WO2022087331A1

WO2022087331A1 - A method for measuring concentration of human milk oligosaccharides (hmo) molecules using glocometer

Info

Publication number: WO2022087331A1
Application number: PCT/US2021/056140
Authority: WO
Inventors: Kamal Ahmadi MAHMOUD
Original assignee: BASF SE
Current assignee: BASF SE
Priority date: 2020-10-22
Filing date: 2021-10-22
Publication date: 2022-04-28
Anticipated expiration: 2023-04-22

Abstract

The present invention is concerned with detection and/or quantification methods for the detection and/or quantification of one or more human milk oligosaccharides. The invention furthermore allows to correct for the presence of glucose in an analyte. The invention is also concerned with materials and devices for use in such detection and/or quantification, and in the processing of results of such detections and/or quantifications, in particular for product quality monitoring.

Description

A METHOD FOR MEASURING CONCENTRATION OF HUMAN MILK OLIGOSACCHARIDES (HMO) MOLECULES USING GLOCOMETER

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Patent Application No. 63/104,429, filed October 22, 2020.

TECHNICAL FIELD

The present invention is concerned with detection and/or quantification methods for the detection and/or quantification of one or more human milk oligosaccharides. The invention furthermore allows to correct for the presence of monosaccharides, e.g. glucose and/or lactose in an analyte. The invention is also concerned with materials and devices for use in such detection and/or quantification, and in the processing of results of such detections and/or quantifications, in particular for product quality monitoring.

BACKGROUND OF THE INVENTION

Human milk oligosaccharides (HMOs) are considered valuable nutrients for infants and older human beings. They are associated with a variety of physiological benefits, in particular prebiotic effects manifesting in the development or reconstitution of a beneficial gut microflora, immune system resilience and in cognitive and neural development. Notable HMOs include, for example, 2'-fucosyllactose, 3-fucosyllactose, lacto-N-triaose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-hexaose, lacto-N-neohexaose, lacto-N- fucopentaose, lacto-N-neofucopentaose, lacto-N-neooctaose, lacto-N-fucopentaose, lacto- N-difucohexaose, lacto-N-neodifucohexaose, 3'-sialyllactose, 6'-sialyllactose, 3'- galactosyllactose, 6'-galactosyllactose, lactodifucotetraose, 2',3-difucosyllactose, 3'-sialyl- 3-fucosyllactose and sialyl-lacto-N-tetraose.

For development, production and sales of products comprising one or more HMOs (herein: "HMO product") reliable and fast determination and quantification of HMOs is required. Analysis of complex sugars is notoriously difficult. This leads to grossly differing quantification results depending on the chosen method and further constituents of the sampled analyte. For an overview see van Leeuwen, Nutrients 2019, 2684 et seq.

SUBSTITUTE SHEET (RULE 26) Most accepted methods rely on HPLC and LC/MS to detect and/or quantify HMOs. These methods are, however, time consuming and expensive. They are thus only of limited applicability. In particular they are unsuitable to perform production monitoring due to the delay between sampling and result output. Furthermore, such methods are also unsuitable for any application in the field where the required specific and expensive laboratory equipment is not readily available.

There is thus a need to provide improved devices and methods for the detection and/or quantification of HMOS. The methods should avoid the need for expensive laboratory equipment, they should be fast enough for process monitoring, performing the methods should not be expensive and accuracy should be acceptable, in particular for process monitoring and quality control purposes.

SUMMARY OF THE INVENTION

The invention accordingly provides a human milk oligosaccharide (HMO) detection and/or quantification method, comprising the steps: i) obtaining a first monosaccharide dependent signal of a sample of an analyte, ii) hydrolysing glycosidic bonds, if present, in the same or a further sample of the analyte, and iii) measuring a monosaccharide dependent signal in the sample obtained in step ii).

The invention also provides a human milk oligosaccharide (HMO) test pad, comprising a test area containing reagents to create a monosaccharide dependent signal, preferably an optical or electrical signal, further comprising one or more reagents for hydrolysing glycosidic bonds, preferably one or more enzymes, even more preferably one or more enzymes selected for releasing glucose from an HMO molecule, preferably selected from

SUBSTITUTE SHEET (RULE 26) The invention also provides a human milk oligosaccharide (HMO) detection and/or quantification device, comprising an HMO test pad receptacle for recording a first and/or second monosaccharide dependent signal when the receptacle has received an HMO test pad, a computer system for determining the concentration or presence of one or more human milk oligosaccharides on the test pad in view of at least the second monosaccharide dependent signal, preferably in view of the first and second monosaccharide dependent signal.

And the invention provides a method of product quality control of a human milk oligosaccharide containing product (HMO product), comprising the steps of i) obtaining specification data for an HMO product lot, wherein the specification data comprise expected values and/or expected variability ranges for a first and/or second monosaccharide dependent signal performed on a sample of the HMO product lot and/or the concentration or presence of one or more human milk oligosaccharides in a sample of the HMO product lot, ii) providing said specification data together with a packaging unit of the HMO product lot, preferably by machine readable recording on the packaging or package insert of the packaging insert and/or in an electronic database accessible by recipients of the packaging unit.

Further details and benefits of the invention will be apparent to the skilled person, in particular in view of the detailed description, figures and examples.

DESCRIPTION OF FIGURES

Figure 1 A-D shows glucometric calibration curves for four HMOs. Tested solutions were made by adding 2 pl of HMO molecules with different concentration to a total 10 pl reaction.

SUBSTITUTE SHEET (RULE 26) Figure 2 shows a comparison of LNT and LNT II contents of fermentation samples wherein HMO content had been measured by HPLC and glucometrically. The fermentation samples contained: Free lactose, byproduct LNT II, and the desired LNT. As a result 3 test tubes for each sample were prepared: (1) Test tube 1: sample + lactase, to measure glucose coming from free lactose content, (2) test tube 2: sample + P-N-acetylglucosaminidase + lactase, to measure glucose liberated from (free lactose + LNT II), (3) test tube 3: sample + beta 1-3 galactosidase + P-N-acetylglucosaminidase + lactase, to measure glucose liberated from (free lactose + LNTII + LNT). The content of LNT II is then the difference between test tube 2 and test tube 1, the content of LNT is calculated as the difference between test tube 3 and (test tube 1 + test tube 2).

Figure 3 shows a comparison of 6'SL contents of fermentation samples wherein HMO content had been measured by HPLC and glucometrically. The fermentation samples contain: Free lactose, and the desired 6’SL. As a result two test tubes for glucose determination per sample are used: (1) Test tube 1: sample + lactase, to measure glucose coming from free lactose content, and (2) test tube 2: sample + neuraminidase + lactase, to measure glucose coming from (free lactose + 6’SL). The content of 6’SL is then the difference between test tube 2 and test tube 1.

Figure 4 shows the detection of 2'FL in commercial infant formulas. The formulas contain lactose, so we two test tubes for each formula are used: (1) Test tube 1: sample + lactase, to measure free glucose content, (2) test tube 2: sample + lactase + fructosidase, to measure potential glucose coming from 2’F1.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a human milk oligosaccharide (HMO) detection and/or quantification method. Within the context of the present invention, the term "detection" means the act of ascertaining the presence of absence of one or more HMOs, and the term "quantification" means the act of determining the concentration of one or more HMOs in an analyte sample.

SUBSTITUTE SHEET (RULE 26) According to the invention, any type of analyte can be examined. Preferably the analyte is a liquid, even more preferably a water-based liquid, even more preferably a solution in water. When the material to be examined is not a liquid, then preferably a sample of the material is dissolved in water. For example, according to the invention a sample of defined weight and/or volume of a solid, e.g. a powder, is dissolved in a defined quantity of water to obtain an analyte sample of defined concentration of the material to be examined.

More preferably the analyte is a liquid consisting essentially of water, optionally one or more HMOs and optionally further sugars. It is particularly preferred that the content of components other than water, HMOs and other sugars is at most 50% by weight of the sample, even more preferably at most 40 by weight of the sample, even more preferably at most 30% by weight of the sample, even more preferably at most 20% by weight of the sample, even more preferably at most 10% by weight of the sample, even more preferably at most 5% by weight of the sample, even more preferably at most 3% by weight of the sample, even more preferably at most 1% by weight of the sample. According to the invention it is particularly preferred that the analyte does not prevent the detection and/or quantification of HMOs, and thus preferably does not contain a disruptive concentration of substances that inhibits enzyme activity such as high salt concentration, uric acid, surfactants, protein denaturing agents, acetaminophen (paracetamol), acetylsalicylic acid,, ascorbic acid, DMSO, dopamine, maltose, mannitol and proteases, which prevent or substantially delay the hydrolysis of HMO glycosidic bonds or the generation of a signal based on the detection and/or quantification of free sugars, preferably glucose and/or galactose and most preferably glucose.

Preferably the analyte is an HMO product or, even more preferably, a defined quantity of an HMO product dissolved in a defined quantity of water. A preferred HMO product is a pure HMO, a mixture of an HMO and a non-HMO sugar, e.g. saccharose a mixture of two or more HMOs, a mixture of two HMO and a non-HMO sugar, e.g. saccharose a fermentation broth in heterologous production of HMO molecules a reaction mixture in synthetic production of HMO molecules a commercial HMO-containing formula or supplement,

SUBSTITUTE SHEET (RULE 26) and optionally further comprises one or more components selected from sweeteners, aromas, colorants, anti-caking agents, salts, amino acids, nucleic acids, lipids and fatty acids.

The method of the invention entails generating a monosaccharide dependent signal. It is a particular advantage of the present invention that any method for generating such signal can be employed. The monosaccharide signal according to the present invention preferably is a glucose and/or a galactose dependent signal, more preferably a glucose dependent signal. According to the invention, all pairs of monosaccharide dependent signals are dependent on the same monosaccharide, preferably glucose and/or galactose, and most preferably glucose. This way the second monosaccharide dependent signal can be corrected by the first monosaccharide dependent signal, as described herein. The detection and/or quantification of free glucose is long established in everyday laboratory practice, it can be performed with high accuracy in a diverse set of media and at low cost. Furthermore, colorimetric tests for free galactose are also commercially available. Thus, the generation of a monosaccharide dependent signal instead of an HMO dependent signal offers the aforementioned advantages and beneficially facilitates HMO detection and/or quantification compared to HPLC and/or LC/MS-based techniques without significant losses in accuracy. Due to the widespread use of glucose detection and/or quantification materials and methods, e.g. blood glucose measuring kits and devices, whenever the term ■■monosaccharide dependent signal” is used in view of the present invention, the term preferably is understood to mean “glucose dependent signal”.

The signal can be any kind of measurable quantity translating the presence and/or concentration of free glucose in a sample. In particular the signal can be an optical signal and/or an electrical signal. Preferred optical signals are a change in luminescence intensity, fluorescence intensity, colour hue, colour intensity and/or colour saturation, or the change in location of such colour signal. Non-limiting examples of optical signals are the darkening and/or hue change of an indicator patch, the movement of coloured microbeads and surface plasmon resonance. Preferred electrical signals are a change in voltage, cunent, phase and/or frequency. Non-limiting examples of electrical signals are signals generated

SUBSTITUTE SHEET (RULE 26) by potentiometry, field effect transistors, impedance spectroscopy and enzymatic electron transfer.

The signal can be an endpoint signal or a rate signal. According to the invention, an endpoint signal is a signal whose value is read after a predetermined time or at signal constancy, preferably once the signal value changes by at most 5% per minute, more preferably by at most 3% per minute, more preferably by at most 1% per minute, more preferably by at most 0.8% per minute. Furthermore according to the invention a rate signal is the first derivative of a base signal. For example, a when at a specific point in time a base signal (e.g. colour intensify) changes by 1% per minute, then the endpoint signal would be the actual value of the base signal at that point in time and the rate signal would be the slope of the base signal, i.e. "1% per minute". Preferred rate signals are maximum rate, duration to maximum rate and duration to signal constancy.

Preferably the signal is smoothed. Preferred methods of soothing an endpoint or rate signal is (1) by calculation of the simple moving average of at least 3 signal values, more preferably at least 4 values, more preferably at least 5 values, more preferably at least 10 values, (2) by calculation of the median of at least 3 signal values, more preferably at least 4 values, more preferably at least 5 values, more preferably at least 10 values, (3) by calculation of an exponentially moving average, preferably having a decay factor alpha of at most 0.5, more preferably at most 0.4, more preferably at most 1/3, more preferably at most 0. 182, (3) Kalman filtering.

According to the invention a first monosaccharide dependent signal is obtained of a sample of an analyte and a second monosaccharide dependent signal in a treated sample of the analyte as described below. It is a particular advantage of the present invention that the time between obtaining the first and second monosaccharide dependent signal is generally of no concern. The time is of no concern in particular where the concentration of free glucose in the analyte does not change between obtaining of the first and second monosaccharide dependent signal, or where the change of free glucose concentration in the analyte in the period between obtaining of the first and second monosaccharide dependent signal can be predicted with sufficient accuracy. Thus, for non-decaying products the

SUBSTITUTE SHEET (RULE 26) invention allows to obtain the first monosaccharide dependent signal from one sample of an analyte, e.g. a product containing one or more HMOs, and to compare this signal with several second monosaccharide dependent signals, e.g. second monosaccharide dependent signals obtained by various customers which analyse their respective package of the product batch. As described above, the second monosaccharide dependent signals are dependent on the same monosaccharide, preferably glucose and/or galactose and most preferably glucose. Thus, the method of the present invention particularly lends itself to fast, reliable and easy product quality control and customer satisfaction applications.

Accordingly, in the method of the present invention preferably a) a sample of the analyte is provided, and step i) is performed on the sample and steps ii) to iii) are performed on the sample after step i) has been started or preferably completed, or b) two samples of the analyte are provided, wherein on a first sample step i) is performed and on the second sample steps ii) and iii) are performed.

Variant a) is particularly suitable to those analytes wherein the concentration of free glucose can vary significantly during the lifetime of the analyte. For example, in liquid HMO products decay of HMOs is frequently observed. Thus, the content of free monosaccharide, e.g. glucose, in such products increases during the lifetime of the product. When the delay between generation of the first and second monosaccharide dependent signal is long, then in such situations the difference between first and second monosaccharide dependent signal does not reflect the initial HMO concentration in the product. Thus, by analysing one sample to obtain a first monosaccharide dependent signal and a second monosaccharide dependent signal preferably shortly after completion of step i), preferably within 60 minutes after completion of step i), more preferably within 30 minutes after completion of step i), more preferably within 15 minutes after completion of step i), more preferably within 10 minutes after completion of step i), more preferably within 5 minutes after completion of step i), more preferably within 2 minutes after completion of step i), more preferably immediately after completion of step i), the discrepancy between real HMO content and the content inferred by comparison of the first and second monosaccharide dependent signal, is kept low.

SUBSTITUTE SHEET (RULE 26) Variant b) is particularly suitable for non-decaying analytes as described above.

The second monosaccharide dependent signal is obtained from a sample of the analyte in which glycosidic bonds have been hydrolysed. The sample for obtaining the second monosaccharide dependent signal may be the sample of step i). However, preferably the sample for obtaining the second monosaccharide dependent signal is another sample of the analyte. As indicated above, generating the second monosaccharide dependent signal from a further sample of the analyte is particularly beneficial in circumstances wherein the development of free glucose concentration is known, can be reasonably predicted or wherein the concentration of free glucose is not expected to change between obtaining the first and the further sample of the analyte. By not requiring two samples to be taken from an analyte for each analysis, e.g. a product comprising one or more HMOs, repeated analysis is expedited, thereby contributing to the advantageously fast, reliable and easy application of the method of the present invention for detection and/or quantification of HMO.

Hydrolysis of glycosidic bonds in step ii) preferably comprises or consists of enzymatic hydrolysis and/or, less preferably, acid hydrolysis. An advantage of the latter method is that acids useful for such hydrolysis are generally very stable as such under standard industrial operation conditions, like hydrochloric acid. However, preferably hydrolysis of glycosidic bonds in step ii) comprises or consists of enzymatic hydrolysis. The intention of the hydrolysis step is to liberate any further monosaccharide, e.g. glucose, moiety from at least one HMO to render it detectable for the generation of the second monosaccharide dependent signal, such that the difference in free monosacchande (e.g. glucose) and liberated monosaccharide (again e.g. glucose) concentration leads to a detectable difference between the first and second monosaccharide dependent signal. This difference is influenced by the type of hydrolysed HMO (number of liberated glucose moieties per HMO molecule) and by the concentration of the hydrolysed HMO. When nothing is known about the sample in advance, then the difference is indicative of the total of HMOs hydrolysable under the selected hydrolysis conditions, preferably the one or more enzymes used for hydrolysis. When the hydrolysed HMO is known in advance, then the signal difference immediately reflects the concentration of hydrolysed HMO. This is generally the case for fermentatively obtained HMO products. Thus, the method of the present invention

SUBSTITUTE SHEET (RULE 26) allows to obtain a fast and reliable detection and/or quantification of HMOs advantageously for the economically most relevant instances of HMO detection/quantification needs, i.e. the analysis of HMO fermentation processes and the analysis of products comprising one or more fermentatively obtained HMOs.

Accordingly the invention provides a human milk oligosaccharide (HMO) detection and/or quantification method, comprising the steps: i) obtaining a first monosaccharide dependent signal of a sample of an analyte, ii) enzy matically hydrolysing glycosidic bonds, if present, in the same or a further sample of the analyte, and iii) measuring a second monosaccharide dependent signal in the sample obtained in step ii), and iv) repeating steps ii) and iii) sequentially or in parallel, wherein the enzymatic hydrolysis in each repetition is adapted to hydrolyse at least one other HMO species.

As described above it is an advantage of enzy matic hydrolysis in the method according to the present invention that enzymatic action is specific for the respective enzymes' substrates. Different enzymes or enzyme combinations will lead to a difference (1) in the species of hydrolysable HMOs - because some HMOs may not be hydrolysable with a given enzyme or enzyme combination -, and/or (2) in the speed of HMO hydrolysis and thus in the development of the respective second monosaccharide dependent signal. Thus, by analysing, sequentially or, preferably, in parallel, several samples of the analyte the invention allows to differentiate between the concentrations of various HMOs even though only a single parameter, i.e. glucose, is used for generating the second monosaccharide dependent signals.

Enzymatic hydrolysis of glycosidic bonds according to the invention preferably comprises or consists of exposing the analyte sample to one or more enzymes for releasing glucose from an HMO molecule, preferably selected from:

SUBSTITUTE SHEET (RULE 26)

The aforementioned enzyme combinations are particularly adapted to release glucose from the respective HMO molecule. The enzymes of the aforementioned combinations, in particular lactase, are well known to the skilled person. The enzymes indicated above are well studied and commercially available in sufficient quantities and purity.

Preferably one or more reagents for hydrolysing glycosidic bonds in step ii), more preferably one or more, preferably all, enzymes for releasing glucose from an HMO molecule, is provided to the sample by delayed release, preferably by dissolving an encapsulation of said one or more reagents or by release of a factor to facilitate the glycosidic bond hydrolysing effect of the one or more reagents. The skilled person is aware of various methods of enzyme encapsulation or binding of enzymes to a earner such that the encapsulated enzymes or the enzymes bound to a carrier are released at will, preferably by contact with a water-based analyte sample. It is a particular advantage that the method of the present invention is not dependent on a particular type of release delay and in particular is not dependent on specific encapsulants or carriers. Thus, the second monosaccharide dependent signal can be created in the same location and using the same signal generation means as for the first monosaccharide dependent signal. This is particularly advantageous when the method is performed according to the above variant a). It is particularly preferred that step iii), more preferably steps ii) and iii), even more preferably steps i) to iii), are performed on a single or limited use glucose test device, preferably a blood glucose test pad. Measurement of glucose concentrations in complex samples has been established in blood glucose applications reliably and at small costs compared to HPLC-based methods, corresponding devices and reagents are well established. Non-limiting examples of materials and devices for blood glucose detection and corresponding methods for generation of a monosaccharide dependent signal, in

SUBSTITUTE SHEET (RULE 26) particular glucose dependent signal, are described in each of WO2012028281, W02017087051, WO2017087916, W02020036906, W02020049561, W02020086934 and W02020086994.

Even more preferably at least the second monosaccharide dependent signal is generated on a single use glucose test device, preferably a blood glucose test pad; the first monosaccharide dependent signal is then also a glucose dependent signal and is preferably also obtained using a single use glucose test device. Such devices are produced as staple goods with consistent glucose detection characteristics. They can be delivered with or attached to HMO product containers and used days or weeks after the first monosaccharide dependent signal has been created. Most preferably both the first and the second monosaccharide dependent signal is obtained on a single use glucose test device, preferably a blood glucose test pad, wherein preferably a first single use glucose test device, preferably a first blood glucose test pad, is used to obtain the first monosaccharide dependent signal and a second single use glucose test device, preferably a second blood glucose test pad, is used to obtain the second monosaccharide dependent signal. When steps ii) and iii) are repeated as described above, for example to perform different hydrolysis reactions in parallel or sequentially, the appropriate number of second single use glucose test devices - preferably blood glucose test pads - are employed. Preferred test pads for generation of the first and/or second monosaccharide dependent signal are described herein.

Accordingly the invention furthermore provides a human milk oligosaccharide (HMO) test pad, comprising a test area containing reagents to create a monosaccharide dependent signal, preferably an optical or electrical signal, further comprising one or more reagents for hydrolysing glycosidic bonds, preferably one or more enzymes, even more preferably one or more enzymes selected for releasing glucose from an HMO molecule, preferably selected from

SUBSTITUTE SHEET (RULE 26)

Such test pads are particularly adapted to materialise the aforementioned advantages of the method of the present invention.

As described above preferably one or more reagents for hydrolysing glycosidic bonds is provided on the test pad according to the present invention in a delayed release form, preferably by encapsulation in a matrix or shell dissolvable by the analyte.

The invention also provides a human milk oligosaccharide (HMO) detection and/or quantification device. The device comprises an HMO test pad receptacle for recording a first and/or second monosaccharide dependent signal when the receptacle has received an HMO test pad. According to the invention, an HMO test pad is an object comprising all reagents and structural components, e.g. electrical conductors, for creating a monosaccharide dependent signal. The detection and/or quantification device according to the present invention thus reads the respective first and/or second monosaccharide dependent signal from an HMO test pad. The test pad can be inserted partially or completely into the device. This is particularly advantageous when the monosaccharide dependent signal is relayed electrically to the device, such that appropriate electric connections are formed between the test pad and the device. The test pad can also be brought into defined proximity to the device. This is particularly suitable when the respective first and/or second monosaccharide dependent signal is an optical signal or otherwise relayed to the device in a contact-free way. However, the test pad can also be inserted partially or completely into the device to have the monosaccharide dependent signal read out optically or otherwise in a contact-free way. In such cases partial or, if applicable, complete insertion of the test pad serves to appropriately align any reader component of the device with the signal displaying section, preferably the test area, of the test pad.

SUBSTITUTE SHEET (RULE 26) The HMO test pad according to the present invention preferably comprises two or more test areas, wherein a first test area is adapted for generating a first monosaccharide dependent signal, and the further test area(s) is/are adapted for generation of the second monosaccharide dependent signal(s), preferably by providing a different composition of one or more enzymes selected for releasing glucose from an HMO molecule. The analyte is preferably transferred to the test areas by suction or, even more preferably, by capillary action. Thus, the test pad only needs to be exposed to a liquid analyte once for obtaining several samples thereof and distribution of the samples to the test areas. When the HMO test pad comprises two or more test areas, the HMO detection and/or quantification device of the present invention preferably comprises a corresponding number of signal reading components. However, when the monosaccharide dependent signal generated at two or more test areas is an electrical signal, then the test pad may comprise a computing component to combine the electrical signals into a combined signal, e.g. a telegram of ordered signals or a telegram comprising records composed of test area name and test area signal. The combined signal can be deconvoluted by the detection and/or quantification device, e.g. according to the order of signals or in view of the name attached to each test area signal.

The human milk oligosaccharide (HMO) detection and/or quantification device furthermore comprises a computer system for determining the concentration or presence of one or more human milk oligosaccharides on the test pad in view of at least the second monosaccharide dependent signal, preferably in view of the first and second monosaccharide dependent signal. Thus, the detection and/or quantification device is particularly adapted to materialise the advantages described above. In particular, when the test pad is based on blood glucose test pads, then the device can be based on state of the art blood glucose readers. Such devices are thus particularly robust and trustworthy in view of the well-established materials and methods for generating and obtaining the monosaccharide dependent signals. Furthermore, the incorporation of enzyme compositions for HMO hydrolysis as described above does not require a complete change materials and techniques of monosaccharide dependent signal generation; the enzyme compositions can instead be incorporated into such test pads reliably and easily.

SUBSTITUTE SHEET (RULE 26) The human milk oligosaccharide (HMO) detection and/or quantification device preferably also comprises a database for storing calibration data for calculating the concentration or deciding on the presence of one or more human milk oligosaccharides on the test pad in view of the second and/or first and second monosaccharide dependent signal. The database is preferably adapted to the respective enzyme composition for hydrolysis of glycosidic bonds and/or to accommodate for influences exerted by further components of the analy te on the generation of the first and/or second monosaccharide dependent signal. Advantageously such database can be prepared in advance and copied to several HMO detection and/or quantification devices. Thus, the devices are particularly suitable for detection and/or quantification of one or more HMOs in separate packages of a common analyte, preferably a batch of an HMO product.

Preferably the HMO detection and/or quantification device also comprises transmission means, preferably wireless transmission means, for communicating the recorded first and/or second monosaccharide dependent signal and/or the result of the determination of the concentration or presence of the one or more human milk oligosaccharides to a central computer system. Such devices are particularly well adapted for quality monitoring and control applications. For example the device may be connected to a fermenter for monitoring the fermentative production of one or more HMOs; in this case the central computer system obtains information about the progress of the fermentation process and can direct changes to the fermentation, e.g. addition of nutrients, to maintain optimal HMO fermentation conditions. Another example are settings wherein an HMO product is packaged for distribution and individual packages are analysed before sending them out to customers or at the respective recipients thereof. Analysis of HMO product packages before distribution allows to maintain a consistent product quality; analysis of HMO product packages at the recipient allows to confirm that the package received was not harmed during transport.

The HMO detection and/or quantification device preferably comprises a reader for reading analyte specification data for a first and/or second monosaccharide dependent signal and/or

SUBSTITUTE SHEET (RULE 26) the concentration or presence of one or more human milk oligosaccharides.

Such analyte specification data can be provided remotely, e.g. via communication with an analyte specification server, and/or together with a package of the analyte. Preferably the analyte specification data is a machine-readable tag, e.g. an optical sign and/or an RFID- type tag, preferably attached to the analyte package or delivered together with such packaging, e.g. on a package leaflet. Preferably the HMO test pad is specifically adapted to the respective packaged analyte and comprises a machine-readable analyte identifier, e.g. an identifier of the respective product batch. The machine-readable tag identifies the analyte and is used to obtain the analyte specification data from a central computer system, preferably by remote or wireless access.

Accordingly the present invention also provides a method of product quality control of a human milk oligosaccharide containing product (HMO product), comprising the steps of i) obtaining specification data for an HMO product lot, wherein the specification data comprise expected values and/or expected variability ranges for a first and/or second monosaccharide dependent signal performed on a sample of the HMO product lot and/or the concentration or presence of one or more human milk oligosaccharides in a sample of the HMO product lot, ii) providing said specification data together with a packaging unit of the HMO product lot, preferably by machine readable recording on the packaging or package insert of the packaging insert and/or in an electronic database accessible by recipients of the packaging unit.

The above quality control method allows to materialise the advantages of the present invention. As described above the package unit, package insert or transportation documents can, for example, display at least one machine readable sign. Without limitation, such sign can be an optical sign, e.g. a QR code, bar code or machine readable identifier text (for example in the form of a https link), or the sign can be encoded in a radio readable signal, for example an active or passive RFID tag. The information relayed by the sign can be the analyte specification data, as described above, and/or an access to an electronic database comprising the analyte specification data relevant for the package or product lot to which

SUBSTITUTE SHEET (RULE 26) the package belongs. The access preferably is a database record identifier which, when submitted to the electronic database, yields the relevant analyte specification data. Likewise, the access can be provided as a direct machine-translatable access code, e.g. a https link, to immediately access the corresponding analyte specification data record without having to enter the record identifier into a database search.

Further aspects of the invention are hereinafter further described by way of illustrative examples. The examples are not intended to limit the scope of the present invention.

EXAMPLES

LNT, LNTII, and 6-SL molecules were obtained from Glycom. 2’FL was a BASF product. We used a CVS Health Advanced Glucose Meter and test strips in this work. Enzymes listed in the tablel were purchased from New England Biolabs (NEB) and Sigma Aldrich. Table 1: Correspondence table of HMO to be detected and enzymes needed for hydrolysis

HPLC quantification of HMO molecules

HMO molecules were analysed as described in Anne Stovlbaek Christensen et al, J Food Sci. 2020 Feb; 85(2): 332-339, doi: 10.1111/1750-3841.15005.

Using a glucose meter for HMO quantification

10 mg/ml HMO molecules stock solutions were prepared by dissolving 10 mg of each molecule in tablel in 1 ml of deionized water Stock with lower concertation were then made by serial dilution of the initial 10 mg/ml stock. The glucometric calibration curves for HMOs molecules (figure 1) were constructed by mixing 2ul of HMO stock solutions, lul of 8 mg/ml lactase, lul of appropriate enzymes (based on table 1), 1 pl of provided buffer,

SUBSTITUTE SHEET (RULE 26) and water to final volume of 10 ul. The mixture is then incubated at 37C for 1 hour. 1 JJ.1 of the reaction was then applied to the test strip using a pipette measure by the glucose meter. The method was then tested to quantify HMO molecules in fermentation broth following the details below.

1) Microbial culture for production of LNT contains: Free lactose, LNT II, and the desired LNT. For each sample we set up three test tubes. Test tube 1 : 2 pl sample + 1 pl of 8 mg/ml lactase, lul buffer and 7 pl water (this will be used to measure glucose coming from free lactose content) Test tube 2: 2 pl sample + 1 pl of 8 mg/ml lactase, lul buffer, 1 pl of P-N-acetylglucosaminidase, and 5ul water (this will be used to measure glucose coming from free lactose + LNT II). Test tube 3: 2 pl sample, 1 pl of 8 mg/ml lactase, lul buffer, lul of beta 1-3 Galactosidase, 1 pl of P-N-acetylglucosaminidase , and 4 pl of water ( to measure glucose from (free lactose+ LNTII + LNT). Tubes were incubated at 37C for 2 hours, and then 2 pl of reaction were measured on glucose meter. Considering glucose and HMO molecular weight we calculated amount of LNT II using the difference between test tube 2 and test tube 1. Amount of LNT was then calculated using the difference between test tube 3 and (test tube 1+ test tube 2). The estimated concentrations were compared to HPLC measurements (Figure 2).

2) Microbial culture for production of 6’SL contains free lactose, and the desired 6’SL. As a result, we use two test tubes to analysis this sample. Test tube 1: 2 pl sample + 1 pl of 8 mg/ml lactase, lul buffer and 7 pl water, (this will be used to measure glucose coming from free lactose content). Test tube 2: 2 pl sample + 1 pl of 8 mg/ml lactase, lul buffer, and lul Neuraminidase (this will be used to measure glucose coming from free lactose + 6’SL). Tubes were incubated at 37C for 2 hours, and then 2 pl of reaction were measured on glucose meter. Considering glucose and HMO molecular weight we calculated amount of 6’SL using the difference between test tube 2 and test tube 1. The estimated concentrations were compared to HPLC measurements (Figure 3).

SUBSTITUTE SHEET (RULE 26) The method was also used to validate presence of 2’F1 HMO molecule in a commercial infant formula. The control sample was another brand that does not contain any HMO molecules. The infant’s formula contains lactose, so we used two test tubes for each formula. Test tube 1: 1 pl sample (10 g/1 of formula) ,1 pl of 8 mg/ml lactase, lul buffer and 8 pl water (this will be used to measure glucose coming from free lactose content). Test tube 2: 1 pl sample (10 g/1 of formula), ,1 pl of 8 mg/ml lactase, 1 pl of Fructosidase, lul buffer and 7 pl water (this will be used to measure potential glucose coming from 2’F1). Tubes were incubated at 37C for 3 hours, and then 2 pl of reaction were measured on glucose meter. Figure 3 shows that there is a significant difference between reads from the two tubes in the formula with 2’FL but not for the formula without the 2’FL (Figure 4).

SUBSTITUTE SHEET (RULE 26)

Claims

1. A human milk oligosaccharide (HMO) detection and/or quantification method, comprising the steps: i) obtaining a first monosaccharide, preferably glucose, dependent signal of a sample of an analyte, ii) hydrolysing glycosidic bonds, if present, in the same or a further sample of the analyte, and iii) measuring a second monosaccharide, preferably glucose, dependent signal in the sample obtained in step ii).

2. The method of claim 1, wherein hydrolysis of glycosidic bonds in step ii) comprises or consists of enzymatic hydrolysis and/or, less preferably, acid hydrolysis.

3. The method of claim 2, wherein enzymatic hydrolysis of glycosidic bonds comprises or consists of exposing the analyte sample to one or more enzymes for releasing glucose from an HMO molecule, preferably selected from

4. The method of any of the previous claims, wherein a) a sample of the analyte is provided, and step i) is performed on the sample and steps ii) to iii) are performed on the sample after step i) has been started or preferably completed, or b) two samples of the analyte are provided, wherein on a first sample step i) is performed and on the second sample steps ii) and iii) are performed.

5. The method according to claim 4, wherein one or more reagents for hydrolysing glycosidic bonds in step ii), preferably one or more enzymes for releasing glucose from an HMO molecule, is provided to the sample by delayed release, preferably by

SUBSTITUTE SHEET (RULE 26) dissolving an encapsulation of said one or more reagents or by release of a factor to facilitate the glycosidic bond hydrolysing effect of the one or more reagents. The method of any of the previous claims, wherein the first and/or second monosaccharide dependent signal is an optical signal or an electronic signal, wherein the optical signal preferably is a change in luminescence, fluorescence, colour hue, colour intensity, colour saturation, or a change in location of such colour signal, and wherein the electronic signal preferably is selected from a change in voltage, current, phase and/or frequency, and wherein the first and/or second monosaccharide dependent signal preferably is an endpoint signal or a rate signal. The method according to any of the previous claims, wherein step iii), more preferably steps ii) and iii), even more preferably steps i) to iii), are performed on a single or limited use glucose test device, preferably a blood glucose test pad. A human milk oligosaccharide (HMO) test pad, comprising a test area containing reagents to create a monosaccharide, preferably glucose, dependent signal, preferably an optical or electrical signal, further comprising one or more reagents for hydrolysing glycosidic bonds, preferably one or more enzymes, even more preferably one or more enzymes selected for releasing glucose from an HMO molecule, preferably selected from

The test pad of claim 8, wherein one or more reagents for hydrolysing glycosidic bonds is provided on the test pad in a delayed release form, preferably by encapsulation in a matrix or shell dissolvable by the analyte.

SUBSTITUTE SHEET (RULE 26) A human milk oligosaccharide (HMO) detection and/or quantification device, comprising an HMO test pad receptacle for recording a first and/or second monosaccharide, preferably glucose, dependent signal when the receptacle has received an HMO test pad, a computer system for determining the concentration or presence of one or more human milk oligosaccharides on the test pad in view of at least the second monosaccharide dependent signal, preferably in view of the first and second monosaccharide dependent signal. The HMO detection and/or quantification device according to claim 10, further comprising a database for storing calibration data for calculating the concentration or deciding on the presence of one or more human milk oligosaccharides on the test pad in view of the second and/or first and second monosaccharide dependent signal. The HMO detection and/or quantification device according to any of claims 10 to 11, further comprising transmission means, preferably wireless transmission means, for communicating the recorded first and/or second monosaccharide dependent signal and/or - the result of the determination of the concentration or presence of the one or more human milk oligosaccharides to a central computer system. The HMO detection and/or quantification device according to any of claims 10 to 12, further comprising a reader for reading analyte specification data for a first and/or second monosaccharide dependent signal and/or the concentration or presence of one or more human milk oligosaccharides. A method of product quality control of a human milk oligosaccharide containing product (HMO product), comprising the steps of i) obtaining specification data for an HMO product lot, wherein the specification data comprise expected values and/or expected variability ranges for

SUBSTITUTE SHEET (RULE 26) - a first and/or second monosaccharide, preferably glucose, dependent signal performed on a sample of the HMO product lot and/or the concentration or presence of one or more human milk oligosaccharides in a sample of the HMO product lot, ii) providing said specification data together with a packaging unit of the HMO product lot, preferably by machine readable recording on the packaging or package insert of the packaging insert and/or in an electronic database accessible by recipients of the packaging unit.

SUBSTITUTE SHEET (RULE 26)