Rdd 750 Final presentation By Sana Naz 2008bb50027

Objectives Industrial uses Materials Reaction Rice bran distillate composition Reactant characterisation Acid catalysed reaction Equipment diagram Optimum reaction conditions from literature Procedure Analysis acid value test and tlc Experiment table

Enzyme catalysed reaction Literature Mechanism Procedure Experiment table Conclusion Issues References

1. Production of DG by glycerolysis between rice bran distillate and glycerol. 2. To quantify the optimum reaction conditions in terms of I. Molar ratio of glycerol to rice bran II. Type of catalyst - enzyme or acid catalyst III. Optimum catalyst loading IV. Temperature

I. II. III.

70% OF THE emulsier usage in food industry Oil in water type emulsion food -mayonnaise and salad dressings Water in oil type emulsion foods - margarine, spreads, butter cream fillings and icings used in baking and confectionary industries As an ice cream coated fat - softer and less brittle but had quicker and smoother meltdown, than cocoa butter based coating fats As a starting material for the production of other surface active chemicals Cosmetics and pharmaceutical industry - to make creams

IV.

V.

VI.

REACTANTS
i. ii.

Rice bran distillate (obtained from m/s sangrur agro ltd. Sagur.) Glycerol

CATALYST :
i.

ii.

Enzyme catalysed system Lipozyme tlim (thermomyces lanuginose) Novozyme 435 (lipase from candida antarctica) Acid catalysed system HCl (1% and 5%)

Glycerolysis is the transesterification of glycerol with triglycerides to give mono and diglycerides

Contains 15-23% lipids and a significant amount of neutraceutical compounds The majority of the lipids are the nonstarch lipids.

MAJOR FATTY ACIDS - palmitic, linoleic, oleic acids

MINOR FATTY ACIDS - monoacylglycerols, diacylglycerols, and sterols. GLYCOLIPIDS - diglycosyl monoacylglycerols and monoglycosyl monoacylglycerols.

COMPONENT SUGARS - galactose and glucose.

 Done

by acid value test -titration with 0.1N, KOH

an average acid value of 216

Indicated Value

found in literature is close to 205-210

 Acid

catalyzed reactions are much slower.

Acidic

catalysts are preferred with feed containing high amounts of free fatty acids. A high temperature of 250c is most suitable for this reaction, however such a temperature could be used in an oil bath, not in a water bath. Acid loadings of 1%, 2% and 5% are most commonly seen in the literature.

I.

Add measured amount of rice bran distillate, glycerol and optimum amount of HCl catalyst, in a round flask with a flat bottom.
Place a magnetic stirrer inside the flask.

II.

III.
IV.

Set the desired temperature and rpm.

Let the reaction run for three hours

V.

Carry out the analysis of the product obtained by tlc or acid value test.
If layering is observed then separate the layers first and the perform the tlc of both the layers.

VI.

 Acid

value (or "neutralization number" or "acid number" or "acidity") is the mass of potassium hydroxide (koh) in milligrams that is required to neutralize one gram of chemical substance. The acid number is a measure of the amount of carboxylic acid groups in a chemical compound, such as a fatty acid, or in a mixture of compounds.

 Titration

with 0.1N, KOH was performed after taking a measured weight of rice bran/product sample and dissolving it in ethyl alcohol, phenolphthalein was taken as the indicator. Acid value was calculated according to the formula: Acid value = (56.1 * v * n) / w

 Fast

and simple Can be carried out on micro scale A high degree of accuracy and precision. Extremely sensitive Permits quatitative estimation of as little as 0.1 % of a single component in a mixture.

 MG,

DG were used as references along with the product sample The solvents hexane/di ethyl ether/acetic acid(8:2:0.5, v/v/v) were used as elution system Bands were visualized with iodine vapor. I. Triglycerides (Rf = 1.69) II. Monoglycerides (Rf = 0.13) III. Diglycerides (Rf = 0.56).

Temperature and rpm

The enzyme( both lipozyme tlim and novozyme 435) require, an optimum temperature of 40 - 70c, for a solvent free system. The reaction mixture is viscous, hence a high rpm (200-300) is desirable.

Best enzyme

The best enzyme for the production of dag with different reactants, was found as novozyme 435 since it gave high yield with lesser reaction time. Tlim which was initially used in experiments gives poor yield, in more reaction time. Optimum reaction time was found to be 17-24 hours for tlim, while novozyme 435 was known to give a better 1,3-dag yield in just 2-3 hours.

Molar ratio

Also literature suggested that when the glycerolysis reaction was carried out with sunflower oil, with novozyme 435, the molar ratio of 7:1 (glycerol:oil) was apt.)

 I. II.

Diglyceride is a product midway between mono- and triglyceride A high accumulation of 1,3-diglyceride is based on the idea A fast further esterication of monoglyceride A retarded formation of triglyceride. TG + G <> DG + MG DG + G <> 2MG TG + MG <> 2DG

The former largely depends on a higher esterication-catalytic activity from the intrinsic nature of the enzyme, ecient interaction between substrates and enzyme, and a rapid dehydration leading to the shift of equilibrium

The latter results from the acylmigration of 1,3-diglyceride from 1(3)- to 2-position of glycerol backbone and the further acylation of the resulting product.

I.

II. III.

Add a measured amount of rice bran distillate, glycerol and enzyme to a flask. Close the mouth of the flask by a cotton plug. Place this flask in a shaker, after setting the desired value of temperature and rpm. Stop the reaction after 20-24 hours in case of tlim, and 2/3 hours in case of novozyme 435.

Analysis
Carry out the analysis of product by TLC. If layering is seen, separate the two layers and perform TLC for both the layers

DIFFERENT RATIOS OF RICE BRAN : GLYCEROL

Two molar ratios (rice bran : glycerol :: 1:3) and (rice bran : glycerol :: 1:6 ) were taken. Better result was obtained for the case of 1:6 ratio. Hence in subsequent systems 1:6 ratio was maintained.

DIFFERENT TEMPERATURE SYSTEMS WITH DIFFERENT RPMS

Initially the reaction was carried out at a low temperature of 28.5c and low rpm of 110 with different enzyme loadings. Time of reaction was kept as 24 hours. However product was not obtained in this case. Hence in subsequent system the temperature was increased to 33c and rpm to 300 (since the reactants form a highly viscous mixture- so for better mixing a higher rpm was considered). Still no significant product formation was observed.

Later on another system with a temperature of 50c and rpm of 200, with a reaction time of 24 hours was taken. In this system layer separation was observed a thick solid layer on top of a viscous liquid layer was seen. The two layers were separated and analysed by means of tlc.

Analysis revealed that product comes only in the lower layer. The reason for product formation in this case might be better enzyme activity due to high temperature and better mixing due to high rpm.

DIFFERENT ENZYME LOADINGS 5% AND 10%

The above reaction set was performed with two different enzyme loadings 5% and 10%. Among these two loadings product came in the lower layer, in case of 10% enzyme loading.

NET RESULT, WHEN ENZYME TLIM WAS USED

Best system comprised of rice bran and glycerol, taken in 1:6 molar ratio, with t= 50c, rpm = 200 and reaction time 24 hours. The enzyme loading 10% was more appropriate than 5% enzyme loading

ENZYME CATALYSED SYSTEM NOVOZYME 435

Analysis was carried out by TLC and it was observed that a significant amount of DAG and MAG were produced at time = 2 hours. At time = 3,4 hours a reduction in the quantity of DAG was observed.

COMPARISON BETWEEN TLIM AND NOVOZYME 435

A reaction time of 2 hours was sufficient with Novozyme 435, as against a reaction time of 20-24 hours with TLIM. Also, only 5% novozyme 435 gave good result, while 5% of TLIM at best conditions (50c, 200rpm, 24 hours) did not give good results (though layering was observed), 10% TLIM gave better results with product coming in the lower layer.

ENZYME

The best enzyme for the production of dag with different reactants, from literature was found as novozyme. However due to cost considerations ( novozyme 435 1 kg costs 2.5 lakhs) tlim was mostly used. Later on 1 experiment setup, using 5% novozym 435, was setup.

TEMPERATURE AND RPM

During initial experiments, an appropriate shaker which could be used for such a high temperature was not found. Hence the initial reactions were carried out at temperatures close to 30c. Later on, when an appropriate shaker was found, we carried out the reaction at 50c, but not at higher temperatures, since it was an old equipment and the reaction time was 24 hours.

ACID CATALYSED REACTION

The

desired temperature for this reaction was above 200c, but for such a high t, we required an oil bath system instead of a system with water. Hence a temperature of 90c was used, which gave good results for 5% acid loading.

QUANTITATIVE ANALYSIS
Hplc

analysis could not be carried out, due to the unavailability of the desired column.

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