ENZYMATIC HYDROLYSIS OF BITTER CASSAVA AND GADUNG STARCHES WITH DIFFERENT COMPOSITIONS AT LOW TEMPERATURE

The effect of compositions of bitter cassava (Manihot glaziovii) and gadung (Dioscorea hispida Dennst) starches on reducing sugar during hydrolysis using granular starch hydrolyzing enzyme (GSHE) was studied. All hydrolyses were conducted at concentration of substrate was 200 g.L, while concentration of enzyme was 1.5 % (w/w), during of hydrolysis time 24 h, at 30°C. Mass compositition of bitter cassava and gadung starches were 9:1 to 1:9 The increase gadung starch compositions will decrease the reducing sugar. The optimum condition of the process using concentration of substrate 200 g.L with compositions of bitter cassava and gadung starches was 9:1 at 18 h. It was found that reducing sugar was 50.20 g.L. The concentration of reducing sugar mainly depend on starch content on bitter cassava, it is much bigger than the gadung starch.


Introduction
In Indonesia, bitter casava (Manihot glaziovii) and bitter yam (Dioscoreae hispida Dennst) which locally known as gadung are commonly found in secondary forest.They grow under shaded areas or near sreams, not consumed by humans because of the presence of poisonous alkaloids known as cyanogens and dioscorin [1], which can seriously impact the health of the people who consump the tuber.Lambri et al. [2] reported that the total cyanide content of cassava root for sweet white and bitter white were 374 and 442 mg/kg (d.w), respectively.Djazuli and Bradbury [3] reported of 14 samples of cyanogen content of cassava starch in Indonesia.The maximum value of cyanogens content in cassava starch and cassava flour were 12 ppm and 149 ppm, respectively.
The conventional conversion of starch to glucose requires a two-step process, namely, liquification and saccharification.During liquefaction, gelatination of starch is promoted by applying high temperatute (90-100 ºC), α-amylase is added in order to convert starch into dextrins, maltose and maltotriose.The resulting mash is then cooled to 60 °C and glucoamylase is added to convert dextrins to fermentable sugars as glucose [4][5][6][7].Granular starch hydrolyzing enzyme (GSHE) is a mixture of α-amylase and glucoamylase which hydrolyzes granular starch directly into fermentable sugars and works at low temperature (30 to 48°C) and pH (4.0 to 4.5) [8].This process is an energy-intensive, and therefore increases the production cost.It has been estimated that about 30-40% of the total energy is demanded during ethanol production from starch [5].The use of GSHE presents others advantages, as the GSHE will negate liquefaction and saccharification.
Previous researches were commonly conducted via conventional of hydrolysis and hydrolysis without heating a single substrate, for example the hydrolysis of corn, wheat, tapioca, sago, sorghum starches, etc, only few researches were conducted on hydrolysis enzymatic of double substrate.Uthumporn et al. [9] also reported the hydrolysis of maize, sago and sweet potato starches using α-amylase and glucoamylase, at 35°C to 50°C.Li et al. [10] reported the hydrolysis of corn and triticle starches at 30-50°C using α-amylase and glucoamylase.Shariffa [11] investigated hydrolysis of tapioca and sweet potato starches by α-amylase and glucoamylase, at 35-65°C.
The objective of this study is to investigate the effect of bitter cassava and gadung starches compositions and on reducing sugar.

Cassava and gadung tubers
Ten month old of bitter cassava was called Pandemir (Manihot glaziovii) tuber was obtained from Wonogiri district in Indonesia, while gadung tuber with 9 month old was obtained in Godean district in Indonesia.The extraction method applied to the gadung is the same as the bitter casava extraction method.The method of extraction of the bitter cassava starch, physicochemical properties of the bitter cassava and gadung used in this study were the same as the those used in the previous research conducted by Hargono [12].Potassium sodium tartrate tetrahydrate and 3,5-Dinitrosalicylic acid, sodium hydroxide (98%, Merck), sodium sulfite MATEC Web of Conferences 156, 01009 (2018) https://doi.org/10.1051/matecconf/201815601009RSCE 2017 (98.5%, Merck), sulfuric acid (98.5%,Merck), sodium acetate buffer, glucose (99.5%), were purchased from their authorized distributor.Granular starch hydrolyzing enzyme as Stargen TM 002, which is a mixture of αamylase and glucoamylase was produced by Genencor (Palo Alto, USA).The activity and optimal pH range declared by the producer are 570 GAU/g and 4.0-4.5, respectively [8].

Analytical methods
The starch content was determined by AOAC method [13].The water content in cassava starch was determined by standard drying method in oven at 105 °C to constant mass [14].The total cyanide analysis by the acid hydrolysis method [15].During the cassava starch hydrolysis, the content of reducing sugar was measured using dinitrosalicylic acid method [16].Reagent consisting of aqueous solution of 1% 3,5-dinitrosalicylic acid, 0.05% Na sulfit, 20% Na-K tartrate and 1% NaOH solution was added in the ratio 3:1 to the samples in glass tubes, shaken in incubated in a boiling water bath for 8 min.Reacted samples were cooled in an ice water bath for 5 min, prior to measuring absorbance at 540 nm by using a UV/visible spectrophotometer (UV-160A, SHIMADZU, Kyoto, Japan).Glucose (0 to 60 g.L -1 ) was used as standard, therefore reducing sugar concentrations was reported as g.L -1 .

Determination of reducing sugar from bitter cassava and gadung starches hydrolysis
The reducing sugars obtained from bitter cassava and gadung starches by Stargen TM 002 with concentration 1.5 % (w/w), and concentration of starch 200 g.L -1 , at 30ºC and pH 4, during of hydrolysing time 0 to 24 hours are given in Figure 1.Over the hydrolysis time from 0 to 24 h, product of the reducing sugar obtained from of bitter cassava starch was higher than gadung starch.Production hydrolysis from 0-18 h showed a maximum reducing sugar concentrations (bitter cassava starch 52.80 g.L -1 and gadung starch 24.65 g.L -1 ), while production rate showed of bitter cassava starch 2.93 g.(Lh) -1 and gadung starch 1.37 g.(Lh) -1 , respectively.Furthermore after 18 hours, the hydrolysis of both of cassava and gadung starches decelerated, and then followed by constant rate to 24 h.The differences of the reducing sugar concentrations, it is because the starch content in gadung is much lower than the bitter cassava starch, as shown in Table 1 and so probably amylose content in bitter cassava starch is much lower than gadung starch.Sharma et al. [17] investigated effect amylose:amylopectin ratios on ethanol production from maize starch using GSHE.They conclused the amylose content in starch increased, the amount of ethanol produced decreased.Wu et al. [18] reported the result showed that the amylose content in starch had a significant effect on the ethanol conversion efficiency.Conversion efficiency increased as the amylose content decreased.So in addition the cyanide content in gadung starch is much higher than bitter cassava starch (Table 1), as a consequence the cyanide inhibit the enzyme leading to decrease of enzyme activity.Hargono et al. [12] investigated of effect of cyanide on reducing sugar from bitter cassava and gadung flours, we conclused increasing of cyanide concentration during hydrolysis process, decreased enzyme activity, as a consequence amount of reducing sugar decreased.Shanavas et al. [19] [20], that the during time 8 to 24 h hydrolysis native tapioca starch was increasing reducing sugar, as indicated by increasing of dextrose equivalent (DE) 18 to 35.7% .

Conclusion
The experiments conducted an attempt is made to present kinetic expression for the hydrolysis of bitter cassava and gadung starches compositions, at concentration of substrate 200 g.L -1 , concentration of Stargen TM 002 1.5 % (w/w), pH 4 and 30 °C.The results showed that at various compositions of bitter cassava to gadung starches (9:1 to 1:9) during 24 h.It was found the maximum of concentration of reducing sugars at 18 h at ratio 9:1 was 50.20 g.L -1 , while the minimum of concentration of reducing sugars at 18 h at ratio 1:9 was 27.12 g.L -1 .The release of reducing sugar increased when bitter cassava to gadung starches compositions increased, the concentration of reducing sugar mainly depend on starch content on bitter cassava, it is much bigger than the gadung starch.

Fig 1 .
Fig 1. Hydrolysis profile of bitter cassava and gadung starches at concentration of substrate 200 g/L -1 , concentration of Stargen TM 002 1.5 % (w/w), pH 4 and 30°C.Each value represents the mean of three independent measurements.Deviation from the mean below 5% for all values displayed.

Fig 2 .
Fig 2.Hydrolysis profile of bitter cassava and gadung starches compositions (9 :1 to 1:1) w/w, at concentration of substrate 200 g.L -1 , concentration of Stargen TM 002 1.5 % (w/w), pH 4 and 30°C.Each value represents the mean of three independent measurements.Deviation from the mean below 5% for all values displayed.

Fig 3 .
Fig 3. Hydrolysis profile of bitter cassava and gadung starches compositions (1:9 to 1:1.5) w/w, at concentration of substrate 200 g.L -1 , concentration of Stargen TM 002 1.5 % (w/w), pH 4 and 30°C.Each value represents the mean of three independent measurements.Deviation from the mean below 5% for all values displayed reported the reducing sugar formed from cassava starch by varying