CN1225185C - Process for preparation of protein hydrolysate from milk protein - Google Patents

Abstract

The present invention provides a method for preparing protein hydrolysate from milk protein. The method has the steps that the milk protein is treated by fungal proteinase for 30 minutes to 2 hours under the conditions of pH between 7.5 and 8.5 and a temperature of 40(+/-)5 DEG C; the materials are heated for at least 3 minutes at 65 to 70 DEG C; clarified supernatant liquid is separated by an existing method, and the clarified liquid is dried, so the protein hydrolysate is obtained.

Description

Method for preparing protein hydrolyzate from milk protein

technical field

The present invention relates to a method for preparing protein hydrolyzate from milk protein using fungal protease. More specifically, the present invention provides a method for preparing a protein hydrolyzate from casein using a fungal protease obtained from Aspergillus sp.

Background technique

Casein is an excellent protein with a balanced amino acid composition. Thus, casein has limited functional properties. Casein makes up about 80% of milk protein. Casein is a phosphoprotein in which phosphorus is covalently bound to the polypeptide chain by serine ester bonds and casein is composed of heterogeneous α-casein, β-casein, κ-casein and other minor proteins. In the food industry, casein is used to prepare artificial meat, wine and beer clarifiers and protein-rich dairy products (Evans, E.W. Uses of milk proteins in formulated foods) Food protein development ( Developments in Food Proteins) 1st ed., B.J.F. Hudson, Applied Science Publishers, London, pp. 131-169). It is also used in biscuits, snacks and other food preparations to add functional and nutritional properties. Use casein hydrolyzate in food.

Acidic, alkaline or enzymatic methods can be used to open peptide bonds to reduce the molecular size of proteins. Acid and alkaline hydrolysis of proteins leads to a reduction in nutritional value due to the destruction and racemization of essential amino acids, producing toxic components such as lys-alanine.

Enzymatic hydrolysis selectively completes the hydrolysis of proteins without causing structural changes in the amino acids that make up proteins. Peptide profiles generated by enzymatic methods are well illustrated. Proteins hydrolyzed by enzymes retain better nutritional value than any conventional acid/alkaline hydrolyzed protein. Casein and whey proteins are well known to form bitter hydrolysates. Almost all development issues, especially the casein hydrolysis process revolve around the problem of bitterness. Hydrolysis by enzymes is a versatile tool for obtaining and modifying proteins. Hydrolysis is precise and unique to a particular protease-protein system. The degree of hydrolysis determines properties such as solubility, function and taste of the hydrolyzed protein. Protein hydrolyzates can be great additives to improve the functional properties and nutritional value of the final product. Most of the protein hydrolysates commercially available in India are acidic protein hydrolysates. Some disadvantages of acidic hydrolyzate are the formation of humins, the need for high temperature participation, brown color formation, high salt concentration, destruction of some essential amino acids and low yield. In the process of acid hydrolysis of vegetable protein, chlorohydrins are produced, and the liquid hydrolyzate needs to be steam-distilled under reduced pressure to remove chlorohydrins.

Due to the excellent nutritional value of the substrate casein, it is a protein widely used in the preparation of hydrolysates. Casein hydrolyzates have traditionally been used in therapeutic foods. Casein hydrolyzates are used in infant formulas and special nutritional formulations.

You can refer to the following publications, title: Enhancing the functionality of food proteins by enzymatic modification, Panyan, D. and Kilara, A.Trends Food Sci.Tech., 7(4), 120 -125, where enzymatic hydrolysis affects the emulsifying ability and hydrophobicity of the protein. The disadvantage of this method is that there is no definite degree of hydrolysis.

You can refer to the following publications, title: dietary enzymic hydrolysates of protein with reduced bitterness (dietary enzymic hydrolysates of protein with reduced bitterness) Clegg, K.M. and McMillan, A.D. (1974) J.Food Tech.9 (1), 21-29 , in which egg white and casein were studied as protein substrates and treated with papain, optionally chloroform followed by endopeptidase, and porcine kidney tissue was used as the source of endopeptidase. The hydrolyzate is relatively free of bitterness and yields continuous small peptides and more than 50% free amino acids. The disadvantage of this method is the low yield (60.6-86.5%). Furthermore, the method includes multiple steps, a two-enzyme system and the use of proteases of animal origin.

You can refer to the following publications, title: bitterness removal and nutritional value-added (debittering and nutritional upgrading of enzymic caseinhydrolysates) Cogan, V., Moshe, M., Mokady, S. (1981) J.Sci. Food.Agric.32(5), 459-466, wherein the casein hydrolyzate obtained by digestion with papain and pepsin was mainly carried out according to the method of Clegg and McMillan (1974). First, the casein solution (pH 7.2) was treated with papain at 40° C. at an enzyme concentration of 4.0%, and 0.2% toluene was added to prevent microbial growth. At the end of 18 hours of incubation, the pH was adjusted to 3.0 with hydrochloric acid, and further treatment with 0.5% pepsin was continued for 22 hours at 37°C.

Rhozyme enzymes were treated with specific enzyme concentrations at the following pH (adjusted with 1M NaOH) and temperature conditions: 50°C, pH 8.5, Rhozyme P-11 and Rhozyme 41 concentrates; 60°C, pH 7.5, Rhozyme P-53 concentrate; 60°C, pH 8.3, Rhozyme 62 concentrate.

The extent of proteolytic digestion was determined as previously described except that the digested samples were diluted 21-fold with 10% trichloroacetic acid solution. Enzymatic digestion was fully accomplished by incubating (37° C.) a 100 ml solution containing 298 mg casein and 6 mg protease for 15 hours. At the end of the incubation period, when the solutions were clear, their absorbance was estimated at 280 nm using appropriate protein and enzyme blanks.

Bitterness can be further reduced by treatment with "0.5g activated carbon/g hydrolyzate". The disadvantage of this method is that it involves multiple steps, sequential and continuous use of multiple enzymes and the use of multiple solvents to obtain the hydrolyzate, which ultimately leads to an increase in the cost of the process. In the process of producing casein hydrolyzate, additional activated carbon is added to reduce bitterness.

You can refer to the following publications, title: Casein hydrolysate produced using a formel-in-place membrane reactor (Casein hydrolysate produced using a formel-in-place membrane reactor) Chland, W.D., Cordle, C.T., Thomas, R.L.J.Food.Sci .60, 1349-1352, 1995. The reported degree of hydrolysis was between 4-51%. The hydrolysis time varied from 18-66 hours. The shortcoming of this method is that its hydrolysis process time is long.

Reference can be made to the following publications, title: Clegg, K.M., Smith, G. and Walker, A.L., 1974J, Food Tech. , 9(4), 425-431, 1974, wherein a laboratory method for the hydrolysis of casein is described. 12 kg of commercially available casein were suspended in 220 liters of water at pH 6.2 and digested with papain at 40°C for 8 hours. Then, pig kidney homogenate was treated at pH 7.8-8.0 for 24 hours, and chloroform was used instead of toluene as a preservative. The hydrolyzate was passed through a Russell separator to remove insoluble matter, and then pasteurized at 83-88°C for 3-5 minutes. The product is spray dried. The whole process takes 60 hours to complete. Bacteria counts were also satisfactory. The disadvantage of this method is that the hydrolysis process is too long. Also, different enzymes are used and the hydrolysis is multi-step, thus costly and time consuming.

Reference may be made to British patent 1595499 (patentee VEB Berlin-Chemie, 1981), where casein is hydrolyzed by boiling with dilute sulfuric acid and spray dried. The powder (100 g) was heated at reflux in 200 ml of methanol for 10 minutes and the insolubles were filtered off, washed twice with 50 ml of methanol and dried to yield 80 g of the hydrolyzate as a colorless, odorless, tasteless powder with all amino acids. A disadvantage of the present invention is that it uses mineral acids, which may cause racemization of amino acids and produce toxic components.

Reference can be made to Japanese Patent No. 5134465, Morinaga Milk Industry Co., Ltd. (1976), which describes the preparation of a concentrated liquid containing an enzymatically degraded component of casein. The disadvantage of this method is that the final product is in liquid form, which is difficult to handle.

Reference may be made to US Patent 5405637, Martinez, S.B., Leary, H.L.J., Nichols, D.J. (1995), which describes a milk protein hydrolyzate prepared by enzymatic hydrolysis and an infant formula prepared from said hydrolyzate. The hydrolyzate has reduced antigenicity and is prepared from a mixture of whey protein and casein. The degree of hydrolysis is between 4% and 10%. The disadvantage of this method is the low degree of hydrolysis.

Reference may be made to European Patent 0321603A1, Jost, R., Meister, N. and Monti, J.C. (1989) wherein hypoallergenic whey protein hydrolyzates are prepared by mixing the initial protein hydrolyzate at 80-100 °C, heat treatment at pH 6-8 for 3 to 10 minutes; cooling to 40-60 °C; second enzymatic hydrolysis (with trypsin, chymotrypsin, or trypsin/chymotrypsin/pancreatin mixture) . Then, heat the proteolytic enzyme to 75-85°C to inactivate it. Hypoallergenic protein hydrolyzates can be added to dietary preparations, baby food, etc. Disadvantages of this method are the use of very high temperatures and proteolytic enzymes of animal origin.

Can refer to U.S. Patent 4293571, Olofsson, M., Buhler, M., Wood, R. (1981), wherein described a kind of method for preparing purified protein hydrolyzate, a typical example of this method is: the dried egg white The aqueous solution is sterilized by steam injection at 115°C for 10-30 seconds; after cooling to 55°C, adjust the pH to 7.2 with calcium hydroxide (Ca(OH) ₂ ) and add 8% trypsin preparation; at 50-55°C, The hydrolysis was allowed to proceed for 5 hours. Then, the pH is adjusted to 6.7 with phosphoric acid (H ₃ PO ₄ ), followed by heat treatment, e.g., 98°C, for 30 minutes to denature the protein, followed by ultrafiltration to remove the denatured protein from the heat-treated hydrolyzate , leaving the filtrate constituting the purified protein hydrolyzate. The hydrolyzate is suitable for baby food, therapeutic food and rehabilitation food. Disadvantages of this method are the use of very high temperatures and proteolytic enzymes of animal origin.

Reference may be made to Swiss patent 570121, Mueller, H., which describes a process for the preparation of a light-colored, high-glutamate condiment hydrolyzate from milk proteins. For this method, whey containing an average lactose content of about 4.5% is fermented under aerobic conditions with a yeast strain having a high glutamic acid content. After concentration, the fermentation product is hydrolyzed with hydrochloric acid (HCl) and can then be dried and the hydrolyzate neutralized, filtered and optionally concentrated using conventional methods. Concentrating the fermented broth by ultrafiltration keeps the lactoglobulins and whey proteins in the concentrate, which increases the yield after hydrolysis by 15-25%. If ultrafiltration is performed prior to fermentation, the retained whey protein is added to the previously concentrated yeast prior to hydrolysis. The light-colored hydrolyzate has no yeasty smell. Disadvantages of this method are the types of organisms and enzymes involved. Fermentation is also different.

Reference may be made to U.S. Patent 3,778,514, Olson, F.C., (1973), which describes a nutritional product of whey protein and collagen hydrolyzate, the description of which covers whey concentrate and collagen hydrolyzate (as Various mixtures obtained from steam refining of fats (tank float waste). In one embodiment, an artificial milk product is obtained by mixing 9 parts lactose reduced whey concentrate (40% solids) with 1 part collagen hydrolyzate (40% solids). The product contains 33.6% protein, 42.0% lactose, 19.1% minerals and 4.2% moisture and its amino acid profile is between that of soy milk and casein.

Contents of the invention

The main purpose of the present invention is to provide a method for preparing protein hydrolyzate from milk protein by using fungal protease.

Another object of the present invention is to provide a process for the preparation of protein hydrolyzates from whey/casein using fungal proteases obtained from Aspergillus.

Another object of the present invention is to provide a protein hydrolyzate with a high degree of hydrolysis at a temperature of 40±5°C.

A further object of the present invention is to provide a method for preparing protein hydrolyzates soluble in a wide range of pH (ie pH 2-11).

Therefore, the present invention provides a method for preparing protein hydrolyzate from milk, the method comprising: using fungal protease to treat milk protein for 30 minutes to 2 hours at pH 7.5-8.5 at a temperature of 40±5°C; Protein hydrolyzate is obtained by heating at 70°C for at least 3 minutes, separating the clarified supernatant by known methods and drying the clarified liquid.

Therefore, the present invention provides a method for preparing protein hydrolyzate from milk protein, the method comprising: using fungal protease to treat milk protein for 30 minutes to 2 hours at pH 7.5-8.5 at a temperature of 40±5°C; Heating at 70° C. for at least 3 minutes, separating the clear supernatant by known methods and drying the clear liquid, thereby obtaining a protein hydrolyzate.

In one embodiment of the present invention, said fungal protease is obtained from Aspergillus.

In another embodiment of the present invention, the Aspergillus fungus is selected from the group consisting of A. flavus, A. japonicus, A. niger and A. awamori.

In another embodiment of the invention, the milk protein is selected from whey and casein.

In another embodiment of the invention, to maximize the activity of the fungal protease, the pH is maintained between 7.6 and 8.0.

In a further embodiment of the present invention, the working temperature during the hydrolysis of protein is 40-45°C.

In a further embodiment of the invention, the hydrolysis process is carried out for 1.5 to 2 hours.

In one embodiment of the invention, drying is performed by freeze drying, spray drying and drum drying.

In another embodiment of the present invention, the protein hydrolyzate has a perceived threshold bitterness of 0.4-0.5%.

In another embodiment of the invention, a high yield of protein hydrolyzate with a degree of hydrolysis of 50% is obtained from the raw materials used.

In another embodiment of the present invention, the obtained protein hydrolyzate is milky white.

In another embodiment of the invention, 95% protein hydrolyzate is obtained.

In another embodiment of the present invention, the protein hydrolyzate contains 11.5-12.5% nitrogen and 4.5-5% ash.

In one embodiment of the invention, the protein hydrolyzate is soluble in water in all pH ranges.

In another embodiment of the invention, the amino acid composition of the protein hydrolyzate is similar to that of the starting material.

In another embodiment of the invention, the protein hydrolyzate retains all the nutritional value of the starting material.

In a further embodiment of the invention, enzyme activity is destroyed and terminated by simultaneously adjusting the pH and temperature to the point where the protease enzyme system is most sensitive to thermal damage and placing the slurry at a high temperature of 65-70°C for 3-5 minutes. Hydrolysis process.

The invention also provides a milky white protein hydrolyzate.

In one embodiment of the invention, 95% protein hydrolyzate is obtained.

In another embodiment of the present invention, the protein hydrolyzate contains 11.5-12.5% nitrogen and 4.5-5% ash.

In another embodiment of the present invention, the protein hydrolyzate has a perceived threshold bitterness of 0.4-0.5%.

In another embodiment of the present invention, the protein hydrolyzate is soluble in water in all pH ranges.

In another embodiment of the invention, the amino acid composition of the protein hydrolyzate is similar to that of the starting material.

In another embodiment of the invention, the protein hydrolyzate retains all the nutritional value of the starting material.

In one embodiment of the invention, a protein hydrolyzate having a degree of hydrolysis of 50% is obtained.

Detailed ways

The present invention comprises the following process steps:

casein

Casein is a phosphoprotein containing 0.7-0.9% phosphorus covalently attached to polyphenolic chains by serine ester bonds. The structure also contains calcium and citrate. Acid casein is obtained by precipitation of milk with an acid such as HCl, sulfuric acid or lactic acid. Sweet casein is obtained due to the action of rennet. Low viscosity casein obtained by treating milk protein with proteolytic enzymes and/or acid. Regardless of the kind of casein produced, the basic operations in producing casein are the same. These operations include the settling of the curd, its washing, pressing and drying. In many countries, large quantities of casein are obtained through the process of acid hydrolysis. Casein for protein hydrolyzates must be edible and bacteriologically pure.

enzyme

The enzyme used was a fungal protease with an enzyme activity of 30,000 units/gram.

Detection of degree of hydrolysis

The trinitrobenzenesulfonic acid (TNBS) method is an accurate, reproducible, and commonly used method for determining the degree of hydrolysis of food protein hydrolysates. The protein hydrolyzate was dissolved/dispersed in hot 1% sodium dodecyl sulfate to a concentration of 0.25-2.5×10 ^-3 amino equivalents/L. A sample of the solution (0.25 ml) was mixed with 2 ml of 0.2125 M sodium phosphate buffer (pH 8.2) and 2 ml of 0.1% trinitrobenzenesulfonic acid, and then incubated at 50° C. in the dark for 60 minutes. The reaction was stopped by adding 4 ml of 0.100N HCl. Absorbance values were read at 340 nm using a 1.5 mM L-leucine solution as a standard. The detected leucine amino equivalents were converted to the degree of hydrolysis by a standard curve for each specific protein substrate (see: Jens Adler-Nissen, J. Agr. Food Chem. Vol. 27, No. 6, 1979).

The casein was dispersed in water with a suitable solvent to solute ratio and the pH of the dispersion was adjusted to 7.6-8.0 with 15N sodium hydroxide. Stirring was continued for a few minutes using a mechanical stirrer, then the temperature was raised to 40-45°C. At this stage, 1% fungal protease based on the protein content of the raw material is added and stirring is continued for 1.5-2 hours. At the end of the above period, the temperature of the slurry was raised to 65-70°C for 3-5 minutes. The slurry was then cooled to room temperature and the insoluble material in the dispersion was removed by centrifugation. The clarified protein hydrolyzate was spray dried to obtain protein hydrolyzate.

The following examples are provided by way of illustration of the invention and therefore, should not be construed as limiting the scope of the invention.

Example 1

200 g of casein were dispersed in 2 L of water and the pH of the dispersion was adjusted to 7.8 with 15% sodium hydroxide (NaOH) while maintaining the slurry at 40°C. To the slurry was added fungal protease dissolved in water. After 1.5 hours, the temperature of the slurry was raised to 65°C for 3 minutes before it was cooled and centrifuged. The clear supernatant was spray dried. The degree of hydrolysis of the product was 45% and the yield was 90%.

Example 2

1 kg of casein was dispersed in 10 L of water and the pH was adjusted to 7.8 with 15% NaOH while maintaining the slurry at 40°C. To the slurry was added fungal protease dissolved in water. After 2 hours, the temperature of the slurry was raised to 65°C for 3 minutes before it was cooled and centrifuged. The clear supernatant was spray dried. The degree of hydrolysis of the product was 45% and the yield was 95%.

Example 3

5 kg of casein was dispersed in 50 L of water and the pH was adjusted to 7.8-8.0 with 15% NaOH while maintaining the slurry at 45°C. To the slurry was added fungal protease dissolved in water. After 2 hours, the temperature of the slurry was raised to 70°C for 5 minutes before it was cooled and centrifuged. The clear supernatant was spray dried. The degree of hydrolysis of the product was 50% and the yield was 93%.

Example 4

5 kg of casein was dispersed in 50 L of water and the pH of the dispersion was adjusted to 8.0 with 15% NaOH while maintaining the slurry at 45°C. To the slurry was added fungal protease dissolved in water. After 2 hours, the temperature of the slurry was raised to 70°C for 5 minutes, then cooled and centrifuged. The clear supernatant was spray dried.

The degree of hydrolysis of the product was 50% and the yield was 95%. The obtained casein hydrolyzate was milky white, had a milky taste, and had a yield of 90% (based on protein). The product contains 11.5-12.5% nitrogen, 4.5-5% ash content and a degree of hydrolysis of 45-50%. The product is highly soluble in water in all pH ranges. Protein hydrolyzates have a perceived threshold bitterness of 0.4-0.5%. It retains all the nutritional value of the starting material.

Main advantage of the present invention:

1. The ratio of water to protein is relatively low, and there is only one-step pH adjustment, and the salt content is minimal.

2. The fungal protease derived from the fungus used is commercially available, and the hydrolysis time of the protease is very short.

3. Use one enzyme and obtain a high degree of hydrolysis (45-50%) in a short time.

4. The yield is very high at 90-95%, and has a relatively high nitrogen content of 11.5-12.5%.

5. Since the hydrolysis process is completed in 1.5-2 hours, there is no need to add antibacterial agents to prevent the growth of microorganisms.

6. The nutritional value of the starting material is maintained with minimal loss of essential amino acids.

Claims (17)

1, a kind ofly prepare the method for protolysate by lactoprotein, described method comprises: at pH7.5-8.5, under 40 ± 5 ℃ of temperature, utilize available from the fungal proteinase of aspergillus fungi and handled lactoprotein 30 minutes to 2 hours; 65-70 ℃ of heating at least 3 minutes, utilize the supernatant of known method separating clarifying and, obtain protolysate thus the liquid dried of clarifying.

2, the method for claim 1, wherein said aspergillus fungi is selected from aspergillus flavus, aspergillus japonicus, aspergillus niger and aspergillus awamori.

3, the method for claim 1, wherein said lactoprotein is selected from whey and casein.

4, the method for claim 1 wherein for making the active maximum of fungal proteinase, remains on the pH value between the 7.6-8.0.

5, the method for claim 1, wherein operating temperature is 40-45 ℃ in the hydrolytic process of protein.

6, the method for claim 1, wherein said hydrolytic process were carried out 1.5 to 2 hours.

7, the method for claim 1 is wherein carried out described drying by freeze drying, spray-drying or drum drying.

8, the method for claim 1, wherein said protolysate have the appreciable threshold value bitter taste of 0.4-0.5%.

9, the method for claim 1 wherein obtains the protolysate of 45-50% hydrolysis degree from the raw material that uses.

10, the method for claim 1, wherein the described protolysate of Huo Deing is a milky.

11, the method for claim 1 has wherein obtained 95% protolysate.

12, the method for claim 1, wherein said protolysate contain the nitrogen of 11.5-12.5% and the ash content of 4.5-5%.

13, the method for claim 1, wherein said protolysate is water-soluble in all pH scopes.

14, the method for claim 1 wherein came destructive enzyme active and stop hydrolytic process in 3-5 minute by regulating pH and temperature simultaneously and slurries being placed under the 65-70 ℃ of high temperature.

15, a kind of available from the protolysate of method according to claim 1.

16, protolysate as claimed in claim 15, this protolysate are milky.

17, protolysate as claimed in claim 15, this protolysate contain the nitrogen of 11.5-12.5% and the ash content of 4.5-5%.