A Process for the Production of Hydrolyzed Vegetable Proteins Using Gaseous Hydrochloric Acid and the Product Therefrom

Europaisches Patentamt European Patent Office © Publication number: 0 495 391 A1 Office europeen des brevets

EUROPEAN PATENT APPLICATION

@ Date of filing: 09.01.92

© Priority: 14.01.91 US 641037 © Applicant: CPC INTERNATIONAL INC. International Plaza P.O. Box 8000 @ Date of publication of application: Englewood Cliffs New Jersey 07632(US) 22.07.92 Bulletin 92/30 @ Inventor: Hamm, Donald J. © Designated Contracting States: 57 Commonwealth Avenue AT BE CH DE DK ES FR GB GR IT LI LU NL PT New Providence, New Jersey 07974(US) SE

A process for the production of hydrolyzed vegetable proteins using gaseous hydrochloric acid and the product therefrom.

@ A process for the production of hydrolyzed vegetable proteins containing no detectable level of monocholorodihydroxypropanol and substantial flavor enhancement characteristics by using enzymatic hydrolysis of the protein followed by mild acid hydrolysis with gaseous hydrochloric acid and the product therefrom.

Rank Xerox (UK) Business Services 1 EP 0 495 391 A1 2

BACKGROUND OF THE INVENTION not for the purpose of flavor enhancement. What is taught in the existing art is generally directed to- FIELD OF THE INVENTION ward producing functionally improved proteins, such as eliminating bitter peptide formation during This invention relates to a process for the pro- 5 enzyme hydrolysis as shown in U.S. Patent No. duction of hydrolyzed vegetable proteins, contain- 4,636,388. Specifically, the patent discloses a low ing no detectable levels of monochlorodihydrox- ash protein product which is particularly adapted ypropanol. The resultant hydrolyzed vegetable pro- for enzymatic hydrolysis. A dispersion of protein is tein is clean and bland in flavor and exhibits sub- gelled and then washed in particulate form in a stantial flavor enhancement characteristics. io liquid in order to allow a portion of the non-proteinaceous material to defuse from the gel into the DESCRIPTION OF THE PRIOR ART liquid, and then the liquid is separated from the gel. The pretreated product is then hydrolyzed en- The preparation of conventional hydrolyzed zymatically, preferably with fungal protease and vegetable proteins (HVPs) is generally carried out 75 pancreatin. by acid hydrolysis with hydrochloric acid under U.S. Patent No. 4,757,007, discloses and refluxing conditions, specifically using 6M hydro- claims a process for the preparation of hydrolyzed chloric acid at 109°C and atmospheric pressure. It products of soy protein by partially hydrolyzing soy has been demonstrated that hydrolyzing vegetable protein with protease, and then separating the re- proteins at these conditions results in the chlorina- 20 suiting hydrolyzed products by using a 5% aque- tion of glycerol, which is derived from the residual ous solution of trichloroacetic acid. The portion of fatty substances present in the crude protein, to hydrolyzed protein with low solubility possesses produce monochlorodihydroxypropanols (MCDPs) excellent emulsifying properties, while the one with and dichloropropanols (DCPs). As MCDPs and high solubility possesses excellent foaming prop- DCPs exhibit questionable properties and charac- 25 erties. teristics, their presence is not desired in food pro- In U.S. Patent No. 3,830,942, a soluble protein ducts. DCPs are readily removed during the evap- product is produced which is particularly useful in oration or concentration steps of standard pro- highly acidic foods, and an insoluble protein prod- cesses. Unfortunately, MCDPs are not removed, uct is prepared which is used in preparing protein but are concentrated in the finished product, and 30 enriched bakery goods. The patent discloses the therefore, additional processing steps must be tak- method for producing the two products by forming en to remove the MCDPs from the finished prod- an aqueous solution of defatted oleaginous seed uct. materials, adjusting the pH of the slurry to the In a conventional acid hydrolysis process for isoelectric point of the seed materials, heating the preparing HVPs, the formation of MCDPs and 35 slurry to elevated temperatures, adding an enzyme DCPs can be avoided by using sulfuric or phos- to the slurry, agitating the mixture during hydrolysis phoric acid in place of hydrochloric acid. However, of the material and thereafter, separating the the HVPs produced by hydrolyzing with sulfuric or hydrolyzed and unhydrolyzed portions of the pro- phosphoric acid are of an inferior quality in that tein product. they exhibit a bitter flavor. 40 In U.S. Patent No. 4,665,158 to Armamet et al., The specific problem is that MCDP is derived a process is disclosed for hydrolyzing dehydrated during conventional acid hydrolysis from the protein materials with gaseous hydrochloric acid. chlorination of the glycerol derived from the resid- Partial hydrolysis is achieved by the process when ual fatty substances which are present in crude a protein material is impregnated with hydrochloric proteins. As an example, vital wheat gluten which is 45 acid and left to rest at a selected temperature until approximately 75% protein, also contains 5.0 to the desired degree of hydrolysis is obtained. If 9.5% fat and other lipid materials, is an abundant complete hydrolysis is desired, then the protein- source of glycerol in the form of a complex mixture hydrochloric acid combination is subjected to heat of mono-, di- and tri-glycerides, phospholipids and under pressure in an autoclave. glycolipids. Numerous factors which are believed to 50 Although enzyme hydrolysis and acid hydroly- effect the formation of MCDP include the presence sis are generally separate procedures, one refer- of high concentrations of chloride ions, high ence has been found which discloses the combina- amounts of excess acid, high refluxing tempera- tion of acid and enzyme hydrolysis to obtain a tures and long reaction times. It is thought that the protein hydrolyzate. In USSR Patent Application bound glycerol is more active in forming MCDPs 55 No. 442800, a method of obtaining a preparation than unbound glycerol. for parenteral protein feeding is taught. A method is Much is also known about the use of enzymes disclosed wherein the raw protein material under- to hydrolyze vegetable proteins for food use, but goes enzymatic cleavage, followed by acid hy-

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drolysis with 5.0% sulfuric acid (4.0N), in a carbon content. Specifically, the acid hydrolysis is carried dioxide atmosphere. Thereafter, the hydrolyzate is out at significantly lower acid concentrations, at passed through an anion exchange column, treated lower temperatures, at lower moisture contents and with aluminum hydroxide and passed through a for shorter periods of time than the conventional column containing cation exchange resin. The acid 5 hydrolysis process. By controlling the conditions, hydrolysis takes place at about 100°C for about deamidation preferentially occurs; ie, the amide seven (7) hours. linkages are hydrolyzed, but the peptide bond hy- Many attempts have been made over the years drolysis is controlled or minimized. It is believed to produce hydrolyzed vegetable protein products that these conditions, combined with the reduced which are used for various purposes, however, to io fat levels from the enzyme hydrolysis, are respon- date no process has been taught which produces a sible for the lack of formation of MCDPs in the hydrolyzed vegetable protein with reduced or non- finished product. existent levels of MCDP or DCP due to preventing MCDP and DCP production by controlling the pa- DETAILED INVENTION rameters of the acid hydrolysis, and which exhibits 15 substantial flavor enhancement characteristics. The present process comprises a number of steps for the hydrolyzing of a protein to a product SUMMARY OF THE INVENTION which contains no detectable level of MCDP. The term "no detectable level" as used herein means The present invention relates to the production 20 that there is no detectable level as measured by of hydrolyzed vegetable proteins which contain no gas chromotagraphy (GC) with a sensitivity to lev- detectable levels of MCDP. This result is achieved els as low as 1 ppm. by a unique process which combines two methods According to the process, a vegetable protein of hydrolysis of the vegetable protein, enzymatic is hydrolyzed by adding it to an aqueous solution hydrolysis followed by mild acid hydrolysis with 25 of at least one protease. The protein can be any gaseous hydrochloric acid. The hydrolyzates which one of the available proteins, such as, but not result from this process are clean and bland in limited to, oil seed proteins (soy, peanut, sunflower, flavor, exhibit substantial flavor enhancement char- cotton seed), leaf proteins, grain proteins, or any acters, and contain substantial amounts of mon- combination thereof. The preferred protein for pro- osodium glutamate, up to 36% w/w of the starting 30 ducing savory flavors with substantial flavor en- protein. hancing properties is wheat gluten, due to its high The production of hydrolyzed vegetable pro- glutamic acid content, present mostly as glutamine. teins with no detectable level of MCDP begins with The protein is added to an aqueous solution of the hydrolyzing of the protein by adding it to an at least one endoprotease, which can be acidic, aqueous solution of at least one protease. The 35 neutral or alkaline in form. The protease is chosen resulting hydrolyzed soluble protein is then sepa- dependent upon a number of parameters for the rated from the insoluble mass. Thereafter, gaseous particular enzyme/substrate combination, such as hydrochloric acid is added and the mixture is heat- a) what the proper pH would be for the optimum ed, providing an acidified hydrolyzate, which is proteolytic activity; b) the peptide bond specificity, then neutralized. 40 which is best suited to meet the end product re- It is believed that the enzyme hydrolysis step quirements; and c) whether or not the substrate contributes to reducing the MCDP and DCP forma- requires debittering. The preferred enzyme for the tion by solubilizing the protein away from the ma- protein wheat gluten is a neutral endoprotease, jority of the non-proteinaceous components of the specifically Prozyme 6 (Amano International En- crude protein. This is believed to result in a signifi- 45 zyme, Troy, Virgina). cant reduction in the level of available glycerol The enzyme hydrolysis of the protein occurs at containing fatty substances and thereby reduces a temperature of from about 25 to about 75 ° C and the level of key substrates required for MCDP at a pH of from about 5.5 to about 8.5, with a formation during the subsequent acid hydrolysis neutral enzyme present in the amount of from step. Another function of the enzyme hydrolysis is 50 about 0.1% to about 2.0% by weight of the sub- to act on the protein to release small peptides and strate. Again, these conditions will vary depending amino acids. on the protein-protease combination. For example, The acid hydrolysis step also contributes to the the pH is dependent upon the type of enzyme decrease in MCDP level, as it is gaseous hy- used. If an acidic enzyme is used, the pH will be in drochloric acid which is used. The conditions at 55 the range of from about 1 .5 to about 4.0 and if an which the acid hydrolysis, or deamidation take alkaline enzyme is used, the pH will be in the place, are significantly milder than those used in range of from about 7.0 to about 12.0. The present conventional processes, and at a lower moisture pH range is based on the use of a neutral protease.

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For the preferred case of wheat gluten and tion of the hydrolyzed soluble protein from the Prozyme 6, a neutral protease, the enzyme hy- insoluble mass. The acid may be added once the drolysis is carried out at a temperature of from hydrolyzate has reached the desired degree of about 40 to about 50 ° C, preferably 45 ° C, and at a solubility and degree of hydrolysis. Specifically, the pH of from about 6.5 to about 7.0, with a preferred 5 degree of solubility should, for economic reasons, level from about 0.5% to 1 .0% by weight Prozyme be at least 60%, with a preferred level of at least 6. The time during which the enzyme hydrolysis 90%. The degree of hydrolysis should be in the takes place is dependent upon quite a number of range of from about 10 to about 70%, preferably factors, specifically, the enzyme concentration about 20% to about 50%. used, the pH, the temperature of the reaction, the io The hydrolyzed vegetable protein is then sepa- substrate level and the desired degree of hydroly- rated from the insoluble mass by any suitable, sis. For the preferred embodiment, a time period of conventional method, such as filtration or centrifu- about four (4) hours is suggested. gation or combinations thereof. The substrate level also plays an important role Thereafter, the hydrolyzed soluble protein is in the present process. The desired level is from is concentrated or dried to lower the moisture content about 1.0 to about 30% by weight of the total to a level of from about 25 to about 40% by weight. batch, with the preferred level from about 22 to The resulting concentrate may be agglomerated, about 26% by weight. These levels are exception- flaked, or dried on an inert but porous substrate in ally high, and generally cannot be achieved by order to provide a large surface area for good conventional methods. In order to reach to desired 20 contact with the gaseous hydrochloric acid. levels, the substrate is added to the enzyme, in- Thereafter, the concentrated hydrolyzate is stead of the conventional method of adding the subjected to mild a gaseous acid hydrolysis by the enzyme to the substrate. addition of gaseous hydrochloric acid. This mild The enzyme hydrolysis is designed to accom- acid hydrolysis is designed to maximize deamida- plish a major portion of the peptide bond hydroly- 25 tion of free amino acids and peptides and minimize sis, which is necessary to release the flavor active the formation of pyroglutamic acid. This deamida- peptides and amino acids. It does not release the tion step is carried out at a temperature not to glutamic acid, or monosodium glutamate, from exceed 95 ° C. Since the reaction is exothermic, it glutamine, nor does it act on the amide bonds of may be necessary to provide a cooling means the glutamine which is bound to the peptides. As 30 during this step to prevent overheating which might stated above, quite a range of commercially avail- increase the risk of MCDP formation. The acidified able endoproteases and exoproteases may be hydrolyzate from the deamidation is then neutral- used to achieve the desired result. Specific ex- ized to a pH of from about 5.0 to about 7.0. Any oproteases, which contain leucine amino peptidase, number of known food grade bases can be used, may be used if it is desired to reduce the bit- 35 but the preferred one is sodium hydroxide. terness from hydrophobic peptides which are The resultant neutralized hydrolyzate may then present in the hydrolyzed vegetable protein. be further processed, if desired, to get it into a It must be pointed out that an endoprotease is more usable form. The hydrolyzate can be sub- absolutely necessary to carry out the initial enzyme jected to decolorization and deodorization pro- hydrolysis. Therefore, if only one protease is used, 40 cesses. This is conventionally carried out by the it must be an endoprotease. If more than one use of activated carbon. The decolorized, deodor- enzyme is used to hydrolyze the vegetable protein ized hydrolyzate may then be concentrated. This the enzymes may be any combination of en- can be performed by any number of methods doproteases and exoproteases, and they may be currently known, such as spray drying, vacuum used either simultaneously or sequentially. 45 tray drying or evaporation, for example by a falling At this point, the enzymatic process may be thin film evaporator. stopped at the desired stage by the addition of During the mild acid deamidation, all the acid to the aqueous solution. This step is not a glutamine produced by the initial enzymatic pro- necessary one, although it is part of the preferred cess is converted to monosodium glutamate process, and the hydrolyzed soluble protein can be 50 (MSG). Therefore, the amount of MSG present in separated from the insoluble mass without it. How- the final product is substantially determined by the ever, addition of a food grade organic or inorganic enzymatic process followed and the substrate acid to bring the aqueous solution to a pH of from used. For example, if wheat gluten is the vegetable about 2.0 to about 4.0 will stop the enzymatic protein in use and the enzymatic process goes to reaction, thereby providing precise control of the 55 total conversion, the level of MSG in the final end point, and providing microbiological stability to product can be as high as about 36% w/w of the the hydrolyzate. Addition of a food grade acid at starting protein. the desired time will also provide for better separa- The following are examples of the present in-

4 7 EP 0 495 391 A1 8 vention, and are not meant to be limiting in any 250g/batch on a dry weight basis. The wet filters way: are then loaded into a special holder constructed of glass which is slotted to hold the filters in vertical EXAMPLE 1 position and separated from each other during the 5 deamidation. The holder and wet filters are then Enzyme Hydrolysis placed in a vacuum oven at low heat (50-70 ° C) to reduce the moisture content of the suspended pro- Each sample is subjected to enzyme hydroly- tein to about 30% (dry basis). The holder and dried sis in a New Brunswick scientific MICROFERM enzyme hydrolyzate is then transferred to the reac- fermenter equipped with a 14 liter vessel and a io tion chamber described above and 100 mL (1.0 standard configuration. The general procedure fol- moles or a 25% excess relative to the amide lowed for conducting enzyme hydrolysis of wheat content of the batch) of concentrated (36.5% w/w) gluten is to first charge the reaction vessel with 65 food grade hydrochloric acid is placed in an open to 90% of the total water to be charged. While the glass tray below the filter holder. The chamber is vessel is being brought up to temperature, the pH is then sealed and flushed with dry nitrogen gas to electrode is standardized and the autotitrator is substantially eliminate oxygen from the chamber. charged with 4.0M sodium hydroxide. The titrator is Once the chamber is flushed, it is placed in a set to the target pH and the enzyme solution (10% standard laboratory forced air oven set at 95 ° C enzyme w/w in D.I. water) is prepared. where it is held for about 1 hour to affect the 2400 grams of wheat gluten (Manildra Milling 20 deamidation. At the end of the reaction, the cham- Corp., Shawnee Mission, Kansas 66205) are added ber is removed from the oven and allowed to cool. to 24 grams of Prozyme 6 (Amano Enzymes, U.S. The chamber is then again flushed with inert gas agent: Mitsubishi International Corp., New York, (into a suitable trap) to remove the excess gaseous New York 10022) in 7500 grams of water, over a hydrogen chloride to prepare the chamber to be period of 15 to 20 minutes with constant agitation. 25 opened safely. The enzymatic hydrolysis, maintained at pH 7 and The deamidated enzyme hydrolyzate is then at a temperature of 45 ° C, is allowed to proceed for collected by washing the filters with deionized wa- 4 hours. ter. The resultant acidic product is then neutralized After 4 hours, the hydrolyzate is rapidly titrated with 4M NaOH and brought to a known weight with to pH 2 with 20 Baume (10.0 N) food grade hy- 30 deionized water. The final hydrolyzate will contain drochloric acid (HCI). The acidified hydrolyzate is substantial levels of MSG but no detectable level of then pumped through tubing immersed in ice water MCDP. into collection vessels and immediately refriger- ated. The soluble phase is recovered by centrifug- Claims ing the entire hydrolyzate for 15 minutes in a 35 centrifuge at 16,000xG, and the recovered super- 1. A process for the production of hydrolyzed nate is set aside for deamidation. vegetable proteins containing no detectable level of monochlorodihydroxypropanol, which Gaseous Deamidation comprises: 40 (a) hydrolyzing a protein by adding it to an The gaseous deamidation is carried out on a aqueous solution of at least one protease; bench scale in a glass chamber constructed from 6 (b) separating the hydrolyzed soluble pro- inch diamter Kimax brand glass process pipe. A tein from any insoluble mass; standard beaded compression coupling equipped (c) concentrating the hydrolyzed soluble with a teflon seal is used to hermetically seal the 45 protein to a low moisture content; chamber during the reaction. The chamber is also (d) contacting the hydrolyzed soluble pro- equipped with inlets (with glass stopcocks) at each tein with gaseous hydrochloric acid to sub- end to allow for flushing the chamber with an inert stantially deamidate the hydrolyzate; and gas and to provide a port for monitoring reaction (e) neutralizing the deamidated hydrolyzate. pressure and a means of pressure release in case 50 excessive pressure builds up during the reaction. 2. The process of claim 1 which further com- To prepare the sample for deamidation, 50 prises addition of an acid to the aqueous solu- glass fiber pads (4X4 in.; CEM Corporation, Indian tion in step (a) to stop the enzymatic reaction. Trail, NC; Cat No. 200150) are loaded with about 25 grams each of the soluble phase (containing 55 3. The process of claim 1 which further com- about 20% solids, about 15% protein and about prises deodorization and decoloration of the 0.75 moles of amide groups) from the enzyme hydrolyzate from step (d). hydrolysis. This represents a total sample load of

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4. The process of claim 3 which further com- proteins containing no detectable level of mon- prises concentrating the deodorized and decol- ochlorodihydroxypropanol, which comprises: orized hydrolyzate. (a) hydrolyzing a protein by adding it to an aqueous solution of at least one protease; 5. The process of claim 1 wherein the protease in 5 (b) adding an acid to the aqueous solution step (a) is an endoprotease. of step (a) to stop the enzymatic reaction; (c) separating the hydrolyzed soluble pro- 6. The process of claim 5 wherein the en- tein from the insoluble mass; doprotease is acidic, neutral or alkaline. (d) concentrating the hydrolyzed protein 10io from step (c) to a lower moisture content; 7. The process of claim 1 wherein the protein in (e) contacting the hydrolyzed soluble pro- step (a) is chosen from the group consisting of tein with gaseous hydrochloric acid to pro- oil seed proteins, leaf proteins, grain proteins vide a deamidated hydrolyzate; or combinations thereof. (f) neutralizing the deamidated hydrolyzate; 15is (g) decolorizing and deodorizing the 8. The process of claim 1 wherein the hydrolysis hydrolyzate from step (f); and in step (a) takes place at a temperature of from (h) concentrating the hydrolyzate from step about 25 to about 75 ° C and a pH of from (g) . about 5.5 to about 8.5. 20 19. The process of claim 18 wherein the protein in 9. The process of claim 8 wherein the hydrolysis step (a) is chosen from the group consisting of in step (a) takes place at a temperature of from oil seed proteins, plasma proteins, leaf pro- about 40 to about 50 ° C and a pH of from teins, grain proteins or combinations thereof. about 6.5 to about 7.0. 25 20. The process of claim 19 wherein the protein in 10. The process of claim 1 wherein the separation step (a) is wheat gluten. in step (b) is by filtration, centrifugation or combinations thereof. 21. The process of claim 18 wherein the deodorization and decolorization of step (g) is carried 11. The process of claim 1 wherein the 30 out by the use of activated carbon. hydrolyzate is concentrated to a moisture content of from about 25 to about 40% by weight. 22. The process of claim 18 wherein the acid in step (b) is a food grade mineral acid. 12. The process of claim 1 wherein the reaction in step (d) takes place at a temperature of not 35 23. The process of claim 22 wherein the food more than about 95 ° C. grade mineral acid is chosen from the group consisting of hydrochloric acid, phosphoric 13. The process of claim 12 wherein the reaction acid, sulphuric acid and combinations thereof. in step (d) takes place at a temperature of about 95 °C. 40 24. The product of the process of claim 1 .

14. The process of claim 12 wherein the reaction 25. The product of the process of claim 18. in step (d) takes place for not more than four (4) hours. 26. A hydrolyzed vegetable protein which has no 45 detectable level of monochlorodihydrox- 15. The process of claim 1 wherein the neutraliza- ypropanol which is clean and bland in flavor tion of step (d) takes place at a pH of from and which exhibits substantial flavor enhance- about 5.0 to about 7.0. ment characteristics.

16. The process of claim 1 wherein the hydrolysis 50 27. The hydrolyzed vegetable protein of claim 26 in step (a) is effected by the sequential addi- which comprises substantial amounts of mon- tion of at least two proteases. osodium glutamate, up to about 36% w/w of the starting protein. 17. The process of claim 1 wherein the hydrolysis in step (a) is effected by the simultaneous addition of at least two proteases.

18. A process for the production of hydrolyzed

6 European Patent Number EUROPEAN SEARCH REPORT Application Office

EP 92 10 0260 DOCUMENTS CONSIDERED TO BE RELEVANT Category Citation of document with indication, where appropriate, Relevant CLASSIFICATION OF THE of relevant passages to claim APPLICATION ant. CI.S ) P.X EP-A-0 408 063 (CPC INTERNATIONAL INC) 1-27 A23J3/34 * the whole document * A23J3/32

EP-A-0 361 595 (UNILEVER NV) 26 * claim 1 *

PATENT ABSTRACTS OF JAPAN 1-27 vol. 12, no. 301 (C-521)16 August 1988 & JP-A-63 074 465 ( NISHIMURA NOBUAKI ) 4 April 1988 * abstract *

US-A-4 757 007 (MASAAKI SATOH) 1-27 * claims 1-2; examples 1-2 *

EP-A-0 169 166 (BATTELLE MEMORIAL INSTITUTE) 1.18 * claim 1 *

TECHNICAL FIELDS SEARCHED (tat. CI. 5 )

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The present search report has been drawn up for ail claims Place of March Date of CMfrfelkw of Ike Karck THE HAGUE 13 FEBRUARY 1992 SANTOS DIAZ A CATEGORY OF CITED DOCUMENTS T : theory or principle underlying the invention E : earlier patent document, but published on, or X : particularly relevant if taken alone after the filing date Y : particularly relevant if combined with another D : document cited in the application document of the same category L : document cited for other reasons A : technological background O : non-written disclosure & : member of the same patent family, corresponding P : intermediate document document