Lipase is an enzyme that the organic structure uses to interrupt down fats in nutrient into fatty acids and glycerin so that they can be absorbed in the micro villi in little bowel into the blood stream. Lipase is chiefly produced in the pancreas but is besides in the oral cavity and tummy. Most people produce adequate pancreatic lipase, but people with cystic fibrosis, Crohn ‘s disease, and celiac disease may non hold adequate lipase to acquire the nutrition they need from nutrient.[ 1 ]Similar like other enzymes, lipases aid to modulate biochemical reactions in human organic structure. Many of those reactions might go on without the enzyme but at really slow rate. However, if an enzyme is present reaction is faster and more efficient.[ 2 ]Role of lipase is represented on figure 1 holla[ 3 ]:
“ One of the many enzymes pancreatic juice contains is lipase. As a consequence of the alkalinity of the gall salts, the pH of the duodenum is about 7.0, which is besides the optimal pH for pancreatic lipase. Having been to the full digested in the duodenum, the lipid-soluble fatty acids and glycerin diffuse through the phospholipid bilayer of the plasma membranes doing up the epithelial cells of the little bowel. Milk is a white liquid composed largely of H2O ( 87.3 % ) , with little sums of fats ( 3.9 % ) , and non-fat solids such as proteins and lactose ( 8.8 ) . Milk contains more fat than most liquids and a bulk of these lipoids are classed as triglycerides which therefore makes milk a suited liquid to be used for this experiment. Using solid fat such as lard would be impractical because the enzyme lipase would merely be able to adhere with lipoids on the surface of the lard, significance there would be an highly slow reaction rate. The globules of fat found in the milk gives the lipoids a larger surface country and provides more ‘surfaces ‘ that the lipase enzyme can adhere to. ”[ 4 ]
Three sorts of tea, oolong, green and black, have been widely used for their purported wellness belongingss from ancient times all over the universe, particularly to forestall fleshiness and lipid metamorphosis.[ 5 ]In vitroA surveies with green tea infusions incorporating 25 % of catechins have shown its capacity ( in conditions similar to physiological 1s ) to significantly suppress the stomachic lipase, and in a lower extent besides the pancreatic lipase. Therefore, the lipolysis of long-chain triglycerides is reduced in a 37 % .[ 6 ]A
In this experiment, we will look into the consequence of green tea infusion ( inhibitor ) on the pancreatic lipase activity in milk ( Alpsko mleko with 3.3 % of fat ) . Therefore our research inquiry will be:
How make different sums ( 0.5mL, 1mL, 2.5mL, 5mL, 10mL ) of green tea ( Camellia sinensis ) extracts which can move as lipase inhibitor, affect the rate of reaction catalysed by pancreatic lipase?
As can be seen from the research inquiry, our independent variable will be the sum of green tea infusion added to a reaction and the depended variable will be the rate reaction, which will be expressed as alteration in pH per unit of clip.
VOLUME OF MILK: milk serves as beginning of substrate that is needed for lipase action. In order to guarantee the same sum of substrate ( lipoids ) will be available at different sum of inhibitor, 5mL of milk will be added to the trial tubing at every test.
% OF FAT IN MILK: as fats in milk are indispensable for examined reaction, we will utilize the milk with the same per centum of incorporating fat for all measurings.
ROOM Temperature: as besides temperature can impact the rate of reaction, all the measurings will be done in the same schoolroom, off from any heat beginning to guarantee equal conditions for all tests. Record the room temperature several times, during the experiment to do certain, that temperature is non changing.
LIPASE CONCENTRATION: 5 % solution of lipase will be prepared and used at all measurings into order to guarantee the same enzyme concentration at all tests
APPARATUS USED: because different research lab equipment have different uncertainnesss, make certain that you will utilize the same balance/pipette/micropipette etc. for the measuring of the same reagent.
lipase pulverization from porcine pancreas[ 7 ]
milk ( Alpsko mleko with 3.5 % of fat )[ 8 ]
electronic balance ( A± 0.001g )
computing machine with LoggerPro package
electronic thermometer ( A±0.5A°C )
pipette ( 5mL A± 0.006mL )
pipette ( 10mL A± 0.006mL )
micropipette ( 1000I?L A± 0.0006 I?L )
mensurating cylinder ( 200mL A± 0.5mL )
250mL beaker with stopper
volumetric flask ( 100mLA± 0.1mL )
25 trial tubings ( 20mL )
trial tubing base
stop watch on computing machine ( A±0.5 )
3 PRELIMINARY PROCEDURES
Preparation of green tea infusion
In order to fix tea infusion, step 200mL of deionised H2O utilizing a measurement cylinder and furuncle it. Then steep 5 tea bags into poached H2O in order to obtain concentrated green tea infusion. After 8 proceedingss take the tea bags out and pour the infusion into the 250mL beaker. Seal the beaker and allow the infusion to chill down.
Preparation of 5 % porcine pancreas lipase solution
Put the 100mL volumetric flask on the balance and reset it. Then, step precisely 5.00 gms of lipase from porcine lipase into the volumetric flask. After that, add deionised H2O to the conelike flask up to the border for 100mL. Seal the volumetric flask and twirl it 5 times.
Connect LoggerPro interphase and pH investigation to computing machine. Launch LoggerPro package.
Travel to “ data aggregation icon ” and set the length of measuring to 3 proceedingss at trying rate 10 measurements/minute.
Measure 5.00mL of milk into the trial tubing with a pipette.
Add three beads of phenolphthalein index to the reagents mixture. Solution will go of purple/pink coloring material. Stir the trial tubing.
Topographic point the trial tubing into the base and set the pH investigation into it.
With appropriate pipette/micropipette, add 0mL/ 0.5mL/ 1mL/ 2.5mL/ 5mL or10mL of 5 % lipase solution into the trial tubing incorporating milk and Na2CO3 solution and imperativeness “ Start measuring ” instantly after adding the lipase.
After you will get down the measuring observe what will go on with the coloring material of the solution. During pH measuring, the clip will be monitored invariably on your computing machine screen. Record the clip at which pink coloring material will vanish into table 1.
Make 5 repetitions for each sum of lipase added on the same graph.
Repeat the process for all sums of green tea added.
Developing A METHOD FOR DATA COLLECTION
Table 1: The clip needed for the trial tubing mixture to turn from pink/purple coloring material to colourless
V ( milk ) ( A±0.006 ) [ mL ]
V ( Na2CO3 ( aq ) ) ( A±0.0006 ) [ I?L ]
V ( green tea infusion ) ( A±0.006 ) [ mL ]
Time needed for the trial tubing mixture to go colourless
( A±0.5 ) [ s ]
In LoggerPro, you will obtain 6 graphs with 5 curves stand foring the alteration of pH per unit of clip. Make additive arrested development for group of curves at each sum of green tea infusion added.
Write down the equations of the additive arrested development lines and secret plan merely that lines into the separate graph. Remark on any evident tendencies that you notice. Explain the relationship between the sums of the inhibitor ( green tea infusion ) added on the pH. Deduce under which conditions, the rate of examined reaction ( a?†pH/time ) was the fastest and where the slowest. Justify your reply with the account from the literature.
If the index phenolphthalein changed from pink/purple coloring material to colourless, what type of biochemical reaction took its topographic point?
Calculate the mean times needed for violet coloring material of the solution to vanish. Plot the graph: volume of the inhibitor added versus the mean clip needed for violet coloring material of the solution to vanish and infer how presence of green tea infusion as an inhibitor affect the rate of lipase catalysed reaction
Measure the method and suggest possible betterments.