WO2017154867A1 - ホエイタンパク質の分画方法、α-ラクトアルブミンを含む組成物の製造方法及びβ-ラクトグロブリンを含む組成物の製造方法 - Google Patents
ホエイタンパク質の分画方法、α-ラクトアルブミンを含む組成物の製造方法及びβ-ラクトグロブリンを含む組成物の製造方法 Download PDFInfo
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- whey protein
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- 0 *CCC1C2=C(C=C3C(CC*4CC4)=C(C4CCCCCCCC4)C=C3)/C=C(\CC(C3)CC33CCCC3)/C=C/C(/CC(C3)C33CCCC3)=C12 Chemical compound *CCC1C2=C(C=C3C(CC*4CC4)=C(C4CCCCCCCC4)C=C3)/C=C(\CC(C3)CC33CCCC3)/C=C/C(/CC(C3)C33CCCC3)=C12 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/20—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey
- A23J1/205—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey from whey, e.g. lactalbumine
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/14—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment
- A23C9/142—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment by dialysis, reverse osmosis or ultrafiltration
- A23C9/1425—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment by dialysis, reverse osmosis or ultrafiltration by ultrafiltration, microfiltration or diafiltration of whey, e.g. treatment of the UF permeate
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/14—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment
- A23C9/146—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment by ion-exchange
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/20—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/04—Animal proteins
- A23J3/08—Dairy proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C2210/00—Physical treatment of dairy products
- A23C2210/20—Treatment using membranes, including sterile filtration
- A23C2210/206—Membrane filtration of a permeate obtained by ultrafiltration, nanofiltration or microfiltration
Definitions
- the present invention relates to a method for fractionating whey protein, a method for producing a composition containing ⁇ -lactalbumin, and a method for producing a composition containing ⁇ -lactoglobulin.
- Whey protein is a protein contained in milk, and whey protein in milk contains about 50% by weight ⁇ -lactoglobulin and about 20% by weight ⁇ -lactalbumin.
- the composition containing ⁇ -lactalbumin can be used for nutritional compositions such as breast milk substitutes and enteral nutrients having a protein composition similar to human milk.
- ⁇ -lactoglobulin can be used to control the physical properties of various foods and enhance nutrition. Therefore, a method for fractionating whey protein, a method for obtaining a composition containing ⁇ -lactalbumin, and a method for obtaining a composition containing ⁇ -lactoglobulin have been studied and disclosed so far.
- Patent Document 1 as a method for efficiently fractionating ⁇ -lactoglobulin, ⁇ -lactalbumin and lactoferrin from various milk whey, NaCl is added to whey and proteins other than ⁇ -lactoglobulin are used as hydrophobic chromatography resins.
- ⁇ -lactoglobulin which accounts for about 50% of whey protein, and other proteins are separated first, then ⁇ -lactoglobulin and lactoferrin adsorbed on the resin are eluted and fractionated by ultrafiltration. The method of doing is disclosed.
- Patent Document 2 the pH of unsterilized cheese whey or unsterilized lactic acid whey is adjusted to 6.3 to 7.0 and the temperature is adjusted to 30 to 60 ° C.
- a permeate obtained by treatment with a filtration membrane is further diafiltered using an ultrafiltration membrane having a molecular weight cut-off of 1,200 or 2,000 to obtain a fraction containing a high amount of ⁇ -lactalbumin.
- a method is disclosed.
- the composition obtained here contains 56% by weight of ⁇ -lactalbumin and 37% by weight of ⁇ -lactoglobulin, which has the same protein composition as breast milk and enteral nutrition used for intensive care. It is disclosed that it can be used as an agent.
- Patent Document 3 uses whey or purified whey protein, and after adjusting the pH to 4 to 6 when the ash content is 0 to 1%, and to 6 to 8 when the ash content is 1 to 3%, Disclosed is a method in which ⁇ -lactoglobulin is adsorbed to the ion exchanger by contacting with a strongly basic anion exchanger, and then ⁇ -lactoglobulin is eluted from the ion exchanger with a solution having a high ionic strength.
- Patent Document 3 does not disclose the content of ⁇ -lactoglobulin and ⁇ -lactalbumin in the obtained composition.
- JP-A-7-203863 United States Patent 4,711,953 JP-A-3-19654
- An object of the present invention is to provide a method for fractionating whey protein, a method for producing a composition containing ⁇ -lactalbumin, and a method for producing a composition containing ⁇ -lactoglobulin.
- the present invention includes the following configurations. (1) preparing a solution containing the first whey protein and the second whey protein; and separating the first whey protein and the second whey protein with a membrane; Is a complex formed with at least one of carbonate ion, hydrogen carbonate ion, light metal ion and transition metal ion, and the second whey protein comprises the following (i) and (ii) (I) does not form a complex with either carbonate ion or bicarbonate ion, (Ii) A method for fractionating whey protein that satisfies at least one of a light metal ion and a transition metal ion that do not form a complex.
- the total concentration of the first whey protein and the second whey protein is 0.001 wt% to 35 wt%, and the total concentration of carbonate ions and hydrogen carbonate ions is 0.001 wt% to 35 wt%. %.
- the concentration of the whey protein in the solution is 0.001% to 35% by weight, the total concentration of carbonate ions and bicarbonate ions in the solution is 0.001% to 35% by weight, and the pH of the solution is 5 or more.
- (10) a step of preparing a solution containing whey protein containing ⁇ -lactoglobulin, carbonate ions and / or hydrogen carbonate ions, light metal ions and / or transition metal ions; and a molecular weight cutoff of 300,000 or less Treating the solution with a membrane of: a solution containing ⁇ -lactalbumin as the membrane permeate, and ⁇ -lactoglobulin, carbonate and / or bicarbonate ions, light metal ions and / or as the membrane concentrate A solution containing a complex formed of a transition metal ion; and a step of treating the solution containing ⁇ -lactalbumin with a membrane having a fractional molecular weight of 14,000 or less.
- a method for preparing a composition comprising. (11)
- the concentration of whey protein in the solution is 0.001% to 35% by weight
- the total concentration of carbonate ions and bicarbonate ions in the solution is 0.001% to 35% by weight
- the pH of the solution is 5 or more.
- the method for preparing a composition containing ⁇ -lactalbumin according to (10) wherein the total concentration of light metal ions and transition metal ions in the solution is 0.00001 wt% or more and 10 wt% or less.
- the concentration of whey protein in the solution is 0.001% to 35% by weight, the total concentration of carbonate ions and bicarbonate ions in the solution is 0.001% to 35% by weight, and the pH of the solution is 5 or more. 10.
- the present invention provides a method for fractionating whey protein, a method for producing a composition containing ⁇ -lactalbumin, and a method for producing a composition containing ⁇ -lactoglobulin.
- FIG. 1 is a flowchart of a method for fractionating whey protein / a method for producing a composition containing LA.
- FIG. 2 is a flowchart of a method for producing a composition containing LG.
- ⁇ -lactalbumin may be referred to as “LA”, ⁇ -lactoglobulin as “LG”, whey protein concentrate as “WPC”, and purified whey protein as “WPI”.
- complex 1 When the present inventors mix (a) whey protein and (b) carbonate ion and / or hydrogen carbonate ion, (b) whey protein- (b) carbonate ion and / or hydrogen carbonate ion complex (Hereinafter referred to as “complex 1”) is formed, but it has been found that LA is not involved in the formation of complex 1 and does not form complex 1. That is, the first aspect of the present invention is made by finding the property that the complex 1 does not contain LA.
- the present inventors mixed (i) whey protein, (b) carbonate ion and / or bicarbonate ion, and (c) light metal ion and / or transition metal ion, (B) Whey protein-(b) Carbonate ion and / or bicarbonate ion-(C) Light metal ion and / or transition metal ion complex, and / or Complex 1- (I) Whey protein- (B) Carbonate ion and / or bicarbonate ion- (C) Complex of light metal ion and / or transition metal ion (hereinafter referred to as “Complex 2”)
- Complex 2 Complex of light metal ion and / or transition metal ion
- the method for fractionating whey protein comprises a step of preparing a solution containing a first whey protein and a second whey protein; and a step of separating the first whey protein and the second whey protein with a membrane.
- the first whey protein forms a complex with at least one of carbonate ion, hydrogen carbonate ion, light metal ion, and transition metal ion
- the second whey protein includes the following (1) (2) (1) Does not form a complex with either carbonate ion or bicarbonate ion, (2) At least one of a light metal ion and a transition metal ion that does not form a complex is satisfied.
- the above (first and second) protein materials only need to contain whey protein, are derived from mammals such as cattle, buffalo, sheep, goats, horses, etc., and satisfy the above requirements. Any material can be used, and one or a plurality of raw materials can be used in combination. Specifically, acid such as lactic acid is added to milk such as whey, cow, buffalo, sheep, goat, horse obtained when producing cheese using milk from cow, buffalo, sheep, goat, horse, etc.
- the whey obtained when the whey is obtained those obtained by pulverizing these whey, and those obtained by reducing the lactose and minerals in these whey and concentrating the whey protein are exemplified.
- the protein material used in the method for fractionating whey protein and the method for producing a composition containing LA has few contaminating components such as lactose and fat, and the total of LA and LG is 50 with respect to the total solid content.
- WPC or WPI of weight% or more is preferable.
- ⁇ -lactoglobulin can be mainly exemplified.
- the second whey protein include mainly ⁇ -lactalbumin.
- the carbonate ion and bicarbonate ion will be described.
- the carbonate ion and bicarbonate ion used in the method for fractionating whey protein and the method for producing the composition containing LA can be generated using any component or method as long as these ions are generated in an aqueous solution.
- the carbonate ion and / or bicarbonate ion may be generated by combining one or more compounds and / or methods exemplified below. Method of adding carbon dioxide such as blowing in gaseous carbon dioxide, adding liquid or solid carbon dioxide, adding hydrogen carbonate such as sodium hydrogen carbonate or potassium hydrogen carbonate, or carbonate such as sodium carbonate or potassium carbonate And a method of adding carbonated water.
- the light metal ion and transition metal ion will be described.
- the light metal ion and the transition metal ion may be any light metal or transition metal monovalent to trivalent cation, and one or more metal ions may be used.
- these metal ion sources inorganic salts and organic salts of light metals and / or transition metals can be used.
- LA-containing compositions in foods and pharmaceuticals use sodium, potassium, magnesium, calcium, manganese, iron, copper, zinc, etc. present in foods and humans as light metal ions and / or transition metal ions Is preferred.
- addition of a metal ion source is not essential.
- sodium hydrogen carbonate is used as a material for carbonate ions and / or hydrogen carbonate ions
- sodium or other light metal or transition metal inorganic or organic salts may or may not be added. May be.
- any membrane can be used as long as it has a molecular weight cut-off of 50,000 or less and can penetrate LA.
- any material can be used as long as it has a fractional molecular weight of 300,000 or less and can permeate LA.
- any membrane When concentrating a composition containing LA obtained as the permeation fraction of the above membrane treatment, any membrane can be used as long as it has a fractional molecular weight of 14,000 or less and can concentrate LA.
- any membrane material or membrane filtration method may be used as long as it is generally used.
- the material of the membrane is organic membrane such as polyacrylamide, regenerated cellulose, polyethylene, tetrafluoroethylene, polypropylene, cellulose acetate, polyacrylonitrile, polyimide, polysulfone, polyethersulfone, aluminum oxide, zirconium oxide, titanium oxide, stainless steel, glass
- An inorganic film such as Examples of the membrane module include a pleated module, a spiral module, a monolith module, and a tube module.
- Examples of the membrane filtration method include a total filtration method, a cross flow method, a diafiltration method, and the like.
- FIG. 1 is a flowchart of a method for fractionating whey protein / a method for producing a composition containing LA.
- a method for fractionating whey protein and a method for producing a composition containing LA will be described with reference to the flowchart of FIG.
- the method example of FIG. 1 is used for description, the present invention is not limited to this method example.
- Step 101 Solution preparation, a solution containing (first and second) whey proteins is prepared.
- the solution containing whey protein is preferably prepared in step 101a when it does not contain metal ions and step 101b when it contains metal ions.
- Step 101a When the solution containing whey protein does not contain metal ions, the total concentration of (first and second) whey proteins is 0.001 wt% or more and 35 wt% or less of carbonate ions and / or bicarbonate ions. It is preferable to prepare an aqueous solution using the raw materials described above so that the total concentration is 0.001 wt% or more and 35 wt% or less and the pH is 5 or more and 10 or less.
- Step 101b When the solution containing whey protein contains metal ions, the total concentration of the (first and second) whey proteins is 0.001 wt% or more and 35 wt% or less, and the total amount of carbonate ions and / or bicarbonate ions The above-mentioned raw materials are adjusted so that the concentration is 0.001% to 35% by weight, the total concentration of light metal and / or transition metal ions is 0.00001% to 10% by weight, and the pH is 5 to 10%. It is preferable to use to prepare an aqueous solution.
- the complex 2 is formed by the interaction of whey protein excluding LA, carbonate ions and / or bicarbonate ions, and light metals and / or transition metal ions.
- LA is not involved in the formation of this complex 2, and LA does not form complex 2.
- it is not preferable that only a metal ion source that reacts with carbonate ions and / or hydrogen carbonate ions to form a precipitate is dissolved.
- the aqueous solution may be added in any order, and no precipitate containing protein is observed during or after the preparation of the aqueous solution using the aforementioned raw materials. It is not necessary to adjust the temperature of the solution, but it is preferably 0 ° C. or higher and 15 ° C. or lower in consideration of the growth of microorganisms.
- Step 103 Concentration Processing A, concentration processing is performed using a membrane that can concentrate the solution obtained in Step 101.
- the complex 1 is concentrated when an aqueous solution containing LA and the complex 1 is prepared
- the complex 2 is concentrated when an aqueous solution containing the LA and the complex 2 is prepared.
- a membrane having a molecular weight cut-off of 50,000 or less it is preferable to use a membrane having a fractional molecular weight of 300,000 or less. Note that one or both of the steps 103a and 103b can be performed depending on the content of the complex 1 and the complex 2.
- the concentrated liquid 1 obtained by this treatment contains a large amount of the complex 1 when an aqueous solution containing the complex 1 is prepared, and when the aqueous solution containing the complex 2 is prepared.
- the permeate that is, the LA-containing permeate 2 contains a large amount of LA, where LA and LG are fractionated, and a composition containing LA can be obtained.
- the processing conditions such as sample temperature, average operating pressure, membrane surface flow rate, and concentration ratio during this treatment are such that the LA content in the permeate and / or the LG content in the concentrate is about 50 to 100% by weight per total solid content. It can adjust suitably so that it may become.
- Step 105 In the concentration process B, the LA-containing permeate 2 can be concentrated using a membrane that can concentrate LA. Thereby, solid content etc. of the composition containing LA can be made into a desired thing.
- the concentration treatment B it is preferable to use a membrane capable of concentrating LA having a molecular weight cut-off of 14,000 or less.
- the concentrate obtained by this treatment that is, the LA concentrate 3 is a composition containing LA.
- the permeate that is, the permeate 4 contains almost no protein.
- the processing conditions such as the sample temperature, the average operating pressure, the membrane surface flow rate, and the concentration ratio during the concentration treatment may be appropriately adjusted so that the composition containing LA can be efficiently concentrated.
- the concentration treatment of the composition containing LA in Step 105 may use a method such as freeze concentration or reduced pressure evaporation concentration in addition to the above-described concentration using a membrane.
- Step 108 In the DF treatment C, the concentrated solution 1 (the fraction containing the complex 1 or the complex 2) obtained in step 103 is diafiltered according to the purity or recovery rate of the desired LA. (Hereinafter, referred to as “DF”) processing.
- the “DF treatment” is a method in which an appropriate liquid is appropriately added to the concentrate 1 using a membrane capable of concentrating the concentrate 1.
- DF treatment generally includes filtered water, ion-exchanged water, distilled water, ultrapure water, a permeate generated in a membrane treatment process, a solution with adjusted pH, ionic strength, or the like, or a plurality of these. Can be used.
- the fraction retained in the membrane that is, the concentrated solution 5 has a high content of the complex 1 or the complex 2 per total solid content.
- a composition containing LA is obtained as the LA-containing permeate 6 which is a permeate of the membrane.
- the processing conditions such as sample temperature, average operating pressure, membrane surface flow rate, concentration ratio, etc. during DF processing C in step 108 are appropriately adjusted so that the LA content in the permeate is about 50 to 100% by weight per total solid content. can do.
- Step 110 In the concentration process D, the LA-containing permeate 6 can be subjected to the same concentration process as in step 105.
- the concentrated solution obtained by this treatment that is, the LA concentrated solution 7 is a composition containing LA.
- the permeate 8 contains almost no protein, like the permeate 4 obtained in step 105.
- steps 103, 108, 105, and 110 concentration process A, DF process C, concentration process B, or concentration process D
- concentration process A, DF process C, concentration process B, or concentration process D concentration process A, DF process C, concentration process B, or concentration process D
- concentration process D concentration process D
- the desired LA content or LA recovery rate or What is necessary is just to combine suitably according to the objectives, such as equipment, energy cost, drainage control.
- the obtained LA-containing composition can be sterilized by a conventional method as necessary, and then dried by a conventional method to obtain a powder state.
- the temperature of the protein raw material production process used in the present method and the process of the preparation method of the composition containing LA described above is included by setting the temperature of the protein raw material production process used in the present method and the process of the preparation method of the composition containing LA described above to a temperature that does not significantly affect the denaturation of LA (for example, less than 62 ° C.). It is possible to obtain a composition containing LA in which most of the LA to be produced is unmodified (for example, 70% or more).
- a method for fractionating whey protein / a method for producing a composition comprising LA comprises one or more steps selected from a concentration step, a diafiltration step, a sterilization step, a freezing step, a drying step, and a powdering step. Can be included.
- the method for producing a composition containing LG comprises (a) whey protein mixed with (b) carbonate ion and / or hydrogen carbonate ion, and (b) whey protein- (b) carbonate ion and / or hydrogen carbonate.
- An ion complex (hereinafter referred to as “LG-containing complex 1”) is formed, but LA is not involved in the formation of LG-containing complex 1 and does not form LG-containing complex 1. It was completed based on knowledge. That is, the present invention has been made by finding the property that LA is not contained in the complex 1 containing LG.
- the manufacturing method of the composition containing LG mix blends (i) whey protein, (b) carbonate ion and / or hydrogen carbonate ion, and (c) light metal ion and / or transition metal ion, B) Whey protein-(b) Carbonate ion and / or hydrogen carbonate ion-(c) Complex of light metal ion and / or transition metal ion and / or Complex containing LG 1-
- Whey protein-( B) A carbonate ion and / or bicarbonate ion- (c) a complex of light metal ions and / or transition metal ions (hereinafter referred to as “complex 2 containing LG”) is formed. It does not participate in the formation of the complex 2 containing LG and does not form the complex 2 containing LG. That is, the present invention has been made by finding the property that LA is not contained in the complex 2 containing LG.
- the protein material, carbonate ion and hydrogen carbonate ion, light metal ion and / or transition metal ion, and membrane used in the method for producing a composition containing LG are used in the method for fractionating whey protein and the method for producing a composition containing LA. It can be the same as the one.
- FIG. 2 is a flowchart of a method for producing a composition containing LG. A method for producing a composition containing LG will be described with reference to the flowchart of FIG. In addition, although the method example of FIG. 2 is used for description, this invention is not limited to this method example.
- step 201 solution preparation, a solution containing (first and second) whey proteins is prepared.
- the solution containing whey protein is preferably divided into step 201a when it does not contain metal ions and step 201b when it contains metal ions, and such a solution is preferably prepared.
- Step 201a When the solution containing whey protein does not contain metal ions, the total concentration of (first and second) whey proteins is 0.001 wt% or more and 35 wt% or less of carbonate ions and / or bicarbonate ions. It is preferable to prepare an aqueous solution using the raw materials described above so that the total concentration is 0.001 wt% or more and 35 wt% or less and the pH is 5 or more and 10 or less.
- Step 201b When the solution containing the whey protein contains metal ions, the total concentration of the (first and second) whey proteins is 0.001% by weight to 35% by weight, and all of the carbonate ions and / or bicarbonate ions.
- the above-described raw materials are adjusted so that the concentration is 0.001% to 35% by weight, the total concentration of light metal and / or transition metal ions is 0.00001% to 10% by weight, and the pH is 5 to 10%. Use to prepare an aqueous solution.
- whey proteins excluding LA and carbonate ions and / or carbonates are prepared.
- the complex 2 containing LG is formed by the interaction between the hydrogen ion and the light metal and / or transition metal ion.
- LA is not involved in the formation of the complex 2 containing LG, and LA does not form the complex 2 containing LG.
- a metal ion source that reacts with carbonate ions and / or bicarbonate ions to form a precipitate is dissolved, but except for this,
- Each material may be added in any order, and no protein-containing precipitate is observed during or after the preparation of the aqueous solution using the raw materials described above. It is not necessary to adjust the temperature of the solution, but it is preferably 0 ° C. or higher and 15 ° C. or lower in consideration of microbial growth.
- Step 203 Concentration Processing A, concentration processing is performed using a membrane that can concentrate the solution obtained in Step 201.
- step 201 when the aqueous solution containing the complex 1 containing LG is prepared, the complex 1 containing LG is concentrated, and when the aqueous solution containing the complex 2 containing LG is prepared, the complex 2 containing LG is concentrated. It will be.
- fractionating the complex 1 containing LG and LA it is preferable to use a membrane having a fractional molecular weight of 50,000 or less (step 203a).
- the complex 2 containing LG and LA are fractionated, it is preferable to use a membrane having a fractional molecular weight of 300,000 or less (step 203b).
- the steps 203a and 203b can be performed depending on the content of the composite 1 containing LG and the composite 2 containing LG.
- LA is transferred to the LA-containing permeate 2 which is mainly a permeate.
- the concentrated solution 1 is obtained by preparing the complex 1 containing LG when the aqueous solution containing the complex 1 containing LG or the complex 2 containing LG when the aqueous solution containing the complex 2 containing LG is prepared.
- a composition containing LG can be obtained.
- the processing conditions such as sample temperature, average operating pressure, membrane surface flow rate, and concentration ratio during this treatment can be adjusted as appropriate so that the LG content in the concentrate is about 50 to 100% by weight per total solid content. .
- Step 208 In the DF treatment C, the concentrated solution 1 (the fraction containing the complex 1 containing LG or the complex 2 containing LG) obtained in step 203 in accordance with the purity and recovery rate of the desired LG Can be subjected to DF processing.
- DF includes filtered water, ion-exchanged water, distilled water, ultrapure water, a permeate produced in a membrane treatment process, a solution with adjusted pH and ionic strength, or a plurality of these. A mixture can be used.
- the fraction retained in the membrane, that is, the concentrated solution 3 has a high content of the complex 1 containing LG or the complex 2 containing LG per total solid content.
- the LA-containing permeate 5 that is the permeate of the membrane is a composition containing LA.
- the processing conditions such as sample temperature, average operating pressure, membrane surface flow rate, and concentration ratio during DF treatment can be adjusted as appropriate so that the LG content in the concentrate is about 50 to 100% by weight per total solid content. .
- Step 210 In the metal removal treatment, the concentrate 3 (the fraction containing the obtained complex 1 containing LG or the complex 2 containing LG) is added with a component having a metal chelating action as necessary or The component which has the effect
- a composition containing LG with reduced metal can be obtained.
- the chelating component include citric acid, ascorbic acid, ethylenediaminetetraacetic acid and the like.
- components that lower the pH include hydrochloric acid, sulfuric acid, and nitric acids.
- step 214 sterilization treatment
- step 216 dry to obtain a powder state by a conventional method.
- the obtained dry powder can also be used as a metal solubilizer.
- a composition in which most of LG contained (for example, 70% or more) is unmodified by setting the temperature of the protein raw material used in the present method and the above-described steps of the composition containing LG to less than 78 ° C. Can be obtained.
- steps 203, 208, 212, 214, and 216 concentration process A, DF process B, and demetallization process, sterilization process, and drying process
- steps 203, 208, 212, 214, and 216 concentration process A, DF process B, and demetallization process, sterilization process, and drying process
- steps 203, 208, 212, 214, and 216 concentration process A, DF process B, and demetallization process, sterilization process, and drying process
- the method for producing a composition containing LG can include one or a plurality of steps selected from a concentration step, a diafiltration step, a sterilization step, a freezing step, a drying step, and a powdering step.
- the processing temperature of all processes can be less than 72 degreeC, and the composition containing (beta) -lactoglobulin in which 70% or more of (beta) -lactoglobulin contained in a composition is undenatured can be obtained.
- Whey powder 1 and whey powder 2 were used as whey protein materials.
- Whey powder 1 is obtained by subjecting cheese whey to ion exchange and ultrafiltration treatment so that the whey protein content is 93.9% by weight.
- the whey powder 2 is obtained by subjecting the acid whey to ion exchange and ultrafiltration treatment so that the whey protein content is 93.5% by weight.
- Sodium bicarbonate was used as a material for carbonate ions and / or bicarbonate ions.
- Light metal ion material is magnesium chloride hexahydrate, potassium chloride, transition metal ion material is manganese chloride tetrahydrate, ferrous sulfate heptahydrate, ferric chloride hexahydrate, ferric sulfate Copper sulfate pentahydrate was used.
- Sodium hydroxide was used for the preparation of the comparative example. All the water used in the examples and comparative examples was ion-exchanged water.
- aqueous solutions according to Examples 1 to 20 and Comparative Examples 1 to 5 shown in Table 1 were prepared.
- the pH of the prepared aqueous solution was 7.8 to 8.3. No precipitate was observed during or after preparation of the aqueous solution.
- the prepared aqueous solution was filtered by a total filtration method using a centrifuge at 4000 G for 15 minutes.
- the filtration membrane used was a material having a polyether sulfone and a molecular weight cut off of 50,000, 100,000, and 300,000, and a material having a regenerated cellulose and a molecular weight cut off of 50,000.
- the solution that permeated the filtration membrane was collected, and the amounts of LA and LG contained in the permeate were measured.
- the amounts of LA and LG were measured using high performance liquid chromatography. Measurement conditions were according to the method of Bordin et al. (Journal of Chromatography A. (2001) 928, 1, 63-76).
- the LA / LG ratio was high in Examples 1 to 20 in which the permeate obtained by forming the complex 2 and filtering through a membrane was obtained as a permeate containing LA. That is, the composite 2 did not permeate the filtration membrane, and the tendency of LA to permeate the filtration membrane was recognized, and LA and the composite 2 were fractionated, and a composition containing LA as a permeate was obtained.
- the LA / LG ratio was high when the filter membrane had a molecular weight cut-off of 50,000. On the other hand, the material of the membrane was not different between regenerated cellulose and polyethersulfone.
- Test Example 2 Fractionation of LA and LG due to formation of complex 1
- Materials Whey powder 1 and whey powder 2 were used as whey protein materials.
- sodium bicarbonate was used as a material for carbonate ions and / or bicarbonate ions
- ion-exchanged water was used as the water.
- ion-exchanged water (carbonated water) saturated with carbon dioxide was used as a material for carbonate ions and / or bicarbonate ions.
- aqueous solutions according to Examples 21 to 23 shown in Table 2 were prepared. No precipitate was observed during or after preparation of the aqueous solution.
- the prepared aqueous solution was filtered by a total filtration method using a centrifuge at 4000 G for 15 minutes.
- the filter membrane was made of polyethersulfone and a molecular weight cut off of 50,000.
- the solution that permeated the filtration membrane was collected, and the amounts of LA and LG contained therein were measured by high performance liquid chromatography. Measurement conditions were according to the method of Bordin et al. (Journal of Chromatography A. (2001) 928, 1, 63-76).
- the LA / LG ratio of the resulting permeate was increased by adding whey protein and components that generate carbonate ions and / or bicarbonate ions, and filtering the solution containing these components through a membrane. . That is, the composite 1 did not permeate the filtration membrane, and the tendency of LA to permeate the filtration membrane was recognized, and LA and the composite 1 were fractionated, and a composition containing LA as the permeate was obtained. Further, in Examples 21 to 23 shown in Table 2, the LA / LG ratio was higher than that of the comparative example before treatment and the comparative example shown in Table 1 and fractionated efficiently under all conditions.
- the LA / LG ratio of the permeate obtained by allowing the aqueous solution before filtration to stand at 10 ° C. for 24 hours and then filtering in the same manner as described above is 95 to 101% of the value shown in Table 2, It was almost the same (data not shown). In addition, no precipitate was observed in each solution after standing for 24 hours.
- Whey powder 3 was used as a whey protein material.
- Whey powder 3 is obtained by subjecting cheese whey to ion exchange and ultrafiltration treatment so that the whey protein content is 95.1%.
- Sodium bicarbonate was used as a material for carbonate ions and / or bicarbonate ions.
- Copper sulfate pentahydrate was used as the light metal / transition metal ion material.
- Water used was ion exchange water.
- a 1 mol / L hydrochloric acid aqueous solution of Wako Pure Chemical Industries, Ltd. was used for adjusting the pH.
- the pH of the aqueous solution was measured according to a conventional method.
- aqueous solutions according to Examples 24 to 26 shown in Table 3 were prepared. No precipitate was observed during or after preparation of the aqueous solution.
- the prepared aqueous solution was filtered by a total filtration method using a centrifuge at 4000 G for 15 minutes.
- the filtration membrane was made of regenerated cellulose and the molecular weight cut off was 50,000.
- the composite 2 did not permeate the filtration membrane, and the tendency of LA to permeate the filtration membrane was recognized, and LA and the composite 2 were fractionated, and a composition containing LA as a permeate was obtained.
- the recovery rates of LA in Examples 24 to 26 were 35%, 39%, and 29%, respectively.
- Example 27 30 kg of an aqueous solution according to Example 27 shown in Table 4 was prepared using the materials described above. No precipitate was observed during or after preparation of the aqueous solution.
- This aqueous solution was subjected to a filtration process by a cross flow method using a filtration membrane (polyacrylonitrile material, molecular weight cut off 50,000, membrane area 0.20 square meters).
- the conditions for the filtration treatment were a temperature of 10 ° C. and an average operating pressure of 0.2 MPa. By this treatment, 10 kg of concentrated liquid and 20 kg of permeated liquid (composition containing LA) were obtained.
- the concentrate obtained in the previous paragraph was subjected to DF treatment.
- the filtration membrane and operating conditions used are the same as in the previous paragraph.
- the DF treatment was completed when the permeate (composition containing LA) reached 18 kg.
- the concentrated solution in the previous paragraph and the permeated solution in the previous paragraph were heat-sterilized according to a conventional method, and then freeze-dried to obtain a composition containing LA in powder form (Example 27).
- the LA / LG ratio of the composition containing dry powdery LA was 6.8, which was significantly higher than 0.3 of whey powder 1 before treatment.
- the recovery rate of LA was 85%.
- Raw milk was processed with a centrifuge to obtain skim milk from which fat had been removed.
- This skim milk is filtered using a filtration membrane (ceramic material, fractional molecular weight 0.1 ⁇ m, membrane area 1.05 square meters) by a cross flow method, and a permeate containing 0.05% by weight of whey protein is obtained. Obtained.
- the aqueous solution was filtered by a cross flow method using a filtration membrane (polyacrylonitrile material, molecular weight cut off 50,000, membrane area 0.20 square meters).
- the conditions for the filtration treatment were a temperature of 10 ° C. and an average operating pressure of 0.2 MPa.
- 10 kg of concentrated liquid and 20 kg of permeate were obtained.
- This permeate had an LA / LG ratio of 4.13, which was significantly higher than 0.5 before the treatment.
- DF treatment was performed on the concentrate obtained in the previous paragraph.
- the filtration membrane and operating conditions used are the same as described in the previous paragraph.
- the DF treatment was terminated when the permeate (composition containing LA) reached 18 kg.
- the permeate obtained in the previous paragraph and the previous paragraph was subjected to a filtration process by a cross flow method using a filtration membrane (polyethersulfone material, molecular weight cut off 10,000, membrane area 1.00 square meter).
- the conditions for the filtration treatment were a temperature of 10 ° C. and an average operating pressure of 0.4 MPa. By this treatment, 4 kg of concentrated liquid (composition containing LA) and 34 kg of permeate were obtained. There was no transfer of LA and LG to the permeate.
- Example 28 The concentrated solution in the previous paragraph was freeze-dried according to a conventional method to obtain a composition (Example 28) containing dry powdery LA.
- the LA / LG ratio of Example 28 was 4.1 and the denatured LA was 1%.
- the recovery rate of LA was 62%.
- aqueous solution 300 g of an aqueous solution according to Example 29 shown in Table 5 was prepared. No precipitate was observed during or after preparation of the aqueous solution.
- This aqueous solution was subjected to a filtration process by a cross flow method using a filtration membrane (polyethersulfone material, molecular weight cut off 50,000, membrane area 0.050 square meter).
- the conditions for the filtration treatment were a temperature of 10 ° C. and an average operation of 2.5 bar. By this treatment, 90 g of concentrated liquid and 200 g of permeated liquid (composition containing LA) were obtained.
- the concentrate obtained in the previous paragraph was subjected to DF treatment.
- the filtration membrane and operating conditions used are the same as described in the previous paragraph.
- the DF treatment was terminated when the permeate (composition containing LA) reached 180 g.
- the concentrated solution was freeze-dried according to a conventional method to obtain a composition containing dry powdery LA (Example 29).
- the LA / LG ratio in the dry powder was 5.2 and the modified LA was 31%.
- the recovery rate of LA was 96%.
- the prepared aqueous solution was filtered by a total filtration method using a centrifuge at 4000 G for 15 minutes.
- the filtration membrane was made of regenerated cellulose and the molecular weight cut off was 50,000.
- the solution that permeated the filtration membrane was collected, and the amounts of LA and LG contained in the permeate were measured.
- the amounts of LA and LG were measured using high performance liquid chromatography. Measurement conditions were according to the method of Bordin et al. (Journal of Chromatography A. (2001) 928, 1, 63-76).
- the LA / LG ratio of Examples 30 to 36 in which the permeate obtained by forming the complex 2 and filtering with a membrane as a permeate containing LA, was high. That is, the composite 2 did not permeate the filtration membrane, and the tendency of LA to permeate the filtration membrane was recognized, and LA and the composite 2 were fractionated, and a composition containing LA as a permeate was obtained.
- the LA / LG ratio was higher than that of the comparative example before treatment and the comparative example shown in Table 1 and fractionated efficiently under all conditions. The recovery rate of LA was 33% to 45%.
- Whey powder 1 and whey powder 2 were used as whey protein materials.
- Whey powder 1 is obtained by subjecting cheese whey to ion exchange and ultrafiltration treatment so that the whey protein content is 93.9% by weight.
- the whey powder 2 is obtained by subjecting the acid whey to ion exchange and ultrafiltration treatment so that the whey protein content is 93.5% by weight.
- Sodium bicarbonate was used as a material for carbonate ions and / or bicarbonate ions.
- Light metal ion material is magnesium chloride hexahydrate, potassium chloride, transition metal ion material is manganese chloride tetrahydrate, ferrous sulfate heptahydrate, ferric chloride hexahydrate, ferric sulfate Copper sulfate pentahydrate was used.
- Sodium hydroxide was used for the preparation of the comparative example. All the water used in the examples and comparative examples was ion-exchanged water.
- Aqueous solutions according to Examples 37 to 56 and Comparative Examples 6 to 10 shown in Table 7 were prepared.
- the pH of the prepared aqueous solution was 7.8 to 8.3. No precipitate was observed during or after preparation of the aqueous solution.
- the prepared aqueous solution was filtered by a total filtration method using a centrifuge at 4000 G for 15 minutes.
- the filtration membrane used was a material having a polyether sulfone and a molecular weight cut off of 50,000, 100,000, and 300,000, and a material having a regenerated cellulose and a molecular weight cut off of 50,000.
- Examples 37 to 56 and Comparative Example 10 to Comparative Example 10 were obtained as concentrated liquids retained on the filtration membrane, and the amounts of LG and LA contained in the concentrated liquids were measured.
- the amounts of LG and LA were measured using high performance liquid chromatography. Measurement conditions were according to the method of Bordin et al. (Journal of Chromatography A. (2001) 928, 1, 63-76).
- the LG / LA ratio was high. That is, the composite 2 containing LG does not permeate the filtration membrane, and LA tends to permeate the filtration membrane.
- the composite 2 containing LG and LA are fractionated, and the composition contains LG as a concentrate. was gotten.
- the LG / LA ratio is higher than that of the comparative example in which only the light metal is added, and Examples 37 to 56 are more efficient than Comparative Examples 6 to 10 in terms of LG. And LA were fractionated.
- the LG / LA ratio was high when the molecular weight cut off of the filtration membrane was 50,000. On the other hand, the material of the membrane was not different between regenerated cellulose and polyethersulfone. LG / LA of the permeate obtained by filtering the aqueous solutions of Example 37 to Example 56 and Comparative Example 6 to Comparative Example 10 before filtration after standing for 24 hours at 10 ° C. The ratio was 97-102% of the values shown in Table 7 and was similar (data not shown). In addition, no precipitate was observed in each solution after standing for 24 hours.
- L-ascorbic acid was added to the concentrated solution treated with DF so as to be 1% by weight, and after gently stirring for 60 minutes at room temperature, this was further subjected to filtration. Filtration was performed using a filtration membrane (polyacrylonitrile material, molecular weight cut off 50,000, membrane area 0.20 square meters) under conditions of 10 ° C. and an average operating pressure of 0.2 MPa.
- a filtration membrane polyacrylonitrile material, molecular weight cut off 50,000, membrane area 0.20 square meters
- Example 10 Materials Whey powder 1 and whey powder 2 were used as whey protein materials.
- sodium hydrogen carbonate was used as a material for carbonate ions and / or hydrogen carbonate ions
- ion-exchanged water was used as the water.
- ion-exchanged water (carbonated water) saturated with carbon dioxide was used as a material for carbonate ions and / or bicarbonate ions.
- aqueous solutions according to Examples 58 to 60 shown in Table 9 were prepared. No precipitate was observed during or after preparation of the aqueous solution.
- the prepared aqueous solution was filtered by a total filtration method using a centrifuge at 4000 G for 15 minutes.
- the filter membrane was made of polyethersulfone and a molecular weight cut off of 50,000.
- the concentrated solution was collected, and the amounts of LG and LA contained therein were measured.
- the amounts of LG and LA were measured using high performance liquid chromatography. Measurement conditions were according to the method of Bordin et al. (Journal of Chromatography A. (2001) 928, 1, 63-76).
- the whey protein and components that generate carbonate ions and / or bicarbonate ions were added, and the solution containing these was filtered through a membrane, resulting in an increase in the LG / LA ratio of the resulting concentrate. . That is, the composite 1 containing LG does not permeate the filtration membrane, and LA tends to permeate the filtration membrane.
- the composite 1 containing LG and LA are fractionated, and the composition contains LG as a concentrate. was gotten. Further, in all the conditions of Examples 58 to 60 shown in Table 9, the LG / LA ratio is higher than that of the pre-treatment and Comparative Examples 6 to 10 shown in Table 7 and LG and LA are efficiently reduced. It was fractionated.
- Example 60 was pulverized by freeze-drying (Example 61).
- the water content of Example 61 stored for 1 month at 10 ° C. was 5.1% by weight. This was dissolved in ion-exchanged water to prepare a 1% by weight aqueous solution.
- Ferric chloride hexahydrate was added to this aqueous solution so that it might become 0.01 weight%, and after stirring, it heat-sterilized on the conditions of 95 degreeC reach
- Raw milk was processed with a centrifuge to obtain skim milk from which fat had been removed.
- This skim milk is filtered using a filtration membrane (ceramic material, molecular weight cut off 0.1 ⁇ m, membrane area 1.05 square meters) in a cross-flow manner, and a permeate containing 0.05% by weight of whey protein is obtained. Obtained.
- Example 62 30 kg of an aqueous solution according to Example 62 was prepared by adding 0.05% by weight of sodium bicarbonate and ferric chloride hexahydrate to 0.005% by weight as iron ions to the aqueous solution containing whey protein. did. No precipitate was observed during or after preparation of the aqueous solution.
- the aqueous solution was subjected to a filtration process by a cross flow method using a filtration membrane (polyacrylonitrile material, molecular weight cut off 50,000, membrane area 0.20 square meters).
- the conditions for the filtration treatment were a temperature of 10 ° C. and an average operating pressure of 0.2 MPa. By this treatment, 20 kg of a permeate and 10 kg of a composition containing LG as a concentrate were obtained.
- the LG / LA ratio of this concentrate was 5.22, which was significantly higher than 2.14 before the treatment.
- This concentrated solution was further subjected to DF treatment.
- the DF treatment was finished when a permeate amount of 18 kg was obtained.
- L-ascorbic acid was added to the concentrated solution treated with DF so as to be 1% by weight, and after gently stirring for 60 minutes at room temperature, this was further subjected to filtration. Filtration was carried out using a filtration membrane (polyacrylonitrile material, molecular weight cut off 50,000, membrane area 0.20 square meters) under conditions of 10 ° C. and an average operating pressure of 0.2 MPa by a cross flow method.
- a filtration membrane polyacrylonitrile material, molecular weight cut off 50,000, membrane area 0.20 square meters
- the concentrated solution in the previous paragraph was freeze-dried according to a conventional method to obtain a composition containing dry powdery LG (Example 62).
- the LG / LA ratio in the dry powder was 4.98, and the modified LG was 1%.
- the recovery rate of LG was 65%.
- the prepared aqueous solution was filtered by a total filtration method using a centrifuge at 4000 G for 15 minutes.
- the filtration membrane was made of regenerated cellulose and the molecular weight cut off was 50,000.
- the solution retained on the filtration membrane was collected, and the amounts of LG and LA contained in the concentrate were measured.
- the amounts of LG and LA were measured using high performance liquid chromatography. Measurement conditions were according to the method of Bordin et al. (Journal of Chromatography A. (2001) 928, 1, 63-76).
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Abstract
Description
(1)第一のホエイタンパク質及び第二のホエイタンパク質を含む溶液を調製する工程と;第一のホエイタンパク質及び第二のホエイタンパク質を膜により分離する工程と;を備え、第一のホエイタンパク質は、炭酸イオン、炭酸水素イオン、軽金属イオン及び遷移金属イオンの少なくとも1つと複合体を形成するものであり、第二のホエイタンパク質は、下記(i)、(ii)
(i)炭酸イオン及び炭酸水素イオンのいずれとも複合体を形成しない、
(ii)軽金属イオン及び遷移金属イオンのいずれとも複合体を形成しない、の少なくとも1つを満たすホエイタンパク質の分画方法。
(2)第一のホエイタンパク質が主としてβ‐ラクトグロブリンを含むホエイタンパク質であり、第二のホエイタンパク質が主としてα‐ラクトアルブミンを含むホエイタンパク質である(1)に記載のホエイタンパク質の分画方法。
(3)膜の分画分子量が50,000以下である(1)又は(2)に記載のホエイタンパク質の分画方法。
(4)膜の分画分子量が300,000以下である(1)又は(2)に記載のホエイタンパク質の分画方法。
(5)溶液は、第一のホエイタンパク質及び第二のホエイタンパク質の全濃度が0.001重量%以上35重量%以下、炭酸イオン及び炭酸水素イオンの全濃度が0.001重量%以上35重量%以下、pHが5以上10以下である(1)から(4)に記載のホエイタンパク質の分画方法。
(6)軽金属イオン及び遷移金属イオンの全濃度が0.00001重量%以上10重量%以下である(1)から(5)に記載のホエイタンパク質の分画方法。
(7)濃縮工程、ダイアフィルトレーション工程、殺菌工程、凍結工程、乾燥工程、粉末化工程から選択される1つ、又は複数の工程を含む(1)から(6)のいずれか1つに記載のホエイタンパク質の分画方法。
(8)β‐ラクトグロブリンを含むホエイタンパク質と、炭酸イオン及び/又は炭酸水素イオンと、を含む溶液を調製する工程と;分画分子量が50,000以下の膜を用いて溶液を処理する工程と;膜の透過液としてα‐ラクトアルブミンを含む溶液と、膜の濃縮液としてβ‐ラクトグロブリン、炭酸イオン及び/又は炭酸水素イオンで形成された複合体を含む溶液と、を得る工程と;α‐ラクトアルブミンを含む溶液を分画分子量が14,000以下の膜で処理する工程と;を含むα‐ラクトアルブミンを含む組成物の調製方法。
(9)溶液のホエイタンパク質の濃度が0.001重量%以上35重量%以下、溶液の炭酸イオン及び炭酸水素イオンの全濃度が0.001重量%以上35重量%以下、溶液のpHが5以上10以下である(8)に記載のα‐ラクトアルブミンを含む組成物の調製方法。
(10)β‐ラクトグロブリンを含むホエイタンパク質と、炭酸イオン及び/又は炭酸水素イオンと、軽金属イオン及び/又は遷移金属イオンと、を含む溶液を調製する工程と;分画分子量が300,000以下の膜を用いて溶液を処理する工程と;膜の透過液としてα‐ラクトアルブミンを含む溶液と、膜の濃縮液としてβ‐ラクトグロブリン、炭酸イオン及び/又は炭酸水素イオン、軽金属イオン及び/又は遷移金属イオンで形成された複合体を含む溶液と、を得る工程と;α‐ラクトアルブミンを含む溶液を分画分子量が14,000以下の膜で処理する工程と;を含むα‐ラクトアルブミンを含む組成物の調製方法。
(11)溶液のホエイタンパク質の濃度が0.001重量%以上35重量%以下、溶液の炭酸イオン及び炭酸水素イオンの全濃度が0.001重量%以上35重量%以下、溶液のpHが5以上10以下、溶液の軽金属イオン及び遷移金属イオンの全濃度が0.00001重量%以上10重量%以下、である(10)に記載のα‐ラクトアルブミンを含む組成物の調製方法。
(12)濃縮工程、ダイアフィルトレーション工程、殺菌工程、凍結工程、乾燥工程、粉末化工程から選択される1つ、又は複数の工程を含む(8)から(11)のいずれか1つに記載のα‐ラクトアルブミンを含む組成物の調製方法。
(13)(8)から(12)に記載の全ての工程の処理温度を62℃未満することを特徴とする組成物に含まれるα‐ラクトアルブミンの70%以上が未変性α‐ラクトアルブミンである組成物の調製方法。
(14)β‐ラクトグロブリンを含むホエイタンパク質と、炭酸イオン及び/又は炭酸水素イオンと、を含む溶液を調製する工程と;分画分子量が50,000以下の膜を用いて溶液を処理する工程と;膜の透過液としてα‐ラクトアルブミンを含む溶液と、膜の濃縮液としてβ‐ラクトグロブリン、炭酸イオン及び/又は炭酸水素イオンで形成された複合体を含む溶液と、を得る工程と;を含むβ‐ラクトグロブリンを含む組成物の調製方法。
(15)溶液のホエイタンパク質の濃度が0.001重量%以上35重量%以下、溶液の炭酸イオン及び炭酸水素イオンの全濃度が0.001重量%以上35重量%以下、溶液のpHが5以上10以下である(14)に記載のβ‐ラクトグロブリンを含む組成物の調製方法。
(16)β‐ラクトグロブリンを含むホエイタンパク質と、炭酸イオン及び/又は炭酸水素イオンと、軽金属イオン及び/又は遷移金属イオンと、を含む溶液を調製する工程と;分画分子量が300,000以下の膜を用いて溶液を処理する工程と;膜の透過液としてα‐ラクトアルブミンを含む溶液と、膜の濃縮液として、β‐ラクトグロブリン、炭酸イオン及び/又は炭酸水素イオン、軽金属イオン及び/又は遷移金属イオンで形成された複合体を含む溶液と、を得る工程と;を含むβ‐ラクトグロブリンを含む組成物の調製方法。
(17)溶液のホエイタンパク質の濃度が0.001重量%以上35重量%以下、溶液の炭酸イオン及び炭酸水素イオンの全濃度が0.001重量%以上35重量%以下、溶液のpHが5以上10以下、溶液の軽金属イオン及び遷移金属イオンの全濃度が0.00001重量%以上10重量%以下、である(16)に記載のβ‐ラクトグロブリンを含む組成物の調製方法。
(18)濃縮工程、ダイアフィルトレーション工程、殺菌工程、凍結工程、乾燥工程、粉末化工程から選択される1つ、又は複数の工程を含む(14)から(17)のいずれか1つに記載のβ‐ラクトグロブリンを含む組成物の調製方法。
(19)金属イオンを低減させる工程を含む(16)から(18)に記載のβ‐ラクトグロブリンを含む組成物の調製方法。
(20)(14)から(19)に記載の全ての工程の処理温度を72℃未満にすることを特徴とする組成物に含まれるβ‐ラクトグロブリンの70%以上が未変性β‐ラクトグロブリンである組成物の調製方法。
なお、以下α-ラクトアルブミンを「LA」、β-ラクトグロブリンを「LG」、ホエイタンパク質濃縮物を「WPC」、精製ホエイタンパク質を「WPI」と記載することもある。
(イ)ホエイタンパク質―(ロ)炭酸イオン及び/又は炭酸水素イオン-(ハ)軽金属イオン及び/又は遷移金属イオンの複合体、及び/又は、
複合体1-(イ)ホエイタンパク質―(ロ)炭酸イオン及び/又は炭酸水素イオン-(ハ)軽金属イオン及び/又は遷移金属イオンの複合体(以下、これらを「複合体2」と記載する)が形成されるが、LAは複合体2の形成には関与せず複合体2を形成しないことを知見した。すなわち、本発明の第二の態様は、複合体2にはLAが含まれない性質を見出してなされたものである。
ホエイタンパク質の分画方法は、第一のホエイタンパク質及び第二のホエイタンパク質を含む溶液を調製する工程と;第一のホエイタンパク質及び第二のホエイタンパク質を膜により分離する工程と;を備える。
ここで、第一のホエイタンパク質は、炭酸イオン、炭酸水素イオン、軽金属イオン及び遷移金属イオンの少なくともいずれか1つと複合体を形成し、また第二のホエイタンパク質は、下記(1)(2)
(1)炭酸イオン及び炭酸水素イオンのいずれとも複合体を形成しない、
(2)軽金属イオン及び遷移金属イオンのいずれとも複合体を形成しない、の少なくとも1つを満たすものである。
ホエイタンパク質の分画方法及びLAを含む組成物の製造方法に用いるタンパク質材料について説明する。
上記(第一、第二の)タンパク質材料は、ホエイタンパク質を含むものであればよく、牛、水牛、羊、ヤギ、馬等の哺乳類に由来するものであり、上述の要件を満たすものであればどのようなものでも用いることができ、1種類あるいは複数の原材料を組み合わせて用いることもできる。
具体的には、牛、水牛、羊、ヤギ、馬等の乳を用いてチーズを製造する際に得られるホエイ、牛、水牛、羊、ヤギ、馬等の乳に乳酸などの酸等を添加した際に得られるホエイ、これらのホエイを粉末にしたもの、これらのホエイ中の乳糖やミネラル等を低減してホエイタンパク質を濃縮したもの等を例示できる。この中で、ホエイタンパク質の分画方法及びLAを含む組成物の製造方法に用いるタンパク質材料としては、乳糖や脂肪分等の夾雑成分が少なく、LAとLGの合計が全固形分に対して50重量%以上のWPCやWPIが好ましい。
ホエイタンパク質の分画方法及びLAを含む組成物の製造方法に用いる炭酸イオンと炭酸水素イオンは、水溶液中でこれらのイオンを生じるものであればどのような成分や方法を用いて生成させてもよく、下記に例示した1種類あるいは複数の化合物及び/又は方法を組み合わせて炭酸イオン及び/又は炭酸水素イオンを生じさせればよい。
気体状の二酸化炭素の吹き込み、液状あるいは固体状の二酸化炭素の添加といった二酸化炭素を添加する方法、炭酸水素ナトリウムや炭酸水素カリウムなどの炭酸水素塩や、炭酸ナトリウムや炭酸カリウムなどの炭酸塩を添加する方法、炭酸水を添加する方法等が例示される。
上記軽金属イオンと遷移金属イオンは、軽金属あるいは遷移金属の1価から3価の陽イオンであればどのようなものでもよく、1種類又は複数の金属イオンを用いることができる。これらの金属イオン源として、軽金属及び/又は遷移金属の無機塩や有機塩を用いることができる。
LAを含む組成物を食品や医薬品に用いる場合は、軽金属イオン及び/又は遷移金属イオンとして、食品やヒト中に存在するナトリウム、カリウム、マグネシウム、カルシウム、マンガン、鉄、銅、亜鉛などを用いることが好ましい。
なお、上記の炭酸イオン及び/又は炭酸水素イオンを生じさせる際に、炭酸イオンや炭酸水素イオンの塩を使用する場合は、別途金属イオン源の添加は必須ではない。例えば、炭酸イオン及び/又は炭酸水素イオンの材料として炭酸水素ナトリウムを用いた場合は、別途、ナトリウム、あるいはその他の軽金属や遷移金属の無機塩や有機塩を添加してもよいし、添加しなくてもよい。
膜の材質はポリアクリルアミド、再生セルロース、ポリエチレン、4フッ化エチレン、ポリプロピレン、酢酸セルロース、ポリアクリロニトリル、ポリイミド、ポリスルホン、ポリエーテルスルホン等の有機膜や、酸化アルミニウム、酸化ジルコニウム、酸化チタン、ステンレス、ガラス等の無機膜を例示できる。
膜モジュールの種類は、プリーツ型モジュール、スパイラル型モジュール、モノリス型モジュール、チューブ型モジュール等を例示できる。
膜濾過の方式は全量濾過方式、クロスフロー方式、ダイアフィルトレーション方式等を例示できる。
図1はホエイタンパク質の分画方法/LAを含む組成物の製造方法のフローチャートである。ホエイタンパク質の分画方法、及びLAを含む組成物を製造する方法について図1のフローチャートを用いて説明する。なお、説明には図1の方法例を用いるが、本発明はこの方法例に限定されるものではない。
pHが5以上10以下で上記した濃度のホエイタンパク質と炭酸イオン及び/又は炭酸水素イオンを含む水溶液中を調製すると、LAを除くホエイタンパク質と、炭酸イオン及び/又は炭酸水素イオンとの相互作用により複合体1が形成される。LAはこの複合体1の形成には関与せず、LAは複合体1を形成しない。前記した原材料を用いた水溶液の調製中または調製後に、タンパク質を含む沈殿物は観察されない。タンパク質原料と炭酸イオン及び/又は炭酸水素イオン源となる材料はどのような順番で添加してもよい。溶液の温度調整は不要であるが、微生物の繁殖を考慮して0℃以上15℃以下とすることが好ましい。
LAを除くホエイタンパク質と、炭酸イオン及び/又は炭酸水素イオンと、軽金属及び/又は遷移金属イオンとの相互作用により複合体2が形成される。LAはこの複合体2の形成には関与せず、LAは複合体2を形成しない。
複合体2の調製において、炭酸イオン及び/又は炭酸水素イオンと、反応して沈殿物を生じるような金属イオン源のみが溶解している状態は好ましくないが、これを除いては、各材料はどのような順番で添加してもよく、前記した原材料を用いた水溶液の調製中または調製後に、タンパク質を含む沈殿物は観察されない。
溶液の温度調整は不要であるが、微生物の繁殖を考慮して0℃以上15℃以下とすることが好ましい。
この処理により得られる濃縮液1には、複合体1を含む水溶液を調製した場合は複合体1、複合体2を含む水溶液を調製した場合は複合体2が多く含まれる。
一方、透過液、即ちLA含有透過液2には、LAが多く含まれるものとなり、ここでLAとLGが分画され、LAを含む組成物を得ることができる。
この処理の際の試料温度、平均運転圧力、膜面流速、濃縮倍率等の処理条件は、透過液中のLA含量及び/又は濃縮液中のLG含量が全固形分あたり50~100重量%程度となるよう適宜調整することができる。
この処理により得られる濃縮液、即ちLA濃縮液3は、LAを含む組成物である。一方、透過液、即ち透過液4はタンパク質をほとんど含まないものとなる。
濃縮処理の際の試料温度、平均運転圧力、膜面流速、濃縮倍率等の処理条件は、LAを含む組成物が効率よく濃縮できるよう適宜調整すればよい。
ステップ105のLAを含む組成物の濃縮処理は、上記した膜による濃縮のほかに、凍結濃縮や減圧蒸発濃縮等の方法を用いてもよい。
このDF処理によって、膜に保持される画分、即ち濃縮液5は、全固形分あたりの複合体1、あるいは複合体2の含量が高くなる。一方、膜の透過液であるLA含有透過液6として、LAを含む組成物が得られる。
ステップ108のDF処理Cの際の試料温度、平均運転圧力、膜面流速、濃縮倍率等の処理条件は、透過液中のLA含量が全固形分あたり50~100重量%程度となるよう適宜調整することができる。
LGを含む組成物の製造方法は、(イ)ホエイタンパク質と、(ロ)炭酸イオン及び/又は炭酸水素イオンと、を混合すると、(イ)ホエイタンパク質―(ロ)炭酸イオン及び/又は炭酸水素イオンの複合体(以下、「LGを含む複合体1」と記載する)が形成されるが、LAはLGを含む複合体1の形成には関与せずLGを含む複合体1を形成しないという知見に基づいて完成されたものである。すなわち、本発明はLGを含む複合体1にはLAが含まれない性質を見出してなされたものである。
図2はLGを含む組成物の製造方法のフローチャートである。LGを含む組成物を製造する方法について図2のフローチャートを用いて説明する。なお、説明には図2の方法例を用いるが、本発明はこの方法例に限定されるものではない。
LGを含む複合体2の調製において、炭酸イオン及び/又は炭酸水素イオンと、反応して沈殿物を生じるような金属イオン源のみが溶解している状態は好ましくないが、これを除いては、各材料はどのような順番で添加してもよく、前記した原材料を用いた水溶液の調製中または調製後に、タンパク質を含んだ沈殿物は観察されない。
溶液の温度調整は不要であるが、微生物の繁殖を考慮して0℃以上15℃以下とすることが好ましい。
この処理によりLAは主に透過液であるLA含有透過液2に移行する。これにより、濃縮液1は、LGを含む複合体1を含む水溶液を調製した場合はLGを含む複合体1、LGを含む複合体2を含む水溶液を調製した場合はLGを含む複合体2が多く含まれるようになり、ここでLGを含む組成物を得ることができる。
この処理の際の試料温度、平均運転圧力、膜面流速、濃縮倍率等の処理条件は、濃縮液中のLG含量が全固形分あたり50~100重量%程度となるよう適宜調整することができる。
この処理によって、膜に保持される画分、即ち濃縮液3は、全固形分あたりのLGを含む複合体1、あるいはLGを含む複合体2の含量が高くなる。一方、膜の透過液であるLA含有透過液5はLAを含む組成物となる。
DF処理の際の試料温度、平均運転圧力、膜面流速、濃縮倍率等の処理条件は、濃縮液中のLG含量が全固形分あたり50~100重量%程度となるよう適宜調整することができる。
これを膜あるいは電気透析等の処理に供することにより、金属を低減したLGを含む組成物を得ることができる。
キレート作用を有する成分としては、クエン酸、アスコルビン酸、エチレンジアミン四酢酸類等が例示できる。
pHを低下させる成分としては、塩酸、硫酸、硝酸類等を例示できる。
材料
ホエイタンパク質材料はホエイ粉1とホエイ粉2を用いた。ホエイ粉1は、チーズホエイをイオン交換と限外濾過処理に供し、ホエイタンパク質含量を93.9重量%としたものである。ホエイ粉2は、酸ホエイをイオン交換と限外濾過処理に供し、ホエイタンパク質含量を93.5重量%としたものである。
炭酸イオン及び/又は炭酸水素イオンの材料は炭酸水素ナトリウムを用いた。
軽金属イオンの材料は塩化マグネシウム6水和物、塩化カリウム、遷移金属イオンの材料は塩化マンガン4水和物、硫酸第一鉄7水和物、塩化第二鉄6水和物、硫酸第二鉄、硫酸銅5水和物を用いた。
比較例の調製には水酸化ナトリウムを用いた。実施例及び比較例に使用した水は全てイオン交換水を用いた。
調製した水溶液は遠心機を用いて、4000G、15分の条件で全量濾過方式により濾過した。濾過膜は材質がポリエーテルスルホンで分画分子量が50,000、100,000、及び300,000のものと、材質が再生セルロースで分画分子量が50,000のものを用いた。
濾過膜を透過した溶液を回収し、透過液に含まれるLAとLGの量を測定した。LAとLGの量は高速液体クロマトグラフィーを用いて測定した。測定条件はBordinらの方法(Journal of Chromatography A.(2001)928、1、63―76)に従った。
なお、濾過前の実施例1から実施例20と比較例1から比較例5の水溶液を、10℃で24時間静置してから、上記と同様に濾過して得られる透過液のLA/LG比は、表1に示す値の98~103%であり、ほぼ同様であった。また、24時間静置後の各溶液に沈殿物は観察されなかった。
材料
ホエイタンパク質材料としてホエイ粉1とホエイ粉2を用いた。
実施例21には炭酸イオン及び/又は炭酸水素イオンの材料として炭酸水素ナトリウムを用い、水はイオン交換水を用いた。
実施例22と実施例23には炭酸イオン及び/又は炭酸水素イオンの材料として二酸化炭素を飽和させたイオン交換水(炭酸水)を用いた。
調製した水溶液は遠心機を用いて4000G、15分の条件で全量濾過方式により濾過した。濾過膜は材質がポリエーテルスルホンで分画分子量が50,000のものを用いた。
濾過膜を透過した溶液を回収し、ここに含まれるLAとLGの量を高速液体クロマトグラフィーで測定した。測定条件はBordinらの方法(Journal of Chromatography A.(2001)928、1、63―76)に従った。
また、表2に示した実施例21から実施例23は全ての条件において、処理前及び表1に示した比較例よりもLA/LG比が高く、効率よく分画されていた。
ホエイタンパク質材料としてホエイ粉3を用いた。ホエイ粉3はチーズホエイをイオン交換と限外濾過処理に供し、ホエイタンパク質含量を95.1%としたものである。
炭酸イオン及び/又は炭酸水素イオンの材料は、炭酸水素ナトリウムを用いた。軽金属/遷移金属イオンの材料は、硫酸銅5水和物を用いた。水はイオン交換水を用いた。pHの調整には、和光純薬工業(株)の1mol/L塩酸水溶液を用いた。水溶液のpHは常法に従い測定した。
調製した水溶液は遠心機を用いて4000G、15分の条件で全量濾過方式により濾過した。濾過膜は材質が再生セルロースで分画分子量が50,000のものを用いた。
材料
ホエイタンパク質材料はホエイ粉1を用いた。炭酸イオン及び/又は炭酸水素イオンの材料は炭酸水素ナトリウムを用いた。軽金属及び/又は遷移金属イオンの材料は硫酸第一鉄7水和物を用いた。水はイオン交換水を用いた。
LAとLGの量は高速液体クロマトグラフィーを用いて測定した。測定条件はBordinらの方法(Journal of Chromatography A.(2001)928、1、63―76)に従った。
この水溶液を、濾過膜(ポリアクリロニトリル素材、分画分子量50,000、膜面積0.20平方メートル)を用いてクロスフロー方式で濾過処理を行なった。濾過処理の条件は温度10℃、平均運転圧力0.2MPaであった。この処理により濃縮液10kgと透過液20kg(LAを含む組成物)を得た。
乾燥粉末状のLAを含む組成物のLA/LG比は6.8であり、処理前のホエイ粉1の0.3から著しく高かった。LAの回収率は85%であった。
材料
タンパク質源として生乳(全固形分14%、脂肪分4.1%)を用いた。
炭酸イオン及び/又は炭酸水素イオンの材料は炭酸水素ナトリウムを用いた。軽金属イオン及び/又は遷移金属の材料は塩化第二鉄6水和物を用いた。水はイオン交換水を用いた。
LAとLGの量は高速液体クロマトグラフィーを用いて測定した。測定条件はBordinらの方法(Journal of Chromatography A.(2001)928、1、63―76)に従った。
材料
ホエイタンパク質材料としてホエイ粉2を用いた。
炭酸イオン及び/又は炭酸水素イオンの材料は炭酸水素ナトリウムを用いた。軽金属/遷移金属イオンの材料は硫酸銅5水和物を用いた。水はイオン交換水を用いた。
LAとLGの量は高速液体クロマトグラフィーを用いて測定した。測定条件はBordinらの方法(Journal of Chromatography A.(2001)928、1、63―76)に従った。
この水溶液を、濾過膜(ポリエーテルスルホン素材、分画分子量50,000、膜面積0.050平方メートル)を用いてクロスフロー方式で濾過処理を行なった。濾過処理の条件は温度10℃、平均運転2.5barであった。この処理により濃縮液90gと透過液200g(LAを含む組成物)を得た。
材料
ホエイタンパク質材料はホエイ粉2を用いた。炭酸イオン及び/又は炭酸水素イオンの材料は炭酸水素ナトリウムを用いた。軽金属イオンの材料は塩化マグネシウム6水和物、遷移金属イオンの材料は硫酸第二鉄を用いた。水はイオン交換水を用いた。上記材料を用いて表6に示す実施例30から実施例36に係る水溶液を調製した。水溶液の調製中あるいは調製後に沈殿物は観察されなかった。
濾過膜を透過した溶液を回収し、透過液に含まれるLAとLGの量を測定した。LAとLGの量は高速液体クロマトグラフィーを用いて測定した。測定条件はBordinらの方法(Journal of Chromatography A.(2001)928、1、63―76)に従った。
また、表6に示した実施例30から実施例36は全ての条件において、処理前及び表1に示した比較例よりもLA/LG比が高く、効率よく分画されていた。LAの回収率は33%から45%であった。
材料
ホエイタンパク質材料はホエイ粉1とホエイ粉2を用いた。ホエイ粉1は、チーズホエイをイオン交換と限外濾過処理に供し、ホエイタンパク質含量を93.9重量%としたものである。ホエイ粉2は、酸ホエイをイオン交換と限外濾過処理に供し、ホエイタンパク質含量を93.5重量%としたものである。
炭酸イオン及び/又は炭酸水素イオンの材料は炭酸水素ナトリウムを用いた。
軽金属イオンの材料は塩化マグネシウム6水和物、塩化カリウム、遷移金属イオンの材料は塩化マンガン4水和物、硫酸第一鉄7水和物、塩化第二鉄6水和物、硫酸第二鉄、硫酸銅5水和物を用いた。
比較例の調製には水酸化ナトリウムを用いた。実施例及び比較例に使用した水は全てイオン交換水を用いた。
調製した水溶液は遠心機を用いて4000G、15分の条件で全量濾過方式により濾過した。濾過膜は材質がポリエーテルスルホンで分画分子量が50,000、100,000、及び300,000のものと、材質が再生セルロースで分画分子量が50,000のものを用いた。
濾過膜に保持された濃縮液として実施例37から実施例56と比較例6から比較例10を取得し、濃縮液に含まれるLGとLAの量を測定した。LGとLAの量は高速液体クロマトグラフィーを用いて測定した。測定条件はBordinらの方法(Journal of Chromatography A.(2001)928、1、63―76)に従った。
なお、濾過前の実施例37から実施例56と比較例6から比較例10の水溶液を、10℃で24時間静置してから、上記と同様に濾過して得られる透過液のLG/LA比は、表7に示す値の97~102%であり、ほぼ同様であった(データは示していない)。また、24時間静置後の各溶液に沈殿物は観察されなかった。
材料
ホエイタンパク質材料としてホエイ粉1を用いた。
炭酸イオン及び/又は炭酸水素イオンの材料として炭酸水素ナトリウムを用いた。軽金属/遷移金属イオンの材料として硫酸銅5水和物を用いた。水はイオン交換水を用いた。
LGとLAの量は高速液体クロマトグラフィーを用いて測定した。測定条件はBordinらの方法(Journal of Chromatography A.(2001)928、1、63―76)に従った。
この水溶液を、濾過膜(ポリアクリロニトリル素材、分画分子量50,000、膜面積0.20平方メートル)を用いてクロスフロー方式で濾過処理を行なった。濾過処理の条件は温度10℃、平均運転圧力0.2MPaであった。この処理により濃縮液10kg(LGを含む組成物)と透過液20kgを得た。
材料
ホエイタンパク質材料としてホエイ粉1とホエイ粉2を用いた。
実施例58の調製には炭酸イオン及び/又は炭酸水素イオンの材料として炭酸水素ナトリウムを用い、水はイオン交換水を用いた。
実施例59と実施例60の調製には炭酸イオン及び/又は炭酸水素イオンの材料として二酸化炭素を飽和させたイオン交換水(炭酸水)を用いた。
調製した水溶液は遠心機を用いて4000G、15分の条件で全量濾過方式により濾過した。濾過膜は材質がポリエーテルスルホンで分画分子量が50,000のものを用いた。
濃縮液を回収し、ここに含まれるLGとLAの量を測定した。LGとLAの量は高速液体クロマトグラフィーを用いて測定した。測定条件はBordinらの方法(Journal of Chromatography A.(2001)928、1、63―76)に従った。
また、表9に示した実施例58から実施例60は全ての条件において、処理前及び表7に示した比較例6から比較例10よりもLG/LA比が高く、効率よくLGとLAが分画されていた。
実施例60を凍結乾燥により粉末化した(実施例61)。10℃で1ヶ月間保存した実施例61の水分含量は5.1重量%であった。これをイオン交換水に溶解させ、1重量%の水溶液を調製した。この水溶液に塩化第二鉄6水和物を0.01重量%となるように添加し、攪拌後、95℃達温の条件で加熱殺菌した。加熱殺菌後、直ちに10℃に冷却し、冷却直後と10℃で3日間保存した後の水溶液の状態を目視で観察した。
その結果、冷却直後と10℃保存3日間後のいずれにおいても、沈殿物や白濁は認められず、粉末化したLGを含む組成物は、金属可溶化能を有していた。
タンパク質源として生乳(全固形分14%、脂肪分4.1%)を用いた。
炭酸イオン及び/又は炭酸水素イオンの材料は炭酸水素ナトリウムを用いた。軽金属イオン及び/又は遷移金属の材料は塩化第二鉄6水和物を用いた。水はイオン交換水を用いた。
LGとLAの量は高速液体クロマトグラフィーを用いて測定した。測定条件はBordinらの方法(Journal of Chromatography A.(2001)928、1、63―76)に従った。
この濃縮液をさらにDF処理に供した。DF処理は透過液量18kgが得られた時点で終了した。
材料
ホエイタンパク質材料はホエイ粉2を用いた。炭酸イオン及び/又は炭酸水素イオンの材料は炭酸水素ナトリウムを用いた。軽金属イオンの材料は塩化マグネシウム6水和物、遷移金属イオンの材料は硫酸第二鉄を用いた。水はイオン交換水を用いた。上記材料を用いて表10に示す実施例63から実施例69に係る水溶液を調製した。水溶液の調製中あるいは調製後に沈殿物は観察されなかった。
濾過膜に保持された溶液を回収し、濃縮液に含まれるLGとLAの量を測定した。LGとLAの量は高速液体クロマトグラフィーを用いて測定した。測定条件はBordinらの方法(Journal of Chromatography A.(2001)928、1、63―76)に従った。
Claims (20)
- 第一のホエイタンパク質及び第二のホエイタンパク質を含む溶液を調製する工程と、
前記第一のホエイタンパク質及び前記第二のホエイタンパク質を膜により分離する工程と、を備え、
前記第一のホエイタンパク質は、炭酸イオン、炭酸水素イオン、軽金属イオン及び遷移金属イオンの少なくとも1つと複合体を形成するものであり、
前記第二のホエイタンパク質は、下記(1)、(2)
(1)炭酸イオン及び炭酸水素イオンのいずれとも複合体を形成しない、
(2)軽金属イオン及び遷移金属イオンのいずれとも複合体を形成しない、
の少なくとも1つを満たすことを特徴とするホエイタンパク質の分画方法。 - 前記第一のホエイタンパク質が主としてβ‐ラクトグロブリンを含むホエイタンパク質であり、前記第二のホエイタンパク質が主としてα‐ラクトアルブミンを含むホエイタンパク質であることを特徴とする請求項1に記載のホエイタンパク質の分画方法。
- 前記膜の分画分子量が50,000以下であることを特徴とする請求項1又は請求項2に記載のホエイタンパク質の分画方法。
- 前記膜の分画分子量が300,000以下であることを特徴とする請求項1又は請求項2に記載のホエイタンパク質の分画方法。
- 前記溶液は、前記第一のホエイタンパク質及び前記第二のホエイタンパク質の全濃度が0.001重量%以上35重量%以下、前記炭酸イオン及び/又は前記炭酸水素イオンの全濃度が0.001重量%以上35重量%以下、pHが5以上10以下であることを特徴とする請求項1から請求項4に記載のホエイタンパク質の分画方法。
- 前記軽金属イオン及び/又は前記遷移金属イオンの全濃度が0.00001重量%以上10重量%以下であることを特徴とする請求項1から請求項5に記載のホエイタンパク質の分画方法。
- 濃縮工程、ダイアフィルトレーション工程、殺菌工程、凍結工程、乾燥工程、粉末化工程から選択される1つ、又は複数の工程を含むことを特徴とする請求項1から請求項6のいずれか1項に記載のホエイタンパク質の分画方法。
- ホエイタンパク質と、炭酸イオン及び/又は炭酸水素イオンと、を含む溶液を調製する工程と、
分画分子量が50,000以下の膜を用いて前記溶液を処理する工程と、
前記膜の透過液としてα‐ラクトアルブミンを含む溶液と、前記膜の濃縮液としてβ‐ラクトグロブリン、炭酸イオン及び/又は炭酸水素イオンで形成された複合体を含む溶液と、を得る工程と、
前記α‐ラクトアルブミンを含む溶液を分画分子量が14,000以下の膜で処理する工程と、を含むことを特徴とするα‐ラクトアルブミンを含む組成物の調製方法。 - 前記溶液の前記ホエイタンパク質の濃度が0.001重量%以上35重量%以下、
前記溶液の炭酸イオン及び/又は炭酸水素イオンの全濃度が0.001重量%以上35重量%以下、
前記溶液のpHが5以上10以下であることを特徴とする請求項8に記載のα‐ラクトアルブミンを含む組成物の調製方法。 - ホエイタンパク質と、炭酸イオン及び/又は炭酸水素イオンと、軽金属イオン及び/又は遷移金属イオンと、を含む溶液を調製する工程と、
分画分子量が300,000以下の膜を用いて前記溶液を処理する工程と、
前記膜の透過液としてα‐ラクトアルブミンを含む溶液と、前記膜の濃縮液として前記β‐ラクトグロブリン、炭酸イオン及び/又は炭酸水素イオン、軽金属イオン及び/又は遷移金属イオンで形成された複合体を含む溶液と、を得る工程と、
前記α‐ラクトアルブミンを含む溶液を分画分子量が14,000以下の膜で処理する工程と、を含むことを特徴とするα‐ラクトアルブミンを含む組成物の調製方法。 - 前記溶液のホエイタンパク質の濃度が0.001重量%以上35重量%以下、
前記溶液の炭酸イオン及び/又は炭酸水素イオンの全濃度が0.001重量%以上35重量%以下、
前記溶液のpHが5以上10以下、
前記溶液の軽金属イオン及び/又は遷移金属イオンの全濃度が0.00001重量%以上10重量%以下、であることを特徴とする請求項10に記載のα‐ラクトアルブミンを含む組成物の調製方法。 - 濃縮工程、ダイアフィルトレーション工程、殺菌工程、凍結工程、乾燥工程、粉末化工程から選択される1つ、又は複数の工程を含むことを特徴とする請求項8から請求項11のいずれか1項に記載のα‐ラクトアルブミンを含む組成物の調製方法。
- 請求項8から請求項12に記載の全ての工程の処理温度を62℃未満することを特徴とする組成物に含まれるα‐ラクトアルブミンの70%以上が未変性α‐ラクトアルブミンである前記組成物の調製方法。
- ホエイタンパク質と、炭酸イオン及び/又は炭酸水素イオンと、を含む溶液を調製する工程と、
分画分子量が50,000以下の膜を用いて前記溶液を処理する工程と、
前記膜の透過液としてα‐ラクトアルブミンを含む溶液と、前記膜の濃縮液として前記β‐ラクトグロブリン、炭酸イオン及び/又は炭酸水素イオンで形成された複合体を含む溶液と、を得る工程と、を含むことを特徴とするβ‐ラクトグロブリンを含む組成物の調製方法。 - 前記溶液の前記ホエイタンパク質の濃度が0.001重量%以上35重量%以下、
前記溶液の炭酸イオン及び/又は炭酸水素イオンの全濃度が0.001重量%以上35重量%以下、
前記溶液のpHが5以上10以下であることを特徴とする請求項14に記載のβ‐ラクトグロブリンを含む組成物の調製方法。 - ホエイタンパク質と、炭酸イオン及び/又は炭酸水素イオンと、軽金属イオン及び/又は遷移金属イオンと、を含む溶液を調製する工程と、
分画分子量が300,000以下の膜を用いて前記溶液を処理する工程と、
前記膜の透過液としてα‐ラクトアルブミンを含む溶液と、前記膜の濃縮液として、前記β‐ラクトグロブリン、炭酸イオン及び/又は炭酸水素イオン、軽金属イオン及び/又は遷移金属イオンで形成された複合体を含む溶液と、を得る工程と、を含むことを特徴とするβ‐ラクトグロブリンを含む組成物の調製方法。 - 前記溶液の前記ホエイタンパク質の濃度が0.001重量%以上35重量%以下、
前記溶液の炭酸イオン及び/又は炭酸水素イオンの全濃度が0.001重量%以上35重量%以下、
前記溶液のpHが5以上10以下、
前記溶液の軽金属イオン及び/又は遷移金属イオンの全濃度が0.00001重量%以上10重量%以下、であることを特徴とする請求項16に記載のβ‐ラクトグロブリンを含む組成物の調製方法。 - 濃縮工程、ダイアフィルトレーション工程、殺菌工程、凍結工程、乾燥工程、粉末化工程から選択される1つ、又は複数の工程を含むことを特徴とする請求項14から請求項17のいずれか1項に記載のβ‐ラクトグロブリンを含む組成物の調製方法。
- 前記軽金属イオン及び/又は前記遷移金属イオンを低減させる工程を含むことを特徴とする請求項16から請求項18に記載のβ‐ラクトグロブリンを含む組成物の調製方法。
- 請求項14から請求項19に記載の全ての工程の処理温度を72℃未満することを特徴とする組成物に含まれるβ‐ラクトグロブリンの70%以上が未変性β‐ラクトグロブリンである前記組成物の調製方法。
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US20190124945A1 (en) | 2019-05-02 |
EP3427594A1 (en) | 2019-01-16 |
NZ740624A (en) | 2020-07-31 |
EP3427594A4 (en) | 2019-09-18 |
AU2017231471A1 (en) | 2018-04-05 |
JP6932687B2 (ja) | 2021-09-08 |
AU2017231471B2 (en) | 2018-07-26 |
JPWO2017154867A1 (ja) | 2018-12-27 |
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