CA1078664A - Fortification of foodstuffs with amino acids and derivatives - Google Patents

Abstract

Abstract of the Disclosure Proteinaceous foodstuffs which are characterized by a nutritionally limiting content of sulfer-containing amino acids and threonine are fortified with selected N-acyl derivatives of the L stereoisomeric form of such sulfer-containing amino acids and with threonine to provide com-positions having nutritional values equivalent to animal derived proteins.

Description

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BACKGROUND OF THE INVENTION
-This invention relates to the fortification of proteinaceous foodstuffs which are deficient in both threonine and sulfur-containing amino acids.
United States patent 3,878,305, R.A. Damico and R. W. Boggs, issued April 15, 1975 describes a method of fortifying certain proteinaceous foodstuffs which are char-acterized by a nutritionally limiting content of sulfur-containing amino acids with selected N-acyl derivatives of the L stereoisomeric form of such sulfur-containing amino acids, and products so fortified.

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As pointed out in great detail in U.S. 3,878,305, there is considerable impetus to satisfy the protein re-¢uirements in the diet of humans and commercially important animals with proteins derived exclusively from vegetable sources. Unfortunately, proteins derived from plant species (including microbially-derived proteins) are not of comparable nutritional quality to animal-derived protein sources. This is evident, in part, by examina-tion of the amino acid content of various plant proteins.
Nutritionists have applied the term "limiting~ to such amino acids and have noticed that a protein characterized by a particular limiting essential amino acid is effectively leveled in nutritive value to the cont:ent of the first limiting amino acid. For example, if a certain vegetable lS protein is relied upon for the sole source of dietary ~tein, and ir that protein is chara~terized by an amino acid profile revealing certain limiting amino acids, it would be necessary to consume in the cliet an excessive amount of that protein in order to insure the nutritionally-required intake of those limiting essentia] amino acids.
In U.S. Patent 3,878,305 it is reported thatmethionine is an essential, but limiting, amino acid in vegetable proteins such as soybean protein, microbially-dèrived proteins, and like protein sources not of animal origin. This methionine de~iciency cannot be alleviated by simply adding methionine to such deficient proteins, inasmuch as methionine can result in the formation of bad tastes and foul odors in the food products fortified therewith. Patentees found that certain N-ac~lated ~78~
methionine derivatives do not suffer from this defect and are nutritionally available and suitable for use as a fortification means. Fortification of vegetable proteins with N-acyl L-methionine results in a very substantial increase in the nutritive value of vegetable proteins such as soy protein and raises its nutritive value to about 74% that of egg white proteins, which are recognized as being an excellent source of essential amino acids.
It has now been determined that threonine is a second limiting amino acid in vegetable proteins and that supplementation of such proteins with both N-acyl L-methionine and threonine results in a protein having a nutritional level of essential amino acids some 88%
lS that of egg white proteins.
The proteins prepared and foxtifi~d irl the m-nncr of this invention are fully equivalent to meat and cheese proteins derived ~rom animal sources.

:~378G~;4 SI~M~ARY OF THE INVENTION
The present invention relates to proteinaceous foodstuffs comprising:
i) an edible protein which is deficient in both threonine and sulfur-containing amino acids;
ii) a nutritionally supplemental amount of threonine; and iii) a nutritionally supplemental amount of an - .. N-acyl L-methionine derivative, or ester or salt 10 - thereof, wherein the N-acyl substituent is derived from fatty acids, said ~oodstuffs being essentially free of ~-acyl D-methionine.
The present invention also encompasses compositions for fortifying foodstuffs comprising N-acyl L-methionine and threonine~ , - ~le present invention also encompasses a method of fortifying protein foodstuffs w~lich. are deficient in both methionine and threonine by adding thereto a nutri- i tionally efective amount o~ N-acyl L-methionine and a 20 nutritionally effective amount of threonine. :~

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DESCRIPTION QF THE PREFER~ED EMBODIMENTS
As disclosed hereinabove, the present compositions comprise proteins which are deficient in sulfur-containing amino acidsO Such sulfur-containing amino acids include methionine, cystine and cysteine. While the compositions herein a~n be additionally supplemented with N-acyl-cystine and -cysteine derivatives without substantially impairing their desirable odor and flavor qualities (which the "free"
cystine and cysteine would do) this is not necessary in the practice of this invention since it is well recognized that methionine can supply the nutritional requirements for cystine and/or cysteine, but that cystine and/or cysteine cannot replace methionine in the diet. Accordingly, supple mentation of the compositions with acylated cystine and/or cysteine derivatives is not necessary to meet nutritional requirements and is entirely optional ~erein.
The N-acyl L-methionine employed herein can be used in its free acid form, in the form o an edible salt, or as an N-acyl L-methionine ester which hydrolyzes and de-acylates in the gut to provide L-methionine. The term "N-acyl L-methionine derivative" as employed herein is in-tended to encompass such edible, digestible salts and este~s, as well as the free acid. A more complete descrip-tion of the N~acyl L-methionine esters suitable for dietary supplementation is contained in Boggs et al. U.S. Patent 3,952,115, issued April ~0, 1976. ~-The method fox the identification and preparation of the N-acyl derivatives of amino acids employed herein c~'~t' `~
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and an experimental procedure for evaluating the palat-ability of foodstuffs fortified in accordance with this invention are fully disclosed in U.S. Patent 3,878,305, referred to hereinabove n . ' ' S The present compositions comprise proteins which are also deficient in threonine. Threonine, its edible salts and its esters which hydrolyze in the gut to provide "free" threonine, can be used herein. At the present time, L-threonine is believed to be the only form of this amino acid which is metabolized by humans. Accordingly, current-governmental regulations specify that only L-threonine can be used to fortify foodstuffs intended for human consumption.
D,L-threonine (racemic mixtures) can be used in animal feeds.
The term "threonine" as employed herein encompasses the free acid and the edible salts and esters; the free acid is preferred.
The classes of foodstufs cont~emplated by this invention comprise any food source which, for reasons of nutrition, require fortification with selected amino acids. This invention finds it5 principal application in foodstu~fs which contain protein derived substantially from vegetable sources, e~g., the oil seeds and leguminous seeds, principally the soybean, but other sources such as .

microbially derived proteins are also fully contemplated herein. Specific examples of such proteinaceous food- -stuff products where the protein component is substantially derived from the above sources include: textured vegetable prote1n meat analogs, vegetable protein cheese analogs, beverages, nut butters, breakfast cereal products, proteinaceous snack products, pet foods and animal ~eeds.

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A detailed discussion of ~oodstuffs of the type employed in this invention also appears in U.S. 3,878,305~
As noted hereinabove, the present compositions supply a nutritionally supplemental ~or "effective") amount of N-acylated methionine and threonine to the foodstuffs. No absolute meaning can be attached to the term "nutritionally supplemental or effective amount"
ln the abstract. However, a full discussion of this term, as it relates to methionine, appears in U.S. Patent 3,878,305 and the same rationale applies to threonine supplementation.
The threonine used in the human Eoodstuffs herein is L-threonine (preferred) or its salts, e.g., sodium salt, or esters, e~g., ethyl ester.
The N-acylated methionines herein include the N-acyl L-methionine wherein the acyl group is derived from Cl, and higher, especially Cl-C24, fatty acids, and the salts and esters of such N-acylated L-methionines. Typical examples of such compounds are set forth in U.S. Patent 3,878,305, and include N-formyl L-methionine, N-acetyl L-methionine (highly preferred~, N-stearoyl L-methionine, and the like.
Preerred compositions herein comprise mixtures of N-acetyl L-methionine plus threonine, each in nutritionally supplemental amounts, and proteinaceous foodstu~fs, especially those derived from soybeans, fortified therewith.
The following animal tests demonstrate that threonine is the second limiting amino acid in important vegetable-~:

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derived proteins. By supplementing such proteins with threonine and N-acyl L-methionine, good tasting, nutritious proteinaceous foodstuffs are secured.

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Animal Testinq The usual tes~ for determining the nutritional benefits of a protein foodstuff involves the protein efficiency ratio (PER) test. In general terms, this test involves feeding a diet comprising ca. 10% protein to rats. The weight gain in the animals divided by the protein consumed over a four-week period is the PER. T~e results of a typical test involving composi-tions of the present type are displayed in Table 1.

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_ _ ComPOsitiOn PER
Soybean protein isolate 1.6 1 0.2 Soybean protein isolate + NALM* 2.6 i 0.2 Casein (standard) 2.5 ~ 0.2 15 Meat 2.7 (defined) Egg 3.3 * 0.2 Cheese ca. 2.8 ~ 0.2 Soybean protein isolate + 2.7 ~ 0.2 NALM + lysinè

20 Soybean protein isolate -~ 2.7 ~ 0.2 NALM ~ threonine *N-acetyl L-methionine Tests of the foregoing type indicated that PER
studies are not sensitive enough to determine the second limiting amino acid in soybean protein. (However, it was noted that soybean protein fortified with NALM and threonine, in the presence or absence of lysine, resulted in .
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increased food consumption and increased growth rate in the rats.) On the basis of PER tests of the foregoing type, it was determined that a more sensitive test of the nutritive value of protein was required in order to determine the second limiting amino acid.
A more sensitive test, the Slope Ratio Test (a standard procedure), compares the weight gain in the animals versus the percentage protein in the diet or ~ -versus the percentage protein consumed. In the Slope Ratio Test, the weight gain of the animals is measured over a range of protein intake levels. In general terms, the test involves the use o a reference protein, such as egg, at three levels in the diet. The test protein is aiso used at three levels. By selecting protein levels in the range wherein weight gain and protein uptake are roughly linear, it is possible to determine, graphicallys the ratio of the slope of the test protein response line to the slope o~ the reference protein response line. (The reference protein line gives the greatest weight gain per protein consumed, i~e., the greatest slope, and is defined as "100%".~
The foregoing procedure was employed to test compositions of the present type versus reference proteins and other test proteins. The proteins were tested at the ?5 10%, 8% and 6% dietary levels. Under this situation, which is quite stressful to the animals, diferences in the nutritiva qualities of the protein due to their differences in amino acid content are readily seen.
~ Results from the Slope Ratio Test appear in Table 2.

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Protein Relative Value Egg 100 (Reference) -Soy protein isolate 48 Soy protein isolate + NALM . 74 Soy protein isolate + NALM ~ 74 lysine Soy protein isolate -~ NALM + 88 threonine Soy protein isolate ~ NALM ~ 88 lysine + threonine Reference Proteins Meat 82 !.
Cheese 88 .
As can be seen fxom the foregoi.ng experiment, threonine is the second limiting amino acid of soy isolate protein and addition of this amino acid to soy isolate-~AI~ compositions raises their nutritive value to that o~ meat and cheese proteins.
20 ~ The important feature of this invention is that ~oodstuffs based on vegetable proteins,.and the like, can ~ ~ .
now be fortified to provide nutritional levels fully ;:
equivalent to meat, cheese, and the like, without undesir- :
able odors and flavors, even after storage or cooking.

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The following foodstuff compositions are prepared in the same manner as that described in U.S. Patent 3,878,305, with the exception that threonine is added to provide the additional nutritive benefits disclosed hereinabove. The examples are typical of the composi-tions which can be made according to the practices of this invention but are not intended to be limiting - thereof.

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EXAMPLE I
A meat analog product is made according to the ollowing formulation and procedure.

ComponentParts by Weiqht ~oyhe~n isolate 5~.0 Water ~ 40.0 N-acetyl-L-methionine1.8 Threonine (L form)2.5 Fat 10.0 Starch 10.0 Flavoring, color and salt 6.0 The components are blended to form a dough, passed through a Brabender extruder at a temperature of ca. 150C and at a pressure of 450 psi. This produces a cylindrical product from the 4 mm. exit die. The extruded product is dried and ground to particle sizes having an average diameter of ca. 1/~ inch.

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A product prepared in the manner of Example I
is hydrated and shaped into hamburger-like patties and fried in an oiled pan. The composition has a nutritive value (with respect to essential amino acids) comparable S to that of cooked hamburger. No off-flavors attributable to the degradation products of methionine are observed.
In Example I the N-acetyl L-methionine is replaced by a nutritionally equivalent amount of N-formyl L-methionine, ~-propionoyl L-methionine, N-stearoyl L-methionine, N-palmitoleoyl L-methionine and N-linolenoyl L-methionine, respectively, and an excellent, nutritive food product is secured.

EXAMPLE II
A fortified peanut butter composition is prepared h' adding 1.5 p~x~s b~ wcight L-thrconine ~extrac~ed from drled egg white) and 0.5 parts ~y weight N-acetyl L- ;
methionine to a commercial peanut butter of the following composition.

InqLredient Parts by Weiqht ;
Peanut paste gO,o Salt 1.2 Sucrose 5.8 Molasses 0.5 Soybean monoglyceride - 0 7 ~25 Soybean oil (iodine value 2) 0.84 Soyb-an oil (iodine value 107) 0.40 . , . . ............ , .. . ' - . . . ' .
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The peanut butter composition prepared in the foregoing manner provides nutritionally effective amounts of methionine and threonine.
A sample of the fortified peanut butter composi-tion of Example I~ is heated in a water bath at 95C ~-for 2.5 hours to simulate shelf storage. No off-flavors or odors attributable to the sulfur-containing amino acids are noted.

EXAMPLE III
-A nutritionally complete cattle feed composition encompassed by the present invention :is prepared by grinding and mixing the following ingxedients. ~The composition employs the relatively inexpensive D,L-threonine mixture which is ~cceptahle for US9 in animal feeds.) , .
Inqredient Parts_by Weiqht Timothy hay 960 Dehydrated alfalfa 40 Yellow corn 600 Corn starch 300 Iodized salt 10 Bone meal 20 Soyhean meal 30 Biuret 28 N-acetyl-L-methionine 1.0 D,L-threonine 1.0 ~.

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;4 The composition of Example III is fed to steers at the rate of approximately 12 pounds per day. The animals on this diet exhibit excellent growth rate and the feed is characterized by the absence of potentially S toxic degradation products of the methionine supplement.

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As can be seen from the foregoing, the present invention encompasses compositions for fortifying food-stuffs de~icient in sulfur-containing amino acids and threonine comprising a nutritionally supplemental amount of threonine and a nutritionally supplemental amount of an N-acyl L-methionine derivative. Under current govern-ment regulations, compositions for human use are those wherein the threonine is L-threonine, whereas compositions -for animal use can employ either L-threonine or D,L-threonine mixtures. The compositions herein typically comprise threonine and N-~cyl L-methionine derivatives at a weig~t ratio in the range from about lO l to about 1:10.
As noted hereinabove, the N-acyl L-methionine "derivatives" useful herein include the "free" acid form, lS as well as esters and edible salts of the N-acyl ~-luethionine. L-methionine is acylated on the nitrogen (N) atom to reduce objectiona~le odors and ~lavors. With threonine, which is not a sulfur-containing amino acid, N-acylation and/or esterification is not necessary since odor and flavor problems, if any, are slight. However, N-acylated and/or esterified threonines are useful herein, inasmuch as such materials are edible (especially when the N-acyl derivative is a Cl-C24 fatty acid moiety and when the esterification is carried out using a Cl-C24 alcohol) and all such materials and edible salts are encompassed by the term "threonine" as employed herein.
As can also be seen from the foregoing detailed descrip-tion, the present invention also encompasses proteinaceous .

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foodstuffs selected from edible proteins and/or protein sources which are deficient in both threonine and sulfur-containing amino acids. Such foodstuffs are fortified with nutritionally supplemental amounts of S the N-acyl L-methionine derivatives and threonine, as defined hereinabove. Under current governmental regula-tions, foodstuffs for human use are fortified using the I.-threonine, whereas foodstuffs for animal use can be . for$ified with either L-threonine or D,L-threonine mix- ~.
tures.
In all of the foregoing compositions, N-acetyl L-methionine is preferred .as the sulfur-containing amino acid source.
The present invention also encompasses methods for fortifying foodstufs which are deficient in sulfur- .
~ontaining amlno acids c~nd ~llreonine. ~uch fortification methods comprise simply admixing with such foodstuf~s, especially proteinaceous foodstufs such as soybeans, a nutritionally supplemental amount of the compositions herein which consist essentially of a nutritionally supplemental amount of threonine and a nutritionally .
supplemental amount of an N-acyl L-methionine derivative, ..
all as described in detail hereinabove.
It will be appreciated by nutritionists and formu-lators that the requisite amount of threonine and N-acyl L-methionine derivatives employed in the compositions and methods herein will depend on the amino acid content of the nutritionally-deficient protein sources being - fortified.... For most purposes, in order to bring ve~etable :
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(including microbially-derived) protein sources up to meat, c~eese and/or egg values, from about 0.01% to about 10%, more pre~erably from about 0.5% to about 5%, b~ weight, of the N-acyl L-methionine derivative ~cal-~ulated on the basis of the total protein content of theprotein source) is suf~icient to provide a nutritionally supplemental amount of sulfur-containing amino acids.
Likewise, fortification of proteinaceous ~ood-stuffs with nutritionally supplemental amounts of threonine involves the use of from about 0~01% to about 10%, more preferably from about 0.5% to about 5%, by weight, of threonine, based on total protein present in said foodstuff.
What is claimed is:

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Claims (15)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A composition for fortifying foodstuffs deficient in sulfur-containing amino acids and threonine comprising a nutritionally supplemental amount of threonine and a nutritionally supplemental amount of an N-acyl L-methionine derivative.

2. A composition for human use according to Claim 1 wherein the threonine is L-threonine.

3. A composition for animal use according to Claim 1 wherein the threonine is a member selected from the group consisting of L-threonine and D,L-threonine mixtures.

4. A composition according to Claim 1 wherein the N-acyl L-methionine derivative is a C1-C24 fatty acid amide derivative of L-methionine.

5. A composition according to Claim 4 wherein the weight ratio of threonine to N-acyl L-methionine derivative is in the range from about 10:1 to about 1:10.

6. A composition according to Claim 1 wherein the N-acyl L-methionine derivative is N-acetyl L-methionine.

7. A proteinaceous foodstuff, comprising:
i) an edible protein which is deficient in both threonine and sulfur-containing amino acids;
ii) a nutritionally supplemental amount of threonine; and iii) a nutritionally supplemental amount of an N-acyl L-methionine derivative, wherein the N-acyl substituent is derived from fatty acids.

8. A foodstuff for human use according to Claim 7 wherein the threonine is L-threonine.

9. A foodstuff for animal use according to Claim 7 wherein the threonine is a member selected from the group consisting of L-threonine and D,L-threonine mixtures.

10. A foodstuff according to Claim 7 wherein the N-acyl L-methionine derivative is a C1-C24 fatty acid amide derivative of L-methionine.

11. A foodstuff according to Claim 10 wherein the weight ratio of threonine to N-acyl L-methionine derivative is in the range from about 10:1 to about 1:10.

12. A foodstuff according to Claim 7 wherein the N-acyl L-methionine derivative is N-acetyl L-methionine.

13. A method for fortifying foodstuffs deficient in sulfur-containing amino acids and threonine comprising admixing therewith a nutritionally supplemental amount of a composition consisting essentially of a nutritionally supplemental amount of threonine and a nutritionally supplemental amount of an N-acyl L-methionine derivative.

14. A method according to Claim 13 wherein the N-acyl L-methionine derivative is a C1-C24 fatty acid amide derivative of L-methionine.

15. A method according to Claim 14 wherein the N-acyl L-methionine derivative is N-acetyl L-methionine.