IMPROVEMENTS RELATING TO THE PREPARATION OF FERMENTS
Year: 1885

page_text: I, MORITZ BLUMENTHAL, Doctor of Philosophy, subject of the King of Prussia,
residing at Grünau near Berlin, Prussia, German Empire do hereby declare the
nature of my invention for "IMPROVEMENTS RELATING TO THE PREPARATION OF
FERMENTS," to be as follows:-
This invention relates to the separation of pure ferments from the organisms or
substances containing the same.
There are at the present time few methods of separating ferments from the
materials or organisms enclosing the same. This invention has for its object to
enable ferments to be isolated from the glandular organs of the organism and other
substances surrounding the same, in a pure state, as precipitates which are soluble
in water; and also to enable different ferments which occur side by side and have
an injurious action upon one another to be separated; for instance for separating
chymosin (from the fourth stomach of ruminating animals) and pepsin, pancreatin
and diastase.
There exist in commerce several forms of animal ferments, such as the extracts
from runnet and pepsin, obtained by various methods. The former (the ordinary
commercial extract from the maws of calves which is used in the manufacture of
sweet-milk cheese) produce a yellow-brown liquid which is obtained by treating the
maws of calves with water and adding preserving media.
This extract has especially two disadvantages for practical use.
First It contains, besides the active ferment from the runnet, various other slimy
albuminous parts of the maw in solution, which parts cause the colour of the
extract to vary from yellow to brown, and at the same time give it an unpleasant
smell which in use is imparted to the cheese to be produced.
Second: It contains a comparatively large quantity of preserving media (boracic
acid and the like) as not only the ferment existing merely in minimum quantities
in the extract must be preserved, but also the slimy extract substances of the
extract from runnet which are far anore decomposable and exist in a predominant
quantity. This increased proportion of preserving media, however acts injuriously
upon the ripening process of the cheese and retards the same, consequently the
extract as heretofore obtained, for instance for the manufacture of Emmenthal cheese
cannot be used.

page_text: The pepsins known in commerce are extraordinarily dissimilar in condition and
action. Many are entirely inactive, and hitherto pepsin has never been obtained
pure, and from the product obtained solutions or mixtures, whose proportion of
pure ferment are known cannot be obtained. The methods of separating the
ferment of the gastric juice, for instance, from the extract of the maws of pigs are
based, on the one hand, upon a precipitability of the ferment by alcohol, on the
other hand upon a precipitability with acids; finally it is stated by Scheffer that
by solutions of common salt the pepsin is precipitated from its solutions. These
methods of separation have the defect that numerous indifferent substances are
precipitated with the ferment causing material injury to the fermenting power;
and the precipitation, for instance by common salt solutions takes place so imper-
fectly that the remaining lyes show considerable digesting power. The latter
process is therefore commercially useless, because the ferment is only partially
isolated and is therefore too costly.
By the present invention chymosin and pepsin, as well as the ferment from the
pancreatic gland in the form of colourless gelatin, precipitate so completely from
the extracts of the glandular organs that the mother-lyes of these ferments, which
remain after filtration possess no fermenting power, and the ferments themselves
produce in water soluble, colourless, odourless, and tasteless substances, further use
of which can be made with perfect safety.
My process is based on the following observations and determinations:
Chymosin, pepsin and pancreatin are soluble in feeble solutions of common salt.
These ferments also dissolve in solutions of common salt which are kept slightly
acid, but in various degrees. Acid solutions of common salt take up only few slimy
substances from organs containing ferment. Pure solutions of common salt dissolve
considerable quantities of slime besides the ferments. From solutions of common
salt which are kept slightly acid, the ferments are separated as gelatinous white
sediments by introducing salts (in the form of powder and soluble in water) of the
alkalies or alkaline earths such as chloride of sodium, chloride of potassium, chloride
of magnesium, chloride of calcium and the like until supersaturation is reached and
a permanent precipitate of the addition of salt is formed, which sediments, if violent
agitation takes place during the separation, are so completely precipitated that the
remaining supersaturated lye scarcely possesses any traces of fermenting power..
Of these properties of the ferments, which I have proved by many experiments
to be correct, it has only through Soxhlet's investigations become known that in
the treatment of extracts from fourth stomachs with a salt solution, a slimy pre-
cipitate containing ferment is produced; the other properties, especially those
relating to the purification, have never been inquired into; It has not therefore
been known whether, and under what circumstances, the ferment contained in theso
slime substances retains or loses its efficiency when being dried, and whether it
remains active or not in the preservation.
What has been heretofore called dry runnet powder is nothing but the dried,
prepared mucous membrane of the fourth stomach, and under no circumstances a
ferment which is soluble in water and free from slime. This is evident from the
coefficients of action, inasmuch as the dry runnet powder shows an action of about
1: 300,000, whereas the pure ferment produced according to my method is active
in the degree of dilution of 1: 1000000.
According to the present invention the production of pure ferments from the
glandular organs is preceded by a preparatory production of raw solutions con-
taining ferment. This can be effected as follows (it being always understood that
by "salt" or "salt solutions" are meant the soluble salts of the alkalies or alkaline
earths such as chlorides, sulphates, nitrates, carbonates, phosphates and the like or
their solutions :-
a by treating the reduced glandular organs with weak salt solutions, preserving
media being added, such as boracic acid, borax, glycerine, thymot, ethereal oils or

page_text: b by treating with slightly acid liquids (mineral or organic acids), salt being
added.
c by treating with pure salt solutions, that is to say, without acids and preserving
media.
d by treating with comparatively strong salt solutions, adding quantities of com-
paratively strong acids, and after filtering, with pure salt solutions.
e by treating with glycerine with or without the addition of salt.
Of these preparatory processes those mentioned under c and d have not been
used at all for the production of solutions containing ferment whilst those mentioned
under ȧ. b. e. are already known. The further treatment of the extracts for isolating
the pure ferment is new. This treatment varies however according to the nature
of the preparatory treatment.
I have observed that aqueous solutions of raw ferments containing slime, which
are extremely difficult to filter and therefore offer great difficulties to a treatment
on an extensive scale with filtering apparatus, can be easily filtered, if the slimy
substances can be removed. This removal of the slimy substances can, however,
be carried out as stated by reason of the ferments and the slime precipitating under
different conditions in acids. If a slimy solution of ferment is acidified, the first
precipitate is nearly pure slime free from ferment, the ferment precipitating later.
Accordingly, by gradually adding acid according to experience conformably to the
degree of concentration, of the raw ferment solution, almost all slime can be
separated from the said solution, a little ferment being lost therein. The solution
of ferment which is free from slime can be very easily separated from the slime
precipitate by means of filter presses. A clear liquid is thus obtained, which when
supersaturated with common salt in the form of powder and then agitated and
allowed to stand for several days, permits the precipitation of ferment which is
soluble in water, colourless, free from slime and pure. The common salt forms
therein so to speak a bearer of the ferment and has, at the same time, a preserving
action. Instead of common salt chloride of potassiam, Glanber salt saltpetre,
sulphate of potash, sugar, or in fact any salt acting without injury, soluble in
water, and preserving the ferment, but otherwise unaffecting the latter, can
be used.
For the treatment of precipitates of raw ferment, which contain mixtures of
chymosin and pepsin, which are generally produced when extracts of fourth
stomachs containing both ferments are operated upon, a special method is required
for separating the pepsin from the chymosin. The pepsin dissolves with much
greater difficulty in salt solutions than chymosin, but is easily soluble in acids.
If therefore the extraction is effected with comparatively strong acid salt solutions,
the pepsin dissolves, but not the chymosin. In the same manner, as long as the
acid reaction of the solution of raw ferment is kept up in the precipitation of the
slime, the pepsin remains in solution. If the acid mother-lye filtered from the pre-
cipitate of chymosin obtained in a surplus of common salt is saturated with
carbonate of sodium, or with carbonate of calcium and phosphate of sodium, the
pepsin contained in the solution is obtained, which pepsin can be thrown down
with indifferent precipitates produced in the solution, such as phosphate of calcium
cholesterine, and the like. Upon these observations is based the process of further
treating the extracts of ferment from the glandular organs.
I The extracts obtained according to the preliminary treatment a, and b, are
strained and then filtered. To the filtrate is added an alkali or salt of alkaline
earths in the form of powder and soluble in water, preferably a chloride, until super-
saturation is reached, during continual agitation. The liquid is then left to stand for
several days, during which a precipitate containing slime is formed which contains
nearly all the raw ferment. This precipitation of raw ferment can be effected,
especially in the case of chymosin which is little soluble in acids, by the addition of
acids. The raw ferment obtained is further treated by first dissolving in water and
then acidifying the latter, thereby precipitating the slime and increasing the solu-
bility of the pepsin by the acidification. The solution which is freed from slime by
+

page_text: filtration is next mixed with common salt in the form of powder. By agitating the
liquid for a certain time and then allowing it to stand for several days, a solution
which is almost free from ferment is produced on the one hand and a precipitate of
ferment which is free from slime on the other hand. The acid solution is next
neutralized thereby permitting pepsin to precipitate. If supersaturation is effected
with carbonate of sodium, the pepsin can, after adding lime salts, be precipitated
mechanically with carbonates of lime.
II The pure salt solution obtained according to c is strained and acidified, so
that slime precipitates with a little ferinent. The precipitate can be easily filtered,
and the filtrate is then supersaturated with salt powder (salts of the alkalies or
alkaline earths which are soluble in water).
Under the same conditions as div I, ferment which is free from slime precipitates,
and, if obtained from runnet extracts, chymosin which is free from pepsin.
III The extract obtained according to div. d supplies first in the case of fourth
stomachs principally pepsin, in the salt solutions obtained after acid extraction
however almost pure, chymosine is obtained. The pepsin extracts are neutralized
and either precipitated by supersaturation with salt powder, or by the presence of
indifferent precipitates in the neutralized liquid. The solutions of chymosin are
treated further exactly in accordance with the process described in div. I.
IV The raw ferment can also be separated from the solutions of glycerine either
by comparatively strong acidification or by alcohol or by an excess of salt powder.
The purification of the raw ferment must then take place as described above.
The precipitates of ferment which are free from slime, as obtained in the manner
stated above are finally washed and pressed with a saturated solution of common
salt for the complete removal of the adherent acids and of the mother-lye. The
precipitates of slime containing little ferment can be used directly for runnet
extracts or runnet solutions or can after collection be converted into pure ferment.
The ferments which are pure and free from slime, as obtained with the above
methods of treatment, can be dried in a vacuum or at a low temperature, or they
can be brought into commerce in a dissolved form as pure, clear solutions of
ferment which are free from slime. In the drying it is well, for the purposes
of convenient quantitative determination, to rub amongst the ferment and adinix
therewith an indifferent material, such as common salt. If the ferment is to be
chemically pure for scientific purposes, this can be attained with facility by dialysing
the dry mixture of salt ferment.
Pancreatin is, similarly to chymosin, nearly insoluble. Also diastase can be
almost completely isolated from malt extracts by means of super-saturation
'with salt.
"
By " salt
is invariably meant in the preceding, if nothing is mentioned
particularly, a salt of the alkalies or alkaline earths which is soluble in water.
Dated this 4th day of May 1885
HASELTINE, LAKE & Co.,
For the Applicant.

page_text: I MORITZ BLUMENTHAL, of Grünau, Berlin, Prussia, German Empire, do hereby
declare the nature of my invention for "IMPROVEMENTS RELATING TO THE
PREPARATION OF FERMENTS " and in what manner the same is to be performed,
to be particularly described and ascertained in and by the following statement:-
This invention relates to the separation of pure ferments from the organisms or
substances containing the same.
There are at the present time few methods of separating ferments from the
materials or organisms enclosing the same. This invention has for its object to
enable ferments to be isolated from the glandular organs of the organism and other
substances surrounding the same, in a pure state, as precipitates which are soluble
in water, and also to enable different ferments which occur side by side and have
an injurious action upon one another to be separated, for instance chymosin (from
the fourth stomach of ruminating animals) and pepsin, pancreatin and diastase.
There exist in commerce several forms of animal ferments, such as the extract
from runnet and pepsin, obtained by various methods. The former (the ordinary
commercial extract from the naws of calves which is used in the manufacture of
sweet milk cheese) produces a yellow brown liquid which is obtained by treating
the maws of calves with water and adding, preserving media, such as boracic acid,
borax, glycerine, alcohol, and thymnol.
This extract has especially two disadvantages for practical use, first it contains,
besides the active feriment from the runnet, various other mucous albuminous parts
of the maw in solution, which parts cause the color of the extract to vary from
yellow to brown and at the same time give it an unpleasant smell which in use is
imparted to the cheese to be produced.
Second. It contains a comparatively large quantity of preserving media (boracic
acid and the like), as not only the ferment existing merely in minimum quantities
in the extract must be preserved, but also the mucous extract substances of the
extract from runnet which are far more decomposable and exist in a predominant
quantity. This increased proportion of preserving media however acts injuriously
upon the ripening process of the cheese and retards the same, consequently the
extract as heretofore obtained, for instance, for the manufacture of Emmenthal
cheese, cannot be used.
The pepsins known in commerce are extraordinarily dissimilar in condition and
action. Many are entirely inactive, and hitherto pepsin has never been obtained
pure, and thus from the product obtained solutions or mixtures, whose proportion
of pure ferment are known cannot be obtained. The methods of separating the
ferment of the gastric juice for instance from the extract of the maws of pigs are
based, on the one hand, upon a precipitability of the ferment by alcohol, and on the
other hand, upon a precipitability with acids; finally it is stated by Scheffer that
by solutions of common salt the pepsin is precipitated from its solutions. These
methods of separation have the defect that numerous indifferent substances are
precipitated with the ferment, causing material injury to the fermenting power;,

page_text: and the precipitation, for instance by common salt solutions, takes place so
imperfectly that the remaining lyes show considerable digesting power. The latter
process is therefore commercially useless, because the ferment is only partially
isolated and is therefore too costly.
By the present invention chymosin and pepsin as well as the ferment from the
pancreatic gland in the form of colourless gelatine, precipitate so completely from
the extracts of the glandular organs that the mother lyes of these ferments, which
remain after filtration possess no fermenting power, and the ferments themselves,
produce in water, soluble, colourless, odourless, and tasteless substances, further use
of which can be made with perfect safety.
My process is based on the following observations and determinations.
Chymosin, pepsin, and pancreatin are soluble in feeble solutions of common salts.
These ferments also dissolve in solutions of common salt, which are kept slightly
acid, but in various degrees. Acid solutions of common salt take up only few
mucous substances from organs containing ferment. Pure solutions of common salt
dissolve considerable quantities of slime besides the ferments. From solutions of
common salt which are kept slightly acid, the ferments are separated as gelatinous
white sediments by introducing salts (in the form of powder and soluble in water)
of the alkalies or alkaline earths such as chloride of sodium, chloride of potassium,
chloride of magnesium, chloride of calcium and the like, until supersaturation is
reached, and a permanent precipitate of the additional salt is formed, which
sediments, if violent agitation takes place during the separation, are so completely
precipitated that the remaining supersaturated lye scarcely possesses any traces of
fermenting power.
Of these properties of the ferments, which I have proved by many experiments
to be correct, it has only through Soxhlets investigations become known that in the
treatment of extracts from fourth stomachs with a salt solution, a mucous preci-
pitate containing ferment is produced; the other properties, especially those relating
to the purification have never been enquired into. It has not therefore been known
whether, and under what circumstances the ferment contained in the mucous sub-
stances retains or loses its efficiency when being dried, and whether it remains
active or not in the preservation.
What has been heretofore called dry runnet powder is nothing but the dried,
prepared mucous membrane of the fourth stomach, and under no circumstances a
ferment which is soluble in water and free from mucus. This is evident from the
coefficient of action, inasmuch as the dry runnet powder shows an action of about
1:300,000, whereas the pure ferment, produced according to my method is active
in the degree of dilution of 1: 1000,000.
According to the present invention the production of pure ferments from the
glandular organs is preceded by a preparatory production of raw solutions con-
taining ferment. This can be effected as follows (it being always understood that
by "salt" or "salt solutions" are meant the soluble salts or the alkalies or alkaline
earths, such as chlorides, sulphates, nitrates, carbonates, phosphates, and the like or
their solutions).
(a) by treating the reduced glandular organs with weak salt solutions, preserving
media being added, such as boracic acid, borax, glycerine, thymol, ethereal oils, or
alcohol.
(b) by treating with slightly acid liquids (mineral or organic acids) salt being
added.
(c) by treating with pure salt solutions, that is to say, without acids and
preserving media.
(d) by treating with comparatively strong salt solutions, adding quantities of
comparatively stronger acids, and, after filtering, with pure salt solutions.
(e) by treating with glycerine, with or without the addition of salt.
2
Of these preparatory processes those mentioned under (c) and (d) have not 5
been used at all for the production of solutions containing ferment, whilst those
mentiqued under (a) (b) (e) are already known. The further treatment of the

page_text: This treatment varies however
extracts for isolating the pure ferment is new.
according to the nature of the preparatory treatinent.
I have observed that aqueous solutions of raw ferments containing mucus, which
are extremely difficult to filter, and therefore offer great difficulties to a treatment
on an extensive scale with filtering apparatus can be easily filtered, if the mucous
substances can be removed. This removal of the mucous substances can however
be carried out as stated by reason of the ferments and the mucus precipitating
under different conditions in acids. If a mucous solution of ferment is acidified,
the first precipitate is nearly pure mucus free from ferment, the ferment precipi-
tating later.
Accordingly by gradually adding acid according to experience
conformably to the degree of concentration, of the raw ferment solution, almost all
mucus can be separated from the said solution, a little ferment being lost therein.
The solution of ferment which is free from mucus can be very easily separated
from the mucous precipitate by means of filter presses. A clear liquid is thus
obtained which when supersaturated with common salt in the form of powder, and
then agitated and allowed to stand for several days, permits the precipitation of
ferment which is soluble in water, colourless, free from mucus and pure. The
common salt forms therein, so to speak a bearer of the ferment and has at the
same time a preserving action. Instead of common salt chloride of potassium,
Glauber salt, saltpetre sulphate of potash, sugar, or in fact any salt, acting without
injury, soluble in water, and preserving the ferment, but otherwise unaffecting the
latter, can be used.
For the treatment of precipitates of raw ferment, which contain mixtures of
chymosin and pepsin, which are generally produced when extracts of fourth.
stomachs, containing both ferments are operated upon, a special method is required
for separating the pepsin from the chymosin. The pepsin dissolves with much
greater difficulty in salt solutions than chymosin, but is easily soluble in acids. If
therefore the extraction is effected with.comparatively strong acid salt solutions,
the pepsin dissolves, but not the chymosin. In the same manner, as long as the
acid reaction of the solution of raw ferment is kept up in the precipitation of the
mucus, the pepsin remains in solution. If the acid mother lye filtered from the
precipitate of chymosin obtained in a surplus of common salt is saturated with
carbonate of sodium or with carbonate of calcium and phosphate of sodium, the
pepsin contained in the solution is obtained, which pepsin can be thrown down
with indifferent precipitates produced in the solution, such as phosphate of
calcium, cholesterine, and the like. Upon these observations is based the process
of further treating the extracts of ferment from the glandular organs.
I. The extracts obtained according to the preliminary treatments (a) and (b) are
strained and then filtered. To the filtrate is added during continual agitation an
alkali or salt of alkaline earths in the form of powder and soluble in water,
preferably a chloride, until super-saturation is reached. The liquid is then left to
stand for several days, during which a precipitate containing mucus is formed
which contains nearly all the raw ferment. This precipitation of raw ferment can
be effected, especially in the case of chymosin, which is little soluble in acids, by
the addition of acids. The raw ferment obtained is further treated by first
dissolving in water and then acidifying the latter, thereby precipitating the mucus
and increasing the solubility of the pepsin by the acidification. To the solution
which is freed from mucus by filtration is next mixed common salt in the form of
powder. By agitating the liquid for a certain time, and then allowing it to stand
for several days, a solution which is almost free from ferment is produced on the
one hand and a precipitate of ferment which is free from mucus on the other hand.
The acid solution is next neutralized thereby permitting pepsin to precipitate. If
supersaturation is effected with carbonate of sodium, the pepsin can, after adding
lime salts, be precipitated mechanically with carbonates of lime.
II. The pure salt solution obtained according to (c) is strained and acidified, so
that mucus precipitates with a little ferment. The precipitate can be easily

page_text: filtered, and the filtrate is then supersaturated with salt powder (salts of the
alkalies or alkaline earths which are soluble in water). Under the same conditions
as div I, ferment which is free from mucus precipitates, and if obtained from
runnet extracts, chymosin which is free from pepsin.
III. The extract obtained according to (d) supplies first, in the case of fourth
stomachs, principally pepsin; in the salt solutions obtained after acid extraction
however, almost pure chymosin is obtained. The pepsin extracts are neutralized
and the pepsin is precipitated either by supersaturation with salt powder, or by
the presence of indifferent precipitates in the neutralized liquid. The solutions of
chymosin are treated further exactly in accordance with the process described in
div I.
IV. The raw ferment can also be separated from the solutions of glycerine,
either by comparatively strong acidification or by alcohol, or by an excess of salt
powder. The purification of the raw ferment must then take place as described
above. The precipitates of ferment which are free from mucus, and obtained in the
manner stated above, are finally washed and pressed with a saturated solution of
common salt for the complete removal of the adherent acids and of the mother
lye. The precipitates of mucus containing little ferment can be used directly for
runnet extracts or runnet solutions or can, after collection, be converted into pure
ferment..
The ferments which are pure and free from mucus, as obtained with the above
methods of treatment, can be dried in a vacuum or at a low temperature, or
they can be brought into commerce in a dissolved form as pure, clear solutions of
ferment which are free from mucus. In the drying it is well for the purposes of
convenient quantitative determination, to rub amongst the ferment and admix
therewith an indifferent material such as common salt. If the ferment is to be
chemically pure for scientific purposes this can be attained with facility by
dialysing the dry mixture of salt ferment.
Pancreatin is, similarly to chymosin, nearly insoluble. Also diastase can be
almost completely isolated from malt extracts by means of supersaturation with
salt.
"
By salt" is invariably meant in the preceding, if nothing is mentioned
particularly, a salt of the alkalies or alkaline earths which is soluble in water.
Having now particularly described and ascertained the nature of my said
invention and in what manner the same is to be performed, I declare that what
I claim is
•
First. The process hereinbefore described of separating pepsin, chymosin, pan-
creatin, and diastase from the organisins or substances, containing the saine, by
mixing with solutions of these ferments during violent agitation an excess of a salt
of an alkali or alkaline earth (especially common salt) which is soluble in water
and in the form of a powder, the mixture being then allowed to stand for several
days.
Second. The process of separating from the raw strained liquids containing the
ferment, the mucus and other impurities which render the said liquids difficult to
filter or incapable of filtration, by acidifying the same with mineral or strong
organic acids until the mucus is separated prior to the precipitation of the ferment,
in order to obtain a pure solution of ferment which can be easily filtered.
Third. The process of separating mixtures of pepsin and chymosin obtained
from extracts of runnet and solutions of the precipitate of raw ferment mixtures
obtained from runnet solutions, by first adding acids to the extract or solution in
order to separate the mucus, then saturating with the soluble salt of an alkali or
alkaline earth (especially common salt) for separating the chymosin, and finally
producing from the acid solution super-saturated with salt after neutralization a
precipitate of pepsin, or permitting the pepsin to separate by precipitation with
indifferent substances.
. 1:

page_text: Fourth. The process for separating mixtures of pepsin and chymosin obtained
from the sediment of raw ferment, by washing the mixtures with diluted acid
containing salt, the chymosin remaining behind.
Fifth. The treatment of raw ferments extracted from the glandular organs,
partly by acid or neutral salt solutions with or without preserving media; partly
by extraction of glycerine, raw precipitates being produced from them; partly by
salts; and partly by acids, the precipitates being further treated according to the
methods described under sections I, II, III IV.
Sixth. The purification of the feriments which contain mucus, or are free from it.
and are produced from an acid solution, by washing with the concentrated solution
of a salt of the alkalies or alkaline earths (especially common salt) which is soluble
in water, for the purpose of removing acids and adherent mother lyes.
Seventh. The chymosin produced in accordance with the methods described in
claiming clauses 1, 2, 3 and 4 and which is absolutely free from pepsin, is soluble,
clear and is free from mucus, in combination with an indifferent and preserving
substance (salt, sugar, or the like) in a dry or dissolved form.
Dis oo
P
1007
HASELTINE, LAKE & Co.,
For the Applicant.