HU202762B - Process for producing compositions containing lizozime and vitamines for stock-raising utilizaiton - Google Patents

Abstract

Veterinary compsns. contain lysozyme (I) or its salts and one or more lipophilic vitamins (II) or their salts. (I) is used in hydrochloride form. (II) are selected from vitamin A, vitamin A propionate, vitamin D3, vitamin E and vitamin E acetate. The compsn. are formulated as aq. emulsions contg. propylene glycol (PG), a nonionic surfactant and opt. antioxidants and/or preservatives. USES - The compsn. are esp. useful for relieving stress in poultry, giving general improvement in health and growth.

Description

The present invention relates to a process for the preparation of lysozyme enzyme and vitamin compositions for animal husbandry.

It is known that oral administration of vitamins is now essential in large-scale animal husbandry and rearing. At the same time, the use of chemotherapeutic agents and antibiotics, which have been used extensively in recent decades, has become increasingly resistant to the use of active ingredients and professionals. Biologically active substances of natural origin (such as enzymes and the like) are increasingly being used for the natural protection of the animal body and for the prevention of disease.

One such natural environmentally friendly enzyme is lysozyme (also known as muramidase) (Biological Lexicon

II. Volume 622 / Academic Publisher, Budapest, 19757). Lysozyme consists of 129 amino acid residues, has a molecular weight of 14,600, and is a basic, water-soluble protein. Its richest source is egg white.

It is known (excerpted from the Biological Lexicon) that lysozyme is able to break down the glucoside bonds between the mucopolysaccharides that make up the cell wall of bacteria and the lysine, thereby killing the bacteria. On this basis, Japanese researchers have developed anti-inflammatory and antibacterial ointments for the treatment of superficial inflammations, which contain as active ingredient lysozyme enzyme in admixture with conventional ointment bases (lanolin, paraffin, etc.) and Japanese Patent Application No. 78.94,007. Examples of antioxidants include tocopherol and vitamin C monopalmitate. The formulations of the cited Japanese Patent Application contain a very small amount of antioxidant, 0.03 parts by weight based on the weight of lysozyme. The antioxidant is intended solely to enhance the shelf life of the ointment formulation; there is no indication in the publication that the antioxidant would in any way alter the known antibacterial activity of lysozyme.

It has been discovered that formulations containing the lysozyme enzyme and vitamin E or the vitamin E ester derivative or vitamin or vitamin C salt are very useful in animal husbandry as preventive, anti-stress and weight gain agents and in the treatment of certain bacterial infections. The use of these types of formulations is particularly important when the use of a particular, potent drug is not yet desired or warranted or prohibited (e.g., during the so-called "waiting time" before slaughter). It has also been found that in these formulations, when administered orally, vitamin E or its ester derivative or vitamin C or its salt synergistically enhances the biological activity of lysozyme.

The present invention therefore relates to a process for the preparation of a composition (preferably a feed additive) containing lysozyme and optionally vitamin C salt for use in animal husbandry, comprising lysozyme 0.4-10 wt% or a salt thereof with 0.2-10 wt% vitamin E or The vitamin E ester derivative or vitamin C or vitamin C salt is mixed with 80-99.4% by weight of an additive such as a carrier and / or diluent, optionally one or more other vitamins and optionally a or in combination with a number of excipients, preferably surfactants, antioxidants and / or preservatives, for oral use in animal husbandry, bactericidal, preventive, anti-stress and weight gaining agents.

The compositions of the present invention preferably contain lysozyme hydrochloride as the lysozyme salt

Preferably, the ester derivative of vitamin E in the compositions is vitamin E acetate, and the salt of vitamin C is, for example, an alkali metal salt of vitamin C. Vitamin E and its ester derivatives, and vitamin C and its salts synergistically enhance the favorable biological activity of the lysozyme enzyme.

The formulations of the present invention may contain other vitamins, in addition to Evitamin (or its ester derivative) or Vitamin C (or its salt), as additional active ingredients. Examples of other vitamins include: , Vitamin D3, Vitamin B2, Vitamin B2, Vitamin B2, Vitamin B2, Vitamin K, and Vitamin K3. They may conveniently be used in an amount of 0.05 to 2% by weight of the total weight of the composition.

Both solid and liquid formulations can be prepared according to the invention. Solid compositions may contain as carrier or diluent, for example, calcium carbonate, calcium phosphate, glucose, sucrose, talc and / or silica. In liquid compositions, the carrier is preferably water and / or a water-miscible non-toxic organic solvent.

A special type of liquid formulation is stable colloidal solutions containing lipophilic vitamins. In our experience, a stable colloidal solution (hydrosol) can be prepared from lysozyme enzyme (or its salt) and lipophilic vitamins (or derivatives thereof) only in a mixture of propylene glycol and water in the presence of a nonionic surfactant. In the presence of a different carrier medium or other type of surfactant, the prepared mixture will not be stable, will break in a short time, and precipitates and precipitates will be observed, which will prevent accurate weighing and hence high efficiency in the use. Preferably, 100 ml of aqueous colloidal solution may contain 0.5 to 50 g of propylene glycol and 5 to 30 g of nonionic surfactant. The nonionic surfactant is preferably Cremophor EL (polyoxyethylene glycerol tri-ricinoleate).

Preferably, the stable colloidal solutions are prepared as follows: Using half of the calculated amount of distilled water, an aqueous solution of 5-10% by weight of lysozyme hydrochloride is prepared at 75-80 'Con and the solution is sterile filtered. At the same time, the lipophilic vitamins or their derivatives are dissolved in 55-65 'Con in the presence of nonionic surfactant, propylene glycol and other auxiliaries (such as antioxidants, preservatives), and the solution is mixed with the previously prepared lysozyme hydrochloride. containing an aqueous solution. The resulting colloidal solution is filtered to remove any mechanical carbon-2 contamination, and the filtrate is filled into sterilized vials and the vials are sealed.

The compositions of the present invention exhibit significant anti-stress activity in animal husbandry, especially poultry, and are therefore more advantageous than hitherto known in the field of preventive animal health protection. The compositions of the present invention are also useful in preventing and / or treating bacterial infections in animals.

The invention will be further described without limiting the invention in the following examples.

Example 5

The following formulation is prepared as a sterile, stable colloidal solution:

Vitamin A propionate (2,500,000 IU) 1,200 g

Vitamin D3 10g

Vitamin E acetate 3000 g

Lysozyme (20,000 IU) 2000 g

Propylene glycol 20000 g

CremophorEL 15000 g

Butylhydroxytoluene (antioxidant) 500 g

Benzyl alcohol 1000 g

Selecton B2 (EDTA-Na) 1000g

Distilled water Add 100 liters of distilled water to a duplicator and, while stirring, dissolve 2000 g of lysozyme hydrochloride in the same amount as lysozyme hydrochloride and the above amount of EDTA-Na while heating the solution to 75 ° C. After reaching the desired temperature, the solution is cooled to 60 ° C. The solution is then removed from the solution by filtration to remove mechanical impurities and filtered through a 0.22 μm membrane filter or filter plug to germinate.

Weigh the lipophilic vitamins, Cremophor EL, butylhydroxytoluene and benzyl alcohol into the sterile container, heat the mixture to 60 ° C with rapid stirring, then add the 60 ° C lysozyme solution and, if necessary, propylene glycol purified from mechanical impurities. Make up to 100 liters with distilled water.

The resulting solution was filtered to remove mechanical impurities, and the filtrate was filled into sterilized vials and the vials were immediately sealed.

Example 2

All were carried out as described in Example 1, except that 2500 g of propylene glycol and 30,000 g of non-ionic Cremophor EL were weighed.

Example 3

The following powder composition is prepared

live: Vitamin A propionate 1560 yrs D3 Ilg Vitamin E acetate 3300g Cremophor 2000 yrs Lysozyme hydrochloride 2200g Selecton B2 50g AerosU200 5000g glucose ad 100000g

Glucose, Selecton B2, Aerosil 200a and lysozyme hydrochloride were weighed into a VIANI acid-proof powder mixer and the materials were thoroughly homogenized. Vitamin A propionate, vitamin D3 and vitamin E acetate are mixed separately, Cremophor EL is added and the resulting mixture is added dropwise to the powder mixture while stirring. After a further 20 minutes of mixing, the drug-bound drug mixture will turn into a well-treatable, non-sticky powder product. The powder mixture is packaged in 50 g portions in black polyethylene foil pouches. The powder mixture may be stored for at least 12 months without change.

The powder composition of the above composition is preferably used in the following doses for the treatment of farm animals:

day-old poultry: 20-40 g / 1000 livestock and adult poultry: 40-60 g / 1000 livestock other species: 8-12 g / 100 kg live weight

Example 4

Example 3 is prepared as follows.

hand powder composition: Vitamin E acetate 0.4 g Vitamin A palmitate 0.1 g D3 0.01 g K-vi tanain 0.1 g Vitamin Bi 0.2g Vitamin B2 0.4 g Bi2-vi tanain 0.2 mg Lysozyme hydrochloride 2.2 g Nicotinic acid amide 20g folic Acid LMG Biotin (vitamin H) 0.5 g Cremophor 5g Ronoxan A (an antioxidant containing 98% by weight of lecithin and 2% by weight of tocopherols) 0.02 g glucose ad 100 g Example 5 Example 3 is prepared as follows. hand powder: Evitan acetate 3.40 g Lysozyme hydrochloride (20,000 IU / mg) 2.20 g Cremophor l.OOg Aerosil 200 3.00 g RonoxanA 0,02g glucose ad 100 g Example 6 By mixing the components, a Vitamin C powder 4.00 g Lysozyme hydrochloride (20,000 IU / mg) 2.20 g Sodium citrate 4.00 g Vitamin K3 0.10 g Cremophor 0,10g Butylhydroxytoluene (antioxidant) 0.01 g glucose ad 100 g

Example 7

By mixing the components, a

-3EN 202762 A

powder composition having the following composition: Vitamin C 1.00 g Lysozyme hydrochloride 0.5 g Vitamin Bi2 0.01 mg Vitamin K3 0.1 g Sodium citrate 1.00 g Butyl hydroxy toluene 0.0 lg Cremophor 0,10g glucose ad 100 g

Example 8

Example 3 is prepared as follows.

hand powder composition: Vitamin E acetate 9.5 g Lysozyme hydrochloride 8.5 g Cremophor 3.0g Aerosil 200 10.0 g Butylated hydroxytoluene 0,05g glucose ad 100 g

Example 9

The efficacy of the composition of Example 3 was tested in broiler chickens. Four groups of 500 chickens were included in the experiment. The animals were observed for 45 days. On days 3, 17, 31 and 38 of the experimental period, the

Group I animals were treated with 3 g of the composition of Example 3 per 100 animals; the preparation was mixed with the drinking water of the animals. At the same times, a. Group IV animals with a composition similar to that of Example 3, but containing the same amount of glucose instead of lysozyme hydrochloride, were used in Example IV. and animals in Group II were treated with the same formulation as in Example 3, but with the same amount of glucose instead of vitamins, in the same manner and dose. The Π. The animals in Group II were given non-active drinking water at all times. All four groups of animals were otherwise housed under the same conditions and fed the same feed. Treatment results were evaluated at day 45. The results are reported in Table I below

Table I

Group death, % Body weight,% ^X Feed utilization,% ^x I 2.9 113.5 109.2 Π. 8.5 100.0 100.0 ΙΠ. 4.9 103.5 102.8 ARC. 5.2 105.4 104.9

X

Compared to untreated control (group Π)

From the data in Table I it can be seen that the composition of the present invention synergistically increases the weight gain of the animals.

Example 10

The 45 anti-stress activities of the formulation of Example 4 were tested in broiler chickens. The animals were observed for 4 weeks and the animals were maintained for 3 weeks during the observation period.

Treated on days 17 and 22 as follows:

Group I animals were dosed with 0.1 g / 50 kg of the composition of Example 4.

AII. Group I animals were administered 0.1 g / kg body weight of the same formulation as Example 4, but containing glucose instead of vitamin E acetate.

The ΠΙ. Group I animals were administered 0.1 g / kg body weight of the same formulation as Example 4, but containing glucose instead of lysozyme hydrochloride. 60

The formulations used for treatment were mixed with the drinking water of the animals.

AIV. Group V and Group V animals served as untreated controls and were provided with potable water at all times. 65

I-IV. group animals were housed in highly crowded, stressful conditions with 24 animals per 1 m ² . In Group V, congestion was alleviated; here only 20 animals per 1 m ^z area. The mean weight gain over 4 weeks was Π. in Table.

II. spreadsheet

Group Average body weight -gyarapodás g I 82 Π. 35 m. 22 ARC. 18 V 69

AII. It can be concluded from Table

ARC. In contrast, untreated animals in Group II undercut under stressful conditions In contrast, animals treated with the composition of the present invention (Group I) had a remarkably good body weight gain under these stress conditions, and even in the larger living space in Group V. so the essence of stress-effects also outperformed the growth of less exposed animals. The I-ΙΠ. In this case, synergistic enhancement can also be observed.

Example 11

The formulation of Examples 5 and 6 has been used very successfully in preventing and treating bacterial infections in young calves. Experiments were performed on groups of 20-20 calves under standardized conditions. The animals were dosed with 5 g / animal / week in the following compositions:

Group I is the composition of Example 5

Π. Group 6 is the composition of Example 6

ΙΠ. Group 5 is a formulation of Example 5 but containing glucose instead of Evamin acetate

TV. Group 6 is a formulation of Example 6 but containing glucose instead of Vitamin

Group V is a formulation of Example 5 but containing glucose instead of lysozyme hydrochloride

Group VL is a formulation of Example 6 but containing glucose instead of lysozyme hydrochloride

VII. group of untreated controls.

The healing rate is a. in Table.

III. spreadsheet

Group Healing rate,% I 79 Π. 76 m. 34 ARC. 37 V 31 VI. 29 VII. 22

A m. It can be seen from the data in Table II, that the formulations according to the invention have a significant antibacterial activity and a synergistic enhancement is obtained when lysozyme hydrochloride is used in combination with vitamin E and vitamin C respectively.

Claims (7)

A process for the preparation of a composition for use in animal husbandry comprising lysozyme and optionally vitamin C salt, characterized in that: 0.4-10% by weight of lysozyme or a salt thereof is mixed with 0.2-10% by weight of vitamin E or an ester derivative of vitamin E or vitamin C or a salt of vitamin C and the mixture is 80-99.4% by weight supplemented with an excipient such as a carrier and / or diluent, optionally one or more other commonly used vitamins and optionally one or more excipients, preferably a surfactant, antioxidant and / or preservative, for bactericidal, preventive, anti-stress and weight gain purposes is formulated for oral administration. (Priority: June 20, 1990) 2. A process for the preparation of a lysozyme formulation for use in animal husbandry, comprising mixing 0.4-10 wt.% Lysozyme or a salt thereof with 0.2-10 wt. 99.4% by weight of an additive such as a carrier and / or diluent, optionally one or more other commonly used vitamins and optionally one or more excipients, preferably a surfactant, antioxidant and / or preservative, for use in animal husbandry, bactericidal, preventive, anti-stress and transforming it into an orally administrable weight gaining composition. (Priority: 1988. 12.) 3. A process according to claim 2, wherein the vitamin E ester derivative is vitamin E acetate. (Priority: 1988. 12.) Process according to claim 2 or 3, characterized in that the lysozyme salt is lysozyme hydrochloride. (Priority 12/10/1988) 5. A process according to claim 2, wherein 3-4% by weight of vitamin E acetate, 2-2.5% by weight of lysozyme hydrochloride, 1-2% by weight of other vitamins, 1.5-2.5% wt% surfactant, 0.010.08 wt% preservative, and the remainder carrier (Priority: 10/1988). 12.) 6. A process according to claim 2, characterized in that 3-4% by weight of vitamin E acetate, 2-2.5% by weight of lysozyme hydrochloride, 0.5-1.5% by weight of surfactant, 0.01% -0.03% by weight of a preservative and the remainder of the carrier. (Priority 12/10/1988) The process according to claim 1, characterized in that 2-6% by weight of vitamin C, 2-2.5% by weight of lysozyme hydrochloride, 2-6% by weight of a preservative, 0.05-0.2% by weight of other vitamin, 0.05-0.2% by weight surfactant, and the remainder with carrier. (Priority: June 20, 1990)