Recovery of protein, protein isolate and/or starch from cereal grains

The protein/starch bond is broken mechanically by wet attrition milling rather than by cooking or with chemicals alone. The grain particles are milled to a particle size sufficiently small to break the bond between starch and protein and sufficiently large to retain substantially all of the starch granules intact. The protein is then extracted with ethanol and alkali solvents, separated and dried to form protein and/or protein isolate. The intact starch granules are cleaned and dried.

FIELD AND BACKGROUND OF THE INVENTION 
This invention relates generally to the production of protein or protein 
isolates and/or starch from cereal grains. In particular, this invention 
relates to a technique in which the bond between starch, on the one hand, 
and protein on the other is broken mechanically rather than by the 
conventional process of cooking. As a result, there is realized an 
increased recovery of a higher quality protein and intact granules of a 
higher quality pure starch. 
It has been proposed heretofore to obtain a protein isolate from corn or 
other cereal grains. As is well known to nutritionists, agricultural 
commodities, such as cereal grains, may contain complete or nearly 
complete proteins and thus, if the protein were isolated, would be capable 
of providing a balanced diet for humans. 
Heretofore, the extraction of isolated proteins from agricultural 
commodities has been made difficult by the chemical bonding which exists 
between protein and the starch constituents of the commodities. In prior, 
well known processes which involve some biochemical treatment or steps 
using one of these constituents, such as the yeast fermentation of corn or 
grains to produce ethanol, the starch/protein bond is broken by hydrolysis 
through cooking. However, breaking the bond by cooking, while making the 
starch and sugar content available for other uses, destroys the 
possibility of recovering the protein in a form useful as food. 
James T. Lawhorn proposed, in U.S. Pat. No. 4,624,805, that, after an 
initial grinding step in which the grain is ground to "meal" size (250-600 
microns), protein be isolated prior to the use of starches and sugars in 
an ethanol fermentation process. The protein is extracted with an 
alkali/alcohol solution either with or without sonification. Lawhorn 
teaches that the protein is recovered by ultrafiltration, with the 
dissolved sugars and starches in a permeate being concentrated by reverse 
osmosis for use in alcohol production. 
It has been discovered by attempts to practice the Lawhorn process that the 
process as disclosed in the patent simply does not work on a commercial 
scale. First, by relying on chemicals and sonification to break the 
protein/starch bond, only minimal percentages of the available protein is 
extracted. Secondly, the ultrafiltration membranes described by Lawhorn 
foul and clog, and the desired continuous operation does not occur. 
However, the Lawhorn process does propose the separation of protein and 
other components of agricultural commodities by a process which avoids 
cooking and thereby would permit the recovery of a food graded protein 
isolate, if it worked on a commercial scale. 
On a dry basis, the endosperm of corn makes up approximately 83% of the 
kernel and contains the bulk of the starch. Other cereal grains may vary 
somewhat in percentages of endosperm but are conceptually similar. The 
starch is present as granules of roughly 5-35 microns in size with the 
space therebetween filled with protein. This protein/starch matrix has to 
be broken to be able to recover either or both starch or protein. Intact 
starch granules are much more valuable than starch granules which are not 
intact. It should be noted that the structure of the granules themselves 
depends on the way in which amylose and amylopection are associated by 
intermolecular hydrogen bonds. Strong bonds give rise to crystalline areas 
and weak bonds to amorphous areas. Normal corn starch has a crystallinity 
in the range of 15-39%. 
In the known processes for separating protein from starch, either the 
usefulness of protein and starch have been destroyed by the cooking and/or 
steeping process, or else the protein has been ineffectively recovered in 
the Lawhorn process. In my prior application Ser. No. 487,739, now 
abandoned, while a technique for mechanically breaking the starch/protein 
matrix, the starch granules are generally not left intact. Therefore, a 
process which effectively recovers quality protein and maintains starch 
granules intact has not been heretofore accomplished. 
BRIEF DESCRIPTION OF THE INVENTION 
With the foregoing in mind, the present invention reduces the grain kernels 
to particles of an optimum size to be treated more effectively with a 
solvent to extract the protein from the starch. Toward this end, it has 
been found experimentally that particle sizes in the range of 1-100 
microns realize enhanced recovery of the available protein (greater than 
80%). Further, after subsequent microfiltration, the end product has a 
protein concentration of over 90%, qualifying it as protein isolate, a 
much more valuable type of protein suitable for use in baby food, food for 
the elderly, as a fortifying agent for snack food, milk shakes, 
nutritional drinks, pizza, and the like, as well as a substitute yeast in 
hamburger meat. 
It has also been determined that if such particle sizes are in the higher 
end of the acceptable range (35-100 microns), all of the starch granules 
may remain intact, which makes them more valuable, as they are suitable 
for new industrial and commercial uses such as, for example, in 
biodegradable plastics, surgical dusting powders, antiperspirant sprays, 
disposable sanitary products, instant laundry starches and others. Other 
applications are also envisioned where cross-linking for instance 
overcomes the sensitivity of the granules to disruption and improves the 
strength of swollen granules. 
In the process of the present invention, the grain is first subjected to a 
dry milling process to reduce the particle size to a range that can be 
easily introduced to the second operation after defatting: disk attrition 
wet milling. It is in this operation that the starch/protein matrix is 
broken. In the attrition mill (Koruma, for example), two carborundum disks 
are used, one stationary and one rotating at a high speed. The dry ground 
kernels are fed in aqueous slurry form through the center of the upper 
disk, and grinding occurs in the horizontal plane between the disks. The 
clearance between the disks is adjustable, as well as the rate of feed. 
Thus, the particle size can be closely controlled. The clearance between 
mill disks, speed of feed, and pumping pressure are adjusted to yield in 
conjunction with sufficient feed pressure, particle sizes of 1-50 microns, 
if the resulting starch is to be used in the production of ethanol where 
disruption of the starch granule is not a factor. However, where it is 
desirable to maintain the starch granule intact, the spacing between the 
disks should be above the particle size of the largest granules (between 
35 and 100 microns and preferably approximately 50 microns). It is desired 
to set spacing between the disks at slightly above the largest particle 
size expected (depending on the type of grain being processes. 
After the conventional extraction phase in which one or more solvents are 
added, and after settling (with or without centrifugation), the clear 
supernatant containing the protein is decanted and the starch cake 
remains. The starch cake is washed to remove further protein and then 
slurried in water and cleaned up in a set of hydroclones in which all 
non-starch particles are separated. Thereafter the slurry is centrifuged 
to 40-70% by weight dry solids and dried in a flash dryer or drum dryer or 
other dryer. The pH is reduced to 6-7 with hydrochloric or other acid. The 
end result is a high quality pure starch of intact granules. 
The protein containing supernatant is processed through a continuous 
cross-flow microfilter having inorganic membranes. Preferably the 
membranes are aluminum oxide or other inorganic membranes. (Note that 
there are also presently being developed but not yet commercially 
available some forms of organic membranes which may become available in 
the future.) The pH of the protein or protein isolate is reduced to any 
desired value through diafiltration with deionized water, which 
simultaneously increases the purity. 
Protein isolate is that material which has a protein purity of 90% or more. 
Protein purity below 90% is considered to be protein concentrate. In corn, 
for example, the available protein is made up of approximately 10% 
albumins and globulins (soluble respectively in water and neutral salt 
solutions), 35+% glutelins (soluble in alkali) and 50+% prolamines (mainly 
zein and soluble in high ethanol concentrations). The system of the 
present invention recovers most of the prolamines and glutelins, as well 
as some of the globulins. The overall extraction efficiency of protein is 
greater than 80%. 
With the foregoing in mind, it is an object of this invention to isolate 
from cereal grains the food value protein and starch present while 
enabling the maximum recovery of other useful by-products. In realizing 
this object, the starch constituents are separated from the protein 
constituents mechanically in a two-step grinding process. In the first 
step, the kernels are subjected to a dry milling operation in which the 
kernels are broken down to a particle size on the order of 5/64" or less 
in diameter. There follows a wet attrition milling process which reduces 
the particle size of the commodity to a very fine range. During this 
operation, the protein/starch matrix is broken resulting in a more 
efficient extraction of protein. 
A further object of this invention is to produce protein isolate from corn 
or other grain while enabling use of the remaining constituents of the 
corn or other grain in further process which yield useful by-products, 
such as bacterial or yeast fermentation. In realizing this object of the 
present invention, the separation of protein and other constituents is 
accomplished while avoiding cooking of the agricultural commodity which 
would otherwise limit the usefulness of the constituents of the commodity. 
Yet another object of this invention is to separate the protein and starch 
constituents of cereal grains in such a manner that the protein recovery 
is maximized without destroying the integrity of the starch granules. 
In the case of corn, current varieties conventionally processed are 
normally deficient in lysine and tryptophan. A high lysine corn is known 
(Crow's Opaque 2) which is only slightly deficient in tryptophan. However, 
new varieties of corn will become available soon with high protein content 
and all eight amino acids essential for human nutrition present in 
sufficient quantities to meet FAO/WHO standards. While the process to be 
described hereinafter will work with any variety of corn, it is 
contemplated as being most useful with such high protein content, 
essentially complete protein corn.

DETAILED DESCRIPTION OF THE INVENTION 
While the present invention will be described more fully hereinafter with 
reference to the accompanying drawing, in which a preferred embodiment of 
the present invention is shown, it is to be understood at the outset of 
the description which follows that persons of skill in the appropriate 
arts may modify the invention here described while still achieving the 
favorable results of this invention. Accordingly, the description which 
follows is to be understood as being a broad, teaching disclosure directed 
to persons of skill in the appropriate arts, and not as limiting as in the 
present invention. 
A process for isolating protein from an agricultural commodity such as 
cereal grains in accordance with this invention preferably begins with a 
suitable commodity which contains all eight essential amino acids in 
sufficient quantities to provide a complete protein as understood in 
accordance with the standards of the Food and Agricultural Organization of 
the United Nations. Such a commodity may be whole corn or some other 
cereal grain or vegetable matter. Such vegetable matter, or a suitable 
grain, is provided as the first step in the process, indicated at 10 in 
the drawing. It is contemplated that the process may possibly be practiced 
with other commodities which might supply less than complete protein. 
However, the maximum benefit is to be obtained where the commodity used 
does provide a complete protein, and thus that is the preferred practice 
of this invention. As will be appreciated, the process may begin with 
whole corn, in which event protein is produced from both the germ and 
endosperm, or with degerminated corn. Generally, a protein isolate 
produced from the germ alone will be a better quality isolate. 
The corn (in the preferred practice) or other grains or vegetable matter is 
first dry ground in a hammer mill or the like to a particle size of from 
about one sixteenth inch to three sixteenth inch nominal diameter. The 
ground corn is now defatted. The ground and defatted corn or other 
particulate vegetable matter containing bound-together protein and starch 
is then mixed with water to form a slurry in which the vegetable matter 
constitutes up to at least about 35 percent of the slurry by weight. The 
slurry is then fed to a special attrition mill for wet grinding which 
reduces the particles to a size in the range of 1-100 microns such that 
the bond between starch and protein constituents is broken mechanically. A 
suitable mill is manufactured by Koruma Maschinenbau of the Federal 
Republic of Germany and sold for use in the cosmetic and drug industries. 
Such a mill also disperses the vegetable oils contained in the starting 
vegetable matter, such as corn oil, if not removed, to such an extent that 
no serious membrane fouling of significance occurs in subsequent process 
steps to be described hereinafter. Both wet and dry milling can proceed at 
essentially ambient room temperatures. In cases where the starch 
constituent is to be used for the production of ethanol, the spacing 
between the disks of the mill, the pumping pressure, and the speed of the 
feed are adjusted to yield particles in the range of approximately 1-10 
microns. Where intact starch granules are desired, the parameters are 
adjusted to yield particles in the range of 35-100 microns. 
After wet milling, the slurry is subjected to protein extraction. The 
specific sequence of the protein extraction steps may vary depending upon 
the specific nature of the starting vegetable matter, and the person of 
skill in the applicable arts practicing this invention will discover that, 
as starting materials vary from those described here as most preferred, at 
least certain process variables must be varied to maximize yields. 
Attention will be directed to some of these matters hereinafter. 
In the presently preferred form of this invention, the temperature of the 
slurry is raised to about 125.degree. Fahrenheit for the extraction steps 
which follow. Such heating will be a side effect of the wet milling and/or 
may be accomplished by supplying heat to the slurry. It should be noted 
that prior to the step of adding alcohol, which will be discussed below, 
the temperature of the slurry is adjusted to about 125.degree. Fahrenheit, 
and the temperature of the slurry and the high liquid content portion 
thereof is maintained within 5 degrees of 125.degree. Fahrenheit 
throughout the process. Temperatures much above 125.degree. will result in 
denaturing the proteins rendering the process ineffective for recovering 
high quality protein with good functionality. 
In the preferred process, a two-step extraction technique is utilized. The 
first step of extraction involves the solubilization of the glutelins. 
This step involves adjusting the pH of the slurry to a range of from about 
9 to about 12 by the addition of a suitable alkali. The step of adjusting 
the pH may comprise adding at least one of sodium hydroxide and potassium 
hydroxide in a quantity sufficient for adjusting the pH of the slurry to 
the specified range. Where it is important that a low sodium end product 
be obtained, then potassium hydroxide is the preferred agent. The slurry 
is agitated during the addition of the pH adjusting hydroxide. 
The pH adjusted water/corn slurry is then allowed to dwell while being 
agitated for a predetermined interval of time to allow for the 
solubilization of glutelin. A residence time of from thirty to sixty 
minutes is generally sufficient. 
In the second step, the slurry is then stirred and sufficient alcohol 
(preferably ethanol) is added to achieve a solution in which the ethanol 
constitutes at least about 50% of the slurry by weight. The upper limit of 
alcohol content preferably is not more than 80% by weight. 
The alcohol/water/corn slurry is then allowed to dwell while being agitated 
for a predetermined interval of time. At this point in the process, the 
prolamines or zein containing proteins enter solution. A residence time of 
from thirty to sixty minutes is generally sufficient. However, the slurry 
may be allowed to dwell for a time interval ranging from about thirty 
minutes to about two hours, during which time the slurry is agitated. 
These two steps for extracting prolamines and glutelins and globulins may, 
with some starting vegetable matter, produce a higher yield of end product 
if reversed in sequence. That is, while the preferred sequence for a high 
lysine corn is as described hereinabove, it is contemplated that other 
starting materials may provide a higher yield if the first extraction is 
for glutelin and the second for prolamines. Further, while here described 
as two separate and sequential steps, it may be possible with some 
materials to essentially combine the two treatment steps and permit only a 
single period of residence or resting for extraction of all proteins. 
During the extraction steps in the process for producing protein and intact 
starch granules and cereal grains, the temperature is maintained within 5 
degrees of 125.degree. F. Further, the slurry is permitted to dwell for an 
interval of time in the range of from about thirty minutes to about two 
hours, during which time the slurry is agitated. 
In any event, the steps of extraction are followed by a step of separating 
the slurry into two portions, a high solids content portion and a high 
liquids content portion. The two portions are here described in that 
manner out of a recognition that it is not easily possible to complete the 
entire separation into wet and dry portions in a single step. Thus any 
attempt to separate solids from the slurry will of necessity carry a small 
quantity of liquids, and vice versa. The separation should be by settling, 
possibly with the use of aids such as biopolymers and rapid settling 
equipment, and may be with the further aid of devices such as a 
centrifuge. The high liquid portion, or supernatant, is withdrawn 
following the separation process. 
The withdrawn supernatant contains the proteins which are in the process of 
becoming separated, and is next purified in a cross-flow microfiltration 
step with inorganic or organic membranes. At the present time, an 
inorganic membrane, namely aluminum oxide, is preferred. This 
microfiltration yields a retentate with a high purity protein content, 
which upon undergoing diafiltration will yield protein isolates with 
purities exceeding 90%. The zeta-potential of the aluminum oxide membrane 
is zero around pH 8-9, and of the prevailing high pH-values of 9-12, both 
the membrane and the proteins are charged negatively, thus repelling one 
another and yielding the retentate as being the product. The retentate is 
then concentrated until the degree of concentration is acceptable for 
spray drying or some other drying procedure which will yield a dry powder 
isolate useful as a foodstuff or food supplement. A liquid protein 
concentrate or isolate may also be produced for use in, for instance, 
nutritional beverages or pharmaceutical applications. 
Alternatively, after separating the high liquid content portion and the 
high solids content portion, the high liquid content portion is 
centrifuged at a g-force in the range of 10,000-15,000.times.g to recover 
dispersed particulate matter. 
After recovery of the ethanol solvent before or after microfiltration, for 
concentration and recycling, the high solids content portion of the slurry 
is concentrated and, where ethanol is the desired end product, then passed 
to a fermentation process as a feedstock. The fermentation process may be 
any of those known to persons skilled in the applicable arts, including a 
bacterial fermentation process for the production of butanol or a yeast 
fermentation process for the production of ethanol. Such processes and 
their other by-products being known from other disclosures, they will not 
be here discussed at length. The fermentation process may be concluded 
with distillation or with other separation procedures such as 
pervaporation to yield the product streams desired. 
Alternatively, when intact starch granules is the desired end product, the 
high solids content portion of the slurry is washed, cleaned, dried, and 
the pH reduced to approximately 6. 
If the corn or other starting material used has an incomplete amino acid 
balance, it is, of course, possible to add some natural or synthetic amino 
acid of the deficient type to bring the end product into appropriate 
balance. Such additions may be made to the dry isolate powder along with 
any other materials necessary or appropriate for flowability, handling or 
the like. In regular corn, the ethanol soluble protein fraction is larger 
than the alkali soluble fraction, with the two together making up some 
80-90% of the total protein of the corn. In a high lysine corn, the Opaque 
2 gene changes both proportions of these protein fractions as well as the 
lysine content within the various protein groups. It is for this reason 
that, with various starting materials, various process parameters must be 
adjusted to optimize the yield obtained from the particular starting 
material. 
EXAMPLE I 
In development of this invention, a quantity of Crow's high lysine corn was 
ground in a roller mill to a particle size of about one sixteenth inch and 
six pounds of such dry ground corn was mixed with 5.1 liters of pure water 
to form a slurry. The corn contained 9.5% moisture and 8.56% protein, thus 
starting the example with 233 grams of protein in the slurry. The slurry 
is then passed through an attrition mill where the corn was further ground 
to a sufficiently small particle size (approximately 1-50 micron) that the 
bond between the protein and starch in the starch/protein matrix was 
broken. The slurry was stirred, heated to 125.degree. Fahrenheit, the pH 
adjusted to 11.4 with NaOH, and sufficient ethanol added to increase the 
total volume to 18 liters. The slurry was agitated for thirty-five minutes 
for protein extraction, and was then allowed to settle for two hours, 
fifteen minutes. Subsequently, the pH of the resulting slurry was adjusted 
to about 8 by adding sulfuric acid. Careful decantation yielded over 10 
liters of supernatant containing 18.9 g/L of protein (an 82% extraction), 
which was then fed to a microfiltration module. The microfiltration module 
was equipped with a 0.2 micron aluminim oxide membrane. During filtration, 
temperature was maintained at 123.degree. Fahrenheit and feed pump 
pressure at about 90 psig. The cross-velocity was 8.36 M/sec and average 
transmembrane pressure was about 60 psig. An 18 second backwash with 
120.degree. water at pH 12 was applied every four minutes. The flux 
obtained was 170 L/M.sup.2 /H. The retentate from the process contained 
5.8 g/L of protein with a purity of 75%. The remaining impurities were 
minerals and some fat which were removed by dialysis to bring the protein 
purity to 90.4%. The retentate was then concentrated to about 50 g/L in a 
rotary vacuum evaporator and spray dried to form a powder isolate with 
about 12% moisture content and over 90% purity. A small amount of a silica 
based antistatic agent was added to facilitate the spray drying step. 
After the process was completed, the filter membrane was regenerated to 
achieve the same flux using water as had been the case prior to the 
process illustrated. 
A later study with defatted (as described in Example II) corn resulted in 
the extraction of 83.4% of the protein originally present in the corn in 
the two-step extraction process (60 minutes at 125.degree. F. and pH 11.6; 
60 minutes at 125.degree. F. and 68% v/v ethanol). After settling and 
centrifugation of the supernatant (15 minutes at 15,100.times.g) a 68.2% 
pure protein solution was obtained. Following microfiltration with a 0.2 
micron aluminum oxide membrane and backflush with compressed air plus 
diafiltration with 5.6 times the original volume of feed as DI-water, the 
retentate yielded a 98% purer protein isolate at pH 6.9. Spray drying 
resulted in a 98% pure protein isolate with 7.1% moisture. 
EXAMPLE II 
13 pounds of whole corn (hi-ly Opaque 2 from Crow) with 8.5% (weight to 
weight) protein on an as is basis were ground in a hammer mill with a 
5/64" screen. Total available protein was thus 502 grams. 
The corn was defatted with n-hexane under high vacuum in a modified Crown 
Iron Works machine at temperatures not exceeding 52.degree. C. 
(125.6.degree. F.) so as not to denature the protein. Ethanol or other 
solvents may be used for the defatting operations. The ground and defatted 
corn was mixed with deionized water into a 31 weight % slurry, which was 
then milled in a Koruma attrition mill to a particle size of 50-100 
microns. In doing so, the protein/starch matrix was broken. 
Next, under continuous stirring and at temperatures not exceeding 
125.degree. F., the protein was extracted in a two-step process. In the 
first step, the pH was raised to 11.3 with sodium hydroxide and a 45 
minute extraction time allowed. In the second step, ethanol was added to a 
final concentration of 60% by volume and another 45 minute extraction time 
allowed. The mixture was allowed to settle for 2 hours, and the 
supernatant decanted. 
A total of 82.1% of the available protein had been obtained in solution. Of 
the supernatant, 12.0 liters were centrifuged at 17,000.times.g. The 
centrate contained 17.43 g/kg of total solids with 10.97 g/kg of total 
protein (determined as total Kjeldahl nitrogen times 6.25). Its purity was 
62.9%. There followed cross-flow microfiltration and dialysis with a 0.2 
micron aluminum oxide membrane (Alcoa) yielding retentate (product in this 
case) of 6.5 liters with 15.58 g/kg of total solids and 14.08 g/kg of 
protein. Hence, the purity was 90.4%. The pH of the retentate was 7.3 
after 63.6 liters of DI-water were used for dialysis. This product was 
spray dried concurrently at exit temperatures of 218.degree. F., yielding 
a fluffy powder with 8.0% moisture. 
The protein in the permeate was not recovered here, however, that may be 
done in a second cross-flow microfiltration unit having a membrane with a 
smaller pore size. Utilizing a 100 Angstrom aluminum oxide membrane, a 
total of approximately 85% of the protein in the feed was recovered. 
Depending upon the protein content and make-up of the protein in the corn, 
from three to over four pounds of protein isolate product may be produced 
per bushel. The product pH may be at or near neutral. By controlling the 
purity through diafiltration until the proper pH (an almost linear 
relationship) is obtained, products with different functionalities may be 
produced. Functional properties as viscosity, water and oil absorption, 
gel strength, emulsification, solubility, wettability, etc. can be varied 
this way. 
The starch is cleaned and dried (spray dried). Due to the mild processing 
conditions, the starch granules are still intact. This is determined by 
electron microscopy, x-ray diffraction, digital scanning calorimetry, 
electron microscopy and Congo red application to a sample under a 
microscope. Such intact starch granules are appropriate raw material for 
new biodegradable plastics which will contain up to 75% starch. It may be 
used to produce pyrodextrins. It may be cross-linked to overcome the 
sensitivity of the granules to disruption while it improves the strength 
of the swollen granules. This way it can be used in surgical dusting 
powders, disposable sanitary products, antiperspirant sprays, etc. It may 
be extruded to yield instant laundry starches or pregelatinized for 
specialty food applications, etc. 
As will be understood, the present invention has disclosed processes 
capable of producing protein isolates, intact starch granules, and other 
by-products. The other products include butanol, ethanol, corn oil, 
dietary fiber, etc. and are produced by applying otherwise known 
technology. In the drawing and specifications, there has been set forth a 
preferred embodiment of the invention and, although specific terms are 
used, the description thus given uses terminology in a generic and 
descriptive sense only and not the purposes of limitation.