Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21004—Trypsin (3.4.21.4)
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J3/00—Working-up of proteins for foodstuffs
  • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
  • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
  • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
  • A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
  • A23J3/343—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins of dairy proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
  • A23L33/18—Peptides; Protein hydrolysates
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

• the present invention relates to a process for the production of a hypoallergenic whey protein hydrolysate and to the product of such a process.
• the hypoallergenic whey protein hydrolysate finds use in the manufacture of infant formulae and special dietetic foodstuffs.
• infant formulae are manufactured based on the nutritional profile of human milk, where the relative concentrations of casein nitrogen, whey protein, nitrogen and non-protein nitrogen are 35%, 40% and 25% respectively (Packard, 1982).
• Bovine milk (78% casein, 17% whey protein and 5% NP-N) is the usual substitute for human milk but it requires several modifications.
• the ratio of casein to whey protein nitrogen for instance must be adjusted to 40:60. This is generally carried out using demineralised whey powder which also advantageousl increases the lactose concentration to that of breast milk.
• Bovine milk proteins however, are known to give rise to antigenic responses in a small percentage of the population; estimates range from 0.1% to 8% (Clein, 1954, and Collins-Williams, 1962). However, Frier and Kletter (1970) found the incidence to be only about 0.5% when stricter criteria were adopted for the survey. Common allergenic response include diarrhoea, vomiting, intestinal disorders, respiratory problems, dermatitis, irritability, restlessness and loss of appetite beta-Lactoglobulin (absent in human milk) is the most frequent cause o milk sensitivity.
• a survey by Saperstein and Anderson (1962) revealed that a variety of infant formulae on the market contained all the majo whey protein and casein antigens. Soybean preparations have been used as substitutes for cows milk in baby formulae but can be at least as antigenic as milk protein. (Eastham and Grady, 1978).
• casein treated with pancreatic protease enzymes was shown to be devoid of antigenicity (Takase et aJ 1977).
• Pahud et a l (1985) and Jost et a l (1987) produced hypoallergenic whey protein hydrolysates using trypsin.
• Asselin et a J (1989) demonstrated that hydrolysis of whey proteins with pepsin followed by alpha-chymotryps was the most efficient combination of enzymes to reduce allergenicity of alpha-lactalbumin and bata-Lactoglobulin.
• a preliminary heat treatme may be desirable to make susceptible bonds more accessible to proteolytic degradation.
• the advantage of enzymic hydrolysis over he treatment alone is the increased solubility of the product over a bro pH range but sensoric properties may be affected due to increased bitterness.
• the European Patent Specification No. 0226221.B describes a whey based protein hydrolysate characterised by having a molecular weight profile of not greater than 6,000 Daltons and being free of allergenic substances and lactose.
• An enzyme hydrolysis process is used to produce the hydrolysate, in which the peptides are harvested from the crude hydrolysate using ultrafiltrat on (m.w.c.o. 6,000) and/or diafiltration. It is evident from the description of the process that NaOH is added as the sole base to control pH. However, this would lead to excessive levels of Na in the final product for inclusion in infant formulae.
• the end product is described as being particularly suitable for allergies and for humans with lactose malabsorption.
• European Patent Specification No. 353 122 A discloses a hypoallergenic whey protein hydrolysate of a defined molecular weight profile, i.e. not containing peptides greater than 5,000 Daltons with 35-40% between 1500 and 500 Daltons and a process for producing the same.
• the proces involves emzymatic hydrolysis of whey proteins using an enzyme mixture consisting of chymotrypsin and trypsin with relative activities of fro 1.5 to 3.0.
• the crude hydrolysate is fractionated by ultrafiltra ion/diafiltration using membranes with a threshold of less than 10,000 Daltons.
• UK Patent Specification No. 2043651 B describes a process for preparing purified protein hydrolysates, for use in the dietetic field, from animal proteins (meat, fish), vegetable proteins, microbial proteins and milk proteins. The process involves hydrolysing the proteins, heat treating the product of hydrolysis to denature the proteins and then ultrafiltering to remove the denatured proteins. A range of ultrafiltration membranes is described for purifying the crude hydrolysates having cut-off zones of 1,000 to 10,000 Daltons.
• PCT Publication No. 087/03785 describes a process in which casein is eliminated from the whey protein material, the casein-free whey protein is hydrolysed with at least one proteinase and the hydrolysate is ultrafiltered through a membrane with a cut-off value of not greater than 20,000 Daltons.
• a product suitable for use in an hypoallergenic formulae can be produced if a membrane with a cut-off value of 6,000 Daltons is used.
• European Patent Specification No. 0065663A describes the preparation of a protein hydrolysate for use in an enteric diet in which whey protein is digested with a fungal protease to produce a product in which not more than 25% of the resultant polypeptides contain 10 or more amino-acids.
• hypoallergenic lactoserum hydrolysate A process for preparing an hypoallergenic lactoserum hydrolysate is described in European Patent Specification No. 0322589 A, in which lactoserum is subjected to a two step enzymatic hydrolysis.
• the specification defines "hypoallergenic to mean having no detectable protein of molecular weight greater than 10,000 Daltons".
• the constituent proteins or p ' olypeptides must be smaller than 10,000 Daltons.
• ultrafiltration is used to remove unhydrolysed protein and aggregated peptides.
• ultrafiltration which excludes peptides of greater than 10,000 Daltons was thought to be necessary to achieve the desired reduction i allergenicity.
• microfiltration membranes which allow the passage of much larger peptides, will also allow the production of a hypoallergenic product, when used in the process of the present invention.
• Microfiltration offers considerable advantages over ultrafiltration in terms of product yield. In general microfiltration membranes are less prone to fouling and can be run for longer periods without cleaning.
• the crude hydrolysate of this invention consists of both highly aggregated unhydrolysed proteins which are removed by microfiltration, and a mixture of polypeptides ranging in size from 50,000 Daltons to free amino acids which pass through the membrane and are hypoallergeni
• Lactose is present in human milk at about 7% and contributes about 40% of the caloric intake of the milk.
• Humanised milk/infant formulae are designed to mimic the composition of human milk and therefore lactose must be included.
• the lactose used When formulating a hypoallergenic baby formulae the lactose used must be of a quality such that residual protein, remainin in the lactose following the crystallization step in manufacture, is low enough to satisfy the immune-response specification for hypoallergenic formulae. Generally it is impossible to obtain commercial edible and refined lactose to satisfy this specification an thus the production of a hypoallergenic infant formula has been very difficult.
• an hypoallergenic whey protein hydrolysate comprising hydrolysing a substrate with a proteolytic enzyme, thermally inactivating the enzyme and microfiltering the product of hydrolysis.
• the microfiltration step allows the passage of molecules having a range of sizes from free amino-acids to molecules of about 50,000 Daltons.
• the pore size of the microfiltration membrane may be between 0.02 and 0.3 i, preferably 0.1 urn.
• the substrate may be selected from lactalbumin, whey protein concentrate, de ineralized whey powder or a mixture thereof.
• lactose may be added to the substrate before hydrolysis.
• the proteolytic enzyme may be a pancreatic, fungal, bacterial or plant protease and may be acidic, neutral or alkaline. The pH and temperature conditions of the process are then suitably adjusted to the optimum requirements of the enzyme.
• the invention also provides an hypoallergenic whey protein hydrolysate comprising peptides which range in molecular weight from free amino acids to 50,000 Daltons.
• the hydrolysate may also comprise lactose.
• the hypoallergenic whey protein hydrolysate of the present invention is suitable for use in infant formulae.
• the products of the invention consist of hydrolysed whey protein with a defined molecular weight profile ranging from 50,000 Daltons to free amino acids.
• An advantage of the present invention is the optional inclusion of lactose which is also subjected to proteolytic treatment to hydrolyse residual proteins present in commercially available lactose, which are known to give rise to antigenic responses.
• the mineral levels of the products described are maintained within the specifications for infant formulae.
• the products are 98-100% soluble across a broad pH range and have a very acceptable flavour at neutral pH. Bitterness is not detectable when the hydrolysate is reconstituted in water at a concentration of 1% protein.
• the product of the present invention reconstitutes to a clear solution which is very low in colour.
• the process of the invention utilises a range of whey protein substrates, i.e., lactalbumin, whey protein concentrate (WPC), demineralised whey powder, or a mixture thereof.
• whey protein substrates i.e., lactalbumin, whey protein concentrate (WPC), demineralised whey powder, or a mixture thereof.
• Protein that is no already in a denatured form is given a preliminary heat treatment before addition of the enzyme solution, and before the optional addition of lactose.
• commercial food grade lactose may be added to the hydrolysis mixture before enzyme hydrolysis.
• the quantity of lactose added is calculated such that the final concentration of lactose and protein in the finished product is 70% and 22% respectively. This embodiment is suitable for use as a hypoallergenic baby formulae. Without additional lactose a high protein (80%) hydrolysate is produced instead.
• the preferred enzyme Pancreatic Trypsin, Novo Industie A/S, Novo All 2880 Bagsvaerd, Denmark
• the preferred enzyme has been carefully selected from a range of food grade proteases to give the required degree of hydrolysis, molecular weight distribution of polypeptides and to give rise to a non-bitter hydrolysate when used under the conditions described. It is desirable to have a certain proportion (appr ⁇ x 8 - 15%) of the total polypeptide mixture in the region of 50,000 to 5,000 Daltons to provide emulsion stability in th final infant formulae.
• Bitter flavour of protein hydrolysates depends on the degree to which protein is hydrolysed (% DH) and on the amino acid composition of the peptides produced. Peptides containing a high percentage of hydrophobic amino acids (i.e. Phe, Pro, Val, Trp, Leu, lie) have been positively correlated with increased bitter flavour. At low degrees hydrolysis, the hydrophobic amino acids in peptides are unavailable f interaction with the taste buds due to folding and existence of "hairpin" loops within large peptide structures which shield the hydrophobic amino acids. At high degrees of hydrolysis the extent to which hydrophobic interactions and folding of polypeptides occur is limited and hydrophobic amino acids are 'forced' to exist at the surface of peptides and therefore become available for interaction with taste buds - hence bitter flavour.
• pancreatic enzyme preparation from Novo (PTN 3.OS) satisfied the above criteria and was successfully used to achieve a hydrolysate which was judged by a trained taste panel to have a very low bitterness.
• a novel approach has also been adopted for the enzyme hydrolysis step by way of pH control.
• a titrant mixture of potassium hydroxide and sodium hydroxide is formulated such that a ratio of 1:3 (w/w) of Na+ and K+ exists in the finished product.
• the quantity of titrant used to maintain pH during the enzyme hydrolysis step is calculated such that the final concentration of Na. and K in the finished product does not exceed the specifications for infant or hypoallergenic formulae; in the case of infant formulae that being 10 mg and 30 mg respectively per gram of pure protein.
• the crude hydrolysate is then clarified using a microfiltration membrane of pore size 0.02 to 0.3im, preferably O.lum.
• Microfiltration advantageously removes unhydrolysed protein and aggregated peptides from the crude hydrolysate to yield a clear permeate containing a mixture of polypeptides, free amino acids and lactose which can be evaporated and spray dried.
• Enzyme Linked Immunosorbent Assay [ELISAj is well described in the literature (Voller, 1976, Ishikava et al 1981, Wisdom 1981). In this instance a sandwich ELISA technique was used to determine the level of whey protein antigens remaining in protein hydrolysates, afte processing. In principle the sandwich technique involves binding anti-whey antibodies to the ELISA plate and reacting the bound materia with protein hydrolysate. This is followed by incubation with anit-whey antibodies conjugated to the enzyme HRP. Enzyme substrate i then added and the amount of colour developed by enzyme-catalysed conversion of substrate is a measure of the amount of antigenic material in the protein hydrolysate.
• results from the ELISA reactivity experiments against a total whey protein antisera showed that the antigenicity of the whey protein hydrolysates produced according to the present invention was reduced b at least 4 orders of magnitude and in most cases by at least 5 orders of magnitude when compared with a standard whey protein concentrate.
• a slurry of lactalbumin (Alatal 560, New Zealand) was prepared by adding 18.75 kg Alatal (86% protein) to 175 litres of pasteurised wate in a 210 litre batch stirred tank reactor (BSTR). The temperature of the slurry was adjusted to 50 C and the pH increased to 8.0 with automatic addition of a titrant mixture consisting of 2.56 M K0H and 1.44 M NaOH (3:1 w/w K + /Na + ), using an industrial pH stat. The total quantity of titrant mixture added to the hydrolysis reaction was calculated so that the final concentrations of Na and K in the crude hydrolysate mixture did not exceed 7mg and 21mg per gram of pure protein respectively. At a protein permeation rate of approximately 70% through the microfiltration membrane this should yield a final product with less than 10 mg and 30 mg of Na and K respectively per gram of pure protein.
• the volume of the reaction mixture was made up to 195 litres with deionised water.
• the proteolytic enzyme mixture was added once the lactalbumin slurry had equilibrated at 50°C and pH 8.0' for a minimum of 30 minutes.
• the enzyme [320 g of food grade Trypsin (PTN 3.OS, Novo)] was dissolved in 5 litres of deionised water before addition to the reaction mixture. In this instance the enzyme to substrate ratio (E/S) was equivalent to 2%.
• the pH stat was immediately activated and the remaining KOH/NaOH mixture continuously added to maintain pH at 8.0. When the allowable concentration of K and Na was reached in the crude hydrolysate, the enzyme reaction was continued without pH control.
• the reaction was stopped (by thermal inactivatio ⁇ of the enzyme) when the required DH (Degree of Hydrolysis) and molecular weight profile had been achieved which typically took 5-6 hours .
• Thermal inactivation was achieved by increasing the temperature of the crude hydrolysate to 80°C and maintaining this temperature for 20 minutes. The hydrolysate was then chilled to 4°C and held overnight for further processing.
• the temperature of the crude hydrolysate was adjusted to 50 C and transferred to the balance tank of the microfiltration unit.
• the microfiltration module consisted of an Abcor hollow fibre
• Microfiltration was continued in batch mode with the retentate recycled to the feed tank, the permeate was collected in 25 litre plastic containers, weighed and pooled to form a bulk permeate. 150 litres of permeate was collected, which represents a volume concentration reduction (VCR) of 4. The pooled permeate was heated to 75°C for 15 mins (which resulted in less foaming during the evaporation step), evaporated to 40% T.S. , and spray dried to yield a 'clarified' lactalbumin hydrolysate powder.
• the physico-chemical properties of the powder are outlined in Table 1, and the molecular weight profile is shown in Table 2. The hydrolysate satisfied the criteria for hypoallergenic baby formulae.
• Example 2 Essentially the same process as in Example 1 was used except that lactose was added to the reaction mixture before enzyme addition. Additional lactose was added to give a final ratio of 2.2:1 lactose to protein in the crude hydrolysate mixture. This was calculated taking into account the relative permeation rates of the protein and lactose through the microfiltration membrane, i.e. 70% and 95% are typical permeation rates for protein and lactose respectively when using the O.lum Abcor spirally wound membrane. The aim was to have a protein content in the finished product of 22% and 70% lactose.
• Lactose is added to the hydrolysis mixture before enzyme addition so that any residual protein remaining from the crystallisation process during the manufacture of lactose is hydrolysed along with the lactalbumin proteins.
• a slurry of lactalbumin (Alatal 560, New Zealand) was prepared by adding 18.75 kg Alatal (86% protein) to 150 litres of pasteurised water in a 210 litre batch stirred tank reactor (BSTR). The temperature of the slurry was adjusted to 50°C.
• the volume of the reaction mixture was made up to 195 litres with deionised water.
• the proteolytic enzyme mixture was added once the lactalbumin slurry had equilibrated at 50°C and pH 8.0 for a minimum of 30 minutes.
• the enzyme [320g of food grade Trypsin (PTN 3.OS, Novo)] was dissolved in 5 litres of deionised water before addition to the reaction mixture. In this instance the enzyme to substrate ratio (E/S) was equivalent to 2%.
• the pH stat was immediately activated and the remaining KOH/NaOH mixture continuously added to maintain pH at 8.0. When the allowable concentration of K and Na was reached in the crude hydrolysate, the enzyme reaction was continued without pH control.
• the reaction was stopped (by thermal inactivation of the enzyme) when the required DH and molecular weight profile had been achieved, which typically took 5-6 hours.
• Thermal inactivation was achieved by increasing the temperature of the crude hydrolysate to 80°C and maintaining this temperature for 20 minutes. The hydrolysate was then chilled to 4°C and held overnight for further processing.
• the temperature of the crude hydrolysate was adjusted to 50°C and transferred to the balance tank of the microfiltration unit.
• the microfiltration module consisted of an Abcor hollow fibre tangential flow membrane, 5m , and with a nominal pore size of 0.1 ⁇ .m
• Microfiltration was continued in batch mode with the retentate recycle to the feed tank, the permeate was collected in 25 litre plastic containers, weighed and pooled to form a bulk permeate. 150 litres of permeate was collected, which represents a volume concentration reduction (VCR) of 4. The pooled permeate was heated to 75°C for 15 mins, evaporated to 40% T.S., and spray dried to yield a 'clarified' lactalbumin hydrolysate powder.
• the physico-chemical properties of th powder are outlined in Table 3, and the molecular weight profile is shown in Table 4.
• the hydrolysate satisfied the criteria for hypoallergenic baby formulae.
• the antigenicity of the whey protein hydrolysate was reduced by approximately 5 orders of magnitude compare with a standard whey protein concentrate not subjected to any modifications.
• Table 3 Product profile of a spray dried hypoallergenic whey protein hydrolysate powder prepared from lactalbumin with added lactose.
• Table 4 Molecular weight profile of a hypoallergenic whey protein hydrolysate powder prepared from lactalbumin with added lactose.
• a solution of whey protein concentrate (WPC 80, Milei, West Germany) was prepared by adding 20 kg WPC 80 (80% protein) to 150 litres of deionised water in a 210 litre batch stirred tank reactor (BSTR) at 50°C with the aid of a SiIverson mixer.
• the concentrated WPC solution was allowed to dissolve completely with continuous stirring a 50°C for 2 hours.
• the solution of WPC was then heat denatured by increasing the temperature to 95 C and maintaining this temperature for 30 minutes. The mixture was stirred vigorously during the heat treatment to prevent clumping of aggregates. After the heat treatment the temperature of the WPC slurry was reduced to 50°C.
• the pH of the mixture was increased to pH 8.0 with automatic addition of a titrant mixture consisting of 2.56 M K0H and 1.44 M NaOH (3:1 w/w, K + /Na + ), using an industrial pH stat.
• the total quantity of titrant mixture added to the hydrolysis reaction was calculated so that the final concentration of Na and K in the crude hydrolysate mixture did not exceed 7 mg and 21 mg per gram of pure protein respectively.
• a protein permeation rate of approximately 70% through the microfiltration membrane this should yield a final product " with less tthhaann 1100 mg and 30 mg of Na and K respectively per gram of pure protein.
• the volume of the reaction mixture was made up to .195 litres with deionised water.
• the proteolytic enzyme mixture was added once the heat denatured WPC slurry had equilibrated at 50°C and pH 8.0 for a minimum of 30 minutes.
• the enzyme [320 g of food grade trypsin (PTN 3.OS, Novo)] was dissolved in 5 litres of deionised water before addition to the reaction mixture. In this instance the enzyme to substrate ratio (E/S) was equivalent to 2%.
• the pH stat was immediately activated and the remaining KOH/NaOH mixture continuously added to maintain pH at 8.0. When the allowable concentration of K and Na was reached in the crude hydrolysate, the enzyme reaction was continued without pH control.
• the reaction was stopped (by thermal inactivation) when the required DH and molecular weight profile had been achieved, which typically took 5-6 hours.
• Thermal inactivation was achieved by increasing the temperature of the crude hydrolysate to 80°C and maintaining this temperature for 20 minutes. The hydrolysate was then chilled to 4 C held overnight for further processing.
• the temperature of the crude hydrolysate was adjusted to 50°C and transferred to the balance tank of the microfiltration unit.
• the microfiltration module consisted of an Abcor hollow fibre
• Microfiltration was continued in batch mode with the retentate recycled to the feed tank, the permeate was collected in 25 litre plastic containers, weighed and pooled to form a bulk permeate. 150 litres of permeate was collected, which represents a volume concentration reduction (VCR) of 4. The pooled permeate was heated to 75°C for 15 mins, evaporated to 40% T.S., and spray dried to yield a 'clarified' whey protein hydrolysate powder. The physico-chemical properties of the powder are outlined in Table 5, and the molecular weight profile is shown in Table 6. The hydrolysate satisfied the criteria for hypoallergenic baby formulae. The antigenicity of the whey protein hydrolysate was reduced by approximately 5 orders of magnitude compared with a standard whey protein concentrate not subjected to any modifications.
• Table 5 Product profile of a spray dried hypoallergenic whey protein hydrolysate powder prepared from WPC.
• Table 6 Molecular weight profile of a hypoallergenic whey protein hydrolysate powder prepared from WPC.
• Example 3 Essentially the same process as in Example 3 was used except that lactose was added to the reaction mixture after the heat denaturation step. Additional lactose was added to give a final ratio of 2.2:1 lactose: protein in the crude hydrolysate mixture. This was calculated taking into account, the permeation rates of the protein and lactose through the microfiltration membrane, i.e. 70% and 95% are typical permeation rates for protein and lactose respectively when using the O.lum Abcor spiral membrane. It was aimed to have a protein content in the finished product of 22% protein and 70% lactose.
• Lactose is added to. the hydrolysis mixture before enzyme addition so tha ⁇ any residual protein remaining from the crystallisation process during the manufacture of lactose is hydrolysed along with the heat denatured WPC.
• a solution of whey protein concentrate (WPC 80, Mieli West Germany) was prepared by adding 20 kg WPC 80 (80% protein) to 150 litres water in a 210 litre batch stirred tank reactor (BSTR) at 50°C with the aid of a Silverson mixer.
• the concentrated WPC solution was allowed to dissolve completely with continuous stirring at 50°C for 2 hours.
• the solution of WPC was then heat denatured by increasing the temperature to 95°C and maintaining this temperature for 30 minutes. The mixture was stirred vigorously during the heat treatment to prevent clumping of aggregates. After the heat treatment the temperature of the WPC slurry was reduced to 50°C.
• Commercial food grade lactose 35.45 kg was then added to the reaction mixture and allowed to dissolve thoroughly.
• the pH of the mixture was increased to pH 8.0 with automatic addition of a titrant mixture consisting of 2.56 M KOH and 1.44 M NaOH (3:1 w/w, K /Na ), using an industrial pH stat.
• the total quantity of titrant mixture added to the hydrolysis reaction was calculated so that the final concentrations of Na and K in the crude hydrolysate mixture did not exceed 7 mg and 21 mg per gram of pure protein respectively.
• a protein permeation rate of approximately 70% through the microfiltration membrane this should yield a final product with less than 10 mg and 30 mg for Na and K respectively per gram of pure protein.
• the volume of the reaction mixture was made up to 195 litres with deionised water.
• the proteolytic enzyme mixture was added once the slurry of heat-denatured WPC/lactose had equilibrated at 50°C and pH 8.0 for a minimum of 30 minutes.
• the enzyme [320 g of food grade trypsin (PTN 3.OS, Novo)] was dissolved in 5 litres of deionised water before addition to the reaction mixture. In this instance the enzyme to substrate ratio (E/S) was equivalent to 2%.
• the pH stat was immediately activated and the remaining KOH/NaOH mixture continuously addeo to maintain pH at 8.0. When the allowable concentration of K and Na was reached in the crude hydrolysate, the enzyme reaction was continued without pH control.
• the reaction was stopped by thermal inactivation of the enzyme when the required DH and molecular weight profile had been achieved, which typically cook 5-6 hours. Thermal inactivation was achieved by increasing the temperature of the crude hydrolysate to 80°C and maintaining this temperature for 20 minutes. The hydrolysate was then chilled to 4°C and held overnight for further processing.
• the temperature of the crude hydrolysate was adjusted to 50°C and transferred to the balance tank of the microfiltration unit.
• the microfiltration module consisted of an Abcor hollow fibre tangential flow membrane, 5m , and with a nominal pore size of 0.1 ⁇ m (Koch International).
• Microfiltration was continued in batch mode with the retentate recycled to the feed tank, the permeate was collected in 25 litre plastic containers, weighed and pooled to form a bulk permeate. 150 litres of permeate was collected, which represents a volume concentration reduction (VCR) of 4. The pooled permeate was heated to 75°C for 15 mins, evaporated to 40% T.S., and spray dried to yield a 'clarified' whey protein hydrolysate powder. The physico-chemical properties of the powder are outlined in Table 7, and the molecular weight profile is shown in Table 8. The hydrolysate satisfied the criteria for hypoallergenic baby formulae.
• Table 8 Molecular weight profile of a hypoallergenic whey protein hydrolysate powder prepared from WPC with added lactose.
• Example 4 Essentially the same process as in Example 4 was used except that an enzyme/substrate ratio of 0.5% was used, and the hydrolysis reaction - 21 - was continued for 16 hours.
• the enzyme 80g food grade trypsin (PTN 3.05, Novo)] was dissolved in 5 litres of deionised water before addition to the reaction mixture. Apart from the lower enzyme/substrate ratio and increased hydrolysis time, all other proces variables were identical to Example 4.
• the physico-chemical properties of the powder are outlined in Table 9 and the molecular weight profile is shown in Table 10.
• the hydrolysa satisfied the criteria for hypoallergenic baby formulae.
• the antigenicity of the whey protein hydrolysate was reduced by approximately 5 orders of magnitude compared with a standard whey protein concentrate not subjected to any modifications.
• the concentration of enzyme is optional. Where a lower concentration is used, a longer reaction time is necessary to achieve the desired degree of hydrolysis, and vice versa.
• Table 9 Product profile of a spray dried hypoallergenic whey protein hydrolysate powder prepared from WPC with added lactose.
• hypoallergenic whey protein hydrolyate of the present invention contains molecules of molecular weight up to 50,000 daltons, a lower degree of hydrolysis is required in the manufacturing process than is required to produce previously known hypoallergenic hydrolysates.
• a feature of the hydrolysates produce by this invention is the excellent flavour of the hydrolysate.
• a panel of trained tastes judged the product to have very low bitterness or other off-flavour.
• the level of bitterness was assessed by a scaling method.
• a taste panel was presented with two standards representing the extremes of bitterness to be tasted. These were water and a solution of caffeine (0.03%) and were allocated a score of 0 and 10 respectively.
• Various reference samples of different bitterness were then presented and the panel was asked to allocate a score from 0 to 10 to them. When the panel were confident in their ability to rank the reference samples consistently, they were presented with the test hydrolysate samples ( solution in water) and asked to allocate them a score in the same way The samples consistently scored 2 or less, and on that basis were adjudged to have a very low level of bitterness.
• a further benificial property is that the product forms a solution which is visually clear.

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• 1992
  • 1992-08-28 EP EP92918198A patent/EP0604467A1/de not_active Withdrawn
  • 1992-08-28 WO PCT/IE1992/000006 patent/WO1993004593A1/en not_active Application Discontinuation
  • 1992-08-28 AU AU24682/92A patent/AU2468292A/en not_active Abandoned
  • 1992-08-31 NZ NZ244157A patent/NZ244157A/en unknown

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