Protein compositions with high isoelectric proteins

Abstract

The present invention is in the field of protein compositions, in particular protein compositions for infant milk formula. The protein compositions according to the invention comprise whey protein, casein and high isoelectric point proteins, such as those having an isoelectric point of at least 6.0. The present invention further relates to the uses of the protein compositions.

Claims

1. A composition comprising casein, whey protein and at least one high isoelectric protein from a non-dairy source, said high isoelectric protein having an isoelectric point of between 6.0 and 11, wherein the weight ratio of the sum of casein and whey protein to the high-isoelectric protein in the composition is between 95:5 and 75:25, wherein the composition is an infant formula comprising 0.8-3.0 g/100 ml protein when in liquid form.

2. The composition according to claim 1, wherein the casein and whey protein are present in a weight ratio between 30:70 and 70:30.

3. The composition according to claim 1, wherein the casein comprises -caseins in an amount of between 35 and 60 wt. % of total casein content; and/or wherein the casein comprises -caseins in an amount of between 20 and 60 wt. % of total casein content; and/or wherein the casein comprises k-caseins in an amount of between 15 and 30 wt. % of total casein content.

4. The composition according to claim 1, wherein the whey protein comprises -lactalbumin in an amount of between 20 and 80 wt. % of total whey protein content; and/or wherein the whey protein comprises -lactoglobulin in an amount of between 20 and 70 wt. % of total whey protein content.

5. The composition according to claim 1, wherein the high isoelectric protein is one or more proteins selected from the group consisting of lysozyme, ovotransferrin, canola protein, rapeseed protein, or mixtures thereof.

6. The composition to according to claim 1, wherein the at least one high isoelectric protein is in intact form.

7. The composition according to claim 1, wherein only one high isoelectric protein having an isoelectric point between 9.0 and 11 is present.

8. The composition according to claim 1, wherein the composition comprises two high isoelectric proteins from a non-dairy source having an isoelectric point between 6.0 and 11, wherein one of the isoelectric proteins has an isoelectric point between 9.0 and 11.

9. The composition according to claim 1, consisting of casein, whey protein and at least one high isoelectric protein, including one high isoelectric protein from a non-dairy source.

10. A composition comprising casein, whey protein and at least one high isoelectric protein from a non-dairy source, said high isoelectric protein having an isoelectric point of between 6.0 and 11, wherein the weight ratio of the sum of casein and whey protein to the high-isoelectric protein in the composition is between 95:5 and 75:25, wherein the composition is an infant formula, wherein the infant formula comprises lipid, protein and digestible carbohydrates, with 5 to 20 energy % protein, based on total calories provided by the composition, and wherein the infant formula comprises 50 to 90 kcal/100 ml when in liquid form.

11. The composition according to claim 10, wherein the infant formula comprises 60 to 70 kcal/100 ml when in liquid form.

12. A composition comprising casein, whey protein and at least one high isoelectric protein from a non-dairy source, said high isoelectric protein having an isoelectric point of between 6.0 and 11, wherein the weight ratio of the sum of casein and whey protein to the high-isoelectric protein in the composition is between 95:5 and 75:25, wherein the composition is an infant formula in liquid form, comprising 0.8-3.0 g/100 ml protein.

13. A composition comprising casein, whey protein and at least one high isoelectric protein from a non-dairy source, said high isoelectric protein having an isoelectric point of between 6.0 and 11, wherein the weight ratio of the sum of casein and whey protein to the high-isoelectric protein in the composition is between 95:5 and 75:25, wherein the composition is an infant formula in liquid form, wherein the infant formula comprises lipid, protein and digestible carbohydrates, with 5 to 20 energy % protein, based on total calories provided by the composition, and wherein the infant formula comprises 50 to 90 kcal/100 ml when in liquid form.

14. The composition according to claim 13, wherein the infant formula comprises 60 to 70 kcal/100 ml liquid.

Description

(1) FIG. 1: shows a comparison between total NH2 appearance over time under intestinal digestion conditions of fresh human milk and a commercial infant formula.

(2) FIGS. 2, FIG. 3 and FIG. 4: show the protein digestibility of standard infant formula (Ref-IMF), formulations having casein and protein (50:50) (Ref-P and Ref-P-beta) and compositions A-G according to the invention under intestinal digestion conditions

EXAMPLES

Example 1

Protein Composition and Digestibility

(3) Protein compositions as described in Table 1 were prepared as follows:

(4) Whey proteins and/or high pI proteins were dissolved in demineralised water at room temperature under mild stirring for 2 h. Caseins were dispersed in 50 C. demineralized water and homogenized at 350+50 bar. Final protein formulations were obtained by mixing dissolved protein ingredients in appropriate ratios to reach final protein concentration of 1.3% (w/w). The amount of high pI proteins is expressed in weight percentage based on total proteins.

(5) TABLE-US-00001 TABLE 1 Protein Casein:whey composition Components weight ratio Ref. IMF Standard casein.sup.1 40:60 Standard whey protein.sup.2 Ref. P Standard casein.sup.3 50:50 Standard whey protein.sup.2 Ref. P-beta beta-casein enriched casein.sup.5 50:50 Standard whey protein.sup.2 A Standard casein.sup.3 50:50 alpha-lactalbumin enriched whey protein.sup.4 15 wt. % ovotransferrin.sup.6 3 wt. % lysozyme.sup.7 B beta-casein enriched casein.sup.5 50:50 alpha-lactalbumin enriched whey protein.sup.4 15 wt. % oyotransferrin.sup.6 3 wt. % lysozyme.sup.7 .sup.1skim milk powder (Omia) .sup.2whey protein concentrate, 80% demineralized (Friesland Campina) .sup.3micellar casein isolate (Friesland Campina) .sup.4Lacprodan (Arla; amount of alpha-lactalbumin 60%) .sup.5Ultranor HT (Kerry Foods; amount of beta-casein 40%) .sup.6pl = 6.7, non-dairy source (Bioseutica Group) .sup.7pl = 10.4, non-dairy source (Bioseutica Group)

(6) Protein compositions of Table 1 were digested using static INFOGEST in vitro digestion model adapted for infants as shown in table 2 below:

(7) TABLE-US-00002 TABLE 2 Gastric phase (60 min) Intestinal phase (120 min) Ratio Meal: Simulated Gastric Ratio Meal: Simulated Gastric Fluid of 63:37 Fluid of 39:61 Enzymes: Pepsin (268 U/ml) Enzymes: Porcine and Lipase (19 U/ml) from Pancreatin (lipase 90 U/ml) Rabbit Gastric Extract and Bovine bile (3.1 mM) pH 4.5, T = 37 C. pH 6.6, T = 37 C.

(8) Digestions of 100 ml volumes were performed in 250 ml Duran bottles. A water bath (MixBathPC, 2mag Magnetic Notion) and magnetic stirrer (Mixcontrol 40, 2mag Magnetic Notion) were set at 37.5 C. and 100 rpm, respectively. For the gastric phase, the protein solution set at 37 C. and a Simulated Gastric Fluid (SGF) were mixed in a ratio 63:37. The pH was adjusted to 4.5 using a 0.25 M HCl, gastric digesta was sampled at times 5, 15, 30 and 60 minutes. After the gastric phase, gastric digest was mixed with a Simulated Intestinal Fluid (SIF) in a ratio 39:61. The pH was adjusted to 6.6 using a 0.25 M NaOH/0.25 M NaHCO3. Intestinal digesta was sampled at times 2, 6, 10, 15, 22, 30, 40, 55, 70, 80, 90, 105 and 120 minutes.

(9) Sampling was performed by mixing 1 ml digesta with 1 ml of 0.1 M phosphate buffer at pH 7.2 (gastric samples) or 5.5 (intestinal samples) to stop digestion, turraxed (T10 Basic Utraturrax, IKA) for 25 seconds at power 5.5 to enhance protein dispersibility, snap frozen in liquid nitrogen and stored at 20 C. until analysed. Each digestion was performed in triplicate.

(10) Collected samples were subjected to analysis of generation of primary amino groups by ortho-phtaldialdehyde (OPA). The method is based on the specific reaction between OPA and free primary amino groups in proteins in the presence of 2-(dimethylamino)ethanethiol hydrochlorid (DMA) to give alkyl-iso-indole derivatives that show an absorbance at 340 nm. Digesta samples were mixed with 2% SDS at ratio 1:1, boiled at 95 C. for 5 minutes and left to cool down at room temperature. Aliquots of 5 l was mixed with 200 l of either OPA working reagent or OPA blank solution and incubated for 25 minutes. Signal was read using Biowave plate reader, the results were analysed using Gen5 2.09 software. Free NH2 groups were quantified using L-Alanine solution (0, 1, 2, 4, 6, 8 and 10 mM) calibration curve. Measurements were performed in duplicates.

(11) Digestion results were expressed to degree of hydrolysis (%) as a function of time. Digestibility parameter was obtained by fitting data to equation as described previously (Deng, van der Veer et al. 2017), and are shown in FIG. 2.

(12) Ref. IMF represents the protein digestibility profile of standard infant formula. Ref. P and Ref. P-beta are used as a reference to protein digestibility profile of formulations having casein and whey (50:50) without high pI proteins. Both Ref. P and Ref. P-beta present lower digestibility than Ref. IMF., regardless the casein source.

(13) Improved digestibility (lower) is observed in compositions according to the invention (A, B), i.e., comprising high isoelectric point proteins, with even improved results when casein source is enriched in beta-casein.

Example 2

Comparison of High pI Protein

(14) Composition B of Table 1 (18 wt. %, based on total protein, of proteins having pI of at least 6.0; 50:50 ratio of casein:whey; and using a beta-casein enriched casein source) was reproduced using different sources and ratios of high isoelectric point proteins. The compositions were prepared in the same manner as described in Example 1, and also had a final protein concentration of 1.3% (w/w).

(15) TABLE-US-00003 TABLE 3 Protein composition Components (wt. % based on total proteins) C 18 wt. % ovotransferrin.sup.1 D 18 wt. % lactoferrin.sup.2 E 18 wt. % rapeseed protein.sup.3 F 18 wt. % lysozyme.sup.4 G 9 wt. % ovotransferrin 9 wt. % lysozyme .sup.1pl = 6.7 (Bioseutica Group) .sup.2pl = 8.7 (FrieslandCampina) .sup.3pl = 7-11 (Synthite Corporate) .sup.4pl = 10.4 (Bioseutica Group)

(16) The results are given in FIG. 3 (effect of single high pI proteins) and FIG. 4 (effect of two high pI proteins). FIG. 3 shows that the higher the pI of the protein used, the lower the protein digestibility. FIG. 4 shows that a composition with a 50:50 ratio of two high pI proteins presents a synergistic effect in lowering protein digestibility.