate cellulose residues. Filtered liquid (agar) was frozen overnight and thawed the next day. Agar was refiltered to remove pigments on samples and dried at 60C and fined to powder.
PREPARATION OF WHEAT STARCH-AGAR (GRACILARIA CHANGII) MIXTURE

Wheat starch-agar samples underwent gelatinization and retrogradation using DSC 822 c, Mettler Toledo, Schwezenbach, Switzerland. About 102 mg mixture of samples were weighed in a 40 L pan and hermatically sealed. Scanning was performed from 20C to 90C at a constant rate of 10C/minute. Empty pan was used as reference and DSC was calibrated using indium. Thermal changes were reported as onset temperature (To), peak temperature (Tp), conclusion temperature (Tc) and entalphy (H) was calculated using the software (Suphantharika & Chaisawang, 2005). Gelatinization range (Rg) and peak height index (PHI) were calculated using formula 2(Tp-To) and H/(Tp-To) respectively (Singh & Sandhu 2007). Retrogradations of gelatinized samples were determined after 7 days of storage at 4C. Retrogradation range (Rret) was calculated as 2(Tp-To) and retrogradation percentage (%R) was calculated using formula below:

Scanning Electron Microscope (SEM) Initially, samples were heated in the water bath at 64C. SEM observation was done as described by Barrera et al. (2002) with modifications. Samples were fixed in 2% glutaraldehyde for 24 hours and dried in a critical point dryer (model CPD-030, BAL-TEC Company). Images were observed by SEM (Phillips XL-accelerated voltage was operated at 10 kV).
STATISTICAL ANALYSIS

The agar was mixed at 0.1%, 0.2%, 0.4% and 0.8% on substitution basis to 6% of wheat starch using 25 mL of distilled water. Sample preparation is simplified in Table 1.
PROXIMATE ANALYSIS

The data reported in all tables are average of triplicate observations and were subjected to one-way analysis of variance (ANOVA) and DUNCAN test. Statistical analyses were performed using SAS version 9.1. RESULT & DISCUSSION In this study, 24.7% of agar was isolated from Gracilaria changii. Previous research on extraction of agar was

After the isolation process, agar and wheat starch underwent a series of proximate analysis to determine the moisture, ash, protein and fat in accordance to the standard
TABLE

1. Level of wheat starch with agar concentration WS WS+A1 5.9 0.1 WS+A2 5.8 0.2 WS+A3 5.6 0.4 WS+A4 5.2 0.8

Wheat starch (%) Agar (%)

WS = Wheat starch only

6 0

WS+A1 = Wheat starch with 0.1 % agar WS+A2 = Wheat starch with 0.2 % agar WS+A3 = Wheat starch with 0.4 % agar WS+A4 = Wheat starch with 0.8 % agar

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reported to have the range of 12% to 25% (Phang et al. 1996). Composition of agar and wheat starch is shown in Table 2. Moisture content obtained was higher than previously reported by Selby and Wynne (1973) who also stated that maximum moisture guideline for commercial agar is 20%. Solute such as agar affects the structure of the water. Interaction of water exists when special force between solute and water molecules appears. Charged ions whether small or big create stronger solute-water bond within the dipoles than water-water hydrogen bond (Fennema 1985). Lower ash (which referred to the percentage of inorganic materials presence in samples) was observed as compared to Selby and Wynne (1973). Commercial agar usually has sodium ion and magnesium besides calcium. Protein and fat presented in lower percentage, as low as 1.01% and 0.12% respectively. Lower percentage of non agar compound such as nitrogen and fat was desired because those compounds could lead to disruption of agar interaction with solvent and thus affect agars properties during gelling. Maximum protein nitrogen agreed is 0.32 % in commercial agar (Selby & Wynne 1973). The amount of wheat starch obtained from alkaline extraction was slightly similar to the findings reported by Verwimp et al. (2004) namely 80.21%. Wheat starch extracted has low proximate value. The moisture content obtained was 35.79%, higher than the moisture standard for starch set by SIRIM, namely 12.0-13.0%. Protein content in wheat starch isolated is 0.67% which is higher than value reported by Verwimp et al. (2004) (0.31%) in commercial wheat flour. The presence of protein on starch granules affects food product hardness (Thomas & Atwell 1999).
TABLE

2. Proximate composition of wheat starch and agar Wheat 35.796.22 0.600.58 0.670.19 0.130.09 Gracilaria changii 35.634.93 1.040.35 1.010.26 0.120.06

Composition (%) Moisture Ash Protein Fat

The fat content of wheat starch obtained was lower than 0.32% reported for commercial wheat flour (Verwimp et al. 2004) and 0.90% for wheat starch (Davies 1995). Starch lipids are saturated in starch granules and the lipid structure enable starch to form complexes by bonding fatty acids in the core of amylose helix (Fennema 1985). Ash content is higher in wheat starch isolated as compared to MALAYSIAN STANDARD (1994) and it maybe due to factors such as sources of raw material, farming methods, milling procedure and chemical modification of starch (Thomas & Atwell 1999). The addition of 0.4% of polysaccharide agar on wheat starch caused formation of bridging by agar surrounding the starch granules. Elevation of agar till 0.8 % showed an extensive bridging with more granules forming a packed structure as shown in Figure 1(c). The result shows similarity with effect of 0.35% xanthan gum on starch (Suphantharika & Chaisawang 2005). According to Mandala et al. (2002), xanthan gum formed film on tapioca starch granules but no aggregation occurs. Starch dispersed in guar gum could adsorb more water which increased swelling ability and viscosity of starch-guar gum complex. On the other hand, xanthan gum lowered the adsorption of water in starch which leads to lower swelling ability and a lower viscosity of starch (Mandala et al. 2002). The gelatinization temperatures, enthalpy of gelatinization, peak height index and gelatinization range for mixtures measured using DSC are presented in Table 3. Significant changes (p<0.05) were observed in To, Tp and Tc for all samples. Sample at 0.2%-0.8% agar displayed lower To. Overall, gelatinization temperature ranges from 65.27C to 78.91C. These values showed similarity with those of Suphantharika and Chaisawang (2005) for tapioca starch with guar gum and tapioca starch with xanthan. In this study, no significant increase was recorded in gelatinization transition temperature. The difference in Hgel reflects melting of amylopectin crystallites. The variations in Hgel could represent differences in bonding forces between the double helices that form the amylopectins crystallites which resulted in different alignment of hydrogen bonds within starch molecules (McPherson & Jane, 1999). PHI is a measurement of uniformity in gelatinization in which

(a)
FIGURE

(b) 1. Scanning electron microscopy of (a) Wheat starch without agar, (b) with 0.4 % agar & (c) 0.8 % agar

(c)

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TABLE

3. Gelatinization properties of mixture of agar Gracilaria changii and wheat starch To (C) Tp (C) Tc (C) Hgel (J/g) Rgel 9.42c 9.99b 9.41c 10.27a PHI

Sample

WS

WS+A1 WS+A2 WS+A3 WS+A4

a

65.70a
65.72a 65.27c 65.36c 65.47b

70.46b
70.43b 70.26c 70.07d 70.60a

78.91a
77.98b 77.29d 77.87c 76.43e

0.84b
0.64d 0.88b 0.77c 1.03a

9.51c

0.18b
0.14d 0.18b 0.16c 0.20a

TO = Onset temperature, TP = Peak temperature and TC = Conclusion temperature TABLE 4.

Means within same column sharing same letter were not significantly (p>0.05) different

Retrogradation properties of mixtures of agar Gracilaria changii and wheat starch after 7 days of storage at 4C Tp (C) 58.85a 58.72a 58.42b 58.82a 58.68a Tc (C) 65.23a 56.40a 64.22a 64.69a 64.25a Hret (J/g) 0.80a 0.37d 0.54c 0.64b 0.39d Rret 34.68a 23.25b 23.92b 34.39a 23.47b %R 94.24a 58.81c 61.99c 82.12b 37.86d

Sample WS WS+A1 WS+A2 WS+A3 WS+A4

a

To (C) 41.51b 47.10a 46.46a 41.63b 46.95a

Means within same column sharing same letter were not significantly (p>0.05) different TO = Onset temperature, TP = Peak temperature and TC = Conclusion temperature

the whole treatments shows fluctuation in the uniformity of gelatinization. Sample with 0.8% agar exhibited the highest Rgel (p<0.05) Starch retrogradation is the event which occurs when starch molecules begin to associate into ordered structures. In its initial phase, two or more molecules may form a simple juncture point which then can develop into more extensive ordered regions. Ultimately, under favourable conditions, crystalline order appears (Zobel & Kulp 1996). The retrogradation properties of mixtures are presented in Table 4. Retrogradation (%) for all sampels was in the range of 37.66% to 94.56% after 7 days of storage. According to Yamin et al. (1997), retrogradation (%) of 50% to 60% was reported on corn starch. However, retrogradation (%) in this study showed lower value than those of corn starch. Retrogradation enthalpies for all samples were lower (p<0.05) than their native counterpart. This may be due to the weaker starch crystallinity of retrograded starch (Sasaki et al. 2000). Hret for corn starch range from 4.4 to 6.9 J/g (Narpinder & Kawaljit 2007). Enthalpy of sample increases as the agar level increased. Hret for all samples range from 0.37 to 0.80 J/g. After 7 days of storage, all samples showed significant changes in transition temperature excluding Tc. Similar changes also reported by Singh & Sandhu (2007) on corn starch. This might be due to recrystallization of amylopectin branch chains occurred in less ordered manner in stored gel, as it is present in native form (Narpinder & Kawaljit 2007). Significant (p<0.05) changes for retrogradation (%) were found for all samples after 7 days of storage in which

0.8% agar showed the lowest percentage. Meanwhile, control sample showed the highest retrogradation (%). Lower retrogradation percentage stabilized freeze-thaw of starch which has wide applications in food. In general, all samples showed fluctuation retrogradation range (Rret). The differences in Hret indicate different tendency towards retrogradation in which control sample observed to display the highest retrogradation. CONCLUSION Addition of agar from Gracilaria changii affects gelatinization properties of wheat starch. Our results shows that retrogradation temperature transition (To, Tp and Tc) of all samples were lower than the gelatinization temperature after 7 days of storage. In conclusion, addition of Gracilaria changii agar could significantly minimize the retrogradation of wheat starch.
ACKNOWLEDMENT

The authors are grateful to the Ministry of Science, Technology and Innovation of Malaysia (IRPA) and Fishery Research Institute (FRI, Ban Merbok) for the support.
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MALAYSIA

H. Rohasmizah, B. Abdul Salam, A.S. Norrakiah* Food Science Programme Centre of Chemical Sciences and Food Technology Faculty of Science and Technology National University of Malaysia 43600, UKM Bagi Selangor, D.E.
MALAYSIA

*Corresponding author, email: norra@ukm.my Received: 18 January 2008 Accepted: 15 September 2008