Modification of Porang ( Amorphophallus oncophyllus ) Flour by Acid and Thermal Process using Conventional Heating in Waterbath and Microwave Irradiation

Modified porang flour by lactic acid and conventional heating using waterbath (PAW) and microwave radiation (PAM) were determined its functional group using FTIR, the chemical structure were studied using HNMR, molecular weight were measured using HPLC-SEC and its microstructure were studied using SEM, particles size were measured using PSA and rheology characteristics were determined using Rheometer. The results showed that modified porang either PAW and PAM showed changes on functional group peaks compared to one of native porang flour included OH, CH3, C-C, C = O, C-O-C, mannose and glucose groups. HNMR spectra of modified porang flour (PAW and PAM) were similar mainly in proton of CH3 and CH2, molecular weight were higher at PAW 2.0×10 and PAM 3.0×10, while particles size of PAW and PAM were smaller. The microstructure of PAW and PAM were porous and spongy indicated that PAW and PAM had higher moisture accessability on the amorph site. Storage modulus (G’) dan loss modulus (G”) PAW and PAM decreasing with the increased temperature. Lower G compared to G and modulus (G’ and G”) did not showed crossing indicated that PAW and PAM have a stable visco-elasticity. It can be concluded that functional group peaks of modified porang flour (PAW and PAM) changed differently compared to native porang flour, H-NMR spectra of modified porang flour were similar, with bigger moleculer weight of PAM than PAW, the microstructure were porous and spongy and rheology characteristics indicated that an unchanged visco-elastic system by thermal treatments.


INTRODUCTION
Porang (Amorphophallus oncophyllus) is included in the tuber family of Araceae, one genus with konjac (Amorphophallus konjac) with major component of both plant are mannan and have composition variation between 8-10%.The other components also found were starch, protein, lipid and mineral.The mannan particles are distributed in whole tuber and encapsulated by protoplasma like a thin film and the space between mannan cell is parenchym.This parenchym tissue consist of thin wall cells containing starch granules.The konjac mannan cell filled with glucomannan polysaccharide covered by cell wall with size variations from 160 to 650 µm (Takigami et al., 1997).
Konjac flour contain about 50-70% glucomannan, while konjac glucomannan extract could contain more than 90% as reported by Takigami (2000).According to Arifin (2001) porang flour contain glucomannan up to 64.98% in the form of polymer of D-mannose and Dglucose.Konjac flour also contain some impurities which most are insoluble starch and cellulosa included protein and lipid and these impurities originated from "sack" that encapsulated konjac flour in the tuber (Tatirat et al., 2013).
Konjac glucomannan were found in copolymer form of β-(1-4) D-glucose and D-mannose binding with molar ratio 1: 1.6 and low rate acetyl group (approximately 1 acetyl group in each 17 residue) at C-6 position (Maeda et al., 1980;Katsuraya et al., 2003).Gao andZhang (2001a, 2001b) reported that as some hydroxyl group (-CH 2 OH) at C-6 position each unit was constitutionally very active, hence some chemical reactions could occured at this position such as esterification, etherification and nitrition and according to Chen et al. (2005) graft Polymeration could also takes place in this site.The problem related to the thermal process of polysaccharide was a strong interchain interaction could occured related to the extensive hydrogen bonding.This interaction produced polymer system specifically could go to thermal decomposition before reaching the melting point (Xiao et al., 2001).
The drawback of porang (Amorphophallus oncophyllus) flour was low solubility in water (Irawan and Widjanarko, 2013), while the lack of konjac glucomannan included low solubility, low sol stability and low solution mobility (Pan et al., 2013), beside porang flour could not formed colloid system with milk.As increasing a new source energy requirements, therefore the improvement of porang flour characteristics are very crucial.Pan et al. (2013) noted that physical, chemical and biological modification of konjac glucomannan had improved its functional properties.
One of the methods to disperse supramolecules physically had been developed i.e., by increasing the energy in polymer chain which was successfully to make the polysaccharide solution homogen in water as reported by Tao and Xu (2008).This method was using sonification, γ -irradiation, autoclave, pressure/heat and microwave.
The advantage of heating technique using microwave oven is time and energy efficiency and easy in application.Sample heating in microwave oven are due to the interaction of electromagnetic environment with chemical components insolution.This interaction could rise a fast heat because of molecular friction and exitation.The utilization of microwave irradiation fasten the organic reaction to produce an efficient internal heating or volumetric heating in nucleus and directly combined with microwave energy and available cell i.e., solute, reagent or catalyst in reaction mixture (Zhang and Zhao, 2009).
Heat and radiation treatments could be combined with lactic acid to modify the physicochemical of a polysaccharide.Gong et al. (2006) had been successfully modified the physicochemical of maize flour using thermal treatment combined with lactic acid through lactic acid copolymerization graft reaction to starch.Therefore, the objective of this study was to investigate the modification of porang flour by conventional heating using waterbath and microwave radiation combined with lactic acid to obtain a better physicochemical quality modified porang flour.

MATERIALS AND METHODS
Sample preparation: Porang flour were obtained from P.T. Perhutani Pare and 3 g of porang flour were added with 100 mL lactic acid solution in 250 mL Erlenmeyer flask and stirred thoroughly for 15 min at room temperature, then the mixture were heated in water bath at 90°C for 30 min (PAW) and radiated in microwave (Sharp R200J) for 10 min PAM) respectively.Furthermore the porang flour solution were cooled down to room temperature before centrifuged for 10 min to separate undiluted components (Gong et al., 2006;Xu et al., 2009;Tatirat and Charoenrein, 2011;Yang et al., 2013).
FTIR analysis: 0.01 g sample was homogenised with 0.01 g anhydrous KBr using a mortar and this mixture was vacuum hydraulic pressed at 1.2 psi before scanned in the absorption area at 600 up to 4000 cm -1 (Widjanarko et al., 2011).
Particle size measurement: Particle Size Instrument (Malvern ZEN3690, Malvern), equipped with 10 mW HeeNe laser at 633 nm was used for measuring the particle size and the measurements were carried out at 25°C and 90° scattering angle which will give the particle diameter distribution estimation (Riou et al., 2002;Wang et al., 2005).

Morphology structure determination by Scanning
Electron Microscope (SEM): Morphology structure of porang flour surface were determined using Scanning Electron Microscope (SEM).The samples were coated with gold in vacuum condition and the determination were carried out at magnification of 2000-8000 times (Wang et al., 2011).

Rheology test:
The AR-G2 Rotary Rheometer equipped with cone plate geometry diameter 40 mm (cone angle 4°, gap 108 micro m) was used for rheology test at varied temperature in linear viscoelastic regime.The samples surface were covered with silicon oil to prevent from drying.Temperature sweep test was done by gel heating from 75±1°C with 1.0°C/min increment using 1% strain.Data G' and G" were recorded (He et al., 2012).

Type of vibration
Broad spectrum (cm -1 ) Group

Type of vibration
Broad spectrum (cm -1 ) Group Fig. 1: FTIR spectra of porang flour and modified porang flour porang flour (P), lactic acid-waterbath modified porang flour (PAW) and lactic acid-microwave radiation modified porang flour (PAM) as broad bands were in the range of 4000-500 cm -1 .The band peak of porang flour (P) was 3390.63 cm -1 which indicated the presence of hydrogen bond i.e., O-H group from glucomannan of porang flour, while band of 2927.74 cm -1 indicated C-H bond from methyl group and asymetric streching vibration was observed at 2156.74 cm -1 from cumulative double bond.C = O group from asetyl was observed at 1724.24 cm -1 and 1639.38 was retention of C-O which related to hydroxyl.The infra spectra bended of CH3, CH, C-O-C, C-C were at 1448.44 cm -1 , 1379.01 cm -1 , 1249.79 cm -1 and 1149.50 cm -1 , 1120.56 cm -1 respectively and stretching broad spectra at 1027,99 cm -1 for C-O-C, 896,84 cm -1 and 806,19 cm -1 for mannose.
The FTIR spectra of porang flour (P) in this study were similar with glucomannan konjac as reported by Huang et al. (2010) and Xia et al. (2010) in the range of 4000-500 cm -1 .While Xia et al. (2010), Meng et al. (2014) and Jin et al. (2014) observed that strectching vibration of O-H group of carboxylate were in the range of 3600-3300 cm -1 .According to Huang et al. (2010), Ha et al. (2011), Meng et al. (2014) and Jin et al. (2014) the stretching broad of C-H group at about 2900 was methyl group of glucomannan.Pan et al. (2013) noted that asymetric stretching broad vibration of cumulative double bond indicated by peak at 2150 cm -1 .Xu et al. (2008) and Liu et al. (2009) also reported that stretching broad vibration of C-O of acetyl group was at 1736 cm -1 and Jin et al. (2014) observed that stretching vibration of C-O related to hydroxyl group was at 1638 cm -1 , while CH 3 bending was observed at 1447 cm -1 (Pivsa- Art et al., 2014).
Furthermore, Chua et al. (2012) noted broad spectrum at 1370 cm -1 was for CH bending and Xu et al. (2009) found the stretching broad of C-O bending was at 1250 cm -1 .Broad spectrum of 1150 cm -1 was for C-O-C as reported by Chua et al. (2012) and Yu et al. (2007) observed the stretching broad spectrum of C-O-C was at 1150 cm -1 while stretching broad spectrum of 1027 cm -1 was for C-O-C as noted by Widjanarko et al. (2011).According to Huang et al. (2010) broad spectra of mannose were at 876 cm -1 and 808 cm -1 , while Meng et al. (2014) observed broad spectra of mannose at 871 cm -1 and 804 cm -1 .
The modification treatments of PAW and PAM porang flour resulted a OH group stronger spectra indicated by sharper tip of peak and according to Huang et al. (2010) this condition showed an hydrogen intermoleculr bond of native porang flour which have a partial breakage.The increased of hydroxyl group in glucomannan konjac indicated bigger opportunity to form hydrogen intermolecular bond (Pan et al., 2011).
Methyl group of porang flour (P) which found at spectra band of 2927.74 cm -1 , shifted into two peaks for PAW and PAM namely 2638.44 cm -1 and 2557.43 cm -1 also at 2635 cm -1 and 2553.58cm -1 each in its sequence.It is possible due to methyl group could react in specific way with lactic acid.
The stronger C-C bonding at PAW (1130.21cm -1 ) and PAM (1130.21cm -1 ), according to Xu et al. (2009) are possibly due to specific interaction between lactic acid and glucomannan of porang flour.Peak intensity of C-O increased from 1724.24 cm -1 (P) to 1725.17 cm -1 (PAW) and 1726.17cm -1 (PAM) and this condition according to Gårdebjer et al. (2015) indicated modification had occured on porang flour i.e., by addition number of C = O group from lactic acid.While Gong et al. (2006) noted that stretching absorption of carbonyl from lactic acid was at 1749 cm -1 with a strong peak indicated occurence of graft copolymer of maize starch and lactic acid.
Peak of CH 3 on porang flour (P) increased from 1448.44 cm -1 to 1456.16 cm -1 (PAW) and 1456.16cm -1 (PAM) indicated the addition of CH 3 group from lactic acid to glucomannan molecule from porang flour.While peak increasing of C-O-C on porang (P) flour from 1027.99 cm -1 to 1047.27 cm -1 (PAW) and 1045.35cm -1 (PAM) and also C-O-C on porang (P) flour from 1149.50 to 1234.36 cm -1 (PAW) and 1234.35cm -1 (PAM) also indicated addition of C-O-C group from lactic acid to glucomannan molecules of porang flour.
The abundance amount of very active hydroxyl group (-CH 2 OH)at C-6 position for each unit of glucomannan component resulted some chemical reactions occured at this position such as esterification, etherification, nitration and branch polymerization (Xu et al., 2009).Unfortunately broad spectra of 806 and 896 at PAW dan PAM samples were not appeared and this is condition according Xu et al. (2009) are possibly due to deformation of mannose molecule present in porang flour.
Modified porang structure: Two similar signal were observed as showed by the NMR spectra as presented in Fig. 2, although there were no differences on signals of either modified porang using waterbath (PAW) and microwave irradiation (PAM).PAM resulted a signal at 1.3 ppm with integral values of 3.011 and 2.999 indicated the proton from CH 3 , while PAW at same signal with integral value of 3.090 and 3.081 indicated proton from CH 3 as well.However, at signal of 4.3 ppm either of PAM and PAW indicated proton of CH 2 group although there is a posibility also indicated proton from mannose and glucose at same signal.The H 1 NMR spectra of porang flour and modified porang flour either PAW and PAM are presented in Fig. 2. Pandey et al. (2008) noted that strong signal at 1.52 ppm observed as methyl group, while Choi et al. (2013)

Molecular weight of modified porang flour:
Molecular weights of modified porang flour PAW and PAM are presented in Table 2 and Fig. 3 and it was found that molecular weight of modified porang flour (PAW) was 2.0×10 6 while molecular weight of PAM was 3.0×10 6 .Li and Xie (2003) reported that molecular weight of glucomannan were in the range of 1.033×10    of their sample was 4.81×10 5 .Furthermore, Wang et al. (2012) noted molecular weight of glucomannan was 1.34×10 6 , while Luo et al. (2012) reported molecular weight of their sample was 1.07×10 6 .This condition indicated a reaction between lactic acid and glucomannan component of porang flour.
Particles size of modified porang flour: The results of particle size measurement are presented in Table 3 and Fig. 4 and heating treatment in waterbath and microwave resulted reduction of particle size compared to native porang flour particle size.Particle size of native porang flour was 8.4902 µm became 0.1434 µm for particles of microwave irradiation modified porang flour (PAM) and 0.1164 µm for particles of waterbath heating modified porang flour (PAM).While distribution of biggest particle size of native porang flour was 831.2676 µm and reduced to 39.3229 µm (PAM) and 20.5980 µm (PAW).According to Tatirat and Charoenrein (2011) and Tatirat et al. (2012) particle size of Konjac flour were around 285.31 µm and 112.4 µm.These results are possibly due to heating porang flour in acid condition at 90°C which caused breakage of particles and microwave irradiation could separate the lamella from porang flour granula, release of polymers as consequence of tissue changes.Kratchanova et al. (2004), reported that microwave treatment could modified capiler porosity of plant tissue, intermolecular friction which resulted from microwave heating resulting in internal cell pressure caused cell rupture hence cell components were flown out.
Microstructure of modified porang flour: Figure 5 showed the results of surface microstructure of porang flour (P) using Low SEM and a crystal like structure was observed while modified porang flour in waterbath reported that glucomannan have moisture accessibility to higher amorph site.The smooth surface structure of either PAW and PAM indicated a homogen mixture formation between lactic acid and porang flour where interfacial interaction were occured properly between lactic acid and porang flour.

Rheology of modified porang flour:
The effect of temperature on visco-elasticity traits of modified porang flour with lactic acid and heating in waterbath (PAW) and microwave irradiation (PAM) were investigated using dynamic temperature oscillatory test as presented in Fig. 6.Storage modulus (G') and loss modulus (G") of PAW and PAM observed decreasing with the increased of temperature.This condition possibly in relation to increase activity of molecular chain and weakness of moisture interaction with porang flour hence a decrease of intermolecular hydrogen bond which support the flow capacity of solution as also reported by Du et al. (2012), Luo et al. (2013) and Jin et al. (2014).
It is interesting to note that storage modulus (G ' ) of PAW and PAM were much lower compared to loss modulus (G " ) either at low or high temperatures.Crossing of modulus (G' dan G") from PAW and PAM did not occur indicated that as noted by Wang et al. (2012) either PAW and PAM had an unchange viscoelastis system and according to Jin et al. (2014) had a thermo-irreversible gel characteristic.Furthermore, during heating gel formation of PAW and PAM did not happen and this condition according to Chen et al.
(2011) indicated there was an acetyl group play an important role on gelation chracteristic of PAW and PAM.Hydrogen bond and hydrophobic interaction both play an important role in network formation, although hydrophobic interaction had a bigger effect on konjac glucomannan gelation characteristic.Therefore, either hydrogen bond and hydrophobic interaction could act as moleculer strength in konjac glucomannan gelation formation (Chen et al., 2011).

CONCLUSION
It can be concluded that functional group peaks of modified porang flour (PAW and PAM) changed differently compared to native porang flour included OH, CH 3 , C-C, C = O, C-O-C groups as well as glucose and mannose groups.H 1 -NMR spectra of modified porang flour (PAW and PAM) were similar to the proton original of CH 3 and CH 2 , with bigger moleculer weight i.e., PAW was 2.0 ×10 6 and PAM was 3.0×10 6 and smaller in particles size.The PAW and PAM surface microstructure were observed porous and spongy and rheology characteristics indicated that PAW and PAM had an unchanged visco-elastic system by thermal treatments.

Fig. 4 :Fig. 5 :Fig. 6 :
Fig. 4: Particles size distribution of native and modified porang flour; (a): Particles size distribution of native porang flour; (b): Particles size distribution of modified porang flour by heating in waterbath (PAW); (c): Particles size distribution of modified porang flour by microwave irradiation (PAM) Ministry of National Education and Culture Republic of Indonesia.