GB2252906A - Biocidal use of naturally occurring oils - Google Patents

Abstract

A method of protecting a product against bacterial spoilage comprises adding a naturally occurring biocidal oil, preferred oils being neem oil, malkanguni oil, darudi oil and karanja oil. The oils may be added in an amount up to 10% by weight and may alternatively be added in a suitable carrier.

Description

BIOCIDAL COMPOUNDS This invention relates to biocidal compounds and, more particularly, to naturally occurring biocidal oils and to a method of protecting a product using such an oil.

Biocidal, or antimicrobial agents or preservatives, as they are also known are used extensively in a wide range of industries. Any production, formulation or system which incorporates water is liable to contamination or spoilage by micro-organisms, either bacteria, yeasts or moulds.

Industrial biocides are used to prevent such contamination or spoilage and may typically be used as preservatives in cosmetics and toiletries, in water based paints, inks and adhesives, to prevent microbial contamination in paper production, leather tanning and textile production.

Extensive use of biocides to prevent microbial attack may also be seen in industrial water treatment to prevent slime formation or the growth of legionnaires disease bacteria, and in oil and gas production to prevent microbial corrosion.

All of the industrial biocides commonly used are synthetic chemicals or blends of synthetic chemicals.

Examples of the chemical compounds used are alkyl aryl trimethyl ammonium chloride, dialkyl dimethyl ammonium chloride, dodecylguanidine hydrochloride, polyhexamethylene biguanide, benzalkonium chloride, 2 (thiocyanomethylthio)-benzothiazole, methylene bisthiocyanate, 1, 2-benzisothiazolin-3-one, isothiazolin-3one, bis(2-hydroxy-5-chlorophenyl)sulphide, zinc pyrithione, hexahydro-1,3,5-tris (2-hydroxyethyl)-S-triazine, chloroallyl) 3:5:7- triaza-azonia adamantane hydrochloride, ("imidazidinyl urea"), O-phenylphenol, p-hydroxybenzoic acid esters and Na salts, sodium pentachlorophenate, 2-bromo-2 nitropropane-1,3-diol, 2-hydroxyethyl-2,3-dibromopropionate, 1,4-bisbromoacetoxybutene, 1, 2-dibromo-2 , 4-dicyanobutane, phenyl mercuric salts and esters.

However, there are many disadvantages with the use of these synthetic biocidal compounds, for example, their toxicity to higher life forms and their dependence upon the petrochemical industry for their raw material sourcing and production.

In view of these disadvantages, there is a desirability for naturally occurring biocides for industrial use which are safe to man and which may be regarded as a renewable resource.

Naturally occurring oils such as neem oil, malkanguni oil, karanja oil and darudi oil have been used in native medicine to treat diseases such as skin diseases. However, it has now surprisingly been found that these oils are active against a wide range of industrial spoilage microorganisms and are effective as biocidal agents in a wide range of products to prevent spoilage.

The present invention seeks to provide naturally occurring biocidal agents which alleviate the disadvantages of synthetic chemical agents.

In one aspect, the invention provides a method of protecting a product against bacterial spoilage comprising adding to the product a naturally occurring biocidal oil.

Preferably, the oil is a fatty oil and contains at least one limonoid group, and preferably additionally contains at least one flavinoid, alkaloid or glycoside group.

Desirably, the oil is either neem, malkanguni, darudi or karanja oil.

In another aspect, the invention provides a biocidal composition comprising a naturally occurring biocidal oil and a suitable carrier.

A further aspect of the invention provides a product which has been protected against bacterial spoilage by the addition of a naturally occurring biocidal oil. The products for use in the present invention are generally industrially produced and are not obtained from animals.

The products all include industrial reagents, processing fluids etc., intermediate products and apparatus and equipment.

The oils used in the present invention are all naturally occurring plant extracts. Neem oil is extracted from Azadirachta indica, N.O. Meliaceae, malkanguni oil is extracted from Celastrus paniculata N.O. Celastraceae, karanja oil is extracted from Ponqamia glabra or Garandupa indica N.O. Papilionnacae and darudi oil is extracted from Argenione maxicana N.O. Papaveraceae. The oils are conveniently obtained by steam distillation from the crushed seeds of the plants.

The chemical composition of the oils is not entirely known. However, all of the oils are fatty oils and appear to contain fatty acids which are typical of plant oil. The fatty acids found to be present in neem oil are myristic, palmitic, stearic, arachidic, behenic, lignoceric, palmitoleic, oleic and linoleic acids. The fatty acid content of the other three oils is similar to that of neem oil.

It is not known exactly what component is responsible for the biocidal activity of the four oils. However, all four oils contain limonoid, or tetranortriterpoid groups, sometimes called "bitter principles" and it is thought that these are probably responsible for the activity of the oil.

The following limonoid groups have been found to be present in neem oil Nimbin, Nimbinin, = Epoxyyazadiradione, Meliantriol, Azadirachtol, Azadirachnol, Salannin, Azadirone, Azadiradione, 17-Epi-Azadiradione, Gedunin, 7 Deacetylgedunin, 7-Deacetylazadiradione 7-benzoate, Epoxyazadiradione 7-benzoate, Gedunin 7-benzoate, nimocin, Nimbocinol, Meldenin, Vepenin, 1,3-Diacetylvilasinin, 1 Tigloyl-3-acetylvilasinin, 17ss-Hydroxyazadiradione, Nimolicinol, lÁ-Methoxy-1,2-dihydro-epoxyazadiradione, lss,2ss-Diepoxyazadiradione, 7-Acetylneotrichilenone, 3 Deacetylsalannin, Salannol, Salannol acetate, Salannolactam (21), Salannolactam-(23), Desacetylnimbin, Azadirachtin, 22,23-Dihydro-23R-methoxy-azadirachtin, 3 Tigloylazadirachtol, Salannolide, Nimbinene, 6 Deacetylnimbinene, Nimbandiol, 6-0-Acetylnimbandiol, 3 Deacetyl-ll-desoxyazadirachtin, 3-Acetoxy-7-tigloylvilasinin lactone, l-Tigloyl-3-acetyl-ll-methoxy-azadirachtinin, Salannin lactone, Nimbin lactone, l,3-Diacetyl-ll,19-deoxa- 11-oxo-meliacarpin, 3-Deacetyl-3-cinnamoyl-azadirachtin, Nimbolide, Nimbic acid, Nimbidic acid, Nimbidin.

The principle limonoid components of neem oil are: Compound Nimbinin = Expoxyazadiradione 4.0 Azadiradione 2.6 Azadirone 2.5 Salannin 0.95 Gedunin 0.37 Nimbin 0.19 17ss-Hydroxyazadiradione 0.19 Karanja oil has been found to contain the limonoids pongamol and karanjin. Small amounts of the sterols ss- sitosterol and cholesterol have been reported to be present in neem oil. All four oils contain various flavinoid groups.

It is thought that the oils may contain some alkaloid groups. The alkaloids berbarine and protoprine have been found in darudi oil. It is also thought that the oils may contain some glycoside groups.

It has been found that the four oils are active against a wide range of the micro-organisms commonly responsible for spoilage of products in industry, including Gram-positive and Gram-negative bacteria, yeasts and fungi. Activity of an oil against a particular micro-organism is assessed by measuring its minimum inhibitory concentration (m.i.c.).

The m.i.c. test may either be carried out by tube dilution or on agar plates. In the present invention, m.i.c. tests were carried out on agar plates according to the following procedure Preparation of test organisms Bacteria - From stock culture slopes, inoculate 10 ml volume of tryptone soya broth. Incubate at 320C for 24 hours. This should result in turbid broths indicating approximately 109 organisms/ml broth. This is used as test inoculum.

Yeast - As for bacteria Moulds - From stock culture slopes inoculate sterile Sabouraud dextrose agar slopes by streaking with an inoculating loop Incubate inoculated slopes for 5 days at 250C. Resultant growth should be confluent on the slope.

Wash off the growth by 3 mls of sterile peptone water and sterile glass beads. Gently shake to remove surface mould mycelial growth. Avoid breaking up the agar slope.

This is used as test inoculum.

Test Procedure Give each sample to be tested a test number.

Make doubling dilutions of the sample in 3 ml volumes of sterile distilled water by taking 2 ml of sample and adding to a sterile 3 ml volume of distilled water, 3 ml of this is then taken after mixing into the next bottle and so on.

Label three Petri dishes with each dilution level and sample details.

Place 1 ml of each dilution into each of the Petri dishes.

Add 15 ml of previously sterilised and tempered (to 480C) tryptone soya agar to two of the Petri dishes and 15 ml of previously sterilised and tempered Sabouraud dextrose broth to the other Petri dish. This is repeated for all dilutions of the sample.

Allow agar to set in the Petri dish and dry surface moisture by inverting the dishes slightly open in the 320C incubator for 1 hour.

Remove the Petri dishes from the incubator and segregate into three stacks: Tryptone soya agar - Bacteria Tryptone soya agar - Yeast Sabouraud dextrose agar - Moulds Inoculate the surface of the plates labelled Bacteria with one drop delivered from a Pasteur pipette (delivery 50 drops/ml) of each bacterial suspension. Allow to dry and incubate at 320C for 36 hours.

Inoculate the surface of the plates labelled Yeasts in the same way as Bacteria.

Inoculate the surface of the plates labelled Moulds in the same way as Bacteria except the plates are incubated for 5 days at approximately 250C.

After incubation the plates are visually assessed for growth or no growth of organisms on the inoculated drop.

Results Note whether there is growth or not on the area of the inoculated drop for each plate.

The m.i.c. is determined as the highest dilution of sample giving "no growth" for each organism of each group.

This dilution is multiplied by 15 to account for the agar volume. This gives the highest dilution of the sample inhibiting microbial growth. Devide one million by the reciprocal of the dilution, to give the m.i.c. in parts per million.

Example Calculation For a highest dilution showing no growth of 1 64 1 x 15 = 1 64 960 1,000,000 = 1041 ppm m.i.c.

960 Using the above method, neem oil was found to have the following m.i.c. levels Micro-organism m.i.c.

Bacteria: Staphylococcus aureus 65ppm S. Epidermidis 65ppm Lactobacillus buchneri 16ppm E.Coli 260ppm Klebsiella pneumophila 260ppm S.marcescens 1000ppm L.monocytoqenes 16ppm Bacillus subtilis 16ppm Yeasts: Candida albicans 1000ppm C.bordinii 260ppm C.parapsilosis 520ppm Saccharomyces cerevisiae 1000ppm Fungi: Aspergillus flavus 2000ppm A.niqer 8000ppm A.funicatus 65ppm A.glaucus 65ppm Penicillium natatum 8000ppm Cladosporum herbarum 8000ppm Trichothecium viride 8000ppm Myrothecium verucaria 4000ppm Alternaria alternata 260ppm It has been found that neem oil, malkanguni oil, darudi oil and karanja oil are effective biocidal agents and may be used to prevent spoilage in a wide range of products.The effectiveness of the oil in a particular product is typically tested using the standard B.P.1988 Challenge Test, details of which are given in the Examples.

The oils are most commonly added to the product to be protected in an amount of up to 10%, preferably up to 5%, more preferably between 0.1% and 1% or 0.1% and 0.5%. All percentages given are by weight unless otherwise specified.

The amount of oil used will vary depending on the particular oil used and the product to be protected. Alternatively, the oil may be used in a suitable carrier, which will depend on the product to be protected. Suitable carriers include, alcohols such as methanol and ethanol, glycols, water/surfactant solutions or other oils. Suitable surfactants would be those sold by Hulls under the name "Marlowet LVX" and "Marlowet LVS".

As mentioned above, the range of products which may be protected by the natural biocidal oils according to the present invention is very varied. The oils may be used at stages during industrial processes and also to protect and clean apparatus. The oils may be used as preservatives in toiletries and cosmetics. It has been found that neem oil, for example, is effective against the accepted range of spoilage organisms when incorporated at levels between 0.1% and 0.5%.

The oils may be used as preservatives in domestic products, such as washing-up liquids, liquid laundry detergents, liquid polishes, institutional cleaners, industrial cleaners. It has been found that, for example, neem oil gives protection against microbial spoilage when incorporated at between 0.1 and 0.5%.

In water-based paints, printing inks and adhesives, it has been found that the oil, for example neem oil can prevent microbial spoilage "in-can" at levels between 0.1 and 1.0% and may be able to provide long-term protection for the dried film of paints and adhesives. Engineering cutting oils may become contaminated by both spoilage and pathogenic organisms and it has been found that most of these may be controlled by the addition of an oil, for example 0.1 to 1.0% of neem oil.

During the production of leather, hides are frequently transported in the "wet-blue" state. In this state they are very susceptible to microbial spoilage, so it is customary to add biocides to them. The use of an oil, added to the processing fluids, for example 0.1 to 1.0% of neem oil should prevent microbial contamination without the handling difficulties associated with synthetic biocides.

At many stages of its production, timber is exposed to microbial attack, which may cause a variety of problems from staining to actual disintegration. The oils may be used as a timber preservative by soaking, pressure impregnation or painting on the surface. They may also be applied in a wide variety of carriers such as alcohols, glycols, water/surfactant solutions, or as the concentrated oil diluted in traditional oils such as linseed oil, tech oil, etc.

In the Oil and Gas industry, the oils may be used to counteract both slime forming aerobic bacteria and corrosion-causing anaerobic bacteria in a wide range of applications in oil and gas production: Drilling muds - 0.1% to 1.0% neat or in suitable carrier Injection waters - 0.1% to 0.5% in water/surfactant solution Fracturing fluids - 0.1% to 0.5% neat or in a suitable carrier such as alcohol Blow-out preventor fluids - 0.1% to 0.5% neat or in suitable carrier such as an alcohol Gas pipelines - dissolved in methanol and injected at the wellhead Oil and fuel tanks - dissolved in a suitable alcoholic solvent. The oil will discourage microbial growth in the water layer at the bottom of tanks.

In all of these applications, the oils may be used in combinations of one or more and this may enable the use of less oil in total due to synergism between the oils being used. The oils may also be used with a synthetic biocide such as those listed earlier in the specification.

It may be possible to increase the activity of the oils before use. The oil may be ref fluxed in ethanol for a period of up to 4 hours or the fatty acid components of the oil may be saponified. This latter treatment would lead to improved water solubility and surface activity which would enhance the ease of use of the oil as well as its biocidal activity.

The invention will now be demonstrated by means of the following non-limiting examples.

Example 1 The efficacy of the oils in a particular product was assessed using the BP 1988 Appendix XVIC Challenge test, the general method of which is as follows: Test organisms The following organisms are used in the test: Aspergillus niger (IMI 149 007) Candida albicans (NCPF 3179) Pseudomonas aeruginosa (NCIB 8626) Staphylococcus aureus (NCTC 10788) Maintenance of cultures Organisms should be maintained as recommended by the Curator of the appropriate culture collection.

Preparation of inoculum For each bacterial organism, inoculate the surface of soya tryptone agar from a suitable, recently-prepared stock culture of the organism; for yeasts and moulds, use Sabouraud agar.

Several subcultures may be needed after revival before the organism is in its optimal state. Incubate bacterial cultures at 300 to 350 for 18 to 24 hours and the culture of Candida albicans at 200 to 250 for 48 hours and the culture of Aspergillus niger at 200 to 250 for 7 days. Harvest the bacteria and Candida albicans using 0.1% peptone water to wash the surface growth into a suitable receptable and then dilute with the same liquid to reduce the microbial count to about 108 organisms per ml. To harvest Aspergillus niger, use 0.1% peptone water containing 0.05% w/v of polysorbate 80, filter through glass wool if necessary and adjust to a microbial count of about 108 organisms per ml using 0.1% peptone water. Suspensions should be used immediately.

Test procedure Introduce into portions of the product being examined sufficient of a suspension of the micro-organism to achieve a final concentration of about 106 organisms per g or per ml; use separate portions for each micro-organism. At the same time, introduce the same volume of inoculum into separate equivalent quantities of 0.1% peptone water for each organism (peptone-water controls). Ensure that the micro-organisms are evenly dispersed throughout the portion of the product being examined. The volume of the inoculum should not exceed 1% of the sample weight or volume. Store the inoculated product at 20 to 250, protected from light.

Removed l-g or l-ml quantities of the inoculated products at zero hour, 6 hours, 48 hours, 7 days, 14 days and 28 days as appropriate. The peptone-water controls should be examined at the same time as the zero-hour sample of the inoculated product, and using the same procedure, to determine the suitability of the media used for the growth of the test organism.

Introduce each portion into 9 ml of 0.1% peptone water.

This may contain up to 10% w/v of polysorbate 80 but when concentrations of the surface-active agent in excess of 1% are used the ability of the medium to support the growth of viable organisms should be confirmed by the use of appropriate controls. Specific antagonists or neutralising agents for preservatives present in the product may also be incorporated but where such materials are used the ability of the medium to support the growth of viable organisms should be confirmed by the use of appropriate controls.

Specific antagonists or neutralising agents for preservatives present in the product may also be incorporated but where such materials are used the ability of the system to support the growth of the test organisms should be confirmed by the use of appropriate controls.

Disperse the material, using sterile glass beads if necessary. Dilute to an appropriate extent using 0.1% peptone water (which may contain preservative antagonists or neutralising agents) and use 1-ml aliquots of these dilutions to prepare, in duplicate, pour-plates using soya tryptone agar or Sabouraud agar (either of which may also contain preservative antagonists or neutralising agents).

Alternatively an appropriate volume of the final dilution may be spread on the surface of a solid soya tryptone agar or Sabouraud agar plate.

If the preservative system present in the product cannot be suitably antagonised or neutralised, it should be eliminated either by suitable dilution or by membrane filtration to prevent carry-over of inhibitory concentrations of preservative into the agar. When dilution procedures are employed, due allowance should be made for the reduced sensitivity in the recovery of small numbers of viable organisms. When membrane filtration is employed the following procedure should be used for each sample taken and the volume should be adjusted to give results that are directly comparable with those that would be obtained using the pour-plate technique described above. Moisten a membrane filter, having a nominal pore size not greater than 0.45 Ìm, with a small quantity of 0.1% peptone water.

Transfer the measured volume of the sample, diluted and dispersed as necessary, to the membrane and filter immediately. Wash the membrane by filtering through it three or more successive quantities each of suitable volume of 0.1% peptone water, wash. Transfer the membrane to the surface of soya tryptone agar or Sabouraud agar plate.

Incubate the plates at 300 to 350 for not less than 3 days for bacteria and at 200 to 250 for not less than 5 day for moulds and yeasts.

Determine the average number of colony-forming units of each test organism recovered after not less than 3 days incubation and, for moulds and yeasts, additionally at not less than 5 days and estimate the total number of viable test organisms per ml or per g of the original product taking into account the subsequent dilutions.

Separate note should be made of any colony-forming units not having the usual morphological characteristics of the test organism. Such organisms should be investigated and if they are manifestly contaminants they should be disregarded.

Interpretation Use the results obtained for the zero-hour peptonewater control to provide the initial number of organisms per ml or per g which is the baseline for the purposes of estimating the reduction in the number of organisms with time.

A product may be considered to be effectively preserved if laboratory and production batches satisfy the relevant criteria stated below over the claimed shelf life and the likely period of use.

While effective product preservation, as defined by these criteria, is highly desirable, it is only one aspect of pharmaceutical formulation. It is recognised that for a number of products (for example certain preparations of biological origin) the criteria are unlikely to be achieved except at the expense of some other property of equal or greater importance. The following criteria of acceptance should therefore be applied after due account has been taken of any special considerations relevant to specific products.

Parenteral preparations supplied in multidose containers BACTERIA The number of organisms recovered per ml is reduced by a factor of not less than 103 within 6 hours of challenge and no organism is recovered from 1 ml at 24 hours and thereafter.

MOULDS AND YEASTS The number of organisms recovered per ml is reduced by a factor of not less than 102 within 7 days of challenge and there is no increase thereafter.

Opthalmic preparations and preparations for use in the ear, supplied in multidose container BACTERIA The number of organisms recovered per ml or per g is reduced by a factor of not less than 103 within 6 hours of challenge and no organism is recovered from 1 ml or 1 g at 24 hours and thereafter.

MOULDS AND YEASTS The number of organisms recovered per ml or per g is reduced by a factor of not less than 102 within 7 days of challenge and there is no increase thereafter.

Topical preparations BACTERIA The number of organisms recovered per ml or per g is reduced by a factor of not less than 103 within 48 hours of challenge and no organism is recovered from 1 ml or 1 g at 7 days and thereafter.

MOULDS AND YEASTS The number of organisms recovered per ml or per g is reduced by a factor of not less than 102 within 14 days of challenge and there is no increase thereafter.

Oral liquid preparations BACTERIA The number of organisms recovered per ml is reduced by a factor of not less than 102 within 7 days of challenge and there is not increase thereafter.

MOULDS AND YEASTS There is no increase in the number of organism recovered per ml within 14 days of challenge or thereafter Media Soya Tryptone Agar Pancreatic digest of casein 15.0 g Papaic digest of soya bean 5.0 g Sodium chloride 5.0 g Agar 15.0 g Water 1000 ml Dissolve the ingredients in the water, filter, adjust to pH 7.2 to 7.4 and sterilise by heating in an autoclave at 1210 for 15 minutes.

Sabouraud Aqar D-Glucose monohydrate 40.0 g Peptone 10.0 g Agar 15.0 g Water 1000 ml Dissolve the ingredients in the water, filter through cotton gauze, adjust to pH 5.4 to 5.6 and sterilise by heating in an autoclave at 1210 for 15 minutes.

0.1% Peptone Water Peptone 1.0 g Sodium chloride 8.9 g Water 1000 ml Dissolve the ingredients in the water, filter, adjust to pH 7.2 to 7.4 and sterilise by heating in an autoclave at 1210 for 15 minutes.

Example 2 Using the procedure of Example 1, the efficacy of neem seed oil as a preservative in shampoo was tested Base formula of shampoo: Empicol TM TC40 50g Emplilan TM MAA 15g Distilled Water to lOOg Results TABLE 1

CHALLENGE REDUCTION IN MICROBIAL CHALLENGE AT ORGANISM STATED CONTACT TIME 0HR 6HR 24HR 48HR 7DAY 14DAY 28DAY STAPHYLOCOCCUS 1.0% 0.5 x 10 0.5 x 10 1.0 x 105 1.0 x 105 1.0 x 105 NOR NOR AUREUS NCTC 10788 0.1% 1.0 x 10 1.0 x 105 1.0 x 105 1.0 x 105 1.0 x 105 NOR NOR PSEUDOMONAS 1.0% 1.0 x 10 1.0 x 105 1.0 x 105 1.0 x 105 1.0 x 105 NOR NOR AERUGINOSA NC1B 8626 0.1% 5.0 x 10 1.0 x 105 1.0 x 105 1.0 x 105 8.0 x 104 NOR NOR CANDIDA 1.0% 9.0 x 10 5.0 x 10 5.0 x 10 NOR NOR NOR NOR ALBICANS NCPF 3179 0.1% 1.0 x 10 5.0 x 10 5.0 x 10 NOR NOR NOR NOR ASPERGILLUS 1.0% NO NO 8.0 x 100 5.0 x 100 2.0 x 10 5.0 x 10 NOR NIGER REDUCTION REDUCTION IMI 149007 NO NO 8.0 x 100 NO 1.0 x 10 1.0 x 10 NOR 0.1% REDUCTION REDUCTION REDUCTION NOR = No organism recovered It was found that the neem oil satisfied the BP challenge test for Candida Albicans and Aspergillus Niger and marginally failed for Staphylococcus Aureus and Pseudomonas Aeruginosa.

From the results shown in Table 1, it will be seen that the neem oil produces a fast and significant reduction in all of the organisms detected in the shampoo and is thus a highly effective preservative agent in shampoo.

Example 3 Using the procedure of Example 1, the efficacy of neem seed oil as a preservative is cosmetic cream was tested Base formulation of the cream: Polawax 15% by weight Light Mineral Oil 10% by weight Demineralised water 75% by weight Results TABLE 2

CHALLENGE REDUCTION IN MICROBIAL CHALLENGE AT ORGANISM STATED CONTACT TIME 0HR 6HR 24HR 48HR 7DAY 14DAY STAPHYLOCOCCUS 1.0% NO 1.0 x 10 1.0 x 105 NOR NOR NOR AUREUS REDUCTION NCTC 10788 0.1% NO 1.0 x 104 1.0 x 105 8.0 x 105 NOR NOR REDUCTION PSEUDOMONAS 1.0% 9.0 x 10 5.0 x 104 5.0 x 106 NOR 1.0 x 105 1.0 x 104 AERUGINOSA NC1B 8626 0.1% 5.0 x 10 1.0 x 104 NOR 2.0 x 107 9.0 x 105 2.0 x 105 CANDIDA 1.0% NO NO 2.0 x 10 6.0 x 10 8.0 x 10 1 x 10 ALBICANS REDUCTION REDUCTION NCPF 3179 0.1% NO 1.0 x 10 1.0 x 10 1.0 x 10 1 x 10 4.0 x 10 REDUCTION ASPERGILLUS 1.0% NO 8.0 x 100 8.0 x 10 8.0 x 10 1.0 x 10 1.0 x 10 NIGER REDUCTION IMI 149007 NO 8.0 x 100 1.0 x 10 8.0 x 10 1.0 x 10 1.0 x 10 0.1% REDUCTION NOR = No organism recovered It was found that neem oil satisfied the BP Challenge Test for Staphylococcus Aureus, Candida Albicans and Aspergillus Niger. For Pseudemonas Aeruginosa, the organism was reduced to non-recverable levels after 48hrs but there was then found to be sifnigicant re-entry of the organism.

However, it can be seen from the results in Table 2 that the neem oil produced a significant reduction in all organism and is therefore a highly effective preservative in cosmetic cream. The preservation was not found to be quite as good as in the shampoo formulation and it is thought that this may be due to some inherent microbial activity of the base formulation.

Example 4 Using the procedure of Example 1, the efficacy of neem oil as a preservative in a latex resin was tested. The latex resin comprised a carboxylated styrene/butadiene copolymer.

Results TABLE 3

CHALLENGE REDUCTION IN MICROBIAL CHALLENGE AT ORGANISM STATED CONTACT TIME 0HR 6HR 24HR 48HR 7DAY 14DAY 28DAY STAPHYLOCOCCUS 1.0% NO NO 1.0 x 10 1.0 x 104 NOR NOR NOR AUREUS REDUCTION REDUCTION NCTC 10788 0.1% NO NO 1.0 x 10 1.0 x 105 NOR NOR NOR REDUCTION REDUCTION PSEUDOMONAS 1.0% NO 1.0 x 104 1.0 x 105 1.0 x 106 NOR NOR NOR AERUGINOSA REDUCTION NC1B 8626 0.1% NO 1.0 x 10 1.0 x 104 NOR NOR NOR NOR REDUCTION CANDIDA 1.0% NO 1.0 x 10 1.0 x 10 1.0 x 10 NOR NOR NOR ALBICANS REDUCTION NCPF 3179 1.0% NO 1.0 x 10 1.0 x 10 1.0 x 10 NOR NOR NOR REDUCTION ASPERGILLUS 1.0% NO 1.0 x 10 1.0 x 10 1.0 x 10 NOR NOR NOR NIGER REDUCTION IMI 149007 NO 1.0 x 10 1.0 x 10 1.0 x 10 NOR NOR NOR 0.1% REDUCTION NOR = No organism recovered The neem oil satisfied the BP Challenge Test for all four organisms.The BP test is not actually appropriate for the latex resin. However, the criteria in the BP Challenge Test are more stringent than those required for a produce such as the latex resin tested. The fact that the neem oil was found to satisfy the criteria of the BP test demonstrates its high efficacy as a preservative in the latex resin, Example 5 Using the procedure of Example 1, the efficacy of neem oil as a preservative in washing-up liquid was tested Base formulation of the washing-up liquid:: Coconut oil base cleaner Milk Whey Sea Salt Citrus oil Calendula extract Chamomile extract Citric Acid Results TABLE 4

CHALLENGE REDUCTION IN MICROBIAL CHALLENGE AT ORGANISM STATED CONTACT TIME 0HR 6HR 24HR 48HR 7DAY 14DAY STAPHYLOCOCCUS 1.0% 1.0 x 10 1.0 x 10 1.0 x 10 1.0 x 10 NOR NOR AUREUS NCTC 10788 0.1% 1.0 x 10 1.0 x 10 1.0 x 10 1.0 x 10 5.0 x 10 1.0 x 10 PSEUDOMONAS 1.0% 5.0 x 10 1.0 x 104 1.0 x 106 1.0 x 107 NOR NOR AERUGINOSA NC1B 8626 0.1% 5.0 x 10 1.0 x 10 5.0 x 10 3.0 x 10 5.0 x 10 1.0 x 10 CANDIDA 1.0% 0.5 x 100 5.0 x 10 1.0 x 10 1.0 x 106 NOR NOR ALBICANS NCPF 3179 0.1% 1.0 x 10 5.0 x 10 6.0 x 10 2.0 x 10 1.0 x 10 5.0 x 10 ASPERGILLUS 1.0% 1.0 x 106 1.0 x 10 1.0 x 10 1.0 x 10 1.0 x 10 1.0 x 104 NIGER IMI 149007 1.0 x 10 1.0 x 10 5.0 x 10 5.0 x 10 1.0 x 10 5.0 x 10 0.1% NOR = No organism recovered The neem oil was found to satisfy the test for all organisms at a level of 1.0% but 0.1% neem oil was found to satisfy the test for Candida Albicans and Aspergillus Niger but failed the test for Staphylococcus Aureus and Pseudomonas s eudomona s Aeruqinosa. However, the results in Table 4 show that neem oil at a level of 1.0% is a highly effective preservative in washing-up liquid.

Example 6 Using the procedure of Example 1, the efficacy of neem oil as a preservative in a hard surface cream cleaner was tested Composition of the cleaner: Finely ground chalk Coconut oil based cleaner Liquid soap from palm Coconut oil Linseed oil Clay Citrus oil Results TABLE 5

CHALLENGE REDUCTION IN MICROBIAL CHALLENGE AT ORGANISM STATED CONTACT TIME 0HR 6HR 24HR 48HR 7DAY 14DAY STAPHYLOCOCCUS 1.0% NO 5.0 x 10 6.0 x 10 1.0 x 105 NOR NOR AUREUS REDUCTION NCTC 10788 0.1% 0.5 x 100 1.0 x 10 5.0 x 10 CONTAMINATION# PSEUDOMONAS 1.0% NO 1.0 x 10 1.0 x 106 1.0 x 106 NOR NOR AERUGINOSA REDUCTION NC1B 8626 #CONTAMINATION# 0.1% 1.0 x 10 1.0 x 10 1.0 x 10 1.0 x 10 1.0 x 10 1.0 x 10 CANDIDA 1.0% 1.0 x 10 1.0 x 10 1.0 x 105 5.0 x 105 NOR NOR ALBICANS NCPF 3179 0.1% 7.0 x 100 2.0 x 10 7.0 x 10 7.0 x 10 7.0 x 10 1.0 x 10 ASPERGILLUS 1.0% 1.0 x 10 2.0 x 10 7.0 x 10 1.0 x 10 NOR NOR NIGER IMI 149007 1.0 x 10 2.0 x 10 5.0 x 10 5.0 x 10 5.0 x 10 5.0 x 10 0.1% NOR = No organism recovered The cleaner sample was found to be contaminated with approximately 5000 organisms/g pseudomonads. It was found that neem oil incorporated at a level of 1.0% satisfied the test for all of the organisms and that neem oil incorporated at a level of 0.1% satisfied the test for Candida Albicans and Aspergillus Niger, thus eliminating the challenge organisms and the contamination. However the contamination was not eliminated by 0.1% neem oil for Staphylococcus Aureus or Pseudomonas Aeruginosa.

The natural contamination is expected to be adapted to survive and grow in the product and would therefore be more difficult to eradicate. The ability of neem oil incorporated at 1.0% to eradicate both the challenge organisms and the contamination shows the effectiveness of neem oil as a preservative in hard surface cream cleaner.

Claims (18)

1. A method of protecting a product against bacterial spoilage comprising adding to the product a naturally occurring biocidal oil.

2. A method according to claim 1 wherein the oil is a fatty oil and contains at least one limonoid group.

3. A method according to claim 1 or 2 wherein the oil additionally contains at least one flavinoid, alkaloid or glycoside group.

4. A method according to any one of the preceding claims wherein the oil is either neem, malkanguni, darudi or karanja oil.

5. A method according to any one of the preceding claims wherein the oil is added in an amount up to 10%.

6. A method according to claim 5 wherein the oil is added in an amount up to 5%.

7. A method according to claim 6 wherein the oil is added in an amount from 0.1% to 1%.

8. A method according to any one of the preceding claims wherein the oil is added in a suitable carrier.

9. A biocidal composition comprising a naturally occurring biocidal oil and a suitable carrier.

10. A composition according to claim 9 wherein the oil is a fatty oil and contains at least one limonoid group.

11. A composition according to claim 9 or 10 wherein the oil additionally comprises at least one flavinoid, alkaloid or glycoside group.

12. A composition according to any one of claims 9 to 11 wherein the oil is either, neem, malkanguni, darudi or karanja oil.

13. A composition according to any one of claims 9 to 12 wherein the oil is present in an amount from 0% to 10%.

14. A compositions according to claim 13 wherein the oil is present in an amount from 0% to 5%.

15. A composition according to claim 14 wherein the oil is present in an amount from 0.1% to 1%.

16. A product which has been protected against bacterial spoilage by the addition of a naturally occurring biocidal oil.

17. A product according to claim 16 wherein the oil is a fatty oil and contains at least one limonoid group.

18. A product according to claim 16 or 17 which is a cosmetic product, a toiletry product, a domestic product such as washing up liquid, liquid laundry detergent, liquid polish, institutional and industrial cleaners, a water-based paint, printing ink or adhesive, an engineering cutting oil, leather hides, timber, drilling mud, injection waters, fracturing fluids, blow-out preventor fluids, gas pipelines or oil and fuel tanks.