WO2019160402A1 - Protéine de lactosérum native pour réduire une allergie - Google Patents

Protéine de lactosérum native pour réduire une allergie Download PDF

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Publication number
WO2019160402A1
WO2019160402A1 PCT/NL2018/050103 NL2018050103W WO2019160402A1 WO 2019160402 A1 WO2019160402 A1 WO 2019160402A1 NL 2018050103 W NL2018050103 W NL 2018050103W WO 2019160402 A1 WO2019160402 A1 WO 2019160402A1
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WO
WIPO (PCT)
Prior art keywords
infant formula
stream
whey protein
formula product
milk
Prior art date
Application number
PCT/NL2018/050103
Other languages
English (en)
Inventor
Betty LOBATO-VAN ESCH
Gerrit HOLS
Original Assignee
N.V. Nutricia
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by N.V. Nutricia filed Critical N.V. Nutricia
Priority to PCT/NL2018/050103 priority Critical patent/WO2019160402A1/fr
Priority to US16/968,167 priority patent/US20210368816A1/en
Priority to AU2019220341A priority patent/AU2019220341A1/en
Priority to CN201980025897.4A priority patent/CN112004424A/zh
Priority to EP19714887.7A priority patent/EP3752009A1/fr
Priority to PCT/NL2019/050102 priority patent/WO2019160416A1/fr
Publication of WO2019160402A1 publication Critical patent/WO2019160402A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C21/00—Whey; Whey preparations
    • A23C21/06—Mixtures of whey with milk products or milk components
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/14—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment
    • A23C9/142—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment by dialysis, reverse osmosis or ultrafiltration
    • A23C9/1422—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment by dialysis, reverse osmosis or ultrafiltration by ultrafiltration, microfiltration or diafiltration of milk, e.g. for separating protein and lactose; Treatment of the UF permeate
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/15—Reconstituted or recombined milk products containing neither non-milk fat nor non-milk proteins
    • A23C9/1512—Reconstituted or recombined milk products containing neither non-milk fat nor non-milk proteins containing isolated milk or whey proteins, caseinates or cheese; Enrichment of milk products with milk proteins in isolated or concentrated form, e.g. ultrafiltration retentate
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/19—Dairy proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C2210/00—Physical treatment of dairy products
    • A23C2210/20—Treatment using membranes, including sterile filtration
    • A23C2210/206—Membrane filtration of a permeate obtained by ultrafiltration, nanofiltration or microfiltration
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C2210/00—Physical treatment of dairy products
    • A23C2210/20—Treatment using membranes, including sterile filtration
    • A23C2210/208—Removal of bacteria by membrane filtration; Sterile filtration of milk products
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C2210/00—Physical treatment of dairy products
    • A23C2210/25—Separating and blending
    • A23C2210/252—Separating a milk product in at least two fractions followed by treatment of at least one of the fractions and remixing at least part of the two fractions

Definitions

  • the present invention relates to the field of infant formula products, in particular for reducing and/or preventing allergic response.
  • WO2013/01 1040 (Ludwig-Maximilians-Universitat, Austria) discloses a dehydrated raw milk preparation that is expressly mentioned not to be heat-treated but obtained via freeze-drying. It also discloses that raw milk is rich in microorganisms and that it is conceivable that the microorganisms stimulate expression of innate immunity receptor genes. It concludes that it remains an open question whether increased expression of Toll-Like Receptors (TLRs) associated with raw milk consumption reflects a relevant pathway underlying allergic diseases development or whether it is merely an indicator of exposure to microbes. It also concludes that the study underlying the claims ofWO2013/01 1040 does not allowto answer whether the up-regulation of innate immune receptors directly modulates the development of allergic disease or whether it is a marker for the effect of genes and environment on allergic disease.
  • TLRs Toll-Like Receptors
  • WO2013/068653 (Valio LTD, Finland) discloses a method for the production of an infant formula base relying on a series of filtration steps to keep protein nativity levels high. Also, WO2013/068653 teaches that whey protein nativity is retained for more than 90% when skim milk is subjected to a pasteurization step. And WO2013/068653 teaches to hydrolyze proteins to enable a hypoallergenic infant formula base to be produced.
  • the present invention provides in the need in the art for an intact, native whey protein fraction to be used for reducing the severity or magnitude of allergic response.
  • the native, intact whey protein is comprised in a nutritional composition, preferably an infant formula product.
  • the present invention concerns the use of the native, intact whey protein according to the invention for reducing and/or preventing allergic response and an infant formula product comprising the native whey protein according to the invention.
  • the invention concerns an infant formula product obtainable by a process comprising:
  • step (ii) subjecting the permeate originating from step (i) to microfiltration over a membrane capable of retaining casein and permeating whey proteins, to provide a casein stream as retentate and a permeate comprising whey protein;
  • step (iii) fractionating the permeate originating from step (ii) into a whey protein stream and a lactose stream;
  • step (a) originating from step (a) and a lactose source to obtain a recombined stream;
  • step (c) optionally pasteurization of the recombined stream from step (b),
  • step (d) using the recombined stream originating from step (b) or (c) in the manufacture of the infant formula product
  • the invention concerns an infant formula product comprising whey protein, wherein the whey protein is intact and native for use in reducing and/or preventing allergic response.
  • the infant formula product of the invention contains native whey protein.
  • the whey proteins have a nativity value of more than 92%, preferably more than 94%, more than 95% or even more than 98%.
  • the infant formula product of the present invention contains intact whey proteins. Intact means that the whey proteins have not been subjected to a hydrolysis step.
  • the infant formula is pasteurized.
  • the inventors of the present invention have surprisingly shown that an infant formula product with intact whey protein, obtained according to the present invention that includes a pasteurization step, can be used for reducing and/or prevention of an allergic response.
  • the infant formula is substantially free of alkaline phosphatase activity.
  • the infant formula is a liquid, ready- to-feed infant formula which is substantially free of alkaline phosphatase activity.
  • the term substantially free of alkaline phosphatase activity means that, when measured using a liquid, ready-to-feed infant formula, the alkaline phosphatase activity is below 350 mU/L.
  • the infant formula product is not pasteurized.
  • the inventors of the present invention have surprisingly shown that an infant formula product with intact whey protein, obtained according to the present invention without the inclusion of a pasteurization step, can be used for reducing and/or prevention of an allergic response.
  • the infant formula contains alkaline phosphatase activity.
  • the infant formula product is a liquid, ready-to-feed product and contains alkaline phosphatase activity or is considered alkaline phosphatase positive.
  • the alkaline phosphatase activity of the infant formula product in this embodiment is above 350 mU/L.
  • the invention relates to an infant formula product comprising whey protein, wherein the whey protein is intact and native, as defined by a nativity value of at least 90 %, for use in reducing and/or preventing allergic response.
  • the infant formula product is preferably pasteurized and substantially free of alkaline phosphatase activity.
  • the infant formula product is not pasteurized and contains alkaline phosphatase activity.
  • the native whey protein is comprised in a nutritional composition, preferably an infant formula product.
  • the inventors have developed a process for preparing infant formulae products, containing native whey protein.
  • the composition according to the invention is a nutritional composition, preferably an infant formula product.
  • infant formula product refers to milk-based nutritional compositions suitable for feeding infants, which typically are in the form of a reconstitutable powder or a ready-to-feed liquid composition, or refers to infant formula bases, which are suitable for making infant formulae and which comprise all or almost all essential ingredients in the required amounts for infant nutrition.
  • the composition is an infant formula, a follow-on formula, a growing-up milk, or a base therefore.
  • the composition is an infant formula.
  • the infant formula product may be a powder, preferably a spray- dried powder, intended to be reconstituted into a liquid infant formula, or a liquid infant formula.
  • composition according to the invention can be defined in two distinct ways.
  • the composition according to the invention is defined by the process of preparing the composition.
  • the composition is defined by the presence of a whey protein which is intact and native.
  • the composition according to the invention is defined by the process of preparing the composition and by the presence of a whey protein which is intact and native.
  • the composition according to the invention comprises a whey protein fraction obtainable by the process according to the invention as defined below, in particular step (a) and optionally step (c).
  • the composition according to the present invention comprises a whey protein fraction which is obtainable as a whey protein stream via the process of the present invention as defined below, in particular step (a).
  • the whey proteins are obtainable by subjecting a defatted, debacterialized milk to microfiltration over a membrane capable of retaining casein and permeating whey proteins to provide a permeate comprising whey protein and fractionating the permeate into a whey protein stream and a lactose stream, wherein debacterization is preferably performed by microfiltration or pasteurization.
  • the whey proteins are present in the thus obtained ultrafiltration retentate.
  • the whey protein is obtained by the process defined herein. It is well-known to the skilled person how to obtain such an ultrafiltration retentate that contains native whey proteins starting from defatted milk.
  • the composition according to the invention is defined by the process of preparing the composition. This process is herein referred to as the process according to the invention.
  • the process according to the invention comprises:
  • step (ii) subjecting the permeate originating from step (i) to microfiltration over a membrane capable of retaining casein and permeating whey proteins, to provide a casein stream as retentate and a permeate comprising whey protein;
  • step (iii) fractionating the permeate originating from step (ii) into a whey protein stream and a lactose stream;
  • step (b) combining at least part of the casein stream, at least part of the whey protein stream originating from step (a) and a lactose source to obtain a recombined stream;
  • step (c) optionally pasteurization of the recombined stream from step (b), (d) using the recombined stream originating from step (b) or (c) in the manufacture of the infant formula product.
  • defatted milk is treated to produce an infant formula product.
  • a certain stream or composition is mentioned to “originate from” a certain process step, such as from the recombined stream originating from step (b), said stream or composition can be the composition which is directly obtained by said process step.
  • additional processing steps such as partial evaporation and/or supplementation of additional water or other components, the stream or composition is also regarded to originate from that specific process step.
  • step (b) if the recombined stream of step (b) would be partially evaporated prior to it is entered in the pasteurization step (c), the incoming stream of step (c) is still regarded to be the recombined stream originating from step (b).
  • the term“stream” refers to a liquid composition, although the presence of some solid material is not excluded, e.g. as in a suspension, as long as the composition can be handled by conventional dairy plants.
  • the present process uses milk as starting material in step (a).
  • Defatted milk preferably defatted cow’s milk
  • “defatted milk” refers to milk having a reduced fat content compared to whole milk.
  • the fat content of the defatted milk is in the range of 0 - 2 wt%, preferably 0 - 1 wt%, more preferably 0 - 0.2 wt%, most preferably 0 - 0.05 wt%, based on total weight of the defatted milk.
  • the defatted milk is skim milk.
  • the present process employs milk, which refers to non-human milk, preferably cow’s milk.
  • the process comprises a step of defatting milk to obtain the defatted milk, which is subsequently subjected to step (a).
  • defatting milk is subjected to the defatting step.
  • the defatting step affords the defatted milk.
  • the defatted milk is the sole protein source for the infant formula product.
  • the defatted milk is processed or fractioned into a casein stream, a whey protein stream and a lactose stream.
  • the casein stream is a liquid composition comprising casein, which is enriched in casein compared to the casein content in the incoming defatted milk
  • the whey protein stream is a liquid composition comprising whey protein, which is enriched in whey protein compared to the whey protein content in the incoming defatted milk
  • the lactose stream is a liquid composition comprising lactose, which is enriched in lactose compared to the lactose content in the incoming defatted milk.
  • step (a) is defined that the content of the enriched component, based on dry weight, is increased in one stream compared to another stream.
  • the casein stream is enriched in casein, i.e. has a higher casein content, based on dry matter, compared to the incoming defatted milk.
  • the fractionation of step (a) is accomplished by membrane filtration techniques and involves a combination of microfiltration and ultrafiltration.
  • the casein stream originates from the microfiltration as retentate
  • the whey protein stream originates from the ultrafiltration as retentate
  • the lactose stream originates from the ultrafiltration as permeate.
  • Suitable membrane filtration processes are known in the art, e.g. as disclosed in WO 2013/068653, WO 2013/137714 and WO 2015/041529. More specifically, step (a) includes:
  • step (ii) subjecting the permeate originating from step (i) to microfiltration over a membrane capable of retaining casein and permeating whey proteins, to provide a casein stream as retentate and a permeate comprising whey protein;
  • step (iii) fractionating the permeate originating from step (ii) into a whey protein stream and a lactose stream.
  • the incoming defatted milk is subjected to debacterization (bacterial removal) in step (i).
  • Debacterization may be performed by filtration or by pasteurization.
  • debacterization is performed by bacterial filtration (e.g. microfiltration (MF)).
  • MF microfiltration
  • the microfiltration of step (i) may be performed by microfiltration over a membrane capable of retaining bacteria and permeating milk proteins, to provide a debacterialized milk as permeate.
  • the microfiltration of step (i) comprises ceramic microfiltration.
  • the MF membrane preferably has a pore size of between 1 .8 and 0.6 mhi, preferably between 1 .4 and 0.8 m ⁇ h.
  • the MF process of step (i) is preferably executed at a temperature of between 4 and 20 °C, more preferably between 8 and 15 °C, most preferably at a temperature of about 10 °C.
  • step (i) is performed by pasteurization.
  • Pasteurization of defatted milk in order to reduce the bacterial load of the milk is well-known in the art. Pasteurization and preferred embodiments thereof are described in more detail below in the context of step (c), which equally applies here.
  • the debacterialized milk originating from step (i) is fractioned into two distinct streams, each enriched in a particular protein type; a casein enriched MF retentate (MFR) and a whey protein enriched MF permeate (MFP) are produced.
  • the MF step (ii) is performed over a membrane that enables fractionation of casein and whey proteins.
  • a membrane typically has a porosity of between 0.05 and 0.5 m ⁇ ti, more preferably between 0.08 - 0.35 mhi.
  • the membrane used in step (ii) may have a molecular weight cut-off in the range of 250 - 1500 kDa, preferably in the range of 500 - 1000 kDa.
  • a ceramic membrane or a spiral wound (organic) membrane is used.
  • Microfiltration of step (ii) is preferably performed with a volume concentration factor (VCF) in the range of 1 .5 - 10, preferably 2 - 5, which has been found to provide the most optimal results in terms of the composition of the MF retentate, especially in terms in terms of casein content.
  • VCF volume concentration factor
  • volume concentration factor is the factor at which a liquid composition is concentrated upon filtration, i.e. the total volume of the incoming stream prior to filtration divided by the total volume of the retentate after filtration, irrespective of the total solid content.
  • VCF volume concentration factor
  • microfiltration of step (ii) is enhanced with diafiltration (DF).
  • Diafiltration may be accomplished by diluting the retentate of the MF at least once with an amount of water, or by diluting the incoming debacterialized milk with an amount of water and subjecting the diluted milk to MF.
  • the DF water may be added to the incoming debacterialized milk or MFR at once, or the total amount of DF water may be added in several fractions. After each addition of DF water to the incoming skim milk or MFR, the diluted liquid composition is subjected to MF.
  • Step (iii) is preferably performed by ultrafiltration (UF).
  • UF ultrafiltration
  • most of the liquid and small solutes end up in the UF permeate (UFP), while the UF retentate (UFR) comprises substantially all whey protein, in a smaller volume.
  • Small molecules which permeate through the UF membrane are for example lactose, monovalent and polyvalent ions.
  • the ultrafiltration of step (iii) can be carried out with any UF membrane known in the art, including ceramic membranes, tubular and organic spiral wound membranes.
  • the UF membrane is an organic spiral wound membrane.
  • the UF membrane has a molecular weight cut-off of that enables proteins, preferably whey proteins, to remain in the retentate, and allow small solutes, for example lactose, to permeate through the membrane.
  • the UF step (iii) preferably is carried out with a membrane having a molecular weight cut-off of at most 25 kDa, more preferably at most 10 kDa, and preferably of at least 2.5 kDa, more preferably at least 5 kDa.
  • the UF step (iii) is preferably carried out with a volume concentration factor (VCF) in the range of 20 - 200, preferably 50 - 150, which has been found to provide the most optimal results in terms of the composition of the UF retentate.
  • VCF volume concentration factor
  • Step (a) may further comprise one or more concentration steps, such as concentration of the MFR originating form step (ii) and/or the UFR originating form step (iii). Concentration is preferably performed by reverse osmosis (RO), nanofiltration (NF) and/or evaporation. NF is most preferred, as NF concentrates the stream and at the same time lowers the monovalent ion content, which are able to permeate the NF membrane. Such lowering of the monovalent ion content is typically desirable in the production of infant formula products.
  • concentration steps such as concentration of the MFR originating form step (ii) and/or the UFR originating form step (iii). Concentration is preferably performed by reverse osmosis (RO), nanofiltration (NF) and/or evaporation. NF is most preferred, as NF concentrates the stream and at the same time lowers the monovalent ion content, which are able to permeate the NF membrane. Such lowering of the monovalent ion content is typically desirable in
  • the protein fraction of the casein stream originating from step (a) typically comprises very little whey protein, preferably less than 15 wt%, more preferably less than 10 wt%, based on the weight of the protein fraction of the casein stream, and is high in casein.
  • the protein fraction comprises at least 85 wt% casein, more preferably at least 90 wt% casein.
  • the content of total solids in the casein stream typically ranges from 5 to 30 wt%, preferably from 7 to 30 wt%, most preferably from 17 to 24 wt%, based on total weight of the casein stream.
  • the casein stream may also be referred to as a casein concentrate, casein isolate, micellar casein concentrate or micellar casein isolate (MCI).
  • the whey protein stream is typically a liquid composition having a total solid content of 5 - 35 wt%, preferably of 10 - 30 wt%, most preferably of 20 - 30 wt%, and typically comprises 25 - 90 wt%, preferably 60 - 85 wt% whey proteins based on total dry weight.
  • the whey protein stream may also be referred to as an aqueous composition comprising whey proteins.
  • the whey protein stream is enriched in whey protein compared to the incoming defatted milk, it may still contain substantial amounts of casein, depending on the exact conditions at which the fractionation between casein and whey protein by ultrafiltration, is performed.
  • the whey protein stream comprises at most 40 wt%, preferably 5 - 20 wt% casein, based on total weight of the protein.
  • the amount of casein used in combining step (b) can be adapted such that the infant formula product has a whey protein: casein ratio that falls within the preferred ratio of 90:10 to 40:60.
  • the lactose stream is typically a liquid composition having a total solid content of 3 - 30 wt%, preferably of 5 - 22 wt%.
  • the lactose content in the lactose stream originating from step (a) is typically at least 75 wt%, preferably at least 90 wt% or even at least 95 wt%, based on total dry weight.
  • the process according to the invention preferably comprises a demineralization step, wherein the lactose source, or one or more components thereof, is/are demineralized prior to being subjected to step (b).
  • Demineralization is thus typically performed on at least part of the lactose stream originating from step (a) prior to being subjected to step (b).
  • Demineralization is particularly preferred for the manufacture of infant formula products, for which it is typically required to lower the mineral content as compared to the incoming milk.
  • step (a) at least part of the lactose stream originating from step (a), preferably the UFP originating from step (iii), is subjected to demineralization prior to being used as (part of) the lactose source in step (b).
  • Demineralization of the lactose source may be performed by any technique known in the art, such as electrodialysis, ion exchange, salt precipitation, lactose crystallization, membrane filtration techniques such as nanofiltration, optionally enhanced with diafiltration, or combinations thereof.
  • demineralization comprises at least one of salt precipitation, electrodialysis, lactose crystallization and ion exchange, optionally in combination with nanofiltration, more preferably demineralization comprises nanofiltration in combination with at least one of salt precipitation, electrodialysis, lactose crystallization and ion exchange.
  • demineralization comprises at least electrodialysis and/or salt precipitation.
  • demineralization comprises at least nanofiltration in combination with electrodialysis and/or salt precipitation.
  • the inventors found that when only nanofiltration is used for demineralization, especially for demineralization of an ultrafiltration permeate as lactose source in the preparation of infant formula products, the content of divalent ions, such as calcium and phosphate, is typically insufficiently reduced to obtain a final infant formula product within legal requirement.
  • Demineralization is preferably performed such that at least 20 wt%, or preferably 50 wt%, more preferably at least 70 wt% or at least 80 wt%, most preferably at least 90 wt% of the polyvalent ions and/or such that at least 20 wt% of the monovalent ions are removed, more preferably at least 35 wt% or at least 50 wt%, most preferably at least 60 wt% of the monovalent ions, present in the lactose stream, e.g.t the UFP originating from step (iii), are removed.
  • step (b) at least part of the casein stream, at least part of the whey protein stream originating from step (a) and a lactose source are combined to obtain a recombined stream.
  • This recombined stream is used to manufacture the infant formula product in step (d), optionally after a pasteurization step (c).
  • the combining of step (b) affords a composition having a protein fraction comprising both casein and whey protein in a certain weight ratio.
  • the combining of step (b) may involve additional components.
  • the combining is preferably done such that the whey protein to casein weight ratio in the recombined stream is in the range of 90:10 to 40:60, more preferably in the range of 80:20 to 50:50, even more preferably in the range of 75:25 to 50:50, most preferably in the range of 70:30 to 55:45.
  • the whey protein to casein weight ratio in the recombined stream is about 60:40.
  • the exact ratio is typically determined by the type of infant formula product that is being produced, and can be adjusted as known in the art.
  • much attention in the art is given to the amino acid profile of infant formula products.
  • the process according to the invention provides optimal flexibility in targeting a specific desired amino acid profile, e.g. by adjusting the ratio in which the whey protein and casein streams are combined or in varying the specific process conditions of the microfiltration of step (a). As such, optimal amino acid profiles resembling those found in human milk are obtainable with the process according to the invention.
  • step (b) 10 - 50 wt%, preferably 12 - 25 wt%, based on total weight of the casein, ofthe casein stream originating from step (a) is subjected to step (b). Most preferably, about 16 wt%, based on total weight of the casein, of the casein stream originating from step (a) is subjected to step (b).
  • the amount of the casein stream originating from step (a) that is subjected to step (b) is advantageously governed by the desired whey protein to casein weight ratio in the recombined stream. Preferably, all of the whey protein stream originating from step (a) is subjected to the combining of step (b).
  • step (b) 0 - 50 wt%, preferably 5 - 25 wt%, based on total weight of the lactose, of the lactose stream originating from step (a) is subjected to step (b) as (part of) the lactose source.
  • the amount of the lactose stream originating from step (a) that is subjected to step (b) as (part of) the lactose source is advantageously governed by the amount of lactose required for step (d). In case the amount of lactose in the lactose stream originating from step (a) that is subjected to step (b) would be insufficient for infant formula product manufacture, additional lactose can be used.
  • part of the casein stream is combined with all of the whey protein stream and part of the lactose stream. In one embodiment, part of the casein stream is combined with all of the whey protein stream and all of the lactose stream. In one embodiment, part of the casein stream is combined with all of the whey protein stream and nothing of the lactose stream. In one embodiment, part of the MFR originating from step (ii) is combined with at least part of the UFR originating from step (iii) and at least part of the UFP originating from step (iii).
  • step (b) three or more streams are recombined into one stream.
  • This recombining may occur at once (streams are combined simultaneously) or step-wise (streams are combined consecutively).
  • Combining can be performed as wet mixing or as dry mixing or even as a combination of both.
  • the combining occurs as wet mixing, wherein liquid compositions are mixed in the appropriate amounts.
  • the process according to the invention may contain a pasteurization step, although omitting the pasteurization step also affords suitable products. If a pasteurization step is performed, it may be performed as step (i) or as step (c). In a preferred embodiment, a pasteurization step is performed, since this is a requirement for infant formula products in many jurisdictions from a food safety perspective. In a preferred embodiment, the process of the invention contains only a single pasteurization step to ensure the obtained product is sufficiently heat-treated with regards to prevention of microbial or bacterial contaminations but on the other hand ensures preservation of protein nativity.
  • step (i) is a pasteurization step and step (c) is not performed, or step (i) is a filtration step and step (c) is performed.
  • the incoming defatted milk may be pasteurized in step (i)
  • no pasteurization step is performed and step (i) is performed by filtration and step (c) in omitted.
  • step (c) is performed in case debacterialization in step (i) is achieved by microfiltration.
  • Pasteurization is known in the art and may e.g. involve HTST, ESL or UHT.
  • the pasteurization step as meant herein has the purpose of reducing the microbial load to such an extent that the resulting infant formula product is free from microorganisms and safe for consumption by infants.
  • it is safe with regards to Bacillus cereus and Enterobacter sakazakii , for instance, such as laid down in European Regulation No 2073/2005 dated 2007, corrigendum No. 1441 /2007.
  • pasteurization involves heating at 72 - 74°C for 15 to 30 seconds, or, alternatively, a heat-treatment equivalent thereto, meaning that the same heat load is applied, as is known to the skilled person.
  • the equivalent heat-treatment results in the same reduction in bacterial load and preserves the protein nativity to the same extent as a pasteurization step at 72 - 74°C for 15 to 30 seconds, resulting in whey proteins having a nativity value of more than 90%, preferably more than 95 or even more than 98%.
  • step (d) the recombined stream originating from step (b) is used to manufacture the infant formula product.
  • Such manufacturing is known in the art and typically involves one or more of drying, concentrating, supplementing with vitamins, minerals, lipids and/or dietary fibres, heat treatment, homogenisation, packaging.
  • step (d) does not involve heat treatment, and involves one or more of drying, concentrating, supplementing with vitamins, minerals, lipids and/or dietary fibres and packaging.
  • step (d) involves at least a drying step, most preferably it involves all of the above mentioned steps.
  • a drying step is performed directly after step (b) or (c), most preferably directly after step (c).
  • step (b) the recombined stream originating from step (b) is dried, preferably spray- dried.
  • the process according to the invention comprises only a single drying step, wherein in step (d) the recombined stream is dried, preferably by spray-drying. Due to the inherently limited heat-load as a consequence of low water activity of droplets produced during spray-drying, protein nativity remains substantially the same and is not significantly impacted during spray-drying.
  • the spray-drying step is preferably executed with an inlet temperature of less than 250 °C, preferably less than 220 °C, more preferably less than 200 °C.
  • the spraydrying step is executed such that the wet bulb temperature is kept below 80°C, preferably below 70° C or even below 50 °C.
  • the recombined stream is concentrated, preferably prior to being dried. Such concentration may be accomplished by any means known in the art, such as by reverse osmosis (RO), nanofiltration (NF) and/or evaporation.
  • supplementation of certain components may be desired.
  • Such supplementation can be performed either prior to, during or after combining step (b) and/or optionally prior to or after a drying step.
  • the skilled person is aware of the requirements of particular types of infant formula products, e.g. from EU directive 91/321 /EEC or EU directive 2006/141/EC or US Food and Drug Administration 21 CFR Ch 1 part 107, and is able to adjust the composition of the recombined stream in order to meet those requirements.
  • the composition according to the invention is an infant formula product comprising whey protein, wherein the whey protein is intact and native.
  • the composition according to this embodiment is obtainable by the process according to the invention as defined above.
  • the infant formula may be a spray-dried powder, in which case it is preferred that that the spray-drying step is executed with an inlet temperature of less than 250 °C, preferably less than 220 °C, more preferably less than 200 °C. It is preferred that the whey proteins have undergone a pasteurization step preferably a single pasteurization step.
  • the whey protein being“native”, is herein defined as having a nativity value of at least 90 %, preferably at least 94 %, most preferably at least 96 %.
  • the nativity value is in the range of 90 - 100 %, preferably in the range of 94 - 99 %, more preferably in the range of 96 - 99 %.
  • the nativity value is in the range of 90 - 99 %, preferably in the range of 91 - 96 %, more preferably in the range of 92 - 94 %.
  • nativity values are known in the art and can be determined by any means available to the skilled person.
  • the nativity value refers to the percentage of native protein of a particular type based on the total amount of protein of the same type.
  • the nativity value of the whey protein refers to the amount of native whey protein based on the total amount of protein.
  • the nativity value is determined according to the procedure in example 3.
  • the invention concerns the composition according to this embodiment.
  • This composition may be referred to as a hypoallergenic infant formula, since the severity of allergic response is significantly reduced.
  • the infant formula product shows a reduced allergic skin response, preferably a reduced food allergy response.
  • composition according to the invention irrespective whether it is defined by the process of manufacture or by the presence of whey protein which is intact and native.
  • the composition according to the invention is defined by the process of preparing the composition and by the presence of an intact whey protein.
  • the composition according to the invention is defined by the process of preparing the composition and by the presence of a native whey protein, as defined herein.
  • the composition according to the invention is defined by the process of preparing the composition and by the presence of a native and intact whey protein, as defined herein.
  • the composition is an infant formula product, it is typically nutritionally complete for infants, and contains all necessary macronutrients and micronutrients for infant formula products as known in the art.
  • the infant formula product preferably contains casein, in addition to the native and intact whey protein.
  • the whey protein to casein weight ratio in the infant formula product is preferably in the range of 90:10 to 40:60, more preferably in the range of 80:20 to 50:50, even more preferably in the range of 75:25 to 50:50, most preferably in the range of 70:30 to 55:45.
  • the whey protein to casein weight ratio in the in the infant formula product is about 60:40.
  • the exact ratio is typically determined by the type of infant formula product that is being produced, and can be adjusted as known in the art.
  • the whey protein, preferably all protein has not been subjected to a hydrolysis step, wherein the protein is partly or fully hydrolysed.
  • the process for obtaining the infant formula product does not contain a hydrolysis step, wherein the whey protein, preferably all protein, is partly or fully hydrolysed.
  • the composition according to the invention show a negative reaction to an alkaline phosphatase (ALP) activity test.
  • ALP alkaline phosphatase
  • Tests for alkaline phosphate activity are known in the art and are used as standard for defining the activity (or lack of activity) of the enzymes in an infant formula product.
  • the law for example European Regulation 2074/05, requires the ALP activity to be below 350 mU/L.
  • the ALP activity can be defined as mU/g (typically for powders, or for liquids based on dry weight) or mU/L (typically for liquids, including reconstituted powders).
  • the ALP activity of the composition according to the invention when in liquid form, is typically below 450 mU/L, preferably below 350 mU/L, or, when in powder form, is typically below 450 mU/L, preferably below 350 mU/L, after reconstitution as common in the art of infant formula products.
  • the composition according to the invention is liquid or reconstituted powder and has an ALP activity in the range of 0 - 450 mU/L, preferably 100 - 350 mU/L, more preferably 200 - 350 mU/L, most preferably 250 - 320 mU/L. In one embodiment, the composition according to the invention has an ALP activity of at most 20 mU/g, preferably at most 5 mU/g, or the composition according to the invention has an ALP activity in the range of 0 - 20 mU/g, preferably 0.1 - 10 mU/g, more preferably 0.2 - 7 mU/g, most preferably 0.5 - 5 mU/g, based on dry weight of the composition.
  • the composition according to the invention has an ALP activity of at least 25 mU/g, preferably at least 30 mU/g, or the composition according to the invention has an ALP activity in the range of 25 - 150 mU/g, preferably 30 - 50 mU/g, based on dry weight of the composition.
  • the ALP activity is determined by ISO standard 1 1816-1 .
  • the ALP activity is determined by the following procedure. A solution of the whey protein, typically as a 10 wt% protein solution, is mixed with an equal amount of 1 -butanol, and the mixture is then centrifuged between 2500-3500 g for 30 min. The aqueous phase is collected from beneath the fat layer and diluted between 1/5 - 1/200. These sample solutions were added in the wells of an enzyme-linked immunosorbent assay (ELISA) plate, coated with a monoclonal antibody specific to the alkaline phosphatase found in cow’s milk, together with control and standard solutions.
  • ELISA enzyme-linked immunosorbent assay
  • the plates are incubated for 1 h at 18 - 25 °C, after which the solutions are removed from the wells and substrate solution is added to each well.
  • the plates are incubate for 2 h at 35 - 38 °C. After stopping the incubation, the plate is imaged at a wavelength of 405 nm, and the ALP activity is determined by comparison of the optical density of the sample with that of the standard.
  • the composition according to the invention is capable of reducing and/or preventing allergic response.
  • the allergic response is related to food allergy, in particular milk allergy, whey protein allergy or bovine milk allergy.
  • the allergic response may be direct or indirect.
  • the allergic response is a direct or immediate response.
  • the allergic response is allergic skin response.
  • the allergic skin response is related to food allergy, in particular milk allergy, whey protein allergy or bovine milk allergy.
  • the allergic skin response may be direct or indirect.
  • the allergic skin response is a direct or immediate skin response.
  • the invention concerns an infant formula product obtainable by a process comprising:
  • step (ii) subjecting the permeate originating from step (i) to microfiltration over a membrane capable of retaining casein and permeating whey proteins, to provide a casein stream as retentate and a permeate comprising whey protein;
  • step (iii) fractionating the permeate originating from step (ii) into a whey protein stream and a lactose stream;
  • step (b) combining at least part of the casein stream, at least part of the whey protein stream originating from step (a) and a lactose source to obtain a recombined stream;
  • step (c) optionally pasteurization of the recombined stream from step (b),
  • step (d) using the recombined stream originating from step (b) or (c) in the manufacture of the infant formula product
  • the invention according to this aspect can also be worded as a use of defatted milk for the manufacture of an infant formula product for use in reducing and/or preventing allergic response, wherein the infant formula product is obtainable by a process comprising:
  • step (ii) subjecting the permeate originating from step (i) to microfiltration over a membrane capable of retaining casein and permeating whey proteins, to provide a casein stream as retentate and a permeate comprising whey protein;
  • step (iii) fractionating the permeate originating from step (ii) into a whey protein stream and a lactose stream;
  • step (b) combining at least part of the casein stream, at least part of the whey protein stream originating from step (a) and a lactose source to obtain a recombined stream; (c) optionally pasteurization of the recombined stream from step (b),
  • step (d) using the recombined stream originating from step (b) or (c) in the manufacture of the infant formula product.
  • the invention according to this aspect can also be worded as a method for reducing and/or preventing allergic response, comprising administering to the subject an infant formula product obtainable by a process comprising:
  • step (ii) subjecting the permeate originating from step (i) to microfiltration over a membrane capable of retaining casein and permeating whey proteins, to provide a casein stream as retentate and a permeate comprising whey protein;
  • step (iii) fractionating the permeate originating from step (ii) into a whey protein stream and a lactose stream;
  • step (b) combining at least part of the casein stream, at least part of the whey protein stream originating from step (a) and a lactose source to obtain a recombined stream;
  • step (c) optionally pasteurization of the recombined stream from step (b),
  • step (d) using the recombined stream originating from step (b) or (c) in the manufacture of the infant formula product.
  • the invention according to the first aspect concerns an infant formula product comprising whey protein, wherein the whey protein is intact and native, as defined by a nativity value of at least 90 %, for use in reducing and/or preventing allergic skin response.
  • the invention according to the first aspect concerns the use of whey protein for the manufacture of an infant formula product for use in reducing and/or preventing allergic skin response, wherein the whey protein comprised in the infant formula product is intact and native, as defined by a nativity value of at least 90 %.
  • the invention according to the first aspect concerns a method for reducing and/or preventing allergic response, comprising administering to the subject an infant formula product comprising whey protein, wherein the whey protein is intact and native, as defined by a nativity value of at least 90 %.
  • the invention relates to an infant formula product, defined by the process of manufacture and/or by the presence of whey protein which is intact and native, for use in preventing or treating atopic dermatitis and/or eczema.
  • the invention according to this aspect is referred to as use according to the invention, which is equally applicable to composition for use according to the invention and the method according to the invention, as defined above.
  • the subject of the use according to the invention is typically an infant, preferably a human infant.
  • the infant is 0 - 36 months of age, more preferably 0 - 24 months of age, even more preferably 0 - 12 months of age, most preferably 0 - 6 months of age.
  • the subject is in need of reducing and/or preventing allergic response.
  • the subject suffers from allergy.
  • the subject is at risk of developing allergy.
  • the allergy is preferably food allergy, more preferably milk allergy and/or whey protein allergy, most preferably whey protein allergy.
  • the milk allergy is typically bovine milk allergy
  • the whey protein allergy is typically bovine whey protein allergy, especially in case defatted bovine milk is used as starting material of the process according to the invention.
  • the subject suffers from direct or immediate skin response or is at risk of developing direct or immediate skin response.
  • the infant formula product according to the invention is typically suitable as complete nutritional product for infants, like regular infant formula. Administration of the infant formula product according to the invention this occurs as (part of) the regular feeding regime of the infant. In one embodiment, the use according to the invention is further for providing nutrition to the infant.
  • WPC70 products (i) native WPC70, (ii) deactivated WPC70, and (iii) denatured WPC70, were prepared according to the following process. Milk and subsequent fractions were stored at 4 °C throughout production. Whole raw milk (purchased from Dairygold) was skimmed using typical GEA Westfalia Separator @ 55 °C and cooled to 4 °C. Skim milk was subjected to microfiltration to separate casein from both whey and lactose. Microfiltration membrane used was a 0.08 mM Synder membrane FR (PVDF 800kDa) spiral wound membrane.
  • the microfiltration retentate (MFR) was kept as the casein fraction and the microfiltration permeate (MFP) contained whey, lactose and ash.
  • the operating temperature was 10 °C and volume concentration factor (VCF) was 3. This VCF factor was optimal to obtain the required final concentration of casein protein in the MFR.
  • the MFP was then subjected to ultrafiltration to separate whey protein from lactose at operating temperature of 10 °C with VCF of 90. This VCF factor gave an optimal final concentration of whey protein in ultrafiltration retentate (UFR).
  • a native WPC70 was produced.
  • the ultrafiltration membrane used was a 10kDa Synder membrane ST (PES 10kDa) spiral wound membrane.
  • Diafiltration medium was added to improve separation efficiency of membranes (200% of original starting skim milk volume).
  • Concentrated liquid WPC70 (DM 1 1 %) was stored at 4 °C until further handling.
  • the WPC70 was heated to 30 °C and spray dried at 1 1 % DM.
  • the spray-dryer used was a single stage pilot scale dryer operated with an inlet temperature of 185 °C and outlet temperature of 90 °C. This sample is referred to as the native WPC70 and represents a highly native, alkaline phosphatase positive sample.
  • Deactivated WPC70 was prepared to represent a highly native, pasteurized protein sample which can be included in an infant formula.
  • Denatured WPC70 was prepared by re-hydrating the native WPC70 in 40 °C RO water using a high speed mixer for 30 min, resulting in a total solids content of 10% and a protein content of about 7 %. This solution was heat treated at 100 °C / 60 s using a Microthermics tubular heat exchanger (MicroThermics, North Carolina, USA). The heat-treated WPC was then freeze-dried resulting in a WPC70 powder with whey protein nativity value of ⁇ 30 %.
  • composition of the three WPC70 products is given in the table below (in wt% based on dry weight):
  • IMF products (i) Native IMF, (ii) Deactivated IMF, and (iii) denatured IMF, were prepared according to the following process.
  • the wet phase of the infant milk formulation was prepared by first dissolving lactose powder in 90 °C RO water with agitation provided by a high speed silverson mixer (Silverson®, Chesham Bucks, U.K).
  • the solution was cooled to 45 °C, micellular casein concentrate (MCC, MFR obtained in Example 1) and native whey protein concentrate (native WPC70 obtained in Example 1) were added to the solution, allowing for a final casein: whey ratio of 40:60 (similar to the ratios observed in mothers milk), and re-hydrated under high speed mixing for 20 min.
  • GOS galcto-oligossaccharide
  • Micronutrient components were added to the macronutrients as per a pre-determined recipe. All ingredients were added and agitated at high speeds for 20 min.
  • IMF native IMF
  • the wet phase was directly combined with a pre-prepared oil blend and homogenized via the addition of soy lecithin powder and high speed agitation for 20 min.
  • This completed IMF 50-55 % TS
  • WEC water evaporation capacity
  • Deactivated IMF was manufactured by pasteurizing the wet phase using a Microthermics tubular heat exchanger (MicroThermics, North Carolina, USA) at 73 °C / 30 s.
  • the pasteurized wet phase was combined with a pre-prepared oil blend and homogenized via the addition of soy lecithin powder and high speed agitation for 20 min.
  • This pasteurized compound was dried using a Single stage pilot dryer (WEC 10 kg/hr) which produced a deactivated IMF with a whey protein nativity value of > 95 % and inactivated bioactive components as indicated by the inactivation of the enzyme alkaline phosphatase.
  • Denatured IMF was prepared by rehydration of native IMF powder to a protein content of about 10 %. This compound was mixed at high speed for 30 min to ensure full dissolution. The compound was then heat-treated using a Microthermics tubular heat exchanger (MicroThermics, North Carolina, USA) at 100 °C / 60 s. The heat-treated compound was collected and freeze-dried to produce a denatured IMF with a whey protein nativity value of ⁇ 40 %.
  • composition of the three IMF products is given in the table below (in wt% based on dry weight):
  • TN total nitrogen
  • NPN non-protein nitrogen
  • NCN non-casein nitrogen
  • the theoretical whey fraction is based on the casein / whey protein ratio of the product, from the recipe of the product.
  • Example 4 Allergenicity native and denatured WPC70 products obtained in Example 1
  • mice Four-week-old, specific pathogen free, female C3H/HeOuJ mice were purchased at Charles River Laboratories (The Netherlands) and housed at the animal facility of the Utrecht University on a 12 h light/dark cycle with access to food and water ad libitum. All animal procedures were conducted according to governmental guidelines and approved by the Ethical Committee for Animal Research of the Utrecht University, Utrecht, The Netherlands (CCD: AVD108002015346).
  • CT cholera toxin
  • mice Five days after the last sensitization (day 33), mice were challenged intradermally (i.d.) in the ear pinnae of both ears with 10 pg denatured WPC70 of Example 1 in 20 pi PBS to determine the acute allergic skin response by locally measuring swelling of the skin of the ears via the thickness of the skin. On the same day, mice were challenged i.g. with 50 mg denatured WPC70 in 0.5 mL PBS. 18 h after the oral challenge blood samples were collected and centrifuged at 10.000 rpm for 10 min. Serum was obtained and stored at -20°C until further analysis. Mice were killed by cervical dislocation and samples were obtained for ex vivo analysis.
  • Single cell splenocyte suspensions were obtained by passing spleen samples through a 70-pm nylon cell strainer using a syringe.
  • the splenocyte suspension was rinsed with RPMI 1640 medium (Lonza, Verviers, Belgium) and incubated with lysis buffer (8.3 g NH4CI, 1 g KHC3O and 37.2 mg EDTA dissolved in 1 L demi water, filter sterilized) to remove red blood cells.
  • the reaction was stopped by adding RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Bodinco, Alkmaar, The Netherlands), penicillin (100 U/mL)/streptomycin (10 mg/mL; Sigma-Aldrich) and b-marcaptoethanol (20 pM; Thermo Fisher Scientific, Paisley, Scotland). Splenocytes were subsequently resuspended in this culture medium.
  • FBS heat-inactivated fetal bovine serum
  • penicillin 100 U/mL
  • streptomycin 10 mg/mL
  • b-marcaptoethanol 20 pM
  • splenocytes 8 x 10 s cells/well
  • IL-5 and IL-13 were performed by means of ELISA according to the protocol described above for IgE.
  • Purified rat anti-mouse antibodies (1 pg/mL for IL-5 and 2 pg/mL for IL-13), recombinant mouse cytokines and biotinylated rat anti-mouse antibodies (1 pg/mL for IL-5 and 400 ng/mL for IL-13) were purchased at BD Biosciences.
  • results on IgE levels generated in the present model are indicative of a lower systemic sensitivity to the allergen used.
  • Low levels of IL5 and IL13 are indicative of reduced allergenicity of the tested WPC70 and thus also an infant formula containing these isolated whey proteins.
  • Results similar to the denatured WPC70 were obtained when a standard infant formula purchased from the supermarket was used. In particular, significantly higher ear swelling was measured, as well as high IgE levels and increased mast cell degranulation for the presently marketed infant formula. The results are depicted in the table below:
  • Example 5 Allergenicity IMF products obtained in Example 2
  • mice Four-week-old, specific pathogen free, female C3H/HeOuJ mice were purchased at Charles River Laboratories (The Netherlands) and housed at the animal facility of the Utrecht University on a 12 h light/dark cycle with access to food and water ad libitum. All animal procedures were conducted according to governmental guidelines and approved by the Ethical Committee for Animal Research of the Utrecht University, Utrecht, The Netherlands (CCD: AVD108002015346).
  • IMF infant milk formula
  • mice Five days after the last sensitization (day 33), mice were challenged intradermally (i.d.) in the ear pinnae of both ears with 10 pg denatured WPC70 in 20 pi PBS to determine the acute allergic skin response by locally measuring swelling of the skin of the ears via the thickness of the skin. On the same day, mice were challenged i.g. with 50 mg denatured WPC70 in 0.5 mL PBS. 18 h after the oral challenge blood samples were collected and centrifuged at 10.000 rpm for 10 min. Serum was obtained and stored at -20°C until further analysis. Mice were killed by cervical dislocation and samples were obtained for ex vivo analysis.
  • Results indicate that the native and deactivated (i.e. pasteurized) IMF samples elicit lower allergenic responses to antigen exposure in an in vivo setting.
  • the results are depicted in the table below:
  • Alkaline phosphatase (ALP) activity in native and heat treated WPC and IMF products was determined using an immunocapture assay using specialized ALP assay kits (IDBiotech, Rue Marie Curie, Issoire, France).
  • the kit contained an enzyme-linked immunosorbent assay (ELISA) plate coated with a monoclonal antibody specific to the alkaline phosphatase found in cow’s milk. 1 - butanol is the solvent used for enzyme extraction. Enzyme activity is expressed as equivalent- milliunit per litre (Eq.mU/l).
  • Sample preparation 3 ml of WPC (10 % protein) or IMF (1 0 % protein) was mixed with 3 ml of 1 -butanol, capped and mixed using a vortex for 30-40 s. Samples were then centrifuged between 2500-3500 g for 30 min. The aqueous phase was collected from beneath the fat layer and diluted between 1/5 - 1/200 (recommended) using the dilution buffer provided.
  • a standard solution was prepared as per the instructions within the assay kit, resulting in a working solution of 15,000 Eq.mU/l which in turn was diluted using the provided dilution buffer to create standards varying in concentration from 5,000-100 Eq.mU/l:
  • each well of the ELISA strips was washed with 300 pi of wash buffer, which was removed by inverting the plate. This is repeated 4 times. 100 pi of standard, control and sample solutions were added to the corresponding wells, the plate was covered and was shaken gently for 1 min and incubated for 1 hour at 18 - 25 °C. After incubation, the standard, control and sample solutions were removed from the wells (by inversion of the plate) and the wash step as described above was performed. 100 pi of substrate solution was added to each well. The plate was covered, shaken gently for 1 min and incubate for 2 hrs at 35- 38 °C. A yellow colour develops.
  • stop solution 50 pi of stop solution (provided in the kit) was added to all wells after incubation. The plastic cover was removed and the plate was read at 405 nm using a microplate reader. A calibration curve was obtained by plotting the optical density reading for the standard samples and this curve was used to determine the alkaline phosphatase activity in the WPC and IMF products.

Abstract

L'invention concerne un produit de formule pour nourrissons comprenant une protéine de lactosérum intacte et native destinée à être utilisée pour réduire ou prévenir une réponse allergique. Le produit de formule pour nourrissons comprend une protéine de lactosérum ayant une valeur de nativité d'au moins 90% et/ou qui peut être obtenue par un procédé consistant à : (a) traiter du lait dégraissé en un courant de caséine, un courant de protéine de lactosérum et un flux de lactose, par (i) soumission du lait dégraissé à une microfiltration sur une membrane capable de retenir les bactéries et d'assurer la perméation de protéines du lait ou à une étape de pasteurisation, en vue d'obtenir un lait débactérialisé ; (ii) soumission du perméat provenant de l'étape (i) à une étape de microfiltration sur une membrane pouvant retenir la caséine et assurer la perméation des protéines de lactosérum, pour obtenir un flux de caséine sous la forme d'un rétentat et d'un perméat comprenant une protéine de lactosérum ; et (iii) fractionnement du perméat provenant de l'étape (ii) en un flux de protéines de lactosérum et en un flux de lactose ; (b) combiner une partie du flux de caséine, d'au moins une partie du flux de protéines de lactosérum provenant de l'étape (a) et d'une source de lactose pour obtenir un flux recombiné ; (c) en option, pasteuriser le flux recombiné de l'étape (b), (d) utiliser le flux recombiné provenant de l'étape (a) ou (c) dans la fabrication du produit de formule pour nourrissons.
PCT/NL2018/050103 2018-02-15 2018-02-15 Protéine de lactosérum native pour réduire une allergie WO2019160402A1 (fr)

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PCT/NL2018/050103 WO2019160402A1 (fr) 2018-02-15 2018-02-15 Protéine de lactosérum native pour réduire une allergie
US16/968,167 US20210368816A1 (en) 2018-02-15 2019-02-15 Native whey protein for reducing allergy
AU2019220341A AU2019220341A1 (en) 2018-02-15 2019-02-15 Native whey protein for reducing allergy
CN201980025897.4A CN112004424A (zh) 2018-02-15 2019-02-15 用于减少过敏的天然乳清蛋白
EP19714887.7A EP3752009A1 (fr) 2018-02-15 2019-02-15 Protéine de lactosérum native pour réduire l'allergie
PCT/NL2019/050102 WO2019160416A1 (fr) 2018-02-15 2019-02-15 Protéine de lactosérum native pour réduire l'allergie

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