GB2386946A - Detecting microorganisms - Google Patents

Abstract

Microorganisms are detected in e.g. foodstuff, pharmaceutical or cosmetic products by admixing with a compound which exhibits luminescence in the presence of the microorganism which is typically a fungus. The compound which exhibits luminescence may be a fluorophore, fluorochrome or a phosphor.

Description

1 2386946

METHOD

Field Of The Invention

5 The present invention relates to a method for detecting a microorganism in a product. The present invention further relates to a method for detecting a thermoresistant mould in a product.

The present invention yet further relates to a composition.

The present invention further relates to a kit for detecting a microorganism in a product. Background To The Invention

Spoilage of products for example thermally processed fruits and fruit products, such as those comprising a pectin, by microorganisms, in particular so thermoresistant moulds, has been recognised in several countries (see Beuchat et a/ 1979 Byssochlamys spp. and their importance in processed fruits - Advan.

Food Res. 25: 237; Fravel et al 1986 Estimation of United States and world distribution of Talaromyces flavus - Mycologia 78: 684; Hellinger 1960 The spoilage of bottled grape juice by Monascus purpureus - Went. Ann. Inst.

25 Pasteur, Lille 11: 183; Hocking etal1984, Food spoilagefungi. Heatresistant fungi - CSIRO Food Res. Quart. 44: 73; Hull 1933-34 Investigation of the control of spoilage of processed fruit by Byssochlamys flu va - In "Annual Report of the Fruit and Vegetable Preservation Research Station" p. 64. Univ.

of Bristol Campden, England; Jesenska et a/ 1984, On the problems of moulds so on some products of canning industry - Cs. Hyg. 29: 102; King et al 1979 Non logarithmic death rate calculations for Byssochlamys fulva and other microorganisms - Appl. Environ. Microbiol. 37: 596; McEvoy ef al 1970

MA Temperature tolerance of Aspergillus fischeri var. glaber in canned strawberries - Irish J. Agric. Res. 9: 59; Put et al 19647 Disintegration and organoleptic deterioration of processed strawberries caused by the mould Byssochlamys nivea - J. Appl. Bacteriol. 27: 53. Most frequentlyencountered 5 thermoresistant moulds which cause spoilage are those from the Ascomycetes class and include the following species: Byssochlamys fulva, Byssochlamys nivea, Neosafforya fischeri, Talomyces flavus, Talomyces bacillisporus and Eupenicillium brefeldianum. Byssochlamys species have been recognised as spoilage moulds in canned fruit since the early 1930's and have been extensively to studied (see Bayne et al 1979 Heat resistance of Byssochlamys ascospores -

Appl. Environ. Microbiol. 37: 449; Beuchat et al 1979 Byssochlamys spp. and their importance in processed fruits - Advan. Food Res. 25: 237; Jensen 1960 Experiments on the inhibition of some thermoresistant moulds in fruit juices -

Ann. Inst. Pasteur., Lille 11: 179; King et al 1969 Control of Byssochlamys 15 and related heat-resistant fungi in grape products - Appl. Microbiol. 18: 166; Put 1964 A selective method for cultivating heat resistant moulds, particularly those of the genus Byssochlamys, and their presence in Dutch soil - J. Appi.

Bacteriol. 27: 59).

o Fungi from the Ascomycetes class, such as thermoresistant moulds, are characterised by the production of ascospores that frequently show high heat resistance which enables them to survive the thermal processing used in the preparation of products, for example pharmaceuticals, cosmetics and foodstuffs, such as plant products or products derived from plants. Germination of :5 ascospores may result in visible growth of mycelia on the products. In fruit products for example, the production of pectic enzymes by byssochlamys species, for example, can result in complete breakdown of texture in fruits and can also result in producing malodours which may impair product flavour. In addition, fungi from the Ascomycetes class, particularly thermoresistant moulds, o may produce deleterious effects on consumption or use of the product, as a result of the production of mycotoxins. For example, thermoresistant moulds of the Byssochlamys genus produce mycotoxins such as patulin, byssotoxin A or

byssochlamic acid and the thermoresistant moulds of the species Neosafforya fscheri produce mycotoxins such as fumitremorgin A7 B or C or verruclogen.

Thermoresistant moulds are widely distributed in soil for example, particularly 5 vineyards orchards and fields in which plants are grown. As a consequence,

these moulds may become contaminants on fruit and other vegetation upon contact with soil, before delivery to the processing plant.

There is a general need in industry, such as the food, pharmaceutical and JO cosmetic industries, for rapid methods to detect, enumerate and identify microorganisms, particularly viable microorganisms. In particular, in the food industry, robust, reliable testing methods are needed that provide results more rapidly than conventional methods, with similar or greater sensitivity and specificity. Conventional methods may grossly underestimate the numbers of specific target microorganisms in a sample. One of the obstacles to the development and application of rapid, direct methods for the detection of specific microorganisms in products such as foods, pharmaceuticals and cosmetics is to the need to concentrate and separate the target microorganism from suspensions of the product. For example, the number of ascospores present on products such as fruits is low, typically less than 1 per gram of fruit or 1 to 10 per 1 00g or ml of processed fruit. Thus, for their effective detection, it is important to analyse relatively large samples which may be centrifuged to provide a 25 concentrated sample (which may be frozen for storage).

The need for such a method and problems associated with known methods for detection, identification and enumeration of viable bacteria are set forth in Weaver et al., U.S. Pat. No. 4,959,301. Conventional methods for detection JO and/or enumeration of viable microorganisms are slow and generally labour intensive. Many of these methods measure some average property of a large number of cells which, under well-defined conditions, correlates with a count,

but which under other conditions generally does not correlate accurately with a viable count.

Known cell analysis methods involve two major classes of assays. The first 5 class detects and identifies specific cells directly from a primary sample. The most widely used in this class are specific ligand binding assays, e.g. immunoassays and genetic probes. However, they require many cells, and do not distinguish between dead and viable cells. This restricts their use to samples in which sufficient numbers of cells are present.

The second class of assays is used for viable cell determinations either directly using the primary sample, or using a subculture of the primary sample. The most traditional and widely used method is the plate count, which allows determination of single cell viability, based on growth, under many test 15 conditions (see, for example, Hattori The Viable Count: Quantitative and Environmental Aspects, Brock/Springer, Madison, 1988). An important attribute of viable plate enumeration is that the time required to obtain a determination is independent of the concentration of the cell in the sample, as formation of each colony proceeds from an initial single cell. The technique is so associated with being time-consuming (as typical determinations require about two weeks depending upon the microorganisms being assayed). In addition, the storage of the product over extended periods in specialized storage facilities whilst the test is being conducted is disadvantageous.

s However, viable plating is a well-established, important method for qualitatively determining the growth of cells, particularly the presence or absence of growth under given conditions, and is often based on the growth of initial cells into distinct colonies. Viable plating typically involves the spreading of a suspension of cells onto the surface of a gelcontaining petri dish, with or so without the pouring of a gel layer over the first gel surface.

The solid media are provided with nutrients, such that following an incubation period at a suitable temperature, many generations of growth occur, which leads to formation of visible colonies. For many microorganisms formation of visible colonies requires growth for 22 to 30 generations and therefore s produces colonies containing 107 to 109 cells. (See Sharpe, in Mechanizing Microbiology, A. N. Sharpe and D. S. Clark (Eds.) Charles C. Thomas, Springfield, 19-40, 1978).

Some microorganisms, in particular some ascospores of thermoresistant to moulds may become activated by heat, which promotes cell growth. In this regard, the composition of the heating medium may also influence the rate and extent of activation. For example, it has been taught that maximum activation of B. fulva and N. fischeri var. glaber ascospores may occur by heating samples at 70 C for 30 minutes in grape juice; in distilled water, 120 minutes 15 may be required for B. fulva, although only 1% of the N. fischeri ascospores may be activated (Splittstoesser, D. F. and Splittstoesser, C. M. 1977, Ascospores of Byssochlamys fulva compared with those of a heat-resistant Aspergillus. J. Food Sci. 42: 685). It is further taught that different strains within the same species may require different treatment times and to temperatures to achieve maximal activation. Thus, in thermally-processed foods, microorganisms such as thermoresistant moulds, may be activated.

The analysis of products such as plant products, for example fruit products, for contamination by thermally resistant moulds is typically laborious and time 25 consuming, requiring incubation of samples for at least 7 days and ideally up to 30 days (Hocking and Pitt, Food Spoilage Fungi II, Heat Resistant Fungi, CAIRO Food Res. Q44: 73-82, 1984). For example, impedance monitoring has been established as a useful tool for detection of bacterial contamination of a wide range of foods. The fundamental work conducted in this area by Williams and so Wood (Impedimetric Estimation Of Moulds, in Methods For The Mycological Examination Of Food, eds. A.D King, J.J Pitt, L.R Beuchst and J.E.L Corry, pages 230-238, New York Plenum Press) focused on the detection of yeasts in

various products: fruit mix (Fleischer et al; Estimation Of Yeast Members In Fruit Mix For Yoghurt, J.Soc. Dairy Technol, 37, pages 63-65, 1984), orange juice (Zindulis, J; A Medium For The Impedimetric Detection Of Yeast In Foods, Food Microbiol, 1:159-167,1984) and wine (Henschke and Thomas; Detection Of 5 Wine-Spoiling Yeasts By Electronic Methods, J.Appl. Bacteriol, 123-133, 1988). A detailed study of impedance induced by food spoilage moulds has been conducted by Watson-Craik et al (Development & Evaluation Of A Medium For the Monitoring Of Food-Borne Moulds By Capacitance Changes, Food Microbiol, 7: 129-145, 1990) which has enabled the development of a medium for general to impedimetric examination of food products. However, even these techniques are considered to take too much time from initiation to final determination of the presence and enumeration of microorganisms (typically 7 to 30 days).

A more recent method for the detection and enumeration of fungal hyphae and s conidia has been developed by the US Department of Energy (An Improved Method For Direct Fungi Identification And Enumeration; M.M. franck, R.L Brigmon, P.C McKinsey, M.A Heitkamp and C.B Fliermans; Westinghouse, Savannah River Company, Aiken, South Carolina - paper available from US Department Of Commerce, National Technical Information Service, 5285 Port ho Royal Road, Springfield, VA 22161). Adhesive tape impressions were made on

suspected fungal growth surfaces and the tapes stained using two stains, FUN-1 and Fluorescence brightener 28, together with potassium hydroxide A disadvantage of such a method is that it does not enable the determination of dormant spores which need to be activated. in addition, in order to examine the 25 samples by microscopy, an adequate amount of fungal material is required.

The present invention seeks to provide a useful method to detect microorganisms in a product.

i Summary Aspects

A seminal finding of the present invention is that microorganisms, in particular thermoresistant moulds, can be detected and/or enumerated in a product, in s particular a food stuff, or a sample thereof by exposure of the product to a compound which is capable of luminescing. In particular, the compound which is capable of luminescing is admixed with the product or a sample thereof and is surprisingly taken up by the microorganism thus enabling the detection thereof.

lo In a broad aspect, the present invention relates to the use of a compound capable of luminescing for the detection of a microorganism in a product.

Statements Of The Invention

Is Aspects of the invention are presented in the accompanying claims and in the following description.

In a first aspect the present invention relates to a method of detecting a microorganism in a product said method comprising admixing said product or a no sample thereof and a compound capable of luminescing such that said compound or a portion thereof is taken up by said microorganism in said product or a sample thereof, and detecting microorganisms comprising a luminescing compound.

25 In a second aspect, the present invention relates to a method of detecting a thermoresistant mould in a product said method comprising admixing said product or a sample thereof and a compound capable of luminescing such that said compound or a portion thereof is taken up by said thermoresistant mould in said product or a sample thereof, and detecting said thermoresistant mould so comprising a luminescing compound.

In a third aspect the invention relates to a composition comprising a foodstuff is in a form suitable for human and/or animal consumption or a sample thereof and a compound capable of luminescing.

5 In a fourth aspect the invention relates to a kit for detecting microorganisms in a product or a sample thereof said kit comprising: a) a composition comprising a foodstuff or a sample thereof and a compound capable of luminescing; and b) means for detecting microorganisms comprising a luminescing compound.

In a fifth aspect the invention relates to a method of identifying whether a product meets regulatory requirements, wherein said method comprises a step of forwarding the methodology of the method according to the present invention to a regulatory authority for approval.

For ease of reference, these and further aspects of the present invention are now discussed under appropriate section headings. However, the teachings under each section are not necessarily limited to each particular section.

20 Preferable Aspects Preferably the present invention relates to a method of detecting and/or enumerating a microorganism in and/or on a product or a sample thereof.

25 Preferably the present invention relates to a method of detecting and/or enumerating a viable microorganism within a product or a sample thereof.

Preferably the present invention relates to a method wherein the compound capable of luminescing does not affect the growth of the microorganism being so detected. In other words, preferably in the present invention the microorganism grows equally well in presence or absence of said compound capable of luminescing.

Preferably the product or a sample thereof and compound capable of luminescing are admixed prior to, during and/or after processing of said product or a sample thereof.

s In a preferred aspect the microorganism of the present invention is detected by optical and/or illumination means.

Preferably the microorganism of the present invention is detected by optical to and/or illumination means which may utilise one or more of the following: fluorescence microscopy, light microscopy, transillumination and/or direct visual examination. Preferably the microorganism of the present invention is detected by excitation of 15 the compound capable of luminescing by a light source at a specific wavelength; optionally together with a barrier filter to filter the luminescence and to selectively distinguish the luminescence caused by the specific compound capable of Iumnescng. to In a preferred aspect the microorganism as mentioned herein is a fungus.

In a more preferred aspect the microorganism as mentioned herein is from the class Ascomycetes or is an anamorph of a microorganism from the class Ascomycetes. As is readily apparent to a skilled person, the anamorph stage of :5 a microorganism from the class Ascomycetes may be classified in a different class. For example, Paecilomyces variotti is the anamorph'stage of the microorganism Themoascus crustaceus (of the Ascomycetes class). However, Paecilomyces variotti can also be classified in the class Deuteromycetes.

so In a further aspect the microorganism as mentioned herein is a mould.

In a further aspect the microorganism as mentioned herein is a thermoresistant mould. In a further preferred aspect the microorganism as mentioned herein is selected 5 from one or more of the genera: Byssochlamys, Eurotium, Neosatorya, Monascus, Talaromyces and Paecilomyces, including both the anamorph and/or teleomorph stage of any one thereof.

In yet a further preferred aspect the microorganism as mentioned herein is one o or more of the species: Byssochlamyces nivea, Eurotium amstelodami, Eurotium herbarorium, Monascus ruber, Neosafforya fischeri, Neosafforya pseudofischeri, Paecilomyces variotti, Talaromyces flavus and Talaromyces macrosporus. 15 Much by preference the microorganism as mentioned herein is viable.

Preferably, the microorganism is viable prior to, during and/or after the detection method according to the present invention.

Preferably the product or a sample thereof as mentioned herein is, or comprises, so any product capable of sustaining growth of a microorganism.

Preferably the product or a sample thereof as mentioned herein is a foodstuff, pharmaceutical product or cosmetic product.

25 In a preferred aspect the product or a sample thereof as mentioned herein may comprise a polysaccharide or a derivative thereof, for example the product may be a foodstuff comprising a polysaccharide or a derivative thereof or the product may be a pharmaceutical product comprising a polysaccharide or derivative thereof. By way of example only, such a polysaccharide, for example pectin, may so be used in combination with Kaolin for treating diarrhoca (Chenworth et a/ Metabolism and Physiological Effects of Pectins- in Hodge: Physiological effects of food carbohydrates, ACS. Symp. Series (1975) pp 312-324).

In yet a further preferred aspect the product or a sample thereof as mentioned herein is a foodstuff or a sample thereof.

s Preferably the product or a sample thereof as mentioned herein is a thermally-

processed product or a sample thereof.

In a preferred aspect the compound capable of luminescing as mentioned herein is a luminophore.

In a yet further preferred aspect the luminophore as mentioned herein is one or more of a fluorophore, a fluorochrome or a phosphor.

Much by preference the compound capable of luminescing as mentioned herein 15 iS a fluorochrome.

Preferably the compound capable of luminescing as mentioned herein is selected from one or more of the following fluorochromes; Primulin, Uvitox BOPT or Calcofluor White M2R.

Preferably, the compound capable of luminescing is a compound which may luminesce before, during and after being taken-up by a microorganism, for example a thermoresistant mould. Following uptake, into the hyphal tips and buds, the compound capable of luminescing may luminesce more intensely, 25 particularly in an transilluminator for example.

Suitably, however, the compound capable of luminescing is a pro-moiety which may only luminesce upon metabolism, i.e. by a microorganism. Example of pro-

moieties include entities that have certain protected group(s) and which may not so possess luminescent activity as such, but may, in certain instances, be taken up by a microorganism and thereafter be metabolised to form a luminescent compound. In particular, the microorganisms as mentioned herein may take up

the compound capable of luminescing and metabolise it, which results in the microorganism becoming more visible to visual examination, particularly in a transilluminator. 5 In a preferred aspect the compound capable of luminescing as mentioned herein may be conjugated. in particular the compound may be conjugated with a lectin for example. The term "conjugated" as used herein means a compound which may attached directly or indirectly to one or more further molecules.

to Preferably the method as mentioned herein is used in the preparation of a product or a sample thereof.

In a preferred aspect the compound capable of luminescing as mentioned herein may be taken up by a microorganism by endocytosis and/or by ingestion, for 15 example during nutrient absorption by fungal hyphae.

In a preferred aspect the detection of microorganisms according to the method of the present invention is rapid.

go In a more preferred aspect microorganisms may be detected according to the method of the present invention within five days after initiation.

In a more preferred aspect microorganisms may be detected according to the method of the present invention within three days after initiation.

In a more preferred aspect microorganisms may be detected according to the method of the present invention within two days after initiation.

In a preferred aspect the product or a sample thereof and the compound capable so of fluorescing are incubated.

Preferably the product or a sample thereof and the compound capable of fluorescing are incubated for up to five days, preferably up to three days.

Preferably the product or a sample thereof and the compound capable of 5 fluorescing are incubated under temperature and/or humidity and/or nutrient conditions which allow for the growth of a microorganism.

Preferably the product or a sample thereof and the compound capable of fluorescing are in a medium.

Preferably the product or a sample thereof and the compound capable of fluorescing are in an incubation medium.

Preferably the product or a sample thereof and the compound capable of 15 fluorescing are in a medium comprising nutrients and agar.

Preferably the product or a sample thereof is added to a medium prior to addition of the compound capable of fluorescing.

no Preferably, the method according to the present invention comprises a step wherein the microorganism, particularly the ascospores of thermoresistant mouids, is/are activated.

Preferably, the method according to the present invention comprises a step of 25 heating the microorganism.

Preferably, the method according to the present invention comprises a heating step in order to activate ascospores and/or to eliminate non-heat resistant flora (and mycelia of thermoresistant moulds if any) Preferably, the method according to the present invention comprises a step of heating the ascospores of thermoresistant moulds. Preferably, the heating step

is at a suitable temperature and for a sufficient time to activate dormant ascospores within the product or a sample thereof.

Preferably the heating step comprises heating to 75 C for 30 minutes.

Preferably the product or a sample thereof, or medium comprising same, is heated to 75 C for 30 minutes prior to addition of the compound capable of fluorescing. lo Some Advantanes Some advantages of the present invention are presented in the following commentary. 15 By way of example, a compound capable of luminescing according to the present invention is taken up, for example by endocytosis and/or ingestion, by the microorganism and thus a plurality of microorganisms may be detected using a single compound without the need to prepare a specific compound for each specific microorganism, i.e. as may be required by some prior art

so methods wherein the compound capable of luminescing is attached to a probe which is specific for a single or limited number of microorganisms.

In addition, the method according to the present invention is advantageous because there is a provided a rapid method for the detection and/or 25 enumeration of microorganisms, preferably viable microorganisms, in and/or on a product or a sample thereof, preferably a foodstuff or a sample thereof.

In particular, the method according to the present invention may allow for the early detection of the microorganism within a product or a sample thereof so without the need to wait until the microorganism is visible on the surface of the product or a sample thereof.

In particular, the method according to the present invention allows for detection and/or enumeration of the microorganism within approximately 5 days, preferably within about 3 days, more preferably within about two days, after initiation of the method.

In addition, the method and/or kit according to the present invention is/are advantageous because there is provided a method for rapidly and routinely monitoring products for quality control or grading purposes.

to By way of further example, the method according to the present invention is advantageous because it reduces the time of storage required for products awaiting testing compared to that required when using traditional detection techniques such as plating techniques.

15 By way of further example, the method according to the present invention is advantageous because it reduces the amount of storage and time required for storing test samples during the method as compared to that required when using traditional detection techniques such as plating techniques.

20 In addition, the method according to the present invention is advantageous because it provides a rapid detection method which may be automated and may comprise video image analysis technology and/or cell counting technology, such as a cytometer.

25 Microorganism Microorganisms for detection in the present invention may be any microorganism capable of colonising a product or a sample thereof, in particular a foodstuff or a sample thereof. In particular, microorganisms for so detection in the present invention are fungi, preferably moulds, particularly those from the Ascomycetes class or an anamorph of a microorganism from the class Ascomycetes. Much by preference the microorganism for detection in

the present invention are thermoresistant moulds, particularly from the class Ascomycetes, (including both anamorph and/or teleomorph stages of said thermoresistant moulds).

5 The class Ascomycetes comprise fungi which includes among its members a number of useful, familiar, and important eukaryotic organisms.

The class Ascomycetes includes fungi from the following genera: Byssochlamys, Chaetomium, Emencella, Eurotium, Neosafforya, Monascus, Talaromyces, lo Paecilomyces and Xeromyces.

Preferably, the microorganism detected by the present method is found in one or more of the following genera: Byssochlamys, Eurotium, Neosafforya, Monascus, Talaromyces or Paecilomyces, including both the anamorph and/or teleomorph 15 stage of any one thereof.

The fungal species found in the class Ascomycetes are identifiable by their ability to produce ascospores. Ascospores are formed in the asci after meiosis. Asci are usually enclosed in ascomata (ascocarps), that is fruit-bodies o which occur singly or aggregated in or on a stroma (a mass of vegetative hyphae). The ascospores, when placed on a suitable medium, may germinate and produce hyphae which absorb nutrients from the medium. Hyphal growth, 25 nuclear division and branching may occur to form a colony. The hyphae of a growing colony may form a mycelium throughout the medium.

Preferably, the method of the present invention is capable of detecting the mycelium of a microorganism growing on or within a product or a sample o thereof. More preferably, the method of the present invention is capable of detecting the mycelium of a microorganism growing within a product or a sample thereof.

Thermoresistant Mould As used herein the term 'thermoresistant mould"relates to moulds which are 5 capable of producing propagules (ascospores) which can survive heat treatments typically associated with the processing of food-stuffs, for example a heat treatment of 75 C for 30 minutes. Such moulds may, thus, survive food processing wherein heating is employed lo Viable As used herein the term "viable" means a living microorganism, i.e. a, microorganism which is capable of living, capable of developing and/or of surviving parturition. The term "viable" further encompasses dormant 15 microorganisms, such as spores.

Product Suitable products for use in the present invention include, but are not limited do to, a foodstuff, a pharmaceutical product or a cosmetic product or a sample of any one thereof.

Suitably, the method of the invention may be used to detect microorganisms on equipment used for handling, processing, packaging or storing a foodstuff, 25 a pharmaceutical product or a cosmetic product or a sample of any one thereof. By way of example, the method of the invention may be used to detect microorganisms in a rinse solution taken from a package or a container suitable for a foodstuff, a pharmaceutical product or a cosmetic product or a sample of any one thereof.

Suitably the method of the invention may be used to detect microorganisms in one or more components of a foodstuff, a pharmaceutical product or a

cosmetic product or a sample of any one thereof. By way of example the method of the invention may be used to detect microorganisms in a component such as an emulsifier.

5 As used herein the term "product" further relates to a component for use in the preparation of a product or a sample thereof or to a final product or a sample thereof for animal or human use and/or consumption.

The product or a sample thereof as mentioned herein may be admixed with a To compound capable of luminescing prior to, during or after processing said product or a sample thereof.

As used herein the term "processing" relates to one or more mechanical or chemical operations typically applied to a product or a sample thereof in order to 15 change or preserve said product or a sample thereof.

Sample Suitably the method of the invention may be used to detect microorganisms in 20 a sample taken from a product. As used herein the term "sample" means a portion of a product. Preferably the sample is a representative part of a product, for example a part of the product presented for inspection or analysis.

Solid / Liquid Suitably the foodstuff, pharmaceutical product or cosmetic product or a sample of any one thereof, may be a liquid or a solid.

In the case where the foodstuff, pharmaceutical product or cosmetic product or so a sample of any one thereof, is placed in a medium, suitably the medium may be a liquid or a solid.

In the case where the foodstuff, pharmaceutical product or cosmetic product or a sample of any one thereof and the compound capable of fluorescing are placed in an incubation medium, suitably the incubation medium, when analysed, is in a solid form.

Suitably the compound capable of fluorescing is added to a foodstuff, pharmaceutical product or cosmetic product or a sample of any one thereof, when the foodstuff, pharmaceutical product or cosmetic product or a sample of any one thereof is in a liquid medium.

Suitably the method of the present invention may be used to detect microorganisms in solid foodstuffs or a sample thereof.

The term "solid" as used herein includes firm gels and/or soft gels.

Foodstuff Preferably the product or a sample thereof for use in the present invention is a foodstuff or a sample thereof.

The term ''foodstuff'' as used herein may include food for animal and/or human consumption. Typical foodstuffs include plant products or algae products and products processed from plants and/or algae, fruit products or products processed from fruit such as preserves (for example jam), dairy products such as 25 milk, desserts (such as mousse), yoghurt or cheese, cereal and seed products for example those comprising wheat or maize, meat products, poultry products, fish products and bakery products. The foodstuff or sample thereof may even be a beverage. The beverage can be a drinking yoghurt, a flavoured milk, a fruit juice or a beverage comprising whey or soy protein. The term "foodstuff" so encompasses animal feed.

As used herein the term "foodstuff" may include any food for animal and/or human consumption that may be restructured and/or reconstituted prior to use.

The term "foodstuff' may refer to the food or feed prior to and/or after reconstitution. Preferably the foodstuff or a sample thereof as mentioned herein is a plant product or product processed from plants.

In a further preferred aspect the foodstuff or a sample thereof as mentioned to herein comprises a polysaccharide such as carrageenan, alginate, locust bean gum, or a pectin or a derivative thereof.

As used herein the term "foodstuff" further relates to a component for use in the preparation of a foodstuff or to a final product for animal or human consumption.

The foodstuff or a sample thereof as mentioned herein may be admixed with a compound capable of luminescing prior to, during or aver processing said foodstuff or a sample thereof.

so As used herein the term "processing" relates to one or more mechanical or chemical operations typically applied to a foodstuff in order to change or preserve said foodstuff.

As used herein the term "thermally-processed" relates to heating a foodstuff or a 25 sample thereof in order to change or preserve said foodstuff or a sample thereof.

If deemed appropriate, prior to carrying out the method of the invention it may be necessary to acidify the foodstuff or a sample thereof and/or add one or more antibacterial agents, such as chloramphenicol for example, to said foodstuff or a so sample thereof.

Derived from Pectin The term "derived from pectin" includes derivatised pectin, degraded (such as partially degraded) pectin and modified pectin. An example of a modified pectin 5 iS pectin that has been prior treated with an enzyme such as a PME. An example of a pectin derivative is pectin that has been chemically treated, e.g. amidated. Compound Capable Of Luminescino As used herein the term "a compound capable of luminescing" relates to compounds which are luminophores, i.e. capable of fluorescing, phosphorescing and/or chemiluminescing.

15 The compound capable of luminescing may be, for example, one or more of the following compounds: a fluorogenic compound such as a fluorochrome and/or a fluorophore, for example polyaromatic hydrocarbons or heterocycles, which fluoresce in the presence of certain stimuli, for example an incandescent lamp or a laser or when present in a specific environment; a chemiluminescent to compound which is capable of producing visible light as a result of a chemical reaction; a phosphor which emits visible light as a result of absorbing energy derived from exciting radiation of a shorter wavelength.

The compound capable of luminescing may be localised within a specific 25 region of a biological specimen.

In the presence of a stimulus the compound capable of luminescing may undergo conformational changes or may be subject to several possible interactions with its molecular environment.

Suitable stimuli for the compounds capable of luminescing in the present invention may include, for example, a change in the compound's environment

on being taken up by the microorganism, metabolism by the microorganism, conformational modification during metabolism, enzymatic degradation, or cellular mechanisms.

5 Preferably, the compound capable of luminescing may luminesce, preferably fluoresce, all of the time, i.e. prior to, during and after being taken up by the microorganism. More preferably, the compound capable of luminescing may luminesce, to preferably fluoresce, more intensely after uptake by a microorganism as compared with immediately prior to uptake. The intensity of the luminescence may change due to the compound binding with one or more cell components in said microorganism.

15 Suitably, the compound capable of luminescing may only luminesce, preferably fluoresce, when taken up by a microorganism.

As used herein the terms "fluoresce, fluorescing and fluorescence" relate to a type of luminescence that consists of the emission by a substance of ho electromagnetic radiation, especially visible light, as a result of and immediately (typically 10-100 ns) after the absorption of energy derived from exciting radiation of another, usually shorter, wavelength or from incident subatomic particles (especially electrons or alpha particles).

25 To date, no one has suggested the direct use of a compound capable of luminescing, in particular fluorescing, for the detection of a microorganism in a product or a sample thereof, such as a foodstuff. In particular, prior to the present invention it had not been contemplated to admix a foodstuff with a compound capable of luminescing such that the compound or part thereof is -,o taken up by the microorganism, and perhaps metabolized thereby, thus facilitating the detection and/or enumeration of said microorganism. By way of example, reference can be made to the following teachings.

Mansour et al., U.S. Pat. No. 4,693,972 teaches a composition and method for rapid detection of microorganisms in clinical samples. The method for detection of microorganisms in a body fluid sample includes detecting a s microorganism after treatment of the sample with a Iysing agent in order to dissolve sample components other than microorganisms, and staining with a fluorescent dye.

Wolf et ale U.S. Pat. No. 4,972,258 discloses a scanning laser microscope lo system and methods of use. The patent teaches an indirect immunofluorescence assay in which a target microorganism is labelled with a primary non-conjugated antibody specific for microorganisms containing a target surface antigen. The microorganisms with bound antibody are fluorescently labelled using a fluorescein isothiocyanate conjugated secondary s antibody which recognises the primary antibody. The labeiled microorganisms are then placed on an appropriate surface and imaged using a scanning laser microscope. A filter is used which reflects the laser light and passes the fluorescence light.

so Melnicoff et al., U.S. Pat. No. 5,256,532 discloses methods, reagents and test kits for determining populations of biological entities. The patent discloses coupling of a detectable reporter substance nonselectively to prokaryotic cells and contacted with a substance which specifically binds to the cells. Following separation of the components of the test sample, the occurrence of the 25 reporter substance in the sample may be detected by fluorescence.

Waggoner et al. U.S. Pat. No. 5,268,486 is directed to a water soluble luminescent dye for use in immunoassays and which can be used for the detection of bacteria.

Chemical Abstracts Vol. 102 (1985) 42486t discloses a solid phase immunoassay for detecting bacteria. The immunoassay uses an

immunoreactant attached to cells such as bacteria and a fluorescent or phosphorescent label.

Sheridan et al teach an immunofluorescence technique used for the detection s of viable Listeria (Sheridan, J.J., I. Walls, J. McLaughlin, D. McDowell & R. Welch. 1991. Use of a microcolony technique combined with an indirect immunofluorescence test for the rapid detection of Listeria in raw meat. Lett.

Appl. Microbiol. 13:140-144) and Salmonella (Roderigues, U.M. 8 R.G. Kroll.

1990. Rapid detection of salmonellas in raw meats using a fluorescent JO antibody-microcolony technique. J. Appl. Bacteriol. 68:213-223).

Hahn et al teach of fluorescently labelled oligonucleotide probes which have been used for detection of microorganisms in soil (Hahn, D., R.l. Amann, W. Is Ludwig, A.D.L. Akkermans & K.-H. Schleifer. 1992 Detection of micro-

organisms in soil after in situ hybridization with rRNA-targeted, fluorescently labelled oligonucleotides. J. Gen. Microbiol. 138:879-887).

As used herein the term "fluorochrome" means any substance exhibiting Jo fluorescence, especially one used to impart fluorescence to another molecule or to a specific structure in a biological specimen.

As one skilled in the art would readily appreciate a single type of compound capable of luminescing may be used. However, suitably more than one 25 compound capable of luminescing may also be used in combination.

The compound capable of luminescing as mentioned herein may be suitable for animal and/or human use or consumption, i.e. may be a food grade compound.

Uptake Of A Compound Capable Of Luminescing By Microorganisms As used herein the term "uptake" means the taking in of a substance, in particular a compound capable of luminescing may be taken up by the s microorganism by mechanisms such as endocytosis and/or ingestion, for example during nutrient absorption by fungal hyphae.

Metabolism to As used herein the term "metabolism" relates to one or more chemical reactions or physical processes on the compound capable of luminescing by a microorganism, in particular a thermoresistant mould.

Application Of A Compound Canabie Of Luminescinn If the compounds capable of luminescing, preferably fluorescing, are used during the preparation of products or samples thereof for animal or human use and/or consumption they may be indirectly or directly applied to the product or a sample thereof.

An example of the application methods which may be used in the present invention, includes admixing the compound capable of luminescing with the product or a sample thereof.

25 Much by preference the product or a sample thereof is placed in a medium which is admixed with a further medium comprising the compour'd capable of fluorescing. Suitably the medium comprises the product or a sample thereof which may be the same as or different from the medium comprising the compound capable of fluorescing.

Suitably, the compound capable of luminescing may be included in the emulsion or raw ingredients of a product, or a sample thereof, or to the interior

of a solid product, or a sample thereof, formed therefrom, by injection or tumbling. Amount of Compound Capable Of Luminescinn The optimum amount of the compound capable of luminescing to be used in the present invention will depend on the product or a sample thereof to be treated and/or the method of contacting the product or a sample thereof with JO the compound capable of luminescing. The amount of compound capable of luminescing should be in a sufficient amount to be effective to detect all or substantially all of the viable microorganisms in a product or a sample thereof following admixing of the product or a sample thereof and the compound capable of luminescing.

As a skilled person would be readily aware, the quantity of compound capable of luminescing applied to the product or a sample thereof should be in an amount to be effective and to remain effective to detect a microorganism, such as a thermoresistant mould, for a suitable length of time. This length of time for so effectiveness should extend at least from the time of contacting to, and including, the time of detection of the microorganism. Preferably, the compound capable of luminescing is stable and visible for at least up to 14 days in incubation at 30 C. Suitably, a solution of the compound capable of luminescing may be stable for several months when kept in the dark at room as temperature.

Typically the compound capable of luminescing is applied to a foodstuff in an amount of from 10 to 100 parts per million, preferably from 30 to 70 parts per million, more preferably from 55 to 65 parts per million, more preferably 60 30 parts per million.

If deemed appropriate, the compound capable of luminescing may remain effective throughout the normal "shelf-life" of the product or a sample thereof.

In such circumstances, the compound could be used to detect product spoilage or spoilage of a sample of product caused by microorganisms during 5 the shelf-life of the product or a sample thereof. The desired lengths of time and normal shelf-life will vary from product to product and those of ordinary skill in the art will recognise that shelf-life times will vary depending upon, for example, the type of product or a sample thereof, storage temperatures, processing conditions, and packaging material used. In this way, it may be JO possible to detect the growth of microorganisms in a product or a sample thereof, in particular a foodstuff for example jam, during the shelf life of said product or a sample thereof.

Detecting Microorganisms Comprising a Luminescing Compound Methods for quantifying luminescence, for example fluorescence, are well known in the art for example U.S. patent 4,626,684 and U.S. patent 4,501,970.

In general, fluorescence detection systems comprise four elements: go (1) an excitation source, (2) a fluorochrome, (3) wavelength filters to isolate emission photons from excitation photons and (4) a detector that registers emission photons and produces a recordable output, usually as an electrical signal or a photographic image.

25 Microorganisms comprising a luminescing compound as mentioned herein may be detected by optical and/or illumination means.

Preferably the microorganisms of the present invention are detected by optical and/or illumination means which may utilise one or more of the following: 30 fluorescence microscopy, light microscopy, transillumination, illumination and/or direct examination.

Suitably an additional step of the methods of the present invention may comprise an identification step, suitably by the use of microscopic means.

Suitably the optical and/or illumination means as mentioned herein may be 5 used quantitatively with a flow cytometer.

Suitably the optical and/or illumination means as mentioned herein is automated and may comprise video image analysis technology and/or a cytometer, thus enabling detection and/or counting of the one or more JO microorganisms.

Suitably the compound capable of luminescing is detectable by direct visual examination. 15 The optical and/or illumination means for use in the present invention may be enhanced by treating or exposing the microorganisms to at least one staining process. Representative stains which may be suitable for detecting microorganisms Jo comprising a luminescing compound according to the present invention include stains indicative of biological composition, stains indicative of enzyme activity, and stains indicative of cell membrane integrity. Such stains are generally selected to have readily measurable properties such as fluorescent stains, light absorbance stains and light scattering stains, and can be further selected according to the class of biological material which is stained, including, therefore, stains such as nucleic acid stains, protein stains, lipid stains, cell membrane stains, cell wall stains, stains responsive to enzyme activity, stains responsive to transmembrane potentials and cell surface receptor stains.

Preparation Of A Composition The composition of the present invention may be prepared by admixing the compound capable of luminescing directly or indirectly with a foodstuff or a 5 sample thereof. Mixtures of compounds capable of luminescing may also be used and may be applied separately, simultaneously or sequentially.

All publications mentioned in the above specification are herein incorporated by

reference. Various modifications and variations of the described methods and to system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of 15 the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be

within the scope of the following claims.

The present invention will now be described only by way of example.

EXAMPLES

EXAMPLE 1

25 Determination of the inhibitory effect of fluorochromes on the growth of thermoresistant moulds in a regular TRM medium using a nonconventional method. 1.1 METHOD

Thermoresistant mould (TRM) strains Byssochlamyces nivea, Eurotium amstelodami, Monascus ruder, Neosafforya fischeri, Neosartorya pseudofischeri, Paecilomyces variotti, and Talaromyces macrosporus were grown in Malt Extract Agar for 25 days at 30 C. From each strain, ascospores

were harvested with a sterilized needle and distributed in 1 ml of dilution water (19 peptone, 8.5 g sodium chloride and 1g Tween 80/L). The moulds were squashed against the tube wall several times and left for 5 minutes at room temperature to disrupt the asci and free ascospores. One tenth of the liquid 5 was diluted ten fold and heat treated for 15 minutes at 75 C.

The solution comprising the activated ascospores was then inoculated (using a needle) into two regular TRM media as detailed in Table 1.

o Table 1

Comparison of medium composition.

Composition, g per litre IngredientControl TRM MediumTRM Medium with Fluorochrome Malt Extract Broth35.535 5 Peptone7.07 0 Dextrose25.025.0 Agar-agar7.07 0 Pectin28.028.0 Calcofluor White0.00.06 15 The cultures were incubated at 30 C for five days and the colony diameter measured daily. Three replications were used for each medium and the data obtained were analysed by ANOVA.

The difference between this method and the conventional TRM method is that 20 the heat treatment at 75 C lasts for a full 30 minutes in the conventional TRM method whereas it only lasts for 15 minutes in the above identified non-

conventional method. Thus, in contrast to the conventional method, i.e. heat treatment for 30 minutes at 75 C, in the non-conventional method detailed above the mycelia of the thermoresistant moulds may not been killed.

25 However, the non-conventional method used the same "TRM7' medium as the conventional method and shows that the presence of fluorochrome in the medium does not affect the growth of the thermoresistant moulds therein.

1.2. RESULTS

The Results are presented in Table 2.

The TRM mould growth was observed after the first incubation day in both the control TRM medium and TRM medium containing a fluorochrome. (This occurred due to the "non-conventional" method used (i.e. heated for 15 minutes at 75 C) which did not fully kill the fungal mycelia. The fungal colonies Jo would not normally be observed, using the conventional TRM method (i.e. heated for 30 minutes at 75 C) within the first approximately 7 days without the use of a fluorochrome). Almost all the mould colonies showed good growth with the exception of Eurotium amstelodami (which is xerophilic and requires low water activity content).

ANOVA analysis demonstrated that the presence of a fluorochrome in the regular TRM medium did not have a significant effect (P>0.001) on mould growth. 20 Table 2

Colony growth of TRM in two media incubated at 30 C for five days (colony diameter, mm).

Regular TRM Medium, mm Regular TRM Medium with Fluorochrome Strain day1 day2 days day4 Day 5 day 1 day2 days day4 day 5 Bissochlamyces nivea 17 28 48 71 78 22 27 35 60 64 Eurotiumamstelodami 2 3 5 7 9 2 4 4 6 6 Monascus ruder 14 22 25 30 41 8 10 19 20 25 Neosartorya fischeri 15 25 36 47 55 16 26 34 45 58 Neosartorya pseudofischeri 12 24 37 65 67 28 37 40 50 58 Paecilomyces varioffi 20 24 30 38 45 17 26 33 42 46 Talaromyces macrosporus 12 23 33 41 49 14 27 37 45 53

Table 3 demonstrates that the growth of mould using the non-conventional method (as detailed above), measured as colony diameter, provided similar results in the control regular TRM medium and the regular TRM medium containing a fluorochrome (P>0.001).

Table 3

Analysis of variance for colony diameter from TRM cultivated in two different medium, control regular TRM medium and regular TRM medium with fluorochrome. Source Sum of Squares Of Mean Squares F-ratio P-value MAIN EFFECTS

A: Incubation period 9847.51 4 2461.88137.56 0.0000 B: Medium composition 41.6571 1 41.65712.33.1402 C: TRM strain 11191.8 6 1365.3104. 23.0000 INTERACTIONS

AB 140.486 4 35.12141.96 0.1326

AC 2310.49 24 96.27025.38 0.0001

BC 369.343 6 61.66713.44.0136

RESIDUAL 429.514 24 17.8964

TOTAL (CORRECTED) 24330.8 69

Initial investigations showed that the growth of thermoresistant mould was similar in both the control regular TRM medium and regular TRM medium containing a fluorochrome. Thus, the presence of fluorochrome does not affect 15 thermoresistant mould growth in regular TRM medium.

The results obtained using a regular TRM medium comprising a'fluorochrome were in accordance with those obtained using the control regular TRM medium, using the non-conventional method.

1.3 CONCLUSION

The results thus demonstrate that the addition of a fluorochrome does not affect mould growth.

EXAMPLE 2

Determination of growth of thermoresistant moulds in pectin.

2.1 DCMXT METHOD

Abstract Jo A pectin solution was aseptically heat treated 30 minutes at 75 C for activating ascospores and to eliminate non-heat resistant flora (and mycelia of thermoresistant moulds if any). Subsequently the emulsion was enriched with Malt Extract Medium containing a fluorochrome. The resulting emulsion was incubated for nine days at 30 C and analysed on day three and day nine for Is fluorescent mycelia in a transilluminator equipped with white and UV light.

2.1.1 Reagents Dextrose solution: to Dissolve 25 grams of dextrose food grade in 600 ml of deionized water and Autoclave 15 min at 121 C.

Malt Extract Medium: Dissolve 35.5 g of Malt Extract Broth (OXOID No. CM57) and 7.0 g of Agar agar purified and free from inhibitors MERCK No. 1.01614.1000 in 400 ml of 25 water. Autoclave 15 min at 121 C.

Fluorochrome solution: Under aseptic conditions dissolve 1 gram of Calcofluor White SIGMA No. F 3543 in 1000 ml of deionized sterilised water. This solution is stable for 4 months. Dissolve 1:10 in deionized sterilised water the day you used in the Jo medium, use 60 mis per sample.

Shear's mounting fluid: Dissolve 3 g potassium acetate, 150 ml water, 60 ml glycerine, 90 ml ethanol (95%) Lactic acid 85%

Ethanol 95%.

2.1.2 Apparatus Stomacher 3500 Lab System (BIKMAN, USA).

5 Plastic container, NALGENE No. 2105-0032 (NALGENE, USA).

Blue cap flask, Schot Duran 1000 ml capacity.

Balance. Water bath with recycling water at 75 C Whirl-Pak bag, 52 ounce, 1560 ml capacity, No. B01239WA (NASCO, USA) to Ultraviolet Fluorescence Analysis Cabinet, SPECTROLINE Mod. CC-80 (SPECTROLINE, USA)

Barrier filter 530 nm, SIRCHIE Mod. BMS200 (SIRCHIE, USA) White light lamp.

Ultraviolet lamp, long wave 365 nm.

15 Petri dishes 150 x 15 mm.

Incubators at 30 C pH meter Thermometer Microscope go Microscope slide Cover glass.

2.1.3 Procedure 25 28 grams of the pectin sample were weighed aseptically in a Whirl-Pack bag and 600 ml of dextrose solution at 50 C added. The pectin was dissolved in a stomacher for 3-4 minutes after which time the stomacher bag was heat-

treated in a water bath at 75 C. After 30 minutes the sample was cooled to about 55 C in water at room temperature. 60 ml of fluorochrome solution were so added aseptically to the sample (in a cabinet), and the bag placed on the stomacher for 3 minutes. The content of the stomacher bag was added aseptically (within a cabinet) to a flask containing 400 ml of the malt extract medium at 50-55 C and homogenized thoroughly. The final emulsion was then poured into Petri dishes (within the cabinet).

The samples were placed in an incubator at 30 C and examined after three and nine incubation days in a transilluminator using UV and white light. 5 Where no visible fluorescent mould structures were observed, the samples

were incubated up to nine days. If, after the nine days, no visible structures were observed, the sample was classified as Not Found TRM/25 g.

On observation of fluorescent mould structures, the mould was classified as to confirming a positive TRM result.

For samples providing a positive result, a piece of fungus (from the colony centre) was transferred to a microscope slide with a sterile needle and a drop of water or Shear's mounting fluid or lactic acid 85% (in a flow laminar 15 cabinet). The preparation was covered with a cover glass. (In samples where there was not enough fungal material for microscopic identification more incubation days were needed.) 2.1.4 Microscopic Identification Ascospores, fruit-bodies and mycelia of mould species were identified (Plates 12 to 23 of Samsom, R. A., Hoekstra, E. S. Frisvad, J. C., and Filtenborg, O. 2000 - Introduction to Food-And Airborne fungi. CBS, Utrecht; The

Netherlands. ISBN 90-70351-42-0 (the contents of which are incorporated 25 herein by reference) were used for comparative purposes.

In samples classified as positive in which it was not possible to identify the TRM species, the samples were incubated for a total period of 14 days and then re-examined under the microscope.

2.1.5. CONCLUSION

The presence of a fluorochrome in media does not significantly affect the growth of thermoresistant mould.

The presence of thermoresistant moulds, such as Paecilomyces variotti and Bissochlamyces nivea, can be determined at least 3 days after incubation. The Paecilomyces spp. colonies had sufficient mycelia on the third incubation day 5 to be detected.

EXAMPLE 3

Comparison between two TRM determination methods; a conventional TRM 10 method (Samsom) and the DCMXT determination method of the present invention, using alpinate, carreaeenan, locus bean gum, yogurt and pectin samples. In order to compare the detection time between a conventional TRM method 15 (Samsom) and the DCMXT method of the present invention, a comparison was carried out using several food-stuff samples. Samples of pectin, alginate, carrageenan, locus bean gum, pectin and a commercial yogurt sample were spiked with thermoresistant mould (TRM) spores (approximately 100 + 50 spores for each 25 grams). The samples were the divided and detection of go TRM growth determined in each sample using both the detection method of the invention and that of Samsom.

3.1 Conventional TRM method (Samsom) [control methods 25 Detection of TRM growth was conducted using the method according to Samsom, R. A., Hoekstra, E. S. Frisvad, J. C., and Filtenborg, O. in Introduction to Food-And Airborne fungi. CBS, Utrecht; The Netherlands.

ISBN 90-70351-42-0, 2000, page 288, Enumeration of heat resistant fungi'.

30 Duplicate 12.59 samples were dissolved in 250 ml of Ringer's solution, heat treated for 30 minutes at 75 C, and cooled at 50 . The content of the stomacher bag was then transferred aseptically to a bottle with 250 ml of

double strength Malt Extract Agar (50 C). Antibiotics were added and mixed thoroughly. The samples were then poured into sterile plastic 14 cm diameter Petri-dishes (approx. 8) and incubated at 28 C.

5 3.2 DCMXT method 25g samples of alginate, carrageenan, locus bean gum, yogurt or pectin were weighed aseptically in a Whirl-Pack bag and 600 ml of dextrose solution at 50 C added. The samples were dissolved in a stomacher for 3-4 minutes after lo which time the stomacher bag was heattreated in a water bath at 75 C. After 30 minutes the sample was cooled to about 55 C in water at room temperature (preventing the medium and sample from solidifying). 60 ml of fluorochrome solution were added aseptically to each sample (in a cabinet). The stomacher bags were placed on the stomacher for 3 minutes. The contents of each stomacher bag were added aseptically (within a cabinet) to flasks containing 400 mi of malt extract medium at 50-55 C and homogenized thoroughly. The final emulsion was then poured into Petri dishes (within the cabinet).

* Samples from each method were placed in an incubator at 30 C and examined 20 every twelve hours up to three incubation days in a transilluminator using UV and white light.

The method was run in duplicate and two readings were taken independently in order to agree whether a mould structure was detected and not something 25 other than a would structure.

3.3 RESULTS

TRM detection is faster by the method of the present invention (DCMXT) than so by the conventional TRM detection method (Samsom). In each foodstuff sample mould structures were observed after 36 hours by the DCMXT method compared to detection after 60 incubation hours by the Samsom method.

Addition of fluorochrome to the medium enhances mould structure detection in the transilluminator in which small point mould structures are detectable as "little stars". Such material is not visible by eye at such early incubation periods 5 using the conventional TRM method (Samsom) (Tables 5 and 6).

Table 4. Detection of Thermoresistant moulds in several foodstuffs spiked with Thermoresistant Mould spores using the conventional TRM (Samsom).

Incubation Time (hrs) Sample 12 24 36 48 60 72 Alginate _ Carrageenan + Locus Bean + + Yoghurt + + Pectin + + lo - no mould structure is observed.

+ Small mould structure, difficult to observe and more growth is required to confirm presence + Small mould structures observed.

++ Mould structure is observed and identified easily

Table 5. Detection of Thermoresistant moulds in several foodstuffs spiked with Thermoresistant Mould spores using the DCMXT method of the present invention. Incubation Time (hrs) Sample 12 24 36 48 60 72 AIginate + + ++ ++ Carrageenan Locus Bean + + ++ ++ Yoghurt ++ ++ Pectin An additional observation made in the course of conducting the present comparison was the presence of a brown opaqueness in samples due to the presence of malt agar. As a result it has been found that the use of thin layers of agar are advantageous for observing the growth of mould structures within a to medium. Results from both the conventional TRM detection method and the DCMXT method of the present invention have been compared using approximately the same depth of medium in the petri-dishes.

CONCLUSIONS

The comparison between the conventional TOM detection method (Samson) and the DCMXT method of the present invention (as described in EXAMPLE 2 "Determination of growth of Thermoresistant moulds in pectin") demonstrate that the method of the present invention allows conditions for easier and faster so detection of Thermoresistant Mould in foodstuffs, or a sample thereof, such as alginate, carrageenan, locus bean gum, pectin and commercial yoghurt samples.

The use of a fluorochrome in a sample medium and a transilluminator provides a convenient and rapid method for detecting the presence of small mould structures in a product, e.g. a foodstuff, or a sample thereof. In addition, growth of small mould structures may be observed more rapidly using a 5 fluorochrome in a sample medium, for example in a sample medium poured to the depth of a thin layer within a petri-dish.

Claims (29)

1. A method of detecting a microorganism in a product said method 5 comprising admixing said product or a sample thereof and a compound capable of luminescing such that said compound or a portion thereof is taken up by said microorganism in said product, and detecting microorganisms comprising a luminescing compound.

to

2. A method according to claim 1 wherein the luminescing microorganisms are detected by optical and/or illumination means.

3. A method according to claim 2 wherein the optical and/or illumination means may be one or more of fluorescence microscopy, light microscopy, transillumination, illumination or direct visual examination.

4. A method according to claim 1 wherein the microorganism is a fungus.

5. A method according to claim 4 wherein the fungus is from the class 20 Ascomycetes or is an anamorph of a microorganism from the class Ascomycetes.

6. A method according to claim 4 or claim 5 wherein the fungus is a mould.

25

7. A method according to any one of claims 4 to 6 wherein the fungus is a thermoresistant mould.

8. A method according to any one of claims 4 to 7 wherein the fungus is one or more of the genera: Byssochlamys, Eurotium, Neosafforya, Monascus, so Talaromyces or Paecilomyces, including both the anamorph and/or teleomorph stage of any one thereof.

9. A method according to any one of claims 4 to 8 wherein the fungus is one or more of the species Byssochlamyces nivea, Eurotium amstelodami, Eurotium herbarorium, Monascus ruber, Neosafforya fischeri, Neosafforya pseudofischeri, Paecilomyces variotti, Talaromyces flavus and Talaromyces 5 macrosporus.

10. A method according to any one of claims 1-9 wherein the product or a sample thereof is any one or more of the following: a foodstuff, a pharmaceutical product, a cosmetic product or a sample of any one thereof.

11. A method according to any one of claims 1-9 wherein the product or a sample thereof is a foodstuff or a sample thereof.

12. A method according to claim 10 or claim 11 wherein the foodstuff or a 15 sample thereof is a plant product or product processed from plants.

13. A method according to any one of claims 10 to 12 wherein the foodstuff or a sample thereof comprises a polysaccharide such as carrageenan, alginate, locust bean gum or a pectin or a derivative thereof.

14. A method according to any one of claims 10 to 13 when dependent upon claims 7 to 9 wherein the foodstuff or a sample thereof is thermally processed.

15. A method according to any one of the preceding claims wherein the 25 compound capable of luminescing is a luminophore.

16. A method according to claim 15 wherein the luminophore is one or more of a fluorophore, a fluorochrome or a phosphor.

so

17. A method according to claim 15 or claim 16 wherein the luminophore is a fluorochrome.

18. A method according to claim 16 or claim 17 wherein the fluorochrome may be one or more of Primulin, Uvitox BOPT or Calcofluor White M2R.

19. A composition comprising a foodstuff in a form suitable for human and/or 5 animal consumption or a sample thereof and a compound capable of luminescing.

20. A composition according to claim 19 wherein the foodstuff or a sample thereof is thermally processed.

21. A composition according to claim 19 or claim 20 wherein the foodstuff or a sample thereof is one or more of the following: algae products; products processed from algae; plant products; products processed from plants; fruit products; products processed from fruit; dairy products; cereal products; seed 15 products, meat products, poultry products, fish products and bakery products.

22. A composition according to any one of claims 19 to 21 wherein the foodstuff or a sample thereof comprises a polysaccharide such as carrageenan, alginate, locust bean gum or a pectin or a derivative thereof.

23. A composition according to any one of the preceding claims wherein the compound capable of luminescing is a luminophore.

24. A composition according to claim 23 wherein the luminophore is one or s more of a fluorophore, a fluorochrome or a phosphor.

25. A composition according to claim 23 or claim 24 wherein the luminophore is a fluorochrome.

so

26. A composition according to claim 24 or claim 25 wherein the fluorochrome may be one or more of Primulin, Uvitox BOPT or Calcofluor White M2R.

27. A method substantially as described herein.

28. A composition substantially as described herein.

5

29. A method of identifying whether a product meets regulatory requirements, wherein said method comprises a step of forwarding the methodology of the method according to claim 1 to a regulatory authority for approval.