Preparation method of casein peptone

Abstract

The invention relates to the technical field of food processing, in particular to a preparation method of casein peptone. According to the preparation method provided by the invention, firstly, raw milk is taken as an initial raw material to prepare a casein solution, then specific compound enzymes are used for enzymolysis, the compound enzymes comprise an enzyme A and an enzyme B, the enzyme A is selected from at least one of neutral protease, trypsin, papain and bromelain, and the enzyme B is selected from at least one of amino enzyme and carboxypeptidase. The complex enzyme enzymolysis product can be prepared into a high-quality casein peptone product through the steps of centrifugation and dehydration without acid precipitation. The method provided by the invention provides a new way for producing and processing casein peptone.

Description

Technical Field

[0001] The invention relates to the technical field of food processing, and in particular to a method for preparing casein peptone. Background Art

[0002] Casein peptone is a powder made from casein as raw material, which is hydrolyzed, neutralized, filtered, concentrated and spray-dried by protease. The difference between casein peptone and general peptone lies in the different enzymatic hydrolysis process and protein raw materials. Casein peptone has a higher degree of hydrolysis and a balanced molecular weight distribution. It has good physical properties such as light color, easy solubility, transparency, and no precipitation. It contains rich nitrogen sources and amino acids. Casein peptone is generally used as a nitrogen source for microbial test culture media internationally. It is widely used in microbiological testing, medical and health research, pharmaceutical industry production, and biological product manufacturing. For example, the culture, separation, proliferation, and identification of bacteria, as well as the preparation of culture media for bacterial biochemical characteristics tests such as sterile test culture media and anaerobic culture media. At present, commercially available casein peptone generally uses acid precipitation or membrane filtration casein powder as raw material to prepare casein peptone. Casein powder needs to be re-dissolved, which has problems such as poor solubility, affecting the subsequent enzymatic hydrolysis effect, and unstable casein peptone product quality. Chinese patent CN118388622A discloses a method for preparing casein peptone, which hydrolyzes casein with a single alkaline protease, and after enzymolysis, acid solution is used to adjust the pH for acid precipitation, and then diatomaceous earth is added for filtration and spray drying for powdering, which is complicated. Therefore, studying a new method for preparing casein peptone is of great significance to the development of casein peptone products. In view of this, the present invention is proposed. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a method for preparing casein peptone, which uses raw milk as raw material, and the enzymatic hydrolysis product does not need to be precipitated with acid, so that a high-quality casein peptone product can be obtained.

[0004] Specifically, the technical solution of the present invention is as follows: The invention provides a method for preparing casein peptone, comprising the steps of enzymolyzing a casein solution by using a composite enzyme, wherein the casein solution is prepared by taking raw cow milk as a raw material; the composite enzyme comprises an enzyme A and an enzyme B, the enzyme A is selected from at least one of neutral protease, trypsin, papain and bromelain, and the enzyme B is selected from at least one of amino enzyme and carboxypeptidase; and the enzymolysis product is subjected to centrifugation and dehydration to obtain the casein peptone.

[0005] The invention uses raw milk as the initial raw material, first prepares a casein solution, then uses the prepared casein solution as an enzymatic substrate, combines with a specific composite enzyme for enzymatic hydrolysis, and the enzymatic hydrolysis product does not need to be acid-precipitated, and can obtain a high-quality casein peptone product through centrifugation and dehydration.

[0006] Preferably, the casein solution is made from raw cow milk and is obtained by defatting, microfiltration and ultrafiltration concentration; the conditions for the ultrafiltration concentration include: ultrafiltration membrane pore size 30-100kDa, concentration temperature 10-20°C, transmembrane pressure 0.1-1MPa, concentration to 5-10%; the amount of washing water in the ultrafiltration concentration: the feed amount is 2:1-4:1.

[0007] Preferably, the degreasing temperature is 45-55°C.

[0008] Preferably, the microfiltration conditions include: microfiltration membrane pore size 0.1-0.15 μm, separation temperature 30-50° C., and transmembrane pressure 0.1-0.2 MPa.

[0009] Preferably, a sterilization step is included before microfiltration, and the sterilization parameters are: temperature 60-90°C, time 10-60s.

[0010] Preferably, the enzymatic hydrolysate is not subjected to acid addition, but directly precipitated by centrifugation and then subjected to falling film concentration and spray drying.

[0011] Preferably, the temperature of the centrifugal separation is 5-15°C and the rotation speed is 5000-15000 r / min.

[0012] Preferably, the falling film concentration conditions are: the first-effect falling film concentration temperature is 75-85°C during concentration; the second-effect falling film concentration temperature is 60-70°C; and the falling film concentration is performed until the total solid content is ≥20%.

[0013] Preferably, the spray drying conditions include: air inlet temperature 160-220°C, air outlet temperature 60-85°C.

[0014] Preferably, the degree of hydrolysis of the enzymatic hydrolysis product prepared by the preparation method of the present invention is ≥35%, and the amino acid nitrogen is ≥3.8%.

[0015] Beneficial effects:

[0016] The present invention provides a method for preparing casein peptone. The preparation method provided by the present invention first prepares a casein solution using raw milk as an initial raw material, and then uses a specific composite enzyme for enzymolysis, wherein the composite enzyme includes an enzyme A and an enzyme B, wherein the enzyme A is selected from at least one of neutral protease, trypsin, papain, and bromelain, and the enzyme B is selected from at least one of amino enzyme and carboxypeptidase. The composite enzyme enzymolysis product does not need to be acid-precipitated, and a high-quality casein peptone product can be obtained through centrifugation and dehydration steps. The method provided by the present invention provides a new approach for the production and processing of casein peptone. DETAILED DESCRIPTION

[0017] The invention provides a method for preparing casein peptone. The method comprises taking raw milk as a raw material, and preparing a casein solution through processes such as defatting, microfiltration and ultrafiltration. Taking the casein solution as a raw material, a specific protease combination is used, and the casein peptone is prepared through enzymolysis, centrifugal separation, concentration and drying.

[0018] In a more specific and preferred embodiment, the present invention provides a method for preparing casein peptone comprising the following steps: (1) Preparation of casein solution: Raw milk is skimmed, pasteurized, microfiltered, and ultrafiltered to obtain the solution. The skimming conditions are as follows: skimming temperature is 45-55°C. The pasteurization conditions are as follows: temperature is 60-90°C, time is 10-60s. The microfiltration conditions are as follows: microfiltration membrane pore size is 0.1-0.15 microns, separation temperature is 30-50°C, and transmembrane pressure is 0.1-0.2MPa. The ultrafiltration conditions are as follows: ultrafiltration membrane pore size is 30-100kDa, concentration temperature is 10-20°C, transmembrane pressure is 0.1-1MPa, and concentration is to 5-10%; the amount of washing water: feed amount is 2:1-4:1.

[0019] (2) Preparation of casein peptone, including: casein enzymatic hydrolysis, centrifugal separation, pH adjustment, falling film concentration and spray drying. Wherein, casein enzymatic hydrolysis: using the above casein solution as raw material, adding protease, and enzymatic hydrolysis under suitable temperature, pH and other conditions. The protease includes an endoprotease and an exoprotease combination, the endoprotease is one or two of neutral protease, bromelain, papain, and trypsin; the exoprotease is one or two of aminopeptidase and carboxypeptidase. The conditions for lactoproteinase hydrolysis are: substrate concentration of 5-10%, endoprotease addition of 1000-5000U / g protein, exoprotease addition of 200-2000U / g protein, hydrolysis at 45-60℃ for 2-5h, and then pasteurization to inactivate the enzyme, the enzyme inactivation temperature is 85-120℃, and the enzyme inactivation time is 30s-300s. The centrifugal conditions for centrifugal separation and precipitation are: centrifugal temperature of 5-15°C, centrifugal speed of 5000-15000r / min. The falling film concentration conditions are: the first effect falling film concentration temperature is 75-85°C during concentration; the second effect falling film concentration temperature is 60-70°C; the falling film concentration is concentrated to a total solid content of ≥20%. The spray drying conditions are: inlet air temperature: 160-220°C, outlet air temperature: 60-85°C.

[0020] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0022] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0023] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0024] In the following examples, the present invention uses the degree of hydrolysis and amino acid nitrogen as indicators to determine the optimal conditions for enzymatic hydrolysis.

[0025] In the following embodiments, the result detection method involved includes: Total nitrogen detection method: follow the method specified in Part IV Nitrogen Determination Method (General Rule 0704) of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0026] Amino acid nitrogen detection method: in accordance with the method specified in GB 20886.

[0027] Moisture detection method: in accordance with the method specified in GB 5009.3.

[0028] Ash content testing method: in accordance with the method specified in GB 5009.4.

[0029] Hydrolysis degree detection method: O-phthalaldehyde (OPA) method is used for determination. The specific steps are as follows: (1) Prepare OPA reagent: 7.62g sodium tetraborate and 200mg sodium dodecyl sulfate are dissolved in ultrapure water, 160mg OPA (dissolved in 4mL ethanol) and 176mg thiothreitol (dissolved in ultrapure water) are added, and the volume is finally adjusted to 200mL and stored in the dark. (2) Preparation of serine standard solution: 50mg L-serine is dissolved in 500mL ultrapure water (0.9516meqv / L). (3) Under light-proof conditions, add 3mL OPA reagent and 400μL of the test solution (serine standard solution or goat milk protein hydrolysate) to the test tube, mix for 5s, react at room temperature for 2min, and measure the absorbance at a wavelength of 340nm. Serine is used as the standard solution and ultrapure water is used as the blank control.

[0030] The calculation formula is: .

[0031] Where: DH is the degree of protein hydrolysis, %; h is the number of peptide bonds hydrolyzed in milk protein, meqv / g; h tot is the total number of peptide bonds in milk protein, meqv / g.

[0032] The calculation formula of h is: .

[0033] Where: serine-NH2 is the amount of serine-NH2 in each gram of protein, meqv / g; β and α are constants, which are 0.40 and 1.00 respectively.

[0034] The calculation formula of serine-NH2 is: .

[0035] Where: V is the sample volume, L; X is the sample mass, g; P is the protein content in the sample, %.

[0036] Example 1 This embodiment provides a method for preparing casein peptone.

[0037] The steps of the preparation method provided in this embodiment are: (1) Preparation of casein solution: Raw milk is skimmed, pasteurized, microfiltered, and ultrafiltered to obtain the solution. The skimming conditions are: skimming temperature is 45°C. The pasteurization conditions are: temperature is 60°C, time is 60s. The microfiltration conditions are: microfiltration membrane pore size is 0.1 micron, separation temperature is 30°C, and transmembrane pressure is 0.2MPa. The ultrafiltration conditions are: ultrafiltration membrane pore size is 50kDa, concentration temperature is 10°C, transmembrane pressure is 0.3MPa, and concentration is to 5%; the amount of washing water: feed amount is 2:1.

[0038] (2) Preparation of casein peptone, including: casein enzymatic hydrolysis, centrifugal separation, pH adjustment, falling film concentration and spray drying. Casein enzymatic hydrolysis: using the casein solution prepared in step (1) as raw material, adding protease, and performing enzymatic hydrolysis under appropriate temperature, pH and other conditions; the enzyme combination used for enzymatic hydrolysis is: neutral protease, trypsin, aminopeptidase, and the enzymatic hydrolysis conditions are: substrate concentration is 5%, the amount of neutral protease added is 1000U / g protein, the amount of trypsin added is 1000U / g protein, and the amount of aminopeptidase added is 500U / g protein. Hydrolysis is carried out at 45°C for 3h. After completion, pasteurization is performed to inactivate the enzyme at a temperature of 85°C and a time of 300s. Centrifugal separation and precipitation: the centrifugal temperature is 10°C and the centrifugal speed is 15000r / min. Falling film concentration: the temperature of the first effect falling film concentration is 75°C; the temperature of the second effect falling film concentration is 60°C; the falling film concentration is performed until the total solid content is ≥20%. Spray drying: air inlet temperature: 220℃, air outlet temperature: 85℃.

[0039] Example 2 This embodiment provides a method for preparing casein peptone.

[0040] The steps of the preparation method provided in this embodiment are: (1) Preparation of casein solution: Raw milk is obtained by defatting, pasteurizing, microfiltration, and ultrafiltration concentration. The defatting conditions are: defatting temperature is 55°C. The pasteurizing conditions are: temperature is 70°C, time is 45s. The microfiltration conditions are: microfiltration membrane pore size is 0.15 microns, separation temperature is 50°C, and transmembrane pressure is 0.15MPa. The ultrafiltration conditions are: ultrafiltration membrane pore size is 30kDa, concentration temperature is 20°C, transmembrane pressure is 0.2MPa, and concentration is to 7%; the amount of washing water: feed amount is 3:1.

[0041] (2) Preparation of casein peptone, including: casein enzymolysis, centrifugal separation, pH adjustment, falling film concentration and spray drying. Casein enzymolysis: using the casein solution prepared in step (1) as raw material, adding protease, and performing enzymolysis under appropriate temperature, pH and other conditions; the enzyme combination used for enzymolysis is: bromelain and aminopeptidase, and the enzymolysis conditions are: substrate concentration is 7%, bromelain addition amount is 3000U / g protein, aminopeptidase addition amount is 1000U / g protein, hydrolysis is carried out at 50℃ for 3h, and then pasteurization is performed to inactivate the enzyme at a temperature of 95℃ and an enzyme inactivation time of 200s. Centrifugal separation and precipitation: centrifugal temperature is 15℃, and centrifugal speed is 10000r / min. Falling film concentration: the first effect falling film concentration temperature is 80℃; the second effect falling film concentration temperature is 65℃; the falling film is concentrated to a total solid content of ≥20%. Spray drying: inlet air temperature: 260℃, outlet air temperature: 70℃.

[0042] Example 3 This embodiment provides a method for preparing casein peptone.

[0043] The steps of the preparation method provided in this embodiment are: (1) Preparation of casein solution: Raw milk is skimmed, pasteurized, microfiltered, and ultrafiltered to obtain the solution. The skimming conditions are: skimming temperature is 50°C. The pasteurization conditions are: temperature is 90°C, time is 10s. The microfiltration conditions are: microfiltration membrane pore size is 0.1 micron, separation temperature is 40°C, and transmembrane pressure is 0.1 MPa. The ultrafiltration conditions are: ultrafiltration membrane pore size is 100 kDa, concentration temperature is 15°C, transmembrane pressure is 0.8 MPa, and concentration is to 8%; the amount of washing water: feed amount is 3:1.

[0044] (2) Preparation of casein peptone, including: casein enzymatic hydrolysis, centrifugal separation, pH adjustment, falling film concentration and spray drying. Casein enzymatic hydrolysis: using the casein solution prepared in step (1) as raw material, adding protease, and performing enzymatic hydrolysis under appropriate temperature, pH and other conditions; the enzyme combination used for enzymatic hydrolysis is: papain and carboxypeptidase, and the enzymatic hydrolysis conditions are: substrate concentration is 8%, papain addition amount is 2000U / g protein, carboxypeptidase addition amount is 1000U / g protein, hydrolysis is carried out at 55℃ for 4h, and then pasteurization is performed to inactivate the enzyme, the enzyme inactivation temperature is 115℃, and the enzyme inactivation time is 200s. Centrifugal separation and precipitation: the centrifugal temperature is 5℃, and the centrifugal speed is 12000r / min. Falling film concentration: the first effect falling film concentration temperature is 80℃; the second effect falling film concentration temperature is 70℃; the falling film concentration is performed to a total solid content of ≥20%. Spray drying: inlet air temperature: 200℃, outlet air temperature: 75℃.

[0045] Example 4 This embodiment provides a method for preparing casein peptone.

[0046] The steps of the preparation method provided in this embodiment are: (1) Preparation of casein solution: Raw milk is obtained by defatting, pasteurizing, microfiltration, and ultrafiltration concentration. The defatting conditions are: defatting temperature is 45°C. The pasteurizing conditions are: temperature is 80°C, time is 30s. The microfiltration conditions are: microfiltration membrane pore size is 0.15 microns, separation temperature is 45°C, and transmembrane pressure is 0.15MPa. The ultrafiltration conditions are: ultrafiltration membrane pore size is 50kDa, concentration temperature is 20°C, transmembrane pressure is 1MPa, and concentration is to 9%; the amount of washing water: feed amount is 4:1.

[0047] (2) Preparation of casein peptone, including: casein enzymatic hydrolysis, centrifugal separation, pH adjustment, falling film concentration and spray drying. Casein enzymatic hydrolysis: using the casein solution prepared in step (1) as raw material, adding protease, and performing enzymatic hydrolysis under appropriate temperature, pH and other conditions; the enzyme combination used for enzymatic hydrolysis is: trypsin, aminopeptidase, carboxypeptidase, and the enzymatic hydrolysis conditions are: substrate concentration is 9%, trypsin addition amount is 5000U / g protein, aminopeptidase addition amount is 400U / g protein, carboxypeptidase addition amount is 400U / g protein, hydrolysis is carried out at 50℃ for 2h, and then pasteurization is performed to inactivate the enzyme at a temperature of 90℃ and an enzyme inactivation time of 200s. Centrifugal separation and precipitation: the centrifugal temperature is 10℃ and the centrifugal speed is 15000r / min. Falling film concentration: the first effect falling film concentration temperature is 85℃; the second effect falling film concentration temperature is 65℃; the falling film concentration is performed until the total solid content is ≥20%. Spray drying: air inlet temperature: 180℃, air outlet temperature: 65℃.

[0048] Example 5 This embodiment provides a method for preparing casein peptone.

[0049] The steps of the preparation method provided in this embodiment are: (1) Preparation of casein solution: Raw milk is skimmed, pasteurized, microfiltered, and ultrafiltered to obtain the solution. The skimming conditions are: skimming temperature is 50°C. The pasteurization conditions are: temperature is 75°C, time is 30s. The microfiltration conditions are: microfiltration membrane pore size is 0.1 micron, separation temperature is 35°C, and transmembrane pressure is 0.2MPa. The ultrafiltration conditions are: ultrafiltration membrane pore size is 100kDa, concentration temperature is 15°C, transmembrane pressure is 0.6MPa, and concentration is to 10%; the amount of washing water: feed amount is 3:1.

[0050] (2) Preparation of casein peptone, including: casein enzymolysis, centrifugal separation, pH adjustment, falling film concentration and spray drying. Casein enzymolysis: using the casein solution prepared in step (1) as raw material, adding protease, and performing enzymolysis under appropriate temperature, pH and other conditions; the enzyme combination used for enzymolysis is: bromelain, aminopeptidase, carboxypeptidase, and the enzymolysis conditions are: substrate concentration is 10%, bromelain addition amount is 1000U / g protein, aminopeptidase addition amount is 1000U / g protein, carboxypeptidase addition amount is 1000U / g protein, hydrolysis is carried out at 60℃ for 5h, and then pasteurization is performed to inactivate the enzyme at a temperature of 120℃ and an enzyme inactivation time of 30s. Centrifugal separation and precipitation: the centrifugal temperature is 15℃ and the centrifugal speed is 8000r / min. Falling film concentration: the first effect falling film concentration temperature is 80℃; the second effect falling film concentration temperature is 60℃; the falling film concentration is performed to a total solid content of ≥20%. Spray drying: air inlet temperature: 190℃, air outlet temperature: 80℃.

[0051] Comparative Example 1 This comparative example provides a method for preparing casein peptone, which is different from Example 5 in that the casein solution is reconstituted with commercially available casein powder.

[0052] Comparative Example 2 This comparative example provides a method for preparing casein peptone. Compared with Example 5, the only difference is that in step (1) the casein solution preparation process, there is no diafiltration process.

[0053] Comparative Example 3 This comparative example provides a method for preparing casein peptone. Compared with Example 1, the only difference is that the enzyme combination used in step (2) enzymatic hydrolysis is: neutral protease and trypsin, and no exonuclease is added; wherein, the amount of neutral protease added is 1000U / g protein, and the amount of trypsin added is 1000U / g protein.

[0054] Comparative Example 4 This comparative example provides a method for preparing casein peptone. Compared with Example 1, the only difference is that the enzyme used in the enzymatic hydrolysis in step (2) is aminopeptidase; the amount of aminopeptidase added is 500 U / g protein, and no endonuclease is added.

[0055] The test results of Examples 1-5 and Comparative Examples 1-4 are shown in Table 1 below.

[0056] Table 1

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing casein peptone, comprising the step of enzymolyzing a casein solution using a composite enzyme, characterized in that: The casein solution is prepared from raw milk; the complex enzyme comprises enzyme A and enzyme B, wherein the enzyme A is selected from at least one of neutral protease, trypsin, papain and bromelain, and the enzyme B is selected from at least one of amino enzyme and carboxypeptidase; the enzymatic hydrolysis product is centrifuged and dehydrated to obtain casein peptone.

2. The method for preparing casein peptone according to claim 1, wherein The casein solution is obtained from raw milk through defatting, microfiltration and ultrafiltration concentration; the conditions of the ultrafiltration concentration include: ultrafiltration membrane pore size 30-100kDa, concentration temperature 10-20°C, transmembrane pressure 0.1-1MPa, concentration to 5-10%; the amount of washing water in the ultrafiltration concentration: the feed amount is 2:1-4:

1.

3. The method for preparing casein peptone according to claim 2, characterized in that: The degreasing temperature is 45-55°C.

4. The method for preparing casein peptone according to claim 3, characterized in that: The microfiltration conditions include: microfiltration membrane pore size 0.1-0.15 μm, separation temperature 30-50° C., and transmembrane pressure 0.1-0.2 MPa.

5. The method for preparing casein peptone according to claim 4, characterized in that: A sterilization step is included before microfiltration, and the sterilization parameters are: temperature 60-90°C, time 10-60s.

6. The method for preparing casein peptone according to claim 1, characterized in that: The enzymatic hydrolysate is not subjected to acid addition, but is directly precipitated by centrifugation and then subjected to falling film concentration and spray drying.

7. The method for preparing casein peptone according to claim 6, characterized in that: The temperature of the centrifugal separation is 5-15°C, and the rotation speed is 5000-15000r / min.

8. The method for preparing casein peptone according to claim 7, characterized in that: The falling film concentration conditions are as follows: during concentration, the first effect falling film concentration temperature is 75-85° C.; the second effect falling film concentration temperature is 60-70° C.; and the falling film concentration is performed until the total solid content is ≥20%.

9. The method for preparing casein peptone according to claim 6, characterized in that: The spray drying conditions include: air inlet temperature of 160-220°C and air outlet temperature of 60-85°C.

10. The method for preparing casein peptone according to any one of claims 1 to 9, characterized in that: The degree of hydrolysis of the enzymatic hydrolysis product is ≥35%, and the amino acid nitrogen is ≥3.8%.

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