A method for fractionating a high calcium-chelating casein phosphopeptide

Abstract

The application discloses a kind of high chelating calcium ability casein phosphopeptide fractionation methods, comprising the following steps: heating skim milk, stirring and adjusting the pH of skim milk, standing and precipitating, filtering separation, washing with distilled water, to obtain casein wet solid;Casein wet solid is added to distilled water, and the pH of enzyme system is adjusted;Add trypsin and hydrolyze, adjust the pH of enzyme system again, and separate to obtain enzyme solution;The pH of enzyme solution is adjusted, then calcium chloride and anhydrous ethanol are added, and CPPs-1 solid and ethanol supernatant 1 are collected by centrifugation;CPPs-2 solid and ethanol supernatant 2 are collected by centrifugation after adding calcium chloride to ethanol supernatant 1;CPPs-3 solid is collected by centrifugation after adding calcium chloride to ethanol supernatant 2, and high calcium chelating ability casein phosphopeptide is obtained by drying;The product prepared by the method has strong calcium chelating ability, high yield, high purity, good biological activity and is beneficial to human body absorption.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of casein phosphopeptide preparation, in particular to a method for fractionation of casein phosphopeptide with high calcium chelating capacity. BACKGROUND

[0002] Calcium, as a constant element, is one of the essential mineral elements for the survival of human beings. The content of calcium in the human body accounts for 1-2% of the total weight of the human body, and about 99% of the calcium exists in the bones and teeth, and about 1% of the calcium exists in the blood and soft tissues, which plays an important role in the regulation of life activities such as nerve regulation, muscle contraction, mitosis, blood coagulation, etc. Calcium deficiency has become one of the major nutritional problems in the world today, especially among children, pregnant women and the elderly. Long-term calcium deficiency can cause many symptoms of calcium deficiency, such as rickets, pathological fractures, osteoporosis and hypertension. The development of calcium supplement products includes three stages: the first stage of calcium preparation is mainly inorganic calcium salt, although the calcium content is high, but the solubility is poor, the utilization is low, and a large amount of gastric acid will be consumed in the stomach, which is not suitable for taking a large amount; the second stage of calcium preparation is mainly organic acid calcium, which has greatly improved the solubility and utilization rate, but still has certain toxic and side effects on the body; the third stage of calcium preparation includes amino acid chelated calcium and short-chain peptide chelated calcium, which has the advantages of good solubility, high calcium utilization rate, easy absorption by the body, etc.

[0003] The peptide calcium chelate with a bioactive structure is the third stage of calcium supplement products, and the calcium source includes casein phosphopeptide, ovalbumin phosphopeptide and other active polypeptide chelates. The bioactive peptide can combine with calcium ions to form a stable peptide calcium chelate, and the peptide calcium chelate has its own absorption and transport mechanism, which can be completely absorbed in the form of chelate, and can effectively release calcium ions. Casein phosphopeptide (CPPs) is a short peptide containing phosphoserine, which can prevent calcium from precipitating in the small intestine, significantly promote calcium absorption, and have a positive effect on calcium metabolism, with good calcium absorption rate, high bioavailability, etc. At the same time, it is found that the calcium binding sites of casein phosphopeptide are mainly located at the carbonyl group of Glu-2 and the phosphate groups of Ser-15, Ser-18 and Ser-18, and the hydrophobic interaction is the main driving force for calcium binding. The activity of casein phosphopeptide in promoting calcium absorption is mainly affected by the phosphate group on serine, the composition of amino acids and the molecular structure.

[0004] CN200310107554.3 provides a process for continuously preparing casein bioactive polypeptides by enzymatic hydrolysis and membrane filtration integration, which takes casein as raw material, and uses at least one protease to prepare bioactive polypeptides with multiple functions in a multi-stage enzyme-membrane reactor composed of an enzymatic hydrolysis tank and ultrafiltration and nanofiltration membranes with different molecular weight cut-off. However, the product is crude casein phosphopeptide, the length of the peptide chain is not uniform, the purity of the casein phosphopeptide is not enough, the casein phosphopeptide with high chelating ability cannot be enriched, the calcium chelating ability is low, and the utilization rate is low. SUMMARY

[0005] In order to solve the problems in the prior art, the purpose of the present application is to provide a method for fractionally separating casein phosphopeptide with high calcium chelating ability, by fractionally adding calcium chloride, separating and precipitating casein phosphopeptide, and enriching casein phosphopeptide containing high acidic amino acid components, so as to provide more high-affinity sites for calcium binding, thereby separating a casein phosphopeptide with high purity and high calcium chelating ability.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] A method for fractionally separating casein phosphopeptide with high calcium chelating ability, comprising the following steps:

[0008] (1) Preparing casein solid from skim milk: heating skim milk to 40-50℃, stirring and adjusting the pH of the skim milk, standing and precipitating, filtering and separating, and washing 2-3 times with distilled water to obtain casein wet solid with a protein content of more than 95%;

[0009] (2) Enzymatic hydrolysis and separation of casein wet solid: adding casein wet solid to distilled water, adjusting the pH of the enzymatic hydrolysis system, adding trypsin, stirring and enzymatic hydrolysis at 35-50℃ for 2-6h, adjusting the pH of the enzymatic hydrolysis system again, standing, centrifuging and separating to obtain an enzymatic hydrolysate;

[0010] (3) Fractional separation of casein phosphopeptide: adjusting the pH of the enzymatic hydrolysate obtained in step (2), then adding calcium chloride and anhydrous ethanol, standing and precipitating for 6-10h, centrifuging to collect CPPs-1 solid and ethanol supernatant 1; adding calcium chloride to the ethanol supernatant 1, standing and precipitating for 6-10h, centrifuging to collect CPPs-2 solid and ethanol supernatant 2; adding calcium chloride to the ethanol supernatant 2, standing and precipitating for 6-10h, centrifuging to collect CPPs-3 solid, and drying to obtain casein phosphopeptide with high calcium chelating ability.

[0011] Preferably, in step (1), the pH of the skim milk is adjusted to 4.5-4.8.

[0012] Preferably, in step (2), the ratio of casein wet solid to distilled water is 1:10-1:30 (m / v).

[0013] Preferably, in step (2), the pH of the enzymatic system is adjusted to 7-9 for the first time and to 4.5-4.8 for the second time.

[0014] Preferably, in step (2), the dosage of trypsin is 0.01-0.1 g / g casein wet solid.

[0015] Preferably, in step (2), the stirring speed is 60-200 r / min.

[0016] Preferably, in step (3), the pH of the enzymatic solution obtained in step (2) is adjusted to 6-8.

[0017] Preferably, in step (3), the dosage of calcium chloride is 0.01-1 wt%, 0.01-1 wt% and 0.01-1 wt% of the enzymatic solution, respectively.

[0018] Preferably, in step (3), the concentration of ethanol in the system is 40-70 wt%.

[0019] The present application also claims a high calcium-binding ability casein phosphopeptide prepared by the fractionation method for use in calcium-fortified dairy products, fruit juice beverages, protein beverages, instant beverages, sports foods, candies and nutrient supplements.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] 1) The present application provides a fractionation method for high calcium-binding ability casein phosphopeptide, which uses fractionation to obtain a casein phosphopeptide with high calcium-binding ability, high yield, high purity and good biological activity, which is beneficial to human body absorption and has simple process, simple equipment and convenient operation.

[0022] 2) The present application provides a fractionation method for high calcium-binding ability casein phosphopeptide, which separates and precipitates casein phosphopeptide by adding calcium chloride in stages, so as to enrich casein phosphopeptide containing high acidic amino acid components and provide more high-affinity sites for calcium binding, thereby obtaining a casein phosphopeptide with high purity and higher calcium-binding ability. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described in detail below with reference to examples. Of course, the specific examples described herein are only used to explain the present application and do not limit the present application.

[0024] Unless otherwise specified, the chemicals and materials in the present application are purchased through market channels or synthesized from raw materials purchased through market channels.

[0025] Example 1

[0026] A method for fractionating casein phosphopeptide with high calcium chelating capacity, comprising the following steps:

[0027] (1) Preparation of casein solid from skim milk: the skim milk is heated to 45℃, and the pH of the skim milk is adjusted to 4.6 while stirring, and then precipitated and separated by filtration, and washed with distilled water for 3 times to obtain casein wet solid with a protein content of more than 95%;

[0028] (2) Enzymatic separation of casein wet solid to obtain enzymatic hydrolysate: the casein wet solid is added to distilled water at a solid-liquid ratio of 1:20, the pH of the enzymatic system is adjusted to 8, and trypsin is added at a dosage of 0.03 g / g casein wet solid, and then enzymatic hydrolysis is carried out at 45℃ and 60 r / min for 4 h, and then the pH of the enzymatic system is adjusted to 4.6, and then precipitated, centrifuged and separated to obtain the enzymatic hydrolysate;

[0029] (3) Fractionation of casein phosphopeptide: the pH of the enzymatic hydrolysate obtained in step (2) is adjusted to 7, and then 0.1 wt% calcium chloride and anhydrous ethanol are added to the enzymatic hydrolysate to make the ethanol concentration 55 wt%, and then precipitated for 6 h, and then centrifuged to collect CPPs-1 solid and ethanol supernatant 1; 0.1 wt% calcium chloride is added to the ethanol supernatant 1, and then precipitated for 6 h, and then centrifuged to collect CPPs-2 solid and ethanol supernatant 2; 0.1 wt% calcium chloride is added to the ethanol supernatant 2, and then precipitated for 6 h, and then centrifuged to collect CPPs-3 solid, and then the solid is dissolved and dried by spray drying to obtain casein phosphopeptide with high calcium chelating capacity.

[0030] Example 2

[0031] A method for fractionating casein phosphopeptide with high calcium chelating capacity, comprising the following steps:

[0032] (1) Preparation of casein solid from skim milk: the skim milk is heated to 45℃, and the pH of the skim milk is adjusted to 4.6 while stirring, and then precipitated and separated by filtration, and washed with distilled water for 3 times to obtain casein wet solid with a protein content of more than 95%;

[0033] (2) Enzymatic separation of casein wet solid to obtain enzymatic hydrolysate: the casein wet solid is added to distilled water at a solid-liquid ratio of 1:20, the pH of the enzymatic system is adjusted to 8, and trypsin is added at a dosage of 0.03 g / g casein wet solid, and then enzymatic hydrolysis is carried out at 45℃ and 60 r / min for 4 h, and then the pH of the enzymatic system is adjusted to 4.6, and then precipitated, centrifuged and separated to obtain the enzymatic hydrolysate;

[0034] (3) Fractionation of CPPs: adjust the pH of the enzyme solution obtained in step (2) to 6, then add 0.1 wt% calcium chloride and absolute ethanol to the enzyme solution to make the ethanol concentration 55 wt%, stand for 6 h, centrifugal collection of CPPs-1 solid and ethanol supernatant 1; add 0.1 wt% calcium chloride to the ethanol supernatant 1, stand for 6-10 h, centrifugal collection of CPPs-2 solid and ethanol supernatant 2; add 0.12 wt% calcium chloride to the ethanol supernatant 2, stand for 6 h, centrifugal collection of CPPs-3 solid, and the solid is dissolved and dried by spray drying to obtain CPPs with high calcium chelating capacity.

[0035] Example 3

[0036] A method for fractionating CPPs with high calcium chelating capacity, comprising the following steps:

[0037] (1) Preparation of casein solid from skim milk: warm skim milk to 45°C, adjust the pH of the skim milk to 4.6 while stirring, stand for precipitation, filter and separate, and wash with distilled water 3 times to obtain casein wet solid with a protein content of more than 95%;

[0038] (2) Enzymatic separation of casein wet solid to obtain enzyme solution: add casein wet solid to distilled water at a solid-liquid ratio of 1:20, adjust the pH of the enzyme solution to 8, add trypsin at a dosage of 0.03 g / g casein wet solid, and enzymatically hydrolyze at 45°C and 60 r / min for 4 h, adjust the pH of the enzyme solution to 4.6, stand, centrifugal, and separate to obtain enzyme solution;

[0039] (3) Fractionation of CPPs: adjust the pH of the enzyme solution obtained in step (2) to 7, then add 0.08 wt% calcium chloride and absolute ethanol to the enzyme solution to make the ethanol concentration 55 wt%, stand for 6 h, centrifugal collection of CPPs-1 solid and ethanol supernatant 1; add 0.1 wt% calcium chloride to the ethanol supernatant 1, stand for 6-10 h, centrifugal collection of CPPs-2 solid and ethanol supernatant 2; add 0.12 wt% calcium chloride to the ethanol supernatant 2, stand for 6 h, centrifugal collection of CPPs-3 solid, and the solid is dissolved and dried by spray drying to obtain CPPs with high calcium chelating capacity.

[0040] Example 4

[0041] A method for fractionating CPPs with high calcium chelating capacity, comprising the following steps:

[0042] (1) Preparation of casein solid from skim milk: The skim milk was warmed to 45℃, and the pH of the skim milk was adjusted to 4.6 while stirring, and then the skim milk was allowed to stand and precipitate, and then filtered and separated, and then washed with distilled water three times to obtain casein wet solid with a protein content of greater than 95%;

[0043] (2) Enzymatic separation of casein wet solid to obtain an enzymatic hydrolysate: The casein wet solid was added to distilled water at a solid-to-liquid ratio of 1:20, and the pH of the enzymatic hydrolysis system was adjusted to 8, and then trypsin was added at a dosage of 0.03 g / g of casein wet solid, and then the enzymatic hydrolysis was performed at 45℃ and 60 r / min for 4 h, and then the pH of the enzymatic hydrolysis system was adjusted to 4.6, and then the system was allowed to stand and centrifuged to separate the enzymatic hydrolysate;

[0044] (3) Fractionation of casein phosphopeptide: The pH of the enzymatic hydrolysate obtained in step (2) was adjusted to 7, and then 0.05 wt% calcium chloride and anhydrous ethanol were added to the enzymatic hydrolysate to obtain an ethanol concentration of 55 wt%, and then the system was allowed to stand and precipitate for 6 h, and then centrifuged to collect the CPPs-1 solid and ethanol supernatant 1; 0.05 wt% calcium chloride was added to the ethanol supernatant 1, and then the system was allowed to stand and precipitate for 6 h, and then centrifuged to collect the CPPs-2 solid and ethanol supernatant 2; 0.2 wt% calcium chloride was added to the ethanol supernatant 2, and then the system was allowed to stand and precipitate for 6-10 h, and then centrifuged to collect the CPPs-3 solid, and then the solid was dissolved and dried by spray drying to obtain casein phosphopeptide with high calcium-chelating capacity.

[0045] Comparative Example 1

[0046] A method for separating casein phosphopeptide, comprising the following steps:

[0047] (1) Preparation of casein solid from skim milk: The skim milk was warmed to 45℃, and the pH of the skim milk was adjusted to 4.6 while stirring, and then the skim milk was allowed to stand and precipitate, and then filtered and separated, and then washed with distilled water three times to obtain casein wet solid with a protein content of greater than 95%;

[0048] (2) Enzymatic separation of casein wet solid to obtain an enzymatic hydrolysate: The casein wet solid was added to distilled water at a solid-to-liquid ratio of 1:20, and the pH of the enzymatic hydrolysis system was adjusted to 8, and then trypsin was added at a dosage of 0.03 g / g of casein wet solid, and then the enzymatic hydrolysis was performed at 45℃ and 60 r / min for 4 h, and then the pH of the enzymatic hydrolysis system was adjusted to 4.6, and then the system was allowed to stand and centrifuged to separate the enzymatic hydrolysate;

[0049] (3) Fractionation of casein phosphopeptide: The pH of the enzymatic hydrolysate obtained in step (2) was adjusted to 7, and then 0.05 wt% calcium chloride and anhydrous ethanol were added to the enzymatic hydrolysate to obtain an ethanol concentration of 55 wt%, and then the system was allowed to stand and precipitate for 6 h, and then centrifuged to collect the CPPs-1 solid and ethanol supernatant 1; 0.05 wt% calcium chloride was added to the ethanol supernatant 1, and then the system was allowed to stand and precipitate for 6 h, and then centrifuged to collect the CPPs-2 solid and ethanol supernatant 2; 0.2 wt% calcium chloride was added to the ethanol supernatant 2, and then the system was allowed to stand and precipitate for 6-10 h, and then centrifuged to collect the CPPs-3 solid, and then the solid was dissolved and dried by spray drying to obtain casein phosphopeptide with high calcium-chelating capacity.

[0050] Comparing the casein phosphopeptide prepared by example 1-4 and comparative example 1, it is found that the yield, purity and in vitro calcium binding capacity of the casein phosphopeptide prepared by the separation method provided by the present application are all significantly improved, and the specific data are shown in Table 1.

[0051] Table 1

[0052]

[0053]

[0054] The above merely provides the preferred embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art, according to the technical solution and the inventive concept of the present application, makes equivalent replacement or change within the technical range disclosed by the present application, should be covered within the protection scope of the present application.

Claims

1. A method for fractionating high calcium-binding casein phosphopeptides, characterized by, The method comprises the following steps: (1) preparing casein solid from skim milk: heating skim milk to 40-50℃, stirring and adjusting the pH of the skim milk, precipitating, separating by filtration, washing 2-3 times with distilled water, and obtaining casein wet solid with a protein content of more than 95%; (2) enzymatic separation of casein wet solid: adding casein wet solid to distilled water, adjusting the pH of the enzymatic system, adding trypsin, stirring and enzymatic hydrolysis at 35-50℃ for 2-6 h, adjusting the pH of the enzymatic system again, and obtaining an enzymatic hydrolysate by standing, centrifuging and separating; (3) fractionation of casein phosphopeptide: adjusting the pH of the enzymatic hydrolysate obtained in step (2), then adding calcium chloride and anhydrous ethanol, standing for 6-10 h, and centrifuging to collect CPPs-1 solid and ethanol supernatant 1; adding calcium chloride to the ethanol supernatant 1, standing for 6-10 h, and centrifuging to collect CPPs-2 solid and ethanol supernatant 2; adding calcium chloride to the ethanol supernatant 2, standing for 6-10 h, and centrifuging to collect CPPs-3 solid, and drying to obtain casein phosphopeptide with high calcium chelating capacity; In step (3), the pH of the enzymatic hydrolysate obtained in step (2) is adjusted to 6-8; In step (3), the amount of calcium chloride added is 0.05-0.1 wt%, 0.05-0.1 wt% and 0.1-0.2 wt% of the enzymatic hydrolysate, respectively; In step (3), the concentration of ethanol in the system is 40-70 wt%.

2. The fractionation method of claim 1, wherein, In step (1), the pH of the skim milk is adjusted to 4.5-4.

8.

3. The fractionation method of claim 1, wherein, In step (2), the ratio of casein wet solid to distilled water is 1:10-1:30 (m / v).

4. The fractionation method of claim 1, wherein, In step (2), the pH of the enzymatic system is adjusted to 7-9 for the first time, and to 4.5-4.8 for the second time.

5. The fractionation method of claim 1, wherein, In step (2), the amount of trypsin used is 0.01-0.1 g / g of casein wet solid.

6. The fractionation method of claim 1, wherein, In step (2), the stirring speed is 60-200 r / min.

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