JPH0331421B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a method for producing a casein partial decomposition product having new functionality with excellent emulsion stability, foaming property, and solubility. It is effectively used as a food material for foods, drinks, cheese-like foods, etc. Technical Background Casein has conventionally been used as a protein material for various foods by taking advantage of its functional properties, such as emulsifying properties, foaming properties, and solubility. However, these functional properties of casein cannot be said to be satisfactory for use as a food material.
Further, the functional properties of casein can be improved by decomposing it with enzymes, but enzymatic decomposition products of casein are often considered unsuitable as food materials because they produce a bitter taste. Problems to be Solved by the Invention The present invention aims to improve the partial decomposition of casein, which has excellent functional properties, so that casein, which is a nutritionally superior main component of milk protein, can be effectively used as a food material as a protein source. The goal is to provide a manufacturing method for obtaining products. The present inventor has developed a method for improving emulsion stability by partially decomposing casein by applying an immobilized proteolytic enzyme that is stable in a neutral to alkaline region and has excellent heat resistance to casein. The present inventors succeeded in obtaining a casein partial decomposition product that has excellent functional properties such as foamability and foaming stability, and has no bitter taste, leading to the present invention. The present invention will be explained in detail below. Structure of the Invention The present invention is characterized in that casein is partially decomposed using an immobilized protease that is stable in a neutral to alkaline region and has excellent heat resistance. Means for Solving the Problems The immobilized enzyme used in the present invention acts stably and effectively in a neutral to alkaline region, particularly at pH 6 to 9, and has excellent heat resistance with an optimal operating temperature range of 50 to 60°C. An example of such a proteolytic enzyme is Proleather, which is a commercially available enzyme agent. Proleather is a proteolytic enzyme originating from Bacillus bacteria and has almost no peptidase activity. For immobilization of proteolytic enzymes, chitosan or alumina is used as a carrier, and a mixture of the enzyme and glutaraldehyde is immobilized on the carrier, or the enzyme is immobilized on chitosan or alumina treated with glutaraldehyde. Alternatively, the enzyme can be immobilized on a carrier made of chitosan, or by mixing the enzyme with glutaraldehyde and reacting with a carrier made of chitosan to immobilize the enzyme, followed by further treatment with NaBH ₄ . However, from the viewpoint of the stability of the immobilized enzyme, that is, the amount of enzyme released over time during the reaction, the mixture of the enzyme and glutaraldehyde is immobilized by reacting with a chitosan carrier, and then treated with _NaBH4 . The use of the immobilized enzyme is preferred because it has the lowest amount of the enzyme released and has the highest stability. In addition, using alumina as a carrier for the above enzyme,
There is also a method of immobilization using bovine serum albumin (BSA) as a spacer, but the immobilized enzyme obtained in this way is not preferred because the enzyme is easily released during the reaction and has poor stability. Next, the results of examining the stability of each of the immobilized enzymes obtained by the various immobilization methods described above are shown. An immobilized enzyme was prepared by the method shown in Table 1 using the commercially available enzyme agent Proleather as the enzyme.

[Emulsifying capacity]

A 10% solution of casein as the raw material was decomposed using immobilized Proleather and free Proleather, and the resulting decomposition products were freeze-dried.A solution in which casein was dissolved to a concentration of 1% by partial decomposition was prepared.
Emulsification was carried out at 10,000 rpm using a homogenizer (manufactured by Nippon Seiki Co., Ltd.) equipped with a rotational speed control mechanism, and the limit oil amount was measured using the above device to determine the emulsification capacity. Also, regarding the emulsification capacity at each PH,
The emulsifying capacity of the undegraded casein solution whose pH was adjusted with McIlvaine buffer was measured (see Figure 3), and the ratio of the emulsifying capacity of the partially decomposed casein to the emulsifying capacity of the undegraded casein at each pH was determined (see Figure 4). (see Figures and Figure 5). As a result, as shown in Figures 3 to 5,
In the case of partial decomposition products with free proleather, the decomposition rate is
At 1.4%, the emulsifying capacity was higher than that of undegraded casein at pH 6.5 or lower, and at pH 5.5 or lower at degradation rates of 4.2% and 5.6%, but at decomposition rates of 10% or higher, the emulsifying capacity decreased at any pH. was lower than that of undegraded casein. On the other hand, the partially decomposed products of immobilized Proleather showed emulsification capacities equivalent to or about 10% higher than undecomposed casein at pH 5.0 and 5.5, even though the decomposition rates were high at 17.1% and 22%. [Foaming property] Foam was generated using each of the solutions in which the partially decomposed casein was dissolved at a concentration of 1.0% using the apparatus shown in FIG. 2, and changes in the height of the foam over time were examined. Note that the height of the bubbles was expressed as a ratio to the height of the bubbles immediately after formation. The results are shown in the attached Figures 6 and 7. The rate of decrease in foam height with respect to time, that is, the rate of foam decrease, was higher than that of casein with a decomposition rate of 15% compared to undegraded casein. However, it can be seen that partially decomposed casein is slower than other caseins, especially one with a decomposition rate of 22%. Furthermore, at PH7.0, both free Proleather and the partially decomposed product of immobilized Proleather showed high foaming power values due to undecomposed casein, and these foaming powers were almost constant regardless of the decomposition rate. It was hot. The foaming power of the partially decomposed product of free Proleather was the same as that of undecomposed casein at pH 6.0, or was slightly lower. As for foaming stability, as shown in Figure 8, the highest decomposition rate is 22% for the partially decomposed product made of free Proleather, while the highest decomposition rate is 15% for the product made of immobilized Proleather. It showed a high value. [Osmotic pressure] Osmotic pressure is proportional to the number of molecules contained in the solution. Therefore, the osmotic pressure of the partially degraded product is higher than that of undegraded casein, and its freezing point is lowered. Therefore, we measured the osmotic pressure, considering the possibility of its application as a freezing point depressant. The results are shown in Table 2, and the osmotic pressure of the partially decomposed product by immobilized Proleather increased as the decomposition rate increased;
On the other hand, with free Proleather, the osmotic pressure increased up to a decomposition rate of 15%, but at higher decomposition rates it became lower than that of undegraded casein.

[Solubility]

The relationship between the solubility of partially decomposed casein (amount of nitrogen in the supernatant liquid/amount of nitrogen in the dispersion liquid) and the decomposition rate is
Shown in Figure 0. As shown in the figure, the decomposition rate of partially decomposed casein using immobilized ProLeather was 22.2%.
However, when free Proleather was used, insoluble matter was rapidly generated at a decomposition rate of 20%, and it was observed that the amount increased as the decomposition rate increased. As can be seen from the above experimental results, according to the present invention, an immobilized enzyme obtained by immobilizing casein with a proteolytic enzyme that is stable in a neutral to alkaline region and has excellent heat resistance, such as Proleather. Partially decomposed casein having desired functional properties can be obtained by performing partial decomposition using a method and controlling the layer decomposition rate. That is, the partially decomposed casein obtained by the present invention is
It can be seen that the emulsifying capacity, foaming power and foaming stability are improved compared to undecomposed casein, while the viscosity is also reduced. Although partially degraded casein obtained by partially decomposing casein using a free, unimmobilized proteolytic enzyme has improved overall functional properties compared to undegraded casein, There is a substantial difference compared to when using In other words, when using free enzymes, the emulsifying capacity is higher than undegraded casein at a low degradation rate of 1.4 to 5.6%, but as the degradation rate advances, the emulsifying capacity actually decreases, whereas when using immobilized enzymes, emulsifying capacity decreases as the degradation rate progresses. It exhibits higher emulsifying capacity than undegraded casein with a wide range of degradation rates from 8.3 to 22.2%,
It can be seen that the emulsifying power is excellent. Furthermore, casein partially degraded by immobilized enzymes shows almost 100% solubility even when the degradation rate reaches 22%.
It can also be seen that the solubility is excellent when using an immobilized enzyme, since when using a free enzyme, an insoluble substance consisting of a polymer is rapidly generated at a decomposition rate of 20%. In addition, as mentioned above, there are differences between the two in terms of functionality other than the above, but this means that the molecular weight of the partially degraded product obtained when decomposing with an immobilized enzyme and with a free enzyme is This is thought to be due to the difference in distribution, and even at the same degradation rate, the amount of residual undegraded casein and the amount of low-molecular-weight decomposition products produced are greater in the partially degraded product using the immobilized enzyme. This difference in decomposition is due to the fact that in a free enzyme, both the enzyme and the substrate can freely diffuse, so the enzyme cleaves the substrate from the easily cleavable part, whereas in the case of an immobilized enzyme, the enzyme cleaves from the part of the substrate that is easy to cleave. It is also thought that the substrates that are restrained and have entered the pores of the carrier are also restricted from diffusing, so that the substrates that have entered the pores are completely decomposed and many low-molecular-weight peptides are produced. Next, FIG. 11 shows the molecular weight distribution of each partially decomposed casein and undegraded casein prepared from free Proleather and immobilized Proleather. As shown in the figure, a peak was observed at a molecular weight of 160,000 for undegraded casein, but when this casein was degraded with free Proleather, the peak shifted to approximately 8,000 and 35,000, indicating that these decomposed casesin had a peak at a molecular weight of 160,000. It became higher as the rate increased. Also, the decomposition rate is 15
%, most of the undegraded casein disappeared. In contrast, with partially decomposed casein produced by immobilized ProLeather, undecomposed casein has a decomposition rate of 14%, which is approximately 1/2 that of undecomposed casein.
It was also observed that at a decomposition rate of 1%, about 1/3 remained. Similar to the partial decomposition product of free Proleather, there was a peak at a molecular weight of approximately 35,000, and a peak was also observed at a lower molecular weight that was not observed in free Proleather.
In addition, in order to compare the amount of peptides on the low molecular side,
Table 3 shows the results of measuring the amount of nitrogen in supernatants prepared with varying TCA concentrations.

[Table] As shown in Table 3, the decomposition rates are different for partially decomposed casein produced by free Proleather and immobilized Proleather. % or higher TAC concentration showed high values. Next, the influence of substrate concentration on the reaction rate in partial decomposition of casein in the present invention will be explained. The following experiment was conducted to investigate the above effects. Experimental method: Acid casein at 4%, 6%, 8%, 10% and 12%
Using substrates prepared at various concentrations, immobilized Proleather was reacted with each substrate 1 at 50 °C under stirring at 247 rpm, and the relationship between the substrate concentration and the amount of partially decomposed casein produced over time was determined. Ta. Fixed Proleather is made by reacting a mixture of Proleather and glutaraldehyde with a chitosan carrier, fixing it, and then treating it with NaBH ₄ .
Stabilized by reacting with 10% casein solution three times,
Then, it was washed with water and used. The results of the above experiment are shown in FIG. As seen from the figure, the amount of partially decomposed casein produced over time, that is, the production rate of soluble nitrogen, decreased as the substrate concentration increased. In addition, 4%
Solutions with a substrate concentration of 12% differ widely in viscosity, with the latter being about 38 times that of the former. In addition, in the above experiment, the results of investigating the production rate of the above soluble nitrogen when changing the stirring rotation speed during the reaction are shown in Figure 14.
At a substrate concentration of 4%, there was no change in the production rate of soluble nitrogen depending on the rotational speed, but at a substrate concentration of 10%, the production rate tended to increase as the rotational speed increased. That is, this shows that as the concentration of the casein solution as a raw material increases, the viscosity also increases, and therefore the reaction is greatly influenced by diffusion from the outside. In the present invention, when casein is partially decomposed using the above-mentioned immobilized enzyme, when a casein solution is passed through a reactor in which the immobilized enzyme is set for a long time, bacterial contamination of the enzyme may occur because casein is a protein. This poses a practical problem. In order to prevent this, it is necessary to determine operating conditions that make it difficult for contaminating bacteria to grow and to carry out periodic sterilization treatments. However, using strong acids or strong alkalis for sterilization is undesirable because it deactivates enzymes, so the present invention According to the results of tests conducted by researchers, it can be said that sterilization using nonionic surfactants is suitable. As described above, according to the present invention, it is possible to obtain partially decomposed casein having functional properties not seen before, and by selecting the conditions for partial decomposition of casein, it is possible to obtain particularly excellent emulsifying capacity and foaming power. Since partially decomposed casein can be obtained, it can be said to be useful for the use of milk protein as a food material. EXAMPLES The present invention will be specifically described below with reference to Examples. Example 1 Proleather (17 units/g, wet) immobilized on alumina (pore size: 100-500 Å) was packed into a column, and a 10% casein solution was added to it at 50°C, pH 7,
A partially decomposed product with a decomposition rate of 20% was prepared by passing the solution through SV18. This partially decomposed product has a pH higher than that of undegraded casein.
The emulsifying capacity at PH5.5 is 1.1 times, the emulsifying capacity at PH5.0 is 1.05 times, and the foaming power at PH7 is 1.2
The foaming stability is twice as high. Example 2 Proleather (17.1 unit/g, wet) immobilized on chitosan (pore size: 3μ) was packed in a column,
A 10% casein solution was passed through this at 50°C, PH7, and SV34 to prepare a partially degraded product with a decomposition rate of 6.8%. This partially decomposed product has the same emulsifying capacity at pH 5.5 and foaming power at pH 7 as undecomposed casein, but has three times the foaming stability.

[Brief explanation of drawings]

Figure 1a shows the relationship between reaction time and decomposition rate when an enzyme treatment solution and a casein solution are reacted for enzymes immobilized by various immobilization methods. Indicates stability. Figure 1b shows the relationship between the retention time and the decomposition rate when a casein solution is passed through a column packed with immobilized enzymes and then held at a constant temperature, indicating the degree of enzyme detachment. . FIG. 2 illustrates an apparatus for measuring the foamability of a partially decomposed product obtained according to the present invention. Figure 3 shows the relationship between the emulsification capacity of undegraded casein and PH, Figure 4 shows the relationship between the decomposition rate of casein using free enzymes and the emulsification capacity of partial degradation products, and Figure 5 shows the relationship between emulsification capacity and PH when immobilized enzyme is used. Figure 6 shows the relationship between foaming power and casein decomposition rate of partial decomposition products when free enzymes are used, and Figure 7 shows the relationship between foaming power and decomposition rate when immobilized enzymes are used. are shown respectively. FIG. 8 shows the relationship between foaming stability and decomposition rate when using free enzyme and immobilized enzyme. FIG. 9 shows the relationship between viscosity and decomposition rate when both of the above enzymes are used, and FIG. 10 similarly shows the relationship between solubility and decomposition rate. FIG. 11 shows the relationship between the molecular weight distribution and decomposition rate of partial decomposition products obtained when free enzyme and immobilized enzyme were used. FIG. 12 shows the relationship between the concentration of casein used as a substrate and the amount of partial decomposition products produced, that is, the production rate of soluble nitrogen, with respect to the reaction time, when an immobilized enzyme is used.
Figure 3 shows the relationship between casein concentration, rotation speed, and production rate of solubilized nitrogen when an immobilized enzyme is used.

Claims (1)

[Scope of Claims] 1. A functional property characterized by partially decomposing casein with an immobilized protease that is stable in a neutral to alkaline region and has excellent heat resistance. A method for producing a casein partial decomposition product having the following. 2. A mixture of a proteolytic enzyme, in which the immobilized enzyme acts particularly effectively at a pH of 6 to 9 and a temperature of 50 to 60°C, and has almost no peptidase activity, is mixed with glutaraldehyde and is mixed with a carrier made of alumina or chitosan. After reacting and immobilizing,
The method according to claim 1, which is prepared by treatment with sodium borohydride (NaBH ₄ ). 3. The production according to claim 1 or 2, in which a 10% solution of casein is passed through a column packed with a proteolytic enzyme immobilized on an alumina carrier, and partially decomposed to a decomposition rate of 5 to 20%. Law. 4. The manufacturing method according to claim 1 or 2, in which a 10% solution of casein is passed through a column packed with a protease immobilized on a chitosan carrier, and partially decomposed to a decomposition rate of 2.5 to 10%. .