Cross-linked enzyme aggregates (CLEA) from germinating mustard (Brassica juncea) seeds as sturdy biocatalysts for the interesterification of pili (Canarium ovatum Engl) nut kernel oil in the preparation of cocoa butter equivalent (CBE)

Date

4-2014

Degree

Bachelor of Science in Chemistry

College

College of Arts and Sciences (CAS)

Adviser/Committee Chair

Milagros M. Peralta

Restrictions

Restricted: Not available to the general public. Access is available only after consultation with author/thesis adviser and only to those bound by the confidentiality agreement.

Abstract

A lipase from germinating mustard seeds was investigated as a biocatalyst in the preparation of cocoa butter equivalent (CBE) from pili nut kernel oil. Mustard seeds were germinated using the ragdoll method and the crude lipase was extracted from the homogenized 6day old seedlings using Tris buffer with L-cysteine. Specific lipase activity was determined by a titrimetric method using olive oil as the substrate and the Lowry method of protein determination. The crude lipase thus obtained was found to have a specific activity of 0.039 U/mg protein and was then purified using ammonium sulfate (AS) precipitation. It was found that maximum lipase activity was concentrated in the 95 % AS supernatant fraction which had a specific activity of 117.43 U/mg protein, corresponding to 3011X purification-fold relative to the crude lipase. It was also noted that the 90 % AS supernatant fraction as well as the precipitate obtained from the 95 % saturation cut exhibited significant lipase activites (30.51 U/mg protein and 17.688 U/mg protein for the 90 % AS supernatant and 95 % AS precipitate fractions, respectively). These data indicate that the mustard seed lipase seems to be a hydrophilic protein since high ammonium sulfate concentrations were necessary for salting out the enzyme. Further analysis using sodium dodecyl sulfate ? polyacrylamide gel electrophoresis (SDSPAGE) confirmed that the 90 % AS supernatant, 95 % AS precipitate and the 95 % AS supernatant fractions showed similar protein MW profiles. Because of this, the 90 % AS supernatant fraction was used for cross-linking to produce the cross-linked enzyme aggregate (CLEA) lipase. Crosslinking of the lipase was found to be optimum at pH 7.5 and room temperature. The activity of the optimized CLEA was found to 26.41 U/mg representing an 880 % increase in activity relative to the crude lipase. This was used in the interesterification of pili nut oil with palmitic and stearic acids to produce a cocoa butter equivalent. Using the CLEA lipase at a 20 % enzyme load from germinating mustard seeds, studies on the interesterification reaction in the preparation of cocoa butter equivalent (CBE) gave the following conditions for optimum yield and characteristics of the CBE: 1:3:6 (w/w/w) of pili nut oil: palmitic acid:stearic acid reaction temperature ? ambient reaction time = 24 hours. Under these optimum conditions, the melting point of the CBE was found to be 35-38 oC using the Fischer Johns melting point apparatus. The fatty acid profile of the CBE from pili nut kernel oil using this method was 36.42 % stearic acid (S), 30.74 % palmitic acid (P), 30.42 % oleic acid (O) and 2.43 % linoleic acid (L). The triglyceride profile of the CBE was 40.50 % at position number (PN) 48, 38.90 % at PN 50, 16.68 % at PN 52 and 3.91 % at PN 46. Based on the fatty acid profile, the possible dominant triglyceride species found in CBE were 19.50% OOO and SOL with carbon number (CN) 54 at PN 48 and 17.88 POS with CN 52 at PN 50. The mustard seed CLEA lipase was found to retain much of its activity after 5 cycles of olive oil hydrolysis, indicating its potential for recycling and its robustness as a biocatalyst. Considering that the melting point of the interestified triglycerides from pili nut kernel oil was very close to that of cocoa butter (literature MP = 35 ? 37 oC), these studies show the potential utility of germinating mustard seed CLEA lipase for the preparation of cocoa butter equivalent from pili nut kernel oil.

Language

English

Location

UPLB Main Library Special Collections Section (USCS)

Call Number

LG 993.5 2014 C42 /S55

Document Type

Thesis

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