Techniques for in SITU breastmilk assessment

Abstract

Described herein are methods and kits for assaying the rancidity of human milk, in particular breastmilk. Specifically, methods and kits thereof for determining whether a subject is a high-lipase producer of breastmilk by assessing lipase activity levels and determining whether a breastmilk sample has become rancid by assessing the extent of lipolysis (or potential rancidity) by measuring glycerol accumulation are provided. Kits that use test strips are also described.

Description

Atty. Dkt. No.: 136671-0110 TECHNIQUES FOR IN SITU BREASTMILK ASSESSMENT CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No.63 / 657,086 filed June 6, 2024, which is incorporated herein by reference, in its entirety for any and all purposes. FIELD

[0002] The present technology generally relates to methods and kits for assaying the rancidity of human milk, in particular breastmilk. Specifically, the present disclosure provides methods and kits thereof for determining whether a subject is a high-lipase producer of breastmilk by assessing lipase activity levels and determining whether a breastmilk sample has become rancid by assessing the amount of accumulated glycerol. BACKGROUND

[0003] Established guidelines for infant nutrition assert that infants be provided nothing other than human milk for the first 6 months of age. That is, no other food source or water should be given to infants in that time window. This guidance makes human milk the sole recommended calorie source over this critical window of life. Human milk can be augmented with or replaced by infant formula, when desired.

[0004] Nursing parents pump, store, and freeze excess human milk as a practical matter. The milk is later thawed and used. However, human milk often becomes rancid following storage making it unpalatable to infants. The rancid milk has an undesirable odor / flavor, leading to avoidance by the newborn / infant.

[0005] This issue is a major cause of concern for parents and providers as it forces suboptimal or unwanted feeding regimes (i.e., resorting to commercial formulas despite knowing them to be imperfect substitutes for expressed human milk in terms of nutritional value). It is known that the oxidation lipids, which results in production of free fatty acids in expressed human milk increases during storage, which subsequently results in a rancid flavor, reduced shelf life,Atty. Dkt. No.: 136671-0110 and, ultimately, infant undernutrition. Oxidizing agents and lipolytic proteins (lipases) are substances present in expressed human milk that break down lipids and convert them into free fatty acids. The exact chemical composition of expressed human milk, including the abundance and / or activity of oxidizing agents and lipases, is person-specific and, within a single individual, varies over time, making it difficult to predict in a home setting when stored expressed human milk will become rancid. Moreover, there is currently no method to reverse the rancidity of human milk after it spoils, and there is no proven solution to prevent rancidity from occurring prior to storage that can be performed without advanced equipment / know-how (ultra pasteurization).

[0006] Methods of inhibiting rancidity are set forth in U.S. Published Patent Application 2020 / 0323225 A1, published October 15, 2020, U.S. Provisional Application No.63 / 445,776, filed February 15, 2023, and U.S. Non-Provisional Application No.18 / 442,060 filed February 14, 2024, each of which is incorporated by reference in their entireties as if fully set forth herein, including for the methods and compositions therein.

[0007] The changes human milk undergoes during storage and ways to maintain its nutritional value are not fully understood. Specifically, there are currently no methods for parents to determine whether and when breastmilk will go rancid as inter-individual differences in milk composition make it difficult to establish guidelines. Thus, there exists a need to develop methods, assays, and kits that relate to determining whether breastmilk is likely to go rancid in days rather than weeks or months and whether breastmilk, in particular after storage, has become rancid.

[0008] This disclosure addresses these needs by providing methods and kits for assaying the rancidity of human milk, in particular breastmilk. Specifically, the present disclosure provides methods and kits thereof for determining whether a subject is a high-lipase producer of breastmilk (e.g., likely to produce milk that will go rancid in days rather than weeks / months) by assessing lipase activity levels and determining whether a breastmilk sample (e.g., such as those after storage) has become rancid by measuring the amount of accumulated glycerol.Atty. Dkt. No.: 136671-0110 SUMMARY

[0009] Provided in one aspect is an in vitro method for determining whether a subject is a high-lipase producer of breastmilk, the method comprising: contacting a milk sample from the subject with a substrate comprising 1,2-O-dilauryl-rac- glycero-3-glutaric acid-(6'-methylresorufin) ester, a β-naphthyl ester, indoxyl acetate, or 5-bromo-4-chloro-indoxyl caprylate that generates a color change from an initial color that measures the amount lipase present in the breastmilk within a given period of time following contact, wherein the color change indicates the amount of lipase present in the milk sample.

[0010] In some embodiments, the period of time is at least about 2 minutes. In some embodiments, the period of time is from about 2 minutes to about 30 minutes, optionally from about 3 minutes to about 10 minutes.

[0011] In some embodiments, the amount of lipase present in the milk sample is greater than about 0 U / L, optionally at least about 1.0 U / L. In some embodiments, the amount of lipase present in the milk sample is from about 0 U / L to about 5,000 U / L, optionally from about 0.1 U / L to about 5,000 U / L. In some embodiments, the subject is a high lipase producer of breastmilk when the amount of lipase present in the milk sample is from about 100 U / L to about 5,000 U / L, optionally from about 300 U / L to about 1,000 U / L.

[0012] In some embodiments, the substrate further comprises a carrier matrix that is a bibulous material, optionally a cellulosic filter paper.

[0013] In some embodiments, the substrate further comprises a carrier matrix that is a test strip.

[0014] In some embodiments, the test strip comprises: (a) a base membrane in contact with the test strip; (b) a first layer disposed on the base membrane, the first layer comprising a buffer; and (c) a second layer overlaying the first layer, the second layer comprising 1,2-O- dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin).Atty. Dkt. No.: 136671-0110

[0015] In some embodiments, the color change of the substrate from the initial color to red or purple indicates that the subject is a high-lipase producer. In some embodiments, the color change of the substrate from the initial color to yellow or orange indicates that a subject is not a high-lipase producer.

[0016] In some embodiments, the test strip comprises: (a) a base membrane in contact with the test strip; (b) a first layer disposed on the base membrane, the first layer comprising a buffer; and (c) a second layer overlaying the first layer, the second layer comprising a β- naphthyl ester, optionally 2-naphthyl caprylate, and a color indicator.

[0017] In some embodiments, the test strip comprises: (a) a base membrane in contact with the test strip; (b) a first layer disposed on the base membrane, the first layer comprising a buffer; and (c) a second layer overlaying the first layer, the second layer comprising indoxyl acetate or 5-bromo-4-chloro-indoxyl caprylate, and a color indicator.

[0018] In some embodiments, the β-naphthyl ester, indoxyl acetate, or 5-bromo-4-chloro- indoxyl caprylate reacts with the lipase present in the milk sample to provide a compound that reacts with the color indicator to generate the color change.

[0019] In some embodiments, the color indicator comprises a diazonium salt, optionally selected one or more of 4-benzamido-2,5-diethoxybenzene-1-diazonium (Fast Blue BB salt), bis(2-methoxy-4-nitrophenyldiazonium) 1,5-naphthalenedisulfonate (Fast Red B Salt 1,5- naphthalenedisulfonate), 1-diazo-2-naphthol-4-sulfonic acid, and 4-diazo-3- methoxydiphenylamine sulfate.

[0020] Provided in another aspect is a kit for determining whether a subject is a high-lipase producer of breastmilk, the kit comprising:Atty. Dkt. No.: 136671-0110 a substrate comprising 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester, a β-naphthyl ester, indoxyl acetate, or 5-bromo-4-chloro-indoxyl caprylate that generates a color change after contacting a breastmilk sample from a subject, wherein the color change indicates the amount of lipase present in the breastmilk sample.

[0021] In some embodiments, the amount of lipase present in the breastmilk sample is greater than about 0 U / L, optionally at least about 1.0 U / L. In some embodiments, the amount of lipase present in the breastmilk sample is from about 0 U / L to about 5,000 U / L, optionally from about 0.1 U / L to about 5,000 U / L. In some embodiments, the subject is a high lipase producer of breastmilk when the amount of lipase present in the milk sample is from about 100 U / L to about 5,000 U / L, optionally about 300 U / L to about 1,000 U / L.

[0022] In some embodiments, the substrate further comprises a carrier matrix that is a bibulous material, optionally a cellulosic filter paper.

[0023] In some embodiments, the substrate further comprises a carrier matrix that is a test strip.

[0024] In some embodiments, the test strip comprises: (a) a base membrane in contact with the test strip; (b) a first layer disposed on the base membrane, the first layer comprising a buffer; and (c) a second layer overlaying the first layer, the second layer comprising 1,2-O- dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester, a β-naphthyl ester, indoxyl acetate, or 5-bromo-4-chloro-indoxyl caprylate, and optionally a color indicator.

[0025] In some embodiments, the buffer comprises one or more of Tris buffer, a borate buffer, Bicine buffer and TAPS (N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) buffer, optionally the buffer comprises a borate buffer.

[0026] In some embodiments, the first layer further comprises one or more of a lipase accelerator a polymer binder, a surfactant, a stabilizer, a reaction accelerator, and a buffer.Atty. Dkt. No.: 136671-0110

[0027] In some embodiments, the β-naphthyl ester, indoxyl acetate, or 5-bromo-4-chloro- indoxyl caprylate reacts with the lipase present in the milk sample to provide a compound that reacts with a color indicator to generate the color change.

[0028] In some embodiments, the color indicator comprises a diazonium salt, optionally selected from one or more of 4-benzamido-2,5-diethoxybenzene-1-diazonium (Fast Blue BB salt), bis(2-methoxy-4-nitrophenyldiazonium) 1,5-naphthalenedisulfonate (Fast Red B Salt 1,5-naphthalenedisulfonate), 1-diazo-2-naphthol-4-sulfonic acid, and 4-diazo-3- methoxydiphenylamine sulfate.

[0029] In some embodiments, the second layer further comprises one or more of an organic acid, a stabilizer, a reaction accelerator, and a buffer. In some embodiments, the reaction accelerator comprises one or more of a ferrocyanide and a ferricyanide.

[0030] In some embodiments, the test strip comprises: (a) a base membrane in contact with the test strip; (b) a first layer disposed on the base membrane, the first layer comprising a buffer; and (c) a second layer overlaying the first layer, the second layer comprising 1,2-O- dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester.

[0031] In some embodiments, the test strip comprises one or more of: (a) the buffer comprises tris buffer; (b) the first layer further comprises one or more of bile salts and calcium chloride; and (c) the second layer further comprises an organic acid and a stabilizer.

[0032] In some embodiments, the test strip comprises: (a) a base membrane in contact with the test strip; (b) a first layer disposed on the base membrane, the first layer comprising a buffer; and (c) a second layer overlaying the first layer, the second layer comprising the β- naphthyl ester, optionally 2-naphthyl caprylate and a color indicator.Atty. Dkt. No.: 136671-0110

[0033] In some embodiments, the test strip comprises one or more of: (a) the buffer comprises a borate buffer, optionally sodium borate buffer; (b) the first layer further comprises polyvinylpyrrolidone; (c) the color indicator comprises a diazonium salt, optionally 4-benzamido-2,5- diethoxybenzene-1-diazonium (Fast Blue BB salt) or 1-diazo-2-naphthol-4-sulfonic acid; and (d) optionally, the second layer further comprises a stabilizer.

[0034] In some embodiments, the test strip comprises: (a) a base membrane in contact with the test strip; (b) a first layer disposed on the base membrane, the first layer comprising a buffer; and (c) a second layer overlaying the first layer, the second layer comprising indoxyl acetate or 5-bromo-4-chloro-indoxyl caprylate, optionally indoxyl acetate, and a color indicator.

[0035] In some embodiments, the test strip comprises one or more of: (a) the buffer comprises a borate buffer, optionally sodium borate buffer; (b) the first layer further comprises a reaction accelerator, optionally a ferrocyanide, further optionally potassium ferrocyanide; (c) the color indicator comprises a diazonium salt, optionally diazo-3- methoxydiphenylamine sulfate; and (d) the second layer further comprises a stabilizer.

[0036] In some embodiments, the second layer comprises indoxyl acetate and the indoxyl acetate and the color indicator are present in a weight ratio of from about 20:1 to about 5:1, optionally a weight ratio of about 10:1.

[0037] Provided in another aspect is an in vitro method for determining whether a breastmilk sample has become rancid, the method comprising: contacting the breastmilk sample with a substrate comprising an enzyme mixture that measures the amount of glycerol present in the breastmilk by generating a color change after a given period of time,Atty. Dkt. No.: 136671-0110 wherein the color change indicates the amount of glycerol present in the breastmilk sample.

[0038] In some embodiments, the method further comprises: comparing the color change of the substrate to a first color change, which is a color change of a control sample comprising no glycerol, and a second color change, which is a color change of a sample comprising an amount of glycerol sufficient to indicate that the breastmilk sample has become rancid, wherein the color change of the substrate to an intermediate color between (i) a color resulting from the first color change and (ii) a color resulting from the second color change of the sample indicates that the breastmilk sample is not rancid.

[0039] In some embodiments, the enzyme mixture comprises glycerol kinase, glycerol phosphate oxidase, and a peroxidase.

[0040] In some embodiments, the enzyme mixture further comprises adenosine triphosphate (ATP), 4-aminoantipyrine (4-AAP) and an indicator. In some embodiments, the indicator is N-ethyl-N-(3-sulfopropyl)-m-anisidine, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5- dimethoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline, 3-(N-ethyl-3- methylanilino)-2-hydroxy-propanesulfonic acid, N,N-bis(4-sulfobutyl)-3,5-dimethylaniline, or a salt thereof. In some embodiments, the indicator is the sodium salt of N-ethyl-N-(2- hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline.

[0041] In some embodiments, the period of time is at least about 2 minutes. In some embodiments, the period of time is from about 2 minutes to about 30 minutes, optionally from about 2 minutes to about 5 minutes.

[0042] In some embodiments, the amount of glycerol to indicate that the breastmilk sample has become rancid is less than about 100 mg / L, wherein the breastmilk sample is fresh. In some embodiments, the amount of glycerol to indicate that the breastmilk sample has become rancid is from about 2.5 mg / L to about 100 mg / L.

[0043] In some embodiments, the amount of glycerol to indicate that the breastmilk sample has become rancid is greater than about 1000 mg / L, wherein the breastmilk sample has beenAtty. Dkt. No.: 136671-0110 stored in a household refrigerator or freezer for a period of from about 3 days to about 12 months.

[0044] In some embodiments, the amount of glycerol to indicate that the breastmilk sample has become rancid is from about 101 mg / L to about 1000 mg / L, wherein the breastmilk sample has been stored in a household refrigerator or freezer for a period of from about 3 days to about 12 months.

[0045] In some embodiments, the substrate further comprises a carrier matrix that is a test strip.

[0046] In some embodiments, the enzyme mixture further comprises one or more of a polymer binder, a surfactant, a buffer, a stabilizing sugar, an enzyme cofactor, and a color indicator.

[0047] In some embodiments, the test strip comprises: (a) a base membrane in contact with the test strip; and (b) one or more layers disposed on the base membrane, the one or more layers comprising 4-aminoantipyrine (4-AAP), glycerol kinase, glycerol phosphate oxidase, a peroxidase, and a color indicator.

[0048] In some embodiments, the one or more layers may further comprise one or more of a polymer binder, a surfactant, a buffer, a stabilizing sugar, and an enzyme cofactor.

[0049] Provided in another aspect is a kit for determining whether a breastmilk sample has become rancid comprising: a substrate comprising an enzyme mixture that measures the amount of glycerol present in the breastmilk sample by generating a color change after contacting a breastmilk sample from a subject.

[0050] In some embodiments, the enzyme mixture comprises glycerol kinase, glycerol phosphate oxidase, and a peroxidase.

[0051] In some embodiments, the enzyme mixture further comprises adenosine triphosphate (ATP), 4-aminoantipyrine (4-AAP), and an indicator. In some embodiments, the indicator is N-ethyl-N-(3-sulfopropyl)-m-anisidine N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5- dimethoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline, 3-(N-Ethyl-3-Atty. Dkt. No.: 136671-0110 methylanilino)-2-hydroxy-propanesulfonic acid, N,N-bis(4-sulfobutyl)-3,5-dimethylaniline, or a salt thereof, optionally the sodium salt of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5- dimethoxyaniline.

[0052] In some embodiments, the color change indicates the amount of glycerol present in the breastmilk sample.

[0053] In some embodiments, the substrate further comprises a carrier matrix that is a test strip. In some embodiments, the enzyme mixture further comprises one or more of a polymer binder, a surfactant, a buffer, a stabilizing sugar, an enzyme cofactor, and a color indicator.

[0054] In some embodiments, the test strip comprises: (a) a base membrane in contact with the test strip; and (b) one or more layers disposed on the base membrane, the one or more layers comprising 4-aminoantipyrine (4-AAP), glycerol kinase, glycerol phosphate oxidase, a peroxidase, and a color indicator.

[0055] In some embodiments, the one or more layers further comprise one or more of a polymer binder, a surfactant, a buffer, a stabilizing sugar, and an enzyme cofactor. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Features, aspects, and advantages of the present disclosure will become apparent from the following description and the accompanying non-limiting examples shown in the drawings, which are briefly described below.

[0057] FIG. 1 depicts a flow chart showing an in vitro method for determining whether a subject is a high-lipase producer of breastmilk in accordance with the disclosure.

[0058] FIGS.2A and 2B depict an embodiment of the test strips and corresponding method of use in accordance with the disclosure.

[0059] FIGS.3A and 3B depict the results of the assay described in Example 1, where the tips of cotton applicators comprising a lipase substrate were contacted with milk samplesAtty. Dkt. No.: 136671-0110 comprising a high level of lipase a milk samples comprising a low level of lipase. The samples with a high level of lipase produced a red / purple color change while those with a low level of lipase produced an orange / yellow color change.

[0060] FIG.4 depicts the results of the assay described in Example 2, where milk samples containing varying amounts of glycerol were treated with an enzyme mixture, resulting in a couple enzymatic reaction system that generates a color change to indicate the amount of glycerol present in the samples. DETAILED DESCRIPTION

[0061] Embodiments according to the present disclosure will be described more fully hereinafter. Aspects of the disclosure may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided for completeness, and will fully convey the scope of the present technology to those skilled in the art. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0062] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0063] The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0064] The practice of the present technology will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, chemical engineering, and cell biology, which are within the skill of the art.Atty. Dkt. No.: 136671-0110

[0065] Unless the context indicates otherwise, it is specifically intended that the various features of the disclosure described herein can be used in any combination. Moreover, the disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C (or A, B, and / or C), it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0066] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 1.0 or 0.1, as appropriate, or alternatively by a variation of + / - 15 %, or alternatively 10%, or alternatively 5%, or alternatively 2%. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about”. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0067] As used herein and in the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well as well as the singular form, unless the context clearly indicates otherwise.

[0068] Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0069] As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term.

[0070] As used herein, the terms “acceptable,” “effective,” or “sufficient” refer to the selection of any components, ranges, dose forms, etc. disclosed herein intend that said component, range, dose form, etc., is suitable for the disclosed purpose.Atty. Dkt. No.: 136671-0110

[0071] Approximately 50% of all infants in the U.S. are exclusively breastfed through 3 months, half of which (25% total) remain exclusively breastfed through 6 months (per WHO and UNICEF recommendations). Given that human milk is the only matter many infants ingest for months, ensuring they consume proper amounts of high nutrient value human milk is essential to their ability to meet the most critical of early milestones in life. Human milk oligosaccharides (HMOs) are key nutrients present exclusively in expressed human milk that cannot be sourced from other mammals nor can they be synthesized, making human milk the gold standard for infant nutrition. In today’s work-life paradigm, nursing mothers may return to work while their infants still require human milk. Thus, nursing mothers use breast pumps to enable the acquisition of expressed milk which can be stored in disposable freezer bags to thaw for use at later dates. Current commercially available freezers in most homes (cooling to -20°C) do not adequately preserve the complex emulsified structure of human milk, and as a result, thawed human milk is frequently rancid, leading to it being rejected by some infants.

[0072] In view of the above, there exists a need for parents to determine whether / when their milk will go rancid. However, there are currently no methods for parents to determine whether / when their milk will go rancid. This is attributed to the scientific understanding of the changes that human milk undergoes during storage being lacking and to inter-individual differences in milk composition making it difficult to establish guidelines.

[0073] Described herein methods and kits for assaying the rancidity of human milk, in particular breastmilk. Specifically, the present disclosure provides methods for determining whether a subject is a high-lipase producer of breastmilk (e.g., likely to produce milk that will go rancid in days rather than weeks / months) and determining whether a breastmilk sample has become rancid (e.g., those that have been stored prior to use). As described herein such methods also provide corresponding kits that are specifically developed for at-home use, such as those that use test strips. High-Lipase Producer of Breastmilk

[0074] Lipases, such as lipoprotein lipase (LPL), are substances present in expressed human milk that break down lipids (triglycerides) and convert them into free fatty acids, contributing to changes in flavor and odor often described as rancidity, which can lead to infant rejection ofAtty. Dkt. No.: 136671-0110 stored milk. The activity level of these lipases varies between individuals and potentially over time for a single individual. This disclosure recognizes that individuals producing milk with higher lipase activity may find their stored milk becomes rancid more quickly (e.g., within days or weeks) compared to those with lower lipase activity (whose milk might be stable for longer periods). Thus, such milk should be used immediately and is not suitable for long term storage. Determining whether an individual is a “high-lipase producer” (e.g., higher than a nominal or average lipase producer) can inform storage practices.

[0075] As demonstrated in the Examples, an exemplary method for determining whether a subject is a high lipase producer involves using an assay that allows for the qualitative determination of lipase present in the milk sample (e.g., breastmilk sample) by a color test. The assay includes a chromogenic lipase substrate, such as those disclosed in U.S. Patent 4,847,376, which is incorporated herein by reference in its entirety for the disclosure of such compounds. As disclosed herein, illustrative examples of a suitable lipase substrate include, but are not limited to, 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester (DGGR), a β-naphthyl ester, indoxyl acetate, or 5-bromo-4-chloro-indoxyl caprylate.

[0076] As shown in the Examples and FIG. 1, the in vitro methods described herein begin with obtaining a milk sample (e.g., breastmilk sample) from a subject. Then contacting the lipase substrate with a milk sample will generate a color change after a sufficient period of time that indicates the amount of lipase present in the sample. For instance, if the milk sample contains a high concentration of lipase (e.g., the subject is a high-lipase producer), then the color change of the substate will be from an initial color (or initial state), wherein the color change indicates the amount of lipase present in the milk sample. The initial state may be characterized by a white or neutral color. In some embodiments, a color change from a first color to a second color indicates an extent of lipase production. In other embodiments, the color change of the substrate may be compared to a color change of control sample comprising no lipase, a color change sample comprising an amount of lipase sufficient to indicate that the milk sample is from a subject who is a high-lipase producer, and / or a color change of a sample comprising an amount of lipase sufficient to indicate that the milk sample is from a subject who is not high-lipase producer. This then allows for determining whether the subject is a high lipase producer. A color change may be visually observed, e.g., as a differentiation in at leastAtty. Dkt. No.: 136671-0110 one of a color hue, a color value or a color intensity. As described herein and in the Examples, colorimetric test strips may be used for visual observation of such color changes.

[0077] In some embodiments, a high-lipase producer is characterized by having lipase in an amount at or above a threshold, whereas a low-lipase producer is characterized by having lipase in an amount below the threshold. In some embodiments, a high-lipase producer may be characterized by having lipase in an amount at or above a threshold such that there is an increased risk of rejection of the milk by a recipient infant or baby, whereas a low-lipase producer may be characterized by having lipase in an amount below the threshold such that there is a reduced risk of rejection of the milk by a recipient infant or baby. A high-lipase producer’s milk may only allow storage for a matter of days (e.g., 7 days or less) before exhibiting rancidity, whereas a low-lipase producer’s milk may allow storage for weeks or months before exhibiting rancidity.

[0078] In some embodiments, control samples may be used, such as a milk sample containing a high concentration of lipase and a milk sample containing no lipase or a low concentration of lipase. The color changes of these control samples or control samples having a known amount lipase may be used as reference samples and used to compare with the color change of the test milk sample to qualitatively assess the amount of lipase present in the test milk sample.

[0079] DGGR Method - One approach for determining whether a subject is a high-lipase producer of breastmilk by assessing lipase activity levels is to use 1,2-O-dilauryl-rac-glycero- 3-glutaric acid-(6'-methylresorufin) ester (DGGR) as a lipase substrate. This substrate is cleaved by lipase at a suitable pH to release methylresorufin, which is a colorimetric indicator that produces a purple color. In some embodiments, a color change of the substrate from an initial color to red or purple indicates the subject is a high-lipase producer. In other embodiments, a color of the substrate from an initial color to yellow or orange indicates that the subject is not a high-lipase producer.

[0080] β-Naphthol Method - A second approach for determining whether a subject is a high- lipase producer of breastmilk by assessing lipase activity levels is to use a β-naphthyl ester, such as 2-naphthyl caprylate, as a lipase substrate. This substrate reacts with lipase to release β-naphthol, which can then react with a diazonium salt to provide a colored azo (540 nm).Atty. Dkt. No.: 136671-0110

[0081] Indoxyl Acetate Method - A third approach for determining whether a subject is a high-lipase producer of breastmilk by assessing lipase activity levels is to use indoxyl acetate (IA) or 5-bromo-4-chloro-indoxyl caprylate, as a lipase substrate. Lipase hydrolyzes IA or 5- bromo-4-chloro-indoxyl caprylate to produce the corresponding indoxyl, which can react with a diazonium salt or undergo oxidative dimerization to form indigo, a blue dye (peaks observed around 570-630 nm depending on the diazonium salt).

[0082] Provided in one aspect is an in vitro method for determining whether a subject is a high-lipase producer of breastmilk, the method comprising contacting a milk sample from the subject with a substrate comprising 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6'- methylresorufin) ester, a β-naphthyl ester, indoxyl acetate, or 5-bromo-4-chloro-indoxyl caprylate that generates a color change from an initial color that measures the amount lipase present in the breastmilk within a given period of time following contact, wherein the color change indicates the amount of lipase present in the milk sample.

[0083] Provided in one aspect is an in vitro method for determining whether a subject is a high-lipase producer of breastmilk, the method comprising contacting a milk sample from the subject with a substrate comprising 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6'- methylresorufin)ester that generates a color change within a given period of time following contact, wherein a color change of the substrate from an initial color to red or purple indicates the subject is a high-lipase producer.

[0084] In some embodiments, the period of time is at least about 2 minutes. In some embodiments, the period of time is from about 2 minutes to about 30 minutes, including about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes. In some embodiments, the period of time is from about 3 minutes to about 10 minutes.Atty. Dkt. No.: 136671-0110

[0085] In some embodiments, the period of time is at least about 10 minutes. In some embodiments, the period of time is from about 10 minutes to about 20 minutes, including about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, and about 20 minutes.

[0086] In some embodiments, the amount of lipase present in the milk sample is greater than about 0 U / L. In some embodiments, the amount of lipase present in the milk sample is at least about 1.0 U / L, including at least about 10 U / L, at least about 20 U / L, at least about 30 U / L, at least about 40 U / L, at least about 50 U / L, at least about 60 U / L, at least about 70 U / L, at least about 80 U / L, at least about 90 U / L, at least about 100 U / L, at least about 110 U / L, at least about 120 U / L, at least about 130 U / L, at least about 140 U / L, at least about 150 U / L, at least about 160 U / L, at least about 170 U / L, at least about 180 U / L, at least about 190 U / L, at least about 200 U / L, at least about 210 U / L, at least about 220 U / L, at least about 230 U / L, at least about 240 U / L, at least about 250 U / L, at least about 260 U / L, at least about 270 U / L, at least about 280 U / L, at least about 290 U / L, at least about 300 U / L, at least about 310 U / L, at least about 320 U / L, at least about 330 U / L, at least about 340 U / L, at least about 350 U / L, at least about 360 U / L, at least about 370 U / L, at least about 380 U / L, at least about 390 U / L, at least about 400 U / L, at least about 410 U / L, at least about 420 U / L, at least about 430 U / L, at least about 440 U / L, at least about 450 U / L, at least about 460 U / L, at least about 470 U / L, at least about 480 U / L, at least about 490 U / L, at least about 500 U / L, at least about 1,000 U / L, at least about 2,000 U / L, at least about 3,000 U / L, and at least about 4,000 U / L.

[0087] In some embodiments, the amount of lipase present in the milk sample is from about 0 U / L to about 5,000 U / L, including about 0 U / L, about 1.0 U / L, about 10 U / L, about 20 U / L, about 30 U / L, about 40 U / L, about 50 U / L, about 60 U / L, about 70 U / L, about 80 U / L, about 90 U / L, about 100 U / L, about 110 U / L, about 120 U / L, about 130 U / L, about 140 U / L, about 150 U / L, about 160 U / L, about 170 U / L, about 180 U / L, about 190 U / L, about 200 U / L, about 210 U / L, about 220 U / L, about 230 U / L, about 240 U / L, about 250 U / L, about 260 U / L, about 270 U / L, about 280 U / L, about 290 U / L, about 300 U / L, about 310 U / L, about 320 U / L, about 330 U / L, about 340 U / L, about 350 U / L, about 360 U / L, about 370 U / L, about 380 U / L, about 390 U / L, about 400 U / L, about 410 U / L, about 420 U / L, about 430 U / L, about 440 U / L, aboutAtty. Dkt. No.: 136671-0110 450 U / L, about 460 U / L, about 470 U / L, about 480 U / L, about 490 U / L, about 500 U / L, about 1,000 U / L, about 2,000 U / L, about 3,000 U / L, about 4,000 U / L, and about 5,000 U / L. In some embodiments, the amount of lipase present in the milk sample is from about 0.1 U / L to about 500 U / L. In some embodiments, the amount of lipase present in the milk sample is from about 0.1 U / L to about 5,000 U / L. In some embodiments, the amount of lipase present in the milk sample is from about 1.0 U / L to about 500 U / L. In some embodiments, the amount of lipase present in the milk sample is from about 1.0 U / L to about 5,000 U / L.

[0088] In some embodiments, the subject is a high lipase producer of breastmilk when the amount of lipase present in the milk sample is from about 100 U / L to about 5,000 U / L, optionally from about 300 U / L to about 1,000 U / L.

[0089] The substrate comprising the lipase substrate, such as 1,2-O-dilauryl-rac-glycero-3- glutaric acid-(6'-methylresorufin) ester, a β-naphthyl ester, indoxyl acetate, or 5-bromo-4- chloro-indoxyl caprylate, may contain a carrier matrix that is a bibulous material (e.g., a cellulosic filter paper or a cotton swab). In some embodiments, the carrier matrix is a cellulosic filter paper. In some embodiments, the carrier matrix is a cotton swab. In other embodiments, the substrate comprising the lipase substrate, such as 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin)ester, a β-naphthyl ester, indoxyl acetate, or 5-bromo-4-chloro- indoxyl caprylate, may contain a carrier matrix that is a test strip. In some embodiments, the carrier matrix is a test strip.

[0090] In some embodiments, the method further comprises comparing the color change of the substrate to a first color change, which is a color change of a control sample comprising no lipase, a second color change, which is a color change of a sample comprising an amount of lipase sufficient to indicate that the milk sample is from a subject who is a high-lipase producer of breastmilk, and / or a third color change, which is a color change of a sample comprising an amount of lipase sufficient to indicate that the milk sample is from a subject who is not high- lipase producer of breastmilk

[0091] Also provided herein are kits for determining whether a subject is a high-lipase producer of breastmilk. Such kits may be those that are suitable for at-home use. As shown in the Examples, the kit may comprise a bibulous material, such as a cellulosic filter paper or aAtty. Dkt. No.: 136671-0110 cotton swab, comprising the lipase substrate, which upon contact with a breastmilk sample, generates a color change within a given period of time (e.g., at least 10 minutes) that correlates to the amount of lipase in the sample. The kit may also comprise a test strip comprising the lipase substrate, which upon contact with a breastmilk sample, generates a color change within a given period of time (e.g., at least 2 minutes) that correlates to the amount of lipase in the sample. The determination of whether a subject is a high-lipase producer will allow for the parent to determine if the subject’s breastmilk is likely to go rancid in days versus months / years if stored.

[0092] Provided in another aspect is a kit for determining whether a subject is a high-lipase producer of breastmilk, the kit comprising: a substrate comprising 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester, a β-naphthyl ester, indoxyl acetate, or 5-bromo-4-chloro-indoxyl caprylate that generates a color change after contacting a breastmilk sample from a subject, wherein the color change indicates the amount of lipase present in the breastmilk sample.

[0093] Provided in another aspect is a kit for determining whether a subject is a high-lipase producer of breastmilk, the kit comprising: a substrate comprising 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6'- methylresorufin)ester that generates a color change after contacting a breastmilk sample from a subject.

[0094] In some embodiments, the color change indicates the amount of lipase present in the breastmilk sample. In some embodiments, the amount of lipase present in the breastmilk sample is greater than about 0 U / L. In some embodiments, the amount of lipase present in the breastmilk sample is at least about 1.0 U / L, including at least about 10 U / L, at least about 20 U / L, at least about 30 U / L, at least about 40 U / L, at least about 50 U / L, at least about 60 U / L, at least about 70 U / L, at least about 80 U / L, at least about 90 U / L, at least about 100 U / L, at least about 110 U / L, at least about 120 U / L, at least about 130 U / L, at least about 140 U / L, at least about 150 U / L, at least about 160 U / L, at least about 170 U / L, at least about 180 U / L, at least about 190 U / L, at least about 200 U / L, at least about 210 U / L, at least about 220 U / L, at least about 230 U / L, at least about 240 U / L, at least about 250 U / L, at least about 260 U / L, atAtty. Dkt. No.: 136671-0110 least about 270 U / L, at least about 280 U / L, at least about 290 U / L, at least about 300 U / L, at least about 310 U / L, at least about 320 U / L, at least about 330 U / L, at least about 340 U / L, at least about 350 U / L, at least about 360 U / L, at least about 370 U / L, at least about 380 U / L, at least about 390 U / L, at least about 400 U / L, at least about 410 U / L, at least about 420 U / L, at least about 430 U / L, at least about 440 U / L, at least about 450 U / L, at least about 460 U / L, at least about 470 U / L, at least about 480 U / L, at least about 490 U / L, at least about 500 U / L, at least about 1,000 U / L, at least about 2,000 U / L, at least about 3,000 U / L, and at least about 4,000 U / L.

[0095] In some embodiments, the amount of lipase present in the breastmilk sample is from about 0 U / L to about 5,000 U / L, including about 0 U / L, about 1.0 U / L, about 10 U / L, about 20 U / L, about 30 U / L, about 40 U / L, about 50 U / L, about 60 U / L, about 70 U / L, about 80 U / L, about 90 U / L, about 100 U / L, about 110 U / L, about 120 U / L, about 130 U / L, about 140 U / L, about 150 U / L, about 160 U / L, about 170 U / L, about 180 U / L, about 190 U / L, about 200 U / L, about 210 U / L, about 220 U / L, about 230 U / L, about 240 U / L, about 250 U / L, about 260 U / L, about 270 U / L, about 280 U / L, about 290 U / L, about 300 U / L, about 310 U / L, about 320 U / L, about 330 U / L, about 340 U / L, about 350 U / L, about 360 U / L, about 370 U / L, about 380 U / L, about 390 U / L, about 400 U / L, about 410 U / L, about 420 U / L, about 430 U / L, about 440 U / L, about 450 U / L, about 460 U / L, about 470 U / L, about 480 U / L, about 490 U / L, about 500 U / L, about 1,000 U / L, about 2,000 U / L, about 3,000 U / L, about 4,000 U / L, and about 5,000 U / L. In some embodiments, the amount of lipase present in the milk sample is from about 0.1 U / L to about 500 U / L. In some embodiments, the amount of lipase present in the breastmilk sample is from about 1.0 U / L to about 500 U / L. In some embodiments, the amount of lipase present in the milk sample is from about 1.0 U / L to about 5,000 U / L.

[0096] In some embodiments, when the subject is not a high-lipase producer, the breastmilk may be stored (e.g., between about -80°C to about 4°C for at least 5 years).

[0097] In some embodiments, a substrate comprising 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester, a β-naphthyl ester, indoxyl acetate, or 5-bromo-4-chloro- indoxyl caprylate contains a carrier matrix that is a bibulous material, optionally a cellulosic filter paper or a cotton swab. In other embodiments, a substrate comprising 1,2-O-dilauryl- rac-glycero-3-glutaric acid-(6'-methylresorufin) ester contains a carrier matrix that is aAtty. Dkt. No.: 136671-0110 bibulous material, optionally a cellulosic filter paper or a cotton swab. In some embodiments, the bibulous material is a cellulosic filter paper. In some embodiments, the bibulous material is a cotton swab.

[0098] In some embodiments, a substrate comprising 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester, a β-naphthyl ester, indoxyl acetate, or 5-bromo-4-chloro- indoxyl caprylate contains a carrier matrix that is a test strip. In other embodiments, a substrate comprising 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester contains a carrier matrix that is a test strip. In some embodiments, a substrate comprising a β-naphthyl ester contains a carrier matrix that is a test strip. In some embodiments, a substrate comprising indoxyl acetate contains a carrier matrix that is a test strip. In some embodiments, a substrate comprising 5-bromo-4-chloro-indoxyl caprylate contains a carrier matrix that is a test strip.

[0099] FIG.2A shows an exemplary embodiment of a test strip 100. The test strip comprises a base membrane 101 in contact with the test strip, a first layer 102 disposed on the base membrane, and a second layer 103 overlaying the first layer. The first layer 102 may comprise a buffer while the second layer 103 may comprise the lipase substrate (e.g., 1,2-O-dilauryl-rac- glycero-3-glutaric acid-(6'-methylresorufin), β-naphthyl ester, indoxyl acetate, and 5-bromo- 4-chloro-indoxyl caprylate) and optionally a color indicator. In some embodiments, where the lipase substrate is β-naphthyl ester, indoxyl acetate, or 5-bromo-4-chloro-indoxyl caprylate, lipase reacts with the substrate to provide a compound that reacts with the color indicator, such as a diazonium salt, to generate the color change.

[0100] FIG. 2B shows an exemplary embodiment of using a test strip as described herein. After contacting the test strip comprising the lipase substrate (e.g., 1,2-O-dilauryl-rac-glycero- 3-glutaric acid-(6'-methylresorufin) ester, a β-naphthyl ester, indoxyl acetate, or 5-bromo-4- chloro-indoxyl caprylate) with a milk sample, the test strip will produce a color change after a sufficient period of time (e.g., within 5 minutes in some embodiments). This color change indicates the amount of lipase present in the sample, thereby allowing for the determination of whether the subject is a high lipase producer.

[0101] Test Strips using DGGR Method – As described in the Examples, initial formulations involved a two-coat system on a suitable membrane substrate, with a first coatAtty. Dkt. No.: 136671-0110 containing a buffer and a second coat containing DGGR dissolved in a volatile organic solvent. Instability of the DGGR substrate under the basic conditions for desired lipase activity was observed, leading to auto-hydrolysis and background color development even in the absence of lipase (0 U / L). To stabilize the DGGR substrate, an organic acid (e.g., citric acid) was added to the formulation. While adding citric acid improved the stability of the zero reading (less background color, strip appeared yellow / orange as expected), the resulting acidic microenvironment significantly inhibited lipase activity, leading to poor color development and reduced sensitivity or resolution in the desired lipase activity range (e.g., 0-500 U / L). Further optimizations included increasing the DGGR concentration, removing the organic acid, and adjusting the buffer molarity.

[0102] Test Strips Using β-Naphthol Method – As described in the Examples, a test formulation involved a first coat containing a buffer and a second coat containing β-NC and the diazonium salt in an organic solvent. Initial screening showed this reaction was very slow at room temperature, requiring several hours for noticeable color development, although 1- diazo-2-naphthol-4-sulfonic acid appeared more reactive than Fast Blue BB. Heat may be used to achieve a practical reaction speed.

[0103] Test Strips Using Indoxyl Acetate Method – As described in the Examples, initial formulations involved a two-coat system on a suitable membrane substrate. The first coat typically contained a buffer and optionally an accelerator. The second coat contained indoxyl acetate (IA) and a diazonium salt indicator dissolved in an organic solvent.

[0104] Additional studies conducted by the inventors of the present application found that the approximate 3:1 molar ratio of IA to diazonium salt was optimal for signal generation, 1- diazo-2-naphthol-4-sulfonic acid was highly reactive and sensitive but proved unstable at 0 U / L lipase. 4-Diazo-3-methoxydiphenylamine sulfate showed good reactivity and, importantly, stability at 0 U / L lipase for at least 5 minutes. Testing in pasteurized milk showed that increasing IA significantly improved color development in milk samples containing 500 U / L lipase. Potassium ferricyanide and potassium ferrocyanide were tested as potential accelerators – using both at a 1:1 ratio or individually in the first coat. Ferrocyanide (e.g., potassium ferrocyanide) was found to be effective. It was also found that increasing the buffer concentration was also implemented to enhance buffering capacity.Atty. Dkt. No.: 136671-0110

[0105] An exemplary formulation for detecting lipase in milk using the indoxyl acetate method comprises a base membrane, a first layer that was obtained from applying a first coat containing sodium borate and potassium ferrocyanide (no PVP) to the base membrane, and a second layer that was obtained from applying a second coat containing indoxyl acetate in an organic solvent to the first layer. A preferred concentration of the diazonium salt, such 4-diazo- 3-methoxydiphenylamine sulfate, was found to be 1 mg / g. This formulation demonstrated stability at 0 U / L and a detectable color change indicative of lipase activity in the 0-500 U / L range in pasteurized milk within approximately 3-5 minutes.

[0106] The test strip described herein may comprise (a) a base membrane in contact with the test strip; (b) a first layer disposed on the base membrane, the first layer comprising a buffer; and (c) a second layer overlaying the first layer, the second layer comprising 1,2-O-dilauryl- rac-glycero-3-glutaric acid-(6'-methylresorufin) ester, a β-naphthyl ester, indoxyl acetate, or 5- bromo-4-chloro-indoxyl caprylate, and optionally a color indicator.

[0107] The base membrane as described herein may comprise a microporous membrane. The base membrane may include but are not limited to one or more polymer based materials, such as polyester, nylon, and polysulfone, glass fibers, cotton linters, cotton linter fibers, and cellulose. The base membrane described herein may have a thickness of about 0.15 mm to about 1 mm, including about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, and about 1 mm. In some embodiments, the base membrane has a thickness of 0.5 mm. In some embodiments, the base membrane has a thickness of from about 0.15 mm to about 0.6 mm.

[0108] A buffer as used herein may include but are not limited to one or more of Tris buffer, a borate buffer, Bicine buffer and TAPS (N-tris(hydroxymethyl)methyl-3- aminopropanesulfonic acid) buffer. In some embodiments, the buffer comprises a borate buffer, such as sodium borate.

[0109] In some embodiments, the first layer further comprises one or more of a lipase accelerator (e.g., bile salts), a polymer binder (e.g., PVP) , a surfactant, a stabilizer (e.g., calcium chloride), and a reaction accelerator. In some embodiments, the first layer further comprises a lipase accelerator (e.g., bile salts). In some embodiments, the first layer furtherAtty. Dkt. No.: 136671-0110 comprises a polymer binder (e.g., PVP). In some embodiments, the first layer further comprises a surfactant. In some embodiments, the first layer further comprises a stabilizer (e.g., calcium chloride). In some embodiments, the first layer further comprises a reaction accelerator (e.g., a ferrocyanide and a ferricyanide).

[0110] The color indicator may comprise a diazonium salt. Examples of suitable color indicators include but are not limited to, one or more of 4-benzamido-2,5-diethoxybenzene-1- diazonium (Fast Blue BB salt), bis(2-methoxy-4-nitrophenyldiazonium) 1,5- naphthalenedisulfonate (Fast Red B Salt 1,5-naphthalenedisulfonate), 1-diazo-2-naphthol-4- sulfonic acid, and 4-diazo-3-methoxydiphenylamine sulfate.

[0111] In some embodiments, the second layer further comprises one or more of an organic acid (e.g., citric acid, malic acid, and oxalic acid), a stabilizer, a reaction accelerator, and a buffer. In some embodiments, the second layer further comprises one or more of an organic acid (e.g., citric acid. In some embodiments, the organic acid is citric acid. In some embodiments, the second layer further comprises a stabilizer. In some embodiments, the second layer further comprises a reaction accelerator, such as one or more of a ferrocyanide and a ferricyanide. In some embodiments, the second layer further comprises a buffer.

[0112] In some embodiments, the test strip comprises: (a) a base membrane in contact with the test strip; (b) a first layer disposed on the base membrane, the first layer comprising a buffer; and (c) a second layer overlaying the first layer, the second layer comprising 1,2-O- dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester.

[0113] In some embodiments, the test strip comprises one or more of the following: (a) the buffer comprising tris buffer; (b) the first layer further comprising one or more of bile salts and calcium chloride; and (c) the second layer further comprising an organic acid (e.g., citric acid) and optionally, a stabilizer.

[0114] In some embodiments, the test strip comprises:Atty. Dkt. No.: 136671-0110 (a) a base membrane in contact with the test strip; (b) a first layer disposed on the base membrane, the first layer comprising a buffer; and (c) a second layer overlaying the first layer, the second layer comprising the β- naphthyl ester, optionally 2-naphthyl caprylate and a color indicator.

[0115] In some embodiments, the test strip comprises one or more of the following: (a) the buffer comprising a borate buffer, optionally sodium borate buffer; (b) the first layer further comprising polyvinylpyrrolidone; (c) the color indicator comprising a diazonium salt, optionally 4-benzamido-2,5- diethoxybenzene-1-diazonium (Fast Blue BB salt) or 1-diazo-2-naphthol-4-sulfonic acid; and (de) optionally, the second layer further comprising a stabilizer.

[0116] In some embodiments, the test strip comprises: (a) a base membrane in contact with the test strip; (b) a first layer disposed on the base membrane, the first layer comprising a buffer; and (c) a second layer overlaying the first layer, the second layer comprising indoxyl acetate or 5-bromo-4-chloro-indoxyl caprylate, optionally indoxyl acetate and a color indicator.

[0117] In some embodiments, the test strip comprises one or more of the following: (a) the buffer comprising a borate buffer, optionally sodium borate; b) the first layer further comprising a reaction accelerator, optionally a ferrocyanide, further optionally potassium ferrocyanide; (c) the color indicator comprising a diazonium salt, optionally diazo-3- methoxydiphenylamine sulfate; and (d) the second layer further comprising a stabilizer.

[0118] In some embodiments, indoxyl acetate is present in excess of the color indicator.

[0119] In some embodiments, indoxyl acetate and the color indicator are present in a weight ratio of from about 20:1 to about 5:1, optionally a weight ratio of about 10:1.Atty. Dkt. No.: 136671-0110

[0120] In some embodiments, the color indicator is applied to the second layer in a solution comprising a volatile organic solvent and is present in a concentration of from about 0.01 mg / g to about 1 mg / g. In some embodiments, the indoxyl acetate is applied to the second layer in a solution comprising a solvent carrier and is present in a concentration of from about 1 mg / g to about 30 mg / g.

[0121] In some embodiments, the reaction accelerator is applied to the first layer in an aqueous solution and is present in a concentration of about 0 to about 1% (w / v). In some embodiments, the buffer is applied to the first layer in an aqueous solution and is present in a concentration of about 1 to 10% w / v. Rancidity of Breastmilk Sample

[0122] The rancidity of the human milk may be determined by the detection of fat breakdown via glycerol accumulation. As shown in the Examples, one method for determining whether a milk sample (e.g., breastmilk sample) has become rancid is by testing if glycerol is present in the milk sample by a color change test, where the color change indicates the amount of glycerol present in the milk sample. Such an assay would allow for the determination of whether milk samples (e.g., such as those that have been stored) have gone rancid and are therefore no longer suitable for use.

[0123] As shown in the Examples, the amount of glycerol present in a milk sample can be determined by using a coupled enzymatic reaction system, such as the one shown in Scheme 1. Glycerol is phosphorylated by glycerol kinase to produce glycerol-3-phosphate and adenosine-5’-diphosphate (ADP) (eq. 1). Glycerol-3-phosphate is oxidized by glycerol phosphate oxidase to produce dihydroxyacetone phosphate and hydrogen peroxide (H2O2) (eq. 2). Peroxidase catalyzes the redox-coupled reaction of H2O2 with 4-aminoantipyrine (4-AAP) and an indicator, such as N-ethyl-N-(3-sulfopropyl)-m-anisidine, to produce a purple or blue color depending on the indicator, wherein the intensity / shade of the purple / blue color indicates the amount of glycerol present in the sample.Atty. Dkt. No.: 136671-0110 Scheme 1. Glycerol Enzymatic Reaction System

[0124] Control samples containing a no glycerol and / or known amounts of glycerol (such as the minimum amount of glycerol for a sample to be considered “rancid”) may be used. The color changes of these control samples may be used as reference samples. Further, the color change of one or more control samples can be used to compare with the color change of the test milk sample to qualitatively assess the amount of glycerol present in the test milk sample.

[0125] Provided in one aspect is an in vitro method for determining whether a breastmilk sample has become rancid, the method comprising: contacting the breastmilk sample with a substrate comprising an enzyme mixture that measures the amount of glycerol present in the breastmilk by generating a color change after a given period of time, wherein the color change indicates the amount of glycerol present in the breastmilk sample.

[0126] In some embodiments, the method further comprises: comparing the color change of the substrate to a first color change, which is a control change of a control sample comprising no glycerol, and a second color change, which is a color change of a sample comprising an amount of glycerol sufficient to indicate that the breastmilk sample has become rancid, wherein the color change of the substrate to an intermediate color between (i) a color resulting from the first color change (ii) a color resulting from the second color change of the sample indicates that the breastmilk sample is not rancid.

[0127] In some embodiments, the enzyme mixture comprises glycerol kinase, glycerol phosphate oxidase, and a peroxidase. In some embodiments, the enzyme mixture furtherAtty. Dkt. No.: 136671-0110 comprises adenosine triphosphate (ATP), 4-aminoantipyrine (4-AAP) and an indicator. In some embodiments, the indicator is N-ethyl-N-(3-sulfopropyl)-m-anisidine, N-ethyl-N-(2- hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5- dimethylaniline, 3-(N-ethyl-3-methylanilino)-2-hydroxy-propanesulfonic acid, N,N-bis(4- sulfobutyl)-3,5-dimethylaniline, or salt thereof. In some embodiments, the indicator is the sodium salt of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline.

[0128] In some embodiments, the sufficient period of time is at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, including at least about 6 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 11 minutes, at least about 12 minutes, at least about 13 minutes, at least about 14 minutes, at least about 15 minutes, at least about 16 minutes, at least about 17 minutes, at least about 18 minutes, at least about 19 minutes, at least about 20 minutes, at least about 21 minutes, at least about 22 minutes, at least about 23 minutes, at least about 24 minutes, at least about 25 minutes, at least about 26 minutes, at least about 27 minutes, at least about 28 minutes, at least about 29 minutes, and at least about 30 minutes.

[0129] In some embodiments, the sufficient period of time is from about 2 minutes to about 30 minutes, including about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, and about 30 minutes. In some embodiments, the sufficient period of time is from about 3 minutes to about 30 minutes. In some embodiments, the sufficient period of time is from about 5 minutes to about 20 minutes. In some embodiments, the sufficient period of time is from about 2 minutes to about 5 minutes. In some embodiments, the sufficient period of time is from about 3 minutes to about 5 minutes.

[0130] In some embodiments, the sufficient amount of glycerol to indicate that the breastmilk sample has become rancid is less than about 100 mg / L, wherein the breastmilk sample is fresh. In some embodiments, the sufficient amount of glycerol to indicate that the breastmilk sample has become rancid is from about 2.5 mg / L to about 100 mg / L.Atty. Dkt. No.: 136671-0110

[0131] In some embodiments, the sufficient amount of glycerol to indicate that the breastmilk sample has become rancid is greater than about 1000 mg / L, wherein the breastmilk sample has been stored (e.g., in a household refrigerator or freezer for a period of from about 3 days to about 12 months).

[0132] In some embodiments, the sufficient amount of glycerol to indicate that the breastmilk sample has become rancid is from about 101 mg / L to about 1000 mg / L, wherein the breastmilk sample has been stored (e.g., in a household refrigerator or freezer for a period of from about 3 days to about 12 months).

[0133] The substrate may comprise anything suitable for storing human milk. Non-limiting examples include plastic cups, plastic human milk storage bags, or a plastic biological collection container, suitable for collecting biological samples and biological fluids. In some embodiments, the substrate is a milk storage bag. As with the earlier methods described herein, the substrate may comprise a carrier matrix that is a test strip.

[0134] Also provided herein are kits that are suitable for determining whether a breastmilk sample has become rancid. In some embodiments, the enzyme mixture that measures the amount of glycerol present in the milk sample is embedded into a solid material (such as the inside of a storage bag, vessel or cap). This would allow for the determination of whether the milk sample has become rancid (especially if the milk sample has been stored) and therefore unsuitable for use.

[0135] Provided in one aspect is a kit for determining whether a breastmilk sample has become rancid comprising: a substrate comprising an enzyme mixture that measures the amount of glycerol present in the breastmilk sample by generating a color change after contacting a breastmilk sample from a subject.

[0136] In some embodiments, the enzyme mixture comprises glycerol kinase, glycerol phosphate oxidase, and a peroxidase. In some embodiments, the enzyme mixture further comprises adenosine triphosphate (ATP), 4-aminoantipyrine (4-AAP) and an indicator. In some embodiments, the indicator is N-ethyl-N-(3-sulfopropyl)-m-anisidine, N-ethyl-N-(2- hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5- dimethylaniline, 3-(N-Ethyl-3-methylanilino)-2-hydroxy-propanesulfonic acid, N,N-bis(4-Atty. Dkt. No.: 136671-0110 sulfobutyl)-3,5-dimethylaniline, or salt thereof. In some embodiments, the indicator is the sodium salt of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline.

[0137] In some embodiments, the color change indicates the amount of glycerol present in the breastmilk sample. In some embodiments, the substrate contains a milk storage container such as a milk storage bag or vessel. In some embodiments, the substrate contains a milk storage bag configured to be stored between about -80°C to about 4°C for at least 5 years.

[0138] In some embodiments, the milk storage bag is configured to be stored for between about 1 day to about 5 years. In some embodiments, the milk storage bag is configured to be stored for between about 1 day to about 2 days, between about 1 day to about 3 days, between about 1 day to about 4 days, between about 2 days to about 3 days, between about 2 days to about 4 days, between about 2 days to about 5 days, between about 3 days to about 4 days, between about 3 days to about 5 days, between about 3 days to about 6 days, between about 4 days to about 5 days, between about 4 days to about 6 days, between about 4 days to about 7 days, between about 5 days to about 6 days, between about 5 days to about 7 days, between about 6 days to about 7 days, between about 1 week to about 2 weeks, between about 1 week to about 2 weeks, between about 1 week to about 3 weeks, between about 2 weeks to about 3 weeks, between about 2 weeks to about 4 weeks, between about 3 weeks to about 4 weeks, between about 1 month to about 1.5 months, between about 1 month to about 2 months, between about 1.5 months to about 2 months, 2 months to about 4 months, between about 2 months to about 6 months, between about 2 months to about 8 months, between about 4 month to about 6 months, between about 4 months to about 8 months, 4 months to about 10 months, between about 6 months to about 8 months, between about 6 months to about 10 months, between about 6 months to about 12 months, between about 8 months to about 10 months, between about 8 months to about 12 months, between about 8 months to about 14 months, between about 10 months to about 12 months, between about 10 months to about 14 months, between about 10 months to about 16 months, between about 12 months to about 14 months, between about 12 months to about 16 months, between about 12 months to about 18 months, between about 14 months to about 16 months, between about 14 months to about 18 months, between about 14 months to about 20 months, between about 16 months to about 18 months, between about 16 months to about 20 months, between about 16 months to about 22 months,Atty. Dkt. No.: 136671-0110 between about 18 months to about 20 months, between about 18 months to about 22 months, between about 18 months to about 24 months, between about 20 months to about 22 months, between about 20 months to about 24 months, between about 22 months to about 24 months, between about 2 years and about 2.5 years, between about 2 year and 3 years, between about 2 years and 3.5 years, between about 2.5 years and about 3 years, between about 2.5 years and 3.5 years, between about 2.5 years and 4 years, between about 3 years and about 3.5 years, between about 3 years to about 4 years, between about 3 years and about 4.5 years, between about 3.5 years to about 4 years, between about 3.5 years to about 4.5 years, between 3.5 years to about 5 years, between about 4 years to about 4.5 years, between about 4 years to about 5 years, or between about 4.5 years to about 5 or more years.

[0139] In some embodiments, the milk storage bag is configured to be stored at a temperature of about 0 °C, about -2 °C about -4 °C about -8 °C, about -10 °C, about -12 °C, about -14 °C, about -16 °C , about -18 °C, or about -20 °C or less. In some embodiments, the milk storage bag is configured to be stored at a temperature of at least 0 °C, at least -2 °C at least -4 °C at least -8 °C, at least -10 °C, at least -12 °C , at least -14 °C , at least -16 °C , at least -18 °C, or at least -20 °C or less. In some embodiments, the milk storage bag is configured to be stored at a temperature of greater than -20 °C. In some embodiments, the milk storage bag is configured to be stored at a temperature of at least -30 °C, at least -40 °C, at least -50 °C, at least -60 °C, at least -70 °C, at least -80 °C. In some embodiments, the milk storage bag is configured to be stored at a temperature of greater than -80 °C. In some embodiments, the milk storage bag is configured to be snap frozen. In some embodiments, the milk storage bag is configured to be stored in liquid nitrogen.

[0140] In some embodiments, the milk storage bag is configured to be stored at a temperature of 4 °C. In some embodiments, the milk storage bag is configured to be stored at a temperature at about 2°C, about 4 °C, about 6 °C, about 8 °C, about 10 °C, about 12 °C, about 14 °C, about 16 °C or more. In some embodiments, the milk storage bag is configured to be stored at a temperature of at least 2 °C, at least 4 °C, at least 6 °C, at least 8 °C, at least 10 °C, at least 12 °C, at least 14 °C, at least 16 °C or more.

[0141] In some embodiments, the milk storage bag is configured to be stored at room temperature.Atty. Dkt. No.: 136671-0110

[0142] In some embodiments, the substrate is a test strip. Referring to FIG.2A, the test strip 100 may comprise a base membrane 101 in contact with the test strip; and one or more layers (102 and 103) disposed on the base membrane. In some embodiments, the layers comprise a combination of 4-aminoantipyrine (4-AAP), glycerol kinase, glycerol phosphate oxidase, a peroxidase, and a color indicator.

[0143] In some embodiments, the test strip for the glycerol assay as described herein comprises a mixture of 4-aminoantipyrine (4-AAP), glycerol kinase, glycerol phosphate oxidase, a peroxidase, and a color indicator. In some embodiments, the mixture may further comprise one or more of a polymer binder, a surfactant, a buffer, a stabilizing sugar, and an enzyme cofactor. In some embodiments, the mixture may further comprise one or more of a polymer binder (e.g. polyvinyl alcohol). In some embodiments, the mixture may further comprise one or more of a surfactant. In some embodiments, the mixture may further comprise one or more of a buffer. In some embodiments, the mixture may further comprise one or more of a stabilizing sugar (e.g. mannitol or D-mannitol). In some embodiments, the mixture may further comprise one or more of an enzyme cofactor (e.g., magnesium dichloride).

[0144] In some embodiments, the test strip comprises: (a) a base membrane in contact with the test strip; and (b) one or more layers disposed on the base membrane, the one or more layers comprising 4-aminoantipyrine (4-AAP), glycerol kinase, glycerol phosphate oxidase, a peroxidase, and a color indicator.

[0145] In some embodiments, the one or more layers further comprise one or more of a polymer binder, a surfactant, a buffer, a stabilizing sugar, and an enzyme cofactor.

[0146] In some embodiments, the test strip comprises one or more of the following: (a) the indicator is applied in an aqueous solution and is present in a concentration from about 0.1 mg / g to about 10 mg / g; (b) ATP is applied an aqueous solution and is present in a concentration from about 1 mM to about 30 mM; (c) glycerol kinase is applied in an aqueous solution and is present in a concentration from about 50 U / g to about 1500 U / g;Atty. Dkt. No.: 136671-0110 (d) glycerol phosphate oxidase is applied in an aqueous solution and is present in a concentration from about 50 U / g to about 1500 U / g; (e) the peroxidase is applied in an aqueous solution and is present in a concentration from about 50 U / g to about 1500 U / g; and (f) 4-aminoantipyrine (4-AAP) is applied in a aqueous solution and is present in a concentration of from 0.2 mg / g to about 20 mg / g.

[0147] The present technology, thus generally described, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present technology. EXAMPLES Example 1: Detection of active lipase (cotton swab)

[0148] Sterile, 6” cotton-tipped applicators (VWR 10192-490) were soaked (approximately 1 minute) in a glass dish containing a solution of 60 mM 1,2-Di-O-lauryl-rac-glycero-3- (glutaric acid 6-methylresorufin ester) (Sigma 30058-50MG-F) in methanol. The soaked applicators were removed from the glass dish and dried at 37 °C for approximately 1 h, after which they were considered ready to use. Testing consisted of exposing (by submerging) the 1,2-Di-O-lauryl-rac-glycero-3-(glutaric acid 6-methylresorufin ester)-stained applicator into a 500 µL aliquot of human milk. The tip of the cotton applicator, which becomes saturated with milk within several seconds of exposure to the milk, was transferred to a dry polystyrene FlowTube (VWR 76449-670) and allowed to sit at room temperature for at least 10 minutes. Lipolysis was detected as a change in color from orange to purple of the 1,2-Di-O-lauryl-rac- glycero-3-(glutaric acid 6-methylresorufin ester) stained applicator tips due to reaction with lipases in the milk. Test samples used to establish the proof-of-concept consisted of aliquots of human milk having varying amounts of lipase.

[0149] FIGS.3A and 3B depict the results of the assay described in Example 1, where the tips of cotton applicators comprising a lipase substrate were contacted with milk samples comprising a high level of lipase a milk samples comprising a low level of lipase. The samples with a high level of lipase produced a red / purple color change while those with a low level ofAtty. Dkt. No.: 136671-0110 lipase produced an orange / yellow color change. The test milk samples for FIG.3A from left- to-right are: first three are milk samples having a high level of lipase including 100 U lipoprotein lipase from bovine milk (Sigma L2254-1KU) and the last three are milk samples having a low level of lipase. The test milk samples for FIG.3B from left-to-right are: a milk sample having a high level of lipase and a milk sample having low level of lipase. Accordingly, these results demonstrate that the methods of the present technology are useful for determining whether a subject is a high-lipase producer of breastmilk. Example 2: Detection of fat breakdown via glycerol accumulation in stored milk (glycerol)

[0150] This assay utilized reagents based on a commercial kit (Cayman Chemical, item no. 10010755). A glycerol enzyme mixture (Cayman 10010962), containing lyophilized glycerol kinase, glycerol phosphate oxidase, peroxidase, ATP, 4-aminoantipyrine (4-AAP), and N- ethyl-N-(3-sulfopropyl)-m-anisidine, was reconstituted in deionized water. 50 µL aliquots of the reagent mixture were dispensed into tubes. 5 µL of human milk samples (previously stored, with varying expected glycerol levels) were added to the reagent mixture. The mixtures were incubated at room temperature.

[0151] Color development (purple) was observed, indicating the presence of glycerol. The reaction was observed to be substantially complete within approximately 3 to 3.5 minutes, even for a sample known to contain >1000 mg / L glycerol based on laboratory analysis. Replicates showed good consistency (%Reflectance at 600 nm measured at 3 minutes). Samples with lower expected glycerol content (<50 mg / L) showed significantly less color development compared to high-glycerol samples (>100 mg / L), demonstrating the assay's ability to differentiate glycerol levels indicative of potential rancidity due to lipolysis during storage.

[0152] FIG.4 depicts the results of the assay described in Example 2, where milk samples containing varying amounts of glycerol were treated with an enzyme mixture, resulting in a coupled enzymatic reaction system that generates a color change to indicate the amount of glycerol present in the samples. The test milk samples for FIG.4 from left-to-right are: Sample A, yielding >100 mg / L glycerol; Sample B, yielding >100 mg / L glycerol; Sample C, yielding >100 mg / L glycerol; Sample D, yielding <50 mg / L glycerol; Sample E, yielding <50 mg / LAtty. Dkt. No.: 136671-0110 glycerol; Sample F, yielding <50 mg / L glycerol; Sample G, yielding <50 mg / L glycerol; and Sample H (untreated), yielding >100 mg / L glycerol. Example 3: Detection of Active Lipase using Indoxyl Acetate Test Strips

[0153] For lipase detection using indoxyl acetate, a carrier matrix / base membrane (e.g., one comprising a cotton linter material) was impregnated by applying the first coating composition formulated in accordance with components listed in the below table. After allowing the first coating composition to dry, the second coating composition formulated with the components indicated in the below table was then applied to the dried first coat.Atty. Dkt. No.: 136671-0110

[0154] Testing consisted of applying a sample of human milk (pasteurized milk spiked with known concentrations of Lipoprotein Lipase, LPL, ranging from 0 U / L to 500 U / L) to the test strip. Color development was observed over time (e.g., 3 minutes and 5 minutes) at room temperature.

[0155] Results indicated that the strip remained stable (minimal color change) when exposed to the 0 U / L sample for at least 5 minutes. Samples containing LPL produced a blue color, with the intensity of the blue color increasing with higher LPL concentrations (e.g., 500 U / L sample showing a distinct blue color compared to the 0 U / L sample within 3-5 minutes). These results demonstrate the method's utility for detecting lipase activity in milk. Example 4: Detection of Active Lipase using DGGR Test Strips

[0156] For lipase detection using 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6’- methylresorufin) ester (DGGR), a carrier matrix / base membrane (e.g., cellulose filter paper) was impregnated by applying the first coating composition formulated in accordance with components listed in the below table. After allowing the first coating composition to dry, the second coating composition formulated with the components indicated in the below table was then applied to the dried first coat.Atty. Dkt. No.: 136671-0110Example 5: Detection of Active Lipase using β-Naphthol Test Strips

[0157] For lipase detection using β-Naphthol, a carrier matrix / base membrane (e.g., cellulose filter paper) was impregnated by applying the first coating composition formulated in accordance with components listed in the below table. After allowing the first coating composition to dry, the second coating composition formulated with the components indicated in the below table was then applied to the dried first coat.Atty. Dkt. No.: 136671-0110Example 6: Detection of Glycerol using Test Strips

[0158] For glycerol detection, a carrier matrix / base membrane (e.g., cellulose filter paper) was impregnated by applying the components indicated in the below table.Atty. Dkt. No.: 136671-0110

[0159] Results indicated that the strip remained stable (minimal color change) when exposed to the 0 mg / L sample for at least 5 minutes. Samples containing glycerol produced a light blue color, with the intensity of the blue color increasing with higher glycerol concentrations (e.g., 200 mg / L sample showing a distinct blue color compared to the 0 mg / L sample within 3-5 minutes). This demonstrates the method's utility for detecting glycerol concentration in milk.

[0160] While certain embodiments have been illustrated and described, it should be understood that changes and modifications can be made therein in accordance with ordinary skill in the art without departing from the technology in its broader aspects as defined in the following claims.

[0161] The embodiments, illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. Additionally, the phrase “consisting essentially of” will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase “consisting of” excludes any element not specified.

[0162] The present disclosure is not to be limited in terms of the particular embodiments described in this application. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and compositions within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. TheAtty. Dkt. No.: 136671-0110 present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds, or compositions, which can of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0163] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0164] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.

[0165] All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

[0166] Other embodiments are set forth in the following claims.

Claims

Atty. Dkt. No.: 136671-0110 WHAT IS CLAIMED IS:

1. An in vitro method for determining whether a subject is a high-lipase producer of breastmilk, the method comprising: contacting a milk sample from the subject with a substrate comprising 1,2-O-dilauryl-rac- glycero-3-glutaric acid-(6'-methylresorufin) ester, a β-naphthyl ester, indoxyl acetate, or 5-bromo-4-chloro-indoxyl caprylate that generates a color change from an initial color that measures the amount lipase present in the breastmilk within a given period of time following contact, wherein the color change indicates the amount of lipase present in the milk sample.

2. The in vitro method of claim 1, wherein the period of time is at least about 2 minutes.

3. The in vitro method of claim 2, wherein the period of time is from about 2 minutes to about 30 minutes, optionally from about 3 minutes to about 10 minutes.

4. The in vitro method of any one of claims 1-3, wherein the amount of lipase present in the milk sample is greater than about 0 U / L, optionally at least about 1.0 U / L.

5. The in vitro method of claim 4, wherein the amount of lipase present in the milk sample is from about 0 U / L to about 5,000 U / L, optionally from about 0.1 U / L to about 5,000 U / L.

6. The in vitro method of claim 5, wherein the subject is a high lipase producer of breastmilk when the amount of lipase present in the milk sample is from about 100 U / L to about 5,000 U / L, optionally from about 300 U / L to about 1,000 U / L.

7. The in vitro method of any one of claims 1-6, wherein the substrate further comprises a carrier matrix that is a bibulous material, optionally a cellulosic filter paper.Atty. Dkt. No.: 136671-0110 8. The in vitro method of any one of claims 1-6, wherein the substrate further comprises a carrier matrix that is a test strip.

9. The in vitro method of claim 8, wherein the test strip comprises: (a) a base membrane in contact with the test strip; (b) a first layer disposed on the base membrane, the first layer comprising a buffer; and (c) a second layer overlaying the first layer, the second layer comprising 1,2-O- dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin).

10. The in vitro method of claim 9, wherein the color change of the substrate from the initial color to red or purple indicates that the subject is a high-lipase producer.

11. The in vitro method of claim 9, wherein the color change of the substrate from the initial color to yellow or orange indicates that a subject is not a high-lipase producer.

12. The in vitro method of claim 8, wherein the test strip comprises: (a) a base membrane in contact with the test strip; (b) a first layer disposed on the base membrane, the first layer comprising a buffer; and (c) a second layer overlaying the first layer, the second layer comprising a β- naphthyl ester, optionally 2-naphthyl caprylate, and a color indicator.

13. The in vitro method of claim 8, wherein the test strip comprises: (a) a base membrane in contact with the test strip; (b) a first layer disposed on the base membrane, the first layer comprising a buffer; and (c) a second layer overlaying the first layer, the second layer comprising indoxyl acetate or 5-bromo-4-chloro-indoxyl caprylate, and a color indicator.Atty. Dkt. No.: 136671-0110 14. The in vitro method of claims 12 or 13, wherein the β-naphthyl ester, indoxyl acetate, or 5-bromo-4-chloro-indoxyl caprylate reacts with the lipase present in the milk sample to provide a compound that reacts with the color indicator to generate the color change.

15. The in vitro method of claim 14, wherein the color indicator comprises a diazonium salt, optionally selected one or more of 4-benzamido-2,5-diethoxybenzene-1-diazonium (Fast Blue BB salt), bis(2-methoxy-4-nitrophenyldiazonium) 1,5- naphthalenedisulfonate (Fast Red B Salt 1,5-naphthalenedisulfonate), 1-diazo-2- naphthol-4-sulfonic acid, and 4-diazo-3-methoxydiphenylamine sulfate.

16. A kit for determining whether a subject is a high-lipase producer of breastmilk, the kit comprising: a substrate comprising 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester, a β-naphthyl ester, indoxyl acetate, or 5-bromo-4-chloro-indoxyl caprylate that generates a color change after contacting a breastmilk sample from a subject, wherein the color change indicates the amount of lipase present in the breastmilk sample.

17. The kit of claim 16, wherein the amount of lipase present in the breastmilk sample is greater than about 0 U / L, optionally at least about 1.0 U / L.

18. The kit of claim 17, wherein the amount of lipase present in the breastmilk sample is from about 0 U / L to about 5,000 U / L, optionally from about 0.1 U / L to about 5,000 U / L.

19. The kit of claim 18, wherein the subject is a high lipase producer of breastmilk when the amount of lipase present in the milk sample is from about 100 U / L to about 5,000 U / L, optionally about 300 U / L to about 1,000 U / L.

20. The kit of any one of claims 16-19, wherein the substrate further comprises a carrier matrix that is a bibulous material, optionally a cellulosic filter paper.Atty. Dkt. No.: 136671-0110 21. The kit of any one of claims 16-19, wherein the substrate further comprises a carrier matrix that is a test strip.

22. The kit of claim 21, wherein the test strip comprises: (a) a base membrane in contact with the test strip; (b) a first layer disposed on the base membrane, the first layer comprising a buffer; and (c) a second layer overlaying the first layer, the second layer comprising 1,2-O- dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester, a β-naphthyl ester, indoxyl acetate, or 5-bromo-4-chloro-indoxyl caprylate, and optionally a color indicator.

23. The kit of claim 22, wherein the buffer comprises one or more of Tris buffer, a borate buffer, Bicine buffer and TAPS (N-tris(hydroxymethyl)methyl-3- aminopropanesulfonic acid) buffer, optionally the buffer comprises a borate buffer.

24. The kit of any one of claims 22-23, wherein the first layer further comprises one or more of a lipase accelerator a polymer binder, a surfactant, a stabilizer, a reaction accelerator, and a buffer.

25. The kit of any one of claims 22-24, wherein the β-naphthyl ester, indoxyl acetate, or 5- bromo-4-chloro-indoxyl caprylate reacts with the lipase present in the milk sample to provide a compound that reacts with a color indicator to generate the color change.

26. The kit of claim 25, wherein the color indicator comprises a diazonium salt, optionally selected from one or more of 4-benzamido-2,5-diethoxybenzene-1-diazonium (Fast Blue BB salt), bis(2-methoxy-4-nitrophenyldiazonium) 1,5-naphthalenedisulfonate (Fast Red B Salt 1,5-naphthalenedisulfonate), 1-diazo-2-naphthol-4-sulfonic acid, and 4-diazo-3-methoxydiphenylamine sulfate.Atty. Dkt. No.: 136671-0110 27. The kit of any one of claims 22-26, wherein the second layer further comprises one or more of an organic acid, a stabilizer, a reaction accelerator, and a buffer.

28. The kit of claim 27, wherein the reaction accelerator comprises one or more of a ferrocyanide and a ferricyanide.

29. The kit of any one of claims 22-28, wherein the test strip comprises: (a) a base membrane in contact with the test strip; (b) a first layer disposed on the base membrane, the first layer comprising a buffer; and (c) a second layer overlaying the first layer, the second layer comprising 1,2-O- dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester.

30. The kit of claim 29, wherein one or more of: (a) the buffer comprises tris buffer; (b) the first layer further comprises t one or more of bile salts and calcium chloride; and (c) the second layer further comprising an organic acid and a stabilizer.

31. The kit of any one of claims 22-28, wherein the test strip comprises: (a) a base membrane in contact with the test strip; (b) a first layer disposed on the base membrane, the first layer comprising a buffer; and (c) a second layer overlaying the first layer, the second layer comprising the β- naphthyl ester, optionally 2-naphthyl caprylate and a color indicator.

32. The kit of claim 31, wherein one or more of: (a) the buffer comprises a borate buffer, optionally sodium borate buffer; (b) the first layer further comprises polyvinylpyrrolidone;Atty. Dkt. No.: 136671-0110 (c) the color indicator comprises a diazonium salt, optionally 4-benzamido-2,5- diethoxybenzene-1-diazonium (Fast Blue BB salt) or 1-diazo-2-naphthol-4-sulfonic acid; and (d) optionally, the second layer further comprises a stabilizer.

33. The kit of any one of claims 22-28, wherein the test strip comprises: (a) a base membrane in contact with the test strip; (b) a first layer disposed on the base membrane, the first layer comprising a buffer; and (c) a second layer overlaying the first layer, the second layer comprising indoxyl acetate or 5-bromo-4-chloro-indoxyl caprylate, optionally indoxyl acetate, and a color indicator.

34. The kit of claim 33, wherein one or more of: (a) the buffer comprises a borate buffer, optionally sodium borate buffer; (b) the first layer further comprises a reaction accelerator, optionally a ferrocyanide, further optionally potassium ferrocyanide; (c) the color indicator comprises a diazonium salt, optionally diazo-3- methoxydiphenylamine sulfate; and (d) the second layer further comprises a stabilizer.

35. The kit of claims 33 or 34, wherein the second layer comprises indoxyl acetate and the indoxyl acetate and the color indicator are present in a weight ratio of from about 20:1 to about 5:1, optionally a weight ratio of about 10:

1.

36. An in vitro method for determining whether a breastmilk sample has become rancid, the method comprising: contacting the breastmilk sample with a substrate comprising an enzyme mixture that measures the amount of glycerol present in the breastmilk by generating a color change after a given period of time,Atty. Dkt. No.: 136671-0110 wherein the color change indicates the amount of glycerol present in the breastmilk sample.

37. The in vitro method claim 36, wherein the method further comprises: comparing the color change of the substrate to a first color change, which is a color change of a control sample comprising no glycerol, and a second color change, which is a color change of a sample comprising an amount of glycerol sufficient to indicate that the breastmilk sample has become rancid, wherein the color change of the substrate to an intermediate color between (i) a color resulting from the first color change and (ii) a color resulting from the second color change of the sample indicates that the breastmilk sample is not rancid.

38. The in vitro method of claims 36 or 37, wherein the enzyme mixture comprises glycerol kinase, glycerol phosphate oxidase, and a peroxidase.

39. The in vitro method of any one of claims 36-38, wherein the enzyme mixture further comprises adenosine triphosphate (ATP), 4-aminoantipyrine (4-AAP) and an indicator.

40. The in vitro method of claim 39, wherein the indicator is N-ethyl-N-(3-sulfopropyl)-m- anisidine, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(2- hydroxy-3-sulfopropyl)-3,5-dimethylaniline, 3-(N-ethyl-3-methylanilino)-2-hydroxy- propanesulfonic acid, N,N-bis(4-sulfobutyl)-3,5-dimethylaniline, or a salt thereof, optionally the sodium salt of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5- dimethoxyaniline.

41. The in vitro method of any one of claims 36-40, wherein the period of time is at least about 2 minutes.

42. The in vitro method of claim 41, wherein the period of time is from about 2 minutes to about 30 minutes, optionally from about 2 minutes to about 5 minutes.Atty. Dkt. No.: 136671-0110 43. The in vitro method of any one of claims 36-42, wherein the amount of glycerol to indicate that the breastmilk sample has become rancid is less than about 100 mg / L, wherein the breastmilk sample is fresh.

44. The in vitro method of claim 43, wherein the amount of glycerol to indicate that the breastmilk sample has become rancid is from about 2.5 mg / L to about 100 mg / L.

45. The in vitro method of any one of claims 36-42, wherein the amount of glycerol to indicate that the breastmilk sample has become rancid is greater than about 1000 mg / L, wherein the breastmilk sample has been stored in a household refrigerator or freezer for a period of from about 3 days to about 12 months.

46. The in vitro method of any one of claims 36-42, wherein the amount of glycerol to indicate that the breastmilk sample has become rancid is from about 101 mg / L to about 1000 mg / L, wherein the breastmilk sample has been stored in a household refrigerator or freezer for a period of from about 3 days to about 12 months.

47. The in vitro method of any one of claims 36-46, wherein the substrate further comprises a carrier matrix that is a test strip.

48. The in vitro method of claim 47, wherein the enzyme mixture further comprises one or more of a polymer binder, a surfactant, a buffer, a stabilizing sugar, an enzyme cofactor, and a color indicator.

49. The in vitro method of claim 47, wherein the test strip comprises: (a) a base membrane in contact with the test strip; and (b) one or more layers disposed on the base membrane, the one or more layers comprising 4-aminoantipyrine (4-AAP), glycerol kinase, glycerol phosphate oxidase, a peroxidase, and a color indicator.Atty. Dkt. No.: 136671-0110 50. The in vitro method of claim 49, wherein the one or more layers may further comprise one or more of a polymer binder, a surfactant, a buffer, a stabilizing sugar, and an enzyme cofactor.

51. A kit for determining whether a breastmilk sample has become rancid comprising: a substrate comprising an enzyme mixture that measures the amount of glycerol present in the breastmilk sample by generating a color change after contacting a breastmilk sample from a subject.

52. The kit of claim 51, wherein the enzyme mixture comprises glycerol kinase, glycerol phosphate oxidase, and a peroxidase.

53. The kit of claims 51 or 52, wherein the enzyme mixture further comprises adenosine triphosphate (ATP), 4-aminoantipyrine (4-AAP), and an indicator.

54. The kit of claim 53, wherein the indicator is N-ethyl-N-(3-sulfopropyl)-m-anisidine), N- ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(2-hydroxy-3- sulfopropyl)-3,5-dimethylaniline, 3-(N-Ethyl-3-methylanilino)-2-hydroxy- propanesulfonic acid, N,N-bis(4-sulfobutyl)-3,5-dimethylaniline, or a salt thereof, optionally the sodium salt of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5- dimethoxyaniline.

55. The kit of any one of claims 51-54, wherein the color change indicates the amount of glycerol present in the breastmilk sample.

56. The kit of any one of claims 51-55, wherein the substrate further comprises a carrier matrix that is a test strip.

57. The kit of claim 56, wherein the enzyme mixture further comprises one or more of a polymer binder, a surfactant, a buffer, a stabilizing sugar, an enzyme cofactor, and a color indicator.Atty. Dkt. No.: 136671-0110 58. The kit of claim 56, wherein the test strip comprises: (a) a base membrane in contact with the test strip; and (b) one or more layers disposed on the base membrane, the one or more layers comprising 4-aminoantipyrine (4-AAP), glycerol kinase, glycerol phosphate oxidase, a peroxidase, and a color indicator.

59. The kit of claim 58, wherein the one or more layers further comprises one or more of a polymer binder, a surfactant, a buffer, a stabilizing sugar, and an enzyme cofactor.

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