WO2009080563A1

WO2009080563A1 - Use of cationic surfactants against fish parasites

Info

Publication number: WO2009080563A1
Application number: PCT/EP2008/067425
Authority: WO
Inventors: Xavier Rocabayera Bonvila; Sergi Figueras Roca; Roger Segret Pons; Eva Piera Eroles
Original assignee: Laboratorios Miret, S.A.
Priority date: 2007-12-21
Filing date: 2008-12-12
Publication date: 2009-07-02
Also published as: AR069424A1; PE20091097A1; CL2008003859A1; UY31528A1

Abstract

Cationic surfactants derived from the condensation of fatty acids and esterified dibasic amino acids, such as from lauric acid and arginine, in particular the ethyl ester of the lauramide of the arginine monohydrochloride (LAE), may be used for the treatment of fish which is infected with parasites. The cationic surfactants of this type are effective against the larvae of parasites such as Anisakis.

Description

USE OF CATIONIC SURFACTANTS AGAINST FISH PARASITES.

The present application relates to a novel use of cationic surfactants.

Cationic surfactants are known as preservatives used in food, cosmetic and pharmaceutical industry. Cationic surfactants have turned out to be highly effective against microbial proliferation and at the same time safe for intake in humans and mammals in general. For all of this, cationic surfactants are an attractive tool in the industry.

!t has been demonstrated that cationic surfactants according to formula (1 ) derived from the condensation of fatty acids and esterified dibasic amino acids are highly effective protective substances against microorganisms.

©

where:

X^" js, a counter ion derived from an organic or inorganic acid, preferably Br^', Cl^" or HSO₄ ^", or an anion on the basis of a phenolic compound;

Ri: is a straight aikyl chain from a saturated fatty acid or hydroxyl acid having from 8 to 14 atoms linked to the α-amino acid group via an amidic bond;

R₂: is a straight or branched alkyl chain from 1 to 18 carbon atoms or an aromatic group; R₃: is — NH,

or

where n is from 0 to 4.

The organic acids which may be the source of the counter ion X^" can be citric acid, lactic acid, acetic acid, fumaric acid, maleic acid, gluconic acid, propionic acid, sorbic acid, benzoic acid, carbonic acid, glutamic acid or other amino acids, lauric acid and fatty acids such as oleic acid and linoleic acid, whereas the inorganic acids can be phosphoric acid, nitric acid and thiocyanic acid.

The phenolic compound which may be the basis of the anion X^" is for instance butylated hydroxyanisole (BHA) and the related butylated hydroxytoluene, tertiary butyl hydroquinone and parabens such as methylparaben, ethylparaben, propylparaben and butylparaben.

The most preferred compound of the above class of compounds is the ethyl ester of the lauramide of the arginine monohydrochloride, hereafter referred to as LAE (CAS No. 60372-77-2). This compound is now well-known for its use as an antimicrobial agent. In practical use LAE turned out to be well tolerated and to display a very low toxicity to human beings. LAE has the chemical structure of formula (2) displayed hereafter.

The compound LAE is remarkable for its activity against different microorganisms, like bacteria, moulds and yeasts which can be present in food products (WO 03/034842) and also in cosmetic formulations and preparations (WO 03/013453, WO 03/013454 and WO 03/043593).

The general preparation of the cationic surfactants is described in Spanish patent ES 512643 and international patent applications WO 96/21642, WO 01/94292 and WO 03/064669.

LAE, also known as lauric arginate, is manufactured by Laboratorios Miret, S.A. (LAMiRSA, Spain). Lauric arginate is listed by the FDA (Food and Drug Administration) as being a GRAS substance (Generally Recognized As Safe) under GRN 000164. The USDA (United States Department of Agriculture) has approved its use in meat and poultry products (FSIS Directive 7120.1 ). The metabolism of the above cationic surfactant of formula (2) in rats has been studied, these studies have shown a fast absorption and metabolisation into naturally-occurring amino acids and the fatty acid lauric acid, which are eventually excreted as carbon dioxide and urea. Toxicological studies have demonstrated, that LAE is completely harmless to animals and humans.

Therefore, LAE and related compounds are particularly suitable to be used in the preservation of all perishable food products. LAE and related 0 compounds are equally suitable for use in cosmetic products.

As has been remarked above, the cationic surfactants are remarkable for their inhibitory action over the proliferation of different microorganisms, such as bacteria, fungi and yeasts. The minimum inhibitory concentrations of 5 LAE are shown in the following table 1.

Table 1

It is preferred to dissolve the compound directly before use in one of the following preferred solvents of food grade: water, ethanol, propylene glycol, isopropyl alcohol, other glycols, mixtures of glycols and mixtures of glycols and water, diacetin, triacetin, glycerol, sorbitol, mannitol and xylitol. If the treatment shall be performed at a specific pH value the use of a corresponding buffer solution may be recommendable. On the other hand the compound can be easily used in its solid form.

For the cationic surfactants of the above formula (1 ) the antibacterial 0 activity and the biological activity against other microorganisms such as fungi and yeasts is well documented. Fish parasites are a major problem for the health of humans and marine mammals which are consuming infected fish. The parasites which are of most concern are those of the Anisakidae family, which includes the genus Anisakis, Phocanamea or Pseudoterranova and Contracaecum. Anisakiasis is the disease caused by infection with Anisakis worms. The disease is frequently reported in areas of the world where fish is consumed raw, lightly pickled or salted. The areas of highest prevalence are Scandinavia (from eating cod livers), Japan (after eating sushi and sashimi), the Netherlands (by eating infected fermented herrings), and along the Pacific coast of South America (from eating ceviche). Heating to 60 ⁰C, or freezing to below -20 ⁰C has been reported to be an effective method of killing Anisakis. Within hours after ingestion of infective larvae, violent abdominal pain, nausea, and vomiting may occur. Occasionally the larvae are coughed up. If the larvae pass into the bowel, a severe eosinophilic granulomatous response may also occur 1 to 2 weeks following infection, causing symptoms mimicking Crohn's disease.

Diagnosis can be made by gastroscopic examination during which the 2 cm larvae are visualized and removed, or by histopathologic examination of tissue removed at biopsy or during surgery.

Anisakiasis is a serious public health concern and an economic problem for the fish-processing industry (Adams et al., Revue Scientifique et Terchnique de I' Office International des Epizooties, 1997; 16: 652-660). In Spain, the consumption of marinated anchovies is the main route of transmission of Anisakis spp. from fish to humans. This traditional dish is widely consumed by millions of Spaniards and visitors to Spain, creating a public health problem of considerable importance. The traditional marinating process requires that the fish be left in a solution of commercial vinegar and salt, usually for less than 24 h. Under these conditions, however, Anisakis larvae are not killed. Current European regulations (Regulation 853/2004) require the freezing of this type of products at -20⁰C during at least 24 hours. Nevertheless, freezing of anchovies before marinating has disadvantages because the high fat content makes difficult a suitable freezing and especially the thawing process modifies their texture, aroma and flavour, inducing a reduction of the quality of the product. In Europe, as in Germany and Denmark, elaboration of marinated products concretely fillets of herring are processed in acetic acid solution at 5-7%, but require long treatment times to destroy Anisakis larvae (5 or 6 weeks). Nevertheless, these marinades have traditionally involved the use of hydrogen peroxide, this product is very effective, but it is forbidden by the European Union. Other inactivation processes have been applied with great effectiveness, like high pressure, although mainly for economic reasons, its industrial use is limited.

No treatment has turned out to represent an ideal solution for the treatment of Anisakiasis. There is in the fish industry a persistent need for improved methods to avoid the occurrence of Anisakiasis.

It is an object of the present invention to provide a further method for the treatment of fish, in order to kill the parasites which are present in the fish before its consumption by humans.

It has been the surprising result of investigations performed by the present inventors that the cationic surfactants according to the above formula (12) display a biological activity against larvae in fish. An activity of the cationic preservatives against parasites in fish had not been described before.

The activity of the cationic surfactants is observed against larvae of Anisakis, but the wanted biological activity is also observed on larvae of Cod or Seal Worm Pseudoterranova (Phocanema, Terranova) clecipiens, Contracaecum spp. and Hysterothylacium (Thynnascaris) spp. The cationic surfactant which is used in the present invention is derived from the condensation of fatty acids and esterified dibasic amino acids, having the above formula (1 ), the most preferred species of the cationic surfactants of formula (1 ) being the ethyl ester of lauric arginate of above formula (2).

The cationic surfactants may be applied to the fish as a bath of water, as a spray, in a bath of ice or in a solid form. The cationic surfactants may be administered most conveniently as a solution in a suitable solvent, but it is also possible according to a further preferred embodiment to perform the treatment of the fish through application of the solid form.

The most preferred cationic surfactant in the context of the present invention is LAE. The further preferred embodiments apart from LAE are provided by changing the anion (other than CT), forming mixed micelles, constituting liposome, encapsulated in a form that would release LAE at high temperatures or by friction, or encapsulated with beta-cyclodextrines. Microencapsulated and nanoencapsulated forms are also possible.

Application of the cationic surfactant of formula (1 ), more in particular of

LAE as the preferred embodiment, should lead to a concentration in the treated fish of a level which is sufficient to achieve the wanted biological action. Such a sufficient level of concentration would be expected in the range of 2 to 20,000 ppm, more preferred 100 to 10,000 ppm and even more preferred 200 to 2,000 ppm, containing the cationic surfactant of the above formula (1 ), according to the preferred embodiment containing LAE. These concentrations are given in terms of the final concentration of the cationic surfactant in the fish.

If surfaces are treated with a preparation of the cationic surfactant of formula (1 ), the amount which is applied shall be such that the amount of the cationic surfactant of formula (1 ) is in the range of 0.01 to 10,000 mg/dm², preferably an amount of 0.5 to 5,000 mg/dm², and more preferably an amount of 1 to 100 mg/dm². Concentrations in the higher range are required if the parasites are present within the muscular tissue, the need to achieve a certain minimum concentration in the tissue leads to more severe treatment conditions.

When the cationic surfactants are applied as a solution, the liquid basis of the solution may be any liquid which is suitable for use in the preparation of food. Such liquids are water, propylene glycol, ethano!, or glycerine. Mixtures of these liquids are possible as well.

Water may refer to tap water, demineraiised water, distilled water, or solutions of any suitable salt in water.

Solutions in aqueous solutions are preferred. As the vehicle for the solution, water, such as tap water or demineraiised water, is the most suitable, solutions in brine are also possible.

Addition of further solvents are possible, such as any organic solvent, as long as this further added solvent does not cause any negative effect on later consumption by human consumers. In general, there is no specific advantage in adding further solvents and the administration of a solution in tap water is sufficient for usual purposes.

For the wanted effect on the larvae of the parasites a sufficient concentration of the cationic surfactant needs to be achieved. It has been observed that such sufficient concentration is achieved when the solution contains the cationic surfactant in a concentration of 0.001 to 50 % by weight. A more preferred concentration is in the range of 0.1 to 2.5 % by weight and the most preferable concentration in the range of 0.1 to 1.5 % by weight.

The usual concentration in a bath with a solution which is preferably on the basis of water is 1 % by weight. The solution of cationic surfactant may contain an additional compound that is a buffering agent in order to guarantee the stability of the solution between the pH 1-9, although the preferred pH range is between 5-7.

For the purpose of achieving a result on the larvae quickly after the initiation of the treatment, it is a preferred embodiment of the present invention to combine the cationic surfactant, the preferred one is LAE (formula 2), with acetic acid. Solutions of acetic acid in water or brine are most effective in the concentration range of the acetic acid of 10% by weight (w/w) to 40% by weight, for practical reasons concentrations of 10% by weight and 20 % by weight are regularly used. In a combination of the cationic surfactant with acetic acid, the acetic acid is preferably present in the concentration range of

2% by weight to 25 % by weight, preferably 4% by weight to 15 % by weight, more preferably 6% by weight to 12% by weight.

Other preferred embodiment of the present invention is the combination of the cationic surfactant, the preferred one is LAE (formula 2), with a saline solution. The most effective concentration of the saline solution of NaCI combined with LAE is in the range of 5-15%. It has been observed that this combination is highly effective against the larvae than using only the cationic surfactant or the saline solution alone.

Another preferred embodiment of the present invention is the combination of the cationic surfactant with the solution of acetic acid along with the saline solution of NaCI, This combination results in an improved synergic effect against the larvae.

Further compound of these combinations is the presence of surfactants with a high HLB (i.e.: hydrophil/lipophilic (hydrophobe) balance) such as polysorbates, the preferred one is Tween 20, in order to avoid the formation of precipitates. Further additional components may be preservatives, antioxidants, surfactants, thickeners, enzymatic inhibitors, organic and inorganic salts, organic and inorganic acids, liquid and solid carriers. Their addition depends on the circumstances, such as the products to be treated and the degree of contamination.

The required duration of the treatment varies due to the concentration of the cationic surfactant which is used. The preferred cationic surfactant is ethyl ester of the lauramide of the arginine monohydrochloride, with this preferred species a treatment period of between 12 hours up to 6 days is usually sufficient to achieve the finally wanted result. A treatment during 12 hours yields a good efficacy effect but the preferred one is the treatment for 1 day, which is usually long enough to achieve the wanted reduction of the number of larvae, whereas treatment for more than 6 days is not economic, since by then the effect is practically complete and further treatment is not necessary.

The duration of the treatment with the cationic surfactant also depends on the type of preparation which shall be treated, whole fish shall be treated longer than smaller parts excised from fish.

The treatment may be initiated on different points in the course of treatment of the products. The application of the cationic surfactants may be done immediately after catching the products, such as catching the fish from the sea, or may even be done immediately before the offering of the products for sale. The only aspect to be considered is to provide sufficient time for achieving the wanted effect.

After completion of the treatment, the cationic surfactant material may be wanted to be washed out. The presence of small amounts of the cationic surfactant is usually no problem for consumers, neither in terms of tolerance or safety, nor in terms of taste. In case of a combination with other products, such as in a preferred embodiment with acetic acid, a more complete removal is wanted in terms of taste.

In some cases, after the completion of the treatment it has been observed that the larvae is not killed but it has been enough affected by the treatment that the ingestion of it through the treated fish does not develop an infection to mammals. For this case, the most surprising effect is that when the treatment is unable to kill the larvae, it is practically reduced the pathogenity of it along with its capacity to migrate to the intestines of the mammals that are eating the treated fish. Thus, in this occasion, the negative effect of the ingestion of larvae is totally nullified due to the previous treatment with the cationic surfactant.

Example 1.

Material and methods

The larvae of Anisakis were obtained from blue whiting (Micromesistius poutassou) (not eviscerated). Abdominal cavity was opened from opercula to the anus. Peritoneum and viscera was visually examined for evaluating the presence of coiled and encapsulated larvae. Unencapsulated larvae were also observed on the surface of the viscera. The larvae were carefully extracted with plastic forceps and maintained in a saline solution (NaCI, 0.9%, w/vol) at room temperature (20°C±3°C).

LAE is produced by LAMIRSA.

Peptone water is prepared by Biomedics.

_Suryjyal_of_Anisakis in LAE

The effect of the LAE at 1 % dissolved in peptone water at different times was evaluated during 96 hours at room temperature. The individual test was made with 135 larvae unit. Differentiation of ;_. viable jnd_^dead larvae

Several methods can be used to differentiate between dead and live larvae. The observation of motility is one of the more widely used methods. Spontaneous motility or motility after stimulation with plastic clamps to minimize the alteration of its external structure was performed (Dong et al., J. Food Protection, 2003; 66: 1924-1926). Thereafter, the elution test was made, the larvae were introduced in a sieve n° 4, and partially submerged in physiological solution at 0.9% during 18-24h at room temperature. This period of time allows alive larvae to migrate towards the container with physiological solution.

Finally, the colour uptake of the larvae in a 0.1 % solution of methylene blue was assayed for selected samples. Larvae were placed in a cuvette with this solution for several hours at room temperature. The larvae were removed and superficially dried an filter paper prior to observation of the blue colour of dead nematodes (Molina-Garcia and Sanz, J. Food Protection 2002; 65: 383-388). Finally, a microscopic observation was made, previously larvae were fixed with a solution of alcohol-formaldehyde-acetic acid (85 ml ethanol, 10 ml formaldehyde, 5 mL glacial acetic acid).

Larvae treated with a solution of 1 % LAE in 1 % peptone water showed external changes. Colour of larvae was very white colour and light lacerations in cuticle were observed. After colouring of larvae with methylene blue, larvae considered dead were stained completely because the structure of the cuticle was affected. Also, observation with microscope shows altered cuticle.

The experiment was performed during 96 hours in a solution of 1 % peptone water with 1 % LAE.

As it is observed in Table 2, the survival of Anisakis decreased significantly (36%) during the first 24 hours, next it decreased gradually (approximately 1 1-13 %) at 48, 72 and 96 hours. At 96 hours, the larvae lethality was complete.

Table 2.

Percentage of living larvae in 1 % peptone water solution with 1 % LAE at room temperature during 96 hours

Time (hours) Dead Larvae Living Larvae Larvae surviving

(%)

0,5 0 135 100,00

1 0 135 100,00

2 0 135 100,00

3 9 126 93,33

4 8 118 87,41

5 6 112 82,96

6 6 106 78,52

7 6 100 74,07

8 7 93 68,89

9 6 87 64,44

10 5 82 60,74

24 33 49 36,30

27 3 46 34,07

30 0 46 34,07

33 3 43 31,85

48 11 32 23,70

51 2 30 22,22

54 0 30 22,22

57 4 26 19,26

72 7 19 14,07

75 1 18 13,33

78 1 17 12,59

81 2 15 11,11

96 15 0 0,00

Claims

CLAIMS:

1. Use of a cationic surfactant derived from the condensation of fatty acids and esterified dibasic amino acids, according to the following formula (1) :

where:

X^" is, a counter ion derived from an organic or inorganic acid, preferably Br^", Ci^" or HSO₄ ^", or an anion on the basis of a phenolic compound;

R₁: is a straight alkyl chain from a saturated fatty acid or hydroxyl acid having from 8 to 14 atoms linked to the α-amino acid group via an amidic bond;

R₂: is a straight or branched alkyl chain from 1 to 18 carbon atoms or an aromatic group;

R₃: is

— NH₃

or

where n is from 0 to 4, for the treatment of parasitic infection in seafood.

2. The use of claim 1, in which the cationic surfactant of the formula (1) is the ethyl ester of the lauramide of the arginine monohydrochloride (LAE) of the formula (2):

3. The use of claim 1 or 2, for the treatment of infection with larvae of Anisakis.

4. The use of any of the previous claims, in which the final concentration of the cationic surfactant in fish is 2 to 20,000 ppm, more preferred 100 to 10,000 ppm and more preferred 200 to 2,000 ppm.

5. The use of any of the previous claims, in which the treatment is performed in a solid form.

6. The use of any of the claims 1 to 4, in which the treatment is performed a solution such as a bath of water or as a spray or in a bath ice of the cationic surfactant.

7. The use of claim 6, in which the solvent for the preparation of the solution is water, propylene glycol, ethanol, glycerine or a mixture of these liquids.

8. The use according to claim 6 or 7, in which the solution contains a further component such as preservatives, antioxidants, surfactants, thickeners, enzymatic inhibitors, organic and inorganic salts, organic and inorganic acids, liquid and solid carriers and antifreeze,

9. The use of ciaim 8, in which the further component is acetic acid.

10. The use of any of the claims 6 to 9, in which the concentration of the cationic surfactant in the solution is in the range of 0.001 to 50 % by weight.