CN116987170A - Beta-casein and preparation method and application thereof - Google Patents
Beta-casein and preparation method and application thereof Download PDFInfo
- Publication number
- CN116987170A CN116987170A CN202310960181.1A CN202310960181A CN116987170A CN 116987170 A CN116987170 A CN 116987170A CN 202310960181 A CN202310960181 A CN 202310960181A CN 116987170 A CN116987170 A CN 116987170A
- Authority
- CN
- China
- Prior art keywords
- casein
- beta
- solution
- sodium
- preparation
- Prior art date
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Links
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- 102000011632 Caseins Human genes 0.000 title claims abstract description 95
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 6
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- 238000000034 method Methods 0.000 claims description 50
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 20
- 239000011780 sodium chloride Substances 0.000 claims description 15
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 13
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 13
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 13
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 13
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- 239000001509 sodium citrate Substances 0.000 claims description 13
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- 238000011068 loading method Methods 0.000 claims description 12
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- 229910052791 calcium Inorganic materials 0.000 abstract description 25
- BZQFBWGGLXLEPQ-REOHCLBHSA-N phosphoserine Chemical compound OC(=O)[C@@H](N)COP(O)(O)=O BZQFBWGGLXLEPQ-REOHCLBHSA-N 0.000 abstract description 14
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- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
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- VZBXBGHHDNVFGP-UHFFFAOYSA-N barium;ethanol Chemical compound [Ba].CCO VZBXBGHHDNVFGP-UHFFFAOYSA-N 0.000 description 2
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- 229940055695 pancreatin Drugs 0.000 description 2
- 229940111202 pepsin Drugs 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
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- 239000000758 substrate Substances 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
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- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4732—Casein
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
- A23C9/1526—Amino acids; Peptides; Protein hydrolysates; Nucleic acids; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/20—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey
- A23J1/202—Casein or caseinates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/04—Animal proteins
- A23J3/08—Dairy proteins
- A23J3/10—Casein
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Nutrition Science (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the technical field of beta-casein, in particular to beta-casein and a preparation method and application thereof. The preparation method comprises degreasing camel milk; the whey part after degreasing is subjected to ammonium sulfate precipitation treatment; mixing the ammonium sulfate precipitate into an ion solution for treatment, centrifuging, and drying supernatant to obtain freeze-dried powder; and (3) carrying out ion exchange chromatography treatment on the freeze-dried powder, and collecting elution peaks to obtain the beta-casein. The beta-casein prepared by the preparation method of the beta-casein provided by the invention has high purity, and the phosphorus content and the phosphoserine content are both obviously higher than those of the comparative example, so that the beta-casein has the application prospect of promoting calcium absorption and providing calcium bioavailability.
Description
Technical Field
The invention relates to the technical field of beta-casein, in particular to beta-casein and a preparation method and application thereof.
Background
Casein is a phosphorylated protein produced by mammary gland acinar epithelial cells. Can be precipitated from skim milk under the conditions of isoelectric point of protein and proper temperature. It is derived from various animal milks and is one of the important constituent components of milk proteins. Milk is a product secreted by the mammary gland of mammals and is a complex component biological fluid. The components in the milk are mixed in a proper proportion, so that the milk has high nutritional value, and meanwhile, the milk has irreplaceable functions on human and animal growth regulation and immune protection, so that the milk is the best nutritional substance for newborn mammals.
The proportion of casein in milk is about 30% -35% of total protein, mainly beta-casein and kappa-casein, wherein the proportion of beta-casein is larger. Beta-casein consists of 226 amino acid residues and has a molecular weight of 25382Da. Each molecule can bind 5 phosphates, with binding sites concentrated predominantly at peptide chains 16-33. There are 6 levels of phosphorylation, from 0 (beta-casein-0P) to 5 (beta-casein-5P). There are studies comparing the higher content of beta-casein combined with two phosphates (beta-casein-2P) and the four phosphates (beta-casein-4P) in beta-casein, and the results show that both account for about 70% of the total beta-casein, each about 35%. The beta-casein obtained in the above examples and comparative examples was subjected to enzymatic hydrolysis using pancreatin, trypsin, pepsin, etc., to obtain a characteristic peptide fragment containing phosphoserine: the core structure of the phosphoserine peptide (CPP) is-Ser (P) -Ser (P) -Ser (P) -Glu-Glu, and because the phosphoserine peptide contains clustered phosphoserine residue [ -Ser (P) ], calcium ions can be chelated to form a soluble phosphate complex, and calcium is effectively prevented from forming calcium phosphate precipitation under neutral or alkaline conditions, so that the absorption of calcium is promoted and the bioavailability of the calcium is provided.
Thus, when the content of phosphoserine in beta-casein is higher, the content of serine sulfate peptide obtained by enzymolysis is also higher, which is beneficial to promoting calcium absorption. It is thus clear that it is of great importance how beta-casein with hyperphosphorylated serine can be obtained.
Disclosure of Invention
In view of this, the present invention provides a method for preparing beta-casein of hyperphosphorylated serine and uses thereof.
The invention provides a preparation method of beta-casein, which comprises the following steps:
taking camel milk for degreasing treatment;
the whey part after degreasing is subjected to ammonium sulfate precipitation treatment;
mixing the ammonium sulfate precipitate into an ion solution, treating for 60-180 min at 2-8 ℃, centrifuging, leaving supernatant, and freeze-drying to obtain freeze-dried powder;
and (3) carrying out ion exchange chromatography treatment on the freeze-dried powder, and collecting elution peaks to obtain the beta-casein.
In the preparation method, the ionic solution comprises sodium citrate, sodium acetate, potassium hydrogen phthalate, potassium dihydrogen phosphate and sodium chloride, and the pH value of the ionic solution is 5.6-6.5.
In the preparation method, the ionic solution further comprises ethanol.
In the preparation method, the ionic solution comprises 1-5 mM sodium citrate, 1-5 mM sodium acetate, 1-5 mM potassium hydrogen phthalate, 1-12.5 mM potassium dihydrogen phosphate and 1-20 mM sodium chloride.
In the preparation method, the ionic solution is a solution containing 5mM sodium citrate, 5mM sodium acetate, 5mM potassium hydrogen phthalate, 12.5mM potassium dihydrogen phosphate, and 20mM sodium chloride, wherein the pH=6.2.
In the preparation method, the ionic solution comprises 1-5 mM sodium citrate, 1-5 mM sodium acetate, 1-5 mM potassium hydrogen phthalate, 1-12.5 mM potassium dihydrogen phosphate, 1-20 mM sodium chloride and 10-20% v/m ethanol.
In the preparation method, the ionic solution is a ph=6.2 solution containing 5mM sodium citrate, 5mM sodium acetate, 5mM potassium hydrogen phthalate, 10mM potassium dihydrogen phosphate, 18mM sodium chloride and 15% v/m ethanol.
In the preparation method, the step of ion exchange chromatography includes:
preparing the freeze-dried powder into a sample loading liquid;
loading the loading liquid into a chromatographic column filled with DEAE-Sepharose Fast Flow resin, mixing mobile phase A liquid and mobile phase B liquid for gradient elution, and collecting separation components by using UV280nm as detection wavelength; wherein the mobile phase A liquid is a solution containing 20mmol/LTris and 3.3mol/L urea, and the pH value is 9.5; the mobile phase B solution is a solution of 20mmol/LTris,3.3mol/L urea and 0.3mol/LNaCl, and the pH is 9.5;
beta-casein was identified and obtained from the isolated fractions.
One of the purposes of the present invention is to provide the beta-casein produced by the process.
The invention provides an application of the beta-casein prepared by the preparation method in preparing a product for promoting bone growth and development.
Compared with the prior art, the invention has at least one of the following beneficial effects:
the beta-casein prepared by the preparation method of the beta-casein provided by the invention has high yield and high purity. And the phosphorus content and the phosphoserine content of the beta-casein prepared by the method are both obviously higher than those of the comparative example, and the beta-casein has application prospects of promoting calcium absorption and bioavailability.
In addition, the freeze-dried powder of the beta-casein prepared by the method has excellent fluidity and can be used as raw materials of formula milk powder and formula milk tablets through bulk density, tapping density, hausner ratio and Carr's index analysis.
The Caco-2 monolayer cell experiment proves that the beta-casein prepared by the method has excellent calcium transport promoting property, can promote the proliferation of osteoblasts, and has activating and promoting effects on the enzyme activity of osteoblast alkaline phosphatase. Therefore, the beta-casein prepared by the method provided by the invention can improve the alkaline phosphatase activity of osteoblasts, promote the osteoblasts to produce mineralized crystals, and is beneficial to bone growth and development.
Drawings
FIG. 1 is a SDS-PAGE of examples 1 to 5 and comparative examples 1 to 3 (lanes 1 to 8).
FIG. 2 is a graph showing the results of Western-Blot identification provided in examples 1 to 5 and comparative examples 1 to 3 (lanes 1 to 8).
FIG. 3 is a graph showing the CPP content results obtained by enzymatic hydrolysis of beta-casein produced in examples 1 to 5 and comparative examples 1 to 3.
FIG. 4 is a graph showing the results of phosphorus content in the beta-casein produced in examples 1 to 5 and comparative examples 1 to 3.
FIG. 5 shows Hausner ratios of the lyophilized powders of beta-casein obtained in examples 1 to 5 and comparative examples 1 to 3.
FIG. 6 shows the Carr's index of the lyophilized powder of beta-casein obtained in examples 1 to 5 and comparative examples 1 to 3.
FIG. 7 shows the solubility of 30% ethanol solutions of the lyophilized powders of beta-casein obtained in examples 1 to 5 and comparative examples 1 to 3.
FIG. 8 is a graph showing the results of calcium transport amounts of beta-casein produced in examples 1 to 5 and comparative examples 1 to 3 in Caco-2 monolayer cells.
FIG. 9 is a graph showing the proliferation effect of beta-casein produced in examples 1 to 5 and comparative examples 1 to 3 on osteoblasts.
FIG. 10 is a graph showing the results of ALP activity detection on osteoblasts by beta-casein produced in examples 1 to 5 and comparative examples 1 to 3.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. The reagents not specifically and individually described in the present invention are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.
All numbers expressing quantities, percentages, and other values used in the present invention are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Preparation of beta-casein
One example 1 discloses the preparation of beta-casein as follows:
(1) Degreasing
10mL of camel milk is taken, the sample is centrifuged for 30min at the temperature of 4 ℃ and 10400g, the sample is divided into three layers, wherein the milk fat part is at the upper layer, the whey part is at the middle layer, the milk grain part is at the bottom, and the middle layer is taken.
(2) Ammonium sulfate precipitation
Taking the intermediate layer solution, adding ammonium sulfate until the concentration of the solution reaches 35%, standing at 4 ℃ for about 4 hours, and centrifuging to precipitate for later use.
(3) Solution treatment
Mixing the precipitate obtained in the ammonium sulfate precipitation step into a solution with pH=6.2 containing 5mM sodium citrate, 5mM sodium acetate, 5mM potassium hydrogen phthalate, 12.5mM potassium dihydrogen phosphate and 20mM sodium chloride, treating at 4deg.C for 120min, centrifuging at 100000g for 60min, collecting supernatant, and lyophilizing to obtain lyophilized powder.
(4) Ion exchange chromatography
Ion exchange chromatography is carried out on the freeze-dried powder obtained by solution treatment, and the specific steps are as follows:
1) Preparation of sample solution
The above-mentioned freeze-dried powder was dissolved in 50mL of equilibration buffer (containing 20mM Tris and 3.3M urea), pH was adjusted to 9.5 with 1mol/LNaOH, intermittently stirred in a water bath at 20℃for 0.5 to 1 hour, after complete dissolution, centrifuged at 4℃for 30min at 10000g, the supernatant was filtered with a 0.45 μm filter membrane, and the filtrate was collected for use at 4 ℃.
2) Preparation of ion exchange chromatography column
Pretreatment of DEAE-Sepharose Fast Flow ion exchange resin: 20mL of DEAE-Sepharose Fast Flow is taken, 50mL of ultrapure water is poured, the mixture is gently stirred by a glass rod, after the resin is precipitated, the broken rubber and the supernatant are poured out, and the step is repeated for 5 to 6 times until the supernatant is free of broken rubber.
And (3) column loading: the column was vertically fixed to an iron frame, and the pretreated DEAE-Sepharose Fast Flow resin was poured into the column along a glass rod, the column had a specification of 16mm X60 cm and a bed height of 55cm. The gel was allowed to slowly and uniformly settle into a column. After the chromatographic column is connected to the protein purifier, the column is flushed with water at a pressure of 0.3MPa to improve the column efficiency.
Balance: after column packing, the column was equilibrated overnight with 20mmol/L Tris-HCl containing 3.3mol/L urea, pH=9.5, for the next day.
3) Loading and elution
Before loading, the solution A is used: the operation is automatically zeroed when 20mmol/L Tris-HCl containing 3.3mol/L urea and having pH=9.5 is eluted to A280<0.3, and the sample is loaded after the baseline is stable. Mobile phase a liquid: pH 9.5, 20mmol/LTris,3.3mol/L urea; and (2) liquid B: pH 9.5, 20mmol/L Tris,3.3mol/L urea, 0.3mol/L LNaCl. Elution procedure: and (3) linearly gradient eluting with 0-100% B solution. The flow rate was 1.5mL/min. Column temperature 4 ℃, detection wavelength: UV280nm.
4) Collecting
The different fractions were collected separately based on the absorbance at 280nm (A280 nm) of the AKTApurifier 100 protein chromatograph.
5) Regeneration of ion exchange columns
The column is washed by 2mol/LNaCl until the A280 of the effluent liquid is less than 0.02, and then the NaCl in the column is washed by ultrapure water until the conductivity is constant, thus finishing the regeneration of the ion exchange column.
6) Confirmation of the respective Casein peaks
The fractions obtained were detected by 12% SDS-PAGE and compared with the pre-stained protein Marker to determine the peak of interest and the relative percentage. And (3) dialyzing and desalting the target eluting peak collecting liquid by using a dialysis bag with the interception amount of 14kDa at 4 ℃, replacing the dialyzate for several times in the middle, and dialyzing until the conductivity is constant. And finally, freeze-drying the different components, and storing the different components in a refrigerator at the temperature of minus 20 ℃.
One example 2 discloses the preparation of beta-casein as follows:
(1) Degreasing
10mL of camel milk is taken, the sample is centrifuged for 30min at the temperature of 4 ℃ and 10400g, the sample is divided into three layers, wherein the milk fat part is at the upper layer, the whey part is at the middle layer, the milk grain part is at the bottom, and the middle layer is taken.
(2) Ammonium sulfate precipitation
Taking the intermediate layer solution, adding ammonium sulfate until the concentration of the solution reaches 32%, standing at 4 ℃ for about 4 hours, and centrifuging to precipitate for later use.
(3) Solution treatment
The procedure is as in example 1.
(4) Ion exchange chromatography
The procedure is as in example 1.
One example 3 discloses the preparation of beta-casein as follows:
(1) Degreasing
The procedure is as in example 1.
(2) Ammonium sulfate precipitation
The procedure is as in example 1.
(3) Solution treatment
Mixing the precipitate obtained in the ammonium sulfate precipitation step into a solution with pH=6.2 containing 5mM sodium citrate, 5mM sodium acetate, 5mM potassium hydrogen phthalate, 12.5mM potassium dihydrogen phosphate and 20mM sodium chloride, treating at 4deg.C for 120min, centrifuging at 100000g for 60min, collecting supernatant, and lyophilizing to obtain lyophilized powder.
(4) Ion exchange chromatography
The procedure is as in example 1.
One example 4 discloses the preparation of beta-casein as follows:
(1) Degreasing
The procedure is as in example 1.
(2) Ammonium sulfate precipitation
The procedure is as in example 1.
(3) Solution treatment
Mixing the precipitate obtained in the ammonium sulfate precipitation step into a solution with pH=6.2 containing 5mM sodium citrate, 5mM sodium acetate, 5mM potassium hydrogen phthalate, 10mM potassium dihydrogen phosphate, 18mM sodium chloride and 15% v/m ethanol, treating at 4deg.C for 120min, centrifuging at 100000g for 60min, collecting supernatant, and lyophilizing to obtain lyophilized powder.
(4) Ion exchange chromatography
The procedure is as in example 1.
One example 5 discloses the preparation of beta-casein as follows:
(1) Degreasing
The procedure is as in example 1.
(2) Ammonium sulfate precipitation
The procedure is as in example 1.
(3) Solution treatment
The procedure is as in example 1. A step of
(4) Ion exchange chromatography
The preparation of the loading solution and ion exchange chromatography was the same as in example 1.
Before loading, eluting with solution A until A280 is less than 0.3, automatically zeroing, and loading after the base line is stable. Mobile phase a liquid: pH 9.5, 20mmol/LTris,3.3mol/L urea; and (2) liquid B: pH 9.5, 20mmol/LTris,3.3mol/L urea, 0.3mol/LNaCl. Elution procedure: after 0-50% of the B solution is eluted linearly, 50% of the B solution is eluted for 2 column volumes, and 50-100% of the B solution is eluted linearly continuously. The flow rate was 1.5mL/min. Column temperature 4 ℃, detection wavelength: UV280nm.
One of the preparation processes of beta-casein disclosed in comparative example 1 is as follows:
(1) Degreasing
The procedure is as in example 1.
(2) Ammonium sulfate precipitation
The procedure is as in example 1.
(3) Ion exchange chromatography
The precipitate obtained from ammonium sulphate was subjected to ion exchange chromatography, the procedure being as in example 1.
One of the preparation processes of beta-casein disclosed in comparative example 2 is as follows:
(1) Degreasing
The procedure is as in example 1.
(2) Isoelectric precipitation
Taking 50mL of a degreasing part, regulating the pH value by using 1mol/LHCl to enable the pH value to be respectively 4.3 to precipitate casein, centrifuging the sample at 4 ℃ for 30min and 40000g after 1h, collecting casein, washing for three times, discarding supernatant, and collecting precipitate.
(3) Ion exchange chromatography
Ion exchange chromatography was performed on the precipitate obtained by isoelectric precipitation of casein, and the procedure was as in example 1.
One of the preparation processes of beta-casein disclosed in comparative example 3 is as follows:
(1) Degreasing
The procedure is as in example 1.
(2) Precipitation of casein by salting-out
Respectively 1.08mol/LCaCl 2 0.47mL of the solution was added to 8mL of the defatted portion obtained in step 1 at 25℃and pH 4.3, and vortexed for 20min. Samples were centrifuged at 4℃and 40000g for 30min, casein was collected, washed three times, the supernatant was discarded, and the pellet was collected.
(3) Ion exchange chromatography
Ion exchange chromatography was performed on the precipitate obtained by isoelectric precipitation of casein, and the procedure was as in example 1.
Protein concentration determination
The protein concentrations obtained in each of the above examples 1 to 5 and comparative examples 1 to 3 were measured by the Bradford method, and bovine serum albumin was used as a standard protein.
(1) 0.10g Bovine Serum Albumin (BSA) was weighed by a balance, dissolved in deionized water, and after sufficient dissolution, a 10mL standard protein solution was prepared, the concentration of the solution was 10mg/mL.
(2) 100, 80, 60, 40, 20. Mu.L of BSA solution was taken with a pipette, placed in a 1.5mL EP tube, and 900, 920, 940, 960, 980. Mu.L of deionized water was added, respectively, to prepare 1mL of solution, and the solution was mixed uniformly by shaking. A set of BSA solutions were obtained at concentrations of 1.0,0.8,0.6,0.4,0.2mg/mL, respectively.
(3) The pipette was used to remove 100. Mu.L of the freshly prepared set of BSA solutions, and the solutions were mixed well by shaking with a 1.5mL EP tube, each with 900. Mu.L deionized water. A set of BSA solutions were obtained at concentrations of 0.10,0.08,0.06,0.04,0.02mg/mL, respectively. A further 1mL of deionized water (BSA solution concentration of 0 mg/mL) was taken for control.
(4) mu.L of the prepared BSA solution was removed by a pipette, and the solution was added dropwise to the well plate, and 200. Mu.L of Coomassie Brilliant Blue (CBB) was added, respectively. After 10min of standing, the optical density values of the set of BSA solutions at 595nm were measured with an enzyme-labeled instrument. The protein concentration of the sample was calculated from the standard curve.
The protein concentration in the intermediate layer obtained in the degreasing step in examples 1 to 5 and comparative examples 1 to 3 was measured by the above-mentioned steps and the amount of protein therein was calculated, the amount of protein in the lyophilized product of the peak fraction of beta-casein collected by ion exchange chromatography was measured, the a562nm value of the sample to be measured was measured, and the beta-casein yield = the amount of beta-casein obtained after chromatography/the amount of protein after degreasing was calculated, and the obtained result is shown in fig. 1. As a result, it was found that the yields of beta-casein produced in examples 1 to 5 were higher, respectively, and the yield of example 4 was the highest.
TABLE 1
Identification of beta-Casein by Western-Blot method
1. SDS-PAGE electrophoresis
Electrophoresis gel was prepared and subjected to SDS-PAGE, and the preparation method of SDS-PAGE was described in the technical Manual of protein. The concentration of the poured separating gel is 12%, the concentration of the concentrated gel is 5%, and the gel dyeing adopts a Coomassie brilliant blue dyeing method. As shown in FIG. 1, lanes 1 to 8 are the beta-casein electrophoresis strips of examples 1 to 5 and comparative examples 1 to 3, respectively, which are consistent with the target size and have no impurity strips, indicating that the purity of the prepared beta-casein is higher.
2. Transfer film and color development
(1) After electrophoresis, the gel strips were cut to appropriate size and equilibrated with transfer buffer for 5min×3 times.
(2) Film treatment: filter paper and PVDF membrane with the same size as the adhesive tape are cut in advance, firstly soaked in 100% methanol for 1min, and then immersed in the transfer buffer solution for 10min.
(3) Transferring: the membrane transferring device sequentially places a cathode carbon plate, 24 layers of filter paper, gel, PVDF membrane, 24 layers of filter paper and an anode carbon plate from bottom to top, the filter paper, the gel and the PVDF membrane are precisely aligned, air bubbles are removed in each step, a weight of 500g is pressed, and redundant liquid on the carbon plate is sucked to be dry. The transfer conditions were constant current 100mA for 25min.
(4) Color development: to detect the success of transfer, ponceau staining can be used. The membrane is put into PBS for washing once, then put into ponceau dyeing working solution, and is shake-dyed for 5min at room temperature, and a large amount of water is used for washing the membrane until the water becomes clear and colorless protein strips are clear.
3. Closure of a film
The membrane was washed three times with 0.01mol/LPBS (0.1 mol/LNa2HPO4/NaH2PO4 buffer, pH 7.4) for 5min each to wash out SDS on the transfer membrane as much as possible, preventing the influence of the subsequent antibody binding. Blocking with 0.01mol/L PBS of 5% skimmed milk powder at room temperature for 2 hr, washing with 0.01mol/LPBS three times for 5min each time, and incubating with antibody.
4. Incubation with primary antibody
The rabbit anti-human beta-casein polyclonal antibody was diluted with PBS buffer at a volume ratio of 1:400 and incubated overnight at room temperature. Excess unbound antibody was then washed with 0.01mol/LPBS 3 times for 5min each at room temperature.
5. Second antibody incubation
The horseradish peroxidase-labeled goat anti-rabbit antibody and PBS buffer solution are diluted according to the volume ratio of 1:3000, and are shaken for 2 hours at room temperature. Followed by washing 3 times with 0.01mol/LPBS for 5min each.
6. Protein detection
According to the enhanced HRP-DAB substrate chromogenic kit operation, 1mL of 1 XHRP reaction buffer is added into a test tube, and then 50 μL of reagent A, 50 μL of reagent B and 50 μL of reagent C are sequentially added and mixed uniformly. And (3) adding the prepared color development liquid on a PVDF film, developing the color for about 20 minutes in a dark place until a strip appears, and putting the PVDF film into double distilled water to terminate the reaction.
7. Analysis of results
Scanning the PVDF film: the scanner converts the negative film into a picture, and the picture is scanned and stored as a computer file.
The results of Western-Blot identification of the beta-casein obtained in each of examples 1 to 5 and comparative examples 1 to 3 are shown in FIG. 2. The figure shows that the protein can be combined with rabbit anti-human beta-casein antibody specifically, i.e. the protein is proved to be beta-casein, and has no impurity band, thus indicating that the prepared beta-casein has higher purity.
Detection of related indexes of beta-casein
The experiment adopts pancreatin, trypsin, pepsin and the like to carry out enzymolysis on the beta-casein obtained in the example and the comparative example, and can obtain a characteristic peptide segment containing phosphoserine: the core structure of the phosphoserine peptide (CPP) is-Ser (P) -Ser (P) -Ser (P) -Glu-Glu, and because the phosphoserine peptide contains clustered phosphoserine residue [ -Ser (P) ], calcium ions can be chelated to form a soluble phosphate complex, and calcium is effectively prevented from forming calcium phosphate precipitation under neutral or alkaline conditions, so that the absorption of calcium is promoted and the bioavailability of the calcium is provided. Therefore, in order to evaluate the phosphoserine content in the β -casein produced in each of examples 1 to 5 and comparative examples 1 to 3 described above, it is necessary to enzymatically convert it into a polypeptide, and the phosphoserine content in the β -casein can be indirectly detected by detecting the Content of Phosphoserine Peptide (CPP) therein.
1. Enzymolysis
Beta-casein prepared in each of examples 1 to 5 and comparative examples 1 to 3 was precisely weighed, a 50mM Tris-HCl buffer solution with pH=8 was used to prepare a 2g/L solution, 12g of trypsin (CAS No. 9002-07-7, T9201, > 7500BAEE U/mg, sigma-Aldrich) was added in a mass ratio of enzyme/beta-casein of 1:15, acetic acid (final concentration of 0.3%) and acetonitrile (final concentration of 25%) were added at the same time, and after thoroughly vortexing and mixing, the mixture was placed in a constant temperature incubator at 37℃for incubation for 6 hours, and after concentration, freeze-drying (i.e., casein hydrolysate) was obtained.
2. CPP detection
CPP content detected by national standard barium ethanol method GB31617-2014 Casein phosphopeptide as food safety national Standard food nutrient supplement: 1.5g of the dried casein hydrolysate was accurately weighed and placed in a 50mL centrifuge tube, 15mL deionized water was added, and the mixture was completely dissolved by shaking. The pH of the sample was adjusted to 4.6 with 1.87M hydrochloric acid and centrifuged at 600 rpm at 4℃for 30min. The supernatant was taken in a 50mL centrifuge tube which was previously dried and of constant weight, the pH was adjusted to 6.8 with 2M sodium hydroxide, 1.5mL of barium chloride solution was added, vortexed for 10s, and then added with absolute ethanol previously refrigerated to 4℃to 50mL, shaken well and refrigerated overnight (at least 12 h) at 4 ℃. Centrifuging at 6000rpm at 4deg.C for 30min, removing supernatant to obtain precipitate, placing the precipitate in a 60deg.C oven for 1 hr, and oven drying at 105deg.C to constant weight, and calculating CPP content in casein hydrolysate according to the method provided by national standard barium ethanol method GB 31617-2014. As a result, as shown in FIG. 3, the CPP content obtained in examples 1 to 5 was significantly higher than that obtained in comparative examples 1 to 3.
3. Phosphorus content detection
The phosphorus content is measured by referring to GB5413.22-2010 method of measuring phosphorus in infant food and dairy products which are national standards for food safety. The method mainly comprises the following steps: precisely weighing 0.5g of the beta-casein prepared in each of examples 1 to 5 and comparative examples 1 to 3, and placing in a triangular flask; adding a proper amount of nitric acid, and placing the triangular flask on an electric heating plate to heat and digest the sample; adding proper amount of perchloric acid to continue heating and digestion; if the digestion liquid is not clear and transparent, nitric acid is added to continue digestion until the digestion liquid becomes colorless or light yellow. 10mL of digestion solution diluted by a certain multiple is taken, a small amount of deionized water is added, 2 drops of dinitrophenol indicator are added, the mixture is firstly adjusted to yellow by sodium hydroxide solution, then adjusted to colorless by nitric acid solution, and finally adjusted to slight yellow by sodium hydroxide solution. The phosphorus content of the sample solution was calculated from the standard curve with the blank solution zeroed. As a result, as shown in FIG. 4, the phosphorus contents obtained in examples 1 to 5 were significantly higher than those in comparative examples 1 to 3.
4. Bulk density, tap density, hausner ratio, carr's index
The determination of bulk, tap density, hausner ratio, carr's index is referred to published methods (Chen Chengjun et al, 2013). An appropriate amount of the beta-casein samples prepared in examples 1 to 5 and comparative examples 1 to 3 were precisely weighed, and a 100mL measuring cylinder was filled with a funnel at a constant speed, the sample volume was precisely recorded, the operation was repeated 6 times, and the bulk density (. Rho.b) was calculated from the weight and the volume. The measuring cylinder containing the CPP sample (sample with measured bulk) was then allowed to fall freely from 2cm from the bench height, repeated 300 times, the powder volume was recorded, the measurement was repeated 6 times, and the tap density (ρt) was calculated. The Hausner ratio=ρt/ρb and the Carr's index= (ρt- ρb)/ρb are calculated according to the formula using the bulk density (ρb) and tap density (ρt) results.
Hausner ratio is used to indicate the compressibility and flowability of the powder, which reflects the degree of compressibility, agglomeration and flowability of the powder. Powder is generally considered to have good flowability and filling properties when the Hausner ratio is less than 1.2, and can be used for production, while powder is poor in flowability and filling properties when the Hausner ratio is greater than 1.5, and when the Hausner ratio is close to 1.6, powder is difficult to flow out freely from the container. When the Hausner ratio approaches 2.0, the powder does not flow (Wu Fuyu, 2014). It was found that the flowability of the powder was at an intermediate level when the Carr's index was between 18% and 21%, a Carr's index greater than 21% indicated a gradual deterioration in the flowability of the powder, a Carr's index less than 18% indicated a gradual improvement in the flowability of the powder, and a Carr's index between 5% and 10% indicated an excellent flowability of the powder. As shown in FIGS. 5 to 6, the fluidity of the beta-casein obtained in examples 1 to 5 was significantly better than that of comparative examples 1 to 3.
5. Dissolution index
According to the process for producing calcium tablets, the dissolution index of beta-casein prepared in each of examples 1 to 5 and comparative examples 1 to 3 in 30% aqueous ethanol was examined. Refer to published literature methods and make modifications as the case may be (Wu et al 2015). Solubility is one of the important properties of proteins, directly affecting the application value of the proteins. The calcium tablets were prepared from the beta-casein-bound calcium sources and auxiliary materials prepared in examples 1 to 5 and comparative examples 1 to 3, respectively, and were analyzed for plasmid standards. As a result, as shown in FIG. 7, the 30% aqueous ethanol solutions of beta-casein obtained in examples 1 to 5 were significantly better in solubility than those of comparative examples 1 to 3.
Cell experiment
1. Establishment of Caco-2 monolayer cell model
Selecting 30-60 generation Caco-2 cells (Yubo organism) to build small intestineEpithelial monolayer cell model. The formula of the cell complete culture medium comprises 77% MEM,20% fetal bovine serum, 1% diabody, 1% Glutamax,1% nonessential amino acid and 1% sodium pyruvate (the proportions are all volume fractions), and the culture environment is 37 ℃ and the volume fraction is 5% CO 2 Concentration. When the cells grew to 80%, at 2X 10 per ml 5 Density of the cells were seeded into the cells in the upper layer of a 12-well transwell transfer plate, the volume of the upper layer medium was 0.5mL, and 1.5mL of complete medium was supplemented in the lower layer. Culturing for 21d, and changing liquid every 2d until the cells form complete membranes. The integrity of the cell monolayer is determined by measurement of the transmembrane resistance when the resistance exceeds 500 Ω/cm 2 When a monolayer film was used for the transport experiments.
2. Determination of calcium transport
The method reported earlier in the laboratory is adopted to measure the calcium transport amount, and the method is slightly modified according to the specific experimental condition. After 21d of Caco-2 cell culture, the medium was discarded, and the monolayer cells were immediately washed twice with HBSS (free of calcium and magnesium) at 37℃and then transferred to a new 12-well plate for co-incubation with HBSS in an incubator for 30min.
Sample preparation: 100. Mu.g of the lyophilized powder of beta-casein obtained in each of examples 1 to 5 and comparative examples 1 to 3 was mixed with 300. Mu.g of calcium chloride, and 300. Mu.g of calcium chloride was used as a control group.
Samples were added to the upper layer of the transwell chamber, and at various time points (20, 40, 60, 90, 120 min) 0.5mL of lhbss buffer was collected from the lower layer chamber to measure calcium ion concentration while 0.5mL of fresh HBSS buffer was replenished to maintain a constant volume. The calcium concentration at each time point was measured using flame atomic absorption. The calcium content of each well was calculated as follows: bn=1.5×an+0.5×Σak; wherein Bn is the calcium content in 1.5mL HBSS buffer solution in the lower layer of each hole at each time point, and μg/hole; an is the calcium ion concentration of HBSS at the lower layer of each hole at different time points, mug/mL; n is a natural number 1, 2, 3, 4, 5, and represents 20, 40, 60, 90, 120min respectively.
The specific flow of measuring the content of the calcium ions by using the flame atomic absorption spectrometry is the same as that of the flame atomic absorption spectrometry in GB5009.92-2016 national food safety Standard for measuring the calcium in food, and an aqua regia digestion method is selected to digest a sample, wherein the volume ratio of the aqua regia to the sample is 1:1.
As a result, as shown in FIG. 8, the calcium transport amount of each group of cells was increased continuously within 0 to 90 minutes, and after 90 minutes, the calcium transport was substantially completed. And the beta-casein prepared in examples 1-5 is mixed with calcium chloride for transportation, and the final transportation quantity is obviously higher than that of a comparison example and a comparison group, which shows that the beta-casein provided by the invention can obviously promote calcium transportation.
4. Proliferation of osteoblasts
Taking MC3T3-El cells (SNL-518, shang En Bio) in logarithmic growth phase of 3 rd generation at 10 per ml 4 The density of each was inoculated in 96-well plates, 6 wells per group, 100. Mu.L of complete culture broth per well. After the cells were completely adhered, the medium was removed, and 100. Mu.L of complete medium containing 10. Mu.g/mL of the samples (. Beta. -casein prepared in examples 1 to 5 and comparative examples 1 to 3, respectively) was added, respectively.
After 72h of incubation, 20. Mu.LMTT (0.5 mg/mL) was added to each well, incubation was stopped at 37℃in the absence of light for 4h, the supernatant was carefully aspirated off, 150. Mu.L of dimethyl sulfoxide was added to each well, and shaking was performed on a shaker for 5min at low speed to allow the crystals to dissolve well. Absorbance values for each well were measured at 570nm on an enzyme-linked immunosorbent assay. The same conditioned culture was performed with the same cell wells without added sample as the control group. Cell proliferation rate = (experimental group OD-control group OD)/control group od×100% was calculated. As a result, as shown in FIG. 9, the proliferation effect of the beta-casein provided in examples 1 to 5 on osteoblasts was remarkable, whereas comparative examples 1 and 3 had no remarkable effect.
5. Alkaline phosphatase (ALP) assay
Taking MC3T3-El cells in logarithmic growth phase at 2X 10 per ml 6 The density of each was inoculated in 6-well plates, 4-well per group, and 2mL of complete culture broth per well. The next day the medium was carefully removed and 2mL of induction medium containing 10. Mu.g/mL of the samples (the. Beta. -casein prepared in examples 1-5 and comparative examples 1-3, respectively) was added, respectively, and the solution was changed every other day. On day 7, the medium was carefully removed and the cells were washed 3 times with 4℃pre-chilled PBS, and after harvesting the cells with a cell scraper, the cells were lysed using a sonicator (power 300W, in ice water bath, 1 time every 3-5 s ultrasonic, 4 times at intervals, 30s at intervals). The BCA method determines protein content, and the kit is used to determine alkaline phosphatase content after quantification.
As a result, as shown in FIG. 10, after the beta-casein provided in examples 1 to 5 was allowed to act on osteoblasts, the alkaline phosphatase activity was significantly higher than that in comparative example 2, whereas the alkaline phosphatase activity was not detected in comparative examples 1 and 3. Alkaline phosphatase is a key enzyme for the formation of extracellular matrix of osteoblast, and can hydrolyze naturally-occurring phosphate-containing substrate to release free phosphate ions, and participate in mineralization and deposition of extracellular matrix. Therefore, the beta-casein prepared by the method provided by the invention can improve the alkaline phosphatase activity of osteoblasts, promote the osteoblasts to produce mineralized crystals, and is beneficial to bone growth and development.
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.
Claims (10)
1. A method for preparing beta-casein, comprising the steps of:
taking camel milk for degreasing treatment;
the whey part after degreasing is subjected to ammonium sulfate precipitation treatment;
mixing the ammonium sulfate precipitate into an ion solution, treating for 60-180 min at 2-8 ℃, centrifuging, leaving supernatant, and freeze-drying to obtain freeze-dried powder;
and (3) carrying out ion exchange chromatography treatment on the freeze-dried powder, and collecting elution peaks to obtain the beta-casein.
2. The method according to claim 1, wherein the ionic solution comprises sodium citrate, sodium acetate, potassium hydrogen phthalate, potassium dihydrogen phosphate, and sodium chloride, and the ionic solution has a pH of 5.6 to 6.5.
3. The method of claim 2, wherein the ionic solution further comprises ethanol.
4. The method according to claim 2, wherein the ionic solution comprises 1 to 5mM sodium citrate, 1 to 5mM sodium acetate, 1 to 5mM potassium hydrogen phthalate, 1 to 12.5mM potassium dihydrogen phosphate, and 1 to 20mM sodium chloride.
5. The method according to claim 4, wherein the ionic solution is a solution having ph=6.2 containing 5mM sodium citrate, 5mM sodium acetate, 5mM potassium hydrogen phthalate, 12.5mM potassium dihydrogen phosphate, and 20mM sodium chloride.
6. The preparation method according to claim 2, wherein the ionic solution comprises 1 to 5mM sodium citrate, 1 to 5mM sodium acetate, 1 to 5mM potassium hydrogen phthalate, 1 to 12.5mM potassium dihydrogen phosphate, 1 to 20mM sodium chloride and 10 to 20% v/m ethanol.
7. The method of claim 6, wherein the ionic solution is a ph=6.2 solution containing 5mM sodium citrate, 5mM sodium acetate, 5mM potassium hydrogen phthalate, 10mM potassium dihydrogen phosphate, 18mM sodium chloride and 15% v/m ethanol.
8. The method of claim 1, wherein the step of ion exchange chromatography comprises:
preparing the freeze-dried powder into a sample loading liquid;
loading the loading liquid into a chromatographic column filled with DEAE-Sepharose Fast Flow resin, mixing mobile phase A liquid and mobile phase B liquid for gradient elution, and collecting separation components by using UV280nm as detection wavelength; wherein the mobile phase A solution is a solution containing 20mmol/L Tris and 3.3mol/L urea, and the pH value is 9.5; the mobile phase B solution is a solution of 20mmol/L Tris,3.3mol/L urea and 0.3mol/L NaCl, and the pH is 9.5;
beta-casein was identified and obtained from the isolated fractions.
9. The beta-casein produced by the production process according to any one of claims 1 to 8.
10. Use of the beta-casein produced by the production method according to any one of claims 1 to 8 for producing a product for promoting bone growth and development.
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| US6121421A (en) * | 1998-08-21 | 2000-09-19 | Abbott Laboratories | Methods for isolating recombinant β-casein |
| CN102675448A (en) * | 2012-06-08 | 2012-09-19 | 广西大学 | Method for isolating casein components in milk |
| CN105254714A (en) * | 2015-10-16 | 2016-01-20 | 中国农业大学 | Casein-derived antioxidant peptide and preparation method thereof |
| CN110973345A (en) * | 2019-12-26 | 2020-04-10 | 吉林大学 | Method for continuously separating and preparing functional lactoprotein in colostrum |
| CN112625110A (en) * | 2019-09-24 | 2021-04-09 | 泰州医药城国科化物生物医药科技有限公司 | Purification preparation method of beta-casein in cow milk |
| CN116217705A (en) * | 2023-03-27 | 2023-06-06 | 新疆大学 | Separation and purification method and detection method for alpha-lactalbumin in camel milk |
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| US6121421A (en) * | 1998-08-21 | 2000-09-19 | Abbott Laboratories | Methods for isolating recombinant β-casein |
| CN102675448A (en) * | 2012-06-08 | 2012-09-19 | 广西大学 | Method for isolating casein components in milk |
| CN105254714A (en) * | 2015-10-16 | 2016-01-20 | 中国农业大学 | Casein-derived antioxidant peptide and preparation method thereof |
| CN112625110A (en) * | 2019-09-24 | 2021-04-09 | 泰州医药城国科化物生物医药科技有限公司 | Purification preparation method of beta-casein in cow milk |
| CN110973345A (en) * | 2019-12-26 | 2020-04-10 | 吉林大学 | Method for continuously separating and preparing functional lactoprotein in colostrum |
| CN116217705A (en) * | 2023-03-27 | 2023-06-06 | 新疆大学 | Separation and purification method and detection method for alpha-lactalbumin in camel milk |
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