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Preparation of Starch Nanoparticles From Indonesia’s Local Starches

Titi Candra Sunarti1 ,Christina Winarti 2, Nur Richana2 1Agroindustrial Technology Department, Faculty of Agricultural Technology, Bogor Agricultural University, Bogor 16002, Indonesia 2Indonesian Center for Agricultural Postharvest Research and Development (ICAPRD) Indonesian Agency for Agricultural Research and Development (IAARD) Jl. Tentara Pelajar 12, Bogor 16114, Indonesia Email: titi-cs@ipb.ac.id (corresponding author)

Keywords: Tapioca starch, arrowroot starch, ethanol precipitation, butanol complex

Abstract Indonesia has abundant local starch-source crops which have potential to be

developed as industrial materials by modification into nano-sized particles to extend its application. Type of starch with different granule size and crystallinity might result different structure. The research aims to find out the characteristics of two different starch sources namely arrowroot and tapioca by ethanol and butanol precipitation. Pretreatment of starch has carried out to increase their characteristics using acid hydrolysis (lintnerization) for 2; 4; 6 and 24 hours to produce dextrin. Type of starch affected the resulted starch nanoparticles. Lintnerization duration also influenced size particles distribution, WAC and OAC. Starch morphology exhibited porous and fine structure that improving their absorption capacity. The absorption capacity reaches the highest level at lintnerization duration for 4 h at arrowroot starch and 6 h at cassava starch. The crystalline pattern (XRD) showed that butanol precipitated starch nanoparticles produced different pattern of crystallinity compared to ethanol precipitation.

INTRODUCTION Recently, a trend of minimizing the impact of chemicals on the environment as well

as looking for alternatives to depleting petrochemical resources has generated high demand for bio-based polymers (Cadar et al. 2012). Starch is a promising candidate and has been extensively investigated. Starch has been widely used in various industries such as food, textile, and pharmacy. It is used in many applications including surface sizing, as a food emulsifier, fat replacer, excipients for tabletting and drug delivery carriers (Mahkam, 2010).

Starch is mainly composed of amylase (AM) and amylopectin (AP), which are two homopolymers having the same repeat units that is linked in linear and branched way, respectively (Pareta et al. 2006). There are a lot of hydroxyl groups on the backbone. The primary and secondary hydroxyl groups at C-2, C-3, and C-6 of each glucose residue make starch hydrophilic and available for further modification (Tomasik., 2004). Preparation into starch nanoparticles is one kind of modification to enhance starch performance (Xu et al. 2010). A starch nanoparticle has benefits in terms of higher surface area, lower viscosity at higher concentration and higher entrapment of active ingredients (Chen et al. 2006).

Ethanol precipitation produces porous starch that is used as reinforcing effect and filler in nanocomposites, as well as a potential carrier matrix for the active ingredients that are difficult to dissolve in water. According to Devesvaran et al. (2012) porous starch has a nanoporous structure, low density, large surface area and large pore volume. Non-solvent precipitation will produce nanostructures because it is a process that involves the addition of

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dilute polymer into a non-solvent to the polymer precipitated in nano size (Tan et al. 2009), or the addition of non-solvent in the polymer. Tan et al. (2009) and Amelia et al. (2007) use acetone while Ma et al. (2008) using ethanol, moreover, Patindol et al. (2012) use ethanol on gelatinized starch and rice flour to produce starch with high porosity.

Research on preparation of starch having nano-sized particles by a butanol complex has been carried out by some researchers such as Herbert and Chancy (1994); Kim et al, (2009); Kim and Lim (2010). Butanol can only form a complex with amylose and precipitates, but not with amylopectin. Production of nanosize particle by butanol complex require preparation of starch into shorter and more crystalline amylose, such as acid hydrolysis (lintnerization) or acid-alcohol hydrolysis. Kim et al, (2009); Kim and Lim, (2010).

Lintnerization or acid hydrolysis is one of starch modification processes that are often used for initial preparation as reported Gunaratne and Corke (2007); Kim and Lim (2010). The botanical origin of starch which have different properties may produce different characteristics of nanoparticles. The purpose of the study was to determine the characteristics of pecipitated of lintnerized starch from two kinds local starch using ethanol and butanol precipitation pretreated with different levels of acid hydrolysis.

MATERIALS AND METHODS The raw materials that used are arrowroot with Creole varieties that orginating from

the area around Bogor, tapioca obtained from tapioca industry Sentul, Bogor. Chemicals that used for this process among others HCl, ethanol, butanol, methanol, and other chemicals used for analysis.

Production and Characterization of Cristalline Starch by Acid hydrolysis Production of crystalline starch carried out by acid hydrolysis/lintnerization

(Jayakody and Hoover, 2002). The starch is made suspension in HCl 2.2 N with ratio 1:2 and incubated at temperature 35C for 2, 4, 6, and 24 hours using rocking waterbath. Starch suspension that has hidrolyzed by acid and has neutralized by NaOH 1 N, then washed with ethanol and distilled water and then dried at temperature 40C for 24 hours.

Production and Characterization of Starch Nanoparticles Starch nanoparticles production was carried out via complex formation of lintnerized

starch with ethanol precipitation (Ma et al. 2008) and butanol precipitation (Kim & Lim, 2010). For ethanol precipitation, the lintnerized starch was dispersed with distilled water at ratio of starch: distilled water 1:20 (w/v) and then heated until it was gelatinized (approx. 30 minutes) while stirring with rapid rate on the hotplate stirrer. After that ethanol was added drop wise with a dropping rate of about 3 ml/min for about 60 minutes with rapid stirring. The complex/precipitated was washed several times with ethanol and then filtered, dried with a freeze dryer and analyzed.

Meanwhile, for butanol precipitation, the lintnerized starch (40 g) was dispersed in hot distilled water and the suspension was autoclaved at 121C for 20 min. The solution was cooled to 70C and about 20% of n-butanol was slowly added to the solution to form a separated butanol phase from the starch solution. The solution was then stirred gently (100 rpm) at 35C for 3 days. The solution was centrifuged at 5000 rpm for 20 min, and then the precipitates were dried by freeze dryer. The weight of the precipitates was measured to calculate the yield of the complex.

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Treatments for ethanol complex preparation involved: lintnerization duration for 2, 4, 6 and 24 hours, meanwhile for butanol precipitation involved: Lintnerization durations (H2 = 2 h and H24 = 24 h) and lintnerized starch concentration (B5 = 5% and B10 = 10%). The research design used was Randomly Complete Design with three replicates.

The parameters observed in starch nanoparticles include yield, water and oil absorption capacity (Leach et al. 1959), digestibility (in vitro) using pancreatin amylase), morphology (SEM and TEM), particle size (PSA) and cristallinity pattern (XRD).

RESULT AND DISCUSSION

Yield, Digestibility, and Water and Oil Absorption The yield of starch hydrogel prepared from arrowroot and tapioca starch by ethanol

precipitation showed that the longest duration of lintnerization (24 h) revealed the lowest yield of starch hydrogel prepared by ethanol precipitation (less than 40%). However, lintnerization for 4, 6 h showed higher yield (about 80%) compared to 2 (74%) and 24 h lintnerization ( 40%).

Digestibility and absorption of water and oil are presented in Table 2. The results showed that materials undergo prolonged lintnerization decreased their digestibility but to some level but then increased again. Unlike the tapioca starch, both materials have an increased the digestibility of the 4 hours lintnerization, then decreased again at 6 and 24 hours lintnerization. Factors affected the digestibility of starch is retrogradation process in which starch recrystalled and formed a rigid structure to caused a decline in digestibility. The occurrence of such events may cause the short chain amylose retrogradated fraction forming a double helix chain to form a compact crystallites, where the intermolecular bonds are formed again to be more resistant to destruction by the enzyme α - amylase .

Water and oil absorption showed an increase until 4 hours lintnerization for arrowroot starch and 6 hours for tapioca starch, but then decreased after 24 hours. Treatment of 4 h acid hydrolysis resulted increasement to more than 900% compared to the dried sample. Moreover, for other two starches increase about 800% (Table 1). Factors affecting the absorption capacity of starch including botanical source, granule size, ratio of crystalline and amorphous parts, amylose content. The granule size of tapioca (5-40 m) and arrowroot starch (5-70m) (Tester and Karkalas, 2002). Moreover, Swinkels (1985) reported that the amylose content in starch can also affect the absorption of oil and water. According to Wei et al. (2012) higher amylose content in starch super absorbent will absorb more water because of the higher the grafting ratio and grafting efficiency and the high mobility of starch chains. The longer duration of lintnerization namely 24 h showed less water and oil capacity because of the higher crystallinity of the starch. As already mentioned acid attack mainly on the amorphous region and left the crystalline parts (Srichuwong et al., 2005).

The longer lintnerization process caused increasing oil absorption capacity. The lower WAC and OAC at long duration of lintnerization may related to its higher crystallinity level. Das et al (2010) mentioned that the decrease of OAC occurred because of the reduction of the amorphous region in starch granule, that lead to reduction the number of available binding sites for oil. As mentioned before at the 24 hour lintnerization, acid hydrolyzed mainly on the amorphous region so that this region reduced and the crystalline region remaining.

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SEM Morphology The observation of surface morphology by Scanning Electron Morphology (SEM)

showed different surface morphology among the differed lintnerization duration and starch origin, in which for 2 hours resulted in agglomerated structures as shown in Fig 2. While the starch treated longer lintnerization produce a finer structure. The process was done by ethanol precipitation in gelatinized starch the dropping rate of about 3 ml/min. By setting dropping rate, starch hydrogel produced finer consistency. During the process of temperature and precipitation starch experienced strong mechanical treatment that caused decreasing particle size. Klingler et al. (1986) suggested that the temperature and mechanical energy that occurs during the process can break the covalent bonds and intermolecular hydrogen bonding of the starch.

Observations with the SEM image did not reveal nano structure, but after analyzed by Transmission Eelectron Mycroscopy (TEM) showed that starch treated for 24 hours lintnerization resulted in a much different structure and nano in sized namely below 100 nm while the shorter duration of acid hydrolysis treatment (4 hours) did not produce nano size particles (Figure 3).

Size Distribution Result of particle size distribution showed that lintnerization for 24 hours revealed

much smaller particles than that of shorter lintnerization duration. However, the polidispersity index still quite large (0.88) showing that the particles were very disperse, not homogenous. For ethanol precipitation prepared by lintnerization of arrowroot starch for 4 and 6 hours have not produced nanosize particles yet (Table 3) with the particle size ore than 1m. Result from Ma et al (2008) for corn starch prepared with ethanol precipitation produced nanosized particles about 100 – 300 nm. Treatment of butanol precipitation resulted smaller and more homogenous particle size as shown at Table 4.

XRD Starch treated with ethanol precipitation did not produce a crystal structure as shown

in the figure 4 which showed that ethanol precipitation changed into amorphous semi-crystalline structure. Changes in the pattern of crystallinity in the precipitated arrowroot starch occured due to the opening of the double helix structure of the crystalline regions. In addition, the process also occured from changing double helix crystalline forms into the short amylose chains that decline the degree of crystallinity. When ethanol was added dropwise to a solution of starch paste which has tergelatinisasi, nano precipitated starch. Gelatinizations change the crystal structure of type A arrowroot starch into the amorphous starch crystallinity loss. This result is slightly different from the study Ma et al. (2008) in which starch ethanol precipitation results nanosize particles having V type crystallinity. Butanol complex precipitation produced different pattern of crystallinity, compared to ethanol treatment, in which buthanol precipitation shift the crystalline pattern from A-type to V-type.

CONCLUSION Preparation of precipitated starch by ethanol and butanol precipitation from different

lintnerization duration from tapioca and arrowroot starch could produce modified starch nanoparticles. The morphology of starch nanoparticles still agglomerated and produced nanosized particles for 24 h lintnerization pretreatment. The absorption capacity reaches the highest level at lintnerization duration for 4 h at arrowroot starch and 6 h at cassava starch.

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From the crystalline pattern (XRD) showed that butanol precipitation produced different pattern of crystallinity compared to ethanol precipitation.

ACKNOWLEGEMENT This work was financially supported by the Indonesian Agency for Agricultural

Research and Development, Ministry of Agriculture, through KKP3T project fiscal year 2012.

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Tables

Table 1. Enzymatic digestibility, WAC and OAC of ethanol precipitated starch at different lintnerization duration from arrowroot and tapioca starch.

Sample Enzymatic Digestibility (%) WAC (%) OAC

Arrowroot Starch (native)

H2E H4E H6E H24E

74.11±0.13b 69.29±0.36b 61.97±0.53a 69.91±0.33b 71.30±0.25b

230.30± 13.37a 589.33±15.18b

687.40±106.00b 574.89±103.21b 355.56±44.53a

264.91±37.13a 528.68±4.63c 512.32±14.28c 522.55±20.09c 386.58±90.06b

Tapioca Native

H2E H4E H6E H24E

72.17±3.16ab 72.90±2.17b 71.30±1.22ab 67.67±3.00a

478.50±42.57a 620.00±42.00b 797.67±67.56c 448.33±20.51a

130.67±16.07ab 146.83±28.92ab 81.67±22.72a

202.17±88.79b

Table 2. Enzymatic digestibility, WAC and OAC of butanol precipitated starch at different lintnerization duration from arrowroot and tapioca starch.

Samples Enzymatic digestibility (%) WAC (%) OAC

Native H2B5 H2B10 H24B5 H24B10

74.12±0.11b 64.63±0.12ab 57.63±0.13a 59.08±0.16a 53.68±0.15a

230.30± 13.37 432.45±13.22 402.05±58.47 421.05±67.38 408.65±37.47

264.91±37.13 365.65±27.65 388.00±17.11 363.60±25.31 389.10±10.32

Table 3. Size distribution and polydispersity index (PDI) of arrowroot starch nanoparticles by ethanol precipitation

Sample Average size (nm) Size range (nm) PDI

H4E H6E H24E

1806.4±42.9 1457.5±153.6 181.6±27.9

1255,7 - 2526 1333.6 - 2727. 61.68 - 137.63

0.71 0.44 0.88

Table 4. Size distribution and polydispersity index of arrowroot starch nanoparticles by butanol precipitation

Sample Particle size (nm) Size range (nm) PDI

H2B5 538.7 127.6 424.0 – 739.6 0.50 H2B10 316.2 28.0 94.2 – 418.6 0.63 H24 B5 324.9 6.3 120.5 – 580.7 0.45 H24 B10 152.9 39.0 119.3 – 187.4 0.51

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Figure 2. Surface morphology of starch resulted from hydrolyzed and ethanol precipitated

arrowroot treatment H6E(a) and H24E (b) and tapioca starch (c and d); and butanol precipitated tapioca (e) arrowroot starch (f).

a b

c d

e f

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Figure 3. Transmission morphology (TEM) of arrowroot starch resulted from treatment

H4E (a) and H24E (b) followed by ethanol precipitation and butanol precipitation for H2B (c) and H24B (d) with magnificant 8000x.

Figure 4. Crystalline pattern of precipitated arrowroot starch resulted from lintnerization

for 4 h (a), 6 h (b) dan 24h (c).

Inte

nsita

s

a

b

c

a b

c d

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Figure.5 Crystalline pattern of precipitated tapioca starch resulted from lintnerization for

4 h (a), 6 h (b) dan 24h (c)