Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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1 The present invention pertains to processes for the utiliza-
tion of cellulosic starch and protein-containing substances such
as wheat and cereals of all kinds as well as bran from cereal and
flour mills and broken pieces and crumbs of baked goods from
bakeries by converting the starches therein into protein-contain-
ing nutriments or concentrates that can be used in human nutrition
and as supplements for animal feeds and that are richer in proteins
and accordingly more valuable than the original cellulosic substan-
ces from which they were produced. The processes of the present
invention are accordingly adapted for converting cereals such as
wheat, rice, barley, rye, sorghum and grains and plants of all kinds
into more valuable protein-containing concentrates or nutriments.
....
In the production of cereal products, large quantities of
waste cellulosic substances containing starches and proteins are
produced which are used almost exclusively for animal feed.s that
nonetheless have inferior nutritional valueO Considerable quanti-
ties of such cellulosic starch and protein-containing waste prod-
ucts are also produced, for example, in the production of baked
goods such as biscuits and crackers in bakeries.
Processes are known for the conversion of starches and starch-
like substances into protein-containing plants,such.as yeast cells
or solids. One such process, which is described in United States
Patent No. 3,105,799, consists in symbiotically cultivating in a
substrate consisting essentially of potatoes or similar vegetable
material containing starch with two microorganisms, namely, a : .
microorganism which hydrolyzes s*arch, such as Endomycopsi.s fibul-
igera, and a sugar-fermenting yeast such as Torulopsis utilis, and
separating and using the yeast that were grown therein during
the fermentation as a nutriment~ Such cereals and waste cellulosic
~0 substances, however, also contain a substantial proportion of highly
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1 valuable proteins which had heretofore been overlooked or ignored.
Processes have also been described for the recovery of pro-
teins by ultrafiltration but such processes have been heretofore
used chiefly in the dairy industry, for example, for recovery of
proteins from whey, for example, in an apparatus such as is de-
scribed in German published application No. 2,220,308.
The principal object of the present invention is to provide a
; process for the production of valuable pro-teins or protein-contain-
ing concentrates or nutriments from the aforementioned cellulosic
: 10 starch and protein-containing substances that can be used in human
as well as animal nutrition. Other ob~ects and advantages of the
invention, some of which are referred to hereinafter, will be ob-
vious to those skilled in the art to which it perta.ins.
The foregoing object is achieved in accordance with the pro-
cesses of the present invention, in which processes the cellulosic
starch and protein-containing substance is first subjected to
hydrolysis to convert the starches thereinto into glucose or other
fermentable sugars. The resulting slurry is then subjected to
ultrafiltration to separate the sugar or glucose-containing liquid
from the protein-containing solids that remain therein. These :
protein-containing solids are also valuable nutriments. Therea:Eter
the sugar or glucose-containing liquid is inoculated with a yeast
such as Candida utilis (also known as Torulopsis utilis), Saccharo-
myces cerevisiae or Hansenula polymorpha, or a species of Lacto-
bacillus or Aerobacter bacteria that has been approved by the ap-
propriate governmental authority for the production of nutriments
for use in human nutrition, and the yea~tor bacterium is aerobic-
ally cultivated therein. The yeast or bacterium that has been grown
therein is then recovered by conventional methods. Both the yeasts
: 30 and bacteria that are thus grown, as well as the proteln--
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1 containing solids that have been recovered in the ultrafiltration
step, are valuable sources of protein and may be used not only
for the production of animal feeds and fodder but also in human
nutrition.
The invention is described further in connection with the e~-
amples and description which follow, which were selected solely for
the purposes of illustration and consequently are not to be con-
strued as restrictive of the invention or its scope.
EXAMPLE 1
The general procedure for carrying out the process that is
described in this example is a preferred method and was used in
all of the subsequent examples that are referred to hereinafter.
Ten hundred (1000) grams of comminuted defatted zwiebach crumbs
was stirred into 3 liters of water in an autoclave to which 30
milliliters of concentrated hydrochlorio acid (32~i by weight of
hydrogen chloride) was then added and the resulting slurry was then
heated at a temperature of 120C for a period of approximately 2
hours, during which period it was stirred vigorously. At the end of
; this period, no starch could be detected in the mixture, indicating
that it had been completely hydrolyzed. The resulting hydrolyzed
mixture contained between 500 and 700 grams of glucose which was
identified by chromatography. This mixture was then diluted with
tap water to a volume of approximately 20 liters and subjected to
ultrafiltration to separate and recover the protein-containing
~solids therefrom. Suitable ultrafiltration apparatus that lS com-
' mercially available was used for this purpose. The filtrate con- ~
tained between 500 and 700 grams of fermentable sugar and had a -
concentration of between 25 and 30 grams per liter.
Three (3) liters of this sugar solution was then placed into
a fermentor having a capacity of 7 liters and 15 milliliters of
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1 concen~rated phosphoric acid (85% by weight of orthophosphoric acid)
and nutrient salts that are required for growth were added thereto.
Ammonium hydroxide was then added to the solution in such an amount
as to bring the hydrogen-ion concentration of the solution to a
value corresponding to a pH of 4.0 and the mixture was sterilized
by heating it for 20 minutes at a temperature of 121C, after which
it was cooled to a temperature between 28 and 32C and inoculat~d
with a culture of Candida utilis yeast in an amount equivalent to be-
tween 2 and 5 grams (dry weight) of the yeast. The fermentation was
then continued at a temperature of approximately 32C while air at
a space velocity between 0.5 and 1.0 volume per volume per minute
was bubbled through the fermenting culture for a period of approxim-
ately 25~ hours, or until all of the sugar had been completely con-
sumed by the yeast for its growth and additional yeast cells had
been produced.
After the fermentation had been completed, the entire mass of
solid living yeast cells was separated in a centrifuge, washed with
water, and formed into a slurry having a solids content between ap-
proximately 16 and 20~ on a dry-weight basis. The yeast slurry was -
then dried to a solids content of approximately 93~ in a drum
drier. The drying could alternatively have been effected by spray-
drying.
The yield of solid yeast cellular matter on a dry-weight basis
was 50 grams, which is between 45 and 48% relative to the weight of
the sugar that was fermented. The yeast thus produced by cultiva-
tion of Candida utilis contained 7% by weight of water, approximately
50% by weight of protein (determined by the Kjeldahl method), ap-
proximately 30% by weight of nonnitrogenous compounds, 5.1% by weight
of fat, and 7.6~ by weight of ash. It had a nucleic acicl content
of 9.3% by weight. The amino acids of the proteins and the percentages
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1 by weight in which they were present in the yeast were as follows:
Aspartic acid 10.4%, threonine 5.8%, acrine 6.0~, glutamic
acid 12.4%, proline 3.5%, glycine 5.1%, alanine 6.7%, valine 5.3%,
methionine 2.0%, isoleucine 4.7%, leucine 7.9%, tyrosine 5.3%, phen-
ylalanine 7.1%, lysine 8.0%, histidine 2.5%, arginine 5.8% and
tryptophen 0.8%.
~ The yeast solids thus produced can be used as a protein-rich
concentrate or supplement for addition to poultry, cattle and hog
feeds in amounts between 10 and 30% by weight. It is also suitable
for use as a yeast nutrient or supplement in human nutrition.
EXA~PLE 2 .
Ten hundred (1000) grams of finely ground defatted æwiebach
crumbs were stirred into 20 liters of water and the slurry was then
made homogeneous by vigorous stirring. A small amount of alpha-
amylase enzyme was then added to the suspension and the mixture was
allowed to stand for such a period and at such a temperature that
the starch therein was completely hydrolyzed. The resulting hydro-
lyzate was then subjected to ultrafiltration as in Example 1 and
the filtrate was inoculated with a culture of Candida utilis yeast
20 as in Example 1. The fermentation and recovery of the yeast solids
from the ~ermentation brew were completed as described hereinbefore
in connection with Example 1,
EXAMPLE 3
Instead of using zwiebach as in Example 1, 1000 grams of fine-
ly ground defatted cake crumbs which on a weight basis consisted of
4.9% of water, 16.0% sucrose, 1.5% of sodium chloride, and 77.6%
of starch, were substituted therefor, and in all other respects the
procedure described in Example 1 was repeated.
The yeast solids that were recovered did not di.ffer to any
substantial extent from the product that was described in Example 1
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1 and the amount that was obtained was equivalent to 55 grams on
a dry-weight basis.
EXAMPLE 'l
Ten hundred (1000) grams of defatted wheat bran were sub-
stituted for the zwiebach crumbs that were used in Example 1 and
the procedure therein described was repeated with essentially the
same results.
, EXAMPLE 5
One hundred (100) kilograms of wheat kernels are ground
together with 400 liters of water and 6 liters of concentrated
hydrochloric acid are added thereto and the slurry is heated at a
temperature between 110 and 125C in a sealed autoclave for a per-
iod between 1.5 and 2.5 hours, thereby hydrolizing the starches
therein to soluble sugars.
The same results are obtainable when dry whole wheat flour is
used in preparing the slurry and when at least 6 and at most 12
liters of concentrated hydrochloric acid is used for the hydrolysis.
The course of the hydrolysis is followed by periodically test-
ing the mixture of unhydrolyzed starch with a solution of iodine
prepared by dissolving iodine crystals in an aqueous solution of
' potassium iodide.
After the wheat has thus been hydrolyzed, the slurry is di-
luted with water until it has a concentration of approximately 60
grams of soluble sugars per liter. The mixture is then filtered
through an ultrafilter consisting of a plurality of ultrafiltration
membranes at a temperature between 30 and 60C. The residue is
washed thoroughly with water until most of the soluble sugars are
extracted therefrom. The aqueous extracts are collected and com-
bined with the ultrafiltrate which then will contain between 35
~ and 40 grams of soluble sugars per liter.
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1 The ultrafiltrate is then inoculated with a suitable yeast
culture nutrient salts and other substances that are required in
accordance with conventional fermentation methods, and is aerobically
fermented in conventional manner. At the conclusion of the fermenta-
tion, the beer contains between 17 and 20 grams per liter of yeast
based on its dry weight. This yeast is recc,vered by centrifugation
and dried. It has a protein content of approximately 50~ by weight
and a gross dry weight of between 25 and 32 kilograms.
The li~uid remaining after removing the yeast cells from the
beer by centrifugation contains the major portion of the proteins
that were originally present in the starch and protein-containing
cellulosic substance together with some carbohydrates. The solids
that are obtained when this liquid is evaporated have a protein
content between approximately 30 and 50% by weight. E'rom 100 kilo-
grams of wheat kernels, between 25 and 50 kilograms of these pro-
tain-containing solids or concentrates can be obtained in accordance
with this process.
EXAMPLE 6
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Five (5) kilograms of whole wheat flour containing approximate-
ly 12~ protein, between 60 and 70% starch, approximately 15% moisture,
approximately 1% ash, and 0.5% fibrous substances (all percentages
being in percentages by weight) is stirred into 15 liters of water.
; Then 3.5 grams of an enzyme -- alpha-amylase, type HT-1000 Miles
Laboratories was used -- is added to the resulting slurry and the
slurry is allowed to stand while it is maintained at a temperature
of 80C until substantially all starches therein are hydrolyzed to
` dextrin. Subsequently, the dextrin therein is converted to glucose
or other soluble sugars by adding a second enzyme thereto -- amylo-
slucosidase, Diazyme L-100 Miles Laboratories was used -- and al-
lowing the slurry to stand for several more hours.
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1 The resulting hydrolyzate is then diluted with water to a
final volume of 65 liters and its hydrogen--ion concentration is
adjusted to a value corresponding to a pH of ~.5.
The solid portion of the hydrolyzate which includes proteins
that were originally present in the flour are separated by ultra-
filtration from the liquid portion or ultrafiltrate which consists
essentia]ly of a solution of sugars.
The concentration of total solids in the 65 liters of the
diluted hydrolyzate, including the solid portion as well as the
solids dissolved in the liquid portion, is 54 grams per liter, which
corresponds to 3.27 kilograms or 65.4% by weight of the original
whole wheat flour.
The solid portion of the hydrolyzate that is thus separated
:is a protein~containing concentrate which, after washing twice
with minial portions of water and drying, contains between approx-
imately 40 and approximately 70% by weight of the gluten and other
proteins that were originally present in the whole wheat flour,
depending upon the degree of washing and hydrolysis. The less washing
or hydrolysis, the higher the residual soluble salts and the higher
the percentage of ash after dry:ing, so that the protein content is
lower.
The ultrafiltrate or liquid portion of the hydrolyæate contains
39.2 grams of dissolved solids per liter, of which 77% by weight
are sugars. This corresponds to a 72% conversion of the starches
in the whole wheat flour to sugars. This ultrafiltrate on analysis
was found to have a nitrogen content of only 0.11%, which is
equivalent to 0.7% by weight of proteins which may consist of
peptides or amino acids that passed through the ultrafiltrate mem-
branes. Since this amount corresponds to only 1~ grams of the original
5 kilograms of the whole wheat flour, it may consequently be dis-
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1 regarded.
The sugar-containing ultrafiltrate is then inoculated with a
yeast culture and the yeast is grown there:Ln in accordance with
conventional aerobic fermentation methods such as are referred to
hereinbefore and the yeast that is thus produced is recoverëd ~from
the brew in conventional manner. A 50% yield of dry yeast sub-
stance, as related to the sugar in the filtrate, was obtained. A
quantity of 72 kg sugar was obtained per 100 kg of starches used,
and 36 kg dry yeast substance was obtained per these 72 kg of sugar,
i.e. 36% of yeast as related to the starch. Dry yeast substance
should be understood to be 100~ yeast, i.e. completely dry yeast. -
In the foregoing manner, the whole wheat flour is converted
into two products, each containing a high percentage of protein,
one being the solid concentrate containing gluten and other pro-
teins that was direc-tly separated from the hydrolyzate by ultra-
filtration, and the other being the yeast produced by fermentation
;~ of the sugars in the liquid portion of the hydrolyzate or ultra-
filtrate.
In this example, the enzymatic hydrolysis can be completed
within a relatively short period so that the solids remaining in
the slurry that are separated :in the subsequent ultrafiltration
step, consist, for example~ of approximately equal parts by weight
of carbohydrates and proteins. By longer or more vigorous hydroly-
sis, the carbohydrates remaining in the solids can be almost com-
pletely converted to soluble sugars which remain dissolved in the
li~uid portion of the hydrolyzate and can be separated from the
solid portion of the hydrolyzate in the processes of the present
invention in the ultrafiltrate.
The processses of the present invention are illustrated further
in connection with the accompanying drawing wh:ich is a schematic
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1 flow sheet of a preferred embodiment of the invention.
The starch and protein-containing cellulosic substances are
crushed or milled in the pulverizer or grinder l that is represent-
ed in the accompanying drawing to form a slurry with water which is
then passed into one of the hydrolysis kettles 2 in which the
starches that are present in the cellulosic substances are hydro-
lyzed to sugars. The resulting hydrolyzate is then passed into
and through a series of ultrafilter membranes 3 in which the
protein-containing solids are separated from the liquid portion con-
taining dissolved sugars. The separated solids, which include theproteins that were originally present in the cellulosic substance,
are dried in a dryer 4 and are used as such or combined with other
proteins that are produced in the process.
The ultraEiltrate containing the dissolved sugars is passed
to a tank 5 and nutrient salts and any other substances that are
required to produce a culture medium upon which the preselected
yeast is to be grown are added thereto and mixed therewith. This
culture medium is then passed to the fermentor 6 in which it is
inoculated with a culture of the preselected yeast, which is then
aerobically grown therein.
After conclusion of the fermentation in the fermentor 6, the
; brew is passed into a separator 7 in which the-yeast cells are
separated from the accompanying liquor and are eventually washed
with water. The slurry of yeast cells which has a dry-solid con-
tent between 16 and 20% by weight is then collected in the tank 8.
From this tank the slurry is then passed to a dryer 9 in which the
solids are dried and the dried solids are then packaged into sacks
or other containers.
Although alpha-amylase was used in Example 2, it is to be
understood that other known starch-hydrolyzing enzymes may be sub-
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1 stituted therefor.
Furthermore, instead of using an enzyme, the hydrolysis may
be effected by cultivation in the slurry of a microorganism that
is known to produce an enzyme such as alpha-amylase that is cap-
able of hydrolyzing starch, such as microorganism of the genus
Endomycopsis, such as, for example, the species Endomycopsis fib-
uligera~ The microorganism is then separated from the hydrolyzate
before the hydrolyzate is subjected to ultrafiltration or is separ-
ated with the other solids during the ultrafiltration step.
Other acids such as orthophosphoric and sulfuric ac:ids can
be substituted for the hydrochloric acid that was used for the
hydrolysis step of the process in the Eoregoing Examples 1, 3, 4
and 5.
When enzymes such as amylases are used in the hydrolysis
step, none or at most only small quantities of the proteins that
are present in wheat and other cellulosic substances containing
starches and proteins are converted to products that are soluble
in the hydrolyzate. Acids have a tendency to degrade proteins
to amino acids.
; 20 Instead of separating the solids from the liquid portion of
the hydrolyzates by ultrafiltration and fermenting only the ultra-
filtrate, as illustrated in the foregoing examples, the entire
hydrolyzate as such containing the desired sugars as well as the
~, proteins and other slids can be fermented. The proteins that are
originally present in the cellulosic substance are practically not
effected or converted during the fermentation and can be recovered
together with the yeast cells in the subsequent ultrafiltration
step. The~ultrafiltrate then contains only dissolved salts that
have been washed out of the solids, which solids are rPtained on
the ultrafilter membranes. Such ultrafiltrates have a biochemical
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1 oxidation demand of only 1000 parts per million or less.
When the cultivation or fermentation is conducted in a con-
tinuous manner that is by continuously withdrawing brew and adding
fresh culture medium, a residence period of the brew in the ferment-
er of approximately 4 to 5 hours must be a]lowed for completion of
the fermentation with yeasts, whereas a residence period of 2 hours
at most is required in a bacterial fermentation.
When bacteria axe used instead of yeasts, the fermentation
is completed after a period of 20 hours at most.
The processes of the present invention are advantageous since
the amino acids that are present in -the yeasts and in the original
protein-containing cellulosic substances are an extremely valuahle
combination from a physiological and nutritional standpoint. Thus,
the amino acids that are present in the proteins of the starch and
protein-containing cellulosic substances may compensa-te for in-
adequate amounts of methionine and cystine that are supplied by
the yeast. On the other hand, the yeasts may supply asparagine
and other amino acids that may be present in inadequate amounts or
may be absent in the proteins of the starch and protein-containing
cellulosic substances.
If comminuted plants such as wheat which has not been pre-
liminarily threshed, or grains or cereals which still include large
portions of fibrous substances in the Eorm of chaff husks, cobs,
or bran, are to be used as the starting starch and protein-contain-
in~ cellulosic substance in the processes of the present invention,
such f~brous substances can be separated preliminarily from the
starch or protein-containing portions of the cellulosic substances
by con~entional straining or centrifuging procedures. On the other
hand, if the starch and protein-conkaining cellulosic substances
include fibrous substances that can be hydrolyzed to pentoses, such
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1 fibrous substances can be included along with the other components
of the cellulosic substance that is to be hydrolyzed in the pres-
ence of an acid and be subse~uently fermented together with the other
sugars or hexoses that are present in -the hydroly~ate to produce
additional ~uantities of yeast and need not be preliminarily sep-
arated from the other fibrous substances.
Hydrolysis of starch may take place in the presence of an
organic acid, such as citric acid o~r acetic acid.
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