Milk-based protein hydrolysates and compositions made thereof

Abstract

A process for preparing a milk protein hydrolysate comprising hydrolysing a milk-based proteinaceous material with a microbial alkaline serine protease in combination with bromelain, a protease from Aspergillus and a protease from Bacillus.

Claims

1. A process for preparing a milk protein hydrolysate, the process comprising hydrolyzing a milk-based proteinaceous material with subtilisin Carlsberg, bromelain, leucine aminopeptidase from Apergillus oryzae, aspergillopepsin 1 from Apergillus oryzae, and neutral proteinase from Bacillus subtilis.

2. The process according to claim 1 comprising: a first hydrolysis step comprising hydrolysing the milk-based proteinaceous material with subtilisin Carlsberg; and a second hydrolysis step comprising hydrolysing the milk protein with the bromelain, the leucine aminopeptidase, the Aspergillopepsin 1 and the neutral proteinase from Bacillus subtilis.

3. The process according to claim 1, wherein the milk-based proteinaceous material is selected from the group consisting of whey protein, casein and mixtures thereof.

4. The process according to claim 1, wherein the milk-based proteinaceous material is whey protein.

5. The process according to claim 1 further comprising subjecting the hydrolyzed milk-based proteinaceous material to a process selected from the group consisting of enzyme inactivation, microfiltration and ultrafiltration.

6. The process according to claim 1, wherein the milk protein hydrolysate is an extensively hydrolyzed product, and the extent of hydrolysis (NPN/TN %) is greater than 95%.

7. The process according to claim 1, wherein the extent of hydrolysis (NPN/TN %) is greater than 99%.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Shows the amino acid profiles of UF permeate using the enzyme combinations according to the invention, determined in two different trials (left-hand bar 1.sup.st trial, right-hand bar 2.sup.nd trial).

DETAILED DESCRIPTION OF THE INVENTION

(2) The milk-based protein hydrolysate of the present invention is obtained by the treatment of a solution of a milk-based proteinaceous material with the proteases referred to herein.

(3) Milk-Based Proteinaceous Material

(4) The milk-based protein hydrolysate is preferably a milk-based proteinaceous material. It may be a whey-based proteinaceous material, casein or mixtures of whey-based proteinaceous material and casein.

(5) The casein source may be acid casein or non-fat milk solids.

(6) Preferably the milk-based proteinaceous material is whey based.

(7) The whey based proteinaceous material may be a whey from cheese making, particularly a sweet whey such as that resulting from the coagulation of casein by rennet, an acidic whey from the coagulation of casein by an acid, or the acidifying ferments, or even a mixed whey resulting from coagulation by an acid and by rennet. This starting material may be whey that has been demineralized by ion exchange and/or by electrodialysis and is known as demineralised whey protein (DWP).

(8) The source of such whey-based proteinaceous material may be sweet whey from which the caseino-glycomacropeptide (CGMP) has been totally or partially removed. This is called modified sweet whey (MSW). Removal of the CGMP from sweet whey results in a protein material with threonine and trytophan contents that are closer to those of human milk. A process for removing CGMP from sweet whey is described in EP 880902. The starting material may be a mix of DWP and MSW. It may be a concentrate wherein the whey protein is 35-80% protein (WPC) or an isolate if the whey protein concentration is more than 95% protein (WPI). An example of WPC is WPC 87 Lacprodan available from Aria Foods, Denmark and an example of WPI is Bipro from Davisco Foods International (Minnesota USA).

(9) Preferably the milk-based proteinaceous material is whey protein isolate (WPI).

(10) The milk based proteinaceous material may be in solution or suspension, and may be, for example, present at a concentration of 2-30% by weight of proteinaceous material, more preferably 5-20%, more preferably 6-10%. In one embodiment the milk based proteinaceous material is present at a concentration of about 6%.

(11) Addition of lactose to starting material for hydrolysis has the advantage that any residual protein contained in the lactose is hydrolysed. Lactose may be present in concentrations from 0.05-30% w/w, preferably 0.10-20% w/w, or in cases where a lower lactose content is preferred, 0.10 to 1%, preferably 0.10 to 0.20% (w/w). In the latter case the final product may be destined for subjects with a low lactose tolerance. Lactose may be removed, for example, by ultrafiltration (yielding UF whey), optionally followed by dialysis. In one embodiment the lactose is present at a concentration of about 2%.

(12) The starting material may be in the form of a true or colloidal aqueous solution, or in the form of a powder. In the latter case, the powder is dissolved in preferably demineralised water to form an aqueous solution

(13) Enzymes Used in Hydrolysis

(14) Microbial Alkaline Serine Protease

(15) The microbial alkaline serine protease is preferably derived from a Bacillus species, more preferably from Bacillus licheniformis.

(16) In a preferred embodiment the alkaline serine protease is a subtilisin.

(17) Examples of subtilisins are those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin BPN subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279) and Protease PD138 (WO 93/18140). Examples are described in WO 98/020115, WO 01/44452, WO 01/58275, WO 01/58276, WO 03/006602 and WO 04/099401.

(18) An example of an alkaline serine protease is subtilisin Carlsberg enzyme Uniprot P00780 or a variant thereof.

(19) In a particularly preferred embodiment the alkaline serine protease for use in the invention is Alcalase.

(20) Bromelain

(21) The term bromelain is well known in the art. Bromelain may be referred to as an extract derived from members of the Bromeliaceae family which comprises various thiol proteases and is known to have proteolytic activity in vitro and in vivo.

(22) Preferably the extract is derived from the stem of Ananas comosus (pineapple). The extract may include elements such as cysteine proteases, amylase, acid phosphatase, peroxidases and cellulases.

(23) The enzyme may have the following EC Number: EC 3.4.22.32.

(24) Bromelain may comprise the: stem bromelain (UniProt P14518) or a variant thereof.

(25) Aspergillus Proteases

(26) Leucine Amino Peptidase

(27) Preferably an Aspergillus leucine amino peptidase is used in the present invention. More preferably, an Aspergillus oryzae leucine amino peptidase is used in the present invention.

(28) The enzyme may have the following EC Number: EC 3.4.11.1.

(29) A leucine amino peptidase preferably catalyses the hydrolysis of residues at the N-terminus of peptides, preferably the hydrolysis of leucine residues.

(30) Examples of leucine amino peptidase enzymes which are expressed by Aspergillus oryzae include LAPA (UniProt Q2U1F3), LAP1 (UniProt Q2PIT3) and LAP2 (UniProt Q2ULM2)).

(31) Aspergillopepsin 1

(32) Preferably Aspergillopepsin 1 derived from Aspergillus species is used in the present invention. More preferably Aspergillus oryzae Aspergillopepsin 1 is used in the present invention. Other names for Aspergillopepsin 1 commonly used in the art include, inter alia, Aspergillopepsin A, Aspergillopepsin F and Aspergillopeptidase A.

(33) The enzyme may have the following EC Number: EC 3.4.23.18.

(34) Aspergillopepsin 1 enzymes preferably catalyse the hydrolysis of polypeptides with a broad specificity, preferably the hydrolysis of peptide bonds between hydrophobic residues.

(35) Bacillus Protease

(36) A protease from Bacillus species is preferably used in the process of the present invention. Preferably Bacillus subtilis neutral proteinase is used in the process of the present invention. Other names for Bacillus subtilis neutral proteinase commonly used in the art include, inter alia, bacillolysin, Bacillus metalloendopeptidase, megateriopeptidase, Bacillus neutral protease and Bacillus extracellular neutral metalloprotease.

(37) Preferably the Bacillus subtilis neutral proteinase is from Bacillus subtilis.

(38) The Bacillus protease may have the following EC Number: 3.4.24.28.

(39) An example of a Bacillus protease is NPRE (UniProt P68763) or a variant thereof.

(40) The Hydrolysis Process

(41) The typical conditions for carrying out the hydrolysis process have been described in the prior art. The temperature may range from about 40 C. to 60 C., for example about 55 C. The reaction time may be, for example, from 1 to 10 hours and pH values before starting hydrolysis may, for example, fall within the range 6.5 to 8.5, preferably 7.0 to 8.0.

(42) The pH may be adjusted with known agents, for example Ca(OH).sub.2.

(43) In one embodiment, the process comprises: (i) a first hydrolysis step comprising hydrolysing the milk-based proteinaceous material with the microbial alkaline serine protease; and (ii) a second hydrolysis step comprising hydrolysing the milk protein with bromelain in combination with the proteases from Aspergillus and the protease from Bacillus.

(44) Step (i) may be performed, for example, for about four hours and step (ii) may be performed, for example, for about six hours.

(45) Irrespective of how the hydrolysis is carried out, the hydrolysis product undergoes a heat treatment, which inactivates the enzyme carrying out the hydrolysis. This heat treatment preferably comprises preheating the hydrolysate to a temperature of or above 75 C. (for example 75 C. to 90 C.) and keeping it at that temperature for about 0.1 to 30 minutes to promote auto-digestion of the enzyme. This treatment may be followed by sterilization, preferably at ultra-high temperature, for example at 125 C.-135 C. for 30 seconds to 3 minutes by injection of steam or in a heat exchanger.

(46) The hydrolysate thus obtained may be clarified, microfiltered and/or ultrafiltrated to remove residual protein large fragments. It may also be concentrated, for example by reverse osmosis. It may then be dried, for example by lyophylisation, spray drying, or by freeze drying for different applications, or may even be subsequently treated. In the latter case, the enzyme may be inactivated during the subsequent treatment.

(47) The hydrolysates of the invention may have an extent of hydrolysis that is characterised by NPN/TN % content. NPN/TN % ratio means the Non Protein Nitrogen divided by the Total Nitrogen100. The non-protein Nitrogen is the nitrogen fraction obtained after acid precipitation of proteins. NPN/TN % may be measured as detailed in Adler-Nissen J-, 1979, J. Agric. Food Chem., 27 (6), 1256-1262.

(48) Alternatively, the extent of hydrolysis may be characterized by the amount of amino nitrogen released upon hydrolysis; free amino nitrogen can react with a reagent such as trinitrobenzenesulfonic acid (TNBS).

(49) In general, extensive hydrolysates are characterised as having a NPN/TN % of greater than 95%, whereas partially hydrolysed hydrolysates are characterized as having a NPN/TN % in the range 75%-85%. In a preferred embodiment the hydrolysates of the invention are extensive hydrolysates having an NPN/TN % in the range of greater than 95%, 96%, 97%, 98% or 99%.

(50) These hydrolysates may also be characterised in that at least 95% their protein/peptide population has a molecular weight of <1000 Daltons.

(51) The molecular weight distribution of the peptides in the protein hydrolysate obtained may be determined, e.g., by size exclusion chromatography (SEC). In a preferred embodiment the hydrolysate of the invention has a peptide weight distribution similar or substantially identical to that of Alfar. Preferably the hydrolysate of the invention has a peptide weight distribution similar to or substantially identical to that of a hydrolysate made with porcine pancreatin in place of the bromelain and Aspergillus and Bacillus proteases (in particular, in place of bromelain, a leucine aminopeptidase from Apergillus oryzae, Aspergillopepsin 1 from Apergillus oryzae and Bacillus subtilis neutral proteinase) referred to herein. Put another way, the bromelain and Aspergillus and Bacillus protease blend performs substantially the same hydrolytic activity in the context of the present invention as porcine pancreatin.

(52) In a preferred embodiment, the hydrolysate of the invention is an extensive hydrolysate and is comprised of peptides having a median molecular weight of 300 Da to 370 Da, preferably 320 Da to 360 Da.

(53) The residual antigenicity of the hydrolysates may be evaluated using standard immunoassays such ELISA tests. Preferably the hydrolysates of the invention present a residual -lactoglobulin (BLG) of <0.1 mg BLG equivalent/g protein equivalent, and most preferably <0.01 mg BLG equivalent/g protein.

(54) The hydrolysates of the invention may be incorporated into infant formula, follow-on formula, a baby food, infant cereals, growing-up milk, infant or child's food supplement or an adult nutritional composition, i.e. all preparations treatment of allergy, as well as any other benefits that protein hydrolysates could provide to humans. Preferably, the hydrolysates are used in starter infant formula.

(55) Current hypoallergenic formulas composed of such cows' milk proteins hydrolysates aimed at allergy prevention also comprise other nutrients such as animal oils, vegetable oils, starch, maltodextrin, lactose and sucrose.

(56) In one embodiment of the invention, the hydrolysates of the invention are used in combination with selected probiotics, for example in infant formula. The selected probiotics can be any of the probiotics conventionally used in infant formula. Preferably the probiotics are those able to provide additional or synergistic effect on allergies.

(57) Examples of suitable probiotic micro-organisms which may be used in the present invention include yeasts such as Saccharomyces, Debaromyces, Candida, Pichia and Torulopsis, moulds such as Aspergillus, Rhizopus, Mucor, and Penicillium and Torulopsis and bacteria such as the genera Bifidobacterium, Bacteroides, Clostridium, Fusobacteum, Melissococcus, Propionibacteum, Streptococcus, Enterococcus, Lactococcus, Staphylococcus, Peptostrepococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus, Oenococcus and Lactobacillus. Specific examples of suitable probiotic microorganisms are: Saccharomyces cereviseae, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Enterococcus faecium, Enterococcus faecalis, Lactobacillus acidophilus, Lactobacillus alimentarius, Lactobacillus casei subsp. casei, Lactobacillus casei Shirota, Lactobacillus curvatus, Lactobacillus delbruckii subsp. lactis, Lactobacillus farciminus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus reuteri, Lactobacillus rhamnosus {Lactobacillus GG), Lactobacillus sake, Lactococcus lactis, Micrococcus varians, Pediococcus acidilactici, Pediococcus pentosaceus, Pediococcus acidilactici, Pediococcus halophilus, Streptococcus faecalis, Streptococcus thermophilus, Staphylococcus carnosus, and Staphylococcus xylosus.

(58) Preferred probiotic bacterial strains include Lactobacillus rhamnosus ATCC 53103 obtainable from Valio Oy of Finland under the trade mark LGG, Lactobacillus rhamnosus CGMCC 1.3724, Lactobacillus paracasei CNCM 1-2116, Bifidobacterium lactis CNCM 1-3446 sold inter alia by the Christian Hansen company of Denmark under the trade mark Bb 12 and Bifidobacterium longum ATCC BAA-999 sold by Morinaga Milk Industry Co. Ltd. of Japan under the trade mark BB536.

(59) The probiotics may, for example, be present in an amount of 10.sup.3 to 10.sup.12 cfu/g, more preferably 10.sup.6 to 10.sup.11 cfu/g, even more preferably 10.sup.4 to 10.sup.9 cfu/g, most preferably 10.sup.7 to 10.sup.9 cfu/g composition or per mL of composition.

(60) Those skilled in the art will understand that they can freely combine all features of the present invention described herein, without departing from the scope of the invention as disclosed.

(61) Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples.

(62) The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; and E. M. Shevach and W. Strober, 1992 and periodic supplements, Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. Each of these general texts is herein incorporated by reference.

EXAMPLES

(63) Renovate Alfar and Althra are extensively hydrolysed infant formulas intended for babies with food allergies and intolerances.

(64) Alfar& Althra make use of pig pancreatin preparation during manufacturing process.

(65) A preblend of plant and microbial proteases has unexpectedly been identified as a suitable alternative to replace pancreatin in EHP productions while maintaining the biological & physiological properties of hydrolysed products, particularly Extensively Hydrolysed Products (EHP). In particular, the use of a combination of bromelain, a microbial alkaline serine protease, proteases from Aspergillus and a protease from Bacillus has been identified as a suitable alternative to pancreatin.

(66) The present inventors have established a process for preparing a milk protein hydrolysate comprising hydrolysing a milk-based proteinaceous material with the aforementioned agents.

Example 1Methods

(67) 500 kg Bipro (whey isolate) was hydrolysed with Alcalase, bromelain, leucine aminopeptidase from Apergillus oryzae, aspergillopepsin 1 from Apergillus oryzae and Bacillus subtilis neautral proteinase from Bacillus subtilis.

(68) Enzyme inactivation was achieved by heat treatment (90 C. for 5 minutes) and all residual intact proteins and large peptides were removed by micro filtration and ultrafiltration.

Example 2Extent of Hydrolysis

(69) NPN/TN %

(70) The extent of hydrolysis was determined using the ratio between Non Protein Nitrogen and Total Nitrogen (NPN/TN %). Non Protein Nitrogen fraction is obtained by acid precipitation of proteins. Upon protein hydrolysis, resulting peptides will fall into the Non Protein Nitrogen part. Thus, NPN/TN content is increasing with increasing hydrolysis extent. The target is NPN/TN>=95% in the hydrolysate and NPN/TN>=99% after filtration.

(71) SDS-PAGE

(72) The extent of hydrolysis was also determined by SDS-PAGE electrophoresis using Phastsystem and silver staining, to identify any residual intact protein and large peptides (above 10 kDa) in the hydrolysate and after filtration. The total protein and peptides present in the sample are separated using a polyacrylamide gel after denaturation (treatment with SDS and heat), reduction (treatment with DTT to reduce disulfide bridges) and acylation (addition of iodoactamide to block thiols groups) of the sample. Although some intact protein and large peptides may still be present after hydrolysis, no band related to residual protein and large peptide are detected after filtration by loading on the gel a sample solution containing 2 g Nitrogen/L solution.

(73) BLG-ELISA

(74) The absence of residual antigenicity was assessed through determination of beta-lactoglobulin antigenicity using a commercially available enzyme-linked immunosorbent assay (ELISA) kit specific to beta-lactoglobulin form r-Biopharm. The target is below the limit of detection of this kit in a whey based hydrolysate, i.e. 0.01 mg beta-lactoglobulin equivalent/g protein.

(75) Peptide Profile

(76) Peptides generated though hydrolysis and present in the final product after filtration were characterized based on their molecular weight using size-exclusion chromatography (SE-HPLC). Molecular weight distribution (and Median value) of soluble peptides was obtained after sample dissolution in 0.1% TFA in water and using Superdex Peptide 10/300 GL Size-Exclusion column with 0.1% v/v TFA, 30% v/v ACN in water as mobile phase. Molecular weight distribution of peptides was determined in following ranges: peptides>2400 Da, 1200-2400 Da, 600-1200 Da, 240-600 Da and <240 Da. Median value is the molecular weight at which 50% of the peptides have molecular weight above this value.

(77) Free AA/Total AA %

(78) The release of free amino acids upon hydrolysis was determined using the ratio between free amino acid and total amino acids (free AA/Total AA %). Free amino acids content is obtained by separation of the free amino groups present in an aqueous sample extract by ion exchange chromatography (IEC) and photometric detection after post-column derivatization with ninhydrin reagent. Total amino acids content is obtained by hydrolysis of the test portion in 6 mol/L hydrochloric acid (HCl) under nitrogen (a peroxidation of cystine to cysteic acid and methionine to methionine-sulfone is applied before hydrolysis to quantify the acid stable amino acids) and separation of individual amino acids by ion-exchange chromatography as described above.

(79) The results of three experiments using the enzyme blend of the present invention are shown in Table 1.

(80) TABLE-US-00001 TABLE 1 Peptide SDS profile Residual Page MED Free BLG mg No (50%) AA/Total eq./g prot NPN/TN residual Da AA (Elisa) Combination Targets >95% band 350 max 25% <0.01 mg/g Alcalase; UF permeate 99.3 OK 352.8 10.8 n.q. Bromelain; leucine amino- peptidase and Aspergillopepsin 1 from Apergillus oryzae; Bacillus subtilis neutral proteinase from Bacillus subtilis As above UF permeate 100.0 OK 354.0 11.4 n.q As above UF permeate 100.5 OK 354.9 11.6 n.q

(81) All results were within the target values.