Spallanzani's Digestion Experiment — Can We Build an Artificial Stomach?
Classic Biology Experiments | Hemel Private Tuition | GCSE and A-Level Biology
What happens to your food after you swallow it? Does your stomach simply dissolve everything in acid, or is something much more interesting taking place?
Imagine placing a piece of boiled egg into a test tube, adding a little liquid, and leaving it at body temperature. A few hours later, the egg white has started to disappear.
Now imagine repeating the experiment with exactly the same egg white but changing just one ingredient. This time, almost nothing happens.
Why?
The answer takes us back more than 240 years to an Italian scientist who helped change our understanding of digestion: Lazzaro Spallanzani.
In my teaching laboratory at Hemel Private Tuition, I enjoy taking historical experiments and adapting them so that students can investigate the science themselves. This is a particularly good example because it combines biology, chemistry, scientific history and experimental design.
We are going to build a simple artificial stomach, investigate the enzyme pepsin, and discover why the acidic conditions inside the stomach matter so much.
1. The Scientist Who Challenged the Old Ideas About Digestion
During the eighteenth century, scientists did not fully understand what happened to food inside the stomach.
Some believed digestion was primarily a mechanical process, rather like grinding food into smaller pieces. Others thought fermentation or putrefaction might be responsible.
Spallanzani wanted experimental evidence.
Building on the work of earlier investigators, including René-Antoine Ferchault de Réaumur, he carried out a remarkable series of experiments involving food and gastric juices.
He investigated digestion both inside and outside the body. Some of his experiments involved swallowing small containers of food and subsequently examining their contents. By modern standards, some of these methods were decidedly unpleasant!
But the question he was investigating was extremely important:
Could food be broken down by the chemical action of gastric juice, even without the mechanical movements of the stomach?
His investigations helped establish that digestion was a chemical process and that gastric juice could act on food outside the body.
This was a major step towards modern digestive physiology.
There is an important historical distinction, however. Spallanzani did not discover pepsin. That came later, with Theodor Schwann's work in 1836. Our artificial stomach is therefore a modern investigation inspired by Spallanzani's approach, rather than an exact recreation of his original experiments.
2. What Actually Happens Inside Your Stomach?
When we eat a meal, several processes occur.
Our teeth break food into smaller pieces, increasing the surface area available for digestion. Saliva begins the chemical digestion of starch, and swallowing transports the food into the stomach.
Inside the stomach, muscular contractions mix the food with gastric juice.
Gastric juice contains several important substances, but two are particularly relevant to our experiment.
Hydrochloric acid
Hydrochloric acid creates the strongly acidic conditions found inside the stomach.
The stomach contents can have a pH of approximately 1–3, although the pH changes during digestion, particularly after eating.
These acidic conditions help to unfold proteins, activate pepsinogen to pepsin, and support the action of pepsin.
The acid also helps reduce the survival of many microorganisms entering with food.
Pepsin
Pepsin is a protease, an enzyme that breaks down proteins.
Proteins are large molecules made from amino acids joined together by peptide bonds.
Pepsin catalyses the hydrolysis of some of these bonds, producing smaller polypeptides and peptides. Further digestion takes place in the small intestine.
The important point is that pepsin works particularly effectively in a strongly acidic environment.
At approximately neutral pH, its activity is much lower.
This gives us an ideal question to investigate experimentally.
Is the digestion of protein caused by stomach acid, pepsin, or a combination of the two?
3. Building Our Artificial Stomach
We can construct a simple model of the stomach using test tubes, cooked egg white, pepsin and carefully prepared solutions.
The egg white is especially useful because it contains protein and is easy to obtain.
Heating the egg changes its protein structure. The proteins denature and aggregate, producing the familiar white, firm material found in a hard-boiled egg.
When pepsin breaks down these proteins, the structure may gradually soften or partly disappear.
This makes the digestion process potentially visible.
Equipment
Five or more test tubes or small laboratory sample containers
Test-tube rack
Hard-boiled egg white
Laboratory-grade pepsin solution
Prepared dilute hydrochloric acid or an appropriate pH 2 buffer
Neutral pH 7 buffer
Distilled water
Water bath maintained at 37°C
Thermometer or digital temperature probe
pH meter or suitable pH indicator strips
Measuring cylinders or pipettes
Forceps, small knife and ruler
Stopwatch or timer
Protective glasses and gloves
For accurate results, use commercially supplied pepsin suitable for educational laboratory experiments, following the supplier's instructions.
The acidic solutions must be prepared and checked by the supervising teacher. Pupils should not handle concentrated hydrochloric acid, and no one should taste or consume any of the experimental materials.
The five experimental conditions
The most useful arrangement is to keep the temperature at 37°C while changing whether pepsin is present and whether the solution is acidic.
Tube | Conditions | Purpose |
|---|---|---|
A | Pepsin + pH 2 buffer | Artificial stomach |
B | Pepsin + pH 7 buffer | Does pepsin work at neutral pH? |
C | pH 2 buffer, no pepsin | Can acid digest protein on its own? |
D | Water, no pepsin | Negative control |
E | Pepsin + pH 2 buffer at 20°C | How does lower temperature affect digestion? |
Ensure that the final volume of liquid is the same in every tube. Where pepsin is omitted, replace its solution with an equal volume of an appropriate control liquid. Check the actual pH after all reagents have been mixed.
The fifth tube is a temperature comparison, so its other conditions should match those of Tube A.
This simple arrangement enables students to explore two fundamental aspects of enzyme activity: pH and temperature.
4. Carrying Out the Experiment
Step 1 — Prepare the protein
Hard-boil an egg, allow it to cool, and separate the white from the yolk.
Cut the egg white into uniform pieces, perhaps approximately 5 mm cubes.
This sounds straightforward, but it introduces an important experimental issue: surface area.
A large cube contains more protein than a small cube, but also has a different surface-area-to-volume ratio.
If one test tube contains a much smaller piece of egg, it might appear to digest more quickly simply because more of its protein is exposed relative to its volume.
For this reason, the pieces should be as similar as possible.
For more quantitative work, use a balance to select pieces of approximately equal initial mass.
Step 2 — Prepare the test tubes
Label the tubes clearly.
Add equal amounts of the appropriate solutions, then add the same mass of egg white to each tube.
A practical starting point is to use 10 ml of total liquid and a small piece of cooked egg white in each tube. The concentration of pepsin should follow the educational reagent supplier's guidance, and preliminary testing will help determine whether a visible change can be obtained within the lesson period.
Step 3 — Control the temperature
Place Tubes A–D in a water bath at 37°C.
Keep Tube E at approximately 20°C.
Why 37°C?
Because it is close to normal human core body temperature.
The stomach is a warm environment, and the activity of digestive enzymes is influenced by temperature.
A controlled water bath is preferable to a beaker of warm water because the temperature remains much more stable.
Step 4 — Observe the changes
Record observations at regular intervals.
For example:
0 minutes
30 minutes
60 minutes
120 minutes
240 minutes
Photographing each tube from the same angle, with the same lighting and a ruler beside it, makes the comparisons more useful.
Depending on the pepsin activity, protein preparation and conditions, measurable changes might take several hours. The experiment can also be left for a supervised longer observation period using suitable laboratory procedures.
Step 5 — Analyse the results
Students should record whether the egg white has:
changed shape;
softened;
fragmented;
reduced in apparent size;
or remained largely unchanged.
For stronger scientific evidence, we need to move beyond simple visual observations. We will return to quantitative measurements shortly.
5. What Results Should We Expect?
ILLUSTRATIVE EXPECTED RESULTS — NOT ACTUAL EXPERIMENTAL DATA
Hypothetical visual breakdown scores after four hours, from 0 meaning no visible change to 5 meaning extensive breakdown. Actual results depend on enzyme activity and substrate preparation.
Tube A should generally show the greatest digestion because both the enzyme and favourable acidic conditions are present.
Tube B contains pepsin, but at neutral pH, where its activity is greatly reduced.
Tube C contains acid but no pepsin. The acid may affect the structure of the protein, but it does not carry out the same catalytic hydrolysis as pepsin.
Tube D should provide a useful comparison because neither the enzyme nor the acid is present.
Tube E should usually digest more slowly than Tube A, because the lower temperature reduces the rate of enzyme-catalysed reactions.
However, students should be prepared for some surprising results.
Small pieces of solid boiled egg white do not always show dramatic visible changes within a single lesson. Enzyme concentration, available surface area, the physical condition of the protein and the duration of the experiment all affect the outcome.
If stronger results are required, a prepared cloudy egg-white protein suspension can offer a more sensitive method. As protein particles are digested, the suspension may become clearer. This approach has been used in published biology teaching investigations.
6. Why Does Pepsin Need Acid?
This is probably the most important scientific question in the entire investigation.
Enzymes are proteins with specific three-dimensional structures.
Their activity depends on the shape and chemical properties of the active site, where substrate molecules bind and the reaction is catalysed.
Changes in pH can alter the electrical charges of particular amino-acid groups within an enzyme.
These changes may affect the shape of the protein, the binding of the substrate and the chemical steps of catalysis.
Pepsin has evolved to function in the unusual environment of the stomach, where many other enzymes would perform poorly.
For A-level students, this leads to a more sophisticated discussion about how the ionisation of amino-acid side chains affects enzyme function.
There is another important detail: pepsin is initially secreted as an inactive precursor called pepsinogen.
The acidic environment helps convert pepsinogen into active pepsin, with active pepsin then helping activate additional pepsinogen.
This arrangement helps protect the stomach's own cells from premature enzyme activity.
It is therefore misleading to say simply that stomach acid dissolves food.
The stomach is a controlled chemical environment in which acid and enzymes work together.
7. Turning the Experiment into a Proper Scientific Investigation
One of the differences between demonstrating an experiment and teaching students how to become scientists is the quality of the measurements.
It is easy to place egg white into a series of test tubes and observe what happens.
It is much more interesting to ask how we can obtain reliable, quantitative evidence.
Investigation A — Measuring the loss of protein mass
We could measure the initial mass of each egg-white sample.
After a fixed period, the remaining solid could be removed, gently rinsed using a standardised procedure, blotted consistently and weighed again.
The percentage change in mass can be calculated using:
Percentage mass loss = (Initial mass − Final mass) / Initial mass × 100
For example, suppose an egg-white sample initially weighs 0.50 g and the remaining mass after digestion is 0.32 g.
Percentage mass loss = (0.50 − 0.32) / 0.50 × 100
Percentage mass loss = 36%
This would indicate that the mass of the recovered solid has decreased by 36%.
But there is a problem.
Some protein may break into small fragments, making recovery difficult. The egg white may also absorb liquid or lose material through handling.
Consequently, mass loss is only an approximate measure of digestion, not a direct measurement of the number of peptide bonds broken.
That limitation is itself an excellent discussion point for A-level Biology.
Investigation B — Using a light sensor or colorimeter
A more sophisticated approach involves using a cloudy suspension of heat-denatured egg-white protein.
As the pepsin breaks down suspended protein particles, the liquid may become more transparent.
A light sensor can measure the light transmitted through the suspension.
This is particularly interesting in my laboratory because I can use PASCO data-logging equipment to record changes over time, rather than relying entirely on what students can see.
A simple arrangement would include a constant LED light source, a sample container and a light sensor aligned on the opposite side.
We would need to control the following:
The wavelength and intensity of the light source
The distance between the light source and detector
The size and position of the sample container
The initial concentration of the protein suspension
The temperature of the reaction
The pH and pepsin concentration
The result could be a graph of transmitted light intensity against time.
A rising light intensity would suggest increasing transparency, consistent with digestion of suspended protein particles.
However, light transmission is an indirect measurement. It can be influenced by sedimentation, bubbles and changes in particle size. Appropriate blanks, controls and repeated trials would therefore be important.
A published classroom investigation demonstrates this general approach using egg-white protein and light measurements.
Investigation C — Finding the optimum pH
Instead of comparing only pH 2 and pH 7, we could investigate a range of pH values.
For example:
pH 1, 2, 3, 4, 5, 6 and 7.
Keep temperature, pepsin concentration and protein quantity constant.
Using appropriate buffer solutions, students could measure the initial rate of digestion at each pH and plot their results.
This could produce a characteristic enzyme activity curve, with a maximum in strongly acidic conditions.
For A-level students, the shape of this curve can be related to the enzyme's structure and catalytic mechanism.
Investigation D — What happens when the temperature increases?
We could compare enzyme activity at:
20°C, 30°C, 37°C, 45°C and 60°C.
The rate of an enzyme-catalysed reaction often increases with temperature initially because molecules possess more kinetic energy and collisions occur more frequently.
However, at sufficiently high temperatures, the structure of the enzyme becomes disrupted and its activity decreases.
Pepsin, like other enzymes, can undergo thermal inactivation.
There is a useful distinction here.
At low temperatures, an enzyme usually works more slowly. At sufficiently high temperatures, it may become irreversibly inactivated.
A particularly interesting extension would be to expose one pepsin sample to an elevated temperature for a controlled period, cool it to 37°C, and then test its activity against an unheated control.
If the previously heated enzyme has lost activity, cooling alone may not restore it.
That helps students distinguish between slowing a reaction and damaging an enzyme.
8. The Examination Connection — GCSE and A-Level Biology
This experiment provides an opportunity to teach considerably more than the function of the stomach.
At GCSE, students should understand enzymes, digestion, optimum conditions and the effect of temperature and pH on activity.
They also need to understand fair testing and how to identify variables.
For this experiment:
Variable type | Example |
|---|---|
Independent variable | pH of the reaction mixture |
Dependent variable | Rate of protein digestion |
Control variables | Temperature, enzyme concentration, protein mass, surface area and reaction volume |
We could ask students to explain why the experiment includes a tube containing acid but no pepsin.
A strong answer would explain that it allows us to distinguish the effect of acid alone from the action of pepsin in acidic conditions.
At A-level, the investigation becomes considerably richer.
Students can consider enzyme structure, active sites, hydrolysis, reaction rates, repeatability and the limitations of the chosen measurement method.
They might also investigate why pepsin is adapted to the stomach while other digestive enzymes, such as trypsin, function in the different pH environment of the small intestine.
A challenge for students
Suppose a student carries out the experiment and discovers that egg white in Tube B, containing pepsin at pH 7, has lost some mass.
Does that prove pepsin is working efficiently at neutral pH?
No.
The student would need to consider experimental uncertainty, physical fragmentation, changes in water content and the possibility that the pH changed during the experiment.
One observation does not automatically establish the explanation.
This is exactly the type of scientific reasoning that separates a basic descriptive answer from a high-quality examination response.
9. What If the Experiment Does Not Work as Expected?
One lesson I have learnt from teaching practical science over many years is that real experiments seldom behave quite as neatly as the diagrams in textbooks.
Sometimes the change is slower than expected.
Sometimes a test tube becomes cloudy.
Sometimes a sample appears to swell instead of shrinking.
And sometimes there seems to be no observable difference between two conditions.
These are not necessarily failures.
They are opportunities to ask better questions.
Perhaps the pepsin concentration was too low.
Perhaps the egg-white cubes had too little exposed surface area.
Perhaps the temperature was not maintained consistently.
Or perhaps the solution was not at the pH that we thought it was.
I particularly enjoy asking students what they would change if they repeated the investigation.
This encourages them to think about the method rather than treating practical work as a sequence of instructions that must produce a predetermined answer.
In examination questions, students are often asked to identify experimental limitations or suggest improvements.
These questions are much easier to understand when students have actually encountered such problems.
10. Safety in the Teaching Laboratory
Although this is a straightforward biology investigation, it requires sensible laboratory precautions.
Pepsin is a protein enzyme and should be treated as a potential sensitiser. Powdered enzyme should not be handled in a way that creates dust or aerosols.
For school-age students, I would use a prepared pepsin solution wherever possible.
Dilute hydrochloric acid can still irritate or damage skin and eyes depending on its concentration, so the teacher should prepare the solutions, check the relevant safety data sheets and carry out a suitable risk assessment.
The important precautions include using eye protection, avoiding skin contact, using small quantities and keeping all materials clearly labelled.
A thermostatically controlled water bath is preferable to direct heating or naked flames.
All egg and enzyme mixtures should be treated as laboratory materials and disposed of appropriately after the investigation. They must never be eaten or tasted.
I would also avoid leaving protein mixtures at 37°C overnight without suitable laboratory controls because unwanted microbial growth can occur. For longer investigations, the teacher should plan storage, supervision and disposal before starting.
The original historical methods involving swallowing or retrieving objects from the digestive system are not appropriate to reproduce.
Our modern artificial stomach gives us an effective way to investigate the same scientific principles without resorting to those historical procedures.
11. Why This Experiment Is More Interesting Than Simply Learning About Enzymes
A student can learn the following sentence from a textbook:
Pepsin is a protease that digests proteins in the acidic conditions of the stomach.
That is an important fact.
But what does it really mean?
When the student places identical protein samples into different test tubes, they begin to see why the conditions matter.
The acid-only tube shows why acid is not the complete explanation.
The neutral-pH tube demonstrates the importance of the enzyme's working environment.
The lower-temperature tube introduces reaction rates.
And the water-only control establishes a baseline.
The student is no longer simply being told that pepsin works in the stomach.
They are gathering evidence to explain how and why it works.
This is the real value of practical science.
Could we build an even more realistic artificial stomach?
Our simple test-tube model does not reproduce everything that happens in the human body.
The real stomach is a muscular organ that continually mixes food and gastric secretions.
It also regulates the secretion of acid and enzymes, protects its lining with mucus and transfers partially digested food into the small intestine.
A more ambitious model could introduce gentle stirring to represent mechanical mixing.
We could compare stationary samples with samples mixed at a constant speed.
Would the stirred samples digest more quickly because fresh substrate surfaces are continually exposed to the enzyme?
This introduces another variable: the relationship between mechanical processes and chemical digestion.
Interestingly, it brings us back to the questions that occupied Spallanzani and the early investigators of digestion.
The difference is that we now have the equipment and scientific understanding to investigate them quantitatively.
12. Practical Biology at Hemel Private Tuition
One of the advantages of teaching science in my own laboratory in Hemel Hempstead is that students can move beyond diagrams and videos.
We can build the apparatus, conduct experiments, take measurements and discuss the results together.
With data logging, microscopy, digital imaging and laboratory equipment, we can investigate many aspects of GCSE and A-level Biology that students may otherwise encounter only on paper.
I believe there is a fundamental difference between watching someone else perform an experiment and actually taking responsibility for conducting it yourself.
Students learn to handle equipment, make decisions, interpret unexpected observations and explain the evidence they have collected.
Those skills are valuable not only for examinations but for anyone who wants to understand how science works.
At Hemel Private Tuition, I offer individual tuition in Biology, Chemistry, Physics and Mathematics, together with other GCSE and A-level subjects.
Lessons can focus on examination preparation, strengthening understanding or gaining experience of practical science.
For students who find biology difficult because it seems to involve too many disconnected facts, practical investigations provide a way to bring those ideas together.
Conclusion — A Stomach in a Test Tube
More than two centuries ago, Lazzaro Spallanzani helped scientists understand that digestion involved chemical action rather than simply grinding or fermenting food.
Today, we can investigate that central idea using little more than egg white, an enzyme, carefully prepared solutions and a controlled water bath.
Yet behind this apparently simple experiment lie some of the most important principles in biology:
Enzymes are catalysts. Their activity depends on their environment. Experimental controls matter. Scientific explanations must be supported by evidence.
Perhaps the most valuable part of the experiment is not watching the egg white disappear.
It is understanding why it disappears in one test tube but not another.
Science becomes much more memorable when students discover the explanation for themselves.
If you are studying GCSE or A-level Biology and would benefit from a more practical, investigative approach, visit Hemel Private Tuition to find out more.
