Note: Descriptions are shown in the official language in which they were submitted.
8~
The invention relates to novel and useful food com-
positions comprising an edible fat and a source of protein,
said source of protein comprising 0 to 50% by weight of a
milk protein and 50 to 100~ by weight of a microbial source
of protein selected from the group consisting of microbial
protein, certain metal salts of microbial protein, certain
derivatives of microbial protein, heat denatured microbial
protein and mixtures of microbial protein and whey powder and
preparations thereof.
The invention further relates to novel and useful marsh-
mallow, non-dairy but~ermilk, cake and textured vegetable
protein food compositions wherein microbial proteins, certain
derivatives and mixtures thereof with whey powder are
advantageously employed.
There have been numerous publications in both the
technical and patent literature dealing with methods of
production of microbial cells for application in foods as
: well as processes for isolating protein-enriched fractions
from these cells. By the terms "microbial protein" and
"microbial protein isolate" as used herein is meant the
protein-enriched fraction obtained from disrupted cells of
yeasts, bacteria and fungi which fraction has been treated
such that it is relatively f~ee of other cell components.
Se~eral methods have been described in the art for disruption
of ~he cell walls of microorganisms to release cell components
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followed by isolation of the microbial protein released.
These proteins have been recognized as being nutritious and
their use in human food has been proposed.
United States 3,784,536 and United States 3,833,552,
disclose methods of isolating microbial proteins by treatment
of cells in aqueous or aqueous ethanolic medium containing
mineral acid at a controlled temperature. The latter patent
provides a water soluble whippable protein for food use.
United States Patent 3,821,080 provides a process
for extracting protein from microorganism cells which com-
prises rupturing the cells by mechanical disintegration in a
strongly alkaline medium and thereafter recovering the
extracted proteins. United States Patent 3,845,222 discloses
a process whereby an aqueous paste of microbial cells is
heated while applying a shearing force to the hot paste,
which is then extruded to obtain a product which resists
dispersion in water.
Yeast protein is produced by the method of United
States Patent 3,867,555 by separating the soluble fraction
obtained from ruptured yeast cells from the cell wall debris
followed by hydrolysis of the nucleic acid with alkali at pH
9.5-12.5 and 50~120C., after which the protein is isolated
by isoelectric precipitation. United States Patent 3,887,431
utilizes endogenous nuclease to hydrolyze nucleic acids pres-
ent in alkaline solution after cells are ruptured; followingthis treatment the protein is precipitated.
In United States 3,891,772 a process is set forth
for extraction of undesirable flavor and odor components
from microbial cells by treatment in a slurry of aqueous
alcohol containing 60~ to 80% by volume of an alcohol having
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1 to 3 carbon atoms, wherein the weight ratio of alcoholic
solution to cells is in the range from 3:1 to 7:1. The
extracted cells are then recovered.
British Patent 1,372,870 provides a method for
improving the nutritional and functional properties of
protein derived from unicellular microorganisms by maintain-
ing an ammoniacal slurry of cells at 60 to 140C. and at a
pH of 8.0 to 11Ø The isolated product has reduced nucleic
acid con~ent.
In Japanese Patent 9,124,292, methods are described
for extraction of protein from microbial cells. The cells
are first defatted by an organic solvent, gel filtered to
remove low molecular weight substances, the protein is ex-
tracted into aqueous alkali, then precipitated.
Japanese Patent 9,001,790 discloses a method by
which microbial cells are heated at 80-220C., under high
pressure, greater than 10 atmospheres, of a gas containing
more than 22~ oxygen to improve the digestability of
microbial protein. By the process of Japanese Patent
4,014,760 the extraction of protein from yeast cells is
improved by homogenizing ~he cells in an alkaline medium
under a pressure above 100 kg./cm2. A method for rupturing
cells grown on hydrocarbon feed stock by autolysis is dis-
closed in United States 3,268,412 wherein autolysis is
brought about by means of enzymes contained in the micro-
organism or by addition of other enzymes.
Various methods for disrupting microbial cells and
equipment used for this purpose are reviewed in "Methods in
Microbiology" edi~ed by Norris and Ribbons, acad~mic Pr`ess, ~eW York,
N. Y., 1971, Volume 5A, pp. 363-368; Volume 5B, pp. 1-54.
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Compositions o demineralized dry whey solids and
either monosodium phosphate or insoluble metaphosphate which
exhibit functional properties in food compositions are dis-
closed in United States 3,615,662 and comparable products
employing alkali polyphosphates are provided by United States
3,620,757. Undenatured lactalbumin phosphate food composi-
tions wherein said phosphate derivative replaces a portion
of the egg white normally contained is set forth in United
States 3,706,575.
Acetylated animal and vegetable proteins for food
use are previously described in United States 3,619,206;
United States 3,764,~11; United States 3,782,~71 and
British Patent l,294,664.
The novel food compositions of the invention employ
a microbial source of protein to replace from about 20 to
100% by weight of certain protein-containing ingredients
ordinarily used in such food compositions. The microbial
sources of protein are especially effective in replacing
vegetable proteins such as soy protein, wheat gluten, egg
white, gelatin, proteinaceous foaming agents and milk protein
sources such as nonfat dry milk, sodium caseinate, calcium
caseinate and magnesium caseinate. In replacin~ these
ordinary food proteins, the microbial source of protein can
replace both the functional properties and the food value of
the conventional food proteins, i.e., the microbial source of
protein serves as a functional pro~ein and is nutritious.
By the term "microbial source of protein", within
the context of this in~ention, we mean microbial proteins as
de~ined above, as well as certain metal salts o microbial
0 proteins, derivatives of microbial proteins, denatured
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microbial proteins and mixtures of microbial protein and whey
powder. Preferred metal salts are the sodium, calclum and
magnesium salts of microbial proteins. Preferred derivatives
are the acetylated derivatives of microbial proteins and the
S phosphate complexes of microbial proteins. Preferred de-
natured microbial proteins are the heat denatured microbial
proteins. Preferred mixtures of microbial protein and whey
powder are those containing from about 20 to 80% of whey
powder by weight; especially preferred are mixtures of
microbial protein and whey powder containing about 50% whey
powder by weight.
By the term 1I functional protein" we mean proteins
which primarily serve to improve the physical and/or organo-
leptic properties of a food and their nutritive value may be
of secondary importance in a particular food. Examples of
physical and organoleptic properties of foods which are
improved by a functional protein are water binding, fat bind-
ing, protein binding, emulsification, whippability, texture,
volume, dispersibility, gelation, viscosity, flavor, aroma,
color and the like. Examples of proteins known in the art
and the functions they ordinarily improve in foods are egg
albumen for whlppability, color or as a binder of other
pr~teins; egg yolk for emulsification, color or flavor; soy
protein ~or water binding, fat binding, tex~ure and
whippability; gelatin for gelation; milk proteins and their
salts for water bindingt fat binding, flavor, texture;
whippability, emulsification and heat stability: and wheat
gluten for water binding, texture and flavor.
The microbial sources of protein of the invention
0 have now been found to replace functional proteins known in
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the prior art such as the vegetable proteins, egg white,
gelatin, edible proteinaceous foaming agents, milk protein
and milk protein salts.
~ y the term "edible proteinaceous foaming agents"
we mean commercially available edible proteins as well as
edible mixtures containing proteins, or modified proteins
and optionally containing other ingredient~ such as carbo-
hydrate~, chemically modified carbohydrate~, ~alts and
stabilizers, used as foaming and frothing agent~ in edible
compositions such as marshmallow, whipped toppings and in
packaged bar mixes for alcoholic beverages such as whiskey
sour and daiquiri mixes. Examples of such foaming agents
are egg white and mixtures of modified or unmodified
vegetable proteins with egg white, gelatin and gums. Ex-
amplee of such foaming agents which are ~old under tradenames
are Hyfoma*, a product of ~enderink and Co., Schiedam, the
Netherlands, which is a mixture of vegetable proteins, egg
white, sucrose and stabilizer; and Gunthers Foaming Protein*
1026 which i~ a mixture of modified 80y protein and gelatin
available from A. E. Staley Mfg Co., Proteln Division
Decatur, Illinois 62525. *Trademarks.
The microbial sources o~ prot2in of the invention
have now been ~ound to replace the above-mentioned prior art
functional proteins to provide superior food compositions.
The microbial sources of protein di6closed herein have now
been found to be surprisingly effective in their ability to
func~ion as fat binding and water binding agents. According-
ly, they have been found to be e~pecially effective in fat
containing food compo ition~ in which they repLace at lea t
a substantial portion of the conventional proteins used for
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their fat binding ability. The novel food compositions dis-
closed herein containing edible fat and a microbial source of
protein have been found to have improved physical and organo-
leptic properties when compared with their counterparts con-
taining conventional food proteins. Preferred food composi-
tions of the invention are those comprising an edible fat
and a source of protein in a weight ratio of said fat to said
source of protein of from about 25:1 to about 1:6, said
source of protein comprising from about 0 to 50% by weight of
at least one milk protein source selected from the group con-
sisting of nonfat dry milk, sodium caseinate, calcium casein-
ate and magnesium caseinate and from 50 to 100% by weight at
least one microbial source of protein selected from the group
consisting of microbial protein, sodium, calcium and magnesium
salts of microbial protein, acetylated microbial protein,
microbial protein phosphate complex, heat denatured microbial
protein and mixtures of microbial protein and whey powder con-
taining from about 20 to 80% whey powder by weiyht.
Classes of such food compositions included within
the scope of the present invention are baked goods, processed
meat products, chocolate flavored beverages, non-dairy
fermented milk products and desserts as well as whipped
toppings and coffee whiteners.
Examples of the above mentioned baked goods include
both yeast leavened and chemically leavened baked goods such
as bread, rolls, buns, doughnuts, pastries and cheese cake.
Examples of the above mentioned processed meat products are
meat loaf, frankfurters, sausages and meat~containing pet
foods. Examples of such non-dairy fermented milk products
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are yogurt, sour cream, cream cheese, process cheese, cheese
spread and cheese food products. Examples of such desserts
include bo~h frozen desserts such as ice cream, sherbet, ice
milk, mellorine and milk sherbet; non-frozen desserts such as
puddings; and confections such as cake icings or frostings,
caramel, fudge and cream filling.
In referring to the food compositions of the inven-
tion the term "source of protein" refers to -the microbial
source of protein and the conventional food proteins being
partially or completely replaced by the microbial source of
protein. ~owever, a given food composition may contain other
food proteins or protein-containing ingredients not included
in said source of protein.
In the food composi-tions of the invention compris-
li ing an edible fat and a source of protein, the edible fatincludes that derived from relatively pure sources of edible
fat such as animal and vegetable fats, vegetable oils,
shortenings, lard and the like, as well as the fatty portion
of other fat-containing food ingredients which contribute
about 1% or more to the total weight of edible fat in the
food composition. The fat content of said fat-containing
; food ingredients was taken from data set forth in i'Composi-
tion of Foods, Raw Processe , Prepared", Agricultural
H~ndbook No. 8, Agricultural Research Service, U.S. Depart-
ment of Agriculture, 1963.
In food compositions of the invention comprising
edible fat and a source of protein wherein said source of
protein comprises from about 0 to 50% by weight of at least
one milk protein selected from the group consisting of nonfat
dry milk, sodium caseinate, calcium caseinate and magnesium
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caseinate and from about 0 to 100% of at least one microbial
source of protein, the weight ratio of edible fat to said
microbial source of protein plus said milk protein may vary
from about 22:1 in cakes in which the entire source of
protein is from microbial protein and said microbial protein
is used at one-halE the level of the nonfat dry milk
ordinarily used; to a ratio of about 1:6 in non-dairy yogurt
compositions in which the protein source is entirely from a
microbial protein or mixtures of microbial protein and whey
powder containing about 50% whey powder by weight. Other
such food composi~ions of the invention contain edible fat
and said source of protein in ratios between 22:1 to 1:6.
In food compositions of the invention comprising
edible fat and a source of protein wherein said sources of
protein comprises from about 0 to 50~ nonfat dry milk and
from about 50 to 100% of a microbial source of protein, an
especially preferred microbial source of protein is a mix-
ture of microbial protein and whey powder containing from
about 20 to 80~ of whey powder by weight. ~specially prefer-
red mixtures of microbial protein and whey powder are thosecontaining 50% whey powder by weight. Said mixtures are
preferred because of their efficacy in replacing nonfat dry
milk and for reasons of economy. Classes of such food com-
positions employing said mixtures of microbial protein and
whey powder are baked goods, processed meat products,
chocolate flavored beverages, non-dairy fermented milk pro-
ducts and desserts. Examples of such baked goods include
both yeast leavened and chemically leavened baked goods such
as bread, rolls, buns, doughnuts, pastries and cheese cake.
Examples of such processed meat products are meat loaf,
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frankfurters, sausages and meat-containing pet foods Ex-
amples of such non-dairy fermented milk products are yogurt
and sour cream. Examples of such desserts are frozen des-
serts such as ice cream, sherbet, ice milk, mellorine, milk
sherbet; non-frozen desserts such as puddings; and confec-
t-i~ns-such as cake icings or frostings, caramel, fudge and
cream filling.
Any microbial protein and particularly those
proteins isolated from microorganisms classified as yeasts,
bacteria and fungi may serve to replace conventional proteins
in the food compositions of the invention. By way of illus-
tration, examples of genera of microorganisms suitable for
providing microbial proteins are yeasts of the genera
Saccharomyces, Candida, Hansenula and Pichia; bacteria of
the genera Pseudomonas, Lactobacillus, Streptococcus, Micro-
coccus, Cellulomonas, Arthrobacter, Bacillus, Hydrogenomonas
and Aerobacter; and fungi of the genera Trichoderma~ Fusarium,
Penicillium, Aspergillus, Neurospora and Endomycopsis. Ex-
amples of species of microorganisms suitable for providing
microbial proteins are listed in the following table:
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O ~ h
a~ 1~1
;~ a a
~ a~
00l~0l a~l~a~al~U~
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Preferred microbial proteins useful in the ood
compositions of the invention are those isolated from the
yeasts Saccharomvces carlsbergensis, Saccharomyces cerevisiae,
Saccharomyces fragilis and Candida utilis; the bacteria
Pseudomonas methylotropha, Lactobacillus bul~aricus,
Streptococcus lactis, Micrococcus cerificans and Cellulomonas
cartalyticum; and the fungi Trichoderma veride, Fusarium
solani, Penicillium chryso~enum, Aspergillus niger,
Aspergillus oryzae and Neurospora crasa.
The microbial proteins isolated after cell disrup-
tion may be further reacted to form certain derivatives to pro-
vide products which are also useful in many of the food com-
positions of the invention.
One such class of derivatives are the phosphate
complexes which result when an aqueous solution or suspension
of a microbial protein is contacted with an aqueous solution
of an alkaline phosphate salt such as potassium metaphosphate,
sodium hexametaphosphate, sodium polyphosphate, potassium
polyphosphate, monosodium phosphate and mixtur~s thereof.
Any of the above alkaline phospha~e salts and mixtures of
such salts may be contacted with any of the microbial proteins
in varying proportions of phosphate salt to microbial protein,
to provide microbial protein phosphate complexes which are
useful in the food compositions of the invention. However,
the preerred microbial protein phosphate complexes are those
obtained by contacting, in a~ueous solution, one part by
weight of a mixture of sodium hexametaphosphate and potassi~m
metaphosphate in a weight ratio from about ~:1 to 10:1 with
about 10 to 15 parts by weight of microbial protein.
The phosphate complexes of the above microbial
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proteins are especially useful in the whipped topping,
coffee whitener, cake frosting, angel food cake and meringue
topping food compositlons of the invention.
Another useful class of derivatives of the above
speci~ied microbial proteins which serve as a microbial
source of protein are the acetylated microbial proteins
which are obtained when the microbial proteins are acetylated
to varying degrees of acetylation by methods known in the art
for acetylation of proteins employing reagents such as acetic
anhydride, acetyl chloride and acetyl bromide; see for
example United States 3,619,206; United States 3,764,711 and
United States 3,782,971. While any of the acetylated
microbial proteins are useful in the food compositions of the
invention, we prefer those obtained by carrying out the
acetylation by dissolving one o~ the microbial proteins in
water containing a polyvalent salt such as trisodium citrate
which acts as a protein stabilizer. The resulting solution
is then adjusted to a pH in the range of about 5 to ~ and
treated with an acetylating agent such as acetic anhydride
or acetyl chloride while maintaining the pH in the above
specified range by periodic addition of strong alkali such
as sodium hydroxide solution. Especially preferred acetylat-
ed microbial proteins are those obtained by reacting microbial
protein and acetic anhydride in a weight ratio o~ from about
1:1 to 1:0.1 at a temperature in the range of about 15 to
35C.; an especially preferred range for the weight ratio of
microbial protein and acetic anhydride is from about 1:0.6
to 1:0.2.
The acetylated microbial proteins of the invention
are especially useful in fat containing food compositions of
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the invention such as whipped toppinys and coffee whiteners
in which they display enhanced fat binding functionality.
A further microbial source of protein which is of
use in food compositions of the invention comprising an edible
fat and said source of protein are the heat denatured
microbial proteins obtained by heat denaturing any of the
microbial proteins. While the heat denaturing of microbial
proteins may be carried out with satisfactory results at
temperatures as low as 50C. or at temperatures up to 150C.
or higher to obtain a source of protein useful in the food
compositions of the invention, we prefer the heat denatured
microbial proteins obtained by heat denaturing a microbial
protein at a temperature in the range of about 50~ to 100C.
In denaturing microbial proteins at tem~eratures within the above
range, it should be kept in mind that the denaturation of the
protein will proceed more rapidly at the higher temperatures
and slower at the lower temperatures in accordance with the
laws of thermodynamics. For example, at 100C. denatuxation
is essentially complete in about 5 minutes. While at 50C.,
one hour or more may be required. While the denaturation may
be carried out in the absence of solvent with satisfactory re-
sults, it may also be carried out conveniently by heating a
suspension or solution of microbial protein in waterO
The heat denatured microbial proteins thus obtained
are especially useful in the whipped topping and meringue
food compositions of the invention.
Also included as a microbial source of protein
within the scope of the present invention are mixtures of
the above described microbial protein isolates with up to
0 80% or more of whey powder. Preferred mixtures are those
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containing from about 20 to 80% by weiyht of whey powaer andespeciall~ said mixtures which contain about 50% whey powder
by weight.
Whey powder is a readily available food ingredient
commercially produced on a large scale from whole liquid
whey by modern evaporation techniques. ~n illustrative and
generally representative approximate compositiQn of whey
powder is:
Weight,
Protein 12.5
Fat 1.0
Moisture 4.5
Ash g.o
Lactose 73.0
100.0
For the purposes of this invention, whey powder of
the above approximate composition or any of the specially
treated whey powders such as demineralized whey powder and
whey protein concentrates obtained by techniques such as ion
exclusion, ultrafiltration, electrodialysis, reverse osmosis
or enzymatic methods utilizing, e.g., lactase, are also
suitable.
In many of the food compositions of the invention,
the microbial protein-whey powder mixtures perform as well or
better than the undiluted microbial proteins in replacing
conventional food proteins. Said mixtures-are especially
useful in replacing whole milk powder and nbnf~t dry milk in
food compositions. Nonat dry milk is also referred to here-
in as NFDM. ~In replacing e.g., NFDM, they are ordinarily
used to replace an equal weight of the conventional milk
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866
protein source. However, in certain cases they may be used
in an amount approximate~y one-half to twice that of the
protein being replaced to obtain superior food compositions.
The microbial protein-whey powder mixtures, which
are also referrad to herein as "nonfat dry milk substitute",
may be prepared by simply blending the two dry ingredients
in the desired proportions. However, to insure intimate mix-
ture of the components and to prevent separation due to
differences in particle size of the components, it is pre-
ferred to blend them first in water by mixing followed byhomogeni~.ation in a standard food homogenizer and pasturiza-
tion. The resulting mixture i5 then dried to a powder by
standard drying techniques used in the food industry such as
freeze drying, spray drying or vacuum drum drying.
Whipped toppings are substituted for whipped cream
which have found wide commercial acceptance in manufactured
baked goods and in the home as toppings for desserts. Whipp-
ed toppings are air-liquid emulsions in which the function
of proteins, such as the sodium caseinate ordinarily
employed, is to disperse the fat and water and to bind each
of these components, as well as to facilitate air entrapment
and stabilize the air so entrapped. It is also very important
that the protein employed performs these functions at the low
~eratures normally used for making and storing whipped
toppings. Sodium caseinate is widely used for this purpose
and soy protein is sometimes used, but is considered inferior
to the caseinates due to its inadequate functionality and
off-flavor. We have now found that the microbial sources of
protein of the invention and especially the above described
preferred acetylated microbial proteins, the preferred
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rnicrobial protein phosphate complexes and the preferred heat
denatured microbial proteins, can replace from about 50 to
100% by weight of the sodium caseinate in conventional
whipped toppings to provide superior products. The whipped
toppings containing said microbial sources of protein were
found to give significantly greater overrun as determined by
methods known in the art and also had a more creamy texture
and superior low temperature stability. A particularly effec-
tive microbial source of protein in whipped toppings is the
phosphate complex of C. utilis protein.
Coffee whiteners have been marketed for several
years and have proven to be effective substitutes for milk
and cream in coffee and tea. These products enjoy the
advantage of being less expensive than the corresponding
natural dairy products and the more popular dry coffee
whiteners have the further advantage of being stable upon
storage at room temperature, whereas milk and cream must be
refrigerated.
Proteins such as sodium caseinate are commonly used
in coffee whiteners to encapsulate fat or oil, to prevent
separation of fat and its coalescence to form fat globules
- on the surface of the hot beverage. The proteins also serve
to provide whitening power and reduce the acrid taste of
cof~ee. Proteins employed in coffee whiteners must perform
these functions under fairly harsh conditions, i.e., at a
pH in the range of about 4-5.5 and temperatures of about 50-
80C. Furthermore, the protein employed should perform
these functions with minimal heat denaturation in the hot
beverage which results in precipitation or feathering of the
protein. Very fëw proteins have been found to work adequate-
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866
ly in such a food system. Sodium caseinate is most widelyused for this purpose and has been considered to be the best
protein for this application. Soy protein has been used to
some extent but its performance is generally poorer and it
does not perform adequately in dry powdered coffee whitener
formulations. We have surprisingly fou~d that the microbial
sources of pro~ein of the invention perform well in coffee
whiteners. The coffee whitener compositions of the invention
have less tendency to feather and have improved whiteness and
flavor when compared with controls containing sodium caseinate
only. ParticuLarly effective as the microbial source of
protein in the coffee whitener compositions of the invention
are the microbial protein phosphate complexes and the acety~-
ated microbial proteins; said phosphata complex being obtain-
ed by contacting, in a~ueous solution, one part by weight ofa mixture of sodium hexametaphosphate and potassium meta-
phosphate in a weight ratio of from about 6:1 to 10:1 with
10 to 15 parts by weight of microbial protein; and said
acetylated microbial protein being obtained by reacting
microbial protein and acetic anhydride in a weight ratio of
from about 1:0.6 to 1:0.2 at a temperature in the range o~
about 15 to 35C.
The microbial sources of protein of the invention
and especially the microbial proteins, their sodium, calcium
and magnesium salts and their mixtures with whey powder have
been found to provide novel non-dairy fermented milk products
such as yogurt, sour cream, buttermilk, cream cheese, process-
ed cheese, cheese food produc~s and the like~ By "non-dairy
fermented milk products" within the context of this inven-
tion we mean a food composition made from reconstituted milk
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containing one or more proteins selected from the group of
milk proteins such as the caseinates, whey protein and nonfat
dry milk; vegetable proteins such as soy protein and
microbial proteins and salts thereof; optionally containing
other ingredients such as vegetable fat, butterfat,
saccharides such as sucrose or corn syrup solids, whey
powder, stabilizers, emulsifiers, food acids and the like;
and are only optionally fermented.
The consumption of both dairy and non-dairy ferment-
ed milk products has been increasiny steadily. The protein
ingredients in products like yogurt, sour cream, cream cheese
and buttermilk are responsible or the nutritional quality
of these products and the formation of acid curd. During the
lactic acid fermentation, which is the key reaction for the
lS manufacture of these products, the proteins precipitate and
form a gel. Milk proteins, namely, caseinates and whey
proteins (lactalbumin and lactoglubulin), present in fluid
milk and nonfat dry milk, are generally employed for this
purpose. Unexpectedly, it has been found that the microbial
sources o protein of the in~ention and especiall~ the above
me~tioned micro~ial proteins, the sodium, calcium and
magnesium salts of microbial proteins and the above mentioned
mixtures of micrGbial protein with whey powder can be sub-
stituted for the case ~ tes and nonfat dry milk to provide
superior quality non-dairy fermented milk products. In pre-
paring these products, the microbial protein, salts, or
mixtures thereof with whey powder, may optionally be treated
with a food acceptable acid such as lactic or citric acids,
or a mixture of such acids with the milk clotting enzyme,
rennet.
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The consumption of cheeses and cheese products has
increased steadily over the years. Recently, non-dairy
cheeses have gained increasing acceptance in school lunch
programs and by large manufacturers of frozen pizzas. Non-
dairy cheeses can be used effectively to lower the cost ofingredients and sodium, calcium and magnesium caseinate are
currently being used for this purpose along with vegetable
oils, minerals, vitamins and flavorings. We have found that
the microbial proteins of the invention and their sodium,
calcium and magnesium salts can be employed to partially or
totally replace caseinate salts in non-dairy cheeses and non-
dairy cheese products. These microbial sources of protein,
because of their superior water binding and fat binding
properties, afford non-dairy cheeses and non-dairy cheese
products with improvad texture and mouthfeel.
Especially effective in replacing sodium, calcium
and magnesium caseinate in the non-dairy ermented food com-
positions of the invention are the above mentioned microbial
pro~eins and the sodium, calcium and magnesium salts of said
~0 microbial proteins. Especially effective in replacing NFDM
in the non dair~ fermented milk products of the invention are
the above mentioned microbial proteins and mixtures of said
microbia~ proteins with whey powder containing from about 20
to 80% whey powder by weight and especially said mixtures
containing about 50~ whey powder by weight. In non-dairy
yogurt formulations said microbial proteins can effectively
replace 50% or more of both the sodium caseinate and NFDM.
When the above mentioned salts of the microbial
proteins are the preferred source o microbial protein for
use in the food compos~tions o~ the invention, they are pre-
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pared from the microbial proteins by methods well known in
the art. ~or example, the sodium, calcium or magnesium salts
are readily obtained by dissolving or dispersing the appropri-
ate microbial protein in water, followed by addition of base
such as ~odium hydroxide, calcium hydroxide or magnesium
oxide with stirring to adjust the pH of the resulting mixture
to ahout 9. The resulting dispersion or solution may be used
as is or can be dried to a powder by conventional dry~ing
methods.
In the above mentioned baked goods of the inven-
tion comprising an edible at and a source of protein, the
microbial sources of protein can replace NFDM to provide
baked goods with improved physical and organoleptic properties
such as increased volume, higher moisture content, improved
color, taste and mastication. Especially effective microbial
sources of protein in baked goods are the above mentioned
microbial proteins and their mixtures with whey powder con-
taining from about 20 to 80% whey powder by weight. In
employing said microbial proteins to replace NFDM in baked
~0 goods such as bread and cake we have surprisingly found that
as little as one-half the amount of microbiaI protein can
effectively replace all of the NFDM used in the control
products.
In many processed meat produ~ts nonfat dry milk is
incorporated during manufac~ure to reduce the amount of
shrinkage of these meat products during cooking. The shrink-
age, which i5 due primarily to loss o water and fat from the
mea~, is also reduced by the microbial proteins of the inven-
tion and their blends with whey powder. ~n processed meat
products as meat loaf, frankfurters, bologna, sausage and
-22-
: . , .-
~0~ 6
meat containing pet foods incorporation of the instant
microbial proteins and blends thereof with whey powder are
superior to NFDM on an equal weight basis and in some cases
can perform satisfactorily when used at one-half the level
of NFDM.
In chocolate flavored beverages NF~M is often used
at levels of 9% or more by weight for its functional
properties as well as serving as a nutritive protein source.
The microbial sources of protein of the invention and
especially the above mentioned microbial proteins and their
mixtures with 20-80% of whey powder can totally replace the
NFDM in such beverages to obtain a product of improved
~uality. Of course, mixtures of NFDM and the above mentioned
; ~ microbial sources of protein can also be satisfactorily used
in this application and others in which NFDM is being replaced.
In standard dessert formulations containin~ NFDM or
~ whole milk such as ice cream, sherbet, mellorine, ice milk,
- pudding and confections, the instant microbi~l sources of
protein can replace the milk solids or NFDM to provide
products which have surprising advantages when compared with
the controls. For example, with puddings such as starch
thickened puddings, the miGrobial protein containing foods
; show definite improvement in texture, mouthfeel and syneresis
upon evaluation when the microbial protein is used in an
amount equal to the NFDM replaced. When the microbial protein
used is only half of the amount of NFDM replaced the test
pudding shows no syneresis and is comparable to the control
pudding in all other characteristics evaluated. When the
microbial sources of protein pxovided herein are substituted
for NFDM in confection compositions such as for caramel candy,
-23-
'
,
fudge, cake icings, cream filling, chocolate and the like,
the products are superior in texture and gloss to their
counter-parts containing NFDM. Microbial sources of protein
that are particularly effective in dessert compositions of
the invention are the microbial proteins and mixtures of
microbial protein with whey powder containiny from about 20
to 80~ whey powder by weight. Especially preferred are the
proteins isolated from C. utilis and S. fragilis and their
mixtures with whey powder containing from 20 to 80% whey
powder by weight.
Another food composition in which the microbial
protein~ of the invention can replace NFDM without loss of
quality in the final product is pancakes in which L.
bulgaricus protein, as well as _. utilis, S. cerevisiae,
S. lactis, M. cereficans, C. cartalytlcum, T. viride,
P. chrysogenum, A. niger, F. solani and Cellulobacillus
mucosis pr~tein and their blends with whey powder, are
particularly useful.
In addition to the above described food compositions
comprising edible fat and a source of protein in a weight
ratio of said fat to said source of protein of from about
22:1 to about 1:6, wherein said source of protein comprises
from about 0 to 50% by weight of at least o~e milk protein
selected from the group consisting of NF~M and sodium,
calcium and magnesium caseinates and from 50 to 100~ by weight
of at least one microbial source of protein, the present
invention discloses novel and useful marshmallow and non-
dairy buttermilk compositions in which certain microbial
proteins and their mixtures with whey powder replace a sub-
-24-
. .
86 6i
stantial portion of the ordinarily employed food proteins
such as the NFDM, gelatin and edible proteinaceous foaming
agents ordinarily employed in marshmallow compositions and
the NFDM and sodium caseinate ordinarily employed in non-
dairy buttermilk compositions. Also disclosed are novelimproved cake compositions comprising sodium hexametaphosphate
and a source of protein in a weight ratio of from 1:13 to
1:38 wherein said source of protein comprises from about 0 to
30% by dry weight of egg white and from about 70 to 100~ by
dry weight of a microbial source of protein selected from the
group consisting of S. fragilis protein, L. bulgaricus
protein, P. chrysogenum protein and the preferred phosphate
complexes of each. Examples of superior cakes provided
according to this composition are white cake and angle food
cake.
Additionally, we have unexpectedly found that in
~ood compositions containing textured vegetable protein and
a protein binding agent, certain microbial proteins of the
invention can effectively replace from about 20 to 100% of
the egg white, soy flour and wheat gluten ordinaril~ employed
as protein binding agents.
Standard formulations for marshmallow employ, in
addition to sugarssuch as sucrose and glucose, protein sources such as
NFDM, gelatin and edible proteinaceous foaming agents such as
egg white and vegetable proteins or commercial foaming agents
containing, for example, mixtures of vegetable proteins, egg
white and other non-protein ingredients. The proteins employ-
ed aid in formation and stabilization of the foam and ef~ect
such properties as e.g., texture, taste, odor, smoothness and
spreadability. We have found that the microbial proteins of
-25-
66
the invention and their mixtures with whey powder can replace
substantial amounts of the above-mentioned protein sources
ordinarily used in marshmallow compositions to provide
superior products. Especially preferred are those marsh-
mallow compositions comprising a source of protein consistingof from about 25 to 50% by weight of a microbial source of
protein, 25 to 50% by weight of an edible proteinaceous foam-
ing agent and 25 to 50% by weight of at least one member
selected from the group consisting of NFDM and gelatin; said
microbial source of protein being a member selected from the
group consisting of C. utilis protein, S. fragilis protein,
P. meth~lotropha protein, F. solani protein and mixtures of
C. utilis, S. fragilis, P. methylotropha or F. solani protein
with whey powder containing from abou~ 20 to 80% whey powder
by weight.
In typical formulations for non-dairy buttermilk
the reconstituted milk employed contains NFDM and sodium
caseinate. The reconstitu~ed milk adjusted to pH 5.5 to
6.0 is pasteurized, homogenized, inoculated with a buttermilk
culture and allowed to ferment to obtain the desired flavor
and appearance. We have found that the microbial sources of
protein of the invention can replace a substantial portion of
the above milk proteins to provide non-dairy buttermilk with
improved properties such as texture and mouthfeel and have
comparable flavor and appearance when compared to controls
containing only the abo~e milk proteins. Preferred non-
dairy buttermilk compositions of the invention are those com- .
prising a microbial source of protein and at least one milk
protein source selected from the group consisting of NFDM and
0 sodium caseinate, said microbial source of protein and said
-26-
.
, :
6 ~
milk protein source being in a weight ratio of from about1:1.5 to 1.5:1 and said microbial source of protein being a
member selected from the group consisting of C. utilis
protein, S. fragilis protein and a mixture of C. utilis
protein or S. fragilis protein with whey powder containing
about 50% whéy powder by weight.
In the above mentioned cake compositions comprising
sodium hexametaphosphate and a source of protein, certain
microbial proteins and certain phospha~e complexes of
microbial protein can replace all or at least a substantial
portion of the egg white ordinarily employed in such cake
compositions as white cake and angel food cake. The microbial
proteins can also replace egg white and other protein contain-
ing ingredients ordinarily used as protein binders in textur-
ed vegetable protein food compositions. Examples of suchother protein containing ingredients are soy flour and wheat
gluten.
In food compositions containing textured vegetable
protein and a protein binding agent, such as, for example,
in simulated meat products, the textured vegetable proteins
are impregnated with a suitable binde~, the impregnated
vegetable proteins are then cooked ~o cause the binder to set
to form a continuous mass that can be cut or shaped into
pieces of sui~able form. We have found that many of the
microbial proteins of the invention can be substituted ~or
the above mentioned protein binders ordinarily employed to
afford improved textured vegetable protein food compositions.
Wnile the above mentioned microbial proteins may
be employed advantageouslv as protein binding agents in food
compositions containing any of the known textured vegetable
-27-
,
. , . :.
:: .: .
: - . ~ ,
, . -: : .
~4866
proteins such as textured soy protein, textured wheat protein,
textured peanut protein, textured cottonseed meal and the
like, such food compositions wherein the textured vegetable
protein is textured soy protein are preferred for reasons of
economy and availability. Preferred such food compositions
are those containing textured vegetable protein and a protein
binding agent consisting of from about 20 to 100% by dry
weight of microbial protein and from about 0 to 80% by dry
weight of up to two additional ingredients selected from the
group consisting of egg white, soy flour and wheat gluten.
Preferred microbial proteins for use as protein binding agents
in the above food compositions are those microbial proteins
isolated from the yeasts S. fragilis, S. carlsbergensis,
S. cerevisiae and C. utilis; from the bacteria P. methylo-
tropha, _. bulgaricus, S~ lactis, M. cerificans and C._
cartalyticum; and from the fungi T. viride, F. solani, P.
chrysogenum and A niger.
_ .
The snack food industry has grown rapidly in recent
years. The basic raw materials utilized include corn meal,
wheat flour, potato flour, oat flour, tapioca and modified
starches. These high starch foods can be fabricated into a
wide variety of textures and shapes by extrusion methods.
Extrusion is known to be a means of converting cereal products
under controlled temperature and molsture conditions into
~ 25 expanded snack food products. Often, milk solids and soy
- protein are included to upgrade the nutritional quality and
handling characteristics of the snack foods. The other major
processing operation used to prepare snack foods is deep fat
frying. Surprisingly, the microbial protein isolates de-
scribed above can be used to replace soy protein, milk solids
-28-
: .
~g~6~
or both of these protein sources in snack food cornpositions
to obtain products having acceptable taste and textural charac-
teristics. In snack food compositions, mixtures of microbial
proteins containing about 50% whey powder are also effective
in replacing milk solids and N~DM.
The snack food compositions containing the microbial
proteins of the invention cause less spattering during deep
fat frying than those containing soy protein and milk solids,
indicating that the microbial proteins are superior in bind-
ing water at frying temperatures. In addition, superior
flavor and texture are observed in the snack food products
of the invention.
EXAMPLE 1
Microbial Protein Isolated by Mechanical Disruption
.
Candida _ilis cells, 130 g. dry weight, were
dispersed in l000 ml. of water and the pH adjusted to ll.5
by addition of lN NaOH. The cells were homogenized at
633 Kg./cm2. ~9000 psi.) at 30C. for one hour. The result-
ing mixture was centrifuged and the supernate decanted. The
remaining solids were repulped in one liter of water and
centri~g~d. The supernates were combined, adjusted to pH
4.0 with lN hydrochloric acid and the precipitate collected
by centrifugation. The solid product was washed with water,
then suspended in water, adjusted to pH 7.0 with sodium
hydroxide solution and freeze dried to obtain 39 g. of
protein isolate.
When the above process i5 repeated but using an
equal weight of cells of Saccharomyces cerevisiae,
Saccharomyces carlsbergensis, Saccharomyces fragilis,
Asperqillus oryzae or L obacillus buL~aricus in place of
-29-
, -
'
8fi~
_. utilis cells, the results are substantially the same.
When the above process is repeated but homogeniza-
tion is carried out at 200 Kg./cm2. and 80C. the results are
essentially unchanged.
When cells were homogenized at a pressure of 1000
Kg./cm2. and a temperature of 5C. the result was essential-
ly the same.
EXAMPLE 2
Microbial Protein from Cells Disrupted by Treatment in Aqueous
Acid
Saccharomyces fragilis, 500 g. dry weight, was
suspended in 2.5 liters of normal hydrochloric acid to afford
a slurry of pH 1. The slurry was stirred while heating to
95C. The mixture was then cooled to 25C., adjusted to pH
11.5 with sodium hydroxide solution and centrifuged. The
supernate was decanted and the debris was repulped with one
liter of water and centxifuged. The supernates were combined
and adjusted to pH 4.0 with hydrochloric acid to precipitate
the protein. The protein was collected by centrifuga~ion,
repulped in water and centrifuged again. The supernate was
discarded and the solid suspended in fresh water, adjusted
to pH 7.0, centrifuged again and the solids dried in the
vacuum oven at 65~C. to afford 115 g. of S. ~ ilis protein.
When cells o~ Candida utilis, C. lipolx_ica,
C. pulcherima, Saccharomyces cerevislae, S. carlsber~ensis,
Neurospora crasa, Aspergillus ~ , A. oryzae, Penicillium
chrysogenum or Lactobacillus bulgaricus are used in place of
S. fragilis cells the results are essentially unchanged.
-
When the above process is repeated by heating in
0.1 molar hydrochloric acid at 100C. for 3 hours; heating in
-30-
, , ' '
6~
5M sulfuric acid at 30C. for 30 minutes or heating in 5M
phosphoric acid at 70~C. for one minute, the results are sub-
stantially unchanged~
EXAMPLE 3
Isolation of Microbial Protein bY D:isrupting Cells in Aqueous
Alkali
One hundred grams of dry Saccharomyces cerevisiae
cells were suspended in 500 ml. water and adjusted to pH
11.5 with aqueous potassium hydroxide solutlon. The mixture
was stirred while heating at 85C. for one hour. The mix-
ture was then cooled to 259C., centrifuged and the supernate
decanted. The residue was repulped in water (350 ml.) and
recentrifuged. The combined supernates were adjusted to pH
4.0 with dilute sulphuric acid and the resulting precipitate
collected by centrifugation, repulped in water and isolated
again by centrifugation. The solids were suspended in water,
adjusted to pH 7.0 and freeze dried to obtain 16 grams of
product.
When the process is repeated:
a) with Candida utilis cells at pH 12 and at a tempera-
ture of 10C. for 15 hours;
b) with S. c_rlsbergensis cells at pH 8 and 100C. for
5 hours;
c) with Streptococcus lactis cells at pH 12, 100C. for
one hour;
d) with Trichoderma viride cells at pH 11 and 90C. for
two hours;
e) with Cellumonas cartalyticum cells at pH 12 and 80C.
for 3 hours;
f) with Penisilllum chryso~enum cells at pH 11.5 and
-31-
:
- ~0~4~36~i
85C. for two hours;
g) with Asper~illus ~y~ cells at pH 11.5 and 90C.
for two hours;
the results are substantially the same.
EX~UPLE 4
M
Candida utilis cells, 350 g. dry weight were
su~pended in 1500 ml. water and adjusted to pH 5Ø The
slurry wa6 stirred at 50C. for 90 hours. The resulting
mixture was cooled to 30C., adjusted to pH 11.5 with aqueous
potassium hydroxide solution and centrifuged at 12,000 r.p.m.
for 10 minutes. The supernate was decanted and the cell
debris washed with water tone liter) and recentrifuged, The
combined supernates were adjusted to pH 4.0 and diluted with
an equal volume of 95% ethanol. The precipitated protein was
collected by c~ntrifugation and wa hed with an equal volume
of water. The solids were suspended in water, adjusted to
pH 7.0 and freeze dried to obtain 57 g. of protein isolate.
EXAMPLE S
A wet paste, 1000 g., of ~ ra~ilis
cells ~650 g. dry weight) at p~ lloO and moist~re content of
35~ was extruded employiny a laboratory model extruder having
length to diameter ratio of 12:1. The extruqion was carried out
at 120C. and at a maxlmum pressure of 35 Kg./cm~. Excel-
lent c011 disruption is noted upon examination under tha
microscope.
The extruded material wa3 then dissolved in water
at pH 11.5. The de~ris was removed by centri~ugation and
wa~hed with water. The protein in th~ combined supernates
-32-
4~36~
was precipitated by isoelectric precipitation at pH 3.5-4~5,
washed with water and dried in the vacuum oven at 70C. to
obtain 78 g. of product which assayed 87% protein (N x 6.25).
When the above procedure is repeated with Candida
utilis cells and extruding at a temperature of 80C. and at
a maximum pressure of 200 Kg./cm2. the results are sub-
stantially the same. Alternatively, the process is also
carried out with equal facility using S. cervisiae cells at
300C. and 10 Kg./cm .
E~AMPLE 6
Disruption of Cells by Treatment at ~i~h Temperatures and
Pressure
Ten liters of a 5% by weight slurry of S. fragilis
cells are heated in an autoclave at 1S0-150C. and at a pres-
sure of 50-80 p.s.i. (3.8-6.1 Kg./cm2) for six hours. The
autoclave is allowed to cool to room temperature overnight
after which the contents àre adjusted to pH 11 with potassium
hydroxide solution and centrifuged. The supernate is de-
canted and the remaining solids repulped in 5 liters of water
and centrifuged. The supernates are combined, adjusted to
pH 4.0 with hydrochloric acid and the precipitate is collect-
ed by centrifugation. The solid product is washed with
water, then suspended in water and adjusted to pH 7.0 with
potassium hydroxide solution. The solid protein is obtained
by spray dryingO
When the above process is repeated but employing a
temperature of 60C. for 20 hours or a temperature of 200C.
for 2 hours, the results are substantially unchanged.
-33-
,
- ~19~66
EXAMPLE 7
To a 2% suspension of Micrococcus cerificans cells
is added sufficient concentrated sulfuric acid ~o make the
suspension 0.2M with respect to H2SO4. The resulting mixture
is heated at 70C. for 30 minutes then cooled and adjusted
to pH ll with sodium hydroxide solution. This solution is
homogenized by means of a RIBI cell disintegrator under a
pressure of 600 Kg./cm2. and worked up as described in
Example 1.
E~AMPLE 8
Candida utilis cells, 350 g. dry weight are suspend-
ed in 1500 ml. of water and adjusted to pH 5Ø The slurry
is autolyzed at 50C. for 90 hours then homogenized and
worked up as described in Example l.
EXAMPLE 9
Microbial Protein-Phos~hate Complex
Twenty-five grams of C. utilis protein was added to
500 ml. of water and the mixtur~ warmed to effect solution.
To the warm solution was then added 20 ml. of a solution ~on-
taining 1.78 g. of sodium hexametaphosphate and 0.22 g. of
potassium metaphosphate. The resulting mixture was stirred
while allowing to cool to room temperature then centrifuged
to remove the precipitated phosphate complex; yield, 26 g.
When the above is repeated with 20 g. of S.
fragilis protein or 30 g. of P methylotropha protein in
place of the 25 g. of C. utilis protein, the results are
substantially unchanged.
When the above procedure employing 25 g. of C.
utilis protein is repeated but employing a total of 2.0 g.
of sodium hexamétaphosphate and potassium metaphosphate in
-34-
~9~i~66
weight ratios of 6:1 and 10:1 the results are essentially
the same.
EXAMPLE 1 0
Heat Denaturation of Isolated Microbial Protein
Fifty grams of the product of Example 1 is
suspended in 2 liters of water at pH 7.0 and heated at 80C.
for twenty minutes. After cooling to room temperature and
adjusting the p~I to 4.0 the mixture is centrifuged, the
solid is suspended in a small amount of water, adjusted to
pH 7.0 and lyophilized.
When the above procedure is carried out at 50C.
for one hou~ or at 100C. for 5 minutes the results are
unchanged.
The heat denaturation is also carried out success-
fully when the heating period is carried out at pH 6, pH 11
or at intermediate pH's.
When the above procedure is carried out with the -~
products obtained in Example 2 through 9 the results are sub-
stantially the same.
EXAMPLE 11
Ethanol Treatment o~ Microbial Protein Isolates
A) One hundred grams of dry microbial protein isolate
of Example 4 and 750 ml. of 95% ~by volume) ethanol are
stirred at 30~C. for one hour then centrifuged. The solids
~5 are slurried again with a fresh portion of 95% ethanol,
centrifuged and the solids dried in the vacuum oven at 50C.
overnight. The ethanol treated protein is judged to have an
improved flavor quality, davoid of bitterness and exhibited
improved functional properties in certain food applications
such as in whipped toppings.
-35-
/
- ~ :
: . . .~, . . .
:~
8~;
When the above procedure is carried out using 20
to 100~ ethanol (by vo ~me) in place of 95% ethanol or at
temperatures of 20C. or 60C. a significant improvement in
protein flavor ls noted in each case.
When any of the protein products of the preceding
examples are treated by the above procedure, a definite
improvement in Elavor qualit~ and/or functional properties
of the alcohol treated protein is noted.
B) Candida utilis cells, 130 g. dry weight, are dis-
persed in 1000 ml. of water and the pH adjusted to 11.5 by
addition of lN NaOH. The cells are homogenized at 633 Kg./-
cm2. (9000 p.s.i.) at 30C. for one hour. The resulting mix-
ture is centrifuged and the supernate decanted. The remain-
ing solids are repulped in one liter of water and centrifuged.
The supernates are combined, adjusted to pH 4.0 with lN
hydrochloric acid and the precipitate collected by centrifuga-
tion. The solid product is washed with water, then resuspend-
ed in 100 ml. of waterO To the slurry 400 ml. of pure
ethanol is added and the resulting mixture warmed to 30C.
and stirred for one hour at this temperature. The mixture
is then cooled, centrifuged and the solid product is washed
with water, then suspended in water, adjusted to pH 7.0 and
freeze dried.
EXAMPLE 12
~5 Acetylated Microbial Protein
A) A suspension of 50 grams of ~he C. utilis protein
isolate of Example 3 in 1000 ml. of water was adjusted to
pH 5.7, 10 y. of trisodium citrate was added and the mixture
stirred for 30 minutes. The resulting solution was adjusted
to pH 7.0 with lN sodium hydroxide and 30 g. of acetic
-36-
`~
ar~ydride was added dropwise over a 60 minute period while
maintaining the reaction mixture at pH 7-8 by periodic addi-
tion of sodium hydroxide (lN). The reaction mixture was
stirred at room temperature for one hour after the addition,
then stored at 20C. for use on an as is basis. If desired
the solid product can be isolated by conventional methods
such as lyophilization.
B) When the above procedure is repeated, but using
50 g. of S. cerevisiae protein in place of C. utilis protein
and 10 g. of acetic anhydride, an acetylated protein with
commensurately lower degree of acetylation is obtained.
EXAMPLE 13
Non-Fat Dry Milk S_bstitute
A) C. utilis protein isolate of Example 1, 100 g. and
an equal weight of whey powder were blended by mixing in
1000 ml. of water at pH 7Ø The resulting mixture was then
homogenized at 2000 p.s.i., pasteurized and concentrated to
a paste. The paste was then freeze-dried.
B) S. fragilis protein isolate of Example 2, 40 g.
~and whey powder, 160 g. were blended by mixing in 1000 ml.
of water at pH 7Ø The resulting mixture was then treated
as described in Part A, above, to obtain a mixture containing
20% microbial protein and 80% whey powder.
C) S. ~ ~rotein isolate of Example 5, 160 g.
and whey powder, 40 g., were blended by mixing in 1000 ml.
of water at pH 7Ø The resulting mixture was then treated
as described in Part A, above, to obtain a mixture containing
80% microbial protein isolate and 20% whev powder.
E~AMPLE 14
Standard white bread control loaves containing non-
-37-
fat dry milk w0re prepared according to Formulation A. In
Formulation B the bread contained microbial protein-whey
powder blend of Example 13A in place of non-fat dry milk.
The microbial protein-whey powder blend is used at a level of
80% of the NFDM of the control. Formulation C contains the
protein isolate of Example 1 at 50% of the level of the NFDM
which it replaces.
-38-
_ __ O U~ O O LO O In ~ o o o o~
o ~ o Lr) r~ ) N oo Lt~ O u-) O
S~ C) O ~D O II ~ U~ O 0~ i U~
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.~
~ o ~ oO Ln "., ~ ~ In o U
. ~ O ~ O ~ ~ ~ In O 00 ~ i ~D
~r
' ~: _ .
o n o o u~ O Lr~ ~ o o o o~
¢ o ~1:\ o u) ~ o co ~ i
__ 01 ~ ~
8 ..............
" _ 'd
~r ~
,i! ):1 m . . ~ ~ -. ., ' , ~ . ,' .
~ . . . . a)
~1 3 . . ~ ' ~ ' ' ' ' , ,
''~X .,,,~
' ' ~ O
,1 . . . . ~ X ~ O ~ O ~ ~ ' ~
~ ~
,1 . . . . ~ ~ . ~ . ~ . .
: ~ Q o R o
; : O
¢~ 3 ~ 1
~ ~ Q, ~ S-l X~
S~
~ O ~1 ~ Ql 0 ~3 0
Q~ 1
~1 ~ ~ Q~~1 ~ u~ ~ R
,~ 1 ~ O
td ~ ~ rl n3 E~ R ~ ,1 . u~
O rl ' ~
O ~ r' ~1 ~ ~ o ~ ~ E3 ~
æ ~ ~ ~ o ~ ~ o
.
--39--
~,
.
,, :: .
For each formulation~ all ingredients were combined
in a ~obart Mixer at #1 speed for 2-3 minutes then mixed at
#3 speed for a~ additional 5 minutes. In resulting dough was
transferred to greased bread pans, cc~vered and allowed to
raise at about 60C. for 2.5 hours. The dough was then
kneaded, divided into two approximately equal portions, each
of which was placed in greased pans. The dough was again
allowed to raise at 60C. for 2.5 hours then=baked at 430F.
(221C.) for 15-20 minutes and allowed to cool to room tem-
perature. The loaf volume and water loss during baking werethen determined by standard methods, after which the loaves
were evaluated by experienced judges.
Water loss Loaf
Formulation during baking, % Volume, ml.
A 12 800
B 8 1100
C 7 1100
Sensor ~Evaluation
~ _ , , y
Perfect Formulation
Characteristics Score _ A_ _ _ B _ C
Loaf Volume 108.5 10.0 10.0
Crust Color 86.0 8.0 80 0
Symmetry 32.5 3.0 3.0
E~enness of
Baking 32~0 2.S 3.0
Character o
Crust 31.0 3.0 3.0
Grain Color
of crumb 1010.0 10.0 9.5
Aroma 1010.0 10.0 9.5
Taste 1514.0 14.5 14.0
Mastication 108.0 10.0 9.5
-40-
: ' . ' , :: ' :
..
Sensor Evaluation lCont'd)
Y
Perfect Formulation
Characteristics Score A B C
Texture 15 15.0 15.0 14.5
Total 87 77.0 86.0 84.0
As the above data indicates, Formulations B and C,
containing products of the invention produced higher quality
bread than the control, Formulation Ao It is especially
significant that a better quality of bread was obtained in
Formulation C, in which our protein isolate was employed in
amounts equal to half the NFDM of the control bread. Further-
more the experimental loaves were of superior volume and show-
ed improved water binding.
When the C. utilis protein-whey powder blend in
Formulation B is replaced with a similar mixture of S.
fragilis protein containing 80% by weight of whey powder, or
a mixture of P. m~y~ 2e~ protein and whey powder contain-
ing 20~ whey powder by weight, comparable breads are obtained.
Also, when ~he C utilis protein of Formulation C
is replaced with an equal amount of one of the following
microbial proteins, the rssults are essentially unchanged:
T. viride protein, A. oryzae protein, L. bulgaricus protein,
- M. cerificans protein, P. methylotropha protein.
,
E~AMPLE 15
Control cakes, D and cakes containing the protein
isolates of the invention, E, F, G, were formulated as shown
below and all were prepared and evaluated by the same pro-
cedures.
-41-
. .
~o~
EXA~PLE 15 (cont'd) Baked Cake
Weight, Grams
Formulation D E F G
Cake flour112.00 112.00 112.00112.00
Sucrose 112.00 112.00 112.00112.00
NFDM 6.50 ---- ---- ----
Microbial Protein-
Whey blend* ---- 6.50 ---- ----
Microbial Protein
Isolate$ ---- ---- 6.50 3.25
Vegetable Shortening 61.60 61.6061.60 61.60
Fresh eggs 46.20 46.20 46.2046.20
Emulsifying Agent# 3.30 3.30 3.30 3.30
Baking soda 0.75 0.75 0.75 0.75
lS Vanillan 5.50 5.50 5.50 5.50
Water 46.20 46.20 46.2046.20
Total `394.05394.05394.05 390.80
* Product of Example 13A
Derived from S. fragilis by procedure of Example 1.
# Mixed mono- and diglycerides
The sugar, non-fat dry milk or the indicated
replacement for non-fat dry milk, vegetable shortening and
emulsifying agent were combined and blended in a Hobart mixer
for 2-3 minutes. The eggs were added in two, approximately
equal portions with 2-3 minutes between additions. Half the
water was addad and mixing continued for 2 minutes. The
remainder of the water was then added with continuous mixing,
after which the flour and baking soda were added and the
mixture blended un~il it was smooth. The batter was poured
into greased cake pans and baked at 375F. ~191C.) for 35
minutes. The cakes were then allowed to cool to room tem-
-42-
.
.
. -. . : ...
366
perature and functional performance data, as follows, was
obtained by standard procedures. Sensory evaluation was
carried out by an experienced panel of j~udges.
Formulation
S Test D E F G
Water 105s
during baking, % 6.7 5.7 5.6 6.2
Volume, ml~ 830 810 830 840
Moistur~ content, ~12.012.7 11.812.5
Sensory Evaluation of Baked Cakes
Perfect
Color of Crust 8 8 8 7 7
Evenness of Bake 3 3 3 3 2.5
Character of Crust ~3 3 3 2.5 2.5
Grain texture 15 15 15 13 14.5
Aroma 10 10 10 10 10
Taste 15 15 15 15 15
Mastication 10 10 10 10 10
Grain 10 9 9.5 8 9.5
Total74 73 73.568.5 71.0
As the above da~a reveals, the products of the
invention in Formulations E, F and G effectively replace
non-fat dxy milk in cakes. It is esp cially noteworthy that
Formulation G, in which microbial protein isolate is used at
half the level of NFDM of Formulation D (the control) pro-
duced a high quality cake of superior volume and similar
sensory characteristics.
When 5. cerevisiae protein isolated by the procedure
of Example 1, Neurospora crasa protein isolated by the pro-
cedure of Example 8, L. bulgaricus protein isolated by the
-43-
' " ' ', '
procedure of Example 6 or the mixtures of any of these
proteins with an equal weight of whey powder are substituted
for NFDM in the above procedure, the resulting cakes are also
found to have an improved score when compared with the NFDM
control.
EXAMPLE 16
Microbial protein isolates were used in a standard
pudding formulation as a replacement for whole milk solids.
Whole milk contains approximately 3.3~ protein, 4.0% fat and
5.0% carbohydrates for a total solids content of 12.3~.
Control Test
Pudding,Pudding,
Inaredient Grams Grams
12~ solution by weight of
nonfat dr~ milk 84.00
12% solution by weight of
Microbial protein isolate* --- 84.00
Stabilizer and Emulsifier 0.14 0.14
Sucrose 10.40 10.40
Corn starch 3.00 3.00
Salt 0-04 0O04
Vegetable fat 2.08 2.08
Buffering agent 0.28 0.28
Flavor q.s. q.5.
* Derived from S. fragilis by the procadure of Example 5.
All ingredients were combined and mixed at 120-
130F. (49-54C.) and then pasteurized at 165F. (73C.) for
15-30 minutes. The resulting mixture was then packaged in
cups and chilled in the refrigerator at 40F. (4.5C.) over-
night. They were then evaluated or comparative functional
and sensory characteristics using a rating scale ranging from
5, for excellent to 1, for poor. A second test pudding was
-44-
:' '
36~
prepared in the same manner, except that a 6% solution of
microbial protein isolate was used.
Test Test
Characteristic Pudding Puddiny I Pudding II
Texture 3.5 5 3.5
Mouth feel 4.0 5 4.0
Syneresis yes no no
Taste 5 5 5
From the data it appears that the microbial protein
isolate is 1.5-2.0 times more potent than nonfat dry milk in
puddings.
When the above procedure was repeated using
Candida utilis protein isolates by ~he procedure of Example
1, the results were substantially the same. Similarly, when
the proteins listed below are used in place of S. fragilis
protein of Example 5, the results are substantially the same.
Microorganism Isolation Procedure
S. carlsbergensis Example 1
., " " " 5
A. orYzae " 3
M. ceriicans " 5
C. cartaly _ um " 3
P. chrysogenum " 2
T. viride " 4
When a 12% solution of a 1:1 mixture of C. utilis
protein and whey powder prepared accordin~ to the procedure
of Example 13A is used in place of NFDM in the above pudding,
the resulting product is also of high quality. Similar
results are obtained when equal weight mixtures of whey powder
and the proteins derived from A~ oryzae, _. cerificans and
-45-
- ,
T. viride are substituted for NFDM.
EXAMPLE 17
Control ice cream and test ice cream in which the
microbial proteins of the invention replaced NFDM were pre-
pared accordiny to the following formulations and procedure.
Ice Cream
Waight, grams
Ingredient Control Test
Heavy cream 200.0 200.0
Nonfat dry milk 42.0 ---
Microbial protein -- 42.0
70% Sucrose solution128.5 128.5
Gelatin 3.0 3.0
Atmos-150 (Atlas Chem. Co.) 1.5 1.5
Water 212.0 212.0
Flavor 9.5 9.5
Total596.5 596.5
PROCEDURE
Mix in all the ingredients except gelatin and
flavoring until uniformly mixed. Bring the temperature of
the mix up to 145-150F. (62.5-65.5C.) and add gelatin
solution with constant stirring. Pasteurize the mix at
165F. (74C.) for 20-30 min. Add the flavoring to the mix.
Homogenize the mix through a two-stage homogenizer using
2500 p.s.i. at the first stage and 500 p.s.i. on the second
stage homogenization valve. Cool the mix to 40F. (4.5C.
rapidly and let it age overnight at 40F. (4.5C.) Freeze
the ice cream mix and whip it using acetone-dry ice bath
until a desired overrun is obtained using normal ice cream
mix freezing principles. Store the ice cream in suitable
containers in ice cream freezer.
-46-
:
After storing for 24-48 hours in the freezer, th~
control and test ice creams were compared for functional and
sensory characteristics. When the test ice cream was prepared
using microbial protein obtained from S. fra~ilis cells by
the procedure of Example 1, for example and vanilla flavor-
ing, a good quality test ice cream was obtained which was
judged to be superior in texture to the control product using
NFDM. The test ice cream and control gave similar overruns,
but the test ice cream demonstrated slower melting at room
temperature with improved retention of incorporated air.
Comparable results are obtained employing Candida
lipolytica protein isolated by the procedure of Example 2;
Sacaharomyces cerevisiae protein, A. oryæae protein or
Pseudomona~ methylotropha protein isolated by the procedure
of-Example 3-; ~eurospora crasa protain, Lactobacillus
protein, Trichoderma viride protein or As~er~illus
niger protein using the procedure of Example 5, in place of
C. utilis protein.
Comparable quality ice creams are also obtained
when the C. utilis protein of the above test ice cream is
replaced by a l~l by weight mixture of C. u~ilis protein and
whey powder, a 1:4 by weight mixture or S. fragilis protein
and whey powder or a 4:1 by weight mixture of P. methylo-
~ protein and whey powder.
EXAMPLE 18
Oran~ She bet or Ice Milk
When orange sherbets are prepared by standard
method~ using the following formulation, employing either
non-fat dry milk (control), equal amounts of the microbial
protein products of the invention or a mixture of one such
-47-
- :'. - .
-: :
' .
36~
microbial protein with whey powder prepared according to
Example 13A-C, the experimental products are judged to be of
better texture than the control and show improved retention
of incorporated air.
Ingredient3 Weight, %
Non-fat dry milk or Microbial
protein or microbial protein-whey
powder mixture 5.5
Sucrose 21.0
Vegetable shortening 2.9
Orange juice 11.4
Water 51.4
Lemon juice 5.7
Stabilizer 2.0
Color and flavor 0.1
Total 100.0
EXAMPLE 19
Mellorines or Milk Sherb~x~
Another application for the microbial protein iso-
lates of the invention are in frozen desserts such as mel-
lorines or milk sherbets in which non-fat dry milk is
ordinarily employed to bind water, encapsulate fat droplets
and stabiliæe air incorporated into the mixture. When the
non-fat dry milk of the control mellorine is replaced with
either the above-described microbial protein or microbial
protein-whey powder mixtures the experimental products are
of superior quality when compared to the NFDM control.
Ingredients Weight % (Range)
Non-fat dry milk
or microbial protein isolate
or microbial protein~whey
powder mixture 1.5 20.0
~48-
18G6
Ingredients Weight ~ (Range)
Sucrose 0 -25.0
Corn syrup solids 0 -25.0
Vegetable shortening 5 -15.0
Stabilizers and emulsifiers 0.1- 3.0
Flavor, color, salt and water as needed to bring
to 100%
Either the non-fat dry milk, microbial protein
isola~e or microbial protein-whey mixture is blended with
the sucrose and stabilizer. The water is placed in a steam-
jacketed kettle and the dry ingredients are added with stir-
ring. The corn syrup solids are then added followed by the
shortening, emulsifier and salt. With continued stirring
the mixture is heated to 160F. (71C.). The hot solution is
homogenized in a two stage homogenizer at 2500/500 p.s.i.
The homogenizer liquid is cooled to 35-40F. (2-4.5C.) and
aged overnight. The aged solution is frozen in a conven-
tional ice cream freezer a~ -10 to ~20F. (-22 to -7C.) for
about 12 hours~
EXAMPLE 20
Cheese Cake
Cheese cakes were prepared according to the formula-
tion and procedure described below.
Weight! %
In~redie ts Control Test
Cream cheese 31.0 31.0
Water 18.5 18.5
Whole eggs 18.2 18.2
Sucrose 16.8 16.8
Cot~age cheese 8.5 8.5
Egg albumin 2.9 2.9
-49-
.
~0~ i6
Weight, %
Ingredients Control Test
NFDM 4 0 ~-
Microbial protein-whey
powder product of
Example 13C --- 4.0
Flavor 0.1
100.0 100.0
Blend the water, cottage cheese, NFDM or microbial
protein-whey powder mixture and flavor in a blender on high
speed until smooth and homogenize the blend in two stages at
1000/500 p.s.i. Then add the ~ugar and egg albumin and mix
well. The remaininy ingredients are added with continued
mixing and the resulting mixture is poured into a graham
cracker crust and baked at 350F. (177C.) for 40 minutes.
The test cheese cake is found to be of superior
texture and volume and equivalent to the control in all
other functional and sensory characteristics.
EXAMPLE 21
Other Yeast Leavened Baked Goods
When bread rolls, buns, doughnuts a~d sweet rolls
; (danish pastry) are prepared by standard proceduxes using
the illustrative formulations given below, the test food
items containing the products of the invention are found to
be superior to the controls in functional and organoleptic
properties.
Rolls or Buns
Weigh~ Grams
Ingredients_ Control Test A Test B
NFDM 57
Microbial protein of Example 4 -- 57 --
-50-
: '
Weight, Grams
Ingredients Control Test A Test B
Microbial pro~ein-whey powder
blend of Example 13A -- -- 57
Shortening 142 142 142
Sugar 170 170 170
Salt 28 28 28
Eggs 114 114 114
Water 910 910 910
Yeast 8S 85 85
: Bread flour 1535 1535 1535
Total30413041 3041
Yeast Raised ~oughnuts (cake type)
Weight, Grams
Ingredients Control Test A Test B
Sugar 228 228 228
Salt 28 28 28
: NFDM 85
Microbial Protein Isolate -- 85 --
Product of Example 13B -- -- 85
Shortening 170 170 170
Eggs 170 170 170
Water 682 682 682
Vanilla 28 28 28
Yeast 228 228 228
Water 681 681 681
Bread flour 1590 1590 1590
Total38903890 3890
-51-
, - , -,,, :~
Danish Pastr~
, Weight, Grams
In~redients Control Test A Test B
Yeast 114 114 114
Water 454 454 454
sugar 454 454 454
Salt 28 28 28
NFDM 114
Microbial Protein Isolate -- 114 --
Product of Example 13C ~~ -~ 114
Shortening 228 228 228
Eggs 454 454 454
Water 454 454 454
Vanilla Flavor 28 28 28
Flour 1817 1817 1817
Shortening 909 909 909
Total5054 5054 5054
When C. utilis protein isolated by alkaline treat-
ment of cells as described in Example 3, M. cerificans
protein isolated by the procedure of Example 1, A. niger
protein isolated by the procedure of Example 5 or their
appropriate blends with whey powder according to Example
13A-C are substituted for NFDM in the above formulakions for
rolls, doughnuts and Danish pastry, the rPsulting baked goods
5 are also found to be better than the NFDM controls.
EXAMPLE 2_
Test pancakes containing Lactobacillus bulgaricus
protein isolated by the procedure of Example 5 in place of
non-fat dry milk were judged to have superior texture and
0 volume when compared to the control pancakes. Both control
-52-
''
8~
and test pancakes were prepared identically by the formula-
tion and procedure set forth below.
Weight, Grams
Ingredients Control Test
Flour 109.0 109.0
Sugar 45.0 45.0
Sodium bicarbonate 1.0 1.0
Glucono-delta-lactone 2.0 2.0
Non-fat dry milk 10.0 ---
Microbial protein isolate --- 10.0
Fresh whole eggs 50.0 50.0
Salt 2.5 2.5
Water 69.0 69.0
Total 288.6 288.6
PROCEDURE
In the two-quart bowl o a Hobart Mixer equipped
with a flat paddle premix at #l speed flour, sucrGse, sodium
bicarbonate, glucono-delta-lactone and non-fat dry milk or
microbial protein isolate un~il a homogeneous mixture is
obtained, requiring about 5 minutes. Add the water and mix
at #2 speed for an additional 2-3 minutes. Scrape bottom and
side of bowl and paddle. Finally add whole eggs and continue
mixing for an addifional 2-3 minutes. Pour two full table-
spoons on pre-greased, pre-heated (350F.) pancake griddle.
Allow to cook until bubbles appear on surface of pancakes
(2-3 minutes). Turn and brown on opposite side for approxim
ately the same amount of time.
Pancakes of excellent quality are also obtained
when the L. bulgaricus protein is replaced by protein obtain-
ed from C. u lis, S. cerevisiae, Streptococcus lactis,
-53
, !
'
Micrococcus cexificans, Cellumonas cartalyticum, Trichoderma
viride, Penicilllum chrysogenum, As~ergillus niger, Fusarium
solani and Cellulobacillus mucosis.
EXAMPLE 23
Meat Loaf
Meat loaves were prepared uslng the microbial
proteins of the invention to replace the non-fat dry milk
ordinarily used in commercial practice. Mixtures of
microbial protein isolate and whey powder were found to be
especially effective in this application. The loaves were
prepared following the formulation and procedure d~scribed
below.
Wei ht, Lbs.
Ingredient Con~rol Tes~ A Test
Fresh beef 100 100 100
Grated onions 2 2 2
Tomato catsup 10 10 10
Salt 3 3 3
NFDM 12 -- --
Microbial Protein-Whey
powder mix~ure* -- 12 6
White pepper 0.5 0.5 0.5
Ground bay leaf 0.125 0.125 0.12S
Worcester hire sauce0.188 0.188 0.188
*S. cerevi1iae protein isolated by the procedure of
Example 3 and blended with an equal weight of whey powder
by the method of Example 13~.
PROCEDURE
The beef was ground through a 1/8-1/2 inch plate,
co~ered with water in a steam-jacketed v~ssel and brought to
boiling. All ingredients except NFDM or the mlcrobial
~54-
. ; . , ~ . . ,
':
:: :
8~
protein-whey powder mi~ture were then added and the mixture
boiled until the meat was tender. The meat was transferred
to a mixer, the NFDM or microbial protein-whey powder mixture
and 50 g. of cooking broth from above were added and the
mixture blended. The resulting mix was divided in 475 g.
portions, placed in pans ancl chilled.
Water and fat loss during processing, as well as
loaf volume, were then determined by standard methods known
in the art and ~he meats were subjected to sensory evaluation
lO by an experienced panel. The results are summarized below.
Water
Loss Fat Loss
Protein g- H2~/- g. fat/- Sensory
Ingredient 100 g. lO0 g. Loaf Evaluation
Used, Starting Starting Volume, l 5
g./lQ0 g. beef Material Material mls. (5-Best)
Nonfat dry
milk, 12 g. 8.6 7.5 450 5
Microbial pro-
tein-whey
powder mix-
ture, 12 g. 6.5 6.5 500 5
Microbial pro-
tein-whey
powder mix-
ture, 6 g. 9.0 7.9 450 5
As the data indicates, the loaves prepared with
microbial protein-whey powder mixture were equal to or better
than loaves containiny the nonfat dry milk. Furthermore, the
amount of microbial protein~whey powder mix~ure can be reduc-
ed to half that of the NFDM to obtain a substantially
equivalent meat loaf. When used at the same level as NFDM,
the microbial protein-whey powder mixture significantly re-
duced at and mois~ure ~ss during processing and increased
loaf volume.
When S. cerevisiae protein alone is substituted
-5S-
,
6~
for an equal weight of NFDM in the above meat loaf formulatiGn,
the resulting products also show improvement in water and fat
loss due to cooking and are of excellent sensory quality.
When proteins derived from S. carlsbergensis,
P. methylotropha, M. cerificans, F. solani, A. o~yzae and
Neurospora crasa are substituted for S. cerevisiae protein
in the above procedures comparable results are obtained.
EXAMPLE 24
Frankfurters and liver sausages prepared according
to the following formulations but with the instant microbial
proteins or instant mixtures of microbial protein and whey
powder in place of NFDM are of better quality than the com-
mercial products employing an equal amount of nonfat dry
milk.
Frankfurters
Ingredients Lb. Oz.
Regular pork trimmings 90 --
Lean beef 10 --
Ice 30
NFDM 3 --
Spices -- 15
Sodium nitrate -- 0.25
Liver Sausa~e
Ingredients Lb. Oz.
Pork livers 50 --
Pork ~rimmings 40 --
Veal 10 --
Fresh onions 5 --
NFDM 3 8
Salt 2 8
White pepper -- 4
Sodium nitrate -- O.S
-56-
,
,
66
EXAMPLE 25
Soft moist p t foods of high quality are prepared
according to the following formulation.
Ingredients Wt. %
Tripe 18.0
Fish 6.0
Beef trimmings 6.0
Soy flakes 31.5
Corn syrup solids21.4
Soy hulls 3.0
Nonfat dry milk2.5
Bone meal 2.4
Dicalcium phosphate 1.4
Propylene glycol2.0
Sorbitol 2.0
Tallow 2.0
Mono & diglycerides 1.0
NaCl 0.6
Preservatives 0.3
Minerals & vitamins etc. 0.3
When the microbial proteins of the invention or
their blends with whey powder prepared according to the pro-
cedures of Example 13~-C, are substituted for nonfat dry
milk on an equal weight basis the pet foods obtained are of .
improved quality when compared with the nonfat dry milk
controls.
EXAMPLE 26
Chocolate cake icings were prepared according to
the following formulations.
-57-
- . ' ' - :-' ` ' ~ :
~ .
6~
Weight, Grams
Ingredients Control Test A Test B
Vegetable fat 26.6 26.6 26.6
Sucrose 60.1 60.1 60.1
Tapioca starch 0.5 0.5 0.5
NFDM 3.8
Yeast protein isolate* -- 1.9 --
Yeast protein-whey powder
(1:1) $ ~~ 3.8
Cocoa 5.8 5.8 5~8
Vanillan 0.1 0.1 0.1
Salt 0.08 0.08 0.08
Chocolate color 0.04 0.04 0.04
Water 56.9 5609 56.9
153.92 152.02 153.92
* Candida utilis protein of Example 4
$ C. utilis protein obtained by the procedure of
Example 3 and blended with whey powder as described in Ex-
ample 13A
All dry ingredients were blended in a mixing bowl
for 3-5 minutes. The water, heated to boiling, was added
and the mixture stirred until homogeneous. It was then
whipped in the Sunbeam mixer at high speed for 15-20 minutes.
The functional and sensory characteristiss were then
evaluated. The results are tabulated below. Except for
specific gravity, all ratings were made on an a~hltrary scale
of 1-5, where 1 is poor, 5 is excellent.
-58-
' ~
.
8~
Peaks
Specific Character- Spread-
gravity istics ibility Gloss Dryness
Control 0.40 4.5 5.0 3.0 5.0
Test A 0.40 5.0 5.0 5.0 4.0
Test B 0.64 3.0 4.0 4.0 4.0
From the above it is apparent that the protein
products of the invention are about 1.5-2 times as potent as
non~fat dry milk in this application.
The following microbial proteins may be substituted
for the C. u ilis protein and CO utilis whey powder blend in
the above formulations with substantially ~he same results.
Protein Isolated Isolation Procedure
From of Example No.
S. ~ 6
S. cerevisiae 9
M~ cerificans 7
A. niger 8
C. utilis 12B
P. chrysogenum 10
S. cerevisiae 12A
.~
EXAMPLE 27
White Cake Frosting
Frosting base mixes were prepared as follows:
Weight, Grams
Control Test
Sugar 318.5 318.5
Corn syrup solids 83.0 83.0
Non-fat dry milk 13.5 --
Microbial Protein
Isolate of Example llB -- 13.5
Kaokreme~(I-2975,728/71) 75.0 75.0
4~0.0 490.0
-59-
.,,
The inyredients are stirred in a Waring Blender at
low speed until homogeneous Then 98.0 g. of the frosting
bases, 2.0 g. gelatin (225 Bloom) and 64.4 g. of water are
combined and whipped ~t ~9 speed in a Sunbeam Mixmaster for
ten minutes. The resulting cake frostings are poured into
beakers and refrigerated overnight. The test frosting is
found to have superior overrun and to be comparable to the
control upon organoleptic evaluation. When 13.5 g. of the
product of Example 13A is employed in place of the isolate
of Example llB in the above test frosting, the results are
substantially the same.
*Kaokreme i5 a vegetable shortening manufactured by
Durkee Famous Foods CorpO The word "Xaokreme" is a Trademark.
EX~MPLE 28
Bakers Marshmallow
A standard method for preparing bakers marshmallow
is as follows:
Grams
A. ~yfoma * 2.0
NFDM 1.0
Water 15.0
Confectionary sugar 15.0
; B. Gelatin 1.0
Water 3.0
C. Sugar 91.0
Glucose 11.0
Water 31.0
Total 170.0
Mix ingredients in Part A and beat until a stiff
i 30 foam is obtained. Dissolve gelatin in water ~Part B) and add
s 60-
.
, .... .
."''~ .. ' , ~
i6
to Part A with continuou~ mixing. Dissolve sugar and
glucose in water, heat to a boil, then add to the blend of
Parts A and B. Then beat the resulting mixture in the Sun-
beam mixer until stiff peaks are obtained.
Test I was carried out using the C. utilis protein
isolate of Example 4 to replace the gelatin in Part B.
Test II was carried out using the C. utilis protein
isolate of Example 4 to replace half of the Hyfoma of Part A.
The products of Test I and Test II were compared
with the control marshmallow for function and organoleptic
properties. The results are summarized below. Scoring was
carried out on a 1 (poor) to 5 (excellent) scale.
* A commercial foaming agent containing 64% protein
(vegetable protein and egg white), 8% sucrose and 20%
stabilizers by weight. It is available from Landerink and
Co., Schiedam, the Netherlands.
Properties Control Test I Test II
Specific gravity 0.52 0.44 0.58
Texture 3.0 5.0 5.0
Gloss 5.0 5.0 5.0
Taste 3.0 5.0 5.0
Odor 2.0 4.5 5.0
Smoothness 2.0 4.5 5.0
Spreadability 3.0 4.5 5.0
Color 4.5 4 5 5 0
Both test marshmallows show significant improve-
ment in texture, taste, odor, smoothness and spreadability.
When Tests I and II are repeated, but using S. fragilis
protein, P. methylotroEha protein or F. sol _ i protein instead
0 of C. utilis protein as replacements for gelatin and Hyfoma,
-61-
84~,6
respectively, the test marshmallows are also found to besuperior to the controls.
An improved product is also obtained when, in the
above formulation, the microbial protein isolates of Examples
1 through 8 or their mixtures containing from about 20 to
80% by weight of whey powder prepared by the procedures of
Example 13A-C are used to replace:
a) The NFDM in Part A.
b) The NFDM plus half the Hyfoma of Part A.
C) Half the Hyfoma in Part A plus the gelatin in Part B.
Especially effective are C. utilis protein, S.
fragilis protein, P. methylotropha protein and F. solani
protein; as well as mixtures of these proteins with whey
powder containing from about 20 to 80% whey powder by weight
I5 as prepared by the procedures of Example 13A-C.
When the Hyfoma in the above tests is replaced by
either an equal weight of dry egg white, a mixture of equal
parts by weight of soy pro~ein and dry egg white, or Gunthers
Foaming Protein 1026 available fxom A. E. Staley Mfg. Co.,
Protein Division, Decatur, Illinois 62525, the results are
substantially unchanged.
EXAMPLE 29
Superior quality confections are obtained using the
formulations below, in which the microbial proteins or
microbial protein-whey powder mixtures of the invention
replace the NFDM ordinarily used and following procedures
well known in the art of candy manufacture.
-62-
',,
EXAMPLE_29 (cont'd)
Grams
Pan Cream
Ingredients CaramelFudye Filling
Sugar 32 73 72
Corn syrup 32 8 10
Fat 10 5 5.5
Microbial Protein or
Microbial Protein-whey powder 15 6 10
Water 21 30 --
Fondant -- 8 --
Frappe 11 -- 7.5
Salt -~ -- 15
Totals121 130 120
EXAMPLE 30
A chocolate flavored beverage of improved quality
is obtained when the instant microbial protein or instant
microbial protein whey powder blends are employed to replace
the nonfat dry milk ordinarily employed.
Chocolate Beverage
Ingredients Wt., %
Microbial protein isolate
or mixtures with whey powder 9
Vegetable fat 2
Cocoa or chocolate liquor 1.0-2.0
Vanillan 0.1 0.2
Sugar 5-8
Water as needed to
make 100%
EXAMPLE 31
Standard whipped toppings were prepared according
to the following formulation and procedure.
-63-
EXAMPLE 31 (cont'd)
Wt., grams
Sodium caseinate 6.0
Fat 35.0
Corn syrup solids 5.0
Stabilizers 0.8
Emulsifiers 1.0
Salts 0.15
Water 64.0
Total 111.95
PROCEDURE
Heat half of the water to 125F. (52C.) and add
salts and stabilizers with constant mixing. Blend all other
ingredients except fat and emulsifiers in the remaining
water, then combine the two blends. Melt the fat and
emulsifiers together at low temperature and add with stirring
to the water phase. Pasteurize at 160F. (72C.) for 30
minutes and homogenize in a two stage homogenizer at 1000-
6000 p.s.i. in the first phase and 500 p.s.i. in the second
phase. Cool as rapidly as possible to 40~F. (4.5C.) and
whip in Sunbeam Mixmaster for 2 minutes at #3 speed.
When the sodium caseinate was replaced in the above
formulation with an equal weight of the microbial protein
products tabulated below, superior overrun values were
obtained as shown by the following data.
Overrun, %
Sodium Caseinate
(Control) 125
S. fragilis protein
by procedure o Example 5 155
-64-
',
,
~C3 9~6
Overrun, %
C. utilis protein by
procedure of Example l 167
_. utilis protein-
phosphate complex 185
The microbial protein containing toppings were
judged to have a more creamy texture and superior low tem-
perature stability when compared with the control topping.
When the microbial prote.in isolates tabulated
below are employed to replace from 50 to 100% by weight of
the sodium caseinate of the control, whipped toppings with
improved properties are also obtained.
Microbial Protein Heat Den~tured Micro-
Microbial Proteins Acetylated Phosphate Complexes bial Proteins by the
15 by the Procedures of Example by the Procedure of Procedure of E~ple
12A or 12B Example 9 10
C. utilis S. fragilis C. utilis
M. cerificans M. cerificans S. fragilis
P. chrysogenum C. cartalyticum S. cerevisiae
S. fragilis F. solani L. bulgaricus
S. cerevisiae P. chrysoyenum P. methylotrop_a
P. methylotropha _~ oryzae M. cerificans
A. niger S. carlsbergensis T. viride
S. lactis F. solani
N. crasa
EXAMPLE 32
Coffee whiteners were prepared according to the
following general formulation and evaluated for feathering,
whitening power and flavor.
Ingredients Grams
Sodium Caseinate, microbial
protein isolate or microbial
protein isolate derivative 5-10
-65-
6~,
EXAMPLE 32 (cont'd)
Ingredients Grams
Vegetable fat 20-25
Corn syrup solids 1S-20
Potassium dihydrogen phosphate 0.25-0.5
Emulsifiers 0.5-1.0
Stabilizers 0.5-1.0
Flavor and color q.s.
Water 75-150
PROCEDURE
The potassium dihydrogen phosphate was dissolved
in the water, then the other dry ingredients were added with
mixing adequate to insure proper dispersion. The remaining
ingredients were then added and the mixture heated rapidly
to 160F. (71C.). The mixture was pasteuri2ed at 165F.
(74C.) for 15 minutes, then homogenized in a two stage
homogenizer at 2500/500 p.s.i. and spray dried.
Coffee whiteners prepared by the above formulation
in which the sodium caseinate content is from 0 to 50% by
Z0 weight of the total of sodium caseinate plus microbial
protein or microbial protein derivatives are found to have a
reduced tendency to feather and have improved whiteness and
flavor when compared to control coffee whiteners either made
by the above formulation with sodium caseinate alone or a
commercially available coffee whitener employing sodium
caseinate. Microbial proteins which may be employed are
those derived from C. utilis, S. fragilis, A. oryzae and S.
lact_s. Especially effective in this food composition are
the following microbial protein derivatives.
-66-
',' ~'' ~ .' '
~48~;
Microbial Protein Derivatives
Phosphate ComplexQs Acetylated Derivatives
by the Procedure of by the Procedure'of
Example 9 E mples 12A or 12B
C. utilis C. utilis
S. fragilis S. fragilis
S. cerevisiae P. methylotropha
S. carlsbergensis S. lactis
P. methylotropha M. cerificans
M. cerificans C. cartal~ticum
C. cartalyticum T. viride
T. viride F. solani
F. solani A. niger
P. chrysogenum A. oryzae
_. oryzae N. crasa
EXAMPLE 33
Replacement of NFDM in Non-Dairy Yogurt
A standard formulation and procedure for non-dairy
yogurt in which the weight ratio of fat to protein source is
about 1:6 is as follows:
Ingredients Weight, Grams
Nonfat dry milk 18
Vegetable fat 3
Sucrose 5
Stabilizer 0.5
Water 73.5
Lactic Acid, dilute as needed to adjust
to pH 4.0
All ingredients except lactic acid are mixed with
the water and pasteurized at 65C. ~or 15 minutes then
homogenized using 2500 p.s.i. pressure at the first stage and
-67-
. . .
' ~
500 p.s.i. on the second stage. The mix is cooled to room
temperature and adjusted to pH 4.0 with dilute lactic and
refrigerated.
In yogurts made by the above formulation separation
of solid discrete curds and whey commonly occurs when the mix
is adjusted to pH 4Ø This can be minimized, but not
eliminated, by adjusting the pH after cooling. However, when
an equal weight of the microbial protein products of the
invention are used in place of nonfat dry milk in the above
formulation this problem was completely alleviated. The
product was stable without separation of curd and whey.
Furthermore, the product was stable during storage at 4C.
and had superior mouthfeel when compared to the yogurts made
with nonfat dry milk.
Microbial proteins of the invention which are use-
ful in preparing yogurts in this manner are those derived
from the following microorganisms: C. utilis, S. fragilis,
S. cerevisiae, S. carlsber~ensis, P. me~hylotropha, S. lactis,
L. bulgaricus, M. cerificans, C. cartalyticum, T. viride,
F. solani, P. chryso~enum, A. niger and mixtures of any one
of the above microbial proteins with whey powder according
to the procedures of Example 13A-C.
Especially effective in this use are the yeast
protein isolates derived from C. utilis and S. fragilis as
well as mixtures of C. utilis or S. fragilis with an equal
weight of whey powder.
EX~NPLE 34
Replacement of Sodium Caseinate in Non-Dairy Yogurt
A non-dairy ~ogurt con~ol is prepared by the following
0 formulation and procedure. When the sodium caseinate is
-68-
,: ~ ' ' ', ,
8~
replaced by an equal weight of one of the microbial protein~of the invention an improved yogurt is obtained which is
judged to have improved mouthfeel and shows little or no
syneresis upon storing at 4C.
Ingredients Weight, Grams
Sodium caseinate 45
Nonfat dry milk 30
Vegetable fat 15
Sucrose 25
Flavor q.s.
Water 385
Mix all ingredients in 300 ml. of water, adjust
the pH to 5.5-6.0 with citric acid, then add the remainder
of the water. Pasteurize at 65C. for 30 minutes and
homogenize through a double stage homogenizer at 2500/500
p.s.i. Cool to 45C. and innoculate with yogurt starter
culture. Divide into 4 equal portions and allow to stand in
draft free area for 4-5 hours during which time the mixture
thickens. Store at 4C.
The yeast isolates derived from C. utilis, S.
fragilis and 5. cerevisiae are especially effective replace-
ments for sodium caseinate.
EXAMPLE 35
Microbial Protein as a Replacement for both Sodium Caseinate
and NFDM in Non-Dairy Yo~urt
When 50 to 100% by weight of each of the sodium
caseinate and nonfat dry milk in the formulation for non-
dairy yogurt given in Example 34 are replaced with the
microbial proteins of the invention, significant improvement
is observed in the mouthfeel and syneresis of ~he resulting
-69-
products prepared by the procedure given therein. Especially
effective are C. utilis protein and S. fragilis protein.
E~AMPLE 36
Replacement of NFDM in Non-Dairy Sour Cream
When the nonfat dry milk in the following formula-
tion is replaced with the instant microbial proteins or a 1:1 (w/w)
blend of the microbial protein and whey powder, superior non-
dairy (or imitation) creams are obtained which show improved
water and fat binding characteristics
Ingredients Weight, ~
Sodium caseinate 0.5 - 5.0
NFDM, microbial protein
or microbial protein-
whey powder blend (1:1) 2.0 - 10.0
Vegetable fat 10.0 - 25.0
Corn syrup solids 5.0 - 15.0
Stabilizers and Emulsifiers 0.1 - 0.2
Water to bring to 100%
Flavor q.s.
Sour cream culture q.s.
PROCEDURF.
To the warm (32-43C.) water, add the sodium caseinate
and ei~ ~ NFDM, microbial probein or microbial protein-whey powder
blend and mdx to dissol~e. Add o~her ingredients then pasteurize at
about 73C. for 30 minutes. Homogenize twice at 2500 p.s.i.
(one stage only) while keeping the temperature above about
71C. Cool to 22C. and innoculate with a suitabLe sour
cream culture. Hold at 22C. until acidity reaches 0.8%
(about 13-20 hrs.), then store at 2-4C.
-70-
, '- .'
8~
EXAMPLE 37
Microbial Protein as a Replacement for Sodium Caseinate in
Non-Dair~ Sour Cream
When non-dairy sour creams are made according to
the following formulation and procedure and compared with
sour creams in which the S. ragilis protein isolated by the
procedure of Example 2 is used to replace sodium caseinate
in the formulation and procedure given below, the microbial
protein-containing sour creams show signiicantly improved
texture and mouthfeel~ Also, no whey separation was noted
in the sour creams containing S. fra~_lis protein, whereas
the sodium caseinate controls show considerable whey separa-
tion.
Range,
Ingredients ~ by Weight
Sodium caseinate 5 - 15
Vegetable oil 10 - 25
Sugar or corn syrup solids 5 - 15
Stabilizers and Emulsifiers 0.1 - 0.2
Flavor and Color q.s.
Water to bring to 100%
Sour cream culture q.s.
Warm the water to 32-43C., add sodium caseinate
and stabilizer then stir ~o dissolve them. Then add the oil
and emulsifier and pasteurize while stirri~g at 74C. for
thirty minutes. Homogenize twice at 2500 p.s.i. while keep-
ing temperature above 71C. Cool to 22C. and add the
culture to be used (e.g., 3~ buttermilk or a commercial
starter). Maintain at 224C. until acidity reaches 0.8%
~about 18-20 hours), then store at 2-4C.
When the sodium caseinate in the above formulation
-71-
~, : . .... : .,,., ~
6~
is replaced by an equal weight of the microbial protein
isolated from one of the following microorganisms, similarly
improved non-dairy sour creams are obtained: S. cerevisiae,
C. cartalyticum, S. lactis, N. crasa, A. niger, A. oryzae.
_
EXAMPLE 38
-
Replacement of NFDM in Non Dairy Buttermilk
A standard formulation and procedure for non-dairy
buttermilk is given below.
Ingredients Weight, Grams
Sodium caseinate 45
NFDM 30
Flavor q.s.
Water 400
Mix all ingredients and adjust to pH 5.5-6.0 with
lactic acid. Pasteurize at 65C. for 30 minutes then pass
through a two stage homogenizer at 2500/500 p.s.i. Cool to
45C. and innoculate with a buttermilk culture (e.g., 3%
buttermilk or a commercial culture). Allow to stand at 20C.
overnight then store at 4C.
When the instant microbial proteins and their
blends with an equal weight of whey powder prepared as de-
scribed in Example 13A are used to replace all of the nonfat
dry milk in the above formulation, the resulting buttermilk
samples are judged to be comparable in flavor and appearance
and have superior texture and mouthfeel when compared to the
NFDM control. The yeast protein - whey blends (1:1) and
especially those derived from C. u~ilis and S. fragilis
protein are particularly effective.
.
. .
-
8~
EXAMPLE 39
Microbial Protein as a Replacement for Sodium Caseinate in
Non-Dairy Buttermilk
Fourty-five grams C. utilis protein, isolated by
the procedure of Example 3, is slurried in 200 ml. of water
and the mixture is adjusted to p~ 8.0-8.5 by addition of
normal sodium hydroxide. The resulting solution is then
mixed with a solution of 30 y. of nonfat dry milk in 200 ml.
of water. The resulting mixture is then carried through the
procedure of Example 38 to provide a non-dairy buttermilk.
When this is compared to a control containing sodium caseinate
according to Example 38, the two are judged to be comparable
in flavor and appearance.
When S. fragilis protein isolated by the extrusion
method described in ~xample 5 is substituted for the above
C. utilis protein, the results are substantially unchanged.
EXAMPLE 40
Microbial Protein as a Replacement for Sodium Caseinate in
Non-Dairy Cream Cheese
Non-dairy cream cheeses were made by the following
formulations and procedure.
Control Test
Sodium caseinate 9.5 ~~
C. utilis protein of
Example 1 -- 9.5
Corn syrup solids 3.8 3.8
Emulsifier 0.4 0.4
Stabilizer 2.0 2.0
Butter or ~egetable Fat 30.5 30.5
Water 53.8 53.8
Total 100.0 100.0
~73-
.
.
~48~6
Blend all dry ingredients in water and then add
melted fat with emulsifier. Acidify to pH 4.0-5.0 and
pasteurize at 65C. for 15 minutes. Homogenize at 3000
p.s.i. single stage. Cool to 4~C. and evaluate.
The control cream cheese is difficult to pasteurize
after adjusting the pH to 4.0-5.0 as the material tends to
give whey separation and curd formation. This results in a
cream cheese with a 1I chewy" texture which is undesirable. To
avoid this difficulty with the sodium caseinate-containing
product the pH was adjusted on cooling following homogeniza-
tion. This results in a non-homogeneous cream cheese which
is also undesirable. By contrast, the test cream cheese is
readily prepared without these difficulties; it was found
to have superior texture and mouthfeel, showed no whey
separation and appeared to be completely homogeneous.
When an equal weight of protein isolated fr~m S.
fragilis, S. carlsbergensis, S. cerevisiae, A. oryzae, or
L. bulgaricus are employed in place o~ C. utilis protein in
the test formulation the results are essentially unchanged.
EXAMPLE 41
Sodium, Calcium and Magnesium Salts o Microbial Protein as
Repla_ements for Caseinates in Non-Dair~ _rocess Cheese
A standard formulation and procedure for preparing
heat processed non~dairy cheese is as follows:
Ingredients Grams
Sodium, Calcium
or Magnesium
caseinate 20-25
Sodium chloride 2.15
Adipic acid 0.5-1.0
Sorbic acid 0.1-0.2
-74-
.: .
~4~
EXAMPLE 41 (cont'd~
Ingredient Grams
Vege~able fat 20-25
Cheese flavor powder 2-5
Waterto make up to 100 g.
Mix all ingredients until a very uniform mixture
is obtained. The blend is then quickly heated to 75-80C.
with continuous mixing, after reaching this temperature it
is packaged and chilled.
The product obtained resembles mozzarella type
cheese and has a milk cheddar cheese flavor.
From 50 to 100% by weight of the above caseinate
salts may be replaced by an equal weight o~ the sodium,
calcium or magnesium salts of the microbial protein products
of the invention to obtain a variety of processed non-dairy
cheese products that resemble either soft textured cheeses
such as mozzarella, blue and camembert or hard cheeses such
as cheddar and brick by varying the amount of microbial
protein, salt and acids within ~he range or the above formula-
tion and, in the case of the hard cheese, by additlon ofsmall amounts (e.g., about 0~1 g.) of edible gums such as
gum tragacanth, gum ghatti or sodium carboxyme~hylcellulose.
The non-dairy process cheeses containing the abov~
salts of the microbial proteins showed water binding, fat
binding and emulsification characteristics superior to those
containing caseinate only. The microbial protein cheeses
were also judged to have enhanced flavor.
The sodium, calcium and magnesium salts of the
microbial protein products of the invention are readily
-75
- . .. ~: .., ~, .
: . ~ , , . ~. -
,
8~ 6i
obtained by dissolving or dispersing the microbial proteinin water, e.g., the water used in the above formulation and
add~j~ion of sodium hydroxide, calcium hydroxide or magnesium
oxide with stirring to adjust to pH 8.5-9Ø The aqueous
dispersion or solution can be used as is or the salt can be
obtained by drying, for example, by lyophilization.
EXAMPLE 42
Microbial Protein and Microbial Protein Salts as Replacements
for Caseinate Salts in Non-DairY Cheese Food Pr~ducts
-
In the following formulations, the test non-dairy
cheese food products containing the indicated products of the
invention are of overall good quality and are superior to the
sodium or calcium caseinate products with respect to water
and fat binding, viscosity and texture even when the gelatin
was omitted from the non-dairy cheese spread.
Non-Dalry Cheese Spread
Grams
IngredientsControl Test A Test B
Calcium caseinate 15 -- --
Microbial Protein isolate* -- 15 15
VegetabIe Fat 30 30 30
; Starch 3 3 3
Nonfat dry milk 5 5 5
Gelatin 2 2 --
Blend of lactic, citric
and acetic acids ~ 0.5 0.5 0.5
Water 60_ 60 _ 60
Total115.5 115.5 113.5
All ingredients are mixed until a uniorm paste is
obtained, then heated at 70-75C. for 5 minutes, packaged and
chilled.
~76-
: : ~ :::, - : '
,: ~
'
8~
EXAMPLE 42 (cont'd)
Non-Dairy Cheese Food Product
_ Grams
Ingredients Control Test ATest B
Calcium caseinate 20 -- --
Calcium salt of microbial
protein* -- 20 --
Magnesium salt of
microbial protein* -- -- 20
Soluble starch 2 2 2
Sodium chloride 3 3 3
Adipic acid
Citric acid 0.5 0.50.5
: Sorbic acid 0.1 0.10.1
lS Vegetable oil 25 25 25
Gelatin
Agar 0.5 0.50.5
Emulsifier 0.05 0.05 0.05
Water 46.85 46.85 46.85
Total 100.00 100~00 100.00
*C utilis or S. fragilis protein isolated by any of
the procedures of Exa~pIes 1, 3, 4, 5, 6 and llA are prefer-
: ably used. The calcium and magnesium salts were obtained as
described in Example 41.
~ Lactic acid (50%) 96.33~
Citric acid 3.60%
Acetic acid, glacial 0.07%
EXAMPLE_43
Low Calorle Non~Dairy Cheese Spread
~ Grams
Water 58.8
' ~
~0~66
EXAMPLE 43 (cont'd)
ow Calorie Non-Dairy Cheese Spread
In~redient Grams
Cheddar cheese 25.0
Microbial protein isolate* 10.0
Edlble gums 4.5
Sodium chloride 0.7
Dehydrated cheddar cheese 0.7
Lactic acid 0.2
Fla~or 0.1
Total 100.0
A good quality cheese spread was obtained using
the above formulation and the procedure of Example 42.
Microbial proteins which may be employed are those derived
from C. utilis, S. fragilis, A. niger, T. viride, L.
bulgaricus and S. lactis cells by the procedures of Examples
1, 3, 4, 5 and 6 and the corxesponding sodium, calcium, or
magnesium salts of these proteins.
* Alternately an acid or rennet curd of the microbial
protein may be used here. The curd may be prepared from a
solution of the microbial protein in water by suitable addi-
tion of acid, (e.g., lactic acid) or calf rennet b~ standard
methods.
EXAMPLE 44
__.
Micro~a1 Protein as a Replacement for Egg Wh te in Cake
Typical white cakes are prepared according to the
following formulations and procedure.
-78-
.-.: :, .. . :
, . . . .
i6
EXAMPLE 44 (cont'd)
Wei ht %
g
Ingredients Control cake Cake A Cake B
Flour 25.52 25.52 25.52
Salt 0.89 0.89 0.89
Baking powder 1.47 1.47 1.47
Nonat dry milk 3.50 3.50 3.50
Sugar 32.56 30.38 30.~8
Shortening 12.76 12.76 12.76
Dried Egg White 2.10 1.09 --
Microbial Protein --- 2.90 3.99
Sodium ~exametaphospha~e --- 0.29 0.29
Water 21.20 21.20 21.20
___
Total100.00 10Q.00 100.00
PROCEDURE
1. Blend dry ingredients.
2. Add shortening and mix until evenly distributed.
3. Add dried egg white and/or microbial protein and mix
well.
4. Add water and mix un~il batter is smooth.
5. Bake at 375F. ~190C.) for 20 minutes.
Upon evaluation, all three cakes are found to have
good 1avor; however, cakes A and B have greater volume than
the con~rol cake and are judged to have superior texture and
are more moist tasting. CaXe B is found t~ be especially
moist.
The S. fra~ilis and L. ~ proteins of the
invention are especially effective in this application.
Th~ test cake is found to have appreciably greater
volume, improved tex~ure and to be more moist tasting than
-79-
- , .
,
~ .
.
-
109~86G
the control. The two cakes are judged to be of equivalent
flavor.
The S. fragilis, L. bulgaricus and P. chrysogenum
proteins, as well as the phosphate complexes of each prepared
according to the procedure of Example 9, are especially
effective in the above angel food cake formulation.
EXAMPI,E 45
Replacement of Egg White in Angel Food Cake
Angel food cakes are made according to the follow-
ing fomulations and directions.
I~redients Control Test
-
Mixture A
Sugar 9.80 8.69
Dried Egg white 5.77 1.54
Microbial protein -- 4.19
Sodium hexametaphosphate -- 0.15
Nonfat dry milk 0.52 0.52
Cream of tartar 0.39 0.39
Salt 0.13 0.13
Mixture B
-
Sugar 25.94 25~94
Starch 8.34 8.34
Flour 6.34 6.34
Raking soda 0.15 0.15
Salt 0.13 0.13
Corn sugar 0.84 0.84
Cream of tartar 0.13 0.13
Water 41.52 41.52
Total lO0.00 lQ0.00
-80~
Directions
1. Pour water in the mixing bowl, add Mixture A and
blend one minute at low speed and then at high speed until
peaks are ormed.
2. Blend in Mixture B at low speed.
3. Bake at 375F. (190C.) for 25 minutes.
EX~MPLE 46
Microbial Protein as a Replacement for Egg White as a Protein
Binding Agent
A typical simulated food product in which egg white
is successfully replaced by the microbial proteins o the
invention as a protein binder is set forth below in which
soy protein fibers, 100 g., containing 60-65% ~f water (by
weight) are impregnated with the following ingredients:
Ingredients Control Test
Dried egg white20.0 g. --
Microbial protein -- 20.0 g.
Flour 10.0 g. 10.0 g.
Nonfat milk solids 10.0 g. 10.0 g.
Sodium chloride8.0 gO 8.0 g.
Yellow onion powder 1.0 g. 1.0 g.
Monosodium glutamate 0.5 g. 0.5 g.
Red Dye (2% in water) 1.5 ml. 1.5 ml.
Water 150.0 ml. 150.0 ml.
The impregnated fibers are baked at 350F. (177C.)
for 20 minutes, cooled and cut into squares to aford the
final product. Upon evaluation, the test product is found to
have superior texture and 1avor.
S. fra~ilis protein, S. cerevisiae protein and
S. lactis protein are especially effective in this use.
-81-
:- .
,
:'
, -- , . . .
~g~6~
E ~PLE 47
A simulated food product based on soy pro~ein fibers
which demonstrates the superiority of the microbial proteins
of the invention as protein binding agents is prepared accord-
5 ing to the following formulation and procedure.
Inyredients Parts by Weight
Vegetable oil 50
Glyceryl lactopalmitate 4
Water (hot) 160
Monosodium glutamate 3
Yellow onion powder 18
Brown sugar 7
Red dye (1% aqueous solution) 6
Egg white, dried or microbial protein10
; 15 Toasted, defatted soybean flour (200 mesh~ 20
Fresh gluten (33~ solids by weight) 90
Sodium chloride 25
Water 40
; The soy protein fiber (35-40% solids by weight~,
100 parts, is impregnated with 200 parts of an emulsion of
the above ingredients. The emulsion is prepared by first
mixing ths vegetable oil, hot water, glyceryl lactopalmitate,
mon~sodium;glutanate, onion powder, sugæ and dye. In the 40 g. portion of
cold water containing the salt are dispersed the dried egg
white or microbial protein isolate of Example 2, soybean
flour and gluten. This dispersion is then added to the other
ingredients with high speed stirring to form a free flowing
emulsion.
The impregnated soy protein fibers are heated at
-82-
, "
8~6
340F. (171C.) for 30 minutes, cooled and cut into strips.
EXAMPLE 48
Microbial Protein as a Binding Agent for Textured Protein
Meat patties containing one of the microbial
proteins of the invention as a binding agent for textured
protein, water and fat were prepared using the following
formulations and procedure.
Weight, Grams
_ Test Patties
IngredientsControl A B C D
Hydrated TVP ~ 100 100 100100 100
Vegetable fat 20 20 20 20 20
Dried egg white 5 -- 5 -- --
Microbial Protein* -- 5 -- 5 5
Soy flour 10 10 -- -- -- -
Microbial Protein* -- -- 10 10 10
Wheat gluten 10 10 10
Microbial Protein* ~ -- 10 --
~ater 25 25 25 25 25
Total 170 170 170170 160
* S. fragilis protein
~ Hydrated textured vegetable protein (TVP) is prepared
by blending the following ingredients and allowing to stand
overnight.
Ingr_dientsWeight, Grams
Meat flavor 25.0
Monosodium gluta-
mate 8.0
Sodium chloride12.5
Water 300.0
-83
'; ,
36~
EXAMPLE 48 (cont'd)
IngredientsWeight, Grams
White pepper 0.5
Textured vegetable
protein, dry 125.0
471.0
PROCEDURE
Melt the vegetable fat in a beaker and add the
water mix in the appropriate protein binders (i.e., egg
white, gluten, flour, microbial protein) and stir for 5
minutes. Blend the above mixture with the 100 g. of hydrated
TVP and allow to stand for 1~3 hours. Shape into patties and
broil in oven at 375F. (190C.) for 15 minutes. The patties
are then evaluated for functional and sensory properties.
Total Grams of Water and
Protein Binders Fat Loss
Meat (Gram Microbial on Cooking, Sensory t
Patties Protein) % Evaluation
Control 25 (0) 19.7 5.0
Test A 25 (5) 16.1 4.5
Test B 25 (10) 15.1 5.0
Test C 25 (25) 12.2 5.0
Test D 15 (15) 13.4 4.5
In~lude color, texture, mouthfeel and tas~e properties.
Evaluated on arbitrary scale range from 1 for very poor
to 5 for excellent.
Thus, the microbial protein shows good protein
binding characteristic when compared with egg white, soy
flour and wheat gluten. The loss of fat and water upon cook-
ing the meat patties decreases directly with increased amounts
o~ microbial protein.
When other microbial proteins of the invention
-84-
,,
, , , ,:,
,
.
,
- :,
isolated from cells of the following yeasts, bacteria and
fungl are employed in the above procedure in place of the
S. fra~ilis protein, similar results are obtained.
Yeasts Bacteria Fungi
S. carlsbergensis P. methylotropha T. viride
S. cerevisiae L. bulgaricus F. solani
C. utilis S. lactis P. chrysogenum
M. cerificans A. niger
C. cartalyticum
EXAMPLE 49
Microbial Protein as a Replacement for Gelatin in Low Calorie
Gelatin Dessert
Ingredients Weight, Grams
Soluble polyglucose tartrate* 72.00
Gelatin (225 Bloom) or Microbial Protein 9.00
Citric acid 2.40
Sodium citrate 0.40
Sodium chloride 0.40
Strawberry flavor, powder 0.24
Strawberry FDC color 0.06
9:1 Sodium cyclamate-Sodium saccharine 0.50
85.00
Water to make 16 oz. volume
The dry inyredients are blended and dissolved in
8 oz. of boiling water. The solution is diluted to 16 ounces
with cold water and refrigerated to gel.
Strawberry flavored gelatin desserts are prepared
using gelatin (control) and the microbial proteins of the
invention in place of gelatin. The con~rol desserts and
those containing microbial protein are found to be of equal
-85-
quality when evaluated for functional and sensory properties.
* Soluble polyglucose tartrate prepared as described in
United States 3,766,165.
-86-
,
. .... ~
