CA2092953C - Enzymatic hydrolysis - Google Patents
Enzymatic hydrolysis Download PDFInfo
- Publication number
- CA2092953C CA2092953C CA002092953A CA2092953A CA2092953C CA 2092953 C CA2092953 C CA 2092953C CA 002092953 A CA002092953 A CA 002092953A CA 2092953 A CA2092953 A CA 2092953A CA 2092953 C CA2092953 C CA 2092953C
- Authority
- CA
- Canada
- Prior art keywords
- hydrolysis
- tank
- tube
- substrate
- enzyme
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 230000007071 enzymatic hydrolysis Effects 0.000 title claims abstract description 13
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 title claims abstract description 13
- 230000007062 hydrolysis Effects 0.000 claims abstract description 96
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 96
- 239000000758 substrate Substances 0.000 claims abstract description 69
- 238000000034 method Methods 0.000 claims abstract description 66
- 238000002156 mixing Methods 0.000 claims abstract description 53
- 230000003068 static effect Effects 0.000 claims abstract description 43
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 28
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 28
- 102000035195 Peptidases Human genes 0.000 claims abstract description 10
- 108091005804 Peptidases Proteins 0.000 claims abstract description 10
- 102000004190 Enzymes Human genes 0.000 claims description 56
- 108090000790 Enzymes Proteins 0.000 claims description 56
- 229940088598 enzyme Drugs 0.000 claims description 56
- 235000018102 proteins Nutrition 0.000 claims description 26
- 239000000376 reactant Substances 0.000 claims description 19
- 108010046377 Whey Proteins Proteins 0.000 claims description 18
- 102000007544 Whey Proteins Human genes 0.000 claims description 14
- 238000011144 upstream manufacturing Methods 0.000 claims description 10
- 235000021119 whey protein Nutrition 0.000 claims description 10
- 239000005862 Whey Substances 0.000 claims description 8
- 230000000694 effects Effects 0.000 claims description 7
- 108090000631 Trypsin Proteins 0.000 claims description 5
- 102000004142 Trypsin Human genes 0.000 claims description 5
- 230000001580 bacterial effect Effects 0.000 claims description 5
- 239000005018 casein Substances 0.000 claims description 5
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 5
- 235000021240 caseins Nutrition 0.000 claims description 5
- 239000012588 trypsin Substances 0.000 claims description 5
- 239000004365 Protease Substances 0.000 claims description 4
- 239000007900 aqueous suspension Substances 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 235000013336 milk Nutrition 0.000 claims description 4
- 210000004080 milk Anatomy 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 241000251468 Actinopterygii Species 0.000 claims description 3
- 230000002378 acidificating effect Effects 0.000 claims description 3
- 239000008267 milk Substances 0.000 claims description 3
- 239000007858 starting material Substances 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 2
- 108090000317 Chymotrypsin Proteins 0.000 claims description 2
- 241001465754 Metazoa Species 0.000 claims description 2
- 108010019160 Pancreatin Proteins 0.000 claims description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 2
- 235000013351 cheese Nutrition 0.000 claims description 2
- 229960002376 chymotrypsin Drugs 0.000 claims description 2
- 235000009508 confectionery Nutrition 0.000 claims description 2
- 230000002349 favourable effect Effects 0.000 claims description 2
- 235000013312 flour Nutrition 0.000 claims description 2
- 230000002538 fungal effect Effects 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 229940055695 pancreatin Drugs 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 229960001322 trypsin Drugs 0.000 claims description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 28
- 108090000765 processed proteins & peptides Proteins 0.000 description 20
- 238000012360 testing method Methods 0.000 description 18
- 229910052757 nitrogen Inorganic materials 0.000 description 14
- 239000000047 product Substances 0.000 description 14
- 230000000875 corresponding effect Effects 0.000 description 12
- 150000001412 amines Chemical class 0.000 description 10
- 108010056079 Subtilisins Proteins 0.000 description 9
- 102000005158 Subtilisins Human genes 0.000 description 9
- 238000001816 cooling Methods 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 8
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 7
- 239000004472 Lysine Substances 0.000 description 7
- 230000002779 inactivation Effects 0.000 description 7
- 239000012141 concentrate Substances 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 230000000774 hypoallergenic effect Effects 0.000 description 5
- 102000004196 processed proteins & peptides Human genes 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 241000194108 Bacillus licheniformis Species 0.000 description 3
- 108091005658 Basic proteases Proteins 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 229920002401 polyacrylamide Polymers 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 238000007693 zone electrophoresis Methods 0.000 description 3
- 241001136782 Alca Species 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 238000010924 continuous production Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 238000010979 pH adjustment Methods 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000003531 protein hydrolysate Substances 0.000 description 2
- 229940076279 serotonin Drugs 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 108010028690 Fish Proteins Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 238000010793 Steam injection (oil industry) Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000005904 alkaline hydrolysis reaction Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229940000425 combination drug Drugs 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- IJKVHSBPTUYDLN-UHFFFAOYSA-N dihydroxy(oxo)silane Chemical compound O[Si](O)=O IJKVHSBPTUYDLN-UHFFFAOYSA-N 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- ZZUFCTLCJUWOSV-UHFFFAOYSA-N furosemide Chemical compound C1=C(Cl)C(S(=O)(=O)N)=CC(C(O)=O)=C1NCC1=CC=CO1 ZZUFCTLCJUWOSV-UHFFFAOYSA-N 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 210000003200 peritoneal cavity Anatomy 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002250 progressing effect Effects 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 230000007065 protein hydrolysis Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000003381 solubilizing effect Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- 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
- A23J3/346—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/18—Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/06—Tubular
Abstract
A process and apparatus for the enzymatic hydrolysis of proteins, in which a proteolytic enzyme and a protein substrate are mixed, a first hydrolysis step is carried out in a stirred tank and a second hydrolysis step is carried out in a tube equipped with static mixing elements.
Description
This invention relates to a process for the enzymatic hydrolysis of proteins and to an apparatus for carrying out this process.
There are various known processes for the enzy-matic hydrolysis of proteins which differ from one another, for example, in the choice of the substrate, the enzyme, the degree of hydrolysis and/or the required peptide profile. In cases where, for example, a rela-tively well-defined peptide profile, more particularly a narrow oligopeptide profile, is required for reasons of assimilation of the hydrolyzate by the intestinal mucosa, known hydrolysis processes generally comprise at least one hydrolyzate filtration or screening step.
For example, EP 226 221 describes a process for the preparation of hypoallergenic peptides having a molecular weight in the range from 2000 to 6000 by one or more enzymatic protein hydrolysis steps each carried out discontinuously in a fermentation tank and each terminating in an ultrafiltration step.
US 4,212,889 describes a process for solubilizing fish proteins, in which a mixture of fish flesh and enzyme is continuously passed through an installation comprising several hydrolysis tanks connected in series.
The problem addressed by the present invention was to provide a hydrolysis process which, preferably carried out continuously, would enable the efficiency of discontinuous hydrolysis in a tank to be equalled or even increased and which would enable a protein hydroly-zate having a well-defined and reproducible degree of hydrolysis and/or peptide spectrum to be obtained.
To this end, the process according to the inven-tion for the: enzymatic hydrolysis of proteins, in which a protein substrate is subj ected to hydrolysis with a proteolytic enzyme, comprises a first enzymatic hydroly-sis step i:n a stirred tank and a second enzymatic hydrolysis :step in a tube. In a preferred embodiment, the process according to the invention is carried out continuously and the second enzymatic hydrolysis step is carried out in a tube equipped with static mixing elements.
Simi7.arly, the apparatus for carrying out the process according to the invention comprises a double-jacketed stirred hydrolysis tank which is connected upstream to a substrate metering unit and to an enzyme metering unit and downstream to at least one hydrolysis tube. In a preferred embodiment, the tube is equipped with static mixing elements.
It has been found that it is thus possible to produce a protein hydrolyzate having a well-defined degree of hydrolysis and/or peptide spectrum in a highly efficient and reproducible, preferably continuous manner.
By virtue of the process and apparatus according to the invention, it is possible in particular to work with a tank of relatively small dimensions which may be filled completely without leaving any head space and with a tube of relatively large dimensions. It is thus possible to carry out, preferably continuously, a first relatively ;short step, i.e. a hydrolysis initiation step, in a relatively small tank, and a second relative-ly long step, i . a . a hydrolysis completion step, in a tube of relatively large volume. The reaction time, i. e. the re:~idence time of the substrate in the total volume represented by the sum of the tank volume and the tube volume, can thus be controlled in a precise and simple manner, for example by means of volumetric pumps.
If, for comparison, it is desired to carry out a hydrolysis in a single hydrolysis tank of large dimen sions, the residence time of a unit volume of hydroly zate cannot be precisely defined. This is true of discontinuous hydrolysis where the times required to establish given pH and/or temperature conditions and even to empty the tank, for example, are considerable.
However, the same is even truer of continuous hydro-lysis, in which case is it only possible to define a mean residence time. Even in a process of the type described in the above-cited US 4,212,889, the residence time can scarcely be defined any more precisely.
By contrast, it has been found that, with the process and apparatus according to the present inven-tion, the residence time of a unit volume of hydrolyzate can be defined in a remarkably precise manner, the flow of hydrolyzate through the hydrolysis tube - preferably equipped with static mixing elements - having a very flat front.
In t:he present specification, the degree of hydrolysis is defined via the quantity of non-protein nitrogen (NPN) determined as the percentage of total nitrogen which cannot be precipitated with 13% trichlo-roacetic aced.
The nitrogen contents are determined by the Kjeldahl mei:hod.
The amine nitrogen (free a-NHZ) contents are determined by reaction with ninhydrin after alkaline hydrolysis.
The serotonin relaxation tests using tritium-labelled exogenous serotonin (serotonin-3H) are carried out on normal mastocytes of the peritoneal cavity of rats by thEa method described by R. Fritsche and M.
Bonzon in Int. Arch. Allerg. Immunol. 93, 289-293 (1990).
The ELISA inhibition tests are carried out with rabbit antibodies specific of f3-lactoglobulin (BLG), bovine serum albumin (BSA) and casein (CAS). The sensi ,.~. 4 2092953 tivity of the method, i.e. the concentration detection limit, is 20 ng/ml.
The high performance liquid chromatography analyses (HPLC tests, peptide profiles) are carried out under non-denaturing conditions on gel based on type TSK-62000-SWT"' silica (a product of Toyo Soda), of which the fractionation range extends from 500 to 50,000 dalton, in a Biorad BIOSIL SEC-125T"~ column. The results are expre:~sed in % surface distribution of the peaks read at 2:20 nm in a 0.1 M phosphate solution + 0.4 M
NaCl at pH 6.80.
The: analyses by zone electrophoresis in poly acrylamide~ gel (SDS-PAGE tests) are carried out by the method described by Laemmli in Nature 227, 680 et seq.
(1970) .
The blockage of lysine is determined by HPLC and is expressed as % blocked lysine relative to the total lysine of the hydrolyzate.
"Static mixing elements" are understood to be undulating crossmembers or strips of metal or plastic which intersect or which are interlocked in one another and which divide the space defined by the tube into a plurality of intersecting passages progressing along the axis of the tube. Elements of the type in question are marketed by Sulzer A.G. of CH-8401 Winterthur, for example under the trade-marks SMV, SMX or SMXL.
Finally, it is important to appreciate that the tubes provided with static mixing elements are sys tematically equipped with a double jacket even when this is not specifically ment-Toned.
The process according to the invention may be carried out. using any starting material rich in proteins as the prot=ein substrate, such as flours or semolina of oil seeds or oil seed cakes, food-quality yeasts or bacteria, minced animal or fish flesh or milk or milk derivative:, for example in the form of particles in aqueous suspen-sion or aqueous suspensions.
The protein substrate is preferably a whey substrate containing whey proteins, more particularly a 5 sweet whey i°rom cheese production or an acidic whey from casein production either as such or in demineralized or lactose-free, liquid or reconstituted form.
In another preferred embodiment, the enzyme is selected from the group consisting of trypsin, chymo trypsin, pancreatin, bacterial proteases, fungal prote ases and mixtures thereof.
The proteolytic enzyme and the substrate may be mixed in a quantity of enzyme having an activity of 0.1 to 12 Anson units (AU) per 100 g substrate dry matter.
The first hydrolysis step is preferably carried out over a period of 10 to 60 minutes at a pH and a temperature adjusted to values favourable to the acti-vity of the enzyme while the second hydrolysis step is preferably carried out over a period of 1 to 8 h at a temperature equal to or above, particularly 0 to 10°C
above, the 'temperature of the first step.
Intermediate or complementary steps may be included, more particularly a preliminary mixing step preferably carried out in a tube equipped with static mixing elements; a thermal denaturing step before, in the middle of or after the first hydrolysis step, particularly using a heat exchanger or a tube equipped with static mixing elements; one or more enzyme inacti-vation steps, particularly after the second hydrolysis step, more especially using a heat exchanger and/or a steam injector and/or a tube equipped with static mixing elements; a:nd/or a cooling step carried out in particu-lar after a denaturing step, more particularly using a heat exchanger or preferably a tube equipped with static mixing elements for example.
The enzyme may be inactivated in one or preferab-ly two steps, a first step corresponding more precisely to autodigestion of the enzyme and a second step corre-sponding more precisely to sterilization.
The first hydrolysis step in a tank may also be divided into at least two parts carried out in at least two tanks connected in series. Similarly, the second hydrolysis step carried out in a tube may be divided into at least two parts carried out in at least two tubes connected in series. In the latter case, a pH
adjustment and/or an addition of enzyme may be carried out between two successive tubes.
For the pH adjustment(s), it is preferred to use a suitable reactant which may either be alkaline, such as KOH, NaOH or Ca(OH)z, or acidic, such as HC1 or HP04 for example.
In one preferred embodiment of the process according to the invention, the enzyme is a bacterial alkaline protease, more particularly that produced by Bacillus licheniformis and marketed by the Novo company under the trade-mark of "Alcalase", more particularly "Alcalase ~).6 L" or "Alcalase 2.4 L" for example.
It has been found that, with this preferred embodiment, it was possible to obtain a hydrolysate having a p<~rticularly high NPN and particularly reduced al lergenic:ity .
To this end, the first hydrolysis step is carried out at a p~H value of 7.0 to 10.0 and at 50 to 80°C and preferably at 63 to 73°C while the second hydrolysis step is carried out at a pH value of 6.5 to 8.0 and at 55 to 80°C' and preferably at 65 to 73°C. A thermal denaturing step may be carried out either after the first hydrolysis step or between the two hydrolysis steps over a period of 30 s to 10 rains. and preferably over a period of 4 to 6 rains. at a temperature of 80 to n 120°C and preferably at a temperature of 85 to 95°C.
The enzyme may then be inactivated by an autodigestion step carried out over a period of 10 s to 20 mins. and preferably over a period of 2 to 8 mins. at 70 to 110°C
and preferably at 85 to 90°C, followed by a steriliza-tion step ~~arried out over a period 5 s to 5 mins. and preferably over a period of 30 s to 2 mins. at 110 to 150°C and preferably at 120 to 130°C.
In another preferred embodiment of the process according to the invention, the enzyme used is a com bination of, on the one hand, a bacterial alkaline protease, more particularly that produced by Bacillus licheniformis and marketed by the Novo company under the name of "Alcalase", more particularly "Alcalase 0.6 L"
or "Alcala:~e 2.4 L", and on the other hand a pancreatic enzyme, more particularly trypsin for example.
In this other preferred embodiment, two sub-strates, more particularly two whey substrates, may each be separately subjected to a separate hydrolysis with one of the.>e two enzymes by the process according to the invention up to a common step, preferably up to a common sterilization step following two separate autodigestion steps of the two different enzymes. The same substrate may also be successively subjected to the action of one and then 'the other of these two enzymes. This is because it has been found that a hydrolysis product of whey proteins, for example, obtained by this combination can show particularly good stability in storage.
To carry out the process according to the inven tion with a pancreatic enzyme, particularly trypsin for example, above all in the above-mentioned combination, the pH and temperature conditions described in EP 322 589 may be used with advantage.
The: apparatus for carrying out the process s~
according t:o the invention thus comprises a double-jacketed stirred hydrolysis tank which is connected upstream to a substrate metering unit and to an enzyme metering unit and downstream to at least one hydrolysis tube equipped with static mixing elements.
In this apparatus, the tube may be vertically arranged, its lower end being connected to the tank and its upper e:nd opening into an outlet pipe. It may also be arranged horizontally or in any other position. In a preferred embodiment, it has a length of greater than 4 times its diameter.
In another preferred embodiment, the substrate and enzyme metering units each comprise a feed vessel connected to the hydrolysis tank by a volumetric pump.
The apparatus may also comprise a reactant metering unit comprising a feed vessel connected to the hydrolysis 'tank by a volumetric pump controlled by a pH
meter.
The apparatus may also comprise several tanks connected in series instead of a single tank, more particularly two tanks of which one may be used as a prehydrolysis tank.
The apparatus may also comprise several hydroly sis tubes equipped with static mixing elements connected in series downstream of the tank by connecting pipes which may be connected upstream to the enzyme metering unit and to the reactant metering unit.
A tube equipped with static mixing elements may also be provided between the enzyme, substrate and/or 3 0 reactant metering units and the tank or even between two successive tanks where the apparatus comprises several tanks.
The apparatus for carrying out the process according to the invention is described in more detail hereinafter with reference to the accompanying drawings which illustrate three examples of embodiment and in which:
Figure 1 diagrammatically illustrates a first embodiment of the apparatus comprising a tank and a tube equipped with static mixing elements.
Figure 2 diagrammatically illustrates a second embodiment of the apparatus comprising a tank and several hydrolysis tubes equipped with static mixing elements.
Figure 3 diagrammatically illustrates a third embodiment of the apparatus comprising two tanks and several hydrolysis tubes equipped with static mixing elements.
Referring to Fig. 1, the present apparatus com prises a hydrolysis tank 1 with a double jacket 2 and a stirrer 3 driven by a motor 4. The tank is closed in fluid-tight manner by a cover 5 through pass various pipes and the shaft of the stirrer 3.
The hydrolysis tank 1 is connected upstream by a pipe 6 to a substrate metering unit 7-11, by a pipe 12 to an enzyme metering unit 13-17 and by a pipe 18 to a reactant mei~ering unit 19-24.
The substrate metering unit comprises a substrate feed vessel 7 with a double jacket 8 and a stirrer 9 driven by a motor 10. The vessel 7 is connected to the hydrolysis tank 1 by the volumetric pump 11 connected to the pipe 6.
The enzyme metering unit comprises an enzyme feed vessel 13 with a double jacket 14 and a stirrer 15 driven by a motor 16. The vessel 13 is connected to the hydrolysis tank 1 by the volumetric pump 17 connected to the pipe 12..
The :reactant metering unit comprises a reactant feed vessel 19 connected to the hydrolysis tank 1 by a volumetric pump 20 connected to the pipe 18. The volumetric pump 18 is controlled by a pH meter 21 of which the measuring electrode 24 dips into the tank 1 through the cover 5 and which is electrically connected (chain line 23) to an electronic device for controlling 5 the pump 20 (not shown).
The lhydrolysis tank 1 is connected downstream to a hydrolysis tube 25 with a double j acket 26 equipped with static mixing elements 27 consisting of metal or plastic crosspieces interlocked in one another. The 10 tank 1 is connected to the tube 25 by a pipe to which is connected a three-way valve 29 designed to enable samples of :hydrolyzate to be removed from the tank.
The 'tube 25 is vertically arranged, its lower end being connected to the tank 1 and its upper end opening into an outlet pipe 30.
The temperature of a fluid circulating in each of the double jackets is regulated by a device shown symbolically at 31 for the tank 1, at 32 for the vessel 7, at 33 for the vessel 13 and at 34 for the tube 25.
In F:ig. 2, the elements of this second embodiment of the apparatus which correspond to the elements of the first embodiment shown in Fig. 1 are denoted by the same reference numerals.
In this second embodiment, the apparatus com prises several hydrolysis tubes 25, 35, 36 equipped with static mix~.ng elements 27, 37, 38 and connected in series downstream of the tank 1 by connecting pipes 39, 40 connected upstream to the enzyme feed vessel 13 by pipes 41, ~42 which rejoin the pipe 12 to which the volumetric ;pump 17 is connected.
The connecting pipes 39, 40 are also connected upstream to the reactant feed vessel 19 by pipes 43, 44 which rejoin the pipe 18 to which the volumetric pump 20 is connected.
In 'this second embodiment of the apparatus according to the invention, a mixing tube 45 equipped with static mixing elements is again provided between the enzyme, substrate and reactant metering units and the tank 1.
The various enzyme, substrate and reactant feed vessels are connected to the tube 45 by pipes 46, 47 and 48 to which a volumetric pump 49 and the volumetric pumps 11 and 20 are respectively connected.
In Fig. 3, the elements of this third embodiment of the apparatus which correspond to the elements of the first two embodiments shown in Figs. 1 and 2 are again denoted by 'the same reference numerals.
In this third embodiment, the apparatus comprises several hydrolysis tubes 25, 35, 36, 50 equipped with static mixing elements and connected in series down stream of the hydrolysis tank 1. The hydrolysis tank is connected upstream to a second tank, in the present case a prehydrol:ysis tank 51, by a denaturing tube 52 equip-ped with static mixing elements.
In this third embodiment, it is the prehydrolysis tank 51 which is connected upstream to the substrate and reactant feed vessels 7 and 19 while the enzyme feed vessel 13 is connected downstream by the pipes 56, 12 and 41, respectively, to the prehydrolysis tank 51, the hydrolysis tank 1 and the pipe 39 connecting the hydro-lysis tubes 25 and 35.
In this embodiment, inactivation tubes 53 , 54 and a cooling tube 55 equipped with static mixing elements and connected in series downstream of the last hydroly-sis tube 50 are again provided.
The process according to the present invention is illustrated by the following Examples in which parts and percentages are by weight.
Example 1 The process according to the invention is carried out in an apparatus similar to that described with reference t~o Fig. 1, in which the hydrolysis tank has a volume of 30 1 and the hydrolysis tube equipped with static mixing elements has a volume of 180 1 for a height of 3 m.
The :substrate used is a partly demineralized whey protein concentrate having a dry matter content of 20%
and respective contents (in % based on dry matter) of approximately 23~ proteins, 1.9% fats, 73% lactose and 1.3~ ash.
Porcine trypsin having an activity of 3 AU/g is used as the enzyme in a quantity of 1 g enzyme to 100 g substrate dry matter, i.e. 3 AU to 100 g substrate dry matter.
2N K~OH is used as reactant.
The tank is first filled with substrate and, after mixing in the enzyme, the discontinuous hydrolysis process is initiated at pH 7.3/60°C for 15 minutes, after which the hydrolyzed substrate has an NPN of 40~.
The process is then resumed continuously at such a rate that the mean residence time of the substrate in the tank is. 30 minutes and the residence time of the hydrolyzate in the tube is 3 h. A temperature of 60°C
and a pH of 7.3 are maintained in the tank. A tempera ture of 60°C is maintained in the tube, the pH being allowed to float so that it falls spontaneously from approximately 7.3 at the tube entrance to approximately 6.9 at the 'tube exit.
The lzydrolyzate has an NPN of 65% on leaving the tube.
If, :Eor comparison, the same substrate is hydro-lyzed discontinuously with the same enzyme in the same enzyme-to-substrate ratio for approximately 7 h at pH
There are various known processes for the enzy-matic hydrolysis of proteins which differ from one another, for example, in the choice of the substrate, the enzyme, the degree of hydrolysis and/or the required peptide profile. In cases where, for example, a rela-tively well-defined peptide profile, more particularly a narrow oligopeptide profile, is required for reasons of assimilation of the hydrolyzate by the intestinal mucosa, known hydrolysis processes generally comprise at least one hydrolyzate filtration or screening step.
For example, EP 226 221 describes a process for the preparation of hypoallergenic peptides having a molecular weight in the range from 2000 to 6000 by one or more enzymatic protein hydrolysis steps each carried out discontinuously in a fermentation tank and each terminating in an ultrafiltration step.
US 4,212,889 describes a process for solubilizing fish proteins, in which a mixture of fish flesh and enzyme is continuously passed through an installation comprising several hydrolysis tanks connected in series.
The problem addressed by the present invention was to provide a hydrolysis process which, preferably carried out continuously, would enable the efficiency of discontinuous hydrolysis in a tank to be equalled or even increased and which would enable a protein hydroly-zate having a well-defined and reproducible degree of hydrolysis and/or peptide spectrum to be obtained.
To this end, the process according to the inven-tion for the: enzymatic hydrolysis of proteins, in which a protein substrate is subj ected to hydrolysis with a proteolytic enzyme, comprises a first enzymatic hydroly-sis step i:n a stirred tank and a second enzymatic hydrolysis :step in a tube. In a preferred embodiment, the process according to the invention is carried out continuously and the second enzymatic hydrolysis step is carried out in a tube equipped with static mixing elements.
Simi7.arly, the apparatus for carrying out the process according to the invention comprises a double-jacketed stirred hydrolysis tank which is connected upstream to a substrate metering unit and to an enzyme metering unit and downstream to at least one hydrolysis tube. In a preferred embodiment, the tube is equipped with static mixing elements.
It has been found that it is thus possible to produce a protein hydrolyzate having a well-defined degree of hydrolysis and/or peptide spectrum in a highly efficient and reproducible, preferably continuous manner.
By virtue of the process and apparatus according to the invention, it is possible in particular to work with a tank of relatively small dimensions which may be filled completely without leaving any head space and with a tube of relatively large dimensions. It is thus possible to carry out, preferably continuously, a first relatively ;short step, i.e. a hydrolysis initiation step, in a relatively small tank, and a second relative-ly long step, i . a . a hydrolysis completion step, in a tube of relatively large volume. The reaction time, i. e. the re:~idence time of the substrate in the total volume represented by the sum of the tank volume and the tube volume, can thus be controlled in a precise and simple manner, for example by means of volumetric pumps.
If, for comparison, it is desired to carry out a hydrolysis in a single hydrolysis tank of large dimen sions, the residence time of a unit volume of hydroly zate cannot be precisely defined. This is true of discontinuous hydrolysis where the times required to establish given pH and/or temperature conditions and even to empty the tank, for example, are considerable.
However, the same is even truer of continuous hydro-lysis, in which case is it only possible to define a mean residence time. Even in a process of the type described in the above-cited US 4,212,889, the residence time can scarcely be defined any more precisely.
By contrast, it has been found that, with the process and apparatus according to the present inven-tion, the residence time of a unit volume of hydrolyzate can be defined in a remarkably precise manner, the flow of hydrolyzate through the hydrolysis tube - preferably equipped with static mixing elements - having a very flat front.
In t:he present specification, the degree of hydrolysis is defined via the quantity of non-protein nitrogen (NPN) determined as the percentage of total nitrogen which cannot be precipitated with 13% trichlo-roacetic aced.
The nitrogen contents are determined by the Kjeldahl mei:hod.
The amine nitrogen (free a-NHZ) contents are determined by reaction with ninhydrin after alkaline hydrolysis.
The serotonin relaxation tests using tritium-labelled exogenous serotonin (serotonin-3H) are carried out on normal mastocytes of the peritoneal cavity of rats by thEa method described by R. Fritsche and M.
Bonzon in Int. Arch. Allerg. Immunol. 93, 289-293 (1990).
The ELISA inhibition tests are carried out with rabbit antibodies specific of f3-lactoglobulin (BLG), bovine serum albumin (BSA) and casein (CAS). The sensi ,.~. 4 2092953 tivity of the method, i.e. the concentration detection limit, is 20 ng/ml.
The high performance liquid chromatography analyses (HPLC tests, peptide profiles) are carried out under non-denaturing conditions on gel based on type TSK-62000-SWT"' silica (a product of Toyo Soda), of which the fractionation range extends from 500 to 50,000 dalton, in a Biorad BIOSIL SEC-125T"~ column. The results are expre:~sed in % surface distribution of the peaks read at 2:20 nm in a 0.1 M phosphate solution + 0.4 M
NaCl at pH 6.80.
The: analyses by zone electrophoresis in poly acrylamide~ gel (SDS-PAGE tests) are carried out by the method described by Laemmli in Nature 227, 680 et seq.
(1970) .
The blockage of lysine is determined by HPLC and is expressed as % blocked lysine relative to the total lysine of the hydrolyzate.
"Static mixing elements" are understood to be undulating crossmembers or strips of metal or plastic which intersect or which are interlocked in one another and which divide the space defined by the tube into a plurality of intersecting passages progressing along the axis of the tube. Elements of the type in question are marketed by Sulzer A.G. of CH-8401 Winterthur, for example under the trade-marks SMV, SMX or SMXL.
Finally, it is important to appreciate that the tubes provided with static mixing elements are sys tematically equipped with a double jacket even when this is not specifically ment-Toned.
The process according to the invention may be carried out. using any starting material rich in proteins as the prot=ein substrate, such as flours or semolina of oil seeds or oil seed cakes, food-quality yeasts or bacteria, minced animal or fish flesh or milk or milk derivative:, for example in the form of particles in aqueous suspen-sion or aqueous suspensions.
The protein substrate is preferably a whey substrate containing whey proteins, more particularly a 5 sweet whey i°rom cheese production or an acidic whey from casein production either as such or in demineralized or lactose-free, liquid or reconstituted form.
In another preferred embodiment, the enzyme is selected from the group consisting of trypsin, chymo trypsin, pancreatin, bacterial proteases, fungal prote ases and mixtures thereof.
The proteolytic enzyme and the substrate may be mixed in a quantity of enzyme having an activity of 0.1 to 12 Anson units (AU) per 100 g substrate dry matter.
The first hydrolysis step is preferably carried out over a period of 10 to 60 minutes at a pH and a temperature adjusted to values favourable to the acti-vity of the enzyme while the second hydrolysis step is preferably carried out over a period of 1 to 8 h at a temperature equal to or above, particularly 0 to 10°C
above, the 'temperature of the first step.
Intermediate or complementary steps may be included, more particularly a preliminary mixing step preferably carried out in a tube equipped with static mixing elements; a thermal denaturing step before, in the middle of or after the first hydrolysis step, particularly using a heat exchanger or a tube equipped with static mixing elements; one or more enzyme inacti-vation steps, particularly after the second hydrolysis step, more especially using a heat exchanger and/or a steam injector and/or a tube equipped with static mixing elements; a:nd/or a cooling step carried out in particu-lar after a denaturing step, more particularly using a heat exchanger or preferably a tube equipped with static mixing elements for example.
The enzyme may be inactivated in one or preferab-ly two steps, a first step corresponding more precisely to autodigestion of the enzyme and a second step corre-sponding more precisely to sterilization.
The first hydrolysis step in a tank may also be divided into at least two parts carried out in at least two tanks connected in series. Similarly, the second hydrolysis step carried out in a tube may be divided into at least two parts carried out in at least two tubes connected in series. In the latter case, a pH
adjustment and/or an addition of enzyme may be carried out between two successive tubes.
For the pH adjustment(s), it is preferred to use a suitable reactant which may either be alkaline, such as KOH, NaOH or Ca(OH)z, or acidic, such as HC1 or HP04 for example.
In one preferred embodiment of the process according to the invention, the enzyme is a bacterial alkaline protease, more particularly that produced by Bacillus licheniformis and marketed by the Novo company under the trade-mark of "Alcalase", more particularly "Alcalase ~).6 L" or "Alcalase 2.4 L" for example.
It has been found that, with this preferred embodiment, it was possible to obtain a hydrolysate having a p<~rticularly high NPN and particularly reduced al lergenic:ity .
To this end, the first hydrolysis step is carried out at a p~H value of 7.0 to 10.0 and at 50 to 80°C and preferably at 63 to 73°C while the second hydrolysis step is carried out at a pH value of 6.5 to 8.0 and at 55 to 80°C' and preferably at 65 to 73°C. A thermal denaturing step may be carried out either after the first hydrolysis step or between the two hydrolysis steps over a period of 30 s to 10 rains. and preferably over a period of 4 to 6 rains. at a temperature of 80 to n 120°C and preferably at a temperature of 85 to 95°C.
The enzyme may then be inactivated by an autodigestion step carried out over a period of 10 s to 20 mins. and preferably over a period of 2 to 8 mins. at 70 to 110°C
and preferably at 85 to 90°C, followed by a steriliza-tion step ~~arried out over a period 5 s to 5 mins. and preferably over a period of 30 s to 2 mins. at 110 to 150°C and preferably at 120 to 130°C.
In another preferred embodiment of the process according to the invention, the enzyme used is a com bination of, on the one hand, a bacterial alkaline protease, more particularly that produced by Bacillus licheniformis and marketed by the Novo company under the name of "Alcalase", more particularly "Alcalase 0.6 L"
or "Alcala:~e 2.4 L", and on the other hand a pancreatic enzyme, more particularly trypsin for example.
In this other preferred embodiment, two sub-strates, more particularly two whey substrates, may each be separately subjected to a separate hydrolysis with one of the.>e two enzymes by the process according to the invention up to a common step, preferably up to a common sterilization step following two separate autodigestion steps of the two different enzymes. The same substrate may also be successively subjected to the action of one and then 'the other of these two enzymes. This is because it has been found that a hydrolysis product of whey proteins, for example, obtained by this combination can show particularly good stability in storage.
To carry out the process according to the inven tion with a pancreatic enzyme, particularly trypsin for example, above all in the above-mentioned combination, the pH and temperature conditions described in EP 322 589 may be used with advantage.
The: apparatus for carrying out the process s~
according t:o the invention thus comprises a double-jacketed stirred hydrolysis tank which is connected upstream to a substrate metering unit and to an enzyme metering unit and downstream to at least one hydrolysis tube equipped with static mixing elements.
In this apparatus, the tube may be vertically arranged, its lower end being connected to the tank and its upper e:nd opening into an outlet pipe. It may also be arranged horizontally or in any other position. In a preferred embodiment, it has a length of greater than 4 times its diameter.
In another preferred embodiment, the substrate and enzyme metering units each comprise a feed vessel connected to the hydrolysis tank by a volumetric pump.
The apparatus may also comprise a reactant metering unit comprising a feed vessel connected to the hydrolysis 'tank by a volumetric pump controlled by a pH
meter.
The apparatus may also comprise several tanks connected in series instead of a single tank, more particularly two tanks of which one may be used as a prehydrolysis tank.
The apparatus may also comprise several hydroly sis tubes equipped with static mixing elements connected in series downstream of the tank by connecting pipes which may be connected upstream to the enzyme metering unit and to the reactant metering unit.
A tube equipped with static mixing elements may also be provided between the enzyme, substrate and/or 3 0 reactant metering units and the tank or even between two successive tanks where the apparatus comprises several tanks.
The apparatus for carrying out the process according to the invention is described in more detail hereinafter with reference to the accompanying drawings which illustrate three examples of embodiment and in which:
Figure 1 diagrammatically illustrates a first embodiment of the apparatus comprising a tank and a tube equipped with static mixing elements.
Figure 2 diagrammatically illustrates a second embodiment of the apparatus comprising a tank and several hydrolysis tubes equipped with static mixing elements.
Figure 3 diagrammatically illustrates a third embodiment of the apparatus comprising two tanks and several hydrolysis tubes equipped with static mixing elements.
Referring to Fig. 1, the present apparatus com prises a hydrolysis tank 1 with a double jacket 2 and a stirrer 3 driven by a motor 4. The tank is closed in fluid-tight manner by a cover 5 through pass various pipes and the shaft of the stirrer 3.
The hydrolysis tank 1 is connected upstream by a pipe 6 to a substrate metering unit 7-11, by a pipe 12 to an enzyme metering unit 13-17 and by a pipe 18 to a reactant mei~ering unit 19-24.
The substrate metering unit comprises a substrate feed vessel 7 with a double jacket 8 and a stirrer 9 driven by a motor 10. The vessel 7 is connected to the hydrolysis tank 1 by the volumetric pump 11 connected to the pipe 6.
The enzyme metering unit comprises an enzyme feed vessel 13 with a double jacket 14 and a stirrer 15 driven by a motor 16. The vessel 13 is connected to the hydrolysis tank 1 by the volumetric pump 17 connected to the pipe 12..
The :reactant metering unit comprises a reactant feed vessel 19 connected to the hydrolysis tank 1 by a volumetric pump 20 connected to the pipe 18. The volumetric pump 18 is controlled by a pH meter 21 of which the measuring electrode 24 dips into the tank 1 through the cover 5 and which is electrically connected (chain line 23) to an electronic device for controlling 5 the pump 20 (not shown).
The lhydrolysis tank 1 is connected downstream to a hydrolysis tube 25 with a double j acket 26 equipped with static mixing elements 27 consisting of metal or plastic crosspieces interlocked in one another. The 10 tank 1 is connected to the tube 25 by a pipe to which is connected a three-way valve 29 designed to enable samples of :hydrolyzate to be removed from the tank.
The 'tube 25 is vertically arranged, its lower end being connected to the tank 1 and its upper end opening into an outlet pipe 30.
The temperature of a fluid circulating in each of the double jackets is regulated by a device shown symbolically at 31 for the tank 1, at 32 for the vessel 7, at 33 for the vessel 13 and at 34 for the tube 25.
In F:ig. 2, the elements of this second embodiment of the apparatus which correspond to the elements of the first embodiment shown in Fig. 1 are denoted by the same reference numerals.
In this second embodiment, the apparatus com prises several hydrolysis tubes 25, 35, 36 equipped with static mix~.ng elements 27, 37, 38 and connected in series downstream of the tank 1 by connecting pipes 39, 40 connected upstream to the enzyme feed vessel 13 by pipes 41, ~42 which rejoin the pipe 12 to which the volumetric ;pump 17 is connected.
The connecting pipes 39, 40 are also connected upstream to the reactant feed vessel 19 by pipes 43, 44 which rejoin the pipe 18 to which the volumetric pump 20 is connected.
In 'this second embodiment of the apparatus according to the invention, a mixing tube 45 equipped with static mixing elements is again provided between the enzyme, substrate and reactant metering units and the tank 1.
The various enzyme, substrate and reactant feed vessels are connected to the tube 45 by pipes 46, 47 and 48 to which a volumetric pump 49 and the volumetric pumps 11 and 20 are respectively connected.
In Fig. 3, the elements of this third embodiment of the apparatus which correspond to the elements of the first two embodiments shown in Figs. 1 and 2 are again denoted by 'the same reference numerals.
In this third embodiment, the apparatus comprises several hydrolysis tubes 25, 35, 36, 50 equipped with static mixing elements and connected in series down stream of the hydrolysis tank 1. The hydrolysis tank is connected upstream to a second tank, in the present case a prehydrol:ysis tank 51, by a denaturing tube 52 equip-ped with static mixing elements.
In this third embodiment, it is the prehydrolysis tank 51 which is connected upstream to the substrate and reactant feed vessels 7 and 19 while the enzyme feed vessel 13 is connected downstream by the pipes 56, 12 and 41, respectively, to the prehydrolysis tank 51, the hydrolysis tank 1 and the pipe 39 connecting the hydro-lysis tubes 25 and 35.
In this embodiment, inactivation tubes 53 , 54 and a cooling tube 55 equipped with static mixing elements and connected in series downstream of the last hydroly-sis tube 50 are again provided.
The process according to the present invention is illustrated by the following Examples in which parts and percentages are by weight.
Example 1 The process according to the invention is carried out in an apparatus similar to that described with reference t~o Fig. 1, in which the hydrolysis tank has a volume of 30 1 and the hydrolysis tube equipped with static mixing elements has a volume of 180 1 for a height of 3 m.
The :substrate used is a partly demineralized whey protein concentrate having a dry matter content of 20%
and respective contents (in % based on dry matter) of approximately 23~ proteins, 1.9% fats, 73% lactose and 1.3~ ash.
Porcine trypsin having an activity of 3 AU/g is used as the enzyme in a quantity of 1 g enzyme to 100 g substrate dry matter, i.e. 3 AU to 100 g substrate dry matter.
2N K~OH is used as reactant.
The tank is first filled with substrate and, after mixing in the enzyme, the discontinuous hydrolysis process is initiated at pH 7.3/60°C for 15 minutes, after which the hydrolyzed substrate has an NPN of 40~.
The process is then resumed continuously at such a rate that the mean residence time of the substrate in the tank is. 30 minutes and the residence time of the hydrolyzate in the tube is 3 h. A temperature of 60°C
and a pH of 7.3 are maintained in the tank. A tempera ture of 60°C is maintained in the tube, the pH being allowed to float so that it falls spontaneously from approximately 7.3 at the tube entrance to approximately 6.9 at the 'tube exit.
The lzydrolyzate has an NPN of 65% on leaving the tube.
If, :Eor comparison, the same substrate is hydro-lyzed discontinuously with the same enzyme in the same enzyme-to-substrate ratio for approximately 7 h at pH
7.3/60°C in a 200 litre tank, a hydrolyzate having an NPN of 60% is obtained.
Example 2 The procedure is the same as described in Example 1 except for the fact that, during three separate tests, the useful 'volume of the tank is varied so that the NPN
obtained after the passage of the substrate through the tank is 15, 35 and 45%, respectively.
Hydrolyzates having respective NPN's of 59, 63 and 66~ are thus obtained at the tube exit.
For comparison, an NPN of 60~ is obtained in approximately 7 h under the same conditions discontin-uously in a tank, i.e. at pH 7.3/60°C with a substrate having a dry matter content of 20% and a quantity of enzyme having an activity of 3 AU per 100 g substrate dry matter.
In other words, it is possible by the present process continuously to obtain an NPN higher than that which would be discontinuously obtained if the substrate had an NPN above 35% at the tube exit.
Example 3 The ;procedure is the same as described in Example 1 except that a pH value of 7.8 as opposed to 7.3 and a temperature of 55°C as opposed to 60°C are maintained in the tank.
A hydrolyzate having an NPN of 70% is obtained at the tube exit.
Example 4 The ;process according to the invention is carried out using an apparatus similar to that described with reference to Fig. 2.
A whey protein concentrate having a dry matter content of :33~, including 7.5% proteins, is used as the substrate.
The enzyme used is a bacterial alkaline protease produced by Bacillus licheniformis and marketed by the Novo company under the name of "Alcalase 2.4 L" which has an activity of 2.4 AU/g. This enzyme is used in a total quantity of 4 to 8.6% based on protein, i.e. 2.2 to 4.7 AU per 100 g substrate dry matter.
2N Kc~H is used as the reactant.
After the process has been suitably initiated, it is resumed continuously. The throughput of substrate and the dimensions of the tube and the tank are deter-mined in such a way that the residence times of the substrate or the hydrolyzate are, respectively, 5 to 10 minutes in a preliminary mixing tube equipped with static mixing elements preceding the tank, 5 to 8 minutes in a thermal denaturing tube equipped with static mixing elements connected in series between the preliminary mixing tube and the tank, 25 to 40 minutes in the tank (first hydrolysis step), 15 to 25 minutes in a first tube A equipped with static mixing elements following the tank (tube A of the second hydrolysis step), 15 to 25 minutes in a second tube B equipped with static mixing elements (tube B of the second hydrolysis step), 0 to 100 minutes in a third tube C equipped with static mixing elements (tube C of the second hydrolysis step), 5 to 20 minutes in an inactivation tube equipped with static mixing elements connected in series to the remainder o:E the tube C and 5 to 15 minutes in a cooling tube equipped with static mixing elements.
The total quantity of enzyme is divided into 4 parts, namely a first part of 5 to 15% of the total mixed with t:he substrate in the preliminary mixing tube, a second part of 30 to 40% of the total mixed with the substrate in the tank, a third part of 20 to 30~ of the total mixed with the substrate in the tube A and a fourth part of 20 to 30% of the total mixed with the substrate i:n the tube B.
The ;pH value of the substrate is adjusted to 7.3 5 as far as the tube B from which the pH is left to float.
The temperature is adjusted to 75°C in the preliminary mixing tube, to 85°C in the thermal denatur ing tube, to 70°C in the tank, to 71°C in the tube A and the tube B, to 80-105°C in the activation tube and to 2 10 8°C in the cooling tube.
The :hydrolyzate thus produced is collected after the cooling tube.
Example 5 15 The present process is carried out using an apparatus of the type similar to that described with reference to Fig. 3.
A whey protein concentrate having a dry matter content of 28%, including 7% proteins, is used as the substrate.
Alcalase 2.4 L is used as the enzyme in a total quantity of 2 to 6% based on protein, i.e. 1.2 to 3.6 AU
per 100 g substrate dry matter.
2N K~~H is used as the reactant.
After the process has been suitably initiated, it is resumed continuously. The throughput of substrate and the dimensions of the tubes and the tanks are determined in such a way that the successive steps take place as follows.
In a preliminary mixing tube equipped with static mixing elements preceding a prehydrolysis tank, 33% of the total quantity of enzyme is mixed with the substrate at pH 8.7/10°C.
A first phase of the first hydrolysis step is carried out in the prehydrolysis tank for 15 minutes at 65°C.
In a thermal denaturing tube equipped with static mixing elements connected in series between the pre-hydrolysis tank and a hydrolysis tank, the temperature is increased to 92°C for 5 minutes followed by cooling to 65°C.
In t:he hydrolysis tank, the remaining 66% of the total quantity of enzyme is added and a second phase of the first hydrolysis step is carried out over a period of 45 minutes at pH 7.4/65°C.
In three tubes equipped with static mixing elements and connected in series downstream of the tank, the second :hydrolysis step is carried out over a period of 195 minutes at 65°C, i.e. for 65 minutes in each tube. The pH is adjusted to 7.5 at the entrance of each tube and then floats.
In an inactivation tube equipped with static mixing elements connected in series after the three hydrolysis tubes, the enzyme is autodigested for 5 minutes at 87°C.
In a steam injection heating unit connected in series after the inactivation tube, the hydrolyzate is sterilized for 1 minute at 125°C.
The :hydrolyzate is then collected after cooling.
Example 6 The present process is carried out using an apparatus similar to that described with reference to Fig. 1, in which the hydrolysis tank has a volume of 2.8 1 and the hydrolysis tube equipped with static mixing elements ha;s a volume of 11.6 1 for a length of approxi-mately 5 m.
A whey protein concentrate having a dry matter content of 33%, including 7.5% proteins, is used.
Alcalase 2.4 L is used as the enzyme in a total quantity of 6.3% based on protein, i.e. 3.4 AU per 100 g substrate dry matter.
2N KOH is used as reactant.
The tank is first filled with substrate and, after mixing in the enzyme, the discontinuous hydrolysis process is initiated for 25 minutes at pH 7.3/70°C.
The process is then resumed continuously at such a rate that the total residence time of the hydrolyzate in the apparatus is 240 mins. (47 mins. in the tank and 193 mins. in the tube). A temperature of 70°C and a pH
of 7.3 are maintained in the tank. A temperature of 70°C is maintained in the tube, the pH being allowed to float so that it falls spontaneously from approximately 7.3 at the tube entrance to approximately 6.72 at the tube exit.
Samples are taken for analysis at the tube exit at times of 0, 60, 120 and 180 minutes counting from 240 minutes afi~er the start of the continuous process.
These samples have the pH values and the amine nitrogen contents shown in Table I below where the corresponding quantity of KOH used to keep the pH at 7.3 in the tank is also shown.
Table I
Time pH Amine nitrogen KOH
(mins.) (%) (g/h) 0 6.71 0.26 124 60 6.72 0.25 123 120 6.73 0.26 125 180 6.71 0.26 125 The quantities of KOH indicated in g/h correspond to an average consumption of 44.375 g per 1 of the tank for a residence time of 47 minutes.
It can be seen from Table I that the characteris-tics of the hydrolyzate hardly vary irrespective of the time the samples are taken for analysis from the tube exit. It i:: also possible to verify by zone electropho-resis in polyacrylamide gel (SDS-PAGE method) that the advantageous peptide profile of these samples, mostly small peptides, also remains remarkably constant.
For comparison, the same substrate is subjected to enzymatic: hydrolysis with the same enzyme in the same enzyme-to-substrate ratio discontinuously for 47 minutes in a 2 litre tank at 70°C and at a pH kept at 7.3.
After these first 47 minutes, the pH is left to float.
Samples are taken for analysis after 47 minutes counting from the bs~ginning of hydrolysis and then at various times up to and beyond 240 minutes. These samples have the pH value's and amine nitrogen contents shown in Table II below.
Table II
Time pH Amine nitrogen (mins.) (%) 47 7.30 0.21 67 7.0 0.22 140 6.77 0.24 197 6.75 0.27 240 6.72 0.26 300 6.68 360 6.65 The quantity of KOH used to keep the pH at 7.3 during the first 47 minutes is 45.5 g per litre of the tank.
It can be seen from Table II that the charac-teristics o7. the hydrolyzate obtained discontinuously in a tank vary rapidly, again after the time of 240 minutes corresponding to the continuous residence time in the apparatus used in Example 6.
This demonstrates one of the advantages of the process according to the invention in which there is no danger of development of the product comparable with that occurring during the time required to empty the tank in a discontinuous process.
Example 7 The present process is carried out using an apparatus similar to that described with reference to Fig. 1, in 'which the hydrolysis tank has a volume of 5 1 and the hydrolysis tube equipped with static mixing elements ha:~ a volume of 9.6 1 for a length of approxi-mately 5 m.
A whey protein concentrate having a dry matter content of :33%, including 7.5% proteins, is used as the substrate.
Alca:lase 2.4 L is used as the enzyme in a total quantity of 8% based on protein, i.e. 4.4 AU per 100 g substrate d:ry matter. Of these 8%, 2% are used in the tank and 6% are added at the tube entrance.
2N KOH is used as reactant.
In two separate tests, the tank is first filled with substrate and, after mixing in the enzyme, the discontinuous hydrolysis process is started at pH 7.3 and at two different temperatures of 72.5°C and 74°C for 40 minutes.
Each process is then resumed continuously at such a rate that the total residence time of the hydrolyzate in the apparatus is 116 minutes (40 minutes in the tank and 76 minutes in the tube). Respective temperatures of 72.5°C and '74°C and a pH of 7.3 are maintained in the tank for each of the two tests. A temperature of 72°C
is maintained in the tube, the pH being allowed to float.
The hydrolyzates thus obtained corresponding to 5 the temperatures of 72.5°C and 74°C in the tank have respective NPN's of 97.2% and 91.4% and necessitated the use of respective quantities of 205 g/h and 198 g/h KOH
to keep the pH at 7.3 in the tank. In addition, zone electrophoresis in polyacrylamide gel (SDS-PAGE method) 10 shows that they have a relatively narrow peptide pro-file.
For comparison, the same substrate is subjected to enzymati~~ hydrolysis with Alcalase 2.4 L in a total quantity of 4% based on protein, i.e. 2.2 AU per 100 g 15 substrate dry matter, continuously for 200 minutes in a 5 liter tan)H; at 70°C and at respective pH values of 6.4, 6.8, 7.3 and 7.8 in four separate tests.
The lhydrolyzates thus obtained have NPN's of 80 to 83% and necessitated the use of respective quantities 20 of KOH (in g/h) of 33.4, 50.1, 60.2 and 77.2 to keep their pH values at 6.4, 6.8, 7.3 and 7.8. In addition, they have respective amine nitrogen contents of 0.17, 0.20, 0.21 and 0.22%. The SDS-PAGE test shows that they have a relai~ive broad peptide profile.
This demonstrates another advantage of the process according to the invention insofar as it is possible to obtain a product having a high degree of hydrolysis .and a relatively narrow peptide profile by comparison with a product obtained by continuous enzy-matic hydro:Lysis in a tank which has a lower degree of hydrolysis <~nd a relatively broad peptide profile.
Example 8 The present process is carried out using an apparatus similar to that described with reference to Fig. 1, in which the hydrolysis tank has a volume of 2.8 1 and the hydrolysis tube equipped with static mixing elements has a volume of 11.6 1 for a length of ap-proximately 5 m.
A whey protein concentrate having a dry matter content of :33%, including 7.5% proteins, is used as the substrate.
Alca:Lase 2.4 L is used as the enzyme in a total quantity of 7% based on protein, i.e. 3.8 AU per 100 g substrate d:ry matter. Of these 7%, 2% are used in the tank and 5% are added at the tube entrance.
2N K()H is used as the reactant.
The tank is first filled with substrate and, after mixing in the enzyme, the discontinuous hydrolysis process is :started at pH 7.8/70°C for 25 minutes.
The process is then resumed continuously at such a rate that the residence time of the hydrolyzate is 45 minutes in 'the tank and 170 minutes in the hydrolysis tube, i.e. a total of 215 minutes. A temperature of 70°C and a pH of 7.8 are maintained in the tank. A
temperature of 70 ° C is maintained in the tube, the pH
being allowed to float so that it falls spontaneously from approximately 7.8 at the tube entrance to approxi-mately 6.67 at the tube exit.
In an inactivation tube equipped with static mixing elements and connected in series with the exit of the hydroly~;is tube, the hydrolyzate is inactivated for 18 minutes at 90°C. In a cooling tube equipped with static mixing elements connected in series downstream of the inactivation tube, the hydrolyzate is cooled to ambient temperature.
At the exit of the cooling tube, samples are taken for analysis at times of 0, 60, 120, 180 and 240 minutes counted at the exit of the hydrolysis tube from 215 minutes after the start of the continuous process.
These samples have the pH values, amine nitrogen con-tents, lysine blockages and NPN's shown in Table III
below where the corresponding quantity of KOH used to keep the pH at 7.8 in the tank is also shown.
Table III
Time pH Amine KOH Lysine NPN
nitrogen blockage (mins.) (%) (g/h) (%) (%) 0 6,.67 0.26 125 16.3 95 60 6..68 0.25 124 16.2 96 120 6..67 0.26 128 16.3 94 180 6..67 0.27 124 16.2 95 240 6..67 0.26 127 16.1 96 It can be seen from Table III in the same way as from Table I of Example 6 that the characteristics of the hydroly~:ate hardly vary irrespective of the time at which the samples are taken for analysis at the exit of the hyrolysis tube.
The peptide profile and the hypoallergenic properties of the product obtained under the conditions of the present Example are also examined by subjecting it to the HPLC, ELISA and serotonin-3H tests of which the results are set out in Tables V, VI and VII below.
For comparison, 160 kg of the same substrate are subjected to discontinuous enzymatic hydrolysis with the same enzyme in a total quantity of 7% based on protein in a tank at, 70°C. Of these 7% of enzyme, 2% are used for a first hydrolysis phase for 45 minutes at pH 7.8, after which the pH is left to float. After 60 minutes, the remaining 5% enzyme are added and hydrolysis is continued at: 70°C and at a floating pH up to and beyond 215 minutes.
20 kg hydrolyzate are removed after 120 minutes counting from the beginning of hydrolysis. Further quantities of 20 kg are taken after 150, 180, 200, 250, 300 and 360 mins. A sample is taken for analysis after 215 minutes' hydrolysis.
The hydrolyzates corresponding to the various removals and samples are immediately inactivated (for 18 minutes at 90°C in a heat exchanger) and then cooled to l0 ambient temj?erature (in a heat exchanger) and analyzed.
They have i~he pH values, amine nitrogen contents (%
based on powder containing 97% dry matter), lysine blockages and NPN's shown in Table IV below.
Table IV
Time pH Amine Lysine NPN
nitrogen blockage (mins.) (%, powder) (%) (%) 60 7 ,. 56 120 6..97 0.60 15.3 150 6,.87 0.63 17.2 90 180 6..82 0.66 18.2 94 200 6..80 0.68 18.3 95 215 6..80 0.68 18.9 95 250 6..79 0.69 19.4 96 300 6..77 0.71 19.5 96 360 6..76 0.74 19.6 97 It can be seen from Table IV in the same way as from Table :CI in Example 6 that the characteristics of the hydrolyzate obtained discontinuously in a tank vary rapidly again after the 215 minutes corresponding to the continuous residence time in the apparatus used in Example 8.
This confirms one of the advantages of the present process in which there is no danger of develop-ment of the product comparable to that which occurs during the time required to empty the tank in a discon-tinuous process.
The peptide profile and the hypoallergenic properties ~of the product obtained under the conditions of the above Comparison Example are also examined after 215 minutes by subjecting the product to the HPLC, ELISA
and serotonin-3H tests of which the results are set out in Tables V, VI and VII below.
Analogous tests are carried out on the product obtained continuously in a tank under the conditions corresponding to pH 7.3 presented for comparison with Example 7, the results also being set out in Tables V, VI and VII :below.
Table 0 Peptide profile (HPLC test) Product Percentage of peptides in the ranges acc. to within the molecular weight limits expressed in kDalton >14 14-6 6-3.5 3.5-1.0 < 1 Example 8 5 7 9 30 49 Comparison 4 7 10 31 48 (disc. tank) Comparison 21 13 9 24 33 ( cont . tank;
The test results set out in Table V clearly illustrate the fact that a hydrolyzate continuously obtained by the process according to the present inven-tion can have a peptide profile at least as narrow and centred on ithe small peptides as a hydrolyzate obtained for compari:aon in a discontinuous tank whereas a hydro-5 lyzate obtained for comparison in a continuous tank has a much broader peptide profile displaced towards the large peptides.
Table VI ELISA inhibition test Product Residual antigenicity expressed in acc. to ug antigen per g protein BLG BSA CAS
Example 8 53 20 150 Comparison 41 7 141 (disc. tank;) Comparison 111 > 1000 319 ( cont . tank;) Table VII Serotonin-3H relaxation test Product Residual antigenicity expressed in acc. to ug of BLG equivalent for the relaxation per g protein equivalent Example 8 20 Comparison 5 (disc. tank;l Comparison 50 ( cont . tank;~
The test results set out in Tables VI and VII
illustrate 'the fact that a hydrolyzate obtained by the process according to the present invention can be at least as hypoallergenic as a hydrolyzate obtained for comparison p_n a discontinuous tank whereas a hydrolyzate obtained for comparison in a continuous tank is not hypoallergenic.
Example 9 The ;present process is carried out in the same way as described in Example 7 under conditions corre-sponding to a temperature of 72 . 5 ° C in the tank. The tube is divided into nine segments. Samples are taken between two successive segments as the continuous hydrolysis progresses after the discontinuous initiation phase. Samples are taken at the tube exit at the same intervals when the duration of the continuous hydrolysis reaches the time corresponding to the residence time of the product in the tube.
The amine nitrogen content of the samples is determined. Each of these contents is divided by the equilibrium content towards which the hydrolyzate tends.
On a system of coordinates, the quotients obtained are plotted as ordinates while the quotients of the sampling times divided by the residence time of the hydrolyzate in the apparatus are plotted on the abscissa.
A sic~moid curve is obtained, crossing from the abscissa 0.8, intersecting the vertical of the abscissa 1.0 at two thirds of its maximum value and reaching its maximum value, in other words touching the horizontal of the ordinate: 1.0, at the vertical of the abscissa 1.2.
For comparison, the test is carried out in the same way except for the fact that the tube used is empty and, in addition, has the same dimensions as the tube equipped with static mixing elements.
Samp7les are taken under the same conditions, the same quotients are established and the corresponding curve is drawn in the same way.
A sigmoid curve is obtained, crossing from the abscissa O.E>, intersecting the vertical of the abscissa 1.0 at half' its maximum value and only reaching its maximum value, i.e. 1.0, beyond the vertical of the abscissa 1. F3 .
This demonstrates another two advantages of the process according to the invention, namely on the one hand the rapidity with which steady-state conditions can be established and, on the other hand, the homogeneity of the hydrolyzate on leaving the apparatus.
Example 2 The procedure is the same as described in Example 1 except for the fact that, during three separate tests, the useful 'volume of the tank is varied so that the NPN
obtained after the passage of the substrate through the tank is 15, 35 and 45%, respectively.
Hydrolyzates having respective NPN's of 59, 63 and 66~ are thus obtained at the tube exit.
For comparison, an NPN of 60~ is obtained in approximately 7 h under the same conditions discontin-uously in a tank, i.e. at pH 7.3/60°C with a substrate having a dry matter content of 20% and a quantity of enzyme having an activity of 3 AU per 100 g substrate dry matter.
In other words, it is possible by the present process continuously to obtain an NPN higher than that which would be discontinuously obtained if the substrate had an NPN above 35% at the tube exit.
Example 3 The ;procedure is the same as described in Example 1 except that a pH value of 7.8 as opposed to 7.3 and a temperature of 55°C as opposed to 60°C are maintained in the tank.
A hydrolyzate having an NPN of 70% is obtained at the tube exit.
Example 4 The ;process according to the invention is carried out using an apparatus similar to that described with reference to Fig. 2.
A whey protein concentrate having a dry matter content of :33~, including 7.5% proteins, is used as the substrate.
The enzyme used is a bacterial alkaline protease produced by Bacillus licheniformis and marketed by the Novo company under the name of "Alcalase 2.4 L" which has an activity of 2.4 AU/g. This enzyme is used in a total quantity of 4 to 8.6% based on protein, i.e. 2.2 to 4.7 AU per 100 g substrate dry matter.
2N Kc~H is used as the reactant.
After the process has been suitably initiated, it is resumed continuously. The throughput of substrate and the dimensions of the tube and the tank are deter-mined in such a way that the residence times of the substrate or the hydrolyzate are, respectively, 5 to 10 minutes in a preliminary mixing tube equipped with static mixing elements preceding the tank, 5 to 8 minutes in a thermal denaturing tube equipped with static mixing elements connected in series between the preliminary mixing tube and the tank, 25 to 40 minutes in the tank (first hydrolysis step), 15 to 25 minutes in a first tube A equipped with static mixing elements following the tank (tube A of the second hydrolysis step), 15 to 25 minutes in a second tube B equipped with static mixing elements (tube B of the second hydrolysis step), 0 to 100 minutes in a third tube C equipped with static mixing elements (tube C of the second hydrolysis step), 5 to 20 minutes in an inactivation tube equipped with static mixing elements connected in series to the remainder o:E the tube C and 5 to 15 minutes in a cooling tube equipped with static mixing elements.
The total quantity of enzyme is divided into 4 parts, namely a first part of 5 to 15% of the total mixed with t:he substrate in the preliminary mixing tube, a second part of 30 to 40% of the total mixed with the substrate in the tank, a third part of 20 to 30~ of the total mixed with the substrate in the tube A and a fourth part of 20 to 30% of the total mixed with the substrate i:n the tube B.
The ;pH value of the substrate is adjusted to 7.3 5 as far as the tube B from which the pH is left to float.
The temperature is adjusted to 75°C in the preliminary mixing tube, to 85°C in the thermal denatur ing tube, to 70°C in the tank, to 71°C in the tube A and the tube B, to 80-105°C in the activation tube and to 2 10 8°C in the cooling tube.
The :hydrolyzate thus produced is collected after the cooling tube.
Example 5 15 The present process is carried out using an apparatus of the type similar to that described with reference to Fig. 3.
A whey protein concentrate having a dry matter content of 28%, including 7% proteins, is used as the substrate.
Alcalase 2.4 L is used as the enzyme in a total quantity of 2 to 6% based on protein, i.e. 1.2 to 3.6 AU
per 100 g substrate dry matter.
2N K~~H is used as the reactant.
After the process has been suitably initiated, it is resumed continuously. The throughput of substrate and the dimensions of the tubes and the tanks are determined in such a way that the successive steps take place as follows.
In a preliminary mixing tube equipped with static mixing elements preceding a prehydrolysis tank, 33% of the total quantity of enzyme is mixed with the substrate at pH 8.7/10°C.
A first phase of the first hydrolysis step is carried out in the prehydrolysis tank for 15 minutes at 65°C.
In a thermal denaturing tube equipped with static mixing elements connected in series between the pre-hydrolysis tank and a hydrolysis tank, the temperature is increased to 92°C for 5 minutes followed by cooling to 65°C.
In t:he hydrolysis tank, the remaining 66% of the total quantity of enzyme is added and a second phase of the first hydrolysis step is carried out over a period of 45 minutes at pH 7.4/65°C.
In three tubes equipped with static mixing elements and connected in series downstream of the tank, the second :hydrolysis step is carried out over a period of 195 minutes at 65°C, i.e. for 65 minutes in each tube. The pH is adjusted to 7.5 at the entrance of each tube and then floats.
In an inactivation tube equipped with static mixing elements connected in series after the three hydrolysis tubes, the enzyme is autodigested for 5 minutes at 87°C.
In a steam injection heating unit connected in series after the inactivation tube, the hydrolyzate is sterilized for 1 minute at 125°C.
The :hydrolyzate is then collected after cooling.
Example 6 The present process is carried out using an apparatus similar to that described with reference to Fig. 1, in which the hydrolysis tank has a volume of 2.8 1 and the hydrolysis tube equipped with static mixing elements ha;s a volume of 11.6 1 for a length of approxi-mately 5 m.
A whey protein concentrate having a dry matter content of 33%, including 7.5% proteins, is used.
Alcalase 2.4 L is used as the enzyme in a total quantity of 6.3% based on protein, i.e. 3.4 AU per 100 g substrate dry matter.
2N KOH is used as reactant.
The tank is first filled with substrate and, after mixing in the enzyme, the discontinuous hydrolysis process is initiated for 25 minutes at pH 7.3/70°C.
The process is then resumed continuously at such a rate that the total residence time of the hydrolyzate in the apparatus is 240 mins. (47 mins. in the tank and 193 mins. in the tube). A temperature of 70°C and a pH
of 7.3 are maintained in the tank. A temperature of 70°C is maintained in the tube, the pH being allowed to float so that it falls spontaneously from approximately 7.3 at the tube entrance to approximately 6.72 at the tube exit.
Samples are taken for analysis at the tube exit at times of 0, 60, 120 and 180 minutes counting from 240 minutes afi~er the start of the continuous process.
These samples have the pH values and the amine nitrogen contents shown in Table I below where the corresponding quantity of KOH used to keep the pH at 7.3 in the tank is also shown.
Table I
Time pH Amine nitrogen KOH
(mins.) (%) (g/h) 0 6.71 0.26 124 60 6.72 0.25 123 120 6.73 0.26 125 180 6.71 0.26 125 The quantities of KOH indicated in g/h correspond to an average consumption of 44.375 g per 1 of the tank for a residence time of 47 minutes.
It can be seen from Table I that the characteris-tics of the hydrolyzate hardly vary irrespective of the time the samples are taken for analysis from the tube exit. It i:: also possible to verify by zone electropho-resis in polyacrylamide gel (SDS-PAGE method) that the advantageous peptide profile of these samples, mostly small peptides, also remains remarkably constant.
For comparison, the same substrate is subjected to enzymatic: hydrolysis with the same enzyme in the same enzyme-to-substrate ratio discontinuously for 47 minutes in a 2 litre tank at 70°C and at a pH kept at 7.3.
After these first 47 minutes, the pH is left to float.
Samples are taken for analysis after 47 minutes counting from the bs~ginning of hydrolysis and then at various times up to and beyond 240 minutes. These samples have the pH value's and amine nitrogen contents shown in Table II below.
Table II
Time pH Amine nitrogen (mins.) (%) 47 7.30 0.21 67 7.0 0.22 140 6.77 0.24 197 6.75 0.27 240 6.72 0.26 300 6.68 360 6.65 The quantity of KOH used to keep the pH at 7.3 during the first 47 minutes is 45.5 g per litre of the tank.
It can be seen from Table II that the charac-teristics o7. the hydrolyzate obtained discontinuously in a tank vary rapidly, again after the time of 240 minutes corresponding to the continuous residence time in the apparatus used in Example 6.
This demonstrates one of the advantages of the process according to the invention in which there is no danger of development of the product comparable with that occurring during the time required to empty the tank in a discontinuous process.
Example 7 The present process is carried out using an apparatus similar to that described with reference to Fig. 1, in 'which the hydrolysis tank has a volume of 5 1 and the hydrolysis tube equipped with static mixing elements ha:~ a volume of 9.6 1 for a length of approxi-mately 5 m.
A whey protein concentrate having a dry matter content of :33%, including 7.5% proteins, is used as the substrate.
Alca:lase 2.4 L is used as the enzyme in a total quantity of 8% based on protein, i.e. 4.4 AU per 100 g substrate d:ry matter. Of these 8%, 2% are used in the tank and 6% are added at the tube entrance.
2N KOH is used as reactant.
In two separate tests, the tank is first filled with substrate and, after mixing in the enzyme, the discontinuous hydrolysis process is started at pH 7.3 and at two different temperatures of 72.5°C and 74°C for 40 minutes.
Each process is then resumed continuously at such a rate that the total residence time of the hydrolyzate in the apparatus is 116 minutes (40 minutes in the tank and 76 minutes in the tube). Respective temperatures of 72.5°C and '74°C and a pH of 7.3 are maintained in the tank for each of the two tests. A temperature of 72°C
is maintained in the tube, the pH being allowed to float.
The hydrolyzates thus obtained corresponding to 5 the temperatures of 72.5°C and 74°C in the tank have respective NPN's of 97.2% and 91.4% and necessitated the use of respective quantities of 205 g/h and 198 g/h KOH
to keep the pH at 7.3 in the tank. In addition, zone electrophoresis in polyacrylamide gel (SDS-PAGE method) 10 shows that they have a relatively narrow peptide pro-file.
For comparison, the same substrate is subjected to enzymati~~ hydrolysis with Alcalase 2.4 L in a total quantity of 4% based on protein, i.e. 2.2 AU per 100 g 15 substrate dry matter, continuously for 200 minutes in a 5 liter tan)H; at 70°C and at respective pH values of 6.4, 6.8, 7.3 and 7.8 in four separate tests.
The lhydrolyzates thus obtained have NPN's of 80 to 83% and necessitated the use of respective quantities 20 of KOH (in g/h) of 33.4, 50.1, 60.2 and 77.2 to keep their pH values at 6.4, 6.8, 7.3 and 7.8. In addition, they have respective amine nitrogen contents of 0.17, 0.20, 0.21 and 0.22%. The SDS-PAGE test shows that they have a relai~ive broad peptide profile.
This demonstrates another advantage of the process according to the invention insofar as it is possible to obtain a product having a high degree of hydrolysis .and a relatively narrow peptide profile by comparison with a product obtained by continuous enzy-matic hydro:Lysis in a tank which has a lower degree of hydrolysis <~nd a relatively broad peptide profile.
Example 8 The present process is carried out using an apparatus similar to that described with reference to Fig. 1, in which the hydrolysis tank has a volume of 2.8 1 and the hydrolysis tube equipped with static mixing elements has a volume of 11.6 1 for a length of ap-proximately 5 m.
A whey protein concentrate having a dry matter content of :33%, including 7.5% proteins, is used as the substrate.
Alca:Lase 2.4 L is used as the enzyme in a total quantity of 7% based on protein, i.e. 3.8 AU per 100 g substrate d:ry matter. Of these 7%, 2% are used in the tank and 5% are added at the tube entrance.
2N K()H is used as the reactant.
The tank is first filled with substrate and, after mixing in the enzyme, the discontinuous hydrolysis process is :started at pH 7.8/70°C for 25 minutes.
The process is then resumed continuously at such a rate that the residence time of the hydrolyzate is 45 minutes in 'the tank and 170 minutes in the hydrolysis tube, i.e. a total of 215 minutes. A temperature of 70°C and a pH of 7.8 are maintained in the tank. A
temperature of 70 ° C is maintained in the tube, the pH
being allowed to float so that it falls spontaneously from approximately 7.8 at the tube entrance to approxi-mately 6.67 at the tube exit.
In an inactivation tube equipped with static mixing elements and connected in series with the exit of the hydroly~;is tube, the hydrolyzate is inactivated for 18 minutes at 90°C. In a cooling tube equipped with static mixing elements connected in series downstream of the inactivation tube, the hydrolyzate is cooled to ambient temperature.
At the exit of the cooling tube, samples are taken for analysis at times of 0, 60, 120, 180 and 240 minutes counted at the exit of the hydrolysis tube from 215 minutes after the start of the continuous process.
These samples have the pH values, amine nitrogen con-tents, lysine blockages and NPN's shown in Table III
below where the corresponding quantity of KOH used to keep the pH at 7.8 in the tank is also shown.
Table III
Time pH Amine KOH Lysine NPN
nitrogen blockage (mins.) (%) (g/h) (%) (%) 0 6,.67 0.26 125 16.3 95 60 6..68 0.25 124 16.2 96 120 6..67 0.26 128 16.3 94 180 6..67 0.27 124 16.2 95 240 6..67 0.26 127 16.1 96 It can be seen from Table III in the same way as from Table I of Example 6 that the characteristics of the hydroly~:ate hardly vary irrespective of the time at which the samples are taken for analysis at the exit of the hyrolysis tube.
The peptide profile and the hypoallergenic properties of the product obtained under the conditions of the present Example are also examined by subjecting it to the HPLC, ELISA and serotonin-3H tests of which the results are set out in Tables V, VI and VII below.
For comparison, 160 kg of the same substrate are subjected to discontinuous enzymatic hydrolysis with the same enzyme in a total quantity of 7% based on protein in a tank at, 70°C. Of these 7% of enzyme, 2% are used for a first hydrolysis phase for 45 minutes at pH 7.8, after which the pH is left to float. After 60 minutes, the remaining 5% enzyme are added and hydrolysis is continued at: 70°C and at a floating pH up to and beyond 215 minutes.
20 kg hydrolyzate are removed after 120 minutes counting from the beginning of hydrolysis. Further quantities of 20 kg are taken after 150, 180, 200, 250, 300 and 360 mins. A sample is taken for analysis after 215 minutes' hydrolysis.
The hydrolyzates corresponding to the various removals and samples are immediately inactivated (for 18 minutes at 90°C in a heat exchanger) and then cooled to l0 ambient temj?erature (in a heat exchanger) and analyzed.
They have i~he pH values, amine nitrogen contents (%
based on powder containing 97% dry matter), lysine blockages and NPN's shown in Table IV below.
Table IV
Time pH Amine Lysine NPN
nitrogen blockage (mins.) (%, powder) (%) (%) 60 7 ,. 56 120 6..97 0.60 15.3 150 6,.87 0.63 17.2 90 180 6..82 0.66 18.2 94 200 6..80 0.68 18.3 95 215 6..80 0.68 18.9 95 250 6..79 0.69 19.4 96 300 6..77 0.71 19.5 96 360 6..76 0.74 19.6 97 It can be seen from Table IV in the same way as from Table :CI in Example 6 that the characteristics of the hydrolyzate obtained discontinuously in a tank vary rapidly again after the 215 minutes corresponding to the continuous residence time in the apparatus used in Example 8.
This confirms one of the advantages of the present process in which there is no danger of develop-ment of the product comparable to that which occurs during the time required to empty the tank in a discon-tinuous process.
The peptide profile and the hypoallergenic properties ~of the product obtained under the conditions of the above Comparison Example are also examined after 215 minutes by subjecting the product to the HPLC, ELISA
and serotonin-3H tests of which the results are set out in Tables V, VI and VII below.
Analogous tests are carried out on the product obtained continuously in a tank under the conditions corresponding to pH 7.3 presented for comparison with Example 7, the results also being set out in Tables V, VI and VII :below.
Table 0 Peptide profile (HPLC test) Product Percentage of peptides in the ranges acc. to within the molecular weight limits expressed in kDalton >14 14-6 6-3.5 3.5-1.0 < 1 Example 8 5 7 9 30 49 Comparison 4 7 10 31 48 (disc. tank) Comparison 21 13 9 24 33 ( cont . tank;
The test results set out in Table V clearly illustrate the fact that a hydrolyzate continuously obtained by the process according to the present inven-tion can have a peptide profile at least as narrow and centred on ithe small peptides as a hydrolyzate obtained for compari:aon in a discontinuous tank whereas a hydro-5 lyzate obtained for comparison in a continuous tank has a much broader peptide profile displaced towards the large peptides.
Table VI ELISA inhibition test Product Residual antigenicity expressed in acc. to ug antigen per g protein BLG BSA CAS
Example 8 53 20 150 Comparison 41 7 141 (disc. tank;) Comparison 111 > 1000 319 ( cont . tank;) Table VII Serotonin-3H relaxation test Product Residual antigenicity expressed in acc. to ug of BLG equivalent for the relaxation per g protein equivalent Example 8 20 Comparison 5 (disc. tank;l Comparison 50 ( cont . tank;~
The test results set out in Tables VI and VII
illustrate 'the fact that a hydrolyzate obtained by the process according to the present invention can be at least as hypoallergenic as a hydrolyzate obtained for comparison p_n a discontinuous tank whereas a hydrolyzate obtained for comparison in a continuous tank is not hypoallergenic.
Example 9 The ;present process is carried out in the same way as described in Example 7 under conditions corre-sponding to a temperature of 72 . 5 ° C in the tank. The tube is divided into nine segments. Samples are taken between two successive segments as the continuous hydrolysis progresses after the discontinuous initiation phase. Samples are taken at the tube exit at the same intervals when the duration of the continuous hydrolysis reaches the time corresponding to the residence time of the product in the tube.
The amine nitrogen content of the samples is determined. Each of these contents is divided by the equilibrium content towards which the hydrolyzate tends.
On a system of coordinates, the quotients obtained are plotted as ordinates while the quotients of the sampling times divided by the residence time of the hydrolyzate in the apparatus are plotted on the abscissa.
A sic~moid curve is obtained, crossing from the abscissa 0.8, intersecting the vertical of the abscissa 1.0 at two thirds of its maximum value and reaching its maximum value, in other words touching the horizontal of the ordinate: 1.0, at the vertical of the abscissa 1.2.
For comparison, the test is carried out in the same way except for the fact that the tube used is empty and, in addition, has the same dimensions as the tube equipped with static mixing elements.
Samp7les are taken under the same conditions, the same quotients are established and the corresponding curve is drawn in the same way.
A sigmoid curve is obtained, crossing from the abscissa O.E>, intersecting the vertical of the abscissa 1.0 at half' its maximum value and only reaching its maximum value, i.e. 1.0, beyond the vertical of the abscissa 1. F3 .
This demonstrates another two advantages of the process according to the invention, namely on the one hand the rapidity with which steady-state conditions can be established and, on the other hand, the homogeneity of the hydrolyzate on leaving the apparatus.
Claims (16)
1. A process for the enzymatic hydrolysis of proteins, in which a protein substrate is subjected to hydrolysis with a proteolytic enzyme comprising as a first step carrying out the enzymatic hydrolysis in a stirred tank and as a second step carrying out the enzymatic hydrolysis in a tube.
2. A continuous enzymatic hydrolysis process as claimed in claim 1, in which the second hydrolysis step is carried out in a tube equipped with static mixing elements.
3. A process as claimed in claim 1, in which the substrate is a starting material rich in proteins.
4. A process as claimed in claim 3, in which the starting material rich in proteins comprises flours or semolina of oil. seeds or oil seed cakes, food-quality yeasts or bacteria, minced animal or fish flesh or milks or milk derivatives in the form of particles in aqueous suspension or an aqueous suspension.
5. A process as claimed in claim 1, in which the protein substrate is a whey substrate containing whey proteins.
6. A process as claimed in claim 5, in which the whey substrate containing whey proteins comprises a sweet whey from cheese production or an acidic whey from casein production in its normal form or in demineralized or lactose-free, liquid or reconstituted form.
7. A process as claimed in claim 1, in which the proteolytic enzyme is selected from the group consisting of trypsin, chymotrypsin, pancreatin, bacterial proteases, fungal proteases and mixtures thereof.
8. A process as claimed in claim 1, in which the proteolytic enzyme and the substrate are mixed in such proportions that the enzyme activity is 0.1 to 12 AU per 100 g substrate dry matter.
9. A process as claimed in claim 1, in which the first step is carried out for 10 to 60 minutes at a pH and temperature adjusted to values favourable to the activity of the enzyme and the second step is carried out for 1 to 8 h at a temperature adjusted to a value equal to or above the temperature of the first step.
10. The process of claim 9, wherein the second step's temperature is 0 to 10°C above the first step's temperature.
11. An apparatus for carrying out the process claimed in claim 1, comprising a stirred hydrolysis tank connected upstream to a substrate metering unit and an enzyme metering unit arid connected downstream to at least one hydrolysis tube.
12. An apparatus as claimed in claim 11, in which the hydrolysis tube is equipped with static mixing elements.
13. An apparatus as claimed in claim 12, in which the tube is vertically arranged, its lower end being connected to the tank and its upper end opening into an outlet pipe.
14. An apparatus as claimed in claim 12, in which the substrate and enzyme metering units each comprise a feed vessel connected to the hydrolysis tank.
15. An apparatus as claimed in claim 14, additionally comprising a reactant metering unit with a feed vessel connected to the hydrolysis tank by a volumetric pump controlled by a pH meter connected to the tank.
16. An apparatus as claimed in claim 15, comprising several hydrolysis tubes equipped with static mixing elements and connected in series downstream of the tank by connecting pipes connected upstream to the enzyme metering unit and to the reactant metering unit.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CH1160/92A CH683695A5 (en) | 1992-04-09 | 1992-04-09 | enzymatic hydrolysis. |
| CH1160/92-4 | 1992-04-09 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2092953A1 CA2092953A1 (en) | 1993-10-10 |
| CA2092953C true CA2092953C (en) | 2000-05-30 |
Family
ID=4203885
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002092953A Expired - Fee Related CA2092953C (en) | 1992-04-09 | 1993-03-30 | Enzymatic hydrolysis |
Country Status (12)
| Country | Link |
|---|---|
| EP (1) | EP0566877B1 (en) |
| JP (1) | JP2788837B2 (en) |
| AT (1) | ATE228571T1 (en) |
| AU (1) | AU659925B2 (en) |
| BR (1) | BR9301500A (en) |
| CA (1) | CA2092953C (en) |
| CH (1) | CH683695A5 (en) |
| DE (1) | DE69332512T2 (en) |
| MY (1) | MY109153A (en) |
| NZ (1) | NZ247275A (en) |
| PT (1) | PT566877E (en) |
| ZA (1) | ZA932226B (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5618689A (en) * | 1995-05-25 | 1997-04-08 | Nestec S.A. | Enhanced procedures for preparing food hydrolysates |
| DE10054516A1 (en) * | 2000-11-03 | 2002-05-16 | Henkel Kgaa | Extruded protein hydrolyzate, process for its preparation and its use |
| DK175501B1 (en) * | 2002-12-02 | 2004-11-15 | Green Earth | Plant and process for continuous hydrolysis of a proteinaceous animal or vegetable feedstock |
| EP2072622A4 (en) * | 2006-10-12 | 2012-01-25 | Kaneka Corp | Method for production of l-amino acid |
| CN103875844B (en) * | 2014-04-13 | 2015-07-01 | 周午贤 | Improved green tea enzymolysis device |
| CN104304643A (en) * | 2014-09-22 | 2015-01-28 | 江苏麦凯乐生物科技有限公司 | Production process of wheat hydrolyzing protein |
| GB201419096D0 (en) * | 2014-10-27 | 2014-12-10 | Firmenich & Cie | Improved apparatus and method for hydrolysing a product |
| CN108697144A (en) * | 2016-01-22 | 2018-10-23 | 利乐拉瓦尔集团及财务有限公司 | Method and apparatus for hydrolyzed infant formula |
| GB2548386A (en) | 2016-03-17 | 2017-09-20 | Alkymar As | Mixing and processing apparatus |
| CN107674835A (en) * | 2017-09-11 | 2018-02-09 | 广西肽王生物科技有限公司 | Moringa protease method hydrolysis device |
| CN108753886B (en) * | 2018-06-15 | 2021-07-06 | 黑龙江省野生动物研究所 | Deer bone active peptide extraction device and extraction method |
| US10941432B1 (en) | 2019-08-15 | 2021-03-09 | Santiago De Urbina Gaviria | Method for the preparation of low molecular weight porcine lympho-reticular polypeptides |
Family Cites Families (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3442656A (en) * | 1965-10-20 | 1969-05-06 | Dennis A Meadors | Method of separating protein into a food concentrate and chemical separator therefor |
| JPS5176443A (en) * | 1974-12-26 | 1976-07-02 | Denki Kagaku Kogyo Kk | Budotono renzokutekiiseikahoho |
| JPS6025112B2 (en) * | 1981-12-02 | 1985-06-17 | 日東電工株式会社 | immobilized enzyme reactor |
| JPS59162873A (en) * | 1983-03-09 | 1984-09-13 | Hitachi Ltd | Immobilized enzyme reactor |
| US4464509A (en) * | 1983-07-20 | 1984-08-07 | Marathon Oil Company | Apparatus and method for preparing polymers |
| JPS6098993A (en) * | 1983-11-04 | 1985-06-01 | Fuji Oil Co Ltd | Production of oligopeptide mixture |
| JPS60192599A (en) * | 1983-11-30 | 1985-10-01 | Fuji Oil Co Ltd | Production of oligopeptide mixture |
| IE58568B1 (en) * | 1984-11-15 | 1993-10-06 | Suiker Unie | Method and device for the carrying out of a microbiological or enzymatic process |
| DD251539B1 (en) * | 1986-07-31 | 1988-08-31 | Leuna Werke Veb | PROCESS FOR PREPARING HYDROXYLAMMONIUM SULFATE |
| FR2608051B1 (en) * | 1986-12-15 | 1989-04-07 | Bellon Labor Sa Roger | PROCESS FOR THE MANUFACTURE OF AN ENZYMATIC HYDROLYSATE OF PROTEINS RICH IN DI- AND TRI-PEPTIDES, FOR USE IN PARTICULAR IN ARTIFICIAL NUTRITION AND DIETETICS |
| CH670743A5 (en) * | 1987-04-06 | 1989-07-14 | Nestle Sa | |
| JP2507748B2 (en) * | 1987-06-30 | 1996-06-19 | 農林水産省 食品総合研究所長 | Enzyme-immobilized membrane reactor |
| JPH0198494A (en) * | 1987-10-09 | 1989-04-17 | Agency Of Ind Science & Technol | Continuous reaction process with immobilized lipase |
| JPH01167200U (en) * | 1988-05-06 | 1989-11-24 | ||
| CA2020461A1 (en) * | 1989-07-14 | 1991-01-15 | Donald J. Hamm | Process for the production of hydrolyzed vegetable proteins and the product therefrom |
| US5174651A (en) * | 1991-03-12 | 1992-12-29 | Gaddis Petroleum Corporation | Low shear polymer dissolution apparatus |
| JP3167723B2 (en) * | 1991-05-31 | 2001-05-21 | ダンマーク プロテイン アクティーゼルスカブ | Method for producing whey protein hydrolyzate |
-
1992
- 1992-04-09 CH CH1160/92A patent/CH683695A5/en not_active IP Right Cessation
-
1993
- 1993-03-22 AT AT93104638T patent/ATE228571T1/en not_active IP Right Cessation
- 1993-03-22 PT PT93104638T patent/PT566877E/en unknown
- 1993-03-22 EP EP93104638A patent/EP0566877B1/en not_active Expired - Lifetime
- 1993-03-22 DE DE69332512T patent/DE69332512T2/en not_active Expired - Fee Related
- 1993-03-26 AU AU35523/93A patent/AU659925B2/en not_active Ceased
- 1993-03-29 NZ NZ247275A patent/NZ247275A/en not_active IP Right Cessation
- 1993-03-29 ZA ZA932226A patent/ZA932226B/en unknown
- 1993-03-30 CA CA002092953A patent/CA2092953C/en not_active Expired - Fee Related
- 1993-04-06 MY MYPI93000622A patent/MY109153A/en unknown
- 1993-04-09 JP JP5083449A patent/JP2788837B2/en not_active Expired - Fee Related
- 1993-04-12 BR BR9301500A patent/BR9301500A/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| EP0566877A3 (en) | 1994-11-23 |
| DE69332512D1 (en) | 2003-01-09 |
| DE69332512T2 (en) | 2003-04-10 |
| AU3552393A (en) | 1993-10-14 |
| NZ247275A (en) | 1995-07-26 |
| AU659925B2 (en) | 1995-06-01 |
| JPH0686639A (en) | 1994-03-29 |
| ZA932226B (en) | 1993-10-15 |
| EP0566877B1 (en) | 2002-11-27 |
| BR9301500A (en) | 1993-10-13 |
| ATE228571T1 (en) | 2002-12-15 |
| CH683695A5 (en) | 1994-04-29 |
| MY109153A (en) | 1996-12-31 |
| EP0566877A2 (en) | 1993-10-27 |
| JP2788837B2 (en) | 1998-08-20 |
| CA2092953A1 (en) | 1993-10-10 |
| PT566877E (en) | 2003-03-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2092953C (en) | Enzymatic hydrolysis | |
| US5523237A (en) | Protein preparations | |
| CN1121732A (en) | A method for hydrolysing proteins | |
| EP1914298B1 (en) | Process for production of beer or beer-like beverage | |
| AU2942392A (en) | Casein hydrolyzate and method for production of such casein hydrolyzate | |
| Galvão et al. | Controlled hydrolysis of cheese whey proteins using trypsin and α-chymotrypsin | |
| Boudrant et al. | Continuous proteolysis with a stabilized protease. II. Continuous experiments | |
| Gallagher et al. | Hydrolysis of casein: a comparative study of two proteases and their peptide maps | |
| US5192677A (en) | Alkali and heat stable protease from thermomonospora fusca | |
| Doi et al. | Effects of limited proteolysis on functional properties of ovalbumin | |
| CHIANG et al. | Casein hydrolysate produced using a formed‐in‐place membrane reactor | |
| CN101487002A (en) | Alkaline protease | |
| Hartnett et al. | THE FORMATION OF HEAT AND ENZYME INDUCED (PLASTEIN) GELS FROM PEPSIN‐HYDROLYZED SOY PROTEIN ISOLATE 1 | |
| Ge et al. | Control of the degree of hydrolysis of a protein modification with immobilized protease by the pH‐drop method | |
| AU4348699A (en) | Method of producing iron-whey-proteolysate complex | |
| CN101487003A (en) | Acid protease | |
| EP0626007B1 (en) | Continuous membrane reactor and use thereof in biotechnical processes | |
| JPH02234642A (en) | Low-molecular peptide composition and production thereof | |
| GIUSTI et al. | Horse urinary kallikrein, I. Complete purification and characterization | |
| JP3364551B2 (en) | Method for producing low allergenic milk protein | |
| JPH025829A (en) | Method for removing bitterness from protein hydrolysate and obtained product | |
| CN112126669B (en) | Claudinate for immunoregulation and preparation method thereof | |
| CN101492664A (en) | Acid protease | |
| Watanabe et al. | The plastein reaction: fundamentals and applications | |
| Einhorn‐Stoll et al. | Enzymatic modification of yeast protein isolates |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKLA | Lapsed |