ENZYMES

MEETING HELD AT THE KING’S HEAD HOTEL, SHEFFIELD,
ON WEDNESDAY, OCTOBER 28th, 1903.

Mr. J. W. Potter in the Chair.

The following paper was read and discussed:—

Enzymes
by H. Johnson, D.Sc.

The whole brewing process, from beginning to end, hinges upon the chemical changes which enzymes occasion. Malting, for instance, may be considered a process by which the enzymes are developed in barley. As germination begins, and their secretion takes place, their presence becomes manifest by a rise in temperature of the grain, and by a commencement of the rendering soluble of the reserve materials.

The activity of the enzymes is then checked by kilning the grain, by which means the excess moisture necessary for their development is removed.

During mashing, their activity is again awakened by bringing the mash into contact with water at suitable temperatures. The importance of this stage of the action of diastase on starch is so well known that I have no need here to further refer to it.

Again, when we come to fermentation, the complex changes by which wort is transformed into beer are all accomplished through the direct agency of enzymes, secreted by yeasts or other micro-organisms, introduced into the worts accidentally or by design.

As it will be impossible, in a short paper like this, to make anything like a complete study of brewing enzymes, I shall restrict myself to first giving a few general ideas about these bodies, and then going on to describe one or two experiments dealing with enzymic action, and which have a more practical bearing on ordinary brewing routine.

General Characters of Enzymes
The living cell is the seat of countless reactions. Bodies of extremely complicated natures are being continually formed and split up again. In former times, the way in which these changes took place, was a cause of much speculation to observers, who finally found an explanation for them by supposing that there existed in living matter a special form of energy termed vital force. However, as general knowledge of plant and animal physiology progressed, the theory concerning vital force was gradually abandoned. Most of the phenomena which take place in animal and vegetable life can now be explained on purely chemical and physical grounds.

This result has been largely brought about by the extension of our knowledge of the enzymes. Life itself is now sometimes defined by physiologists as a series of fermentations, because investigation has shown that the living cell, “nature’s laboratory,” accomplishes most of its work through the agency of enzymes, or allied substances, which have the peculiarity of setting up fermentations.

Thus the act of breathing in the human body is accomplished by the aid of an enzyme secreted in the cells which form the lung tissue. The oxygen inhaled into the lungs enters into a loose combination with this enzyme, which then passes it on the red corpuscles of the blood. Without this enzyme breathing would be impossible. In the same way growing barley absorbs oxygen, and gives out carbon dioxide gas. The enzyme which enables the barley to do this appears identical with the oxidising diastase found in the lungs.

Although it is only within late years that our knowledge of enzymes has become at all precise, yet the existence of bodies, with the properties of enzymes, was suspected many hundred years ago.

In the sixteenth century Spallanzani showed that meat could be liquefied by the action of gastric juices. Spallanzani contrived to make birds of prey swallow small sponges tied to strings. He then recovered the sponges from the digestive canals of the birds, and showed that the liquids expressed from them displayed digestive properties. This and other experiments of n similar nature carried out at a comparatively early date, when scientific research did not receive the attention given to it in later times, were soon more or less forgotten. But in 1822 the question of enzymes was revived by the researches of Dubrunfant, who showed that the gluten of barley owed its saccharifying power to the presence of a small quantity of a particular active body, and that when the grain was submitted to germination the active body increased greatly in quantity, at the expense of the inactive gluten. This active body is now generally known by brewers as malt diastase. The researches of Dubrunfant were afterwards taken up by Payen, who made the important discovery that the active substance in germinated malt could be precipitated from an aqueous solution, in a more or less pure state, by the addition of alcohol. A general reagent for the separation of enzymes was now in the hands of investigators, and in due course of time active substances were separated from the saliva, and from the gastric and pancreatic juices, and many other sources. The enzyme of saliva, which somewhat resembles ordinary diastase, and saccharifies starch, received the name of ptyalin. The enzymes separated from the gastric juices are characterised by their property of attacking and liquefying albuminoids, transforming insoluble albumin into a series of simple bodies termed alburnoses, peptones, and amides. Later on a similar enzyme was found in germinating barley and in many other seeds and fruits.

It is interesting to note that the changes produced by enzymes appeared to the early observers identical in nature to those set up by yeast and other micro-organisms, and so the enzymes received the name of soluble ferments, a name by which they are still sometimes known.

Pasteur and his pupils, however, refused to admit that alcoholic and other fermentations set up by micro-organisms were similar to the changes produced by enzymes. To Pasteur fermentation appeared an essentially vital phenomenon, and, to his last days, he upheld the view that fermentation could only take place simultaneously with the growth and development, in short, the vital function of yeast. It remained for Buchner to show that the real agent of fermentation was an enzyme which could be extracted by suitable methods from the yeast, and that fermentation could take place in the absence of organized cells.

Up to the present I have only made mention of one type of enzyme, that is the destructive type which splits up bodies of simple composition into a series of simple substances. However, in all animal and vegetable tissues there exists side by side with the destructive enzymes a group of bodies which possess the opposite function, that of building up complex bodies from others of more simple structure. It therefore often happens that when a particular action is being produced by one enzyme, another is working against it in an opposite direction. A well-known case in point can be formed in the saccharification of starch under the action of malt extract; the ordinary diastase transforms the sugar into maltose and dextrins of various kinds, while the constructive enzyme elaborates a more complex body from the maltose. This often leads to much confusion as to the exact nature of the various products produced by any special enzyme. It also leads to the curious result that chemical reactions set up by enzymes are never complete. Before the destructive enzymes have finished their action the constructive enzymes have set to work, synthesising the products of disagregation.

The way in which enzymes are formed in living tissue is not yet very well understood.

It is probable that most of them are formed from insoluble albumin; by some process of oxidation; their composition appears to be closely allied to that of the soluble and coagulable albuminoids. Like these latter they are very sensitive to the influence of temperature, and if heated above a certain point their composition undergoes a change and their power of inducing chemical action is completely lost. Enzymes, in general, work very slowly at the ordinary temperature—60° F. Below this, their activity becomes slower and slower, the nearer the temperature approaches the freezing point. This is why vegetation practically comes to a standstill during the winter months in the temperate and arctic regions of the world. Above 60° F. the activity of the enzymes increases until it reaches its maximum power towards 125—132° F. After the maximum point has been passed the activity of the enzymes slowly declines, until 160—166^o F. is reached, at which temperature it is completely destroyed.

The rate at which chemical action takes place is, generally speaking, proportional to the amount of enzyme present; in all cases the actual quantity present is very small, since an enzyme is capable of affecting many thousand times its own weight of the substance acted on.

They are also extremely sensitive to the presence of foreign substances; many antiseptics in small proportions completely arrest their action.

In connection with this it has always seemed to me rather a dangerous practice to add antiseptics—sulphites—to the mash-tun, as it is so often done. I have not any data at hand to show the exact effect produced on the progress of saccharification by the addition of sulphites to the mash; at the same time, I think conversion must be considerably retarded, and possibly extract lost, if the quantity of sulphite added is at all considerable.

Mr. Greaves tells me that he has tried the effect of sulphites in the mash as far back as 1873; and that the results obtained were by no means satisfactory, the beers produced being flat, with poor production of yeast during fermentation. This then is a point which I think requires further looking into.

The principal enzymes with which the brewer has to deal are, firstly those met with in malted barley, and, secondly, those secreted by yeast.

In the first group we have:—

Amylase, the liquefying and saccharifying enzyme of starch.

Peptase, or the proteolytic enzyme which digests and renders soluble vegetable albumin.

Cytase, the enzyme which digests certain forms of cellulose.

Oxidase, the enzyme which carries out the respiratory functions of germinating barley, and by whose medium the oxygen of the air is absorbed and carbon dioxide given off.

In the second group—enzymes secreted by yeasts—we have:—

Zymase, the ferment which splits up sugar into alcohol and carbon dioxide; and also many other enzymes, whose functions are to digest unassimilable food and bring it into a form in which it can be assimilated and used either as material for building up the cells or else for furnishing bodies capable of undergoing fermentation. Among the latter there are invertase, which transforms cane into invert sugar, maltase, which hydrolyses maltose, and other enzymes, capable of degrading malto-dextrins and other sugars to the state of fermentable glucose.

As I stated at the beginning of this paper, it is impossible to deal in a short paper with all these enzymes. I shall, therefore, now proceed to discuss one or two experiments which deal with the more practical side of brewing.

The Proteolylie Enzyme of Malt
When ground malt is infused with water at suitable temperatures, the acidity of the infusion increases perceptibly on standing. Moreover, it will be found that the actual amount of acid formed in a given time depends to a large extent on the temperature at which the infusion is made and maintained. The most favourable heats for the formation of acidity lie between 105—130° F. The amount of acid also slowly increases in an infusion kept at 140—145″ F., but ceases entirely towards 155^o F. This formation of acidity is generally supposed to be due to a lactic acid fermentation set up by germs from the malt. A few experiments, however, soon prove this view to be erroneous, and lead to the conclusion that the real cause of the formation of acid is due to the activity of an enzyme in the malt. Experiments to prove this should be carried out as follows:— 10 per cent, infusions, made with ground malt and distilled water, are left to digest for some time at various temperatures. A second batch of infusions, similar to the first, and kept at the same temperatures, are then left to digest, after that a small proportion of chloroform has been added to them. To a third lot, lactic ferments are added, and to a fourth, lactic ferments together with chloroform. Acting in this way, I obtained the following results. Acidity is expressed in cubic centimetres of decinormal alkali per 100 c.c. of filtered infusion:—

These figures show that the acidity of a malt infusion increases as it stands, so long as the temperature does not go above 140° F. At 158° F. there is no increase; the amount of acid extracted in ½ hour is the same as that extracted in 6 hours.

Chloroform has no retarding action on formation of normal acidity. The introduction of lactic ferments causes a very large increase of acidity. Chloroform, however, completely arrests the formation of lactic acid; the total amount of acid formed in an infusion to which have been added chloroform and lactic ferments is the same as in the case of pure malt. Further, we have to note that the amount of acid formed at 104—140° F. is far greater than that obtained at 158^o F.

The action cannot, therefore, be a purely extractive one, since most bodies are more soluble in hot than in cold water.

The formation of acidity must, therefore, be due to enzymic action, since antiseptics, such as chloroform, do not hinder the normal formation of acidity whilst they arrest lactic fermentation; and since we have more acid produced at 122° F. than at any other point, this again is a well-marked characteristic of the influence that the temperature bears on enzymic action.

A curious observation which I have made in these experiments is that the acidity of a filtered wort does not increase, even when left standing for 6—8 hours, at a temperature of 105—140° F. It would, therefore, seem that either the enzyme is practically insoluble in water, or else that the acid substances are produced at the expense of insoluble bodies, which only enter into solution under the influence of the enzyme.

The acid substances which are produced are known as albumoses, peptones and amides. They are formed from insoluble vegetable albuminoids under the influence of the proteolytic enzymes. The amides have the simplest composition, and at the same time appear to be the most acid of these three classes of compounds; 1 gram of asparagin, a typical amido-acid amide, requires 18 c.c. of decinormal alkali for its complete neutralisation. I have not yet been able to fix the exact amount of alkali required to neutralise 1 gram of peptone or albumose, owing to the difficulty of obtaining these bodies in a sufficient state of purity. They appear, however, to be less acid than the amides.

Now these facts appear to me to possess considerable importance. In the first place, the acidity of a malt can only be correctly estimated by extracting the acid bodies at a temperature not lower than 158° C. Below this point the amount of acid extracted depends merely on the temperature at which the infusion is made and the length of time allowed for extraction; it also depends on the activity of the proteolytic enzymes.

Secondly, the percentage of acid in a malt, correctly estimated, is capable of yielding very valuable information, since, roughly speaking, it measures the amount of soluble nitrogenous bodies.

The increase of acidity which takes place during germination must be put down to the formation of amides, peptones, &c.; the proteolytic enzyme during germination digests the insoluble albuminoids just as diastase renders starch soluble. If the malt is forced, that is grown at temperatures which are too high, the enzymes render soluble too great a proportion of the reserve material, with the result that the malt yields la high cold water extract. The most advanced product of digestion, the amides, provide the necessary yeast food of a wort; if the proportion of these is too great, part of them remains in the finished beer, and furnishes an easily digestible food for bacteria or other disease organisms, and thus contribute towards instability.

The peptones and albumoses, bodies of high molecular weight, which diffuse slowly into the yeast cells, are less easily assimilated, and remain in the finished beer, contributing viscosity and “head”-forming capacity.

From the table we see that low mashing temperatures favour the activity of the enzyme and lead to large production of amides, while high temperatures restrict its action and lead to the formation of a larger percentage of albumoses and peptones, at the same time limiting the total percentage of soluble nitrogen. English brewers, with their particular type of beer and well-germinated malts, have always found that the best results arc to be obtained by the use of high initial mashing temperatures, which restrict the action of the enzyme, and prevent the formation of amides in the mash. High initial temperatures, however, not unfrequently lead to yeast weakness, owing to the lack of amides which the ferment finds at its disposal. Where this is the case, yeast weakness can generally be removed by slightly lowering the initial mashing heat.

Continental and American brewers, on the other hand, who work on the low fermentation system, find it advisable to mash at low initial temperatures, 120° F., so that an advanced peptonisation of albuminoids may take place.

This method of working is found useful in their case because, when wort is fermented at low temperatures, yeast requires plenty of ready formed and easily assimilable food; the low temperatures greatly hinder the digestive properties of the yeast, which will soon become sluggish in its action unless provided with previously digested food.

In the English system of fermentation the temperatures are sufficiently high to enable the yeast to digest unassimilable food by means of the enzymes it secretes. At the same time, the yeast should not be unduly taxed in this direction, for if it has to digest the greater part of its food through want of sufficient amide bodies, it will fall off in fermentative power and become weak. Another reason for advanced peptonisation demanded by low fermentation beers, is that imperfectly digested albuminoids are thrown out of solution by the cold temperatures employed, and therefore, unless they are well digested, great difficulties are experienced in obtaining beers which are absolutely brilliant.

This is especially the case with American beers, which are drunk at extraordinary low temperatures, and which, at the same time, must possess perfect brilliancy. I think in view of these facts that English brewers might find it desirable to mash at lower initial temperatures than are now used for the production of chilled beers.

We often hear that chilled beers have no body and lack greatly in palate fulness. This might well be obviated by adopting low initial mashing temperatures for this particular type of beer. If a good peptonisation was obtained, the deposit thrown down by cooling would be greatly lessened, and the beers would gain much in brilliancy and fulness.

I wish it to be clearly understood that, generally speaking, I do not advocate low initial mashing temperatures for ordinary English beers, racked or bottled in the usual way, although I see no reason why a suitable method of mashing, with low initials, should not give good results. The subject, however, is one which would require to be handled with considerable care. At present I only suggest that low initial heats might be suitable for chilled beers.

Before leaving this subject, I wish to point out that the activity of the proteolytic enzyme of a malt might perhaps be easily measured from the amount of acid that can be formed in a given time at a fixed temperature. Our present methods of estimating the activity of the enzyme are cumbrous and unsatisfactory, and I think that better results might be obtained in the way I suggest. Unfortunately I have not yet had the leisure to work out a process. I make the suggestion, however, for what it is worth, hoping that someone else may take up the subject if I am unable to continue it myself.

Liquefying Diastase
Amylase or ordinary diastase consists of a mixture of two enzymes. Ono of these is called a “liquefying diastase”, and produces by carbinol hydrolysis of starch, a body which only differs from starch in that it is of less complicated structure and is soluble in water. The other enzyme produces carbonyl hydrolysis of starch, in brewing terms “saccharification,” yielding maltose and dextrins of various types which reduce Fehling’s solution.

The saccharifying enzyme exists in considerable quantities in unmalted barley; the liquefying enzyme, however, is only secreted daring germination. The relative amounts of the two enzymes in a finished malt appear quite independent of each other. Certain samples which I have examined contain a high proportion of saccharifying diastase, and but little of the liquefying agent. In other samples the proportions are reversed.

The presence of the liquefying enzyme can be easily demonstrated by allowing a small proportion of malt extract to act on a thick starch paste at a temperature of 175° F. In a few minutes the paste loses all viscosity, becoming liquid and transparent. It should be noted that at 175^o F. the saccharifying enzyme is totally destroyed.

In ordinary mashing operations, where malt is the only material used, the action of the liquefying enzyme is apt to be overlooked. The starch which has been partially prepared by the action of enzymes during germination is quickly liquefied and saccharified at temperatures between 142° F. and 150° F. When raw grain, however, is used, in which the starch granules are intact, it is found necessary to heat such material to a point considerably above 150° F. in order to obtain its complete solution.

Practical experience has proved to me that the most favourable temperature for producing the liquefaction or “conversion” of raw grain, maize or rice grits, is one of 175° F., the quantity of malt required being about 20 per cent, of that of the raw grain.

Under the combined action of the heat and the liquefying diastase the starch cells are quickly burst and rendered soluble at a temperature of 175^o F.

If we look in the technical manuals we shall find that maize and rice starch are attacked by diastase at a much lower heat than 175° F. We must remember, however, that in such experiments the starch was in a complete state of disagregation, in which it is easily attacked.

Such conditions are not met with in the brew-house, where the state of division or the relative size of the portions of the material used have to be taken into consideration.

All raw grain should therefore be subjected to a digestion with malt, for about £ hour at a temperature of 175° F., before being submitted to ebullition.

Final saccharification in the mash-tun will then take place easily and completely, and no difficulty will be obtained in producing a wort, with a suitable polarimetric value, which attenuates normally during fermentation.

Enzymes of Yeast
Invertase.—This is one of the best studied enzymes of yeast. It possesses the well-known property of splitting up cane sugar into equal quantities o! dextrose and levulose.

The enzyme displays a fair amount of activity at ordinary fermentation temperatures, so that cane sugar, when introduced into a brewer’s wort, soon undergoes inversion, and ferments.

In order, however, to bring about the inversion, the yeast has to expend a certain amount of energy, which causes a falling-off in fermentative power. This illustrates a well-known peculiarity of enzymic action: enzymes are only secreted in appreciable quantity when actually required to do active work. The amount of invertase found in yeast is very small at ordinary times, and only becomes considerable when the yeast is brought into contact with cane sugar.

It is plain that the manufacture of invertase in the cell must take place at the expense of other enzymes, and, therefore, produces a falling-off in the quantity of zymase, available for setting up fermentation.

The brewer, therefore, generally takes the precaution of previously inverting cane sugar, when this material is used, before adding it to the fermenting vessel, so as not to overtax the strength of the yeast actually engaged in producing fermentation.

The inversion can be performed with case and simplicity in the brewery by simply digesting yeast, at a suitable temperature, in a solution of cane sugar. This process, of course, is by no means new, but comparatively few brewers, I think, avail themselves of it. By buying raw cane sugar, and inverting it at the brewery, it is possible to make an economy of about £2 the ton.

I have lately been making a few experiments to find out what is the exact temperature, and the amount of yeast required, to bring about a good inversion.

Most authors, who have experimented with pure invertase, give 122—127° F. as the temperature at which the enzyme exerts its optimum activity,

I find, however, when experimenting with yeast, that inversion proceeds much more quickly at the temperature generally adopted, 133° F. To carry out the process efficiently, I should advise a quantity of 1 gram of yeast per 100 c.c. of solution, containing 40 grams of sugar. To put it into brewery figures, to each barrel of solution, containing 144 lbs. of sugar, and having-a gravity of about 1150, add 3 lbs. of liquid yeast for each cwt. of sugar put in. The mixture should be then digested at exactly 133° F., for 1½—2 hours, at the end of which time conversion will be found to be practically complete. The solution can then be used directly in the copper. The quantity of yeast mentioned, 3 lbs. to the cwt. of sugar, does not make the solution very thick, and nothing is to be gained by attempting to get rid of the yeast.

The Influence of Enzymes on Attenuation
Zymase, the acting fermenting principle of yeast, is only capable of fermenting simple sugars of the glucose type, containing six atoms of carbon.

It therefore happens that the fermentative power of a yeast, introduced into a solution containing a mixture of sugars of various types, depends to a very large degree on the enzymes secreted.

An ordinary malt wort containing small quantities of cane sugar, glucose, maltose, and various malto-dextrins or dextrins, is capable of yielding five distinct degrees of attenuation, according to the type of yeast introduced.

These types are generally classified as Apiculatus, Saaz, Frohberg, Burton, and Logos yeasts.

Of these, Apiculatus produces the smallest attenuation in a wort, since it secretes no invertase or other hydrolysing diastases, and is therefore only capable of fermenting glucose. Saaz yeast carries down the attenuation further, inverting and fermenting maltose. Next in order we have the Frohberg type, which degrades dextrins of simple composition to the state of glucose. Following Frohberg comes the Burton type, which is a strongly attenuative yeast, capable of inverting and fermenting fairly complex dextrins. Last in order we have Logos yeast, isolated from a South American distillery, which will carry down the attenuation of a wort farther than any other of the yeasts mentioned.

A simple experiment will suffice to prove that the different attenuations obtained with these five types of yeast are really due to their varying capacities for attacking diverse kinds of sugars; for if we inoculate solutions containing glucose only, we shall obtain the same attenuation with each type of yeast, on the condition that the quantity of alcohol produced during fermentation is not sufficient to prove harmful to the yeasts.

A further peculiarity with regard to attenuation is that once a yeast has reached the point where all the available sugars have disappeared, it will not produce any further fermentation even if left in the solution for a year or more. That fermentation comes to a stop owing to lack of fermentable sugar can again be easily proved by introducing a little glucose into the solution, when fermentation will at once begin again and the glucose will disappear. Each type of yeast, therefore, possesses a definite “limit attenuation,” which it will always reach under suitable conditions of temperature and concentration, and beyond which it will never pass. The idea has often occurred to me, whether it would not be possible to obtain more control over brewery fermentations by the use of a specially selected type or types of yeast employed for pitching purposes, from which powerfully attenuating types were eliminated. Primary fermentation could then be allowed to proceed until the “attenuation limit” was reached.

Given, a wort of definite composition; with a certain polarimetric value, it would be found possible to control attenuation within very narrow limits. The primary yeasts should then be removed from the beer, and a more powerfully attenuating type capable of producing aroma should be added either in the storage or the carriage casks. It would then be possible to obtain the conditioning of the beer, with more certainty than is now the case, without risking either flatness or frets due to secondary fermentations set up by wild yeasts.

This practice has been carried out for some time in a low fermentation brewery, where it has given excellent results. Mr. Greaves also tells me that for years he was in the habit of introducing fresh yeast into the casks, and obtained from it excellent results. As, however, the ordinary pitching yeast contains many types which cannot possibly produce any result when added to the cask, it seems a more rational method to separate the useful types, by appropriate methods of culture, and use these only for secondary pitching. Perhaps some of you who have listened to this paper tonight have tried adding yeast to the cask, and if so should be very pleased to hear what results have been obtained.

An interesting discussion followed the reading of the paper.