ACETYLATION OPTIMIZATION OF SAGO (METROXYLON SAGU ROTT) STARCH FOR EDIBLE FILM PRODUCTION.

Rosniyati Suwarda 1,3 , Tun Tedja Irawadi 2 , Prayoga Suryadarma 3 And Indah Yuliasih 3 . 1. Maluku Assessment Institute for Agricultural Technology (Maluku AIAT), Indonesia. 2. Departement of Chemistry, Bogor Agricultural University, Dramaga, Bogor-West Java, Indonesia. 3. Departement of Agroindustrial Technology, Bogor Agricultural University, Dramaga, Bogor-West Java, Indonesia. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 24 March 2019 Final Accepted: 26 April 2019 Published: May 2019


ISSN: 2320-5407
Int. J. Adv. Res. 7 (5), 1207-1217 1208 more resistant to water vapor transport than other lipid or nonlipid coatings. However, lipid-based coatings produce a more oily surface (Guilbert, 1986), low permeability of O2, CO2, and ethylene (Baldwin, 1994). Also, wax coatings are difficult to obtain because it is expensive and petroleum-based waxes are non-edible and toxic. This indicates that starch is a potential alternative material in the development of edible films and coatings.
The requirement of starch as edible film, it should have high amylose content (30%) (Bae et al. 2008). Amylose composition of several types of sago starch from Maluku are quite high compared to sago starch from other regions including sago tuni (Metroxylon rumphii Mart.), Ihur (M. sylvestre Mart.), and molat (M. sagu Rott.) (Polnaya et al 2008). The high amylose composition in the three types of sago starch has great potential to be explored in the development of edible films and coatings. Starch-based films such as sago starch have low barrier properties to moisture due to their hydrophilic properties (Mali et al. 2005). To increase the strength of the film, the desired film should have low water absorption.
The low hydrophobicity can cause edible films and coatings to easily absorb water vapor and cause the growth of microorganisms, such as fungal growth on the surface of the film, and reduce the stability of the film (Garcia et al. 2011). This can shorten the shelf life of the coated product. The hydrophobicity of edible film and sago starch-based coatings can be improved by increasing starch substitution (DS) by acetylation. However, the increase in DS values can reduce the tensile strength of edible films. The reactant concentration, temperature, reaction time, particle size and characteristics of native granule structure will affect the amount of DS starch produced (Sun and Sun, 2002;Huang et al. 2007). Also, catalyst concentrations also contribute to the value of DS produced (Tupa et al. 2015). DS values for edible films ranged from 0.01 -0.2 (Matti et al. 2004). Methylated starch has superior physical-chemical properties such as gelatinization temperature, swelling power, solubility, and high clarity of pasta and has better storage and cooking stability than native starch (Sodhi and Singh, 2005). Therefore, the purpose of this study was to investigate the characteristics of sago starch as an edible film and to obtain the optimum condition of the acetylation process in improving its hydrophobicity.
Characterisation of sago starch : Sago starch characterization data includes physicochemical analysis of moisture, ash, protein, fat, and amylose content (AOAC, 1995), granule size determined by SEM, functional analysis including solubility of swelling power, clarity of paste, freeze-thaw stability (Perez et al. 1999) and amylographic properties.

Starch Acetylation.
The acetylation procedure was performed according to the Wurzburg method (1964). The desired DS value is 0.01 -0.20, to obtain the degree of substitution (DS) value, the treatment of reactant concentration (44-88%), reaction time (30-60 minutes) and pH (7.5 -8.5) was investigated. The parameters observed were percent acetyl content and degree of substitution (DS) determined according to Whistler and Daniel (1995).

Preparation of edible film.
The process of edible film production uses a modified method by Lopez et al. (2008) and Parra et al. (2004). A sample sago starch acetate of 5 g was dispersed in 80 mL distilled water, the solution was stirred for 15 minutes. Dispersed starch was heated at 80 -85 o C for 15 minutes while stirring with a stirrer. Glycerol (1%, w/w) and 20 mL of aquadest are added as plasticizers, then the suspension is reheated at 80 -85 o C for 15 minutes. The coating solution (18 g) is casting on acrylic plates (Ø 8.5 cm) and dried at 40 o C for 24 hours. The parameters observed were tensile strength (TS) according to ASTM D 638 (2005) and contact angle (CA) (ASTM D5946-04, 2005). Before being tested, the sample is conditioned at 27 °C, 65% RH for 24 hours.

Statistical Analysis.
Data from the research on the optimization of the acetylation factor for sago starch were analyzed using the response surface method (RSM). First-order experiment used a full factorial 23 with three replications at center point, namely the concentration of acetic acid anhydride/reactant (X 1 = A), reaction time (X 2 = B) and pH (X 3 = C) ( Table 1) to determine effect of acetylation factors on percent acetyl, DS, TS, and CA. The second-order experiment used a 1209 central composite design (CCD) to determine the optimum conditions for two parameters of response to tensile strength and contact angle. Data processing was carried out with the help of Design-Expert software 7. The stages of this research can be found in Figure 1.   The quality of native sago starch used in this study was in accordance with the SNI 01-3729-1995 and SIRIM (1972) quality standards as food ingredients except for color and crude fiber quality. The results of the quality analysis of sago starch presented in Table 2. The crude fiber content of sago starch not in accordance with quality standards as 1210 food ingredients but still in accordance with SIRIM-468 (1976) as an industrial ingredient which is a maximum of 1%.

Physicochemical Properties of Sago Starch (Metroxylon sagu Rott.)
The physicochemical and functional properties of native sago starch compared to some types of native starch from other sources presented in Table 3, while the granular shape can be found in Figure 2. Granule size and amylose content of sago starch were greater than rice, corn, and potato starch. The swelling power of sago starch and potato starch was greater than that of low amylose starch such as rice and corn. This is related to the larger size of the sago starch and potato granules.  Fat and protein content of sago starch was greater than potato starch but smaller than rice and corn starch. Fat and protein content are minor components of starch, but these minor components greatly determine the functional properties of starch. Both of these minor compositions will form a hydrophobic layer on the surface of starch granules causing inhibition of starch granule swelling so that the value of solubility and swelling power of rice and 1211 corn starch is low. This makes the viscosity of paste higher and has an impact on the formation of the film (filmforming) when casting because it will form a thick film, rough surface and not transparent (opaque).   Table 4. Based on observations of the sago starch gelatinization temperature at 74.1 o C (Table 4) as indicated by pasting temperature parameters, the gelatinization temperature of sago starch was relatively the same as the results of Polnaya's research (2008). When sago starch gelatinized, it will begin to increase the viscosity of the sago starch granules which are irreversible. As a result, when sago starch gelatinized, maltose cross (birefiregrance) from the sago starch granules will disappear (Kusnandar 2010). At the time of gelatinization, the amylose starch structure will begin to diffuse out of the granule due to the breaking of the hydrogen bond between amylose and amylopectin. This will be followed by an increase in viscosity to its peak so that the structure of the sago starch granules will break, forming a paste.  Sandhu et al. (2015) Sago starch has higher final viscosity (3502 cP, Table 4) than rice and corn starch and lower than potatoes. Starch which is high in amylose content generally has a higher final viscosity (viscosity at 50 C) and can be used as raw material for gelling and film to be used as raw material for making vermicelli and noodles (Winger et al. 2014), while starch the high amylopectin content will have a lower viscosity (viscosity at 50 C) making it suitable for thickening agents (Trappey et al. 2015).

Effect of Acetylation Factors on Tensile Strength of Edible Film
Edible films produced from the acetylation of sago starch produce tensile strength (TS) values ranging from 1.23 -4.08 MPa. This TS of the edible film was influenced by the concentration of reactants and pH. The ANOVA of a full factorial design results for TS edible film sago starch acetate presented in Table 6. Based on the results of the regression analysis the determination coefficient (R 2 ) TS was 96.79%, this shows that the equation for TS response (Equation 3) has a degree of conformity high with experimental data. From the ANOVA test, we obtained the regression equation model: TS = 2.041 -0.65A -0.04B -0.29C As shown in Table 6, TS response decreased along with the increased in reactant concentration indicated by a negative coefficient of -0.65 with a significance of 97.88% (p <0.05) and a reaction pH of -0.29 with a significance of 90.71% (p <0.1). The higher the concentration of reactants and reaction pH will produce a low TS value, this is related to percent acetyl and DS produced from the acetylation process. Percent acetyl and DS of sago starch acetate were strongly influenced by the concentration of reactants ( Table 6). The higher the concentration of reactants the greater the two values. Percent acetyl and DS value ranged from 0.07 to 0.27, the value of which increased from both parameters will reduce the TS of edible film properties because there is an acetyl group in acetate sago starch. The acetyl group will prevent bonding between molecules and OH groups. The results of the same study were reported by Colussi et al. (2017) that the acetylation of starch with acetic acid anhydride increases the value of DS which has an impact on low tensile strength (TS) of edible films. The acetylation process of sago starch decreases the elasticity of the edible film. Edible films of methylated sago starch become less flexible or easier to break. The lower the TS value, the more the material is brittle. The process of substitution of the hydroxyl (OH) group to the acetyl group has reduced the elasticity of the material. This decrease in TS value is due to the acetyl group has greater molecular weight and more molecular shape compared to OH. Acetyl which enters sago starch makes sago starch denser and harder which results in a decrease in elasticity.    Table 7, the concentration of reactants has a very positive effect on CA values of edible film with a oefficient value of 10.46 and a significance of 99.4% (p <0.05), as well as pH, has a positive effect on CA values of edible film with a coefficient of 1.71 and significance 82.1% (p <0.1). The higher the concentration of reactants and pH the greater the CA value of the edible film. The CA value of the edible film was influenced by the magnitude of the DS value, the DS value also affects the tensile strength of edible film `previously described. Acetyl percent and DS of acetate starch were strongly influenced by the concentration of reactants (Table 5) Figure  3). The entry of the acetyl group on starch converts the starch's polarity more towards hydrophobicity. The acetyl group (CH 3 COO) is less polar when compared to the OH group that has been replaced. The more OH groups substituted, the more starch the hydrophobic properties will be. The difference in polarity will make a greater interaction between starch and measuring surface resulting in a greater contact angle.

Optimization of Acetylation on Tensile Strength and the Contact Angle Properties Responses of Edible film:-
Results of analysis of variance (ANOVA) in Table 5 and Table 7 showed that the reaction time had no effect on TS and CA of edible film properties with significance of 30.80% (p> 0.05 and 82.10% (p> 0.05), then the reaction time factor was eliminated for second-order RSM analysis with CCD (Central Composite Design) ( Table 8). Contour plots of TS and CA of edible film test results with response surface methodology can be seen in Figure 4. Contour plot TS (Figure 4a) of the edible film showed that the greater concentration of reactants and pH, the lower TS value, but increases CA value (Figure 4b)   The determination of the optimum formula was determined based on superimposed contour plots from the parameters of tensile strength and contact angle of edible film using Design-Expert software. The optimization value was done using the maximum value criteria for TS and CA of edible film responses. The optimum formula area obtained can be seen from the superimposed results of combining contour plots of each parameter used for optimization ( Figure 5). Superimposed graphs obtained show areas that provide optimum response according to the desired goal.   1216 response values of edible film meet the 95% confidence interval that the program presents. The results of predictions and validation values of responses at optimum conditions of the acetylation process can be seen in Table 9.