CHANGES IN ANTIOXIDANT AND PHYSICOCHEMICAL PROPERTIES OF INDONESIAN BLACK RICE FLOUR ( VAR . BANJARNEGARA AND BANTUL ) DURING NO-DIE EXTRUSION COOKING

This research aimed to evaluate the effect of extrusion cooking conditions (barrel temperature and feed moisture content) on the changes in the physicochemical properties and antioxidant activity of the Indonesian black rice flour (var. Banjarnegara and Bantul). The rice flours were extruded using a no-die twin screw extruder at various barrel temperatures (110 and 140°C) and moisture content of 15, 20, 25% (wb). The total phenolic content (TPC), total anthocyanin content (TAC), and antioxidant activity generally decreased by 29, 46, and 19%, respectively. During extrusion cooking, the higher moisture content resulted in a higher retention of anthocyanins hence increased the antioxidant activity as measured by DPPH assay. Increasing temperature produced less retention of both anthocyanins and phenolics, hence lowering the antioxidant activity. The water absorption of the products also increased as the moisture content and barrel temperature increased, while the water solubility of the products became lower as the moisture content increased. Following a no-die extrusion cooking, both varieties of the black rice experienced changes with regard to the physicochemical properties and antioxidant activity. Due to the high antioxidant activity (DPPH value of 510.4 mg Trolox equiv/100 g) and FRAP value of 2340.9 mg Trolox equiv/100 g), the black rice var. Banjarnegara is recommended for further development. No-die extrusion cooking conditions at 110C and moisture content of more than 25% is selected to achieve fully gelatinized flour with high antioxidant activity.


INTRODUCTION1
Rice (Oryza sativa L.) is one of the staple foods for more than 3 billion people in more than 100 countries of t he world (Birla et al., 2017).B ased on the bran color, rice is classified into non -pigment ed rice and pigmented rice (Goufo and Trindade, 2014).Pigmented rice cultivars are reported as pot ent sources of antioxidants and are encouraged as a functional food (Reddy et al., 2016).Raw black rice has the highest antioxidant activity as it is the richest source of phenolic acids, flavonoids and anthocyanins compared to brown, red, and purple rice (Goufo and Trindade, 2014).Previous studies have shown that anthocyanins in black rice possessed anti-canc er properties as well as anti-inflammatory and antiatherogenic properties to prevent cardiovascular diseases (Min et al., 2010;Hui et al., 2010).Thus, black rice is one of the promising functional foods to meet the demands of health conscious consumers.Pratiwi and Purwesti (2017) revealed that there were 24 varieties of Indonesian black rice; six varieties from Central Java, five varieties from Yogy akarta, four varieties from Nusa Tenggara Timur (NTT), six varieties from Sulawesi, and one variety each from Kalimantan and West Java.The black rice varieties were named after t heir origin area, for instance the Bantul black rice from Bantul area in Yogyakarta province (K ristamtini et al., 2014).However, there were very rare data on Indonesian black rice production except the production of Waja Loka and Laka black rice variety from NTT cultivated in irrigated organic paddy field that only reac hed 6 tons/ha and 8 tons/ha in dry season, respectively (Budiman et al., 2012).Black rice is still rarely consumed in Indonesia, but it has been sold as unmilled rice.The number of research on Indonesian black rice is still growing, and the res earches considering antioxidants in Indonesian black rice are also still limited to the antioxidant activity in raw flour, cooked rice, and beverages from its cooking water (Hart ati, 2013;Handayani et al., 2014).
The growing demand of black rice creates the opportunity for the new product development, such as instant porridge, rice noodle, and rice snacks.These processed foods production utilize extrusion cooking, which is a rapid processing method involving high temperature and pressure in a short time.Extrusion cooking is able to change the amount and profiles of food nutritional and functional components, which may be either beneficial or deleterious.These changes are mostly caused by extrusion cooking conditions, such as barrel temperature, feed moisture, screw speed, and flow rate (Brennan et al., 2011). Sompong et al. (2011) reported that feed moisture only affected the retention of total phenolic and did not affect the retention of anthocyanin and antioxidant activities in the extrusion cooking of Thailand black rice.There has been no report on t he extrusion cooking of Indonesian black rice, especially the effect of its process conditions.
The objective of this research was therefore t o investigate the influence of extrusion cooking conditions to obtain pre-gelatinised flours for ingredient purposes, especially barrel temperature and feed moisture content, on the retention of total phenolics, anthocyanins, and antioxidant activity of the black rice flour.Moreover, the changes on the physicoche-mical properties, such as water solubility, water absorption, and color, were also evaluated.

Materials
Black rice (Oryza sativa L. indica) cultivar Banjarnegara and Bant ul black rice were purchas ed from farmer association in Cigudeg, West Java, Indonesia.According to our sample characterization, the amylose content of Banjarnegara black rice is 1.31% and Bant ul black rice is 2.04%.Based on the classification by Sompong et al. (2011), Banjarnegara black rice was classified as waxy (<2%) and Bantul black rice as very low amylose (2-9%).

Sample preparation
Preparation of rice flour for extrusion cooking experiments was done by husking the black rice grains twice using rice huller (Yanmar rice huller model W-60A, Japan) and milling them by passing through 80-mesh sieve in a stainless steel disk mill (Agrowindo stainless steel disk mill model AGC 21, Indonesia).The flours obtained were stored at divided into 1 kg portions for each application, packaged in vac uum aluminum foil bags and stored at -18°C prior to use.

Extrusion cooking
Rice flour samples were extruded in duplicat e using a no-die twin-screw extruder (B erto BE X-DS-2256, Indonesia) with a screw length of 900 mm and diameter of 64.5-50 mm.Feed rate for all experimental results was set at 19.7 kg/h.No die applied for the extrusion cooking, so that no expansion of the extrudates occurred as the aim of this study was not to ac hieve well expanded extrudates but pregelatinised flours for ingredient purposes.
The screw speed was maintained at 80 rpm.The temperature profile in the first two barrel sections from the feed was maintained at 50 and 70 °C, while the third barrel section was set at 110 or 140°C as the variable of processing.The moisture content of each black rice flours was determined prior to extrusion cooking (AOAC, 2012) and lat er adjusted by adding water to reach three different levels of moisture content (15, 20, and 25%; wet basis).The addition of water was conducted by spraying then the flours were stored at -18°C in a vacuum foil bag to reach equilibrium for 24 h before extrusion.Pre-gelatinis ed flours obtained from extrusion were packed into 1 kg portion each and stored at -18°C in airtight aluminum foil bags for further analysis.

Extraction of phenolic compounds
Phenolic compounds of unprocessed and extruded rice flour samples were extracted using the method by Zhang et al. (2010).Briefly, 5 g of black rice flour was blended with 50 mL of chilled acidified Methanol using 95% Methanol (Merck, Germa-ny) and 1 M HCl (Merck, Germany ) in ratio of 85: 15 (v/ v).The mixture then was centrifuged (IEC Centra-8 Centrifuge, Waltham, MA) at 2500g for 10 min.The supernatant was removed, and the remaining pellet was again extracted with 50 mL of chilled acidified Met hanol.The s upernatant was pooled and made up to a final volume of 100 mL with chilled acidified methanol.The extracts were stored at -18°C in dark bottles until analysis.

Determination of total phenolic content
The total phenolic content (TPC) of unprocessed and extruded rice flour extract was determined by Folin-Ciocalt eu spectrometric method (Ti et al., 2015) with a slight modification.The phenolic extracts of Banjarnegara and B antul black rice flour were first diluted 100-fold and 50-fold respectively in acidified Methanol (95% Methanol and 1 M HCl 85:15, v/ v).An aliquot of extract (600 µL) was reacted with 3. 0 mL freshly dilut ed (10-fold) Folin-Cioc alteu reagent (Merck, Germany).The mixture was allowed to equilibrate for 10 min and then neut rallized with 2.4 mL of sodium carbonate (Sigma Aldrich, USA) solution (75 g/L).After incubation at a room temperature (25°C) for 90 min, the absorbance of the resulting solution was measured at 747 nm using UV-Vis spectrometer (Shimadzu, UV-1800, Japan).Samples were assayed in duplicate and G allic acid (Sigma Aldric h, USA) was used as the standard.TP C was expressed as mg of G allic acid equivalents (GAE) per 100 g of dry weight of sample.

Determination of total anthocyanin content
Determination of t otal anthocyanin content (TAC) was conducted using a spectrophotometric pH differential method by Lee et al. (2016).The phenolic extracts of Banjarnegara and Bantul black rice flour were first diluted 100-fold and 50-fold respectively in acidified Met hanol (95% methanol and 1 M HCl 85: 15, v/ v).The extracts were mixed thoroughly with 0.025 M Potassium chloride (Merck, Germany) buffer (pH 1).The abs orbance of the mixture was then measured using UV-Vis spectrometer (Shimadzu, UV-1800, Japan) at 515 and 700 nm against distilled water blank.Similarly, the above extracts were dissolved with Sodium acetate (Merck, Germany) buffer (pH 4.5) and the absorbance of these solutions was measured at the s ame wavelengt hs.The anthocyanin content was calculated by: where, is the molar absorptivity of Cyanidin-3glucoside (Cy-3-G) = 26.900;C is the concentration of the buffer in mg/mL; A is abs orbance = (A 515 -A 700 ) pH 1.0 -(A 515 -A 700 ) pH 4.5 ; MW is the molecular weight of Cy-3-G = 449.2;and DF is the dilution factor.The anthocyanin c ontent was expressed as the mean of mg Cy-3-G equivalents per 100 g dry sample ± SD for duplicate samples.

Determination of DPPH free radical scavenging activity
Analysis of 2,2-Diphenyl-1-picrylhydrazyl (DPP H) free radical scavenging capacity was performed using 96-well plate assay method from B obo-garc ía et al. ) in methanol-water (80:20, v/ v) and shaken for 60 s in a 96-well microplate (Iwaki, Japan).Th e absorbance was measured at 515 nm in the ELISA microplate reader (Biotek Instruments, Winooski, USA) after 40 min incubation in the dark at room temperature.Trolox (Sigma Aldrich, USA) was used as a standard at 100-500 µmol L -1 and the % DPPH quenched was calculated by: where, A sample is the absorbance at 515 nm of 20 µl of extract or standard with 180 µL DPPH solution after 40 min; A blank is the absorbance at 515 nm of 20 µL of water with 180 µl Met hanol-water (80:20, v/ v) after 40 min, A control is the absorbance at 515 nm of 20 µL of water with 180 µL DPPH solution after 40 min.The DPPH antioxidant activity was expressed as the mean of mg Trolox equivalent (TE) per 100 g dry sample for duplicate samples.

FRAP analysis
Determination of Ferric Reducing Ability Power (FRAP) of the free phenolic extracts from black rice flours and their extrudates was conducted following the method from Handayani et al. (2014).FRAP reagent was prepared by mixing 300 mM acetate buffer (pH 3.6) containing 16 mL acetic acid (Merck, Germany) per litre of the buffer solution, 10 mM 2,4,6,-Tripyridil-s-triazine (TP TZ) (Sigma Aldrich, USA) in 40 mM HCl and 20 mM FeCl 3 .6H 2 O (Merck, Germany) in a ratio of 1:1:10.The 2.7 mL freshly prepared FRAP reagent then were incubated to 37°C.Then, the extract of sample (150 µL) and 210 µL of distilled water were added to FRAP reagent then allowed to stand for 10 min.The FRAP assay and acidified methanol as reagent blank absorbance were read at 593 nm using UV-Vis spectromet er (Shimadzu, UV -1800, Japan) and Trolox (Sigma

Experimental design and statistical analysis
The experimental design was carried out for three independent variables, rice variety (Banjarnegara and Bantul black rice), temperature (T, 110°C and 140°C), and moisture content (15; 20; and 25% in wet basis) by applying a 223 factorial design.All statistical analyses of dat a were carried out using SPSS version 22.0 statistical software for analysis of variance.Significant differenc es bet ween means were determined by the univariate analysis.Significant differences bet ween means were determined by u ' u R .Correlation coefficients amongst data obt ained were calculat ed u P ' ff r).

Total phenolic content
The total phenolic content (TPC) from t he phenolic extracts of unprocessed and extruded Bantul and Banjarnegara black rice flour were exhibited in Figure 1.The TPC values of waxy Banjarnegara black rice flour was significantly higher (P<0.001)than low amylose Bantul rice, which was in opposite to the findings by Tang et al. (2015) who report ed that non-waxy black rice had higher TPC than waxy black rice.In addition, the TP C values of unprocessed Bantul black rice and Banjarnegara rice (36.01 mg GAE/100 g and 114.06 mg GAE/100 g, respectively) were lower than that of black rice from Thailand whose TP C was 351.In this studies, the TPC of Banjarnegara black rice flour decreased significantly (P<0.05) to its corresponding control (unprocessed flour) sample.This result is consistent with previous black rice extrusion studies carried out by Sompong et al. (2011) andTi et al. (2015).However, in this study, the highest reduction of TPC was 29.3%, which was not as high as reported in the two previous studies (59.8-79.5% of reduction).Rice variety, barrel temperature, and their interaction affected TP C significantly (P<0.001).Although no-die extrusion cooking generally exhibited a negative effect on TPC, the degree of the influence between Banjarnegara rice and Bant ul black rice was different.The difference bet ween rice varieties most likely was caused by the difference in amylose content.Extrusion gave more declines of TP C values to waxy Banjarnegara rice flour than low amylose B antul rice flour.In Bantul rice flour, within all the moisture content and barrel temperature given, there were no significant differences in their TP C values.These results were in opposite with the findings reported by Tang et al. (2015), in which waxy black rice had no significant change in TP C values upon two different cooking method (i.e.cooked rice and porridge) while non-waxy black rice showed significant (P<0.05)changes in TPC values after the two cooking treatments.
Increasing barrel temperature from 110°C t o 140°C within the same moisture content resulted in significant (P<0.001)reduction of TP C in Banjarnegara black rice by 12.2-22.9%.These results are consistent with those reported by Sharma et al. (2012) when the t emperature was increased from 150 to 180°C for the extrusion of barley cultivars.The reduction of TPC owing to decarboxylation of phenolic compounds in high extrusion temperature, leading to the reduction in their chemical reactivity or their extractability due to certain degree of polymerization (Altan et al. 2009).
Increased moisture content alone affected the TP C insignificantly (P>0.05).These results were in contrast to the black rice extrusion studies by Sompong et al. (2011) when increasing moisture content from 12 to 16% and with Sarawong et al., (2014) who reported 17.5-42.8%reduction of TP C for extruded green banana flour when increasing moisture content (from 20 to 50% ).However, there was a significant effect (P<0.05) of combined int eraction between moisture content, rice variety, and barrel temperature.Within this interaction, high feed moisture content may promot e polymerization of phenols, leading to reduced extractability and antioxidant activity.As the combined interaction between moisture content, rice varieties, and barrel temperature affected the TPC values, there might be optimum TPC result achieved from one condition applied from the three variables.Therefore, due to no-die extrusion cooking, the optimum TPC result (106.13 mg GAE/100 g) was obtained by extruding Banjarnegara black rice with barrel temperature 110°C and 15% (wb) of moisture content as the extrusion cooking condition.

Total anthocyanin content
The total anthocyanin content (TA C) from the phenolic extracts of Banjarnegara and Bantul unprocessed and extruded black rice flour were exhibit ed in Figure 2. TA C of unprocessed Banjarnegara black rice was 179.30 mg Cy-3-G equiv/ 100 g and unprocessed Bantul black rice was 70.30 mg Cy-3-G equiv/100 g.Meanwhile, Kristamtini et al. (2014) reported that the TA C value of Banjarnegara black rice was 165.78 mg Cy-3-G equiv/ 100 g and unprocessed Bantul black rice was 90.22 Cy-3-G equiv/ 100 g.The differences in ant hocyanin content among black rices were might be because of differences in growing conditions and extraction method among the studies (Tang et al., 2015).Banjarnegara had significantly (P<0.001) higher TA C value than Bantul, in agreement with TA C based Indonesian black rice cultivars classification made by Kristamtini et al. (2014), which classify Banjarnegara black rice in cultivar group II and Bantul black rice in cultivar group III.Numerous studies have suggested that Cyanidin-3-glucoside (Cy-3-G) is the most dominant anthocyanin in black rice, and the minor one is Peonidin-3-glucoside (Pe-3-G), in which each rice varieties had different composition of anthocyanin compounds (Tang et al., 2015;Goufo and Trindade, 2014).
In general, no-die extrusion cooking caused significant decrease in TAC values of both rice flour extrudat es, with reduction range from 8.6 to 46.2% for extruded Banjarnegara black rice flour and 7.1 to 46.5% for extruded Bantul rice flour.These results were consistent with the studies by Sompong et al. (2011) andTi et al. (2015).All condition variable (moisture content, barrel t emperature, and rice varieties) and their interactions affected the TA C values significantly (P<0.001).Although no-die extrusion cooking generally exhibited a negative effect on TA C, the degree of the influence bet ween Banjarnegara and Bantul black rice was different.Extrusion gave more declines of TA C values to low amylose Bantul ric e flour than waxy Banjarnegara rice flour.The difference between rice varieties most likely was because of the difference in their anthocyanins composition, in which each of anthocyanins had different degradation rate (Tang et al., 2015).
Increasing barrel temperature caused significant (P<0.001) less retention of anthocyanins by 1.8-29.8% in Banjarnegara black rice and up to 17.2% in Bantul rice.The losses of anthocyanins may be attributed to the degradation of Cyanidin-3glucoside into protocatechuic acid, arising from thermal processing (Hiemori et al., 2009).Patras et al. (2010) also reported that thermal degradation of Cyanidin-3-Glucoside might obtained Phloroglucinaldehyde and 4-hydroxy-benzoic acid through deglycosylation and cleavage mechanism.
Within the constant barrel t emperature, increased moisture cont ent caused inc rease anthocyanin retention significantly (P<0.001) up to 4.9% in Bantul rice flour extrudates and up to 38.4% in Banjarnegara rice flour extrudates.Higher moisture content has protective effect on bioactive compounds as high moisture content increases shear stress, resulting in less dissipating heating (Hirth et al., 2014).There was a significant effect (P<0.001) of combined interaction between moisture content, rice varieties, and barrel temperature.As the consequence of the influence from combined interaction between moisture content, rice varieties, and barrel temperature, there also might be optimum TA C result achieved from one condition applied from t he three variables.Therefore, due to no -die extrusion cooking, the optimum TAC result (163.86 mg Cy-3-G equiv/100 g) was obtained by extruding Banjarnegara black rice with barrel temperature 110°C and 25% (wb) of moisture content as the extrusion cooking condition.

Antioxidant activity
DPPH free radical scavenging capacities of the unprocessed and processed black rice were mentioned in Figure 3A.The DPP H value of unprocessed waxy Banjarnegara black rice (510.43 mg TE/100 g) was significantly higher (P<0.001)than that of non-waxy Bantul black rice (251.96mg TE/ 100 g).After no-die extrusion cooking, both two varieties (non-waxy and waxy) exhibited a decreasing tendency.However, different moisture content can trigger significant diversity of the DPPH values.
In general, DPPH values were reduced due to no-die extrusion cooking since there were mostly loss of phenolics and anthocyanins as antioxidant components that have been discussed above.Different rice variety caused significant difference (P< 0.001) of DPP H values obtained.This means that Banjarnegara and Bantul black rice flour had different DPPH values in nature and also exhibited different antioxidant activity (measured by DPP H assay) changes behavior upon no-die extrusion cooking.This phenomenon can be explained by the difference of TPC and TAC values owned by both varieties.
Within constant barrel temperat ure, higher moisture content applied mostly resulted in increase of DPP H values (P<0.05).These results may be caused by either the less retention of anthocyanins due t o higher moisture content that was explained in the previous section, which was support ed with the strong correlation between DPP H antioxidant activity and TA C (r=0.805, P<0.001).Moreover, the DPPH antioxidant activity also affected by TPC owing to their strong correlation (r=0.950,P<0.001), which was in opposite to the low correlation bet ween DPPH and TP C in eight barley cultivars observed by Sharma et al. (2012).The increase in antioxidant activity also could be explained by the formation of antioxidative Maillard browning products which enhanced the antioxidant activity (Sharma et al., 2012).This is influenced by many factors, including reactant concentration, temperature, time, and water activity.
Barrel temperature alone caused insignificant reduction (P>0.05) of DPP H values in the extrudates.These results were in opposite with the observation of eight barley cultivars which were extrusion cooked at 150C and 180°C (Sharma et al., 2012).But when temperature was combined with moisture content and either with moisture content and rice varieties, there were significant reduction (P<0.05) of DPP H observed.The reduction of DPPH antioxidant activity due to higher barrel temperature might be correlated with the loss of anthocyanins and phenolics since there was a strong positive correlation between DPP H antioxidant activity and TA C (r=0.805, P<0.01), as well as DPPH along with TPC (r= 0.950, P<0.001).
There was a significant effect (P<0.05) of combined interaction between moisture cont ent, rice varieties, and barrel temperature.As the consequence of the influence from combined interaction bet ween moisture cont ent, rice varieties, and barrel tempera-

A
Bantul Banjarnegara ture, it can be seen that there also might be optimum antioxidant activity measured by DPPH assay achieved from one condition applied from the three variables.Therefore, due to no-die extrusion cooking, the optimum DPP H value (537.57mg TE/100 g) can be obtained by extruding Banjarnegara black rice with barrel temperature 110°C and 25% (wb) of moisture content as the extrusion cooking condition.
The FRAP values of unprocessed and extruded Banjarnegara and Bant ul black rice were present ed in Figure 3B.The FRAP values of unprocessed waxy Banjarnegara black rice and non-waxy Bantul black rice were measured individually and result ed in 537.56 mg TE/100 g and 2340.96mg TE/100 g, respectively.Hence, relatively stronger antioxidant activity profiles were shown in waxy Banjarnegara black rice rather than non-waxy Bantul black rice.A similar result had been also gained from the determination of radical DPPH scavenging activity.

Water absorpti vity index and water solubility index
Water absorptivity index (WAI) has been commonly referred to as the dispersion of starch excess water and is an indicator of the ability of flour to absorb water or for the swelling ability of starch.It depends on the availability of hydrophilic groups, which bind water molecules and on the gel forming capacity of macromolecules.WAI is increased by the degree of starch damage due to gelatinization and extrusion-induced fragmentation, i.e. molecular weight reduction of amylose and amylopectin.WSI is generally used as an index of degradation of molecular components, and thus is a measure for the degree of starch degradation during extrusion cooking (Mesquita et al., 2013).The WAI and WSI of unprocessed and extruded Banjarnegara and Bantul black rice flours.WAI of black rice flours and their extrudat es were relatively low (range 1.43-2.05g/g).Extrusion processing condition (temperature in °C, moisture content in %)

B
Bantul Banjarnegara WAI was significantly (P<0.05)increased (1.8-13.9%)by increasing barrel temperature for only Banjarnegara rice (Figure 4A ), indicating that WAI was affected by rice variety and barrel temperature (P<0.05).For B antul rice, WAI was increased significantly (P<0.05) after extruded at 140°C and the moisture content did not affect the WAI.These results were in agreement with t he studies conducted by Sompong et al. (2011) and Sarawong et al. (2014).According to Sobukola et al. (2013), barrel temperature had significant effect to WAI, since at higher temperature, starch granule is disrupted and more water is bound to the starch molecule which resulting in increased WAI.In Banjarnegara black rice, eventhough not significant (P>0.05),but there were increases of WAI when higher moisture was applied.At higher moisture content, viscosity of starch would be low, allowing the starch molecules to move freely and thereby enhancing the penetration of heat as a result great er gelatinisation.In addition, Sobukola et al. (2013) interpret ed that higher moisture content imparts less shearing action in the barrel causing less mechanical damage to starch, thus increase WAI.However, there was no significant effect (P>0.05) of the interaction between moisture content and barrel temperature to the WAI.
Water solubility index (WSI) is an indicator of the degradation of molecular components, in which represents the amount of soluble polysaccharide released from the starch after extrusion (Ding et al., 2005).WSI of black rice flours and their extrudates were relatively low (range 2.43-4.18%)(Figure 15b).Since high amylose starch had high WSI values (Stojceska et al., 2009), it is easy to comprehend that low amylose and waxy black rice would have low WSI values.Due to no-die extrusion cooking, WSI value of Banjarnegara black rice was affected significantly (P<0.001) by barrel temperat ure and moisture content.A higher moisture content caus ed significant (P<0.001)dec reased of WSI by 0.3-28.2%.Higher moisture content during extrusion resulting in a lower degree of starch gelatinization and probably acts as a plasticizer caused by reduc ed starch degradation, also due to less shearing taking place, thus resulting in a decrease in WSI (Seth et al., 2015).Bantul black rice flours as the effect of rice variety, barrel temperature, and moisture content Within constant moisture content, higher barrel temperature also increased WSI significantly (P< 0.001) by up to 19.9%.This could be due t o starch degradation at higher temperat ure exposure to product inside the barrel and greater shearing action of the blend.Sobukola et al. (2013) reported that increased barrel temperature increases WSI, due to increased solubility of starch molecules.The WSI value is also an indication of state of protein in food or extrusion blends.Since the proportion of prot ein in Banjarnegara and Bantul black rice is high, the increase of WSI might be due to partial prot ein denaturation at higher temperature (Oikonomou and Krokida, 2011).Ito et al. (2015) stated that desirable maximum WSI can be achieved with minimum moisture content (20% ) and high screw s peed (200 rpm).The desired high WSI could not be achieved since the screw speed applied in this study was low (80 rpm).Meanwhile, though WSI value of Bantul black rice was dec reased after extrusion, the variation of temperature and moisture c ontent did not affect the WSI value significantly (P>0.05).
There was a significant effect (P<0.001) of combined interaction between moisture content, rice varieties, and barrel temperature.As the consequence of the influence from combined interaction between moisture content, rice varieties, and barrel temperature, there also might be optimum WSI value ac hieved from one condition applied from t he three variables.Therefore, due to no-die extrusion cooking, the optimum WSI value (3.73% ) can be obtained by extruding Bantul black rice with barrel temperature 140°C and 15% (wb) of moisture content as the extrusion cooking condition.Since high value of WSI is desirable in extrusion cooked products such as breakfast cereals and porridge (Oikonomou and Krokida, 2011), extended extrusion cooking studies of black rice are necessary to improve the physical properties of the products.

CONCLUSION
The heat of no-die extrusion cooking decreased the total phenolic content of waxy Banjarnegara black rice flour but not affected low amylose Bantul black rice flour.No-die extrusion decreased the total anthocyanin content of both Banjarnegara and Bantul black rice flours and higher feed moisture content gave less retention of the total anthocyanin content.DPPH antioxidant activity decreased due to no-die extrusion cooking, but increased when high feed moisture content (25% ) applied for both Bantul and B anjarnegara black rice flours.The heat of nodie extrusion cooking decreased the FRAP antioxidant activity of B anjarnegara black rice flour but not affected Bantul black rice flour.Increasing heat of no-die extrusion cooking increased the water ab-sorptivity index of Bantul black rice flour, but decreased the water absorptivity index of B antul black rice flour.In Banjarnegara black rice flour, increasing temperature of no-die extrusion cooking inc reas ed water solubility index but increasing feed moisture content dec reas ed its water solubility index.Meanwhile, temperature and feed moisture content of nodie extrusion cooking did not affect the water solubility index of B antul black rice flour.For future development, it is recommended to utilize Banjarnegara black rice as its antioxidant is higher than Bantul black rice.The processing condition may apply a nodie extrusion cooking at 110°C of barrel temperature and more than 25% of moisture content to achieve fully gelatinised flour with high antioxidant activity.
(2014).The phenolic extracts of Banjarnegara and Bant ul black rice flour were first diluted 100-fold and 50-fold respectively in acidified Methanol (95% methanol and 1 M HCl 85:15, v/ v).A total of 20 µL of diluted sample was reacted with 180 µL of DPPH (Sigma Aldrich, USA) solution (150 µmol L -1 (temperature in °C, moisture content in %) Bantul Banjarnegara Aldrich, USA) was used as standard.The measurements were done in duplicate and the results were expressed as mg Trolox equivalent (TE ) per 100 g of dry sample.Water absorption index and water solubility indexWater absorption index (WAI) and wat er solubility index (WSI) of samples were determined by a modified method from Sompong et al.(2011).Each flour (2.5 g) was dispersed in 30 mL of distilled wat er at room temperature for 30 min with a gentle stirring every 5 min.Then the samples were centri -fuged (IE C Centra-8 Centrifuge, Waltham, MA) at 1500×g for 20 min.The supernat ant was decanted int o a tared aluminum pan and dried at 105°C until constant weight.The weights of the gel remaining in the centrifuge t ubes were noted.The measurements were done in duplicate.WAI were calculated as weight gain of gel/dry weight of sample and WSI were calculat ed as weight of dry solid in s upernatant × 100/dry weight of samples.
23 mg/100g (Sompong et al. 2011) and from China (Oryza sativa var.Heiyounian) whose TP C was 545.8 mg GAE/100g (Ti et al., 2015).Possibly, black rice growing under different environments and regions produce different physical characteristics and chemical compositions.Apart from this reason, the method of extraction plays an important role to the various obtained TPC values.

Figure 1 .
Figure 1.Total phenolic content (TP C) of Banjarnegara and Bantul black rice flours as the effect of rice variety, barrel temperature, and moisture content

Figure 2 .
Figure 2. Total anthocyanin content (TA C) of B anjarnegara and Bantul black rice flours as the effect of rice variety, barrel temperature, and moisture content (temperature in °C, moisture content in %)

Figure 3 .
Figure 3. (A) DPPH antioxidant activity and (B ) FRAP antioxidant activity of Banjarnegara and Bantul black rice flours as the effect of rice variety, barrel temperature, and moisture content

Figure 4 .
Figure 4. (A) Water absorptivity index (WAI) and (B) Water solubility index (WSI) of Banjarnegara and Bantul black rice flours as the effect of rice variety, barrel temperature, and moisture content