10urnal of Scientific & Industrial Research Vol . 60, April 200 1 , pp 298-3 1 0

Using Enzymes for Oil Recovery from Edible Seeds

v C Kalia " Rashmi, Sadhana Lal Centre for Biochemical Technology (CSIR), Delhi University Campus, Mall Road, Delhi 1 1 0 007, India

Phone: 9 1 - 1 1 - 725 72 98; 725 61 56, Fax: 9 1 - 1 1 - 725 74 7 1 and 74 1 64 89; e-mail : vckal ia@cht.res. in and

M N Gupta Department of Chemistry, Indian Institute of Technology, Hauz Khas, New Delhi 1 1 0 0 1 6, India

Oilseeds and their products are the most valuable agricultural crops in the world trade with, ever-increasing demand for oil from edible oilseeds all over the world. India accounts for 9.6 per cent of the world's oilseeds production. The demand for vegetable oils is increasing at 5 1akh t1y while the production is increasing at 2 1akh t1y only. The present demand over supply gap, in edible oils, is 1 . 8 million t needing to produce additional S.4 million t oilseeds/y. Hydraulic, and expeller pressing, and solvent extraction are the three most common processes for oil recovery from oil seeds. Enzyme based oilseed processing technologies emerge as one of the most eeo-friendly processing methods. The enzymes have specific mode of action, therefore, cellulase, hemieellulase and pectinase and even proteases are the most favourable enzymes for degrading the cell wall in oilseeds to loosen oil sacs embedded in the structures. The enzyme treatment has been found useful in conventional solvent extraction process also. Different factors like temperature, pH, moisture, grinding and size reduction of oi lseeds are required by enzymatic processes which intluence the efficiency of extraction, recovery of oi l, that also helps maintain higher nutritive value. The usage of en­zymes reduces environmental pollution with consequent reduction in BOD (Biological Oxygen Demand) and COD (Chemical Oxygen Demand) of the residues and wastewaters along with reduction in acid development and oxidation during further pro­cessing and storage. High cost and specificity of enzymes limit the enzyme usage for different oilseeds.

All over the world, an ever-increasing demand for oil from edible oilseeds is being witnessed. Since the be­ginning of history, people have made use of oils obtained from oilseeds and used principally as food. Oil consti­tutes a major portion of agricultural products . The sole objective is to increase productiv ity of oilseeds, in the wake of ever-increasing urbanization and industrializa­tion.

India accounts for 9.6 per cent of world's total oil­seeds production from over 25 mil lion ha of land, the largest area under oilseeds in the world. The average yield of oilseeds i s only 900 kg/ha against the world av­erage of 1 ,275 kg/ha and 2,500 kg/ha in USA.

Groundnut, rapeseed, mustard, castorseed, sesamum, nigerseed, linseed, safflower, sunflower and soybean are the major oil seeds produced in the country with ground­nut, rapeseed/mustard and soybean accounting for a major chunk of the output. The production of oil seeds in India rose dramatically from 1 08 lakh t in 1 985-86 to 2 1 5 1akh t in 1 993-94 with a target of 270 lakh t in 1 998-99. The growth in oilseeds was 1 2 per cent per annum during 1 985-86 to 1 993-94 which dropped to just three

* Author for correspondance

per cent per annum during 1 997-98.

The demand for vegetable oils is increasing at five lakh tly while the production is increasing at two lakh t/ y only. In the last six years, a demand over supply gap of 1 8 lakh t has been created and the gap stands at 1 . 8 mil­lion tones at present. In order to meet the gap, additional 5 .4 mil lion t/y oi lseeds are required ' .

The ever-increasing demand, the gap o f demand over production, and increased production of oilseeds would necessitate a critical examination of look into oil extrac­tion and processing technologies . It entails adoption of most modem technologies in extraction and processing of oils and also providing necessary technological in­puts to the existing extraction and processing plants. Extraction technology, e g, alternative solvents for ex­traction of oil including supercritical fluids (CO ), the enzymes assisted aqueous extraction and membran� tech­nology for upgradation of protein quality, are the tech­nologies that have a scope in the future2•

According to oil technologists all around the world, it is clear that more land wil l not be available for oi lseed crop production. On the other hand, more pressure can­not be put on agricultural biotechnologists and scien-

KAllA et al. : USING EMZYMES FOR OIL RECOVERY FROM EDIBLE OIL SEEDS 299

Table I - Enzymatic hydrolysis of oil Seeds for enhancing oil extraction.

Sl .no. I .

Material a Melon seeds') b Ground soyabean c Rape seeds

Effect of hydrolysis (i) Extra oil released (ii) Better oil quality

2. a Crushed Soyabeanlt' b Cotton Seeds

Enhanced release of extractable oil

c Castor Bean

3 . Canol a Flakesl1 (i) Enhanced release of extractable oil (ii) Reduction in oil extraction time

4. Soyabean'K (i) Enhanced release of extractable oil (ii) Enhance oil recovery

5 . Groundnut'Y Enhance oil recovery

tists to increase crop yields. With rising values of oils, h igher demand for quality in oil , coupled with several years of unfavorable c limatic conditions in growing re­gions, there has been a noticeable increase in trials of enzyme based processing for a wider range of oilseeds. Enzyme based technologies have emerged as one of the most eco- friendly, in recent times .

A method for treatment of oi lseeds with enzymes for softening and increasing the porosity of oilseeds to en­hance the rate of oi l recovery, has to be developed to minimise residual oil in oi lseed cake and refining of high free fatty acid and dark colored oi l . Besides increasing the efficiency of extraction, the method is environment friendly, e g, a reduction of 75 per cent in BOD and 35-45 per cent in COD levels of wastewater were achieved after enzymatic treatment3. 4 . The article aims at review­ing the results of different extraction processes and then examine the potential of the eco-friendly approach for oil recovery from oilseeds.

Structure of Oilseeds For a better understanding of the possible role of en­

zymes, it is essential to consider the structure of oil-bear­ing materials . The oilseed cotyledon is the existence of discrete cellular organel les called l ipid and protein bod­ies, which contains, respectively, most of the oil and pro­tein in the grain . Protein bodies (aleurone grains) vary in size depending on the oilseeds, e g, the average size of the protein body is 8- I O !lm in soy and peanut5, and 2-20 !lm in other oi lseeds('·'J . The protein bodies contain 60-70 per cent of the protein in oi lseeds9. Lipid bodies

(also known as spherosomes and oleosomes) are the prin­c ipal repository sites of l ipid reserves in oi l seeds 10. I I

which are 1 -2 !lm in most oilseeds5. 9. 10, although varia­tion of 0.2-0.5 !lm in soybean9• 12 to as large as 4 !lm in cotton seeds" have been observed.

Scanning electron microscopy (SEM) showed that l ipid bodies of soybeansl4 and peanuts5 are enmeshed in a kind of cytoplasmic network presumably composed of proteinsl 4. The spaces between protein bodies in cotyle­don cel ls are filled with l ipid body and cytoplasmic net­work.5• 1 2 The walls, which surround the cells, are prima­rily composed of cel lulose, hemi-cellulose and l ignin in addition to pectin'!. Oil i s usually inside of vegetative cel ls, l inked with other macromolecules, such that par­tial hydrolysis of structural polysaccharide, the constitu­ent of the cell wall and lipid body membrane of oilseeds lS­

I '! (Table 1 ) by enzymes makes the cell structure more permeable (Figure 1 ) .

Oil Extraction Processes

There are many processes to extract oil w ith their as­sociated merits and demerits (Table 2). H istorically, the three most common processes fot recovering oil from oilseeds are hydraulic pressing, expeller pressing and solvent extraction20. 2 1 .

Hydraulic Pressing

Hydraulic pressing is the earliest process, which origi­nated in Europe2 1 • It is simple in operation, the ground seed material is placed in layers, with each layer sepa­rated from the other by a cloth. Pressure is applied, slowly

300 1 SCI IND RES VOL 60 APRIL 200 I

Table: 2 - Methods used in Oil Extraction

Process of extraction Advantages Disadvantages

Physical grinding Conventional and easy operation Oil remain in the cake

Aqueous extraction Environmentally cleaner technology Low in yield

Solvent extraction Easy and instant High solvent cost Uneconomical and Environment hazardous

CELL WALL

LIPID BODIES

PROTEIN GRAINS

ENZVMA TIC HYDROLYSIS J

�:i;���-- HYDROLYZED CEU

Figure I -Enzymatic hydrolysis of oilseeds wall

at first, and then increased as the oil remaining in the tissue decreases. A maximum total pressure of 2000 psi is generally applied requiring one h for the extraction of oil . As the hydraul ic pressing was labor intensive and slow in processing large volumes of oilseeds, continu­ous physical extraction methods were developed, viz the French screw press and Anderson expeller. These de­vices function effectively for oilseeds containing large large fraction of oil , 30-50 per cent, such as in corn germ and sunflower seed, but are not effective for oilseed such as soybeans having only 20 per cent oil . Pressing or ex­pelling may also be used before and in conjunction with solvent extraction techniques. However, being labor in­tensive, its use declined over the years and is no longer in use. Continuous screw presses such as expeller re­placed the hydraulic equipment22 .

Expeller Pressing The process is used for oilseeds containing more than

35 per cent oil, e g, groundnut, flaxseed, safflower and sunflower. A two-step process is carried out which com­prises of pre-pressing, that allows solvent extraction to

be applied to oleaginous materials, which would be quite difficult to process by direct extraction methods. Sol­vent requirement is also reduced for soybeans, where a single solvent extraction is sufficient, due to lower oil content. An expeller can exert much greater pressure on the seedcake than a hydraulic press. The increased pres­sure permits recovery of a larger proportion of the oil present in oilseeds. Generally, 3-4 per cent oil is left in the cake when extracted with an expeller, compared to 4-6 per cent with a hydraulic press. Expellers vary in size, from machines that process 1 00 kg to more than 1 0 t oilseed / h . Solvent Extraction

Solvent extraction of oil was a batch process in Eu­rope in late n ineteenth century. Technological advances after World War I led to the development of continuous solvent extraction systems which proved to be excellent in processing oleaginous materials, including low oil content seeds2 1 . The modern solvent based extraction process consists of successive counter current wash ex­traction with hexane, of the previously cracked, flaked, ground or pressed oleaginous material. Hexane is then removed, from oil, by evaporation and finally by vacuum distillation23 (Figure 2).

Properties of Solvent Used in Oil Extraction

Nearly all known oilseed extraction plants use hex­ane as the solvent, however, the industry is constantly looking for ideal solvent, one with high solubility at el­evated temperatures and low solubil i ty at ambient tem­perature which can be easily recovered from meal and oil , is plentiful , economical and finally that entai ls low energy costs, with high product yield, purity and market value. Solvent stabil ity is the single most desired qual­ity and it should not react with equipment.

Solvents, generally used in oil seed extraction, are hydrocarbon naphthas, trichloro ethylene, ethanol and ethanol-benzene. Recently the research focused on etha-

KALlA e/ at. : USING EMZYMES FOR OIL RECOVERY FROM EDIBLE OIL SEEDS 301

Ollseeos

I GRlND1NG I

ENZYMATIC HYDROLYSIS

SOL VENT EXTRACTION

r SOLVENT + OIL]

r DISTILLATION 1 .,

1 SOLVENT

Figure 2-Steps involved in enzymatic treatment and solvent extraction combination process for oil extraction from oilseeds

nol, isopropanol, methylene chlorides, acetone and hex­ane. However, in the last few decades, the l ight paraf­finic petroleum fractions such as hexane, heptanes and pentanes were the most common solvents used. Hexane became the choice solvent as it evaporates easily and leaves no residual obnoxious odors or taste. Later the decision was further supported by Eaves and cowork­ers24 who investigated the extraction of cottonseed by five commercial solvents, viz, hexane, benzene, ethyl ether, acetone and butanone, and found that none com­pared favorably w ith hexane for the oil extraction. Sol­vent extraction is routinely used to extract oil from soy­bean, canola, sunflower, and other oilseeds25.

Physico-chemical Methods

Elevated gas pressure is employed for transferring oil from particulate solids (oilseeds) to a transfer l iquid (water) with the help of supercritical fluid (CO ) which is used as a solvent for the displacement26. Physicfil crack­ing of hul l and beans is done as a pretreatment by heat­ing to 60°C in a microwave dryer prior to oi l extrac­tion27. Screw pressing is a physical force employed on vegetable oilseed, as a primary step prior to extraction in a common worm shaft2H. 2Y rotating within a perfo­rated barrel wall section and a non-perforated barrel wal l section connected by an annular membrane with water injection nozzles, and then meal enters at inlet and oi l is extracted through the wall . Other physico-chemical meth­ods are used to increase porosity of oleaginous material such as rice bran, wheat mil l feed, rapeseed, amaranth and other simi lar materials by stabil izing oil contained in them30. Whereas removal of hull from oilseed by em­ploying upward flows of gases for extended period of time such that the oilseed maintained within the plenum" faci l itates crisping of hul l .

I n another method oilseeds were treated with counter current flow of CO , ethane, ethene and/or propane at 40- 1 1 0°C at pressur�s of 250-750 bar for 0.5-2.5 h32 with solvent ratios of 5-30 kg solvent/kg cake. Among the chemical methods, 2-25 per cent acetic acid (v/v) was used along with hexane to prepare glanded cottonseed" for oil extraction by a solvent. As acetic acid in solvent increased, the concentration of total l ipids, phosphol ip­ids and neutral oil in miscella also increased. The solu­bi lity of protein in 0.02 N NaOH did not decrease unti l the acetic acid used to prepare the meal increased to 4-1 0 per cent.

The major l imitation of the conventional extrusion­expell ing methods is the large residual o i l left in the cake after extraction. Using hexane for soaking oilseeds, as a treatment, and subsequent recovery of hexane from the miscella (hexane and oi l mixture) and from the marc (residue left after oi l extraction) are both energy inten­sive processes that require extensive capital equipment.

Oil extraction by conventional mechanical methods do not ensure complete recovery of oip4 and the cake residues still contain 6- 1 0 per cent oil . It leads to recur­ring loss of oil , running into mill ions of tones/yo Oil ex­traction by different methods l ike soxhlet, automated soxhlet and sonication, microwave, SFE, ASE (acceler­ated solvent extraction) consumed 1 5-500 mL solvent/ kg and average extraction time varied from 1 2 min to 48 h.

302 J SCI IND RES VOL 60 'APRIL 200 1

Table 3 - Parameters suitable for enzyme action on oil seeds.

Oilseed pH Temperature, "C

Rapeseed1H• 1Y 6.6 70

Soyabean4o. 41 4.5

Sunflower1K. 42 5.0 Room temperature

Peanut3K. 41-16 4- 1 0 60-65

Aqueous Enzymatic Oil Extraction (AEOE)

Aqueous extraction process (AEP) using water alone as the medium, was an alternative to solvent extraction of oil in 1 950s as a safer and cheaper method. AEP used a distinctive principle compared to the usual solvent extraction process based on the capacity of oil to dis­solve in and be extracted by a solvent. In AEP, as oil does not have high chemical affinity for the extraction medium, i e, water, therefore, there was no chemical potential for oil dissolution.

Extraction of oil by AEP attracted attention for sev­eral years as it was based more on the insolubil ity of oil in water than on the dissolution of oi 1'-'. The water soluble components of oil crops diffused in water rather than in oil , thereby releasing oil, previously bound in the origi­nal structure. The unit operations involved in AEP are :

(i) Size reduction (grinding) of seed/fruit material, (ii) Extraction, (iii) Solid-liquid separation, (iv) Separation of oil rich phase, (v) De-emulsification for further recovery of oil, and (vi) Drying to remove moisture.

Grinding was carried out with a pestle and mortar, and the ground meal was boiled in water, liberating oil, which floated on to the surface. The oil was then care­ful ly scooped out from the water surface, and dried to remove moisture36.

In the modern aqueous process, centrifuges have re­placed the gravity separation. For the application of AEP, different oilseeds require specific changes in conditions such as pH and temperature due to differences in chemi­cal composition and the physical s tructure of o i l seeds17· 41i (Table 3). It naturally makes sense to use all cell-wall

Enzyme Extracted oi l , per cent

Protease, a- I , 4 7 8 galacturonide acids

Proteolytic 86

Cellulase, a- I , 4 5 2 galacturonide acids

Protease, Cellulase 74-78

degrading enzymes to faci litate the release of oil . The approach has several advantages, some inherent in the AEP, and some arising out of the use of enzymes.

(i) It nearly eliminated use of organic solvents re­sulting in (a) savings on the cost of such sol­vents, and (b) possibly low investments in in­frastructure.

( i i ) S imultaneous recovery of oil and protein frac­tions. It was one of the early dri ving forces which motivated people to explore AEP.

(i i i) Quality of Oil-It is now well establ ished that use of AEOE resulted in oil composition, which was very d ifferent from the one, obtained by 'conventional ' means. A good illustrative ex­ample is the recent work by Ranalli et at. 47 on olive oils where the oil obtained by enzymatic process had (a) higher contents of phenolics, to­copherols, trans-2-hexanal and other volatile aromatics, (b) enhanced oxidative stability, (c) lower turbidity values, and (d) h igher ratios of 1 ,2 diglycerides/ l ,3 diglycerides, campesteroll stigmasterol and trans-2-hexanal/total aroma. Overall , better qual itative characteristics and higher oil yields were reported. 'Cytolase 0' (Gist-Brocades, France) was the enzyme used in the process that mostly possessed pectinase and cellulase activities.

(iv) Degumming may not be needed.

Phospholipids, especially phosphatides, in oils, cause 'gumminess ' . Lecithin and cephalin are common phos­pholipids found in edible oils . Soybean, corn, cotton­seed and rapeseed are some of the major oils , which con­tain significant levels of phosphatides. Degumming i s

KALlA el ai. : USING EMZYMES FOR OIL RECOVERY FROM EDIBLE OIL SEEDS 303

Table 4 - Enzymatic extraction for different oil bearing material

Seeds Use level, Enzymes per cent (w/w)

Rapeseeds2 0. 1 to 3 Pectinase, Cellulase Soybean41 0.2 Protease CoconutS) 0. 1 p-Glucanase, Pectinase,

a-Amylase and Protease Avocados5 1 .0 a-Amylase Sunflower)K 1 .5 (Ultrazyme) Cellulase,

a-Galacturnido-glicano hydrolase

Peanut)K 3 .0 Cellulase, Protease 1 .5 Cellulase, Protease, and

Ultrazymes

carried out for the fol lowing reasons4X : (i) To produce oil which during shipping and storage does not produce settled material, ( i i ) To recover phosphatides, ( i i i ) To remove emulsifying agents (i .e. phosphat ides and muci­laginous gums) that increase loss of neutral oil when the oil is alkal i refined, and (iv) In further oil processing, gumminess causes problems.

Some phosphatides are removed by water degumming, e g, phosphatidylcholine, phosphatidylethanolamine and phosphatidyl inositol present in soybean oil , hydrate with water49 and swell , and the gel thus formed, precipitates out from oil . Incidentally, thi s simple step forms the ba­sis of food grade lecithin with an estimated annual world­wide production of 1 00,000 t49.

Treatment with salt solution or dilute acid, l ike phos­phoric acid, was also used in degumming of oi l . The non-hydratable phospholipids, remaining i n the de­gummed oil , were generally removed during the refin­ing stage. Recently, membrane filtration was used for degumming of crude palm oi ]5() that reduced phospho­l ipids to less than 0.3 ppm, but membrane fouling re­mained the bottleneck in adopting membranes to most of such applications. The enzymax degumming process51 for soybean oi l used lecitase, theNovo Nordisk's trade name for phospholipase A , which cost less than two per cent of the sale price of oir. If al l soya oi l was processed using the enzymatic process, the market s ize for phos­pholipase A would be $ 1 50 mill ion that would make the enzyme 1narket for oi l processing, the largest food area outside of starch hydrolysis.

It should be possible to use other phosphol ipases l ike phospholipase A , Phospholipase C and Phospholipase D for degumming purpose.

Removal of phosphatides also removes bound iron with it and as such the oil obtained after enzymatic de­gumming is reported to be more stable. A new degum­ming process has been developed using a natural enzyme as a catalyst for phosphorus removal that can be used on all types and qualities of vegetable oils to reduce phos­phorus to less than 1 0 ppm and thus potentially treated before physical refin ing.

The use of enzymes, from 0.1 to 3 .0 per cent (w/w) in rapeseed52, soybean4 1 , coconut5" avacad054, sunflower3x and peanut40 resulted in h igher oil y ields in AEP (Table 4). Selected enzymes that include cellulase, pectinase, protease, �-glucanase and a-amylase, when used on dif­ferent types of oilseeds, resulted in h igher oi l extraction yields.

The Quality of Protein Residue

High temperatures involved in the conventional pro­cess are associated with decreased nutritional availabil­ity of some essential amino acids, e g, l ysine, due to Maillard reaction between free amino group in proteins and carbonyl group in reducing sugars, resulting in de­creased biological value of such proteins.

Ambient temperatures used in AEP avoided protein denaturation. 'Food grade' , instead of 'feed grade' , pro­tein residues were obtained after AEP as: (i) it either inactivated or removed anti nutritional factors present in oilseeds, (ii) it removed b itter, poisonous alkaloids from lupin seeds and non-gossipol pigments from cottonseed]5, ( i i i) it removed toxic goitrogenous sulfur compounds in the case of rapeseed2, ( iv) it also removed phytic acid and other toxins35, and (v) caffeic and chlorogenic acids which cause sunflower seed meal to turn dark green or brown, were effectively removed in the aqueous pro­cessss. Such polyphenols produced O-quinones, an ex­tremely reactive class of compounds. Chemistry of the reaction of O-quinones w ith proteins and its nutritional consequences has also been reported56-5x.

Factors Affecting Oil Yield

Conditions required for oil extraction varied accord­ing to the oilseed composition and structureS9. Factors which influence the efficiency of extraction include grinding, size reduction of oilseeds, pH, moisture, tem­perature, time, solid:water ratio, degree of agitations9. m and the number of extraction stages. Sometimes, just simple stirring was sufficient to obtain h igh yield as in the case of peanut40 and sunflower42.

304 J SCI IND RES VOL 60 APRIL 200 I

Grinding

Grinding has been considered a critical step in ex­traction process that d irectly affected oi I yieldsf>O· fi l . Smaller particle size allowed easier diffusion of water­soluble components leading to disintegration of the origi­nal structure and faci l itated oil release; and it enhanced enzyme diffusion rates.

The grinding operation may be carried out either wet or dry depending on the oilseed. The choice between wet or dry grinding was dependent on several factors, such as in itial moisture content, chemical composition and the physical structure of oilseed(,(). In case of materi­als with high moisture content, e g, coconut, wet grind­ing was more appropriate resulting in less then 5 per cent of oil in the residue after extraction. Thus avoiding additional intensive drying step before grinding. In case of material with low initial moisture content l ike pea­nuts, rapeseed and soybean, dry grinding was more suit­able. Excessive grinding favored cel l rupture and in­creased the efficiency of oil extraction, however, it also produced smaller o i l globules, which made de-emulsifi­cation more d ifficult. Insufficient grinding, on the other hand, resulted in loss of oil in residue(,()' 62 .

Size Reduction of Oilseeds

The extraction of oil from oilseeds, either by mechani­cal pressing or by solvent extraction proceeded more efficiently, if the seed was first flaked or ground. Opin­ion was divided whether grinding or flaking was more effective i n rupturing oil cells. Flaked oilseeds yielded a larger fraction of 'easily extractable' oil on treatment with solvents and a smaller fraction (usually 1 0-30 per cent of total oil) that was difficul t to extract22•

pH

Extraction pH varied with the oil-bearing material . The highest oil extraction yields general ly coincided with those for the highest protein yields. Therefore, the high­est oil extraction y ield occurred at pH values correspond­ing to maximum protein extractabi lity in aqueous sys­tem and the lowest y ield was obtained when the protein solubil i ty was also at its lowest, i e, around the isoelec­tric point. In the case of rapesed3x, 3Y, soybean40, 4 1 , sun­flowe�x, 42 and peanut3X, 43.46, the pH values correspond­ing to the least oil extraction, which resulted in the small­est proportion of oil free after the downstream separa­tion, step shown in Table 3 .

Moisture

Control of moisture was important for efficient press­ing. Moisture content varied with oilseed and method used for pressing. Moisture control was also important in solvent extraction. Generally, intermediate moisture levels (4-7 per cent) resulted in efficient extraction of oil from oilseeds. Low moisture levels usually resulted in lower efficiency due to the lower solubility of phos­phatides, while high moisture could cause swelling of proteins with subsequent reduction of flake porosity and solvent diffusion rate. As with pressing, moisture varied with the material being extracted, approximately 7- 1 0

per cent for cottonseed and 8- I 2 per cent for soybean22.

Temperature

It also showed considerable effect on oi l extraction yields, though some authors considered it important in the hot water floatation process, and they ensured that boiling water was used for the extraction . However, pro­longed boiling might not affect yield in certain oilseeds61 . The extraction temperature was not critical for protein recovery but important for oil extraction5Y. The maxi­mum oil recovery occurred between 40-60 °C (Table 3) .

Solid: Water Ratio

The highest possible solid:water ratios were desirable in the extraction step to obtain less stable emulsion and generate less effluent. However, to obtain the h ighest extraction rate and extraction yield, the use of large quan­tities of water was necessary. Recommended solid :water ratios3Y, 42-44. 59. 63, 64 are shown in Table 5 .

SWOT Analysis of Oil Extraction

SWOT (Strength , Weakness, Opportun it ies and Threats) analysis has been an excellent, and fast tool for exploring the possibi l ities in in itiating new programs. It has also been used as a decision making aid. The SWOT approach requires an internal survey to record strengths and weaknesses and an external survey to enlist threats and, opportun ities65. It i s a general tool, mostly used in the preliminary stages of decision-making and also as a precursor to strategic planning in a variety of appl ica­tions66. 67 .

Strengths and Weaknesses

These are internal (endogenous) characteristics that can be usually controlled or managed around and include

KALlA et ai. : USING EMZYMES FOR OIL RECOVERY FROM EDIBLE OIL SEEDS 305

Table 5 - Solid water ratio in extraction of oil from oil seeds

S No. Oil seeds Solid water ratio Oil yield*

Peanut4J. 44. 6J. 64 1 :5 to 1 : 1 2 89

2. SunflowerA2 1 : 1 0

3 . Soybean5� 1 : 1 2 86

4. RapeseedJ� I :2.5 to 1 :3 .5 90

*based on the extracted oil from the seed.

factors such as geographic resources, climate, financial resources and the tax environment, human resources and skill information system, research and development ca­pability, infrastructure/utilities, and the role of govern­ment and the regulatory environment.

Opportunities and Threats These refer to external factors that enhance economic

and human development. These factors include markets and trade, customer, industry trends, economic factors and technology.

The Strengths Scientific and production innovations were applied

which rapidly increased productivity in oilseeds. It would enable the industry to further expand and diversify its production base to capitalise on export market opportu­n ities . Further, the enzymatic process has many advan­tages :

( i ) It is eco-friendly, simple, and relatively inexpen­sive and produces l ittle waste.

(ii) There is no need for degumming as the process is carried out in an aqueous medium, and phos­pholipids are also separated from oil .

(iii) It removes undesirable constituents presents in oilseeds.

(iv) It helped refine free fatty acid (FFA) and dark colored oils.

(v) It reduces BOD and COD levels, acid develop­ment, and oxidation during further processing and storage.

(vi) The use of enzymes has the potential to increase productivity, efficiency and quality in agro-in­dustrial processing operation in many develop­ing countries.

(vii)Enzymes enhance extraction and separation pro­cesses, eliminate toxic and anti-nutritional fac­tors, catalyse carbohydrates, proteins, and lipids conversion through anti-oxidant and bio-catalytic activity.

(viii) It has reputations for supply of h igh quality, clean, dry products.

The Weakness (i) Necessary enzymes are not available at low

enough cost. (ii) Capacity to produce, process and store quality

oil, on a commercial scale, is currently l imited. (iii) Recovery of hexane is energy intensive process

and requires extensive capital equipment. (iv) There is insufficient work done to draw any defi­

n ite conclusions on the possibility of using en­zymes in softening seed coat and the impact of such a pretreatment on oil recovery.

The Opportunities (i) A Technology Mission on Oilseeds (TMO) was

established in May 1 986 for harnessing the best of production, processing and management tech­nologies.

(ii) Intensification of research efforts to increase pro­duction of oilseeds.

(iii) Increasing areas under non-traditional oilseeds crops.

(iv) S et t ing .up of necessary process ing and infrastructural facilities to keep pace with the production programme of oilseeds .

(v) Better incentive to producers through fixation of minimum support prices of major oilseeds.

To encourage industry modernization and technologi­cal upgradation, certain equipment considered necessary for modernization has been granted concessional rate of custom duty.

The Threats (i) Rising costs of production and processing. (ii) Government intervention in trade and produc­

tion policies. (iii) Reduction of R&D funding and other govern­

ment support. (iv) Technological barriers due to limited informa­

tion available about most of the oilseeds. (v) Conservative nature of oil extraction and pro­

cessing industries. (vi) There is excessive control and regulation by the

Government which is difficult to justify in mod­ern economy.

Enzyme Based Oil Extraction

Enzyme treatment is probably the most important in oil extraction as it digests the complex cell wall of oil-

306 J SCI IND RES VOL 60 APRIL 200 1

seeds, altering permeability favouring oil extraction. After enzymatic treatment individual components, viz, protein, oi l and polysaccharide could be conveniently separated with further processing22. The treatment ap­peared to increase the productivity, efficiency and pro­vide quality output in agro-industrial processing in many developing countries. Enzymes enhance extraction and separation processes, eliminate toxic and anti-nutritional factors, catalyse carbohydrate, protein and l ipid conver­sion through their antioxidant and biocatalytic activities. The energy costs associated with processing were re­duced, the nutritional quality and safety of foods im­proved, and the processing time were shortened. New products may be generated and alternative application for several agricultural product may be realized after enzymatic treatment6X•

Enzymatically treated oilseeds showed an increase in the oil yield in comparison to untreated samples. Re­gardless of the type of enzyme, the quality of the oil was good and its composition was not affected by the treat­ment6Y. Enzyme may either be directly incorporated or may be immobilized on inert support matrices (immobi­l ized enzymes) and allowed to act.

Enzymatic hydrolysis as a pretreatment of oil seeds have been shown to be effective for quick softening of seed coat which opened up oil cell walls and also broke up the complex l ipoprotein and l ipo-polysaccharide molecules (not extractable for oil) into simple molecules releasing oil from l ipid bodies enmeshed with carbohy­drate and protein structures. Seed lipid bodies contained abundant proteins termed oleosins, which seem to play a major role in stabilizing these bodies. The structure of oleos ins is generally the same for most oilseeds; it con­sists of low molecular weight proteins in the range of 1 5000 to 26000 1 1 . 70. The enzyme mediated oil extrac­tion process would reduce loss of oil that normally re­mained in the seedcake after extraction (Figure 1 ) .

The oilseed composition determined choice of en­zymes. However, it was very difficult to compare results reported in different studies with a view to select the best enzyme combination for a given oilseed as most of the studies employed different extraction conditions42. 4x. 53 . Different oil yields obtained with different enzymes for the same oil-seed reflected differences in the enzyme ff' . . h t t 0' 1 elease3x. 40, 42-46, 52 An en-e IClency WIt respec 0 I r - .

zyme system developed for one type of oilseed material could not be adopted to another oilseed. Also, the num­ber of oilseeds tested so far has been very limited. In future, enzyme technology would play a big role in im-

Table 6 - Enzyme involved in pretreatment of oil seeds and their sources

Enzymes

Cellulase I; Hemicellulase Protease Extract Cellulase'6 Hemicellulase Extract

Extract'K Cellulase'9

Sources

Trichoderma viridi

Aspergilus niger

Bacillus licheniformis Bacillus subtilis

Sigma Chern. Co. USA Sigma Chern. Co. USA Aspergillus Jumigatus Sporotrichulll thermophile Humicola lanuginosa Aspergillus Jumigatus Sigma Chern. Co. USA

proving the yield and quality of oils. I t has been found that use of proteases, cellu lases and

hemicellulases helped release more oil both during sol­vent extraction as well as when expellers were used. Proteolytic enzymes mainly hydrolyzed proteins in cell membranes as well as inside the cytoplasm I 1 . 39, 4 1 (Table 6). Pectinase and cellulase were the most effective en­zymes that increased oil y ield by 20 per cent. Consider­ing the specificity of each carbohydrase, a rational choice of the enzyme for a given oilseed could only be made after gaining an understanding of the complex arrange­ment of polysaccharides in the cel l wal l . Primary cell walls of a variety of higher plants contained cellulosic fibres to which strands of hemicelluloses are attached42. 5 1 , therefore, enzyme preparations capable of attacking cell walls would necessarily contain a m ixture of cellu­lases, hemicellulases, pectinases and even proteases5 1 .

Enzyme mixtures gave better results compared to that with individual enzymes, e g, the oil extraction yield from coconut was as h igh as 80 per cent with the com­bined treatment of polygalacturonase, a-amylase and protease53-69, 7 1 . Aqueous hydrolysis72 of dehulled rape­seed by a mixture of three cell wall degrading enzymes, pectinase, cellulase and hemicellulase increased perme­abil ity of cell wall allowing more efficient extraction of the oi145 . The effect of different carbohydrases was evalu­ated on the extraction time and yield of canola oil I X .

Oil extraction yields above 90 per cent were obtained using galactomanase combined with a polysaccharide enzyme-degrading complex. These enzymes degraded components of structural cell wall which contained man­nan, galactomannan, arabinoxylogacton and cellu lose73. The recovery of oil from oilseeds could also be increased

KALlA et at. : USING EMZYMES FOR OIL RECOVERY FROM EDIBLE OIL SEEDS 307

Table 7 - Effect of enzymatic treatment on oil yield

Oilseed, fruit

Soybean41 Palm3� Coconut53 Avacado54

Enzyme

Proteolytic Pectinase Polygalacto-uraonase a-Amylase

by addition of microbial enzymes 15 . Enzymes obtained from thermophilic moulds were much superior due to greater thermostability74. Because of this, higher ambi­ent temperature (45 to 60 °C), in industrial processes could be used with less contamination and cooling costs. Various crude enzymes have been isolated from differ­ent sources, viz, A spergillus Jumigatus, Humicola lanuginosa, and Sporotrichum thermophile and used in oil extraction 15. 10. IX. 19 (Table 7).

The recovery of oil increased up to 2-5 per cent with H. lanuginosa in cottonseed while in sunflower and soy­bean oil it increased up to 4.2 per cent with Aspergillus Jumigatus lO• The solvent oil extraction yield increased by around 20 per cent when kernels of shea tree were pre-treated with a mixture of protease and carbohy­drases7o. 75. In soybeans, the enhancement in expelled oil was up to 2.8 per cent of moisture free oil when pre­treated with crude enzyme preparations obtaimed from Aspergillus Jumigatu. The gain corresponded to about 1 1 .7 per cent more oil recovery; equivalent to 63.5 per cent of the total extractable oil 17 • In groundnut kernels, pretreatment with cellulase resulted in 2.7 per cent more oil yield l9 . Enzyme preparation from Aspergillus gave higher yield, and that from Bacillus solubilised 35 per cent crude protein and released 1 6 per cent of total weight of crude isolates as extra oil.

Production of Enzymes for Enzyme Assisted Oil

Extraction

It is apparent from the foregoing that while the indi­vidual enzyme formulations, best suited for a particular system, had to be experimentally worked out, and gen­erally proteases, cellulases, pectinases, hemicellulases and phospholipases were needed as obtained from vari­ous sources (Table 7). As the volumes of enzymes in­volved in the application are rather large, the cost of enzymes becomes a critical factor in the adaptation of

Oil yield, per cent With enzyme

86.0 97.7 80-90 75.0

Without enzyme

62.0 9 1 . 1

65.0

this technology on a commercial scale. A large compo­nent of the high cost is due to downstream processing. The pretreatment of oilseeds and other such materials for loosening the structural matrix to facilitate oil re­covery, apparently, does not demand high purity levels in enzyme preparations, it should, therefore, be possible to develop specific strategies for enzyme purification, which are efficient, economical and suitable for such applications36• Less elaborate, toned down purification protocols with fewer steps and high selectivity might just be suitable30. 76. Several relatively new techniques like expanded bed affinity chromatography77, perfusion chromatography 7X, three phase partitioning79 and affin­ity precipitationX() that emerged in recent years, along with some more well established approaches like two phase affinity extractionXI have been increasingly uti­lized to develop less costly procedures for obtaining en­zymes. Use of fusion tags like oligohistidine residue to recover the largest protein via metal affinity has been increasingly used36 and appear to have the potential to prove useful in obtaining commercial grade enzymes for bulk applications like oil release.

It is quite possible that the three factors would com­bine in future to increase the use of enzyme in this area at an industrial level. First is, of course, the cost of en­zymes. Second is the increase in oil yield upon enzyme treatment. However, environmental concerns in all prob­ability will exert greater pressure for the adoption of enzyme assisted oil extraction by the trade in addition to the enumerated benefits. Also, the possibility of simul­taneous oil and protein recovery, and energy saved by eliminating need for organic solvent stripping and fur­ther cost saved in process monitoring (as investment in volatile organic compound emission monitoring and con­trol is not needed) may offset to a significant extent the, high cost oj enzymes factor. Hence, one would actually see this green technology being increasingly used in oil extraction.

308 J SCI IND RES VOL 60 APRIL 2001

Conclusions

The use of enzymes has the potential to increase pro­ductivity, efficiency and quality of oil obtained. It re­quires a simple manufacturing base with low capital in­vestment and relatively small amounts of energy con­sumption in comparison to other methods of oi l recov­ery and processing.

Although basic information on most enzymatic pro­cesses is available, it is generally restricted to labora­tory-scale studies. Enzymes and enzyme-catalyzed pro­cesses, however, need to be more fully explored and exploited in the light of benefits that may accrue from their use. Systematic process engineering investigations and economic evaluation of these processes appear nec­essary before venturing into scale- up studies.

Acknowledgements

We thank TMOP&M, CSIR for financial support for the projects.

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