Note: Descriptions are shown in the official language in which they were submitted.
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. ~
' ~ :' .. -':
-2- :
....... ... .. . . . ~ . . :
~Q884Z5 : `
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
-3-
.. . . .. - . . . . . .
- :t.Q8~ Z~ ` ~
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 , ~
,, ,,, ,: ' '.
i ',"'".' .,
l. .. , .. ; ,,. . ,.......:. . .. , .: . .. ~ . :. : .
~Q88~Z~i
' ` ':
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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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.
. :
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
.
`, ;'~ '
- 6- ~
;
1~8~4;~5
...., .. . ...
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.
..
--7--
~}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
"~. ~'. '.
'~ '' ` ~
- 8-
34Z5
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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-10- ;'','.','','"
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:
~H
7 9.1 g- KH2PO4 ~19. 7 g- Ma2HPO4 per liter.
~ 1 1-
... . . .
.. . . . .
~ 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 ~
. .
which have been considered essential to the proper functioning of the
"" ~
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-12-
: , . . , . . - . ~ . , . . , ,,, , . . . . : ~
34Z~ ~
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,
,
-13-
. : : ; , ,: .: .
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.
' .', ':
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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- 14-
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1~88~Z~ ~
.............................................................................. .... ... .... .... ... .:
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
- 1 5-
.
~Q~Z~ : ~
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
7 -1. 129
-16-
. . :: . . , . , :.
~8B4~S
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
--17--
-, . . , ~ , . . . .
~8fl~2~
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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ro ~ ~ ~ ~ ~ ~ a~ ~ v
V s: s~ ~--I ~ _ a) ra 5~ ~ Q .` i ~ - :
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j~ O .:~ a ~ o a~
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& ~ ~ c a)1
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V ~ ~ _, o - ~ ~ ' ,'
a~ "' .' ,:
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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.
: - -.
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
, '; ' .
. .: '.:
- 2 0~
' '. :, :'' ' .
.~8
' ' ": :
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.
,, .'.. , "'` "
.
- 2 1-
' ' ,. . '
: ::
Z5
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%
'~ ''""' ' ',''
, .. .
--2 2--
. ., ,:
.. .,, ,..... ... , ... ... . , .. , ~ . ..... ...... . ..
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 ;
.-' ''','`,'.'. ';' .
--2 3--
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.
': '; - '
.' :. ':
- 2 5-
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 ;
-26--
~ . . .. ~ , . . . .
~ 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
-27-
.... .. , ~ . ., , . .. ., . . ~ - . . . - . .: . .
- - ~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.
- 2 8-
~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
--30--
'',:~
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. ~ ~
" , . .. .
--31--
;~,,:.
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
-32-
.~
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
-33-
.... ..
. . .. , , . ,.. : . - .. .. ::
:
~ 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
.
--3 4--
: :
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.
- 3 5-
.. .. . . .... ...
~ ~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
.,.~,~
.. -'
. ~
--3 6-- - -
.
4Z5
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
.~ ~ .
-38-
'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
, .
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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
; .
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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
. , " ` '
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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 ~ ~
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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 ;
' J . i . . ' .
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1 ~43-
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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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~81~
-
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
,
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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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-46-
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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 .
.
- 52-
. ' ~.. - .
~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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