JPH07102126B2 - Method for culturing microorganism by mixed medium of animal blood cell-derived peptide and amino acid - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for culturing microorganisms, and more particularly to a method for culturing microorganisms using a medium material containing a nitrogen source and a growth factor.

[0002]

2. Description of the Related Art Generally, a nitrogen source is required for culturing microorganisms. As the nitrogen source in this case, milk protein, soybean protein, animal meat protein, gelatin, etc. are usually decomposed by means such as enzymatic and acid decomposition Various peptones obtained by hydrolysis are used. However, the above-mentioned various raw materials are expensive, and especially when industrially culturing a large amount of microorganisms using them, the high cost of the medium containing a nitrogen source has been a problem. On the other hand, in terms of culture medium manufacturing, blood has been used for a long time and is used for various culture media. But,
Most of the uses are expected to remove the inherent antibacterial substances as blood characteristics, impart a vitamin-like effect, and discriminate against bacteria.

In addition, blood pigment containing excess iron is present in blood, and due to the low availability of bacteria due to the presence of blood protein as it is, a large amount of nitrogen source or peptone is used. It was not meant to be used as a source.
Furthermore, in a small part, blood is hydrolyzed with pepsin and trypsin, and like the various peptones described above, there is a method of using blood for the nutritional effect. And since undegraded protein coexists,
It cannot be sterilized by heat, and its use requires an extremely complicated operation of aseptically adding a hydrolyzed solution to other medium components that have been sterilized by heat in advance after cooling. Further, this hydrolyzed liquid is merely a nutritional supplement, and the possibility that it may be the main nitrogen source or peptone source for a wide range of microorganisms has not yet been examined. .

Here, as one solution to various problems in using blood components as a medium material, for example, Japanese Patent Laid-Open No. 59-59
-159772 publication is proposed. This publication discloses culturing yeast belonging to Saccharomyces bacterium by using a decomposition solution obtained by hydrolyzing blood or the like in slaughter liquid as a main nitrogen source or a carbon source. However, its content is derived from blood or plasma or serum, which mainly seeks its effect on the plasma part of blood, and is not currently applied to the blood cell part, which has the lowest utilization. Absent. Further, the target microorganism is only the genus Saccharomyces, and there is no mention of a detailed hydrolysis method for blood or the like, which can be a nitrogen source of widely-used microorganisms. In addition, there is no blood bleaching process, so its use is very limited.

On the other hand, British Patent GB2018 121A describes a method for hydrolyzing blood cells. However, this is a description of a decomposition method for obtaining a relatively high-molecular peptide as a food material, and no description is given of a microorganism culture medium material. In addition, Japanese Patent Publication No. 1-15 by the inventors
Japanese Patent No. 277 discloses a method of using as a medium material of blood cells hydrolyzed after iron removal, but the present invention particularly shows an effect against Gram-negative bacteria, Did not support the development of. The present invention has been made in view of the above circumstances, and provides an inexpensive medium material for culturing a wide range of microorganisms,
It is an object of the present invention to provide a method for culturing a microorganism that enables effective use of blood cells, which is an unused resource.

[0006]

Means for Solving the Problems The present invention is to hemolyze a blood cell part in blood and remove iron after hydrolysis or use a decomposed product hydrolyzed after iron removal as a medium material. , It was found that it is possible to make a medium material suitable for the growth of a wide range of microorganisms by mixing an enzyme hydrolyzate containing other nutrients and growth factors with an acid hydrolyzate having a complementary effect as a nitrogen source. It was made based on.

The blood cells used in the present invention are obtained by centrifuging blood of animals such as cows and pigs, after adding an anticoagulant in the usual manner. As a method of hemolyzing blood cells, 1 part by weight of blood cells is mixed with the same or 3 parts by weight of water. Here, if the amount of water added is smaller than this, hemolysis becomes insufficient, and if it is larger than this, recovery of the hydrolyzate becomes difficult. For the hydrolysis of hemolyzed blood cells, a method of hydrolyzing a protein can usually be used. For example, when hydrolyzing with an enzyme, it is possible to select the optimum conditions of the enzyme to be used as appropriate as the condition, but the molecular weight of the resulting hydrolyzate must be 5,000 or less. . Where the molecular weight is
If a decomposition method exceeding 5,000 is adopted, the iron content in the blood cells will be insufficiently removed, and the final decomposed product will not only be colored reddish brown but also its yield will be reduced. or,
Since it is a polymer, its ability to assimilate microorganisms is also reduced.

The type of enzyme used for the hydrolysis of hemolyzed blood cells may be any ordinary proteolytic enzyme, but it has a strong decomposing power for blood cells, and the degree of removal of iron and the molecular weight thereof.
Considering the high yield for obtaining a decomposition product of 5,000 or less, pepsin and microbial alkaline enzyme are particularly preferable. After the reaction is completed, the decomposed solution may be divided into a supernatant containing the blood cell protein hydrolyzate and a precipitate containing the undegraded protein and hemoglobin by the usual solid-liquid separation method after deactivating the enzyme.
In this case, for example, the operation by centrifugation is suitable,
000 rpm-20 minutes is enough. Further, in order to complete the removal of iron, the supernatant may be treated with activated carbon. In this case, the amount of activated carbon added is 1 to the supernatant.
2% is desirable. And, the supernatant portion after solid-liquid separation can be used as a nitrogen source in the medium in the state of the decomposition solution as it is, but in terms of storability and ease of use, if this is powdered by a treatment such as spray drying. More desirable.

On the other hand, in the case of hydrolyzing with an acid, an acid such as hydrochloric acid or sulfuric acid is used, but it is necessary that most blood cell proteins be decomposed into amino acids. The iron removal operation after decomposition is the same as that described in the section of enzymatic decomposition, but these sequences may be hydrolyzed after iron removal, for example.
An example of this is hydrolysis using globin protein prepared from blood cells. The enzyme hydrolyzate and the acid hydrolyzate of the blood cells obtained by the above method are appropriately mixed and used, but the mixing ratio varies depending on the type of microorganism. The present invention will be described below in more detail.

[0010]

Example 1 Preparation of Enzymatic Hydrolyzate After adding 3 l of water to 1 l of porcine blood cell fraction to hemolyze, the pH was adjusted to 8.5. Furthermore, alkaline protease
14 g was added and hydrolyzed at 55 ° C. for 2 hours. After disassembly,
The enzyme was inactivated by heating at 80 ° C. for 30 minutes, and the centrifugal supernatant (8,000 rpm-20 minutes) was treated with activated carbon and spray-dried. By this operation, 210 g of dry powder was obtained, which was designated as enzymatic hydrolyzate A. Similarly, 180 g of dry powder was obtained using 4.5 g of acidic protease (pH 5.5, reaction at 50 ° C. for 2 hours), and this was designated as enzymatic hydrolyzate B. In addition, 1 g of pepsin was added to 1 liter of a 1% globin solution (including iron removal operation) prepared from pig blood cells by the hydrochloric acid-acetone method to carry out enzymatic decomposition (pH 2.0, 35 ° C.-3 hours). After neutralization,
Further, 1 g of actinase was added for enzymatic decomposition to finally obtain 8 g of dry powder, which was designated as enzymatic decomposed product C.

Example 2 Preparation of Oxidized Hydrolyzate Hydrochloric acid was added to 1 liter of a 25% pig blood cell solution so that the final concentration was 6 N, and the mixture was decomposed under reduced pressure at 110 ° C. for 48 hours. After the decomposition, decolorization, dehydrochlorination, and concentration under reduced pressure were performed to obtain 150 ml of a decomposition solution having a nitrogen concentration of 4.5%, which was used as an acid hydrolyzate.

Example 3 Single application example of the prepared decomposed product 0.39% of each decomposed product (nitrogen conversion), 1% of glycerin,
A medium consisting of 2.4% sodium chloride (pH 0.7) was prepared and a culture test was conducted. The test bacteria are Eschericia coli.
hia coli IFO 3301), Bacillus subtilis (Bacill)
us subtilis IFO 3336) and Lactobacillus plantarum IFO 3070), in the case of Lactobacillus, the glycerin in the medium was changed to 1% glucose. 1 to 3 show growth curves of test bacteria that were shake-cultured at 30 ° C. In addition, FIG. 1 shows Escherichia using the enzyme hydrolysates A to C and the acid hydrolyzate, respectively.
2 and 3 are growth curves of Bacillus subtilis and Lactobacillus plantarum by the same method. As is clear from the figure, the growth state varied greatly depending on the test bacteria or the degradation products used. That is,
The enzyme hydrolyzate A has a good promoting effect on the growth of Escherichia coli and Bacillus, but it is not found in Lactobacillus, and the oxygen hydrolysates B and C and the acid hydrolyzate are growth of the test bacteria. Was generally suppressive.

Example 4 Esci by mixing enzyme hydrolyzate B and acid hydrolyzate
Growth-promoting effect of Elysia The ratio of enzyme hydrolyzate B to acid hydrolyzate in each of the decomposed products of the medium used in Example 3 was 100: 0,75: 25,50: 50,25:
Each was adjusted to 75, 0: 100, and the culture test of Escherichia was conducted in the same manner. The results are shown in Fig. 4, and the test bacteria showed that the enzyme hydrolyzate B and the acid hydrolyzate were 75:25.
Good growth was shown by mixing in the range of ~ 25: 75. From this result, the growth promoting action of the enzymatic hydrolyzate B on Escherichia and the complementary effect as a nitrogen source of the acid hydrolyzate are shown.

Example 5 Batyl by Mixing Enzymatic Hydrolyzate C and Acid Hydrolyzate
Growth-promoting effect of sucrose A mixed medium of enzyme hydrolyzate C and acid hydrolyzate was prepared in the same manner as in Example 4, and a Bacillus culture test was conducted. The results are shown in Fig. 5, and the test bacteria showed good growth by mixing the enzyme hydrolyzate C and the acid hydrolyzate in the range of 75:25 to 25:75. From this result, the growth promoting action of the enzyme hydrolyzate C on Bacillus and the complementary effect of the acid hydrolyzate as a nitrogen source are shown.

Example 6 Lacto by mixing enzyme hydrolyzate C and acid hydrolyzate
Growth promoting effect of Bacillus A mixed medium of enzymatic hydrolyzate C and acid hydrolyzate was prepared in the same manner as in Example 5, and a lactobacillus culture test was conducted. The results are shown in FIG. 6, and the test bacteria were enzyme hydrolyzate C.
Good growth was demonstrated by mixing the acid hydrolyzate with the acid hydrolyzate in the range of 75:25 to 25:75. From this result, the growth promoting action of the enzymatic hydrolyzate C on Lactobacillus and the complementary effect as a nitrogen source of the acid hydrolyzate are shown.

[0016]

As described above, according to the present invention, a blood cell portion in blood is enzymatically and acid hydrolyzed, and both are mixed to provide a very effective medium material for culturing a wide range of microorganisms. be able to. Needless to say, blood cells are much cheaper than the peptone raw material conventionally used for the same purpose.

[Brief description of drawings]

FIG. 1 is a growth curve diagram of Escherichia coli using enzyme hydrolysates A to C and acid hydrolysates, respectively.

FIG. 2 is a growth curve diagram of Bacillus subtilis similar to FIG.

FIG. 3 is a growth curve diagram of Lactobacillus plantarum similar to FIG.

FIG. 4 is a growth curve diagram of Escherichia coli when the enzyme hydrolyzate B and the acid hydrolyzate are mixed at various ratios.

FIG. 5 is a growth curve diagram of Bacillus subtilis when enzyme hydrolyzate C and acid hydrolyzate are mixed at various ratios.

FIG. 6 is a growth curve diagram of Lactobacillus plantarum similar to FIG.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Toyo Nakamura Inventor 1-2 Kubogaoka, Moriya-cho, Kitasoma-gun, Ibaraki Itoham Co., Ltd. Central Research Laboratory (56) Reference Japanese Patent Publication 1-15277 (JP, B2)

Claims (2)

[Claims] 1. A blood cell component in animal blood is used as a raw material, which is used as a raw material.
Separately hydrolyze with an enzyme and an acid , respectively, and then remove iron or iron and then hydrolyze them to obtain an enzymatic hydrolyzate and an acid hydrolyzate.
Obtained respectively, and mix each of the above hydrolysates as appropriate to make a medium material
A method for culturing a microorganism in a mixed medium of an animal blood cell-derived peptide and an amino acid, which is characterized in that 2. The enzymatic hydrolyzate and the acid hydrolyzate are 7
The method for culturing a microorganism in a mixed medium of an animal blood cell-derived peptide and an amino acid according to claim 1 , wherein the mixing is performed in the range of 5:25 to 25:75 .