NZ265247A

NZ265247A - Method for hydrolysing vegetable or animal protein

Info

Publication number: NZ265247A
Application number: NZ265247A
Authority: NZ
Inventors: Per Munk Nielsen; Peter Hvass; Kim Hansen; Gitte Budolfsen
Original assignee: Novo Nordisk As
Priority date: 1993-04-26
Filing date: 1994-04-25
Publication date: 1996-07-26
Also published as: AU681653B2; AU6564094A; CN1090675C; JPH08509366A; DK46793D0; CN1121732A; EP0700433A1; WO1994025580A1; KR960701993A

Description

<div class="application article clearfix" id="description"> New Zealand No. International No. 265247 PCT/DK94/00165 Priority Date{s): Complete Specification f iled Class: Publication Da*??:.. ... 2 R -JUL-1996 | P.O. Journal No: ;NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION ;Title of Invention: ;A method for hydrolysing proteins ;Name, address and nationality of applicant(s) as in international application form: ;NOVO NORDISK A/S, of Novo Alle, DK-2880 Bagsvaerd, Denmark ;O 94/25580 ;1 ;PCT/DK94/00165 ;265247 ;A METHOD FOR HYDROLYSING PROTEINS TECHNICAL FIELD ;The present invention relates to a method of hydrolysing proteins, a protein hydrolysate obtained by the 5 method, and food and non-food products containing the protein hydrolysate. ;BACKGROUND ART ;Methods of hydrolysing proteins have been described in e.g. GB Patent 1,507,380, US Patent 3,723,250, and io International Patent Application Nos. WO 89/00272, WO 92/13964, and WO 90/05462. ;There is a need for methods of hydrolysing proteins leading to a high degree of protein hydrolysis and to hydrolysates with excellent organoleptic properties. ;15 SUMMARY OF THE INVENTION ;It has now been found that a proteolytic enzyme preparation derived from Aspergillus oryzae and supplied from Novo Nordisk A/S under the tradename Flavourzyme™ possesses excellent protein hydrolysing properties, for example is it 20 possible to obtain a high degree of hydrolysis and a non-bitter hydrolysate. ;Accordingly, the present invention provides a method for hydrolysis of proteins by incubation with a proteolytic preparation derived from Aspergillus oryzae and supplied by 25 Novo Nordisk A/S, Denmark under the tradename Flavourzyme™. ;In its second aspect, the invention provides protein hydrolysates obtained by the method of the invention. ;In further aspects, the invention provides food and non-food products comprising a protein hydrolysate of the 30 invention. ;WO 94/25580 ;PCT/DK94/00165 ;2 ;BRIEF DESCRIPTION OF DRAWINGS ;The present' invention is further illustrated by reference to the accompanying drawings, in which: ;Fig. 1 shows the degree of hydrolysis (%DH) vs. time s of hydrolysis (hours) achieved by the method of the invention applied to sodium caseinate in the pH range of from pH 5 to pH 9 (■: pH 5, a pH 6, O pH 7, DpH 8, • pH 9); ;Fig. 2 shows the degree of hydrolysis (%DH) of soy protein isolate after 22 hours of hydrolysis according to the io method of invention (■: Flavourzyme™, ▲ : Corolase™ 7092, O: Corolase™ 7093, ♦: Alcalase™, □: Neutrase™); and ;Fig. 3 shows the degree of hydrolysis (%DH) of Na-caseinate after 22 hours of hydrolysis according to the method of invention (■: Flavourzyme™, a : Corolase™ 7092, O: Corolase™ is 7093, ♦: Alcalase™, □: Neutrase™). ;DETAILED DISCLOSURE OF THE INVENTION ;Characterization of Flavourzyme™ showed that the proteolytic Aspergillus oryzae preparation comprises several proteolytic components. It appeared that the preparation 20 comprises five or more proteolytic enzyme components, each of which may have any of the following approximate molecular weights: 23 kD, 27 kD, 31 kD, 32 kD, 35 kD, 38 IcD, 42 kD, 47 kD, 53 kD, and 100 kD. ;Accordingly, the present invention provides a method 25 for hydrolysing a protein by incubating with a proteolytic enzyme preparation which is derived from Aspergillus oryzae and comprises at least five proteolytic components having an approximate molecular weight, respectively, selected from 23 kD, 27 kD, 31 kD, 32 kD, 35 kD, 38 kD, 42 kD, 47 kD, 53 kD, and 30 100 kD. ;In a preferred embodiment, the protein is incubated with a proteolytic preparation derived from Aspergillus oryzae and comprising at least five proteolytic components having the approximate molecular weights 23 kD, 31 kD, 35 kD, 38 kD, and 35 53 kD, respectively. ;WO 94/25580 ;PCT/DK94/00165 ;3 ;The molecular weight of the protease components in the proteolytic preparation was determined by using SDS polyacrylamide gel electrophoresis (SDS-PAGE) in a manner known to persons skilled in the art. In this way, the molecular 5 weight of each protease component was determined. ;The method of the invention is able to perform extensive protein hydrolysis of proteins, and the method leads to non-bitter hydrolysates and hydrolysates having pronounced soup-flavour/meat-flavour. ;io The extent of the protein hydrolysis may be determined by the degree of hydrolysis achieved. In the context of this invention, the degree of hydrolysis (DH) is defined by the following formula: ;Number of peptide bonds cleaved is DH = ■ x 100 % ;Total number of peptide bonds ;The DH may be calculated according to Adler-Nissenj Enzymic Hydrolysis of Food Proteins; Elsvier Applied Science Publishers Ltd. (1986), p. 122. ;20 By using the method of the invention it may be possible to obtain a DH which is higher than 35%, preferably higher than 60%, more preferably higher than 70%, most preferably higher than 80%. ;By the method of the invention it may also be 25 possible to obtain a high degree of protein solubility. The degree of protein solubility may be described by way of a Protein Solubility Index (PSI) as described by Adler-Nissen, op. cit. ;In a preferred embodiment, a protein solubility 30 higher than 50% PSI, preferably higher than 70% PSI, more preferably higher than 90% PSI, is obtained. ;The protein or proteinaceous material which may advantageously be hydrolysed by the present method may be any vegetable protein such as soy protein, grain proteins, e.g. 35 wheat gluten or zein, rape seed protein, alfalfa protein, pea protein, fabaceous bean protein, cotton seed protein or sesame seed protein, or any animal protein or proteinaceous material ;WO 94/25580 ;PCT/DK94/00165 ;such as milk protein, whey protein, casein, meat protein, fish protein, blood protein, egg white or gelatin. ;To obtain a satisfactory degree of hydrolysis, the proteolytic enzyme may suitably be added to the protein or 5 proteinaceous material in an amount of 0.05-15 AU/100 g of protein, in particular 0.1-8 AU/100 g of protein. ;The incubation may be performed at a pH from between - about 4 and about 10, preferably between about 5 and about 9. ;As shown in Example 2, the method of the invention performs io excellent even at extreme pH conditions, i.e. at pH values in all of the range of 5 to 9. ;The incubation may be performed at any convenient temperature at which the enzyme preparation does not become inactivated, i.e. in the range of from about 20°C to about is 70°c. ;In accordance with established practice, the proteolytic enzyme preparation may suitably be inactivated by increasing the temperature of the incubation mixture to above about 70°C, or by decreasing the pH of the incubation mixture 20 to below about 4.0. ;In yet another preferred embodiment, incubation of a protein or proteinaceous substrate may be performed with a combination of Flavourzyme™ and one or more other protease preparations. ;25 Preferred protease preparations comprises neutral or alkaline proteases. Examples of suitable neutral proteases are neutral proteases derived from Bacillus, preferably from Bacillus subtilis, such as the enzyme preparation supplied by Novo Nordisk, Denmark, under the tradename Neutrase™. Examples 30 of suitable alkaline proteases are alkaline proteases derived from Bacillus, preferably from Bacillus licheniformis, such as the enzyme preparation supplied by Novo Nordisk, Denmark, under the tradename Alcalase™ and containing Subtilisin A (Subtilisin Carlsberg) as the active component. ;35 The incubation carried out in the method of the invention may also be performed with a combination of Flavourzyme™ and one or more other lipase preparations. ;WO 94/25580 ;PCT/DK94/00165 ;s ;Preferred lipase preparations comprises fungal lipases. Examples of suitable fungal lipases are lipases derived from Mucor, preferably from Rhizomucor miehei, such as the enzyme preparation supplied by Novo Nordisk, Denmark, under 5 the tradename Palatase™M; and lipases derived from Aspergillus, preferably from Aspergillus niger such , as the enzyme preparation supplied by Novo Nordisk, Denmark, under the tradename Palatase^A. ;In another aspect, the present invention provides a protein 10 hydrolysate obtained by the method of the invention. ;Determination of AU ;The proteolytic activity may be determined with haemoglobin as substrate. ;In the Anson-Haemoglobin method for the determination i5 of proteolytic activity denatured haemoglobin is digested, and the undigested haemoglobin is precipitated with trichloroacetic acid (TCA). The amount of TCA soluble product is determined with phenol reagent, which gives a blue colour with tyrosine and tryptophan. ;20 one Anson Unit (AU) is defined as the amount of enzyme which under standard conditions (i.e. 25°C, pH 7.5 and 10 min. reaction time) digests haemoglobin at an initial rate, so that there is liberated per minute an amount of TCA soluble product which gives the same colour with phenol reagent as one 25 milliequivalent of tyrosine. ;A folder AF 4/5 describing the analytical method in more detail is available from Novo Nordisk A/S, DK-2880 Bagsvaerd, Denmark, upon request which folder is hereby included by reference. ;3° Determination of LU ;Assay for lipase activity : ;A substrate for lipase was prepared by emulsifying glycerine tributyrat (MERCK) using gum-arabic as emulsifier. ;WO 94/25580 ;PCT/DK94/00165 ;6 ;Lipase activity was assayed at pH 7 using pH stat method. One unit of lipase activity (LU/mg) is defined as the amount needed to liberate one micromole fatty acid per minute. ;Step 1:- Centrifuge the fermentation supernatant, discard the s precipitate. Adjust the pH of the supernatant to 7 and add gradually an equal volume of cold 96 % ethanol. Allow the mixture to stand for 30 minutes in an ice bath. Centrifuge and discard the precipitate. ;Step 2:- Ion exchange chromatography. Filter the supernatant io and apply on DEAE-fast flow (Pharmacia TM) column equilibrated with 50 mM tris-acetate buffer pH 7. Wash the column with the same buffer till absorption at 280 nm is lower than 0.05 OD. Elute the bound enzymatic activity with linear salt gradient in the same buffer (0 to 0.5 M NaCl ) using five column volumes. 15 Pool the fractions containing enzymatic activity . ;Step 3:- Hydrophobic chromatography. Adjust the molarity of the pool containing enzymatic activity to 0.8 M by adding solid Ammonium acetate. Apply the enzyme on TSK gel Butyl- Toyopearl 650 C column (available from Tosoh Corporation Japan) which 20 was pre-equilibrated with 0.8 M ammonium acetate. Wash the unbound material with 0.8 M ammonium acetate and elute the bound material with distilled water. ;Step 4:- Pool containing lipase activity is diluted with water to adjust conductance to 2 mS and pH to 7. Apply the pool on 25 High performance Q Sepharose (Pharmacia) column pre-equilibrated with 50 mM tris -acetate buffer pH 7. Elute the bound enzyme with linear salt gradient. ;Food Products ;In a further aspect, the present invention relates to 30 food products comprising a protein hydrolysate of the invention. ;The amount of protein hydrolysate incorporated in the food product will typically be in the range of 1-30% by weight. ;WO 94/25580 PCT/DK94/00165 ;An important food product of the present invention is an ingredient of a mother milk substitute for infants. Due to the high degree of hydrolysis obtained by the method of the invention, the protein hydrolysates of the invention may advan-5 tageously be incorporated in mother milk substitutes, the hydrolysate having a significantly lower allergenicity than unhydrolysed milk proteins. The milk substitute may be formulated in substantially the same way as that indicated in the prior literature for products of this type (cf. for instance EP io Patent Application 322,589) with the exception that the protein hydrolysate included in the known products is replaced by the protein hydrolysate of the present invention. ;The food product of the invention may also include the protein hydrolysate of the invention as a protein supple-i5 ment or to provide other properties of the food product. Thus, the protein hydrolysate incorporated in the food product may for instance be based on meat or scrap meat (e.g. the so-called mechanically recovered meat, i.e. meat remaining on bones after the regular pieces of meat have been carved from animal 20 carcases in the slaughterhouse. For a more detailed description of the general procedure, see applicant's co-pending International Patent Application WO 90/05462) rendered from bone by subjecting crushed bone to the method of the invention. Other proteinaceous by-products from the meat industry may also 25 be used. ;The resulting protein hydrolysate may then suitably be added to emulsified meat products, e.g. sausages or pat£s, or as flavour ingredients in sOups or other food products. ;A food product obtained from cow milk by the method 30 of invention is preferably a cheese flavour product. The extensive hydrolysis of the milk protein surprisingly leads to flavour compounds with very distinct cheese flavour. Such cheese flavour products of the invention preferably find application in snack products, imitation cheese products, or as 3s enhancers of cheese flavour in general. The cheese flavour may be adjusted by use of lipases according to well established practice. ;WO 94/25580 ;PCT/DK94/00165 ;8 ;A traditional way of producing Hydrolysed Vegetable Protein (HVP) for use as flavour products is based on cooking in hydrochloric acid at a high temperature and for a long time.. This is known to cause unwanted formation of chloric containing 5 compounds. By the process of the invention it is now possible to obtain enzymatically produced HVP products, which are considered more healthy products. The high DH obtainable by the process of the invention leads to desirable flavour characteristics and flavour enhancing properties. 10 The process of- the invention may also be used for protein enrichment of dietetic products due to the non-bitter flavour obtained by the process. Also advantageous is the very high protein solubility in a very broad pH range that is associated with the process of the invention. 15 It has surprisingly been found that an excellent fermentation medium may be produced by subjecting milk to the method of the present invention. Such milk hydrolysates may be used for acceleration of the fermentation in yoghurt production or in the production of lactic acid starter bacteria cultures, 20 by addition in relatively small amounts. The shortening of process time increases production capacity and reduces the risk of infection, e.g. bacteriophage infection. ;Accordingly, the method of the invention may be used in combination with a fermentation process, preferably a 25 fermentation process for the production of a food product. Thus, the proteolytic enzyme preparation, e.g. Flavourzyme™, used in the present method or the protein hydrolysate of the invention or both may be added in a fermentation process, preferably a food fermentation process, for enhancing the 30 productivity of the fermentation process. Examples of such fermentation processes are processes involving or fermenting fish (fish sauce), cocoa beans or soy (such as soy sauce, tempeh, miso). The addition of the proteolytic enzyme preparation or the addition of protein hydrolysate may be 35 useful for decreasing the total process time, i.e. increasing the rate of fermentation. ;WO 94/25580 ;PCT/DK94/00165 ;9 ;Non-food products ;The process of the invention may also find application in the non-food area. The flavour characteristics obtained by the method of invention may advantageously be used for production s of pet food. ;Production of very high DH-hydrolysates from gelatine improves gelatine products for incorporation into cosmetics, e.g. creams and shampoos. ;The beneficial effect as fermentation medium mentioned 10 above is expected to make the hydrolysates of the invention useful for other fermentations as well. ;The following examples further illustrate the present invention, and they are not intended to be in any way limiting to the scope of the invention as claimed. ;is EXAMPLE 1 ;Beef Hydrolysis ;In this example two experiments were carried out using the following enzyme preparations (indicating % w/w based on protein content): ;20 Preparation A) 2% Flavourzyme™ (2.30 AU/g) ;Preparation B) 1% Flavourzyme™, 2% Neutrase™ 0.5L, and 0.15% Alcalase™ 2,4L. ;All enzymes are available from NOVO NORDISK A/S, Denmark. Flavourzyme™ is a proteolytic preparation derived from 25 Aspergillus oryzae, Neutrase™ 0.5L is a proteolytic preparation derived from Bacillus subtilis, and Alcalase™ 2.4L is a proteolytic preparation derived from Bacillus licheniformis. ;408 g of beef were minced twice and mixed with 392 g of water to a protein content of 10 % w/w. The mixture was 30 blended for 30 sec. and heated to 55°C. Initial osmolality (mOsm) and soluble dry matter (°Brix) were measured, and the subsequent hydrolysis was monitored by these values. ;After four hours of incubation the enzymes were inactivated by heating at 90°C for 30 min. The hydrolysates ;WO 94/25580 ;PCT/DK94/00165 ;10 ;were subjected to centrifugation at 3 000 xG for 15 min. and weighed. The centrifugate was stored in a refrigerator until the next day, and the lipid phase separated off and weighed. pH in centrifugate was 5.85. ;s The results are shown in Tables 1 and 2 below. ;Table 1 ;Initial Osmolality (mOsm) and Soluble Drv Substance fBrix^ ;Time Preparation A Preparation B ;min. mOsm/kg AraOsm/kg °Brix mOsm/kg AmOsm/kg °Brix ;0 ;203 ;0 ;3.9 ;202 ;0 ;4.5 ;15 ;360 ;158 ;9.9 ;45 ;490 ;287 ;11.1 ;484 ;282 ;11.1 ;60 ;534 ;331 ;11.6 ;90 ;576 ;373 ;12.3 ;534 ;332 ;12.3 ;120 ;623 ;420 ;12.7 ;579 ;377 ;12.8 ;180 ;685 ;482 ;13.5 ;624 ;422 ;13.3 ;24 0 ;726 ;523 ;13.8 ;663 ;461 ;13.7 ;20 Table 2 ;Preparation A ;Mixture Weight Protein Protein Protein yield DS* g % g % % 25 Meat/Water 800 10 80 15 Defatted centrifugate 689 9.46 65.2 81.5 10.6 Dry Substance 30 As calculated from the Tables, the dosages used in these experiments Flavourzyme™ increase yields from usually 45% WO 94/25580 PCT/DK94/00165 11 to 81.5%, and the solubility increases from usually 57% to 89.9% PSI. A degree of hydrolysis 70.2% DH was achieved. The hydrolysates were served before a tasting panel in a 4% solution. The meat flavour was most pronounced in s hydrolysates obtained with Preparation A. EXAMPLE 2 Hydrolysis at Various pH In this experiment the method of' the invention was performed at pH values in the range of 5-9 with sodium io caseinate as substrate. An 8% (w/w) solution of sodium caseinate (Miprodan™ 30, obtained from MD FOODS Amba, Denmark) was prepared by heating to 80°C. To 800 g of this solution 1 g methyl-4-hydroxybenzoate and 0.15 g propyl-4-hydroxybenzoate were added, is and the temperature of the solution was lowered to 50°C. pH was adjusted to the desired value with 4 N NaOH or 4 N HC1. 1% w/w based on the protein content Flavourzyme™ (2.30 AU/g, NOVO NORDISK A/S, Denmark) was added the solution, and the incubation was allowed to proceed to maximal degree of 20 hydrolysis (about 22 hours) . At pH 9.00, 8.00, and 7.00 the hydrolysis was carried out in a pH stat, and the incubation monitored by consumption of NaOH. At pH 6.00 and 5.00 (initial pH) the hydrolysis was monitored by decreased pH and increased osmolality. 25 Samples were obtained after 0.25, l, 2, 4.5, and 22 hours of incubation. The enzyme was inactivated by heating to 85°C for 3 minutes, followed by cooling in ice-water. The results are presented in Fig. 1. As evident from this Figure, the process of the invention is capable of 30 exerting hydrolysis in the pH range of from pH 5 to pH 9. WO 94/25580 PCT/DK94/00165 EXAMPLE 3 Comparison Example In this experiment the degree of hydrolysis (DH) obtained by the process of invention (incubation with 5 Flavourzyme™) was compared to the DH obtained by incubation with other known proteolytic preparations. The experiment was carried out at 50°C as a non-pH stat hydrolysis (no adjustments of pH during hydrolysis), starting at pH 7.00 adjusted with 4N NaOH. To ensure equal io enzyme dosage each hydrolysis was carried out using the same proteolytic activity as expressed in AU/g (cf. above for determination of AU). Two protein raw materials were tested: 1) Na-caseinate (supplied by MD Foods Amba, Denmark) is 2) Soy protein isolate (supplied by Protein Technologies International, U.S.A.). 2500 g of an 8% (w/w based on protein content) solution of protein raw material was prepared. The protein solution was heat treated at 85 °C for 3 minutes and cooled to 20 hydrolysing temperature 50°C. Samples of 400g were prepared for hydrolysis. The following enzymes and enzyme dosages were used: Enzyme Activity, AU/g Dosage per 400g 25 Flavourzyme f Novo Nordisk A/S 3.36 0.321 g Neutrase 0.5L, Novo Nordisk A/S 0.484 2.231 g Alcalase 2.4L, Novo Nordisk A/S 2.58 0.419 g 30 Corolase 7093, Rohm GmbH 0.094 11.489 g Corolase 7092, Rohm GmbH 1.54 1.426 g WO 94/25580 PCT/DK94/00165 13 The hydrolysis was carried on for 22 hours. The degree of hydrolysis was measured after 5 and 22 hours of hydrolysis, respectively. The results are shown in fig. 2 (soy protein isolate) and fig. 3 (Na-caseinate) . From 5 the results it is evident that hydrolysis by using Flavourzyme clearly results in the highest degree of hydrolysis. EXAMPLE 4 Cheese Flavour Production Whole milk was heat treated for 2 min at 100°C and io subsequently cooled to 50 °C. Four portions of 200 ml each were prepared (indicating % w/w based on protein content): 1) 1% Flavourzyme™ (2.30 AU/g) 2) 1% Flavourzyme™ (2.30 AU/g) + 0.15% Palatase™ M 200L (200 LU/g from NOVO NORDISK A/S, Denmark) 15 3) 0.15% Palatase™ M 200L (200 LU/g from NOVO NCnDTSK T./S, Denmark) 4) Control The hydrolysis was carried out for 20 hours at 50 °C. Intensive cheese flavour developed in samples 1, 2, and 3, 20 while the control was almost neutral. The flavour-samples 1, 2, and 3, were now diluted in cream cheese, and a fourth sample was prepared as a control. 1.1) 0.5 g of sample No. 1 + 20 g of cheese 2.1) 6.0 g of sample No. 2 + 20 g of cheese 25 3.1) 6.0 g of sample No. 3 + 20 g of cheese 4.1) 6.0 g of fresh milk + 20 g of cheese. Sample No. l.l (Flavourzyme™) appeared to have a very strong and intense flavour and a taste of well ripened cheese. Sample No. 2.1 (Flavourzyme™ + Palatase™) appeared to have a 30 well balanced cheese flavour. Compared to sample No. 1.1 and No. 2.1, sample No. 3.1 turned out to give the weakest taste impression. The reference, sample No. 4.1 was evaluated being sour/sourish. In conclusion it is possible to add the enzymes 35 Flavourzyme™ and the combination of Flavourzyme™ and Palatase™ directly into the milk to form excellent cheese flavour. WO 94/25580 PCT/DK94/00165 14 The traditional way of producing cheese flavour is carried out by adding lipases and proteases to cheese. That is, the use of Flavourzyme™ and Flavourzyme™ in combination with Palatase™ creates a new and much more simple way of producing 5 cheese flavour. EXAMPLE 5 Accelerated Yoghurt Fermentation Whole milk was heat treated for 2 min at 100°C and" subsequently cooled to 50°C. Two portions of 200 ml were io prepared (indicating % w/w based on protein content): 1) 1% Flavourzyme™ (2.30 AU/g) 2) Control The hydrolysis was carried out for 20 hours at 50°C. Then the enzymes were denatured by heat treatment at 8 0°C for u 5 min. Skim milk was heat treated at 90 °C for 2 min and three samples of 200 ml were prepared. 1.1) 200 ml of milk + 10 ml of sample No. 1 2.1) 200 ml of milk + 10 ml of sample No. 2 20 3.1) 200 ml of milk The temperature was adjusted to 41°C, and yoghurt culture YC DVS from Chr. Hansens Lab. was added to all three samples in a level of 106 bacteria per g. The fermentation course was followed by measuring pH, 25 cf. Table 4, below. WO 94/25580 PCT /DK94/00165 15 Table 4 Acidification Course (pH) 1) Flavour- 2) Blind 3) Blind/Blind ,TM zyme' Initial pH value 6.6 6.5 6.5 pH after 200 min 5.9 6.4 6.4 pH after 360 min 4.4 5.9 6.3 10 It appears from the pH measurements that the addition of Flavourzyme™ hydrolysed milk accelerates the fermentation, meaning that the production time of fermented milk products can be reduced considerably. PCT/DK94/00165 265247 1. A method for hydrolysing a protein by incubating with a proteolytic enzyme preparation, characterized in that the proteolytic preparation is derived from Aspergillus oryzae 5 and comprises at least five proteolytic components each having an approximate molecular weight, respectively, selected from 23 kD, 27 kD, 31 kD, 32 kD, 35 kD, 38 kD, 42 kD, 47 kD, 53 kD, and 100 kD. 2. The method according to claim 1 in which the io proteolytic preparation comprises at least five proteolytic components having the approximate molecular weights 23 kD, 31 kD, 35 kD, 38 kD and 53 kD, respectively. 3. The method according to claim 1 or 2, by which there is obtained a degree of protein hydrolysis (DH) higher is than 35%. 'WO 94/25580 </div>

Claims

<div class="application article clearfix printTableText" id="claims">
CLAIMS
4. The method according to claim 1, by which there is obtained a degree of protein solubility (Protein Solubility Index (PSI)) higher than 50%PSI.
5. The method according to any one of claims 1-4, in which the protein is a vegetable protein.
6. The method according to any one of claims in which the protein is an animal protein.. t 

1-4, 

■ WO 94/25580 

PCT /DK94/00165 

17 

265247
7. The method according to any one of claims 1-6, in which the incubation is performed at a pH of between about 4 and about 10. , ■ <
8. The method according to any one of claims 1-7, in 

5 which the protein is incubated with the proteolytic preparation in combination with one or more other protease preparations.
9. The method according to claim 8, in which the protease preparation is a neutral protease derived from Bacillus.
>o 10. The method according to claim 8, in which the protease preparation is an alkaline protease derived from Bacillus.
11. The method according to any one of the claims 1-10, in which the protein is incubated with the proteolytic is preparation in combination with one or more lipase preparations.
12. The method according to claim 11, in which the lipase preparation comprises a fungal lipase derived from Mucor.
20 13. The method according to claim 11, in which the lipase preparation comprises a fungal lipase derived from Aspergillus.
14. The method according to any one of the claims 1-13, wherein the protein is hydrolysed and simultaneously fermented is to a food product.
15. A protein hydrolysate obtained by the method according to any one of the claims 1-13. 

N .Z. OFFICE 

1 5 MAY 1396 

RECEIVED
16. The protein hydrolysate according to claim 15, wherein the hydrolysed protein is an animal protein selected from the group consisting of milk protein, whey protein, casein, meat proteins, fish protein, blood protein, egg white and gelatin.
17. The protein hydrolysate according to claim 15, wherein the hydrolysed protein is a vegetable protein selected from the group consisting of soy protein; grain proteins; rape seed protein; a,lfalfa protein; pea protein; fabaceous bean protein; cotton seed protein; and sesame seed protein.
18. A food product comprising a protein hydrolysate according to any one of the claims 15-17.
19. The food product according to claim 18, being an ingredient of mother milk substitute; a cheese flavour product; an enzymatically produced HVP; a protein enriched dietetic product; a soup-, bouillon- or meat-flavour product; a meat extract product; or a hydrolysate for improving the production of a starter culture.
20. A non-food product comprising a protein hydrolysate according to any one of! the claims 15-17.
21. The non-food product according to claim 20, being a pet food, a cosmetic, or a fermentation broth.
22. A method for hydrolysing a protein by incubating with a proteolytic enzyme preparation substantially as herein described with reference to the Examples.
23. A protein hydrolysate obtained by the method of claim 1 substantially as herein described with reference to the Examples.
24. A food product of claim 18 substantially as herein described.
25. A non-food product of claim 20 substantially as herein described. 



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