JPH08238059A - Production of milk protein having low allegenicity - Google Patents

Abstract

PURPOSE: To obtain a milk protein having low allergenicity, reduced in decomposition of β-casein, excellent in nutritional property and useful as a protein source for various foods by reacting a specific protease with a milk protein solution and selectively decomposing α-casein in the milk protein. CONSTITUTION: A protease produced by a bacterium belonging to the genus Mucor selected from a group comprising Mucor angulisporus, Mucor hiemalis, Mucor janssein, Mucor ramannianus, Mucor rouxianus and Mucor rouxii and having optimum activity near pH3 is reacted with a milk protein solution and αS-casein in the milk protein is selectively decomposed to provide the objective milk protein having low allergenicity.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a hypoallergenic milk protein obtained by selectively decomposing αs-casein contained in milk protein. The milk protein obtained in the present invention has a low allergenicity because αs-casein is selectively decomposed, and is nutritionally excellent, and can be used as a protein source for various foods.

[0002]

BACKGROUND OF THE INVENTION Proteins in milk are roughly classified into casein and whey protein, and the casein is mainly αs-.
It is composed of casein, β-casein, and κ-casein. The whey protein is mainly composed of α-lactalbumin and β-lactoglobulin. Among the components of these proteins, αs-casein and β-lactoglobulin are proteins that are rarely present in human milk, and a nutritional composition for childcare using milk protein as a protein source may be an allergen for artificial nutrition. is there. For this reason, studies have been made so far to reduce the above αs-casein and β-lactoglobulin from milk proteins as much as possible by means such as fractionation or decomposition.

For example, as a method for fractionating αs-casein from casein, a divalent salt is added to a casein fraction containing αs-casein and β-casein as main components at a temperature of 0 to 10 ° C. to obtain αs-casein. Casein-based fraction and β-
A method of dividing into fractions containing casein as a main component (JP-A-59-9)
1849) or a method of fractionating casein by gel filtration.
(RD Hill et al .; J. Dairy Res., 31, 291 (196
4)), and also a method for selectively hydrolyzing αs-casein, which is a casein component, by a proteolytic enzyme (JP-A-6-
261691) is known. On the other hand, there is also known a method for selectively degrading β-lactoglobulin by adding an animal-derived or microbial-derived proteolytic enzyme to an aqueous whey protein solution in order to decompose β-lactoglobulin (JP-A-2-265441). Has been.

[0004]

Conventionally, αs-casein, which is one component of allergen in milk protein, was fractionated by allowing it to react with a divalent salt and then precipitating or gel filtration. . Therefore, there is a problem in terms of production that mass production is not possible or cost is high. Further, the obtained product itself was reacted with a divalent salt and then precipitated and fractionated, resulting in a problem of a β-casein fraction having a high salt concentration.

On the other hand, recently, a method of selectively hydrolyzing αs-casein in milk protein by a proteolytic enzyme has been attempted, but a crude product produced by a Mucor sp. Or Cladosporium sp. Proteolytic enzymes are
Its main component is a protease having an optimum activity at pH 6, and it has a decomposing power for β-casein as well as αs-casein in milk protein, so that β
-There was a drawback that the residual rate of casein was significantly reduced. Therefore, there is still a demand for an enzyme that selectively decomposes only αs-casein from milk proteins.

[0006]

Means for Solving the Problems As a result of intensive studies to solve this problem, the present inventors have found that a crude proteolytic enzyme produced by a Mucor bacterium has a protease having an optimum activity around pH 6. And a protease having an optimal activity around pH 3 coexist, and the protease having an optimal activity around pH 3 selectively decomposes αs-casein in milk protein but has an optimal activity around pH 6. It was found that protease decomposes not only αs-casein but β-casein to the same extent. Therefore, by newly purifying a crude proteolytic enzyme produced by a bacterium belonging to the genus Mucor, a protease having an optimal activity in the vicinity of pH 6 contained in the crude enzyme is removed as much as possible to reach a pH of around 3. After obtaining a protease fraction having an appropriate activity, αs-casein is selectively decomposed by adding a protease having an optimal activity in the vicinity of pH 3 to a milk protein solution,
It was possible to obtain a hypoallergenic milk protein with reduced αs-casein, and completed the present invention. That is, the present invention causes a milk protein solution to act on a milk protein solution with a protease produced by a bacterium belonging to the genus Mucor and having an optimal activity around pH 3 (hereinafter referred to as "a protease having an optimal activity around pH 3" of the present invention). A method for producing a hypoallergenic milk protein characterized by selectively degrading αs-casein in milk protein.

As the milk protein used as a starting material in the present invention, whole milk milk, skim milk or casein fractionated from these milk materials can be used, but the preferred milk protein is casein. is there.

Next, the milk protein solution is subjected to enzyme treatment by adding a protease having an optimum activity in the vicinity of pH 3 of the present invention as a protein hydrolase. The condition of the enzyme treatment is not particularly limited as long as the protease having an optimal activity around pH 3 of the present invention maintains the activity, and is not particularly limited, but more preferably at around neutral pH. 30 to 70
It is better to treat at 30 ° C for 30 minutes to 30 hours.

The strain producing a protease having an optimum activity in the vicinity of pH 3 of the present invention belongs to the genus Mucor. For example, the strain Mucor anglycus (Muc
or angulisporus), Mucor hiemalis
lis), Mucor janssein, Mucor ramannianus, Mucor rouxianus, and Mucor rouxii.

Of these microorganisms, for example, M. janssein is used for solid culture to prepare a crude enzyme, which is then purified to have optimum activity around pH 3 of the present invention. The method for obtaining the protease is exemplified below, but the protease having the optimum activity in the vicinity of pH 3 of the present invention can be obtained from other Mucor spp.

150 g of water was added to 180 g of bran, and after sterilization, seed koji of Mucor janssein IAM 6100 was inoculated and cultured at 30 ° C. for 4 days. Water (500 ml) was added to the koji obtained after culturing, and the mixture was extracted overnight at a low temperature to obtain 300 ml of a crude enzyme solution. The pH 3 protease activity of this crude enzyme solution was 16.7 u / ml, and the pH 6 protease activity was 6.3.
It was u / ml. Then, 300 ml of the obtained crude enzyme solution was salted out with ammonium sulfate twice to remove contaminating proteins, desalted with a membrane, and the desalted solution was buffered with phosphate buffer (pH 7.0) to DEAE-. After adsorbing to a Toyopearl column and washing, elution was performed with a concentration gradient method using 0 to 600 mM NaCl to separate active fractions 1, 2 and 3. A protease solution having an optimal activity around pH 3 was obtained from active fraction category 1 (protease activity at pH 3 was 7.4 u / ml), and pH 6 was obtained from each of active fraction categories 2 and 3. A protease solution having optimum activity was obtained in the vicinity (protease activities at pH 6 were 4.2 u / ml and 6.4 u / ml, respectively.) The DEAE-Toyopearl chromatograph of the obtained purified enzyme and The optimum pH curve of each active fraction obtained by chromatography is shown in FIGS. 1 and 2.

The enzyme chemical properties of the protease having the optimum activity around pH 3 of the present invention, which is derived from the active fractionation category 1, thus prepared are as follows. (1) Action: It acts on milk proteins such as casein to produce peptides. (2) Substrate specificity: It decomposes αs-casein in milk proteins well, but β-casein has a weak decomposition power. (3) Optimum pH: around 3.0 (4) Optimum temperature: around 60 ° C (5) pH stability: Stable at pH 2.5 to 6.0. (6) Heat resistance: 75% remains after treatment at 45 ° C for 10 minutes. (7) Isoelectric point: PI = 6.1 (by isoelectric point chromatography)

In the present invention, the above-mentioned enzyme is used, and 0.01 to 100 units of protease activity at pH 7 is added per g of casein as an added amount to treat β-casein in milk protein for decomposition. Less, α
S-casein can be selectively hydrolyzed. The αs-casein-decomposed milk protein obtained by such a method can be used as it is or after being dried and powdered, as a protein source for foods, infant nutrition compositions and the like. And these foods and infant nutrition compositions are allergen-reduced and nutritionally excellent. When used in a nutritional composition for infants, since αs-casein in casein protein has been decomposed with an enzyme in advance,
A soft curd forms in the stomach, making it easier to digest.

Casein was prepared by using each of the components of protease activity fractionation category 1, activity fractionation category 2 and activity fractionation category 3 having optimum activity around pH 3 of the present invention obtained as described above. Degradation rate of αs-casein in proteins and β-
The results of comparison of casein decomposition rates are shown in Test Example 1.

Test Example 1 Reaction of milk protein solution A 5% casein solution adjusted to pH 7.0 was added to DEAE-Toyopearl chromatograph with active fractionation category 1 (protease having optimum activity near pH 3 of the present invention) and activity. The enzyme solution of each of the fraction 2 and the active fraction 3 (casein g
Per 1 unit of protease activity of pH 7) and reacted at 40 ° C for 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes and 180 minutes, and then heated at 90 ° C for 15 minutes. Then, the reaction was stopped, and the relationship between the proteolysis rate and the residual rates of αs-casein and β-casein was investigated. The results are shown in FIGS. 3, 4 and 5. The degradation rate of protein, the residual rate of αs-casein and β-casein and the protease activity (protease activity at pH 3, pH 6 and pH 7) were measured according to the following methods.

(1) Method of measuring decomposition rate 7.5 ml of purified water was added to 2.5 ml of the decomposition solution, and the mixture was placed in a constant temperature bath at 40 ° C.
Leave for a minute. After that, add 1 ml of 10% acetic acid solution, 40 ℃
After allowing to stand for 5 minutes at room temperature, add 1 ml of 1N sodium acetate solution and
After standing at ℃ for 10 minutes, make up to 50 ml with purified water and filter with Toyo filter paper No. 5C. For this filtrate and the casein solution before decomposition, the Folin reagent reaction was carried out to calculate the decomposition rate.

(2) Method for measuring residual ratio of αs-casein and β-casein A sample is treated with a tris-hydrochloric acid buffer solution containing 4.5M urea and mercaptoethanol, and then a polyacrylamide gel containing 4.5M urea is applied. Was used for electrophoresis. After electrophoresis, staining and decolorization were performed, densitometry was performed, and the residual ratio was determined from each peak area of αs-casein and β-casein.

(3) Method for measuring protease activity Preparation of a casein solution pH3.0 for measuring protease activity at pH 3: 1.5 g of milk casein (Hammerstein No. 2242 manufactured by Merck) was weighed and 60 ml of 0.1 M lactic acid solution was added. In addition, the mixture is heated and dissolved at 90 to 95 ° C and then cooled.
After cooling, adjust the pH to 3.0 with dilute sodium hydroxide test solution (0.1N), and add 0.1 M lactic acid / sodium hydroxide buffer (pH3.0) 20
Add ml and water to make 100 ml.

Preparation of pH 6.0 Protease Activity Substrate Casein Solution pH 6.0: Milk Casein (Merck
Weigh 1.5 g of Hammerstein No.2242), add 20 ml of dilute sodium hydroxide test solution (0.1N), dissolve by heating at 90-95 ℃, and then cool. After cooling, adjust the pH to 6.0 with 0.1N phosphoric acid solution, and add 20 ml of 0.1 M phosphate buffer (pH 6.0) and water to make 100 ml.

Preparation of pH 7.0 Protease Activity Measuring Substrate Casein Solution pH 7.0: Milk Casein (Merck
Weigh 1.5 g of Hammerstein No.2242), add 20 ml of dilute sodium hydroxide test solution (0.1N), dissolve by heating at 90-95 ℃, and then cool. After cooling, adjust to pH 7.0 with 0.1N phosphoric acid solution and add 20 ml of 0.1 M phosphate buffer (pH 7.0) and water to make 100 ml.

Method for measuring each protease activity: Take 1 ml of a 1.5% milk casein solution (pH 3.0, pH 6.0 or pH 7.0) in a test tube, put it in a constant temperature water bath at 37 ° C., leave it for 5 minutes, and then sample it. Add 1 ml of solution, mix well and immediately at 37 ° C.
Put in the constant temperature water bath of and react for 30 minutes. To this, 2 ml of 0.4 M trichloroacetic acid solution was added, mixed well, allowed to stand at room temperature for 10 minutes, filtered with filter paper (Toyo No. 131.7 cm), and filtrate 1
Take 5 ml of the solution in a test tube, add 5 ml of 0.55 M sodium carbonate solution and 1 ml of Folin's test solution, mix well, react for 5 minutes in a constant temperature water bath at 50 ° C to develop color, and then measure the absorbance At at wavelength 660 nm using water as a control. To do. Separately, as a blank test (blank), take 1 ml of milk casein solution in a test tube, add 2 ml of 0.4 M trichloroacetic acid solution, mix well, and add exactly 1 ml of sample solution. To measure.

Activity indication: Reaction filtrate 1 within 60 minutes under these conditions
The amount of enzyme that produces a colored substance corresponding to 100 μg of tyrosine in ml is defined as 1 unit of protease activity and calculated by the following formula. Protease (u / g) = (At-Ab) x F x 1/100 x n x 2 F: Tyrosine amount (μg) when the absorbance difference obtained from the tyrosine calibration curve is 1.0 F = 100 2, 1/100 : Unit conversion coefficient n: Sample solution dilution factor

As is clear from FIG. 3, the protease having the optimum activity in the vicinity of pH 3 of the present invention is α in milk protein.
It can be seen that s-casein is decomposed well, but β-casein is hardly decomposed. On the other hand, as is clear from FIGS. 4 and 5, both the active fractions 2 and 3 of the DEAE-Toyopearl chromatograph mainly containing the protease having the optimum activity around pH 6 are αs-
It can be seen that casein has a weak decomposition power and that αs-casein and β-casein are decomposed to the same extent. Therefore, the protease having the optimum activity in the vicinity of pH 3 of the present invention obtained by removing the protease having the optimum activity in the vicinity of pH 6 as much as possible from the crude proteolytic enzyme produced by Mucor spp. It can be seen that it can be efficiently used for the purpose of the present invention for selectively degrading αs-casein in proteins.

Next, a crude proteolytic enzyme (a mixture of a protease having an optimum activity around pH 6 and a protease having an optimum activity around pH 3) prepared according to the description in JP-A-6-261691. And the protease of the present invention having an optimum activity around pH 3 were used to compare the degree of αs-casein decomposition and the degree of β-casein decomposition in casein proteins. The results are shown in Test Example 2.

Test Example 2 Reaction of milk protein solution Crude proteolytic enzyme solution prepared according to the method described in JP-A-6-261691 and 100 g of a 5% casein solution adjusted to pH 6.5 or pH 7.0 and the present invention A protease solution having an optimal activity (each 0.5 unit of protease activity at pH 7 per g of casein) was added at around pH 3 and reacted at 40 ° C. for 30 minutes, and then heated at 90 ° C. for 15 minutes to stop the reaction. The residual rate of αs-casein and the residual rate of β-casein are shown in FIGS. 6, 7, 8 and 9.

As is apparent from FIGS. 6 and 7, Japanese Patent Laid-Open No.
When a crude proteolytic enzyme prepared according to the description of 6-261691 is used, a large amount of protease having an optimum activity is present around pH 6, and the protease has a pH of 6.5 or 7.0. Even under the reaction conditions of αs-
It is understood that not only casein but also β-casein is degraded. However, as is clear from FIGS. 8 and 9, when the protease of the present invention having an optimum activity in the vicinity of pH 3 is used, it is effective under both reaction conditions of pH 6.5 and pH 7.0. Degradation of αs-casein progresses over time, but degradation of β-casein progresses slowly. Then, when the difference between β-casein and αs-casein remaining in the reaction product after the reaction for a certain period of time is taken, it is found that a constant value of about 40 to 50% continues to be exhibited. That is, from the results of comparing FIG. 6, FIG. 7 and FIG. 8, FIG. 9, by using the protease having the optimum activity in the vicinity of pH 3 of the present invention, the βs-casein in the milk protein is left and αs- It can be seen that casein can be selectively degraded.

In addition, the allergenicity of the degradation product (degradation rate of about 13%) by the protease having the optimum activity around pH 3 of the present invention is determined by an ELISA (ELI) using anti-αs-casein serum.
SA: Enzyme linked immunosorbent assay) As a result of measurement by an inhibition test method, the antigenicity was reduced to 1/100. The allergenicity test was carried out by the following method.

Test method for allergenicity Antigenicity is determined by enzyme-linked immunosol assay.
nt assey (ELISA) inhibition test method. 96-well plate (manufactured by Nunc) was coated with αs-casein and washed, and a mixture of rabbit anti-αs-casein serum and hydrolyzate sample was supplied to the plate wells for reaction, and after washing, alkaline phosphatase-labeled goat After reacting with anti-rabbit IgG antibody (Zemed Laboratory), wash and p-
Add sodium nitrophenyl phosphate and, after 30 minutes, add 5N
The reaction was stopped by adding sodium hydroxide and the reaction product was measured with a microplate reader. (Journal of Japanese Society of Pediatric Allergy, Vol. 1, p. 36, 1987)

Examples of the present invention will be shown below, but the present invention is not limited to these examples.

Example 1 A 5% casein solution adjusted to pH 7.0 was added to a protease solution having an optimal activity near pH 3 derived from M. janssein IAM 6100 (pH 7 per g of casein).
0.5 units of protease activity) and added at 50 ° C for 6
After reacting for 0 minutes, the reaction was stopped by heating at 90 ° C. for 15 minutes. The residual rates of αs-casein and β-casein were 23% and 72%, respectively. In addition, the antigenicity using αs-casein as an index decreased to 1/100 or less. The proteolytic solution thus treated was spray-dried at a hot air temperature of 173 ° C. and an exhaust air temperature of 98 ° C. to obtain a protein food powder, which was used as a protein source for infant nutrition composition.

Example 2 In a 5% casein solution adjusted to pH 6.5, pH 3 derived from M. angulisporus IAM 6151 was added.
Add a protease solution with optimum activity (0.6 units of protease activity at pH 7 per g of casein) near the
After reacting at 0 ° C for 90 minutes, the reaction was stopped by heating at 90 ° C for 15 minutes. The residual rates of αs-casein and β-casein were 22% and 65%, respectively. In addition, the antigenicity using αs-casein as an index decreased to 1/100 or less.

Example 3 A 5% casein solution adjusted to pH 7.0 was added to a protease solution having an optimum activity around pH 3 derived from M. hiemalis IAM 6090 (pH 7 per g of casein).
0.5 units of protease activity) and added at 50 ℃
After reacting for 0 minutes, the reaction was stopped by heating at 90 ° C. for 15 minutes. The residual rates of αs-casein and β-casein were 25% and 69%, respectively. In addition, the antigenicity using αs-casein as an index decreased to 1/100 or less. The proteolytic solution thus treated was spray-dried at a hot air temperature of 173 ° C. and an exhaust air temperature of 98 ° C. to obtain a protein food powder, which was used as a protein source for infant nutrition composition.

Example 4 A 5% casein solution adjusted to pH 6.5 was added to a protease solution having optimum activity around pH 3 derived from M. ramannianus IAM 6128 (per g of casein).
Add 0.4 units of protease activity at pH7), 40 ℃
After reacting for 120 minutes at 90 ° C., the reaction was stopped by heating at 90 ° C. for 15 minutes. The residual rates of αs-casein and β-casein were 28% and 68%, respectively. In addition, the antigenicity using αs-casein as an index decreased to 1/100 or less.

Example 5 A 5% casein solution adjusted to pH 7.0 was added to a protease solution having an optimum activity around pH 3 derived from M. rouxianus IAM 6132 (pH per g of casein).
0.5 unit of protease activity of 7) was added and reacted at 45 ° C. for 60 minutes and then heated at 90 ° C. for 15 minutes to stop the reaction. The residual rates of αs-casein and β-casein were 30% and 71%, respectively. In addition, the antigenicity using αs-casein as an index decreased to 1/100 or less. The proteolytic solution thus treated was spray-dried at a hot air temperature of 173 ° C. and an exhaust air temperature of 98 ° C. to obtain a protein food powder, which was used as a protein source for infant nutrition composition.

Example 6 A 5% casein solution adjusted to pH 6.5 was added to a protease solution having an optimum activity around pH 3 derived from M. rouxii IAM1066 (0.6 unit of protease activity at pH 7 per g of casein). Was added and reacted at 45 ° C for 100 minutes, and then heated at 90 ° C for 15 minutes to stop the reaction. The residual rate of αs-casein and β-casein is 27%.
And was 69%. In addition, the antigenicity using αs-casein as an index decreased to 1/100 or less.

[0036]

Industrial Applicability The protease of the present invention having an optimum activity around pH 3 is allowed to act on a milk protein solution to reduce the decomposition of β-casein and selectively hydrolyze αs-casein. It is possible, the production cost can be reduced, and the hydrolyzate obtained is excellent in nutrition as well as having reduced allergens. The milk protein treated in this way is usefully utilized as a protein source in foods or infant nutrition compositions.

[Brief description of drawings]

FIG. 1 is a pattern diagram of a purified proteolytic enzyme eluted with a saline solution after adsorbing a crude proteolytic enzyme solution obtained by culturing Mucor Jansein IAM 6100 onto a DEAE-Toyopearl column. The dotted line in the figure shows the protease activity at pH 3, and the solid line shows the protease activity at pH 6.

FIG. 2 shows an optimum pH curve of each active fraction category of DEAE-Toyopearl chromatograph.

[Explanation of symbols]

In the figure, the white squares show the pH fractions of the active fractions 1, the white circles show the activity fractions 2 and the black circles show the pH fractions of the active fractions 3, respectively.

FIG. 3 shows the relationship between the rate of degradation of milk protein decomposed by a protease having optimum activity around pH 3 of the present invention in Test Example 1, the residual rate of αs-casein, and the residual rate of β-casein.

[Explanation of symbols]

The black circles in the figure represent the residual ratio of αs-casein, and the open triangles represent the residual ratio of β-casein.

FIG. 4 shows the relationship between the milk protein degradation rate and the αs-casein residual rate and β-casein residual rate that were degraded by the DEAE-Toyopearl active fractionation category 2 in Test Example 1.

[Explanation of symbols]

In the figure, black circles are residual rates of αs-casein and open triangles are residual rates of β-casein.

FIG. 5 shows the relationship between the degradation rate of milk protein and the residual rate of αs-casein and the residual rate of β-casein, which were degraded by DEAE-Toyopearl active fractionation category 3 in Test Example 1.

[Explanation of symbols]

In the figure, black circles are residual rates of αs-casein and open triangles are residual rates of β-casein.

FIG. 6 shows the residual ratio of αs-casein in the digested milk protein, which was decomposed at pH 6.5 by a crude proteolytic enzyme prepared according to the description in JP-A-6-211691 in Test Example 2, and The residual rate of β-casein is shown.

[Explanation of symbols]

In the figure, black circles are residual rates of αs-casein and open triangles are residual rates of β-casein.

FIG. 7 shows the residual ratio of αs-casein in a milk protein hydrolyzate decomposed at pH 7.0 by a crude proteolytic enzyme prepared according to the description in JP-A-6-211691 in Test Example 2 and The residual rate of β-casein is shown.

[Explanation of symbols]

In the figure, black circles are residual rates of αs-casein and open triangles are residual rates of β-casein.

FIG. 8 shows the residual rate of αs-casein and the residual rate of β-casein in a digested milk protein product, which was decomposed at pH 6.5 by a protease having optimum activity around pH 3 of the present invention in Test Example 2.

[Explanation of symbols]

In the figure, black circles are residual rates of αs-casein and open triangles are residual rates of β-casein.

FIG. 9 shows the residual rate of αs-casein and the residual rate of β-casein in a degraded protein of milk protein, which was degraded at pH 7.0 by a protease having optimum activity around pH 3 of the present invention in Test Example 2.

[Explanation of symbols]

The black circles in the figure represent the residual ratio of αs-casein, and the open triangles represent the residual ratio of β-casein.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location // (C12P 21/06 C12R 1: 785) (72) Inventor Tetsuro Nakamura Shimofujisawa, Iruma City, Saitama Prefecture 580-5 (72) Kenichi Hirano Kenichi Hirano, Nishiharu Kasugai-gun, Aichi Kunitsubo Nishijo Yashiki 51, Amano Pharmaceutical Co., Ltd. Central Research Laboratory

Claims (2)

[Claims] 1. A low allergen characterized in that a protease produced by a bacterium belonging to the genus Mucor and having an optimum activity in the vicinity of pH 3 is allowed to act on a milk protein solution to selectively decompose αs-casein in the milk protein. Method for producing sex milk protein. 2. A bacterium belonging to the genus Mucor is a bacterial species selected from the group consisting of Mucor angrisporus, mucor hiemalis, mucor jeansein, mucor lamannianus, mucor ruxianus and mucor ruxii. The manufacturing method described.