M&MriahandM'llhds

cEmmsmmmms3.1 Material sourceRice bran and rice bran oil were obtained from Rice Mills and Solvent Extraction Plants of Rudrapur, Distt. Udham Singh Nagar (Uttarakhand). The acid activated bleaching earth was obtained from M/s Amrit Vanaspati Company Limited, Gaziabad (UP.). All other raw materials were purchased from local market. All the reagents used in the investigation were of AR/GR Grade.3.2 Stabilization of rice branFresh rice bran obtained from the rice mill was stabilized at 120C for 45 minutes by dry heat method in hot air oven in a shallow pan according to method described by Sharma and Chauhan (2002). The proximate composition of stabilized rice bran was determined following standard methods.3.3 Extraction of rice bran oilRice bran oil was extracted from the stabilized rice bran with n-hexane as solvent at 60C using Soxhlet Apparatus for 6 hours. Both the oil samples obtained by Soxhlet extraction method and from solvent extraction plants were analysed for physico-chemical characteristics.3.4 Refining of rice bran oilRefining of rice bran oil was carried out according to flow diagram outlined in Fig. 3.1.Crude rice bran oilIDewaxing1Crude waxGumsSoap stockImpuritiesPigmentsVolatileconstituents

Dewaxed oiliDegumming1Degummed oiliNeutralizationNeutralized oiliBleaching11Bleached oil1DeodorizationiRefined rice bran oil42

3.4.2 Degumming44

Fig. 3.1: Flow diagram of rice bran oil refining process3.4.1 DewaxingDewaxing of rice bran oil was done following the cold-hot extraction and hot extraction methods described by Bhattacharya and Bhattacharya (1987a).3.4.1.1 Cold-hot extraction methodThe gum was precipitated from crude rice bran oil miscella with n-hexane at 5-7C for 6-8 hours. The wax was precipitated in the miscella which was removed by centrifugation at 1500 rpm for 3 minutes. The miscella, thus obtained was heated to 60C and allowed to stand for 2 hours at this temperature. The micelle was cooled to 5C and the precipitate formed of remaining wax was removed by centrifugation at 1500 rpm for 3 minutes or by filtration.3.4.1.2 Hot extraction methodThe effect of method of dewaxing on the recovery of oil, wax, oil percentage in wax and recovery of oil from wax were calculated the miscella concentration, temperature and time after dewaxing were also optimized. The crude rice bran oil in miscella form was taken in a clean conical flask and heated to a temperature of 60C for 10 hours and then cooled. The separated wax removed by centrifugation at 1500 rpm for 3 minutes after cooling to 5C..The oil obtained after dewaxing by both the methods were analysed for physico-chemical characteristics.The dewaxed rice bran oil was degummed by the method of Bhattacharya and Bhattacharya (1985). The dewaxed rice bran oil (100 gm) was taken in a 500 ml conical flask and following quantities of degumming agents were added.SI.No.Deguming agent% amount added (v/v)

1.Water2

3

4

2.Phosphoric acid0.25

0.5

1.0

3.Water + phosphoric acid3+0.25

3+0.5

3 + 0.75

3+1.0

4.Control-

The degumming of rice bran oil was done at 70C for 30 minutes with continuous stirring on a magnetic stirrer. The hydrated gums were separated by centrifugation at 10000 rpm for 3 minutes.The effect of method of degumming on the recovery of oil and gum was determined. The oil obtained was analysed for its physico-chemical characteristics.The degummed oil was neutralized by the method described by Goenka (1987). The following volumes of lye of different strengths were used for miscella neutralization.SI.No.Strength of lye(%)Volume of lye solution (ml)

1.1010

15

20

2.1210

15

20

3.1410

15

20

4.1610

15

20

5.1810

15

20

3.4.3.1 Miscella neutralization3.4.3 Neutralization45

46

47

One hundred ml of degummed rice bran oil was taken in a 500 ml separating funnel. To which 66 ml. n-hexane and 18 ml ethanol were added and mixed properly by shaking. The different volumes of different strength of lye wasthen added to the separating funnel and mixed. A contact time of 30 minutes was kept constant for all the treatments. The heavier sodium soap and other impurities settled in the bottom layer leaving the lighter phase of neutralized oil with hexane in upper layer. The traces of n-hexane was removed from the oil by distillation of upper layer.3.4.3.2 Alkali neutralizationOne hundred ml of degummed rice bran oil was transferred to 500 ml separating funnel and different strength and volume of lye were added. The contents were mixed thoroughly and allowed to stand for 30 minutes. The heavier phase of sodium soap and impurities were separated out from bottom leaving the lighter upper layer of neutralized rice bran oil.3.4.3.3 Caustic soda neutralizationThe method described by Forster and Harper (1983) was used to neutralize the degummed rice bran oil. One hundred ml degummed rice bran oil was taken into a 500 ml round bottom flask and neutralized by a dilute solution of caustic soda. The contents were transferred to a 500 ml separating funnel and allowed to stand for layer separation. The lower layer of sodium soap and impurities was separated out. The neutralized oil was dried under vacuum to remove the volatile material.3.4.4 Chemical interesterificationThe chemical interesterification was done by the method of Khatoon (1991). One hundred ml of neutral oil was taken in a 500 ml vacuum flask and heated to 90C, after addition of 0.2% sodium methoxide (30% w/v), the contents were kept under vacuum for 45 minute at 30C. The oil was then washed with 50 ml portions hot water 2-3 times in a separating funnel.3.4.5 BleachingThe neutralized rice bran oil was bleached by the method of Nechaev et al. (1973). Neutralized oil (100 ml) was taken in a 500 ml conical flask and 5 ml of saturated aqueous solution of organic acid (oxalic acid/citric acid) was added. The contents were mixed and kept at room temperature for 30 minutes. The contents were heated to 60C and then allowed to settle. The oily layer is washed with hot water untill free of acid and dried under vacuum. The oil thus obtained was treated with a mixture of activated bleaching earth and activated charcoal at 110C in different ratios for 20 minutes (1:1, 2:1, 3:1, 4:1 and 5:1). The amount of bleaching mixture was added at the rate of 1, 2, 2.5, 3, 3.5 and 4 per cent. After cooling the oil was filtered.3.4.6 DeodorizationThe bleached rice bran oil was deodorized according to the method described by Dudrow (1983) using flash evaporator. One hundred ml of oil was taken in a flask and heated to 240C with an absolute pressure of 4 mm Hg. The steam was passed to it for a residence time of 10 minutes. The oil was then cooled.3.5 Physico-chemical analysis3.5.1 Rice branRice bran samples were analysed for proximate composition.3.5.1.1 Moisture contentMoisture content was determined by AOAC (1984) procedure. The sample (10 g) was weighed accurately and transferred to a cleaned, dried and weighed aluminum moisture dish. The contents were dried at 102C until a constant weight. After cooling, the loss in weight was taken as moisture content and expressed in terms of percentage.Moisture %- Wdght f Sample ~Weight of dried samPles .:100Weight of sample3.5.1.2 ProteinProtein estimation was done by Macro-Kjeldhal Method (AOAC, 1984). Accurately weighed 2 g rice bran sample was transferred to a digestion flask followed by addition of 3 g of digestion mixture (K2 SO4 : CuS04 : Se02 in 100:20:5 ratio) and 40 ml of concentrated sulphuric acid. The contents were then digested till a bluish-green transparent liquid was obtained. The contents were then transferred to a 100 ml volumetric flask and the volume was made up to 100 ml using glass distilled water. A 10 ml aliquot of this solution was distilled with excess of 40 per cent sodium hydroxide and the liberated ammonia was collected in 30 ml of 4 per cent boric acid solution containing 2-3 drops of mixed indicator (10 ml of 0.1% bromocresol green + 2 ml of 0.1% methyl red + 88 ml95% ethanol). The entrapped ammonia was liberated against 0.1 N hydrochloric acid. A blank was simultaneously digested and distilled. Nitrogen content in the sample was calculated as follows:/ Nitrogens (sample titre-blank tite)xNormalityof Hcl x Volumanadeupof digest^ ^1 OOQx Weightof sampl^g)x Aliquotof digest taknfordistillatira% Protein = % Nitrogen x 5.713.5.1.3 FatThe fat content in rice bran was determined by Soxhlet Method (AOAC, 1984). Accurately weighed 10 g rice bran dried at 102C for 3 hours was transferred to a thimble. The thimble was placed in a soxhlet apparatus and fat was extracted for 8-10 hours with petroleum ether (B.P. 60/80C). The ether was then evaporated and collected. The extracted fat was dried at 100C and weighed. The fat per cent was calculated by the following expression._Weight of ether extract ...Fat % =-x 100Weight of the sample3.5.1.3 AshThe ash content in the sample was determined according to AOAC (1984) method. A 10 g sample of rice bran was weighed in a silica dish, charred and ignited on the flame till the smoke stops. The silics dish was kept in muffle furnace at 550C until carbon free ash was obtained. The dish was then cooled in desiccator and weighed.Ash%=WeightfthereSidU-ex100Weight of the sample3.5.1.4 CarbohydrateThe sum total of per cent moisture, protein, fat and ash contents were subtracted from hundred to obtain carbohydrate per cent in the rice bran sample by difference.3.5.1.5 Crude fiberThe crude fiber content was determined by AOAC (1984) method. The residue (2 g) obtained from fat determination was transferred to a 500ml digestion flask and 200 ml of boiling 1.25 per cent sulphuric acid solution was added. The contents were refluxed for exactly 30 minutes and then filtered through linen cloth placed over a funnel. The residue was washed with hot water until acid free. The residue was again transferred to the digestion flask and 200 ml of boiling 1.25 per cent sodium hydroxide solution was added. The content were again refluxed for 30 minutes, filtered through linen cloth and washed with hot water until alkali free. Finally, the residue was washed with 15 ml of ethyl alcohol and dried in oven at 110C until constant weight. The residue was then ignited in a muffle furnace at 550C until carbon free. The loss in weight represents crude fibre.Weight of residue Weight of^ j r-u o/ after digestionashCrude fiber %x 100Weight of the sample3.5.2 Rice bran oilRice bran oil samples were analyzed for physico-chemical characteristics using following methods.3.5.2.1 Specific gravityThe specific gravity of rice bran oil was determined by Pycnometer methods as described by Rangana (1991). Pycnometer was cleaned, dried to constant weight and filled with recently boiled and cooled distilled water.Pycnometer was stoppered, kept in water both at 30C for 30 minutes and weighed after removing surface moisture. The same Pycnometer was filled with oil after removing water and drying. The Pycnometer was stoppered and kept in water both at 30C for 30 minutes and weighed after removing surface water. The specific gravity was calculated by following formula:Specific gravity at 30C = 2*1Weight of water3.5.2.2 ViscosityThe viscosity of rice bran oil was determined by the method described byRangana (1991) using Brook field Synchrolectric Viscometer (LVT) model. Rice bran oil (100 ml) was taken in a 150 ml beaker and spindle No. 2 was immersed into it upto the desired depth. The spindle was rotated and dial reading was noted at different speeds. The viscosity was calculated by following formula.Viscosity (centipoises) = Dial reading x Conversion factorThe conversion factors for spindle No. 2 at different rpm are as follows:Speed (rpm) 6 12 30 60

50

51

Conversion factor 50 25 10 53.5.2.3 Refractive indexRefractive index of rice bran oil was measured following the method described by Rangana (1991) using Butyrorefractometer. 2-3 drops of rice bran oil was placed on the lower prism of Butyrorefractometer and the prism was closed and tightened with the screw.The refractometer was allowed to attain the desired temperature of 40C by passing boiled distilled water through it. Butyrorefractometer, reading was noted and refractive index was calculated using conversion table or following expression.R = R-K(T-T)Where,R = The reading reduced to temperature T,R = Reading at TT = The standard temperatureT = Temperature at which reading R was taken3.5.2.4 Melting pointMelting point of rice bran oil was determined by the method of Lees (1975) using Toshniwal melting point apparatus. A fine glass capillary was filled with rice bran oil and kept in ice after closing one end using paraffein wax. The solidified oil capillary and thermometer was placed in the melting point apparatus. The bulb was switched on and the temperature was slowly raised till the sampleof oil in the capillary become clear. This temperature is referred as melting pointof oil.3.5.2.5 Smoke pointThe smoke point of rice bran oil was determined by the methods of AOCS (1964). On hundred gram of oil was taken in a clean and dried 250 ml beaker and heated till the smoke began to come out of the sample. The temperature of oil at this point was recorded as smoke point.3.5.2.6 ColourThe colour of the oil samples was measured as per cent transmittance by the method of AOCS (1964) using spectronic-20. A mixture of 20 per cent oil in acetone was filled in the Curette at 30C and per cent transmittance was measured at 570 nm using acetone as blank.3.5.2.7 Iodine valueThe iodine value of rice bran oil was estimated according to the method described by AOCS (1964) one gram of filtered oil was taken in a 250 ml stoppered conical flask, to which 10 ml of carbon tetrachloride and 25 ml of wijs solution was added. Mixed and stored in dark for 30 minutes. Then 15 ml of 10 per cent potassium iodine solution and 100 ml distilled water were added. After thorough mixing, contents were titrated with 0.1 N sodium thiosulphate using starch as an indicator at the end points. A blank was also run simultaneously., ,. , (Blank titre (ml) - Sample titre (ml) )x Normality of sodium thiosulphateIodine value = -.;* luu

weight of sample (g)The saponification value was estimated according to AOCS (1964) method. One gram of oil was taken in a 250 ml conical flask to which 25 ml of 0.5 N alcoholic potassium hydroxide solution and few pieces of glass beads were added. The contents were refluxed on a water bath for 45 minutes using air condenser and then cooled. It was titrated with 0.1 N hydrochloric acid using phenolphthalein as indicator. A blank was also run in similar way./, , . , ,s , . , * Normality of alcoholic potassium . (Blank titre(ml)-Sampletitre(ml) )x Saponificationhydroxide=x5601valueweight of sample (g)3.5.2.9 Acid value and free fatty acidThe acid value and free fatty acids were determined by AOAC (1964) methods. Five gram rice bran oil was taken in a 250 ml conical flask to which 50 ml of hot neutralized ethanol was added and titrated with 0.1 N sodium hydroxide using phenolphthalein as indicator. The solution was kept warm and shaken vigorously during titration. Acid value and free fatty acids were calculated as follows:...cNormality of sodiumVolume of titre ( ml) x , ' .,Acid value =hydroxidex ^ Q1(as oleic acid)weight of sample (g)% Free fatty acids = Acid value x 0.503The unsaponifiable matter in rice bran oil was estimated using AOCS (1964) method. Five gram of sample of rice gram oil was taken into a reflux flask and saponified by boiling under the reflux with 50 ml of 0.5 N alcoholic potassium hydroxide solution for one hour. The condenser was washed with 10 ml of ethylalcohol, cooled and contents were transferred quantitatively to a 250 ml separating funnel using 50 ml ethyl ether. The lower layer was transferred to another separating funnel. Extraction was repeated thrice. The ether extracts were combined in a separating funnel and washed thrice with 25 ml portions of 0.5 N aqueous potassium hydroxide and twice with distilled water. The ether layer was then transferred to tarred flask containing few pieces of pumice stone and evaporate dryness on a water both followed by drying in a hot air oven at 80C for one hour. After weighing the residue was dissolved in 50 ml neutral ethanol and titrated with 0.1 N sodium hydroxide using phenolphthalein as indicator.The unsaponifiable matter in oil was calculated as follows:% Fatty acid content ml of sodium Normality in the ether extract = hydroxide used x of sodium x 0.282 (as oleic acid)in titrati on hydroxidegm% Unsaponifiable Weight of residue (g) - Weight of fatty acids residue (g) ,,|qq matterWeight of samaple (g)3.5.2.11 Peroxide valueThe peroxide value was estimated according to the AOCS (1964) method. One gram of rice bran oil was weighed into a conical flask, to which 25 ml of the peroxide solvent glacial acetic acid + chloroform (2:1) and 1 ml of saturated potassium iodide solution were added. The contents were mixed by shaking and allowed to stand for one minute. After adding 35ml of water, the liberated iodine was titrated with 0.1 N sodium thiosulphate using starch as an indicator. A blank was also run simultaneously and the peroxide value was calculated as follows:,(Sample titre (ml) - blank titre (ml)) x Nonmlity of sodiumPeroxide value_thiasulphate(milli equivalent per 1000 g of oil)weight of sample(g)X3.5.2.12 Thiobarbituric acid test (TBATest)3.5.2.8 Saponification value

3.5.2.10 Unsaponifiable matter

The T.B.A. Test was conducted by the method described by Rangana (1991). One gram rice bran oil was dissolved in 10 ml of carbon tetrachloride, to which 10 ml of TBA reagent (1 gm TBA dissolved in 75 ml of 0.1 N sodium hydroxide and volume made up to 100 ml with distilled water) was added and shaked thoroughly for 4 minutes. The contents were transferred to a 50 ml separating funnel. The aqueous lower layer was collected in a test tube and immersed in boiling water bath for 30 minutes. After cooling, the optical density was measured at 530 nm using spectronic-20 and 0.1 N sodium hydroxide as blank.Fatty acid composition of rice bran oil was determined by method of Gupta and Sand (1993) using high pressure liquid chromatography (HPLC).The methyl esters were prepared according to the method described by Ackman (1967). One gram rice bran oil was taken into a 250ml refluxing flask to which 25 ml of 3 per cent H2SO4 in methanol (v/v) was added and refluxed for 3 hours. After neutralization with saturated sodium bicarbonate solution, the contents were transferred into a 250 ml separating funnel and 50 ml of hexane was added. The content was shaked well and allowed to stand for 20 minutes. The lower layer was removed and upper organic layer was washed with 10 ml hot distilled water. The organic layer was collected, one gram anhydrous sodium sulphate added and filtered. The filtrate was dried in the presence of nitrogen and dissolved in 2 ml acetonitrite.The fatty acid composition was determined by Beckman model 322 HPLC equipped with model 100 A pump for 20 [i processor controller, mixer, 210 injector, model 156 refractive index detector and Kitts-zonen B, D-40 recorder. A stainless steel column (4.5 mm internal diameter x 150 mm) packed with C-18 (5 H silica) bonded with acetyl, deacylsilica was used. A mobile phase 100 per cent acitonitrile at constant flow rate of 1.5 ml/minute was used. The detector response was recorded on a 100 milli volt recorderThe sample were loaded on to the column with the help of a Hemiton \i\ syringe fitted with a 22 gauge and type needle. Standard methyl esters of fatty acids obtained from Sigma Chemical Company, USA were used for identification of the peaks. Per cent fatty acids content in the sample were calculated by using the following expression given by Murthi and Deudhara (1988).Peakheight concentralon of Volume of Final volume XXxpatt ac jd _ f sample standard([ig/|al) standard injected(jal) of sample (ml) ^ ^Peakheight weight of Volume of sample xxof standard sample (g) injected(|il)3.5.,2.14 Wax estimationThe wax content of rice bran oil was estimated by the method of MOrrison (1982) with some modification using colorimeter instead of turbidity meter. The optical density of the oil at 30%, 40% and 50% concentration level in chloroform was measured at 570 nm at 4C using chloroform as blank. The standard curve of optical density versus wax concentration was prepared using 2 to 10 per cent carnauba wax in chloroform solution using chloroform as blank at 4C (Fig. g.2). The optical density of oil sample was matched with the standard curve and wax content of oil was calculated.3.5.2.15 PhospholipidsAshing of oil for the determination of total phospholipids was done by thp method of AOCA (1964). Rice bran oil (5 g) was taken in a silica dish to which 0.5 g zinc oxide was added and heated slowly till the sample thickened followed by complete carrying at high temperature. The silica dish was then placed for ashing in a muffle furnace at 550C for 2 hours, cooled and boiled for 5 minutes after adding 5 ml distilled water and 5 ml hydrochloric acid to the silica dish ThA

0.2 0.4 0.6 0.811.2 1.4 1.6Wax (g)Fig. 3.2: Standard curve for wax estimation

0.3Phosphorus (mg)Fig. 3.3: Standard curve for phosphorus estimation

3.5.2.13 Fatty acid composition

b 9

solution was filtered into a 100 ml volumetric flask, cooled and neutralized by adding 50 per cent potassium hydroxide solution.Hydrochloric acid was added to it until the zinc precipitate was dissolved. Volume was made upto 100 ml with distilled water and mixed.Phosphorus content in the ash solution was estimated according to the procedure described by Rangana (1991), 5 ml of ash solution was taken in a 50 ml volumetric flask to which 5 ml of 0.5 per cent ammonium molybdate solution and 2 ml of 0.2 per cent amino nephthol sulphuric acid was added. The volume made upto 50 ml with distilled water, mixed and allowed to stand for 10 minutes. The colour was measured at 650 nm using spectronic-20.A standard was also prepared by dissolving 0.439 g potassium dihydrogen phosphate and 10 ml of 10 N sulphuric acid in distilled water and the volume was made to one litre. 5 ml of this was treated in the same way as sample (Fig. 3.3).The phosphorus content was calculated as follows:mg of phosphorus Total volume ofN in digest of ash ash solution (ml)Phosphorus (mg /100g) =-, -{ x 100yjjr . * /* , Solution taken forWeight of sample x , ,*analysis (ml)Phospholipids mg/100 g = Phosphorus x 303.6 Sensory analysis3.6.1 Preparation of potato chips

#

61

Potatoes were peeled, sliced (1.25 mm thickness) and dipped in a 2 per cent brine solution to avoid browning. The slices were then dried at 60C for 4 hours in order to remove surface moisture prior to frying. A batch of 50 g potato slices were fried at 180C 2C in deep fat frier for 2 minutes. The oil was drained from the chips. The chips were then cooled, packaged in high density polyethylene bags and stored at room temperature.3.6.2 Sensory evaluationThe sensory evaluation of potato chips fried in refined rice bran oils, refined groundnut oil, Dhara vegetable oil was carried out by a taste panel consisting of 10 panelists. The panelists were asked to evaluate the potato chips for the sensory attributes namely colour and appearance, taste and flavour, mouth feel and overall acceptability on following 9 point Hedonic Scale.Like extremely9Dislike slightly4

Like very much8Dislike moderately3

Like moderately7Dislike very much2

Like slightly6Dislike extremely1

Neither like nor dislike5

2.7 Determination of frying characteristicsFrying characteristics of rice bran oil were determined by frying 50 g of potato chips in 100 g of oil at 1802C. The amounts of oil recovered and absorbed were noted. The refined groundnut oil and vegetable oil (Dhara) were taken as reference oils. All the oil samples were subjected to physico-chemical analysis namely free fatty acids content, peroxide value, viscosity and refractive index before and after frying.The fried potato chips in all the types of oils were subjected to sensory analysis on 9 point Hedonic scale. The potato chips were evaluated for colour and appearance, taste and flavour, mouth feel and over all acceptability.3.8 Storage studiesStorage stability of rice bran oil was determined following the method described by Ohlson and Svensson (1976). Refined rice bran oil and crude rice bran oil (200 ml each) were taken into 500 ml beakers and kept open at 60C for 120 hours. The samples were analyzed for rancidity, colour intensity, free fatty acid content TBA value and peroxide value at an interval of 12 hours.2.9 Statistical analysisStatistical procedures as described by Snedecor and Cochran (1977) were used to analyze the data for the interpretation of results. Analysis of variance (ANOVA), split plot design in completely randomized design (CRD) were used for analysis of results.