SINCE the fundamental activity of an organism, tissue respiration, is the setting free of energy from food, generally by oxidation, it follows that all organisms must, if only to have a source of energy for this process. This, incidentally, gives one definition of a food as a substance containing potential energy which can be liberated and utilized during tissue respiration. Such substances are of three types, carbohydrates, oils and fats and proteins. Proteins, however, are not normally directly used for respiration but are utilized for the growth and repair of protoplasm. Other material requirements are oxygen, water, salts and vitamins, so that although the above definition of a food may not be a wide enough one, it is the most simple and most suitable for the present purpose.
As substances termed enzymes commonly occur in organisms and have a marked effect on chemical changes, especially those occurring to food, we shall deal with these first. An enzyme is a biological catalyst, that is to say, it is a substance which alters the rate of a chemical change but which remains unchanged at the end of the reaction ; it is produced by the living organism, but is not itself alive. Four important kinds of enzymes are :—
Amylases, which ’attack ’starches and other carbohydrates.
Cellulases and Cytases, which ’attack ’cellulose.
Lipases, which ’attack ’lipides.
Proteases, which ’attack ’proteins.
The Characters of Enzymes.
Enzymes hasten the speed of reactions which otherwise proceed with extreme slowness. In the case of reversible actions, e.g. sugar -starch and starch sugar, the reaction is acoelerated in either direction.
The action of any particular enzyme is specific, I.e. it is limited to one particular reaction—e.g. diastase will only act in the case of starch being converted to sugar and vice versa.
Enzymes resemble inorganic catalysts, e.g. platinum, in that a small amount of enzyme can produce a large amount of chemical change without being affected themselves. However, unlike inorganic catalysts, enzymes do tend to become ’used up ’during the course of a reaction.
Enzymes act best at a moderately warm temperature, but are destroyed on heating to temperatures over 6o° C.
Some enzymes work only in acid solutions ; others in alkaline.
A few require the presence of another substance known as a co-enzyme.
CLASSES OF FOODS
These are compounds of carbon, hydrogen and oxygen such that there are always twice as many atoms of hydrogen as there are of oxygen.
Experiment 25—To show that Carbohydrates contain Carbon, Hydrogen and Oxygen
A little starch is heated in a dry test-tube. It turns black and steam condenses near the mouth. If a little of this condensed liquid is poured on to white anhydrous copper sulphate a blue colour results, confirming the presence of water. Since water contains hydrogen and oxygen, starch must also contain these elements. The black residue is carbon. The presence of carbon can also be confirmed by mixing some starch with black copper oxide and heating the mixture. The copper oxide is reduced to reddish metallic copper, while the issuing gases when passed through lime water turn it ’milky, ’ showing the presence of carbon dioxide and hence of the presence of carbon in starch. Similar results are obtained with the other carbohydrates.
Glucose is found in dried raisins as hard brownish nodules, and, mixed with fructose, occurs in the juices of nearly all sweet fruits, in theand of many plants, and in honey. In animals possessing blood, carbohydrate is carried in the blood stream as glucose, being known in this case as blood sugar.
The simple sugars are sweet-tasting, although not so sweet as cane sugar. They dissolve slowly in water and will diffuse through a parchment-paper membrane.
They are probably the form in which carbohydrate is used during tissue respiration according to the equation :—
C6H1206 + 602 = 6C02 + 6H20 + Free Energy.
Experiment 26—To test for a Simple Sugar
On boiling a simple sugar with Fehling ’s solution, the blue liquid first turns green and finally produces an orange or rust-coloured precipitate. The test will only work in an alkaline solution. The simple sugar reduces the blue copper ion derived from black cupric oxide CuO to form red insoluble cuprous oxide Cu20. This indicates the ease with which simple sugar removes oxygen and is itself oxidized at the same time.
Ripe sugar cane contains 15 to 20 per cent, of cane sugar, whereas sugar beet contains up to 16 per cent, of the same sugar, known commercially as beet sugar. The milk of mammals contains about 4 per cent, of lactose. Another complex sugar is maltose, found in malt extract.
The complex sugars are colourless and crystalline, being approximately three times more soluble in water than the simple sugars, although they diffuse rather more slowly than simple sugars. They are readily converted into simple sugars by traces of acid, or by enzymes, e.g. invertase in the case of sucrose and maltase in the case of maltose or malt sugar.
Experiment 27—Tests for Complex Sugars
Complex sugars also affect Fehling ’s solution, with the exception of cane sugar.
If a solution of cane sugar be boiled first with hydrochloric acid coloured red with litmus, then made alkaline with caustic soda, it will give the characteristic precipitate with Fehling ’s solution on further boiling.
Provided the quantity present is not too small, cane sugar will give a characteristic red solution on adding concentrated hydrochloric acid and heating to boiling-point.
These compounds are found as granules in the cells of plants, their appearance varying with the species of plant. Those in a potato are large and show concentric markings. The presence of starch in Spirogyra can be demonstrated by the iodine test. Glycogen is present in the muscles and liver of vertebrate animals.
The starches are white solids and are typical storage substances, being completely insoluble in water. They are slowly hydrolyzed by mineral acids, being converted first into complex sugars and then into simple sugars. Whe.1 boiled with much water a cloudy blue solution results. A more concentrated solution is bluish white and sticky, being a typical colloid solution.
Experiment 28—Tests for Starch
Starch granules slowly turn blue in aqueous iodine.
After boiling in water, and cooling by adding much water, traces of starch are readily detected by the blue solution produced on adding aqueous iodine.
After hydrolyzing with boiling dilute hydrochloric acid for some minutes then making alkaline, a red precipitate will be obtained with Fehling ’s solution on boiling, owing to the conversion of the starch to sugar. In nature this change is brought about by diastase, an enzyme present especially in germinatingand in the saliva in the mouth.
Digestion of Starch.
Experiment 29—To show the Action of the Enzyme Diastase on Starch Four tubes are set up :—
Containing 5 per cent, starch solution only.
Containing 5 per cent, starch solution plus a saltspoonful of diastase. ’
Containing 5 per cent, starch solution plus a saltspoonful of diastase plus a few cubic centimetres of 1 per cent, sodium carbonate solution.
Containing 5 per cent, starch solution plus a saltspoonful of diastase plus a few cubic centimetres of dilute hydrochloric acid. At intervals of 25 sees, a drop of each solution is added to a spot of iodine solution on a white tile.
With a blue colour is produced each time showing no change in the starch.
If a commercial preparation of diastase cannot be obtained, some saliva from the mouth may be used instead.
With a series of colour changes from blue to purple is observed, due to the formation of dextrin—an intermediate product in the breakdown of starch. Finally a yellow colour results, showing the disappearance of the starch.
With the same as in, though faster, provided the alkalinity is not too strong.
With a blue colour is produced each time, showing that diastase will not work in a strongly acid solution.
Finally, the remaining solution in all the tubes should be tested by Fehling ’s test: and give a red precipitate of CutO, showing the presence of sugar.
Experiment 29a—To show the Conversion of Starch to Sugar in Germinating
Barley grains which have germinated are ground up in a mortar and boiled with water. The liquid is filtered and the filtrate boiled with Fehling ’s solution. A red precipitate of Cu20 is produced, showing that the starch in the barley grains has been partly converted to glucose.
Experiment 30—To show Digestion and Diffusion
Three porous paper ’thimbles ’‘ are soaked in a solution of collodion in ether. The ether is then allowed to evaporate, leaving the paper impregnated with collodion, which acts as a semi-permeable membrane. Each ’thimble ’is stood in a beaker flask with the lower end immersed in water to a depth of 1 in.
In the first thimble is placed a little starch solution.
In the second thimble is placed a little glucose solution.
In the third thimble is placed a little starch solution plus some taka diastase.
After twenty-four hours the water in the first beaker flask is tested with iodine. No blue colour appears. Therefore starch cannot diffuse through a semipermeable membrane. The water in the second beaker flask is boiled with Fehling ’s solution. A red precipitate of cuprous oxide is formed, showing that glucose can diffuse through a semi-permeable membrane.
The water in the third beaker flask is also boiled with Fehling ’s solution, and again a red precipitate is obtained. In this case the starch has been digested to form sugar which has diffused.
Starch must be digested before it can diffuse into an animal ’s body or pass from cell to cell in a plant.
The cell wall of all young plants is made of cellulose, this becoming strengthened later by deposits of other compounds, for example the lignin of wood. Its industrial importance may be judged from the following :—
Esparto grass from which are made textiles, paper, cordage, artificial silk, cellophane, cellulose enamel, gun-cotton, celluloid and collodion. Cellulose is never made by animals. Pure cellulose, e.g. filter paper, is white, insoluble in water and in common chemical reagents. It is not attacked by animal enzymes, but moulds, mildews and bacteria produce enzymes called celiulases which dissolve the cellulose in food on which these organisms live. In germinating cereals other enzymes termed cytases are found which will also break down cellulose into simpler soluble carbohydrates. The value of cellulose as a skeletal material lies in its properities of flexibility and elasticity.
Experiment 31—To test for Cellulose
Streak a little cone. H2S04 on a filter paper with a glass rod and then a little iodine solution across these streaks. A blue colour results.
Soak thin slices of date stones in iodine solution and then transfer to cone. H0SO4. Again a dark-blue colour results.
Lipides are mixtures of similar compounds which, like carbohydrates, contain carbon, hydrogen and oxygen only, but unlike them, contain very little oxygen, e.g. olive oil is impure triolein, C57H104O6, beef fat is impure tristearin, C57H110OG, containing also tripalmitin, C51H9806. All are completely insoluble in water and have similar chemical properties, their physical state, however, depending upon the proportions of the constituents : olive oil is fluid because it consists almost entirely of the fluid triolein ; beef fat is solid at ordinary temperatures as a high proportion of solid tristearin is present, although at body temperature it is in a semi-fluid condition ; lard, however, at ordinary temperatures is a pasty mass because it contains a higher proportion of triolein.
When made liquid by a suitable temperature, they are emulsified by alkalis, I.e. they become split up into tiny droplets which remain permanently dispersed throughout the alkaline liquid, the mixture being termed an emulsion. As droplets in the emulsion the oil exposes a tremendously increased surface to any chemical reagent, thereby enabling any chemical reaction to proceed more rapidly. They are readily split into simpler molecules by the enzymes termed lipases, e.g.:— C„H110O„ +3H,0 = 3C17H3B.COOH +C3H6 or, representing the fat according to its chemical nature :— 3C3H, +3H20 =3C„H3B.COOH +C3H8 a, 3C3HB +3H20 = 3C17H33.COOH +C3H, s.
Lipases are present, for example, in germinating caster oiland in pancreatic juice of vertebrates as steapsin.
Since the reaction in all cases is one of splitting with water, we may say that lipases hydrolyze lipides. All the acids produced by such reactions are insoluble in water, but the glycerin is soluble.
This process is termed saponification.
Because of their insolubility, lipides form useful storage substances. They are, however, not readily used for tissue respiration. They have the advantage that when oxidized they liberate much heat energy, although more oxygen is required for the process than in the case of carbohydrate, e.g. a carbohydrate when completely oxidized yields 4-0 Calories per gm., whereas a lipide yields 9-0 Calories per gm. This is readily understood when it is seen that there is sufficient oxygen in a carbohydrate to oxidize all hydrogen present to water, so that extra oxygen has to be supplied for the oxidation of carbon only, whereas in the case of lipide. oxygen is required to oxidize the outstanding hydrogen also, and this hydrogen on oxidizing to form water liberates much heat energy.
Certain vegetable oils dry and harden on exposure to air and light. This fact may explain how leaves produce their waterproof cuticle and how cork is rendered waterproof by the production of suberin.
Experiment 32—Tests for Fats and Oils
If the specimen to be tested is crushed on filter paper, any lipide present makes the paper permanently translucent. The transluccncy readily disappears on immersing the paper in ether with subsequent drying on the radiator.
Plant sections readily show oil droplets on examining after immersing in Sudan III and washing in water; the oil present is stained red.
Sections may be treated with osmic acid which stains oil black.
Proteins are extremely complex compounds of carbon, hydrogen, oxygen and nitrogen, with a relatively small quantity of sulphur, and in the case of those present in the nuclei of cells, phosphorus also. Examples are legumelin in peas and beans, gluten in wheat-flour, making it sticky when made into a paste with water, albumin in white of egg and myosin in muscle. Very few of them are soluble in water. They are, however, soluble in solutions containing acids, salts or alkalis, I.e. in electrolytes. The solution in all cases is a colloidal one, I.e. the dissolved protein diffuses extremely slowly, if at all, and will not, in the very great majority of cases, pass through a membrane. This property emphasizes the fact that their molecules are enormous, being actually the most complex molecules known, e.g. white of egg consists largely of ovalbumin, m. wt. probably 34,500, each molecule possessing at least 3,000 atoms. It is also of biological importance, since proteins cannot pass through cell membranes as such. Many are coagulated by heat.
The group in the bracket:— is common to all peptides and is termed the peptide linkage. Glycyl-glycine is a dipeptide.
The more complex compounds so built up are termed polypeptides or peptones which differ from the amino-acids only in that they have much larger molecules and do not therefore diffuse very readily. Proteins are still largerof amino-acids linked together as above.
In organisms peptones react with sulphates in a way not yet understood to produce the simpler proteins containing the element sulphur.
More complex proteins are formed by reactions between peptones or simple proteins, and phosphates so that the final products contain the element phosphorus, and are therefore termed phosphoproteins.
The changes whereby the molecules are made more and more complex are all cases of condensation : during digestion the reverse process of hydrolysis occurs, generally in the presence of enzymes termed proteases.
Experiment 33—Tests for Proteins
When heated, preferably after being mixed with water, some proteins are coagulated, e.g., white of egg.
Proteins after drying, when heated with sodium hydroxide, decompose to give ammonia gas, readily detected by its turning damp red litmus paper blue, and by the white fumes produced on a concentrated hydrochloric acid stopper.
Boiled with concentrated nitric acid, they turn yellow. On pouring away the acid and adding excess of ammonium hydroxide, the colour changes to orange.
If mixed first with caustic soda, and then one drop of copper sulphate solution added, proteins give a purple coloration, whilst peptones give a rose colour.
Millon ’s Test
When Millon ’s reagent is added to a soluble protein, e.g. white of egg, a white precipitate is formed which turns brick-red on heating.
Digestion of Proteins
Proteins are digested in stages by the action of proteases, the sequence being : proteins peptones -polypeptides amino-acids.
Peptones and polypeptides have smaller molecules than those of proteins, consisting of smaller complexes of amino-acids. Soluble proteins give the biuret test, but peptones give a rose colour instead of a violet one when the biuret test is applied to them.
Experiment 34—To show the Conversion of Protein to Peptone by the Action of the Protease Pepsin To some fibrin in a test-tube is added some pepsin solution made by dissolving 0-25 gm. of pepsin powder in 25 c.c. of water and then an equal volume of 0-4 per cent. HCI solution. The tube is left standing in warm water, and it will be noticed how the fibrin swells up, becomes transparent and then appears to dissolve. The biuret test should then be applied to the liquid, when a rose colour will appear showing the presence of peptone.
The experiment can be repeated using water, 1 per cent, solution of sodium carbonate in place of the HCI. It will be found that the pepsin acts only in acid solution.
Experiment 35—To show the Digestion of White of Egg by Trypsin
A small cube of hard-boiled egg white is placed in a glass tube or basin, and to it is added a 1 per cent, solution of trypsin and an equal volume of 1 per cent, sodium carbonate solution. A small crystal of thymol is added to prevent decay. The egg white will be seen to gradually break up and dissolve.
On repeating the experiment with water, 1 per cent. HCI solution in place of the sodium carbonate, it will be found that the trypsin will only-digest the egg white in an alkaline solution due to the sodium carbonate.
Experiment 35^—To show the Digestion of White of Egg by Pepsin
Experiment 35 can be repeated using pepsin instead of trypsin. Digestion is then found to occur only in the acid solution and not in the alkaline one.