Protein isolate having low solubility characteristics and process for producing same

The present invention provides a process for the production of a protein isolate having low solubility with excellent functional properties as a complete or partial replacement for milk co-precipitate in a non-aqueous, "creme" filling used in cookies, snacks and the like. The process comprises; forming an aqueous slurry of an isolated soy protein; controlling the pH of the slurry to between about 4.5 to 5.8 by the addition of a monovalent alkali reagent; heating said slurry to a temperature of between about 170.degree.-240.degree. F.; and neutralizing said slurry to a pH of 6.8 to 7.2 in the presence of an alkaline earth cation to provide a low solubility protein isolate.

BACKGROUND OF THE INVENTION 
This invention relates to a low solubility vegetable protein isolate and a 
process for producing the same. 
Vegetable protein isolates, especially soy protein isolates have found a 
wide degree of utility as food ingredients. Typically, soy isolates are 
produced by processes which provide products with a relatively high 
nitrogen solubility index (NSI). Isolates with high NSI values generally 
have good dispersibility characteristics in aqueous systems and are 
preferred in food products where the protein isolate must also have a 
certain degree of functionality in the food product in addition to simply 
providing a source of protein. Functionality refers to the ability of the 
protein isolate to contribute to or interact with other ingredients of the 
food product in order to improve or maintain the desired chemical or 
physical characteristics in the food product. An example of this would be 
the addition of a protein isolate to an emulsified meat system wherein the 
protein isolate serves as a "binder" for the moisture and fat thereby 
improving the physical properties or texture of the meat system besides 
increasing or maintaining the desired level of protein. 
By contrast, there are certain food product applications of protein 
isolates where a high degree of solubility is not desirable. An example of 
this is a non-aqueous, "creme" filling of the type normally found in 
cookies or snacks. These fillings usually comprise a mixture of a milk 
co-precipitate, shortening and sugar. The filling must be very spreadable, 
yet retain its softness for a prolonged period of time. The use of a 
highly soluble protein isolate or one having a relatively high NSI results 
in the formation of a very hard and brittle filling when used as a 
replacement for a portion of the milk protein. Why this is the case is not 
completely understood, however, if a low solubility isolate or one having 
a low NSI value is employed, the filling material is usually much better 
with desirable softness and spreadability. On the other hand, insolubility 
is not the sole criteria since if the protein becomes too insoluble, a 
"gritty" undesirable texture results in the filling. It would, therefore, 
be desirable if a protein isolate could be produced which has selective 
insolubility characteristics to provide a filling with the above 
properties. 
It is, therefore, an object of the present invention to provide a low 
solubility protein isolate having the ability to function in a non-aqueous 
filling material. 
It is also an object to provide a low solubility isolate having the 
physical ability to form a smooth, creamy, and spreadable filling. 
It is an object to produce an isolate which will function in this fashion 
by a process that imparts selective insolubility characteristics to the 
isolate. 
It is also an object to produce such an isolate by a process which is 
reliable and convenient to practice on a commercial basis. 
SUMMARY OF THE INVENTION 
These and other objects are achieved in the present invention by a process 
for producing a low solubility protein isolate, that functions quite well 
in a non-aqueous creme filling of the type noted above. 
The low solubility protein isolate of the present invention having a 
nitrogen solubility index (NSI) below 20 is produced by a process 
comprising; forming an aqueous slurry of an isolated soy protein; 
controlling the pH of the slurry to between about 4.5 to 5.8 by the 
addition of a monovalent alkali reagent; heating said slurry to a 
temperature of between about 170.degree.-240.degree. F.; and neutralizing 
said slurry to a pH of 6.8 to 7.2 in the presence of an alkaline earth 
cation. The temperature range for heating of the slurry, the type of 
alkali used to adjust the pH prior to heating, and the stage of the 
process at which the alkaline earth cation is present in the heated slurry 
are all critical parameters which collectively provide the protein isolate 
with the desired degree of insolubility to serve as a partial or complete 
replacement for milk protein in products that require good softness, 
smoothness, and spreadability. 
Certainly, the application of more heat to the slurry can also insolubilize 
the protein, however, even if the nitrogen solubility index is reduced to 
below 20 by the use of more heat, the protein tends to form a "gritty" 
texture in a non-aqueous "creme" filling. Likewise, if the alkaline earth 
is added prior to the application of heat, even when the slurry is heated 
to within the above temperature range, the addition of alkaline earth 
cation at this point in the process results in a filling which is also 
handicapped by a gritty texture. 
Therefore, the above combination of steps and the order thereof, result in 
a protein isolate having a low nitrogen solubility index (NSI), which 
functions well in a non-aqueous creme type filling, providing such a 
product with good smoothness, spreadability and overall appearance. These 
functional characteristics are believed to be achieved by the selective 
degree and manner in which the protein source has been insolubilized. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The isolated soy protein employed as the starting material in the present 
process is obtained from whole soybeans in the following manner. The whole 
soybeans or vegetable protein material which form the starting material of 
the instant invention are preferably defatted and the oil is extracted to 
leave the soybean meal or flakes. More specifically, the soybeans may be 
crushed or ground and passed through a conventional oil expeller. However, 
it is preferable to remove the oil by solvent extraction techniques, using 
aliphatic hydrocarbons such as hexane, or azeotropes thereof, and these 
have been conventionally employed for this purpose. Following extraction 
to remove residual lipids and oil, vegetable protein flakes having a high 
degree of dispersibility are obtained. 
Following grinding or treatment of the vegetable protein material, the 
resulting solids, which in the case of soybeans are commonly referred to 
as soybean flakes, contain many ingredients including complex proteins, 
sugars, fibers, and various other types of materials. Initially, these 
proteins and many of the soluble sugars are dissolved from the other 
constituents of the soybean meal including cellulosic fibers and the like. 
This is accomplished by placing the flakes in an aqueous bath to provide a 
mixture having a pH of at least about 6.5 and preferably between about 7.0 
and 10.0. Typical alkaline reagents which may be employed if it is desired 
to elevate the pH above 6.5 include sodium hydroxide, potassium hydroxide, 
calcium hydroxide, or other commonly accepted food grade alkaline reagents 
and the present invention is not intended to be limited in this respect. A 
pH of above about 7.0 is generally preferred since an alkaline extraction 
facilitates solubilization of the protein, although the exact pH which may 
be employed should not be considered as limiting the present invention. 
Typically, the pH of the aqueous extract will be at least about 6.5 and 
preferably between about 7.0 to 10. The ratio by weight of the aqueous 
extractant to the vegetable protein material that is typically employed in 
the instant process is between about 5 to 20 to 1 and preferably is 
employed at a ratio of about 10:1. 
It is also desirable in the process of the instant invention that an 
elevated temperature be employed during the aqueous extraction step to 
facilitate solubilization of the protein, although ambient temperatures 
are equally satisfactory if desired. The exact temperature which may be 
employed is not intended to limit the instant process for the isolation of 
protein, although the actual temperature of extraction may range from 
ambient up to 120.degree. F. with a preferred temperature of 90.degree. F. 
The period of time for extraction with the aqueous medium is further not 
intended to limit the present invention and a period of time between about 
5 and 120 minutes may be conveniently employed with a preferred time of 
about 30 minutes. 
Following extraction of the vegetable protein material, the aqueous extract 
of protein can preferably be stored in a holding tank or suitable 
container while a second extraction is performed on the insoluble or 
residual solids from the first aqueous extraction step. It is desirable to 
carry out a second aqueous extraction of the insoluble solids remaining 
after the first extraction step in order to remove any residual protein 
which may not have been removed during the initial extraction and thereby 
increase the efficiency and yield of the instant process. If so desired, 
however, this additional step may be completely avoided and one may 
proceed, if desired, immediately to the precipitation of the protein. 
The insoluble solids from the first extraction is extracted with a second 
portion of an aqueous extractant to provide a second aqueous protein 
extract. The second extraction of the spent flakes or insoluble solids is 
carried out in a similar manner to the first extraction step at a pH of at 
least about about 6.5 or preferably between about 7.0 to 10.0. In the 
second extraction, however, it is preferred that a ratio by weight of 
extractant to the ground protein material is between about 4 to 15 to 1 
and preferably is at a ratio of about 6:1. Otherwise, the conditions of 
the second extraction are essentially the same as that of the first 
extraction and following extraction, the second aqueous protein extract is 
separated from the residual material and combined with the first aqueous 
protein extract prior to the precipitation step which is hereafter 
described. 
The combined aqueous protein extracts at a pH of at least 6.5 or preferably 
7.0 to 10.0 are then precipitated by adjustment of the pH of the extracts 
to at or near the isoelectric point of the protein to thereby form an 
insoluble precipitate. This would typically be between about 4.0 and 5.0 
and preferably between about 4.4 and 4.6. The precipitation step may be 
conveniently carried out with the addition of a common food grade acidic 
reagent typically such as acetic, sulfuric, phosphoric, hydrochloric acid, 
or with any other suitable acidic reagent. The isolated or precipitated 
soy protein is then formed into an aqueous slurry at a proteinaceous 
solids level of between about 4 to 25% by weight, preferably 10 to 20% by 
weight. The pH of the slurry is adjusted or controlled to a pH range of 
4.5-5.8, preferably 5.0 to 5.7 by the addition of a food grade 
neutralizing reagent. Typically, such a reagent will be a monovalent 
alkali reagent such as an alkali metal hydroxide such as sodium or 
potassium hydroxide, or another hydroxide such as ammonium hydroxide. 
Alternatively, various other monovalent alkali reagents such as monovalent 
alkali carbonates and the like may be used. As noted, hereafter and as 
will be shown in the Examples, it is essential not to employ a divalent 
alkali such as calcium hydroxide and the like at this stage of the 
process, since the addition of this cation prior to heating results in 
undesirable textural properties in the isolate produced by such a process. 
Typically, the slurry is formed at room temperature or heated to about 
70.degree. F. to form a relatively uniform slurry. 
Following formation of the slurry, the slurry is heated to a temperature of 
between about 170.degree.-240.degree. F., preferably 
180.degree.-200.degree. F. The slurry can be heated by the injection of 
steam into the slurry by any suitable steam injection device or can be 
heated by an indirect heating drive such as a heat exchanger and the like 
wherein direct contact between the heating medium and the slurry is 
avoided. The particular means used to heat the slurry is not critical 
relative to the practice of the present invention. 
Upon achieving the noted temperature, an alkaline earth cation is added to 
the slurry. The alkaline earth cation may be added directly to the heated 
slurry or alternatively the slurry may be cooled to 160.degree. F. or so 
to minimize bacterial propagation followed by neutralization. As 
previously noted, it is important that the alkaline earth cation be added 
subsequent to heating of the slurry within the desired temperature range. 
If the alkaline earth cation is added prior to heating, the protein is 
insolubilized to an NSI value below 20, however, when used in the "creme" 
type filling a "gritty" texture is achieved, indicating perhaps that the 
protein has reacted with the cation in same fashion to provide a product 
that lacks the desired textural properties. 
The addition of the alkaline earth cation can be carried out in several 
different ways. Since neutralization of the slurry to a pH of 6.8-7.2 
takes place following heating, a preferred means of adding the required 
percentage of alkaline earth cation is simply to use an alkaline earth 
hydroxide such as calcium or magnesium hydroxide to adjust the pH of the 
slurry to within the desired range of 6.8-7.2. In this manner, the 
required level of alkaline earth cation of 0.25 to 1.0% by weight of the 
solids is achieved simultaneously with neutralization of the slurry. 
Alternatively, an alkaline earth cation such as Mg.sup.++ or Ca.sup.++ 
can be added in the form of a salt to achieve the desired level of 0.25 to 
1.0% alkaline earth cation by weight of the solids in the slurry without a 
substantial change in the pH of the slurry, followed by neutralization to 
the pH range of 6.8-7.2 by the addition of any type of food grade 
neutralizing agent such as sodium or potassium hydroxide. The present 
invention is, therefore, not intended to be limited by the order in which 
alkaline earth cation addition or pH adjustment is carried out subsequent 
to heating. 
Following neutralization of the slurry, the slurry or suspension is then 
dried to a powder having a moisture content of about 3-5% preferably by a 
flash drying process such as spray drying. 
The dried product is a protein isolate of relatively low solubility having 
a nitrogen solubility index of below about 20. The product has unusual 
properties in the whole or partial replacement of milk co-precipitate in 
non-aqueous fillings for cookies, snacks and the like. As previously 
noted, such a filling must be smooth, soft, and spreadable and 
additionally must retain these properties upon storage. The above product 
provides the filling with this unique combination of properties.

To insure a complete understanding of the present invention, the following 
Examples are set forth to illustrate the process by which the product of 
the present invention is obtained, the criticality of certain steps in the 
process, and the unique properties of the product of the present invention 
in certain product applications. 
EXAMPLE 1 
An aqueous slurry of insolated soy protein was formed having a pH of 4.5 
and a solids level of 17% by weight. The slurry was maintained at a 
temperature of 70.degree. F. and the pH of the slurry was adjusted to 5.5 
by the addition of sodium hydroxide. 
Steam was injected into the slurry until the slurry reached a temperature 
of about 200.degree. F. The slurry was held at the noted temperature for a 
period of 15 minutes. The heated slurry was homogenized at 2,000 psi and 
adjusted to a pH of 6.8 by the addition of 1.5% by weight of the total 
solids of calcium hydroxide. The slurry was spray dried to a moisture 
level of below about 3% by weight. 
Analysis of the spray dried product showed the following average values. 
______________________________________ 
% Protein % Moisture NSI* 
______________________________________ 
89.6% 2.49% 9.7 
______________________________________ 
*The nitrogen solubility index (NSI) values expressed herein are based on 
the following general test procedure. 
A 10-gm. aliquot of the sample to be tested is added to a Waring Blendor 
cup having 200 ml. of distilled water. 
The blended slurry is transferred quantitatively to a 400 ml. beaker, using 
small rinses when needed. The pH of the transferred slurry is adjusted, if 
necessary, to a pH of 6.7-6.9 with 1 N NaOH or 1 N HCI. The suspension is 
then stirred for 15 minutes at slow speed with care so as to avoid 
creating a vortex action or stirring in air. The slurry is diluted to 500 
ml. with distilled water. 
A 250 ml. aliquot is centrifuged, using a 250 ml. cup, for 10 minutes at 
2,500 r.p.m. (1600.times.g). 
A 50.0 ml. aliquot of supernatant free of suspended particles (if 
necessary, filter) is transferred to a Kjeldahl flask and the nitrogen 
content is determined by the Kjeldahl method. (A smaller aliquot may be 
used if the soluble nitrogen content is unusually high). 
The percent by weight of soluble nitrogen of the sample is calculated based 
on 1/10 of the weight of the material initially used, as follows: 
##EQU1## 
To evaluate the effectiveness of the above isolate as a partial replacement 
for milk co-precipitate in creme type fillings, a sample of the above 
product was used in preparing such a filling, followed by an examination 
of the textural properties of the filling material. 
The formula used in preparing the non-aqueous "creme" filling was as 
follows: 
______________________________________ 
Ingredient % by weight 
______________________________________ 
Isolated Soy Protein 14.86% 
Milk Co-precipitate 14.86% 
Shortening 34.69% 
Powdered Sugar 35.59% 
______________________________________ 
The filling was prepared by forming a "creme" of the sugar and shortening 
by mixing for 3 minutes with a paddle type mixer. The temperature of the 
"creme" was raised to 80.degree. F., with the isolated soy protein and 
milk co-precipitate being immediately added, followed by mixing for 3 
minutes. 
The filling was evaluated subjectively for spreadability and was determined 
to spread well. The filling had an overall satisfactory color and 
appearance with a smooth mouthfeel. The filling also had a softness which 
corresponds to a filling prepared with milk co-precipitate as the only 
proteinaceous ingredient. On a subjective evaluation scale of 1 to 3 with 
1 being the very best and 3 having the poorest properties, the filling 
containing the isolated soy protein had a rating of 1. 
EXAMPLE 2 
To illustrate the importance of certain steps in the process of the present 
invention, the following products were prepared. 
600 lbs. of isolated soy protein having a pH of about 4.5 was formed into 
an aqueous slurry at a temperature of 70.degree. F. having a solids 
content of 20% by weight. 
1.2% by weight of the solids of calcium hydroxide was added to adjust the 
pH of the slurry to 5.5. 
Steam was injected into the slurry to heat the slurry to a temperature of 
200.degree. F. The slurry was held at this temperature for 15 minutes and 
then was split into two portions designated IA and IB. 
Heated protein IA was neutralized to a pH of 6.8 with calcium hydroxide. 
Following neutralization the solids level was reduced to 16-17% by the 
addition of water and spray dried. 
Heated portion IB was neutralized to a pH of 6.8 by using a 50% solution of 
sodium hydroxide. Following neutralization, the solids level was reduced 
to 16-17% by the addition of water and spray dried. 
Another 600 lb. batch of isolated soy protein having a pH of about 4.5 was 
formed into an aqueous slurry having a solids content of 17% by weight and 
a temperature of 70%. 
The pH of the slurry was adjusted to 5.5 by the addition of a 50% solution 
of sodium hydroxide. Steam was injected into the slurry to heat the slurry 
to a temperature of 200.degree. F. The slurry was held at this temperature 
for 15 minutes, the solids level was reduced to about 15% by the addition 
of water and then was split into two portions designated IIA and IIB. 
Heated portion IIA was neutralized to a pH of 7.0 by the addition of 
calcium hydroxide. Following neutralization, the slurry was spray dried. 
Heated portion IIB was neutralized to a pH of 6.8 by the addition of a 50% 
solution of sodium hydroxide. Following neutralization, the slurry was 
spray dried. 
Each of the products from Portions IA, IB, IIA, and IIB were subjected to 
the following proximate analyses set forth in Table I together with the 
various processing variables involved. 
TABLE I 
______________________________________ 
Proximate Analysis of Products 
Sam- Moisture Protein 
ple Processing Variables 
% % NSI 
______________________________________ 
IA Before heat addition of Ca.sup.++ 
4.38 88.8 3.5 
Neutralized to pH 6.8 with Ca.sup.++ 
IB Before heat addition of Ca.sup.++ 
4.0 89.3 5.7 
Neutralized to pH 6.8 with Na.sup.+ 
IIA Before heat addition of Na.sup.+ 
3.83 89.7 7.4 
Neutralized to pH 6.8 with Ca.sup.++ 
IIB Before heat additon of Na.sup.+ 
3.99 88.6 16.3 
Neutralized to pH 6.8 with Na.sup.+ 
______________________________________ 
Each of the products from Runs IA, IB, IIA, and IIB were evaluated as a 
partial replacement for milk coprecipitate in a non-aqueous creme filling 
as set forth in Example 1 and these results are set forth in Table II. 
TABLE II 
______________________________________ 
Evaluation of Products in "Creme" Filling 
Sample Spreadability 
Mouthfeel Overall Ranking* 
______________________________________ 
IA OK Gritty 3 
IB OK OK 2 
IIA OK OK 1 
IIB OK OK 2 
______________________________________ 
*Scale of 1 to 3 with 1 being best and 3 being poorest. 
It may be seen from the above evaluation that the best filling was obtained 
from an isolate produced by a process which employed neutralization 
subsequent to heating with an alkaline earth hydroxide and avoided the use 
of an alkaline earth cation to adjust the pH prior to heating. 
EXAMPLE 3 
400 lb. of isolated soy protein having a pH of about 4.5 was formed into a 
slurry having 15% solids. The pH of the slurry was adjusted to 6.1 by the 
addition of a 50% solution of sodium hydroxide. 
The slurry was heated by steam injection to a temperature of 305.degree. 
F., followed by neutralization of the heated slurry with calcium hydroxide 
to a pH of 6.8. The neutralized slurry was spray dried to a powder having 
the following proximate analyses. 
______________________________________ 
Protein % Moisture NSI 
______________________________________ 
89.4 3.69% 70.9 
______________________________________ 
To evaluate the effectiveness of the above product in a non-aqueous "creme" 
type filling, such a product was prepared as set forth in Example 1. The 
filling was evaluated for textural properties as set forth in Table III. 
TABLE III 
______________________________________ 
Spreadability Mouthfeel Overall Ranking* 
______________________________________ 
Failed Very dry 3 
______________________________________ 
*Scale of 1 to 3, with 1 being best and 3 being poorest. 
It may be seen that when the temperature range for heating of the slurry 
prior to neutralization is exceeded that the isolate obtained has poor 
textural properties when employed in a non-aqueous creme filling. 
EXAMPLE 4 
An aqueous slurry of 200 lbs. isolated soy protein having a pH of 4.5 was 
formed having a solids level of 20% by weight. The slurry was maintained 
at a temperature of 70.degree. F. and the pH of the slurry was adjusted to 
5.5 with sodium hydroxide. 
Steam was injected into the slurry until the slurry reached a temperature 
of about 200.degree. F. The slurry was held at the noted temperature for a 
period of 15 minutes. The heated slurry was then neutralized by the 
addition of calcium hydroxide to a pH of 6.8. The slurry was then spray 
dried into a product having the following proximate analyses. 
______________________________________ 
Protein % Moisture % NSI 
______________________________________ 
88% 4.39 5.2 
______________________________________ 
The above product was evaluated in a non-aqueous "creme" type filling of 
the type set forth in Example 1. The filling was evaluated for the 
textural properties set forth in Table IV. 
TABLE IV 
______________________________________ 
Spreadability Mouthfeel Overall Rating* 
______________________________________ 
OK OK 1 
______________________________________ 
*Scale of 1 to 3, with 1 being best and 3 being the poorest. 
It may be seen that an isolate produced according to the critical 
parameters set forth in the present invention provided the desired 
textural properties in the non-aqueous filling which were not achieved 
when isolates not processed pursuant to the present invention were 
employed. 
While the above Examples set forth specific embodiments of the present 
invention, it is to be understood that reasonable modifications or 
substitutions may be made without departing from the spirit or scope of 
the present invention.