trypsin experiment with milk

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Published experiments

Investigating effect of temperature on the activity of lipase, class practical.

Phenolphthalein is an indicator that is pink in alkaline solutions of about pH10. When the pH drops below pH 8.3 phenolphthalein goes colourless. Here, an alkaline solution of milk, lipase and phenolphthalein will change from pink to colourless as the fat in milk is broken down to form fatty acids (and glycerol ) thus reducing the pH to below 8.3. The time taken for this reaction to occur is affected by temperature .

Lesson organisation

This investigation could be carried out as a demonstration at two different temperatures, or in a group of at least 5 students with each student working at a different temperature. This would allow students to collect repeat data at their allocated temperature. Or it could be an investigation carried out by one student.

Apparatus and Chemicals

For each group of students:.

Test tube rack

Measuring cylinder (or syringe), 10 cm 3 , 2

Beaker, 100 cm 3 , 2 (for milk and sodium carbonate solution)

Beaker, 250 cm 3 , 2 (to act as water baths for temperatures below room temperature)

For each temperature: Thermometer

Syringe, 2 cm 3

Stop clock/stopwatch

For the class – set up by technician/ teacher:

Milk, full-fat or semi-skimmed, 5 cm 3 per student per temperature assessed

Phenolphthalein in a dropper bottle ( Note 2 )

5% lipase solution, 1 cm 3 per student per temperature assessed

Sodium carbonate solution, 0.05 mol dm – 3 , 7 cm 3 per student per temperature assessed

Electric hot water baths set to a range of temperatures, each containing a thermometer, a test-tube rack and a beaker of lipase solution.

Health & Safety and Technical notes

Sodium carbonate solution, 0.05 M. Make with 5.2 g of anhydrous solid, or 14.2 g of washing soda per litre of water. See CLEAPSS Hazcard; it is an IRRITANT at concentrations over 1.8 M.

Ethanol (IDA) in the phenolphthalein indicator is described as HIGHLY FLAMMABLE on the CLEAPSS Hazcard (flash point 13 °C) and HARMFUL (because of presence of methanol).

Glassware is breakable.

Electric water baths should be safety checked in accordance with your employer’s instructions.

Take care with thermometers and brief students how to react if they are broken.

Read our standard health & safety guidance

1 Lipase solution is best freshly made, but it will keep for a day or two in a refrigerator. Don’t try to study different temperatures on different days for the same investigation; the activity of the enzyme will change and it will not be a fair test.

2 Phenolphthalein is described as low hazard on CLEAPSS Hazcard. Refer to Recipe card (acid-base indicators): Dissolve 1 g in 600 cm 3 of IDA then make up to 1 litre with water. Label the bottle highly flammable. Suppliers of phenolphthalein solution may not use IDA; it also may be diluted. Follow any hazard warning on supplier’s bottles.

SAFETY: Keep the phenolphthalein solution away from sources of ignition.

Wear eye protection and quickly rinse any splashes of enzyme solution or sodium carbonate from the skin.

a Make up lipase solution and suitable quantities of the other solutions.

b Set up the water baths at a range of temperatures and put a beaker of lipase, containing a 2 cm 3 syringe into each water bath. Cover a range of temperatures up to around 60°C. An ice-bath will maintain a temperature of 0°C, until all the ice is melted.

Investigation

c Label a test tube with the temperature to be investigated.

d Add 5 drops of phenolphthalein to the test tube.

e Measure out 5 cm 3 of milk using a measuring cylinder (or syringe) and add this to the test tube.

f Measure out 7 cm 3 of sodium carbonate solution using another measuring cylinder (or syringe) and add this to the test tube. The solution should be pink.

g Place a thermometer in the test tube. Take care as the equipment could topple over.

h Place the test tube in a water bath and leave until the contents reach the same temperature as the water bath.

i Remove the thermometer from the test tube and replace it with a glass rod.

j Use the 2 cm 3 syringe to measure out 1 cm 3 of lipase from the beaker in the water bath for the temperature you are investigating.

k Add the lipase to the test tube and start the stopclock/ stopwatch.

l Stir the contents of the test tube until the solution loses its pink colour.

m Stop the clock/ watch and note the time in a suitable table of results.

Teaching notes

The quantities used should take approximately 4 minutes to change from pink to white at normal laboratory temperature. If this is not the case, change the concentration of enzyme to alter the speed of the reaction (more enzyme will reduce the time or increase the speed). Students will need to use the same volume at each temperature.

Digestion of fat produces fatty acids (and glycerol) that neutralise the alkali, sodium carbonate, thus lowering the pH and changing phenolphthalein from pink to colourless. You could use a pH probe or data logger, or another indicator.

You could add washing-up liquid to the solution (1 or 2 drops per 250 cm 3 ), to emulsify the fats which will provide a larger surface area for enzyme action. This will demonstrate the effect of bile salts. Or bile salts could be used.

Other factors to test:

• This protocol is based on a pH dependent result, so is not suitable for assessing the effect of different pHs on lipase.
• It would be possible to vary the concentration of the lipase and look at the effect of enzyme concentration on the breakdown of fat in milk.
• Different types of milk could be used Jersey, full cream, semi-skimmed and skimmed, to explore the effect on the reaction of changing fat concentration (substrate concentration).

Question 6 on the student question sheet opens the doors to a more extensive piece of research on this enzyme.

Health and safety checked, September 2008

Related experiments

Investigating an enzyme-controlled reaction: catalase and hydrogen peroxide concentration

Investigating effect of concentration on the activity of trypsin

Investigating the effect of pH on amylase activity

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The effect of temperature on the action of the enzyme trypsin

Biology Coursework – Enzymes

To investigate the activity of trypsin under the change of temperature.

Introduction

Enzymes are catalysts in the body that speed up the breakdown of food, and are essential in the digestive system. Although food can be broken down by molecules colliding with it, the process is speeded up greatly by enzymes, a type of catalyst. We know from previous knowledge that enzymes are proteins, and they require the presence of other compounds, or co factors, before their catalytic ability can be exerted. Enzymes can be used more that once and are replaced only after a period of time when they are denatured. They are all specific to certain foods, but some are more specific than others. The lock and key theory of enzymes is that each enzyme has a specific ‘key’ shape, which will only fit into one sort of ‘lock’, or substrate. This is illustrated in the below diagram.

We also know they are denatured by high temperatures, certain salts, solvents and other reagents, where they lose their ‘lock and key’ shape, making them useless. Enzymes work by attaching themselves to a bond in the substance and breaking the bond between them. It is because of these reasons we chose to examine enzymes in this experiment. The enzyme we are using is trypsin, which breaks down amino acids in the body.

I hypothesise that as the temperature is increased, the rate of reaction will increase. However, when higher temperatures are reached, enzyme reaction rate will drop rapidly as the enzymes are denatured. I have drawn out a hypothesis graph, which I believe will be what the final graph will look like approximately. I have also labelled a number of stages, and explained what will happen to them, as known from past experience and research.

We know that even without enzymes in the solution, the rate of reaction would still increase, due to the kinetic theory. The higher the temperature, the more heat energy. Heat energy is converted into kinetic energy, making the water molecules move around more quickly, hence colliding more often with the substrate, helping break it down. We also know that if enzymes are present, when the temperature is higher, the substrate will move more quickly into the active site. At stage 1 on the graph, the rate of reaction increases at a similar rate to the temperature, that is, the increase is roughly proportional. At stage 2, the enzyme activity is at its peak, I believe around 40 degrees. At stage 3, the enzyme is becoming denatured from the high temperatures, and is losing its unique shape, which allows it to catalyse substances. At stage 4, the high temperatures have completely denatured the enzyme, making it ineffective as a catalyst. 20 o

There are a number of different types of variable in this experiment. The independent variable in this test is the temperature. We ensured that this stayed exactly as we wanted it by checking the temperature before inserting the photographic film. This is the only variable we should be changing and have direct control of. The dependant variable is the result we want, that is, the rate of reaction. If we perform the experiment correctly, the only thing that should be affecting the dependant variable is the independent variable, the temperature. All other variables, such as pH and the size of the photographic film, should be kept constant to ensure a fair test. If we change the pH, the enzyme’s bonding will change and cause it to lose its active site. This would make the test unfair, so we added buffer to the solution to keep the pH constant. If the photographic film size was not constant, it would take longer for the enzymes to break down the gelatine on some of the film, while the experiments with smaller pieces of photographic film would be broken down faster. To ensure all conditions are identical apart from temperature in each test, we kept the size of the photographic film to exactly 4mm squared. We also ensured that all tubes were given 10 minutes to acclimatise to the appropriate temperature. This is discussed later in detail. The temperature, the main variable we will be controlling, we have decided to test the trypsin at 0 degrees, 20 degrees, 40 degrees, 60 degrees and 80 degrees. We decided to choose this range, as it should provide a large spectrum of results, and are at the same time not too far apart in temperature, so we can hypothesise what results between them will be once we have our set of results. We made the temperatures precise by using water baths set to exactly the correct temperature, or an icebox at exactly 0 degrees. We also used a thermometer to ensure the temperatures were correct just before starting the experiment.

Procedure for Preliminary Test

• Test tube rack
• 10 Test Tubes
• Ice for 0 degrees experiment
• Thermometer
• Water baths at 40, 60 and 80 degrees.
• Trypsin solution
• 10 pieces of photographic film
• 10 splints with cut ends
• Stopwatches

This is a preview of the whole essay

Before beginning, we ensured the area was safe by wearing safety goggles and clearing the nearby area of books or obstacles. We placed 10 test tubes in the test tube rack. 5 were control tubes, so we added exactly 3ml of water to each of the 5. We then added 3ml of trypsin solution to the other 5. We inserted the photographic film squares, of 2mm squared in size, into each of the 10 splints. There were two tubes, one control and one test, at each temperature. We placed two test tubes, one filled with water, the other filled with trypsin, in the icebox. We placed two in a rack to stay at room temperature. Two were placed in the 40-degree water bath, two in 60 degree water bath, and two in the 80-degree water bath. Each one was allowed to acclimatise to the appropriate temperature in its water bath/environment for 10 minutes. This also ensured that the trypsin would denature if it were at too high a temperature, discussed in detail later. Stopwatches were started at the point when the splints were placed into the tube. Every 10 seconds the film would be examined. The timer was stopped only when the film was clear, so that all the film had to reach the same stage (eliminating the possibility of human error as to judging when the enzyme has completed it’s job). We then recorded all our results in a table, as shown below the ‘Fair Test’ Section. Finally, we all washed our hands to ensure any trypsin on them was washed off.

Ensuring that the experiment was a fair test was one of the most important parts of the experiment; if each test were not fair, then the results would be incorrect. The first thing we had to be sure of was that we did not contaminate the trypsin with dirt or bacteria that may have been on our fingers, as this may have affected the rate at which the enzyme works. We also made sure that all the test tubes reached their correct temperature and were allowed to acclimatise for 10 minutes. This is important for two reasons, the first being that if we did not ensure the test tube was at the correct temperature, then the results would not be a correct reflection of what we had hoped to achieve. Also, it is important to remember that at high temperatures, enzymes work at accelerated speeds for short periods of time before denaturing (when the enzymes lose their ‘key’ shape so they cannot fit in the ‘lock’ of the substrate), whereby they are useless. We can see this in commercial industry, where enzymes are used at extremely high temperatures when they work very quickly, and then denature and are removed for another batch of enzymes to work. It is also important we keep pH constant, as if the pH changes, the bonding of the enzyme would change, causing it to lose it’s active site. This could affect the results and therefore our final conclusion, so we used buffer to regulate the pH. We also decided to keep the photographic film size at exactly 2mm squared. If photographic film were at different sizes, then in some test tubes the trypsin would have to work for longer to break down the larger piece of photographic film, hence increasing the result time and making the test unfair. To ensure complete accuracy, we checked our stopwatch every ten seconds instead of twenty or longer, so that we could pinpoint exactly when the photographic film had become transparent.

Results from Preliminary Experiment

The results we obtained were tabulated in a table below:

As we can see, the control experiment showed no change, so I did not need to put the results into a graph. I then created a graph from my results and to ensure that a curve was made in the correct direction, I divided 1 by the time taken in seconds. This ensured that as the rate of reaction was faster, the value would be higher. Values were in seconds and degrees. The graph is shown below:

Preliminary Experiment Evaluation

As one can see from the above graph, it is very similar to my hypothesized graph. The slight difference may be due to human error, or the length of time in between each check of the tube. As we can see, up to 40 degrees, the rate of reaction steadily increases, and, as I hypothesised, after this point it begins to drop as the enzymes are denatured. We can also see that at 80 degrees the enzymes cannot break down the gelatine at all or very little as they have been completely denatured by the high temperature. It was important that we allowed the trypsin to acclimatise; else we may have found that the enzymes worked quickly on the photographic film and made it transparent before they became denatured. I saw no anomalies in my work; this is a sign of good planning, but I must continue to work effectively in the final experiment to ensure there are no anomalies then. However, after doing the experiment once, there are a number of changes I will make to my work. Instead of checking the tubes every ten seconds, I will check them every five seconds. This will ensure more accuracy, as my results will be more precise. We have obtained 4mm squared photographic film as opposed to 2mm squared photographic film, so that it would take longer for the enzyme to break down the gelatine, and therefore meaning that it give us a more accurate result; it is more easy to accurately measure a long period of time than it is a very short one. This will affect our spread of results, and ensured all tests were accurate. We will also triple the concentration of trypsin to speed the results, but at the same time, as a group, we have all decided we will not stop the stopwatch until the photographic film is completely transparent, without exception. This will regulate our results further as we are all stopping the timer at a predefined point. To ensure complete precision of results, in my next experiment I will use two test tubes filled with trypsin at each temperature instead of one. In this way we can cancel out anomalous results. In this way I hope I can further improve the accuracy of our results, so that my final table is a good reflection of the way that enzyme reaction changes.

Final Experiment

• 15 Test Tubes
• 15 pieces of photographic film
• 15 splints with cut ends

We ensured the area was safe by wearing safety goggles and clearing the nearby area of books or obstacles. We placed 15 test tubes in the test tube rack. 5 were control tubes, so we added exactly 3ml of water to each of the 5. We then added 3ml of trypsin solution to the other 10. We inserted the photographic film squares, of 4mm squared in size, into each of the 15 splints. There were three tubes, one control and two tests, at each temperature. We placed three test tubes, one filled with water, the other two filled with trypsin, in the icebox. We placed three in a rack to stay at room temperature. Three were placed in the 40-degree water bath, three in 60 degree water bath, and three in the 80-degree water bath. Each one was allowed to acclimatise to the appropriate temperature in its water bath/environment for 10 minutes. This also ensured that the trypsin would denature if it were at too high a temperature as explained before. Stopwatches were started at the point when the splints were placed into the tube. Every 5 seconds the film would be examined. The timer was stopped only when the film was clear, so that all the film had to reach the same stage (eliminating the possibility of human error as to judging when the enzyme has completed it’s job). We then recorded all our results in a table, and averaged out the results of the two trypsin test tubes at each temperature. Finally, we all washed our hands to ensure any trypsin on them was washed off.

Observations

Finally, I made an “S -1 x1000” row, where 1 was divided by the average, and then the result was multiplied by 1000 for a more manageable number. This means that I had some values that correspond to the rate of reaction. For those results which showed no reaction, they were given a result of 0.

From these average results, we can construct a graph to display our information:

Analysis and Conclusion

The graph was constructed by taking the number of seconds the enzyme took to completely clear the photographic film, and called it X. The formula we used to plot each graph point was “1 divided by X times 1000” or S -1  x 1000. This was so as the time taken was shorter, the graph value was higher, hence reflecting the true rate of reaction. We can see a number of things from this graph. The basic observation, is simply that as the temperature increases, as does the rate of reaction, to a point round 42-44 degrees. As with my hypothesis, I believe this is because the heat energy the substrate has is converted into kinetic energy, moving it more quickly into the enzyme’s active site. Water molecules also move around at higher speeds, colliding with substrates, perhaps making them break apart. At this point, the optimum rate, it then begins to drop, and as the enzyme denatures, we can see the rate of reaction steadily drop until at 80 degrees it is 0. The high heat makes the enzyme, which is only a protein, lose it’s unique key shape, so it can no longer fit in the ‘lock’ of the substrate, as shown below.

As we can see, the high temperature has stopped the enzyme being able to fit into the substrate’s ‘keyhole’ shape as it is a protein and has denatured. This is why we saw no change at 80 degrees; we had allowed a short period of time for the temperature to denature the enzymes. This meant that when the photographic film was inserted, all the enzymes were denatured and useless. At 60 degrees, only some of the enzymes are denatured, leaving the others to remove the gelatine from the photographic film. We can clearly see that at around 40 degrees there is an optimum temperature. It is also interesting to notice that this is near body temperature, however body temperature is slightly lower as they body would need more energy input to maintain a higher temperature, so it is more energy efficient to work at a slightly lower temperature. At 20 degrees we can see the the rate of reaction still increases after this temperature, showing that the optimum enzyme temperature is obviously over 20 degrees. At 0 degrees there is no reaction. The reason why 20 degrees at 0 degrees ended up with lower reaction times or times about the cut off point is because the enzyme and substrate have less heat energy, therefore less kinetic energy, so the substrate will move into the enzyme’s active site more slowly. This means that it will take longer to break down the enzyme. The graph, and these descriptions support my hypothesis completely, with very little difference between my hypothesised graph and my real graph, bar minor differences. My hypothesis was that as temperature increased, as would rate of reaction, to a certain point whereby the enzymes would denature and the rate of reaction would start to drop till it reached zero. This is the exact same conclusion that I have drawn below. There is a very secure support of my first prediction, and this shows not only that I was correct, but at exactly what points the optimum trypsin level is.

I can also say that our results are very accurate, they are very similar to my planning results, and the two numbers from which I took my average were close together. I can put the fact that the planning experiment figures being lower than the final experiment figures down to the fact that we decided to stop the timer only when the photographic film was completely clear. I also found no anomalies in my results, which shows that my planning was effective and my experiment accurate.

We can therefore conclude from all of this, that the rate of reaction for trypsin is a curve, peaking at around 40 degrees, and dropping to zero around 80 degrees. We have firm support now for the kinetic and lock and key theories, and I have proved my hypothesis correct.

The experiment went very smoothly, and we worked efficiently and quickly from the beginning of the experiment. The results fit the prediction perfectly, backing up my hypothesis completely. We found no anomalies in our results, and they were all fairly accurate. We didn’t get any anomalous results because we were precise with our timing, we allowed the enzymes to acclimatise, and we ensured all variables, bar the temperature, were kept constant. We did not have to ignore any results, as they were all relatively accurate to what they should have been. We also know that none of the variables such as pH change or contaminations with fingertips occurred, since there were no anomalies. However, there is always the problem of human error, as they human eye is not a measuring device and works on what the brain deems to be correct rather than precisely measuring what is correct. We could have used a photosensor, which measures how much light passes through it. If we place this behind the photographic film, our results could have been accurate to a fraction of a second. There would also be no problems with human judgement. A light dependent diode could also have been used, and, when connected to a voltmeter, we could work out at how many volts the timer should be stopped (as the LDD works as when the light changes, as does the voltage), also giving us more accurate results, and would be viable to do in a school environment. More accuracy could also be employed with our range of temperatures; the main problem with our graph, is our curve is a hypothesis of what should be in between each number; we do not actually know what the curve looks like in between values. Therefore, for much more accuracy and a more accurate graph curve, we could have done tests every 5 degrees rather than every 20 degrees. This would ensure our graph’s line of best fit is the correct shape. We could also have extended our investigation by using different substrates and enzymes, examining how each substance breaks down, and which substrates break down more quickly. For further investigation, we could have even used pH as our independent variable (the one that did not change). However, under the circumstances, I believe that I did fairly well and produced an accurate table of numbers.

Document Details

• Word Count 3514
• Page Count 9
• Level AS and A Level
• Subject Science

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• Supporting Biology

Trypsin and milk

• Thread starter Marie
• Start date Feb 7, 2017
• Feb 7, 2017

Hi Was wondering if anyone could help with this question we often run this experiment looking at the effect of temperature on trypsin using 2 water baths one at 40C and one at 80C. The 80C one is supposed to inhibit the trypsin so it doesn't work but this doesn't happen, would anyone have any idea why this is the case? We are using powdered milk.  

Michael Clee

I don't know why it doesn't work but you could try diluting the trypsin in the 80C water bath to fix the results?  

GeorgetheScienceTech

Marie said: Hi Was wondering if anyone could help with this question we often run this experiment looking at the effect of temperature on trypsin using 2 water baths one at 40C and one at 80C. The 80C one is supposed to inhibit the trypsin so it doesn't work but this doesn't happen, would anyone have any idea why this is the case? We are using powdered milk. Click to expand...

Michael Clee said: I don't know why it doesn't work but you could try diluting the trypsin in the 80C water bath to fix the results? Click to expand...

Jack of all trades master of several.

• Feb 8, 2017

How long do you leave the trypsin in the water bath? Go with at very least ten munities. The number of times enzymes come up when I get the chance I think I will start a enzymes top tips. ChrisN  

The trypsin needs leaving in the water bath to denature before the milk is added than it should work - at least 10 mins as ChrisN suggests (we've had this problem too)  

• Feb 9, 2017

Thanks very much for the help, after another discussion with the teacher sounds like they haven't been leaving the trypsin in the water bath long enough before adding the milk. We will try to get them to do it correctly. Thanks Marie  

I see if I can test it before hand as well just to make sure.  

Marie said: I see if I can test it before hand as well just to make sure. Click to expand...

• Feb 24, 2017

This is one enzyme experiment I actually like, because it always seems to work! In fact, we ran it yesterday with a BTEC group, and as folks have already said, it is important to leave the enzyme in the water bath BEFORE mixing with the milk - about 5 mins seems to be long enough if it's in a test tube. We use Marvel milk powder (4%) and Trypsin at 0.5%. I'll try attaching the method we use - it's a very old worksheet, but it does the job, and if you don't want the students all setting up and maintaining their own individual water baths, it's fine to pre-set thermostatically controlled ones around the lab. It can also be interesting to set up a tube in a bath of iced water. It doesn't do much for ages, but when brought back to room temp, the reaction then works as normal. Shows that the enzyme is just slowed down at low temps as opposed to being denatured at high temps.  

Attachments

• Effect of temperature on enzymes (milk & trypsin).pdf 1.2 MB · Views: 86

• Jan 31, 2019

Hi, we just did an experiment, using Trypsin with different concentration of Milk, the time needed for the reaction to finish was higher with higher concentration, but the teacher said it shouldn`t keep increasing. Any idea why? or have you done this before? Thank you  

Lvl 39 Alchemist

May Aziz said: Hi, we just did an experiment, using Trypsin with different concentration of Milk, the time needed for the reaction to finish was higher with higher concentration, but the teacher said it shouldn`t keep increasing. Any idea why? or have you done this before? Thank you Click to expand...

mike wilson

The teacher's education is sadly lacking. Trypsin is an enzyme. Enzymes are unchanged through a reaction, so can react with more substrate. It will keep reacting until it denatures, no matter how much substrate you throw at it.  

• Feb 1, 2019

Although the enzyme is not changed in this reaction I think in theory a substrate concentration will be reached where all the enzyme active sites are saturated so the graph will level off at a maximum rate.  

• Feb 4, 2019

Rate yes - but May Aziz was talking about time. The time will keep increasing unless the substrate concentration denatures the enzyme in some way or you can't increase the concentration any more due to limits of solubility.  

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• v.34(2); 2014

Effects of Concentration and Reaction Time of Trypsin, Pepsin, and Chymotrypsin on the Hydrolysis Efficiency of Porcine Placenta

Kyung-hun jung.

1 Department of Bio-Industrial Technologies, Konkuk University, Seoul 143-701, Korea

Ye-Chul Choi

Ji-yeon chun, sang-gi min.

This study investigated the effects of three proteases (trypsin, pepsin and chymotrypsin) on the hydrolysis efficiency of porcine placenta and the molecular weight (Mw) distributions of the placental hydrolysates. Because placenta was made up of insoluble collagen, the placenta was gelatinized by applying thermal treatment at 90 ℃ for 1 h and used as the sample. The placental hydrolyzing activities of the enzymes at varying concentrations and incubation times were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel permeation chromatography (GPC). Based on the SDS-PAGE, the best placental hydrolysis efficiency was observed in trypsin treatments where all peptide bands disappeared after 1 h of incubation as compared to 6 h of chymotrypsin. Pepsin hardly hydrolyzed the placenta as compared to the other two enzymes. The Mw distribution revealed that the trypsin produced placental peptides with Mw of 106 and 500 Da. Peptides produced by chymotrypsin exhibited broad ranges of Mw distribution (1-20 kDa), while the pepsin treatment showed Mw greater than 7 kDa. For comparisons of pre-treatments, the subcritical water processing (37.5 MPa and 200 ℃ of raw placenta improved the efficiency of tryptic digestions to a greater level than that of a preheating treatment (90 ℃ for 1 h). Consequently, subcritical water processing followed by enzymatic digestions has the potential of an advanced collagen hydrolysis technique.

Introduction

Collagen is an abundant protein in animal skin and organs. Collagen is composed of a triple-helix of chain structures repeated with glycine-X-Y where X and Y are predominantly proline and hydroxyproline ( Miller, 1988 ). Collagen has not attracted interest from a nutritional perspective because of its poor composition of sulfur-containing amino acids. However, collagen-derived hydrolysates are considered as an important functional food as well as the pharmaceutical and cosmetic industries ( Zhang et al ., 2006 ).

It is a relatively new concept that low molecular weight (Mw) peptides possess various advantageous nutritional and physiological functions. These functions include physicochemical properties (solubility, emulsifying, water binding, and foaming capacity), antioxidant activity, antihypertensive activity, antimicrobial activity and antianemia activity ( He et al ., 2013 ). Besides these functions, moisturizing, softening and skin reproducing functions have enabled collagen hydrolysates to be a focus in bio-industry ( Yorgancioglu and Bayramoglu, 2013 ).

Until now, commercial collagen hydrolysis has been achieved by acid or alkali treatment, although proteases have also been used to obtain low Mw collagen peptides. Acid or alkali hydrolysis is an approved economical process ( Denis et al ., 2008 ); however, acid-hydrolyzed collagen must be neutralized and desalted. In addition, acid and alkali hydrolysis require a long processing time (about 24 h). Enzymatic processing is an alternative to produce commercial collagen peptides. Various proteases including trypsin, pepsin, chymotrypsin, alcalase, collagenase, and papain are applicable depending on the purpose of the final products ( Gómez-Guillén et al ., 2011 ).

Applying subcritical water is a novel technique to degrade organic compounds, including proteins, carbohydrates and lipids; hence, it is commercially used for waste processing ( Yang et al ., 1997 ). The critical point of water is 374℃ at 22 MPa at which water ionizes readily to hydrogen and hydroxide ions and the hydrogen ions cause the disruption of peptide bonds ( Brunner, 2009 ; Watchararuji et al ., 2008 ). We found in a previous study that subcritical water processing hydrolyzed animal by-product collagen ( Lee et al ., 2013 ). Both conversion of collagen to gelatin and partial hydrolysis of gelatin occur during subcritical water processing, however, the majority of the gelatin hydrolysates have Mw > 10 kDa, which limits subcritical water processing as a collagen hydrolyzing technology.

Collagen peptides of 1-2 kDa are recommended as functional food or cosmetic ingredients and enzymatic digestion is essential ( Chai et al ., 2010 ). Numerous proteases can be used for protein hydrolysis, but the actual activity and efficiency of these enzymes on animal by-product collagen have rarely been compared. Therefore, we compared the effects of three representative enzymes (trypsin, pepsin, and chymotrypsin) on hydrolysis of porcine placenta and Mw distribution of the placental hydrolysates. In addition, effect of subcritical water processing on the enzymatic digestion and Mw characteristics of placental peptides was also explored.

Materials and Methods

Frozen porcine placenta was donated by Samwoo Husbandry (Korea). The frozen placenta was thawed in running water for 4 h and washed to remove residual blood. All visible fat was trimmed, and the placenta was cut into 5 cm lengths. Crude protein (5.8%) and moisture contents (92.2%) of the placenta were determined by Kjeldahl (% N ×6.25) and a 102℃ air drying AOAC (1990) method, respectively. The placenta was vacuum-packaged and frozen at ˗50℃ prior to use (within 2 mon). Trypsin (T4549), pepsin (P6887), and chymotrypsin (C4129) were purchased from Sigma-Aldrich Co. (USA), and used without further processing ( Table 1 ). All chemicals were analytical grade.

Pretreatment

Placenta was thawed at 4℃ overnight and homogenized using a SMT homogenizer (SMT Co., Ltd., Japan) at 14,000 rpm for 5 min. A preliminary study indicated that raw placenta does not undergo hydrolysis in various concentrations of trypsin ( Fig. 1 ), hence, the placenta was pretreated to convert the collagen to soluble gelatin. The raw collagen suspension was transferred to a 50 mL test tube and heated in a 90℃ water bath for 1 h, then cooled to 30℃ in water. Subcritical water processing was conducted using a high pressure device as described previously ( Lee et al ., 2013 ). Raw placenta was inserted into the pressure vessel and pressurized to 37.5 MPa. The temperature was increased to 200℃ while maintaining pressure. When the inside of the vessel reached the target temperature (~90 min), the vessel was cooled to 40℃ in ice.

Hydrolysis procedure

Selected enzyme stock was prepared by dissolving an adequate enzyme concentration in water. The enzyme solutions were mixed with pretreated placenta suspensions and incubated for 24 h at 37℃. Based on the product information, the trypsin stock was prepared to contain 25 BAEE unit/mg enzyme in the mixture, whereas pepsin and chymotrypsin were prepared to contain 40 units/mg in the mixture. Enzyme concentration was controlled by diluting the stock solution with distilled/deionized water. At given times, the mixture was removed from the incubator and heated to 70℃ for 30 min to inactivate the enzyme. The sample was kept at ambient temperature for 1 h and used for analysis.

Gel electrophoresis

The pretreated placenta and enzyme mixture was transferred to a test tube and vortexed vigorously. Aliquots of 100 μL of sample were diluted with 400 μL of 8 M urea (final protein concentration, 4 mg/mL). Peptide profiles of samples were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12% acrylamide gels (EzWay TM PAG, KOMA Biotech Inc., Korea) based on the method of Laemmli (1970) . Samples were mixed with one part sample buffer (KTG020, KOMA Biotech Inc., Korea), consisting of 10% glycerol, 2% SDS, 0.003% bromophenol blue, 5% β-mercaptoethanol, and 63 mM Tris (pH 6.8). The sample was boiled for 2 min and 20 μL of the sample mixture was loaded into gel wells. Peptide separation was performed at a constant voltage of 140 V. Pre-stained marker (K18000, EzWay TM , protein-preblue marker, KOMA Biotech Inc., Korea) was used as Mw standard.

Molecular weight distribution

The Mw distribution of the placenta hydrolysates was determined by the method of Gu et al . (2011) with minor modifications. The pretreated placenta and enzyme mixture was centrifuged at 10,000×g for 5 min. Gel permeation chromatography (GPC) was performed on the supernatant using a YL 9100 high performance liquid chromatography system (Younglin Instrument Co. Ltd., Korea) equipped with three Ultrahydrogel TM 120 columns (7.8× 3,000 mm, Waters, USA). The mobile phase was distilled/ deionized water at a flow rate of 1 mL/min, and the Mw distributions of the collagen peptides were monitored using a YL 9100 refractive index detector (YL Instrument Co. Ltd., Korea) at 40℃. A Mw standards kit (106- 20,100 Da, Polymer Standards Service, Mainz, Germany) was used as the standards.

Statistical analysis

A completely randomized design was adopted to evaluate the effect of enzyme concentration, incubation time as well as subcritical water processing using a SAS statistical program (SAS Institute, USA). Each determination was performed in triplicate, and the entire experiment was repeated three times. Representative data are presented. A p <0.05 was considered significant.

Results and Discussion

Trypsin-catalyzed placental hydrolysis.

As depicted in Fig. 2A , trypsin displayed good hydrolyzing activity for porcine placenta. The porcine placenta was composed of four peptides near 200 and 100 kDa. The former indicates α-chains which have M w of 116 kDa and the latter were β-chains (205 kDa) as revealed in other studies ( Ahmad et al ., 2010 ; Klomklao et al ., 2006 ; Liu et al ., 2012 ). The collagen peptides completely disappeared when the sample was reacted with trypsin for 1 h, indicating that the placenta acted as a good substrate for trypsin. The catalytic action of trypsin hydrolyze the placenta was verified when enzyme concentration was reduced. No peptide bands were detected even if the enzyme concentration was decreased from 25.0 to 6.25 BAEE unit/mg. It is well recognized that trypsin has optimum activity at pH 7-9 ( Sipos and Merkel, 1970 ). The placental suspension (~pH 6.3) provided an optimum environment for trypsin activity, resulting in good tryptic placental digestion.

The peptide profiles of the placental hydrolysates revealed that trypsin produced mainly two groups of peptides, one group with a relatively high Mw (>20 kDa) and the other with a Mw of about 626 and 106 Da after a 1 h incubation ( Fig. 2B ). Tryptic hydrolysis continued with increasing incubation time and the high Mw peptide peaks shifted toward the lower Mw range and the number of low Mw peptides increased. After 12 h incubation, the predominant peptide peaks obtained were <1 kDa. In particular, the main peptide peaks were identified at 106 and 500 Da. Based on the Mw calculation, these peptides seemed to be composed of 1-5 amino acids. These results were identical when enzyme concentration was reduced. The lower the enzyme concentration, the higher the Mw distribution in the high Mw peptide group. However, the main Mw peaks were obtained at 106 and 500 kDa regardless of enzyme concentration.

Consequently, trypsin showed good activity, producing low Mw collagen peptides (< 1 kDa) for use in functional foods. This activity resulted from specific cleavage site in substrates. Although, trypsin cleaves the C-terminal to arginine and lysine residues ( Olsen et al ., 2004 ), Rodriguez et al . (2008 ) postulated that it tends to cleave before proline. Proline is the most abundant amino acid in porcine placenta followed by glycine ( Lee et al ., 2013 ) which increased susceptibility to tryptic digestion.

Pepsin-catalyzed placental hydrolysis

The peptide bands in the placenta were not affected by pepsin treatment until 6 h of incubation, and hydrolysis occurred after 12 h ( Fig. 3A ). The peptide bands detected at < 36.5 kDa disappeared if incubation time was extended to 24 h. Maximum activity of pepsin occurs at about pH 2, and pepsin is inactivated in neutral or alkaline conditions ( Johnston et al ., 2007 ). Based on the pH of the placental suspension (~pH 6.3), pepsin seemed to be inadequate to hydrolyze the porcine placenta. According to the SDS-PAGE pattern, two peptide bands (20 and 37 kDa) were detected after 24 h incubation. The GPC pattern also indicated that low Mw peptide peaks (< 20 kDa) were not observed after a 6 h incubation ( Fig. 3B ). A new Mw peak was detected near 10 kDa after 12 h of incubation and the major peaks were present at >20 kDa. Low Mw peak hydrolysates were observed at 7 kDa and < 626 Da after 24 h incubation, however, the main portion of the peptides still existed at > 20 kDa.

Because of the poor collagen hydrolyzing activity of pepsin, decreasing the enzyme concentration resulted in relatively high Mw peptides according to both SDS-PAGE and GPC. Therefore, pepsin must be excluded as a collagen- hydrolyzing enzyme at near neutral conditions. The poor activity of pepsin could be explained by its protein cleaving sites, i.e., pepsin specifically cleaves the peptide bonds between hydrophobic and aromatic residues ( Fruton, 1970 ; Kageyama, 2004 ), and showed limited and slow peptic digestion of the placenta. Consequently, the results indicate that pepsin cannot efficiently cleave porcine collagen because of the limited number of aromatic amino acids and inappropriate pH ( Johnston et al ., 2007 ; Lee et al ., 2013 ).

Chymotrypsin-catalyzed placental hydrolysis

Chymotrypsin had potential application as a placental hydrolysis enzyme (Fig. 4A ). Chymotrypsin-catalyzed collagen hydrolysis occurred after 1 h incubation, and the collagen chains revealed a different susceptibility to this enzyme. According to the SDS-PAGE pattern, the α-chains of placental collagen (~116 kDa) digested gradually and completely disappeared after 6 h incubation. Meanwhile, β-chain (~205 kDa) digestion was initiated faster than that of the α-chains, and β-chain band intensity was smeared when new peptide bands appeared at < 69 kDa after 1 h incubation. The hydrolysate bands disappeared completely after the 6 h incubation with the exception of a 37 kDa peptide that lost its intensity after 12 h incubation. No visual difference in the SDS-PAGE pattern between 30 and 40 units/mg concentrations was observed. However, decreasing the enzyme concentration to 20 unit/mg resulted in minor bands at 20-30 kDa, and the bands were intense at an enzyme concentration of 10 units/mg.

The peptides hydrolyzed by chymotrypsin had relatively high Mw (> 20 kDa) until 6 h of incubation ( Fig. 4B ). After 12 h incubation, a new peak was generated at 7 kDa, though the major Mw peaks were still detected at > 20 kDa. Increases in the low Mw peaks (1.4 kDa and < 434 Da) were obtained, but the half peak dimension belonged to the 20 kDa peptides. The lower the enzyme concentration, the higher the Mw distribution of placental hydrolysates.

Although chymotrypsin exhibited less impact on collagen hydrolysis compared to trypsin, it was difficult to directly compare the hydrolyzing activity between the two enzymes due to differences in enzyme concentrations. Chymotrypsin preferentially cleaves peptide bonds connected with tyrosine, tryptophan, and phenylalanine ( Appel, 1986 ; Ma et al ., 2005 ; Vajda and Szabo, 1976 ). It is likely that the different substrate specificity of this enzyme caused different trypsin hydrolyzing activities, and an increase in enzyme concentration was required to observe the effect of chymotrypsin on placental hydrolysis. However, it should be noted that the commercial chymotrypsin product had a maximum activity of 40 units/mg. Consequently, it was expected that chymotryptic digestion would be successful by increasing the enzyme concentration, which warrants further exploration.

Substrate processing

As discussed earlier, insoluble collagen is not hydrolyzed by proteases; thus, all of the placenta were initially preheated to convert insoluble collagen to soluble gelatin. This preheating treatment was eventually replaced with subcritical water processing, and the samples were treated with trypsin ( Fig. 5 ). The results revealed that subcritical water processing alone was unable to produce low Mw peptides, and the major peak was observed at about 10 kDa, reflecting a limited collagen hydrolyzing effect of subcritical processing ( Lee et al ., 2013 ). Tryptic digestion of the subcritical water processed placenta produced a major peak at 626 Da with a minor peak at about 1 kDa. Although no peak was observed at 106 Da (free amino acids), subcritical processing seemed to be a better placental pre-treatment than preheating to produce low Mw placental hydrolysates.

Concluding Remarks

The present study demonstrated the effects of proteases on hydrolysis activity of porcine placenta. The insoluble collagen had to be converted to soluble gelatin for the enzymes to act. This conversion procedure is traditionally conducted by thermal treatment, however, subcritical water processing allowed the protease to hydrolyze the placenta effectively. Because of differences in substrate specificity and optimum conditions, trypsin was the best enzyme to hydrolyze placenta. However, pepsin and chymotrypsin might be applicable if experimental conditions are optimized for these enzymes, which warrant further exploration.

Acknowledgments

Financial support for this study was obtained from the Korean Institute of Planning and Evaluation for Technology in Food, Agriculture, Forest, and Fisheries, Korea (iPET Project No. 311029-3).

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