METHOD FOR PRODUCTION OF A VEGETABLE PROTEIN HYDROLYZATE

Abstract: The method for production of a vegetable protein hydrolyzate comprises a combination of i.a. an ultrafiltration and a non-pH-stat hydrolysis of a vegetable protein. Hereby a protein hydrolyzate is generated, which exhibits satisfactory organoleptic properties and which can be produced in high yield.

Description: METHOD FOR PRODUCTION OF A VEGETABLE PROTEIN HYDROLYZATE The invention comprises a method for production of a vegetable protein hydrolyzate. Methods of this kind usually comprise a pretreatment of a raw protein, in order to remove non-protein components, a hydrolysis, and a posttreatment in order to purify the protein hydrolyzate. An example of the pretreatment appears from US 4,420,425, an example of the hydrolysis appears from US 4,324,805 and 4,100,024, and an example of the pretreatment and posttreatment appears from American Chemical Society Symposium No. 154, Synthetic Membranes, Vol. II, Hyper- and Ultrafiltration Uses. Many methods for production of a protein hydrolyzate with good organoleptic properties can be carried out with a low yield only. Thus, it is the purpose of the invention to indicate a method for production of a protein hydrolyzate with good properties, which can be carried out with a relatively high yield. Surprisingly, according to the invention it has been found that a certain combination of an ultrafiltration and a non-pH-stat hydrolysis provides a process for production of a well tasting and organoleptically acceptable product in high yield. Thus, the method according to the invention for production of a vegetable protein hydrolyzate is characterized by the fact 1 ) that vegetable protein and water is mixed to a slurry with a protein content up to about 20%, preferably up to 10%, 2) that the pH of the slurry from step 1 ) is adjusted to a value, which is more than 3 pH-units from the isoelectric point of the protein, 3) that after dissolution or substantial dissolution of the protein the solubilized proteins are separated from the slurry, 4) that the supernatant from step 3 is ultrafiltered with an ultrafiltration unit with a cut-off value of above 20,000 Daltons, 5) that the reteπtate from step 4) is heat-treated during such time period i al Liie proteins are denatured, 6) that the denatured proteins in the retentate are proteolytically hydrolyzed by means of at least one protease at pH values and temperatures close to the optimum pH values and temperatures for the protease(s), by means of a non- pH-stat method to a DH of between 15 and 30%, 7) that the hydrolysis is terminated by inactivation of the enzyme (s), 8) that the effluent from step 7 is concentrated on an ultrafiltration unit with cut-off value above 5,000 to the maximum value or approx. the maximum value of DS in the retentate, whereafter a diafiltration with water is carried out until the percentage of DS in the permeate is below 0.9%, 9) that the permeate from step 8) is heated to between 130 and 140°C and immediately thereafter fiashcooied to around 75°C and then cooled in a heat exchanger to between 50 and 70°C, and 10) that the effluent from step 9) is concentrated and desalinated by nanofiltration at a temperature between 50 and 60°C, whereafter the retentate is collected as the protein hydrolyzate solution. It is to be understood that the vegetable protein used as raw material in step 1 can be any vegetable protein, e.g. soy protein, sesame protein, pea protein, rape seed protein, and faba bean protein. Also, it is to be understood that some of the above steps can be omitted under certain circumstances. Thus, step 3) can be omitted, if a lower ratio protein/dry matter can be accepted, step 5) can be omitted, if protein denaturation at this stage (i.e. before hydrolysis) is unnecessary, step 7) can be omitted, if inactivation of the enzyme(s) takes place during step 8) - 10), step 9) can be omitted, if the organoleptic properties of the protein hydrolyzate is already satisfactory, and step 10) can be omitted, if no concentration of the hydrolyzate is wanted, and if a high salinity and osmolality can be accepted. Furthermore, the steps 1-10 indicated above are not necessarily performed consecutively. Thus, step 3) can be performed immediately after step 4), step 6), step 7) or step 8); step 4) can be performed immediately after step 5); step 7) can be performed immediately after step 8), step 9) or step 10); and step 9) can be performed immediately before step 7) or step 8). A preferred embodiment of the method according to the invention comprises that the protein in step 1) is soy meal with high PSI (> 50% at pH 6.5). Soy meal with high PSI (> 50% at pH 6.5) is a readily available raw material well suited for the method according to the invention. PSI is Protein Solubility Index. A preferred embodiment of the method according to the invention comprises that the soy meal is defatted. Defatted soy meal is cheap, and with this raw material the process runs smoothly. A preferred embodiment of the method according to the invention comprises that the slurry in step 1) has a protein content of around 8%. In this manner the equipment is utilized optimally, and also, the viscosity is not too high for handling. A preferred embodiment of the method according to the invention comprises that the temperature during the steps 1) to 4) is above 60°C. In this manner bacterial growth will be limited. A preferred embodiment of the method according to the invention comprises that the separation in step 3) is carried out by means of gravity separation, preferably centrifugation. This separation is efficient and cheap. A preferred embodiment of the method according to the invention comprises that the protease or one of the proteases used during step 6) is a Bacillus protease. In this manner use can be made of cheap, commercial enzymes. A preferred embodiment of the method according to the invention comprises that the Bacillus protease is a Bacillus licheniformis protease. In this manner a simple process and a good yield is provided. A preferred embodiment of the method according to the invention comprises that at least two proteases are used during step 6), i.e. a Bacillus licheniformis protease and a Bacillus subtilis protease. In this manner a very high yield and a good taste of the end product is provided. A preferred embodiment of the method according to the invention comprises that the mixture at the end of step 7) is treated with activated carbon for more than 5 minutes at between 50 and 70°C in an amount corresponding to between 1 and 5% carbon, calculated in relation to soluble protein hydrolyzate. Hereby an end product with better organoleptic properties is achieved: better taste, no off-flavor, and better color. This embodiment is specially preferred in those cases, where the retentate by-product can not be utilized. A preferred embodiment of the method according to the invention comprises that the mixture at the end of step 10) is treated with activated carbon for 5 more than 5 minutes at between 50 and 70°C in an amount corresponding to between 1 and 5% carbon, calculated in relation to soluble protein hydrolyzate, whereafter the activated carbon is removed, and the filtrate is collected as the protein hydrolyzate solution. Hereby an end product with better organoleptic properties Is achieved: better taste, no off-flavor, and better color. 10 A preferred embodiment of the method according to the invention comprises that the protein hydroiyzate solution from step 10) is spray-dried to a water content below 6.5%. In this manner a stable product is achieved, both microbially and organoleptically. A preferred embodiment of the method according to the invention 15 comprises that 11) that the protein hydrolyzate solution from step 10) is sterile filtered, 12) that the sterile filtrate from step 11) is concentrated to a concentration of between 40 and 60 total DS, 13) that the concentrate from step 12) is pasteurized, and 20 14) that the pasteurized concentrate from step 13) is spray-dried to a water content of below 6.5%. If a spray-drying tower for treatment of the effluent for step 10) is not available in the factory at the appropriate time, steps 11), 12), and 13) may be carried out, whereafter step 14) can be performed, when the spray-drying tower 5 becomes available. Danish patent application no. 1498/87 describes a process with vegetable seeds as a starting material, in which some of the process steps are similar to the process steps used in the process according to the invention. However, the end product of the prior art process is a protein isolate, i.e. not a 0 protein hydrolyzate, and this protein isolate is an insoluble coagulate and thus not a soluble protein hydrolyzate as in relation to the process according to the invention. EP 325986 describes a method for the hydrolysis of food grade proteins by means of a special combination of proteolytic enzymes. However, the non-pH- stat-hydrolysis and the ultrafiltration used in the method according to the invention is not used in the prior art method. Also, according to the prior art method a product is produced which is not fully soluble, in contradistinction to the product produced by means of the method according to the invention. EXAMPLE 1 Mixing Untoasted defatted soy meal with a PSI of 55% at pH 6.5 and water are mixed to a dry matter content of 10% at a temperature of 62-63°C. The pH of the slurry is adjusted to 8.5 with 4N NaOH. Extraction After 30 minutes holding time the soluble proteins are extracted from the sludge by means of two centrifugation steps whereby an extraction efficiency of approx. 90% is obtained. After the first centrifugation the sludge is rediluted with deionized water, still at 62-63°C, and passed over the second centrifugation step whereafter the sludge is disposed. The centrifugate from both centrifugations are collected in the feed tank to the first ultrafiltration unit. It is very important that the temperature of the process liquid during mixing, extraction and ultrafiltration 1 is always above 60°C in order to limit bacteria! growth and that the temperature is below 64-65°C during mixing and extraction in order to prevent protein denaturation, excess coloring and degradation of the Ultrafiltration 1 The centrifugate is ultrafiltered in order to wash out carbohydrates and salts from the protein extract. The ultrafiltration unit is run at 65°C. The centrifugate is concentrated to maximum 5.5% DS and diafiltered by addition of deionized water until % DS ( iip-ermeate) L— - o.09 % DS (retentate) Then the retentate is concentrated to 9-10% DS. The permeate is disposed. Heat treatment The retentate is heat treated at 85°C for 1 minute in order to denature the proteins, thereby facilitating the hydrolysis. At the same time the bacterial counts in the process liquid is lowered. Hydrolysis The heat treated retentate is delivered to the hydrolysis tank at 55°C, and pH is adjusted to 8.5 by means of 4N NaOH. The hydrolysis is started by addition of Alcalase ® 2.4 L corresponding to E/S = 2%, when pH has passed 7.0. Neutrase ® 0.5 L corresponding to E/S = 1% is added, and the hydrolysis takes place during the next 10-12 hours obtaining a % DH (TNBS-method) of around 20%. The degree of hydrolysis can easily be followed by measuring the increase in osmolality during the hydrolysis. Inactivation and enzyme treatment The hydrolysis is stopped by lowering the pH to 4.2 by addition of 30% HCI. Then Viscozyme 120L corresponding to E/S = 0.1% is added for enzyme treatment. Carbon treatment Activated carbon (Picatif 120 FGV EWN) is mixed in the inactivated/enzyme treated hydrolysis mixture and should react for 30 minutes with slow agitation at 55°C. The carbon treatment is performed in order to improve the color and the organoleptic properties of the hydrolyzate. The dosage of activated carbon is calculated as 3% of dry matter measured as ° Brix. Ultrafiltration 2 The slurry from the carbon treatment is heated to 65°C and is concentrated on the ultrafiltration unit to approx. 8-9% DS followed by diafiltration by addition of deionized water until % DS in the permeate is below 0.9%. At last the retentate is concentrated as much as possible in order to maximize the yield. The retentate is disposed. Flash The permeate from ultrafiltration 2 is heated to 135°C by steam injection and within few seconds flash cooled to approx. 75°C followed by cooling in a plate heat exchanger to 55°C. The flash process is improving the organoleptic properties, and furthermore a positive effect on the bacterial counts is obtained. Nanofiltration The flashed process liquid is concentrated and desalinated by nanofiltration at 55°C. On AFC 30 membranes from PCI Membrane Systems the osmolality after concentration will be below 180 mOsm/kg H 2 0 at 7.5° Brix without diafiltration. In case lower osmolality is desired diafiltration with addition of deionized water can be performed before the final concentration. The nanofiltration is stopped at 30°C Brix because of low flux. Sterilizing filtration The concentrate from nanofiltration is filtered at approx. 50°C on Supra EKS sheets rinsed with citric acid solution (50 l/m 2 at pH = 4.2) and deionized water to neutral pH before steaming. The filter sheets are precoated with 0.25 kg Hyflo Super Cel and 0.25 kg Clarcel CBL-3 per m 2 . Evaporation The protein hydrolyzate is further concentrated to 60° Brix by vacuum evaporation at Tin/Tout = 70/40°C. Pasteurization The concentrated protein hydrolyzate is pasteurized in a plate heat exchanger for 4 seconds at 85°C. The pasteurized concentrate is cooled to 4-8°C and stored into a sterile tank until drying. Sorav-drving The protein hydrolyzate is spray-dried and agglomerated at Tin 200 = C. The water content in the spray-dried powder should be below 6.5% to obtain satisfactory stability of the powder. EXAMPLE 2 Mixing 30 kg of sesame flour with a protein content of 44.6% is mixed with 270 I of demineralized water at 65°C. Ultrafiltration 1 The mixture is ultrafiltrated to remove soluble carbohydrates. Diafiltration with 2 volumes of water and concentration to 8% protein. The permeate is disposed. Heat treatment The retentate is heat treated to 85°C for 5 minutes. Hydrolysis The heat treated retentate is delivered to the hydrolysis tank at 55°C, and pH is adjusted to 8.0 by means of Ca(OH) 2 . The hydrolysis is started by addition of Alcalase ® 2.4 L corresponding to E/S = 2%, when pH has passed 7.0. Neutrase ® 0.5 L corresponding to E/S = 1 % is added, and the hydrolysis takes place during the next 10-12 hours obtaining a % DH (TNBS-method) of around 20%. The degree of hydrolysis can easily be followed by measuring the increase in osmolality during the hydrolysis. Inactivation and enzyme treatment The hydrolysis is stopped by lowering the pH to 4.2 by addition of 30% HCI. Ultrafiltration 2 The mixture from the inactivation is heated to 65°C and is concentrated on the ultrafiltration unit to approx. 8-9% DS followed by diafiltration by addition of deionized water until % DS in the permeate is below 0.9%. At last the retentate is concentrated as much as possible in order to maximize the yield. The retentate is disposed. Flash The permeate from ultrafiltration 2 is heated to 135°C by steam injection and within few seconds flash cooled to approx. 75°C followed by cooling in a plate heat exchanger to 55°C. The flash process is improving the organoleptic properties, and furthermore a positive effect on the bacterial counts is obtained. Nanofiltration The flashed process liquid is concentrated and desalinated by nanofiltration at 55°C. On HC 50 membranes from DDS the osmolality after concentration will be below 180 mOsm/kg H 2 0 at 7.5° Brix without diafiltration. In case lower osmolality is desired diafiltration with addition of deionized water can be performed before the final concentration. The nanofiltration is stopped at 30°C Brix because of low flux. Carbon treatment Activated carbon (Picatif 120 FGV EWN) is mixed in the inactivated/enzyme treated hydrolysis mixture and should react for 30 minutes with slow agitation at 55°C. The carbon treatment is performed in order to improve the color and the organoleptic properties of the hydrolyzate. The dosage of activated carbon is calculated as 3% of dry matter measured as ° Brix. Sterilizing filtration The concentrate from carbon treatment is filtered at approx. 50°C on Supra EKS sheets rinsed with citric acid solution (50 t/m 2 at pH = 4.2) and deionized water to neutral pH before steaming. The filter sheets are precoated with 0.25 kg Hyflo Super Cel and 0.25 kg Clarcel CBL-3 per m 2 . Sprav-drving The protein hydrolyzate is spray-dried and agglomerated at Tin 200°C. The water content in the spray-dried powder should be below 6.5% to obtain satisfactory stability of the powder.

Claims: CLAIMS 1. Method for production of a vegetable protein hydrolyzate, characterized by the fact 1) that vegetable protein and water is mixed to a slurry with a protein content up to about 20%, preferably up to 10%, 2) that the pH of the slurry from step 1) is adjusted to a value, which is more than 3 pH-units from the isoelectric point of the protein, 3) that after dissolution or substantial dissolution of the protein the solubiiized proteins are separated from the slurry, 4) that the supernatant from step 3 is ultrafiltered with an ultrafiltration unit with a cut-off value of above 20,000 Daltons, 5) that the retentate from step 4) is heat-treated during such time period that the proteins are denatured, 6) that the denatured proteins in the retentate are proteolytically hydrolyzed by means of at least one protease at pH values and temperatures close to the optimum pH values and temperatures for the protease(s), by means of a non- pH-stat method to a DH of between 15 and 30%, 7) that the hydrolysis is terminated by inactivation of the enzyme (s), 8) that the effluent from step 7 is concentrated on an ultrafiltration unit with cut-off value above 5,000 to the maximum value or approx. the maximum value of DS in the retentate, whereafter a diafiltration with water is carried out until the percentage of DS in the permeate is below 0.9%, 9) that the permeate from step 8) is heated to between 130 and 140°C and immediately thereafter flashcooled to around 75°C and then cooled in a heat exchanger to between 50 and 60°C, and 10) that the effluent from step S) is concentrated and desalinated by nanofiltration at a temperature between 50 and 70°C, whereafter the retentate is collected as the protein hydrolyzate solution. 2. Method according to Claim 1 , characterized by the fact that the protein in step 1) is soy meal with high PSI (> 50% at pH 6.5). 3. Method according to Claim 2, characterized by the fact that the soy meal is defatted. 4. Method according to Claims 1 - 3, characterized by the fact that the slurry in step 1) has a protein content of around 8%. 5. Method according to Claims 1 - 4, characterized by the fact that the temperature during the steps 1) to 4) is above 60°C. 6. Method according to Claims 1 - 5, characterized by the fact that the separation in step 3) is carried out by means of gravity separation, preferably centrifugation. 7. Method according to Claims 1 - 6, characterized by the fact that the protease or one of the proteases used during step 6) is a Bacillus protease. 8. Method according to Claim 7, characterized by the fact that the Bacillus protease is a Bacillus licheniformis protease. 9. Method according to Claim 8, characterized by the fact that at least two proteases are used during step 6), i.e. a Bacillus licheniformis protease and a Bacillus subtilis protease. 10. Method according to Claims 1 - 9, characterized by the fact that the mixture at the end of step 7) is treated with activated carbon for more than 5 minutes at between 50 and 70°C in an amount corresponding to between 1 and 5% carbon, calculated in relation to soluble protein hydrolyzate. 11. Method according to Claims 1 - 9, characterized by the fact that the mixture at the end of step 10) is treated with activated carbon for more than 5 minutes at between 50 and 70°C in an amount corresponding to between 1 and 5% carbon, calculated in relation to soluble protein hydrolyzate, whereafter the activated 5 carbon is removed, and the filtrate is collected as the protein hydrolyzate solution. 12. Method according to Claims 1 - 11, characterized by the fact that the protein hydrolyzate solution from step 10) is spray-dried to a water content below 6.5%. 13. Method according to Ciaims 1 -11, characterized by the fact 10 11) that the protein hydrolyzate solution from step 10) is sterile filtered, 12) that the sterile filtrate from step 11) is concentrated to a concentration of between 40 and 60 total DS, 13) that the concentrate from step 12) is pasteurized, and 14) that the pasteurized concentrate from step 13) is spray-dried to a water content 15 of below 6.5%.

Inventors: ERIKSEN SVEND, HANSEN OLE REGNAR, KRISTENSEN SVEND ERIK, NIELSEN PER MUNK, ERIKSEN, SVEND, HANSEN, OLE, REGNAR, KRISTENSEN, SVEND, ERIK, NIELSEN, PER, MUNK
Applicants: NOVONORDISK AS, NOVO NORDISK A/S
Application year: 1992
Main classification: A23J3
IPC codes: A23J3, C12P21