Native whey protein composition for improving gastro-intestinal tolerance
20230380438 · 2023-11-30
Assignee
Inventors
Cpc classification
A23C9/1422
HUMAN NECESSITIES
A23L33/40
HUMAN NECESSITIES
A23C21/06
HUMAN NECESSITIES
International classification
A23C21/06
HUMAN NECESSITIES
Abstract
The invention concerns a native whey protein composition with profound amounts of native whey protein and with beta-casein for use in the treatment and/or prevention of gastrointestinal intolerance. The inventors found that the native whey protein composition relatively high in beta-casein and low in kappa- and alpha-casein provides a beneficial effect on gastrointestinal intolerance.
Claims
22. A nutritional composition comprising 3 to 7 g lipid/100 kcal, 1.25 to 5 g protein/100 kcal and 6 to 18 g digestible carbohydrate/100 kcal, wherein the protein fraction consists of whey protein and beta-casein, and is substantially devoid of alpha-casein and kappa-casein, and wherein the formula optionally comprises added free amino acids, wherein the ratio of whey protein to beta-casein is in the range between 85:15 and 55:45, wherein the protein fraction has been pasteurized, and wherein the whey protein has a nativity of more than 80%.
23. The nutritional composition according to claim 22, comprising less than 6 wt % of the sum of alpha-casein and kappacasein, based on the total protein weight of the protein fraction.
24. The nutritional composition according to claim 22, wherein the ratio beta-lactoglobulin to alphalactalbumin is below 7:3.
25. The nutritional composition according to claim 22, wherein the ratio of whey protein to beta-casein is in the range between 80:20 and 60:40.
26. The nutritional composition according to claim 22, wherein the whey protein has a nativity of more than 90%.
27. The nutritional composition according to claim 22, wherein the nutritional composition is selected from a preterm formula, an infant formula and a follow-on formula.
28. The nutritional composition according to claim 22, wherein the native whey protein composition has a protein solubility of more than 55% based on the total amount of protein in the native whey protein composition at acidic pH conditions.
29. The nutritional composition according to claim 22, wherein the native whey does not originate from acid whey or from sweet whey.
30. The nutritional composition according to claim 22, wherein the native whey protein composition is obtainable by cold membrane-filtration based technology.
31. The nutritional composition according to claim 22, wherein the native whey protein composition has been pasteurized at 72-74° C. for 15 to 30 seconds.
32. The nutritional composition according to claim 22, wherein the nutritional composition exhibits an alkaline phosphatase activity of at most 350 mU/L.
33. The nutritional composition according to claim 22, wherein the native whey protein composition and optionally at most 2 wt % of added free amino acids based on the total weight of protein in the nutritional composition are the sole protein sources for the nutritional composition.
34. The nutritional composition according to claim 22, 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: subjecting the defatted milk to a debacterialization treatment, to provide a debacterialized milk; (ii) subjecting the debacterialized milk originating from step (i) to cold 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 and β-casein; (iii) fractionating the permeate originating from step (ii) into a whey protein stream comprising 1) whey protein and β-casein and 2) a lactose stream; (b″) optionally spray-drying the whey protein stream originating from (a)-(iii) followed by dissolving; wherein at least one of the debacterialization treatment of step (a)-(i) or the stream originating from step (a)-(iii) after optional step (b″) is subjected to pasteurization; and; (c″) optionally freeze-drying the stream, and wherein the debacterialization treatment (i) if not pasteurization, involves subjecting the defatted milk to microfiltration over a membrane capable of retaining bacteria, wherein the debacterialized milk is in the permeate.
35. The nutritional composition according to claim 34, wherein 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.
36. The nutritional composition according to claim 35, wherein ultrafiltration step (iii) operates with a volume concentration factor in the range of 20-200.
37. A method for administering a nutritional composition comprising beta-casein and whey protein for reducing the rate and/or extent of coagulation in the stomach; and/or increasing the rate of gastric emptying; and/or improving intestinal transit in a subject that is not at imminent or at increased risk of gastrointestinal intolerance, wherein the nutritional composition is a composition according to claim 1, and wherein administration of the composition to the subject reduces the rate and/or extent of coagulation in the stomach; and/or increases the rate of gastric emptying; and/or improves intestinal transit in the subject.
38. The method according to claim 37, wherein administration of the composition to the subject prevents, reduces or treats regurgitation and/or reflux.
39. The method according to claim 34, wherein the subject is an infant selected from preterm infants, infant that is small for gestational age, infant with a low birth weight, very young infant and/or infant suffering from reflux and/or mild regurgitation.
Description
DESCRIPTION OF THE FIGURES
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EXAMPLES
[0200] The following examples illustrate the invention.
Example 1: WPC Preparation
[0201] Two whey protein compositions comprising whey protein and casein [WPC] products, (i) mildly-pasteurized WPC [MP-WPC], and (ii) extensively heat-treated WPC [EH-WPC], 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 cold to separate casein from both whey and lactose. Microfiltration membrane used was a 0.08 M 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, β-casein, lactose and ash. The operating temperature was 10° C. and volume concentration factor (VCF) was 3. 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 WPC was produced. The ultrafiltration membrane used was a 10 kDa Synder membrane ST (PES 10 kDa) spiral wound membrane. Diafiltration medium was added to improve separation efficiency of membranes (200% of original starting skim milk volume). Concentrated liquid WPC (DM 11%) was stored at 4° C. until further handling. The WPC 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 native WPC.
[0202] The spray-dried WPC was then prepared to represent a mildly treated highly native, pasteurized protein sample which can be included in an infant formula. It was prepared by re-hydrating 100 g/L of native WPC 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% (about 70 g protein per liter). This solution was heat-treated at 73° C./30 s using a MicroThermics tubular heat exchanger (MicroThermics, North Carolina, USA) and then freeze-dried resulting in MP-WPC powder with whey protein nativity of >95%, determined according to example 2.
[0203] MP-WPC powder was dissolved in demineralized water at 100 g/L, corresponding to 68.3 g protein per liter. pH was adjusted to 7.1 by addition of 1M NaOH, to prevent gelation during heating. Half of the MP-WPC solution was used without further processing (MP-WPC). The other part was thermally treated to denature whey protein using a shaking water bath at 80° C. for 20 min: extensively heated-WPC (EH-WPC). Temperature was monitored and reached 80° C. after 14 min, thus the effective thermal treatment at 80° C. was 6 min. The EH-WPC was then freeze-dried resulting in a WPC powder with whey protein nativity of <30%.
Example 2: Soluble Protein Fraction Determination and Nativity Calculation
[0204] To determine the soluble protein fraction, WPC protein solution at pH 7.1 was centrifuged at 15000×g for 30 min and the supernatant was collected for protein quantification. The level of protein denaturation was determined by precipitation of the aggregated and unfolded proteins at pH 4.6 (adjusted by 0.1 M HCl) followed by centrifugation at 15000×g for 30 min to collect the supernatant as described by: Delahaije, R. J Journal of agricultural and food chemistry 2016, 64, 4362-4370. Crude protein (N×6.25) quantification in the total protein solution, the soluble fraction and the native fraction was performed by DUMAS as described in Chibnall A C, The Biochemical journal 1943, 37, 354-359, and soluble and native protein were expressed as % (w/w) of total protein.
[0205] Extensive heating only mildly affected protein solubility at pH 7.1, with 98% for MP-WPC compared with 87% for EH-WPC. In contrast, protein solubility at pH 4.6 was highly affected by the extensive heat treatment, with 72% for MP-WPC compared with 21% for the EH-WPC.
[0206] Since all milk proteins except native whey proteins precipitate at pH 4.6 and between 24-28% of the protein in the WPC fractions was β-casein (table 1), it follows that the whey protein fraction can be considered close to 100% native in the MP-WPC and only 30% native in EH-WPC.
[0207] 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 nativities of the whey proteins in Example 1 was calculated as follows: [0208] (a) Casein fraction=(TP−NPN)−NCN [0209] (b) Whey fraction=NCN−NPN [0210] (c) Nativity=measured whey fraction (b)/theoretical whey faction*100%
The theoretical whey fraction is based on the casein/whey protein ratio of the product, from the recipe of the product.
Example 3: Protein Profile Analysis
[0211] The protein composition of both fresh and digested EH-WPC and MP-WPC was evaluated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on NuPAGE™ 4-12% bis-tris midi protein gel (ThermoFisher Scientific, Amsterdam, the Netherlands). Equal levels of protein per sample were loaded, as determined by BCA reaction. Analysis was performed under reducing (with 2-mercaptoethanol (2ME)), and also under non-reducing conditions, to evaluate the protein composition of aggregates held together by disulfide bridges. As a result of heat treatment, disulfide bond formation can occur between two cysteine residues present in the proteins, creating aggregation of proteins. Proteins and peptides were identified using the PageRuler™ Plus protein ladder (10-250 kDa, #26619, ThermoFisher Scientific, Amsterdam, the Netherlands), and included bovine serum albumin (BSA) 69 kDa, immunoglobulin G heavy chain polypeptide (IgG HC) 53 kDa, β-casein ((3CAS) 24 kDa, (i-lactoglobulin ((3LG) 18 kDa and α-lactalbumin 14 kDa. Gels were scanned using Gel Doc Universal Hood II (Bio-rad, California, US) and densitometric analysis was performed in Quantity One version 4.6.9 (Bio-Rad, California, US). Each peak intensity displayed on the densitogram corresponds to the amount of soluble proteins that migrated through the gel to their specific protein size.
[0212] Both BSA and IgG (HC) were found to be aggregated, as these bands disappeared under non-reducing conditions for both EH-WPC and MP-WPC. BSA consists of mixed disulfides that can form polymers, which react very rapidly to give aggregates upon heat treatment, even after mild pasteurization only. IgG heavy chains are connected by disulfide bridges to form dimers in normal conformation. The effect of extensive heat treatment was most striking for α-lactalbumin and to a smaller extent also for β-lactoglobulin, with a decrease in band intensity under non-reducing conditions for EH-WPC only. This data implies that α-lactalbumin and β-lactoglobulin in the MP-WPC are present freely without a covalent link to other proteins, but upon extensive heating become a covalently linked part of protein aggregates/polymers. Beta-casein was found to be present at a weight percentage of about 24-28%, no other types of caseins were observed in the SDS-PAGE.
[0213] The protein composition in WPC was determined by SDS-PAGE analysis as percentage of the total protein.
TABLE-US-00001 TABLE 1 Protein composition whey protein compositions comprising native whey protein and casein [WPC] % of total protein Whey protein composition [WPC] β-lactoglobulin 37%-39% α-lactalbumin 20%-23% β-casein 24%-28% Other whey proteins (a.o. BSA, IgG) 13%-16%
Example 4: Protein Maillardation Determination
[0214] To gain insights in the degree of protein Maillardation, the level of carboxymethyllysine (CML), an advanced glycation end-product, was determined by UFLC/Flu method. Protein was hydrolysed in 6M hydrochloric acid, and CML in the hydrolysate was quantified in μg/ml by UFLC using a pre-column derivatization with o-phtaldialdehyde and fluorimetry as detection.
[0215] Compared with EH-WPC, there was a significantly lower level of CML detected in the MP-WPC; EH-WPC had a CML level of 18.8 μg/ml ±1.6 and the MP-WPC had a CML level of 16.1 μg/ml ±0.4 respectively.
Example 5: In Vivo Effect on Gastrointestinal Tolerance
[0216] A MP-WPC 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 gastrointestinal intolerance prevention, gastric emptying and upper gastrointestinal coagulation behaviour. Gastric parameters in near-term and preterm piglets were compared to an identical whey protein product which was denatured to a nativity level of below 40% by extensive heating obtained according to the process of Example 1.
[0217] Piglet study: Preterm and near-term pigs (Danish Landrace×Large White×Duroc) were delivered from sows by caesarean section at 90% gestation (106 days of gestation, n=34, 2 litters) and 96% gestation (113 days of gestation, n=18, 1 litter) respectively. Piglets were transferred to the intensive care unit and housed individually in heated incubators with air and oxygen supply. Piglets were passively immunized and surgically prepared with an orogastric feeding tube and an arterial catheter for parenteral nutrition (PN). Piglets from each litter were block randomized according to birthweight into two groups of enteral diets: 1) EH-WPC group and 2) MP-WPC group. During the study, investigators were all blinded for type of diet. The Danish National Committee on Animal Experimentation approved all procedures, which is in accordance with the EU Directive 2010/63/EU Article 23.2 and the Danish executive order no 2014-15-0201-00418. Piglets received minimal enteral nutrition for 5 days based on EH-WPC or MP-WPC, via an orogastric tube (6F, Portex, UK). Each formula consisted of 80 g/L WPC, 50 g/L Pepdite (infant milk formula containing non-milk derived low molecular weight peptides, essential amino acids, carbohydrates, fats, vitamins, minerals and trace elements), 50 g/L Liquigen (medium-chain fatty acids) and 30 g/L Calogen (long-chain fatty acids) (all obtained from Nutricia advanced medical nutrition). Macronutrient composition of the formulas was as follows: 3629 kJ/L energy, 59 g/L protein, 52 g/L fat, 39 g/L carbohydrate (of which 21 g/L maltodextrin, 2.7 g/L maltotriose and 1.8 g/L maltose and 16 g/L lactose). During the study period, piglets received the enteral nutrition via the orogastric tube in increasing dose as indicated in Table 2 below, with additional continuous parenteral nutrition support (Kabiven, Fresenius Kabi) through an umbilical catheter. 1 h prior to euthanasia on day 5, the pigs received a last bolus EN of 15 ml/kg bodyweight. Additional enteral boluses via the orogastric tube included galactose on day 3, lactose and x-ray contrast fluid (Iodixnol, Visipaque) on day 4 and lactulose/mannitol at day 5, as indicated in Table 2.
TABLE-US-00002 TABLE 2 Feeding regimen Enteral Parenteral Time Nutrition Nutrition Bolus Day 1 6 ml/kg/3 h 4 ml/kg/h — Day 2 8 ml/kg/3 h 4 ml/kg/h — Day 3 8 ml/kg/3 h 4 ml/kg/h 5% galactose: 15 ml/kg Day 4 10 ml/kg/3 h 4 ml/kg/h 10% lactose: 15 ml/kg Day 5 10 ml/kg/3 h 4 ml/kg/h 5% lactulose/mannitol: 15 ml/kg 3 hrs. before euthanasia last bolus of EN: 15 ml/kg 1 hr before euthanasia
[0218] Clinical evaluation and sample collection: Pigs were continuously monitored for clinical symptoms of feeding intolerance, vomiting, abdominal distention, haemorrhagic diarrhoea and/or respiratory distress during the entire study. Gastric residuals were measured and sampled to investigate the value of gastric residual as a marker of feeding tolerance. Aspirates were taken three times a day, prior to EN feeding (i.e., 3 h after the previous bolus). One ml of air was put into the feeding tube, after which gastric content was pulled back up until vacuum was reached. The total volume of gastric aspirate was measured and, when possible, 1 ml of aspirate was collected and stored at −80° C. Remaining gastric aspirate was returned into the stomach gently via the tube. Fecal assessment was performed twice a day based on stool frequency and stool consistency, according to Table 3 below. Both clinical and fecal assessment were performed by personnel blinded for the diet.
TABLE-US-00003 TABLE 3 Fecal assessment. Fecal assessment was performed twice a day blinded for diet. Date and time for first meconium after birth were also registered. For analysis, score ≥3 was classified as a piglet with diarrhoea. Score Feces 0 no stool 1 firm feces 2 pasty feces 3 Droplets of watery feces/diarrhoea 4 Moderate amounts of diarrhoea 5 Large amounts of diarrhoea
[0219] Fecal scores, based on stool frequency and consistency (Table 3), showed minimal to no feces on day 1, 2 and 3 for any of the preterm or near-term piglets, but an extensive increase on days 4 and 5 (
[0220] An important indicator of feeding intolerance in preterm infants used in the clinic is the presence of gastric residuals, which is assessed by taking gastric aspirates. During this study, remarkable differences in gastric residual volumes 3 h after an enteral bolus were observed. In general, the volume of gastric residuals decreased over the days, with detectable gastric residuals in most piglets at day 1 (
[0221] In preterm piglets, the volume of gastric residuals was higher in the piglets receiving MP-WPC compared with EH-WPC on all days (
GI Transit by X-Ray Analysis
[0222] On day 4 in the evening, gut transit time was assessed by x-ray photography after oral intake of a contrast solution, as described previously by Chen, Pediatric Research, 2020. Each piglet received an enteral bolus of 4 ml/kg contrast fluid (4 ml/kg, Idoixnol, Visipaque®, GE Healthcare, Brondby, Denmark) after 2 h of fasting, to mimic clinical practices. X-ray images of the GI tract were taken using Mobilett XP Hybrid (Siemens, Germany) at 20 min, 1, 2 and 4 h and subsequent every second hour till the contrast fluid was cleared from the GI tract or maximally 14 hours, which ever came first. The piglets were placed on their back while the x-ray was taken and returned to their home cage between screenings. During the x-ray examination, piglets received enteral nutrition according to the regular feeding schedule (i.e., every 3 h). X-ray images were interpreted by both a neonatologist and a radiologist, blinded to the types of diet. For analysis, time of contrast to be cleared from the stomach (StEmpty), to be cleared from the small intestine (SlEmpty), to first appear at caecum (ToCaecum) and to first appear in the rectum (ToRectum) were recorded.
[0223] When assessing stomach emptying (StEmpty) of contrast fluid by x-ray on day 4, there were no differences between MP-WPC and EH-WPC in either preterm or near-term piglets (data not shown). The contrast fluid was emptied from the stomach in similar rate for both diets, suggesting that the differences in gastric emptying observed were mainly caused by the coagulation and not due to differences in motility.
[0224] Coagulation of proteins can delay gastric emptying and impact GI transit, therefore the small intestine (SI) and colon content was determined (
Sample Collection
[0225] Piglets were euthanized on day 5 for sample collection, or when clinical symptoms of feeding intolerance, vomiting, abdominal distention, haemorrhagic diarrhoea and/or respiratory distress appeared during the study and the humane endpoint was reached. The piglets were first anaesthetized with an intramuscular injection of Zoletil mix (0.1 mL/kg, Virbac, Kolding, Denmark) and subsequently mL of 20% pentobarbital (Euthanimal, Scanvet, Denmark) was injected intracardiac to euthanize the piglet. Weight of full and empty stomach was recorded to determine the volume of the gastric content, and samples of gastric content were collected and stored at −80° C. until further analysis.
[0226] To decipher whether the MP-WPC was emptied slower from the stomach or whether the EH-WPC residual was too thick for aspiration via the gastric tube, the volumes of the gastric contents were measured during necropsy at day 5, 1 h after a 15 mL/kg bolus of EN. In contrast to the gastric aspirates, the volume of gastric contents was significantly lower in the piglets receiving MP-WPC compared with EH-WPC for both preterm and near-term piglets (
Analysis of Digesta
[0227] To measure pH levels in the gastric content, 0.5 gram of gastric content was diluted in 1 ml of dH2O, vortexed and subsequently pH was recorded with the SensION+PH3 meter (HACH).
[0228] The gastric contents were poured over sequentially placed analytical sieves, with a mesh width of 2 mm, 1 mm and 0.25 mm (Retsch, VWR, Amsterdam, Netherlands). Coagulates were separated according to their particle diameter in four fractions: larger than 2 mm (D>2 mm), between 1 and 2 mm (2>D>1 mm), between 0.25 and 1 mm (1>D>0.25 mm) and the permeate which is smaller than 0.25 mm (D<0.25 mm). After 30 min, wet weight of the sieves and permeate was recorded to determine the wet weight of the fractions.
[0229] The pH levels of the gastric content tended to be higher in the MP-WPC compared with EH-WPC for preterm piglets (
[0230] The gastric content of 6 near-term piglets (n=3 for each diet) were further analyzed to determine the formation and size of coagulates. The coagulates in the gastric content of piglets fed MP-WPC were smaller in weight and size compared with the coagulates in the gastric content of piglets fed EH-WPC (
[0231] Taken together, although the volumes of gastric aspirates were significantly higher, the gastric content were significantly lower in preterm piglets fed MP-WPC compared with EH-WPC, indicating that the gastric EH-WPC residuals were too thick for complete aspiration and thereby lead to an underestimation of the real gastric residual volume. Near-term piglets showed similar lower gastric content for MP-WPC, with a lower volume of coagulates in the gastric content present compared with EH-WPC.
Example 6. In Vitro Gastric Digestion Assay
[0232] Digestion of the EH-WPC and MP-WPC was simulated in vitro in the semi-dynamic digestion model (SIM) described previously by van den Braak, Clinical Nutrition, 2013, 32, 765-771, with minor adaptations to mimic preterm infant digestion. In detail, gastric digestion was simulated over a period of 120 min at 37° C. in multi fermenter fed-batch bioreactors (Dasgip A G, Julich, Germany). Bioreactors were filled with a bolus of 150 ml WPC (100 g/L) and were mixed by short and gentle orbital shakings of the bioreactor every 10 min. At the start of the digestion, 25 ml of simulated saliva fluid (SSF) was added to each bioreactor. During the digestion, simulated gastric fluid (SGF) was added in a dynamic manner, with 9 ml in the first 3 min followed by a continuous addition of 22.5 ml/h. To decrease pH in time hydrochloric acid (1M, Sigma-Aldrich, Zwijndrecht, the Netherlands) was added in a pre-determined dynamic flow to result in a final pH of 4.3 after 120 min. SSF (pH 6.3) consisted of 0.1 M NaCl, 30 mM KCl (Merck, VWR International), 2 mM CaCl.sub.2.Math.2H.sub.2O, 14 mM NaHCO.sub.3, 0.06% (w/v) α-amylase (from Aspergillus oryzae, A9857) (all Sigma-Aldrich, Zwijndrecht, the Netherlands). SGF (pH 4.0) contained 50 mM NaCl, 15 mM KCl (Merck, VWR, Amsterdam, the Netherlands), 1 mM CaCl.sub.2.Math.2H.sub.2O, 0.005% (w/v) pepsin (from porcine gastric mucosa, P7125), 0.013% (w/v) lipase (from Rhizopus oryzae, 80612) (all Sigma-Aldrich, Zwijndrecht, the Netherlands).
[0233] In vitro obtained digesta were poured over sequentially placed analytical sieves, with a mesh width of 2 mm, 1 mm and 0.25 mm (Retsch, VWR, Amsterdam, Netherlands). Coagulates were separated according to their particle diameter in four fractions: larger than 2 mm (D>2 mm), between 1 and 2 mm (2>D>1 mm), between 0.25 and 1 mm (1>D>0.25 mm) and the permeate which is smaller than 0.25 mm (D<0.25 mm). After 30 min, wet weight of the sieves and permeate was recorded to determine the wet weight of the fractions. Protein concentration in the separated fractions was measured by BCA
[0234] After 2 h of in vitro digestion, the digesta were poured over sequentially placed sieves separating the digesta based on particle size. Coagulates >1 mm were nearly undetectable in the gastric digesta of MP-WPC nor EH-WPC (
[0235] It was impossible to sample coagulates with size >2 mm and 2-1 mm for MP-WPC due to minimal digesta retained on the sieves. However, protein concentrations of the coagulated fraction 1>D>0.25 mm tended to be lower for MP-WPC compared than EH-WPC (
[0236] Comparing the coagulates from MP-WPC with the coagulates from EH-WPC revealed significant differences in protein composition. The concentration of β-lactoglobulin in the coagulates with particle size 1>D>0.25 mm was significant lower in digested MP-WPC compared with digested EH-WPC (
[0237] In summary, MP-WPC shows lower level of protein coagulation after in vitro simulated digestion compared with EH-WPC, with lower levels of β-lactoglobulin and α-lactalbumin in the coagulates.
Example 7. Infant Formula Containing Native Whey Protein Concentrate (WPC)
[0238] An infant formula containing the native whey protein composition according to the invention is exemplified as follows. The main nutrients of this infant formula are as follows:
TABLE-US-00004 Units Per 100 ml RTF Per 100 kcal Energy value kcal 66 100 Protein g 1.3 2 Whey g 0.94 1.44 beta-casein g 0.36 0.56 free tyrosine mg 7.92 12 Weight ratio 64:36 β-lactoglobulin:α-lactalbumin 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
[0239] The infant formula is intended for feeding of term infants aged 0 to 6 months. In terms of energy value, the infant formula contains 8 En % protein, 44 En % carbohydrate, 46 En % fat. Minerals and vitamins and other micronutrients 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%.
Example 8. Preterm Formula Containing Native Whey Protein Concentrate (WPC)
[0240] A preterm formula containing the native whey protein composition according to the invention is exemplified as follows. The main nutrients of this preterm formula are as follows.
TABLE-US-00005 Units Per 100 ml RTF Per 100 kcal Energy value kcal 78 100 Protein g 2.6 3.3 Whey g 1.87 2.37 beta-casein g 0.56 0.92 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 Minerals g 0.2 0.2
[0241] 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 and 44 En % fat. RTF=Ready-To-Feed. Minerals and vitamins and other micronutrients 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%.
Example 9. In Vitro Gastric Digestion Assay
[0242] Gastric physicochemical behaviour of a native whey protein concentrate, wherein 30% of the protein was beta-casein (WPC-B) was compared to a native whey protein concentrate wherein 30% of the protein was a mixture of alpha- and beta-caseins (WPC-AB) and a whey protein isolate containing only whey proteins (WPI). Gastric simulation tests were performed in duplicate. Pre-term infant gastric conditions were simulated by the method of Example 6. Multi-fermenter fed-batch bioreactors (Dasgip A G, Jülich, Germany) were placed at 37° C., filled with a bolus of 150 ml WPC/WPI (62.5 g/L) and were mixed by short and gentle orbital shakings of the bioreactor. Simulated gastric electrolyte (SGE) was added in one shot of 100 ml, afterwards the pH was decreased to pH 4.3 in twenty minutes using hydrochloric acid (1M) under magnetic stirring (150 rpm). SGE (pH 4.0) contained 50 mM NaCl, 15 mM KCl and 1 mM CaCl2.Math.2H2O.
[0243] In vitro obtained digesta were poured over sequentially placed analytical sieves, with a mesh width of 2 mm, 1 mm and 0.25 mm (Retsch, VWR, Amsterdam, Netherlands). Coagulates were separated according to their particle diameter in three fractions: larger than 2 mm (D>2 mm), between 1 and 2 mm (2>D>1 mm) and between 0.25 and 1 mm (1>D>0.25 mm). After 30 min, wet weight of the sieves and permeate was recorded to determine the wet weight of the fractions.
[0244] The results are shown in