Native whey protein for treating and/or preventing intestinal infection

Abstract

The invention concerns native whey protein for use in the treatment and/or prevention of intestinal infection or inflammation, in particular necrotizing enterocolitis. The inventors found that native whey protein provides a beneficial effect on intestinal infection or inflammation.

Claims

1-20. (canceled)

21. A method for treatment and/or prevention of intestinal infection or inflammation in a subject in need thereof, comprising administering to the subject a native whey protein.

22. The method according to claim 21, wherein the intestinal infection is necrotizing enterocolitis.

23. The method according to claim 21, wherein the subject is a vulnerable subject.

24. The method according to claim 21, wherein the subject is an infant.

25. The method according to claim 21, wherein the subject is a preterm infant.

26. The method according to claim 21, wherein the intestinal infection or inflammation is an acute infection or inflammation.

27. The method according to claim 21, wherein the infection or inflammation is of an immature intestine.

28. The method according to claim 21, wherein the native whey protein has a nativity of more than 80%.

29. The method according to claim 28, wherein the native whey protein has a nativity of more than 90%.

30. The method according to claim 29, wherein the native whey protein has a nativity of more than 95%.

31. The method according to claim 21, wherein at least 70% of the α-lactalbumin is native and/or at least 70% of the β-lactoglobulin is native.

32. The method according to claim 31, wherein 75-95% of the α-lactalbumin is native and/or 80-100% of the β-lactoglobulin is native.

33. The method according to claim 31, wherein 78-85% of the α-lactalbumin is native and/or 85-95% of the β-lactoglobulin is native.

34. The method according to claim 21, wherein the treatment and/or prevention occurs with respect to the occurrence of intestinal infection or inflammation in a subject fed the same whey protein which is made non-native.

35. The method according to claim 21, wherein the intestine includes at least the colon.

36. The method according to claim 21, wherein the native whey protein is bovine whey protein obtained by a membrane-filtration based technology to retain the nativity of the whey protein.

37. The method according to claim 21, wherein the native whey protein is obtainable by a process comprising: (a) processing defatted milk into a casein stream, a whey protein stream and a lactose stream, by: (i) subjecting the defatted milk to microfiltration over a membrane capable of retaining bacteria and permeating milk proteins or to a pasteurization step, to provide a debacterialized milk; (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; (iii) fractionating the permeate originating from step (ii) into a whey protein stream and a lactose stream.

38. The method according to claim 21, wherein the native whey protein is comprised in a nutritional composition obtainable by a process comprising: (a) processing defatted milk into a casein stream, a whey protein stream and a lactose stream, by: (i) subjecting the defatted milk to microfiltration over a membrane capable of retaining bacteria and permeating milk proteins or to a pasteurization step, to provide a debacterialized milk; (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; (iii) fractionating the permeate originating from step (ii) into a whey protein stream and a lactose stream; (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 the recombined stream from step (b), (d) using the recombined stream originating from step (b) or (c) in the manufacture of the nutritional composition.

39. The native whey protein for use according to any one of claim 38, wherein one or more of the following applies: the defatted milk is defatted bovine milk; at least part of the lactose stream originating from step (a) is used as lactose source in step (b); step (iii) is performed by ultrafiltration over a membrane capable of retaining whey proteins and permeating lactose, to provide a whey protein stream as retentate and a permeate comprising lactose; step (iii) is performed by ultrafiltration with a volume concentration factor in the range of 20-200; the defatted milk is the sole protein source for the infant formula; the manufacturing of step (d) includes at least one of drying, concentrating, supplementing with vitamins, minerals, lipids and/or dietary fibres, packaging; the nutritional composition is a powder obtained by spray-drying; the nutritional composition is a powder obtained by spray-drying as part of step (d).

40. The native whey protein for use according to claim 21, wherein the nutritional composition is not subjected to heat treatment and/or wherein the infant formula exhibits an alkaline phosphatase activity of at least 25 mU/g, or the nutritional composition is a pasteurized nutritional composition and/or wherein the nutritional composition exhibits an alkaline phosphatase activity of at most 20 mU/g.

Description

DESCRIPTION OF THE FIGURES

[0109] The figures depict the results of Example 6.

[0110] FIG. 1A depicts the Ki67 positive proliferating cells per crypt, observed for the preterm group (PT) and the near term group (NT). FIG. 1B depicts the number of animals with a histological positive alkaline activity, observed for the preterm group (PT) and the near term group

[0111] (NT), wherein negative is white and positive is shaded. Herein, P=PAST-WPC diet according to the invention and D=DENAT-WPC control diet.

[0112] FIG. 2A depicts the ALP activity (in units) for the preterm group (PT) and the near term group (NT). FIG. 2B depicts the same ALP activity, but after specific blockage of iALP (pre-incubation with L-PHE). FIG. 2C depicts the ALP activity after exclusion of samples from the NEC positive piglets. Herein, P=PAST-WPC diet according to the invention and D=DENAT-WPC control diet.

[0113] FIG. 3 depicts the relative expression of (A) CD14 and (B) IL-8 as observed in the colon tissue of the preterm group (PT) and the near term group (NT). Herein, P=PAST-WPC diet according to the invention and D=DENAT-WPC control diet.

[0114] FIG. 4 depicts the neutrophil response based on MPO staining (y-axis) for the near term group. Herein, P=PAST-WPC diet according to the invention and D=DENAT-WPC control diet.

EXAMPLES

[0115] The following examples illustrate the invention.

Example 1: WPC70 Preparation

[0116] Three 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 μM Synder membrane FR (PVDF 800 kDa) 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 10 kDa Synder membrane ST (PES 10 kDa) spiral wound membrane.

[0117] Diafiltration medium was added to improve separation efficiency of membranes (200% of original starting skim milk volume). Concentrated liquid WPC70 (DM 11%) was stored at 4° C. until further handling. The WPC70 was heated to 30° C. and spray dried at 11% 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.

[0118] Deactivated WPC70 was prepared to represent a highly native, pasteurized protein sample which can be included in an infant formula. It 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 73° C./30 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 inactivated bioactive components, indicated by the inactivation of alkaline phosphatase, and whey protein nativity of >95%.

[0119] 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 of <30%.

[0120] The composition of the three WPC70 products, as a 7% protein solution (see Example 3), is given in the table below (in wt % based on dry weight):

TABLE-US-00001 Total True Protein NPN NCN protein Casein Whey Nativity Native WPC70 7.13 0.15 5.33 6.97 1.65 5.30 .sup. 100% Deactivated WPC70 7.11 0.15 5.23 6.96 1.73 5.08 95.73% Denatured WPC70 6.70 0.16 1.16 6.54 5.38 1.14 21.43%

Example 2: IMF Preparation

[0121] Three 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. Galacto-oligossaccharide (GOS) syrup was added to the mix once the casein and whey protein powders had sufficiently hydrated and mixed for 15 min. 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.

[0122] For 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) was subjected directly to a multi-stage Anhydro spray-dryer (water evaporation capacity (WEC) 30 kg/hr) operated with an inlet temperature of 185° C. and an outlet temperature of 90° C., resulting in a powdered native IMF with <4% moisture.

[0123] 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 of >95% and inactivated bioactive components as indicated by the inactivation of the enzyme alkaline phosphatase.

[0124] 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 of <40%.

[0125] The composition of the three IMF products, as a 10% protein solution (see Example 3), is given in the table below (in wt % based on dry weight):

TABLE-US-00002 Total True Protein NPN NCN protein Casein Whey Nativity Native IMF 10.19 0.41 6.44 9.78 3.34 6.38 .sup. 100% Deactivated IMF 10.05 0.40 6.25 9.65 3.40 6.19 97.03% Denatured IMF 9.98 0.40 2.43 9.59 7.15 2.37 37.16%

Example 3: Nativity Calculation

[0126] The total nitrogen (TN), non-protein nitrogen (NPN) and non-casein nitrogen (NCN) were determined via kjeldahl analysis, as per the ISO 8968-3/IDF 20-3:2004 standard (Milk—Determination of nitrogen content—Part 3: Block-digestion method (Semi-micro rapid routine method), 2004), using an automatic Kjeltec 8400 unit (FOSS, Warrington, U.K). The nativitys of the whey proteins in Examples 1 and 2 were calculated as follows:

(a) Casein fraction=(TP−NPN)−NCN
(b) Whey fraction=NCN−NPN
(c) Nativity=measured whey fraction (b)/theoretical whey faction*100% [0127] The theoretical whey fraction is based on the casein/whey protein ratio of the product, from the recipe of the product.

Example 4: Determination of Alkaline Phosphatase Activity

[0128] 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.Math.mU/I).

[0129] Sample preparation: 3 ml of WPC (10% protein) or IMF (10% 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.

[0130] A standard solution was prepared as per the instructions within the assay kit, resulting in a working solution of 15,000 Eq.Math.mU/I which in turn was diluted using the provided dilution buffer to create standards varying in concentration from 5,000-100 Eq.Math.mU/I: [0131] STD1 5000 Eq.Math.mU/I: 125 μl (15,000 Eq.Math.mU/I)+250 μl dilution buffer; [0132] STD2 3000 Eq.Math.mU/I: 75 μl (15,000 Eq.Math.mU/I)+300 μl dilution buffer; [0133] STD3 1000 Eq.Math.mU/I: 25 μl (15,000 Eq.Math.mU/I)+350 μl dilution buffer; [0134] STD4 500 Eq.Math.mU/I: 50 μl (STD1)+450 μl dilution buffer; [0135] STD5 300 Eq.Math.mU/I: 50 μl (STD2)+450 μl dilution buffer; [0136] STD6 100 Eq.Math.mU/I: 50 μl (STD3)+450 μl dilution buffer.

[0137] To run the assay: each well of the ELISA strips was washed with 300 μl of wash buffer, which was removed by inverting the plate. This is repeated 4 times. 100 μl 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 μl 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. 50 μl 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.

[0138] The results are given in the table below:

TABLE-US-00003 ALP activity Native WPC70 195 mU/g Deactivated WPC70 Not determined Native IMF 33 mU/g Deactivated IMF <3 mU/g

Example 5: Determination of Alkaline Phosphatase Activity

[0139] The alkaline phosphatase (ALP) activity in native WPC and IMF products according to the invention (without and with denaturation) was determined in mU/L via ISO standard 11816-1 (version valid in October 2018). Solutions were prepared and tested according to the test protocol. The results for all four products at 1.3 wt % protein (based on total weight), which is the protein content of standard infant formulae, are given in the table below:

TABLE-US-00004 ALP activity Native WPC70 1.8 × 10.sup.4 mU/L Denatured WPC70 <20 mU/L Native IMF 2.1 × 10.sup.4 mU/L Denatured IMF <20 mU/L

Example 6: Effect on Necrotizing Enterocolitis (NEC)

[0140] A deactivated whey protein product with high nativity of over 90%, obtained according to the process of Example 1 was analysed with respect to its properties to prevent intestinal infection or inflammation exemplified by the occurrence of NEC in piglets. NEC symptoms in piglets were compared to an identical whey protein product which was denatured to a nativity level of below 40% by extensive heating as detailed below.

[0141] Piglet study: Preterm pigs (Danish Landrace×Large White×Duroc) were delivered from sows by caesarean section at approximately 90% gestation (2 litters, preterm group) and approximately 96% gestation (1 litter, near term group). Surgical preparation with an oro-gastric feeding tube and a vascular catheter for parental nutrition (PN) and passive immunization took place as previously described (Cilieborg M S, Boye M, Thymann T, et al., J Parenter Enteral Nutr. 2011; 35:32-42). The pigs from each litter were stratified according to birth weight into 2 groups receiving 2 types of enteral diets (n=7-9/group/per litter): 1) PAST-WPC group received formula based on deactivated WPC (i.e. pasteurized WPC by heating at 73° C. for 30 seconds which maintains proteins in their native form; native whey protein formula group) and 2) DENAT-WPC group received formula with WPC that was pasteurized and additionally heat-treated (i.e. heated 73° C., 30 sec+80° C., 6 min, heated whey protein formula group), which results in extensive protein denaturation. The WPC used in this study was prepared from raw cow's milk by the process of Example 1 which did not involve heating. Each formula consisted of 80 g/L whey protein concentrate, 50 g/L Pepdite, 50 g/L Liquigen and 30 g/L Calogen. Macronutrient composition of the formulas was as follows: 3629 kJ/L Energy, 59 g/L Protein, 52 g/L Fat, 39 g/L Carbohydrate, 16 g/L Lactose. During the study period, pigs in each group received enteral nutrition as described in table below. In addition, all pigs received parenteral nutrition (PN), 4 ml/kg/h from day 1 to day 5. The piglets in both the preterm and near term litter had the same weight gain regardless the dietary group they were assigned (data not shown). PN solution was based on a commercially available product (Kabiven, Fresenius Kabi) and adjusted in nutrient composition to meet the requirement of pigs. On day 5 pigs were anaesthetized and blood was collected. Subsequently, pigs were euthanized followed by collection of urine and intestinal tissues.

TABLE-US-00005 TABLE 1 Feeding regimen Time Enteral Nutrition Feeding Regime Day 1 6 ml/kg/3 h Day 2 8 ml/kg/3 h Day 3 8 ml/kg/3 h Day 4 10 ml/kg/3 h Day 5 10 ml/kg/3 h

[0142] Clinical evaluation and sample collection: Pigs were continuously monitored and sacrificed when clinical symptoms of NEC appeared during the study or on day 5 for tissue collection. Tissues of the stomach, 3 regions of small intestine [proximal, middle, and distal] and colon were macroscopically evaluated for incidence and severity of necrotizing enterocolitis (NEC) (see table below & as described previously in Cilieborg M S, Boye M, Thymann T et al., J Parenter Enteral Nutr. 2011; 35:32-42). Pigs with macroscopic lesion score of in any of the segments were diagnosed as NEC.

TABLE-US-00006 TABLE 2 Macroscopic evaluation scores Score Macroscopic evaluation 1 No or minimal hyperemic gastroenterocolitis 2 Mild focal gastroenterocolitis 3 Moderate local extensive gastroenterocolitis 4 Severe focal gastroenterocolitis 5 Severe local extensive hemorrhagic and necrotizing gastroenterocolitis 6 Extensive hemorrhagic and necrotizing gastroenterocolitis

TABLE-US-00007 TABLE 3 Microscopic evaluation scores, NEC scoring and immunohistochemistry: Paraformaldehyde fixed tissues from colon were processed for microscopy/histology to determine microscopic tissue damage and the NEC severity score. Score Epithelium Edema Infiltration Erythrocytes 1 Normal None None Absent/Normal 2 Disorganized Some Between crypts In lamina propria 3 Discontinuous Throughout In lamina Patchy between propria crypts 4 Absent Extreme Transmural Extreme

[0143] To localize proliferating epithelial cells (i.e. Ki67 staining), enteroendocrine cells (i.e. serotonin (5HT) staining), neutrophils (i.e. myeloperoxidase (MPO) staining) and to determine brush border expression of iALP (i.e., iALP staining) immunohistochemistry was performed as described previously (Marit Navis, Tania Martins Garcia, Ingrid B Renes et al., EMBO Reports (2018), DOI 10.15252/embr.201846221). The following antibodies were used: rabbit polyclonal anti-Ki67 (1:8000, Abcam, ab15580), rabbit polyclonal anti-MPO (1:100, Abcam, ab9353) and mouse monoclonal anti-5HT-H209 (1:100, ThermoFisher Scientific MA5-12111). Moreover, iALP brushborder activity on tissue slides was determined with NBT/BCIP conversion as described previously (Srivillibhuthur M, Verzi et al., DOI: 10.1016/j.ydbio.2018.04.015) (Schneeberger K, Middendorp S et al., DOI: 10.1073/pnas.1516672112).

[0144] Additionally, tissue from the colon was homogenized and activity of iALP was determined by spectrophotometry with a diethanolamine assay (EC 3.1.3.1) measuring pNPP hydrolysis according to manufacturer's instruction (Phosphatase substrate, ThermoFisher

[0145] Scientific) and previous described (Arnal M E, Lallès J P et al., DOI: 10.1371/journal.pone.0118092), in presence of L-phenylalanine (Gosh N K and Fishman W H, PMID: 5911626).

[0146] Additionally, mRNA was isolated from colon to determine gene expression levels of intestinal inflammation markers by quantitative-PCR analysis. In brief, RNA was isolated with TRI-reagent (Sigma-Aldrich) and purified with the Bioline ISOLATE II RNA Mini kit (BIO-25073, Bioline) according to manufacturer's protocol. 1.0 μg of RNA was transcribed using Revertaid reverse transcriptase (Fermentas, Vilnius, Lithuania). Quantitative RT-PCR was performed on a BioRad iCycler using sensifast SYBR No-ROX Kit (GC-biotech Bio-98020) according to manufacturer's instructions. From a panel of 7 genes, most stable reference genes were identified by GeNorm. Relative expression levels were calculated with NO values obtained by LinRegPCR and normalized to reference genes. Primers used were specific for pig IL8 and CD14, and validated based on melting curves and product size.

[0147] Results: The piglets experienced regular body weight gain which did not differ significantly between both treatment groups nor did the weight of the colon of the piglets differ (data not shown). Macroscopic evaluation of the colon of the preterms showed a mean score of 2 in the native whey protein formula fed piglets and a mean score of 3 in the heated whey protein formula fed piglets. In the near term piglets a significant difference in macroscopic scores was observed between the two diet groups with the native whey protein formula fed piglets having a mean score of 1 and the heated whey protein formula fed piglets having a mean score of 3 (p<0.05). Pigs with macroscopic lesion score of in any of the segments were diagnosed as NEC. The table below sets out the incidence of NEC in the different treatment groups. The incidence was significantly different between the two dietary inventions when including all regions analysed (two-way ANOVA p<0.05)

TABLE-US-00008 TABLE 4 Incidence of NEC Preterm piglets Near term piglets Native Heated Native Heated n 17 17 8 9 NEC incidence* 59% 82% 0% 56% NEC lesion score stomach 2.24 3.06 1.00 2.33 NEC lesion score prox SI 1.11 1.71 1.00 1.00 NEC lesion score mid SI 1.41 1.94 1.11 1.00 NEC lesion score dist SI 1.76 1.94 1.00 1.56 NEC lesion score colon 2.53 3.13 1.67 2.44

[0148] Microscopic analysis of colon sections showed that in the preterm piglets the mean histology score was 5 in the native whey protein formula fed piglets, versus 8 in the heated whey protein formula fed piglets. In the near term piglets a significantly lower mean histology score of 3 was observed for the native whey protein formula fed piglets whereas the heated whey protein formula fed piglets had a mean histology score of 9 (p<0.05). Overall microscopic evaluation of intestinal epithelial tissues obtained from sacrificed piglets shows lower grades of inflammatory signs and lesions for the native whey protein formula vs the heated whey protein formula samples (data not shown). On histological slides a significantly decreased number of Ki67 positive proliferating cells per crypt were observed in the native whey protein treated piglets as compared to the heated whey protein formula treated piglets both in the preterm (p<0.001) and the term ((p<0.05) groups (FIG. 1A). The lower level of proliferating cells per crypt is indicative of a more differentiated phenotype of the colon and improved gut maturation in the native whey protein formula fed piglets. Histological colon slides from native whey protein treated piglets had prominent apical brush border ALP activity staining (data not shown). A significantly increased number of piglets in the preterm group treated with the native whey protein formula had a positive iALP staining compared to the piglets fed the heated whey protein formula (p<0.01). A comparable trend is observed in the near term piglets with more native whey protein formula fed piglets having a positive histological iALP score than the heated whey protein formula fed piglets (FIG. 1B). The increased iALP score is indicative of increased intestinal ALP activity in the colon of the native whey protein formula fed piglets and a more mature immune function of the gut. iALP activity is known to be associated with the deactivation of intraluminal lipopolysaccharide (LPS) a.o. in the presence of pathogenic bacteria and may thereby prevent the mucosal damage at the onset of NEC.

[0149] Spectroscopic analysis of intestinal alkaline phosphatase (iALP) activity in colon homogenates showed a significantly increased iALP activity in the preterm piglets fed the native whey protein formula as compared to those piglets born preterm and fed the heated whey protein formula. In the near term piglets fed the native whey protein formula there is a trend towards an increased iALP activity as compared to the piglets fed the heated whey protein formula (FIG. 2a). No significant difference was observed in ALP activity in the samples after specific blockage of iALP between the different diet groups in either the preterm or the near term piglets (FIG. 2b). Increased iALP enzyme activity in the animals fed the native whey protein composition is suggestive for a more mature immune function of the gut. Upon exclusion of samples from the NEC positive piglets from the data analysis a similar but more pronounced pattern of increased iALP activity in samples from piglets fed the native whey protein formula is observed (FIG. 2c)

[0150] The relative expression of CD14, a pro-inflammatory cytokine, was significantly decreased in both the preterm (p<0.01) and the near term (p<0.01) piglets that were fed the native whey protein formula compared to the expression in colon tissue of the piglets fed the heated whey protein formula (FIG. 3A). The expression of IL-8, also a pro-inflammatory cytokine and clinically associated with NEC severity (Maheswari A et al. Pediatr. Res. 2014 July 76(1); 100-108), was likewise significantly decreased in both the preterm (p<0.01) and the near term (p<0.05) piglets that were fed the native whey protein formula compared to the expression in colon tissue of the piglets fed the heated whey protein formula (FIG. 3B). The pro-inflammatory mediators mentioned above are capable of attracting further inflammatory cells, induce the production of reactive oxygen species and thereby inflict damage to the intestinal barrier, intestinal epithelial damage ultimately triggering apoptosis and mucosal necrosis. (Hunter et al. pathophysiology 21 (2014) 55-65). Overall, there is a clear indication that the animals fed the native whey protein formula according to the invention have decreased relative expression levels of pro-inflammatory cytokines, accordingly a cytokine pattern that is less pro-inflammatory than the animals fed the heated whey protein formula, and as such a lower risk of developing NEC. The neutrophil response was significantly decreased for the near term piglets that were fed with the native whey protein formula, compared to the response of piglets fed with the heated whey protein (FIG. 4), again indicative of a reduced inflammation in subjects administered with the composition according to the invention.

Example 7. Infant Formula Containing Native Whey Protein Concentrate (WPC)

[0151] An infant formula containing native whey protein according to the invention is exemplified as follows. The main nutrients of this infant formula are as follows:

TABLE-US-00009 Units Per 100 ml RTF Per 100 kcal Energy value kcal 66 100 Protein g 1.3 2 Whey g 0.8 1.2 Casein g 0.5 0.8 Carbohydrate g 7.3 11.1 of which sugars g 7.2 10.9 Glucose g 0.2 0.3 Lactose g 7.0 10.6 Galactose g 0.01 0.02 Polysaccharides g 0.01 0.02 Fat g 3.4 5.1 Vegetable g 3.3 5 Animal g 0.1 0.1 Saturated g 1.5 2.2 Monounsaturated g 1.4 2.1 Polyunsaturated g 0.6 0.8 Dietary fibre g 0.6 0.9

[0152] The infant formula is intended for feeding of term infants aged 0 to 3 months. In terms of energy value, the infant formula contains 8 En % protein, 44 En % carbohydrate, 46 En % fat and 2 En % dietary fibre. Minerals and vitamins are included according to prevailing nutritional guidelines to produce a complete enteral infant feed. The indicated totals may not be reached due to rounding off of values. RTF=Ready-To-Feed. Whey protein is present in the infant formula with a nativity of more than 90%. The ALP activity of the RTF infant formula is either not higher than 350 mU/L and considered ALP negative or ALP positive in which case ALP activity is above 350 mU/L. Microbial safety of the ALP positive infant formula is ensured by microfiltration over a membrane capable of retaining bacteria.

Example 8. Preterm Formula Containing Native Whey Protein Concentrate (WPC)

[0153] A preterm formula containing native whey protein according to the invention is exemplified as follows. The main nutrients of this preterm formula are as follows.

TABLE-US-00010 Units Per 100 ml RTF Per 100 kcal Energy value kcal 78 100 Protein g 2.6 3.3 Whey g 1.5 2.0 Casein g 1.0 1.3 Carbohydrate g 8.2 10.4 of which sugars g 6.1 7.7 Glucose g 0.3 0.4 Lactose g 5.5 6.9 Maltose g 0.2 0.3 Polysaccharides g 2.1 2.6 Fat g 3.8 4.8 Vegetable g 3.4 4.2 Animal g 0.3 0.5 Saturated g 1.6 2.0 Monounsaturated g 1.4 1.8 Polyunsaturated g 0.8 1.0 Dietary fibre g 0.6 0.7 Minerals g 0.2 0.2

[0154] The preterm formula is intended for feeding of preterm infants, meaning infants born before the 37.sup.th week of gestation. The protein amount is increased compared to infant formula intended for feeding of term infants for reasons related to catch-up growth which is intended to occur in preterm-born infants. In terms of energy value, the preterm formula contains 13 En % protein, 42 En % carbohydrate, 44 En % fat and 1 En % dietary fibre. RTF=Ready-To-Feed. Minerals and vitamins are included according to nutritional guidelines to produce a complete enteral preterm feed. The indicated totals may not be reached due to rounding off of values. Whey protein is present in the preterm formula with a nativity of more than 90%. The ALP activity of the RTF preterm formula is not higher than 350 mU/L and considered ALP negative.