EP4623105A1

EP4623105A1 - Treatment of milk samples for improved analysis

Info

Publication number: EP4623105A1
Application number: EP23810090.3A
Authority: EP
Inventors: Lea HOLRITZ; Winnie Edith Svendsen; Maria DIMAKI; Susan Ibi PREUS
Original assignee: Danmarks Tekniske Universitet
Current assignee: Danmarks Tekniske Universitet
Priority date: 2022-11-25
Filing date: 2023-11-24
Publication date: 2025-10-01
Also published as: WO2024110621A1

Abstract

The present invention relates to the preparation of milk samples with bile salts and lipase to allow the detection of analytes and/or pathogens.

Description

Treatment of milk samples for improved analysis

Technical field

Background

Quality analysis of milk samples plays an important role in numerous settings, from neonatal units to dairy industry. In neonatal units, identifying undesired components such as pathogens, chemicals, and hormones plays an important role in the assessment of the quality of donor milk. In the dairy industry, mastitis plays a great economic role and may affect milk quality as well as animal welfare. Screening for mastitis in e.g. cow milk is hence of outmost interest. Quality analysis, such as identification of analytes or pathogens, is however hampered by the nature of milk samples.

Milk is a viscous and complex fluid. Milk fat in human milk ranges from 1.8-8.9 g/dL. The composition of milk changes throughout stages of lactogenesis, hindmilk versus foremilk, from donor to donor, and even depending on what time of the day the milk is produced. In human milk, the reference standard related to the fat content is assumed to be 3.5 g/dL. However, numerous studies provide diverging results.

Efforts to create sample homogeneity of milk samples for analysis in the literature include centrifuging, filtering, or diluting the milk samples before analysis. By centrifuging and filtering, larger molecules are separated from the serum and are, therefore, not included in the sample to be analyzed. Bacteria are known to attach to the surface of the milk fat globules; thus, by excluding parts of the sample matrix, some analytes are likely to be lost, thus decreasing the sensitivity of the test.

Point-of-use technologies, such as lateral flow assays (LFA), for detection of quality parameters are currently hampered by the high fat percentage in raw milk. Diluting the samples leads to a significant decrease in sensitivity, and simply upscaling the milk sample size in a LFA is not favorable due to the limitations of the paper media. The pore distribution in nitrocellulose membranes is chaotic and non-homogeneous. Thus, LFA is currently limited by the nature of the paper platform, where larger molecules tend to stick and clog the membrane. Even in lower concentrations, milk fat clogs the pores of the paper membranes. Therefore, LFA is not a standard or recommended method for milk analysis.

Improved methods with reduced costs and allowing for reproducible, easy and fast analysis of milk samples are thus needed.

Summary

The main object of the present disclosure is to provide a method for preparing a mammalian milk sample in order to facilitate the analysis of pathogens and other analytes potentially present in the milk.

Thus, in a first aspect of the invention, the present disclosure concerns a method for preparing a mammalian milk sample for detecting at least one analyte and/or a pathogen, the method comprising: a) obtaining the milk sample from a mammal; and b) contacting said milk sample with lipase and one or more bile salts, each at a concentration above 2 mg/mL and below 120 mg/mL, thereby obtaining a treated milk sample.

In another aspect of the invention, the present disclosure concerns a method for detecting at least one analyte and/or a pathogen in a mammalian milk sample, the method comprising: a. obtaining the milk sample from a mammal; b. contacting said milk sample with lipase and one or more bile salts at concentrations above 2 mg/mL and below 120 mg/mL, thereby obtaining a treated milk sample; and c. subjecting the treated milk sample obtained in b) to analysis, thereby allowing detection of said analyte and/or pathogen.

In another aspect of the invention, the present disclosure concerns a method of diagnosing mastitis in a mammal, the method comprising performing the method described herein, wherein the analysis is for detecting a pathogen causing mastitis. In another aspect of the invention, the present disclosure concerns a method of assessing the quality of a milk sample of mammalian origin, such as a milk sample from a cow or from a human , the method comprising performing the method described herein, wherein the analysis is for detecting a pathogen or an analyte.

In another aspect of the invention, the present disclosure concerns a kit comprising a. lipase in an amount allowing concentrations above 2 mg/mL and below 120 mg/mL to be added to a milk sample, preferably said milk sample has a volume of above 500 pL and below 700 pL; b. one or more bile salts in an amount allowing concentrations above 2 mg/mL and below 120 mg/mL to be added to said milk sample; and c. instructions for use.

Description of Drawings

Figure 1 Initial analysis, where the protocol was dependent on broth as a buffer. Time is recorded in minutes. means that the sample is never fully absorbed. BS: Bile salts, L: lipase

Figure 2 LFA analysis of different matrices, a) untreated milk, b) ultrapure water, c) milk serum collected from centrifuged samples, d) chemically treated raw milk.

Figure 3. LFA test with 100 pL spiked milk. From left: 10⁵ CFU/mL, 10⁸ CFU/mL

Figure 4. The effect of the preparation protocol on a raw milk sample with fat concentration of 7.6%. The sample turned transparent with a faint yellow colour.

Figure 5. From left: raw human milk sample, treated human milk sample approximately 3 minutes after applying the protocol), and picture of the glass slide without added sample. 20x magnification. The raw milk sample has a fat percent of 7.6%.

Figure 6. Tracking a milk fat cell. T0= 15 s after applied protocol. Data obtained through Ocelloscope. Figure 7. The visual effect of the preparation protocol in a subclinical mastitis cow's milk sample.

Figure 8. The visual effect of the preparation protocol after 5 minutes. From left to right: samples from a healthy cow, a cow suffering from subclinical mastitis, and a cow suffering from acute mastitis.

Figure 9. Visual appearance of the milk 5 minutes after treatment, ‘c’ refers to treatment with lipase from Candida rugosa. ‘p’ refers to treatment with lipase from porcine pancreas.

Figure 10. Microscope image of milk without prior treatment.

Figure 11. Microscope image of milk treated with lipase from Candida rugosa.

Figure 12. Microscope image of milk treated with lipase from Porcine pancreas.

Figure 13. (Upper panel) The ability of whole milk facilitating through a paper-based membrane of nitrocellulose. is milk with no treatment, ‘c’ is milk treated with Candida rugosa. lipase and ‘p’ is milk treated with porcine pancreatic lipase. (Lower panel) Dipsticks in no treatment, ‘c’: C. rugosa, and ‘p’: porcine pancreatic lipase. The dipstick comprises a nitrocellulose membrane and an absorption pad.

Detailed description

The present disclosure describes a method or a kit for preparing a mammalian milk sample in order to facilitate the analysis of pathogens and analytes. Specifically, milk samples are contacted with lipase and one or more bile salts. This ensures breakdown of the fat molecules in the milk sample and prevents the fat from re-accumulating. The breakdown of fat in the milk sample allows the sample to be analysed by analytical methods in a straightforward, rapid, reproducible and low-cost manner.

In a first aspect of the invention, the present disclosure concerns a method for preparing a mammalian milk sample for detecting at least one analyte and/or a pathogen, the method comprising: a) obtaining the milk sample from a mammal; and b) contacting said milk sample with lipase and one or more bile salts, each at a concentration above 2 mg/mL and below 120 mg/mL, thereby obtaining a treated milk sample.

In another aspect of the invention, the present disclosure concerns a method for detecting at least one analyte and/or a pathogen in a mammalian milk sample, the method comprising: a) obtaining the milk sample from a mammal; b) contacting said milk sample with lipase and one or more bile salts at concentrations above 2 mg/mL and below 120 mg/mL, thereby obtaining a treated milk sample; and c) subjecting the treated milk sample obtained in b) to analysis, thereby allowing detection of said analyte and/or pathogen.

The milk sample

The milk sample may be obtained from various sources, including but not limited to human donors and the dairy industry. Depending on the source of the milk sample, detection of various analytes and/or pathogens may be of interest as well as the location for the analysis. The milk sample may e.g. be prepared and subjected to analysis by the milk provider, such as the donor of the milk sample or a farmer. In another non-limiting example, the milk sample is obtained and prepared by the milk provider and transported to a specialist, where the treated milk is subjected to analysis. In another non-limiting example, the milk sample is obtained by the milk provider and transported to a specialist, where the milk sample is prepared and subjected to analysis.

The "milk", as referred to herein, refers to animal milk, which may be low fat milk, full fat milk, raw milk, lactose-free milk (produced by hydrolyzing the lactose by lactase enzyme to glucose and galactose, or by other methods such as nanofiltration, electrodialysis, ion exchange chromatography and centrifugation technology), concentrated milk and/or dry milk. As used herein, the term "milk" is also intended to encompass milks from mammals. The milk may originate from mammals including, but not limited to, the group of mammals, such as from a cow, such as from a buffalo, such as from a goat, such as from a sheep, such as from a camel, such as from a horse, or from a donkey. In addition, the term "milk" refers not only to whole milk, but also to skim milk or any liquid component derived therefrom. "Raw milk" is used as a term generally recognized in the art, and means unprocessed milk. "Raw milk" is sometimes also referred to as "fresh milk", and both terms are considered and used as equivalents for the purpose of the present disclosure.

In some embodiments, said mammal is a human being.

In some embodiments, said mammal is a non-human mammal, such as a cow, such as a buffalo, such as a goat, such as a sheep, such as a camel, such as a horse, or a donkey.

In some embodiments, said milk sample comprises between 1.7% and 7.7% fat, such as between 2% and 7.7%, between 2% and 7%, between 2% and 6%, between 2% and 5%, between 2% and 4%, between 2% and 3%, between 1.7% and 2%, between 3% and 7.7%, between 3% and 7%, between 3% and 6%, between 3% and 5%, between 3% and 4%, between 1.7% and 3%, between 4% and 7.7%, between 4% and 7%, between 4% and 6%, between 4% and 5%, between 1.7% and 4%, between 5% and 7.7%, between 5% and 7%, between 5% and 6%, between 1.7% and 5%, between 6% and 7.7%, between 6% and 7%, between 1.7% and 6%, between 7% and 7.7%, or between 1.7% and 7% fat.

Once a milk sample has been obtained from a mammal, the sample is contacted with lipase and one or more bile salts as described herein below.

Upases

The "lipase", as referred to herein, denotes an enzyme that catalyzes the hydrolysis of lipids to glycerol and simple fatty acids. Examples of lipases suitable for the present invention include, but are not limited to, animal lipase (e.g., porcine lipase, or human lipase), bacterial lipase (e.g., Pseudomonas lipase and/or Burkholderia lipase), fungal lipase, plant lipase, recombinant lipase (e.g., produced via recombinant DNA technology by a suitable host cell, selected from any one of microorganisms, bacteria, yeast, fungi, plants, insects or mammalian host cells in culture, or recombinant lipases which include an amino acid sequence that is homologous or substantially identical to a naturally occurring sequence, lipases encoded by nucleic acid sequences homologous or substantially identical to naturally occurring lipase-encoding nucleic acid sequences, etc.), synthetic lipases, chemically-modified lipases, and mixtures thereof. In some embodiments, said lipase is a pancreatic lipase, such as a lipase from porcine pancreas, or such as a lipase from human pancreas, or a plant lipase such as a lipase from wheat, such as a wheat germ lipase. In some embodiments, the lipase is selected from a lipase from porcine pancreas, a plant lipase such as a wheat lipase, in particular a wheat germ lipase, and a microbial lipase, such as a yeast lipase or a bacterial lipase, for example the lipase is selected from a lipase from porcine pancreas, a wheat germ lipase and a Candida lipase. In some embodiments, the lipase is selected from a lipase from a porcine pancreas lipase and a Candida rugosa lipase.

In some embodiments, the milk sample is contacted with lipase, wherein the lipase concentration is between 2 and 120 mg/mL, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, or between 100 and 120 mg/mL, preferably wherein the lipase concentration is between 2 and 40 mg/mL, such as between 10 and 40 mg/mL, such as between 20 and 40 mg/mL, such as 10, 15, 20, 25, 30, 35 or 40 mg/mL.

In some embodiments, said lipase is a lipase from porcine pancreas. In some embodiments, the milk sample is contacted with a lipase from porcine pancreas, wherein the lipase concentration is between 2 and 120 mg/mL, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, or between 100 and 120 mg/mL, preferably wherein the lipase concentration is between 2 and 40 mg/mL, such as between 10 and 40 mg/mL, such as between 20 and 40 mg/mL, such as 10, 15, 20, 25, 30, 35 or 40 mg/mL. In some embodiments, said lipase is a lipase from Candida rugosa. In some embodiments, the milk sample is contacted with a lipase from Candida rugosa, wherein the lipase concentration is between 2 and 120 mg/mL, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, or between 100 and 120 mg/mL, preferably wherein the lipase concentration is between 2 and 40 mg/mL, such as between 10 and 40 mg/mL, such as between 20 and 40 mg/mL, such as 10, 15, 20, 25, 30, 35 or 40 mg/mL.

In some embodiments, said lipase is a lipase from human pancreas. In some embodiments, the milk sample is contacted with a lipase from human pancreas, wherein the lipase concentration is between 2 and 120 mg/mL, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, or between 100 and 120 mg/mL, preferably wherein the lipase concentration is between 2 and 40 mg/mL, such as between 10 and 40 mg/mL, such as between 20 and 40 mg/mL, such as 10, 15, 20, 25, 30, 35 or 40 mg/mL.

In some embodiments, the milk sample is contacted with lipase, wherein the concentration of the lipase is 5 mg/mL. In some embodiments, the lipase is from porcine pancreas and is contacted with the milk sample at a concentration of 5 mg/mL.

In some embodiments, the lipase is not a lipase derived from a plant. In some embodiments, the lipase is not a lipase derived from wheat such as wheat germ lipase. Bile salts

In addition to being contacted with a lipase, the milk sample is, in the present methods, also contacted with one or more bile salts.

The one or more bile salts as referred to herein includes reference to a bile acid or a salt thereof. Therefore, the terms "bile acid," "bile salt," "bile acid/salt," "bile acids," "bile salts," and "bile acids/salts" are, unless otherwise indicated utilized interchangeably herein. The at least one bile salt as used herein, includes steroid acids (and/or the carboxylate anion thereof), and salts thereof, found in the bile of an animal (e.g., a human, or a pig), including cholic acid, cholate, deoxycholic acid, deoxycholate, hyodeoxycholic acid, hyodeoxycholate, glycocholic acid, glycocholate, taurocholic acid, taurocholate, chenodeoxycholic acid, ursodeoxycholic acid, ursodiol, a tauroursodeoxycholic acid, a glycoursodeoxycholic acid, a 7-B-methyl cholic acid, a methyl lithocholic acid, chenodeoxycholate, lithocholic acid, lithocolate, and the like. Furthermore, pharmaceutically acceptable bile acid esters are optionally utilized as the "bile acids" described herein, e.g., bile acids/salts conjugated to an amino acid (e.g., glycine or taurine). Other bile acid esters include, e.g., substituted or unsubstituted alkyl ester, substituted or unsubstituted heteroalkyl esters, substituted or unsubstituted aryl esters, substituted or unsubstituted heteroaryl esters, or the like. For example, the term "bile acid" includes cholic acid conjugated with either glycine or taurine: glycocholate and taurocholate, respectively (and salts thereof). Any reference to a bile acid used herein includes reference to an identical compound naturally or synthetically prepared. Moreover, as used herein, bile acid/salt mimics or mimetics described herein are compounds that mimic the agonist signaling properties of the bile acid/salt.

Examples of bile acid analogues useful in the context of the present disclosure include e.g. fluorinated compounds such as described in WO 2010/014836, in the section ‘Detailed description of the invention’. In some embodiments, bile acid mimetics include triterpenoids, such as oleanoic acid, ursolic acid, or the like.

Alternatives to bile salts such as detergents may in principle be used for solubilizing milk fat and proteins. However, utilizing detergents in replacement of bile salts requires 1) that these do not interfere with the enzyme, and 2) that they do not interfere with the diagnostic target (i.e. bacteria) or with the assay for detecting the target, which assay may require the use of antibodies: the sensitivity of the assay and/or the apparent affinity of the antibodies used in the assay can be affected by the use of detergents. 1) Detergents such as SDS (sodium dodecyl sulfate) and polysorbate (commonly referred to as “Tween”) can affect enzyme activity and apparent affinity of antibodies. These detergents are often used in biochemical and molecular biology research to solubilize and stabilize proteins, including enzymes. However, their impact on enzyme activity varies depending on the specific detergent, its concentration, and the enzyme in question. SDS is a strong anionic detergent commonly used to denature proteins by disrupting their structure and separating them into individual polypeptide chains, thus SDS can inhibit the activity of some enzymes by denaturing or inactivating them by disrupting their natural conformation. This is concentration dependent, and enzymespecific. SDS up to a concentration of 20% increases lipase activity, however higher concentrations decrease enzyme activity. Polysorbate is a nonionic detergent that is less denaturing than SDS. It is often used to solubilize hydrophobic proteins and maintain their native conformation. In some cases, polysorbate can enhance enzyme activity by stabilizing the enzyme’s structure, preventing aggregation, and maintaining enzyme activity in solution. However, the effect of polysorbate on enzyme activity can vary depending on the enzyme and the specific conditions of the assay. Overall, detergents like SDS and polysorbate can either enhance or inhibit enzyme activity, depending on the concentration of the detergent and the specific enzyme.

2) The impact of these detergents on bacteria varies depending on the concentration, bacterial species, and the specific application. Detergents like SDS can disrupt bacterial cells while polysorbate can aid in the dispersion of hydrophobic substances. The interaction is both concentration and time-dependent.

Thus, as the interaction of detergents with a species is dependent on both species, the specific detergent and the concentration thereof, and without being bound by theory, it less likely to find a detergent which does not interfere with the enzyme or the diagnostic target or assay which could give the same results as using a bile salt.

Methods based on detergents can be expected to lack sensitivity as they may interfere with the enzyme, diagnostic target (i.e. bacteria) and/or with the assay for detecting the target.

Thus in preferred embodiments, no detergent is used in the present methods. In some embodiments, the milk sample is contacted with one or more bile salts, wherein the concentration of the one or more bile salts is between 2 and 120 mg/mL, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 50 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, preferably wherein the concentration of the bile salts is between 80 and 120 mg/mL, such as between 90 and 110 mg/mL, such as between 95 and 105 mg/mL, such as 80, 85, 90, 95, 100, 105, 110, 115 or 120 mg/mL.

In some embodiments, said one or more bile salts comprise cholic acid sodium salt and deoxycholic acid sodium salt. In some embodiments, the milk sample is contacted with one or more bile salts, wherein the concentration of the at least one bile salt is 100 mg/mL. In some embodiments, said one or more bile salts comprise cholic acid sodium salt and deoxycholic acid sodium salt, and the concentration of bile salts is between 2 and 120 mg/mL, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 50 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, preferably wherein the concentration of the bile salts is between 80 and 120 mg/mL, such as between 90 and 110 mg/mL, such as between 95 and 105 mg/mL, such as 80, 85, 90, 95, 100, 105, 110, 115 or 120 mg/mL. In some embodiments, the bile salts comprise cholic acid sodium salt and deoxycholic acid sodium salt and the concentration of bile salts is 100 mg/mL.

Preparing the milk sample

The use of both lipase and one or more bile salts ensures breakdown of the fat molecules and prevents the fat from re-accumulating. In some embodiments, the milk sample is contacted with said lipase and said one or more bile salts simultaneously.

In some embodiments, the milk sample is first contacted with said lipase and then contacted with said one or more bile salts.

In some embodiments, the bile salts comprise or consist of cholic acid sodium salt and deoxycholic acid sodium salt and/or the lipase is lipase from porcine pancreas. In other embodiments, the bile salts comprise or consist of cholic acid sodium salt and deoxycholic acid sodium salt and/or the lipase is lipase from porcine pancreas. In some embodiments, the milk sample is contacted with bile salts at a concentration of between 2 and 120 mg/mL, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 50 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, preferably wherein the concentration of the bile salts is between 80 and 120 mg/mL, such as between 90 and 110 mg/mL, such as between 95 and 105 mg/mL, such as 80, 85, 90, 95, 100, 105, 110, 115 or 120 mg/mL, and with lipase at a concentration of between 2 and 120 mg/mL, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, or between 100 and 120 mg/mL, preferably wherein the lipase concentration is between 2 and 40 mg/mL, such as between 10 and 40 mg/mL, such as between 20 and 40 mg/mL, such as 10, 15, 20, 25, 30, 35 or 40 mg/mL.

In some embodiments, the milk sample is contacted with lipase and one or more bile salts, wherein the concentration of the one or more bile salts is 100 mg/mL and/or the concentration of the lipase is 5 mg/mL. In some embodiments, the bile salts are cholic acid sodium salt and deoxycholic acid sodium salt and/or the lipase is lipase from porcine pancreas and the concentration of the one or more bile salts is 100 mg/mL and/or the concentration of the lipase is 5 mg/mL.

In some embodiments, the concentrations of the lipase and the one or more bile salts are the same or different.

In some embodiments, the milk sample is contacted with lipase and one or more bile salts, wherein the concentrations are selected from: a) 2 mg/mL of the one or more bile salts and 2 mg/mL of the lipase; b) 10 mg/mL of the one or more bile salts and 10 mg/mL of the lipase; c) 20 mg/mL of the one or more bile salts and 20 mg/mL of the lipase; d) 50 mg/mL of the one or more bile salts and 50 mg/mL of the lipase; e) 2 mg/mL of the one or more bile salts and 100 mg/mL of the lipase; f) 10 mg/mL of the one or more bile salts and 100 mg/mL of the lipase; g) 20 mg/mL of the one or more bile salts and 100 mg/mL of the lipase; h) 50 mg/mL of the one or more bile salts and 100 mg/mL of the lipase; i) 100 mg/mL of the one or more bile salts and 100 mg/mL of the lipase; j) 100 mg/mL of the one or more bile salts and 2 mg/mL of the lipase; k) 100 mg/mL of the one or more bile salts and 10 mg/mL of the lipase; l) 100 mg/mL of the one or more bile salts and 20 mg/mL of the lipase; or m) 100 mg/mL of the one or more bile salts and 50 mg/mL of the lipase.

In some embodiments, the milk sample is contacted with lipase from porcine pancreas, cholic acid sodium salt and deoxycholic acid sodium salt simultaneously.

In some embodiments, said contacting the milk sample with lipase and one or more bile salts is performed for less than 5 minutes, such as less than 4 minutes, such as less than 3 minutes, such as 2 minutes, such as 1 minute, such as 30 seconds, such as 20 seconds, such as 10 seconds, or such as 1 second.

In some embodiments, said contacting is performed at a room temperature In some embodiments, said contacting is performed at a temperature between 18°C and 30°C, such as between 18°C and 28°C, between 18°C and 26°C, between 18°C and 24°C, between 18°C and 22°C, between 18°C and 20°C, between 20°C and 30°C, between 20°C and 28°C, between 20°C and 26°C, between 20°C and 24°C, between 20°C and 22°C, between 22°C and 30°C, between 22°C and 28°C, between 22°C and 26°C, between 22°C and 24°C, between 24°C and 30°C, between 24°C and 28°C, between 24°C and 26°C, between 26°C and 30°C, between 26°C and 28°C, or between 28°C and 30°C.

In some embodiments, said lipase and one or more bile salts are suspended in water prior to said contacting, thereby obtaining a preparation solution, preferably wherein the water is distilled water or ultrapure water. The preparation solution is a water suspension comprising said lipase and one or more bile salts.

In some embodiments, said preparation solution is mixed with said milk sample in a preparation solution to milk sample ratio between 1:1 and 1 :3, such as between 1 :1 and 1 :2.5, between 1:1 and 1 :2, between 1 :1 and 1 :1.5, between 1 :1.5 and 1:3, between 1 :1.5 and 1 :2.5, between 1:1.5 and 1:2, between 1 :2 and 1:3, between 1 :2 and 1 :2.5, or between 1 :2.5 and 1:3.

In some embodiments, the preparation solution is stored prior to contacting the milk sample with the preparation solution. In some embodiments, the storing is in a refrigerator for up to 20 days, such as up to 18 days, up to 16 days, up to 14 days, up to 12 days, 10 day, up to 8 days, up to 7 days, up to 6 days, up to 4 days, up to 2 days, or up to 1 day. In some embodiments, the storing is in a freezer for up to 8 weeks, such as up to 7 weeks, such as up to 6 weeks, such as up to 5 weeks, such as up to 4 weeks, such as up to 3 weeks, such as up to 2 weeks, or such as up to 1 week.

It may be desirable to analyse the milk sample for the presence of pathogens, e.g. bacteria. The milk sample contacted with lipase and bile salts can be inoculated and incubated in a growth medium supporting growth of suspected pathogens in order to amplify the number of bacterial cells and facilitate detection. Thus, in some embodiments, said lipase and one or more bile salts are suspended in bacterial growth medium prior to said contacting, thereby obtaining a medium suspension, preferably wherein the bacterial growth medium is LB medium.

In some embodiments, said medium suspension is mixed with said milk sample in a medium suspension to milk sample ratio between 1 :1 and 1:3, such as between 1 :1 and 1 :2.5, between 1:1 and 1:2, between 1 :1 and 1 :1.5, between 1:1.5 and 1:3, between 1 :1.5 and 1 :2.5, between 1:1.5 and 1:2, between 1 :2 and 1:3, between 1 :2 and 1 :2.5, or between 1 :2.5 and 1 :3, thereby obtaining a growth suspension.

In some embodiments, said growth suspension is incubated at room temperature.

In some embodiments, said growth suspension is incubated at temperatures above 16°C and below 42°C, such as between 16°C and 40°C, between 16°C and 38°C, between 16°C and 36°C, between 16°C and 34°C, between 16°C and 32°C, between 16°C and 30°C, between 16°C and 28°C, between 16°C and 26°C, between 16°C and 24°C, between 16°C and 22°C, between 16°C and 20°C, between 16°C and 18°C, between 18°C and 42°C, between 18°C and 40°C, between 18°C and 38°C, between 18°C and 36°C, between 18°C and 34°C, between 18°C and 32°C, between 18°C and 30°C, between 18°C and 28°C, between 18°C and 26°C, between 18°C and 24°C, between 18°C and 22°C, between 18°C and 20°C, between 20°C and 42°C, between 20°C and 40°C, between 20°C and 38°C, between 20°C and 36°C, between 20°C and 34°C, between 20°C and 32°C, between 20°C and 30°C, between 20°C and 28°C, between 20°C and 26°C, between 20°C and 24°C, between 20°C and 22°C, between 22°C and 42°C, between 22°C and 40°C, between 22°C and 38°C, between 22°C and 36°C, between 22°C and 34°C, between 22°C and 32°C, between 22°C and 30°C, between 22°C and 28°C, between 22°C and 26°C, between 22°C and 24°C, between 24°C and 42°C, between 24°C and 40°C, between 24°C and 38°C, between 24°C and 36°C, between 24°C and 34°C, between 24°C and 32°C, between 24°C and 30°C, between 24°C and 28°C, between 24°C and 26°C, between 26°C and 42°C, between 26°C and 40°C, between 26°C and 38°C, between 26°C and 36°C, between 26°C and 34°C, between 26°C and 32°C, between 26°C and 30°C, between 26°C and 28°C, between 28°C and 42°C, between 28°C and 40°C, between 28°C and 38°C, between 28°C and 36°C, between 28°C and 34°C, between 28°C and 32°C, between 28°C and 30°C, between 30°C and 42°C, between 30°C and 40°C, between 30°C and 38°C, between 30°C and 36°C, between 30°C and 34°C, between 30°C and 32°C, between 32°C and 42°C, between 32°C and 40°C, between 32°C and 38°C, between 32°C and 36°C, between 32°C and 34°C, between 34°C and 42°C, between 34°C and 40°C, between 34°C and 38°C, between 34°C and 36°C, between 36°C and 42°C, between 36°C and 40°C, between 36°C and 38°C, between 38°C and 42°C, between 38°C and 40°C, or between 40°C and 42°C.

In some embodiments, the method comprises a step of diluting the milk sample in a milk sample to diluent ratio of at most 1:1, such as at most 1 :0.9, such as 1 :0.8, such as 1 :0.7, such as 1 :0.6, such as 1 :0.5, or such as 1 :0.4.

As described elsewhere in this application, during centrifugation and filtration, larger molecules are separated from the serum and, therefore, not included in the sample to be analyzed. Bacteria are known to attach to the surface of the milk fat globules; thus, by excluding parts of the sample matrix, some analytes are likely to be lost, thus decreasing the sensitivity of the test. Thus, in some embodiments, the method does not comprise a step of filtering the sample. In some embodiments, the method does not comprise a step of centrifuging the sample. In some embodiments, the method does not comprise a step of filtering the sample, and does not comprise a step of centrifuging the sample.

Protease

Protein accumulation, such as fibrin accumulations, is known in the art to be found in a subclinical and acute mastitis sample. These protein accumulations make the milk more likely to be viscous. Proteases, such as plasmin, known in the art to break down fibrin, may hence be employed in the preparation to ensure an even more homogenous preparation of the milk sample.

In some embodiments, said contacting comprises contacting with a lipase, one or more bile salts and a protease capable of degrading fibrin.

In some embodiments, said protease is plasmin.

In some embodiments, the bile salts are cholic acid sodium salt and deoxycholic acid sodium salt and the protease is plasmin. In some embodiments, the lipase is lipase from porcine pancreas and the protease is plasmin. In some embodiments, the bile salts are cholic acid sodium salt and deoxycholic acid sodium salt, the lipase is lipase from porcine pancreas and the protease is plasmin.

The concentration of lipase and bile salts can be as described herein above. In some embodiments, the milk sample is contacted with bile salts at a concentration of between 2 and 120 mg/mL, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 50 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, preferably wherein the concentration of the bile salts is between 80 and 120 mg/mL, such as between 90 and 110 mg/mL, such as between 95 and 105 mg/mL, such as 80, 85, 90, 95, 100, 105, 110, 115 or 120 mg/mL, a lipase at a concentration of between 2 and 120 mg/mL, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, or between 100 and

120 mg/mL, preferably wherein the lipase concentration is between 2 and 40 mg/mL, such as between 10 and 40 mg/mL, such as between 20 and 40 mg/mL, such as 10, 15, 20, 25, 30, 35 or 40 mg/mL, and a protease capable of degrading fibrin, preferably plasmin.

In some embodiments, the concentration of said protease is between 1 pg/mL and 100 pg/mL, such as between 2 and 90 pg/mL, between 40 and 100 pg/mL, between 1 and 40 pg/mL, such as 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 pg/mL.

In some embodiments, the milk sample is contacted with bile salts at a concentration of between 2 and 120 mg/mL, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 50 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, preferably wherein the concentration of the bile salts is between 80 and 120 mg/mL, such as between 90 and 110 mg/mL, such as between 95 and 105 mg/mL, such as 80, 85, 90, 95, 100, 105, 110, 115 or 120 mg/mL, a lipase at a concentration of between 2 and 120 mg/mL, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, or between 100 and 120 mg/mL, preferably wherein the lipase concentration is between 2 and 40 mg/mL, such as between 10 and 40 mg/mL, such as between 20 and 40 mg/mL, such as 10, 15, 20, 25, 30, 35 or 40 mg/mL, and a protease at a concentration of between 1 pg/mL and 100 pg/mL, such as between 2 and 90 pg/mL, between 40 and 100 pg/mL, between 1 and 40 pg/mL, such as 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 pg/mL.

Analytical methods

Lateral flow assay (LFA) is a low-cost and fast alternative to the methods of centralized laboratories for analyte identification. LFA is by now a commonly used method for point-of-use testing. Both centralized laboratories and LFA rely upon a qualitative result.

A lateral flow assay device includes a strip of absorbent or porous material (such as a microporous membrane), which, in some instances, can be made of different substances each joined to the other in zones, which may be abutted and/or overlapped. In some examples, the absorbent strip can be fixed on a supporting non- interactive material (such as nonwoven polyester), for example, to provide increased rigidity to the strip. Zones within each strip may differentially contain the specific binding partner(s) and/or other reagents required for the detection and/or quantification of the particular analyte being tested for. Thus these zones can be viewed as functional sectors or functional regions within the test device.

In some embodiments, the sample is prepared as described herein above, and said analysis comprises the step of performing a lateral flow assay.

In some embodiments, said lateral flow assay comprises a membrane.

In some embodiments, said membrane is a porous membrane such as a nitrocellulose membrane, such as a cellulose membrane, or such as a cellulose membrane with embedded glass fibres.

Milk samples treated by the methods disclosed herein can however also be analysed by other means than LFA.

In embodiments where detection of pathogens is desired, the milk sample contacted with lipase and bile salts can be inoculated and incubated in a growth medium, as described herein above.

In some embodiments, said analysis comprises the step of performing an analytical method, such as MALDI-TOF, such as ELISA, such as electrochemical platforms, such as microfluidic platforms, or such as colony forming unit analysis.

In some embodiments, the time-to-result is between 1 and 30 minutes, such as between 1 and 25 minutes, between 1 and 20 minutes, between 1 and 15 minutes, between 1 and 10 minutes, between 5 and 30 minutes, between 5 and 25 minutes, between 5 and 20 minutes, between 5 and 15 minutes, between 5 and 10 minutes, between 10 and 30 minutes, between 10 and 25 minutes, between 10 and 20 minutes, between 10 and 15 minutes, between 15 and 30 minutes, between 15 and 25 minutes, between 15 and 20 minutes, between 20 and 30 minutes, between 20 and 25 minutes, or between 25 and 30 minutes.

Storage

The workflow of the end user may necessitate storage of the milk sample prior to preparing or after the preparing, prior to analysis. The method described in the present disclosure comprises three steps: a) obtaining the milk sample from a mammal; b) contacting said milk sample with lipase and one or more bile salts; and c) subjecting the treated milk sample obtained in b) to analysis.

In some embodiments, the method comprises a step of storing the milk sample obtained in step a) before step b) is performed. In other embodiments, the method comprises a step of storing the treated milk obtained in step b) before step c).

In some embodiments, the storing is in a refrigerator or in a freezer.

In some embodiments, the storing is at temperatures above 0°C and below 10°C, such as between 0°C and 8°C, between 0°C and 6°C, between 0°C and 4°C, between 0°C and 2°C, between 2°C and 10°C, between 2°C and 8°C, between 2°C and 6°C, between 2°C and 4°C, between 4°C and 10°C, between 4°C and 8°C, between 4°C and 6°C, between 6°C and 10°C, between 6°C and 8°C, or between 8°C and 10°C.

In some embodiments, the storing is in a fridge at a temperature between 0°C and 6°C.

In some embodiments, the storing is for up to 4 days, such as up to 3 days, such as up to 2 days, or such as up to 1 day.

In some embodiments, the storing is at a temperature above -80°C and below -20°C, such as between -80°C and -20°C, between -80°C and -30°C, between -80°C and - 40°C, between -80°C and -50°C, between -80°C and -60°C, between -80°C and -70°C, between -70°C and -10°C, between -70°C and -20°C, between -70°C and -30°C, between -70°C and -40°C, between -70°C and -50°C, between -70°C and -60°C, between -60°C and -10°C, between -60°C and -20°C, between -60°C and -30°C, between -60°C and -40°C, between -60°C and -50°C, between -50°C and -10°C, between -50°C and -20°C, between -50°C and -30°C, between -50°C and -40°C, between -40°C and -10°C, between -40°C and -20°C, between -40°C and -30°C, between -30°C and -10°C, between -30°C and -20°C, or between -20°C and -10°C.

In some embodiments, the storing is in a freezer at -20°C.

In some embodiments, the storing is in a freezer at -80°C. In some embodiments, the storing is in a freezer for up to 12 months, such as up to 11 months, such as up to 10 months, such as up to 9 months, such as up to 8 months, such as up to 7 months, such as up to 6 months, such as up to 5 months, such as up to 4 months, such as up to 3 months, such as up to 2 months, or such as up to 1 month.

In some embodiments, the storing is in a refrigerator for up to 20 days, such as up to 18 days, up to 16 days, up to 14 days, up to 12 days, 10 day, up to 8 days, up to 7 days, up to 6 days, up to 4 days, up to 2 days, or up to 1 day.

In some embodiments, the storing is in a freezer for up to 8 weeks, such as up to 7 weeks, such as up to 6 weeks, such as up to 5 weeks, such as up to 4 weeks, such as up to 3 weeks, such as up to 2 weeks, or such as up to 1 week.

Analytes

The term "analyte" broadly refers to any substance to be analyzed, detected, measured, or quantified. Examples of analytes include, but are not limited to, proteins, peptides, hormones, haptens, antigens, antibodies, receptors, enzymes, nucleic acids and combinations thereof.

In some embodiments, said pathogen is selected from the group consisting of: a virus; a bacterium; a fungus; an algae; and a yeast.

In some embodiments, the virus is cytomegalovirus, Hepatitis B, or human immunodeficiency virus.

In some embodiments, the fungus is Candida albicans.

In some embodiments, the pathogen belongs to the genus of Streptococcus, Enterococcus, Staphylococcus, Micrococcus, Escherichia, Klebsiella, Serratia, Pseudomonas, Proteus, Pasteurella, Nocardia, Prototheca, Corynebacterium, Arcanobacterium, Mycobacterium, Bacillus, Mycoplasma, such as Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus, coagulase-negative Staphylococci, Escherichia coli, Bacillus cereus, Corynebacterium bovis, Arcanobacterium pyogenes, Mycoplasma bovis, Mycoplasma californicum, or Mycoplasma bovigenitalium.

In some embodiments, the pathogen belongs to the familily Enterobacteriaceae.

In some embodiments, said analyte is a chemical, a toxin or a hormone.

In some embodiments, the chemical is per- and polyfluoroalkyl substances, chemicals from clothing, chemicals from perfumes, chemicals from lotions, medicine traces, toxic chemicals from food, alcohol, or drugs.

In some embodiments, the present disclosure concerns the method for detecting at least one analyte and/or a pathogen in a mammalian milk sample, the method comprising: a) obtaining the milk sample from a mammalian such as a human or a cow; b) contacting said milk sample with the bile salts is cholic acid sodium salt and deoxycholic acid sodium salt at concentrations above 2 mg/mL and below 120 mg/mL, thereby obtaining a treated milk sample; and c) subjecting the treated milk sample obtained in b) to analysis by lateral flow assay, thereby allowing detection of said analyte and/or pathogen.

In some embodiments, the present disclosure concerns a method for detecting at least one analyte and/or a pathogen in a mammalian milk sample, the method comprising: a) obtaining the milk sample from a mammal; b) contacting said milk sample with lipase and one or more bile salts at concentrations above 2 mg/mL and below 120 mg/mL, thereby obtaining a treated milk sample; and c) subjecting the treated milk sample obtained in b) to analysis, thereby allowing detection of said analyte and/or pathogen.

In some embodiments, the mammal is a human; in other embodiments, the mammal is a a non-human mammal, such as a cow, such as a buffalo, such as a goat, such as a sheep, such as a camel, such as a horse, or a donkey. In some embodiments, the milk sample is contacted with bile salts at a concentration of between 2 and 120 mg/mL, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 50 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, preferably wherein the concentration of the bile salts is between 80 and 120 mg/mL, such as between 90 and 110 mg/mL, such as between 95 and 105 mg/mL, such as 80, 85, 90, 95, 100, 105, 110, 115 or 120 mg/mL. In some embodiments, the milk sample is contacted with lipase at a concentration of between 2 and 120 mg/mL, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, or between 100 and 120 mg/mL, preferably wherein the lipase concentration is between 2 and 40 mg/mL, such as between 10 and 40 mg/mL, such as between 20 and 40 mg/mL, such as 10, 15, 20, 25, 30, 35 or 40 mg/mL. In some embodiments, the milk sample is contacted with a protease capable of degrading fibrin, preferably plasmin.

In some embodiments, the milk sample is contacted with bile salts at a concentration of between 2 and 120 mg/mL, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 50 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, preferably wherein the concentration of the bile salts is between 80 and 120 mg/mL, such as between 90 and 110 mg/mL, such as between 95 and 105 mg/mL, such as 80, 85, 90, 95, 100, 105, 110, 115 or 120 mg/mL, a lipase at a concentration of between 2 and 120 mg/mL, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, or between 100 and 120 mg/mL, preferably wherein the lipase concentration is between 2 and 40 mg/mL, such as between 10 and 40 mg/mL, such as between 20 and 40 mg/mL, such as 10, 15, 20, 25, 30, 35 or 40 mg/mL and/or a protease capable of degrading fibrin, preferably plasmin.

Methods of diagnosis

In another aspect, the present disclosure concerns a method of diagnosing mastitis in a mammal, the method comprising performing the method described herein, wherein the analysis is for detecting a pathogen causing mastitis. The mammal may for example be a cow or a human.

In some embodiments, the present disclosure concerns a method of diagnosing mastitis in a mammal, the method comprising performing the method described herein, wherein the analysis is for detecting a pathogen causing mastitis, wherein if the pathogen is detected, this indicates that mastitis is present.

In some embodiments, the present disclosure concerns a method of diagnosing mastitis in a mammal, the method comprising performing the method as described herein, wherein the analysis is for detecting a pathogen causing mastitis, wherein if mastitis is not present, the milk sample can be used for production of dairy products, such as cheese, such as milk for consumption, such as yoghurt, such as butter, or such as cream.

In another aspect, the present disclosure concerns a method of assessing the quality of a milk sample of mammalian origin, such as a milk sample from a cow or from a human, the method comprising performing the method described herein, wherein the analysis is for detecting a pathogen or an analyte. In some embodiments, the present disclosure concerns a method of assessing the quality of a milk sample of mammalian origin, such as a milk sample from a cow or from a human, the method comprising performing the method described herein, wherein the analysis is for detecting a pathogen or an analyte, wherein if the pathogen or the analyte is detected, this indicates low quality of said milk sample.

In some embodiments, the present disclosure concerns a method of assessing the quality of a milk sample of mammalian origin, such as a milk sample from a cow or from a human, the method comprising performing the method as described herein, wherein the analysis is for detecting a pathogen or an analyte, wherein if the milk sample is not of low quality, i.e. if the pathogen or analyte is not detected, the milk sample can be used for feeding infants.

Kit

In another aspect, the present disclosure concerns a kit comprising a. lipase in an amount allowing concentrations above 2 mg/mL and below 120 mg/mL to be added to a milk sample, preferably said milk sample has a volume of above 500 pL and below 700 pL; b. one or more bile salts in an amount allowing concentrations above 2 mg/mL and below 120 mg/mL to be added to said milk sample; and c. instructions for use.

In some embodiments, the volume of said milk sample is above 550 pL and below 700 pL, such as above 550 pL and below 650 pL, such as above 550 pL and below 600 pL, such as above 600 pL and below 700 pL, such as above 600 pL and below 650 pL, or such as above 650 pL and below 700 pL.

The lipase, milk sample, and one or more bile salts, may be as described herein.

In some embodiments, the kit comprises bile salts in an amount allowing concentrations of between 2 and 120 mg/mL to be added to a milk sample, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 50 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, preferably wherein the concentration of the bile salts is between 80 and 120 mg/mL, such as between 90 and 110 mg/mL, such as between 95 and 105 mg/mL, such as 80, 85, 90, 95, 100, 105, 110, 115 or 120 mg/mL to be added to a milk sample.

In some embodiments, the kit comprises lipase in an amount allowing concentrations of between 2 and 120 mg/mL to be added to a milk sample, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, or between 100 and 120 mg/mL, preferably wherein the lipase concentration is between 2 and 40 mg/mL, such as between 10 and 40 mg/mL, such as between 20 and 40 mg/mL, such as 10, 15, 20, 25, 30, 35 or 40 mg/mL to be added to a milk sample.

In some embodiments, the kit comprises protease capable of degrading fibrin, preferably plasmin.

In some embodiments, the kit comprises bile salts in an amount allowing concentrations of between 2 and 120 mg/mL to be added to a milk sample, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 50 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, preferably wherein the concentration of the bile salts is between 80 and 120 mg/mL, such as between 90 and 110 mg/mL, such as between 95 and 105 mg/mL, such as 80, 85, 90, 95, 100, 105, 110, 115 or 120 mg/mL to be added to a milk sample, lipase in an amount allowing concentrations of between 2 and 120 mg/mL to be added to a milk sample, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, or between 100 and 120 mg/mL, preferably wherein the lipase concentration is between 2 and 40 mg/mL, such as between 10 and 40 mg/mL, such as between 20 and 40 mg/mL, such as 10, 15, 20, 25, 30, 35 or 40 mg/mL to be added to a milk sample, and/or protease capable of degrading fibrin, preferably plasmin.

The kit can be used to prepare a milk sample for analysis to detect an analyte or a pathogen as described herein above.

In some embodiments, the kit further comprises a protease as described herein.

In some embodiments, the lipase is not a lipase derived from a plant. In some embodiments, the lipase is not a wheat lipase, in particular the lipase is not a wheat germ lipase.

Examples

Example 1 The effect of the preparation protocol for Lateral flow assay Firstly, it was investigated whether different concentrations of pancreatic lipase and bile salts enable analysis of a milk sample by paper-based analysis. Thus, the aim was to test whether the preparation protocol facilitated break down of triglycerides and prevented re-accumulating in milk samples with high fat concentration.

Materials and methods:

The experiments were performed using dipsticks, comprising a reaction membrane attached to an absorption pad by adhesives. In the reaction membrane, a capture antibody was immobilized, which reacted with a detection antibody conjugated to gold nanoparticles. This allowed verification that the components migrated through the membrane. Reaction membrane: Whatman Nitrocellulose membrane: Prima 40. (Cytiva) Geometry: 5x27 mm

Absorption pad: Whatman cotton linter membrane: CF7. (Cytiva) Geometry: 5x30 mm Capture antibody: ab31499 (abeam)

Detection antibody ab6702 (Abeam) conjugated to 40 nm gold nanoparticles, 20 OD: ab154873 (Abeam)

Human milk samples were thawed in a fridge 10-15 hours before use.

Experimental setup: dipsticks were placed in ELISA wells, where the milk samples (50 pL) subjected to different treatments were placed. The time for complete absorption of the sample from the well was recorded.

The investigated parameters were the time, the colour development of the assay, and the apparent fat accumulation on the dipstick surface.

The treatments tested were:

1. No treatment

2. Dilutions only

3. Lipase only

4. Bile salts only

Treatment of different variations of bile salts and lipases on milk samples with fat content between 2.2% and 7.6% were tested (Table 1).

Table 1 Human milk protocol development.

In total:

Human milk samples: 10: 3 low, four medium, three high Four different treatments n=2, N=80

Results

Initial analyses on a pooled human milk sample of unknown milk fat content, suggested that a combination of bile salts and lipases was necessary (Figure 1).

Different sample matrices were applied to the paper-based dipsticks to analyze the effect of the preparation protocol. The results are presented in Figure 2. The same type of membranes of the same batch was used. The result of an untreated milk sample is presented in Figure 2a. In the untreated milk sample, fat accumulation was noticed at the bottom of the LFA, where the paper was immersed in the sample, and the LFA was incapable of absorbing the whole sample. The color development was limited in the LFA, meaning a low signal and sensitivity. Figure 2b presents a result where ultrapure water is the sample. Within 3 minutes, the sample was absorbed, and the color was developed. Compared to Figure 2a, the result was strong and clear. In Figure 2c, the milk serum was centrifuged and applied on an LFA. No milk fat was accumulated on the bottom of the paper membrane. The sample was absorbed within 5 minutes, and although the signal was not as strong as Figure 2b, the result was markedly stronger than Figure 2a. Figure 2d displays the results obtained with a milk sample that was treated with the protocol disclosed in this application. No milk fat was accumulated on the bottom of the membrane, meaning that the treatment decreased size of the milk fat globules enough to allow mitigation through the paper membrane. The sample was fully absorbed in approximately 15 minutes, which was longer than both Figure 2b and Figure 2c. The color development was strong and similar to Figure 2b, meaning that the fat does not appear to interfere with the signal.

Next, the broth was substituted with demineralized water. The optimal protocol was tested and calibrated to milk samples with 7.6 % milk fat. For this sample, gradually increasing concentrations of bile salts and lipase were tested. The samples with higher milk fat content required higher concentrations of lipase and bile salts (Table 2).

Table 2. Time absorbent for three milk samples representing low, middle, and high-fat content:

Average absorption time of the milk samples (50 pL) undergoing different treatments. BS = bile salts, L= Lipase, all diluted in demineralized water (50 pL). *not diluted. Means not absorbed after 2 hours. Time was recorded in minutes, +/-standard deviation. n=3, N=81.

Next, it was analysed whether bacteria can be detected in raw human milk containing fat percent 4.7% (Figure 3). To this end, Escherichia coli were added to a milk sample and were detected through a surface protein of E. coli. The preparation protocol allows detection of bacteria in raw milk samples.

Conclusions

The preparation protocol enables raw milk analysis on paper-based analytical platforms, eliminating the need for external equipment such as centrifuges.

The optimal treatment parameters were: 5 mg/ml lipase, 100 mg/ml bile salts in demineralized water, mixed 1 :1 with raw milk samples. There was a clear correlation between fat content and concentrations of the lipase and bile salts required to homogenize the sample. The optimal protocol is therefore based on the highest fat percent available milk sample, which was 7.6 %. Furthermore, it was concluded that bacteria can be detected in spiked milk samples.

Example 2 The effect of the preparation protocol visualized through the naked eye in a microscope and the Ocelloscope. (instrument from Biosense solutions).

Materials and methods

• Optical microscope

• Ocelloscope

• Glass slide/cover slide

• Whole milk samples: fat percent 7.6%

• Preparation protocol: bile salts 5 mg/mL, lipase 100 mg/mL Human milk samples were thawed in a fridge 10-15 hours before use.

Results

The effect of the preparation protocol was visual by eye (Figure 4) and was similarly visually apparent on micro-scale (Figure 5). The image to the left shows the sample composition before treating the milk sample, and the middle picture presents the result after 3 minutes. A picture of the glass slide before adding anything is presented in the image to the right in Figure 5.

The same sample was also subjected to Ocelloscope readings, where one fat cell was tracked during the preparation. The results are presented in figure 7.

Conclusion

The preparation protocol obtained through Example 1 works on milk samples with a fat content of 7.6%.

Example 3 The effect of the preparation protocol applied to cow's milk

It was investigated whether the protocol applies to milk samples of other mammals and hence whether the protocol allows the preparation of milk samples from non-human mammals for diagnostic purposes and quality assurance. Materials and methods

• Milk samples of 1) a healthy cow (milk fat content: 2.7%), 2) a cow with known subclinical mastitis (milk fat content: 2.4%), and 3) a cow with severe haemolytic acute mastitis (milk fat content: 2.2%).

• Optimal preparation protocol obtained in Example 1.

• LFA dipsticks, the same composition as in Example 1.

Cow milk samples were donated from organic Danish milk farms. The samples were frozen upon arrival to the laboratory and thawed in the fridge 10-15 hours before use. The fat content was measured to range from 2.2 to 2.7%.

Results

Visual results of the sample

The effect of the preparation protocol on a sample originating from a cow with subclinical mastitis was similar to effect observed in Example 2 (Figure 7). However, the milk sample was not as transparent, which may arise from somatic cells, more specifically fibrin accumulations in the milk. These make the milk more likely to be clumpy and sticky. An image of milk samples originating from healthy-, subclinical mastitis-and acute mastitis- cows, respectively, is presented in Figure 8.

The visual effects of the preparation protocol on healthy cow samples were similar to the effect on human milk samples, presented in Example 2. The subclinical samples also turned more transparent, but appeared more viscous. The acute mastitis milk samples had higher contents of somatic cells, which results in a clumpy appearance. LFA analysis revealed that the protocol allows analysis of healthy cows and cows with subclinical mastitis (Table 3).

Table 3. Results from applying the samples onto a paper-based system

Flow results from LFA investigation. The preparation protocol was based on the concentrations identified in Example 1. Time was measured in minutes. means that the sample was not fully absorbed in 2 hours. Conclusion

The preparation protocol work on both healthy and subclinical mastitis cow's milk samples.

Example 4 The effect of the preparation protocol for growing bacteria milk samples for faster results.

The application spectrum for the preparation protocol was investigated.

Materials and methods:

• Raw human milk sample: 3.7% fat

• E. coli stock

• LB agar plate

E. coli stock at ~ 10⁹ CFU/mL, diluted ~1 O'², or diluted ~10'⁴ were added to raw milk sample and plated on LB agar in either upon treatment or no treatment.

Results

Table 4 displays growth of bacteria on a LB agar plate, upon treatment or no treatment.

Table 4: Bacterial growth on LB agar plate.

Conclusion

It appeared that with the preparation protocol, the bacteria colonies were visible at a faster rate. Example 5: The effect of the preparation protocol for increasing the sample size: Determining the limiting factor

The sensitivity of lateral flow assays is not very high compared to other analytical techniques. One way to increase it is to increase the sample size and thus increase the amount of analyte.

Materials and methods:

• Nitrocellulose: Prima 40: geometry: ranging from 5x25mm to 10x 25 mm

• Fusion 5 (cotton linters + glass fibers imbedded): geometry: ranging from 5x25 mm to 10x 25 mm

• Absorbent pad: CF7: geometry: ranging from 5x30mm to 10x50mm, (two layered on top of each other)

• Raw human milk sample: 4.9% fat

• Preparation protocol obtained in Example 1

By changing the dimensions of the paper strip and/or substituting the reaction membrane with another material, the sample size was increased, and the limiting factor was determined.

Results

By changing the parameters of the paper system while keeping the preparation protocol the same, it was identified that it was not the sample composition but the absorption pad being the limiting factor. 1500 pL of sample was absorbed in a nitrocellulose membrane in 20 minutes and 2000 pL in 21 minutes in Fusion 5 membrane (not shown) (Table 5).

Table 5: The effect of the preparation protocol for increasing the sample size.

Conclusion

By including a preparation step, it appears that it is no longer the sample matrices that are the limiting factor but rather the material composition and geometry. Example 6: Storage conditions/shelf life: A continuous investigation

Preparation solutions have been prepared and tested over time to investigate the stability of the solution.

Materials and methods

• Preparation solutions containing bile salts and lipase in ultrapure water (established in example 1) in Eppendorf tube.

• Fridge/freezer

• LFA dipstick

• Microscope

Twenty preparation solutions were prepared, ten were placed in the fridge, and ten were placed in the freezer.

Every other day for the first eight days a milk sample was subjected to a preparation solution, stored in a fridge, followed by LFA analysis and analysis by microscopy upon 5 minutes of incubation at room temperature (Table 6).

Once a week, a test was made for the frozen preparation solution. The solutions were thawed 10-15 hours in the fridge and, once thawed, a milk sample was subjected to the solution, followed by LFA and microscopy (Table 7).

Results

Table 6: stability of the preparation solution in fridge.

Table 7: stability of the preparation solution in freezer. For the preparation solutions stored in the fridge, the quality was too low for LFA analysis at day 20, compared to days 1-14. When the preparation solutions were stored in the freezer, the quality remained the same.

Conclusions The preparation solution appears stable for 14 days at 5 degrees Celsius or eight weeks in -20 degrees Celsius.

Example 7: Different lipases for milk pretreatment enabling paper-based analysis

Different lipases were analyzed in the established milk pretreatment protocol for investigating whether more than one type of lipase can be used for the protocol. Materials and methods:

Lipases used in the experiments

• Human milk, fat percentage: 3,4%

• ELISA well plate

• Eppendorf tubes

• Paper-based materials: Prima40 and CF7

• Optical microscope means no treatment, only raw (human) milk

‘c’ means treated with lipase from Candida rugosa ‘p’ means treated with lipase from porcine pancreas

Concentrations used in the experiment: 5 mg/ml Lipase, 100 mg/ml Bile salts

Results

The visual appearance of the treated milk after 5 minutes is displayed in Figure 9. Samples were further analyzed by microscopy (Figure 10-12).

Lastly, paper-based flow test was conducted on samples. In this experiment, the ability of whole milk facilitating through a paper-based membrane of nitrocellulose was analysed. All milk samples treated with ‘p’ were facilitated through the membrane within 10 minutes. 1/3 of the milk samples ‘c’ reached full absorption. had almost no uptake, and the majority of the milk sample remains in the ELISA well (Figure 13, upper panel).

Dipsticks in no treatment, ‘c’: Candida, and ‘p’: porcine pancreatic lipase. The dipstick comprises a nitrocellulose membrane and an absorption pad. On the absorption pad, it is easy to see how the milk has been absorbed, and which membrane who absorbed all and those who did not (Figure 13, lower panel).

Conclusions

In conclusion, both lipase from porcine pancreas and lipase from Candida rugosa can be used in the present methods.

References

WO 2010/014836 Items

1. A method for preparing a mammalian milk sample for detecting at least one analyte and/or a pathogen, the method comprising: a) obtaining the milk sample from a mammal; and b) contacting said milk sample with lipase and one or more bile salts, each at a concentration above 2 mg/mL and below 120 mg/mL, thereby obtaining a treated milk sample.

2. A method for detecting at least one analyte and/or a pathogen in a mammalian milk sample, the method comprising: a) obtaining the milk sample from a mammal; b) contacting said milk sample with lipase and one or more bile salts at concentrations above 2 mg/mL and below 120 mg/mL, thereby obtaining a treated milk sample; and c) subjecting the treated milk sample obtained in b) to analysis, thereby allowing detection of said analyte and/or pathogen.

3. The method according to item 2, wherein said analysis comprises a step of performing a lateral flow assay.

4. The method according to item 3, wherein said lateral flow assay comprises a membrane.

5. The method according to item 4, wherein said membrane is a porous membrane such as a nitrocellulose membrane, such as a cellulose membrane, or such as a cellulose membrane with embedded glass fibres.

6. The method according to any one of items 1 to 2, wherein said analysis comprises the step of performing an analytical method, such as MALDI-TOF, such as ELISA, such as electrochemical platforms, such as microfluidic platforms, or such as colony forming unit analysis.

7. The method according to any one of the preceding items, wherein said lipase and one or more bile salts are suspended in water prior to said contacting, thereby obtaining a preparation solution, preferably wherein the water is distilled water or ultrapure water.

8. The method according to item 7, wherein said preparation solution is mixed with said milk sample in a preparation solution to milk sample ratio between 1 :1 and 1 :3, such as between 1:1 and 1:2.5, between 1 :1 and 1:2, between 1:1 and 1 :1.5, between 1:1.5 and 1 :3, between 1 :1.5 and 1 :2.5, between 1:1.5 and 1 :2, between 1 :2 and 1 :3, between 1:2 and 1 :2.5, or between 1:2.5 and 1:3.

9. The method according to any one of the preceding items, wherein said lipase and one or more bile salts are suspended in bacterial growth medium prior to said contacting, thereby obtaining a medium suspension, preferably wherein the bacterial growth medium is LB medium.

10. The method according to item 9, wherein said medium suspension is mixed with said milk sample in a medium suspension to milk sample ratio between 1 :1 and 1 :3, such as between 1 :1 and 1:2.5, between 1 :1 and 1:2, between 1:1 and 1 :1.5, between 1:1.5 and 1 :3, between 1 :1.5 and 1 :2.5, between 1:1.5 and 1 :2, between 1 :2 and 1:3, between 1 :2 and 1 :2.5, or between 1 :2.5 and 1 :3., thereby obtaining a growth suspension.

11. The method according to item 10, wherein said growth suspension is incubated at room temperature.

12. The method according to any one of the preceding items, wherein the method comprises a step of diluting the milk sample in a milk sample to diluent ratio of at most 1 :1 , such as at most 1 :0.9, such as 1 :0.8, such as 1 :0.7, such as 1:0.6, such as 1:0.5, or such as 1 :0.4.

13. The method according to any one of the preceding items, wherein the method comprises a step of storing the milk sample obtained in step a) before step b).

14. The method according to item 13, wherein the storing is in a refrigerator for up to 14 days. 15. The method according to item 13, wherein the storing is in a freezer for up to 12 months.

16. The method according to any one of the preceding items, wherein the method comprises a step of storing the treated milk obtained in step b) before step c).

17. The method according to item 16, wherein the storing is in a refrigerator for up to 20 days, such as up to 18 days, up to 16 days, up to 14 days, up to 12 days, 10 day, up to 8 days, up to 7 days, up to 6 days, up to 4 days, up to 2 days, or up to 1 day.

18. The method according to item 16, wherein the storing is in a freezer for up to 8 weeks.

19. The method according to item 7, wherein the preparation solution is stored prior to step b).

20. The method according to item 19, wherein the storing is in a refrigerator for up to 20 days, such as up to 18 days, up to 16 days, up to 14 days, up to 12 days, 10 day, up to 8 days, up to 7 days, up to 6 days, up to 4 days, up to 2 days, or up to 1 day.

21. The method according to item 19, wherein the storing is in a freezer for up to 8 weeks.

22. The method according to any one of the preceding items, wherein said mammal is a human being.

23. The method according to any one of the preceding items, wherein said mammal is a non-human mammal, such as a cow, such as a buffalo, such as a goat, such as a sheep, such as a camel, such as a horse, or a donkey.

24. The method according to any one of the preceding items, wherein said milk sample comprises between 1.7% and 7.7% fat, such as between 2% and 7.7%, between 2% and 7%, between 2% and 6%, between 2% and 5%, between 2% and 4%, between 2% and 3%, between 1.7% and 2% between 3% and 7.7%, between 3% and 7%, between 3% and 6%, between 3% and 5%, between 3% and 4%, between 1.7% and 3%, between 4% and 7.7%, between 4% and 7%, between 4% and 6%, between 4% and 5%, between 1.7% and 4%between 5% and 7.7%, between 5% and 7%, between 5% and 6%, between 1.7% and 5%, between 6% and 7.7%, between 6% and 7%, between 1.7% and 6%, between 7% and 7.7%, or between 1.7% and 7% fat. The method according to any one of the preceding items, wherein the lipase concentration is between 2 and 120 mg/mL, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, or between 100 and 120 mg/mL, preferably wherein the lipase concentration is between 2 and 40 mg/mL, such as between 10 and 40 mg/mL, such as between 20 and 40 mg/mL, such as 10, 15, 20, 25, 30, 35 or 40 mg/mL. The method according to any one of the preceding items, wherein the concentration of the one or more bile salts is between 2 and 120 mg/mL, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 50 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, preferably wherein the concentration of the bile salts is between 80 and 120 mg/mL, such as between 90 and 110 mg/mL, such as between 95 and 105 mg/mL, such as 80, 85, 90, 95, 100, 105, 110, 115 or 120 mg/mL. 27. The method according to any one of the preceding items, wherein the concentrations of the lipase and the one or more bile salts are the same or different.

28. The method according to any one of the preceding items, wherein the concentrations are selected from: a) 2 mg/mL of the one or more bile salts and 2 mg/mL of the lipase; b) 10 mg/mL of the one or more bile salts and 10 mg/mL of the lipase; c) 20 mg/mL of the one or more bile salts and 20 mg/mL of the lipase; d) 50 mg/mL of the one or more bile salts and 50 mg/mL of the lipase; e) 2 mg/mL of the one or more bile salts and 100 mg/mL of the lipase; f) 10 mg/mL of the one or more bile salts and 100 mg/mL of the lipase; g) 20 mg/mL of the one or more bile salts and 100 mg/mL of the lipase; h) 50 mg/mL of the one or more bile salts and 100 mg/mL of the lipase; i) 100 mg/mL of the one or more bile salts and 100 mg/mL of the lipase; j) 100 mg/mL of the one or more bile salts and 2 mg/mL of the lipase; k) 100 mg/mL of the one or more bile salts and 10 mg/mL of the lipase; l) 100 mg/mL of the one or more bile salts and 20 mg/mL of the lipase; or m) 100 mg/mL of the one or more bile salts and 50 mg/mL of the lipase.

29. The method according to any one of the preceding items, wherein the concentration of the one or more bile salts is 100 mg/mL and the concentration of the lipase is 5 mg/mL.

30. The method according to any one of the preceding items, wherein said lipase is a pancreatic lipase, such as a lipase from porcine pancreas, or such as a lipase from human pancreas.

31. The method according to any one of the preceding items, wherein said lipase is from yeast, such as wherein the lipase is from Candida, such as wherein the lipase is from C. rugosa.

32. The method according to any one of the preceding items, wherein said lipase is from a plant, such as wherein the lipase is from wheat, such as wherein the lipase is from wheat germ. 33. The method according to any one of the preceding items, wherein said one or more bile salts comprise cholic acid sodium salt and deoxycholic acid sodium salt.

34. The method according to any one of the preceding items, wherein the milk sample is contacted with said lipase and said one or more bile salts simultaneously.

35. The method according to any one of the preceding items, wherein the milk sample is first contacted with said lipase and then contacted with said one or more bile salts.

36. The method according to any one of the preceding items, wherein said contacting the milk sample with lipase and one or more bile salts is performed for less than 5 minutes, such as less than 4 minutes, such as less than 3 minutes, such as 2 minutes, such as 1 minute, such as 30 seconds, such as 20 seconds, such as 10 seconds, or such as 1 second.

37. The method according to any one of the preceding items, wherein said pathogen is selected from the group consisting of: a virus; a bacterium; a fungus; an algae; and a yeast.

38. The method according to item 37, wherein the virus is cytomegalovirus, Hepatitis B, or human immunodeficiency virus.

39. The method according to item 37, wherein the fungus is Candida albicans.

40. The method according to item 37, wherein the pathogen belongs to the genus of Streptococcus, Enterococcus, Staphylococcus, Micrococcus, Escherichia, Klebsiella, Serratia, Pseudomonas, Proteus, Pasteurella, Nocardia, Prototheca, Corynebacterium, Arcanobacterium, Mycobacterium, Bacillus, Mycoplasma, such as Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus, coagulase-negative Staphylococci, Escherichia coli, Bacillus cereus, Corynebacterium bovis, Arcanobacterium pyogenes, Mycoplasma bovis, Mycoplasma californicum, or Mycoplasma bovigenitalium.

41. The method according to item 37, wherein the pathogen belongs to the family Enterobacteriaceae.

42. The method according to any one of the preceding items, wherein said analyte is a chemical, toxin or a hormone.

43. The method according to item 42, wherein the chemical is per- and polyfluoroalkyl substances, chemicals from clothing, chemicals from perfumes, chemicals from lotions, medicine traces, toxic chemicals from food, alcohol, or drugs.

44. The method according to any one of the preceding items, wherein the time-to- result is between 1 and 30 minutes, such as between 1 and 25 minutes, between 1 and 20 minutes, between 1 and 15 minutes, between 1 and 10 minutes, between 5 and 30 minutes, between 5 and 25 minutes, between 5 and 20 minutes, between 5 and 15 minutes, between 5 and 10 minutes, between 10 and 30 minutes, between 10 and 25 minutes, between 10 and 20 minutes, between 10 and 15 minutes, between 15 and 30 minutes, between 15 and 25 minutes, between 15 and 20 minutes, between 20 and 30 minutes, between 20 and 25 minutes, or between 25 and 30 minutes.

45. The method according to any one of the preceding items, wherein the method does not comprise a step of filtering the sample.

46. The method according to any one of the preceding items, wherein the method does not comprise a step of centrifuging the sample.

47. The method according to any one of the preceding items, wherein said contacting is performed at a temperature between 18°C and 30°C, such as between 18°C and 28°C, between 18°C and 26°C, between 18°C and 24°C, between 18°C and 22°C, between 18°C and 20°C, between 20°C and 30°C, between 20°C and 28°C, between 20°C and 26°C, between 20°C and 24°C, between 20°C and 22°C, between 22°C and 30°C, between 22°C and 28°C, between 22°C and 26°C, between 22°C and 24°C, between 24°C and 30°C, between 24°C and 28°C, between 24°C and 26°C, between 26°C and 30°C, between 26°C and 28°C, or between 28°C and 30°C, such as at room temperature.

48. The method according to any one of the preceding items, wherein said contacting comprises contacting with a lipase, one or more bile salts and a protease capable of degrading fibrin.

49. The method according to item 48, wherein said protease is plasmin.

50. The method according to any one of items 48 to 49, wherein the concentration of said protease is between 1 pg/mL and 100 pg/mL, such as between 2 and 90 pg/mL, between 40 and 100 pg/mL, between 1 and 40 pg/mL, such as 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 pg/mL.

51. A method of diagnosing mastitis in a mammal, the method comprising performing the method according to any one of the preceding items, wherein the analysis is for detecting a pathogen causing mastitis.

52. The method of diagnosing mastitis in a mammal according to item 51 , wherein if the pathogen is detected, this indicates that mastitis is present.

53. The method of diagnosing mastitis in a mammal according to item 51 , wherein if mastitis is not present, the milk sample can be used for production of dairy products, such as cheese, such as milk for consumption, such as yoghurt, such as butter, or such as cream.

54. A method of assessing the quality of a milk sample of mammalian origin, such as a milk sample from a cow or from a human, the method comprising performing the method according to any one of the preceding items, wherein the analysis is for detecting a pathogen or an analyte. 55. The method of assessing the quality of a milk sample of human origin according to item 53, wherein if the pathogen or the analyte is detected, this indicates low quality of said milk sample.

56. The method of assessing the quality of a milk sample of human origin according to item 54, wherein if the milk sample is not of low quality, the milk sample can be used for feeding infants.

57. A kit comprising a. lipase in an amount allowing concentrations above 2 mg/mL and below 120 mg/mL to be added to a milk sample, preferably said milk sample has a volume of above 500 pL and below 700 pL; b. one or more bile salts in an amount allowing concentrations above 2 mg/mL and below 120 mg/mL to be added to said milk sample; and c. instructions for use.

58. The kit according to item 57, wherein said milk sample is above 550 pL and below 700 pL, such as above 550 pL and below 650 pL, such as above 550 pL and below 600 pL, such as above 600 pL and below 700 pL, such as above 600 pL and below 650 pL, or such as above 650 pL and below 700 pL.

59. The kit according to any one of items 57 to 58, wherein said lipase, milk sample, one or more bile salts, and analyte are as defined in any one of items 1 to 47.

60. The kit according to any one of items 57 to 58, wherein the kit further comprises a protease as defined in any one of items 48 to 50.

Claims

3. The method according to claim 2, wherein said analysis comprises: a. performing a lateral flow assay, such as wherein said lateral flow assay comprises a membrane, such as wherein said membrane is a porous membrane such as a nitrocellulose membrane, such as a cellulose membrane, or such as a cellulose membrane with embedded glass fibres; and/or b. performing an analytical method, such as MALDI-TOF, such as ELISA, such as electrochemical platforms, such as microfluidic platforms, or such as colony forming unit analysis.

4. The method according to any one of the preceding claims, wherein said mammal is a human being, or wherein said mammal is a non-human mammal, such as a cow, such as a buffalo, such as a goat, such as a sheep, such as a camel, such as a horse, or a donkey.

5. The method according to any one of the preceding claims, wherein the lipase concentration is between 2 and 120 mg/mL, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, or between 100 and 120 mg/mL, preferably wherein the lipase concentration is between 2 and 40 mg/mL, such as between 10 and 40 mg/mL, such as between 20 and 40 mg/mL, such as 10, 15, 20, 25, 30, 35 or 40 mg/mL, and/or the concentration of the one or more bile salts is between 2 and 120 mg/mL, such as between 2 and 100 mg/mL, between 2 and 80 mg/mL, between 2 and 60 mg/mL, between 2 and 50 mg/mL, between 2 and 40 mg/mL, between 2 and 20 mg/mL, between 20 and 120 mg/mL, between 20 and 100 mg/mL, between 20 and 80 mg/mL, between 20 and 60 mg/mL, between 20 and 40 mg/mL, between 40 and 120 mg/mL, between 40 and 100 mg/mL, between 40 and 80 mg/mL, between 40 and 60 mg/mL, between 60 and 120 mg/mL, between 60 and 100 mg/mL, between 60 and 80 mg/mL, between 80 and 120 mg/mL, between 80 and 100 mg/mL, preferably wherein the concentration of the bile salts is between 80 and 120 mg/mL, such as between 90 and 110 mg/mL, such as between 95 and 105 mg/mL, such as 80, 85, 90, 95, 100, 105, 110, 115 or 120 mg/mL, preferably, the concentrations are selected from: a) 2 mg/mL of the one or more bile salts and 2 mg/mL of the lipase; b) 10 mg/mL of the one or more bile salts and 10 mg/mL of the lipase; c) 20 mg/mL of the one or more bile salts and 20 mg/mL of the lipase; d) 50 mg/mL of the one or more bile salts and 50 mg/mL of the lipase; e) 2 mg/mL of the one or more bile salts and 100 mg/mL of the lipase; f) 10 mg/mL of the one or more bile salts and 100 mg/mL of the lipase; g) 20 mg/mL of the one or more bile salts and 100 mg/mL of the lipase; h) 50 mg/mL of the one or more bile salts and 100 mg/mL of the lipase; i) 100 mg/mL of the one or more bile salts and 100 mg/mL of the lipase; j) 100 mg/mL of the one or more bile salts and 2 mg/mL of the lipase; k) 100 mg/mL of the one or more bile salts and 10 mg/mL of the lipase; l) 100 mg/mL of the one or more bile salts and 20 mg/mL of the lipase; or m) 100 mg/mL of the one or more bile salts and 50 mg/mL of the lipase. The method according to any one of the preceding claims, wherein said lipase is a pancreatic lipase, such as a lipase from porcine pancreas, or such as a lipase from human pancreas, and/or wherein said one or more bile salts comprise cholic acid sodium salt and deoxycholic acid sodium salt. The method according to any one of the preceding claims, wherein said lipase is from yeast, such as wherein the lipase is from Candida, such as wherein the lipase is from C. rugosa, and/or wherein said one or more bile salts comprise cholic acid sodium salt and deoxycholic acid sodium salt. The method according to any one of the preceding claims, wherein said pathogen is selected from the group consisting of: a virus; a bacterium; a fungus; an algae; and a yeast, such as a virus selected from the group consisting of cytomegalovirus, Hepatitis B, and human immunodeficiency virus, or such as a fungus, preferably Candida albicans, or such as a pathogen belonging to the genus of Streptococcus, Enterococcus, Staphylococcus, Micrococcus, Escherichia, Klebsiella, Serratia, Pseudomonas, Proteus, Pasteurella, Nocardia, Prototheca, Corynebacterium, Arcanobacterium, Mycobacterium, Bacillus, or Mycoplasma, preferably Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus, coagulase-negative Staphylococci, Escherichia coli, Bacillus cereus, Corynebacterium bovis, Arcanobacterium pyogenes, Mycoplasma bovis, Mycoplasma californicum, or Mycoplasma bovigenitalium, such as wherein the pathogen belongs to the familily Enterobacteriaceae. The method according to any one of the preceding claims, wherein said analyte is a chemical, toxin or a hormone, such as wherein the chemical is per- and polyfluoroalkyl substances, chemicals from clothing, chemicals from perfumes, chemicals from lotions, medicine traces, toxic chemicals from food, alcohol, or drugs. 10. The method according to any one of the preceding claims, wherein the time-to- result is between 1 and 30 minutes, such as between 1 and 25 minutes, between 1 and 20 minutes, between 1 and 15 minutes, between 1 and 10 minutes, between 5 and 30 minutes, between 5 and 25 minutes, between 5 and 20 minutes, between 5 and 15 minutes, between 5 and 10 minutes, between 10 and 30 minutes, between 10 and 25 minutes, between 10 and 20 minutes, between 10 and 15 minutes, between 15 and 30 minutes, between 15 and 25 minutes, between 15 and 20 minutes, between 20 and 30 minutes, between 20 and 25 minutes, or between 25 and 30 minutes.

11. The method according to any one of the preceding claims, wherein the method does not comprise either or both of steps of filtering the sample, and centrifuging the sample.

12. The method according to any one of the preceding claims, wherein said contacting comprises contacting with a lipase, one or more bile salts and a protease capable of degrading fibrin, such as plasmin.

13. A method of diagnosing mastitis in a mammal, the method comprising performing the method according to any one of the preceding claims, wherein the analysis is for detecting a pathogen causing mastitis, preferably wherein if the pathogen is detected, this indicates that mastitis is present.

14. A method of assessing the quality of a milk sample of mammalian origin, such as a milk sample from a cow or from a human, the method comprising performing the method according to any one of the preceding claims, wherein the analysis is for detecting a pathogen or an analyte, preferably wherein if the pathogen or the analyte is detected, this indicates low quality of said milk sample.

15. A kit comprising a. lipase in an amount allowing concentrations above 2 mg/mL and below 120 mg/mL to be added to a milk sample, preferably said milk sample has a volume of above 500 pL and below 700 pL; b. one or more bile salts in an amount allowing concentrations above 2 mg/mL and below 120 mg/mL to be added to said milk sample; and c. instructions for use,

Optionally wherein said milk sample is above 550 pL and below 700 pL, such as above 550 pL and below 650 pL, such as above 550 pL and below 600 pL, such as above 600 pL and below 700 pL, such as above 600 pL and below 650 pL, or such as above 650 pL and below 700 pL; and/or optionally wherein said lipase, milk sample, one or more bile salts, and analyte are as defined in any one of claims 1 to 11; and/or optionally wherein the kit further comprises a protease as defined in claim 12.