WO2005068502A1

WO2005068502A1 - Method of lactoferrin separation and purification

Info

Publication number: WO2005068502A1
Application number: PCT/JP2005/000804
Authority: WO
Inventors: Hiroyoshi Inoue
Original assignee: Kurume University
Priority date: 2004-01-20
Filing date: 2005-01-17
Publication date: 2005-07-28

Abstract

A method of lactoferrin separation and purification, comprising the step of mixing an aqueous solution of lactoferrin, an organic solvent and an anionic surfactant or anionic lipid together under agitation and recovering an organic phase; and the step of adding an alkali aqueous solution to the organic phase, agitating the mixture and thereafter recovering a water phase. Lactoferrin of high purity can be easily obtained by virtue of this method. The lactoferrin obtained by this method is suitable for use as a raw material for food, medicine, cosmetic or the like.

Description

Method of separating and purifying rata toferin

Technical field

The present invention relates to a method for separating and purifying lactofurin. Light

Background art

Rice field

Lactoferrin is an iron-binding glycoprotein with a molecular weight of about 800,000, which binds two irons in one molecule. Lactoferrin is present in the body fluids of many mammals, for example, in milk. In particular, it is known that the colostrum of breast milk contains 5 to 10 g / L, which accounts for 30% to 70% of the total protein contained. Lactoferrin is an important protein for maintaining and developing the health of infants, and has recently been shown to have antibacterial and antibacterial activities, and is used in various fields besides the food industry. ing.

Lactoferrin is generally extracted from colostrum, normal milk, cheese whey (residue generated during cheese production), etc. (for example, Mamoru Tomita, MRC News 21, 1998, p. 247; and Mamoru Tomita, Foods Food Ingredients J. Jpn, Vol. 181, 1999, pp. 33-41). In cheese whey, which has a pH of about 6, ratatoferrin is cationic, whereas most other proteins are anionic, and this property is used for extraction. For example, Mamoru Tomita, MRC News 21, 1998, p. 247, states that whey is contacted with a cation exchange resin to adsorb ratatoferin on the cation exchange resin, and the resin is concentrated in a high-concentration salt solution. It describes that ratatoferrin is removed by washing with a water filter, and then the lactoferrin-containing eluate is desalted by ultrafiltration to obtain a lactoferrin concentrate. However, this method has many steps and is not efficient. Furthermore, this method In this case, operations such as regeneration of the ion-exchange resin are complicated, and the extraction efficiency is about 50%.

In addition, various methods for separating and purifying small amounts of ratatoferin at the laboratory level have been studied. For example, Clovis K. Chiu and Mark R. Etzel, Journal of Food Science, Vol. 62, No. 5, 1997, pp. 996-1001, describe that cheese whey was prepared by a simple diffusion method using a cation exchangeable cellulose membrane. A method for separating lactoferrin from lactoferrin is described. However, this method is time-consuming and inefficient in separating. Other methods include separation by electrophoresis (Hurly WL et al., J Dairy Sci, vol. 76, 1993, p. 377) and separation by affinity chromatography (MK Walsh and SH Nam, Prep. Biochem. Biotechnol. , Vol. 31, No. 3, 2001, pp. 229-240) and a separation method by capillary electrophoresis (Peter Riechel et al., Journal of Chromatography A, Vol. 817, 1998, pp. 187-193).

Reverse micelle extraction is known as a method for separating and purifying proteins. Reverse micelles are molecular assemblies of amphiphilic compounds formed in organic solvents. In general, biomolecules such as proteins may be denatured in a hydrophobic environment such as in an organic solvent, but among the above-described reverse micelles, in particular, reverse micelles prepared with an ionic surfactant are used. It is known that the protein can be safely dissolved in an organic solvent by using. So far, it has been reported that, for example, c-chymotrypsin, lipase, cytochrome c, hemoglobin, etc. can be extracted by the reverse micelle extraction method (Yamada et al., J. Chem. Eng. No. 1994, 404-409; Ziyi Hu and Erdogan Gulari, Biotechnol. Bioeng., 50, 1996, 203-206; and Goto et al., Biotechnol. Bioeng., 54, 1997, 26-32). In these methods, various surfactants such as anionic surfactants, cationic surfactants, and nonionic surfactants are used. And the relationship between reverse micelles and proteins Investigations have not been conducted sufficiently, and the reverse micelle extraction method has not yet been put to practical use. Disclosure of the invention

An object of the present invention is to provide a method for easily separating and purifying high-purity lactoferrin.

The method for separating and purifying lactofurin according to the present invention comprises the steps of: mixing and stirring a lactofurin-containing aqueous solution, an organic solvent, and an anionic surfactant or an anionic lipid, and recovering an organic phase; and After adding an aqueous alkaline solution to the mixture and stirring, and then collecting an aqueous phase.

In a preferred embodiment, in the step of recovering the organic phase, the stirring is performed using ultrasonic treatment.

In a preferred embodiment, the pH of the ratatoferrin-containing aqueous solution is 5-7.

In a preferred embodiment, the pH of the alkaline aqueous solution is 8-9.

In a preferred embodiment, the method further comprises the step of adjusting the pH of the aqueous phase to 0.5 to 3.0.

According to the method of the present invention, ratatoferrin can be easily separated and purified in two steps, and the obtained ratatofurin has high purity. Further, by adjusting the pH of the aqueous phase to 0.5 to 3.0, it is possible to obtain ratatoferin containing a high proportion of iron-free lactoferrin (aporatatoferin) having a high antibacterial property. it can. Brief Description of Drawings

FIG. 1 shows the SDS-polyacrylic acid for the aqueous phase obtained by the method of the present invention. It is a photograph of luamide gel electrophoresis.

FIG. 2 is an HPLC chart of the analysis of ratatoferin in cheese whey (left) and the aqueous phase obtained by the method of the invention (right).

FIG. 3 is a schematic diagram of an extraction device used in the method of the present invention.

FIG. 4 is a graph showing a time-dependent change in the concentration of lactoferrin in an aqueous phase when lactoferrin is extracted from cheese whey using the apparatus shown in FIG. BEST MODE FOR CARRYING OUT THE INVENTION

The principle of the method of the present invention is based on the reverse micelle extraction method described above. That is, when an organic solvent and an anionic surfactant or an anionic lipid are added to an aqueous solution containing ratatoferin, which is a raw material, and stirred, ratatoferin becomes anionic surfactant or anionic lipid. It is retained in the hydrophilic part inside the reverse micelle formed by the above and moves to the organic phase. Lactoferrin has an isoelectric point of 7.5 to 8.0, so if an alkaline aqueous solution is added to the recovered organic phase and the mixture is stirred again, ratatofurin transfers to the aqueous phase. Usually, other proteins coexisting with ratatoferin do not show such behavior, so that only lactoferrin can be recovered with high purity.

Therefore, in the method of the present invention, after mixing and stirring the aqueous solution containing ratatoferin, the organic solvent, and the anionic surfactant or anionic lipid, collecting the organic phase, and adding an alkaline aqueous solution to the organic phase. After stirring, ratatoferin is separated and purified by the step of collecting the aqueous phase.

The ratatofurin-containing aqueous solution used in the method of the present invention is not particularly limited as long as it is an aqueous solution containing lactoferrin. Examples of such an aqueous solution include secretions of mammals such as milk (eg, milk); processed milk products such as skim milk and whey; microorganisms (including ratatoferin transgenic bacteria) Alternatively, a cell culture solution or cell supernatant may be used. Here, the ratatofurin concentration of the lactoferrin-containing aqueous solution is not particularly limited. Milk and whey are preferred because they contain large amounts of lactoferrin and are readily available, and whey is more preferred in terms of waste utilization. For example, whey typically contains about 10 Omg / L lactoferrin.

The pH of the ratatoferrin-containing aqueous solution is preferably 5 to 7. The pH can be adjusted by, for example, sodium hydroxide aqueous solution, dilute hydrochloric acid, phosphoric acid, acetic acid, succinic acid, or citric acid. This is done by adding a regulator. If the pH of the ratatoferrin-containing aqueous solution is 5 to 7, it can be used as it is as a ratatoferrin-containing aqueous solution. For example, whey has a pH of about 6. There is no particular limitation on the salt concentration in the aqueous solution containing ratatofurin. Considering the extraction efficiency of ratatoferrin, a low concentration is preferred. For example, in an aqueous solution of ratatofurin, the concentration of sodium chloride is preferably 10 O mM or less.

The organic solvent used in the method of the present invention is not particularly limited as long as it is an organic solvent that is phase-separated without being mixed with water. Preferred are aliphatic hydrocarbons and fatty acid alcohols, for example, octanol, decanol, hexane, heptane, isooctane and the like. Octanol is preferred in that the yield of ratatoferin is good, and isooctane is preferred in that the solvent odor attached to ratatoferrin obtained is small. The above organic solvents may be used alone or in combination. For example, an organic solvent in which isooctane and decanol are mixed at a weight ratio of 1: 0.02 to 1: 0.1 has a good yield of lactoferrin and a low solvent odor of ratatoferin. It is particularly suitable.

The anionic surfactant or anionic lipid used in the method of the present invention has no particular limitation on the length of the alkyl chain which is a hydrophobic moiety. Anionic surfactant Alternatively, the anionic lipid can be appropriately selected depending on the purpose of use of lactoferrin.For example, when ratatoferrin is used in food, anionic surfactant or anionic lipid that can be used as a food additive is used. Is preferred. Examples of anionic surfactants include sodium di-2-ethylhexylsulfosuccinate (AOT), sodium di (trihexyl) sulfosuccinate (ATR), sodium dioctylsulfosuccinate (AOC), and sodium dioleinosulfosuccinate (AOC). AOL), sodium bis (2-ethylhexyl) phosphate (NaDEHP), 1,2-dioleoyl-sn-glyceric acid 13-phosphatidic acid (DOLPA), sodium tetraphenylborate (TPBNa), laurylbenzenesulfone Acid sodium (LB SNa) and the like. Examples of anionic lipids include long chain fatty acid salts, and typically include palmitate, oleate (eg, sodium oleate), linoleate, caprylate, stearate, and araquine. Acid salts, behenates, lignocerates, cerotates, arachidates, melinates, erucinates, linolenates, arachidonates, eicosapentaenoates, and the like. These anionic surfactants or anionic lipids may be used alone or in combination.

In the method of separating and purifying lactoferrin of the present invention, first, an aqueous solution containing ratatoferin, an organic solvent, and an anionic surfactant or an anionic lipid are mixed and stirred. From the viewpoint of preventing the formation of the intermediate phase and promoting the phase separation, a modifier such as tributyl phosphate may be mixed in advance if necessary.

The mixing ratio of the lactoferrin-containing aqueous solution, the organic solvent, and the anionic surfactant or anionic lipid is not particularly limited as long as reverse micelles can be formed in the organic solvent. For organic solvents, the higher the amount, the higher the yield of ratatoferrin. For example, aqueous solutions containing ratatofurin are usually The organic solvent may be mixed with 100 parts by mass to 100 parts by mass with respect to 100 parts by mass of the liquid, and may be mixed with 200 parts by mass to 1000 parts by mass. The anionic surfactant or anionic lipid is usually mixed with 0.1 part by mass to 100 parts by mass with respect to 100 parts by mass of the lactoferrin-containing aqueous solution, and preferably the lactoferrin used is mixed. Depending on the type of the phosphorus-containing aqueous solution, 0.1 to 50 parts by mass, 0.1 to 20 parts by mass, or 0.1 to 10 parts by mass can be mixed. When the lactopurine-containing aqueous solution is whey, for example, 100 parts by mass of whey, 2 parts by mass to 100 parts by mass of an organic solvent, 100 parts by mass of an anionic surfactant or an anion 0.1 to 20 parts by weight, preferably 0.1 to 10 parts by weight, of the active lipid may be mixed.

Stirring is performed by stirring with a stirrer or a stirring blade, shaking, ultrasonic treatment, or the like. In particular, in this step, sonication is preferred in terms of extraction efficiency. The temperature for stirring by these means is usually 4 ° C. to 37 ° C., preferably room temperature. In the case of sonication, the output, frequency, etc. commonly used by those skilled in the art can be employed. The processing time is usually 3 minutes to 30 minutes. On the other hand, when stirring or shaking, preferably 60 r ρ π! Can be performed at ~ 100 rpm, for 5 minutes to 60 minutes.

After stirring, the organic phase is separated into an organic phase and an aqueous phase for recovery. Although the phase separation can be achieved by allowing the mixture to stand still, centrifugation or the like may be performed in order to perform processing in a short time. Centrifugation can be performed, for example, at 2000 rpm for 2 minutes. After phase separation, the organic phase can be recovered by liquid separation means commonly used by those skilled in the art. By this operation, ratatoferin is taken into the hydrophilic portion (water pool) or the micelle interface inside the reverse micelle formed in the organic phase by electrostatic interaction, and is transferred (extracted) to the organic phase. At the same time, some of the proteins other than lactoferrin contained in the aqueous solution containing lactoferrin are retained in the reverse micelles and transferred to the organic phase. Next, an alkaline aqueous solution is added to the collected organic phase, followed by stirring. By this operation, the reverse micelles holding lactoferrin become unstable, lactoferrin detaches from the reverse micelles, and ratatoferin transfers to the aqueous phase. At this time, the protein transferred to the organic phase together with lactoferrin by the previous operation hardly transfers to the aqueous phase.

The aqueous solution is adjusted to a pH around the isoelectric point of lactoferrin so as to weaken the interaction between ratatoferin and anionic surfactants or anionic lipids. Preferred pH is 8-9, more preferably 8-8.5. If the pH is higher than 9, ratatoferin may be denatured, which is not preferable. For pH adjustment, pH adjusters commonly used by those skilled in the art can be used.

Stirring is performed by stirring, shaking, ultrasonic treatment, or the like using a stirrer or a stirring blade as described above. Here, any stirring means may be used. After adding an alkaline aqueous solution to the organic phase and stirring, the aqueous phase is separated into an organic phase and an aqueous phase in order to recover the aqueous phase. Although the phase separation can be achieved by allowing the mixture to stand, centrifugation or the like may be performed in order to perform processing in a short time.

The aqueous phase obtained after adding the aqueous solution to the organic phase and stirring the mixture contains ratatoferin. The proportion of lactofurin in the total protein contained in the aqueous phase is very high, preferably 70% by mass or more, more preferably 90% by mass or more of the total protein. Lactoferrin becomes apolactoferin when iron is released, and exerts an antibacterial effect. Then, in order to further release iron from ratatofurin contained in the aqueous phase, the pH of the recovered aqueous phase is adjusted to preferably 0.5 to 3.0, more preferably 2. As the pH adjuster, for example, hydrochloric acid, phosphoric acid, phthalic acid, glycine and the like are used.

The aqueous phase containing ratatoferin is then concentrated, dried, And can be formulated through processing such as powdering. In addition, the amount of ratatofurin is measured by a method commonly used by those skilled in the art, such as an immunochemical method using an anti-lactofurin antibody and HPLC.

Lactoferrin obtained by the method of the present invention has a very high purity and can be used as a raw material for foods, pharmaceuticals, cosmetics and the like.

(Example)

(Example 1)

ゥ -Silactoferrin (Sigma) was dissolved in 50 g of pasteurized milk to obtain lOOg / g. Add AOT so that the final concentration is 2 OmM.

(Tokyo Kasei Co., Ltd.) and 150 g of isooctane containing 4% by mass of decanol were added, followed by vigorous shaking at room temperature. After allowing this mixture to stand for 10 minutes, the organic phase was recovered. Next, 20 g of an aqueous sodium hydroxide solution adjusted to pH 8.5 was added to the obtained organic phase, and the mixture was shaken and stirred at room temperature for 5 minutes. After the mixture was allowed to stand for 10 minutes, the aqueous phase was recovered. The same operation was performed twice in total, and the obtained aqueous phases were designated as aqueous phases 1 and 2, respectively.

(Example 2)

Instead of using 150 g of isooctane containing AOT to a final concentration of 2 OmM and containing 4% by mass of decanol, containing AOT and 4% by mass of decanol to a final concentration of 2 OmM An aqueous phase was obtained in the same manner as in Example 1 except that 300 g of isooctane was used (aqueous phase 3). (Example 3: Confirmation of ratatoferin by SDS-PAGE)

The proteins contained in the aqueous phases 1 to 3 obtained in the above Examples 1 and 2, It was confirmed by SDS-PAGE under reducing conditions. Aqueous phases 1 to 3 were applied to two lanes, 100 L each (lanes 4 to 9 in Fig. 1). In addition, molecular weight markers (lane 1 in Fig. 1), raw pasteurized milk (lanes 2 and 3 in Fig. 1), and an aqueous solution containing ratatoferin (200 µg / mL) (lanes 10 and 1 in Fig. 1) 1) was applied in 10 L increments (10 times the amount of the aqueous phase). The results are shown in Figure 1.

As shown in Fig. 1, various bands were detected in the pasteurized milk as a raw material (lanes 2 and 3), whereas in the aqueous phase 1-3 (lanes 4 to 9), the target was A single band was observed at around 80,000, the same molecular weight as the product ratatoferrin. This indicates that the method of the present invention can easily separate and purify ratatoferin. In particular, the aqueous phase 3 (lanes 8 and 9) has a stronger band than the aqueous phases 1 and 2, and it is considered that the recovery of lactoferrin is increased by using a larger amount of the organic solvent. (Example 4: Determination of lactoferrin by HP LC)

The amount of ratatoferin in the aqueous phase 1 obtained in Example 1 was quantified by HPLC as follows. Using an HP LC system (Gilson), a Mono S5 50 GL column (strong acidic cation exchange column, Amersham Biosciences) was used as the column, and 0.3 M NaCl was used as the mobile phase. A 0.2 M sodium phosphate buffer (pH 7.5) was used. The flow rate was set at 2. OmLZ and the detection wavelength was set at 280 nm. After injecting 200 ^ L of each sample prepared by diluting the aqueous phase with ion-exchanged water 5 to 20 times, the concentration of NaCl in the mobile phase was adjusted to 0.3 M for 8 minutes and 0.9 M for 4 minutes. Eluted for 2 min, 2.0 M for 4 min, and 0.3 M for 8 min. Lactoferrin eluted with 0.9 M NaCl (retention time about 10 minutes). A calibration curve for the standard sample was created based on the elution peak area, and the amount of lactoferrin contained in the aqueous phase was measured. It was calculated from the peak area obtained. As a result, the amount of lactoferrin in the aqueous phases 1 to 3 was all about 290 g. The recovery rate of lactoferrin calculated from the amount of lactoferrin added was 4.7%. Since lactoferrin was contained in pasteurized milk in a small amount and contained no power, ratatoferrin could not be quantified after the operation.

Furthermore, the total protein in the aqueous phase was quantified by the Lowry method, and the purity of lactoferrin relative to the total protein in the aqueous phase was calculated. The result was 97.2%. (Example 5)

Perilla tofferin (Sigma) was dissolved in 50 g of pasteurized milk so as to be 100 // gZ g. In addition, 150 g of isooctane containing AOT (Tokyo Kasei Co., Ltd.) and 4% by mass of decanol to give a final concentration of 2 OmM, and an ultrasonic disperser (TA-4021, Riki Ijo Sonic) The sample was treated at a power of 50 W and a frequency of 28 kHz for 5 minutes. After the obtained mixture was allowed to stand for 10 minutes, the organic phase was recovered. An aqueous sodium hydroxide solution was further added, and the mixture was shaken and stirred at room temperature for 5 minutes, and then the aqueous phase was recovered. Lactoferrin in the aqueous phase was quantified by HP LC in the same manner as in Example 4 above. The recovery of ratatoferin was 11.4%.

(Example 6)

A water phase was prepared in the same manner as in Example 1 except that another anionic surfactant, sodium laurylbenzenesulfonate (LBSNa: Nacalai Tester Co., Ltd.) was used instead of AOT. Was recovered. Lactoferrin in the aqueous phase was quantified by HPLC in the same manner as in Example 4 above. Ratataferin recovery rate was 2.8. /. Met. (Example 7)

A water phase was prepared in the same manner as in Example 1 except that sodium tetraphenylborate (TPBNa: Nacalai Tesque, Inc.), another anionic surfactant, was used instead of AOT. Was recovered. Ratatoferin in the aqueous phase was quantified by HPLC in the same manner as in Example 4 above. The recovery of ratatoferrin was 1.1%.

(Comparative Example 1)

Instead of AOT, use a nonionic surfactant, 3 — [(3-colamid dope pill) 1-dimethinoleammonio] 1-1-propanesnolefonic acid (CHAP S: Nakarai Testa Co., Ltd.) A water phase was recovered in the same manner as in Example 1 except for the above. Lactoferrin in the aqueous phase was quantified by HP LC in the same manner as in Example 4 above. As a result, lactoferrin was not detected in the aqueous phase.

(Comparative Example 2)

The aqueous phase was recovered in the same manner as in Example 1, except that benzyltriethylammonium chloride (Nacalai Tester Co., Ltd.), which is a cationic surfactant, was used instead of AOT. Ratatoferin in the aqueous phase was quantified by HP LC in the same manner as in Example 4 above. As a result, ratatofulin could not be detected in the aqueous phase.

(Example 8: Examination of amount of organic solvent)

ゥ Dissolve silatatoferin (Sigma) in 9 mL of water, add 1 mL of 1 M HC1, and add 1 mL of 200 / ig Zml or 1 mg / mL rata. Multiple toferin solutions were prepared. To these, anionic surfactants or anionic lipids were added to a final concentration of 5 mM or 0.5 mM, as described in Table 1 below. Then, isooctane containing 20 OmM decanol was calorie from 2 OmL to 100 OmL as shown in Table 1 below, and stirred for 30 minutes. After allowing to stand for 5 minutes, the supernatant was taken out, and the aqueous solution of 119 was added with 5111 and stirred at room temperature for 10 minutes. After allowing to stand for 5 minutes, only the lower liquid (aqueous phase) is removed, and lactoferrin is assayed by ELISA using the Bovine Lactoferrin ELISA Kit (BETHYL LABORATORIES, INC). Quantified. Table 1 shows the results. As can be seen from Table 1, the greater the amount of organic solvent, the higher the recovery of ratatoferin in the aqueous phase. The recovery of lactoferrin was highest when sodium oleate was used as the anionic substance.

table 1

AOT: sodium di-2-ethylhexyl sulfosuccinate (Tokyo Kasei Kogyo Co., Ltd.) DOLPA: 1, 2-dioleoil-1 sn-glycemic acid 3-phosphatidic acid

(BIOMOL INTERNATIONAL, LP)

TPBNa: Sodium tetraphenylborate (Nacalai Tesque, Inc.)

LBSNa: Sodium laurylbenzenesulfonate (Nacalai Tesque, Inc.)

Na oleate: sodium oleate (Nacalai Tesque, Inc.) (Example 9: Recovery of lactoferrin from cheese whey) Cheese whey (manufactured by Ohm Dairy Co., Ltd.) was prepared, and the lactoferrin concentration in the whey was measured by ELISA using a ゥ lactoferrin measurement kit. Quantified. 2 OmL of this cheese whey was collected and sodium tetraphenylborate (TPBNa) was added to a final concentration of 5 mM or sodium oleate to 0.5 mM. Further, 10 OmL of isooctane containing 20 OmM decanol was added, and the mixture was stirred at room temperature for 30 minutes. After standing for 5 minutes, only the supernatant was taken out, 1 OmL of an aqueous solution of pH 9 was added, and the mixture was stirred at room temperature for 10 minutes. After allowing to stand for 5 minutes, only the lower liquid (aqueous phase) was taken out and quantified using the above-mentioned ゥ silatatoferin measurement kit. Table 2 shows the results. Table 2

At the same time, other proteins were quantified by HP LC. The conditions for HPLC are as follows:

Column: CAPCELL PAK C ₈ SG300 S5 (Shiseido)

Mobile phase: (A) CH ₃ CNZO.5M NaCl / trifluoroacetic acid = 50/450 / 0.15 (v / v / v)

(B) CH ₃ CN / 0.5 NaCl / trifluoroacetic acid = 500/450 / 0.3 (v / v / v) A / B (v / v): 50/5 0 → 10/9 0 (20 minutes) Gent flow rate: 0.8 mL / min

Detection: UV detection (280 nm) Figure 2 shows the HP LC chart. In FIG. 2, peak 1 shows immunoglobulin G (IgG), and peak 2 shows lactoferrin. By the extraction operation, proteins in the cheese whey were removed, and proteins other than ratatoferin were hardly detected in the aqueous phase. The purity of the extracted lactoferrin was over 96%.

(Example 10: Temporal change of lactoferrin extraction)

Using a device as shown in Fig. 3, the time-dependent change of ratatoferin extraction was examined. The apparatus is configured such that phases I and III are completely separated, phases I and III are each in contact only with phase II, and each phase is provided with a stirring means.

First, cheese whey (manufactured by Ohm Dairy Co., Ltd.) was prepared, and the ratatoferin concentration in the whey was measured by the ELISA method using Siratatoferin measurement kit. Add 2 OmL of this cheese whey to phase I of the device in Fig. 3 and add sodium tetraphenylborate (TPBNa) to a final concentration of 5 mM or sodium oleate to a final concentration of 0.5 mM. I got calories. Phase III contained 2 OmL of an aqueous solution of pH 9. On top of phases I and III, 10 OmL of isooctane containing 20 OmM decanol was placed as phase II.

Each of the phases was stirred at 120pmpm, and the ratataferin concentration of phase III was measured over time using the above-mentioned ゥ silatatofurin measurement kit. Figure 4 shows the results. As shown in Fig. 4 (1), the concentration of lactoferrin in phase III (aqueous phase) increased with the elapse of the stirring time. Next, the extracted whey (phase I) and the aqueous phase containing ratatofurin (phase III) were taken out, a new whey and aqueous phase were charged, and the mixture was stirred as before. The results are shown in Fig. 4 by a triangle. By repeating this operation, the whey can be continuously reduced from whey. It was found that ferrin could be recovered. Industrial applicability

According to the method of the present invention, lactoferrin can be separated and purified by a simple step of performing liquid-liquid extraction twice, and high-purity ratatoferin can be easily obtained. Furthermore, since continuous extraction can be performed, extraction of ratatoferin from a large amount of whey can be performed more easily. Ratatofurin obtained by the present invention is suitable for use as a raw material for foods, pharmaceuticals, cosmetics and the like.

Claims

The scope of the claims

1. A method for separating and purifying lactoferrin,

Mixing and stirring the ratatoferin-containing aqueous solution, the organic solvent, and the anionic surfactant or anionic lipid, and then collecting the organic phase, and adding an alkaline aqueous solution to the organic phase, stirring, and then adding water. Recovering the phase.

2. The method according to claim 1, wherein in the step of recovering the organic phase, the stirring is performed using ultrasonic treatment.

3. The method according to claim 1, wherein the pH of the ratatofurin-containing aqueous solution is 5 to 7.

4. The method according to any one of claims 1 to 3, wherein the pH of the alkaline aqueous solution is 8 to 9.

5. The method according to any one of claims 1 to 4, further comprising the step of adjusting the pH of the aqueous phase to 0.5 to 3.0.