vol. 04: december, 2016 issn 2222-1247 doi: 10.24081...

Enzymatic Treatment of Rice bran to Improve Extraction of Oil

1Abdul Mateen Khan*, 2 2M. Shahzad Zafar, 1Shahid Raza Malik, Faisal Rehman 1NFC institute of Engineering and Fertilizer Research Faisalabad, Pakistan

2Department of Chemical and Polymer Engineering, Faisalabad Campus, University of Engineering and Technology Lahore, Faisalabad, Pakistan

*Corresponding Author: [email protected]

Abstract—A modification in the process involving oil extraction from rice bran is presented, this is to introduce multiple enzymatic reactions prior to solvent extraction. Thermal treatment is applied to the rice bran for the deactivation of lipase. At first the oil extraction was carried out with solvent extraction technique only by using a solvent mixture composed of 50% acetone and 50% ethanol. The oil extraction yield was found to be 80.502 %. In another process the rice bran was first treated with a combination of enzymes comprising of Protease, Cellulase, Hemi-cellulose & α-Amylase and then after enzymatic activity the dried rice bran was subjected to solvent extraction using 50% acetone and 50%. It was found that extraction got an increase of 6.51% when rice bran is treated with enzymes prior to solvent extraction in such a way that the enzymatic concentration was maintained as 3% by weight of rice bran, contact time for enzymatic reaction as 4hrs and keeping moisture to rice bran ratio as 7 during the enzymatic activity.

Index Terms—Protease, Cellulase, Hemi-Cellulase, α-Amylase

I. INTRODUCTION

Rice bran is a byproduct formed during the milling process of rice which involves the removal of upper brown layer from the brown rice. After milling the rice bran gets separated from the rice surface and we get white rice. Due to its unique properties, rice bran is considered as a very good source of antioxidants and minerals [1]. Different factors affects the composition of rice bran like climate, soil composition, precipitation, type of crop and the rice processing procedures adopted [2]. On an average, rice bran may be composed of 12 to 25% oil, which can fulfill some portion of oil needs of Pakistan, which is an agricultural country. Oil extracted from rice bran find its utilization both in edible and nonedible industries [3]. If we see the edible application, it finds it use in pharmaceutical industry as an ingredient for making medicines and as vegetable oil [4]. While, it is also used as ingredient in cosmetics and soap industry as a non-edible application [5].

There has been various methods remained in service for extraction of oil from rice bran. If we see the mechanical methods, it involves the pressing of rice bran and the physical methods involve the utilization of solid-liquid extraction technique [6]. Other method involve the use of solvents like Subcritical water and subcritical carbon dioxide [7]. Hexane has been the most attractive solvent to be used in solid liquid extraction method when it comes the extraction of rice bran oil to get maximum yield [8]. But, due to its hazardous nature, as

reported recently [9], hexane is finding limited utilization as a solvent. This has created an opportunity for the researchers to look for other environmental friendly methods to extract oil from rice bran.

Presence of discrete cellular organelles is a unique property of the cotyledon of oil seeds. These cellular organelles which are basically proteins and lipids in nature occupies almost all the oil and protein in it. If we see the cell wall composition, it is made up of cellulose, hemi-cellulose, lignin and pectin. Further, the lipoprotein layer envelops over the lips bodies. In order to break the cell wall network, hydrolysis by the use of enzymes like cellulases, hemicellulases, and pectinases are proved to be useful. Further, protease can be used to break the protein layer. This breakdown of cell wall and protein layers helps releasing all the oil present in it, which otherwise remains in a stabilized condition and a limited extraction could be possible [12],[27]. It is also important to note that protein layers near the cell wall also plays very significant role in stabilizing cell wall. Hence, a combination of photolytic enzymes along with other enzymes selected according to the cell wall properties can be used to breakdown the cytoplasmic network that is mainly composed of protein and also the cell wall surrounding the oil [28]. This will cause the oil to become more exposed for extraction. After the enzymatic step the oil can easily be recovered by using solvent extraction method in which solvent other than hexane can be used.

It’s been long since the of utilization of enzymes in food processing is understood, with an aim to improve the product yield along with reducing the by-products, and to get escape from operational conditions that may be sever to practice. It is reviewed recently that enzymes have been useful in extracting oil and protein many oil containing seeds [10-12]. By using this method, oil from oleaginous seed like coconut [14-17], avocado [13], corn germ [18], rapeseed [19], soya bean [20, 21] and sunflower. [20, 22-24] It was found that the oil yieldsgot an increase when rice bran was treated with Cellulase andpectinase and then subjected to extraction with n-hexane [25].However, the utilization of enzymatic treatment alone for theextraction of oil did not result in high yields [26].

In present study, effect of treatment with enzymes like Protease, Cellulase, Hemi-cellulose & α-Amylase on rice bran oil extraction was studied. Also the complete process parameters like enzyme concentration, incubation time &

VOL. 04: DECEMBER, 2016 ISSN 2222-1247

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DOI: 10.24081/nijesr.2016.1.0003

solvent to rice bran ratio for getting maximum yield were studied. The effect of the parameters on oil yield is presented and discussed in the sections to follow. Further the quality of oil extracted was analyzed by estimating the fatty acid composition, saponification number and Acid number.

II. MATERIALS AND METHODS

A. Materials

For the purpose of oil extraction, rice bran was collected from Iqbal Rice mill, near Chiniot, Pakistan. Reagents like Ethanol and Acetone were Laboratory grade 99% pure and were purchased from Merck, Darmstadt Germany. A solvent mixture comprising 50% Ethanol and 50% Acetone (v/v) was prepared. Enzymes were provided by Institute of Biotechnology and Genetic Engineering, Faisalabad, Pakistan, as free samples with the proteolytic activity expressed IU/ml in Table I.

B. Equipment:

All enzymatic reactions and oil extraction were carried out with incubator Shaker, Bibby Scientific Ltd UK, Model Sl600C (sturat®), having provision of temperature and RPM control of. After enzymatic treatment & oil extraction the oil was separated from solvent with laboratory scale batch distillation apparatus [29].

C. Feed Preparation

Foreign material, like paddy kernels, broken grains and the pigments of hulls were removed by screening it through 100 mesh Tyler series [29]. After screening the rice bran was subjected to stabilization which is necessary to deactivate the activity of an enzyme called lipase which otherwise, can convert the useful oil to free fatty acids [30]. Thus increasing the rancidity of oil. Stabilization was done by placing 500 grams of freshly screened rice bran in oven, Memmert Germany at 1500C for 10min [31].

TABLE I (ENZYME CONCENTRATION)

Enzyme Type Concentration (IU/ml)

Cellulase 10

Hemi- Cellulase 8

α-Amylase 100

Protease 35

The stabilized product was then placed at 0OC in

refrigerator until further use

D. Initial Feed Analysis

TABLE II (FEED ANALYSIS)

Component %age

Moisture Content 10.2

Crude Oil Content 22.27

Crude Protein 17.96

Ash Content 7.04

Carbohydrates & fibers 42.53

The feed analysis of rice bran is presented above in table

II. For analysis of moisture content, AOAC 930.15 method was used while ash contents were determined using AOAC 924.05. Oil & protein present in rice bran sample were determined by using Soxhlet extraction (AOAC 920.39) and Kjeldahl method (AOAC 988.05) respectively [32].

E. Solvent Extraction Process

For solvent extraction process, 200ml of a solvent mixture containing 50% Acetone and 50% Ethanol on volume basis was taken in the flask. The mixture was heated up to a temperature of 50OC for a time period of 30min. 40 grams rice bran was then added to the heated mixture to achieve a solvent to bran ratio as 7 (v/w) and is allowed to shake in an orbital shaker at 200 RPM for the next 50min with temperature maintained at 60OC and pH at 8.5. While heating the solvent, special care was taken while heating the mixture as flash point of acetone is very low. Fire extinguisher was placed during the experimentation to mitigate any unpleasant happening. Furthermore the heating was done in an explosion proof vessel. The mixture was then filtered through a filter paper to separate the fat free rice bran from the oil and solvent mixture.

After recovering the solvent and oil, the mixture was subjected to batch distillation in which the oil remains as residue and solvent gets distilled out.

The percentage of oil recovery was estimated by the following formula:

Percentage Oil Recovery = (Weight of oil extracted/Weight of oil in bran) * 100 [5]

F. Enzymatic Treatment Followed By Solvent Extraction:

The process starts by adding a calculated amount of water in rice bran sample to achieve the desired moisture to rice bran ratio. The mixture achieved is mixed well and then enzymes were added to the mixture as %age of weight of rice bran sample. The mixture is maintained at a temperature of 550C and pH of 6.5 as necessary for the enzymes to carryout activity. Citrate buffer was also added to the mixture to maintain pH during the enzymatic activity. After the enzyme reaction the temperature was raised and maintained at 800C to destroy the activity of enzymes [25]. The mixture was then centrifuged at 15000 RPM for 10min and the meal was


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recovered by removing the supernatant and filtering through filter paper (Whatman no. 1). The filter paper was placed in open air for 3hrs and then it was placed in a draft air oven at 600C. The recovered meal was allowed to dry in open air for the next 24hrs. The dried meal was then subjected to solvent extraction using 50% Ethanol and 50% Acetone (v/v) at the same conditions mentioned earlier to get the oil extracted. III. This procedure was repeated thrice by changing the

parameters like enzyme concentration, time and moisture to rice bran ratio. Enzyme concentration was changed as 1%, 2% and 3%. Incubation time to carry out the enzymatic was practiced as 2hrs, 4hrs and 6hrs. Moisture to rice bran content were varied as 5, 7 & 10. The results were discussed in the following section.

IV. RESULTS AND DISCUSSION

A. Effect of Enzyme Concentration on Extraction

Figure No. 01 demonstrates that if we increase the enzymatic concentration from 1% to 3% than the oil extraction increases. This is because of the fact that as the concentration of the enzyme increases in the solution there

Fig. 1 Effect of Enzyme Concentration on Extraction

appears to be more breakage of cell walls surrounding the oil due to better interaction. This facilitates to free more oil from the oil containing bodies.

There is a sudden increase in the oil extraction as the concentration of enzyme increases from 1% to 2% due to increase in potential of enzyme to cause more breakage. But, after 2% and until 3% the increase in oil extraction is very low, moreover in case of contact time 6hrs, the extraction even goes down a little bit. This is because a high enzymatic treatment may lead to that level where the oil starts flowing from the oil bodies to the solution, although this flow might be very low quantitatively but it will be lost in the solution as it cannot be recovered even centrifuging at high speed.

In the case of enzymatic treatment for 6hrs, the same thing happens as discussed earlier, the oil is lost in the enzymatic treatment step and there remains lesser oil to be recovered for the solvent extraction step.

It can also be observed that oil extraction is higher if the incubation time is selected as 4hrs. This is also due to the reason that at lower incubation time as 2hrs, the contact is not sufficient to carryout complete cell wall breakage to free oil bodies. But, at higher incubation time the contact is in such a way that oil extraction start happening in the first step which

actually should happen in the second step, i.e. solvent extraction.

B. Effect of Contact Time on Extraction:

Figure No. 02 demonstrates that contact time plays an important role in extraction. At low contact time the extraction of oil is low, this is because a certain time is required for the activity of enzyme to take place and to carryout cell wall rupture.

Fig. 2 Effect of Contact Time on Extraction

A higher value of contact time brings adverse effect on oil extraction. This is because after the enzyme is done with the cell wall breakage, the oil that is now in free condition and starts flowing from the oil containing bodies to solution and becomes part of it. Hence there remains lesser oil left for extraction in the next step.

As the enzymatic activity takes places in aqueous solution, at very low moisture content the paste is so thick that it not workable and it would be very hard for the enzyme to come in contact with each and every particle of rice bran. So a minimum moisture level was selected as 5 times the weight of rice bran so that the contact of enzyme could be easier.

It can be observed from the graph that by increasing the moisture contents from 5 ~ 7 time the oil extraction increases which is due to the ease of interaction of enzyme that facilitates more cell wall breakage. Now, if we continue increasing the moisture content of the system, this will bring adverse effect on oil extraction which is due to the fact that higher moisture contents will make the oil to come out of its pockets and get dissolved in it due to large concentration gradient.

C. Effect of Moisture to Rice Bran Ratio on Extraction:

Moisture is the most significant parameter that effects the oil recovery. This is because of the fact that enzyme acts with moisture and also more moisture present in the mixture provides more contact between rice bran and enzymes. We can see from figure No. 03 that when we increase the moisture to rice bran ratio from 5 to 7 then the oil gets increased. This happens because the contact between rice bran and enzymes increases due to increase in surface area and cell wall degradation increases which leads to better recovery of oil in the second, solvent extraction step.

We further see that if the moisture content are increased further from 5 times to 7 then it brings adverse effect on oil


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extraction. That is, the extraction decreases. This happens because after the cell wall breakage the oil is not free to move from the rice bran into the water and if there may be higher quantities of moisture then the concentration gradient will drive the oil to move into water due to which the oil is lost.

As the lost oil is in very low quantity, its recovery even at ultracentrifuge is not favorable because during the enzymatic treatment step many other byproducts like sugars and protein

Fig. 3 Effect of Moisture to Rice Bran Ratio on Extraction

extract are also produced due to which the oil gets contaminated with these byproduct and recovery of such contaminated oil leaves us with no benefit.

So we have to limit the use the moisture in enzymatic treatment step to save oil from mixing in to water and to get a better recovery in the next step.

D. Oil Quality

Fatty acid contents of recovered oil was estimated through gas chromatography using Agilent 7890A with chemstation software and using N2 at 16 psi as carrier gas. The results were as under in table No. III.

Similar results have been reported recently [33]. Also the saponification number & acid number values were determined using titrimetric method [34, 35] as 180 & 23. These results were found comparable with findings of other researchers. [29]

TABLE III (OIL QUALITY)

Fatty Acid Composition

Palmitic Acid 21 %

Stearic acid 1.91%

Oelic Acid 40.23 %

Linolic Acid 36.86 %

V. CONCLUSION

Enzymatic treatment prior to solvent extraction can increase the yield of rice bran oil. Three significant parameters like enzymatic concentration, moisture to rice bran ratio and contact time increases the oil recovery An increase of 6.51% is experienced during experimentation if rice bran is treated

with enzymes prior to solvent extraction in such a way that the enzymatic concentration was maintained as 3% by weight of rice bran, contact time for enzymatic reaction as 4hrs and keeping moisture to rice bran ratio as 7 during the enzymatic activity.

E. Acknowledgment

The technical support of Institute of Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan is gratefully acknowledged.

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