ANALYTICAL INVESTIGATIONS TO COMPARE THE ENZYME-ASSISTED EXTRACTION OF VEGETABLE OILS WITH CONVENTIONAL METHODS

By

SAJID LATIF
M. Sc. (UAF)

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY In CHEMISTRY

DEPARTMENT OF CHEMISTRY & BIOCHEMISTRY FACULTY OF SCIENCES UNIVERSITY OF AGRICULTURE FAISALABAD 2009

Contents
Chapters
Chapter 1 Chapter 2 2.1 2.2 2.3 Chapter 3 3.1 3.2 3.3 3.4 3.4.1 3.4.2 3.4.2.1 3.4.3 3.4.3.1 3.5 3.5.1 3.5.2 3.5.3 3.6 Introduction Literature Review Oilseed Cell Wall Structure Processes for Oil Extraction from Seeds Factors Affecting Oil Yield During EACP and EAAE Materials and Methods Procurement of Oilseeds Analytical Instruments Used Throughout the Work Reagents, Chemicals, Standards and Enzymes Extraction of Oil from Seeds Solvent Extraction Enzyme-assisted Cold Pressing (EACP) Optimization of Parameters for EACP Enzyme-assisted Aqueous Extraction (EAAE) Optimization of Parameters for EAAE Quality Evaluation of Oils and Oilseed Residues Proximate Composition of Oilseed Residues Analysis of Oils Antioxidant Activity of Extracted Oils Statistical Analysis

Titles

Page No.
1-4 5-30 5 9 27 31-45 31 31 32 32 32 34 34 36 37 40 40 41 43 45

Chapter 4 Chapter 5 5.1 5.2 5.3 Chapter 6

Results Discussion Factors Affecting Oil Yield during Enzyme-assisted Cold Pressing (EACP) Factors Affecting Oil Yield during Enzyme-assisted Aqueous Extraction (EAAE) Comparison of the Quality of Extracted Oils and Oilseed Residues Summary References

46-134 139-160 139 144 153 161-163 164-178

Serial No.
2.1 2.2 2.3 3.1 4.1.1a 4.1.1b 4.1.2a 4.1.2b 4.1.3a 4.1.3b 4.1.4a 4.1.4b 4.1.5a 4.1.5b 4.1.6a 4.1.6b 4.2.1a 4.2.1b 4.2.2a 4.2.2b 4.2.3a 4.2.3b

LIST OF TABLES Titles

Typical Composition of Selected Seed Cell Wall Polysaccharides Oil Extraction by Aqueous Extraction Process Literature Cited for Enzyme-assisted Oil Extraction from Selected Seeds Activity and Supplier of Enzyme Preparations Comparison of Proximate Composition of Enzyme-assisted Cold Pressed Cottonseeds Comparison of Proximate Composition of Enzyme-assisted Aqueous Extracted Cottonseeds Comparison of Physico-chemical Properties of Enzyme-assisted Cold Pressed Cottonseed Oils Comparison of Physico-chemical Properties of Enzyme-assisted Aqueous Extracted Cottonseed Oils Comparison of Oxidative State of Enzyme-assisted Cold Pressed Cottonseed Oils Comparison of Oxidative State of Enzyme-assisted Aqueous Extracted Cottonseed Oils Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzymeassisted Cold Pressed Cottonseed Oils Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzymeassisted Aqueous Extracted Cottonseed Oils Comparison of Tocopherol of Enzyme-assisted Cold Pressed Cottonseed Oils Comparison of Tocopherol of Enzyme-assisted Aqueous Extracted Cottonseed Oils Comparison of Antioxidant Activity of Enzyme-assisted Cold Pressed Cottonseed Oils Comparison of Antioxidant Activity of Enzyme-assisted Aqueous Extracted Cottonseed Oils Comparison of Proximate Composition of Enzyme-assisted Cold Pressed Hempseeds Comparison of Proximate Composition of Enzyme-assisted Aqueous Extracted Hempseeds Comparison of Physico-chemical Properties of Enzyme-assisted Cold Pressed Hempseed Oils Comparison of Physico-chemical Properties of Enzyme-assisted Aqueous Extracted Hempseed Oils Comparison of Determination of Oxidative State of Enzyme-assisted Cold Pressed Hempseed Oils Comparison of Determination of Oxidative State of Enzyme-assisted Aqueous Extracted Hempseed Oils

Page No.
8 19 30 33 48 48 49 50 51 51 52 53 54 54 55 55 56 56 57 58 59 59

4.2.4a 4.2.4b 4.2.5a 4.2.5b 4.2.6a 4.2.6b 4.3.1a 4.3.1b 4.3.2a 4.3.2b 4.3.3a 4.3.3b 4.3.4a 4.3.4b 4.3.5a 4.3.5b 4.3.6a 4.3.6b 4.4.1a 4.4.1b 4.4.2a 4.4.2b 4.4.3a

Comparison of Fatty acid (FA) Composition (g/100g FA) of Enzymeassisted Cold Pressed Hempseed Oils Comparison of Fatty acid (FA) Composition (g/100g FA) of Enzymeassisted Aqueous Extracted Hempseed Oils Comparison of Tocopherol of Enzyme-assisted Cold Pressed Hempseed Oils Comparison of Tocopherol of Enzyme-assisted Aqueous Extracted Hempseed Oils Comparison of Antioxidant Activity of Enzyme-assisted Cold Pressed Hempseed Oils Comparison of Antioxidant Activity of Enzyme-assisted Aqueous Extracted Hempseed Oils Comparison of Proximate Composition of Enzyme-assisted Cold Pressed Sunflower Seeds Comparison of Proximate Composition of Enzyme-assisted Aqueous Extracted Sunflower Seeds Comparison of Physicochemical Properties of Enzyme-assisted Cold Pressed Sunflower Seed Oils Comparison of Physicochemical Properties of Enzyme-assisted Aqueous Extracted Sunflower Seed Oils Comparison of Oxidative State of Enzyme-assisted Cold Pressed Sunflower Seed Oils Comparison of Oxidative State of Enzyme-assisted Aqueous Extracted Sunflower Seed Oils Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzymeassisted Cold Pressed Sunflower Seed Oils Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzymeassisted Aqueous Extracted Sunflower Seed Oils Comparison of Tocopherol in Enzyme-assisted Cold Pressed Sunflower Seed Oils Comparison of Tocopherol in Enzyme-assisted Aqueous Extracted Sunflower Seed Oils Comparison of Antioxidant Activity of Enzyme-assisted Cold Pressed Sunflower Seed Oils Comparison of Antioxidant Activity of Enzyme-assisted Aqueous Extracted Sunflower Seed Oils Comparison of Proximate Composition of Enzyme-assisted Cold Pressed Sesame Seeds Comparison of Proximate Composition of Enzyme-assisted Aqueous Extracted Sesame Seeds Comparison of Physico-chemical Properties of Enzyme-assisted Cold Pressed Sesame Seed Oils Comparison of Physico-chemical Properties of Enzyme-assisted Aqueous Extracted Sesame Seed Oils Comparison of Oxidative State of Enzyme-assisted Cold Pressed Sesame Seed Oils

60 61 62 62 63 63 64 64 65 66 67 67 68 69 70 70 71 71 72 72 73 74 75

4.4.3b 4.4.4a 4.4.4b 4.4.5a 4.4.5b 4.4.6a 4.4.6b 4.5.1a 4.5.1b 4.5.2a 4.5.2b 4.5.3a 4.5.3b 4.5.4a 4.5.4b 4.5.5a 4.5.5b 4.5.6a 4.5.6b 4.6.1a 4.6.1b 4.6.2a 4.6.2b

Comparison of Oxidative State of Enzyme-assisted Aqueous Extracted Sesame Seed Oils Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzymeassisted Cold Pressed Sesame Seed Oils Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzymeassisted Aqueous Extracted Sesame Seed Oils Comparison of Tocopherol of Enzyme-assisted Cold Pressed Sesame Seed Oils Comparison of Tocopherol of Enzyme-assisted Aqueous Extracted Sesame Seed Oils Comparison of Antioxidant Activity of Enzyme-assisted Cold Pressed Sesame Seed Oils Comparison of Antioxidant Activity of Enzyme-assisted Aqueous Extracted Sesame Seed Oils Comparison of Proximate Composition of Enzyme-assisted Cold Pressed Canola Seeds Comparison of Proximate Composition of Enzyme-assisted Aqueous Extracted Canola Seeds Comparison of Physico-chemical Properties of Enzyme-assisted Cold Pressed Canola Seed Oils Comparison of Physico-chemical Properties of Enzyme-assisted Aqueous Extracted Canola Seed Oils Comparison of Oxidative State of Enzyme-assisted Cold Pressed Canola Seed Oils Comparison of Oxidative State of Enzyme-assisted Aqueous Extracted Canola Seed Oils Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzymeassisted Cold Pressed Canola Seed Oils Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzymeassisted Aqueous Extracted Canola Seed Oils Comparison of Tocopherol of Enzyme-assisted Cold Pressed Canola Seed Oils Comparison of Tocopherol of Enzyme-assisted Aqueous Extracted Canola Seed Oils Comparison of Antioxidant Activity of Enzyme-assisted Cold Pressed Canola Seed Oils Comparison of Antioxidant Activity of Enzyme-assisted Aqueous Extracted Canola Seed Oils Comparison of Proximate Composition of Enzyme-assisted Cold Pressed Moringa oleifera Seeds Comparison of Proximate Composition of Enzyme-assisted Aqueous Extracted Moringa oleifera Seeds Comparison of Physico-chemical Properties of Enzyme-assisted Cold Pressed Extraction Moringa oleifera Seed Oils Comparison of Physico-chemical Properties of Aqueous Enzymeassisted Extraction Moringa oleifera Seed Oils

75 76 77 78 78 79 79 80 80 81 82 83 83 84 85 86 86 87 87 88 88 89 90

4.6.3a 4.6.3b 4.6.4a 4.6.4b 4.6.5a 4.6.5b 4.6.6a 4.6.6b 4.7.1 4.7.2 4.7.3 4.7.4 4.7.5

Comparison of Oxidative State of Enzyme-assisted Cold Pressed Moringa oleifera Seed Oils Comparison of Oxidative State of Enzyme-assisted Aqueous Extracted Moringa oleifera Seed Oils Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzymeassisted Cold Pressed Moringa oleifera Seed Oils Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzymeassisted Aqueous Extracted Moringa oleifera Seed Oils Comparison of Tocopherol of Enzyme-assisted Cold Pressed Moringa oleifera Seed Oils Comparison of Tocopherol of Enzyme-assisted Aqueous Extracted Moringa oleifera Seed Oils Comparison of Antioxidant Activity of Enzyme-assisted Cold Pressed Moringa oleifera Seed Oils Comparison of Antioxidant Activity of Enzyme-assisted Aqueous Extracted Moringa oleifera Seed Oils Comparison of Proximate Composition of Aqueous Enzyme-assisted Moringa concanensis Seeds Comparison of Physico-chemical Properties of Enzyme-assisted Aqueous Extracted Moringa concanensis Seed Oils Comparison of Oxidative State of Enzyme-assisted Aqueous Extracted Moringa concanensis Seed Oils Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzyme-assisted Aqueous Extracted Moringa concanensis Seed Oils Comparison of Tocopherol of Enzyme-assisted Aqueous Extracted Moringa concanensis Seed Oils

91 91 92 93 94 94 95 95 96 96 97 97 97

Serial No.
2.1 2.2 3.1 3.2 4.1.1a 4.1.1b 4.1.1c 4.1.1d 4.1.1e 4.1.1f 4.1.2a 4.1.2b 4.1.2c 4.1.2d 4.1.2e 4.1.2f 4.2.1a 4.2.1b 4.2.1c 4.2.1d 4.2.1e 4.2.1f 4.2.2a 4.2.2b 4.2.2c

LIST OF FIGURES Titles

Structure of an Oilseed General Structure of a Primary Seed Cell Wall Flow Chart for Enzyme-assisted Cold Pressing (EACP) Flow Chart for Enzyme-assisted Aqueous Extraction (EAAE) Effect of Temperature on Oil Recovery from Cottonseeds during EACP Effect of pH on Oil Recovery from Cottonseeds during EACP Effect of Moisture on Oil Recovery from Cottonseeds during EACP Effect of Hydrolysis Time on Oil Recovery from Cottonseeds during EACP Effect of Enzyme Concentration on Oil Recovery from Cottonseeds during EACP Enzymes Offering Optimum Oil Recovery from Cottonseeds during EACP Effect of Temperature on Oil Recovery from Cottonseeds during EAAE Effect of pH on Oil Recovery from Cottonseeds during EAAE Effect of Water/Seed Ratio (w/w) on Oil Recovery from Cottonseeds during EAAE Effect of Hydrolysis Time on Oil Recovery from Cottonseeds during EAAE Effect of Enzyme Concentration on Oil Recovery from Cottonseeds during EAAE Enzymes Offering Optimum Oil Recovery from Cottonseeds during EAAE Effect of Temperature on Oil Recovery from Hempseed during EACP Effect of pH on Oil Recovery from Hempseed during EACP Effect of Temperature on Oil Recovery from Hempseed during EACP Effect of Hydrolysis Time on Oil Recovery from Hempseed during EACP Effect of Enzyme Concentration on Oil Recovery from Hempseed during EACP Enzymes Offering Optimum Oil Recovery from Hempseeds during EACP Effect of Temperature on Oil Recovery of Hempseeds during EAAE Effect of pH on Oil Recovery of Hempseeds during EAAE Effect of Water/Seed Ratio (w/w) on Oil Recovery of Hempseeds during EAAE

Page No.
7 7 35 38 99 99 100 100 101 101 102 102 103 103 104 104 105 105 106 106 107 107 108 108 109

4.2.2d 4.2.2e 4.2.2f 4.3.1a 4.3.1b 4.3.1c 4.3.1d 4.3.1e 4.3.1f 4.3.2a 4.3.2b 4.3.2c 4.3.2d 4.3.2e 4.3.2f 4.4.1a 4.4.1b 4.4.1c 4.4.1d 4.4.1e 4.4.1f 4.4.2a 4.4.2b 4.4.2c 4.4.2d

Effect of Hydrolysis Time on Oil Recovery of Hempseeds during EAAE Effect of Enzyme Concentration on Oil Recovery of Hempseeds during EAAE Enzymes Offering Optimum Oil Recovery from Hempseeds during EAAE Effect of Temperature on Oil Recovery from Sunflower Seeds during EACP Effect of pH on Oil Recovery from Sunflower Seeds during EACP Effect of Moisture on Oil Recovery from Sunflower Seeds during EACP Effect of Hydrolysis Time on Oil Recovery from Sunflower Seeds during EACP Effect of Enzyme Concentration on Oil Recovery from Sunflower Seeds during EACP Enzymes Offering Optimum Oil Recovery from Sunflower Seeds during EACP Effect of Temperature on Oil Recovery of Sunflower Seeds during EAAE Effect of pH on Oil Recovery from Sunflower Seeds during EAAE Effect of Water/Seed Ratio (w/w) on Oil Recovery from Sunflower Seeds during EAAE Effect of Hydrolysis Time on Oil Recovery from Sunflower Seeds during EAAE Effect of Enzyme Concentration on Oil Recovery from Sunflower Seeds during EAAE Enzymes Offering Optimum Oil Recovery from Sunflower Seeds during EAAE Effect of Temperature on Oil Recovery from Sesame Seeds during EACP Effect of pH on Oil Recovery from Sesame Seeds during EACP Effect of Moisture on Oil Recovery from Sesame Seeds during EACP Effect of Hydrolysis Time on Oil Recovery from Sesame Seeds during EACP Effect of Enzyme Concentration on Oil Recovery from Sesame Seeds during EACP Enzymes Offering Optimum Oil Recovery from Sesame Seeds during EACP Effect of Temperature on Oil Recovery of Sesame Seeds during EAAE Effect of pH on Oil Recovery of Sesame Seeds during EAAE Effect of Water/Seed Ratio (w/w) (w/w) on Oil Recovery of Sesame Seeds during EAAE Effect of Hydrolysis Time on Oil Recovery from Sesame Seeds during EAAE

109 110 110 111 111 112 112 113 113 114 114 115 115 116 116 117 117 118 118 119 119 120 120 121 121

4.4.2e 4.4.2f 4.5.1a 4.5.1b 4.5.1c 4.5.1d 4.5.1e 4.5.1f 4.5.2a 4.5.2b 4.5.2c 4.5.2d 4.5.2e 4.5.2f 4.6.1a 4.6.1b 4.6.1c 4.6.1d 4.6.1e 4.6.1f 4.6.2a 4.6.2b 4.6.2c 4.6.2d

Effect of Enzyme Concentration on Oil Recovery of Sesame Seeds during EAAE Enzymes Offering Optimum Oil Recovery from Sesame Seeds during EAAE Effect of Temperature on Oil Recovery from Canola Seeds during EACP Effect of pH on Oil Recovery from Canola Seeds during EACP Effect of Moisture on Oil Recovery from Canola Seeds during EACP Effect of Hydrolysis Time on Oil Recovery from Canola Seeds during EACP Effect of Enzyme Concentration on Oil Recovery from Canola Seeds during EACP Enzymes Offering Optimum Oil Recovery from Canola Seeds during EACP Effect of Temperature on Oil Recovery from Canola Seeds during EAAE Effect of pH on Oil Recovery from Canola Seeds during EAAE Effect of Water/Seed Ratio (w/w) on Oil Recovery from Canola Seeds during EAAE Effect of Hydrolysis Time on Oil Recovery from Canola Seeds during EAAE Effect of Enzyme Concentration on Oil Recovery from Canola Seeds during EAAE Enzymes Offering Optimum Oil Recovery from Canola Seeds during EAAE Effect of Temperature on Oil Recovery from Moringa oleifera Seeds during EACP Effect of pH on Oil Recovery from Moringa oleifera Seeds during EACP Effect of Moisture on Oil Recovery from Moringa oleifera Seeds during EACP Effect of Hydrolysis Time on Oil Recovery from Moringa oleifera Seeds during EACP Effect of Enzyme Concentration on Oil Recovery from Moringa oleifera Seeds during EACP Enzymes Offering Optimum Oil Recovery from Moringa oleifera Seeds during EACP Effect of Temperature on on Oil Recovery from Moringa oleifera Seeds during EAAE Effect of pH on Oil Recovery from Moringa oleifera Seeds during EAAE Effect of Water/Seed Ratio (w/w) on Oil Recovery from Moringa oleifera Seeds during EAAE Effect of Hydrolysis Time on Oil Recovery from Moringa oleifera Seeds during EAAE

122 122 123 123 124 124 125 125 126 126 127 127 128 128 129 129 130 130 131 131 132 132 133 133

4.6.2e 4.6.2f 4.7.1 4.7.2 4.7.3 4.7.4 4.7.5 4.7.6 4.7.7

Effect of Enzyme Concentration on Oil Recovery from Moringa oleifera Seeds during EAAE Enzymes Offering Optimum Oil Recovery from Moringa oleifera Seeds during EAAE Typical GLC Chromatogram Showing the Separation of FAs of Enzyme-assisted Aqueous Extracted Sunflower Oil Typical GLC Chromatogram Showing the Separation of FAs of Enzyme-assisted Cold Pressed Canola Oil Typical HPLC Chromatogram Showing the Separation of Tocopherol Standards Typical HPLC Chromatogram Showing the Separation of Tocopherols of Enzyme-assisted Cold Pressed Hempseed Oil Typical HPLC Chromatogram Showing the Separation of Tocopherols of Enzyme-assisted Cold Pressed Canola Seed Oil Typical Rancimat Profile Showing Rancimat Analysis of Enzymeassisted Cold Pressed Cottonseed Seed Oil Typical Rancimat Profile Showing Rancimat Analysis of Enzymeassisted Aqueous Extracted sesame seed oil

134 134 135 135 136 136 137 137 138

ACKNOWLEDGEMENTS
I have no words to praise Almighty ALLAH, the Beneficent, the Merciful, Whose blessings and exaltation flourished my thoughts, thrived my ambitions and enabled me to contribute the sacred wealth of knowledge. Special praises for His last Prophet of Islam Hazrat MUHAMMAD (P.B.U.H) who is a bonfire of knowledge and guidance for humanity.

I would like to express my sincere and immense gratitude to Dr. Farooq Anwar, Assistant Professor, Department of Chemistry and Biochemistry, for providing guidance, unwavering support, and encouragement. To me he has been and will always be a great mentor. I am also very thankful to the other two members of my supervisory committee, Dr. Ijaz Ahmad Bhatti, and Prof. Dr. Tahira Iqbal who gave valuable and constructive suggestions throughout the program.

I would also like to record my appreciation to Higher Education Commission (HEC) of Pakistan for providing me HEC Indigenous and HEC International Research Support Initiative Program (IRSIP) awards to finance my studies. My thanks go to the Prof. Levente L. Diosady, University of Toronto for his technical guidance. Those who contributed to make it a memorable experience were members of my lab including: Abdullah Ijaz Hussain, Umer Rashid, Dr. Bushra Sultana, and Maleeha Manzoor for readily assisting me during my research work.

Lastly and certainly not in the least, I would like to thank my parents, my wife and the rest of my family for offering their words of encouragement, undying support and patience. I could not have done anything without them.

Sajid Latif

CHAPTER 1
INTRODUCTION AND OBJECTIVES

1.1 Introduction Oil and fats are the most concentrated source of energy in our diet and also act as carrier for liposoluble vitamins viz A, D, E, and K. Vegetable oils are an important ingredient for several foodstuffs and non-food products. These are also recognised as one of the richest source of essential fatty acids (C18:2 and C18:3). Extraction of oils from oilseeds is a key step for their commercialization. The extraction process has a direct effect on the quality and quantity of protein and oils obtained (Rosenthal et al., 1996). Various methods like cold pressing, solvent, super-critical fluid and aqueous extractions are in practice for oilseed extraction. The use of solvents for oil recovery has shown several drawbacks such as high safety hazard (Kingsbaker 1983), high energy input (Johnson and Lusas 1983; Sosulski and Sosulski 1993), low quality oil (Diosady et al., 1983; Usuki et al., 1984), environmental risk (Wolff 1983), toxicological effects (Grant et al., 1983) and low quality meal (Diosady et al., 1987). Conventionally, hexane has been used for decades to extract oil from oilseeds with massive construction and operational costs. Because of the safety, environmental issues and potential health risks, the edible oil industry is in urgent need of replacing hexane-extraction (Bhattacharjee et al., 2006) with some suitable and environmentfriendly techniques. In 2001, the U.S. Environmental Protection Agency issued strict guidelines for hexane emissions by vegetable oil extraction facilities (EPA 2001), providing new incentives to develop alternative methods of edible oil extraction. The new tendency to avoid the use of toxic organic solvents in large installations has renewed interest in alternative extraction processes (Johnson and Lusas 1983). Continuous efforts are being made to develop new and efficient processes alternatives to hexane as the extracting solvent for production of betterquality edible oils, with simultaneous recovery of valuable nutrients (Chen and Diosady 2003). Like other bio-renewable solvents (alcohols, supercritical fluids), the use of water as the most economical extracting agent is gaining interest, especially

with the aim of replacing toxic solvents (Sineiro et al., 1998). Aqueous oil extraction has emerged as a promising technique for extraction of oil from certain oilbearing materials e.g. peanut (Rhee et al., 1972; Shi et al., 1998), coconut (Hagenmaier et al., 1973). Cold pressing and aqueous oil extractions are found to be the most suitable alternative for solvent extraction, nevertheless these processes have low oil yield. Recently, enzymatic pre-treatment has emerged as a novel and an effective means to improve the oil yield in cold pressing and aqueous extraction techniques (Rosenthal et al., 1996). The use of enzymes in oil extraction process has been studied by several researchers (Dominguez et al., 1994; Che Man et al., 1996; Latif et al., 2007; Sharma et al., 2001; Tano-Debrah and Ohta 1997). The main purpose of using the enzymes in oil extraction process is to hydrolyze the structural polysaccharides which form the cell wall of oilseeds or the proteins which form the cell and lipid body membrane. The enzymes most frequently reported in the literature, used for the oil extraction, are cellulase, -amylase, and pectinase (Rosenthal et al., 1996). There are two general approaches for enzymatic oil extraction: enzymeassisted aqueous extraction (EAAE) and enzyme-assisted cold pressing (EACP) which may offer a number of advantages as compared to conventional extraction. In enzyme-assisted aqueous process, the enzymatic action is reported to improve the oil recovery by degrading the seed cell wall, and rupturing the polysaccharide-protein colloid which may cause emulsion formation, resulting in low yield. However, in EACP technique, the enzymes only facilitate the hydrolysis of seed cell wall, because in this non-aqueous system there is no polysaccharide-protein colloid (Concha et al., 2004). Some quality attributes including free fatty acid content, refractive index, and peroxide value of the enzyme-extracted oils have been reviewed by Dominguez et al. (1994). The quality of oils obtained by enzyme treatment is relatively good as compared with hexane-extracted oils (Ranalli 1997). EACP, because of its nontoxic and noninflammable standing, seems to be an ideal alternative for oilseed extraction (Bhattacharjee et al., 2006). The enzymeassisted pressing has been employed for extraction of oils from palm, canola, soybean, chilean hazelnut and rosehips (Sosulski et al., 1988; Cheah et al., 1987;

Smith et al., 1993; Zuniga et al., 2001a,b). In these studies, an enhancement of oil yields and, in most of the cases, an improvement in product quality was observed. Aqueous enzymatic methods have been developed for extraction of oils from various seeds (Hanmoungjai et al., 2001; Rosenthal et al., 1996; Rosenthal et al., 2001; Sharma et al., 2002). Karlovic et al. (1994) investigated an aqueous enzymatic method for extraction of oil from corn germs using cellulase which resulted in 80% of the oil recovery. Enzymatic aqueous process facilitate the removal of phospholipids from the oil thus leading to skipping the need for degumming, thereby, reducing the overall cost of oils processing (Christensen 1991). As the need exists for the development of eco-friendly technologies for the extraction of vegetable oilseeds and in this connection enzyme-assisted extraction methods are gaining much recognition. Some of the needs triggering technology innovation in the oil extraction sector such as cost savings, environmental and safety concerns, and nutrition issues seem to be achievable by successful development of enzyme-based processes. There is an extensive need to develop optimized and comprehensive protocols for the extraction of oilseeds offering enhanced oil recoveries. Therefore, the present project was undertaken with the following aims and objectives.

1.2 Aims and Objectives of the Present Study Exploration of enzyme-assisted methods for the extraction of conventional and non-conventional vegetable oilseeds. Optimization of experimental parameters (such as pH, temperature, water/seed ratio, enzyme concentration and time of hydrolysis) leading to development of a comprehensive protocol for improving oil recovery and oil quality Detailed characterization of enzyme-extracted vegetable oils for physicochemical and antioxidant attributes Quality evaluation and comparison of enzyme-extracted vegetable oils with those of control and produced by conventional extraction methods

CHAPTER 2
LITERATURE REVIEW

Oilseeds are mainly composed of oil and protein. For better oil extraction an in-depth understanding of the complex arrangement of polysaccharides in the oilseeds cell wall is a prerequisite. The oil and protein are stored in cotyledon cell of the oilseed (Rosenthal et al., 1996). The lipids and proteins are associated with various complex substances in the cell walls (Christensen 1991).

2.1 Oilseed Cell Wall Structure The general structure of a plant cell wall is presented in Fig. 2.1. The primary cell wall of an oilseed consists of cellulose fibre attached with hemicellulose. The fibres are combined in a pectic substances complex connected to structural protein (Christensen 1991). While the secondary cell wall is dominated by cellulose and hemicellulose (Dominguez et al., 1994). The cell walls of different materials have different physico-chemical structure. The cell wall composition of coconut, corn germ, rapeseed, rice bran and soybean are summarized in Table 2.1. Cellulose and hemicellulose were found to be the dominant constituents. These components can be broken by using mechanical or chemical methods and enzymatic application in order to extract oil and protein. Enzyme pre-treatment is considered to be very important, the enzyme selected and its activity must be appropriate to the oilseed cell wall composition. The oilseed cotyledon cells contain discrete cellular organells called lipid and protein bodies (Fig 2.1). The oil bodies of seed contain abundant proteins termed oleosins which play an important role in stabilizing these bodies (Huang 1994; Murphy 1993). Protein bodies contain 60-70% of the total protein in the oilseeds (Rosenthal et al., 1996). The spaces between protein bodies in the cotyledon cells are filled with lipid bodies and a cytoplasmic network which also consists of proteins (Young and Schadel 1990; Huang 1994). The oleosins have low molecular weight proteins (15000-26000Da) which play a major role in the stability of the lipid bodies

(Huang 1992). These also maintain the integrity of the lipid bodies during the desiccation that accompanies seed maturation by preventing interaction and possible coalescence between these bodies (Murphy 1993; Huang 1992). The walls surrounding the cell are primarily composed of cellulose, hemicellulose, lignin and pectin (Snyder and Kwon 1987). In the conventional process, the grain is flaked, to rupture the cell wall that exposes the oil to the solvent. In aqueous processes, comminuted material is used which serves the same purpose of exposing and releasing the oil and protein. The soluble components diffuse into water, and the released oil forms a separate liquid phase or partially emulsified with water (Rosenthal et al., 1996). Rosenthal et al. (1996) divided the seed cell wall structure into three basic domains: (1) Amphipathic domain at the amino terminus of oleosins which are most probably associated with the oil body surface (2) Central hydrophobic domain which contains around 70 non polar amino acids in succession that may strongly interact with the triacylglycerol matrix of the lipid bodies (3) Amphipathic domain at or near the carboxyl terminus which interacts with the surface of a phospholipic monolayer surrounding the triacylglycerol matrix

Fig. 2.1 Structure of an Oilseed (Rosenthal et al., 1996)

Fig. 2.2 General Structure of a Primary Seed Cell Wall (Keegstra et al., 1973) 7

Table 2.1 Typical Composition of Selected Seed Cell Wall Polysaccharides Polysaccharide Components Cellulose Hemicellulose Pectic substances Mannan Glactomannan Arabinogalactans Xyloglycan Arabioxylan Other substances -Data not shown
a

Content (%) Rapeseeda 22 39 8 29 2 Corn germa 39 <1 40 10 Coconuta Soybeanb 13 61 26 Some 20 50 30 -

Christensen (1991) Mustakas (1980)

2.2 Processes for Oil Extraction from Seeds Since the dawn of human history, several oil extraction methods have been developed but wet rendering, mechanical pressing and solvent extraction are the basic methods for oil extraction. Mechanical (hydraulic and screw) pressing has been practiced for thousands of years (Bargale 1997). Oilseed material is boiled in the wet rendering method for partial separation then the oil is skimmed off (Bredeson 1983). The third and the most conventionally used method is the solvent extraction (Bargale 1997).

2.2.1 Solvent Extraction Solvent extraction of oil from oilseeds is the most efficient and attractive method for oilseeds having low oil content (Anjou 1972; Caviedes 1996). This is the most economical, efficient and widely used process for high oil content seeds (e.g. sunflower, peanut, canola) and also for medium oil content seeds (cottonseed and corn germ) (Norris 1964; Ward 1976). Therefore, vegetable oils are mostly extracted by using solvent extraction (Kemper 2005). Solvent extraction was originated as a batch process in Europe in 1870 and developed to the continuous solvent extraction systems by technological developments (Mustakas 1980). Extraction is normally carried out in a counter-current fashion to maximize oil recovery while minimizing solvent use (Balke 2006). The most common type of extractor used is the percolation type in which the solvent flows by gravity through a bed of meal (Anjou 1972; Beach 1983). The hexane leaves the extractor with an oil concentration of 25-30% and the defatted meal with a solvent content of 30-35% (Anjou 1972). The hexane in the meal is removed in a desolventizer-toaster which consists of steam heated trays, the meal is first heated by indirect heat in the upper trays to remove most of the solvent, followed by a direct steam injection in the middle trays, and finally by indirect heat in the lower trays to dry the meal (Anjou 1972; Beach 1983). The meal has dark colour with 6-11% moisture content and 300 to 1800 ppm hexane (Beach 1983; Dahlen and Lindh 1983). This desolventization of meal is the main source of solvent losses (Dahlen and Lindh 1983). 9

The solvent from the oil is removed in rising film evaporators and vacuum distillation (Serrato 1981). The hexane containing oil (miscella) is sent to a double effect shell-and-tube rising film evaporator, where 90% of the hexane is removed (Anjou 1972). The rest of the hexane and moisture in the oil is removed in stripping tower under vacuum (100 torr) at 110 oC (Anjou 1972; Beach 1983). The extracted oil is sent for degumming which removes the phosphatides by treating the oil with a small amount of water (2%) or a dilute acid solution (Caviedes 1996). Mostly the oil is super degummed (acid degummed) by using 2500 ppm H3PO4, or 1500 ppm citric acid or maleic acid and the oil is separated in a continuous centrifuge which is dried in vacuum at 80 oC (Caviedes 1996). Generally, a two-step process is used for high oil contents (above 35%) materials which consists of a continuous pre-press followed by solvent extraction. The pre-pressing allows solvent extraction to be applied to the oilseeds that are difficult to process by direct extraction methods and it also lowered the solvent requirements (Mustakas 1980). A single solvent extraction is employed for soybeans (Mustakas 1980). Despite recent developments in the techniques for minimizing VOC (Volatile organic compounds) emissions, the food industry is still responsible for about 7.5% of the mass emissions from stationary sources (Valentin 1992). Vegetable oil sector is mainly responsible for the high VOC emission level. According to Mustakas (1980), solvent losses in American soybean oil plants was 4-8 litres/ton of seed processed. On this basis the annual level of hexane emission from the soybean oil industries could be 210-430 million litres. It is also reported that a well-designed plant in Canada lost about 1.5 litre hexane/ton of processed rapeseed (Embong and Jelen 1977). It is estimated that the edible oil sector, in U.K. produced 10kt/a VOC emissions (Marlowe et al., 1991). Besides environmental problems, hexane is highly inflammable and in spite of elaborate precautions, there is still danger of severe accidents (Mustakas 1980). During solvent oil extraction the drastic thermal treatment of oilseeds reduces the quality of extracted oil and proteinaceous materials (Yoon et al., 1991). The 10

excessive heating in the conventional process decreased the nutritional availability of some essential amino acids (mainly lysine) which results from the Maillard reaction between free amino groups from proteins and carbonyl groups from reducing sugars (Rosenthal et al., 1996). Bargale (1997) summarized the limitations of the solvent extraction method. The process is not chemically safe for human health. Although desolventization removes most of the solvent, but some traces are always present in the meal. The solvents used for the oil extraction are highly flammable. High initial capital and operating costs are required for handling even smaller quantities of oilseeds. Toxic meals may be produced. Natural flavour cannot be extracted. The energy requirements are high and a lower quality of oil (with high content of phosphatides) is recovered. As a consequence of these drawbacks of solvent extraction method, several new approaches have been tried in order to develop a better technology.

2.2.1a Two-phase Solvent Extraction For production of high quality canola meal with low glucosinolate and phenolic content and improved quality of the oil with lower phosphatide content (gum) two phase solvent extraction method was developed (Rubin et al., 1984; Diosady et al., 1985a; Diosady et al., 1985c; Rubin et al., 1986; Shahidi et al., 1988). It produced good results in mustard seed (Shahidi et al., 1988), Chinese rapeseed (Liu et al., 1994) and flaxseed (Varga and Diosady 1994). The detoxified meal with traces of glucosinolates (Diosady et al., 1985b; Rubin et al., 1986) was found to be close to the meal produced by Sosulski et al. (1976), Eapen et al. (1969), and Gorrill et al. (1974). The residual oil and protein content in the meal was about 8% and 94%, respectively of the original amount in the seed (Rubin et al., 1986). Two phases in this method consists of polar phase and non-polar phase, the polar phase consists of a lower alkanol containing ammonia (0-14% w/w) and water (0-15% v/v) with hexane as a non-polar phase (Caviedes 1996). Methanol ammonia was used to facilitate the lipid extraction by breaking the cell wall but it also removed glucosinolates and polyphenols (Rubin et al., 1986). Water addition to the polar phase

11

improved the process because the glucosinolate content in the meal was reduced and the hexane as non-polar phase extracts the oil from the crushed seed (Caviedes 1996). The canola meals produced by this method have superior functional properties (Naczk el al., 1985). It has 25% more crude protein than the meal produced by hexane extraction. The meal was almost bland flavour and light colour. The water absorption and hydration capacity of the glucosinolate-free meal was similar to the air desolventized meal and higher than the commercial canola meal. A significant increase in fat absorption was observed in methanol-ammonia-water treated meal which was about three times more than the commercial soybean and canola meals. The improvements of fat and water absorption may be due to the unfolding of protein (Naczk et al., 1985). In this process the protein solubility is much lower than the hexane extracted meals (Tzeng et al., 1990; Rubin et al., 1986) which can be increased from 50 to 87% by using sodium dodecyl sulfate (SDS) (Igor et al., 1993). But this process is impractical because it requires high amounts of SDS which can be hard to remove by compatible methods to food and the products obtained will have low purity due to the interactions between SDS and protein (Caviedes 1996). The involvement of the two solvents in this procedure increases the complexity and energy requirements. The meal desolventizing step is unavoidable and the problems associated with use of solvents are major drawback of this technology.

2.2.1b Enzyme-assisted Solvent Extraction Some studies have been reported regarding the enzyme-assisted solvent extraction. The effect of enzymatic hydrolysis to enhance oil extraction with hexane on canola was demonstrated by Sosulski et al. (1988). They observed that hydrolysis of canola with carbohydrase increased oil yield and reduced time for oil extraction by solvent extraction. They incorporated flaking, autoclaving and optimized moisture to 30% including 0.12% enzyme concentration and incubation for 12 h at 50 oC followed by decreasing moisture content to 4%. Hexane extraction was found to be effective by further grinding of treated flakes. The enzyme-mixtures with mixed activity was found to be superior than individual enzymes to enhance the oil extraction efficiency. 12

Dominguez et al. (1995b) conducted a similar study on soybean and reported an increase in oil extractability by 8-10% and up to 4% in the case of sunflower oil (Dominguez et al., 1993) with the help of different commercial enzymes. In another study, cellulase and protease enzymes were used for soybeans (Fullbrook 1983; Rosenthal et al., 1995). Enzyme used to release protein and oil whereas hexane was added to the aqueous slurry to improve the oil extraction up to 90% (Fullbrook 1983). An increased amount of solvent extractable oil was observed by Bhatnagar and Johari (1987) for some oilseeds after treatment with enzymes originating from thermophilus moulds. The cottonseed oil recovery was increased up to 5% with previous enzymatic action while sunflower oil yield was increased by 4.2% after using enzymes. Twenty percent increase was observed in the solvent oil extraction by pre-treating shea tree kernels with a mixture of protease and carbohydrases (TanoDebrah and Ohta 1994; 1995a,b).

2.2.2 Supercritical Fluid Extraction A new method of oil extraction from oilseeds was developed by using carbon dioxide without using organic solvents is supercritical fluid extraction (SCE) (Bulley 1984; Eggers et al., 1985; Eggers and Sievers 1989; Reverchan 1994). Carbon dioxide is a non-flammable and harmless medium instead of organic solvents such as hexane. This method is as efficient as solvent extraction and the extracted oil has better quality than the solvent-extracted oil especially with regard to negligible phosphatides contents and lower peroxide value (Eggers and Sievers 1989). Pre-pressing can be merged with SCE because pressed cake is only extracted with SCE carbon dioxide that can eliminate the unnecessary conventional distillation and desolventizing steps (Eggers and Sievers 1989). The oil saturated with CO2 can be released by lowering the pressure or reducing the temperature (Eggers and Sievers 1989; Reverchan 1994). A low temperature during the process and without desolventizing step a good quality meal with lower protein de-naturation than the conventional process meal can be produced (Eggers and Sievers 1989). 13

This technology also has some disadvantages for example the capital investment of a SCE extraction plant is higher than that of conventional plant (Eggers and Sievers 1989; Reverchan 1994). Although the operating costs of this process could be reduced which would be even higher than that of a solvent extraction plant (Eggers and Sievers 1989; Reverchan 1994).

2.2.3 Pressing Mechanical pressing (hydraulic and screw) is the oldest and simplest method for oil extraction from oleaginous seeds. No chemical is used for oil extraction and therefore the residue is free of chemical residue. The first records of oilseed presses are from Sanskrit near 500 BC (Achaya 1994) whereas in 1795, hydraulic pressing was created in Europe (Mustakas 1980). Its a labour intensive technique and its use declined over the years. Continuous screw-presses (expellers) have replaced the hydraulic equipment (Bargale 1997). It consists of an extruder with a perforated body (slots or holes), a helical screw is used to convey and press out the oil (Balke 2006). A major drawback of this process is the lower oil extraction. Heat pre-treatment improves the malleability of the seed, lower the shattering and denature some proteins to improve oil extractability (Balke 2006). Before pressing, the seed are flaked to crush the seed shell and for better oil diffusion (Ward 1982). The extracted oil is of superior quality but intense pressure and heat damages the seed protein (Balke 2006). This method is popular in developing countries for low operating costs than the solvent extraction (Bargale 1997). The meal obtained is very compact and unsuitable for solvent extraction therefore, this method has been eliminated by modern expressors (Achaya 1994; Head and Swetman 1999). But high oil content seeds (e.g. sunflower and rapeseed) are pressed before solvent extraction to maximize oil recovery (Ward 1982). The extreme heat generation during this method cause darkening of the oil due to burning of the meal and loss of protein amino acids (Bargale 1997). The choking and plugging problem results in loss of production capacity and increases in energy, 14

labor and other resource requirements (Rosenthal et al., 1996). The characteristic rapeseed and mustard flavoured oils are produced due to the presence of water (Achaya 1994; Head and Swetman 1999). On crushing the oilseeds, toxic compounds are produced when the enzymes (e.g. myrosinase in rapeseed) contact with substrates (e.g. glucosinolates) (Bargale 1997). So after flaking, it is recommended to cook the seeds to inactivate enzymes and reduce the production of free fatty acids (Ward 1982).

2.2.3a Enzyme-assisted Cold Pressing (EACP) The oilseed cell walls have many common features and very similar structure consisting of cellulose fibers to which strands of hemicelluloses are attached (Christensen 1989; Keegstra et al., 1973; Talmadge et al., 1973). The enzyme preparations capable of attacking cell walls must necessarily contain a mixture of cellulases, hemicellulases, pectinases, and even proteases (Christensen 1989). Commercial preparations containing these enzymes are often unable to hydrolyze specific components and some more development may be required to degrade, complex polysaccharides in the cell wall of plant material (Christensen 1989). Cheah et al. (1990) reported 97.7% from the pectinase-treated mesocarp of palm by using a hydraulic press compared to 91.1% obtained from the control. Similarly, Bouvier and Entressangles (1992) utilized a cellulase preparation to reduce the palm oil losses by 3% and 18% from press fibers and crude juice respectively during clarification as compared with the traditional process (without enzyme). Moisture free soybean oil yield was increased up to 2.78% which corresponded to an increase of about 11.7% in oil recovery by using mixed-activity crude enzyme (Smith et a1., 1993). The enzyme treatment conditions of whole and flaked canola seeds to enhance oil recovery during expeller pressing were optimized by Sosulski and Sosulski (1990). The enzymatic pre-treatment of canola, prior to pressing on the expeller, reduced the pressing time and increased the oil recovery from 75% to 92% of the total oil in single pressing. The residual oil content in the cake was reduced to 6.2% and protein content was slightly increased with high lysine availability. The fiber content was also 15

reduced by 1.1 to 2.6 percentage units and the digestibility of organic matter of treated press cake was improved by 8-21 percentage units over that of the control. The carbohydrase and mixed activity enzyme (Novo Industri SP-249) were used for an expeller process and increased the oil extraction from 72-78% (for the untreated seed) to 90-92% (for the hydrolyzed seeds) (Sosulsky and Sosulsky 1993). The expeller performance was improved as the material throughput was increased by 51% and the oil flow by 84%. This process produced better quality oil than the solvent extraction process with lower energy requirements. In another study, Sosulski and Sosulski (1993) investigated the use of carbohydrase enzymes to enhance oil recovery in screw pressing. Enzyme-treated seeds at 6% moisture content were pressed in the expeller at full press conditions. Control seeds were also pressed at the same pressure but without enzyme treatment. The enzyme treatment improved throughput of the expeller, increased oil flow rate and oil recovery. Material throughput was increased by 30-50%, and the oil recovery was increased from 72% (control samples) to 90-93% (enzyme-treated samples). The residual oil content in press cakes from enzyme treated seeds was 7.4%. The oil quality was inferior to the cold pressed control but was much better than that of solvent extracted oil. The feasibility of enzymatically treated soybean and sunflower seed to improve oil extractability was studied by Dominguez et al. (1993). They emphasised on particle size, moisture content, enzyme concentration and hydrolysis time parameters. The size reduction was found to be favourable during enzymatic treatment because better accessibility of the enzyme to the cellular wall. By reducing size from 2 mm to 1 mm, the oil extraction efficiency increment was more than 35% for soybean and 50% for sunflower. The moisture content required during the enzyme treatment was 50% for soybean and 20-30% for sunflower. An increase in moisture had little positive effect on oil extractability even with a very low concentration of enzyme (0.01g/100g of seed), the increased oil extractability could clearly be observed. The higher enzyme concentration of up to 1g/100g seed for soybean and 2g/100g seed of sunflower, increased the oil extraction and the hydrolysis time of 6 h was found to be optimum for both oilseeds. 16

Enzymatic hydrolysis of soybean seeds followed by mechanical pressing was studied by Smith et al. (1993). Response surface methodology was used to optimize the process parameters for maximum oil extractability. The result indicated that the enzyme treatment enhanced the oil extractability by 1.5%. The optimum process conditions obtained were: 23.0% (wet basis) moisture content during hydrolysis; enzyme concentration, 11.8% (vol/wt.); incubation period, 13.24 h; moisture content during pressing, 9.4% (wet basis); pressing pressure, 75 MPa; and pressing time, 5.36 min. These parameters had no interactive effects on expelled oil. They observed that under these optimum conditions, dehulled cracked enzyme-treated soybean expelled oil by static pressing at room temperature (18 oC) was 11.7% higher than that obtained from untreated samples under the same pressing conditions. They also predicted that the oil recovery could be increased by adding one or more conventional pre-treatments to the enzymatic hydrolysis pre-treatment.

2.2.4 Aqueous Extraction The aqueous extraction process was developed as an alternative to the solvent oil extraction process in the 1950s (Rosenthal et al., 1996). It was thought to be safe and cheap with simultaneous recovery of oil and protein from oil-bearing materials (Cater et al., 1974). The hot water flotation method for oil extraction from oilseed is a traditional process used in the rural areas of many developing countries (Southwell and Harris 1991). It includes five basic steps: (1) heat conditioning of the seed, (2) grinding, (3) extraction by boiling, (4) oil recovery, and (5) drying (Rosenthal et al., 1996). Aqueous extraction of peanuts was first described by Sugarman (1956). Peanuts were flaked and wet ground with an addition of water and alkali to maintain a pH of 7.5. The slurry was diluted with water at constant pH, heated to 80 C and passed through a centrifuge to separate solids, emulsion phase and the aqueous protein solution. The solids were dried and the protein from the aqueous solution was precipitated by lowering the pH and separating the protein precipitate by centrifugation or filtration. The emulsion was broken by heating; controlling its pH and moisture content. High shear and pressure stress can be used to break the 17

emulsion. The stream was then sent to another centrifuge where oil, solids and aqueous phase were obtained. Subrahmanyan et al. 1959 and Bhatia et al. 1966 extended Sugarman extraction system with slight modification in two points: milling was used to extract most of the oil, and the conditions under which oil was recovered. In this process 86% of oil and 71% of protein was recovered. The low oil recovery was attributed to a low efficiency in the three phase separator where 5% of the oil was not recovered from the aqueous solution. The oil had good quality with low colour and free fatty acid (FFA) contents. The protein product had a composition of 85% protein, 9% fat, 6% moisture and 0.4% ash and the carbohydrate meal had 16.2% protein, 6.1% fat, 12% moisture, 41.6% starch, 10.3% crude fiber and 6% ash contents. Bhatia et al. (1966) improved this process and reported 91-94% oil recovery and protein products with 4% fat. Aqueous extraction of oil and protein from oilseeds has been studied for peanuts (Sugarman 1956; Subrahmanyan et al., 1959; Bhatia et al., 1966; Rhee et al., 1972, 1973a,b: Lawhon et al., 1981a), coconuts (Hagenmaier et al., 1972, 1973; Kumar and Bhowrnick 1995), soybeans (Lawhon et al., 1981b; Omosaiye and Cheryan 1979a,b), cottonseed (Sugarman 1956), sunflower (Hagenmaier 1974) and rapeseed (Embong and Jelen 1977; Staron and Guillaumin 1979). The results of various studies on aqueous extraction process have been reported in Table 2.2. Al1 the other extraction processes are primarily concerned with the production of edible oil with no attention to the utilization of the protein but with this process both protein and oil can be utilized (Cater et al., 1974). Hagenmaier et al. (1972) reported that the de-emulsification step and the coconut protein recovery were the key factors for a successful process for high oil and non-denatured protein recovery. Although grinding is vital for high protein and oil extraction but it also contributes to the formation of stabilized oil-in-water emulsions. Hagenmaier et al. (1972) depicted the optimal grinding conditions that can produce 10m diameter oil globules. The protein extraction from the aqueous phase is important step and is strongly affected by the pH. For coconut protein, isoelectric pH is 4.0 and binds more oil at this pH (Hagenmaier el al., 1972). At pH 8.0 much lower oil content was observed in the protein product, the oil recovery was strongly affected 18

by the type of centrifuge used. De-emulsification of the cream phase is an important step, 90% of the oil in the cream was obtained as clear oil by the phase inversion technique (Sugarman 1956). On pilot plant-scale, this process produced 85% clear oil and 2% in the protein and skim milk whereas, 85% of the protein was also obtained in these two products.

Table 2.2 Oil Extraction by Aqueous Extraction Process

Extraction Conditions Oilseed/fruit pH Sunflower Sunflower Soybean Coconut Palm kernel Peanut Peanut Lupin
a

Temp. ( C) 100 50 80 45 60 60 65
o

Time (min) 45 30 15 20 30 40 40

Oilseed to water ratio 1:10 1:4 1:10 1 :3 1 :6 1 :5 1 :15

Oil yielda (%) 86 64 60 90 85 98 91 70

Reference Hagenmaier (1974) Southwell and Harris (1992) Rosenthal et al., (1998) Hagenmaier (1973) Kim (1989) Rhee et al., (1972), Rhee et al., (1973a) Rustom et al., (1991) Aguilera et al., (1983)

10 2 8 7 7 8 8 8

- Data not reported. Based on total extractable oil in the oilseed/fruit. Rhee et al. (1972, 1973a. 1973b) introduced a different approach for the separation of oil from the aqueous solution by following the basic steps reported by Sugarman (1956). The optimum conditions used to maximize the oil and protein yield were: fine grinding, solid-to solvent ratio of 1:6 (w/v), extraction time of 30 min., temperature of 60-64 C, and pH of 8.0. Precipitation was done at pH 4.25 which produced the highest oil recovery and the lowest emulsion formation. The oil extraction had significantly affected by the particle size and wet or dry grinding, but protein was not strongly affected by these parameters. Two processes were tested, one for the production of protein concentrates, and the other for protein isolates. The first process was carried out at pH 4.0 in the beginning, to bring the oil into the aqueous

19

solution, and almost all the protein in the solid phase with the fiber and insoluble carbohydrates. The oil and protein yields were 96% and 94% respectively. While, in the second process the extraction was done at pH 8.0 which extracted most of the oil and protein, the aqueous phase from the residue was separated by centrifugation. The clear oil and protein from the water phase were separated by precipitating the protein at pH 4.0. A pilot plant-scale production of peanut protein concentrates was tested with few modifications in method reported by Rhee et al. (1973a). Modern aqueous process consists of centrifuges instead of gravity separation and the emphasis is on operating conditions for the highest oil and protein recovery with the least damage to the nutritional value of food proteins (Embong and Jelen 1977; Hagenmaier et al., 1972). Because of different chemical compositions and physical structure of the seeds specific conditions (like pH and temperature of extraction) are required for different oilseeds (Lusas et al., 1982). The unit operations of this process may vary with the nature of the oilseed, but generally consist of grinding, solid-liquid separation, centrifugation, demulsification and drying (Cater et al., 1974; Lusas et al., 1982). The centrifugation separates into oil, solid, aqueous phases and the protein may be recovered as concentrate in the solid phase or as isolate in the aqueous phase depending on the pH (Lusas et al., 1982). The final recovery from protein isolate can be achieved by isoelectric precipitation (Cater et al., 1974; Lusas et al., 1982) and the oil can be recovered by breaking the emulsion (Cater et al., 1974). The type of separation after aqueous extraction has been reported to affect oil recovery in sunflower seed (Hagenmaier 1974). A batch centrifuge had lower oil recovery than a basket centrifuge over a wide range of pH values. Alkaline extraction at pH 10.0 and at 25 oC was done for simultaneous protein and oil extraction from the ground seeds. The aqueous slurry was passed through a basket centrifuge to remove fibrous material. The solution was then separated into cream, protein solution and solids. The oil was recovered from the cream by phase inversion (Sugarman 1956), and protein from the aqueous phase by isoelectric precipitation. The protein product contained 68% of the protein whereas, 86% was extracted.

20

The quantity and quality of the aqueous-extracted rapeseed oil was investigated by Embong and Jelen (1977). They did not consider the quality and yield of protein. They reported that the oil yield is affected by liquid-to-seed ratio, pH, temperature of stirring, time of stirring, blending, and centrifugation procedure. They observed 1:3.35 an optimum liquid-to-seed ratio. More contact time caused an increase in oil yield but stabilized oil-in water emulsion. Similarly a longer stirring time increases the coalescence rate of the oil droplets. pH had a limited effect on the oil yield but a marked effect on the amount of free oil in the emulsion phase. At pH 7.0 maximum oil extraction was observed, and 90% oil was extracted by heating at 80
o

C. The significant variation was observed by centrifugation on the oil yield. The

rapeseed oil obtained by this process had lower free fatty acids and phospholipid content than industrial and Soxhlet-extracted oil, and there is no need for degumming (Embong and Jelen 1977). The unsaponifable matter was not affected but the sulfur content was slightly higher than that of Soxhlet-extracted oil and much lower than industrial rapeseed oil. This might be attributed to the quick deactivation of myrosinase. Three processes which include (full-fat, low-fat and intermediate-fat protein products) for protein and oil extraction from soybeans were developed by Lawhon et al. (1981b). The dry ground soybeans were extracted with water at three different pH levels and solvent to-seed ratios. The full-fat process gave protein products with 32% oil and 66% protein and the intermediate-fat product had only 9.8% oil and 78-92% of protein whereas the low-fat process produced a protein product with 1.9% oil and the same protein content as in the intermediate fat product. Aqueous extraction allows inactivation or removal of anti-nutritional factors and other undesirable substances in some oilseeds (Cater et al., 1974; Lawhon et al., 1981a,b; Lusas et al., 1982). The undesirable components such as caffeic and chlorogenic acids, which cause sunflower seed meal to turn dark green or brown when wetted, can be removed by aqueous extraction (Dominguez et al., 1995a; Sosulski and McCleary 1972). Similarly, toxic sulfur compounds (goitrogens) in rapeseed can be removed by aqueous extraction (Sosulski et al., 1972). The addition of some chemicals such as hydrogen peroxide and sodium hypochlorite in the aqueous extraction medium has proved to be effective for destroying aflatoxins in peanuts 21

(Rhee et al., 1977). The bitter, poisonous alkaloids from lupin seeds and non-gossipol pigments from cottonseed can be effectively removed with this process (Aguilera et al., 1983; Lusas and Jividen 1987). Sugars and other compounds which cause the problems of flatulence and off-flavour to soybean might be removed by using this technique (Cater et al., 1974). The physico-chemical studies in terms of free fatty acids, refractive index, color on Lovibond scale, saponification value, iodine value, Reichert value, Polenske value, Kirschner value, and the fatty acid composition of the extracted oil were conducted by Gunetileke and Laurentius (1974) but no significant variation was observed. Hagenmaier et al. (1973) reported 0.4% free fatty acids in the coconut oil which was superior to the oil obtained by crushing copra with 5% free fatty acid content. Kim and Yoon (1990) did not find any significant differences in aqueous and hexane extracted soybean oils in terms of iodine, acid, and saponification values; unsaponificable matters; iron; sterol, tocopherol, and phosphorus contents; and fatty acid composition. Yoon et al. (1991) also reported that aqueous-extracted soybean oil was slightly darker than the hexane-extracted. In a comparative study, the aqueous extracted rapeseed oil was found slightly superior to the Soxhlet-extracted oil, but much superior to the industrial oil in terms of the sulfur content and peroxide value (Embong and Jelen 1977). Aqueous processes avoid serious damage to the proteins of the seed which allows the production of food-grade instead of feed-grade protein products (Cater et al., 1974). The environmental issues, especially the increasing concern about volatile organic compounds (VOCs) have also proved to be a driving force for the development of an environmental friendly extraction technique. It is therefore necessary to find an alternative extraction method which can eliminate or reduce the use of all VOCs. Aqueous extraction process is considered to be a potential alternative to which we can claim as environmentally clean process (Marlowe et al., 1991). The low extraction efficiency of this process can be overcome by the use of hydrolytic enzymes (Fullbrook 1983; Lanzani et al., 1975; Sosulski et al., 1988). It eliminate the solvent safety problem and resulting in low fire hazard (Lusas et al., 1982; Lanzani et al., 1975; Cater et al., 1974; Lusas and Jividen 1987; Embong and 22

Jelen 1977; Lawhon et al., 1981a,b; Erickson 1983). It may be more economical because the investments relating to solvent recovery, process safety, and solvent loss control systems will be much lower.

2.2.4a Enzyme-assisted Aqueous Extraction (EAAE) Aqueous enzymatic oil extraction is an emerging technology in the fats and oil industry, and it may offer many advantages as compared to conventional extraction (Rosenthal et al., 1996; Table 2.1). It eliminates solvent consumption, which may lower investment costs (Barrios et al., 1990; Lusas et al., 1982) and energy requirements (Barrios et al., 1990). Simultaneous oil and protein recovery from most oilseeds is possible. Degumming operation can be eliminated and it may allow the removal of some toxins or antinutritional compounds from oilseeds (Caragay 1983). Several studies have been carried out on aqueous processing of oilseeds (Eapen et al., 1966; Hagenmaier et al., 1973; Kim 1989; Rhee et al., 1972; Subrahmanyan et al., 1959). The oil globules are associated with proteins and a wide range of carbohydrates inside plant cells surrounded by a thick cell wall which has to be ruptured to release the protein and oil. Enzymatic hydrolysis of cell wall is an option for pre-treatment of oilseeds as it hydrolyses the complex lipoprotein and lipopolysaccharides molecules into simple molecules, thus releasing extra oil for extraction. Fullbrook (1983) first isolated the crude protein from melon seeds by enzymatic hydrolysis and released extra oil. Then this process was utilized for the processing of ground soybean and rapeseed. Bhatnagar and Johri (1987) reported an enhanced release of oil in crushed soybean, cottonseed and castor bean hydrolyzed in the presence of hexane. Hydrolytic enzyme was initially investigated by Sherba et al. (1972) for partially releasing oil from full fat, heat-treated soya meal. Fullbrook (1983) hydrolyzed oilseeds in aqueous medium followed by solvent extraction. He also tried hydrolysis in the presence of a solvent to simultaneously extract the released oil. He observed that yields could be considerably improved if hydrolysis of the finely ground flour of soybean and rapeseed was carried out in the presence of solvent. By using 3% 23

enzymatic mixture obtained from Aspergillus niger, 50% more oil was obtained for rapeseed and 90% of the original extractable oil for soybeans (Fullbrook 1983). Olsen (1988) also performed aqueous hydrolysis of dehulled rapeseed with enzyme preparation including: pectinase, cellulase, and hemicellulase followed by extraction of the residual oil with petroleum ether. In these studies the enzymatic hydrolysis increased the permeability of the seed cell wall for more efficient extraction of the oil. Enzymatic processes had been tried on the seed cell walls to facilitate oil extraction (Cintra et al., 1986; Sosulski et al., 1988; Frevert et al., 1990; Ho et al., 1992; Ohlson 1992; Sosulski and Sosulski 1993; Rosenthal et al., 1995). The use of hydrolytic enzymes such as cellulases, hemicellulases, and pectinases in aqueous enzyme-assisted extraction process break the structure of cotyledon cell walls which makes the structure more permeable. Protease and carbohydrase enzymes are mainly used for this process. The proteins in the cell membranes and inside the cytoplasm are hydrolyzed by the proteolytic enzymes (Rosenthal et al., 1996). Proteolytic enzymes can potentially hydrolyze the lipid body membranes and can also affect the cytoplasmic network thus make the inner structure less tightly bound/compact which facilitates the protein and lipid removal from the cell (Rosenthal et al., 1996). The released oil can be easily separated from the cotyledon cells by an aqueous medium. An enzyme-aided process has also been used for soybeans (Rosenthal et al., 1995) and coconuts (Cintra et al., 1986). The enzyme efficiency based on oil yield enhancement was: mixed activity enzyme > -glucanase > pectinase > hemicellulase > cellulase (Rosenthal et al., 1996). For palm oil recovery from the centrifuge sludge of a palm oil mill cellulase, -amylase, pectinase and protease enzymes were utilized (Ho et al., 1992). A surfactant (SDS) was tried to release the oil into the aqueous phase because the oil remains attached to the plant material even with high speed centrifugation (Ho et al., 1992). Although they optimized conditions for cellulase, pH 4.8 and 30 C for 5 hr with and enzyme concentration of 0.5% (w/v), followed by a washing with 0.03 M SDS solution but still it was not economical viable. A mixed solution of -amylase, protease and polygalacturonase (PG) was used for coconut oil extraction (Cintra et al., 1986). Under optimal conditions: an 24

enzymatic digestion with 0.1 % (w/v) -amylase, 0.1 % (w/v) PG and 0.1 % (w/v) protease; (4:1) water to coconut ratio; 80% of oil was extracted with an enzymatic treatment at 40 C for 10 min.; and, centrifugation for 10 min at l2300 x g. An excellent quality of oil was obtained by this process. The hydrolysis was carried out at 50 C for 4 hr using 2% (w/w) enzyme and the oil recovered by this process had a very good quality. Rosenthal et al. (1995) used proteolitic and cell-wall degrading enzymes for protein and oil extraction from soybeans but did not observed a significant improvement similar to those reported by Fullbrook (1983) and Ho et al. (1992). The particle size and the extraction steps significantly affected the oil and protein extraction from soybeans. The particle size smaller than 150 m and three or more extraction stages were found to be the optimal conditions for 75% oil and 80% protein. The highest protein and oil extraction yield of both were observed with protease for the hydrolysis of heat treated flour, which depicts that the solubility of protein is very important in the enzymatic extraction of soybean oil. Enzymatic extraction of rapeseed oil had been widely studied (Fullbrook 1983; Sosulski et al., 1988; Sosulski and Sosulski 1993). Fullbrook (1983) was able to extract 70% of the original oil present in the seed, by using hexane, and extracted only 13%, when the enzymes were used alone. Sosulski et al. (1988) used carbohydrase to degrade cell walls to enhance oil recovery during solvent extraction but the best performance was observed with a mixed activity enzyme (Novo Industri SP-249). The enzymatic digestion was carried out at 50 C, for 12 hr at pH of 5.5. An aqueous enzymatic process was developed by Novo Industries NS (Bagsvaerd, Denmark) in which multi-activity enzymes were used to extract the oil, producing a high quality oil, rape flour and molasses (Ohlson 1992). McGlone et al. (1986) compared the quality of coconut oil obtained by aqueous enzymatic process with the Official Mexico Standards. Without any further purification, the oil obtained after reaction and centrifugation was reported to be an excellent quality and with a simple deodorization process it could be readily used in existing food applications.

25

In contrast, Bocevska et al. (1993) obtained corn germ oil with a lighter yellow color using an enzymatic aqueous process as compared to the commercial product; they concluded that the aqueous product may be refined with less bleaching earth. It was also suggested that a thermal discoloration may be useful. In the same study, it was also shown that the quality of corn germ oil obtained by an aqueous process was similar to the oil extracted by solvent in terms of free fatty acids content, primary and secondary oxidation product levels in the oil by solvent extraction. The aqueous product also showed good stability especially when extraction was preceded by hydrothermal pretreatment to inactivate the lipase in the grain. Aqueous enzymatic process appears to be an attractive approach compared to the conventional hexane-based process. Several reports suggest that oils obtained from aqueous or enzymatic extraction show superior quality (Bocevska et al., 1993; Dominguez et al., 1994; Che Man et al., 1996). However, Dominguez et al. (1994) reported that the composition and characteristics of oils obtained from enzymeassisted process was similar to the oil obtained by solvent extraction but the color of the oil obtained by aqueous enzymatic process was lighter than solvent-extracted oil. It has several potential advantages over a conventional plant; no need for solvents, lower capital investment, higher safety standards, simple process, simultaneous recovery and purification of protein and oil, and less pollution (Cater et al., 1974; Kumar and Bhowmick 1995; Rosenthal et al., 1995). However, lower efficiency in oil extraction, higher risk of microbial contamination, need for a de-emulsification step and treatment of the resulting aqueous effluent have discouraged its commercial application. As discussed earlier in comparison with n-hexane, the aqueous-based process is safer and more environmentally friendly. On the other hand, lower oil yields, the need for more difficult oil recovery steps and production of significant volumes of aqueous effluent are some of the disadvantages. In order to reduce aqueous effluent and save energy consumption, water used for extraction can be recovered and reused (Southwell and Harris 1992). Furthermore, recycling enzymes with water circulation can help to cut down the cost of enzyme (Barrios et al., 1990). Dominguez et al. (1995c) reported that 30% of active enzyme was eliminated in the effluent. Of course, it is only possible to recycle and reuse enzyme if its activity was not dropped 26

significantly in the process. Furthermore, it can be seen that enzymatic process might give a lower oil yield than solvent extraction. Protein obtained as a by-product, can be a valuable food ingredient. To improve the extraction yields more data have to be investigated.

2.3 Factors Affecting Oil Yield During EACP and EAAE The unit operations in the aqueous process reported by Cater et al. (1974) and Lawhon et al. (1981c) include grinding, extraction parameters, solid-liquid separation and product recovery.

2.3.1 Grinding of Oilseeds The whole oilseeds or fruits must be ground to fine particles for more efficient extraction of water-soluble components (Cater et al., 1974; Lawhon et al., 1981c). Wet or dry grindings are normally used depending on the type of oleaginous materials (Cater et al., 1974). For high moisture content oleaginous materials (coconuts and palm kernel) wet grinding is used (Cater et al., 1974; Kim 1989) and dry grinding is suitable for low moisture oilseeds sunflower seeds, peanut and soybean (Hagenmaier 1974; Rhee et al., 1972; Rustom et al., 1991; Rosenthal et al., 1998). It has been reported that significantly higher protein and oil extraction yields was obtained from smaller particles of non-heat treated soybean flour than the larger sizes (Rosenthal et al., 1998). Although for peanuts, maximum recovery was observed with fine particle size but a very stable emulsion was also observed in aqueous media (Rhee et al., 1972).

2.3.2 Extraction Parameters In enzymatic extraction the ground seeds are mixed with water, and then agitated to increase extraction. The main parameters that influence the enzymatic extraction yields include enzyme mixture, enzyme concentration, particle size of oilseed, pH, solid-liquid ratio, temperature and time (Cater et al., 1974; Lawhon et al., 1981c). Buenrostro and Lopez-Munguia (1986) reported better oil recovery for avocado with 1% (w/w) -amylase, paste-to-water ratio of 1:5 at 65 oC for 1.5h was 27

used. The pH during the process depends on the composition of oilseed. In aqueous process, the aim is to obtain high oil and protein yields but its not possible at a pH value near the isoelectric point of protein, because at this pH, protein can bind the oil in a very stable emulsion (Rosenthal et al., 1996). For peanut, a pH of 4-5 was found to be suitable for oil extraction with no emulsion phase and protein can be extracted easily at a pH of 8.0 (Rhee 1972). Different studies on enzymatic processes were conducted at different temperatures and the optimum temperature range (40-60 oC) was reported for several oilseeds (Rhee et al., 1972; Lawhon et al., 1981c; Kim 1989; Rustom et al., 1991). Rosenthal et al. (1998) reported that the protein yield was decreased slightly when temperature was higher than 50 oC. No significant increase in the palm oil yield was reported when extraction temperature was raised above 45 oC (Kim 1989). For peanut oil extraction, a temperature range of 60-64 oC, and a lower temperature of 40-44 oC found to be suitable for protein extraction (Rhee et al., 1972). An unfavourable effect of temperatures greater than 60 oC was observed on protein extraction yield from peanuts (Rustom et al., 1991). This increased temperature may cause denaturation of protein. The extraction time specially depends not only on the oilseeds type and temperature but also on pH during the extraction process. According to Rhee et al. (1972) maximum oil and protein yields from peanut were obtained within 30 min, while Rustom et al. (1991) reported that increasing extraction times from 15-40 min had no significant effect on the yields. In another study, it was mentioned that an extraction time of 15 min was sufficient for protein and oil extraction from soybean flour (Rosenthal et al., 1998). Solid-to-liquid ratio is another important parameter that allows the products to be dispersed in the aqueous media. A high amount of water will produce a less stable emulsion and will ultimately simplify the separation process (Rosenthal et al., 1996). The review of past studies as discussed in this chapter and Table 2.3 revealed that development of enzymatic processes for extraction of vegetable oil is now possible by application of efficient plant cell wall-degrading enzymes. While the method has been extensively studied for solvent extraction of oilseeds, the 28

information on aqueous and mechanical pressing of enzyme treated samples is scarce and therefore this aspect needs the attention of researchers. The enzyme-assisted process represents a feasible alternative for the oil extraction from oilseeds but it must provide a real advantage over the conventional process to make it more attractive.

29

Table 2.3 Literature Cited for Enzyme-assisted Oil Extraction from Selected Seeds
Oilseed
Coconut

Enzyme
Control (aqueous without enzyme) Pectinase (Clarex) + alpha-amylase (Tanase) + protease (HT-proteolytic) Pectinase (Irgazyme) + alpha-amvlase (Tanase) + protease (HT-proteolytic) Pectinase (Petcimex) + pectinase (Clarex) + alphaamylase (Tanase) + protease (HT-proteolytic) Pectinase (Clearzyme) + pectinase (Clarex) + alphaamylase (Tanase) + protease (HT-proteolytic) Pectinase (Rohapec) + alpha-amylase (Tanase) + protease (HT-proteolytic) Beta-glucanase (brew-n-zyme) Beta-Glucanase (brew-n-zyme) + pectinase Clarex) + alpha-amylase (Tanase) + protease (HT-proteolytic) Control (aqueous without enzyme) Alpha-amylase (Tanase) Alpha-amylase + protease Alpha-amylase + cellulose Cellulase + protease + alpha-amylase Control (aqueous without enzyme) Protease (pepsin-Merck) Cellulase (CGA) Alpha I,4 galacturonide glicano-hydrolase (Ultrazym) Protease (pepsin-Merck) + cellulase (CGA) Protease (pepsin-Merck) + alpha I,4 galacturonide glicano-hydrolase (Ultrazym) Cellulase (CGA) + alpha 1,4 galacturonide glicano-hydrolase (Ultrazym) Protease (pepsin-Merck) + cellulase (CGA) + alpha I,4 gaiacturonide glicano-hydrolase (Ultrazym) Control (aqueous without enzyme) Cellulase (CGA) Alpha I,4 galacturonide glicano-hydrolase (Ultrazym) Cellulase (CGA) + alpha I,4 galacturonide glicano-hydrolase (Ultrazym) Control (aqueous without enzyme) Pectinase (Pectinex ultra-sp) Cellulase Multi-carbohydrases (Viscozyme 120L) Pectinase (Novozyme 249) Cellulase (Novozyme 465) Pectinase (Novozyme 249) + Cellulase (Novozyme 465) Control (aqueous without enzyme) Protease (Alcalase) Protease (Sigma)

Oil Yield (%)

12.0 80.0 89.3 87.6 89.4 83.5 14.4 93.8 2.0 70.0 67.0 67.0 62.0 72.0 78.0 75.0 74.0 78.0 76.0 74.0 78.0 30 44.0 44.0 52.0 63.9 71.4 55.4 71.3 70.0 54.2 80.2 62.0 84.0 86.0

Reference
Barrios (1990) ,, ,, ,, ,, ,, ,, ,, Buenrostro and Lopez-Munguia (1986) ,, ,, ,, ,, Lanzani et al., (1975) ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, ,, Deng et al., (1992) ,, ,, ,, ,, ,, ,, Yoon et (1991) ,, ,, al.,

Avocado

Peanut

Sunflower

Rapeseed

Soybean

30

CHAPTER 3
MATERIALS AND METHODS

The experimental work done so far the present Ph.D. dissertation was conducted in the laboratories of Department of Chemistry & Biochemistry, Department of Botany, University of Agriculture, Faisalabad, Pakistan and Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Canada.

3.1 Procurement of Oilseeds Vegetable oilseeds: canola-, sunflower-, sesame-, and cotton-seeds were procured from Ayub Agricultural Research Institute (AARI), Jang Road, Faisalabad, Pakistan. While hemp-, and Moringa oleifera-seeds were obtained from open market of Faisalabad, Pakistan whereas, Moringa concanensis seeds were collected from the vicinity of Therparker, Sindh, Pakistan. All the seed samples were further identified and authenticated by Professor Dr. Muhammad Ashraf, Professor of Botany and Dean Faculty of Basic Sciences, University of Agriculture, Faisalabad, Pakistan. The seeds were stored in plastic bags at 4 oC on the day before use.

3.2. Analytical Instruments Used Throughout the Work 3.2.1. Magnetic Stirring Hot Plates (PC101, Corning) 3.2.2. Rotary Vacuum Evaporator (EYELA, N-N Series, Rikakikai Co. Ltd. Japan) 3.2.3. Small Volume Batch Centrifuge (IEC Centra 4 Tabletop, Fisher Scientific) 3.2.4. Large Batch Centrifuge (Beckman J20XP, Beckman Scientific) 3.2.5. Spectrophotometer (U-2001, Hitachi Instruments Inc. Tokyo, Japan) 3.2.6. Refractometer (RX-7000, Atago co., Ltd. Japan) 3.2.7. Commercial Blender (TSK-949, Westpoint France) 3.2.8. Orbital Shaker (Gallenkamp, UK) 3.2.9. Hot Air Oven (IM-30, Irmeco, Germany) 3.2.10. Lovibond Tintometer (Tintometer Ltd., Salisbury, Wiltshire, UK) 31

3.2.11. Rancimat (743, Metrohm Rancimat, Switzerland) 3.2.12. HPLC (LC-10A, Shimadzu, Japan) 3.2.13. GC (17-A, Shimadzu, Kyoto, Japan) 3.3 Reagents, Chemicals, Standards and Enzymes Pure standards of tocopherols [DL--tocopherol, (+)--tocopherol, (+)-tocopherol], fatty acid methyl ester (FAME), Folin-Ciocalteu reagent, 2, 2-Diphenyl1-picrylhydrazyl (DPPH), Linoleic acid and synthetic antioxidant butylated hydroxytoluene (BHT), and gallic acid were obtained from Sigma Chemical Co. (St. Louis, MO). All other chemicals reagents and solvents used were of analytical grade obtained from Merk (Darmstadt, Germany). The enzyme preparations used in this research were; Protex 7L, Multifect Pectinase FE, Multifect CX 13L, Alcalase 2.4L, Viscozyme L, Kemzyme, Natuzyme, Feedzyme, Phytezyme, Allzyme. The activity and supplier of these enzymes are stated in Table 3.1.

3.4 Extraction of Oil from Seeds After removal of seed hull and other impurities, seeds were crushed using a coffee grinder. The material that passed through 80-mesh sieve was used for extraction purposes.

3.4.1 Solvent Extraction The ground seed (15 g) was fed to a Soxhlet extractor fitted with a 0.5 L round-bottom flask and a condenser. The extraction was executed on a water bath for 6 h with 0.3 L of n-hexane. The solvent was distilled off under vacuum in a rotary evaporator (Eyela, N-N Series, Rikakikai Co. Ltd. Tokyo, Japan) at 45 oC. The extracted oil was stored under refrigerator (4 oC), until used for further analyses.

32

Table 3.1 Activity and Supplier of Enzyme Preparations S. No. 1. 2. 3. 4. 5. Enzyme Protex 7L Multifect Pectinase FE Multifect CX 13L Alcalase 2.4L Viscozyme L Main Activities Protease pectinase complex, cellulase and hemicellulase Cellulase, hemicellulase, -glucanase, and arabinoxylans Protease carbohydrases, including arabanase, cellulase, glucanase, hemicellulase, and xylanase alpha-amylase, betaglucanase, cellulosecomplex, hemicellulasecomplex, protease and xylanase cellulose, xylanase, phytase, alpha-amylase, pectinase xylanase beta glucanase cellulase and hemicellulase phytase, acid phosphatase, amylase, xylanase, betaglucanase, celulase, protease phytase, protease, celulase, beta-glucanase, pectinase Suppliers Genencor (Rochester, NY USA) Genencor (Rochester, NY USA) Genencor (Rochester, NY USA) Novozymes Bagsvaerd, Denmark Novozymes Bagsvaerd, Denmark Kemin Europa N.V., Belgium

6.

Kemzyme

7. 8. 9.

Natuzyme Feedzyme Phytezyme

Bioproton Pty Ltd, Australia Agil, England B & G, Co. Ltd., Seoul, Korea Alltech, Nicholasville, Kentucky KY, USA

10.

Allzyme

33

3.4.2 Enzyme-assisted Cold Pressing (EACP) Ground seeds were conditioned at 100 oC for 20 min. Enzymatic hydrolysis was carried out for 4, 5, 6, 7, and 8h period at 35, 40, 45, and 50 oC with enzyme concentrations (0.5, 1.0, 1.5, and 2.0% of seed weight basis) of each of the selected enzyme-mixtures at 3, 4, 5, 6, 7 pH with 35, 40, 45, 50, and 55% moisture. After hydrolysis the enzymes were inactivated for 20 min at 100 oC (Zuniga et al., 2001a). A flow chart showing the schematic process of hydrolysis is presented in Fig. 3.1 Prior to the pressing operation, samples were dried in a vacuum oven (VOC300 SD; EYELA, Tokyo, Japan) at 60 oC up to 3-4% of moisture. Pressing was done in a manual laboratory Hydraulic Press (Carver Press, USA) for 20 min at pressure range of 29.449.0 MPa (Moure et al., 2002). The same set of conditions without the addition of enzyme was used for control.

3.4.2.1 Optimization of Parameters for EACP Enzyme mixtures (Table 3.2) with different enzyme activity were selected for this study. Keeping in view, the literature available and our preliminary trials different enzyme-mixtures were used for each oilseed to find out the best enzyme-mixture during EACP. The enzyme activity for EACP is influenced by the enzyme concentration, temperature, hydrolysis time, moisture, and the pH. These operational parameters were optimized for maximum oil recovery.

3.4.2.1.1 Effect of Moisture The effects of moisture content was analysed at five different levels: 35, 40, 45, 50 and 55% (w/w of seed). For each run, the desired moisture levels were set by pre-dissolving the optimum level of enzyme in an appropriate amount of water and adding it to 25g of the ground seed. The pH of the mixture was maintained with 0.5N NaOH and 0.5 N HCl. The mixture was then incubated in an oven for a period of 6 h at 45 oC.

34

3.4.2.1.2 Effect of Enzyme Concentration The effect of enzyme concentration was observed by using 0.50, 1.00, 1.50, and 2.00 % enzyme on seed weight basis. The enzyme mixture was mixed in distilled water and added to 25 g of dry-ground meal and mixed with a glass rod to get 45% moisture. The sample mixture was incubated in an oven for a period of 6 h at 45 oC. The effect was measured in terms of oil extraction yield.

Seed

Dehulling + Milling

Thermal Treatment (110 oC)

Water

Enzymatic Treatment

Enzymes & Water

Drying

Water

Pressing

Oil

Cake

Fig. 3.1 Flow Chart for Enzyme-assisted Cold Pressing (EACP)

35

3.4.2.1.3 Effect of Temperature The effect of temperature during incubation was evaluated by using five different temperatures: 35, 40, 45, 50, and 55 oC. For each determination, an optimum concentration of each enzyme was pre-dissolved in an appropriate amount of water and adding it to 25 g of gry-ground meal to get 45% moisture content. The pH was adjusted to an optimum level for each enzyme and the sample was placed in an oven for 6 h at temperature varying from 35-55 oC.

3.4.2.1.4 Effect of Hydrolysis Time The effect of hydrolysis time was studied over a period of 4, 5, 6, 7, and 8 hours. An optimum enzyme concentration was pre-dissolved in a suitable amount of water and added to 25 g of dry-ground meal to obtain 45% moisture. The pH was adjusted to an optimum level for each enzyme and the sample was placed in an oven at 45 oC.

3.4.2.1.5 Effect of pH For each run, 25 g of ground seed sample was mixed with an optimum amount of enzyme in an appropriate amount of water to get 45% moisture levels. The pH was set to 3, 4, 5, 6, and 7 by using 0.5 N NaOH and 0.5 N HCl to observe the effect of pH. The mixture was mixed homogenously and then incubated in an oven at 45 oC for a period of 6 h.

3.4.3 Enzyme-assisted Aqueous Extraction (EAAE) Distilled water was mixed with ground seed at different water to seed (w/s) ratios (5:1, 6:1, 7:1, 8:1, and 9:1). The mixture was boiled for 5 min and allowed to cool down to room temperature (Abdulkarim et al., 2005). The pH was adjusted at (3, 4, 5, 6, and 7) for each enzyme mixture with 0.5 N NaOH and 0.5 N HCl. Different enzyme concentrations (0.50, 1.00, 1.50, and 2.00 % seed weight basis) for each of the enzymes were used. The mixture was stirred for different periods of time (1.0, 1.5,

36

2.0, 2.5, and 3 h) and temperature was maintained at 35, 40, 45, 50, and 55 oC. A flow chart of this method is described in Fig. 3.2. After incubation, mixture was centrifuged (7000 rpm, 30 C) for 15 minutes using a centrifuge machine (Sigma, 3K 30, Osterode am Harz, Germany) to separate creamy (or emulsion), supernatant and solid phase (or meal). The creamy phase floating atop was collected using a micro-pipette and transferred to a beaker. The cream left on the wall of the centrifuge tubes was also rinsed with fresh warm water and transferred to the beaker. The wet meal was mixed, weighed and sampled for the determination of moisture content and dry meal. The remaining wet meal was dried overnight in a vacuum oven (VOC-300 SD; EYELA, Tokyo, Japan) at 90-95 oC. The dried meal was ground in a coffee grinder and analysed for residual oil and protein content. The control samples were also preceded under the same set of conditions, except the enzyme addition.

3.4.3.1 Optimization of Parameters for EAAE Various analytical and commercial enzyme-mixtures (Table 3.2) were utilized to evaluate the effect of enzyme on oil recovery from seeds during EAAE. Each of the oilseeds was treated with five enzymes at optimum experimental parameters to find the best enzyme-mixture. The enzyme activity during EAAE is strongly affected by the enzyme concentration, temperature, hydrolysis time, w/s ratio, and the pH. All these operational parameters were optimized for better oil recovery.

3.4.3.1.1 Effect of Water/Seed (w/s) Ratio For each determination, ground seed was mixed with the required distilled water to have w/s ratio: 5:1, 6:1, 7:1, 8:1, and 9:1 ratios. Stirring was done on a magnetic stirring plate at 45 oC with optimum pH and enzyme concentration for 2 h.

37

Seed

Dehulling + Milling

Thermal Treatment (110 oC) Dist. Water at diff. ratios Boiling for 5 min. pH Adjustment Enzymatic Treatment Addition of Enzyme at diff. Conc. Centrifugation

Oil

Oilseed Residue

Fig. 3.2 Flow Chart for Enzyme-assisted Aqueous Extraction (EAAE)

38

3.4.3.1.2 Effect of Enzyme Concentration For each run, 25 g of the ground seed was mixed with distilled water to obtain optimum water/seed ratio and pH in a 500 mL beaker by stirring on a magnetic hot plate at 45 oC for 2 h. Four different enzyme concentrations 0.50, 1.00, 1.50, and 2.00 % on seed weight basis were used to evaluate the effect of enzyme concentration on the oil yield during EAAE.

3.4.3.1.3 Effect of Temperature Ground seed was mixed with distilled water. The effect of temperature was studied with an optimum pH for each enzyme mixture at optimum level of water/seed ratio and enzyme concentration. The temperature was controlled at 35, 40, 45, 50, and 55 oC with a magnetic stirring hot plate and the extraction was carried out for 2 h.

3.4.3.1.4 Effect of Hydrolysis Time Experiments were carried out for five different extraction times: 1, 1.5, 2.0, 2.5, and 3 hours. The ground seed was mixed with distilled water to get optimum water/seed ratio. The temperature was maintained at 45 oC with an optimum pH of each enzyme mixture for a period of 2 h.

3.4.3.1.5 Effect of pH The effect of pH was studied at five different pH levels: 3, 4, 5, 6, and 7. For each trial, ground seed sample was mixed with distilled water to make desired w/s ratio with an optimum concentration of the enzyme mixture. The temperature was maintained at 45 oC on a magnetic stirring hot plate for 2 h.

39

3.5 Quality Evaluation of Oils and Oilseed Residues All the extracted oils and the residues, left after oil extraction were characterized by using various analytical methods.

3.5.1 Proximate Composition of Oilseed Residues After oil extraction, the residue was analyzed for protein, fiber, and ash contents.

3.5.1.1 Protein Protein content (N x 6.25) was determined according to the AOAC (1990) method 954.01.

3.5.1.2 Fiber Fiber content was estimated according to method 5983 (ISO 1977). A finely ground sample (2.5 g) of meal was weighed and freed from fat by extraction with 15mL n-hexane. The test portion was boiled with sulfuric acid (0.255 mol L-1), followed by separation and washing of the insoluble residue. The residue was then boiled with sodium hydroxide (0.313 mol L-1), followed by separation, washing, and drying. The dried residue was weighed and ashed in a muffle furnace (TMF-2100, Eyela, Tokyo, Japan) at 600 oC, and the loss of mass determined.

3.5.1.3 Ash Ash content was determined according to method 749 (ISO 1977). Two grams of the test portion was taken and carbonized by heating on a gas flame. The carbonized material was then ashed in an electric muffle furnace (TMF-2100, Eyela, Tokyo, Japan) at 550 oC until a constant mass was achieved.

40

3.5.2 Analysis of Oils

3.5.2.1 Physico-chemical Parameters of Oils Iodine value, density, unsaponifiable matter, peroxide and saponification values of the control, solvent and enzyme-extracted oils were determined by various standard AOCS (1997) methods. The color and refractive index of the oils were determined by a Lovibond tintometer (Tintometer Ltd., Salisbury, Wiltshire, United Kingdom) using a 1-inch cell and Refractometer (RX-7000, Atago co., Ltd. Japan), respectively.

3.5.2.2 Oxidative Stability The magnitude of oxidative deterioration in oils was assessed by the measurement of induction period (IP), peroxide value (PV), conjugated dienes (CD), conjugated trienes (CT) and para-anisidine (p-anisidine) values.

3.5.2.2a Induction Period (IP) An automated Metrohm Rancimat apparatus, model 743 capable of operating over a temperature range of 50200 C, was used to determine induction periods of the oils. Testing was carried out at 120 0.1 C, and oxidative stability was measured following a procedure described elsewhere (Anwar et al., 2003). Briefly, oil (2.5 g) was carefully weighed into each of the six reaction vessels and analyzed simultaneously. Induction periods of the samples were recorded automatically and corresponded to the break point in the plotted curves.

3.5.2.2b Peroxide Value (PV) The PV is a titration measure of all peroxides and lipid oxidation products that will oxidize the potassium iodide under operating conditions. Five grams of the oil sample was poured into a 250 mL flask. Thirty milliliters of glacial acetic acid/chloroform (3:2, v/v) solutions were added and stirred. A stopper was inserted and the flask was shaken for 1 min and left for 5 min in the dark at 1525 oC. Thirty 41

milliliters of distilled water was added, and the librated iodine was titrated with 0.01 N Na2S2O3, using starch as indicator. The PV was calculated following the (IUPAC) method (1987).

3.5.2.2c Para-anisidine (p-anisidine) Value The determination of p-anisidine value of the oil samples was carried out following an IUPAC method (1987). The oil samples dissolved in isooctane were allowed to react with p-anisidine solution in acetic acid (0.25% w/v) for 10 minutes to produce a colored complex. The absorbance of the coloring mixture value was noted at 350 nm by using spectrophotometer.

3.5.2.2d Cojugated Diene (CD) and Conjugated Triene (CT) The contents of conjugated dienes and trienes, in terms of measurement of specific extinctions at 232 and 268 nm were determined using spectrophotometer (U2001, Hitachi Instrument Inc. Tokyo, Japan). Oil samples were diluted with isooctane to bring the absorbance within limits (0.2-0.8) and [1% 1cm ()] were calculated following an IUPAC method (1987).

3.5.2.3 Fatty Acid (FA) Composition Fatty acid methyl esters (FAMEs) were prepared according to IUPAC method 2.301 (1987) and were analyzed on a Shimadzu (Kyoto, Japan) gas chromatograph, model 17-A, fitted with a methyl-lignocerate-coated film thickness 0.20 m) SP-2330 polar capillary column (30 m 0.32 mm; Supelco Inc., Supelco Park Bellefonte, PA), and a flame ionization detector (FID). Oxygen-free nitrogen was used as a carrier gas at a flow rate of 3.0 mL min-1. Other conditions were as follows: initial oven temperature, 180 C; ramp rate, 5C min-1; final temperature, 220 C; injector temperature, 230 C; detector temperature, 250 C; and temperature hold, 2 min before and 10 min after the run. A sample volume of 1.5 L was injected using split mode (split ratio 1:75). FAMEs were identified by comparing their relative and absolute retention times to those of authentic standards. A data-handling program, Chromatography Station for Windows (CSW32; Data APEX LTD. CZ-158 00 Pague 42

5, The Czech Republic), was used for quantification. The FA composition was reported as percentages.

3.5.2.4 Determination of Tocopherol Tocopherols (, , and ) were analyzed using an HPLC following the CPFA (Current Protocols in Food Analytical Chemistry) methods (CPFA 2003). Oil (0.1 g) and 0.05 g ascorbic acid were weighed accurately into a 16125-mm test tube. Five milliliters of 90.2% ethanol and 0.5 ml of 80% aqueous KOH solution were added to the test tube and vortexed for 30 s. The test tube was flushed with nitrogen, capped and incubated in a water bath (70 oC) for 30 min with periodical vortexing. The tubes were placed in an ice bath for 5 min then 3 ml deionized water and 5 ml n-hexane were added and vortexed for 30 s followed by centrifugation for 10 min at 1,000g (room temperature). The upper hexane layer was transferred to another test tube. The aqueous layer and the residue were re-extracted by repeating the same procedure. The upper hexane layers from both the extractions were combined and evaporated to dryness under nitrogen streaming. One milliliter of mobile phase was added to the tube and vortexed 30 s to re-dissolve the extract and then transferred to an HPLC sample vial. A 20-L sample was injected into a Supelcosil LC-Si column (250 4.6 mm, Supelco Inc. Supelco Park, Bellefonte, USA). A mobile phase of ethyl acetate/acetic acid/hexane (1:1:198, v/v/v) was used at the rate of 1.5 mL min-1. Detection was monitored at 295 nm. Tocopherols were identified by comparing the retention times with those of pure standards of -, -, and - tocopherols, and were quantified on the basis of peak area of the unknowns with those of pure standards (Sigma Chemical Co.). Quantification was based on an external standard method. A D-2500 Hitachi (Hitachi Instruments, Inc., Tokyo, Japan) Chromatointegrator model with a built-in computer program for data handling was used for quantification.

3.5.3 Antioxidant Activity of Extracted Oils

3.5.3.1 Extraction Oil antioxidant constituents were extracted with 80:20 MeOH:H2O v/v. Briefly, one gram of oil was weighed into a test tube and then 3 mL of solvent was 43

added (Parry et al., 2005). The test tube was vortexed and then centrifuged at 6000 rpm for 5 min and the supernatant was collected. The same procedure was repeated further twice and the three extractions were combined. The final volume was brought to 10 mL with the extraction solvent. The resulting antioxidant solution was then kept in the dark under N2 until further analysis.

3.5.3.2 Evaluation of Antioxidant Activity

3.5.3.2a Determination of Total Phenolics (TP)

FolinCiocalteu reagent method was used to determin the amount of TP as described by Chaovanalikit and Wrolstad (2004). 0.5 mL of oil extract solution (0.05 g/5 mL) was mixed with 0.5 mL of 2N FolinCiocalteu reagent and 7.5 mL deionized water. The mixture was kept at room temperature for 10 min. Then 1.5 mL of 20% sodium carbonate (w/v) was added. The mixture was heated on a water bath at 40 oC for 20 min followed by cooling in an ice bath. Finally the absorbance was recorded at 755 nm. The amount of TP was calculated using a calibration curve for Gallic acid (10-130 ppm) and the results were expressed as Gallic acid equivalents (GAE) mg/100g of oil.

3.5.3.2b DPPH Radical Scavenging Assay The antioxidant activity of the oil extracts was assessed by measuring the scavenging ability to 2,2-diphenyl-1-picrylhydrazyl stable radical. The DPPH assay was performed as described by Bozin et al. (2006). The oil extract samples (0.5 to 15.5 g mL-1) were mixed with 1 mL (90 M DPPH solution) and filled up with 95% MeOH, to make a final volume of 4 mL. The absorbance of this solution and the blank were recorded after keeping for 1 h at room temperature. Butylated hydroxytoluene (BHT) was used as a positive control. Three replicates were performed for each sample. The disappearance of DPPH was determined spectrophotometrically by measuring absorbance at 515 nm using a spectrophotometer (U-2001, Hitachi Instruments, Inc., Tokyo, Japan). Scavenging of free radical (DPPH) in percent (%) was calculated by the following formula:

44

I (%) = 100 x (Ablank - Asample/Ablank) Where Ablank is the absorbance of the control reaction mixture excluding the test compounds, and Asample is the absorbance of the test compounds. IC50 values, which represented the minimum concentration of oil extracts required to neutralization 50% of DPPH radicals, were calculated from the plot of inhibition percentage against concentration.

3.5.3.2c Percent Inhibition in Linoleic Acid System The antioxidant activity of oil extract was determined by inhibition of linoleic acid oxidation, following the method described by Singh and Marimuthu (2006) with slight modification. The oil extract test sample (50 L) was dissolved in 1 mL of ethanol, mixed with linoleic acid (2.5%, v/v), 99.5% ethanol (4 mL) and 4 mL of 0.05 M sodium phosphate buffer (pH 7). Then the solution was incubated at 40 C for 175 hrs. The degree of oxidation was measured by peroxide value using the colorimetric method described by Yen et al., (2000). To 0.2 mL sample solution, 10 mL of ethanol (75%), 0.2 mL of an aqueous solution of ammonium thiocyanate (30%) and 0.2 mL of ferrous chloride solution (20 mM in 3.5% HCl) were added sequentially. After stirring for 3 min, the absorbance was measured at 500 nm, using a spectrophotometer (U2001, Hitachi Instruments, Inc., Tokyo, Japan). A control was performed with linoleic acid without the sample. Butylated hydroxytoluene (BHT) was used as positive control. Percent inhibition of linoleic acid oxidation was calculated as follows: % inhibition of linoleic acid oxidation = 100 [(Abs. increase of sample at 175h / Abs. increase of control at 175h) 100]

3.6 Statistical Analysis Values are reported as mean SD of three seed samples for each enzyme, analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05). One way ANOVA was used to determine significant differences between groups, considering a level of significance of less than 5% (P < 0.05) by using the statistical software STATISTICA 5.5 (Stat Soft Inc, Tulsa, Oklahoma, USA). 45

CHAPTER 4 RESULTS

46

Part I Characterization

47

Table 4.1.1a Comparison of Proximate Composition of Enzyme-assisted Cold Pressed Cottonseeds* Parameter (%) Oil content Protein content Fiber content Ash content Solventextracted 16.2 0.3a 27.1 0.6a 21.1 0.5a 4.7 0.2a Enzyme-assisted Natuzyme Kemzyme 12.1 0.4c 12.9 0.3b 24.7 0.7b 25.1 0.5b 20.8 0.6a 20.1 0.6b 4.7 0.2a 4.7 0.3a Control 8.5 0.3e 24.9 0.8b 20.9 0.8a 4.4 0.2a

Phytezyme 11.7 0.3c 25.8 0.8ab 20.2 0.7b 4.4 0.3a

Allzyme 9.7 0.3d 27.9 0.7a 20.5 0.6ab 4.6 0.3a

Feedzyme 11.7 0.5c 25.8 0.6ab 19.7 0.6b 4.5 0.3a

Values are means SD, calculated as percentage on dry seed weight basis for three cottonseed samples for each enzyme analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Table 4.1.1b Comparison of Proximate Composition of Enzyme-assisted Aqueous Extracted Cottonseeds* Parameter (%) Oil content Protein content Fiber content Ash content Solventextracted 16.2 0.3a 27.1 0.6a 21.1 0.5a 4.7 0.2a Enzyme-assisted Allzyme Natuzyme 5.5 0.1c 5.3 0.2c f 17.3 1.0 18.3 1.0e 20.8 0.6bc 20.8 0.4b 4.6 0.3b 4.5 0.2bc Control 4.4 0.2d 24.4 0.9b 21.0 1.2a 4.6 0.2a

Phytezyme 4.2 0.2ed 18.8 0.8d 20.9 0.6ab 4.5 0.2c

Kemzyme 6.4 0.1b 19.5 0.9c 20.8 1.1b 4.5 0.3bc

Feedzyme 4.2 0.1e 18.4 0.7ed 20.7 0.9c 4.5 0.1bc

Values are means SD, calculated as percentage on dry seed weight basis for three cottonseed samples for each enzyme analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

48

Table 4.1.2a Comparison of Physico-chemical Properties of Enzyme-assisted Cold Pressed Cottonseed Oils* Parameter Refractive Index (40 oC) Density (24 oC g mL-1) Saponification value (mg KOH/g oil) Free fatty acids contents (% as oleic acid) Iodine value (g I/ 100g oil) Unsaponifiable matter (% w/w) Color (1-in. cell) Red units Yellow units Solventextracted 1.4642 0.03a 0.93 0.01a 179 3a 4.90 0.09a 103 7a 0.57 0.03a 13.1 r 0.4c 60.3 y 1.8a Enzyme-assisted Natuzyme 1.4637 0.01a 0.93 0.02a 174 4b 3.30 0.15c 106 11a 0.53 0.08a 25.4 r 1.2ab 60.1 y 1.5a Control 1.4637 0.02a 0.94 0.03a 174 7b 3.22 0.14c 105 9a 0.54 0.05a 24.4 r 1.4ab 60.2 y 1.6a

Phytezyme 1.4635 0.01a 0.94 0.03a 175 2b 3.80 0.24b 105 8a 0.53 0.04a 24.5 r 1.3ab 50.1 y 2.0b

Allzyme 1.4636 0.04 a 0.94 0.02a 176 3ab 3.24 0.07c 105 8a 0.54 0.02a 23.2 r 1.9b 49.7 y 0.8b

Kemzyme 1.4639 0.02a 0.93 0.03a 176 2ab 3.90 0.08b 105 8a 0.54 0.04a 23.2 r 1.0b 60.4 y 1.2a

Feedzyme 1.4637 0.03a 0.93 0.01a 176 4ab 3.60 0.12b 105 8a 0.54 0.07a 26.1 r 1.3a 59.8 y 1.8a

Values are means SD, of three cottonseed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

49

Table 4.1.2b Comparison of Physico-chemical Properties of Enzyme-assisted Aqueous Extracted Cottonseed Oils* Parameter Refractive Index (40 oC) Density (24 oC g mL-1) Saponification value (mg KOH/g oil) Free fatty acids contents (% as oleic acid) Iodine value (g I/ 100g oil) Unsaponifiable matter (% w/w) Color (1-in. cell) Red units Yellow units Solventextracted 1.4642 0.03a 0.93 0.01a 179 3a 4.90 0.09a 103 7a 0.57 0.03a 13.1 r 0.4a 60.3 y 1.8a Enzyme-assisted Natuzyme 1.4638 0.03a 0.93 0.02a 173 4c 4.19 0.12cd 105 8a 0.53 0.03a 11.2 r 0.7b 50.6 y 2.4b Control 1.4636 0.02a 0.93 0.01a 175 6bc 4.18 0.18cd 105 3a 0.54 0.06a 10.3 r 0.4bc 45.5 y 1.3c

Phytezyme 1.4637 0.02a 0.93 0.03a 176 6b 4.24 0.07bc 104 4a 0.54 0.06a 10.5 r 0.3bc 50.2 y 1.6b

Allzyme 1.4635 0.03a 0.93 0.02a 175 3bc 4.18 0.08d 106 5a 0.55 0.02a 9.3 r 0.5c 45.8 y 1.5c

Kemzyme 1.4636 0.02a 0.93 0.01a 174 8bc 4.22 0.13c 104 6a 0.55 0.07a 10.5 r 0.4bc 45.9 y 1.8c

Feedzyme 1.4637 0.03a 0.93 0.03a 175 3bc 4.27 0.09b 103 4a 0.54 0.04a 11.7 r 0.3b 50.8 y 1.7b

Values are means SD, of three cottonseed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

50

Table 4.1.3a Comparison of Oxidative State of Enzyme-assisted Cold Pressed Cottonseed Oils* Parameter Conjugated diene 1% 1cm (232) Conjugated triene 1% 1cm (270) Peroxide value (milli eq/kg) p-anisidine value
Induction Period Rancimat method (h)

Solventextracted 2.61 0.05a 0.96 0.02a 1.88 0.01a 2.15 0.04a 1.85 0.06c
*

Phytezyme 1.98 0.02b 0.91 0.03a 1.56 0.01b 1.83 0.05c 1.94 0.05a

Allzyme 2.04 0.09b 0.94 0.03a 1.50 0.02b 1.87 0.03b 1.92 0.06b

Enzyme-assisted Natuzyme Kemzyme 2.10 0.06b 0.96 0.02a 1.52 0.01b 1.80 0.02c 1.94 0.04a 2.00 0.09b 0.93 0.03a 1.54 0.02b 1.84 0.04bc 1.93 0.04a

Feedzyme 1.96 0.06b 0.91 0.02a 1.56 0.01b 1.85 0.05bc 1.94 0.05a

Control 2.12 0.10b 0.92 0.03a 1.52 0.01b 1.82 0.03c 1.93 0.05a

Values are means SD, of three cottonseed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Table 4.1.3b Comparison of Oxidative State of Enzyme-assisted Aqueous Extracted Cottonseed Oils* Parameter Conjugated diene 1% 1cm (232) Conjugated triene 1% 1cm (270) Peroxide value (milli eq/kg) p-anisidine value
Induction Period Rancimat method (h)

Solventextracted 2.61 0.05a 0.96 0.02a 1.88 0.01a 2.15 0.04a 1.88 0.06c
*

Phytezyme 2.17 0.11c 0.82 0.05b 1.54 0.07bc 1.91 0.06bc 1.93 0.07ab

Allzyme 2.25 0.10b 0.81 0.06b 1.52 0.09bc 1.94 0.05b 1.92 0.04b

Enzyme-assisted Natuzyme Kemzyme 2.14 0.09c 0.84 0.04b 1.51 0.08c 1.92 0.04bc 1.95 0.06ab 2.23 0.10b 0.82 0.06b 1.56 0.07b 1.89 0.06c 1.91 0.05b

Feedzyme 2.28 0.07b 0.83 0.04b 1.53 0.06bc 1.93 0.05b 1.96 0.05a

Control 2.24 0.11b 0.82 0.04b 1.55 0.05b 1.95 0.04b 1.97 0.06a

Values are means SD, of three cottonseed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

51

Table 4.1.4a Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzyme-assisted Cold Pressed Cottonseed Oils* FA C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 Solventextracted 0.54 0.02d 23.17 0.53a 0.45 0.01d 3.74 0.06a 21.90 0.52c 49.72 1.39a 0.11 0.02b 0.21 0.01a Enzyme-assisted Phytezyme 0.61 0.01c 23.11 0.39a 0.53 0.02c 3.13 0.05b 23.33 0.68ab 48.87 1.27a 0.13 0.01b 0.15 0.01b
*

Allzyme 0.83 0.03a 22.65 0.63b 0.44 0.01d 3.12 0.07b 23.79 0.43a 48.54 0.97a 0.15 0.01ab 0.19 0.02a

Natuzyme 0.64 0.01c 23.07 0.47a 0.67 0.02a 3.24 0.09b 22.96 0.34b 48.93 1.16a 0.14 0.02ab 0.18 0.02ab

Kemzyme 0.76 0.02b 22.84 0.23ab 0.59 0.03b 3.07 0.06b 23.13 0.27a 49.12 1.31a 0.17 0.03a 0.20 0.01a

Feedzyme 0.52 0.01d 22.79 0.59b 0.54 0.01c 3.16 0.03b 23.38 0.41ab 49.27 1.67a 0.11 0.01b 0.19 0.02a

Control 0.65 0.03c 23.10 0.32a 0.65 0.02a 3.19 0.08b 23.14 0.56b 48.76 1.28a 0.12 0.01b 0.17 0.03ab

Values are means SD, of three cottonseed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

52

Table 4.1.4b Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzyme-assisted Aqueous Extracted Cottonseed Oils* FA C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 Solventextracted 0.54 0.02ab 23.17 0.53a 0.45 0.01ab 3.74 0.06ab 21.90 0.52bc 49.72 1.39bc 0.11 0.02b 0.21 0.01a Enzyme-assisted Phytezyme 0.53 0.01b 23.09 0.45a 0.51 0.02a 3.68 0.08bc 22.14 0.32a 50.16 1.17a 0.14 0.02bc 0.18 0.01
*

Allzyme 0.56 0.02ab 22.94 0.64b 0.53 0.01a 3.71 0.06b 22.12 0.42a 49.84 0.98b 0.13 0.01bc 0.17 0.02bc

Natuzyme 0.57 0.02a 22.96 0.21b 0.48 0.02ab 3.76 0.04a 21.94 0.24b 49.82 1.33b 0.11 0.03b 0.19 0.01bc

Kemzyme 0.55 0.03ab 23.12 0.36a 0.44 0.02b 3.66 0.07c 21.97 0.53b 49.68 1.24c 0.12 0.01b 0.15 0.03b

Feedzyme 0.58 0.01a 23.11 0.38a 0.45 0.02b 3.72 0.05b 21.84 0.66c 49.91 1.26b 0.16 0.02a 0.20 0.01a

Control 0.54 0.02ab 23.15 0.54a 0.47 0.01ab 3.78 0.07a 21.92 0.57bc 49.76 1.38bc 0.15 0.03a 0.18 0.02bc

Values are means SD, of three cottonseed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

53

Table 4.1.5a Comparison of Tocopherols in Enzyme-assisted Cold Pressed Cottonseed Oils* Tocopherols (mg kg-1) Total Solventextracted 240 9ab 380 7a 14.2 0.3b 634.2 Enzyme-assisted Control Phytezyme 250 10a 490 6a 9.7 0.4e 749.7 Allzyme 130 9d 422 7a 18.3 0.6a 570.3 Natuzyme 230 11ab 370 5a 12.9 0.8c 612.9 Kemzyme 160 10c 390 16a 14.5 0.4b 564.5 Feedzyme 220 13b 460 19a 10.6 0.5e 690.6 145 9cd 440 14b 12.1 0.3d 597.1

Values are means SD, of three cottonseed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Table 4.1.5b Comparison of Tocopherols in Enzyme-assisted Aqueous Extracted Cottonseed Oils* Tocopherols (mg kg-1) Total Solventextracted 240 9c 380 7c 14.2 0.3b 634.2 Enzyme-assisted Natuzyme 280 14ab 390 21bc 10.1 0.5c 680.1 Control 260 8b 394 17bc 14.7 1.2ab 668.7

Values are means SD, of three cottonseed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Phytezyme 290 10a 410 6a 15.9 0.7a 715.9

Allzyme 295 16a 417 9a 15.4 0.8b 727.4

Kemzyme 256 10b 410 15a 16.6 0.6a 682.6

Feedzyme 272 13ab 405 15b 16.2 1.3a 693.2

54

Table 4.1.6a Comparison of Antioxidant Activity of Enzyme-assisted Cold Pressed Cottonseed Oils* Parameter TPC (mg GAE/100 g) DPPH, IC50 (g mL-1) Inhibition in linoleic acid system (%) after 175 h incubation at 40C Solventextracted 1.2 0.1 a 65.3 3.2 23.6 0.8
*

Phytezyme a 1.4 0.2 c 58.6 2.7 44.7 0.7

b

Allzyme a 1.5 0.1 c 58.9 4.1 45.2 0.9

b

Enzyme-assisted Natuzyme Kemzyme b a 1.2 0.3 1.5 0.2 c c 58.9 3.7 58.7 3.5 44.8 0.8
b

Feedzyme ab 1.3 0.1 b 59.8 4.2 45.9 1.3

b

Control 1.4 0.3 a 65.2 2.9 45.6 0.8

b a

47.5 0.7

Values are means SD, of three cottonseed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Table 4.1.6b Comparison of Antioxidant Activity of Enzyme-assisted Aqueous Extracted Cottonseed Oils* Parameter TPC (mg GAE/100 g) DPPH, IC50 (g mL-1) Inhibition in linoleic acid system (%) after 175 h incubation at 40C Solventextracted 1.2 0.1 a 65.3 3.2 23.6 0.8
*

Phytezyme c 1.5 0.3 b 58.9 1.5 45.1 1.3

b

Allzyme b 1.4 0.2 bc 57.7 1.3 46.4 1.8

b

Enzyme-assisted Natuzyme Kemzyme c a 1.6 0.1 1.7 0.4 b c 59.1 1.9 56.5 1.8 46.0 1.5
b

Feedzyme b 1.5 0.2 bc 58.2 2.3 47.3 0.9

a

Control 1.3 0.3 a 64.6 3.2 46.2 1.4

b c

48.7 1.2

Values are means SD, of three cottonseed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

55

Table 4.2.1a Comparison of Proximate Composition of Enzyme-assisted Cold Pressed Hempseeds* Parameter (%) Oil content Protein content Fiber content Ash content Enzyme-assisted Viscozyme L 32.8 0.3a 24.7 0.5ab 17.3 0.4b 5.4 0.3a Control 26.7 0.6d 26.0 0.4a 17.5 0.5b 5.4 0.3a

Kemzyme 32.3 0.5a 24.1 0.4b 17.1 0.6b 5.4 0.5a

Protex 7L 28.4 0.4c 25.2 0.5ab 17.4 0.3b 5.4 0.2a

Feedzyme 30.3 0.4b 26.5 0.7a 18.2 0.6a 5.4 0.3a

Natuzyme 28.9 0.5c 25.0 0.6ab 18.1 0.5a 5.4 0.4a

Values are mean SD, calculated as percentage on dry seed weight basis for three hempseed samples for each enzyme, analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Table 4.2.1b Comparison of Proximate Composition of Enzyme-assisted Aqueous Extracted Hempseeds* Parameter (%) Oil content Protein content Fiber content Ash content Enzyme-assisted Viscozyme L Feedzyme 29.1 0.6a 28.3 0.5bc 15.9 0.2c 15.8 0.4d bc 17.2 0.4 17.4 0.6b 5.3 0.2a 5.4 0.1a Control 25.8 0.6e 16.5 0.3a 17.6 0.3a 5.4 0.2a

Kemzyme 28.2 0.3c 15.8 0.4cd 17.5 0.5ab 5.3 0.1a

Protex 7L 27.4 0.4d 16.2 0.3b 17.1 0.7c 5.3 0.1a

Natuzyme 28.5 0.7b 15.9 0.2c 17.4 0.5b 5.3 0.1a

Values are mean SD, calculated as percentage on dry seed weight basis for three hempseed samples for each enzyme, analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

56

Table 4.2.2a Comparison of Physico-chemical Properties of Enzyme-assisted Cold Pressed Hempseed Oils* Parameter Refractive Index (40 oC) Density (24 oC g mL-1) Saponification value (mg KOH/g of oil) Free fatty acids contents (% as oleic acid) Iodine value (g of I/ 100g of oil) Unsaponifiable matter (% w/w) Color (1-in. cell) Red units Yellow units Blue units Enzyme-assisted Viscozyme L 1.4703 0.02a 0.93 0.01a 184 4a 1.87 0.02a 155 6ab 0.24 0.01a 5.3 r 0.1a 49.7 y 2.2a 4.8 b 0.2a Control 1.4699 0.03a 0.93 0.02a 186 3a 1.75 0.07ab 155 5ab 0.27 0.02a 4.4 r 0.1b 39.8 y 1.2b
..

Kemzyme 1.4703 0.02a 0.93 0.03b 183 3ab 1.73 0.04b 152 5b 0.26 0.01a 5.3 r 0.2a 50.3 y 1.5a 4.8 b 0.2a
*

Protex 7L 1.4702 0.01a 0.93 0.02a 181 2b 1.84 0.05a 154 4ab 0.25 0.02a 5.5 r 0.2a 50.4 y 2.6a 4.7 b 0.1a

Feedzyme 1.4701 0.01a 0.93 0.02ab 185 7a 1.79 0.09ab 158 4a 0.27 0.03a 5.4 r 0.2a 50.3 y 1.6a 4.8 b 0.1a

Natuzyme 1.4702 0.02a 0.93 0.01ab 182 2b 1.86 0.06a 157 3ab 0.26 0.02a 5.4 r 0.3a 50.5 y 1.9a 4.8 b 0.1a

Values are mean SD of three hempseed oils, analyzed individually in triplicate Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

57

Table 4.2.2b Comparison of Physico-chemical Properties of Enzyme-assisted Aqueous Extracted Hempseed Oils* Parameter Refractive Index (40 oC) Density (24 oC g mL-1) Saponification value (mg KOH/g of oil) Free fatty acids contents (% as oleic acid) Iodine value (g of I/ 100g of oil) Unsaponifiable matter (% w/w) Color (1-in. cell) Red units Yellow units Enzyme-assisted Viscozyme L 1.4702 0.01a 0.93 0.02a 182 8a 1.81 0.04b 152 3bc 0.21 0.03b 3.5 r 0.1a 31.8 y 1.9a Control 1.4703 0.02a 0.93 0.03a 182 7a 1.85 0.06a 154 3bc 0.25 0.03a 3.2 r 0.2b 30.4 y 2.6a

Kemzyme 1.4701 0.01a 0.93 0.02a 184 6a 1.86 0.03a 155 4b 0.24 0.02ab 3.1 r 0.2b 31.4 y 1.7a

Protex 7L 1.4703 0.02a 0.93 0.03a 181 4a 1.84 0.02a 158 9a 0.22 0.01b 3.2 r 0.2b 32.3 y 1.4a

Feedzyme 1.4702 0.03a 0.93 0.01a 181 5a 1.83 0.03ab 156 6b 0.25 0.02a 3.1 r 0.2b 29.7 y 1.3a

Natuzyme 1.4701 0.01a 0.93 0.02a 183 3a 1.82 0.04ab 151 5c 0.26 0.01a 3.4 r 0.2a 31.3 y 2.2a

Values are mean SD of three hempseed oils, analyzed individually in triplicate Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

58

Table 4.2.3a Comparison of Oxidative State of Enzyme-assisted Cold Pressed Hempseed Oils* Parameter Conjugated diene 1% 1cm (232) Conjugated triene 1% 1cm (270) Peroxide value (milli eq/kg) p-anisidine value
Induction Period Rancimat method (h)

Kemzyme 3.77 0.14a 0.62 0.02a 1.84 0.04a 2.11 0.03a 1.49 0.05b
*

Enzyme-assisted Protex 7L Viscozyme L Feedzyme 3.76 0.17a 0.58 0.01b 1.57 0.05c 1.86 0.04bc 1.52 0.05ab 3.78 0.12a 0.61 0.03a 1.59 0.06bc 1.83 0.05c 1.53 0.06ab 3.79 0.09a 0.59 0.02b 1.62 0.03b 1.88 0.02b 1.51 0.04ab

Natuzyme 3.80 0.15a 0.62 0.03a 1.56 0.01c 1.87 0.02bc 1.56 0.03a

Control 3.78 0.11a 0.63 0.01a 1.57 0.01c 1.89 0.03b 1.55 0.30a

Values are mean SD of three hempseed oils, analyzed individually in triplicate Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Table 4.2.3b Comparison of Oxidative State of Enzyme-assisted Aqueous Extracted Hempseed Oils* Parameter Conjugated diene 1% 1cm (232) Conjugated triene 1% 1cm (270) Peroxide value (milli eq/kg) p-anisidine value
Induction Period Rancimat method (h)

Kemzyme 3.65 0.07a 0.56 0.04ab 1.84 0.05a 2.11 0.07a 1.42 0.06b
*

Enzyme-assisted Protex 7L Viscozyme L Feedzyme 3.62 0.06b 0.52 0.03b 1.59 0.04c 1.88 0.05bc 1.46 0.07a 3.67 0.06a 0.57 0.02ab 1.63 0.06bc 1.91 0.05b 1.43 0.08b 3.65 0.12a 0.56 0.03ab 1.62 0.06bc 1.89 0.07bc 1.47 0.09a

Natuzyme 3.61 0.14b 0.54 0.03b 1.58 0.04c 1.92 0.05b 1.45 0.08ab

Control 3.64 0.09ab 0.59 0.01a 1.66 0.05b 1.87 0.06c 1.43 0.12b

Values are mean SD of three hempseed oils, analyzed individually in triplicate Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

59

Table 4.2.4a Comparison of Fatty acid (FA) Composition (g/100g FA) of Enzyme-assisted Cold Pressed Hempseed Oils* FA Kemzyme C16:0 C18:0 C18:1 C18:2 C18:3 C18:3 C20:1 6.14 0.14a 2.24 0.10ab 12.49 0.24b 57.45 1.17a 18.31 0.20b 1.91 0.05a 0.91 0.04a
*

Protex 7L 6.16 0.15a 2.21 0.04ab 12.63 0.25b 55.18 1.28b 18.56 0.15ab 1.86 0.03ab 0.88 0.02a

Enzyme-assisted Viscozyme L 6.11 0.17a 2.26 0.08ab 12.74 0.27ab 54.22 1.24b 18.65 0.14a 1.85 0.07b 0.92 0.03a

Feedzyme 6.07 0.13a 2.19 0.07b 12.87 0.22a 55.19 1.20b 18.73 0.15a 1.87 0.05ab 0.89 0.05a

Natuzyme 5.91 0.19b 2.31 0.06a 12.85 0.28a 57.89 1.00a 18.29 0.27b 1.84 0.06b 0.88 0.04a

Control 5.95 0.12b 2.25 0.06ab 12.55 0.19b 56.61 1.15ab 18.38 0.19b 1.85 0.07b 0.87 0.03a

Values are mean SD of three hempseed oils, analyzed individually in triplicate Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

60

Table 4.2.4b Comparison of Fatty acid (FA) Composition (g/100g FA) of Enzyme-assisted Aqueous Extracted Hempseed Oils* FA Kemzyme C16:0 C18:0 C18:1 C18:2 C18:3 C18:3 C20:1 6.04 0.12ab 2.22 0.07b 12.86 0.19a 55.21 1.24ab 18.72 0.16ab 1.86 0.02ab 0.88 0.01ab
*

Protex 7L 6.12 0.15a 2.26 0.04a 12.51 0.16c 57.43 1.17a 18.29 0.19b 1.93 0.03a 0.91 0.02a

Enzyme-assisted Viscozyme L 6.17 0.18a 2.22 0.03b 12.53 0.24c 55.28 2.13ab 18.52 0.15ab 1.89 0.04a 0.86 0.03ab

Feedzyme 5.95 0.11b 2.27 0.05a 12.54 0.18c 56.71 2.29a 18.28 0.12b 1.81 0.02b 0.91 0.01a

Natuzyme 6.14 0.16a 2.23 0.04b 12.77 0.21b 54.19 2.09b 18.68 0.14a 1.83 0.01b 0.94 0.02a

Control 6.05 0.18ab 2.19 0.02b 12.89 0.15a 55.29 1.42ab 18.63 0.17a 1.84 0.02ab 0.83 0.01b

Values are mean SD of three hempseed oils, analyzed individually in triplicate Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

61

Table 4.2.5a Comparison of Tocopherols in Enzyme-assisted Cold Pressed Hempseed Oils* Tocopherols (mg kg-1) Total Enzyme-assisted Viscozyme L 40 3c 672 14bc 24.7 1.4c 736.7 Control 43 3b 615 8c 33.2 1.5ab 691.2

Kemzyme 53 8a 685 12b 35.8 2.1a 773.8

Values are mean SD of three hempseed oils, analyzed individually in triplicate Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Protex 7L 42 5b 654 16bc 28.4 1.6b 724.4

Feedzyme 38 6c 693 10b 24.1 1.7c 755.1

Natuzyme 35 4c 725 13a 28.4 0.9b 788.4

Table 4.2.5b Comparison of Tocopherols in Enzyme-assisted Aqueous Extracted Hempseed Oils* Tocopherols (mg kg-1) Total Enzyme-assisted Viscozyme L 55 4a 697 19a 38.3 1.7b 790.3 Control 41 6c 641 23c 34.4 2.8d 716.4

Kemzyme 47 5ab 629 11d 38.4 1.9b 714.4

Values are mean SD of three hempseed oils, analyzed individually in triplicate Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Protex 7L 49 2a 688 13ab 35.7 2.5c 772.7

Feedzyme 48 7ab 692 15a 34.5 1.5d 774.5

Natuzyme 46 3b 674 14b 41.6 1.9a 761.6

62

Table 4.2.6a Comparison of Antioxidant Activity of Enzyme-assisted Cold Pressed Hempseed Oils* Parameter TPC (mg GAE/100 g) DPPH, IC50 (g mL-1) Inhibition in linoleic acid system (%) after 175 h incubation at 40C Enzyme-assisted Protex 7L Viscozyme L Feedzyme ab a b 2.5 0.4 2.6 0.2 2.8 0.5 c c b 51.6 1.4 51.5 1.8 52.6 2.1 53.8 1.3
a

Kemzyme b 2.6 0.3 b 52.3 1.2 54.3 0.9

*

Natuzyme a 2.6 0.3 c 51.7 1.5 54.8 1.8

ab

Control 2.4 0.6 a 58.4 1.1 52.4 1.4

a b

56.1 1.7

55.3 1.5

Values are mean SD of three hempseed oils, analyzed individually in triplicate Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Table 4.2.6b Comparison of Antioxidant Activity of Enzyme-assisted Aqueous Extracted Hempseed Oils* Parameter TPC (mg GAE/100 g) DPPH, IC50 (g mL-1) Inhibition in linoleic acid system (%) after 175 h incubation at 40C Enzyme-assisted Protex 7L Viscozyme L Feedzyme ab a b 2.5 0.3 2.8 0.5 2.6 0.6 c c bc 51.7 1.5 51.4 1.9 52.2 2.4 54.9 1.5
a

Kemzyme ab 2.5 0.4 b 53.6 1.1 56.4 1.4

*

Natuzyme a 2.6 0.3 c 52.4 1.3 54.5 1.2

a

Control 2.4 0.4 a 57.9 1.1 53.2 1.6

ab b

55.6 2.1

55.8 1.9

Values are mean SD of three hempseed oils, analyzed individually in triplicate Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

63

Table 4.3.1a Comparison of Proximate Composition of Enzyme-assisted Cold Pressed Sunflower Seeds* Parameter (%) Oil content Protein content Fiber content Ash content Solventextracted 45.5 1.7a 18.7 0.8a 3.2 0.4a 2.8 0.3a Enzyme-assisted Alcalase 2.4L Viscozyme L 36.4 1.5c 39.2 1.6b a 18.6 1.3 18.7 0.9a 3.1 0.1a 3.2 0.2a a 2.8 0.2 2.7 0.1a Control 23.4 1.4d 18.5 1.2a 3.2 0.3a 2.7 0.1a

Protex 7L 37.2 1.5c 18.8 1.1a 3.1 0.2a 2.9 0.1a

Kemzyme 38.6 1.7bc 18.7 0.9a 3.0 0.1a 2.7 0.3a

Natuzyme 38.1 1.7bc 18.8 1.4a 3.1 0.1a 2.6 0.2a

Values are mean SD, calculated as percentage on dry seed weight basis for three sunflower seed samples for each enzyme, analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Table 4.3.1b Comparison of Proximate Composition of Enzyme-assisted Aqueous Extracted Sunflower Seeds* Parameter (%) Oil extracted Protein (residue) Fiber content Ash content Solventextracted 45.5 1.7a 18.7 0.8a 3.2 0.4a 2.8 0.3a Enzyme-assisted Alcalase 2.4L Viscozyme L 26.6 1.3d 39.7 1.4ab 15.0 1.1d 14.6 1.2d 3.1 0.3a 3.0 0.2a a 2.8 0.2 2.8 0.1a Control 18.3 1.3e 16.1 0.6b 3.1 0.1a 2.8 0.2a

Protex 7L 28.3 1.4cd 13.5 0.7e 3.0 0.2a 2.7 0.1a

Kemzyme 32.2 1.3c 15.2 0.9c 3.2 0.2a 2.9 0.3a

Natuzyme 35.5 1.6b 15.1 0.7cd 3.2 0.2a 2.6 0.3a

Values are mean SD, calculated as percentage on dry seed weight basis for three sunflower seed samples for each enzyme, analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

64

Table 4.3.2a Comparison of Physicochemical Properties of Enzyme-assisted Cold Pressed Sunflower Seed Oils* Parameter Refractive Index (40 oC) Density (20 oC g mL-1) Saponification value (mg KOH/g of oil) Free fatty acids contents (% as oleic acid) Iodine value (g of I/ 100g of oil) Unsaponifiable matter (% w/w) Color (1-in. cell) Red units Yellow units Solventextracted 1.4682 0.02a 0.92 0.03a 190 3a 0.94 0.08a 127 9c 0.51 0.04a 1.9 r 0.4c 19.5 y 0.5c Enzyme-assisted Alcalase 2.4L 1.4679 0.03a 0.92 0.01a 184 3b 0.78 0.04b 129 3bc 0.46 0.06ab 2.6 r 0.1ab 26.4 y 0.7ab Control 1.4678 0.03a 0.92 0.04a 186 8b 0.79 0.02b 135 7a 0.44 0.02b 2.7 r 0.2a 26.9 y 0.7a

Protex 7L 1.4679 0.01a 0.92 0.02a 186 6b 0.82 0.02b 132 4ab 0.43 0.03b 2.5 r 0.1ab 23.2 y 0.8b

Kemzyme 1.4681 0.01a 0.92 0.03a 182 5b 0.81 0.05b 131 6b 0.45 0.02ab 2.3 r 0.3b 22.7 y 0.8b

Viscozyme L 1.4678 0.02a 0.92 0.03a 188 7b 0.79 0.07b 134 8a 0.45 0.02ab 2.5 r 0.2ab 25.6 y 0.6ab

Natuzyme 1.4677 0.02a 0.92 0.02a 185 5b 0.81 0.03b 133 4ab 0.43 0.05b 2.8 r 0.1a 28.3 y 0.9a

Values are means SD, of three sunflower seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

65

Table 4.3.2b Comparison of Physicochemical Properties of Enzyme-assisted Aqueous Extracted Sunflower Seed Oils* Parameter Refractive Index (40 oC) Density (20 oC g mL-1) Saponification value (mg KOH/g of oil) Free fatty acids contents (% as oleic acid) Iodine value (g of I/ 100g of oil) Unsaponifiable matter (% w/w) Color (1-in. cell) Red units Yellow units Solventextracted 1.4682 0.02a 0.92 0.03a 190 3a 0.94 0.08a 127 9a 0.51 0.04b 1.9 r 0.4a 19.5 y 0.5a Enzyme-assisted Alcalase 2.4L 1.4676 0.02b 0.92 0.03a 187 5ab 0.66 0.04bc 124 2ab 0.47 0.04a 1.6 r 0.3b 16.6 y 0.4b Control 1.4678 0.02b 0.92 0.03a 187 3ab 0.68 0.04b 120 4a 0.54 0.04ab 1.5 r 0.2b 15.7 y 0.3b

Protex 7L 1.4678 0.03b 0.92 0.02a 187 4ab 0.69 0.02b 122 4a 0.45 0.03b 1.7 r 0.3ab 17.3 y 0.4ab

Kemzyme 1.4677 0.01b 0.92 0.02a 186 2b 0.65 0.03c 121 3b 0.47 0.02ab 1.5 r 0.2b 15.1 y 0.2b

Viscozyme L 1.4676 0.01b 0.92 0.02a 185 4b 0.64 0.02c 121 5a 0.45 0.03ab 1.6 r 0.2b 16.4 y 0.3b

Natuzyme 1.4677 0.03b 0.92 0.04a 187 6ab 0.67 0.03bc 123 3ab 0.43 0.02ab 1.7 r 0.2ab 17.2 y 0.2ab

Values are means SD, of three sunflower seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

66

Table 4.3.3a Comparison of Oxidative State of Enzyme-assisted Cold Pressed Sunflower Seed Oils* Parameter Conjugated diene 1% 1cm (232) Conjugated triene 1% 1cm (270) Peroxide value (milli eq/kg) p-anisidine
Induction Period Rancimat method (h)

Solventextracted 3.28 0.09a 0.82 0.04a 1.78 0.06a 1.93 0.05a 1.82 0.12c
*

Protex 7L 3.09 0.12c 0.71 0.04b 1.35 0.14bc 1.68 0.08bc 1.87 0.05a

Kemzyme 3.07 0.12c 0.73 0.03b 1.38 0.16b 1.65 0.07c 1.88 0.05a

Enzyme-assisted Alcalase 2.4L Viscozyme L 3.11 0.09bc 0.68 0.01bc 1.32 0.08c 1.66 0.05c 1.86 0.04ab 3.14 0.08b 0.69 0.02bc 1.38 0.06b 1.71 0.08b 1.87 0.06a

Natuzyme 3.08 0.14c 0.74 0.03b 1.33 0.07c 1.69 0.06bc 1.85 0.08b

Control 3.12 0.09b 0.66 0.01b 1.36 0.11bc 1.72 0.07b 1.86 0.05ab

Values are means SD, of three sunflower seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Table 4.3.3b Comparison of Oxidative State of Enzyme-assisted Aqueous Extracted Sunflower Seed Oils* Parameter Conjugated diene 1% 1cm (232) Conjugated triene 1% 1cm (270) Peroxide value (milli eq/kg) p-anisidine
Induction Period Rancimat method (h)

Solventextracted 3.28 0.09a 0.82 0.04a 1.78 0.06a 1.93 0.05a 1.82 0.12c
*

Protex 7L 3.14 0.11bc 0.68 0.03bc 1.31 0.13c 1.74 0.13bc 1.94 0.09ab

Kemzyme 3.15 0.08bc 0.62 0.02c 1.33 0.08bc 1.75 0.06bc 1.93 0.13b

Enzyme-assisted Alcalase 2.4L Viscozyme L 3.18 0.13b 0.72 0.03b 1.25 0.10d 1.76 0.11bc 1.95 0.15a 3.10 0.12c 0.69 0.01b 1.37 0.13b 1.71 0.14c 1.93 0.12b

Natuzyme 3.11 0.10c 0.64 0.04bc 1.32 0.14c 1.78 0.13b 1.96 0.14a

Control 3.23 0.14c 0.72 0.02b 1.36 0.13cd 1.79 0.07c 1.84 0.06ab

Values are means SD, of three sunflower seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

67

Table 4.3.4a Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzyme-assisted Cold Pressed Sunflower Seed Oils* FA C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 Solventextracted 6.89 0.13c 0.22 0.01a 4.31 0.07c 28.32 0.43a 59.44 1.19a 0.13 0.05c 0.21 0.07c 0.16 0.03a Enzyme-assisted Alcalase 2.4L 7.21 0.09ab 0.22 0.01a 4.64 0.06a 27.61 0.34b 58.72 2.39a 0.13 0.02c 0.24 0.01bc 0.16 0.01a Control 7.25 0.11a 0.18 0.01ab 4.53 0.06ab 27.96 0.45ab 58.89 1.87a 0.18 0.01b 0.23 0.02c 0.17 0.01a

Protex 7L 7.13 0.16b 0.14 0.02 4.61 0.09a 28.12 0.39a 59.19 1.32a 0.26 0.03a 0.38 0.01a 0.08 0.02c
*

Kemzyme 7.15 0.11b 0.19 0.03ab 4.56 0.07ab 27.86 0.42ab 58.92 2.11a 0.19 0.01b 0.29 0.02b 0.15 0.02ab

Viscozyme L 7.19 0.14ab 0.13 0.01 4.38 0.08bc 27.65 0.48b 59.24 2.17a 0.17 0.02b 0.19 0.02c 0.12 0.01b

Natuzyme 7.27 0.17a 0.16 0.02b 4.48 0.04b 27.82 0.52ab 59.18 1.94a 0.21 0.01ab 0.26 0.01bc 0.14 0.02ab

Values are means SD, of three sunflower seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

68

Table 4.3.4b Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzyme-assisted Aqueous Extracted Sunflower Seed Oils* FA C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 Solventextarcted 6.89 0.13d 0.22 0.06a 4.31 0.07d 28.32 0.43a 59.44 1.19a 0.13 0.05d 0.21 0.07cd 0.16 0.03a
*

Protex 7L 7.29 0.11ab 0.15 0.08b 4.76 0.07ab 27.39 0.41b 58.18 1.45b 0.18 0.07ab 0.24 0.05c 0.12 0.04b

Kemzyme 7.22 0.06b 0.11 0.02c 4.53 0.11bc 28.12 0.52a 58.31 1.26b 0.22 0.09a 0.28 0.03b 0.16 0.02a

Enzyme-assisted Alcalase 2.4L 7.16 0.09bc 0.13 0.04c 4.41 0.07c 27.64 0.45b 59.45 0.89a 0.15 0.06c 0.19 0.06d 0.14 0.01ab

Viscozyme L 7.11 0.16c 0.16 0.05b 4.79 0.12a 28.14 0.42a 59.21 1.38a 0.24 0.04a 0.35 0.07a 0.11 0.03b

Natuzyme 7.35 0.14a 0.21 0.03a 4.84 0.08a 27.76 0.69b 58.53 1.54ab 0.21 0.05ab 0.33 0.04a 0.17 0.05a

Control 7.24 0.18b 0.19 0.06ab 4.63 0.11b 27.58 0.53b 58.22 0.58b 0.15 0.07c 0.23 0.06bc 0.15 0.03a

Values are means SD, of three sunflower seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

69

Table 4.3.5a Comparison of Tocopherols in Enzyme-assisted Cold Pressed Sunflower Seed Oils* Tocopherols (mg kg-1) Total Solventextracted 579 17c 217 9d 3.4 0.2b 799.4 Enzyme-assisted Kemzyme Alcalase 2.4L Viscozyme L a 614 19 611 25c 598 14b 257 13a 248 16b 242 21b 6.7 0.1a 7.3 0.6a 5.5 0.4ab 877.7 866.3 845.5 Control 583 22bc 229 11c 4.8 0.5b 816.8

Values are means SD, of three sunflower seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Protex 7L 607 23ab 243 14b 4.2 0.4b 854.2

Natuzyme 592 26b 256 13a 6.4 0.2a 854.4

Table 4.3.5b Comparison of Tocopherols in Enzyme-assisted Aqueous Extracted Sunflower Seed Oils* Tocopherols (mg kg-1) Total Solventextracted 579 17a 217 9d 3.4 0.2b 799.4 Enzyme-assisted Alcalase 2.4L Viscozyme L 582 14a 516 19c ab 254 22b 259 19 3.6 0.5b 4.5 0.7b 845.5 833.6 Control 537 11b 238 7c 3.4 0.4b 775.4

Values are means SD, of three sunflower seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Protex 7L 524 21bc 268 13a 0 842

Kemzyme 517 18c 266 24a 6.3 0.9a 849.3

Natuzyme 523 12bc 261 16ab 5.2 0.6a 849.2

70

Table 4.3.6a Comparison of Antioxidant Activity of Enzyme-assisted Cold Pressed Sunflower Seed Oils* Parameter TPC (mg GAE/100 g) DPPH, IC50 (g mL-1) Inhibition in linoleic acid system (%) after 175 h incubation at 40C Solventextracted 0.8 0.1 a 66.4 1.7 48.5 1.3
*

Protex 7L a 1.5 0.1 bc 59.7 1.8 51.4 0.9

b

Kemzyme a 1.4 0.3 c 58.8 1.4 52.6 1.2

ab

Enzyme-assisted Alcalase 2.4L Viscozyme L b a 1.6 0.1 1.7 0.2 bc c 59.9 1.3 58.3 1.7 50.2 1.8
b

Natuzyme ab 1.4 0.2 bc 59.8 1.1 49.5 1.5

c

Control 1.3 0.3 b 64.2 1.3 47.9 1.3

c ab

bc

54.3 1.6

Values are means SD, of three sunflower seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Table 4.3.6b Comparison of Antioxidant Activity of Enzyme-assisted Aqueous Extracted Sunflower Seed Oils* Parameter TPC (mg GAE/100 g) DPPH, IC50 (g mL-1) Inhibition in linoleic acid system (%) after 175 h incubation at 40C Solventextracted 0.8 0.1 a 66.4 1.7 48.5 1.3
*

Protex 7L c 1.6 0.2 bc 59.6 1.5 51.8 1.4

b

Kemzyme b 1.4 0.2 c 58.6 1.2 53.4 0.9

ab

Enzyme-assisted Alcalase 2.4L Viscozyme L c a 1.5 0.1 1.7 0.3 bc c 59.8 1.3 57.8 1.9 50.2 1.4
b

Natuzyme c 1.5 0.2 bc 58.7 1.6 49.8 1.8

c

Control 0.9 0.1 b 64.5 1.8 48.5 1.2

bc b

bc

55.2 1.3

Values are means SD, of three sunflower seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

71

Table 4.4.1a Comparison of Proximate Composition of Enzyme-assisted Cold Pressed Sesame Seeds* Parameter (%) Oil extracted Protein (residue) Fiber content Ash content Solventextracted 30.2 0.8a 29.4 0.7b 11.2 0.3a 5.7 0.2a Enzyme-assisted Protex 7L Alcalase 2.4L 28.2 0.7b 26.7 0.6c a 30.1 1.2 29.2 0.8b 11.1 0.2a 11.2 0.3a a 5.6 0.2 5.5 0.2a Control 25.3 0.9d 29.5 0.7ab 11.2 0.2a 5.6 0.1a

Natuzyme 27.6 0.6bc 29.6 0.8ab 11.3 0.4a 5.6 0.2a

Kemzyme 26.5 1.5c 29.8 0.7a 10.8 0.3a 5.8 0.1a

Viscozyme L 27.8 0.7bc 29.4 1.6b 10.9 0.3a 5.7 0.2a

Values are mean SD, calculated as percentage on dry seed weight basis for three seasame seed samples for each enzyme, analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Table 4.4.1b Comparison of Proximate Composition of Enzyme-assisted Aqueous Extracted Sesame Seeds* Parameter (%) Oil extracted Protein (residue) Fiber content Ash content Solventextracted 30.2 0.8a 29.4 0.7a 11.2 0.3a 5.7 0.2a Enzyme-assisted Protex 7L Alcalase 2.4L 18.7 0.3d 24.8 0.4b 19.2 0.5d 21.4 0.3cd 11.2 0.2d 11.4 0.3a ab 5.5 0.1 5.7 0.1a Control 12.3 0.3f 24.2 0.6b 11.2 0.9a 5.5 0.1ab

Natuzyme 16.8 0.3e 24.3 0.4b 10.9 0.2a 5.4 0.2b

Kemzyme 16.5 0.8e 23.7 0.4bc 11.1 0.6a 5.6 0.2a

Viscozyme L 21.4 1.2c 22.5 0.4c 10.8 0.3a 5.4 0.2b

Values are mean SD, calculated as percentage on dry seed weight basis for three sesame seed samples for each enzyme, analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

72

Table 4.4.2a Comparison of Physico-chemical Properties of Enzyme-assisted Cold Pressed Sesame Seed Oils* Parameter Refractive Index (40 oC) Density (20 oC g mL-1) Saponification value (mg KOH/g of oil) Free fatty acids contents (% as oleic acid) Iodine value (g of I/ 100g of oil) Unsaponifiable matter (% w/w) Color (1-in. cell) Red units Yellow units Solventextracted 1.4664 0.02a 0.92 0.04a 169 6a 0.54 0.03c 107 5ab 1.31 0.11a 1.4 r 0.1b 21.3 y 0.6a Enzyme-assisted Protex 7L Alcalase 2.4L 1.4662 0.03a 0.92 0.02a 163 2b 0.56 0.03b 108 6a 1.14 0.17b 1.8 r 0.3a 20.4 y 0.7a 1.4663 0.02a 0.92 0.02a 161 3b 0.59 0.08ab 105 2b 1.08 0.12c 1.5 r 0.1b 21.3 y 0.9a Control 1.4663 0.03a 0.92 0.01a 162 9b 0.47 0.13b 108 5a 1.15 0.05b 1.2 r 0.1b 21.5 y 0.5b

Natuzyme 1.4663 0.01a 0.92 0.01a 162 4b 0.58 0.07ab 109 5a 1.16 0.09b 1.7 r 0.2ab 17.6 y 0.9ab

Kemzyme 1.4664 0.01a 0.92 0.03a 159 7b 0.62 0.05a 106 2ab 1.12 0.06bc 1.6 r 0.1ab 16.2 y 0.5b

Viscozyme L 1.4664 0.01a 0.92 0.01a 159 6b 0.58 0.02ab 104 3b 1.06 0.07c 1.6 r 0.2ab 18.9 y 0.8ab

Values are means SD, of three sesame seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

73

Table 4.4.2b Comparison of Physico-chemical Properties of Enzyme-assisted Aqueous Extracted Sesame Seed Oils* Parameter Refractive Index (40 oC) Density (20 oC g mL-1) Saponification value (mg KOH/g of oil) Free fatty acids contents (% as oleic acid) Iodine value (g of I/ 100g of oil) Unsaponifiable matter (% w/w) Color (1-in. cell) Red units Yellow units Solventextracted 1.4664 0.02a 0.92 0.04a 169 6a 0.54 0.03a 107 5a 1.31 0.11a 1.4 0.1a 21.3 0.6ab Enzyme-assisted Protex 7L Alcalase 2.4L 1.4663 0.01a 0.92 0.03a 161 1.8c 0.51 0.04ab 108 6a 1.16 0.17b 1.3 r 0.2ab 20.8 y 0.8b 1.4663 0.02a 0.92 0.05a 164 1.4b 0.46 0.03b 105 5b 1.15 0.06b 1.2 r 0.1b 19.7 y 0.6b Control 1.4664 0.01a 0.92 0.03a 159 2.1cd 0.48 0.02b 106 3b 1.14 0.13b 1.3 r 0.2ab 21.4 y 0.6ab

Natuzyme 1.4662 0.01a 0.92 0.02a 158 2.3cd 0.47 0.06b 104 7b 1.13 0.08bc 1.3 r 0.1ab 20.5 y 0.8b

Kemzyme 1.4664 0.02a 0.92 0.06a 162 1.5c 0.44 0.02c 109 4a 1.09 0.14c 1.4 r 0.2a 20.1 y 0.7b

Viscozyme L 1.4662 0.01a 0.92 0.02a 156 1.9d 0.44 0.03c 103 6b 1.08 0.04c 1.3 r 0.1ab 23.6 y 0.8a

Values are means SD, of three sesame seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

74

Table 4.4.3a Comparison of Oxidative State of Enzyme-assisted Cold Pressed Sesame Seed Oils* Parameter Conjugated diene 1% 1cm (232) Conjugated triene 1% 1cm (270) Peroxide value (milli eq/kg) p-anisidine
Induction Period Rancimat method (h)

Solventextracted 1.92 0.06a 0.89 0.03a 1.5 0.05a 0.44 0.01a 1.73 0.04c
*

Natuzyme 1.84 0.08bc 0.73 0.02bc 1.2 0.04ab 0.32 0.02c 1.79 0.06a

Kemzyme 1.79 0.05c 0.71 0.04bc 1.1 0.07b 0.36 0.01b 1.76 0.05b

Enzyme-assisted Protex 7L Alcalase 2.4L 1.83 0.04bc 0.75 0.03b 1.3 0.05ab 0.35 0.01c 1.78 0.06a 1.81 0.07bc 0.72 0.05bc 0.9 0.06b 0.33 0.02 1.77 0.04ab

Viscozyme L 1.78 0.04c 0.69 0.02c 1.2 0.04ab 0.37 0.01b 1.79 0.05a

Control 1.86 0.05b 0.76 0.04b 1.4 0.05a 0.35 0.01bc 1.78 0.07a

Values are means SD, of three sesame seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Table 4.4.3b Comparison of Oxidative State of Enzyme-assisted Aqueous Extracted Sesame Seed Oils* Parameter Conjugated diene 1% 1cm (232) Conjugated triene 1% 1cm (270) Peroxide value (milli eq/kg) p-anisidine
Induction Period Rancimat method (h)

Solventextracted 1.92 0.06a 0.89 0.03a 1.5 0.05a 0.44 0.01a 1.73 0.04b
*

Natuzyme 1.82 0.08bc 0.68 0.02d 0.9 0.06b 0.37 0.02b 1.75 0.07ab

Kemzyme 1.86 0.05b 0.74 0.03bc 1.3 0.04ab 0.33 0.02c 1.77 0.15a

Enzyme-assisted Protex 7L Alcalase 2.4L 1.84 0.11b 0.71 0.04c 1.4 0.06a 0.36 0.01b 1.76 0.04a 1.78 0.06c 0.77 0.02b 1.1 0.07b 0.35 0.03bc 1.75 0.06ab

Viscozyme L 1.85 0.08b 0.66 0.02d 1.2 0.05ab 0.33 0.02c 1.77 0.13a

Control 1.88 0.07ab 0.75 0.03bc 1.3 0.08ab 0.32 0.01c 1.76 0.07a

Values are means SD, of three sesame seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

75

Table 4.4.4a Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzyme-assisted Cold Pressed Sesame Seed Oils* FA C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 C22:0 Solventextracted 0.11 0.01b 9.53 0.25a 5.62 0.14ab 38.94 0.64a 44.37 0.83b 0.82 0.02ab 0.45 0.01a 0.23 0.01b Enzyme-assisted Protex 7L 0.13 0.02ab 9.46 0.19ab 5.62 0.17ab 38.47 0.76ab 43.85 1.18bc 0.85 0.02a 0.37 0.01b 0.25 0.02ab Control 0.16 0.02a 9.49 0.16ab 5.63 0.12ab 38.57 0.61ab 44.55 1.13ab 0.84 0.02ab 0.38 0.01ab 0.16 0.01c

Natuzyme 0.14 0.02ab 9.43 0.18b 5.64 0.11a 38.26 0.59b 43.61 1.17bc 0.83 0.01ab 0.42 0.01ab 0.17 0.02c
*

Kemzyme 0.17 0.01a 9.52 0.22a 5.58 0.09b 37.88 0.82c 44.54 0.94ab 0.78 0.01b 0.45 0.02a 0.22 0.01b

Alcalase 2.4L 0.17 0.01a 9.45 0.27ab 5.65 0.15a 38.73 0.84ab 43.16 0.85c 0.82 0.01ab 0.44 0.01a 0.18 0.02c

Viscozyme L 0.15 0.02a 9.53 0.23a 5.57 0.08b 37.92 0.55bc 44.81 1.09a 0.87 0.01a 0.35 0.02b 0.27 0.01a

Values are means SD, of three sesame seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

76

Table 4.4.4b Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzyme-assisted Aqueous Extracted Sesame Seed Oils* FA C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 C22:0 Solventextracted 0.11 0.01c 9.53 0.25a 5.62 0.14ab 38.94 0.64a 44.37 0.83c 0.82 0.02b 0.45 0.01a 0.23 0.01ab Enzyme-assisted Protex 7L 0.14 0.03b 9.43 0.18c 5.52 0.09c 36.84 0.55ab 45.28 0.72ab 0.78 0.01c 0.43 0.01ab 0.16 0.02c Control 0.22 0.01a 9.41 0.21c 5.54 0.14bc 38.42 0.48ab 44.78 0.85bc 0.85 0.03ab 0.41 0.02b 0.23 0.02ab

Natuzyme 0.16 0.02b 9.54 0.19a 5.57 0.17b 37.33 0.57c 45.15 0.75b 0.84 0.01ab 0.46 0.01a 0.21 0.01b
*

Kemzyme 0.19 0.01ab 9.47 0.24b 5.64 0.16ab 37.24 0.62c 45.41 0.89a 0.87 0.03a 0.34 0.02c 0.18 0.01bc

Alcalase 2.4L 0.21 0.02a 9.56 0.27a 5.55 0.13bc 37.65 0.71bc 45.53 0.84a 0.79 0.02c 0.37 0.01bc 0.25 0.01a

Viscozyme L 0.17 0.02ab 9.52 0.15ab 5.68 0.07a 38.29 0.62b 44.65 0.91bc 0.86 0.01a 0.39 0.01bc 0.17 0.01c

Values are means SD, of three sesame seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

77

Table 4.4.5a Comparison of Tocopherols in Enzyme-assisted Cold Pressed Sesame Seed Oils* Tocopherols (mg kg-1) Total Solventextracted 2.4 0.7d 578 12d 3.7 0.6d 584.1 Enzyme-assisted Natuzyme 3.6 0.4b 615 14bc 4.2 0.5ab 622.8 Kemzyme 3.4 0.5c 623 16b 3.4 0.8c 626.4 Protex 7L 4.7 0.6a 596 12c 3.5 0.9c 604.2 Alcalase 2.4L 3.8 1.1ab 638 22a 3.8 0.6b 641.8 Viscozyme L 3.3 0.8c 614 18bc 4.5 0.2a 621.8 Control 2.9 0.9cd 589 13cd 3.8 0.9b 595.7

Values are means SD, of three sesame seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Table 4.4.5b Comparison of Tocopherols in Enzyme-assisted Aqueous Extracted Sesame Seed Oils* Tocopherols (mg kg-1) Total Solventextracted 2.4 0.1d 578 12d 3.7 0.2e 584.1 Enzyme-assisted Protex 7L Alcalase 2.4L 4.5 0.2a 3.9 0.4b 618 17b 612 13bc 4.8 0.9b 3.9 0.4d 627.3 619.8 Control 2.6 0.1d 596 19c 4.7 0.2b 603.3

Values are means SD, of three sesame seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Natuzyme 4.4 0.3a 624 10ab 4.5 0.8c 628.5

Kemzyme 3.6 0.8c 632 14a 5.6 0.6a 641.2

Viscozyme L 3.7 0.7bc 605 15c 4.1 0.3d 612.8

78

Table 4.4.6a Comparison of Antioxidant Activity of Enzyme-assisted Cold Pressed Sesame Seed Oils* Parameter TPC (mg GAE/100 g) DPPH, IC50 (g mL-1) Inhibition in linoleic acid system (%) after 175 h incubation at 40C Solventextracted 1.7 0.3 a 62.2 1.6 56.4 1.2
*

Natuzyme b 1.9 0.1 bc 55.9 1.2 58.9 1.9

b

Kemzyme c 2.2 0.4 bc 55.7 1.8 59.3 1.3

ab

Enzyme-assisted Protex 7L Alcalase 2.4L b b 2.1 0.3 1.9 0.2 bc c 55.8 1.1 54.5 1.9 59.7 0.8
ab

Viscozyme L a 2.3 0.3 c 54.9 1.2 59.8 1.1

a

Control 1.8 0.4 b 59.1 1.7 57.5 1.9

ab c

ab

58.4 1.2

Values are means SD, of three sesame seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Table 4.4.6b Comparison of Antioxidant Activity of Enzyme-assisted Aqueous Extracted Sesame Seed Oils* Parameter TPC (mg GAE/100 g) DPPH, IC50 (g mL-1) Inhibition in linoleic acid system (%) after 175 h incubation at 40C Solventextracted 1.7 0.3 a 62.2 1.6 56.4 1.2
c c

Natuzyme b 1.9 0.1 b 55.6 1.9 62.1 1.3

*

Kemzyme b 1.8 0.3 b 55.4 1.4 59.5 1.8

bc

Enzyme-assisted Protex 7L Alcalase 2.4L b a 2.2 0.2 2.1 0.4 c b 54.6 1.8 55.3 1.2 61.7 1.6
c

Viscozyme L a 2.4 0.3 c 54.1 1.1 61.8 1.9

ab

Control 1.8 0.2 bc 58.1 1.5 57.3 1.8

b b

62.5 1.4

Values are means SD, of three sesame seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

79

Table 4.5.1a Comparison of Proximate Composition of Enzyme-assisted Cold Pressed Canola Seeds* Solventextracted 43.1 0.9a 37.2 0.6a 7.0 0.3a 5.7 0.2a Enzyme-assisted Multifect Multifect CX Pectinase FE 13L b 28.4 1.7 28.7 1.5b bc 29.1 1.6 28.6 1.7c 7.1 0.4a 6.8 0.3b c 5.6 0.3 5.5 0.2d

Parameter (%) Oil content Protein content Fiber content Ash content
*

Protex 7L 24.6 1.6c 28.9 1.9b 6.8 0.5b 5.6 0.4c

Kemzyme 26.9 1.4bc 29.3 1.8bc 6.9 0.3a 5.7 0.7c

Natuzyme 27.5 1.4bc 28.4 1.1c 6.9 0.4ab 5.6 0.5ab

Control 24.8 1.3c 30.3 1.8b 7.0 0.3a 5.7 0.6b

Values are mean SD, calculated as percentage on dry seed weight basis for three canola seed samples for each enzyme, analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05)

Table 4.5.1b Comparison of Proximate Composition of Enzyme-assisted Aqueous Extracted Canola Seeds* Solventextracted 43.1 0.9a 37.2 0.6a 7.0 0.3a 5.7 0.2a Enzyme-assisted Multifect Multifect CX Kemzyme Pectinase FE 13L 23.2 0.6c 22.2 0.4 c 26.0 0.4b 17.6 0.3b 14.5 0.2b 15.1 0.2b 6.9 0.2b 6.9 0.3ab 6.9 0.3b a ab 5.7 0.2 5.6 0.3 5.7 0.2ab

Parameter (%) Oil extracted Protein (residue) Fiber content Ash content

Protex 7L 23.4 0.5c 12.4 0.3c 6.9 0.2ab 5.6 0.2ab

Natuzyme 22.7 0.5c 14.8 0.3b 6.9 0.2 5.6 0.3b

Control 16.5 0.3d 14.2 0.2b 7.0 0.2a 5.7 0.2a

Values are mean SD, calculated as percentage on dry seed weight basis for three canola seed samples for each enzyme, analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05)

80

Table 4.5.2a Comparison of Physico-chemical Properties of Enzyme-assisted Cold Pressed Canola Seed Oils* Solventextracted 1.4650 0.03a 0.92 0.02a 189 2a 0.81 0.04c 117 1c 0.56 0.03a 1.9 r 0.1c 32.1 y 0.5ab Enzyme-assisted Multifect Pectinase FE 1.4648 0.01a 0.92 0.02a 185 4b 0.91 0.04ab 119 3bc 0.55 0.02ab 2.4 r 0.1a 32.1 y 1.6ab

Parameter Refractive Index (40 oC) Density (24 oC g mL-1) Saponification value (mg KOH/g of oil) Free fatty acids contents (% as oleic acid) Iodine value (g of I/ 100g of oil) Unsaponifiable matter (% w/w) Color (1-in. cell) Red units Yellow units

Protex 7L 1.4649 0.01a 0.92 0.01a 186 2ab 0.92 0.03a 121 2b 0.54 0.07ab 2.1 r 0.2b 34.3 y 1.7a

Kemzyme 1.4651 0.02a 0.92 0.03a 187 6ab 0.89 0.06b 123 3ab 0.53 0.04b 2.4 r 0.1a 33.8 y 1.1bc

Multifect CX 13L 1.4650 0.01a 0.92 0.01a 186 2ab 0.87 0.03b 126 6a 0.52 0.06b 2.5 r 0.2a 28.3 y 0.9b

Natuzyme 1.4651 0.03a 0.92 0.01a 184 3b 0.93 0.07a 124 4ab 0.54 0.03ab 2.4 r 0.1a 33.7 y 1.5a

Control 1.4650 0.02a 0.92 0.02a 185 5b 0.91 0.05ab 125 2a 0.55 0.05a 2.2 r 0.1ab 30.4 y 1.3ab

Values are means SD, of three canola seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05)

81

Table 4.5.2b Comparison of Physico-chemical Properties of Enzyme-assisted Aqueous Extracted Canola Seed Oils* Solventextracted 1.4650 0.03a 0.92 0.02a 189 2b 0.81 0.04a 117 1a 0.56 0.03a 1.9 r 0.1a 32.1 y 0.5a Enzyme-assisted Multifect Pectinase FE 1.4651 0.02a 0.92 0.01a 182 1.8a 0.54 0.01b 116 1.4a 0.53 0.04b 1.6 r 0.1bc 26.8 y 0.5bc

Parameter Refractive Index (40 oC) Density (24 oC g mL-1) Saponification value (mg KOH/g of oil) Free fatty acids contents (% as oleic acid) Iodine value (g of I/ 100g of oil) Unsaponifiable matter (% w/w) Color (1-in. cell) Red units Yellow units

Protex 7L 1.4652 0.03a 0.92 0.03a 185 1.7c 0.57 0.02b 114 1.8b 0.55 0.01a 1.7 r 0.2b 27.6 y 0.4b

Kemzyme 1.4653 0.01a 0.92 0.02a 184 2.2c 0.56 0.03b 115 2.1b 0.54 0.02a 1.8 r 0.2ab 28.4 y 0.5b

Multifect CX 13L 1.4651 0.04a 0.92 0.02a 181 2.4c 0.52 0.02b 116 1.8a 0.52 0.02b 1.6 r 0.3bc 26.5 y 0.4bc

Natuzyme 1.4651 0.03a 0.92 0.03a 188 2.3b 0.55 0.04b 117 1.3a 0.54 0.03a 1.7 r 0.2b 27.2 y 0.2b

Control 1.4652 0.02a 0.92 0.03a 188 1.7b 0.56 0.03c 114 1.2b 0.52 0.04b 1.5 r 0.1c 25.3 y 0.4c

Values are means SD, of three canola seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05)

82

Table 4.5.3a Comparison of Oxidative State of Enzyme-assisted Cold Pressed Canola Seed Oils* Enzyme-assisted SolventParameter Multifect Multifect CX extracted Protex 7L Kemzyme Natuzyme Pectinase FE 13L Conjugated diene 2.96 0.07a 2.42 0.14b 2.40 0.17b 2.43 0.16b 2.46 0.09b 2.44 0.11b 1% 1cm (232) Conjugated triene 0.76 0.04a 0.54 0.02c 0.52 0.03c 0.56 0.01bc 0.55 0.03bc 0.58 0.04b 1% 1cm (270) Peroxide value 1.29 0.02a 0.76 0.03c 0.82 0.04b 0.77 0.02c 0.79 0.06bc 0.81 0.03b (milli eq/kg) p-anisidine 1.86 0.06a 1.67 0.05bc 1.65 0.07c 1.71 0.06b 1.68 0.08bc 1.66 0.05c
Induction Period Rancimat method (h)

Control 2.43 0.13b 0.57 0.03b 0.80 0.05b 1.67 0.09bc 1.81 0.08b

1.78 0.13c
*

1.84 0.06a

1.83 0.08ab

1.85 0.05a

1.82 0.09b

1.83 0.07ab

Values are means SD, of three canola seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05)

Table 4.5.3b Comparison of Oxidative State of Enzyme-assisted Aqueous Extracted Canola Seed Oils* Enzyme-assisted SolventParameter Control Multifect Multifect CX extracted Protex 7L Kemzyme Natuzyme Pectinase FE 13L Conjugated diene 2.44 0.04b 2.52 0.15b 2.48 0.11b 2.40 0.07b 2.53 0.05b 2.51 0.06b 2.96 0.07a 1% 1cm (232) Conjugated triene 0.76 0.04a 0.53 0.01bc 0.50 0.04c 0.57 0.03b 0.54 0.02bc 0.49 0.01c 0.55 0.03b 1% 1cm (270) Peroxide value 1.29 0.02a 0.70 0.01b 0.68 0.05bc 0.64 0.04c 0.72 0.03b 0.71 0.04b 0.69 0.03bc (milli eq/kg) p-anisidine 1.86 0.06a 1.69 0.05bc 1.72 0.07b 1.71 0.09b 1.68 0.07c 1.72 0.06b 1.67 0.05c
Induction Period Rancimat method (h)

1.78 0.13b
*

1.82 0.08ab

1.81 0.06ab

1.84 0.05a

1.83 0.11a

1.82 0.08ab

1.83 0.07a

Values are means SD, of three canola seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05)

83

Table 4.5.4a Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzyme-assisted Cold Pressed Canola Seed Oils* FA C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C22:1 Solventextracted 3.27 0.05a 1.73 0.02a 60.40 0.82a 20.32 0.38a 11.20 0.19a 1.50 0.03b 1.03 0.02b 0.20 0.01a Enzyme-assisted Multifect Multifect CX Pectinase FE 13L ab 3.23 0.08 3.19 0.11b 1.69 0.04a 60.24 1.18a 20.24 0.41a 11.27 0.17a 1.46 0.05c 0.94 0.02b 0.17 0.04ab 1.72 0.05a 60.18 1.23a 20.49 0.55a 11.31 0.14a 1.48 0.03c 1.10 0.02ab 0.22 0.05a

Protex 7L 3.18 0.07b 1.74 0.05a 59.82 0.98a 20.43 0.47a 11.16 0.21a 1.58 0.04a 1.08 0.05ab 0.16 0.03b
*

Kemzyme 3.21 0.04ab 1.70 0.03a 59.96 1.12a 20.17 0.59a 11.18 0.26a 1.54 0.06ab 0.96 0.03b 0.19 0.02ab

Natuzyme 3.24 0.09ab 1.67 0.03a 60.52 1.09a 20.14 0.51a 11.23 0.22a 1.51 0.04b 1.14 0.04a 0.15 0.03b

Control 3.20 0.14ab 1.68 0.06a 60.21 1.27a 20.36 0.44a 11.15 0.26a 1.52 0.06b 1.12 0.03a 0.18 0.02ab

Values are means SD, of three canola seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

84

Table 4.5.4b Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzyme-assisted Aqueous Extracted Canola Seed Oils* FA C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C22:1 Solventextracted 3.27 0.05ab 1.73 0.02a 60.40 0.82a 20.32 0.38a 11.20 0.19a 1.50 0.03a 1.03 0.02a 0.20 0.01ab Enzyme-assisted Multifect Multifect CX Pectinase FE 13L b 3.19 0.07 3.30 0.04ab 1.67 0.01a 59.80 1.09a 20.96 0.41a 11.17 0.21a 1.45 0.05a 0.94 0.02b 0.20 0.02ab 1.70 0.04a 59.97 1.15a 20.52 0.29a 11.05 0.24a 1.46 0.01a 0.99 0.04ab 0.18 0.01b

Protex 7L 3.38 0.09a 1.72 0.03a 60.32 1.32a 20.40 0.36a 10.99 0.15a 1.52 0.02a 1.05 0.01a 0.19 0.02ab
*

Kemzyme 3.23 0.05ab 1.69 0.02a 60.17 2.1a 20.74 0.45a 10.84 0.18a 1.54 0.03a 1.10 0.01a 0.21 0.01ab

Natuzyme 3.29 0.08ab 1.75 0.06a 60.23 0.77a 20.68 0.35a 11.23 0.17a 1.49 0.04a 0.97 0.03ab 0.22 0.03a

Control 3.24 0.06ab 1.74 0.03a 60.12 1.20a 20.67 0.42a 11.28 0.16a 1.48 0.03a 0.96 0.02ab 0.21 0.01ab

Values are means SD, of three canola seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

85

Table 4.5.5a Comparison of Tocopherols in Enzyme-assisted Cold Pressed Canola Seed Oils* Tocopherols (mg kg-1) Total Solventextracted 239 3c 487 8c 13.4 0.2c 739.4 Enzyme-assisted Protex 7L 272 9a 514 13b 15.8 1.5ab 801.8
*

Kemzyme 269 11ab 528 16ab 18.2 1.7a 815.2

Multifect Pectinase FE 274 10a 519 17b 19.3 1.5a 812.3

Multifect CX 13L 265 15ab 521 21ab 14.9 0.8b 800.9

Control Natuzyme 271 9a 539 18a 14.1 0.9b 824.1 254 8b 492 15c 12.2 1.4c 758.2

Values are means SD, of three canola seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Table 4.5.5b Comparison of Tocopherols in Enzyme-assisted Aqueous Extracted Canola Seed Oils* Tocopherols (mg kg-1) Total Solventextracted 239 3a 487 8c 13.4 0.2d 739.4 Enzyme-assisted Multifect Pectinase FE 180 9c 620 7a 19.1 0.2a 819.1

Protex 7L 198 5b 590 16b 17.2 0.1b 805.2

*

Kemzyme 194 3b 611 6ab 13.9 0.3c 818.9

Multifect CX 13L 176 7c 603 11ab 14.7 0.3c 793.7

Natuzyme 184 3c 583 18b 16.3 0.2bc 783.3

Control 164 5d 421 14d 13.2 0.3d 598.2

Values are means SD, of three canola seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

86

Table 4.5.6a Comparison of Antioxidant Activity of Enzyme-assisted Cold Pressed Canola Seed Oils* Solventextracted 1.3 0.2 a 59.6 1.5 57.3 1.9
*

Parameter TPC (mg GAE/100 g) DPPH, IC50 (g mL-1) Inhibition in linoleic acid system (%) after 175 h incubation at 40C

Protex 7L 1.4 0.3 c 52.9 1.1 62.5 1.5

ab c

Enzyme-assisted Multifect Multifect CX Kemzyme Pectinase FE 13L a b d 1.6 0.1 1.5 0.2 1.3 0.1 bc bc bc 53.2 1.4 53.1 1.6 53.2 1.3 61.6 1.7
a

Natuzyme 1.4 0.3 c 52.9 1.1 61.8 1.5

a c

Control 1.4 0.2 b 57.4 1.4 58.4 1.8

ab c

61.9 1.5

ab

59.7 1.8

ab

Values are means SD, of three canola seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05)

Table 4.5.6b Comparison of Antioxidant Activity of Enzyme-assisted Aqueous Extracted Canola Seed Oils* Solventextracted 1.3 0.2 a 59.6 1.5 57.3 1.9
*

Parameter TPC (mg GAE/100 g) DPPH, IC50 (g mL-1) Inhibition in linoleic acid system (%) after 175 h incubation at 40C

Protex 7L 1.4 0.1 bc 53.1 1.7 60.7 1.8

ab b

Enzyme-assisted Multifect Multifect CX Kemzyme Pectinase FE 13L a b b 1.6 0.1 1.5 0.3 1.7 0.2 c bc bc 52.9 1.1 53.2 1.4 53.3 2.1 62.5 1.4
a

Natuzyme 1.6 0.1 c 52.8 2.4 61.9 1.5

a b

Control 1.4 0.3 b 57.9 1.7 58.9 1.6

b b

59.3 1.2

ab

60.8 1.9

ab

Values are means SD, of three canola seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05)

87

Table 4.6.1a Comparison of Proximate Composition of Enzyme-assisted Cold Pressed Moringa oleifera Seeds* Parameter (%) Oil content Protein (residue) Fiber content Ash content Solventextracted 32.4 0.8a 34.2 1.0a 4.1 0.2a 4.2 0.1a Enzyme-assisted Feedzyme 17.5 0.8c 23.1 1.1d 3.9 0.2c 4.0 0.1b Control 9.4 0.5d 29.41 1.2b 4.1 0.1b 4.1 0.1ab

Natuzyme 19.3 0.6bc 24.2 0.9c 4.1 0.3b 4.2 0.2a

Kemzyme 20.5 1.5bc 23.4 0.7cd 4.2 0.1a 4.0 0.1b

Protex 7L 19.7 0.5bc 20.3 1.2e 4.1 0.3b 4.1 0.2ab

Viscozyme L 21.6 1.4b 23.2 0.8d 4.2 0.2b 4.2 0.1a

Values are means SD, calculated as percentage on dry seed weight basis for three moringa oleifera seed samples for each enzyme analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05)

Table 4.6.1b Comparison of Proximate Composition of Enzyme-assisted Aqueous Extracted Moringa oleifera Seeds* Parameter (%) Oil content Protein content Fiber content Ash content Solventextracted 32.4 0.8a 34.2 1.0b 4.1 0.2a 4.2 0.1a Enzyme-assisted Feedzyme 21.8 0.8bc 33.7 0.7c 4.1 0.3b 4.1 0.2ab Control 19.3 0.6d 34.1 1.5b 4.1 0.2a 4.2 0.4a

Natuzyme 21.3 0.7bc 34.2 1.6b 4.1 0.5b 4.1 0.3ab

Kemzyme 22.1 0.6b 34.8 0.7a 4.0 0.2ab 4.1 0.5b

Protex 7L 20.9 0.7c 34.3 0.9b 4.1 0.2a 4.1 0.3b

Viscozyme L 22.5 0.6b 33.8 0.8c 4.0 0.1b 4.2 0.2a

Values are means SD, calculated as percentage on dry seed weight basis for three moringa oleifera seed samples for each enzyme analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05)

88

Table 4.6.2a Comparison of Physico-chemical Properties of Enzyme-assisted Cold Pressed Moringa oleifera Seed Oils* Parameter Refractive Index (40 oC) Density (24 oC g/ mL) Saponification value (mg KOH/g oil) Free fatty acids contents (% as oleic acid) Iodine value (g of I/ 100g oil) Unsaponifiable matter (% w/w) Color (1-in. cell) Red units Yellow units Solventextracted 1.4574 0.02a 0.87 0.03a 164 4a 0.56 0.02c 67 2a 0.63 0.04a 2.3 r 0.2b 23.4 y 0.9b
*

Natuzyme 1.4553 0.01b 0.87 0.05a 159 2b 0.61 0.04b 73 5a 0.56 0.06b 2.6 r 0.1a 24.6 y 1.4b

Kemzyme 1.4551 0.03b 0.87 0.02a 160 5ab 0.59 0.02bc 77 4a 0.54 0.02bc 2.7 r 0.1a 27.3 y 0.8a

Enzyme-assisted Feedzyme 1.4548 0.01b 0.87 0.03a 158 3b 0.62 0.06b 69 2a 0.55 0.03b 2.6 r 0.2a 26.5 y 1.6a

Protex 7L 1.4554 0.02b 0.87 0.02a 156 4b 0.66 0.03a 69 8a 0.52 0.05c 2.6 r 0.1a 24.7 y 1.2b

Viscozyme L 1.4549 0.03b 0.87 0.05a 159 3b 0.58 0.05bc 70 4a 0.57 0.04b 2.7 r 0.2a 27.2 y 0.9a

Control 1.4550 0.01b 0.87 0.02a 161 5bc 0.61 0.03b 69 6a 0.52 0.02c 2.4 r 0.2b 24.5 y 1.5b

Values are means SD, of three moringa oleifera seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

89

Table 4.6.2b Comparison of Physico-chemical Properties of Enzyme-assisted Aqueous Extracted Moringa oleifera Seed Oils* Parameter Refractive Index (40 oC) Density (24 oC g/ mL) Saponification value (mg KOH/g oil) Free fatty acids contents (% as oleic acid) Iodine value (g of I/ 100g oil) Unsaponifiable matter (% w/w) Color (1-in. cell) Red units Yellow units Solventextracted 1.4574 0.02a 0.87 0.03a 164 4a 0.56 0.02a 67 2a 0.63 0.04a 2.3 r 0.2a 23.4 y 0.9a
*

Natuzyme 1.4562 0.01b 0.87 0.05a 156 8b 0.43 0.05b 76 5a 0.54 0.06c 1.8 r 0.1b 18.6 y 0.7b

Kemzyme 1.4564 0.01b 0.87 0.02a 158 5b 0.41 0.02bc 73 11a 0.56 0.02bc 1.7 r 0.1bc 17.9 y 1.1bc

Enzyme-assisted Feedzyme 1.4563 0.02b 0.87 0.04a 155 8b 0.39 0.02c 75 8a 0.58 0.07b 1.8 r 0.2b 18.7 y 0.8b

Protex 7L 1.4565 0.01b 0.87 0.03a 159 6b 0.38 0.04c 74 4a 0.55 0.04bc 1.6 r 0.2c 16.9 y 0.7c

Viscozyme L 1.4563 0.02b 0.87 0.07a 156 4b 0.42 0.02b 76 7a 0.57 0.02b 1.8 r 0.1b 17.9 y 0.9b

Control 1.4562 0.01b 0.87 0.02a 158 8b 0.42 0.06b 70 8a 0.56 0.03bc 1.7 r 0.2bc 19.2 y 1.4bc

Values are means SD, of three moringa oleifera seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

90

Table 4.6.3a Comparison of Oxidative State of Enzyme-assisted Cold Pressed Moringa oleifera Seed Oils* Parameter Conjugated diene 1% 1cm (232) Conjugated triene 1% 1cm (270) Peroxide value (m eq/kg of oil) p-anisidine
Induction Period Rancimat method (h)

Solventextracted 1.87 0.07a 0.54 0.02a 2.09 0.13a 1.85 0.07a 1.52 0.12c
*

Natuzyme 1.62 0.08b 0.46 0.04bc 1.61 0.09b 1.68 0.09b 1.58 0.06ab

Kemzyme 1.58 0.09c 0.49 0.02b 1.56 0.07c 1.64 0.05bc 1.60 0.13a

Enzyme-assisted Feedzyme Protex 7L 1.64 0.06b 0.44 0.02c 1.58 0.05c 1.65 0.07bc 1.57 0.06b 1.54 0.07c 0.42 0.03d 1.59 0.08c 1.62 0.09c 1.59 0.08a

Viscozyme L 1.57 0.05c 0.45 0.02c 1.54 0.07c 1.64 0.06bc 1.56 0.05b

Control 1.65 0.09b 0.47 0.01bc 1.59 0.09b 1.62 0.07c 1.58 0.08ab

Values are means SD, of three moringa oleifera seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Table 4.6.3b Comparison of Oxidative State of Enzyme-assisted Aqueous Extracted Moringa oleifera Seed Oils* SolventEnzyme-assisted Parameter Control extracted Natuzyme Kemzyme Feedzyme Protex 7L Viscozyme L Conjugated diene 1.87 0.07a 1.64 0.06b 1.61 0.09bc 1.63 0.05b 1.59 0.07c 1.62 0.11bc 1.63 0.09b 1% 1cm (232) Conjugated triene 0.45 0.02b 0.46 0.02ab 0.44 0.03b 0.47 0.03ab 0.48 0.02ab 0.54 0.02a 0.48 0.03ab 1% 1cm (270) Peroxide value (m 2.09 0.13a 1.58 0.12c 1.56 0.11c 1.61 0.10bc 1.63 0.08b 1.59 0.05bc 1.60 0.08bc eq/kg of oil) p-anisidine 1.85 0.07a 1.71 0.05b 1.68 0.06bc 1.67 0.07bc 1.72 0.09b 1.70 0.05b 1.66 0.08c
Induction Period Rancimat method (h)

1.52 0.12c
*

1.56 0.09a

1.57 0.05a

1.55 0.06ab

1.54 0.12b

1.55 0.09ab

1.56 0.11a

Values are means SD, of three moringa oleifera seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

91

Table 4.6.4a Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzyme-assisted Cold Pressed Moringa oleifera Seed Oils* FA C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C20:0 C20:1 C22:0 C24:0 Solventextracted 3.11 0.05b 7.93 0.24a 1.71 0.03b 5.18 0.21a 68.94 1.77a 0.64 0.02a 2.98 0.12b 2.35 0.03b 5.06 0.15a 1.67 0.03a Enzyme-assisted Feedzyme 3.26 0.12a 6.84 0.15b 1.77 0.04ab 4.78 0.18c 70.42 1.93a 0.51 0.01c 3.16 0.07a 2.38 0.06ab 4.94 0.12ab 1.66 0.04a Control 3.16 0.11ab 7.21 0.19b 1.95 0.05a 4.92 0.12b 69.71 2.17a 0.62 0.02b 3.04 0.09ab 2.30 0.07b 4.82 0.15b 1.57 0.03b

Natuzyme 3.21 0.13ab 6.89 0.22b 1.84 0.04ab 4.71 0.15c 70.36 1.89a 0.54 0.03bc 3.12 0.08a 2.41 0.05ab 4.96 0.17ab 1.64 0.02ab

Kemzyme 3.19 0.08ab 6.92 0.21b 1.80 0.06ab 4.76 0.11c 70.28 2.51a 0.57 0.02bc 3.08 0.11ab 2.46 0.04a 4.87 0.13ab 1.62 0.03ab

Protex 7L 3.18 0.09ab 6.88 0.17b 1.82 0.03ab 4.84 0.21bc 70.53 2.58a 0.59 0.02bc 3.04 0.04ab 2.36 0.08b 4.82 0.17b 1.63 0.07ab

Viscozyme L 3.24 0.15a 6.86 0.28b 1.78 0.02ab 4.79 0.18c 70.25 2.41a 0.54 0.01bc 3.11 0.06a 2.31 0.03b 4.88 0.23ab 1.65 0.05a

* Values are means SD, of three moringa oleifera seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

92

Table 4.6.4b Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzyme-assisted Aqueous Extracted Moringa oleifera Seed Oils* FA C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C20:0 C20:1 C22:0 C24:0 Solventextracted 3.11 0.05b 7.93 0.24a 1.71 0.03b 5.18 0.21a 68.94 1.77a 0.64 0.02a 2.98 0.12b 2.35 0.03b 5.06 0.15b 1.67 0.03a
*

Natuzyme 3.17 0.07ab 6.96 0.21bc 1.81 0.05a 4.84 0.17c 70.16 1.92a 0.57 0.01b 3.14 0.06a 2.38 0.05ab 5.12 0.11a 1.58 0.04c

Kemzyme 3.15 0.11ab 7.11 0.18b 1.77 0.02ab 4.86 0.19c 70.12 2.15a 0.61 0.02a 3.08 0.07ab 2.42 0.04a 4.98 0.09bc 1.62 0.06b

Enzyme-assisted Feedzyme 3.21 0.09a 7.15 0.24b 1.75 0.04ab 4.95 0.16bc 69.42 1.83a 0.58 0.01ab 2.94 0.15b 2.41 0.02a 4.85 0.07c 1.56 0.03c

Protex 7L 3.19 0.06a 6.98 0.22bc 1.79 0.02ab 5.11 0.09ab 69.84 1.86a 0.62 0.02a 2.96 0.05ab 2.33 0.03b 5.14 0.12a 1.59 0.04bc

Viscozyme L 3.17 0.07ab 7.12 0.14b 1.81 0.03a 4.88 0.05bc 69.62 2.13a 0.56 0.01b 3.17 0.08a 2.42 0.05a 4.89 0.08bc 1.61 0.05b

Control 3.16 0.13ab 6.92 0.16c 1.83 0.02a 5.09 0.08b 70.13 2.22a 0.58 0.02ab 3.04 0.07ab 2.35 0.02b 4.95 0.13bc 1.58 0.03bc

Values are means SD, of three moringa oleifera seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

93

Table 4.6.5a Comparison of Tocopherols in Enzyme-assisted Cold Pressed Moringa oleifera Seed Oils* Tocopherols (mg kg-1) Total Solventextracted 76 8d 55 4d 48.3 1.4b 179.3
*

Natuzyme 89 6b 72 7ab 48.8 2.3b 209.8

Kemzyme 96 11a 80 5b 48.1 1.5b 224.1

Enzyme-assisted Feedzyme 87 8bc 75 9a 54.3 2.4a 216.3

Protex 7L 84 6c 89 4c 41.7 3.3c 214.7

Viscozyme L 83 14c 87 6a 51.9 3.5ab 221.9

Control 84 9c 81 7b 49.4 1.3b 214.4

Values are means SD, of three moringa oleifera seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05)

Table 4.6.5b Comparison of Tocopherols in Enzyme-assisted Aqueous Extracted Moringa oleifera Seed Oils* Tocopherols (mg kg-1) Total Solventextracted 76 8c 55 4c 48.3 1.4a 179.3
*

Natuzyme 95 6ab 88 7b 37.8 2.8b 220.8

Kemzyme 92 5ab 94 12bc 42.5 2.3ab 228.5

Enzyme-assisted Feedzyme 88 4b 92 9a 41.7 3.1ab 221.7

Protex 7L 94 7ab 81 6bc 46.5 6.4a 221.5

Viscozyme L 96 11a 93 13bc 39.3 1.9a 228.3

Control 87 9b 88 7c 41.9 2.5ab 216.9

Values are means SD, of three moringa oleifera seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05)

94

Table 4.6.6a Comparison of Antioxidant Activity of Enzyme-assisted Cold Pressed Moringa oleifera Seed Oils* Parameter TPC (mg GAE/100 g) DPPH, IC50 (g mL-1) Inhibition in linoleic acid system (%) after 175 h incubation at 40C Solventextracted 1.2 0.2 a 64.2 2.2 48.6 0.9
*

Natuzyme b 1.5 0.1 bc 57.8 1.2 54.3 1.3

b

Kemzyme b 1.3 0.2 bc 57.6 1.3 55.5 1.6

b

Enzyme-assisted Feedzyme Protex 7L c a 1.4 0.1 1.4 0.1 c bc 56.4 1.7 57.6 1.2 53.2 1.4
b

Viscozyme L a 1.6 0.3 c 56.3 1.8 53.6 1.6

a

Control 1.2 0.2 b 61.7 1.9 49.2 1.5

c c

55.4 0.8

Values are means SD, of three moringa oleifera seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Table 4.6.6b Comparison of Antioxidant Activity of Enzyme-assisted Aqueous Extracted Moringa oleifera Seed Oils* Parameter TPC (mg GAE/100 g) DPPH, IC50 (g mL-1) Inhibition in linoleic acid system (%) after 175 h incubation at 40C Solventextracted 1.2 0.1 a 64.2 2.2 48.6 0.9
*

Natuzyme c 1.5 0.1 b 57.4 1.7 57.4 1.5

bc

Kemzyme b 1.4 0.2 b 56.8 1.3 58.9 1.1

b

Enzyme-assisted Feedzyme Protex 7L d b 1.3 0.3 1.4 0.1 b b 57.5 1.2 56.7 1.9 63.5 1.7
c

Viscozyme L a 1.6 0.3 b 56.4 1.2 68.4 2.4

a

Control 1.3 0.1 b 62.6 1.8 53.8 1.4

c c

65.6 1.6

Values are means SD, of three moringa oleifera seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

95

Table 4.7.1 Comparison of Proximate Composition of Aqueous Enzyme-assisted Moringa concanensis Seeds* Parameter (%) Oil content Protein content Fiber content Ash content Solventextracted 38.4 1.0a 30.2 0.9a 6.1 0.1a 7.2 0.1a Enzyme-assisted Kemzyme 27.5 0.6b 19.4 0.8c 6.1 0.1a 7.0 0.1a Control 15.4 0.3d 25.4 0.8b 6.1 0.1a 7.1 0.1a

Natuzyme 25.3 0.6bc 20.2 1.0c 6.1 0.1a 7.2 0.1a

Feedzyme 23.5 0.4c 19.1 1.0c 5.9 0.1a 7.0 0.1a

Values are means SD, calculated as percentage on dry seed weight basis for three moringa concanensis seed samples for each enzyme analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05)

Table 4.7.2 Comparison of Physico-chemical Properties of Enzyme-assisted Aqueous Extracted Moringa concanensis Seed Oils* Parameter Refractive Index (40 oC) Density (24 oC g mL-1) Saponification value (mg KOH/g oil) Free fatty acids contents (% as oleic acid) Iodine value (g of I/ 100g oil) Unsaponifiable matter (% w/w) Color (1-in. cell) Red units Yellow units
*

Solventextracted

Natuzyme

Enzyme-assisted Kemzyme

Feedzyme

Control 1.4624 0.02a 0.87 0.03a 176 8a 0.18 0.03b 67 5a 0.67 0.03a 1.2 r 0.1b 14.1 y 0.5b

1.4644 0.02a 1.4627 0.03a 1.4625 0.02a 1.4622 0.03a 0.87 0.04a 180 3a 0.32 0.01a 67 3a 0.76 0.02a 2.1 r 0.2a 19.2 y 0.4a 0.87 0.02a 174 6a 0.19 0.02b 68 6a 0.71 0.01a 1.2 r 0.1b 14.4 y 0.4b 0.87 0.06a 175 4a 0.17 0.02b 68 7a 0.69 0.02a 1.3 r 0.2b 13.8 y 0.3b 0.87 0.01a 173 9a 0.20 0.01b 67 2a 0.70 0.04a 1.2 r 0.1b 12.4 y 0.5c

Values are means SD, of three moringa concanensis seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

96

Table 4.7.3 Comparison of Oxidative State of Enzyme-assisted Aqueous Extracted Moringa concanensis Seed Oils* SolventEnzyme-assisted Parameter Control extracted Natuzyme Kemzyme Feedzyme Conjugated diene 3.17 0.24a 2.92 0.22b 2.88 0.15 b 2.94 0.23b 2.95 0.19b 1% 1cm (232) Conjugated triene 0.67 0.02a 0.59 0.01bc 0.62 0.01b 0.57 0.02c 0.60 0.01bc 1% 1cm (270) Peroxide value (m 1.73 0.14a 1.22 0.13b 1.12 0.09c 1.08 0.11c 1.24 0.13b eq/kg of oil) p-anisidine 1.82 0.06a 1.65 0.04b 1.61 0.08b 1.62 0.11b 1.59 0.09b
Values are means SD, of three moringa concanensis seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).
*

Table 4.7.4 Comparison of Fatty Acid (FA) Composition (g/100g FA) of Enzymeassisted Aqueous Extracted Moringa concanensis Seed Oils* Enzyme-assisted SolventFA Control extracted Natuzyme Kemzyme Feedzyme C16:0 C16:1 C18:0 C18:1 C18:2 C20:0 C20:1 C22:0
*

11.92 0.21a 2.44 0.05a 3.64 0.18a 67.30 1.72a 1.77 0.01a 3.65 0.08a 1.79 0.05a 7.58 0.18a

10.88 0.19a 2.57 0.06a 2.87 0.12b 68.72 1.85a 1.62 0.02bc 3.75 0.11a 1.85 0.07a 7.48 0.15a

10.91 0.24a 2.53 0.04a 2.68 0.14b 68.64 2.50a 1.65 0.02bc 3.83 0.09a 1.90 0.09a 7.39 0.12a

10.83 0.17a 2.50 0.03a 2.84 0.16b 68.78 1.95a 1.59 0.01c 3.71 0.06a 1.82 0.06a 7.46 0.14a

11.20 0.21a 2.68 0.07a 2.86 0.13b 68.07 2.11a 1.70 0.02b 3.79 0.07a 1.74 0.08a 7.34 0.16a

Values are means SD, of three moringa concanensis seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).

Table 4.7.5 Comparison of Tocopherols in Enzyme-assisted Aqueous Extracted Moringa concanensis Seed Oils* Tocopherols Enzyme-assisted SolventControl (mg kg-1) extracted Natuzyme Kemzyme Feedzyme 79 4b 74 7c 82 5a 72 7c 69 2c 11.2 0.5c 19.4 0.6ab 16.9 0.8b 21.3 0.8a 17.1 0.5b b b b a 30.4 1.2 31.2 1.7 28.8 1.2 34.6 1.5 29.7 1.3b Total 120.6 124.6 127.7 127.9 115.8
Values are means SD, of three moringa concanensis seed oils analyzed individually in triplicate. Mean values in the same row followed by the same superscript letters are not significantly different (P > 0.05).
*

97

Part II Optimization of Parameters

98

14 12 10 8 6 4 30 35 40 45 50 55 60 Natuzyme Kemzyme Phytezyme Allzyme Feedzyme

Oil Yield (%)

Temperature ( o C)

Fig. 4.1.1a Effect of Temperature on O il Recove ry from Cottonse eds during EACP

14 12 Natuzyme O il Yield (%) 10 8 6 4 2 3 4 5 pH Fig. 4.1.1b Effect of pH on O il Recovery from Cottonseeds during EACP 6 7 8 Kemzyme Phytezyme Allzyme Feedzyme

99

14 12 O il Yield (%) 10 8 6 4 30 35 40 45 50 55 60 Moisture (%)

Natuzyme Kemzyme Phytezyme Allzyme Feedzyme

Fig. 4.1.1c Effect of Moisture on O il Recovery from Cottonseeds during EACP

14 12 O il Yield (%) 10 8 6 4 3 4 5 6 Time (h) 7 8 9 Natuzyme Kemzyme Phytezyme Allzyme Feedzyme

Fig. 4.1.1d Effect of Hydrolysis Time on O il Recovery from Cottonseeds during EACP

100

14 12 O il Yield (%) 10 8 6 4 0 0.5 1 1.5 2 2.5 3

Natuzyme Kemzyme Phytezyme Allzyme Feedzyme

Enzyme Conc. (% seed basis)

Fig. 4.1.1e Effect of Enzyme Concentration on O il Recovery from Cottonseeds during EACP

14 12 10 Oil Yield (%) 8 6 4 2 0 Natuzyme Kemzyme Phytezyme Enz yme s Fig. 4.1.1f Enz yme s O ffe ring O ptimum O il Re cove ry from C ottonse e ds during EACP Allzyme Feedzyme

101

7 6 5 4 3 2 30 35 40 45 50 55 60 Te mperature ( o C) Natuzyme Kemzyme Phytezyme Allzyme Feedzyme

Oil Yield (%)

Fig. 4.1.2a Effect of Temperature on O il Recove ry from Cottonse eds during EAAE

7 6 Natuzyme O il Yield (%) 5 4 3 2 2 3 4 5 pH Fig. 4.1.2b Effect of pH on O il Recovery from Cottonseeds during EAAE 6 7 8 Kemzyme Phytezyme Allzyme Feedzyme

102

7 6 O il Yield (%) 5 4 3 2 4 5 6 7 8 9 10 Water/Seed Ratio (w/w) Natuzyme Kemzyme Phytezyme Allzyme Feedzyme

Fig. 4.1.2c Effect of Water/Seed Ratio on O il Recovery from Cottonseeds during EAAE

7 6 Natuzyme O il Yield (%) 5 4 3 2 0.5 1.5 Time (h) 2.5 3.5 Kemzyme Phytezyme Allzyme Feedzyme

Fig. 4.1.2d Effect of Hydrolysis Time on O il Recovery from Cottonseeds during EAAE

103

7 6 O il Yield (%) 5 4 3 2 0 0.5 1 1.5 2 2.5 3 3.5 Enzyme Conc. (% seed basis)

Natuzyme Kemzyme Phytezyme Allzyme Feedzyme

Fig. 4.1.2e Effect of Enzyme Concentration on O il Recovery from Cottonseeds during EAAE

7 6 5 Oil Yield (%) 4 3 2 1 0 Natuzyme Kemzyme Phytezyme Enz yme s Fig. 4.1.2f Enz yme s O ffe ring O ptimum O il Re cove ry from Cottonse e ds during EAAE Allzyme Feedzyme

104

35 33 31 Oil Yield (%) 29 27 25 23 30 35 40 45 50 55 60 Temperature (o C) Natuzyme Kemzyme Viscozyme L Protex 7L Feedzyme

Fig. 4.2.1a Effect of Temperature on O il Recove ry from He mpse ed during EACP

34 32 O il Yield (%) 30 28 26 24 2 3 4 5 pH Fig. 4.2.1b Effect of pH on O il Recovery from Hempseed during EACP 6 7 8 Natuzyme Kemzyme Viscozyme L Protex 7L Feedzyme

105

34 32 O il Yield (%) 30 28 26 24 30 35 40 45 50 55 60 Natuzyme Kemzyme Viscozyme L Protex 7L Feedzyme

Moisture (%) Fig. 4.2.1c Effect of Temperature on O il Recovery from Hempseed during EACP

34 32 Natuzyme O il Yield (%) 30 28 26 24 3 4 5 6 Time (h) Fig. 4.2.1d Effect of Hydrolysis Time on O il Recovery from Hempseed during EACP 7 8 9 Kemzyme Viscozyme L Protex 7L Feedzyme

106

34 32 O il Yield (%) 30 28 26 24 0 0.5 1 1.5 2 2.5 3 Enzyme Conc. (% seed basis) Fig. 4.2.1e Effect of Enzyme Concentration on O il Recovery from Hempseed during EACP Natuzyme Kemzyme Viscozyme L Protex 7L Feedzyme

34 30 26 Oil Yield (%) 22 18 14 10 Natuzyme Kemzyme Viscozyme L Enz yme s Fig. 4.2.1f Enz yme s O ffe ring O ptimum O il Re cove ry from He mpse e ds during EACP Feedzyme Protex 7L

107

30

28 Natuzyme Oil Yield (%) 26 Kemzyme Protex 7L Feedzyme 24 Viscozyme L

22 30 35 40 45 50 55 60

Temperature (o C) Fig. 4.2.2a Effe ct of Tempe rature on O il Re covery of Hempsee ds during EAAE

30

28 O il Yield (%)

Natuzyme Kemzyme Viscozyme L Protex 7L Feedzyme

26

24

22 2 3 4 5 pH Fig. 4.2.2b Effect of pH on O il Recovery of Hempseeds during EAAE 6 7 8

108

30

28 O il Yield (%) Natuzyme 26 Kemzyme Viscozyme L 24 Protex 7L Feedzyme 22 4 5 6 7 8 9 10 Water/Seed Ratio (w/w) Fig. 4.2.2c Effect of Water/Seed Ratio on O il Recovery of Hempseeds during EAAE

30

28 O il Yield (%)

Natuzyme Kemzyme Viscozyme L Protex 7L Feedzyme

26

24

22 0.5 1.5 Time (h) 2.5 3.5

Fig. 4.2.2d Effect of Hydrolysis Time on O il Recovery of Hempseeds during EAAE

109

30

28 O il Yield (%)

Natuzyme Kemzyme Viscozyme L Protex 7L Feedzyme

26

24

22 0 0.5 1 1.5 2 2.5 3 3.5 Enzyme Conc. (% seed basis) Fig. 4.2.2e Effect of Enzyme Concentration on O il Recovery of Hempseeds during EAAE

35 30 Oil Yield (%) 25 20 15 10 Natuzyme Kemzyme Viscozyme L Enz yme s Fig. 4.2.2f Enz yme s O ffe ring O ptimum O il Re cove ry from He mpse e ds during EAAE Feedzyme Protex 7L

110

40 38 36 34 32 30 30 35 40 45 50 55 60

Oil Yield (%)

Natuzyme Kemzyme Viscozyme L Protex 7L Alcalase 2.4L

Te mperature ( o C) Fig. 4.3.1a Effect of Temperature on O il Re covery from Sunflower Se eds during EACP

40 38 36 34 32 30 2 3 4 5 pH Fig. 4.3.1b Effect of pH on O il Recovery from Sunflower Seeds during EACP 6 7 8 Natuzyme Kemzyme Viscozyme L Protex 7L Alcalase 2.4L

O il Yield (%)

111

40 38 36 34 32 30 30 35 40 45 50 55 60 Natuzyme Kemzyme Viscozyme L Protex 7L Alcalase 2.4L

O il Yield (%)

Moisture (%) Fig. 4.3.1c Effect of Moisture on O il Recovery from Sunflower Seeds during EACP

40 38 Natuzyme O il Yield (%) 36 34 32 30 3 4 5 6 Time (h) Fig. 4.3.1d Effect of Hydrolysis Time on O il Recovery from Sunflower Seeds during EACP 7 8 9 Kemzyme Viscozyme L Protex 7L Alcalase 2.4L

112

40 38 Natuzyme O il Yield (%) 36 34 32 30 0 0.5 1 1.5 2 2.5 3 Kemzyme Viscozyme L Protex 7L Alcalase 2.4L

Enzyme Conc. (% seed basis) Fig. 4.3.1e Effect of Enzyme Concentration on O il Recovery from Sunflower Seeds during EACP

40

35 Oil Yield (%)

30

25

20 Natuzyme Kemzyme Viscozyme L Enz yme s Fig. 4.3.1f Enz yme s O ffe ring O ptimum O il Re cove ry from Sunflowe r Se e ds during EACP Alcalase 2.4L Protex 7L

113

42 38 34 30 26 22 30 35 40 45 50 55 60 Natuzyme Kemzyme Viscozyme L Protex 7L Alcalase 2.4L

Oil Yield (%)

Te mperature ( o C) Fig. 4.3.2a Effect of Temperature on O il Recove ry of Sunflower Seeds during EAAE

42 38 O il Yield (%) 34 30 26 22 2 3 4 5 pH 6 7 8 Natuzyme Kemzyme Viscozyme L Protex 7L Alcalase 2.4L

Fig. 4.3.2b Effect of pH on O il Recovery from Sunflower Seeds during EAAE

114

42 38 O il Yield (%) 34 30 26 22 4 5 6 7 8 9 10 Natuzyme Kemzyme Viscozyme L Protex 7L Alcalase 2.4L

Water/Seed Ratio (w/w) Fig. 4.3.2c Effect of Water/Seed Ratio on O il Recovery from Sunflower Seeds during EAAE

42 38 34 30 26 22 0.5 1 1.5 2 Time (h) Fig. 4.3.2d Effect of Hydrolysis Time on O il Recovery from Sunflower Seeds during EAAE 2.5 3 3.5

Natuzyme Kemzyme Viscozyme L Protex 7L Alcalase 2.4L

O il Yield (%)

115

40 36 Natuzyme O il Yield (%) 32 28 24 20 0 0.5 1 1.5 2 2.5 3 Enzyme Conc. (% seed basis) Fig. 4.3.2e Effect of Enzyme Concentration on O il Recovery from Sunflower Seeds during EAAE Kemzyme Viscozyme L Protex 7L Alcalase 2.4L

40 35 30 Oil Yield (%) 25 20 15 10 Natuzyme Kemzyme Viscozyme L Enz yme s Fig. 4.3.2f Enz yme s O ffe ring O ptimum O il Re cove ry from Sunflowe r Se e ds during EAAE Alcalase 2.4L Protex 7L

116

30 28 26 24 22 20 30 35 40 45 50
o

Natuzyme Kemzyme Viscozyme L Protex 7L Alcalase 2.4L

Oil Yield (%)

55

60

Temperature ( C) Fig. 4.4.1a Effect of Te mperature on O il Recove ry from Sesame See ds during EACP

30

28 O il Yield (%)

Natuzyme Kemzyme Viscozyme L Protex 7L Alcalase 2.4L

26

24

22 2 3 4 5 pH Fig. 4.4.1b Effect of pH on O il Recovery from Sesame Seeds during EACP 6 7 8

117

30

28 Natuzyme O il Yield (%) 26 Kemzyme Viscozyme L 24 Protex 7L Alcalase 2.4L

22 30 35 40 45 50 55 60 Moisture (%) Fig. 4.4.1c Effect of Moisture on O il Recovery from Sesame Seeds during EACP

30

28

Natuzyme Kemzyme Viscozyme L Protex 7L Alcalase 2.4L

O il Yield (%)

26

24

22 3 4 5 6 Time (h) Fig. 4.4.1d Effect of Hydrolysis Time on O il Recovery from Sesame Seeds during EACP 7 8 9

118

30

28 Natuzyme O il Yield (%) 26 Kemzyme Viscozyme L Protex 7L 24 Alcalase 2.4L

22 0 0.5 1 1.5 2 2.5 3 Enzyme Conc. (% seed basis) Fig. 4.4.1e Effect of Enzyme Concentration on O il Recovery from Sesame Seeds during EACP

30

25

Oil Yield (%)

20

15

10 Natuzyme Kemzyme Viscozyme L Enz yme s Fig. 4.4.1f Enz yme s O ffe ring O ptimum O il Re cove ry from Se same Se e ds during EACP Alcalase 2.4L Protex 7L

119

26 24 22 Oil Yield (%) 20 18 16 14 30 35 40 45 50 55 60 Te mperature ( o C) Fig. 4.4.2a Effect of Temperature on O il Re covery of Sesame Seeds during EAAE Natuzyme Kemzyme Viscozyme L Protex 7L Alcalase 2.4L

26 24 22 O il Yield (%) 20 18 16 14
2 3 4 5 6 7 8

Natuzyme Kemzyme Viscozyme L Protex 7L Alcalase 2.4L

pH Fig. 4.4.2b Effect of pH on O il Recovery of Sesame Seeds during EAAE

120

26 24 22 O il Yield (%) 20 18 16 14 4 5 6 7 8 9 10 Water/Seed Ratio (w/w) Fig. 4.4.2c Effect of Water/Seed Ratio on O il Recovery of Sesame Seeds during EAAE Natuzyme Kemzyme Viscozyme L Protex 7L Alcalase 2.4L

26 24 22 O il Yield (%) 20 18 16 14 0.5 1 1.5 2 Time (h) Fig. 4.4.2d Effect of Hydrolysis Time on O il Recovery of Sesame Seeds during EAAE 2.5 3 3.5 Natuzyme Kemzyme Viscozyme L Protex 7L Alcalase 2.4L

121

26 24 22 O il Yield (%) 20 18 16 14 12 0 0.5 1 1.5 2 2.5 3 Natuzyme Kemzyme Viscozyme L Protex 7L Alcalase 2.4L

Enzyme Conc. (% seed basis) Fig. 4.4.2e Effect of Enzyme Concentration on O il Recovery of Sesame Seeds during EAAE

30 25 20 Oil Yield (%) 15 10 5 0 Natuzyme Kemzyme Viscozyme L Enz yme s Fig. 4.4.2f Enz yme s O ffe ring O ptimum O il Re cove ry from Se same Se e ds during EAAE Alcalase 2.4L Protex 7L

122

30 28

Natuzyme Kemzyme Multifect Pectinase FE Protex 7L Multifect CX 13L

26 Oil Yield (%) 24

22 20 30 35 40 45 50 55 60

Tempe rature (oC) Fig. 4.5.1a Effect of Temperature on O il Re covery from Canola Se eds during EACP

30 28 26 O il Yield (%) 24 22 20 2 3 4 5 pH Fig. 4.5.1b Effect of pH on O il Recovery from Canola Seeds during EACP 6 7 8

Natuzyme Kemzyme Multifect Pectinase FE Protex 7L Multifect CX 13L

123

30 28 O il Yield (%) 26 24 22 20 30 35 40 45 50 55 60 Moisture (%) Natuzyme Kemzyme Multifect Pectinase FE Protex 7L Multifect CX 13L

Fig. 4.5.1c Effect of Moisture on O il Recovery from Canola Seeds during EACP

30 28 26 24 22 20 3 4 5 6 Time (h) Fig. 4.5.1d Effect of Hydrolysis Time on O il Recovery from Canola Seeds during EACP 7 8 9 Natuzyme Kemzyme O il Yield (%) Multifect Pectinase FE Protex 7L Multifect CX 13L

124

30 28 26 24 22 20 0 0.5 1 1.5 2 2.5 3 Enzyme Conc. (% seed basis) Fig. 4.5.1e Effect of Enzyme Concentration on O il Recovery from Canola Seeds during EACP Natuzyme Kemzyme O il Yield (%) Multifect Pectinase FE Protex 7L Multifect CX 13L

30 26 Oil Yield (%) 22 18 14 10 Natuzyme Kemzyme Multifect Pectinase FE Enz yme s Fig. 4.5.1f Enz yme s O ffe ring O ptimum O il Re cove ry from C anola Se e ds during EAC P Multifect CX 13L Protex 7L

125

28 Natuzyme 26 Kemzyme Oil Yield (%) 24 22 20 18 30 35 40 45 50 55 60 Multifect Pectinase FE Protex 7L Multifect CX 13L

Tempe rature ( o C) Fig. 4.5.2a Effect of Temperature on O il Re covery from Canola See ds during EAAE

28 26 O il Yield (%) 24 22 20 18 2 3 4 5 pH 6 7 8

Natuzyme Kemzyme Multifect Pectinase FE Protex 7L Multifect CX 13L

Fig. 4.5.2b Effect of pH on O il Recovery from Canola Seeds during EAAE

126

28 26 O il Yield (%) 24 22 20 18 4 5 6 7 8 9 10 Water/Seed Ratio (w/w)

Natuzyme Kemzyme Multifect Pectinase FE Protex 7L Multifect CX 13L

Fig. 4.5.2c Effect of Water/Seed Ratio on O il Recovery from Canola Seeds during EAAE

28 26 O il Yield (%) 24 22 20 18 0.5 1 1.5 2 Time (h) 2.5 3 3.5

Natuzyme Kemzyme Multifect Pectinase FE Protex 7L Multifect CX 13L

Fig. 4.5.2d Effect of Hydrolysis Time on O il Recovery from Canola Seeds during EAAE

127

28 26 O il Yield (%) 24 22 20 18 0 0.5 1 1.5 2 2.5 3

Natuzyme Kemzyme Multifect Pectinase FE Protex 7L Multifect CX 13L

Enzyme Conc. (% seed basis) Fig. 4.5.2e Effect of Enzyme Concentration on O il Recovery from Canola Seeds during EAAE

30

25 Oil Yield (%)

20

15

10 Natuzyme Kemzyme Multifect Pectinase FE Enz yme s Fig. 4.5.2f Enz yme s O ffe ring O ptimum O il Re cove ry from C anola Se e ds during EAAE Multifect CX 13L Protex 7L

128

24 22 20 18 16 14 30 35 40 45 50 55 60 Natuzyme Kemzyme Viscozyme L Protex 7L Feedzyme

Oil Yield (%)

Temperature (o C) Fig. 4.6.1a Effe ct of Tempe rature on O il Re covery from Moringa oleifera See ds during EACP

24 22 20 18 16 14 2 3 4 5 pH Fig. 4.6.1b Effect of pH on O il Recovery from Moringa oleifera Seeds during EACP 6 7 8 Natuzyme Kemzyme Viscozyme L Protex 7L Feedzyme

O il Yield (%)

129

24 22 Natuzyme O il Yield (%) 20 18 16 14 30 35 40 45 50 55 60 Kemzyme Viscozyme L Protex 7L Feedzyme

Moisture (%) Fig. 4.6.1c Effect of Moisture on O il Recovery from Moringa oleifera Seeds during EACP

24 22 O il Yield (%) 20 18 16 14 3 4 5 6 Time (h) Fig. 4.6.1d Effect of Time of Hydrolysis on O il Recovery from Moringa oleifera Seeds during EACP 7 8 9 Natuzyme Kemzyme Viscozyme L Protex 7L Feedzyme

130

24 22 O il Yield (%) 20 18 16 14 0 0.5 1 1.5 2 2.5 3 Natuzyme Kemzyme Viscozyme L Protex 7L Feedzyme

Enzyme Conc. (% seed basis) Fig. 4.6.1e Effect of Enzyme Concentration on O il Recovery from Moringa oleifera Seeds during EACP

25

20 Oil Yield (%)

15

10 Natuzyme Kemzyme Viscozyme L Enz yme s Fig. 4.6.1f Enz yme s O ffe ring O ptimum O il Re cove ry from Moringa oleifera Se e ds during EACP Feedzyme Protex 7L

131

24

22 Natuzyme Viscozyme L Protex 7L 18 Feedzyme Kemzyme 16 30 35 40 45 50 55 60

Oil Yield (%)

20

Tempe rature ( o C) Fig. 4.6.2a Effect of Tempe rature on on O il Recovery from Moringa oleifera See ds during EAAE

24

22 Natuzyme O il Yield (%) 20 Kemzyme Viscozyme L 18 Protex 7L Feedzyme

16 2 3 4 5 pH Fig. 4.6.2b Effect of pH on O il Recovery from Moringa oleifera Seeds during EAAE 6 7 8

132

24

22 Natuzyme O il Yield (%) 20 Kemzyme Viscozyme L Protex 7L 18 Feedzyme

16 4 5 6 7 8 9 10

Water/Seed Ratio (w/w) Fig. 4.6.2c Effect of Water/Seed Ratio on O il Recovery from Moringa oleifera Seeds during EAAE

24

22 Natuzyme O il Yield (%) 20 Kemzyme Viscozyme L Protex 7L 18 Feedzyme

16 0.5 1 1.5 2 Time (h) Fig. 4.6.2d Effect of Hydrolysis Time on O il Recovery from Moringa oleifera Seeds during EAAE 2.5 3 3.5

133

24

22 Natuzyme O il Yield (%) 20 Kemzyme Viscozyme L Protex 7L 18 Feedzyme

16 0 0.5 1 1.5 2 2.5 3 Enzyme Conc. (% seed basis) Fig. 4.6.2e Effect of Enzyme Concentration on O il Recovery from Moringa oleifera Seeds during EAAE

25

20 Oil Yield (%)

15

10 Natuzyme Kemzyme Viscozyme L Enz yme s Fig. 4.6.2f Enz yme s O ffe ring O ptimum O il Re cove ry from Moringa oleifera Se e ds during EAAE Feedzyme Protex 7L

134

Part III Some Typical Profiles

Fig 4.7.1. Typical GLC Chromatogram Showing the Separation of FAs of Enzymeassisted Aqueous Extracted Sunflower Oil

Fig 4.7.2. Typical GLC Chromatogram Showing the Separation of FAs of Enzymeassisted Cold Pressed Canola Oil

135

Fig 4.7.3. Typical HPLC Chromatogram Showing the Separation of Tocopherol Standards

Fig 4.7.4. Typical HPLC Chromatogram Showing the Separation of Tocopherols of Enzyme-assisted Cold Pressed Hempseed oil

136

Fig 4.7.5. Typical HPLC Chromatogram Showing the Separation of Tocopherols of Enzyme-assisted Cold Pressed Canola Seed Oil

Fig 4.7.6. Typical Rancimat Profile Showing the Induction Period of Enzymeassisted Cold Pressed Cottonseed Seed Oil

137

Fig 4.7.7. Typical Rancimat Profile Showing the Induction Period of Enzymeassisted Aqueous Extracted of Sesame Seed Oil

138

CHAPTER 5
DISCUSSIONS

The experimental parameters such as pH, type of enzyme, enzyme concentration, water/seed (w/s) ratio or moisture content, temperature, and time of extraction were found to be the important factors which affected the oil yield during enzymatic oilseed extraction. The effects of these parameters varied with relative to the type of oilseed due to the differences in oilseed composition. In the present study, we evaluated the effects of these variables on oil recovery during optimization of enzyme-assisted aqueous extraction (EAAE) and enzyme-assisted cold pressing (EACP) of canola-, sunflower-, sesame-, cotton-, hemp-, Moringa oleifera-, and Moringa concanensis-seeds. The quality attributes of the oils produced with enzyme adjuvant were evaluated and compared with control and solvent-extracted oils.

Section 1
5.1 Factors Affecting Oil Yield during Enzyme-assisted Cold Pressing (EACP) Five enzymes were selected for each of the tested oilseed on the basis of literature available and our initial lab trials. In this experimental section we separately determined the effect of each variable, keeping the others constant at the most favourable level. Experiments were carried out mainly to find the optimal levels of the most important variables: moisture, enzyme concentration, pH, temperature, time of hydrolysis, and type of the enzyme for oil recovery. The optimization of conditions affecting oil recovery enzymatic hydrolysis of oilseeds during EACP of seeds is discussed below.

139

5.1.1 Temperature The effect of temperature (35-55 oC) on the oil yield during EACP is displayed in Figs 4.1.1a to 4.6.1a. It was noted that the enzyme activity strongly depend on the temperature. The maximum oil yield was observed at 45 oC for most of the enzymemixtures however Kemzyme (Figs 4.1.1a to 4.6.1a), and Natuzyme (Figs 4.1.1a to 4.6.1a) offered maximum oil recovery at 40 oC. This may be due to the adaptability of the enzyme mixtures at specific temperature ranges. A reduction in oil yield above 45 oC could be caused by an increase in the soluble reducing sugars content caused by enzymatic hydrolysis which would caramelize in the subsequent drying stage (Concha et al., 2004). Other factors could be enzyme inhibition whose concentration would be raised by low moisture conditions (Zuniga et al., 2003). Zuniga et al. (2003) also reported similar effect of temperature on hydrolysis enzymatic oil extraction from Guevina avellana mol.

5.1.2 pH Although there is a range of optimum pH for each enzyme mixture however its effect varies in some cases with the type of oilseed. Figs (4.1.1b to 4.6.1b) depict the variation of oil recovery with a change in pH of the sample mixture. The enzyme activity was strongly affected by the pH variation. Kemzyme, Feedzyme, Natuzyme, Phytezyme, Allzyme, and Viscozyme L exhibited maximum oil recovery at pH = 5 for all the oilseeds tested (Fig 4.1.1b to 4.6.1b), whereas Feedzyme showed maximum activity at pH = 4 for cottonseed (Fig 4.1.1b). For sunflower (Fig 4.3.1b) and sesame (Fig 4.4.1b) an optimum pH for Kemzyme was 6. Alcalase 2.4L and Protex 7L offered potent activity at pH = 6. In similar studies, Dominguez et al. (1993) extracted oil at the natural pH = 6.6 of the soybean seeds suspension whereas, Santamaria et al. (2003) conducted this process at pH= 6.2 for oil extraction from Chilean Hazelnut.

140

5.1.3 Moisture (%) Water is required to moisten the seeds for effective action of the enzymatic formulation. A higher level of moisture could reduce the reaction time required to obtain higher oil yields by facilitating the enzymatic degradation of seed cell walls (Dominguez et al., 1993). However, a high moisture content increases the energy costs involved in the subsequent drying stage, which is necessary for adequate performance of the pressing operation (Concha et al., 2004). In order to determine the effect of moisture on the enzymatic activity, several experiments at moisture levels ranging from 35 to 55% (seed weight basis) were conducted in the present study. An optimum enzyme concentration, pH and a hydrolysis time of 6 h were employed in these assays. An increase in the oil yield was observed by increasing the moisture content from 35 to 45%, and then a decrease in the oil yield was recorded for samples with moisture content > 45% (Figs 4.1.1c to 4.6.1c). The moisture content (45%) was found to be optimum for Feedzyme, Kemzyme, Natuzyme, Phytezyme, and Allzyme whereas, Protex 7L, Viscozyme L, Alcalase 2.4L offered maximum oil recovery at 40% moisture level (Figs 4.1.1c to 4.6.1c). Similar studies were also conducted for the olive oil extraction with 10% moisture content and with an addition of the enzymatic solution in buffered media. Whereas, Montedoro and Petruccioli (1973) reported that enzymes offered their maximum activity during olive oil extraction with a 35% or more water content. Some authors (Sosulski and Sosulski 1993; Smith et al., 1993; Zuniga et al., 2003) reported an optimal moisture level of less than 45% for enzymatic hydrolysis during EACP. In another study, Smith et al. (1993) observed optimal moisture of 23% in soybean oil extraction using an enzyme-aided pressing process. In G. avellana mol oil extraction, the best oil yield resulted when the enzymatic reaction moisture was 45% w/w (Zuniga et al., 2003). Additionally, during canola oil extraction with the help of enzymes, 42% oil was extracted at moisture levels between 30 and 50% (w/w) during enzymatic hydrolysis (Sosulski and Sosulski 1993).

141

5.1.4 Time of Hydrolysis The hydrolysis time affected the oil extraction yield during EACP. For these experiments, moisture content of 45%, an optimum enzyme concentration, pH and 45 oC temperature were kept constant during incubation. By comparing Figs (4.1.1d to 4.6.1d) it can be observed that longer hydrolysis periods more than intermediate values (around 6 h) did not favour extractability of oil from seeds, rather it may produce undesirable effects on odour of oil due to the amount of water present during prolonged incubation (Dominguez et al., 1993). With few exceptions, Viscozyme L, Protex 7L, Alcalase 2.4L, Multifect Pectinase FE, and Multifect CX13L provided maximum oil recovery at 5h (Figs 4.2.1c to 4.6.1c), while Kemzyme, Natuzyme, Feedzyme, and Allzyme exhibited maximum oil recovery at 6h (Figs 4.1.1c to 4.6.1c). In case of Phytezyme 7h was found to be the optimum hydrolysis time (Figs 4.1.1c). Similar findings were reported by Dominguez et al. (1993), although reducing sugar production is more affected by the length of the hydrolysis time, but there is no favourable action on the oil extractability therefore, a period around 6 h should be chosen for the treatment. In contrast to our findings, Zuniga et al. (2003) reported no significant effect of incubation temperature on the oil recovery from Guevina avellana mol seeds.

5.1.5 Enzyme Concentration It is very important to determine whether a higher enzyme concentration could exert a more efficient action for oil extraction. For this purpose, seeds in the present experiment were treated for a period of 6 h with the enzymatic preparations by properly diluting to keep final moisture of 45% at five enzyme concentrations 0.5, 1.0, 1.5, 2.0, and 2.5% of the seed weight. Figs (4.1.1e to 4.6.1e) show the effect of this variable on oil yield. For Allzyme, Phytezyme, Feedzyme, Natuzyme, and Kemzyme 2% enzyme concentration (on seed weight basis) was found to be the best concentration whereas, 1.0% was found to be optimum for Viscozyme L, Multifect Pectinase FE, Multifect CX13L, Protex 7L and 1.5% for Alcalase 2.4L (Figs 4.1.1e to 4.6.1e). The effect of

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enzyme concentration on the oil extractability was more marked for the range between 0.5 and 1.0 g/100 g seed, than when it is raised from 1.0 to 2.5%. An additional increase of this variable does not imply a parallel improvement in oil yield, but an increase of the costs. As the oil extractability is enhanced by increasing the enzyme concentration, the net reducing sugar production is also increased, which were caramelised in the consecutive drying stages, limiting oil release (Dominguez et al., 1993). Browing caramelisation can be easily detected by simple visual observation of the samples after drying, although the drying stage was carried out under vacuum conditions. Zuniga et al. (2003) observed browning reactions for canola and rose hip enzyme assisted oil extraction process. During EACP, the increased oil recovery may be due to the different enzymatic activities of the enzymes on seed cell wall. Even the lower enzyme concentration up to 1.0% has a marked effect on the oil extractability. However, the oil extractability remains almost unaffected or increased slightly when the enzyme concentration is raised above 1.0%. So the enzyme concentration should compromise between the improvement of the oil extractability and the cost of enzyme (Zhang et al., 2007). Under the optimized set of conditions Kemzyme was found to be the most effective enzyme mixture for EACP of cotton- (Fig 4.1.1f), and canola- (Fig 4.5.1f) seeds offering 12.9 and 28.7% oil yields, respectively whereas Viscozyme exhibited best activity for hemp- (Fig 4.2.1f), sunflower- (Fig 4.3.1f), M. oleifera-seeds (Fig 4.6.1f) offering 32.8, 39.2, and 21.6% oil yields, respectively. Protex 7L was efficient for sesame seeds (Fig 4.4.1f) with 28.2% oil recovery. It was observed that the enzyme efficiency was varied with respect to oilseed type. It is generally considered that oils are locked within the matrix of the interacting macromolecules and this close association can be successfully disrupted by using different enzymes to digest the various components resulting more oil release from the matrix (Abdulkarim et al., 2006). In the present study, it was observed that enzyme mixtures offered higher oil recovery as against the single enzymes which may be due to their combined effect on seeds colloidal and lipoproteic structures (Dominguez et al. 1994).

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In agreement to our present study an improvement in lipid extraction from soybean was observed by using proteolytic enzymes compared to the control (Yoon et al., 1991). A combination of polygalacturonase, -amylase, and protease was employed to increase the coconut oil yield upto 80% (McGlone et al., 1986; Barrios et al., 1990) but 90% yield was observed by using a galactomannase combined with a polysaccharideenzyme degrading complex. Freitas et al. (1993) reported an improvement in avocado oil yield by using mixtures of commercial preparations with different carbohydrase activities.

5.2 Factors Affecting Oil Yield during Enzyme-assisted Aqueous Extraction (EAAE) Most of the studies on enzymatic oil extraction have been performed by aqueous extraction process. In EAAE process, the enzymes improve oil recovery by degrading the seed cell wall, resulting in rupturing the polysaccharideprotein colloid system. Similar to the EACP, five enzymes were selected for each of the oilseeds on the basis of previous studies and our preliminary lab trials. Experiments were carried out mainly to preevaluate the feasibility and interest of the enzymatic treatment of oilseeds and select the operational range of the most important variables. Several assays using the more adequate formulations found in previous experiments were carried out. The effects of pH, water/seed ratio, and time of hydrolysis on oil yield by EAAE were investigated. The effects of these parameters allowed us to define the operational characteristics of the process. We proceeded by changing the variables one by one, keeping the others constant at the most favourable value found in previous experiments.

5.2.2 Temperature The values of this parameter should be in the range of maximum enzyme activity, but it must be ensured that the quality of the oil extracted should not be affected. The effect of temperature on oil yields during EAAE is shown in Figs (4.1.2a to 4.6.2a). When the extraction temperature was raised from 30 to 45 oC, the oil yield increased

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significantly. A decrease was observed at higher temperature except the Multifect CX 13L treated sample due to its wide operational temperature range. Kemzyme, Natuzyme, Feedzyme, Allzyme, Phytezyme, Viscozyme L, Protex 7L, Alcalase 2.4L, and Multifect Pectinase FE offered maximum oil recovery at 45 oC (Figs 4.1.2a to 4.6.2a) whereas, Viscozyme L (Figs 4.4.2a) exhibited potent activity at 40 oC for sesame seeds. For hemp(Figs 4.2.2a), sunflower- (Figs 4.3.2a), sesame- (Figs 4.4.2a), and canola-seeds (Figs 4.5.2a), 40 oC temperature was found to be the optimum when Natuzyme, and Kemzyme were employed. No enhancement in the oil yield was observed above 50 oC. It is also expected that the starch would have gelatinised in the pre-heating step at 90 oC and the mixture viscosity would increase due to solubilization of other components. At high temperatures it is difficult to maintain homogeneity during extraction and also it results in protein denaturation. Therefore, a temperature range of 40-50 oC was deemed to be satisfactory for oil extraction. The temperature seems to exhibit considerable effect on the oil yields from different oilseeds during hot water floatation method (Southwell and Harris 1991). Lusas et al. (1982) reported that the temperature is critical for oil extraction from soybeans during aqueous extraction process. They observed maximum oil recovery between 40-60 C. In some other studies, different temperatures have been employed in aqueous extraction process with other oilseeds. Hagenmaier (1974) extracted sunflower oil at room temperature. A temperature range (60-65 C) was selected for the extraction of peanut oil (Subrahmanyan et al., 1959; Rhee 1972) and 70 C for the extraction of rapeseed oil (Embong and Jelen 1977) whereas 80C was maintained during coconut oil extraction (Hagenmaier et al., 1972). Lanzani et al., (1975) treated sunflower, rapeseed and peanut using an increasing temperature (40 C, 50 C and 65 C) for a period of 3 h during enzyme-assisted aqueous extraction. Whereas, Fullbrook (1983) utilized a temperature of 60 min at 50 C, 120 min at 63 C and a short period (for 13 min) at 80 C to inactivate enzymes for rapeseed and soybean during aqueous extraction. Aparna et al. (2002) used temperature of 37, 40, 50 and 60 oC during the enzyme-assisted extraction of peanut oil and observed a temperature

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of 40 oC as the optimum for having yielded the best oil recovery. Barrios et al. (1990) used temperature of 40, 50, 55 and 60 oC for coconut oil extraction and reported the highest oil recovery at 50 oC.

5.2.3 pH The enzymes activities are strongly dependent upon the pH. The pH range of 4-6 was found to be generally suitable in the present study. Figs (4.1.2b to 4.6.2b) show the effect of pH of the extraction medium on oil extraction yield during EAAE. NaOH/HCl was occasionally added to the dispersion to maintain the pH. The data showed that the oil yield increased significantly (P < 0.05) at optimum pH of each enzyme. It has been observed that the effect of pH is more pronounced in EAAE than the EACP. The pH of extraction medium seems to be a key process variable here. Feedzyme, and Multifect Pectinase FE exhibited maximum activity at pH 4.0, Kemzyme, Phytezyme, Allzyme showed maximum efficiency at pH 5.0, whereas pH of 6.0 was found to be most appropriate for Viscozyme L, Protex 7L, and Alcalase 2.4L (Figs 4.1.2b to 4.6.2b). Higher pH can result in higher viscosity due to cells degradation. This makes it more difficult to separate solid-liquid phase by centrifuging which results a decrease in the oil yield. It may also result in deterioration of oil quality through saponification (Rhee et al., 1973b) and cause several changes to the amino acids, such as, the formation of lysinoalanine, lanthionine (De Groot and Slump 1969). The optimum extraction pH varies quite sensitively with changing the oil-bearing material. In aqueous extraction of oil from certain seeds can be regarded as a process primarily aimed at solubilizing proteins with release of oil. A close relationship exists between the oil and protein extraction in some oilseeds because the conditions for the maximum oil extraction generally coincide with the highest protein yields (Rosenthal et al., 1996). Highest oil extraction yield occurs at pH values corresponding to maximum protein extractibility in the aqueous system and the lowest yield is obtained when the protein solubility is at its lowest, i.e., around the isoelectric point (Rosenthal et al., 1996).

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But the isoelectric pH values for soybean, sunflower, and coconut, corresponded to the least oil extraction. In general for most oilseeds, maximum oil and protein yields is obtained at a pH away from the isoelectric point. Optimum pH values for oil extraction were 4.0, 7.0, or 10.0 for peanut (Subrahmanyan et al., 1959; Rhee et al., 1972; Hagenmaier et al., 1973; Rhee et al., 1973b) 10.0 for sunflower (Rhee et al., 1973b) and 6.6 for rapeseed (Embong and Jelen 1977).

5.2.4 Water/Seed (w/s) Ratio The effect of w/s ratio (w/w) on oil recovery is shown in Figs 4.1.2c to 4.6.2c. The optimum w/s ratio in the present study was generally ranged from 6:1 to 8:1. However, Phytezyme-, and Allzyme-treated seeds showed maximum oil yield at 8:1 w/s ratio whereas, 7:1 w/s ratio was found to be optimum for Feedzyme-, Natuzyme, and Kemzyme-treated seeds. For Viscozyme L, Protex 7L, Alcalase 2.4L, Multifect Pectinase FE, and Multifect CX 13L 6:1 w/s ratio was the most appropriate for highest oil recovery from the oilseed materials (Figs 4.1.2c to 4.6.2c). However, at the higher w/s ratio (9:1), the viscosity of the mixture was increased. This made it difficult to maintain mixture homogeneity and decreased the oil recovery. In the present study, a w/s ratio of 7:1 was found to be adequate for this process. The amount of water added during the treatment affects the oil recovery depending on the oilseed. Buenrostro and Lopez-Munguia (1986) reported that the maximum yield of avocado oil was obtained with a 5:1 w/s ratio, while for coconut the best yields were obtained at a 4:1 w/s ratio (Cintra et al., 1986). The oil yield was reported to be increased slightly with dilution, but it decreased again at higher dilutions (Figs 4.1.2c to 4.6.2c). This may be attributed to both emulsion formation and emulsion stability. When the w/s ratio is decreased, the oil yield decreased dramatically which may be in due part to the difficulty of keeping the mixture in suspension during treatment. In agreement to our findings, McGlone et al. (1986) also reported similar trends. However, Picuric-Jovanovic et al. (1997) investigated that the extraction yields depend on the ratios, because the enzyme accessibility to the cell walls should be more effective at lower ratios.

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The w/s ratio is selected so as to obtain less stable emulsions and generate less effluent; however, to obtain the highest extraction rates and extraction yields, it is usually necessary to use large quantities of water. Literature reports reveal that w/s ratios from 5:1 to 12:1 for peanut oil extraction (Subrahmanyan et al., 1959; Rhee et al., 1972; Sugarman 1956; Bhatia et al., 1966), 10:1 for sunflower (Hagenmaier 1974) and 12:1 was found as the optimum water seed ratio for soybeans (Lusas et al., 1982). Embong and Jelen (1977) reported an optimum ratio varying between 2.5:1 and 3.5:1 for rapeseed oil extraction.

5.2.5 Hydrolysis Time The effects of hydrolysis time on EAAE are shown in Figs (4.1.2d to 4.6.2d). It can be seen that hydrolysis time has appreciable effect on oil extraction yield at time higher than 1.5 h but the oil yield decrease after 2.5 h. As evident in the Figs (4.1.2d to 4.6.2d), an increase in the incubation time from 2.5 to 3 h did not improve oil extraction because the levels of reducing sugar increased when carbohydrases were used. It can be seen that oil extraction yields increased slightly when extraction time increased. The effect of hydrolysis time on the oil extraction yield with an optimum enzyme concentration and pH at 45 oC were observed. The maximum oil yield for Kemzyme-, Natuzyme-, and Feedzyme-treated seed was observed at 2 h incubation time whereas for Allzyme-, and Phytezyme-treated seed it was 2.5 h. For Viscozyme L, Protex 7L, Alcalase 2.4L, Multifect Pectinase FE, and Multifect CX 13L 1.5 h hydrolysis time was found to be sufficient for maximum oil recovery. It has been investigated that more stable emulsions are formed in a prolonged extraction process. Kim (1989) reported 30 min period as an optimum time for palm oil extraction, and 40 min period for extracting both soybean oil and protein (Lusas et al., 1982) whereas, Embong and Jelen (1977) found 15 min blending followed by a prolonged period of l-4 h of stirring more appropriate for rapeseed oil extraction. Still,

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there is lack of information on the kinetics of aqueous extraction from different oilseeds which therefore hampered rational process design and development. In contrast to our findings, Abdulkarim et al. (2006) reported increase in the oil recovery with an increase in the incubation time. A very sharp increase in oil recovery was observed from 0 to 2 h, after which the rate slowed down. Similarly, Dominguez et al. (1994) also reported that 0.332.00 h was sufficient to exhibit a significant increase in oil recovery. A gradual increase in the reaction time increased the oil recovery to an optimum level. But the optimal recovery time selected for this study was 36 h and is considered too long if this process is to be commercially employed. Some other concerns such as energy expenditure and undesirable by-products will make it economically unattractive. Therefore, a shorter incubation time will be more advantageous in industrial extraction processes (Abdulkarim et al., 2006).

5.2.6 Enzyme Concentration Figs 4.1.2e to 4.6.2e illustrate the effect on oil yield from oilseeds with increasing the enzyme concentration. The optimum enzyme concentration for Natuzyme, Kemzyme, Feedzyme, Allzyme was found to be 2.0%, whereas it was 2.5% and 1.5% for Phytezyme and Alcalase 2.4L, respectively. For Viscozyme L, Protex 7L, Multifect Pectinase FE, and Multifect CX 13L, 1.0% was found to be the best enzyme concentration for oilseed materials under investigation (Figs 4.1.2e to 4.6.2e). Similar to EACP mixed activity enzymes were found to be more efficient than those of pure enzymes for oil extraction. The reason lies in the nature of the cell wall of oilseeds, which is a complex substrate, more easily degraded by a mixture of enzymes of varing activities than pure enzymes. It has been observed that the oil yield increases with enzyme concentration, but not continuously. The enzyme efficiency was considerably reduced at high and low concentrations. Also, the high enzyme cost associated with high enzyme concentration is a major obstruction for the economy of the process.

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Depending on the enzyme and the experimental conditions, different oil yields are obtained. The enzyme concentration even in the range of 1-3 g/100 g seed has considerable effects on oil yields depending on the seed nature. For rapeseed 2 g/100 g appears to be the optimum (Dominguez et al., 1994). Fullbrook (1983) reported increment in melon seed oil recovery over 100% with respect to control samples during aqueous extraction. Bhatnagar and Johari (1987) made an improvement (more than 20 % of the total oil) in cottonseed oil extraction yield, while for castor and sunflower the increment was less than 10%. It is known that an increase in the enzyme concentration increases the rate at which the oil is separated, but the optimum level must be established. Cellulase (at 2530 g/100 kg) enhanced the oil extraction of olive, but its efficiency was considerably reduced at higher or lower concentrations. Dominguez et al. (1994) reported 50 g/100 kg as an optimum concentration for acid protease. Tano-Debrah and Ohta (1995a) observed a rapid increase in extracting shea fat at enzyme concentration (0.0 to 1.0%). Similarly, Dominguez et al. (1995) used 0.54% (w/w) enzyme (Celluclast + Pectinex) concentration to extract sunflower oil and found that a 2% (w/w) enzyme concentration was most favorable. No increment in the oil recovery was observed beyond this concentration. Abdulkarim et al. (2006) reported a gradual increase in oil recovery with the increase in the enzyme concentration from 0.5 to 2.0% (v/w) and obtained highest M. oleifera seeds oil recovery with 2.0% (v/w) enzyme. No increase in the oil yields were observed on further increase in enzyme concentration to 2.5% (v/w). In the present study, the enzyme pre-treatment appreciably increased the oil yield at optimized set of experimental conditions as shown in the Figs (4.1.2f to 4.6.2f). Enzymes are substrate specific therefore there activity varies according to nature of the oilseeds. The effect of enzymatic pre-treatment depends on the oilseed structure and the cell wall composition therefore, it varies according to the kind of oilseed and type of enzyme used (Dominguez et al., 1994). An increase of about 30-50% in enzymeextracted oils with reference to the controls was observed. Kemzyme, Alcalase 2.4L, and Multifect CX 13L were found to be the best enzyme-mixtures for cotton- (Fig 4.1.2f),

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sesame- (Fig 4.4.2f), and canola-seeds (Fig 4.5.2f) offering 6.4, 24.8, and 26.0% oil recoveries, respectively. Viscozyme L was found to be the most efficient enzyme for hemp- (Fig 4.2.2f), sunflower- (Fig 4.3.2f) and M. oleifera-seeds (Fig 4.6.2f) extraction producing 29.1, 39.7, and 22.5% oil yields, respectively. As expected, enzyme mixtures offered higher oil recovery due to their combined effect on colloidal and lipoproteic seed structures (Dominguez et al., 1994). This can be attributed to the presence of several potent components in these enzyme mixtures, e.g. arabanase, cellulase, -glucanase, hemicellulase, xylanase and pectinase (in Viscozyme). Similar results as ours have also been reported by Hernandez et al. (2000) for rice bran. In agreement to our results that enzyme mixtures were found to be more effective than the single enzyme, Tano-Debrah and Ohta (1995a, 1995b) also examined combined enzymatic effects of acid protease, cellulase, hemicellulase and glucanase, and Sumizymes LP (protease), cellulase and hemicellulase and obtained recoveries of 74.1 and 72.7% for the extraction of shea and cocoa fat, respectively. A 70% recovery during aqueous-enzymatic extraction of sunflower-kernel oil was obtained by using a combination of cellulase and pectinase (Novozymes) (Dominguez et al., 1995a). The palm mesocarp was treated with cellulase preparation and 57% palm oil was recovered during the aqueous process (Cheah et al., 1990). Lanzani (1975) reported peanut oil yields of 74-78% by aqueous extraction using protease, cellulase, and -1,4-galacturonide glycano hydrolase as compared to the control (72%). Olsen (1988) utilized pectinase, cellulase, and hemicellulase combinations to degrade rapeseed during aqueous extraction process. By using cellulolytic enzymes on steamed rapeseed flour, 35% of the oil was extracted (Marek et al., 1989). Dominguez et al. (1995) observed an enhancement up to 30% in the sunflower oil recovery during aqueous extraction by using mixtures of cellulase and pectinase. A carbohydrase complex was found to be the most effective enzyme when a group of commercial enzymes were utilized for aqueous extraction of corn germ oil (Bocevska et al., 1993). Similarly, 80% oil recovery has been reported by using a commercial carbohydrase complex in the aqueous process of rapeseed (Deng et al. 1992).

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Although enzyme mixtures were found to be the most effective enzyme adjuvant however protease (Alcalase 2.4L) was noted to be effective for sesame seeds. This may be due to the proteolytic activity which may break up the cytoplasmic network, composed of structural fibrous proteins encapsulating oil globules, and enable easy removal of oil from the cells (Rosenthal et al., 1996). Several researchers have also reported the use of proteases either alone or in conjunction with other enzymes for improving oil extraction yields from soybean (Bargale et al., 2000; Rosenthal et al., 2001), coconut (McGlone et al., 1986; Barrios et al., 1990; Che Man et al., 1996), Shea fat (Tano-Debrah and Ohta, 1995b), cocoa fat (Tano-Debrah and Ohta 1995a) and avocado (Buenrostro and LopezMunguia 1986).

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Section 2
5.3 Comparison of the Quality of Extracted Oils and Oilseed Residues

5.3.1 Proximate Composition of Oilseeds Data for proximate composition of enzyme-treated and control oilseed are summarized in Tables 4.1.1a to 4.6.1a. The oil content of the enzyme-assisted cold pressed (EACP) oilseeds was found to be significantly (P < 0.05) higher than the control, but lower with respect to hexane-extracted seeds. Depending on the cell wall composition of the oilseeds, enzyme activities were noted to be varied. Kemzyme, Protex 7L, and Multifect CX 13L were found to be the most efficient enzyme mixtures for cotton-, sesame-, and canola-seeds offering 12.9, 28.2, and 28.7% oil yields, respectively whereas, Viscozyme L was effective for hemp-, sunflower-, and M. oleifera-seeds with 32.8, 39.2, and 21.6% oil yields, respectively. The higher oil yield in the present enzymeassisted extraction can be ascribed to better solubilization and hydrolysis of proteins by enzymes, which possibly caused a breakdown in the protein network characteristic of the cotyledon cells cytoplasm, and in the protein (oleosin)-based membranes surrounding the lipid bodies, so liberating the oil (Bair and Snyder 1980; Tzen and Huang 1992; Huang 1994). Soto et al. (2004) reported an enhancement in borage oil recovery by enzymatic cold pressing. Similar results have also been found in the enzyme-assisted oil extraction of sesame, groundnut and sunflower seed oils (Singh et al., 1999). Similarly enzyme-assisted aqueous (EAA)-extracted oil yield was found to be significantly (P < 0.05) higher than the control (without enzyme). However, it was lower (P < 0.05) than that achieved by solvent extraction (Tables 4.1.1b to 4.6.1b, 4.7.4). Alcalase 2.4L, and Multifect CX 13L were found to be the most efficient enzyme mixture for sesame-, and canola-seeds, offering 24.8%, and 26.0% oil yields, respectively whereas, Kemzyme showed highest activity for cotton-, and M. concanensis-seeds with 6.4, and 27.5% oil yields, respectively. Viscozyme L was effective for hemp-, sunflower-, and M. oleifera-seeds, recovering 29.1, 39.7, and 22.5% oil yields, respectively. The

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higher oil extraction efficiency can be explained by enzymatic action that improves oil recovery by degrading seed cell wall (Concha et al., 2004). The multiple activity complexes and enzyme mixtures are especially effective for degradation of seed cell walls, due to their synergistic action thus making more oil available for extraction (Sineiro et al., 1998). The protein content of the oilseeds treated with enzymes during EACP was found to be comparable (P < 0.05) with that of the control and solvent-extracted oilseeds (Tables 4.1.1a to 4.6.1a). Whereas, for EAAE process, the residues remaining after aqueous extraction (with or without enzymes) revealed significantly (P < 0.05) lower protein contents (N x 6.25) as against the solvent-extracted residues which may be attributed to the extraction of some proportion of protein into the aqueous and emulsion phases (Tables 4.1.1a to 4.6.1a, 4.7.4). The highest protein in the aqueous phases of cotton- and canola-seeds was extracted with Allzyme, and Multifect CX 13L, respectively. Therefore, the protein content in the cotton-, and canola-seeds was found to be lower than the solvent-extracted seeds. Feedzyme was found to be the best enzyme mixture for protein extraction in the aqueous phase for hemp-, M. concanensis-, and M. oleifera-seeds whereas, Protex 7L was found to be effective for sunflower-, sesame-, and canola- seeds. The extracted proteins can potentially be used as food ingredients. No significant (P > 0.05) variations were observed for the fiber and ash contents among enzyme-treated and control oilseed residues (Tables 4.1.1a to 4.6.1a and 4.1.1b to 4.6.1b, 4.7.4).

5.3.2 Physicochemical Characteristics of Oils Various physical and chemical parameters of the solvent-extracted, EAC-pressed, and control oils in Tables 4.1.2a to 4.6.2a are given. There were no significant (P > 0.05) differences for the refractive index, density, and saponification values between the enzyme-, and solvent-extracted oils. A slightly higher value of unsaponifiable matter in the solvent-extracted oil as compared with those of the control and EAC-pressed oils was

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observed. This may be due to the ability of the solvent to extract other lipid-associated substances such as sterols, fat-soluble vitamins, hydrocarbons and pigments (Abdulkarim et al., 2005). The level of free fatty acids was found to be significantly (P < 0.05) higher in all the enzyme-extracted seed oils as compared to solvent-extracted oils. This increase in the free fatty acids may be attributed to the longer hydrolysis time and enzymatic effect during the EACP. The color of the enzyme-extracted oils was found to be significantly higher (P < 0.05) with respect to solvent-extracted oil. The intense color in enzymeassisted cold (EAC)-pressed oil may be due to enhanced release of coloring pigments from the seed tissues (Ranalli et al., 2003). For EAA-extracted oils, the analyzed physical and chemical parameters are given in Tables (4.1.2b to 4.6.2b, 4.7.4). As expected, the refractive index, density, and iodine value of the oils were not affected by the extraction methods. As the oil was extracted under mild and non-oxidizing temperature so the free fatty acid content of the EAAextracted oil was significantly lower (P < 0.05) as compared to solvent-extracted oil where accelerated temperature conditions were applied. These results are in accordance with the findings reported by Abdulkarim et al. (2005) and Hanmoungjai et al. (2001).

5.3.3 Oxidative State The oxidation parameters of the EAC-pressed, control, and solvent-extracted oils are shown in Tables (4.1.3a to 4.6.3a). The specific extinctions at 232 and 270 nm, which revealed the oxidative deterioration and purity of the oils (Anwar et al., 2006), for the enzyme-extracted oils were found to be comparable with that of control but significantly (P < 0.05) lower than the solvent-extracted oil. The conjugated diene contents, as represented by absorption at 232 nm, of the enzyme-extracted oils were found to be comparable with that of the control, but significantly (P < 0.05) lower than solventextracted oils. Similarly, the level of conjugated trienes as determined at 270 nm was significantly (P < 0.05) lower for the enzyme-extracted oils as against the solventextracted oils. The peroxide and p-anisidine values of enzyme-extracted seed oil were comparable with those of the control; however, these levels were significantly (P < 0.05)

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lower than those of solvent-extracted oils. The enzyme-extracted oils showed higher oxidative stability in terms of induction period as against the solvent-extracted oils. The induction period (Rancimat: 20L/h, 120 C), is a characteristic of the oxidative stability of oils and fats (Anwar et al., 2003). In agreement to our findings, Ranalli et al., (1997) observed higher induction period in the olive oils obtained with the enzyme processing aid. The oxidative stability parameters of EAA-extracted oils are presented in Tables (4.1.3b to 4.6.3b, 4.7.4). The specific extinctions, at 232 and 270 nm, of aqueous enzymeextracted oils, were comparable to that of control but significantly (P < 0.05) lower with regard to solvent-extracted oil. The peroxide and p-anisidine values of aqueous-enzymeextracted oil were similar to that of control, however, these were significantly (P < 0.05) lower as comapred with solvent-extracted oil. The induction period (h) measured by the Rancimat also confirmed better oxidative stability of EAC-pressed oils than that of solvent-extracted oils. A noticeably lower level of different oxidation parameters and thus good oxidative stability exhibited in the present analysis of enzyme-extracted oil as compared to the solvent-extracted oil might be attributed to the extraction technique applied. In contrast to our findings, the oxidation indices such as carbonyl index, peroxide index and UV indices (K232, and K270) of olive oil are generally not influenced by the enzyme actions (Ranalli et al., 2004; Ranalli et al., 1998). The conventional vegetable oilseed extraction process is performed by means of organic solvents, where the high operational temperature might affect the oil quality and its composition, particularly with regard to the oxidation state.

5.3.4 Fatty Acid Composition Tables (4.1.4a to 4.6.4a) present the fatty acid composition of the control, EACpressed and solvent-extracted oils. Analytical data (with respect to the control and

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solvent-extracted oils), showed that enzyme-assisted extraction had no significant (P > 0.05) influence on the composition and contents of major fatty acids. Similarly, the fatty acid composition of the oils obtained by EAAE, control and solvent extraction methods was not affected by the extraction process (Tables 4.1.4b to 4.6.4b, 4.7.4). The results of the present investigations of fatty acid profiles of extracted oils are in agreement to those of Abdulkarim et al. (2005) and Concha et al. (2006), who showed no significant variation in the major fatty acid contents of the enzyme- and solvent-extracted M. oleifera-, and rosehip-seed oils respectively. Ranalli et al. (1997; 1998) also reported that fatty acid composition of the oils was not significantly (P < 0.05) affected by the enzyme treatment of the olive pastes. Enzymatic process can be favorably recommended for the extraction of oils with high degree of un-saturation such as hempseed oil which is very prone to oxidation during accelerated solvent extraction.

5.3.5 Tocopherol Contents Tables (4.1.5a to 4.6.5a) show the levels of different tocopherols in the control, EAC-pressed and solvent-extracted seed oils. The concentration of tocopherols (-, -, and -) in the oils obtained by the EAAE and other processes are presented in Tables (4.1.5b to 4.6.5b, 4.7.5). The -tocopherol contents, which exhibit the highest vitamin E activity (Anwar et al., 2006), of oils extracted by EACP and EAAE methods were found to be appreciably higher than control and solvent-extracted oils. The concentration of tocopherol, which exhibits potent antioxidant activity than either -, -tocopherols (Anwar et al., 2006), in the enzyme-extracted oils was also higher than that of solventextracted oils. The values of -tocopherol were substantially higher in all the oils, produced with enzyme mixture, revealing the efficacy of the tested enzymes towards greater release of this particular isomer of tocopherol. This variation in the contents of tocopherols is consistent with the improved oxidation levels, in terms of measurements of peroxide, conjugated diene and triene values as observed in enzyme-extracted oils. Overall, the oils extracted by EACP and EAAE in the present analysis were significantly (P < 0.05) richer in total tocopherols than the control, which may be

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attributed to the enzymatic pretreatment. Our these findings are in accord with the investigations of Ranalli et al. (2003) who reported that the use of enzyme during olive oil extraction resulted in higher release of tocopherols. The use of enzymatic preparations containing cell wall degrading enzymes during seed extraction results in release of greater amounts of tocopherols and phenolics due to hydrolysis of seed cell wall, resulting in higher availability of such bioactive components into the oil (Ranalli et al., 2003; Qiu et al., 2003). In some cases, the increase in the amounts of tocopherols and other bioactive components in enzyme-extracted oils could be attributed to a reduced complexation of such compounds with the seed polysaccharides and consequent enhanced partitioning into the oil phase (Ranalli et al., 2005).

5.3.4 Antioxidant Activity Total phenolic contents (TPC), DPPH produced scavenging capacity and inhibition of linoleic acid peroxidation of the oils obtained by EACP, and solvent extraction methods are shown in Tables (4.1.6a to 4.6.6a). While the antioxidant profiles of the oils obtained by EAAE are presented in Tables (4.1.6b to 4.6.6b, 4.7.4).

5.3.4a Total Phenolic Contents Estimation of total phenolic contents is often employed for determination of antioxidant activity of a plant material (Anwar et al., 2007). Total phenolic contents (TPC) in the present work were determined using Folin-Ciocalteu reagent (FCR) method and were expressed as gallic acid equivalents (GAE). The total phenol assay is frequently used due to its convenience, simplicity, and reproducibility, regardless of undefined chemical nature of FCR (Waterman and Mole 1994). Several studies applied the FCRbased assay to determine the TPC of plant materials and often established excellent linear correlations between the total phenolic profile and antioxidant activity (Huang et al., 2006).

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TPC in the oils produced by EACP and EAAE methods were found to be higher than the solvent extracted oils (Tables 4.1.5a to 4.6.5a and 4.1.6b to 4.6.6b, 4.7.4). The improved TPC and antioxidant efficacy of the enzyme-extracted oils might be due to greater release of oil and other phenolic bioactive constituents which dissolve in the oily phase (Ranalli et al., 2003).

5.3.4b DPPH Scavenging Activity DPPH is a very stable organic free radical with deep violet color which give absorption maxima at 515-528 nm, upon receiving proton from any hydrogen donor species mainly, phenolics loses this absorption, resulting in visually noticeable color change from deep violet to yellow. As the concentration of phenolic compounds or degree of hydroxylation of the phenolic compounds increases DPPH scavenging activity increases, hence antioxidant activity increases (Sanchez-Moreno, 2002). As this radical is very sensitive to active ingredients at low concentrations and can accommodate a large number of samples in a very short time, therefore it has been extensively used for measuring radical scavenging activity of different plant extracts. In the DPPH assay, the ability of the examined extracts to donate hydrogen atoms or electrons for the transformation of DPPH into its reduced form (DPPH-H) was investigated. The examined extracts were able to reduce the stable, purple-colored radical DPPH into yellow-colored DPPH. The free radical scavenging capacity of examined extracts increased in a concentration dependent manner. In the DPPH assay, enzyme-extracted oils exhibited more ability to reduce DPPH in terms of lower IC50 as compared to solvent extracted oils. Lower the IC50 value of extract, more effective it will be for the inhibition of DPPH. The values for 50% inhibition (IC50) of oil extracts are shown in Tables 4.1.5a to 4.6.5a and 4.1.6b to 4.6.6b, 4.7.4. The IC50 values of enzyme-extracted oils were lower which showed the higher scavenging capacity among the control and solvent extracted oils. No previous reports are available in the literature to compare the results of DPPH radical scavenging activity in terms of IC50 values in the preset analysis of enzyme-extracted oils.

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5.3.4c % Inhibition of Linoleic Acid Peroxidation Inhibition of linoleic acid peroxidation was also used to assess the antioxidant activity of the oils extracted by different extraction methods. Antioxidant activity of different oil extracts was determined by inhibition of peroxidation in linoleic acid system by using thiocyanate method (Yen et al., 2000). Linoleic acid is a polyunsaturated fatty acid, upon oxidation peroxides are formed which oxidize Fe+2 to Fe+3 , the later forms complex with SCN--, concentration of which is determined spectrophotometrically by measuring absorbance at 500 nm. Higher the absorbance, higher the concentration of peroxides formed during reaction due to lower antioxidant activity. Inhibition of linoleic acid oxidation determined for oil extracts from different extraction methods are shown in Tables 4.1.5a to 4.6.5a and 4.1.6b to 4.6.6b, 4.7.4. The levels of % inhibition of linoleic acid of the enzyme-extracted oils were observed to be significantly (P < 0.05) higher as against solvent extracted oils. This may be attributed to the enzymatic treatment. The enzyme treatment offered an enhanced incorporation of minor components (phenols, tocopherols, volatiles, carotenes, xanthophylls and chlorophylls) into the oil phase with consequent significant improvement of analytical parameters related to typicality, flavour and shelf-life (Chiacchierini et al., 2007).

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CHAPTER 6
SUMMARY

Industrial processes for the extraction of edible oils from oilseeds generally involve the use of n-hexane as extracting solvent. In solvent extraction, the oil has to pass through elevated heat treatment for a longer period which may destroy its important nutritional components e.g. tocopherols and essential fatty acids. The flammability, explosiveness and environmental concerns of hexane-extraction method have urged the edible oil industry to seek some suitable alternative techniques for extraction of oils. Enzyme-assisted oil extraction is considered as a good alternative for this purpose. Oilseeds accumulate oil in intracellular vacuoles, so the oil extraction can be improved by the hydrolytic action of enzymes.

In the present study, effectiveness of five different enzyme preparations was tested for oil extraction from seven indigenously available oilseeds (canola-, sunflower-, sesame-, cotton-, hemp-, Moringa oleifera-, and Moringa concanensis-seeds). Experiments were conducted to investigate the effects of enzyme concentration, temperature, hydrolysis time, water/seed ratio (moisture), and pH on the oil recoveries during enzyme-assisted cold pressing (EACP) and enzyme-assisted aqueous extraction (EAAE). The quality of the extracted oils was assessed by using some conventional and state-of-the-art analytical techniques and compared with control and solvent-extracted oils.

An enhancement in oil recovery was observed by using enzyme adjuvant during cold pressing and aqueous extraction. Enzyme concentrations within the range 0.1-2.0 g/100 g seeds exhibited an appreciable effect on oil extractability. The enzyme concentration > 2.0 g/100 g seeds did not further improve the oil recovery. It is suggested that a final decision about the enzyme concentration must be based on an economical

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balance between the increase in efficiency and the cost of enzyme. The experimental parameters such as, enzyme concentration, temperature, water/seed ratio, pH and time significantly affected the oil recovery during EACP and EAAE. However, in EACP, the influence of these experimental variables varied from those exhibited in EAAE.

So far as the quality attributes of oils produced through enzyme-adjuvant is concerned, the free fatty acid content and color values were lower than those of hexaneextracted oil. However, slightly higher levels of free fatty acids and color were observed in the EAC-pressed oils than those in the solvent-extracted oils. Iodine and saponification values of the enzyme-extracted oils were found to be comparable with those of hexaneextracted oil. When compared with the solvent-extracted oils, superior oxidative stability as measurements of peroxide, para-anisidine values, conjugated dienes, conjugated trienes, and induction periods were exhibited by the enzyme-extracted oils. An increased amount of tocopherols and antioxidants in the enzyme-extracted oils were established. No significant variation was observed in the fatty acid profiles of the extracted oils by either means. During this study, it was observed that enzymatic treatment enhanced the oil yield with respect to the control, but the yield as offered by the solvent extraction was not achieved. A higher oil recovery was observed with EACP than that with EAAE. However, the aqueous phase obtained through the EAAE method may be explored as a potential source of water soluble protein, for value-addition in beverages and other food commodities. Whereas, the dry meal obtained by EACP and EAAE can be used for animal feeding. The water recovered from the EAAE process after precipitation of protein can be effectively reused in further extractions. This strategy can be adopted to minimize the environmental impact of the process. From the present results, it could be concluded that enzyme-assisted process allows an increase in the oil recovery relative to the control, making this technology as an environment-friendly alternative to conventional hexane oil extraction. Finally, the oils

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obtained by employing enzyme preparation offered superior quality indices, higher antioxidants levels, and tocopherol contents reflecting better nutritive value. Two major drawbacks in this process are the cost of enzyme and the low oil yield relative to hexane-extraction. To make this process economically viable, further work is recomended with applications of some modified mixtures of enzymes, which can efficiently break the seed cell wall to further facilitate the liberation of oil. The use of commercial enzymes as used in the present study may cover up the cost of analytical enzymes. Efforts should also be made to simplify the oil recovery process so as to come up with the problem of complex emulsion formation and isolation of oil phase. More studies should be conducted on aqueous enzymatic processes, for production of nutritionally improved functional hydrolysates. Pilot plant scale investigation should be undertaken to evaluate the commercial potential of the process.

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References
Abdulkarim, S. M., K. Long, O. M. Lai, S. K. S. Muhammad, and H. M. Ghazali. 2005. Some physico-chemical properties of Moringa oleifera seed oil extracted using solvent and aqueous enzymatic methods. Food Chemistry. 93: 253-263. Abdulkarim, S. M., O. M. Lai, S. K. S. Muhammad, K. Long, and H. M. Ghazali. 2006. Use of enzymes to enhance oil recovery during aqueous extraction of Moringa oleifera seed oil. Journal of Food Lipids. 13: 113130. Achaya, K. T. 1994. Ghani: a traditional method of oil processing in India. Food, Nutrition and Agriculture. 11: 23-28. Aguilera, J. M., M. F. Gerngross, and E. W. Lusas. 1983. Aqueous processing of lupin seed. Journal of Food Technology. 18: 327-333. American Oil Chemists Society (AOCS). 1997. Official and Recommended Practices of the American Oil Chemists Society, 5th edn., AOCS Press, Champaign, Illinois, USA. Association of Official Analytical Chemist (AOAC). 1985. Official Methods of Analysis, 14th Ed., Association of Official Analytical Chemists, Arlington, VA. Anjou, K. 1972. Manufacture of rapeseed oil and meal. In: Rapeseed: cultivation, composition, processing and utilization (edited by L. Appelqvist and R. Ohlson). Elsevier Publishing Co., New York. Anwar, F., M. I. Bhanger, and T. G. Kazi. 2003. Relationships of Rancimat and AOM values at varying temperatures for several oils and fats. Journal of American Oil Chemists Society. 80: 151155. Anwar, F., S. Latif, and M. Ashraf. 2006. Analytical characterization of hemp (Cannabis sativa) seed oil from different agro-ecological zones of Pakistan. Journal of American Oil Chemists Society. 83: 323-329. Anwar, F., S. Latif, R. Przybylski, B. Sultana and M. Ashraf. 2007. Chemical composition and antioxidant activity of seeds of different cultivars of mungbean (Vigna radiata). Journal of Food Science. 72(7): S503-S510. Aparna, S., S. K. Khare, and M. N. Gupta. 2002. Enzyme-assisted aqueous extraction of peanut oil. Journal of American Oil Chemists Society. 79: 215218. 164

Association of Official Analytical Chemists (AOAC). 1985. Official methods of analysis. 14th edn., Arlington, VA. Bair, C. W., and H. E. Snyder. 1980. Electron microscopy of soybean lipid bodies. Journal of American Oil Chemists Society. 57: 279-282. Balke, D. T. 2006. The production of higher value food ingredients from white mustard via aqueous extraction. Ph.D. Thesis. Department of Chernical Engineering and Applied Chemistry, University of Toronto, Canada Bargale, P. C. 1997, Mechanical oil expression from selected oilseeds under uniaxial compression. Ph.D. thesis, University of Saskatchewan, Canada. Barrios, V. A., D. A. Olmos, R. A. Noyola, and C. A. Lopez-Munguia. 1990. Optimization of an enzymatic process for coconut oil extraction. Oleagineux. 45: 35-42. Beach, D. 1983. Rapeseed crushing and extraction. In high and low erucic acid rapeseed oils, eds. J.K.G. Kramer, P.D. Sauer and W.J. Pigden, Toronto ON: Academic Press. 181-197. Bhatia, D. S., H. A. B. Darpra, and B. P. Balga. 1966. Peanut protein isolate-production and properties. Journal of Food Science and Technology. 3: 2-5. Bhatnagar, S., and B. N. Johari. 1987. Microbial enzymes in the processing of oilseeds. Current Science. 56: 775-776. Bhattacharjee, P., R. S. Singhal, and S. R. Tiwari. 2006. Supercritical carbon dioxide extraction of cottonseed oil. Journal of Food Engineering. 79: 892-989. Bocevska, M., D. Karlovic, J. Turkulov, and D. Pericin. 1993. Quality of corn germ oil obtained by aqueous enzymatic extraction. Journal of American Oil Chemists Society. 70: 1273-1277. Bouvier, F., and B. Entressangles. 1992. Utilization of cellulases and pectinases in the extraction of palm oil. Revue Fruncaise des Corps Gras. 39: 245-252. Bozin, B., N. Mimica-Dukic, N. Simin, and G. Anackov. 2006. Characterization of the volatile composition of essential oil of some lamiaceae species and the antimicrobial and antioxidant activities of the entire oils. Journal of Agriculture and Food Chemistry. 54: 1822-1828.

165

Bredeson. 1983. Mechanical oil extraction. Journal of American Oil Chemists Society. 60: 211-213. Buenrostro, M., and C. A. Lopez-Munguia. 1986. Enzymatic extraction of avocado oil. Biotechnology Letters. 8: 505-506. Bulley, N. R. 1984. Supercritical fluid extraction of vegetable oilseeds. Journal of American Oil Chemists Society. 61: 1362-1365. Chaovanalikit, A., and R. E. Wrolstad. (2004). Total anthocyanins and total phenolics of fresh and processed cherries and their antioxidant properties. Food Chemistry and Toxicology. 69: 6772. Caragay, A. B. 1983. Pacing technologies in the fats and oils industry. Journal of American Oil Chemists Society. 60: 1641-1644. Cater, C. M., K. C. Rhee, R. D. Hagenmaier, and K. F. Mattil. 1974. Aqueous extractionan alternative oilseed milling process. Journal of American Oil Chemists Society. 51: 137-141. Caviedes, J. 1996. Aqueous processing of rapeseed (canola). M.Sc. Thesis. Department of Chernical Engineering and Applied Chemistry, University of Toronto, Canada. Che Man Y. B., A. B. Suhardiyono, A. B. Asbi, M. N. Azudin, and L. S. Wei. 1996. Aqueous enzymatic extraction of coconut oil. Journal of American Oil Chemists Society. 73: 683-686. Cheah, S. C., M. A. Augustin, and L. C. Ooi. 1987. Enzymic extraction of palm oil. Palm Oil Research Institute of Malaysia (PORIM), Kuala Lumpur, Bulletin No. 20: 1214. Cheah, S. C., M. A. Augustin, and L. C. L. Ooi. 1990. Enzymic extraction of palm oil. Palm Oil Research Bulletin Malaysia Bulletin. 20: 30-36. Chen B-K., and L. L. Diosady. 2003. Enzymatic aqueous processing of coconuts. International Journal of Applied Science and Engineering. 1: 55-61. Chiacchierini, E., G. Mele, D. Restuccia, and G. Vinci. 2007. Impact evaluation of innovative and sustainable extraction technologies on olive oil quality. Trends in Food Science and Technologies. 18: 299-305. Christensen, F. M. 1989. Enzyme technology versus engineering technology in the food industry. Biotechnology Applied Biochemistry. 11: 249-265.

166

Christensen, F. M. 1991. Extraction by aqueous enzymatic processes. Inform. 2: 984. Cintra, M. O., A. Lopez-Munguia, and J. Vernon. 1986. Coconut oil extraction by a new enzymatic process. Journal of Food Science. 51: 695-697. Concha, J., C. Soto, R. Chamy, and M. E. Zuniga. 2004. Enzymatic pretreatment on Rose-Hip oil extraction: hydrolysis and pressing conditions. Journal of American Oil Chemists Society. 81: 549-552. Dahlen, J. A. H., and L. A. Lindh. 1983. Mass balance of hexane losses in an extraction plant. Journal of American Oil Chemists Society. 60: 2009-2010. De Groot, A. P., and Slump, P. 1969. Effects of severe alkali treatment of proteins on amino acid composition and nutritive value. Journal of Nutrition. 98: 45-48. Deng, Y., D. L. Pyle, and K. Niranjan. 1992. Studies of aqueous enzymatic extraction of oil from rapeseed. Agriculture Engineering and Rural Development. I Conference Proceeding. Vol. 1 Zhang, W., P. W. Giro, S. W. Zhang, Eds. International Academic Publishers. Beijing, 190-195. Diosady, L. L., N. Rosen, L. J. Rubin, and D. Paton. 1985c. Degradation of wheat starch in single screw extruder mechano-kinetic breakdown of cooked starch. Journal of Food Science. 50: 1697-1700. Diosady, L. L., L. J. Rubin, C. R. Phillips, and M. Naczk. 1985b. Effect of alkanolammonia-water treatment on the glucosinolate content of rapeseed meal. Canadian Institute of Food Science and Technology Journal. 18: 311. Diosady, L. L., C. G. Tar, L. J. Rubin, and M. Naczk. 1987. Scale-up of the production of glucosinolate-free canola meal. Acta Alimentaria 162: 169182. Diosady, L. L., M. Naczk, and L. J. Rubin. 1985a. The effect of ammonia concentration on the properties of canola meals produced by the ammonia methanol/hexane extraction system. Food Chemistry. 18: 121-130. Diosady, L. L., P. Sleggs, and T. Kaji. 1983. Degumming of canola oils, in 7th progress Report on research on canola seed, oil, meal and meal fractions. Canola council of Canada. Winnipeg. pp 186-191. Dominguez, H., M. J. Nunez, and J. M. Lema. 1993. Oil extractability from enzymatically treated soybean and sunflower: range of operational variables. Food Chemistry. 46: 277-284.

167

Dominguez, H., M. J. Nunez, and J. M. Lema. 1994. Enzymatic pretreatment to enhance oil extraction from fruits and oilseeds: a review. Food Chemistry. 49: 271 286. Dominguez, H., M. J. Nunez, and J. M. Lema. 1995a. Aqueous processing of sunflower kernels with enzymatic technology. Food Chemistry. 53: 427-434. Dominguez, H., M. J. Nunez, and J. M. Lema. 1995b. Enzyme-assisted hexane extraction of soya bean oil. Food Chemistry. 54: 223-231. Dominguez, H., M. J. Nunez, and J. M. Lema. 1995c. Aqueous processing of soybeans with enzymatic technology-oil extraction and production of isolates. Grasas Aceites. 46: 11-20. Eapen, K. E., S. S. Kalbag, and V. Subrahmanyan. 1966. Operations in the wet-rendering of peanut for the separation of protein, oil, and starch. Journal of American Oil Chemists Society. 43: 585-589. Eggers, R., U. Sievers, and W. Stein. 1985. High pressure extraction of oil seed. Journal of American Oil Chemists Society. 62: 1222-1230. Eggers, R., and U. Sievers. 1989. Processing oilseed with supercritical carbon dioxide. Journal of Chemical Engineering of Japan. 22: 641-649. Eapen, K. E., N. W. Tape, and R. P. A. Sims. 1969. New process for the production of better quality. rapeseed oil and meal. II: Detoxification and dehulling of rapeseeds feasibility study. Journal of American Oil Chemists Society. 46: 52-55. Embong, M. B., and P. Jelen. 1977. Technical feasibility of aqueous extraction of rapeseed oil-a laboratory study. Canadian Institute of Food Science and Technology Journal Aliment. 10: 239-243. Environmental Protection Agency (EPA). 2001. National emissions standards for hazardous air pollutants: Solvent extraction for vegetable oil production; 40 CFR Part 63. Final Rule, Federal Register 66:19005-19026. Erickson, D. R. 1983. Soybean oil: Update on number one. Journal of American Oil Chemists Society. 60: 303A-308A. Freitas, S. P., R. C. A. Lago, F. H. Jablonka, and L. Hartman. 1993. Aqueous enzymatic extraction of avocado oil from fresh pulp. Revue Francaise des Corps Gras. 40: 365-371.

168

Frevert, J., R. Frische, J. Hart, and J. Wittkind. 1990. Enzymatic solventless recovery of oils from plant materials. Ger. Offen. DE 3843027. Fullbrook, P.D. 1983. The use of enzymes in the processing of oilseeds. Journal of American Oil Chemists Society. 60: 428A-430A. Gorrill, A. D. I., D. M. Waker, and J. D. Jones. 1974. Rapeseed protein sources and amino acid supplementation of diets for weanling rats. Canadian Journal of Animal Science. 54: 659-667. Grant, O. G., R. L. Eager, J. M. Pepper, and J. F. Mathews. 1983. Factors affecting the desolventization of canola meal. Journal of American Oil Chemists Society. 60: 1867-1875. Gunetileke, K. G., and S. F. Laurentius. 1974. Conditions for the separation of oil and protein from coconut milk emulsion. Journal of Food Science. 39: 230-233. Hagenmaier, R. D. 1974. Aqueous processing of full-fat sunflower seeds; yields of oil and protein. Journal of American Oil Chemists Society. 51: 470-471. Hagenmaier, R. D., C. M. Cater, and K. F. Mattil. 1972. Critical unit operations of the aqueous processing of fresh coconuts. Journal of American Oil Chemists Society. 49, 178-181. Hagenmaier, R. D., C. M. Cater, and K. F. Mattil. 1973. Aqueous processing of fresh coconuts for recovery of oil and coconut skim milk. Journal of Food Science. 38: 516-518. Hanmoungjai, P., D. L. Pyle, and K. Niranjan. 2001. Enzymatic process for extracting oil and protein from rice bran, Journal of the American Oil Chemists Society 78: 817821. Head, S., and A. Swetman. 1999. Traditional methods for processing oilseeds. Inform. 10: 151-154. Hernandez, N., M. E. Rodriguez-Alegria, F. Gonzalez, and Lopez-Munguia. 2000. Enzymatic treatment of rice bran to improve processing. Journal of American Oil Chemists Society. 77: 177-180. Ho, C. C., M. C. Chow, and S. H. Ong. 1992. Recovery of residual oil from centrifuge sludge palm oil mill: effect of enzyme digestion and surfactant treatment. Journal of American Oil Chemists Society. 69: 276-282.

169

Huang, A. H. C. 1992. Oil bodies and oleosins in seeds. Annual Reviews in Plant Physiology and Plant Mololecular Biology. 43: 177-200. Huang, A. H. C. 1994. Structure of plant seed oil bodies. Current Opinion in Structural Biology 4: 494-498. Huang, D. J., H. J. Chen, Wen-chi Hou, Chun-Der Lin, and Yaw-Huei Lin. 2006. Sweet potato (Ipomoea batatas [L.] LamTainong 57) storage root mucilage with antioxidant activities in vitro. Food Chemistry. 98: 774-781. Igor, S. O., L. L. Disoady, and L. J. Rubin. 1993. Catalytic deamidation of canola proteins. Acta Alimentaria. 22: 325-336. International Organization for Standardization (ISO). 1977. Oilseed residue determination of total ash. Standard No.749, ISO, Geneva, Switzerland. International Union of Pure and Applied Chemistry (IUPAC). 1987. Standard methods for the analysis of oils, fats and derivatives, 7th rev. edn., C. Paquot and A. Hautfenne (Ed.), Blackwell Scientific, London, 1987. Johnson, L. A., and E. W. Lusas. 1983. Comparison of alternative solvents for oil extraction. Journal of American Oil Chemists Society. 60: 229-242. Karlovic, D. J., M. Bocevska, J. Jakolevic, J. Turkulov. 1994. Corn germ oil extraction by a new enzymatic process. Acta Aliment. 23: 389-402. Keegstra, K., K. W. Talmadge, W. D. Bauer, and P. Albersheim. 1973. The structure of the wall of suspension-cultured sycamore cells based on the interconnections of the macromolecular components. Plant Physiology. 51: 188-196. Kemper, T. G. 2005. Oil extraction. In F. Shahidi (Ed.) Baileys Industrial Oil and Fat Product. Vol. 5, 6th edn., p. 1. John Wiley & Sons, New York. Kim, H. K. 1989. Aqueous extraction of oil from palm kernel. Journal of Food Science. 54: 491-492. Kim, I. -H., and S. H. Yoon. 1990. Effect of extraction solvents on oxidative stability of crude soybean oil. Journal of American Oil Chemists Society. 67: 165-167. Kingsbaker, C. L. 1983. Recent safety experiences. Journal of American Oil Chemists Society. 60: 245-247. Kumar, N. S. K. and D. N. Bhowmick. 1995. A fresh look at the coconut and its processing. Inform. 6: 1217.

170

Lanzani, A., M. C. Petrini, O. Cozzoli, P. Gallavresi, C. Carola, and G. Jacini. 1975. On the use of enzymes for vegetable-oil extraction. A preliminary report. La Rivista Italiana Della Sostanze Grasse LII-Loglio, 226-229. Latif, S., F. Anwar, and M. Ashraf. 2007. Characterization of enzyme-assisted cold pressed cottonseed oil. Journal of Food Lipid. 14: 424-436. Lawhon, J. T., L. J. Manak, K. C. Rhee, and E. W. Lusas. 1981a. Production of oil and protein food products from raw peanuts by aqueous extraction and ultrafiltration. Journal of Food Science. 46: 391-395. Lawhon, J. T., L. J. Manak, K. C. Rhee, K. S. Rhee, and E. W. Lusas. 1981b. Combining aqueous extraction and membrane isolation techniques to recover protein and oil form soybeans. Journal of Food Science. 46: 912-916. Lawhon, J. T., K. C. Rhee, and E. W. Lusas. 1981c. Soy protein ingredients prepared by new processes-aqueous processing and industrial membrane isolation. Journal of American Oil Chemists Society. 58: 377-384. Liu, J., M. Shi, L. L. Diosady, and L. J. Rubin. 1994. Three-phase extraction of Chinese rapeseed using the Karr Column. Journal of Food Engineering. 24: 35-45. Lusas, E. W., and G. M. Jividen. 1987. Glandless cottonseed: A review of the first 25 years of processing and utilization research. Journal of American Oil Chemists Society. 64: 839-854. Lusas, E. W., J. T. Lawhon, and K. C. Rhee. 1982. Producing edible oil and protein from oilseeds by aqueous processing. Oil Mill Gazet. 4: 28-34. Marek, E., E. Schalinatus, E. Weigelt, G. Mieth, G. Kerns, and J. Kude. 1989. Interbitech 89-Mathematical Modelling Biotechnol. Vol. 6 (Blazej, A. and Ottova, A., Eds.). Elsevier, Amsterdam, 471-474. Marlowe, I. T., T. J. Giddings, S. J. Richardson, and A. Stentiford. 1991. U.K. industry and ozone pollution from volatile organic compound emissions. II. Update to October 1991. Warres Spring Laboratory-The Environmental Technology Executive Agency of the Department of Trade and Industry, London, Report 878 (PA). McGlone, O. C., C. A. Lopez-Munguia, and J. V. Carter. 1986. Coconut oil extraction by a new enzymatic process. Journal of Food Science. 51: 695-697.

171

Montedoro, G., and G. Petruccioli. 1974. Trattamenti con additive enzimatici e detannizzanti alle paste di olive sottoposte a processi di estrazione per pressione unica e percolamento: I. Effetti sui rendimenti in olio e su alcuni parametri operativi. La Riv. Ital. dell Sostance Grasse, 51: 378- 85. Moure, A., H. Dominguez, M. E. Zuniga, S. Carmen, and R. Chamy. 2002. Characterization of protein concentrates from pressed cakes of Guevina avellana (Chilean hazelnut). Food Chemistry. 78: 179-186. Murphy, D. J. 1993. Structure, function, and biogenesis of storage lipid bodies and oleosins in plants. Progess in Lipid Research. 32: 247-280. Mustakas. G. C. 1980. Recovery of oil from soybeans. In: Handbook of Soy Oil Processing and Utilization (Erickson, D. R.. Ed.). American Soybean Association and American Oil Chemists Society, St. Louis. 49-65. Naczk, M., L. L. Diosady, and L. J. Rubin. 1985. Functional properties of canola meals produced by the two-phase solvent extraction system. Journal of Food Science. 50: 1685-1692. Norris, F.A. 1964. Extraction of fats and oils. In: Baileys Industrial Oil and Fat Products (edited by D. Swern), 3rd ed., Pp 637-718. London Interscience Publishers. Omosaiye, O., and M. Cheryan. 1979a. Low-phytate, full-fat soy protein product by ultrafiltration of aqueous extracts of whole soybeans. Cereal Chemistry. 56: 5862. Omosaiye, O., and M. Cheryan. 1979b. Ultrafiltration of soybean water extracts: processing characteristics and yields. Journal of Food Science. 44: 1027-1031. Ohlson, R. (1992). Modern processing of rapeseed. Journal of American Oil Chemists Society. 69: 195-198. Olsen, H. S. Aqueous enzymatic extraction of oil from seeds. In: Asian food conference proceedings Bankgok, Thailand, 1988. Reprinted by: Novo Industry A/S, pp A0604la Parry, J., L. SU, M. Luther, K. Zhou, M. P. Yurawecz, P. Whittaker, and L. YU. 2005. Fatty acid composition and antioxidant properties of cold-pressed marionberry, boysenberry, red raspberry, and blueberry seed oils. Journal of Agriculture and Food Chemistry. 53: 566-573.

172

Picuric-Jovanovic, K., Z. Vrbaski, and M. Milovanovic. 1997. Aqueous-enzymatic extraction of plum kernel oil. Fett-Lipid. 99: 433-135. Qiu, X., H. Hong, N. Dalta, D. W. Reed, M. Truska, Z. Hu, P. S. Covello, and S. L. MacKenzie. 2003. Production of nutraceutical fatty acids in oilseed crops. In: Advanced research on plant lipids (edited by N. Murata, M. Yamada, I. Nishida, H. Okuyama, J. Sekiya, W. Hajime), Pp. 403406. Kluwer Academic, Dordrecht, The Netherlands. Ranalli, A., A. Malfatti, L. Lucera, S. Contento, and E. Sotiriou. 2005. Effects of processing techniques on the natural colorings and the other functional constituents in virgin olive oil. Food Research International. 38: 873-878. Ranalli, A., L. Lucera, S. Contento, N. Simone, P. Del Re. 2004. Bioactive constituents, flavours and aromas of virgin oils obtained by processing olives with a natural enzyme extract. European Journal of Lipid Science and Technology. 106: 187197. Ranalli, A., L. Pollastri, S. Contento, and E. Iannucci. 2003. The glyceridic and nonglyceridic constituents of virgin olive oil after use of a novel method of enzyme extraction. International Journal of Food Science and Technology. 38: 1727. Ranalli, A., and G. De Mattia. 1997. Characterization of olive oil produced with a new enzyme processing aid. Journal of American Oil Chemists Society. 74: 11051113. Ranalli, A., G. De Mattia, and M. L. Ferrante. 1998. The characterization of percolation olive oils produced with a new processing enzyme aid. International Journal of Food Science and Technology. 32: 247-258. Reverchan, E. 1994. Comparison of processes for the supercritical carbon dioxide extraction of oil from soybean seeds. Journal of American Oil Chemists Society. 71: 1007-1012. Rhee, K. C., C. M. Cater, and K. F. Mattil. 1973a. Aqueous process for pilot plant-scale production of peanut protein concentrate. Journal of Food Science. 38: l26-128. Rhee, K. C., C. M. Cater, and K. F. Mattil. 1973b. Effect of processing pH on the properties of peanut protein isolates and oil. Cereal Chemistry. 50: 395-404.

173

Rhee, K. C., C. M. Cater, and K. F. Mattil. 1972. Simultaneous recovery of protein and oil from raw peanuts in an aqueous system. Journal of Food Science. 37: 90-93. Rhee, K. C., K. R. Natarajan, C. M. Cater, and K. F. Mattil. 1977. Processing edible peanut protein concentrates and isolates to inactivate aflatoxins. Journal of American Oil Chemists Society. 54: 245A-249A. Rosenthal, A., D. L. Pyle, and K. Niranjan. 1995. Simultaneous aqueous enzymatic extraction of oil and protein from soybean. Personal communication. University of Reading, Reading, England. Rosenthal, A., D. L. Pyle, and K. Niranjan. 1998. Simultaneous aqueous extraction of oil and protein from soybean: mechanisms for process design. Trans Institute of Chemical Engineers. 76: 224-230. Rosenthal, A., D. L. Pyle, and Niranjan, K. 1996. Aqueous and enzymatic processes for edible oil extraction. Enzyme Microbial Technology 19: 402420. Rosenthal, A., D. L. Pyle, K. Niranjan, S. Gilmour, and L. Trinca. 2001. Combined effect of operational variables and enzyme activity on aqueous enzymatic extraction of oil and protein from soybean. Enzyme and Microbial Technology. 28: 499-509. Rubin, L. J., L. L. Diosady, and C. R. Phillips. 1984. Solvent extraction of oil-bearing seeds. U. S. Patent No. 4 460 504. Rubin, L. J., L. L. Diosady, M. Naczk, and M. Halfani. 1986. The alkanol-ammoniaWater/hexane treatment of canola. Canadian Institute of Food Science and Technology Journal. l9: 547-561. Rustom, I. Y. S., M. H. Lopez-Leiva, and B. M. Nair. 1991. A study of factors affecting extraction of peanut (Arachis hypogaea L.) solids with water. Food Chemistry. 42: 153-165. Sanchez-Moreno. 2002. Review: Methods used to evaluate the free radical scavenging activity in foods and biological systems. Food Science and Technology International. 8: 121-137. Serrato, A. G. 1981. Extraction of oil from soybeans. Journal of American Oil Chemists Society. 3: 157-159.

174

Shahidi, F., M. Naczk, L. J. Rubin, and L. L. Diosady. 1988. A novel processing approach for rapseed and mustard seed - removal of undesirable constituents by methanol ammonia. Journal of Food Protection. 51: 743-749. Sharma, A., S. K. Khare, and M. N. Gupta. 2001. Enzyme-assisted aqueous extraction of rice bran oil. Journal of American Oil Chemists Society. 78: 949-951. Sharma, A., S. K. Khare, and M. N. Gupta. 2002. Enzyme-assisted aqueous extraction of peanut oil. Journal of American Oil Chemists Society. 79: 215218. Sherba, S. E., R. B. Steigerwalt, W. T. Faith, and C. V. Smythe Jr. 1972. Soybean fractionation employing a protease. U.S. patent 3,640,725. Shi, L., J. Lu, G. Jones, P. A. Loretan, and W. A. Hill. 1998. Characteristics and composition of peanut oil prepared by an aqueous extraction method. Life Support Bioscience. 5: 225229. Sineiro, J., H. Dominguez, M. J. Nunez, and J. M. Lema. 1998. Optimization of the enzymatic treatment during aqueous oil extraction from sunflower seeds. Food Chemistry. 61: 467474. Singh, G., and P. Marimuthu. 2006. Antioxidant and biocidal activities of Carum nigrum (seed) essential oil, oleoresin, and their selected components. Journal of Agriculture and Food Chemistry. 54: 174-181. Singh, R. K., B. C. Sarker, and B. K. Kumbhar. 1999. Response surface analysis of enzyme assisted oil extraction factors for sesame, groundnut and sunflower seeds. Journal of Food Science and Technology. 36: 511514. Smith, D. D., Y. C. Agrawal, B. C. Sarkar, and B. N. P. Singh. 1993. Enzymatic hydrolysis pretreatment for mechanical expelling of soybeans. Journal of the American Oil Chemists' Society.70: 885-890. Snyder, H. E., and T. W. Kwon. 1987. Morphology and composition. In: Soybean Utilization, Snyder, H. E., and T. W. Kwon, Eds. Van Nostrand Reinhold Co. Inc., New York, 19-70. Sosulski, K., and F. W. Sosulski. 1990. Enzyme pre-treatment to enhance oil extractability in canola. In: Canola and Rapeseed: Production, Chemistry, Nutrition and Processing Technology (edited by F. Shahidi), Pp 277-289. New York NY: Van Nostrand Reinhold Publishers.

175

Sosulski, K., and F. W. Sosulski. 1993. Enzyme-aided vs. two-stage processing of canola: technology, product quality and cost evaluation. Journal of the American Oil Chemists' Society. 70: 825-829. Sosulski, F. W., E. S. Humbert, and J. D. Jones. 1976. Functional properties of rapeseed flours, concentrates and isolates. Journal of Food Science. 41: 1349-1352. Sosulski, K., F. W. Sosulsky, and E. Coxworth. 1988. Carbohydrate hydrolysis of canola to enhance oil extraction with hexane. Journal of American Oil Chemists Society. 65: 357-361. Sosulski, F. W., F. S. Soliman, and R. S. Bhatty. 1972. Diffusion extraction of glucosinates from rapeseed. Canadian Inst. Journal of Food Science and Technology. 5: 101-104. Sosulski, F. W., and C. W. McCleary. 1972. Diffusion extraction of chlorogenic acid from sunflower kernels. Journal of Food Science. 37: 253-256. Soto, C. G., R. Chamy, and M. Zuniga. 2004. Effect of enzymatic application on Borage (Borago officinalis) oil extraction by cold pressing. Journal of Chemical Engineering of Japan. 37: 326331. Southwell, K. H., and R. V. Harris. 1991. Extraction of oil from oilseeds using the hot water flotation method. Tropical Science. 32: 217-221. Southwell, K. H., and R. V. Harris. 1992. Extraction of oil from oilseed using the hot water flotation method. Tropical Science. 32: 251-262. Subrahmanyan, J., D. S. Bhatia, S. S. Kalbag, and N. Subramanian. 1959. Integrated processing of peanut for the separation of major constituents. Journal of American Oil Chemists Society. 36: 66-70. Sugarman, N. 1956. Process for simultaneously extracting oil and protein from oleaginous material. U. S. Patent No. 2 762 820. Staron, T., and A. Guillaumin. 1979. A method for extracting rapeseed protein and oil by water. Reveu Francais des Corps Gras. 26 : 441-446. Talmadge, K. W., K. Keegstra, W. D. Bauer, and P. Albersheim. 1973. The structure of plant cell walls I. The macromolecular components of the walls of suspensioncultured sycamore cells with a detailed analysis of the pectic polysaccharides. Plant Physiology. 51: 158-173.

176

Tano-Debrah, K., and Y. Ohta. 1994. Enzyme-assisted aqueous extraction of fat from kernels of the shea tree Butyrospermum parkii. Journal of American Oil Chemists Society. 71: 979-983. Tano-Debrah, K., and Y. Ohta. 1995a. Enzyme-assisted aqueous extraction of shea fat: A rural approach. Journal of American Oil Chemists Society. 72: 251-256. Tano-Debrah K., and Y. Ohta. 1995b. Application of enzyme assisted aqueous fat extraction to cocoa fat. Journal of American Oil Chemists Society. 72: 14091411. Tano-Debrah K., and Y. Ohta. 1997. Aqueous extraction of coconut oil by an enzymeassisted process. Journal of the Science of Food and Agriculture. 74: 497-502 Tzen, J. T. C., and A. H. C. Huang. 1992. Surface structure and properties of plant seed oil bodies. Journal of Biological Chemistry. 117: 327-335. Tzeng, Y. -M., L. L. Diosady, and L. J. Rubin. 1990. Production of canola protein materials by alkaline extraction, precipitation and membrane processing. Journal of Food Science. 55: 1147. Usuki, R., T. Suzuli, Y. Endo, and T. Kaneda. 1984. Residual amount of chlorophylls and pheophytins in refined edible oils. Journal of American Oil Chemists Society. 61: 785-788. Valentin, F. H. H. 1992. Volatile organic compounds and odors. Environmental Health. Aug. 224-227. Varga, T. K., and L. L. Diosady. 1994. Simultaneous extraction of oil and antinutritional compounds from flaxseed. Journal of American Oil Chemists Society. 71: 603607. Ward, J. A. 1976. Processing high oil content seeds in continuous screw presses. Journal of American Oil Chemists Society. 53: 261-264. Ward, J. A. 1982. Prepressing of oil from rapeseed and sunflower. Journal of American Oil Chemists Society. 61: 1358-1361. Waterman, P. G., and S. Mole. 1994. Analysis of phenolic plants metabolites. Blackwell, Scientific Oxford pp:116-133. Wolff, J. P. 1983. Residual hexane in meals. Journal of American Oil Chemists Society. 60: 220-223.

177

Wrolstad, R. E. 2003. Analysis of tocopherols and tocotrienols. In R. E. Wrolstad (Ed.) Current protocols in food analytical chemistry (CPFA). John Wiley & Sons, UK. Yen, G. C., P. D. Duh, and D. Y. Chuang. 2000. Antioxidant activity of anthraquinones and anthrone. Food Chemistry. 70: 307-315. Yoon, S. -H., I. -H. Kim, S. -H. Kim, and T. -W. Kwon. 1991. Effects of enzyme treatments and ultrasonification on extraction yields of lipids of protein from soybean by aqueous process. Korean Journal of Food Science and Technology. 23: 673-676. Young, C. T., and W. E. Schadel. 1990. Microstructure of peanut seed: A review. Food Structure. 9: 317-328. Zhang, S. B., Z. Wang, and S. Y. Xu. 2007. Optimization of the aqueous enzymatic extraction of rapeseed oil and protein hydrolysates. Journal of American Oil Chemists Society. 84: 97-105. Zuniga, M. E., R. Chamy, and J. M. Lema. 2001a. Canola and Chilean hazelnut products obtained by enzyme-assisted cold-pressed oil extraction. In Proceedings of the world conference and exhibition on oilseed processing and utilization (R.F. Wilson, ed.). AOCS Press, Champaign, pp 210-213. Zuniga, M. E., Concha, J., Soto, C., and R. Chamy. 2001b. Enzyme formulation effect of the rosehip oil cold-pressed extraction process. In Proceedings of the world conference and exhibition on oilseed processing and utilization (R.F. Wilson, ed.). AOCS Press, Champaign, pp. 203209. Zuniga, M. E., C. Soto, A. Mora, R. Chamy, and J. M. Lema. 2003. Enzymatic Pretreatment of Guevina avellana mol Oil Extraction by Pressing, Process Biochemistry. 39: 5157.

178