RU2634859C1 - Method for lactoferrin isolation and purification from dairy raw materials - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: method for lactoferrin isolation and purification from dairy raw materials - cow's milk, comprising one-stage chromatography of lactoferrin from dairy raw material in a column with a sorbent equilibrated with sodium phosphate buffer, lactoperoxidase elution with sodium phosphate buffer, lactoferrin elution in a gradient of sodium chloride, desalting and lyophilic drying.

EFFECT: expansion of the arsenal of methods for lactoferrin isolation and purification from dairy raw materials and obtaining highly purified lactoferrin, while maintaining its enhanced biological activity.

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Description

The field of technology.

The invention relates to the dairy, biotechnological, medical, pharmaceutical, food industries. A chromatographic one-stage industrial method for the isolation and purification of cow lactoferrin from dairy raw materials is proposed, which makes it possible to simultaneously extract at least 90% of the lactoferrin contained in it, free of lactoperoxidase and endotoxins active against microorganisms, using an ion-exchange sorbent based on a hydrophilic macroporous acrylic polymer. The invention allows to selectively obtain highly purified lactoferrin, while maintaining its biological (bactericidal) activity, while the milk raw materials used to produce lactoferrin remain suitable for further processing in the food industry.

The level of technology.

In the food (dairy) industry, chromatography is a generally accepted method for isolating lactoferrin from milk and milk raw materials.

The prior art method for the production of lactoferrin from dairy raw materials (Patent RU 2579661). The method involves the extraction of raw milk on a cation exchange resin using a concentrated solution of sodium chloride. Get a solution of basic milk protein containing lactoferrin, lactoperoxidase and other impurities. The solution of the main milk protein is purified on a cation exchange resin balanced with acetate buffer with a concentration of 50 mM at pH values from 4 to 9. Elution is carried out with solutions of acetate buffer with a concentration of 50 mM at different concentrations of sodium chloride solution - from 0.02 to 1.5 M The difference between this method and the claimed one is the presence of a greater number of cleaning steps and the fact that the remaining raw materials may not be suitable for further use in the food industry. The disadvantage is the fact that the raw material used is milk that has not been processed at a temperature higher than 55 ° C.

A known method of purification of milk raw materials in order to obtain lactoferrin, consisting of sorption of proteins on an ion exchanger, chromatographic separation, elution, determination of fractions with optical density at a wavelength of 280 nm more than 0.1 units, dialysis of the obtained protein fraction against physiological saline, phosphate buffer or distilled water. Stabilization and microbiological purification is carried out by microfiltration through semi-permeable membranes of 0.22-0.45 microns. The disadvantage of this invention is the relative complexity associated with the phased purification of protein, as well as the inability to reuse dairy raw materials for consumption (Patent RU 2366294).

A known method of purification of dairy raw materials in order to obtain lactoferrin (Patent RU 2400106). The method includes the purification of milk raw materials, separation, casein precipitation, two-stage ultrafiltration, chromatographic separation of proteins, dialysis, microfiltration with pore selectivity of 0.2-0.3 μm, mixing the selected monofractions in a ratio similar to their content in freshly milked cow milk, lyophilic drying. At the first stage of ultrafiltration, the immunoglobulin fraction is separated from the serum through 100 kDa membranes. Permeate is subjected to repeated ultrafiltration through 30 kDa membranes. The protein concentrate thus obtained is subjected to ion-exchange chromatography, eluting with a NaCl gradient from 0.35 to 1 M to obtain lactoferrin and lactoperoxidase fractions. The disadvantage of this invention is the relative complexity associated with the phased purification of the protein, the need for separation of ballast proteins, and the inability to reuse dairy raw materials for consumption.

A known method of producing lactoferrin from human milk, including degreasing, removal of caseins, fractionation with ammonium sulfate, dialysis and lyophilization of the target product. Fractionation is carried out in steps: first, ammonium sulfate is added to a concentration of 35-45% of saturation, the precipitate is removed, then ammonium sulfate is added to the supernatant to a concentration of 70-80% of saturation, and before dialysis, the precipitate from fractionation is dissolved in distilled water, incubated from 1, 3-3.7 M sodium chloride at pH 3.6-4.4 and the precipitate formed is removed by filtration (Patent RU 1709606). The disadvantage of this invention is the relative complexity associated with the phased purification of the target protein, as well as the inability to reuse dairy raw materials for consumption.

There is also a method of producing lactoferrin from cottage cheese or cheese whey by ion exchange chromatography using ion exchange cellulose. The method consists in the following: milk whey obtained in the production of cottage cheese by acid or acid rennet methods is neutralized to pH 6.5 with a solution of sodium hydroxide, centrifuged to separate immunoglobulins and calcium phosphate; Further, the prepared serum is fed to a column with a sorbent carboxymethyl cellulose balanced with a pH 6.5 buffer solution containing 50 mmol sodium phosphate. Elution of lactoferrin is carried out using 0.05 M phosphate buffer containing 1.0 M sodium chloride. The resulting solution is desalted by ultrafiltration, subjected to microfiltration and freeze-dried (TI, TU 9224-001-93467423-06). The disadvantage of this method is that curd or cheese whey is used as a raw material, the content of lactoferrin in which is extremely limited, and also that the resulting lactoferrin in its composition contains impurities of other whey proteins.

The prior art also known technology for producing recombinant human lactoferrin from the milk of goat producers (patent RU 2554742). The method includes ion exchange chromatography on a strong cation exchanger, elution of lactoferrin in a gradient of sodium chloride of 0.3-1.0 M, desalination, freeze drying. The method is characterized in that milk of transgenic goats is used as milk raw material, chromatography is carried out on a strong cation exchanger carrying sulfopropyl groups, sodium acetate buffer is used, stepwise washing of the sorbent with 20 mM sodium acetate buffer pH 7.0 containing 0.3 M is used sodium chloride and 10% ethanol to remove LPS bound to lactoferrin.

Closest to the claimed is a method for producing lactoferrin from raw cow's milk, which is passed through a chromatographic column with a sorbent (prototype, patent RU 23902531). The method involves the use of milk as raw milk; chromatography of lactoferrin is carried out in a column with a sorbent carboxymethyl cellulose balanced with 0.05 M sodium phosphate buffer at pH 7.7, the lactoferrin is eluted with 0.05 M sodium phosphate buffer pH 7.7 in a concentration gradient of 0.1-1 sodium chloride, 2 M, when eluting, fractions of a lactoferrin solution with an optical density of 0.5 units are collected. at a wavelength of 280 nm and direct them to a second chromatography with similar parameters, and then the separated fractions are sent to ultrafiltration, desalination and lyophilization. The method allows to obtain native lactoferrin with a purity of not less than 98%. The disadvantage of this invention is the relative complexity associated with the phased purification of the target protein, as well as the inability to reuse dairy raw materials for consumption.

A known method for producing lactoferrin concentrate, which involves the use of an alternative method of membrane adsorption (Plate et al., 2006). Pre-treatment of serum in a microfiltration unit is carried out to isolate insoluble particles and milk fat that impede the operation of cation exchange membrane modules. The permeate obtained in the microfiltration stage is sent to a membrane adsorber. Active groups located on the inner surface of ion-exchange membranes are sulfonic acid radicals. Membrane modules are produced in various designs with sizes from 15 cm ² (0.41 ml) to 8 m ² (2.2 L) each. The dynamic exchange capacity of the spiral membrane module (15 turns) for lactoferrin is 0.2 mg / cm ² (7.3 mg / ml). The disadvantage of this method is the high cost of imported membrane cassettes and the possibility of particles of membrane fibers entering milk raw materials during protein isolation.

A known method for the purification of dairy raw materials (Patent RU 2400106). The method includes the purification of milk raw materials, separation, casein precipitation, two-stage ultrafiltration, chromatographic separation of proteins, dialysis, microfiltration with pore selectivity of 0.2-0.3 μm, mixing the selected monofractions in a ratio similar to their content in freshly milked cow milk, lyophilic drying. At the same time, at the first stage of ultrafiltration, the immunoglobulin fraction is separated from the serum through 100 kDa membranes. Permeate is subjected to repeated ultrafiltration through 30 kDa membranes. The protein concentrate thus obtained is subjected to ion exchange chromatography, illusory with a NaCl gradient of 0.35 to 1 M, to obtain lactoferrin and lactoperoxidase fractions. Mixing the obtained fractions - immunoglobulin, lactoferrin and lactoperoxidase is carried out in a ratio of 91: 5: 4. A feature is that the method does not provide for the separation of fractions and obtaining pure lactoferrin.

A known method of producing enzymes from the group of hydrolases and oxidases, lactoferrin and albumin (RU 2013144910). The method is characterized in that when carrying out the process according to a flexible technology using the same technological equipment in the form of sorption columns with macroporous sulfocathionite, when varying the conditions of isolation and purification, various target substances with a high yield can be obtained. The extraction is carried out with aqueous solutions with different pH and ionic strength for 0.5-3.0 hours, an ammonia solution with a pH of 9.0-11.0 with different ionic strength is used as an eluent.

Thus, technical solutions that coincide with the totality of the essential features of the claimed invention from the prior art have not been identified, which allows us to conclude that the claimed invention meets such a patentability condition as “novelty”.

Terms and Definitions

- The term “milk raw materials” means any raw materials selected from the group: whole milk, semi-skim milk, skim milk, buttermilk, colostrum, low and low fat milk, non-fat milk, whey (milk plasma), including including after removal of casein, a milk-based drink, yogurt, including frozen yogurt, drinking yogurt, cream, milk protein concentrate. The temperature of milk raw materials should not exceed 60 degrees Celsius.

- The term "sorbent" means a strongly acidic ion-exchange sorbent based on a hydrophilic acrylic macroporous polymer with an average particle size of 160 microns, bearing sulfopropyl groups.

Brief Description of the Drawings

FIG. 1. Comparison of technological methods for extracting cow lactoferrin.

FIG. 2. Sorbent - hydrophilic acrylic macroporous polymer with an average particle size of 160 microns, bearing sulfopropyl groups, under a microscope. It can be seen that the particle size of the sorbent is quite uniform, which ensures its satisfactory hydrodynamic properties.

FIG. 3. Growth curve of the Staphylococcus aureus clinical isolate strain using bovine lactoferrin compared to the control.

Fig 4. Identification of the target protein. Electrophoresis: 1 - standards (the positions of standard proteins with a known molecular weight in kDa are indicated on the left), 2 and 3 - obtained samples of bovine lactoferrin; Western blot (4-6): 4 and 5 - obtained samples of cow lactoferrin, 6 - standard cow lactoferrin (Sigma Aldrich)

Disclosure of the invention.

The proposed method is economically advantageous for industrial use, significantly reduces the time of isolation of the target protein and allows you to effectively get rid of ballast proteins. A significant advantage is the fact that the proposed method maintains the conditionality of milk raw materials with the aim of its further industrial processing.

Lactoferrin is a bactericidal protein. It is obtained under industrial conditions and is used for the production of functional and dietary foods, including children’s products, various hygiene products and a large number of parapharmaceutical medical products. Modern technologies for isolating cow's lactoferrin from cow's milk use two-stage chromatography, in which lactoferrin is removed from the sorbent together with lactoperoxidase (LPS), followed by the stage of their separation, which inevitably leads to technological losses of the target protein, increases the process time and increases financial costs.

A one-stage method for the isolation of cow lactoferrin can significantly simplify the technology of its preparation from dairy raw materials. As is known, the proportion of cow lactoferrin (about 0.1 g / l) is extremely small in cow's milk, which dictates the processing of large volumes of dairy raw materials during industrial processing.

The basis of the proposed method for the isolation of lactoferrin from dairy raw materials is the use of a special sorbent that satisfies the following properties:

a) the sorbent must be a strong cation exchanger containing sulfopropyl groups for faster and more durable protein binding. This allows milk raw materials to pass through the sorbent at high speeds, since it is known that the quantitative sorption of protein directly depends on the speed of pumping a protein solution through it;

b) the sorbent must have good hydrodynamic properties that allow it to work at high speed;

c) sufficient mechanical strength;

d) high sorption capacity.

The proposed separation and purification technology uses a column with a strongly acidic ion-exchange sorbent based on a hydrophilic macroporous acrylic polymer containing sulfopropyl groups balanced with 20 mM sodium phosphate buffer, pH 7.5. The sorbent used is a hydrophilic acrylic macroporous polymer satisfying the above properties with an average particle size of 160 μm (Fig. 2). It has a good sorption capacity (20 mg Lf / ml sorbent), hydrodynamic properties that allow it to work at high speeds (50-100 cm / h), as well as mechanical strength, which can withstand the pressure that occurs at these speeds.

The method of isolation and purification of lactoferrin from dairy raw materials is as follows. First, lactoperoxidase is eluted using a buffer containing 0.25 M NaCl, then lipopolysaccharides are removed by washing the sorbent with a buffer with 0.25 M NaCl and 10% ethanol, followed by elution of lactoferrin in a gradient of 0.25-1.0 M NaCl. The obtained fractions of proteins are desalted and dried. Milk passing through the chromatographic column does not change its composition and is suitable for further processing into traditional products (Table 1).

Since lactoferrin is able to bind to endotoxins (lipopolysaccharides, fragments of the cell wall of gram-negative bacteria), the problem of removing lipopolysaccharides from lactoferrin samples was simultaneously solved, which helps to preserve its antimicrobial activity.

The proposed technology for the isolation of lactoferrin using an ion-exchange sorbent based on a hydrophilic macroporous acrylic polymer satisfies all of the above requirements and, being a one-step process, allows to reduce industrial costs by more than 30%. This ensures a high level of extraction of lactoferrin of high purity, free of endotoxins, with high bactericidal and bacteriostatic activity, while dairy raw materials remain suitable for further processing into traditional products in the food industry.

A comparison of most methods with the proposed method for isolating lactoferrin is shown in FIG. one.

The technical result, manifested by using the proposed method for the isolation and purification of lactoferrin, is to obtain highly purified lactoferrin with a purity of at least 95% from milk and (or) milk raw materials through the use of a one-step chromatographic technology using a sorbent based on a hydrophilic acrylic macroporous polymer with an average size particles of 160 μm bearing sulfopropyl groups, including elution of lactoperoxidase using a buffer containing 0.25 M NaCl, removal of lipopolis charides by washing the sorbent with a buffer with 0.25 M NaCl and 10% ethanol, eluting the lactoferrin in a gradient of 0.25-1.0 M NaCl, followed by desalting and drying the fraction of the target protein.

Another technical result of the proposed method for the isolation and purification of lactoferrin is the expansion of the arsenal of funds for this purpose.

The claimed essential features that predetermine the receipt of the specified technical result, do not explicitly follow from the prior art, which allows us to conclude that the claimed invention meets such a patentability condition as "inventive step".

The patentability condition "industrial applicability" is confirmed by examples of specific applications by which the claimed invention is illustrated, but not exhausted.

Examples of carrying out the invention

Example 1. Isolation and purification of lactoferrin from dairy raw materials

10 l of skimmed cow's milk is passed through a column with a sorbent (100 ml) balanced with 20 mM sodium phosphate buffer pH 7.5 containing 0.1 M sodium chloride at a speed of 50-100 cm / h. After washing with a balancing buffer (3 column volumes), lactoperoxidase is eluted with 20 mM sodium phosphate buffer pH 7.5 containing 0.25 M sodium chloride, then the sorbent is washed with 20 mM sodium phosphate buffer pH 7.5 containing 0.25 M sodium chloride and 10% ethyl alcohol (2 column volumes). Elution of cow lactoferrin is carried out in a gradient of sodium chloride 0.25-1.0 M. Fractions containing cow lactoferrin and lactoperoxidase are desalted by dialysis or ultrafiltration or gel filtration, freeze-dried. Get the target protein with a purity of 95%, free from endotoxins with preserved bactericidal activity.

Example 2. Evaluation of the antimicrobial and antifungal activity of lactoferrin

Evaluation of antimicrobial activity was carried out in relation to a strain - a clinical isolate of Staphylococcus aureus. Used an automated system for monitoring the growth of microorganisms in real time. Isolation and cultivation of a microorganism strain was carried out in accordance with the previously described standard protocol (Davydova et al., 2013).

The growth dynamics of microorganisms was studied in two parallels, which was reflected in the graphs of the growth curves of bacterial populations in the presence of bovine lactoferrin. As a control, bacterial growth was assessed, which was characterized by the onset of the logarithmic growth phases after 2-3 hours and reached a maximum with the transition to the stationary growth phase after 12 hours from the start of cultivation. The stationary phase growth rate was significantly higher (from 6.0 to 7.2 OD) than when using lactoferrin. In the study of drug concentrations (10 mg / ml and higher) in the first 1-3 days after the introduction of lactoferrin, bacterial growth was not observed or it was very late compared to the control (Fig. 3).

Additionally, the bactericidal and antifungal activity of the lactoferrin obtained by the method against S. sangius, P. gingivalis and C. albicans was evaluated. Similarly to the results of the above experiment, lactoferrin also exhibits high inhibitory activity.

Example 3. Identification of the target protein

To test samples, lactoferrin was separated by gel electrophoresis under denaturing conditions. A membrane was cut out (Hybond-P, Amersham Biosciences) to fit the gel. Wet it with methanol, washed with distilled water. The membrane and gel were balanced in TGB transfer buffer with methanol (25 mM Tris-HCl, 250 mM glycine, 20% methanol) for 15 minutes on a rocking chair. The membrane and gel were placed in a BioRad Mini Trans-Blot Cell wet transfer cassette. Carried out the transfer for an hour with cooling and a current strength of 350 mA. After transfer, the membrane was placed in 5% skimmed milk powder diluted in PBS-T (1 × PBS, 0.1% Tween-20). Incubated for 1 hour on a shaker at room temperature. The membrane was placed in a solution of purified polyclonal antibodies with 3% milk in PBS-T. The membrane was incubated with a solution of secondary antibodies specifically recognizing rabbit immunoglobulins with 3% milk in PBS-T. Washed with PBS-T on a shaker at room temperature for 20 minutes, then two more times for 5 minutes. Developed using the Amersham ECL Plus Western Blotting Detection System (GE Healthcare), after which an image was obtained using a gel documentation system, indicating the presence of a highly purified target protein (Fig. 4).

Example 4. A comparative study of milk raw materials before and after passing through a chromatographic column.

A comparative study of milk raw materials before and after passing through a chromatographic column with a sorbent. The data are shown in table 1.

Comparison of milk data in the process of isolating cow lactoferrin showed that the composition of milk raw materials does not undergo any significant changes, except for the content of lactoferrin itself. After passing through a chromatographic column, unwanted impurities are not washed out from the sorbent. According to the above analyzes, raw milk after lactoferrin isolation corresponds to the regulatory documentation for milk TP ТС 033/2013 and TP ТСЧ 021/2011 and is suitable for use in processing for traditional dairy products.

Bibliography

1. Davydova M.M., Plakhtiy L.Ya. Tsarev V.N. Methods of microbiological research used in dentistry // in the book. Microbiology, virology, immunology of the oral cavity / ed. Prof. V.N. Tsareva. - M.: GEOTAR-Media. - 2013 .-- S. 223-268).

, Reif O, Ulber R, Scheper T. Isolation of bovine lactoferrin, lactoperoxidase and enzymatically prepared lactoferricin from proteolytic digestion of bovine lactoferrin using adsorptive membrane chromatography. J Chromatogr A. 2006 Jun 2; 1117 (1): 81-6.2. Plate K, Beutel S, Buchholz H, Demmer W,

, Reif O, Ulber R, Scheper T. Isolation of bovine lactoferrin, lactoperoxidase and enzymatically prepared lactoferricin from proteolytic digestion of bovine lactoferrin using adsorptive membrane chromatography. J Chromatogr A. 2006 Jun 2; 1117 (1): 81-6.

Claims (1)

The method of isolation and purification of lactoferrin from dairy raw materials - cow's milk, including chromatography of lactoferrin from dairy raw materials in a column with a sorbent balanced with sodium phosphate buffer, elution of lactoperoxidase with sodium phosphate buffer, elution of lactoferrin in a gradient of sodium chloride, desalting, different drying that chromatography is carried out in one stage, a strongly acidic ion-exchange sorbent based on hydrophilic acrylic macroporous poly is used as a sorbent an era with an average particle size of 160 μm bearing sulfopropyl groups balanced with 20 mM sodium phosphate buffer pH 7.5 containing 0.1 M sodium chloride at a speed of 50-100 cm / h, subsequent washing with a balancing buffer, elution of lactoperoxidase with 20 mM sodium -phosphate buffer pH 7.5, containing 0.25 M sodium chloride, followed by its removal, further washing the sorbent with 20 mm sodium phosphate buffer pH 7.5, containing 0.25 M sodium chloride and 10% ethyl alcohol, eluting lactoferrin in a gradient of sodium chloride 0.2 5-1.0 M, desalting the lactoferrin fraction by dialysis, or ultrafiltration, or gel filtration and freeze drying.

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