CA1088425A - Bio-available essential metals - Google Patents

Abstract

ABSTRACT

Essential metals are more readily assimilated into the body of a warm blooded animal when administered in the form of metal chelates containing at least two ligands wherein the ligands are protein hydrolysates selected from the group consisting of tripeptides, dipeptides and naturally occurring amino acids. For greater effectiveness the metal chelates may be formulated into a composition comprising and acid-base buffer which will buffer the composition to a relatively constant pH between about 7 and 11.

Description

zs Certain metals are known to be essential to the proper functioning of the body. Ca~cium is present in the body in greater abundance than any other metal and is found primarily in the bones and teeth but also plays an important part in blood clotting. Many enzymes require magnesium.
Magnesium deficiencies lend to vasodilation and hyperirritability of the ' nervous system. Magnesium is not easily absorbed into the body and is ~
largely excreted in the feces. Hence most magnesium salts are ;`
laxatives or cathartics. ~i Iron is essential in the formation of hemoglobin and in metabolic and resplratory functlons of the body. Lack of lron absorption ls a ;
prlmary cause of anemla. In lnorganlc form very llttle lron ls absorbed and most lron lngested ls ellmlnated ln the feces.
Copper ls an lmportant metal in the functioning of many enzymes and is assoclated wlth hemoglobin formation and enzyme functions.
Agaln lnorganic copper is excreted via the bowels.
Cobalt is a component of vitamin B12 and has been used successfully ., .: , .
ln the treatment of certaln types of nutrltlonal anemia. ;
Another essentlal element ls manganese. Manganese deflciencles i~.
are essential to growth and vlrillty. Manganese ls also lnvolved ln 20 activating several enzymes. Again, manganese is largely excreted in the feces.
Zinc has been found to be essantial in the actuation of enzymes ~;
relating to cell divlslon and is a constituent of insulin. Excretion of zinc is largely through the digestive tract.
Molybdenum is another element found in certain enzymes.
Other trace metals such as chromium and vanadium are also thought to be essential. ~
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All of the above elements are capable of existing in bivalent ;;
form. By llbivalent" is meant the metals may assume an ionic or oxidation state of at least ~2 or higher. This assumes that copper will be in the cupric or ~2 state.
Since, in general, these metals, as inorganic or organic salts, are absorbed with difficulty it would be desirable to find a formulation whereby these metals could be effectively assimilated into the body.
Salts ionize in the gastric juices of the stomach and enter the small intestine, where most absorption takes place. The intestinal walls are llned with electrlcal charges whlch have a strong tendency to repal the positlve metal cations whlle allowlng the anlons to pass through.
The essentlal metals are thus dlscharged through the bowel after causing diarrhea. The anions passing through the intestinal wall often are excreted via the urine and often act as diuretics.
Various attempts utllizing organic salts have been made with llmited success. Chelates utllizing ligands formed from EDTA
(ethylenedlamlnetetraacetlc acid) and derivatives have also been utlllzed. Chelates formed from EDTA normally blnd the metal so tightly that it ls not made readlly avallable to the body.
It would therefore be beneficial to prepare a formulation of an essential bivalent metal in a form whereby the effects of the electrical charges of the intestinal lining of the metal were minimized and whereby the metal could be made available in a readily assimilable form.
In the past it has been known to utilize certain protein hydrolysates as chelating agents or ligands to increase the assimilation of metals into -biological tissues. It has been found however that certain protein

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hydrolysates are poor ligands due to their size and sterochemistry. Long chain polypeptides when used as ligands do not form as strong a bond ~ -with a metal ion in chelate formation as do aDlino acids, dip~liides and tripeptides. It is therefore axiomatic that chelates formed from metals and long chain polypeptides are more easily destroyed in the acidic gastric juice of the stomach.
Protein hydrolysates is a term generally used for any form of ~ -hydrolyzed protein ranging from the above mentioned long chain polypeptide down to the basic protein building blocks, i.e. amino acids. These ;
hydrolysates are commonly formed utilizing acidic or baslc hydrolysis or a comblnatlon of both. Since many different amino acids are essentlal to the body there is a dlstlnat disadvantage to the utilizatlon of either form of hydrolysis. Acidic hydrolysis destroys the amino acids tryptophan serine and threonine. On the other hand basic hydrolysis racemlzes the amino acids into their D, L forms and destroys arginine threonlne, serine, and cystine. Naturally occurring amino acids belong only to the L-series. Moreover aaidic and basic hydrolytic processes require neutralizatlon and this results in the formation of inorganic salts whlch often remaln with~the hydrolyzed product.
Chelates formed from essential bivalent metals and protein hydrolysates are referred to as metal proteinates. -!, It is an object of the present invention to provide a metal proteinate ' in a form that ls readily assimilated into the body of a warm blooded animal .
It is also an object of the present invention to provide a metal proteinate utilizing as a ligand a naturally occurring amino acid, a , ~

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dipeptide or a tripeptide.
Another object of the present invention is to provide a product that will increase the metal content in biological tissues.
An additional object of this invention is to provide a method for ;;
increasing the level of essential bivalent metals in biological tissues.
A still further object of this invention is to provide a method of correcting met~l deficiencies in the tissues of animals.
Also, an object of this invention is to provide metal proteinates in a sufficiently stable form that they will pass intact through the stomach of a warm blooded animal and be assimilated into the body through the intestinal walls. - -Also, an object of this invention is to provide a compositian for raisiny the lev~l of essential bivalent metals in tissues of ammals comprising a carrier containing an effective amount of metabolically assimilable metal proteinates, said proteinates being in the form of chelates of said metals with at least two protein hydrolysate ligands selected from the group consisting of tripeptides, dipeptides and naturally occurring amino acids, wherein the ligand to metal ratio lS between 2 and 16.
These and other objects may be attained by means of the invention as fully described as follows.
It has now been fcund that essential bivalent metals are mDre effeckively administered to animals in the form of a chelate formed from tripeptides, dipeptides and naturally occurring amino acids wherein the ligand to metal mole ratio is between tWD ànd sixteen. Preferably the mole ratio of ligand to metal wdll be between tw~ and eight. Especially -preferred are ratios between two and four. Each ligand will have a molecul æ
weight varying between abcut 75 and 750.
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The ligands will advantageously ke formed by the enzymatic ...
hydrolysis of protein sources such as collagen, fish meal, meat, isolated soya, yeast, casein, albumin, gelatin and the like. Enzymatic :
hydrolysis utilizing enzymes, such as trypsin, pepsin and any other .
protease may be used is not detrimental to the formation of L-amino : :
acids. Ligands prepared by enzymatic hydrolysis do not contain inorganic ,. ~ .. . .

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~31813~25 salts that ligands from basic or acidic hydrolysis may have. However ~he use of ligands formed from acidic and basic hydrolysis still may be utilized but on a less preferred basis. The term naturally occurring amino acids also includes synthetically produced amino acids having the same stero configuration as those which occur in nature. Proteins yield about twenty amino acids including glyclne, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, se~ine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, arginine, histidine, cystine, cysteine, methionine, proline ancl hydroxy proline. A dipeptide is a combination of two amino acids with a peptide (-CO NH-) bond and a trlpeptide ls a combination o three amino aaids wlth two peptlde bonds. The llgands bonded to a metal ion can be the same or diffeEent.
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When making the chelates the shorter the chain length of the ligand i the easier it is to form chelates and the stronger the chelation bonds will be. As used herein the term chelates and proteinate may be used `
interchangeably unless a non-protein derived ligand is referred to.
It is bene1cial to irst hydrolyze a protein source well so that the ma~or portion of the hydrolysates will be amino acids, dipeptldes 20 and tripeptides. Thus, the subsequent formation of the metal proteinate or chelate can form a product that can ~e actively transported through the body tissues. Large protein entities such as metal salts of gelatinates, caseinates or albuminates must be broken down or hydrolyzed before ... .
transport can take place. It is beli~ed that unh~tdrolyzed protein ;
salts, in general, are unabsorbed from the intestinal walls. ThereEore, in the present invention the protein molecules are hydrolyzed to a .
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tripeptide, dipeptide or amino acid stage prior to mixing with the metal salt to form a proteinate. In order to form a metal proteinate the proper amounts of constituents must be present at the right conditions. The -mineral to be chelated must be in soluble form and the tripeptides, dipeptides or amino acids must be free from interfering protons, i.e., non-intact, in the chelation process so that chemical bonds can be formed between the proteinate ligand and the metal involved. Since chelates, by definition, are molecular structure~in which a heterocyclic ring can be formed by the unshared electrons of nelghboring atoms, it is 10 essential that before a protein hydrolysate can complex with a metal lon to form coordinate bonds, that the protons In the chelating agent, 1. e., the amino acld dipeptide or tripeptide be removed. Again, by definition, a chelating agent is considered to be an organic compound in which the atoms contain donor electrons which form more than one :

coord~lat~l-3 bonds with metal ions in solution. Thus it is essential .
that the chelation process take place in solution. Once the chelating mineral salt is completely soluble and the amino acids or peptides ~ -are su~flclently soluble, the p~I must be adjusted to a point that ls sufficiently basic to remove lnterfering protons from both the amine 20 groups and the carboxyl groups of the ligands. While a pH of 7. 5 may be sufficient a pH in the range of 8 - 10 is preferred. This allows the heterocyclic rings to form bonds between the metal and lone pairs -of electrons left behind on the amine groups. Thus~ the mere mixing of intact amino acids or intact peptides with water in the presence of a metal salt will not result tln a chelate or proteinate because the protons on the carboxyl and amine groups interfere with ~helate formation.

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~}8E~425 When combining protonated or intact peptides or amino acids with a soluble metal salt either no reaction takes place or a salt may be formed from the metal with the peptide or amino acid, which salt may be soluble or may precipitate. The metal proteinates formed as described precipitate in basic solutions and are insoluble or only partially soluble in water solutions. Moreover, metal proteinates or chelates are heterocyclic complexes and are vastly different from metal salts of peptides or ,1 amino acids. The metal proteinates are more readily assimilated making the metal more readily available to the body tissue.

In general the stabllity of the metal proteinate lncreases as the oxldatlon state of the metal ion increases. Also shorter arnlno -acld groups produce stronger proteinates; hence amino acids are preferred over peptides.
The coordlnation number, which is quite different from the valance tells how many coordinate covalent linkages a metal mayl have, Using iron as an example, the number may vary from two to eight although other values are also known. The ferrous ion, may have four waters of hydratlon. Slnce most metals have coordlnation numbers of two to elght thelr ions become incased within hydration ~`

20 shells made up of waters of hydration whereln each electronegative oxygen atom of each water molecule is attracted to the positively - -charged metal ion.
Every bond between the metal, in this case, iron and oxygen ;~
atoms consists of two electrons. The water molecules are said to be coordinated to the metal because each oxygen atom contributes both electrs)ns that make up the coordinate covalent or simply covalent "~. ~'. '.
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bonds .
A complex is formed when a substance other than water forms a coordinate or covalent bond. A chelate may be a fi~omplex but not all complexes are chelates. Oxygen and nitrogen are electronegative elements that cl~ntribute electron pairs to form covalent bonds. Amino acid and peptide ligands contain both of these electronegative elements. :
A ligand may refer to the part of a molecule which contains these negatlvely charged elements and is the site where complex formation occurs. The term "ligand" ls also used to designate the metal bindir~
molecules themselves. As previously stated the term chelate or protelnate refers to a metal containing at least two ligands. ~hen a metal proteinate has two or more llgands lt may be referred to as a bi-, tri-, quadri-, quinque-, or sexadentate proteinate, etc. A metal proteinate is a complex containing two or more heterocyclic rings. The term "chelation", therefore refers to a particular kind of metal binding ~
where the amino acid function of the molecule is clamped onto the metal ;
at two or more sites, thus producing the heterocyclic ring. Obviously a metal cannot ~o~m a coordination complex with an intact amino acid or peptide. Therefore it is important to metal chelate formation that the interfering protons be removed from the carboxyl and amino agroups before chelation can take place.
To form a metal proteinate the coordinated water molecules will be replaced by the ligand.
Chelates differ from salts and other complexes due to their closed ring structure in which the metals are tightly held. Generally five membered chelate rings are most stable. It is to be remembered .

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1C1~3~4;~5 that while covalent bonds are most stable and are preferred some ionic bonding may be formed in each chelate. The transition metals tend to form more stable covalent bonds whereas alkaline earth metals are more likely to contain some ionic bonds.
Sability constants may be determined by polorography wherein it is shown the chelates are of the magnitude of 103 to 1013 times more stable than the corresponding salts or complexes.
When forming a metal proteinate the pH of the solution drops upon the addition of the ligand. It is important that the protons on the llgand be removed so that they do not lnterfere wlth chelate ~ormation by competlng wlth the metal ion.
Uslng glycine, the slmpllst amino acld, as a model and iron as the metal ion containing four waters of hydration the reaction would be as follows. ~ -C - a~ H O~ Fe~ H201 ~C ~ ~ F~e2 ~C ' NICH3~ L ~ ~--t CE12 E12O NEI

Thus a complex ls formed which may or may not be soluble in the aqueous reaction media. Upon the addition of more base such -as NaOH the product becomes ~C~ ~ /~C~O ~ ~' H2 C ~NH2 H~ ~CH2 This represents a true metal proteinate wherein all of the protons on the ligand have been removed and thus heterocyclic rings i ~ . .
have been formed. Note that each ring has five members which are :,... :., :
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found to be most stable. From the above it is imperative that the ligand protons be removed and full chelation be carried out under basic reation conditions.
Metal proteinates are relatively insoluble in basic solutions but depending upon the concentration, metal proteinates at low concentrations are soluble or partially soluble in slightly acidic to a~aline solutions.
VVhen administering metal proteinates orally to warm blooded .. ~. ,.
animals a portion of the product may be destroyed by the acidic media of the stomach thus deprlvlng the animal Erom the full benefit of the dosage administered. It has been shown, however, that a maJor proportlon of the metal proteinates are passed lntact into the intestlne for absorption.
The metal protelnates can be stabilized and thus pass more readily ;
through the stomach when buffered to a constant pH between about 7 and 11. The buffer system may ch~nge according to the metal proteinate belng admlnlstered, and one or more metal proteinates may be admlnistered at one time.
The choice of buffer system wlll depend upon the pH deslred.
20 Awh~oo aclds alone wlll reacti wlth bases such as sodium hydroxide to form buffered systems. Typlcal ~uffer solutions include phosphate, carbonate and bicarbonate anions or combinations thereof. Either :''~
alkali or alkallne earth met als may be used as cations in the buffer systems. Examples of typlcal buffering systems in the pH range of 7 to 11 are as follows:
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7 9.1 g- KH2PO4 ~19. 7 g- Ma2HPO4 per liter.

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~ 8~5 7 50 ml. M/5 KH2PO4 ~ 22.4 ml. M/5 NaOH diluted to 200 ml.
8 50 ml. M/5 KI12PO4 ~ 46.1 ml. M/5 NaOH diluted to 200 ml.
6.5 g. NaHCO3 + 13. 2 Na2CO3 per liter. ~ -11 11- 4 g- Na2Hpo4 ~ 19 7 g. Na3PO4 per liter.

The present invention encompasses any combination of organic or inorganic substituents which will buffer or maintain a system at a pH range of 7 to 11. There are numerous other buffering systems readily available to one with ordinary skill in the art and mere enumeration of them would be redundant. What is important to the invention ls that a bufferlng system be selected whlch will not only stablll~e a metal proteinate but will aL~o be non-toxlc and assist ln the asslmilatlon of the metal protelnate lnto the body.
The term warm blooded animal is intended to encompass all species of the animal kingdom including man.
The dosage to be administered will depend upon the type of formulation, the animal species, weight, age, sex and the metal proteinate or mlxture of metal protelnates belng adminlstered. It may flrst be deslrable to determlne the metal deflciencles in an animal by 20 assaying hair, nails, blood, urine, skin or sallva from the animal and comparing the results with a standard representing a normal healthy `
animal of the same species. A corrective formulation can then be made ~;
up. On the other hand it may be desirable to administer the RDA of' each rnetal in one or more dosages. For example the RDA for an average adult is, Iron 18 mg.; Zinc 15 mg.; Copper 2 mg.; Magnesium 400 mg. ~ -and Calcium 1000 mg. There have been no RDA's set for other minerals ~
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which have been considered essential to the proper functioning of the "" ~
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body.
The metal proteinates can be given in various ways as long as a biologically effective amount is administered. When administered -`
orally the metal proteinates can be admixed with a suitable carrier ~ -and given in the form of tablets, pills, c~psules, emulsions, syrups or admixed with foodstuffs. When given to domestic form animals or pets, the metal proteinate will preferably be admixed with, `
sprinkled on or poured over the anlmal's food. Composition of metal prc)~inates may also be given by stomach tube or by a valve.
When given to a human the metal proteinates may be administered as a tablet, syrup, capsule or the like or may be admixed with or placed on the surface of food such as meats, stews, bakery products, candies, deep fried foods or placed in seasonings or spices.
To further demonstrate the stability of metal proteinates, t~ie'ir -ability to raise the level of metals in biological tlssues and lllustrate vari~us compositions and methods of administration the following examples are given. It is to be noted however that the examples are not lntended to be self limiting but are for purposes of lllustratlon only.

EXAMPLE I
A mixture of 200 lbs. of caseln and 1000 lbs. of water was ; -mixed in a ~acketed tank and stirred. Six pounds of sodium hydroxide was added to neutralize and b~!ing the casein into solution. A mixture .....
of three pounds each of the enzymes papain, bacterial protease and fungal protease was added along with a preservative such as sodium -~
benzoate. The mixture was covered and stirred for a period of si~
days to produce a hydrolysate containing over 85% tripeptides, , . : : ; , ,: .: .

dipeptides and amino acids. The enzymatic action was stopped by bringing the solution to a boil for about 15 to 20 minutes and filtered while hot through muslin. The filtered solution was then ready for use in preparing metal proteinates according t~the following examples. Other proteiin~ sources such as gelatin, collagen, yeast, fish meal, soya and the like could be used in the place of casein.
It will be noted that in most cases the metal content of the proteinate formed will vary from about 5 to 15 percent by weight with 10 percent being the average.

EX~MPLE II
To a flltrate similar to that obtained from Example I was added 33 pounds of zlnc chlorlde. To this solutlon was added sufflcient sodium hydroxide to raise the pH to about 8. 5 which produced a precipitate containing about 14 percent zinc as a zinc proteinate. The `
ligand to mole ratio was found to be two to one.

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EXAMPLE III
Example II was repeated using 75 pounds of ferrous sulfate ;
(FeSO4 ' 7H2O). The washed and dried precipltate produced about 175 pounds of iron proteinate containing nine percent by weight iron.

EXAMPLE IV
Again Example II was followed using 41 pounds of cupric chloride (CuC12 2H2O) plus 1000 pounds of methyl alcohol. The collected precipitate contained about 180 pounds of a copper proteinate containing ~;
eleveh percent by weight copper. ~
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EXAMPLE V
The prodedure of Example II was again repeated using 56 pounds of manganese chloride (MnC12 4H2O). The precipitated manganese proteinate contained about eight percent manganese and weighed about 170 pounds.

EX~MPLE VI
An acid hydrolyzed soy protein reduced to the tripeptide, dipeptide and amino acid stage was prepared by mixing 16 pounds of water with -5. 7 pounds of concentrated hydrochloric acld. Ten pounds of isolated soya was added wlth mlxing and the mixture was heated to 130C for

4 hours to hydrolyze the proteln. The hydrolysate was neutralized with 4 pounds of zinc carbonate and the pH was adjusted to 8. 5 by the addition of sodium hydroxide. The precipitated zinc proteinate was ~-washed and dried to obtain a product containing about 15 percent by w~ight zinc.
In each of the above examples the ligand to metal mole ratio ~`
was at least two to one. !` `
Other acids and bases could be used ln the hydrolysis process such as phosphoric acid and sulfuric acid and sodium hydroxlde.

EXA~PLE VII
The stability of metal proteinates, exemplified by zinc methionate is demonstrated as follows.
Traces of Zn65C12 were mixed with nQn radioactive ainc chloride and chelated with methionine as an amino acid. To demonstrate structure and how tightly the methionine was bound to the zinc, a .

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polarographic study was made. A solution was prepared containing -. 0001 moles of zinc per 100 mls of solution of ZnC12, and there was added thereto sufficient 0.2 M methionine to produce a solution having a molar ratio of amino acidsto zinc as follows:

Solution Mls of 0. 2 M Moles Ratio Methionine Methionine Zinc 0 ml 2 2.5 ml 0. 5 3 3 5. 0 ml 1. 0 4 10.0 ml 2. 0 20.0 ml 4.0 6 40.0 ml 8.0 7 80. O ml 16. 0 To each of the above solutions was added 10 mls of 1 M potassium ~ ;
nltrate (KN03) as an electrolyte and 10 mls of a 0.1% gelatin solution.
E(ach solution was corrected to a pH of 7 by the addition of a few ;
drops of concentrated (6N) sodium hydroxide(NaOH) solution.
Using a Metrohm E 261 polarograph wlth a silver/sllver chloride (Ag/AgCl~ reference electrode, the Eollowing E1/2 - S were 2 0 recorded:
Solution No. E~/2 :: ~- -. .
-1. 008 2 -1. 0~3 3 - 1 . 05 7 4 -1. 079 -1. 090 6 - '~D. llO

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A plot of the log of the proteinate ligand concentration against the El/2 gives a sloped line which is indicative of the number of ligands in the complex. It was found that the Zn~ ion complexes with two molecules of methionine.
While not wlshing to be bound by any specific theory, it is believed that at higher ligand concentrations and at a higher pH
(more basic) the complex is probably a bicyclic complex. ;
By knowing the number of ligands / the stability constant at different concentrations and pH's can be determined. It has been found that the logarithm of the stability constant equals: ;
059 ~;/2 P log ~igand] . 059~

where p = the number of ligands and [lgand~r~fers to the concentration of the ligand.
Solution lNumber 7 (16 moles of methionine per mole of zinc) was found to have a stability constant equal to 4. 94 x 107 at pH 7.
The same solution was adjusted to a pH of 9 and the stability constant was found to be 4. 41 x 1012. In cother words, by changing the pH of a zine methionate solution of the same coneentratlon from 7 to 9 an inerease in stability of 105 or 100,000 was obtained. Similar results 20 ean be demonstrated with copper, iron, chromium, calcium,manganese, magnesium, vanadium and otherressential metals.

EXAMPLE VIII
In this series of experiments, day-old chicks were divided ;
into groups and each group, control and treated, was fed the same commercial chick-starter ration. For example, one commercial -, . . , ~ , . . . .

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chick-starter ration has the following composition: soy bean meal, meat meal, ground corn, ground milo, salt, fat, dicalcium carbonate, limestone, and an inorganic trace mineral m~x. This chick-starter ration had 22% protein, and a fat content giving a metabolizable `.
caloric content of 1250 calories per pound. The ration also contained vitamins in addition to the standard inorganic mineral supplement.
Each group of chicks referred to as "treated" received a different predetermined quantity of metal proteinate (as set forth in the following tables) in addition to the above commerical chick-starter ration. Ghicks fed the commercial chick-starter ratlon plus correspondlng amounts of lnorganic metals serv~d as the control.
The comparlson results are reported as a T/C ratlo to in~lcate , .
the concentration of metal ln treated chlcks as compared to the concentration of metal in control chicks. Accordingly, a T/C - ~;
ratio greater than one indicates that there is a greater concentration of metal in the tissue of the treated chick as compared to the concentration of metal in a similar sample oE tlssue from the control ~ : .
chlck. A T/C ratlo less than one lndicates the reverse.
The purpose of the comparison was to demonstrate the lncrease in metals asslmilation by chicks which were fed metal proteinates as compared to chlcks fed lnorganlc metals.
Table I reflects the T/C ratio for treated and control chicks ~ -in various tissues 10 days after treatment commenced.
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~''. ':' , ~' EXAMPIE ~
The beneficial stimulation of growth by the addition of metal ~;
proteinates to the diet of turkey poults was also determined.
Turkey poult hens (one-day old) were divided into five groups ;
and each group was placed on a commercially available pre-starter ration. The first group served as the control and received no supplementary metals. The remaining four groups were treated as follows:
Group 2: Given a (~) capsule (see Table II) daily -by mouth .
Group 3: Glven a (O.IX) capsule (see Table II) daily by mouth.
Group 4: Glven a (O.OI~) capsule (see Table Il)daily by mouth.
Group 5: Given 0. OIX level of metal proteinate blend -ln the pre=starter ration which was prepared by mixlng 10 grams of (IX) metal protelnate blend wlth 990 grams of pre-starter ration.
~i The me~als dosage per capsule ls al(~o glven in Table II.
Dilutlon of the starting amount (~) was made in factors of ten (O.IX) and (0. OIX) with lactose dlluent.
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TABLE II
METAL~)SAGE PER CAPSULE
'I (1~) (O . IX)(O . OIX~ ,.
Metal mq/caPsule m~/caPsulem~/caPsule Co 0. 0296 0. 002960. 00029~i , '; ' .
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Cu 0. 0459 0. 00459 0. 000459 Mn 0.115 v~ u1150. 0115 0. 00115 Fe 0. 230 0. 0230 0. 00230 Zn 0. 492 0. 0492 0. 00492 Ca g . 592 0 . 459 Or 0459 Mg 4. 592 0. 459 0. 0459 The average daily weights for each group was then determined and is repprted in Table III. -. .
TABLE III
AVERAGE DAILY WEIGHTS OF TURKEY
POULTS
(All VV elghts In Grams) 1st 2nd 3rd 4th 5th ~y ~y Day DaY Day Group 1 -(Control) 48. 7 51. 4 60. 0 64. 3 74. 6 Group 2 (I~) Capsule 50.7 50.5 65.8 73.4 80.9 Group 3 (0.D{) Capsule 50.6 53.4 64.2 65.3 77.9 Group 4 (O. OlX) Capsule 50.2 50.4 65. 6 75.5 85.0 GrouP 5 (O. OIX) in Feed 51. 8 53. 0 66. 6 68. 6 84. 6 ~`

Of partlcular lnterest in the results set forth above is that, in general, the treated turkey poults posted significant welght increases over the control group for each dilution of the metal proteinate, whether . .
the metal protelnate was forcefed or amended to the diet.
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' ' ,. . ' : ::

As a further note of signific~nt interest in the foregoing evaluation using turkey poults, deaths of some of the birds was experienced. -~
Death losses which were diagnosed as due to para-colon appeared in the control group and at the lower dosage levels of metal proteinate (Groups 1, 3, 4 and 5). The excellent condition of poults in Group 2 ~ ~
suggests a possible beneficial effect of higher levels of the metal ` ;
proteinate in increasing resistance to para-colon.
Additional benefits to be derived from the discovery that metal deficiencies can first be ~iagnosed and then corrected through this lnventlon are set forth below in the followlng examples.

EXAMPL~ X
Ten thousand laylng hens were chosen and separated into two groups of five thousand each. The same commercial layer ration was fed both groups. Feathers from representative samples of both groups were assayed to determine the metals profile of the hens. The metals prof~Le thus obtained was compared to a standard profile obtained on the basls of data complled from assay of feathers from young, healthy, ~ ;
laylng hens. Deflciencles were observed and on the basls of this comparison, the commerclal feed canpositlon was amended with metal proteinates blended according to Table IV. Controls were fed corresponding -~
amounts of inorganic metals. ~ -TABLE IV
METAL COMPOSITION OR FEED BLEND
Calcium 4. 5%
: ... ...
Magneslum 4 . 6%
'~ ''""' ' ','' , .. .

. ., ,:

.. .,, ,..... ... , ... ... . , .. , ~ . ..... ...... . ..

34~5 Zinc 4%
Iron . 2%
Manganese . 1%
Copper . 04%
Cobalt . oz%

The foregoing metal feed blend was thoroughly mixed with the commercial layer ration on the basis of two pounds of metal feed blend per ton of commercial layer ration.
Group 1 received the metals of Table IV as protelnates of each -me tal. Group 2 recelved the same amount and ratlo of metals blended per ton commercial feed ratlon as fed Group 1. However, the metals were ln the form oE lnorganlc metals. In a sixty-day period, the hens of Group 1 which were treated with metal proteinate of the mentioned formulation layed 18,210 more eggs than hens treated with the inorganlc metals. Moreover, a very Ea~orable effect on the quality of the eggs was found to result. Eggs from Group 1 require an average of 1. 7 pounds more pressure to break the egg shall than eggs from Group 2. Also, the llnlng of the eggs of Group 1 showed greater tensile strength. An analysls of egg yolk ~etermined that there was 11.14%
more zinc, 10.50% more iron and 6.0% more copper in eggs laid by Group 1. ~ - - The increase in the number of eggs produced by Group 1 . ~ ., ~ .
resulted not only from a higher lay rate but also from increased capacity to lay over!allonger time span. Table V sets iorth~3 the results , , measured as the percentage of hens laying an average of one egg per day (lay rate) at the peak lay period for the group and six months after ;

.-' ''','`,'.'. ';' .

88gLZ5 ,`~

the peak. Hens in both groups started laying at 23 weeks of age.
' , TABLE V
Group 1 Group 2 (Trea ted) (Control) Peak Lay Rate 85% 75%
. - . . ' .
6 Months After Peak 80% 64%

The hens were forced to molt after the sixty-day period. Just : ~.. .... ..
prlor to molt, assays of the feathers showed that Group 1 averaged 10 - 17% hlgher metal levbls than the control Group 2.

EXAMPLE XI
Twenty-i~lve hLIndred laylng hens, whlch had been ln peak lay three month~, were selected and twenty-five randomly-selected chlcks reproduced by these laying hens were chosen as controls.
.
The average hemoglobin level in gm% of the chicks was 8. 7gm%.
The hens were then placed on a diet supplemented with the metal protelnate blend of Example DC. Forty-three days later eggs from the treated hens were set and tw~nty-five randomly-selected (treated) chlcks from those eggs were assayed for Jhemoglobln. The average hemoglobin of the second group of chicks was 9. 4 gms% as compared `
, 20 to 8. 7 gms% of the control chicks. Significantly, the death loss in the first seven days of life between the control chicks and the treated chicks decreased from 2. 0% to ~. 8%. ;
~ccordingly, it was found that supplementing the diet of the laying hens with metal proteinates resulted in improved hemoglobin lev~s in their offspring and decreased death loss of chicl~s. :
.: : .. .
:; .,:
..., ~,...

-24~
., ~ .
, . . . . , . , : : . . . .

EXAMPLE XII
Five hundred laying hens diagnosed as having avian leucosis were given metal proteinates along with normal feed ingredients. The formulation of the metal proteinates was the same as set forth in Table IV with calcium, magnesium and zinc the pred~minant ingredients.
The yellow appearance of the combs and the wattles which normally attend this disease disappeared within thirty days and the com~ and wattle returned to the normal red color. Also, the ~irds took on a healthy appearance and the death loss became negliglble. Egg production 10 and the quality of the egg shell returned to the normal range within thls thlrty-day period whlle egg breakaye in the nests was decreased 97%.

EXAMPLE XIII ~ -~
Five races of fingerling cut throat trout, each race having approximately fifty thousand fish, were each given a feed ration with an addltion of one-half per cent per ton of a metal proteinate formulatloil (see Table IV). The flve races of f~ish were compared with a race of control fish glven the same feed ration without the metal protelnate additlon. Samples of the fish in each race were 20 weighed every two weeks for approximately one year. It was discovered that the feed conversion, i. e., the amount of food required to produce one pound of me~t, was h~erhigher in the fish fed with rations having the metal proteinate addition. The treated fish consumed an average of 1. 2 pounds of feed per pound of weight gained while the control trout consumes an average of 4. 2 pounds of feed per pound of weight gained.

': '; - ' .' :. ':

8~Z5 EXAMPLE XIV
Anemia in baby pigs due to iron deficiency has historically been a problem when sows and their offspring are confined without access to the soil or pasture. The baby pigs are particularly susceptible to this type of anemla because of their high rate of growth, low body reserves of iron at birth, and low iron levels in sow's milk. A normal growth rate for baby plgs means an increase in body weight of four to five t~mes their birth weight at the end of only three weeks. A growth rate of this magnitude requires the retention of about 7 mgs. of iron 10 per day. However, sow's mllk supplies only about 1 mg. per day ancl baby pigs consume little feed other than sow mllk for the first three or four weeks. Accordingly, the need for an iron dietary supplement is readily apparent.
..... . . .
Inorganic lron supplements fed to the sow both before and after . - . . .
farrowlng has proven ineffectlve in either raising the iron content of the sow's milk significantly or lncreasing the iron levels of the baby pigs at blrth. Treating the baby pigs is routinely accomp~lshed either by (a) :
oral administration of 400 to 500 mgs. inorganic iron as tablets o~ ;
solutio~s within four days of birth and again at two weeks, or (b) at least two injectionss of 100 to 200 mgs. iron-complex solution during the early growth period. The foregoing has proven inadequate since ~ ;
: . :.: .
eve~ frequent treatment by these steps is insufficient to allow the piglets to achieve maximum growth. This shortcoming is further compounded by the handling requirement ~of large numbers of animals which renders the procedure impractical. Accordingly, it is most ~;
desirabl~ to assay tissue (e.g. blood) from the animal and amend the ;

~ . . .. ~ , . . . .

~ 384;~5 diet of the sow with appropriate metals and in sufficient amounts to enable the sow ~o pass the metals to the lpiglets through the sow's milk .
After suitable analysis, the following formulation of Table VI
was prepared as a dietary supplement for sows. The metals were formulated as proteinates.

TABLE VI
ASSAY OF METAL PROTEINATE FOR SWINE
Metal Percent Composition Mg 6. 80%
Fe 1. 86 Zn 1. 2 6 Cu o. 05 Co 0. 0024 .', The foregoing formulation of Table VI was fed to one group pf sows at a rate of five pounds per ton of feed and the same amount of metals as lnorganlc metals was fed to a second group of sows. This second group of sows served as a control. A random sample of both the treated and control feed rations analyzed for metal content showed 14 mg. iron/100 grams.
Blood hemoglobln levels were determined initially (thirty days --prlor to antlcipated farrowing) and continued until the piglets were weaned at about sixty days of age. Piglet weights and blood hemoglobin levels were determined at birth and upon weaning. The results of hemoglobin determinations and piglet weights are set forth in Tables .... .. , ~ . ., , . .. ., . . ~ - . . . - . .: . .

- - ~03~ 2S
.: .

VII and VIII below:
~, .:. ' TAB~E VII
HEMOGLOBIN (Hb) LEVELS (gm/100~
GrouD Control Treated Sows Hb Ran~e Average Hb Ranae Avera~e Initially 12-15 13. 5 12-~ 13. 5 ~ -At farrowing 12-14 13. 3 13-15 14.3 Litters ;
At brith 7.5-9.8 9.0 9.6-12.0 11.0 At weaning 10. 7-12. 0 11. 7* 11. 9-12. 812. 2 *some piglets requlred iron injections TABLE VIII
LITTER WEIGHTS
~verage in Pounds) Control Treated ;
Birth 3 . G 0 lbs . 3 . 5 3 lbs .
Weaning 37.25 lbs. 39.25 lbs.
Gain 33. 00 lbs. 35. 65 lbs.

It should be particularly emphasi~ed that in the foregoing example some of the control piglets required iron-complex injections to prevent loss of the piglets and, therefore, the hemoglobin level for the control piglets may be artifically high. Even with injections given to the control piglets, the piglets which nursed on treated sows had higher hemoglobin levels and heavier weights at weaning.

~i31342S

EXAMPIE XV
A group of 58 sows with similar breading history was randomly divided into three groups. Group I sows received normal gestation and lactation diets and no form of iron supplementat:ion was given to their offspring. Group II sows also received normal gestation and lactation diets, but their piglets were given 100 mg. of iron dextran by intramuscular injection at one and fifteen days of age. Grot~
sows received normal gestation and lactation diets to which 500 PPM ;
of iron as an iron proteinate containing about 10% by weight iron was 10 added. No other form of iron supplement was given to the piglets farrowed by Group III sows. The results were as follows in Table IX.

TABLE IX

Group I Group II Group III I -tNo Iron) tIron Dextran) tIron Proteinate) No. Sows 13 27 18 ;
No. Plglets Farrowed 144 285 168 Ave. Hbofof Sows at Farrowing (gm/100 mls) 12.03 13.5 14. 0 Ave. Hb or plglets at Farrowing (gm/100 mls) 11.41 11.12 11.35 Ave. Piglet birth weight tlbs . ) 2. 86 2 . 82 2 . 99 No. of Piglets Weaned 11~ 233 157 Ave. Hb at Weaning ' tgm/100 mls) 7. 32 11. 69 10. 83 ~ -Ave. ~IVeaning Welght tlbs.) t37 days) 14.96 16.53 17.43 Percent Mortality 20.1 18.3 6.5 Total Weaning wt. /
Piglet Farrowed tlbs. ) 11. 95 13. 51 16. 28 I ,~, '' ',.:

-29- ~

~38~25 .
The above table shows that piglets farrowed by Group I sows showed lower hemoglobin (Hb) and weight at weaning and higher mortality than Group II and III. A careful observation of Groups II
and III show impressive benefits of iron proteinate supplementation - -to the sow. Both Groups II and III show hemoglobin levels well above .
the anemia level of 9. 0 at birth and at weaning. The average birth weight of Group III piglets was 0.17 lbs. heavier than Group II piglets.
Replicated studies consistently show a difference between 0.15 and 0. 25 lbs. heavier blrth rate when supplementing the sows diet with lron proteinates . Group III plglets also averaged 0. 9 lbs. or 5. 2%
greater weanlng weight than Group II piglets. The difference ln mortality was the most signiflcant bene1t derived from iron proteinate administration. In thls study 2.82 times as many pigs on the average died in the group (Group II) receiving iron dextran injections than in Group III piglets receiving their iron supplementation through the sows diet. The final entry shownn in Table IX is a figure which combines the effects of mortality and weaning weights. This entry shows Group III
having a 17% improvement over Group II or 2. 7~ lbs. gr~eater weaning weight per piglet farrowed.
-, 20 From the above it is clear that the iron proteinate provided higher birthweights, lower mortality and higher weaning weights than piglets that received iron dextran by injection.
From the foregoing, it is clear that surprisingly beneficial ;~ effects result from making essential metals available to animals in `, a blologically acceptable form. With these essential metals readily available to the animals, the animal is no longer required to synthesize '',:~

1~389~Z~ii its own metalsproteinate from inorganic metals and thus individual ~ ;
differences in biological capability of animals are no longer responsible for an inadequate nutritional level of metaIs.
Furthermore, a determination of the metal level in the tissue of a healthy animal to establish a standard and comparison of the metal level of a selected animalswith that standard enables one to -suitably prescribe a corrective dietary supplement. ~

:: .
EXAMPLE XVI
It has been taught that metal proteinates are formed in basic solutlons at a pH above about 7. 5 and preferably between 8 and 10.
The followlng composltlons were made to demonstrate the vlablllty of these teachlngs.
T Ten mlcroliters of 0.073M ZnC12 containing 912 microcuries of radioactlve Zn65 were mixed as follows:
A. Ten microliters of 0. 076M L-leuclne (equamolar ;
ratios of amino acid and zinc) was combined w;th with the zlnc at a pH of about 3. 5 and adjusted to pH 7 wlth 20 mlcrollters of sodlum hydroxlde B. Twenty mlcroliters of 0. 076M L-leuclne (2:1 ratio of amino acid to metal) was combined with the zinc solutionaat a pH of about 3. Twenty microliters -of wa ter wa s added .
C. Twenty microliters of 0. 076M L-leucine and twenty microliters of 0.13M Na2CO3 were combined with ;
the æinc solution to form a metal proteinate at a pH
of 9. ~ ~

" , . .. .

;~,,:.

1~842S ::

Each of the above formulations represent a unit dosage.
Six white male rats weighing 160g. ~ 8 g. were matched to provide equal weights for each set of two rats. The three solutions of each formulation were carefully mixed to prepare a dose. Each rat was -mildly sedated with ether and was dosed orally using an Eppendorf ;
pipet. Each rat swallowed the mixture readily except one being dosed with mixture C, who swallowed most of the mixture after considerable ;
coaxing. The rats had been fasted for 16 hours prior to dosing and were returned to normal feed and water immediately after dosing.
.' After 46 hours the rats were sacrificed and dissected to remove blood, llver, left kidney, heart, skeletal muscle, and braln from each anlmal. One anlmal glven mlxture B had a left kidney only 1/20 normal slze. No Zn65 was detected in this rat's muscle or brain and therefore the results from that rat were not utllized. The samples of tissue were dried, weighed and then counted using a ni~clear Chicago Model 8731 rate meter and a Nuclear Chicago Model 8770 digital scaler.
The values given in Table IX are the corrected counts per minute per mg. and are the average of two anlmals except for Mlxture B.

TABLE X
2 0 A _ C --~ Blood 0. 90 1. 31 1. 64 `
Liver 5.15 6.20 8.65 Kidney 5 . 45 6. 46 8. 55 Heart 6. 42 5 . 23 6. 32 l!~uscle 2. 41 3.10 3 D 88 Brain 1. Z2 3. 86 _ 2 41 Total cc/min/mg. 21. 55 26.16 31. 45 .~
7 ~ ' ' ' . ' ~. . ' ', ', . '' ' ' . ' . .. ' ,' . . ' ' I ' ~ 8~4;~5 ~ ~

By dividing C by A and B it can be seen that the zinc proteinate ~ -~C) provided 1. 45 times greater metal absorption than mixture A and 1.20 times greater absorption than mixture B.

EXAMPLE XVII
The procedure of Example XV was followed to using radioactive iron (Fe59). Each unit dosage was as follows-.. ...
A. Ten microliters of 0.050M FeSO4 7H2O
containing the microcuries of radioactive Fe59. Ten microliters of 0. 05DM L-glumatic acid Forty microliters of NaOH to brlng the solution to a pH of 6.
B. Ten mlcrollters of 0. 050 M FeSO4 7H2O contalning ten mlcrocurles of radioactive Fe59 twenty mlcroliters of 0. 076M L-leucine forty mlcroliters of water to obtain a pH of 3.
C. Ten mlcroliters of 0. 050M FeSO4 7H2O containing ten microcuries of radioactive Fe59 twenty microllters of 0. 076M L-leuclne forty microllters of 0.13M Na2CO3 to Eorm an iron ;
proteinate at a pH of 9.

Six white male rats weighing 133 g. ~ 5g. were matched and .. . . .
dosed as in Example XV and the tissues were removed, dried and .",~., .,, ~..
counted with the results being reported in Table XI .

TABLE XI

A B _C
Blood 36.1 34. 0 83. 0 .... ..

. . .. , , . ,.. : . - .. .. ::

:

~ 389~
Liver 11. 7 16.1 38. 4 Kidney 4. 5 5. 5 8. 5 ~eart 2. 6 4. 7 9. 7 Muscle . 3 1. 7 2. 7 Brain 0 2.1 1. 5 Total cc/min/mg. 55.1 64.1 143.4 Again by dividing C by A and B it can be seen that the iron proteinate (C) provided 2. 60 ~imes greater absorption o iron than mixture A and 2. 24 times greater iron absorption than mixture B.
In both Examples XV and XVI mixtures A and B were not metal protelnates although in mLxture B, ln each case, the amino acid to metal ratio was at least 2:1. -.: ' ' .
EXAMPLE XVIII
The following study was done to illustrate the way animals ~-absorb~buffered metal proteinates. White laboratory rats were used ;
as experimental anlmals, and each rat received the sawe amount oE
tagged zinc chlorlde by dosing wlth a pipette direcd~y into the rat's stomach. The molar ratio of zinc to methionine was one to two for Rat II and III, and the pH was adjusted according to the following tab~:

TABLE XIII

Ra t I Ra t II Ra t III
24 mlcoliters Zn65C12 24 microliters Zn65C12 24 mic~Eoliters Zn65C12 75 microliters H2O 25 microliters H O con- 25 microliters H O
taining NaHC03~Na2C03 containing NaOl~ to to pH 10 pH Y
.

: :
50 microliters methionine 50 microliters methionine ~ ---solution 2:1 molar ratio --solution 2:1 molar with Zn~ ratio with Zn~
The rats were placed in metabolic cages on a normal diet and were observed for one week during which time the feces were collected.
At the end of the week, the rats were sacrificed, and the total excreta measured by scintillation count for radioactivity as compared to a blank.
The following amounts of Zn65 were excreted by each of the rats as `
measured by the collected fec~s for the week:
% of Total Dose Excreted Rat I 52%
Rat II~ 12%
Ra t III 3 6%
, More than half o~ the Zn65 in the control animal was lost. The ~ ~;
Zn65 methionate ret~ntion in '~at II administered at pH 10 was signiflcantly ~;
better than the Zn65 methionate retsEItiOn in Rat III administered at pH 7.
' However, both showed marked improvement in Zn retention over Rat I.
'::
EXAMPLE XIX
Example XVII was essentially repeated using Fe59SOa~ as the control. The solution was orally administered by pipette into the stomach. Each rat received 36. 7 micrograms of Fe59 in 20 microliters of solution. Rat II was administered a methionine solution and Rat III
a glycine solution, both buffered to a pH of 10 in a molar ratio of one to two metal to amino acid. At the end of a week, the rats were sacrificed and parts of various organs analyzed for Fe59 by ~cintillation -count.

.. .. . . .... ...

~ ~8~

The following results were obtained:

TABLE XIII
CORRECTED COUNTS PER MINUTE PER GRAM
Tissue Rat I Rat II Rat III
FeSO ~ FeMet FeGlY
-Heart 63. 151. 83.
Liver 136. 243. 83.
Gastroc 2. 54. 83.
~.. . ...
Masseter 14. 138. 65.
Brain 31. ~0. 142.
Kidney 2. 327. 150. ;
Testes 20. 109. 75. ;
Serum 700. 1, 797. 840.
Cells 742. 2, 076. 773.
Blood 1,335. 4b215. 1,602.
Feces 302,400. 214,000. 205,800. ~-Urlne 490. 370. 690.
Rat I Rat II Rat III
Feces 45.8% Lost 32.4%~ost 31.2% Lost ;;

The results reported above are very dramatic. The amounts of Fe59 retained by Rats II and III administered the buffered Fe59 proteinate were significantly higher than in Rat I as demonstrated by the feces analysis. The amounts of metal retained in the tissues were also significantly higher in almost every lnstance. However, detailed results were not computable because the complete organ .,.~,~
.. -' . ~

.

was not removed for analysis.
Buffering systems which were used in the above examples include amino acid-NaOH solutions and a solution of 6. 5 grams of sodium bicarbonate (NaHCO3) and 13.2 grams of sodium carbonate (Na2CO3) ;~¦
per liter of solution which will produce a buffered solution of about pH
10 . ' ' :'', EXAMPLE }~X
To further substantiate the effects of buffering metal proteinates, - ~
the following tests were conducted on rats which had been fasted over ~ -night. Each rat was given the following dosage of radloactive calcLum by ln~ection into the duodenum~
Ra t,~
250 microliters of CaC12 solution (1 mg. Ca) in dlstilled H2O
40 microliters of distllled H2O (40 mcC Ca45)* as Ca45C12 *mcG--microcuries Rat II
250 microliters of CaC12 solution (1 mg. Ca) in distilled H2O
buffered to pH 7 with NaOH and the amino acids and containing in molar ratio wlth calcium 2 moles of aspartic acid, 2 moles of glyclne and 1 mole of methionine 40 microllters of distilled H2O (40 mcC Ca45) a~Ca45C12 Rat III -Same as Rat II except buffered to pH 10 with NaHCO3/Na2CO3.

~; The rats were fed a normal diet for one week, and the feces were collected. At the end of one week the rats were sacrificed and the total : .

- 3 7- ~ ~ ' ~8~34~25 feces and portions of the tissues were analyzed by scintillation count.

The results obtained are as follows~
: ,.' TABLE XIV

CORRECTED COUNTS PER MINUTE PER GRAM

Tissues Rat I Rat IIRat III
.... . ..
....
- Frontal Bone 3682 5878 5772 Massater 602 844 904 Gastroc (muscle) 614 620 1206 Heart 642 598 932 Liver 664 546 742 R. Cer~brum 698 726 804 Kldney 686 656 730 Lung 676 672 648 Serum) 8.4 39.631.0 100 microliters -Blood Cells) 18. 6 0 13.2 Total Blood 27. 0 39.644.2 . ~ . .
It is evident from the tlssue counts that much more of the calclu~ protelnate was assimllated into the tissues at the buffered 20~- pH of 10 than at pH 7. However, it is also evident that more of the ;
calcium proteinate was absorbed at the buffered pH 7 than was the caIcium salt control.
Insofar as the feces is concerne~1, it was found that about four times as mcch calcium was excreted in the simple organic salt c~ntrol (Rat I) than ln the buffered (pH 10) calcium proteinate. Moreo~er, the pH 10 proteinate was appro,~dmately twice as effective in retaining .~ ~ .

'laF~8425 ; ~. ,. ~. .
calcium than was the buffered (~H7) proteinate. It would thus appear ~ ;
that buffering undoubtedly assists in both promoting stability of the metal proteinate solution and in improving its assimilation into a host ~ ~ -: .. .
of various tissues.

EXAMPLE XXI : ~ .
,;,1 :
The above examples tend to show that a buffer system at about . . . .
pH 10 will improve certain metal assimilation into living tissues. This pH however is not optimum for all metals. Some metals actually are `
. ~ . .
better absorbed at a lower buffered range. The object is to find and maintain the optimum pH range for the metal to be administered. This ~
.... .......
may be empirically established for each metal protelnate.
Manganese, for example, ls absorbed better as a protelnate at : . .. .
a pEI of abou~ 7 or more baslc systems it tends to form Mn (OH) 2 Manganese, and calcium also do not function as well with :.:: , . .
carbonate buffered solutions in that they tend to form insoluble carbonates.
::: . - ,., .:, The absorptive capacities of manganese proteinates at a buffered ~ -pH of 7 are demonstrated below. Two solutions utilizing Mn54 were made up as follows:
Solutlon I
250 mlcroliters dlstllled H2O contalning 100 mg. of Mn as MnC12 50 microliters MnS4 solutlon (14.3 mcC) as Mn54C1 Solution II
250 microliters distilled H O containing 100 mg. of Mn as MnC12 - ~ -50 microliters Mn54 solution (I4.3 mcC) as Mn54C12 Based on a molar ration of total manganesç, the solution contained per mole of manganese, 2 moles each of the amino acids --methionine, glycine, aspartic acid and glutamic acid. The solution was buffered to a pH 7 with NaOH interacting with the , .
: . .
-39- ;
. . .' ' ' : ' .;'' '. '' ' ' ': " ': ' .' .' ' :' . ., " '-. : ". .' '" ,.' ', '' lq~B84ZS
amino acids.
The solutions prepared were injected into the duodenum of laboratory rats (iabeled Rat I and Rat II according to solution given) which were fed a normal diet for one week and then sacrificed. The tissues were then measured by scintillation count asan indication of manganese proteinate uptake. The results are as follows:

:
TABLE XV

CORRECTED COUNTS PER MINUTE PER GRAM

cc/min/qm drY wt I II

Heart 3 7 0 119 0 Kldney 470 i 600 Brain 620 1170 Ga s troc 8 0 0 6 60 ;; Masseter 270 310 Liver 760 1070 , Lung 720 330 Frontal Bone 350 780 Duodenum 170 480 As will be noted from the above table, almost all counts were . higher in Rat II administered the manganese proteinate than in control Rat I. Counts in urlne and feces from these animals were not obtained.

EXAMPLE XXII

The above examples illustrate the assimilation of buffered metal chelate complexes with isolated amino acids or limited combinations ; .
': ,.;

' a~o- , . ~.

:
~181~4;~S :~:
; ,. .: .; ` . ..
of acids. This example demonstrates that hydrolyzed protein (in the form of di- and tripeptides) may be used as effectively. This example further demonstrates the placental transfer of stabilized metal proteinates from the mother to the unborn fetus. Mink were chosen for these te~ts and iron was chosen for the metal proteinate. This was done because many authorities in mink production believe that mink have difficulty in placental transfer of iron from m~other to young.
Two pregnant mink individually housed were fasted for twenty to twenty-four hours and were then given 24.17 milligrams of iron j ` `
containing 5 mcC of Fe59 radioactive isotope. Mink No. 1 was given `
the iron in the form of Fe59S04 which had been chelated into hydrolyzed . .
proteln in the form of dl- and trlpeptldes and buffered with a NaHC03/
Na2C03 solutlon to a pH of 10. Mink No. 2 was glven the same amount of lron as Fe59S04. In each case, the lsotopes were mixed wlth 25 grams of food whlch was consumed by the mlnk by ingesltion. The iron was administered to each mink 15 days before whelping. Feces and urine from each mlnk were collected to determine the amount of Fe excreted. Measurements were recorded 4 days after doslng and at the tlme of sacrlflce. Flfteen days after doslng each mink was sacrlficed and the varlous biologlcal tlssues measured for radlo actlve iron by sclntlllatlon count. Measurements were also made of the hemoglobln and hematocrit of the mother and the kits. The data obtained are given in the two following tables.
'`' ', ,:
TABLE XVI `:
` ~
GENERAL DATA
Mink No. 1 Mink Mo. 2 Total % Fe Retained 70. 4 42. 7 . , " ` ' ; ~
. :,:

` ~18~42S

Total % Fe~+ Excreted in feces 24.4 29. 6 ; .
Total % Fe~+ Excreted in urine 5.17 27. 7 % Fe~+ Excreted in Feces 4 days after . ~ :
dosing 21. 5 23. 8 : -% Fe~+ Passed on to yo~ng (kits) 0. 03 (7. 3 micrograms) 0 Hemoglobin Mother-gm% 20. 5 20. 0 : .
Hematocrit % 45 44 Average Hemoglobin of yo~ing (kits) gm% 19. 5 19 Average Hematocrit of young (kits) 53 50 Whole Body Counts Without Organs -Mother ~corrected counts/minute) 112.4 68.1 Average Body Counts Per Kit . .
(Corrected Counts/mlnute) 42.3 TABLE XVII
CORRECTED COUNTS PER MINUTE PER GRAM
Tlssue Mink No. 1 Mink No. 2 Masseter 7. 81 12. 00 Pectoralis Major 1.22 5. 02 Spleen 15 . 3 10 . 60 Braln 9. 7 7. 57 Lung 6. 4 4. 05 . :. . -Heart 2 . 7 5 .12 Liver 4.98 4.73 ~ -Neck Fur and Skin 6.24 3. 82 . --.
Scalp 5. 74 6. 33 From the above data several conclusions can be drawn. It is ~ ~
.,, ,; ~' , . ''' --42-- :. : .
':j. ;-~ `
10i~84~5 ::
' ':'~'`
at once evident that the amount of Fe retained in Mink No. 1, d~osed : . .::- , with the buffered iron proteinate was 65% ~reater than the amount ret~ined in Mink No. 2 dosed with Fe2SO4. Stating it another way about 70% of the iron in the buffered iron proteinate was metabolized whereas only 42 . 7% was retained in the mink treated with Fe2SO4.
Comparing the amounts of iron excreted after 4 days with the flnal analysis, it is evident that in Mink No. 2, 33. 5% of the iron initial~y dosed was absorbed but not metabolized, and was eventually ~ .

eliminated between the fourth and fifteenth day after dosing. In Mink k No. 1 only about 8% of the absorbed iron proteinate was later eliminated and not metabollzed. The data show that a measurable amount of Fe as lron proteinate was carrled to the kits from Mlnk No. 1 by placental transfer (42. 3 cc/mln) whereas the FeS9 in the klts from Mink No. 2 was barely evident (1 cc/mln). The hemoglobin and hematocrit measurements were hlgher from the iron proteinate dosed mink than from the control. The iron proteinate is utilized in the blood, skin and ~ ;
organs more readily as shown by tissue counts. The spleen, which is 90~ blood, contains aboutr~0% more irDn from the buffered iron protelnate than from the Fe2SO4 control. This is important as it demonstrates that -:
20 the lron protelnate is better for bullding hemoglobin than the aorrespondlng ..
Fe2SO . Flnally, the data show that hydrolyzed protein in the form of tripeptides and dipeptides are effective as ligands for complexing with buffered metals to form metaI protelnates for transport of metal into the blood stream from the intestinal tract as are individual amino acids. -! The following examples lllustrate the lncorporation of metal proteinates in having at least two tripeptide, dipeptide or amirlo acid ;

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1(18~4~5 ligands into or onto foodstuffs. The metal proteinates may be utilized in a communited form.
When used in flours or bakery products the metal content of the product available as a proteinate may vary from about . 00001 to . - .
. 001% by weight.

EXAMPLE ~XIII
A flour containing 50 mg. of iron as iron proteinate per poudd of flour was placed in a Jacksonville Flour Stabllity Cabinet. One week in this cabinet is equal to one month under ambient conditions. At 10 the end of 18 weeks no rust spots developed and the metal proteinate and 1Our maintalned complete stabillty.

EXAMPLE X~I\I
The bio-availabi~ity and stability of iron chelated into tripeptides, dipeptides and amino acids was compared against iron sulfate which is considered to be the standard in the industry and was proven to be more readily available and more stable than lron sulfate in each experiment.
'.'.
EX~MPLE XXV
In order to prove the appllcability of lron protelnates two loaves 20 of bread were prepared and sliced. One l~af had 4 mg. of iron as a proteinate in each slice and the other had 8 mg. of iron as a proteinate 1n each slice. Each loaf was calculated to contain 1. 5 pounds and - -yield 24 slices representing 2 slices per serving. The RDA in iron ~
is 18 mgs. per day. -The bread was prepared as follows:

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-FORMULA

1 package of acti~7e dry yeast 1/2 cup of water 2 cups of scalded milk 2 tablespoons of sugar 2 teaspoons of salt 1 tablespoon of shortening 6 cups of sifted white flour The mixture was divided into equal parts and to one part was mixed 96 mgs. of iron as an iron proteinate. The other mixture contained 192 mgs. of iron as an iron proteinate.
The recipe was combined and processed in a normal manner and allowed to rise. The dough was baked at 400 F. for 35 minutes. After baking and cooking the bread wasaanalyzed for rust spots. Mone were found and the bread was then stored. After storage the bread was , : , , superlor to bread contalning no lron fortlfication at all.
. . .
Other metal chelates could also be used in the place of iron . . . .
such as magnesium, manganese, copper, zinc, cobalt, calcium or any other~ essential blvalent metal.
This fortification does not apply to bread alone. For example :
the metal chelates could be added to enriched f}our, enriched self rising flour, enriched brominated flour, prepared or ready to cook 20 breakfast cereals, poultry stuffing, rice, soya, cornmeal, corn grits, enriched bread, rolls, buns, cookies, cakes and pastries.
' The metal proteinates may be incorporated into cooking oils and be absorbed or absorbed from the cooking oil into or onto foods cook~alin said oils. Typical of such foods are potato chips, corn chips, french fries, donuts and other deep fried pastries and batter - :
covered meats such as corn dogs, deep fried prawns and chicken , - ~L 5--... ~ ~ : ; . . ,. - ,, ~.;

1~8l~5 The metal proteinates may be used in amounts generally ranging f~om . 2 to 2 . 0 grams of metal per gallon of cooking oil. At these concentrations the metal proteinates have been found to inhibit rancidity in the cooking oil. The rate of absorption or absorption of metal proteinates into or onto the foods is thought to be approximately proportionate to the amount proteinates in the amount of oil absorbed. Since the proteinates are substantially insoluble in oil the oil must constantly be stirred or agitated.
Both a vegetable and animal oils may be used. Typical of such olls are corn, palm, coconut, peanut, and safflower oils and rendered anlmal fats both hydrogenated and non-hydrogenated.
. :
The followlng examples are illustratlve.

EXAMPLE XXVI
.: :
Into a vat equlpped wlth a stirrer containlng a ml~ture of hydrogenated palm and cocr~nut oils maintained at 375F. is added a -mlxture of iron, zlnc, and copper proteinates containing about 1. 5 grams of zlnc, 1.8 grams of lron, and .2 grams of copper per gallon of oil. Potatoes sllced on a commercial potato chip sllcer are cooked in the hot oll untll crl~p and a llght golden brown whereupon the cobked 20 potato chips are removed from the oil and allowed to drain. Upon analysls the chips are found to contain 6. 0 mg. of zinc, 7.2 mg. of iron and . 8 mg. of copper in the form of proteinates per 2 ounce serving 1. , .
of potato chips. ;~ - -.

` EXAMPLE X~VII

Into a small container equlpped with a stirrer containing hydrogenated ,~ .
,. ... .

. . .

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corn oil is added a mixture of iron magnesium and zinc proteinates in an amount sufficient to provide 1. 5 grams of iron, . 5 grams of magnesium and 1. 2 grams of zinc per gallon of oil. The oil is maintained at a temperature of about 400F. and is constantly agitated. A yeast leavened donut dough is cooked in the hot oil until done and the donuts are allowed to drain. Upon analysis it is shown that the donuts contained about 9 . 7 mg. of iron, 3. 2 mg. of magnesium and 7. 8 mg. of zlnc for .
4 ouncerserving.

: :. '.''' EXAMPLE ~VIII ~ -Into a vat that constantly strains and recirculates the oil is added a mi~ture of lron, manganese, copper and calcium protelnates ln an amount ~ufficlent to provlde about . S grams of each metal in the form of a protelnate ln the oil. The oil is maintained at a temperature `
of about 325-350~. Chlcken legs are di~pped in a batter and placed in the oil until cooked and are then removed and allowed to drain.
Upon analysis it is shown that the proteinates are absorbed onto the ;
surface of the batter 1~ an amount of which is approximately proportionate . : .
to the concentratlon of the proteinates in the cooking oil.
The meat and meat flavored products which can be fortified 20 by the metal proteinates are too numerous to enumerate. Virtually any recipe may be used as may any type or kind of meat, both domestic and wild. Typical of such products are swiss steaks, stews, processed --.
meats such as bologna, salami and canned or potted meats, steaks, ;~
chops, roasts, hamburger, sau~age, textured vegetable protein and like products. Such products may contain about . 00001 to . 01% by weight metal as a metal proteinate.
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'~' ~ .' ,' iO~38425 The metal proteinates may enhance the flavor of the product but are not to be considered as substitutes for seasonings, spices or other flavor enhancing materials.
The following examples are illustrative.
.::
EXAMPLES XX~

A ten ounce beef steak was prepared for broiling by seasoning . .,.,~
with two grams of salt and adding to the steak a mixture of comminuted metal proteinates wherein the metal content in the proteinates was 7. 5 mg. zinc, 9 mg. iron and 1 mg. copper. The steak was seasoned and then brolled until cooked. The steak contained flve percent of the RDA of each mineral added as a metal protelnate.

E~C~MPLE X~
A stew was made by mixing together 2 lbs. of stew meat; 1 cup water; 2 teaspoonsful meat drippings; 4 chopped green peppers;
6 carrots thickly sllced; dash of thyme, rosemary and basil; 1 head cauliflower; 2 onions; 2 bay leaves; 1 can tomato sauce; and 5~1 mgs.
zinc ~5 mgs. iron and 6 mgs. copper in the form of zinc, iron and copper protelnates. The stew was allowed to simmer until cooked.
The stew served six people each servlng containlng five percent of the ~DA of each metal added as a proteinate.
:
EXAMPLE XXXI -A "Chili Pepper Surprise" was prepared from wild game by combining 2 lbs. venison, .5 lbs. processed cheese, 2 medium chopped onicns, pepper to taste, 1 can green chili peppers, .5 can condensed milk, .25 teaspoon garlic powder, .5 teaspoon ceIery ', ., ' ~,' .'. ' :'.
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-48- ~ ~

~884~5 salt and 45 mgs. iron as an iron amino acid chelate. The mixture was cooked and divided into six servings. Each serving containe~
five percent of the RDA of iron as an iron proteinate. If desired 2 -lbs. of a meat flavored soyaaflour extender could be used instead - -of venison.
While many more examples or recipes could be added it is believed that the above are sufficient to properly describe this portion of the invention. However, many more applications will be obvious . ,, :.
to one skilled in the art. t Another convenient way of adding essential bivalent metals to foods is by combining metal proteinates with seasonings and splces. ~ `
~lmost any seasonlng or spice may be used. However alkali metal `
salts ~uch as sodium ahloride (bakers salt), potassium chlorlde and monosodium glutamate are preferred. Bakers salt may be flavored with ;~
onlon, garllc and other conventlonal f~avorings. The metal proteinate may contain a metal content for each metal present as a proteinate ;
ranging from .001 to 0.1% by weight of the seasoning or spice.
As lt will be seen from the following examples bivalent metals chelated wlth trlp~ptldes, dlpeptides, and amlno aclds are stable with nutrlents.
Potato chlps, corn chips, nuts, spice cookles, or cakes or virtually any other foodstuff upon which a seasoning or spice is placed may be used.
In certain cases the metal proteinates delays spoilage. That ls, convey longer shelf life to a product than corresponding products not containing the metal proteinates.
The following examples are included for illustration.

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E~AMPLE ~XII
Potato chips are made by peeling and slicing potatoes into the desired sizes. They are then deep fried in an oil such as hydrogenated palm oil. After they are removed from the cooking oils, they are flavored to taste with a fortified bakers ~lt. The following experiment was carried out in a commercial potato chip plant utili~ing commercial seasoning equipment. Since a serving of potato chips approximates ~ ~-two ounces (57 grams) that was used as a standard. Onto each two -ounces of hot cooked potato chips was added a mixture consisting of ;~
1.14 grams of NaCl being fortlfied wlth amino acid chelates sufficient to provide 7. 5 mg. zlnc, 9. 0 mg. iron and 1. 0 mg. copper. Each metal therefore constitutes approximately 5% of the RDA (recommended dally allowance) per servlng.
Paltability or taste studies were conducted with fifteen volunteers on a double blind basis so that those administering the tests and those tasting the potato chips did not know which were the sa~t fortifled potato chips and which were those having plain sodium chloride. After a series of tests 97% of the testing clata resultedt in an indication that the fortifled potato chips had a superior taste quality over the controlled chips. Only 3% of the tests detected no difference.
In addition to taste superiority, storage and rancidity studies on ; the treated chips as compared to the controlled chips demonstrated that the fortified or treated chips had two weeks longer shelf life than the cont~3lled chips.

EXAMPLE ~XIII
Compatability studies for a period of twelve months with bakers .. . . ~
' .

, - 50-~)88~¢Z~
, .
salt fortified with an iron amino acid chelate showed no oxidation or reduction of the chelates. Moreover, the chelates are not hydroscopic or deliquescent . -The above studies could be equally applied to other seasonings such as bacon flavored, barbecued flavored and onion flavored salts applied to potato chips or other materials.
A category of food products deficient in essential metals are those consisting primarily of sugar. As it will be seen from the following ;
examples bivalent metals chelated with tripeptides, dipeptides, and amlno acids are stable wlth candles, jams, jellles, syrups, marmalades, topplngs, or vlrtually any other sugar product.
f In certaln cases the metal protelnates may delay spollage, That is, convey longer shelf llf~ to a product than corresponding products not containing the metal protein~es. The amount of each metal in the product in the form of a metal proteinate may vary from about . 00l to l. 0% by weight of the product.
The following examples are included for lllustratlon.

EXAMPLE ~WCIV
A caramel candy was made by buttering the sldes of a large kettle and mixing together 2 cups of sucrose and one pint of cream. ~;
The mixture was brought to a boil for (3) three minutes in the kettle.
One more pint of cream was slowly added to the hot mixture until the :! mixture was homogeneous. There was then added 2 oz. coconut butter and . 5 teaspoons salt. One and one thirds cup of hot glucose was added with stirring and the mixture was cooked to a temperature .~ , . .

, -51-,,. . ~ ~ ..... . :, of about 250F. The hobmixture was removed and allowed to cool slightly whereupon 1 teaspoon of vanilla was added along with 9.1 grams of zinc proteinate containing 10% zinc and 37.1 grams of an iron proteinate containing 12% iron. The vanilla and metal proteinates were blended in to form a uniform mixture. The caramel thus formed was poured on a cool oiled platter and rolled into rolls about one inch in diameter and sliced into one-half inch slices. ;

EXAMPLE }WN ~ ~
. . .
A caramel syrup for use as a topping waspprepared by boiling 10 one quart of mapl~ syrup. One pint of cream was added to the hot syrup wlth constant stirring. One cup of bolling glucose was then added with mixlng. The syrup was cooked to 200F. and was allowed to partlally cool whereupon 1 teaspoon of vanllla was added along wlth 30. 3 grams of a copper protelnate containlng 15% copper. The syrup ~
was poured into containers ahd chilled. - ;

EXAMPLE XXXVI ~
:; ~
An apple jelly was prepared by covering sliced apples wlth peel lntact wlth water and cooklng until the apple slices were soft. The ;
cooked apple - water mlxture was pressed through a ~elly bag to produce 20 a clear apple juice. Four cups of ~uice and 3 cups of sucrose were heated and boiled rapidly to 220F. and then removed from the ;~
heat. A zinc proteinate containing 10% zinc was added to produce a ~`
jelly having . 005% zinc ~ontent was poured lnto clean sterilized containers and sealed wlth a paraffin wax .

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~8842S ~

EXAMPLE XXXVII ~ ;
Peach jam was prepared by cooking well ripened peach slices -until the peaches were easily crushed into a pulp. Equal volumes of peach-pulp and sucrose were mixed and cooked over low heat for 20 to 30 minutes to obtain the desired consistency. A mixture of zinc and manganese proteinates was added to give a peach jam having a zinc content of . 01% and a manganese content of . 005%. The hot jam was sealed with paraffin wax in sterilized glass jars.
While the above examples are many and diverse they are thought 10necessary to illustrate the invention in its various emboi~lments. All `;
are dlrected to methods and formulatlons designed t~ raise the level of essentlal blvalent metals in blologlcal tlssues in a safe and effective manner. The l~ventlon ls not to be llmlted to the examples, however, but is to be accorded the full scope of the appended clalms and all equlvalents thereof.
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Claims (57)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A composition for raising the level of essential bivalent metals in tissues of animals comprising a carrier containing an effective amount of metabolically assimilable metal proteinates, said proteinates being in the form of chelates of said metals with at least two protein hydrolysate ligands selected from the group consisting of tripeptides, dipeptides and naturally occurring amino acids, wherein the ligand to metal ratio is between 2 and 16.

2. A composition according to claim l wherein the metals in the metal proteinates are selected from the group consisting of calcium, magnesium, zinc, iron, manganese, copper, cobalt, molybdenum, chromium and vanadium.

3. A composition according to claim 1 wherein the composition is admixed with a carrier and formulated in the form of a tablet, capsule, syrup, powder granule or pellet.

4. A composition according to claim 3 wherein the composition additionally contains a buffer capable of maintaining the composition at a relatively constant pH in solution.

5. A composition according to claim 4 wherein the buffer is selected from the group consisting of phosphates, carbonates, bicarbonates, amino acid base combinations and mixtures thereof.

6. A composition according to claim 5 wherein the buffer is capable of maintaining the metal proteinates at a relatively constant pH between 7 and 11.

7. A composition according to claim 6 wherein the buffer is a mixture of sodium carbonate and sodium bicarbonate.

8. A composition according to claim 6 wherein the buffer is a mixture of one or more amino acids with sodium hydroxide.

9. A bakery product containing an effective amount of an essential metal as a metal proteinate, said proteinate being in the form of a chelate of said metal with at least two protein hydrolysate ligands selected from the group consisting of tripeptides, dipeptides and naturally occurring amino acids, wherein the ligand to metal ratio is between 2 and 16.

10. A bakery product according to Claim 9 wherein the metal is selected from the group consisting of iron, zinc, copper, magnesium, calcium, cobalt, manganese, molybdenum, chromium, vanadium and mixtures thereof.

11. A bakery product according to Claim 10 wherein each metal is present in an amount ranging from .00001 to .001% by weight.

12. A bakery product according to Claim 11 wherein the metal proteinate is contained within the flour.

13. A bakery product according to Claim 10 wherein the bakery product is bread.

14. A bakery product according to Claim 11 wherein the bakery product is a cereal that has to be cooked.

15. A bakery product according to Claim 11 wherein the bakery product is a prepared cereal.

16. A bakery product according to Claim 11 wherein the bakery product is selected from the group consisting of pastry, cookies, and cakes.

17. A cooking oil containing a stabilizing amount of at least one bivalent metal proteinate wherein said metal proteinate is a chelate of a metal with at least two ligands which ligands are protein hydrolysates consisting of tripeptides, dipeptides and naturally occurring amino acids wherein the ligand to metal ratio is between 2 and 16.

18. A cooking oil according to Claim 17 wherein the metal proteinates are selected from the group consisting of iron, copper, cobalt, manganese, magnesium, calcium, molybdenum, chromium and vanadium proteinates.

19. A cooking oil according to Claim 17 wherein the metal content of each metal in the metal proteinate may vary from about .2 to 2.0 grams per gallon of oil.

20. A method of inhibiting rancidity in a cooking oil which comprises adding an inhibiting amount of an essential bivalent metal in the form of a metal proteinate to the cooking oil wherein said metal proteinate is a chelate of a metal with at least two ligands which ligands are protein hydrolysates consisting of tripeptides, dipeptides, and naturally occurring amino acids, wherein the ligand to metal ratio is between 2 and 16.

21. A method of inhibiting rancidity of cooking oil according to Claim 20 wherein the metal proteinates are selected from a group consisting of iron, zinc, copper, cobalt, manganese, magnesium, calcium, molybdenum, chromium and vanadium proteinates.

22. A method of inhibiting rancidity an a cooking oil according to Claim 21 wherein the metal content of each metal and the metal proteinate may vary from about .2 to 2.0 grams per gallon of oil.

23. An oil cooked edible food containing an effective amount of an essential bivalent metal in the form of a metal proteinate wherein said metal proteinate is absorbed onto said food from the cooking oil wherein said metal proteinate is a chelate of a metal ion with at least two ligands which ligands are protein hydrolysates consisting of tripeptides, dipeptides, and naturally occurring amino acids, wherein the ligand to metal ratio is between 2 and 16.

24. An oil cooked edible food according to Claim 23 wherein the metal proteinates are selected from the group consisting of iron, zinc, copper, cobalt, manganese, magnesium, calcium, molybdenum chromium and vanadium proteinates.

25. An oil cooked edible food according to Claim 24 wherein the cooking oil contains about .2 to 2.0 grams of metal per gallon and the effective amount of metal chelate in the food will be approximately pro-q to the concentration of the metal proteinate in the oil absorbed by the food.

26. An edible food according to Claim 25 wherein the food is potato chips.

27. An edible food according to Claim 25 wherein the food is french fries.

28. An edible food according to Claim 25 the food is a pastry.

29. An edible food according to Claim 25 wherein the food is a batter covered meat product.

30. A method of inhibiting rancidity in oil cooked foods which com-prises cooking said foods in an oil containing an inhibiting amount of a metal proteinate wherein said metal proteinate is a chelate of an essential bivalent metal with at least tow ligands which ligands are protein hydrolysates consisting of tripeptides, dipeptides, and naturally occurring amino acids, wherein the ligand to metal ration is between 2 and 16.

31. A method of inhibiting rancidity in oil cooked foods according to Claim 30 wherein the metal proteinates are selected from the group consisting of iron, zinc, copper, cobalt, manganese, magnesium, calcium, molybdenum, chromium and vanadium proteinates.

32. A method of inhibiting rancidity in oil cooked foods according to Claim 31 wherein the foods are cooked in an oil having a metal content of from .2 to 2.0 grams per gallon of oil.

33. A processed or cooked product comprising a meat or meat flavored vegetable derivative containing an effective amount of an essential bivalent metal in the form of a metal proteinate or mixture of metal proteinates wherein said metal proteinates is a chelate of an essential bivalent metal containing at least two ligands which ligands are selected from the group consisting of tripeptides, dipeptides and naturally occurring amino acids, wherein the ligand to metal ratio is between 2 and 16.

34. A processed or cooked product according to Claim 33 wherein the metal in the metal proteinate is selected from the group consisting of zinc, iron, copper, manganese, magnesium, cobalt, calcium, molybdenum, chromium and vanadium.

35. A processed or cooked product according to Claim 34 wherein the metal content of each metal contained in said product. as a proteinate may vary from .00001 to .01 percent by weight of said product.

36. A processed or cooked product according to Claim 35 wherein said product contains meat without any meat flavored vegetable derivative.

37. A processed or cooked product according to Claim 35 wherein said product contains a mixture of meat and meat flavored vegetable derivatives.

38. A processed or cooked product according to Claim 35 wherein said product contains a meat flavored vegetable derivative without any meat.

39. Seasoning salts and spices which have been fortified with an effective amount of a bivalent metal proteinate wherein said metal proteinate is a chelate of a metal with at least two ligands which ligands are protein hydrolysates consisting of tripeptides, dipeptides and naturally occurring amino acids, wherein the ligand to metal ratio is between 2 and 16.

40. Seasoning and spices s according to Claim 39 wherein the metal proteinate is selected from the group consisting of iron, zinc, copper, cobalt, manganese, magnesium, calcium, molybdenum, chromium and vanadium proteinates.

41. Seasoning salts and spices according to Claim 40 wherein the metal content of each metal in the metal proteinate may vary from about .001 to 0.1% by weight of the seasoning or spice.

42. A season according to Claim 41 wherein said seasoning is a sodium or potassium salt.

43. A seasoning salt according to Claim 42 wherein the metal salt is selected from the group consisting of sodium chloride, potassium chloride, and monosodium glutamate.

44. A seasoning salt according to Claim 43 wherein the seasoning salt is sodium chloride.

45. A spice according to Claim 41.

46. A food product containing on the surface thereof a seasoning salt which has been fortified with an effective amount of at least one metal proteinate wherein said metal proteinate is a chelate of a metal with at least two ligands which ligands are protein hydrolysates consisting of tripeptides, dipeptides, and naturally occurring amino acids, wherein the ligand to metal ratio is between 2 and 16.

47. A food product according to Claim 46 wherein the metal proteinates are selected from the group consisting of iron, zinc, copper, cobalt, manganese,magnesium, calcium, molybdenum, chromium, and vanadium proteinates.

48. A food product according to Claim 47 wherein the seasoning salt is selected from the group consist of sodium chloride, potassium chloride and monosodium glutamate.

49. A food product according to Claim 48 wherein the seasoning salt is sodium chloride.

50. A food product according to Claim 49 wherein the food product is a potato chip.

51. A food product comprising a sugar as the principal ingredient and containing a biologically effective amount of at least one bivalent metal proteinate or mixture of proteinates wherein said metal proteinate is a chelate of a metal with at least two ligands which ligands are protein hydrolysates consisting of tripeptides, dipeptides and naturally occurring amino acids, wherein the ligand to metal ratio is between 2 and 16.

52. A food product according to Claim 51 Wherein the metal proteinates are selected from the group consisting of iron, zinc, copper, cobalt, manganese, magnesium, calcium, molybdenum, chromium and vanadium proteinates.

53. A food product according to Claim 52 wherein the metal content of the metal proteinate may vary from .001 to 1.0% by weight of the food product.

54. A food product according to Claim 52 wherein the food product is a candy.

55. A food product according to Claim 52 wherein the food product is a jelly.

56. A food product according to Claim 52 wherein the food product is a jam.

57. A food product according to Claim 52 wherein the food product is a syrup or topping.