WO2022087331A1 - Procédé de mesure de la concentration de molécules d'oligosaccharides du lait maternel (hmo) à l'aide d'un glocomètre - Google Patents

Procédé de mesure de la concentration de molécules d'oligosaccharides du lait maternel (hmo) à l'aide d'un glocomètre Download PDF

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WO2022087331A1
WO2022087331A1 PCT/US2021/056140 US2021056140W WO2022087331A1 WO 2022087331 A1 WO2022087331 A1 WO 2022087331A1 US 2021056140 W US2021056140 W US 2021056140W WO 2022087331 A1 WO2022087331 A1 WO 2022087331A1
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hmo
monosaccharide
sample
signal
glucose
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Kamal Ahmadi MAHMOUD
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BASF SE
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BASF SE
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
    • A23L2/52—Adding ingredients
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates

Definitions

  • 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.
  • HMOs Human milk oligosaccharides
  • 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, lactodifucotetraos
  • HMO product 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.
  • 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).
  • HMO human milk oligosaccharide
  • 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
  • HMO human milk oligosaccharide
  • 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.
  • HMO human milk oligosaccharide
  • 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.
  • HMO product human milk oligosaccharide containing product
  • 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.
  • FIG. 26 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.
  • test tube 1 sample + lactase, to measure glucose coming from free lactose content
  • test tube 2 sample + P-N-acetylglucosaminidase + lactase, to measure glucose liberated from (free lactose + LNT II)
  • 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.
  • Test tube 1 sample + lactase, to measure glucose coming from free lactose content
  • 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.
  • the invention provides a human milk oligosaccharide (HMO) detection and/or quantification method.
  • HMO human milk oligosaccharide
  • detection means the act of ascertaining the presence of absence of one or more HMOs
  • quantification means the act of determining the concentration of one or more HMOs in an analyte sample.
  • any type of analyte can be examined.
  • the analyte is a liquid, even more preferably a water-based liquid, even more preferably a solution in water.
  • a sample of the material is dissolved in water.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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 signal can be any kind of measurable quantity translating the presence and/or concentration of free glucose in a sample.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the second monosaccharide dependent signals are dependent on the same monosaccharide, preferably glucose and/or galactose and most preferably glucose.
  • the method of the present invention particularly lends itself to fast, reliable and easy product quality control and customer satisfaction applications.
  • 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.
  • concentration of free glucose can vary significantly during the lifetime of the analyte.
  • the content of free monosaccharide, e.g. glucose, in such products increases during the lifetime of the product.
  • 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.
  • 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).
  • the sample for obtaining the second monosaccharide dependent signal is another sample of the analyte.
  • 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.
  • 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.
  • 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.
  • the difference is indicative of the total of HMOs hydrolysable under the selected hydrolysis conditions, preferably the one or more enzymes used for hydrolysis.
  • the signal difference immediately reflects the concentration of hydrolysed HMO. This is generally the case for fermentatively obtained HMO products.
  • 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.
  • 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.
  • HMO human milk oligosaccharide
  • Enzymatic hydrolysis of glycosidic bonds preferably comprises or consists of exposing the analyte sample to one or more enzymes for releasing glucose from an HMO molecule, preferably selected from:
  • 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.
  • 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.
  • 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.
  • 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.
  • SUBSTITUTE SHEET (RULE 26) particular glucose dependent signal, are described in each of WO2012028281, W02017087051, WO2017087916, W02020036906, W02020049561, W02020086934 and W02020086994.
  • 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.
  • a single use glucose test device preferably a blood glucose test pad
  • 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.
  • 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.
  • a single use glucose test device preferably a first blood glucose test pad
  • a second single use glucose test device preferably a second blood glucose test pad
  • 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
  • HMO human milk oligosaccharide
  • test pads are particularly adapted to materialise the aforementioned advantages of the method of the present invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the HMO detection and/or quantification device of the present invention preferably comprises a corresponding number of signal reading components.
  • 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.
  • the detection and/or quantification device is particularly adapted to materialise the advantages described above.
  • the test pad is based on blood glucose test pads
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • HMO product human milk oligosaccharide containing product
  • the package unit, package insert or transportation documents can, for example, display at least one machine readable sign.
  • 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
  • the access preferably is a database record identifier which, when submitted to the electronic database, yields the relevant analyte specification data.
  • 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.
  • 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
  • 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.
  • 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 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,
  • Microbial culture for production of LNT contains: Free lactose, LNT II, and the desired LNT.
  • 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).
  • 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).
  • 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).

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Abstract

La présente invention concerne des procédés de détection et/ou de quantification destinés à assurer la détection et/ou la quantification d'un ou de plusieurs oligosaccharides du lait maternel. L'invention permet en outre d'effectuer une correction pour tenir compte de la présence de glucose dans un analyte. L'invention concerne également des substances et des dispositifs destinés à être utilisés dans une telle détection et/ou quantification, et dans le traitement de résultats de telles détections et/ou quantifications, en particulier pour la surveillance de la qualité d'un produit.
PCT/US2021/056140 2020-10-22 2021-10-22 Procédé de mesure de la concentration de molécules d'oligosaccharides du lait maternel (hmo) à l'aide d'un glocomètre Ceased WO2022087331A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090117106A1 (en) * 2005-07-20 2009-05-07 Glykos Finland Oy Cancer Specific Glycans and Use Thereof
US20100003699A1 (en) * 2008-01-18 2010-01-07 Glykos Finland Ltd. Tissue carbohydrate compositions and analysis thereof
WO2013025104A1 (fr) * 2011-08-16 2013-02-21 Friesland Brands B.V. Compositions nutritionnelles comprenant des oligosaccharides de lait humain et leurs utilisations
WO2018106845A1 (fr) * 2016-12-06 2018-06-14 Kaleido Biosciences, Inc. Polymères glycane et procédés associés
US10100299B2 (en) * 2006-12-21 2018-10-16 Basf Enzymes Llc Amylases and glucoamylases, nucleic acids encoding them and methods for making and using them
US20190037899A1 (en) * 2016-02-05 2019-02-07 The Institute Of Food Research Intramolecular Trans-Sialidase

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090117106A1 (en) * 2005-07-20 2009-05-07 Glykos Finland Oy Cancer Specific Glycans and Use Thereof
US10100299B2 (en) * 2006-12-21 2018-10-16 Basf Enzymes Llc Amylases and glucoamylases, nucleic acids encoding them and methods for making and using them
US20100003699A1 (en) * 2008-01-18 2010-01-07 Glykos Finland Ltd. Tissue carbohydrate compositions and analysis thereof
WO2013025104A1 (fr) * 2011-08-16 2013-02-21 Friesland Brands B.V. Compositions nutritionnelles comprenant des oligosaccharides de lait humain et leurs utilisations
US20190037899A1 (en) * 2016-02-05 2019-02-07 The Institute Of Food Research Intramolecular Trans-Sialidase
WO2018106845A1 (fr) * 2016-12-06 2018-06-14 Kaleido Biosciences, Inc. Polymères glycane et procédés associés

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