JP5537578B2 - Method for producing lactoferrin hydrolyzate - Google Patents

Description

  The present invention relates to a method for producing a lactoferrin hydrolyzate with low bitterness and low antigenicity, and a lactoferrin hydrolyzate provided by the production method.

Limited hydrolysis (fragmentation) using a protein hydrolase (protease) or acid has been performed as one of the means for new expression and improvement of functional properties of food proteins. It has been clarified that the peptide thus obtained is superior in absorption rate and amino acid balance after absorption from the viewpoint of digestion and absorption (Non-patent Document 1). However, it has been pointed out that limited hydrolysis (fragmentation) produces bitter and bitter-tasting peptides, resulting in materials with poor palatability and difficulty in palatability. Low degradation products often have a particularly bitter taste and can be an obstacle when ingested. For that reason, it is also true that usage is limited.
On the other hand, it has become clear that allergic patients caused by dietary proteins have rapidly increased, and particularly in infants, for example, allergies caused by whey proteins, particularly β-lactoglobulin, have occurred frequently (Non-patent Document 1). Therefore, there is a demand for reduction of antigenic substances in foods and substantial removal of antigenicity in proteins and peptides.
Thus, unpleasant taste such as bitterness and antigenicity are suppressed, and development of a peptide material useful in food processing is awaited.

  Lactoferrin is a non-heme iron-binding glycoprotein that is present in mammalian milk, saliva, tears, semen, various mucus, etc., and has many iron adsorption, cell proliferation, immunoregulatory, and antibacterial effects. It is a functional protein. Beef lactoferrin can be obtained easily and in large quantities from raw skim milk or cheese whey routinely handled in dairy factories and can be used immediately as a commercial product.

  And the lactoferrin hydrolyzate obtained by hydrolyzing lactoferrin and the lactoferrin peptide which has a specific amino acid sequence in lactoferrin, for example, lactoferricin (registered trademark by this applicant), are antibacterial activity (patent document 1, non-patent document) Many physiological activities such as patent document 2), tyrosinase activity (patent document 2), apoptosis inducer (patent document 3), oral cancer metastasis inhibitor (patent document 4), liver function improving agent (patent document 5), etc. Are known.

  Thus, since lactoferrin hydrolysates and lactoferrin peptides are peptides derived from natural products that do not contain chemical substances and chemically synthesized amino acid derivatives, they are healthy and safe for humans and animals. . Therefore, lactoferrin hydrolysates and lactoferrin peptides are safe and effective medicines, foods, feeds, etc., for example, eye drops, oral preparations, cosmetics, skin preparations, infant foods, clinical foods, functional foods, pets It can be used for a wide variety of products such as products and has extremely great value.

JP-A-9-124504 JP-A-5-320068 Japanese Patent Laid-Open No. 10-45618 JP 10-59864 A International Publication No. 00/06192 Pamphlet

Dairy Science / Food Research, Vol.39, A-283, 1990 Antimicrobial Agents and Chemotherapy, Vol. 41, No. 1, 1997, pp. 54-59

  An object of the present invention is to provide a method for producing a lactoferrin hydrolyzate with less bitterness and low antigenicity (lower allergen), and a lactoferrin hydrolyzate provided by the production method.

  In order to provide a lactoferrin hydrolyzate that can be used in various fields such as pharmaceuticals, foods, and feeds, the present inventors have overcome the problem of bitterness caused by peptides, which has been a problem in the past. As a research. And, as a result of intensive investigations aimed at application to the food field having good flavor with functionality, hydrolyzing lactoferrin using an enzyme composition containing chymosin which is a kind of protein hydrolase As a result, it was found that a lactoferrin hydrolyzate having significantly reduced bitterness and low antigenicity as compared with the prior art was obtained.

  1st invention of this application is a manufacturing method of the lactoferrin hydrolyzate whose antigenicity is 0.1% or less compared with lactoferrin, Comprising: Lactoferrin is an enzyme containing 80 mass% or more chymosin with respect to all the proteins. It is a method including the process of hydrolyzing on condition of pH2-4, reaction temperature 35-55 degreeC, and reaction time 6-24 hours by a composition.

The above-described method has a preferable aspect in which the antigenicity is evaluated as a lactoferrin concentration measured by an immunoassay using an anti-lactoferrin antibody with lactoferrin as a standard substance.
Moreover, the said method makes it the preferable aspect that the quantity of an enzyme composition is 0.001 to 5% of range as a protein quantity with respect to the mass of lactoferrin.

  Since the lactoferrin hydrolyzate produced by the production method of the present invention has little bitterness even if it is contained in the mouth, it does not affect the flavor of the whole food when added to food, and the antigenicity is also kept low. It can be provided as a raw material suitable for the production of functional foods and drinks imparted with various physiological activities of lactoferrin peptides.

  Next, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the following preferred embodiments, and can be freely changed within the scope of the present invention.

In the present invention, lactoferrin used as a starting material is commercially available lactoferrin, mammals (eg, humans, cows, sheep, goats, horses, etc.) colostrum, transitional milk, normal milk, end milk, etc. Lactoferrin separated from non-fat milk, whey, etc. as processed products by conventional methods (for example, ion exchange chromatography, etc.), apolactoferrin obtained by removing iron from them using hydrochloric acid, citric acid, etc. A metal saturated lactoferrin chelated with a metal such as, or a mixture thereof (hereinafter, these may be collectively referred to as LF).

The lactoferrin hydrolyzate according to the present invention (hereinafter sometimes referred to as “LF hydrolyzate”) is obtained by hydrolyzing the lactoferrin with an enzyme composition containing chymosin.
When lactoferrin is hydrolyzed by the enzyme composition, lactoferrin is dissolved in water, purified water, physiological saline, buffer solution, etc. at a concentration of 0.5 to 20% by mass, preferably 2 to 15% by mass. A lactoferrin (LF) solution is prepared, and the pH of the LF solution is adjusted to 2 to 4, preferably 2.5 to 3.5, particularly preferably 3. Thereafter, the enzyme composition containing chymosin is added for hydrolysis.

The enzyme composition used for hydrolysis of lactoferrin may contain 80% by mass or more of chymosin (Chymosin, Enzyme Code: 3.4.23.4) with respect to the total amount of protein contained in the composition. There is no restriction | limiting in particular in components other than, Other proteolytic enzymes may be contained. Examples of such proteolytic enzymes include pepsin or proteolytic enzymes derived from microorganisms such as filamentous fungi such as Aspergillus. Chymosin is isolated and purified from a mixture of digestive enzymes produced in the stomach of mammals. It is possible to use a prepared chymosin preparation or a crude product, and a recombinant chymosin produced by a genetic recombination method may be used.

  In the present invention, the chymosin content contained in the enzyme composition containing chymosin is preferably 80% by mass or more, more preferably 84% by mass or more, more preferably 88% by mass or more, and 92% by mass with respect to the total amount of protein. % Is particularly preferred. Further, the protein contained in the enzyme composition containing chymosin may be composed entirely of chymosin, that is, an enzyme composition containing 100% by mass of chymosin. The enzyme composition may contain components other than protein, such as moisture, saccharides and the like.

  As the enzyme composition containing chymosin, a mixture of digestive enzymes produced in the stomach of a mammal, that is, rennet (coagulase enzyme) can be used as long as the chymosin content is 80% by mass or more. It is. In this case, it is preferable to use calf-rennet (usually containing chymosin 88 to 94 mass% and pepsin 6 to 12 mass%, respectively) derived from calf.

  The amount of the enzyme composition used for the hydrolysis is preferably in the range of 0.001 to 5%, preferably in the range of 0.01 to 4% as the amount of protein with respect to the mass of lactoferrin as the substrate. It is more preferable, and it is particularly preferable that the content be in the range of 0.1 to 3%.

(Method for producing lactoferrin hydrolyzate)
In hydrolyzing lactoferrin by the method of the present invention, the lactoferrin (LF) solution is adjusted to pH 2 to 4, preferably 2.5 to 3, with an acid such as hydrochloric acid, citric acid or acetic acid before the enzyme reaction treatment. It is particularly preferable that the pH is 3.
For example, in the LF solution adjusted to pH 3, after the enzyme composition containing the chymosin is added in a desired amount, the temperature of the enzyme reaction is 35 to 55 ° C, preferably 40 to 50 ° C, more preferably 42. The lactoferrin is hydrolyzed with stirring for 6 to 24 hours, preferably 12 to 18 hours, maintained at ˜48 ° C.
Next, for example, the reaction solution is heated to 80 ° C. and maintained for 10 minutes to inactivate the enzyme by heating. Further, preferably, an alkaline solution such as sodium hydroxide solution is added to adjust the pH to 5-7.
For example, adjust to 6.
In addition, although the reaction solution (lactoferrin hydrolyzate) after pH adjustment may be in the form of a solution, it is preferably pulverized by lyophilization or the like. In addition, the lactoferrin degradation product may be fractionated by chromatography, ultrafiltration, or the like.

The lactoferrin hydrolyzate obtained as described above has an antigenicity of 0.1% (1/1000) or less compared to lactoferrin (undegraded lactoferrin having the same mass as the lactoferrin hydrolyzate). Antigenicity can be evaluated by, for example, reactivity with anti-lactoferrin antibodies. The reactivity to the anti-lactoferrin antibody can be measured as a lactoferrin concentration measured by an immunoassay using the anti-lactoferrin antibody, for example, ELISA (Enzyme-linked immunosorbent assay). The lactoferrin concentration can be measured using a standard curve prepared using a known amount of lactoferrin as a standard substance. The anti-lactoferrin antibody may be a monoclonal antibody or a polyclonal antibody. Details of the ELISA method will be described later.
In addition, the lactoferrin hydrolyzate obtained as described above has less bitterness than the lactoferrin hydrolyzate obtained using other proteolytic enzymes such as pepsin.

Hereinafter, specific methods for evaluating antigenicity and bitterness are exemplified.
(Antigenicity test)
For the antigenicity test, a normal immunoassay can be employed. For example, an enzyme-linked immunosorbent assay (ELISA, Kawase Kozo et al., Toho Medical Journal, 35: 506, 1989) In accordance with the above, lactoferrin is used as a control, and the reactivity of the lactoferrin hydrolyzate to the lactoferrin antibody can be evaluated.

  Specifically, for example, preparation of reagents and the like used in the ELISA method and measurement using them can be performed as follows, but the present invention is not limited to these and can be appropriately modified.

1) Washing solution 1 L of 100 tablets of Dulbecco's PBS (DULBECCO'S PBS: Phosphate Buffer Saline) solution (DULBECCO'S PBS TABLET (DS PHARMA BIOMEDICAL, DSBN200))
5 mL of Tween (registered trademark) 20 (polysorbate 20, polyoxyethylene sorbitan monolaurate) was added to 1 L of 1 milliliter of Milli-Q water to prepare a Tween-PBS 10-fold concentrated solution. did. Tw
A 10-fold concentrated solution of een-PBS is further diluted 10-fold with milli-Q water and used as a washing solution.

2) Block liquid
REAGENT DILUENT CONCENTRATE 2 (10X) (R & D systems, DY995)
A 10-fold diluted Milli-Q water filtered through a 2.2 μm filter is used as a blocking solution.

3) Primary antibody solution 1 ml of anti-bovine lactoferrin affinity goat antibody (Goat anti-Bovine Lactoferrin affinity Purified 1.0 mL @ 1.0 mg / mL) (Bethyl, A10-126A) was added to PBS solution (pH
7.2) A solution diluted 4,000 times by Gibco (20012) is used as a primary antibody solution.

4) Diluted solution The Tween-PBS 10-fold concentrated solution, the block solution, and Milli-Q water are set to Tween-PBS 10-fold concentrated solution: Block solution: Milli-Q water = 1: 1: 8, respectively. Mix to make the diluted solution.

5) Calibration curve sample Lactoferrin is dissolved in the above-mentioned diluted solution so as to have concentrations of 3.125 ng / mL, 6.25 ng / mL, 12.5 ng / mL, 25 ng / mL, 50 ng / mL, and 100 ng / mL, respectively. Prepare a calibration curve sample.

6) LF degradation product sample Lactoferrin hydrolyzate is dissolved in a diluent to obtain a LF degradation product sample (maximum concentration: 4000 ng / mL).

7) Secondary antibody solution 1mL of 1mL of peroxidase-labeled anti-bovine lactoferrin HRP conjugated 1.0 mL @ 1.0 mg / mL (Bethyl, A10-126P) with the above dilution The solution diluted in step 2 is used as the secondary antibody solution.

8) Coloring solution
ABTS Peroxidase Sulfate (ABTS: 2,2'-Azido.bis-3-ethyl benzthiazoline sulfonic acid) and Peroxidase Solution B (H ₂ O ₂ ) (KSP, 50-62-00) in the same amount (1: 1)
The mixture is used as a color developing solution.

9) Reaction stop solution 20 g of sodium lauryl sulfate (SDS: sodium dodecyl sulfate) is dissolved in 50 mL of milli-Q water, and 50 ml of dimethylformamide (DMF) is added thereto to obtain a reaction stop solution.

10) Measurement procedure i) Dispense 100 μL of the primary antibody solution into the wells of the microplate and leave it at room temperature for 1 hour to overnight, then wash the wells with 300 μL of the washing solution three times, and dispense 200 μL of the blocking solution into the wells. To do. After leaving still at room temperature for 1-2 hours, it wash | cleans 3 times with 300 microliters of washing | cleaning liquids.

ii) A calibration curve sample or 100 μL of an LF degradation product is put in a well, left at room temperature for 2 to 3 hours, and then washed 3 times with 300 μL of a cleaning solution.

iii) Add 100 μL of the secondary antibody solution to the well, let stand for 2 to 3 hours at room temperature, and then wash 3 times with 300 μL of the cleaning solution.
iv) Add 100 μL of the color developing solution to the well and let stand at room temperature for 5 to 15 minutes, and then add 50 μL of the reaction stop solution.

v) The absorbance (405 nm) of the reaction solution is measured. The absorbance can be measured using, for example, a microplate reader (Corona Electric Co., Ltd., MTP-32). A calibration curve is created by plotting the concentration and absorbance of the calibration curve sample. The lactoferrin concentration of the LF degradation product sample is measured from the absorbance of the reaction solution of the LF degradation product sample and a calibration curve.

11) Antigenicity Antigenicity can be calculated based on the following formula.

  Antigenicity (%) = [Lactoferrin concentration of LF degradation product sample measured by ELISA method (W / V)] × 100 / [Peptide concentration of LF degradation product sample (W / V)]

If the proteolytic enzyme contained in the LF degradation product sample is negligible compared to lactoferrin and lactoferrin hydrolyzate, the denominator of the above formula can be replaced with the lactoferrin concentration of the lactoferrin solution before hydrolysis.
The peptide concentration of the LF degradation product sample is, for example, the ultraviolet absorption method, Bradford method, Lowry
It can be measured by a method such as a method.

  The lactoferrin concentration in the LF degradation product sample measured as described above is an undegraded lactoferrin concentration, an apparent lactoferrin concentration due to a hydrolyzed peptide reactive with an anti-lactoferrin antibody, or its Although it is not clear whether both are present, in any case, a low lactoferrin concentration measured by an immunoassay indicates a low reactivity to the anti-lactoferrin antibody, ie, low antigenicity.

(Bitter taste test)
The bitterness of the lactoferrin hydrolyzate can be evaluated by a sensory test. Specifically, for example, a test solution in which each sample is dissolved in purified water at a concentration of 1% by mass is kept at 20 ° C. and sensuously flavored by a panel of 20 men and women from 20 to 40 years old. Are evaluated in four stages: no bitterness (0 points), slightly bitter (1 point), bitter (2 points), and strong bitterness (3 points). From the average value of the evaluation points, for example, less than 0.5 points is “no bitterness”, 0.5 points to less than 1.5 points is “slightly bitter”, 1.5 points to less than 2.5 points is “bitterness” Yes "and 2.5 points or more and less than 3.0 points can be determined as" strongly bitter ".

The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples.
[Example 1]
Bovine lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.) was dissolved in purified water to 5% by mass, and the pH was adjusted to 3 with a hydrochloric acid solution to prepare a lactoferrin solution. The prepared lactoferrin solution was heated to 45, and then 3% calfrennet (produced by RENCO, containing 92% by mass of chymosin and 8% by mass of pepsin) was added to the mass of lactoferrin while stirring. It was made to react for 24 hours and hydrolyzed.
After completion of hydrolysis, the reaction solution was heated to 80 and kept for 10 minutes to deactivate the enzyme.
The reaction solution was ice-cooled, sodium hydroxide solution was added to adjust the pH of the reaction solution to 6, and then the reaction solution was lyophilized to produce lactoferrin hydrolyzate powder.

[Test Example 1]
This test was conducted to examine the antigenicity of the lactoferrin hydrolyzate of the present invention.

  The lactoferrin hydrolyzate produced in Example 1 was tested for antigenicity based on the method described in the antigenicity test (ELISA method).

  As a result, the peptide concentration of the solution obtained by dissolving the lactoferrin hydrolyzate produced in Example 1 in water was 8000 ng / mL, the lactoferrin concentration measured in the antigenicity test was 3.6 ng / mL, and the lactoferrin hydrolyzate Antigenicity was confirmed to be 0.045% (1/1000 or less) compared to the same amount of lactoferrin.

[Test Example 2]
This test was conducted to examine the bitter taste of the lactoferrin hydrolyzate of the present invention and the lactoferrin hydrolyzate digested by other hydrolases.

(Test sample)
As an enzyme used for hydrolysis of lactoferrin, a lactoferrin hydrolyzate using a chymosin (100% by mass: manufactured by DSM), a lactoferrin hydrolyzate using Sample 1 and an enzyme composition containing 92% by mass of chymosin, respectively. Sample 2, lactoferrin hydrolyzate using an enzyme composition containing 88% by mass of chymosin, sample 3, lactoferrin hydrolyzate using an enzyme composition containing 50% by mass of chymosin, sample 4, bovine rennet (RENCO Lactoferrin hydrolyzate using chymosin 5% by mass, pepsin 95% by mass, sample 5, lactoferrin hydrolyzate using porcine pepsin as control 1, lactoferrin hydrolyzate using molsin F as control 2, A lactoferrin hydrolyzate using Sumiteam AP was subjected to a bitterness test as Control 3. The enzyme composition used for the preparation of Samples 2 to 4 was prepared by mixing the chymosin and bovine rennet used for the preparation of Sample 1 and Sample 5, respectively, so as to have a predetermined chymosin content. Sumiteam AP is a protease derived from Aspergillus niger, and Morsin F is a protease derived from Aspergillus saitoi.

(Test method)
When hydrolyzing lactoferrin, lactoferrin was adjusted to 5% by mass with purified water and hydrolyzed.
Moreover, each enzyme was added so that all enzyme amounts might be 3% with respect to the mass of lactoferrin, and the enzyme reaction was performed on the conditions of pH 3 about all the samples.
The reaction temperature was set to 45 ° C. for Samples 1 to 5, and for Controls 1 to 3, the temperature was set to the optimum temperature for each enzyme used, and all the reaction times were set to 6 hours for hydrolysis. .
The bitterness was evaluated based on the above-described four-step sensory evaluation.

(Test results)
The results of this test are as shown in Table 1. As a result, the enzyme used for hydrolysis of lactoferrin contains chymosin, 92% by mass of chymosin, or an enzyme composition containing 88% by mass of chymosin. In this case, no bitterness was detected by this test.
In contrast, when the enzyme used for hydrolysis of lactoferrin contained 50% by mass of chymosin, the enzyme composition contained a little bitter taste, and more clearly when the enzyme composition contained 5% by mass of chymosin. The result was a bitter taste.
In addition, when the enzyme used for hydrolysis of lactoferrin was pepsin, it was confirmed that it felt bitter, and when it was Morcine F or Sumiteam AP, the result was a strong bitter taste.

  That is, it has been found that the hydrolase used for lactoferrin is preferably an enzyme composition containing at least about 80% by mass of chymosin under the above reaction conditions from the viewpoint of suppressing the bitter taste of lactoferrin hydrolyzate.

[Test Example 3]
This test was conducted to examine the bitterness and antigenicity of the lactoferrin hydrolyzate obtained by a method having different enzyme reaction conditions from those of the present invention.

Hoek, KS et al. (Antimicrobial Vial Agents and Chemotherapy, Vol. 41, No. 1, 1997, 54-59.
Page) shows that the enzyme used for the hydrolysis of lactoferrin is chymosin and the pH is 3, and the production method of the lactoferrin hydrolyzate of the present invention is in common with the conditions, but the enzyme reaction time is in the present invention. It is described that it is shorter (reaction time: 1 hour).
Accordingly, the lactoferrin hydrolyzate prepared based on the method for producing a lactoferrin hydrolyzate described in Reference 1 is referred to as Control 4, and the Control 4 and the lactoferrin hydrolyzate produced in Example 1 of the present invention are as follows. Were subjected to antigenicity test and bitterness test.

The results are shown in Table 2. As is clear from Table 2, the bitterness of Example 1 of the present invention and Control 4 was not felt.
On the other hand, the antigenicity (%) was 0.045 in Example 1 and 1.624 in Control 4, and the lactoferrin hydrolyzate described in Hoek, KS et al. It was found that the antigenicity was 36 times as high as that of the degradation product.

In addition, as described in Reference 1, in the preparation of the sample of Control 4, chymosin corresponding to 4% of the mass of lactoferrin was used for the amount of enzyme, and the temperature condition of the enzyme reaction was 60 ° C. This is different from the present invention.
That is, although the amount of chymosin enzyme was 1.45 times that of the present invention and the enzyme reaction temperature was 15 ° C. higher, the antigenicity was higher than that of the present invention.

Therefore, in order to produce a lactoferrin hydrolyzate with reduced bitterness and sufficiently low antigenicity, the enzyme used for hydrolysis is an enzyme composition having a chymosin content of 80% by mass or more, and at least 6 hours or more It has been found that it is important to carry out the enzyme reaction preferably for 24 hours.

Claims (3)

A method for producing a lactoferrin hydrolyzate having an antigenicity of 0.1% or less compared to lactoferrin and having a reduced bitter taste, the enzyme composition comprising lactoferrin in an amount of 80% by mass or more based on the total protein A process comprising a step of hydrolysis under conditions of pH 2 to 4, reaction temperature 35 to 55 ° C., and reaction time 6 to 24 hours.   The method according to claim 1, wherein the antigenicity is evaluated as a lactoferrin concentration measured by an immunoassay using an anti-lactoferrin antibody with lactoferrin as a standard substance. The method according to claim 1 or 2, wherein the amount of the enzyme composition is in the range of 0.001 to 5% as the amount of protein with respect to the mass of lactoferrin .