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Chemicals And Lipases And Experimental Methodology

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Chemicals and Lipases

All chemicals and solvents used were of analytical or chromatographic grade and purchased from SD Fine Chemicals (Mumbai, India). Lipozyme TL IM was obtained as gift from Novo Nordisk Ltd. (Bagsvaerd, Denmark). Soluble Thermomyces lanuginosus lipase [TLL] was purchased from DK Enzymes & Chemicals (India). Indion® PA 500 resin was purchased from Ion Exchange, (India). TLL was indigenously immobilized at DBT-ICT Centre for Energy Biosciences, Mumbai, India. Reference standards of tristearin and triolein(>99.0%) were purchased from Sigma-Aldrich (USA). High oleic canola oil and fully hydrogenated soybean oil used as substrates for interesterification reaction were gifted by General Mills Industries (USA). All chemicals and solvents were used without any modification/purification.

Experimental methodology

Interesterification reaction in batch mode

Interesterification between high oleic canola oil and fully hydrogenated soybean oil blends were carried out in 100 ml stoppered conical flasks. The reaction mixture contained varying amounts of starting materials and appropriate amount of enzyme was added start the reaction . The optimization of the reaction parameters such as substrate mole ratio, enzyme concentration, moisture content of enzyme, temperature, reaction time was carried out. The reaction was performed in an orbital shaker at 200 rpm. After specified time, the interesterified sample was removed and solvent was evaporated. The sample was dried under vacuum and subjected to further analysis.

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Interesterification reaction in continuous mode

Interesterification reaction between high oleic canola oil and fully hydrogenated soybean oil blends was performed in PBR. Ratio of height to diameter of glass reactor was kept at 8:1. 30 g PyLip lipase was uniformly packed in water jacketed glass column and was equilibrated with hexane before passing substrate feed and temperature of reactor and substrate mixture was maintained at 60 C.

Analysis of TAGs by High-performance liquid chromatography

The analysis of TAG species of initial blends and final interesterified sample was conducted by high performance liquid chromatography (HPLC) in an Agilent 1200 Chromatography System (CA, USA) equipped with a quaternary solvent delivery module, and an Agilent evaporative light scattering detector (ELSD) operated at 60 C with nitrogen pressure of 3.5 bar. Each sample, 10 mg/mL, was dissolved in chloroform (10mL) and ten microliters of sample was automatically injected on Agilent Zorbax XDB ODS 5 μM, 4.6 ×150 mm column. Separation was obtained by isocratic elution using acetonitrile and dichloromethane as solvent system. The flow rate was maintained at 1mL/min and total run time was kept at 20 min. Calibration curve was plotted for reference standard of tristearin and triolein and equations were calculated that was used for determination of percent reduction of these TAGs.

Slip Melting point determination by Capillary tube method (AOCS Official Method Cc 3-25)

The extent of interesterification reaction was monitored by determining the slip melting point (SMP) using AOCS Official Method Cc 3-25. The SMP is an index of the temperature at which fat softens and becomes sufficiently fluid to slip in an open capillary tube. In summary, the method involves melting of fat sample, then a glass capillary tube of 1 mm i.d. is dipped in the liquid fat sample till the liquid rises up to 10 mm high in the tube. The capillary tube is then placed in a refrigerator at 4 C for 16 hr. The capillary tube is then attached to a thermometer such that lower end of the capillary tube is in line with the bottom of the thermometer. The thermometer along with capillary tube is then placed in a beaker filled with water. The entire assembly is then placed in a water bath which has a heating rate of 1 C per min. Heating is continued until the fat melts and begins to rise up in the tube. This temperature is recorded as slip melting point of sample.

Acid value estimation

Acid value estimation was carried out using AOCS Official Method Cd 3d-63. Appropriate amount of fat was weighed and dissolved in 1:1 ratio of solvent mixture (isopropyl alcohol/toluene) followed by titration using 0.1N potassium hydroxide. Acid value was determined using following equation:

Acid value = (mL of KOH consumed*56.11*Normality of KOH×1000)/(g of sample)

Trans fatty acid analysis

Trans fatty acid analysis of final interesterified sample was conducted by high performance liquid chromatography (HPLC) in an Agilent 1200 Chromatography System (CA, USA) equipped with a quaternary solvent delivery module, and an Agilent evaporative light scattering detector (ELSD) operated at 60 C with nitrogen pressure of 3.5 bar. Interesterified sample was subjected to methylation using BF3-methanol to produce fatty acid methyl esters (FAMEs) using method described by Whitney et al; [19]. FAMEs were then subjected to HPLC separation and retention times of FAMEs were compared to standard elaidic acid methyl ester (C18 trans fatty acid methyl ester) to determine presence of any trans fatty acid content in the interesterified product.

Differential scanning calorimetry (DSC)

Differential scanning calorimetry (DSC) was used to obtain the thermograms of melting of the physical blend and corresponding interesterified product. An empty aluminium pan was used as a reference and each sample was accurately weighed (5 ± 0.1 mg) for DSC analysis. The sample was heated to 80 C and held for 10 min. Thereafter, the temperature was decreased at 5 C /min to−40 C. After holding for 10 min at −40 C, the melting curve was obtained by heating to 80 C at 5◦C/min.

Polymorphism by X-ray diffraction spectroscopy

Melted fat samples were placed on rectangular plastic molds, and tempered at 24 °C for 16 h. Polymorphic forms of the samples were determined by XRD-6100 X-ray diffractometer with a fine copper X-ray tube, operating at 40 kV and 35 mA. The short spacing was observed in the 2θ range of 5 to 40°, and scan rate was set at 2°/min. The β form was identified with 2 strong spacing at approximately 3.8 and 4.2 °A whereas β form was identified with the strong spacing at approximately 4.6 °A.

Crystal Microstructure

The crystal morphology was observed by means of polarized light microscopy using Olympus BX51 Microscope (Olympus Optical Co., Ltd., Japan). Samples were completely melted and then 1 drop of melted samples was placed on a glass microscope slide. A glass cover slip was placed over the samples to give a homogenous distribution. The samples were cooled at room temperature (25 C) for 1 h.

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