Note: Descriptions are shown in the official language in which they were submitted.
,1 , :~ 1
The present invention relates to the recovery of proteins
from microorganisms, particularly to the obtaining of protein
having a reduced nucleic acid content from such materials.
I~ is known that the population of the world is rapidly
increasing and therefore an acute shortage of food is to be
expected in a few centuries. In many countries this situa-
tion already prevails, Protein deficiency constitutes the
main problem. ~an's daily protein requirement amo~nts to
approximately 70 to lO0 g. In underdeveloped countries this
protein requirement is covered mainly by vegetable foods. In
contrast to animal protein, however, vegetable protein contains
very little of the two essential amino acids, lysine and
methionine. I~ only vegetable protein is available, therefore,
severe deficiency diseases can occur. For this reason attempts
have been made for years to utilize microbial protein in
addition to animal and vegetable protein to cover the protein
needs of the world~population. Microbial protein has the
advantage that it has an amino acid composition similar to
that of animal protein. In a diet of microbial protein
there is no need to fear the occurrence of the deficiency
diseases which can arise in the case of an all-vegetable diet.
.
For decades attempts have been made to use microorgan-
isms for the production of foods for human beings and animals.
These minute forms of life have the advantage that they grow,
under favorable conditions, much more rapidly than plants or
animals. The following table illustrates this.
:
~ .
7777~
DOUBLING TIME FOR CERTAIN FORMS OF LIFE
Organisms Biomass Doubling Time
5 Yeasts and bacteria 20 - 120 minutes
Microorganisms such as
algae 1 - 48 hours
Grass 1 - 2 weeks
Chicks 2 - 4 weeks
Swine 4 - 6 weeks
Cattle 1 - 2 months
.
The rapid proliferation of microorganisms shows that
more protein can be obtained from them in 1 to 2 hours than
5 can be obtained from swine or cattle in 4 to 8 weeks.
However, this kind of protein production entails a
number of difficulties:
1. A very great technical investment (gigantic fer
menters) is required for the growth of microor-
ganisms;
2. Thq separation of the organisms from the culture
broths on the required scale presents technical
difficulties;
3. Undesirable substances (e.g., nucleic acids and
toxins) have~to be removed from the microrganisms. -~
--3--
:
1. ~,
`
7772
In spite of these difficulties, microorganisms are
already being cultivated on a large scale for the feeding
of domestic animals (fodder yeast). For human nutrition,
however, this protein from microorganisms has hitherto been
used either not at all or only to a limited extent. The
high ribonucleic acid content of this protein, for example,
constitutes a serious problem. ~ibonucleic acld (~A) is
decomposed in the mammalian organism to uric acid, which in
turn can cause gout. The ~NA content of microorganlsm
protein must therefore be less than 2%, and preferably less
than 0.5%.
One of the most important aims, therefore, is to pro-
duce the microbial protein free of ribonucleic acid. This
has not been accomplished in a satisfactory manner to date.
Attempts have been made to breed microorganisms of an espe-
cially low RNA content. These microorganisms have the dis-
!
, advantage, however, that they grow considerably more slowly.
It is known that microorganisms of high RNA content grow more
rapidly than those of low RNA content. This results in areal dilemma: the desired fast growth of the microorganisms
is to be equated with a high RMA content. If slower produc-
tion can be accepted, microorganisms of low RNA content can
be cultivated.
The second possibility for eliminating RNA from micro-
rganism protein, which is frequently referred to as single-
1~t7777Z
cell protein ~SCP)), is based upon the extraction of theRNA followed by separation of the insoluble protein. Sev-
eral possibilities for such extraction of RNA have been
described as follows:
5 a) Alkaline extraction - German Offenlegungsschrift
16 70 110.3
b) Acid extraction
i c) Treatment with high salt concentration (NaCl, KCl,
NH4Cl) ~ ,
d) Treatment with detergents (dodecyl sulfate etc.).
All of these methods have the disadvantage that the
microorganisms in some cases have to be processed in a very
complicated manner, and the protein is partially hydrolyzed,
racemized or otherwise altered by the treatment and by the
various additives and thus becomes less suitable if not use-
less for nutritive purposes. For example, alkaline extrac-
tion modifies the protein in its structure such that it
forms colloidal solutions. Separatlon is possible only
after neutralization and accordingly necessitates the re-
moval of the neutral salts that are formed. Furthermore, the
dissolved RNA becomes partially denatured, and can no longer
be quantitatively cleaved with enzymes to form soluble mono-
nucleotides.
~ 25
- From U.S. Pat, 3t720,585 it is furthermore known, in
~ ~he case of two specific types of yeast, to perform a brief
.. . .
1 .
.
3
--5--
}
7777Z
thermal shock treatment consisting of heating at 60 to 70C
for two to twenty seconds, followed by about 20 minutes of
heating at 45 to 50~C at pH values between 4.5 and 7. This
process has the disadvantage that it liberates proteases
5 which cleave the useful (digestible) protein and conse-
quently the protein yield is considerably reduced. The
residual RNA content is reduced but is still greater
than the preferred range of 2%.
T~E INVENTION
The invention is therefore addressed to the problem OL
creating a method for the production of single-cell protein,
which will not have the above-described disadvantages of
5 the known methods. In particular it is an aim of the in-
vention to create a simple process which can be performed
on a large technical scale, requires no expensive apparatus,
can be performed without the addition of foreign substances,
requires no previous treatment for the killing and digestion
f the microorganisms, and does not harm the environment.
This problem is solved in accordance with"the invention
by a method of obtaining protein of reduced nucleic acid
content from microorganisms by thermal treatment, which is
25 characterized in that an aqueous suspension of the micro-
organisms, with the addition in some cases of a material
having ribonuclease activity, is heated at a pH between 7
--6--
;' :
'
' :
.
" ~
1077772
and 805 at a temperature of 63 to 67C, until the content of
5'-nucleotides in the medium has reached a certain value,
then the medium is briefly boiled, and the insoluble crude
protein is separated from the suspension.
In the performance of the process of the invention, the
-~ nucleic acid is cleaved exclusively into 5'-nucleotides
which appear in the medium, while no 2'- or 3'-nucleotides
are formed. Depending on the nucleic acid content of the
microorganism and the desired final content of nucleic acid,
it is therefore easy to determine in advance at what 5'-
nucleotide content in the medium the nucleic aci~ content
will be reduced to the desired leve] and therefore the heat
treatment at 63 to 67C can be terminated. It is decisively
important that the precise temperature limits between 63 and
67C, and preferably a 65C temperature, be maintained, and
that at the same time the pH value remain always in the
stated range. Since the p~ decreases in the course of the
thermal treatment due to the liberation of the nucleotides,
the stated pH range is maintained by the addition of bases,
preferably soda lye, potash lye or ammonia.
Endogenous nucleases are activated by the process of
the invention and this results in the cleavage of khe nucleic
acid with the formation of the 5'~nucleotides which are
highly desirable as by-products: adenosine monophosphate
(AMP), guanosine monophosphate (GMP), cytosine monophosphate
,'' '.
;~ -7-
.,
~ .
.
.
.
~77q~
(CMP) and uridine monophosphate (UMP). The heating time
required depends on the microorganism used, especially on
its content of endogenous nucleases. Since the nuclease
content can differ greatly, the heating time will also differ
greatly and generally will range between about 20 minutes and
about 20 hours.
In the course of the heat treatment of the invention,
the protein is largely converted to the insol~uble state.
The precipi-tation of the protein is then completed by the
brief boiling, i.e., by heating between about 90 and 100C
at normal pressure, or higher temperatures at elevated pres-
sure. Then the insoluble crude protein is separated from
the suspension, for example by centrifugation or filtration,
15 and can then be put out for use as a nutrient or can be
further processed in any desired manner.
All kinds of microorganisms and protozoa are suitable
for the process-of the invention, such as, for example,
yeasts, bacteria, molds and the like. Examples of suitable
microorganisms are Saccharomyces cerevisiae, Saccharomyces
carlsbergensis and Candida boidinii as yeasts, ~ocardia
erythropolis as a mycobacterium, Bacillus cereus and Bacil-
lus subtilis as bacillaceae, Streptococcus faecalis as an
example of streptococci, Pseudomonas fluorescens as an ex-
ample of pseudomonads,~ and Aspergillus niger as an example
~f a mold. As previously mentioned, the microorganisms are
-8-
~777~Z
used preferably in freshly harvested, viable form, i.e.,
in the form in which it is harvested from the culture medium.
However, protozoa treated by known methods for the preserva-
tion of living microorganisms can also be used, such as
5 especially dried or lyophilized protozoa.
'
In a special embodiment of the process of the invention~
; it is possible to reduce the heating time by the addition of
a material having a ribonuclease activity. This includes
both purified ribonuclease preparations and semirefined or
crude biological preparations which contain ribonuclease.
Preferred material having ribonuclease activity are germs of
grasses and cereal species/ especially malt germ
~ or extracts thereof, preferably malt germ extract. In this
- 15 embodiment, the process of the invention is applicable also
. . .
- to ribonuclease-free microorganisms, such as for example the
E. coli MRE 600 mutant.
.. :
As a rule, the process of the invention permits a re-
duction of the ribonucleic acid content in single-cell
protein to from 0 to 0.2% by weight. The protein losses are
slight, and particularly the cleavage of protein and the
loss of value due to racemization are negligible.
., .
:; 25 The protein yield in the method of the in~ention at-
tains 90% and more. A special advantage of the method con-
sists in the fact that the ribonucleic acid is cleaved vir- ;
:
, .
_g _
,.
,~ . '
~6~7777'~
tually quantitatively to the 5'-nucleotides, which them~
selves are valuable substances. It is therefore possible
to prepare the nucleotides from the filtrate after the sep-
aration of the crude protein. It is especially advantageous,
5 however, to reuse the fi]trate for the cultivation of micro-
organisms. The microorganisms use the nucleotides to build
the necessary nucleic acid again, so that in this manner a
virtual recycling of the nucleotides is achieved. At the
same time, the rest of the dissolved cell components, the
remainder of the still soluble protein, and the like, are
utilized by the microorganisms for the fresh growth. Con-
sequently, the addition of biotin or yeast extract, which
is otherwise common in the cultivation of microorganisms,
can be wholly or largely dispensed witho
1 5
By the method of the invention, a purer, more digest-
ible protein is obtained, which is free of deter~ents, salts
or RNA. On the other hand, in the formerly common methods
;of cell digestion with alkali or acids, some of the amino
acids in the protein are destroyed or racemized.
A speclal effect of the method of the invention fur-
thermore lies in the fact that the peculiar odor of the
protein, which is especially pronounced in the case of the
- 25 yeasts, is destroyed by it~ This, too, makes the single-
cell protein of the invention more suitable for use as a
~ood.
~7777~
The removal of the RNA is performed in an especially
simple and economical manner. The 5'-nucleotides can be
recovered as by-products. Additional economies can be
achieved in the culture media by recycling the filtrate into
5 the culture broths. The process is also environmentally
harmless, since the cell waste can be burned without dif-
ficulty, which is not the case with a waste material that
has been treated with salt, acid or alkali.
;~ 15
` 20
. I ,
', :
3
,~ -11 -
~37777Z
.;
.
EXAMPLES
The following examples will further explain the in-
vention.
Examples 1 7
200 liters of an aqueous suspension of each of the
microorganisms listed in Table 1~ with a dry mass content of
about 15 to 20%, is heated at 65C and maintained at this
temperature for 15 hours with gentle stirring. The pH is
maintained constant between 7 and 8.5 by the addition of
soda lye. Then the mixture is briefly boiled and the crude
protein is separated by centrifugation and dried. The yield
5 is between 25 and 32 kg of crude protein. Table 1 shows the
RNA content of the starting material and the RNA cleavage
produced in it.
,:'
;
,.: ,. '
'
:.:
~, :
~
: ~ ,
` ~'',
::
-12-
.~ .
' ' - ' ~ ~ , ' '
.
~ 7777;Z c :"
~_ o
o a:l
rq
~ ~ o
~ o . ~
.r, ~ m
3 ~ U~ .
a) ~ ~ ~
O ' .
o
.,~
~ a) ~ ~ o ..
~I) C,) bO
~1 ~ o~O o~O o~
QI¢ ~I oo~Oo~O o d~ Od~
,az tlS O O ~D O O OO .
E~P; ~ ,~a) ~ ~ ~ ~r~ V
,~ V
m ~
O~ ~ N ~+~
a1 ~I h
O
oP ~ ta
~O ~ d~
Od~ o\ c\ Oo~
Z ~I ~ D O
K C~ ,~o ~~I t` ~I ,J ~ ~ O
~ ~O
_~ o~O V
+ ~ In t~
~ . +~
IO~o0~o 0~o0~o 0~o 0~o ~a :
~C ~ 1~100 ~ ~D ' ~ ~~i ~ ~
Z O . . .. o
~I ~ `J Nu~
. ~ ~4
~ ~1 h
~1 0 U'1:1 0
S~ ~ U
U UOo ~0
S:~
U~ ~ ~ ~) Q ,~
U~ U)Ul U~ O
1~ Idla ql U C) h O O
~1 a
~1 ~ , ~ ~ V a~
. ~ . ' O O
O ~ ,
O O U~ - ~ ~ h
U U ~ rl ~ ~1 a) r-l U U) U~
" a~ ~ aJ o ~ ~1 U U 1~ ~ 3
E~ ~ Q - ~ O
o-,l o ~ ta U ~n o u Q --`
ut ; J u~ X ~1 ~ ,a o ,~
( Q ~ O ~ r~ o ~ ~ o a) ~ ~ (X)
rl h
~ C) ~ U ~ ~ ~ ~ O ~ ~ ~
O V h U h 1~ U ~ ~ O 0 ~1 ~ '~I ~
~ ~ 15 o Id ~ f ~ ~'
M . U~ U u~ V U ~ U~ P~
~l
~; a~
~ ~ ~ .
..
X E~ ~ ~1 .
.
-13-
i ' '. : '~ : . .
,: . ~
777~;~
As Table 1 shows, the process of the invention pro-
duces very good yields of 5'-nucleotides from fresh micro-
organisms. When the microorganisms have been standing for
a comparatively long time, the yield is lower, and this is
5 attributed to the reduced ribonuclease content.
Examples 8 to 14
-
a) 3 kg of malt germs are ground; 30 1 of water is added,
and the mixture is held for 40 minutes at 65C. The
suspension obtained is used either directly or after
removal of the insoluble malt germ fragments in the
following process.
' :
5 b) 200 liters of a suspension of each of the same micro-
organisms as in Examples 1 to 7, with the same dry
mass content as described therein, are heated to about
65C, the malt germ suspension obtained in a) or the
extract thereof is added, and then the mixture is
heated for between 2 and 15 hours at 65C. The proces-
sing is then performed as described in Examples 1 to
7. The yield is between 25 and 35 kg of crude protein,
and the average pure protein content is 70O.
~ .
Table 2 shows the RNA cleavage obtained. The yield
of the 5'-nucleotides~amounts in each case, for an RNA con-
tent in the dry substance o~ about 10o ~ to about 700 to 800
-14-
77'772
grams of each of the four different 5'-nucleotides; where
~ the RNA content is lower, the yield is lower according to
-~ the starting material.
Example 15
As described in the preceding examples, a suspension
of 100 g of dry Saccharomyces cerevisiae with a content of
~' about 45% protein and 1.5% to 3% RNA in the dry substance,
was heated at 65C without the addition of any material hav-
ing a ribonuclease activity, and was boiled and the crude
protein was separated. The composition of the filtrate and
of the crude protein was determined and is given in Table
,; 3 below. The values stated show that the protein content
' 15 in the crude protein ,was enriched from 45% to 56%.
I In the case of microorganisms of hlgher initial protein
,', content, the yield of crude protein can increase up to 85%.
,, 20
: ,
.' . ~
. . .
,~ 25
,'~ . '.
' , .
-15-
'
77~77
~c)
~ ~a
C~ a
~ ~ ~J
~ ~:~
I Q) ~ ~ .
o I ~ X
a) o ~ Q) O o\O
~ a ~ ~, o
~ o ~ .~ ~ o Q~
-~ h ~ ~ h
$ P~
h
~ ~ .
~a ~ ~
~ o a) O~o
h h ,1 Cl~
O ~ ct:~
$~ : '
h ~ ~
Ll~ :
~ a-~
.~ ~ U
~ ~ .,
a) o o ~
.~ e~ h !~ o
~ ~ o ~a c~ ~ .
E~ u~ ~ . - ,
~ ~ a)
.~ ~
_ ~ ~ ~
s~ a) o ~
~ .~ 0\O ,q ' .'
~ .~ ~ ':'
O _l h .. : .
~ m ~ ~a
a) h U
~1 ~: ~ ~ h ~J
h $ ~ ~ o ~a ~
~ O ~: o\O ~ Q
,- ~a h O
~a
~a a
$ ~ ~
~ 5~
~: O ~ ~ . ~
C) ~o5 ~
e~ ~ : -
X .,
-16-
.
', ' .
~L077~7Z
It will be understood that the specification and examples
are illustrative but not limitative of the present invention
and that other embodiments within the spirit and scope of the
invention will suggest themselves to those skilled in the art.
. .
.
;'' ' ' ~
' ' , ,
, ' .
.,
.
. .~ ,.
.', .
, ~ .
'`,
'~'
~ . . .
~ -17-
,
:,,, , . , . :
. . .
..... .
