298

THE INSTITUTE OF BREWING RESEARCH SCHEME.

THE EFFECT OF HYDROGEN IONS IN BREWING PROCESSES.

By G. Haoues, B.Sc., A.I.C.
The study of the influence of hydrogen ions on bio-chemical processes
has become of great importance during the last few years. Chemical
changes, such as those which occur in brewing and fermentation, are now
attributed to the action of a group of substances known as enzymes.
These bodies have the power of accelerating the velocity of certain
organic reactions, which otherwise would proceed very slowly. They
assist the reaction to a rapid completion when often several decades or
centuries would be necessary in order to detect any change in the
reacting substances. The chemical character of an enzyme is supposed
to remain unaltered throughout the reaction, and hence these substances
have become known as " The Organic Catalysts."
Although great difficulties are encountered in the isolation of an
enzyme, many very active preparations have been reported. Neverthe
less, it is doubtful if a pure specimen has ever been obtained, for the
substances cannot be induced to crystallise, and we have, therefore, no
definite criterion of their purity.
Enzymes are colloidal in character : they do not, as a rule, form true
solutions, but exist as suspensions of fine particles. The peculiar proper
ties of colloids in solution arc attributed to the large surface of the
particles in proportion to their size. They have the power of adsorbing
substances from the solution, and it is believed that the adsorbed
substances accumulate on the surface of the particles, where they are
existent in a far more highly concentrated condition than in the solution.
As the velocity of a reaction is proportional to the concentrations of the
reacting substances, it is thus evident that the velocity of a reaction,
which takes place almost unapprcciably in ordinary solution, can be
greatly accelerated by the presence of a suitable enzyme. But although
the effect is, to a large degree, of this purely catalytic nature, yet it is
not entirely so, for the action is also selective. An enzyme will only
catalyse reactions of particular substances, and it is a curious fact that
each enzyme appears to require a definite steric configuration in its
substrate. Each enzyme has its own narrow limits of temperature and
hydrogen-ion concentration, outside of which the action is greatly
inhibited.
Emslander (Zeitschr. ges. Brauw., 1914, 37, 2 ; this Journ., 1914,
 HAOUES : THE EFFECT OF HYDROGEN IONS, ETC. 299

20, 136) has shown that the colloids of beer and wort migrate towards'
the cathode when subjected to an electric field. The particles arc
therefore positively charged, and it is probable that the charge is due
to hydrogen ions which they have adsorbed.
Recently, van Lacr (Ann. Brass, el Disl., 1920, 19, 19, 23; this
Journ, 1920, 26, 568) has put forward the view that the action of
hydrolytic enzymes is merely a modification of the normal catalytic
action of the hydrogen ions. It is well known that acids will catalyse
many non-biochemical organic reactions, such as the decomposition
of diazo acetic ester, the inversion of cane sugar, and many others,
and van Laer believes that one stage in the mechanism of those
enzyme reactions which can also be catalysed by hydrogen ions alone, ■
is the adsorption of hydrogen ions by the colloidal enzymo particles
from the suspending acid medium. Thus a high concentration of hydro
gen ions will be produced on the surface of the particles, and this will
have the effect of rapidly hydrolysing any substrate which may have been
adsorbed from the solution. The velocity of the reaction will depend on
the product of the concentrations of the substrate and hydrogen ions
in the surface zone of the enzyme particles, and not on the concentrations
in the solution. A definite relation, however, will exist between the
hydrion concentration in the solution and that on the colloidal surface.
The velocity of the reaction will attain a maximum value when the
surface is completely saturated. Beyond the optimum hydrion con
centration the particles coagulate, and hydrolysis is arrested.
According to Compton (Proc. Roy. Soc., 1914, 87b, 245 ; 88b, 258,408),
the optimum temperature for enzyme action is independent of the con
centrations of substrate and enzyme; it is, however, affected by the
hydrion concentration in the medium. Moreover, the entire activity
of enzymes appears to be intimately connected with hydrogen ions,
and the study of enzymes is gradually reducing itself to a study of
pB values. It is therefore evident that this free acidity is one of the
important factors to be considered in brewing processes.
Each brewery is dependent on the water to be found in its vicinity,
and it follows that waters of different alkalinities are used in the
mashing process in various parts of the country. Chabot (Ann. de la
Brass., 1923, 310) has shown that this alkalinity has a significant effect
on the yield of extract obtained from the malt. By acidifying a natural
water to methyl-orange neutrality he found that there was an increase
of about 2 per cent, in the extract. The acidity of the mash was also,
thereby increased, the pu value falling from 6-35 to 5*8.
u 2
300 HAOUES : THE EFFECT OF HYDROGEN IONS, ETC.

Of the many enzymes to be found in brewing materials, diastase

merits our first attention by reason of its early control of the process.
The optimum hydrion concentration for diastatic activity has been
investigated by numerous workers and found to be situated between
4-4—4-5. Perhaps the most conclusive evidence is that of Sherman
Thomas and Baldwin [Journ. Avter. Chetn. Soc., 1919, 41, 231 ; this
Journ., 1919, 25, 180), who determined the activity of a very pure
malt amylase over a wide range of pu values. Their results are shown
graphically in Fig. 1. The curve has a maximum at pa 4-4—4-5.

x
«CTIVIIY
•H

tt- so

Fig. 1. Fio. 2.

The only notable exceptions to this optimum value are those of

Adler (Biochem. Zeitschr., 1916, 77, 150) and Euler and Svanberg
(Wocknschr. Bmu., 1921, 38, 111; this Journ., 1921, 27, 429). Adler
found that diastatic action was greatest at pB 4-9 (Fig. 2). Phosphate
buffer mixtures were used to control the pu value in the experiments
summarised in Fig. 1, whilst the graphs I and II in Fig. 2 were obtained
by the use as buffer solutions, of phosphate mixtures and acetate
mixtures respectively. Adler's value of 4-9 is in agreement with the
observations of Euler and Svanberg, who, in their study of the dynamics
■of diastatic action and the use of a totally different method, obtained
the optimum free acidity as pu 5-0. These results indicate that the
action is appreciable between pB 3 — 9, and is very effective between
the limits of pu 4-6 — 5-2.
Adler found that neutral salts were not without effect on the diastatic
activity of malt, but that the influence was small compared with that of
the hydrion concentration. It is a curious fact that when malt amylase
 HAGUES : THE EFFECT OF HYDROGEN IONS, ETC. 301

is carefully purified by dialysis so as to deprive it of electrolytes, the

activity becomes abnormally low (Ford and Guthrie, Journ. Ghem. Sao.,
1906,89,76; this Journ., 1906,12,1606—Sherman and Thomas, Journ.
Amer. Chun. Soc., 1915,37,623; this Journ., 1915,21,358). The purified
diastase can be activated by the addition of strong or weak acids or
salts. The increase in the case of acids and acid salts was found to be
in some instances as much as thirtyfold. The maximum stimulating
influence corresponded with a pa value which lay between 4-2—4*6.
Further addition of acid, to produce greater hydrion concentrations,
had a marked depressing influence on the activity of the enzyme.
There is little doubt that malt amylase is of a very composite nature.
Biedermann (Fcrmentforsch, 1921,4, 258 ; this Journ., 1921, 27, 430)
suggests that it consists of a therinolabilc component (zymogen pro
enzyme), and a thermostable complement (co-enzyme-activator), and
that the ions of a great variety of salts can function as the latter. From
his experiments he concludes that, of simple neutral salts, sodium
chloride and potassium thiocyanate are the most powerful activators.
The flat portion near the maximum of the curve in Fig. 1 suggests that
in all probability the curve represents the superimposed properties cf at
least two enzymes, which may be present in the diastase.
The reactions which take place during mashing are numerous, and
still very obscure. During the initial stages of the process the complex
starchy bodies arc acted upon by the diastase and the molecules broken
down to the simpler ones of soluble starch. Windisch, Dietrich and
Beyer {Wochcnschr. Brau., 1923,40, 49, 55, 61, 67 ; this Journ., 1923,
29,331) have worked out a method for determining the starch-liquifying,
power as distinct from the saccharifying power of malt diastase.
They found that the optimum activity coincided with pa 5 f03, which is
quite near the optimum for saccharification. Investigations on different
malts showed that the relative liquifying and saccharifying powers vary
considerably, and it is therefore unlikely that the two actions are the
work of one enzyme. Ohlsson {Gomptes rend. Soc. Biol., 1922, 87,
1183; this Journ., 1923, 29, 481) recognises the existence of two
enzymes in malt amylase, which he calls doxtrinogenase and
saccharogenase. The former is destroyed if the acidity of the malt
extract is adjusted to pa = 4, and the latter when the medium shows a
reaction of pa = 10. The dextrinogenase converts the starch only to
dextrin, whilst saccharogenase hydrolyses starch or dextrin to maltose.
During the extraction of ground malt with water, there is a gradual
increase of acidity in the latter. Only a very small percentage of this
302 HAGUES : THE EFFECT OF HYDROGEN IONS, ETC.

is due to the preformed acid in the grain, the greater part being pro
duced by the action of a group of enzymes known as phosphatases. They
attack the complex insoluble organic phosphates and break them down
into organic and inorganic phosphates. Northrop and Nelson (Brewing
Trade Review, 1921, Feb.) have been successful in isolating from starch
one of these complex phosphates containing 4-9 per cent, of phosphorus
in the organic state. The temperature most favourable to the phos
phatases, according to Adler {Biochem. Zeilschr., 1915, 70, 1), is 58° C,
and their action is comparatively vigorous between 42° and 60° C.
Graphs I and II (Fig. 3) illustrate the effect of' temperature on the
percentage " Total dissolved phosphoric acid," and " dissolved inorganic
phosphoric acid " respectively.
Adler attributes the action to at least two phosphatases, one of which
acts on the insoluble complex phosphates splitting them up into simpler

Fig. 3.

soluble organic phosphates, whereas the other influences the formation

of inorganic phosphates. His results indicate that the process takes
place in stages. The enzyme which produces the organic phosphates
ceases to have any effect after about 5 hours, whereas the one forming
inorganic phosphates is appreciably active at the end of 14 hours.
The concentration of the solution affects the action of the phosphatases
to a certain degree ; a high viscosity and the presence of the inorganic
phosphates produced by the reaction have a harmful influence. An
extract made from 1 part of malt to 20 parts of water was found to give
the best results. By far the most important controlling factor,
however, is the hydrion concentration of the medium.
At a pH value of 5-4 the whole of the phosphoric acid of the
malt is brought into solution, and of this, 93 per cent, is in the form of
 HAOUES : THE EFFECT OF HYDROGEN IONS, ETC. 203

inorganic phosphates. When the />„ value of the medium is 2, phytin,

an organic phosphorus compound, is dissolved out of the malt. The
phosphatases are secretion enzymes, and act extraccllularly.
The presence of phosphoric acid in wort is of extreme importance.
It exists in solution as a mixture of primary and secondary potassium
phosphates, which has the power of controlling the free acidity of the
medium and of keeping the hydrion concentration at a comparatively
constant value. The mixture thus acts as a buffer, and it usually
happens that the hydrion concentration is maintained at the constant
value whioh is the optimum for the action of the enzymes. Other
substances which assist in this buffer action are the amino acids,

t
IOC

\ i 3 is « ' „ •

Fig. 4.

which are formed as decomposition products of the proteins of the

malt. This degradation is known as proteolysis, and is the result of the
action of the proteolytic enzymes of the malt.
Lundin {Biochem. Zeilschr., 1922, 131, 193; this Journ., 1922,
28, 941) has determined the rate of autolysis of the malt proteins
by estimating the rate at which amino nitrogen is produced—that
is, by the rate of hydrolysis of the peptide group (—CO. NH—).
This was done by Fonnol titration, the total nitrogen of the malt
flour being estimated by Kjeldahl's method. He carried out his
experiments at 110° F., a temperature favourable to the proteolytic
 304 HAG UBS : THE EFFECT OF HYDROGEN IONS, ETC.

enzymes, but not to diastase. His results indicate that the optimum
pa value for these enzymes is 4-4—4-5, thus confirming the observations
of Fernbach and Hubert {Compt. rend., 1900, 130, 1783; 131, 293)
that the optimum acidity is the same for the amylolytic and proteolytic
actions of malt extract.
According to Schjerning, the various reactions which take place in
the autolysis of the original protein matter, may be represented by the
following scheme.
Insoluble Albumin II Albumin I v „ ,
ProWn (EdKilln) (Uuooeln) ~^~ Peptones->-nnlmo add* ■§». ammonia.
■

Sjluble albuminoids.

The principal enzymes, or groups of enzymes, effecting'this change

are peptase and tryptase. The action of the peptase is to split the

Fig. 5.

leucosin into substances such as denuclcin, albuiuoses and peptone,

and tryptase is the agent producing animo acids and ammonia.
Further experiments by Lundin were carried out investigating the
composition of the autolysis mixture at varying intervals of time. He
determined:—
(a) The total nitrogen by Kjeldahl's method.
(6) Albumin nitrogen.
(c) Ammo nitrogen and ammonia by Formol titration.
(d) Ammoniacal Nitrogen by Folin's method.
His results for these estimations at the optimum j>n value of 4-4 are
summarised in the following graph.
 31 AGUES : THE EFFECT OF HYDROGEN IONS, ETC. 305

By considering the alteration of position of the four graphs with varia

tion of the pa value of the medium, he comes to the conclusion that it is
the peptase which acts in acid solution, because the amount of albumin
nitrogen remains almost constant in a netural medium, whereas in the
presence of acid it rapidly diminishes. The action of tryptase, however,
is favoured by neutral solutions, for the peptide nitrogen gives the reverse
results. At the pa value of 4 • 4 both enzymes act with the same intensity.
Lundin finds that the optimum acidity for the action of peptase is pa

So
TOTAL NITRCOSN
t

7o

60-

SO

40

30

20

10

40 80 120 ISO 200

autolysis time in hours.

Fig. 6.

3-7—4-3 in the case of kilned malt, and 3-2 in the case of green malt.
Tiypsin has its region of maximum action between pu 6*2—6-4, and
the optimum pu value for autolysis is 4 -4, which is intermediate between
those for the two enzymes.
The presence of cmulsin and lipase in malt is suggested by Van Laer
(Gomples rend. Soc. Bid., 1921, 84, 471, 473 ; this Journ., 1921,
27, 321). His evidence is based on the comparison of the amounts
of reducing sugar formed in a malt extract, treated with 1 per cent.
306 HAGUES : THE EFFECT OF HYDROGEN IONS, ETC.

of ainygdalin and a little toluene for eight days, and in a blank

experiment containing no amygdalin. Analogous experiments were
done, using esters instead of amygdalin, to prove the presence of lipase.
He finds that the optimum pB value for both these enzymes is 4-4.
Finally, to this already long list of enzymes to be found in malt, he adds
several others, namely, invertase, phytasc, cytase, maltose, trehalase,
glycogenase and pectinasc.
Turning to the practical side of the brewing industry; great advances
apparently have been made recently on the Continent, whereby the
extract from the mash, as carried out by the decoction method,
has been substantially increased. Chabot (loc. cit.) has found that
normally an increase of 6—8 per cent, may be obtained, and under
exceptional circumstances as much as 10 per cent. The method is to
determine the alkalinity of the mash to methyl orange, and add acid
so that half to three-quarters of the alkalinity is neutralised. The
mash is also allowed to peptonise for a longer time (four hours).
Although the usual pa value of a mash is acid to absolute neutrality
(Ph 7), yet it is alkaline to methyl-orange neutrality (4*4). It will be
noticed that an acidity of pH = 4-4 is optimum for most of the malt
enzymes, but nevertheless it is not the optimum medium for the produc
tion of maximum extract. The reason is that at methyl-orange
neutrality saccharification becomes quite difficult. At acidities slightly
higher than p,t 4-4, saccharification is entirely inhibited. The optimum
pu for maximum extract, according to Chabot, is about 5-5. By
analysing the extract, Van Laer (this Journ., 1923, 29, 202) finds that
the increase is due. one-third to nitrogenous bodies, and two-thirds to
sugars obtained from starch cells embedded in nitrogenous substances,
which yield only to proteolysis. Thus all the increased extract is not
formed at the expense of nitrogenous bodies, and the stability of the
beer is not affected. Chabot favours " acidification and peptonisation,"
not only because of the increased yield of extract, but also for the
reason that it stabilises and regulates the free acidity by bringing into
solution amino. acids, which act as buffer substances. Van Laer
emphasises the fact that although the greater free acidity in the beer
does not make it absolutely immune from infection, the resisting power
is increased. Another advantage of this greater acidity in the wort
(Petit Brasserie a Malterie, 1923, 13, 65; this Journ., 1923, 29,
574) is that a better " break" takes place in the copper. In
several French breweries it has been found (Petit ibid, 1921, 10,
321: this Journ., 1921, 27, 130) that if acid is added to the
 HAGUE8 : THE EFFECT OF HYDROGEN IONS, ETC. 307

mashing water to neutralise its alkalinity, difficult saccharificatiou,

and excessive bitterness, which is greatly influenced by presence of
alkaline salts in the water, are overcome. However, the science of
pu adjustment in the initial stages of brewing seems to be only in its
infancy, for there arc many difficulties which arise in consequence of
this treatment. Cauwenberge (Petit Joum. du Brass., 1922, 30, 254,
486) finds that the " acidification and peptonisation " process leads
to filtration trouble by producing a viscous layer of proteins; and
according to Godart {ibid, 1923, 13, 58; this Journ., 1923, 29, 574),
the method is useful for poor malts, but when dealing with well
modified malts the disadvantages outweigh the advantages. The
following table of some typical results of Chabot and Van Laer
(Bull. Soc. Chim. Beige, 1921, 30, 253 ; this Journ., 1922, 28, 15)
illustrates the effect of acidifying the mash, and indicates the
principles on which this new method is based.

Alkalinity of the Malt = -274 grm.

Time of
Method employed. Extract. [oh. Saccharifi-
cation.

mms.

1. Conventional Method 07-8(1 1151) 40

2. Ditto (line grinding) 71-28 113-0
3. Ditto -f 4 hours iwjitonisntion 72-1M 105-4
4. Ditto -f 0-7 c.cs. H,SO4 (X) 73-08 102-3
5. Ditto, ditto + 1 -4 c.cs. ditto 74-20 100-1
0. Ditto, ditto + 2-1 ecu. ditto 75-00 1)8
75-08 07-0 45
7. Ditto, ditto + 2-8 c.cs. ditto
75-70 100-7 75
8. Ditt/', ditto 4- 3-5 c.cs. ditto

We have seen that the dissolved solids present in brewing waters

have a marked effect on the pa value of the wort. This influence is
due to the chemical action on the mixture of substances which produce
the "buffer" action. According to Windisch and Goldacker
(Wochenschr. Brau., 1919, 36, 1, 12, 19, 30, 37, 46, 52, 62, 66 ; this
Journ., 1919,25, 275), the suits which pass into solution during mashing
are chiefly the phosphates and organic acid salts of potassium, mag
nesium and calcium. If a solution of potassium dihydrogen phosphate,
as a typical substance, is taken and titrated with a caustic potash solution
of about the same strength, the variation of the pu value of the phosphate
solution i's influenced as is indicated in the diagram.
 308 HAGUES : THE EFFECT OF HYDROGEN IONS, ETC.

It will be noted that one portion of the curve (about pa 7) is practically

a straight line, and that the pu value in this region alters only slightly on
the addition of acid or alkali. This constitutes the " buffer " effect of
a mixture of the primary and secondary phosphates of potassium, and
the efficiency of this buffer action depends on the relative amounts of the
two substances in the mixture. The buffer action of wort may be taken
to be analogous to this, but the effect will not be so sharply defined, and
will extend over a wider range of pu value, since many other buffer sub
stances arc present.

The effect of alkaline substances, such as calcium, carbonate, in brew

ing water will be to.neutralise a considerable quantity of the more
acidic constituents of the buffer mixture, thus lowering the free acidity

KOH TmATion

Fig. 7.

of the mash. Potassium dihydrogen phosphate may be taken as a typical

acid constituent, and the reactions which occur may be represented,
according to Windisch (Wochenschr. Brau., 1919, 36, 9, 15 ; this
Journ., 1919, 25, 214), by the two equations :—
4KH2PO4 + 3CoC03 = Cas(PO4)2 -f 2K2HPO4 + 3H.CO.,
2KH2PO4 + C«C03 = CaHPO4 + K2HPO4 + HoCO^
calcium carbonate thus lowers the free acidity by converting some of the
primary phosphate into secondary.
On the other hand, the presence of gypsum in the mashing water
has the reverse effect; namely, of lowering the ptt value or increasing
the free acidity. In the reaction which takes place calcium phosphate is
 HAGUES : THE EFFECT OF HYDROGEN IONS, ETC. 309

precipitated, whereby some of the secondary phosphate is changed

into primary.
4K2HPOj + 3C«SO4 = C«a(PO4)2 + 2KH2PO4 -f- 3K2SO4.

Similar equations maybe written down expressing the reactions taking

place with the organic buffer substances. Whilst an excess of gypsum is
detrimental to the final beer, producing a bitter taste, due to the forma
tion of magnesium sulphate, a small amount is advantageous. It
precipitates a certain amount of phosphoric acid as calcium phosphate,
which is removed in the hop-back, and which would otherwise exert
an excessive restraining influence on the free acidity, preventing the
latter from attaining the optimum pu value for the yeast. As a rule,
carbonates in brewing water are harmful because they lower the hydrion
concentration of the wort and give rise to conditions favourable to
the subsequent formation of the potassium salts of tannin and phloba-
phene. These substances, which decompose very slowly in the more acid
medium of the beer, give rise to free tannin and phlobaphene. The
latter combine with protein matter and produce a protein haze which
often separates long after the beer has been made.
The pH of a mash is approximately 5-5, and during the sterilisation
of the wort in the boiling stage the free acidity is further increased,
due to the concentration of the substances already present and to
the extraction of others from the hops. It so happens that this slight
increase of free acidity is favourable to protein coagulation, the optimum
pa value for the " break " being 5-2 (Lucre, Zeilschr. ges. Brau. 1920,
43, 51, 58; this Journ., 1920, 26, 385). The tannin dissolved from
the hops also assists in this precipitation.

• * * * *

Alcoholic fermentation has been known since the first historical

accounts of man were recorded, and the process has been studied more
than any other chemical reaction. The products of the action of
yeast or glucose arc numerous and varied. Pasteur found that succinic
acid, glycerol and fusel oil invariably accompanied the process, and
more recently acetic, formic and other acids, together with various
aldehydes and esters have been detected. By using pressed yeast
juice, it was found that many of these substances were decomposition
products of the cell walls. Although the yield of some of the other by
products is insignificantly small under normal conditions, yet it has
been shown by Neubcrg and his co-workers that by adjusting the
conditions of the fermentation, the yield of these substances, which
310 HAOUES : THE EFFECT OF HYDROGEN IONS, ETC.

above-certain concentrations are detrimental to beer, may be increased

in some instances so as to become the principal products of the process.
Also, as beer is subject to infection of bacteria and wild yeasts from the
atmosphere, the conditions under which fermentation takes place are
therefore of great importance to the brewer, and of these the hydrion
concentration is one of the principal factors. Thus Arzberger, Peterson
and Fred (Journ. Biol. Chem. 1920, 44, 465 ; this Journ., 1920,27, 44)
found that the optimum reaction for the production of acetone by
Bacillus aceto elhylicum is an acidity of pH 5-8—6-0, whilst a slightly
alkaline medium gives rise to large quantities of volatile acids. In
general, it may be said that the ptt value favourable to yeast is
unfavourable to bacteria and wild yeasts, although not totally
inhibitive; and that as the yeast is present in relatively pre
ponderating quantities and tends to produce its own optimum free
acidity, it greatly reduces the activity of the foreign ferments and
bacteria. The resulta of Van Laer and Merton (Comples rend. Soc.
BioL, 1922,87, 990) illustrate this fostering action of the hydrogen ion.
too

8o

60.

40

a©.

W'«-O YtAST

5-5
Pm
Fig. 8.

They found that the optimum reaction for reproduction of Saccharo-

bacittus pastorianus and Bac. viscosus was pn 6-8 in hopped wort and
pH 5-5 in unhoppcd wort. The optimum pn value for yeast is usually
much more acid than those of wild yeasta and bacteria, und for ordinary
brewers' yeast is situated between 4-76—4-46. Pn 4-5 is usually taken
as the most representative value (Emslandcr, Zeilschr. ges. Brau.,
1919, 42, 127, 135 ; this Joum., 1919, 25, 371). In the brewery, the
yeast quickly attains its optimum free acidity, Luers (ibid, 1914, 37,
 IIAGUES : THE EFFECT OF HYDROGEN ION8, ETC. 311

79 ; this Journ., 1914, 20, 221) has shown, by fermenting sugar solu
tions of different hydrion concentrations, with and without nutrient salts,
that the same pu value was finally attained. With brewery worts this
was not always the case, and he suggests that the phenomenon is
possibly due to the great buffer action of the wort, and to adsorption of
hydrogen ions by the colloids, thus preventing the pa value from ever
reaching the optimum. This property of producing its own favourable
hydrion concentration does not depend on the living state of the yeast,
since free zymasc behaves similarly. Emslander finds that all enzymes
and yeasts have an analogous action, but, contrary to the above sugges
tion of Luere, he also states that the pa value for the optimum reaction
of yeast varies considerably with the different nutrient solutions. For
worts which are brewed from carbonate waters the pu optimum is
4-5, and for those from gypseous waters 4-2. Emslandcr's results
arc supported by the observations of Van Laer (Petit Journ. du Brass.,
1922, 30, 887; this Journ., 1922, 28, 907) who has shown that the
optimum hydrion concentration for yeast development depends not
only on the chemical constitution and nutritive values of the medium,
but also on the presence or absence of antiseptics, such as hops. His
method was to seed different culture media with equal quantities of a
pure brewery yeast, and to count the number of cells per cc. of solution
after several days' fermentation. In this way he found that the
optimum pu values for Pasteur mineral (a liquid), unhopped wort (50 per
cent, malt -f- 50 per cent, raw grain) and hopped wort were 6*8,4-6 and
6-0 respectively, and that the yeast could support more acidity the
more nutritive the medium. According to Fodor (Fermentforsch., 1920,
3, 193 ; this Journ., 1920, 26, 599) the optimum pu value depends on
the' individual properties of the ferment colloids and the amphoteric
substrate, on their mutual relationships, and in particular on the
difference in acidic nature between colloid and substrate. This influence
of the composition of the medium undoubtedly accounts for the various
values for the optimum hydrion concentration reported by different
investigators. Thus by measuring the rate of evolution of carbon
dioxide, Euler and Heintze {Arkiv fur Kemi Mineralogi och geologi,
1917, 7, 5, 21) studied the fermentation of cane sugar and glucose by
yeast at 28° C. in the presence of sodium phosphate and hydrochloric
acid. They carried out experiments with and without the use of nutrient
nitrogenous substances, and they give the pa value as 5. Similar
investigations were made by Euler and Emberg (Zeilschr.fur Bid., 1919,
69, 349 ; this Journ., 1919, 25, 314) using phosphoric acid instead of
 312 HAGOES : THE EFFECT OF HYDROGEN IONS, ETC.

hydrochloric acid to produce the required free acidity, and their value
is the same (pH5), but the limits of pa for rapid action are more
narrow, as is shown by the graphs. Svanberg (Zeitschr. tech. Bid., 1920,
8, 1) gives the pu values for the optimum growth of yeasts, whether
grown in beer wort or solutions of ammonium salts with phosphates,
as : top yeast, pu 3-6 ; bottom yeast, pa 4-6. The optimum tempera
ture for reproduction, according to Zikes (Zenlralb. Bait. II, 1919,49,
353 ; this Journ., 1920, 26, 530) is 25° C.
It is well known that the presence of phosphates in wort contributes
to the efficiency of the alcoholic fermenting power of yeast. Harden
and Young {Proc. Roy. Soc. B., 1908, 80, 299 ; this Journ., 1908, 14,
547) found that the addition of a phosphate to a fermenting mixture
of glucose and yeast juice not only produced a temporary acceleration
00. COj Relative
per boar. reaction
velocity.

El'LER AND HeINTZ. ElLKR AND ElIBEUG.

Fig. 9. Fio. 10.

in the rate of fermentation, but also increased the total fermentation,

and it seems probable that the presence of phosphate is necessary to the
reaction. They explain that the role played by the phosphate in the
process is to form an intermediate hexoscphosphate compound
according to the equation :—
2C8H12O0+2R2HPO4 = 2C02+2CoH00+CaH100.1(P04R2)2+2H20.
This view has recently been confirmed experimentally by Euler and
Nordlund (Zeitschr. Physiol. Ghem., 1921,116, 229), who found that the
optimum pa value for the formation of fructose diphosphatc by a
bottom fermentation yeast was 6-2—6-6. This value was approxi
mately the optimum for phosphate formation by all sugars. According
to Harden and Young, the presence of a co-ferment is also necessary
to this reaction.
 HAGUE3 : THE EFFECT OF HYDROGEN IONS, ETC. 313

The next stage is the hydrolysis of the hexosephosphate.

C6H10O4(PO4R2)2 + 2H2O = C0H,.,O6 + 2R,HPO4.

The rate of fermentation is determined by the rate of the latter

reaction, which is the slower of the two. Eulcr also favours the view
that a co-enzyme is necessary in these reactions, since alcoholic fer
mentation is greatly accelerated by activators (Zeitschr. tech. Biol.,
1919, 7, 155). The activation of dried yeast by a co-enzyme is almost
independent of the acidity of the solution in the region of pu 3—7.
Euler and Tholin {Zeitschr. physiol. Chcm., 1916,97, 269 ; this Journ.,
1916, 22, 501), working with both dried and living yeast, found that
the rate of evolution of carbon dioxide was greatly accelerated in acid
solution (pa 4 • 5) by the addition of potassium phosphate, but in alkaline
solution (j>H 8) the phosphate retarded the production of the gas. ■
New light has been thrown on the subject of fermentation by Warden
(Amer. Journ. Physiol, 1921, 57, 454; this Journ., 1922, 28, 742),
who has been able to produce an artificial zymasc from the fats of
yeast. Thus a mixture of glucose, a moist fibrin and the sodium
salts of complex fat acids isolated from yeast exhibited a true fer
mentation with the evolution of carbon dioxide. The fat acids could
be replaced in the mixture by oleic acid or linolcic acid or almost any
insaturated fatty acid. The optimum temperature for the action was
15—25° C, and the fermentation would not take place outside the
limits of pl{ 6*5 and 8-5. In addition to glucose this system would
ferment cane sugar, maltose and also lactose, but not laevulose.
Warden believes that alcoholic fermentation is produced by catalysis on
the surfaces of the yeast cells, or on the colloidal surfaces of the yeast
juice ; also that it is purely a phenomenon of surface action, and
that artificial surfaces formed by complex mixtures similar to those in
the yeast cell can produce fermentation.
There are many enzymes which have already been detected in yeast,
and which act conjointly to produce beer from wort. Of these, invertase,
maltase and zymase have long been recognised. Invcrtase and maltase
convert the disaccharoses, sucrose and maltose into glucose and other
monosaccharoses, and the sugars are then in a form fermentable by the
zymase. Invertase, which is the most stable of these enzymes, has
been studied extensively, and interesting investigations on the effect of
hydrion concentration on its activity have been made by Euler and
Emberg (Zeitschr. Biol., 1919, 69, 349; this Journ., 1919, 25, 314).
They fermented a solution of cane sugar and disodium phosphate with
x
314 HAGUES : THE EFFECT OF HYDROGEN IONS, ETC.

fresh yeast, and determined the inversion constant by observing the

alteration of the rotation of the solution from time to time. It was
found that variations of pn value had the same effect on the invertase
in the living yeast as on the enzyme in the free state. Their results are
given in the graph:—

Fraction
Unxlm.
Action.

The optimum hydrion concentration appears to be about pa 4*5. In

other experiments, they found that the maltase activity of living yeast
was completely inhibited by a very small concentration of hydroxyl ions

The action of the proteolytic enzymes of yeast has been studied by

Dernby (Biochem. Zeitschr., 81, 107) by methods similar to those of
Lundin (see page 303). He finds that the optimum pn for autolysis is
Pa6"0—6-2 and that the limits between which autolysis takes place
are pB 7—i. The amounts of the various nitrogenous bodies present at
different times during an autolysis are given in Fig. 12.

Fiu. 12.
 IIAGUES I THE EFFECT OF HYDROGEN IONS, KTO. 315

He has proved that the autolysiB by yeast is due to at least three

proteolytic enzymes, which he separated from plasmolysed yeast by
dialysis:
(1) Yeast pepsin, which degrades proteins to peptones, and the
optimum action of which takes place in a medium of j>H 4-45.
(2) Yeast tryptase, which docs not act on yeast proteins, but which
can degrade bodies such as acid albumin, gelatin and casein into peptides
and amino acids at an optimum reaction of pB 7.
(3) Yeast ereptase, which decomposes peptones and polypeptidcs into
amino acids, and has an optimum pB value of 7-8 . His results are
represented diagrammatically in the following manner:—

Fig. 13.

The optimum action of autolysis thus takes place at an average value of

those of the separate enzymes.
During the fermentation of wort, the yeast cells bud and reproduce,
and at the same time that the yeast is bringing about the alcoholic
conversion of the sugars, it is living on the assimilable nitrogen and other
nutritive substances present. Hence the increase of nitrogenous tissue
due to the formation .of new cells takes place at the expense of the
nitrogenous bodies of the wort. Nevertheless, there is from 2J to 3 times
as much assimilable nitrogen in wort as is required, and there is always
ample, however low the nitrogen content may be (Hulton, this Journ.,
1922, 28, 4). From this fact, Fembach (Ann. Brass, el Dist., 1923,
21, 289; this Joum., 1923, 29, 576) contends that the removal of
nitrogen by yeast is normally insufficient to ensure stability of the
beer, for according to Emslander (Zeitschr. ges. Brau., 1914, 37, 164 ;
this Journ., 1914, 20, 555) the greater the amount of protein present
in beer, the less is the stability, and the less the hydrion concentration.
X2
 316 HAOUES : THE EFFECT OF HYDROGEN IONS, ETC.

Tim dependence of hydrion concentration on protein content is

exemplified by Emslander's method of the electrometric titration of
beer. When beer is titrated with alkali, the change of pa value is pro
portional to the volume of alkali added within certain limits (4-4—9).
These limits correspond with the extremities of the straight line
portion of the buffer curve (cf. fig. 7). The electrometric titration
graph is thus a straight line.

7. -7-O7

6.

S-

4 6 I* 16 cc. alkali.

Fig. 14.

If dialyscd protein is added to the beer, a different straight line graph

is obtained which starts at a higher pH value, but the point where these
straight lines cut the line of absolute neutrality (pa 7-07) is independent
of the protein added. They all mutually meet the 7-07 line in one and
the same point at which the total acidity is just neutralised. The angle
made by the pu titration line to the horizontal is known as the " titra
tion angle " ; and the less the angle the greater is the amount of protein
present in proportion to the total content of the beer, the greater is the
palatefulness of the beer, and the less its stability. Incidentally, the
total acidity of the beer may be determined electrometrically by this
method. Thus if the pa of a beer is 4-48, and after the addition of
N
10 cc. of — alkali per 100 cc. it changes to 6-03, the amount of alkali
required to produce absolute neutrality (pa 7-07) is
/ 7-07—4-48

Since the stablity of a beer decreases with increasing nitrogen content,

it follows that beers produced by the " acidification and peptonisation "
process will not be as stable as those brewed in the normal way, because
they will contain a greater excess of nitrogen unassimilated by the
yeast. For this reason it is doubtful if this new method is really more
 HAGUES : THE EFFECT OF HYDROGEN IONS, ETC. 317

advantageous than the one in general use, except when large proportions
of diluents deficient in protein matter, such as flaked maize, arc added
to the malt (this Journ., 1923, 29, 576).
Another factor which is influenced by the hydrion concentration is
the production of oxalic acid. According to Geys (Zeilschr. ges. Brau.,
1922,2, 9 ; this Journ., 1922,28, 417), the more frequent appearance
of calcium oxalate in beer recovered from yeast than in beer from
the vats, is due to the lower acidity of the former. In a particular
case vat beer was found to have a pa of 4-64 as compared with
pn 6-54 for beer from the yeast. Thus for almost all the maladies of
beer there seems to be one diagnosis; namely, lack of free acidity
during the process.
Finally, we must consider the head retention and the taste of the beer.
Berry (this Journ., 1909, 15, 391) tried the effect of varying acidities
on the time of duration of the " head " of samples of the same beer.
He found that as a beer increases in acidity the carbon dioxide bubbles
become larger, giving a coarser head. With -05—1 per cent, of acid
there was no decrease in the head retention, but as soon as this value
was exceeded the retentivity fell until the limit of -3 per cent, was
attained. According to Rohland {Wochensch. Brau., 1923, 40, 23, 27)
only those liquids which contain colloids, together with free acidity
or alkalinity, will give a strong permanent head. These conditions
are present in wort and beer, but not in aerated waters.
Many and futile have been the experiments carried out on the subject
of taste. The opinions of different people on the taste of buffer solutions
are by no means alike. Harvey (S.A.S.G., 1920, 42 (1), 712) has pub
lished an interesting paper in which a serious attempt is made to illustrate
graphically how taste varies with the free and total acidities to the
solution. There is no doubt that beers of high protein content have
the greatest palatefulness, but although the hydrion concentration
contributes to the sensation produced on the palate, taste is not tts yet
a quantitative science, and its dimensions as a mathematical quantity
are still unknown.
*****

I should like to thank Messrs. W. H. Bird, J. S. Ford, and D. McCand-

lish for their valuable assistance in the collection of the subject matter
of this paper, and for helpful suggestions.
Tetley's Brewery,
Leeds.
28th F&ruary, 1924.