WO2002000027A1

WO2002000027A1 - Use of azoxystrobin for preservation of foodstuffs

Info

Publication number: WO2002000027A1
Application number: PCT/EP2001/006979
Authority: WO
Inventors: Alfredo Martini; Piergiorgio Gobbi
Original assignee: Individual
Current assignee: Individual
Priority date: 2000-06-28
Filing date: 2001-06-20
Publication date: 2002-01-03
Anticipated expiration: 2002-12-28
Also published as: IT1318599B1; EP1294233A1; AU2001285767A1; ITMI20001447A0; ITMI20001447A1

Abstract

Use of azoxystrobin and/or its derivatives as anti-mould agents for preservation of foodstuffs, in particular of cheese and salami, to be applied, in appropriate formulations, on the surface of alimentary products. The duration of action of azoxystrobin and its derivatives on the foods is sufficient for their good preservation. Azoxystrobin is active at low doses, which applied to the external surfaces of foodstuff result in concentrations on internal edible parts comparable to the amounts admitted as residuals in the food, due to its use as a phytosanitary product. It neither alters the organoleptic characteristics of the treated food, nor interferes with the maturation processes of cheese and salami.

Description

USE OF AZOXYSTROBIN FOR PRESERVATION OF FOODSTUFFS Technical Field

The present invention concerns the use of azoxystrobin for preservation of foodstuffs and/or of its derivatives, as anti-mould agents, for the preservation of alimentary products, particularly of cheese and salami.

Moreover, this invention refers to formulations for food preservation that include azoxystrobin and/or its derivatives, and a polymeric agent forming an insoluble water film to assure adhesion and an uniform distribution of the active substance on the surface of the treated food. Background art

The control of mould growth on food is an important economical and sanitary problem. From the economical point of view it is sufficient to mention that every year a huge amount of foodstuff must be disposed due to the spoilage by moulds. As far as it concerns the sanitary aspects, it is necessary point out that the presence of moulds may be a serious health threat, even when the mould growth is partial, or the presence of moulds is scarcely evident on an approximate examination. In fact, it is well known, that many of the moulds, that attack organic substances, and particularly the alimentary goods, may produce very toxic mycotoxins, harmful for the health, even when assumed in very small doses. It is also known, that many mycotoxins (e.g. aflatoxins) are potent carcinogens, that may cause the onset of malignant tumours (e.g. hepatocarcinoma), even after many years. The threat for human health, represented by aflatoxins, appears particularly dangerous, when we consider, that such substances (like for many other carcinogenic and/or mutagenic compounds and physical agents) do not have a threshold dose: i.e. a dose below which the neoplastic risk reduces to zero.

In order to restrain mould growth on the alimentary products, to improve food preservation and to restrict the risk deriving from mycotoxins, those alimentary products, that are particularly exposed to mould attack, are treated with preservative agents, endowed with an anti-mould activity. At present very few substances are recognised as active and safe to be admitted for control of moulds spread on foodstuff without becoming risky for consumer health. The difficulties associated with the very costly research for such substances have determined, that new anti-mould molecules, useful for food preservation, did not appear for more than 30 years. The large use of the admitted anti-mycotic products and the continuous exposure of the moulds to an extremely limited number of active ingredients rise the risk of a progressive development of mould resistance mechanisms towards these anti-fungal agents. During the last years, therefore, new strains of fungi appeared less and less sensitive to the available treatments, and this increased the need to use progressively growing doses to overcome mould attacks.

There exists a great concern, that the progressive selection of more and more resistant fungal strains, might lead to development of strains endowed with an absolute resistance to the anti-fungal agents admitted for alimentary use, or, might anyway increase the levels of resistance requiring doses higher than accepted and/or considered safe for the consumers.

It appears evident from the above statements, that there is a need to identify, develop and introduce novel molecules with anti-mould activity, useful for food treatment without any cross resistance with the currently available anti-mould products; the high economical and social importance of such possible technological progress is obvious.

Azoxystrobin, CAS number: 131860-33-8, IUPAC: methyl-(E)-(2-{2[6- (2-cyanophenoxy)-pyrimidin-4-iloxy]-phenyl}-3-methoxyacrylate is a phytosanitary product with fungicide activity belonging to the class of the strobilurines and is disclosed, together with numerous other compounds in EP-B-0 382375.

Azoxystrobin is broadly applied for treatment of plants and trees in agriculture (grapevine, tobacco, forages, fruit trees, decorative plants, etc.) to control pathology of mycotic origin. It is listed in the Annex I of the European Union Directive 91/ 414/ concerning the commercial application of the phytosanitary products.

The molecule of azoxystrobin is rather labile in the ordinary conditions of use, being rapidly degraded by light and easily metabolised by the microbial flora. Its half -life in the soil is of about 2 weeks.

Once applied to the plants, azoxystrobin mainly acts as a surface product; however, it is partly absorbed by the leaves (cytotropic and translaminar motion) and transported across the lymphatic vessels of the xylem into the vegetation of novel formation and, in a relevant measure, this systemic activity contributes to its effectiveness.

In fact, a part of the product, absorbed by the plants, persists in active form for a long time, because it is not washed away and photodegraded, but it acts on the plant compartments not easily available to the treatment by atomizer. The lability of the product and the limits, deriving from the requirement to use the product at accepted concentrations, considered safe both for the different crops treated, and for the environment, leads to treatment repetition at intervals of about 10-12 days, in order to get the best control of pathogenic fungi, particularly in the cases of peronospora and grapevine oidium. Treatments must be repeated at short intervals even if, as it has been mentioned above, the activity of azoxystrobin becomes more effective and protracted, due to the systemic activity after absorption of the molecule by the plants.

As it results from EP-B-O 382 375, azoxystrobin and the other chemically correlated compounds, disclosed in the patent too, are active against many types of fungi pathogenic for the plants. However, there is no indication concerning the possible activity of azoxystrobin against those particular moulds and yeasts that more frequently grow on cheese, salami and alimentary goods and, in general, put at risk their preservation. As example of such saprophytic fungi the following species may be quoted: Absidia, Aspergillus, Cladosporium, Geotricum, Monilia, Mucor,

Saccharomyces, Rhizopus, and Wallemia.

Up to now, due to the above mentioned characteristics and, in particular, due to its short half-life on surfaces after application and to its systemic activity on the treated vegetation, azoxystrobin has been used only in agriculture in the treatment of mycotic pathologies of the plants during the various phases of the vegetative cycle.

The use of azoxystrobin as an anti-mould preservative, intended to protract the duration of products for human and/or animal nutrition, was not deemed feasible up to now, because its anti-fungal activity for such use is not known, and also because the nutrition products are generally treated with anti-mould substances or other preservatives only once during their production and/or packaging and the duration of such protection must be quite long (often of several months). Disclosure of the Invention

It has been unexpectedly found, that azoxystrobin and/or derivatives as hereinafter specified, suitably formulated, can be used as appropriate anti- mould products for the preservation of foodstuffs and particularly of cheese and salami. In fact, it has been observed, that the protective activity of azoxystrobin on food continues much longer than expected, considering the relatively easy decomposition of this molecule. The same observation is valid for the derivatives of azoxystrobin.

The doses of azoxystrobin, found as optimal for an effective anti-mould protection of the foodstuffs for an adequately long period of food preservation, are sufficiently low and do not change the organoleptic characteristics of the treated food, being compatible with the consumer's health protection.

Studies on animals have demonstrated that azoxystrobin is scarcely toxic after oral ingestion (acute DL₅₀ in the mouse is higher than 5000 mg/kg of body weight; no toxic effects were evidenced in the rat at the dose of 18 mg/kg of body weight per day, administered for indefinite time). In addition, azoxystrobin is neither mutagenic, nor carcinogenic and does not have reproductive effects.

Azoxystrobin does not accumulate in the organisms when repeatedly assumed over time, nor it accumulate when introduced in the alimentary chain by the ingestion of contaminated plants.

Azoxystrobin is applied in water suspension to the surface of the alimentary products, using adjuvants such as emulsifiers, thickeners and film forming agents for alimentary use. The techniques of application could be of various types: they include, for example, dipping of the treated product into a bath containing the active substance, or its application by spray or by smearing.

Due to the scarce water solubility of azoxystrobin and of its derivatives, and, in order to use them efficiently, extending the duration of perishable foods and protecting them from mould attacks, it is necessary to apply a water suspension of azoxystrobin and/ or of its derivatives on the food surface together with film forming polymeric agents and possibly with emulsifiers and/or the thickeners, in order to obtain water insoluble films and to warrant a good adherence and an uniform distribution of the active substance on the surface of the treated products. Polivinylacetate and ethylene co-polymers with vinyl monomers like vinyl acetate, acrylic acid and vinyl alcohol are particularly suitable as film forming agents.

Polivinylacetate and co-polymers of vinyl-ethylene-acetate are the preferred filmogenic agents. The useful suspending and thickening agents are of known type and include, for instance, carboxymethylcellulose, polyvinylpyrrolidone and gums like xantan and gellan.

The thickeners, suspending and filmogenic agents are used in concentrations ranging from 0.01 to 900 g/1. The formulations could also include, for maintenance of these formulations, suitable antibacterial products that are chosen, for example, among sodium chloride, potassium chloride, benzoic acid and its salts, para- hydroxybenzoates, in concentrations ranging between 0.01 and 900 g/1. There could be present also anti-mycotic acids, for instance benzoic acid, propionic acid, sorbic acid or their salts in concentrations between 0.01 and 300 g/1, or antibiotic polyenes, preferably natamycin, in concentrations between 0,001 and 30 g/1.

Azoxystrobin and/ or its derivatives could be used in mixture with one or more compounds of the strobilurine group. The concentration of azoxystrobin and/ or its derivatives in water suspensions is generally between 0,001 and 30 g/1.

It has been verified, that dipping of the tough-pulp cheese into water emulsions of vinyl-ethylene-acetate co-polymer containing azoxystrobin, can preserve the cheese from getting mouldy and to warrant its good storage for all the time required for its ripening and marketing (example 1).

Azoxystrobin, at the used doses, does not change the organoleptic characteristics of the treated cheese; in particular, there is no alteration of the taste, flavour, and colour or of cheese texture. Moreover, azoxystrobin does not interfere with the process of cheese maturation and neither affects its duration, nor its final result.

In comparative tests, azoxystrobin has shown to be active, at equal doses, at least as natamycin (or pimaricin, CAS 7681-93-8) - the only available product for treatment of cheese with prolonged maturation - (examples 2 and 3); azoxystrobin was also better than the salts of the sorbic acid (example 2). The increase of vinyl acetate co-polymer concentration in the dipping bath increases the anti-mycotic activity of azoxystrobin, which becomes fully active at 0,025% concentration (example 4). The same outcome can be achieved using a more complex dipping bath, associating the vinyl acetate co-polymer with polyvinylpyrrolidone and carboxymethylcellulose (example 5). However, the same concentration of azoxystrobin (0.025%), just described in example 1, was not equally effective.

Azoxystrobin has proven to be a product of sure interest for the preservation of salami, too. Azoxystrobin, used as an anti-mould treatment for preservation of the" Neapolitan type" sausage is equivalent to natamycin, both when the two products are used as single agent, and/or when they are used in association with calcium sorbate (example 6). It seems to be of importance to stress that the association of natamycin with calcium sorbate, utilized as reference in examples 6, is considered by experts in this field as being the optimal formulation to control mould growth on sausages. The useful derivatives of azoxystrobin have the same basic azoxystrobin structure, except for the cyano group in the phenoxy structure, bound to the pyrimidine ring, which is substituted by a hydrogen, chlorine or fluorine atom or by a trifluormethyl, thiocarbamoyl, nitro, alkyl or isoalkyl group (with 1- 4 carbon atoms). Moreover, the phenyl group bound in position 2 of the methoxymethylacrylate is substituted by an atom of chlorine or fluorine, or by a methyl, methoxy, nitro or cyano group. All compounds indicated above are endowed with a phytosanitary pesticide activity. Compounds of this type are disclosed in EP-B-382375, the description of which regarding the methods for their preparation is herewith enclosed by reference . Quantitative determination of azoxystrobin by GC-MS on the rind and at different depth of tough-pulp cheese, after a surface treatment with a suspension of azoxystrobin 0.25%, demonstrated that the molecule scarcely penetrate cheese (pulp to rind ratio being in the order of 1 to 1000).

Azoxystrobin concentration in the internal edible part of cheese resulted always less than 0.2 ppm (0.2 mg/kg). Accordingly, since the Commission of the European Communities recognized as acceptable the amount of 0.1 mg/kg body weight per day of this phytotherapeutic product, a person weighing 50 kg could consume each day, life-long, up to 25 kg of cheese, without exceeding the acceptable daily intake (ADI) of azoxystrobin. The following examples are provided as explanatory, but are not limiting the invention.

EXAMPLE 1.

Fresh "provoletta" cheeses of about 1 kg, just taken out of the pickle, have been dipped in baths containing vinyl acetate co-polymer diluted in water (concentration 50 g/1) and azoxystrobin in different concentrations: 0% (control with co-polymer, label MIC0), 0.025% (label MIC1), 0.1% (label MIC2) and 0.25% (label MIC3). One "provoletta" cheese was dipped in each bath. The "provoletta" cheeses, hanging on the fastening pack-thread, have been then left to dry in the ripening room, at controlled temperature and humidity (the relative humidity was between 80% and 85% and the temperature was between 10 °C and 15 °C) and checked for mould presence at 30 day intervals, during the 3 months used for ripening of this type of cheese.

For comparison, one "provoletta" cheese was kept in the same experimental conditions, but without any surface treatment

The results are reported in Table I.

Table 1. Mould development on the cheese surface

EXAMPLE 2

The fresh "provoletta" cheeses of about 1 kg, just taken out of the pickle, have been dipped into a bath of vinyl acetate co-polymer diluted in water (concentration 50 g/1) and azoxystrobin in different concentrations: 0% (control with co-polymer only, labelled X), 0.1% (labelled A) and 0.25% (labelled C) or, for comparison, in anti-mould products widely used in keeping cheese like natamycin (E235) at the concentration of the 0.1% (labelled B), or calcium sorbate (E203) at 3.0 % (labelled F). One "provoletta" cheese was dipped in each bath.

The "provoletta" cheeses, hanging on the fastening pack-thread, have been dried in the ripening room, at controlled temperature and humidity (the relative humidity was between 80% and 85% and the temperature was between 10 °C and 15 °C) and checked for mould presence at 30 day intervals during the 3 months needed for ripening of this type of cheese. For comparison, one "provoletta" cheese was kept in the same experimental conditions, without any surface treatment.

The results are shown in Table 2.

Table 2- Mould development on the cheese surface.

EXAMPLE 3

Fresh "provoletta" cheeses of about 1 kg, just taken out of the pickle, have been dipped in bath containing vinyl acetate co-polymer diluted in water (450 g/1) (labelled I) and azoxystrobin at the 0.1% concentration was added (label H) or, natamycin (E235) also at the 0.1% concentration was used for comparison (labelled G). One "provoletta" cheese was dipped in each bath.

The "provoletta" cheeses, hanging on the fastening pack-thread, have been kept in the ripening room, at controlled temperature and humidity (the relative humidity was between 80% and 85% and the temperature was between 10°C and 15°C) and checked for mould presence at 30 day intervals during the 3 months, adopted for ripening of this cheese type.

For comparison, one provoletta cheese was kept in the same experimental conditions, however, without any surface treatment.

The results are given in Table 3.

Table 3. Mould development on the cheese surface.

EXAMPLE 4 Fresh "provola" cheeses of about 10 kg, just taken out of the pickle, have been dipped in prepared bath of vinyl acetate co-polymer in water (at concentration 450 g/1) and azoxystrobin in different concentrations: 0% (control containing only the co-polymer, labelled FLO), 0,025% (labelled FLl), 0.1% (labelled FL2) and 0.25% (labelled FL3). One "provola" cheese was dipped in each bath.

The "provola" cheeses, hanging on the fastening pack-thread, have been dried in the ripening room, at controlled temperature and humidity (the relative humidity was between 80% and 85% and the temperature was between 10 °C and 15 °C) and checked for mould presence at 30 day intervals during the 3 months adopted for ripening of this type of cheese. For comparison, one "provola" cheese was kept in the same experimental conditions, without any surface treatment

The results are shown in Table 4.

Table 4- Mould development on the cheese surface.

EXAMPLE 5

Fresh "provola" cheeses of about 10 kg, just taken out of the pickle, have been dipped in bath of vinyl acetate co-polymer in water (at concentration 300 gΛ), polyvinylpyrrolidone (at 8 g/1), carboxymethylcellulose (5 g/1) and azoxystrobin in different concentrations: 0% (control with only co-polymer, labelled F00), 0,025% (labelled F01), 0.1% (labelled F02) and 0.25% (labelled F03). One "provola" cheese was dipped in each bath.

The "provola" cheeses, hanging on the fastening pack-thread, have been left to dry in the ripening room, at controlled temperature and humidity (the relative humidity was between 80% and 85% and the temperature was between 10 °C and 15 °C) and checked for mould presence at 30 day intervals during the 3 months adopted for ripening of this type of cheese. For comparison, one "provola" cheese was kept in the same experimental conditions, but without any treatment of surface.

The results are shown in Table 5.

Table 5. Mould development on the cheese surface.

EXAMPLE 6

Fresh sausages of Neapolitan type, immediately after their preparation, have been dipped in bath containing a water solution of xantan gum (0,025%), azoxystrobin in different concentrations (0.0625%, 0,125% and 0.25%) or natamycin (0,125%) or calcium sorbitol (5%) or natamycin (0,125%) with calcium sorbitol (5%) or azoxystrobin (0,125%) with calcium sorbitol (5%). One sausage has been dipped in each bath.

The sausages have been dried in the maturation room, at monitored humidity and temperature and the presence of moulds was checked daily for 15 days, sufficient for the maturation of this type of product. One sausage, without any surface treatment, has been kept for comparison in the same experimental conditions. The results are reported in Table 6.

Table 6. Mould development on the sausage surface.

The disclosures in Italian- Patent Application No. MI2000A001447 from which this application claims priority are incorporated herein by reference.

Claims

1. Use of methyl (E)-2{-2[-6-(2-cyanophenoxy)-pyrimidin-4-

.1 iloxyphenyl]}-3- methoxyacrylate (azoxystrobin) and/or of its derivatives, in which the phenoxy group, bound to the pyrimidine ring is substituted in position 2 by atoms of hydrogen, chlorine, fluorine, or by trifluormethyl-, thiocabamoyl-, or nitro-, or alkyl- or isoalkyl- groups, or with groups containing 1- 4 carbon atoms and the phenyl group, bound at the position 2 of methylmethoxyacrylate substituted with atoms of chlorine or fluorine or with methyl-, nitro- or cyano- groups, as anti-mould products for surface treatment aimed at preservation of alimentary products.

2. The utilisation, according to the claim 1, wherein the alimentary products are selected from the group consisting of various types of cheese and salami.

3. The use, according to the claims 1 and 2, wherein azoxystrobin and/or its derivatives are applied in water suspension.

4. Azoxystrobin formulations in water suspensions, that include filmogenic polymer agents for alimentary use forming water insoluble films and guarantee adhesion and uniform distribution of the active substance on the surface of the treated products.

5. Formulations according to the claim 4, in which the filmogenic polymer agents are selected from the group consisting of polyvinyl acetate and ethylene-vinyl acetate co-polymers.

6. Formulations according to any one of claims 4 and 5, including an emulsifier and/or a thickener.

7. Formulations according to claim 6, in which the emulsifiers and the thickeners are selected from the group consisting of carboxymethylcellulose, polyvinylpyrrolidone, gellan and xantan gums and with mixtures thereof.

8. Formulations according to any one of claims 4-7, in which the filmogenic agents, the emulsifiers and the thickeners are used in concentrations ranging between 0.01 and 900 g/1.

9. Formulations according to any one of claims 4-8, that include also antibacterial products selected from the group consisting of sodium chloride, potassium chloride, benzoic acid and its salts, para-hydroxybenzoates in concentrations between 0.01 and 900 g/1, used for preservation of the same formulations.

10. Formulations according to any one of claims 4-9, including anti- mycotic acids selected from the group consisting of benzoic acid, propionic acid, sorbic acid or their salts in concentrations between 0.01 and 300 gΛ.

11. Formulations according to any one of claims 4-10, including polyenic antibiotics in concentrations between 0,001 and 30 g/1.

12. Formulations according to any one of claims 4-11, in which azoxystrobin and/ or its derivatives are associated with one or more compounds belonging to the class of strobilurines.

13. Formulations in which azoxystrobin and/or its derivatives and the ingredients, as recited in claims 4 to 12, in the form of concentrated suspensions, powders or granules, by water addition reconstitute formulations equivalent to those recited in claims 4 to 12.