CASEIN HYDROLYSATE

Abstract

A casein hydrolysate formed by controlled hydrolysis of a casein substrate by an aspergillus-derived (fugal) proteolytic preparation is described. The controlled hydrolysis employs a FlavorPro-Whey formulation and a degree of hydrolysis (% DH) of from 5% DH to 15% DH. The Hydrolysate has at least a 98% reduction in antigenicity compared to intact sodium caseinate and a mean bitterness score of less than 30%. The invention also provides a low molecular weight fraction of casein hydrolysate of the invention which is substantially free of peptides having a molecular weight greater than 5 kDa.

Claims

1. A casein hydrolysate formed by hydrolysis of a casein substrate by a aspergillus-derived proteolytic preparation to a degree of hydrolysis (% DH) of from 5% DH to 15% DH, the hydrolysate having a peptide profile that exhibits at least a 98% reduction in antigenicity compared to intact sodium caseinate and a mean bitterness score of less than 30%.

2-29. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0029] FIG. 1 is Table showing antigenicity values of a series of lab-scale casein hydrolysates, including four casein hydrolysates of the invention.sup.a% sample residual antigenicity calculated by dividing intact sodium caseinate antigenicity (given a nominal value of 1) by relative decrease in sample antigenicity (column 3) and multiplying the resulting fraction by 100 to give the % residual activity remaining in the sample compared to that of intact NaCN. The % decrease in antigenicity was obtained by subtracting the % residual activity from 100% (Antigenicity of intact NaCN). [0030] EXAMPLE6.46% DH sample [0031] % sample residual antigenicity=(1/56.23)100%=1.7784% 1.78% [0032] % reduction in sample antigenicity [0033] compared to antigenicity of intact NaCN=100%1.78%=98.22% [0034] i.e. 98.22% of the antigenicity has been removed=1.75 log reduction=56.23 relative decrease;

[0035] FIG. 2 is a Table comparing antigenicity values of a casein hydrolysate of the invention (Crude Hydrolysate14.16% DH) with a low molecular weight fraction of the Crude Hydrolysate (5 kD permeate fraction).sup.a % sample residual antigenicity calculated by dividing intact sodium caseinate antigenicity (given a nominal value of 1) by relative decrease in sample antigenicity (column 3) and multiplying the resulting fraction by 100 to give the % residual activity remaining in the sample compared to that of intact NaCN

[0036] The % decrease in antigenicity was obtained by subtracting the % residual activity from 100% [0037] (Antigenicity of intact NaCN) [0038] EXAMPLE5 kD permeate fraction [0039] % sample residual antigenicity=(1/1000000)100%=0.0001% [0040] % reduction in sample antigenicity [0041] compared to antigenicity of intact NaCN=100%0.0001%=99.9999% [0042] i.e. 99.9999% of the antigenicity has been removed=6 log reduction=1,000,000 relative decrease.

[0043] FIG. 3 is a Table showing the molecular mass distribution profile of intact sodium caseinate, a casein hydrolysate of the invention (Crude Hydrolysate14.16% DH), and a low molecular weight fraction of the Crude Hydrolysate (5 kD permeate);

[0044] FIG. 4 is a Table showing the nitrogen solubility indices of a lab scale casein hydrolysate of the invention, a pilot scale casein hydrolysate of the invention, and a low molecular weight fraction (5 kD permeate) of the pilot scale casein hydrolysate;

[0045] FIG. 5 is a graph comparing the Mean Bitterness Scores of (a) a commercial casein hydrolysate (comparative), (b) an Alcalase casein hydrolysate (comparative), (c) a low molecular weight fraction of the Alcalase casein hydrolysate (comparative), (d) a casein hydrolysate of the invention (14.16% DH), and (c) a low molecular weight fraction of the casein hydrolysate of the invention;

[0046] FIG. 6 is a graph showing the Nitrogen Solubility Index of sodium caseinate hydrolysates generated with FlavorPro Whey protease preparations at different % DH values as a function of pH;

[0047] FIG. 7 is a graph showing Heat Coagulation Time (HCT) of sodium caseinate hydrolysates generated by FlavorPro Whey at different % DH values as a function of pH;

[0048] FIG. 8A and 8B are Clarity Profiles at 660 nm and 4 C. (FIG. 8A) and 20 C. (FIG. 8B) of (a) semi-pilot scale FlavorPro Whey generated crude hydrolysate (14.16% DH), (b) a low molecular weight fraction (5 kD permeate) of (a), and (c) lab scale FlavorPro Whey generated crude hydrolyate (10.76% DH);

[0049] FIG. 9 is a Table showing the sequences and molecular weight profile of peptides in a low molecular weight fraction (<5 kD) of a FlavorPro Whey generated 14.16% DH casein hydrolysate of the invention in which the Alpha Si peptides 1 to 23 correlate with SEQUENCE ID NO'S 1 to 22 and 55, Alpha S2 casein peptides 1 to 9 correlate with SEQUENCE ID NO'S 23 to 31, Beta casein peptides 1 to 12 correlate with SEQUENCE ID NO'S 32 to 43, and Kappa

[0050] FIG. 10 is a Table showing the sequences and molecular weight profile of peptides in a FlavorPro Whey generated 14.16% DH casein hydrolysate of the invention in which the Alpha SI peptides 1 to 19 correlate with SEQUENCE ID NO'S 46 to 64, Alpha S2 casein peptides 1 to 7 correlate with SEQUENCE ID NO'S 65 to 71, and Beta casein peptides I to 12 correlate with SEQUENCE ID NO'S 72 to 83.

DETAILED DESCRIPTION OF THE INVENTION

[0051] The invention provides a casein hydrolysate, and its use as a food ingredient, typically but not limited to foods such as infant formula, geriatric food products, sports and nutritional foods and supplements, beverages, and food products for immunocompromised individuals. The casein hydrolysate is formed by controlled hydrolysis of a casein substrate (for example, to a degree of hydrolysis of about 5% DH to 15% DH) using a proteolytic/peptideolytic formulation derived from an aspergillus fungus. Ideally, the controlled hydrolysis employs a FlavorPro-Whey proteolytic formulation. The molecular weight profile of the peptides in a casein hydrolysate of the invention is shown in FIG. 10 and the Sequence Listing appended hereto. The invention also provides a low molecular weight fraction of a casein hydrolysate of the invention, and its use as a food ingredient, typically in foods such as infant formula, geriatric food products, sports and nutritional foods and supplements, and food products for immunocompromised individuals. The low molecular weight fraction of a casein hydrolysate of the invention is shown below to have highly reduced antigenicity compared to sodium caseinate, and sodium caseinate hydrolysates. The molecular weight profile of the peptides in the low molecular weight fraction of the casein hydrolysate of the invention is shown in FIG. 9 and the Sequence Listing appended hereto.

[0052] The term low molecular weight fraction should be understood as meaning that the hydrolysate is substantially free of peptides having a MW greater than 7 kDa, 6 kDa, and ideally 5 kDa. Generally, it is obtained by separation of a hydrolysate of the invention to remove peptides or proteins have a MW greater than a cut-off value, ie 5 KD, and retaining the permeate. In one embodiment, the low molecular weight fraction is substantially free of peptides having a MW greater than 4 kDa, 3.5 kD and ideally 3 kDa Methods of removing peptide and protein fractions having a MW greater than a specified cut-off value are well known in the art, for example ultrafiltration and dialysis.

[0053] The term substantially free of peptides having a molecular weight of greater than XkDa should be understood to mean that the hydrolysate contains less than 2% (by weight of total peptide in hydrolysate) of peptides or proteins having a MW greater than XkDa, and ideally less than 1.5% or 1% (w/w) of peptides having a MW of greater than XkDa. In this regard, it should be noted that the hydrolysate of the invention, and fractions thereof, may contain trace amounts of peptides derived from other milk proteins, for example lactoglobulins.

[0054] In this specification, the term controlled hydrolysis should be understood to mean that the casein substrate is hydrolysed to a degree of hydrolysis of up to 15% (15% DH), preferably up to 14% DH, 13% DH or 12% DH. Ideally, the term should be understood to mean a degree of hydrolysis of at least 5% DH, 6% DH, 7% DH, 8% DH, 9% DH or 10% DH. In one embodiment, the term should be understood to mean a % DH of from 5% DH to 15% DH, 6% DH to 14% DH, 7% DH to 13% DH, or 8% DH to 12% DH.

[0055] % DH is measured using the technique described in below.

[0056] The term aspergillus-derived proteolytic preparation should be understood to mean a protease, or protease formulation, derived from aspergillus fungus, for example aspergillus oryzae. Examples of suitable protease activities include aspergillus-derived serine proteases and aspergillus-derived calcium-dependent proteases. In one embodiment, the proteolytic preparation employs more than one protease, for example at least two, three, four or five proteases. In a preferred embodiment, an aspergillus-derived serine protease and an aspergillus-derived calcium-dependent protease are employed. Ideally, the aspergillus-derived protease comprises a protease preparation of the FLAVORPRO-WHEY family, for example FLAVORPRO-WHEY 750-P or FLAVORPRO-WHEY 192-P.

[0057] The casein substrate comprises a casein salt, examples of which will be well known to those skilled in the art and include sodium caseinate. In a preferred embodiment, the casein substrate is a milk casein, ideally a bovine milk casein. Typically, the casein substrate (and/or the casein hydrolysate) is substantially free (i.e. less than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1% (w/w) of other milk proteins, for example whey proteins. The casein substrate employed for the hydrolysis typically has a concentration of 5-15%, 8-12%, and ideally about 9-11% (w/v)

[0058] Typically, the ratio of protease to casein substrate (w.w) is from 0.1 to 1.0%, preferably 0.2 to 0.8%, more preferably 0.4 to 0.6%, and ideally at about 0.6%. (0.625-0.658% (w:w)see generation of hydrolysates section). Typically, the protease employed has an activity of >55 Casein Protease units/gram.

[0059] Suitably, hydrolysis of the casein substrate with protease is carried out at a temperature of from 45 C. to 55 C., preferably from 49 C. to 51 C. , and ideally at about 50 C. Typically, hydrolysis of the casein substrate with protease is carried out at a pH of from 5 to 9, suitably at a pH of from 5.5 to 8, preferably at a pH of from 6 to 8, and ideally at a pH of about

[0060] In a preferred embodiment, the casein hydrolysate of the invention has a peptide profile substantially similar to that shown in FIG. 10. In this regard, the term substantially similar should be understood to mean comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the casein peptides of FIG. 10SEQUENCE ID NO's 46 to 83 (for example. comprising 35 of the 38 peptides listed in FIG. 10)

[0061] In a preferred embodiment, the low molecular weight fraction of the casein hydrolysate of the invention has a peptide profile substantially similar to that shown in FIG. 9. In this regard, the term substantially similar should be understood to mean comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the casein peptides of FIG. 9SEQUENCE ID NO's 1 to 45 and 55 (for example. comprising 40 of the 46 casein-derived peptides listed in FIG. 9).

[0062] Thus, in a preferred embodiment, the invention provides a low molecular weight fraction of a casein hydrolysate fraction formed by hydrolyzing a milk (typically bovine milk) casein substrate to a degree of hydrolysis of 5% DH to 15% DH, 6% DH to 14% DH, 7% DH to 13% DH, or 8% DH to 12% DH, by a mixture of an aspergillus-derived serine protease and an aspergillus-derived calcium dependent protease, preferably a FlavorPro Whey protease preparation, in which the hyrolysate is substantially free of peptides having a MW of greater than 5 kDa.

[0063] In another embodiment, the invention provides a casein hydrolysate formed by hydrolyzing a casein substrate to a degree of hydrolysis of from 8% DH to 14% DH by a mixture of an aspergillus-derived serine protease and an aspergillus-derived calcium dependent protease, preferably a FlavorPro Whey protease preparation, in which the hyrolysate is substantially free of peptides having a MW of greater than 7 kDa, 6 kDa, or ideally 5 kDa.

[0064] The invention also relates to a casein hydrolysate of the invention in a dehydrated form, for example a powder. Methods of dehydrating the casein hydrolysate of the invention will be well known to those skilled in the art, and include fluidized bed drying, drum drying and spray drying.

[0065] Preferably, the casein hydrolysate of the invention comprises of peptides spread over the following distribution, as a function of their molecular mass: [0066] >10 kD0 to 30% [0067] 5-10 kD0.1 to 8% [0068] <5 kD60 to 99.7%.

[0069] Preferably, the low molecular weight fraction of the casein hydrolysate of the invention comprises of peptides spread over the following distribution, as a function of their molecular mass: [0070] >10 kD0 to 1% [0071] 5-10 kD0.1 to 1% [0072] <5 kD98 to 99.9%.

Hydrolysate Generation-Lab Scale

[0073] Sodium caseinate (NaCN, 85.92% w/w protein) was provided by Arrabawn Co-op Society Ltd., Tipperary, Ireland. NaCN solutions (4.8 L) of 9.3% (w/v) were prepared the day before hydrolysis experiments by adding the appropriate quantity of NaCN powder to 4.5 L of distilled water. The protein was allowed to hydrate at 50 C. with gentle stirring in a sealed reaction vessel using a Heidolph overhead stirrer for 2 to 3 hours before being stored at 4 C. overnight. The following day, these solutions were subdivided into different aliquots for the generation of hydrolysates at different DH values.

TABLE-US-00001 TABLE 1 Preparation of NaCN hydrolysates using FlavorPro Whey Sample DH (%) .sup.4 = E:S ratio, % 0 (Intact NaCN) 0.40 0.031 1.35 0.313 2.50 0.625 6.46 7.54 44 9.66 44 10.76 44 * %(w:w) = % of enzyme powder/weight of NaCN protein present

[0074] Different E:S ratios were used to generate casein hydrolysates having different DH values. An E:S ratio of 0.625% (w:w) was used to generate the FlavorPro Whey 10.76% DH hydrolysate (Table 1).

[0075] Upon addition of the commercial proteolytic preparation, the pH of the protein solution was kept constant at pH 7 using a Titrino 718 pH stat. The temperature of the hydrolyzing NaCN solutions was maintained at 50 C. Proteolytic activity was inactivated by bringing the temperature of the hydrolysate samples to 80 C. and maintaining the samples at this temperature for a further 20 mins.

Hydrolysate Generation-Semi-Pilot Scale

[0076] A 300 L solution of 8.85% (w/v) NaCN at pH 7 and 50 C. was prepared by mixing with a Silverson high shear mixer in a sealed water jacketed 300 L tank. FlavorPro Whey (174.70 g) was added to the stirred solution at an enzyme powder:substrate (E:S) ratio of 0.658% (w:w). The solution was incubated for 4 h at 50 C. in the 300 L tank and its pH was maintained at pH 7.0 with the addition of NaOH. The proteolytic activity was inactivated by bringing the temperature of the 300 L solution to 80 C. and maintaining it at that temperature for 20 mins. An aliquot of the hydrolysate was spray dried using a Niro Minor spray dryer. The remaining crude hydrolysate solution was then cooled to 5 C. and stored overnight. The following morning, the solution was reheated to 50 C. and following clarification was ultrafiltered through a membrane having an effective molecular mass cut off of 5 kDa mounted on a DSS MemProc membrane filtration unit. The resulting permeate and retentate were then spray dried using a Niro Minor spray dryer.

Quantification of Degree of Hydrolysis Using TNBS

[0077] Aliquots of the NaCN hydrolysates were diluted to0.09% or 0.05% (w/v) protein with 1% (w/v) SDS and heated for 20 min at 80 C. to fully disperse the hydrolyzed protein. The Degree of hydrolysis (DH, %) of the NaCN hydrolysates was then determined using the TNBS method of Adler-Nissen (1979), as described by Spellman et al., (2003).

Residual Antigenicity

[0078] This was quantified using a sandwich format casein ELISA protocol employing polyclonal IgG (purified from serum) raised in rabbit against unhydrolyzed sodium caseinate. The purified IgG was coated onto standard medium bind 96 well microtitre plates, and was also conjugated to horse radish peroxidase (HRP) to make the antibody conjugate. A calibration curve was constructed using the sodium caseinate used to immunize the rabbits for the calibration standard. The calibration curve range was between 10 and 10,000 ng/m/L which was plotted as 1 to 4 Log ng/mL Aliquots of the NaCN hydrolysate samples were analysed for residual antigenicity to intact casein using the polyclonal antisera raised in rabbits. Samples were prepared at concentrations of 10, 1, 0.1 and 0.01 mg/mL (7, 6, 5 and 4 Log ng/mL) based on their protein content. Plots of ELISA response .sub.(A450/600) versus Log protein concentration were created to determine the relative reduction in residual antigenicity to intact casein (expressed as Log reduction and relative decrease).

Gel Permeation Chromatography and Molecular Mass Distributions

[0079] Gel permeation HPLC (GP-HPLC) was performed using the Waters HPLC system as essentially described by Spellman et al. (2005). A calibration curve was prepared from the average retention times of standard proteins and peptides (Smyth & FitzGerald, 1998). Molecular Mass Distributions were generated by integrating the GP-HPLC chromatograms using Breeze version 3.30 software, using the retention times corresponding to 5000 Da and 10000 Da to divide the chromatogram into 3 sections: 0-5000 Da, 5000-10000 Da and >10000 Da. The integrated area of each section was expressed as a percentage of the integrated area of the entire chromatogram obtained at 214 nm.

Bitterness Evaluation

[0080] Hydrolysate samples, made up in non sparkling mineral water (B allygo w an) at a protein equivalent of 4.5 g/L, were randomly presented in triplicate to a 10-member sensory panel which had been trained to detect and quantify bitterness using caffeine solutions that were also made up in non-sparkling water. Panelists were trained to assign bitterness scores to unknown solutions based on a 0-100% scale, where a 100% bitter solution was taken to have a bitterness value equivalent to 1 g caffeine/L. Non sparkling mineral water was used as the 0 bitterness standard. At each sitting, panelists were presented with solutions of 0.00, 0.25, 0.50, 0.75 and 1.00 g caffeine/1, which had been labeled as 0, 25, 50, 75 and 100 and the test hydrolysates. The panelists were first required to taste the 0, 25, 50, 75 and 100 solutions in order to familiarise themselves with their taste intensities. They were then required to taste hydrolysates and based on their evaluations of the taste intensities of the 0, 25, 50, 75 and 100 solutions and then rank the taste intensities of the test hydrolysates out of 100. Between tasting each of the bitterness standards and hydrolysates, panelists were asked to eat a piece of non-salted cracker and to rinse their mouths thoroughly with non-sparkling mineral water (Spellman et al. (2005)). Results were expressed as the mean bitterness scorestandard error of the mean (SEM).

Determination of Nitrogen Solubility Index

[0081] This was carried out essentially according to Flanagan and FitzGerald (2002). Nitrogen was determined on the supernatants of 4 g 100 g.sup.1 aqueous protein solutions following centrifugation (1620 g; 15 min), using a modification of the macro-Kjeldahl method (IDF, 1993). Kjeldahl catalyst tablets were used instead of potassium and copper sulphate, and the end-point of the titration step was reached at pH 4.6. All nitrogen solubility analyses were carried out induplicate.

[0082] Mass Spectral Characterization of Test Samples

[0083] The MS and tandem MS experiments were run on a MicrOTOF II mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with an electrospray ion source, controlled by the MicrOTOF Control software(version 2.3, Bruker Daltonics). The data were acquired over a

mass/charge (m/z) range of 300-2500. A full scan MS spectrum was acquired, followed by tandem mass spectra using collision-induced dissociation (CID) of the five most intense precursor ions present in the MS scan. Electrospray conditions were as follows: capillary temperature, 130C; Capillary voltage, 1500V; Dry Gas flow, 6.0 L min.sup.1 Data analysis software (version 40, Bruker Daltonics), BioTool (version 3.1, Bruker Daltonics) and MASCOT (Perkins et al., 1999) were used for data processing and evaluation of peptide sequencing.

Clarity Determination

[0084] Clarity was determined at both room (20 C.) and storage temperature (4 C.) by measuring the absorbance at 660 nm of aqueous hydrolysate solutions using distilled water as blank essentially as described by Sin et al., (2006)

Heat Stability Determination

[0085] The pH of aliquots of aqueous NaCN hydrolysates (2.0% (w/v) protein equivalent) were adjusted to between 2.0 and 8.0, using 0.1 M and 1 M HCl or NaOH. Samples were then allowed to equilibrate for at least 1 h at room temperature. The pH of the samples was then re-measured and re-adjusted, if necessary, prior to heat stability analysis Immediately after final pH adjustment, samples (2 ml) were placed in glass tubes (10 mm i d_120 mm, AGB Scientific, Dublin, Ireland), sealed with silicone bungs, immersed in an oil bath thermostatically controlled at 140 C. (Elbanton BV, erkdriel,The Netherlands), with continuous rocking at motor speed setting 3. The heat coagulation time (HCT) was taken as the length of time in min that elapsed between placing the sample in the oil bath and the onset of coagulation (Ryan et al., 2004). These analyses were performed in triplicate.

[0086] The invention is not limited to the embodiment hereinbefore described in detail which may be varied in construction, detail and process step without departing from the spirit of the invention.

References

[0087] Adler-Nissen J (1979). Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid. Journal of Agricultural and Food Chemistry 27: 1256-1262

[0088] Flanagan, J. and FitzGerald, R. J. (2002). Physicochemical and nitrogen solubility properties of Bacillus lj;ljl;kj falkgsfdjs;ldgjas;lgakjsgal;sdgjkasdg asdg Flanagan, J. and FitzGerald, R. J. (2002). Physicochemical and nitrogen solubility properties of Bacillus proteinase hydrolysates of sodium caseinate incubated with transglutaminase pre- and post-hydrolysis. J. Agric. Food Chem. 50(19): 5429-5436.

[0089] IDF. (1993). Block Digestion (Macro) Method. International Standard 20B, Part 2. Brussels, Belgium: International Dairy Federation

[0090] Perkins, D. N. , Creasey, D. M. and Cottrell, J. S. (1999). Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis, 20,3551-3567.

[0091] Ryan, M., McEvoy, E., Duignan, S., Crowley, C., Fenelon, M. )' Callaghan, D. M. and FitzGerald, R. J. (2008). Thermal stability of soy protein isolate and hydrolysate ingredients. Food Chem. 108:503-510.

[0092] Sin, H. N., Yusof, S., Hamid, N. Sheikh Abdul and Rahman, R. Abd. (2006). Optimization of enzymatic clarification of sapodilla juice using response surface methodology. Journal of Food Engineering 73: 313-319.

[0093] Smyth M & FitzGerald R. J. 1998. Relationship between some characteristics of WPC hydrolysates and the enzyme complement in commercially available proteinase preparations. Int. Dairy Journal 8: 819-827

[0094] Spellman, D. O'Cuinn, G. and FitzGerald, R. J. (2005) Physicochemical and sensory characteristics of whey protein hydrolysates generated at different total solids levels. J. Dairy Res. 72: 138-143.

[0095] Spellman D, McEvoy E, O'Cuinn G & FitzGerald R J (2003). Proteinase andexopeptidase hydrolysis of whey protein: Comparison of the TNBS, OPA and pH stat methods for the quantification of degree of hydrolysis. International Dairy Journal 13: 447-453