Method for producing lactoferrin-containing aqueous solution

Abstract

To provide means for sterilizing lactoferrin at a high temperature in a short time while maintaining the activity of the lactoferrin.SOLUTION: The present technique provides a method for producing a lactoferrin-containing aqueous solution, including a sterilization step of heat-sterilizing a lactoferrin-containing aqueous solution in which the total mass content of proteins other than lactoferrin is 1 / 12 or less of the mass content of lactoferrin, at a temperature of 100°C or higher. The present technique also provides a lactoferrin-containing aqueous solution in which the total mass content of proteins other than lactoferrin is 1 / 12 or less of the mass content of lactoferrin and which does not contain living bacteria.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present technology relates to a method for producing a lactoferrin-containing aqueous solution, and in particular to a method for producing a lactoferrin-containing aqueous solution that includes a sterilization step. [Background technology]

[0002] Sterilization is carried out to ensure the shelf life or safety of foods. For many foods, heating is used to kill microorganisms for sterilization. Several methods have been proposed for heat sterilization of lactoferrin, a food material that has been attracting attention in recent years, while maintaining its activity.

[0003] For example, Patent Document 1 below discloses a method for sterilizing an aqueous lactoferrin solution by heating after adjusting the ionic strength of the solution to satisfy a specific formula. Patent Document 2 below discloses a method for adjusting the pH of a liquid containing lactoferrin to 1.0 to 6.5 and heating it at a temperature of 60°C or higher.

[0004] In addition, the following Patent Document 3 discloses a lactoferrin composition having heat stability, which is composed of lactoferrin and at least one stabilizer selected from glycerol fatty acid ester, sodium caseinate, and lecithin. The Patent Document 3 states that the composition can be heated at 90°C or higher without inactivating LFs. The following Patent Document 4 discloses a heat-stable protein composition composed of a milk basic protein fraction and at least one stabilizer selected from soybean polysaccharides, xanthan gum, pectin, gum arabic, gum ghatti, carrageenan, locust bean gum, sodium caseinate, lecithin, and carboxymethylcellulose. The Patent Document 4 states that the composition can be heated at 90°C or higher without inactivating the milk basic protein fraction. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 2-108629 [Patent Document 2] Japanese Patent Application Publication No. 3-215500 [Patent Document 3] JP 2010-180219 A [Patent Document 4] JP 2014-193125 A Summary of the Invention [Problem to be solved by the invention]

[0006] Among the bacteria to be sterilized, some are difficult to kill. For example, spore-forming bacteria are difficult to kill. In order to sterilize bacteria that are difficult to kill, a high sterilization temperature may be adopted in heat sterilization. Examples of such heat sterilization methods include ultra-high temperature heat treatment (UHT) (heat treatment at 120 to 150°C for 1 to 5 seconds) and high temperature short time sterilization (HTST) (heat treatment at 72 to 75°C for 15 seconds). In addition, when a high sterilization temperature cannot be adopted, for example, a low temperature is adopted and the heat treatment time is extended. Examples of low temperature sterilization methods include low temperature hold sterilization (LTLT) (heat treatment at 62 to 65°C for 30 minutes).

[0007] The sterilization conditions to be adopted vary depending on the type of food. When producing foods that can be stored for a long time and distributed at room temperature, sterilization at particularly high temperatures is required. For example, when heating retort foods that can be stored for a long time, sterilization conditions of 120°C for 4 minutes or more are required to kill the spores of Clostridium botulinum, which is known to be the cause of food poisoning. In addition, the UHT method is the mainstream technology for heat sterilization of liquid foods such as soft drinks, milk drinks, and mineral water.

[0008] If lactoferrin could be sterilized at high temperature in a short time while maintaining its activity, it would be extremely useful for utilizing lactoferrin as a food ingredient.

[0009] As mentioned above, several methods for sterilizing lactoferrin have been disclosed. However, in the method described in Patent Document 1, in order to set the sterilization temperature to 120°C or higher, the ionic strength must be theoretically 0.1 μM or less in terms of salt. Even if the method described in Patent Document 2 is adopted, the activity of lactoferrin cannot be maintained by sterilization at a high temperature of 120°C or higher. The compositions of Patent Documents 3 and 4 contain a stabilizer, which changes the flavor and / or physical properties, and is expensive in terms of cost.

[0010] Therefore, a new method is required for sterilizing lactoferrin at high temperature in a short time while maintaining the activity of lactoferrin. [Means for solving the problem]

[0011] The present inventors have discovered a new method for sterilizing lactoferrin at high temperature in a short period of time while maintaining the activity of lactoferrin.

[0012] That is, the present technology provides the following: [1] A method for producing an aqueous solution containing lactoferrin, comprising a sterilization step of heat-sterilizing an aqueous solution containing lactoferrin, the aqueous solution having a total mass of proteins other than lactoferrin that is 1 / 12 or less of the mass of lactoferrin, at 100°C or higher. [2] The method of producing the lactoferrin-containing aqueous solution described in [1], wherein the salt concentration of the lactoferrin-containing aqueous solution subjected to the sterilization step is 5 mM or less. [3] The manufacturing method described in [1] or [2], wherein the lactoferrin concentration of the lactoferrin-containing aqueous solution subjected to the sterilization step is 400 mg / ml or less. [4] The method according to any one of [1] to [3], wherein the heat sterilization temperature in the sterilization step is 120°C or higher. [5] The method according to any one of [1] to [4], wherein the temperature and time of the heat sterilization in the sterilization step are 120° C. or higher and 4 minutes or longer, respectively. [6] The method for producing a lactoferrin-containing aqueous solution according to any one of [1] to [5], wherein the lactoferrin-containing aqueous solution subjected to the sterilization step does not contain a stabilizer. [7] The method for producing a lactoferrin according to any one of [1] to [6], wherein the COMT inhibitory activity of lactoferrin in the lactoferrin-containing aqueous solution subjected to the sterilization step is maintained at 80% or more after the sterilization step. The technology also provides the following: <1> A preparation step of preparing a lactoferrin-containing aqueous solution having a total content of proteins other than lactoferrin of 1 / 12 or less of the lactoferrin content by mixing a lactoferrin material with water; A sterilization step of heat-sterilizing the lactoferrin-containing aqueous solution itself at 120°C or higher for 4 minutes or more. A method for producing a lactoferrin-containing aqueous solution comprising the steps of: <2> The lactoferrin-containing aqueous solution, the total mass of which is 1 / 12 or less of the mass of lactoferrin, is sterilized by heating at 120°C or higher for 4 minutes or more. The lactoferrin-containing aqueous solution to be subjected to the sterilization step has not been pH-adjusted by the addition of hydrochloric acid, sodium hydroxide, or primary lactic acid. A method for producing a lactoferrin-containing aqueous solution. <3> The lactoferrin-containing aqueous solution, the total mass of which is 1 / 12 or less of the mass of lactoferrin, is sterilized by heating at 120°C or higher for 4 minutes or more. The lactoferrin-containing aqueous solution to be subjected to the sterilization step is not pH-adjusted by adding hydrochloric acid or sodium hydroxide, and The lactoferrin concentration of the lactoferrin-containing aqueous solution subjected to the sterilization step is 30 mg / ml or less. A method for producing a lactoferrin-containing aqueous solution. <4> The lactoferrin-containing aqueous solution to be subjected to the heat sterilization contains only the lactoferrin material and water. <1> The manufacturing method described in <5> The salt concentration of the lactoferrin-containing aqueous solution subjected to the sterilization step is 5 mM or less. <1> ~ <4> 13. The method for producing a semiconductor device according to claim 12, <6> The lactoferrin concentration of the lactoferrin-containing aqueous solution subjected to the sterilization step is 400 mg / ml or less. <1> or <2> The manufacturing method described in <7> The lactoferrin-containing aqueous solution to be subjected to the sterilization step does not contain a stabilizer. <1> ~ <6> 13. The method for producing a semiconductor device according to claim 12, <8> The COMT inhibitory activity of lactoferrin in the lactoferrin-containing aqueous solution subjected to the sterilization step is maintained at 80% or more after the sterilization step. <1> ~ <7> 13. The method for producing a semiconductor device according to claim 12,

[0013] The technology also provides: [8] A lactoferrin-containing aqueous solution in which the total mass of proteins other than lactoferrin is 1 / 12 or less of the mass of lactoferrin and which does not contain viable bacteria. [9] The lactoferrin-containing aqueous solution described in [8], having a salt concentration of 5 mM or less.

[10] A lactoferrin-containing aqueous solution according to [8] or [9], having a lactoferrin concentration of 400 mg / ml or less.

[11] The lactoferrin-containing aqueous solution according to any one of [8] to

[10] , which does not contain a stabilizer. Effect of the Invention

[0014] According to the present technology, lactoferrin can be sterilized at high temperature in a short time while maintaining the activity of lactoferrin. Note that the effects of the present technology are not limited to the effects described here, and may be any of the effects described in this specification. [Brief description of the drawings]

[0015] [Figure 1] 1 is a graph showing the results of measuring the turbidity of an aqueous lactoferrin-containing solution after autoclave sterilization. [Diagram 2] 1 is a graph showing the results of measuring the turbidity of an aqueous lactoferrin-containing solution after autoclave sterilization. [Diagram 3] 1 is a graph showing the results of measuring the turbidity of an aqueous lactoferrin-containing solution after autoclave sterilization. [Figure 4] 1 is a graph showing the relationship between the COMT inhibitory activity and turbidity of an aqueous solution containing lactoferrin. [Diagram 5] 1 is a graph showing the results of HPLC analysis of an aqueous solution containing lactoferrin. [Figure 6] 1 is a graph showing the results of measuring the COMT inhibitory activity of an aqueous solution containing lactoferrin. [Figure 7] 1 is a graph showing the results of measuring the antibacterial activity of an aqueous solution containing lactoferrin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Preferred embodiments of the present technology will be described below. However, the present technology is not limited to the following preferred embodiments and can be freely modified within the scope of the present technology.

[0017] 1. Method for producing lactoferrin-containing aqueous solution

[0018] (1) Sterilization process

[0019] The production method according to the present technology includes a sterilization step of heat-sterilizing an aqueous lactoferrin solution in which the total mass of proteins other than lactoferrin is 1 / 12 or less of the mass of lactoferrin at 100° C. or higher. This sterilization step allows lactoferrin to be sterilized at high temperature in a short time while maintaining its activity.

[0020] Lactoferrin is an iron-binding glycoprotein present in body fluids such as milk, tears, saliva, and blood, and is found in the milk of mammals, such as sheep, goats, pigs, mice, buffalo, camels, yaks, horses, donkeys, llamas, cows, or humans. The lactoferrin used in the present technology may be derived from mammalian milk. From the viewpoint of content and availability, for example, cow's or human milk is preferred as the source for obtaining lactoferrin, and particularly cow's milk is preferred as the source for obtaining lactoferrin. That is, for example, the lactoferrin used in the present technology may be derived from cow's or human milk, preferably from cow's milk. Milk may be any of colostrum, transitional milk, normal milk, and terminal milk. Commercially available lactoferrin may be used in the present technology. The lactoferrin used in the present technology may be lactoferrin separated by a standard method (e.g., ion chromatography, etc.) from skim milk or whey obtained by processing mammalian milk. The lactoferrin used in the present technology may be non-glycosylated or glycosylated.

[0021] Lactoferrin used in the food and beverage industry is usually milk-derived lactoferrin (particularly cow's milk-derived lactoferrin). Milk-derived lactoferrin is often obtained from whey. The milk-derived lactoferrin thus obtained also contains proteins other than lactoferrin. That is, lactoferrin used in the food and beverage industry is usually a composition containing lactoferrin and proteins other than lactoferrin. The present inventors have found that by reducing the content of proteins other than lactoferrin in an aqueous lactoferrin-containing solution to be subjected to heat sterilization, sterilization can be achieved at high temperature in a short period of time while maintaining the activity of lactoferrin. The above Patent Documents 1 and 2, which relate to methods for sterilizing lactoferrin, are not considered to be applied at temperatures above 100°C. The techniques described in the above Patent Documents 3 and 4, which are considered to be applied at temperatures above 100°C, are premised on the inclusion of a stabilizer for improving the heat resistance of lactoferrin. In this technique, by reducing the content of impurity proteins other than lactoferrin, it becomes possible to apply high temperatures of 100°C or higher, particularly high temperatures of 120°C or higher, to sterilization without the inclusion of the stabilizer.

[0022] The total mass of proteins other than lactoferrin in the lactoferrin-containing aqueous solution subjected to the sterilization step is 1 / 12 or less of the mass of lactoferrin. By adjusting the total mass of proteins other than lactoferrin in this manner, lactoferrin does not precipitate and can maintain its activity even when heated at a temperature of 100°C or higher. The proteins included in the present invention include proteins derived from milk such as casein and whey, proteins derived from microorganisms and airborne contaminating proteins in the lactoferrin purification process, proteins found in bovine blood, and proteins derived from medium components when lactoferrin is produced as a recombinant protein by E. coli. The total mass of proteins other than lactoferrin in the lactoferrin-containing aqueous solution subjected to the sterilization step is 1 / 12 or less of the lactoferrin mass, preferably 1 / 14 or less, more preferably 1 / 16 or less, even more preferably 1 / 18 or less, and particularly preferably 1 / 20 or less. By further reducing the total mass of proteins other than lactoferrin in this way, when heated at a temperature of 100°C or higher, especially when heated at a temperature of 120°C or higher, precipitation is less likely to occur and activity is more likely to be maintained. The total mass of proteins other than lactoferrin and the mass of lactoferrin can be measured by separation by HPLC. In detail, first, the lactoferrin-containing aqueous solution is separated by reversed-phase HPLC using Alliance e2695 (manufactured by Waters), and then the fractions that have a peak of absorption at 280 nm are separated into a fraction containing lactoferrin and a fraction containing proteins other than lactoferrin. The fractions containing lactoferrin can be confirmed, for example, by using a commercially available antibody against lactoferrin. The former fraction and the latter fraction are collected, the solvent is removed by freeze-drying, and the fractions are dissolved in water again. The amount of protein contained in each of the former fraction and the latter fraction is measured by protein assay using a dye method, so that the content of lactoferrin and proteins other than lactoferrin can be measured.

[0023] The lower limit of the total content of proteins other than lactoferrin in the lactoferrin-containing aqueous solution to be sterilized may be, for example, 0% by mass, or may be more than 0% by mass from the viewpoint of the production efficiency of the lactoferrin material used to prepare the aqueous solution. For example, the total content of proteins other than lactoferrin in the lactoferrin-containing aqueous solution to be sterilized may be, for example, 1 / 100,000 or more, 1 / 10,000 or more, 1 / 5,000 or more, 1 / 1,000 or more, or 1 / 100 or more of the lactoferrin content.

[0024] The temperature of heat sterilization in the sterilization step is 100°C or higher. Since the present technology can adopt such a high temperature, sterilization can be performed in a short time. The temperature of heat sterilization in the sterilization step is preferably 105°C or higher, more preferably 110°C or higher, even more preferably 112°C or higher, 114°C or higher, 116°C or higher, or 118°C or higher, and particularly preferably 120°C or higher. By adopting such a high temperature according to the present technology, bacteria that are difficult to kill can be sterilized in a short time. For example, the present technology can apply the heating conditions required for retort foods (120°C for 4 minutes or more) or the UHT method to lactoferrin.

[0025] The temperature of the heat sterilization in the sterilization step is preferably 180° C. or less, more preferably 160° C. or less, even more preferably 155° C. or less, or 150° C. or less, and particularly preferably 140° C. or less. Too high a temperature may cause water evaporation, reducing the amount of the obtained lactoferrin-containing aqueous solution.

[0026] According to a preferred embodiment of the present technology, the salt concentration of the lactoferrin-containing aqueous solution that is subjected to the sterilization process is 5mM or less.This salt concentration makes precipitation less likely to occur and maintains activity better.The salt concentration of the lactoferrin-containing aqueous solution that is subjected to the sterilization process can be preferably 4mM or less, more preferably 3mM or less, and even more preferably 2mM or less. From the viewpoint of production efficiency of the aqueous solution, the salt concentration of the lactoferrin-containing aqueous solution subjected to the sterilization step may be, for example, more than 0 μM, 0.1 μM or more, or more than 0.1 μM, 0.5 μM or more, 1 μM or more, 1.5 μM or more, or 10 μM or more. The salt concentration of the lactoferrin-containing aqueous solution to be subjected to the sterilization step is measured by IPC-MS (inductively coupled plasma mass spectrometry).

[0027] The lactoferrin concentration of the lactoferrin-containing aqueous solution subjected to the sterilization step is preferably 400 mg / ml or less, more preferably 200 mg / ml or less or 100 mg / ml or less, even more preferably 30 mg / ml or less, even more preferably 10 mg / ml or less, and particularly preferably 1 mg / ml or less. If the lactoferrin concentration is too high, the lactoferrin may not dissolve. The lactoferrin concentration of the lactoferrin-containing aqueous solution to be subjected to the sterilization step may be more than 0 mg / ml. The lactoferrin concentration of the lactoferrin-containing aqueous solution to be subjected to the sterilization step may be, for example, 0.00001 mg / ml or more, 0.0001 mg / ml or more, 0.001 mg / ml or more, or 0.01 mg / ml or more.

[0028] The time for heat sterilization in the sterilization step may be set by those skilled in the art depending on factors such as the sterilization temperature and the sterilization method, etc. The time for heat sterilization in the sterilization step may be, for example, 0.5 seconds to 15 minutes, preferably 1 second to 10 minutes, and more preferably 1 second to 8 minutes. When the sterilization method is the UHT method, the time for heat sterilization in the sterilization step is preferably 0.5 seconds to 10 seconds, more preferably 0.8 seconds to 8 seconds, and even more preferably 1 second to 5 seconds. When the sterilization method is retort sterilization, the time for heat sterilization in the sterilization step is preferably 2 to 10 minutes, more preferably 3 to 8 minutes, and particularly preferably 4 to 7 minutes. In the present technology, the heat sterilization time in the sterilization step refers to the time during which the temperature of the central part of the lactoferrin-containing aqueous solution subjected to the sterilization step is maintained at the temperature to be achieved (sterilization temperature).

[0029] According to a particularly preferred embodiment of the present technology, the temperature and time of the heat sterilization in the sterilization step are 120° C. or higher and 4 minutes or longer, respectively, which satisfies the sterilization conditions required for retort sterilization.

[0030] The lactoferrin-containing aqueous solution to be subjected to the sterilization step preferably does not contain a stabilizer. The stabilizer refers to a component added to improve the heat resistance of lactoferrin, separate from the lactoferrin material used to prepare the lactoferrin-containing aqueous solution. The present technology can maintain the activity of lactoferrin without the stabilizer, and also suppresses the formation of precipitates due to heating. Furthermore, by not containing the stabilizer, changes in the flavor and / or physical properties of the lactoferrin-containing aqueous solution produced are suppressed. Examples of the stabilizer include those described in the above Patent Documents 3 and 4. That is, the lactoferrin-containing aqueous solution subjected to the sterilization step does not contain one or more stabilizers selected from the group consisting of glycerin fatty acid esters, sodium caseinate, lecithin, soybean polysaccharides, xanthan gum, pectin, gum arabic, gum ghatti, carrageenan, locust bean gum, and carboxymethylcellulose. In particular, the lactoferrin-containing aqueous solution subjected to the sterilization step does not contain any of glycerin fatty acid esters, sodium caseinate, lecithin, soybean polysaccharides, xanthan gum, pectin, gum arabic, gum ghatti, carrageenan, locust bean gum, and carboxymethylcellulose as stabilizers.

[0031] The lactoferrin material used to prepare the lactoferrin-containing aqueous solution to be subjected to the sterilization step may contain a small amount of sodium caseinate. The lactoferrin material used to prepare the lactoferrin-containing aqueous solution to be subjected to the sterilization step may contain sodium caseinate at a content of, for example, 5% by mass or less, particularly 2% by mass or less, more particularly 0.5% by mass or less, based on the mass of the lactoferrin material. The sodium caseinate originally contained in the lactoferrin material is not the stabilizer. The lactoferrin-containing aqueous solution to be subjected to the sterilization step may have a sodium caseinate concentration of, for example, 20 mg / ml or less, more preferably 10 mg / ml or less or 5 mg / ml or less, even more preferably 1.5 mg / ml or less, even more preferably 0.5 mg / ml or less, and particularly preferably 0.05 mg / ml or less, but particularly preferably the lactoferrin-containing aqueous solution to be subjected to the sterilization step does not contain sodium caseinate.

[0032] In the production method of the present technology, after the sterilization step, the activity of lactoferrin that was present before the sterilization step is maintained. As an indicator of the maintenance of lactoferrin activity, for example, the antibacterial activity and / or catechol-O-methyltransferase (hereinafter also referred to as COMT) inhibitory activity of lactoferrin may be used. In the present technology, maintaining activity may mean that a certain activity of lactoferrin before the sterilization step is maintained, for example, at 50% or more, preferably at 60% or more, more preferably at 70% or more, and even more preferably at 80% or more after the sterilization step. Also, maintaining activity in the present technology may mean that a certain activity of lactoferrin before the sterilization step is maintained, for example, at 90% or more after the sterilization step. In addition, in the manufacturing method of the present technology, the activity of lactoferrin that was present before the sterilization step may be enhanced after the sterilization step.For example, the COMT activity and antibacterial activity of lactoferrin, particularly the antibacterial activity of lactoferrin, can be enhanced by the sterilization step in the manufacturing method of the present technology. That is, in the manufacturing method of the present technology, after the sterilization step, the activity of lactoferrin that was present before the sterilization step may be maintained or may be enhanced.

[0033] According to the present technology, the COMT inhibitory activity of lactoferrin in the lactoferrin-containing aqueous solution subjected to the sterilization step is preferably maintained at, for example, 50% or more, preferably 60% or more, more preferably 70% or more, and even more preferably 80% or more after the sterilization step. That is, the sterilization step in the present technology may preferably maintain, for example, 50% or more, preferably 60% or more, more preferably 70% or more, and even more preferably 80% or more of the COMT inhibitory activity of lactoferrin before the sterilization step after the sterilization step. COMT is expressed in various organs, with high activity in the liver and kidney. COMT is also expressed in the intestine, particularly in the intestinal mucosa. The physiological role of COMT in the metabolism of neurotransmitters has attracted attention in the pharmaceutical field. In the present technology, the COMT inhibitory activity may be measured according to the method described in Ikeda et al., Inhibitory Effect of Bovine Lactoferrin on Catechol-O-Methyltransferase, Molecules. 2017, 22, 1373. The specific method for measuring the COMT inhibitory activity is as described in the following test examples.

[0034] According to the present technology, preferably, the antibacterial activity of lactoferrin in the lactoferrin-containing aqueous solution that is subjected to the sterilization process is maintained or enhanced even after the sterilization process.Maintaining antibacterial activity may mean that the lactoferrin-containing aqueous solution inhibits the proliferation of bacteria, such as E. coli, to the same extent before and after the sterilization process.In addition, enhancing antibacterial activity may mean that the lactoferrin-containing aqueous solution after the sterilization process inhibits the proliferation of bacteria, such as E. coli, to a greater extent than the lactoferrin-containing aqueous solution before the sterilization process.

[0035] According to a preferred embodiment of the present technology, the total protein content of the lactoferrin-containing aqueous solution subjected to the sterilization step is preferably 420 mg / ml or less, more preferably 31.5 mg / ml or less, or 10.5 mg / ml or less, even more preferably 1.05 mg / ml or less, and particularly preferably 0.2 mg / ml or less, based on the mass of the aqueous solution. The protein content that satisfies such a numerical range can further suppress the formation of protein precipitation.

[0036] According to a preferred embodiment of the present technology, the content of water in the lactoferrin-containing aqueous solution subjected to the sterilization process may be, for example, 95% by mass or more, preferably 96% by mass or more, more preferably 97% by mass or more, even more preferably 98% by mass or more, even more preferably 99% by mass or more, and particularly preferably 99.5% by mass or more, based on the mass of the aqueous solution. That is, the content of components other than water in the lactoferrin-containing aqueous solution subjected to the sterilization process may be, for example, 5% by mass or less, preferably 4% by mass or less, more preferably 3% by mass or less, based on the mass of the aqueous solution. Such a content of water makes lactoferrin less likely to precipitate. In addition, in the present technology, the water is preferably pure water such as deionized water and Milli-Q water. By using pure water, the salt concentration can be easily adjusted. Furthermore, according to a preferred embodiment of the present technology, the solids concentration of the lactoferrin-containing aqueous solution subjected to the sterilization step may be, for example, 45 w / v% or less, particularly 3 w / v% or less, and more particularly 0.1 w / v% or less. Furthermore, according to a preferred embodiment of the present technology, the viscosity of the lactoferrin-containing aqueous solution subjected to the sterilization step may be, for example, at 25°C, 800 mPa·s or less, particularly 300 mPa·s or less, and more particularly 7 mPa·s or less.

[0037] The method of heat sterilization of the lactoferrin-containing aqueous solution in the sterilization step may be appropriately selected by those skilled in the art. Examples of heat sterilization methods include UHT method and retort sterilization. The device used for heat sterilization may be appropriately selected by those skilled in the art according to the heat sterilization method adopted. For example, in the case of UHT method, heat sterilization can be performed by using a plate-type sterilizer. In the case of retort sterilization, heat sterilization can be performed by filling a retort container with the lactoferrin-containing aqueous solution and then performing retort sterilization using a retort sterilizer. The sterilization method is not limited to these. The sterilization process in the present technology may be carried out in accordance with, for example, the Standards for Manufacturing (Sterilization) of Soft Drinks, Food Sanitation Law, Ministry of Health, Labor and Welfare Notification No. 213 of 1986.

[0038] (2) Preparation process

[0039] The production method according to the present technology may include a preparation step of preparing a lactoferrin-containing aqueous solution to be subjected to the sterilization step. The preparation step includes, for example, a step of mixing a lactoferrin material with an aqueous medium (e.g., water or an aqueous solution) and, optionally, other components. The lactoferrin contained in the lactoferrin material is dissolved in the water or aqueous medium.

[0040] In the preparation step, the lactoferrin-containing aqueous solution may be prepared so that the total mass of proteins other than lactoferrin is 1 / 12 or less, preferably 1 / 14 or less, more preferably 1 / 16 or less, even more preferably 1 / 18 or less, and particularly preferably 1 / 20 or less of the lactoferrin mass.To prepare the lactoferrin-containing aqueous solution, for example, a lactoferrin material with high lactoferrin purity may be dissolved in water or an aqueous solution.As the lactoferrin material, for example, a lactoferrin material with a lactoferrin purity of 90% by mass or more, preferably 92% by mass or more, more preferably 94% by mass or more, even more preferably 95% by mass or more, and particularly preferably 96% by mass may be used.

[0041] In the preparation step, the lactoferrin-containing aqueous solution may be prepared so that the salt concentration of the lactoferrin-containing aqueous solution is preferably 5 mM or less, more preferably 4 mM or less, even more preferably 3 mM or less, and particularly preferably 2 mM or less. Such a salt concentration can be achieved by using a lactoferrin material with a low salt content.

[0042] A lactoferrin material with high lactoferrin purity and low salt content can be produced, for example, by subjecting skim milk or whey containing lactoferrin to a cation exchange resin adsorption treatment and an ultrafiltration treatment. For example, by contacting skim milk or whey with a cation exchange resin, lactoferrin is adsorbed to the cation exchange resin. The lactoferrin adsorbed to the cation exchange resin is separated from the cation exchange resin by using saline. Next, desalting is performed using an ultrafiltration membrane to obtain a lactoferrin material with high lactoferrin purity and low salt concentration.

[0043] The ion exchanger used in the cation exchange resin adsorption treatment preferably has a carboxymethyl (CM) functional group. An example of an ion exchanger having a CM functional group is CM-Sepharose FF (GE Healthcare Japan), but is not limited thereto. The concentration of the saline solution for separating the lactoferrin adsorbed on the cation exchange resin from the resin may be, for example, 5 to 15% by mass, preferably 6 to 14% by mass, and more preferably 8 to 12% by mass.

[0044] The molecular weight cutoff of the ultrafiltration membrane may be, for example, 5,000 to 50,000, preferably 10,000 to 30,000, and more preferably 15,000 to 25,000. An example of the ultrafiltration membrane is GR61PP (manufactured by Alfa Laval, molecular weight cutoff 20,000), but is not limited thereto.

[0045] Preferably, in the preparation of the lactoferrin-containing aqueous solution to be subjected to the sterilization step, no proteinaceous material other than the lactoferrin material is used. In the present technology, the proteinaceous material includes proteins and peptides, and more particularly means only proteins and peptides. Since no proteinaceous material is used as a material for the preparation of the lactoferrin-containing aqueous solution, the content ratio of impurity proteins contained in the lactoferrin-containing aqueous solution can be easily adjusted. In addition, the formation of precipitates in the sterilization step is more suppressed, and activity is more maintained and / or enhanced.

[0046] Examples of the optional other components include flavors, colorants, sweeteners, acidulants, and fruit juices. By preparing an aqueous lactoferrin-containing solution containing the optional other components in the preparation step, the aqueous lactoferrin-containing solution that has been subjected to the sterilization step can be used as a beverage (e.g., a soft drink) as is. The optional other components are preferably not proteinaceous materials.

[0047] The flavorings may include, for example, fruit flavors (such as strawberry flavor, banana flavor, and peach flavor), coffee flavors, and tea flavors. The coloring agents include, for example, natural coloring agents and synthetic coloring agents. The sweeteners may include natural sweeteners such as glucose, fructose, and sucrose, as well as high-intensity sweeteners such as aspartame, acesulfame potassium, and sucralose. The acidulants can include, for example, citric acid, sodium citrate, malic acid, and tartaric acid. Examples of the fruit juice include strawberry juice, banana juice, and peach juice.

[0048] (3) Filling process

[0049] The manufacturing method according to the present technology may include a pre-sterilization filling step of filling a container with the lactoferrin-containing aqueous solution to be subjected to the sterilization step, or a post-sterilization filling step of filling a container with the lactoferrin-containing aqueous solution after the sterilization step. The filling method in these steps may be appropriately selected by those skilled in the art.

[0050] For example, in the pre-sterilization filling step, the lactoferrin-containing aqueous solution to be subjected to the sterilization step is filled into a retort pack, and retort sterilization is performed in the sterilization step, thereby producing a lactoferrin aqueous solution that can be stored for a long period of time and is filled into a retort pack.

[0051] Alternatively, for example, in the post-sterilization filling step, the lactoferrin-containing aqueous solution after the sterilization step is filled into a container under aseptic conditions, thereby producing a lactoferrin aqueous solution that can be stored for a long period of time and is filled into a container.

[0052] As described above, a lactoferrin-containing aqueous solution filled in a container and sterilized can be provided according to the manufacturing method of the present technology. The lactoferrin aqueous solution thus produced can be provided to consumers or food and beverage manufacturers as a beverage or food and beverage ingredient in which the activity of lactoferrin is maintained and the lactoferrin content is increased.

[0053] In addition, the lactoferrin aqueous solution produced in this manner is heat sterilized, so it can be stored for a long period of time.For example, the lactoferrin aqueous solution product can be stored for, for example, 3 months or more, preferably 4 months or more, and more preferably 5 months or more.The upper limit of the storage period can be, for example, 24 months or less, preferably 12 months or less, more preferably 10 months or less, and even more preferably 9 months or less.

[0054] 2. Lactoferrin-containing aqueous solution

[0055] This technology provides an aqueous solution containing lactoferrin, which contains no more than 1 / 12 of the total mass of proteins other than lactoferrin and does not contain viable bacteria. Since the aqueous solution does not contain viable bacteria, it can be distributed as a beverage as is or can be used as a food and beverage ingredient without the need for heat sterilization.

[0056] In the present technology, the absence of viable bacteria may be determined based on the presence of viable Clostridium botulinum bacteria. The presence of viable Clostridium botulinum bacteria may be determined according to the method described in the Food Sanitation Inspection Guideline (Microorganisms) 2004 (Japan Food Sanitation Association).

[0057] Preferably, the total mass of proteins other than lactoferrin in the lactoferrin-containing aqueous solution according to the present technology is 1 / 12 or less, preferably 1 / 14 or less, more preferably 1 / 16 or less, even more preferably 1 / 18 or less, and particularly preferably 1 / 20 or less of the lactoferrin mass, which makes precipitation less likely to occur in the aqueous solution. The lower limit of the total content of proteins other than lactoferrin in the lactoferrin-containing aqueous solution according to the present technology may be, for example, 0% by mass, or from the viewpoint of production efficiency, may be more than 0% by mass. For example, the total content of proteins other than lactoferrin in the lactoferrin-containing aqueous solution according to the present technology may be, for example, 1 / 100,000 or more, 1 / 10,000 or more, 1 / 5,000 or more, 1 / 1,000 or more, or 1 / 100 or more of the lactoferrin content.

[0058] The lactoferrin-containing aqueous solution according to the present technology preferably has a salt concentration of 5 mM or less, more preferably 4 mM or less, even more preferably 3 mM or less, and particularly preferably 2 mM or less, which makes it difficult for precipitation to occur in the aqueous solution. Furthermore, from the viewpoint of production efficiency of the aqueous solution, the salt concentration of the lactoferrin-containing aqueous solution according to the present technology may be, for example, more than 0 μM, 0.1 μM or more, or more than 0.1 μM, 0.5 μM or more, 1 μM or more, 1.5 μM or more, or 10 μM or more.

[0059] The lactoferrin concentration of the lactoferrin-containing aqueous solution according to the present technology may be preferably 400 mg / ml or less, more preferably 200 mg / ml or less or 100 mg / ml or less, even more preferably 30 mg / ml or less, even more preferably 10 mg / ml or less, and particularly preferably 1 mg / ml or less. The lactoferrin concentration of the lactoferrin-containing aqueous solution according to the present technology may be more than 0 mg / ml. The lactoferrin concentration of the lactoferrin-containing aqueous solution subjected to the sterilization step may be, for example, 0.00001 mg / ml or more, 0.0001 mg / ml or more, 0.001 mg / ml or more, or 0.01 mg / ml or more.

[0060] The lactoferrin-containing aqueous solution according to the present technology preferably does not contain a stabilizer.The lactoferrin-containing aqueous solution according to the present technology does not contain one or more stabilizers selected from the group consisting of glycerin fatty acid esters, sodium caseinate, lecithin, soybean polysaccharides, xanthan gum, pectin, gum arabic, gum ghatti, carrageenan, locust bean gum, and carboxymethylcellulose.In particular, the lactoferrin-containing aqueous solution does not contain any of glycerin fatty acid esters, sodium caseinate, lecithin, soybean polysaccharides, xanthan gum, pectin, gum arabic, gum ghatti, carrageenan, locust bean gum, and carboxymethylcellulose as stabilizers.

[0061] The lactoferrin-containing aqueous solution according to the present technology preferably contains active lactoferrin. The active lactoferrin may be, for example, lactoferrin having COMT inhibitory activity and / or antibacterial activity. The lactoferrin-containing aqueous solution containing active lactoferrin is preferably obtained by the above-mentioned "1. Method for producing a lactoferrin-containing aqueous solution".

[0062] The lactoferrin-containing aqueous solution according to the present technology may contain other optional ingredients such as flavorings, colorings, sweeteners, acidulants, and fruit juices, etc. The optional other ingredients are as described above in "1. Method for producing a lactoferrin-containing aqueous solution."

[0063] The total protein content of the lactoferrin-containing aqueous solution according to the present technology is preferably 420 mg / ml or less, more preferably 210 mg / ml or less or 105 mg / ml or less, even more preferably 31.5 mg / ml or less, even more preferably 10.5 mg / ml or less, and particularly preferably 1.05 mg / ml or less, relative to the mass of the aqueous solution. This makes lactoferrin less likely to precipitate.

[0064] The water content of the lactoferrin-containing aqueous solution according to the present technology may be, for example, 95% by mass or more, preferably 96% by mass or more, more preferably 97% by mass or more, based on the mass of the aqueous solution. That is, the content of components other than water in the lactoferrin-containing aqueous solution according to the present technology may be, for example, 5% by mass or less, preferably 4% by mass or less, more preferably 3% by mass or less, based on the mass of the aqueous solution. Furthermore, according to a preferred embodiment of the present technology, the solids concentration of the lactoferrin-containing aqueous solution subjected to the sterilization step may be, for example, 45 w / v% or less, particularly 3 w / v% or less, and more particularly 0.1 w / v% or less. Furthermore, according to a preferred embodiment of the present technology, the viscosity of the lactoferrin-containing aqueous solution subjected to the sterilization step may be, for example, at 25°C, 800 mPa·s or less, particularly 300 mPa·s or less, and more particularly 7 mPa·s or less.

[0065] The lactoferrin-containing aqueous solution according to the present technology can be produced by the production method described in "1. Production method of lactoferrin-containing aqueous solution" above. In this production method, the activity of lactoferrin is maintained. That is, according to the present technology, it is possible to provide a lactoferrin-containing aqueous solution that maintains the activity of lactoferrin and does not contain viable bacteria.

[0066] The present technology will be described in more detail below with reference to examples, but the present technology is not limited to these examples. EXAMPLES

[0067] [Test Example 1]

[0068] Lactoferrin (Morinaga Milk Industry Co., Ltd., lactoferrin purity 96% by mass or more) was added to deionized water to prepare a lactoferrin-containing aqueous solution at a final concentration of 30 mg / mL. The lactoferrin was a lactoferrin material produced as described in Example 3 below. Alpha casein, bovine albumin (BSA), alpha lactalbumin (alpha LA) and sodium caseinate (Na caseinate) were prepared as additional proteins to be added to the lactoferrin-containing aqueous solution. Any of these additional proteins was mixed with the lactoferrin-containing aqueous solution at final concentrations of 0.3 mg / mL, 0.6 mg / mL, 1.2 mg / mL and 2.4 mg / mL to obtain four samples for each additional protein. The total mass of proteins other than lactoferrin relative to the mass of lactoferrin in the sample is 1 / 19 or less, 1 / 16 or less, 1 / 12 or less, and 1 / 8 or less (total mass of proteins other than lactoferrin / mass of lactoferrin) for each additional protein concentration based on the purity and concentrations. A lactoferrin-containing aqueous solution to which no additional protein was added was also prepared as a sample. The total mass of proteins other than lactoferrin relative to the mass of lactoferrin in the lactoferrin-containing aqueous solution sample to which no additional protein was added is 1 / 24 or less.

[0069] A total of 17 samples obtained above (four samples at four concentrations for each of the four additional proteins, and one sample without additional protein) were autoclaved for 10 minutes. The turbidity after the autoclave sterilization was measured using a Hitachi U-3900 spectrophotometer (Hitachi). The measurement wavelength was 660 nm. The turbidity measurement results are shown in Table 1 and FIG. 1.

[0070] [Table 1]

[0071] As shown in Table 1 and Figure 1, the addition of additional protein caused an increase in turbidity during heat treatment. Furthermore, the higher the concentration of additional protein, the greater the increase in turbidity. In other words, the lower the mass content of proteins other than lactoferrin protein, the more suppressed the increase in turbidity during heat treatment of the lactoferrin-containing aqueous solution. Therefore, it is considered that the lower the mass content of proteins other than lactoferrin protein, the less likely protein precipitation occurs when heated at 120°C. Furthermore, it is considered that the less precipitation occurs, the more lactoferrin activity is maintained.

[0072] As shown in Table 1 and Figure 1, when the added protein concentration is 1.2 mg / ml or less, that is, when the total mass content of proteins other than lactoferrin relative to the mass content of lactoferrin is 1 / 12 or less, the turbidity after heat treatment is about 0.1 or less. It is preferable that the quality change before and after heat treatment is small, and it is particularly desirable for the lactoferrin-containing aqueous solution to have a turbidity of about 0.1 or less after heat treatment. For example, if the turbidity of the lactoferrin-containing aqueous solution is low even after heat treatment, it is easy to check the appearance of the product for microbial contamination or changes in the product (precipitation or increase in turbidity) in quality control. Therefore, if the total mass content of proteins other than lactoferrin relative to the mass content of lactoferrin is 1 / 12 or less, the desired state of the lactoferrin-containing aqueous solution is maintained even when heat sterilized at 120°C, and in particular, precipitation does not occur. It is also considered that the activity of lactoferrin is well maintained by the total mass content of proteins other than lactoferrin relative to the mass content of lactoferrin being 1 / 12 or less.

[0073] Furthermore, when no proteinaceous material other than lactoferrin was included, i.e., when the total mass of proteins other than lactoferrin relative to the mass of lactoferrin was 1 / 24 or less, the turbidity after heat treatment was 0.032, indicating that a particularly preferable lactoferrin-containing aqueous solution was obtained in this case.

[0074] [Test Example 2]

[0075] Lactoferrin (Morinaga Milk Industry Co., Ltd., lactoferrin purity 96% or more by mass) was added to deionized water to a final concentration of 30 mg / mL to prepare a lactoferrin-containing aqueous solution. NaCl, CaCl2, or MgCl2 was mixed with the lactoferrin-containing aqueous solution. Of these salts, NaCl and CaCl2 were added to a final concentration of 1.25 mM, 2.5 mM, 5 mM, 10 mM, 15.6 mM, or 20 mM to obtain samples with six different salt concentrations for each salt. MgCl2 was added to a final concentration of 1.25 mM, 2.5 mM, 5 mM, 7.6 mM, 10 mM, or 20 mM to obtain samples with six different salt concentrations.

[0076] A total of 18 samples (six samples of salt concentrations for each of the three salts) obtained above were autoclaved for 10 minutes at 121°C. The turbidity after the autoclave sterilization was measured. The measurement wavelength was 660 nm. The turbidity measurement results are shown in Table 2 and Figure 2 below.

[0077] [Table 2]

[0078] As shown in Table 2 and Figure 2, it was found that the addition of salt caused an increase in turbidity during heat treatment. In addition, the higher the salt concentration, the greater the increase in turbidity. In other words, the lower the salt concentration, the more suppressed the increase in turbidity during heat treatment of the lactoferrin-containing aqueous solution. Therefore, it is considered that the lower the salt concentration, the less likely protein precipitation occurs.

[0079] A lactoferrin-containing aqueous solution having a turbidity of about 0.1 or less after heat treatment is desirable. As shown in Table 2 and Figure 2, when the salt concentration is 5 mM or less, the turbidity after heat treatment is less than 0.1. Therefore, by keeping the salt concentration at 5 mM or less, the desirable state of the lactoferrin-containing aqueous solution is maintained even when heat sterilized at 120°C, and in particular, no precipitation occurs.

[0080] Furthermore, from Table 2 and Figure 2, it can be seen that the lower the salt concentration, the lower the turbidity. Therefore, if a similar test is performed on a sample to which no salt is added, it is expected that the turbidity will be even lower than when the salt concentration is 1.25 mM.

[0081] [Test Example 3]

[0082] It was shown that the degree of turbidity formation differs depending on the lactoferrin material. Two types of lactoferrin materials were prepared (material 1: manufactured by Tatua, material 2: manufactured by Fontilla). Each of these lactoferrin materials was added to Milli-Q water to a final concentration of 30 mg / mL to prepare two types of lactoferrin-containing aqueous solutions (material 1 dissolved in sample 1 and material 2 dissolved in sample 2). Each of samples 1 and 2 was autoclaved for 10 minutes at 121°C. The turbidity after the autoclave sterilization was measured. The measurement wavelength was 660 nm. The turbidity measurement results are shown in Table 3 and Figure 3 below.

[0083] [Table 3]

[0084] As shown in Table 3 and FIG. 3, some types of lactoferrin materials experience an increase in turbidity upon heat treatment.

[0085] [Test Example 4]

[0086] In this test example, the relationship between the increase in turbidity of an aqueous lactoferrin solution due to heat treatment and the COMT inhibitory activity was confirmed as follows. Lactoferrin (Morinaga Milk Industry Co., Ltd., lactoferrin purity 96% by mass or more) was added to deionized water to a final concentration of 30 mg / mL to prepare a lactoferrin-containing aqueous solution. NaCl, albumin (BSA), or sodium caseinate was added to the lactoferrin-containing aqueous solution. NaCl was added to a concentration of 0 mM, 10 mM, 15 mM, or 20 mM. BSA and sodium caseinate were added to a concentration of 0 mg / mL, 0.6 mg / mL, 2.4 mg / mL, or 4.8 mg / mL.

[0087] A total of 12 kinds of solutions were sterilized in an autoclave at 121° C. for 10 minutes. The turbidity after the autoclave sterilization was measured. The measurement wavelength was 660 nm.

[0088] In addition, each solution after the autoclave sterilization treatment was centrifuged at 150,000xg for 20 minutes, and the COMT inhibitory activity of the supernatant was measured. The COMT inhibitory activity was measured as follows: 50 mM phosphoric acid (pH 7.8), 2 mM MgCl2, 14 A 22.5μL reaction solution was prepared containing 11.2μM SAM (S-adenosyl-L-methionine) labeled with C radioisotope, 1mM DTT (dithiothreitol), 4μg / mL COMT enzyme, and each solution. Each solution was diluted to a concentration of 0.1mg / mL before reaction and heated at 100℃ for 10 minutes. The enzyme reaction was reacted at 37℃ for 10 minutes, and then 2.5μL of DBA (dihydroxybenzoic acid) was added as a substrate, and reacted at 37℃ for 10 minutes. The reaction was stopped by adding 12.5μL of 1M HCl. After the reaction was stopped, 300μL of isoamyl alcohol:toluene=3:7 solution was added for extraction, and the radiation dose was counted using a liquid scintillation counter. Next, the amount of solution required for 50% COMT activity inhibition was calculated for each of the 12 types of solutions. If the amount of solution required for 50% activity inhibition in the case of lactoferrin alone is taken as 1, the amount of solution required for 50% inhibition is greater than 1 because the COMT inhibitory activity of lactoferrin-containing aqueous solutions to which salt or protein has been added is reduced. Therefore, the COMT inhibitory activity (relative potency) of the supernatant was calculated using the following formula: "relative potency" = "amount of solution autoclaved with lactoferrin alone" / "amount of solution autoclaved with salt or the like added, required for 50% inhibition." The relationship between the calculated relative potency and the turbidity after autoclaving is shown in Table 4 and Figure 4.

[0089] [Table 4]

[0090] As shown in Table 4 and Figure 4, the COMT inhibitory activity of the supernatant after centrifugation weakened with increasing turbidity. Therefore, it is considered that there is a relationship between turbidity and COMT inhibitory activity. Therefore, it is considered that the activity of lactoferrin is maintained along with the suppression of precipitation observed in Test Examples 1 and 2.

[0091] Furthermore, taking into consideration the results of Test Examples 1 and 2 and Test Example 4, in order to maintain the COMT inhibitory activity at approximately 80% compared to before heat sterilization (i.e., COMT inhibitory activity of approximately 0.8), it is considered particularly preferable to set the total mass of proteins other than lactoferrin to 1 / 20 or less of the mass of lactoferrin and to set the salt concentration to 5 mM or less.

[0092] [Test Example 5]

[0093] Lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd., lactoferrin purity 96% by mass or more) was dissolved in ion-exchanged water to obtain lactoferrin-containing aqueous solutions of 1 mg / mL, 10 mg / mL, and 30 mg / mL concentrations. These lactoferrin-containing aqueous solutions were subjected to retort heat treatment (121°C, 7 minutes) and stored at temperatures of 5°C, 25°C, or 37°C for 44 or 98 days. Then, each lactoferrin-containing aqueous solution was added to a 0.08% by volume TFA (trifluoroacetic acid) solution so that the lactoferrin concentration was 0.1 mg / mL to obtain a sample for HPLC analysis. HPLC analysis was performed using 40 μL of the obtained sample. In the HPLC analysis, a Cosmosil 5C4-MS (4.6 mm x 250 mm) column was used, and elution was performed with a concentration gradient of acetonitrile containing 0.1% by volume TFA, and lactoferrin was detected at 280 nm. The detection rate of lactoferrin (the ratio of the absorbance after the retort heat treatment to the absorbance of the sample before the treatment, which is taken as 100%) was calculated. The detection rates obtained are shown in Table 5 and FIG.

[0094] [Table 5]

[0095] From the results shown in Table 5 and Figure 5, even after long-term storage after retort processing, the amount of lactoferrin detected by HPLC analysis was the same as that before retort processing. Therefore, it can be seen that the quality of the lactoferrin-containing aqueous solution sterilized according to this technology does not change much even after long-term storage.

[0096] [Test Example 6]

[0097] Lactoferrin (Morinaga Milk Industry Co., Ltd., lactoferrin purity 96% by mass or more) was dissolved in deionized water to a concentration of 10 mg / mL to prepare a lactoferrin-containing aqueous solution, which was then retorted at 121°C for 7 minutes. After the retort treatment, the aqueous solution was stored at 5°C, 25°C, or 37°C for 98 days. After storage, the COMT inhibitory activity of each aqueous solution stored at each temperature was measured. The count value of radioactive substances was used as a measure of COMT inhibitory activity. In addition, the COMT inhibitory activity was measured in the same manner for a lactoferrin-containing aqueous solution that had not been subjected to retort treatment and for a solution to which no lactoferrin was added. The measurement results are shown in Table 6 and FIG.

[0098] [Table 6]

[0099] As shown in Table 6 and Figure 6, when comparing the lactoferrin-containing aqueous solution that was not retorted with the solution without lactoferrin, COMT was inhibited by lactoferrin. In addition, when comparing the lactoferrin-containing aqueous solution that was not retorted with the three types of aqueous solutions that were retorted, all three types of aqueous solutions had the same COMT inhibitory activity even after retort treatment as when no retort treatment was performed. Therefore, it can be seen that the lactoferrin-containing aqueous solution produced according to the present technology maintains its COMT inhibitory activity despite being subjected to a sterilization process.

[0100] [Test Example 7]

[0101] Lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd., lactoferrin purity 96% by mass or more) was dissolved in deionized water to a concentration of 30 mg / mL to obtain a lactoferrin-containing aqueous solution. The lactoferrin-containing aqueous solution was autoclaved at 121°C for 10 minutes, and the antibacterial activity of this solution was examined as follows. Escherichia coli was cultured in 5 mL of M9 liquid medium, and the cells were collected by centrifugation. The collected cells were suspended in PBS (phosphate buffer pH 7.4), and two 100 μL suspensions with OD (630 nm) of 0.2 were prepared. On the other hand, PBS was added to the lactoferrin-containing aqueous solution so that the lactoferrin concentration was 2 mg / mL to obtain a diluted lactoferrin-containing aqueous solution. An equal amount (100 μL) of the diluted lactoferrin-containing aqueous solution was added to each of the two 100 μL suspensions to prepare two solutions containing lactoferrin and cells at a final concentration of 1 mg / mL. The lactoferrin-containing aqueous solution that had not been autoclaved was also added to 100 μL of two suspensions prepared in the same manner to prepare two solutions containing lactoferrin and bacterial cells at a final concentration of 1 mg / mL. Two 100 μL suspensions were also prepared without the addition of the lactoferrin-containing aqueous solution. The above six samples were incubated at 37°C for 1 hour and then centrifuged. The precipitate obtained by centrifugation was suspended in PBS, and the suspension was applied to an M9 plate. The plate was incubated overnight at 37°C to allow colonies to form, and the number of colonies was counted. The average number of colonies counted is shown in Table 7 and Figure 7.

[0102] [Table 7]

[0103] From the results shown in Table 7 and Figure 7, it can be seen that the lactoferrin-containing aqueous solution has antibacterial activity by comparing the case without lactoferrin with the case of the lactoferrin-containing aqueous solution that has not been autoclaved. Also, it can be seen that the antibacterial activity is enhanced by autoclaving by comparing the case of the lactoferrin-containing aqueous solution that has not been autoclaved with the case of the lactoferrin-containing aqueous solution that has been autoclaved. Therefore, it can be seen that the antibacterial activity of the lactoferrin-containing aqueous solution produced according to the present technology is enhanced by the sterilization process.

[0104] [Example 1]

[0105] Lactoferrin (Morinaga Milk Industry Co., Ltd., lactoferrin purity 96% by mass or more) was dissolved in deionized water to a concentration of 10 mg / ml, and 0.1% (W / V) strawberry flavor (Hasegawa Fragrance) and 0.02% (W / V) colorant (Hasegawa Fragrance) were added to the solution to obtain an aqueous solution containing lactoferrin. The resulting aqueous solution containing lactoferrin was sterilized by heating and holding at 131°C for 30 seconds using a plate sterilizer (Morinaga Engineering Co., Ltd.). After cooling, the solution was filled into containers under aseptic conditions to obtain a lactoferrin-containing beverage that can be stored for a long period of time.

[0106] [Example 2]

[0107] Lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd., lactoferrin purity 96% by mass or more) concentration 30 mg / ml and NaCl concentration 2 mM were dissolved in deionized water to obtain 10 L of lactoferrin-containing aqueous solution. The obtained lactoferrin-containing aqueous solution was dispensed into retort packs. After dispensing, the retort packs containing the aqueous solution were sterilized in a retort sterilizer at 120°C for 7 minutes to obtain a lactoferrin-containing aqueous solution that can be stored for a long period of time.

[0108] [Example 3]

[0109] A lactoferrin material with high lactoferrin purity and low salt content was obtained as follows. First, 1 L of an ion exchanger (CM-Sepharose FF (GE Healthcare Japan)) was packed into a column, and 2 L of 0.1 N hydrochloric acid was passed through it, followed by washing with water to equilibrate the ion exchanger. Subsequently, the ion exchanger was removed from the column, and then the ion exchanger was added to 100 L of skim milk at pH 6.7 that had been cooled to 4°C, and then stirred at 4°C for 6 hours. The skim milk was removed by filtration, and the ion exchanger was collected, and then packed into a column. The column was washed with deionized water to remove the skim milk content. Then, 5 L of 10% by mass saline was passed through the column to separate the components adsorbed on the ion exchanger, and 5 L of a recovered liquid was obtained. The recovered liquid obtained was circulated at a circulation flow rate of 8 L / min and an average pressure of 30 kg / cm using an ultrafiltration membrane GR61PP (Alfa Laval) having a molecular weight cutoff of 20,000. 2 Ultrafiltration was carried out at 100°C. Deionized water was further added, and ultrafiltration was repeated to perform desalting. The resulting solution was freeze-dried to obtain 3.5 g of powdered lactoferrin. In this way, lactoferrin material with a purity of 96% by mass or more and a sodium salt content of 70 mg per 100 g was obtained.

Claims

1. A preparation step of preparing a lactoferrin-containing aqueous solution having a total mass of proteins other than lactoferrin of 1 / 12 or less of the lactoferrin mass by mixing a lactoferrin material with water; The lactoferrin-containing aqueous solution itself is subjected to a sterilization step of heat sterilization at 120° C. or higher and 180° C. or lower for 4 to 7 minutes, A method for producing an aqueous lactoferrin-containing solution, wherein the salt concentration of the aqueous lactoferrin-containing solution subjected to the sterilization step is 5 mM or less.

2. The lactoferrin-containing aqueous solution, the total mass of which is 1 / 12 or less of the mass of lactoferrin, is subjected to a sterilization step of heat sterilizing the aqueous solution at 120° C. or more and 180° C. or less for 4 to 7 minutes, The salt concentration of the lactoferrin-containing aqueous solution subjected to the sterilization step is 5 mM or less; The lactoferrin-containing aqueous solution to be subjected to the sterilization step has not been pH-adjusted by the addition of hydrochloric acid, sodium hydroxide, or primary lactic acid. A method for producing a lactoferrin-containing aqueous solution.

3. The lactoferrin-containing aqueous solution, the total mass of which is 1 / 12 or less of the mass of lactoferrin, is subjected to a sterilization step of heat sterilizing the aqueous solution at 120° C. or more and 180° C. or less for 4 to 7 minutes, The salt concentration of the lactoferrin-containing aqueous solution subjected to the sterilization step is 5 mM or less; The lactoferrin-containing aqueous solution to be subjected to the sterilization step is not pH-adjusted by adding hydrochloric acid or sodium hydroxide, and The lactoferrin concentration of the lactoferrin-containing aqueous solution subjected to the sterilization step is 30 mg / ml or less. A method for producing a lactoferrin-containing aqueous solution.

4. The method according to claim 1 , wherein the lactoferrin-containing aqueous solution subjected to the heat sterilization contains only the lactoferrin material and water.

5. 3. The method according to claim 1, wherein the lactoferrin concentration of the lactoferrin-containing aqueous solution subjected to the sterilization step is 0.00001 mg / ml or more and 400 mg / ml or less.

6. The method according to any one of claims 1 to 5, wherein the lactoferrin-containing aqueous solution subjected to the sterilization step does not contain a stabilizer.

7. The method according to any one of claims 1 to 6, wherein the COMT inhibitory activity of lactoferrin in the lactoferrin-containing aqueous solution subjected to the sterilization step is maintained at 80% or more after the sterilization step.

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