JPH0241313B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention does not require any special coupling means.
The present invention relates to a method for very easily preparing a card using immobilized protease directly adsorbed onto an adsorbent. Generally, in the production of cheese, one of the important steps is to coagulate the proteins in animal milk such as cow's milk or goat's milk (i.e., curds) to obtain coagulated proteins (curds). teeth,
It originally requires rennin (a milk-clotting enzyme secreted from the abomasum of calves 1 to 4 weeks old). However, in recent years, while cheese production has increased significantly, demand for beef cattle has increased due to a global shortage of beef cattle, and the number of calves used to collect rennin has decreased significantly, leading to a serious rennin shortage. I'm getting used to it. Therefore, a milk-clotting enzyme (rennet) that could replace calf rennin was widely searched for, and so-called microbial rennet produced by microorganisms such as Mucor rennet appeared.At present, microbial rennet, or microbial rennet and proteases such as pepsin, are now available. Mixtures now account for the majority of milk-clotting enzymes in practical use. However, calf rennin is a protease with extremely high substrate specificity for katsupercasein (k-casein), which is the key to curdling milk, and hardly degrades other casein components (αs-, β-casein). It has the characteristic of having no power. On the other hand, microbial rennet and other substitute enzymes can be used for caseins other than k-casein.
Because they have some decomposition power, when these substitute enzymes are used, the recovery rate of curd is low and the flavor of the finished cheese is inferior (bitterness may occur due to the decomposition of β-casein). It has the disadvantage that it is inferior to calf rennin in terms of its texture (changes in texture). This is the reason why many other proteases, although having milk curdling activity, cannot act as milk curdling enzymes due to their strong hydrolytic activity. There are many attempts at cloning. In other words, many proteases other than rennin can relatively quickly degrade k-casein, which triggers curdling, and curd milk. However, most other proteases have broader substrate specificity than calf rennin, so they also hydrolyze other caseins (αs-, β-casein), which are the main components of curd. This will reduce the card collection rate,
This will cause the finished cheese to deteriorate. That is,
The amount of β-casein controls the hardness of the curd.
When β-casein is decomposed, it becomes a soft cheese, and some peptides, which are the decomposition products of β-casein, have a bitter taste and deteriorate the quality of the curd. In another direction, attempts have been made to reduce costs and streamline operations by using calf rennin repeatedly or continuously. It is an attempt to immobilize calf rennin on a suitable carrier. In this case, among the many immobilization methods, covalent bonding methods, ion adsorption methods, and crosslinking methods are used, but these methods require complicated operations during immobilization and are not suitable for food applications. It requires the use of appropriate reagents and tends to cause deactivation of the enzyme, and in addition, it has several drawbacks such as deactivation during use, and has not yet reached a practical stage. In addition, soy milk is a typical example of other aggregating proteins. Coagulating soy milk yields tofu, which can also be produced using immobilized protease. Conventionally, tofu is coagulated by adding a coagulating agent such as bittern (calcium salt, etc.) to hot soymilk, or by adding glucono delta-lactone to soymilk once cooled and heating it. On the other hand, if an immobilized protease is used, tofu can be produced without the use of a coagulant. If we can create an excellent immobilized protease, we will not only be able to use rennin repeatedly, but also use not only mucor rennet but also many proteases with strong hydrolyzing power as a substitute for calf rennin. However, as a result of intensive research based on the assumption that it could be used as a milk-clotting enzyme, we found that an excellent immobilized protease could be produced by directly contacting the protease with a porous adsorbent in an aqueous solvent and adsorbing it. He succeeded in obtaining it. The present invention was completed based on this knowledge, and includes immobilized protease produced by contacting and adsorbing protease with a porous adsorbent having hydrophobic binding strength in an aqueous medium. By contacting animal milk or aggregating proteinaceous substances,
This is a method for preparing curd, which is characterized by obtaining curd by coagulation. The adsorbent for immobilized protease used in the present invention is a porous adsorbent with hydrophobic binding strength, examples of which are styrene synthetic adsorbents HP-10, 20, 21, 30, 40, 50 (manufactured by Mitsubishi Chemical Industries, Ltd.) and methacrylic acid ester synthetic adsorbents
Examples include HP-2MG (manufactured by Mitsubishi Chemical Industries, Ltd.) or porous glass beads (Corning, Glass, Worker, Fuji, Davison, Chemicals). The protease to be adsorbed may be any so-called protease enzyme, and examples thereof include rennin, pepsin, trypsin, papain, Aspergillus alkaline protease, and actinomycete protease. During adsorption, each enzyme is adsorbed onto an adsorbent in an aqueous solvent. Generally, rennin, pepsin, trypsin, papain, Aspergillus alkaline protease, Streptomyces protease, etc. are dissolved in water.
Contact with adsorbent. Furthermore, since these enzymes tend to be preferentially adsorbed to adsorbents, any liquid containing each enzyme can be adsorbed as is. For example, since raw soy sauce contains a large amount of Aspergillus alkaline protease, by directly contacting this raw soy sauce with an adsorbent, an adsorbent adsorbing Aspergillus alkaline protease, that is, an immobilized enzyme can be obtained. In the case of rennin, immobilized rennin can be obtained by contacting a crude rennin-containing liquid obtained from calves with an adsorbent. These enzymes may be used alone, but they can also be adsorbed in combination. For example, as a group of milk-clotting enzymes, rennin and mucorrennet can be appropriately mixed to form an aqueous solution, and an adsorbent can be brought into contact with this to obtain an adsorbent adsorbing rennet and mucorrennet, that is, a composite immobilized enzyme. .
Furthermore, if you want to curdle milk more quickly, it is also possible to add acidic protease to these and adsorb the three enzymes at the same time. To adsorb the protease, simply contact the adsorbent with the protease-containing aqueous solvent.
This method of adsorption is thought to be mainly based on physical adsorption power, and is the mildest immobilization method, and requires an extremely simple operation, i.e., simply combining an enzyme solution and a synthetic adsorbent. All you have to do is make contact with it. Specifically, the protease solution is passed through a column packed with adsorbent,
Immobilized protease can be obtained by adding an adsorbent to a container containing a protease solution and stirring gently. Adsorption of protease onto the adsorbent is carried out at a temperature that does not cause deactivation of each enzyme, for example, 45°C or lower,
And it is carried out in a wide PH range, for example PH4-10,
Moreover, it can be carried out regardless of ionic strength. The immobilized protease obtained is extremely stable and does not easily detach even after use. In the present invention, an agglutinated curd can be obtained very easily and in extremely high quality by contacting the immobilized protease obtained here with animal milk, skim animal milk, or an aggregating proteinaceous substance. The recovery rate of the curd can be equal to or higher than that of the conventional method using rennin, and the quality of the protein in the curd can be almost the same as that using rennin. can. The material to be flocculated is animal milk such as cow's milk, goat's milk,
Proteinaceous substances that aggregate, such as sheep milk, various skim milks skimmed from these, various caseins separated from these, regenerated milk regenerated from these, regenerated skim milk, and emulsions containing vegetable proteins such as soy milk. Any of them can be used. During the aggregation treatment, these animal milks, etc., are brought into contact with the immobilized protease either as they are or after being diluted. Contact with a container containing animal milk, etc.
It is also possible to use a batch method in which a wire mesh basket containing immobilized protease is immersed for a short period of time, or animal milk, etc. is injected into a container containing immobilized protease for a short period of time and then taken out. A continuous system may be used in which animal milk or the like is continuously passed through the tank. The contact conditions may be room temperature, heating, or cooling, and the contact time will vary depending on the amount of enzyme, so determine the amount of enzyme in advance depending on the quality of the curd you are looking for, and adjust the contact time accordingly. It is preferable to decide. After the contact, the animal milk or the like is allowed to stand or is heated to cause aggregation, and curds can be obtained in batches or continuously. As described above, the method of the present invention uses immobilized protease, and the milk curd reaction can be appropriately controlled by simply changing the flow rate of milk, so it is possible to prepare a desirable curd according to the purpose. Furthermore, high-quality and expensive rennin can be used for a long period of time, and homogeneous cards can be manufactured. Next, experimental examples and examples of the present invention will be shown, and the measurement methods used here are as follows. Method for measuring milk curd activity Skim milk 10% with 0.012% _CaCl2
(Adjusted to PH5.8 with 0.05M acetate buffer) solution as a substrate, 10ml of enzyme solution was added to 10ml of substrate kept at 35℃.
was added, and the time (seconds) during which curd fragmentation occurred was measured. (The standard unit for flocculation activity is 200 mg of enzyme and 40 minutes (2400 seconds) of milk curdling time.) If this time is t, the milk curdling activity is expressed by the following formula. Milk curd activity = 2400/t x (200mg/added enzyme (mg)
) The activity of the immobilized enzyme was expressed as the amount of enzyme activity added to the adsorbent minus the amount of enzyme activity that flowed out without being adsorbed. Recovery rate of curd protein: The curd produced by the curd was filtered through a filter paper, thoroughly washed with water, and the T-N was determined by the Kjeldahl method.Additionally, 10 ml of the substrate skim milk solution was added with a TCA mixture (0.11M trichloroacetic acid, 0.22M sodium acetate,
The recovery rate of curd protein was determined by adding 10 ml of a solution containing 0.33M acetic acid and setting the TN (total nitrogen) of the resulting curd as 100%. SDS-electrophoresis of curd: After passing the curd produced by the curds through a filter paper and washing thoroughly with water, a small amount is placed in a test tube and subjected to SDS-electrophoresis.
B-Buffer (2%, SDS, 50% glycerol,
0.02M sodium phosphate buffer (PH7.2), 3%2
- 0.5 ml of mercaptoethanol-containing solution) was added and converted to SDS at 100°C for 5 minutes. The amount of sample was added so that the amount of protein was approximately 100 μg per electrophoresis column, and electrophoresis was carried out in a conventional manner. Measurement of aggregation activity of soymilk The prepared soymilk was centrifuged using a conventional method to remove Okara residue, and this was used as a substrate (T-N, 0.56%).
As in the case of milk curdling activity, the time until aggregation was measured by acting on immobilized protease. Experimental Example 1 Regarding the milk curdling properties of six types of proteases (Aspergillus alkaline protease, calf rennin, pepsin, trypsin pronase (manufactured by Kaken Chemical Co., Ltd., trade name), and protrichuase (manufactured by Ueda Chemical Co., Ltd., trade name)) investigated. Table 1 shows the recovery rate of curd protein obtained after adding an enzyme solution whose concentration was adjusted so that the curdling time was about 5 minutes to the substrate skim milk and leaving it for 20 hours. Furthermore, the SDS electrophoresis pattern of the obtained card is shown in FIG. In Figure 1, Aspergillus alkaline protease is calf rennin, pepsin is
is an SDS electrophoresis pattern of cards derived from trypsin protriciphasase.

【table】

[Table] From Table 1, the six enzymes examined here were all found to have milk curdling activity, although their activities varied in magnitude. Furthermore, although the recovery rate of curd protein differs depending on the enzyme, it was found that a recovery rate of 30% or more could be obtained in all cases. In Figure 1, the right side of the figure is a high-molecular protein and the left side is a low-molecular protein, so compared to the protein obtained from rennin, the protein obtained from each protease in a solution state has a lower molecular weight. (the pattern is shifted to the left), indicating that it is overresolved. Experimental Example 2 The six types of enzymes used in Experimental Example 1 were adsorbed onto various adsorbents to prepare immobilized enzymes. That is, a column (φ: 10 x 200 mm) was filled with 15 ml each of porous adsorbents HP-20, 30, and 2MG (all manufactured by Mitsubishi Chemical Industries, Ltd., trade names), and approximately 100 ml of the enzyme dissolved in water was packed.
200 mg was passed through at room temperature and adsorbed on an adsorbent to obtain an immobilized enzyme column. When water was passed through the immobilized enzyme obtained here, the effluent showed no flocculating activity, indicating that immobilized protease had been prepared. At this time, the immobilized enzyme activity is shown in Table 2.

[Table] Pass the substrate skim milk solution through the immobilized protease obtained here at a rate of about 10 ml/min at room temperature, take the effluent into a test tube, and immediately place it in a thermostat at 35°C to increase the curdling time. It was measured. In addition, the recovery rate of curd protein was checked after leaving the milk for 20 hours after curdling. These results are shown in Table 3 below.

[Table] Furthermore, in order to confirm the presence or absence of excessive decomposition of the curd, the SDS-electrophoresis pattern of the protein of the curd obtained by allowing it to stand for an additional 20 hours is shown in FIGS. 2 and 3. In Figure 2, TCA curd protein (curd protein precipitated by trichloroacetic acid: control)
′ is HP-20 adsorbed Aspergillus alkaline protease,
′ is HP-20 adsorbed rennin, ′ is HP-20 adsorbed pepsin, ′ is HP-20 adsorbed trypsin, ′ is
HP-20 adsorbed pronase, ′ is the production of curd protein by HP-20 adsorbed protrichuase
The S′DS′ electrophoresis pattern is shown. In Figure 3, "" is HP-2MG adsorbed Aspergillus alkaline protease, "" is HP-2MG adsorbed rennin, and "" is HP-2MG adsorbed Aspergillus alkaline protease.
HP-2MG adsorbed pepsin, ``HP-2MG adsorbed trypsin,'' HP-2MG adsorbed pronase,
'' indicates the pattern of curd protein produced by HP-2MG adsorbed protriciphaase. As can be seen from Experimental Examples 1 and 2, the recovery rate of curd protein can be increased by immobilizing protease according to the present invention. , also the second
As can be seen from the figure and Figure 3 (the same result was obtained when immobilized on HP-30, and the figure is omitted), by immobilization, the curd protein curdled by each protease becomes TCA curd protein or It can be seen that the effect is almost the same as that of immobilized rennin and that over-decomposition of curd protein is prevented. The details of the rationale behind the effectiveness of the immobilized protease according to the present invention in the production of cards will have to be studied in the future, but for the time being, the protease can be immobilized on a porous immobilization carrier with hydrophobic binding strength. Although it is conceivable that the degradability of α,β-casein may have changed due to the immobilization of α, β-casein, this is still a speculation. Example 1 Approximately 5 ml of synthetic adsorbent HP-2MG was added to a column (φ10×
60 mm) and fill it with ammonium sulfate fraction from Aspergillus oryzae bran, Cephadex G-25, and Bio-Gel P-.
Approximately 0.25 g of an aqueous solution of the alkaline protease fraction (unpurified) obtained by gel filtration of No. 150 was passed through the column at room temperature, and thoroughly washed with water to form immobilized protease, and the column temperature was maintained at 30°C. A 10% skim milk solution was passed through this at a flow rate of 10 ml/min at room temperature, and the effluent was collected in 20 ml portions. These fractionated effluents were immediately transferred to a constant temperature bath at 35°C, and the curd time and recovery rate of the curd protein obtained at that time are shown in Table 4.

[Table] Example 2 Approximately 5 ml of synthetic adsorbent HP-20 was added to a column (φ10×60
mm) and add it to the calf rennin (Difco・mm).
About 1.25 g of aqueous solution (manufactured by Laboratories, USA) was passed through at room temperature to form immobilized rennin, and the column temperature was
The temperature was maintained at 30°C, and a 10% skim milk solution was passed through the tube at a flow rate of 10 ml/min, and the effluent was collected in 20 ml portions. These fractionated effluents were immediately transferred to a constant temperature bath at 35°C, and the curd time and recovery of the curd protein obtained at that time are shown in Table 5.

[Table] Example 3 About 10 ml of porous synthetic adsorbent HP-40 was packed into a column (φ10 x 120 mm), and about 0.1 g of Amano-P (trade name of Amano Pharmaceutical Co., Ltd.), which is Aspergillus alkaline protease, was packed into a column (φ10 x 120 mm). to obtain the immobilized protease. Next, at room temperature, soymilk was passed through the column at both speeds of 20 ml/min to collect 20 ml of the treated solution, which was immediately transferred to a 35° C. constant temperature bath, and the time until aggregation was measured. The results are shown in Table-6.

[Table] After that, pass through the soy milk and add 100% of the treated solution.
ml, divide this into 50 ml portions and store unheated and at 80℃.
Calculate the amount of curd protein after heating for 30 minutes.
After passing through HP-40 without immobilized enzyme, 100% of the curd protein was precipitated with TCA.
It is shown in Table 7.

【table】 [Brief explanation of the drawing]

FIG. 1 is a diagram showing the SDS-electrophoresis pattern of each card obtained using solution protease in Experimental Example 1. FIG. 2 is a diagram showing the SDS-electrophoresis pattern of each card obtained by using HP-20 adsorbed protease in Experimental Example 2. Figure 3 shows the SDS of each card obtained by HP-2MG adsorbed protease in Experimental Example 2.
It is a figure showing an electrophoresis pattern.

Claims (1)

[Scope of Claims] 1 Immobilized protease obtained by contacting and adsorbing protease with a porous adsorbent having hydrophobic binding strength in an aqueous medium, and animal milk, skim animal milk, or an aggregating proteinaceous substance. By contacting
A method for preparing curd, characterized by obtaining curd by coagulation. 2 The protease is rennin, pepsin, trypsin, papain, Aspergillus alkaline protease,
Claim 1 selected from the group consisting of protease enzymes such as actinomycete protease.
Preparation of curd as described in section. 3. The method for preparing a card according to claim 1, wherein the porous adsorbent having hydrophobic bonding strength is a styrene-based synthetic adsorbent, a methacrylic acid ester-based synthetic adsorbent, or porous glass beads. 4. The curd preparation method according to claim 1, wherein the animal milk or skim milk is cow's milk or skim milk. 5. The curd preparation method according to claim 1, wherein the aggregating proteinaceous substance is soy milk.