TECHNICAL FIELD

The present invention relates to the use of chloramine-T in feedstuff as a bactericidal, antiviral and mould-inhibiting active ingredient. BACKGROUND ART

As known, feedstuff must contain specific bactericidal agents or, in general, disinfectants.

By way of example, Carbadox, Olaquindox, Nitrovin are some of the bactericidal agents commonly used for this purpose. Chloramine-T, i.e., N-chloro-4-methylbenzenesulfonamide sodium salt is a disinfectant generally used for application to floors, walls, medical apparatus, skin, wounds, and for water disinfection.

In the alimentary field, it is also used as a bactericide in milk and derivatives thereof, e.g., ice creams, cheese, eggs, in chicken- and mincemeat, in fish, etc .

It is also used on fish to treat the bacterial branchial disease (DE-

A-432488 ⁱ ) and on cows to disinfect udders before milking.

W096/16555 describes a method to disinfect food by exposing it to an electrically charged mist of disinfectants having a redox potential. EP-A-490567 describes the use of chloramine-T as stabilizer in 3- isothiazolone compositions. JP-B-47000319 and JP-B-45033737 describe the use of chloramine-T in food preservation.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 Time preceding fungal alteration, detected by calory production (self- heating dT ^β C) , in a feedstuff, containing the following concentrations

of chloramine-T : 0 , 0. 1 , 0 . 2% . ( initial water content 14 . 5% ; temperature 21 .5 °C ) .

Figure 2

Time preceding fungal alteration, detected by CO2 production in a feedstuff, containing the following concentrations of chloramine-T: 0,

0.1, 0.2%. (initial water content 14.5%; temperature 21.5°C).

DISCLOSURE OF THE INVENTION

The present invention relates to the use of chloramine-T as a bactericidal, antiviral and mould-inhibiting active principle in feedstuff for cattle, sheep, pigs, and birds.

In order to exert a suitable activity without toxicity to the animals, chloramine-T must be present in the feedstuff at a concentration comprised between 0.05% and 1% by weight.

The feedstuff according to the present invention are in the form typically foreseen for animal feeding, i.e. essentially solid and dry, thus differing from fresh food.

The present composition is especially adapted to prevent bacterial, viral or fungal contamination under the typical stockage conditions of feedstuffs, i.e contained in non-hermetic packages, silos, sacks, in proximity of highly polluted areas like animal sheds, stalls, under variable ambient temperature and humidity, and is adapted for long term storage.

The present composition is also adapted to provide no toxicity or chloramine buildup in the animal tissues; this prevents chloramine from entering the food chain.

Chloramine-T is added to feeds in the form of concentrated solid compositions, the so-called premix, or in the form of liquid or solid

additives also containing preservatives, or in the form of an aqueous solution, where the solvent is thereafter evaporated. The solid or liquid concentrated compositions are prepared by addition of chloramine-T to the other ingredients following procedures well-known in the art: e.g. by mixing the components in a solvent under stirring, or by spraying the liquid components into a rotating pan containing the solid components.

The final feedstuff is prepared from the concentrated composition by dilution with further feed and evaporation of the solvent, if present, according to procedures known in the art.

The present invention also extends to concentrated solid compositions or premix including chloramine-T as an active ingredient for the preparation of the above feedstuff. The premix composition must have a chloramine-T content such to provide a final feedstuff containing the aforesaid amounts of chloramine-T.

Examples of premix compositions are those described and prepared according to European patents EP 197188 and EP 363733; furthermore, they preferably include the following components: a) a support selected from meals, bran, or by-products thereof, gluten, sugars, proteic flours of animal or vegetable origin derived from fermentation, yeasts, wastes of industrial processes, derived from fermentation, or materials of mineral origin, such as vermiculite or calcium carbonate; b) other known preservatives, if any, such as, e.g., propionic acid, formic acid, lactic acid, and/or the salts thereof with alkali metals or alkaline earth metals.

c) non-ionic surfactants, such as propylene glycol monoesters with fatty acids used in the alimentary field (acetic acid, lactic acid, citric acid, tartaric acid, etc.); d) antioxidants such as polyvinylpyrrolidone. The decontaminant activity of chloramine-T and its effective use in feedstuffs is further described in the following experimental part. EXPERIMENTAL PART

In-vitro test of fungistatic effects of chloramine-Chloramine-T is dissolved in water at different concentrations, and is added to a sterylised colture medium. The medium is then infected with the following fungal strains, chosen among the most common feedstuff contaminants, responsible for mycosis, mycotoxicose, food intolerance. Aspergillus flaυus, Aspergillus fumigatus, Aspergillus ochraceus, Aspergillus niger , Penicillum cyclopium, Penicillum granulatum, Penicillum roqueforti , Fusarium monili forme , Byssochlamys niυea, Bhizopus nigricans, Aspergillus gr.glaucus, Cladosporium herbarum, Wallemia ic hthyophaga

Table 1: results after 8 days

Strain 1% 0.5% 0.25% 0.12% 0.1% control

A.flavus ++ ++++

A.fumigatus ++ ++++

A.ochraceus +(D +++ ++++

A.niger ++ ++++

A.glaucus +(2) +++ ++++

P.cyclopium ++ ++++

.P.granulatum ++ ++++

P.roqueforti ++ ++++

F.monilifor e ++ ++++

B.nivea +(3) +++ ++++

R.nigricans ++ ++++

C.herbarum ++ ++++

W.ichthyophaga ++ ++++

- : absence of growth +/++++: degree of growth (1): 58% compared to control (2): 23% compared to control (3): 15% compared to control

Table 2: inhibitory concentration 50% calculated at 12 days

Strain IC 50

A. flavus 1.11/1000

A. fumigatus 1.00/1000

A.ochraceus 1.43/1000

A.niger 1.00/1000

A.gr.glaucus 1.25/1000

P.cyclopium 1.25/1000

P.granulatum 1.00/1000

P.roqueforti 1.25/1000

F. oni1iforme 1.11/1000

B.nivea 1.25/1000

R.nigricans 1.43/1000

C.herbarum 1.00/1000

W.ichthyophaga 1.00/1000

As show in tables 1-2, fungistatic effects can be observed at the concentration of 0.1%. With 0.12% chloramine-T, only three strains were moderately developed compared to the controls at 8 days. Two other species appeared at 12 days. With 0.25% chloramine-T, the fungostatic effect is total.

The ICCQ range is comprised between 1 and 1.43/1000.

Similar fungostatic effects at the same concentrations could be observed when tested on natural substrate (feedstuff) as shown in figures 1-2. 2. Phannacokinetics of chloramine-T in rats after single intravenous and oral administration

One hundred and sixty male Wistar rats (Charles river Inc., Margate, Kent, UK) each weighing 180-200 g were used. The animals were housed in polycarbonate cages with sawdust bedding and were maintained in environmentally controlled rooms (22±2°C and 50±10% relative humidity) with a 12 hr light/dark cycle (light from 8:00 to 20:00 hr) . Food (aθ4 rodent diet, Panlab SL) an water were available ad l ibitum . These rats were divided into two groups of 80 animals each to receive chloramine- T given either oral or i.v.. Group 1 rats were deprived of food, but allowed water, for 12 hr before the single oral administration of chloramine-T (100 mg/Kg body weight). Group 2 rats were given a single i.v. injection of chloramine-T (30 mg/Kg body weight) into the lateral tail vein. Groups of 8 animals each where then killed by exanguination at 0.08, 0.17. 0.25, 0.50, 1.00, 2.00, 4.00, 6.00. 8.00 and 12.00 hr after dosing. Blood samples were withdrawn and collected in heparinized tubes. Plasma was separated after centrifugation and stored frozen

until analized. After oral dose, the liver, kidney, brain, lung, fat and ileum were rapidly removed at 0.08, 0.17, 0.25, 0.50, 1.00, 2.00,

4.00, 6.00, 8.00 and 12.00 hr after dosing. Each tissue sample was weighed and kept frozen until analized. The concentration of chloramine-T in plasma and tissue samples was determined using HPLC as described by Beljaars and Rondags

(J.Assoc.Off.Anal.Chem. 61(6), 1978, 1415). These concentration values were used for all pharmacokinetic determinations.

Results and discussion The dosages chosen in this experiment provide a chloramine intake similar to that obtained in animals eating normal quantities of the feedstuff disclosed in the present invention.

The kinetics of chloramine-T after 30mg/Kg i.v or 100 mg/Kg oral in rats were best described by a two-compartment open model, with a rapid elimination phase (tl/2 beta = 1.4l or I.98 hr, respectively). These values suggest a good elimination of chloramine-T in body fluids including intracellular water ( _jj / _gg V 0.23 L).

The bioavailability of chloramine-T was relatively low (F 50.64%) following oral administration. Although chloramine-T was distributed widely in the body, the time course analysis of the tissue concentrations revealed a rapid elimination half-life in all tissues investigated.

In conclusion, chloramine-T offers the possibility of effective treatment of feedstuff without displaying toxicity to the animal organism and without entering the food chain.