NL2031571B1 - Binding buffer for lactoferrin extraction, preparation method and application thereof, and method for detecting bovine lactoferrin - Google Patents
Binding buffer for lactoferrin extraction, preparation method and application thereof, and method for detecting bovine lactoferrin Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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Abstract
The present invention provides a binding buffer for lactoferrin extraction, a preparation method and an application thereof, and a method for detecting bovine lactoferrin, and belongs to the technical field of protein detection. The binding buffer of the 5 present invention, used as an extracting solution for lactoferrin, can ensure the binding ability of an affinity column to lactoferrin in dairy products, realize the enrichment and detection. of lactoferrin. in the sample, improve the extraction rate and recovery rate of lactoferrin in the products, and 10 overcome the influence of sample matrix on the target substance. Therefore, the binding buffer is applicable for extracting lactoferrin in different types of dairy products, and can meet the quantitative detection of lactoferrin in different types of dairy products; besides, all the recovery rates can reach above 90% with 15 stable results, and there is a good peak shape and fewer impurity peaks after elution. (+ Fig. l)
Description
BINDING BUFFER FOR LACTOFERRIN EXTRACTION, PREPARATION METHOD AND
APPLICATION THEREOF, AND METHOD FOR DETECTING BOVINE LACTOFERRIN
The present invention relates to the technical field of pro- tein detection, in particular to a binding buffer for lactoferrin extraction, a preparation method and an application thereof, and a method for detecting bovine lactoferrin.
Lactoferrin (LF) is an important iron-binding glycoprotein in milk with a relative molecular mass of 70-80 kDa. It is mainly ex- pressed and secreted by mammary epithelial cells and widely dis- tributed in mammalian milk, especially in colostrum. Lactoferrin is also contained in various other tissue secretions (saliva, tears or bile), providing the first most important line of defense for the mucosal system invaded by a large number of pathogenic mi- croorganisms. Lactoferrin not only participates in the transport of iron, but also has powerful biological functions such as broad- spectrum antibacterial effect, anti-oxidation, anticancer effect and regulation on the immune system. Thus it is deemed as a new antibacterial and anticancer drug, as well as a food and feed ad- ditive with great development potentials.
Lactoferrin has seven major functions: regulation on a body's immune response, regulation on the gastrointestinal absorption of iron, broad-spectrum antibacterial effect, antiviral effect, anti- oxidation, anticancer effect and synergistic effects with drugs.
Because of such seven major effects of lactoferrin, many scien- tists and medical scientists have begun to apply lactoferrin clin- ically and explored lactoferrin in the process of preventing and treating diseases, and lactoferrin has been recognized by clinical medical experts.
With the increasing improvement of people's living standards, domestic dairy products are developing in a direction of higher quality. The evaluation indicators for dairy products include al-
kaline phosphatase, peroxidase, lactoferrin, lactalbumin, lacto- globulin, lactulose and furosine. Among those indicators, lac- toferrin is one of the key indicators, so it is particularly im- portant to establish a method that can accurately and quickly de- tect a lactoferrin content in a sample. At present, the detection methods for lactoferrin in dairy products mainly include: immuno- logical methods (immunocolloidal gold method, enzyme-linked im- munosorbent assay (ELISA)), high-performance capillary electropho- resis, atomic absorption spectrophotometry, gel imaging system
SDS-PAGE method and high-performance affinity and chromatography.
Wherein, the immunocolloidal gold method has the advantages of convenient and quick operations and low cost, but it cannot ac- curately quantify a lactoferrin content in dairy products. The im- munocolloidal gold method has the characteristic of high sensitiv- ity to lactoferrin in dairy products, but it has a cumbersome op- eration process and a difficultly controllable deviation of re- sults. The high-performance capillary electrophoresis features high efficiency, high sensitivity and high degree of automation, but it is a little inconvenient for detecting a high lactoferrin content in dairy products due to a small injection volume and poor separation reproducibility. The atomic absorption spectrophotome- try is usually used for detecting metal elements and some non- metal elements, and it is rarely used in the field of protein de- tection for dairy products. In addition, the gel imaging system
SDS-PAGE is not as sensitive as the ELISA and not as accurate as high-performance liquid chromatography (HPLC) for detection of lactoferrin; besides, it is complicated in operation process and time-consuming.
The high-performance affinity chromatography realizes an ob- jective of separation using the reversible binding of some rela- tively specific molecules, such as antigens and antibodies, en- zymes and substrates. Two molecules that specifically bind to each other are ligands, and a ligand that specifically binds to a sam- ple to be tested is immobilized on a water-insoluble carrier to prepare an affinity column, a target substance specifically binds with the affinity column to achieve an objective of separating and purifying the target substance, and HPLC is combined for detec-
tion. This method is more accurate and simple to operate, and can satisfy the purification, detection and analysis requirements of most samples. Wherein the affinity column plays a decisive role in the binding ability of the target substance in a sample, which is indispensable from a pre-treatment process of the sample. At pre- sent, in a method of the existing group standard T/TDSTIA 006- 2019, a recovery rate of lactoferrin in dairy products is low and the quantitative result of lactoferrin is inaccurate.
The present invention is intended to provide a binding buffer for lactoferrin extraction, a preparation method and an applica- tion thereof, and a method for detecting bovine lactoferrin; and the binding buffer has an excellent effect of recovering lactofer- rin.
In order to realize the above-mentioned purpose, the present invention adopts the following technical solution:
The present invention provides a binding buffer for lactofer- rin extraction, including the following raw materials: disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate di- hydrate, an extracting reagent and water, wherein a concentration of the disodium hydrogen phosphate dodecahydrate in the binding buffer is 30.083 g/L; a concentration of the sodium dihydrogen phosphate dihydrate in the binding buffer is 2.496 g/L; a volume concentration of the extracting reagent in the binding buffer is 20%; and the extracting reagent is an aqueous mixture of sodium chloride and Tween-20.
Preferably, a mass concentration of the sodium chloride in the extracting reagent is 146.1 g/L, and a volume concentration of the Tween-20 in the extracting reagent is 25%.
The present invention provides a method for preparing the binding buffer for lactoferrin extraction described in the above- mentioned technical solution, including the following steps:
According to a concentration ratio of the raw materials, disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phos- phate dihydrate and extracting reagent are mixed and dissolved in water to obtain a binding buffer for lactoferrin extraction; and the the extracting reagent is an aqueous mixture of sodium chlo- ride and Tween-20.
The present invention provides an application of the binding buffer for lactoferrin extraction described in the above-mentioned technical solution or the binding buffer for lactoferrin extrac- tion prepared by the preparation method described in the above- mentioned technical solution.
The present invention provides a method for detecting bovine lactoferrin, including the following steps: a sample containing bovine lactoferrin is mixed with a bind- ing buffer, and extraction is performed to obtain an extracting solution; and the binding buffer is the binding buffer for lac- toferrin extraction described in the above-mentioned technical so- lution or the binding buffer for lactoferrin extraction prepared by the preparation method described in the above-mentioned tech- nical solution; the extracting solution is purified using a heparin affinity column to obtain a purified solution containing purified lactofer- rin; and the purified solution containing purified lactoferrin is de- tected by HPLC to obtain a lactoferrin content.
Preferably, a dosage ratio of the sample containing bovine lactoferrin to the binding buffer is (1-20) g:50 mL.
Preferably, a number of extraction times is 1-2; and a tem- perature for each extraction is 4°C, and the extraction duration is 10 min.
Preferably, the conditions of the HPLC detection include: gradient elution procedure: at 0-5 min, mobile phase A is 70%, and mobile phase B is 30%; at 5.0-10 min, mobile phase A is 45%, and mobile phase B is 55%; at 10.00-12 min, mobile phase A is 40%, and mobile phase B is 60%; at 12.00-16 min, mobile phase A is 70%, and mobile phase B is 30%; the mobile phase A is TFA with a mass con- centration of 0.1%; and the mobile phase B is acetonitrile.
Preferably, the conditions of the HPLC detection further in- clude: detection wavelength: ultraviolet 280 nm; column tempera- ture: 30°C; injection volume: 30 pL; and flow rate: 1 mL/min.
Preferably, the lactoferrin content= (instrument detection concentration of HPLC detectionx4 (elution volume of the heparin affinity column)xconstant volume of the heparin affinity col- umn) / (injection volume of the heparin affinity column x weighed sample amount x 1,000) mg/g, wherein a unit of ug/mL is used for 5 the instrument detection concentration; a unit of mL is used for the elution volume, the constant volume and the injection volume; and a unit of g is used for the weighed sample amount.
The present invention provides a binding buffer for lactofer- rin extraction, including the following raw materials: disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate di- hydrate, an extracting reagent and water, wherein a concentration of the disodium hydrogen phosphate dodecahydrate in the binding buffer is 30.083 g/L; a concentration of the sodium dihydrogen phosphate dihydrate in the binding buffer is 2.496 g/L; a volume concentration of the extracting reagent in the binding buffer is 20%; and the extracting reagent is an aqueous mixture of sodium chloride and Tween-20. In the binding buffer for lactoferrin ex- traction of the present invention, the extraction reagent includes sodium chloride and Tween-20, and sodium ions and calcium ions in dairy products produce a synergistic effect in the binding of lac- toferrin to heparin, which can improve the binding capacity of lactoferrin to heparin. As a surfactant, Tween 20 can reduce the non-specific adsorption of the heparin affinity column and enhance the specific binding of heparin to lactoferrin, and provide a pro- tective effect for the lactoferrin. The binding buffer, used as the extracting solution for lactoferrin, can ensure the binding ability of the affinity column to lactoferrin in dairy products, realize the enrichment and detection of lactoferrin in the sample, improve the extraction rate and recovery rate of lactoferrin in the products, and overcome the influence of sample matrix on the target substance. Therefore, the binding buffer is applicable for extracting lactoferrin in different types of dairy products, and can meet the quantitative detection of lactoferrin in different types of dairy products.
The binding buffer for lactoferrin extraction provided by the present invention has a good applicability to various samples, such as milk powder, sterilized milk, bovine colostrum and yogurt;
the recovery rate can reach above 90% with stable results, and there is a good peak shape and fewer impurity peaks after elution.
The commercially available affinity column is purified in the form of a prepacked column which requires the assistance of exter- nal mechanical pressure to complete the processes of injection, rinsing and elution of a sample, producing a long detection time and complicated operation. In the present invention, a gravity column is adopted for sample injection without a need of addition- al external force, thereby providing a more convenient operation process and improving the efficiency of purification.
FIG. 1 shows a standard curve of lactoferrin.
The present invention provides a binding buffer for lactofer- rin extraction, including the following raw materials: disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate di- hydrate, an extracting reagent and water, wherein a concentration of the disodium hydrogen phosphate dodecahydrate in the binding buffer is 30.083 g/L; a concentration of the sodium dihydrogen phosphate dihydrate in the binding buffer is 2.496 g/L; a volume concentration of the extracting reagent in the binding buffer is 20%; and the extracting reagent is an aqueous mixture of sodium chloride and Tween-20.
Unless otherwise noted, all the raw materials or reagents in the present invention are commercially available products familiar to those skilled in the art.
The binding buffer for lactoferrin extraction provided by the present invention includes disodium hydrogen phosphate dodecahy- drate; and a concentration of the disodium hydrogen phosphate do- decahydrate in the binding buffer is 30.083 g/L.
The binding buffer for lactoferrin extraction provided by the present invention includes sodium dihydrogen phosphate dihydrate; and a concentration of the sodium dihydrogen phosphate dihydrate in the binding buffer is 2.496 g/L.
The binding buffer for lactoferrin extraction provided by the present invention includes an extracting reagent which is an aque- ous mixture of sodium chloride and Tween-20; and a volume concen- tration of the extracting reagent in the binding buffer is 20%.
In the present invention, a mass concentration of the sodium chloride in the extracting reagent is preferably 146.1 g/L, and a volume concentration of the Tween-20 in the extracting reagent is preferably 25%. In the present invention, the roles of the ex- tracting reagent include reducing the non-specific adsorption of a heparin affinity column, improving the specific adsorption of a target substance, ensuring the binding ability of the affinity column to lactoferrin in dairy products, realizing the enrichment and detection of lactoferrin in a sample, and improving an extrac- tion rate and a recovery rate of lactoferrin in the products.
The present invention has no special limitation on the pro- cess for preparing the extracting reagent, and the extracting so- lution meeting the above concentration requirement can be prepared according to a process well known in the art. In an embodiment of the present invention, a specific process for preparing the ex- tracting reagent is as follows: 2.922 g of sodium chloride was weighed and dissolved in 10 mL of deionized water, then 5 mL of
Tween-20 was added, and water was added until the volume reached 20 mL.
In the present invention, an amount of water is to meet the above concentration requirements of components in the binding buffer.
The present invention provides a method for preparing the binding buffer for lactoferrin extraction described in the above- mentioned technical solution, including the following steps: according to a concentration ratio of the raw materials, disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phos- phate dihydrate and extracting reagent are mixed and dissolved in water to obtain a binding buffer for lactoferrin extraction; and the extracting reagent is an aqueous mixture of sodium chlorideand
Tween-20.
The present invention has no special limitation on the pro- cess of mixing and dissolving the disodium hydrogen phosphate do- decahydrate, the sodium dihydrogen phosphate dihydrate and the ex-
tracting reagent in water, and the binding buffer of a correspond- ing concentration can be obtained according to a process well known in the art.
In an embodiment of the present invention, a specific process for preparing the binding buffer for lactoferrin extraction is as follows: 30.083 g of disodium hydrogen phosphate dodecahydrate and 2.496 g of sodium dihydrogen phosphate dihydrate were weighed, 20 mL of extracting solution was added, 900 mL of deionized water was added for dissolution, and water was added until the volume reached 1,000 mL.
The present invention provides an application of the binding buffer for lactoferrin extraction described in the above-mentioned technical solution or the binding buffer for lactoferrin extrac- tion prepared by the preparation method described in the above- mentioned technical solution.
The present invention provides a method for detecting bovine lactoferrin, including the following steps: a sample containing bovine lactoferrin is mixed with a bind- ing buffer, and extraction is performed to obtain an extracting solution; and the binding buffer is the binding buffer for lac- toferrin extraction described in the above-mentioned technical so- lution or the binding buffer for lactoferrin extraction prepared by the preparation method described in the above-mentioned tech- nical solution; the extracting solution is purified using a heparin affinity column to obtain a purified solution containing purified lactofer- rin; and the purified solution containing purified lactoferrin is sub- jected to HPLC detection to obtain a lactoferrin content.
In the present invention, a sample containing bovine lac- toferrin is mixed with a binding buffer, and extraction is per- formed to obtain an extracting solution. In the present invention, the sample containing bovine lactoferrin preferably includes bo- vine colostrum, raw milk, sterilized milk, fermented milk or in- fant formula milk powder. The present invention has no special limitation on the source of the sample containing bovine lactofer- rin, and commercially available products well known in the art can be used.
The present invention has no special limitation on the pro- cess of mixing the sample containing bovine lactoferrin with the binding buffer, and the materials can be mixed evenly according to a process well known in the art.
In the present invention, a dosage ratio of the sample con- taining bovine lactoferrin to the binding buffer is preferably (1- 20) g:50 mL, and more preferably (2.5-10) g:50 mL.
In the present invention, a number of extraction times is preferably 1-2; and a temperature for each extraction is prefera- bly 4°C, and the extraction duration is preferably 10 min; and the extraction is preferably performed by centrifugation, with a pref- erable rotating speed of 12,000 r/min.
Upon completion of the centrifugation, a liguid part of a middle layer of the resulting mixture is preferably sucked out us- ing a pipette to obtain an extracting solution; an upper layer of the resulting mixture is lipid, and a lower layer is sediment, sucking lipid from the upper layer and sediment from the lower layer is avoided to prevent the lipid and the sediment from block- ing an affinity column when they pass through the column.
After the extracting solution is obtained, the extracting so- lution is purified using a heparin affinity column to obtain a pu- rified solution containing purified lactoferrin. In the present invention, an injection volume of the extracting solution is pref- erably 5-20 mL, and more preferably 10-15 mL.
The present invention has no special limitation on the hepa- rin affinity column, and any heparin affinity column well known in the art can be used.
In the present invention, before the purification, the hepa- rin affinity column is preferably pretreated and equilibrated as follows: the heparin affinity column is taken out, a syringe bar- rel penetrating a plunger is connected with the heparin affinity column, and a plug at a lower end of the affinity column is re- moved, and an preservation solution in the column is dripped out by gravity {or poured out); 1 mL of binding buffer is added to the heparin affinity column with a pipette, then the syringe barrel is connected to the heparin affinity column, and 4 mL of binding buffer is added to the syringe barrel to equilibrate the affinity column.
Upon completion of the pre-treatment and equilibration, after a liquid level of the binding buffer is at a distance of about 5 mm from an upper sieve plate of the heparin affinity column, the extracting solution is injected and purified. In the present in- vention, an injection volume of the extracting solution is prefer- ably adjusted according to the actual situation of different sam- ples; when the injection volume of the extracting solution is greater than a maximum capacity of the syringe barrel, the ex- tracting solution is preferably added barrel by barrel; and there is no special limitation on the specific process of adding the ex- tracting solution barrel by barrel, and it is ensured that all the extracting solution can be added.
After the extracting solution is completely added dropwise and the liquid level is at a distance of about 5 mm from the upper sieve plate, the binding buffer is preferably added into the hepa- rin affinity column to wash off non-target substances; after the binding buffer is added and the liquid level is flush with the sieve plate, the residual liquid on an inner wall of a column tube is wiped away with absorbent paper, then eluent is added into the heparin affinity column and dripped out by the gravity flow rate until the last drop, and all the eluent is collected. The present invention has no special limitation on the specific dosages on the binding buffer and the eluent, and the dosages can be adjusted ac- cording to the injection volume of the extracting solution.
In the present invention, a specific process for preparing the eluent is preferably as follows: 7.52 g of disodium hydrogen phosphate dodecahydrate, 0.624 g of sodium dihydrogen phosphate dihydrate and 29.2 g of sodium chloride were weighed, 400 mL of deionized water was added for dissolution, and water was added un- til the volume reached 500 mL.
In the present invention, after the eluent is obtained, the eluent is centrifuged or filtered, and a middle layer or filtrate is taken to obtain a purified solution containing purified lac- toferrin; a rotation speed of the centrifugation is preferably 12,000 rpm, and the duration is preferably 5 min; and a membrane used for the filtration is preferably an aquo-system filter mem- brane. The present invention has no special limitation on the spe- cific specification of the aquo-system filter membrane, and the aquo-system filter membrane, which does not absorb lactoferrin, well known to the art can be used.
In the present invention, when the heparin affinity column is to be used again after being used, in case of short-term storage, 5 mL of binding buffer is added to the heparin affinity column for washing, then 2 mL of binding buffer is added, and the lower plug and the plunger are installed; in case of long-term storage, 5 mL of 20% alcohol is add into the heparin affinity column for wash- ing, then 2 mL of 20% alcohol is added, and the lower plug and the plunger are installed; and in order to ensure the accuracy of the experiment, the affinity column is not reused, and it can be dis- carded after use.
In the present invention, after the purified solution con- taining purified lactoferrin is obtained, the purified solution containing purified lactoferrin is subjected to HPLC detection to obtain a lactoferrin content.
The present invention has no special limitation on a concen- tration of the purified solution containing purified lactoferrin, and the concentration can be adjusted according to actual samples.
In the present invention, when the HPLC detection is per- formed, a ratio of a mobile phase A to a mobile phase B at the in- itial elution is used as a standard, and the injection starts af- ter a baseline becomes stable. In the present invention, the con- ditions of the HPLC detection preferably include: gradient elution procedure: at 0-5 min, mobile phase A is 70%, and mobile phase B is 30%; at 5.0-10 min, mobile phase A is 45%, and mobile phase B is 55%; at 10.00-12 min, mobile phase A is 40%, and mobile phase B is 60%; at 12.00-16 min, mobile phase A is 70%, and mobile phase B is 30%; the mobile phase A is TFA with a mass concentration of 0.1%; and the mobile phase B is acetonitrile.
In the present invention, the conditions of the HPLC detec- tion preferably further include: detection wavelength: ultraviolet 280 nm; column temperature: 30°C; injection volume: 30 pL; flow rate: 1 mL/min; and chromatographic column: C4 column, 250 mmx4.6 mmx5 um, 300 A.
After the HPLC detection is completed, an ultraviolet absorp- tion peak area of lactoferrin is obtained; in the present inven- tion, a lactoferrin concentration in a sample is preferably deter- mined according to a standard curve of lactoferrin, i.e., the in- strument detection concentration.
In the present invention, a process of creating a standard curve of lactoferrin preferably includes:
Lactoferrin reference substance: CAS: 112163-33-4, purity 2 85.0%.
Lactoferrin standard stock solution (10 mg/mL): the lactofer- rin reference substance is weighed after conversion according to the purity provided by a reference substance certificate, and wa- ter is added until the volume reaches 10 mL. The solution is stored at -20°C, with a validity of 4 months.
Lactoferrin standard intermediate solution (200 mg/L): 200 uL of lactoferrin standard stock solution (10 mg/mL) is accurately pipetted, and water is added until the volume reaches 10 mL. It is prepared before use.
Lactoferrin standard series solutions: the eluent used is that used in the elution process of the heparin affinity column, in order to keep the HPLC detection system consistent with a final solution system of the target, accordingly ensuring the accuracy of the HPLC detection results.
Standard series solution 6: 1.5 mL of lactoferrin standard intermediate solution is accurately pipetted, and 1.5 mL of eluent is added to 3 mL (equivalent to a 100 mg/L standard solution).
Standard series solution 5: 1,600 pL of 100 mg/L lactoferrin standard intermediate solution is accurately pipetted, and 400 pL of eluent is added {equivalent to an 80 mg/L standard solution).
Standard series solution 4: 750 uL of 80 mg/L lactoferrin standard intermediate sclution is accurately pipetted, and 250 pL of eluent is added (equivalent to a 60 mg/L standard solution).
Standard series solution 3: 500 uL of 80 mg/L lactoferrin standard intermediate solution is accurately pipetted, and 500 pL of eluent is added (equivalent to a 40 mg/L standard solution).
Standard series solution 2: 500 uL of 40 mg/L lactoferrin standard intermediate solution is accurately pipetted, and 500 pL of eluent is added (equivalent to a 20 mg/L standard solution).
Standard series solution 1: 500 uL of 20 mg/L lactoferrin standard intermediate solution is accurately pipetted, and 500 pL of eluent is added (equivalent to a 10 mg/L standard solution).
In the present invention, each of the standard series solu- tions 1-6 is subjected to HPLC detection to obtain a corresponding ultraviolet absorption peak area; then by taking the concentration of the lactoferrin standard solution as an abscissa and taking the ultraviolet absorption peak area as an ordinate, a standard curve of lactoferrin is created and obtained, and a linear equation is y=7603.7x-15699, R°=1, as shown in FIG. 1. In the present inven- tion, the conditions of the HPLC detection in the process of cre- ating the standard curve are the same as those for the purified solution containing purified lactoferrin, which will not be re- peated herein.
After the standard curve is obtained, the ultraviolet absorp- tion peak area obtained from the HPLC detection of the purified solution containing purified lactoferrin is preferably introduced into the standard curve of lactoferrin to determine a lactoferrin concentration in the purified liquid, namely an instrument detec- tion concentration, and then a lactoferrin concentration in an ac- tual sample is calculated.
In the present invention, the lactoferrin content={({instrument detection concentration of HPLC detectionx4 (elution volume of the heparin affinity column) xconstant volume of the heparin affinity column) / (injection volume of the heparin affinity column * weighed sample amount x 1,000) mg/g; a unit of pg/mL is used for the in- strument detection concentration; a unit of mL is used for the elution volume, the constant volume and the injection volume; and a unit of g is used for the weighed sample amount.
The technical solutions in the present invention will be clearly and completely described below in combination with the em- bodiments of the present invention. It is obvious that the de- scribed embodiments are only parts of, rather than all of, the em- bodiments of the present invention. Based on the embodiments of the present invention, all the other embodiments obtained by those skilled in the art without creative efforts will fall within the protection scope of the present invention.
In the following examples, a method for preparing the binding buffer was as follows: 30.083 g of disodium hydrogen phosphate do- decahydrate and 2.496 g of sodium dihydrogen phosphate dihydrate were weighed, 20 mL of extracting reagent was added, 900 mL of de- ionized water was added for dissclution, and water was added until the volume reached 1,000 mL; a method for preparing the extracting solution was as fol- lows: 2.922 g of sodium chloride was weighed and dissolved in 10 mL of deionized water, then 5 mL of Tween-20 was added, and water was added until the volume reached 20 nL; a process for preparing the eluent was as follows: 7.52 g of disodium hydrogen phosphate dodecahydrate, 0.624 g of sodium dihy- drogen phosphate dihydrate and 29.2 g of sodium chloride were weighed, 400 mL of deionized water was added for dissolution, and water was added until the volume reached 500 mL; and the conditions of the HPLC detection were as follows: gradi- ent elution procedure: at 0-5 min, mobile phase A was 70%, and mo- bile phase B was 30%; at 5.0-10 min, mobile phase A was 45%, and mobile phase B was 55%; at 10-12 min, mobile phase A was 40%, and mobile phase B was 60%; at 12.00-16 min, mobile phase A was 70%, and mobile phase B was 30%; the mobile phase A was TFA with a mass concentration of 0.1%; and the mobile phase B was acetonitrile.
Detection wavelength: ultraviolet 280 nm; column temperature: 30°C; injection volume: 30 pL; flow rate: 1 mL/min; and chromato- graphic column: C4 column, 250 mmx4.6 mmx5 um, 300 A.
In the following examples, a process of creating a standard curve of lactoferrin included:
Lactoferrin reference substance: CAS: 112163-33-4, purity 2 85.0%
Lactoferrin standard stock solution (10 mg/mL): the lactofer- rin reference substance was weighed after conversion according to the purity provided by a reference substance certificate, and wa- ter was added until the volume reached 10 mL. It was stored at - 20°C, with a validity of 4 months.
Lactoferrin standard intermediate solution (200 mg/L): 200 uL of lactoferrin standard stock solution (10 mg/mL) was accurately pipetted, and water was added until the volume reached 10 mL. It was prepared before use.
Lactoferrin standard series solutions: the eluent used was that used in the elution process of the heparin affinity column;
Standard series solution 6: 1.5 mL of lactoferrin standard intermediate solution was accurately pipetted, and 1.5 mL of elu- ent was added to 3 mL (equivalent to a 100 mg/L standard solu- tion).
Standard series solution 5: 1,600 uL of 100 mg/L lactoferrin standard intermediate solution was accurately pipetted, and 400 pL of eluent was added (equivalent to an 80 mg/L standard solution).
Standard series solution 4: 750 pL of 80 mg/L lactoferrin standard intermediate solution was accurately pipetted, and 250 pL of eluent was added (equivalent to a 60 mg/L standard solution).
Standard series solution 3: 500 uL of 80 mg/L lactoferrin standard intermediate solution was accurately pipetted, and 500 pL of eluent was added (equivalent to a 40 mg/L standard solution).
Standard series solution 2: 500 pL of 40 mg/L lactoferrin standard intermediate solution was accurately pipetted, and 500 pL of eluent was added {equivalent to a 20 mg/L standard solution).
Standard series solution 1: 500 pL of 20 mg/L lactoferrin standard intermediate solution was accurately pipetted, and 500 pL of eluent was added (equivalent to a 10 mg/L standard solution).
Each of the standard series solutions 1-6 was subjected to
HPLC detection to obtain a corresponding ultraviolet absorption peak area; then by taking the concentration of the lactoferrin standard solution as an abscissa and taking the ultraviolet ab- sorption peak area as an ordinate, a standard curve of lactoferrin was created and obtained, and a linear equation was y=7603.7x- 15699, R‘=1, as shown in FIG. 1;
In the field of HPLC detection, an average peak height of a baseline noise fluctuation selected for HPLC detection is A, and a peak height of a sample is B; when a signal-to-noise ratio B/A is greater than or equal to 3, it is a minimum detection limit, and the minimum detection limit of a lactoferrin standard substance is 1 ppm.
Example 1
Different brands of commercially available sterilized milk (Satine pure skim milk, Yili pure skim milk, Wondersun pure skim milk, Mengniu pure milk, Deluxe pure skim milk and Satine full cream pure milk) were selected, 10 g (accurate to 0.01 g) of each brand of sterilized milk was weighed and placed in a 50 mL centri- fuge tube, a binding buffer was added until the volume reached 50 mL, and the mixture was mixed evenly; then the mixture was centri- fuged at 4°C and 12,000 r/min for 10 min, then a liquid part of a middle layer was sucked out using a wide-range pipette, and 10 mL was taken as a sample injected to a heparin affinity column; after sample injection, the heparin affinity column was washed with 10 mL of binding buffer and then eluted with 4 mL of eluent, and af- ter centrifugation at 4°C and 12,000 r/min for 5 min, the obtained purified liquid was subjected to HPLC detection; and a blank con- trol (with a standard addition amount of 0, that is, an actual sample was detected without additional lactoferrin standard sub- stance added) and a standard addition experiment (with a standard addition amount of 50 ng/g, a lactoferrin standard stock solution was added) were set for each type of the sterilized milk. The re- sults are as shown in Table 1.
Table 1 Detection Results of Different Brands of Sterilized
Milk
Sample Name Appearance | Peak Content Detected by Standard Addi- | Recovery
Time {min) Area HPLC (ppm) tion Amount Rate (%) (mAU*mi (ppm) n) eee
It can be seen from Table 1 that the lactoferrin is 1 ppm lower than the minimum detection limit for the HPLC in the blank control groups of all the brands of commercially available steri- lized milk described above. This is because after the finished sterilized milk is sterilized by high temperature, there is no lactoferrin with physiological activity, and no lactoferrin is de- tected.
For the different brands of sterilized milk treated by the binding buffer of the present invention, a relatively ideal recov- ery effect can be achieved after the sterilized milk passes through a heparin affinity column, and the binding buffer can be applicable for extracting lactoferrin from most of the brands of commercially available sterilized milk.
Example 2
Different brands of commercially available solid milk powder (Yili PRO-KIDO (stage 1), Ausnutria Aiyou (stage 2), Beingmate
Tongxiang (stage 1), Firmus Zhenai Beihu (stage 3) and Junlebao
Lezhen {stage 1)} were selected, 2.5 g (accurate to 0.01 g) of each sample was weighed and placed in a 50 mL centrifuge tube, 30 mL of binding buffer was firstly added, vortex oscillation was performed until the sample was completely dissolved, then the binding buffer was added until the volume reached 50 mL, and the mixture was mixed evenly; then the mixture was centrifuged at 4°C and 12,000 r/min for 10 min, then a liquid part of a middle layer was sucked out using a wide-range pipette, and 10 mL was taken as a sample injected to a heparin affinity column; upon completion of the sample injection, the heparin affinity column was washed with 10 mL of binding buffer and finally eluted with 4 mL of eluent, and after centrifugation at 4°C and 12,000 r/min for 5 min, the obtained purified liquid was subjected to HPLC detection; and a blank control {with a standard addition amount of 0) and a stand- ard addition experiment (with a standard addition amount of 50 ppm, a lactoferrin standard stock solution was added) were set for each brand of the milk powder.
Table 2 Detection Results of Different Brands of Commercially
Available Solid Milk Powder
Standard
Appear- Recov-
Content Detected by | Addition
Sample Name ance Peak Area ery
HPLC {ppm} Amount
Time Rate (ppm)
Yili PRO-KIDO (stage 7.663 109,847 72.3 1)
Yili PRO-KIDO (stage 7.661 211,102 132.6 500 97% 1)
Ausnutria Aiyou 7.663 136,505 88.1 {stage 2)
Ausnutria Aiyou 7.670 240,718 150.2 500 99% (stage 2}
Beingmate 7.669 114,799 75.2
Tongxiang (stage 1}
Beingmate 7.663 214,908 134.8 500 95%
Tongxiang (stage 1)
Firmus Zhenai Bei- 7.665 114,815 75.2 hu {stage 3}
Firmus Zhenai Bei- 7.666 223,890.68 140.2 500 104% hu {stage 3)
Junlebao Lezhen 7.661 110,746 72.8 {stage 1)
Junlebao Lezhen 7.662 217,764.52 136.6 500 102% (stage 1}
It can be known from Table 2 that the the binding buffer pro- vided by the present invention can be used for effectively ex- tracting lactoferrin from milk powder, and the recovery rates of standard addition are all within a normal range (80-1203}, which meets the criteria of standard addition recovery. In addition, the detection values of the brands of milk powder are all in line with the corresponding labeling values of lactoferrin on their respec- tive packages.
Example 3
Different brands of commercially available fermented milk (yogurt) (Bright plain yogurt, Yili Ambrosial plain yogurt, Jiabao plain yogurt, Mengniu Guanyi milk, Sanyuan plain yogurt, Junlebao
Jianchun yogurt, Yili Yixiao flavored fermented milk and Jiabao probiotic fermented milk) were selected, 20 g (accurate to 0.01 9) of each brand of sterilized milk was weighed and placed in a 50 mL centrifuge tube, the binding buffer was added until the volume reached 50 mL, and the mixture was mixed evenly; the mixture was centrifuged at 4°C and 12,000 r/min for 10 min, then 30 mL of the middle layer was sucked out using a wide-range pipette, centrifu- gation was performed again at 4°C and 12,000 r/min for 10 min, 15 mL of the middle layer was gently taken out, 10 mL was taken as an injection sample and injected to a heparin affinity column; after injection, the heparin affinity column was washed with 10 mL of binding buffer and finally eluted with 4 mL of eluent; and after centrifugation at 4°C and 12,000 r/min for 5 min; the obtained pu- rified liquid was subjected to HPLC detection; and a blank control (with a standard addition amount of 0) and a standard addition ex- periment (with a standard addition amount of 50 ug/g, a lactofer- rin standard stock solution was added) were set for each type of the yogurt.
Table 3 Detection Results of Different Brands of Fermented
Milk
Sample Name Appear- Peak Area | Content Detected by Standard Recov- ance Time HPLC {ppm} Addition ery Rate
Amount (ppm) seen [OJ Joo
Yili Ambrosial Plain me
Yili Ambrosial Plain 8.094 350,341 47.0 50 94% ll A
JobsoPlainomut | Jo fe 0 f° ewes [ov Jo 5
Sram ee
Junlebao Jianchun me
Junlebao Jianchun 8.098 351,924 47.5 50 95% i lA
Yili Yixiao Flavored
Fermented Milk And
Jiabao Probiotic Fer- mented Milk
Yili Yixiao Flavored 8.116 364,006 48.6 50 97%
Fermented Milk And
Jiabao Probiotic Fer- mented Milk
Jiabao Probiotic Fer-
Fl NN A A A te [oP 8.118 377,340 50.1 mented Milk
It can be seen from Table 3 that the lactoferrin is 1 ppm lower than the minimum detection limit for the HPLC in the blank control groups of all the brands of fermented milk described above. For the different brands of fermented milk (yogurt) treated by the binding buffer of the present invention, a relatively ideal recovery effect can be achieved after the fermented milk is puri- fied by a heparin affinity column, and the binding buffer can be applicable for extracting lactoferrin from most of the brands of commercially available fermented milk.
Comparative Example 1
A method of the group standard T/TDSTIA 006-2019 was adopted to extract different brands of sterilized milk (Satine pure skim milk, Yili pure skim milk, Wondersun pure skim milk, Mengniu pure milk, Deluxe pure skim milk and Satine full cream pure milk): 10 g of sterilized milk was weighed as a sample, a phosphate buffer I (23.85 g of disodium hydrogen phosphate and 4.99 g of so- dium dihydrogen phosphate were weighed and dissolved by an appro- priate amount of water, a pH value was adjusted to 7.50 by a sodi- um hydroxide solution, and a volume was added until the volume reached 1 L) was added until the volume reached 50 mL, the mixture was mixed evenly and centrifuged for 15 min, 25 ml of the sample of the middle layer was pipetted into another 50 mL centrifuge tube and centrifuged for 15 min, the supernatant was pipetted for purification; a heparin affinity column was activated using 10 mL of phosphate buffer solution II ( 5.96 g of disodium hydrogen phosphate, 0.96 g of sodium dihydrogen phosphate and 5.84 g of so- dium chloride were weighed and dissolved by an appropriate amount of water, a pH value was adjusted to 7.50 by a sodium hydroxide solution, and a volume was added until the volume reached 1 L), 10 mL of supernatant was accurately pipetted to pass through the col- umn, washed with 10 mL of phosphate buffer solution II, and eluted with 4.5 mL of phosphate buffer solution III (5.96 g of disodium hydrogen phosphate, 2.50 g of sodium dihydrogen phosphate and 119.30 g of sodium chloride were weighed and dissolved by an ap- propriate amount of water, a pH value was adjusted to 7.50 by a sodium hydroxide solution, and the volume reached 1 L}), and the mixture was mixed evenly by vertex and sieved using a 0.22 um mi- croporous membrane, and then HPLC detection was performed on an instrument; and a blank control (with a standard addition amount of 0, that is, an actual sample was detected without additional lactoferrin standard substance added) and a standard addition ex- periment (with a standard addition amount of 50 ppm, a lactoferrin standard stock solution was added) were set for each type of the sterilized milk. The results are as shown in Table 4.
Table 4 Comparison of Detection Results Between Example 1 and
Comparative Example 1
Content De- Standard
Extraction Peak tected in Lig- Addition Recovery
Sample Name Peak Area
Method Time uid Phase Amount Rate oe EE 9 0
Saine ure Skim vi Comparative |- 0 00 00
Example 1 30,067
Example 1 ik 7997
Comparative [- [0 jo [0 em ee mee 10 9
Wondersun Pure 49.4
Skim Milk Comparative ee a
Se —
Example 1
Mengniu Pure ik Comparative 0 jo 10 ra
Example 1
Deluxe Pure skim ik Comparatve 0 f° 0°
Sample amper 10 1° 10
Satine Full Cream 50 101%
It can be seen from Table 4 that the lactoferrin is 1 ppm lower than the minimum detection limit for the HPLC in the blank control groups of all the brands of commercially available steri- lized milk described above; moreover, by using the method of the present invention for extracting lactoferrin from different brands of sterilized milk, the standard addition recovery rate is better than the existing group standard, the method is stabler on the whole with a good purification effect, and it is more suitable for extracting lactoferrin from different brands of sterilized milk.
In addition, only two solutions (i.e., the binding buffer and the eluent) are used in the method of the present invention, and thus the operation process is simpler and faster; whereas, three buffer salt solutions are used for processing in the group standard.
The above-mentioned descriptions are only some of the embodi- ments of the present invention. It should be noted that for those of ordinary skill in the art, some improvements and modifications may be made without departing from the principle of the present invention. These improvements and modifications should also fall within the protection scope of the present invention.
Claims (10)
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| WO1995022258A2 (en) * | 1994-02-16 | 1995-08-24 | Pharming Bv | Isolation of lactoferrin from milk |
| CN113063860A (en) * | 2021-02-04 | 2021-07-02 | 浙江方圆检测集团股份有限公司 | Method for determining lactoferrin content in infant milk powder |
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| PARRÓN JOSÉ ANTONIO ET AL: "Antirotaviral potential of lactoferrin from different origin: effect of thermal and high pressure treatments", BIOMETALS, KLUWER ACADEMIC PUBLISHERS, NL, vol. 31, no. 3, 26 February 2018 (2018-02-26), pages 343 - 355, XP036511443, ISSN: 0966-0844, [retrieved on 20180226], DOI: 10.1007/S10534-018-0088-4 * |
| SALMON V ET AL: "PRODUCTION OF HUMAN LACTOFERRIN IN TRANSGENIC TOBACCO PLANTS", PROTEIN EXPRESSION AND PURIFICATION, ACADEMIC PRESS, SAN DIEGO, CA, vol. 13, no. 1, 1 June 1998 (1998-06-01), pages 127 - 135, XP000863470, ISSN: 1046-5928, DOI: 10.1006/PREP.1998.0886 * |
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