JPS5991841A - Preparation of curd by immobilized protease - Google Patents

Abstract

PURPOSE:To prepare a curd using an immobilized protease, easily, by contacting animal milk, defatted animal milk or coagulable proteinous substance with an immobilized protease obtained by adsorbing protease to an adsorbent. CONSTITUTION:An immobilized protease is prepared by contacting and adsorbing a protease in an aqueous solvent to a hydrophobic porous adsorbent having bonding power. The immobilized protease is made to contact with animal milk, defatted animal milk or coagulable proteinous substance to obtain coagulated curd. The protease to be adsorbed may be any protease enzymes, e.g. rennin, pepsin, trypsin, papain, aspergillus alkaliprotease, etc. Any coagulable proteinous substances can be used as the raw material for coagulation, e.g. animal milk such as cow's milk, defatted milk prepared therefrom, various caseins separated therefrom, milky emulsion containing vegetable proteins such as soya milk, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a very simple method for preparing cards using immobilized proteases directly adsorbed onto an adsorbent without the use of special binding means.

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 milk (i.e., curds) to obtain coagulated proteins (curds). Essentially, rennin (a milk-clotting enzyme secreted from the abomasum of 1-4 week old calves) is required for this purpose. However, in recent years, while the production of cheese has increased significantly, the demand for beef cattle has increased due to the global shortage of beef cattle, and the number of calves used to collect rennin has decreased significantly, resulting in a serious shortage of rennin. It's coming.

Therefore, a milk-clotting enzyme (rennet) that could replace calf rennin was widely searched for, and microorganisms such as mucor rennet were produced. So-called microbial rennet has appeared, and at present, microbial rennet or a mixture of microbial rennet and proteases such as zubusin account for the majority of milk-clotting enzymes in practical use.

However, calf rennin is a protease with extremely high substrate specificity for kappa casein (k-casein), which is the key to curdling milk, and has little degrading power for other casein components (α, -2β-casein). It has a characteristic that it does not have.

On the other hand, microbial rennet and other substitute enzymes have some ability to decompose casein other than casein, so when these substitute enzymes are used, the recovery rate of curd is low and the finished product is The flavor of the cheese is inferior (
It has the disadvantage that it is inferior to calf rennin in that it produces bitter taste and changes in texture due to the decomposition of β-casein. This is the reason why many other proteases have milk curdling activity but cannot be used as milk curdling enzymes due to their strong hydrolytic activity. Many attempts are being made to clone Rennin.

In other words, many proteases other than rennin can cause milk to curd by relatively quickly decomposing casein, which triggers curdling. but,
Many other proteases have broader substrate specificity than calf rennin, so carP
Other caseins (αs-, β-casein) that are the main components of
It also hydrolyzes the card, reducing the recovery rate of the card.
This will cause the finished cheese to deteriorate. In other words, the amount of β-casein controls the hardness of the curd, and when β-casein is decomposed, it becomes soft cheese, and some of the decomposition products of β-casein give butide a bitter taste. , which deteriorates the quality of the card.

In another direction, attempts have been made to reduce costs and streamline operations by using calf rennin either 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.
The immobilization process is complicated, or the enzyme is likely to be deactivated due to the use of unsuitable reagents for food applications. It has shortcomings and has not yet reached a practical stage.

In addition, soy milk is a typical example of other aggregating proteins. If you coagulate soy milk, you can get tofu.
This can also be produced by immobilized protease.

Conventionally, tofu is made 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 to coagulate it.

On the other hand, if immobilized protease is used, tofu without a coagulant as described above can be produced.

The present inventors have developed an excellent immobilized protease (5)
Heh.

If it can be produced, not only can rennin be used repeatedly, but also mucorrennet, as well as many proteases with strong hydrolyzing power, can be used as a milk-clotting enzyme as a substitute for calf rennin. As a result of intensive research based on this assumption, they succeeded in obtaining an excellent immobilized protease by bringing the protease into direct contact with a porous adsorbent in an aqueous solvent and adsorbing it.

The present invention was completed based on this knowledge, and involves contacting and adsorbing protease with a porous adsorbent having hydrophobic binding strength in an aqueous solvent. This method of preparing curd is characterized by obtaining curd by coagulating it by contacting with animal milk or an aggregating proteinaceous substance.

The adsorbent for immobilized protease used in the present invention is
It is a porous adsorbent with hydrophobic bonding strength, such as HP-10 and HP-20, which are styrene-based synthetic adsorbents.
, 21, 30, 40, 50 (manufactured by Mitsubishi (6) Chemical Industries, Ltd.), methacrylic acid ester synthetic adsorbent HP-2MG (manufactured by Mitsubishi Chemical Industries, Ltd.) or porous glass beads (Corning, Glass, Worker). Fuji, Davison, and Chemicals).

The protease to be adsorbed may be any so-called protease enzyme, such as rennin, bapsin, trypsin, papain, Aspergillus alkaline zolothiase,
Examples include actinomycete protease.

During adsorption, each enzyme is adsorbed onto an adsorbent in an aqueous solvent. Generally speaking, rennin, psin, trypsypeno'y? '%in, Aspergillus alkaline protease, Actinomycete protease, etc. are dissolved in water and brought into contact with the adsorbent.

Furthermore, since these enzymes tend to be preferentially adsorbed onto adsorbents, any enzyme-containing solution can be directly adsorbed. 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 shusei 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 is brought into contact with this to obtain an adsorbent that has adsorbed rennet and mucorrennet, that is, a composite immobilized enzyme. can.

Furthermore, if milk is to be curdled more quickly, acidic protease can be added to the milk to adsorb the three enzymes at the same time.

To adsorb the protease, simply contact the adsorbent with the protease-containing aqueous solvent.

This adsorption method is thought to be mainly based on physical adsorption power, and is the mildest immobilization method, and
It is a very simple operation, ie simply bringing the enzyme solution into contact with the synthetic adsorbent. Specifically, a protease solution is passed through a column filled with an adsorbent, the adsorbent is poured into a container containing a droplet of protease, and the mixture is gently stirred to obtain immobilized protease. I can do it.

Adsorption of protease onto the adsorbent should be carried out at a temperature that does not cause deactivation of each enzyme, for example, 45°C or lower, and should be carried out in a wide range of pH ranges, for example, pH 4 to 10, and should be carried out regardless of ionic strength. I can do it.

The immobilized protease obtained is extremely stable and does not easily detach even after use.

In the present invention, an agglutinating card can be obtained very easily and in extremely high quality by contacting the immobilized protease obtained here with animal milk, skimmed animal milk, or an agglutinating 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 about the same as that using rennin. can do.

The materials to be agglomerated include animal milk such as cow's milk, goat's milk, sheep's milk, various skimmed milks skimmed from these, various caseins separated from these, regenerated milk regenerated from these, regenerated skim milk, and even soy milk. Any proteinaceous substance that can be flocculated, such as a vegetable protein-containing emulsion, can be used. In the flocculation treatment, these animal milks can be brought into contact with the immobilized protease either directly or after dilution. Contact can be made by immersing a wire mesh basket containing immobilized protease in a container containing animal milk, etc. for a short period of time, or by injecting animal milk, etc. into a container containing immobilized protease for a short period of time, and then removing it. Alternatively, it may be a continuous method in which animal milk or the like is continuously passed through nine columns filled with immobilized protease.

The contact conditions may be room temperature, heating, or cooling, and the contact time varies depending on the amount of enzyme, so it is best to determine the amount of enzyme in advance depending on the quality of the card you are looking for, and then determine the contact time accordingly. preferable.

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 a patch or continuous manner.

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 therein are as follows.

Method for measuring milk curd activity: 10% skim milk containing 0.012 g of Ca (J?t)
Using a PI (adjusted to 5,8 with 0.05 M acetate buffer) solution as a substrate, 10 m/ml of enzyme solution was added to substrate 101 kept at 35° C., and the time required for curd fragmentation to occur was measured. (The standard unit for flocculating activity is enzyme 200 ~ and milk curdling time 40 minutes (2400 seconds)) If this time is t, milk curdling activity is expressed by the following formula 1%. It was expressed as the amount of enzyme activity minus the amount of enzyme activity that flowed out without being adsorbed.

Recovery rate of curd tannolyte: The curd produced by the curds was filtered with a filter paper, thoroughly washed with water, and the T-N was determined by the Kjeldahl method. The recovery rate of curd protein was determined by adding 1 Qmt of a solution containing lychloroacetic acid, 0.22 M sodium acetate, and 0.33 M acetic acid, and setting the TN (total nitrogen) of the resulting curd as 100%.

5O8-electrophoresis of curd: After filtering the curd produced by the curdled milk with a filter paper and washing it thoroughly with water, a small amount was placed in a test tube, and 5ns-B-buffer (
2%, 8D 8.50 tiglycerol, 0.02 M sodium phosphate buffer (pH 72), 3 ml of solution containing 2-mercaptoethanol) Q, add 5 ml, 10
It was converted to SDa at 0°C for 5 minutes. Samples were added in an amount of approximately 100 ag of protein per electrophoresis column, and electrophoresis was performed in a conventional manner.

Measuring the coagulation activity of soymilk The prepared soymilk is centrifuged to remove okara residue using a conventional method, and this is used as a substrate (T-N, 0.56%) to act on immobilized protease to determine the coagulation activity. As in case K
, the time to aggregation was measured.

Example 6 types of proteases (■ Aspergillus alkaline protease, ■
The milk curdling properties of Shusei rennin, ■Hafusin, ■Trypsin, ■Pronase (manufactured by Kaken Kagaku (A, trade name); ■Prototrichuifase (manufactured by Ueda Chemical, trade name)) were 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 8D8 electrophoresis pattern of the obtained card is shown in FIG.

In Fig. 1, ■ indicates Aspergillus alkaline protease, ■ indicates modified rennin, ■ indicates pepsin, ■ indicates trypsin, and ■ indicates the SDS electrophoresis pattern of the card derived from protrichuifase.

Table 1: Milk curdling properties of solution proteases From Table 1, the six enzymes examined here were all found to have milk curdling activity, although their activities varied in magnitude. In addition, although the recovery rate of curd protein differs depending on the enzyme, it was found that a recovery rate of 30 or more was obtained in all cases.

In Figure 1, the right side of the figure is a high-molecular protein.

Since the protein on the left is a low-molecular protein, compared to the protein obtained from rennin, the protein obtained from each protease in a solution state has a much lower molecular weight (the pattern is to the left), and It can be seen that it has been disassembled.

EXAMPLE The six types of enzymes used in Experimental Example 1 were adsorbed onto various adsorbents to adjust the immobilization content and substance. That is, porous adsorbents I (P-20, 30, and 2MG (all manufactured by Mitsubishi Chemical Corporation, trade names) were placed in columns (φ: 1
ox2oomg) and dissolved in water.
~ 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 2 Immobilized Enzyme Activity A substrate skim milk solution was passed through the immobilized protease obtained here at a rate of about 10 m/l'/min at room temperature.
Take the effluent into a test tube and immediately! The milk was placed in a constant temperature bath at 15°C and the curdling time was measured.

I cut it. After curdling, the milk was left to stand for 20 hours, and the recovery rate of curd protein was observed. These results are shown in Table 3 below.

(15) (17) (16) Furthermore, in order to confirm whether or not the card has been overly decomposed,
8D of the protein of the curd left for 0 hours and obtained at that time
The S-' electrophoresis pattern is shown in FIGS. 2 and 3.

In Figure 2, ■ is TCA curd protein (curd tannin precipitated by trichloroacetic acid: control) ■ 1dHP-
20 adsorbed Aspergillus alkaline protease, (Y is HP-2
11 adsorbed renin, (V is HP-2 [] adsorbed pepsin, (d is 1 (P-20 adsorbed trypsin, ■ is HP-20 adsorbed trypsin)
adsorbed pronase, (6Y indicates the s'DFI electrophoresis pattern of card protein by HP-20 adsorbed protoriquiphases. In Fig. 3, σ indicates TAP-2
MG adsorption koji cocoon alkaline zolothiase, ■ “HP-2M
G adsorption rennin, Vuke HP -2M G suction d to i shiy
, (gitrp-2MG adsorbed trypsin, (5'/
' is HP-2MG adsorbed pronase, ω' is HP-2M
() Shows the pattern of curd pulp due to adsorbed protrichuifase.

By immobilizing tease, the recovery rate of curd protein can be increased, and as can be seen from Figures 2 and 3 (the results are similar when immobilized on HP-30, the figures are omitted). By immobilization, the curd protein curdled by each protease becomes TCA.
It can be seen that the curd protein is almost the same as the curd tannin ξ substance ■ or the immobilized rennin ■, and has the effect of preventing over-decomposition of curd protein.

Example 1 Synthetic adsorbent HP-2MG about 5 mm was placed in a column (φ10 x 60
About 0.25 g of an aqueous solution of the alkaline protease fraction (unpurified) obtained by gel filtration of ammonium sulfate fraction from Aspergillus oryzae bran, Sephadex G-25, and Bio-Kel P-150. was passed through the column at room temperature and thoroughly washed with water to immobilize the protease, and the column temperature was maintained at 30°C.

A 10 part skim milk solution was passed through this at a flow rate of 10 d/min at room temperature, and the effluent was collected in 20 d/min portions.

These fractionated effluents were immediately transferred to a constant temperature bath at 35°C, and the curdling time and the recovery rate of the curd protein obtained at that time are shown in Table 4.

Table 41 (Recovery rate of curd milk and curd protein by continuous reaction of P-2M G Aspergillus alkaline protease Example 2 Synthetic adsorbent)
)+m), and a water bath solution of approximately 1.25% of rennin (manufactured by Difco Laboratories, USA) was passed through it at room temperature to obtain immobilized rennin.The column temperature was maintained at 30°C, and a 104 skim milk solution was added. It was passed through at a flow rate of 10 d/min and the effluent was collected in 20 mj 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 5.

Table-5 Milk curd activity of HP-20 immobilized rennin Example 3 Porous synthetic adsorbent) (about 10 mj of P-40 was
10 x 120 m), and this was filled with koji alkaline protease, Amanor P (trade name, Ohno Pharmaceutical Co., Ltd.), about 0.
One gram of aqueous solution is passed through to obtain immobilized protease.

Next, the soymilk was passed through the column at a flow rate of 20 g/min at room temperature, and 2 orul of the treated solution was collected, immediately transferred to a 35° C. constant temperature bath, and the time until aggregation was measured. The results are shown in Table-6.

Table 6: Aggregation time of soymilk by HP-40 immobilized protease.Also, soybean milk was passed backwards to obtain 100 ml of the treated solution, which was divided into 501 portions and mixed without heating.

'C, determine the amount of curd protein after heating for 50 minutes, enzyme is not immobilized) (after passing through P-40,
The curd pulp precipitated with TCA is shown in Table 7 as a percentage of 100.

Table-7

[Brief explanation of drawings]

FIG. 1 is a diagram showing the 5DS-electrophoresis pattern of each card obtained by solution protease in Experimental Example 1. FIG. 2 is a diagram showing the 5ns-electrophoresis pattern of each card obtained by HP-20 adsorbed protease in Experimental Example 2. FIG. 3 is a diagram showing the 8DS-electrophoresis pattern of each card obtained by HP-2MG adsorbed protease in Experimental Example 2. Representative Patent Attorney 1) Parent Male (25) Figure 1

Claims (1)

[Scope of Claims] (1) Immobilized protease obtained by contacting a porous adsorbent with hydrophobic binding force with a protease in an aqueous solvent and adsorbing it to animal milk, skim animal milk, or aggregating protein. By coming into contact with sexual substances. A method for preparing curd, characterized by obtaining curd by coagulation. 121 Proteases include rennin, hafcin, trypsin, papain, and Aspergillus alkaline protease. The method for preparing a curd according to claim 1, wherein the curd is selected from the group consisting of protease enzymes such as actinomycete protease. (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. (41) The curd preparation method according to claim 1, wherein the animal milk and skimmed animal milk are cow's milk and skim milk. (5) The curd preparation method according to claim 1, wherein the aggregating protein substance is soy milk. Preparation method.