WO2007063043A1

WO2007063043A1 - Improved anti-microbial composition

Info

Publication number: WO2007063043A1
Application number: PCT/EP2006/068932
Authority: WO
Inventors: Jacobus Stark; Nicole Liliane Dutreux
Original assignee: Dsm Ip Assets B.V.
Priority date: 2005-11-29
Filing date: 2006-11-27
Publication date: 2007-06-07

Abstract

The present invention describes a composition comprising epsilon-poly-L-lysine and an anti-fungal agent. The composition has improved anti-microbial activity. Also covered are methods for preserving foodstuffs, agricultural products, adhesives, paints, inks, cosmetics and pharmaceutical products by applying the composition of the invention. The composition of the invention is stable and effective against a broad-spectrum of microbe.

Description

IMPROVED ANTI-MICROBIAL COMPOSITION

Field of the invention

The present invention discloses anti-microbial compositions to prevent microbial spoilage in foodstuffs, agricultural products, pharmaceutical products, adhesives, paints, inks and cosmetics.

Background of the invention

The prevention and control of microbial growth, especially bacterial growth is an important issue in the food industry, particularly with the increasing trend for fresh good tasting convenience foods.

The current challenge for food producers is to satisfy the consumer demand for good tasting high-quality food products. Despite substantial improvements in the field of food preservation and hygiene, many food products are still exposed to microbial spoilage, in particular to pathogenic bacteria and as a consequence, the number of food poisonings is still increasing. Food-borne microbial infections represent a serious threat to human health and therefore food manufacturers need to deliver safe products. Many bacterial species may contaminate and grow in foodstuffs. Some of these species are pathogenic or produce toxins causing a range of food-poisoning diseases. There is much concern about the growth of pathogenic species such as Salmonella, Campylobacter, Listeria monocytogenes and the heat resistant spore formers such as Bacillus cereυs. Also shelf life of food products are often limited by the presence of spoilage bacteria such as Pseudomonas fluorescens, Citrobacter freundi and lactic acid bacteria.

Examples of fresh food products particularly sensitive to bacterial spoilage are dairy products (e.g. cheese, cottage cheese, ricotta, cream cheese, sour cream and yogurt), meat or fish products (e.g. hamburgers, sausages and pate), (processed) eggs, (processed) fruits, (processed) vegetables or grains such as boiled or steamed rice, spaghetti and cooked vegetables. Mostly, processed foods are not eaten directly after processing thereby permitting bacteria, which survived the production process (e.g. spore formers) or bacteria introduced by post-contamination to grow. Since toxins might be present or consumption may occur without sufficient reheating of the processed foods, there is a risk of food poisoning. Furthermore, the recent trend for minimally processed foods with the intrinsic nutritional and sensory qualities of raw and fresh foods has raised a new safety risk. Milder preservation treatments proved to be successful but rely on effective hurdles i.e. cold chain and addition of natural anti-microbial. There has been extensive research conducted to develop stable and effective broad-spectrum antimicrobial compositions. However there is still an increasing need to develop economical, natural and effective food preservative systems.

Description of the invention The invention provides novel natural improved anti-microbial compositions and methods for preserving foodstuffs, agricultural products, adhesives, paints, inks, cosmetics and pharmaceutical products by applying the composition of the invention. Unexpectedly, we have found that the composition of the invention is stable, and effective against a broad- spectrum of microbe.

According to a first aspect, the invention provides a composition comprising an epsilon-poly- L-lysine and an anti-fungal agent, wherein the anti-fungal agent is selected from the group consisting of natamycin, sorbic acid, propionic acid, benzoic acid, and salts of these compounds. In this composition, the combination of the components together works synergistically and is more effective against microbial growth than each agent taken separately.

The composition of the invention may contain a further anti-fungal agent, wherein the further anti-fungal agent is selected from the group consisting of: 1. a weak acid preservative such as sorbic acid, propionic acid, benzoic acid, a p- hydroxybenzoic acid, lactic acid, citric acid, acetic acid or an alkali metal or alkali earth metal salt thereof or a polyphosphate;

2. a polyene anti-microbial compound, preferably natamycin;

3. a sulphur dioxide or a sulphite;

4. a nitrate or a nitrite;

5. a salt, preferably sodium chloride;

6. dimethyl dicarbonate; 7. a biphenyl-, a diphenyl-, an orthophenylphenol- or a thiabendazole- ;

8. an inorganic acid, such as hydrochloric acid;

9. an imidazole such as imazalil; and/or

10. an anti-microbial compound known in the art for use as a preservative for food products, crop protection or after-harvest treatment of fruits, vegetables or cereals, pharmaceutical or cosmetic products.

In this composition, the combination of these components together works synergistically such that the combination is more effective against microbial growth than each agent taken separately.

In one embodiment of the invention, the composition of the invention consists of a ε-Poly-L- lysine and an anti-fungal agent, wherein the anti-fungal agent is selected from the group consisting of natamycin, sorbic acid, propionic acid, benzoic acid, and salts of these compounds.

In the context of the invention, epsilon-poly-L-lysine is equivalent to ε-Poly-L-lysine. ε- Poly-L-lysine is a homo-poly amino acid in which the carboxyl group is linked to the ε- amino group of L-lysine, which is an essential amino acid. Epsilon-poly-L-lysine contains in general from 25 to 35 L-lysine residues. In the context of the invention, an epsilon poly-L-lysine means at least one epsilon poly-L-lysine having from 25 to 35 L-lysine residues. Epsilon-Poly-L-lysine is a stable natural fermentation compound. This compound is produced during aerobic fermentation of Streptomyces albulus. It is on the market under the brand name Save-Ory® (Chisso, Japan). The anti-bacterial activity of ε-Poly-L-lysine is based on the electrostatic absorption of ε-Poly-L-lysine to the cell surface on the basis of its poly-cationic properties. Epsilon-Poly-L-lysine is particularly effective against Gram-positive bacteria, especially of the genus Bacillus. But, in practice it is not efficient enough to prevent the growth of Gram-negative bacteria in foods. Only at very high concentrations some inhibition is observed.

In the context of the invention, natamycin refers to the anti-fungal agent also known as pimaricin. Natamycin has been used for more than 30 years to prevent outgrowth of fungi on cheeses and sausages. Natamycin is on the market under the brand name of Delvocid® or Natamax®, a powder composition containing 50% (w/w) of natamycin and 50% (w/w) of lactose. Natamycin has a MIC (Minimal Inhibition Concentration) of less than 10 ppm for most food born fungi while its solubility in water is from 30 to 50 ppm. Natamycin can easily be applied to prevent spoilage by fungi in food products. However, the low solubility of natamycin has limited its use to the surface treatment of cheese and fluids e.g. juices, lemonades, wine and yoghurt.

Salts of benzoic acid, propionic acid and ascorbic acid are well-known in the art, especially their methyl, calcium, sodium and potassium salts. In one embodiment, potassium sorbate is used.

In one embodiment, the composition of the invention comprises: ε-Poly-L-lysine preferably in an amount of 20 to 1 000 000 ppm, more preferably 100 to 300 000 ppm and natamycin preferably in an amount of 5 to 50 000 ppm, more preferably in an amount of 10 to 20 000 ppm, or potassium sorbate, preferably in an amount of 1000-500 000 ppm, more preferably in an amount of 5000 to100 000 ppm.

In another embodiment, the composition of the invention comprises: ε-Poly-L-lysine preferably in an amount of 20 to 1 000 000 ppm, more preferably

100 to 300 000 ppm and acetic acid, sodium acetate, preferably in an amount of 0.001 to 3% (w/w) each, more preferably in an amount of 0.05 to 0.3% (w/w) and/or sodium chloride preferably in an amount of 1 to 99% (w/w) and/or natamycin preferably in an amount of 5 to 50 000 ppm, more preferably in an amount of 10 to 20 000 ppm, or potassium sorbate, preferably in an amount of 1000-500 000 ppm, more preferably in an amount of 5000 to 100 000 ppm.

The composition of the invention can be used for any type of applications in food products, agricultural products, adhesives, paints, inks, cosmetic products or pharmaceutical products. The composition may be a liquid composition, a coating emulsion, a dry formulation such as a powder, a granulate or a tablet. Each constituent of the composition of the invention may be applied separately to the product to be treated. Alternatively, the composition of the invention is first prepared, subsequently added to the product to be treated. Depending on the type of product to be treated by the composition of the invention, the skilled person would know how to choose the best suited concentration for each constituent of the composition.

Preferably, a liquid composition is used in an immersion treatment or a spraying treatment. In principle, the immersion liquid may be of any kind. When an aqueous system is used, the addition of a surfactant may be of advantage, in particular for treating objects with a hydrophobic surface.

In another embodiment of the invention, the composition of the invention is a composition suitable for treating agricultural products such as fruits, vegetable, flower bulbs and seed. Preferred fruits are citrus fruit, bananas, pine apple, apple, peer, strawberry and the like. A preferred citrus fruit is orange.

In another embodiment of the invention, the composition of the invention is a pharmaceutical preparation suitable for topical application. Examples of suitable pharmaceutical preparations are lotions, creams and ointments.

In yet another embodiment of the invention, the composition of the invention is a glue, preferably a water-based or water-thinned glue, including glues based on starch or modified starch and casein.

In yet another embodiment of the invention, the composition of the invention is an ink or paint, preferably a water-based or water-thinned ink or paint. The composition may be used to protect the ink or paint itself, but can also be used to protect the surface to which the ink or paint is applied.

Therefore, in another embodiment of the invention, the composition of the invention is used in a coating. The coating may for example be applied to domestic appliances, work surfaces for domestic and industrial use, architectural and other engineering products, garden furniture and marine vessels. The composition of the invention may further comprise water and/or salt and/or any component selected from the group consisting of a solvent, a surfactant, a carrier, a thickener. A preferred carrier is fumed silica. Preferred solvents are an alcoholic solvent comprising one or more lower alcohols (C1-C4), an aqueous acid, an aqueous alkali, glacial acetic acid or mixtures of an alcoholic solvent comprising one or more lower alcohol with either an aqueous acid, an aqueous alkali or glacial acetic acid. Preferred surfactants are sodium lauryl sulphate, dioctyl sulphosuccinate, calcium chloride, or surfactants of the non-ionic types, for example those, which are known under the brand names Tween™, Span™, Brij™, and Myrj™. A thickener may be any thickener known in the art for use in food products. Preferred thickeners are hydroxypropylmethylcellulose (HPMC), and/or gums, and/or carrageenan and/or methylcellulose. Preferred gums are xanthan gum and/or gellan gum and/or arabic gum.

According to a further aspect, the invention provides a method for treating a product with the composition of the invention. The composition can be added to the product to be treated at any moment or step of processing of the product. The composition can be added to the end product before packaging, during processing or to any ingredients used to prepare the products. Each constituent of the composition of the invention can be added separately to the product to be treated. Alternatively, the composition is first prepared and subsequently added to the product to be treated. The advantage of the present invention is that it allows the production of microbiologically stable and safe products with low concentration of anti-microbials. The innovative composition can be used to preserve microbiological safety and/or stability in all kind of products. Preferably the product is a food product such as a dairy product, e.g. ice cream mixes, yoghurt, any type of cheese, e.g. cottage cheese, ricotta, cream cheese, sour cream. Other preferred food products are hot-baked flour products, spreads, margarines, sauce, dressings, any rice-containing product or any other foodstuffs distributed at ambient or chilled temperatures. Other preferred food products are beverages like fruit juice, wine. The addition of the composition of the invention to food products is furthermore expected to reduce or completely eliminate moulds, yeasts and bacterial outgrowth on the food products in the time frame comprised between the end of the processing of the product, size reduction included, and commercial sale. This time frame varies with the type of food, the distribution and sale conditions. Preferably, the product to be treated is such that its surface will be in contact with oxygen at the end of its processing and/or later on if a reduction of size occurs.

According to a further aspect of the invention, a product is provided which has been treated with a composition of the invention. In one embodiment of the invention, the product is a water-containing product for example a food product, which comprises the composition of the invention.

According to another embodiment, the product treated with the composition of the invention comprises: - ε-Poly-L-lysine preferably in an amount of 2 to 2 000 ppm, more preferably 10 to

300 ppm and natamycin preferably in an amount of 0.5 to 50 ppm, more preferably in an amount of 1 to 20 ppm, or potassium sorbate, preferably in an amount of 100-3000 ppm, more preferably in an amount of 400 to 2500 ppm.

According to yet another embodiment, the product treated with the composition of the invention comprises: ε-Poly-L-lysine preferably in an amount of 2 to 2 000 ppm, more preferably 10 to 300 ppm and acetic acid, sodium acetate, preferably in an amount of 0.001 to 3% (w/w) each, more preferably in an amount of 0.05 to 0.3% (w/w) and/or sodium chloride preferably in an amount of 1 to 99% (w/w) and/or natamycin preferably in an amount of 0.5 to 50 ppm, more preferably in an amount of 1 and 20 ppm, or potassium sorbate, preferably in an amount of 100 to 3000 ppm, more preferably in an amount of 400 to 2500 ppm.

According to a further aspect of the invention, there is provided the use of the composition of the invention to obtain a product, for example a food product, comprising the composition of the invention.

According to a further aspect of the invention, there is provided a method for treating a product, for example a food product, with the composition of the invention.

According to a further aspect, the invention relates to a method for protecting a product against microbial spoilage by applying the composition of the invention. The product may be a food product, an agricultural product, a cosmetic product, a water- based paint, a water-based ink, or a water-based glue. The composition may be applied by any suitable means, be it by mixing with, incorporation in or surface application onto the earlier mentioned products. Before, during or after the addition of the composition to the product, other ingredients such as colorants, texturals etc. may be added as well to the product.

The invention will further now be illustrated by examples, which should not be construed as limiting the scope of the invention.

EXAMPLES

Example 1 : Inhibition of Aspergillus niger by natamycin, ε-Poly-L-lysine and a combination thereof The behaviour of the mould Aspergillus niger (DSM strain 402) was studied in the presence of natamycin and ε-Poly-L-lysine. Two commercially available products were used as sources of the products tested: Delvocid (DSM; 50% natamycin) and Save-Ory PL-50™ (Chisso; 50% polylysin). The effect of these products on the growth of A. niger was tested in Potato Dextrose Broth (Difco, pH 5 [PDB]). PDB was supplemented with various levels of the products mentioned.

Subsequently, the broths were inoculated with mould spores, which had been stored frozen prior to use (final level 5x10² spores/ml). During storage in multiwell plates at 25°C, the behaviour of A. niger was studied using the microscope.

The results, presented in table 1 , indicate that the combination of low levels of natamycin and polylysin inhibit growth of A. niger in PDB, whereas the individual components did not inhibit growth of the mould.

Table 1 : Behaviour of A. niger in the presence of natamycin and/or ε-Poly-L-lysine

^a: Concentration of active components: natamycin and polylysin +: Growth observed -: No growth observed Example 2: Inhibition of Kluyveromyces marxianus by natamycin, ε-Poly-L- lysine and a combination thereof

The behaviour of the yeast Kluyveromyces marxianus (strain CBS 397) was studied in the presence of natamycin and ε-Poly-L-lysine. Two commercially available products were used as sources of the products tested: Delvocid (DSM; 50% natamycin) and Save-Ory GA-30™ (Chisso; 1.75% polylysin). The effect of these products on the growth of K. marxianus was tested in Potato Dextrose Broth (Difco, pH 5 [PDB]).

PDB was supplemented with various levels of the products mentioned. Subsequently, the broths were inoculated with yeast cells, which had been cultured overnight in PDB at 25°C, prior to use (final level 8x10² cells/ml). During storage in multiwell plates at 25°C, the behaviour of K. marxianus was studied using OD measurements (turbidity indicating growth).

The results, presented in table 2, indicate that the combination of low levels of natamycin and polylysin, individually not sufficient to inhibit growth of K. marxianus, is able to inhibit growth of the yeast.

Table 2: Behaviour of K. marxianus in the presence of natamycin and/or ε-Poly-L-lysine

: Concentration of active components: natamycin and polylysin +: Growth observed -: No growth observed

Example 3: Inhibition of Aspergillus niger by K-sorbate, ε-Poly-L-lysine and a combination thereof

The behaviour of the mould Aspergillus niger (DSM strain 402) was studied in the presence of sorbate and ε-Poly-L-lysine. Two commercially available products were used as sources of the products tested: K-sorbate (Aldrich, 99% K-sorbate) and Save- Ory GA-30™ (Chisso; 1.75% polylysin). The effect of these products on the growth of A. niger was tested in Potato Dextrose Broth (Difco, pH 5 [PDB]). PDB was supplemented with various levels of the products mentioned. Subsequently, the broths were inoculated with mould spores, which had been stored frozen prior to use (final level 5x10² spores/ml). During storage in multiwell plates at 25°C, the behaviour of A. niger was studied using the microscope.

The results, presented in table 3, indicate that the combination of low levels of K- sorbate and polylysin inhibit growth of A. niger in PDB, whereas significantly higher levels of the individual components did not inhibit growth of the mould.

Table 3: Behaviour of A. niger in the presence of K-sorbate and/or ε-Poly-L-lysine

^a: Concentration of active components: K-sorbate and polylysin +: Growth observed -: No growth observed

Example 4: Inhibition of Kluyveromyces marxianus by K-sorbate, ε-Poly-L- lysine and a combination thereof

The behaviour of the yeast Kluyveromyces marxianus (strain CBS 397) was studied in the presence of K-sorbate and ε-Poly-L-lysine. Three commercially available products were used as sources of the products tested: K-sorbate (Aldrich, 99% K- sorbate), Save-Ory PL-50™ (Chisso; 50% polylysin) and Save-Ory GA-30™ (Chisso; 1.75% polylysin). The effect of these products on the growth of K. marxianus was tested in Potato Dextrose Broth (Difco, pH 5 [PDB]).

PDB was supplemented with various levels of the products mentioned. Subsequently, the broths were inoculated with yeast cells, which had been cultured overnight in PDB at 25°C, prior to use (final level 8x10² cells/ml). During storage in multiwell plates at 25°C, the behaviour of K. marxianus was studied using OD measurements (turbidity indicating growth). The results, presented in table 4, indicate that combinations of low levels of K- sorbate and polylysin, individually not sufficient to inhibit growth of K. marxianus, are able to inhibit growth of the yeast.

Table 4: Behaviour of K. marxianus in the presence of K-sorbate and/or ε-Poly-L-lysine

: Concentration of active components: K-sorbate and polylysin +: Growth observed -: No growth observed

Claims

1. A composition comprising an epsilon-poly-L-lysine and an anti-fungal agent selected form the group consisting of natamycin, sorbic acid, propionic acid, benzoic acid, and salts of these compounds.

2. A composition according to claim 1 , wherein the anti-fungal agent is natamycin, sorbic acid, or a salt of one of these compounds.

3. A composition according to claim 1 or 2, wherein the anti-fungal agent is natamycin or potassium sorbate.

4. A composition according to claim 1- 3 comprising (i) 20 to 1 000 000 ppm ε-poly- L-lysine and (ii) 5 to 50 000 ppm natamycin or 1000 to 500 000 ppm potassium sorbate.

5. A composition according to claim 1- 3 comprising (i) 2 to 2000 ppm ε-poly-L- lysine and (ii) 0.5 to 50 ppm natamycin or 100 to 3000 ppm potassium sorbate.

6. A method for treating a product which method comprises applying a composition according to any of claims 1- 5 to the product.

7. A product treated with the composition according to claims 1 - 5.

8. A product according to claim 7 wherein the product is a food product, an agricultural product, a cosmetic product, a water-based or water-thinned paint, ink or glue.

9. A method for protecting a product against microbial spoilage, the method comprising applying a composition according to claims 1 - 5 to the product.

10. The method according to claim 9 wherein the product is a food product, an agricultural product, a cosmetic product, a water-based or water-thinned paint, ink or glue.