WO2006080424A1

WO2006080424A1 - Low allergen milk obtained using electrical energy

Info

Publication number: WO2006080424A1
Application number: PCT/JP2006/301291
Authority: WO
Inventors: Tomoaki Matsumoto; Kowashi Yosioka; Isao Inoue
Original assignee: Kumamoto University; Institute Of National Colleges Of Technology, Japan
Priority date: 2005-01-28
Filing date: 2006-01-27
Publication date: 2006-08-03
Also published as: JP4769949B2; JPWO2006080424A1

Abstract

It is intended to produce a dairy product for treatment of milk allergy or a dairy product for prevention of onset of milk allergy by reducing the allergenicity of whey components, particularly β-lactoglobulin protein that has the highest allergenicity among them, without the loss of safety and an organoleptic aspect as food. A whey raw material or β-lactoglobulin protein whose allergenicity is reduced, which can be obtained by putting electrical energy into the whey raw material or β-lactoglobulin protein, is provided.

Description

Low allergen milk using electric energy

[0001] The present invention relates to a whey raw material with reduced allergenicity, β-ratatoglobulin protein and dairy product, which can be used for the treatment of immune diseases caused by intake of milk components, in particular, allergy to milk and prevention of the onset thereof. And a manufacturing method thereof. More specifically, the present invention provides whey by electrical energy without using foreign substances such as proteolytic enzymes.

In particular, the allergenicity of j8-lactoglobulin protein with the highest allergenic activity (Haru Adachi, Shuichi Uenogawa. Specific IgE antigen recognition in milk allergy. Clinical allergy (1997), 3 卷. 144-151) The present invention relates to a whey raw material, β-lactoglobulin protein, and a dairy product obtained by reducing the amount of milk. The present invention relates to a medical dairy product that is safe and retains its flavor, and a method for producing the same.

Background art

[0002] As a method of inactivating the allergenicity of milk components, the peptide bond of milk protein is cleaved using porcine spleen or microorganism-derived proteolytic enzyme, and allergic antibody (IgE antibody) cannot be bound. , OOODa) It is widely used to reduce the molecular weight below (Caff arelli et al., Et al., Determination of aliergenicity to three cow s milk hydroiysates and an amino acid-derived formula in children with cow's milk allergy. Exp. Immuno 1., (2002), vol. 32, p74-79; Rosendal A. and Barkholt V., Detection of potentially all ergenic material in 12 hydorolyzed milk formulas. J. Dairy Sci., (2000), vol 83, p22 00—2210; and Oldaus G. et al., Cow's milk IgE and IgG antibody responses to cow's milk formulas. Allergy, (1999), vol., 54, p352-357). Dairy products produced by this technology are commercially available as hydrolyzed milk for treating milk allergy patients or preventing the development of milk allergies. In experiments using Inu, cleaved disulfide (S-S) bond of j8-lactoglobulin protein, which has the highest allergen activity among whey components, was cleaved using thioredoxin, a redox-regulated small molecule protein. There is a report that the allergenic attenuation of vagina was obtained del Val G. et al., Thioredoxin treatment increases digestibility and lowers allergenicity of milk.J. Allergy Clin. Immunol., (1999), vol. 103, p690-697) _o

[0003] Dairy products produced by these methods have several problems from the viewpoint of health when considering long-term drinking. The first is that these substances used in the hypoallergenic reaction remain in the dairy product. Since these hydrolyzed milks are taken by infants over a long period of time, there is concern that porcine spleen or microbial proteolytic enzymes may become new allergens for infants with an allergic predisposition. In addition, these substances retain their biological activity even after they have been taken, so that continued ingestion in infants with immature organ development can be endangered (Carrocclo A). et al. Evaluation of pancrea tic lunction development after hydrolyzed protein-based and soy-based formulas in unweaned infants. Scand. J. Gastroenterol. (1997), vol. 32, p273-277). In addition, these methods obtain low allergens by changing the primary structure of milk protein, and the inherent flavor of milk as a food is lost.

[0004] Further, JP-A-2003-18956 discloses that the deterioration of the quality of milk components can be prevented over a long period of time by subjecting a liquid containing milk components and / or a preparation to electrolysis or energization treatment. It is described that it is suitable for the production of canned milk coffee and milk tea beverages sold by vending machines and can warmers. In Patent Document 1, a liquid containing a milk component is allowed to flow continuously between two electrodes, or in a stationary state, a voltage is applied between the electrodes, and a current of 0.1 A to 50 A flows. It is described. However, Patent Document 1 teaches and suggests that allergenicity can be reduced by injecting electric energy into whey raw materials or j8-ratoglobulin protein. .

Disclosure of the invention

Problems to be solved by the invention

[0005] The present invention has been made for the purpose of responding to the needs of the medical community seeking a new low allergen koji manufacturing method because the conventional milk component low allergen koji manufacturing method has the above-mentioned problems. . That is, the present invention is intended to reduce the whey ingredients, especially the allergenicity of j8-latatobrovulin protein, which has the highest allergen activity, in terms of food safety and sensory loss. The problem to be solved was to produce milk products for the treatment of milk allergies and milk products for preventing the onset of milk allergies.

Means for solving the problem

[0006] In order to achieve the above object, the present inventors have studied from various directions, and as a result, the physical components of the milk component are not determined by adding a foreign substance such as an enzyme to the milk component and performing a chemical reaction. Extensive research was conducted to obtain allergenicity reduction.

[0007] First, the present inventors focused on the fact that j8-latatoglobulin, which has the highest allergen activity among milk proteins, is a typical globular protein, and its higher-order structure is recognized as an allergen. Generally, protein conformation can be changed by physical and chemical methods, but j8-lactoglobulin is heat-stable and is allergenic even at 1 hour at 90 ° C. Britt- Mane E. et al., Modification of IgE Dinding during heat proces sing of the cow's milk allergen beta- lactoglobulin. J Agric food Chem., (2004), vol. 52, pl398-1403) o also because it is a food In addition, chemical treatment using an acid-alkali solution cannot be performed. Therefore, the inventors have confirmed that when a certain amount of electric energy is injected into whey or isolated | 8-lactoglobulin protein, their allergen activity is remarkably attenuated. The present invention has been completed based on these findings.

[0008] That is, according to the present invention, there is provided a whey raw material or j8-lactoglobulin protein with reduced allergenicity obtained by injecting electric energy into the whey raw material or j8-lactoglobulin protein. The

[0009] Preferably, the whey raw material or j8-lactoglobulin protein of the present invention is used for treating a milk allergy patient or preventing the onset of a milk allergy.

[0010] Preferably, the whey raw material or β-ratatoglobulin protein of the present invention injects electric energy by energizing a solution containing the whey raw material or β-lactoglobulin protein, and recovers the cathode-side solution. It is obtained by this.

[0011] According to another aspect of the present invention, there is provided a dairy product for treating milk allergy patients and a dairy product for preventing allergy onset prepared by using the whey raw material or β-lactoglobulin protein of the present invention described above. Provided.

[0012] According to still another aspect of the present invention, the whey raw material or 13 lactoglobulin protein is electrically There is provided a method for producing whey raw materials or j8-ratoglobulin protein with reduced allergenicity, including injection of energy.

[0013] According to still another aspect of the present invention, the allergenicity includes: injecting electrical energy by energizing a solution containing a whey raw material or β-lactoglobulin protein and recovering the cathode side solution. A method for producing reduced whey ingredients or) 8-lactoglobulin protein is provided.

BEST MODE FOR CARRYING OUT THE INVENTION

[0014] Milk is a basic nutrient source for infants. However, its allergenic activity is the second most common food cause of food allergies in Japan and is observed in 2% of infants (Yoji Iikura, the epidemiology of immediate food allergies. Academic Journal (2002), 16 pp. 139-143). In these infants, dairy products cause atopic dermatitis, dyspnea, diarrhea, and vomiting, and rarely disturbed consciousness by ingesting dairy products (Tomoaki Matsumoto) An easy-to-understand allergic disease in small children, Kinyoshido Publishing, 2003, 1 151). Nutrition disorders occur in infants due to the inability to consume dairy products over time.

[0015] Infants who have severe gastrointestinal symptoms after ingestion of dairy products that are not the cause of milk allergies have also been reported, and this is called food protein-induced enterocolitis caused by dairy products. (Akihiko Kawase, Yuichi Kondo, Tomoaki Matsumoto. A case of low birth weight infant considered to be food protein-induced ente rocolitis syndrome. Journal of the Japan Pediatric Society (2004), 108 卷, 635-638). It also causes eosinophilic gastroenteritis (Matsumoto T. et al. Markedly high eosinophilia and an elevated serum IL— 5 level in an infant wit h cow milk allergy. Ann. Allergy Asthma Immunol., (1999 ), vol. 82, p253-256).

[0016] Hydrolyzed milk has been conventionally provided as milk for patients with milk allergies, but has been associated with health problems described below. Therefore, as a result of diligent efforts based on a completely new idea of using electric energy, the applicant was able to produce a therapeutic milk that solved these problems.

[0017] In the present invention, whey raw material or j8-lactoglobulin protein with reduced allergenicity can be produced by injecting electric energy into whey raw material or β-lactoglobulin protein. Build.

[0018] The whey raw material referred to in the present invention means any raw material containing a milk component, and may be, for example, raw milk (cow milk), processed milk, skim milk powder, whole milk powder, skim milk, concentrated milk, and the like. Further, a liquid containing these, a liquid obtained by restoring powder with water or hot water, a concentrated liquid or a diluted liquid of these liquids may be used. The whey raw material preferably contains j8-latatoglobulin protein.

In the present invention, electric energy is injected into whey raw material or β-lactoglobulin protein. Examples of the method for injecting electric energy include energization processing (or electrolysis processing). In the present invention, the energization process can be performed as follows.

[0020] The apparatus used for conducting the energization treatment is not particularly limited as long as it is a normal electrolysis apparatus used for electrolyzing a normal aqueous solution or the like. It is also possible to use an electrolysis method using an ion exchange membrane or a neutral membrane. For example, prepare two glass tubes of appropriate size, connect the central part with a glass tube into a bowl shape, and place it on the base. The bottom of the two left and right tube ports are each plugged with rubber fitted with a platinum electrode plate and a liquid sampling glass cock. This device is filled with whey raw material or j8-lactoglobulin protein solution and can be energized between circuits in which two platinum electrodes are connected in series.

[0021] In addition to the platinum electrode, for example, a ferrite electrode, a platinum-plated titanium electrode, or the like can be used as an anode, and a stainless electrode, a platinum-plated titanium electrode, or the like can be used as a cathode. Can also be used.

[0022] In the energization, a whey material or a liquid containing β-lactoglobulin protein is retained between the two electrodes, or a direct current is energized in a state in which the whey is continuously flowing between the two electrodes. Can be done.

[0023] The amount of current is preferably 0.1 A or more, more preferably 0.1 to 20 mm, and particularly preferably 0.5 to 5 mm. The amount of current can be appropriately set according to the electrical conductivity, the distance between the electrodes, the temperature, and the like.

[0024] The energization time is a force that can be performed in any time from several seconds to several hours, preferably 1 minute to 3 hours, more preferably 5 minutes to 1 hour.

[0025] The total electric energy can be set by adjusting the amount of current and voltage and the energization time. The total electrical energy injected by the method of the present invention is 10 kilojoules or more. 20 kilojoules or more is preferable, for example 10 to 500 kilojoules, more preferably 20 to 300 kilojoules, and even more preferably 30 to 200 kilojoules.

In the present invention, after the energization treatment, the cathode side liquid may be collected and used, or the entire treatment tank may be collected and used, but preferably the cathode side liquid is used. Can be collected and used.

In the present invention, a dairy product can be produced using a whey raw material or β-lactoglobulin protein with reduced allergenicity. The dairy product as referred to in the present invention means a product made from milk or a part thereof, which is covered with the raw material, such as cream, butter, butter oil, cheese, ice cream, concentrated milk, defatted concentrated milk, no milk. Sugared milk, sugar-free skim milk, sweetened milk, sweetened skim milk, whole milk powder, skim milk powder, sweetened powdered milk, prepared milk powder, fermented milk, lactic acid bacteria beverage, and milk beverage (in addition to raw milk and reduced milk, coffee The ability to include beverages in which an extract or fruit juice is added to raw materials) is not limited to these. The dairy product of the present invention has reduced allergenicity and can be used for treatment of patients with milk allergies and prevention of the development of milk allergies.

The ability to explain the present invention more specifically by the following examples The present invention is not limited by the examples.

Example

[0028] Example 1

As one embodiment of the present invention, electric energy is injected into the milk component, and a raw milk (milk), processed milk, or skim milk powder solution is used as the milk component-containing liquid. Add acetic acid to this, add 1/5 volume of ether to the whey obtained after precipitating casein protein, shake twice in a separating funnel and degrease twice. Place the resulting liquid in a dialysis membrane and dialyze twice using dialysate. The resulting liquid (defatted whey) and the isolated and purified ι8-lactoglobulin protein (derived from milk, sold by SIGMA, USA, purity 80%) were each dissolved in 1% by weight. Add% sodium chloride and inject electric energy.

[0029] Electric energy injection into skim whey and β-lactoglobulin protein is as follows: I went there. Prepare two glass tubes with a length of 300 mm and a diameter of 30 mm, and connect the central part with a glass tube with a length of 40 mm and a diameter of 25 mm to form an H-shape, which is installed on the base. The bottom of the two left and right tube ports are each plugged with rubber fitted with a platinum electrode plate and a liquid sampling glass cock. This apparatus is filled with the above-described skim whey solution and 250 ml of 1% J8-lactoglobulin protein solution, and energized between circuits in which two platinum electrodes are connected in series.

[0030] When a non-fat whey was energized at a voltage of about 83.5 V, the optimum current value was 1. OA, and the electrical resistance of the non-fat whey was about 84 ohms. Under this condition, power was applied for up to 30 minutes and total electric energy was injected to about 150 kilojoules. The electrical resistance of the 1 ° / ₀ J8-lactoglobulin protein lysate was about 140 ohms, and the optimum current value at a voltage of about 83.5 V was 0.6 A. Under this condition, power was applied for up to 30 minutes, and the total electrical energy was injected to approximately 90 kilojoules. Each glass tube was wrapped with a paper towel, and cold water was constantly immersed to keep the solution at 55 ° C or lower. The sample was collected by twisting the glass cock for collecting liquid at the bottom of the tube mouth. Figure 1 shows an overall photograph of the device energizing the j8-lactoglobulin protein (A) and an enlarged photograph of the platinum electrode plate below the tube port and the glass cock for collecting liquid (B).

[0031] Example 2

Electric energy was injected into the skim milk as described above, and the liquid collected on the anode side and the cathode side was placed in a dialysis membrane and dialyzed twice using the dialysate. A sensory test was conducted by relying on five health care workers. As a result, it was found that the unique flavor of milk was not impaired.

[0032] Example 3

Changes in pH, acid-reduction potential (ORP), and protein concentration by injecting electrical energy into skim whey and j8-lactoglobulin solution were measured. Protein concentration was measured by the Lowry method (Fryer H. J. L., Davis G. E. Lowry protein assay using an automatic microtiter plate spectrophotometer. Anal. Biochem. (1986), vol. 153, p262—266.). As a result, the pH change was small in skimmed whey, and the ρΗ value changed significantly in β-lactoglobulin solution. In both solutions, the acid potential increased on the anode side and the reduction potential increased on the cathode side. Although the protein concentration decreased, there was no significant difference between the anode and cathode (Table 1).

[0033] [Table 1] Table 1 Changes in pH, ORP, and protein concentration of skim whey and i3-lactoglobulin (/ 3 LG)

[0034] Example 4

J8-lactoglobulin protein was added to the sera of milk allergy patients, and the amount of anti-β-lactoglobulin-specific IgE antibody contained in the patient sera was measured (RAST suppression (Yman L. et al., RAST -based allergen assay methods. Dev. Biol. Stand., 1975), vol. 29, pl51-165). To determine the allergenicity of proteins, it is common to examine the skin reaction in actual allergic patients and infer the positive reaction threshold power. However, the RAST suppression test described here is used when a patient cannot perform such a biopsy (biological experiment) for reasons such as being a child. This is because, if allergen protein is added to the serum prior to measuring the amount of allergic antibody (IgE antibody) in the patient's serum, the IgE antibody in the serum is bound and consumed. The principle is that the number of antibodies decreases, that is, the measured value decreases. On the contrary, the amount of allergen protein can be estimated from the decrease of IgE antibody in serum.

The milk allergy patient serum used here was selected from milk allergy patients who visited the Department of Developmental Pediatrics, Kumamoto University Hospital. The purpose of this study was well explained, and the strengths of seven people who had consent from the patients themselves or their parents were collected. When blood was collected in normal outpatient practice, an additional 2 ml was collected. When various j8 lactoglobulin proteins are added to serum mixed with equal amounts of them, the anti-lactoglobulin-specific IgE antibody in the mixed serum is adsorbed and the antibody concentration decreases. It was calculated by applying it to the formula. The anti-j8-ratatoglobulin-specific IgE antibody titer is determined by the fluorescent enzyme antibody method (Falma Measured by Shia).

[0036] Specific IgE antibody titer reduction rate (%) = (1-β IgE antibody titer in serum with added lactoglobulin protein Ig IgE antibody titer in serum without adding anything) X 100

[0037] When the 1% β-ratatoglobulin protein solution was added to the mixed serum, it was diluted 10-fold, 100-fold, and 1,000-fold with physiological saline in advance. The 8 1 was mixed with 0.4 ml of mixed serum and stirred for 1 hour at 37 ° C., and the anti- | 8-exoglobulin specific IgE antibody titer was measured. As a result, the IgE antibody titer decreased most when the 10-fold solution was added (Table 2). For this reason, we decided to use a 10-fold dilution for subsequent experiments to observe IgE antibody adsorption.

[0038] [Table 2]

Table 2 Changes in serum specific IgE antibody titer by addition of lactoglobulin (j3LG)

[0039] Thus, IgE antibodies in serum are apparently reduced by binding to the allergen portion of j8-lactoglobulin. It was decided to evaluate the allergen activity of β-latatoglobulin by measuring the rate of decrease. That is, from the reduction rate obtained by adding j8-lactoglobulin before treatment, j8-ratatoglobulin collected on the anode side and the cathode side by energization, or j8 treated with acid or alkali -Subtracting the rate of decrease obtained when each ratatoglobulin was added, the difference was used as an index of attenuation of β-lactoglobulin protein allergen activity.

[0040] Specific allergenic attenuation rate of specific IgE antibody (%) = [Non-treated j8—IgE reduction rate when lactoglobulin was prepared (%)] [Various treatments were performed / 3 Rate of IgE reduction when lactoglobulin is picked up (%)]

[0041] When electric energy was injected into a β-lactoglobulin solution, changes in pH and ORP value occurred, so whether or not allergenicity of 13 lactoglobulin ¾H or ORP was also affected. As shown in Table 1, the pH of the solution obtained on the anode side is 3.50, ORP is 182 mV, and the pH of the solution obtained on the cathode side is 11.20, ORP is 208 mV It is. Therefore, 1 drop of 1 mol of acetic acid was added to 30 ml of 1% β-ratatoglobulin protein solution to prepare a solution with pH 3.50 (ORP, 132 mV). In addition, 150 1 drops of 1M sodium hydroxide solution was added dropwise to prepare a pHl.20 (ORP, -256mV) solution.

[0042] Acid treatment by adding 0.4 ml of untreated / 3 lactoglobulin protein solution, liquid collected on the anode side for 30 minutes, solution collected on the cathode side, acetic acid to 0.4 ml of mixed serum of milk allergy patients The solution prepared by adding sodium hydroxide solution and alkali-treated solution was diluted 10 times and added 81. After stirring at 37 ° C for 1 hour, anti-j8-ratatogloline specific IgE antibody titer was measured by a fluorescent enzyme antibody method. As a result, a significant reduction in allergen activity was observed in the j8-latatoglobulin protein obtained by injecting electric energy on the cathode side. This reduction in allergenicity was not seen in j8-lactoglobulin protein with calorie sodium hydroxide so that the pH and ORP were at the same level, so allergens found on the cathode side when electric energy was injected. It was found that the property-reducing property was not simply a phenomenon associated with changes in pH and ORP (Table 3).

[0043] [Table 3]

Table 3 Changes in lactoglobulin (J3 L G) allergen activity in terms of adsorption rate to specific IgE antibodies

[0044] Example 5

Non-fat whey and evaluation of allergenic activity of j8-lactoglobulin protein were performed on skin allergic reactions in patients with milk allergy. In conducting this test, Kumamoto University Ethics Committee reviewed the clinical research. From the milk allergy patients who visited the Department of Developmental Pediatrics at Kumamoto University Hospital, the contents of the study were well explained to the patients themselves or the parents of the patients, and a total of 15 people who obtained consent were examined. For the test solution, inject the above skimmed whey and the electrical energy for 30 minutes, then the anode side, negative side The solution obtained on the extreme side, β-ratatoglobulin protein solution, and electric energy are injected into the solution for 10 to 30 minutes. The solution collected on the anode and cathode sides, and physiological saline as a negative reaction reference The solution was a histamine 1,000-fold solution as a positive reaction control.

[0045] 201 drops were applied to the inner skin of each forearm and punctured with a child's brick needle (manufactured by Lincoln, USA), and the maximum diameter of wheal appearing on the surface after 20 minutes was measured. The rate at which the skin allergic reaction was reduced by injecting electric energy into skim whey and β-latatoglobulin solution was determined by the following formula, and this value was used as an index of allergenic attenuation.

[0046] Decrease rate of skin allergic reaction (%) = (1 (wheal diameter after electric energy injection, wheal diameter of control liquid) / (wheal diameter before electric worker energy injection, wheal diameter of negative control liquid)) X 100 (%)

FIG. 2 shows a typical example of an allergic reaction that occurs when skim whey (left) and β-lactoglobulin solution (right) are used on the inner skin of the forearm of a milk allergy patient. V, in either case, the reaction to the cathode-side collected solution drawn on the skin (-) is (before) or (i8 LG) and the solution before applying the treatment drawn on the skin, or the anode written (+). It can be seen that it is significantly weaker than the reaction to the side sample.

[0048] Skin reactions using skimmed whey were measured in a total of 8 patients. When electric energy was injected into skim whey for 30 minutes under the above conditions, the allergen activity was reduced by an average of 48% on the cathode side. On the other hand, on the anode side, the average decrease was only 28% (Table 4).

[0049] [Table 4]

Table 4 Changes in cutaneous wheal response due to electric energy injection into skim milk whey

* Average person 1 standard deviation [0050] Skin allergic reactions to solutions collected by injecting electrical energy into β-lactoglobulin for 10 minutes under the above conditions were evaluated in three patients. In this energization time, there was no reduction in allergen activity on both the cathode and anode sides (Table 5).

[0051] [Table 5]

Table 5 / 3—Electric energy applied to lacto globulin solution 10 Changes in skin wheal response after 10 minutes of application

* Average ± 1 standard deviation

[0052] β-lactoglobulin was injected with electrical energy for 30 minutes, and skin allergic reactions to the collected solutions were evaluated in 15 patients. When the solution collected on the cathode side was used, the diameter of the wheal was suppressed by 71% on average compared to the solution before energization, and the allergen activity was significantly reduced. On the other hand, the mean effusion diameter was suppressed to 10% on the anodic side collection solution (Table 6).

[0053] [Table 6]

Table 6 Changes in cutaneous eruption response after 30 minutes of injection of electrical energy into a single lactopurine solution

Anode side Cathode side

Before control solution (% decrease) (% decrease)

A 4 21 21 (0) 13 (47)

B 2 23 16 (33) 3 (95)

C 4 17 22 (0) 20 (0)

D 4 13 12 (11) 6 (78)

E 3 9 9 (0) 3 (100)

F 2 5 5 (0) 3 (67)

G 3 25 24 (5) 5 (91)

H 1 11 11 (0) 5 (60)

I 2 15 14 (8) 2 (100)

J 2 15 9 (46) 8 (54)

K 4 28 17 (46) 5 (96)

L 2 4 4 (0) 2 (100)

M 5 16 15 (0) 8 (73)

N 4 15 24 (0) 7 (73)

0 3 6 16 (0) 5 (33)

(10 ± 16) * (71 ± 28)

* Average ± 1 standard deviation

[0054] Common milk that is generally marketed! (Milk and listed in Table 7), hydrolyzed milk marketed for allergies (hydrolyzed milk and listed in Table 7), and | 8-lactoglobulin Table 7 shows the results of a skin brick test using a solution collected on the cathode side after injecting electric energy for 30 minutes (collected on the cathode side and listed in Table 7). The magnitude of the skin wheal reaction is given in millimeters. Calculates whether wheal was reduced summer much, illustrated with it and inhibition rate ^_0/0.

[0055] [Table 7]

Table 7

* Maximum diameter of wheal (wake)

* * Mean ± 1 standard deviation

[0056] Example 6

Changes in amino acid composition of lactoglobulin by electric energy injection. β-Lactoglobulin 1. / ₀ solution 0. 5 ml to 12 N hydrochloric acid 0. 5 ml Ka卩E was 20 hours in Kyupeto at 100 ° C. The amount of free amino acid was measured using a high-performance liquid chromatograph (L-8500, Hitachi) dedicated to amino acid analysis after dilution 20-fold with lithium citrate buffer. Milk-derived β-latatoglobulin has a molecular weight of about 18 kDa and is composed of 20 types of 162 amino acids. J8-Lactoglobulin used in this study (SIGMA, USA) has a purity of 80%. Asparagine (N) was changed to aspartic acid (D) and dartamine (Q) was changed to glutamic acid (E) by strong hydrochloric acid treatment, but tributofan (W) was destroyed and was not detected.

[0057] Table 8 shows the measurement results in percentage. With 30 minutes injection of electrical energy, cysteine (C), methionine (M), and tyrosine (Y) were below the detection limit on both the cathode and anode sides. Lysine (K) also decreased, but decreased particularly on the cathode side. On the other hand, aspartic acid (D), threonine (T), glutamic acid (E), proline (P), and leucine (L) are both on the cathode and anode sides. Increased. Except for lysine, there was no significant difference in amino acid composition between the anode and cathode. Therefore, the remarkable allergenic attenuation of j8-lactoglobulin seen on the cathode side did not seem to be related to the change in the amino acid composition of the protein.

[Table 8]

Changes in amino acid composition of lactoglopurin by electric energy injection

* Amino acids with marked decrease

[0059] Example 7

β-latatoglobulin has five cysteine residues, four of which form intramolecular disulfide (S-S) bonds at two locations. The remaining one cysteine residue remains as a thiol group (-SH) or forms an intermolecular disulfide bond, so | 8-latatoglobulin is usually a simple force dimer! /. When the amount of thiol groups was measured by the Ehleman method, it was found to be almost the same on both the cathode and anode sides (Table 9). The significant allergenic attenuation of j8-lactoglobulin seen on the cathode side is due to del Val G. et al., Thioredoxin treatment increases digestibility and lowers allergenicity of milk. J. Allergy Clin. I mmunoL, (1999), vol. 103, p690-697, the mechanism was different from that using thioredoxin.

[0060] [Table 9]

Table 9 Changes in thiol group content of lactoglopurine (; 3 L G) by electric energy injection

[0061] Example 8 Changes in the molecular weight of 13 lactoglobulin caused by electrotechnical energy injection were investigated by polyacrylamide gel electrophoresis. Each j8-lactoglobulin solution was desalted and a non-reducing buffer (8% SDS, 40% glycerol, BPB, 200 mM Tris—HCL pH 6.8) was added to obtain a protein concentration of lmgZml. The 101 was electrophoresed on a 16% polyacrylamide gel for 90 minutes at a constant current of 16 mA. Staining was done with Kumasi Brilliant Blue.

[0062] Only one band with a molecular weight of around 18 kDa was observed in the j8-lactoglobulin solution before treatment, and this band was considered to be a single | 8-lactoglobulin (Figure 3). When electric energy was injected for 10 minutes under the above conditions, the band around 18 kDa became thinner on both the anode and cathode sides, and a new wide shadow was created in the polymer region. However, after 30 minutes, these polymer region shadows disappeared on both the anode and cathode sides. Only a band near 18 kDa was observed again on the anode side, and this band near 18 kDa almost disappeared on the cathode side. From this, it was considered that β-lactoglobulin alone and dimer disappeared on the cathode side when electric energy was injected into β-latatoglobulin for 30 minutes.

[0063] Example 9

When a certain amount of electric energy was injected, the fact that lactoglobulin alone disappeared on the gel at the cathode side was divided, so we investigated whether this protein became polymerized and disappeared. . That is, ultrafiltration was performed at a molecular fraction of 30 kDa to obtain a high molecular weight side solution and a low molecular side solution, and polyatalide amide gel electrophoresis was performed again together with the solution before ultrafiltration. As a result, the high molecular weight side solution and the solution before ultrafiltration were almost the same migration image, and the band formation was not observed at all in the low molecular side solution. Therefore, β-lactoglobulin was polymerized and appeared on the gel. It disappeared because it disappeared (Figure 4).

Industrial applicability

[0064] By injecting a certain amount of electrical energy into whey and / 3 lactoglobulin, its allergen activity is significantly attenuated on the negative side. By this method, milk products for treating milk allergy and milk products for preventing the onset of milk allergies can be produced without using a heterologous protein or chemical substance such as proteolytic enzyme thioredoxin. Dairy products obtained by this method are safe and do not lose their flavor. This dairy product is a source of dairy ingredients. Contribute to medical treatment aimed at the treatment and prevention of immune diseases that occur in humans, especially milk allergies.

[0065] Whey is an industrial waste in the cheese manufacturing process, most of which is swine feed. In this experiment, 50 ml was collected after injecting electric energy into 250 ml of each solution. As shown in Table 1, the concentration of skim whey protein before treatment is 1,460 mgZdl, and the concentration of protein on the cathode side after 30 minutes is 930 mgZdl. Therefore, the recovery rate was (50 + 250) X (930 + 1460) = 12.7%. In addition, the protein concentration of the β-latatoglobulin solution before treatment is 728 mgZdl, and the concentration of protein on the cathode side after 30 minutes energization is 264 mgZdl. Therefore, the recovery rate was (50 + 250) X (264 + 728) = 7.3%. If the recovery rate is this level, it is considered that there is no problem in industrial mass production in terms of cost.

Brief Description of Drawings

[0066] FIG. 1 shows an overall image (A) of the large electrolyzer used in the example, and an enlarged image (B) of the platinum electrode and the liquid sampling cock part.

[Figure 2] Figure 2 shows typical allergic reactions that occurred when using skim whey (left; whey) and j8-lactoglobulin solution (right; j8 LG) on the inner skin of the forearm of a milk allergy patient. An example is shown.

[Fig. 3] Fig. 3 shows the change in the molecular weight of β-lactoglobulin protein by electric energy injection. Μ: Molecular weight marker, 1: j8-lactoglobulin solution before treatment, 2: anode collection solution after 10 minutes of energy injection, 3: cathode collection solution after 10 minutes of injection, 4: 30 minutes after injection Anode-side sampling solution, 5: Cathode-side sampling solution 30 minutes after injection

[FIG. 4] FIG. 4 shows an electrophoresis image after ultrafiltration (30 kDa) of the β-lactoglobulin cathode-side collected solution. M: Molecular weight marker, 1: High molecular weight solution, 2: Low molecular weight solution, 3: Ultrafiltration solution

Claims

The scope of the claims

[1] A whey raw material or j8-lactoglobulin protein with reduced allergenicity, obtained by injecting electric energy into a whey raw material or β-lactoglobulin protein.

[2] Claims used for treatment of milk allergy patients or prevention of milk allergy

The whey raw material or j8-lactoglobulin protein according to 1.

[3] The whey raw material or β-lactoglobulin according to claim 1, obtained by injecting electric energy by energizing a whey raw material or a solution containing β-lactoglobulin protein and collecting the cathode-side solution. Protein.

[4] A milk product for treating milk allergy patients and a milk product for preventing the onset of milk allergy, prepared by using the whey raw material according to any one of claims 1 to 3 or β-lactoglobulin protein.

[5] A method for producing a whey raw material or β-lactoglobulin protein with reduced allergenicity, comprising injecting electric energy into the whey raw material or β-lactoglobulin protein.

[6] Whey raw materials with reduced allergenicity, including injecting electric energy by energizing a whey raw material or a solution containing β-lactoglobulin protein, and collecting the solution on the cathode side A method for producing a lactoglobulin protein.