DE2755709A1 - PROCEDURE FOR INCREASING METAL CONTENT IN FABRIC - Google Patents

Description

Patent attorney DIPL-PHYS. DR. W. LANGHOFF «EcursAiswALr B. LANGHOFF *

B MUNICH 81 WISSMANNSTRASSE 14 TELEPHONE 83 27 74 TELEGRAM ADDRESS:

Munich, December 13, 1977 Our reference: 42 - 1695

Patent application for

Harvey Harold ASHMEAD, 719 East Center, Kaysville, Utah 84037

United States

Method of increasing the metal content in tissue

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rtreter npif.H § 46 Pat An wO. approved by the regional courts MOnct> tin I and M

The invention relates to a method for increasing the content of vital divalent metals in the tissue of warm-blooded animals, especially animals.

Certain metals are vital for the body. Calcium, for example, is present in the body in greater excess than other metals and is found mainly in bones and teeth, but also plays an important role in blood clotting. Some enzymes require magnesium. A magnesium deficiency leads to vasodilation and excessive irritability of the nervous system. magnesium is not only absorbed by the body, but also excreted to a large extent with the excrement. Most of the magnesium salts are hence laxatives.

Iron is important for the formation of hemoglobin and for the metabolic and respiratory functions of the body. A lack of iron absorption is the leading cause of anemia. Little iron is absorbed in inorganic form. Most of it is unused again excreted with the excrement.

Copper is an important metal for many enzymes and is interrelated with hemoglobin formation and enzyme functions. Inorganic copper is again excreted through the intestine.

Cobalt is part of vitamin B ₁₂ and has been used successfully to treat certain types of nutritional deficiencies.

Another vital element is manganese. Manganese deficiency is detrimental to growth and virility. It is further involved in the activation of various enzymes. Manganese is also excreted to a large extent with the excrement.

Zinc is important for the activity of the enzymes in cell division and forms part of insulin. The excretion Zinc takes place to a large extent through the digestive tract.

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Molybdenum is another element in certain enzymes. Other metals, such as chromium and vanadium, are also essential for life held.

All of the aforementioned elements can occur in a divalent form. With bivalent are meant metals that have an ionic, i.e. can assume an oxidation state of at least +2 or higher. For copper, this is the cupric state.

Since these metals are generally difficult to absorb as inorganic or organic salts, there is a desire to achieve a Find the composition of matter in which these metals are used by the body easily assimilated. Salts have an ionizing effect in gastric juices and reach the small intestine, where absorption takes place mainly takes place. The intestinal walls carry electrical charges, which have a strong tendency, the positive metal cations hold back while letting anions through. The vital metals are therefore excreted again through the intestine, by causing diarrhea. The anions that have passed through the intestinal walls are often excreted in the urine and often form diuretics.

Various attempts have been made to use organic salts as carriers for the metals, but with limited ones Success. Chelates have also been used with ligands formed from ethylenediaminetetraacetic acid (EDTA) and derivatives thereof. However, these chelates bind the metal so tightly that it is not immediately unlocked for the body can.

The invention is based on the object of a method for lifting the content of essential metals in tissues of warm-blooded animals as well as a composition of matter for Performing the procedure, which the body metals in higher

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Measures and in an easily assimilable form by reacting less to the electrical charges of the intestinal walls.

It is true that certain protein hydrolysates have already been used as chelating agents or ligands to prevent absorption of metals in living tissue to improve. However, it has been found that certain protein hydrolyzates are poor Ligands are great because of their size and stereochemical structure.

By the term protein hydrolyzate is meant any form of hydrolyzed Protein is meant, from long-chain polypeptides to basic protein building blocks, e.g. amino acids. These hydrolyzates are commonly formed by using acidic or basic hydrolysis or the combination in the. Since many different amino acids are vital for the body, any form of hydrolysis is disadvantageous. One acidic hydrolysis destroys the amino acids tryptophan, serine and threonine. On the other hand, the basic hydrolysis racemizes converts the amino acids into their D and L forms and destroys arginine, threonine, serine and cystine. Naturally occurring amino acids have the L-shape. Also, acidic and basic hydrolysis processes require neutralization, which in turn leads to formation leads to inorganic salts, which often remain in the hydrolysis product.

Chelates from essential divalent metals and protein hydrolyzates are referred to as metal proteinates.

The invention is based on the object of a method for increasing the content of vital divalent metals in the tissue of warm-blooded animals and a composition of matter for Perform the procedure to create. The composition of matter should be able to pass unhindered through the stomach of a warm-blooded animal and be absorbed through the intestinal walls.

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The solution to this problem is to be seen in the fact that one gives the warm-blooded animals an effective amount of a metabolically assimilable metal proteinate administered in the form of chelates of these metals with at least two protein hydrolyzate ligands from the group of tripeptides, dipeptides and naturally occurring amino acids. The molar ratio of ligands to metal should be between 2 and 16 and preferably between 2 and 8. Particularly suitable are molar ratios between 2 and 4. Each ligand should preferably have a molecular weight between 75 and 750 .

The ligands are preferably formed by enzymatic hydrolysis of protein sources such as collagen, fish meal, meat, soy extract, yeast, casein, albumin, gelatin and the like. Enzymatic hydrolysis can be used with enzymes such as trypsin, pepsin or any other protease, which is not harmful to the formation of L-amino acids. the Ligands produced by enzymatic hydrolysis do not contain any inorganic salts, as in the case of the basic or aeidic Hydrolysis. However, ligands obtained from aeidic and basic hydrolysis can be used with less success. Of the The term "naturally occurring amino acid" also includes synthetically produced amino acids having the same stereo structure as the natural amino acids. Proteins contain around 20 amino acids, such as glycine, alanine, valine, leucine, isoleucine, phenylalanine, Tyrosine, tryptophan, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, arginine, histidine, cystine, methionine, Proline and hydroxyprolyn. A dipeptide is a combination of two amino acids with a peptide bond (-CO-NH-), and a tripeptide is a combination of three amino acids with two peptide bonds. The ligands bound to a metal ion can be the same or different.

In the production of chelates, the smaller the chain length of the ligands, the easier the production, while the chelation becomes stronger. In the following, the terms chelate and proteinate are used interchangeably, mfo § '$ ³ f $ V ^fl t) tf $ * 3 ^{the case that on not}

protein derived ligands is referred to.

It is advantageous to hydrolyze a protein starting material so that the main component of the hydrolyzate is formed from amino acids, dipeptidones and tripeptides. The subsequent formation of the metal proteinates, that is, metal chelates, results in a substance addition that is easily transported through the tissue. Large blocks of protein, such as the metal salts of gelatinates, caseinates or albumins, must first be broken up or hydrolyzed before they can be transported. Not hydro! Synthesized protein salts are generally not absorbed by the intestinal walls. Therefore, according to the invention, the protein molecules are hydrolyzed down to tripeptides, dipeptides or amino acids before they are able to form a proteinate with the metal salt. In order to form a metal proteinate, the correct amounts of the constituents must be present under the correct conditions so that the mineral to be chelated must be soluble and the tripeptides, dipeptides or amino acids must be free of protons which interfere with the chelate bond so that there is no bond between The proteinate ligand and the relevant H (MiIJ can be produced. Since chelates, by definition, have structures in which a heterocytic ring is formed by the undivided electrons of neighboring atoms, it is uo .'- ent 1 i ch in that before the Compl exbildunq of a protein hydro lysate: - with a metal ion, the protons i in the chelating Stoi, ie the amino acid, dipeptides or tripeptides, removed WfJi (Ki a chelatbi1dendcs means del ini ti on sg oma β a. organic '. "<ι bond in which the atoms contain donor electrons, which my- .ils a looidinative bond with metal ions in solution c; η belongs: η. Es :.; - i da! tpr knowingly that ai ie chelation in I, "). si; 'ig or]' ·, l'if. If iias Cl ic1 atmi nera J na 1 ζ is completely soluble ■:] · "di '· Ami no. ^R ; ä u] -i η and peptides hi nrei cItcikI are soluble, Γ.Γίβ i" l ·; · "Ü-value can be set sufficiently basic to the 5rt'V 'nil ·."' I * Ά. a v: n di: n Amincjriij.ipeii as at ch from the carboxyl

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remove groups of ligands. A pH of 7.5 is sufficient for this, but a pH of 8 to 10 is preferable. This allows the heterocyclic rings to form bonds between the metal and the individual pairs of electrons that remained on the amine groups. Simply mixing the intact amino acids or intact peptides with water in the presence a metal salt does not yet produce a chelate or proteinate, since the protons on the carboxyl and amine groups form the chelate disturb.

If you have the protonized, i.e. intact peptides or amino acids combined with a soluble metal salt, either no reaction takes place or a salt is formed from the metal and the peptide or amino acid, which salt is soluble or precipitates. Those formed as described above Metal proteinates precipitate in basic solution and are insoluble or only partially soluble in aqueous solutions. In addition, metal proteinates or "chelates" are heterocyclic complexes and quite different from metal salts of peptides or amino acids. The metal proteinates are easier to assimilate, see above that the metal is more readily available to the body tissue.

In general, the stability of the metal proteinates increases as the oxidation state of the metal ion increases. Even shorter ones Amino acid groups create more stable proteinates. Amino acids are preferable to peptides.

The coordination number, which is completely different from the valence, indicates how many coordinative covalent bonds Metal can have. If, for example, one assumes iron, the coordination number can be between 2 and 8, although other values are also possible have been observed. The ferrous ion can have four water molecules in the hydrated state. Since most metals have coordination numbers between 2 and 8, their ions are covered by hydration surrounded by the water of hydration, each electronecjative

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Oxygen atom of each water molecule from a positively charged one Metal ion is attracted.

Every bond between the metal, in this case iron, and oxygen atoms comprises two electrons. The water molecules are coordinated in the metal, since each oxygen atom contributes both electrons, which are the coordinative covalent or simply covalent Establish a bond.

A complex bond is formed when a substance other than water forms a coordinative, covalent bond. A chelate can be a complex compound, but not all chelates are complex compounds. Oxygen and nitrogen are electronegative elements that contribute electron pairs for covalent bonds. Amino acid and peptide ligands both contain these electronegative elements. A ligand can refer to the part of a molecule that contains these negatively charged elements, and this is where complex formation takes place. The term "ligand" is also used to refer to the metal binding molecules themselves. As mentioned earlier, the term "chelate" or "proteinate" refers to a metal compound which contains at least two ligands. When a metal proteinate has two or more ligands, it is referred to as bi-, tri-, quadruple, five-fold or six-fold proteinate , and so on. A metal proteinate is a complex with two or more heterocyclic rings. Hence the term "CheLieriuuj" refers to a particular kind of metal bond, wherein the amino acid function of the HoleküLs at two or more locations is linked with the metal, so that a Heterocyc 1 ischer ring is formed. Obviously, a metal cannot form a coordinative complex with an intact amino acid or peptide. It is therefore important for the Meta LLche latbi Lduruj that interfering protons are removed from the carboxyl and amino groups before the chelation can take place.

A Mn t; .- i 1 1 i> r) te ina t bildtm, the kcv "rdiruit i van V <. ι

'U'küli. lur.-h in I.iq ir: <ifii t-i "-: ·· (*.

H I) ^f ) Fl 2 Γ 'Π R /. 1

IS

Chelates differ from salts and other complexes because of their closed ring structure in which the metals be held. In general, five-membered chelate rings are the most stable. It should be remembered that covalent bonds While they are most stable and should be preferred, some ionic bonds also occur in chelates can. The transition metals tend to be more stable, more covalent To enter into bonds, whereas alkaline earth metals tend to have some ionic bonds.

The stability constants can be determined by polarography, it is found that chelates are 10 to 10 times more stable than the corresponding salts or complexes.

When a metal protein is formed, the pH of the solution drops as the ligand is added. It is important that the protons on the ligand are removed so that the chelation is not disturbed by competition with the metal ions.

If one uses the simplest amino acid, glycine, and iron as the metal ion with four water hydration molecules, then this is

Reaction as follows:

C - Ο ^θ +! H O- Fe - Η ₉ θ | - ^ CH ₂

I I

H ₀ OI NH- H ₀ O CH,

This forms a complex in the aqueous reaction medium is soluble or not. When adding lye, e.g. NaOH, the result is:

C 1 Fe; CI ₂ C - NH ₂ H ₂ N - CH,

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This represents a real metal proteinate, with all protons of the ligand have been removed and so heterocyclic rings are formed. Each ring consists of five links, which are extremely stable. From the foregoing it can be seen that it is extremely important to remove the protons of the ligands and to carry out the chelation under basic reaction conditions.

Metal proteinates are relatively insoluble in basic solutions, however, depending on the concentration, they are partial or completely soluble in slightly acidic to alkaline solutions.

When the metal proteinates are administered orally to warm-blooded animals, some of them can be destroyed by gastric acid, so that the full dosage is not available. It has been shown, however, that a substantial part of the metal proteinates reaches the intestinal tract undamaged and is absorbed there will.

Metal proteinates can be stabilized and therefore pass through the stomach more easily when they are at a pH between 7 and 11 are buffered. The buffer system may differ depending on the metal proteinate administered, and it one or more metal proteinates can also be administered at the same time.

The choice of buffer system also depends on the desired pH. Amino acids alone react with bases, such as sodium hydroxide, and form buffer systems. Typical buffer solutions include phosphate, carbonate, and bicarbonate anions or combinations the same. Alkali or alkaline earth metals can be used as cations. Examples of typical buffer systems in the pH range from 7 to 11 are the following:

OBiGiNAL 1N6PECTEO

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7 9.1 g. KH ₂ PO ₄ + 19.7 g. Na ₂ HPO ₄ per liter

7 50 ml. M / 5 KH ₂ PO ₄ + 22.4 ml. M / 5 NaOH diluted to 200 ml

8 50 ml. M / 5 KH ₃ PO ₄ + 46.1 ml. M / 5 NaOH diluted to 200 ml

10 6.5 g. NaHCO ₃ + 13.2 Na ₃ CO ₃ per liter

11 11.4 g. Na ₃ HPO ₄ + 19.7 g. Na ₃ PO ₄ per liter.

The invention includes the use of any combinations organic or inorganic substituents that keep a system at a pH of 7 to 11. There are numerous other buffer systems which are known per se and therefore do not need to be listed. Important for the implementation of the invention is that the buffer system is selected so that it not only stabilizes a metal proteinate, but is also non-toxic and helps the body to assimilate the metal protein.

The term "warm-blooded" is intended to refer to both animals and humans.

The amount to be administered depends on the type of composition, the type of animal, weight, age, sex and metal proteinate or mixtures thereof away. It is desirable to first find out the metal deficiency in a body by examining hair, nails, blood, urine, skin or saliva figuring out and using the default values of normal to compare healthy animals. A special composition of matter can then be produced. On the other hand it is desirable to find out the RDA for each metal in one odor in multiple doses. For example is the RDA value for an adult human: iron 18 mg, zinc 15 mg, copper 2 mg, magnesium 100 mg and calcium 1000 mg. L's No RDA is set for other minerals yet

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are seen as vital to the proper functioning of a body.

The metal proteinates can be given in different ways, as long as only a biologically effective amount is administered. When administered orally, the metal proteinates can with a Suitable carriers are mixed and in the form of tablets, pills, capsules, emulsions, syrup or as an addition to feed or food. For use in domestic animals, metal proteinates are preferably added to the feed or sprayed or poured over it. The metal proteinate compositions can also be entered through a gastric catheter or valve.

For the administration of metal proteinates in humans, tablets, syrups, capsules or the like or admixtures come into question to food such as meat, cookware, bakery products, Sugar confectionery, frozen food, herbs or spices in question.

In order to demonstrate the stability of the metal proteinates, one can study their ability to determine the metal content in biological Tissues, and the following are various compositions of matter and methods and modes of administration given for example.

Π e i s ρ ι e L I

One, ¹ mix of 100k <; Casein and ^r ) 0 () k <j water were mixed in a jacketed tank and stirred. L! ;; became ! kg of sodium hydroxide added to the milk egg and casein solution. Then became one; Mixture of 1, ^r > kg each of the Kiu'.yine I'apoin, bacterial protease and P i Izprot ease added

η u ^c ) η 2 ^r ) / η ο 4; i

with a preservative such as sodium benzoate. This mixture was covered and stirred for six days at which time a hydrolyzate resulted with over 85% tripeptides, dipeptides and amino acids. The enzymatic reaction was terminated by boiling the solution for about 15 to 20 minutes and filtering the same in the hot state through a gauze fabric. The filtered solution was then ready to make the metal proteinate according to the following examples. Other protein sources such as gelatin, collagen, yeast, fish meal, soy or the like can also be used in place of casein. In most cases the metal content was the one formed Protein between 5 and 15% by weight and on average by 10%.

Example II

26.5 kg of zinc chloride were added to a filtrate according to Example I. Sodium hydroxide was added to this solution in an amount that raised the pH to about 8.5, thereby forming a precipitate originated with about 14% zinc as zinc proteinate. The ligand to metal ratio was two to one.

Example III

Example II was repeated using 37.5 kg of ferrous sulfate (FeSO ^. 7H ₂ O). The washed and dried precipitate gave 87.5 kg of iron proteinate with a content of 9% by weight of iron.

Example IV

The starting point was again Example II and 20.5 kg of cupric chloride (CuCl _2. 2H ₂ O) in conjunction with 500 kg of methyl alcohol.

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The precipitate contained 90 kq of copper proteinate with a content of copper of 11 wt.

Example V

The procedure according to Example TI was repeated using of 28 kg of manganese chloride (MnCl ,,. 4H ~ O). The precipitated Manqonprotoinat contained 8 wt. Manganese and weighed 85 kg.

Example VI

One by acid hydrolysis to tripeptides, dipeptides and amino acids Reduced soy protein was made by mixing 8 kg of water with 2.85 kg of hydrochloric acid. It was 5 kg

isolated soy was added and the whole thing was heated to 130 ° C. for four hours to hydrolyze the protein. The hydrolyzate was neutralized with 2 kg of zinc carbonate and the pH was adjusted to 8.5 by adding sodium hydroxide. The precipitated zinc proteinate was washed and dried and contained 15% by weight zinc.

In each of the examples given above, the molar ratio of liquid to metal was at least two to one. For hydrolysis Other acids and bases can also be used, such as phosphoric acid and sulfuric acid and sodium hydroxide.

Example VII

The stability of metal proteinates can be shown using zinc methionate as an example. Sol traces of Zn Cl ₂ πι it non-radioactive zinc chloride were mixed and methionine as an amino

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acid chelates. To demonstrate the structure and the solid Binding of the zinc to the methionine became a polarocfraphic Investigation made. A solution was prepared with a content of 0.0001 mol of zinc per 100 ml of ZnCl- and solution then added enough 0.2 M methionine to make a solution with an amino acid to zinc molar ratio as follows :

ml of 0.2 M molar ratio ^ methionine methionine zinc

1 0 ml 0

2 2.5 ml 0.5 1 5.0 ml 1.0

4 10.0 ml 2.0

5 20.0 ml 4.0

6 40.0 ml 8.0

7 80.0 ml 16.0

10 ml of 1 M sodium nitrate (KNO ₃ ) as an electrolyte and 10 ml of 0.1% gelatin solution were added to each of the above solutions. Each solution was adjusted to a pH of 7 by adding a few drops of concentrated (6N) sodium hydroxide solution.

Using the Metrohm E 261 polarograph with a silver / silver chloride (Ag / AgCl) reference electrode, the results were obtained the following values E. ":

Solution no. E., "

1 - 1.008

2 -1.055 i - 1.057

■ \ - 1.07 ')

5 -1.0 9 0

6 - t , 1 1 0

7-1, 129

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The protein concentration was plotted against the E .., ₂ ~ value, which resulted in a sloping line which is a measure of the number of ligands in the complex. It has been found that the Zn ion with ^r: forming a complex white Methioninmolekülen.

It can be assumed without going into any particular theory be that ions at higher ligand concentrations and at a higher, i.e. more basic pH, the complex is likely to be a bicyclic complex.

Knowing the number of ligands can be the stability constant at different concentrations and pH values to be determined. It has been found that the logarithm the stability constant has the following value:

log K = 2 E _{1 ΙΊ} - ρ log ligand , 059 ,, o59 ^U "2

where ρ is the number of ligands and ligand is the concentration des Licjanden means.

The solution no. 7 (16 molecules of methionine per zinc molecule) has a S t ab i L ί ta't skons tante of 4.04 χ 10 at a pH of 7. The same solution was adjusted to a pH of 9 and then gave a Stability constant of 4.4 1 χ 10. In other words, by changing the pH of a zinc methionate solution with the same concentration from 7 to 9, the stability around the e'ctor 10 can be increased. Similar results are obtained
when using copper, iron, chromium, calcium, manganese, magnesium, vanadium or other vital metals.

Ei (■ i ·; ρ ί e I V [IE

In dii'- ^; he ί; - - LIi = "* '. ^R ou ^l .' · · Ι \ ;; ΐ ·, - 'η · ί; ν; ιΐ ['. 1 · Μΐ -.tritMi Ί'α ι old Ki'iken in ( ! r 11 ρ ρ ι ■ η 11 i 11 · ■ ι ■ ί ■ i 1 t 111 ..! ^; ■ ■ d ■ ■; ', t "u ρ: ■ ι ■:, ι; · h 1 '. > r 11 r η 1 1 «■. 11 · <1 i ι * ■ harr!

H 0 '■) 8 2', / ί) B ', Ί

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with the same standard chick starter ration. A standard starter feed ration has the following composition, for example: Soy meal, meat meal, ground corn, ground miIo, salt, fat, dicalcium carbonate, lime and inorganic mineral Trace substances. This starting ration contained 22% protein and a fat content such that the total caloric value was 2760 cal / kg. The ration also contained vitamins. Each group of chicks designated as treated received a different predetermined amount of a metal proteinate according to the following tables in addition to the usual starting chick feed ration. Chicks who received this ration in addition to corresponding amounts of an inorganic metal, served as a control group.

The comparison results are given as the T / C ratio, which is the ratio of the concentration of the metal in the treated Chick compared to the concentration of the metal in the control chick is meant. A T / C ratio greater than that so as one indicates that there is a greater concentration of the metal in the tissue of the treated chicks than in that of the control chick. A T / C ratio less than one then means the opposite.

The purpose of the comparative experiments was to show that there is an increase in metal assimilation in chicks that were fed with metal proteins had been fed instead of inorganic metal compounds.

Table I shows the metal concentration ratio T / C after ten days of treatment.

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TABLE I.

Effect of the metal protein in chick feed after 10 days

(T / C ratio)

tissue 2% zinc
Proteinate 2% magnesium
Proteinate 2% calcium
proteinat 0.5% manganese
proteinat 0.5% cobalt
proteinat brain 1.43 1.35 0.48 zero zero heart 1, 36 0.92 1, 00 on recognizable Height raised liver 25.77 1.41 1, 00 0.5 at a recognizable height
raised O chest 2.17 1, 20 1, 09 zero zero CO
OD skin 2.32 1, 60 1, 62 reduced at a recognizable height
raised 5/08 whole leg 5.46 1.31 8.69 1, 00 at a recognizable height
raised

cn cn -J O CD

as

Example IX

The beneficial effect on the stimulation of growth by adding metal proteinates to the diet feed of turkeys has been shown also determined.

Day-old turkeys were divided into five groups and each group was given a commercial starter rearing feed fed. The first group served as a control group and did not receive any additional metals. The remaining four groups were like treated as follows:

Group 2: one capsule (IX) per day was given by the

Beak inserted (see Table II). Group 3: one capsule (0.IX) daily by the

Beak inserted (see Table II). Group 4: one capsule (0.0IX) daily by the

Beak inserted (see Table II).

Group 5: became an initial metal proteinate culture produced with the metal proteinate content of dosage 0.0IX by mixing 10 g metal proteinate (IX) with 99o g of the initial culture. The metal dosage per capsule is given in Table II. In Dosages following one line in a row are each diluted by a factor of 10 with a lactose dilution.

TABLE II
Metal dosage per capsule

(IX) (0.IX) (0.0IX) metal mg / capsule mg / capsule L. iroj / KapseL Co 0.0296 0.00296 0.000296 Cu 0.0459 0.00459 0.000459 Mn 0, 1 15 0.0115 0.001 I5 Fe 0.230 0.0230 0.002J0 Zn 0.492 0.0492 0.00 192 Approx 4,592 0.459 0.04 5 9 Mg 4,592 0.459 0.0459

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The average daily weights of each group were then measured and are summarized in Table III.

TABLE III

Average daily weights of turkey hens (all weights in grams)

1st day 2nd day 3rd day 4th day 5th day

Group 1

(Control) 48.7 51.4 60.0 64.3 74.6

Group 2
(IX) capsule 50.7 50.5 65.8 73.4 80.9

Group 3

(0.IX K.) 50.6 53.4 64.2 65.3 77.9 Group 4

(O.OIXK.) 50.2 50.4 65.6 75.5 85.0

Group 5
(0.00IX K) 51.8 53.0 66.6 68.6 84.6

Of particular interest in these test results is the fact that in general the turkey hens treated are one showed considerable weight gain over the control group at each level of dilution of the metal proteinate, independently whether the metal proteinate is forcibly fed or the Diet food was added.

Of particular importance in the experiments with turkey hens was that some of them came in. The diagnosis revealed para-colon in the control group and lower in the groups Metal protein dosage (groups I, 3, 4 and 5). The excellent one The condition of the turkey hens in Group 2 suggests that Schluli that higher Meta 1! prote inatante lie an increased resistance to Para-colon conditional.

Additional advantages result from the fact that a lack of metal, first diagnosed and then corrected according to the invention as set out in the following UuLS cases.

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Example X

10,000 laying hens were selected and divided into two groups of 5,000 each. Commercial feed was given to both groups allocated. The feathers of representative samples from both groups were examined to determine the metal content in the hens determine. This was compared with the metal content of feathers from young healthy laying hens. When a defect is found If, on the basis of the comparisons, metal proteinate according to Table IV was added to the commercial feed. as Control tests were used to add inorganic metal compounds.

TABLE IV
Metal content of the feed mixture

Calcium 4.5% magnesium 4.6% zinc 0.4% iron 0.2% manganese 0.1% copper 0.04 cobalt 0.02

The above metal-containing mixture was thoroughly mixed with a standard laying hen feed ration in the size of 1 kg metal-containing mixture per ton of commercial laying hen feed.

Group 1 received the metals according to Table IV as proteinates. Group 2 received the same amount and proportions of Metals per ton of feed like group 1, but the metals in the form of inorganic compounds. During a period of The hens in group 1 were treated with the metal proteins for sixty days and lay 18,210 more eggs during this time

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than the hens treated with the inorganically bound metals. In addition, the quality of the eggs was better. Group 1 eggs required an average of 0.77 kg higher pressure to break the eggshell than group 2 eggs. In addition, group 1 eggs exhibited a greater tensile strength. The analysis of the egg yolk showed 11.14% more zinc, 10.5% more iron and 6.0% more copper in the eggs of group 1.

The increased number of eggs laid in Group 1 resulted not only from a higher laying rate, but also from one longer length of time the hens lay. Table V shows the results as a percentage based on a lay rate of one egg per day per hen during the main laying period of a group and six months after the main laying period. In both groups the hens started laying at 20 weeks of age.

TABLE V Group 1
(treated) Group 2
(Control group) 85% 75% highest Laying rate 80% 64% 6 months thereafter

The hens were brought to moult after the sixty days.

Shortly before the moult, the feathers were examined, with feathers

from group 1 had 10 to 70% higher metal contents

than that of the control group 2.

Example XI

2500 laying hens, which had their main laying activity for three months, and 25 randomly selected chicks were examined

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these laying hens were chosen as the control group. The average Hemoglobin content in gm% in the chicks was 8.7 gm%. The hens were then placed on a diet containing a metal proteinate mixture according to Example IX. Forty-three days later, eggs were incubated from the treated hens, and twenty-five Randomly selected (treated) chicks were selected and examined for hemoglobin. The average hemoglobin content the second group of chicks was 9.4 gms% compared to 8.7 gms% for the control chicks. The percentage of chicks one year old in the first seven days of life was 0.8% versus 2% in the control chicks.

It has therefore been found that a supplement to the diet of laying hens with metal proteinates a higher hemoglobin content in the resulting chicks and a lower intake rate exhibited.

Example XII

500 laying hens were diagnosed with avian leukemia and treated with metal proteinates in the normal feed. The composition of the metal proteinates corresponded to that of Table IV, with calcium, magnesium and zinc being the predominant ones Components were. The yellow appearance of the combs and tails, which is typical of this disease, disappeared after 30 days and turned the normal red color. The animals also looked healthy again, and the failure rate from death was negligible. The egg production and the quality of the eggshell were back to normal, with the egg breakage in the nests around 97% decreased.

Example XIII

Five breeds of young trout were taken, each breed including about 5000 fish. These 5 groups were each

raised on a feed ration with the addition of 0.5% of a metal protein (Table IV). The five breeds of fish were compared with a control breed, which contained the same feed, but without the addition of metal protein. Then samples of the fish from each breed were weighed every two weeks for one year. It was found that the feed turnover, i.e. the amount of feed required to produce a 1 kg weight gain is much higher than feeding the fish without adding metal protein. The treated fish consumed an average of 1.2 kg of feed per kg of weight gain, while the control trout consumed an average of 4.2 kg of feed per kg of weight gain.

Example XIV

Exfoliation in piglets due to iron deficiency is a well-known problem when sows and their offspring do not have access to pasture or soil. Piglets are particularly susceptible to this type of bleaching because of their high growth rate and the low reserves of iron in the body shortly after giving birth, and also because of the low iron content in the milk of the sows. The normal growth rate in piglets entails an increase in body weight of four to five times the weight at birth after three weeks. A growth rate of this size requires maintenance of about 7 mg iron per day. However, the milk of sows only provides 1 mg per day, while on the other hand, take the pig except the milk of the sows in the first three or four weeks little extra food to him. Therefore, there is a need for an additional iron diet.

Sows fed with inorganically bound iron as an additive did not have any before or after piglets

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increased iron content in the milk, and the iron content in the piglets at birth was not increased either. The treatment the piglets were routinely given either a) oral administration of 400 to 500 mg of inorganically bound iron as tablets or by solutions within 5 days of birth and again after two weeks, or b) by at least two injections from 100 to 200 mg of iron complex solution during the early growing season. This has proven to be insufficient even with frequent treatment, so that no maximum growth of the piglets occurred. This unfavorable circumstance is made even more difficult in treating large numbers of animals, making the process impractical. It is therefore highly desirable to study the tissues of animals, e.g., blood, and then the diet of the sows with adequate metal levels to enrich, so that the metals are transferred from the sows to the piglets via the milk.

After appropriate analysis, it became the following composition in Table VI produced as an additive to the sow feed. The metals were added as proteinates.

TABLE VI Metal proteinate experiment for pigs

Metal percent d. composition

Mg 6.80%

Fe 1.86

Zn 1.26

Cu 0.05

Co 0.0024

The composition according to Table VI was added to a group of sows in an additional amount of 2.3 kg per ton of feed, while a second group of sows added the same metal content in the form of inorganically bound metal

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3a

became. The second group of sows served as a control group. A randomly selected sample of the proteinate feed and the control feed showed an iron content of 14 mg / 100 g in both cases.

The hemoglobin content in the blood was initially determined, ie 30 days before the expected piglet date, up to a point in time of 60 days after the piglet had been given. The weight and blood hemoglobin content of the piglets was determined at birth and after weaning. The results are given in Tables VII and VIII.

TABLE VII
Hemoglobin content (Hb) in gm / 100 ml

Group

Sows

at first
when throwing

Control pit Hb area Average Hb area

12-15
12-14

13.5

13.3

12.14 13-15

Average

piglet

at d. Birth 7.5-9.8 9.0

at d. Weaning 10.7-12.0 11.7

* some piglets need iron injections.

9.6-12.0 11.9-12.8

at d. birth
after weaning
Increase:

TABLE VIII
Throwing weights

(Average in pounds)

Control group behande l te Gr u ppe

3.60 3.53

37.25 39.25

33.00 35.65

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It should be noted that in the previous examples, some of the control piglets were given iron complex injections needed to keep them from dying, and that is why the hemoglobin content of the control pigs was for this reason was high. Even with these injections, the piglets suckling from treated sows had a higher hemoglobin content and a greater weight when weaning.

example

XV

A group of 58 sows with similar pregnancy history was randomly divided into three groups. The group I received a normal pregnancy and lactation diet and the offspring received no iron. The swine of the group II received the same gestation and suckling diet, however the piglets received by intramuscular injection at the age of one and 15 days 100 mg iron dextran. Group III sows received a normal pregnancy and suckling diet with one Addition of 500 PPM iron as iron proteinate, which contained about 10% by weight iron. The piglets were not given any additional Iron administered. The results are shown in Table IX.

TABLE IX Number of d. Sows Group I.
(No iron) 11, 41 Group II
(Iron-
Dextran) Group III
(Iron-
Proteinate Number of littered piglets 13th 2.86 27 18th 144 Number of weaned
Piglet 8098 ^ / 0843 285 168 avg. Hemoglobin (Hb) 12.03
d.Sows while throwing (gm / 1ooml) 13.5 14.0 avg. Hb the piglet
when throwing (gm / 1oo mis) 11.2 11, 35 avg. Birth weight
the piglet in pounds 2.82 2.99 233 157

14th , 96 16 , 53 17th , 43 20th ,1 18th , 3 6th , 5

avg. Hb when weaning 7.32 11.69 10.83 (gm / 100 mis)

avg. Weight at weaning (37 days) in pounds Percentage of intake Total weaning weight /

Weaned piglets (pounds) 11.9 13.51 16.28

The table above shows that the piglets from Group I sows had a lower hemoglobin content (Hb) and a lower weight when weaning and had a higher intake rate than that of groups II and III. Careful study of this last group showed clear benefits of adding iron proteinate. Also the hemoglobin content when throwing and weaning was significantly higher than 9.0. The average litter weight of piglets from Group III sows was 80g higher than that of the piglets from group II. Repeated examinations showed a weight difference of 70 to 110 g at litter, if the sows had been fed iron proteinate. The piglets of sows in group III had a weight of around 0.41 kg when weaning, i.e. 5.2% greater weight than that of Group II. The difference in incorporation rate was considerable. In group II 2.82 times more pigs died on average despite iron dextran injections than in group III, in which the piglets had the Iron obtained by feeding the supplements to the sows. The last fourth in Table IX show the combined effect mortality and weaning weights. In group III there is an improvement of 17% compared to group II, i.e. the weaning weight is 1.25 kg higher.

From the above it is clear that iron proteinate becomes one higher weight of the littered piglets, lower mortality and higher weight during weaning results in im Compared to iron dextran injections.

The surprising advantage that results from the biologically detectable form of the

3S

essential metals. With these additions, the animals no longer need their own metal proteinates from inorganic synthesize bound metals, and therefore there are individual differences in biological capabilities and properties of the animals are no longer of importance if the metal content is unsuitable. It also enables an investigation of the tissue of healthy animals the establishment of standard and comparison lists from which the respective metal content can be seen, which should be adhered to and which can be achieved through a corrective diet supplement.

Example XVI

It has already been mentioned that metal proteinates from basic solutions at a pH value above 7.5 and preferably between 8 and 10 are formed. The following compositions serve to underpin this.

Ten microliters of 0.073 M ZnCl- with a content of 912 micro-

65
curie radioactive Zn were mixed as follows:

A. 10 microliters of 0.076 M L-leucine (equimolar ratios of amino acid and zinc) were combined with zinc at a pH of about 3.5 and to pH 7 with 20 microliters of sodium hydroxide set.

B. 20 microliters of 0.076 M L-leucine (2: 1 ratio of amino acid to metal) was combined with a zinc solution at a pH of 3. 20 microliters of water were added.

C. 20 microliters of 0.076 M L-leucine and 20 microliters of 0.13

M Na-CO, were combined with a zinc solution to make metal proteinate arose at pH 9.

Each of the above recipes represents a dosing unit.

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6 male rats weighing 160 g + 8 g were put together in pairs according to the same weights as possible. Three solutions of each composition were carefully mixed to make up a dose. Each rat was lightly anesthetized with ether and then dosed orally with an Eppendorf pipette. Every rat swallowed the mixture immediately, with the exception of one rat, which was supposed to be given Mixture C, and which only swallowed most of the mixture after considerable experimentation. The rats had starved for 16 hours before the administration and were fed and watered normally again after the administration.

After 46 hours, the rats were dissected and the blood, liver, left kidney, heart, skeletal muscle and brain of each animal were removed. One animal that received Mixture B had a left kidney only 1/20 normal size. No Zn was detected in the muscles or the brain of this rat, so the results related to this rat were not used . The tissue samples were dried, weighed, and then examined with a Model Chicago 8731 and Model 8770 radiation counter. The values are summarized in Table X and represent the counts per minute per mg as the average of two animals, with the exception of mixture B.

TABLE X B. C. A. 1.31 1, 64 blood 0.90 6.20 8.65 liver 5.15 6.46 8.55 kidney 5.45 5.23 6.32 heart 6.42 3.10 3.88 muscle 2.41 3.86 2.41 brain 1.22 26.16 31.45 Total cc / min / mg 21, 55

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3?

By dividing C by A and B, it can be seen that the zinc proteinate (C) gives 1.46 times greater metal absorption than Mixture A and 1.2 times greater absorption than that Mixture B.

Example XVII

The method according to Example XV is expanded using

generation of radioactive iron (Fe). The following dosing units were used used:

A. 10 microliters of 0.05 M FeSO. . 7H-0 with a grade of

59
10 microcuries of radioactive Fe, 10 microliters of 0.05 M L-glutamic acid, 40 microliters of NaOH to bring the solution to pH 6.

B. 10 microliters of 0.05 M FeSO ₄ . 7H ₉ O with a content of

59
10 microcuries of radioactive Fe, 20 microliters of 0.076 M L-leucine, 40 microliters of water to adjust to pH 3.

C. 10 microliters of 0.05 M FeSO. . 7H ₉ O with a content of

59
10 microcurie of radioactive Fe, 20 microliters of 0.076 M L-leucine, 40 microliters of 0.13 M Na ₉ CO- for the formation of iron proteinate at pH 9.

Six female rats weighing 133 g + _ 5 g were paired and the dosage according to Example XV was used and the tissue removed, dried and counts made, the results being summarized in Table XI.

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TABLE XI

ABC

blood 36, 1 34.0 83.0 liver 11.7 16.1 38.4 kidney 4.5 5.5 8.5 heart 2.6 4.7 9.7 muscle 0.3 1.7 2.7 brain 0 2.1 1.5 Total cc / min / mg 55.1 64.1 143.4

If you divide C by A and B again, you can see that iron proteinate (C) gives a 2.6 times greater absorption of iron than the mixture A and a 2.24 times greater absorption of iron than the mixture B. In the two examples XV and XVI Mixtures A and B did not consist of metal proteinates, although in mixture B the ratio of amino acids in each case to metal was at least 2: 1.

Example XVIII

The following study was carried out to show how animals absorb buffered metal proteinates. Laboratory white rats were used as the test animals and each rat received the same amount of labeled zinc chloride by pipetting directly into the rats' stomach. The molar ratio of zinc to methionine was one to two in Rats II and III, and the pH was adjusted according to the following table.

TABLE XIII

Ratt e I e II Ratt rat III

24 microliters Zn ⁶⁵ Cl ₂ 24 microliters Zn ⁶⁵ Cl ₂ 24 microliters Zn ⁶⁵ Cl ₂

75 microliters H ₂ O 25 microliters HO with 25 microliters H ₂ O

NaHOWNa ₂ CO ₃ au """" ~ "* - · - * - a pH of 10

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NaHC0 / Na ₂ C0, egg-adf with NaOH to a pH of 1 NEN 0 pH 7

50 microliters 50 microliters

Methionine solution methionine solution

in molar ratio in molar ratio

2: 1 with Zn ⁺ 2: 1 with Zn

The rats were caged for metabolism studies and placed on a normal diet and observed for a week in which the feces were collected. At the end of Week the rats were sacrificed and the total excrement by means of scintillation counting in comparison to lack of marking measured for radioactivity. Each of the rats had the following amounts of Zn in the feces for one week accumulated:

% of the total dose in the excrement

Rat I 52%

Rat II 12%

Rat III 36%

More than half of the Zn in the control animal was lost. The storage of the Zn methionate in the rat II, which at pH 10 administered was considerably greater than that Storage of the Zn methionate in rat III to which the composition was administered at pH 7. Both rats however, showed a significant increase in the Zn content the rat I.

Example XIX

Example XVII was essentially repeated using

59
application of Fe SO. as a control. The solution was given orally into the stomach using a pipette. Every rat received

59

36.7 micrograms of Fe in 20 microliters of solution. The rat II was administered a methionine solution and the rat III one Glycine solution, both of which were buffered to a pH value of 10 with a molar ratio of metal to amino acid accordingly One to two. At the end of a week the rats were

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to

59

sacrificed and parts of various organs on Fe "" "by scintillation counting analyzed. The results are compiled in the following table:

TABLE XIII
Corrected counts per minute in grams

tissue Rat i 1 Rat II Rat III FeSO ₄ 2 FeMet FeGIy heart 64.0 4th 151, 0 83.0 liver 136.0 214 243.0 83.0 stomach 2.0 54.0 83.0 Masseter muscle 14.0 138.0 65.0 brain 31, 0 130.0 142.0 kidney 2.0 327.0 150.0 Testicles 20.0 109.0 75.0 serum 700.0 .797.0 840.0 Cells 742.0 .076.0 773.0 blood 1,335.0 .215.0 1,602.0 Excrement 302,400.0 .000.0 205,800.0 urine 490.0 370.0 690.0 Rat i Rat II Rat III

Excrement

45.8% lost 32.4% lost

31.2% lost

The above results are extremely instructive. The salary

59
on Fe, the rats II and II, which with buffered

59
Fe proteinate treated, was considerably higher than in Rat I, as revealed by examination of the excrement. The amounts of metal stored in the tissue were also significantly higher in almost all cases. However, more precise results could not be calculated because the entire organs had not been removed.

The buffer systems used were amino acid NaOH solutions and a solution of 65 g sodium bicarbonate (NaHCO ₃ ) and 13.2 g sodium carbonate (Na ^ CO,.) Per liter of solution, which means a pH of the buffered solution of e

Example XX

In order to further sub stant the effect of the buffered metal proteinates, the following studies were carried out on rats who fasted during the night. Each rat received the following dose of radioactive calcium by injection in the duodenum:

Rat I :

250 microliters of CaCl ₂ solution (1 mg Ca) in distilled H ₂ O, 40 microliters of distilled H ₃ O (40 mcC Ca ⁴⁵ ) as Ca ⁴⁵ Cl ₂ (mcC = Microcurie)

Rat II:

250 microliters of CaCl ₂ solution (1 mg of Ca) in distilled H ₂ O, buffered to pH 7 with NaOH and the amino acids and with a molar ratio to calcium of 2 moles of aspartic acid, 2 moles of glycine and 1 mole of methionine, and 50 microliters of distilled H. ₂ O (40 mcC Ca ⁴⁵ ) as Ca ⁴⁵ Cl ₂

Rat III :

the same as for rat II, but buffered to pH 10 with NaHCO / Na ₂ CO ₃ .

The rats were fed normally for one week and the excrement collected. At the end of the week the rats were sacrificed and the entire excrement and parts of the tissue analyzed by scintillation counting. The results are compiled in the following table:

TABLE XIV
Corrected counts per minute in grams

Tissue rat I rat II rat III

Frontal bone 3682 5878 5772 Masseter muscle 602 844 904 Stomach (muscle) 614 620 1206 heart 642 598 932

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liver 664 right brain 698 kidney 686 lung 676 serum 100 microliters of blood 8.4 Cells 18.6 Total blood 27.0

546 742 726 804 656 730 672 648 39.6 31, 0 0 13.2

39.6 44.2

It is evident from the tissue count that much more calcium proteinate had been assimilated in the tissue when buffered to pH 10 rather than pH 7. on the other hand it is found that more calcium proteinate was absorbed at pH 7 than with the calcium control salt.

The excrement showed a four-fold higher calcium excretion for the simple organic control salt (rat I) compared to the calcium proteinate buffered to pH 10. In addition, the pH 10 proteinate was about twice as effective in terms of efficiency the storage of calcium like the proteinate buffered to pH 7. So the stability can be increased by buffering of metal proteinate solution and increase assimilation in various tissues.

Example XXI

The examples given above seem to show that a buffer system with a pH of 10 assimilates some Metals improved in living tissue. However, this pH is not optimal for all metals. Some metals will usually better absorbed at a lower pH. So it's about finding the optimal pH range for that too

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the metals to be administered. This can be done empirically for each metal proteinate.

Manganese, for example, is better absorbed as a proteinate at a pH value of about 7 and tends to in more basic systems Formation of Mn (OH) -.

Manganese and calcium also do not work favorably with carbonate buffered Solutions together as they tend to form insoluble carbonates. The absorption capacity of male proteinates, buffered to pH 7 is illustrated below. Two solutions were prepared using

54 tion of Mn:

Solution I:

250 microliters of distilled HO with a content of 100 mg Mn as MnCl ₂ , 50 microliters of Mn solution (14.3 mcC) as Mn ⁵⁴ Cl ₂

Solution II :

250 microliters of distilled HO with a content of 100 mg Mn as MnCl ₂ , 50 microliters of Mn54 solution (14.3 mcC) as Mn ⁵⁴ Cl _2. The solution contained 2 moles of each of the amino acids methionine, glycine, aspartic acid and glutamic acid per mole of total manganese. The solution was buffered to pH 7 with NaOH, which reacted with the amino acids.

The solutions prepared in this way were injected into the duodenum of examination rats, the rats correspondingly the investigated solutions were marked. The rats were fed the normal diet for one week and then sacrificed. The tissue was then analyzed by scintillation counting to find out how much manganese proteinate was taken up had been. The following results were obtained:

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TABLE XV Corrected Counts Per Minute Per Gram

cc / min / gm dry weight

heart

kidney

brain

stomach

Masseter muscle

liver

lung

Frontal bone

Duodenum

370 470 620 800 270 760 720 350 170

II

1190 600

1170 660 310

1070 330 780 480

It can be seen from the table above that almost all of the counts in rat II, which had received low-protein proteinate , are higher than in control rat I. No urine and excrement counts were carried out.

Example XXII

The above examples demonstrate the assimilation of buffered metal chelate complexes with isolated amino acids or limited combinations of acids. The example also shows that hydrolyzed protein (in the form of di- and tripeptides) can be used with just as good success. The example also shows the transfer of stabilized metal proteinates through the placenta from the mother to the still unborn fetus. Mink was used for these studies and iron was used as the basis for the metal proteinates, because some experts in mink breeding believe that there are difficulties in mink in the placental transfer of iron from mother to young.

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Two pregnant mink were caged individually and subjected to a fasting treatment for 20 to 24 hours. and then both were given 24.17 mg of iron, which

59
ches 5mcC contained Fe as a radioactive isotope. The mink no.

59
received the iron in the form of Fe SO ₄ , which had been converted into hydrolyzed protein in the form of dipeptides and tripeptides and buffered to pH 10 _{with NaHCO- / Na 0 CO, solution.}

59 Mink No. 2 received the same amount of iron as Fe SO ·. In each case, the isotopes were mixed with 25g of feed, which was picked up by the mink. The iron was given to each mink 50 days prior to throwing. Feces and urine of each mink were collected to determine the amount of Fe excreted. The measurements were made four days after dosing recorded as well as when sacrificing the animals. Fifteen days after dosing, the mink were sacrificed and various biologicals Measure tissue for radioactive iron by scintillation counting. The measurements were also made with respect to Hemoglobin and hematocrit in dams and offspring. The test results are compiled in the two tables below.

TABEL Total percentage
contained Fe ⁺⁺ XVI . 1 mink No. 2 GENERAL Total percentage
precipitated Fe ⁺⁺
in the excrement DATA 42.7 Total percentage in
Urine excreted Fe ⁺⁺ Mink No. 29.6 % of the excrement in the excrement
separated Fe ⁺⁺ 4 days after
the treatment 70.4 27.7 % Fe ⁺⁺ passed to the
Young animals 24.4 23.8 Hemoglobin content d. Dams
gm% 5.17 (7.3 micro- °
gram) 809825 21.5 20.0 0.03 20.5 / 0843

- « ft

Hematocrit% 45 44

avg. Hemoglobin content

of young animals in gm% 19.5 19

avg. Hematocrit content

d. Young 53 50

Counts d. Total body

without mother's organs

(corrected counts / minute) 112.4 68.1

avg. Counts of the

Body d. Young animals

(corrected counts / minute) 42.3 1

TABLE XVII Corrected Counts Per Minute Per Gram

tissue Mink No. 1 Mink No. 2 Masseter muscle 7.81 12.00 large pectoral muscle 1, 22 5.02 spleen 15.3 10.60 brain 9.7 7.57 lung 6.4 4.05 heart 2.7 5.12 liver 4.98 4.73 Throat fur and skin 6.24 3.82 Scalp 5.74 6.33

Several conclusions can be drawn from the above data. It is first apparent that the amount of iron remaining in No. 1 mink which received the buffered iron proteinate was 65% greater than that of No. 2 mink containing only Fe-SO. had received. In other words, 70% of the iron in the buffered iron proteinate was metabolically processed, whereas only 4 2.7% of the mink treated _{with Fe 3} SO _{4 was processed.}

If one compares the quantities of iron excreted after four days with the final analysis, one recognizes that with the

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Mink No. 2 had absorbed 33.5% of the iron initially given, but had not been metabolized and may have been excreted between the fourth and fifteenth days after dosing. In mink no.1, only 8% of the absorbed iron protein was later excreted and not metabolized. The payment show that a measurable amount of iron passed over the placenta as iron proteinate in the descendants of mink No. 1

Was 59 (42.3 cc / min), whereas the Fe in the descendants of mink No. 2 was practically negligible (1cc / min). The hemoglobin and hematocrit values were also higher in the mink that had received iron proteinate than in the comparison animal. The iron proteinate is contained in the blood, skin tissue and organs to a greater extent, as the scintillation measurements on the tissue have shown. The spleen, which is 90% blood, contains about 50% more iron with buffered iron proteinate compared to Fe-SO .. This is important because it shows that the iron proteinate is better for building hemoglobin than the Fe ₃ SO ₄ . Ultimately, the data show that hydrolyzed protein in the form of tripeptides and dipeptides are effective as ligands for complex formation with buffered metals for the formation of metal proteinates, which transfer the metal into the bloodstream from the digestive tract, as with the individual amino acids.

The following examples show the inclusion of metal proteinates, which contain at least two tripeptides, dipeptides or amino acids as ligands, in or on food and feed. The metal proteinates can be in a finely ground form. When used in flour or baked goods the metal content of the product in the form of a proteinate can be between 0.00001 and 0.001% by weight.

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Example XXIII

A flour containing 110 mg iron as iron proteinate per kg flour was placed in a Jacksonville wheat stability cabinet. Storage in this cabinet for a week is equivalent to storage for one month under ambient conditions. After 18 weeks no rust had developed and the metal protein and flour were still completely stable.

Example XXIV

The bioavailability and stability in tripeptides, di peptides and amino acids in chelate iron present was compared with ferrous sulfate, which is considered in the industry as standard. The superiority of iron in chelate binding was shown in every experiment.

Example XXV

To test the usability of iron proteins, two loaves of bread were baked and cut into slices. One loaf contained 4 mg of iron as proteinate in each slice and the other 8 mg of iron as proteinate. Each bread weighed 0.68 kg and made 24 slices with two slices per meal. The iron requirement (RDA) is 18 mg per day. The bread was made as follows :

RECIPE :

1 packet of active dry yeast 1/2 cup of water

2 cups of boiled milk 2 tablespoons of sugar

2 teaspoons of salt

1 tablespoon of spices 6 cups of sifted bleached flour.

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ts

The mixture was divided into equal parts, and 96 mg of iron as iron proteinate was added to one part, and 192 mg of iron, also as iron proteinate, was added to the other mixture. The ingredients were stirred and processed in the usual manner and left to rise. The dough was baked at 205 ^° C. for 35 minutes. After the bread had been baked and cooked, it was analyzed for rust spots. When none could be found, the bread was stored. After storage, the bread was superior to bread that did not contain iron as an additive.

Other metal chelates can also be used. Instead of iron can be magnesium, manganese, copper, zinc, cobalt, calcium or any other divalent vital metal can be used.

The addition is not only possible with bread. For example, metal chelates can also be added to fortified (nutritionally enhanced) flour, fortified self-rising flour, fortified brominated flour, prepared or ready-to-cook Breakfast cereals, poultry farce, rice, soy, corn flour, corn semolina, fortified bread, croissants, rolls, Biscuits, cakes and baked goods.

The metal proteinates can also be added to edible oil and absorbed or adsorbed by the edible oil in or on foods treated in such oils. Typical such foods are potato chips, corn chips, donuts and other fried candies and breaded meats, corn dumplings, fried shrimp and chicken. The metal proteinates can be used in amounts of 50 to 500 mg per liter of edible oil. At these concentrations, metal proteinates prevent the cooking oil from going rancid. The rate of absorption of the metal proteinates in or onto the food is approximately proportional to the amount of proteinates in the amount of oil absorbed. Since the proteinates are essentially insoluble in oil, the oil must be constantly stirred or agitated. 809825/0843

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Both vegetable and animal oils can be used. Typical such oils are corn oil, palm oil, coconut oil, peanut oil, safflower oil, and rendered animal fats hydrogenated as not hydrogenated. The following examples are given for this.

Example XXVI

In a bucket equipped with a stirrer, a mixture of hydrogenated palm and coconut oil was ^{heat-treated at 190 °} C. and a mixture of iron, zinc and copper proteinate was added with a content of about 400 mg zinc, 475 mg iron and 53 mg copper per Liter of oil. Potatoes were sliced on a commercially available slicer and cooked in the hot oil until crispy and slightly golden brown, after which they were removed from the oil and the oil allowed to drain. After analyzing the chips, they contained 6 mg zinc, 7.2 mg iron and 0.8 mg copper in the form of proteinates for a meal of 60 g.

Example XXVII

In a small container equipped with a stirrer, hydrogenated corn oil, containing a mixture of iron, Magnesium and zinc proteinates had been added so that the content of iron was 400 mg, of magnesium 132 mg and of zinc Was 320 mg per liter of oil. The oil was at one temperature held at about 205 ° and constantly stirred. A piece of pastry puffed up by yeast was fried in the hot oil and then removed the baking value and drained the oil. The analysis of the finished baked goods resulted in 9.7 mg Iron, 3.2 mg magnesium and 7.8 mg zinc for a meal of 120 g.

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Example XXVIIi

In a vessel in which oil is constantly circulated and filtered a mixture of iron, manganese, copper and calcium proteinate was added so that about 0.5 of each metal in the form of proteinate was present in the oil and maintained at a temperature of about 162 to 177 ° C. It became chicken thighs turned in batter and dipped in the oil until cooked, then removed and drained. The analysis found that the proteinates had been absorbed from the surface of the batter in such an amount that was roughly proportional to the concentration of proteinates in the edible oil.

Meat and meat-tasting products can also be enhanced with metal proteins. This can be practical any recipe can be used as is used in the preparation of pet meat and game, etc. Typical such products are Swiss steaks, stews, processed meat such as Bologna sausage, salami, Canned meat, steaks, minced meat, grilled meat, hamburgers, Sausages, textured vegetable protein and the like. From about 0.00001 to 0.01 weight percent metal can be used in such products be present as a metal proteinate. The metal proteinates may, but are, improve the taste of the products No substitute for spices, herbs or other taste-influencing Fabrics.

Example XXIX

A beef steak weighing 300 g was seasoned with 2 g of salt and a mixture of metal proteins, the Metal content was 7.5 mg zinc, 9 mb iron and 1 mg copper. The finished steak contained 5% of the RDA of each added Minerals as metal proteinate.

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Example XXX

A pot roast was made with 1 kg of braised meat, a cup of water, 2 teaspoons of meat juice, 4 ground green peppercorns, 6 carrots cut into thick slices, a pinch of thyme, rosemary and basil, a head of cauliflower, two onions, two bay leaves, 1 Can of tomato sauce and 54 mg of zinc, 45 mg of iron and 6 mg of copper in the form of proteinates. The pot roast was lightly cooked until cooked and then served to six people, each serving containing 5% of the recommended daily allowance of each metal added as proteinate.

Example XXXI

A "Chili Pepper Surprise" was made with one kg of game meat, 230 g of processed cheese, 2 medium-sized chopped onions, pepper to taste, 1 can of green chilli pepper, half a can of condensed milk, 1/4 teaspoon of garlic powder, 1/2 teaspoon of celery salt and 45 mg of iron as an iron amino acid chelate. The mixture was boiled and divided into six servings. Each serving contained 5% of the Recommended Daily Allowance (RDA) of iron in the form of iron proteinate. Instead of venison, the same amount of meat -flavored soy flour can be used as an extender.

The above examples can be expanded as desired and show the broad field of application of the invention.

The vital divalent metals can also be added to food by adding them as metal proteins to spices and herbs. Alkali metal salts such as sodium chloride (baker's salt), potassium chloride and monosodium glutamate are particularly suitable for this purpose. Sodium chloride can be used with onions,

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Garlic or other common spices. The metal proteinate should be metal in a range of 0.001 Contain up to 0.1% by weight of the spices or herbs. The following examples show that divalent, with tripeptides, dipeptides and / or amino acid chelated metals are stable with nutrients. Potato chips, corn chips, nuts, spice biscuits or biscuits or virtually any other food that contains spices or herbs can be used. In some cases, a metal proteinate also delays food spoilage. So it leads to a longer allowable Storage time compared to non-metal protein containing products. The following examples may illustrate this.

Example XXXII

Potato chips are made by peeling and cutting into slices of potatoes to the desired size. The chips are then fried in hydrogenated palm oil. After this Remove from the oil they are flavored with flavored food Table salt. The following experiment was carried out in a commercial plant for the production of potato chips using common spices. Since a meal of potato chips is about 57g, this was taken as the standard. A mixture of 1.14 g table salt with enough amino acid chelate was added to each such serving of potato chips that 7.5 mg zinc, 9 mg iron and 1 mg copper were present. Each of these metals represented about 5% of the recommended Daily consumption in one portion. On 15 test persons Taste studies were carried out on the basis of double-blind experiments to find out how the addition of Table salt improved with metal chelates has an effect compared to table salt alone. The tests showed that in 97% of the test data the potato chips improved with metal proteinate had an outstanding taste quality compared to the control chips. Only 3% of the trials showed no difference. Besides the better taste quality it showed

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St.

that also the shelf life and the tendency to not go rancid had been influenced more favorably compared to the control chips. The shelf life was compared to these two Weeks longer.

Example XXXIII

Investigations were carried out over a period of twelve months into the compatibility of table salt enriched with iron amino acid chelate had been. There was no oxidation or reduction of the chelates. These are also not hydroscopic or melting.

Such experiments can also be carried out with other spices, e.g. with ham-flavored salts, barbecue-flavored or onion-flavored potato chips or the like.

Foods that mainly contain sugar tend to contain too few essential metals. The following examples show that divalent metals chelated with tripeptides, dipeptides and amino acids in confectionery, jams, Syrups, coatings and other sugar products are stable. In certain cases, the metal proteinates delay this Spoilage. So they lead to a longer permissible shelf life of the products compared to those that do not contain metal proteinates contain. The proportions of each metal in the metal proteinate product can be between 0.001 and 1% by weight of the product lie.

Example XXXIV

Caramel candies were made by spreading the Walls of a large kettle with butter, mixing 2 cups of sucrose with 0.47 1 cream. The mixture was for 3 minutes

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boiled up. Another 1/2 liter of cream was then slowly added to the hot mixture until it was homogeneous. Then, 60 g of coconut butter and 1/2 teaspoon salt was added, then 1 1/3 cups of hot glucose with stirring, whereupon the mixture was boiled at a temperature of about 120 ⁰ C. The hot mixture was removed and cooled slightly, then 1 teaspoon of vanilla was added, as well as 9.1 g of zinc proteinate with a content of 10% zinc, 37.1 g of iron proteinate with a content of 12% iron. The vanilla
and the metal proteinates were mixed into a uniform mixture. The caramel mass produced in this way was poured onto a cold, oiled plate, rolled out into rolls about 2.5 cm in diameter and then cut into slices about 1.2 cm thick.

Example XXXV

A caramel syrup was produced as the topping composition by boiling 1 liter of maple syrup to which 0.5 liter of cream was added in the hot state with constant stirring. 1 cup of boiling glucose was also added while stirring and the syrup was then ^{boiled at 94 °} C., partially cooled, then 1 teaspoon
Vanilla added together with 30.3 g of copper proteinate with a content of 15% copper. The syrup was then poured into containers and allowed to cool.

Example XXXVI

An apple jelly was made by pouring sliced apples over which the peel was not
removed with water and boil until the apple slices were soft. The boiled apple-water mixture
was then squeezed through a cheesecloth so that a clear
Apple juice was created. 4 cups of apple juice were made with 3 cups

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SG

Sucrose is heated and quickly brought to a temperature of 104 ⁰ C and the heat is then removed. Zinc proteinate containing 10% zinc was added so that the jelly contained a total of 0.005% zinc. The whole was then poured into sterilized containers and sealed with paraffin wax.

Example XXXVII

Peach jam was made by cooking well ripen peach slices until they can be easily crushed into pulp. There were equal amounts of peach pulp and Sucrose mixed and cooked on low heat for 20 to 30 minutes until the desired consistency was achieved. This pulp a mixture of zinc and manganese proteinate was added so that the peach jam had a zinc content of 0.01% and had a manganese content of 0.005%. The hot jam was poured into sterilized mason jars and covered with paraffin wax locked.

The above examples show the variety of possible applications the invention. All processes and recipes are aimed at the content of vital divalent metals in living tissue in a safe and effective manner.

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Claims (37)

Claims 1. Procedure for increasing the salary of essentials divalent metals in the tissue of warm-blooded animals, in particular animals, characterized in that the warm-blooded animals administered an effective amount of a metabolically assimilable metal proteinate and that the metal proteinate Used in the form of chelates of these metals is made with at least two protein hydrolyzate ligands the group of tripeptides, dipeptides and naturally occurring amino acids. 2. The method according to claim 1, characterized in that the metal proteinates are metals selected from the group calcium, magnesium, zinc, iron, manganese, copper, cobalt, molybdenum, chromium and vanadium will. 3. The method according to claim 1 or 2, characterized in that the metal proteinates with a Carriers in the form of tablets, capsules, syrup, feed additives and dietary supplements. 4. The method according to claim 1 to 3, characterized in that g e k e η η that each of the metal proteinates is administered in an amount sufficient to cause a metal deficiency balance in the animal body during the administration of the metal proteinates. 5. The method according to claim 4, characterized in that the metal deficiency by tissue analysis of the 809826/0843 Animal is found to be administered the Hetallproteinate should be. 6. The method according to claim 1, for treating bleaching of piglets, characterized in that of the farrowing sow during the last stage of pregnancy and during lactation, metal proteinates according to claim 3 can be administered. 7. The method according to claim 6, characterized in that that an iron proteinate is used as the metal proteinate. 8. The method according to claim 6, characterized in that that the metal proteinate is a mixture of an iron proteinate and other metal proteinates from the group the metals magnesium, zinc, copper and cobalt are selected, 9. The method according to claim 1 to 8, characterized in that the metal proteinates in the form of buffered Metal proteinates are administered, which are kept at a practically constant pH. 10. The method according to claim 9, characterized in that that a buffer is used from the group of phosphates, carbonates, bicarbonates, amino acid-base combinations and mixtures thereof. 11. The method according to claim 9 or 10, characterized in that the buffer is chosen so that the Metal proteinates are kept at a pH between 7 and 11. 12. The method according to claim 11, characterized e t that a mixture of sodium carbonate and sodium bicarbonate is used as the buffer. 809825/0843 13. The method according to claim 11, characterized in that that a mixture of one or more amino acids with sodium hydroxide is used as a buffer. 14. Composition for performing the method according to claim 1, characterized by a content of metabolically assimilable metal proteinates in the form of chelates of the metals with at least two protein hydrolyzate ligands from the group of tripeptides, dipeptides and naturally occurring amino acids, optionally in Mixture with a carrier. 15. Composition according to claim 14, characterized in that the metal proteinates from the group the metals calcium, magnesium, zinc, iron, manganese, copper, cobalt, molybdenum, chromium and vanadium can be selected. 16. Composition according to claim 14 or 15, characterized in that it is combined with a carrier substance in the form of tablets, capsules, syrup, powder, granules or tablets. 17. Composition according to claim 14 to 16, characterized by the addition of a buffer substance to keep the pH value constant. 18. Composition according to claim 17, characterized in that g e k e η η, that the buffer substance from the group of phosphates, carbonates, bicarbonates, amino acid-base combinations and mixtures thereof. 19. Composition according to claim 18, characterized in that the buffer substance is dimensioned so that they keep the metal proteinates at a pH between 7 and 11 holds. 809825/0843 20. Composition according to claim 19, characterized in that the buffer consists of a mixture of Is composed of sodium carbonate and sodium bicarbonate. 21. Composition according to claim 19, characterized in that g ek e η η that the buffer consists of a mixture of one or more amino acids with sodium hydroxide. 22. Bakery product with a content of essential metals in the form of at least one metal protein, thereby characterized in that the metal proteinate is selected as a chelate of the metal with at least two protein hydrolyzate ligands from the group of tripeptides, dipeptides and naturally occurring amino acids. 23. Bakery product according to claim 22, characterized in that g e k e η η, that for the metal chelate a metal from the group iron, zinc, copper, magnesium, calcium, Cobalt, magnesium, molybdenum, chromium, vanadium and mixtures of these metals is used. 24. Bakery product according to claim 23, characterized in that each metal product in an amount is present between 0.00001 to 0.001% by weight. 25. Bakery product according to claim 24, characterized in that the metal proteinate in the flour is included. 26. Composition as a stabilizing additive to cooking oil, characterized by at least one divalent metal proteinate in the form of a metal chelate with at least two ligands as protein hydrolysates from the group of tripeptides, dipeptides and of course occurring amino acids. 809825 / OBt 1 ORIGINAL INSPECTED 27. Composition according to claim 26, characterized in that Metallprotexnate with metals of the group iron, zinc, copper, cobalt, manganese, magnesium, calcium, molybdenum, chromium and vanadium are used. 28. Composition according to claim 26 or 27, characterized in that the metal content of each metal in the metal proteinate is between about 50 and 500 mg / l. 29. Use of the composition according to claim 26 to 29 for preventing edible oil from going rancid. 30. Use of the composition according to claim 26 to 28 as an additive for edible oil for preparing fried foods, for example potato slices, french fries, baked goods or breaded meat products. 31. Application of the composition according to claim 26 to 28 as Added to cooking oil to prevent fried foods from turning rancid. 32. Use of the composition according to claim 26 to 28 as an additive to a product consisting of pure meat, a meat-containing product, a meat-like tasting vegetable product, or mixtures thereof. 33. Use of the composition according to claim 32 in an amount of 0.00001 to 0.01% by weight of the product. 34. Use of the composition according to claim 14 to 21 as an additive to table salt or spices, in particular to one Sodium salt or a potassium salt or monosodium glutamate. 809825/0843 35. Use of the composition according to claim 34 in an amount of 0.001 to 0.1% by weight. 36. Use of the composition according to claim 14 to 21 as an additive to foods containing sugar as the main ingredient, such as jam, syrup, sugar candy. 37. Application according to claim 36, characterized in that it is identified indicates that the additive is used in an amount of 0.001 to 1% by weight. 809825/084 3