NUTRITIONAL COMPOSITION

Abstract

The invention relates to a nutritional composition comprising carbohydrates, protein and a fat composition, wherein: (a) the nutritional composition has a total calcium content on dry matter of at least 3.5 g/kg; (b) the protein comprises casein; (c) the nutritional composition has a content of protein-bound calcium of 7.5 mmoles or less per 10 grams of casein; and (d) the ratio NPN to TN is 0.7 or less, with:—NPN meaning non-protein nitrogen in the nutritional composition in grams per 100 grams of nutritional composition (g/100 g); and—TN meaning total nitrogen (TN) in the nutritional composition in g/100 g. The invention further relates to a process for the preparation of the nutritional composition in powder form and to the composition for use in the prevention of gut discomfort and constipation in human subjects, in particular human subjects of 0 to 36 months of age

Claims

1. Nutritional composition comprising carbohydrates, protein and a fat composition, wherein: (a) the nutritional composition has a total calcium content on dry matter of at least 3.5 g/kg; (b) the protein comprises casein; (c) the nutritional composition has a content of protein-bound calcium of 7.5 mmoles or less per 10 grams of casein; and (d) the ratio NPN to TN is 0.7 or less, with: NPN meaning non-protein nitrogen in the nutritional composition in grams per 100 grams of nutritional composition (g/100 g); and TN meaning total nitrogen (TN) in the nutritional composition in g/100 g.

2. Nutritional composition according to claim 1, wherein the fat composition comprises at least 20% by weight, preferably at least 45% by weight, based on total weight of the fat composition of triacylglycerols (TAG) originating from a bovine milk fat source.

3. Nutritional composition according to claim 2, wherein the bovine milk fat source is whole milk, cream or anhydrous milk fat.

4. Nutritional composition according to claim 1, wherein the fat composition comprises: (a) 0.5-2.2% by weight, preferably 0.6-2.0% by weight, based on total weight of fatty acid acyl groups in TAG of butanoate groups (C4:0); and (b) 18.0-35.0% by weight, preferably 19.0 to 32.0% by weight, based on total weight of fatty acid acyl groups in TAG of long chain saturated fatty acid acyl groups having a chain length of 12 or more carbon atoms at the sn-1 and sn-3 position in TAG.

5. Nutritional composition according to claim 1, which further comprises at least one human milk oligosaccharide, preferably a fucosylated lactose, more preferably 2′-fucosyllactose.

6. Nutritional composition according to claim 5, wherein the at least one human milk oligosaccharide is present in an amount of 0.05 to 2.0 grams per 100 grams of the nutritional composition on dry matter.

7. Nutritional composition according to claim 1, in powder form.

8. Process for the preparation of a nutritional composition according to claim 7, the process comprising: (a) preparing a mixture comprising a whey protein source, a casein source, a fat source, a carbohydrate source and salts; (b) spray drying the mixture resulting from step (a) into a powder; and (c) optionally dry-blending further salts into the powder resulting from step (b) to obtain the nutritional composition, wherein the salts comprise (i) a calcium-binding acid or water-soluble salt thereof; and (ii) at least one calcium salt selected from calcium phosphate and calcium carbonate, and wherein the calcium-binding acid or water-soluble salt thereof is added in step (a) and the at least one calcium salt is added in step (a) and/or in step (c).

9. Process according to claim 8, wherein the at least one calcium salt is added in step (c).

10. Process according to claim 8, wherein the calcium-binding acid or water-soluble salt thereof is selected from citric acid, sodium citrate, potassium citrate and any combination of two or more of these, preferably a combination of sodium citrate and potassium citrate.

11. A method of preventing gut discomfort and constipation in a human subject, the method comprising administering to the human subject an effective amount of the nutritional composition of claim 1, wherein the human subject is preferably 0 to 36 months of age.

Description

DESCRIPTION OF FIGURES

[0060] FIG. 1 is a schematic representation of the procedure for determining protein-bound calcium.

[0061] FIG. 2 shows the set-up of the in vitro gastric digestion model used.

[0062] FIG. 3 shows photographic images after in-vitro gastric digestion of two samples according to the invention and one comparative sample at different pH values.

[0063] FIG. 4 shows SDS-PAGE electrophoretograms of the model formulations tested.

[0064] FIG. 5 shows photographic images after in-vitro gastric digestion of a nutritional composition according to the invention.

[0065] FIG. 6 shows SDS-PAGE electrophoretograms of the nutritional composition of Example 3.

EXAMPLES

Example 1—Preparation of Model Formulations

[0066] Model formulations with different levels of protein-bound calcium were prepared and tested in vitro to demonstrate the effect of protein-bound calcium on curd formation in the stomach during gastric digestion. Milk samples with differing degrees of casein mineralization were prepared using the method described by Pyne & McGann (Pyne, G. T., & McGann, T. C. A. (1960). The colloidal phosphate of milk: II. Influence of citrate. Journal of Dairy Research, 27(1), 9-17).

[0067] For this purpose, a batch of 50 kg of pasteurized skim milk was obtained. Three subsamples (200 g each) were taken, one of which (Sample 2) was kept at its original pH, whereas Sample 1 was adjusted to pH 5.7 by the addition of 1 M hydrochloric acid (acid) and Sample 3 was adjusted to pH 8.0 by the addition of 1 M sodium hydroxide (base). The pH adjustment was carried out at 5° C., with milk samples as well as acid and base equilibrated at this temperature for 1 hour prior to pH adjustment.

[0068] Due to the pH adjustment the salt balance in the milk had changed. In order to re-equilibrate the serum composition of the adjusted milk the samples are dialyzed. Accordingly, Samples 1, 2 and 3 were subsequently exhaustively dialyzed against the original pasteurized skim milk using a dialysis membrane with a nominal molecular weight cut-off of 14 kDa. The first dialysis was carried out for 24 h at 5° C. whereby for each of Samples 1, 2 and 3 200 g milk was dialyzed against 5000 g of the original pasteurized milk with gentle agitation. Following this dialysis stage, the dialysis tubes were transferred to another container containing 5000 g of the original pasteurized skim milk at 5° C. and dialysis was again conducted under the same conditions for 24 h. Following this dialysis step, samples were removed from the dialysis tubing.

[0069] The pH of the dialyzed Samples 1DIA (dialyzed sample obtained from Sample 1), 2DIA (dialyzed sample obtained from Sample 2) and 3DIA (dialyzed sample obtained from Sample 3) was 6.8 as measured at 20° C. using standard equipment.

[0070] Samples 1DIA, 2DIA and 3DIA were subsequently used to prepare Model Formulations MF1, MF2 and MF3, respectively, by mixing 21 g of the respective dialyzed sample with 21 g of demineralized whey (DEMINAL® 90 Liquid ex FrieslandCampina Ingredients, having total protein content on dry matter of 13.5 wt %, lactose content on dry matter of 84.5 wt %, calcium content on dry matter of 0.040 wt % and total solids content of 28 wt %) and 58 g of milk permeate produced by ultrafiltration of pasteurized skimmed milk at 50° C. using a 10 kDa membrane and having TN of less than 0.05 g/100 g, a lactose content of 4.9 wt %, and calcium content 0.028 wt %.

[0071] Model formulations MF1, MF2 and MF3 were subsequently analyzed for total nitrogen content (TN), non-protein nitrogen content (NPN), casein nitrogen content (CN) and calcium content (Ca). The ratio protein-bound calcium to casein (PBCa/Cas) was subsequently calculated in mmoles Ca per 10 g casein.

[0072] TN was determined using the Kjeldahl method as described in method ISO 8968-1/IDF 020-1 (Milk and milk products—Determination of Nitrogen Content—Part 1: Kjeldahl Principle and Crude Protein Calculation). NPN was determined as described in method ISO 8968-4/IDF 020-4 (Milk and milk products—Determination of Nitrogen Content—Part 4: Determination of non-protein-nitrogen content).

[0073] CN was determined as described in ISO17997/IDF 29-1—Determination of casein nitrogen content—Part 1—Indirect method (Reference method). Casein content of the dialyzed sample is then calculated as CN*6.38.

[0074] Calcium content was determined using Inductively Coupled Plasma Atomic Mass Spectrometry (ICP-MS) as described in standard method ISO 21424 I IDF 243:2018 (Milk, milk products, infant formula and adult nutritionals—Determination of minerals and trace elements—Inductively coupled plasma atomic mass spectrometry (ICP-MS) method).

[0075] PBCa was determined as described hereinbefore with reference to FIG. 1. Accordingly, samples of MF1, MF2 and MF3 were first equilibrated at 20° C. for 1 hour and centrifuged at 200×g for 15 minutes at 20° C. to remove any insoluble calcium salts, followed by separation of the pellet and supernatant (SUP). The SUP is subsequently equilibrated at 20° C. for 2 hours prior to centrifugation at 100,000×g for 60 minutes at 20° C. The resulting liquid serum layer is separated from the cream layer and pellet and subsequently filtered through a 10 kDa membrane to obtain the 10 kDa-permeable fraction of the sample (PER10 kD). Total calcium (<Ca>) concentration in grams per kilogram (g/kg) in the SUP and the PER10kD was determined by the ICP-MS method described above. The concentration of protein-bound calcium (PBCa, in g/kg) was calculated as:

PBCa=<Ca>in SUP−<Ca>in PER10 kD

[0076] Key parameters of model formulations MF1, MF2 and MF3 are indicated in Table 1.

TABLE-US-00001 TABLE 1 Compositional parameters of model formulations MF1, MF2 and MF3 Sample Sample Sample MF1 MF2 MF3 Ca (g/kg) 0.40 0.45 0.47 NPN (g/100 g) 0.03 0.03 0.03 TN (g/100 g) 0.25 0.26 0.25 CN (g/100 g) 0.09 0.09 0.09 NPN/TN 0.12 0.12 0.12 CN/TN 0.4 0.4 0.4 PBCa/Cas (mmol/10 g casein) 5.4 7.1 8.0

Example 2—In Vitro Gastric Digestion

[0077] In-vitro gastric digestion of formulations MF1, MF2 (both according to the invention) and MF3 (comparative) was conducted using a semi-dynamic digestion model at NIZO (Ede, The Netherlands) with digestive conditions adapted to mimic infant gastric conditions.

[0078] For the in-vitro gastric digestion the samples, a system as outlined in FIG. 2 was used. For typical experiments, 0.83 g 30 mM HCl was placed in a 100 mL bottle placed in an agitating waterbath set at 37° C. and ˜120 rpm. The sample was also equilibrated at 37° C. 20 mL of the sample was subsequently fed into the bottle at a rate of 1 mL/min, whereas gastric juice (30 mM HCl containing 250 U/mL pepsin and 8.75 U/mL lipase) was added at a rate of 0.13 mL/min. Gastric juice was kept on ice to prevent losses in enzyme activity. Porcine pepsin (Sigma) was used as the pepsin source in the gastric juice, whereas Amano lipase A (Amano) was the standard lipase used.

[0079] Standard conditions for the in-vitro gastric digestion are summarized in Table 2.

TABLE-US-00002 TABLE 2 Standard conditions for in-vitro gastric digestion Gastric Juice 30 mM HCl 8.75 U/mL lipase A 250 U/mL pepsin Production rate 0.4 mL/min Enzymes Amano lipase A from Aspergullus niger Pepsin from porcine gastric mucosa Fasting gastric pH 3.5 Product feeding rate 1.0 mL/min Feeding time 20 min Amount of product 20 mL

[0080] To allow sampling at specified pH values, buffering curves of samples were first determined. For this purpose, samples were mixed with different volumes of 30 mM HCl and the pH was determined. Based on the amount of 30 mM HCl required to reach a certain pH and the pumping speed, the time point at which the sample should be taken could be calculated. To inhibit pepsin activity after sampling, a pepstatin A stock solution (0.02 g Pepstatin A in 18 mL methanol +2 mL glacial acetic acid) was added at a level of 50 μL/10 mL of digested sample.

[0081] At set points during the digestion (pH 6.0, 5.0, 4.5, 3.5) a product was removed (a separate product was used for each pH point) and the contents of the container were poured into a petri-dish and photographed, for visual observation of curd formation and breakdown. Subsequently, samples were centrifuged at 4000×g for 10 min and pellet and supernatant were separated by decanting. Both fractions were weighed and freeze-dried and subsequently analyzed by SDS-PAGE under reducing conditions.

[0082] FIG. 3 shows photographic images of the model formulations after the in-vitro gastric digestion at different pH values. Diameter of the container in which the samples were photographed was 88 mm. FIG. 3 shows that visible curd formation was not observed in Sample MF1 (according to the invention) at any pH. In Sample MF2 (according to the invention), visible coagulation was not observed at pH 6.0, but as digestion progressed coagulation was observed at pH 5.0. Upon further progression, however no residual curd particles were observed in this sample, indicating the breakdown of curd particles formed initially. For Sample MF3 (comparative), strong coagulation was already observed at pH 6.0 and although some breakdown of particles was observed over time, even at the end of digestion (pH 3.5; >120 min) large residual curd particles were still observed.

[0083] The samples MF1, MF2 and MF3 were also centrifuged, yielding a pellet and a supernatant (serum), and analyzed by SDS-PAGE under reducing conditions. As outlined in FIG. 4, intact caseins, observed on SDS-PAGE gels in the range 25-35 kDa, were only observed in the original samples and in the pellets formed.

[0084] Residual intact caseins were observed in the pellet of all samples MF1, MF2 and MF3 at pH 6.0; however, at pH 5.0, no residual intact casein was observed anymore in sample MF1, whereas samples MF2 and MF3 still showed residual intact casein. At lower pH the intact casein is no longer observed in sample MF2. In sample MF3, however, even at the end of the digestion process (pH 3.5) residual intact casein was still observed, probably contained within the large curd particles still observed in these samples as well (see FIG. 3). Hence, it is apparent that the size of the curd particles formed is a key factor determining the rate of casein breakdown in the samples. If large particles are formed, the total particle surface area is lower and with diffusion of digestive enzymes through the particles likely a rate-limiting step, casein breakdown is slow.

Example 3—Nutritional Composition

[0085] A composite blend was prepared from thermised whole milk, cream and demineralized whey. To that effect 145 kg of the whole milk was blended with 163 kg of the demineralized whey and 16 kg of the cream. TN, NPN, total calcium content and casein content of each ingredient and of the composite blend were determined as described above. In addition, and protein-bound calcium (PBCa) of the milk and the composite blend was determined as described above. Fat content was determined using the Rose Gottlieb method (ISO 1211/IDF 1, Milk—Determination of fat content—Gravimetric method (Reference method). The results are indicated in Table 3.

TABLE-US-00003 TABLE 3 Properties PBCa/ Cas TN NPN NPN/ Fat Ca (mmol/ Sample (g/100 g) (g/100 g) TN (g/100 g) (g/kg) 10 g) Milk 0.54 0.03 0.06 4.3 1.20 7.3 Cream 0.33 0.02 0.06 42.2 0.61 — Whey 0.56 0.06 0.11 0.2 0.056 — Blend 0.54 0.05 0.09 4.1 0.60 6.3

[0086] The composite blend was subjected to an evaporation treatment and further ingredients were added (see Table 4 for recipe). The resulting mixture was pasteurized, homogenized and spray-dried, yielding a spray-dried base powder with a moisture content of 2.5%. To the spray-dried base powder, further ingredients were added by dry blending, which are shown in Table 4.

[0087] The final nutritional composition powder had a moisture content of <3.0%.

TABLE-US-00004 TABLE 4 Nutritional composition recipe Amount added (kg/ Ingredient 100 kg final products) Added to evaporated composite blend: Fat blend 13.4 Potassium Citrate 0.55 Calcium carbonate 0.37 Magnesium chloride 0.21 TriCalcium phosphate 0.15 Sodium citrate 0.09 Sodium chloride 0.15 Calcium hydroxide 0.03 Dry-blended: Lactose 5.6 Galacto-oligosacharides powder 4.4 Premixes Vitamins, Trace elements, 0.4 Nucleotides 2′-Fucosyllactose 0.30

[0088] The powdered nutritional composition was reconstituted in demineralized water at 40° C. for 60 min at a level of 13 g powder per 90 g of water. The level of casein mineralization (i.e. protein-bound calcium, PBCa/Cas) in the product was determined using the methods described above.

[0089] The nutritional composition was analyzed using the methods described hereinbefore. Casein content was determined via TN, NPN and amino acid composition as described hereinbefore. Results are indicated in Table 5.

TABLE-US-00005 TABLE 5 Key characteristics of nutritional composition Total protein (6.38 * TN) g/100 g 11.4 Casein (CN * 6.38) g/100 g 4.2 NPN g/100 g 0.18 NPN/TN 0.10 Fat g/100 g 27 2′-FL g/100 g 0.25 GOS g/100 g 3.0 Lactose g/100 g 51.7 Ca mg/100 g 421 PBCa/Cas mmol/10 g casein 5.2

[0090] The sample was subsequently subjected to in-vitro digestion using the method described in Example 2, with samples taken at pH 6.5, 6.0, 5.5, 4.5 and 3.5. Photographic images of samples after reaching these pH values are shown in FIG. 4, SDA-PAGE electrophoretograms of are shown in FIG. 6.

[0091] As can be seen from FIGS. 5 and 6 at pH below 6.0 no large coagulates were observed and no intact casein (see range 25-35 kDa) is observed anymore, clearly indicating that under gastric conditions (pH<5) no curd formation takes place and digestion takes place effectively.