JPH0322150B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for producing a milk-clotting enzyme used in milk-clotting processing of cheese and the like.

(Prior art) Conventionally, as a method for producing milk-clotting enzymes using filamentous fungi, Mucor luxii is cultured in a solid rice medium to produce an enzyme with milk-clotting power. It has been reported that Mucor is unsuitable for the production of milk-clotting enzymes, and from this point of view, Mucor-based enzymes have been proposed as a method for industrially producing milk-clotting enzymes with high productivity and extremely useful in cheese production at low cost. 7 parts psilum lint to 10 parts wheat bran
By culturing under aerobic conditions in a medium sprayed with water and heated and steamed, or in a liquid medium containing wheat bran and dissolved nutrients such as a carbon source, nitrogen source, and inorganic salts, it is possible to produce wheat that is suitable for industrial production. An invention is disclosed in which a high-unit milk-clotting enzyme can be obtained. (Tokuko Akira
40-18830).

There is also a known method for producing milk coagulating enzyme by culturing a specific bacterial strain in a medium made by adding various nutritional components to a starchy substrate prepared by decomposing starch such as rice flour with bacterial amylase before sterilization. . (Tokuko Showa 50-
Publication No. 37276).

(Problems to be Solved by the Invention) According to the above conventional invention, the titer of the enzyme solution is at the level described in the Examples.

In particular, as disclosed in the above-mentioned Japanese Patent Publication No. 50-37276, if a starchy substrate such as rice flour is used as a component of the culture medium and has been previously decomposed with bacterial amylase, the titer of the milk-clotting enzyme solution produced is You can only get a low value. Moreover, in a nutrient-rich medium containing nitrogen, phosphoric acid, potassium, and other various nutrient sources, even if filamentous fungi are cultured with shaking or aeration, a high-titer milk-clotting enzyme solution will not be produced. I can't get it. Therefore, from this point of view, the starch decomposition treatment using enzymes is a wasteful process and increases production costs.

The present invention provides a method for producing milk-clotting enzyme having an extremely high titer compared to conventional methods.
The method is characterized in that a rice aqueous solution medium is prepared by adding and dissolving ~3wt%, and filamentous fungi are cultured with shaking or aeration using this medium.

(Example) Next, examples of the present invention will be described.

Bacteria belonging to the genus Rhizopus, Rhizomucor, and Mucor are used as filamentous fungi, and have traditionally been used for alcoholic fermentation, organic acid fermentation,
Filamentous fungi used in the fermentation and production of miso, soy sauce, etc., for example, strains used as Aspergillus oryzae for sake brewing, and strains distributed from universities, research institutes, etc. can be used. For example, Rhizopus oryzae isolated from Jiyawa koji.
Oryzae), R.Arrhizus isolated from China koji, Rhizomucor pusillus (Lint), and Mucor luxii are readily available from Institute for Fermentation (IFO), American Type Culture Collection (ATCC), etc. available at.

In an attempt, we inoculated steamed rice (10g of rice) with 2 to 3 platinum loops of each of Rhizopus oryzae and Rhizopus alrizus and tried solid culture at 30°C ± 5°C for 4 days, but the curds extracted from this The titer of the enzyme solution was almost zero in Soxhlet units. The same results were obtained when culturing was carried out in the same manner using 10 g of each solid medium of wheat bran, rolled barley, corn, and potato. Next, add 10g of rice flour to 400ml of water,
A rice aqueous solution obtained by dissolving by boiling and cooling to 30°C to 35°C was used as a culture medium, and the above strain was inoculated into it, and the rice was heated at 30°C.
After static culture at ±5°C for 4 days, centrifuge the
When the obtained liquid was concentrated and the titer of the milk-clotting enzyme was measured, it was found that there were only a few Soxhlet units, and it was almost zero. Instead of rice, add 10g of each of wheat bran, rolled barley flour, and potato flour, or a mixture of these powders, to 400ml of water, boil and dissolve, then boil at 30°C.
Each aqueous solution prepared by cooling to ~35°C was used as a culture medium, and 2 to 3 platinum loops of each of the above-mentioned Rhizopus oryzae and Rhizopus arulis strains, such as the 6155 strain and 5378 strain obtained from IFO, were inoculated into the medium. ,
Although it was statically cultured in the same manner as the rice aqueous medium, the titer of milk-clotting enzyme in the obtained concentrate was so low that it could not be measured in Soxhlet units.

Next, 10g of rice made as above/400ml of water
(2.5 wt%) and in which the rice is not subjected to decomposition treatment by bacterial amylase and a similar culture substrate aqueous solution medium other than rice, each of the strains of Rhizopus oryzae and Rhizopus alurisus is added. Inoculate 2 to 3 platinum loops of 30℃±5℃
When cultured with shaking in a rice aqueous medium for 4 days, the titer of the milk-clotting enzyme solution was 20,000 units/ml in Soxhlet units. This is 10
When converted to gram of rice, it is 500,000 units. On the other hand, in the various aqueous media other than rice, even when cultured with shaking, the titer was so low that the Soxhlet unit could not be measured.

In addition, Soxhlet unit = M / E × 2400 / T, M: amount of milk used in the test, E: amount of enzyme,
T: Represents the time until card fragmentation occurs.

As is clear from the above, by using an aqueous solution of undecomposed rice as a medium, inoculating the filamentous fungus into this, and shaking culture, the enzyme titer can be increased by at least several times the maximum enzyme titer compared to the maximum enzyme titer obtained by the conventional method. It was confirmed that the enzyme could be produced with a significantly high Soxhlet unit titer of about 7 cultures. In addition, a similar remarkable effect was obtained by aerated culture.

Therefore, various conditions for shaking culture using this rice aqueous solution as a culture medium were investigated. That is,
In the case of sticky rice, non-glutinous rice, or a mixture of both, an aqueous solution of rice alone is best, and generally
It was found that an enzyme solution with the maximum titer could be obtained by culturing with shaking at 30±5°C for 3 to 4 days.
Furthermore, it was surprisingly found that when rice was dissolved in a synthetic medium such as LCA medium or Czapetsk's solution, the titer of milk-clotting enzyme did not rise to Soxhlet units. It has also been found that rolled barley and corn are effective when used in combination with rice. That is, for example, in the case of a medium in which 5 g of rice and 5 g of rolled barley or 5 g of corn, or 2.5 g each of rolled barley and corn are dissolved in 400 ml of water, the same amount as in the case of a medium in which 10 g of rice alone is dissolved in 400 ml of water. A milk-clotting enzyme solution with a titer of 80% of the titer could be obtained. The amount of rolled barley or corn dissolved in water is preferably in the range of 1 to 3 wt%, respectively. The mixing ratio for rice is generally in the range of 0.5 to 1 part for rice.

As a result of examining the relationship between the dissolved concentration of rice in water and the obtained enzyme titer, we found that the dissolved concentration of rice in water was approx.
The maximum titer can be obtained at 1wt% or more, but from an economical point of view it is preferable to limit its use to about 3%. On the other hand, if it is less than 1 wt%, the titer will gradually decrease, but even if it is 0.5%, an enzyme solution with about 60% of the maximum titer can be obtained, which is not practical.

Generally, it is practically preferable to use an aqueous rice solution containing about 1 to 3 wt% of the rice as a medium. In this way, the laborious and uneconomical disadvantages of using various synthetic media as in the past can be eliminated.

In addition, in actual production, the above filamentous fungi are
Although pre-culture may be performed under aerobic conditions using a medium other than rice, it is necessary to use an aqueous rice solution as the medium for the main culture, and perform shaking culture or aeration culture. However, it is of course possible to use an aqueous rice solution as a medium and inoculate 2 to 3 loopfuls of rice for pre-cultivation.

Example 1 Add 10g of ground glutinous rice to 400ml of water,
After melting by heating at ℃, it was cooled and then Rhizopus alrizus was added to it at 30℃±5℃.
IF06155 strain 2 to 3 platinum loops were inoculated as IF06155 (Arrhizus) and cultured with horizontal shaking (shaking width 20°C) for 90 to 95 hours at 30°C.
mm, shaking rate 100 times/min). After culturing, the bacterial cells and culture solution were centrifuged at 10,000 rpm and 10,000 rpm, and the solution was concentrated to obtain 25 ml of enzyme solution. When I calculated the Soxhlet unit for this, it was 20,000 units/ml. Therefore, an enzyme solution with an enzyme titer of 500,000 units was obtained from 10 g of rice.

Example 2 Example 1 except that a rice aqueous solution prepared by heating and dissolving 5 g of rice and 5 g of rolled barley in 400 ml of water was used as the medium.
As a result of culturing in the same manner as above, 400,000 units of enzyme solution were obtained from 10 g of the culture medium.

Example 3 400 ml of water was added to 10 g of glutinous rice flour, heated to 100°C, dissolved, and cooled. When the temperature reached 30°C ± 5°C, it was added as Rhizopus oryzae.
Inoculate 2 to 3 platinum loops of IF05378 strain, 30℃, 90-95
Shaking culture for a period of time under the same conditions as in Example 1 above,
Next, the bacterial cells and the culture solution were separated by centrifugation, and the solution was concentrated to obtain 30 ml of crude enzyme solution.

When the titer of this enzyme solution was measured, it showed a titer of 24,000 Soxhlet units/ml. That is,
30ml has 720,000 units.

Example 4 5g of glutinous rice flour and 5g of corn flour in 400ml of water
Culture was carried out in the same manner as in Example 3, except that the culture medium was an aqueous rice solution prepared by adding the rice to the rice and dissolving it by heating. The Soxhlet unit of the concentrated enzyme solution is 80 in Example 3.
% titer.

Comparative example: 1000g of glutinous rice was soaked in water for 12 hours, steamed for 40 minutes, then inoculated with Rhizopus alrizus and heated at 30°C.
The cells were cultured for 72 hours. After culturing, some water was added and the culture solution was separated to obtain 1500 ml of crude enzyme solution. When the Soxhlet unit was measured, it was only 13 units/ml.

The concentrated crude enzyme solution obtained by this method is purified by adding 20 to 30 g of common salt and storing it by drying at low temperature, or by salting out with ammonium sulfate.

An example of producing cheese using the milk-clotting enzyme obtained according to the present invention will be shown below.

CaCl ₂ 0.8-1.0 for 2 skim milk and 5 whole milk
After 10 minutes, 5 ml of the crude enzyme solution obtained in Example 1 above was added, stirred, and allowed to stand. Agglutination is observed after 25 minutes. Next, the whey is discharged by a conventional method.

Cheese is manufactured using the following conventional steps.

Furthermore, the enzyme of the present invention can coagulate milk without the conventional addition of CaCl ₂ or lactic acid bacteria starter.
It is also advantageous because it can provide a new food material as a dairy product.

(Effects of the Invention) According to the present invention, rice alone, whose starch has not been subjected to enzymatic decomposition treatment as described above, is added to water at a rate of 1 to 10%.
3wt% added dissolved aqueous solution or such a small amount of rice per 1, rolled barley or/and corn 0.5-1
We used an aqueous solution prepared by adding and dissolving the rice aqueous solution medium as a medium, and cultured filamentous fungi in this aqueous rice solution medium by shaking culture or aeration culture. Since it brings about the production of milk enzymes and does not use other nutrients or ingredients such as synthetic media, it has the effect of enabling easy and economical mass production.

Claims (1)

[Scope of Claims] 1. A rice aqueous medium is prepared by adding and dissolving at least 1 to 3 wt% of undecomposed rice in water as a medium, and culturing filamentous fungi by shaking culture or aerated culture. Characteristic method for producing milk-clotting enzymes. 2 The rice aqueous medium contains 1 to 3 wt% of rice in water,
2 or 3 ingredients obtained by adding and dissolving rolled barley and/or corn at a mixing ratio of 0.5 to 1 to 1 of the rice.
The method for producing milk-clotting enzyme according to claim 1, comprising an aqueous solution medium of the components.