Method of detecting microorganisms

Abstract

The present invention provides a method of detecting microorganisms in a substance. The method includes the steps of providing a fluid sample of a substance containing particles, taking measurements of a characteristic of the particles at a first and second time, calculating the difference between the measurements of the characteristic at the first time and the second time, and determining, based on that difference, the microorganism content in the fluid sample. The method enables the detection of microorganisms in the substance based on measurements of a characteristic of particles in that same substance. Also provided is a method of generating a reference profile for a substance.

Description

[0001] METHOD OF DETECTING MICROORGANISMS

[0002] Related Application

[0003] The present application claims the benefit of, and priority to, GB 2501291.5 filed 29 January 2025 (29.01.2025), the contents of which are hereby incorporated by reference in their entirety.

[0004] Field of the Invention

[0005] The present invention relates to methods of detecting microorganisms in substances. In particular, the present invention relates to methods of detecting microorganisms in food and beverages.

[0006] Background

[0007] Microbial contamination in beverages poses significant challenges for various substances, such as for products made in the food and beverage industry. Contamination by bacteria, yeast, mold or other types of microorganisms can lead to spoilage of food and drinks. This can result in adverse consequences on consumer health and loss of saleable products for manufacturers. It is therefore critical to ensure product safety, and this requires monitoring for microbial presence, such as during manufacture and production and in the final consumer product.

[0008] Current methods of measuring microbial content in the food and beverage industry involve preparing cultures. Typically, a food or beverage sample to be tested is plated onto or into growth media, such as in nutrient agar. The growth media is then incubated at temperatures to promote microbial growth over time. After incubation, the microbial growth can be observed and quantified in terms of, for example, the number of colony forming units per unit volume (CFU / mL).

[0009] However, such methods rely exclusively on the time required for microbial growth, and this can be extensive even when specific growth promoting conditions are used. Microbes which are present in samples at particularly low concentrations may take even longer to detect. This can lead to extended incubation periods, which may not be suitable for industries requiring rapid quality control and assurance.

[0010] An alternative method of detecting microorganisms involves using polymerase chain reactions (PGR). Typically, a sample to be tested is placed in a thermal cycler together with a PGR reaction mixture containing a polymerase enzyme and primers for binding to the target DNA sequence. The thermal cycler performs heating and cooling cycles to make copies of the target DNA sequence, such that the sequences are amplified. The amplified

[0011] 008894123DNA sequences are separated using gel electrophoresis and analysed to identify microorganisms.

[0012] However, PCR relies exclusively on the detection of microorganism DNA. The limitation of this method is that it cannot distinguish between unwanted live microorganisms from dead and inactive ones. This may lead to false positive results whereby dead microorganisms, which may essentially be inconsequential to consumer health or manufacturing quality control, present in substances are nevertheless flagged and identified.

[0013] Consequently, there is a need to provide improved methods of detecting microorganisms in substances, such as in food and beverages. The improved methods may be simpler than existing methods or may provide more rapid detection. The improved methods may provide, in a time-efficient manner, an indication of whether the content of microorganisms is above or below a specific threshold amount. The improved methods may be selective for live microorganisms.

[0014] The present invention aims to solve one or more of the above problems.

[0015] Summary of the Invention

[0016] In a first aspect of the invention, there is provided a method of detecting microorganisms in a substance, the method comprising the steps of:

[0017] (i) providing a fluid sample of a substance, wherein the fluid sample comprises particles;

[0018] (ii) taking a measurement of a characteristic of the particles at a first time;

[0019] (iii) taking a measurement of the characteristic of the particles at a second time, wherein the second time is later than the first time;

[0020] (iv) calculating the difference between the measurements of the characteristic at the first time and the second time;

[0021] (v) determining, based on the difference, the microorganism content in the fluid sample.

[0022] A fluid sample comprises particles which are suspended therein. The particles are typically particles of proteins, enzymes, polyphenols, emulsifiers and carbohydrates. The particles are solid nanoparticles or solid microparticles. The particles typically have a particle size from 2 nm to 200 pm, wherein the particle size is defined further herein. The particles may be native to the substance, such as being inherently occurring in the substance itself, or inherently introduced during the preparation of the product, where the substance is a product. Alternatively, the particles may be externally added to form the fluid sample, such as where the substance does not comprise any particles. Where the substance is a product, the product may be a food, beverage, detergent or a medicinal product.

[0023] 008894123Surprisingly, the present inventors have discovered that characteristics of the particles in a substance are affected by the presence of microorganisms in the same substance over time. Put simply, these characteristics can change over time if there are microorganisms present in the fluid sample. Furthermore, the rate of change of these characteristics of the particles in the presence of microorganisms is typically significantly faster than the rate of change of the microorganisms themselves, such as the rate of change of the microorganism concentration in the same substance. In particular, the present inventors have found that microorganisms can change the characteristics of the particles in a substance over time, and that this change in the characteristics can be monitored and recorded over a duration of time by taking a fluid sample of the substance to determine whether there are any microorganisms present in the fluid sample.

[0024] A change in the measurement of the characteristics of the particles over time can therefore provide a ‘fingerprint’ for microbial activity in the substance, without needing to directly measure the microbial growth over time.

[0025] Without wishing to be bound by theory, the change in the characteristics of the particles due to the presence of microorganisms is believed to occur due to interactions between the microorganism with the particles in the substance. During these interactions, the microorganism can cause changes to the characteristic of the particles. By measuring the characteristic at two different time points, the presence of microorganisms can be detected. Thus, the detection of microorganisms in the substance relates to the measurement of a characteristic of a particle in that same substance, where that characteristic is linked to live microorganisms present in that same substance.

[0026] For example, some microorganisms can cause flocculation and aggregation of particles in a substance over time. This may cause a decrease in the concentration of particles over time. This may also cause an increase in the average particle size over time. Other microorganisms can cause metabolism and degradation of particles in a substance over time. This may cause an increase in the concentration of particles over time. This may also cause a decrease in the average particle size over time. Measuring the characteristic of the particles, such as the particle concentration or average particle size, can allow the determination of whether there are any such microorganisms present in the fluid sample and, by extension, in the substance itself.

[0027] In a typical fluid sample for detection by the method of the invention, the concentration of particles, across a wide range of sizes beyond those of microorganisms, is significantly higher than the concentration of microorganisms. This means the contribution to a measured change from the change in characteristics of particles is significantly higher compared to any contribution to the measurement from any change in the microorganisms themselves. This means that, in practice, any contribution to the measured change from the microorganisms does not need to be resolved (e.g. accounted for) in order for the methods

[0028] 008894123of the invention to provide accurate, reliable results. Put another way, the measurements performed by the method are representative of the (change in) characteristics of the particles alone.

[0029] These changes in the characteristics also typically occur where there are live, viable microorganisms in the fluid sample.

[0030] In this way, the methods of the invention allow for selective detection of live, viable microorganisms over dead, inactive ones.

[0031] These changes in the characteristics can be measured on timescales that are typically shorter than microbial incubation processes.

[0032] In this way, the methods of the invention allow for faster determination of microbial content using simple techniques.

[0033] The method of the invention therefore involves measuring a characteristic of the particles in the fluid sample of the substance at two different times, and then calculating the difference between the two measurements. The difference can be used to detect microorganisms in the substance, or to detect the absence of microorganisms in the substance, such as whether the microorganism content in the substance is above or more than a specific threshold amount.

[0034] The measurement of the characteristic of the particles may be taken using, for example, resistive pulse sensing.

[0035] WO 2021 / 079153 describes devices and methods for the characterisation of microorganisms in a sample using resistive pulse sensing. The device employs the use of microfluidic channels which can allow microorganisms, such as particles of microorganisms, including bacterial cells and fungal cells, to flow therethrough. Under the application of a potential difference between electrodes at the microfluidic channels, the microorganisms can be recorded as a resistive pulse to determine information on their size, shape and flow rate. The methods described in WO 2021 / 079153 involve the direct measurement of the characteristics of the microorganisms themselves, and so do not rely on measuring characteristics of non-microbial particles, or taking measurements of the characteristics of particles overtime.

[0036] Yu et al. describes a bacterial growth monitoring method using resistive pulse sensing. The aim in Yu et al. is to demonstrate that resistive pulse sensing is an improved bacterial cell quantification method over optical density measurements, while not needing extensive time and labour which is typically associated with conventional colony-plating methods. Yu et al. describes the direct analysis of bacterial cells to determine bacterial growth, by using tunable

[0037] 008894123resistive pulse sensing to count total bacterial cell number, but does not describe the analysis of particles (native and / or foreign) as an indicator for bacterial growth, or performing a comparison between a profile of a sample and a reference profile for a reference sample. In some embodiments, the characteristic of the particle is selected from particle concentration, average particle size and particle size distribution. In some embodiments, the method comprises taking a measurement of more than one characteristic of the particles.

[0038] The characteristic of the particles is measured at a first time and at a second time. The difference between the first time and the second time may be referred to as the “total time difference”.

[0039] In some embodiments, the total time difference between the first time and the second time is from 1 to 12 hours.

[0040] In some embodiments, the total time difference between the first time and the second time is from 12 to 48 hours.

[0041] In some embodiments, the method comprises taking at least one measurement of a characteristic of the particles at a third time between the first time and the second time. Thus, in some embodiments, the method comprises taking a measurement of a characteristic of the particles at least three times.

[0042] Where the method comprises taking at least one measurement of a characteristic of the particles between the first time and the second time, the time difference between taking any two consecutive measurements may be referred to as the “individual time difference”.

[0043] In some embodiments, the individual time difference is from 15 minutes to 6 hours.

[0044] In some embodiments, the characteristic of the particles is measured continuously between the first time and the second time.

[0045] A profile may be generated from the measurements of the characteristics of the particles of the fluid sample of the substance. The profile contains information on the measurements of the characteristics of the particles in the fluid sample at the first and second times, and preferably further measurements of the characteristics of the particles at additional times between the first and second times. Thus, the profile comprises the difference between the measurements of the characteristic at the first time and the second time. The profile may be represented in a graphical format.

[0046] The profile may be compared to a reference profile. The reference profile may be generated from the measurements of the characteristics of the particles of a reference fluid sample of the substance. Thus, the reference profile comprises the difference between the

[0047] 008894123measurements of the characteristic at the first time and the second time. Preferably, the reference profile comprises a plurality of measurements of the characteristic at the first time and a plurality of measurements of the characteristic at the second time. In this way, the reference profile may comprise a plurality of differences, where each difference is the difference between the measurements of the characteristics of the particles at each first time and each second time.

[0048] A comparative difference between the profile and the reference profile may be indicative of the presence of unwanted microorganisms in the fluid sample of the substance and, by extension, in the substance. This may be done, for example, by determining whether the profile is within or outside of the standard deviation of the differences between the measurements in the reference profile, where the reference profile comprises a plurality of differences.

[0049] A reference fluid sample is a fluid sample of a substance, where the fluid sample comprises particles and microorganisms at a known amount and / or microorganisms of a known type. The fluid sample and the reference fluid sample share at least the same particles, by virtue of both samples originating from the same type of substance. For example, in the case where the fluid sample is a sample of a batch A of beverage X, a reference fluid sample refers to a sample of a batch B of beverage X with a known microorganism content, where batch A and batch B may be different batches of the same beverage X. The “known microorganism content” may be no microorganisms or may be an acceptable amount of microorganisms for consumption (e.g. an acceptable yeast content for consumption in a beer-based beverage). That is, the reference fluid sample may be a sample of the same substance as the sample to be measured having a known concentration of microorganism. The concentration of microorganisms in the reference fluid sample may be known e.g. by culture testing for microbial content.

[0050] By comparing the profile to a reference profile, the presence of unwanted microorganisms in the substance may be determined, such as the presence of microorganisms which is above a threshold or baseline as defined by the reference fluid sample and reference profile.

[0051] Thus, in some embodiments, the method further comprises the steps of

[0052] generating a profile from the difference between the measurement of the characteristic at the first time and the second time;

[0053] providing a reference fluid sample of the substance, wherein the reference fluid sample and the fluid sample share at least the same particles and wherein the reference fluid sample comprises a known microorganism content;

[0054] taking a measurement of a characteristic of the particles of the reference fluid sample at the first time;

[0055] taking a measurement of a characteristic of the particles of the reference fluid sample at the second time;

[0056] 008894123calculating the difference between the measurement of the characteristic of the particles of the reference fluid sample at the first time and the second time, and generating a reference profile for the reference fluid sample; and

[0057] comparing the profile to the reference profile.

[0058] In some embodiments, the method further comprises the steps of

[0059] taking a plurality of measurements of the characteristic of the particles of the reference fluid sample at the first time;

[0060] taking a plurality of measurements of the characteristic of the particles of the reference fluid sample at the second time;

[0061] calculating the difference between each measurement of the characteristic of the particles of the reference fluid sample at each first time and each second time to obtain a plurality of differences, and generating, based on the plurality of differences, a reference profile for the reference fluid sample; and

[0062] comparing the profile to the reference profile, based on a comparison between the profile and the standard deviation of the plurality of differences of the reference profile.

[0063] The standard deviation may be calculated from the arithmetic average of the plurality of differences of the reference profile. The standard deviation may be a metric for comparing the profile to the reference profile, and determining the microorganism content in the fluid sample relative to the reference fluid sample. For example, the profile may be different to the reference profile where the profile differs from the reference profile by at least two standard deviations from the arithmetic average of the reference profile.

[0064] In some embodiments, the fluid sample comprises native particles and / or foreign particles. Preferably, the fluid sample comprises foreign particles. That is, the substance comprises native particles, and foreign particles are externally added to form the fluid sample.

[0065] Alternatively, the substance does not comprise native particles, and foreign particles are externally added to form the fluid sample. In both cases, the external addition of foreign particles to form the fluid sample results in an increase in the particle concentration.

[0066] In this way, the methods of the invention allow for a greater resolution of observing the changes in the characteristics of the particles over time.

[0067] In some embodiments, the particle concentration in step (ii) is from 100 to 1,000,000,000 particles / mL. The particle concentration in step (ii) may be measured using resistive pulse sensing.

[0068] In some embodiments, the average particle size in step (ii) is from 1 to 10 pm. The average particle size in step (ii) may be measured using resistive pulse sensing.

[0069] 008894123The “average particle size” may refer to the mean value of particle sizes across a detectable population, as described herein. The detectable population is a specific subpopulation of particle sizes which can be observed by the analytical measurement technique.

[0070] In some embodiments, the method is performed at a temperature of at least 20 °C, such as at least 30 °C, such as at least 40 °C.

[0071] In some embodiments, the microorganism is selected from a bacterial cell, a fungal cell and a viral cell.

[0072] In some embodiments, the substance is a fluid.

[0073] In some embodiments, the substance is a product. In some such embodiments, the substance is a food, beverage, detergent, or medicinal product, such as an oral medicinal product. Preferably, the product is a fluid such as a beverage or liquid food product. In some such embodiments, the fluid is a non-alcoholic beverage, such as a non-alcoholic beer. In some such embodiments, the fluid is an alcoholic beverage.

[0074] In a second aspect of the invention, there is provided a method of generating a reference profile for a substance, the method comprising the steps of:

[0075] (i) providing a reference fluid sample of a substance, wherein the reference fluid sample comprises particles and a known microorganism content;

[0076] (ii) taking a measurement of a characteristic of the particles at a first time;

[0077] (iii) taking a measurement of a characteristic of the particles at a second time, wherein the second time is later than the first time;

[0078] (iv) calculating the difference between the measurement of the characteristic of the particles at the first time and the second time, and generating a reference profile for the characteristic of the particles over time.

[0079] Preferred features of the first aspect apply equally to the second aspect, unless otherwise specified.

[0080] These and other aspects and embodiments of the invention are described in further detail below.

[0081] Summary of the Figures

[0082] Figure 1 shows the particle concentration (expressed as “particles / mL”) for four brands of commercially available beers, two brands of commercially available spirits, two brands of commercially available sodas, three brands of commercially available dairy drinks and two brands of commercially available juices.

[0083] 008894123Figure 2 shows the colony forming unit (CFU) over a period of 24 hours for a sample obtained from a brand of commercially available beer each modified with the following foreign cells: no added yeast cells, 10 added yeast cells, 100 added yeast cells, 1,000 added yeast cells and 10,000 added yeast cells. Only CFU data for samples obtained from beer modified with 1,000 added yeast cells and 10,000 added yeast cells can be resolved.

[0084] Figure 3 shows the particle concentration (expressed as “particles / mL”) in the same beer samples used in Figure 2, comprising no added yeast cells, 10 added yeast cells, 100 added yeast cells, 1,000 added yeast cells and 10,000 added yeast cells over a period of 24 hours.

[0085] Figure 4 shows the particle size distribution for a sample obtained from commercially available beer over a period of 48 hours, where the measurements for the beer sample were taken at time intervals of 24 hours.

[0086] Figure 5 shows the variability of Lactobacillus brevis bacterial growth in a sample obtained from a brand of commercially available alcoholic beer and a sample of a brand of commercially available non-alcoholic beer over a period of 48 hours.

[0087] Figure 6 shows the colony forming unit (CFU) over a period of 24 hours for a sample obtained from a brand of commercially available beer modified with the following: no added yeast cells, 10 added yeast cells, 100 added yeast cells, 1,000 added yeast cells and 10,000 added yeast cells.

[0088] Figure 7 shows the particle concentration (expressed as “particles / mL”) over a period of 24 hours in the same beer samples used in Figure 6.

[0089] Figure 8 shows the particle concentration (expressed as “particles / mL”) over a period of up to 72 hours for four samples obtained from three brands of commercially available beers incubated with added Lactobacillus brevis bacteria, one beer sample of which was modified with freeze-dried tomato powder as additional foreign particles, where the particle concentrations are shown in the 1 to 4 pm particle size range.

[0090] Figure 9 shows the change in particle concentration (expressed as “particles / mL”) after a period of 24 hours for 12 samples obtained from 5 brands of commercially available beers, and compared against a baseline (control) commercially available beer having no viable microorganisms.

[0091] Detailed Description of the Invention

[0092] Generally, the present invention relates to a method of detecting microorganisms in a substance. The method uses a fluid sample of the substance, where the fluid sample comprises particles. Measurements of a characteristic of the particles are taken at different

[0093] 008894123times during the method. The difference between the measurements at the different times are calculated to determine the microorganism content in the fluid sample.

[0094] In a first aspect of the invention, there is provided a method of detecting microorganisms in a substance, the method comprising the steps of:

[0095] (i) providing a fluid sample of a substance, wherein the fluid sample comprises particles;

[0096] (ii) taking a measurement of a characteristic of the particles at a first time;

[0097] (iii) taking a measurement of the characteristic of the particles at a second time, wherein the second time is later than the first time;

[0098] (iv) calculating the difference between the measurements of the characteristic at the first time and the second time;

[0099] (v) determining, based on the difference, the microorganism content in the fluid sample.

[0100] Providing Substance and Particles

[0101] A substance, in the context of the present invention, is a substance which is intended for the detection of microorganisms therein. The substance may be a solid or a fluid. Where the substance is a fluid, the substance may be a liquid.

[0102] In some embodiments, the substance is a natural substance. A natural substance is a substance which occurs in nature. A natural substance is typically not manufactured, processed or refined by industrial means. Examples of natural substances include, but are not limited to, rainwater, sea or ocean water, river water and lake water.

[0103] In some embodiments, the substance is a natural substance which is derived from a human. In some embodiments, the substance is a human bodily fluid. In some embodiments, the substance is urine.

[0104] In some embodiments, the substance is a product. A product is a substance which is manufactured or refined for sale.

[0105] In some embodiments, the product is a product for human intake. The product may be a food, beverage or a medicinal product.

[0106] In some embodiments, the product is a food or beverage.

[0107] A food is a solid product for human consumption. Examples of foods include, but are not limited to, bread, cereal, vegetables, nuts, seeds, legumes, meat and rice.

[0108] 008894123A beverage is a fluid product for human consumption. In some embodiments, the product is a beverage. Examples of beverages include, but are not limited to, water, juice, tea, coffee, hot chocolate, milk, syrups, soups, sauces, beer, wine, spirits and yoghurt.

[0109] In some embodiments, the beverage is an alcoholic beverage or a non-alcoholic beverage.

[0110] In some embodiments, the beverage is an alcoholic beverage. Examples of alcoholic beverages include beer, wine and spirits.

[0111] In some embodiments, the beverage is a non-alcoholic beverage. Examples of nonalcoholic beverages include water, juice, tea, coffee, hot chocolate, milk yoghurt and nonalcoholic beer. In some embodiments, the beverage is a non-alcoholic beer.

[0112] In some embodiments, the product is a medicinal product. The medicinal product may be an oral medicinal product, a topical medicinal product or a subcutaneous medicinal product. The oral medicinal product may be a tablet, pill or capsule. The topical medicinal product may be a drop, cream or oil. The subcutaneous medicinal product may be an injection, such as a vaccine.

[0113] In some embodiments, the product is a detergent. A detergent is a substance which comprises a surfactant, or a mixture thereof, with cleansing properties. Detergents typically include anionic detergents, cationic detergents, non-ionic detergents and amphoteric detergents. Examples of detergents include, but are not limited to, dishwashing detergent, laundry detergent, surface cleaner, fabric softener, bleach and soap.

[0114] The method of the invention comprises providing a fluid sample of a substance. Where the substance is a fluid, a fluid sample may simply be a sample of the substance itself. Thus, no further modification may be needed. Where the substance is a solid, a sample of the substance may be mixed with a fluid, such as water, of a sufficient quantity to obtain a fluid sample of the substance. The substance may be dispersed or dissolved in the fluid.

[0115] The method of the invention comprises providing a fluid sample of a substance, wherein the fluid sample comprises particles. The substance may or may not comprise particles.

[0116] A particle, in the context of the present invention, is a solid nanoparticle or solid microparticle. The particle has characteristics which can be changed over time in the presence of a microorganism in the fluid sample, due to mechanisms as described above. The term “particles” refers to the total population of particles present in a substance, or in a fluid sample of the substance.

[0117] 008894123The term “particles” in the present invention does not include microorganisms themselves or particles of microorganisms. Thus, the particle is a non-microbial particle. Suitably, the particle is non-living.

[0118] In a typical fluid sample, the particle concentration is significantly greater than the microorganism concentration. For example, the particle concentration is typically at least one order of magnitude greater than the microorganism concentration for a given fluid sample.

[0119] In some embodiments, the particle concentration in step (ii) is at least 10 times greater than the microorganism concentration in step (ii).

[0120] In some preferred embodiments, the particle concentration in step (ii) is at least 100 times greater than the microorganism concentration in step (ii).

[0121] In some further preferred embodiments, the particle concentration in step (ii) is at least 1,000 times greater than the microorganism concentration in step (ii), preferably at least 10,000 times greater, preferably at least 100,000 times greater, preferably at least 1,000,000 times greater.

[0122] The particles of the present invention are typically organic particles. Examples of organic particles include, but are not limited to, proteins, enzymes, polyphenols, emulsifiers and carbohydrates.

[0123] Examples of proteins include, but are not limited to, hordeins, albumins, gliadins, serpins, dehydrins and globulins.

[0124] Examples of enzymes include, but are not limited to, amylase, endochitinase, lipoxygenase dehydrogenase and kinase.

[0125] Examples of polyphenols include, but are not limited to, tannins, catechins, epicatechins, humulones and proanthocyanidins.

[0126] Examples of emulsifiers include, but are not limited to, phospholipids, gum arabic, guar gum and xanthan gum.

[0127] Carbohydrates may be monosaccharides or polysaccharides.

[0128] Examples of monosaccharides include, but are not limited to, glucose, fructose, maltose, maltriose, dextrins and cyclodextrins.

[0129] Examples of polysaccharides include, but are not limited to, mannans, arabinose, arabinoxylans, p-glucans, cellulose, chitosan, chitins, malt arabinoxylans and starch.

[0130] 008894123Other types of organic particles include humulones, coumarins, benzoic acid, ferulic acid, cinnamic acid, prenylated chaicones, a-acids and iso-a-acids.

[0131] The particle may be a native particle or a foreign particle. A native particle is a particle which is inherently present in the substance due to the nature of occurrence of the substance, or the manufacture or preparation of a product where the substance is a product. Therefore, where a substance comprises particles, that substance comprises native particles. A foreign particle is a particle which is externally added to a fluid sample of the substance, where the substance may or may not already comprise native particles. Therefore, where a substance comprises or does not comprise native particles, the fluid sample of the substance comprises foreign particles. A foreign particle may comprise the same substances as a native particle.

[0132] In some embodiments, the substance comprises native particles and / or foreign particles.

[0133] Where the substance does not comprise particles, or where the substance comprises a concentration of particles below a detectable limit, the method of the present invention may comprise a step of adding foreign particles to a fluid sample of the substance. For example, foreign particles may be added to a fluid sample where the concentration of particles in the substance is at most 1,000 particles / mL, such as at most 500 particles / mL, such as at most 100 particles / mL, such as at most 10 particles / mL.

[0134] An example of a substance which does not comprise particles is pure water. By introducing foreign particles into the fluid sample of the substance, the measurements of the characteristic of the particles, represented by the total population of native particles and foreign particles, may be performed with greater resolution and sensitivity.

[0135] In some embodiments, the particle concentration of the fluid sample is at least 100 particles / mL, such as at least 1,000 particles / mL, such as at least 10,000 particles / mL, such as at least 100,000 particles / mL.

[0136] In some embodiments, the particle concentration of the fluid sample is at most 1,000,000,000 particles / mL, such as at most 100,000,000 particles / mL, such as at most 10,000,000 particles / mL, such as at most 1,000,000 particles / mL.

[0137] The particle concentration may be a particle concentration selected from the lower and upper values from the particle concentrations given above. For example, the particle concentration of the fluid sample may be from 100 to 1,000,000,000 particles / mL, such as from 1,000 to 100,000,000 particles / mL, such as from 10,000 to 10,000,000 particles / mL, such as from 100,000 to 1,000,000 particles / mL.

[0138] 008894123In some embodiments, the particle size of the fluid sample is at least 2 nm, such as at least 10 nm, such as at least 50 nm, such as at least 0.1 pm, such as at least 1 pm.

[0139] In some embodiments, the particle size of the fluid sample is at most 200 pm, such as at most 100 pm, such as at most 50 pm, such as at most 20 pm, such as at most 10 pm.

[0140] The particle size may be a particle size selected from the lower and upper values from the particle sizes given above. For example, the particle size of the fluid sample may be from 2 nm to 200 pm, such as from 10 nm to 100 pm, such as from 50 nm to 50 pm, such as from 0.1 to 20 pm, such as from 1 to 10 pm.

[0141] The particle size may be a particle size as described further herein.

[0142] Measuring Characteristics

[0143] A characteristic of the particles refers to a physical or chemical property of the particles in the substance, where the property can be changed over time in the presence of microorganisms which interact with the particles. The characteristic is the property of the particles which is directly measured in the method of the present invention.

[0144] The method of the present invention relies on a link between the characteristic of the particles and the interactions between the particles and microorganisms, where present. Overtime, microorganisms may change the characteristic of the particles in the substance, and fluid sample thereof, due to various physical or chemical reactions which may take place.

[0145] The rate of change of the characteristics of the particles in the presence of microorganisms is typically significantly faster than the rate of the change of the microorganisms themselves, such as the rate of change of the microorganism concentration in the same substance or fluid sample. In a typical substance or fluid sample for detection by the method of the invention, the particle concentration is significantly higher than the microorganism concentration.

[0146] This means the contribution to a measured change from the change in characteristics of particles is significantly higher compared to any contribution to the measurement from any change in the microorganisms themselves. This means that, in practice, any contribution to the measured change from the microorganisms does not need to be resolved (e.g. accounted for) in order for the methods of the invention to provide accurate, reliable results. Therefore, the measurements performed by the method are representative of the (change in) characteristics of the particles alone.

[0147] 008894123Examples of the characteristics of the particles include, but are not limited to, the particle concentration, the average particle size and the particle size distribution. The method may include taking measurements of two or more different types of characteristics.

[0148] The particle concentration, which is the total concentration of native particles and foreign particles which is present in the fluid sample, can vary depending on the type of the substance tested in the method. Some substances inherently have more native particles than other substances. For example, where the substance is beer, the concentration of particles may be at least 1,000 particles / mL. For example, where the substance is milk, the concentration of particles may be at least 1,000,000,000 particles / mL. Thus, where the substance comprises a particularly small concentration of native particles, foreign particles may be added to a fluid sample of the substance to increase the sensitivity and resolution of the characteristic measurement.

[0149] In some embodiments, the characteristic of the particles is the particle concentration. For example, the particle concentration may be expressed as the number of particles per unit volume, such as millilitre (mL), of fluid sample. The particle concentration may be measured by any standard method for analysing particle concentrations in a fluid sample of a substance. Examples of methods of measuring particle concentration include, but are not limited to, resistive pulse sensing, dynamic light scattering, and turbidity measurement, the measurement protocols of which are described herein.

[0150] In some embodiments, the particle concentration in step (ii) is at least 100 particles / mL, such as at least 1,000 particles / mL, such as at least 10,000 particles / mL, such as at least 100,000 particles / mL.

[0151] In some embodiments, the particle concentration in step (ii) is at most 1,000,000,000 particles / mL, such as at most 100,000,000 particles / mL, such as at most 10,000,000 particles / mL, such as at most 1,000,000 particles / mL.

[0152] The particle concentration may be a particle concentration selected from the lower and upper values from the particle concentrations given above. For example, the particle concentration in step (ii) may be from 100 to 1,000,000,000 particles / mL, such as from 1,000 to 100,000,000 particles / mL, such as from 10,000 to 10,000,000 particles / mL, such as from 100,000 to 1,000,000 particles / mL.

[0153] The particles may be visible or microscopic, depending on their size, to the human eye. The particles may not be necessarily spherical, but may be elongate, ellipsoidal or irregular in shape. Preferably, the particles are predominately spherical in shape or substantially spherical in shape.

[0154] 008894123A particle may be characterised by its largest transverse dimension. For a given individual particle, the largest transverse dimension may be its diameter. Thus, the “particle size” may refer to the largest transverse dimension, such as the diameter, of an individual particle.

[0155] A population of particles may be characterised by an average particle size. The “average particle size” may refer to the mean value of particle sizes across the population. For example, the population comprises particles having an average particle size of 2 nm to 200 pm. The population of particles may refer to the total particles in the fluid sample.

[0156] In the context of the present invention, the “average particle size” may refer to the mean value of particle sizes across a detectable population. For detecting microorganisms, the detectable population is deemed as being representative of the population of the particles in the fluid sample.

[0157] The detectable population is a specific subpopulation of particle sizes which can be observed by the analytical measurement technique. That is, the range of average particle sizes of the detectable population is a subrange of the range of average particle sizes of the population. For example, the detectable population comprises particles having an average particle size of 0.1 to 100 pm, based on the detectable range which can be observed by the analytical measurement technique. Thus, the method involves observing the changes in the measurements of the characteristics of the detectable population of particles in the fluid sample.

[0158] The detectable population of particles may be a typical amount that can be analysed together to determine the average particle size using the analytical measurement technique. The number of particles within a detectable population may be at least a typical amount that can be analysed together, such as at least 50 particles, such as at least 100 particles.

[0159] Examples of analytical measurement techniques for measuring average particle sizes include, but are not limited to, resistive pulse sensing and dynamic light scattering, the measurement protocols of which are described herein.

[0160] In some embodiments, the characteristic of the particles is the average particle size. For example, the average particle size may be expressed as the average particle size in the micrometer (pm) scale.

[0161] In some embodiments, the average particle size in step (ii) is at least 0.1 pm, such as at least 0.2 pm, such as at least 0.3 pm, such as at least 0.4 pm, such as at least 0.5 pm, such as at least 0.6 pm, such as at least 0.7 pm, such as at least 0.8 pm, such as at least 0.9 pm, such as at least 1 pm. In some embodiments, the average particle size of the particle is at least 2 pm.

[0162] 008894123In some embodiments, the average particle size in step (ii) is at most 100 pm, such as at most 90 pm, such as at most 80 pm, such as at most 70 pm, such as at most 60 pm, such as at most 50 pm, such as at most 40 pm, such as at most 30 pm, such as at most 20 pm, such as at most 10 pm. In some embodiments, the average particle size of the particle is at most 8 pm.

[0163] The average particle size in step (ii) may be an average size selected from the lower and upper values from the sizes given above. For example, the average particle size in step (i) may be from 0.1 to 100 pm, such as from 0.5 to 50 pm, such as from 1 to 10 pm, such as from 2 to 8 pm.

[0164] The detectable population of particles may be defined by selecting a detection range of the particle size. For example, the detection range of the particle size does need not overlap with the range of the microorganism cell size present in the sample. This may be particularly useful where the size of the microorganism cells present in the sample are within an average particle size of the particles present in the sample. By adjusting the detection population of particles based on the detection range of the particle size, the method ensures that such microorganism cells fall outside the detection range and are not detected.

[0165] In some embodiments, the detection range of the particle size is less than 2 pm, such as 1 pm or less, such as 0.1 pm or less.

[0166] In some embodiments, the detection range of the particle size is more than 8 pm, such as 9 pm or more, such as 10 pm or more, such as 11 pm or more, such as 12 pm or more, such as 13 pm or more, such as 14 pm or more, such as 15 pm or more.

[0167] The detection range of the particle size may be a detection range selected from the lower and upper values from the detection ranges given above. For example, the detection range of the particle size may be from less than 2 pm and more than 8 pm, such as from 1 pm or less and such as 12 pm or more, such as from 0.1 pm or less to 15 pm or more.

[0168] The particles may be characterised by their particle size distribution. The “particle size distribution” may refer to the distribution of particle sizes across the detectable population, as above.

[0169] In some embodiments, the characteristic of the particles is the particle size distribution. For example, the particle size distribution may be expressed as the number of particles having specific sizes in the micrometer (pm) scale. Percentile values for the particle size distribution may be characterised by D10, D50 and D90 values. These values may be calculated based on the average particle size, as above. That is, D10, D50 and D90 values reported may be based on the detectable population which can be observed by the analytical measurement technique used in the method.

[0170] 008894123D90 is the particle size at which 90% of the particles in the detectable population of particles by mass have a particle size of less than or equal to the D90 particle size.

[0171] In some embodiments, the particles in step (ii) have a D90 of at least 1.5 pm, such as at most 2.0 pm, such as at most 2.5 pm.

[0172] In some embodiments, the particles in step (ii) have a D90 of at most 7.0 pm, such as at most 6.5 pm, such as at most 6.0 pm.

[0173] D90 may be a value selected from the lower and upper values from the values given above. For example, the particles in step (ii) may have a D90 from 1.5 to 7.0 pm, such as from 2.0 to 6.5 pm, such as from 2.5 to 6.0 pm.

[0174] D50 is the particle size at which 50% of the particles in the detectable population of particles by mass have a particle size of less than or equal to the D50 particle size.

[0175] In some embodiments, the particles in step (ii) have a D90 of at least 2.5 pm, such as at least 2.8 pm, such as at least 3.0 pm.

[0176] In some embodiments, the particles in step (ii) have a D90 of at most 4.0 pm, such as at most 3.8 pm, such as at most 3.5 pm.

[0177] D50 may be a value selected from the lower and upper values from the values given above. For example, the particles in step (ii) may have a D50 from 2.5 to 4.0 pm, such as from 2.8 to 3.8 pm, such as from 3.0 to 3.5 pm.

[0178] D10 is the particle size at which 10% of the particles in the detectable population of particles by mass have a particle size of less than or equal to the D10 particle size.

[0179] In some embodiments, the particles in step (ii) have a D10 of at least 1.5 pm, such as at least 1.6 pm, such as at least 1.7 pm.

[0180] In some embodiments, the particles in step (ii) have a D10 of at most 2.2 pm, such as at most 2.1 pm, such as at most 2.0 pm.

[0181] D10 may be a value selected from the lower and upper values from the values given above. For example, the particles in step (ii) may have a D10 from 1.5 to 2.2 pm, such as from 1.6 to 2.1 pm, such as from 1.7 to 2.0 pm.

[0182] The method of the invention comprises measuring a characteristic of the particles at a first time and measuring a characteristic of the particles at a second time. The time elapsed

[0183] 008894123between the first time and the second time (herein also referred to as the “total time difference”) is typically sufficient to resolve the difference in the characteristic of the particles. For example, the total time difference may be dependent on the microorganism content in the fluid sample, the growth rate of the microorganism, and the capability of the microorganism to change the characteristics of the particles over a period of time, such as when compared to a reference profile.

[0184] In some embodiments, the total time difference is at least 1 hour, such as at least 2 hours, such as at least 3 hours, such as at least 4 hours, such as at least 6 hours, such as at least 8 hours, such as at least 10 hours, at least 12 hours, such as at least 14 hours, such as at least 16 hours, such as at least 18 hours, such as at least 20 hours.

[0185] In some embodiments, the total time difference is at most 48 hours, such as at most 44 hours, such as at most 40 hours, such as at most 36 hours, such as at most 32 hours. In some embodiments, the total time difference is at most 12 hours, such as at most 10 hours, such as at most 8 hours, such as at most 6 hours, such as at most 4 hours.

[0186] The total time difference may be a total time difference selected from the lower and upper values from the total time differences given above. In some embodiments, the total time difference is from 12 to 48 hours, such as from 14 to 44 hours, such as from 16 to 40 hours, such as from 18 to 36 hours, such as from 20 to 32 hours. In some embodiments, the total time difference is from 1 to 12 hours such as from 2 to 8 hours, such as from 3 to 4 hours.

[0187] The method of the invention involves taking a measurement of a characteristic at a first time and at a second time. In some embodiments, the method comprises taking at least one measurement of a characteristic of the particles between the first time and the second time. Thus, in some embodiments, the method comprises taking a measurement of a characteristic of the particles at least three times.

[0188] The at least one measurement taken between the first time and the second time may be at least two measurements, such as at least two measurements, such as at least three measurements, such as at least four measurements, such as at least five measurements, such as at least six measurements, such as at least seven measurements, such as at least eight measurements.

[0189] The measurements taken between the first time and the second time may each be taken at a different time. For example, the measurements taken between the first time and the second time may each be taken at regular intervals.

[0190] Where the method comprises taking at least one measurement of a characteristic of the particles at a third time between the first time and the second time, the time difference between taking any two consecutive measurements (herein also referred to as the

[0191] 008894123“individual time difference”) is smaller than the total time difference. The number of measurements taken between the first time and the second time may typically be selected based on the length of the total time difference.

[0192] In some embodiments, the individual time difference is at least 15 minutes, such as at least 30 minutes, such as at least 1 hour.

[0193] In some embodiments, the individual time difference is at most 6 hours, such as at most 5 hours, such as at most 4 hours.

[0194] The individual time difference may be an individual time difference selected from the lower and upper values from the individual time differences given above. In some embodiments, the individual time difference is from 15 minutes to 6 hours, such as from 30 minutes to 5 hours, such as from 1 to 4 hours.

[0195] In some embodiments, the characteristic of the particles is measured continuously between the first time and the second time. The characteristic of the particles may be measured at a high frequency. Thus, the individual time difference may be at most 1 minute, such as at most 1 second, such as at most 0.1 seconds.

[0196] The characteristic of the particle may be measured by any standard method for analysing particle properties. Examples of suitable methods include, but are not limited to, resistive pulse sensing, dynamic light scattering, laser diffraction, flow cytometry, turbidity measurement, microscopy, and spectrophotometry.

[0197] In some embodiments, the method for analysing particle properties is resistive pulse sensing, dynamic light scattering, or turbidity measurement. Such methods are typically suitable for particle sizes in the range from 0.1 to 1,000 pm.

[0198] In some embodiments, the method for analysing particle properties is resistive pulse sensing. Resistive pulses provide detailed characterization of materials from small molecules to nanomaterials on an individual particle basis. They provide information on the particle size, shape, concentration and charge. Importantly, the low cost and high throughput (tens to hundreds of particles per second) of using resistive pulses make it applicable.

[0199] In a typical particle characteristic measurement using resistive pulse sensing, particles of the fluid sample are passed through an aperture, such as a microfluidic channel, containing an electrolyte and with a potential difference across the aperture. Each particle displaces a volume of the electrolyte, and the electrical properties, such as the resistance, at the aperture is changed. The change in the electric property is detected as a voltage or current

[0200] 008894123pulse. The magnitude of the pulse is proportional to the size of the particle. The number of pulses is proportional to the concentration of particles.

[0201] In some embodiments, the method for analysing particle properties is dynamic light scattering. In a typical particle characteristic measurement using dynamic light scattering, particles of the fluid sample are irradiated with a laser light. Laser light is scattered by the particles due to Brownian motion, and fluctuations in the scattered light intensity are analysed overtime to determine particle size distribution.

[0202] In some embodiments, the method for analysing particle properties is measuring the turbidity of the fluid sample comprising particles. Turbidity is an optical measurement showing the presence of suspended particles, such that a higher turbidity represents a greater number of particles as the fluid sample becomes ‘cloudier’. In a typical particle characteristic measurement using turbidity measurement, the fluid sample is exposed to a light beam. The quantity of light reflected or scattered is a measure of the concentration of the particles in the fluid sample.

[0203] Detecting Microorganisms

[0204] A microorganism, or a microbe, is a microscopic organism. A microorganism is typically a single-celled or multi-celled organism. Examples of microorganisms include, but are not limited to, bacteria, fungi, mold, archaea, protozoa and viruses. In some embodiments, the microorganism is selected from a bacterial cell, a fungal cell, mold and a viral cell.

[0205] In the context of the present invention, the microorganism is a live microorganism. The method of the present invention is particularly for detecting live microorganisms in substances. Live microorganisms are capable of changing the characteristic of particles in substances by various interactions as described herein between the microorganisms and the particles. Inactive or dead microorganisms typically do not change the characteristics of particles.

[0206] In the context of the present invention, the detection of microorganisms in the substance relates to the measurement of a characteristic of a particle in that same substance, where that characteristic is linked to live microorganisms present in that same substance.

[0207] In some embodiments, the bacteria cell is a gram-positive bacteria.

[0208] In some embodiments, the gram-positive bacteria is lactic acid bacteria or acetic acid bacteria.

[0209] 008894123Examples of lactic acid bacteria include, but are not limited to, Lactobacillus, Streptococcus, Lactococcus, Enterococcus, Leuconostoc, Pediococcus, Limosilactobacill Fermentum, Oenococcus, and Lactobacillales.

[0210] Lactobacillus bacteria may be selected from Lactobacillus brevis, Lactobacillus brevisimilis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus coryneformis, Lactobacillus curvatus, Lactobacillus lindneri, Lactobacillus malefermentans, Lactobacillus parabuchneri, and Lactobacillus plantarum.

[0211] Pediococcus bacteria may be selected from Pediococcus damnosus, Pediococcus dextrinicus and Pediococcus inopinatus.

[0212] Examples of acetic acid bacteria include, but are not limited to, Acetobacter, Gluconacetobacter, Gluconobacter, Komagataeibacter, A. pasteurianus, and Komagataeibacter europaeus.

[0213] Other examples of gram-positive bacteria include Micrococcus kristinae.

[0214] In some embodiments, the bacteria cell is gram-negative bacteria.

[0215] In some embodiments, the gram-negative bacteria is Enterobacteriaceae bacteria or spiral or curved bacteria.

[0216] Enterobacteriaceae bacteria may be selected from Enterobacter cloacae, Escherichia coli, Escherichia coli 0157:1-17, Hafnia protea, Klebsiella pneumoniae, Salmonella, Salmonella enterica and Shigella sonnei.

[0217] Spiral or curved bacteria include, but are not limited to, Campylobacter coli, Campylobacter jejuni, Helicobacter pylori and Vibrio cholera.

[0218] Other examples of gram-negative bacteria include, but are not limited to, Legionella pneumophila, Mycobacterium spp., Pectinatus cerevisiiphilus, Pectinatus frisingensis, Selenomonas lacticifex, Zymomonas mobilis and Zymophilus raffinosivorans.

[0219] In some embodiments, the fungal cell is yeast.

[0220] Examples of yeast include, but are not limited to, Candida albicans, Candida glabrata, Candida spp. and Saccharomyces spp.

[0221] Examples of mold include, but are not limited to, Aspergillus niger, Aspergillus spp., Byssochlamys spp., Cladosporium cladosporioides, Cladosporium spp., Penicillium citrinum, Penicillium glabrum, Penicillium spp., Rhizopus spp. and Alternaria alternate.

[0222] 008894123Examples of protozoa include, but are not limited to, Cryptosporidium hominis, Cryptosporidium parvum and Cyclospora cayatenensis.

[0223] The microorganism may be characterised by the average microorganism cell size. The “average microorganism cell size” may refer to the mean value of microorganism cell sizes across the population. The population of microorganisms may refer to the total microorganisms in the fluid sample. The average microorganism cell size may be determined using resistive pulse sensing.

[0224] In some embodiments, the average microorganism cell size is at least 0.1 pm, such as at least 0.2 pm, such as at least 0.3 pm, such as at least 0.4 pm, such as at least 0.5 pm, such as at least 0.6 pm, such as at least 0.7 pm, such as at least 0.8 pm, such as at least 0.9 pm, such as at least 1 pm. In some embodiments, the average microorganism cell size is at least 2 pm.

[0225] In some embodiments, the average microorganism cell size is at most 100 pm, such as at most 90 pm, such as at most 80 pm, such as at most 70 pm, such as at most 60 pm, such as at most 50 pm, such as at most 40 pm, such as at most 30 pm, such as at most 20 pm, such as at most 10 pm. In some embodiments, the average microorganism cell size is at most 8 pm.

[0226] The average microorganism cell size may be an average size selected from the lower and upper values from the sizes given above. For example, the average microorganism cell size may be from 0.1 to 100 pm, such as from 0.5 to 50 pm, such as from 1 to 10 pm, such as from 2 to 8 pm.

[0227] The method of the invention relies on the difference between measurements of the particle characteristic, which measurements thereof are taken at two times to identify a change which may be indicative of microorganism presence and activity. This is done by calculating the difference between the measurement of the characteristic of the particles at a first time and the measurement of the characteristic of the particles at a second time. The calculation may be performed by a computer. The method of the invention may provide a rapid detection of microorganisms, and this can be achieved by minimising the time difference.

[0228] The method of the invention may be accelerated by promoting the growth of microorganisms in a fluid sample between the first time and the second time. The growth of microorganisms may be increased by performing the method of the invention at an elevated temperature, such as at a temperature above ambient temperature. The temperature may be selected based on the microorganism of interest to be detected. The temperature at the first time may be the same as the temperature at the second time. The temperature may be constant throughout the method.

[0229] 008894123In some embodiments, the temperature is at least 20 °C, such as at least 24 °C, such as at least 30 °C, such as at least 35 °C, such as at least 40 °C, such as at least 45 °C, such as at least 50 °C.

[0230] In some embodiments, the temperature of the particle is at most 80 °C, such as at most 75 °C, such as at most 70 °C, such as at most 65 °C, such as at most 60 °C, such as at most 55 °C, such as at most 50 °C.

[0231] The temperature may be a temperature selected from the lower and upper values from the temperatures given above. For example, the temperature is from 20 to 80 °C, such as from 24 to 75 °C, such as from 30 to 70 °C, such as from 35 to 65 °C, such as from 40 to 60 °C.

[0232] The detection of microorganism in the fluid sample is based on the difference between the measurement of the characteristic of the particles at the first time and the measurement of the characteristic of the particles at the second time. The difference between the measurements is an absolute difference.

[0233] The magnitude of the difference in the measurement of the particle concentration between the first time and the second time typically provides an indication of microbial activity.

[0234] In some embodiments, the difference in the measurement of the particle concentration between the first time and the second time is at least 20,000 particles / mL, such as at least 40,000 particles / mL, such as at least 60,000 particles / mL, such as at least 80,000 particles / mL, such as at least 100,000 particles / mL, such as at least 120,000 particles / mL, such as at least 140,000 particles / mL, such as at least 180,000 particles / mL.

[0235] In some embodiments, the difference in the measurement of the particle concentration between the first time and the second time is at least 20%, such as at least 50%, such as at least 100%, such as at least 200%, such as at least 300%, such as at least 400%, such as at least 500%.

[0236] The magnitude of the difference in the measurement of the average particle size between the first time and the second time typically provides an indication of microbial activity.

[0237] In some embodiments, the difference in the measurement of the average particle size between the first time and the second time is at least 0.2 pm, such as at least 0.4 pm, such as at least 0.6 pm, such as at least 0.8 pm, such as at least 1.0 pm.

[0238] In some embodiments, the difference in the measurement of the average particle size between the first time and the second time is at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%.

[0239] 008894123The magnitude of the difference in the measurement of the particle size distribution between the first time and the second time typically provides an indication of microbial activity.

[0240] In some embodiments, the difference in the measurement of the D10 between the first time and the second time is at least 0.2 pm, such as at least 0.4 pm, such as at least 0.6 pm, such as at least 0.8 pm, such as at least 1.0 pm.

[0241] In some embodiments, the difference in the measurement of the D10 between the first time and the second time is at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%.

[0242] In some embodiments, the difference in the measurement of the D50 between the first time and the second time is at least 0.2 pm, such as at least 0.4 pm, such as at least 0.6 pm, such as at least 0.8 pm, such as at least 1.0 pm.

[0243] In some embodiments, the difference in the measurement of the D50 between the first time and the second time is at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%.

[0244] In some embodiments, the difference in the measurement of the D90 between the first time and the second time is at least 0.2 pm, such as at least 0.4 pm, such as at least 0.6 pm, such as at least 0.8 pm, such as at least 1.0 pm.

[0245] In some embodiments, the difference in the measurement of the D90 between the first time and the second time is at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%.

[0246] A profile may be generated from taking measurements of the characteristic of the particles in the fluid sample at the first time and the second time. The profile contains information about the measurements of the characteristic of the particles in the fluid sample over the course of the method, preferably containing a plurality of measurements of the characteristic of the particles between the first time and the second time. Thus, the profile contains information about the difference between the measurements of the characteristic at the first time and the second time.

[0247] A visual representation of the profile may be made available to a user for reference. The profile may be represented in a graphical format. The profile may have the change in measurement of the characteristic of the particles between the first time and the second time aligned on one axis, and the fluid samples tested on another axis. The profile may have the measurement of the characteristic of the particles aligned on one axis, and the time aligned

[0248] 008894123on another axis. For example, the profile may have the measurement of the characteristic of the particles aligned on the y-axis, and the time aligned on the x-axis.

[0249] The profile may be regarded as a fingerprint for the fluid sample of the substance, in that it is characteristic of the substance analysed.

[0250] Where the measurements of the characteristic of the particles in the fluid sample is provided in the form of a profile, the profile may be compared to a reference profile.

[0251] A reference profile contains information about the measurements of the characteristic of the particles in a reference fluid sample of the substance at a first time and a second time.

[0252] Thus, the reference profile comprises information about the difference between the measurements of the characteristic at the first time and the second time. The first time and the second time of the reference profile may be the same as the first time and the second time of the profile.

[0253] A reference fluid sample is a fluid sample of a substance, where the fluid sample comprises particles and microorganisms at a known amount and / or microorganisms of a known type. The fluid sample and the reference fluid sample share at least the same particles, by virtue of both samples originating from the same type of substance. For example, in the case where the fluid sample is a sample of a batch A of beverage X, a reference fluid sample refers to a sample of a batch B of beverage X with a known microorganism content, where batch A and batch B may be different batches of the same beverage X. The “known microorganism content” may be no microorganisms or may be an acceptable amount of microorganisms for consumption (e.g. an acceptable yeast content for consumption in a beer-based beverage). That is, the reference fluid sample may be a sample of the same substance as the sample having a known concentration of microorganism. The concentration of microorganisms in the reference fluid sample may be known e.g. by culture testing for microbial content. In some embodiments, the microorganism content in the reference fluid sample is below a certain threshold amount.

[0254] The reference profile may be regarded as a fingerprint for the reference fluid sample of the substance, in that it is characteristic of the substance, analysed, where that fluid sample has a known microorganism content.

[0255] Where that profile closely matches that of a reference profile, then the microorganism content may be regarded as the being same as the microorganism content in the reference fluid sample. Thus, the profile, when matched to its reference profile, can provide a guarantee of the fluid sample of the substance having met a threshold microorganism content as determined by the reference fluid sample. For example, where the reference profile corresponds to a reference fluid sample having a threshold microorganism content, by

[0256] 008894123having such a profile matched to its reference profile, a user can use the substance as intended, and without the need for further characterisation of the substance.

[0257] Where that profile does not closely match that of a reference profile, then the microorganism content in the fluid sample may be regarded as being different to the microorganism content in the reference fluid sample. Thus, the profile, when matched to its reference profile, can provide an indication of the fluid sample of the substance not having met or exceeded a threshold microorganism content. For example, where the reference profile corresponds to a reference fluid sample having a threshold microorganism content, by observing such a profile which does not match to its reference profile, a user may choose either to reduce the microorganism content in the substance, or to dispose of the substance entirely.

[0258] Thus, in some embodiments, the method further comprises the steps of

[0259] generating a profile from the difference between the measurement of the characteristic at the first time and the second time;

[0260] providing a reference fluid sample of the substance, wherein the reference fluid sample and the fluid sample share at least the same particles and wherein the reference fluid sample comprises a known microorganism content;

[0261] taking a measurement of a characteristic of the particles of the reference fluid sample at the first time;

[0262] taking a measurement of a characteristic of the particles of the reference fluid sample at the second time;

[0263] calculating the difference between the measurement of the characteristic of the particles of the reference fluid sample at the first time and the second time, and generating a reference profile for the reference fluid sample; and

[0264] comparing the profile to the reference profile.

[0265] By comparing the profile, having calculated a difference in the measurement of the characteristic, to a reference profile, corresponding to a reference fluid sample having a known microorganism content, it can be possible to determine the microorganism content in the fluid sample. In particular, it can be possible to determine the microorganism content which is above a baseline as defined by the reference fluid sample and reference profile.

[0266] Visual representations of the profile and the reference profile may be useful for determining the microorganism content which is above a baseline as defined by the reference fluid sample and reference profile. For example, the profile and the reference profile may each be represented in a graphical format and overlayed on top of each other. A visual inspection of the profile and the reference profile may be taken, and a difference, if present, may be compared.

[0267] In some embodiments, the method further comprises the steps of

[0268] 008894123taking a plurality of measurements of the characteristic of the particles of the reference fluid sample at the first time;

[0269] taking a plurality of measurements of the characteristic of the particles of the reference fluid sample at the second time;

[0270] calculating the difference between each measurement of the characteristic of the particles of the reference fluid sample at each first time and each second time to obtain a plurality of differences, and generating, based on the plurality of differences, a reference profile for the reference fluid sample; and

[0271] comparing the profile to the reference profile, based on a comparison between the profile and the standard deviation of the plurality of differences of the reference profile.

[0272] A plurality of differences is produced for the reference profile to provide an extent of the natural and / or experimental variation in the measurements between reference fluid samples obtained from the same substance. Such natural and / or experimental variation typically follows a normal distribution around an arithmetic average. The standard deviation may be used as a metric for quantifying this natural and / or experimental variation. The standard deviation may be calculated from the arithmetic average of the plurality of differences of the reference profile. The microorganism content can then be determined from a comparison between the profile and the reference profile, based on the extent to which the profile differs from the standard deviation of the plurality of differences of the reference profile.

[0273] In some embodiments, the profile may match the reference profile where at least one measurement of the characteristic at a time for the fluid sample is within two standard deviations from the arithmetic average of the measurement of the characteristic at the same time for the reference fluid sample.

[0274] In some embodiments, the profile may be different to the reference profile where at least one measurement of the characteristic at a time for the fluid sample is outside of two standard deviations from the arithmetic average of the measurement of the characteristic at the same time for the reference fluid sample.

[0275] Alternatively, the microorganism content can be detected by calculating a comparative difference. The “comparative difference” may refer to the relative difference between (a) the difference in the measurement of the characteristic between the first time and second time for the fluid sample and (b) the difference in the measurement of the characteristic between the first time and second time for the reference fluid sample. The comparative difference may be expressed as a percentage difference between (a) and (b).

[0276] In some embodiments, the profile may match the reference profile where the comparative difference is at most 5%, such as at most 4%, such at most 3%, such as at most 2%, such as at most 1%.

[0277] 008894123In some embodiments, the profile may be different to the reference profile where the comparative difference is at least 10%, such as at least 20%, such at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 100%, such as at least 200%, such as at least 300%, such as at least 400%, such as at least 500%.

[0278] Generating Reference Profiles

[0279] In a second aspect of the invention, there is provided a method of generating a reference profile for a substance, the method comprising the steps of:

[0280] (i) providing a fluid sample of the substance, wherein the reference fluid sample comprises particles and a known microorganism content;

[0281] (ii) taking a measurement of a characteristic of the particles at a first time;

[0282] (iii) taking a measurement of a characteristic of the particles at a second time, wherein the second time is later than the first time; and

[0283] (iv) calculating the difference between the measurement of the characteristic of the particles at the first time and the second time, and generating a reference profile for the characteristic of the particles over time.

[0284] The reference profile contains information about the measurements of the characteristics of the particles in the reference fluid sample at a first time and a second time, where the reference fluid sample comprises particles and a known microorganism content. Suitably, the reference fluid sample and the substance may share at least the same particles and the same known quantity of microorganisms.

[0285] The “known microorganism content” may be no microorganisms or may be an acceptable amount of microorganisms for consumption (e.g. an acceptable yeast content for consumption in a beer-based beverage).

[0286] In some embodiments, the known microorganism content may be no microorganisms.

[0287] Thus, the reference profile of the second aspect is intended to be compared against the profile of the first aspect, to determine the microorganism content in the fluid sample of the substance, and the substance, of the first aspect.

[0288] The method of generating a reference profile may be repeated for different substances, such that a plurality of reference profiles may be generated for each substance. Each reference profile may be specific for each substance.

[0289] The method of generating a reference profile may be repeated for the same substance, such that a plurality of measurements of a characteristic of the particles at a first time, and a plurality of measurements of a characteristic of the particles at a second time, are taken.

[0290] 008894123Thus, in some embodiments, the method further comprises the steps of

[0291] taking a plurality of measurements of the characteristic of the particles at the first time;

[0292] taking a plurality of measurements of the characteristic of the particles at the second time; and

[0293] calculating the difference between each measurement of the characteristic of the particles of the reference fluid sample at each first time and each second time to obtain a plurality of differences, and generating, based on the plurality of differences, a reference profile for the reference fluid sample.

[0294] Preferred features of the first aspect apply equally to the second aspect, unless otherwise specified.

[0295] In some embodiments, the reference fluid sample comprises no microorganisms, such as no live microorganisms.

[0296] In some embodiments, the substance comprises no microorganisms, such as no live microorganisms.

[0297] In some embodiments, the characteristic of the particles is selected from particle concentration, average particle size and particle size distribution.

[0298] The method of generating a reference profile comprises measuring a characteristic of the particles at a first time and measuring a characteristic of the particles at a second time. The method of generating a reference profile may comprise any of the total time differences as described in the method of the first aspect. For example, the total time difference is from 12 to 48 hours, such as from 14 to 44 hours, such as from 16 to 40 hours, such as from 18 to 36 hours, such as from 20 to 32 hours. For example, the total time difference is from 1 to 12 hours such as from 2 to 8 hours, such as from 3 to 4 hours.

[0299] The method of generating a reference profile involves taking a measurement of a characteristic at a first time and at a second time. In some embodiments, the method comprises taking at least one measurement of a characteristic of the particles between the first time and the second time. Thus, in some embodiments, the method comprises taking a measurement of a characteristic of the particles at least three times.

[0300] In some embodiments, the characteristic of the particles is measured continuously between the first time and the second time.

[0301] In some embodiments, the characteristic of the particles is measured using resistive pulse sensing, as described herein.

[0302] 008894123The substance may be a solid or a fluid, as described herein. Suitably, the substance is a fluid.

[0303] In some embodiments, the substance is a product.

[0304] In some embodiments, the product is a food, beverage, detergent, or medicinal product, as described herein. Suitably, the product is a beverage, such as an alcoholic beverage.

[0305] Other Preferences

[0306] Each and every compatible combination of the embodiments described above is explicitly disclosed herein, as if each and every combination was individually and explicitly recited.

[0307] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0308] “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0309] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0310] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above.

[0311] Experimental and Results

[0312] Particle Size and Concentration

[0313] Changes in particle size and particle concentration over time were investigated. Both increases in particle and changes in average particle size were observed in the data.

[0314] However, the focus was placed on observing absolute changes in particle number rather than restricting the analysis to observing increases alone. It was recognized that particle numbers within specific size ranges could decrease, potentially due to smaller particles aggregating into larger ones. This hypothesis was supported by observations where increases in the number of larger particles were accompanied by decreases in the number of smaller particles.

[0315] 008894123Particle sizes analysed were typically within the range of 1 to 20 pm in diameter. However, a narrower size range of 2 to 8 pm was often focused on as the more typical range for beverages. Nevertheless, consistent trends were observed across all ranges of particle sizes.

[0316] Baseline particle numbers vary across different types of alcoholic beverages. For instance, particularly high particle counts were observed in cream liqueurs (e.g., Baileys and Kahlua), and more variable particle concentrations were noted in seasonal beers (e.g., Rudolf and Blitzen).

[0317] Particle concentrations in beverages were shown to span a wide range, depending on the specific type of the beverage. Particle numbers of approximately 1 ,000 particles / mL were observed in some final product beers. Figure 1 shows the concentration of particles (expressed as “particles / mL”) for four brands of commercially available beers. These beers were shown to have concentrations in the range from 100,000 to 1,000,000 particles / mL. For comparison, particle numbers up to 1,000,000,000 particles / mL were shown in dairybased products, such as milk.

[0318] The analysis focussed on the changes in characteristics of the particles from an earlier time to a later time. Notably, in the absence of any microorganisms, or a small content of microorganisms in the fluid sample, particle concentrations were found to remain stable or to exhibit minimal changes over time. Significant changes in particle concentration or average particle size were interpreted as indicators of microbial activity.

[0319] In one illustrative example, the method is applied to a beverage having a total volume of approximately 440 mL. Such beverages may contain particles, for example ingredient particles, at a concentration in the range from 100 to 10,000 particles / mL. In this example, a sample of a volume of less than 50 mL of the beverage is measured. The particles present in the sample are detected, and the characteristics of the particles in the sample are extrapolated to predict the characteristics of the particles in the full beverage volume.

[0320] In this beverage, the microorganism concentration is at a significantly low level, for example fewer than 20 microorganism cells in the total volume. Statistically, this corresponds to around one or two microorganism cells present in the 50 mL sample. In such conditions, the microorganisms represent a very small proportion of the detected signal compared to the particle population (for example, around 0.2% or less of detected entities when using resistive pulse sensing). In the method, even a low concentration of microorganisms may produce a measurable and resolvable change in particle characteristics.

[0321] As a result, any changes in the detected particle characteristics are representative of the presence or activity of microorganisms in the sample, even though the microorganisms themselves are not individually resolved by the method.

[0322] 008894123Microorganism Effect and Analysis

[0323] The beverage was taken and placed into an oven as part of the analytical process. A range of temperatures may be used, and in some cases, the process was performed by leaving the beverage on a benchtop. Nevertheless, any temperature which promoted microorganism growth would be applicable. For example, the same process would have worked by leaving the beverage in a fridge, although it would have taken longer for any changes in the characteristics to be observed.

[0324] Figure 2 shows the colony forming unit (CFU) over a period of 24 hours for a sample obtained from a brand of commercially available beer modified with the following foreign microorganism cells: no added yeast cells, 10 added yeast cells, 100 added yeast cells, 1 ,000 added yeast cells and 10,000 added yeast cells. Only CFU data for samples obtained from beer modified with 1,000 added yeast cells and 10,000 added yeast cells can be resolved.

[0325] Analysis on the same samples used to produce Figure 2 were performed to obtain Figure 3.

[0326] Figure 3 shows the concentration of particles (expressed as “particles / mL”) in the same beer samples used in Figure 2, comprising no added yeast cells, 10 added yeast cells, 100 added yeast cells, 1,000 added yeast cells and 10,000 added yeast cells over a period of 24 hours.

[0327] The solid line represented the sample obtained from beer that had no microbial contamination over the course of the analysis. A small change was observed in this sample. Each coloured dot represented a different level of microbial contamination, and at each time point, the presence of microorganisms resulted in a higher particle count. The magnitude of change, compared to the baseline, was quantified. A greater number of particles (concentration of particles) from the initial number corresponded to a larger change.

[0328] In some cases, microorganisms cause an increase in the number of particles and a change in particle size distribution. Figure 4 shows the particle size distribution over a period of time across three measurements. A darker area represents a measurement taken at a later time, such that the light grey area is the time after 0 hours, the medium grey area is the time after 24 hours and the dark grey area is the time after 48 hours. The results show a significant increase in the number of particles between 4 to 7 pm in size across the 48-hour period, which is indicative of microbial contamination.

[0329] Figure 5 shows the variability of Lactobacillus brevis bacterial growth in two brands of commercially available beers, an alcoholic beer and a non-alcoholic beer, over a period of 48 hours.

[0330] 008894123Bacteria contamination also shows the same trend, and depending upon the strain of bacteria the time taken to see the increase in particle number can vary. Figure 6 shows the colony forming unit (CFU) over a period of 24 hours for a sample obtained from a brand of commercially available beer modified with the following: no added yeast cells, 10 added yeast cells, 100 added yeast cells, 1,000 added yeast cells and 10,000 added yeast cells.

[0331] The method targets specifically live microorganisms. Figure 6 shows that at time zero, 10,000 bacterial cells were added, but after 24 hours, the number had decreased. Thus, the cells were found to be dying and not viable in the drink. When the bacteria were filtered out and placed on agar nutrients, only 1,000 bacterial cells were observed to grow. In this experiment, the bacteria were not alive or growing in the beer, and so little change in particle number was observed in the data.

[0332] Figure 7 shows the concentration of particles (expressed as “particles / mL”) over a period of 24 hours in the same beer samples used in Figure 6. As with Figure 6, Figure 7 shows little to no change in particle numbers. Therefore, the microorganisms typically need to be alive and capable of metabolising particles for a change in the particle concentration to be observed relative to the baseline (solid line).

[0333] Figure 8 shows the concentration of particles (expressed as “particles / mL”) over a period of 72 hours for four samples obtained from three brands of commercially available beers modified with freeze-dried tomato powder as additional foreign “indicator” particles. The total number of native particles at the first time is represented by the left-most bar on each individual plot.

[0334] The data in Figure 8 shows particle concentrations in the 1 to 4 pm particle size range. In the three beer samples (“Brand 1”, “Brand 2” and “Brand 3”) without foreign particles, each had Lactobacillus brevis bacteria added, but the bacteria failed to grow in the beer samples. No bacteria were observed using standard microbiological techniques, and no significant changes in the particle concentration were detected in the analysis. The natural variation in particle concentrations for different beers exposed to air over time was visible, but no significant changes similar to those caused by the presence of microorganisms were observed.

[0335] The last data set in Figure 8 (“Brand 3+indicator particle”) represented samples of “Brand 3” beer to which foreign “indicator” particles, in the form of freeze-dried tomato powder, had been added as a way of providing extra nutrients to microorganisms present in beer. The foreign particles increased the initial particle concentration, as shown on the first bar at a time of 0 hours. These nutrients were found to sustain the bacteria, leading to an increase in particle concentration over time. This increase confirmed the presence of Lactobacillus brevis bacteria. These foreign particles behaved in the same way as the native beer

[0336] 008894123particles, allowing them to be added to water to observe bacterial growth, even when no native particles were initially present.

[0337] Figure 9 shows the change in concentration of particles (expressed as “particles / mL”) after 24 hours for 5 brands of commercially available beers which have been confirmed as being contaminated with microorganisms through standard microbiological tests. The test beers labelled “Brand 1”, “Brand 2”, “Brand 3” and “Brand 3a” are each final product beers obtained from their respective kegs. The test beers labelled “Brand 3” and “Brand 3a” are from the same beer. The test beers labelled “Brand 4” and “Brand 5” are measurements for the respective brands taken at different times or locations along the prefilter, filter and packaging points during beer production.

[0338] The magnitude of change that triggered a positive test for microbial contamination was found to vary, as it depended on the concentration of particles present initially in the beer and the type of microbe present in the beer. Lactobacillus brevis bacteria required a longer time to be detected, whereas yeast was detected within a few hours.

[0339] A specific threshold of difference in the characteristic could be used for a beverage if the baseline was known. The threshold of difference may correspond to an upper limit in the concentration of microorganisms in the beer, such that the beer is deemed to meet an internal standard. Measurements taken at a first time and a second, later time would be compared, and the observed comparative difference in the characteristics would trigger a positive result. In the case of Figure 9, the test beers were compared against a control beer having no microorganisms as the known baseline.

[0340] By establishing the characteristics for beer which has not been contaminated with microbes and providing this information in the form of reference profiles, the characteristics of beer which is being tested for microbial contamination could be measured after 24 hours and compared against the reference profile to detect microorganisms and microbial contamination in the beer sample.

[0341] References

[0342] A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

[0343] WO 2021 / 079153

[0344] Yu, S. et al. Monitoring bacterial growth using tunable resistive pulse sensing with a porebased technique. Appl. Microbiol. Biotechnol. 98, 855-862 (2014)

[0345] 008894123

Claims

Claims:

1. A method of detecting microorganisms in a substance, the method comprising the steps of:(i) providing a fluid sample of the substance, wherein the fluid sample comprises particles;(ii) taking a measurement of a characteristic of the particles at a first time;(iii) taking a measurement of the characteristic of the particles at a second time, wherein the second time is later than the first time;(iv) calculating the difference between the measurements of the characteristic at the first time and the second time;(v) determining, based on the difference, the microorganism content in the fluid sample.

2. The method of claim 1, wherein the characteristic of the particles is selected from particle concentration, average particle size and particle size distribution.

3. The method of claim 1 or 2, wherein the total time difference between the first time and the second time is from 1 to 12 hours.

4. The method of any one of the preceding claims, further comprisingtaking at least one measurement of a characteristic of the particles at a third time between the first time and the second time.

5. The method of claim 4, wherein the individual time difference between taking any two consecutive measurements is from 15 minutes to 6 hours.

6. The method of any one of the preceding claims, wherein the characteristic of the particles is measured continuously between the first time and the second time.

7. The method of any one of the preceding claims, further comprisinggenerating a profile from the difference between the measurement of the characteristic at the first time and the second time;providing a reference fluid sample of the substance, wherein the reference fluid sample and the fluid sample share at least the same particles and wherein the reference fluid sample comprises a known microorganism content;taking a measurement of a characteristic of the particles of the reference fluid sample at the first time;taking a measurement of a characteristic of the particles of the reference fluid sample at the second time;008894123calculating the difference between the measurement of the characteristic of the particles of the reference fluid sample at the first time and the second time, and generating a reference profile for the reference fluid sample; andcomparing the profile to the reference profile.

8. The method of claim 7, further comprisingtaking a plurality of measurements of the characteristic of the particles of the reference fluid sample at the first time;taking a plurality of measurements of the characteristic of the particles of the reference fluid sample at the second time;calculating the difference between each measurement of the characteristic of the particles of the reference fluid sample at each first time and each second time to obtain a plurality of differences, and generating, based on the plurality of differences, a reference profile for the reference fluid sample; andcomparing the profile to the reference profile, based on a comparison between the profile and the standard deviation of the plurality of differences of the reference profile.

9. The method of any one of the preceding claims, wherein the characteristic of the particles is measured using resistive pulse sensing.

10. The method of any one of the preceding claims, wherein the fluid sample comprises native particles and / or foreign particles.

11. The method of any one of the preceding claims, wherein the concentration of particles in step (ii) is from 100 to 1,000,000,000 particles / mL, as measured using resistive pulse sensing.

12. The method of any one of the preceding claims, wherein the average particle size in step (ii) is from 1 to 10 pm, as measured using resistive pulse sensing.

13. The method of any one of the preceding claims, wherein the method is performed at a temperature of at least 20 °C, such as at least 30 °C, such as at least 40 °C.

14. The method of any one of the preceding claims, wherein the microorganism is selected from a bacterial cell, a fungal cell and a viral cell.

15. The method of any one of the preceding claims, wherein the substance is a product, such as a food, beverage, detergent or medicinal product.

16. The method of any one of the preceding claims, wherein the substance is a beverage, such as a non-alcoholic beverage.00889412317. A method of generating a reference profile for a substance, the method comprising the steps of:(i) providing a reference fluid sample of the substance, wherein the reference fluid sample comprises particles and a known microorganism content;(ii) taking a measurement of a characteristic of the particles at a first time;(iii) taking a measurement of a characteristic of the particles at a second time, wherein the second time is later than the first time; and(iv) calculating the difference between the measurement of the characteristic of the particles at the first time and the second time, and developing a reference profile for the characteristic of the particles over time.

18. The method of claim 17, wherein the characteristic of the particles is selected from particle concentration, average particle size and particle size distribution.

19. The method of claim 17 or 18, wherein the total time difference between the first time and the second time is from 1 to 12 hours.

20. The method of any one of claims 17 to 19, further comprisingtaking at least one measurement of a characteristic of the particles at a third time between the first time and the second time.

21. The method of any one of claims 17 to 20, wherein the characteristic of the particles is measured continuously between the first time and the second time.

22. The method of any one of claims 17 to 21, further comprisingtaking a plurality of measurements of the characteristic of the particles at the first time;taking a plurality of measurements of the characteristic of the particles at the second time; andcalculating the difference between each measurement of the characteristic of the particles of the reference fluid sample at each first time and each second time to obtain a plurality of differences, and generating, based on the plurality of differences, a reference profile for the reference fluid sample.

23. The method of any one of claims 17 to 22, wherein the characteristic of the particles is measured using resistive pulse sensing.

24. The method of any one of claims 17 to 23, wherein the substance is a product, such as a food, beverage, detergent or medicinal product.

25. The method of any one of claims 17 to 24, wherein the substance is a beverage, such as a non-alcoholic beverage.008894123

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