Thank you for submitting your article to IJCRBP.

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9/7/2021 Yahoo Mail - Thankyou for Submiting your Article to IJCRBP

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Thankyou for Submiting your Article to IJCRBP

From: IJCRBP (submit@ijcrbp.com) To: a_pahira@yahoo.com

Date: Friday, August 14, 2015, 12:31 AM PDT

Thank you for submitting your article to IJCRBP.

Dear Abdul Rahim,

We are in receipt of your article and reply you in this regard soon.Â We will assign Manuscript number within 24 hours for your manuscript. If not received manuscript number within 24 hours, please contact us with immediate effect and resubmit your manuscript to this email submit@ijcrbp.com.

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International Journal of Current Research in Biosciences and Plant Biology.

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1

CHEMICAL AND FUNCTIONAL PROPERTIES OF ACETYLATED ARENGA

STARCHES PREPARED AT DIFFERENT REACTION TIME RAHIM, A

^1*)

, SYAHRAENI, K

¹⁾

AND JUSMAN

²⁾

1: Faculty of Agricultural, Tadulako University,

Jl. Soekarno Hatta Km 8 No.32 Palu, Central Sulawesi, 94118 Indonesia 2: Faculty of Mathematics and Natural science, Tadulako University, Jl. Soekarno Hatta Km 9 No.32 Palu, Central Sulawesi, 94118 Indonesia

*Corresponding author: Email: a_pahira@yahoo.com; Mobile number: +6281524509635 Ph.: +62451 429738; Fax: +62451 429738

ABSTRACT

Arenga starch obtained from the pith of palm Arenga (Arenga pinnata) and subjected to chemical modification by acetylation at different reaction time. In this study, acetylation of arenga starch was carried out. Arenga starch was reacted with acetic anhydride at 5% (starch basis, sb) in aqueous slurry at pH 8.0 ± 8.2 for 15, 30, 45, 60, 75 and 90 min. The native and modified starches were characterized for acetyl percentage (Ac%), degree of substitution (DS), fourier transform infrared spectroscopy (FT-IR) spectra, crystallinity, water and oil holding capacity (WHC and OHC), swelling power and solubility. The results indicated that Ac% and DS of acetylated arenga starches prepared with 60 min was the highest 2,715% and 0.105 respectively. The modification resulted in the acetyl group incorporation with the starch molecule as shown by absorption of the ester carbonyl group at band 1720 cm

^-1

of the fourier transform infrared spectra. X-ray diffraction revealed that increasing reaction time was related to decreasing crystallinity. The WHC and OHC of acetylated arenga starches increased until 60 min, indicating that acetylation increased both hydrophilicity and hydrophobicity of the modified starches. Swelling power tended to increase and solubility to decrease along with the increase reaction time until 60 min of acetylated arenga starches.

Keywords : Acetylated arenga starches, Reaction time, Chemical properties, Functional properties.

INTRODUCTION

Chemical modifications of starch including esterification are efficacious methods to

improve the properties of starch. The chemical and functional properties achieved, depending on

by chemical substitution, on starch source, reaction conditions, type of substituent, extent of

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degree of substitution (DS), and the distribution of the substituent in the starch molecule [1].

Acetylated starch exhibits increased stability in food applications compared to its native counterpart; therefore, it has been used to increase the stability and resistance of food products to retrogradation [2]. Chemical modification of native starches is often required to improve their properties, as well as to overcome the undesirable changes in product texture and appearance caused by retrogradation or breakdown of starch during storage and processing [3, 4]. Starch acetates are prepared commercially with a low (< 0.1) degree of substitution (DS) through the reaction of an aqueous suspension of starch granules with acetic anhydride [5, 6, 7].

Acetylated starch is a starch ester that has been extensively studied over the last two decades [8, 9,]. In modified starch, part of hydroxyl groups in anhydroglucose units have been converted to acyl groups. Acylated starch with low DS is commonly obtained by esterification of native starch in an aqueous medium in the presence of an alkaline catalyst. A low DS with (0.01- 0.20) acylated starch has been applied in many areas, such as film forming, binding, adhesion, thickening, stabilizing, texturing [10, 11, 2, 12]. In the current study, acetylated starches were prepared by reacting arenga starch with acetic anhydride in aqueous slurry to determine the optimum reaction time and to give the chemical and functional of properties such as acetyl percentage (Ac%), degree of substitution (DS), fourier transform infrared (FT-IR) spectra, cristallinity, water and oil holding capacity (WHC and OHC), swelling power and solubility.

MATERILAS AND METHODS Materials

Arenga starch (Arenga pinnata Merr.) used for this study was obtained from Sigi, Central Sulawesi Provinsi, Indonesia. High-purity acetic anhydride 98% was purchased from Sigma- Aldrich Chemical Co. (St. Louis, MO, USA). The Sodium hydroxide (NaOH) and hydrochloric acid (HCl) were purchased from Merck. The chemicals for analysis used in the study were of analytical grade purchased at local agent.

Acetylation of arenga starch

Acetylated starch was prepared by a modified procedure of [13] with slight modification.

Starch (100 g) was dispersed in 225 ml of distilled water and stirred for 60 min at 25

^o

C. The

acetic anhydride of 5% (starch basis, sb) was added drop-wise to the stirred slurry, while

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3

maintaining the pH 8.0 ± 8.2 using 3.0% NaOH solution. The reaction was allowed to proceed for 15, 30, 45, 60, 75 and 90 min after the completion of acetic anhydride addition. The slurry was then adjusted to pH 4.5 with 0.5 N HCl. After sedimentation, it was washed free of acid, twice with distilled water and once with 95% ethanol, and then oven-dried at 40

^o

C. Determination of acetyl percentage and degree of substitution

The percentage of acetyl groups (Ac%) and degree of substitution (DS) were determined titrimetrically, following the method by [14]. Acetylated starch (1.0 g) and 50 ml of 75%

aqueous ethanol were placed in a 250 ml flask. The loosely stopper flask was agitated, warmed to 50 ◦C for 30 min, cooled and 40 ml of 0.5 M KOH were added. The excess alkali was back- titrated with 0.5 M HCl using phenolphthalein as an indicator. The solution was left to stand for 2 h, and then the alkali leached from the sample was titrated. A blank, using the original unmodified starch, was also used. Initially, the acetyl (%) was calculated as:

Acetyl % = [(Blank – Sample) x Molarity of HCl x 0.043 x 100]

Sample weight

Blank and sample were titration volumes in ml, sample weight was in g. DS is defined as the average number of sites per glucose unit that possess a substituent group.

DS = (162 x Acetyl %) [4300 – (42 x Acetyl %)]

Fourier transform infrared spectroscopy (FT-IR) spectra analysis

Sample preparation and analysis parameters were performed according to [15]. Tablets were prepared from the mixture of the sample with KBr at a ratio of 1:100 (sample: KBr).

Infrared spectra of native and acetylated starches samples were obtained by a Fourier Transform Spectrometer (IR Prestige 21, Shimadzu) in the 5000–400 cm

^-1

region.

XRD analysis

X-ray diffraction of native and acetylated starches was measured using method of [16].

X-ray diffraction analysis was performed with an X’ Pert PRO X-ray powder diffractometer (PANalytical, Almelo, The Netherlands) operating at 40 kV and 30 mA with Cu Kα radiation (λ

= 1.5406 Å). The starch powders were packed tightly in a rectangular glass cell (15 x 10 mm, thickness 0.15 cm) and scanned at a rate of 2

^o

/min from the diffraction angle (2θ) 3

^o

to 80

^o

at room temperature. The crystallinity was calculated according to the equation below:

Xc = Ac

(Aa + A𝑐)

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4

where Xc is the crystallinity, A

c

is the crystalline area and Aa is the amorphous area on the X- ray diffractogram.

Water and oil holding capacity

Water and oil holding capacity (WHO, OHC) of native and acetylated starches was measured using method of [17]. Twenty-five ml of distilled water or commercial olive oil were added to 250 mg of dry sample, stirred and left at room temperature for 1 h. After centrifugation, the residue was weighed WHC and OHC were calculated as g water or oil per g of dry sample, respectively.

Swelling power and solubility

The method of [18] was employed to determine the swelling power and solubility of the starch. Five hundred (500) mg of starch sample was weighed into a centrifuge tube and it was reweighed (W

1

). The starch was then dispersed in 20 ml of water. It was then heated at temperature of 80

^o

C for 30 min in a thermostated water bath. The mixture was cooled to room temperature and centrifuged at 3000g for 15 min. Supernatant was decanted carefully and residue weighed for swelling power determination. The weight of dry centrifuge tube and the residue and the water retained was taken as W

2

.

Swelling power = W

₂

– W

₁

Weight of starch

Aliquots (5 ml) of the supernatant were dried to a constant weight at 110

^o

C. The residue obtained after drying of the supernatant represented the amount of starch solubilized in water.

Solubility was calculated as g per 100 g of starch on dry weight basis.

Statistical analysis

The data reported were the means of triplicate measurements. Statistical analysis were carried out with Duncan’s multiple test (P < 0.05) using SPSS version 18 software (SPSS Institute Inc., Cary, NC).

RESULTS AND DISCUSSION

Acetyl percentage and degree of substitution

Percentage of acetyl groups and degree of substitution of the acetylated arenga starch at

different reaction time (15, 30, 45, 60, 75 and 90 min) are show in Figure 1. The acetylation of

arenga starch promoted the incorporation of acetyl groups in the molecule, resulting in an acetyl

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percentage and degree of substitution between 0.429 to 2.715% (Figure 1a) and DS 0.016 to 0.105 (Figure 1b) respectively, allowing food application. The highest Ac% (2.715%) and DS (0.105) was reached by acetylation at reaction time 60 min. Increasing the reaction time from 15 - 60 min resulted in increase in Ac% and DS, but longer reaction (75 – 90 min) resulted in a decrease in Ac% and DS. An explanation was that, as the reaction progressed, the concentration of anhydride was depleted, due to esterification and hydrolysis reactions similar to that of [19].

The evaluated the effect of at different reaction time in potato and cassava starches.

Acetyl percentage and DS indicated the difference (p < 0.05) in the acetylation reaction time.

The DS for cassava starches was 0.10 and 0.26 at 10 min and 20 min of reaction, respectively; in the case of potato starches DS was 0.10 and 0.18 [20].

FTIR spectra

The FTIR spectra of native and acetylated arenga starches with different reaction time are shown in Figure 2. The peak at 1720 cm

⁻¹

is because of the introduction of ester group which is present in the acetylated arenga straches, which otherwise is absent in native arenga starch.

Similarly, the reduction of the intermolecular hydrogen bonding between 3700-3000 cm

⁻¹

confirms the introduction of the acetyl groups in the arenga starch structure thus replacing the hydroxyl groups. Compared to that of the native starch curve (Figure 2a), new absorption band at 1720 cm

⁻¹

appeared in acetylated arenga starches curves (Figure 2b; 2c; 2d; 2e) is C=O

Acetylated barley starches had strong absorption bands at 1735–1740 cm

^-1

(C=O stretching of acetyl group) with evidence of acetylation. On the other hand, since the intensity of the hydroxyl group peak at 3000–3600 cm

^-1

decreased, it has been suggested that the hydroxyl groups in the starch molecules were converted into acetyl groups [21]. The structures of butyrylated arenga starch at band of 1728 cm

^-1

[22].

Crystallinity

The X-ray diffraction patterns of native and acetylated arenga starch samples are shown

in Figure 3. The peaks observed in present study showed that native and acetylated arenga

starches displayed typical A-type pattern with main peaks at 2θ = 15

^o

, 17

^o

, 18

^o

and 23

^o

. The

described that A-type pattern of starches exhibited sharp peaks at 2θ = 15

^o

, 17

^o

, 18

^o

and 23

^o

[16,

23]. These results suggested that esterification did not change the crystalline pattern of acetylated

arenga starches up to DS 0.105. These were agreed with the findings of [24], who mentioned that

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the esterification occurred primarily in the amorphous regions and did not change the crystalline pattern of starches.

Degree of crystallinity of the acetylated arenga starch granules were lower than that of the native starch. This indicated that the granules of acetylated arenga starches had been damaged to some extent by the modification processes. Intra and intermolecular hydrogen bonds were responsible for the highly ordered crystalline structure. The results were in accordance that report of [21], that showed lower crystallinity when compared to that of native barley starch and by [25], that degree of crystallinity of the acetylated corn starch granules was lower than that of the native starch.

Water and oil holding capacity

WHC and OHC of the native and acetylated arenga starches increased with the increase reaction time until 60 min, then decrease for longer time are show in Figure 4. The results indicate that with increase in time from 15 min to 60 min, the WHC and OHC of acetylated arenga starches was found to increase, however further decrease to 90 min, it did not reflect in increase in WHC and OHC leveling-off in accessibility of arenga starch for the reactants indicating near stagnancy in level of acetylation. These data indicate that either hydrophilicity or hydrophobicity tend to improve after acetylation. Improvement in water and oil absorption was a result of introduction of functional groups to the starch molecules, which facilitated a more enhanced holding capacity [22].

According by [26], that found that WHC of the acetylated sword bean starch at DS 0.14 was higher than that of the native starch, while [27] reported that the OHC of the acetylated banana was higher than that of the raw banana.

Swelling power and solubility

Swelling power and solubility of native and acetylated arenga starch samples are shown

in Figure 5. Swelling power of the acetylated arenga starches increased with the increase

reaction time until 60 min, then decrease for longer time, while solubility of the acetylated

arenga starches to tend constant for longer reaction time. This phenomenon was probably caused

by a weakening of intermolecular association force due to introduction of acetyl groups that

reduced the hydroxyl groups after acetylation. These were similar to the results of [28] that

swelling power of the acetylated oat β-glucans increased with increasing time from 10 min to 20

min.

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Conclusion

The acetylation of arenga starch promoted the incorporation of acetyl groups in the molecule at reaction time from 15 min to 90 min, resulting in a degree of substitution between 0.016 to 0.105, allowing food application. Acetylated of arenga starch with 5% acetic anhydride at reaction time from 15 min to 90 min to improved the chemical and functional properties of the modified starches. This report will be useful to promote the use of the acetylated arenga starch for industrial applications in food production.

Acknowledgement

This research was funded by “Grand Research Program of National strategic” with contract number: 040/SP2H/PPM/DIT.LITABMAS/II/ 2015 from the Ministry of National Education of Republic of Indonesia.

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[2] Singh N, Chawla D and Singh J, Influence of acetic anhidrida on physicochemical, morphological and thermal properties of corn and potato starch, Food Chem., 86, 2004, 601- 608.

[3] Hung PV and Morita N, Effects of granule sizes on physicochemical properties of cross- linked and acetylated wheat starches, Starch/Stärke, 57, 2005, 413–420.

[4] Miladinov VD and Hanna MA, Starch esterification by reactive extrusion, Industr. Crops and Produc.,11, 2000, 51–57.

[5] Biswas A, Shogren R L, Selling G, Salch J, Willett J L and Buchanan M, Rapid and environmentally friendly preparation of starch esters. Carbohydr. Polym.,74, 2008, 137–

141. [6] Shogren RL,Rapid preparation of starch esters by high temperature/pressure reaction, Carbohydr. Polym., 52, 2003, 319–326.

[7] Shogren RL and Biswas A, Preparation of water soluble and water swellable starch acetates

using microwave heating, Carbohydr. Polym., 64, 2006, 16–21.

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[8] Gonzalez Z and Perez E, Effect of acetylation on some properties of rice starch, Starch, 54,

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[9] Singh J, Kaur L and McCarthy OJ, Factors influencing the physico-chemical, morphological, thermal and rheological properties of some chemically modified starches for food applications-a review, Food Hydrocol., 21, 2007, 1–22.

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[11] Matti E, Tomas A, Pasi S, Reino L, Soili P and Sari H, Determination of the degree of substitution of acetylated starch by hydrolysis, 1H NMR and TGA/IR, Carbohydr. Polym., 57, 2004, 261–267.

[12] Sodhi NS and Singh N, Characteristics of acetylated starches prepared using starches separated from different rice cultivars, J. Food Engine.,70, 2005, 117–127.

[13] Phillips DL, Huijum L, Duohai P and Harold C, General application of raman spectroscopy for the determination of level of acetylation in modified starches, Cereal Chem., 76, 1999, 439–443.

[14] Sánchez-Rivera MM, Flores-Ramírez I, Zamudio-Flores PB, González-Soto R, Rodríguez- Ambríz SL and Bello-Pérez LA, Acetylation of banana (Musa paradisiaca L.) and maize (Zea mays L.) starches using a microwave heating procedure and iodine as catalyst: Partial characterization, Starch/Stärke, 62, 2010, 155–164.

[15] Diop C, Li HL, Xie BJ and Shi J, Effects of acetic acid/acetic anhydride ratios on the properties of corn starch acetates. Food Chem., 126, 2011, 1662–1669.

[16] Miao M, Zhang T, Mu W and Jiang Bo, Structural characterizations of waxy maize starch residue following in vitro pancreatin and amyloglucosidase synergistic hydrolysis. Food Hydrocol., 25, 2011, 214–220.

[17] Larrauri JA, Ruperez P, Borroto B and Saura-Calixto S, Mango Peels as a New Tropical Fibre: Preparation and Characterization, Lebensm.Wiss.u.Technol., 29, 1996, 729–733.

[18] Adebowale KO, Henle T, Schwarzenbolz U and Doert T, Modification and properties of African yam bean (Sphenostylis stenocarpa Hochst. Ex A. Rich.) Harms starch I: Heat moisture treatments and annealing, Food Hydroc., 23, 2009, 1947–1957.

[19] Ruan H, Chen QH, Fu ML, Xu Q and He GQ, Preparation and properties of octenyl succinic

anhydride modified potato starch, Food Chem., 114, 2009, 81–86.

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[20] Mbougueng PD, Tenin D, Scher J and Tchiégang C, Influence of acetylation on physicochemical, functional and thermal properties of potato and cassava starches, J. Food Engine.,108, 2012, 320–326.

[21] Halal SMLE, Colussi R, Pinto VZ, Bartz J, Radunz M, Carreño NLV, Dias ARG and Zavareze ER, Structure, morphology and functionality of acetylated and oxidized barley starches. Food Chem., 168, 2015, 247–256.

[22] Rahim A, Haryadi, Cahyanto MN and Pranoto Y, Characteristics of butyrylated arenga starch prepared at different reaction time and butyric anhydride concentration, Int. Food Res. J., 19(4), 2012, 1655-1660.

[23] Chi H, Xu K, Wu X, Chen Q, Xue D, Song C, Zhang W and Wang P, Effect of acetylation on the properties of corn starch, Food Chem., 106,2008, 923–928.

[24] Song XY, He GQ, Ruan H and Chen QH, Preparation and properties of octenyl succinic anhydride modified early indica rice starch, Starch/Stärke, 58, 2006, 109–117.

[25] Lopez OV, Zaritzky NE and Garcia MA, Physicochemical characterization of chemically modified corn starches related to rheological behavior, retrogradation and film forming capacity. J. Food Engine.,100, 2010, 160–168.

[26] Adebowale KO, Afolabi TA and Olu-Owolabi BI, Functional, physicochemical and retrogradation properties of sword bean (Canavalia gladiata) acetylated and oxidized starches, Carbohydr. Polym., 65, 2006, 93–101.

[27] Teli MD and Valia SV, Acetylation of banana fibre to improve oil absorbency, Carbohydr.

Polym., 92, 2013, 328– 333.

[28] Souza NL, Bartz J, Zavareze ER, Oliveira PD, Silva WSV, Alves GH and Dias ARG,

Functional, thermal and rheological properties of oat β-glucan modified by acetylation,

Food Chem.,178, 2015, 243–250.

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Figure 1. Effect of reaction time with 5% acetic anhydride on the acetyl (a) and DS (b).

Numbers in the graph followed by a same letter in common are not significantly different at p < 0.05.

Figure 2. FTIR sfectra of native arenga starch (a) and acetylated arenga starches at different reaction time: 30 min (b), 45 min (c), 60 min (d) and 75 min (e).

0.854a 1.215a 1.283a 2.715b

0.429c 0.643c 0.00

1.00 2.00 3.00 4.00 5.00 6.00

15 30 45 60 75 90

Acetyl (%)

Reaction time (min)

0.033a 0.047a 0.049a 0.105b

0.016a 0.024a 0.00

0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40

15 30 45 60 75 90

DS

(a) (b)

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Figure 3. X-ray diffractograms and cristallinity of native arenga

starch (a) and acetylated arenga starches at different reaction time: 30 min (b), 45 min (c), 60 min (d) and 75 min (e).

Figure 4. Effect of reaction time with 5% acetic anhydride on the WHC and OHC of native and acetylated arenga starches.

Numbers in the graph followed by a same letter in common are not significantly different at p < 0.05.

0.97ab 0.88a

0.99ab 1.04b 1.03b 1.00ab 0.86a

0.89pq 0.96pq

1.02q 1.01q 1.09q

0.86pq 0.77p

0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30

0.00 0.20 0.40 0.60 0.80 1.00 1.20

Native starch

15 30 45 60 75 90

OHC (g/g)

WHC (g/g)

Reaction time (min) WHC OHC

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Figure 5. Effect of reaction time with 5% acetic anhydride on the

swelling power and solubility of native and acetylated arenga starches. Numbers in the graph followed by a same letter in common are not significantly different at p < 0.05.

6.46pq 6.62pq 6.65pq 6.98q 7.12q 6.89q 5.50p 13.92a

12.53a 12.62a 11.92a 11.54a

14.36a 14.78a

0 5 10 15 20

0 2 4 6 8 10 12 14 16

Native strach

15 30 45 60 75 90

Solubility (%)

Swelling power (g/g)

Swelling power Solubility

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Acknowledgement on receipt of your article :: MSS No. assigned :: IJCRBP-2015-09-12:: Reg.

From: Editor-in-Chief IJCRBP (editorinchiefijcrbp@gmail.com) To: a_pahira@yahoo.com

Date: Friday, August 14, 2015, 02:39 AM PDT

Dear Sir/Madam,

We thank you for your interest in our Journal, IJCRBP.

We are hereby acknowledging the receipt of your article entitled "Chemical and functional properties of acetylated arenga starches prepared at different reaction time" submitted to IJCRBP by your good-self.

The paper will be processed under Fast Track reviewing and after obtaining the approval from the *reviewers of the Editorial Board, it will be published in Vol. 2, Issue-09 of International Journal of Current Research in Biosciences and Plant Biology (IJCRBP) to be released in the First Week of September-2015.

Kindly note down Your manuscript no.

*(MSS No: IJCRBP-2015-0 9-12)

for further communication.

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SECTION – I

PART A: Editorial Office Only *Accepted PART B: For Reviewers

Manuscript Number IJCRBP-2015-09-12

Title Chemical and Functional Properties of Acetylated Arenga Starches Prepared at Different Reaction Time

Author(s) Abdul Rahim

^1*

, Syahraeni Kadir

¹

and Jusman

²

International Journal of Current Research in Biosciences and Plant Biology

(IJCRBP)

ISSN: 2349-8072 (Print) ISSN 2349-8080 (Online))

An International, Monthly, Online, Free Access, Peer-Reviewed, Indexed, Fast Track Scientific Research Journal

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IJCRBP-2015-09-12

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Make a tick mark [ ]in the columns as applicable for this research paper.

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Reference(s) cited in the text, but missing in the list of references (to be added to the references list):

---

Uncited References -- references not cited in the text, but listed in References section (to be deleted from the list):

Sánchez-Rivera, M. M., Flores-Ramírez, I., Zamudio-Flores, P. B., González-Soto, R.,

Rodríguez-Ambríz, S. L., and Bello-Pérez, L. A. 2010. Acetylation of banana (Musa

paradisiaca L.) and maize (Zea mays L.) starches using a microwave heating procedure and

iodine as catalyst: Partial characterization. Starch/Stärke 62, 155–164.

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Re: Acceptance Letter :: IJCRBP-2015-09-12 :: PROOF and Documents attached - Reg.

Date: Thursday, August 20, 2015, 08:27 PM PDT

Dear Sir/Madam,

Thank you for sending all the details. We will be contacting you at the earliest possible.

Regards

www.ijcrbp.com

On Fri, Aug 21, 2015 at 8:39 AM, Abdul Rahim <a_pahira@yahoo.com> wrote:

To Editor International Journal of Current Research in Biosciences and Plant Biology (IJCRBP)

Dear Sir/Madam, Dear Sir/Madam,

Herewith I am sending the AUTHOR INFORMATION FORM IJCRBP-2015-09-12, Copyright Form IJCRBP-2015-09-12, Corrections to PROOF IJCRBP-2015-09-12 and Transaction or Payment details. I am looking forward to hearing from you. Thank you very much.

Sincerely yours, Abdul Rahim

Departement of food science at Faculty of Agricultural, Tadulako University Jl. Soekarno Hatta Km 8 No.32 Palu, Central Sulawesi, 94118 Indonesia

From: Editor-in-Chief IJCRBP <editorinchiefijcrbp@gmail.com>

To: a_pahira@yahoo.com Cc: ijcrbpeditor@gmail.com

Sent: Wednesday, August 19, 2015 5:25 PM

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I am pleased to inform you that the below mentioned Manuscript has been

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Manuscript No. Title of the Article

IJCRBP-2015-09-12 Chemical and Functional Properties of Acetylated Arenga Starches Prepared at Different Reaction Time

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TITLE "Chemical and functional properties of acetylated arenga starches prepared at different reaction time"

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Sanchez et al. (2010)

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Cited References -- references cited in the text (line 65 in the PROOF IJCRBP-2015- 09-12.pdf), listed in References section (not deleted from the list):

Sánchez-Rivera, M. M., Flores-Ramírez, I., Zamudio-Flores, P. B., González-Soto, R., Rodríguez-Ambríz, S. L., and Bello-Pérez, L. A. 2010. Acetylation of banana (Musa paradisiaca L.) and maize (Zea mays L.) starches using a microwave heating procedure and iodine as catalyst: Partial characterization. Starch/Stärke 62: 155–164.

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Int. J. Curr. Res. Biosci. Plant Biol. 2015, 2(9): xx-xx

A. Rahim et al. (2015) / Int. J. Curr. Res. Biosci. Plant Biol. 2015, 2(9): xx-xx 1

International Journal of Current Research in Biosciences and Plant Biology

ISSN: 2349-8080 Volume 2 Number 9 (September-2015) pp. xx-xx

www.ijcrbp.com 1

Original Research Article

2

3

Chemical and Functional Properties of Acetylated Arenga Starches Prepared at

4

Different Reaction Time

5

6

Abdul Rahim

^1*

, Syahraeni Kadir

¹

and Jusman

²

7 8

1Faculty of Agricultural, Tadulako University, Jl. Soekarno Hatta Km 8 No.32 Palu, Central Sulawesi, 94118

9

Indonesia

10

2Faculty of Mathematics and Natural science, Tadulako University, Jl. Soekarno Hatta Km 9 No.32 Palu, Central

11

Sulawesi, 94118 Indonesia

12 13

*Corresponding author.

14 15

A b s t r a c t K e y w o r d s

Arenga starch obtained from the pith of palm Arenga (Arenga pinnata) and subjected to chemical modification by acetylation at different reaction time. In this study, acetylation of arenga starch was carried out. Arenga starch was reacted with acetic anhydride at 5% (starch basis, sb) in aqueous slurry at pH 8.0 ± 8.2for 15, 30, 45, 60, 75 and 90 min. The native and modified starches were characterized for acetyl percentage (Ac%), degree of substitution (DS), fourier transform infrared spectroscopy (FT-IR)spectra, crystallinity, water and oil holding capacity (WHC and OHC), swelling power and solubility. The results indicated that Ac% and DS of acetylated arenga starchesprepared with 60 min was the highest 2,715% and 0.105 respectively. The modification resulted in the acetyl group incorporation with the starch molecule as shown by absorption of the ester carbonyl group at band 1720 cm^-1 of the fourier transform infrared spectra. X-ray diffraction revealed that increasing reaction time was related to decreasing crystallinity. The WHC and OHC of acetylated arenga starches increased until 60 min, indicating that acetylation increased both hydrophilicity and hydrophobicity of the modified starches. Swelling power tended to increase and solubility to decrease along with the increase reaction time until 60 min of acetylated arenga starches.

Acetylated arenga starches Chemical properties Functional properties Reaction time

16

Introduction

17 18

Chemical modifications of starch including esterification

19

are efficacious methods to improve the properties of

20

starch. The chemical and functional properties achieved,

21

depending on by chemical substitution, on starch source,

22

reaction conditions, type of substituent, extent of degree

23

of substitution (DS), and the distribution of the

24

substituent in the starch molecule (Hirsch and Kokini,

25

2002). Acetylated starch exhibits increased stability in

26

food applications compared to its native counterpart;

27

therefore, it has been used to increase the stability and

28

resistance of food products to retrogradation (Singh et

29

al., 2004). Chemical modification of native starches is

30

(25)

A. Rahim et al. (2015) / Int. J. Curr. Res. Biosci. Plant Biol. 2015, 2(9): xx-xx 2 often required to improve their properties, as well as to

1

overcome the undesirable changes in product texture and

2

appearance caused by retrogradation or breakdown of

3

starch during storage and processing (Hung and Morita,

4

2005; Miladinov and Hanna, 2000). Starch acetates are

5

prepared commercially with a low (< 0.1) degree of

6

substitution (DS) through the reaction of an aqueous

7

suspension of starch granules with acetic anhydride

8

(Biswas et al., 2008; Shogren, 2003; Shogren and

9

Biswas, 2006).

10 11

Acetylated starch is a starch ester that has been

12

extensively studied over the last two decades (Gonzalez

13

and Perez, 2002; Singh et al., 2007). In modified starch,

14

part of hydroxyl groups in anhydroglucose units have

15

been converted to acyl groups. Acylated starch with low

16

DS is commonly obtained by esterification of native

17

starch in an aqueous medium in the presence of an

18

alkaline catalyst. A low DS with (0.01-0.20) acylated

19

starch has been applied in many areas, such as film

20

forming, binding, adhesion, thickening, stabilizing,

21

texturing (Boutboul et al., 2002; Matti et al., 2004;

22

Singh et al., 2004; Sodhi and Singh, 2005).

23 24

In the current study, acetylated starches were prepared

25

by reacting arenga starch with acetic anhydride in

26

aqueous slurry to determine the optimum reaction time

27

and to give the chemical and functional of properties

28

such as acetyl percentage (Ac%), degree of substitution

29

(DS), fourier transform infrared (FT-IR) spectra,

30

cristallinity, water and oil holding capacity (WHC and

31

OHC), swelling power and solubility.

32 33

Materials and methods

34 35

Arenga starch (Arenga pinnata Merr.) used for this

36

study was obtained from Sigi, Central Sulawesi

37

Provinsi, Indonesia. High-purity acetic anhydride 98%

38

was purchased from Sigma-Aldrich Chemical Co. (St.

39

Louis, MO, USA). The Sodium hydroxide (NaOH) and

40

hydrochloric acid (HCl) were purchased from Merck.

41

The chemicals for analysis used in the study were of

42

analytical grade purchased at local agent.

43 44

Acetylation of arenga starch

45 46

Acetylated starch was prepared by a modified procedure

47

of Phillips et al. (1999) with slight modification. Starch

48

(100 g) was dispersed in 225 ml of distilled water and

49

stirred for 60 min at 25^oC. The acetic anhydride of 5%

50

(starch basis, sb) was added drop-wise to the stirred

51

slurry, while maintaining the pH 8.0 ± 8.2 using 3.0%

52

NaOH solution. The reaction was allowed to proceed for

53

15, 30, 45, 60, 75 and 90 min after the completion of

54

acetic anhydride addition. The slurry was then adjusted

55

to pH 4.5 with 0.5 N HCl. After sedimentation, it was

56

washed free of acid, twice with distilled water and once

57

with 95% ethanol, and then oven-dried at 40^oC.

58 59

Determination of acetyl percentage and degree of

60

substitution

61 62

The percentage of acetyl groups (Ac%) and degree of

63

substitution (DS) were determined titrimetrically,

64

following the method by Sanchez et al. (2010).

65

Acetylated starch (1.0 g) and 50 ml of 75% aqueous

66

ethanol were placed in a 250 ml flask. The loosely

67

stopper flask was agitated, warmed to 50 ◦C for 30 min,

68

cooled and 40 ml of 0.5 M KOH were added. The excess

69

alkali was back-titrated with 0.5 M HCl using

70

phenolphthalein as an indicator. The solution was left to

71

stand for 2 h, and then the alkali leached from the

72

sample was titrated. A blank, using the original

73

unmodified starch, was also used. Initially, the acetyl

74

(%) was calculated as:

75 76

77 78

Blank and sample were titration volumes in ml, sample

79

weight was in g. DS is defined as the average number of

80

sites per glucose unit that possess a substituent group.

81 82

83 84

Fourier Transform Infrared Spectroscopy (FT-IR)

85

spectra analysis

86 87

Sample preparation and analysis parameters were

88

performed according to Diop et al. (2011). Tablets were

89

prepared from the mixture of the sample with KBr at a

90

ratio of 1:100 (sample: KBr). Infrared spectra of native

91

and acetylated starches samples were obtained by a

92

Fourier Transform Spectrometer (IR Prestige 21,

93

Shimadzu) in the 5000–400 cm^-1 region.

94 95

XRD analysis

96 97

X-ray diffraction of native and acetylated starches was

98

measured using method of Miao et al. (2011). X-ray

99

diffraction analysis was performed with an X’ Pert PRO

100

X-ray powder diffractometer (PANalytical, Almelo, The

101

(26)

A. Rahim et al. (2015) / Int. J. Curr. Res. Biosci. Plant Biol. 2015, 2(9): xx-xx 3 Netherlands) operating at 40 kV and 30 mA with Cu Kα

1

radiation (λ = 1.5406 Å). The starch powders were

2

packed tightly in a rectangular glass cell (15 x 10 mm,

3

thickness 0.15 cm) and scanned at a rate of 2^o/min from

4

the diffraction angle (2θ) 3^o to 80^o at room temperature.

5

The crystallinity was calculated according to the

6

equation below:

7

8 9

where Xc is the crystallinity, Ac is the crystalline area

10

and Aa is the amorphous area on the X-ray

11

diffractogram.

12 13

Water and oil holding capacity

14 15

Water and oil holding capacity (WHO, OHC) of native

16

and acetylated starches was measured using method of

17

Larrauri et al. (1996). Twenty-five ml of distilled water

18

or commercial olive oil were added to 250 mg of dry

19

sample, stirred and left at room temperature for 1 h.

20

After centrifugation, the residue was weighed WHC and

21

OHC were calculated as g water or oil per g of dry

22

sample, respectively.

23 24

Swelling power and solubility

25 26

The method of Adebowale et al. (2009) was employed to

27

determine the swelling power and solubility of the

28

starch. Five hundred (500) mg of starch sample was

29

weighed into a centrifuge tube and it was reweighed

30

(W1). The starch was then dispersed in 20 ml of water. It

31

was then heated at temperature of 80^oC for 30 min in a

32

thermostated water bath. The mixture was cooled to

33

room temperature and centrifuged at 3000g for 15 min.

34

Supernatant was decanted carefully and residue weighed

35

for swelling power determination. The weight of dry

36

centrifuge tube and the residue and the water retained

37

was taken as W2.

38

39

Aliquots (5 ml) of the supernatant were dried to a

40

constant weight at 110^oC. The residue obtained after

41

drying of the supernatant represented the amount of

42

starch solubilized in water. Solubility was calculated as

43

g per 100 g of starch on dry weight basis.

44 45

Statistical analysis

46 47

The data reported were the means of triplicate

48

measurements. Statistical analysis was carried out with

49

Duncan’s multiple test (p<0.05) using SPSS version 18

50

software (SPSS Institute Inc., Cary, NC).

51 52

Results and discussion

53 54

Acetyl percentage and degree of substitution

55 56

Percentage of acetyl groups and degree of substitution of

57

the acetylated arenga starch at different reaction time

58

(15, 30, 45, 60, 75 and 90 min) are show in Fig.1. The

59

acetylation of arenga starch promoted the incorporation

60

of acetyl groups in the molecule, resulting in an acetyl

61

percentage and degree of substitution between 0.429 to

62

2.715% (Fig.1a) and DS 0.016 to 0.105 (Fig.1b)

63

respectively, allowing food application. The highest

64

Ac% (2.715%) and DS (0.105) was reached by

65

acetylation at reaction time 60 min. Increasing the

66

reaction time from 15-60 min resulted in increase in Ac%

67

and DS, but longer reaction (75 – 90 min) resulted in a

68

decrease in Ac% and DS. An explanation was that, as the

69

reaction progressed, the concentration of acetic anhydride

70

was depleted, due to esterification and hydrolysis reactions

71

similar to that of (Ruan et al., 2009).

72 73

Fig. 1: Effect of reaction time with 5% acetic anhydride on the acetyl (a) and DS (b). Numbers in the graph followed by a 74

same letter in common are not significantly different at p<0.05.

75

76

(a)

(b)

(27)

A. Rahim et al. (2015) / Int. J. Curr. Res. Biosci. Plant Biol. 2015, 2(9): xx-xx 4 Mbougueng et al. (2012) evaluated the effect of at

1

different reaction time in potato and cassava starches.

2

Acetyl percentage and DS indicated the difference (p <

3

0.05) in the acetylation reaction time. The DS for

4

cassava starches was 0.10 and 0.26 at 10 min and 20 min

5

of reaction, respectively; in the case of potato starches

6

DS was 0.10 and 0.18.

7 8

FTIR spectra

9 10

The FTIR spectra of native and acetylated arenga

11

starches with different reaction time are shown in Fig.2.

12

The peak at 1720 cm⁻¹ is because of the introduction of

13

ester group which is present in the acetylated arenga

14

straches, which otherwise is absent in native arenga

15

starch. Similarly, the reduction of the intermolecular

16

hydrogen bonding between 3700-3000 cm⁻¹ confirms

17

the introduction of the acetyl groups in the arenga starch

18

structure thus replacing the hydroxyl groups. Compared

19

to that of the native starch curve (Fig.2a), new

20

absorption band at 1720 cm⁻¹ appeared in acetylated

21

arenga starches curves (Fig.2b; 2c; 2d; 2e) is C=O

22

stretching vibration of an acetyl group.

23 24

Fig. 2: FTIR sfectraof native arenga starch (a) and 25

acetylated arenga starches at different reaction time: 30 26

min (b), 45 min (c), 60 min (d) and 75 min (e).

27

28 29

Acetylated barley starches had strong absorption bands

30

at 1735–1740 cm^-1 (C=O stretching of acetyl group)

31

with evidence of acetylation. On the other hand, since

32

the intensity of the hydroxyl group peak at 3000–3600

33

cm^-1 decreased, it has been suggested that the hydroxyl

34

groups in the starch molecules were converted into

35

acetyl groups (Halal et al., 2015). The structures of

36

butyrylated arenga starch at band of 1728 cm^-1 (Rahim et

37

al., 2012).

38

Crystallinity

39 40

The X-ray diffraction patterns of native and acetylated

41

arenga starch samples are shown in Fig.3. The peaks

42

observed in present study showed that native and

43

acetylated arenga starches displayed typical A-type

44

pattern with main peaks at 2θ = 15ô, 17ô, 18ô and 23ô.

45

Chi et al. (2008) and Miao et al. (2011) described that

46

A-type pattern of starches exhibited sharp peaks at 2θ =

47

15ô, 17ô, 18ô and 23ô. These results suggested that

48

esterification did not change the crystalline pattern of

49

acetylated arenga starches up to DS 0.105. These were

50

agreed with the findings of Song et al. (2006), who

51

mentioned that the esterification occurred primarily in

52

the amorphous regions and did not change the crystalline

53

pattern of starches.

54 55

Degree of crystallinity of the acetylated arenga starch

56

granules was lower than that of the native starch. This

57

indicated that the granules of acetylated arenga starches

58

had been damaged to some extent by the modification

59

processes. Intra and intermolecular hydrogen bonds

60

were responsible for the highly ordered crystalline

61

structure. The results were in accordance that report of

62

Halal et al. (2015), that showed lower crystallinity when

63

compared to that of native barley starch and by Lopez et

64

al. (2010), that degree of crystallinity of the acetylated

65

corn starch granules was lower than that of the native

66

starch.

67 68

Fig. 3: X-ray diffractograms and cristallinity of native 69

arenga starch (a) and acetylated arenga starches at 70

different reaction time: 30 min (b), 45 min (c), 60 min (d) 71

and 75 min (e).

72

73 74

Water and oil holding capacity

75 76

WHC and OHC of the native and acetylated arenga

77

starches increased with the increase reaction time until

78